# A Comprehensive Review on Phytochemistry and Pharmacological Activities ofClinacanthus nutans (Burm.f.) Lindau

Authors: Leng Wei Khoo; Siew Audrey Kow; Ming Tatt Lee; Chin Ping Tan; Khozirah Shaari; Chau Ling Tham; Faridah Abas
Journal: Evidence-Based Complementary and Alternative Medicine
 (2018)

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## Abstract

Clinacanthus nutans(Burm.f.) Lindau (Acanthaceae), commonly known as Sabah snake grass, is a vegetable and a well-known herb that is considered an alternative medicine for insect bites, skin rashes, herpes infection, inflammation, and cancer and for health benefits. Current review aims to provide a well-tabulated repository of the phytochemical screening, identification and quantification, and the pharmacological information ofC. nutans according to the experimental design and the plant preparation methods which make it outstanding compared to existing reviews. This review has documented valuable data obtained from all accessible library databases and electronic searches. For the first time we analyzed the presence of flavonoids, triterpenoids, steroids, phytosterols, and glycosides inC. nutans based on the results from phytochemical screening which are then further confirmed by conventional phytochemical isolation methods and advanced spectroscopic techniques. Phytochemical quantification further illustrated thatC. nutansis a good source of phenolics and flavonoids. Pharmacological studies onC. nutans revealed that its polar extract could be a promising anti-inflammation, antiviral, anticancer, immune and neuromodulating, and plasmid DNA protective agent; that its semipolar extract could be a promising antiviral, anticancer, and wound healing agent; and that its nonpolar extract could be an excellent anticancer agent.


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## Body

## 1. Introduction

Utilizing plants as a source of medicines has been practiced for a long time, especially in developing countries where drugs are usually inaccessible or costly, obligating people to use traditional remedies. Vast development in investigating the herbal plants as alternative therapeutic agents for different type of diseases is notable, starting from the late 1980s and continuing in recent years. Malaysia, a country with rich biodiversity, has been gifted with valuable medicinal plants resources in its tropical rainforest. Among them, Acanthaceae is considered as one of the leading families of the dicotyledonous flowering plants and consist of 250 genera and approximately 2500 species [1]. This family is mainly distributed in Indonesia, Malaysia, Africa, Brazil, and Central America and possesses numerous high medicinal value species [2, 3].Clinacanthus nutans(Burm.f.) Lindau, which belongs to the Acanthaceae family, is a very well-known traditional herb and vegetable in Southeast Asia countries and has been chosen for this review study [4]. It has a few vernacular names such as “belalai gajah” or “pokok stawa ular” (Malay), “e zui hua” or “you dun cao” (Chinese), “payayor” or “slaed pang pon” (Thai), and “daun dandang gendis” or “kitajam” (Javanese). Since a long time ago, this plant is famous as an antidote for snake bites, herpes infection, skin infection, cancer, burns and scalds, dysentery, and diabetes in Thailand, Indonesia, and Malaysia [5–8]. Numerous studies on this plant have been published by researchers from Thailand since 1967. Since 1987,C. nutans has become one of the herbs recommended for use in hospitals and was included in the Primary Health Care Programme by the Government of Thailand [9, 10]. The most used part of the plant was the leaf, and the common prescription method was decocting the plant with water for oral ingestion or soaking it in the alcohol for a week for topical application to an affected area [11]. In 2011, this plant began gaining popularity in Malaysia, and various types ofC. nutans related studies have been conducted by Malaysian scientists.Authors noticed the publication of reviews on this plant, where some of them also discussed the topics chosen in this paper [2, 12–16]. However, authors target to provide a well-managed summary and possible recommendation on the existing studies so that the readers could acquire much information regarding the current and future direction of this plant from present review just by a glance. Thus, a well-organized and critical review paper on this plant is in demand to reduce redundant studies and allow new insight into the research direction to be made. Therefore, instead of discussing a few selected studies, present review discusses and covers thoroughly the previousC. nutans studies related to the topics chosen. The details such as pre- and postharvesting preparation methods of the plant extract as well as the experiment design applied in the studies of phytochemical screening, identification and quantification, and pharmacological activities ofC. nutans are systematically categorized, compared, and summarized. Information on the preharvesting (plant age and plant origin) and postharvesting (plant part used, drying method, extraction method, extraction solvent, and storage duration) methods of the plant as well as on the experiment design employed was considered and was emphasized in the discussion section which makes present review different from the others. Different plant preparation methods and experimental designs will result in the generation of different conclusions, even for the same plant. Thus, including and correlating all variable factors and parameters in the discussion will help to determine the pro and cons of each study and lead to a better summary in the end. We hypothesized that, through all these efforts, a good summary, precautionary step, challenge, limitation, new idea, or a clearer future perspective may be initiated.


## 2. Phytochemistry

### 2.1. Phytochemical Screening

For the last few decades, the study of plants has progressed rapidly [17]. Plants contain both primary and secondary metabolites. Primary metabolites are those chemical constituents that are inherently present in most organisms and have a direct involvement in plant growth [18]. On the other hand, secondary metabolites are the bioactive phytochemicals that are normally produced by a plant in response to specific environmental stresses and that are considered to possess additional health benefits [18]. Throughout the years, the common practice of researchers was started with the screening of plant. Phytochemical screening tests, thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) are among the most popular methods used as they are easy to conduct, of low cost, and time-effective. These methods are able to provide guidelines on the class and functional groups of the chemical constituents that are present in the plant.Table1 lists the occurrence of chemical classes inC. nutans extract, as determined through specific phytochemical screening tests. The phytochemical screening tests adopted include Mayer’s reagent, modified Dragendorff’s reagent, or the Wagner test for alkaloid detection; the frothing test for saponin, the alkaline reagent, Shinoda, or lead acetate tests for flavonoids; the frothing, ferric chloride, potassium dichromate, or gelatin tests for tannins; the copper acetate test for diterpenes; Salkowski’s test, or Liebermann-Burchard test for phytosterols; the iron (III) chloride test for phenolic compounds; the pseudoindicans test for iridoids; Grignard reagent, Baljet, Keller-Kiliani, or Molisch tests for glycosides; Molisch or Fehling tests for carbohydrates; Salkowski’s test for steroids and sterols; and the biuret test for proteins and amino acids [6, 19–22]. As a summary, flavonoids, triterpenoids, steroids, phytosterols, and glycosides are the phytochemical classes that are most likely present inC. nutans extract when the plant is extracted by a polar solvent, such as water, methanol, ethanol, or an aqueous organic solvents or by a semipolar (e.g., chloroform) solvent. The presence of alkaloids, saponins, and tannins inC. nutans depends on the plant origin and the postharvesting method used. For instance, alkaloids were typically present in water and chloroform leaf extracts and were absent in methanol and aqueous methanol leaf extracts collected from Malaysia. By contrast, saponins were present in methanol extract cultivated in Malaysia and absent in water extract from Indonesia and chloroform and aqueous methanol extracts from Malaysia. Tannins were found in leaves extracted with water, aqueous methanol, and chloroform, but not those extracted with methanol. For the presence of quinones, iridoids, carbohydrates and protein, and amino acid, further confirmation is required as there has been only one study conducted on each of them to date.Table 1
Phytochemical screening test ofC. nutans.
Chemical class Origin Extract Present/Absent Alkaloid Indonesia1 Leaf-water extract1 Present1 Malaysia2,3,5 Leaf-100% methanol extract2,5 Absent2,5 Leaf-100% chloroform extract3 Presence3 Leaf-70% methanol extract4 Absent4 Saponin Indonesia1 Leaf-water extract1 Absent1 Malaysia2,3,4,5 Leaf-100% methanol extract2, 5 Present2,5 Leaf-100% chloroform extract3 Absent3 Leaf-70% methanol extract4 Absent4 Flavonoids Indonesia1 Leaf-water extract1 Present1 Malaysia2,3,4,5 Leaf-100% methanol extract2,5 Present2,5 Leaf-100% chloroform extract3 Present4 Leaf-70% methanol extract4 Present3 Triterpenoids Indonesia1 Leaf-water extract1 Present1 Malaysia5 Leaf-100% methanol extract5 Present2 Diterpenes Malaysia2 Leaf-100% methanol extract2 Present Steroids Indonesia1 Leaf-water extract1 Present1 Malaysia4,5 Leaf-70% methanol extract4 Present2,3 Thailand7 Leaf-100% methanol extract5 Present5 Stem-Light petroleum extract7 Present7 Phytosterol Malaysia2,4 Leaf-100% methanol extract2 Present1 Leaf-70% methanol extract4 Present2 Tannin Indonesia1 Leaf-water extract1 Present1 Malaysia2,3,4,5 Leaf-100% methanol extract2,5 Absent2,5 Leaf-100% chloroform extract3 Present3 Leaf-70% methanol extract4 Present4 Quinone Indonesia1 Leaf-water extract1 Absent Phenolic compound Malaysia2 Leaf-100% methanol extract2 Present Glycosides Malaysia3,4 Leaf-100% chloroform extract3 Present1 Leaf-70% methanol extract4 Present2 Iridoids Thailand6 Leaf6 Present6 Carbohydrates Malaysia4 Leaf-70% methanol extract4 Present4 Protein and amino acids Malaysia4 Leaf-70% methanol extract4 Present4 Reference. 1Nurulita et al. [25]; 2Yang et al. [6]; 3Goonasakaran [26]; 4Sekar and Rashid [22]; 5Abdul Rahim et al. [19]; 6Keawpradub and Purintrapiban [20]; 7Dampawan et al. [27].In 2012, Roeslan and his teams [11] compared leaves from two different sources, Thailand and Indonesia, using TLC separation method. Their findings showed that there was a quality difference and indicated that the quality of the sample from Indonesia was better, as this sample was thicker than the sample from Thailand. Chelyn et al. [23] used the HPTLC method to screen for the presence ofC-glycosyl flavones in threeC. nutans plant samples that underwent the same postharvesting processing but that were from three different geographical locations in Malaysia: Taiping (Perak), Kota Tinggi (Johor), and Sendayan (Negeri Sembilan). All the results indicated that schaftoside was present inC. nutans collected from all the investigated locations, whereas isoorientin, isovitexin, orientin, and vitexin were found only in the plants harvested from Perak and Johor, not in the sample harvested from Negeri Sembilan. Tiew et al. [24] used UV-visible and FTIR spectrophotometry for the functional group identification ofC. nutans leaves dried under the shade and macerated with methanol. Based on the results obtained, the extract showed two major bands 240–400 nm and the presence of an -OH band and a C=O stretch suggests that the constituents in the extract have -OH group and C=O functional groups.


### 2.2. Phytochemical Identification

Phytochemical screening methods can only provide a clue of the chemical class present in the plant. To further validate the identity of the compounds, column chromatography (isolation) and TLC (separation and purity) together with nuclear magnetic resonance (NMR) spectroscopy (identification and structural elucidation) are the most conventionally used techniques. In addition, the emergence of advanced analytical tools such as liquid chromatography mass spectrometry (LCMS), gas chromatography mass spectrometry (GCMS), and NMR spectroscopy provides researchers with useful alternatives for identification.


#### 2.2.1. Phytochemical Identification Using Column Chromatography-Spectroscopy Analysis

A summary of the phytochemicals that have been identified in this plant is provided in Tables2(a) and 2(b). Table 2(a) displays those compounds that have been isolated through column chromatography and identified using spectroscopic analysis, the results from which were tabulated according to the phytochemical class. To date, the pure compounds that have been isolated fromC. nutans include 2 triterpenoids; 6 phytosterols; 8 phenolics, including 7C-glycosyl flavones and 1 phenolic acid; 5 sulfur-containing glucosides; 8 sulfur-containing compounds; 7 lipid related compounds; and 8 chlorophyll derivatives.Table 2
Phytochemicals (primary and secondary metabolites) that presented inC. nutans.
(a)
Phytochemical identification based on column chromatography isolation and spectroscopy structural elucidation
Phytochemical Plant part Postharvesting method Extract/Fraction Plant origin Reference Terpenes-Triterpenoids  Lupeol Stem2 Air Dry; Soxhlet2 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3 Oven Dry; Soaking3 Light petroleum extract2 Seremban Malaysia3 [27]2 Hexane fraction of methanol extract3  [45]3 Betulin Root - - China [46]  Terpenes-Phytosterols  β-sitosterol Stem2 Air Dry; Soxhlet2 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3 Oven Dry; Soaking3 Light petroleum extract2 Seremban Malaysia3 [27]2 Hexane fraction of methanol extract3  [45]3 β-Sitosterol-3-O-β glucopyranoside Stem Sun Dry; Soaking Sub-fraction of methanol extract Vietnam [47] β-Sitosterol-3-O-β glucoside Stem Sun Dry; Soaking Sub-fraction of methanol extract Vietnam [47] Stigmasterol Stem2 Oven Dry; Soaking3 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3  Light petroleum extract2 Malaysia3 [48]2 Hexane fraction of methanol extract3  [45]3 Stigmasterol-β-D-glucoside Aerial Sun Dry; Maceration Methanol fraction of 96% ethanol extract Vietnam [49] Stigmasteryl-3-O-β-D-glucopyranoside Leaf Oven Dry; Soxhlet Chloroform extract Thailand [9]  Phenolics compounds  Shaftoside1,2,3, vitexin1,2, isovitexin1,2, isomollupentin 7-O-β-glucopyranoside1, orientin1,2, isoorientin1,2, gallic acid2, apigenin 6,8-di-C-α-L-arabinopyranoside2 Aerial1,2 Reflux2 Butanol soluble portion of methanol Thailand1 [50]1 Oven Dry; Soaking3 extract1 Seremban Malaysia2,3 [33]2 Leaf3 - 30% ethanol extract2  [45]3 Hexane fraction of methanol extract3  Sulfur-containing glucosides compounds  Clinacoside A, Clinacoside B, Clinacoside C, Cycloclinacoside A1, Cycloclinacoside A2 Aerial - Butanol soluble and aqueous soluble portion of methanol extract Thailand [51]  Sulfur-containing compounds  Clinamides A1, Clinamides B1, Clinamides C1, Clinamides D2, Clinamides E2, 2-cis-entadamide A1, Entadamide A1, Entadamide C1 Aerial1 Air Dry; Soaking1 80% Ethanol extract1 Taiwan1 [52]1 Whole plant2 Sonication2 Methanol extract2 Jelebu, Malaysia2 [53]2  Lipids  Myricyl alcohol - - Petroleum ether extract Thailand [44] 1,2-O-dilinolenoyl-3-O-β-D-galactopyranosyl-glycerol, 1-O-palmitoyl-2-O-linolenoyl-3-O-(α-D-galactopyranosyl-(1′′→6′)-O-β-D-galacctopyranosyl)-glycerol Leaf - - Thailand [54] Monoacylmonogalactosylglycerol, a mixture of nine cerebrosides Leaf - Ethyl acetate-soluble fraction of 95% ethanol extract Thailand [55] Monogalactosyl diglyceride, digalactosyl diglyceride Leaf Soxhlet extraction Chloroform extract Thailand [56]  Chlorophyll a and b derivatives  Purpurin 18 phytyl ester1,2,4 Phaeophorbide a1,2,4  132-hydroxy-(132-S)-chlorophyll b3,4 132-hydroxy-(132-R)-chlorophyll b3,4 132-hydroxy-(132-S)-phaeophytin b1,2,4 132-hydroxy-(132-R)-phaeophytin b3,4 132-hydroxy-(132-S)-phaeophytin a3,4 132-hydroxy-(132-R)-phaeophytin a3,4 Leaf1,2,3,4 Oven Dry; Soxhlet1,2,3,4 Chloroform extract1,2,3,4 Thailand1,2,3,4 [57]1 [17]2 [9]3 [58]4  Others  trans-3-methylsulfinyl-2-propenol Aerial Air Dry; Soaking 80% Ethanol extract Taiwan [52] 3-amino-4,5-dihydroxyfuran-2(3H)-one Aerial Sun Dry; Maceration Methanol fraction of 96% ethanol extract Vietnam [49] polysaccharide–peptide complex Leaf Oven Dry Hot water and 75% ethanol precipitation extract Seremban Malaysia [59]  (b)
Phytochemical identification based on spectrometry and spectroscopy identification
Phyto-constituents Plant part Post-harvesting method Extract Plant origin Analytical tools Reference 2-ethyl-oxetane, 9,12,15-octadecatrienoic acid, 2,3-dimethylpyridine, 3-deoxyd-mannoic lactone, neophytadiene, phytol, 2,3-dihydrobenzofuran,n-hexadecanoic acid Leaf Oven dry; Soaking Abs methanol Kuala Lumpur Malaysia GCMS [19] n-Pentadecanol, eicosane, 1-nonadecene, heptadecane, dibutylphthalate, n-Tetracosanol-1, heneicosane, behenic alcohol, 1-heptacosanol, 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, nonadecyl heptafluorobutyrate, eicosayl trifluoroacetate, 1,2-benzenedicarcoxylic acid, dinonyl ester, phthalic acid, dodecyl nonylester Leaf Oven dry Chloroform Selangor Malaysia GCMS [32] Squalene,β-tocopherol, vitamin E, Campsterol, stigmasterol, γ-sitosterol, β-amyrin, α-amyrin, lupeol, propanoic acid, betulin Leaf, stem Combination of air dry and freeze dry; Maceration Abs methanol, abs ethyl acetate Sabah Malaysia GCMS [29] Lactic Acid, Glycolic acid, 3-Pyridinol, Glycerol, Phosphonic acid, Catechol, Butanedioic acid, Glyceric acid, Erythrono-1,4-lactone, (Z), Malic acid, Benzaldehyde, 3-methoxy-4-[(trimethylsilyl)oxy]-, O-methyloxime, Cyclooctasiloxane, Levoglucosan, Vanillic Acid, D-Ribo-Hexonic acid, 3-deoxy-2,5,6-tris-O-(trimethylsilyl)-, lactone, D-(-)-Fructofuranose, pentakis(trimethylsilyl) ether (isomer 1), Myristic acid, D-Pinitol, Neophytadiene, D-Fructose, 1,3,4,5,6-pentakis-O-(trimethylsilyl)-, O-methyloxime, Syringic acid, D-Fructose, 1,3,4,5,6-pentakis-O-(trimethylsilyl)-, O-methyloxime, d-Glucose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)-, o-methyloxyme, (1Z)-, Gallic acid, Muco-Inositol, D-Glucose, Heptadecanoic acid, Phytol, 9,12-Octadecadienoic acid (Z,Z)-, Oleic Acid, L-Rhamnose, Cyclononasiloxane, octadecamethyl-, Octadecane-1,2-diol, Ricinoleic acid, Arachidic acid, L-Rhamnose, 1,2-Benzenedicarboxylic acid, mono(1-methylheptyl) ester, (+/−),1-Monopalmitin, Cyclononasiloxane, octadecamethyl-, 1-Monolinolein, Glycerol monostearate, Squalene,β-Gentiobiose, B-tocopherol, γ-tocopherol, α-Tocopherol, Campesterol Stem Air dry; Supercritical-soxhlet extraction - Seremban Malaysia GC-QTOF-MS [28] Vitamin E, campesterol, stigmasterol,γ-Sitosterol, β-Sitosterol, β-amyrin, lupeol, betulin Root Freeze dry; Soaking Methanol Sabah Malaysia GCMS [31] Oleic acid, squalene, vitamin E, campesterol, stigmasterol,γ-stosterol, β-sitosterol, lup-20(29)-en-3-one, β-amyrin, lupeol, lup-20(29)-en-3-ol-acetate, betulin Root Freeze dry; Soaking Ethyl acetate Sabah Malaysia GCMS [31] Neophytadiene, iron, 7,9-Dodecadien-1-ol, Myristic acid, Palmitic acid, Palmitic acid-methyl ester, Benzenethanol, Phytol, Squalene, Stearic acid-methyl ester, Margaric acid-ethyl ester, Lupeol, Linoleic acid-ethyl ester, Linolenic acid-methyl ester, 2-Butanol, Butanamide, 2-cyclopenten-1-one-2-hydroxy, Glycine, Pentanal, Isoveraldehyde, Dimethyl trisulfide, Thiophene, Succinic acid, Glycolic acid, Oxazolidine, Thiophene, 9-Azabicyclo (6.1.0) non-4-4en-9-amine, 4-Vinyl-2-methoxy-phenol, Phenol,2,6-dimethoxy, 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, Glycerine, 4-Vinylphenol Aerial Oven dry; MAE, SFE, Soxhlet Aqueous ethanol Kuala Lumpur Malaysia GCMS [30] Isoorientin, orientin, isovitexin, vitexin, schaftoside, 6, 8-apigenin-C-α-L-pyranarabinoside Aerial - 30% Ethanol Seremban Malaysia HPLC LCMS/MS [33] Kaempferol-7-neohesperidoside, Isoschaftoside, Isoorientin, Vitexin, 3′,7-Dimethoxy-3-hydroxyflavone, 2′,6-Dihydroxyflavone, (+)−Catechin, 7-Hydroxyflavone, Gallic acid, Flavanone-7-O-glycoside Aerial Oven dry; MAE, SFE, Soxhlet 86% Ethanol,50% Ethanol KL Malaysia UPLC-ESI-QTOF/MS [30] Schaftoside, Arabinoysl–glucosyl apigenin isomer, Ascorbic acid, Gendarucin A, Gendarucin A isomer, 3, 3-di-O-methylellagic acid, Methyl 2-[cyclohex-2-en-1-yl(hydroxyl) methyl]-3-hydroxy-4-(2-hydroxyethyl)-3-methyl-5-oxoprolinate, Methyl 2-[cyclohex-2-en-1-yl(hydroxyl) methyl]-3-hydroxy-4-(2-hydroxyethyl)-3-methyl-5-oxoprolinate isomer Leaf Air dry; Sonication 70% Ethanol Seremban Malaysia UPLC-MS/MS [4] Gallic acid, 4-hydroxybenzoic acid, caffeic acid, coumaric acid, ferulic acid, schaftoside, vitexin, orientin, isoorientin, isovitexin, luteolin, apigenin, forsythosides H, forsythosides I, diosmetin glycoside, diosmetin. Leaf Oven dry; Soaking 100% Methanol Kuala Lumpur Malaysia UHPLC-MS [19] Primary metabolites: Fructose, α-Glucose, β-Glucose, Sucrose, a mixture of cerebrosides, Monoacylmonogalactosylglycerol, Alanine, Glutamate, Glutamine, Proline, Threonine, Tryptophan, Valine, Citric acid, Formic acid, Fumaric acid, Choline, Adenine, Fatty acid, Ascorbic acid Secondary metabolites: Betulin, Lupeol, Stigmasterol, β-sitosterol, Clinacoside A, Clinacoside B, Clinacoside C, Cycloclinacoside A1, Cycloclinacoside A2, Isoorientin, Isovitexin, Orientin, Vitexin, Schaftoside, Gendarucin A, Catechin, Quercetin, Quercetin 3-O-rhamnoside, Quercetin 3-O-arabinofuranoside, Rutin, Chlorogenic acid, Gallic acid Leaf, Stem Air dry, Oven dry, Freeze dry; Sonication, Soaking 70% Ethanol Seremban Malaysia 1H-NMR [4] 1, 2, 3, 4: in each phytochemical class, every superscript number attached to a particular phytochemical, plant part, postharvesting method, extract/ fraction, or plant origin indicated that such information is extracted from the reference which has attached with the same superscript number.


#### 2.2.2. Phytochemical Identification Using Advanced Spectrometric and Spectroscopic Analysis

Meanwhile, Table2(b) shows a list of compounds identified according to the type of the advanced spectrometric and spectroscopic analytical tools used. There are a total of 6 research teams that have used GCMS for compound identification [19, 28–32], 4 research teams that have used LCMS analytical tool [4, 19, 30, 33], and 1 research team that has used proton andJ-resolved NMR approaches [4]. Based on GCMS identification results, Cheong et al. [29] found a rich variety of triterpenoids and phytosterols in the extract. Yong et al. [32] showed that of the 14 phytochemicals identified in the chloroform extract, 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl)ester, a common plasticizer, was the most abundant compound. Mustapa et al. [30] found that phytol was the main compound in the microwave (MAE) and Soxhlet assisted extracts, while palmitic acid was dominant in the supercritical fluid assisted (SFE) extract. A study by Abdul Rahim et al. [19] identified a total of 39 peaks, but only 8 major compounds have been listed in Table 2(b). In addition, Alam et al. [28] have successfully identified a total of 48 compounds, the most abundant of which were neophytadiene, glycolic acid, phosphonic acid, catechol, butanedioic acid, vanillic acid, gallic acid, pinitol, phytol, squalene, tocopherols, and campesterol. Moreover, Teoh et al. [31] have found that the ethyl acetate root extract possessed more compounds than the methanol root extract. Among the compounds identified, lupeol was the main compound present in both extracts.Using LCMS in negative ionization mode, Huang et al. [33] successfully identified 6C-glycosyl flavones, the research team of Khoo et al. [4] was able to identify 8 compounds, Mustapa et al. [30] team have found 10 phenolic compounds from the aqueous ethanolic extract, and Abdul Rahim et al. [19] have detected 16 phenolic compounds. Khoo et al. [4] have also tentatively identified 20 primary metabolites and 22 secondary metabolites in 70% ethanol extract through proton andJ-resolved NMR spectroscopy.


### 2.3. Phytochemical and Nutrient Content

Environmental condition, geographical location, cultivation practices, genetics, pre- and postharvesting methods practiced and some other unexpected factors will cause phytochemical level variations in a plant even in the same species [18]. Thus, this section reveals the phytochemical content ofC. nutans including the total phenolic content (TPC) in Table 3, the total flavonoid content (TFC) in Table 4, and the nutritional composition ofC. nutans in Table 5. Generally, the phytochemical content ofC. nutans has been evaluated using simple chemical assay or high performance liquid chromatography (HPLC) methods. Compared to other tests, determination of TPC and TFC is preferred due to the interest in the therapeutic benefit imparted by phenolics and flavonoid; in addition, they are easy, time-effective, and cost-effective and well-established methods.Table 3
Total phenolic content (TPC) ofC. nutans.
Extract Plant part Pre- or post-harvesting method Plant origin Results of TPC Extract with highest TPC Extract with lowest TPC Reference Hot water - Oven dry; Heating at 90°C Malaysia 14.70 ± 0.08 mg GAE/g DM - - [37] Hot water Leaf Unfermented and fermented; Microwave-oven dry, freeze dry; Hot boiling water (100°C); Infusion for 1, 2, 5, 10, 15, and 20 min Sabah Malaysia 88.56 ± 4.40 to 177.80 ± 19.10 mg TAE/L Unfermented-microwave oven-20 min infusion Fermented-freeze dry-1 min infusion [36] Distilled water Leaf Combination of air dry and oven dry; Maceration with shaker-0.5, 1, 3, 5, 24 h Thailand 26.53 ± 8.83 to 46.71 ± 9.31 mg GAE/g DW Maceration for 1 h Maceration for 5 h [60] Hot water (70°C), water, methanol, 80% methanol, ethyl acetate, hexane Leaf, stem Freeze dry; Sonication Seremban Malaysia 23.15 ± 2.78 to 73.33 ± 12.18 mg GAE/g extract 80% methanol leaf Hexane stem [42] Hot distilled water, cold distilled water, methanol, ethanol, dichloromethane Leaf Orbital shaker Seremban Malaysia 24.57 ± 0.07 to 48.08 ± 0.04 mg GAE/g DW extract Cold distilled water Ethanol [18] 100% Methanol Leaf Maceration Seremban Malaysia 1.77 ± 0.01 mg GAE/g extract - - [6] 100% Methanol Leaf Air dry; Maceration Seremban Malaysia 0.78 ± 0.01 mg GAE/g DW extract at stock: 10 mg/mL - - [24] Methanol Leaf, stem Oven dry; Sonication Seremban Malaysia 0.12 and 2.68 mg GAE/g DW sample Leaf Stem [61] Methanol Leaf Air dry; Orbital shaker Malaysia, Thailand, Vietnam 8.29 to 72.16 mg GAE/g DW extract Chiang Dao, Thailand Map Khae, Thialand [18] Methanol Leaf Oven dry 40°C, 50°C, 60°C, 70°C, 80°C, 100°C; Orbital shaker Seremban Malaysia 22.44 ± 0.03 to 63.31 ± 0.03 mg GAE/g DW extract Oven dry at 100°C Oven dry at 40°C [18] Methanol Leaf, bud 1, 6 and 12 months old; Freeze dry; Shaking Serdang Malaysia 6.840 ± 0.470 to 15.460 ± 1.231 mg/g DW 6-month-old buds 1-month-old buds [41] Polar (methanol and dichloromethane), non-polar (hexane and diethyl ether) Leaf, stem Soaking Pahang Malaysia 1.43 ± 0.1 to 7.99 ± 0.6 mg GAE/g DM Leaf-polar Stem-non polar [62] Methanol, ethyl acetate, chloroform, hexane fraction - Sonication Malaysia 22.17 ± 0.02 to 119.29 ± 0.07 mg GAE/g DW Chloroform Ethyl acetate [35] 100% Ethanol Leaf Maceration Thailand 4.67 ± 3.60 mg GAE/g wet weight sample - - [63] 100%, 86%, 65%, 50%, 44% Ethanol Aerial Oven dry; MAE, p-MAE, SFE, soxhlet extraction KL Malaysia 5.74 ± 0.29 to 14.56 ± 0.77 mg GAE/g DM p-MAE in 50% ethanol MAE in 86% ethanol [30] 70% Ethanol Leaf Freeze dry; Sonication Perak Malaysia 23.5 mg GAE/g DM - - [64] 70% Ethanol Leaf, Stem Freeze dry, oven dry, air dry; Sonication, soaking Seremban Malaysia 1.04 ± 0.02 to 7.29 ± 0.11 mg GAE/g DW sample Leaf-Air dry-Sonication Stem-Freeze dry-Soaking [4] - Stem Air dry; CO2-Soxhlet, Maceration SerembanMalaysia CO2-Soxhlet: 49.45 mg GAE/g DM, Maceration: 7.54 mg GAE/g DM - - [28] 80% Ethanol Leaf, stem Young and mature; Stored for 1, 2, 3, and 4 days Malaysia Maturity: 60.75 to 117.00 mg GAE/100 g fresh sample Storage duration: 71.08 to 140.92 mg GAE/100 g fresh sample Maturity young leaf Storage 1 day storage Maturity matured stem Storage 4 days storage [38] KL: Kuala Lumpur.Table 4
Total flavonoid content (TFC) ofC. nutans.
Extract Plant part Pre- or post- harvesting method Plant origin Results of TFC Extract with highest TFC Extract with lowest TFC Reference Hot water - Oven dry; Heating at 90°C Malaysia 2.07 ± 0.05 mg QE/g dry material - - [37] Hot water Leaf Unfermented and fermented; Microwave-oven dry, freeze dry; hot boiling water (100°C); Infusion for 1, 2, 5, 10, 15, and 20 min Sabah Malaysia 14.57 ± 0.42 to 22.13 ± 1.53 mg CE/L Fermented leaf-microwave-oven dry-10 min infusion Unfermented leaf-microwave oven dry-1 min infusion [36] Hot distilled water, cold distilled water, methanol, ethanol, dichloromethane Leaf Orbital shaker Seremban Malaysia 7.09 ± 2.98 to 14.66 ± 1.71 mg QE/g of dry extract Cold distilled water Ethanol extract [18] 100% Methanol Leaf Maceration Seremban Malaysia 0.04 ± 0.001 mg QE/g extract - - [6] 100% Methanol Leaf Air dry; Maceration Seremban Malaysia 0.21 ± 0.005 mg QE/g extract - - [24] Methanol Leaf Air dry; orbital shaker Malaysia, Thailand, Vietnam 2.96 ± 0.03 to 58.38 ± 0.19 mg QE/g of dry extract Chiang Dao, Thailand Map Khae, Thailand [18] Methanol Leaf Oven dry 40°C, 50°C, 60°C, 70°C, 80°C, 100°C; orbital shaker Seremban Malaysia 14.02 ± 1.68 to 27.72 ± 0.14 mg QE/g of dry extract Oven dry at 80°C Oven dry at 40°C [18] Methanol Leaf, bud 1, 6 and 12 months old; Freeze dry; Shaking Serdang Malaysia 3.27 ± 0.33 to 6.32 ± 0.74 mg QE/g dry weight 6-month-old buds 1-month-old buds [41] Polar (methanol and dichloromethane), non-polar (hexane and diethyl ether) Leaf, stem Soaking Pahang Malaysia 3.27 ± 1.10 to 16.09 ± 4.20 mg QE/g DM Polar leaf Non-polar stem [62] Methanol, ethyl acetate, chloroform, hexane fraction - Sonication Malaysia 428.67 ± 0.03 to 937.67 ± 0.02 mg BHTE/g Chloroform fraction Methanol fraction [35] 100%, 86%, 65%, 50%, 44% Ethanol Aerial Oven dry; MAE, p-MAE, SFE, Soxhlet extraction KL Malaysia 2.71 ± 0.47 to 5.88 ± 0.22 mg QE/g of dried material SFE p-MAE at 86% ethanol [30] 80% Ethanol Leaf, stem Young and mature; Stored for 1, 2, 3, and 4 days Malaysia Maturity 45.56 to 99.47 mg RE/100 g fresh sample Storage duration 41.48 to 116.36 mg RE/100 g fresh sample Maturity young leaf Storage 1 day storage Maturity matured stem Storage 4 days storage [38] - Stem Air dry; CO2-Soxhlet, maceration Seremban Malaysia CO2-Soxhlet: 43.81 mg RE/g dried material Maceration: 16.83 mg RE/g dried material CO2-Soxhlet Maceration [28]Table 5
Nutritional composition ofC. nutans.
Sample details Carbohydrate Protein Fat Fiber Ash Moisture Vitamin Mineral Reference China- Leaf - 5.73 ± 0.14% 0.50 ± 0.02% 2.71 ± 0.05% - 78.30 ± 0.29% C: 1.57 ± 0.07 mg/100 gB1: 0.27 ± 0.04 mg/100 g - [40] Seremban Malaysia- Leaf 73.27 ± 3.14% 5.16 ± 0.08% 2.21 ± 0.66% - 10.0 ± 0.20% 9.28 ± 0.40%  Potassium: 1097.90 ± 6.93 mg/100 gCalcium: 874.50 ± 31.25 mg/100 g Sodium: 6.78 ± 1.01 mg/100 g Copper: 0.26 ± 0.01 mg/100 g [39]The Folin-Ciocalteu method is the most established method for determining the total phenolic content ofC. nutans and gallic acid or, in some cases, tannic acids are the most common standards used for TPC calibration. Values are normally expressed in mg gallic acid equivalent (GAE)/g dry weight (DW) or dry material (DM). Total phenolic content is based on a colorimetric reaction between easily oxidized polyphenols or hydroxylated aromatic compounds and phosphotungsten-polymolybdic acid [34]. The redox reaction results in the formation of blue chromophores consisting of phosphotungstic-phosphomolybdenum complexes, which have a maximum absorption at 765 nm and are proportional to the total quantity of phenolic compounds [34]. Among the TPC tests conducted, the chloroform fraction prepared by Hamid and Yahaya [35] from a Malaysian farm that underwent sonication was detected to have the highest TPC, with 119.29 ± 0.07 mg GAE/g DW. In addition, Lusia Barek et al. [36] also illustrated that an unfermented leaf subjected to drying in a microwave oven and infusion in water for 20 min retained a high TPC value of 177.80 ± 19.10 mg tannic acid equivalent (TAE)/L.The flavonoid level inC. nutans was mostly determined by the aluminum chloride (AlCl3) method. Essentially, the detection of flavonoid (the colored complexes) is conducted using the reaction between AlCl3 and the carbonyl and hydroxyl groups of flavonoids in an alkaline solution [18]. As shown by the results in Table 4, TFC was determined using quercetin as the positive control and the results were expressed in mg quercetin equivalent (QE)/g dry weight (DW) sample, although there were also some studies that used catechin, rutin, or butylated hydroxytoluene (BHT) as the positive control. Again, the chloroform fraction ofC. nutans that underwent sonicated extraction was able to retain more TFC (937.67 ± 0.02 mg BHTE/g) than those undergoing other combination of pre- and postharvesting methods [35].Other phytochemical content studies onC. nutans included hydroxycinnamic acid content determination using Arnow’s reagent. The results suggested that oven-dried and hot water extractedC. nutans extract possessed 0.91 ± 0.01 mg caffeic acid equivalent/g dry material (mg CAE/g DW) [37]. For the chlorophyll content ofC. nutans, Raya et al. [38] found that, compared to mature stem (32.27 mg/100 g) or plant sample that kept for 4 days (17.97 mg/100 g), a young leaf or plant sample subjected to 1 day of storage possessed a higher chlorophyll content with 64.35 mg/100 g and 78.83 mg/100 g, respectively. By contrast, Mustapa et al. [30] found that the absolute ethanol aerial extract ofC. nutans possessed a higher chlorophyll content (1.30 g/g DM) than absolute acetone aerial extract (1.06 g/g DM). On the other hand, Raya et al. [38] showed thatC. nutans leaves harvested at a young stage had a higher content of ascorbic acid (0.38250 mg/100 g). The results also revealed that increasing the storage duration from 1 day to 4 days caused a reduction in the ascorbic content of the extract from 0.49833 mg/100 g to 0.23083 mg/100 g. For phytosterol determination using Liebermann-Burchard reagent, one study showed that increasing the organic solvent ratio in the solvent mixture will result in a decrease in the phytosterol content [30]. This study also suggested that the supercritical fluid extraction (SFE) method allowed higher phytosterol retention (1.35 ± 0.12 mg of β-sitosterol (BS)/g DM), followed by pressurized-microwave assisted extraction (p-MAE) for the 86% ethanolic extract (1.19 ± 0.22 mg BS/g DM), microwave assisted extraction (MAE) of the 86% ethanolic extract (0.70 ± 0.10 mg BS/g DM), and Soxhlet extraction of the absolute ethanolic extract (0.47 ± 0.20 mg BS/g DM). Additionally, the nutrient composition ofC. nutans has also been determined by Sarega et al. [39] and Yu et al. [40] through proximate, vitamin, and mineral analysis as shown in Table 5.Meanwhile, there were also studies that used HPLC for the quantification of phenolic compounds [23, 39, 41–43] and, in the case of Mustapa et al. [30], for the quantification of a phytosterol compound, β-sitosterol. The HPLC parameters used and the sample details are tabulated into Table 6. Although Wong et al. [43] attempted to quantify 4 phenolics compounds, only quercetin was found (0.25 ± 0.01 μg per mg of dried sample), whereas Chelyn et al. [23] quantified schaftoside, orientin, isovitexin, and vitexin content forC. nutans leaves collected from 3 different sources. The results revealed thatC. nutans collected from Taiping, Perak, Malaysia, possessed the highest amount of all 4 phenolics: 17.43 ± 0.01 mmol/g of schaftoside, 0.86 ± 0.04 mmol/g of orientin, 2.01 ± 0.02 mmol/g of isovitexin, and 0.91 ± 0.03 mmol/g of vitexin. Ghasemzadeh et al. [41] found that samples with different maturity and from different plant parts retained different phenolics and flavonoids. Six-month-old bud extract was found to be able to retain the highest concentration of gallic acid (5.96 ± 0.55 mg/g DW). Sarega et al. [39, 42] analyzed and quantified a total of 8 phenolics in the extract. Both studies found that protocatechuic acid was the most prominent phenolic acid in the hot water, water, and 80% methanol leaf extracts (33.28 ± 0.01 mg/g extract). Finally, aerial extract that underwent SFE possessed the highest amount of β-sitosterol (0.83 ± 0.10 mg/g DM) followed by the 86% ethanolic p-MAE extract (0.65 ± 0.14 mg/g DM), 86% ethanolic MAE extract (0.52 ± 0.10 mg/g DM), and Soxhlet assisted extract (0.23 ± 0.18 mg/g DM) [30].Table 6
HPLC quantification ofC. nutans.
Sample details Stationary phase Mobile phase Detector; Wavelength Flow rate (mL/min) Elution method Standard for Quantification The corresponding concentration Reference Hot water; Oven dry Thermo ScientificODS Hypersil (5 μm, 100 × 4.6 mm) Water with acetic acid (pH 2.74), acetonitrile UV;272–370 nm 0.8 Gradient Gallic acid, catechin, caffeic acid,quercetin 0.25 ± 0.01μg/mg of dried sample [43] Leaf;Taiping (Perak), Kota Tinggi (Johor), Sendayan (Negeri Sembilan); Ethanolic; Oven dry, Sonication Kinetex Pentafluoro-phenyl (PFP)(5 μm, 250 × 4.6 mm) Water with 0.8% (v/v) glacial acetic acid, acetonitrile UV-Vis/DAD;330 nm 0.7 Gradient Shaftoside, orientin, isovitexin, vitexin 17.43 ± 0.01 mmol/g [23] Buds, Leaf; 1, 6 and 12 months old; Methanol; Freeze dry C18(5 μm, 250 × 4.6 mm) 0.03 Mortho-phosphoric acid, methanol UV-Vis;260–360 nm 1.0 Gradient Catechin, kaempferol, luteoli 7-O-β-D-glucoside, quercetin, gallic acidmonohydrate, caffeic acid 5.963 ± 0.545 mg/g DW [41] Aerial; 86% ethanol; Oven dry; MAE, p-MAE,SFE Symmetry C18(5 μm, 150 × 4.6 mm) Methanol, 2-propanol UV-ELSD; 210 nm 0.7 Isocratic β -sitosterol 0.83 ± 0.10 mg/g DM [30] Leaf;Hot water, water, 80%methanol; Freeze dry; Sonication LUNA C18(5 mm, 250 × 4.6 mm) Water with 6% acetic acid (pH 2.27), acetonitrile DAD;320 nm 0.5 Gradient Cinnamic acid,protocatechuic acid, ferulic acid, gallic acid, p-coumaric, chlorogenic acid, vanillic acid, caffeic acid 33.28 mg/g extract [42] Leaf, Aqueous,80%methanol; Sonication LUNA C18(5 mm, 250 × 4.6 mm) Water with 6% acetic acid (pH 2.27), acetonitrile DAD;320 nm 0.5 Gradient Cinnamic acid, protocatechuic acid, ferulic acid, gallic acid,p-coumaric, chlorogenic acid, vanillic acid, caffeic acid 33.38 ± 0.31 mg/g extract [39] The text inbold word indicated that particular extract has the highest concentration of the particular standard.


## 2.1. Phytochemical Screening

For the last few decades, the study of plants has progressed rapidly [17]. Plants contain both primary and secondary metabolites. Primary metabolites are those chemical constituents that are inherently present in most organisms and have a direct involvement in plant growth [18]. On the other hand, secondary metabolites are the bioactive phytochemicals that are normally produced by a plant in response to specific environmental stresses and that are considered to possess additional health benefits [18]. Throughout the years, the common practice of researchers was started with the screening of plant. Phytochemical screening tests, thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) are among the most popular methods used as they are easy to conduct, of low cost, and time-effective. These methods are able to provide guidelines on the class and functional groups of the chemical constituents that are present in the plant.Table1 lists the occurrence of chemical classes inC. nutans extract, as determined through specific phytochemical screening tests. The phytochemical screening tests adopted include Mayer’s reagent, modified Dragendorff’s reagent, or the Wagner test for alkaloid detection; the frothing test for saponin, the alkaline reagent, Shinoda, or lead acetate tests for flavonoids; the frothing, ferric chloride, potassium dichromate, or gelatin tests for tannins; the copper acetate test for diterpenes; Salkowski’s test, or Liebermann-Burchard test for phytosterols; the iron (III) chloride test for phenolic compounds; the pseudoindicans test for iridoids; Grignard reagent, Baljet, Keller-Kiliani, or Molisch tests for glycosides; Molisch or Fehling tests for carbohydrates; Salkowski’s test for steroids and sterols; and the biuret test for proteins and amino acids [6, 19–22]. As a summary, flavonoids, triterpenoids, steroids, phytosterols, and glycosides are the phytochemical classes that are most likely present inC. nutans extract when the plant is extracted by a polar solvent, such as water, methanol, ethanol, or an aqueous organic solvents or by a semipolar (e.g., chloroform) solvent. The presence of alkaloids, saponins, and tannins inC. nutans depends on the plant origin and the postharvesting method used. For instance, alkaloids were typically present in water and chloroform leaf extracts and were absent in methanol and aqueous methanol leaf extracts collected from Malaysia. By contrast, saponins were present in methanol extract cultivated in Malaysia and absent in water extract from Indonesia and chloroform and aqueous methanol extracts from Malaysia. Tannins were found in leaves extracted with water, aqueous methanol, and chloroform, but not those extracted with methanol. For the presence of quinones, iridoids, carbohydrates and protein, and amino acid, further confirmation is required as there has been only one study conducted on each of them to date.Table 1
Phytochemical screening test ofC. nutans.
Chemical class Origin Extract Present/Absent Alkaloid Indonesia1 Leaf-water extract1 Present1 Malaysia2,3,5 Leaf-100% methanol extract2,5 Absent2,5 Leaf-100% chloroform extract3 Presence3 Leaf-70% methanol extract4 Absent4 Saponin Indonesia1 Leaf-water extract1 Absent1 Malaysia2,3,4,5 Leaf-100% methanol extract2, 5 Present2,5 Leaf-100% chloroform extract3 Absent3 Leaf-70% methanol extract4 Absent4 Flavonoids Indonesia1 Leaf-water extract1 Present1 Malaysia2,3,4,5 Leaf-100% methanol extract2,5 Present2,5 Leaf-100% chloroform extract3 Present4 Leaf-70% methanol extract4 Present3 Triterpenoids Indonesia1 Leaf-water extract1 Present1 Malaysia5 Leaf-100% methanol extract5 Present2 Diterpenes Malaysia2 Leaf-100% methanol extract2 Present Steroids Indonesia1 Leaf-water extract1 Present1 Malaysia4,5 Leaf-70% methanol extract4 Present2,3 Thailand7 Leaf-100% methanol extract5 Present5 Stem-Light petroleum extract7 Present7 Phytosterol Malaysia2,4 Leaf-100% methanol extract2 Present1 Leaf-70% methanol extract4 Present2 Tannin Indonesia1 Leaf-water extract1 Present1 Malaysia2,3,4,5 Leaf-100% methanol extract2,5 Absent2,5 Leaf-100% chloroform extract3 Present3 Leaf-70% methanol extract4 Present4 Quinone Indonesia1 Leaf-water extract1 Absent Phenolic compound Malaysia2 Leaf-100% methanol extract2 Present Glycosides Malaysia3,4 Leaf-100% chloroform extract3 Present1 Leaf-70% methanol extract4 Present2 Iridoids Thailand6 Leaf6 Present6 Carbohydrates Malaysia4 Leaf-70% methanol extract4 Present4 Protein and amino acids Malaysia4 Leaf-70% methanol extract4 Present4 Reference. 1Nurulita et al. [25]; 2Yang et al. [6]; 3Goonasakaran [26]; 4Sekar and Rashid [22]; 5Abdul Rahim et al. [19]; 6Keawpradub and Purintrapiban [20]; 7Dampawan et al. [27].In 2012, Roeslan and his teams [11] compared leaves from two different sources, Thailand and Indonesia, using TLC separation method. Their findings showed that there was a quality difference and indicated that the quality of the sample from Indonesia was better, as this sample was thicker than the sample from Thailand. Chelyn et al. [23] used the HPTLC method to screen for the presence ofC-glycosyl flavones in threeC. nutans plant samples that underwent the same postharvesting processing but that were from three different geographical locations in Malaysia: Taiping (Perak), Kota Tinggi (Johor), and Sendayan (Negeri Sembilan). All the results indicated that schaftoside was present inC. nutans collected from all the investigated locations, whereas isoorientin, isovitexin, orientin, and vitexin were found only in the plants harvested from Perak and Johor, not in the sample harvested from Negeri Sembilan. Tiew et al. [24] used UV-visible and FTIR spectrophotometry for the functional group identification ofC. nutans leaves dried under the shade and macerated with methanol. Based on the results obtained, the extract showed two major bands 240–400 nm and the presence of an -OH band and a C=O stretch suggests that the constituents in the extract have -OH group and C=O functional groups.


## 2.2. Phytochemical Identification

Phytochemical screening methods can only provide a clue of the chemical class present in the plant. To further validate the identity of the compounds, column chromatography (isolation) and TLC (separation and purity) together with nuclear magnetic resonance (NMR) spectroscopy (identification and structural elucidation) are the most conventionally used techniques. In addition, the emergence of advanced analytical tools such as liquid chromatography mass spectrometry (LCMS), gas chromatography mass spectrometry (GCMS), and NMR spectroscopy provides researchers with useful alternatives for identification.


### 2.2.1. Phytochemical Identification Using Column Chromatography-Spectroscopy Analysis

A summary of the phytochemicals that have been identified in this plant is provided in Tables2(a) and 2(b). Table 2(a) displays those compounds that have been isolated through column chromatography and identified using spectroscopic analysis, the results from which were tabulated according to the phytochemical class. To date, the pure compounds that have been isolated fromC. nutans include 2 triterpenoids; 6 phytosterols; 8 phenolics, including 7C-glycosyl flavones and 1 phenolic acid; 5 sulfur-containing glucosides; 8 sulfur-containing compounds; 7 lipid related compounds; and 8 chlorophyll derivatives.Table 2
Phytochemicals (primary and secondary metabolites) that presented inC. nutans.
(a)
Phytochemical identification based on column chromatography isolation and spectroscopy structural elucidation
Phytochemical Plant part Postharvesting method Extract/Fraction Plant origin Reference Terpenes-Triterpenoids  Lupeol Stem2 Air Dry; Soxhlet2 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3 Oven Dry; Soaking3 Light petroleum extract2 Seremban Malaysia3 [27]2 Hexane fraction of methanol extract3  [45]3 Betulin Root - - China [46]  Terpenes-Phytosterols  β-sitosterol Stem2 Air Dry; Soxhlet2 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3 Oven Dry; Soaking3 Light petroleum extract2 Seremban Malaysia3 [27]2 Hexane fraction of methanol extract3  [45]3 β-Sitosterol-3-O-β glucopyranoside Stem Sun Dry; Soaking Sub-fraction of methanol extract Vietnam [47] β-Sitosterol-3-O-β glucoside Stem Sun Dry; Soaking Sub-fraction of methanol extract Vietnam [47] Stigmasterol Stem2 Oven Dry; Soaking3 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3  Light petroleum extract2 Malaysia3 [48]2 Hexane fraction of methanol extract3  [45]3 Stigmasterol-β-D-glucoside Aerial Sun Dry; Maceration Methanol fraction of 96% ethanol extract Vietnam [49] Stigmasteryl-3-O-β-D-glucopyranoside Leaf Oven Dry; Soxhlet Chloroform extract Thailand [9]  Phenolics compounds  Shaftoside1,2,3, vitexin1,2, isovitexin1,2, isomollupentin 7-O-β-glucopyranoside1, orientin1,2, isoorientin1,2, gallic acid2, apigenin 6,8-di-C-α-L-arabinopyranoside2 Aerial1,2 Reflux2 Butanol soluble portion of methanol Thailand1 [50]1 Oven Dry; Soaking3 extract1 Seremban Malaysia2,3 [33]2 Leaf3 - 30% ethanol extract2  [45]3 Hexane fraction of methanol extract3  Sulfur-containing glucosides compounds  Clinacoside A, Clinacoside B, Clinacoside C, Cycloclinacoside A1, Cycloclinacoside A2 Aerial - Butanol soluble and aqueous soluble portion of methanol extract Thailand [51]  Sulfur-containing compounds  Clinamides A1, Clinamides B1, Clinamides C1, Clinamides D2, Clinamides E2, 2-cis-entadamide A1, Entadamide A1, Entadamide C1 Aerial1 Air Dry; Soaking1 80% Ethanol extract1 Taiwan1 [52]1 Whole plant2 Sonication2 Methanol extract2 Jelebu, Malaysia2 [53]2  Lipids  Myricyl alcohol - - Petroleum ether extract Thailand [44] 1,2-O-dilinolenoyl-3-O-β-D-galactopyranosyl-glycerol, 1-O-palmitoyl-2-O-linolenoyl-3-O-(α-D-galactopyranosyl-(1′′→6′)-O-β-D-galacctopyranosyl)-glycerol Leaf - - Thailand [54] Monoacylmonogalactosylglycerol, a mixture of nine cerebrosides Leaf - Ethyl acetate-soluble fraction of 95% ethanol extract Thailand [55] Monogalactosyl diglyceride, digalactosyl diglyceride Leaf Soxhlet extraction Chloroform extract Thailand [56]  Chlorophyll a and b derivatives  Purpurin 18 phytyl ester1,2,4 Phaeophorbide a1,2,4  132-hydroxy-(132-S)-chlorophyll b3,4 132-hydroxy-(132-R)-chlorophyll b3,4 132-hydroxy-(132-S)-phaeophytin b1,2,4 132-hydroxy-(132-R)-phaeophytin b3,4 132-hydroxy-(132-S)-phaeophytin a3,4 132-hydroxy-(132-R)-phaeophytin a3,4 Leaf1,2,3,4 Oven Dry; Soxhlet1,2,3,4 Chloroform extract1,2,3,4 Thailand1,2,3,4 [57]1 [17]2 [9]3 [58]4  Others  trans-3-methylsulfinyl-2-propenol Aerial Air Dry; Soaking 80% Ethanol extract Taiwan [52] 3-amino-4,5-dihydroxyfuran-2(3H)-one Aerial Sun Dry; Maceration Methanol fraction of 96% ethanol extract Vietnam [49] polysaccharide–peptide complex Leaf Oven Dry Hot water and 75% ethanol precipitation extract Seremban Malaysia [59]  (b)
Phytochemical identification based on spectrometry and spectroscopy identification
Phyto-constituents Plant part Post-harvesting method Extract Plant origin Analytical tools Reference 2-ethyl-oxetane, 9,12,15-octadecatrienoic acid, 2,3-dimethylpyridine, 3-deoxyd-mannoic lactone, neophytadiene, phytol, 2,3-dihydrobenzofuran,n-hexadecanoic acid Leaf Oven dry; Soaking Abs methanol Kuala Lumpur Malaysia GCMS [19] n-Pentadecanol, eicosane, 1-nonadecene, heptadecane, dibutylphthalate, n-Tetracosanol-1, heneicosane, behenic alcohol, 1-heptacosanol, 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, nonadecyl heptafluorobutyrate, eicosayl trifluoroacetate, 1,2-benzenedicarcoxylic acid, dinonyl ester, phthalic acid, dodecyl nonylester Leaf Oven dry Chloroform Selangor Malaysia GCMS [32] Squalene,β-tocopherol, vitamin E, Campsterol, stigmasterol, γ-sitosterol, β-amyrin, α-amyrin, lupeol, propanoic acid, betulin Leaf, stem Combination of air dry and freeze dry; Maceration Abs methanol, abs ethyl acetate Sabah Malaysia GCMS [29] Lactic Acid, Glycolic acid, 3-Pyridinol, Glycerol, Phosphonic acid, Catechol, Butanedioic acid, Glyceric acid, Erythrono-1,4-lactone, (Z), Malic acid, Benzaldehyde, 3-methoxy-4-[(trimethylsilyl)oxy]-, O-methyloxime, Cyclooctasiloxane, Levoglucosan, Vanillic Acid, D-Ribo-Hexonic acid, 3-deoxy-2,5,6-tris-O-(trimethylsilyl)-, lactone, D-(-)-Fructofuranose, pentakis(trimethylsilyl) ether (isomer 1), Myristic acid, D-Pinitol, Neophytadiene, D-Fructose, 1,3,4,5,6-pentakis-O-(trimethylsilyl)-, O-methyloxime, Syringic acid, D-Fructose, 1,3,4,5,6-pentakis-O-(trimethylsilyl)-, O-methyloxime, d-Glucose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)-, o-methyloxyme, (1Z)-, Gallic acid, Muco-Inositol, D-Glucose, Heptadecanoic acid, Phytol, 9,12-Octadecadienoic acid (Z,Z)-, Oleic Acid, L-Rhamnose, Cyclononasiloxane, octadecamethyl-, Octadecane-1,2-diol, Ricinoleic acid, Arachidic acid, L-Rhamnose, 1,2-Benzenedicarboxylic acid, mono(1-methylheptyl) ester, (+/−),1-Monopalmitin, Cyclononasiloxane, octadecamethyl-, 1-Monolinolein, Glycerol monostearate, Squalene,β-Gentiobiose, B-tocopherol, γ-tocopherol, α-Tocopherol, Campesterol Stem Air dry; Supercritical-soxhlet extraction - Seremban Malaysia GC-QTOF-MS [28] Vitamin E, campesterol, stigmasterol,γ-Sitosterol, β-Sitosterol, β-amyrin, lupeol, betulin Root Freeze dry; Soaking Methanol Sabah Malaysia GCMS [31] Oleic acid, squalene, vitamin E, campesterol, stigmasterol,γ-stosterol, β-sitosterol, lup-20(29)-en-3-one, β-amyrin, lupeol, lup-20(29)-en-3-ol-acetate, betulin Root Freeze dry; Soaking Ethyl acetate Sabah Malaysia GCMS [31] Neophytadiene, iron, 7,9-Dodecadien-1-ol, Myristic acid, Palmitic acid, Palmitic acid-methyl ester, Benzenethanol, Phytol, Squalene, Stearic acid-methyl ester, Margaric acid-ethyl ester, Lupeol, Linoleic acid-ethyl ester, Linolenic acid-methyl ester, 2-Butanol, Butanamide, 2-cyclopenten-1-one-2-hydroxy, Glycine, Pentanal, Isoveraldehyde, Dimethyl trisulfide, Thiophene, Succinic acid, Glycolic acid, Oxazolidine, Thiophene, 9-Azabicyclo (6.1.0) non-4-4en-9-amine, 4-Vinyl-2-methoxy-phenol, Phenol,2,6-dimethoxy, 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, Glycerine, 4-Vinylphenol Aerial Oven dry; MAE, SFE, Soxhlet Aqueous ethanol Kuala Lumpur Malaysia GCMS [30] Isoorientin, orientin, isovitexin, vitexin, schaftoside, 6, 8-apigenin-C-α-L-pyranarabinoside Aerial - 30% Ethanol Seremban Malaysia HPLC LCMS/MS [33] Kaempferol-7-neohesperidoside, Isoschaftoside, Isoorientin, Vitexin, 3′,7-Dimethoxy-3-hydroxyflavone, 2′,6-Dihydroxyflavone, (+)−Catechin, 7-Hydroxyflavone, Gallic acid, Flavanone-7-O-glycoside Aerial Oven dry; MAE, SFE, Soxhlet 86% Ethanol,50% Ethanol KL Malaysia UPLC-ESI-QTOF/MS [30] Schaftoside, Arabinoysl–glucosyl apigenin isomer, Ascorbic acid, Gendarucin A, Gendarucin A isomer, 3, 3-di-O-methylellagic acid, Methyl 2-[cyclohex-2-en-1-yl(hydroxyl) methyl]-3-hydroxy-4-(2-hydroxyethyl)-3-methyl-5-oxoprolinate, Methyl 2-[cyclohex-2-en-1-yl(hydroxyl) methyl]-3-hydroxy-4-(2-hydroxyethyl)-3-methyl-5-oxoprolinate isomer Leaf Air dry; Sonication 70% Ethanol Seremban Malaysia UPLC-MS/MS [4] Gallic acid, 4-hydroxybenzoic acid, caffeic acid, coumaric acid, ferulic acid, schaftoside, vitexin, orientin, isoorientin, isovitexin, luteolin, apigenin, forsythosides H, forsythosides I, diosmetin glycoside, diosmetin. Leaf Oven dry; Soaking 100% Methanol Kuala Lumpur Malaysia UHPLC-MS [19] Primary metabolites: Fructose, α-Glucose, β-Glucose, Sucrose, a mixture of cerebrosides, Monoacylmonogalactosylglycerol, Alanine, Glutamate, Glutamine, Proline, Threonine, Tryptophan, Valine, Citric acid, Formic acid, Fumaric acid, Choline, Adenine, Fatty acid, Ascorbic acid Secondary metabolites: Betulin, Lupeol, Stigmasterol, β-sitosterol, Clinacoside A, Clinacoside B, Clinacoside C, Cycloclinacoside A1, Cycloclinacoside A2, Isoorientin, Isovitexin, Orientin, Vitexin, Schaftoside, Gendarucin A, Catechin, Quercetin, Quercetin 3-O-rhamnoside, Quercetin 3-O-arabinofuranoside, Rutin, Chlorogenic acid, Gallic acid Leaf, Stem Air dry, Oven dry, Freeze dry; Sonication, Soaking 70% Ethanol Seremban Malaysia 1H-NMR [4] 1, 2, 3, 4: in each phytochemical class, every superscript number attached to a particular phytochemical, plant part, postharvesting method, extract/ fraction, or plant origin indicated that such information is extracted from the reference which has attached with the same superscript number.


### 2.2.2. Phytochemical Identification Using Advanced Spectrometric and Spectroscopic Analysis

Meanwhile, Table2(b) shows a list of compounds identified according to the type of the advanced spectrometric and spectroscopic analytical tools used. There are a total of 6 research teams that have used GCMS for compound identification [19, 28–32], 4 research teams that have used LCMS analytical tool [4, 19, 30, 33], and 1 research team that has used proton andJ-resolved NMR approaches [4]. Based on GCMS identification results, Cheong et al. [29] found a rich variety of triterpenoids and phytosterols in the extract. Yong et al. [32] showed that of the 14 phytochemicals identified in the chloroform extract, 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl)ester, a common plasticizer, was the most abundant compound. Mustapa et al. [30] found that phytol was the main compound in the microwave (MAE) and Soxhlet assisted extracts, while palmitic acid was dominant in the supercritical fluid assisted (SFE) extract. A study by Abdul Rahim et al. [19] identified a total of 39 peaks, but only 8 major compounds have been listed in Table 2(b). In addition, Alam et al. [28] have successfully identified a total of 48 compounds, the most abundant of which were neophytadiene, glycolic acid, phosphonic acid, catechol, butanedioic acid, vanillic acid, gallic acid, pinitol, phytol, squalene, tocopherols, and campesterol. Moreover, Teoh et al. [31] have found that the ethyl acetate root extract possessed more compounds than the methanol root extract. Among the compounds identified, lupeol was the main compound present in both extracts.Using LCMS in negative ionization mode, Huang et al. [33] successfully identified 6C-glycosyl flavones, the research team of Khoo et al. [4] was able to identify 8 compounds, Mustapa et al. [30] team have found 10 phenolic compounds from the aqueous ethanolic extract, and Abdul Rahim et al. [19] have detected 16 phenolic compounds. Khoo et al. [4] have also tentatively identified 20 primary metabolites and 22 secondary metabolites in 70% ethanol extract through proton andJ-resolved NMR spectroscopy.


## 2.2.1. Phytochemical Identification Using Column Chromatography-Spectroscopy Analysis

A summary of the phytochemicals that have been identified in this plant is provided in Tables2(a) and 2(b). Table 2(a) displays those compounds that have been isolated through column chromatography and identified using spectroscopic analysis, the results from which were tabulated according to the phytochemical class. To date, the pure compounds that have been isolated fromC. nutans include 2 triterpenoids; 6 phytosterols; 8 phenolics, including 7C-glycosyl flavones and 1 phenolic acid; 5 sulfur-containing glucosides; 8 sulfur-containing compounds; 7 lipid related compounds; and 8 chlorophyll derivatives.Table 2
Phytochemicals (primary and secondary metabolites) that presented inC. nutans.
(a)
Phytochemical identification based on column chromatography isolation and spectroscopy structural elucidation
Phytochemical Plant part Postharvesting method Extract/Fraction Plant origin Reference Terpenes-Triterpenoids  Lupeol Stem2 Air Dry; Soxhlet2 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3 Oven Dry; Soaking3 Light petroleum extract2 Seremban Malaysia3 [27]2 Hexane fraction of methanol extract3  [45]3 Betulin Root - - China [46]  Terpenes-Phytosterols  β-sitosterol Stem2 Air Dry; Soxhlet2 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3 Oven Dry; Soaking3 Light petroleum extract2 Seremban Malaysia3 [27]2 Hexane fraction of methanol extract3  [45]3 β-Sitosterol-3-O-β glucopyranoside Stem Sun Dry; Soaking Sub-fraction of methanol extract Vietnam [47] β-Sitosterol-3-O-β glucoside Stem Sun Dry; Soaking Sub-fraction of methanol extract Vietnam [47] Stigmasterol Stem2 Oven Dry; Soaking3 Petroleum ether extract1 Thailand1,2 [44]1 Leaf3  Light petroleum extract2 Malaysia3 [48]2 Hexane fraction of methanol extract3  [45]3 Stigmasterol-β-D-glucoside Aerial Sun Dry; Maceration Methanol fraction of 96% ethanol extract Vietnam [49] Stigmasteryl-3-O-β-D-glucopyranoside Leaf Oven Dry; Soxhlet Chloroform extract Thailand [9]  Phenolics compounds  Shaftoside1,2,3, vitexin1,2, isovitexin1,2, isomollupentin 7-O-β-glucopyranoside1, orientin1,2, isoorientin1,2, gallic acid2, apigenin 6,8-di-C-α-L-arabinopyranoside2 Aerial1,2 Reflux2 Butanol soluble portion of methanol Thailand1 [50]1 Oven Dry; Soaking3 extract1 Seremban Malaysia2,3 [33]2 Leaf3 - 30% ethanol extract2  [45]3 Hexane fraction of methanol extract3  Sulfur-containing glucosides compounds  Clinacoside A, Clinacoside B, Clinacoside C, Cycloclinacoside A1, Cycloclinacoside A2 Aerial - Butanol soluble and aqueous soluble portion of methanol extract Thailand [51]  Sulfur-containing compounds  Clinamides A1, Clinamides B1, Clinamides C1, Clinamides D2, Clinamides E2, 2-cis-entadamide A1, Entadamide A1, Entadamide C1 Aerial1 Air Dry; Soaking1 80% Ethanol extract1 Taiwan1 [52]1 Whole plant2 Sonication2 Methanol extract2 Jelebu, Malaysia2 [53]2  Lipids  Myricyl alcohol - - Petroleum ether extract Thailand [44] 1,2-O-dilinolenoyl-3-O-β-D-galactopyranosyl-glycerol, 1-O-palmitoyl-2-O-linolenoyl-3-O-(α-D-galactopyranosyl-(1′′→6′)-O-β-D-galacctopyranosyl)-glycerol Leaf - - Thailand [54] Monoacylmonogalactosylglycerol, a mixture of nine cerebrosides Leaf - Ethyl acetate-soluble fraction of 95% ethanol extract Thailand [55] Monogalactosyl diglyceride, digalactosyl diglyceride Leaf Soxhlet extraction Chloroform extract Thailand [56]  Chlorophyll a and b derivatives  Purpurin 18 phytyl ester1,2,4 Phaeophorbide a1,2,4  132-hydroxy-(132-S)-chlorophyll b3,4 132-hydroxy-(132-R)-chlorophyll b3,4 132-hydroxy-(132-S)-phaeophytin b1,2,4 132-hydroxy-(132-R)-phaeophytin b3,4 132-hydroxy-(132-S)-phaeophytin a3,4 132-hydroxy-(132-R)-phaeophytin a3,4 Leaf1,2,3,4 Oven Dry; Soxhlet1,2,3,4 Chloroform extract1,2,3,4 Thailand1,2,3,4 [57]1 [17]2 [9]3 [58]4  Others  trans-3-methylsulfinyl-2-propenol Aerial Air Dry; Soaking 80% Ethanol extract Taiwan [52] 3-amino-4,5-dihydroxyfuran-2(3H)-one Aerial Sun Dry; Maceration Methanol fraction of 96% ethanol extract Vietnam [49] polysaccharide–peptide complex Leaf Oven Dry Hot water and 75% ethanol precipitation extract Seremban Malaysia [59]  (b)
Phytochemical identification based on spectrometry and spectroscopy identification
Phyto-constituents Plant part Post-harvesting method Extract Plant origin Analytical tools Reference 2-ethyl-oxetane, 9,12,15-octadecatrienoic acid, 2,3-dimethylpyridine, 3-deoxyd-mannoic lactone, neophytadiene, phytol, 2,3-dihydrobenzofuran,n-hexadecanoic acid Leaf Oven dry; Soaking Abs methanol Kuala Lumpur Malaysia GCMS [19] n-Pentadecanol, eicosane, 1-nonadecene, heptadecane, dibutylphthalate, n-Tetracosanol-1, heneicosane, behenic alcohol, 1-heptacosanol, 1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, nonadecyl heptafluorobutyrate, eicosayl trifluoroacetate, 1,2-benzenedicarcoxylic acid, dinonyl ester, phthalic acid, dodecyl nonylester Leaf Oven dry Chloroform Selangor Malaysia GCMS [32] Squalene,β-tocopherol, vitamin E, Campsterol, stigmasterol, γ-sitosterol, β-amyrin, α-amyrin, lupeol, propanoic acid, betulin Leaf, stem Combination of air dry and freeze dry; Maceration Abs methanol, abs ethyl acetate Sabah Malaysia GCMS [29] Lactic Acid, Glycolic acid, 3-Pyridinol, Glycerol, Phosphonic acid, Catechol, Butanedioic acid, Glyceric acid, Erythrono-1,4-lactone, (Z), Malic acid, Benzaldehyde, 3-methoxy-4-[(trimethylsilyl)oxy]-, O-methyloxime, Cyclooctasiloxane, Levoglucosan, Vanillic Acid, D-Ribo-Hexonic acid, 3-deoxy-2,5,6-tris-O-(trimethylsilyl)-, lactone, D-(-)-Fructofuranose, pentakis(trimethylsilyl) ether (isomer 1), Myristic acid, D-Pinitol, Neophytadiene, D-Fructose, 1,3,4,5,6-pentakis-O-(trimethylsilyl)-, O-methyloxime, Syringic acid, D-Fructose, 1,3,4,5,6-pentakis-O-(trimethylsilyl)-, O-methyloxime, d-Glucose, 2,3,4,5,6-pentakis-O-(trimethylsilyl)-, o-methyloxyme, (1Z)-, Gallic acid, Muco-Inositol, D-Glucose, Heptadecanoic acid, Phytol, 9,12-Octadecadienoic acid (Z,Z)-, Oleic Acid, L-Rhamnose, Cyclononasiloxane, octadecamethyl-, Octadecane-1,2-diol, Ricinoleic acid, Arachidic acid, L-Rhamnose, 1,2-Benzenedicarboxylic acid, mono(1-methylheptyl) ester, (+/−),1-Monopalmitin, Cyclononasiloxane, octadecamethyl-, 1-Monolinolein, Glycerol monostearate, Squalene,β-Gentiobiose, B-tocopherol, γ-tocopherol, α-Tocopherol, Campesterol Stem Air dry; Supercritical-soxhlet extraction - Seremban Malaysia GC-QTOF-MS [28] Vitamin E, campesterol, stigmasterol,γ-Sitosterol, β-Sitosterol, β-amyrin, lupeol, betulin Root Freeze dry; Soaking Methanol Sabah Malaysia GCMS [31] Oleic acid, squalene, vitamin E, campesterol, stigmasterol,γ-stosterol, β-sitosterol, lup-20(29)-en-3-one, β-amyrin, lupeol, lup-20(29)-en-3-ol-acetate, betulin Root Freeze dry; Soaking Ethyl acetate Sabah Malaysia GCMS [31] Neophytadiene, iron, 7,9-Dodecadien-1-ol, Myristic acid, Palmitic acid, Palmitic acid-methyl ester, Benzenethanol, Phytol, Squalene, Stearic acid-methyl ester, Margaric acid-ethyl ester, Lupeol, Linoleic acid-ethyl ester, Linolenic acid-methyl ester, 2-Butanol, Butanamide, 2-cyclopenten-1-one-2-hydroxy, Glycine, Pentanal, Isoveraldehyde, Dimethyl trisulfide, Thiophene, Succinic acid, Glycolic acid, Oxazolidine, Thiophene, 9-Azabicyclo (6.1.0) non-4-4en-9-amine, 4-Vinyl-2-methoxy-phenol, Phenol,2,6-dimethoxy, 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl, Glycerine, 4-Vinylphenol Aerial Oven dry; MAE, SFE, Soxhlet Aqueous ethanol Kuala Lumpur Malaysia GCMS [30] Isoorientin, orientin, isovitexin, vitexin, schaftoside, 6, 8-apigenin-C-α-L-pyranarabinoside Aerial - 30% Ethanol Seremban Malaysia HPLC LCMS/MS [33] Kaempferol-7-neohesperidoside, Isoschaftoside, Isoorientin, Vitexin, 3′,7-Dimethoxy-3-hydroxyflavone, 2′,6-Dihydroxyflavone, (+)−Catechin, 7-Hydroxyflavone, Gallic acid, Flavanone-7-O-glycoside Aerial Oven dry; MAE, SFE, Soxhlet 86% Ethanol,50% Ethanol KL Malaysia UPLC-ESI-QTOF/MS [30] Schaftoside, Arabinoysl–glucosyl apigenin isomer, Ascorbic acid, Gendarucin A, Gendarucin A isomer, 3, 3-di-O-methylellagic acid, Methyl 2-[cyclohex-2-en-1-yl(hydroxyl) methyl]-3-hydroxy-4-(2-hydroxyethyl)-3-methyl-5-oxoprolinate, Methyl 2-[cyclohex-2-en-1-yl(hydroxyl) methyl]-3-hydroxy-4-(2-hydroxyethyl)-3-methyl-5-oxoprolinate isomer Leaf Air dry; Sonication 70% Ethanol Seremban Malaysia UPLC-MS/MS [4] Gallic acid, 4-hydroxybenzoic acid, caffeic acid, coumaric acid, ferulic acid, schaftoside, vitexin, orientin, isoorientin, isovitexin, luteolin, apigenin, forsythosides H, forsythosides I, diosmetin glycoside, diosmetin. Leaf Oven dry; Soaking 100% Methanol Kuala Lumpur Malaysia UHPLC-MS [19] Primary metabolites: Fructose, α-Glucose, β-Glucose, Sucrose, a mixture of cerebrosides, Monoacylmonogalactosylglycerol, Alanine, Glutamate, Glutamine, Proline, Threonine, Tryptophan, Valine, Citric acid, Formic acid, Fumaric acid, Choline, Adenine, Fatty acid, Ascorbic acid Secondary metabolites: Betulin, Lupeol, Stigmasterol, β-sitosterol, Clinacoside A, Clinacoside B, Clinacoside C, Cycloclinacoside A1, Cycloclinacoside A2, Isoorientin, Isovitexin, Orientin, Vitexin, Schaftoside, Gendarucin A, Catechin, Quercetin, Quercetin 3-O-rhamnoside, Quercetin 3-O-arabinofuranoside, Rutin, Chlorogenic acid, Gallic acid Leaf, Stem Air dry, Oven dry, Freeze dry; Sonication, Soaking 70% Ethanol Seremban Malaysia 1H-NMR [4] 1, 2, 3, 4: in each phytochemical class, every superscript number attached to a particular phytochemical, plant part, postharvesting method, extract/ fraction, or plant origin indicated that such information is extracted from the reference which has attached with the same superscript number.


## 2.2.2. Phytochemical Identification Using Advanced Spectrometric and Spectroscopic Analysis

Meanwhile, Table2(b) shows a list of compounds identified according to the type of the advanced spectrometric and spectroscopic analytical tools used. There are a total of 6 research teams that have used GCMS for compound identification [19, 28–32], 4 research teams that have used LCMS analytical tool [4, 19, 30, 33], and 1 research team that has used proton andJ-resolved NMR approaches [4]. Based on GCMS identification results, Cheong et al. [29] found a rich variety of triterpenoids and phytosterols in the extract. Yong et al. [32] showed that of the 14 phytochemicals identified in the chloroform extract, 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl)ester, a common plasticizer, was the most abundant compound. Mustapa et al. [30] found that phytol was the main compound in the microwave (MAE) and Soxhlet assisted extracts, while palmitic acid was dominant in the supercritical fluid assisted (SFE) extract. A study by Abdul Rahim et al. [19] identified a total of 39 peaks, but only 8 major compounds have been listed in Table 2(b). In addition, Alam et al. [28] have successfully identified a total of 48 compounds, the most abundant of which were neophytadiene, glycolic acid, phosphonic acid, catechol, butanedioic acid, vanillic acid, gallic acid, pinitol, phytol, squalene, tocopherols, and campesterol. Moreover, Teoh et al. [31] have found that the ethyl acetate root extract possessed more compounds than the methanol root extract. Among the compounds identified, lupeol was the main compound present in both extracts.Using LCMS in negative ionization mode, Huang et al. [33] successfully identified 6C-glycosyl flavones, the research team of Khoo et al. [4] was able to identify 8 compounds, Mustapa et al. [30] team have found 10 phenolic compounds from the aqueous ethanolic extract, and Abdul Rahim et al. [19] have detected 16 phenolic compounds. Khoo et al. [4] have also tentatively identified 20 primary metabolites and 22 secondary metabolites in 70% ethanol extract through proton andJ-resolved NMR spectroscopy.


## 2.3. Phytochemical and Nutrient Content

Environmental condition, geographical location, cultivation practices, genetics, pre- and postharvesting methods practiced and some other unexpected factors will cause phytochemical level variations in a plant even in the same species [18]. Thus, this section reveals the phytochemical content ofC. nutans including the total phenolic content (TPC) in Table 3, the total flavonoid content (TFC) in Table 4, and the nutritional composition ofC. nutans in Table 5. Generally, the phytochemical content ofC. nutans has been evaluated using simple chemical assay or high performance liquid chromatography (HPLC) methods. Compared to other tests, determination of TPC and TFC is preferred due to the interest in the therapeutic benefit imparted by phenolics and flavonoid; in addition, they are easy, time-effective, and cost-effective and well-established methods.Table 3
Total phenolic content (TPC) ofC. nutans.
Extract Plant part Pre- or post-harvesting method Plant origin Results of TPC Extract with highest TPC Extract with lowest TPC Reference Hot water - Oven dry; Heating at 90°C Malaysia 14.70 ± 0.08 mg GAE/g DM - - [37] Hot water Leaf Unfermented and fermented; Microwave-oven dry, freeze dry; Hot boiling water (100°C); Infusion for 1, 2, 5, 10, 15, and 20 min Sabah Malaysia 88.56 ± 4.40 to 177.80 ± 19.10 mg TAE/L Unfermented-microwave oven-20 min infusion Fermented-freeze dry-1 min infusion [36] Distilled water Leaf Combination of air dry and oven dry; Maceration with shaker-0.5, 1, 3, 5, 24 h Thailand 26.53 ± 8.83 to 46.71 ± 9.31 mg GAE/g DW Maceration for 1 h Maceration for 5 h [60] Hot water (70°C), water, methanol, 80% methanol, ethyl acetate, hexane Leaf, stem Freeze dry; Sonication Seremban Malaysia 23.15 ± 2.78 to 73.33 ± 12.18 mg GAE/g extract 80% methanol leaf Hexane stem [42] Hot distilled water, cold distilled water, methanol, ethanol, dichloromethane Leaf Orbital shaker Seremban Malaysia 24.57 ± 0.07 to 48.08 ± 0.04 mg GAE/g DW extract Cold distilled water Ethanol [18] 100% Methanol Leaf Maceration Seremban Malaysia 1.77 ± 0.01 mg GAE/g extract - - [6] 100% Methanol Leaf Air dry; Maceration Seremban Malaysia 0.78 ± 0.01 mg GAE/g DW extract at stock: 10 mg/mL - - [24] Methanol Leaf, stem Oven dry; Sonication Seremban Malaysia 0.12 and 2.68 mg GAE/g DW sample Leaf Stem [61] Methanol Leaf Air dry; Orbital shaker Malaysia, Thailand, Vietnam 8.29 to 72.16 mg GAE/g DW extract Chiang Dao, Thailand Map Khae, Thialand [18] Methanol Leaf Oven dry 40°C, 50°C, 60°C, 70°C, 80°C, 100°C; Orbital shaker Seremban Malaysia 22.44 ± 0.03 to 63.31 ± 0.03 mg GAE/g DW extract Oven dry at 100°C Oven dry at 40°C [18] Methanol Leaf, bud 1, 6 and 12 months old; Freeze dry; Shaking Serdang Malaysia 6.840 ± 0.470 to 15.460 ± 1.231 mg/g DW 6-month-old buds 1-month-old buds [41] Polar (methanol and dichloromethane), non-polar (hexane and diethyl ether) Leaf, stem Soaking Pahang Malaysia 1.43 ± 0.1 to 7.99 ± 0.6 mg GAE/g DM Leaf-polar Stem-non polar [62] Methanol, ethyl acetate, chloroform, hexane fraction - Sonication Malaysia 22.17 ± 0.02 to 119.29 ± 0.07 mg GAE/g DW Chloroform Ethyl acetate [35] 100% Ethanol Leaf Maceration Thailand 4.67 ± 3.60 mg GAE/g wet weight sample - - [63] 100%, 86%, 65%, 50%, 44% Ethanol Aerial Oven dry; MAE, p-MAE, SFE, soxhlet extraction KL Malaysia 5.74 ± 0.29 to 14.56 ± 0.77 mg GAE/g DM p-MAE in 50% ethanol MAE in 86% ethanol [30] 70% Ethanol Leaf Freeze dry; Sonication Perak Malaysia 23.5 mg GAE/g DM - - [64] 70% Ethanol Leaf, Stem Freeze dry, oven dry, air dry; Sonication, soaking Seremban Malaysia 1.04 ± 0.02 to 7.29 ± 0.11 mg GAE/g DW sample Leaf-Air dry-Sonication Stem-Freeze dry-Soaking [4] - Stem Air dry; CO2-Soxhlet, Maceration SerembanMalaysia CO2-Soxhlet: 49.45 mg GAE/g DM, Maceration: 7.54 mg GAE/g DM - - [28] 80% Ethanol Leaf, stem Young and mature; Stored for 1, 2, 3, and 4 days Malaysia Maturity: 60.75 to 117.00 mg GAE/100 g fresh sample Storage duration: 71.08 to 140.92 mg GAE/100 g fresh sample Maturity young leaf Storage 1 day storage Maturity matured stem Storage 4 days storage [38] KL: Kuala Lumpur.Table 4
Total flavonoid content (TFC) ofC. nutans.
Extract Plant part Pre- or post- harvesting method Plant origin Results of TFC Extract with highest TFC Extract with lowest TFC Reference Hot water - Oven dry; Heating at 90°C Malaysia 2.07 ± 0.05 mg QE/g dry material - - [37] Hot water Leaf Unfermented and fermented; Microwave-oven dry, freeze dry; hot boiling water (100°C); Infusion for 1, 2, 5, 10, 15, and 20 min Sabah Malaysia 14.57 ± 0.42 to 22.13 ± 1.53 mg CE/L Fermented leaf-microwave-oven dry-10 min infusion Unfermented leaf-microwave oven dry-1 min infusion [36] Hot distilled water, cold distilled water, methanol, ethanol, dichloromethane Leaf Orbital shaker Seremban Malaysia 7.09 ± 2.98 to 14.66 ± 1.71 mg QE/g of dry extract Cold distilled water Ethanol extract [18] 100% Methanol Leaf Maceration Seremban Malaysia 0.04 ± 0.001 mg QE/g extract - - [6] 100% Methanol Leaf Air dry; Maceration Seremban Malaysia 0.21 ± 0.005 mg QE/g extract - - [24] Methanol Leaf Air dry; orbital shaker Malaysia, Thailand, Vietnam 2.96 ± 0.03 to 58.38 ± 0.19 mg QE/g of dry extract Chiang Dao, Thailand Map Khae, Thailand [18] Methanol Leaf Oven dry 40°C, 50°C, 60°C, 70°C, 80°C, 100°C; orbital shaker Seremban Malaysia 14.02 ± 1.68 to 27.72 ± 0.14 mg QE/g of dry extract Oven dry at 80°C Oven dry at 40°C [18] Methanol Leaf, bud 1, 6 and 12 months old; Freeze dry; Shaking Serdang Malaysia 3.27 ± 0.33 to 6.32 ± 0.74 mg QE/g dry weight 6-month-old buds 1-month-old buds [41] Polar (methanol and dichloromethane), non-polar (hexane and diethyl ether) Leaf, stem Soaking Pahang Malaysia 3.27 ± 1.10 to 16.09 ± 4.20 mg QE/g DM Polar leaf Non-polar stem [62] Methanol, ethyl acetate, chloroform, hexane fraction - Sonication Malaysia 428.67 ± 0.03 to 937.67 ± 0.02 mg BHTE/g Chloroform fraction Methanol fraction [35] 100%, 86%, 65%, 50%, 44% Ethanol Aerial Oven dry; MAE, p-MAE, SFE, Soxhlet extraction KL Malaysia 2.71 ± 0.47 to 5.88 ± 0.22 mg QE/g of dried material SFE p-MAE at 86% ethanol [30] 80% Ethanol Leaf, stem Young and mature; Stored for 1, 2, 3, and 4 days Malaysia Maturity 45.56 to 99.47 mg RE/100 g fresh sample Storage duration 41.48 to 116.36 mg RE/100 g fresh sample Maturity young leaf Storage 1 day storage Maturity matured stem Storage 4 days storage [38] - Stem Air dry; CO2-Soxhlet, maceration Seremban Malaysia CO2-Soxhlet: 43.81 mg RE/g dried material Maceration: 16.83 mg RE/g dried material CO2-Soxhlet Maceration [28]Table 5
Nutritional composition ofC. nutans.
Sample details Carbohydrate Protein Fat Fiber Ash Moisture Vitamin Mineral Reference China- Leaf - 5.73 ± 0.14% 0.50 ± 0.02% 2.71 ± 0.05% - 78.30 ± 0.29% C: 1.57 ± 0.07 mg/100 gB1: 0.27 ± 0.04 mg/100 g - [40] Seremban Malaysia- Leaf 73.27 ± 3.14% 5.16 ± 0.08% 2.21 ± 0.66% - 10.0 ± 0.20% 9.28 ± 0.40%  Potassium: 1097.90 ± 6.93 mg/100 gCalcium: 874.50 ± 31.25 mg/100 g Sodium: 6.78 ± 1.01 mg/100 g Copper: 0.26 ± 0.01 mg/100 g [39]The Folin-Ciocalteu method is the most established method for determining the total phenolic content ofC. nutans and gallic acid or, in some cases, tannic acids are the most common standards used for TPC calibration. Values are normally expressed in mg gallic acid equivalent (GAE)/g dry weight (DW) or dry material (DM). Total phenolic content is based on a colorimetric reaction between easily oxidized polyphenols or hydroxylated aromatic compounds and phosphotungsten-polymolybdic acid [34]. The redox reaction results in the formation of blue chromophores consisting of phosphotungstic-phosphomolybdenum complexes, which have a maximum absorption at 765 nm and are proportional to the total quantity of phenolic compounds [34]. Among the TPC tests conducted, the chloroform fraction prepared by Hamid and Yahaya [35] from a Malaysian farm that underwent sonication was detected to have the highest TPC, with 119.29 ± 0.07 mg GAE/g DW. In addition, Lusia Barek et al. [36] also illustrated that an unfermented leaf subjected to drying in a microwave oven and infusion in water for 20 min retained a high TPC value of 177.80 ± 19.10 mg tannic acid equivalent (TAE)/L.The flavonoid level inC. nutans was mostly determined by the aluminum chloride (AlCl3) method. Essentially, the detection of flavonoid (the colored complexes) is conducted using the reaction between AlCl3 and the carbonyl and hydroxyl groups of flavonoids in an alkaline solution [18]. As shown by the results in Table 4, TFC was determined using quercetin as the positive control and the results were expressed in mg quercetin equivalent (QE)/g dry weight (DW) sample, although there were also some studies that used catechin, rutin, or butylated hydroxytoluene (BHT) as the positive control. Again, the chloroform fraction ofC. nutans that underwent sonicated extraction was able to retain more TFC (937.67 ± 0.02 mg BHTE/g) than those undergoing other combination of pre- and postharvesting methods [35].Other phytochemical content studies onC. nutans included hydroxycinnamic acid content determination using Arnow’s reagent. The results suggested that oven-dried and hot water extractedC. nutans extract possessed 0.91 ± 0.01 mg caffeic acid equivalent/g dry material (mg CAE/g DW) [37]. For the chlorophyll content ofC. nutans, Raya et al. [38] found that, compared to mature stem (32.27 mg/100 g) or plant sample that kept for 4 days (17.97 mg/100 g), a young leaf or plant sample subjected to 1 day of storage possessed a higher chlorophyll content with 64.35 mg/100 g and 78.83 mg/100 g, respectively. By contrast, Mustapa et al. [30] found that the absolute ethanol aerial extract ofC. nutans possessed a higher chlorophyll content (1.30 g/g DM) than absolute acetone aerial extract (1.06 g/g DM). On the other hand, Raya et al. [38] showed thatC. nutans leaves harvested at a young stage had a higher content of ascorbic acid (0.38250 mg/100 g). The results also revealed that increasing the storage duration from 1 day to 4 days caused a reduction in the ascorbic content of the extract from 0.49833 mg/100 g to 0.23083 mg/100 g. For phytosterol determination using Liebermann-Burchard reagent, one study showed that increasing the organic solvent ratio in the solvent mixture will result in a decrease in the phytosterol content [30]. This study also suggested that the supercritical fluid extraction (SFE) method allowed higher phytosterol retention (1.35 ± 0.12 mg of β-sitosterol (BS)/g DM), followed by pressurized-microwave assisted extraction (p-MAE) for the 86% ethanolic extract (1.19 ± 0.22 mg BS/g DM), microwave assisted extraction (MAE) of the 86% ethanolic extract (0.70 ± 0.10 mg BS/g DM), and Soxhlet extraction of the absolute ethanolic extract (0.47 ± 0.20 mg BS/g DM). Additionally, the nutrient composition ofC. nutans has also been determined by Sarega et al. [39] and Yu et al. [40] through proximate, vitamin, and mineral analysis as shown in Table 5.Meanwhile, there were also studies that used HPLC for the quantification of phenolic compounds [23, 39, 41–43] and, in the case of Mustapa et al. [30], for the quantification of a phytosterol compound, β-sitosterol. The HPLC parameters used and the sample details are tabulated into Table 6. Although Wong et al. [43] attempted to quantify 4 phenolics compounds, only quercetin was found (0.25 ± 0.01 μg per mg of dried sample), whereas Chelyn et al. [23] quantified schaftoside, orientin, isovitexin, and vitexin content forC. nutans leaves collected from 3 different sources. The results revealed thatC. nutans collected from Taiping, Perak, Malaysia, possessed the highest amount of all 4 phenolics: 17.43 ± 0.01 mmol/g of schaftoside, 0.86 ± 0.04 mmol/g of orientin, 2.01 ± 0.02 mmol/g of isovitexin, and 0.91 ± 0.03 mmol/g of vitexin. Ghasemzadeh et al. [41] found that samples with different maturity and from different plant parts retained different phenolics and flavonoids. Six-month-old bud extract was found to be able to retain the highest concentration of gallic acid (5.96 ± 0.55 mg/g DW). Sarega et al. [39, 42] analyzed and quantified a total of 8 phenolics in the extract. Both studies found that protocatechuic acid was the most prominent phenolic acid in the hot water, water, and 80% methanol leaf extracts (33.28 ± 0.01 mg/g extract). Finally, aerial extract that underwent SFE possessed the highest amount of β-sitosterol (0.83 ± 0.10 mg/g DM) followed by the 86% ethanolic p-MAE extract (0.65 ± 0.14 mg/g DM), 86% ethanolic MAE extract (0.52 ± 0.10 mg/g DM), and Soxhlet assisted extract (0.23 ± 0.18 mg/g DM) [30].Table 6
HPLC quantification ofC. nutans.
Sample details Stationary phase Mobile phase Detector; Wavelength Flow rate (mL/min) Elution method Standard for Quantification The corresponding concentration Reference Hot water; Oven dry Thermo ScientificODS Hypersil (5 μm, 100 × 4.6 mm) Water with acetic acid (pH 2.74), acetonitrile UV;272–370 nm 0.8 Gradient Gallic acid, catechin, caffeic acid,quercetin 0.25 ± 0.01μg/mg of dried sample [43] Leaf;Taiping (Perak), Kota Tinggi (Johor), Sendayan (Negeri Sembilan); Ethanolic; Oven dry, Sonication Kinetex Pentafluoro-phenyl (PFP)(5 μm, 250 × 4.6 mm) Water with 0.8% (v/v) glacial acetic acid, acetonitrile UV-Vis/DAD;330 nm 0.7 Gradient Shaftoside, orientin, isovitexin, vitexin 17.43 ± 0.01 mmol/g [23] Buds, Leaf; 1, 6 and 12 months old; Methanol; Freeze dry C18(5 μm, 250 × 4.6 mm) 0.03 Mortho-phosphoric acid, methanol UV-Vis;260–360 nm 1.0 Gradient Catechin, kaempferol, luteoli 7-O-β-D-glucoside, quercetin, gallic acidmonohydrate, caffeic acid 5.963 ± 0.545 mg/g DW [41] Aerial; 86% ethanol; Oven dry; MAE, p-MAE,SFE Symmetry C18(5 μm, 150 × 4.6 mm) Methanol, 2-propanol UV-ELSD; 210 nm 0.7 Isocratic β -sitosterol 0.83 ± 0.10 mg/g DM [30] Leaf;Hot water, water, 80%methanol; Freeze dry; Sonication LUNA C18(5 mm, 250 × 4.6 mm) Water with 6% acetic acid (pH 2.27), acetonitrile DAD;320 nm 0.5 Gradient Cinnamic acid,protocatechuic acid, ferulic acid, gallic acid, p-coumaric, chlorogenic acid, vanillic acid, caffeic acid 33.28 mg/g extract [42] Leaf, Aqueous,80%methanol; Sonication LUNA C18(5 mm, 250 × 4.6 mm) Water with 6% acetic acid (pH 2.27), acetonitrile DAD;320 nm 0.5 Gradient Cinnamic acid, protocatechuic acid, ferulic acid, gallic acid,p-coumaric, chlorogenic acid, vanillic acid, caffeic acid 33.38 ± 0.31 mg/g extract [39] The text inbold word indicated that particular extract has the highest concentration of the particular standard.


## 3. Pharmacological Activity

### 3.1. Pharmacological Activity ofC. nutans Extracts and Fractions

A number ofC. nutans’ traditional uses have been further verified by laboratory experiments. They have been categorized into antivenom, analgesic, anti-inflammatory, immunomodulating, neuroprotective and neuromodulating function, antidiabetic and α-glucosidase inhibitory, antioxidant, antiviral, antibacterial, antifungal, anticancer, wound healing, plasmid DNA protective, lipid elevated inhibition, and oral mucositis and stomatitis treatment activities. Tables 7(a–o) summarize all the available data regarding the pharmacological activities ofC. nutans extracts and fractions accordingly. All the tables were systematized based on experimental model, followed by type of assay and polarity of extract used. Generally, diverse results can be observed for the same pharmacological activity. The extract preparation method, origin, concentration, and assays type are the primary factors that contributed to the variation in results, and a short summary and point of view have been included in each section.Table 7
Pharmacological activities ofC. nutans.
(a)
Pharmacological activity: antivenom
Experiment design(Experiment model; Venom; Assay; Test subject) Extract Plant part Plant Source Extract dose; Route of administration Result Reference in vitro; Heterometrus laoticusscorpion venom; Pre-incubated extract with 0.2 μg/μL venom-Cell lytic test incubated for 30 min; CEFs Water Leaf Thailand 0.706, 0.406 mg/mL Extract at 0.706 mg/mL give 46.51% of efficiency but the cytotoxic of extract is questionable [65] in vitro; Apis melliferaLinn. bee venom; Pre-incubated extract with 0.6 μg/μL venom-Cell lytic test incubated for 30 min; CEFs Water, 90% and 50% ethanol Leaf Thailand 0.706, 0.406 mg/mL Ineffective [66] in vivo; Laticauda colubrinesnake venom; Mice and Mongrel dog Water; Maceration Leaf SarawakMalaysia 6 mg/kg per mouse (i.p), 20 mg/kg per dog (i.v) Ineffective [7] in vivo; Naja naja siamensis snake venom; Isolated rat phrenic-nerve diaphragm preparations; Mice Water Leaf Thailand p.o, i.p Ineffective [5] in vivo;Snake venom; Mice Water Leaf Thailand - Reduce mortality rate from 100% to 63 ± 3.34% [67] in vivo;Snake venom; Mice 95% alcohol Leaf Thailand 2000 mg/kg; i.v, i.p, p.o Ineffective [67] CEFs: Chick embryo fibroblasts.  (b)
Pharmacological activity: analgesic/antinociceptive
Experiment design(Experiment model; Assay; Test subject) Extract Plant part Plant Source Extract dose; Route of administration; positive control Result Reference in vivo; Acetic acid-induced writhing test; Mice Water, methanol, chloroform,n-butanol Leaf Thailand Phenylbutazone (100 mg/kg) Effective (n-butanol extract at 90 mg/kg) [68, 69] in vivo; Acetic acid-induced abdominal writhing test-Pre-treatment 1 h before test; ICR mice (adult male, 25–30 g) 100% Methanol; Oven dry; Soaking Leaf Kuala Lumpur Malaysia 100, 250, and 500 mg/kg; ASA (100 mg/kg) ED50: 279.3 mg/kg [19] in vivo; Formalin-induced paw licking test-Pre-treatment 1 h before test; Sprague Dawley rat (adult male 150–180 g) 100% Methanol; Oven dry; Soaking Leaf Kuala Lumpur Malaysia 100, 250, and 500 mg/kg; ASA (positive, 100 mg/kg); morphine (5 mg/kg) Early phase: ED50: >500 mg/kg Late phase: ED50: 227.7 mg/kg [19] in vivo; Hot plate test at 50°C- Pre-treatment 1 h before test; ICR mice (adult male; 25–30 g) 100% Methanol; Oven dry; Soaking Leaf Kuala Lumpur Malaysia 100, 250, and 500 mg/kg; morphine (5 mg/kg) 500 mg/kg significant delay response at the interval of 60 to 210 min [19] in vivo; Hot glass jar at 72°C water bath; Albino mice (either sex, 40–50 g) 95% Ethanol; Maceration Leaf Thailand 5 g/kg Ineffective [92] in vivo; Hot water plate test; Mice n-Butanol Leaf Thailand p.o, i.p; morphine Ineffective [68, 69]  (c)
Pharmacological activity: anti-inflammatory
Experiment design(Experiment model; Assay; Test subject) Extract Plant part Plant Source Extract dose; Route of administration; Positive control Result Reference in vitro; fMLP induced elastase release- Pre-treatment; Human neutrophils Methanol; Air dry; Percolation Whole plant Thailand 0.01–100μg/mL; Indomethacin (1–100 μg/mL) IC50: 186.8 ± 20.5 μg/mL elicited a weak, but significant inhibition of human neutrophil elastase release (100 nM) [70] in vitro; fMLP/CB induce elastase release; Human neutrophils 80% ethanol, ethyl acetate,n-hexane; Air dry; Soaking Aerial TaichungTaiwan 10μg/mL 80% ethanol showed highest inhibition: 68.33 ± 5.49% [52] in vitro; fMLP induced neutrophil superoxide anion generation (pre-incubation for 10 min) Methanol; Air dry; Percolation Whole Thailand 0.01–100μg/mL; Indomethacin IC50: 23.4 ± 3.1 μg/mL [70] in vitro; Superoxide anion generation assay; Human neutrophils 80% ethanol, ethyl acetate,n-hexane; Air dry; Soaking Aerial TaichungTaiwan 10μg/mL 80% ethanol showed highest inhibition: 28.52 ± 2.55% [52] in vitro; fMLP induced neutrophil myeloperoxidase (MPO) production Methanol; Air dry; Percolation Whole Thailand 0.01–100μg/mL; Indomethacin IC50: 219.5 ± 25.7 μg/mL Inhibition via reduced neutrophil migration [70] in vitro; Immunoblotting-LPS induced TLR-4 inflammatory proteins; Protein lysate from macrophage Polar; Soaking Leaf, stem Pahang Malaysia 20μg/mL Significantly reduced the LPS induced phosphorylation of p65, p38, ERK1/2, JNK1/2, IRF3 [62] in vitro; LPS induced TLR-4 assay; HEK-Blue-hTLR4 cells Polar, nonpolar; Soaking Leaf, stem Pahang Malaysia 100μg/mL IC50: 21.3 ± 5.0 (leaf polar) to 29.4 ± 9.0 (leaf non-polar) μg/mL [62] in vitro; LPS induced cytokine production assay-1 h pre-treatment; murine macrophages RAW 264.7 cell Polar, nonpolar; Soaking Leaf, stem Pahang Malaysia 100μg/mL Polar leaf (p< 0.05) inhibited TNF-α, IFN-γ, IL-1β, IL-6, IL12p40, IL-17 production [62] in vitro; LPS induced NO- 1 h pre-treatment- Griess assay; Murine macrophages RAW 264.7 cell Polar, nonpolar; Soaking Leaf, stem Pahang Malaysia 100μg/mL IC50: 18.9 ± 3.6 (leaf polar) to 43.1 ± 4.7 (leaf non-polar) μg/mL [62] in vivo; Acetic acid induced vascular permeability model; Mice Water, methanol, chloroform,n-butanol Leaf Thailand Indomethacin (4 mg/kg) Effective,n-butanol at 540 mg/kg as potent as indomethacin [68, 69] in vivo; EPP induced rat ear oedema model- pre-treatment; Sprague Dawley rats (male, 40–60 g) Methanol; Air dry; Percolation Whole plant Thailand 3, 6, 9 mg/20μL acetone per ear; Apply topically; Indomethacin (2 mg/20 μL) At dose 9 mg EPP/ear: 79% oedema inhibition at 15 min, 44.4% MPO reduction after 120 min of induction [70] in vivo; Carrageenan induced paw oedema model-1 h pre-treatment; Sprague Dawley rats (male, 100–120 g) Methanol; Air dry; Percolation Whole plant Thailand 50, 100, 200 mg/kg; p.o; Indomethacin (20 mg/kg) 200 mg/kg of extract inhibit 59% of oedema at 3 h [70] in vivo; Carrageenan induced paw oedema; Albino mice (40–50 g) 95% Ethanol; Maceration Leaf Thailand 5000 mg/kg; p.o 17.73% at 3 h and 36.47% at 6 h of oedema inhibition [92] in vivo; Carrageenan induced paw oedema model; Mice n-Butanol Leaf Thailand p.o; acetylsalicylic acid (100 mg/kg) Effective, n-butanol at 270 mg/kg as potent as ASA [68, 69] in vivo; Granuloma pouch model; Wistar rats (male) Aqueous ethanol cream Leaf Thailand 125 mg cream/rat; Apply topically; Prednisolone (0.25%) Inhibit 48.3% granuloma formation [71] in vivo; Granuloma pouch model; Wistar rats (male) 95% ethanol cream Leaf Thailand 125 mg cream/rat; Apply topically; Prednisolone (0.25%) Inhibit 50.1% granuloma formation [71] in vivo; Granuloma pouch model-1 h pre-treatment; Mice n-Butanol cream Leaf Thailand 270 and 540 mg/kg; Apply topically Ineffective [68, 69] in vivo; Granuloma pouch model; Wistar rats (male) Cold cream Leaf Thailand 125 mg cream/rat; Apply topically; Prednisolone (0.25%) Inhibit 50.98% granuloma formation [71]  (d)
Pharmacological activity: immunomodulating effect
Experiment design(Experiment model; Assay; Incubation period; Test subject; Extract dose; Positive control) Extract Plant part Plant Source Result Reference in vitro; with and without fMLP induced Chemotaxis and Chemokinesis; 45 min; Human neutrophils; 0.1–100 μg/mL; Indomethacin (0.01–100 μg/mL, IC50: 56.3 ± 3.5 ng/ml) Methanol; Air dry; Percolation Whole plant Thailand Chemotaxis: With fMLP induction, IC50: 2.7 ± 0.6 μg/mL, Without fMLP, IC50: >100 μg/mL Chemokinesis: With fMLP induction, IC50: 5.5 ± 0.6 μg/mL, Without fMLP, IC50: 5.0 ± 0.5 μg/mL [70] in vitro; Apoptosis assessment via morphological, flow cytometry-Annexin V binding; 20 h; Human neutrophils; 10–500 μg/mL; Dexamethasone (1 μM, inhibited 54.5% neutrophil apoptosis) Methanol; Air dry; Percolation Whole plant Thailand No significant effect on neutrophil apoptosis [70] in vitro; ConA & LPS-induced IL-10 & TNF-α expression-Real time PCR analysis; 16 h; PBMC; 1.56 mg/mL Methanol; Oven dry; Maceration Leaf, Stem Chiang Mai Thailand Reduce IL-10 mRNA expression, no modulating effect on TNF-α mRNA expression [72] in vitro; IFN-γ expression Splenocyte from ovalbumin-primed BALB/c mice 80% Ethanol; Air dry; Soaking Aerial Taichung Taiwan 0.1μg/mL: upregulation of IFN-γ. 100 μg/mL: down-regulation of IFN-γ [52] in vitro; HaCaT; IFN-γ/TNF-α-induced apoptosis- MTT assay; 1 and 100 μg/mL Ethanol; Maceration Leaf Thailand Significantly(p<0.05) suppressed keratinocytes apoptosis [63] in vitro;Lymphocyte proliferation assay-3H- thymidine-radioactivity; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand 0.5–5μg/mL cause proliferation increase, 2.5, 5 mg/mL cause proliferation decrease [73] in vitro;NK activity-K562 as target cell-cytotoxicity assay; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand 1 and 5 mg/mL-cause NK activity decrease [73] in vitro;IL-2 production-ELISA; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand undetectable IL-2 [73] in vitro;IL-4 production-ELISA; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand 2.5 and 5 mg/mL-increase IL4 production [73] in vitro;Lymphocyte subpopulation-flow cytometry assay; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand no change in the percentages of CD3+, CD4+, CD8+, CD16+/CD56+ and CD19+ cells [73]  (e)
Pharmacological activity: neuroprotective and neuromodulating function
Experiment design(Experiment model; Assay; Incubation period; Test subject) Extract Plant part Plant Source Extract dose; Route of administration; Positive control Result Reference in vitro; OGD–reoxygenation and hypoxic neuronal death, Cell viability assay;Mouse primary cortical neurons, cerebral astrocytes, cerebral endothelial cells 80% Ethanol; Soaking Leaf Singapore 5μg/mL Extract suppressed post-hypoxic HDACs activation and reduce OGD-caused neuronal death [75] in vitro; OGD–reoxygenation; 24 h Human SH-SY5Y neuroblastoma cells 80% Ethanol; Soaking Leaf Singapore 100μg/mL Extract modulated cPLA2 expression induction in SH-SY5Y cells by HDAC inhibitors, MS-275, MC-1568, TSA and inhibited HAT activity. [74] in vitro;OGD–reoxygenation; 12 h and 24 h; Mouse primary cortical neurons 80% Ethanol; Soaking Leaf Singapore 1.6, 6.25μg/mL Extract inhibited levels of cPLA2 mRNA expression in primary cortical neurons subjected to 0.5 h OGD injury [74] in vitro;OGD; CCK-8; up to 12 h; neutron pre-treated with extract 1 h before OGD; Mouse primary cortical neurons from E15.5 Balb/c mouse embryos 80% Ethanol; Soaking Leaf Malaysia 6.25μg/mL Extract treated neurons showed significant increment in cell viability [76] in vitro;OGD–reoxygenation; CCK-8, MMP, apoptosis analysis, transient transfection and chromatin reporter assay; 0.5 h OGD+ 4–24 h Reoxygenation; Neurons treated with extract (1 h before, on the onset or after OGD-reoxygenation); Mouse primary cortical neurons from E15.5 Balb/c mouse embryos 80% Ethanol; Soaking Leaf Malaysia 0.075–20μg/mL Neurons treated with extract before, at the onset or after OGD showed increment in dose dependent manner but protective effect of extract was lesser when applied after OGD.Neurons treated with 6.25 μg/mL extract at the onset of OGD has reduced MMP breakdown, apoptotic death, and pro-apoptotic (caspase-3, PARP-1) and has higher anti-apoptotic (14-3-3ε, p-Bad, Bcl-2) markers.Neurons treated with 2.5–10 μg/mL extract at the onset of OGD suggested a dose dependent increase effect in PPAR-γ mRNA level. [76] in vitro; Anti-spasmotic effect on acetycholine induced rat’s bladder tissue contraction; Isolated tissues of rat’s bladder Ethanol Leaf, stem Malaysia Mebeverine hydrochloride and detrusitol Stem at 120 mg/mL: 79.77%Leaf at 100 mg/mL: 26.62% Detrusitol at 200 μg/mL: 89.86%, Mebeverine at 10 mg/mL: 80.74% of contraction inhibition [93] in vivo; Ellman assay-Acetylcholinesterase activity in brain, liver, kidney, heart; Pretreatment for 14 days (daily treatment (once)); Balb/C mice (male, 25 ± 5 g) Methanol, Air dry, Maceration Leaf Seremban Malaysia 250, 500, 1000 mg/kg bw All doses of extracts caused the higher acetylcholinesterase activity in liver, kidney, and heart compared to control group while they did not caused any difference in brain [77] in vivo; MCA occlusion stroke model; Reperfusion infarct volume, behaviour assessment, MRI; extract treated 1 h before, immediate or 3–24 h after 30 min MCA occlusion or 1 d after 15 min MCA acclusion; Long- Evans rats (male, 7-8 week-old) 80% Ethanol; Soaking Leaf Malaysia 10–60 pg (i.c.v., after 30 min MCA occlusion); 24 mg/kg body wt (i.p, 1 h before or 3–24 h after 30 min or 24 h after 15 min MCA occlusion) I.C.V. treatment of 40 pg extract reduced apoptotic neuronal death, showed a maximal decrease of cerebral infarct volume at 1 day reperfusion. PPAR-γ, C/EBPβ mRNA levels, anti- apoptotic 14-3-3ε, p-Bad, and Bcl-2 levels were upregulated. Pro-apoptotic cleaved PARP-1 and cleaved caspase 3 protein levels were downregulated. Extract improved the functional outcomes based on the behavior observation at day 7 after ischemia (elevated body swing test and Bederson’s postural reflex task) and at day 14 after ischemia (better ladder rung walking test). Extract via i.p treatment also significantly reduced infarct volume except 24 h after 30 min MCA occlusion. Behavior test conducted also showed better functional outcomes and smaller infarct volume via MRI. [76]  (f)
Pharmacological activity: antidiabetic andα-glucosidase inhibition activity
Experiment design(Experiment model; Assay; Test subject) Test sample Plant part Plant Source Extract dose; Route of administration; Positive control Result Reference in vitro;α-glucosidase inhibition assay Hot water; Oven dry - Malaysia 50 mg/mL 88.2% of inhibition [37] in vitro;α-glucosidase inhibition assay Methanol; Oven dry; Sonication Leaf, Stem Seremban Malaysia 5000μg/mL (in stock) 13.57 (leaf), 17.67 (stem) % of inhibition [61] in vitro;α-glucosidase inhibition assay 70% Ethanol; Oven, air, freeze dry; Sonication, soaking Leaf, stem Seremban Malaysia 5000μg/mL (in stock); Quercetin Lowest: 5.31% (Leaf-Freeze dry-Soaking), Highest: 41.70% (Leaf-Oven dry-sonication) [4] in vitro;α-glucosidase inhibition assay CO2-Soxhlet, Maceration Stem Seremban Malaysia 5000μg/mL (in stock); Quercetin CO2-Soxhlet: 95.79% of inhibition Maceration: 58.23% of inhibition [28] in vivo; Alloxan induced model- daily treatment for 9 days;Swiss webstermice (male) Hot water; Sun dry Leaf Bandung Indonesia 50, 100, and 150 mg/kg BW; p.o; Oral glibenclamide 150 mg/kg significantly lower blood glucose serum level from 442 ± 149 mg/dl (day 0) to195±66 mg/dl (day 9) [25] in vivo; High fat and high cholesterol diet (HFHC) induced insulin resistance-7 weeks periods-daily treatment of extract; Sprague Dawleys rat (male, 200–250 g) Water, 80% methanol; Sonication Leaf Seremban Malaysia 500, 250 or 125 mg/kg/day/rat; p.o.; Simvastatin C. nutansattenuated the metabolic effects and transcriptional changes induced by the HFHC diet [39] in vivo; Glucose solution (p.o, 2 g/kg bw);Swiss webstermice (male) Insoluble ethyl acetate fraction from ethanol Leaf Bandung Indonesia 100 mg/kg bw; p.o Decreased 18.4% blood glucose serum level in 3 h [25]  (g)
Pharmacological activity: antioxidant
Test sample Plant part Plant Source Extract dose; Positive control Result Reference (1) Type of assay:in vitro; DPPH scavenging assay  Hot water; Oven dry - Malaysia 1 to 16 mg/mL 16 mg/mL: ~60% inhibition [37] Hot water; Unfermented and fermented; Microwave-oven dry, freeze dry; Infusion for 1, 2, 5, 10, 15, and 20 min Leaf Sabah Malaysia 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL Unfermented possessed higher DPPH inhibition than fermented tea [36] Hot water, water, 80% methanol, methanol, ethyl acetate, hexane; Freeze dry; Sonication Leaf, stem Seremban Malaysia Trolox 6.12% (hexane leaf) to 55.12% (80% methanol leaf) inhibition [42] Water, methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL; Trolox Chloroform > methanol > water864.11 ± 73.49 to 7852.63 ± 449.90 (chloroform) μg Teq/g extract [32] Methanol; Sun dry Aerial Vietnam 0, 20, 40, 60, 80, 100μg/mL IC50: 114.50 μg/mL [49] Methanol; Air dry; Percolation Whole Thailand 1–400μg/mL; Trolox IC50: 240.1 ± 15.3 mg/mL [70] Methanol; Air dry; Maceration Leaf Seremban Malaysia 0.25 to 10 mg/mL; Quercetin IC50: 1.33 ± 0.0001 mg/mL [24] Methanol; Oven dry; Soaking Leaf, stem Seremban Malaysia 5000μg/mL (stock); Quercetin Leaf: IC50: 1126.63 μg/mL Stem: IC50: 1548.89 μg/mL [61] Methanol; 1, 6 and 12 months old; Freeze dry Bud, leaf Serdang Malaysia BHT, caffeic acid,α-tocopherol IC50: 64.6 (1 year old buds) to 112.1 (1 year old leaf) μg/mL [41] Methanol, Petroleum ether, Ethyl acetate; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.2 to 10.0 mg/mL 4 mg/mL: leaf petroleum ether give 82% of DPPH inhibition [88] Methanol, ethyl acetate, chloroform, hexane Whole Jelebu Malaysia 1000μg/mL; BHT 50.50 ± 0.03% (Hexane) to 70.96 ± 0.03% (chloroform) inhibition [53] 98% methanol, ethyl acetate, chloroform, hexane; Sun dry; Soaking Stem Vietnam Vitamin C Ineffective [47] 50% ethanol; Soaking Leaf Thailand 1–300μg/mL; Ascorbic acid I C 50: 110.4±6.59 μg/mL [94] 70% ethanol; Freeze dry; Sonication Leaf PerakMalaysia 200–1000μg/mL; Green tea I C 50 not determined [64] 70% ethanol; Freeze dry, oven dry, air dry; Sonication, soaking Leaf, stem Seremban Malaysia 5000μg/mL (stock); Quercetin 15.44 ± 2.21% (Stem-freeze dry-soaking) to 44.31 ± 3.16% (Leaf-oven dry-sonication) inhibition [4] 80% Ethanol; Young, old; Storage duration (1, 2, 3, and 4 days) Leaf, stem Malaysia - Maturity: 31.24% (matured stem) to 112.12% (young leaf) Storage duration: 31.88% (4 days) to 101.85% (1 day) inhibition [38] Ethanol; Maceration Aerial BangkokThailand 1 mg/mL Ineffective [78] Ethyl acetate Leaf Indonesia BHT I C 50: 178.40 mg/L [95] CO2-Soxhlet, Maceration Stem Seremban Malaysia 5000μg/mL (stock); Quercetin CO2-Soxhlet: 98.92% inhibition Maceration: 63.00% inhibition [28]  (2) Type of assay:in vitro; FRAP assay  Hot water; Unfermented and fermented; Microwave-oven dry, freeze dry; Infusion for 1, 2, 5, 10, 15, and 20 min Leaf Sabah Malaysia 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL 250.70 ± 49 (fermented-microwave oven dry-20 min infusion) to 438.80 ± 94 (unfermented-freeze dry-10 min infusion) mg/L [36] Hot water, water, 80% methanol, methanol, ethyl acetate,Hexane; Freeze dry; Sonication Leaf, stem Seremban Malaysia. Gallic acid ~45 (hexane stem) to ~148 mg (hot water leaf) GAE/g extract [42] Methanol, ethyl acetate, chloroform, hexane Whole Jelebu Malaysia 1000μg/mL; Ascorbic acid hexane and ethyl acetate (not determined) to 56.49 ± 0.05% (methanol) [53] Methanol; 1, 6 and 12 months old; Freeze dry Bud, leaf Serdang Malaysia BHT, caffeic acid, Vitamin C 209μM of Fe (II)/g (1-month-old leaves) to 488 μM of Fe (II)/g (6-month-old buds). [41] 70% Ethanol; Freeze dry; Sonication Leaf PerakMalaysia 200–1000μg/mL; Green tea very low absorbance (ineffective) [64] 50% Ethanol; Soaking Leaf Thailand 1–100μg/mL; Ascorbic acid 17 mg ascorbate/g extract [94]  (3) Type of assay:in vitro; Hydrogen peroxide scavenging activity  Water, methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL; Quercetin Methanol: highest radical scavenging: ~34% at 100μg/mL [32]  (4) Type of assay:in vitro; Metal chelating activity  Hot water; Oven dry - Malaysia 1, 2, 5, 10 mg/mL 10 mg/mL: ~90% inhibition [37]  (5) Type of assay:in vitro; Nitric oxide scavenging assay  Hot water; Oven dry - Malaysia 1, 5, 10 mg/mL 10 mg/mL: ~90% inhibition [37] Water; methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL Water: 32.33% at 100μg/mL [32]  (6) Type of assay:in vitro; ABTS cation radical scavenging assay  Hot water; unfermented and fermented; microwave-oven dry, freeze dry; hot boiling water (100°C); infusion for 1, 2, 5, 10, 15, and 20 min Leaf Sabah Malaysia 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL 50.36 ± 4.07 (fermented-microwave oven dry-10 min infusion) to 74.03 ± 2.26 (unfermented-freeze dry-5 min infusion) mg AEAC/L [36] Hot water, water, 80% methanol, methanol, ethyl acetate,hexane; Freeze dry; Sonication Leaf, stem Seremban Malaysia Trolox 18 (hexane leaf) to 65 (80% methanol-leaf) mg TE/g extract [39]  (7) Type of assay:in vitro; Galvinoxyl radical scavenging activity  Water; methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL; Trolox Chloroform > methanol > aqueousChloroform: 12248.82 μg Teq/g extract [32]  (8) Type of assay:in vitro; Superoxide radical scavenging activity  Methanol; Air dry; Percolation Whole Thailand Direct EPR scavenging effect showed significant direct scavenging activity when the incubation time extended to 60 min [70] 70% ethanol; Freeze dry; Sonication Leaf PerakMalaysia Green tea; by NBT method 3651.5 U/g [64]  (9) Type of assay:in vitro; Phorbol 12-myristate 13-acetate (PMA) induced peroxide production in rat macrophages  50% Ethanol; Soaking Leaf Thailand 10, 30, 100 and 300μg/mL; Tiron Fluorescent intensity: 58.72 ± 5.52 (30μg/mL), 51.92 ± 8.49 (100 μg/mL), 53.50 ± 6.17 (300 μg/mL) [94]  (10) Type of assay:in vitro; Protective effect against peroxyl radicals initiator (AAPH)-induced oxidative hemolysis  50% Ethanol; Soaking Leaf Thailand 200–1000μg/mL; Ascorbic acid IC50: 359.38 ± 14.02 μg/mL [94]  (11) Type of assay:in vivo; Hyperlipidemia-associated oxidative stress model  Water, 80% methanol; Freeze dry; Sonication Leaf, stem Seremban Malaysia 125, 250, 500 mg/kg; Sprague Dawley rats (male, 200–250 g); daily treatment for 49 days; p.o; Simvastatin Both leaf extracts reduce oxidative stress through increasing serum antioxidant enzymes activity and upregulating the expression of hepatic antioxidant genes [42]  (h)
Pharmacological activity: antimicrobial-antiviral
Experiment design(Experiment model; Virus strain; Assay; Test subject; Route of administration/Prescription) Extract Plant part Plant Source Extract dose; Positive control Result Reference Type of virus: Varicella zoster virus (VZV)  in vitro; VZV Kawaguchi strain; (1) DNA hybridization technique, (2) plaque reduction assay-pre, post, direct; WI-38 HEL cells Organic; Soxhlet Leaf Thailand AcyclovirIC50: 107 μM (pre), 5 μM (post), 30 μM (direct) Most effective: Direct inactivation(1) IC50: 1 : 2000 (pre), 1 : 6000 (post), >1 : 18000 (direct) (2) IC50: 1 : 2000 (pre), 1 : 4800 (post), 1 : 9600 (direct) [80] Clinical; Herpes zoster (shingles); Double-blinded, randomized trials; 60 patients; Apply topically 5 times daily for 7 to 14 days 5% extract cream - Thailand Placebo Lesion crusting within 3 days:C. nutans(89.3%), Placebo (0%) Lesion healing within 7 days:C. nutans(100%), Placebo (100%) Pain scores: C. nutansbetter than placebo group [8] Clinical; Herpes zoster (shingles); Randomized trials; 48 patients; Apply topically 5 times daily for 5 days 5% extract cream - Thailand Placebo, Acyclovir Lesion crusting within 3 days:C. nutans(81.3%), Acyclovir (27.2%), Placebo (0%) Lesion healing within 7 days:C. nutans(81.30), Acyclovir (18.20%), Placebo (0%) [82] Clinical; Herpes zoster (shingles); Double-blinded, block randomization; 120 patients; Apply topically 3 times daily from 1 to 26 days 120 patients - Thailand Placebo Lesion crusting within 3 days:C. nutans(27.5%), Placebo (8.6%) Lesion healing:C. nutans(14 days) Placebo (18 days) Symptom reduction: C. nutans better thanplacebo groups [81]  Type of virus: Herpes simplex virus (HSV)  in vitro; HSV-1-KOS strain; Plaque reduction assay-post; Vero cell Methanol, dichloromethane,n-hexane; Soxhlet Leaf Thailand 12.5–100μg/mL; Acyclovir (IC50: 0.09 μg/mL) IC50: 32.05 ± 3.63 (n-hexane) to 64.93 ± 7.00 (methanol) μg/mL [83] in vitro; HSV-1F strain; Plaque reduction assay-pre, post; Vero cell Ethyl acetate; Oven dry; Soxhlet Leaf Thailand 19μg/mL; Acyclovir (5 μg/mL, post), Dextran sulfate (2 μg/mL, pre) Pre: IC50: 7.6 μg/mL significantly reduce plaques Post: ineffective [79] in vitro; HSV-1 strain; Plaque reduction assay-pre, post-incubated for 48 h; Vero cell Chloroform; Soxhlet Leaf Thailand Acyclovir (IC50:  0.64 ± 0.07 μg/mL) Pre: less than 50% inhibition of plaque formationPost: IC50: 115.00 μg/mL [56] in vitro; HSV-2-strain G, 5 clinical HSV-2 isolates; Plaque reduction assay (post), Yield reduction assay (post), Inactivation kinetics (direct, 4 h); Vero cell Methanol; Air dry; Percolation Whole Thailand 2500μg/mL Ineffective [96] in vitro; HSV-2-Baylor 186; Plaque reduction assay-post; Vero cell Methanol, dichloromethane,n-hexane; Soxhlet Leaf Thailand 12.5-100μg/mL; Acyclovir (IC50: 0.43 μg/mL) IC50: 65.13 ± 2.22 (methanol) to 72.62 ± 12.60 (n-hexane) μg/mL [83] in vitro; HSV-2 standard strain; Plaque reduction assay-pre, post, direct; BHK cell Ethanol(A2, C1, C2, C3, C4, C5 fraction) Leaf Thailand - C2, C3, C4: 1 : 2400 dilution cause 100% plaque inhibition, through extracellularly pathway [97] in vitro; HSV-2 strain; Plaque reduction assay-pre, post-incubated for 96 h; Vero cell Chloroform; Soxhlet Leaf Thailand Acyclovir (IC50: 0.80 μg/mL) Post: IC50: 140.00 ± 3.00 μg/mL [56] Clinical; HSV-2-Herpes genitalis; Sequential randomisation; 77 patients; Apply topically 4 times daily for 6 days 5% extract cream - Thailand Placebo, Acyclovir (Zovirax) Lesion crusting within 3 days:C. nutans(98.6%), Acyclovir (80.8%), Placebo (12.5%) Lesion healing within 7 days:C. nutans(88.9%), Acyclovir (84.6%), Placebo (20.8%) C.nutanshas no sticky, burning, stinging pain and side effects. [82, 84] Clinical; HSV-2-Herpes genitalis; Randomized trials; 163 patients; Apply topically 4 times daily for 6 days 5% extract cream - Thailand Placebo, Acyclovir The lesion crusting and healing speed were significantly better in theC. nutansand acyclovir treated groups compared to the placebo groups. No side effect observed C. nutanstreated group. [85]  Type of virus: Fish pathogenic virus  in vitro; IHNV, OMV, IPNV strain; Plaque reduction assay-pre, post, direct; CHSE-214 cells Ethanol; Soxhlet - Thailand 100μg/mL (pre, post), 500 μg/mL (direct) Direct: 100% (IHNV and OMV), 0% (IPNV) of plaque reductionPre: 31% (IHNV), 54% (OMV), 74% (IPNV) of plaque reduction Post: 25% (IHNV), 48% (OMV), 3% (IPNV) of plaque reduction [98]  Type of virus: Crustaceans (shrimp and prawn) infectious virus  in vivo;YRV-RNA virus; Anti-viral test-direct; cultured black tiger shrimp Ethanol; Soxhlet Leaf Thailand 0.1 to 10 mg/mL Minimum inhibition: 1μg/mL [87] in vivo; YRV-RNA virus; Protective efficacy assay-14 days observation; daily twice treatment for 7 days-pre; cultured black tiger shrimp; p.o Ethanol; Soxhlet Leaf Thailand 0, 0.1, 1 and 10 g/kg of pellet 1 g/kg of feed exhibited best protective efficacy with 57.6% [87]  Type of virus: Mosquito-borne virus  in vitro; DENV-2 strain 16681; Western blot assay, ECL detection kit-post; Naïve Huh-7 cells 80% Ethanol; Air dry; Soaking Aerial Taichung Taiwan Ribavirin IC50: 31.04 μg/mL [52]  Type of virus: Poultry and bird contagious virus  in vitro;NDV- La Sota strain; Hemagglutination test-pre, post; CEFs Water, Ethanol; Soxhlet - Thailand 31.25 g/mL (final) Ineffective [86]  (i)
Pharmacological activity: Anti-microbial-anti-bacterial
Experiment design(Experiment model; Bacteria strain; Assay) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro;A. hydrophila; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99] in vitro; B. cereus; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf PerakMalaysia 25, 50 and 100 mg/mL;Ciprofloxacin At 100 mg/mL:15.00±1.00 mm [22] in vitro;B. cereus; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: >12.5 mg/mL [6] in vitro; B. cereus;Microdilution assay Ethyl acetate and its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Ampicillin MIC: Ethyl acetate: 6.31 mg/mL,F7: 1.39 mg/mL [88] in vitro; B. subtilis; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; E. coli; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; E. coli; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf Perak 25, 50 and 100 mg/ml;Ciprofloxacin At 100 mg/ml:17.00±2.00 mm [22] in vitro;E. coli; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: 12.5 mg/mL [6] in vitro; E. coli;Microdilution assay Ethyl acetate and its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Ampicillin MIC: Ethyl acetate: >100 mg/mL,F7: 1.39 mg/mL [88] in vitro; E. coliDC10; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; E. coli(wild); Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; M. luteus; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; MRSA;Disc diffusion assay, microdilution assay Ethanol; Oven dry; Maceration - Thailand 5 mg/mL No detected inhibition zone, MIC and MBC: >5 mg/mL [100] in vitro; MSSA K147; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; N. gonorrhoeae & 11 clinical isolates; Disc diffusion Methanol; Maceration Leaf Thailand 44 mg/mL (final); Norfloxacin Ineffective [101] in vitro; P. acnes; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: >12.5 mg/mL [6] in vitro;P. acnes; Disc diffusion, microdilution assay Ethanol; Maceration Aerial Thailand Mangostin MIC and MBC: >5 mg/mL [102] in vitro; P. aeruginosa; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; P. aeruginosa; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf Perak 25, 50 and 100 mg/ml;Ciprofloxacin At 100 mg/ml:13.00±1.00 mm [22] in vitro; P. aeruginosa187 (wild); Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; S. aureus; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; S. aureus; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf Perak 25, 50 and 100 mg/ml;Ciprofloxacin At 100 mg/mL:26.67±3.51 mm [22] in vitro; S. aureus; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: 12.5 mg/mL [6] in vitro; S. aureus;Disc diffusion assay, microdilution assay Methanol; Sun dry; Maceration Aerial Vietnam 125–1000 mg/mL; Erythromycin (10–50μg/μL) 16.67 mm inhibition zone, MIC: 62.5 mg/mL [49] in vitro; S. aureus;Disc diffusion assay, microdilution assay Ethanol; Oven dry; Maceration - Thailand 5 mg/mL No detected inhibition zone, MIC: 5 mg/mL, MBC: >5 mg/mL [100] in vitro; S. entericaserovar Paratyphi C; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Showed inhibition zone [26] in vitro; S. entericaserovar Paratyphi B; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Chloroform: a larger inhibition zone compare to other solvents. [26] in vitro; S. entericaserovar Typhi; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Least inhibition zone [26] in vitro; S. entericaserovar Typhimurium; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Showed inhibition zone [26] in vitro; S. enterica Typhimurium;Disc diffusion, microdilution assay Methanol; Sun dry; Maceration Aerial Vietnam 125–1000 mg/mL; Erythromycin (10–50μg/μL) 15.67 mm inhibition zone, MIC: 125 mg/mL [49] in vitro; S. enterica Typhimurium;Microdilution assay Ethyl acetate and its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Ampicillin MIC: Ethyl acetate: >100 mg/mL,F7: 1.39 mg/mL [88] in vitro; S. entericaserovar Weltevreden; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Showed inhibition zone [26] in vitro;S. epidermidis; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: >12.5 mg/mL [6] in vitro;S. epidermidis; Disc diffusion, microdilution assays Ethanol; Maceration Aerial Thailand Mangostin MIC and MBC: >5 mg/mL [102] in vitro; S. epidermidis;Disc diffusion, microdilution assays Ethanol; Oven dry; Maceration - Thailand 5 mg/mL No detected inhibition zone, MIC and MBC: 5 mg/mL [100] in vitro;Streptococcussp.; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99] in vitro; V. harveyi; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99] in vitro; V. paraliaemolyticlls; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99]  (j)
Pharmacological activity: antimicrobial-antifungal
Experiment design(Experiment model; Fungal strain; Assay) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro; A. fumigatus; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Nystatin Ineffective [89] in vitro; C. albicans; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Nystatin Ineffective [89] in vitro; C. albicans; Microdilution assay Ethyl acetate & its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Amphotericin B Ethyl acetate: 6.31 mg/mL,F7: MIC: 1.39 mg/mL [88]  (k)
Pharmacological activity: anticancer
Experiment design(Experiment model; Cancer cell(s); Incubation period; Assay; Test subject; Route of administration) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro; Mutated Salmonella typhimurium (T98 and T100) without metabolic activation; 6 days; Mutagenicity assay-Ames test Water; Air dry; Maceration Leaf Penang Malaysia 500μg/well Non-mutagenic activity inS. typhimuriumhistidine auxotrophs [103] in vitro; D24, MM418C1, MCF7, BT474 cancer cells; 24 h and 72 h; CCK-8 assay Hot water, cold water, methanol, ethanol, dichloromethane Leaf Seremban Malaysia 2 mg/mL D24 cell: cold water at 72 h caused 42.9% cell death (EC50: 1.63 mg/mL) MM418C1 and MCF7 cells: hot and cold water showed cytotoxicity BT474 cells: extract ineffective [18] in vitro; K562, HCT 116 cancer cells; MTT cytotoxicity assay Water, 50% methanol, 100% methanol, 50% ethanol, 100% ethanol Leaf Malaysia 100 and 200μg/mL Ineffective [104] in vitro; HepG2, IMR-32, NCI-H23, SNU-1, LS-174T, K-562, HeLa, Raji cancer cells; 72 h; MTT cytotoxicity assay Water, ethanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 3.125 to 100μg/mL Most effective: chloroform with IC50: 47.70 μg/mL (K562), 47.31 μg/mL (Raji) [32] in vitro; Cultured Saos-2 human steosarcoma cells; HIF activity, MTT cytotoxicity assay Methanol Leaf Malaysia 125–2000μg/mL Ineffective [105] in vitro; HeLa cancer cells; 72 h; MTT cytotoxicity assay Methanol; Freeze dry Leaf, buds Serdang Malaysia 10, 20, 40, 80, 160, 320μg/mL; Tamoxifen Most effective: 6-month bud with IC50: 56.8 μg/mL [41] in vitro; D24 melanoma cells; 24 h and 72 h, CCK-8 assay Methanol; Air dry; Soaking with shaker Leaf Malaysia, Vietnam, Thailand 0–2 mg/mL (stock) Chiang Dao Thailand extract has highest cytotoxicity: EC50: 0.95 mg/mL (24 h), 0.77 mg/mL (72 h) [106] in vitro; HepG2, MCF-7, NCI-H460, HeLa cancer cells; 48 h; Sulforhodamine B colorimetric assay Sub-fraction F-III from Methanol extract Stem Vietnam Camptothecin Effective, IC50: 36.80 (HepG2), 57.36 (NCI-H460), 66.57 (MCF-7), 91.08 (Hela) μg/mL [47] in vitro;HepG2 cell; 24 h; MTT cytotoxicity assay Methanol, ethyl acetate, hexane, chloroform fraction - Malaysia 0, 6.25, 12.5, 25, 50, 100μg/mL (stock) IC50: 43.93 (methanol), 55.61 (chloroform), 62.06 (ethyl acetate), 68.38 (hexane) μg/mL [35] in vitro;MCF-7, HeLa cells, 72 h; MTT cytotoxicity assay Methanol, ethyl acetate; Freeze dry; Soaking Root Sabah Malaysia 10–50μg/mL; Camptothecin (0.35 μg/mL) MCF-7 cell: IC50: 35.0 μg/mL (methanol), 30.0 (ethyl acetate) μg/mL HeLa cell: ~25% of inhibition for both extracts [31] in vitro; HeLa, K-562 cells; 24 h and 72 h; MTT cytotoxicity assay Methanol, ethyl acetate, petroleum ether; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.2–10.0 mg/mL Petroleum ether leaf extract showed the strongest cytotoxic activity after 72 h, CC50: 18.0 μg/mL (HeLa), 20.0 μg/mL (K-562) [88] in vivo; MMS induced Allium cepa chromosome assay-post and suppressive treatment;Allium cepa Water, methanol; oven dry Leaf PenangMalaysia 100, 200, 400, 800 mg/kg Extract has repairing and anti-mutagenic effect [90] in vivo; Hepatocarcinoma tumor-bearing mice-daily treatment for 10 days; ICR mice (with normal T/B cells, 6–8 weeks, 18–22 g); Gastric probe 30% Ethanol fraction Aerial SerembanMalaysia 3 and 10 mg/kg; Fluorouracil (20 mg/kg) 8.2% (3 mg/kg) and 58.6% (10 mg/kg) of tumor size and weight reduction, higher reduction rate than positive control (37.1%) [33]  (l)
Pharmacological activity: wound healing ability
Experiment design(Experiment model; Assay; Test subject) Extract Plant part Plant Source Extract dose Result Reference in vitro; Migration rate of HGF at 0 h, 2 h, 4 h, and 6 h after scratching; Human gingival fibroblast (HGF) Chloroform, Hexane; Sun dry Leaf Indonesia 10, 25, 50 and 100μg/mL Culture supplemented with 10μg/mL of chloroform extract give the fastest HGF migration rate and wound recovery [11]  (m)
Pharmacological activity: protective effect on plasmid DNA
Experiment design(Experiment model; Assay; Test Subject) Extract Plant part Plant Source Extract dose Result Reference in vitro; DNA integrity assay; Supercoiled plasmid DNA react with O2•-generated via a riboflavin photoreaction treatment 70% Ethanol; Freeze dry; Sonication Leaf PerakMalaysia 10000, 1000, 100, 10 and 0μg/mL; Green tea Extract reduced DNA cleavages, retained high levels of super-coiled plasmid DNA integrity and exhibited protection up to 50 min [64]  (n)
Pharmacological activity: lipid elevated inhibition activity
Experiment design(Experiment model; Assay) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro; Porcine pancreatic lipase inhibition assay Methanol; Freeze dry; Sonication Leaf Malaysia 100μg/mL; Orlistat Ineffective, −22.56% indicates a promotion of pancreatic lipase activity [21]  (o)
Pharmacological activity: oral mucositis and stomatitis treatment
Experiment design(Experiment model; Infection; Assay; Test subject; Route of administration/Prescription) Extract Plant Source Positive control Result Reference Clinical; Radiation induced oral mucositis; Single-blinded-Randomized trial; 60 patients; Apply 2 drops drip into mouth or on the lesion 3 to 5 times daily from first to last day of radiation Glycerin payayor Thailand Benzydamine hydrochloride(Difflam, 3M, Australia) The time onset of oral mucositis in the payayor group was significantly later, and its severity and pain score were less than those of the benzydamine group throughout the study period. [10] Clinical; Reccurent aphthous stomatitis; Double-blinded-controlled trial; 43 patients; apply to the ulcer 4 times daily C. nutans in orabase Thailand Triamcinolone acetonide, placebo C. nutans in orabase provide better healing of the ulcer as compared to placebo but efficacy was lesser compared to triamcinolone acetonide in orabase [91]


#### 3.1.1. Antivenom Activity

Table7(a) summarizes the antivenom tests that have been carried out thus far onC. nutans. The experiments examined the antivenom properties ofC. nutans’ leaves against snake, scorpion, and bee venoms. There were a total of 2in vitro and 4in vivo antivenom studies, and the extracts tested were prepared from water, ethanol, and aqueous ethanol. From the results of thein vitro studies, the water extract at 0.706 mg/mL exhibited a moderate anti-scorpion venom effect through a direct inactivation mechanism [65] while the aqueous ethanol extracts were ineffective against bee venom [66]. On the other hand, in 3 out of the 4in vivo studies conducted, the extracts (2 water and 1 aqueous ethanol extracts) notably did not exhibit antivenom effects after the test subjects were given different doses (6 to 2000 mg/kg bw) of the extracts through intraperitoneal injection (i.p.), intervenous injection (i.v.), or per os (p.o.) [5, 7, 67]. Although there was 1in vivo study proposing that the water extract ofC. nutans could exhibit a moderate anti-snake venom effect, the study did not include the administration method or the doses given [67]. Thus, defining a suitable extract that is biologically active as an antidote for snake and insect venoms is ambiguous. In summary, among the extracts tested, only water leaf extract exhibited potentialin vitroinhibition effect towards snake and insect venoms via direct inactivation mechanism. Further animal studies that focus on studying the metabolites of water extract and their reaction with the mediators that responsible for the direct inactivation pathway are worth emphasizing. Clinical study that investigates the efficacy of water extract as an antivenom agent through application of topical formulation on the patients might lead to a possible breakthrough.


#### 3.1.2. Analgesic Activity

Table7(b) displays the scientific studies that investigated the analgesic ability ofC. nutans through 3 different types ofin vivo assays: the acetic acid-induced writhing test, formalin-induced paw licking test, and hot plate test. Three studies have used the acetic acid-induced writhing test to examine the analgesic effect ofC. nutans extracts prepared using 4 different types of solvents [19, 68, 69]. The results suggested that treating mice with then-butanol extract at 90 mg/kg as well as with the methanol extract at 279.3 mg/kg was as potent in terms of analgesic properties as treating them with phenylbutazone at 100 mg/kg. On the other hand, the study by Abdul Rahim et al. [19] was the only one that used formalin-induced paw licking test to evaluate the analgesic properties ofC. nutans. The results suggested that the methanol extract, with a half maximal effective concentration (EC50) at 227.7 mg/kg, was able to relieve pain in the late phase (centrally inflammatory induced pain pathway) via opioid/nitric oxide- (NO-) mediated, but cyclic guanosine monophosphate- (cGMP-) independent modulation systems. In addition, the analgesic effects of the absolute methanol, ethanol, andn-butanol extracts have been examined through the hot plate test. The results suggested that 500 mg/kg methanol extract was effective in alleviating the pain response at the interval from 60 to 210 min while both the ethanol and butanol extracts showed no analgesic effects up to 5 g/kg. Overall, methanol extract ofC. nutans exerted potential analgesic activity in both acute and persistent pain tests. The study also suggested that the presence of phenolics particularly gallic acid, caffeic acid, ferulic acid, vitexin, and apigenin which previously reported to exert antinociceptive activity which might be the main activity contributors. Since methanol extract might be a potential analgesic agent, it is important to further investigate the efficacy dosages, sites of action, and the level of the extract to modulate the pain along the pathway suggested. Furthermore, study may also provide information regarding antinociception properties ofC. nutans methanol extract on visceral and neuropathic pains.


#### 3.1.3. Anti-Inflammatory Activity

Table7(c) summarizes the anti-inflammatory effect ofC. nutans. The anti-inflammatory effect ofC. nutans was assessed based onin vitro assays, such as the macrophage activatorN-formyl-methionyl-leucyl-phenylalanine- (fMLP-) induced neutrophil elastase release superoxide anion generation, lipopolysaccharides (LPS) induced toll like receptor 4 (TLR-4), NO Griess, and cytokine production assays. Furthermore, the types ofin vivo experiments used to evaluate the acute anti-inflammatory properties ofC. nutans were the acetic acid-induced vascular permeability model and ethyl phenylpropiolate- (EPP-) induced rat ear oedema and carrageenan-induced paw oedema model, whereas its subchronic anti-inflammatory effect was assessed using the granuloma pouch model, which mimics subchronic inflammation in humans. For thein vitro anti-inflammatory effects, both the methanol and 80% ethanol extracts have been evaluated through the neutrophil elastase release and superoxide anion generation assays [52, 70]. The results showed that 10 μg/mL of 80% ethanol extract inhibited 68.33% elastase release, which was more effective than the methanol extract (<20%), while to inhibit superoxide radical formation, both extracts at 10 μg/mL showed comparable efficacy, as they inhibited approximately 30% superoxide formation [52, 70]. For the LPS-induced TLR-4, NO, and cytokine production assays, the results suggested that the polar (methanol and dichloromethane) extract possessed a better IC50 value (<22 μg/mL) for inhibiting TLR-4, NO and proinflammatory cytokine production than the nonpolar (hexane and diethyl ether) extract [62]. For thein vivo acute anti-inflammatory properties ofC. nutans, the most effective dose was pretreatment of 9 mg EPP/ear of methanol extract on the rat which resulted in 79% oedema inhibition at 15 min and 44.4% myeloperoxidase (MPO) reduction after 120 min of induction [70].Additionally, in a comparison of the methanol, ethanol, andn-butanol extracts, a 1 h pretreatment of 200 mg/kg methanol extract administered via p.o. to carrageenan-induced rats was found to inhibit 59% oedema formation [70]. The acetic acid-induced vascular permeability model showed that, among the water, methanol, chloroform, andn-butanol extracts, the butanol extract at 540 mg/kg was superior at exhibiting an anti-inflammatory effect [68, 69]. Ethanol, aqueous ethanol,n-butanol, and cold creams have been evaluated for their subchronic anti-inflammation properties. The results showed that application of 125 mg ofC. nutans cold cream to a rat topically throughout the experimental period inhibited granuloma formation by as much as 50.98%, which is comparable to the inhibitory effect of 0.25% prednisolone (56.82%) [71]. According to the previousC. nutans anti-inflammatory studies, the extract prepared from polar solvent notably exhibited a promising anti-inflammatory propertiesin vitro(at a dose less than 30 μg/mL) andin vivo (at a dose less than 300 mg/kg). Therefore, it is greatly desired to focus on the relationship between the mode of action ofC. nutanspolar extract in both biological and nonbiological anti-inflammatory systems. In addition, since polar extract exhibited potential anti-inflammatory activity, knowledge of extract prepared from the inorganic polar solvent particularly water is still scarce as regards itsin vivoandin vitro anti-inflammatory activity properties, thus possessing potential research interest.


#### 3.1.4. Immunomodulating Activity

Table7(d) shows the immunomodulating effect ofC. nutans. The methanol extract ofC. nutans has been tested for its immune modulating effect on apoptosis and cytokines expression in experiments on human neutrophils and porcine peripheral blood mononuclear cells (PPBMCs), such as assays for fMLP macrophage activator-induced chemotaxis and chemokinesis, apoptosis, and concanavalin (ConA) and LPS-induced interleukin 10 (IL-10), and tumor necrosis factor-alpha (TNF-α) expression [70, 72]. Wanikiat et al. [70] suggested thatC. nutans methanol extract exhibited dose-dependent suppression of fMLP-induced chemotaxis and chemokinesis of neutrophils without causing the cells to undergo apoptosis.C. nutans was found to cause a reduction in IL-10 expression and to have no effect on TNF-α expression in PBMC [72]. On the other hand, the ethanolic extract ofC. nutans has been tested for its immunomodulating effect on cytokines expression in splenocytes, a human keratinocyte cell line (HaCaT), and human peripheral blood mononuclear cells (HPBMC). Tu et al. [52] suggested that low concentrations of the ethanol extract cause IFN-γ upregulation while higher concentration ofC. nutans cause IFN-γ downregulation. Sriwanthana et al. [73] studied found that low concentrations ofC. nutans resulted in increase in lymphocyte proliferation, while higher concentrations ofC. nutans resulted in decrease of lymphocyte production. The study also found thatC. nutans did not stimulate an interleukin-2 (IL-2) response or affect the lymphocyte subpopulation, such as total T lymphocytes (CD3), T helper/inducer cells (CD4), T suppressor/cytotoxic cells (CD8), natural killer (NK) cells (CD16/CD56), or B lymphocytes (CD19). On the other hand, 2.5 and 5 mg/mL of ethanol extract increased the interleukin-4 (IL-4) production and 1 and 5 mg/mL of extract suppressed NK activity [73]. Furthermore, 1 and 100 μg/mLC. nutans were found to inhibit IFN-γ and TNF-α-induced keratinocytes apoptosis [63]. A similar trend as in the analgesic and anti-inflammatory properties ofC. nutans could be observed, where extract prepared from polar solvents exerted better analgesic, anti-inflammatory, and immunomodulating effects. Phenolics, sulfur-containing glucosides, and sulfur-containing compounds might be the main contributors to these activities, as all the mentioned compounds were primarily isolated from polarC. nutans extracts. In addition, the described therapeutic effects are always well correlated with the immunology results. Thus, investigations on the biological mechanism of the active constituents present in the polar extract and their effects on immunological cells, mediators, and substances deserve special attention. Exploring the immunological function of other polar extracts, such as water, and nonpolar extracts should be encouraged as well.


#### 3.1.5. Neuromodulating Activity

The efficacy ofC. nutans in neuro-related protective and modulating functions has also been studied in recent years, as shown in Table 7(e).In vitro studies suggested that the neuroprotective effect ofC. nutans was promising. From the studies by Tan et al. [74] and Tsai et al. [75], the 80% ethanol extract with a dose concentration less than 10 μg/mL was able to suppress post-hypoxic histone deacetylase (HDAC) activation and hypoxic neuronal death in an oxygen-glucose deprivation- (OGD-) reoxygenation assay. It also decreased the levels of cytosolic phospholipase 2 (cPLA2) mRNA expression in mouse primary cortical neurons subjected to 0.5 h of OGD injury. In addition, Tan et al. [74] found that 100 μg/mL 80% ethanol extract was able to suppress histone acetylase (HAT) activity and regulate cPLA2 expression induction in a human neuroblastoma cell line (SH-SY5Y cells) through HDAC inhibitors such as entinostat (MS-275), MC-1568, and trichostatin A (TSA). The research team of Wu et al. [76] further suggested that treating the primary neurons with 0.15–20 μg/mL of 80% ethanol extract 1 h before, at the onset, or after the OGD induced cell death or OGD-reoxygenation treatment was able to mitigate the neuronal apoptosis and protected primary neurons by activating the antiapoptotic activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ) → 14-3-3ε (antiapoptotic marker), enhancing the C/EBPβ binding to PPAR-γ promoter and amplifying its transcription though the extract was less effective when applied after the OGD treatment. The result also suggested that the protective effect of extract was revoked when cotreated with GW9662 (PPAR-γ antagonist). Thisin vitro screening further complemented with the protective effect of extract in attenuating the ischemic brain damage inin vivo middle cerebral artery occlusion (MCA) stroke model [76]. Both intracerebroventricular (i.c.v.) infusion (10–60 pg extract) and i.p. injection (24 mg/kg body wt extract) of extract demonstrated that the extract possessed the ability in mitigating apoptotic neuronal death, cerebral infarct volume, and behavioral deficiency in the rat MCA occlusion model. The detailed neuroprotective mechanism ofC. nutans was similar toin vitro OGD study and was elaborated in Table 7(e). In addition, anin vivostudy conducted by Lau et al. [77] revealed that the three different tested doses of methanol extract were able to stimulate acetylcholinesterase activity in the heart, liver, and kidney, but not the brain of mice without inducing any signs of toxicity in the mice. As a short summary, similar results have been reported as those regarding the analgesic, anti-inflammatory, and immunomodulating activities, where the polar extract ofC. nutans possessed a great neuroprotective effect. Nevertheless, research on the neuroprotective strength ofC. nutans is still new and therefore holds enormous potential for different research directions. Further investigation of the potential efficacy of polarC. nutans extract inin vivo neurology-inflammation related studies might lead to a breakthrough. Research should fill in gaps about the neuro-related function of nonpolar extract as well.


#### 3.1.6. Antidiabetic andα-Glucosidase Inhibitory Activity

Table7(f) shows a list of studies conducted on the antidiabetic and α-glucosidase inhibition effects ofC. nutans. There have been a total of 4in vitro studies assessing the hyperglycemia inhibitory activity ofC. nutansthrough an α-glucosidase inhibition assay. All the results showed thatC. nutans had a very low inhibitory effect on the α-glucosidase enzyme when the employed extracts were prepared using conventional preparation methods [4, 28, 37, 61]. Although IC50 was not calculated by Alam et al. [28], a lower IC50 value is anticipated as the extract prepared through advanced supercritical extraction exhibited 95.79% of α-glucoside inhibition when the stock was prepared at 5000 μg/mL. DespiteC. nutans not showing particularly pronounced inhibition against α-glucosidase, 3in vivo antidiabetic experiments suggested that the water extract, 80% methanol extract, and the insoluble ethyl acetate fraction from the 80% ethanol extract were able to attenuate the insulin resistance induced by a high fat and high cholesterol diet (HFHC) and serum glucose level [25, 39]. In general, the high α-glucosidase inhibition by SFE treated extracts might indicate that the compounds active against the α-glucosidase enzyme are heat-sensitive or easily degradable. Thus, extra precaution is needed during the extract preparation step. On the other hand, conventional method prepared extracts that mimicked the traditional application showed low inhibition towardsin vitroα-glucosidase tests while traditional uses suggested thatC. nutans is an antidote for diabetes. The contradictory results might be attributed by improper mode of action of diabetes being investigated. Since α-glucosidase inhibition test focuses on mechanism of postprandial hyperglycemia, further study on other mechanism approach, experiment on the reaction of the extract towards β-cell in pancreas, test that involved other enzymes in hyperglycemia such as pancreatic α-amylase, glucose diffusion assay, glucose uptake by yeast cells, and nonenzymatic glycosylation assay are highly recommended.


#### 3.1.7. Antioxidant Activity

Considerable number of analyses have been done on the antioxidant effect ofC. nutans as shown in Table 7(g). Based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay, some studies showed thatC. nutans up to 1 mg/mL is a potential antioxidant agent, while other studies found that the DPPH inhibitory properties ofC. nutans could not be determined [47, 64, 78]. The data in the table showed that, among the polar and nonpolar extracts tested, the highest antioxidant properties ofC. nutanswas detected in the study by Ghasemzadeh et al. [41], whereC. nutans bud extract that was cultivated for 1 year at a farm located in Malaysia and that underwent freeze drying and methanol extraction resulted in IC50 value of 64.6 μg/mL. The second most common antioxidant determination assay selected by researchers was the ferric reducing antioxidant power (FRAP) assay. However, discussion of the FRAP results is difficult, as there is a lack of unit standardization in the measurement. The results from othersin vitro antioxidant assays, such as those for hydrogen peroxide scavenging, metal chelating, nitric oxide scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical scavenging, galvinoxyl radical scavenging activity, superoxide radical scavenging activity, and phorbol 12-myristate 13-acetate (PMA) induced peroxide production in rat macrophages and protective effect against peroxyl radicals initiator- (AAPH-) induced oxidative hemolysis, have been summarized in Table 7(g(3)–g(10)). Overall, polar and semipolar solvent-extractedC. nutans were more likely to exhibit moderate antioxidant activity at concentration varying from 0.0125 to 10 mg/mL than nonpolar extracts. In addition, there was a recent study that evaluated the antioxidant activity ofC. nutans in vivo by using a hyperlipidemia-associated oxidative stress model [42]. The study suggested that both the water and 80% methanol leaf extracts at up to 500 mg/kg bw rats were able to mitigate oxidative stress by improving the activity of serum antioxidant enzymes and expression of hepatic antioxidant genes [42]. In summary, polar and semipolar extracts are more likely to exhibit antioxidant properties. A possible hypothesis is that phenolics, sulfur-containing glucosides, sulfur-containing compounds, chlorophyll derivatives, and some phytosterol derivatives that are isolated fromC. nutans using polar and semipolar solvents might be the contributors. Thus, studies on the antioxidant properties of these isolated compounds and further evaluation of the plant’s antioxidant effect inin vivo studies are required.


#### 3.1.8. Antiviral Activity

The antimicrobial properties ofC. nutans have been further classified into antiviral, antibacterial, and antifungal effects, as shown in Tables 7(h), 7(i), and 7(j), respectively. For the antiviral activity ofC. nutans, the types of virus that have been studied were varicella zoster virus (VZV), herpes simplex virus (HSV), fish pathogenic viruses, crustaceans (shrimp and prawn) infectious viruses, mosquito-borne viruses, and poultry and bird contagious viruses. Notably, the antivirus studies were always accompanied with a prescreening test for cytotoxicity to determine the subtoxic concentration of the test sample and to ensure that the test sample has a killing effect on the virus instead of on the host cell [79]. Researchers have also investigated the mode of action ofC. nutans in terms of its antiviral potential through 3 different stages of treatment. Pretreatment (pre-) studies are those where the test sample is allowed to incubate with the cell for a time of period before it is infected with the virus. This approach reflects the virucidal activity of the test sample, as the test sample interferes with the viral structure to prevent or inhibit the viral penetrating or adsorbing to the host cell [79]. In posttreatment (post-) studies, instead of being preincubated with the test sample, the virus is attached to the host cell that has first only been cultured with the test sample. The test sample is thus considered to have antiviral activity if it successfully inhibits viral DNA replication [79]. By contrast, for the direct inactivation (direct) pathway, the virus is preincubated with the test samples first before being added to the host cell. The test sample is said to have an inactivation ability against the virus if it interferes with or causes damage to the viral glycoproteins, the virus envelope, or the virus structure before they enter the host cell [79].Thus far, only onein vitro study and a total of 3 clinical trials have been conducted to evaluateC. nutans’ anti-VZV activity. Thein vitro result suggested that the organic extract exhibited anti-VZV activity through the direct inactivation stage [80]. In the clinical studies, theC. nutans extracts were formulated into a 5%C. nutans cream prior to testing its ability to combat VZV infections. The result suggested that theC. nutans containing cream was able to exhibit a positive curing effect. All the studies showed that the percentage of patients who experienced lesion crusting within 3 days and lesion healing within 7 days after applying theC. nutans cream topically was better than the percentage in the placebo and acyclovir groups. In addition, the pain score and side effect resulting fromC. nutans cream application were lower [8, 81, 82].For the anti-HSV-1 activity ofC. nutans, the ethyl acetate, methanol, dichloromethane, chloroform, andn-hexane extracts have been evaluated. Thongchai et al. [79] suggested that theC. nutans ethyl acetate leaf extract displayed the best inhibition on HSV strain with IC50 value of 7.6 μg/mL through the pretreatment mechanism. Meanwhile, the methanol, ethanol dichloromethane, chloroform, and hexane extracts were also tested for their anti-HSV-2 activity. TheC. nutans methanol and hexane leaf extracts were found to have better and comparable inhibition against HSV-2-Baylor 186 strain, via posttreatment action, with IC50 value of 65.13 and 72.62 μg/mL, respectively [83]. The anti-HSV-2 activity ofC. nutans has also been evaluated in clinical studies using the 5% extract cream. The results obtained were similar to those from the clinical studies on the anti-VZV activity; when compared to a placebo, the days required for lesion crusting and healing after application ofC. nutans were higher than or the same in terms of efficacy as acyclovir [82, 84, 85]. Regarding its efficacy in inhibiting fish pathogenic viruses, among the virus strains tested, theC. nutansethanolic extract exerted 100% inhibition on plaque formation in infectious hematopoietic necrosis virus (IHNV) andOncorhynchus masouvirus (OMV) strains but not in infectious pancreatic necrosis virus (IPNV) through the direct inactivation stage.On the other hand, it was also found thatC. nutans ethanol extract had an excellent protective effect on cultured black tiger shrimp against yellow head rhabdo-like virus (YRV-RNA) through a direct inactivation mechanism and pronounced virucidal activity against dengue virus (IC50 value of 31.04 μg/mL); however, it was less effective against Newcastle disease virus (NDV) caused by poultry and birds [52, 86, 87]. In summary,C. nutans ethanol extract possessed a very promising antiviral effect. Future research may explicate the antiviral activity of stigmasterol derivative, sulfur-containing compounds, monoacylmonogalactosylgycerol, and all of nine cerebrosides, as, according to previous studies, they are the main compounds isolated from the ethanol extract. Though clinical trial suggested its therapeutic properties, research that optimizes the efficacy dosage and profiles the safety uses of the plant extract is lacking.


#### 3.1.9. Anti-Bacterial Activity

Table7(i) shows the antibacterial properties ofC. nutans. Overall, studies were mainly based onin vitro assays and only polar (water, methanol, and ethanol) and semipolar (chloroform and ethyl acetate) extracts, but nonpolarC. nutans extracts not were evaluated for their anti-bacterial activity. A total of 9 different Gram-positive bacteria have been used in previous studies, includingBacillus cereus, Bacillus subtilis, Staphylococcus aureus, methicillin-resistantStaphylococcus aureus (MRSA), methicillin-sensitiveStaphylococcus aureus (MSSA),Micrococcus luteus, Propionibacterium acnes, Staphylococcus epidermidis, andStreptococcus sp. On the other hand,Aeromonas hydrophila, Escherichia coli and its strains,Neisseria gonorrhoeae,Pseudomonas aeruginosa and its strains,Salmonella enterica and its serovars, andVibrio harveyiandVibrio parahaemolyticus were the Gram-positive bacteria selected for study. As a summary of the antibacterial efficacy ofC. nutans, the most effective extract of the plant regarding antibacterial effect was prepared by oven-drying and undergoing ethyl acetate fractionation. The extract prevented visibleB. cereus, E. coli,andS. enterica Typhimurium growth at 1.39 mg/mL [88].


#### 3.1.10. Antifungal Activity

Concerning the antifungal efficacy ofC. nutans,limited studies had been carried out to date. In the two reported antifungal activity studies, Cheeptham and Towers [89] found thatC. nutans did not exert a fungicidal effect onCandida albicans orAspergillus fumigatuswhen 5 mg/mL 95% ethanol leaf extract was tested. By contrast, a fraction of the ethyl acetate extract at the minimal concentration of 1.39 mg/mL did exhibit an antifungal effect onC. albicans[88]. In summary, based on the availablein vitro studies,C. nutans polar and semipolar extracts exhibited a weak antibacterial and antifungal activities. Any further research should focus on assessing the biological action of the polar and semipolar extracts ofC. nutans in inhibiting bacterial and fungal infections as well as augmenting the limited information on the antibacterial and antifungal potency of the nonpolar extract.


#### 3.1.11. Anticancer Activity

A total of 15 types of cancer cells that are responsible for different types of cancer have been used to evaluate thein vitro anticancer effect ofC. nutans, as summarized in Table 7(k). These include human melanoma cell lines (D24 and MM418C1) for skin cancer, a human osteosarcoma cell line (cultured Saos-2) for bone tumor, human breast adenocarcinoma (MCF7) and human breast carcinoma (BT474) cell line for breast cancer, human lung adenocarcinoma (NCI-H23) and human lung carcinoma (NCI-H460) cell lines for lung cancer, a human liver hepatocellular carcinoma cell line (HepG2) for liver cancer, a human neuroblastoma cell line (IMR-32) for nerve tissue cancer, a human gastric cancer cell line (SNU-1) for gastric cancer, human colon adenocarcinoma (LS-174T) and human colorectal carcinoma cell line (HCT 116) for colon and rectal cancer, a human cervical cancer cell line (HeLa) for cervical cancer, a human erythroleukemia cell line (K562) for acute myeloid leukemia, and a human Burkitt’s lymphoma cell line (Raji) for lymphatic disorder. In addition, there were 2 studies that assessed thein vivo antimutagenic and anticancer effect ofC. nutans [33, 90]. Based on these studies, the most effective extract was the petroleum ether leaf extract, which showed the strongest cytotoxic activity after 72 h of incubation. The concentration of the extract that caused 50% death (CC50) was 18.0 μg/mL in HeLa cell and 20.0 μg/mL in the K-562 cell line [88]. According to the National Cancer Institute (NCI), a crude extract that exhibits an IC50 value <20 μg/mL can be considered as an active anticancer agent [41]. Overall, nonpolar leaf extract possessed a very pronounced cytotoxic effect on cervical and erythroleukemia cancerous cell. Hence, to evaluate the suitability ofC. nutans to involve in anticancer drug development, furtherin vivo clinical experimental studies on cervical cancer and erythroleukemia models are encouraged. The mechanism behinds the activity should be critically analyzed too. On the other hand, from the summarized data, observed inconsistency of incubation period during the experiment might be one of the factors that caused result deviation. Further parameter standardization should be implemented. Furthermore, the effect of the ethanol extract onin vivo hepatocarcinoma tumor-bearing mice exhibited a similar trend as in thein vitro study (methanolic extract) suggesting that both polar extracts possessed similar cytotoxic action towards liver cancerous cells, which is worthy of further investigation.


#### 3.1.12. Wound Healing Activity

In addition to the other well studied pharmacological activities ofC. nutans, there are also a few other potential pharmacological activities which are less studied or that even have been reported only once. Table 7(l) summarizes the wound healing ability ofC. nutans.Between the chloroform extract and hexane extract, the chloroform extract (10 μg/mL) was found to provide the best improvement in human gingival fibroblast (HGF) migration rate and wound recovery in the 6 h of observation [11]. Before labelling it as a potential wound healing agent during inflammation, however, more studies are needed to verify its wound healing effect and determine which of the chlorophyll derivatives, diglycerides, or stigmasterol derivatives are the main factor for this positive activity.


#### 3.1.13. Protective Effect on Plasmid DNA Activity

The protective effect ofC. nutanson plasmid DNA is tabulated in Table 7(m). Yuann et al. [64] suggested that, compared to green tea extract (protection up to 30 min), the 70% ethanol extract had the ability to reduce the number of DNA cleavages, preserve higher levels of supercoiled plasmid DNA integrity, and provide better protection against the riboflavin photoreaction induced superoxide for up to 50 min. In short, the polar extract showed a protective effect on plasmid DNA, but more analysis involving anin vivo model is needed. In addition, it is important to direct research focus towards the protective effect of the nonpolar extract as well.


#### 3.1.14. Lipid Elevated Inhibition Activity

The ability ofC. nutans to attenuate obesity had been assessed as shown in Table 7(n) through anin vitro porcine pancreatic lipase inhibition assay. The result suggested that, instead of an inhibitory effect, the methanol extract exhibited a pancreatic lipase promoting effect [21]. However, as suggested previously, more research involving cellular andin vivoexperimental design are encouraged before reaching any conclusion.


#### 3.1.15. Oral Mucositis and Stomatitis Treatment

C. nutansis also formulated for oral application to treat radiation induced oral mucositis in head and neck cancer patients and in patients suffering from recurrent aphthous stomatitis, as presented in Table 7(o) [10, 91]. The results revealed that, compared to the positive control group, the time onset of oral mucositis was significantly later, and the pain score was significantly lower [10]. Although it is not as efficacious as the positive control group (triamcinolone acetonide), it can be a good alternative to lessen the severity of ulcers [91].


### 3.2. Pharmacological Activity ofC. nutans Isolated Pure Compounds

After understanding the pharmacological properties ofC. nutans extract, it is beneficial to further investigate which compound in a particular extract is responsible for the therapeutic activity of the plant. Table 8 summarizes the pharmacological activities of the isolated compounds fromC. nutans. For anti-inflammatory activity, only cerebrosides from the ethanol extract have been studied, and they were reported as being ineffective in suppressing cyclooxygenase (COX-1 and COX-2) induced prostaglandin E2 generation and HSV-1 infection [55]. The result obtained was inconsistent with the anti-inflammatory effect of the extract observed previously. Nevertheless, the type of experiment conducted might cause this variation, and further attention should focused on the anti-inflammatory effect of other compounds isolated from the polar solvent. Le et al. [45] studied the immunomodulating function of flavones (schaftoside) and terpenes (stigmasterol, β-sitosterol, and lupeol) compounds isolated from the hexane fraction of the methanol extract. The results showed that only stigmasterol and β-sitosterol were able to suppress the T cell proliferation mediated by ConA and that only β-sitosterol exerted an immune suppressive effect on T helper 2 (Th2) cytokines (IL-4 and IL-10). A similar trend was observed with the polar extract ofC. nutans which exerted a better immunomodulating effect than the nonpolar extract. Clinamide D, which was isolated from the methanol extract, was found to have a moderate antioxidant effect with 76.05 ± 0.02% DPPH radical inhibition at 1000 μg/mL which is consistent with the good DPPH inhibition properties of the methanol extract [41, 53].Table 8
Pharmacological activity of isolatedC. nutanscompounds.
Experiment design(Experiment model; Assay; Incubation period; Test subject; Extract dose; Positive control) Isolated compound Result Reference (a) Anti-inflammatory activity  in vitro; Prostaglandin E2 determination assay by the radioimmunoassay Immortalized COX-1 and COX-2 mouse lung fibroblast cell; Aspirin (IC50: 2.06 μg/mL (COX-1), 3.57 μg/mL (COX-2)) Cerebrosides Ineffective [55]  (b) Immunomodulating activity  in vitro; ConA-induced T cell, LPS-induced B cell, evaluation of Th1 cytokines (IL-2 and IFN-γ), Th2 cytokines (IL-4 and IL-10); Splenocytes; Control on Th2 cytokines IL-4 (138.3 pg/mL) and IL-10 (1234.9 pg/mL) ( 1 ) CN1 (shaftoside) (2) CN2 (stigmasterol) (3) CN3 (β-sitosterol)  (4) CN4 (lupeol) CN3 inhibit T lymphocyte proliferation the most (RP: 0.16) followed by CN2 (RP: 0.47), only CN1 inhibit B cell proliferation (RP: 0.77), all ineffective on Th1 cytokines, CN3 inhibit secretion of IL-4 (22.6 pg/mL) and IL-10 (63.9 pg/mL), CN3 significantly reduce activated helper T cells (54.3%) and activated cytotoxic T cells (62.2%) [45]  (c) Anti-oxidant activity  in vitro; DPPH scavenging assay; Vitamin C (IC50: 22.589 μg/mL) ( 1 ) Stigmasterol-β-D-glucoside (2) 3-amino-4,5-dihydroxyfuran-2(3H)-one Compound 1: IneffectiveCompound 2: IC50: 102.949 μg/mL [49] in vitro; DPPH scavenging assay, FRAP assay; 1000 μg/mL (stock); BHT (DPPH), Ascorbic acid (FRAP) ( 1 ) Clinamide D(2) Clinamide E (1) 76.05 ± 0.02%, (2) 72.84 ± 0.01% of DPPH inhibition, (1) 15.47 ± 0.03%, (2) 38.56 ± 0.02% of FRAP inhibition [53]  (d) Anti-viral activity  Anti HSV-1 assay in vitro; HSV-1 virus strain; Vero cell; Acyclovir (IC50: 2–5 μg/mL) Cerebrosides Ineffective [55] Anti HSV-1 assay in vitro; HSV-1F strain; Plaque reduction assay-direct, pre, post; 72 h; Vero cell; Acyclovir, Dextran sulfate (1 mg/mL) ( 1 ) 132-hydroxy-(132-R)-phaeophytin b (2) 132-hydroxy-(132-S)-phaeophytin a (3) 132-hydroxy-(132-R)-phaeophytin a Direct: All exhibited 100% inhibitionIC50: 1.96 nM, 3.11 nM, and 3.11 nM, respectively, Post: 30% of inhibition [107] Anti HSV-1 and HSV-2 in vitro; Plaque reduction assay-pre-, post-; 48 h (HSV-1), 96 h (HSV-2); Vero cells; Acyclovir (IC50: 0.64 μg/mL (HSV-1), 0.80 μg/mL (HSV-2)) ( 1 ) monogalactosyl diglyceride (MGDG) (2) digalactosyl diglyceride (DGDG) Pre: Exhibited < 50% protective effectPost: Exhibited 100% protective effect Post-HSV-1: IC50: 36.00 μg/mL (MGDG), 40.00 μg/mL (DGDG), HSV-2: IC50: 41.00 μg/mL (MGDG), 43.20 μg/mL (DGDG) [56] Anti-dengue virus assay in vitro; DV2 strain 16681; Real time-PCR, immunofluorescence assay; direct, pre-, post-; 5 d; Dextran sulfate (pre-), Ribavirin (post-) ( 1 ) 132-hydroxy-(132-S)-chlorophyll b (2) phaeophorbide A (3) 132-hydroxy-(132-S)-phaeophytin b (4) purpurin 18 phytyl ester Compound 2 inhibit dengue viral 2 replication in direct and post- stages, other compounds ineffective in all stages. [108]  (e) Anti-bacterial activity  in vitro; S. aureus, S. typhimurium; (1) Disc diffusion assay, (2) Microdilution assay; 10 mg/mL; Erythromycin (10–50 μg/μL) ( 1 ) Stigmasterol-β-D-glucoside (2) 3-amino-4,5-dihydroxyfuran-2(3H)-one Compound 1: IneffectiveCompound 2 on S. aureus:18.33 mm inhibition value, MIC: 0.3125 mg/mL Compound 2 on S. typhimurium: 20.33 mm inhibition value, MIC: 0.625 mg/mL [49]  (f) Anti-cancer activity  in vitro; SGC-7901 cancer cells; MTT assay; 48 h; 50, 100 and 200 μg/mL Polysaccharide peptide complex: CNP-1-2 92.34 ± 0.94% of inhibition on cell growth at 200μg/mL in 48 h [59] in vitro; A549 cells; MTT assay; 72 h ( 1 ) 132-hydroxy-(132-S)-chlorophyll b (2) phaeophorbide A (3) 132-hydroxy-(132-S)-phaeophytin b (4) purpurin 18 phytyl ester CC50: (1) 43 μg/mL, (2) 25 μg/mL, (3) 50 μg/mL, (4) 50 μg/mL [108]  (g) Cytotoxicity assay  in vitro; CVS assay; 72 h; Vero cells ( 1 ) 132-hydroxy-(132-R)-phaeophytin b (2) 132-hydroxy-(132-S)-phaeophytin a (3) 132-hydroxy-(132-R)-phaeophytin a Maximum concentration that is not toxic to Vero cell is: Compound 1 (5.89μM), 2 (6.21 μM), 3 (6.21 μM) [107] in vitro; MTT assay; 48 h; Vero cells; 100–15000 μg/mL ( 1 ) monogalactosyl diglyceride (MGDG) (2) digalactosyl diglyceride (DGDG) MGDG: CC50: 955.00 ± 7.00 μg/mL DGDG: CC50: 922.00 ± 4.00 μg/mL [56]Regarding the antiviral activity, 132-hydroxy-(132-R)-phaeophytin b from the chloroform extract was found to possess a better anti-HSV-1 effect with an IC50 value of 1.96 nM through direct inactivation [107]. Pongmuangmul et al. [56] showed that monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) isolated from the chloroform extract exhibited promising anti HSV-1 and anti-HSV-2 properties in the poststep of infection, where the IC50 values were in a range from 36.00 μg/mL to 43.20 μg/mL. Sittiso et al. [108] suggested that phaeophorbide A from the chloroform extract was able to inhibit dengue viral 2 replication in the direct inactivation and postincubation stages with a CC50 of 25 μg/mL. Based on this study, it can be observed that, in antiviral studies, compounds isolated from the chloroform extract are more likely to exhibit antiviral activity whereas the ethyl acetate, methanol, and ethanol extracts were more likely to possess anti-HSV-1 and anti-HSV-2 activities, and the ethanol extract was more likely to possess an anti-dengue inhibitory effect. Thus, it might be a valuable approach to investigate the relationship between that antiviral properties of compounds isolated from the aforementioned extracts. Tinh [49] found that 3-amino-4,5-dihydroxyfuran-2(3H)-one isolated from the ethanol extract exhibited moderate antibacterial activity againstS. aureus(MIC equal to 0.3125 mg/mL). This result was inconsistent with the antibacterial activity of the corresponding extract against the same bacterial strain. This might due to the antibacterial properties of the crude extract being hindered by the other constituents present in the extract. In addition, phaeophorbide A from the chloroform extract was found to possess a promisingin vitroanticancer effect on human lung carcinoma cell (A549 cells) at a CC50 equal to 25 μg/mL [108]. This result was consistent with the study from Yong et al. [32] which found that the chloroform extract at 100 μg/mL showed 55.82% inhibition of human lung cancer cell. A novel polysaccharide-peptide complex (CNP-1-2) isolated by Huang et al. [59] from the 70% ethanol extract also exhibited potential anticancer activity on a human gastric cancer cell line (SGC-7901 cells), where, at concentration of 200 μg/mL, it inhibited cell growth by 92.34 ± 0.94% after 48 h of incubation. By contrast, Yong et al. [32] found that the chloroform extract exhibited a weak killing effect on human gastric cancer cells (31.25 ± 1.09% at 100 μg/mL).


## 3.1. Pharmacological Activity ofC. nutans Extracts and Fractions

A number ofC. nutans’ traditional uses have been further verified by laboratory experiments. They have been categorized into antivenom, analgesic, anti-inflammatory, immunomodulating, neuroprotective and neuromodulating function, antidiabetic and α-glucosidase inhibitory, antioxidant, antiviral, antibacterial, antifungal, anticancer, wound healing, plasmid DNA protective, lipid elevated inhibition, and oral mucositis and stomatitis treatment activities. Tables 7(a–o) summarize all the available data regarding the pharmacological activities ofC. nutans extracts and fractions accordingly. All the tables were systematized based on experimental model, followed by type of assay and polarity of extract used. Generally, diverse results can be observed for the same pharmacological activity. The extract preparation method, origin, concentration, and assays type are the primary factors that contributed to the variation in results, and a short summary and point of view have been included in each section.Table 7
Pharmacological activities ofC. nutans.
(a)
Pharmacological activity: antivenom
Experiment design(Experiment model; Venom; Assay; Test subject) Extract Plant part Plant Source Extract dose; Route of administration Result Reference in vitro; Heterometrus laoticusscorpion venom; Pre-incubated extract with 0.2 μg/μL venom-Cell lytic test incubated for 30 min; CEFs Water Leaf Thailand 0.706, 0.406 mg/mL Extract at 0.706 mg/mL give 46.51% of efficiency but the cytotoxic of extract is questionable [65] in vitro; Apis melliferaLinn. bee venom; Pre-incubated extract with 0.6 μg/μL venom-Cell lytic test incubated for 30 min; CEFs Water, 90% and 50% ethanol Leaf Thailand 0.706, 0.406 mg/mL Ineffective [66] in vivo; Laticauda colubrinesnake venom; Mice and Mongrel dog Water; Maceration Leaf SarawakMalaysia 6 mg/kg per mouse (i.p), 20 mg/kg per dog (i.v) Ineffective [7] in vivo; Naja naja siamensis snake venom; Isolated rat phrenic-nerve diaphragm preparations; Mice Water Leaf Thailand p.o, i.p Ineffective [5] in vivo;Snake venom; Mice Water Leaf Thailand - Reduce mortality rate from 100% to 63 ± 3.34% [67] in vivo;Snake venom; Mice 95% alcohol Leaf Thailand 2000 mg/kg; i.v, i.p, p.o Ineffective [67] CEFs: Chick embryo fibroblasts.  (b)
Pharmacological activity: analgesic/antinociceptive
Experiment design(Experiment model; Assay; Test subject) Extract Plant part Plant Source Extract dose; Route of administration; positive control Result Reference in vivo; Acetic acid-induced writhing test; Mice Water, methanol, chloroform,n-butanol Leaf Thailand Phenylbutazone (100 mg/kg) Effective (n-butanol extract at 90 mg/kg) [68, 69] in vivo; Acetic acid-induced abdominal writhing test-Pre-treatment 1 h before test; ICR mice (adult male, 25–30 g) 100% Methanol; Oven dry; Soaking Leaf Kuala Lumpur Malaysia 100, 250, and 500 mg/kg; ASA (100 mg/kg) ED50: 279.3 mg/kg [19] in vivo; Formalin-induced paw licking test-Pre-treatment 1 h before test; Sprague Dawley rat (adult male 150–180 g) 100% Methanol; Oven dry; Soaking Leaf Kuala Lumpur Malaysia 100, 250, and 500 mg/kg; ASA (positive, 100 mg/kg); morphine (5 mg/kg) Early phase: ED50: >500 mg/kg Late phase: ED50: 227.7 mg/kg [19] in vivo; Hot plate test at 50°C- Pre-treatment 1 h before test; ICR mice (adult male; 25–30 g) 100% Methanol; Oven dry; Soaking Leaf Kuala Lumpur Malaysia 100, 250, and 500 mg/kg; morphine (5 mg/kg) 500 mg/kg significant delay response at the interval of 60 to 210 min [19] in vivo; Hot glass jar at 72°C water bath; Albino mice (either sex, 40–50 g) 95% Ethanol; Maceration Leaf Thailand 5 g/kg Ineffective [92] in vivo; Hot water plate test; Mice n-Butanol Leaf Thailand p.o, i.p; morphine Ineffective [68, 69]  (c)
Pharmacological activity: anti-inflammatory
Experiment design(Experiment model; Assay; Test subject) Extract Plant part Plant Source Extract dose; Route of administration; Positive control Result Reference in vitro; fMLP induced elastase release- Pre-treatment; Human neutrophils Methanol; Air dry; Percolation Whole plant Thailand 0.01–100μg/mL; Indomethacin (1–100 μg/mL) IC50: 186.8 ± 20.5 μg/mL elicited a weak, but significant inhibition of human neutrophil elastase release (100 nM) [70] in vitro; fMLP/CB induce elastase release; Human neutrophils 80% ethanol, ethyl acetate,n-hexane; Air dry; Soaking Aerial TaichungTaiwan 10μg/mL 80% ethanol showed highest inhibition: 68.33 ± 5.49% [52] in vitro; fMLP induced neutrophil superoxide anion generation (pre-incubation for 10 min) Methanol; Air dry; Percolation Whole Thailand 0.01–100μg/mL; Indomethacin IC50: 23.4 ± 3.1 μg/mL [70] in vitro; Superoxide anion generation assay; Human neutrophils 80% ethanol, ethyl acetate,n-hexane; Air dry; Soaking Aerial TaichungTaiwan 10μg/mL 80% ethanol showed highest inhibition: 28.52 ± 2.55% [52] in vitro; fMLP induced neutrophil myeloperoxidase (MPO) production Methanol; Air dry; Percolation Whole Thailand 0.01–100μg/mL; Indomethacin IC50: 219.5 ± 25.7 μg/mL Inhibition via reduced neutrophil migration [70] in vitro; Immunoblotting-LPS induced TLR-4 inflammatory proteins; Protein lysate from macrophage Polar; Soaking Leaf, stem Pahang Malaysia 20μg/mL Significantly reduced the LPS induced phosphorylation of p65, p38, ERK1/2, JNK1/2, IRF3 [62] in vitro; LPS induced TLR-4 assay; HEK-Blue-hTLR4 cells Polar, nonpolar; Soaking Leaf, stem Pahang Malaysia 100μg/mL IC50: 21.3 ± 5.0 (leaf polar) to 29.4 ± 9.0 (leaf non-polar) μg/mL [62] in vitro; LPS induced cytokine production assay-1 h pre-treatment; murine macrophages RAW 264.7 cell Polar, nonpolar; Soaking Leaf, stem Pahang Malaysia 100μg/mL Polar leaf (p< 0.05) inhibited TNF-α, IFN-γ, IL-1β, IL-6, IL12p40, IL-17 production [62] in vitro; LPS induced NO- 1 h pre-treatment- Griess assay; Murine macrophages RAW 264.7 cell Polar, nonpolar; Soaking Leaf, stem Pahang Malaysia 100μg/mL IC50: 18.9 ± 3.6 (leaf polar) to 43.1 ± 4.7 (leaf non-polar) μg/mL [62] in vivo; Acetic acid induced vascular permeability model; Mice Water, methanol, chloroform,n-butanol Leaf Thailand Indomethacin (4 mg/kg) Effective,n-butanol at 540 mg/kg as potent as indomethacin [68, 69] in vivo; EPP induced rat ear oedema model- pre-treatment; Sprague Dawley rats (male, 40–60 g) Methanol; Air dry; Percolation Whole plant Thailand 3, 6, 9 mg/20μL acetone per ear; Apply topically; Indomethacin (2 mg/20 μL) At dose 9 mg EPP/ear: 79% oedema inhibition at 15 min, 44.4% MPO reduction after 120 min of induction [70] in vivo; Carrageenan induced paw oedema model-1 h pre-treatment; Sprague Dawley rats (male, 100–120 g) Methanol; Air dry; Percolation Whole plant Thailand 50, 100, 200 mg/kg; p.o; Indomethacin (20 mg/kg) 200 mg/kg of extract inhibit 59% of oedema at 3 h [70] in vivo; Carrageenan induced paw oedema; Albino mice (40–50 g) 95% Ethanol; Maceration Leaf Thailand 5000 mg/kg; p.o 17.73% at 3 h and 36.47% at 6 h of oedema inhibition [92] in vivo; Carrageenan induced paw oedema model; Mice n-Butanol Leaf Thailand p.o; acetylsalicylic acid (100 mg/kg) Effective, n-butanol at 270 mg/kg as potent as ASA [68, 69] in vivo; Granuloma pouch model; Wistar rats (male) Aqueous ethanol cream Leaf Thailand 125 mg cream/rat; Apply topically; Prednisolone (0.25%) Inhibit 48.3% granuloma formation [71] in vivo; Granuloma pouch model; Wistar rats (male) 95% ethanol cream Leaf Thailand 125 mg cream/rat; Apply topically; Prednisolone (0.25%) Inhibit 50.1% granuloma formation [71] in vivo; Granuloma pouch model-1 h pre-treatment; Mice n-Butanol cream Leaf Thailand 270 and 540 mg/kg; Apply topically Ineffective [68, 69] in vivo; Granuloma pouch model; Wistar rats (male) Cold cream Leaf Thailand 125 mg cream/rat; Apply topically; Prednisolone (0.25%) Inhibit 50.98% granuloma formation [71]  (d)
Pharmacological activity: immunomodulating effect
Experiment design(Experiment model; Assay; Incubation period; Test subject; Extract dose; Positive control) Extract Plant part Plant Source Result Reference in vitro; with and without fMLP induced Chemotaxis and Chemokinesis; 45 min; Human neutrophils; 0.1–100 μg/mL; Indomethacin (0.01–100 μg/mL, IC50: 56.3 ± 3.5 ng/ml) Methanol; Air dry; Percolation Whole plant Thailand Chemotaxis: With fMLP induction, IC50: 2.7 ± 0.6 μg/mL, Without fMLP, IC50: >100 μg/mL Chemokinesis: With fMLP induction, IC50: 5.5 ± 0.6 μg/mL, Without fMLP, IC50: 5.0 ± 0.5 μg/mL [70] in vitro; Apoptosis assessment via morphological, flow cytometry-Annexin V binding; 20 h; Human neutrophils; 10–500 μg/mL; Dexamethasone (1 μM, inhibited 54.5% neutrophil apoptosis) Methanol; Air dry; Percolation Whole plant Thailand No significant effect on neutrophil apoptosis [70] in vitro; ConA & LPS-induced IL-10 & TNF-α expression-Real time PCR analysis; 16 h; PBMC; 1.56 mg/mL Methanol; Oven dry; Maceration Leaf, Stem Chiang Mai Thailand Reduce IL-10 mRNA expression, no modulating effect on TNF-α mRNA expression [72] in vitro; IFN-γ expression Splenocyte from ovalbumin-primed BALB/c mice 80% Ethanol; Air dry; Soaking Aerial Taichung Taiwan 0.1μg/mL: upregulation of IFN-γ. 100 μg/mL: down-regulation of IFN-γ [52] in vitro; HaCaT; IFN-γ/TNF-α-induced apoptosis- MTT assay; 1 and 100 μg/mL Ethanol; Maceration Leaf Thailand Significantly(p<0.05) suppressed keratinocytes apoptosis [63] in vitro;Lymphocyte proliferation assay-3H- thymidine-radioactivity; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand 0.5–5μg/mL cause proliferation increase, 2.5, 5 mg/mL cause proliferation decrease [73] in vitro;NK activity-K562 as target cell-cytotoxicity assay; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand 1 and 5 mg/mL-cause NK activity decrease [73] in vitro;IL-2 production-ELISA; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand undetectable IL-2 [73] in vitro;IL-4 production-ELISA; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand 2.5 and 5 mg/mL-increase IL4 production [73] in vitro;Lymphocyte subpopulation-flow cytometry assay; 72 h; HBMC; 0.5–5000 μg/mL Ethanol; Soxhlet Leaf Thailand no change in the percentages of CD3+, CD4+, CD8+, CD16+/CD56+ and CD19+ cells [73]  (e)
Pharmacological activity: neuroprotective and neuromodulating function
Experiment design(Experiment model; Assay; Incubation period; Test subject) Extract Plant part Plant Source Extract dose; Route of administration; Positive control Result Reference in vitro; OGD–reoxygenation and hypoxic neuronal death, Cell viability assay;Mouse primary cortical neurons, cerebral astrocytes, cerebral endothelial cells 80% Ethanol; Soaking Leaf Singapore 5μg/mL Extract suppressed post-hypoxic HDACs activation and reduce OGD-caused neuronal death [75] in vitro; OGD–reoxygenation; 24 h Human SH-SY5Y neuroblastoma cells 80% Ethanol; Soaking Leaf Singapore 100μg/mL Extract modulated cPLA2 expression induction in SH-SY5Y cells by HDAC inhibitors, MS-275, MC-1568, TSA and inhibited HAT activity. [74] in vitro;OGD–reoxygenation; 12 h and 24 h; Mouse primary cortical neurons 80% Ethanol; Soaking Leaf Singapore 1.6, 6.25μg/mL Extract inhibited levels of cPLA2 mRNA expression in primary cortical neurons subjected to 0.5 h OGD injury [74] in vitro;OGD; CCK-8; up to 12 h; neutron pre-treated with extract 1 h before OGD; Mouse primary cortical neurons from E15.5 Balb/c mouse embryos 80% Ethanol; Soaking Leaf Malaysia 6.25μg/mL Extract treated neurons showed significant increment in cell viability [76] in vitro;OGD–reoxygenation; CCK-8, MMP, apoptosis analysis, transient transfection and chromatin reporter assay; 0.5 h OGD+ 4–24 h Reoxygenation; Neurons treated with extract (1 h before, on the onset or after OGD-reoxygenation); Mouse primary cortical neurons from E15.5 Balb/c mouse embryos 80% Ethanol; Soaking Leaf Malaysia 0.075–20μg/mL Neurons treated with extract before, at the onset or after OGD showed increment in dose dependent manner but protective effect of extract was lesser when applied after OGD.Neurons treated with 6.25 μg/mL extract at the onset of OGD has reduced MMP breakdown, apoptotic death, and pro-apoptotic (caspase-3, PARP-1) and has higher anti-apoptotic (14-3-3ε, p-Bad, Bcl-2) markers.Neurons treated with 2.5–10 μg/mL extract at the onset of OGD suggested a dose dependent increase effect in PPAR-γ mRNA level. [76] in vitro; Anti-spasmotic effect on acetycholine induced rat’s bladder tissue contraction; Isolated tissues of rat’s bladder Ethanol Leaf, stem Malaysia Mebeverine hydrochloride and detrusitol Stem at 120 mg/mL: 79.77%Leaf at 100 mg/mL: 26.62% Detrusitol at 200 μg/mL: 89.86%, Mebeverine at 10 mg/mL: 80.74% of contraction inhibition [93] in vivo; Ellman assay-Acetylcholinesterase activity in brain, liver, kidney, heart; Pretreatment for 14 days (daily treatment (once)); Balb/C mice (male, 25 ± 5 g) Methanol, Air dry, Maceration Leaf Seremban Malaysia 250, 500, 1000 mg/kg bw All doses of extracts caused the higher acetylcholinesterase activity in liver, kidney, and heart compared to control group while they did not caused any difference in brain [77] in vivo; MCA occlusion stroke model; Reperfusion infarct volume, behaviour assessment, MRI; extract treated 1 h before, immediate or 3–24 h after 30 min MCA occlusion or 1 d after 15 min MCA acclusion; Long- Evans rats (male, 7-8 week-old) 80% Ethanol; Soaking Leaf Malaysia 10–60 pg (i.c.v., after 30 min MCA occlusion); 24 mg/kg body wt (i.p, 1 h before or 3–24 h after 30 min or 24 h after 15 min MCA occlusion) I.C.V. treatment of 40 pg extract reduced apoptotic neuronal death, showed a maximal decrease of cerebral infarct volume at 1 day reperfusion. PPAR-γ, C/EBPβ mRNA levels, anti- apoptotic 14-3-3ε, p-Bad, and Bcl-2 levels were upregulated. Pro-apoptotic cleaved PARP-1 and cleaved caspase 3 protein levels were downregulated. Extract improved the functional outcomes based on the behavior observation at day 7 after ischemia (elevated body swing test and Bederson’s postural reflex task) and at day 14 after ischemia (better ladder rung walking test). Extract via i.p treatment also significantly reduced infarct volume except 24 h after 30 min MCA occlusion. Behavior test conducted also showed better functional outcomes and smaller infarct volume via MRI. [76]  (f)
Pharmacological activity: antidiabetic andα-glucosidase inhibition activity
Experiment design(Experiment model; Assay; Test subject) Test sample Plant part Plant Source Extract dose; Route of administration; Positive control Result Reference in vitro;α-glucosidase inhibition assay Hot water; Oven dry - Malaysia 50 mg/mL 88.2% of inhibition [37] in vitro;α-glucosidase inhibition assay Methanol; Oven dry; Sonication Leaf, Stem Seremban Malaysia 5000μg/mL (in stock) 13.57 (leaf), 17.67 (stem) % of inhibition [61] in vitro;α-glucosidase inhibition assay 70% Ethanol; Oven, air, freeze dry; Sonication, soaking Leaf, stem Seremban Malaysia 5000μg/mL (in stock); Quercetin Lowest: 5.31% (Leaf-Freeze dry-Soaking), Highest: 41.70% (Leaf-Oven dry-sonication) [4] in vitro;α-glucosidase inhibition assay CO2-Soxhlet, Maceration Stem Seremban Malaysia 5000μg/mL (in stock); Quercetin CO2-Soxhlet: 95.79% of inhibition Maceration: 58.23% of inhibition [28] in vivo; Alloxan induced model- daily treatment for 9 days;Swiss webstermice (male) Hot water; Sun dry Leaf Bandung Indonesia 50, 100, and 150 mg/kg BW; p.o; Oral glibenclamide 150 mg/kg significantly lower blood glucose serum level from 442 ± 149 mg/dl (day 0) to195±66 mg/dl (day 9) [25] in vivo; High fat and high cholesterol diet (HFHC) induced insulin resistance-7 weeks periods-daily treatment of extract; Sprague Dawleys rat (male, 200–250 g) Water, 80% methanol; Sonication Leaf Seremban Malaysia 500, 250 or 125 mg/kg/day/rat; p.o.; Simvastatin C. nutansattenuated the metabolic effects and transcriptional changes induced by the HFHC diet [39] in vivo; Glucose solution (p.o, 2 g/kg bw);Swiss webstermice (male) Insoluble ethyl acetate fraction from ethanol Leaf Bandung Indonesia 100 mg/kg bw; p.o Decreased 18.4% blood glucose serum level in 3 h [25]  (g)
Pharmacological activity: antioxidant
Test sample Plant part Plant Source Extract dose; Positive control Result Reference (1) Type of assay:in vitro; DPPH scavenging assay  Hot water; Oven dry - Malaysia 1 to 16 mg/mL 16 mg/mL: ~60% inhibition [37] Hot water; Unfermented and fermented; Microwave-oven dry, freeze dry; Infusion for 1, 2, 5, 10, 15, and 20 min Leaf Sabah Malaysia 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL Unfermented possessed higher DPPH inhibition than fermented tea [36] Hot water, water, 80% methanol, methanol, ethyl acetate, hexane; Freeze dry; Sonication Leaf, stem Seremban Malaysia Trolox 6.12% (hexane leaf) to 55.12% (80% methanol leaf) inhibition [42] Water, methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL; Trolox Chloroform > methanol > water864.11 ± 73.49 to 7852.63 ± 449.90 (chloroform) μg Teq/g extract [32] Methanol; Sun dry Aerial Vietnam 0, 20, 40, 60, 80, 100μg/mL IC50: 114.50 μg/mL [49] Methanol; Air dry; Percolation Whole Thailand 1–400μg/mL; Trolox IC50: 240.1 ± 15.3 mg/mL [70] Methanol; Air dry; Maceration Leaf Seremban Malaysia 0.25 to 10 mg/mL; Quercetin IC50: 1.33 ± 0.0001 mg/mL [24] Methanol; Oven dry; Soaking Leaf, stem Seremban Malaysia 5000μg/mL (stock); Quercetin Leaf: IC50: 1126.63 μg/mL Stem: IC50: 1548.89 μg/mL [61] Methanol; 1, 6 and 12 months old; Freeze dry Bud, leaf Serdang Malaysia BHT, caffeic acid,α-tocopherol IC50: 64.6 (1 year old buds) to 112.1 (1 year old leaf) μg/mL [41] Methanol, Petroleum ether, Ethyl acetate; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.2 to 10.0 mg/mL 4 mg/mL: leaf petroleum ether give 82% of DPPH inhibition [88] Methanol, ethyl acetate, chloroform, hexane Whole Jelebu Malaysia 1000μg/mL; BHT 50.50 ± 0.03% (Hexane) to 70.96 ± 0.03% (chloroform) inhibition [53] 98% methanol, ethyl acetate, chloroform, hexane; Sun dry; Soaking Stem Vietnam Vitamin C Ineffective [47] 50% ethanol; Soaking Leaf Thailand 1–300μg/mL; Ascorbic acid I C 50: 110.4±6.59 μg/mL [94] 70% ethanol; Freeze dry; Sonication Leaf PerakMalaysia 200–1000μg/mL; Green tea I C 50 not determined [64] 70% ethanol; Freeze dry, oven dry, air dry; Sonication, soaking Leaf, stem Seremban Malaysia 5000μg/mL (stock); Quercetin 15.44 ± 2.21% (Stem-freeze dry-soaking) to 44.31 ± 3.16% (Leaf-oven dry-sonication) inhibition [4] 80% Ethanol; Young, old; Storage duration (1, 2, 3, and 4 days) Leaf, stem Malaysia - Maturity: 31.24% (matured stem) to 112.12% (young leaf) Storage duration: 31.88% (4 days) to 101.85% (1 day) inhibition [38] Ethanol; Maceration Aerial BangkokThailand 1 mg/mL Ineffective [78] Ethyl acetate Leaf Indonesia BHT I C 50: 178.40 mg/L [95] CO2-Soxhlet, Maceration Stem Seremban Malaysia 5000μg/mL (stock); Quercetin CO2-Soxhlet: 98.92% inhibition Maceration: 63.00% inhibition [28]  (2) Type of assay:in vitro; FRAP assay  Hot water; Unfermented and fermented; Microwave-oven dry, freeze dry; Infusion for 1, 2, 5, 10, 15, and 20 min Leaf Sabah Malaysia 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL 250.70 ± 49 (fermented-microwave oven dry-20 min infusion) to 438.80 ± 94 (unfermented-freeze dry-10 min infusion) mg/L [36] Hot water, water, 80% methanol, methanol, ethyl acetate,Hexane; Freeze dry; Sonication Leaf, stem Seremban Malaysia. Gallic acid ~45 (hexane stem) to ~148 mg (hot water leaf) GAE/g extract [42] Methanol, ethyl acetate, chloroform, hexane Whole Jelebu Malaysia 1000μg/mL; Ascorbic acid hexane and ethyl acetate (not determined) to 56.49 ± 0.05% (methanol) [53] Methanol; 1, 6 and 12 months old; Freeze dry Bud, leaf Serdang Malaysia BHT, caffeic acid, Vitamin C 209μM of Fe (II)/g (1-month-old leaves) to 488 μM of Fe (II)/g (6-month-old buds). [41] 70% Ethanol; Freeze dry; Sonication Leaf PerakMalaysia 200–1000μg/mL; Green tea very low absorbance (ineffective) [64] 50% Ethanol; Soaking Leaf Thailand 1–100μg/mL; Ascorbic acid 17 mg ascorbate/g extract [94]  (3) Type of assay:in vitro; Hydrogen peroxide scavenging activity  Water, methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL; Quercetin Methanol: highest radical scavenging: ~34% at 100μg/mL [32]  (4) Type of assay:in vitro; Metal chelating activity  Hot water; Oven dry - Malaysia 1, 2, 5, 10 mg/mL 10 mg/mL: ~90% inhibition [37]  (5) Type of assay:in vitro; Nitric oxide scavenging assay  Hot water; Oven dry - Malaysia 1, 5, 10 mg/mL 10 mg/mL: ~90% inhibition [37] Water; methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL Water: 32.33% at 100μg/mL [32]  (6) Type of assay:in vitro; ABTS cation radical scavenging assay  Hot water; unfermented and fermented; microwave-oven dry, freeze dry; hot boiling water (100°C); infusion for 1, 2, 5, 10, 15, and 20 min Leaf Sabah Malaysia 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL 50.36 ± 4.07 (fermented-microwave oven dry-10 min infusion) to 74.03 ± 2.26 (unfermented-freeze dry-5 min infusion) mg AEAC/L [36] Hot water, water, 80% methanol, methanol, ethyl acetate,hexane; Freeze dry; Sonication Leaf, stem Seremban Malaysia Trolox 18 (hexane leaf) to 65 (80% methanol-leaf) mg TE/g extract [39]  (7) Type of assay:in vitro; Galvinoxyl radical scavenging activity  Water; methanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 12.5, 25, 50 and 100μg/mL; Trolox Chloroform > methanol > aqueousChloroform: 12248.82 μg Teq/g extract [32]  (8) Type of assay:in vitro; Superoxide radical scavenging activity  Methanol; Air dry; Percolation Whole Thailand Direct EPR scavenging effect showed significant direct scavenging activity when the incubation time extended to 60 min [70] 70% ethanol; Freeze dry; Sonication Leaf PerakMalaysia Green tea; by NBT method 3651.5 U/g [64]  (9) Type of assay:in vitro; Phorbol 12-myristate 13-acetate (PMA) induced peroxide production in rat macrophages  50% Ethanol; Soaking Leaf Thailand 10, 30, 100 and 300μg/mL; Tiron Fluorescent intensity: 58.72 ± 5.52 (30μg/mL), 51.92 ± 8.49 (100 μg/mL), 53.50 ± 6.17 (300 μg/mL) [94]  (10) Type of assay:in vitro; Protective effect against peroxyl radicals initiator (AAPH)-induced oxidative hemolysis  50% Ethanol; Soaking Leaf Thailand 200–1000μg/mL; Ascorbic acid IC50: 359.38 ± 14.02 μg/mL [94]  (11) Type of assay:in vivo; Hyperlipidemia-associated oxidative stress model  Water, 80% methanol; Freeze dry; Sonication Leaf, stem Seremban Malaysia 125, 250, 500 mg/kg; Sprague Dawley rats (male, 200–250 g); daily treatment for 49 days; p.o; Simvastatin Both leaf extracts reduce oxidative stress through increasing serum antioxidant enzymes activity and upregulating the expression of hepatic antioxidant genes [42]  (h)
Pharmacological activity: antimicrobial-antiviral
Experiment design(Experiment model; Virus strain; Assay; Test subject; Route of administration/Prescription) Extract Plant part Plant Source Extract dose; Positive control Result Reference Type of virus: Varicella zoster virus (VZV)  in vitro; VZV Kawaguchi strain; (1) DNA hybridization technique, (2) plaque reduction assay-pre, post, direct; WI-38 HEL cells Organic; Soxhlet Leaf Thailand AcyclovirIC50: 107 μM (pre), 5 μM (post), 30 μM (direct) Most effective: Direct inactivation(1) IC50: 1 : 2000 (pre), 1 : 6000 (post), >1 : 18000 (direct) (2) IC50: 1 : 2000 (pre), 1 : 4800 (post), 1 : 9600 (direct) [80] Clinical; Herpes zoster (shingles); Double-blinded, randomized trials; 60 patients; Apply topically 5 times daily for 7 to 14 days 5% extract cream - Thailand Placebo Lesion crusting within 3 days:C. nutans(89.3%), Placebo (0%) Lesion healing within 7 days:C. nutans(100%), Placebo (100%) Pain scores: C. nutansbetter than placebo group [8] Clinical; Herpes zoster (shingles); Randomized trials; 48 patients; Apply topically 5 times daily for 5 days 5% extract cream - Thailand Placebo, Acyclovir Lesion crusting within 3 days:C. nutans(81.3%), Acyclovir (27.2%), Placebo (0%) Lesion healing within 7 days:C. nutans(81.30), Acyclovir (18.20%), Placebo (0%) [82] Clinical; Herpes zoster (shingles); Double-blinded, block randomization; 120 patients; Apply topically 3 times daily from 1 to 26 days 120 patients - Thailand Placebo Lesion crusting within 3 days:C. nutans(27.5%), Placebo (8.6%) Lesion healing:C. nutans(14 days) Placebo (18 days) Symptom reduction: C. nutans better thanplacebo groups [81]  Type of virus: Herpes simplex virus (HSV)  in vitro; HSV-1-KOS strain; Plaque reduction assay-post; Vero cell Methanol, dichloromethane,n-hexane; Soxhlet Leaf Thailand 12.5–100μg/mL; Acyclovir (IC50: 0.09 μg/mL) IC50: 32.05 ± 3.63 (n-hexane) to 64.93 ± 7.00 (methanol) μg/mL [83] in vitro; HSV-1F strain; Plaque reduction assay-pre, post; Vero cell Ethyl acetate; Oven dry; Soxhlet Leaf Thailand 19μg/mL; Acyclovir (5 μg/mL, post), Dextran sulfate (2 μg/mL, pre) Pre: IC50: 7.6 μg/mL significantly reduce plaques Post: ineffective [79] in vitro; HSV-1 strain; Plaque reduction assay-pre, post-incubated for 48 h; Vero cell Chloroform; Soxhlet Leaf Thailand Acyclovir (IC50:  0.64 ± 0.07 μg/mL) Pre: less than 50% inhibition of plaque formationPost: IC50: 115.00 μg/mL [56] in vitro; HSV-2-strain G, 5 clinical HSV-2 isolates; Plaque reduction assay (post), Yield reduction assay (post), Inactivation kinetics (direct, 4 h); Vero cell Methanol; Air dry; Percolation Whole Thailand 2500μg/mL Ineffective [96] in vitro; HSV-2-Baylor 186; Plaque reduction assay-post; Vero cell Methanol, dichloromethane,n-hexane; Soxhlet Leaf Thailand 12.5-100μg/mL; Acyclovir (IC50: 0.43 μg/mL) IC50: 65.13 ± 2.22 (methanol) to 72.62 ± 12.60 (n-hexane) μg/mL [83] in vitro; HSV-2 standard strain; Plaque reduction assay-pre, post, direct; BHK cell Ethanol(A2, C1, C2, C3, C4, C5 fraction) Leaf Thailand - C2, C3, C4: 1 : 2400 dilution cause 100% plaque inhibition, through extracellularly pathway [97] in vitro; HSV-2 strain; Plaque reduction assay-pre, post-incubated for 96 h; Vero cell Chloroform; Soxhlet Leaf Thailand Acyclovir (IC50: 0.80 μg/mL) Post: IC50: 140.00 ± 3.00 μg/mL [56] Clinical; HSV-2-Herpes genitalis; Sequential randomisation; 77 patients; Apply topically 4 times daily for 6 days 5% extract cream - Thailand Placebo, Acyclovir (Zovirax) Lesion crusting within 3 days:C. nutans(98.6%), Acyclovir (80.8%), Placebo (12.5%) Lesion healing within 7 days:C. nutans(88.9%), Acyclovir (84.6%), Placebo (20.8%) C.nutanshas no sticky, burning, stinging pain and side effects. [82, 84] Clinical; HSV-2-Herpes genitalis; Randomized trials; 163 patients; Apply topically 4 times daily for 6 days 5% extract cream - Thailand Placebo, Acyclovir The lesion crusting and healing speed were significantly better in theC. nutansand acyclovir treated groups compared to the placebo groups. No side effect observed C. nutanstreated group. [85]  Type of virus: Fish pathogenic virus  in vitro; IHNV, OMV, IPNV strain; Plaque reduction assay-pre, post, direct; CHSE-214 cells Ethanol; Soxhlet - Thailand 100μg/mL (pre, post), 500 μg/mL (direct) Direct: 100% (IHNV and OMV), 0% (IPNV) of plaque reductionPre: 31% (IHNV), 54% (OMV), 74% (IPNV) of plaque reduction Post: 25% (IHNV), 48% (OMV), 3% (IPNV) of plaque reduction [98]  Type of virus: Crustaceans (shrimp and prawn) infectious virus  in vivo;YRV-RNA virus; Anti-viral test-direct; cultured black tiger shrimp Ethanol; Soxhlet Leaf Thailand 0.1 to 10 mg/mL Minimum inhibition: 1μg/mL [87] in vivo; YRV-RNA virus; Protective efficacy assay-14 days observation; daily twice treatment for 7 days-pre; cultured black tiger shrimp; p.o Ethanol; Soxhlet Leaf Thailand 0, 0.1, 1 and 10 g/kg of pellet 1 g/kg of feed exhibited best protective efficacy with 57.6% [87]  Type of virus: Mosquito-borne virus  in vitro; DENV-2 strain 16681; Western blot assay, ECL detection kit-post; Naïve Huh-7 cells 80% Ethanol; Air dry; Soaking Aerial Taichung Taiwan Ribavirin IC50: 31.04 μg/mL [52]  Type of virus: Poultry and bird contagious virus  in vitro;NDV- La Sota strain; Hemagglutination test-pre, post; CEFs Water, Ethanol; Soxhlet - Thailand 31.25 g/mL (final) Ineffective [86]  (i)
Pharmacological activity: Anti-microbial-anti-bacterial
Experiment design(Experiment model; Bacteria strain; Assay) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro;A. hydrophila; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99] in vitro; B. cereus; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf PerakMalaysia 25, 50 and 100 mg/mL;Ciprofloxacin At 100 mg/mL:15.00±1.00 mm [22] in vitro;B. cereus; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: >12.5 mg/mL [6] in vitro; B. cereus;Microdilution assay Ethyl acetate and its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Ampicillin MIC: Ethyl acetate: 6.31 mg/mL,F7: 1.39 mg/mL [88] in vitro; B. subtilis; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; E. coli; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; E. coli; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf Perak 25, 50 and 100 mg/ml;Ciprofloxacin At 100 mg/ml:17.00±2.00 mm [22] in vitro;E. coli; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: 12.5 mg/mL [6] in vitro; E. coli;Microdilution assay Ethyl acetate and its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Ampicillin MIC: Ethyl acetate: >100 mg/mL,F7: 1.39 mg/mL [88] in vitro; E. coliDC10; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; E. coli(wild); Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; M. luteus; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; MRSA;Disc diffusion assay, microdilution assay Ethanol; Oven dry; Maceration - Thailand 5 mg/mL No detected inhibition zone, MIC and MBC: >5 mg/mL [100] in vitro; MSSA K147; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; N. gonorrhoeae & 11 clinical isolates; Disc diffusion Methanol; Maceration Leaf Thailand 44 mg/mL (final); Norfloxacin Ineffective [101] in vitro; P. acnes; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: >12.5 mg/mL [6] in vitro;P. acnes; Disc diffusion, microdilution assay Ethanol; Maceration Aerial Thailand Mangostin MIC and MBC: >5 mg/mL [102] in vitro; P. aeruginosa; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; P. aeruginosa; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf Perak 25, 50 and 100 mg/ml;Ciprofloxacin At 100 mg/ml:13.00±1.00 mm [22] in vitro; P. aeruginosa187 (wild); Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Gentamycin Ineffective [89] in vitro; S. aureus; Microdilution assay Hot water; Oven dry - Malaysia 0.4 to 50.0 mg/mL (final); Ampicillin MIC: >50 mg/mL [43] in vitro; S. aureus; Disc diffusion assay 70% Methanol; Air dry; Soxhlet Leaf Perak 25, 50 and 100 mg/ml;Ciprofloxacin At 100 mg/mL:26.67±3.51 mm [22] in vitro; S. aureus; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: 12.5 mg/mL [6] in vitro; S. aureus;Disc diffusion assay, microdilution assay Methanol; Sun dry; Maceration Aerial Vietnam 125–1000 mg/mL; Erythromycin (10–50μg/μL) 16.67 mm inhibition zone, MIC: 62.5 mg/mL [49] in vitro; S. aureus;Disc diffusion assay, microdilution assay Ethanol; Oven dry; Maceration - Thailand 5 mg/mL No detected inhibition zone, MIC: 5 mg/mL, MBC: >5 mg/mL [100] in vitro; S. entericaserovar Paratyphi C; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Showed inhibition zone [26] in vitro; S. entericaserovar Paratyphi B; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Chloroform: a larger inhibition zone compare to other solvents. [26] in vitro; S. entericaserovar Typhi; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Least inhibition zone [26] in vitro; S. entericaserovar Typhimurium; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Showed inhibition zone [26] in vitro; S. enterica Typhimurium;Disc diffusion, microdilution assay Methanol; Sun dry; Maceration Aerial Vietnam 125–1000 mg/mL; Erythromycin (10–50μg/μL) 15.67 mm inhibition zone, MIC: 125 mg/mL [49] in vitro; S. enterica Typhimurium;Microdilution assay Ethyl acetate and its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Ampicillin MIC: Ethyl acetate: >100 mg/mL,F7: 1.39 mg/mL [88] in vitro; S. entericaserovar Weltevreden; Disc diffusion assay Distilled water, 70% ethanol, absolute ethanol, chloroform Leaf Malaysia - Showed inhibition zone [26] in vitro;S. epidermidis; Microdilution assay Methanol; Maceration Leaf Seremban Malaysia 0.1 to 12.5 mg/mL; Erythromycin, chloramphenicol MIC: >12.5 mg/mL [6] in vitro;S. epidermidis; Disc diffusion, microdilution assays Ethanol; Maceration Aerial Thailand Mangostin MIC and MBC: >5 mg/mL [102] in vitro; S. epidermidis;Disc diffusion, microdilution assays Ethanol; Oven dry; Maceration - Thailand 5 mg/mL No detected inhibition zone, MIC and MBC: 5 mg/mL [100] in vitro;Streptococcussp.; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99] in vitro; V. harveyi; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99] in vitro; V. paraliaemolyticlls; Agar dilution assay Ethanol; Soxhlet - Thailand 0 to 10.0 mg/mL MIC: >10 mg/mL [99]  (j)
Pharmacological activity: antimicrobial-antifungal
Experiment design(Experiment model; Fungal strain; Assay) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro; A. fumigatus; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Nystatin Ineffective [89] in vitro; C. albicans; Light mediated disk diffusion assay 95% Ethanol; Soaking Leaf Thailand 5 mg/mL; Nystatin Ineffective [89] in vitro; C. albicans; Microdilution assay Ethyl acetate & its fraction; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.08 to 5 mg/mL (final); Amphotericin B Ethyl acetate: 6.31 mg/mL,F7: MIC: 1.39 mg/mL [88]  (k)
Pharmacological activity: anticancer
Experiment design(Experiment model; Cancer cell(s); Incubation period; Assay; Test subject; Route of administration) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro; Mutated Salmonella typhimurium (T98 and T100) without metabolic activation; 6 days; Mutagenicity assay-Ames test Water; Air dry; Maceration Leaf Penang Malaysia 500μg/well Non-mutagenic activity inS. typhimuriumhistidine auxotrophs [103] in vitro; D24, MM418C1, MCF7, BT474 cancer cells; 24 h and 72 h; CCK-8 assay Hot water, cold water, methanol, ethanol, dichloromethane Leaf Seremban Malaysia 2 mg/mL D24 cell: cold water at 72 h caused 42.9% cell death (EC50: 1.63 mg/mL) MM418C1 and MCF7 cells: hot and cold water showed cytotoxicity BT474 cells: extract ineffective [18] in vitro; K562, HCT 116 cancer cells; MTT cytotoxicity assay Water, 50% methanol, 100% methanol, 50% ethanol, 100% ethanol Leaf Malaysia 100 and 200μg/mL Ineffective [104] in vitro; HepG2, IMR-32, NCI-H23, SNU-1, LS-174T, K-562, HeLa, Raji cancer cells; 72 h; MTT cytotoxicity assay Water, ethanol, chloroform; Oven dry; Soaking Leaf Serdang Malaysia 3.125 to 100μg/mL Most effective: chloroform with IC50: 47.70 μg/mL (K562), 47.31 μg/mL (Raji) [32] in vitro; Cultured Saos-2 human steosarcoma cells; HIF activity, MTT cytotoxicity assay Methanol Leaf Malaysia 125–2000μg/mL Ineffective [105] in vitro; HeLa cancer cells; 72 h; MTT cytotoxicity assay Methanol; Freeze dry Leaf, buds Serdang Malaysia 10, 20, 40, 80, 160, 320μg/mL; Tamoxifen Most effective: 6-month bud with IC50: 56.8 μg/mL [41] in vitro; D24 melanoma cells; 24 h and 72 h, CCK-8 assay Methanol; Air dry; Soaking with shaker Leaf Malaysia, Vietnam, Thailand 0–2 mg/mL (stock) Chiang Dao Thailand extract has highest cytotoxicity: EC50: 0.95 mg/mL (24 h), 0.77 mg/mL (72 h) [106] in vitro; HepG2, MCF-7, NCI-H460, HeLa cancer cells; 48 h; Sulforhodamine B colorimetric assay Sub-fraction F-III from Methanol extract Stem Vietnam Camptothecin Effective, IC50: 36.80 (HepG2), 57.36 (NCI-H460), 66.57 (MCF-7), 91.08 (Hela) μg/mL [47] in vitro;HepG2 cell; 24 h; MTT cytotoxicity assay Methanol, ethyl acetate, hexane, chloroform fraction - Malaysia 0, 6.25, 12.5, 25, 50, 100μg/mL (stock) IC50: 43.93 (methanol), 55.61 (chloroform), 62.06 (ethyl acetate), 68.38 (hexane) μg/mL [35] in vitro;MCF-7, HeLa cells, 72 h; MTT cytotoxicity assay Methanol, ethyl acetate; Freeze dry; Soaking Root Sabah Malaysia 10–50μg/mL; Camptothecin (0.35 μg/mL) MCF-7 cell: IC50: 35.0 μg/mL (methanol), 30.0 (ethyl acetate) μg/mL HeLa cell: ~25% of inhibition for both extracts [31] in vitro; HeLa, K-562 cells; 24 h and 72 h; MTT cytotoxicity assay Methanol, ethyl acetate, petroleum ether; Oven dry; Soaking Leaf, stem Taiping Malaysia 0.2–10.0 mg/mL Petroleum ether leaf extract showed the strongest cytotoxic activity after 72 h, CC50: 18.0 μg/mL (HeLa), 20.0 μg/mL (K-562) [88] in vivo; MMS induced Allium cepa chromosome assay-post and suppressive treatment;Allium cepa Water, methanol; oven dry Leaf PenangMalaysia 100, 200, 400, 800 mg/kg Extract has repairing and anti-mutagenic effect [90] in vivo; Hepatocarcinoma tumor-bearing mice-daily treatment for 10 days; ICR mice (with normal T/B cells, 6–8 weeks, 18–22 g); Gastric probe 30% Ethanol fraction Aerial SerembanMalaysia 3 and 10 mg/kg; Fluorouracil (20 mg/kg) 8.2% (3 mg/kg) and 58.6% (10 mg/kg) of tumor size and weight reduction, higher reduction rate than positive control (37.1%) [33]  (l)
Pharmacological activity: wound healing ability
Experiment design(Experiment model; Assay; Test subject) Extract Plant part Plant Source Extract dose Result Reference in vitro; Migration rate of HGF at 0 h, 2 h, 4 h, and 6 h after scratching; Human gingival fibroblast (HGF) Chloroform, Hexane; Sun dry Leaf Indonesia 10, 25, 50 and 100μg/mL Culture supplemented with 10μg/mL of chloroform extract give the fastest HGF migration rate and wound recovery [11]  (m)
Pharmacological activity: protective effect on plasmid DNA
Experiment design(Experiment model; Assay; Test Subject) Extract Plant part Plant Source Extract dose Result Reference in vitro; DNA integrity assay; Supercoiled plasmid DNA react with O2•-generated via a riboflavin photoreaction treatment 70% Ethanol; Freeze dry; Sonication Leaf PerakMalaysia 10000, 1000, 100, 10 and 0μg/mL; Green tea Extract reduced DNA cleavages, retained high levels of super-coiled plasmid DNA integrity and exhibited protection up to 50 min [64]  (n)
Pharmacological activity: lipid elevated inhibition activity
Experiment design(Experiment model; Assay) Extract Plant part Plant Source Extract dose; Positive control Result Reference in vitro; Porcine pancreatic lipase inhibition assay Methanol; Freeze dry; Sonication Leaf Malaysia 100μg/mL; Orlistat Ineffective, −22.56% indicates a promotion of pancreatic lipase activity [21]  (o)
Pharmacological activity: oral mucositis and stomatitis treatment
Experiment design(Experiment model; Infection; Assay; Test subject; Route of administration/Prescription) Extract Plant Source Positive control Result Reference Clinical; Radiation induced oral mucositis; Single-blinded-Randomized trial; 60 patients; Apply 2 drops drip into mouth or on the lesion 3 to 5 times daily from first to last day of radiation Glycerin payayor Thailand Benzydamine hydrochloride(Difflam, 3M, Australia) The time onset of oral mucositis in the payayor group was significantly later, and its severity and pain score were less than those of the benzydamine group throughout the study period. [10] Clinical; Reccurent aphthous stomatitis; Double-blinded-controlled trial; 43 patients; apply to the ulcer 4 times daily C. nutans in orabase Thailand Triamcinolone acetonide, placebo C. nutans in orabase provide better healing of the ulcer as compared to placebo but efficacy was lesser compared to triamcinolone acetonide in orabase [91]


### 3.1.1. Antivenom Activity

Table7(a) summarizes the antivenom tests that have been carried out thus far onC. nutans. The experiments examined the antivenom properties ofC. nutans’ leaves against snake, scorpion, and bee venoms. There were a total of 2in vitro and 4in vivo antivenom studies, and the extracts tested were prepared from water, ethanol, and aqueous ethanol. From the results of thein vitro studies, the water extract at 0.706 mg/mL exhibited a moderate anti-scorpion venom effect through a direct inactivation mechanism [65] while the aqueous ethanol extracts were ineffective against bee venom [66]. On the other hand, in 3 out of the 4in vivo studies conducted, the extracts (2 water and 1 aqueous ethanol extracts) notably did not exhibit antivenom effects after the test subjects were given different doses (6 to 2000 mg/kg bw) of the extracts through intraperitoneal injection (i.p.), intervenous injection (i.v.), or per os (p.o.) [5, 7, 67]. Although there was 1in vivo study proposing that the water extract ofC. nutans could exhibit a moderate anti-snake venom effect, the study did not include the administration method or the doses given [67]. Thus, defining a suitable extract that is biologically active as an antidote for snake and insect venoms is ambiguous. In summary, among the extracts tested, only water leaf extract exhibited potentialin vitroinhibition effect towards snake and insect venoms via direct inactivation mechanism. Further animal studies that focus on studying the metabolites of water extract and their reaction with the mediators that responsible for the direct inactivation pathway are worth emphasizing. Clinical study that investigates the efficacy of water extract as an antivenom agent through application of topical formulation on the patients might lead to a possible breakthrough.


### 3.1.2. Analgesic Activity

Table7(b) displays the scientific studies that investigated the analgesic ability ofC. nutans through 3 different types ofin vivo assays: the acetic acid-induced writhing test, formalin-induced paw licking test, and hot plate test. Three studies have used the acetic acid-induced writhing test to examine the analgesic effect ofC. nutans extracts prepared using 4 different types of solvents [19, 68, 69]. The results suggested that treating mice with then-butanol extract at 90 mg/kg as well as with the methanol extract at 279.3 mg/kg was as potent in terms of analgesic properties as treating them with phenylbutazone at 100 mg/kg. On the other hand, the study by Abdul Rahim et al. [19] was the only one that used formalin-induced paw licking test to evaluate the analgesic properties ofC. nutans. The results suggested that the methanol extract, with a half maximal effective concentration (EC50) at 227.7 mg/kg, was able to relieve pain in the late phase (centrally inflammatory induced pain pathway) via opioid/nitric oxide- (NO-) mediated, but cyclic guanosine monophosphate- (cGMP-) independent modulation systems. In addition, the analgesic effects of the absolute methanol, ethanol, andn-butanol extracts have been examined through the hot plate test. The results suggested that 500 mg/kg methanol extract was effective in alleviating the pain response at the interval from 60 to 210 min while both the ethanol and butanol extracts showed no analgesic effects up to 5 g/kg. Overall, methanol extract ofC. nutans exerted potential analgesic activity in both acute and persistent pain tests. The study also suggested that the presence of phenolics particularly gallic acid, caffeic acid, ferulic acid, vitexin, and apigenin which previously reported to exert antinociceptive activity which might be the main activity contributors. Since methanol extract might be a potential analgesic agent, it is important to further investigate the efficacy dosages, sites of action, and the level of the extract to modulate the pain along the pathway suggested. Furthermore, study may also provide information regarding antinociception properties ofC. nutans methanol extract on visceral and neuropathic pains.


### 3.1.3. Anti-Inflammatory Activity

Table7(c) summarizes the anti-inflammatory effect ofC. nutans. The anti-inflammatory effect ofC. nutans was assessed based onin vitro assays, such as the macrophage activatorN-formyl-methionyl-leucyl-phenylalanine- (fMLP-) induced neutrophil elastase release superoxide anion generation, lipopolysaccharides (LPS) induced toll like receptor 4 (TLR-4), NO Griess, and cytokine production assays. Furthermore, the types ofin vivo experiments used to evaluate the acute anti-inflammatory properties ofC. nutans were the acetic acid-induced vascular permeability model and ethyl phenylpropiolate- (EPP-) induced rat ear oedema and carrageenan-induced paw oedema model, whereas its subchronic anti-inflammatory effect was assessed using the granuloma pouch model, which mimics subchronic inflammation in humans. For thein vitro anti-inflammatory effects, both the methanol and 80% ethanol extracts have been evaluated through the neutrophil elastase release and superoxide anion generation assays [52, 70]. The results showed that 10 μg/mL of 80% ethanol extract inhibited 68.33% elastase release, which was more effective than the methanol extract (<20%), while to inhibit superoxide radical formation, both extracts at 10 μg/mL showed comparable efficacy, as they inhibited approximately 30% superoxide formation [52, 70]. For the LPS-induced TLR-4, NO, and cytokine production assays, the results suggested that the polar (methanol and dichloromethane) extract possessed a better IC50 value (<22 μg/mL) for inhibiting TLR-4, NO and proinflammatory cytokine production than the nonpolar (hexane and diethyl ether) extract [62]. For thein vivo acute anti-inflammatory properties ofC. nutans, the most effective dose was pretreatment of 9 mg EPP/ear of methanol extract on the rat which resulted in 79% oedema inhibition at 15 min and 44.4% myeloperoxidase (MPO) reduction after 120 min of induction [70].Additionally, in a comparison of the methanol, ethanol, andn-butanol extracts, a 1 h pretreatment of 200 mg/kg methanol extract administered via p.o. to carrageenan-induced rats was found to inhibit 59% oedema formation [70]. The acetic acid-induced vascular permeability model showed that, among the water, methanol, chloroform, andn-butanol extracts, the butanol extract at 540 mg/kg was superior at exhibiting an anti-inflammatory effect [68, 69]. Ethanol, aqueous ethanol,n-butanol, and cold creams have been evaluated for their subchronic anti-inflammation properties. The results showed that application of 125 mg ofC. nutans cold cream to a rat topically throughout the experimental period inhibited granuloma formation by as much as 50.98%, which is comparable to the inhibitory effect of 0.25% prednisolone (56.82%) [71]. According to the previousC. nutans anti-inflammatory studies, the extract prepared from polar solvent notably exhibited a promising anti-inflammatory propertiesin vitro(at a dose less than 30 μg/mL) andin vivo (at a dose less than 300 mg/kg). Therefore, it is greatly desired to focus on the relationship between the mode of action ofC. nutanspolar extract in both biological and nonbiological anti-inflammatory systems. In addition, since polar extract exhibited potential anti-inflammatory activity, knowledge of extract prepared from the inorganic polar solvent particularly water is still scarce as regards itsin vivoandin vitro anti-inflammatory activity properties, thus possessing potential research interest.


### 3.1.4. Immunomodulating Activity

Table7(d) shows the immunomodulating effect ofC. nutans. The methanol extract ofC. nutans has been tested for its immune modulating effect on apoptosis and cytokines expression in experiments on human neutrophils and porcine peripheral blood mononuclear cells (PPBMCs), such as assays for fMLP macrophage activator-induced chemotaxis and chemokinesis, apoptosis, and concanavalin (ConA) and LPS-induced interleukin 10 (IL-10), and tumor necrosis factor-alpha (TNF-α) expression [70, 72]. Wanikiat et al. [70] suggested thatC. nutans methanol extract exhibited dose-dependent suppression of fMLP-induced chemotaxis and chemokinesis of neutrophils without causing the cells to undergo apoptosis.C. nutans was found to cause a reduction in IL-10 expression and to have no effect on TNF-α expression in PBMC [72]. On the other hand, the ethanolic extract ofC. nutans has been tested for its immunomodulating effect on cytokines expression in splenocytes, a human keratinocyte cell line (HaCaT), and human peripheral blood mononuclear cells (HPBMC). Tu et al. [52] suggested that low concentrations of the ethanol extract cause IFN-γ upregulation while higher concentration ofC. nutans cause IFN-γ downregulation. Sriwanthana et al. [73] studied found that low concentrations ofC. nutans resulted in increase in lymphocyte proliferation, while higher concentrations ofC. nutans resulted in decrease of lymphocyte production. The study also found thatC. nutans did not stimulate an interleukin-2 (IL-2) response or affect the lymphocyte subpopulation, such as total T lymphocytes (CD3), T helper/inducer cells (CD4), T suppressor/cytotoxic cells (CD8), natural killer (NK) cells (CD16/CD56), or B lymphocytes (CD19). On the other hand, 2.5 and 5 mg/mL of ethanol extract increased the interleukin-4 (IL-4) production and 1 and 5 mg/mL of extract suppressed NK activity [73]. Furthermore, 1 and 100 μg/mLC. nutans were found to inhibit IFN-γ and TNF-α-induced keratinocytes apoptosis [63]. A similar trend as in the analgesic and anti-inflammatory properties ofC. nutans could be observed, where extract prepared from polar solvents exerted better analgesic, anti-inflammatory, and immunomodulating effects. Phenolics, sulfur-containing glucosides, and sulfur-containing compounds might be the main contributors to these activities, as all the mentioned compounds were primarily isolated from polarC. nutans extracts. In addition, the described therapeutic effects are always well correlated with the immunology results. Thus, investigations on the biological mechanism of the active constituents present in the polar extract and their effects on immunological cells, mediators, and substances deserve special attention. Exploring the immunological function of other polar extracts, such as water, and nonpolar extracts should be encouraged as well.


### 3.1.5. Neuromodulating Activity

The efficacy ofC. nutans in neuro-related protective and modulating functions has also been studied in recent years, as shown in Table 7(e).In vitro studies suggested that the neuroprotective effect ofC. nutans was promising. From the studies by Tan et al. [74] and Tsai et al. [75], the 80% ethanol extract with a dose concentration less than 10 μg/mL was able to suppress post-hypoxic histone deacetylase (HDAC) activation and hypoxic neuronal death in an oxygen-glucose deprivation- (OGD-) reoxygenation assay. It also decreased the levels of cytosolic phospholipase 2 (cPLA2) mRNA expression in mouse primary cortical neurons subjected to 0.5 h of OGD injury. In addition, Tan et al. [74] found that 100 μg/mL 80% ethanol extract was able to suppress histone acetylase (HAT) activity and regulate cPLA2 expression induction in a human neuroblastoma cell line (SH-SY5Y cells) through HDAC inhibitors such as entinostat (MS-275), MC-1568, and trichostatin A (TSA). The research team of Wu et al. [76] further suggested that treating the primary neurons with 0.15–20 μg/mL of 80% ethanol extract 1 h before, at the onset, or after the OGD induced cell death or OGD-reoxygenation treatment was able to mitigate the neuronal apoptosis and protected primary neurons by activating the antiapoptotic activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ) → 14-3-3ε (antiapoptotic marker), enhancing the C/EBPβ binding to PPAR-γ promoter and amplifying its transcription though the extract was less effective when applied after the OGD treatment. The result also suggested that the protective effect of extract was revoked when cotreated with GW9662 (PPAR-γ antagonist). Thisin vitro screening further complemented with the protective effect of extract in attenuating the ischemic brain damage inin vivo middle cerebral artery occlusion (MCA) stroke model [76]. Both intracerebroventricular (i.c.v.) infusion (10–60 pg extract) and i.p. injection (24 mg/kg body wt extract) of extract demonstrated that the extract possessed the ability in mitigating apoptotic neuronal death, cerebral infarct volume, and behavioral deficiency in the rat MCA occlusion model. The detailed neuroprotective mechanism ofC. nutans was similar toin vitro OGD study and was elaborated in Table 7(e). In addition, anin vivostudy conducted by Lau et al. [77] revealed that the three different tested doses of methanol extract were able to stimulate acetylcholinesterase activity in the heart, liver, and kidney, but not the brain of mice without inducing any signs of toxicity in the mice. As a short summary, similar results have been reported as those regarding the analgesic, anti-inflammatory, and immunomodulating activities, where the polar extract ofC. nutans possessed a great neuroprotective effect. Nevertheless, research on the neuroprotective strength ofC. nutans is still new and therefore holds enormous potential for different research directions. Further investigation of the potential efficacy of polarC. nutans extract inin vivo neurology-inflammation related studies might lead to a breakthrough. Research should fill in gaps about the neuro-related function of nonpolar extract as well.


### 3.1.6. Antidiabetic andα-Glucosidase Inhibitory Activity

Table7(f) shows a list of studies conducted on the antidiabetic and α-glucosidase inhibition effects ofC. nutans. There have been a total of 4in vitro studies assessing the hyperglycemia inhibitory activity ofC. nutansthrough an α-glucosidase inhibition assay. All the results showed thatC. nutans had a very low inhibitory effect on the α-glucosidase enzyme when the employed extracts were prepared using conventional preparation methods [4, 28, 37, 61]. Although IC50 was not calculated by Alam et al. [28], a lower IC50 value is anticipated as the extract prepared through advanced supercritical extraction exhibited 95.79% of α-glucoside inhibition when the stock was prepared at 5000 μg/mL. DespiteC. nutans not showing particularly pronounced inhibition against α-glucosidase, 3in vivo antidiabetic experiments suggested that the water extract, 80% methanol extract, and the insoluble ethyl acetate fraction from the 80% ethanol extract were able to attenuate the insulin resistance induced by a high fat and high cholesterol diet (HFHC) and serum glucose level [25, 39]. In general, the high α-glucosidase inhibition by SFE treated extracts might indicate that the compounds active against the α-glucosidase enzyme are heat-sensitive or easily degradable. Thus, extra precaution is needed during the extract preparation step. On the other hand, conventional method prepared extracts that mimicked the traditional application showed low inhibition towardsin vitroα-glucosidase tests while traditional uses suggested thatC. nutans is an antidote for diabetes. The contradictory results might be attributed by improper mode of action of diabetes being investigated. Since α-glucosidase inhibition test focuses on mechanism of postprandial hyperglycemia, further study on other mechanism approach, experiment on the reaction of the extract towards β-cell in pancreas, test that involved other enzymes in hyperglycemia such as pancreatic α-amylase, glucose diffusion assay, glucose uptake by yeast cells, and nonenzymatic glycosylation assay are highly recommended.


### 3.1.7. Antioxidant Activity

Considerable number of analyses have been done on the antioxidant effect ofC. nutans as shown in Table 7(g). Based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay, some studies showed thatC. nutans up to 1 mg/mL is a potential antioxidant agent, while other studies found that the DPPH inhibitory properties ofC. nutans could not be determined [47, 64, 78]. The data in the table showed that, among the polar and nonpolar extracts tested, the highest antioxidant properties ofC. nutanswas detected in the study by Ghasemzadeh et al. [41], whereC. nutans bud extract that was cultivated for 1 year at a farm located in Malaysia and that underwent freeze drying and methanol extraction resulted in IC50 value of 64.6 μg/mL. The second most common antioxidant determination assay selected by researchers was the ferric reducing antioxidant power (FRAP) assay. However, discussion of the FRAP results is difficult, as there is a lack of unit standardization in the measurement. The results from othersin vitro antioxidant assays, such as those for hydrogen peroxide scavenging, metal chelating, nitric oxide scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical scavenging, galvinoxyl radical scavenging activity, superoxide radical scavenging activity, and phorbol 12-myristate 13-acetate (PMA) induced peroxide production in rat macrophages and protective effect against peroxyl radicals initiator- (AAPH-) induced oxidative hemolysis, have been summarized in Table 7(g(3)–g(10)). Overall, polar and semipolar solvent-extractedC. nutans were more likely to exhibit moderate antioxidant activity at concentration varying from 0.0125 to 10 mg/mL than nonpolar extracts. In addition, there was a recent study that evaluated the antioxidant activity ofC. nutans in vivo by using a hyperlipidemia-associated oxidative stress model [42]. The study suggested that both the water and 80% methanol leaf extracts at up to 500 mg/kg bw rats were able to mitigate oxidative stress by improving the activity of serum antioxidant enzymes and expression of hepatic antioxidant genes [42]. In summary, polar and semipolar extracts are more likely to exhibit antioxidant properties. A possible hypothesis is that phenolics, sulfur-containing glucosides, sulfur-containing compounds, chlorophyll derivatives, and some phytosterol derivatives that are isolated fromC. nutans using polar and semipolar solvents might be the contributors. Thus, studies on the antioxidant properties of these isolated compounds and further evaluation of the plant’s antioxidant effect inin vivo studies are required.


### 3.1.8. Antiviral Activity

The antimicrobial properties ofC. nutans have been further classified into antiviral, antibacterial, and antifungal effects, as shown in Tables 7(h), 7(i), and 7(j), respectively. For the antiviral activity ofC. nutans, the types of virus that have been studied were varicella zoster virus (VZV), herpes simplex virus (HSV), fish pathogenic viruses, crustaceans (shrimp and prawn) infectious viruses, mosquito-borne viruses, and poultry and bird contagious viruses. Notably, the antivirus studies were always accompanied with a prescreening test for cytotoxicity to determine the subtoxic concentration of the test sample and to ensure that the test sample has a killing effect on the virus instead of on the host cell [79]. Researchers have also investigated the mode of action ofC. nutans in terms of its antiviral potential through 3 different stages of treatment. Pretreatment (pre-) studies are those where the test sample is allowed to incubate with the cell for a time of period before it is infected with the virus. This approach reflects the virucidal activity of the test sample, as the test sample interferes with the viral structure to prevent or inhibit the viral penetrating or adsorbing to the host cell [79]. In posttreatment (post-) studies, instead of being preincubated with the test sample, the virus is attached to the host cell that has first only been cultured with the test sample. The test sample is thus considered to have antiviral activity if it successfully inhibits viral DNA replication [79]. By contrast, for the direct inactivation (direct) pathway, the virus is preincubated with the test samples first before being added to the host cell. The test sample is said to have an inactivation ability against the virus if it interferes with or causes damage to the viral glycoproteins, the virus envelope, or the virus structure before they enter the host cell [79].Thus far, only onein vitro study and a total of 3 clinical trials have been conducted to evaluateC. nutans’ anti-VZV activity. Thein vitro result suggested that the organic extract exhibited anti-VZV activity through the direct inactivation stage [80]. In the clinical studies, theC. nutans extracts were formulated into a 5%C. nutans cream prior to testing its ability to combat VZV infections. The result suggested that theC. nutans containing cream was able to exhibit a positive curing effect. All the studies showed that the percentage of patients who experienced lesion crusting within 3 days and lesion healing within 7 days after applying theC. nutans cream topically was better than the percentage in the placebo and acyclovir groups. In addition, the pain score and side effect resulting fromC. nutans cream application were lower [8, 81, 82].For the anti-HSV-1 activity ofC. nutans, the ethyl acetate, methanol, dichloromethane, chloroform, andn-hexane extracts have been evaluated. Thongchai et al. [79] suggested that theC. nutans ethyl acetate leaf extract displayed the best inhibition on HSV strain with IC50 value of 7.6 μg/mL through the pretreatment mechanism. Meanwhile, the methanol, ethanol dichloromethane, chloroform, and hexane extracts were also tested for their anti-HSV-2 activity. TheC. nutans methanol and hexane leaf extracts were found to have better and comparable inhibition against HSV-2-Baylor 186 strain, via posttreatment action, with IC50 value of 65.13 and 72.62 μg/mL, respectively [83]. The anti-HSV-2 activity ofC. nutans has also been evaluated in clinical studies using the 5% extract cream. The results obtained were similar to those from the clinical studies on the anti-VZV activity; when compared to a placebo, the days required for lesion crusting and healing after application ofC. nutans were higher than or the same in terms of efficacy as acyclovir [82, 84, 85]. Regarding its efficacy in inhibiting fish pathogenic viruses, among the virus strains tested, theC. nutansethanolic extract exerted 100% inhibition on plaque formation in infectious hematopoietic necrosis virus (IHNV) andOncorhynchus masouvirus (OMV) strains but not in infectious pancreatic necrosis virus (IPNV) through the direct inactivation stage.On the other hand, it was also found thatC. nutans ethanol extract had an excellent protective effect on cultured black tiger shrimp against yellow head rhabdo-like virus (YRV-RNA) through a direct inactivation mechanism and pronounced virucidal activity against dengue virus (IC50 value of 31.04 μg/mL); however, it was less effective against Newcastle disease virus (NDV) caused by poultry and birds [52, 86, 87]. In summary,C. nutans ethanol extract possessed a very promising antiviral effect. Future research may explicate the antiviral activity of stigmasterol derivative, sulfur-containing compounds, monoacylmonogalactosylgycerol, and all of nine cerebrosides, as, according to previous studies, they are the main compounds isolated from the ethanol extract. Though clinical trial suggested its therapeutic properties, research that optimizes the efficacy dosage and profiles the safety uses of the plant extract is lacking.


### 3.1.9. Anti-Bacterial Activity

Table7(i) shows the antibacterial properties ofC. nutans. Overall, studies were mainly based onin vitro assays and only polar (water, methanol, and ethanol) and semipolar (chloroform and ethyl acetate) extracts, but nonpolarC. nutans extracts not were evaluated for their anti-bacterial activity. A total of 9 different Gram-positive bacteria have been used in previous studies, includingBacillus cereus, Bacillus subtilis, Staphylococcus aureus, methicillin-resistantStaphylococcus aureus (MRSA), methicillin-sensitiveStaphylococcus aureus (MSSA),Micrococcus luteus, Propionibacterium acnes, Staphylococcus epidermidis, andStreptococcus sp. On the other hand,Aeromonas hydrophila, Escherichia coli and its strains,Neisseria gonorrhoeae,Pseudomonas aeruginosa and its strains,Salmonella enterica and its serovars, andVibrio harveyiandVibrio parahaemolyticus were the Gram-positive bacteria selected for study. As a summary of the antibacterial efficacy ofC. nutans, the most effective extract of the plant regarding antibacterial effect was prepared by oven-drying and undergoing ethyl acetate fractionation. The extract prevented visibleB. cereus, E. coli,andS. enterica Typhimurium growth at 1.39 mg/mL [88].


### 3.1.10. Antifungal Activity

Concerning the antifungal efficacy ofC. nutans,limited studies had been carried out to date. In the two reported antifungal activity studies, Cheeptham and Towers [89] found thatC. nutans did not exert a fungicidal effect onCandida albicans orAspergillus fumigatuswhen 5 mg/mL 95% ethanol leaf extract was tested. By contrast, a fraction of the ethyl acetate extract at the minimal concentration of 1.39 mg/mL did exhibit an antifungal effect onC. albicans[88]. In summary, based on the availablein vitro studies,C. nutans polar and semipolar extracts exhibited a weak antibacterial and antifungal activities. Any further research should focus on assessing the biological action of the polar and semipolar extracts ofC. nutans in inhibiting bacterial and fungal infections as well as augmenting the limited information on the antibacterial and antifungal potency of the nonpolar extract.


### 3.1.11. Anticancer Activity

A total of 15 types of cancer cells that are responsible for different types of cancer have been used to evaluate thein vitro anticancer effect ofC. nutans, as summarized in Table 7(k). These include human melanoma cell lines (D24 and MM418C1) for skin cancer, a human osteosarcoma cell line (cultured Saos-2) for bone tumor, human breast adenocarcinoma (MCF7) and human breast carcinoma (BT474) cell line for breast cancer, human lung adenocarcinoma (NCI-H23) and human lung carcinoma (NCI-H460) cell lines for lung cancer, a human liver hepatocellular carcinoma cell line (HepG2) for liver cancer, a human neuroblastoma cell line (IMR-32) for nerve tissue cancer, a human gastric cancer cell line (SNU-1) for gastric cancer, human colon adenocarcinoma (LS-174T) and human colorectal carcinoma cell line (HCT 116) for colon and rectal cancer, a human cervical cancer cell line (HeLa) for cervical cancer, a human erythroleukemia cell line (K562) for acute myeloid leukemia, and a human Burkitt’s lymphoma cell line (Raji) for lymphatic disorder. In addition, there were 2 studies that assessed thein vivo antimutagenic and anticancer effect ofC. nutans [33, 90]. Based on these studies, the most effective extract was the petroleum ether leaf extract, which showed the strongest cytotoxic activity after 72 h of incubation. The concentration of the extract that caused 50% death (CC50) was 18.0 μg/mL in HeLa cell and 20.0 μg/mL in the K-562 cell line [88]. According to the National Cancer Institute (NCI), a crude extract that exhibits an IC50 value <20 μg/mL can be considered as an active anticancer agent [41]. Overall, nonpolar leaf extract possessed a very pronounced cytotoxic effect on cervical and erythroleukemia cancerous cell. Hence, to evaluate the suitability ofC. nutans to involve in anticancer drug development, furtherin vivo clinical experimental studies on cervical cancer and erythroleukemia models are encouraged. The mechanism behinds the activity should be critically analyzed too. On the other hand, from the summarized data, observed inconsistency of incubation period during the experiment might be one of the factors that caused result deviation. Further parameter standardization should be implemented. Furthermore, the effect of the ethanol extract onin vivo hepatocarcinoma tumor-bearing mice exhibited a similar trend as in thein vitro study (methanolic extract) suggesting that both polar extracts possessed similar cytotoxic action towards liver cancerous cells, which is worthy of further investigation.


### 3.1.12. Wound Healing Activity

In addition to the other well studied pharmacological activities ofC. nutans, there are also a few other potential pharmacological activities which are less studied or that even have been reported only once. Table 7(l) summarizes the wound healing ability ofC. nutans.Between the chloroform extract and hexane extract, the chloroform extract (10 μg/mL) was found to provide the best improvement in human gingival fibroblast (HGF) migration rate and wound recovery in the 6 h of observation [11]. Before labelling it as a potential wound healing agent during inflammation, however, more studies are needed to verify its wound healing effect and determine which of the chlorophyll derivatives, diglycerides, or stigmasterol derivatives are the main factor for this positive activity.


### 3.1.13. Protective Effect on Plasmid DNA Activity

The protective effect ofC. nutanson plasmid DNA is tabulated in Table 7(m). Yuann et al. [64] suggested that, compared to green tea extract (protection up to 30 min), the 70% ethanol extract had the ability to reduce the number of DNA cleavages, preserve higher levels of supercoiled plasmid DNA integrity, and provide better protection against the riboflavin photoreaction induced superoxide for up to 50 min. In short, the polar extract showed a protective effect on plasmid DNA, but more analysis involving anin vivo model is needed. In addition, it is important to direct research focus towards the protective effect of the nonpolar extract as well.


### 3.1.14. Lipid Elevated Inhibition Activity

The ability ofC. nutans to attenuate obesity had been assessed as shown in Table 7(n) through anin vitro porcine pancreatic lipase inhibition assay. The result suggested that, instead of an inhibitory effect, the methanol extract exhibited a pancreatic lipase promoting effect [21]. However, as suggested previously, more research involving cellular andin vivoexperimental design are encouraged before reaching any conclusion.


### 3.1.15. Oral Mucositis and Stomatitis Treatment

C. nutansis also formulated for oral application to treat radiation induced oral mucositis in head and neck cancer patients and in patients suffering from recurrent aphthous stomatitis, as presented in Table 7(o) [10, 91]. The results revealed that, compared to the positive control group, the time onset of oral mucositis was significantly later, and the pain score was significantly lower [10]. Although it is not as efficacious as the positive control group (triamcinolone acetonide), it can be a good alternative to lessen the severity of ulcers [91].


## 3.1.1. Antivenom Activity

Table7(a) summarizes the antivenom tests that have been carried out thus far onC. nutans. The experiments examined the antivenom properties ofC. nutans’ leaves against snake, scorpion, and bee venoms. There were a total of 2in vitro and 4in vivo antivenom studies, and the extracts tested were prepared from water, ethanol, and aqueous ethanol. From the results of thein vitro studies, the water extract at 0.706 mg/mL exhibited a moderate anti-scorpion venom effect through a direct inactivation mechanism [65] while the aqueous ethanol extracts were ineffective against bee venom [66]. On the other hand, in 3 out of the 4in vivo studies conducted, the extracts (2 water and 1 aqueous ethanol extracts) notably did not exhibit antivenom effects after the test subjects were given different doses (6 to 2000 mg/kg bw) of the extracts through intraperitoneal injection (i.p.), intervenous injection (i.v.), or per os (p.o.) [5, 7, 67]. Although there was 1in vivo study proposing that the water extract ofC. nutans could exhibit a moderate anti-snake venom effect, the study did not include the administration method or the doses given [67]. Thus, defining a suitable extract that is biologically active as an antidote for snake and insect venoms is ambiguous. In summary, among the extracts tested, only water leaf extract exhibited potentialin vitroinhibition effect towards snake and insect venoms via direct inactivation mechanism. Further animal studies that focus on studying the metabolites of water extract and their reaction with the mediators that responsible for the direct inactivation pathway are worth emphasizing. Clinical study that investigates the efficacy of water extract as an antivenom agent through application of topical formulation on the patients might lead to a possible breakthrough.


## 3.1.2. Analgesic Activity

Table7(b) displays the scientific studies that investigated the analgesic ability ofC. nutans through 3 different types ofin vivo assays: the acetic acid-induced writhing test, formalin-induced paw licking test, and hot plate test. Three studies have used the acetic acid-induced writhing test to examine the analgesic effect ofC. nutans extracts prepared using 4 different types of solvents [19, 68, 69]. The results suggested that treating mice with then-butanol extract at 90 mg/kg as well as with the methanol extract at 279.3 mg/kg was as potent in terms of analgesic properties as treating them with phenylbutazone at 100 mg/kg. On the other hand, the study by Abdul Rahim et al. [19] was the only one that used formalin-induced paw licking test to evaluate the analgesic properties ofC. nutans. The results suggested that the methanol extract, with a half maximal effective concentration (EC50) at 227.7 mg/kg, was able to relieve pain in the late phase (centrally inflammatory induced pain pathway) via opioid/nitric oxide- (NO-) mediated, but cyclic guanosine monophosphate- (cGMP-) independent modulation systems. In addition, the analgesic effects of the absolute methanol, ethanol, andn-butanol extracts have been examined through the hot plate test. The results suggested that 500 mg/kg methanol extract was effective in alleviating the pain response at the interval from 60 to 210 min while both the ethanol and butanol extracts showed no analgesic effects up to 5 g/kg. Overall, methanol extract ofC. nutans exerted potential analgesic activity in both acute and persistent pain tests. The study also suggested that the presence of phenolics particularly gallic acid, caffeic acid, ferulic acid, vitexin, and apigenin which previously reported to exert antinociceptive activity which might be the main activity contributors. Since methanol extract might be a potential analgesic agent, it is important to further investigate the efficacy dosages, sites of action, and the level of the extract to modulate the pain along the pathway suggested. Furthermore, study may also provide information regarding antinociception properties ofC. nutans methanol extract on visceral and neuropathic pains.


## 3.1.3. Anti-Inflammatory Activity

Table7(c) summarizes the anti-inflammatory effect ofC. nutans. The anti-inflammatory effect ofC. nutans was assessed based onin vitro assays, such as the macrophage activatorN-formyl-methionyl-leucyl-phenylalanine- (fMLP-) induced neutrophil elastase release superoxide anion generation, lipopolysaccharides (LPS) induced toll like receptor 4 (TLR-4), NO Griess, and cytokine production assays. Furthermore, the types ofin vivo experiments used to evaluate the acute anti-inflammatory properties ofC. nutans were the acetic acid-induced vascular permeability model and ethyl phenylpropiolate- (EPP-) induced rat ear oedema and carrageenan-induced paw oedema model, whereas its subchronic anti-inflammatory effect was assessed using the granuloma pouch model, which mimics subchronic inflammation in humans. For thein vitro anti-inflammatory effects, both the methanol and 80% ethanol extracts have been evaluated through the neutrophil elastase release and superoxide anion generation assays [52, 70]. The results showed that 10 μg/mL of 80% ethanol extract inhibited 68.33% elastase release, which was more effective than the methanol extract (<20%), while to inhibit superoxide radical formation, both extracts at 10 μg/mL showed comparable efficacy, as they inhibited approximately 30% superoxide formation [52, 70]. For the LPS-induced TLR-4, NO, and cytokine production assays, the results suggested that the polar (methanol and dichloromethane) extract possessed a better IC50 value (<22 μg/mL) for inhibiting TLR-4, NO and proinflammatory cytokine production than the nonpolar (hexane and diethyl ether) extract [62]. For thein vivo acute anti-inflammatory properties ofC. nutans, the most effective dose was pretreatment of 9 mg EPP/ear of methanol extract on the rat which resulted in 79% oedema inhibition at 15 min and 44.4% myeloperoxidase (MPO) reduction after 120 min of induction [70].Additionally, in a comparison of the methanol, ethanol, andn-butanol extracts, a 1 h pretreatment of 200 mg/kg methanol extract administered via p.o. to carrageenan-induced rats was found to inhibit 59% oedema formation [70]. The acetic acid-induced vascular permeability model showed that, among the water, methanol, chloroform, andn-butanol extracts, the butanol extract at 540 mg/kg was superior at exhibiting an anti-inflammatory effect [68, 69]. Ethanol, aqueous ethanol,n-butanol, and cold creams have been evaluated for their subchronic anti-inflammation properties. The results showed that application of 125 mg ofC. nutans cold cream to a rat topically throughout the experimental period inhibited granuloma formation by as much as 50.98%, which is comparable to the inhibitory effect of 0.25% prednisolone (56.82%) [71]. According to the previousC. nutans anti-inflammatory studies, the extract prepared from polar solvent notably exhibited a promising anti-inflammatory propertiesin vitro(at a dose less than 30 μg/mL) andin vivo (at a dose less than 300 mg/kg). Therefore, it is greatly desired to focus on the relationship between the mode of action ofC. nutanspolar extract in both biological and nonbiological anti-inflammatory systems. In addition, since polar extract exhibited potential anti-inflammatory activity, knowledge of extract prepared from the inorganic polar solvent particularly water is still scarce as regards itsin vivoandin vitro anti-inflammatory activity properties, thus possessing potential research interest.


## 3.1.4. Immunomodulating Activity

Table7(d) shows the immunomodulating effect ofC. nutans. The methanol extract ofC. nutans has been tested for its immune modulating effect on apoptosis and cytokines expression in experiments on human neutrophils and porcine peripheral blood mononuclear cells (PPBMCs), such as assays for fMLP macrophage activator-induced chemotaxis and chemokinesis, apoptosis, and concanavalin (ConA) and LPS-induced interleukin 10 (IL-10), and tumor necrosis factor-alpha (TNF-α) expression [70, 72]. Wanikiat et al. [70] suggested thatC. nutans methanol extract exhibited dose-dependent suppression of fMLP-induced chemotaxis and chemokinesis of neutrophils without causing the cells to undergo apoptosis.C. nutans was found to cause a reduction in IL-10 expression and to have no effect on TNF-α expression in PBMC [72]. On the other hand, the ethanolic extract ofC. nutans has been tested for its immunomodulating effect on cytokines expression in splenocytes, a human keratinocyte cell line (HaCaT), and human peripheral blood mononuclear cells (HPBMC). Tu et al. [52] suggested that low concentrations of the ethanol extract cause IFN-γ upregulation while higher concentration ofC. nutans cause IFN-γ downregulation. Sriwanthana et al. [73] studied found that low concentrations ofC. nutans resulted in increase in lymphocyte proliferation, while higher concentrations ofC. nutans resulted in decrease of lymphocyte production. The study also found thatC. nutans did not stimulate an interleukin-2 (IL-2) response or affect the lymphocyte subpopulation, such as total T lymphocytes (CD3), T helper/inducer cells (CD4), T suppressor/cytotoxic cells (CD8), natural killer (NK) cells (CD16/CD56), or B lymphocytes (CD19). On the other hand, 2.5 and 5 mg/mL of ethanol extract increased the interleukin-4 (IL-4) production and 1 and 5 mg/mL of extract suppressed NK activity [73]. Furthermore, 1 and 100 μg/mLC. nutans were found to inhibit IFN-γ and TNF-α-induced keratinocytes apoptosis [63]. A similar trend as in the analgesic and anti-inflammatory properties ofC. nutans could be observed, where extract prepared from polar solvents exerted better analgesic, anti-inflammatory, and immunomodulating effects. Phenolics, sulfur-containing glucosides, and sulfur-containing compounds might be the main contributors to these activities, as all the mentioned compounds were primarily isolated from polarC. nutans extracts. In addition, the described therapeutic effects are always well correlated with the immunology results. Thus, investigations on the biological mechanism of the active constituents present in the polar extract and their effects on immunological cells, mediators, and substances deserve special attention. Exploring the immunological function of other polar extracts, such as water, and nonpolar extracts should be encouraged as well.


## 3.1.5. Neuromodulating Activity

The efficacy ofC. nutans in neuro-related protective and modulating functions has also been studied in recent years, as shown in Table 7(e).In vitro studies suggested that the neuroprotective effect ofC. nutans was promising. From the studies by Tan et al. [74] and Tsai et al. [75], the 80% ethanol extract with a dose concentration less than 10 μg/mL was able to suppress post-hypoxic histone deacetylase (HDAC) activation and hypoxic neuronal death in an oxygen-glucose deprivation- (OGD-) reoxygenation assay. It also decreased the levels of cytosolic phospholipase 2 (cPLA2) mRNA expression in mouse primary cortical neurons subjected to 0.5 h of OGD injury. In addition, Tan et al. [74] found that 100 μg/mL 80% ethanol extract was able to suppress histone acetylase (HAT) activity and regulate cPLA2 expression induction in a human neuroblastoma cell line (SH-SY5Y cells) through HDAC inhibitors such as entinostat (MS-275), MC-1568, and trichostatin A (TSA). The research team of Wu et al. [76] further suggested that treating the primary neurons with 0.15–20 μg/mL of 80% ethanol extract 1 h before, at the onset, or after the OGD induced cell death or OGD-reoxygenation treatment was able to mitigate the neuronal apoptosis and protected primary neurons by activating the antiapoptotic activity of peroxisome proliferator-activated receptor-gamma (PPAR-γ) → 14-3-3ε (antiapoptotic marker), enhancing the C/EBPβ binding to PPAR-γ promoter and amplifying its transcription though the extract was less effective when applied after the OGD treatment. The result also suggested that the protective effect of extract was revoked when cotreated with GW9662 (PPAR-γ antagonist). Thisin vitro screening further complemented with the protective effect of extract in attenuating the ischemic brain damage inin vivo middle cerebral artery occlusion (MCA) stroke model [76]. Both intracerebroventricular (i.c.v.) infusion (10–60 pg extract) and i.p. injection (24 mg/kg body wt extract) of extract demonstrated that the extract possessed the ability in mitigating apoptotic neuronal death, cerebral infarct volume, and behavioral deficiency in the rat MCA occlusion model. The detailed neuroprotective mechanism ofC. nutans was similar toin vitro OGD study and was elaborated in Table 7(e). In addition, anin vivostudy conducted by Lau et al. [77] revealed that the three different tested doses of methanol extract were able to stimulate acetylcholinesterase activity in the heart, liver, and kidney, but not the brain of mice without inducing any signs of toxicity in the mice. As a short summary, similar results have been reported as those regarding the analgesic, anti-inflammatory, and immunomodulating activities, where the polar extract ofC. nutans possessed a great neuroprotective effect. Nevertheless, research on the neuroprotective strength ofC. nutans is still new and therefore holds enormous potential for different research directions. Further investigation of the potential efficacy of polarC. nutans extract inin vivo neurology-inflammation related studies might lead to a breakthrough. Research should fill in gaps about the neuro-related function of nonpolar extract as well.


## 3.1.6. Antidiabetic andα-Glucosidase Inhibitory Activity

Table7(f) shows a list of studies conducted on the antidiabetic and α-glucosidase inhibition effects ofC. nutans. There have been a total of 4in vitro studies assessing the hyperglycemia inhibitory activity ofC. nutansthrough an α-glucosidase inhibition assay. All the results showed thatC. nutans had a very low inhibitory effect on the α-glucosidase enzyme when the employed extracts were prepared using conventional preparation methods [4, 28, 37, 61]. Although IC50 was not calculated by Alam et al. [28], a lower IC50 value is anticipated as the extract prepared through advanced supercritical extraction exhibited 95.79% of α-glucoside inhibition when the stock was prepared at 5000 μg/mL. DespiteC. nutans not showing particularly pronounced inhibition against α-glucosidase, 3in vivo antidiabetic experiments suggested that the water extract, 80% methanol extract, and the insoluble ethyl acetate fraction from the 80% ethanol extract were able to attenuate the insulin resistance induced by a high fat and high cholesterol diet (HFHC) and serum glucose level [25, 39]. In general, the high α-glucosidase inhibition by SFE treated extracts might indicate that the compounds active against the α-glucosidase enzyme are heat-sensitive or easily degradable. Thus, extra precaution is needed during the extract preparation step. On the other hand, conventional method prepared extracts that mimicked the traditional application showed low inhibition towardsin vitroα-glucosidase tests while traditional uses suggested thatC. nutans is an antidote for diabetes. The contradictory results might be attributed by improper mode of action of diabetes being investigated. Since α-glucosidase inhibition test focuses on mechanism of postprandial hyperglycemia, further study on other mechanism approach, experiment on the reaction of the extract towards β-cell in pancreas, test that involved other enzymes in hyperglycemia such as pancreatic α-amylase, glucose diffusion assay, glucose uptake by yeast cells, and nonenzymatic glycosylation assay are highly recommended.


## 3.1.7. Antioxidant Activity

Considerable number of analyses have been done on the antioxidant effect ofC. nutans as shown in Table 7(g). Based on the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical assay, some studies showed thatC. nutans up to 1 mg/mL is a potential antioxidant agent, while other studies found that the DPPH inhibitory properties ofC. nutans could not be determined [47, 64, 78]. The data in the table showed that, among the polar and nonpolar extracts tested, the highest antioxidant properties ofC. nutanswas detected in the study by Ghasemzadeh et al. [41], whereC. nutans bud extract that was cultivated for 1 year at a farm located in Malaysia and that underwent freeze drying and methanol extraction resulted in IC50 value of 64.6 μg/mL. The second most common antioxidant determination assay selected by researchers was the ferric reducing antioxidant power (FRAP) assay. However, discussion of the FRAP results is difficult, as there is a lack of unit standardization in the measurement. The results from othersin vitro antioxidant assays, such as those for hydrogen peroxide scavenging, metal chelating, nitric oxide scavenging, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) cation radical scavenging, galvinoxyl radical scavenging activity, superoxide radical scavenging activity, and phorbol 12-myristate 13-acetate (PMA) induced peroxide production in rat macrophages and protective effect against peroxyl radicals initiator- (AAPH-) induced oxidative hemolysis, have been summarized in Table 7(g(3)–g(10)). Overall, polar and semipolar solvent-extractedC. nutans were more likely to exhibit moderate antioxidant activity at concentration varying from 0.0125 to 10 mg/mL than nonpolar extracts. In addition, there was a recent study that evaluated the antioxidant activity ofC. nutans in vivo by using a hyperlipidemia-associated oxidative stress model [42]. The study suggested that both the water and 80% methanol leaf extracts at up to 500 mg/kg bw rats were able to mitigate oxidative stress by improving the activity of serum antioxidant enzymes and expression of hepatic antioxidant genes [42]. In summary, polar and semipolar extracts are more likely to exhibit antioxidant properties. A possible hypothesis is that phenolics, sulfur-containing glucosides, sulfur-containing compounds, chlorophyll derivatives, and some phytosterol derivatives that are isolated fromC. nutans using polar and semipolar solvents might be the contributors. Thus, studies on the antioxidant properties of these isolated compounds and further evaluation of the plant’s antioxidant effect inin vivo studies are required.


## 3.1.8. Antiviral Activity

The antimicrobial properties ofC. nutans have been further classified into antiviral, antibacterial, and antifungal effects, as shown in Tables 7(h), 7(i), and 7(j), respectively. For the antiviral activity ofC. nutans, the types of virus that have been studied were varicella zoster virus (VZV), herpes simplex virus (HSV), fish pathogenic viruses, crustaceans (shrimp and prawn) infectious viruses, mosquito-borne viruses, and poultry and bird contagious viruses. Notably, the antivirus studies were always accompanied with a prescreening test for cytotoxicity to determine the subtoxic concentration of the test sample and to ensure that the test sample has a killing effect on the virus instead of on the host cell [79]. Researchers have also investigated the mode of action ofC. nutans in terms of its antiviral potential through 3 different stages of treatment. Pretreatment (pre-) studies are those where the test sample is allowed to incubate with the cell for a time of period before it is infected with the virus. This approach reflects the virucidal activity of the test sample, as the test sample interferes with the viral structure to prevent or inhibit the viral penetrating or adsorbing to the host cell [79]. In posttreatment (post-) studies, instead of being preincubated with the test sample, the virus is attached to the host cell that has first only been cultured with the test sample. The test sample is thus considered to have antiviral activity if it successfully inhibits viral DNA replication [79]. By contrast, for the direct inactivation (direct) pathway, the virus is preincubated with the test samples first before being added to the host cell. The test sample is said to have an inactivation ability against the virus if it interferes with or causes damage to the viral glycoproteins, the virus envelope, or the virus structure before they enter the host cell [79].Thus far, only onein vitro study and a total of 3 clinical trials have been conducted to evaluateC. nutans’ anti-VZV activity. Thein vitro result suggested that the organic extract exhibited anti-VZV activity through the direct inactivation stage [80]. In the clinical studies, theC. nutans extracts were formulated into a 5%C. nutans cream prior to testing its ability to combat VZV infections. The result suggested that theC. nutans containing cream was able to exhibit a positive curing effect. All the studies showed that the percentage of patients who experienced lesion crusting within 3 days and lesion healing within 7 days after applying theC. nutans cream topically was better than the percentage in the placebo and acyclovir groups. In addition, the pain score and side effect resulting fromC. nutans cream application were lower [8, 81, 82].For the anti-HSV-1 activity ofC. nutans, the ethyl acetate, methanol, dichloromethane, chloroform, andn-hexane extracts have been evaluated. Thongchai et al. [79] suggested that theC. nutans ethyl acetate leaf extract displayed the best inhibition on HSV strain with IC50 value of 7.6 μg/mL through the pretreatment mechanism. Meanwhile, the methanol, ethanol dichloromethane, chloroform, and hexane extracts were also tested for their anti-HSV-2 activity. TheC. nutans methanol and hexane leaf extracts were found to have better and comparable inhibition against HSV-2-Baylor 186 strain, via posttreatment action, with IC50 value of 65.13 and 72.62 μg/mL, respectively [83]. The anti-HSV-2 activity ofC. nutans has also been evaluated in clinical studies using the 5% extract cream. The results obtained were similar to those from the clinical studies on the anti-VZV activity; when compared to a placebo, the days required for lesion crusting and healing after application ofC. nutans were higher than or the same in terms of efficacy as acyclovir [82, 84, 85]. Regarding its efficacy in inhibiting fish pathogenic viruses, among the virus strains tested, theC. nutansethanolic extract exerted 100% inhibition on plaque formation in infectious hematopoietic necrosis virus (IHNV) andOncorhynchus masouvirus (OMV) strains but not in infectious pancreatic necrosis virus (IPNV) through the direct inactivation stage.On the other hand, it was also found thatC. nutans ethanol extract had an excellent protective effect on cultured black tiger shrimp against yellow head rhabdo-like virus (YRV-RNA) through a direct inactivation mechanism and pronounced virucidal activity against dengue virus (IC50 value of 31.04 μg/mL); however, it was less effective against Newcastle disease virus (NDV) caused by poultry and birds [52, 86, 87]. In summary,C. nutans ethanol extract possessed a very promising antiviral effect. Future research may explicate the antiviral activity of stigmasterol derivative, sulfur-containing compounds, monoacylmonogalactosylgycerol, and all of nine cerebrosides, as, according to previous studies, they are the main compounds isolated from the ethanol extract. Though clinical trial suggested its therapeutic properties, research that optimizes the efficacy dosage and profiles the safety uses of the plant extract is lacking.


## 3.1.9. Anti-Bacterial Activity

Table7(i) shows the antibacterial properties ofC. nutans. Overall, studies were mainly based onin vitro assays and only polar (water, methanol, and ethanol) and semipolar (chloroform and ethyl acetate) extracts, but nonpolarC. nutans extracts not were evaluated for their anti-bacterial activity. A total of 9 different Gram-positive bacteria have been used in previous studies, includingBacillus cereus, Bacillus subtilis, Staphylococcus aureus, methicillin-resistantStaphylococcus aureus (MRSA), methicillin-sensitiveStaphylococcus aureus (MSSA),Micrococcus luteus, Propionibacterium acnes, Staphylococcus epidermidis, andStreptococcus sp. On the other hand,Aeromonas hydrophila, Escherichia coli and its strains,Neisseria gonorrhoeae,Pseudomonas aeruginosa and its strains,Salmonella enterica and its serovars, andVibrio harveyiandVibrio parahaemolyticus were the Gram-positive bacteria selected for study. As a summary of the antibacterial efficacy ofC. nutans, the most effective extract of the plant regarding antibacterial effect was prepared by oven-drying and undergoing ethyl acetate fractionation. The extract prevented visibleB. cereus, E. coli,andS. enterica Typhimurium growth at 1.39 mg/mL [88].


## 3.1.10. Antifungal Activity

Concerning the antifungal efficacy ofC. nutans,limited studies had been carried out to date. In the two reported antifungal activity studies, Cheeptham and Towers [89] found thatC. nutans did not exert a fungicidal effect onCandida albicans orAspergillus fumigatuswhen 5 mg/mL 95% ethanol leaf extract was tested. By contrast, a fraction of the ethyl acetate extract at the minimal concentration of 1.39 mg/mL did exhibit an antifungal effect onC. albicans[88]. In summary, based on the availablein vitro studies,C. nutans polar and semipolar extracts exhibited a weak antibacterial and antifungal activities. Any further research should focus on assessing the biological action of the polar and semipolar extracts ofC. nutans in inhibiting bacterial and fungal infections as well as augmenting the limited information on the antibacterial and antifungal potency of the nonpolar extract.


## 3.1.11. Anticancer Activity

A total of 15 types of cancer cells that are responsible for different types of cancer have been used to evaluate thein vitro anticancer effect ofC. nutans, as summarized in Table 7(k). These include human melanoma cell lines (D24 and MM418C1) for skin cancer, a human osteosarcoma cell line (cultured Saos-2) for bone tumor, human breast adenocarcinoma (MCF7) and human breast carcinoma (BT474) cell line for breast cancer, human lung adenocarcinoma (NCI-H23) and human lung carcinoma (NCI-H460) cell lines for lung cancer, a human liver hepatocellular carcinoma cell line (HepG2) for liver cancer, a human neuroblastoma cell line (IMR-32) for nerve tissue cancer, a human gastric cancer cell line (SNU-1) for gastric cancer, human colon adenocarcinoma (LS-174T) and human colorectal carcinoma cell line (HCT 116) for colon and rectal cancer, a human cervical cancer cell line (HeLa) for cervical cancer, a human erythroleukemia cell line (K562) for acute myeloid leukemia, and a human Burkitt’s lymphoma cell line (Raji) for lymphatic disorder. In addition, there were 2 studies that assessed thein vivo antimutagenic and anticancer effect ofC. nutans [33, 90]. Based on these studies, the most effective extract was the petroleum ether leaf extract, which showed the strongest cytotoxic activity after 72 h of incubation. The concentration of the extract that caused 50% death (CC50) was 18.0 μg/mL in HeLa cell and 20.0 μg/mL in the K-562 cell line [88]. According to the National Cancer Institute (NCI), a crude extract that exhibits an IC50 value <20 μg/mL can be considered as an active anticancer agent [41]. Overall, nonpolar leaf extract possessed a very pronounced cytotoxic effect on cervical and erythroleukemia cancerous cell. Hence, to evaluate the suitability ofC. nutans to involve in anticancer drug development, furtherin vivo clinical experimental studies on cervical cancer and erythroleukemia models are encouraged. The mechanism behinds the activity should be critically analyzed too. On the other hand, from the summarized data, observed inconsistency of incubation period during the experiment might be one of the factors that caused result deviation. Further parameter standardization should be implemented. Furthermore, the effect of the ethanol extract onin vivo hepatocarcinoma tumor-bearing mice exhibited a similar trend as in thein vitro study (methanolic extract) suggesting that both polar extracts possessed similar cytotoxic action towards liver cancerous cells, which is worthy of further investigation.


## 3.1.12. Wound Healing Activity

In addition to the other well studied pharmacological activities ofC. nutans, there are also a few other potential pharmacological activities which are less studied or that even have been reported only once. Table 7(l) summarizes the wound healing ability ofC. nutans.Between the chloroform extract and hexane extract, the chloroform extract (10 μg/mL) was found to provide the best improvement in human gingival fibroblast (HGF) migration rate and wound recovery in the 6 h of observation [11]. Before labelling it as a potential wound healing agent during inflammation, however, more studies are needed to verify its wound healing effect and determine which of the chlorophyll derivatives, diglycerides, or stigmasterol derivatives are the main factor for this positive activity.


## 3.1.13. Protective Effect on Plasmid DNA Activity

The protective effect ofC. nutanson plasmid DNA is tabulated in Table 7(m). Yuann et al. [64] suggested that, compared to green tea extract (protection up to 30 min), the 70% ethanol extract had the ability to reduce the number of DNA cleavages, preserve higher levels of supercoiled plasmid DNA integrity, and provide better protection against the riboflavin photoreaction induced superoxide for up to 50 min. In short, the polar extract showed a protective effect on plasmid DNA, but more analysis involving anin vivo model is needed. In addition, it is important to direct research focus towards the protective effect of the nonpolar extract as well.


## 3.1.14. Lipid Elevated Inhibition Activity

The ability ofC. nutans to attenuate obesity had been assessed as shown in Table 7(n) through anin vitro porcine pancreatic lipase inhibition assay. The result suggested that, instead of an inhibitory effect, the methanol extract exhibited a pancreatic lipase promoting effect [21]. However, as suggested previously, more research involving cellular andin vivoexperimental design are encouraged before reaching any conclusion.


## 3.1.15. Oral Mucositis and Stomatitis Treatment

C. nutansis also formulated for oral application to treat radiation induced oral mucositis in head and neck cancer patients and in patients suffering from recurrent aphthous stomatitis, as presented in Table 7(o) [10, 91]. The results revealed that, compared to the positive control group, the time onset of oral mucositis was significantly later, and the pain score was significantly lower [10]. Although it is not as efficacious as the positive control group (triamcinolone acetonide), it can be a good alternative to lessen the severity of ulcers [91].


## 3.2. Pharmacological Activity ofC. nutans Isolated Pure Compounds

After understanding the pharmacological properties ofC. nutans extract, it is beneficial to further investigate which compound in a particular extract is responsible for the therapeutic activity of the plant. Table 8 summarizes the pharmacological activities of the isolated compounds fromC. nutans. For anti-inflammatory activity, only cerebrosides from the ethanol extract have been studied, and they were reported as being ineffective in suppressing cyclooxygenase (COX-1 and COX-2) induced prostaglandin E2 generation and HSV-1 infection [55]. The result obtained was inconsistent with the anti-inflammatory effect of the extract observed previously. Nevertheless, the type of experiment conducted might cause this variation, and further attention should focused on the anti-inflammatory effect of other compounds isolated from the polar solvent. Le et al. [45] studied the immunomodulating function of flavones (schaftoside) and terpenes (stigmasterol, β-sitosterol, and lupeol) compounds isolated from the hexane fraction of the methanol extract. The results showed that only stigmasterol and β-sitosterol were able to suppress the T cell proliferation mediated by ConA and that only β-sitosterol exerted an immune suppressive effect on T helper 2 (Th2) cytokines (IL-4 and IL-10). A similar trend was observed with the polar extract ofC. nutans which exerted a better immunomodulating effect than the nonpolar extract. Clinamide D, which was isolated from the methanol extract, was found to have a moderate antioxidant effect with 76.05 ± 0.02% DPPH radical inhibition at 1000 μg/mL which is consistent with the good DPPH inhibition properties of the methanol extract [41, 53].Table 8
Pharmacological activity of isolatedC. nutanscompounds.
Experiment design(Experiment model; Assay; Incubation period; Test subject; Extract dose; Positive control) Isolated compound Result Reference (a) Anti-inflammatory activity  in vitro; Prostaglandin E2 determination assay by the radioimmunoassay Immortalized COX-1 and COX-2 mouse lung fibroblast cell; Aspirin (IC50: 2.06 μg/mL (COX-1), 3.57 μg/mL (COX-2)) Cerebrosides Ineffective [55]  (b) Immunomodulating activity  in vitro; ConA-induced T cell, LPS-induced B cell, evaluation of Th1 cytokines (IL-2 and IFN-γ), Th2 cytokines (IL-4 and IL-10); Splenocytes; Control on Th2 cytokines IL-4 (138.3 pg/mL) and IL-10 (1234.9 pg/mL) ( 1 ) CN1 (shaftoside) (2) CN2 (stigmasterol) (3) CN3 (β-sitosterol)  (4) CN4 (lupeol) CN3 inhibit T lymphocyte proliferation the most (RP: 0.16) followed by CN2 (RP: 0.47), only CN1 inhibit B cell proliferation (RP: 0.77), all ineffective on Th1 cytokines, CN3 inhibit secretion of IL-4 (22.6 pg/mL) and IL-10 (63.9 pg/mL), CN3 significantly reduce activated helper T cells (54.3%) and activated cytotoxic T cells (62.2%) [45]  (c) Anti-oxidant activity  in vitro; DPPH scavenging assay; Vitamin C (IC50: 22.589 μg/mL) ( 1 ) Stigmasterol-β-D-glucoside (2) 3-amino-4,5-dihydroxyfuran-2(3H)-one Compound 1: IneffectiveCompound 2: IC50: 102.949 μg/mL [49] in vitro; DPPH scavenging assay, FRAP assay; 1000 μg/mL (stock); BHT (DPPH), Ascorbic acid (FRAP) ( 1 ) Clinamide D(2) Clinamide E (1) 76.05 ± 0.02%, (2) 72.84 ± 0.01% of DPPH inhibition, (1) 15.47 ± 0.03%, (2) 38.56 ± 0.02% of FRAP inhibition [53]  (d) Anti-viral activity  Anti HSV-1 assay in vitro; HSV-1 virus strain; Vero cell; Acyclovir (IC50: 2–5 μg/mL) Cerebrosides Ineffective [55] Anti HSV-1 assay in vitro; HSV-1F strain; Plaque reduction assay-direct, pre, post; 72 h; Vero cell; Acyclovir, Dextran sulfate (1 mg/mL) ( 1 ) 132-hydroxy-(132-R)-phaeophytin b (2) 132-hydroxy-(132-S)-phaeophytin a (3) 132-hydroxy-(132-R)-phaeophytin a Direct: All exhibited 100% inhibitionIC50: 1.96 nM, 3.11 nM, and 3.11 nM, respectively, Post: 30% of inhibition [107] Anti HSV-1 and HSV-2 in vitro; Plaque reduction assay-pre-, post-; 48 h (HSV-1), 96 h (HSV-2); Vero cells; Acyclovir (IC50: 0.64 μg/mL (HSV-1), 0.80 μg/mL (HSV-2)) ( 1 ) monogalactosyl diglyceride (MGDG) (2) digalactosyl diglyceride (DGDG) Pre: Exhibited < 50% protective effectPost: Exhibited 100% protective effect Post-HSV-1: IC50: 36.00 μg/mL (MGDG), 40.00 μg/mL (DGDG), HSV-2: IC50: 41.00 μg/mL (MGDG), 43.20 μg/mL (DGDG) [56] Anti-dengue virus assay in vitro; DV2 strain 16681; Real time-PCR, immunofluorescence assay; direct, pre-, post-; 5 d; Dextran sulfate (pre-), Ribavirin (post-) ( 1 ) 132-hydroxy-(132-S)-chlorophyll b (2) phaeophorbide A (3) 132-hydroxy-(132-S)-phaeophytin b (4) purpurin 18 phytyl ester Compound 2 inhibit dengue viral 2 replication in direct and post- stages, other compounds ineffective in all stages. [108]  (e) Anti-bacterial activity  in vitro; S. aureus, S. typhimurium; (1) Disc diffusion assay, (2) Microdilution assay; 10 mg/mL; Erythromycin (10–50 μg/μL) ( 1 ) Stigmasterol-β-D-glucoside (2) 3-amino-4,5-dihydroxyfuran-2(3H)-one Compound 1: IneffectiveCompound 2 on S. aureus:18.33 mm inhibition value, MIC: 0.3125 mg/mL Compound 2 on S. typhimurium: 20.33 mm inhibition value, MIC: 0.625 mg/mL [49]  (f) Anti-cancer activity  in vitro; SGC-7901 cancer cells; MTT assay; 48 h; 50, 100 and 200 μg/mL Polysaccharide peptide complex: CNP-1-2 92.34 ± 0.94% of inhibition on cell growth at 200μg/mL in 48 h [59] in vitro; A549 cells; MTT assay; 72 h ( 1 ) 132-hydroxy-(132-S)-chlorophyll b (2) phaeophorbide A (3) 132-hydroxy-(132-S)-phaeophytin b (4) purpurin 18 phytyl ester CC50: (1) 43 μg/mL, (2) 25 μg/mL, (3) 50 μg/mL, (4) 50 μg/mL [108]  (g) Cytotoxicity assay  in vitro; CVS assay; 72 h; Vero cells ( 1 ) 132-hydroxy-(132-R)-phaeophytin b (2) 132-hydroxy-(132-S)-phaeophytin a (3) 132-hydroxy-(132-R)-phaeophytin a Maximum concentration that is not toxic to Vero cell is: Compound 1 (5.89μM), 2 (6.21 μM), 3 (6.21 μM) [107] in vitro; MTT assay; 48 h; Vero cells; 100–15000 μg/mL ( 1 ) monogalactosyl diglyceride (MGDG) (2) digalactosyl diglyceride (DGDG) MGDG: CC50: 955.00 ± 7.00 μg/mL DGDG: CC50: 922.00 ± 4.00 μg/mL [56]Regarding the antiviral activity, 132-hydroxy-(132-R)-phaeophytin b from the chloroform extract was found to possess a better anti-HSV-1 effect with an IC50 value of 1.96 nM through direct inactivation [107]. Pongmuangmul et al. [56] showed that monogalactosyl diglyceride (MGDG) and digalactosyl diglyceride (DGDG) isolated from the chloroform extract exhibited promising anti HSV-1 and anti-HSV-2 properties in the poststep of infection, where the IC50 values were in a range from 36.00 μg/mL to 43.20 μg/mL. Sittiso et al. [108] suggested that phaeophorbide A from the chloroform extract was able to inhibit dengue viral 2 replication in the direct inactivation and postincubation stages with a CC50 of 25 μg/mL. Based on this study, it can be observed that, in antiviral studies, compounds isolated from the chloroform extract are more likely to exhibit antiviral activity whereas the ethyl acetate, methanol, and ethanol extracts were more likely to possess anti-HSV-1 and anti-HSV-2 activities, and the ethanol extract was more likely to possess an anti-dengue inhibitory effect. Thus, it might be a valuable approach to investigate the relationship between that antiviral properties of compounds isolated from the aforementioned extracts. Tinh [49] found that 3-amino-4,5-dihydroxyfuran-2(3H)-one isolated from the ethanol extract exhibited moderate antibacterial activity againstS. aureus(MIC equal to 0.3125 mg/mL). This result was inconsistent with the antibacterial activity of the corresponding extract against the same bacterial strain. This might due to the antibacterial properties of the crude extract being hindered by the other constituents present in the extract. In addition, phaeophorbide A from the chloroform extract was found to possess a promisingin vitroanticancer effect on human lung carcinoma cell (A549 cells) at a CC50 equal to 25 μg/mL [108]. This result was consistent with the study from Yong et al. [32] which found that the chloroform extract at 100 μg/mL showed 55.82% inhibition of human lung cancer cell. A novel polysaccharide-peptide complex (CNP-1-2) isolated by Huang et al. [59] from the 70% ethanol extract also exhibited potential anticancer activity on a human gastric cancer cell line (SGC-7901 cells), where, at concentration of 200 μg/mL, it inhibited cell growth by 92.34 ± 0.94% after 48 h of incubation. By contrast, Yong et al. [32] found that the chloroform extract exhibited a weak killing effect on human gastric cancer cells (31.25 ± 1.09% at 100 μg/mL).


## 4. Conclusion

Phytochemical identification and quantification have suggested thatC. nutans is a rich source of phenolics, flavonoids, triterpenoids, and chlorophyll derivatives; however, their retention in an extract is largely influenced by the plant preparation methods. On the other hand, pharmacological studies onC. nutans have suggested that its polar extracts can be a promising anti-inflammation, antiviral, anticancer, immune- and neuromodulating, and plasmid DNA protective agents, as well as a moderate antivenom, analgesic, antidiabetic, and antioxidant agent and a weak lipid elevating inhibitor. By contrast, its semipolar extracts can be a promising antiviral, anticancer and wound healing agent, a moderate anti-inflammation and antioxidant agent, and a weak antibacterial and antifungal agent, and its nonpolar extracts can be a strong anticancer agent. However, as insufficient previous scientific studies have been conducted and most experiments were preliminary and fundamentally oriented, more sophisticated evaluation and pathway analyses of the aforementioned biological and therapeutic potential of this plant are urged before implementing it in the pharmaceutical and cosmetics industries. In addition, sincein vitro assays do not fully mimic the physiological environment in animals and humans, additional cellular,in vivoand clinical trials are likewise to fully interpret the effect ofC. nutans on disease inhibition and prevention. Mechanism of action of the extract towards particular treatment should explicate. It is also likely to have more experimental studies that could substantiate and describe the correlation of the isolated phytochemicals fromC. nutanswith their corresponding pharmacological effects. A note for future researchers: it is of utmost importance to provide complete data such as the extract concentration, the extraction solvent used, and the experimental design, which was found to be lacking throughout the data searching. All the aforementioned data could be a crucial point that determines the accuracy of the interpretation of the results, the credibility of the study, and the reproducibility of the work in the future. In conclusion, through this review, the authors hope to provide a more systematic summary of the previousC. nutans works according to the experimental design and plant preparation methods that have been thus far done in phytochemical and pharmacological relevantC. nutans studies. The authors also anticipate to provide some possible idea for researchers regarding the future research perception and direction of this plant.



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*Source: 9276260-2018-07-04.xml*