# Evaluation on Antidiabetic Properties of Medicinal Plants from Myanmar

Authors: Dongdong Zhang; Karuppusamy Arunachalam; Yuehu Wang; Yu Zhang; Jun Yang; Pyae Phyo Hein; Aye Mya Mon; Jianwen Li; Angkhana Inta; Xuefei Yang
Journal: The Scientific World Journal
 (2021)

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## Abstract

Objectives. To explore the effective and safe medicines for treating diabetes. Methods. Hydroalcoholic extracts of 130 medicinal plants belonging to 66 families were evaluated using porcine pancreatic lipase (PPL) inhibition and glucose uptake methods together with a literature review. Results. The extracts of 22 species showed the PPL inhibition activity; 18 extracts of 15 species stimulated glucose uptake in 3T3-L1 adipocytes. Among them, Mansonia gagei J.R. Drumm., Mesua ferrea L., and Centella asiatica (L.) Urb. exhibited both activities. The extracts of Caladium lindenii (André) Madison rhizomes and Azadirachta indica A. Juss. leaves presented the utmost lipase inhibitory activity with IC50 of 6.86 ± 0.25 and 11.46 ± 0.06 μg/mL, respectively. The extracts of Coptis teeta Wall. rhizomes and Croton tiglium L. seeds stimulated the maximum glucose uptake. Ten species are reported to have antidiabetic activity for the first time. Flavonoids and triterpenoids are the dominant antidiabetic compounds in selected medicinal plants from Myanmar. Conclusions. P. zeylanica, L. cubeba, H. crenulate, M. gagei, C. teeta, and M. ferrea are worthy to advance further study according to their strong antidiabetic activities and limited research on effects in in vivo animal studies, unclear chemical constitutes, and safety.


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## Body

## 1. Introduction

Diabetes mellitus (DM) is a chronic metabolic disease and characterized by hyperglycemia that result from defects in insulin secretion, insulin action, or both [1]. Diabetes includes three main types, type I, type II, and gestational diabetes. Type II diabetes mellitus (T2DM) is the most common and accounts for over 90% of the total cases [2, 3]. T2DM has high morbidity and mortality worldwide and brings a large economic burden. Cardiovascular complications are a major cause of morbidity and mortality among people with diabetes [2]. The International Diabetes Federation reported that there were 463 million adults with diabetes in 2019, which have more than tripled over the past 20 years, and diabetes-related healthcare costs (USD 760 billion) are now estimated to account for 10% of the total healthcare budget (https://www.diabetesatlas.org/).Most patients with T2DM are overweight or obese. Pancreatic lipase is an important enzyme for digestion and absorption of dietary lipids, and lipase inhibition is the most widely studied for the discovery of potential antiobesity agents [4]. Monogenic and common forms of obesity initially cause insulin resistance [5]. Insulin resistance stimulates glucose production in the liver and attenuates glucose uptake in muscle and adipose tissue which is one of the important factors resulting in T2DM [2]. As the behavior of 3T3-L1 adipocytes is similar to primary adipocytes and mostly available muscle cell lines are not insulin sensitive in glucose transport, 3T3-L1 adipocytes have become the most preferred cell line to study insulin-stimulated glucose uptake and an excellent cell model to study insulin action and signaling [6].Currently, oral antidiabetic medications are mainly classified as biguanides, sulfonylureas, meglitinides, thiazolidinediones, dipeptidyl peptidase 4 (DPP-4) inhibitors, sodium-glucose cotransporter inhibitors, andα-glucosidase inhibitors [1]. These agents have adverse effects such as leading to hypoglycemia, cardiovascular, and metabolic alterations. People prefer medicinal plants for DM therapy due to low cost, minimal side effects, and inexpensive availability [1, 7]. In many parts of the world, traditional knowledge and biodiversity play an import role in health care, and many widely used plant-based medicines are derived from traditional knowledge [8]. However, only parts of medicinal plants have received scientific evaluation for their efficacy [7]. Thus, it is a challenge and an opportunity to search medicinal plants to treat diabetes and its complications.Myanmar, a tropical country, possesses a rich resource of medicinal plants, which is widely practiced by the majority of the population [9]. However, there are limited pharmacological studies on traditional medicines. Aumeeruddy et al. reviewed the ethnomedicinal plants for the management of diabetes worldwide but lack of information in Myanmar [10]. To explore the pharmacological application of antidiabetic properties of traditional plants in Myanmar and search for new antidiabetic agents, we investigated three main regions of the medicinal plants source and reported their current research status on antidiabetes. These species were screened for the antidiabetic activities by porcine pancreatic lipase (PPL) inhibition and glucose uptake in 3T3-L1 adipocyte assays of selected medicinal plants in Myanmar.


## 2. Materials and Methods

### 2.1. Investigation and Collection of Medicinal Plant Materials

This study was carried out in the markets and villages of three regions including Mandalay, Sagaing, and Shan State in 2015–2017. These places possess affluent medicinal plants according to our survey in Myanmar. Mandalay has the biggest market of medicinal plants, which is Zay Cho market. Shan is the biggest state and the most famous for its traditional medicines in Myanmar [11]. The local name and usage of medicinal plants were recorded in the field survey. Meanwhile, the voucher specimens and materials were collected for identification and further research, respectively. Among them, 93 samples of medicinal plants from Mandalay were screened for antibacterial and anti-T3SS activities in our previous research [12]. The plants were identified by Ms. Jun Yang and Mr. Yu Zhang from the Kunming Institute of Botany based on specimens. The plant names and families were confirmed and conducted at the Plant List (http://www.theplantlist.org).


### 2.2. A Review of Medicinal Plants in Antidiabetic Research

To make clear the status quo on antidiabetes of these medicinal plants, we performed a thorough literature review by using electronic databases (Web of Science, PubMed, Google Scholar, and SciFinder). Specific keywords include “scientific name of species” and “diabetes” until 2021. The used parts of the species were searched about their antidiabetes including traditional use,in vitro assay, in vivo animal studies, clinical trials, and antidiabetic constitutes. Other parts were continued to search if the collected parts did not report any antidiabetic properties, and other species of the same genus were continued to collect information if the species did not report any antidiabetes. We used rigorous inclusion and exclusion criteria to select antidiabetic compounds with IC50 ≤ 50 µM, or dose ≤50 µM in vitro, or dose ≤50 mg/kg in vivo, excluding compounds with name or structure inconsistent with the source. Here, we refer to the antidiabetic activity of compounds with IC50 ≤ 10 µM, dose ≤10 mg/kg in vivo, or dose ≤10 µM in vitro as high and from 11 to 50 as moderate.


### 2.3. Preparation of Plant Extracts

The dried different parts of the plants were powdered in a grinder and then extracted with 70% ethanol at 60°C once for 30 minutes. After filtration, the solvent was removed under reduced pressure at 60°C in a rotary evaporator. The extracts were stored at 4°C before analysis.


### 2.4. Porcine Pancreatic Lipase (PPL) Inhibition Assay

Lipase inhibition activity was conducted as described in literature with some modifications [13]. Briefly, p-NPB was used as a substrate. The reactions were carried out in a 96-well microtiter plate where 5 µL of lipase solution (40 U/mL) in Tris-HCl buffer (100 mM Tris-HCl, 5 mM CaCl2; pH 7.0) was used as enzyme buffer. Then, 1 µL of each extract dissolved in dimethyl sulfoxide (DMSO) (20 mg/mL) and 184 µL of the same Tris-HCl buffer were added and mixed with the enzyme buffer. After incubation at 37°C for 15 min, 10 µL of the substrate solution (10 mM p-NPB in DMSO) was added. Then, enzymatic reactions were performed for 15 min at 37°C. Lipase activity was determined by measuring the hydrolysis of p-NPB to p-nitrophenol at 400 nm using a microplate reader, 630 nm as a reference. Orlistat was used as a positive control. The inhibitory activity (I) was calculated according to the following formula:(1)I%=1−AB×100%.A is the optical density (OD) value of the sample. B is the OD value of negative control.The half-maximal inhibitory concentration (IC50) was calculated by Reed and Muench’s method.


### 2.5. Glucose Uptake in 3T3-L1 Adipocytes Assay

The differentiation of 3T3-L1 adipocytes and glucose uptake assay were carried out using a mild modified method described [14]. The differentiated 3T3-L1 adipocytes were seeded in 96-well plates and preincubated with the same way in reference. After that, they were incubated with 40 μg/mL samples in medium. Insulin (0.1 μmol/L) or berberine (10 μmol/L) was used as positive control. DMSO was added as blank control. After 24 h, 10 μL medium was taken to measure the glucose concentration by the glucose oxidase method. The experiments were repeated three times. Meanwhile, 20 μL MTS was added in the remaining medium of cells and incubated 2 h at 37°C. Then, the absorbance at 492 nm was measured to determine the cytotoxicity of extracts. The value of glucose uptake of the sample was calculated using these formulas:The glucose concentration (mmol/L) = sample absorbance/positive control absorbance × positive control concentration.Glucose uptake (% of difference) = (glucose concentration of blank wells − remaining glucose in the cell-plated wells)/glucose concentration of blank wells ×100%.


## 2.1. Investigation and Collection of Medicinal Plant Materials

This study was carried out in the markets and villages of three regions including Mandalay, Sagaing, and Shan State in 2015–2017. These places possess affluent medicinal plants according to our survey in Myanmar. Mandalay has the biggest market of medicinal plants, which is Zay Cho market. Shan is the biggest state and the most famous for its traditional medicines in Myanmar [11]. The local name and usage of medicinal plants were recorded in the field survey. Meanwhile, the voucher specimens and materials were collected for identification and further research, respectively. Among them, 93 samples of medicinal plants from Mandalay were screened for antibacterial and anti-T3SS activities in our previous research [12]. The plants were identified by Ms. Jun Yang and Mr. Yu Zhang from the Kunming Institute of Botany based on specimens. The plant names and families were confirmed and conducted at the Plant List (http://www.theplantlist.org).


## 2.2. A Review of Medicinal Plants in Antidiabetic Research

To make clear the status quo on antidiabetes of these medicinal plants, we performed a thorough literature review by using electronic databases (Web of Science, PubMed, Google Scholar, and SciFinder). Specific keywords include “scientific name of species” and “diabetes” until 2021. The used parts of the species were searched about their antidiabetes including traditional use,in vitro assay, in vivo animal studies, clinical trials, and antidiabetic constitutes. Other parts were continued to search if the collected parts did not report any antidiabetic properties, and other species of the same genus were continued to collect information if the species did not report any antidiabetes. We used rigorous inclusion and exclusion criteria to select antidiabetic compounds with IC50 ≤ 50 µM, or dose ≤50 µM in vitro, or dose ≤50 mg/kg in vivo, excluding compounds with name or structure inconsistent with the source. Here, we refer to the antidiabetic activity of compounds with IC50 ≤ 10 µM, dose ≤10 mg/kg in vivo, or dose ≤10 µM in vitro as high and from 11 to 50 as moderate.


## 2.3. Preparation of Plant Extracts

The dried different parts of the plants were powdered in a grinder and then extracted with 70% ethanol at 60°C once for 30 minutes. After filtration, the solvent was removed under reduced pressure at 60°C in a rotary evaporator. The extracts were stored at 4°C before analysis.


## 2.4. Porcine Pancreatic Lipase (PPL) Inhibition Assay

Lipase inhibition activity was conducted as described in literature with some modifications [13]. Briefly, p-NPB was used as a substrate. The reactions were carried out in a 96-well microtiter plate where 5 µL of lipase solution (40 U/mL) in Tris-HCl buffer (100 mM Tris-HCl, 5 mM CaCl2; pH 7.0) was used as enzyme buffer. Then, 1 µL of each extract dissolved in dimethyl sulfoxide (DMSO) (20 mg/mL) and 184 µL of the same Tris-HCl buffer were added and mixed with the enzyme buffer. After incubation at 37°C for 15 min, 10 µL of the substrate solution (10 mM p-NPB in DMSO) was added. Then, enzymatic reactions were performed for 15 min at 37°C. Lipase activity was determined by measuring the hydrolysis of p-NPB to p-nitrophenol at 400 nm using a microplate reader, 630 nm as a reference. Orlistat was used as a positive control. The inhibitory activity (I) was calculated according to the following formula:(1)I%=1−AB×100%.A is the optical density (OD) value of the sample. B is the OD value of negative control.The half-maximal inhibitory concentration (IC50) was calculated by Reed and Muench’s method.


## 2.5. Glucose Uptake in 3T3-L1 Adipocytes Assay

The differentiation of 3T3-L1 adipocytes and glucose uptake assay were carried out using a mild modified method described [14]. The differentiated 3T3-L1 adipocytes were seeded in 96-well plates and preincubated with the same way in reference. After that, they were incubated with 40 μg/mL samples in medium. Insulin (0.1 μmol/L) or berberine (10 μmol/L) was used as positive control. DMSO was added as blank control. After 24 h, 10 μL medium was taken to measure the glucose concentration by the glucose oxidase method. The experiments were repeated three times. Meanwhile, 20 μL MTS was added in the remaining medium of cells and incubated 2 h at 37°C. Then, the absorbance at 492 nm was measured to determine the cytotoxicity of extracts. The value of glucose uptake of the sample was calculated using these formulas:The glucose concentration (mmol/L) = sample absorbance/positive control absorbance × positive control concentration.Glucose uptake (% of difference) = (glucose concentration of blank wells − remaining glucose in the cell-plated wells)/glucose concentration of blank wells ×100%.


## 3. Results

### 3.1. PPL Inhibition Activity of Plant Extracts

Twenty-two plant extracts (100μg/mL) showed the PPL inhibition activity (Table 1 and Table S1). Among them, eight species were tested, with the IC50 values according to PPL inhibition rate higher than 50% except for Cuscuta sp. in Table 1. Cuscuta sp. showed the PPL inhibition activity with an IC50 value of 93.52 ± 1.38 μg/mL. Cuscuta sp. was not properly identified because of morphological characteristics without the flower or fruits. It was found in the same spot where C. reflexa was discovered. The ethanolic extracts of C. lindenii rhizomes and A. indica leaves possessed the highest lipase inhibitory activity with IC50 values of 6.86 ± 0.25 and 11.46 ± 0.06 μg/mL, respectively. The ethanolic extracts of P. zeylanica stems, G. glabra roots, C. pareira aerial parts, M. gagei woods, and C. reflexa whole plants showed moderate lipase inhibitory activity with IC50 values of 37−95 μg/mL. Fourteen species showed mild lipase inhibitory activity with 30–47% inhibition (Supplementary Materials, Table S1).Table 1
Ethnobotanical information and the PPL inhibition activity of seven species.
Scientific nameFamilyMyanmar nameCommon namePartaYieldb (%)Traditional use in MyanmarIC50 (μg/mL)Azadirachta indica A. Juss.MeliaceaeTamaNeemL12The decoction of leaves is orally taken for diabetes and antiseptic purpose. Leaves are externally used for skin diseases [9]11.46 ± 0.06Caladium lindenii (André) madisonAraceaeGamon-gyaIndian kaleRh21Undocumented6.86 ± 0.25Plumbago zeylanica L.PlumbaginaceaeKant-gyoke-phyuWhite leadwortSt11Whole plants are used for stimulating palate and digestion and treating diarrhea, gastric diseases, and herpes-like skin disorders [8]39.06 ± 1.11Glycyrrhiza glabra L.LeguminosaeThinbaw-nwechoLiquoriceR20Undocumented58.36 ± 6.58Cuscuta reflexa Roxb.ConvolvulaceaeShwe-nweDodderWP13Whole plants are used to treat irregularities of the blood. The boiling liquid of whole plants is used for inflammation and hardening of the liver. Whole plants are crushed and pasted for itches and rashes [8].78.83 ± 3.68Cissampelos pareira L.MenispermaceaeKywet-nabaungVelvet leafAP20Whole plants are pasted for inflammatory conditions of the eye. Leaves are used for cooling [8].87.71 ± 3.01Mansonia gagei J. R. Drumm.MalvaceaeKala-metBustard sandalwoodW4Woods and roots are used to eliminate phlegm, to treat heart diseases, urinary disorders, and anemia, and to alleviate itches [8]88.91 ± 0.96aL: leaves, AP: aerial parts, W: woods, St: stems, Rh: rhizomes, R: roots, and WP: whole plants. bYield (%) = (weight of extract/weight of dry sample)×100.


### 3.2. Glucose Uptake in 3T3-L1 Adipocytes of Plant Extracts

Seventeen extracts (40μg/mL) and one plant extract reduced from 40 μg/mL to 20 μg/mL as cytotoxicity to cells of 15 species stimulated glucose uptake in 3T3-L1 adipocytes (Figure 1). The ethnobotanical information and pharmacological properties can be found in Table 2. C. teeta rhizomes and C. tiglium seeds (oily and solid phase) stimulated the highest increase in glucose uptake. At concentration 40 μg/mL, their glucose uptakes were higher than positive controls which was insulin (0.1 μM) and berberine (10 μM). Mahonia sp. and L. cubeba fruits stimulated glucose uptake equivalent to berberine (10 μM); M. ferrea leaves were close to insulin (0.1 μM). The seed extracts of C. tiglium and N. sativa have oily and solid phase; the two phases were separated to screen the antidiabetic activities. Their oily phase of the extract showed a higher increase in glucose uptake. Mahonia sp. leaves significantly stimulated glucose uptake compared to the control group (Figure 1). The concentration was lowered to 20 μg/mL for glucose uptake test as the root extracts of H. crenulata showed cytotoxicity against adipocytes at 40 μg/mL. H. crenulata root significantly increased glucose uptake with the value of glucose uptake (21.81 ± 2.49) % at 20 μg/mL compared to the control group (9.31 ± 1.34) %; the value of insulin (0.1 M) and berberine glucose uptake (10M) were (28.95 ± 1.16) % and (25.72 ± 1.45) %. This information was not mentioned in Figure 1.Figure 1
The effects 17 extracts (40μg/mL) of 15 plant species on glucose uptake of 3T3-L1 adipocytes. Data is represented as percentage of glucose uptake, in comparison to the control group (DMSO) and positive control (insulin and berberine).Table 2
Ethnobotanical information and pharmacological properties of species with stimulating glucose uptake.
Scientific nameMyanmar nameFamilyPartaYield (%)Traditional use in MyanmarPharmacological propertiesCroton tiglium L.Kanakho, mai-hkangEuphorbiaceaeS (oily phase)9Oil can be used for stomach disorders, hypertension, fever, inflammation, infections, and diseases of the throat and ear [8]Antioxidant, antitumor, anti-HIV, anticonvulsant, gastrointestinal, anti-inflammatory, andα-amylase inhibitory activities [15, 16]Croton tiglium L.Kanakho, mai-hkangEuphorbiaceaeS (solid phase)7To stimulate appetite; correct imbalances in phlegm and gas; prevent jaundice, fainting, and facial paralysis; also taken as a laxative to rid the body of impurities [8]Antioxidant, antitumor, anti-HIV, anticonvulsant, gastrointestinal, anti-inflammatory, andα-amylase inhibitory activities [15, 16]Mansonia gagei J.R. Drumm.Kala-metMalvaceaeW4To eliminate phlegm; to treat heart diseases, urinary disorders, and anemia; to alleviate itches [8]Anticancer, antifungal, antioxidant, and larvicidal activities [17, 18]Litsea cubeba (lour.) pers.Thing tingLauraceaeFr21To treat stomachache and stomach ulcer; used for postpartum care [19]Antimicrobial, anti-inflammatory, antiasthmatic, anticholinergic, and antiplatelet aggregation activities [20]Valeriana jatamansi jonesKanpaluCaprifoliaceaeRh22Treating for diabetesAnalgesic, anti-inflammatory, myorelaxant and antispasmodic, psychotic, radioprotective, antimicrobial, hypnotic, anthelmintic, cytotoxic, and antioxidant activities [21]Ligusticum officinale (makino) kitag.Kantbalu-u-thaeApiaceaeRh12UndocumentedPancreatic lipase inhibitory, anti-inflammatory, anticancer, and antioxidant activities [22]Canscora diffusa (vahl) R.Br. ex roem. and schult.Kyauk-panGentianaceaeL19UndocumentedAntibacterial activities [23]Caladium lindenii (andré) madisonGamone-gyaAraceaeRh21UndocumentedNot reportedZingiber officinale roscoeGyinZingiberaceaeRh11Laryngitis, chest and respiratory ailments, infected sores, and inflammation caused by injury [8]Antidiabetic, antithrombotic, anti-inflammatory, analgesic, antioxidant, and antimicrobial activities [24]Nigella sativa L.Samon-netRanunculaceaeS (oily phase)3Carminative and galactagogue [8]Antimicrobial, antioxidant, antihyperlipidemic, anticancer, antidiabetic, gastroprotective, nephroprotective, and hepatoprotective activities [25]Nigella sativa L.Samon-netRanunculaceaeS (solid phase)12Used as a carminative and galactagogue [8]Antimicrobial, antioxidant, anti-inflammatory, antihyperlipidemic, anticancer, antidiabetic, cardiovascular protective, gastroprotective, nephroprotective, hepatoprotective, wound-healing activities, the effect on the reproductive system, and immunoprotective activity [25]Baliospermum solanifolium (Burm.) SureshHnat-cho, ya-wo-mo, ya-wo-poEuphorbiaceaeSt4Toothache, purgeRoots have anticancer, immunomodulatory, anti-inflammatory, antioxidant, and hepatoprotective activities [26]Pterocarpus indicus Willd.Pan-padaukLeguminosaeW5Dysentery, diarrheaBarks showedα-glucosidase inhibitory activity [27], antitumor and antibacterial activity, and antiallergic properties [28]Coptis teeta Wall.Khan taukRanunculaceaeR23To relieve constipation, regulate bowel movements, promote digestion, reduce fever, treat malaria, and increase vitality; roots soaked in liquor for malaria [8]Antipathogenic microorganism, antidiabetes, and anticancer activities; protective effects on the cardiovascular system [29]Mesua ferrea L.GuntgawCalophyllaceaeL12To treat snakebites [8]Analgesic and antibacterial activities [30]Hesperethusa crenulata (Roxb.) Roem.Sansph-ka, ThanakaRutaceaeR9Barks are used as a skincare [31].Barks showed antioxidant, anti-inflammatory, and tyrosinase inhibitory activities [31]; leaves showed tyrosinase inhibitory activity [32]Hesperethusa crenulata (Roxb.). Roem.Sansph-ka, ThanakaRutaceaeL5Barks are used as a skincare [31].Barks showed antioxidant, anti-inflammatory, and tyrosinase inhibitory activities [31]; leaves showed tyrosinase inhibitory activity [32]aS: seeds, L: leaves, R: roots, Rh: rhizomes, W: woods, WP: whole plants, St: stems, and Fr: fruits.


### 3.3. Research Progress on Antidiabetic Activities of Medicinal Plants

To make clear the antidiabetic medicines for further study, the research status of medicinal plants in antidiabetes was reported in Supplementary Materials (TableS2) from traditional use to clinical research including traditional use, in vitro assay, in vivo animal studies, and clinical trials. The antidiabetic evidence of relative plants can be found in the Supplement Materials (Table S3).In our research, extracts (100μg/mL) of 22 species showed the PPL inhibition activity; 17 extracts (40 μg/mL) and one plant extract reduced from 40 μg/mL to 20 μg/mL as cytotoxicity to cells of 15 species stimulated glucose uptake in 3T3-L1 adipocytes. Among them, Caladium lindenii (André) Madison, Mansonia gagei J.R. Drumm., and Mesua ferrea L. possessed both activities (Figure 2(a)). Crateva religiosa G. Forst., Antidesma acidum Retz., and Coptis teeta Wall. were the only reported traditional use for treating diabetes without any report on antidiabetic activity that accounted for 10% of research status (Figure 2(b)). Here, Hesperethusa crenulata (Roxb.) M. Roem., M. gagei, A. acidum, Eriobotrya bengalensis Kurz., C. lindenii, Ligusticum officinale (Makino) Kitag., Cajanus volubilis (King) Maesen, Canscora diffusa (Vahl) R.Br. ex Roem. and Schult., Canscora andrographioides Griff. ex C.B. Clarke, and Pterocarpus indicus Willd. were reported for their antidiabetic activities for the first time.Figure 2
(a) The species possessed the PPL inhibition activity and stimulated glucose uptake in 3T3-L1 adipocytes. (b) Research status of traditional plants showing antidiabetic activities in our study. (c) Research status of extracted antidiabetic plants.
(a)(b)(c)There are 82 species of 51 families and 14 species in the genus with antidiabetic properties so that they have high diversity (Supplementary Material,Tables S2 and S3). The Fabaceae family had the largest number of species showed antidiabetic activity (Figure 3). Among them, 7.3% species were already studied in type 2 diabetic patients, 46.9% species were researched antidiabetic activities in vivo, and 11.5% species were only recorded traditional use without any reported pharmacological activities on antidiabetes (Figure 2(c)).Figure 3
Top five plant families reported for antidiabetic properties by survey.


### 3.4. A Literature Review of Antidiabetic Compounds from Selected Medicinal Plants in Myanmar

Forty of the 82 species were reported antidiabetic components with various types including terpenes, alkaloids, flavonoids, lignans, and other types (FigureS1). Among them, flavonoids (28 compounds) and triterpenoids (26 compounds) are the dominant compounds accounting for 61.4%. Flavonoids were mainly reported α-glucosidase inhibition and PPAR-γ-ligand-binding activities. They are found in Oroxylum indicum, Eclipta prostrata, Nigella sativa, Glycyrrhiza glabra, Senna siamea, Boesenbergia rotunda, and Cinnamomum tamala. PPAR-γ-ligand-binding activities in phenolic compounds are affected by the slight differences of substitution groups on the aromatic rings [33].The primary targets of antidiabetic triterpenoids wereα-glucosidase inhibition and lipolytic activity in 3T3-L1 adipocytes. Triterpenoids were isolated from Abrus precatorius, Plumeria rubra, Pongamia pinnata, Entada phaseoloides, Eclipta prostrata, Lagerstroemia speciosa, Trigonella foenum-graecum, Centella asiatica, Brucea javanica. Lupane skeleton and a ketone at C-3 of triterpenes would be essential for exerting a potent PPA inhibition in A. precatorius [34]. Triterpenoid glycosides having glucopyranosyl moiety are favorable for inhibiting PTP1B enzyme from E. prostrata [35]. Bitter-taste receptors of quassinoids may be a target as to induce lipolytic activity, the length of acyl side chain at 15-OH is important for the lipolytic activity of B. javanica [36].


## 3.1. PPL Inhibition Activity of Plant Extracts

Twenty-two plant extracts (100μg/mL) showed the PPL inhibition activity (Table 1 and Table S1). Among them, eight species were tested, with the IC50 values according to PPL inhibition rate higher than 50% except for Cuscuta sp. in Table 1. Cuscuta sp. showed the PPL inhibition activity with an IC50 value of 93.52 ± 1.38 μg/mL. Cuscuta sp. was not properly identified because of morphological characteristics without the flower or fruits. It was found in the same spot where C. reflexa was discovered. The ethanolic extracts of C. lindenii rhizomes and A. indica leaves possessed the highest lipase inhibitory activity with IC50 values of 6.86 ± 0.25 and 11.46 ± 0.06 μg/mL, respectively. The ethanolic extracts of P. zeylanica stems, G. glabra roots, C. pareira aerial parts, M. gagei woods, and C. reflexa whole plants showed moderate lipase inhibitory activity with IC50 values of 37−95 μg/mL. Fourteen species showed mild lipase inhibitory activity with 30–47% inhibition (Supplementary Materials, Table S1).Table 1
Ethnobotanical information and the PPL inhibition activity of seven species.
Scientific nameFamilyMyanmar nameCommon namePartaYieldb (%)Traditional use in MyanmarIC50 (μg/mL)Azadirachta indica A. Juss.MeliaceaeTamaNeemL12The decoction of leaves is orally taken for diabetes and antiseptic purpose. Leaves are externally used for skin diseases [9]11.46 ± 0.06Caladium lindenii (André) madisonAraceaeGamon-gyaIndian kaleRh21Undocumented6.86 ± 0.25Plumbago zeylanica L.PlumbaginaceaeKant-gyoke-phyuWhite leadwortSt11Whole plants are used for stimulating palate and digestion and treating diarrhea, gastric diseases, and herpes-like skin disorders [8]39.06 ± 1.11Glycyrrhiza glabra L.LeguminosaeThinbaw-nwechoLiquoriceR20Undocumented58.36 ± 6.58Cuscuta reflexa Roxb.ConvolvulaceaeShwe-nweDodderWP13Whole plants are used to treat irregularities of the blood. The boiling liquid of whole plants is used for inflammation and hardening of the liver. Whole plants are crushed and pasted for itches and rashes [8].78.83 ± 3.68Cissampelos pareira L.MenispermaceaeKywet-nabaungVelvet leafAP20Whole plants are pasted for inflammatory conditions of the eye. Leaves are used for cooling [8].87.71 ± 3.01Mansonia gagei J. R. Drumm.MalvaceaeKala-metBustard sandalwoodW4Woods and roots are used to eliminate phlegm, to treat heart diseases, urinary disorders, and anemia, and to alleviate itches [8]88.91 ± 0.96aL: leaves, AP: aerial parts, W: woods, St: stems, Rh: rhizomes, R: roots, and WP: whole plants. bYield (%) = (weight of extract/weight of dry sample)×100.


## 3.2. Glucose Uptake in 3T3-L1 Adipocytes of Plant Extracts

Seventeen extracts (40μg/mL) and one plant extract reduced from 40 μg/mL to 20 μg/mL as cytotoxicity to cells of 15 species stimulated glucose uptake in 3T3-L1 adipocytes (Figure 1). The ethnobotanical information and pharmacological properties can be found in Table 2. C. teeta rhizomes and C. tiglium seeds (oily and solid phase) stimulated the highest increase in glucose uptake. At concentration 40 μg/mL, their glucose uptakes were higher than positive controls which was insulin (0.1 μM) and berberine (10 μM). Mahonia sp. and L. cubeba fruits stimulated glucose uptake equivalent to berberine (10 μM); M. ferrea leaves were close to insulin (0.1 μM). The seed extracts of C. tiglium and N. sativa have oily and solid phase; the two phases were separated to screen the antidiabetic activities. Their oily phase of the extract showed a higher increase in glucose uptake. Mahonia sp. leaves significantly stimulated glucose uptake compared to the control group (Figure 1). The concentration was lowered to 20 μg/mL for glucose uptake test as the root extracts of H. crenulata showed cytotoxicity against adipocytes at 40 μg/mL. H. crenulata root significantly increased glucose uptake with the value of glucose uptake (21.81 ± 2.49) % at 20 μg/mL compared to the control group (9.31 ± 1.34) %; the value of insulin (0.1 M) and berberine glucose uptake (10M) were (28.95 ± 1.16) % and (25.72 ± 1.45) %. This information was not mentioned in Figure 1.Figure 1
The effects 17 extracts (40μg/mL) of 15 plant species on glucose uptake of 3T3-L1 adipocytes. Data is represented as percentage of glucose uptake, in comparison to the control group (DMSO) and positive control (insulin and berberine).Table 2
Ethnobotanical information and pharmacological properties of species with stimulating glucose uptake.
Scientific nameMyanmar nameFamilyPartaYield (%)Traditional use in MyanmarPharmacological propertiesCroton tiglium L.Kanakho, mai-hkangEuphorbiaceaeS (oily phase)9Oil can be used for stomach disorders, hypertension, fever, inflammation, infections, and diseases of the throat and ear [8]Antioxidant, antitumor, anti-HIV, anticonvulsant, gastrointestinal, anti-inflammatory, andα-amylase inhibitory activities [15, 16]Croton tiglium L.Kanakho, mai-hkangEuphorbiaceaeS (solid phase)7To stimulate appetite; correct imbalances in phlegm and gas; prevent jaundice, fainting, and facial paralysis; also taken as a laxative to rid the body of impurities [8]Antioxidant, antitumor, anti-HIV, anticonvulsant, gastrointestinal, anti-inflammatory, andα-amylase inhibitory activities [15, 16]Mansonia gagei J.R. Drumm.Kala-metMalvaceaeW4To eliminate phlegm; to treat heart diseases, urinary disorders, and anemia; to alleviate itches [8]Anticancer, antifungal, antioxidant, and larvicidal activities [17, 18]Litsea cubeba (lour.) pers.Thing tingLauraceaeFr21To treat stomachache and stomach ulcer; used for postpartum care [19]Antimicrobial, anti-inflammatory, antiasthmatic, anticholinergic, and antiplatelet aggregation activities [20]Valeriana jatamansi jonesKanpaluCaprifoliaceaeRh22Treating for diabetesAnalgesic, anti-inflammatory, myorelaxant and antispasmodic, psychotic, radioprotective, antimicrobial, hypnotic, anthelmintic, cytotoxic, and antioxidant activities [21]Ligusticum officinale (makino) kitag.Kantbalu-u-thaeApiaceaeRh12UndocumentedPancreatic lipase inhibitory, anti-inflammatory, anticancer, and antioxidant activities [22]Canscora diffusa (vahl) R.Br. ex roem. and schult.Kyauk-panGentianaceaeL19UndocumentedAntibacterial activities [23]Caladium lindenii (andré) madisonGamone-gyaAraceaeRh21UndocumentedNot reportedZingiber officinale roscoeGyinZingiberaceaeRh11Laryngitis, chest and respiratory ailments, infected sores, and inflammation caused by injury [8]Antidiabetic, antithrombotic, anti-inflammatory, analgesic, antioxidant, and antimicrobial activities [24]Nigella sativa L.Samon-netRanunculaceaeS (oily phase)3Carminative and galactagogue [8]Antimicrobial, antioxidant, antihyperlipidemic, anticancer, antidiabetic, gastroprotective, nephroprotective, and hepatoprotective activities [25]Nigella sativa L.Samon-netRanunculaceaeS (solid phase)12Used as a carminative and galactagogue [8]Antimicrobial, antioxidant, anti-inflammatory, antihyperlipidemic, anticancer, antidiabetic, cardiovascular protective, gastroprotective, nephroprotective, hepatoprotective, wound-healing activities, the effect on the reproductive system, and immunoprotective activity [25]Baliospermum solanifolium (Burm.) SureshHnat-cho, ya-wo-mo, ya-wo-poEuphorbiaceaeSt4Toothache, purgeRoots have anticancer, immunomodulatory, anti-inflammatory, antioxidant, and hepatoprotective activities [26]Pterocarpus indicus Willd.Pan-padaukLeguminosaeW5Dysentery, diarrheaBarks showedα-glucosidase inhibitory activity [27], antitumor and antibacterial activity, and antiallergic properties [28]Coptis teeta Wall.Khan taukRanunculaceaeR23To relieve constipation, regulate bowel movements, promote digestion, reduce fever, treat malaria, and increase vitality; roots soaked in liquor for malaria [8]Antipathogenic microorganism, antidiabetes, and anticancer activities; protective effects on the cardiovascular system [29]Mesua ferrea L.GuntgawCalophyllaceaeL12To treat snakebites [8]Analgesic and antibacterial activities [30]Hesperethusa crenulata (Roxb.) Roem.Sansph-ka, ThanakaRutaceaeR9Barks are used as a skincare [31].Barks showed antioxidant, anti-inflammatory, and tyrosinase inhibitory activities [31]; leaves showed tyrosinase inhibitory activity [32]Hesperethusa crenulata (Roxb.). Roem.Sansph-ka, ThanakaRutaceaeL5Barks are used as a skincare [31].Barks showed antioxidant, anti-inflammatory, and tyrosinase inhibitory activities [31]; leaves showed tyrosinase inhibitory activity [32]aS: seeds, L: leaves, R: roots, Rh: rhizomes, W: woods, WP: whole plants, St: stems, and Fr: fruits.


## 3.3. Research Progress on Antidiabetic Activities of Medicinal Plants

To make clear the antidiabetic medicines for further study, the research status of medicinal plants in antidiabetes was reported in Supplementary Materials (TableS2) from traditional use to clinical research including traditional use, in vitro assay, in vivo animal studies, and clinical trials. The antidiabetic evidence of relative plants can be found in the Supplement Materials (Table S3).In our research, extracts (100μg/mL) of 22 species showed the PPL inhibition activity; 17 extracts (40 μg/mL) and one plant extract reduced from 40 μg/mL to 20 μg/mL as cytotoxicity to cells of 15 species stimulated glucose uptake in 3T3-L1 adipocytes. Among them, Caladium lindenii (André) Madison, Mansonia gagei J.R. Drumm., and Mesua ferrea L. possessed both activities (Figure 2(a)). Crateva religiosa G. Forst., Antidesma acidum Retz., and Coptis teeta Wall. were the only reported traditional use for treating diabetes without any report on antidiabetic activity that accounted for 10% of research status (Figure 2(b)). Here, Hesperethusa crenulata (Roxb.) M. Roem., M. gagei, A. acidum, Eriobotrya bengalensis Kurz., C. lindenii, Ligusticum officinale (Makino) Kitag., Cajanus volubilis (King) Maesen, Canscora diffusa (Vahl) R.Br. ex Roem. and Schult., Canscora andrographioides Griff. ex C.B. Clarke, and Pterocarpus indicus Willd. were reported for their antidiabetic activities for the first time.Figure 2
(a) The species possessed the PPL inhibition activity and stimulated glucose uptake in 3T3-L1 adipocytes. (b) Research status of traditional plants showing antidiabetic activities in our study. (c) Research status of extracted antidiabetic plants.
(a)(b)(c)There are 82 species of 51 families and 14 species in the genus with antidiabetic properties so that they have high diversity (Supplementary Material,Tables S2 and S3). The Fabaceae family had the largest number of species showed antidiabetic activity (Figure 3). Among them, 7.3% species were already studied in type 2 diabetic patients, 46.9% species were researched antidiabetic activities in vivo, and 11.5% species were only recorded traditional use without any reported pharmacological activities on antidiabetes (Figure 2(c)).Figure 3
Top five plant families reported for antidiabetic properties by survey.


## 3.4. A Literature Review of Antidiabetic Compounds from Selected Medicinal Plants in Myanmar

Forty of the 82 species were reported antidiabetic components with various types including terpenes, alkaloids, flavonoids, lignans, and other types (FigureS1). Among them, flavonoids (28 compounds) and triterpenoids (26 compounds) are the dominant compounds accounting for 61.4%. Flavonoids were mainly reported α-glucosidase inhibition and PPAR-γ-ligand-binding activities. They are found in Oroxylum indicum, Eclipta prostrata, Nigella sativa, Glycyrrhiza glabra, Senna siamea, Boesenbergia rotunda, and Cinnamomum tamala. PPAR-γ-ligand-binding activities in phenolic compounds are affected by the slight differences of substitution groups on the aromatic rings [33].The primary targets of antidiabetic triterpenoids wereα-glucosidase inhibition and lipolytic activity in 3T3-L1 adipocytes. Triterpenoids were isolated from Abrus precatorius, Plumeria rubra, Pongamia pinnata, Entada phaseoloides, Eclipta prostrata, Lagerstroemia speciosa, Trigonella foenum-graecum, Centella asiatica, Brucea javanica. Lupane skeleton and a ketone at C-3 of triterpenes would be essential for exerting a potent PPA inhibition in A. precatorius [34]. Triterpenoid glycosides having glucopyranosyl moiety are favorable for inhibiting PTP1B enzyme from E. prostrata [35]. Bitter-taste receptors of quassinoids may be a target as to induce lipolytic activity, the length of acyl side chain at 15-OH is important for the lipolytic activity of B. javanica [36].


## 4. Discussion

The potential species were discussed from pharmacological activities and safety extracts were with IC50 < 50 μg/mL in PPL inhibition, higher or closely positive in glucose uptake, or showed these two activities.H. crenulata is known as Thanaka, and the stem bark powder is used as a skincare regiment over one thousand years in Myanmar [31]. It is also a common tropical plant species in the Indian subcontinent and Southeast Asia with various medicinal properties, such as purgative, antidote, stomachic, and sudorific [31]. Coumarins and sitosterol were found in its root bark [37]. Extracts from Thanaka bark showed strong anti-inflammatory, significant antioxidation, and mild tyrosinase inhibition without detectable genotoxicity [31]. We reported the antidiabetic activities of its roots and barks for the first time.The heartwood ofM. gagei showed antibacterial, antifungal, and antioxidant activities [12, 17]. The chemical constitutes of M. gagei revealed antiestrogenic, antifungal, and antioxidant, and anticancer activities [17, 18]. Coumarins and mansonones are the main constituents and active ingredients in M. gagei [17, 18, 38, 39]. Its antidiabetic activities were reported for the first time which was proved with PPL inhibition and increase the glucose uptake to deserve further study about the activity in vivo and its safety.The tuber ofC. lindenii was used for Stingray wounds in Brazil [40]. The phytochemical investigation of Caladium indicated flavonoids, alkaloids, saponins, cardiac glycosides, carbohydrates, and deoxy sugars [41]. There is no reported research about the phytochemical and pharmacological research of C. lindenii. The corm of C. bicolor showed stimulation of glucose uptake in adipocytes and hepatoprotective activity in Hep G2 cells [42]. Methanol leaf extract of C. bicolor showed toxicity to the kidney but no adverse effect on the heart, lungs, spleen, liver, and brain [43].A. indica, known as neem, is a common traditional medicine for the treatment with diabetes mellitus in Africa and India [44, 45]. At 25, 48.4, 93.5, 180.9, and 350 mg/kg body weight, intraperitoneal and oral administration of aqueous leaf extract of A. indica decreased the blood glucose levels in alloxan-induced diabetic mice but not in a dose-related manner [44]. Ten ligands of neem possessed binding properties with T2DM protein enzyme target phosphoenolpyruvate carboxykinase; compound 3-deacetyl-3-cinnamoyl-azadirachtin showed the best binding [46]. Meliacinolin possessed α-glucosidase and α-amylase inhibition activities with IC50 46.7 and 32.2 µg/mL from chloroform extract of A. indica leaves. And orally administered meliacinolin (20 mg/kg body weight) could be able to revert a set of biochemical parameters of streptozotocin STZ-diabetic mice to respective normal values and its mechanism was deduced via its insulinogenic action [45]. Braga et al. reviewed the safety of A. indica extracts and/or isolated compounds and revealed nontoxicity or less toxicity when orally administered but had acute toxicity by intramuscular injection or via the intraperitoneal route [47].The roots and leaves ofP. zeylanica are traditionally used to treat diabetes in India and China [48, 49]. The ethanol extracts of P. zeylanica stem inhibited porcine pancreatic lipase activity with IC50 39.1 μg/mL (Table 1). Oral administration of ethanolic extract of P. zeylanica roots (100 and 200 mg/kg) decreased blood and urine glucose levels, increased hepatic hexokinase activity, and decreased hepatic glucose-6-phosphatase, serum acid phosphatase, alkaline phosphatase, and lactate dehydrogenase in streptozotocin STZ-diabetic rats [50]. Plumbagin is the major bioactive component for antidiabetic activity (15 and 30 mg/kg body weight) by reducing blood glucose and returning other biochemical parameters to normal and enhancing GLUT4 mRNA, protein expression in diabetic rats from P. zeylanica root. The antidiabetic effect is lower than glibenclamide (4 mg/kg) [49]. Oral administration of aqueous extract was found to be safe up to the dose of 2000 mg/kg during the 14 days of observation by acute toxicity profile experiment [51].The rhizome ofC. teeta along with C. chinensis and C. deltoidea is known as “coptidis rhizome” which has been widely used to treat bacillary dysentery, diabetes, pertussis, sore throat, aphtha, and eczema in China [29]. C. chinensis polysaccharide (200 mg/kg body weight) showed significant inhibition in fasting blood glucose level and triglycerides [52]. Alkaloids are the most abundant components and are considered as the main active ingredients [29]. The methanol extract of C. chinensis rhizome significantly inhibits adipocyte differentiation and lipid contents in 3T3-L1 cells. Five alkaloids, berberine, epiberberine, coptisine, palmatine, and magnoflorine were significantly inhibited lipid accumulation in 3T3-L1 cells without affecting cell viability and reduced the expression levels of several adipocyte marker genes including proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer-binding protein-α (C/EBP-α) from methanol extract of C. chinensis rhizome [53]. (3β)-Stigmast-5-en-3-ol stimulates glucose uptake by the PI3K-dependent pathway in L6 myotubes and activates GLUT 4 transport [54, 55]. Berberine was the main compound from the ethanol extract of C. teeta rhizome [54]. Lan et al. extracted 27 clinical trials (2569 patients) and revealed berberine with comparable therapeutic effects on T2DM, hyperlipidemia, and hypertension with no serious adverse reactions [56].C. tiglium seeds were traditionally used for stimulating appetite, imbalances in phlegm and gas, jaundice, fainting, and facial paralysis and taken as a laxative to rid the body of impurities in Myanmar. The seeds oil can be used for stomach disorders, hypertension, fever, inflammation, infections, and diseases of the throat and ear [8]. C. cajucara, C. macrostachys, and C. malambo were used to cure diabetes [57]. The extract of C. tiglium inhibited α-amylase with 55.1% at 80 μg/mL [16]. Oral medication can cause severe gastrointestinal syndrome and even mortality because of irritating oils and croton proteins [58]. Tigliane-type diterpenoids are the predominant secondary metabolite constituents in C. tiglium L. [59].The raw fruits ofL. cubeba were traditionally used to prevent and check for hyperglycemia in India. Its methanol extract inhibited α-amylase and α-glucosidase with IC50 values of 514.9 µg/mL and 1,435.7 µg/mL, respectively. Its ethanol extracts exhibited lower inhibition activity. Phenols and flavonoids were the major phytochemicals in different extracts against diabetes [60]. L. cubeba oil was proved with slightly toxic that the oral LD50, dermal LD50, and inhalation LC50 values were approximately 4,000 mg/kg of body weight [61].The buds ofM. ferrea displayed the mild inhibition on α-glucosidase and α-amylase activity with IC50 values of 128.8 µg/mL and 146.8 µg/mL, respectively [62, 63]. Stamens of M. ferrea produced a dose-dependent reduction in blood sugar (hypoglycemia) at a dose of 150 and 300 mg/kg body weight in alloxan-induced diabetic rats [64]. The methanol extract of M. ferrea flower has shown the antidiabetic activity in streptozotocin-induced diabetic rats and deduced the mechanism [65]. The extract of M. ferrea flower was proved no acute toxicity in an animal model but exhibited mild lymphocytic infiltration and hepatocyte degeneration [65, 66]. Xanthones and coumarins are predominantly secondary metabolites from Mesua species which display antitumor and antimicrobial, antioxidant, anti-inflammatory, or immunomodulating properties [67]. However, there is not any research reported related with antidiabetic activity of its leaves previously.


## 5. Conclusion

L. cubeba, H. crenulate, and M. gagei deserve further study because of the potential antidiabetic activity in in vitro assay and unclear active ingredients and mechanism along with research about antidiabetic effects in in vivo animal studies and unclarity in safety except for L. cubeba with slightly toxic activity. Moreover, C. teeta, P. zeylanica, and M. ferrea are also worth giving attention as the low toxicity and antidiabetic activities of these species or its genus level in research status. The antidiabetic activities and their constitutes of M. ferrea are studied in our research.



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