# Determination of the Chemical Composition, Antioxidant, and Enzyme Inhibitory Activity ofOnosma mollis DC

Authors: Arzuhan Sihoglu Tepe
Journal: Journal of Chemistry
 (2021)

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## Abstract

Onosma species have long been used traditionally for respiratory tract infections, abdominal pain, wound treatment, burns, and constipation. This study aims to investigate the chemical composition and in vitro antioxidant and enzyme inhibitory activities of ethyl acetate (EtOAc), methanol (MeOH), and water extracts of Onosma mollis DC. MeOH extract was richer in both phenolics and flavonoids than other extracts (44.06 mg GAEs/g and 41.57 mg QEs/g, respectively). The findings obtained from the results of the chromatographic analysis also supported the results of the spectrophotometric analysis. The MeOH extract was the richest in terms of most of the phytochemicals screened. Apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol were over 1000.0 μg/g in MeOH extract. The extract in question showed the highest activity in phosphomolybdenum, DPPH, and ABTS radical scavenging and CUPRAC and FRAP reducing power activity assays (2.01, 3.33, 2.30, 1.48, and 0.79 mg/ml, respectively). The water extract presented the highest activity in the ferrous ion chelating assay (1.01 mg/ml). While EtOAc extract showed high activity in acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and α-glucosidase inhibitory activity tests (1.11, 1.49, and 1.07 mg/ml, respectively), MeOH extract showed significant efficacy in tyrosinase and α-amylase inhibitory activity assays (2.94 and 2.08 mg/ml, respectively). There was a high correlation between the total phenolics/flavonoids of the extracts and their antioxidant activities (correlation coefficients were over 0.9). In addition, the phytochemicals mentioned above were found to contribute significantly to the antioxidant activity. It was concluded that a more detailed analysis should be done to determine the compounds responsible for the enzyme inhibitory activities of the extracts.


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## Body

## 1. Introduction

Plants and plant-based nutrients are an indispensable part of the human diet. Many researchers agree that, in addition to fruits and vegetables, many medicinal and aromatic plants that we consume in our daily lives serve as an additional source of natural antioxidants [1–4]. Studies have shown that plant-derived flavonoids, some alkaloids, phenolic compounds, some diterpenoids, and sulfur compounds have a high degree of antioxidant activity [4–6]. These compounds help protect our body from the harmful effects of these compounds by eliminating oxidant substances that occur due to both normal metabolic reactions and some environmental conditions (gamma, UV, and X-ray radiation, etc.). Therefore, plants continue to be of great interest as additional sources of antioxidant compounds [4, 5, 7–10].Alzheimer's disease (AD) is a critical neurodegenerative disorder affecting older people [11]. Various strategies have been developed to treat this health problem. Some of these strategies are targeting amyloid-beta (Aβ) aggregation and modulating enzymes responsible for metabolizing the amyloid precursor protein (APP) or tau phosphorylation [12]. The first pharmacological treatment method approved by the US Food and Drug Administration (FDA) in AD treatment is the use of cholinesterase (ChE) inhibitors. Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) hydrolyze acetylcholine (ACh), impairing signal transduction. This situation is considered among the fundamental reasons for the progression of AD [13]. Some ChE inhibitors such as galantamine, rivastigmine, donepezil, or tacrine have been shown to reduce symptoms in AD patients. However, these agents exhibit some unwanted side effects that have limited their use [14–16]. Therefore, researchers are screening plants for the discovery of new and alternative ChE inhibitors.Melanin is a heteropolymer synthesized by the catalytic activity of tyrosine in melanosomes. Researchers have reported that metal ions TRP-1 and TRP-2 play a critical role in melanin synthesis [17–20]. However, the most crucial task in the melanogenesis process is fulfilled by tyrosinase. The use of tyrosinase inhibitors to treat hyperpigmentation disorders due to excessive melanin synthesis is considered a critical treatment approach. Tyrosinase inhibitors are also frequently used in skin whitening applications and are extremely important for the cosmetic industry. Over the last decades, researchers have been conducting intensive research on plants to discover new and effective tyrosinase inhibitors [21–28].Diabetes is a severe disease that affects a large number of people worldwide [29, 30]. It is evaluated in two categories, Type 1 or Type 2. The leading cause of type 2 diabetes is the imbalance between the transfer of blood sugar to tissues and insulin secretion. Postprandial hyperglycemia is one of the most important reasons for Type 2 diabetes to develop [31]. In the treatment of Type 2, the plasma glucose level is tried to be kept under control. One of the ways to achieve this is to stimulate insulin secretion through medical intervention or diet [30, 32, 33]. Another solution is to slow down the digestion of starch and lower the glucose concentration, which is the end product of digestion. Thus, the amount of glucose absorbed from the small intestine is reduced [33]. This strategy is more practical and economical than controlling insulin secretion [30]. Digestive enzymes such as α-amylase and α-glucosidase must be inhibited to limit glucose production [34–36]. In addition to the biological activities documented above, the researchers also analyze the plants for their antidiabetic activity.Onosma, one of the important genera of Boraginaceae, consists of approximately 150 species distributed worldwide. Literature records show that only 10% of these species are reported in terms of their chemical constituents and biological activity potential. These reports indicate that Onosma species are rich in shikonins and onosmins. These species have long been used traditionally for respiratory tract infections, abdominal pain, wound treatment, burns, and constipation. Onosma species are also used to color foods, medicinal preparations, and wool because of the dye contained in the root [37]. According to literature data, it is known that some Onosma species (such as O. sieheana, O. bourgaei, O. brachytricha, O. hispidum, and O. heterophyllum) have antioxidant, anti-inflammatory, antinociceptive, enzyme inhibitory, hypoglycemic, antimicrobial, etc. activities [38–42]. There is also a report in the literature on the antioxidant activity of methanol (MeOH) extract from the aerial parts of O. mollis [43]. However, in the report in question, ethyl acetate (EtOAc) and water extracts, which include nonpolar compounds, were not evaluated, and the enzyme inhibitory activity of O. mollis was not included.This study aimed to determine the chemical composition, antioxidant, and enzyme inhibitor activities of ethyl acetate (EtOAc), methanol (MeOH), and water extracts obtained from the aerial parts ofOnosma mollis DC.


## 2. Experimental

### 2.1. Plant Material and Extract Preparation

Aerial parts ofO. mollis were collected from Yazihan-Hekimhan highway, Yazihan, Malatya, Turkey (1140 m., 38° 41’ 58”N38° 06’ 54”E). Dr. Olcay Ceylan (MuglaSitkiKocman University) performed the identification of the species.Aerial parts of the plants were used as the study material to obtain solvent extracts. Details of the extraction procedure can be found in the supplementary file.


### 2.2. Determination of the Phenolic Compositions of the Extracts

Details of the spectrophotometric and chromatographic methods are given in theSupplementary file [44, 45].


### 2.3. Biological Activity

The antioxidant [45–49] and enzyme inhibitory activity [41] tests are given in the Supplementary file.


### 2.4. Statistical Analysis

The details of the statistical analysis are presented in theSupplementary file.


## 2.1. Plant Material and Extract Preparation

Aerial parts ofO. mollis were collected from Yazihan-Hekimhan highway, Yazihan, Malatya, Turkey (1140 m., 38° 41’ 58”N38° 06’ 54”E). Dr. Olcay Ceylan (MuglaSitkiKocman University) performed the identification of the species.Aerial parts of the plants were used as the study material to obtain solvent extracts. Details of the extraction procedure can be found in the supplementary file.


## 2.2. Determination of the Phenolic Compositions of the Extracts

Details of the spectrophotometric and chromatographic methods are given in theSupplementary file [44, 45].


## 2.3. Biological Activity

The antioxidant [45–49] and enzyme inhibitory activity [41] tests are given in the Supplementary file.


## 2.4. Statistical Analysis

The details of the statistical analysis are presented in theSupplementary file.


## 3. Results

### 3.1. Chemical Composition of the Extracts

Table1 shows the yields of extracts from O. mollis. The yield of the water extract was found to be higher than the others (14.9%). The yields of MeOH and EtOAc extracts were 4.58 and 3.91%, respectively.Table 1
Total flavonoid and phenolic contents ofO. mollis extracts.
AssaysEtOAcMeOHWaterYield (%)3.914.5814.09Total flavonoids (mg QEs/g extracts)1.69 ± 0.35c41.57 ± 0.60a13.44 ± 0.76bTotal phenolics (mg GAEs/g extracts)20.43 ± 0.46b44.06 ± 3.48a21.80 ± 3.54bQEs: Quercetin equivalent. GAEs: Gallic acid equivalent. The different superscripts within the same row are different statistically at a 5% significance level.The phytochemical composition data of the extracts obtained by spectrophotometric method are also given in Table1. The total amount of phenolic compounds of the extracts was found to be higher than the total amount of flavonoid compounds. The total amounts of phenolic and flavonoid compounds of the MeOH extract were 44.06 mg GAEs/g and 41.57 mg QEs/g, respectively. The EtOAc extract was extremely poor in these compounds (20.43 mg GAEs g and 1.69 mg QEs/g, respectively).The results of the chromatographic analysis are presented in Table2. Apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol were found in MeOH extract at a concentration above 1000 μg/g. In addition, the MeOH extract was found to contain significant amounts of apigenin (679.43 μg/g), luteolin (581.55 μg/g), and 4-hydroxybenzoic acid (553.68 μg/g). On the other hand, vanillic acid and pinoresinol were higher concentrations in water extract than other phytochemicals (896.81 and 841.68 μg/g, respectively). Compared to the others, the EtOAc extract was the poorest in terms of phytochemicals screened. Apigenin 7-glucoside in this extract stood out with its concentration (752.01 μg/g) than other phytochemicals.Table 2
Concentration (µg/g extract) of selected phenolic compounds in O. mollis extracts.
CompoundEtOAcMeOHWaterApigenin 7-glucoside752.01 ± 2.56b22320.63 ± 302.76a27.96 ± 0.71bLuteolin 7-glucoside253.14 ± 1.09b15684.92 ± 77.23a243.7 ± 5.56bRosmarinic acid232.48 ± 9.98b13552.66 ± 144.22a16.18 ± 4.86bVanillic acid48.31 ± 3.41c1623.24 ± 79.44a896.81 ± 46.15bPinoresinol109.14 ± 2.69c1292.32 ± 36.50a841.68 ± 44.49bApigenin123.25 ± 19.25b679.43 ± 16.50a22.52 ± 2.36cLuteolin101.72 ± 5.28c581.55 ± 0.38a459.34 ± 5.47b4-hydroxybenzoic acid27.18 ± 2.47b553.68 ± 5.37a33.04 ± 0.59bFerulic acid12.1 ± 0.14c291.46 ± 2.73a34.15 ± 1.17bChlorogenic acid8.3 ± 0.17b277.63 ± 17.69aNdCaffeic acid1.53 ± 0.07b179.93 ± 0.54a0.66 ± 0.09bHyperoside5.52 ± 1.15b171.48 ± 1.65aNdVanillinnd152.12 ± 0.95Nd2,5-Dihydroxybenzoic acidnd129.41 ± 0.79a3.97 ± 0.33bSyringic acid6.59 ± 0.69b115.61 ± 1.41a115.54 ± 6.99aHesperidin9.75 ± 0.02b90.93 ± 0.49aNdp-Coumaric acid1.98 ± 0.12c68.44 ± 0.41a24.86 ± 0.04bProtocatechuic acid3.01 ± 0.08b65.74 ± 1.01a69.40 ± 2.05aSinapic acidnd37.84 ± 1.99Nd3-Hydroxybenzoic acidnd14.6 ± 0.95a2.40 ± 0.20bGallic acidnd4.21 ± 0.02Nd3,4-Dihydroxyphenylacetic acidndNd6.80 ± 0.69Quercetin2.98 ± 0.14NdNd(+)-CatechinndNdNdPyrocatecholndNdNd(-)-EpicatechinndNdNdVerbascosidendNdNdTaxifolinndNdNd2-Hydroxycinnamic acidndNdNdEriodictyolndNdNdKaempferolndNdNdThe different superscripts within the same row are different statistically at a 5% significance level. nd: Not detected.


### 3.2. Antioxidant Activity Potential of the Extracts

Table3 shows the antioxidant activities of the extracts. MeOH extract showed higher activity than others in all the tested assays except for the ferrous ion chelating activity. The activity data exhibited by the MeOH extract in phosphomolybdenum, DPPH and ABTS radical scavenging, CUPRAC, and FRAP tests were 2.01, 3.33, 2.30, 1.48, and 0.79 mg/ml, respectively. The water extract exhibited higher ferrous ion chelating activity test activity than other extracts (1.01 mg/ml). It was followed by MeOH and EtOAc extracts, respectively (2.40 and 2.58 mg/ml, respectively). None of the extracts exhibited as high antioxidant activity as trolox and EDTA used as positive control agents.Table 3
Antioxidant activity ofO. mollis extracts.
Antioxidant activityEtOAcMeOHWaterTroloxEDTAPhosphomolybdenum(EC50: mg/ml)3.31 ± 0.03c2.01 ± 0.22b3.28 ± 0.14c1.05 ± 0.02a—(mg TEs/g extracts)315.25 ± 3.33b521.22 ± 56.20a317.31 ± 13.31b——DPPH scavenging(IC50: mg/ml)95.732.55c3.33 ± 0.07ab5.78 ± 0.45b0.29 ± 0.01a—(mg TEs/g extracts)2.97 ± 0.07c77.66 ± 1.72a45.09 ± 3.43b——ABTS scavenging(IC50: mg/ml)>20.00c2.30 ± 0.06b2.75 ± 0.28b0.26 ± 0.03a—(mg TEs/g extracts)5.24 ± 0.06b117.79 ± 2.92a98.97 ± 10.08a——CUPRAC reducing(EC50: mg/ml)3.96 ± 0.08d1.48 ± 0.01b2.30 ± 0.14c0.24 ± 0.01a—(mg TEs/g extracts)67.93 ± 1.34c182.03 ± 0.19a117.03 ± 7.09b——FRAP reducing(EC50: mg/ml)2.61 ± 0.10d0.79 ± 0.01b1.44 ± 0.06c0.10 ± 0.01a—(mg TEs/g extracts)38.22 ± 1.48c125.80 ± 0.14a69.12 ± 2.89b——Ferrous ion chelating(IC50: mg/ml)2.58 ± 0.02c2.40 ± 0.18c1.01 ± 0.02b—0.06 ± 0.004a(mg EDTAEs/g extracts)21.11 ± 0.19b22.67 ± 1.71b53.72 ± 1.27a——TEs: trolox equivalent. EDTAEs: ethylene diamine tetra acetic acid (disodium salt) equivalent. The different superscripts within the same row are different statistically at a 5% significance level.


### 3.3. Enzyme Inhibitory Activity Potential of the Extracts

Table4 shows the AChE, BChE, tyrosinase, α-amylase, and α-glucosidase inhibitory activity data. The results were given as IC50 and the equivalent of galantamine, kojic acid, and acarbose. None of the extracts exhibited as high antioxidant activity as the positive control agents.Table 4
Enzyme inhibition activity ofO. mollis extracts.
Enzyme inhibitory activityEtOAcMeOHWaterGalantamineKojic acidAcarboseAChE inhibition(IC50: mg/mL)1.11 ± 0.03a1.41 ± 0.04a11.54 ± 1.08b0.004 ± 0.0003a——(mg GALAEs/g extracts)2.75 ± 0.06a2.16 ± 0.06b0.26 ± 0.03c———BChE inhibition(IC50: mg/mL)1.49 ± 0.01a6.72 ± 0.26b>20.00c0.006 ± 0.0001a——(mg GALAEs/g extracts)3.75 ± 0.04a0.83 ± 0.03b0.26 ± 0.01c———Tyrosinase inhibition(IC50: mg/mL)3.66 ± 0.01c2.94 ± 0.19b4.54 ± 0.34c—0.31 ± 0.01a—(mg KAEs/g extracts)73.34 ± 0.21b91.61 ± 5.98a59.26 ± 4.42c———α-Amylase inhibition(IC50: mg/mL)2.63 ± 0.01b2.08 ± 0.04b16.88 ± 0.30c——1.06 ± 0.03a(mgACEs/g extracts)399.44 ± 2.17b504.24 ± 9.45a59.80 ± 1.12c———α-Glucosidase inhibition(IC50: mg/mL)1.07 ± 0.01a1.55 ± 0.02b4.12 ± 0.07c——1.65 ± 0.07b(mgACEs/g extracts)1630.61 ± 10.78a1123.62 ± 15.90b423.06 ± 7.22c———GALAEs: galantamine equivalent. KAEs: Kojic acid equivalent. ACEs: Acarbose equivalent. The different superscripts within the same row are different statistically at a 5% significance level.The EtOAc extract exhibited 1.11 and 1.49 mg/ml activity in the AChE and BChE inhibitory activity tests, respectively. It was followed by the MeOH extract (1.41 and 6.72 mg/ml, respectively). The water extract exhibited relatively weak inhibitory activity (11.54 and >20.00 mg/ml, respectively). In the case of tyrosinase inhibitory assay, the MeOH extract showed the highest activity (2.94 mg/ml). It was followed by EtOAc and water extracts, respectively (3.66 and 4.54 mg/ml, respectively). In theα-amylase inhibitory activity test, the MeOH extract showed the highest activity (2.08 mg/ml), while the α-glucosidase inhibitory activity test resulted in the superiority of the EtOAc extract (1.07 mg/ml). The water extract showed weaker inhibition in both activity tests than the others (16.88 and 4.12 mg/ml, respectively).


### 3.4. Correlation Coefficients between the Parameters

The correlation between total phenolic/flavonoid data and activity tests is given in Table5. The same table also presents the correlation coefficients between biological activity parameters and phytochemicals found in high amounts in the extracts.Table 5
Correlations among phenolics and assays.
PhosphomolybdenumDPPHABTSCUPRACFRAPFerrous ion chelatingAChEBChETyrosinaseα-amylaseα-glucosidaseDPPH0.815——————————ABTS0.6140.956—————————CUPRAC0.8830.9880.902————————FRAP0.9110.9670.8540.991———————Ferrous ion chelating−0.4290.1180.398−0.036−0.127——————AChE0.282−0.295−0.557−0.146−0.059−0.979—————BChE−0.361−0.822−0.949−0.725−0.662−0.6590.786————Tyrosinase0.8980.4980.2290.6190.671−0.7670.6670.075———α-Amylase0.6520.153−0.1380.3020.385−0.9630.8970.4350.905——α-Glucosidase0.079−0.483−0.713−0.343−0.260−0.9240.9780.8950.5010.791—Total flavonoid0.9350.9510.8200.9850.998−0.1890.006−0.6130.7200.444−0.197Total phenolic0.9310.8420.6680.9150.933−0.4100.236−0.4020.8340.6350.042Apigenin 7-glucoside0.9730.8100.6040.8890.927−0.4870.319−0.3350.8920.7030.120Luteolin 7-glucoside0.9720.8260.6260.9010.937−0.4620.292−0.3610.8790.6830.093Rosmarinic acid0.9730.8180.6160.8950.932−0.4740.305−0.3480.8860.6920.106Vanillic acid0.8090.9940.9420.9870.9760.083−0.270−0.8040.4990.182−0.457Pinoresinol0.7770.9950.9650.9710.9530.178−0.355−0.8560.4340.090−0.538Apigenin0.9540.7360.5100.8310.877−0.5840.423−0.2250.9240.7790.233Luteolin0.6780.9730.9860.9310.8960.311−0.482−0.9170.305−0.049−0.647The correlation coefficients between the total phenolics/flavonoids of the extracts and the results obtained from the phosphomolybdenum, DPPH radical scavenging, CUPRAC, and FRAP assays were over 0.9. According to the data in the table, there was also a high correlation between the antioxidant activities of the extracts with apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, pinoresinol, and apigenin. However, apart from the correlation between apigenin and tyrosinase inhibitory activity (0.924), no significant correlation was found between the phytochemicals in question and enzyme inhibitory activity.


## 3.1. Chemical Composition of the Extracts

Table1 shows the yields of extracts from O. mollis. The yield of the water extract was found to be higher than the others (14.9%). The yields of MeOH and EtOAc extracts were 4.58 and 3.91%, respectively.Table 1
Total flavonoid and phenolic contents ofO. mollis extracts.
AssaysEtOAcMeOHWaterYield (%)3.914.5814.09Total flavonoids (mg QEs/g extracts)1.69 ± 0.35c41.57 ± 0.60a13.44 ± 0.76bTotal phenolics (mg GAEs/g extracts)20.43 ± 0.46b44.06 ± 3.48a21.80 ± 3.54bQEs: Quercetin equivalent. GAEs: Gallic acid equivalent. The different superscripts within the same row are different statistically at a 5% significance level.The phytochemical composition data of the extracts obtained by spectrophotometric method are also given in Table1. The total amount of phenolic compounds of the extracts was found to be higher than the total amount of flavonoid compounds. The total amounts of phenolic and flavonoid compounds of the MeOH extract were 44.06 mg GAEs/g and 41.57 mg QEs/g, respectively. The EtOAc extract was extremely poor in these compounds (20.43 mg GAEs g and 1.69 mg QEs/g, respectively).The results of the chromatographic analysis are presented in Table2. Apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol were found in MeOH extract at a concentration above 1000 μg/g. In addition, the MeOH extract was found to contain significant amounts of apigenin (679.43 μg/g), luteolin (581.55 μg/g), and 4-hydroxybenzoic acid (553.68 μg/g). On the other hand, vanillic acid and pinoresinol were higher concentrations in water extract than other phytochemicals (896.81 and 841.68 μg/g, respectively). Compared to the others, the EtOAc extract was the poorest in terms of phytochemicals screened. Apigenin 7-glucoside in this extract stood out with its concentration (752.01 μg/g) than other phytochemicals.Table 2
Concentration (µg/g extract) of selected phenolic compounds in O. mollis extracts.
CompoundEtOAcMeOHWaterApigenin 7-glucoside752.01 ± 2.56b22320.63 ± 302.76a27.96 ± 0.71bLuteolin 7-glucoside253.14 ± 1.09b15684.92 ± 77.23a243.7 ± 5.56bRosmarinic acid232.48 ± 9.98b13552.66 ± 144.22a16.18 ± 4.86bVanillic acid48.31 ± 3.41c1623.24 ± 79.44a896.81 ± 46.15bPinoresinol109.14 ± 2.69c1292.32 ± 36.50a841.68 ± 44.49bApigenin123.25 ± 19.25b679.43 ± 16.50a22.52 ± 2.36cLuteolin101.72 ± 5.28c581.55 ± 0.38a459.34 ± 5.47b4-hydroxybenzoic acid27.18 ± 2.47b553.68 ± 5.37a33.04 ± 0.59bFerulic acid12.1 ± 0.14c291.46 ± 2.73a34.15 ± 1.17bChlorogenic acid8.3 ± 0.17b277.63 ± 17.69aNdCaffeic acid1.53 ± 0.07b179.93 ± 0.54a0.66 ± 0.09bHyperoside5.52 ± 1.15b171.48 ± 1.65aNdVanillinnd152.12 ± 0.95Nd2,5-Dihydroxybenzoic acidnd129.41 ± 0.79a3.97 ± 0.33bSyringic acid6.59 ± 0.69b115.61 ± 1.41a115.54 ± 6.99aHesperidin9.75 ± 0.02b90.93 ± 0.49aNdp-Coumaric acid1.98 ± 0.12c68.44 ± 0.41a24.86 ± 0.04bProtocatechuic acid3.01 ± 0.08b65.74 ± 1.01a69.40 ± 2.05aSinapic acidnd37.84 ± 1.99Nd3-Hydroxybenzoic acidnd14.6 ± 0.95a2.40 ± 0.20bGallic acidnd4.21 ± 0.02Nd3,4-Dihydroxyphenylacetic acidndNd6.80 ± 0.69Quercetin2.98 ± 0.14NdNd(+)-CatechinndNdNdPyrocatecholndNdNd(-)-EpicatechinndNdNdVerbascosidendNdNdTaxifolinndNdNd2-Hydroxycinnamic acidndNdNdEriodictyolndNdNdKaempferolndNdNdThe different superscripts within the same row are different statistically at a 5% significance level. nd: Not detected.


## 3.2. Antioxidant Activity Potential of the Extracts

Table3 shows the antioxidant activities of the extracts. MeOH extract showed higher activity than others in all the tested assays except for the ferrous ion chelating activity. The activity data exhibited by the MeOH extract in phosphomolybdenum, DPPH and ABTS radical scavenging, CUPRAC, and FRAP tests were 2.01, 3.33, 2.30, 1.48, and 0.79 mg/ml, respectively. The water extract exhibited higher ferrous ion chelating activity test activity than other extracts (1.01 mg/ml). It was followed by MeOH and EtOAc extracts, respectively (2.40 and 2.58 mg/ml, respectively). None of the extracts exhibited as high antioxidant activity as trolox and EDTA used as positive control agents.Table 3
Antioxidant activity ofO. mollis extracts.
Antioxidant activityEtOAcMeOHWaterTroloxEDTAPhosphomolybdenum(EC50: mg/ml)3.31 ± 0.03c2.01 ± 0.22b3.28 ± 0.14c1.05 ± 0.02a—(mg TEs/g extracts)315.25 ± 3.33b521.22 ± 56.20a317.31 ± 13.31b——DPPH scavenging(IC50: mg/ml)95.732.55c3.33 ± 0.07ab5.78 ± 0.45b0.29 ± 0.01a—(mg TEs/g extracts)2.97 ± 0.07c77.66 ± 1.72a45.09 ± 3.43b——ABTS scavenging(IC50: mg/ml)>20.00c2.30 ± 0.06b2.75 ± 0.28b0.26 ± 0.03a—(mg TEs/g extracts)5.24 ± 0.06b117.79 ± 2.92a98.97 ± 10.08a——CUPRAC reducing(EC50: mg/ml)3.96 ± 0.08d1.48 ± 0.01b2.30 ± 0.14c0.24 ± 0.01a—(mg TEs/g extracts)67.93 ± 1.34c182.03 ± 0.19a117.03 ± 7.09b——FRAP reducing(EC50: mg/ml)2.61 ± 0.10d0.79 ± 0.01b1.44 ± 0.06c0.10 ± 0.01a—(mg TEs/g extracts)38.22 ± 1.48c125.80 ± 0.14a69.12 ± 2.89b——Ferrous ion chelating(IC50: mg/ml)2.58 ± 0.02c2.40 ± 0.18c1.01 ± 0.02b—0.06 ± 0.004a(mg EDTAEs/g extracts)21.11 ± 0.19b22.67 ± 1.71b53.72 ± 1.27a——TEs: trolox equivalent. EDTAEs: ethylene diamine tetra acetic acid (disodium salt) equivalent. The different superscripts within the same row are different statistically at a 5% significance level.


## 3.3. Enzyme Inhibitory Activity Potential of the Extracts

Table4 shows the AChE, BChE, tyrosinase, α-amylase, and α-glucosidase inhibitory activity data. The results were given as IC50 and the equivalent of galantamine, kojic acid, and acarbose. None of the extracts exhibited as high antioxidant activity as the positive control agents.Table 4
Enzyme inhibition activity ofO. mollis extracts.
Enzyme inhibitory activityEtOAcMeOHWaterGalantamineKojic acidAcarboseAChE inhibition(IC50: mg/mL)1.11 ± 0.03a1.41 ± 0.04a11.54 ± 1.08b0.004 ± 0.0003a——(mg GALAEs/g extracts)2.75 ± 0.06a2.16 ± 0.06b0.26 ± 0.03c———BChE inhibition(IC50: mg/mL)1.49 ± 0.01a6.72 ± 0.26b>20.00c0.006 ± 0.0001a——(mg GALAEs/g extracts)3.75 ± 0.04a0.83 ± 0.03b0.26 ± 0.01c———Tyrosinase inhibition(IC50: mg/mL)3.66 ± 0.01c2.94 ± 0.19b4.54 ± 0.34c—0.31 ± 0.01a—(mg KAEs/g extracts)73.34 ± 0.21b91.61 ± 5.98a59.26 ± 4.42c———α-Amylase inhibition(IC50: mg/mL)2.63 ± 0.01b2.08 ± 0.04b16.88 ± 0.30c——1.06 ± 0.03a(mgACEs/g extracts)399.44 ± 2.17b504.24 ± 9.45a59.80 ± 1.12c———α-Glucosidase inhibition(IC50: mg/mL)1.07 ± 0.01a1.55 ± 0.02b4.12 ± 0.07c——1.65 ± 0.07b(mgACEs/g extracts)1630.61 ± 10.78a1123.62 ± 15.90b423.06 ± 7.22c———GALAEs: galantamine equivalent. KAEs: Kojic acid equivalent. ACEs: Acarbose equivalent. The different superscripts within the same row are different statistically at a 5% significance level.The EtOAc extract exhibited 1.11 and 1.49 mg/ml activity in the AChE and BChE inhibitory activity tests, respectively. It was followed by the MeOH extract (1.41 and 6.72 mg/ml, respectively). The water extract exhibited relatively weak inhibitory activity (11.54 and >20.00 mg/ml, respectively). In the case of tyrosinase inhibitory assay, the MeOH extract showed the highest activity (2.94 mg/ml). It was followed by EtOAc and water extracts, respectively (3.66 and 4.54 mg/ml, respectively). In theα-amylase inhibitory activity test, the MeOH extract showed the highest activity (2.08 mg/ml), while the α-glucosidase inhibitory activity test resulted in the superiority of the EtOAc extract (1.07 mg/ml). The water extract showed weaker inhibition in both activity tests than the others (16.88 and 4.12 mg/ml, respectively).


## 3.4. Correlation Coefficients between the Parameters

The correlation between total phenolic/flavonoid data and activity tests is given in Table5. The same table also presents the correlation coefficients between biological activity parameters and phytochemicals found in high amounts in the extracts.Table 5
Correlations among phenolics and assays.
PhosphomolybdenumDPPHABTSCUPRACFRAPFerrous ion chelatingAChEBChETyrosinaseα-amylaseα-glucosidaseDPPH0.815——————————ABTS0.6140.956—————————CUPRAC0.8830.9880.902————————FRAP0.9110.9670.8540.991———————Ferrous ion chelating−0.4290.1180.398−0.036−0.127——————AChE0.282−0.295−0.557−0.146−0.059−0.979—————BChE−0.361−0.822−0.949−0.725−0.662−0.6590.786————Tyrosinase0.8980.4980.2290.6190.671−0.7670.6670.075———α-Amylase0.6520.153−0.1380.3020.385−0.9630.8970.4350.905——α-Glucosidase0.079−0.483−0.713−0.343−0.260−0.9240.9780.8950.5010.791—Total flavonoid0.9350.9510.8200.9850.998−0.1890.006−0.6130.7200.444−0.197Total phenolic0.9310.8420.6680.9150.933−0.4100.236−0.4020.8340.6350.042Apigenin 7-glucoside0.9730.8100.6040.8890.927−0.4870.319−0.3350.8920.7030.120Luteolin 7-glucoside0.9720.8260.6260.9010.937−0.4620.292−0.3610.8790.6830.093Rosmarinic acid0.9730.8180.6160.8950.932−0.4740.305−0.3480.8860.6920.106Vanillic acid0.8090.9940.9420.9870.9760.083−0.270−0.8040.4990.182−0.457Pinoresinol0.7770.9950.9650.9710.9530.178−0.355−0.8560.4340.090−0.538Apigenin0.9540.7360.5100.8310.877−0.5840.423−0.2250.9240.7790.233Luteolin0.6780.9730.9860.9310.8960.311−0.482−0.9170.305−0.049−0.647The correlation coefficients between the total phenolics/flavonoids of the extracts and the results obtained from the phosphomolybdenum, DPPH radical scavenging, CUPRAC, and FRAP assays were over 0.9. According to the data in the table, there was also a high correlation between the antioxidant activities of the extracts with apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, pinoresinol, and apigenin. However, apart from the correlation between apigenin and tyrosinase inhibitory activity (0.924), no significant correlation was found between the phytochemicals in question and enzyme inhibitory activity.


## 4. Discussion

According to the literature research, it was determined that the chemical composition ofO. mollis was investigated previously by both spectrophotometric and chromatographic methods [43]. The amounts of phenolic and flavonoid compounds reported in this report were considerably lower than those obtained from the current study. In addition, all of the phytochemicals found in high amounts in the present study were higher than those in the report of Tlili et al. [43]. Although the same plant species were analyzed in both studies, it was seen that the plant species analyzed in these two studies were collected from different altitudes. This situation is thought to cause a difference in climatic conditions and, therefore, in chemical composition.In many studies on the chemical composition of Onosma species, apigenin 7-glucoside [50–53], luteolin7-glucoside [43, 50–53], rosmarinic acid [43, 50–55], vanillic acid [40, 50, 53], and pinoresinol [50–52] were found to be present in high amounts. Therefore, it was understood that the data obtained from the present study support the literature data.The antioxidant activity ofO. mollis was previously reported by Tlili et al. [43]. As in the section above, where chemical composition data were discussed, the antioxidant activity data obtained from the present study were higher than the data in the literature. Tlili et al. [43] reported the antioxidant activity of O. mollis in phosphomolybdenum, DPPH, and ABTS radical scavenging, CUPRAC and FRAP reducing activity, and ferrous ion chelating activity tests as 3.95, 14.22, 8.73, 4.38, 2.53, and 1.95 mg/ml, respectively. The main reason for the difference between the data obtained from the current study and the data published by Tlili et al. [43] is that the polarity gradient of the extracts in the current study is wider. Unlike the previous study, EtOAc and water extracts were also isolated in the current study. While nonpolar phytochemicals are soluble in EtOAc extract, those with high polarity dissolve in water extract. Therefore, these phytochemicals exhibit different antioxidant activity than those with moderate polarity (MeOH extract).Many researchers agree that there is a significant relationship between the chemical composition of plants and their antioxidant activities [56–58]. Some reports in the literature showed apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol, which were found in high concentrations in the current study, may contribute to antioxidant activity [50–53, 59]. These literature data support the data obtained from the current study.Considering the data in Table2, it is thought that apigenin 7-glucoside in EtOAc extract may contribute to cholinesterase inhibitory activity. There are no reports in the literature that this phytochemical has cholinesterase inhibitory activity. However, some reports indicate that some Onosma species containing high amounts of apigenin 7-glucoside exhibit significant cholinesterase inhibitory activity [50–53]. Therefore, it is considered that both the AChE and BChE inhibitory activities of the compound in question should be evaluated in future studies.As can be seen from the section, where the chemical compositions of the extracts were discussed, the MeOH extract was found to be rich in apigenin 7-glucoside, luteolin 7-glucoside, rosmarinic acid, vanillic acid, and pinoresinol. Thus, these phytochemicals were thought to be possible compounds contributing to activity. To have more convincing information about the tyrosinase inhibitory activity potential of these compounds, it is helpful to look at some literature data. A study conducted by Aghraz et al. [60] reported that Bubonic imbricatum and Cladanthus arabicus exhibited significant tyrosinase inhibitory activity, and both aglycon and glycosidic forms of apigenin were among the main components. In another study conducted by Sarikurkcu et al. [50], the MeOH extract of O. ambigens exhibited higher tyrosinase inhibitory activity than other extracts and was rich in apigenin 7-glucoside and luteolin 7-glucoside. There are also some reports in the literature that plant extracts rich in rosmarinic acid [61–63], vanillic acid [64, 65], and pinoresinol [66] show significant tyrosinase inhibitory activity.It is thought that the superiority of the MeOH extract in theα-amylase inhibitor activity test may be due to the compounds indicated in Table 1 and also expressed in the sections above. There are some reports in the literature that these compounds themselves or extracts rich in these compounds have high α-amylase inhibitor activities. In a study conducted by Witkowska-Banaszczak et al. [67], it was reported that Succisa pratensis rich in luteolin and apigenin in glycosidic forms exhibited significant α-amylase inhibitory activity. In another study carried out by Uysal et al. [61], it was reported that the α-amylase inhibitor activity of Salvia ceratophylla might be due to luteolin and rosmarinic acid. In a study in which the α-amylase inhibitory activities of different Brassica oleracea subspecies collected from north-central Italy were analyzed, it was reported that vanillic acid could be one of the compounds contributing to the activity [68]. However, in a study conducted by Mwakalukwa et al. [69], it was reported that the α-amylase and α-glucosidase inhibitory activity of pinoresinol was weak. There is also evidence in the literature that vanillic acid exhibits more than 40% inhibitory activity on glucosidase [70]. This finding is consistent with the data obtained from the current study.To the best of our knowledge, AChE, BChE, andα-glucosidase inhibitory activity of O. mollis have not previously been reported elsewhere. Therefore, data presented on these parameters could be assumed as the first report for the literature.


## 5. Conclusion

In this study, thein vitro antioxidant and enzyme inhibitory activities of the extracts obtained from the aerial parts of O. mollis were analyzed. It was concluded that the extracts have antioxidant activity comparable to positive control agents, and therefore, they could be assumed as one of the sources rich in antioxidants. In addition, it was concluded that MeOH and EtOAc extracts exhibited α-amylase and α-glucosidase inhibitory activity comparable to acarbose and, therefore, could be the source of new and alternative compounds in the treatment of diabetes. However, it is thought that more detailed analysis should be carried out to determine the phytochemicals responsible for the activity in these extracts.



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*Source: 5405365-2021-08-07.xml*