# Coumarins and Polar Constituents fromEupatorium triplinerve and Evaluation of Their α-Glucosidase Inhibitory Activity

Authors: Do Thi Viet Huong; Phan Minh Giang; Vu Minh Trang
Journal: Journal of Chemistry
 (2020)

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## Abstract

In our study of antidiabetic compounds from the leaves ofEupatorium triplinerve Vahl. (Asteraceae), ten compounds were isolated from the methanol leaf extract. They were determined to be β-sitosterol (1), stigmasterol (2), β-sitosterol 3-O-β-D-glucopyranoside (3), ayapanin (4), ayapin (5), thymoquinol 5-O-β-D-glucopyranoside (6), thyrsifloside (8), (E)-4-methoxymelilotoside (9), and kaempferol 3,7-di-O-β-D-glucopyranoside (10) by using ESI-MS, 1D (1H-, 13C-, DEPT) and 2D NMR (HSQC, HMBC, and NOESY) techniques. This is the first report of water-soluble compounds from E. triplinerve and compounds 6–10 were isolated for the first time from E. triplinerve. NMR profiling and HPLC analysis are fast and reliable methods to screen phytochemicals in plant samples. Due to their high concentrations in the leaf extracts of E. triplinerve, coumarins 4 and 5 could be fast screened by NMR profiling and RP-HPLC-PDA analysis. In the in vitro test for α-glucosidase inhibition of compounds 4–9, compounds 4, 5, and 7 showed the enzymatic inhibition of 40%, 46%, and 81%, respectively, at 256 μg/mL. An IC50 value of 58.65 ± 1.20 μg/mL (302 μM) was calculated for compound 7 which is lower than that of the positive control acarbose (IC50 197.33 ± 2.51 μg/mL; 306 μM).


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## Body

## 1. Introduction

The genusEupatorium (family Asteraceae) is a taxonomically complex group of species distributed mainly in Europe, eastern Asia, and North America. Studies on Eupatorium species have revealed diversity of secondary metabolites such as sesquiterpene lactones, flavonoids, diterpenes, benzofurans, pyrrolizidine alkaloids, chromenes, and thymol derivatives [1]. A number of Eupatorium species are employed in traditional medicine in the treatment of different pathologies and as a consequence bioactive natural compounds with cytotoxic, anti-inflammatory, antifungal, and antibacterial activities have been reported from the species. E. triplinerve Vahl. (syn. E. ayapana Vent., Ayapana triplinervis (Vahl.) R.M. King and H. Robinson) is a perennial and aromatic herb, 35–100 cm tall of the family Asteraceae. The plant was originated in the area from northern Brazil to Suriname and was introduced, cultivated, and naturalized long ago in some Caribbean islands, Africa, India, and South-East Asian countries. The leaves of E. triplinerve are used in folk medicine of India and South-East Asian countries as a heart stimulant, laxative, anticoagulant, stimulant, and tonic and for the treatment of yellow fever [2]. In Vietnam, the plant is popularly known as “Bả dột” and the leaves are used to heal wounds, snake bites, and stop bleeding. People also prepare tea from the twigs and leaves [3]. A number of studies investigated essential oils from different parts of E. triplinerve, showing variation of chemotypes such as 2,5-dimethoxy-p-cymene [4], β-caryophyllene [4], selina-4(15),7(11)-dien-8-one [5], and 2-tert-butyl-1,4-methoxybenzene [6]. The leaves emit a distinct coumarin odour, and the isolation of ayapanin (or herniarin, 7-methoxycoumarin) and ayapin (or 6,7-methylenedioxycoumarin) was reported. Ayapanin and ayapin showed toxicity to cancer cells including multidrug resistant cancer cells [6], haemostatic properties [7], blood coagulation, and phytoalexin activity to inhibit or destroy the invading bacteria, insects, and viruses [8, 9]. Ayapanin reduced the number of abdominal constrictions in mice and decreased the time spent in paw licking and biting response in formalin assay [10]. Kumala and Sapitri reported toxicity test using brine shrimp lethality method that showed all of the fractions of methanol extract had toxicity (LC50 < 1000 µg/mL). The water extract had no toxicity. The highest LC50 was for the ethyl acetate (24,42 µg/mL) and the lowest LC50 was for the n-hexane (238,66 µg/mL) extracts [11]. Both ayapanin and ayapin are nontoxic and are effective when applied locally or when administered by subcutaneous injections or by mouth. They have no effect on respiration or on blood pressure [12]. In addition, ayapanin did not show antigenotoxic effects on human lymphocyte DNA damage using single-cell gel electrophoresis [13]. Therefore the reason for the toxicity of the n-hexane and ethyl acetate extracts is complex and may be related to minor nonpolar or volatile compounds which may be produced by plants either constitutively or in response to different biotic or abiotic stresses [14]. The high contents in E. triplinerve enable ayapanin and ayapin to be used as starting materials for pharmacological investigations involving coumarins such as antidiabetic activity [15], affinity to cannabinoid receptors (cannabimimetic ligands) [16] or cognitive enhancing activity through inhibiting oxidative stress and brain inflammation [17, 18]. Till now, there has not been any report on the quantification of the two biologically active coumarins from the leaves of E. triplinerve. In this study, the principles 4 and 5 were analysed in the leaves of E. triplinerve by using proton NMR profiling and HPLC-PDA analysis.Type II diabetes is a chronic metabolite disease caused mainly by excess levels of blood glucose and is a major cause of premature death, blindness, kidney failure, heart attack, stroke, and lower limb amputation [19]. Type II diabetes is characterized by hyperglycaemia as a consequence of insulin resistance and affects over 90% of patients diagnosed with this disease. Oral hypoglycemic medications currently used in the treatment of type II diabetes include sulfonylureas, meglitinides, biguanides, thiazolidinediones, α-glucosidase inhibitors, and dipeptidyl peptidase-IV (DPP-IV) inhibitors. The antidiabetic drugs such as metformin, pioglitazone, thiazolidinedione, and acarbose decrease hepatic glucose output and reduce starch digestibility. Due to the side effects of these agents such as severe hypoglycemia, weight gain, and gastrointestinal disturbances medicinal plants with antidiabetic properties have been investigated for finding safer and cost-effective antidiabetic drugs [20]. Coumarins and cinnamic acid derivatives have been reported as antidiabetic agents [15, 21]. Currently, the inhibition of starch-hydrolysing enzymes such as α-amylase and α-glucosidase are one of the approaches to reduce hyperglycemia by retarding glucose uptake. In the present study, coumarins 4 and 5, thymoquinol glucoside 6, and o-hydroxycinnamic acid derivatives 7–9 were isolated from the leaves of E. triplinerve collected in Vietnam and evaluated for the enzymatic inhibitory activity against α-glucosidase.


## 2. Materials and Methods

### 2.1. General Procedures

Electron-spray ionization-mass spectrometry (ESI-MS) spectra were measured on an Agilent 1100 LC-MSD-Trap-SL system (Agilent Technologies, USA).1H-NMR, 13C-NMR, and DEPT spectra were recorded in CDCl3 using a Bruker Avance 500 NMR spectrometer. The chemical shifts are expressed in ppm relative to tetramethylsilane (TMS) as an internal standard. The 1H-NMR spectrum was recorded with spectrometer frequency (SF) 500.20 MHz. The 13C-NMR spectrum was recorded with SF 125.13 MHz. High-performance liquid chromatography (HPLC) analysis was carried out with a Shimadzu 20A HPLC system (Shimadzu corporation, Japan) equipped with a LC-20AD pump, SIL-20AC auto-sampler, DGU-20A5R degassing unit, SPD-M20A photodiode array (PDA) detector, and CBM-20A controller system. Silica gel 40–63 μm (Merck, Germany), reversed-phase C-18 silica gel 40–63 μm (Merck, Germany), and highly porous Diaion HP-20 (Mitsubishi, Japan) were used for column chromatography (CC). Precoated silica gel Merck 60 F254 aluminum plates were used for thin-layer chromatography (TLC).


### 2.2. Plant Material

The leaves ofE. triplinerve Vahl. were collected in Nghe An province, Vietnam, in July 2016. The plant material was taxonomically identified by Dr. Nguyen Thi Kim Thanh, Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi. A voucher sample (ET-616) was deposited at the same institution.


### 2.3. Extraction and Isolation of Compounds1–10

The fresh leaves (7.5 kg) were air-dried in the shade, then oven-dried at 45°C, and ground into powder. The dry powder (2 kg) was extracted with MeOH at room temperature for 3 days, and the extraction was repeated for three times. The extracts obtained after filtration were concentrated under reduced pressure to yield a methanol extract. The methanol extract was suspended in water and extracted successively withn-hexane and dichloromethane to give the corresponding n-hexane- (86 g) and dichloromethane-soluble (50 g) fractions after in vacuo evaporation. Separation of a part of the n-hexane-soluble fraction (17.2 g) by CC over silica gel with gradient elution of n-hexane-acetone 90 : 1, 49 : 1, 19 : 1, 9 : 1, 3 : 1, 1 : 1, 1 : 2 gave 9 fractions. Fractions 4 (0.62 g) and 5 (0.91 g) were purified by CC over silica gel, eluting with n-hexane-EtOAc 12 : 1, 9 : 1, 7 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1 to give a mixture of 1 and 2 (110 mg and 38.9 mg, respectively). Fraction 6 (6.1 g) was separated by CC over silica gel, eluting with CH2Cl2-MeOH 15 : 1 to give 3 (7 mg) and 4 (109 mg). Fraction 9 (1 g) was separated by CC over silica gel, eluting with n-hexane-EtOAc 7 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2 to give 5 (5.3 mg). A part of the dichloromethane-soluble fraction (1.1 g) was washed with n-hexane and then acetone to give 5 (45 mg). The residue was separated by CC over RP-18, eluting with 70% MeOH-H2O. The fraction eluting with 70% MeOH-H2O (882.5 mg) was separated by CC over silica gel, eluting with CH2Cl2-acetone 90 : 1, 49 : 1, 25 : 1, 15 : 1 to give 4 (13.5 mg). The water phase was concentrated and passed through a Diaion HP-20 column eluting with MeOH-H2O 2 : 3, 3 : 2 and MeOH to give three corresponding fractions 1–3. Fr. 1 (6.6 g) was separated by silica gel CC, eluting with EtOAc-MeOH 30 : 1, 25 : 1, 15 : 1, 12 : 1, 9 : 1, 6 : 1, 3 : 1 to give four subfractions 1–4. Subfr. 1 was separated by silica gel CC, eluting with CH2Cl2-MeOH 20 : 1, 15 : 1, 10 : 1, 7 : 1, 5, 3 : 1, 3 : 1 and purified by Sephadex LH-20, eluting with MeOH to give 6 (5 mg). Fr. 2 (17.2 g) was separated by silica gel CC, eluting with CH2Cl2-MeOH 60 : 1, 50 : 1, 40 : 1, 30 : 1 to give 2 subfractions 1 and 2. Subfr. 1 was purified by silica gel CC, eluting with CH2Cl2-EtOAc 45 : 1, 40 : 1, 30 : 1, 25 : 1, 20 : 1 to give 7 (5 mg). Subfr. 2 (3 g) was separated by CC over Sephadex LH-20, eluting with MeOH to give 8 (5 mg), a mixture of 8/9 (20 mg), and 10 (5 mg).


### 2.4. NMR Profiling of the Methanol or Boiling Water Leaf Extracts

The dried leaf powder (240 g) was extracted with methanol at room temperature for three days or with boiling water for 24 h. The extracts were separately filtered and evaporated to dryness under reduced pressure to give the corresponding methanol or water extract. A small part of the methanol or water extract (30 mg) was dissolved in pure methanol and extracted using a Lichrolut® RP-18 SPE cartridge (Merck, 40–60 μm) with a solvent gradient of 70%, 80%, and 100% MeOH-H2O. The fractions eluting with 70% MeOH-H2O were concentrated under reduced pressure to afford two NMR samples. 5 mg of each sample was dissolved in CD3OD and directly analysed by 1H-NMR spectroscopy at 500 MHz frequency.


### 2.5. HPLC Analysis of the Methanol Leaf Extract

The dried leaf powder (80 g) was extracted with MeOH at room temperature for 1 day. The solution was filtered and then evaporated to dryness under reduced pressure. The methanol leaf extract (220 mg) was dissolved with 2 mL acetonitrile of HPLC grade (Merck, Germany). The reversed-phase solid-phase extraction (RP-SPE) of the solution eluting with MeOH of HPLC grade (Merck, Germany) was performed to remove impurities. The sample was filtered through a Millipore 0.45μm membrane filter. Two standard solutions (100 μg/mL) were prepared by dissolving compounds 4 and 5 in MeOH of HPLC grade (Merck, Germany). The calibration curves were constructed by plotting the peak areas versus the corresponding concentrations (expressed as μg/mL) of each standard. A Shimadzu 20A HPLC system equipped with a photodiode array detector (PDA) was used for the qualitative and quantitative analysis of samples. An analytical column C-18 (4.6 mm × 250 mm, 5 µm), a gradient mobile phase of 44% acetonitrile-H2O (15 min.), a flow rate of 1 mL/min., an injection volume of 10 µL, and column temperature 25°C were used for the HPLC analysis. The UV detection wavelength was set at 306 nm. All the measurements were performed in triplicate.


### 2.6.α-Glucosidase Inhibitory Activity of Compounds 4–9 and 11

The inhibition ofα-glucosidase activity was carried out following the method described by Li et al. [22]. A 2.5 mM solution of p-NPG (p-nitrophenyl α-D-glucopyranoside) (Sigma-Aldrich) and 0.2 U/mL α-glucosidase from Saccharomyces cerevisiae (Sigma-Aldrich) were prepared in 100 mM potassium phosphate buffer pH 6.8. Compounds 4–9 and compound 10 previously isolated from the leaves of E. japonicum [23] were prepared in dimethylsulfoxide (DMSO) (Sigma-Aldrich) and serially diluted in the concentrations of 1, 4, 16, 64, and 256 μg/mL. 10 μL of sample was added to a reaction mixture consisting of 40 μL of 100 mM phosphate buffer pH 6.8 and 25 μL of 0.2 U/mL α-glucosidase in a 96-well microplate and the reaction mixture was incubated for 10 min at 37°C. Then 25 μL of 2.5 mM p-NPG was added and the reaction mixture was further incubated for 20 min at 37°C. After 30 min, 100 mM sodium carbonate solution (100 µL) was added to stop reaction. The absorbance of the mixture was measured at λ 410 nm on an UV-VIS spectrophotometer (Biotek Instruments, USA). To make a control reaction, the tested sample was replaced by 10 µL of 100 mM phosphate buffer (pH 6.8). Acarbose was used as the reference standard. The experiments were repeated three times. The α-glucosidase inhibitory activity was calculated using the following equation, where Acontrol is the absorbance of the control, Asample is the absorbance of the sample: α-glucosidase inhibitory activity (%) = (Acontrol−Asample)/Acontrol × 100. IC50 (half maximal inhibitory concentration) was calculated using Tablecurve software. All analyses were performed in triplicate and data were reported as mean ± SEM.


## 2.1. General Procedures

Electron-spray ionization-mass spectrometry (ESI-MS) spectra were measured on an Agilent 1100 LC-MSD-Trap-SL system (Agilent Technologies, USA).1H-NMR, 13C-NMR, and DEPT spectra were recorded in CDCl3 using a Bruker Avance 500 NMR spectrometer. The chemical shifts are expressed in ppm relative to tetramethylsilane (TMS) as an internal standard. The 1H-NMR spectrum was recorded with spectrometer frequency (SF) 500.20 MHz. The 13C-NMR spectrum was recorded with SF 125.13 MHz. High-performance liquid chromatography (HPLC) analysis was carried out with a Shimadzu 20A HPLC system (Shimadzu corporation, Japan) equipped with a LC-20AD pump, SIL-20AC auto-sampler, DGU-20A5R degassing unit, SPD-M20A photodiode array (PDA) detector, and CBM-20A controller system. Silica gel 40–63 μm (Merck, Germany), reversed-phase C-18 silica gel 40–63 μm (Merck, Germany), and highly porous Diaion HP-20 (Mitsubishi, Japan) were used for column chromatography (CC). Precoated silica gel Merck 60 F254 aluminum plates were used for thin-layer chromatography (TLC).


## 2.2. Plant Material

The leaves ofE. triplinerve Vahl. were collected in Nghe An province, Vietnam, in July 2016. The plant material was taxonomically identified by Dr. Nguyen Thi Kim Thanh, Faculty of Biology, VNU University of Science, Vietnam National University, Hanoi. A voucher sample (ET-616) was deposited at the same institution.


## 2.3. Extraction and Isolation of Compounds1–10

The fresh leaves (7.5 kg) were air-dried in the shade, then oven-dried at 45°C, and ground into powder. The dry powder (2 kg) was extracted with MeOH at room temperature for 3 days, and the extraction was repeated for three times. The extracts obtained after filtration were concentrated under reduced pressure to yield a methanol extract. The methanol extract was suspended in water and extracted successively withn-hexane and dichloromethane to give the corresponding n-hexane- (86 g) and dichloromethane-soluble (50 g) fractions after in vacuo evaporation. Separation of a part of the n-hexane-soluble fraction (17.2 g) by CC over silica gel with gradient elution of n-hexane-acetone 90 : 1, 49 : 1, 19 : 1, 9 : 1, 3 : 1, 1 : 1, 1 : 2 gave 9 fractions. Fractions 4 (0.62 g) and 5 (0.91 g) were purified by CC over silica gel, eluting with n-hexane-EtOAc 12 : 1, 9 : 1, 7 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1 to give a mixture of 1 and 2 (110 mg and 38.9 mg, respectively). Fraction 6 (6.1 g) was separated by CC over silica gel, eluting with CH2Cl2-MeOH 15 : 1 to give 3 (7 mg) and 4 (109 mg). Fraction 9 (1 g) was separated by CC over silica gel, eluting with n-hexane-EtOAc 7 : 1, 5 : 1, 3 : 1, 2 : 1, 1 : 1, 1 : 2 to give 5 (5.3 mg). A part of the dichloromethane-soluble fraction (1.1 g) was washed with n-hexane and then acetone to give 5 (45 mg). The residue was separated by CC over RP-18, eluting with 70% MeOH-H2O. The fraction eluting with 70% MeOH-H2O (882.5 mg) was separated by CC over silica gel, eluting with CH2Cl2-acetone 90 : 1, 49 : 1, 25 : 1, 15 : 1 to give 4 (13.5 mg). The water phase was concentrated and passed through a Diaion HP-20 column eluting with MeOH-H2O 2 : 3, 3 : 2 and MeOH to give three corresponding fractions 1–3. Fr. 1 (6.6 g) was separated by silica gel CC, eluting with EtOAc-MeOH 30 : 1, 25 : 1, 15 : 1, 12 : 1, 9 : 1, 6 : 1, 3 : 1 to give four subfractions 1–4. Subfr. 1 was separated by silica gel CC, eluting with CH2Cl2-MeOH 20 : 1, 15 : 1, 10 : 1, 7 : 1, 5, 3 : 1, 3 : 1 and purified by Sephadex LH-20, eluting with MeOH to give 6 (5 mg). Fr. 2 (17.2 g) was separated by silica gel CC, eluting with CH2Cl2-MeOH 60 : 1, 50 : 1, 40 : 1, 30 : 1 to give 2 subfractions 1 and 2. Subfr. 1 was purified by silica gel CC, eluting with CH2Cl2-EtOAc 45 : 1, 40 : 1, 30 : 1, 25 : 1, 20 : 1 to give 7 (5 mg). Subfr. 2 (3 g) was separated by CC over Sephadex LH-20, eluting with MeOH to give 8 (5 mg), a mixture of 8/9 (20 mg), and 10 (5 mg).


## 2.4. NMR Profiling of the Methanol or Boiling Water Leaf Extracts

The dried leaf powder (240 g) was extracted with methanol at room temperature for three days or with boiling water for 24 h. The extracts were separately filtered and evaporated to dryness under reduced pressure to give the corresponding methanol or water extract. A small part of the methanol or water extract (30 mg) was dissolved in pure methanol and extracted using a Lichrolut® RP-18 SPE cartridge (Merck, 40–60 μm) with a solvent gradient of 70%, 80%, and 100% MeOH-H2O. The fractions eluting with 70% MeOH-H2O were concentrated under reduced pressure to afford two NMR samples. 5 mg of each sample was dissolved in CD3OD and directly analysed by 1H-NMR spectroscopy at 500 MHz frequency.


## 2.5. HPLC Analysis of the Methanol Leaf Extract

The dried leaf powder (80 g) was extracted with MeOH at room temperature for 1 day. The solution was filtered and then evaporated to dryness under reduced pressure. The methanol leaf extract (220 mg) was dissolved with 2 mL acetonitrile of HPLC grade (Merck, Germany). The reversed-phase solid-phase extraction (RP-SPE) of the solution eluting with MeOH of HPLC grade (Merck, Germany) was performed to remove impurities. The sample was filtered through a Millipore 0.45μm membrane filter. Two standard solutions (100 μg/mL) were prepared by dissolving compounds 4 and 5 in MeOH of HPLC grade (Merck, Germany). The calibration curves were constructed by plotting the peak areas versus the corresponding concentrations (expressed as μg/mL) of each standard. A Shimadzu 20A HPLC system equipped with a photodiode array detector (PDA) was used for the qualitative and quantitative analysis of samples. An analytical column C-18 (4.6 mm × 250 mm, 5 µm), a gradient mobile phase of 44% acetonitrile-H2O (15 min.), a flow rate of 1 mL/min., an injection volume of 10 µL, and column temperature 25°C were used for the HPLC analysis. The UV detection wavelength was set at 306 nm. All the measurements were performed in triplicate.


## 2.6.α-Glucosidase Inhibitory Activity of Compounds 4–9 and 11

The inhibition ofα-glucosidase activity was carried out following the method described by Li et al. [22]. A 2.5 mM solution of p-NPG (p-nitrophenyl α-D-glucopyranoside) (Sigma-Aldrich) and 0.2 U/mL α-glucosidase from Saccharomyces cerevisiae (Sigma-Aldrich) were prepared in 100 mM potassium phosphate buffer pH 6.8. Compounds 4–9 and compound 10 previously isolated from the leaves of E. japonicum [23] were prepared in dimethylsulfoxide (DMSO) (Sigma-Aldrich) and serially diluted in the concentrations of 1, 4, 16, 64, and 256 μg/mL. 10 μL of sample was added to a reaction mixture consisting of 40 μL of 100 mM phosphate buffer pH 6.8 and 25 μL of 0.2 U/mL α-glucosidase in a 96-well microplate and the reaction mixture was incubated for 10 min at 37°C. Then 25 μL of 2.5 mM p-NPG was added and the reaction mixture was further incubated for 20 min at 37°C. After 30 min, 100 mM sodium carbonate solution (100 µL) was added to stop reaction. The absorbance of the mixture was measured at λ 410 nm on an UV-VIS spectrophotometer (Biotek Instruments, USA). To make a control reaction, the tested sample was replaced by 10 µL of 100 mM phosphate buffer (pH 6.8). Acarbose was used as the reference standard. The experiments were repeated three times. The α-glucosidase inhibitory activity was calculated using the following equation, where Acontrol is the absorbance of the control, Asample is the absorbance of the sample: α-glucosidase inhibitory activity (%) = (Acontrol−Asample)/Acontrol × 100. IC50 (half maximal inhibitory concentration) was calculated using Tablecurve software. All analyses were performed in triplicate and data were reported as mean ± SEM.


## 3. Results and Discussion

### 3.1. Isolation and MS/NMR Elucidation of Compounds

The dried powdered leaves ofE. triplinerve were extracted with MeOH at room temperature to yield a methanol extract. The methanol extract was fractionated into n-hexane-, dichloromethane-, and water-soluble fractions. Separation of the n-hexane-, dichloromethane-, and water-soluble fractions by repeated CC over different adsorbents such as silica gel, Diaion HP-20, Sephadex LH-20, and RP-18 using stepwise gradient elution gave phytosterols 1–3, coumarins 4 and 5, thymoquinol glucoside 6, o-hydroxycinnamic acid derivatives 7–9, and kaempferol diglucoside 10. Structures of the isolated compounds were determined as β-sitosterol (1) [24], stigmasterol (2) [24], β-sitosterol 3-O-β-D-glucopyranoside (3) [25], ayapanin (4) [26, 27], ayapin (5) [28], thymoquinol 5-O-β-D-glucopyranoside (6) [29], (E)-4-hydroxy-2-methoxycinnamic acid (7) [30, 31], thyrsifloside (8) [32], (E)-4-methoxymelilotoside (9) [33], and kaempferol 3,7-di-O-β-D-glucopyranoside (10) [34] (Figure 1) by comparing their spectroscopic data (MS, 1H-, and 13C-NMR) (Figures S1–S30) with the reported literature values. Compound 8 was isolated in its pure form and compound 9 was isolated in a nonseparable mixture of 8 and 9 (molar ratio 1 : 1 as determined by 1H-NMR intensity). The locations of the substituents of compounds 6, 8, and 9 were assigned by using heteronuclear single-quantum correlation (HSQC), heteronuclear multibond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY) spectra (Figure 2). In compound 6, key HMBC correlations were observed between CH3-7 (δH 2.15) and C-1 (δC 123.3), C-2 (δC 151.8), between H-8 (δH 3.5) and C-3 (δC 113.1), C-5 (δH 149.2), and between Glc-1 (δH 4.72) and C-5 (δC 149.2). In compound 8, key HMBC correlations for the assignment of the glucopyranosyl moiety were observed between H-7 (δH 8.13) and C-2 (δC 154.0), between H-6 (δH 7.13) and C-2, and between Glc-1 (δH 4.85) and C-2. In compound 9, key HMBC correlations for the assignment of the glucopyranosyl moiety and the methoxy group were observed between H-6 (δH 7.57) and C-2 (δC 159.0), between 4-OCH3 (δH 3.87) and C-4 (δC 164.4), and between Glc-1 (δH 4.98) and C-2. Further confirmation was obtained from the NOESY spatial interactions between H-5 (δH 6.67) and 4-OCH3, between 4-OCH3 and H-3 (δH 6.87), and between H-3 and Glc-1. The 1H-NMR spectra of 4 and 5 were also recorded in different NMR solvents (CDCl3, CD3OD, and DMSO-d6) and the signals were assigned to ease NMR identification of the compounds in different NMR solvents. Compounds 6–10 were isolated for the first time from E. triplinerve. The isolation of compounds has not showed the presence of alkaloids, saponins, triterpenoids, depsides, and depsidones which were detected in the previous phytochemical screening by using color reactions [35]. However, high concentration of coumarins and the presence of sterols in the leaves are in line with most of the phytochemical screenings [35]. In the genus Eupatorium, derivatives of 2-O-hydroxycinnamic acid and 2-O-glucopyranosylcinnamic acid were isolated from E. micranthum, while thymols were reported in E. fortunei [1]. Compounds 8 and 9 are glycosidic precursors of compounds 5 and 4, respectively. Under enzymatic conditions, they can be converted into nonglycosidic derivatives 2-hydroxy-6,7-methylenedioxy-trans-cinnamic acid (8a) and 2-hydroxy-4-methoxy-trans-cinnamic acid (9a), respectively. Compound 4 would be formed from 9a and compound 5 from 8a via photoisomerization of the trans double bond to cis and subsequent ring closure to the lactones (Figure 3) [30].Figure 1
Structures of compounds1–10 from E. triplinerve and 11 from E. japonicum.Figure 2
HMBC (⟶) and NOESY (—) correlations of compounds6, 8, and 9.Figure 3
Plausible biosynthesis of4 from 9 and 5 from 8 in E. triplinerve.


### 3.2.α-Glucosidase Inhibitory Activity

Type II diabetes mellitus is as metabolic disease mainly caused by the accumulation of excess sugar in the body. The enzymeα-glucosidase breaks down large starch polysaccharides into monosaccharides or disaccharides. Thus, inhibiting the enzymatic activity of α-glucosidase may reduce absorption of glucose in the body. In the in vitro α-glucosidase inhibitory activity test, the substrate p-nitrophenyl α-D-glucopyranoside (pNPG) is hydrolyzed by α-glucosidase to release p-nitrophenyl which can be monitored at 405 nm. The results are expressed as percentage inhibition (%) and the concentration of an inhibitor required to inhibit 50% of enzyme activity (IC50) is determined [36]. The inhibitory activity of compounds isolated from E. triplinerve against α-glucosidase is reported in Table 1. Compounds 4, 5, and 7 showed the highest enzyme inhibitory activity at 256 μg/mL (40%, 46%, and 81%, respectively). An IC50 of 58.65 ± 1.20 μg/mL (302 μM) was calculated for compound 7, while the positive control acarbose showed a higher IC50 value of 197.33 ± 2.51 μg/mL (306 μM). The other related o-hydroxycinnamic acids including 8, a mixture of 8/9 (molar ratio 1 : 1), and 11 (compound 11 was isolated from E. japonicum [23]) and thymoquinol glucoside 6 did not show any α-glucosidase inhibitory activity (2% inhibitory activity at 256 μg/mL for all compounds investigated). (E)-cinnamic acid does not inhibit α-glucosidase from yeasts, but o-hydroxy or o-methoxy substituents increase the activity of the derivatives and the potency is higher for the methoxy group [15, 35, 36]. The structure of compound 7 possessing a methoxy group at C-2 and a hydroxy group at C-4 matched well the above-mentioned structural requisite. However, the activity was lost in compounds 8, 10, and a mixture of compounds 8/9 (molar ratio 1 : 1) when the 2-hydroxy group was blocked by a glucopyranosyl group in spite of the presence of 4-methoxy group in 9. Thus, the importance of 2-hydroxy or 2-methoxy group in modulating α-glucosidase inhibitory activity of (E)-cinnamic acids was confirmed which is in line with the previously published results [21, 36, 37]. The results of the present investigation are an additional contribution to the development of novel antidiabetic agents derived from (E)-cinnamic acid.Table 1
α-glucosidase inhibitory activity of compounds 4–9 and 11.
Compoundα-glucosidase inhibitory activity (%)IC50 (μg/mL) (μM)256μg/mL64μg/mL16μg/mL4μg/mL1μg/mL44038353025>25654641383019>256620000>25678153.52216058.65 ± 1.20 (302)820000>2568/9∗20000>2561120000>256Acarbose: IC50 197.33 ± 2.51 μg/mL [306 μM]∗1H-NMR: molar ratio 1 : 1.


### 3.3. HPLC Analysis of Coumarins4 and 5

HPLC analysis of the leaf methanol extract revealed coumarins4 and 5 as the major compounds in the extract. An optimized HPLC condition using a mobile phase of 44% acetonitrile in deionized H2O in 15 min was used to effectively separate compounds 4 and 5. With the wavelength of a PDA (photodiode array), detector set at 306 nm the HPLC chromatogram (Figures 4 and S33) showed two main resolved peaks of compound 4 at Rt 5.304 min and compound 5 at Rt 4.628 min. Compounds were identified on the basis of Rt comparison with those of the standard compounds 4 and 5. Polar compounds including 6–10 were not well separated and they appeared as a broad band at Rt about 2.5 min in the HPLC chromatogram. Thus, the HPLC method was suitable for analysing the contents of compounds 4 and 5. The calibration curves of compounds 4 and 5 were constructed, injecting 20 μL of each standard compound (5–100 ppm) (Figure S34). Using the Shimadzu LabSolutions HPLC software, each of the peaks was integrated to get the area values. The calibration curves were constructed by plotting area versus concentration with a correlation coefficient R2 > 0.990. Based on the calibration curve, the methanol leaf extract was determined to contain 16.69% of compound 4 and 2.02% of compound 5. Thus, the amounts of compounds 4 and 5 in the dry leaves of E. triplinerve collected in July in Vietnam were calculated as 2.63% and 0.32%, respectively.Figure 4
HPLC analysis of compounds4 and 5 in the methanol leaf extract.


### 3.4. NMR Profiling of Coumarins4 and 5

At present,1H-NMR spectroscopy has become a frequently used technique for metabolic fingerprinting. In addition, 1H-NMR spectroscopy has been used in the qualitative and quantitative assessment of secondary plant metabolites as well as quality control in food science and technology. One of the major advantages of NMR techniques is their reproducibility with rich structure information and applicability to a wide range of plant metabolites [38–40]. Coumarins have been successfully analysed in a few studies by 1H-NMR profiling [41, 42]. Compounds 4 and 5 could be extracted from the leaves of E. triplinerve by extraction with an alcohol such as methanol and boiling water. The methanol and boiling water extracts from the dry leaves of E. triplinerve were analysed by 1H-NMR spectra in this study. Solid-phase extraction (SPE) of the extracts eluting with 70% MeOH-H2O afforded the analytical samples. The following NMR parameters were used for the analysis: solvent 0.5 mL CD3OD in 5-mm NMR tubes, 16 scans, acquisition time 3.2767999 sec, temperature 299.0 K, spectral width 10000 Hz, and line broadening 0.3 Hz. FIDs were Fourier transformed with FIDRES 0.152588, GB 0, and PC 1.0. Figures S31 and S32 showed the representative 1H-NMR spectra of the methanol or water extracts, respectively. Referencing was to the lock solvent. The 1H-NMR spectra of the standards 4 and 5 were prepared in 0.5 mL of CD3OD in 5 mm NMR tubes. By using high-field 500 MHz NMR spectrometer, the signals of compounds 4 and 5 were resolved in both extracts. The identification match was based on chemical shifts, coupling, peak shape, and peak intensity data for individual compounds. Coumarins 4 and 5 were confirmed to be the abundant compounds in the extracts; their signal intensity ratio was about 15 : 1 in the methanol extract and about 10 : 1 in the water extract. These findings well supported the results obtained via the quantitative HPLC analysis. The concentrations of polar compounds 6–10 may be too low for the reliable detection by NMR techniques. Proton chemical shifts of compounds 4 and 5 in the extracts are summarized in Table S1. The 1H-NMR spectrum of compound 4 in the extracts was in accordance with that of the standard 4 in CD3OD, showing the signals of a lactone ring of the coumarin structure at δH 6.27 (1H, d, J = 9.5 Hz, H-3) and 7.93 (1H, d, J = 9.5 Hz, H-4), three aromatic protons with characteristic splitting of a 1,3,4-trisubstituted benzene ring at δH 6.93 (1H, br s, H-8), 6.96 (1H, dd, J = 8.5 Hz, 2.5 Hz, H-6), and 7.57 (1H, d, J = 8.5 Hz, H-5), and an aromatic methoxy group at δH 3.90 (3H, s, 7-OCH3). The characteristic proton signals of compound 5 in the extracts matched the signals of the standard 5 in CD3OD. The signals of a lactone ring of the coumarin structure at δH 6.29 (1H, d, J = 9.5 Hz, H-3) and 7.90 (1H, d, J = 9.5 Hz, H-4), a 1,3,4,6-tetrasubstituted benzene ring bearing an o-methylenedioxy moiety at δH 6.11 (2H, s, −OCH2O-), and characteristic singlets of the aromatic protons at δH 6.93 (1H, s, H-5), 7.07 (1H, s, H-8) were observed.


## 3.1. Isolation and MS/NMR Elucidation of Compounds

The dried powdered leaves ofE. triplinerve were extracted with MeOH at room temperature to yield a methanol extract. The methanol extract was fractionated into n-hexane-, dichloromethane-, and water-soluble fractions. Separation of the n-hexane-, dichloromethane-, and water-soluble fractions by repeated CC over different adsorbents such as silica gel, Diaion HP-20, Sephadex LH-20, and RP-18 using stepwise gradient elution gave phytosterols 1–3, coumarins 4 and 5, thymoquinol glucoside 6, o-hydroxycinnamic acid derivatives 7–9, and kaempferol diglucoside 10. Structures of the isolated compounds were determined as β-sitosterol (1) [24], stigmasterol (2) [24], β-sitosterol 3-O-β-D-glucopyranoside (3) [25], ayapanin (4) [26, 27], ayapin (5) [28], thymoquinol 5-O-β-D-glucopyranoside (6) [29], (E)-4-hydroxy-2-methoxycinnamic acid (7) [30, 31], thyrsifloside (8) [32], (E)-4-methoxymelilotoside (9) [33], and kaempferol 3,7-di-O-β-D-glucopyranoside (10) [34] (Figure 1) by comparing their spectroscopic data (MS, 1H-, and 13C-NMR) (Figures S1–S30) with the reported literature values. Compound 8 was isolated in its pure form and compound 9 was isolated in a nonseparable mixture of 8 and 9 (molar ratio 1 : 1 as determined by 1H-NMR intensity). The locations of the substituents of compounds 6, 8, and 9 were assigned by using heteronuclear single-quantum correlation (HSQC), heteronuclear multibond correlation (HMBC), and nuclear Overhauser effect spectroscopy (NOESY) spectra (Figure 2). In compound 6, key HMBC correlations were observed between CH3-7 (δH 2.15) and C-1 (δC 123.3), C-2 (δC 151.8), between H-8 (δH 3.5) and C-3 (δC 113.1), C-5 (δH 149.2), and between Glc-1 (δH 4.72) and C-5 (δC 149.2). In compound 8, key HMBC correlations for the assignment of the glucopyranosyl moiety were observed between H-7 (δH 8.13) and C-2 (δC 154.0), between H-6 (δH 7.13) and C-2, and between Glc-1 (δH 4.85) and C-2. In compound 9, key HMBC correlations for the assignment of the glucopyranosyl moiety and the methoxy group were observed between H-6 (δH 7.57) and C-2 (δC 159.0), between 4-OCH3 (δH 3.87) and C-4 (δC 164.4), and between Glc-1 (δH 4.98) and C-2. Further confirmation was obtained from the NOESY spatial interactions between H-5 (δH 6.67) and 4-OCH3, between 4-OCH3 and H-3 (δH 6.87), and between H-3 and Glc-1. The 1H-NMR spectra of 4 and 5 were also recorded in different NMR solvents (CDCl3, CD3OD, and DMSO-d6) and the signals were assigned to ease NMR identification of the compounds in different NMR solvents. Compounds 6–10 were isolated for the first time from E. triplinerve. The isolation of compounds has not showed the presence of alkaloids, saponins, triterpenoids, depsides, and depsidones which were detected in the previous phytochemical screening by using color reactions [35]. However, high concentration of coumarins and the presence of sterols in the leaves are in line with most of the phytochemical screenings [35]. In the genus Eupatorium, derivatives of 2-O-hydroxycinnamic acid and 2-O-glucopyranosylcinnamic acid were isolated from E. micranthum, while thymols were reported in E. fortunei [1]. Compounds 8 and 9 are glycosidic precursors of compounds 5 and 4, respectively. Under enzymatic conditions, they can be converted into nonglycosidic derivatives 2-hydroxy-6,7-methylenedioxy-trans-cinnamic acid (8a) and 2-hydroxy-4-methoxy-trans-cinnamic acid (9a), respectively. Compound 4 would be formed from 9a and compound 5 from 8a via photoisomerization of the trans double bond to cis and subsequent ring closure to the lactones (Figure 3) [30].Figure 1
Structures of compounds1–10 from E. triplinerve and 11 from E. japonicum.Figure 2
HMBC (⟶) and NOESY (—) correlations of compounds6, 8, and 9.Figure 3
Plausible biosynthesis of4 from 9 and 5 from 8 in E. triplinerve.


## 3.2.α-Glucosidase Inhibitory Activity

Type II diabetes mellitus is as metabolic disease mainly caused by the accumulation of excess sugar in the body. The enzymeα-glucosidase breaks down large starch polysaccharides into monosaccharides or disaccharides. Thus, inhibiting the enzymatic activity of α-glucosidase may reduce absorption of glucose in the body. In the in vitro α-glucosidase inhibitory activity test, the substrate p-nitrophenyl α-D-glucopyranoside (pNPG) is hydrolyzed by α-glucosidase to release p-nitrophenyl which can be monitored at 405 nm. The results are expressed as percentage inhibition (%) and the concentration of an inhibitor required to inhibit 50% of enzyme activity (IC50) is determined [36]. The inhibitory activity of compounds isolated from E. triplinerve against α-glucosidase is reported in Table 1. Compounds 4, 5, and 7 showed the highest enzyme inhibitory activity at 256 μg/mL (40%, 46%, and 81%, respectively). An IC50 of 58.65 ± 1.20 μg/mL (302 μM) was calculated for compound 7, while the positive control acarbose showed a higher IC50 value of 197.33 ± 2.51 μg/mL (306 μM). The other related o-hydroxycinnamic acids including 8, a mixture of 8/9 (molar ratio 1 : 1), and 11 (compound 11 was isolated from E. japonicum [23]) and thymoquinol glucoside 6 did not show any α-glucosidase inhibitory activity (2% inhibitory activity at 256 μg/mL for all compounds investigated). (E)-cinnamic acid does not inhibit α-glucosidase from yeasts, but o-hydroxy or o-methoxy substituents increase the activity of the derivatives and the potency is higher for the methoxy group [15, 35, 36]. The structure of compound 7 possessing a methoxy group at C-2 and a hydroxy group at C-4 matched well the above-mentioned structural requisite. However, the activity was lost in compounds 8, 10, and a mixture of compounds 8/9 (molar ratio 1 : 1) when the 2-hydroxy group was blocked by a glucopyranosyl group in spite of the presence of 4-methoxy group in 9. Thus, the importance of 2-hydroxy or 2-methoxy group in modulating α-glucosidase inhibitory activity of (E)-cinnamic acids was confirmed which is in line with the previously published results [21, 36, 37]. The results of the present investigation are an additional contribution to the development of novel antidiabetic agents derived from (E)-cinnamic acid.Table 1
α-glucosidase inhibitory activity of compounds 4–9 and 11.
Compoundα-glucosidase inhibitory activity (%)IC50 (μg/mL) (μM)256μg/mL64μg/mL16μg/mL4μg/mL1μg/mL44038353025>25654641383019>256620000>25678153.52216058.65 ± 1.20 (302)820000>2568/9∗20000>2561120000>256Acarbose: IC50 197.33 ± 2.51 μg/mL [306 μM]∗1H-NMR: molar ratio 1 : 1.


## 3.3. HPLC Analysis of Coumarins4 and 5

HPLC analysis of the leaf methanol extract revealed coumarins4 and 5 as the major compounds in the extract. An optimized HPLC condition using a mobile phase of 44% acetonitrile in deionized H2O in 15 min was used to effectively separate compounds 4 and 5. With the wavelength of a PDA (photodiode array), detector set at 306 nm the HPLC chromatogram (Figures 4 and S33) showed two main resolved peaks of compound 4 at Rt 5.304 min and compound 5 at Rt 4.628 min. Compounds were identified on the basis of Rt comparison with those of the standard compounds 4 and 5. Polar compounds including 6–10 were not well separated and they appeared as a broad band at Rt about 2.5 min in the HPLC chromatogram. Thus, the HPLC method was suitable for analysing the contents of compounds 4 and 5. The calibration curves of compounds 4 and 5 were constructed, injecting 20 μL of each standard compound (5–100 ppm) (Figure S34). Using the Shimadzu LabSolutions HPLC software, each of the peaks was integrated to get the area values. The calibration curves were constructed by plotting area versus concentration with a correlation coefficient R2 > 0.990. Based on the calibration curve, the methanol leaf extract was determined to contain 16.69% of compound 4 and 2.02% of compound 5. Thus, the amounts of compounds 4 and 5 in the dry leaves of E. triplinerve collected in July in Vietnam were calculated as 2.63% and 0.32%, respectively.Figure 4
HPLC analysis of compounds4 and 5 in the methanol leaf extract.


## 3.4. NMR Profiling of Coumarins4 and 5

At present,1H-NMR spectroscopy has become a frequently used technique for metabolic fingerprinting. In addition, 1H-NMR spectroscopy has been used in the qualitative and quantitative assessment of secondary plant metabolites as well as quality control in food science and technology. One of the major advantages of NMR techniques is their reproducibility with rich structure information and applicability to a wide range of plant metabolites [38–40]. Coumarins have been successfully analysed in a few studies by 1H-NMR profiling [41, 42]. Compounds 4 and 5 could be extracted from the leaves of E. triplinerve by extraction with an alcohol such as methanol and boiling water. The methanol and boiling water extracts from the dry leaves of E. triplinerve were analysed by 1H-NMR spectra in this study. Solid-phase extraction (SPE) of the extracts eluting with 70% MeOH-H2O afforded the analytical samples. The following NMR parameters were used for the analysis: solvent 0.5 mL CD3OD in 5-mm NMR tubes, 16 scans, acquisition time 3.2767999 sec, temperature 299.0 K, spectral width 10000 Hz, and line broadening 0.3 Hz. FIDs were Fourier transformed with FIDRES 0.152588, GB 0, and PC 1.0. Figures S31 and S32 showed the representative 1H-NMR spectra of the methanol or water extracts, respectively. Referencing was to the lock solvent. The 1H-NMR spectra of the standards 4 and 5 were prepared in 0.5 mL of CD3OD in 5 mm NMR tubes. By using high-field 500 MHz NMR spectrometer, the signals of compounds 4 and 5 were resolved in both extracts. The identification match was based on chemical shifts, coupling, peak shape, and peak intensity data for individual compounds. Coumarins 4 and 5 were confirmed to be the abundant compounds in the extracts; their signal intensity ratio was about 15 : 1 in the methanol extract and about 10 : 1 in the water extract. These findings well supported the results obtained via the quantitative HPLC analysis. The concentrations of polar compounds 6–10 may be too low for the reliable detection by NMR techniques. Proton chemical shifts of compounds 4 and 5 in the extracts are summarized in Table S1. The 1H-NMR spectrum of compound 4 in the extracts was in accordance with that of the standard 4 in CD3OD, showing the signals of a lactone ring of the coumarin structure at δH 6.27 (1H, d, J = 9.5 Hz, H-3) and 7.93 (1H, d, J = 9.5 Hz, H-4), three aromatic protons with characteristic splitting of a 1,3,4-trisubstituted benzene ring at δH 6.93 (1H, br s, H-8), 6.96 (1H, dd, J = 8.5 Hz, 2.5 Hz, H-6), and 7.57 (1H, d, J = 8.5 Hz, H-5), and an aromatic methoxy group at δH 3.90 (3H, s, 7-OCH3). The characteristic proton signals of compound 5 in the extracts matched the signals of the standard 5 in CD3OD. The signals of a lactone ring of the coumarin structure at δH 6.29 (1H, d, J = 9.5 Hz, H-3) and 7.90 (1H, d, J = 9.5 Hz, H-4), a 1,3,4,6-tetrasubstituted benzene ring bearing an o-methylenedioxy moiety at δH 6.11 (2H, s, −OCH2O-), and characteristic singlets of the aromatic protons at δH 6.93 (1H, s, H-5), 7.07 (1H, s, H-8) were observed.


## 4. Conclusions

The present study isolated phytosterols (1–3), coumarins (4 and 5) in the n-hexane- and dichloromethane-soluble fractions, and polar compounds: thymoquinol glucoside (6), o-hydroxycinnamic acid derivatives (7–9), and kaempferol diglucoside (10) in the water-soluble fraction from the leaves of E. triplinerve growing in Vietnam. Compounds 6–10 were isolated for the first time from E. triplinerve. We provided an efficient approach towards rapid identification of coumarins in E. triplinerve by NMR and HPLC analysis. Coumarins 4 and 5 were confirmed as the main constituents in the leaf extracts by proton NMR profiling and quantitative RP-HPLC-PDA analysis. In the inhibitory activity, test against yeast α-glucosidase of compounds 4–9, coumarins 4 and 5, and (E)-4-hydroxy-2-methoxycinnamic acid (7) showed 40%, 46%, and 81% inhibitory activity, respectively, at 256 μg/mL. The half inhibitory concentration (IC50) of compound 7 was determined as 58.65 ± 1.20 μg/mL (302 μM) which is lower than that of the positive control acarbose (197.33 ± 2.51 μg/mL, 306 μM).



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*Source: 8945063-2020-12-02.xml*