Coumarins and Polar Constituents from Eupatorium triplinerve and Evaluation of Their α-Glucosidase Inhibitory Activity

In our study of antidiabetic compounds from the leaves of Eupatorium triplinerve Vahl. (Asteraceae), ten compounds were isolated from the methanol leaf extract. (ey were determined to be β-sitosterol (1), stigmasterol (2), β-sitosterol 3-O-β-Dglucopyranoside (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 (H-, C-, DEPT) and 2D NMR (HSQC, HMBC, and NOESY) techniques. (is 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).


Introduction
e genus Eupatorium (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 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. e 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-pcymene [4], β-caryophyllene [4], selina-4(15),7(11)-dien-8one [5], and 2-tert-butyl-1,4-methoxybenzene [6]. e leaves emit a distinct coumarin odour, and the isolation of ayapanin (or herniarin, 7-methoxycoumarin) and ayapin (or 6,7methylenedioxycoumarin) 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 (LC 50 < 1000 µg/ mL). e water extract had no toxicity. e highest LC 50 was for the ethyl acetate (24,42 µg/mL) and the lowest LC 50 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.
ey 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]. erefore 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]. e 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. e 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 starchhydrolysing 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.

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

NMR Profiling of the Methanol or Boiling Water Leaf
Extracts. e dried leaf powder (240 g) was extracted with methanol at room temperature for three days or with boiling water for 24 h. e 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-H 2 O. e fractions eluting with 70% MeOH-H 2 O were concentrated under reduced pressure to afford two NMR samples. 5 mg of each sample was dissolved in CD 3 OD and directly analysed by 1 H-NMR spectroscopy at 500 MHz frequency.

HPLC Analysis of the Methanol Leaf Extract.
e dried leaf powder (80 g) was extracted with MeOH at room temperature for 1 day. e solution was filtered and then evaporated to dryness under reduced pressure. e methanol leaf extract (220 mg) was dissolved with 2 mL acetonitrile of HPLC grade (Merck, Germany). e reversedphase solid-phase extraction (RP-SPE) of the solution eluting with MeOH of HPLC grade (Merck, Germany) was performed to remove impurities. e sample was filtered through a Millipore 0.45 μm membrane filter. Two standard solutions (100 μg/mL) were prepared by dissolving compounds 4 and 5 in MeOH of HPLC grade (Merck, Germany). e 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-H 2 O (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. e UV detection wavelength was set at 306 nm. All the measurements were performed in triplicate.

11.
e inhibition of α-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. en 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. e 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. e experiments were repeated three times. e α-glucosidase inhibitory activity was calculated using the following equation, where A control is the absorbance of the control, A sample is the absorbance of the sample: α-glucosidase inhibitory activity (%) � (A control −A sample )/A control × 100. IC 50 (half maximal inhibitory concentration) was calculated using Tablecurve software. All analyses were performed in triplicate and data were reported as mean ± SEM.

α-Glucosidase Inhibitory Activity.
Type II diabetes mellitus is as metabolic disease mainly caused by the accumulation of excess sugar in the body. e enzyme α-glucosidase breaks down large starch polysaccharides into monosaccharides or disaccharides.
us, 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. e results are expressed as percentage inhibition (%) and the concentration of an inhibitor required to inhibit 50% of enzyme activity (IC 50 ) is determined [36]. e inhibitory activity of compounds isolated from E. triplinerve against α-glucosidase is reported in Table 1  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]. e 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 2hydroxy group was blocked by a glucopyranosyl group in spite of the presence of 4-methoxy group in 9. us, 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]. e results of the present investigation are an additional contribution to the development of novel antidiabetic agents derived from (E)-cinnamic acid.  Figure S34). Using the Shimadzu LabSolutions HPLC software, each of the peaks was integrated to get the area values. e calibration curves were constructed by plotting area versus concentration with a correlation coefficient R 2 > 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. us, 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 3: Plausible biosynthesis of 4 from 9 and 5 from 8 in E. triplinerve.   Journal of Chemistry 5 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][39][40]. Coumarins have been successfully analysed in a few studies by 1 H-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. e methanol and boiling water extracts from the dry leaves of E. triplinerve were analysed by 1 H-NMR spectra in this study. Solid-phase extraction (SPE) of the extracts eluting with 70% MeOH-H 2 O afforded the analytical samples. e following NMR parameters were used for the analysis: solvent 0.5 mL CD 3 OD 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 1 H-NMR spectra of the methanol or water extracts, respectively. Referencing was to the lock solvent. e 1 H-NMR spectra of the standards 4 and 5 were prepared in 0.5 mL of CD 3 OD in 5 mm NMR tubes. By using highfield 500 MHz NMR spectrometer, the signals of compounds 4 and 5 were resolved in both extracts. e 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. ese findings well supported the results obtained via the quantitative HPLC analysis. e 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. e 1 H-NMR spectrum of compound 4 in the extracts was in accordance with that of the standard 4 in CD 3 OD, showing the signals of a lactone ring of the coumarin structure at δ H 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 δ H 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 δ H 3.90 (3H, s, 7-OCH 3 ). e characteristic proton signals of compound 5 in the extracts matched the signals of the standard 5 in CD 3 OD. e signals of a lactone ring of the coumarin structure at δ H 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 δ H 6.11 (2H, s, −OCH 2 O-), and characteristic singlets of the aromatic protons at δ H 6.93 (1H, s, H-5), 7.07 (1H, s, H-8) were observed.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.