Bioassay-Guided Isolation of Triterpenoids as α-Glucosidase Inhibitors from Cirsium setosum

Cirsium setosum (C. setosum) has a potential antihyperglycemic effect, but it is unclear what bioactive components play a key role. According to the α-glucosidase inhibition activity, three new taraxastane-type triterpenoids of 3β-hydroxy-30-hydroperoxy-20-taraxastene (1), 3β-hydroxy-22α-methoxy-20-taraxastene (2), and 30-nor-3β,22α-dihydroxy-20-taraxastene (3), as well as five known taraxastane triterpenoids of 3β,22-dihydroxy-20-taraxastene (4), 20-taraxastene-3,22-dione (5), 3β-acetoxy-20-taraxasten-22-one (6), 3β-hydroxy-20-taraxasten-22-one (7), and 30-nor-3β-hydroxy-20-taraxastene (8) were obtained from the petroleum ether-soluble portion of the ethanol extract from C. setosum. All chemical structures of the compounds were elucidated by spectroscopic data analysis and compared with literature data. Compounds 4–8 were identified for the first time from this plant, and compounds 1, 2, 4, and 7 exhibited more potent α-glucosidase inhibitory activity—with IC50 values of 18.34 ± 1.27, 26.98 ± 0.89, 17.49 ± 1.42, and 22.67 ± 0.25 μM, respectively—than acarbose did (positive control, IC50 42.52 ± 0.32 μM).

The purpose of this study is to explore new α-glucosidase inhibitors (AGIs) from C. setosum. In a bioassay-guided fractionation of an EtOH extract of C. setosum, we found that the petroleum ether-soluble fraction showed potent α-glucosidase inhibitory activity. Further separation from the above inhibitory activities component against α-glucosidase resulted in the isolation of eight triterpenoids inhibitors. Among these eight compounds, three are new structures and two are found to be more active than the acarbose that is available clinically. This work elucidates the relationship between triterpenoids constituents and hypoglycemic functions of C. setosum. Findings of this study contain important empirical implications in terms of developing future hypoglycemic functional food and improving its quality standards.

Results and Discussions
The crude extract of stems of C. setosum was suspended in H 2 O and then partitioned with petroleum ether and EtOAc. Our random bioassay revealed that the petroleum ether-soluble portion had the highest activity against α-glucosidase, with an inhibitory rate of 87.6 ± 1.23% (300 µg/mL). Bioassay-guided isolation yielded eleven fractions (Sh1-Sh11) via silica gel column chromatography, eluting with a gradient of acetone (0-100%) in petroleum ether (60-90 • C). Fraction Sh8 showed significant activity against α-glucosidase, with an inhibitory rate of 99.2 ± 2.19% (300 µg/mL). Fraction Sh8 was further isolated by the combination of silica gel column chromatography, low pressure liquid chromatography, Sephadex LH-20 chromatography, and high-performance liquid chromatography (HPLC), generating three new (1-3) and five known (4-8) compounds ( Figure 1).
Molecules 2019, , x FOR PEER REVIEW 2 of 10 The purpose of this study is to explore new α-glucosidase inhibitors (AGIs) from C. setosum. In a bioassay-guided fractionation of an EtOH extract of C. setosum, we found that the petroleum ethersoluble fraction showed potent α-glucosidase inhibitory activity. Further separation from the above inhibitory activities component against α-glucosidase resulted in the isolation of eight triterpenoids inhibitors. Among these eight compounds, three are new structures and two are found to be more active than the acarbose that is available clinically. This work elucidates the relationship between triterpenoids constituents and hypoglycemic functions of C. setosum. Findings of this study contain important empirical implications in terms of developing future hypoglycemic functional food and improving its quality standards.

Structural Elucidation of the Three New Compounds
Compound 1 was obtained as a white amorphous powder. The IR spectrum of 1 suggested that it contained hydroxyl groups (3417 and 3165 cm −1  . The 13 C-NMR spectrum displayed 30 carbon signals, which were classified as seven methyls, ten methylenes (one oxygenated), seven methines (one oxygenated and one olefinic), and six quaternary carbons (one olefinic carbon) on the basis of DEPT and HSQC spectra. These data suggested that 1 was very similar, with one known 30-hydroperoxy-ψ-taraxasteryl acetate [18], except for lacking acetate group located at C-3, which was confirmed by the comprehensive analysis of the 2D NMR spectra of 1, especially 1 H-1 H COSY and HMBC ( Figure 2). carbons, the other functional groups and the above five structural fragments was mainly achieved by the analysis of the HMBC spectrum ( Figure 2). HMBC correlations from 3H-23 (H 1.25) to C-5, C-3 and C-24, and from 3H-24 (H 1.06) to C-4, C-3, C-5, and C-23 indicated that Me-23 and Me-24 were attached to C-4. The HMBC correlations of 3H-25 (H 0.91) to C-5, C-1, C-10, and C-9; 3H-26 (H 1.00) to C-9, C-7, and C-8; 3H-27 (H 0.97) to C-13, C-15, and C-8; 3H-28 (H 0.96) to C-16, C18, and C-22; and 2H-30 (H 4.66 and 4.91) to C-19, C-20, and C-21 not only confirmed the presence of A/B/C/D/Ering systems but also located the Me-25, Me-26, Me-27, Me-28, and -CH2OOH-30 at C-10, C-8, C-14, C-17, and C-20 respectively. The structure of 1 was, therefore, determined as 3β-hydroxy-30hydroperoxy-20-taraxastene. Compound 2 was obtained as a white amorphous powder. The presence of hydroxyl groups (3362 cm −1 ) and a double bond (1673 cm −1 ) functionalities were evident in its IR spectrum. Its molecular formula was deduced as C31H52O2, from the negative HRESIMS at m/z 455.3890 [M − H] − (calcd for C31H51O2 455.3895) and 13 C-NMR spectrum. This indicated six degrees of unsaturation. The 1 H and 13 C-NMR spectra of 2 were very similar to those of compound 4, a known 3β,22α-dihydroxy-20-taraxastene that was also isolated from this plant [19], with the only difference being the replacement of the hydroxyl group by a methoxy moiety at C-22 (Table 1). This inference was confirmed by the HMBC correlation of 3H-OMe/C-22. The configuration of H-22 was assigned as βequatorial on the basis of the coupling constant (5.8 Hz) with the vicinal olefinic proton H-21 and the NOESY correlation with Me-28. Thus, compound 2 was deduced to be 3β-hydroxy-22α-methoxy-20taraxastene.
Compound 3, a white amorphous powder, had the formula of C29H48O2 on the basis of the negative HRESIMS at m/z 427.3585 [M − H] − (calcd for C29H47O2 427.3582) and the 13 C-NMR spectrum. The IR spectrum showed absorption bands at 3656, 3405, 1657, and 1607 cm -1 due to the hydroxyl groups and double bond. The NMR spectra of 3 and a known 3β,22α-dihydroxy-20-taraxastene (compound 4, which was also isolated from this plant) were closely comparable [20], with the only difference being the lack of a methyl group at C-20. The structure of 3 was confirmed by the 2D NMR HSQC, COSY, HMBC, and NOESY data. The NOESY correlation of Me-28 with H-22, and the coupling constant (6.0 Hz) of H-22 with the vicinal olefinic proton H-21 indicated that H-22 was βoriented. The structure for 3 was thus assigned as 30-nor-3β,22α-dihydroxy-20-taraxastene.
Compound 2 was obtained as a white amorphous powder. The presence of hydroxyl groups (3362 cm −1 ) and a double bond (1673 cm −1 ) functionalities were evident in its IR spectrum. Its molecular formula was deduced as C 31 H 52 O 2 , from the negative HRESIMS at m/z 455.3890 [M − H] − (calcd for C 31 H 51 O 2 455.3895) and 13 C-NMR spectrum. This indicated six degrees of unsaturation. The 1 H and 13 C-NMR spectra of 2 were very similar to those of compound 4, a known 3β,22α-dihydroxy-20-taraxastene that was also isolated from this plant [19], with the only difference being the replacement of the hydroxyl group by a methoxy moiety at C-22 (Table 1) The IR spectrum showed absorption bands at 3656, 3405, 1657, and 1607 cm -1 due to the hydroxyl groups and double bond. The NMR spectra of 3 and a known 3β,22α-dihydroxy-20-taraxastene (compound 4, which was also isolated from this plant) were closely comparable [20], with the only difference being the lack of a methyl group at C-20. The structure of 3 was confirmed by the 2D NMR HSQC, COSY, HMBC, and NOESY data. The NOESY correlation of Me-28 with H-22, and the coupling constant (6.0 Hz) of H-22 with the vicinal olefinic proton H-21 indicated that H-22 was β-oriented. The structure for 3 was thus assigned as 30-nor-3β,22α-dihydroxy-20-taraxastene.

α-Glucosidase Inhibitory Activity of the Isolates
All the isolates were evaluated for their α-glucosidase inhibitory activities using p-nitrophenyl-α-dglucopyranoside (p-NPG) as the substrate and acarbose as the positive control ( Table 2). All of the eight compounds that showed inhibitory rates higher than 50% at the concentration of 100 µM, were further evaluated for their IC 50 values. As shown in Figure 3, Figure 4, and Table 2, IC 50 values of the eight compounds were in the range of 18.34 to 80.07 µM. were further evaluated for their IC50 values. As shown in Figure 3, Figure 4, and Table 2, IC50 values of the eight compounds were in the range of 18.34 to 80.07 μM. The tested concentration of all samples was 100 μM, IC50 values represent the concentrations that caused 50% activity loss. The value of each activity is expressed as mean SD (n = 3).  were further evaluated for their IC50 values. As shown in Figure 3, Figure 4, and Table 2, IC50 values of the eight compounds were in the range of 18.34 to 80.07 μM. The tested concentration of all samples was 100 μM, IC50 values represent the concentrations that caused 50% activity loss. The value of each activity is expressed as mean SD (n = 3).

Plant Material
The

General Experimental Procedures
The HRESIMS data were generated on a Thermo QE UPLC-Orbitrap MS spectrometer (Thermo Scientific Inc., Waltham, MA, USA). The specific rotations data were obtained with a Rudolph Research Autopol III automatic polarimeter (Rudolph Research Analytica, Hackettstown, NJ, USA). The UV data and circular dichroism spectra were recorded on a JASCO J-810 circular dichroism spectrometer (JASCO Corporation, Tokyo, Japan). IR spectra were acquired on a Nicolet Impact 400 FT-IR spectrophotometer (Nicolet Instrument Inc., Madison, WI, USA). 1D-and 2D-NMR spectra were acquired in C 5

Extraction and Isolation
The air-dried stems of Cirsium setosum (Willd.) (10 kg) were ground into powder and extracted with 90%, 80%, and 70% aqueous EtOH sequentially at room temperature for 120 min under sonication. The extract was evaporated under reduced pressure to yield a dark brown residue, which was suspended in H 2 O and then partitioned with petroleum ether and EtOAc. The petroleum ether-soluble portion (468.5 g) was fractionated via silica gel column chromatography, eluting with a gradient of acetone (0-100%) in petroleum ether (60-90 • C), to give eleven fractions (Sh1-Sh11).

α-Glucosidase Inhibitory Effect Assay
The α-glucosidase inhibitory assay was carried out spectrophotometrically, according to the previously described method, with slight modifications, in which acarbose was used as the positive control [23].
A total of 200 µL of reaction mixture, containing 70 µL of 0.1 M phosphate buffer (pH 6.8), 10 µL of 1.0 mg/mL reduced glutathione solution, and 10 µL of the sample solution (test concentration at 0.1 mg/mL), was added to each well of a 96-well plate, followed by the addition of 20 µL of 0.5 U/mL α-glucosidase solution. The plate was incubated at 37 • C for 15 min, and then 20 µL of p-Nitrophenyl α-d-glucopyranoside substrate was added to the mixture to start the reaction. The reaction mixture was incubated at 37 • C for 30 min, and then 70 µL of 0.1 M Na 2 CO 3 solution was added to the mixture to terminate the reaction. All samples were analyzed in triplicate with three different concentrations near the IC 50 values. The absorbance (A) was immediately recorded at 400 nm, using a spectrophotometrical method to estimate the enzymatic activity. The inhibition percentage was calculated by the following equation: Inhibitory rate (%) = [1 − (A test − A blank )/(control A test − control A blank )] × 100%.
Here, A test represents the absorbance value of the experimental sample, A blank represents the absorbance value of sample blank, control A test represents the absorbance value of the control, and control A blank represents the absorbance value of the blank.
The results suggest that triterpenoids from C. setosum could be the key and potential functional food ingredients for a new antidiabetic agent. Due to the relatively high contents and potent α-glucosidase inhibitory activity of compounds 1, 2, 4, and 7 in C. setosum, we speculated that those four compounds could be the main bioactive components responsible for the α-glucosidase inhibitory effect of C. setosum. This work provides a scientific basis for the development of C. setosum as a hypoglycemic functional food, and also a theoretical basis for the establishment of a quality test method for the bioactivity factor of C. setosum as a dietary supplement for hypoglycemic products.
Supplementary Materials: The following are available online, IR, UV, HRMS and NMR spectra of compounds 1-8 as well as other supporting data.