Two new phenolic compounds from Boerhavia erecta collected in Vietnam

Abstract Boerhavia erecta is a tropical plant that is widely used in Asian folk medicine. Little is known about the alpha-glucosidase inhibition and antimicrobial properties of compounds from this plant. In the present study, the phytochemical study of the aerial parts of B. erecta collected in Vietnam was conducted using multiple chromatographic methods. The chemical structures of isolated compounds were identified by comprehensive spectroscopic methods. Two new compounds, berectone C (1) and (E)-tetracosyl 3-(3-hydroxy-4-methoxyphenyl)acrylate (4), together with the known compounds boeravinone C (2), liquiritigenin (3), bis(1H-indol-3-yl)methanone (5), and indole-3-carboxylic acid (6) were isolated and structural elucidated. Compounds 1 and 4 were evaluated for alpha-glucosidase inhibition and antimicrobial activity against antibiotic-resistant, pathogenic bacteria Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Compound 1 showed strong inhibition of the alpha-glucosidase enzyme (IC50 43 µg/mL). Only compound 1 exhibited antimicrobial property against A. baumannii, forming an inhibition zone of 11 mm.


Results and discussion
Protons of a 1,2,3-trisubstituted A-ring at d H 7.82 (H-1) and 6.87 (H-2), and a hydroxy group at d H 5.79 (12a-OH), gave HMBC correlations with C-1a, suggesting the presence of 12a-OH group. This hydroxy group provided a further HMBC crosspeak to a ketone carbon at d C 195.9 (C-12) and an oxygenated methine carbon at d C 77.2 (C-6a), confirming its location. The presence of a 4-OH group was defined by HMBC correlations between H-2 and H-3 (d H 6.86) and carbon at d C 146.4. The connection between the A-and B-rings was determined at C-1a and C-4a by HMBC correlations of H 2 -6 with C-4a, of H-6 with C-1a, and of H 2 -6 and H-6 with the same carbon C-12a (d C 66.8).
In the D-ring, HMBC correlations were found of both the methyl at d H 2.02 (10-CH 3 ) and the hydroxy group at d H 12.02 with the same carbons at d C 160.1 and d C 106.0, indicating that this methyl was positioned at C-10. The methoxy group was assigned to C-8 based on HMBC correlation of this group with an upfield carbon at d C 128.2. This is a characteristic feature of related rotenoids coccineone B (Ferrari et al. 1991), boeravinone K (Do et al. 2013), and berectone A (Dao et al. 2021). Full comparisons of the 1D and 2D NMR spectra of 1 with those of boeravinone C (2) (Table S1 and Figure  S1) suggested a basic consistency, except for the absence of 7-OCH 3 and the presence of 8-OCH 3 in 1. This established the structure of 1, which was named berectone C.
Compound 4 was isolated as a white amorphous powder, whose molecular formula of C 34 H 58 O 4 was deduced from the deprotonated molecular ion peak at m/z 529. , and 4-OCH 3 with the same carbon C-4 (d C 146.9) defined the position of the methoxy group. Protons CH 2 -1, H-7, and H-8 gave HMBC correlations with C-9, determining the attachment of the long chain at C-9. Altogether, compound 4 was elucidated as shown in Figure 1.
Compound 5 was produced synthetically by Gucchait and co-workers in 2011. To the best of our knowledge, this is the first time it has been isolated as a natural product.
Compounds 1 and 4 were evaluated for alpha-glucosidase inhibition and antimicrobial activity against the pathogenic antibiotic-resistant bacteria Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Compound 1 exhibited strong alpha-glucosidase inhibition with an IC 50 value of 43 mg/mL, compared with the 162 lg/mL of the acarbose positive control. It also showed moderate inhibition of A. baumannii, with an inhibition zone of 11 mm at a concentration of 50 mg/mL. Compound 4 was inactive in both tests.

General experimental procedures
The NMR spectra were recorded on a Bruker Avance III spectrometer (500 MHz for 1 H-NMR and 125 MHz for 13 C-NMR) using residual solvent signals as internal references: chloroform-d at d H 7.26, d C 77.16. The HR-ESI-MS was recorded using an HR-ESI-MS MicrOTOF-Q mass spectrometer on an LC-Agilent 1100 LC-MSD Trap spectrometer. Thin layer chromatography (TLC) was carried out on precoated silica gel 60 F 254 or silica gel 60 RP-18 F 254S (Merck). Spots were visualized by spraying with 10% H 2 SO 4 solution followed by heating. Gravity-column chromatography was performed on silica gel 60 (0.040-0.063 mm, Himedia).

Plant material
The aerial parts of B. erecta were collected from Binh Thuan Province, Vietnam, in April 2021. The plant material was identified by Dr. Tran Cong Luan, Tay Do University, Can Tho City, Vietnam. A voucher specimen (No UP-011) was deposited in the herbarium of the University's Department of Organic Chemistry.

Alpha-glucosidase inhibition assay
Saccharomyces cerevisiae a-glucosidase (E.C 3.2.1.20), acarbose, and 4-nitrophenyl b-Dglucopyranoside (pNPG) were obtained from Sigma-Aldrich Co (Saint Louis, MI, USA). The a-glucosidase inhibition assay was performed using a slight modification of a published method (Dao et al. 2021). Briefly, solutions of alpha-glucosidase and its substrate (p-nitrophenyl a-D-glucopyranoside pNPG) were prepared in phosphate buffer (100 mM, pH 6.9). DMSO 5% was used as solvent for preparation of the inhibitor solutions. The inhibition assay was conducted by addition 50 lL of inhibitor solution to 40 mL of enzyme solution (0.2 unit/mL) in 100 mM phosphate buffer (pH 6.8). The mixture was left at room temperature for 20 min. After pre-incubation, 40 mL of 3 mM substrate (pNPG) in phosphate buffer was added, initiating an enzymatic reaction. The reaction mixture was incubated at 37 C for 20 min, and the reaction was stopped by addition of 130 mL of 0.2 M Na 2 CO 3 . Acarbose was used as a positive control. The a-glucosidase activity was determined by measuring the p-nitrophenol released from pNPG at 405 nm in a 96-well ELISA Reader. The % inhibition was calculated as Inhibition (%) ¼ [1 À (A sample /A control )] Â 100%. The IC 50 values of the inhibitors were determined using five serial dilutions of a-glucosidase inhibitor. The results were recorded as the concentration of inhibitor at which alpha-glucosidase activity was inhibited by 50%.

Antibacterial activity assay
The agar well diffusion method was used to evaluate the antibacterial activity of the isolated compounds on antibiotic-resistant, pathogenic bacteria E. faecium, S. aureus, and A. baumannii. Three bacterial pathogens were cultured in Nutrient Broth at 37 C for 18 hours. The cultures were diluted with sterile 0.9% NaCl to obtain bacterial solutions of 1.5 Â 10 8 CFU/ml. Then 100 mL of each solution was spread on a Mueller-Hinton agar (MHA) plate. Holes with a diameter of 8 mm were punched aseptically to create wells on the surface of the MHA. The compounds were prepared in DMSO at a concentration of 1 mg/ml and 50 mL of each compound solution was introduced into the wells. The plates were incubated at 37 C for 16-18 hours and the antibacterial activity of each compound was recorded by measuring the diameters of the inhibition zones surrounding the wells. DMSO was used as a control.
Compound 5 is believed to be a newly-reported natural product. Compound 1 showed strong inhibition of the alpha-glucosidase enzyme. Only compound 1 showed antimicrobial activity, showing an inhibition zone of 11 mm against A. baumannii.