Antimicrobial secondary metabolites from the aerial parts of Perilla frutescens

Abstract Phytochemical research of Perilla frutescens aerial parts led to isolation of 12 secondary metabolites, including one new 3-benzoxepin glucoside, perillafrutoside A (1), one new megastigmane glycoside, perillafrutoside B (2), and 10 known compounds. Their chemical structures were identified based on 1D/2D NMR, HRESIMS, and ECD spectroscopic analyses. The structure of 2 was elucidated based on revision of the previously reported stereoisomer, (6R,9R)-blumenyl α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranoside. Evaluation of their antimicrobial effect revealed that compounds 1 and 5–11 inhibit Enterococcus faecalis growth, compounds 6, 7 and 9 suppress Staphylococcus aureus growth, whereas compounds 6 and 11 attenuate Candida albicans growth. This is the first report of the isolation of 3–5, 8–10 and 12 from the genus Perilla and the antimicrobial effect of compounds 3, 8 and 10. Graphical Abstract


Introduction
Perilla frutescens (L.) Britton is an annual herb belonging to Lamiaceae family. This plant is widely cultivated in Asian countries, such as China, India, Japan, Korea, and Vietnam. In addition to the common consumption as a vegetable as well as a food
Compound 1 was isolated as a white amorphous powder. .7 (C-7) and 147.7 (C-9)] and five nonprotonated carbons [d C 127.4 (C-3a), 152.2 (C-5), 126.6 (C-5a), 119.9 (C-10a), and 152.9 (C-10b)] were recognized. Based on the observed spectroscopic evidence, 1 was proposed to have a benzoxepin structural moiety (Liu et al. 2000;Zhang et al. 2022). This was supported by 1 H-1 H COSY correlations between H-6/H-7 and between H-9/H-10 and HMBC cross-peaks from H-4 to C-5a and C-10b, from H-6 to C-5 and C-10a, and from H-10 to C-5a and C-10b ( Figure S1). Of the remaining 12 sp 3 carbon atoms, five oxymethines [d C 103.5 (C-1 0 ), 75.0 (C-2 0 ), 78.0 (C-3 0 ), 71.6 (C-4 0 ) and 78.1 (C-5 0 )] and one oxymethylene [d C 62.9 (C-6 0 )] were assignable to a glucose (Ngan et al. 2020). As the anomeric proton possessed a large coupling constant (J ¼ 7.8 Hz), the glucose was unambiguously defined to have b-configuration. In addition, observations of two sp 3 oxymethines at d C 92.5 (C-2) and 82.6 (C-3) and HMBC correlations from H-3 to C-3a, C-4, and C-10b suggested the presence of a 2,3-dihydrofuran-2,3-diol moiety which was fused with the benzoxepine at C-3a/C-10b. Moreover, signals of a non-protonated carbon at d C 74.3 (C-11), an oxymethylene at d C 67.3 (C-12), and a methyl at d C 20.8 (C-13) were also observed, suggesting the existence of a propan-1,2-diol substituent (Zhang et al. 2022). This was supported by HMBC cross-peaks from H 3 -13 to C-11 and C-12 and from H 2 -12 to C-13. Comparison of the 1 H and 13 C NMR data of 1 with those of the previously reported prenylated 3-benzoxepin perilloxin revealed a close similarity, except for the respective replacement of a propan-2-ol and a methylene in perilloxin by a propan-1,2-diol and an oxymethine in 1 and the additional presence of a glucose in 1 (Liu et al. 2000). The locations of the propan-1,2-diol at C-2 and the glucose at C-3 were deduced by HMBC correlations from both H 3 -13 and H 2 -12 to C-2 and from H-1 0 to C-3, respectively. The relatively small coupling constants of H-2 and H-3 ( 3 J 2,3 ¼ 4.2 Hz) implied that both protons are co-facial. Finally, the absolute configuration 2S3R of 1 was identified by ECD calculation using the TD-DFT method in comparison with the experimental ECD spectrum ( Figure S1, Supplementary material) (O'Boyle et al. 2011;Bruhn et al. 2013;Frisch et al. 2016;Bruhn et al. 2017). However, as the calculated ECD spectra for both 2S3R11R and 2S3R11S were almost identical ( Figure S1), analysis of the NOESY spectrum of 1 based on the conformers (with Boltzmann population > 1%) generated by molecular mechanics MMFF force field using Spartan'18 program (Wavefunction, Inc. Irvine, CA, USA) was performed to suggest the relative configuration of C-11. In the NOESY spectrum, correlations between H 3 -13/H-2 and H-3 and between H-3/H-4 and Ha-12 were observed. Considering the distance and orientation of the protons, the conformers A1ÀA5 of 2S3R11R isomer were shown to be satisfied whereas all B1ÀB7 conformers of 2S3R11S isomer were unsuitable with the observed NOE correlations ( Figure S2), thus suggesting that 1 has 11R Ã relative configuration. Consequently, the chemical structure of 1 was established as shown in Figure 1, named perillafrutoside A.
The antimicrobial effect of all the compounds against the growth of three Grampositive bacteria E. faecalis ATCC299212, S. aureus ATCC25923 and B. cereus ATCC13245, three Gram-negative bacteria E. coli ATCC 25922, P. aeruginosa ATCC 9027 and S. enterica ATCC13076, and a yeast strain C. albicans ATCC 24433. The result showed that compounds 1 and 5-11 suppressed the growth of E. faecalis, with MIC values in a range of 150-300 mM (Table S1). Compounds 6, 7 and 9 additionally inhibited the growth of S. aureus, with MIC values of 300, 300 and 150 mM, respectively. In addition, the growth of C. albicans was attenuated by compounds 6 and 11, with MIC values of 150 and 300 mM, respectively. However, none of the compounds showed antimicrobial against the Gram-negative bacteria.
In conclusion, phytochemical study on the aerial parts of P. frutescens led to the isolation and identification of 12 secondary metabolites, including two new ones, perillafrutosides A (1) and B (2). Compounds 1 and 5-11 inhibited E. faecalis growth, compounds 6, 7 and 9 suppressed S. aureus growth, whereas 6 and 11 attenuated C. albicans growth. It is noted that this is the first time to report the isolation of 3-5, 8-10 and 12 from the genus Perilla and the antimicrobial effect of compounds 3, 8 and 10.

General experimental procedures
Specific optical rotation values were identified using Jasco P2000 polarimeter. UV spectra were obtained on a JASCO V-630 UV-vis spectrophotometer (Jasco, Japan). Circular dichroism (CD) spectra were acquired on a Chirascan spectrometer (Applied Photophysics, UK). NMR spectra of the isolated compounds were conducted on a Bruker AVANCE III 600 MHz spectrometer (Bruker, Germany). HRESIMS analyses were measured on an Agilent 1290 UPLC/6530 Accurate-Mass QTOF LC/MS system. Preparative high-performance liquid chromatography (prep. HPLC) was conducted on an Agilent 1200 system (Agilent Technologies, USA). Column chromatography (CC) was performed using silica gel or reversed phase C 18 as stationary phase. Thin layer chromatography (TLC) was carried out with pre-coated plates (silica gel 60 F 254 or RP-18 F 254S ), and the spots were visualized by UV detection at 254 nm by spraying a 10% aqueous H 2 SO 4 solution, followed by heating.

Plant material
The aerial parts of P. frutescens were obtained from a traditional medicinal plant market in Hanoi, Vietnam in March 2020. The plant material was identified by one of the authors, Dang Viet Cuong. A voucher specimen (No: IMBC-PF.03.2020) was deposited at the Institute of Marine Biochemistry, VAST, Vietnam.

ECD calculation
ECD calculations of 1 and 2 were performed using the Gaussian 16W program (Frisch et al. 2016). Conformational analysis was initially carried out using the MMFF force field (Spartan'18 program, Wavefunction, Inc. Irvine, CA, USA). The conformers were selected for optimization using b3lyp/6-311g(d,p) basic set and conductor-like polarizable continuum model (CPCM) in methanol (O'Boyle et al. 2011). Subsequently, ECD spectra of all the selected conformers were calculated at the same level using TDDFT by Gaussian 16 W (Frisch et al. 2016). Finally, the calculated ECD spectra of 1 and 2 were simulated using SpecDis 1.71 program (Bruhn et al. 2013(Bruhn et al. , 2017.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This study was financially supported by Vietnam Academy of Science and Technology (VAST) under the grant number KHCBHH.02/20-22.