Two new terpenoid glycosides from Isodon macrocalyx

Abstract One previously undescribed tricyclo-diterpenoid saponin (1), one undescribed dammarane triterpenoid saponin (2) and four known triterpenoids (3–6) were isolated from the aerial part of Isodon macrocalyx. Their structures were determined using 1 D- and 2 D-NMR, HRESIMS, and comparison of their spectroscopic data with previously reported data. The isolates were evaluated for their anti-HBV activity in vitro using the HBV transfected Hep G2.2.15 cell line. Compounds 2 and 3 exhibited moderate anti-HBV activity. GRAPHICAL ABSTRACT


Introduction
The genus Isodon (Lamiaceae family) consists of approximately 150 species that are mainly distributed in the tropical and subtropical regions of Asia (Delectis Florae reipublicae popularis sinicae agendae academiae sinicae edita 1977).Some species have been used in traditional medicine, e.g.I. rubescens has been used in traditional Chinese medicine (TCM) for its various activities, including anti-inflammatory, antitumour, antimicrobial, immunological and antioxidant activities (Chen et al. 2022).The roots and aerial parts of I. ternifolius have been used in TCM to treat acute icterohepatitis, diarrhoea, enteritis, and other types of inflammation (Wu and Li 1977;Hong et al. 2007).I. phyllopodus and I. japonica have been traditionally used to treat hepatitis, tumor, gastritis, mastitis, stomach-aches and arthralgia (Lee et al. 2001;Shin et al. 2004;Lee et al. 2007;Yang et al. 2011;Liu et al. 2017a;Deng et al. 2021).To date, more than 60 Isodon species in China have been phytochemically investigated, and a large amount of terpenoids (especially ent-kaurane diterpenoids) have been isolated and characterized from this genus (Wang et al. 1995;Liu et al. 2017b;Hu et al. 2020;Li et al. 2021).
I. macrocalyx Kudo, a plant of the genus Isdodon, is mainly widespread in the south China (Agendae Academiae Sinicae Edita 1977).I. macrocalyx has been used in Chinese popular folk medicine for the treatments of hepatitis, gastricism, mastitis and cancer for a long history (Shi et al. 2007;Wang et al. 2015).In our first study, the ethyl acetate extract of I. macrocalyx showed good anti-HBV activity.To inspire further use of this plant and identify new anti-HBV agents, further investigations were carried out.Two new (1-2) and four known triterpenoids (3-6) were isolated and identified from the EtOAc extract of aerial parts of I. macrocalyx.All compounds (1 À 6) were evaluated for the anti-HBV activities in vitro using the HBV transfected HepG2.2.15 cell line.
The six isolates (1-6) from I. macrocalyx were tested for their abilities to inhibit the secretion of HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in HBV-infected 2.2.15 cells using lamivudine as a positive control (Table S3).The results showed that 2 and 3 exhibited anti-HBV activities with IC 50 values of 60.35 mM and 63.72 mM for HBsAg at no cytotoxicity, and IC 50 values of 83.26 mM and 85.04 mM for HBeAg at no cytotoxicity.

General
Optical rotations were measured on an Anton Paar MCP500 polarimeter (k 589 nm, path length 1.0 cm).NMR experiments were conducted on Bruker Advance 600 MHz spectrometers with residual solvent as the internal standard.HRESIMS were recorded on a Thermo MAT 95XP mass spectrometerSemi-preparative.HPLC separations were conducted on an Agilent 1260 instrument equipped with a DAD and an Agilent Zorbax SB-C 18 column (5 lm, 9.4 Â 250 mm) at a flow rate of 2.5 mL/min.Analytical HPLC was conducted on a Waters 2695 instrument equipped with a PAD detecter and a Waters C 18 column (5 lm, 4.6 Â 250 mm) at a flow rate of 1.0 mL/min.OD values of 96-well were measured with an imark Bio-Rad plate microplate reader.Silica gel (200-300 mesh, Qingdao Marine Chemical CoLtd, China) and ODS C 18 (50 lm, Merck, Germany) were used for the preliminary separation of crude samples, and a sephadex LH-20 column (Amersham Pharmacia Biotech AB, Sweden) as used for the final purification.L-arabinose, D-glucose, L-glucose, and D-apiose were used as sugar standards (Sigma-Aldrich, Munich, Germany).Other chemicals were of analytical or HPLC grade.

Plant material
Dried aerial part (20.0 kg) of I. macrocalyx was obtained from Jinxiu in Guangxi Province, China, in September, 2019 and identified by associate professor Yusong Huang (Guangxi Institute of Botany).A voucher specimen (NoID-20190915) is deposited at the State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, Guangxi Normal University, China.

Hydrolyses of cardiac glycosides to determine the linking sugars
Macrocalyxosides A-B (1-2) were individually dissolved in 1 M HCl (hydrochloric acid) and refluxed at 80 C for 1 h.Each reaction mixture was then fractionated with ethyl acetate (EtOAc, 3 times).The remaining aqueous phase was evaporated to dryness under a vacuum to remove water (H 2 O).Analytical HPLC was conducted on a Waters 2695 instrument (Waters Corporation, Milford, Massachusetts MA, USA) using a Waters 2424 ELSD detector.An Asahipak NH 2 P-50-4E (4.6 mm Â 250 mm, 5 lm) column was used, and the HPLC conditions were as follows: column temp, 35 C; mobile phase, acetonitrile:water, 78:22; flow rate, 1.0 mL/min.D-glucose and L-araboes were confirmed by comparison of their retention times with those of authentic samples.D-glucose (t R ¼ 7.35 min) and D-apiose (t R ¼ 7.87 min) were confirmed as the sugar units of macrocalyxoside B (1) by HPLC with an authentic sugar samples.The presence of Larabinose (t R ¼ 6.95 min) was detected by HPLC with standard sugar in the water phase of macrocalyxoside A (2).
, ten methylene carbons, and five methyl carbons.The 13 C NMR and HSQC spectra showed two anomeric carbon to proton couplings (d C 103.0 with d H 4.90 and d C 110.3 with d H 4.97), which indicated the presence of two sugar units.Analyses of the 1 H-NMR, 13 C-NMR, DEPT, HSQC, 1 H-1 H COSY, and HMBC data of 1 showed that the characteristic signals of aglycone are almost the same to those of ent-3b-hydroxymanoyl oxide . Fr 4.2 (8.8 g) was further purified on a RP C 18 column and eluted with H 2 O-MeOH mixtures (10:90 to 0:100) to give five fractions (Fr 4.2.1 to Fr 4.2.5).Fr 4.2.2 was subjected to Sephadex LH-20 CC (eluted with MeOH) and semi-preparative HPLC (eluted with H 2 O-MeOH, 47:53) to yield compound 5 (t R 23.5 min, 5.6 mg).Fr 4.3 (18.1 g) was applied to a C 18 RP silica gel column and eluted with H 2 O-MeOH mixtures (10:90 to 0:100)) to give five subfractions (Fr 4.3.1 to Fr 4.3.5).Fr 4.3.4(121.0 mg) was separated on a Sephadex LH-20 column and further purified by RP-HPLC eluted with H 2 O-MeOH (55:45) to obtain compounds 2 (t R 16.7 min, 5.3 mg) and 3 (t R 18.2 min, 4.9 mg).Fr 4.6 (3.3 g) was subjected to semi-preparative HPLC (eluted with H 2 O-MeOH, 60:40) to yield compound 1 (t R 31.6 min, 4.1 mg) 1 H and 13 C NMR data, see Table