English

• 1.   Introduction

  Lycopodiella cernua (L.) Pic. Serm, belonging to the family Lycopodiaceae, has been used as a traditional Chinese folk medicine to treat rheumatism, quadriplegia and contusion for centuries.^[1] Palhinhaea cernua (L.) Franco & Vasc. is a synonym of Lycopodiella cernua. Previous phytochemical study of this plant species has led to the isolation of serratane triterpenoids, ^[2-6] alkaloids, ^[7-13] flavonoids, ^[14] and neolignans.^[4] Serratane triterpenoids and lycopodium alkaloids are characteristic components of L. cernua plant. Serratane is a unique class of pentacyclic triterpenoids with a central seven-membered ring C, which is rich in Lycopodiaceae family. Recently, the structural classification, biological activities and hypotheses about biosynthetic pathways of serratane-type triterpenes from Lycopodiaceae family was comprehensively reviewed.^[15] As part of our research on novel serratane triterpenoids from L. cernua, ^{[2, 6]} two serratane triterpenoids, 3β, 14α, 15α, 21β-tetrahydroxyserrat-15-(3'-methoxyl-4'-hydroxybenzoa-te) (1), and 16-oxoserrat-14-en-3β, 21α-diol (2), were isolated from the whole plant of Lycopodiella cernua (Figure 1). Compound 1 was a new one. Herein, we report the isolation, structural identification and anti-influenza A virus (IAV) activity of these two compounds.

  Figure 1

  

  Figure 1.  Structures of compounds 1 and 2

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  2.   Results and discussion

  Compound 1 was obtained as a white amorphous powder. The molecular formula of 1 was determined as C₃₈H₅₈O₇ by positive HRESIMS (m/z [M+H]⁺ 627.4247, calcd for C₃₈H₅₉O₇: 627.4255) in combination with the ¹³C and DEPT NMR analysis. The ¹H NMR spectrum (Table 1) displayed the signals for seven methyls at δ_H 0.74 (s, 3H, H-23), 0.92 (s, 3H, H-24), 0.84 (s, 3H, H-25), 1.07 (s, 3H, H-26), 1.02 (s, 3H, H-28), 0.95 (s, 3H, H-29), 0.87 (s, 3H, H-30), an oxygenated methyl at δ_H 3.92, three methines at δ_H 3.11 (dd, J=11.5, 4.8 Hz, 1H, H-3), 3.36 (t, J=2.5 Hz, 1H, H-21), and 4.94 (dd, J=11.3, 4.6 Hz, 1H, H-15), and a 1, 3, 4-trisubstituted benzene ring at δ_H 7.68 (d, J=1.9 Hz, 1H, H-2'), 6.86 (d, J=8.2 Hz, 1H, H-5'), 7.70 (dd, J=8.2, 1.9 Hz, 1H, H-6'). The ¹³C and DEPT NMR spectra suggested the presence of ten quaternary carbons, ten methines, ten methylenes, and eight methyls. The ¹³C NMR spectra (DEPT) showed signal at δ_C 55.8 for a methylene, which was characteristic signal of C-27 in serratane-type triterpenoid.^[6] By detailed analysis of the HMBC spectrum of compound 1 and comparison of its NMR data with those of 3β, 14α, 15α, 21α-tetrahydroxyserrat-3-(3′-me- thoxyl-4′-hydroxybenzoate), ^[2] the serratane triterpenoid skeleton was assigned as 3, 14, 15, 21-tetrahydroxyserratane. Except 30 carbons signals for a serratane triterpenoid, the remaining signals in the ¹H NMR and ¹³C NMR spectra of compound 1 indicated the presence of a 3'-methoxy-4'- hydroxybenzoate group through comparing corresponding NMR data with 3β, 14α, 15α, 21α-tetrahydroxyserrat-3-(3'- methoxyl-4'-hydroxybenzoate).^[2] The ester linkage between C-15 of serratane triterpenoid and C-7' of 3'-meth- oxy-4'-hydroxybenzoate was established by heteronuclear multiple bond correlation (HMBC) correlation (Figure 2) from H-15 (δ_H 4.94) to C-7′ (δ_C 168.0). The big coupling constant (J=11.5 Hz) of H-3 indicated ax-ax coupling with axial H-2, while the small coupling constant (J=4.8 Hz) of H-3 indicated ax-eq coupling with equatorial H-2. Therefore, H-3 was at axial bond and was α-orientated.^[3] In the same way, H-21 appeared to be equatorial (on the α-face) according to its small coupling constants of 2.5 Hz.^[3] The nuclear overhauser effect spectroscopy (NOESY) correlation (Figure 2) between H-15 and CH₃-26β, confirmed the β configuration of H-15. According to literature, ^[16] osmolation of lycernuic acid A diacetate gave two isomeric glycols. The minor glycol was proven to be 14α, 15α-diol, while the major one to be 14β, 15β-diol. The chemical shifts of C-17 in 14α, 15α-diol and 14β, 15β-diol were at δ_C 48.0 and 40.9, respectively. Compound 1 had a chemical shift of C-17 at δ_C 47.6, which indicated 14α, 15α configurations of two hydroxyl groups in compound 1. Therefore, compound 1 was elucidated as 3β, 14α, 15α, 21β-tetrahydroxyserrat-15-(3′-methoxyl-4′-hydroxybenzo-ate) as shown in Figure 1.

  Table 1

  

  Table 1.  ¹H NMR and ¹³C NMR (600 and 150 MHz, resp.) data for 1 in CD₃OD

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  No.	δH	δC
  1	1.78~1.80 (m, 1H), 0.99~1.01 (m, 1H)	39.9
  2	1.58~1.60 (m, 2H)	28.2
  3	3.11 (dd, 11.5, 4.8, 1H)	79.7
  4		40.0
  5	0.78 (dd, 9.1, 5.2, 1H)	57.1
  6	1.44~1.46 (m, 2H)	19.6
  7	1.24~1.26 (m, 2H)	46.7
  8		38.6
  9	1.13 (dd, 11.2, 2.2, 1H)	63.2
  10		39.3
  11	1.92~1.94 (m, 1H), 1.22~1.24 (m, 1H)	27.8
  12	1.72~1.74 (m, 1H), 1.47~1.49 (m, 1H)	24.6
  13	1.37~1.39 (m, 1H)	59.7
  14		78.1
  15	4.94 (dd, 11.3, 4.6, 1H)	81.0
  16	1.89~1.91 (m, 1H), 1.68~1.70 (m, 1H)	25.3
  17	1.57~1.59 (m, 1H)	47.6
  18		39.8
  19	1.49~1.51 (m, 1H), 1.40~1.42 (m, 1H)	33.7
  20	2.00~2.02 (m, 1H), 1.56~1.58 (m, 1H)	26.2
  21	3.36 (t, 2.5, 1H)	76.7
  22		38.7
  23	0.92 (s, 3H)	28.6
  24	0.74 (s, 3H)	16.1
  25	0.84 (s, 3H)	16.6
  26	1.07 (s, 3H)	21.4
  27	α, 1.36 (d, 15.5, 1H), β, 1.66 (d, 15.5, 1H)	55.8
  28	1.02 (s, 3H)	16.5
  29	0.87 (s, 3H)	22.6
  30	0.95 (s, 3H)	29.0
  1'		122.9
  2'	7.68 (d, 1.9, 1H)	114.0
  3'		148.8
  4'		152.9
  5'	6.86 (d, 8.2, 1H)	116.0
  6'	7.70 (dd, 8.2, 1.9, 1H)	125.5
  7'		168.0
  3'-OCH3	3.92 (s, 3H)	56.6

  Figure 2

  

  Figure 2.  Significant HMBC (from H to C) and NOESY correlations of compound 1

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  The structure of known compound 2 was identified as 16-oxoserrat-14-en-3β, 21α-diol (2) by interpretation of its spectroscopic data, as well as by comparison with reported values.^[17]

  Compounds 1 and 2 were evaluated for their anti-IAV activity against A/WSN/33 (H1N1). Preliminary antiviral screening showed that compounds 1 and 2 displayed 9% and 36% inhibition activity at 50 µmol•L^-1, respectively.

  3.   Conclusions

  In conclusion, two serratane triterpenoids, 3β, 14α, 15α, 21β- tetrahydroxyserrat-15-(3'-methoxyl-4'-hydroxybenzoate) (1) and 16-oxoserrat-14-en-3β, 21α-diol (2), were isolated from the whole plant of Lycopodiella cernua. Compound 1 was a new one. These two compounds were isolated from Lycopodiella genus for the first time.

  4.   Experimental section

  4.1   General

  Optical rotations were measured on a Perkin-Elmer Model 341 polarimeter (Perkin-Elmer, Inc., Waltham, MA). UV spectra were recorded on a Perkin-Elmer Lambda 650 UV-Vis spectrophotometer. Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker DRX-400 NMR spectrometer (Bruker Biospin Gmbh, Rheistetten, Germany) and a Bruker AVANCE 600 instrument. ESIMS was collected on an MDS SCIEX API 2000 LC/MS/MS instrument. HRESI-MS was acquired on a time-of-flight mass spectrometer (Bruker maxis 4G). Medium pressure liquid chromatography (MPLC) and preparative HPLC were carried out on a CXTH P3000 instrument (Beijing Chuang Xin Tong Heng Science and Technology Co., Ltd, Beijing, China) equipped with a UV 3000 UV-vis Detector. A Fuji-C18 column (10 µm, 30 mm×250 mm) was used for preparative HPLC. For column chromatography, silica gel (80~100 mesh and 200~300 mesh Qingdao Haiyang Chemical Co., Qingdao, China), and Sephadex LH-20 (Pharmacia Fine Chemical Co., Ltd., Oppsala, Sweden) were performed. Thin-layer chromatography (TLC) was conducted on pre-coated silica gel plates (HSGF₂₅₄, Yantai Jiang you Silica Gel Development Co., Ltd., Yantai, China) and spot detection was performed by spraying 10% H₂SO₄ in ethanol, followed by heating.

  4.2   Plant material

  The information of L. cernua plant material was published in our previous study.^[2]

  4.3   Extraction and isolation

  The extraction procedure was previously published, ^[2] and six serratane triterpenoids were isolated in our previous study.^[2] The remaining subfractions of L. cernua extracts were isolated in this study. Subfractions A and B were obtained from EtOAc fraction of L. cernua extracts after silica gel column chromatography and medium pressure liquid chromatography (MPLC). Subfraction A was applied to Sephadex LH-20 column chromatography (CHCl₃-MeOH, V:V=1:4), and followed by preparative HPLC (80% aqueous CH₃OH, 10 mL/min, R_t=84 min) to afford compound 1 (11 mg). Subfraction B was separated by Sephadex LH-20 column chromatography (CHCl₃-MeOH, V:V=1:4) to give subfractions B1 and B2. Compound 2^[17] (5 mg) was obtained from subfraction B2 by preparative HPLC (84% aqueous CH₃OH, 10 mL/min, R_t=84 min).

  3β, 14α, 15α, 21β-tetrahydroxyserrat-15-(3'-methoxyl-4'-hydroxybenzoate) (1): White amorphous powder, m.p. 220~222 ℃; $[\alpha ]_{\rm{D}}^{{\rm{20}}}$-12.61 (c 0.33, CH₃OH); UV (CH₃- OH) λ_max [log ε/(L•mol^-1•cm^-1)]: 217.02 (3.16), 224.94 (3.30), 263.32 (2.98), 293.53 (1.72) nm; ¹H NMR and ¹³C NMR see Table 1. HRESIMS (positive) calcd for C₃₈H₅₉O₇ [M+H]⁺ 627.4255, found 627.4247.

  4.4   Antiviral activity

  Two isolated compounds 1 and 2 were evaluated for their anti-IAV activities according to the previously reported 3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, ^[18] using zanamivir (200 nmol•L^-1) as a positive control. Test compounds were preliminary screened at two concentrations of 50 and 10 μmol•L^-1, and those which had more than 50% inhibition of influenza virus infection activity were further experimented for cytotoxicity and IC₅₀ evaluation. In detail, Madin Derby canine kidney (MDCK) cells were cultured overnight in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin in 96 well plates. A/WSN/33 (H1N1) influenza virus (100 TCID₅₀), which was propagated from 8-day-old chicken embryos, was added and incubated for 1 h. Then the supernatant was removed and washed with phosphate-buffered saline (PBS), and MDCK cells infected with influenza virus were exposed to 200 µL of compounds 1 and 2 at 50 or 10 μmol•L^-1. After 48 h of incubation, 100 μL of MTT solution, which was diluted by the medium to 0.5 mg/mL, was added and retained at 37 ℃ for 4 h. Then the supernatant was removed, followed by the addition of 150 μL of dimethyl sulfoxide (DMSO) to dissolve the formazan product. The optical density for each well was measured on the Tecan Genios Pro microplate reader (Bedford, MA, USA) at 570 nm.

  Supporting Information  ¹H NMR, ¹³C NMR, HSQC, HMBC, and NOESY spectra of compounds 1 and 2. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

  
  
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• 

  Figure 1  Structures of compounds 1 and 2

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  Figure 2  Significant HMBC (from H to C) and NOESY correlations of compound 1

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  Table 1.  ¹H NMR and ¹³C NMR (600 and 150 MHz, resp.) data for 1 in CD₃OD

  No.	δH	δC
  1	1.78~1.80 (m, 1H), 0.99~1.01 (m, 1H)	39.9
  2	1.58~1.60 (m, 2H)	28.2
  3	3.11 (dd, 11.5, 4.8, 1H)	79.7
  4		40.0
  5	0.78 (dd, 9.1, 5.2, 1H)	57.1
  6	1.44~1.46 (m, 2H)	19.6
  7	1.24~1.26 (m, 2H)	46.7
  8		38.6
  9	1.13 (dd, 11.2, 2.2, 1H)	63.2
  10		39.3
  11	1.92~1.94 (m, 1H), 1.22~1.24 (m, 1H)	27.8
  12	1.72~1.74 (m, 1H), 1.47~1.49 (m, 1H)	24.6
  13	1.37~1.39 (m, 1H)	59.7
  14		78.1
  15	4.94 (dd, 11.3, 4.6, 1H)	81.0
  16	1.89~1.91 (m, 1H), 1.68~1.70 (m, 1H)	25.3
  17	1.57~1.59 (m, 1H)	47.6
  18		39.8
  19	1.49~1.51 (m, 1H), 1.40~1.42 (m, 1H)	33.7
  20	2.00~2.02 (m, 1H), 1.56~1.58 (m, 1H)	26.2
  21	3.36 (t, 2.5, 1H)	76.7
  22		38.7
  23	0.92 (s, 3H)	28.6
  24	0.74 (s, 3H)	16.1
  25	0.84 (s, 3H)	16.6
  26	1.07 (s, 3H)	21.4
  27	α, 1.36 (d, 15.5, 1H), β, 1.66 (d, 15.5, 1H)	55.8
  28	1.02 (s, 3H)	16.5
  29	0.87 (s, 3H)	22.6
  30	0.95 (s, 3H)	29.0
  1'		122.9
  2'	7.68 (d, 1.9, 1H)	114.0
  3'		148.8
  4'		152.9
  5'	6.86 (d, 8.2, 1H)	116.0
  6'	7.70 (dd, 8.2, 1.9, 1H)	125.5
  7'		168.0
  3'-OCH3	3.92 (s, 3H)	56.6

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