New Serratene Triterpenoids from Palhinhaea cernua and Their Cytotoxic Activity

• Materials and Methods
• Supporting information
• Acknowledgements
• References

Abstract

Four new serratene triterpenoids, 3β,21β,24-trihydroxyserrat-14-en-24-(4′-hydroxybenzoate) (1), 3β,21α,24-trihydroxyserrat-14-en-3-(4′-hydroxybenzoate) (2), 3β,14α,15α,21α-tetrahydroxyserrat-14-en-3-(3′-methoxyl-4′-hydroxybenzoate) (3), and 3β,14α,15α,21α-tetrahydroxyserrat-14-en-21-acetyl-3-(4′-hydroxybenzoate) (4), together with two known ones, 5 and 6, were isolated from whole plants of Palhinhaea cernua. The structures of these new compounds were elucidated by spectroscopic methods. All the six compounds were tested for their in vitro cytotoxicity against three human cancer cell lines (K562, SMMC-7721, and SGC7901). Compound 5 showed cytotoxicity against the three test cell lines with IC₅₀ values of 20.3, 34.0, and 22.5 ug/mL, respectively. Compound 1 showed slight cytotoxicity against K562 cell lines with IC₅₀ value of 56.1 ug/mL, while no obvious inhibitory effects were detected for other compounds.

The serratene group is a unique family of pentacyclic triterpenoids possessing seven to five tertiary methyl groups and a central seven-membered ring C, some of which showed important pharmacological activities [1], [2], [3]. This group of triterpenoids was mainly discovered from plants belonging to the Pinaceae, Huperziaceae, and Lycopodiaceae families, together with a few other plant species, e.g., Primula rosea [4], Imperata cylindrical [5], Polypodium vulgare [6], and Lemmaphyllum microphyllum [7]. Recently, we have studied several plants of the Lycopodiaceae family and revealed some new serratene-type triterpenoids from those species [8], [9], [10], [11]. Palhinhaea cernua, a typical pteridophyte of the Lycopodiaceae family, has been used as a traditional Chinese folk medicine for centuries to treat rheumatism, whooping coughs, hepatitis, nephrolith, and others [12]. Due to a continued interest in searching for potentially new and bioactive natural products from medicinal fern plants, a phytochemical investigation on the EtOAc extract of the whole plants of P. cernua was carried out, which resulted in the isolation of four new serratene-type triterpenoids, 1–4, together with two known ones, 5 and 6 ([Fig. 1]). Herein, we report the isolation and structural elucidation of these compounds and describe their cytotoxicities against three human cancer cell lines.

Compound 1 was obtained as a white powder. Its molecular formula C₃₇H₅₄O₅ was determined by HREIMS m/z 578.3972 (calcd. 578.3966 for C₃₇H₅₄O₅). The ¹H NMR spectrum ([Table 1]) displayed signals readily recognized for six tertiary methyl groups at δ 0.76 (3H), 0.78 (3H), 0.81 (3H, s), 0.93 (3H), 1.15 (3H), and 1.53 (3H), four aromatic protons at δ 7.16 (2H, d, J = 8.6 Hz) and 8.37 (2H, d, J = 8.6 Hz), two oxygenated methine protons at δ 3.63 (1H, dd, J = 11.5, 4.7 Hz), 3.67 (1H, m), and one olefinic proton at δ 5.46 (1H, br.s). The ¹³C NMR spectrum ([Table 2]) in combination with DEPT spectra indicated, besides resonances for a 4-hydroxybenzoic acid moiety, the presence of 30 carbons including six methyls, eleven sp³ methylenes, six sp³ methines, five sp³ quaternary carbons, one sp² methine (δ 122.8), and one sp² quaternary carbons (δ 139.0), among which one sp³ methylene (δ 64.5) and two sp³ methine carbons (δ 75.2 and 80.0) were oxygenated. The spectroscopic data above supported 1 to be a trihydroxyserratene triterpenoid compound with one hydroxyl group esterified by a 4-hydroxybenzoic acid unit. By analysis of 2D NMR spectra (HSQC and HMBC), the ¹H and ¹³C NMR spectroscopic data were well assigned as shown in [Tables 1] and [2], which indicated that the center trihydroxyserratene triterpene unit was closely related to known compound 5 [13]. The observation of ¹H-¹³C long-range correlations of H-24 with C-7′ in the HMBC spectrum further confirmed the ester bond linkage between C-7′ and C-24 (see [Fig. 2]). Therefore, 1 was deduced as 3β,21β,24-trihydroxyserrat-14-en-24-(4′-hydroxybenzoate).

Compound 2 was also obtained as a white powder. Its molecular formula was determined as C₃₇H₅₄O₅, the same as 1, by HREIMS m/z 578.3970 (calcd. 578.3966 for C₃₇H₅₄O₅). In the ¹H NMR spectrum, two sets of signals, one for a unit of 3β,21α,24-trihydroxyserrat-14-en [14] and one for a 4-hydroxybenzoic acid moiety [δ _H 7.16 and 8.37 (each 2H, d, J = 8.4)] were observed ([Table 1]). Just like those for 1, the ¹³C NMR and DEPT spectra of 2 also displayed 37 carbon signals, corresponding to structure units of 3β,21α,24-trihydroxyserrat-14-en and the 4-hydroxybenzoic acid moiety [13]. The existence of the 4-hydroxybenzoic acid moiety in 2 was further supported by the detection of a molecular ion at m/z 577 and fragment ion peaks at m/z 136, 121 in the negative ESIMS. The data above indicated that 2 should contain a basic 3β,21α,24-trihydroxyserrat-14-en unit connected with a 4-hydroxybenzoic acid moiety. In the HMBC spectrum, the long-range correlations of H-3 with C-7′, C-2, and C-4 were exhibited, indicating the ester-bond linkage between C-7′ and C-3. Thus, 2 was established as 3β,21α,24-trihydroxyserrat-14-en-3-(4′-hydroxybenzoate).

Compound 3 was deduced to have the molecular formula C₃₈H₅₈O₇ as determined by HREIMS, m/z 626.4178 (calcd. for C₃₈H₅₈O₇, 626.4177). In its ¹³C-NMR and DEPT spectra, thirty-eight carbon signals (10×C, 9×CH, 11×CH₂, 8×CH₃) were exhibited, including eight methyls, eleven sp³ methylenes, six sp³ methines (three oxygenated, δ 80.7, 78.2, and 77.9), six sp³ quaternary carbons (one oxygenated, δ 76.8), three sp² methine, one carboxyl carbon, and three sp² quaternary carbons. The presence of a 3-methoxy-4-hydroxybenzoate (vanillic acid) moiety in the molecule was suggested by the presence of carbon signals at δ _C 166.8 (C), 153.3 (C), 148.5 (C), 125.1 (CH), 122.2 (C), 116.2 (CH), 113.8 (CH), and 55.7 (CH₃), confirmed by proton signals in its ¹H-NMR spectrum at δ _H 8.04 (1H, H-2′), 7.16 (1H, H-5′), 8.09 (1H, H-6′), and 3.66 (3H, 3′-OCH ₃). Further comparison of the ¹H and ¹³C NMR spectral data with those of 2 showed that compound 3 would have a central serratane triterpenoid unit similar to 2, with the exceptions that the resonances for the hydroxylmethylene group at C-24 and the double bond at C-14(15) were absent in 3. Instead, signals for an additional methyl group at C-24 [δ _C 28.5 and δ _H 1.12 (3H, s)] and two hydroxyl groups, one at C-14 (δ _C 76.8) and the other at C-15 [δ _C 78.2 and δ _H 3.56 (1H, d, 4.6 Hz)], were exhibited. These findings indicated that 3 possessed a basic 3,14,15,21-tetrahydroxyserratane skeleton with one hydroxyl group esterified by a vanillic acid unit in the molecule. The exhibited coupling constants of H-3 (dd, J = 11.3, 4.5 Hz) and H-21 (dd, J = 10.8, 5.3 Hz) in the ¹H-NMR spectrum suggested that the configuration of the two hydroxyl groups at C-3 and C-21 were β and α oriented, respectively [15], [16]. The α orientation of the two hydroxyl groups at C-14 and C-15 were supported by the closely related ¹³C NMR data of C and D rings of 2 with those of lycernuic acid C [3]. The HMBC correlations of H-3 with C-7′, C-4, and C-2 indicated the ester linkage of the vanillic acid unit at C-3. Thus, 3 was determined as 3β,14α,15α,21α-tetrahydroxyserrat-3-(3′-methoxyl-4′-hydroxybenzoate).

Compound 4 was established to have the molecular formula C₃₉H₅₈O₇ on the basis of HREIMS analysis. The similarity of the ¹³C NMR spectral data from C-1 to C-30 with those of 3 indicated that 4 had the same central serratane unit as that in 3. Careful comparison of the ¹H and ¹³C NMR spectral data ([Tables 1] and [2]) with those of 2 indicated that 4 possessed a 4-hydroxybenzoic acid unit the same as that in 2. The acetylation of the hydroxyl group at C-21 was supported by the appearance of NMR signals at δ _C 170.7 (-COCH₃), 21.1 (-COCH₃), δ _H 2.07 (-COCH₃) and the observed chemical shift change at C-21 (+Δ 2.8 ppm from that in 3), resulting from the substituent group effect exerted by the acetyl moiety. The connections of these units were then established by 2D NMR (HSQC and HMBC) analyses with a similar process just like that for 1–3. Eventually, the structure of 4 was deduced as 3β,14α,15α,21α-tetrahydroxyserrat-21-acetyl-3-(4′-hydroxybenzoate).

The other two known compounds, 3β,21β,24-trihydroxyserrat-14-en (5) [17] and 3α,21β,24,29-tetrahydroxy-16-oxoserrat-14-en (6) [10], were identified by comparison of their spectroscopic data with literature values.

All the six compounds were tested for their cytotoxicity against three human cancer cell lines (K562, SMMC-7721, and SGC7901) using the MTT method as described in the literature [17]. Compound 5 was found to show cytotoxicity toward the three cancer cell lines with IC₅₀ values of 20.3, 34.0, and 22.5 ug/mL, respectively, in comparison to the positive control mitomycin C with IC₅₀ values ranging from 13.0 to 22.1 ug/mL. Compound 1 showed cytotoxicity against K562 cell lines with IC₅₀ 56.1 ug/mL, while no obvious cytotoxic activity was detected for other compounds in this assay ([Table 3]). These results suggested that the coexistence of free β-OH groups at C-3 and C-21 in the serratene skeleton might be necessary for this type of serratene triterpenoids to “maintain” their potential cytotoxic activities.

Materials and Methods

The whole plants of Palhinhaea cernua, pre-sprayed with elicitor of coronalon (0.1 mg/mL in water), were collected at Sangzhi County, Hunan Province, PR China, in October 2009, and identified by Prof. Fu-Wu Xing, South China Botanical Garden, the Chinese Academy of Sciences (CAS). A voucher specimen (No: 20090522) has been deposited at the Laboratory of Phytochemistry, South China Botanical Garden, CAS. The powdered material (10 kg) was exhaustively extracted with 90 % EtOH at room temperature, and the ethanol extract was combined and evaporated to near dryness to get a residue, which was resuspended in 3000 mL of MeOH/H₂O (1 : 9) and partitioned with EtOAc (1500 mL × 4) to give an EtOAc fraction (230 g) which was further applied to repeated column chromatography to purify compounds 1-6.

Compound 1: white powder; [α]_D ²³ − 12.4 (c 0.2, CHCl₃); ¹³C and ¹H NMR spectral data, see [Tables 1] and [2]; negative ESIMS m/z: 577 [M – H]⁻, 457 [M – C₇H₅O₂]⁺; HREIMS: 578.3972 [M]⁺ (calcd. for C₃₇H₅₄ O₅, 578.3966).

Compound 2: white powder; [α]_D ²³ + 19.5 (c 0.3, MeOH); ¹³C and ¹H NMR spectral data, see [Tables 1] and [2]; negative ESIMS m/z: 577 [M – H]⁻, 136, 121, 92; HREIMS: 578.3970 [M]⁺ (calcd. for C₃₇H₅₄ O₅, 578.3966).

Compound 3: white powder; [α]_D ²³ − 4.1 (c 0.75, MeOH); ¹³C and ¹H NMR spectral data, see [Tables 1] and [2]; negative ESIMS m/z: 625 [M – H]⁻, 485, 151; HREIMS: 626.4178 [M]⁺ (calcd. for C₃₈H₅₈ O₇, 626.4177).

Compound 4: white powder; [α]_D ²³ − 1.2 (c 0.19, MeOH); ¹³C and ¹H NMR spectral data, see [Tables 1] and [2]; negative ESIMS m/z: 637 [M – H]⁻; HREIMS: 638.4182 [M]⁺ (calcd. for C₃₉H₅₈ O₇, 638.4177).

A detailed description of the cytotoxicity assay is available as Supporting Information. The positive control, mitomycin C (purity ≥ 98 %), was purchased from Kyowa Hakko Kogyo Co. Ltd. Tested compounds were demonstrated to be pure as evidenced by HPLC and NMR analyses (purity ≥ 95 %).

Supporting information

General experimental procedures, details on extraction and isolation, cytotoxicity assay details, and the ¹H and ¹³C NMR spectra for new compounds 1–4 are available as Supporting Information.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (30870248 and 30900125), the Chinese Academy of Sciences (KSCX2-YW-N-0804, Talent Project of SCBG 200733, 200747), and Key Laboratory of Plant Resources Conservation and Sustainable Utilization, South China Botanical Garden, CAS (200920 to J. Y.).

Conflict of Interest

There are no conflicts of interest among all authors of this manuscript.

• References

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Correspondence

• References

• 1 Tanaka R, Minami T, Tokuda H. Anti-initiating activity of 3β-methoxy-13α,14α-epoxyserratan-21β-ol (PJJ-34) from the stem bark of Picea jezoensis Carr. var. jezoensis . Chem Biodivers 2006; 3: 818-824
  CrossrefPubMedSearch in Google Scholar
• 2 Tanaka R, Minami T, Ishikawa Y, Tokuda H, Matsunaga S. Cancer chemopreventive activity of serratane-type triterpenoids from Picea jezoensis . Chem Biodivers 2004; 1: 878-885
  CrossrefPubMedSearch in Google Scholar
• 3 Zhang Z, Elsohly HN, Jacob MR, Pasco DS, Walker LA, Clark AM. Natural products inhibiting Candida albicans secreted aspartic proteases from Lycopodium cernuum . J Nat Prod 2002; 65: 979-985
  CrossrefPubMedSearch in Google Scholar
• 4 Bhutani KK, Kapoor R, Atal CK. Three bridged 14β,26-epoxy-C-chomo-pentacycle triterpenes from Primula rosea . Phytochemistry 1984; 23: 403-406
  CrossrefPubMedSearch in Google Scholar
• 5 Kulshreshtha MJ, Kulshreshtha DK, Rastogi RP. Review article: the triterpenoids. Phytochemistry 1972; 11: 2369-2381
  CrossrefPubMedSearch in Google Scholar
• 6 Ghisalberti EL, DeSouza NJ, Rees HH, Goodwin TW. Biosynthesis of the triterpene hydrocarbons of Polypodium vulgare . Phytochemistry 1970; 9: 1817-1823
  CrossrefPubMedSearch in Google Scholar
• 7 Ageta H, Shiojima K, Masuda K. Fern constituents: Onoceroid, α-onoceradiene, serratene and onoceranoxide, isolated from Lemmaphyllum microphyllum varieties. Chem Pharm Bull 1982; 30: 2272-2274
  CrossrefPubMedSearch in Google Scholar
• 8 Yan J, Zhang XM, Li ZR, Zhou L, Chen JC, Sun LR, Qiu MH. Three new triterpenoids from Lycopodium japonicum Thunb. Helv Chim Acta 2005; 88: 240-244
  CrossrefPubMedSearch in Google Scholar
• 9 Yan J, Yi P, Chen BH, Lu L, Li ZR, Zhang XM, Zhou L, Qiu MH. Five new serratane triterpenoids with poly-hydroxyl from Diphasiastrum complanatum . Phytochemistry 2008; 69: 506-510
  CrossrefPubMedSearch in Google Scholar
• 10 Yan J, Sun LR, Zhang XM, Li ZR, Zhou L, Qiu MH. Serratene triterpenoids from Palhinhaea cernua var. sikkimensis . Chem Pharm Bull 2009; 57: 1381-1384
  CrossrefPubMedSearch in Google Scholar
• 11 Yan J, Sun LR, Li W, Zhou L, Li ZR, Zhang XM, Yang L, Qiu MH. Cytotoxic serratane triterpenes from Diphasiastrum complanatum with a hydroxyl group at C-27. Planta Med 2010; 76: 353-357
  Thieme ConnectPubMedSearch in Google Scholar
• 12 Xiao PG. Modern Chinese materia medica. Beijing: Chemistry and Industry Press; 2002; 3: 135-139
  PubMedSearch in Google Scholar
• 13 Haruo S, Kazuo F, Xu GY, Cao X, Pan DJ. Assignment of the ¹H-and ¹³C-NMR spectra of four lycopodium triterpenoids by the application of new two-dimensional technique, heteronuclear multiple bond connectivity (HMBC). Agric Biol Chem 1988; 52: 1797-1801
  CrossrefPubMedSearch in Google Scholar
• 14 Zhou H, Li YS, Tong XT, Liu HQ, Jiang SH, Zhu DY. Serratane-type triterpenoids from Huperzia serrata . Nat Prod Res 2004; 18: 453-459
  CrossrefPubMedSearch in Google Scholar
• 15 Tanaka R, Ohmori K, Minoura K, Matsunaga S. Two new epoxyserratanes from the cuticle of Picea jeazoensis . J Nat Prod 1996; 59: 237-241
  CrossrefPubMedSearch in Google Scholar
• 16 Tanaka R, Tsujimoto K, Muraoka O, Matsunaga S. Two serratane triterpenoids from the stem bark of Picea jezoensis var. hondoensis . Phytochemistry 1998; 47: 839-843
  CrossrefPubMedSearch in Google Scholar
• 17 Mosmann T. Rapid colourimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65: 55-63
  CrossrefPubMedSearch in Google Scholar

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