Three new triterpenoids from Rubia schumanniana

Introduction

Rubia schumanniana, an endemic species, is mainly distributed in southwest China. As one of the substitutes of traditional Chinese medicine R. cordifolia, its roots have been used for the treatment of tuberculosis, rheumatism, contusion, febrility and menoxenia. Previous studies on this plant have resulted in the isolation of seven quinions and β-sitosterol.^1,2 As part of our continuing research on chemical constituents of medicinal plants from the genus Rubia, a systematic phytochemical investigation of the roots of R. schumanniana was carried out, which led to the isolation of three new triterpenoids (1–3), along with seven known triterpenoids, zamanic acid (4),³ maslinic acid (5),⁴ ursolic acid (6),⁵ rubifolic acid (7),⁶ oleanolic acid (8),⁷ karachic acid (9),⁸ and rubiarbonol K (10).⁹ All compounds were evaluated for cytotoxicity against three human cancer cell lines (Hela, BGC-823, A549). Herein, we report the isolation, structural determination, and cytotoxic activities of these compounds.

Results and Discussion

Compound 1 was obtained as a white powder with a positive specific rotation ([α]_D¹⁶ + 6.5). Its molecular formula, C₃₉H₅₄O₆, was deduced by HRESIMS (m/z 617.3856 [M – H]^–), indicating 13 degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3426 cm^–1), carbonyl (1689 cm^–1) and olefinic (1632 cm^–1) groups. The ¹³C NMR spectrum of 1 (Table 1) exhibited 39 carbons, including one trisubstituted double bond (δ_C 126.7, 139.3), one carboxyl (δ_C 180.3) and one p-hydroxycinnamoyl group, six methyls, ten methylenes (one oxygenated), six methines (one oxygenated), and five quaternary carbons. Comparison of the NMR data of 1 with those of zamanic acid (4) revealed that both compounds are ursane-type triterpenoids. The only difference between them was that the coupling constant of the disubstituted double bond in p-hydroxycinnamoyl group is 13.0 Hz in 1 while 16.0 Hz in 4. The HMBC correlations of H-30 with the ester carbonyl carbon, C-19, C-20, and C-21 enabled the p-Z-hydroxycinnamoyl group to be placed at C-30 (Figure 1). The relative configuration of 1 was deduced from the analysis of its ROESY spectrum (Figure 2). The observed NOE correlations of H-3/H-5 and Me-23, H-5/H-9, and H-9/Me-27 indicated that H-3, H-5, H-9, Me-23 and Me-27 are cofacial and assigned as α-oriented. In turn the cross-peaks of Me-25/Me-24 and Me-26, and H-20/H-18 and Me-29 indicated the β-oriented of H-18, H-20, Me-24, Me-25, Me-26 and Me-29. From the above evidences, the structure of 1 was established as 3β-hydroxy-urs-30-p-Z-hydroxycinnamoyl-12-en-28-oicacid.

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Compound 2 exhibited the same molecular formula C₃₉H₅₄O₆ as 1, as established by HREIMS at m/z 618.3890 [M]⁺. The NMR data of 2 (Table 1) were similar to those of oleanolic acid (8) except for the presence of one p-E-hydroxycinnamoyl group in the downfield region of 2 and the replacement of one methyl in 8 by one hydroxymethyl group (δ_C 74.9). HMBC correlations of H-30 with the ester carbonyl carbon (δ_C 167.7), C-19, C-20, C-21, and Me-29 indicated that the p-E-hydroxycinnamoyl group located at C-30 (Figure 1). The observed NOE correlations (Figure 2) of H-3/H-5 and Me-23, H-5/H-9, and H-9/Me-27 indicated that H-3, H-5, H-9, Me-23 and Me-27 are cofacial and assigned as α-oriented. In turn the cross-peaks of Me-25/Me-24 and Me-26, and H-18/Me-29 indicated the β-oriented of H-18, Me-24, Me-25, Me-26 and Me-29. Thus, the structure of 2 was assigned as 3β-hydroxy-olean-30-p-E-hydroxycinnamoyl-12-en-28-oic-acid.

Compound 3 gave the molecular formula C₃₀H₄₈O₃, based on HRESIMS (m/z 479.3504 [M + Na]⁺), requiring seven degrees of unsaturation. The IR spectrum showed absorption bands for hydroxyl (3442 cm^–1), carbonyl (1705 cm^–1) and olefinic (1640 cm^–1) groups. The ¹³C NMR spectrum data (Table 1) showed the presence of 30 carbon signals due to one trisubstituted double bond (δ_C 119.1, 157.2), one ketone carbon (δ_C 215.8), eight methyls, seven methylenes, seven methines (two oxygenated), and five quaternary carbons. Comparison of the NMR data of 3 with those of ursolic acid suggested that their structures are closely related.¹⁰ The main differences were that one characteristic trisubstituted double bond at C-12/C-13 in conventional ursane-type triterpenoids was absent in 3, while one different trisubstituted double bond, one carbonyl group, and one additional hydroxy group were present. The double bond was placed between C-14 and C-15, as determined by HMBC correlations (Figure 1) of H-15 (δ_H 5.75) with C-17 (δ_C 35.5) and of Me-26 and Me-27 with C-14 (δ_C 157.2). The location of the ketone carbon (δ_C 215.8) at C-12 was elucidated by HMBC correlations of H-9, H-11 and Me-27 with C-12. In addition, the position of the additional hydroxy group at C-6 was deduced by correlations of H-6 with H-5 in the ¹H-¹H COSY spectrum combined with HMBC correlations of H-5 with C-6 (Figure 1). Thus, the planar structure of 3 was established. The α-orientations of H-3, H-5, H-9, and Me-23 were established by NOE correlations of H-3/H-5 and Me-23 and H-5/H-9, and the β-orientations of H-6, H-18, H-20, Me-24, Me-25, Me-26, Me-27, Me-28, Me-29 were deduced by NOE correlations of H-6/Me-24 and Me-25, H-7β/H-6 and Me-26, H-18/Me-27, Me-28 and Me-29, and Me-29/H-20 (Figure 2). Accordingly, the structure of 3 was elucidated as 3β,6α-dihydroxy-urs-14-en-12-one.

All compounds were evaluated for cytotoxicity against three human cancer cell lines, Hela (human cervical carcinoma), BGC-823 (human stomach adenocarcinoma), and A549 (human lung adenocarcinoma), and results indicated that compounds 2–6 showed cytotoxicity with the IC₅₀ values of 10.75~18.87 μg/mL (Table 2).

Experimental Section

General Experimental Procedures. Optical rotations were measured with a Horiba SEPA-300 polarimeter. IR spectra were obtained by a Bruker FT-IR Tensor 27 spectrophotometer using KBr pellets. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. 1D and 2D NMR spectra were recorded on Bruker AX-400, DRX-500, or AV-600 spectrometers with TMS as an internal standard. Chemical shifts (δ) were expresses in ppm with reference to solvent signals. HREIMS were recorded on a Waters Auto Premier P776 spectrometer. HRESIMS were recorded on an API QSTER time-of-flight spectrometer. Analytical or Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax Eclipse-C₁₈ (4.6 mm × 150 mm; 9.4 mm × 250 mm) column. Cloumn chromatographies were performed using silica gel (200–300 mesh, Qingdao Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, China), Sephadex LH-20 (Pharmacia Fine Chemical Co., Uppsala, Sweden), and Lichroprep RP-18 gel (40–63 μM, Merck, Darmstadt, Germany). Fractions were monitored by TLC (GF 254, Qingdao Yu-Ming-Yuan Chemical Co. Ltd., Qingdao, China), and spots were visualized by heating silica gel plates sprayed with 10% H₂SO₄ in EtOH.

Plant Materal. The roots of R. schumanniana were purchased in August 2009 from the Yunnan Lv-Sheng Pharmaceutical Co. Ltd., Kunming, China. The material was identified by Prof. Xi-Wen Li of Kunming Institute of Botany. A voucher specimen (KUN0328859) was deposited at the Herbarium of Kunming Institute of Botany, Chinese Academy of Sciences.

Extraction and Isolation. The dried and powdered roots of R. schumanniana (50 kg) were extracted with 70% aqueous MeOH (40 L × 3) for 12 hours at room temperature. After removal of the solvent under reduced pressure, the MeOH extract (18.6 kg) was suspended in H₂O and partitioned successively with EtOAc and n-BuOH to give an EtOAcsoluble portion (3.7 kg) and a n-BuOH-soluble portion (4.2 kg). The EtOAc part was chromatographed on silica gel column eluting with chloroform-methanol (1:0, 95:5, 9:1, 8:2, 7:3, and 0:1) to afford fractions Ⅰ–Ⅳ. Fraction Ⅰ (9:1, 163 g) was further chromatographed on silica gel using a petroleum ether-acetone gradient (10:1 to 0:1) as the eluent to yield 6 subfractions, Ⅰ-1–Ⅰ-6. Subfractions Ⅰ-1 was chromatographed with RP-18, and then separated by semi-preparative HPLC (CH₃CN:H₂O, 80:20) to yield 1 (12 mg) and 2 (2.6 mg). 4 (23 mg) was purified from subfractions Ⅰ-2 by repeated chromatographed with silica gel. Subfractions Ⅰ-4 was chromatographed on silica gel using a chloroform-methanol gradient (50:1 to 10:1) as the eluent, and then purified over Sephadex LH-20 eluted with chloroform-methanol (1:1), then by semipreparative HPLC (CH₃CN:H₂O, 60:40 and 73:27) to yield 5(22 mg), 6 (8 mg), 8 (35 mg) and 3 (7 mg), respectively. Subfractions Ⅰ-6 was chromatographed on silica gel using a chloroform-acetone gradient (50:1 to 0:1) as the eluent, and then purified by semi-preparative HPLC (CH₃CN:H₂O, 65:35) to yield 7 (15 mg), 9 (10.2 mg) and 10 (6 mg).

3β-Hydroxy-urs-30-p-Z-hydroxycinnamoyl-12-en-28-oicacid (1): white powder; [α]_D¹⁶ + 6.5 (c 0.08, MeOH:CHCl₃ = 1:1); UV (MeOH) λ_max (log ε) 202 (4.16), 312 (4.14) nm; IR (KBr) ν_max 3426, 2965, 2937, 2873, 1689, 1632, 1606, 1514, 1456, 1377, 1311, 1277, 1258, 1202, 1184, 1029, 997, 833, 519 cm^–1; 1H and ¹³C NMR data, see Table 1; negative ESIMS m/z 617 [M – H]^–; negative HRESIMS m/z 617.3856 [M – H]^–(calcd for C₃₉H₅₃O₆, 617.3842).

3β-Hydroxy-olean-30-p-E-hydroxycinnamoyl-12-en-28-oic-acid (2): white amorphous powder; [α]_D¹⁶ + 13.1 (c 0.13, MeOH); UV (MeOH) λmax (log ε) 202 (4.16), 227 (4.01), 313 (4.22) nm; IR (KBr) ν_max 3429, 2938, 2875, 1692, 1632, 1606, 1515, 1456, 1387, 1167, 1027, 996, 833, 519 cm^–1; ¹H and ¹³C NMR data, see Table 1; positive EIMS m/z 618 [M]⁺; positive HREIMS m/z 618.3890 [M]⁺ (calcd for C₃₉H₅₄O₆, 618.3920).

3β, 6α-Dihydroxy-urs-14-en-12-one (3): white amorphous powder; [α]_D²⁰ – 10.7 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 201 (3.41) nm; IR (KBr) ν_max 3442, 2927, 2866, 1705, 1640, 1462, 1382, 1140, 1036, 987 cm^–1; ¹H and ¹³C NMR data, see Table 1; positive ESIMS m/z 479 [M + Na]⁺; positive HRESIMS m/z 479.3504 [M + Na]⁺ (calcd for C₃₀H₄₈O₃Na, 479.3501).

Cytotoxicity Assay. The cytotoxicity of all compounds against Hela, A549, and BGC-823 cancer cell lines was measured using the sulforhodamine B (SRB) assay. Taxol was used as positive control. Cells were plated in 96-well culture plates for 24h before treated with serial dilutions of all compounds. After being incubated for 48 h, cells were fixed with 25 μL of ice-cold 50% trichloroacetic acid and incubated at 4 OC for 1 h. After washing with distilled water and airdrying, the plate was stained for 15 min with 100 μL of 0.4% SRB (Sigma) in 1% glacial acetic acid. The plates were washed with 1% acetic acid and air-dried. For reading the plate, the protein-bound dye was dissolved in 100 μL of 10 mM Tris base. The absorbance was measured at 560 nm. All tests were performed in triplicate, and results are expressed as IC₅₀ values.

Notes

Electronic Supplementary Material

Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s13659-012-0038-8 and is accessible for authorized users.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (U1032602, 91013002, 30725048), the National New Drug Innovation Major Project of China (2011ZX09307-002-02), and the National Basic Reaearch Program of China (2009 CB522300).