Triterpenoid Saponins from the Roots of Cyathula Officinalis and Their Inhibitory Effects on Nitric Oxide Production

Chinese Journal of

Natural Chinese Journal of Natural Medicines 2017, 15(6): 04630466 Medicines

doi: 10.3724/SP.J.1009.2017.00463

Triterpenoid saponins from the roots of Cyathula officinalis and their inhibitory effects on nitric oxide production

JIANG Yun-Tao1, 2, YAN Wen-Jing1, 2, QI Chu-Lu1, 2, HOU Ji-Qin1, 2, ZHONG Yan-Ying1, 2, LI Hui-Jun1, WANG Hao1, 2 *, LI Ping1

1 State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China; 2 Department of Natural Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, China

Available online 20 Jun., 2017

[ABSTRACT] The present study was designed to investigate the chemical constituents of the roots of Cyathula officinalis. Compounds were isolated by silica gel, Sephadex LH-20, ODS column chromatography, and preparative HPLC. Their structures were determined on the basis of 1D and 2D NMR techniques, mass spectrometry, and chemical methods. One new oleanane-type triterpenoid saponin, 28-O-[α-L-rhamnopyranosyl-(1→3)-β-D-glucuronopyranosyl-(1→3)-β-D-glucopyranosyl] hederagenin (1), was isolated from the roots of Cyathula officinalis. The anti-inflammatory activities of the isolates were evaluated for their inhibitory effects against LPS-induced nitric oxide (NO) production in RAW 264.7 macrophages cells. Compounds 2, 4, and 6 exhibited moderate anti-inflammatory activities.

[KEY WORDS] Cyathula officinalis; Amaranthaceae; Triterpenoid saponins; Nitric oxide inhibition [CLC Number] R284 [Document code] A [Article ID] 2095-6975(2017)06-0463-04

 hibitory effects on NO production in LPS-stimulated RAW 264.7 Introduction macrophages cells. Cyathula officinalis Kuan belongs to Amaranthaceae Results and Discussion family and grows in the southwest of China. The roots of C. officinalis as traditional Chinese medicine are widely used for Compound 1 was obtained as a white amorphous powder. removing blood stasis, restoring menstrual ﬂow, easing joint The HR-ESI-MS of 1 showed a quasimolecular ion [M + Na]+ + movement, and inducing diuresis for treating stranguria, ac- at m/z 979.487 1 (Calcd. for [C48H76O19 + Na] 979.487 1), [1] cording to the theory of traditional Chinese medicine . consistent with a molecular formula C48H76O19. The IR spec- Nowadays, the aqueous extract of C. officinalis has been re- trum of 1 displayed absorption bands of hydroxyl group at 3 ported to possess anti-inflammation, antihypertensive and 420 cm–1, carbonyl group at 1 735 cm–1, and double bond at 1 antifertility activities, and its clinical use is significant in 637 cm–1. The 1H NMR spectra showed six angular methyl China [2-4]. Previous studies on this species have resulted in proton signals at δH 0.87, 0.88, 0.91, 0.92, 1.05, and 1.21 (each [5] [6-7] the isolation of glycosides , phytoecdysteroids , and 3H, s), and an olefinic proton at δ 5.35 (1H, br s, H-12). The [8] H polysaccharide . In our continued investigation on 13C NMR spectrum of 1 displayed 48 signals, including six anti-inflammatory constituents from the roots of C. officinalis, tertiary methyl signals at δC14.1 (C-24), 16.6 (C-25), 18.0 a new triterpenoid saponin (1) and six known compounds (2–7) (C-26), 24.1 (C-27), 33.6 (C-29), and 24.1 (C-30), two ole- were isolated. All isolates were evaluated for their in vitro in- finic carbons at δ 122.4 (C-12) and 144.6 (C-13), one oxy- C genated methylene at δC 64.6 (C-23), one oxygenated methine [Received on] 18-Jul.-2016 at δC 73.2 (C-3), and one carbonyl group at δC 176.9 (C-28) (Ta- 1 [Research funding] This work was supported by the National New ble 1). The H NMR data of 1 showed the presence of three

Drug Innovation Major Project of China (No. 2012ZX09304005002), anomeric protons at δH 6.30 (d, J = 8.2 Hz, H-1′of Glc), 5.14 (d, the Fundamental Research Funds for the Central Universities (No. J = 7.2 Hz, H-1′′of GluA), and 6.27 (br s, H-1′′′of Rha), which were JKFP2013003) and National Undergraduate Training Program for correlated with the carbon signals at δ 96.2 (C-1′), 106.5 Innovation and Entrepreneurship (No. G13031). C [*Corresponding author] Tel: 86-25-86185376, Fax: 86-25 (C-1′′), as well as 103.2 (C-1′′′) in the HSQC spectrum, respectively. 85301528; E-mail: [email protected] The oleanane-type aglycone of 1 was revealed by comparison of the These authors have no conflict of interest to declare. 1H and 13C NMR data with those of hederagenin-28-O-

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1 13 a Table 1 H NMR and C NMR data of compound 1 (500 MHz and 125 MHz, C5D5N)

No. δC δH (J, in Hz) No. δC δH (J, in Hz) 1 37.3 0.90 (d, 1.5),1.40 (d, 12.8) 28-Glc 1′ 96.2 6.30 (d, 8.2) 2 26.6 1.24 (o), 2.28 (d, 12.8) 2′ 74.7 4.36 (m) 3 73.2 4.60 (dd, 9.4,2.3) 3′ 82.4 4.27 (m) 4 43.9 - 4′ 76.3 4.10 (m) 5 47.5 1.65 (d, 12.0) 5′ 79.8 4.03 (m) 6 19.1 1.72 (d, 6.1), 1.84 (d, 6.1) 6′ 62.7 4.40 (m), 4.46 (m) 7 33.0 1.62 (m), 1.72 (d, 6.1) GluA 8 40.4 - 1″ 106.5 5.14 (d, 7.2) 9 47.9 1.65 (d, 12.0) 2″ 74.6 4.35 (m) 10 34.5 - 3″ 82.6 4.36 (m) 11 24.3 1.89-1.90 (o) 4″ 79.4 4.29 (m) 12 122.4 5.35 (br s) 5″ 78.1 4.30 (m) 13 144.6 - 6″ 173.7 14 42.6 - Rha 15 28.8 1.13 (d, 12.0), 2.29 (m) 1′″ 103.2 6.27 (br s) 16 23.9 1.89(m), 2.26 (d, 11.5) 2′″ 73.0 4.76 (m) 17 48.6 - 3′″ 71.6 4.34 (m) 18 42.2 3.17 (dd, 13.3,3.4) 4′″ 72.2 4.43 (m) 19 46.6 1.22 (m), 1.71 (d, 6.1) 5′″ 70.2 5.07 (m) 20 31.2 - 6′″ 18.7 1.67 (d, 6.0) 21 33.0 1.62 (m), 1.72(d, 6.1) 22 33.3 0.89(d, 6.9),1.33 (d, 7.0) 23 64.6 3.64(d, 2.4), 4.13 (m) 24 14.1 0.91 (s) 25 16.6 0.88 (s) 26 18.0 1.05 (s) 27 24.1 1.21 (s) 28 176.9 - 29 33.6 0.92 (s) 30 24.1 0.87 (s) a The assignments are based upon 1H–1H COSY, HSQC, HMBC and ROESY experiments. Coupling constants (J) in Hz are given in parentheses. o: the abbreviation foroverlapped

[9] β-D-glucopyranosyl ester . The upfield shift of δC 176.9 (C-28), compound 1 was identified as 28-O-[α-L-rhamnopyranosyl- compared with that of hederagenin [10], indicated that the (1→3)-β-D-glucuronopyranosyl-(1→3)-β-D-glucopyranosyl]- sugar moieties were attached to the aglycone at C-28 position. hederagenin, as shown in Fig. 1. Compound 1 had three hexoses according to the positive-ion The structures of known compounds were characterized + ESI-MS data at m/z 828.43 [M + NH4 – 146] , 652.30 [M + as 3-O-{β-D-glucopyranosyl-(1→2)-[α-L-rhamnopyanosyl- + + NH4 – 146 – 176] , 471.43 [M – H – 146 – 176 – 162] . Acid (1→3)]-(β-D-glucuronopyranosyl)}-28-O-β-D-glucopyranosy hydrolysis of 1 gave L-rhamnose, D-glucose, and D-glucuronic l oleanolic acid (2) [11], 3-O-[α-L-rhamnopyranosyl-(1→3)- acid, confirmed by HPLC-UV analysis of the respective β-D-glucuronopyranosyl]-28-O-β-D-glucopyranosyl oeanolic isothiocyanate derivatives with authentic references. The acid (3) [12], 3-O-β-D-glucuronopy-ranosyl-28-O-β-D-glucopyranosyl linkages and sequence of sugar chain of 1 were corroborated oleanolic acid (4) [13], 3-[α-L-rhamnopyranosyl-(1→3)-β-D- by the following HMBC correlations: δH 6.30 (H-1′of Glc) / glucuronopyranosyl]-28-O-[β-D-xylopyranosyl-(1→2)-β-D- δC176.9 (C-28), δH 5.14 (H-1′′of GluA) / δC 82.4 (C-3′of Glc), glucopyranosyl] oeanolic acid (5) [13], cyasterone (6) [14-15], and δH 6.27 (H-1′′′of Rha) /δC 82.6 (C-3′′of GluA) (Fig. 2). The [16] 1 1H and 13C NMR data were fully assigned by the 1H–1H COSY, and 7- O-Methyl-loganin (7) , by comparisons of their H 13 HSQC, HMBC and ROESY spectra. Based on these evidences, and C NMR data with those reported values. Compounds 2,

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5, and 7 were isolated from this plant for the first time. guanidine (16.60 μmol·L−1), the positive control. The prelimi- nary structure-activity relationship analysis indicated that the linkage of sugar moieties at a C-28 carbonyl group of triterpenoid aglycone decreased anti-inflammatory activities among the oleanane-type saponins 1–5. These findings also suggested that the anti-inflammatory activities of C. officinalis were partly attributed to these sterone and triterpenoid saponins.

Table 2 Inhibitory activities of compounds 1–7 against LPS-induced NO production in RAW264.7 macrophages (n = 3) IC IC Compounds 50 Compounds 50 (μmolL–1) (μmolL–1) 1 81.55 ± 0.54 5 >200 2 22.98 ± 1.35 6 22.51 ± 0.27 3 30.41 ± 0.66 7 >200 4 25.00 ± 0.23 AHa 16.60 ± 0.23 a Aminoguanidine Hydrochloride, the positive control

Experimental

General experimental procedures Melting points were determined on an X-4 melting point microscopic apparatus and were uncorrected. The HR-ESI- MS spectra were measured with an Agilent 6210 Q-TOF mass spectrometer equipped with ESI source. The IR spectra were recorded on a Nicolet Is10 Adv FTIR Spectrometer with KBr pellets. The NMR spectra were recorded on a Bruker Avance 500 MHz instrument, operating at 500 MHz for 1H and 125 MHz for 13C, respectively. Column chromatography was Fig. 1 Structures of compounds 1−7 performed on silica gel (200–300 mesh, Qingdao Marine Chemistry Ltd., China), Sephadex LH-20 (Pharmacia Fine

Chemical Co., Ltd., Germany), and LI Chroprep RP-C18 gel (40−63 μm, Merck, Germany). Preparative HPLC was car-

ried on a Shim-Pack preparative HPLC column (C18, 20 mm × 250 mm, 5µm) equiped with LC-8A pump (Shimadzu, Kyoto, Japan). Plant materials The roots of Cyathula officinalis were collected from Ya'an, Sichuan Province, China, in September 2012. The plant materials were authenticated by Prof. Li Hui-Jun of the De- partment of Pharmacognosy, China Pharmaceutical University. A voucher specimen (No. 20121009) was deposited in the Department of Natural Medicinal Chemistry, China Pharma- ceutical University, Nanjing, China. Fig. 2 Key HMBC correlations of compound 1 Extraction and Isolation The air-dried roots (2 kg) of Cyathula officinalis were The cytotoxic effects of compounds 1–7 were evaluated extracted with 70% EtOH under reflux. After removal of the via the MTT assays to ensure no signiﬁcant toxicity at their solvent under vacuum, the residue was suspended in H2O and effective concentrations (0.01–100 μmol·L−1) required for the partitioned successively with petroleum ether (3 × 1 L), chlo- inhibition of NO production. All isolates were examined for roform (3 × 1.2 L), and n-BuOH (3 × 1.5 L). The n-BuOH their inhibitory effects on the nitric oxide (NO) production in portion (200 g) was fractionated by a silica gel column, elut- [17] LPS-stimulated RAW 264.7 macrophages using Griess assay ing with CHCl3/MeOH (90 : 10→50 : 50). Each fraction was (Table 2). Compounds 2, 3, 4, and 6 showed potent inhibitory subjected to repeated low pressure C18 column with activities in vitro with IC50 values being 22.98, 30.41, 25.00, MeOH/H2O (20: 80→80: 20) as five eluents (Frs. 1−5). Then and 22.51 μmol·L−1, respectively, comparable to that of amino- subfraction (Fr. 2 23 g) was purified by preparative HPLC

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−1 (MeOH/H2O 40: 60, flow rate, 15 mL·min ) to obtain Com- Bidentata Blume and Cyathula Officinalis Kuan combined pounds 1 (36.1 mg, tR = 35.5 min), 2 (121.3 mg, tR = 16.5 min), with herbal prescription [J]. J Gansu Coll Tradit Chin Med, and 3 (106.2 mg, tR = 23.6 min). Compounds 4 (21.0 mg, tR = 2012, 29(6): 67-70. 38.8 min) and 5 (23.7 mg, tR = 21.2 min) were obtained from [2] Bin XY, Shu GM, He GX, et al. Comparing study on an- −1 ti-inflammatory effects of Cyathula officinalis Kuan and its Fr. 4 (18 g) by Rp-HPLC (MeOH/H2O 48: 52, flow rate 15 mL·min ). The subfraction (Fr.3 11 g) was further separated by Sephadex adulterants [J]. Res Dev Market, 2013, 29(4): 341-343. [3] Qi M, Xin G, Zhu G. Effects of Cyathula officinalis Kuan al- LH-20 column (MeOH/H2O 50 : 50) and repeated C18 column cohol extract on blood pressure, ACE activities and cardiac with MeOH/H2O (50 : 50) to obtain compounds 6 (25.4 mg) and 7 (46.2 mg). myocyte diameter in spontaneously hypertensive rat [J]. China Acidic hydrolysis of compound 1 Modern Tradit Chin Med, 2010, 12(6): 34-37. 2M HCl (10 mL) was added into a MeOH (2 mL) solu- [4] Li QW, Ge L. Investigation on anticontraception of Cyathula tion of Compound 1 (2 mg), and the mixture was refluxed at officinalis`s extracts [J]. J Xi'an Med Univ, 1990, 11(1): 27-29. 80 °C for 2 h. The mixture was evaporated to dryness, and the [5] Zhou R, Li BG, Zhang GL, et al. Glycosides from roots of Cyathula officinalis Kuan [J]. Inter Plant Biol, 2005, 47(3): residue was partitioned between CH2Cl2 and H2O. The aqueous layer was concentrated under reduced pressure to give a 368-374. residue. Then, anhydrous pyridine (1 mL) and of D-cysteine [6] Zhou R, Li BG, Zhang GL. Chemical study on Cyathula offi- methyl ester hydrochloride (4 mg) were added to the residue, cinalis Kuan [J]. J Asian Nat Prod Res, 2005, 7(3): 245-252. and heated at 60 ºC for 1 h. Then Isothiocyanate (10 μL) was [7] Chen X, Li WS, Liang Y, et al. Identification of chemical components of Cyathula officinalis Kuan [J]. Chin Tradit added to the mixture and heated at 60 ºC for additional 1 h. Herbal Drugs, 2004, 35(9): 978-979. The reaction mixture was directly analyzed by HPLC eluting [8] Chen XM, Tian GY. Structural features of fructans from the with 23% CH3CN in 0.1% HAc for 40 min. The injection root of Cyathula officinalis Kuan [J]. Chin J Chem, 2003, 21(7): volume was 10 μL and detecting wavelength was 250 nm. 858-863. The absolute configuration of the monosaccharide was con- [9] Yand L, Jiang H, Wang QH, et al. A new feruloyl tyramine firmed to be L-rhamnose, D-glucose, D-glucuronic acid, by glycoside from the roots of Achyranthes bidentata [J]. Chin J comparison of the retention times of monosaccharide derivatives Nat Med, 2012, 10(1): 16-19. with those of standard sample, L-rhamnose (21.8 min), D-glucose [10] Ding HY, Lin HC, Teng CM, et al. Phytochemical and phar- (2.8 min), D-glucuronic acid (25.1 min), respectively. macological studies on Chinese paeonia species [J]. J Chin NO inhibitory activities and cell viability Chem Soc, 2000, 47(2); 381. The murine macrophage RAW 264.7 cells were cultured [11] Bore1 C, Hostettmann K. Molluscicidal Saponins from Swartzia in 96-well plate for 4 h and pre-treated with indicated concentra- madagascariensis [J]. Helv Chim Acta, 1987, 70(3): 570-576. tions of the isolates for 2 h before stimulation with 10 μg·mL−1 [12] Wang XJ, Zhu LZ. Studies on the saponin constituents of Niu LPS for 20 h. Equal volumes of cultured medium and Griess Qi (Achyrathes Bidentata) [J]. J Fourth Milit Med Univ, 1996, reagent (1% sulfanilamidein 5% phosphoric acid and 0.1% 17(6): 427-430. naphtylethylene diamine dihydrochloride in distilled water) [13] Sati OP, Bahuguna S, Uniyal S, et al. A new saopnin from were mixed and the absorbance at 540 nm was determined Deeringia amaranthoides [J]. 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Cite this article as: JIANG Yun-Tao, YAN Wen-Jing, QI Chu-Lu, HOU Ji-Qin, ZHONG Yan-Ying, LI Hui-Jun, WANG Hao, LI Ping. Triterpenoid saponins from the roots of Cyathula officinalis and their inhibitory effects on nitric oxide production [J]. Chin J Nat Med, 2017, 15(6): 463-466

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