Study on Chemical Constituents of Ethanol Extract from Yao Medicine Cissampelopsis spelaeicola

Shuang LIANG, Wenfang MA, Kewen LIANG, Lichun ZHAO, Gang DENG*, Dongyang XIE

1. College of Pharmacy, Guangxi University of TCM, Nanning 530001, China; 2. Guangxi Jianfeng Pharmaceutical Co., Ltd., Nanning 530104, China; 3. Guilin Niutai Biological Technology Co., Ltd., Guilin 541004, China

Abstract [Objectives] The research aimed to study the chemical constituents of ethanol extract from Yao medicine Cissampelopsis spelaeicola. [Methods] The petroleum ether, ethyl acetate and n-butanol fractions from 75% ethanol extract of C. spelaeicola were isolated and purified by silica gel, SephadexLH-20 gel column and AB-8 macroporous resin column, etc. The structures of the compounds were analyzed and identified by physicochemical properties and spectral data (mass spectrometry, hydrogen spectrum and carbon spectrum). [Results] Twelve compounds were isolated and identified from 75% ethanol extract of C. spelaeicola. β-sitostero (I) and stigmasterol (II) were isolated from petroleum ether fraction; p-hydroxybenzoic acid (III), β-daucossterol (IV), protocatechuic acid (V), 6β-hydroxyeremophi-7(11)-en-12, 8β-olide(VI), 10β-hydroxyeremophil-7(11)-en-8, 12-olide (VII), 10β-hydroxyeremophi-7(11), 8(9)-dien-8, 12-olide (VIII), quercetin (IX), hyperin (X) and 4α-hydroxy-eudesman-11-ene (XI) were isolated from ethyl acetate fraction; quercetin-3-O-robinobioside (XII) was isolated from n-butanol fraction. Compounds I-XII were isolated from C. spelaeicola for the first time. [Conclusions] The study can lay material foundation for activity study of C. spelaeicola.

Key words Cissampelopsis spelaeicola, Chemical constituents, Isolation and identification, Structural identification

1 Introduction

Nuomifeng is dry aboveground part ofCissampelopsisspelaeicola(Vant) C. Jeffrey et Y. L. Chen.C.spelaeicolais mainly distributed in Guangxi, Sichuan, Guizhou, and Yunnan, and its main production site is in Yao region of Guangxi, and it is traditional Yao medicine[1].C.spelaeicolahas long history as traditional Yao medicine, and is early recorded in theQualityStandardsofYao’sTraditionalMedicinalMaterialsinGuangxi. Its taste is pungent and slightly bitter, and nature is mild, with the functions of dispelling wind, removing dampness, clearing channels and activating collaterals.C.spelaeicolacould be used to cure rheumatism, rheumatoid arthritis, tendon spasm, infantile fever convulsion, poliomyelitis, polio sequelae and injury by falling[1]. Prior research by our research group found that the yield of 75% ethanol extract ofC.spelaeicolawas the highest. To understand its chemical composition and excavate the material basis of its pharmacological action, silica gel chromatography, SephadexLH-20 gel column chromatography, recrystallization and AB-8 macroporous resin column chromatography were used to separate, purify and identify chemical constituents of 70% ethanol extract fromC.spelaeicola, and the research aimed to provide the basis for further development and utilization of the medicinal material.

2 Materials

2.1Instruments1100 type of liquid chromatograph (USA Agilent Company); Eaquire HCT type of mass spectrometer, AVANCE-500 MHz type of superconducting NMR spectrometer (USA Bruker Daltonics Company); YRT-3 type of melting point instrument (Tianjin Jingtuo Instrument Technology Co., Ltd.); RE-52AA type of rotary evaporator (Shanghai Yarong Biochemical Instrument Co., Ltd.).

2.2MedicinalmaterialsandreagentsNuomifeng was collected in Pingxiang City of Guangxi in October of 2014, and was identified as aboveground part ofC.spelaeicola(Vant)C. Jeffrey et Y. L. Chen by associate research fellow Liang Kewen from Guangxi Jianfeng Pharmaceutical Co., Ltd. Its specimen was reserved in Guilin Yitiancheng Biotechnology Co., Ltd.

SephadexLH-20 gel (18-111 μm, USA Pharmacia Company); silica gel (100-200 and 200-300 meshes, Qingdao Ocean Chemical Plant); AB-8 macroporous resin (0.3-1.25 mm, Dalian Meilun Biotechnology Co., Ltd.); polyamide (30-60 and 80-100 meshes, Sinopharm Chemical Reagent Co., Ltd); β-daucossterol control (lot number: 474-58-8, purity: 98%) and β-sitostero control (lot number 83-46-5, purity: 98%) were all bought from Shanghai Guangrui Biotechnology Co., Ltd. Other reagents were AR, and purified water was used.

3 Extraction and separation

10 kg of dryC.spelaeicolawas crushed, and 10 times (L/kg) of 75% ethanol was added for reflux extraction for 3 times, and 2 h for each time. It was filtrated, and extraction solution was emerged. Solvent was recovered under reduced pressure, and 1 035 g of alcohol extract was obtained. The extract was stirred, mixed and suspended by water, and the obtained solution was extracted successively by petroleum ether (60-90 ℃), ethyl acetate, and n-butanol. Each extraction fraction was merged respectively, and solvent was recovered, obtaining 185 g of petroleum ether fraction, 235 g of ethyl acetate fraction, and 126 g of n-butanol fraction.

100 g of petroleum ether fraction was mixed with 200 g of silica gel (100-200 meshes), and then it was separated via silica gel column chromatography (200-300 meshes). Petroleum ether-acetone (10∶1, 8∶1, 4∶1, 6∶4, 1∶1,V/V) was used for gradient elution, and eluate was merged after identification by TLC, obtaining 5 fractions, namely S1-S5. Among them, white crystal precipitated in fraction S2, and its recrystallization was conducted with petroleum ether, obtaining compound I (22 mg); fraction S4 flew through silica gel column, and petroleum ether-ethyl acetate (50∶1,V/V) was used for elution, obtaining compound II (19 mg).

150 g of ethyl acetate was mixed with 200 g of silica gel (100-200 meshes), and then it was separated via silica gel column chromatography, and petroleum ether-acetone (100∶0, 50∶1, 20∶1, 8∶1, 5∶1, 1∶1，V/V) was used for gradient elution. After identification via TLC, 12 fractions were obtained, namely H1-H12. Fraction H3(750 mg)was separated via silica gel column chromatography, and petroleum ether-ethyl acetate (50∶1-5∶1,V/V) was used for gradient elution, obtaining 72 fractions. It was merged after TLC identification, and 92 mg of extract was obtained. The above extracts were analyzed by silica gel column chromatography, and ethyl acetate-methanol (50∶1-3∶ 1,V/V) was used for gradient elution. Among them, 16 mg of compound III was obtained by elution of ethyl acetate-methanol (5∶1,V/V). Fraction H4(900 mg)flew through silica gel column, and chloroform-methanol (50∶1-5∶1,V/V) was used for gradient elution. Eluate was merged after TLC identification, and solvent was recovered, obtaining subfractions C1-C4. Subfraction C2 flew through silica gel column, and ethyl acetate-methanol (1∶1,V/V) was used for elution, obtaining compound IV (17 mg); subfraction C3 flew through silica gel column, and ethyl acetat-methanol (5∶1,V/V) was used for elution, obtaining compound V (12 mg). Fraction H5 (670 mg) flew through Sephadex LH-20 column, and chloroform-methanol (1∶1,V/V) was used for elution, obtaining subfractions A1-A5. Subfraction A1 flew through silica gel column, and petroleum ether-ethyl acetate (6∶1,V/V) was used for elution, obtaining compound VI (13 mg) and compound VII (20 mg); subfraction A3 flew through polyamide thin layer, and chloroform-methanol (8∶1,V/V) was taken as developing solvent, obtaining compound VIII (9 mg). Fraction H6 (1 200 mg) flew through silica gel column, and chloroform-methanol (50∶1-1∶1,V/V) was used for gradient elution, obtaining subfraction M1-M4. Subfraction M2 flew through Sephadex LH-20 column, and chloroform-methanol (1∶1,V/V) was used for elution, obtaining compound IX (15 mg); subfraction M2 flew through polyamide column, and dichloromethane-methanol (100∶0-3∶1,V/V) was used for gradient elution, in which light yellow crystal precipitated from eluate of dichloromethane-methanol (20∶1,V/V), and compound X (17 mg) was obtained after recrystallization with methanol. Fraction H8 (700 mg) flew through Sephadex LH-20 column, and chloroform-methanol (1∶1,V/V) was used for elution. Elution component flew through silica gel column, and chloroform-methanol (100∶1-1∶1,V/V) was used for gradient elution, obtaining subfractions D1-D3. Subfraction D3 flew through silica gel column, and petroleum ether-acetone (20∶1,V/V) was used for elution. After that, polyamide thin layer chromatography was conducted, and ethanol-water-formic acid (4∶1∶0.1) was taken as developing solvent, obtaining compound XI (18 mg).

100 g of n-butanol fraction flew through AB-8 macroporous resin column, and water and 95% ethanol were used for elution successively, to remove carbohydrates and obtain 46 g of extract. Above extract flew through silica gel column, and chloroform-methanol (100∶1-0∶1,V/V) was used for gradient elution. Eluate was merged after TLC identification, obtaining 7 fractions, namely D1-D7. Among them, fraction D2 (900 mg) flew through silica gel column, and chloroform-methanol (8∶1,V/V) was used for elution. Pale yellow crystal (106 mg) precipitated in eluate, and polyamide thin layer chromatography of the crystal was conducted, and then methanol-formic acid (100∶2,V/V) was taken as developing solvent, obtaining compound XII (10 mg).

4 Structural identification

Structures of compounds I-XII were shown as Fig.1.

Compound I: white crystal (petroleum ether), melting point (mp): 136-137 ℃. Molecular formula: C29H50O, and ESI-MS:m/z413.3[M-H]-. Liebermen-Buchard reaction was positive, and TLC of compound I and β-sitostero was conducted. After treatment by three kinds of deployment systems with different polarity[petroleum ether-ethyl acetate (4∶1,V/V), petroleum ether-dichloromethane-acetone (10∶1∶1,V/V), and petroleum ether-dichloromethane-ethyl acetate (6∶1∶1,V/V)], Rf was consistent, and mp did not decline after mixing. So, the compound was identified as β-sitostero.

Compound II: white powder (petroleum ether), mp: 144-145 ℃. Molecular formula: C29H48O, ESI-MS:m/z413[M+H]+.1H-NMR(CDCl3, 500 MHz)δ: 3.53(1H, m, H-3), 5.14 (1H, m, H-6), 0.66(3H, s, 13-CH3), 1.01(3H, s, 20-CH3), 0.98(3H, s, 10-CH3), 5.15(1H,dd,J=15.2,7.8 Hz,H-22), 5.01(1H, dd,J=15.2, 7.8 Hz, H-23), 0.84-0.81 (6H, m, H-26, 27), 0.83(3H, t,J=7.2 Hz,H-29).13C-NMR(CDCl3,125 MHz) δ: 37.2(C-1), 31.7(C-2), 71.8(C-3), 42.2(C-4), 140.6(C-5), 121.7(C-6), 31.7(C-7), 31.9(C-8), 50.1(C-9), 36.6(C-10), 21.0(C-11), 39.7(C-12), 42.3(C-13), 56.9(C-14), 24.3(C-15), 28.7(C-16), 56.4(C-17), 12.1(C-18), 19.2(C-19), 40.3(C-20), 19.8(C-21), 138.3(C-22), 129.2(C-23), 51.1(C-24), 31.6(C-25), 19.5(C-26), 18.8(C-27), 25.2(C-28), 12.3(C-29). The above data were basically consistent with the literature[2], so the compound was identified as stigmasterol.

Fig.1 Structures of compounds I-XII

Compound III: white crystal (acetone), mp: 213-214 ℃. Molecular formula: C7H6O3, ESI-MS:m/z121[M-OH]+.1H-NMR (CD3OD, 500 MHz)δ:7.96(2H，d，J=8.0 Hz，H-2，6), 6.94(2H, d,J=8.0 Hz, H-3, 5).13C-NMR(CD3OD，125 MHz)δ:122.9(C-1), 163.7(C-4), 131.6(C-2，6), 115.8(C-3, 5), 169.4(1-C=O). The above data were basically consistent with the literature[3], and the compound was identified as p-hydroxybenzoic acid.

Compound IV: white powder (pyridine), mp: 286-287 ℃. Molecular formula: C35H60O6,m/z：577[M+H]+. Liebermann-Burchard reaction was positive, while Molish reaction was negative. TLC of compound IV and β-daucossterol control was conducted, and three kinds of developing systems with different polarity[chloroform-methanol (10∶1,V/V), dichloromethane-methanol (6∶1,V/V), and ethyl acetate-methanol (7∶1,V/V)]were used. After treatment, Rf was consistent, and mp did not decline after mixing. So, the compound was identified as β-daucossterol.

Compound V: white powder (methanol), mp: 200-201 ℃. Molecular formula: C7H6O4, ESI-MS:m/z153[M-H]+.1H-NMR (CD3OD，500 MHz)δ: 7.43(1H，d，J=1.8 Hz，H-2), 7.51(1H，dd，J=8.0，1.5 Hz，H-6), 6.77(1H，d，J=8.4 Hz，5-OH).13C-NMR(CD3OD，125 MHz)δ: 123.9(C-1), 117.5(C-2), 147.1(C-3), 152.5(C-4), 117.1(C-5), 124.3(C-6), and 169.6(C-7). The above data were basically consistent with the literature[4], so the compound was identified as protocatechuic acid.

Compound VI: colorless crystal(petroleum ether ethyl acetate), mp: 177-178 ℃. Molecular formula: C15H22O3，ESI-MS：m/z251[M+H]+.1H-NMR(CD3OD，500 MHz)δ: 4.93(1H，s，H-6), 4.91(1H，m，H-8), 1.96(3H，s，H-13), 0.91(3H，s，H-14), 1.06(3H，d，J=6.5 Hz，H-15).13C-NMR(CD3OD，125 MHz)δ: 28.1(C-1)，19.5(C-2)，28.3(C-3)，32.1(C-4)，45.1(C-5)， 71.1(C-6)，162.9(C-7)，77.5(C-8)，35.2(C-9)，35.4(C-10)，122.9(C-11)，176.0(C-12)，9.1(C-13)，20.4(C-14), 15.0(C-15). The above data were basically consistent with the literature[5], so the compound was identified as 6β-hydroxyeremophi-7(11)-en-12，8β-olide.

Compound VII: colorless crystal (petroleum ether-ethyl acetate), mp: 170-171 ℃. Molecular formula: C15H22O3，ESI-MS:m/z251[M+H]+.1H-NMR(CD3OD，500 MHz)δ: 1.80(3H，s，H-13), 0.92(3H，s，H-14), 0.81(3H，d，J=6.0 Hz，H-15), 2.65(1H，d，J=14.6 Hz，H-6a), 2.48(1H，d，J=14.6 Hz，H-6b)，2.09(1H，dd，J=7.0，12.3 Hz，H-9a), 1.88(1H，dd，J=10.9，13.8 Hz, H-9b).13C-NMR(CD3OD，125 MHz)δ: 36.1(C-1), 22.6(C-2)，28.7(C-3)，33.0(C-4)，45.2(C-5)，30.8(C-6)， 161.9(C-7)，78.5(C-8)，41.3(C-9)，74.7(C-10)，121.2(C-11)，174.6(C-12)，8.8(C-13)，14.2(C-14)，16.5(C-15). The above data were basically consistent with the literature[6], so the compound was identified as 10β-hydroxyeremophil-7(11)-en-8，12-olide.

Compound VIII: colourless powder(petroleum ether-ethyl acetate), mp: 169-171 ℃. Molecular formula: C15H20O3，ESI-MS:m/z249[M+H]+.1H-NMR(CD3OD，500 MHz)δ: 1.78(2H，m，H-1), 1.24(2H，m，H-2), 2.09(2H，m，H-3)，4.11(2H，m，H-6)，1.61(1H，m，H-4)，2.32(1H，brd，J=13.2 Hz，H-6b)，2.38(1H，d，J=13.2 Hz，H-6a)，1.69(3H，s，H-15)，0.88(3H，s，H-13)，0.93(3H，s，H-14).13C-NMR(CD3OD，125 MHz)δ: 27.1(C-1)，30.9(C-2)，32.6(C-3)，44.4(C-4)，47.1(C-5)， 36.2(C-6)，147.2(C-7)，149.3(C-8)，120.5(C-9)，77.6(C-10)，123.7(C-11)，170.9(C-12)，7.9(C-13)，15.8(C-14)，17.3(C-15). The above data were basically consistent with the literature[6], so the compound was identified as 10β-hydroxyeremophi-7(11)，8(9)-dien-8，12-olide.

Compound IX: yellow crystal (methanol), mp：313-315 ℃. Molecular formula: C15H10O7，ESI-MS：m/z303[M+H]+.1H-NMR(CD3OD，500 MHz)δ：6.15(1H，d，J=2.0 Hz，6-OH)，6.35(1H，d，J=2.0 Hz，8-OH)，7.69(1H，d，J=2.0 Hz，H-2′)，7.71(1H，d，J=8.2 Hz，H-5′)，7.59(1H，dd，J= 8.4， 2.0 Hz，H-6′).13C-NMR(CD3OD，125 MHz)δ：147.3(C-2)，175.2(C-4)，156.7(C-5)，99.2(C-6)，164.5(C-7)，93.8(C-8)，160.3(C-9)，104.1(C-10)，122.3(C-1′)，116.4(C-2′)，145.2(C-3′)，147.9(C-4′)，115.1(C-5′)，121.8(C-6′). The above data were basically consistent with the literature[7], so the compound was identified as quercetin.

Compound X: yellow powder (methanol), mp: 225-226 ℃. Molecular formula: C21H20O12, ESI-MS：m/z465[M+H]+.1H-NMR(CD3OD，500 MHz)δ：6.19(1H，d，J=1.8 Hz，6-OH)，6.38(1H，d，J=1.8 Hz，8-OH)，7.54(1H，d，J=1.8 Hz，H-2′)，6.82(1H，d，J=8.2 Hz，H-5′)，7.66(1H，dd，J=8.2，2.0 Hz，H-6′)，5.22(1H，d，J=7.3 Hz，H-1″).13C-NMR(CD3OD，125 MHz)δ：157.1(C-2)，135.7(C-3)，179.2(C-4)，160.5(C-5)，100.5(C-6)，166.3(C-7)，94.3(C-8)，158.2(C-9)，105.5(C-10)，123.3(C-1′)，116.2(C-2′)，145.6(C-3′)，150.1(C- 4′)，117.9(C-5′)，123.7(C-6′)，104.3(C-1″)，72.9(C-2″)， 75.2(C-3″)，70.5(C-4″)，77.3(C-5″)，61.8(C-6″). The above data were basically consistent with the literature[8], so the compound was identified as hyperin.

Compound XI：white crystal (petroleum ether-ethyl acetate)，mp：181-183 ℃. Molecular formula: C15H27O2，ESI-MS：m/z240[M+H]+.1H-NMR(CD3OD，500 MHz)δ：4.66(1H，brs，H-12a)，4.68(1H，brs，H-12b)，1.74(3H，s，H-13)，1.16(3H，s，H-14), 0.88(3H，s，H-15).13C-NMR(CD3OD，125 MHz)δ：41.5(C-1)，19.5(C-2)，42.8(C-3)，72.6(C-4)，54.4(C-5)，25.7(C-6)，46.5(C-7)，26.2(C-8)，44.1(C-9)，35.2(C-10)，150.6(C-11)，110.2(C-12)，21.8(C-13)，23.1(C-14)，18.1(C-15). The above data were basically consistent with the literature[9], so the compound was 4α-hydroxy-eudesman-11-ene.

Compound XII：brownish yellow crystal(methanol)，mp：193-200 ℃. Molecular formula: C27H30O16，ESI-MS：m/z611[M+H]+.1H-NMR(CD3OD，500 MHz)δ：7.87(1H，d，J=2.0 Hz，H-2′)，7.60(1H，dd，J=8.2，2.0 Hz，H-6′)，6.83(1H，d，J=8.2 Hz，H-5′)，6.17(1H，d，J=1.6 Hz，6-OH)，6.36(1H，d，J=1.6 Hz，8-OH)，5.05(1H，d，J=7.0 Hz，H-1″).13C-NMR(CD3OD，125 MHz)δ：158.6(C-2)，136.9(C-3)，177.3(C-4)，160.9(C-5)，99.3(C-6)，165.1(C-7)，94.0(C-8)，156.8(C-9)，105.2(C-10)，123.7(C-1′)，116.1(C-2′)，145.1(C-3′)，149.6(C-4′)，116.7(C-5′)，122.7(C-6′)，104.7(C-1″)，73.4(C-2″)，75.1(C-3″)，70.7(C-4″)，74.6(C-5″)，67.0(C-6″)，101.6(C-1 ?)， 71.3(C-2?)，72.1(C-3?)，73.7(C-4?)，70.3(C-5 ?)，18.5(C-6?). The above data were basically consistent with the literature[10], so the compound was identified as quercetin-3-O-robinobioside.

5 Discussions

Yao medicineC.spelaeicolacontains complex ingredients. At present, there is no related report on its active ingredients and pharmacological actions. It is pointed out thatSenecioL. is the biggest in Compositae, and plants in the genus mainly contain terpenes, alkaloids, phenolic acids, and flavonoids, in which sesquiterpenes and alkaloids are main active ingredients. Additionally, plants in the genus have the functions of antibacterial, antiviral, antitumor, anti-inflammatory, antioxidant, eliminating free radicals and strengthening immunity, and could be used for clinical treatment of eye diseases, skin diseases, enteritis, dysentery, upper respiratory infections, urogenital system and other diseases[6,11]. In this paper, chemical composition of 75% ethanol extract ofC.spelaeicolawas analyzed, and 12 chemical compounds were separated, and they were identified as β-sitostero (I) and stigmasterol (II) were isolated from petroleum ether fraction; p-hydroxybenzoic acid (III), β-daucossterol (IV), protocatechuic acid (V), 6β-hydroxyeremophi-7(11)-en-12, 8β-olide(VI), 10β-hydroxyeremophil-7(11)-en-8, 12-olide (VII), 10β-hydroxyeremophi-7(11), 8(9)-dien-8, 12-olide (VIII), quercetin (IX), hyperin (X) and 4α-hydroxy-eudesman-11-ene (XI), and quercetin-3-O-robinobioside (XII). Above compositions respectively belong to terpenoids, phenolic acids, and flavonoids, which corresponds with basic characteristics of the chemical compounds from the genus[6,11-12]. Additionally, pharmacological research points out that stigmasterol has the functions of antitumor, anti-inflammatory, antioxidant, lowering cholesterol and improving memory[13], while hyperin has the pharmacological actions of anti acute liver injury, antidepressant, anti-inflammatory, antithrombotic, antitumor and antibacterial[14], and following pharmacology research can be carried out with reference to this. In this paper, the obtained compounds I-XII were all isolated from the plant for the first time, which could provide material basis for studying anti-inflammatory, antiviral and other pharmacological activities of Yao medicineC.spelaeicola.