Isolation And Identification Of The Chemical Constituents Of Sanguisorba Officinalis L.and Their Activities In Vitro

The traditional Chinese medicine Sanguisorba officinalis L.was systematically separated and purified,chemical constituents of which were studied and identified in this paper.Meanwhile,the cytotoxic activity and anti-complementary activity of the isolated compounds were tested.S.officinalis is a kind of perennial herb belonging to the genus Sanguisorba and the family Rosaceae.It is widely distributed and grows in north temperate Asia,Europe and China.As a traditional Chinese medicine,S.officinalis,which was first listed in the Chinese medical classic "Shen Nong Ben Cao Jing",has a wide medicinal value and has been used as a medicinal ingredient for a long history,having the functions of stopping bleeding and cooling blood,clearing heat and detoxification,astringent and preventing diarrhea,nourishing Yin and reducing swelling and convergence.There are many kinds of chemical constituents and many pharmacological effects such as hemostatic,hematopoietic promoting,anti-inflammatory,anti-bacterial,and anti-tumor activity in S.officinalis.In order to obtain more clearly the active chemical constituents from the roots of S.officinalis,the dried roots of S.officinalis(9.5 kg)were extracted with 95%EtOH(3×40 L,each for one week)at room temperature.The combined extracts were concentrated under a vacuum to obtain a crude extract(2.2 kg).The crude extract was suspended in H2O(6 L)and partitioned sequentially with petroleum ether,EtOAc and n-BuOH(3×6 L in each case).Seventy-four compounds were isolated from the roots of S.officinalis using silica gel column,octade-cylsilyl(ODS),Sephadex LH-20,preparative middle pressure liquid chromatography(MPLC)and high performance liquid chromatography(HPLC).The chemical structures were elucidated based on extensive spectroscopic experiments,including infrared spectrometry(IR),1D and 2D NMR as well as HR-ESI-MS,and comparison with those reported in the literature,including four new triterpenoids:3-oxo-15?,19?-dihydroxyurs-12-en-28-oic acid(1),3-oxo-7?,19?-dihydroxyurs-12-en-28-oic acid(2),18,19-seco,1?-hydroxyl-3,19-dioxo-urs-11,13(18)-dien-28-oic acid(3),and 3-oxo-urs-11,13(18)-dien-19,28-olide(4),two new phenolic glycosides:methyl 2,6-di-O-galloyl-?-D-glucopyranoside(65),and chavicol 4-O-?-L-arabinopyranosyl-(1?6)-?-D-glucopyranoside(68),the other known compounds were identified as 3-oxo-19?-hydroxyurs-12-en-28-oic acid(5),2?,19?-dihydroxy-3-oxo-12-ursen-28-oic acid(6),3,11-dioxo-19?-hydroxyurs-12-en-28-oic acid(7),fupenzic acid(8),?-amyrin(9),ursolic aldehyde(10),ursolic acid(11),19?-hydroxy ursolic acid(12),euscaphic acid(13),1?-hydroxyeuscaphic acid(14),1?,2?,3?,19?-tretrahydroxyurs-12-en-28-oic acid(15),ursolic acid 3-O-?-L-arabinopyranoside(16),3-O-?-L-arabinopyranosyl pomolic acid(17),28-O-?-D-glucopyranosyl-pomolic acid ester(18),ziyu glycoside I(19),ursolic acid-3-O-behenate(20),2?,3,?,19?-trihydroxyurs-12-en-28-oic acid ?-D-galactopyranosyl ester(21),2?,3?,19?,23-tetrahydroxyurs-12-ene-28-O-?-D-glucopyranosyl ester(22),2?,3?,19?-trihydroxyurs-12-en-28-oic acid ?-D-glucopyranosyl ester(23),2?,3?-dihydroxyurs-12,18-dien-28-oic acid 28-O-?-D-glucopyranosyl ester(24),2?,19?-dihydroxy-3-oxo-12-ursen-28-oic acid ?-D-glucopyranosyl ester(25),2,19?-dihydroxy-3-oxours-1,12-dien-28-oic acid 28-O-?-D-glucopyranosyl ester(26),3?-[(?-L-arabinopyranosyl)oxy]-urs-11,13(18)-dien-28-oic acid ?-D-glucopyranosyl ester(27),3?-[(?-L-arabinopyranosyl)oxy]urs-12,19(29)-dien-28-oic acid 2 8-?-D-glucopyranosyl ester(28),3-O-?-L-arabinopyranosyl-urs-12,18(19)-dien-28-oic acid ?-D-glucopyranosyl ester(29),sanguidioside C(30),?-amyrin acetate(31),3,29-dioxoolean-12-en-27-oic(32),3-oxo-olean-11,13(18)-dien-28,19?-olide(33),2?,3?,9?,24-tetrahydroxyolean-12-en-28-oic acid(34),maslinic acid(35),arjunic acid(36),arjunetin(37),?-sitosterol(38),daucosterol(39),(2E,6Z)-3,7-dimethyl-8-hydroxyoctadien-1-ol(40),8-hydroxygeraniol-1-O-(6-O-galloyl)-?-D-glucopyranoside(41),(2E)-3,7-dimethyl-2,6-octadien-1-y1 6-O-?-L-arabinofuranosyl-?-D-glucopyranoside(42),7-hydroxy-3,7-dimethyloctyl-6-O-?-L-arabinofuranosyl-?-D-glucopyranoside(43),(2E)-7-hydroxy-3,7-dimethyl-2-octenyl 6-O-?-L-arabinopyranosyl-?-D-glucopyranoside(44),(2E)-7-hydroxy-3,7-dimethyl-2-octenyl 6-O-?-L-arabinofuranosyl-?-D-glucopyranoside(45),(+)-catechin(46),(-)-catechin(47),(-)-gallocatechin(48),taxifolin 3-O-glucoside(49),taxifolin 4'-O-?-D-glucopyranoside(50),(-)-aromadendrin 3-O-?-D-glucopyranoside(51),catechin-3-O-?-L-rhamnopyranoside(52),maesopsin-6-O-glucopyranoside(53),bis-8,8'-catechinylmethane(54),bis-6,8'-catechinylmethane(55),?-hydroxypropiovanillone(56),methyl 3-hydroxy-4-methoxybenzoate(57),gallic acid(58),methyl gallate(59),methyl 3-O-methyl-gallate(60),potassium 3,4-dihydroxy-3-methoxybenzoic acid methyl ester-5-sulphate(61),methyl 3-?-D-glucopyranosyloxy)-4-hydroxy-5-methoxybenzoate(62),methyl 4-(?-D-glucopyranosyloxy)-3-hydroxy-5-methoxybenzoate(63),methyl 6-O-galloyl-?-D-glucopyranoside(64),1,2-di-O-galloyl-?-D-glucopyranoside(66),D-threo-guaiacylglycerol 8-O-?-D-(6'-O-galloyl)glucopyranoside(67),chavicol 4-(O-?-L-arabinofuranosyl-(1?6)-?-D-glucopyranoside(69),lyoniside(70),methyl ?-D-glucopyranoside(71),?-L-arabinofuranoside(72),n-butyl-?-D-fructofuranoside(73),and ?-D-fructofuranose(74).Among them,compounds 33,40,53-55,69,72,and 73 were the first to be reported in the family Rosaceae,compounds 8-10,21,24,26,31,48,50-52,56,and 70 were firstly identified from the genus Sanguisorba,and compounds 6,25,and 34-35 were obtained from S.officinalis for the first time.Due to the wide variety of compounds isolated from S.officinalis,different activities in vitro were determined according to different types of compounds.All the triterpenoids(1-37)were assessed for their cytotoxic activities against lung cancer cell line(A549),cervical cancer cell line(HeLa),hepatoma cell line(SK-Hepl).Additionally,two sterols(38-39),six monoterpenoid glycosides(40-45),ten flavonoids(46-55),fourteen phenols(56-69),one lignan(70),and four glycosides(71—74)were evaluated for their anti-complementary activities against the classical pathway(CP).Compounds 1,3,5,7-9,11-12,14,16,18,21-22,25,27-29,31,and 33 showed cytotoxic activities against A549 cells with half-maximal inhibitory concentrations(IC50)values ranging from 42.93 to 91.52 ?M.Compounds 1,5,7-9,12-14,16,18,21-22,25,27—29,33-34,and 36 showed cytotoxic activities against HeLa cells with IC50 values ranging from 41.56 to 91.01 ?M.Compounds 1,3,5,7-9,11,13—14,16,18,21-22,25,27,29,and 33—35 showed cytotoxic activities against SK-Hep1 cells with IC50 values ranging from 41.92 to 92.14 ?M.The compounds 1,5,7-9,14,16,18,21-22,25,27,29,and 33 demonstrated cytotoxic activities against these three human cancer cells,and the compound 1 showed better cytotoxic activities against three human cancer cells than other compounds with IC50 values 42.93± 0.05,41.56±0.66,and 41.92± 0.09 ?M,respectively,suggesting that compound 1 has potential as a prospective inhibitor of three human cancer cells.Compounds 40-42,49-51,58-59,63-66,and 68-69 exhibited anti-complementary effects toward the CP with CH50 values ranging from 0.40 to 2.08 mM.Furthermore,compounds 41,59,66,and 68 and showed significant anti-complementary activities with CH50 values of 0.53± 0.05,0.51 ± 0.07,0.57 ± 0.01,and 0.40 ± 0.03 mM,respectively.The targets of compounds 41,59,66,and 68 in the complement activation cascade were identified using complement-depleted sera.Compound 41 interacted with C2 and C9.Compound 59 only interacted with C9.Compound 66 interacted with C4,C5,and C9,but not with the C2 and C3 components.The C2 component was the target of 68.In particular,41 and 66 acted on diverse targets,making them potential natural complement inhibitors.To sum up,the study not only enriched the chemical constituents of S.officinalis,but also found that the compound 1 showed better cytotoxic activities and the compound 68 had better anti-complementary activities.Of note,the anti-complementary activities of S.officinalis were studied for the first time.It is hoped that this study can be able to provide theoretical support for follow-up research and development of the traditional Chinese medicine S.officinalis and conducive to the discovery of new lead compounds and the use of natural medicine.