The Study On The Chemical Constituents Of Viola Tianshanic Maxim

Viola tianshanica Maxim(Violaceae)is a small perennial herb with violet flowers,widely distributed in the Central Asian region,especially wide-spread in Xinjiang region China.The whole plant is mainly used for all kinds of fever,chills,colds,pleurisy,pneumonia,pharyngitis,swollen boils and carbuncle embolism.It is slightly bitter smell,cool,detoxification,softening,sweating role.Mainly for abnormal bile laxative quality,regulate blood excessive heat,thirst,sweating fever.Its medicinal part is the whole plant,the whole plant was detected in preliminary experiments containing volatile oils,flavonoids,alkaloids,amino acids,phlegmatic saponins,tannins,etc[1].Literature had reported that Viola extract has obvious anti-inflammatory[2],antibacterial[3]and antioxidant[4]activities and so on.Current research on the chemical composition of which is less reported in the literature which contains chemical components are flavonoids[5]and the cyclic peptide[6]ingredients.In this study,the biological activities of Viola are carried by anti-complement activity,antimicrobial activity,antiviral activity and resistance to cytotoxic activity and found that petroleum ether,ethyl acetate fraction anti-complement effect are better,and ethyl acetate extract also has strong anti-bacterial and anti-cytotoxic activity,butanol fraction of 25%with anti-influenza virus activity inhibition.37 compounds were isolated and identified from the active fraction.All compounds were measured on the classical and alternative pathway of anti-complement activity using cell hemolysis and also select flavonoid glycosides and lignan glycosides compounds were investigated on behalf of anti-complement effect targets.In addition,a comparative research between Viola tianshanica Maxim and Viola yedoensis Makino had been studied by using GC-MS to analysis the volatile components and UPLC-QTOF to analysis the solar ingredients to found the differences in the chemical composition,the presence of these different ingredients will lead to differences in treatment effect,it is worth further pharmacodynamic studies.The main findings are as follows:1.The biological activities of Viola tianshanica Maxim.Anti-complement activity was established.We have investigated the medicine Viola extract anti-complement activity in different parts of the extraction method using in vitro cell hemolysis.The results showed that the ethyl acetate extract Viola(CH50=0.011±0.01 mg/ml)anti-complement activity was stronger than petroleum ether extract(CH50=0.023±0.01 mg/ml),n-butanol extract(CH50=0.253±0.01 mg/ml),aqueous extracts of parts(CH50=0.547±0.01 mg/ml).Antibacterial and antiviral activities was determined.The extraction has certain extent inhibitory against Staphylococcus aureus,Escherichia coli,Streptococcus pneumoniae,MRSA and Pseudomonas aeruginosa bacteria.We also found ethyl acetate extracts has better inhibitory activities against Staphylococcus aureus,Pseudomonas aeruginosa activity,and the inhibition zone size 10 mm-15 mm;pneumococcal bacteria and MRSA inhibiting activity,and inhibition zone size of 6 mm-10 mm.N-butanol extract of pneumococcus has good inhibitory activity and inhibition zone size at 10 mm-15 mm.Aqueous extract of Staphylococcus aureus,Streptococcus pneumoniae and Pseudomonas aeruginosa inhibit the activity and inhibition zone size 6 mm-10 mm.Meanwhile extracts in vitro anti-herpes virus type I and in vitro anti-influenza virus activity screening experiments,and found four kinds of extracts of herpes virus type I had no activity,n-butanol extract has in vitro inhibition of influenza virus 25%activity.Anti-cytotoxic activity was determined.,extract for anti-tumor activity in vitro biological screening test,using sulforhodamine B(sulforhodamineB,SRB)protein staining was performed on the cell line A549 activity assay.The results showed that ethyl acetate extract has anti-cytotoxic activity(IC50=68.50 ?g/ml).2.Biological constituents from Viola tianshanica Maxim.37 compounds were isolated and identified from petroleum ether and ethyl acetate extract of Viola tianshanic Maxim,including a new lignan glycosides suh as tianshanoside A(18),including 36 known compounds,such as 17 flavonoids:kaempferol-3,7-two-O-?-D-glucopyranoside(1),kaempferol-3-O-?-D-rutinose(2),isorhamnetin-3-O-?-D-rutinoside(3),kaempferol-3-O-?-D-glucoside(4),kaempferol-3,7-O-a-L-two rhamnosidase(5),tricin(6),sutchuenoside A(7),kaempferol 3-0-(2"-O-?-D-glucose-based)-p-D-rutinoside(8),kaempferol-3-O-{a-L-rhamnose(1?6)-[a-L-rhamnose(1?2)]}-?-D-glucopyranoside(9),kaempferol-3-O-?-D-rutinoside-7-O-?-D-glucoside(10),isorhamnetin-3-O-?-D-rutinoside-7-O-?-D-glucoside(11),kaempferol-3-(2",6"-bis-O-?-L-rhamnose)-?-D-glucoside(12),rutin(13),kaempferol-3-O-?-D-glucoside-7-O-?-D-glucoside(14),isorhamnetin-3-O-?-D-glucose(1?2)-O-?-D-glucose-7-O-?-D-glucoside(15),isorhamnetin-3-O-(2-O-?-L-isorhamnetin-6-O-?-D-glucose)-O-?-D-glucose-7-O-?-D-glucoside(16),isorhamne-tin-3-O-?-D-glucose(17);3 lignan compound:Aketrilignoside B(19),wood bitter glycosides(20),syringin(21);the phenolic compounds:4-(hydroxymethyl)-2,6-dimethoxy-phenyl-O-?-D-glucopyranoside(22),benzyl alcohol-O-?-D-glucoside(23),2,4,6-Ethylparaben(24),2,2'-dithio-bisbenzothiazole(25);two nucleotide compounds:adenosine(26),uridine(27);two steroidal compounds:?-sitosterol(28),daucosterol(29);6 esters and aliphatic compounds:single octadecadienoic acid ester(30),octadecatrienoic acid methyl ester(31),2,3-bis-linolenic acid glycerides cis-1-O-?-D-galactopyranoside(32),gingerglycolipid A(33),2,3-bis-linolenic acid glycerides(34),1,3-bis-linolenic acid glycerides(35),9-hydroxy-4-megastigmen-3-one(36),apigenin-6,8-bis-C-a-L-arabinoside(37).Compounds 19,20 and 21 which were first isolated from Viola tianshanic Maxim.The chemical composition of Viola yedoensis Makino have been isolated terpenes,coumarins,flavonoids,alkaloids,lignans and phenolic compounds,there are differences in the two species.But the preparative separation of the resulting chemical composition is not a complete response in kind of the two plants,requiring other methods to analyze the chemical composition of them.So we choose GC-MS method to analysis the constituents of volatile oil which has anti-inflammatory effects and used UPLC-QTOF method to identify a large solar compound.3.By steam distillation,headspace direct injection and Needle trap GC-MS methods to analysis the constituents of Viola tianshanica Maxim.and Viola yedoensis Makino.Literature has reported that volatile oil inhibited xylene-induced ear edema in mice efficacy,thus it is necessary to study the volatile components.In this paper,the international advanced dynamic needle trap technology(Needle trap)was used to analysis the volatile components from Viola tianshanic Maxim and Viola yedoensis Makino,and also conducted by traditional steam distillation method and the headspace direct injection method comparison;which were found that volatile components obtained by headspace direct injection was low boiling point components,volatile components obtained by steam distillation often high and medium boiling point ingredients.Needle trap method won't destroy the volatile components kind and can reflect the overall volatile components accurately(including low boiling components in various high),with better enrichment and high sensitivity.The volatile components were identified by retention time,retention index and similarity matching,At last volatile 86 volatile components were identified from Viola tianshanic Maxim,105 volatile components were identified from Violayedoensis Makino,33 components were the same,there are more differences in components.4.By UPLC-QTOF-MS/MS method to analysis the constituents of Viola tianshanica Maxim.and Viola yedoensis Makino.For this study the chemical composition of the polar parts of Viola tianshanic Maxim were unknown,and our group have preliminarily found the flavonoid C-glycosides,it is necessary to establish a rapid and effective methods to analysis the chemical composition in the large polar parts of Viola tianshanic Maxim,but also compared with the chemical composition of Viola yedoensis Makino in the large polarity parts.This study established the ultra performance liquid chromatography-photodiode array detection-electrospray ionization-quadrupole-time of flight mass spectrometry for flavonoid glycosides Viola tianshanic Maxim and Viola yedoensis Makino were structural characterization,discusses the cleavage of flavonoid O-glycosides and C-glycosides compounds,found that flavonoid O-glycoside is mainly sugar-based removal,and flavonoid C-glycosides is the cleavage of the sugar ring,combined with differences in flavonoid glycoside oxygen and carbon glycosides PDA feature map,the precise molecular weight difference between the theoretical value of the retention time and resolution.The result of oxygen 27 flavonoid O-glycosides and 4 flavonoid C-glycosides from Viola tianshanic Maxim 75%methanol extract,and 22 flavonoid C-glycosides and 5 flavonoid O-glycosides from Viola yedoensis Makino75%methanol extract.The existence of these differences constituents may cause the difference in the treatment effect,it is worth further study pharmacodynamic studies.5.Anti-complement activity?target research and cytotoxic activity of monomeric compoundsBy the cellular activity of all compounds in vitro anti-complement activity test results showed that among 11 compounds 1,2,3,4,5,6,7,8,21 and 25 with varying degrees of the complement system suppression,the active compounds mainly are flavonoids and lignans compounds;the findings of system targets complement showed that:kaempferol-3-O-?-D-glucoside(4)acted on the complement Clq,C2,C4 and C9 components,tricin(6)acting on the complement Clq,C4 and C9 components,wood bitter glycosides(21)acting on the complement Clq,C3 and C9 components.Lignan compounds were cytotoxic activity,the use of CCK-8 assay of four lignan compounds were isolated from gastric cancer cells and HepG2 cells Ags cytotoxic activity assay,the results showed that these four compounds are not obvious anti-cytotoxic activity.