Study On The Chemical Constituents Of Tibetan Medicine Myrobalan And Its Anti-cancer Activity In Vitro

Terminalia billirica?Gaert.?Roxb of Terminalia Linn.belongs to Combretaceae mainly located in South-Asia and Southeast-Asia,and less distributed in Tibet,Yun-nan and other provinces in China.Its pitted fruits is one of Tibetan common medicine,which has clearing and detoxifying heat,astringing and nourishing blood,harmonizing ingredients of a prescription effect.In addition,T.billirica has a variety of pharmacological activities such as anticancer,antioxidant,antibacterial,anticonvulsions,liver protective,cholesterol reducing,hypoglycemic and prevent myocardial necrosis,etc.According to the theory of Tibetan traditional medicine,T.billirica is generally used together with Phyllanthus emblica.L and T.chebula,called as“Dasan Guo”with the treatment of various“peigen”,“chiba”and“huangshui”pathologic diseases.Until now,only 13 compounds had been isolated from T.billirica as reviews,including gallic acid,ellagic acid,3,3'-dimethyl ellagic acid,2,2'-dimethoxy-3,3'-dihydroxy-5,5'-oxygen-6,6'-biphenylformic anhydride,mucic acid dimethyl ester-2-O-gallate,corilagin,?-sitosterol,tannic acid-3,3'-dimethyl ether-4-O-?-D-xyloside,Termilignan,Thannilignan,Anolignan B and 2?,3?,23,24-tetrahydroxyolean-12-en-28-oic acid,3-O-?-D-galactopyranosyl-?1?4?-O-?-L-rhamnopyranoside.Most of them have well anticancer activity in vitro.Therefore,in order to discover more anti-cancer ingredients,this thesis performed separation and purification of the chemical constituents of pitted fruits of T.billirica,while made detection on anticancer activity of four different polar extracts and 21 compounds isolated from T.billirica in vitro.Phytochemical studies on the pitted fruits of T.billirica,61 compounds were isolated from the 70%acetone extraction by means of silica gel,macroporous adsorption resin,Diaion HP-20,Sephadex LH-20,Toyopearl HW-40?Coarse?and Toyopearl HW-40?Fine?column,MCI gel CHP 20P chromatography and recrystalization.The structures were elucidated on the basis of physicochemical properties and spectral data(¹H-NMR,¹³C-NMR and MS).The PE extract has isolated and identified 8 compounds,including 7 fatty acids:palmitic acid?1?,stearin acid?2?,methyl octadecanoate?3?,3-hydroxydodecanoic acid?4?,myristic acid?5?,methyl behenate?6?and?-linolenic acid?7?;1 diterpenoids:Vitamin E?8?.The CH₂Cl₂ extract has isolated and identified 11compounds,including 4 steroids:?-glucoside?9?,?-sitosterol?10?,stigmasterol?11?,?-sitosterol palmitate?12?;7 triterpenoids:oleanolic acid?13?,maslinic acid?14?,arjunolic acid?15?,arjunic acid?16?,ursolic acid?17?,tormentic acid?18?,betulin?19?.The EtOAc?EA?extract has isolated and identified 36 compounds,including 11 tannins:maplexin C?20?,1,2,3,6-tetra-O-galloyl-?-D-glucopyranonside?21?,1,2,3,4,6-penta-O-galloyl-?-D-glucopyranonside?22?,methylellagic acid-4-O-?-D-xylopyranoside?23?,chebulagic acid?24?,chebulinic acid?25?,chebulic acid?26?,chebulanin?27?,Punicalagin A?28?,Punicalagin B?29?,3',3''',4',4'''-tetra-methylcatetin?4a?8?-3-?-D-glucose?1?4??-D-glucosidea?30?;18 polyphenols:gallic acid?31?,methyl galate?32?,ethyl gallate?33?,propyl gallate?34?,galloyl myristate?35?,ellagic acid?36?,3,3'-di-O-methylellagic acid?37?,3,4-di-O-methylellagic acid?38?,3,3',4-tri-O-methylellagic acid?39?,?+?-lyoniresinol?40?,brevifolincarboxylic acid?41?,syringic acid?42?,sinapic acid?43?,3,4-di-O-methylcinnamic acid?44?,4-hydroxy-benzoic acid?45?,shikimic acid?46?,quinic acid?47?,syringin?48?;1lignans:syringaresinol?49?;6 flavonoids:eriodictyol?50?,eriodictyol-7-O-?-D-glucoside?51?,pinocembrin?52?,chrysoeriol-7-O-?-D-glucopyranoside?53?,luteolin?54?,hispidulin?55?.The H₂O extract isolated and identified 7 compounds,including 1 tannins:corilagin?56?;1 phenolic:salicoside?57?;4 sugars:methyl-?-arabino-furanoside?58?,1,4-di-methyl-?-D-fructopyranose?59?,?-D-fructofuranose?60?and?-D-fructopyranose?61?.Among them,54compounds were isolated from the pitted fruits of T.billirica for the first time;35 compounds were isolated from Terminalia Linn.for the first time;Flavanones were isolated from Terminalia Linn.for the first time.Analyzing constituents of EA and H₂O extracts of the pitted fruits of T.billirica based on UPLC-MSⁿ technique.acquity UPLC ben C₁₈?2.1×100 mm,1.7?m?colum was used,with a binary solvent system which was employed consisting of methanol as solvent A and 0.2%acetic acid in Milli-Q water as solvent B.The gradient elute was as follows:0-5 min,5%A?15%A;5-10 min,15%A?25%A;10-15 min,25%A?60%A;15-25 min,60%A?90%A;25-30 min,90%A?90%A.The flow rate was 0.3 mL·min^-11 and column temperature was 30°C.The mass spectrometer equipped with ESI ionization source,and operated in negative ion mode at 270 nm and 280 nm.The full scan ranged from m/z 100 up to 1500.70 compounds were detected according to the accurate molecular mass provided by ESI-MS,MS/MS fragmentation,relative retention time,the estimated formula?error<±5 ppm?or other reports in the literature.48compounds were identified from EA extract and 22 were from H₂O extract.Among them,14 compounds were isolated from the EA extract.In addition,the fragmentation rules of gallic acid and derivatives,ellagic acid and derivatives and hydrolysable tannins.CellTiter-Blue?Cell Viability Assay was used to evaluate anticancer activity of different polar extracts of T.billirica and 21 compounds isolated from EA extract.10 human cancer cell lines?ZR75-1,Colo-205,BT-474,T-47D,MCF-7,HT-29,SW480,Hec-1-A,LnCap,SH-SY5Y?were chosen to study the anticancer activity of different polar extracts of T.billirica.The result indicated that EA and H₂O extracts could inhibit the proliferation of human cancer cell lines and have obvious dose-dependent relationship.ZR75-1,Colo-205,BT-474and T-47D were much more sensitive to EA extract,the half inhibitory concentration(IC₅₀)were 27.33?g/mL,36.63?g/mL,50.67?g/mL and 53.47?g/mL,while T-47D and Colo-205 were much more sensitive to H₂O extract with the half inhibitory concentration(IC₅₀)were 35.98?g/mL and 55.17?g/mL.ZR75-1and Colo-205 cell lines were selected to evaluate the anticancer activity of 21 compounds isolated from EA extract.The results showed that chebulanin,chebulinic acid,punicalagin,corilagin and 1,2,3,4,6-penta-O-galloyl-?-D-glucopy-ranoside were have a good anticancer activity to ZR 75-1 while the half inhibitory concentration(IC₅₀)were 6.31mM,7.48mM,7.95mM,7.71mM and 25.23 mM,respectively.Chebulanin,chebulinic acid,punicalagin and corilagin were also inhibit the proliferation of Colo-205 significantly.The half inhibitory concentration(IC₅₀)were 6.04 mM,19.69 mM,10.88 mM and 7.58 mM,respectively.To assess the mechanism underlying T.billirica growth inhibition,ZR75-1and Colo-205cell lines were treated with different concentrations of EA extract,and then flow cytometric analysis was performed to detect apoptotic using an Annexin V-FITC/PI apoptosis assay kit.The results indicated that EA extract induced a dose-dependent apoptosis both in early and late stage.The apoptosis rates were 17.58%and 21.33%in early apoptosis,while 29.20%and40.55%in late apoptosis.The effect of apoptosis-related proteins of EA extract were detection by western blot analysis.The results revealed that EA extract treatment significantly increased the expression of cleaved PARP??Cle-PARP?level in a dose-dependent manner when?-actin served as a loading control?P<0.01?.Treatment with 200.0?g/mL of EA extract increased Cle-PARP?expression by 53.44%in ZR75-1 cells and 84.06%in Colo-205 cells,respectively.The thesis provided the basis and rationale by studying the chemical constituents of pitted fruits of T.billirica and laid on foundation for pharmaceutical material basis and quality control through researching on anticancer activity of the different polar extracts and compounds of T.billirica.,enriching the theoretical basis for clinical application of Tibetan medicine.Innovation:1.For the first time,we made systemic photochemical research on the pitted fruits of T.billirica.54 were isolated from the plant for the first time and 35 compounds were firstly isolated from Terminalia Linn.Flavanones and phlobatannins were isolated from Terminalia Linn.at first.2.It's the first time that we analyzed constituents of T.billirica by UPLC-MSⁿ technique.We have identified seventy compounds from the plant,while fourteen have been isolated.3.It's a first CellTiter-Blue assay to evaluate anticancer activity of different extracts and compounds of T.billirica.The results revealed that EA extract could is the main part to inhibit the proliferation of human cancer cell lines,while the biochemical compounds are tannins and phenolic acids,which were worthwhile for the future research of anticancer drug of T.billirica.