Study On Anti-breast Cancer And Quality Standard Of Triterpenoids From The Leaves Of Ilex Latifolia

Objective: To investigate the inhibition of triterpenoids from the leaves of Ilex latifolia on the proliferation of breast cancer in vivo and in vitro and screen an active ingredients of triterpenoids from the leaves of Ilex latifolia,the quality standard of Ilex latifolia was studied in our research.A method for quantities of triterpenoids in the leaves of Ilex latifolia was established by using HPLC.Their triterpenoids differences in the leaves of Ilex latifolia from different habitats and different harvesting periods were discussed with the fingerprint of traditional Chinese medicine,which provided scientific basis for quality control and clinical application of Ilex latifolia.Methods: 1)MTT assay was used to compare the effects of triterpenoids in the Ilex latifolia on the proliferation inhibition of human breast cancer MDA-MB-231 cells and human cervical cancer Hela cells.The ability of 27-O-p-(E)-coumaroyl ursolic acid(C-Ur)and Eto Ac-P to induce apoptosis of MDA-MB-231 cells and Hela cells was detected by flow cytometry.MTT and flow cytometry was used to detect the apoptosis of MDA-MB-231 cells induced by different solubility C-Ur.The expression of C-Ur in vitro and inhibit the expression of EGFR in MDA-MB-231 cells was detected by Wstern-Blot.The inhibitory effect of different solubility C-Ur on EGFR tyrosine kinase was determined.The expression of EGFR in MDA-MB-231 cells supplemented with si RNA EGFR and Lapatinib was determined by flow cytometry.In vivo experimentswere conducted to investigate the effect of C-Ur on tumor formation and expression of related pathways in athymic mice.2)A quantitative analysis method for the content of triterpenoids in Ilex latifolia was established.The content of 6 triterpenoids including ziyuglycoside II,pomolic acid,27-O-p-coumaroyl ursolic acid,daucossterol,3?-Hydroxyurs-11-en-28,13?-olide and uvaol in Ilex latifolia was simultaneous determined by HPLC.Agilent HC-C18(4.6mm×250 mm,5 ?m)column was used for gradient elution with acetonitrile-0.4%phosphoric acid aqueous solution as mobile phase.The detection wavelength was 205 nm,column temperature was 30 ?,flow rate was 1.0 m L/min,and injection volume was 10 ?L.Fingerprint analysis was performed using the Chinese Medicine Chromatographic Fingerprint Similarity Evaluation System(2004A)software.3)A method for simultaneous determination of lupinol,stigmasterol and ?-Amyrin was established on YMC-ODS C18((4.6 mm×250 mm,5 ?m)column with methanol-0.4% phosphoric acid solution(98:2)as mobile phase,flow rate of 1 m L/min,detection wavelength of 205 nm,column temperature of 30 ? and injection volume of20 ?L.Results: 1)MTT assay showed that C-Ur,(20S,24S)-Epoxydammarane-3?,25-diol,25-Deuteriostigmasterol,a-amyrin,pomolic acid 3-O-alpha-L-arabinopyranoside,ursolic acid,oleanolic acid,?-sitosterol,?-daucrol,Eto Ac-P and n-butanol extract of the leaves of Ilex latifolia could inhibit MDA-MB-231 cells and Hela cells,and C-Ur and Eto Ac-P had the best inhibitory effect on both cancer cells.Flow cytometry showed that C-Ur induced apoptosis of MDA-MB-231 cells more effectively than Eto Ac-P,and inhibited the proliferation of MDA-MB-231 cells in a dose-dependent manner.The expression of C-caspase-3 in MDA-MB-231 cells treated with drugs increased parallel to the apoptotic rate of MDA-MB-231 cells treated with drugs,and the expression of PARP1 was significantly inhibited.C-Ur inhibits EGFR overexpression and dose-dependent inhibition of EGFR tyrosine kinase activity in MDA-MB-231 cells withan IC50 of 56.48 nmol,but EGFR-inhibited MDA-MB-231 cells no inhibition.In vivo experiments showed that C-Ur inhibited tumor growth in mice,down-regulated the PI3K/AKT/m TOR and interfered with Ras-Raf-MEK-ERK signaling pathway.2)Six triterpenoids including ziyuglycoside II,pomolic acid,27-O-p-coumaroyl ursolic acid,daucossterol,3?-Hydroxyurs-11-en-28,13?-olide and uvaol from Ilex latifolia have good separation.The linear ranges were 4.70~235.00(r=0.999 9),6.94~347.00(r=1.000 0),12.60~630.00(r = 0.999 6),36.24~1812.00(r=0.999 9),2.08~104.00(r=0.999 7),28.62~1431.00 mg/L(r=0.999 9),respectively.The linear relationship was good,the average recovery was 98.18%~102.64%,and the RSD was less than 3%(n=6).The similarity of fingerprints of 14 batches of the leaves of Ilex latifolia from the different harvesting periods was more than 0.9,which indicated that the samples had good consistency.The similarity of fingerprints of 10 batches of the leaves of Ilex latifolia from different habitats was significantly different.3)Under the above chromatographic conditions,lupeol,stigmasterol and?-Amyrin in the Ilex latifolia were well separated,and in the mass solubility18.88~906.00 mg/L(r=1.000 0,n=6)and 15.06~723.00 mg/L(r=0.999 9,n=6),22.75~1092.00 mg/L(r=0.999 9,n=6)showed a good linear relationship,and the average recovery rate(n=6)was 100.43%(RSD=1.96%),98.72%(RSD=0.88%),102.86%(RSD=2.47%).Conclusion: C-Ur from the leaves of Ilex latifolia can effectively inhibit the proliferation of human breast cancer MDA-MB-231 cells and promote cell apoptosis in vitro.It can act as an EGFR tyrosine kinase inhibitor on MDA-MB-231 cells and induce apoptosis.The two established methods are simple,convenient,accurate and reproducible.They can be used to determine the contents of several triterpenoids in the leaves of Ilex latifolia.Combining the fingerprint analysis of the leaves of Ilex latifolia can provide a more comprehensive theoretical basis for the establishment of the qualitystandard of the leaves of Ilex latifolia.The contents of triterpenoids in the leaves of Ilex latifolia from different habitats and harvesting periods were significantly different.