Studied On The Chemical Components And Antitumor Activity Of Ilex Rotunda Thunb

Objective:Ilex rotunda Thunb.(Aquifoliaceae) was early recorded in "Lin Nan Cai Yao Lu". Now,This medical plant is recorded in "Guangdong Chinese Medicine criterion",used as traditional Chinese medicine in the southern region of the People's Republic of China, also named "Jiu-bi-ying".The property and flavor of Ilex rotunda is cold and bitter.Its efficiency include heat-clearing and detoxicating,cooling blood,stop bleeding, promoting qi circulation to relieve pain,et al.A literature search revealed that the clinical internal used for treatment of cold fever,sunstroke,tonsillitis,swell throat, abdominal distension,diarrhea,dysentery,acute gastroenteritis and rheumatism,at the same time,external used for treatment of fracture,pyocutaneous disease,bleeding wound,scald and burn.Now,a lot of patent medicine in china use this plant as the main ingredient.The patent medicine including "Jiubiying" have good perspective marketing. The original plant of "Jiubiying" is distributed widespread in Guangdong province, China.Because of these reasons,the intensive research and development of this plant is value and meaningful.Pharmacopoeia of the People's Republic of China,edition 1977 (Pharmacopoeia of P.R.C) was recorded this plant as Chinese medicine,but the Pharmacopoeia of P.R.C hadnot been recorded this plant again in the following edition. The main reason is this plant was lacked of TLC identification and assaying,and the chemical composition or active component were unclear.Therefore,for fully quality control and develop a new patent medicine,the systematically phytochemical investigation and pharmacological research are very necessary and significant.Methods:Phytochemical research:The dried root bark of the plant(28.8kg) was extracted with hot 95%ethanol and concentrated under reduced pressure to give a deep brown syrup(12.3kg).The syrup(3.0kg) was partitioned successively as five parts by Soxhlet's apparatus,these parts were concentrated under reduced pressure to give brown syrup respectively.Petroleum ether part(15g),chloroform part(92g),ethyl acetate part (910g),acetone part(950g) and methanol part(157g),which was directly treated by silica gel column chromatography and rechromatographed over Sephadex LH20 or separated on ODS.The structures of these compounds were elucidated on the basis of spectroscopic analysis,including UV,IR,MS,¹H-NMR,¹³C-NMR,DEPT,¹H-¹H-COSY,HSQC and HMBC,et al.Antitumor research:Some bioactivce screening of the part chemical constituents isolated from Ilex rotunda had been done.In vitro experiments,MTT assay was used to determine the cell growth inhibitory rate of the several kind of cancer cell line before and after the compouds treatment,which isolated from the Ilex rotunda.The results were processed by BLISS computer programme to obtain the IC₅₀ of each cell line in every compounds.Results:Studied on the chemical constituents of Ilex rotunda:22 compounds had been isolated from the bark of Ilex rotunda.On the basis of their physiochemical and spectroscopic evidence,their chemical structures were elucidated as follows.Eight triterpenoids:friedelin(Comp.1),3β-OH-oleanane(Comp.2),28-OH-friedelin(Comp.3), rotundic acid(Comp.10),3β,19α-dihydroxyurs-12-en-24,28-dioic acid(Comp.11), peduncloside(Comp.12),19α,24-dihydroxyurs-12-en-3-one-28-oic acid(Comp.17), oleanolic acid(Comp.19).Four compounds based on phenolic glycoside:disyringin ether(Comp.5),rotundaol(Comp.16),syringin(Comp.8),sinapaldehyde glucoside(Comp.9).Two compounds based on diterpene:abbeokutone(Comp.4), sugereoside(Comp.6).Two steroides:β-sitosterol(Comp.21),β-daucosterol(Comp.22). Four other compounds:glucose(Comp.7),inositol(Comp.13),nonadecylic acid(Comp.18),stearic acid(Comp.20).Among these compounds,two are new compounds:Both are phenolic glycoside,Comp.5 and Comp.16,named disyringin ether and rotundaol.Studied on the antitumor activities screening of 12 compouds isolated from Ilex rotunda on 11 kinds of tumor cells line in vitro:two triterpenoid compouds had showed the positive results.There are rotundic acid(Comp.10)against the CNE1,CNE2,HeLa, SW620,Hep3B,A549,MDA-MB-435 tumor cells line and 3β,19α-dihydroxyurs-12-en-24,28-dioic acid(Comp.11)against SW620,Hep3B,LoVo, MDA-MB-435,Bel-7402 tumor cells line.Conclusion:Advanced research on the chemical constituents and the vitro antitumor activities of Ilex rotunda.22 compouds were isolated from the bark of Ilex rotunda.The structures of 8 triterpenoids,4 phenolic glycosides,2 diterpene compouds,2 sterides and 4 other compounds were established on the basis of physicochemical evidence and spectral characteristics.Both the friedelane-type triterpenoid discovered from genus Ilex and the two diterpenes isolated from Ilex rotunda were the first time.The screening of antitumor effect of 12 compouds on 11 tumor cell lines shows two triterpenoids,rotundic acid and 3β,19α-dihydroxyurs-12-en-24,28-dioic acid, have significant antitumor activities.The antitumor effect of two triterpenoids are first reported in this dissertation...