Separation, Identification And Biological Activities Of The Ethyl Acetate Extraction From Rubia Cordiforlia Linn

Rubia Cordifolia L., the dry root and rhizome of the Rubiaceae, is one oftraditional Chinese medicine and widely distributed in our country. Recently,it was reported that Rubia cordifolia L. exhibited some biological activitiessuch as inhibition of the human keratinocytes cells (HaCaT) proliferation,anti-tumor and anti-oxidant properties which had attracted much attention. Atpresent, most of studies on the biological activity of Rubia Cordifolia focusedon its alcohol extraction or water extraction, and only a few literaturesreported the biological activities of ethyl acetate (EA) extraction. Ourprevious research results showed that EA extraction of Rubia Cordifolia L.had better anti-proliferative activity and anti-oxidant activity than that of theextraction from other solvent. However, there is no report on the separation,identification and biological activities of EA extraction of Rubia Cordifolia L..In this thesis, EA extraction of Rubia Cordifolia L. was further separated andpurified. Two hundreds and ten different extractions and twelve purecompounds were obtained. The compounds (1~12) were identified bydifferent kinds of spectroscopy techniques. The anti-proliferative andanti-oxidant activities of the extractions and pure compounds were alsodetermined, which will benefite the study of applications of Rubia cordifoliaL. in clinic treatment.The works are summarized as following:1. The EA extraction of Rubia cordifolia L. was isolated and purified byadsorption column chromatography, thin layer chromatography, filtration andrecrystallization. Two hundreds and ten different extractions and twelvecompounds were obtained. The compounds were identified by highperformance liquid separation (HPLC), infrared spectroscopy (IR), nuclearmagnetic resonance spectroscopy (1H-NMR,13C-NMR) and massspectrometry (MS). Compounds7,10,11and12were assigned to be 1-hydroxy-2-methoxy anthraquinone,2-methyl anthraquinone,1-hydroxy-2-methyl anthraquinone,1,4-dihydroxy-2-methyl anthraquinone,respectively. The identification of other eight compounds needs furtheranalysis.2. The inhibition of HaCaT cell proliferation of the extractions andcompounds (1~12) were determined by MTT assay. The results showed thatⅡJ and ⅡK with low polarity exhibited the higher inhibition activities ofHaCaT cell proliferation among the secondary extractions of ⅡA~ⅡK. ⅡJand ⅡK were further isolated and the inhibition of HaCaT cell proliferation ofisolated extractions were also examined. It was found that ⅡJ2, ⅢKF andIVKFF showed higher activities, which has a IC50value of10,14.5, and12.5μg/mL, respectively. Compounds10and11exhibited higher inhibition of theHaCaT cell proliferation with a IC50value of31.91and20.97μg/mL,respectively.3. The total anti-oxidant activities, inhibition of lipid peroxidationactivities and scavenging of free radical activities of the extractions andcompounds (1~12) were determined by ferric reducing antioxidant power(FRAP) method,2-thiobarbituric acid (TBA) method, and1,1-dipheny-2-picrylhydrazyl (DPPH·) method. The results showed that the totalanti-oxidant activities of the extractions with high polarity were stronger thanthat of the other extractions with low polarity among the extractions ofⅡA~ⅡK. Extraction ⅡA exhibited the highest anti-oxidant activity. Theinhibition of lipid peroxidation of the extractions of ⅡA~ⅡH weresignificantly stronger than that of ascorbic acid (Vc). Extraction ⅢAF hashigher anti-oxidant activity through the activity evaluation of ⅢAA~ⅢAJ,which were isolated from ⅡA. Compound1exhibites the highest anti-oxidantactivity in the isolated pure compounds.