| Ilex dunniana Levl. and Ilex pernyi Franch. are evergreen shrub or small trees of Ilex genus in Aquifoliaceae, widely distributed throughout the southern regions of the Qinling mountains and the provinces northward to the Changjiang River Vally. Many species of Ilex genus have been used as Chinese tradition medicine or beverage to clear away heat and toxic material, to reduce inflammation, to relieve cough and eliminate phlegm or to heal cardiovascular disease.Inflammation is one of the most important pathophysiological factors of many diseases, such as furuncle, pneumonia, intestinal catarrh, infection of external injury and various infectious diseases. Inflammation is the defense reaction of body caused by the damage from outside, in which cardiovascular system is in the central status. In fact, majority of cardio-cerebrovascular diseases including atherosclerosis, stroke, high blood pressure and cardiac insufficiency are chronic inflammatory diseases. With the development of free radical medical study, the important status of oxidation in various diseases was increasingly accepted. Studies demonstrate that oxidative stress is closely involved in many diseases, such as cancer, cardio-cerebrovascular diseases, diabetes, aging, and et al. Consequently, anti-inflammatory and antioxidant activities are key factors deserved to be valued as properties of Chinese traditional medicine which are used to treat diseases mentioned above or related symptoms. In another word, evaluation of the anti-inflammatory and anti-oxidative potential of a plant is an efficient method to set about.1. Screening of active part(s)In this study, anti-inflammatory activities of the 90% EtOH extracts of two Ilex plant leaves were evaluated using mice ear oedama and rats cotton-pellet granuloma tests. The result showed both of the two extract presented the ability of inhibition of inflammation in mice and rats models in dose-dependent mode. Based on this result, two extracts were devided into four portions according to polarity, respectively. Levels of inflammatory and proinflammatory cytokins were valued with two extract and all portions. The results showed that the n-BuOH extracts of I.D. and I.P. were the most active portion. T-AOC, DPPH and hydroxyl radical assays were adopted to evaluate the anti-oxidant and free radical scavenging activities. In these assays, EtOAC and n-BuOH portion of I.P., and n-BuOH portion of I.D. showed better activity than the extracts and the other portions.2. Isolation of compounds from active parts2.1 Compounds from Ilex dunniana Levl.18 compounds were isolated from Ilex dunniana Levl. and they were identified as:dunnianaolide A (ID6), dunnianaolide B (ID12), dunnianaolide C (ID16), rotundioic acid 28-O-?-D-glucopyranoside (ID8), 3-O-?-D-glucopyranosyl-rotundic acid 28-O-?-D-glucopyranoside (ID37), quercetin (ID1), quercetin-3-O-(?-D-glucopyranoside) (ID2), quercetin-3-O-(?-L-rhamnopyranosyl)-(1-6)-?-D-glucopyranoside (ID3), kaempferol-3-O-(?-L-rhamnopyranosyl)-(1-6)-?-D-gluco pyranoside (ID4), kaempferol-3-O-(?-L-rhamnopyranosyl)-(1-6)-?-D-galactose (ID7), kaempferol-4'-ethoxy-3-O-?-D-glucopyranoside (ID9),4-[(erythro)2,3-dihydro-3-hydroxy-methyl-5-(3-hydroxypropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenyl-?-D-gluco pyranoside (ID14), (7S,8R)-dihydrodehydrodiconiferylalcohol-9-?-D-glucopyranoside (ID19), lariciresinol-4-O-?-D-glucopyranoside (ID33),?-sitosterol (ID5),?-daucosterol (ID18),p-hydroxybenzoic acid (ID20), sucrose (ID21).All these compounds were isolated from Ilex dunniana Levl. for the first time. ID6, ID12 and ID16 were new compounds. X-ray data of ID6 was provided in this thesis. Most interesting is the fact that it is to our knowledge one of the first X-ray analysis of a saponin, a class of compounds which poorly crystallize.2.2 Compounds from Ilex pernyi Franch.23 compounds were isolated from Ilex pernyi Franch. and they were identified as:a-amyrin (IP1), uvaol (IP2), ursolic acid (IP3-1), Oleanolic acid, (IP3-2),3a,19a-dihydroxyurs-12-en-28-oic acid (IP4), lupeol (IP5-1), betulinic acid (IP5-2), lup-20(29)-ene-3?,24-diol (IP6),3?-hydroxyurs-11-ene-13?-olide (IP7),?-amyrone (IP8-1), lupeone (IP8-2), urs-12(13)-en-3-one (IP8-3), friedelin (IP9), (3?)-3-(?-D-glucopyranosyloxy)-19,23-dihydroxyurs-12-en-28-oic acid (IP12-1), (3?)-3-(?-D-glucopyranosyl oxy)-19,23-dihydroxyolean-12-en-28-oic acid (IP12-2), quercetin (IP13), quercetin-3-O-(?-D-glucopyranoside) (IP14), rutine (IP17), 5,6,11,12-tetramethyl tritriaconta -5,11,17,23,29-pentaenoate (IP10), heptadecanoic acid (IP11-1), methyl heptadecanoate (IP11-2),?-sitosterol (IP15),?-daucosterol (IP16).IP8-1, IP8-2, IP8-3, IP10, IP11-1, IP11-2 and IP12-1 were isolated from Ilex pernyi Franch. for the first time.3. Inflammatory and antioxidative activities of compoundsLevels of NO, TNF-a, PGE2, IL-1?and IL-6 secreted from RAW 264.7 induced by LPS were tested by ELISA kits treated with several compounds isolated. The results showed that ID6, ID8, ID12 and ID16 presented significant inhibition activities of TNF-?, PGE2 and IL-1?; ID16 inhibited NO secreting from cells and at high dose (12?g/ml) ID16 showed mild inhibition activity of IL-6, as well. In DPPH radical assay, three flavonoids, ID1 (EC506.5?M), ID2 (EC50 8.1?M), ID3 (EC50 7.6?M) and two lignens ID19 (EC50 16.4?M), ID33 (EC50 12.9?M) showed good scavenging activity as compared with Vc (EC50 16.0?M). |
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