| Ilex kudingcha C. J. Tseng (Aquifoliaceae) is an evergreen arbor and its leaves have been used as a traditional Chinese medicine, called ku-ding-cha. Phytochemical investigations on I. kudingcha reported occurrences of triterpenoid saponins, phenolic acid, polysaccharide, essential oil, etc, which has anti-inflammatory, anti-bacterial and cardiovascular pharmacological effects. It’s commonly used in the treatment of hypertension, hyperlipidemia and hyperglycemia. With the purpose of seeking bioactive substances from this plant and elucidating the medicinal effectiveness of its material foundation, a phytochemical investigation of I. kudingcha was performed. On the basis of systematical study on the chemical constituents of I. kudingcha, we further performed a content determination of the representative constituents of I. kudingcha, and the triterpenoid saponins isolated from the leaves of I. kudingcha were screened for antiplatelet aggregation activity in vitro induced by ADP. This research will provide useful data for the deeper digging of the medicinal value of I.kudingcha.We carried out a phytochemical investigation on the leaves of I. kudingcha, and it will provide scientific basis for broadening the scope of its clinical application. The methanol residue was suspended in water and then was partitioned with petroleum ether, chloroform, ethyl acetate and n-butanol. All fractions were further separated by sephadex LH-20 column, silica gel column, ODS column, macroporous resin column and semi-preparative HPLC. Thirty-five compounds were isolated and the structures of all compounds were elucidated on the basis of spectroscopic analysis (1H and 13C-NMR) and physiochemical properties. The compounds were identified as 3-O-β-D-glucopyranosyl(1â†'>3)-α-L-rhamnopyranosyl(1â†'2)]-α-L-arabinopyranosylurs-12,19(29)-dien-28-oic acid 28-O-α- L-rhamnopyranosyl(1â†'2)-β-D-glucopyranosyl ester(1),3-O-β-D-glucopyranosyl(1â†'3)-α-L-rhamnopyranosyl(1â†'2)]-α-L-arabinopyranosyl-19α,20α-dihydroxyurs-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl(1â†'2)-β-D-glucopyranosyl ester(2), ursolic acid(3), kudinoside D(4), kudinoside G(5), latifoloside C(6), latifoloside G(7), latifoloside H(8), kudinoside A(9), latifoloside Q(10), ilekudinoside T(11), kudinoside E(12), kudinoside H(13), ilekudinoside R(14), oleanolic acid(15), ilekudinoside K(16), 3β-(α-L-arabinopyranosyloxy)-19α-hydroxy-olean-12-en-28-oic acid-β-D-glucopyranosyl ester(17),4-hydroxy-3-methoxy-a-methyl benzyl alcohol(18), iso-ferulic acid(19), 2-methoxy-phenylacetic acid(20), caffeic acid(21),3,4-dihydroxy benzoic acid(22), hydroxytyrosol glucoside(23),3-caffeoylquinic acid(24),5-caffeoylquinic acid(25), 3,5-O-dicaffeoylquinic acid methyl ester(26),3,4-O-dicaffeoylquinic acid(27), 3,4-O-dicaffeoylquinic acid methyl ester(28),3,5-O-dicaffeoylquinic acid(29), ferulic acid(30),β-daucosterol(31),β-sitosterol(32), quercetin-3-O-glucoside(33), rutin(34), and 6-hydroxy-7,7a-dihydro-2(6H)-benzofuranone (35). Compounds 1 and 2 were new compounds, and compounds 17,19,33 and 35 were obtained from this species for the first time.Based on the isolation and structure elucidation of triterpene saponins and acids from the leaves of Ilex Kudingcha, a high performance liquid chromatography method was conducted for simultaneous determination of seven triterpene saponins, kudinoside D(4), kudinoside G(5), latifoloside C(6), latifoloside G(7), latifoloside H(8), kudinoside A(9) and kudinoside E(12) for the first time. The separation was carried out on a YMC C 18 column (YMC-pack ODS-A,250×4.6 nm,5 μm) which eluted with a MeCN-H2O gradient system at the flow rate 1 mL/min, the temperature of 30 ℃, and the dection wavelength at the 210 nm.The calibration curves of 4～9 and 12 indicated a good linear at ranges of 0.22～4.95 (r> 0.9999),0.24～5.40 (r> 0.9995),0.20～4.50 (r> 0.9996),1.02-22.95 (r> 0.9999), 0.96～21.60 (r> 0.9999),1.18～26.55 (r>0.9999) and 0.30～6.75 (r> 0.9999) μg/ml, respectively, the average recovery rates in the range of 99.25%to 100.8%, and the RSD was less than 2.0%. In addition, a method was also estblished by HPLC to simultaneously determine the five acids, caffeic acid(21), hydroxyl tyrosol glycosidase(23), 3,4-O-dicaffeoylquinic acid(27),3,5-O-dicaffeoylquinic acid(29) and 6-hydroxy-7,7a-dihydro-2(6H)-benzofuranone(35) from this plant. The chromatographic separation was achieved on a kromasil C18 (kromasil,250x4.6 nm.5 μm) using a mobile phase made up of acetonitrie and 0.02% trifluoroacetic acid water at a flow rate of 1.0 mL/min. The detect wavelength was set at 260 nm, and the column temperature was set at 30℃. The results showed the five compounds (21,23,27,29,35) all have good separating degree, and good linear range of 0.003～0.043(r> 0.9999),0.036～0.450(r> 0.9999),0.052～0.650(r> 0.999 9),0.280～3.500(r> 0.9999),0.400～5.000(r> 0.9999) μg/mL, respetively, and the average recovery rates ranged from 96.37% to 103.3%, with the RSD of the method was not more than 3.0%. This results show that the method is considered to be accurate, simple, good precision and reproducible. The established method is condsidered to have vital significance for evaluting the quality and utilization of the I. Kudingcha and it’s preparations.The triterpenoid saponins isolated from the leaves of I. Kudingcha were screened for antiplatelet aggregation activity in vitro induced by ADP (5 μM), and compounds 1,2,10, 11,13 and 16 showed significant antiplatelet aggregation activity with IC50 values of 14.7, 11.3,20.5,18.9,17.4 and 8.1 μM, respectively. |
PDF Full Text Request