| Akebia trifoliata (Thunb.) Koidz. var. australis (Diels) Rehd, commonly called "Bai Mu Tong", is an liana plant distributed in China. It is used as a diuretic and an antiphlogistic in traditional Chinese clinics. According to Chinese Pharmacopoeia, origin of Caulis Akebiae is legally used from the stems of Akebia quinata (Houtt.) Decne., A. trifoliata (Thunb.) Koidz. or A. trifoliata (Thunb.) Koidz. var. australis (Diels) Rehd (Lardizabalaceae). Although phytochemical analyses were extensively investigated on the stems, pericarps, seeds and callus tissues of A. quinata or A. trifoliata, few chemical papers were published on A. trifoliata var. australis, except the studies reporting the triterpenoid saponins from the fruits of A. trifoliata var. australis. To investigate the chemical difference among three plants above and discover new active compounds, a detailed phytochemical investigation was carried out on the stems of A. trifoliata var. australis, which resulted in the isolation of seven new triterpenoid saponins, together with twenty-five known triterpenoidal saponins, three known phenylethanoid glycosides, two sterol and two fatty acid glycoside.38 Compounds were separated from the 70% ethanol extract of the stems of Akebia trifoliata var. australis and identified on the basis of physochemical properties and spectroscopic analysis. Among them, seven new compounds were elucided as 2a,3β,23-trihydroxy-30-norolean-12-en-28-oic acid β-D-glucopyranosyl ester(BMT-17), 2a,3β,23-trihydroxy-30-norolean-12-en-28-oic acid (3-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyrano syl-(l→4)-O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl ester(BMT-20), 2α,3β,23-trihydroxyurs-12-en-28-oic acid P-D-xylopyranosyl-(1→3)-O-α-L-rhamnopyranosyl-(1→4) -O-β-D-glucopyranosyl-(1→6)-O-β-D-glucopyranosyl ester (BMT-22), 3-β-[(β-D-gluco pyranosyl-(l→3)-O-α-L-arabinopyranosyl)oxy]-23-hydroxy-30-norolean-12-en-28-oic acid a-L-rhamnopyranosyl-( 1→4)-O-P-D-glucopyranosyl-( 1 →6)-O-β-D-glucopyranosy 1 ester (BMT-26), 3-β-[(α-L-xylopyranosyl-(1→2)-O-α-L-arabinopyranosyl)oxy]-30-norolean-12-en-28-oic acid a-L-rhamnopyranosyl-(1→4)-O-β-D-glucopyranosyl-(1→6)-O-β-D-gluco pyranosyl ester(BMT-33), 3-P-[(β-D-xylopyranosyl-(1→2)-O-α-L-arabinopyranosyl)oxy]-30-norolean-12-en-28-oic acid α-L-rhamnopyranosyl-( 1→4)-O-P-D-glucopyranosyl-( 1 —>6) -O-β-D-glucopyranosyl ester(BMT-29), 3-p-[(P-D-glucopyranosyl-(1→2)-O-[P-D-gluco pyranosyl-(l→3)-O-]-a-L-arabinopyranosyl)oxy]-30-norolean-12-en-28-oic acid α-L-rhamnopyranosyl-( 1→4)-O-β-D-glucopyranosyl-( 1 →6)-O-P-D-glucopyranosyl ester(BMT-30-2);Three known phenylethanoid glycosides were isolated from the plants of the Akebia genus for the first time and identified as (3,4-dihydroxyphenyl)-ethyl-6-E-caffeoyl-glucopyrano side(Calceolarioside B, BMT-4), (3,4-dihydroxyphenyl)-ethyl-6-E-feruloyl-glucopyrano side(BMT-7) , 4-hydroxyphenyl-ethyl-6-O-E-caffeoyl-glucopyranoside (BMT-7-2) andother compounds were identified as 3-P-[(p-D-glucopyranosyl-(l—>2)-O-[|3-D-glucopyrano syl-(l-^3)-O-]-a-L-arabinopyranosyl)oxy]-olean-12-en-28-oic acid a-L-rhamnopyranosyl-(1—*4)-O-P-D-glucopyranosyl-(l—>6)-O-P-D-glucopyranosyl ester(Leonticins E, BMT-27), 3-P-[(P-D-gIucopyranosyl-(l-^2)-O-[P-D-glucopyranosyl-(l-^3)-O-]-a-L-arabinopyranosyl) oxy]-olean-12-en-28-oic acid p-D-glucopyranosyl-(l—>6)-O-p-D-glucopyranosyl ester (BMT-28-2), 3-P-[(P-D-glucopyranosyl-(1^2)-O-a-L-arabinopyranosyl)oxy]-olean-12-en-28-oic acid P-D-glucopyranosyl-(l—?6)-O-P-D-glucopyranosyl ester(Ciwujianoside Ai? BMT-24), 3-P-[(P-D-glucopyranosyl-(l—>2)-O-a-L-arabinopyranosyl)oxy]-30-norolean-12-en-28-oic acid p-D-glucopyranosyl-(l—>6)-O-P-D-glucopyranosyl ester(Ciwujianoside A2, BMT-23), 2a,3p,23-trihydroxyolean-12-en-28-oic acid P-D-xylopyranosyl-(l->3)-O-a-L-rhamnopyranosyl-( 1 ->4)-O-p-D-glucopyranosyl-( 1 -?6)-O-P-D-glucopyranosyl ester(BMT-18), 2a,3p,23-trihydroxy-30-norolean-12-en-28-oic acid a-L-rhamnopyranosyl-(l—?4)-O-P-D-glucopyranosyl-( 1 —>6)-O-P-D-glucopyranosyl ester(BMT-19), 2a,3p,23-trihydroxyolean-12 -en-28-oic acid a-L-rhamnopyranosyl-(l-?4)-O-P-D-glucopyranosyl-(l—>6)-O-P-D-glucopyranosyl ester(BMT-31), 2a,3p,23-trihydroxyurs-12-en-28-oic acid a-L-rhamno pyranosyl-(l^>4)-O-P-D-glucopyranosyl-(l—?6)-O-P-D-glucopyranosyl ester (BMT-32), 2a,3P-trihydroxy-olean-12-en-28-oic acid a-L-rhamnopyranosyl-(l-^4)-O-p-D-glucopyrano syl-(l—>6)-O-P-D-glucopyranosyl ester(BMT-21), 30-norolean-12-en-28-oic acid a-L-rhamn opyranosyl-(l^-4)-O-P-D-glucopyranosyl-(l—*6)-O-P-D-glucopyranosyl ester(BMT-30), 3-P-[(P-D-glucopyranosyl-(l->2)-O-[P-D-glucopyranosyl-(l—>3)-a-L-arabinopyranosyl])oxy]-30-norolean-12-en-28-oic acid(BMT-37), 3-p-[(P-D-glucopyranosyl-(l-^2)-O-[P-D-gluco pyranosyl-(l-43)-a-L-arabinopyranosyl])oxy]-olean-12-en-28-oic acid(BMT-36), 3-O-p-D-glucopyranosyl-(l-?3)-a-L-arabinopyranosyl-23-hydroxyolean-12-en-28-oic acid (BMT-15), 3-O-P-D-glucopyranosyl-(l-^2)-a-L-arabinopyranosyl-30-norolean-12-en-28-oic acid (BMT-14), 3-O-P-D-glucopyranosyl-(l—>2)-a-L-arabinopyranosyl-olean-12-en-28-oic acid (saponin PE? BMT-10), 3-O-p-D-glucopyranosyl-(1^3)-a-L-arabinopyranosyl-olean-12-en-28-oic acid(guaianin N, BMT-9), 2a,3p,23-trihydroxy-olean-12-en-28-oic acid P-D-glucopyranosyl ester(BMT-16- I ) , 2a,3p,23-trihydroxyurs-12-en-28-oic acid P-D-gluco pyranosyl ester (BMT-16-II), 2a,3p,23,29-tetrahydroxy-olean-12-en-28-oic acid(BMT-6), 2a,3p-dihydroxy-olean-12-en-28-oic acid(BMT-3c), 2a,3p,23-trihydroxy-olean-12-en-28-oic acid(BMT-3a- I ), 2a,3p,23-trihydroxyurs-12-en-28-oic acid(BMT-3a-II), 2a,3p,23-trihydroxy-30-norolean-12-en-28-oic acid, oleanolic acid, myristic acid P-D-glucopyranoside, a-(a-galactopyranosyl)-a'-(P-galactopyranosyl)-P-dodecane oic acid glyceride, P-sitosterol and daucosterol.The preliminary screening has been established on the isolated compounds in relation to antioxidative activity, anti-tumor, as well as the inhibition against the xanthine oxidase and the acethylcholine in vitro. The DPPH radical scanvenging assay has showed thatcalceolarioside B exhibited the significantly antioxidative activity and scavenging against DPPH radical. This activity maybe contribute to some functions of Caulis Akebia. MTT assay indicated that all tested compounds had no inhibition against the tumor cells A-549 and Bel-7402. Additonally, no inhibition of the tested compounds was observed against the the xanthine oxidase and the acethylcholine. However, the tested compounds may have other pharmacological activity and the screening on them will be further carried out.A stable and repeatable HPLC method was established for the content of calceolarioside B in Caulis Akebiae for the first time. The average recovery and RSD were 97.2% and 2.33%, respectively. 39 Samples, collected from different growing areas, were determined and the contents of calceolarioside B in the stems of Akebia trifoliata, A. trifoliata var. australis and A. quinata.v/ere fluctuated from 0.021% to 0.745%, and furthermore, calceolarioside B was widely distributed in the roots, leaves or fruits of A. trifoliata, A. trifoliata var. Australia and A. quinata. Therefore, the quality requirement of Caulis Akebiae was suggested to add that the contents of calceolarioside B should be not less than 0.1% in pharmacopoeia.A HPLC method was established for the simultaneous determination of saponin Vn and mutongsaponin C in Caulis Akebiae for the first time and 40 crude drug materials were determined from the root, stem, leaf and fruit, collected from different growing areas. As a result, the contents of saponin Pji and mutongsaponin C in the samples originated from the stems of A. trifoliata var. australis and A. trifoliate were varied over the range of 0.021-0.875 % and 0.074-0.290 %, respectively, however, both compounds rarely were found in the stems of A. quinata. Accoedingly, saponin Pji and mutongsaponin C could be regarded as marker compounds distinguishing Akebia trifoliata, A. trifoliata var. australis and A quinata.A HPLC method was also established for the fingerprint of Caulis Akebiae for the first time and 26 crude drug materials, collected from different growing areas, were analyzed. The chemical differences in the stems of Akebia trifoliata, A. trifoliata var. australis and A. quinata were estimated according to the HPLC profiles and the characteristic peaks were identified by LC-MS. The investigation showed that the phenylethanoid glycosides were common in the MeOH extracts of the stems of A. trifoliata, A. trifoliata var. australia andA. quinata, but the composition of triterpenoid saponins was different in the stems of three plants above. 10 Compounds were identified as calceolarioside B, BMT-7, mutongsaponinB, saponin Pji, mutongsaponin C, mutongsaponin D, ciwujianoside A2, BMT-27, mutongsaponin E, ciwujianoside Ai in the stems of A. trifoliata var. Australia. 8 Compounds were identified as calceolarioside B, BMT-7, mutongsaponin B, saponin Pji, mutongsaponin C, BMT-27, mutongsaponin E, ciwujianoside Ai in the stems of A. trifoliata and 3 compounds were identified as calceolarioside B, BMT-7, BMT-27 in the stems of A. quinata.In addition, Caulis Akebiae was investigated referring to the qualitive TLC examination, moisture, ash, as well as the analysis for pesticide residue and heavy metals. So the quality requirement of Caulis Akebiae was suggested to revise that the contents of water should be not less than 8.5 % in pharmacopoeia.The control criterion protocols for Caulis Akebiae trifoliata var. Australia, Caulis Akebiae trifoliata and Caulis Akebiae were established on basis of the chemical investigation and the quantitative and qualitative analysis.Scutellarin, 4',5,6-trihydroxy-flavonon-7-O-P-D-glucuronopyranoside, is a famous Chinese drug, firstly isolated from a folk herbal medicine Erigaron breviseapus (Compositae), having many pharmacological effects such as dilating vessel, improving microcirculation, increasing artery blood flow and inhibiting platelet aggregation, etc. It has been widely used for the treatment of coronary heart disease, cerebral infarction, hemiplegia and apoplexy in the clinical practice. The published studies on scutellarin were mainly focused on pharmacological activity and clinical practice, and recently, there have also been many studies on the determination of scutellarin in the plasma after intravenous injections or oral adminastration of breviscapine in rabbits, mices, rats and dogs by HPLC and LC-MS-MS methods. However, the pharmacokinetics in model animal and metabolites of scutellarin has not been reported.The pharmacokinetics of scutellarin in normal rats and thrombosis model rats induced by carrageenan were investigated by HPLC methods and the metabolites of scutellarin in rats were identified by HPLC-PDA and HPLC-MS-MS. The calibration curve was linear over the range from 0.625 to 80.0ug-mL~1(r=0.9995) and the limit quantitation was 0.312ug-mL"'. The concentration-time curves of scutellarin in normal and thrombosis model rats were fitted to the open two-compartment model, but the pharmacokinetic parameters of scutellarin in normal and model rats were significantly different. The main metabolites in the rat plasma were deduced as 4',5-dihydroxyflavonon-7-O-P-D-glucuronopyranosyl ester(Ml), scutellarin (M2), 7-methoxy-4',5-dihydroxy-flavonon(M3), 7-methoxy-4',5,6-dihydroxy-flavonon(M4), respectively, and scutellarin was proposed in the metabolic pathways of dehydroxylation and methylation.The thrombus model was induced by a single sc dose of 1% carrageenan to a rat and the effect of Scutellarin on hemorheology of thrombus rat was observed after intravenous administration of Scutellarin. The assay showed that scutellarin inhibited significantlyplatelet aggregation, depressed the whole blood viscosity and influenced slightly the plasma viscosity, which indicated certain trends with extended action duration, but it did not affect the level of the hematocrit and the fibrinogen. |
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