Studies On Bioactive Constituents From Three Medical Plants Collected On Taibai Mountain

Studies on the chemical constituents of medicinal plants or traditional folk medicinalplants can lead to the discovery of new lead compounds which contains somepharmacological activity. This is an important way to discover innovative medicines andto provide a scientific basis for the further development and utilization of traditionalChinese medicine resources. There are rich plant resources of which numerous are used asherb drugs can be found in Qinling Mountains. In this thesis, three medical plants(Anemone taipaiensis，Anemone rivularis var. flore-minore and Pachysandra terminalis)collected on Taibai mountain were studied. EtOH extract of these plants were separated byon the normal-phase silica gel, Sephadex LH-20, reversed-phase ODS and HPLCchromatography to yeild54compounds (16were identified as new compounds). On thebasis of chemical evidence and spectral analysis, especially2D NMR techniques, and X-raySingle crystal diffraction, the structures of these compounds were identified. The antitumor activity of triterpenoid saponins were conducted by the methods of MTT, flow cytometry and western blot. The antibacterial activity of alkaloids was tested by three kinds of standard strains and four kinds of drug-resistant bacteria.1. Studies on chemical constituents of A. taipaiensisPhytochemical studies of a large polar fragment of n-butanol extract from70%ethanol extract of the roots of A. taipaiensis lead to the isolation of10oleanane type triterpenoid saponins, including4new compounds. These compounds were3β-O-{α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl} siaresinolic acid28-0-{α-L-rhamno-pyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(T-1),3β-O-{α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl(1→4)]-α-L-arabinopyranosyl} siaresinolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyra-noside}(T-2), Hederagenin28-0-{α-L-rhamnopyranosyl-(1→4)-p-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-a-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-p-D-glucopyranoside}(T-3),3β-O-{β-D-glucopyranosy1(1→4)-β-D-glucopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]-a-L-arabinopyranosyl} siaresinolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(T-4),3β-O-(β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-[a-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranosyl} hederagenin28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucop-yranosyl-(1→6)-β-D-glucopyranoside}(T-5),3β-O-(β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl} hederag-enin28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyr-anoside}(T-6),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)]-α-L-arabinopyranosyl}hederagenin28-O-{α-L-rhamnnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(T-7),3β-0-{α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl} oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(T-8),3P-O-(β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl} hederagenin28-O-{a-L-rhamnopyranosyl-(1→4)-p-D-glucopyranosyl-(1→6)-p-D-glucop-yranosyl-(1→4)-a-L-rhamnopyranosyl-(1→4)-p-D-glucopyranosyl-(1→6)-p-D-glucopyra-noside}(T-9),3β-O-(β-D-xylopyranosyl-(1→3)-α-L-rhammopyranosyl-(1→2)-[P-D-glu-copyranosyl-(1→4)]-α-L-arabinopyranosyl} oleanolic acid28-0-{α-L-rhamnopyran-osyl-(1→4)-p-D-glucopyranosyl-(1→6)-p-D-glucopyranosyl-(1→4)-a-L-rhamnopyranosyl-(1→4)-p-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(T-10). Compounds T-1, T-4, T-9and T-10were identified as new compounds. All these saponins belong oleanane type and are based on three aglycone types:oleanolic acid, hederagenin and siaresinolic acid.2. Studies on chemical constituents of A. rivularis var. flore-minorePhytochemical studies of n-butanol extract from70%ethanol extract of the roots of A. rivularis var. flore-minore lead to the isolation of37oleanane type triterpenoid saponins, including9new compounds. These compounds were β-sitosterol (X-1),3β-O-L-arabinopyranosyl oleanolic acid28-O-β-D-glucopyranoside (X-2), β-daucosterol (X-3),3β-O-{β-D-xylopyranosyl-(1→2)-a-L-arabinopyranosyl} hederagenin (X-4), kalopana-xsaponin A (X-5),3β-O-{β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl} oleanolic acid (X-6), HN-saponin F (X-7), sapindoside B (X-8), pulsatilla saponin D (X-9), sapindoside B (X-10), pulsatilloside D (X-11), prosapogenin CP4(X-12),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-α-L-ar-abinopyranosyl} oleanolic acid (X-13),3β-O-{β-D-glucopyranosyl-(1→2)-a-L-arabinopyranosyl} oleanolic acid28-O-β-D-glucopyranoside (X-14), cussonside B (X-15), pulsatilla saponin C (X-16), cauloside D (X-17),3β-O-(β-D-ribopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl} hederagenin28-O-β-D-glu-copyranoside (X-18),3β-O-{β-D-glucopyranosyl-(1→2)-α-L-arabinopyranosyl} hedera-genin28-O-β-D-glucopyranoside (X-19),3β-O-α-L-arabinopyranosyl oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-20), ciwujianosideA1(X-21),3p-O-{β-D-xylopyranosyl-(1→2)-a-L-arabinopyranosyl} oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-p-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-22), sieboldianoside B (X-23),3β-O-α-L-arabinopyranosyl betulinic acid28-O-{a-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-p-D-glucopyrano-side}(X-24),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-gluco-pyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl} oleanolic acid28-O-p-D-glucopyranoside (X-25),3β-O-{β-D-glucopyranosyl-(1→2)α-L-arabinopyrano-syl} oleanolic acid28-O-(β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-26),3p-O-α-L-arabinopyranosyl gypsogenin28-O-{α-L-rhamnopyranosyl-(1→4)-p-D-gluco-pyranosyl-(1→6)-β-D-glucopyranoside}(X-27),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl} gypsoge-nin28-O-β-D-glucopyranoside (X-28),3β-O-{β-D-xylopyranosyl-(1→3)-α-L-rham-nopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl} gypsogenin28-O-{α-L-rhanmopyranosyl-(1→4)-p-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-29), cauloside F (X-30), hederasaponin B (X-31),3β-O-{β-D-ribopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-arabinopyranosyl} hederagenin28-O-{α-L-rham-nopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-32),3β-O-{a-L-rhamnopyranosyl-(1→2)-[β-D-glucopyranosyl-(1→4)]-a-L-arabinopyranosyl}21a-hydroxy oleanolic acid28-O-{α-L-rhamnopyranosyl-(1→4)-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside}(X-33),3β-O-{β-D-glucopyranosyl-(1→4)-β-D-glucopyranosyl-(1→4)-[a-L-rhamnopyranosyl-(1→2)]-α-L-arabinopyranosyl}21α-hydroxy oleanolic acid28-O-{α-L-rhanmopyranosyl-(1→4)-p-D-glucopyranosyl-(1→6)-p-D-glucopyranoside}(X-34), kalopanaxsaponin B (X-35), sieboldianoside A (X-36) and hederacolchiside E (X-37). Except for two steroidal compounds (X-1and X-3) and one lupane type triterpenoid (X-24), the remaining34compounds were all belong to oleanane type saponins. Compounds X-4, X-18, X-22, X-24, X-25, X-28, X-29, X-33and X-34were identified as new compounds. These triterpenoid saponins are based on five aglycone types: oleanolic acid, hederagenin, gypsogenin,21α-hydroxy oleanolic acid and betulinic.The aglycone types of21α-hydroxy oleanolic acid and betulinic were isolated from this genus for the first time.3. Studies on chemical constituents of P. terminalis The whole plants of P. terminalis were extracted by90%EtOH under reflux. Theextract was treated by hydrochloric acid, ammonia, and extract by CHCl3successively.Phytochemical studies of total alkaloids lead to the isolation of7alkaloids, including3new compounds. These compounds were1-6,10β-dihydroxy-2,3,7,7,10-pentamethyl-2-azabicyclo-[4.3.1]decan-9-one (D-1), pachysamine A (D-2),20-dimethylamino-3β-senecioylamino-16β-hydroxy-pregn-5-ene (D-3),20-dimethylamino-3β-senecioylamino-pregn-5-ene (D-4), Epipachysamine E (D-5), Epipachysamine B (D-6)and20-dimethylamino-pregnane-3β,4-diol (D-7). Compounds D-2~D-7belong to thePachysandra type alkaloid. Compounds D-1, D-3and D-4were identified as newcompounds。4. Bioactivity studiesThe cytotoxicities of the saponins X-4~X-23were evaluated against five humancancer cell lines, including HepG2, HL-60, A549, HeLa and U87MG. The most potentone was X-12with IC50value of8.21M against U87MG cells. The proportions ofapoptotic cells were evaluated by flow cytometric AnnexinV/PI staining analysis. Survivinexpression was down-regulated in U87cells after treated with X-12for24and72h，accompanied with Bcl-2/Bax expression down-regulated and active-caspase3andactive-caspase9up-regulated in western blot assays, which were postulated as themolecular mechanism of apoptosis of the U87MG cell induce by X-12.The antibacterial activity of alkaloids D-2~D-7was evaluated against two standardstrains (S. aureus ATCC29213and of E.coli ATCC25922). The minimum inhibitoryconcentration (MIC) results showed that D-2~D-7were all antibacterial active againstATCC29213. The most potent one was D-3with MIC=32μg/mL. The antibacterialactivity of D-3was futher tested against a standard strain (S. aureus ATCC14990) andfour-resistant bacteria (MRSA, MRSE, VRSAMu50and LAC). D-3showed better activitythan the positive control ceftazidime by comparing their MIC values25μg/mL and64~128μg/mL. The growth curve and sterilization curve resluts indicated that D-3showedfull growth inhibition and a good performance of bactericidal activity agaist the resistant strains (LAC and MRSE), while the positive control ceftazidime was completely resistant.Innovations and significance:1. Systematic phytochemical studies of three medical plants collected on Taibaimountain led to isolation of54compounds, including45triterpenoid saponins,7alkaloidsand2steroids.2.16compounds were elucidated as new compounds of which saponins based on the21-hydroxy oleanolic acid and betulinic acid as aglycone were first obtained from thegenus Anemone. The new alkaloid D-1was identified a new structure type isolated fromthe genus Pachysandra.3. On the base of the anti-tumor and anti-bacterial experiments, several saponins withsignificant cytotoxic activity, as well as several alkaloids with significant anti-bacterialactivity were found.4. The results of this study provided a reasonable ground for further exploitation ofthese three plants and valuable leads compounds for pursuing new anti-tumor andanti-bacterial drugs and also established a homebase for the further development of themedicinal resources on Taibai mountain.