Study On Chemical Constituents And Bioactivity Of Hyptis Rhomboidea

Hyptis rhomboidea belong to Lamiaceae Hyptis, is also called Pycnanthemumdecurrens Blanco, Hyptis decurrens (Blanco) Epling, Hyptis celebica, which used tobe the folk herb for hepatitis, ulcer, edema in China. Accoding to the early screeningantitumor activity of H. rhomboidea, it had a certain antitumor activity, and researchabout this plant was still in the stage of blank, therefore, this thesis regard H.rhomboidea as the research object, main workshop as follows:Objective: Systematically studied the chemical composition on the H. rhomboideagrowed in Hainan province, and in the process of separation to track the activitiessuch as antioxidant activity, antifungal activity, toxicity activity and antitumoractivity in vivo of crude extracts; studied the allelopathice potential of the invasiveplant H. rhomboidea on the ecology; to analysis of various active H. rhomboidea,provide reference basis for its development and utilization.Methods:(1) Quenched the whole whole plant powder75%ethanol, extract bypetroleum ether (PE), ethyl acetate (EtOAc), n-butyl alcohol in order, and get thetotal extracts (A), PE extracts (B), EtOAc extracts (C), n-butyl alcohol extracts(D),n-butyl alcohol extracts was divided into20%methanol extract (E) and40%methanol extract (F) by Diaion HP-20, and EtOAc extract was divided into four parts, Y5（G）, Y6-8（H）, Y6-9（I）, Y7（J） by silica column; isolated and purified theEAc extracts by Toyopearl HW-40, Sephadex LH-20, MCI-Gel CHP-20, RP-18,PTLC and silica column methods, according to the physical and chemical propertiesand structure of modern compound spectra（1H-NMR，13C-NMR，UV，IR，MS，HMQC， HMBC） analysis technical identify these monomer compounds. Theexperiment has identified11monomer compounds.(2) Brine shrimp toxicity experiment and MTT method were used to H.rhomboidea alcohol extracts in vitro antitumor activity. Among the brine shrimptoxicity experiment, the distilled water as a negative control, results were showed byLC50（μg/mL）. MTT method acted on H1299, Lovo, SW480, A2058, results wereshowed by LC50（μg·mL-1）.Using H. rhomboidea alcohol extracts of high, mediumand low doses (13,6.5,3.25mg/10g) on ICR beared H22tumor mice solid tumor,ascites tumor, immunodeficiency model experiment of antitumor activity in vivo.(3) Using DPPH, FRAP, ABTS method of crude extractings from H.rhomboidea A~D, G~J for determination of antioxidant activity, the Trolox as apositive control. Results were showed by the TEAC (mg/100g).(4) Using double dilution method, to evaluate the antifungal activity of A~J andmonomer compounds on Fusarium graminearum(F.g), Fusarium graminearumSchwabe (F.gs), Rhizopus nigricans (R.n), Exserohilum turcicum(E.t), Fusariumoxysporum (F.o), Botrytis cinerea (B.c), Sclerotinia sclerotiorum (S.s), Sclerotiniasclerotiorum（Lib.）de Bary (S.sc), results were showed by MIC.(5) Using Petri dish and "a little cup" methods, to study the seed germination and seedling growth of the stems and leaves aqueous extracts on eight kinds of seeds(Brassica campestris L., Raphanus sativus L, Oryza sativa, Lactuca sativa, Vignaradiate, Triticum aestivum Linn., Capsicum Linn L., Lolium perenne L.), andaqueous extracts acted on brine shrimp toxicity experiment, the distilled water asnegative control, the results were showed by LC50（μg·mL-1）.Results:(1) Eleven compounds were isolated from the EAc extracts of H.rhomboidea75%ethanol extracts and identified as ethyl caffeate (1), ursolic acid(2),oleanolic acid(3), vanillactic acid(4), methyl rosmarinate(5), kaempferol3-O-α-L-rhamnopyranosyl-(1â†'6)-β-D-glucopyranoside(6), kaempferol3-O-α-L-rhamnopyranosyl-(1â†'6)-β-D-glucopyranoside(7), ilexgenin A (8),β-Amyrin(9), kaempferol3-O-β-D-glucopyranoside (astrgalin,10), Cholest-5-ene-3β,4β-diol(11), respectively. Compound1~11were isolated firstly from the plant.(2) The LC50(μg·mL-1) of the A~D, G~J of brine shrimp toxicity experimentwere94.91,276.91,96.86,392.10,446.14,189.95,167.76μg· mL-1respectively.The LC50(μg·mL-1) of the H1299, Lovo，SW480, A2058, PLC of the MTT methodantitumor activity in vitro were8,18,23,12,50μg·mL-1.The inhibition rate of H.rhomboidea alcohol extracts of high, medium and low doseswere68.83%，59.24%，46.40%respectively on ICR beared H22tumor mice solid tumor, both H-r and M-rincreased the spleen index (SI), compared with the model group owned thestatistically significantascites（P＜0.05）. ELISA analysis showed cboth H-r and M-rimproved the contents TNF-α, IL-2, ALT, ASTï¼› according to the results of tumourbiopsies dyeing, model group mice tumor cell proliferated activity with much bubbles, but the CTX, H-r and M-r group mice tumor cell were less than modelgroup with less bubbles. In ascites tumor model, the H-r can prolong the micesurvival time about20d, and improved the quality of life in immunodeficiencygroup.(3) In the antioxidant activity experiment of the H. rhomboidea extracts A~D,G~J, the DPPH method showed that the TEAC of H, I, C, A were greater than Trolox,the H owed the best antioxidant activity; the ABTS method showed that the TEAC ofH, C, A were greater than Trolox, the H owed the best antioxidant activity; the DPPHmethod showed that the TEAC of H, I, A, C, J, D, E, G were greater than Trolox, theH owed the best antioxidant activity either.(4) The antibacterial experiments show that: G~J showed the strong inhibitionon the eight kinds of fungals, especially to F.gs and F.g. Test of the monomercompounds (phenolic acids) has good inhibition on most of fungals. Compound1，4，5showed inhibition on F.gs, F.g, E.t and S.s,4，5owned the strongest inhibitioneffect. compound6only inhibited the S.sc, compound10inhibited the S.s and S.sc.(5) The stems and leaves extracts showed different degrees allelopathicpotential on eight kinds of seeds. Improvd the LS and TA, and inhibited the BC, RS,OS, VR, CL and LP. The inhibited order were LP＞CL＞RS＞BC＞OS≈VR＞LS＞TA.Conclusions：(1)The types of main chemical composition of H. rhomboidea werephenolic acids, flavonoids and terpenoids.(2)H. rhomboidea ethanol extracts owned a certain toxicity activity in vitro, and a certain antitumor activity and important immune activity in vivo.(3) H. rhomboidea ethanol extracts owned a certain antioxidant activity,especially the I showed the strongest activity, and H. rhomboidea can be thecandidate natural antioxidant plant sources.(4) H. rhomboidea ethanol extracts owned a certain antifungal activity.(5) H. rhomboidea owned a strong allelopathic potential to native plants.