Study On Chemical Constituents From Anemarrhenae Rhizoma

Anemarrhenae Rhizoma is the dried roots of Anemarrhena asphodeloides Bge.,which has the functions of reducing internal heat, producing saliva to embellish dryness.Pharmacological studies have shown that the Anemarrhenae Rhizoma has activities onanti-inflammatory, anti-microbial, anti-plate aggregation and prominent curative effectin the treatment of rheumatism and diabetes.The chemical constituents in Anemarrhenae Rhizoma are mainly steroidal saponinsand flavones, in addition to containing phenylpropanoids, alkaloids,organic acids,etc.This experiment had gained10compounds from90%ethanol extract ofAnemarrhenae Rhizoma.by using the main methods of column chromatography ofsilica-gel or ODS, recrystallization,etc. nine ones among the ten compounds had beenidentified by NMR and MS spectroscopy methods as monomethyl-cis-hinokiresinol,2,6,4’-trihydroxy-4-methoxybenzophenone, helichrysetin, vitexin, Timosaponin AⅣ,AnemarrhenasaponinⅠ, Timosaponin B III, Timosaponin AⅠand mangiferin.Among the nine compounds, helichrysetin were obtained from AnemarrhenaeRhizoma for the first time.The paper also established a HPLC-ELSD method to determine the content ofTimosaponin AⅣ in Anemarrhenae Rhizoma. In addition,the ten compounds and theextract obtained from Anemarrhenae Rhizoma were tested for its ability to inhibitDipeptidyl peptidase-Ⅳ activity.1． Extraction,isolation and structural identification of the chemical constituentsof Anemarrhenae Rhizoma1.1Extraction30kg dried Anemarrhenae Rhizoma were crushed and then extracted by heatreflux for three times in90%ethanol. The volume of90%ethanol were10times（V/W） each time. The extracted solution was combined after filtered.After thesolvent was evaporated by rotary evaporators,2.5kg crude extract was obtained.1.2Isolation and purification Isolation methods mainly included recrystallization and column (silica gel andODS column) chromatography and isolation process needed to be repeatedcontinuously. Reagents used as eluents inclued ethyl acetate, ethyl alcohol, petroleumether, methanol, acetone and distilled water. Finally10compounds have been wereisolated from2.5kg crude extract of Anemarrhenae Rhizoma and9ones namedzm-1,zm-2,zm-3,zm-4,zm-5,zm-6,zm-7,zm-8,zm-9were identified.1.3Structural identificationEight compounds had been identified by NMR and MS spectroscopy methods asmonomethyl-cis-hinokiresinol,2,6,4’-trihydroxy-4-methoxybenzophenone,helichrysetin, vitexin, Timosaponin AⅣ, AnemarrhenasaponinⅠ, Timosaponin B III,Timosaponin AⅠand mangiferin.2. Content determination of Timosaponin AⅣ(zm-5) by HPLC-ELSD2.1Instruments and chromatographic conditionsChromatograph-Detector: Agilent1200HPLC-Alltech2000ELSDColumn: Unitary C18（4.6×250mm,5μm）Chromatographic conditions: Mobile phase: Acetonitrile-water(0.2%HAc)(43:57);Column temperature:30℃; Flow rate:1.0mL/min; Injection volume:20μL;conditions of the ELSD detector: drift tube temperature:101℃; gas(air) pressure:2.15bar; impactor: closed.2.2Methodology investigation2.2.1Linear relationWithin the range of0.6μg~30μg, the lgS(S:peak area) and the lgm(m:sample mass)of compound zm-5were in a good linear relationship. Regression equation wasy=1.358x+1.211，R2=0.9990.2.2.2PrecisionThe intra-day and inter-day RSD of the peak area of compound zm-5was1.22%and1.33%respectively. It indicated that the method had a good precision.2.2.3RepeatabilitySix samples were determined and the results showed that the RSD of thecontent of compound zm-5was1.47%(less than2%),which indicated that the method had a good repeatability.2.2.4StabilityThe same sample solution have been analyzed by interval of2h during10hours.The results showed that the RSD of the peak area of compound zm-5was1.24%(lessthan2%), which indicated that the sample solution was stable within10hours.2.2.5RecoveryThe average recovery of compound zm-5was97.01%and the RSD was1.09%,which indicated that the method had a good recovery.2.3Content determinationThe experiment determined the content of Timosaponin AⅣ in AnemarrhenaeRhizoma which purchased from different regions. The results showed that the contentvaried significantly from defferent sources. Among the samples,the highest content ofTimosaponin AⅣ in Anemarrhenae Rhizoma was0.643%,which Purchased fromAnguo in Hebei province,while the lowest one was0.028%, which Purchased fromYixian in Hebei province.3. Inhibition test of the Dipeptidyl peptidase-Ⅳ activityTen compounds obtained from Anemarrhenae Rhizoma and the extract from itwere tested for its ability to inhibit Dipeptidyl Peptidase-Ⅳ activity. The resultshowed that only the compound monomethyl-cis-hinokiresinol can inhibite DPP-Ⅳactivity by30%, others showed no inhibition or weak inhibition.4. SummaryOn the basis of the domestic and foreign studies, the chemical constituents fromAnemarrhenae Rhizoma was deeply investigated in this paper. Ten compounds wereisolated and nine of them were identified, one of them were obtained from this plantfor the first time. In this study, a HPLC-ELSD method was also developed for thedetermination the content of Timosaponin AⅣ in Anemarrhenae Rhizoma. Inaddition,the ten compounds obtained from Anemarrhenae Rhizoma and the extract ofthis herb were tested for their ability to inhibit DPP-Ⅳ activity.The study provided favorable scientific basis for research, development andutilization of Anemarrhenae Rhizoma.