【作者】 黄彬彬；

【导师】 金永日；

【作者基本信息】 吉林大学 ， 分析化学， 2015， 硕士

【摘要】 知母(Anemarrhenae Rhizoma)为百合科植物知母（Anemarrhenaasphodeloides Bge.）的干燥根茎，具有清热泻火、生津润燥之功效。药理研究表明，知母具有抗炎、抗菌、抗血小板凝集等作用，尤其在治疗风湿、糖尿病方面疗效显著。知母的化学成分以甾体皂苷类化合物和黄酮类化合物为主，此外还含有苯丙素类、生物碱类、有机酸类等化合物。本文利用90%乙醇提取、硅胶及ODS柱层析、重结晶等手段从知母干燥根茎中分离出10个单体化合物，利用NMR、MS等波谱学手段鉴定了其中9个化合物的结构，它们分别是：单甲基-顺-扁柏树脂酚、2,6,4’-三羟基-4-甲氧基二苯甲酮、蜡菊亭、牡荆素、知母皂苷AⅣ、知母皂苷Ⅰ、知母皂苷BⅢ、知母皂苷AⅠ、芒果苷，其中蜡菊亭是首次从知母中分离得到。本文还建立了知母中知母皂苷AⅣ的HPLC-ELSD含量测定方法。另外，还对从知母中分离得到的10个单体化合物及知母的不同提取物的DPP-Ⅳ活性抑制作用进行了研究。一、知母化学成分的提取、分离和结构鉴定1.1、提取将干燥知母根茎30kg粉碎，以90%乙醇回流提取3次,每次1h，90%乙醇用量均为10倍(V/W)，过滤，合并滤液，减压回收溶剂得到粗提物2.5kg。1.2、分离及纯化将2.5kg粗提物进行硅胶柱层析分离，以乙酸乙酯-乙醇-水为洗脱液进行梯度洗脱，得到A、B、C、D四个部分。将A部分以石油醚-乙酸乙酯-乙醇-水为洗脱液进行硅胶柱层析分离，得到A1～A5四个组分。将A1组分进行ODS柱层析，以乙醇-水为洗脱液，再经重结晶，得到化合物zm-1单体100mg。A2组分以乙醇-水为洗脱液，经ODS柱层析分离，得到化合物zm-2单体100mg、化合物zm-3单体40mg。A4组分经多次洗涤，最后经重结晶得到化合物zm-4单体30mg。A5组分以乙醇-水为洗脱液，经ODS柱层析和重结晶，得到化合物zm-5单体100mg。B部分分别经硅胶柱层析（乙酸乙酯-乙醇-水为洗脱剂）、ODS柱层析（乙醇-水为洗脱剂）及重结晶，得到化合物zm-6单体100mg。C部分分别经硅胶柱层析（乙酸乙酯-乙醇-水为洗脱剂）、ODS柱层析（丙酮-水为洗脱剂）及重结晶，得到化合物zm-7单体100mg、化合物zm-8单体80mg、化合物zm-10单体40mg。D部分经重结晶得到化合物zm-9单体150mg。1.3结构鉴定利用核磁、质谱等波谱学手段并与文献对照，共鉴定了9个化合物的结构，它们分别是：单甲基-顺-扁柏树脂酚(zm-1)、2,6,4’-三羟基-4-甲氧基二苯甲酮(zm-2)、蜡菊亭(zm-3)、牡荆素(zm-4)、知母皂苷AⅣ(zm-5)、知母皂苷Ⅰ(zm-6)、知母皂苷BⅢ(zm-7)、知母皂苷AⅠ(zm-8)、芒果苷(zm-9)。二、HPLC-ELSD法测定知母中知母皂苷AⅣ（zm-5）的含量2.1仪器和色谱条件Agilent1200高效液相色谱仪；Unitary C18100（4.6×250mm,5μm）色谱柱；Alltech2000蒸发光散射检测器。色谱条件为：流动相：乙腈-0.1%乙酸（43:57）；柱温：30℃；流速：1.0mL/min；进样量：20μL；漂移管温度：101℃；气体压力：2.15bar；撞击器：关闭。2.2方法学考察2.2.1线性关系在进样量为0.6μg～30μg范围内，化合物zm-5的峰面积的对数lgS与进样量的对数lgm呈良好的线性关系。线性方程为：y=1.358x+1.211，R2=0.9990。2.2.2精密度化合物zm-5的日内和日间峰面积的RSD值分别为1.22%和1.33%，表明仪器精密度良好。2.2.3重复性测定6份供试品溶液，化合物zm-5含量的RSD值为1.47%，表明重复性良好。2.2.4稳定性供试品溶液在0～10h内每隔2h测定一次，结果化合物zm-5峰面积的RSD值为1.24%，说明供试品溶液在10h内稳定性良好。2.2.5加样回收率化合物zm-5的平均加样回收率为97.01%，RSD值为1.09%，说明回收率良好。2.3含量测定本文测定了不同产地的知母中化合物zm-5（知母皂苷AⅣ）的含量，结果表明不同产地含量差异显著。其中购自河北安国的第3批知母药材中知母皂苷AⅣ的含量最高，为0.643%；第一批知母药材中知母皂苷AⅣ的含量最低，为0.028%。三、DPP-Ⅳ活性抑制试验本文对从知母中分离得到的10个单体化合物及知母的各种提取物进行了DPP-Ⅳ活性抑制试验。结果表明，化合物单甲基-顺-扁柏树脂酚对DPP-Ⅳ的抑制率达到30%，其余成分均对DPP-Ⅳ无抑制作用。四、总结本论文在国内外学者的研究基础上，对知母的化学成分进行了深入研究，共分离出10个单体化合物，鉴定了其中9个化合物，其中1个为首次从知母中分离得到。本文建立了知母中知母皂苷AⅣ的HPLC-ELSD的含量测定方法。此外，本文还对10个单体化合物及知母药材提取物的DPP-Ⅳ酶活性抑制作用进行了研究。本论文的研究结果对知母的开发利用提供了新的科学依据。更多还原

【Abstract】 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.更多还原