桑叶体外抗RSV及诱导Hela细胞凋亡物质基础研究
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摘要
物质基础研究,即阐明什么药理作用由什么化学物质产生。中药发挥药效的物质基础往往是多成分的,因此要阐明药效物质基础,既要研究是哪些成分在起作用,也要研究他们各自的比例,即解决“是什么”、“有多少”的问题。
桑叶是桑科植物桑Morus alba L的叶子,具有疏风清热、清肝明目的功效,在我国有悠久的栽培和药用历史,资源极其丰富,临床常用于治疗风热感冒、消渴症等,现代药理研究表明,桑叶具有降血糖、抗病毒、抗癌等多种生物活性,桑叶中的多羟基生物碱已被确认为降血糖的主要物质基础,而桑叶中抗病毒、抗肿瘤的物质基础研究尚不充分,为桑叶的综合利用及进一步开发带来了困难。
本研究首先采用高效液相色谱法(HPLC法)建立了桑叶主要活性成分1-脱氧野尻霉素(1-DNJ)、绿原酸、芦丁、槲皮素、r-氨基丁酸等的含量测定方法及指纹图谱,用以追踪主要化学成分在分离过程中的去向及转移率,为分离条件的优化、阐明物质基础提供定量、定性分析方法;采用高效液相色谱电喷雾串联质谱法(HPLC-MS/MS法)对桑叶中的化合物进行了分析,在对比文献的基础上,初步明确桑叶提取物中的主要物质组成,为物质基础的阐明提供基础(即物质基础是哪些化合物);在活性成分的指引下,采用工业生产可行的分离方法对桑叶进行系统分离,得到粗多糖部位、生物碱部位及Ⅰ、Ⅱ、Ⅲ共五个提取部位,并对部位Ⅰ、Ⅱ、Ⅲ进行体外体外抗RSV评价,对部位Ⅲ进行了诱导细胞凋亡实验研究,初步确定桑叶中体外抗RSV、抗肿瘤的药效物质基础。
方法:1、建立桑叶中活性成分的HPLC含量测定方法及指纹图谱。
(1)HPLC法同时测定桑叶中芦丁、绿原酸和槲皮素。色谱柱为Kromasil C18柱(4.6×250 mm,5μm)柱;流动相为乙腈:甲醇:0.01 mol/L醋酸铵液(pH3.50)(20:10:70);流速1.0 mL/min;柱温为25℃,检测波长为350 nm;
(2)HPLC-ELSD法测定桑叶中的r-氨基丁酸。用Kromasil C18柱(4.6mm×250mm,5μm),流动相为水:乙腈(90:10),流速0.6min/ml,柱温25℃,进样10μL;ELSD气化室温度108.3°,氮气流速:3.0L/min。
(3)HPLC-MS/MS法测定桑叶中1-DNJ含量。以乙腈:0.1%甲酸水溶液(12:88,v/v)为流动相,SHIMADZU HRC-NH_2柱分离,采用大气压化学电离源(APCI),164.1/146.1,164.1/110.2两对离子,多反应监测。
(4)桑叶HPLC指纹图谱的建立。色谱柱:C18(4.6mm×250mm,5μm);流动相:乙腈(A)-水(C)-甲醇(B),梯度洗脱,洗脱曲线:0～28min,A 5～35%,B95～65%,C 0%:28～35min,A 35～50%,B 60～0%,C 0～50%;35～60min;A50～60%,B 0%,C 50～40%。流速:1.0ml/mim柱温:25℃;检测波长:350nm;进样量:10μl。
2、HPLC-MS/MS法分析桑叶中的化学成分。桑叶经50%乙醇回流提取,上液质联用进行分析,对各色谱峰质谱信号进行分析,初步确定桑叶提取物中主要化学成分。液相条件:色谱柱:Aglient Zorbax Extend C18柱(50×4.6mm,1.8μm);流动相:A(甲醇)-B(乙腈)-C(水),洗脱曲线为:0～8min,B4%～35,C94%～65,8～11minA0～40%,B35～60%,C65～0%;11～2lmin:A40～45%,B60～55%,流速:600μl/min,梯进样量:5μL,柱温:室温。质谱条件电喷雾电离源(ESI源),负离子扫描,扫描范围m/z 100～1200。小口电压:-80V,入口电压:10V,温度350℃,碰撞气压力:12psi,雾化气压力:30psi。
3、桑叶有效部位的提取分离
将桑叶提取物进行了较为系统的分离,初步分成五个部位,分别为多糖部位、生物碱部位、Ⅰ部位,Ⅱ部位及Ⅲ部位,采用上述建立的分析方法对各分离部位有效成分进行定量分析,采用HPLC-MS/MS对各部位化学成分进行分析。4、以阳性药物利巴韦林注射液作为对照,对桑叶提取物Ⅰ部位,Ⅱ部位及Ⅲ部位进行体外抗呼吸道合胞病毒活性评价。
药物毒性实验方法:Hela细胞常规传代培养,待长满后用胰酶消化,含10%小牛血清的RPMI-1640培养液制成细胞悬液,以8×10~5个/mL的细胞密度,加入96孔板中,每孔200μl,37℃,5%CO_2孵箱中培养24h,待细胞长成单层。吸出培养液,加入各浓度药液200μl,每个浓度8个复孔。正常对照只加入等体积2%的维持液。37℃,5%CO_2孵箱中培养,每24h观察细胞病变情况(CPE),连续观察72h,记录实验结果。72h后,将药液吸出,每孔加入5mg/mLMTT(用PBS配制)200μl,37℃,5%CO_2孵箱中孵育2h后,弃上清,每孔加入DMSO200μl,室温下振荡混匀10min,在490nm下,酶联免疫检测仪测定吸光度值,并计算细胞存活率。采用SPSS13.0统计软件,Regression-probit回归将药物浓度与细胞存活率进行回归分析,计算细胞半数存活浓度。
呼吸道合胞病毒(RSV)毒力测定:采用TCID_(50)微量法,用Reed-Munch公式计算TCID_(50)。
药物抗病毒实验方法:药物在无毒浓度范围内,用2%的细胞维持液对药物进行倍比稀释,加入病毒液使病毒浓度为100TCID_(50),混匀,阳性对照药为利巴韦林注射液(0.1g/mL),用2%细胞维持液稀释,并加入病毒液,使利巴韦林终浓度为100μg/mL,病毒终浓度为100TCID_(50),于37℃,5%CO_2孵箱中孵育1h。已长成单层Hela细胞的96孔板,吸掉培养液,每孔接种200μl各浓度药液与病毒的混合液,每个浓度8个复孔,同时设阳性对照组,病毒对照组和正常对照组,37℃,5%CO_2培养2h后,将药物组和病毒组液体吸出,加入细胞维持液继续培养,每日观察CPE。72h后将药液吸出,每孔加入5mg/mLMTT(用PBS配制)200μl,37℃,5%CO_2孵箱中孵育2h后,弃上清,每孔加入DMSO200μl,室温下振荡混匀10min,在490nm下,酶联免疫检测仪测定吸光度值。并计算药物对病毒的抑制率。
采用SPSS13.0统计软件,Regression-probit回归将药物浓度与病毒抑制率进行回归分析,计算药物的半数有效浓度(IC_(50))、治疗指数(TI)。
采用SPSS13.0统计软件,Onc-Way ANOVA对各药物不同浓度组、阳性对照组、病毒对照组、正常对照组间吸光度均数进行方差分析及组间多重比较,确定各药物组间及其与对照组间病毒抑制率是否有显著性差异。
5、桑叶提取物Ⅱ、Ⅲ部位体外诱导Hela细胞凋亡的研究。常规培养的Hela细胞,待长至80%时,倒掉培养液,用PBS冲洗2次,分别加入用细胞培养液稀释的各浓度药液,正常对照组加入细胞培养液培养,置37℃,5%CO_2孵箱中培养,倒置显微镜观察细胞病变情况,24h、48h后,分别用0.25%胰酶消化收集细胞,1000rpm离心5min,弃上清,细胞用PBS洗2次,离心,弃上清,细胞用4℃,75%的乙醇固定4h以上,离心弃去固定液,PBS重悬5min,500～1000rpm离心5min,弃去PBS,1ml PI染液染色,终浓度为100μg/mL。4℃避光30 min,过400目筛后上流式细胞仪测试,Multicycles软件分析细胞凋亡率,采用SPSS13.0统计软件,One-Way ANOVA(方差分析)对Ⅱ、Ⅲ部位不同浓度组、正常对照组的细胞凋亡率进行方差分析,确定药物组间与对照组间细胞凋亡率是否有显著性差异。常规培养的Hela细胞,待长至80%时,倒掉培养液,用PBS冲洗2次,加入用细胞培养液稀释的各浓度药液,正常对照组加入细胞培养液培养,置37℃,5%CO_2孵箱中培养,倒置显微镜观察细胞病变情况,24h后,用0.25%胰酶消化收集细胞,1000rpm离心5min,弃上清,细胞用PBS洗2次,离心,弃上清,细胞用2.5%戊二醛固定过夜,常规脱水、超薄切片、染色、透射电镜观察细胞形态。
结果:1、建立了桑叶中有效成分的含量测定方法及指纹图谱
(1)建立了HPLC同时测定桑叶中芦丁、绿原酸、槲皮素含量的分析方法。在此条件下,芦丁线性范围为0.196～1.960μg,r=0.9998,样品的平均加样回收率为97.0%,RSD2.69%:绿原酸线性范围为0.158～1.580μg,r=0.9995,样品的平均加样回收率为96.9%,RSD 2.10%;槲皮素线性范围为0.0848～0.848μg,r=0.9992,样品的平均加样回收率为96.2%,RSD 2.90%。
(2)建立了HPLC-ELSD法测定桑叶中r-氨基丁酸的方法,桑叶中r-氨基丁酸在0.476～4.760μg范围内呈线性,平均回收率为99.6%,RSD为2.43%。
(3)建立了桑叶中1-DNJ的HPLC-MS/MS检测方法,1-DNJ保留时间为2.87min,标准曲线在482μg/L～2410μg/L范围内线性关系良好(r=0.9993),平均加样回收率为95.8%,最低检测限为53.6μg·L~(-1)。
(4)建立了桑叶提取物的HPLC指纹图谱,有18个峰出峰时间稳定可以用于定性分析,其中11个峰峰面积稳定,可以用于定量分析。
2、桑叶化学成分分析结果
从桑叶提取物中共检测出49个色谱峰。通过标准品对照、谱图中碎片离子结构分析及文献查阅,鉴定了奎尼酸、绿原酸、新绿原酸、隐绿原酸、东莨宕苷、环桑皮素、槲皮素.双吡喃葡萄糖苷、槲皮素-双吡喃鼠李糖基吡喃葡萄糖苷、槲皮素-双鼠李糖苷、异绿原酸、芦丁、异槲皮苷、Moralbanone,紫云英苷、桑皮酮S、桑根皮素酸A、B、D、F,桑辛素C、桑辛素D、异戊烯羟基藜芦酚等26个化学成分。
3、桑叶提取分离结果
对桑叶提取物进行了系统的分离,得到5个提取部位,分别为多糖部位、生物碱部位、桑叶Ⅰ、Ⅱ、Ⅲ部位,HPLC-MS/MS显示,桑叶Ⅰ部位化学成分主要为奎尼酸、绿原酸及其同分异构体、咖啡酸等化合物;桑叶Ⅱ部位主要为槲皮素—己碳糖苷类、异绿原酸、异槲皮苷、芦丁等化合物;桑叶Ⅲ部位主要为桑辛素及其同分异构体等化合物。
4、桑叶有效部位体外抗RSV研究结果
对桑叶Ⅰ、Ⅱ、Ⅲ部位进行体外抗呼吸道合胞病毒活性筛选,结果桑叶Ⅰ部位表现出较好体外抗RSV活性,TC_(50)=37.98mg/mL,IC_(50)=0.4195mg/mL,TI=90.53;桑叶Ⅱ部位表现出较好体外抗RSV活性,TC_(50)=2.826mg/mL,IC_(50)=0.2493mg/mL,TI=11.32;桑叶Ⅲ部位在此条件下未表现出体外抗RSV活性;
5、桑叶有效部位体外诱导Hela细胞凋亡实验结果
对桑叶Ⅱ、Ⅲ部位进行体外诱导Hela细胞凋亡实验,结果Ⅱ、Ⅲ部位的不同浓度组处理24h,48h的细胞与正常对照组相比均有不同程度的凋亡,与正常对照组比均有显著性差异,且呈现一定的量效关系,说明桑叶Ⅱ、Ⅲ部位能诱导Hela细胞凋亡,电镜观察结果进一步证实。
结论:桑叶体外抗RSV作用是多成分的共同作用,研究表明桑叶Ⅰ部位、Ⅱ部位均有较强的抗RSV的活性。化学成分分析表明:Ⅰ部位主要为奎尼酸、绿原酸、新绿原酸、隐绿原酸等,Ⅱ部位主要为槲皮素的己糖苷、桑素、芦丁、异槲皮苷、桑根皮素A、B、F等,初步确定其为桑叶体外抗RSV的主要物质基础。
桑叶Ⅱ、Ⅲ部位具有较强的抑制Hela细胞生长和诱导细胞凋亡的作用,可能含有潜在的抗肿瘤成分,化学成分分析表明:Ⅱ部位主要含有槲皮素的己糖苷类、桑素、异绿原酸、异槲皮苷等化合物,有文献报道槲皮素的己糖苷类、桑素具有一定的抗肿瘤活性,可能是该部位抗肿瘤活性的主要成分;Ⅲ部位主要含桑辛素或其同分异构体以及分子量在200～400的小分子化合物,可能是该部位抗肿瘤活性的主要成分,具体成分尚需进一步的研究。
It is well know that one essential difference of traditional medicines from chemical drugs is that their effects owe to the joint contribution of multi-components,not only the major ones,so it is very important to find out the bioactive constituents,determine their quantity and explain their pharmacologic action.
Mulberry leaves(Morus alba L.) is a traditional Chinese herb recorded in the Pharmacopoeia of the People's Republic of China(2005 edition) which has been used to treat acute upper respiratory tract infection and diabetes in China from old times. The studies on the pharmacological activities of mulberry leaves showed the extract of mulberry leaves had antihyperglycemic,antiviral,anti-tumor activities.The alkaloid such as deoxynojirimycin(DNJ) have been found to be potent intestinalα-glucosidase inhibitors.the antiviral and anti-tumor constituents have not been reported so far.Thus,the purpose of this study is to find out the constituents with antiviral and anti-tumor activities from mulberry leaves.
Methods:1.The quantitative determination of bioactive compounds in mulberry leaves by HPLC were established and the chromatographic fingerprint of mulberry leaves was developed.
(1) Quantitative determination of rutin,chlorogenetic acid and quercetin in mulberry leaves by HPLC.An Agilent 1100 HPLC system equipped with a quaternary pump,a diode-array detector,an autosampler and a column compartment was used for analysis.The extract were separated on a Kromasil C18 Column(250×4.6mm, 5μm).The mobile phase were CH3CN,0.01mol/L ammonium acetate aqueous solution and methanol(20:10:70,v/v),the mobile folw rate was 1.0mL/min,the column temperature was 25℃,and the chromatogram was recorded at 350nm.
(2) Quantitative determination of r-GABA in mulberry leaves by HPLC-ELSD.An Agilent 1100 HPLC system equipped with a quaternary pump,a diode-array detector, an autosampler and a column compartment was used for analysis.The extract were separated on a Kromasil C18 Column(250×4.6mm,5μm).The mobile phase were CH3CN and water(90:10,v/v),the mobile folw rate was 0.6mL/min,the column temperature was 25℃,the injection volumn was 10ul,the ELSD temperature was 108.3℃,and the N_2 flow rate was 3.0L/min.
(3) Quantitative determination of 1-deoxynojirimycin(1-DNJ) in mulberry leaves using liquid chromatography-tandem mass spectrometry(HPLC-MS) 1-DNJ was isolated from the mulberry leaves extract on a SHIMADZU HRC-NH_2.column using CH_3CN and 0.1%formic acid as a mobile phase.A triple quadrupole mass spectrometry using APCI source in a positive ion mode under multiple reaction monitoring with the[M+H]~+ ions,m/z 164.1 / 146.1 and 164.1/110.2 were used.
(4) Development of the chromatographic fingerprint of mulberry leaves by HPLC. The extract were separated on a Kromasil C18 Column(250×4.6mm,5μm).The mobile phase were CH3CN,water and methanol,the gradient program was as: 0～28min,A:5～35%,B:95～65%,C:0%;28～35min,A:35～50%,B:60～0%, C:0～50%;35～60min.A:50～60%,B:0%,C:50～40%,the mobile folw rate was 1.0mL/min,the column temperature was 25℃,the injection volumn was 10ul,and the chromatogram was recorded at 350nm.
2.Mulberry leaves were extract with 50%ethanol and then filted through a 0.45um membrane before HPLC-MS analysis.An API4000 QTrap LC/MS system was used for analysis equipped with an electrospray ionization(ESI) source.The samples were separated on a Agilent ZORBX Extend C18 Column(.50×4.6mm, 1.8μm).The mobile phase were The mobile phase were CH3CN,water and methanol, the gradient program was as follows:0～8min,B4%～35,,C94%～65, 8～11minA0～40%,B35～60%,C65～0%;11～21min:A40～45,B60～55%,the mobile folw rate was 0.6mL/min,the injection volumn was 5ul.The ESI MS analysis was performed in negative mode using full scan mode and the mass range was set at 100～1200 ainu,the conditions of the ESI source were as follows:DP -80V, temperature,350℃,drying gas(N_2) 12psi,nebulizing gas(N_2) pressure,30psi.
3.The extract of mulberry leaves were separated into five parts,including polysaccharide extration,alkaloid extraction,partⅠ,PartⅡ,PartⅢ,HPLC-MS technique were used to identify the main constituents of each part.
4.In vitro antiviral activities of each partⅠ,Ⅱ,Ⅲof mulberry leaves,as well as control antiviral drug ribovirin against respiratory synytial virus(RSV) were determined using the viral cytopathic effect assay.The cytotoxicity was tested using a colorimetric assay based on the mitochondria metabolisation of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide(MTT).The compounds toxicity was measured on Hela cells in replication,after seeding 1×10~6 cells per well in a volume of 200ul into 96-well microtiter plates.After 24h of incubation at 37℃and 5%CO_2,the culture medium were discharged and 200ul of medium contaning serial dilutions of the tested compounds were added.The cells were incubated and the vira cytopathic effect(CPE) were recorded every 24h,after 3 days of incubation, culture medium was discharged and cell debris were removed by rinsing each well with 100ul of phosphate buffersaline(PBS).A 5mg/mL MTT solution in PBS was added in a volum of 200ul per well,after an incubation of 2h at 37℃and 5%CO_2, the PBS were discharged and 200ul of DMSO were added in each well to dissolve formazan crystals on the bottom of the microtiter plates.The intensity of the colour in correspondence of each well was automatically measured by using a spectrophotometer with a filter of 490nm.The median toxic concentration(TC_(50)) values of each compound was calculated by spss13.0 software Regression-probit analysis.
The respiratory synytial virus were adapted to grow and titrated in Hela cells, RSV titres,calculated by the method of Reed and Muench,TCID_(50) was 10~(-4.5/200μl)
The antiviral activities of the test compounds against RSV were evaluated by the methods as follows:viral solutions were mixed with various concentrations of the compounds and the control antiviral drug ribovirin,the final concentration of the viral was 100 TCID_(50) while the final concentration of the ribovirin was 0.1 mg/mL and then incubated for lh at 37℃and 5%CO_2.Afterwards,samples were added to a confluent monolayer of Hela cells in 96-well microtiter plates,the normal group add culture medium only while the viral group add viral solutions with the concentration of 100TICD_(50),then the cells incubated at 37℃and 5%CO_2,after 2h,the samples were removed,and the cell monolayer was overlaid with RPMI-1640 2%FCS.the vira cytopathic effect(CPE) were recorded every 24h,after 3 days of incubation,culture medium was discharged and cell debris were removed by rinsing each well with 100ul of phosphate buffersaline(PBS).A 5mg/mL MTT solution in PBS was added in a volum of 200ul per well,after an incubation of 2h at 37℃and 5%CO_2,the PBS were discharged and 200ul of DMSO were added in each well to dissolve formazan crystals on the bottom of the microtiter plates.The intensity of the colour in correspondence of each well was automatically measured by using a spectrophotometer with a filter of 490nm.The 50%inhibitory concentration(IC_(50)) values of each compound was calculated by spss13.0 software Regression-probit analysis.The deviation between the groups were analysised by spss13.0 sofeware One-way ANOVA.
5.Seeding 1×10~6 cells per well in a volume of 200ul into 96-well microtiter plates.After 24h of incubation at 37℃and 5%CO_2,the culture medium were discharged and rinsing each well with phosphate buffer saline(PBS),200ul of medium contaning serial dilutions of the tested compounds were added.The cells were incubated and the vim cytopathic effect(CPE) were recorded every 24h,after 48h,the cells were collected and apoptosis was detected by Flow Cytometry and transmission electron microscopy.The deviation between the groups were analysised by spss13.0 sofeware One-way ANOVA.
Results:1.(1)The HPLC method for the quanitative determination of rutin, chlorogenetic acid and quercetin gave high sensitivity and selectivity,the calibration curves of rutin showed good linearity in the range of 0.196～1.960ug,r=0.998,the average recoveries was 97.0%,with RSD of 2.69%.The calibration curves of chlorogenetic acid showed good linearity in the range of 0.158～1.580ug,r=0.998, the average recoveries was 96.9%,with RSD of 2.10%.The calibration curves of quercetin showed good linearity in the range of 0.0848～0.848ug,r=0.992,the average recoveries was 96.2%,with RSD of 2.90%.
(2) The calibration curves of r-GABA showed good linearity in the range of 0.476～4.760ug,r=0.992,the average recoveries was 99.6%,with RSD of 2.43%.
(3) The retention time of 1-DNJ was 2.87min,calibration curves of r-GABA showed good linearity in the range of 482ug/L～2410ug/L,r=0.993,the average recoveries was 95.8%,and limit ofquantitation RSD was 53.6ug/L.
(4)In fingerprint analysis of mulberry leaves,18 peaks were selected as the characteristic peaks to evaluate the similarities of different samples,relative retention time of 18 peaks and relative peak area of 11 peaks were used to identify the common peaks in samples.
2.With the HPLC-MS methods,49 peaks were analysised in the mulberry leaves, in which 26 compounds were identified by comparing their HPLC retention times with the reference standards and MS~n fragmentation behaviors as quinic acid, 3-caffeoylquinic acids,4-caffeoylquinic acids,5-caffeoylquinic acids, dicaffeoylquinic acids,cyclomorusin,kuwanon S,morusimic acid A,B,D,F, moralbanone,rutin,astragalin,scopolin,quercetin- glucopyranoside,isoquerceitrin, moracin C and D,et al.
3.The extract of mulberry leaves was separated into five parts,including polysaccharide extration,alkaloid extraction,partⅠ,PartⅡ,PartⅢ,the main constituents in partⅠwere quinic acid,isomer of caffeoylquinic acids,in partⅡwere quercetin- glucopyranoside,isoquerceitrin,rutin,morusimic acid A,B,D,F et al.in partⅢ,the main constituents were isomer of moracin C and D.
4.The antiviral results showed partⅠof Mulberry leaves had an antiviral effect against RSV with TC_(50)=37.98mg/mL,IC_(50)=0.4195mg/mL,TI=90.53.PartⅡof Mulberry leaves had an antiviral effect against RSV with TC_(50)=2.826mg/mL, IC_(50)=0.2493mg/mL,TI=11.32;PartⅢwas inactive against RSV.
5.Both partⅡand partⅢof Mulberry leaves had an anti-tumor activities.
Conclusion:The antiviral effect of mulberry leaves owe to the joint contribution of multi-components,both the partⅠand partⅡof mulberry leaves had an antiviral effect against RSV.The main constituents among them were quinic acid, 3-caffeoylquinic acids,4-caffeoylquinic acids,5-caffeoylquinic acids,quercetinglucopyranoside, isoquerceitrin,rutin,morusimic acid A,B,D,F et al.PartⅡandⅢof mulberry leaves shown to have potent anti-tumor activities.The main constituents were quercetin- glucopyranoside,moracin C,D and some unknow constituents.
桑叶是桑科植物桑Morus alba L的叶子,具有疏风清热、清肝明目的功效,在我国有悠久的栽培和药用历史,资源极其丰富,临床常用于治疗风热感冒、消渴症等,现代药理研究表明,桑叶具有降血糖、抗病毒、抗癌等多种生物活性,桑叶中的多羟基生物碱已被确认为降血糖的主要物质基础,而桑叶中抗病毒、抗肿瘤的物质基础研究尚不充分,为桑叶的综合利用及进一步开发带来了困难。
本研究首先采用高效液相色谱法(HPLC法)建立了桑叶主要活性成分1-脱氧野尻霉素(1-DNJ)、绿原酸、芦丁、槲皮素、r-氨基丁酸等的含量测定方法及指纹图谱,用以追踪主要化学成分在分离过程中的去向及转移率,为分离条件的优化、阐明物质基础提供定量、定性分析方法;采用高效液相色谱电喷雾串联质谱法(HPLC-MS/MS法)对桑叶中的化合物进行了分析,在对比文献的基础上,初步明确桑叶提取物中的主要物质组成,为物质基础的阐明提供基础(即物质基础是哪些化合物);在活性成分的指引下,采用工业生产可行的分离方法对桑叶进行系统分离,得到粗多糖部位、生物碱部位及Ⅰ、Ⅱ、Ⅲ共五个提取部位,并对部位Ⅰ、Ⅱ、Ⅲ进行体外体外抗RSV评价,对部位Ⅲ进行了诱导细胞凋亡实验研究,初步确定桑叶中体外抗RSV、抗肿瘤的药效物质基础。
方法:1、建立桑叶中活性成分的HPLC含量测定方法及指纹图谱。
(1)HPLC法同时测定桑叶中芦丁、绿原酸和槲皮素。色谱柱为Kromasil C18柱(4.6×250 mm,5μm)柱;流动相为乙腈:甲醇:0.01 mol/L醋酸铵液(pH3.50)(20:10:70);流速1.0 mL/min;柱温为25℃,检测波长为350 nm;
(2)HPLC-ELSD法测定桑叶中的r-氨基丁酸。用Kromasil C18柱(4.6mm×250mm,5μm),流动相为水:乙腈(90:10),流速0.6min/ml,柱温25℃,进样10μL;ELSD气化室温度108.3°,氮气流速:3.0L/min。
(3)HPLC-MS/MS法测定桑叶中1-DNJ含量。以乙腈:0.1%甲酸水溶液(12:88,v/v)为流动相,SHIMADZU HRC-NH_2柱分离,采用大气压化学电离源(APCI),164.1/146.1,164.1/110.2两对离子,多反应监测。
(4)桑叶HPLC指纹图谱的建立。色谱柱:C18(4.6mm×250mm,5μm);流动相:乙腈(A)-水(C)-甲醇(B),梯度洗脱,洗脱曲线:0～28min,A 5～35%,B95～65%,C 0%:28～35min,A 35～50%,B 60～0%,C 0～50%;35～60min;A50～60%,B 0%,C 50～40%。流速:1.0ml/mim柱温:25℃;检测波长:350nm;进样量:10μl。
2、HPLC-MS/MS法分析桑叶中的化学成分。桑叶经50%乙醇回流提取,上液质联用进行分析,对各色谱峰质谱信号进行分析,初步确定桑叶提取物中主要化学成分。液相条件:色谱柱:Aglient Zorbax Extend C18柱(50×4.6mm,1.8μm);流动相:A(甲醇)-B(乙腈)-C(水),洗脱曲线为:0～8min,B4%～35,C94%～65,8～11minA0～40%,B35～60%,C65～0%;11～2lmin:A40～45%,B60～55%,流速:600μl/min,梯进样量:5μL,柱温:室温。质谱条件电喷雾电离源(ESI源),负离子扫描,扫描范围m/z 100～1200。小口电压:-80V,入口电压:10V,温度350℃,碰撞气压力:12psi,雾化气压力:30psi。
3、桑叶有效部位的提取分离
将桑叶提取物进行了较为系统的分离,初步分成五个部位,分别为多糖部位、生物碱部位、Ⅰ部位,Ⅱ部位及Ⅲ部位,采用上述建立的分析方法对各分离部位有效成分进行定量分析,采用HPLC-MS/MS对各部位化学成分进行分析。4、以阳性药物利巴韦林注射液作为对照,对桑叶提取物Ⅰ部位,Ⅱ部位及Ⅲ部位进行体外抗呼吸道合胞病毒活性评价。
药物毒性实验方法:Hela细胞常规传代培养,待长满后用胰酶消化,含10%小牛血清的RPMI-1640培养液制成细胞悬液,以8×10~5个/mL的细胞密度,加入96孔板中,每孔200μl,37℃,5%CO_2孵箱中培养24h,待细胞长成单层。吸出培养液,加入各浓度药液200μl,每个浓度8个复孔。正常对照只加入等体积2%的维持液。37℃,5%CO_2孵箱中培养,每24h观察细胞病变情况(CPE),连续观察72h,记录实验结果。72h后,将药液吸出,每孔加入5mg/mLMTT(用PBS配制)200μl,37℃,5%CO_2孵箱中孵育2h后,弃上清,每孔加入DMSO200μl,室温下振荡混匀10min,在490nm下,酶联免疫检测仪测定吸光度值,并计算细胞存活率。采用SPSS13.0统计软件,Regression-probit回归将药物浓度与细胞存活率进行回归分析,计算细胞半数存活浓度。
呼吸道合胞病毒(RSV)毒力测定:采用TCID_(50)微量法,用Reed-Munch公式计算TCID_(50)。
药物抗病毒实验方法:药物在无毒浓度范围内,用2%的细胞维持液对药物进行倍比稀释,加入病毒液使病毒浓度为100TCID_(50),混匀,阳性对照药为利巴韦林注射液(0.1g/mL),用2%细胞维持液稀释,并加入病毒液,使利巴韦林终浓度为100μg/mL,病毒终浓度为100TCID_(50),于37℃,5%CO_2孵箱中孵育1h。已长成单层Hela细胞的96孔板,吸掉培养液,每孔接种200μl各浓度药液与病毒的混合液,每个浓度8个复孔,同时设阳性对照组,病毒对照组和正常对照组,37℃,5%CO_2培养2h后,将药物组和病毒组液体吸出,加入细胞维持液继续培养,每日观察CPE。72h后将药液吸出,每孔加入5mg/mLMTT(用PBS配制)200μl,37℃,5%CO_2孵箱中孵育2h后,弃上清,每孔加入DMSO200μl,室温下振荡混匀10min,在490nm下,酶联免疫检测仪测定吸光度值。并计算药物对病毒的抑制率。
采用SPSS13.0统计软件,Regression-probit回归将药物浓度与病毒抑制率进行回归分析,计算药物的半数有效浓度(IC_(50))、治疗指数(TI)。
采用SPSS13.0统计软件,Onc-Way ANOVA对各药物不同浓度组、阳性对照组、病毒对照组、正常对照组间吸光度均数进行方差分析及组间多重比较,确定各药物组间及其与对照组间病毒抑制率是否有显著性差异。
5、桑叶提取物Ⅱ、Ⅲ部位体外诱导Hela细胞凋亡的研究。常规培养的Hela细胞,待长至80%时,倒掉培养液,用PBS冲洗2次,分别加入用细胞培养液稀释的各浓度药液,正常对照组加入细胞培养液培养,置37℃,5%CO_2孵箱中培养,倒置显微镜观察细胞病变情况,24h、48h后,分别用0.25%胰酶消化收集细胞,1000rpm离心5min,弃上清,细胞用PBS洗2次,离心,弃上清,细胞用4℃,75%的乙醇固定4h以上,离心弃去固定液,PBS重悬5min,500～1000rpm离心5min,弃去PBS,1ml PI染液染色,终浓度为100μg/mL。4℃避光30 min,过400目筛后上流式细胞仪测试,Multicycles软件分析细胞凋亡率,采用SPSS13.0统计软件,One-Way ANOVA(方差分析)对Ⅱ、Ⅲ部位不同浓度组、正常对照组的细胞凋亡率进行方差分析,确定药物组间与对照组间细胞凋亡率是否有显著性差异。常规培养的Hela细胞,待长至80%时,倒掉培养液,用PBS冲洗2次,加入用细胞培养液稀释的各浓度药液,正常对照组加入细胞培养液培养,置37℃,5%CO_2孵箱中培养,倒置显微镜观察细胞病变情况,24h后,用0.25%胰酶消化收集细胞,1000rpm离心5min,弃上清,细胞用PBS洗2次,离心,弃上清,细胞用2.5%戊二醛固定过夜,常规脱水、超薄切片、染色、透射电镜观察细胞形态。
结果:1、建立了桑叶中有效成分的含量测定方法及指纹图谱
(1)建立了HPLC同时测定桑叶中芦丁、绿原酸、槲皮素含量的分析方法。在此条件下,芦丁线性范围为0.196～1.960μg,r=0.9998,样品的平均加样回收率为97.0%,RSD2.69%:绿原酸线性范围为0.158～1.580μg,r=0.9995,样品的平均加样回收率为96.9%,RSD 2.10%;槲皮素线性范围为0.0848～0.848μg,r=0.9992,样品的平均加样回收率为96.2%,RSD 2.90%。
(2)建立了HPLC-ELSD法测定桑叶中r-氨基丁酸的方法,桑叶中r-氨基丁酸在0.476～4.760μg范围内呈线性,平均回收率为99.6%,RSD为2.43%。
(3)建立了桑叶中1-DNJ的HPLC-MS/MS检测方法,1-DNJ保留时间为2.87min,标准曲线在482μg/L～2410μg/L范围内线性关系良好(r=0.9993),平均加样回收率为95.8%,最低检测限为53.6μg·L~(-1)。
(4)建立了桑叶提取物的HPLC指纹图谱,有18个峰出峰时间稳定可以用于定性分析,其中11个峰峰面积稳定,可以用于定量分析。
2、桑叶化学成分分析结果
从桑叶提取物中共检测出49个色谱峰。通过标准品对照、谱图中碎片离子结构分析及文献查阅,鉴定了奎尼酸、绿原酸、新绿原酸、隐绿原酸、东莨宕苷、环桑皮素、槲皮素.双吡喃葡萄糖苷、槲皮素-双吡喃鼠李糖基吡喃葡萄糖苷、槲皮素-双鼠李糖苷、异绿原酸、芦丁、异槲皮苷、Moralbanone,紫云英苷、桑皮酮S、桑根皮素酸A、B、D、F,桑辛素C、桑辛素D、异戊烯羟基藜芦酚等26个化学成分。
3、桑叶提取分离结果
对桑叶提取物进行了系统的分离,得到5个提取部位,分别为多糖部位、生物碱部位、桑叶Ⅰ、Ⅱ、Ⅲ部位,HPLC-MS/MS显示,桑叶Ⅰ部位化学成分主要为奎尼酸、绿原酸及其同分异构体、咖啡酸等化合物;桑叶Ⅱ部位主要为槲皮素—己碳糖苷类、异绿原酸、异槲皮苷、芦丁等化合物;桑叶Ⅲ部位主要为桑辛素及其同分异构体等化合物。
4、桑叶有效部位体外抗RSV研究结果
对桑叶Ⅰ、Ⅱ、Ⅲ部位进行体外抗呼吸道合胞病毒活性筛选,结果桑叶Ⅰ部位表现出较好体外抗RSV活性,TC_(50)=37.98mg/mL,IC_(50)=0.4195mg/mL,TI=90.53;桑叶Ⅱ部位表现出较好体外抗RSV活性,TC_(50)=2.826mg/mL,IC_(50)=0.2493mg/mL,TI=11.32;桑叶Ⅲ部位在此条件下未表现出体外抗RSV活性;
5、桑叶有效部位体外诱导Hela细胞凋亡实验结果
对桑叶Ⅱ、Ⅲ部位进行体外诱导Hela细胞凋亡实验,结果Ⅱ、Ⅲ部位的不同浓度组处理24h,48h的细胞与正常对照组相比均有不同程度的凋亡,与正常对照组比均有显著性差异,且呈现一定的量效关系,说明桑叶Ⅱ、Ⅲ部位能诱导Hela细胞凋亡,电镜观察结果进一步证实。
结论:桑叶体外抗RSV作用是多成分的共同作用,研究表明桑叶Ⅰ部位、Ⅱ部位均有较强的抗RSV的活性。化学成分分析表明:Ⅰ部位主要为奎尼酸、绿原酸、新绿原酸、隐绿原酸等,Ⅱ部位主要为槲皮素的己糖苷、桑素、芦丁、异槲皮苷、桑根皮素A、B、F等,初步确定其为桑叶体外抗RSV的主要物质基础。
桑叶Ⅱ、Ⅲ部位具有较强的抑制Hela细胞生长和诱导细胞凋亡的作用,可能含有潜在的抗肿瘤成分,化学成分分析表明:Ⅱ部位主要含有槲皮素的己糖苷类、桑素、异绿原酸、异槲皮苷等化合物,有文献报道槲皮素的己糖苷类、桑素具有一定的抗肿瘤活性,可能是该部位抗肿瘤活性的主要成分;Ⅲ部位主要含桑辛素或其同分异构体以及分子量在200～400的小分子化合物,可能是该部位抗肿瘤活性的主要成分,具体成分尚需进一步的研究。
It is well know that one essential difference of traditional medicines from chemical drugs is that their effects owe to the joint contribution of multi-components,not only the major ones,so it is very important to find out the bioactive constituents,determine their quantity and explain their pharmacologic action.
Mulberry leaves(Morus alba L.) is a traditional Chinese herb recorded in the Pharmacopoeia of the People's Republic of China(2005 edition) which has been used to treat acute upper respiratory tract infection and diabetes in China from old times. The studies on the pharmacological activities of mulberry leaves showed the extract of mulberry leaves had antihyperglycemic,antiviral,anti-tumor activities.The alkaloid such as deoxynojirimycin(DNJ) have been found to be potent intestinalα-glucosidase inhibitors.the antiviral and anti-tumor constituents have not been reported so far.Thus,the purpose of this study is to find out the constituents with antiviral and anti-tumor activities from mulberry leaves.
Methods:1.The quantitative determination of bioactive compounds in mulberry leaves by HPLC were established and the chromatographic fingerprint of mulberry leaves was developed.
(1) Quantitative determination of rutin,chlorogenetic acid and quercetin in mulberry leaves by HPLC.An Agilent 1100 HPLC system equipped with a quaternary pump,a diode-array detector,an autosampler and a column compartment was used for analysis.The extract were separated on a Kromasil C18 Column(250×4.6mm, 5μm).The mobile phase were CH3CN,0.01mol/L ammonium acetate aqueous solution and methanol(20:10:70,v/v),the mobile folw rate was 1.0mL/min,the column temperature was 25℃,and the chromatogram was recorded at 350nm.
(2) Quantitative determination of r-GABA in mulberry leaves by HPLC-ELSD.An Agilent 1100 HPLC system equipped with a quaternary pump,a diode-array detector, an autosampler and a column compartment was used for analysis.The extract were separated on a Kromasil C18 Column(250×4.6mm,5μm).The mobile phase were CH3CN and water(90:10,v/v),the mobile folw rate was 0.6mL/min,the column temperature was 25℃,the injection volumn was 10ul,the ELSD temperature was 108.3℃,and the N_2 flow rate was 3.0L/min.
(3) Quantitative determination of 1-deoxynojirimycin(1-DNJ) in mulberry leaves using liquid chromatography-tandem mass spectrometry(HPLC-MS) 1-DNJ was isolated from the mulberry leaves extract on a SHIMADZU HRC-NH_2.column using CH_3CN and 0.1%formic acid as a mobile phase.A triple quadrupole mass spectrometry using APCI source in a positive ion mode under multiple reaction monitoring with the[M+H]~+ ions,m/z 164.1 / 146.1 and 164.1/110.2 were used.
(4) Development of the chromatographic fingerprint of mulberry leaves by HPLC. The extract were separated on a Kromasil C18 Column(250×4.6mm,5μm).The mobile phase were CH3CN,water and methanol,the gradient program was as: 0～28min,A:5～35%,B:95～65%,C:0%;28～35min,A:35～50%,B:60～0%, C:0～50%;35～60min.A:50～60%,B:0%,C:50～40%,the mobile folw rate was 1.0mL/min,the column temperature was 25℃,the injection volumn was 10ul,and the chromatogram was recorded at 350nm.
2.Mulberry leaves were extract with 50%ethanol and then filted through a 0.45um membrane before HPLC-MS analysis.An API4000 QTrap LC/MS system was used for analysis equipped with an electrospray ionization(ESI) source.The samples were separated on a Agilent ZORBX Extend C18 Column(.50×4.6mm, 1.8μm).The mobile phase were The mobile phase were CH3CN,water and methanol, the gradient program was as follows:0～8min,B4%～35,,C94%～65, 8～11minA0～40%,B35～60%,C65～0%;11～21min:A40～45,B60～55%,the mobile folw rate was 0.6mL/min,the injection volumn was 5ul.The ESI MS analysis was performed in negative mode using full scan mode and the mass range was set at 100～1200 ainu,the conditions of the ESI source were as follows:DP -80V, temperature,350℃,drying gas(N_2) 12psi,nebulizing gas(N_2) pressure,30psi.
3.The extract of mulberry leaves were separated into five parts,including polysaccharide extration,alkaloid extraction,partⅠ,PartⅡ,PartⅢ,HPLC-MS technique were used to identify the main constituents of each part.
4.In vitro antiviral activities of each partⅠ,Ⅱ,Ⅲof mulberry leaves,as well as control antiviral drug ribovirin against respiratory synytial virus(RSV) were determined using the viral cytopathic effect assay.The cytotoxicity was tested using a colorimetric assay based on the mitochondria metabolisation of the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide(MTT).The compounds toxicity was measured on Hela cells in replication,after seeding 1×10~6 cells per well in a volume of 200ul into 96-well microtiter plates.After 24h of incubation at 37℃and 5%CO_2,the culture medium were discharged and 200ul of medium contaning serial dilutions of the tested compounds were added.The cells were incubated and the vira cytopathic effect(CPE) were recorded every 24h,after 3 days of incubation, culture medium was discharged and cell debris were removed by rinsing each well with 100ul of phosphate buffersaline(PBS).A 5mg/mL MTT solution in PBS was added in a volum of 200ul per well,after an incubation of 2h at 37℃and 5%CO_2, the PBS were discharged and 200ul of DMSO were added in each well to dissolve formazan crystals on the bottom of the microtiter plates.The intensity of the colour in correspondence of each well was automatically measured by using a spectrophotometer with a filter of 490nm.The median toxic concentration(TC_(50)) values of each compound was calculated by spss13.0 software Regression-probit analysis.
The respiratory synytial virus were adapted to grow and titrated in Hela cells, RSV titres,calculated by the method of Reed and Muench,TCID_(50) was 10~(-4.5/200μl)
The antiviral activities of the test compounds against RSV were evaluated by the methods as follows:viral solutions were mixed with various concentrations of the compounds and the control antiviral drug ribovirin,the final concentration of the viral was 100 TCID_(50) while the final concentration of the ribovirin was 0.1 mg/mL and then incubated for lh at 37℃and 5%CO_2.Afterwards,samples were added to a confluent monolayer of Hela cells in 96-well microtiter plates,the normal group add culture medium only while the viral group add viral solutions with the concentration of 100TICD_(50),then the cells incubated at 37℃and 5%CO_2,after 2h,the samples were removed,and the cell monolayer was overlaid with RPMI-1640 2%FCS.the vira cytopathic effect(CPE) were recorded every 24h,after 3 days of incubation,culture medium was discharged and cell debris were removed by rinsing each well with 100ul of phosphate buffersaline(PBS).A 5mg/mL MTT solution in PBS was added in a volum of 200ul per well,after an incubation of 2h at 37℃and 5%CO_2,the PBS were discharged and 200ul of DMSO were added in each well to dissolve formazan crystals on the bottom of the microtiter plates.The intensity of the colour in correspondence of each well was automatically measured by using a spectrophotometer with a filter of 490nm.The 50%inhibitory concentration(IC_(50)) values of each compound was calculated by spss13.0 software Regression-probit analysis.The deviation between the groups were analysised by spss13.0 sofeware One-way ANOVA.
5.Seeding 1×10~6 cells per well in a volume of 200ul into 96-well microtiter plates.After 24h of incubation at 37℃and 5%CO_2,the culture medium were discharged and rinsing each well with phosphate buffer saline(PBS),200ul of medium contaning serial dilutions of the tested compounds were added.The cells were incubated and the vim cytopathic effect(CPE) were recorded every 24h,after 48h,the cells were collected and apoptosis was detected by Flow Cytometry and transmission electron microscopy.The deviation between the groups were analysised by spss13.0 sofeware One-way ANOVA.
Results:1.(1)The HPLC method for the quanitative determination of rutin, chlorogenetic acid and quercetin gave high sensitivity and selectivity,the calibration curves of rutin showed good linearity in the range of 0.196～1.960ug,r=0.998,the average recoveries was 97.0%,with RSD of 2.69%.The calibration curves of chlorogenetic acid showed good linearity in the range of 0.158～1.580ug,r=0.998, the average recoveries was 96.9%,with RSD of 2.10%.The calibration curves of quercetin showed good linearity in the range of 0.0848～0.848ug,r=0.992,the average recoveries was 96.2%,with RSD of 2.90%.
(2) The calibration curves of r-GABA showed good linearity in the range of 0.476～4.760ug,r=0.992,the average recoveries was 99.6%,with RSD of 2.43%.
(3) The retention time of 1-DNJ was 2.87min,calibration curves of r-GABA showed good linearity in the range of 482ug/L～2410ug/L,r=0.993,the average recoveries was 95.8%,and limit ofquantitation RSD was 53.6ug/L.
(4)In fingerprint analysis of mulberry leaves,18 peaks were selected as the characteristic peaks to evaluate the similarities of different samples,relative retention time of 18 peaks and relative peak area of 11 peaks were used to identify the common peaks in samples.
2.With the HPLC-MS methods,49 peaks were analysised in the mulberry leaves, in which 26 compounds were identified by comparing their HPLC retention times with the reference standards and MS~n fragmentation behaviors as quinic acid, 3-caffeoylquinic acids,4-caffeoylquinic acids,5-caffeoylquinic acids, dicaffeoylquinic acids,cyclomorusin,kuwanon S,morusimic acid A,B,D,F, moralbanone,rutin,astragalin,scopolin,quercetin- glucopyranoside,isoquerceitrin, moracin C and D,et al.
3.The extract of mulberry leaves was separated into five parts,including polysaccharide extration,alkaloid extraction,partⅠ,PartⅡ,PartⅢ,the main constituents in partⅠwere quinic acid,isomer of caffeoylquinic acids,in partⅡwere quercetin- glucopyranoside,isoquerceitrin,rutin,morusimic acid A,B,D,F et al.in partⅢ,the main constituents were isomer of moracin C and D.
4.The antiviral results showed partⅠof Mulberry leaves had an antiviral effect against RSV with TC_(50)=37.98mg/mL,IC_(50)=0.4195mg/mL,TI=90.53.PartⅡof Mulberry leaves had an antiviral effect against RSV with TC_(50)=2.826mg/mL, IC_(50)=0.2493mg/mL,TI=11.32;PartⅢwas inactive against RSV.
5.Both partⅡand partⅢof Mulberry leaves had an anti-tumor activities.
Conclusion:The antiviral effect of mulberry leaves owe to the joint contribution of multi-components,both the partⅠand partⅡof mulberry leaves had an antiviral effect against RSV.The main constituents among them were quinic acid, 3-caffeoylquinic acids,4-caffeoylquinic acids,5-caffeoylquinic acids,quercetinglucopyranoside, isoquerceitrin,rutin,morusimic acid A,B,D,F et al.PartⅡandⅢof mulberry leaves shown to have potent anti-tumor activities.The main constituents were quercetin- glucopyranoside,moracin C,D and some unknow constituents.
引文
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