Studies On The Constituents Of Mulberry Leaves Against Respiratory Synytial Virus And Inducing Hela Cells Apoptosis

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 N₂ 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-NH₂.column using CH₃CN 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（N₂） 12psi,nebulizing gas（N₂） pressure,30psi.3.The extract of mulberry leaves were separated into five parts,including polysaccharide extration,alkaloid extraction,partâ… ,Partâ…¡,Partâ…¢,HPLC-MS technique were used to identify the main constituents of each part.4.In vitro antiviral activities of each partâ… ,â…¡,â…¢of 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 1×10⁶ cells per well in a volume of 200ul into 96-well microtiter plates.After 24h of incubation at 37℃and 5%CO₂,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%CO₂, 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(TC₅₀) 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,TCID₅₀ 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 TCID₅₀ while the final concentration of the ribovirin was 0.1 mg/mL and then incubated for lh at 37℃and 5%CO₂.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 100TICD₅₀,then the cells incubated at 37℃and 5%CO₂,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%CO₂,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(IC₅₀) 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 1×10⁶ cells per well in a volume of 200ul into 96-well microtiter plates.After 24h of incubation at 37℃and 5%CO₂,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 MSⁿ 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,partâ… ,Partâ…¡,Partâ…¢,the main constituents in partâ… were quinic acid,isomer of caffeoylquinic acids,in partâ…¡were quercetin- glucopyranoside,isoquerceitrin,rutin,morusimic acid A,B,D,F et al.in partâ…¢,the main constituents were isomer of moracin C and D.4.The antiviral results showed partâ… of Mulberry leaves had an antiviral effect against RSV with TC₅₀=37.98mg/mL,IC₅₀=0.4195mg/mL,TI=90.53.Partâ…¡of Mulberry leaves had an antiviral effect against RSV with TC₅₀=2.826mg/mL, IC₅₀=0.2493mg/mL,TI=11.32;Partâ…¢was inactive against RSV.5.Both partâ…¡and partâ…¢of Mulberry leaves had an anti-tumor activities.Conclusion:The antiviral effect of mulberry leaves owe to the joint contribution of multi-components,both the partâ… and partâ…¡of 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.Partâ…¡andâ…¢of mulberry leaves shown to have potent anti-tumor activities.The main constituents were quercetin- glucopyranoside,moracin C,D and some unknow constituents.