凉茶的清咽润喉功效研究
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摘要
生产凉茶的原料主要选于药膳同源的中草药,现代医学及药理研究表明,凉茶中所用的药物大多具有抗菌、消炎、解热、抗病毒、调节机体免疫功能等作用。本研究根据凉茶的传统配方组合特点,结合中医药理论,选用了金银花、金银花叶、菊花、甘草、鱼腥草、罗汉果、胖大海、蒲公英、桔梗、淡竹叶共10种中草药原料,对凉茶的清咽润喉功效进行了研究。
本试验是以国家食品药品监督管理局(SFDA)所批准的具有清咽润喉功效的上述常用10种中草药为原料,用80%的乙醇进行提取,得到粗提物。通过体外抑菌试验测试了10种中草药对大肠杆菌和金黄色葡萄球菌两种供试菌的抑制作用,初步筛选出抑菌效果较好的6种中草药。采用正交试验设计法优化了抑菌物质的提取条件,并进一步筛选出抑菌效果较好的4种中草药。对4种中草药提取液的主要生物活性成分含量进行测定,分析了抑菌作用与生物活性成分的关系。根据4种提取液对两种供试菌的最低抑菌浓度(minimun inhibitoryconcentration,MIC)的测定结果,依浓度由高到低进行1:1:1:1复配,得出4种提取液最佳浓度组合配方。通过清咽润喉保健食品功能评价方法,雄性SD大鼠为试验对象,研究了复方中草药液对致炎剂所致大鼠足跖肿胀的抑制作用。主要研究结果如下:
1.具有抑菌效果的中草药的筛选
采用醇热连续回流法提取,通过体外抑菌试验比较了10种中草药提取液的抑菌圈直径和最低抑菌浓度(MIC):筛选出了抑菌活性较强的6种中草药,从初步筛选结果看,单味中药提取液对大肠杆菌的MIC介于12.5～93.0mg生药/ml之间,对金黄色葡萄球菌的MIC介于6.25～93.0mg生药/ml之间。
2.单味中草药抑菌物质提取条件
以初步筛选出的6种中草药为研究对象,采用正交试验优化了乙醇连续回流提取条件,选用正交表L_9(3~4),考察溶剂不同浓度、提取温度、提取时间和溶剂加倍量对抑菌效果的影响。结果表明,各因素对试验结果的影响顺序为乙醇浓度＞提取时间＞溶剂加倍量＞提取温度,根据方差分析得出最佳提取条件为:用70%乙醇提取,料液比为1:10,温度85℃,浸提4.5h。在此提取条件下,单味中药提取液对大肠杆菌的MIC介于1.6～50mg生药/ml之间,对金黄色葡萄球菌的MIC介于3.125～25mg生药/ml之间。其中金银花叶提取液对大肠杆菌的抑菌浓度最低,其MIC为1.6mg生药/ml,甘草提取液对金黄色葡萄球菌的抑菌浓度最低,其MIC为3.125g生药/ml。
3.复配组合抑菌试验研究
通过最佳提取条件,对初筛出的抑菌效果较好的6种中草药进行提取,并测定MIC值,进一步优选出了4种抑菌效果较好的中草药,即金银花、金银花叶、甘草、罗汉果,其中金银花、金银花叶和甘草三种中草药对大肠杆菌的抑制效果较佳;金银花、甘草和罗汉果三种中草药对金黄色葡萄球菌的抑制效果较佳。分别进行复配,通过单因素试验和正交试验得出结果:与单味中草药抑菌效果相比,配伍后单味中草药抑菌力有显著增效作用,配伍的中草药间有协同作用;对大肠杆菌的MIC分别为:金银花3.125mg生药/ml、金银花叶1.6mg生药/ml、甘草6.3mg生药/ml,对金黄色葡萄球菌的MIC分别为:金银花叶3.125mg生药/ml、甘草1.6mg生药/ml、罗汉果12.5mg生药/ml,根据MIC值确定了4种提取液的最佳浓度组合:金银花3.125 mg生药/ml、甘草6.3 mg生药/ml、金银花叶3.125 mg生药/ml、罗汉果12.5 mg生药/ml。
4.四种中草药的主要生物活性成分分析
对体外抑菌试验筛选出的金银花、金银花叶、甘草、罗汉果4种中草药提取液中的主要生物活性物质:绿原酸、甘草酸、罗汉果皂甙V进行了研究。采用紫外可见分光光度法分别确定了最大吸收波长绿原酸为330nm、甘草酸为250nm、罗汉果皂甙V为199nm。结果表明:用紫外可见分光光度法测定三种中草药主要活性成分含量,相对标准偏差较小,说明测定结果重现性好,精密度较高,所得数据精确。金银花、金银花叶提取液中绿原酸的含量分别为1.15mg/ml、2.48 mg/ml;甘草酸提取液中甘草酸含量为29.68mg/ml;罗汉果提取液中罗汉果皂甙V含量为26.54mg/ml。研究表明,单味中草药提取液的抑菌活性与其主要活性成分的含量成正比。
5.动物试验结果表明:(1)由4种中草药提取液配制成复方提取液大、中、小剂量组均能够明显抑制角叉菜胶所致的大鼠足跖肿胀度。在给药后1h即能达到明显的抑制效果,药效可持续到8h以上,与空白组对照,复方提取液大、中、小三个剂量组在各个时段均有极显著的抑制效果(P＜0.01)。(2)复方提取液大、中、小剂量组均能够明显抑制蛋清所致的大鼠足跖肿胀度。在给药后1h即能达到明显的抑制效果,药效可持续到8h以上。在1h时,其大剂量作用效果甚至强于阿司匹林(P＜0.01)。与空白组对照,复方提取液大剂量组在各个时段均有极显著的抑制效果(P＜0.01)。
Herb teas select Chinese medicinal herb as the raw material.Modem medicine research shows that,most Herb tea drugs have the functions of the bacteriostasis,dephlogistication,relieving fever, anti-virus,adjusting organic immunity,etc.Our study according to traditional characteristic of Herb teas,combined Chinese traditional medicine and chosed Lonicera japonica Thunb and Lonicera japonica leaf,Chrysanthemum morifolium Ramat,Glycyrrhiza uralensis Fisch,Houlluynia condata Thunb,Taraxacum mongolicum Hand,Siraitia grosvenori C.Jeffrey,Sterculia lychnophora Hance, Platycodon grandiflorum(Jacq.)A.DC,Lophatherum gracile Brongn all 10 kinds of Chinese herbal medicine as the raw material to study the Qingyan Runhou function.
In this test,all 10 kinds of raw material we chosed were authorized by SFDA.We got crude extract as using 80%alcohol to extract.We tested the bacteriostasis to Escherichia coli and Staphylococcus aureus of those 10 Chinese herbal medicines in vitro,preliminary chosing 6 better kinds.We optimized the extracting condition through orthogonal experiment,and then screenen out 4 better Chinese herbal medicines.The major biological activity ingredients contents in those 4 Chinese herbal medicines were detected and the relations of the bacteriostasis and the biological activity ingredients were analysed.
According to the 4 medicine MIC results to the two bacteria,through the progressing concentration match by 1:1:1:1,the optimized prescription was gained.Througth the appraising methods of the Qingyan Runhou functional food and the SD rats as studying object,we studied the inhibitory actions of combination Chinese herbal medicines to the rat swallened pedis.The results are as follows:
1.Chosing of bacteriostasis Chinese herbal medicine
We used hot alcohol continuous recirculation method to extract,and compared inhibition zone and MIC of 10 Chinese herbal medicines extract by bacteriostasis test in vitro.Chosing 6 Chinese herbal medicine which had better bacteriostasis activity,the results showed that the MIC singleChinese herbal medicine extract to Escherichia coli was between 12.5～93.0mg crude drug/ ml,MIC to Staphylococcus aureus was between 6.25～93.0mg crude drug/ml.
2.The extraction condition of bacteriostasis matters of single Chinese herbal medicine
The preliminary screening 6 Chinese herbal medicines as test object,taking orthogonal experiment to optimize alcohol continuous recirculation extraction,adopting L9(3~4)orthogonal table to investigating the bacteriostasis effect of different concentration solvent,temperature,time and solvent reduplication.The influence degree was:alcohol concentration＞extraction time＞dissolvant reduplication＞extraction temperature.According to the analysis of variance,the best extraction condition was:temperature 85℃,time 4.5h,the material to solvent ratio was 1:10 and the solvent was 75%ethanol.In that extraction condition,the MIC of single Chinese herbal medicine extract to Escherichia coli was between 1.6～50mg crude drug/ml and Staphylococcus aureus was between 3.125～25mg crude drug/ml.Among the 6 meidicines,the extract of Lonicera japonica Thunb floral leaf had the lowest bacteriostasis to Escherichia coli,which MIC was 1.6mg crude drug/ml.
3.The combination bacteriostasis test
Through extracted preliminary screening 6 Chinese herbal medicines by the best extraction condition and detected MIC,we screening 4 Chinese herbal medicine which had better bacteriostasis effect:Lonicerajaponica Thunb,Lonicerajaponica floral leaf,Glycyrrhiza uralensis Fisch and Siraitia grosvenori C.Jeffrey.Among the 4 medicines,Lonicerajaponica Thunb,Lonicerajaponica floral leaf,Glycyrrhiza uralensis Fisch had better bacteriostasis to Escherichia coli,and Lonicera japonica Thunb,Glycyrrhiza uralensis Fisch,Siraitia grosvenori C.Jeffrey had better bacteriostasis to Staphylococcus aureus.We combined the different medicines,through the single test and orthogonal experiment we got:as compared to single tests,the combination medicines had better bacteriostasis effect,the MIC to Escherichia coli were:Lonicera japonica Thunb was 3.125 mg crude drug/ml,Lonicerajaponica floral leaf was 1.6 crude drug/ml,Glycyrrhiza uralensis Fisch was 6.3 crude drug/ml;the MIC to Staphylococcus aureus was:Lonicerajaponica Thunb was 6.3 crude drug/ml,Glycyrrhiza uralensis Fisch was 1.6 crude drug/ml and Siraitia grosvenori C.Jeffrey was 12.5 crude drug/ml.
4.The analysis of biological activity matters in 4 Chinese herbal medicines
We studied the biological activity matters(chlorogenic acid,glycyrrhizic acid and mogrosides V)from screening bacteriostasis test in vitro,there were Lonicera japonica Thunb,Lonicera japonica floral leaf,Glycyrrhiza uralensis Fisch and Siraitia grosvenori C.Jeffrey extracts.By using the ultraviolet spectrophotometry method,we confirmed the max absorption wave length of the chlorogenic acids was 333nm,glycyrrhizic acid was 250nm,and mogrosides V was 199nm.The results showed that the determination method by ultraviolet spectrophotometer was acute,precise and exact.The optimized conditions for the extraction of chlorogenic acide extract was:Lonicera japonica Thunb was 1.15mg/ml,Lonicera japonica floral leaf was 2.48 mg/ml,glycyrrhizic acid was 29.68mg/ml,and mogrosides V was 26.54mg/ml.The results showed that single Chinese herbal medicine extract and its biological activity matter contents to a direct ratio.
5.The result of animal experiment showed that:(1)The combination of those 4 Chinese herbal medicine extracts all could markedly restrain rat swaUened pedis which induced by carrageen after feeded 1h.,and the medicine effect caould persisting over 8h.As comparison to the blank,the combination extracts in big,middle,and small dose all had markedly inhibiting effect(P＜0.01). (2)The combination extracts in big,middle,and small dose all had markedly restrain rat swallened pedis which induced by egg white after feeded 1h.,and the medicine effect caould persisting over 8h.In 1h,the retraining effects of the combination extracts in big dose was stronger than Asplin (P＜0.01).As contrast to the blank,the combination extracts in big dose had the markedly inhibiting effect in every periods(contrast to control group P＜0.01).
本试验是以国家食品药品监督管理局(SFDA)所批准的具有清咽润喉功效的上述常用10种中草药为原料,用80%的乙醇进行提取,得到粗提物。通过体外抑菌试验测试了10种中草药对大肠杆菌和金黄色葡萄球菌两种供试菌的抑制作用,初步筛选出抑菌效果较好的6种中草药。采用正交试验设计法优化了抑菌物质的提取条件,并进一步筛选出抑菌效果较好的4种中草药。对4种中草药提取液的主要生物活性成分含量进行测定,分析了抑菌作用与生物活性成分的关系。根据4种提取液对两种供试菌的最低抑菌浓度(minimun inhibitoryconcentration,MIC)的测定结果,依浓度由高到低进行1:1:1:1复配,得出4种提取液最佳浓度组合配方。通过清咽润喉保健食品功能评价方法,雄性SD大鼠为试验对象,研究了复方中草药液对致炎剂所致大鼠足跖肿胀的抑制作用。主要研究结果如下:
1.具有抑菌效果的中草药的筛选
采用醇热连续回流法提取,通过体外抑菌试验比较了10种中草药提取液的抑菌圈直径和最低抑菌浓度(MIC):筛选出了抑菌活性较强的6种中草药,从初步筛选结果看,单味中药提取液对大肠杆菌的MIC介于12.5～93.0mg生药/ml之间,对金黄色葡萄球菌的MIC介于6.25～93.0mg生药/ml之间。
2.单味中草药抑菌物质提取条件
以初步筛选出的6种中草药为研究对象,采用正交试验优化了乙醇连续回流提取条件,选用正交表L_9(3~4),考察溶剂不同浓度、提取温度、提取时间和溶剂加倍量对抑菌效果的影响。结果表明,各因素对试验结果的影响顺序为乙醇浓度＞提取时间＞溶剂加倍量＞提取温度,根据方差分析得出最佳提取条件为:用70%乙醇提取,料液比为1:10,温度85℃,浸提4.5h。在此提取条件下,单味中药提取液对大肠杆菌的MIC介于1.6～50mg生药/ml之间,对金黄色葡萄球菌的MIC介于3.125～25mg生药/ml之间。其中金银花叶提取液对大肠杆菌的抑菌浓度最低,其MIC为1.6mg生药/ml,甘草提取液对金黄色葡萄球菌的抑菌浓度最低,其MIC为3.125g生药/ml。
3.复配组合抑菌试验研究
通过最佳提取条件,对初筛出的抑菌效果较好的6种中草药进行提取,并测定MIC值,进一步优选出了4种抑菌效果较好的中草药,即金银花、金银花叶、甘草、罗汉果,其中金银花、金银花叶和甘草三种中草药对大肠杆菌的抑制效果较佳;金银花、甘草和罗汉果三种中草药对金黄色葡萄球菌的抑制效果较佳。分别进行复配,通过单因素试验和正交试验得出结果:与单味中草药抑菌效果相比,配伍后单味中草药抑菌力有显著增效作用,配伍的中草药间有协同作用;对大肠杆菌的MIC分别为:金银花3.125mg生药/ml、金银花叶1.6mg生药/ml、甘草6.3mg生药/ml,对金黄色葡萄球菌的MIC分别为:金银花叶3.125mg生药/ml、甘草1.6mg生药/ml、罗汉果12.5mg生药/ml,根据MIC值确定了4种提取液的最佳浓度组合:金银花3.125 mg生药/ml、甘草6.3 mg生药/ml、金银花叶3.125 mg生药/ml、罗汉果12.5 mg生药/ml。
4.四种中草药的主要生物活性成分分析
对体外抑菌试验筛选出的金银花、金银花叶、甘草、罗汉果4种中草药提取液中的主要生物活性物质:绿原酸、甘草酸、罗汉果皂甙V进行了研究。采用紫外可见分光光度法分别确定了最大吸收波长绿原酸为330nm、甘草酸为250nm、罗汉果皂甙V为199nm。结果表明:用紫外可见分光光度法测定三种中草药主要活性成分含量,相对标准偏差较小,说明测定结果重现性好,精密度较高,所得数据精确。金银花、金银花叶提取液中绿原酸的含量分别为1.15mg/ml、2.48 mg/ml;甘草酸提取液中甘草酸含量为29.68mg/ml;罗汉果提取液中罗汉果皂甙V含量为26.54mg/ml。研究表明,单味中草药提取液的抑菌活性与其主要活性成分的含量成正比。
5.动物试验结果表明:(1)由4种中草药提取液配制成复方提取液大、中、小剂量组均能够明显抑制角叉菜胶所致的大鼠足跖肿胀度。在给药后1h即能达到明显的抑制效果,药效可持续到8h以上,与空白组对照,复方提取液大、中、小三个剂量组在各个时段均有极显著的抑制效果(P＜0.01)。(2)复方提取液大、中、小剂量组均能够明显抑制蛋清所致的大鼠足跖肿胀度。在给药后1h即能达到明显的抑制效果,药效可持续到8h以上。在1h时,其大剂量作用效果甚至强于阿司匹林(P＜0.01)。与空白组对照,复方提取液大剂量组在各个时段均有极显著的抑制效果(P＜0.01)。
Herb teas select Chinese medicinal herb as the raw material.Modem medicine research shows that,most Herb tea drugs have the functions of the bacteriostasis,dephlogistication,relieving fever, anti-virus,adjusting organic immunity,etc.Our study according to traditional characteristic of Herb teas,combined Chinese traditional medicine and chosed Lonicera japonica Thunb and Lonicera japonica leaf,Chrysanthemum morifolium Ramat,Glycyrrhiza uralensis Fisch,Houlluynia condata Thunb,Taraxacum mongolicum Hand,Siraitia grosvenori C.Jeffrey,Sterculia lychnophora Hance, Platycodon grandiflorum(Jacq.)A.DC,Lophatherum gracile Brongn all 10 kinds of Chinese herbal medicine as the raw material to study the Qingyan Runhou function.
In this test,all 10 kinds of raw material we chosed were authorized by SFDA.We got crude extract as using 80%alcohol to extract.We tested the bacteriostasis to Escherichia coli and Staphylococcus aureus of those 10 Chinese herbal medicines in vitro,preliminary chosing 6 better kinds.We optimized the extracting condition through orthogonal experiment,and then screenen out 4 better Chinese herbal medicines.The major biological activity ingredients contents in those 4 Chinese herbal medicines were detected and the relations of the bacteriostasis and the biological activity ingredients were analysed.
According to the 4 medicine MIC results to the two bacteria,through the progressing concentration match by 1:1:1:1,the optimized prescription was gained.Througth the appraising methods of the Qingyan Runhou functional food and the SD rats as studying object,we studied the inhibitory actions of combination Chinese herbal medicines to the rat swallened pedis.The results are as follows:
1.Chosing of bacteriostasis Chinese herbal medicine
We used hot alcohol continuous recirculation method to extract,and compared inhibition zone and MIC of 10 Chinese herbal medicines extract by bacteriostasis test in vitro.Chosing 6 Chinese herbal medicine which had better bacteriostasis activity,the results showed that the MIC singleChinese herbal medicine extract to Escherichia coli was between 12.5～93.0mg crude drug/ ml,MIC to Staphylococcus aureus was between 6.25～93.0mg crude drug/ml.
2.The extraction condition of bacteriostasis matters of single Chinese herbal medicine
The preliminary screening 6 Chinese herbal medicines as test object,taking orthogonal experiment to optimize alcohol continuous recirculation extraction,adopting L9(3~4)orthogonal table to investigating the bacteriostasis effect of different concentration solvent,temperature,time and solvent reduplication.The influence degree was:alcohol concentration＞extraction time＞dissolvant reduplication＞extraction temperature.According to the analysis of variance,the best extraction condition was:temperature 85℃,time 4.5h,the material to solvent ratio was 1:10 and the solvent was 75%ethanol.In that extraction condition,the MIC of single Chinese herbal medicine extract to Escherichia coli was between 1.6～50mg crude drug/ml and Staphylococcus aureus was between 3.125～25mg crude drug/ml.Among the 6 meidicines,the extract of Lonicera japonica Thunb floral leaf had the lowest bacteriostasis to Escherichia coli,which MIC was 1.6mg crude drug/ml.
3.The combination bacteriostasis test
Through extracted preliminary screening 6 Chinese herbal medicines by the best extraction condition and detected MIC,we screening 4 Chinese herbal medicine which had better bacteriostasis effect:Lonicerajaponica Thunb,Lonicerajaponica floral leaf,Glycyrrhiza uralensis Fisch and Siraitia grosvenori C.Jeffrey.Among the 4 medicines,Lonicerajaponica Thunb,Lonicerajaponica floral leaf,Glycyrrhiza uralensis Fisch had better bacteriostasis to Escherichia coli,and Lonicera japonica Thunb,Glycyrrhiza uralensis Fisch,Siraitia grosvenori C.Jeffrey had better bacteriostasis to Staphylococcus aureus.We combined the different medicines,through the single test and orthogonal experiment we got:as compared to single tests,the combination medicines had better bacteriostasis effect,the MIC to Escherichia coli were:Lonicera japonica Thunb was 3.125 mg crude drug/ml,Lonicerajaponica floral leaf was 1.6 crude drug/ml,Glycyrrhiza uralensis Fisch was 6.3 crude drug/ml;the MIC to Staphylococcus aureus was:Lonicerajaponica Thunb was 6.3 crude drug/ml,Glycyrrhiza uralensis Fisch was 1.6 crude drug/ml and Siraitia grosvenori C.Jeffrey was 12.5 crude drug/ml.
4.The analysis of biological activity matters in 4 Chinese herbal medicines
We studied the biological activity matters(chlorogenic acid,glycyrrhizic acid and mogrosides V)from screening bacteriostasis test in vitro,there were Lonicera japonica Thunb,Lonicera japonica floral leaf,Glycyrrhiza uralensis Fisch and Siraitia grosvenori C.Jeffrey extracts.By using the ultraviolet spectrophotometry method,we confirmed the max absorption wave length of the chlorogenic acids was 333nm,glycyrrhizic acid was 250nm,and mogrosides V was 199nm.The results showed that the determination method by ultraviolet spectrophotometer was acute,precise and exact.The optimized conditions for the extraction of chlorogenic acide extract was:Lonicera japonica Thunb was 1.15mg/ml,Lonicera japonica floral leaf was 2.48 mg/ml,glycyrrhizic acid was 29.68mg/ml,and mogrosides V was 26.54mg/ml.The results showed that single Chinese herbal medicine extract and its biological activity matter contents to a direct ratio.
5.The result of animal experiment showed that:(1)The combination of those 4 Chinese herbal medicine extracts all could markedly restrain rat swaUened pedis which induced by carrageen after feeded 1h.,and the medicine effect caould persisting over 8h.As comparison to the blank,the combination extracts in big,middle,and small dose all had markedly inhibiting effect(P＜0.01). (2)The combination extracts in big,middle,and small dose all had markedly restrain rat swallened pedis which induced by egg white after feeded 1h.,and the medicine effect caould persisting over 8h.In 1h,the retraining effects of the combination extracts in big dose was stronger than Asplin (P＜0.01).As contrast to the blank,the combination extracts in big dose had the markedly inhibiting effect in every periods(contrast to control group P＜0.01).
引文
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