摘要
目的：借鉴比较医学研究的方法并结合现代实验药理研究方法系统比较暗紫贝母及浙贝母的差异。为2种贝母临床合理应用提供科学依据。
方法：借鉴前瞻性对比研究的方法对暗紫贝母及浙贝母进行了本草考证、对比分析了2者原植物及鳞茎形态特征、化学成分异同、药理及临床应用方面的特点,并运用整体动物实验法系统比较了暗紫贝母及浙贝母粉末及提取物的镇咳、祛痰、平喘作用差异；整体动物引喘实验法、离体器官实验法、体外细胞筛选实验法深入探讨了暗紫贝母及浙贝母总生物碱平喘作用,总碱及4种代表性生物碱单体的平喘作用机理。
结果：
1.“川贝母”和“浙贝母”区分明确起自清代《本草纲目拾遗》,作为川贝母代表基源种的暗紫贝母与浙贝母之间从形态、化学成分、药效及临床应用方面,尤其是临床应用存在差异。
2.暗紫贝母及浙贝母全粉2.0g·kg-1·d-1(小鼠)、1.0g·kg-1·d-1(大鼠)灌胃给药,通过小鼠酚红排痰实验法比较,2个贝母组酚红排泌量均高于阴性对照组,差异有统计学意义(P<0.01)；通过大鼠毛细玻管排痰实验法比较,2个贝母组痰液排出量与阴性对照组比较,差异亦有统计学意义(P<0.05)。
3.暗紫贝母及浙贝母提取物2.0g·kg-1·d-1(小鼠)、1.0g·kg-1·d-1(大鼠)灌胃给药,通过小鼠酚红排痰实验法比较,暗紫贝母水提物组、暗紫贝母醇提物组、浙贝母水提物组酚红排泌量高于阴性对照组,差异有统计学意义(P<0.05),两两比较结果显示：暗紫贝母水提物组酚红排出量略高于暗紫贝母醇提物组及浙贝母水提物组(P<0.05),后2组两两比较,差异无统计学意义；通过大鼠毛细玻管排痰实验法比较,暗紫贝母水提取物组、浙贝母水提取物组、浙贝母醇提物组痰液排出量高于阴性对照组,差异有统计学意义(P<0.05)；暗紫贝母与浙贝母水提物痰液排出量高于其余各组,两两比较结果显示：浙贝母水提物痰液排出量,与其余各组比较,差异有统计学意义(P<0.05)。
4.暗紫贝母及浙贝母全粉2.5g·kg-1·d-1(小鼠)、1.5g·kg-1·d-1(豚鼠)灌胃给药时,2个贝母组延长小鼠氨水引咳ET50不明显,浙贝母组R值为91.39%,暗紫贝母组R值为123.06%；减少豚鼠枸橼酸引咳后5min内咳嗽次数与阴性对照组比较,差异有统计学意义(P<0.05)；
5.暗紫贝母及浙贝母提取物5g·kg-1·d-1(小鼠)、1.5g·kg-1·d-1(豚鼠)灌胃给药时,暗紫贝母醇提物组延长小鼠氨水引咳ETso明显,R值为143.21%,表现出镇咳效应；2种贝母的醇提物减少豚鼠枸橼酸引咳后5min内咳嗽次数与阴性对照组比较,差异有统计学意义(P<0.05)。
6.暗紫贝母及浙贝母全粉1g·kg-1·d-1(豚鼠)灌胃给药时,暗紫贝母组延长哮喘潜伏期,与阴性对照组比较,P<0.05；
7.暗紫贝母及浙贝母提取物1.5g·kg-1·d-1(豚鼠)灌胃给药时,暗紫贝母及浙贝母各提取物组均未表现出明显的平喘作用,哮喘潜伏期与阴性组比较,差异无统计学意义。
8.暗紫贝母总生物碱及浙贝母总生物碱50mg·kg-1灌胃给药,延长豚鼠哮喘潜伏期,与阴性对照组比较,差异有统计学意义(P<0.05),作用强度略弱于沙丁胺醇组(4.5×10-3g·kg-1),两两比较结果显示：暗紫贝母组与阳性组比较差异有统计学意义。
9.基于β2受体通路体外细胞筛选实验结果显示：西贝素、贝母辛、贝母素甲及贝母素乙均未表现出激动β2受体的活性；暗紫贝母及浙贝母总生物碱中亦未发现存在其他可能具有激动β2受体的活性物质。
10.基于M胆碱受体通路离体平滑肌实验结果显示：暗紫贝母总生物碱低(2g·L-1)、中(10g·L-1)、高(50g·L-1)剂量对氯贝胆碱引起的豚鼠离体支气管平滑肌收缩的抑制率分别为：27.5%、26.4%、40.6%,浙贝母总生物碱低(2g·L-1)、中(10g·L-1)、高(50g·L-1)剂量对氯贝胆碱引起的豚鼠离体支气管平滑肌收缩的抑制率分别为：11.4%、22.7%、37.5%；西贝素、贝母素甲、贝母素乙、贝母辛拮抗氯贝胆碱的PA2值分别为：7.32、7.26、9.04、7.76。
结论：
1.暗紫贝母与浙贝母均具有较明显的祛痰作用,暗紫贝母祛痰作用强度略优于浙贝母,其祛痰有效成分主要集中在其水提物部分；
2.2种贝母镇咳有效成分主要集中在其醇提物部分,暗紫贝母镇咳作用略优于浙贝母(小鼠氨水引咳模型)或强度相当(豚鼠枸橼酸引咳模型)；
3.2种贝母全粉给药时,具有相当强度的平喘作用,其有效成分没有明显分布趋势；
4.2种贝母研粉冲服均能使其有效成分较好吸收；
5.2种贝母祛痰作用有效剂量小于其镇咳及平喘有效剂量；
6.2种贝母总生物碱不能完全代表其平喘有效成分；
7.暗紫贝母及浙贝母生物碱成分可能主要通过拮抗支气管平滑肌M3受体实现舒张支气管平滑肌的作用；
Objiect:
A similar comparison method of medical research and modern pharmacolo-gical research methods were carried on to compare the and provide more supp-orts andideas for the reasonabale clinical use of the two Beimus.
Methods:
A similar prospective and comparative study methods were carried on to compare the Fritillaria unibracteata Hisao et K.C.Hsia and Fritillaria Thunber-gii Miq on the herbal textual research,morphological comparison,chemical com-position,pharmacodynamics and clinical use.Variety of experimental techniques methods also used in the experiments.The differences between F. unibracteata Hsiao et K.C.Hsia and F. thunbergii Miq on elimilating cough,sputum and asth-ma and the mechanisms of anti-asthma effect of the two total alkaloids and4representative isosteroidal alkaloids of the two Beimus' were investigated by overall animal experiments, isolated organ experiment and ceel-based screening experimental methods.
Results:
1.Discrimination of "Chuanbeimu" and "Zhebeimu" bagan from "Bencaogangm-ushiyi".There were many differences between Fritillaria unibracteata Hisao et K.C.Hsia and Fritillaria thunbergii Miq in morphous,chemical composition,phar-macodynamics and especially in clinical use.
2.Micro powder of Fritillaria unibracteata Hisao et K.C.Hsia and Fritillaria th-unbergii Miq administered on the dose of2.0g·kg-1·d-1in mice and1.0g·kg-1·d-1in rat.The differences of the amount of phlegm between two Beimus and the negative group were statistically significant(P<0.05)
3.Extracts of Fritillaria unibracteata Hisao et K.C.Hsia and Fritillaria thunber-gii Miq. administered on the dose of2.0g·kg-1·d-1in mice and1.0g·kg-1·d in rat.The amount of phlegm between the aqueous extracts,the ethanol extract of the Fritillaria unibracteata Hisao etK.C.Hsia,the aqueous extracts of the Fritill-aria thunbergii Miq and the negative group were statistically significant(P<0.05) in mice. The aqueous extract of the Fritillaria unibracteata Hisao et K.C.Hsia was more effective than the other two groups in mice, and the differences bet- ween this group to the other two were statistically significant(P<0.05),and there were no significant differrences between the other two groups. The amount of phlegm between the aqueous extracts of the Fritillaria unibracteata Hisao et K. C.Hsia,the aqueous extract,the ethanol extract of the Fritillaria thunbergii Miq and the negative group were statistically significant in rat,the aqueous extract of the Fritillaria thunbergii Miq was more effective than other groups.
4.Micro powder of Fritillaria unibracteata Hisao et K.C.Hsia and Fritillaria th-unbergii Miq administered on the dose of2.5g·kg-1·d-1in mice and l.5g·kg-1·d-1in guinea pigs.The R of Fritillaria thunbergii Miq was91.39%,and the Fritilla-ria unibracteata Hisaoet K.C.Hsia was123.06%.The difference of the counts of cough induced by critic acid in guinea pigs between the two Beimus and the negative group were significant(P<0.05).
5.Extracts of Fritillaria unibracteata Hisao et K.C.Hsia and Fritillaria thunber-gii Miq administered on the dose of5.0g·kg-1·d-1in mice and1.5g·kg-1·d-1in guinea pigs. Fritillaria unibracteata Hisaoet K.C.Hsia's R was143.21%.The di-fference of the counts of cough induced by critic acid in guinea pigs between the two ethanol extracts of the two Beimus and the negative group were signi-ficant(P<0.05).
6.Fritillaria unibracteata Hisao et K.C.Hsia micro powder administered on the dose of1.0g·kg-1·d-1in guinea pigs showed significant anti-athma effect.The di-fferences of athma time between the two groups was statistically significant (P <0.05).
7.Both of the extracts of the two Beimus showed no anti-athma effect.
8.The total alkaloids of the Fritillaria unibracteata Hisao et K.C.Hsia and Friti llaria thunbergii Miq.could increase the athma time when administered on the dose of50mg·kg-1in guinea pigs,the differences between this group to negative one was statistically signifi-cant (P<0.05).
9.Both the total alkaloids of the two Beimus and imperialine,peimisine,peimine and peiminine had no activity on the β2AR.
10.In the organ experiment,the contraction of grips of guinea pigs were investi gated. Inhibition rate of the total alkaloids of Fritillaria unibracteata Hisao et K.C.Hsia was27.5%、26.4%、40.6%,and the inhibition rate Fritillaria thumb-ergii Miq was11.4%、22.7%、37.5%.The PA2value of imperialine,peimisine, peimineand peiminine was:7.327.269.047.76.
Conclusions:
1. Fritillaria unibracteata Hisao et K.C.Hsia and Fritillaria thunbergii Miq had significant eleminating phlegm effect.The Fritillaria unibracteata Hisao et K.C. Hsia was more effective to Fritillaria thunbergii Miq. The aqueous extracts of the two fritillarias could be the active part.
2. The ethanol extracts of the two fritillarias could be the active part of antit-ussive effect.Fritillaria unibracteata Hisao et K.C.Hsia could be more effective than the Fritillaria thunbergii Miq one.
3.Both of the two Beimus' micro powder had similar anti-athma effect.
4. The active ingredient of the two Beimu could be better absorbed by oral of of it's powder.
5.The active dose of eleminating phlegm could be higher than the antitusiive and antiathma one.
6. The total alkaloids of the two Beimus could not be fully representative of the effective ingredients on their antiasthmatic;
7. The total alkaloids and4representative isosteroidal alkaloids of Fritillaria unibracteata HsiaoetK. C. Hsia and Fritillaria thunbergii Miq mainly role by antagonizing the bronchial smooth muscle M3receptor to relax of bronchial smooth muscle.
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