黑曲霉转化桔梗皂苷及其祛痰作用研究
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摘要
桔梗(Platycodon grandiflorum (Jacq.) A.DC)为我国传统中药,始载于《神农本草经》,后以宣肺、利咽、祛痰、排脓等功效被广泛应用于各种方剂中,传承至今。随着研究的深入和研究手段的发展,现代药理学揭示了它具有降血脂,降血糖,抗肥胖,镇静,降低胆固醇等广泛的生物活性。桔梗食药兼用,且资源十分丰富,因此深入研究开发桔梗这一广阔植物资源,提高其食用和药用的利用度,有着重要的意义。
本论文利用常规色谱分离和制备高效液相色谱分离相结合,共从桔梗中分离得到3个单体化合物,应用现代波谱技术(MS, IR,1H-NMR,13C-NMR,2D-NMR等)鉴定已知的3个化合物分别为：deapio-platycodin-D, platycodin-D, polygalancin D。
本文以桔梗总皂苷、去芹糖桔梗皂苷D、桔梗皂苷D、远志皂苷D的含量为考察指标,综合考虑溶剂种类、溶剂浓度、液料比、提取次数、提取时间这五个单因素实验的结果,最终确定的闪式提取法的最优提取条件为：以70%乙醇为溶剂、液料比为1：8、提取3次、每次提取时间为5min。
选择黑曲霉,黑根酶,嗜酸乳杆菌,婴儿双歧杆菌为发酵菌种,分别测定了发酵前后的桔梗皂苷转化情况。对比原样,黑曲霉与黑根霉转化后的桔梗总皂苷HPLC色谱峰明显后移,推断可能是部分皂苷糖链被水解掉而形成次级皂苷或是苷元。在嗜酸乳杆菌和婴儿双歧杆菌发酵培养后,桔梗总皂苷的HPLC谱图没有变化,也就是说,嗜酸乳杆菌和婴儿双歧杆菌都无法水解或是转化桔梗总皂苷或是某单体皂苷。本次实验初步确定了以黑曲霉或黑根霉为发酵菌种的想法。
考虑到高温或许会对桔梗皂苷产生影响,在进行发酵培养前,我们对高温灭菌后的桔梗总皂苷与未经过任何处理的总皂苷进行了比较,发现桔梗皂苷在121℃条件下灭菌20mmin,对其成分无任何影响,可以证明桔梗总皂苷转化为桔梗次级皂苷并不是由于高温导致的。采取液体培养的方法,发现仅是在总皂苷无需任何外界营养物质,黑曲霉亦可很好的生长。
利用酚红作为祛痰效果指示剂,在川贝枇杷糖浆、桔梗总皂苷及黑曲霉转化的桔梗总次皂苷的作用下,使呼吸道黏膜排出的酚红增加,从而证实支气管分泌液增加。研究结果显示,桔梗总皂苷及黑曲霉转化的桔梗总次皂苷的高、中剂量组均能明显增加小鼠呼吸道酚红排泌量,使小鼠呼吸道分泌液明显增加,与空白对照组相比较,有统计学差异(P<0.05)。
曾有学者报道,桔梗皂苷D具有祛痰的作用,是桔梗祛痰作用的关键成分。但本次实验的结果显示,即便黑曲霉将桔梗皂苷D转化殆尽,几组灌胃转化后总皂苷小鼠的酚红排泌量与转化前相比并没有减少。可见桔梗中皂苷类物质的祛痰作用不只是通过桔梗皂苷D来体现的,其他单体皂苷抑或是皂苷以外的成分也可能具有祛痰的作用,关于这一点尚需进一步研究。本实验也进一步验证了桔梗祛痰的功效,今后对其他桔梗皂苷的研究可以着眼于祛痰镇咳这类桔梗的传统功效上。
Platycodi Radix is one kind of Traditional Chinese Medicine, which is originated from the record of Shen Nong's Hebal and has been widely used in various kinds of prescription for its function of opening the inhibited lung-energy, relieving sore-throat, eliminating phlegm, apocenosis. With the development of study means and deepening investigation, extensive bioactivities of Platycodi Radix have been demonstrated in Modern Pharmacology, such as lowering blood fat, blood glucose and cholesterol, anti-obesity, conscious-sedation. Platycodi Radix is fresh drug along with food and rich in natural resource, so it is important to study and improve its edible and medicinal availability.
With the method of conventional chromatographic fractionation and preparation High Performance Liquid Chromatograph, we have separated seven compounds. Three were identified by modern spectrum technology (such as MS,1R,1H-NMR,13C-NMR,2D-NMR) and the rest were deapio-platycodin-D, platycodin-D, polygalancin D.
According to the contents of total saponin, deapio platycodin D, platycodin D and polygalacin D for index of investigation, extraction of flash-type extraction optimize platycodin selection process, considering the solvent, solvent concentration, liquid ratio, extraction times and extraction time the five single factor experimental results, finally flash-type extraction to determine the optimal extraction conditions as follows: 70% ethanol, liquid ratio of 1:8, extract 3 times, each extraction time 5min.
Aspergillus niger, Rhizopus nigricans, Bacillus acidophilus, Bifidobacterium infantis was selected as strain for the fermentation, the secondary saponin and the total saponin were measured by HPLC. The HPLC chromatogram of secondary saponin fermentation by Aspergillus niger and Rhizopus nigricans was backward clearly. The HPLC chromatogram of fermentation Bacillus acidophilus, Bifidobacte-rium infantis have no changed. Therefore, the fermentation of Aspergillus niger and Rhizopus nigricans was Initially identified.
Before the fermentation, compared the total saponin between high-temperature sterilization and untreated, we found total saponin have not affected by high-temperature sterilization. Prove that high-temperature is not the reason of total saponin changed to secondary saponin.
Study on the expectorant effect of total saponin and secondary saponin conversion by Aspergillus niger through animal experiment. Cough modle was induced by phenol red expectoration test, to research on the expectorant effect of total saponin and secondary saponin conversion by Aspergillus niger, Total saponin and secondary saponin conversion by Aspergillus niger had expectorant effect on mice induced by phenol red (P< 0.05). Total saponin and secondary saponin conversion by Aspergillus niger had expectorant effect.
Platycodin D is one of the most important components of the effect of expectorant. The results showed that platycodin D had completed conversion, however, the effect of expectorant is not only because of platycodin D, other single saponin or non-saponin maybe the reason.
本论文利用常规色谱分离和制备高效液相色谱分离相结合,共从桔梗中分离得到3个单体化合物,应用现代波谱技术(MS, IR,1H-NMR,13C-NMR,2D-NMR等)鉴定已知的3个化合物分别为：deapio-platycodin-D, platycodin-D, polygalancin D。
本文以桔梗总皂苷、去芹糖桔梗皂苷D、桔梗皂苷D、远志皂苷D的含量为考察指标,综合考虑溶剂种类、溶剂浓度、液料比、提取次数、提取时间这五个单因素实验的结果,最终确定的闪式提取法的最优提取条件为：以70%乙醇为溶剂、液料比为1：8、提取3次、每次提取时间为5min。
选择黑曲霉,黑根酶,嗜酸乳杆菌,婴儿双歧杆菌为发酵菌种,分别测定了发酵前后的桔梗皂苷转化情况。对比原样,黑曲霉与黑根霉转化后的桔梗总皂苷HPLC色谱峰明显后移,推断可能是部分皂苷糖链被水解掉而形成次级皂苷或是苷元。在嗜酸乳杆菌和婴儿双歧杆菌发酵培养后,桔梗总皂苷的HPLC谱图没有变化,也就是说,嗜酸乳杆菌和婴儿双歧杆菌都无法水解或是转化桔梗总皂苷或是某单体皂苷。本次实验初步确定了以黑曲霉或黑根霉为发酵菌种的想法。
考虑到高温或许会对桔梗皂苷产生影响,在进行发酵培养前,我们对高温灭菌后的桔梗总皂苷与未经过任何处理的总皂苷进行了比较,发现桔梗皂苷在121℃条件下灭菌20mmin,对其成分无任何影响,可以证明桔梗总皂苷转化为桔梗次级皂苷并不是由于高温导致的。采取液体培养的方法,发现仅是在总皂苷无需任何外界营养物质,黑曲霉亦可很好的生长。
利用酚红作为祛痰效果指示剂,在川贝枇杷糖浆、桔梗总皂苷及黑曲霉转化的桔梗总次皂苷的作用下,使呼吸道黏膜排出的酚红增加,从而证实支气管分泌液增加。研究结果显示,桔梗总皂苷及黑曲霉转化的桔梗总次皂苷的高、中剂量组均能明显增加小鼠呼吸道酚红排泌量,使小鼠呼吸道分泌液明显增加,与空白对照组相比较,有统计学差异(P<0.05)。
曾有学者报道,桔梗皂苷D具有祛痰的作用,是桔梗祛痰作用的关键成分。但本次实验的结果显示,即便黑曲霉将桔梗皂苷D转化殆尽,几组灌胃转化后总皂苷小鼠的酚红排泌量与转化前相比并没有减少。可见桔梗中皂苷类物质的祛痰作用不只是通过桔梗皂苷D来体现的,其他单体皂苷抑或是皂苷以外的成分也可能具有祛痰的作用,关于这一点尚需进一步研究。本实验也进一步验证了桔梗祛痰的功效,今后对其他桔梗皂苷的研究可以着眼于祛痰镇咳这类桔梗的传统功效上。
Platycodi Radix is one kind of Traditional Chinese Medicine, which is originated from the record of Shen Nong's Hebal and has been widely used in various kinds of prescription for its function of opening the inhibited lung-energy, relieving sore-throat, eliminating phlegm, apocenosis. With the development of study means and deepening investigation, extensive bioactivities of Platycodi Radix have been demonstrated in Modern Pharmacology, such as lowering blood fat, blood glucose and cholesterol, anti-obesity, conscious-sedation. Platycodi Radix is fresh drug along with food and rich in natural resource, so it is important to study and improve its edible and medicinal availability.
With the method of conventional chromatographic fractionation and preparation High Performance Liquid Chromatograph, we have separated seven compounds. Three were identified by modern spectrum technology (such as MS,1R,1H-NMR,13C-NMR,2D-NMR) and the rest were deapio-platycodin-D, platycodin-D, polygalancin D.
According to the contents of total saponin, deapio platycodin D, platycodin D and polygalacin D for index of investigation, extraction of flash-type extraction optimize platycodin selection process, considering the solvent, solvent concentration, liquid ratio, extraction times and extraction time the five single factor experimental results, finally flash-type extraction to determine the optimal extraction conditions as follows: 70% ethanol, liquid ratio of 1:8, extract 3 times, each extraction time 5min.
Aspergillus niger, Rhizopus nigricans, Bacillus acidophilus, Bifidobacterium infantis was selected as strain for the fermentation, the secondary saponin and the total saponin were measured by HPLC. The HPLC chromatogram of secondary saponin fermentation by Aspergillus niger and Rhizopus nigricans was backward clearly. The HPLC chromatogram of fermentation Bacillus acidophilus, Bifidobacte-rium infantis have no changed. Therefore, the fermentation of Aspergillus niger and Rhizopus nigricans was Initially identified.
Before the fermentation, compared the total saponin between high-temperature sterilization and untreated, we found total saponin have not affected by high-temperature sterilization. Prove that high-temperature is not the reason of total saponin changed to secondary saponin.
Study on the expectorant effect of total saponin and secondary saponin conversion by Aspergillus niger through animal experiment. Cough modle was induced by phenol red expectoration test, to research on the expectorant effect of total saponin and secondary saponin conversion by Aspergillus niger, Total saponin and secondary saponin conversion by Aspergillus niger had expectorant effect on mice induced by phenol red (P< 0.05). Total saponin and secondary saponin conversion by Aspergillus niger had expectorant effect.
Platycodin D is one of the most important components of the effect of expectorant. The results showed that platycodin D had completed conversion, however, the effect of expectorant is not only because of platycodin D, other single saponin or non-saponin maybe the reason.
引文
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