| This research paper, referring to the project "Research on establishment of standardized planting base and the SOP of Alpinia oxyphylla Miq.", which is contained in the regional base project "Optimization and upgrading of standardized planting base and the SOP of7kinds of traditional Chinese medicines" financially supported by the national12th five-year plan regional base project (2011BA101B07). Besides, a part of research contents are from "Establishment of quality control on crude drugs and decoction pieces of the fruits of A. oxyphylla from standardized planting base in Hainan" supported by Hainan special plan for the modernization of traditional Chinese medicines.This dissertation involves five chapters.Chapter1Literature researchIn this part, mainly through summarizing relevant literature, the pharmaceutical usages of the plants belong to Zingiberacea family, the research progress in the plants of Alpinia genus, traditional applications and research advances of chemistry and pharmacology of the fruits of A. oxyphylla were reviewed systematically. Based on the preliminary work, the research ideas and experimental design were put forward.Chapter2Studies on the chemical resources of A. oxyphyllaThis chapter is further divided into two sections.Section one includes three parts.In part1, studies on the chemical constituents from the fruits of A. oxyphylla were included. The objective of this study is to further develop and exploit the rich resources of Hainan famous-region drug-A. oxyphylla, clarify the pharmacological substances based on its traditional efficacy and find its new effective fractions and components. For this reason, the chemical investigation on the fruits of A. oxyphylla were carried out with the combined methods of silica gel column chromatography, preparative TLC, gel permeation chromatography, ODS column chromatography and recrystallization. The structures of the isolated compounds were elucidated on the grounds of their physicochemical constants and NMR spectral data analysis.11compounds were isolated and their structures were determined as9-hydroxy epinootkatol (1), pinocembrin (2), nookatone (3), tectochrysin (4),β-sitosterol (5), yakuchinone A (6), izalpinin (7), β-daucosterol (8),β-sitosterol palmitate (9),(S)-2-pentanol-2-O-β-D-glucopyranoside (10) and protocatechuic acid (11). Compounds1and10were new natural products and compound2was isolated from the fruits of A. oxyphylla for the first time. This study provided scientific basis for the further research and exploitation of the resources of the fruits of A. oxyphylla in the future.In part2, investigation on the chemical constituents from the rhizomes of A. oxyphylla was referred to. Based on the pharmacological substances exploited that were discovered from the rhizomes of A. oxyphylla, the chemical investigation on the rhizomes of A. oxyphylla was carried out with the methods of silica gel column chromatography and polyamide column chromatography. The structures of the isolated compounds were elucidated according to the physicochemical constants and NMR spectral data analysis.14compounds were isolated from the ethanol extract from the rhizomes of A. oxyphylla and their structures were determined as doacontanol (1), izalpinin (2), tectochrysin (3), kaempferol-7,4’-dimethyl mester (4), β-sitosterol (5),p-hydroxylcinnamic acid (6), dihydrokaempferol (7), yakuchinone A (8), chrysin (9), pinocembrin (10), kaempferol-4’-methylmester (11), myristic acid (12), pinostrobin (13). All the compounds were isolated from the rhizomes of A. oxyphylla for the first time. This study lays scientific basis for the exploitation of the resources of rhizomes of A. oxyphylla in the future.In part3, research into the chemical constituents from the stems and leaves of A. oxyphylla was discussed. To study the effective substances in the stems and leaves of A. oxyphylla further, their chemical constituents were isolated and purified by silica column chromatography and polyamide column chromatography. The structures of those compounds were identified and determined based on their physicochemical constants and spectral data analysis. Consequently,16compounds were obtained, and they were elucidated as izalpinin (1), tectochrysin (2), apigenin-7,4’-dimethyl ester (3), kaempferol-4’-methyl mester (4),(E)-p-hydroxy cinnamic acid (5), chrysin (6), pinocembrin (7), apigenin-4’-methyl ester (8), myristic acid (9), docosanoic acid (10), octacosanoic acid (11), septadecanoic acid (12), eicosanoic acid (13), n-hexadecanoic acid (14), pinostrobin (15),5,7,4’-trimethyl flavone (16). All the compounds were isolated from the stems and leaves of A. oxyphylla for the first time. This study provides scientific basis for the exploitation of the resources of stems and leaves of A. oxyphylla in the future.In section two, the chemical resources evaluation of A. oxyphylla was studied. This chapter consists of five parts.In part1, the volatile components in the fruits of A. oxyphylla were tested and analyzed. Chemical compositions in the volatile oils from the fruits of A. oxyphylla collected from different provinces and regions in China, including Yunnan, Guangxi, Guangdong and Hainan, were studied by GC-MS. The results showed that there was a large variation in the components and content of volatile oils among the drugs from different regions. Among the components in the volatile oil, β-panasinsene and humulene are two major compounds. In addition, hierarchical cluster analysis (HCA) was performed to classify all the samples according to the contents of the investigated analytes. The samples from Baisha, Qiongzhong and Tunchang (Hainan province) can be classified into one cluster, which indicates that these regions are similar in the environment, while the samples from Guandong, Guangxi and Yunnan were classified into another cluster, whose contents of volatile oils were different from the three regions mentioned above.In part2, the variation rule about the contents of the constituents in the fruits, rhizomes, stems and leaves of A. oxyphylla at different growth time was studied. Nine compounds, i.e., nootkatone, yakuchinone A, yakuchinone B, nootkatol, tectochysin, izalpinin, chrysin and7,4’-dimethyl apigenin were analyzed by LC-MS/MS.The contents of the resource constituents in the different parts of A. oxyphylla are different. The result shows that the resource constituents of terpenoids, diarylheptanoids are mainly rich in the fruits of A. oxyphylla, while the flavonoids is evenly distributed in the fruits, stems and leaves, and rhizomes of A. oxyphylla in the whole growth period of the fruits.The results showed that the contents of all the compounds in the fruits reached the highest at45-day harvest time. Thus, it is reasonable that the best harvest time for the fruits is set at45days after it bears fruits. With the maturation of the fruits, most of the chemical components accumulated in the fruits at45days. This indicates that it is reasonable for the fruits of A. oxyphylla traditionally used as a medicinal part. At45days, a large quantity of flavonoids and a small amount of terpenoids and diary lheptanoids also accumulated in the stems and leaves of A. oxyphylla, which shows that the stems and leaves can be used as new resources in the future.The best harvest time of the fruits and the transfer rule of the resource constituents among the fruits, rhizomes, stems and leaves, need to be verified.In part3, the assay of nucleobases and nucleosides in the fruits of A. oxyphylla was done. Nucleobases and nucleosides were quantitatively determined in the fruits of A. oxyphylla collected from different cultivation regions. Almost all the samples were rich in nucleobases and nucleosides, but there was a large variation in the content, which might attribute to the factor of harvest time. Nucleobases and nucleosides were also quantitatively determined in the fruits of A. oxyphylla collected at different harvest time. The results showed that the content of thymidine is much higher during the whole research period than that of others, which is in the range of36.79-310.58μg/g. Hypoxanthine is observed to be the least in all the analytes, and its contents in some samples cannot be detected. Additionally, the total amount of both total and individual nucleosides and nucleobases in the fruits of A. oxyphylla decreased with the increase of maturity degree, which indicates that the synthesis of such components is closely relevant to the growth cycle. In part4, the contents of inorganic elements in the fruits, rhizomes, stems and leaves and flowers at different growth stages were assayed. The results showed that the contents of nutrient elements such as Zn、Fe、Mn、Ca and Mg in the fruits of A. oxyphylla were higher than those in the rhizomes or stems and leaves. In the fruits of A. oxyphylla, the contents of heavy metals, i.e., As、Hg、Pb and Cu (except Cd) are lower than those in the rhizomes or stems and leaves.As we all known,"Green trade standards of importing&exporting medicinal plants&preparations", which was awarded by the state department of commerce in2001, has set the lowest content of heavy metals in medicinal plants. The contents of As、Hg、Pb、Cd in the20samples of the fruits of A. oxyphylla, which were collected from different regions, such as Yunnan, Guangxi, Guangdong and Hainan, are in line with the standards. The contents of Cu in the samples from some regions, such as the samples from Changxing village and Maoyang village in Qiongzhong County, Wupo village and Nanlv village in Tunchang County, Hainan province, are in accordance with the standards. While the contents of Cu in the samples from other regions collected from Xishuangbanna, Yunnan province, Nanning, Guangxi, Guangdong and other areas in Hainan province are higher than the limitation set in the standards. The exact content of heavy metals need farther research on atmosphere, soil and irrigate water.It is concluded from the results of the contents of heavy metals and nutrient elements in the20samples that, the sample from Hainan province is the best in the security of medicinal plants, especially from Changxing village and Maoyang village in Qiongzhong county, Wupo village and Nanlv village in Tunchang county in Hainan province. The contents of nutrient elements in the samples from Nanning and Guangxi are the highest of all.A conclusion can be drawn from the changes in the contents of heavy metals and nutrient elements in the different parts of A. oxyphylla at different growth time:the contents of heavy metals in the fruits and rhizomes are lower than those in the stems and leaves, while the contents of nutrient elements in the fruits and rhizomes are higher. The contents of heavy metals are the lowest and the contents of nutrient elements are the highest when the fruit of A. oxyphylla is the45th day.Chapter3Bioactive studies on the extracts and compounds from A. oxyphyllaIn Chapter3, spleen warming, anti-diarrhea, kidney-tonifying and anti-polyuria bioactivities of the fruits of A. oxyphylla were studied. This chapter can be divided into three sections.In Section1, preparation of different extracts of A. oxyphylla was studied. To clarify the bioactive fraction, the volatile oil, the95%ethanol extract, the50%ethanol eluate and the90%ethanol eluate from the fruits of A. oxyphylla were prepared for the bioactive assay. The quality of the four fractions was controlled by HPLC. In Section2, the preliminary research focused on the active fractions from the fruits of A. oxyphylla and the mechanism of spleen warming and anti-diarrhea was included. Acute diarrhea induced by senna-leaf and the mouse model of diarrhea of spleen deficiency induced by rhubarb were used to study the anti-diarrhea effect of the active fractions from the fruits of A. oxyphylla. Four fractions were prepared to study the anti-diarrhea effect of the fruits of A. oxyphylla. The experiment results suggested that the95%ethanol extract and the90%ethanol eluate could prolong the onset time of diarrhea and reduce the wet faeces proportion, however, the50%ethanol eluate had no effect on diarrhea. The volatile oil prolonged the onset time of diarrhea, but showed no effects on the wet faeces proportion and the number of wet faeces. Chemical analysis results displayed that fat-soluble components may be the active ingredients for curing diarrhea. The further research showed the95%ethanol extract and the90%ethanol eluate affect the gastric residual rate and the intestinal propulsion rate, and improve the spleen asthenia syndrome to some extent.The results indicated the95%ethanol extract could improve the spleen asthenia syndrome by increasing the level of plasma LDH, inhibit gastrointestinal motility by increasing plasma NO and reducing gastrointestinal hormone MOT and SS. The90%ethanol eluate can improve the spleen asthenia by increasing the level of plasma LDH and reducing SDH, while its inhibitory effect on gastrointestinal motility is primarily related with the increase in the level of NO and the decrease in the level of gastrointestinal hormone MOT and SS.In Section3, pharmacodynamic screening of different fractions and compounds from A. oxyphylla to ensure the urination reducing activity was investigated. The volatile oil, the95%ethanol extract (non-volatile oil), the eluate of50%ethanol and the eluate of90%ethanol prepared by macroporous resin column chromatography inhibit the bladder detrusor contraction that induced by carbachol.7compounds isolated from A. oxyphylla, i.e., izalpinin, kaempferide, oxyphyllacinol, yakuchinone B, nootkatone, tectochrysin, chrysin were screened for the activity, and found that izalpinin showed inhibitory effect on the bladder detrusor contraction induced by carbachol with a mean EC50(concentration for50%of maximal effect) as0.35±0.05μM.Chapter4Quality standard of the fruits of A. oxyphyllaIn this chapter, the quality standard of the fruits of A. oxyphylla was studied and established. Based on the quality standard of the fruits of A. oxyphyllae in Chinese Pharmacopoeia2010th edition, TLC and HPLC methods were adopted to improve its quality standard. The moisture, total ash, acid-insoluble ash of A. oxyphylla fruits were measured according to the procedures recorded in the Chinese Pharmacopoeia. The contents of nootkatone, tectochrysin and yakuchinone A were determined by HPLC. All these work will establish a solid foundation for the quality standard of new products.Chapter5Exploitation and industrialization of A. oxyphyllaThe fruits of A. oxyphylla are commonly used as herbal medicine and food, which is called "the same source of medicine and food" according to the principle of traditional Chinese medicine. As one of undergrowth vegetation, A. oxyphylla is commonly planted under the trees, such as Areca catechu and Hevea brasiliensis, and the cultivated area is about66.7km2in Hainan province. Therefore, the common foods are more suitable for the exploitation and industrialization for the fruits of A. oxyphylla. We have developed two new powdered drink products (i.e., Yizhi-Yezi tea and Yizhi-Mugua tea) containing the fruits of A. oxyphylla with the financial support from the Hainan provincial special fund for the modernization of traditional Chinese medicines. These products may play important roles in the Hainan provincial tourism service trade and promote the plant industry development of A. oxyphylla. |
PDF Full Text Request