| Background:Herba Nervilia Fordii, the leaves or the whole plant of Nervilia fordii (Hance) Schltr.(Orchidaceae), which is cool in nature, sweet in flavor, is a famous traditional Chinese medicine of removing heat and toxic material, relieving cough and asthma, removing blood statis and stopping pain.It is mainly used for the treatment of pulmonary diseases, especially in treating Children pneumonia. As the folk medicine in the south of China, it has been widely recognized by clinical physicians.It is mainly distributed in Southern China such as Guangdong, Guangxi, Hainan, and it can also be found in Sichuan, Yunnan province and Thailand. Because of its limited distributions and resources, low reproduction coefficient as well as unauthorized mining, resources of wild Nervilia fordii (Hance) Schltr. gradually exhausted.Using plant tissue technology to develop its artificial resource has become the hot spot.There are three commercial species of it as Big-leaf, Middle-leaf and Small-leaf according to the leaf size.In some places, Nervilia plicata (Andr.) Schltr. was used as the substitute.Due to its expensive price, some other adultrants such as Plantago depreessa Willd., Centella asiatica(L) Urb.are usually used to replace it, which makes the application confused.Objective:According to the chemical composition and the effect relationship of Traditional Chinese medicine, Flavonoids have the role of removing heat and toxic material, relieving cough and asthma,which matches Herba Nervilia Fordii’s clinical efficacy.With the completed subject "Nervilia Fordii on the pharmacological effect of the systemic inflammatory response syndrome-Mechaiam of rats with acute lung injury inflammation National (The Natural Science Foundation funded project)" has showed that Flavonoids are material basis of anti-inflammatory, enhancing the immunity, fever, cough and asthma.In order to promote the comprehensive Utilization of its rational development, this thesis is focused on the Flavonoids in Nervilia Fordii, including four aspects:the separation of Flavonoids, HPLC fingerprint, quantitative determination of two flavonoids and extraction technology research.Methods:1.Flavonoids were enriched by macroporous resin column, polyamide resin and so on. After the concentration under the vacuum, the bioactive compounds was determined by the HPLC(with PDA detector).2. According to the results of the pharmacological experiment of previous study of this subject, the bioactive Flavonoid-chemicals were isolated by the technique of Preparative High Performance Liquid and the chemical structure were identified by the data of MS, UV and NMRC.3.Using the Separated flavonoid as the reference, this study established the HPLC fingerprint chromatographic conditions. Chromatographic fingerprints of26batches of Herba Nervilia Fordii were analysed and obtaineded the medicinal pattern.4. A HPLC method for the simultaneous determination of two characteristic Flavonoid ingredients was established. The contents of different batches of Herba Nervilia Fordii were compared preliminarily.5. A UV method for the total Flavonoids was developed, and the process of extracting Total flavonoids from Nervilia fordii by central composite design and response surface methodology was optimized.The contents of flavonoids from different sources were compared as followed.Results:1.Total Flavonoids (about1.14%of the total amount of Nervilia fordii) were enriched by macroporous resin column, polyamide resin and so on. After the concentration under the vacuum, the bioactive compounds was determined by the HPLC(with PDA detector) and UV absorption.The results shows that at the266nm it has the strong absorption.2.Several compounds of flavone were isolated from the total Flavonoids by high liquid preparation HPLC chromatogram.Using the Separated flavone-complanatuoside as the reference, this study established a HPLC fingerprinting chromatographic conditions.26batches of Herba Nervilia Fordii were analysed and obtaineded the HPLC fingerprint pattern, which was calculated by a software named "Similarity evaluation system for chromatographic fingerprint of TCM".Most samples showed high good similarity except for seven, from which we can easily distinguish the Nervilia fordii.3.A HPLC assay was developed for determination of complanatuoside and rhamnocitrin in the Herba Nervilia Fordii.After the methodology validation, Both of them showed good linearity, while the recovery was98.43%and98.10%respectively.This method was successfully applied for determination of Herba Nervilia Fordii. The results suggested that the total amount of two compounds of flavone ranged from0.9582～15.5032mg·g-1and0.3134～7.2406mg.g-1. Two compounds of different origins, varieties and harvest period exist difference.The content of two compounds from Guangxi is higher than other regions, different harvest seasons in September by content is the highest.This may result from environment, ecological environment, Storage time and so on. On the basis of test results, the contents of complanatuoside and rhamnocitrin should not be less than2.0mg·g-1、0.5mg·g-1separately.4.Using the Separated flavonoid--complanatuoside as the reference, a UV method for the total flavonoids was developed. After the method validation, it shows good linearity, while the recovery was95.51%. This method was also successfully applied for determination of total flavonoids. The results suggested that the total amount of flavonoids ranged from18.88～47.44mg·g-1, which indicated the contents of total flavonoids from different origin, varieties and harvest period were different.On the basis of test results, the contents of the above markers should not be less than20.0mg·g-1.The process of extracting Total flavonoids from Nervilia fordii was optimized by central composite design and response surface methodology. Extraction time is tentatively set for two times and five levels experiment was designed using three facors-extraction time, extraction solvent concentration and solvent multiples.At last, the optimized conditions of extraction process were65%ethanol,75minutes for reflux,30fold solvent and two times for extraction.Conclusion:1.In this paper, the chemical constituents of flavonoids-compounds in Nervilia fordii were separated systematacially by the advanced method of high liquid preparation HPLC chromatogram, which enriched the study of chemical constituents and settled the problem that the lack of chemical reference substance which limited its further development.Taking fingerprint and double index component quantitative system, at the same time emboding the flavonoids’general characteristics and individual characteristics, achieving "Spectral efficiency", which can better control the quality of medicinal materials. At the same time, the extraction process of flavonoids was designed to optimize by central composite design and response surface methodology.It can provide the quality standard and reference for the subsequent research and development.2.This research embodies the scientific research idea that "Choosing the Chinese medicine with definite effect as the research object, obtaining pharmacological effects through in body animal testing model, basing on the therapeutic effects of tracking the effective components monomer separation, using advanced and reliable analytical method to monitor quality".Finally establishing a scientific evaluation method for the effective components, which provids a scientific basis for new drugs’development. |
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