Study On The Quality Evaluation And Standard Of Chrysanthemum Pieces 2, Extraction And Purification Of Total Flavonoids From

Chrysanthemum is a kind of Chinese herbal medicine is commonly used in clinic, which has the characteristics for both medicine and food. The Editorial Board of Pharmacopoeia of the People’s Republic of China (2010) contains the content determination of qualitative iden-tification and index components in chrysanthemum, but there are still short comings. Now the classification of chrysanthemums being sold on the market is in disorder, which makes the source judgment of chrysanthemum difficult. This article with chrysanthemum and its decoc-tion pieces as the object of study is based on the Editorial Board of Pharmacopoeia of the People’s Republic of China (2010) and conducts a research on the following series, so as to improve the level of quality control standards of chrysanthemum. Part1. Researches on TLC identificationIn this article we innovatively to used the C18 reversed-phase thin-layer chromatography to identify the chemical components in 5 kinds of chrysanthemums and compared the identifica-tion effect with the normal phase thin-layer chromatography in the Editorial Board of Phar-macopoeia of the People’s Republic of China (2010). The results showed that the C18 re-versed-phase thin-layer chromatography reflected not only retains the original identification informations of ingredients in chrysanthemum, but also added a lot of new informations, and thses informations can reflect the difference between chrysanthemum varieties to a certain extent.On the other hand, the C18 reversed-phase thin-layer chromatography detection has the advantages of high efficiency, rapid process, simple solvent ratio and less toxic. The re-searches on the C18 reversed-phase thin-layer chromatography wil provide a new study direc-tion for qualitative identification and quality control of Chinese herbal medicine. Part 2. Researches on the fingerprints of chrysanthemumWe have used the high performance liquid chromatography method to establish the finger-prints for 5 kinds of chrysanthemums. Among all these 5 kinds of chrysanthemums,ChuJu has 23 common peaks in its common mode fingerprint, BoJu has 20 common peaks in its com-mon mode fingerprint, GongJu has 14 common peaks in its common mode fingerprint, HangJu has 22 common peaks in its common mode fingerprint, QiJu has 22 common peaks in its common mode fingerprint. Last we use the traditional Chinese medicine fingerprint simi-larity evaluation software to summary the peak informations of the 5 kinds of chrysanthe-mums in order to obtain the characteristic fingerprint that can on behalf of the common fea-tures of all the 5 kinds of chrysanthemums including ChuJu, BoJu, GongJu, HangJu, QiJu.In this characteristic fingerprint we have totally established 12 characteristic peaks.By using the high performance liquid chromatography and mass spectrometry technology,we have identi-fied the the components of the characteristic peaks in the chrysanthemum. Part 3. Researches on content determinationWe have set up the content detemination method that can test six kinds of effective compo-nents including Chlorogenic acid, Cynaroside,3,5-O-dicaffeoyl quinic acid, Luteolin, Kaempferol and Acacetin in five kinds of chrysanthemums at the same time as the establish-ing of the characteristic fingerprint of chrysanthemum. The precision, stability, repeatability, accuracy of the established determination method are all qualified, so this content detemina-tion method can be taken as the quality control method for the six kinds of effective compo-nents in five kinds of chrysanthemums on sale.Part 4. Researches on the identification method about the source of chrysanthemumsBased on fingerprint and determination of content, the ingredients of all the collected batches of chrysanthemums were analyzed by principal component analysis in the experiment, Firstly, implemented the principal component factor analysis by SPSS 16.0 to simplify the data informations and received three principal components with cumulative variance contribution rate of 95.882%. Secondly, put the three principal components into the SIMCA-P data analy-sis software to do the clustering analysis that used the varieties of chrysanthemums as classi-fication labels, The coincidence rate of the clustering result and experience identification re-sults was more than 80%. So finally, we determined the main characteristic components group for the classification of the source of chrysanthemum was cynaroside-luteolin.Based on the limiting of the harvest time, the range of ratio of (cynaroside/luteolin) could be used as the evaluation basis to determine the source of the chrysanthemum on sale.Part 5. Researches on the inherent differences between the water decoction and alcohol ex-tractsDue to the use of traditional Chinese medicine and traditional Chinese medicine production are almost extracted by water, so the compositions of extractive may has some differences from those extracted by chemical reagent. In this paper, we made a comparison about the in-gredients between the water decoction and alcohol extractive among the five kinds of chry-santhemums. The results show that:overall, the ingredients content in water decoction were lower than those in alcohol extractive. Among the 6 components mensurated in this experi-ment the content of chlorogenic acid and the 3,5-O-dicaffeoyl quinic acid were higher in wa-ter decoction except for GongJu and ChuJu. In terms of the small polar substances such as Luteolin, Kaempferol and Acacetin, their content were lower in water extractive. This conclu-sion can provide a reference for the use of clinical decoction of traditional Chinese medicine and the manufacture of Chinese patent drug even for the research of pharmacology and toxi-cology on traditional Chinese medicine.The Citrus aurantium L.var.amara Engl (bitter orange flower) as a kind of traditional Chinese medicine that can recuperate Qi wide bosom, appetizing anti-nausea is widely used in the folk due to its obvious curative effect, less toxic side effect, even though it was not included in the Pharmacopoeia of People’s Republic of China. The domestic and abroad related researches showed that the citrus flavonoids have the antioxidant, antibacterial, antiviral, anti-inflammatory and anticancer activity and can play a role for the prevention of atherosclerosis and the intervention treatment effect of the increased cholesterol. Since the Citrus aurantium L.var.amara Engl is also belong to the citrus,it contains abundant flavonoids as well. At present, further research about the flavonoids in the Citrus aurantium L.var.amara Engl is still rare, In order to make further clarify about the flavonoid components and their pharmacological activities of this flower, we firstly need to adopt appropriate methods to gather and then purify the flavonoids from the flower. The purpose of this study is to explore how to extract and purify the flavonoids from the Citrus aurantium L.var.amara Engl more efficiently and conveniently. Part 1. Researches on the extraction method of flavonoids in the bitter orange flowerOn the basis of early preliminary experiment, we comprehensive took the factors such as extraction cost and extraction efficiency into consideration and finally adopted the method of water extract and then alcohol precipitate the impurity as the extraction methods of flavonoids ingredients in bitter orange flower. Adopted the L9(34) orthogonal test table, through the orthogonal experiment, optimized the water extraction process, set the ratio of material to liquid, extraction time and extraction times as the main factors of the orthogonal test according to the production experience to determine factors appropriate under several levels. Take the content of total flavones and transfer rate as the indicators in the extraction to choose the best level of each factor in the L9(34) orthogonal test table to creat a new extraction conditions as the water extraction method, that is 16 times the amount of water, reflux extraction 3 times, each time 2.5 hours. On the basis of the water extraction method in above, we next chose the U6* (64) uniform design table to guide the optimization test of the alcohol precipitation process, and set the relative density of the water extraction before droping the alcohol and the percent concentration of ethanol after the precipitation process as the main factors then chose several right levels according to experience under each factor. Use the same indicators as those in the orthogonal experiment to deside the best level of the factor group in the uniform test.At last, in the form of visual analysis, determined the alcohol precipitating method as the relative density of water extract is 1.17 mg·mL-1, stirring and adding alcohol to make the alcohol precipitation to be 85% in the final ethanol concentration and put it aside at room temperature for 24 hours so we could get the crude extract of total flavonoids in the bitter orange flower. Part 2. Researches of the determination method of total flavonoids in bitter orange flowerMake a comparison about the determination results of the total flavonoids in the flower between the UV Spectrophotometry method and the high performance liquid chromatography method by the way the UV method were compared by using different chromogenic agents (AlCl3 and NaNO2-Al(NO3)3-NaOH).then took the test results of the HPLC method as the standard reference due to its accepted accuracy. Because of the big gap of the final results of UV method and HPLC method, in order to ensure the accuracy of the determination methods of total flavonoids, we chose the HPLC method as the best determination method for the total flavonoids in the bitter orange flower. Part 3. Researches on purification process of total flavonoids in bitter orange flowerSince the macroporous resin is a kind of polymer that is commonly used in the separation and purification of natural products.The permanent adsorption of the macroporous resin is less than other fillers so it can fully guarantee the transfer rate, at the same time, most of the price of the macroporous resin are moderate, so this experiment selected the macroporous resin as the major purification materials of flavonoids. At the beginning of the study we selected 5 kinds of macroporous resins according to different polarities and then we the adopted the static adsorption method, took the adsorption rate and adsorption-desorption rate as the evaluation conditions, the preferred macroporous resin should had larger adsorption rate and adsorption-desorption rate,on the other hand, the price should be suitable. In the screening conditions, we finally choose the AB-8 macroporous resin filler as the most suitable filler. After determining the most appropriate macroporous resin filler, we continued to determine the maximum sample volume, the water purification volume and the alcohol washing volume by dynamic manner, and on the basis of the maximum sample volume, the water purification volume and the alcohol washing volume we tried to optimize the concentration and the flow rate of sample, the concentration and the elution flow of ethanol by single factor optimization experiment. Chose the best level of each factor to combine to establish the purification process for the total flavonoids in the flower. The final technology was on the basis of the maximum loading capacity of 54.89 resins mg · g-1, total flavonoids concentration of the sample that was going to be purified was 10 mg · mL-1, the sample flow rate 4 BV · h-1, water rinse volume was 15 BV, alcohol elution volume was 5 BV, used two ethanol concentrations including the the 20% and the 40% ethanol solution gradient elution at a flow rate of 6 BV·h-1. Collected the eluent of the latter part of 20% gradient parts and all the 40% gradient parts and dry them, so we could get the refined extract whose content of total flavonoids was at the percent content of 60%.