Study On The Purification Of Total Flavones From Nervilia Fordii By Macroporous Resins And Quality Standard Of Emilia Sonchifolia

Part 1 Study on the Purification of Total Flavones from Nervilia fordii by Macroporous ResinsObjective:This paper takes the total Flavones from Nervilia fordii as the research object. To optimize the extraction and purification process of total flavnoids from Nervilia fordii. and formulate the quality standard of extractive. It provides the theoretical basis for the extraction and exploitation of flavonoids in Nervilia fordii.Herba Nervilia fordii, the leaves or the whole plant of Nervilia fordii (Hance) Schltr. (Orchidaceae). It is mainly distributed in the south of China. It cools in nature, sweets 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. These are many researcher have reported the extraction technonlgy of total flavnoids. But the purification of total Flavones from Nervilia fordii by Macroporous Resins have not reported. In this experimental, the method for purification of total flavones from Nervilia fordii was established.Methods:1. The single factor tests and orthogonal design were used in optimizing the extraction process of Nervilia fordii. Orthogonal experiment was designed with the content of single flavonoids, total flavonoids and total solid as index, and with ethanol concentration, the ratio of material and solvent, extracting time and frequency as factors.2. Purification of total flavonoids from Nervilia fordii by adsorption of macro-porous resin was studied in this paper. The procedures with frozen-drying were used to produce the total flavonoids.3. Analysis of the flavonoids in the Nervilia fordii by HPLC-MS. Agilent 6406 triple quad LC-MS, Agilent SB-C18 analytical column (3.0×50 mm, D.2.7μm), mobile phase consisted of solvent A (acetonitrile) and solvent B (0.1% acetic acid) elution profile:0-1 min,20%-30% A,1-3 min,30%-60% A,3-4 min,60%-70%A; flowrate:0.4 ml/min, column temperature25℃. Ionization mode for electrospray ionization, negative ion mode and MRM mode.Results:1. The optimum extracting process for Nervilia fordii is adding 12/10 times amount of 80% alcohol into materials and refluxing for 2 times,1 hour every time.2. The species of flavonoids in samples were identified by color reactions, and the contents of total flavonoids were mesasured through UV spectroscopy using rutin as control. The rusults shows that at the 410nm it has the strong absorption. The AB-8 macroporous resin gave the best effect of separation with the sampling rate 2 BV/h, concentration 1.0 g/L, pH 4 respectively,3 BV water for removing impurities, the concentration of elution was 5 BV 70% alcohol, eluting velocity 2 BV/h. AB-8 resin could be applied to purify total flavonoids from Nervilia fordii.3. The HPLC-MS methods were established for determination of flavonoids.The content of 7 flavonoids was tested, a total of 7 batches of total flavonoids of Nervilia fordii samples were determined. The highest content in the Nervilia fordii is complanatoside. The average content of complanatoside was 51.43 mg/g. The higher content is nervilifordizin C, and it is average content is 12.35 mg/g.There are small differences between each batches, this experiment can be applied to mass production and preparation of total flavonoids of Nervilia fordii.Conclusion:In this paper.Using the method of synthesized multi index analysis. The results show that this method is more effectivem and more just and reasonable. We used the macroporous resin to study the purification process of total flaconoids in Nervilia fordii. In order to make good ues of and enhance the value of Nervilia fordii resources, the suitable methods on extraction and purification of total flavonoids were studied. Analysis of the flavonoids in the Nervilia fordii by HPLC-MS. Above research results has established good foundation for the next step.Results:1. The optimum extracting process for Nervilia fordii is adding 12/10 times amount of 80% alcohol into materials and refluxing for 2 times,1 hour every time.2. The species of flavonoids in samples were identified by color reactions, and the contents of total flavonoids were mesasured through UV spectroscopy using rutin as control. The rusults shows that at the 410nm it has the strong absorption. The AB-8 macroporous resin gave the best effect of separation with the sampling rate 2 BV/h, concentration 1.0 g/L, pH 4 respectively,3 BV water for removing impurities, the concentration of elution was 5 BV 70% alcohol, eluting velocity 2 BV/h. AB-8 resin could be applied to purify total flavonoids from Nervilia fordii.3. The HPLC-MS methods were established for determination of flavonoids.The content of 7 flavonoids was tested, a total of 7 batches of total flavonoids of Nervilia fordii samples were determined. The highest content in the Nervilia fordii is complanatoside. The average content of complanatoside was 51.43 mg/g. The higher content is nervilifordizin C, and it is average content is 12.35 mg/g.There are small differences between each batches, this experiment can be applied to mass production and preparation of total flavonoids of Nervilia fordii.Conclusion:In this paper.Using the method of synthesized multi index analysis. The results show that this method is more effectivem and more just and reasonable. We used the macroporous resin to study the purification process of total flaconoids in Nervilia fordii. In order to make good ues of and enhance the value of Nervilia fordii resources, the suitable methods on extraction and purification of total flavonoids were studied. Analysis of the flavonoids in the Nervilia fordii by HPLC-MS. Above research results has established good foundation for the next step.Part 2 Study on Quality Standard of Emilia sonchifoliaObjective:Emilia sonchifolia (Linn.) DC is a compositae’s annual obiennial herb, which distributes in southern of China such as Guangdong, Guangxi, Fujian, Guizhou, Jiangxi provinces. It is mild-natured, taste bitter and tiny pungent, and can be used for heat-clearing, detoxicating and detumescence. It is a traditional medicine with a great medicinal value. It has the activities of anti-inflammation, antioxidation, antibacterial, analgesic, enhance immunity, antiviral.Emilia sonchifolia is used widely in south of China. There is no uniform standard to evaluating qualitity of Emilia sonchifolia. It has been included in Chinese Pharmacopoeia 1977. Since then it disappeared. In recent years, it appeared in the local standards such as GuangDong, HuNan and FuJian. Today, series of preparations of the Emilia sonchifolia have been developed, but the early quality control of Emilia sonchifolia is too simple, especially lack the qualitative and quantitative analysis method. According to require ments of State Pharmacopoeia Committee to medicinal materials collected in Pharmacopoeia of PRC, the research is to compensate for the defects of the early quality standards of Emilia sonchifolia, which is out-of-date and oversimplified in techniques and unperfectin project standards. We tend to make practical quality control system of Emilia sonchifolia medicinal materials, which conforms to the modern Chinese medicine standard and technology standard, so as to regulate the development and utilization of Emilia sonchifolia.Methods:1. We had characteristic identification and microscopical identification of Emilia sonchifolia.2. Two reference substance were isolated from methanol extracts by using various chromatographic techniques and their structures were elucidated on the basis of extensive spectroscopic analysis.TLC as well as HPLC was used on this study an im-purity control method for these two reference substance.3. This paper describes a method of detecting senkirkine and quercitrin in Emilia sonchifolia by TLC and high-performance liquid chromatography.4. According to the Chinese Pharmacopoei of 2010 edition, the moisture, total ash, acid insoluble ash, water-soluble extract, ethanol soluble extraction, volatile ether soluble extract were checked.Results:1. To overall survey and distinguish on botany morphology, pharmaceutical botany character. Microscopic identification includes transverse section of blade, epidermis of blade, and power of blade.2. The two reference substances were characterized by NMR, IR UV and MS, TLC as well as HPLC was used on this study an im-purity control method for these two reference substance. The purity of product can reach 98%.3. Estblished the TLC method of senkirkine in Emilia sonchifolia. The good separation was taken by using chloroform-methanol (8:1) as the mobile phase, Estblished the TLC method of quercitrin in Emilia sonchifolia. The good separation was taken by using ethyl acetate-acetone-acetic acid-water (4:1:0.5:0.5) as the mobile phase.The chromatographic spots were clear and the Rf value was moderate. The TLC method is adaptable and reproducible, and can be used as one of the exclusive methods to the identification of senkirkine and quercitrin in Emilia sonchifolia. The content of quercitrin was detected by HPLC. The content in the Emilia sonchifolia is about 0.05～0.58 mg/g. The average content is 0.33 mg/g. According to the measured data, the limit of quercitrin content was set as not less than 0.16 mg/g. The content of senkirkine detected by HPLC, The content in the Emilia sonchifolia is about 0.15-0.81 mg/g. The average content is 0.40 mg/g. Do not exceed suggested dosage.4. According to the Chinese Pharmacopoei of 2010 edition,13 batches medicinal meterials were checked. Combined with the measured data, the limits were setas follows: the moisture, total ash and acid insoluble ash should not be over 15.0%,19% and 9%, respectively,water-soluble extract, ethanol soluble extraction and Volatile ether soluble extract should not be less than 15.0%,6% and 0.22%.Conclusion:Afer systematic studied on 13 batches medicinal materials, we formulated the limits of waters, ash, abstracts, increased the safety index of Emilia sonchifolia. In addition, we established the method of determining the content of quercitrin and senkirkine. From both qualitative and quantitative aspects, we perfected the study on qualitu standards of Emilia sonchifolia.