Studies On The Pilot-scale Preparation Technology, And The Determination Of Content Of Total Flavonoids From Bidens Bipinnata L.

Bidens bipinnata L., a species of the genus Bidens (Compositae), commonly known as“po-po-zhen” in China, was first recorded on the “Supplement to the Herbal” written by a famousphysician in the Tang Dynasty one thousand years ago. It is widely distributed in majority provincein China. Bidens bipinnata L. had been traditionally used for many years. It is bitter in taste, neutralin nature and innocuity, and has the effect of heat-clearing, detoxicating and eliminating stasis tosubdue swelling. It contains many chemical compositions, such as flavonoid glycoside, saponin,alkaloid, aetherolea. It was largely used to treat hepatitis, inflammation, malaria, hypertension,diabetes, peptic ulcer, snake bite, smallpox. Flavonoids are rich in Bidens bipinnata L., in which thecontent is ad high as5%in the leave. But so far, the total flavonoids of Bidens bipinnata L. are notfully studied. In addition, in the clinical practice, Bidens bipinnata L. and compound preparationcontaining Bidens bipinnata L. had obvious therapeutic effect on virus hepatitis, which cansignificantly degrade the ALT and AST of hepatopath with no obvious toxicity and side effects. Inorder to develop a new use of Bidens bipinnata L., a flavonoid-rich extract of Bidens bipinnata L.was prepared by our research group. But according to the traditional Chinese medicine withreference to the requirements of the five kinds of new medicine, traditional Chinese medicinecomposition of effective parts content must achieve50%above, so consider this process isoptimized, and enlarge pilot study, make its content with five kinds of traditional Chinese medicineto the requirements of the new drug declaration. The main contents were divided into some sections as follows:1．Pilot-scale study on extraction technology of total flavonoids of Bidens Bipinnata L.We applied orthogonal design to optimize the extraction technique of total flavonoids of Bidensbipinnata L. The best extraction technology was that10-fold and70%ethanol,2hour of reflux, and2times of extraction.Purified by HPD100macroporous resin, decontamination with1.5BV distilledwater then70%ethanol was used to elute the total flavonoids, the eluting volume was3BV, and20BV ethanol ultrasonic extraction2times. On this condition to adsorption and elution, the HPD100resin could be used5times repeatedly. The content of total flavonoids of Bidens Bipinnata L.wasabove50%and the average yield was1%.To establish an HPLC method for the simultaneous determination of hyperoside, isoquercetin，and bipinnatpolyacetylenicloside in Bidens Bipinnata L. and its extracts of continuous productiontechnology. The separation was carried out on an Agilent ZORBAX SB-C18(4.6mm×250mm，5μm)colum at the temperature of30℃; the mobile phase consisted of0.4%acetic acid in acetonitrile(A)and water(B) with gradient elution. The flow rate was1.0mL·min-1，the detection wavelength was setat255nm. The good seperation of three substances was achieved within30min, with the relativestandard deviations (RSD) of intra-day precision≤1.4. Calibration curves of hyperoside, isoquercetinand bipinnatpolyacetylenicloside showed good linear relationship（R2>0.9994, n=6）. The averagerecoveries of hyperoside, isoquercetin and bipinnatpolyacetyleniclosidewere within99.08%~103.52%，RSD≤3.8%. The developed method is simple，accurate，and repeatable，andcan be readily used for the quality control of Bidens Bipinnata L. and its extracts.2. Determination of content of total flavonoids from Bidens Bipinnata L.The quantitative control (QC) is the critical of the whole work. We applying ultravioletspectrophotometry to determine the content of TFB using NaNO2-Al(NO3)-NaOH chromogenicsystem. The optimized process in our study is stable, highly efficient and suitable for industrialproduction.