Study On Preparation Process And Quality Standard Of Total Flavonoids From Leaves Of Murraya Panaculata (L.) Jack

Murraya panaculata (L.) Jack , a well-known medicinal plant, is widely used for treatment of stomachic diseases, inflammation, different body pains, trauma , bleeding caused by internal and external injury etc. It was been also used as a tonic and haemostatic agent. Due to the demand for its important medicinal use in China, it was largely cultivated in Yunnan and Guangxi Provinces.The alkaloids, terpenoids and coumarins in this plant have been reported more before, but the flavonoids reported fewer. Five parts were included in this paper :the extraction process, the purification process; the establishment of quality standard of total flavonoids from leaves of Murraya panaculata (L.) Jack; the isolation, structural determinations of two refrence substances-5,7,3′,4′-tetramethoxyflavone (JA) and 5,7,3′,4′,5′- pentamethoxyflavone (JB); the effect of total flavonoids on blood glucose.1. The extraction process of total flavonoids from leaves of Murraya panaculata (L.) Jack.The solvent and extraction method were selected first, and then three important factors of orthogonal test were inspected. Finally, the optimal extraction condition of total flavonoids was determined as the leaves was refluxed in 85% alcohol (6 times ) for 3 times (60, 45 and 30 min, respectively).2. The purification process of total flavonoids from leaves of Murraya panaculata (L.) Jack.In this part, macroporus resin chromatography had been chosen as purifing method before the ability of static adsorption and desorption of four types macroporus resin (D4020, HP20, AB-8, 860021) have been tested. The result showed that macroporous resin D4020 was the optimal resin, because it possessed the strongest absorption ability and desorption property to the total flavonoids among the 4 resins studied. Some factors, including the loading concentration, loading velocity, eluant, eluting velocity and absorbing time et al. were performanced on D4020 resen, then purification process was concluded as follow: The extracted solution (concentration was 50mg/ml, pH was 6) flowed the macroporous resin chromatographic column by 5BV/h, absorbed for 5 hours, then eluted by distilled water. Finally, eluted by 50% ethanol ,collected the ethanol elution for the decoloration resin chromatographic column.Due to much pigment exists inthe leaves of Murraya panaculata (L.) Jack., so the decoloration resin was needed to decolorate above ethanol elution . Because the decoloration resin D280 possessed the strongest decoloration ability to the pigment among the 5 different resins(D280, D315, D316, D941, 330)studied, so it was selected to tested the factors, which including the effect of absorbing time , absorbing tempreture to the decoloration et al. Eventually, the decoloration process of decoloration resin D280 was decided, viz. the ethanol elution flew the decoloration resin chromatographic column at room tempreture and decolorded for 3 hours. At last, the silica gel column chromatographic column was needed to purify total flavonoids, The mass ratios of sample to silica gel quantity were teseted and the results show that 10 times silica gel can purified the total flovonoids.Through all three purification progresses mentioned above, the contents sum of compound JA and JB in total flavonoids is above 70%, and the recovery of total flavonoids is above 80%.3. The quality standard of total flavonoids from leaves of Murraya panaculata (L.) JackIn this paper, a series determinations were performed concluding moisture determination, ignited residue determination , heavy metal determination et al. The results of these determinations meet the rules of the pharmacopoeia. To determine compounds JA and JB in the total flavonoids from leaves of Murraya panaculata (L.) Jack, the RP-HPLC was used. The separation was performed on ZOBAX SB-C18 column (250mm×4.6 mm,5μm), using 35% acetonitrile and 65% water as mobile phase with the flow rate of 1.2 ml/min at 25℃, the wavelength for measurement was 337nm. The results showed that the linearity was in the range of 0.05μg～5μg(n=6) for both, and all the correlation coefficients were 0.9999. The average recoveries for compound JA and JB were 102.20%(RSD=2.46%)and 97.34%(RSD=0.82%), respectively(n=6). This method is aurate, reliable and reproducible to determine two compounds JA and JB in the total flavonoids from leaves of Murraya panaculata (L.) Jack.4. Study on extraction, isolation and identificationof the reference substances. In the experiment, two flavonoids compounds JA and JB were extracted by heat reflux from leaves of Murraya panaculata (L.) Jack and then was isolated with column chromatography. Two Compounds JA and JB were identified respectively by UV, IR, MS, 1DNMR and 2DNMR as 5,7,3′,4′-tetramethoxyflavone and 5,7,3′,4′,5′- pentamethoxyflavone. They were used for TLC and contents determination as reference substances.5. Study on reduction in boold glucose of total flavonoids from leaves of Murraya panaculata (L.) JackThe result of reduction in boold glucose show that the mechanism of total flavonoids to reduce blood glucose is owning to stimulating the pancreatic islets to release insulin, elevating the sensitivity of tissue to insulin, improving the resistance of insulin; The low incidence rate of adverse effects are the advantage of the total flavonoids, so that the total flavonoids might be developed into a new promising oral antidiabetes drugs.In conclusion, the results of this paper can offer the new preparation process and new items of content determination for quality standard establishment of total flavonoids from leaves of Murraya panaculata (L.) Jack. Also, the results can offer science foundations for the development and utilization from this medicine plant.