Extraction, Separation, Identification And Antioxidant Activities Of Flavonoid And Polysaccharide From Cinnamomum Camphora Leaves

Cinnamomum camphora is an important plant that has been used to produce natural flavor and aromatic oils in China for a long time. In recent years, it was reported that purebred Cinnamomum camphora could be successfully cultivated by tissue culture, which significantly enhanced the value of Cinnamomum camphora. However, considerable components of pharmacological merit in Cinnamomum camphora, e.g. flavonoid, polysaccharide, received little attention. To utilize Cinnamomum camphora trees more reasonably and economically, the techniques for extracting pharmacological components from Cinnamomum camphora trees should be improved and exploited.A large variation of total flavonoid contents and FRAP values in different parts of Cinnamomum camphora trees were observed. The Cinnamomum camphora leaves showed to be of the highest total flavonoid content and FRAP value. A positive linear correlation between FRAP values and total flavonoid contents were established. After the acid-catalyzed hydrolysis of the leaves quercetin(0.39%), kaempferol(0.14%) and isorhmnetin(0.063%) were detected and founded to exist mainly in the form of flavonoid glucosides.By a microwave-assisted extraction technology flavonoid was extracted from the residue of Cinnamomum camphora leaves after essential oil extraction. Macroporous resin adsorption was used for further purification. With single factor and orthogonal designed experiments the optimal extraction conditions were determined as follows: 60% ethanol aqueous solution, ratio of solid to liquid l:12(g/mL), microwave power 320 W and treated for 2 min,. Under such conditions, the yield of the flavonoid reached 2.97%, which was 6.82% higher than that obtained using heat ethanol reflux extraction, and the extraction operation duration of the technology was significantly reduced to 1% of that for the control process. Ten kinds of macroporous resin were then examined for further purification of Cinnamomum camphora flavonoid. DA201 resin was showed to possess the best absorption and desorption behavior. The optimum adsorption and desorption conditions were sample solution flavonoid concentration 13.20 mg/mL, diameter to heighth 1:10, absorption rate 3BV/h; The optimum desorption conditions were volume of 50% ethanol 150 mL (approximately 5 BV) as eluting solvent, eluting rate 3BV/h. The content of flavonoid could be improved to 91.18% from 38.15% after an absorption-desorption operation applied upon a DA201 resin packed column.By HPLC, rutin(123.33 mg/g), quercetin(70.00 mg/g), kaempferol(28.63 mg/g), isorhmnetin(20.81 mg/g) were identfied and determined in the purified flavonoid obtained above. By HPLC-DAD-ESI-MS, six flavonoid glycosides, rutin, quercetin-3-O-β-D-galacoside, kaempferol-3-O-βD-rutinoside, isorhmnetin-3-O-β-D-rutinoside, quercetin-3-O-rhamnoside and kaempferol-3-O-β-D-rhamnoside were primarily identified in the purified flavonoid. Rutin and kaempferol-3-O-β-D-rutinoside were separated from the purified flavonoid by sephadex LH-20 column chromatography.The crude water-soluble polysaccharide (CLPS) was obtained from Cinnamomum camphora leaves by boiled water extraction and ethanol precipitation. DEAE52-cellulose column and Sephacryl-S400 gel filtration column were used to further separate an acid polysaccharide (CLPS-A2) from the crude polysaccharide. The molecular weight of CLPS-A2 was determined to be 244 kDa. UV-VIS spectrum and FTIR analysis showed that CLPS-A2 wasβ-heterosaccharides with pyran and acetyl amino group. Analysis of the hydrolysis products of CLPS-A2 showed that CLPS-A2 was a heterosaccharide composed of rhamnose, arabinose, xylose, glucose and galactose.The antioxidant activities of various extracts from Cinnamomum camphora leaves were evaluted by FRAP assay and DPPH assay in vitro. The result showed that the antioxidant activities of various extracts ranged as the following order: total flavonoid-aglycone>purified flaovnoid>polysaccharide. It was inferred that the potent antioxidant activities of Cinnamomum camphora leaves were mainly attributed to flavonol glycosides.