Application Of Microwave-assisted Multiphase Extraction Combined With Polynary Multidimensional Fingerprint In Quality Control Of Cassia Obtusifolia L.

Cassia obtusifolia L. or Cassia tora L. as one of homology materials of medicine and food by the Minsitry of Health in the first batch, have the effect of benefiting kidney, improving eyesight, relaxing bowels, cooling liver and clearing damp, etc. In China, it has been commonly used in the treatment of severe photophobia tears, headache, diziness, dim vision and other symptoms. Cassia obtusifolia L. mainly contains anthraquinones, flavonoids, naphthalene pyrones, plolysaccharides, fatty acids, amino acids and other constituents, which were often extracted by methods such as reflux extraction, cold-maceration, etc. However, these traditional methods are time-consuming, inefficient, reagent-consuming as well as harmful to the environment. Recently, microwave-assisted extraction(MAE) is a rapid, efficient, and environmental- friendly technique with fewer reagents and less time due to advantages of the thermal and the non-thermal effects between microwave and media. In this study, a novel method for extraction of effective constituents from Cassia obtusifolia L. is developed using(NH4)2SO4/acetone/n-heptane system as the extracting solvent by MAE coupled with three-phase solvent extraction. Based on data of extracts in various phases obtained by chromatographic and spectral analysis, fingerprints of the chemical constituents in each phase were established. In order to comprehensively reflect the characteristic information for this kind of medicinal material, the multi-dimensional fingerprint database of Cassia obtusifolia L. has been also established according to the nature and characteristics of effective components for evaluation and control of the quality of Cassia obtusifolia L.. The main works were as follows:1、The methods of determination of effective constituents in Cassia obtusifolia L. were developed by high performance liquid chromatography(HPLC) with UV and RF detectors, gas chromatography(GC), Fourier transform infrared spectroscopy(FT-IR). Separation of the extracted for the determination of anthraquinones, flavonoids, naphthalene pyrones in Cassia obtusifolia L. were performed by HPLC with acrtonitrile-0.1% phosphoric acid as the mobile phase in gradient elution mode, detected at 278 nm. The fluorescent constituents were determined by a fluorescence detector at the excitation wavelength of 270 nm and the emission wavelength of 440 nm while polysaccharides and their hydrolyzed products were analyzed by FT-IR and the derivative HPLC with 1-phenyl-3-methyl-5-pyrazolone(PMP), respectively. Monosaccharides after hydrolysis separated on a C18 column with acetonitrilephosphate buffer(50mmol/L, 18:82) as mobile phase, and detected at 245 nm by PMP derivatization prior to HPLC. And the nonpolar constituents were analyzed by GC. The methods proposed were of wide applicability with good chromatographic resolution. Precisions, reproducibility and stability were less than 2%, meeting to requirement for qualitative and quantitative analysis.2 、 According to the chemical prosperities,(NH4)2SO4/acetone/n-heptane system was finally screened out for extraction of the effective constituents in Cassia obtusifolia L from several three-phase solvent systems. And the composition of three-phase solvent system was optimized by improved Simplex Optimization Method. Using optimized three-phase solvent system as extracting solvent, the different kinds of constituents were extracted from the herb material by microwave-assisted extraction(MAE) through optimization of MAE condition with Central Composite Design-Response Surface Method(CCD-RSM). The results showed that nonpolar consitituents, anthraquinones, flavonoids, naphthalene pyrones and polysaccarides could be respectively extracted into the top phase(n-heptane), the middle phase(acetone), and the bottom phase((NH4)2SO4 aqueous solution) owing to the difference between three phases in the extraction performance. The higher yields of anthraquinones, flavonoids, naphthalene pyrones and polysaccarides were achieved when the mass percentages of n-heptane, acetone and(NH4)2SO4 were 7.1%, 31.9% and 9.2%, respectively. the extraction time was 16.5min, the extraction temperature was 83℃, and the liquid-to-material ratio was 56:1.3、According to the above extraction conditions, 12 batches of Cassia obtusifolia L. from different areas were extracted, separated and determined by the chromatographic and spectral analysis method for fingerprint data. GC fingerprint in the n-heptane phase, fingerprints of HPLC-UV and HPLC-RF in acetone phase and fingerprints of HPLC-PMP and FT-IR for polysaccharides in aqueous phase were obtained respectively. The classification feature of 12 batches of Cassia obtusifolia L. was analyzed by Principal Component Analysis(PCA) and Cluster Analysis method. The results demonstrated that the similarities of these 12 batches of Cassia obtusifolia L. were higher than 0.9, conforming to Similarity evaluation of fingerprint for traditional Chinese medicine(TCM). HPLC-UV fingerprint is of characteristics of comprehensive information for Cassia obtusifolia L.. On the basis of the cluster analysis results, 12 batches of medicinal materials could be divided into five groups: S2(Guangxi) in Group I; S5(Guangdong) in Group II; S6(Guangdong) in Group III; S7(Guangdong), S10 and S11(Shanghai) in Group IV; S1 and S3(Guangxi), S4(Hubei), S8(Anhui), S9 and S12(Shanghai) in Group V.4、In terms of effective constituents, the five standard fingerprints from GC, HPLC-UV, HPLC-RF, HPLC-PMP and FT-IR were investigated to express the feature for Cassia obtusifolia L.. The polynary multidimensional fingerprint database was established to evaluate the quality of the herb materials. The method of the quality evaluation of Cassia obtusifolia L. was also established, and applied to identify and discriminate three commercial samples. The results of validation suggested that two commercial samples were not qualified while the other did. The quality evaluation standard for Cassia obtusifolia L. is accurate and reliable, and can be applied in the comprehensive quality control of Cassia obtusifolia L..5、To evaluate of the antioxidant activity in vitro, the removal ability of hydroxyl radicals, DPPH free radical and superoxide anion free radical of the polysaccharides in 12 batches of Cassia obtusifolia L. was determined by ultraviolet spectrophotometry. The results showed that, the polysaccharides from 12 batches of Cassia obtusifolia L. had certain antioxidant activity. Within 0.2~1.0mg/mL, as the concentration of polysaccharides increased, the antioxidant ability growed gradually, but the antioxidant activity of 12 batches of polysaccharides were obviously different, According to the antioxidant ability, they were divided into three groups: S1, S2(Guangxi) and S12(Shanghai) in Group I; S9, S10, S11(Shanghai) and S7(Guangdong) in Group II; S3(Guangxi), S4(Hubei), S5, S6(Guangdong) and S8(Anhui) in Group III. Under comprehensive analysis, the antioxidant activity of polysaccharide samples of Cassia obtusifolia L.originated from Guangdong was higher than that from Shanghai and Guangxi.