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OBJECTIVE：To establish a method for the determination of 8 glycosides（astragaloside Ⅰ，Ⅱ，Ⅲ，Ⅳ and calycosin glucopyranoside ，2′-hydroxy-3′，4′-dimethoxy-isoflavan-glucoside and 9，10-dimethoxy-pterocarpan-glucoside） and 4 aglycones（calycosin，formononetin，7，2′-dihydroxy-3′，4′-dimethoxy-isoflavan and 3-hydroxy-9，10-dimethoxy-pterocarpan） in Astragalus membranaceus ，and to investigate the effects of different processing temperatures on the contents of above 12 components. METHODS ：The contents of 12 components in A. membranaceus and samples processed under different temperatures（120，140，160，180，200 ℃）were determined by UPLC-MS/MS. The determination was performed on ACQUITY UPLC HSS T 3 column with mobile phase consisted of 0.1 mol/L formic acid water solution -0.1 mol/L formic acid acetonitrile solution （gradient elution ）at the flow rate of 0.5 mL/min. The column temperature was 30 ℃. The detection wavel ength was 260 nm，and sample size was 2 μL. Electrospray ion source（ESI）was used under positive ion mode （ESI+）. The mass scanning range was mass ratio （m/z）of 50-1 500，with capillary voltage of 2 000 V and ion source temperature of 100 ℃. The desolvation temperature was 400 ℃；flow rate of atomizing gas （N2） was 40 L/h，and that of desolvation was 800 L/h；collision energy （CE）was 20-30 V；data acquisition rate was 0.5 s/scan. RESULTS：The linear range of astragaloside Ⅰ，astragaloside Ⅱ，astragaloside Ⅲ，astragaloside Ⅳ，calycosin-glucopyranoside， calycosin，ononin，formononetin，2′-hydroxy-3′，4′-dimethoxy-isoflavan-glucoside，7，2′-dihydroxy-3′，4′-dimethoxy-isoflavan，9， 10-dimethoxy-pterocarpan-glucoside and 3-hydroxy-9，10-dimethoxy-pterocarpan were 0.001 16-0.232 0，0.000 276-0.055 2， 0.000 22-0.044 0，0.000 225-0.045 0，0.000 734-0.587 0，0.001 17-0.234 0，0.000 742- 0.148 0，0.001 30-0.260，0.003 98-0.795 0， 0.000 476-0.476 0，0.001 89-0.378 0，0.000 336-0.336 0 μg（all R2≥0.999 2），respectively. The limits of detection were 6.2×10－6， 4.8×10－6，3.8×10－6，3.4×10－6，5.8×10－6，4.8×10－6，4.2×10－6，3.2×10－6，5.8×10－6，2.6×10－6，4.2×10－6，6.4×10－6 μg，respectively. The limits of quantitation were 12.6×10－6，16.2×10－6，14.4×10－6，14.8×10－6，18.8×10－6，16.4×10－6，15.4×10－6，10.8×10－6，20.2×10－6， 12.4×10－6，14.6×10－6，23.4×10－6 μg，respectively. RSDs of precision ，stability（24 h）and repetition tests were all lower than 3.0％（n＝6）. The average recoveries were 99.1％，100.2％，98.7％，101.9％，98.6％，102.1％，99.2％，100.3％，98.7％， 99.2％，99.3％ and 100.8％，with the RSDs of 1.9％，2.2％，2.4％，1.8％，2.1％，1.7％，2.3％，1.9％，2.4％，1.8％，2.2％ and 1.9％（n＝6），respectively. The results showed that the contents of astragaloside Ⅰ，Ⅱ and Ⅲ decreased gradually with the increase of processing temperature ；the content of astragaloside Ⅳ increased gradually with the increase of temperature. The content of flavonoid glycosides ，such as calycosin glucopyranoside ，ononin，2′-hydroxy-3′，4′-dimethoxy-isoflavan-glucoside and 9， 10-dimethoxy-pterocarpan-glucoside decreased with the increase of temperature ；the corresponding aglycone components as flavonoid glycosides ，formononetin，3-hydroxy-9，10-dimethoxy- pterocarpan increased firstly and then decreased with the increase ； the content of 7，2′-dihydroxy-3′，4′- dimethoxy-isoflavan decreased with the increase of temperature. CONCLUSIONS ：Established UPLC-MS/MS method can be used for determination of 12 components in A. membranaceus . After processed under different temperature，the contents of glycosides decreased in general ，while the contents of aglycones increased in general.
ABSTRACT
ABSTRACTThe use of cell and plant tissue culture techniques to produce economically important active metabolites has been growing. Among these substances are total limonoid aglycones, which are produced by "pera" orange (Citrus sinensis (L.) Osbeck, Rutaceae) and have received considerable attention because of their anticancer actions. The main objective of the present study was to analyze and compare the levels of limonoid aglycones in seeds, callus cultures (originating from seeds), callus cultures (originating from hypocotyls), cell suspensions from hypocotyls cells, and cell suspensions from cotyledons. The cell cultures or C. sinensis were obtained by inoculating two strains of callus in MS medium supplemented with 2.0 µM 2,4-dichlorophenoxyacetic acid, 7.0 µM benzyl aminopurine, and 3% (w/v) sucrose in the dark. The highest concentrations of limonoid aglycone that were obtained were observed in cotyledon cell lines (240 mg/100 g dry weight) that were produced on day 21 of culture and hypocotyl cell lines on day 7 (210 mg/100 g dry weight). Explants of different origins under the same culture conditions had different limonoid aglycone content. The present results may suggest strategies for enhancing the productivity of biologically important limonoid aglycones and investigating the complex pathways of these secondary metabolites in plant tissue cultures.
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The objective of this study was to investigate the immobilisation efficiency of soybean β-glucosidase (181.6 U/mL; 23.8 mg protein/mL) on activated chitosan beads. Central Composite Rotational Design (CCDR) 23 was used and the application of immobilised enzyme in commercial soy drink was evaluated. The activation of chitosan beads was achieved with established 2.5% glutaraldehyde, pH 7.5, 8 h incubation time (6 h with agitation and 2 h without agitation) at 37ºC. The highest immobilisation efficiency (%) of soybean β-glucosidase on chitosan beads obtained was 37.74 U/mL and 18.84 mg protein/4 chitosan beads at pH 7.5 and 20 h coupling time of enzyme-matrix (7 h with agitation and 13 h without agitation) at 4ºC. The immobilised enzyme incubated at 50ºC, pH 5.5 resulted in 24% increase in the aglycones content in commercial soy drink after 60 min.
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AIM: To use qualitative and quantitative RP HPLC for the sepatation and determination of flavonoids found in the leaves of Acer truncatum bunge in spring, summer and autumn. METHODS: The determination of three kinds of flavonoid aglycones was carried out with Diamonsil TM C 18 column (4.6 mm?250 mm,5 ?m), using methanol water(1% acetic acid) as mobile phase at a flow rate of 1.0 mL?min -1 and detected at the wavelength of 360 nm. RESULTS: The average recovery of quercetin was 100.4%, RSD =1.61%( n =5). The average recovery of kaempferol was 102.5%, RSD =1.77%( n=5 ). The average recovery of isorhamnetin was 102.3% , RSD =1.63%( n=5 ) CONCLUSION: Isorhamnetin and kaempferol in the leaves are reported here for the first time. The methods were simple, rapid and reliable and could be used to control the quality of the herbal medicines prepared from the leaves of Acer Truncatum Bunge.
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AIM: To optimize the extraction of the anthraquinone aglycnes from Rhubarb using supercritical fluid extraction(SFE). METHODS: Orthogonal design and variance analysis were used to optimize five operational variables of SFE. RESULTS: The optimized operational variables of SFE were obtained as following: the temperature was 70 ?C , the pressure was 35 MPa, the static extraction time was 8 min, the dynamic extraction volume 5 mL and methanol were used as modifier. CONCLUSION: SFE is rapid, convenient and accurate, and can be used to extract athraquinone aglycones from Rhubarb.
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AIM:To determine Radix et Rhizoma Rhici in Ganning Granula(Radix et Rhizoma Rhei,Rhizoma Chuanxiong). METHODS: In order to determine five anthraquinone aglycones of Radix et Rhizoma Rhei,the HPLC system consisted of Nucleosil ODS column,methand-0.1% phosphoric acid(85∶15) mixture as mobile phase,with detection wavelength at 430 nm. RESULTS: HPLC showed that the linearity ranges were as follows: aloe-emodin concentration was at the range of 0.041 6-0.291 2 ?g,the rhein concentration was at the range of 0.037 1-0.259 8 ?g,the emodin concentration was at the range of 0.045 8-0.320 3 ?g,the chrysophanol concentration was at the range of 0.049 60.347 2 ?g,the physcion concentration was at the range of 0.020 6-0.144 5 ?g.The average recoveries of five anthraquinone aglycones were between 96.6%-98.4%.CONCLUSION: The method is simple,accurate,sensible and with good repeatability and can be used for quality control of Ganning Granule.