| Herba Desmodii Styracifolii, originating from the dried Desmodium styracifolium (Osb.) Merr. (Leguminosae) plants, is one of the famous herbal medicines for the treatment of urinary calculi. It has been reported that Herba Desmodii Styracifolii are rich in flavonoids which may contribute to the various pharmacological effects, such as anti-inflammatory, antibiosis, antipyretic and diuretic effects.Flavonoids, a kind of important constituent of the plants, played an important role on human health owing to its anti-inflammatory, anticarcinogenic and antioxidant effects.In the present study, six MARs were used to select the optimum resin by comparing their adsorption/desorption capacities and desorption ratios for total flavonoids. The adsorption mechanism was elaborated by analysing the adsorption isotherms models with Langmuir and Freundlich equations, and the purification parameters were optimized by static and dynamic adsorption and desorption tests. Furthermore, the antioxidant activities were evaluated by DPPH radical-scavenging activity and reducing power tests.A high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) method was developed for qualitative analyses of the chemical constituents in the total flavonoids extract from Herba Desmodii Styracifolii (TFEHDS). First,21 standards were studied, and mass spectrometry fragmentation patterns were found. Then, the TFEHDS was analyzed by the combination of multiple-reaction monitoring-information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) mode and precursor scan information-dependent acquisition-enhanced product ion (PREC-IDA-EPI) mode on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 53 constituents were identified by comparing their retention times and mass measurement with references or literature data. Among them,25 compounds were further quantified by HPLC-ESI-MS/MS method.Part one Process optimization for purification of total flavonoids from Herba Desmodii Styracifolii extracs and evaluation of antioxidant activities in vitroObjective:The aim of this research was to investigate the adsorption and desorption behaviours of total flavonoids on macroporous adsorption resins. Furthermore, the antioxidant activities of TFEHDS were evaluated in vitro.Methods:In the present study, six MARs were used to select the optimal resin for the separation and purification of the total flavonoids from Herba Desmodii Styracifolii extracts. The performance of the total flavonoids on MARs including HPD-100, D101, AB-8, DM130, S-8 and NKA-9 was compared according to their adsorption/desorption capacities and desorption ratio. The purification parameters (such as the concentrations of total flavonoids in sample solution, feeding volume, different proportions of ethanol-aqueous solutions and the eluent volume) were investigated by static and dynamic adsorption and desorption tests. The adsorption mechanism of the total flavonoids on DM130 resin was elaborated by Langmuir and Freundlich isotherms models. The antioxidant activities of the PTFE were further investigated in vitro through DPPH radical-scavenging activity and reducing power tests using Vc as the positive control.Results:DM130 resin was chosen for the dynamic adsorption/desorption experiments. The optimal adsorption conditions were as follows: concentration of total flavonoids in sample solution 8.09 mg/mL; feed volume 3 BV. The optimal desorption conditions were as follows:deionized water washed thoroughly, and 10% ethanol 6 BV to wash impurities, then 50% ethanol 6 BV to elute target compounds. After one run treatment with DM130 resin at the optimal conditions, the content of the total flavonoids in the product was increased from 9.48% to 54.30%, with a recovery yield of 80.59%.The DPPH radical-scavenging effect (%) of the PTFE increased from 12.90 ± 0.47% to 91.40 ± 1.37%, when its concentration increased from 0.01 to 0.18 mg/mL, and the scavenging effect (%) at the concentration of 0.09 mg/mL was 71.48 ± 0.83%, which was slightly lower than that of ascorbic acid (74.80 ± 1.07%) at the same concentration. IC50 was 0.046 mg/mL. The absorbance values of the PTFE increased from 0.34 to 1.06 with its concentrations ranging from 0.2 to 1.0 mg/mL, and the absorbance value was slightly lower than ascorbic acid at the same concentration.Conclusion:This adsorption-desorption method is useful in the separation and purification of total flavonoids from Herba Desmodii Styracifolii extracts. The present study also showed that the PTFE exhibited significant antioxidant activities.Part Two Qualitative analysis of the chemical constituents in the total flavonoids extract from Herba Desmodii Styracifolii by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometryObjective:To develop a sensitive and effective liquid chromatography-mass spectrometry (LC/MS) method for identification of chemical constituents in the total flavonoids extract from Herba Desmodii Styracifolii (TFEHDS).Methods:An aliquot (10 mg) of the TFEHDS was accurately weighed into a 10 mL volumetric flask and ultrasonically dissolved with 50% ethanol aqueous, cooled at room temperature, and finally made up to its maximum volume with 50% ethanol aqueous, and filtered through a 0.45μm millipore filter before analysis. The analytes were separated on a ZORBAX SB-C18 (250 mm ×4.6 mm,5.0μm) column at room temperature. The mobile phase consisted of methanol (A) and 0.1‰ acetic acid aqueous solution (B). The following gradient condition was used:0～42 min,25～38% A; 42～70 min, 38～60%A; 70～75 min,60～95% A; 75～80 min, maintained 95% A. The flow rate was 1.0 mL/min and the injection volume was 20μL. The ion spray voltage was set to 5500 V and -4500 V, respectively. The turbo spray temperature was maintained at 650℃. Nebulizer gas (gas1) and heater gas (gas2) was set at 60 and 65 psi, respectively. The curtain gas was kept at 25 psi and interface heater was on. Nitrogen was used in all cases. Then, TFEHDS was analyzed by the combination of two scan modes, i.e., multiple reaction monitoring-information-dependent acquisition-enhanced product ion mode (MRM-IDA-EPI) and precursor scan information-dependent acquisition-enhanced product ion mode (PREC-IDA-EPI) on a hybrid triple quadrupole-linear ion trap mass spectrometer.Results:A total of fifty-three compounds, including 4 phenolic acids,4 flavonoids,24 flavone-C-glycosides,6 flavonoid-O-glycoside,3 flavonol,4 flavonol-O-glycoside,2 flavonone,1 flavonone-O-glycoside,1 isoflavone and 4 isoflavone-O-glycosid were unambiguously or tentatively identified in the TFEHDS based on their retention times, MS data and literature information.Conclusion:A sensitive and effective HPLC-ESI-MS/MS method was established for the identification of chemical composition in the TFEHDS for the first time. This study would facilitate the quality control of TFEHDS. Part three Simultaneous quantification of 25 active constituents in the total flavonoids extract from Herba Desmodii Styracifolii by high performance liquid chromatography coupled with electrospray ionization tandem mass spectrometryObjective:To develop a rapid and accurate LC-MS/MS method for the simultaneous analysis of 25 components (4 phenolic acids,4 flavonoids,5 flavone-C-glycosides,1 flavonoid-O-glycoside,3 flavonol,3 flavonol-O-glycoside,2 flavonone,1 flavonone-O-glycoside,1 isoflavone and 1 isoflavone-O-glycosid) in TFEHDS. Furthermore, the developed method was applied to quantify the 25 active components in six batches of TFEHDS.Methods:An aliquot (25.0 mg) of the TFEHDS was accurately weighed into a 50 mL volumetric flask and ultrasonically dissolved with 15 mL 50% ethanol aqueous, cooled at room temperature, and finally made up to its maximum volume with 50% ethanol aqueous, and filtered through a 0.45μm millipore filter before analysis. The analytes were separated on a ZORBAX SB-C18 (250 mm × 4.6 mm,5.0μm) column at room temperature. The mobile phase consisted of methanol (A) and 0.1‰ acetic acid aqueous solution (B). The following gradient condition was used:0-35 min,25-42% A; 35-42 min, 42-95% A; 42-47 min, maintained 95% A. The flow rate was 1.0 mL/min and the injection volume was 10μL. The ESI source was operated in the negative ion mode using the following conditions:ion spray voltage,-4500 V; curtain gas (CUR),25 psi; nebulizer gas (gas1) and heater gas (gas2),60 and 65 psi; the turbo spray temperature,650℃. The precursor-to-product ion pairs of 25 analytes were 152.9/108.9 for protocatechuic acid,179.0/134.9 for caffeic acid, 193.0/133.9 for ferulic acid,353.1/191.0 for chlorogenic acid,255.0/118.9 for liquiritigenin,269.0/116.9.0 for apigenin,269.1/133.0 for genistein, 431.0/268.0 for genistin,271.1/150.9 for naringenin,283.0/268.0 for acacetin, 284.9/92.8 for kaempferol,285.0/132.9 for luteolin,299.0/284.0 for diosmetin, 607.1/299.0 for diosmin,300.9/150.9 for quercetin,315.0/150.9 for isorhamnetin,463.2/300.1 for isoquercitrin and hyperin,609.0/299.9 for rutin, 609.3/301.1 for hesperidin.431.2/311.1 for vitexin and isovitexin,563.2/353.0 for schaftoside and isoschaftoside and 447.2/357.1 for homoorientin.Results:The calibration curves exhibited good linearity with the correlation coefficients (r) of greater than 0.9977 for all the compounds in the concentration range. Relative standard deviation (RSD) was used to evaluate the precision of the developed method. For the instrument precision, the RSD of the investigated compounds was less than 2.3%, and for the intra-and inter-day variations, the RSD values of the investigated compounds were less than 2.8% and 4.4%, respectively. The recoveries (n=9) of these components were between 98.1% and 104.4% with RSD values less than 4.6%. All analytes exhibited good stability, with an RSD for the peak areas of less than 4.0%.The results indicated that the method was accurate and reproducible. The results indicated that the method was accurate and reproducible. Furthermore, the developed method was successfully applied to quantify the 25 active components in six batches of TFEHDS.Conclusion:The LC-MS/MS method was sensitive, selective and suitable for the simultaneous analysis of 25 constituents in TFEHDS. Thus, this method could be used for the reliable quality control of TFEHDS. |
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