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OBJECTIVE：To estab lish the fingerprint ，analyze the monosaccharide composition and content ，investigate the inhibitory effects of the polysaccharide from Desmodium styracifolium on α-glucosidase in vitro . METHODS ：Polysaccharide from D. styracifolium was prepared by water extraction and ethanol precipitation. After hydrolyzed by TFA and derived by PMP ，HPLC method was adopted to establish the fingerprint （using glucose peak as reference ），and analyze the constituent and content of monosaccharide. The content determination was performed on Phenomenex Luna C 18 column with mobile phase consisted of acetonitrile-0.05 mol/L potassium phosphate （pH adjusted to 6.8 with sodium hydroxide ）in gradient elution at the flow rate of 0.8 mL/min. The detection wavelength was set at 250 nm，and column temperature was set at 30 ℃. The sample size was 10 μL. Using acarbose as control ，PNPG assay was used to investigate the α-glucosidase inhibitory activity of polysaccharide from D. styracifolium. RESULTS ：There were 9 common peaks in HPLC fingerprints of 18 batches of samples ，and the similarity of 15 batches of samples was higher than 0.90. Totally 7 peaks were identified as mannose ，rhamnose，galacturonic acid ，glucose， galactose，xylose and arabinose. The contents of rhamnose ，galacturonic acid ，glucose，galactose and arabinose were 0.471-2.092， 1.379-8.919，2.560-35.679，1.194-6.905，0.566-4.158 mg/g，respectively. Based on rhamnose ，the molar ratios of the other four monosaccharides were 1.58-4.07，2.26-19.95，2.20-4.21 and 1.31-2.86，respectively. The inhibitory activity of polysaccharide from D. styracifolium on α-glucosidase increased with the increase of dose ，and the half inhibitory concentrations of it was 0.70 mg/mL， lower than 7.76 mg/mL of acarbose （positive control ）. CONCLUSIONS ：Glucose is the main component of D. styracifolium polysaccharide in different batches ，and the contents of monosaccharides are different. The polysaccharide from D. styracifolium have significant inhibitory activity on α-glucosidase，which is better than that of acarbose.
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To screen active components of Desmodium styracifolium in protecting calcium oxalate monohydrate (COM) -induced human proximaltubular epithelial cell (HK-2) damage model, and furtherly explore its mechanism of action, total flavonoids of Desmodium styracifolium (TFDS) and eight flavonoids (schaftoside, isoschaftoside, vicenin-2, isovitexin, isoorientin, apigenin, luteolin and genistein) were tested by COM-induced HK-2 damage model. MTT assay was used to detect the effects of different components on the cell viability of COM-induced HK-2 damage model. The lactate dehydrogenase (LDH) release in the cell supernatant and the activity level of superoxide dismutase (SOD) and reactive oxygen species (ROS) of cell were detected by the kit. Western blot was used to detect the expression levels of NLRP3, caspase-1, HMGB1 in HK-2 of different groups. Compared with the model group, the cell activity was significantly increased after 24 h co-culture with TFDS and four flavonoids (isoorientin, apigenin, genistein and luteolin). These active components can reduce the LDH leakage and ROS in cell supernatant and increase the activity of SOD, with regulating the expression of NLRP3, caspase-1, HMGB1. TFDS, apigenin, isoorientin, luteolin and genistein can protect COM-induced HK-2 cell damage, including enhancing cell viability, protecting cell membrane integrity and enhancing oxidative stress, and regulate the expression of proteins related to NLRP3 inflammasome.
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Objective To screen out the key chemical constituents and target protein of essential oil of Desmodium styracifolium for its anti-inflammatory effect. Methods Steam distillation method was used to extract the volatile oils from D. styraci folium, and its chemical constituents were identified by GC-MS, and the relative content of chemical constituents was determined by peak area normalization. The small molecule ligand library was established based on Traditional Chinese Medicine Systems Pharmacology (TCMSP). Reverse target prediction was conducted online using Swiss Target Prediction, the anti-inflammatory pathways were screened by KOBAX 3.0, conducting energy match between the key small molecular and the target protein in the TRP channels by molecular docking (SYBYL2.1). Construction of chemical constituents-targets network model was based on Cytoscape 3.5.1. Results A total of 48 chromatographic peaks were detected from D. styracifolium volatile oils, and 33 kinds of compound structure were determined by searching in mass spectral database and document retrieval, which account for 90.1% of total volatile oils. There were 17 key chemical constituents, and 88 target proteins were selected. TRP channels included 11 potential targets. Through molecular docking, we found that the phytol, hentriacontane, farnesyl acetone, and squalene were the key anti-inflammatory chemical constituents of D. styraci folium volatile oils. TPRV1 (transient receptor potential cation channel, subfamily V, member 1), PRKCB (protein kinase C, beta), and PRKCD (protein kinase C, delta) (degree > 10) are the key anti-inflammatory target protein. Conclusion We preliminarily select the key anti-inflammatory target and active constituents of D. styraci folium volatile oils from this study, and this research provides the theoretical basis for the development and application of its products.
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HPLC analysis was performed on a Phenomenex PS C₁₈（4.6 mm×250 mm, 5 μm）column using methanol -0.2% formic acid (30:70) at a flow rate of 0.8 mL·min⁻¹. The column temperature was 30 °C and the detection wavelength was set at 335 nm. The injection volume was 10 μL. The HPLC fingerprint of Desmodium styracifolium was established with 10 common peaks, and 5 of them were identified as vicenin-1, schaftoside, isoorientin, isoschaftoside and isovitexin, respecivetly. The fingerprints of 21 batches of D. styracifolium samples were analyzed with similarity evaluation, cluster analysis, principal component analysis and partial least squares discriminant analysis. There was no significant difference among the quantitative results of these five ingredients verified by external standard method (ESM) and quantitative analysis of multi-components by single marker (QAMS) method. The application of fingerprint, pattern recognition combined with QAMS can provide more comprehensive references for the quality control and evaluation of D. styracifolium.
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The research on distribution and quality suitability division of Desmodium styracifolium were formulated by Maxent and ArcGIS model based on the content of schaftoside and polysaccharide of D. styracifolium and its field research in the south and southwest areas of China (Guangdong, Guangxi, Hainan and Yunnan), and the most suitable habitats of distribution suitability and quality suitability were screened. The distribution suitability results indicated that average air temperature in April,mean temperature of coldest quarter, soil type, coldness index were found as the four dominant factors contributing to the plant distribution. The quality suitability results indicated that: ①Polysaccharide content and precipitation in April show significant positive correlation;Schaftoside content and mean temperature of April, mean temperature of coldest quarter show significant negative correlation. Schaftoside content shows significant negative correlation with the precipitation in October and November and the sunshine duration in April and May, while there is a significant positive correlation between schaftoside content and precipitation in April and temperature seasonality standard deviation, and a highly significant positive correlation was found between schaftoside content and precipitation in February and March. ②The quality zoning map was drawn depend on general content of polysaccharide and schaftoside as the index of quality. And this research provides scientific location basis for the production regionalization, cultivation bases selection and directive breeding of D. styracifolium.
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Objective: The differences in the compounds between Lysimachia christinae and Desmodium styracifolium were compared in order to provide the references for pharmacodynamic differences of them. Methods: The RP-HPLC-UV and RP-HPLC-ELSD fingerprints of L. christinae and D. styracifolium were established, and the common peaks of two kinds of fingerprints were compared. Results: The HPLC-UV fingerprints of L. christinae were obviously different from the HPLC-ELSD fingerprints, and the strong peaks detected in UV were not the same as what detected in ELSD, so were those of D. styracifolium. There were 14 and 5 common peaks, respectively in the HPLC-UV and HPLC-ELSD fingerprints of L. christinae, while 28 and 18 common peaks, respectively in the HPLC-UV and HPLC-ELSD fingerprints of D. styracifolium. The similarities of chromatograms of D. styracifolium were better than those of L. christinae. Four common peaks were identified in the HPLC-UV fingerprints of L. christinae and D. styracifolium, and two common peaks in their HPLC-ELSD fingerprints. Conclusion: It is better using HPLC-ESLD to establish the fingerprints of L. christinae and D. styracifolium. The chemical constituents of L. christinae were significantly different between the different batches. However, little variation was found in the chromatograms between the different batches of D. styracifolium. The two plants may contain two same compounds with high content, which may have the function as the substantial bases of their treatments for the same indication. However, they may have their special efficacies.
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Objective: To study the chemical constituents from the seeds of Desmodium styracifolium and their activity of scavenging DPPH free radicals. Methods: The compounds were isolated and purified by various column chromatographyic techniques (silica gel, polyamide, sephadex LH-20, etc) and their structures were elucidated by MS and NMR spectral analyses. The activities of scavenging DPPH free radicals from different extracts from the seeds of D. styracifolium were compared. Results: Ten compounds were isolated and identified from the seeds of D. styracifolium, including stigmasterol (1), dibutyl phthalate (2), (+)-catechin (3), benzoic acid (4), 3-hydroxy-2-methyl-4-pyrone (5), 3-O-acetyl (-)-epicatechin (6), afzelin (7), 3'-O-methyl epicatechin (8), kaempferol-7-O-β-D-glucopyranoside (9), and quercetin-3-O-β-D-glucopyranoside (10). Conclusion: Compounds 4-6 and 8 are isolated from the plants of Desmodium Desv. for the first time. Compounds 2, 3, 7, and 9 are isolated from D. styracifolium for the first time. The different extracts from the seeds of D. styracifolium exhibit the certain activity of scavenging DPPH free radicals.
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Objective To study the influence of climate, geographical environment and harvesting time on contents of schaftoside in Desmodium styracifolium Herba. Methods Content of schaftoside in Desmodium styracifolium Herba was determined by using HPLC. ODS-C18 column (4. 6 mmí250 mm,5 μm) was used, the mobile phase was methanol-water (3268), the detection wavelength was 272 nm, the flow rate was 0. 5 mL·min-1, and the column temperature was 35℃. Results The climate type, amount of precipitation, average temperature, duration of sunshine, geographical environment of different province had significant impacts on the content of schaftoside in Desmodium styracifolium Herba. Conclusion Subtropical monsoon climate, temperature from 25 ℃ to 32 ℃, sunshine time of 10 h per day and the average annual rainfall of 1 942 mm are suitable for growth of Desmodium styracifolium. The content of schaftoside in samples cultivated from Guangxi Province is higher than that cultivated from Guangdong Province and Hainan Province. The schaftoside content of sample cultivated in July is higher than that cultivated in other months.
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Objective: To optimize the formulations of microporosity osmotic pump controlled release tablet of solid dispersion for total flavonoids from Desmodium styracifolium (TFDS) by central composite design-response surface methodology. Methods: The independent variables comprised of the amount of lactose, pore-forming agent, and coating weight gain, and the dependent variables involved the cumulative release after 2, 6, and 12 h, and the linear correlation coefficient of cumulative release curve. Design-expert software was used to fit multivariate linear models and quadratic multinomial models for experimental data. Response surface was delineated according to best-fit mathematic models and the optimum formulation was selected by Numerical Optimization. Results: The correlation coefficients of quadratic multinomial models were better than those of multivariate linear models. There was close agreement between the observed and predicted values for the cumulative release, and bias were all less than 5%. Conclusion: The model established by Design-expert Software is better to predict and could be used to optimize the formulations of microporosity osmotic pump controlled release tablets of solid dispersion for TFDS.
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OBJECTIVE:To establish RP-HPLC fingerprints of Desmodium styracifolium and its preparations in order to provide basis for the quality evaluation of them.METHODS:The separation was performed on ODS-C18(250 mm?4.6 mm,5 ?m) column with mobile phase consisted of methanol-20% phosphoric acid(gradient elution).The detection wavelength was set at 360 nm and flow rate was 1.0 mL?min-1.Column temperature was set at 25 ℃.The RP-HPLC fingerprint of D.styracifolium and its preparations were recorded.RESULTS:There were 13 common peak in RP-HPLC fingerprint of D.styracifolium and its preparations.CONCLUSION:The method is accurate,stable and reproducible for basis of quality evaluation and RP-HPLC finger print of D.styracifolium and its preparations.
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AIM:To prepare a compound as the chemical reference substance of Guangjinqiancao Zonghuangtong Capsule.METHODS:To apply general column chromatography combined with preparative HPLC to isolate the target compound,to use analytic HPLC to determine the purity,stability and its content in the capsule,and to employ spectroscopic analysis (UV,IR,ESI-MS,1H-NMR,13C-NMR,DEPT,1H-13CCOSY,1H-1HCOSY,1DHOHAHA.1D.NOE,HMBC) to elucidate the structure of the isolated compound.RESULTS:The obtained compound was identified as isoschaftoside with the purity of over 99%, which was stable within 3 months at ambient temperature.As for isosehaftoside solution.it was stable within 8 h at ambient temperature.Its content in the capsule was above 3.0%.CONCLUSION:Isoschaftoside is a qualified reference substance for analytic assay ofGuangjinqiancao Zonghuangtong Capsule,and can be isolated from Desmodium styracifolium(Osb.)Merr.
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Objective To observe the influence of sowing time and planting density on seed germination percentage and growth of Desmodium styracifolium(Osb.) Merr..Methods After quality inspection of the seed,the seeds planting test for Desmodium styracifolium(Osb.) Merr.was carried out from March to October,and the influences of sowing time and planting density on the seed germination percentage and growth were observed.Results On the planting base of Pingyuan county in Guangdong province,the seeds of Desmodium styracifolium(Osb.) Merr.planted from March to October had a high germination percentage and the seedling grew well.With the increase of planting density,the seed germination percentage decreased,but was up to 80% when the planting density was 0.5～1.0 g /m2.Conclusion The optimum planting time for Desmodium styracifolium(Osb.) Merr.is in autumn and the optimum planting density is 0.5～1.0 g /m2.
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To compare the quality of cultivated species with w ild species of Desmodium styracifolium (Osbeck) Merr. TLC and UV were used to study their qualities. Results showed that there was no obvious di fference between them.
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AIM: The fingerprint chromatograms were established for quality evaluation of Desmodium styracifolium collected from 11 areas by HPLC. METHODS: The analysis was performed on a Phenomsil C_(18) column((4.6 mm)?250 mm,5 ?m) with acetonitrile-carbinol-water as mobile phase in a gradient mode at a flow rate of(1.0) mL/min,and at a column temperature of 25℃.The detection of wavelength was at 272 nm. RESULTS: 11 peaks were selected as the common fingerprint peaks.The relative standard deviations for relative retention values and relative peak areas were less than 3% in the precision and repeated test,the similarity of 11 batches of samples were no less than 0.9. CONCLUSION: The method is reliable and can be helpful on the quality control of Desmodium styracifolium.
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AIM: To develop an RP-HPLC method for preparing the reference substanc of vicenin-2 in Desmodium styracifolium. METHODS: Ethanol-extract of desmodium styracifolium was isolated and purified by RP-HPLC combining solvent extraction with column chromatography and recrystalliztion.The purity and content of vicenin-2 were identified by HPLC. RESULTS: The flvonoids were completely separated under this chromatographic condition.The purity of the reference substance was 99.0% or above. CONCLUSION: The method is simple,accurate,better on repeatability,and effective to yield high-purity product.It can be used as reference substance for the research of herbal medicine.