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Objective: To establish the fingerprint of Althaeae Roseae Flos by HPLC and the molecular identification method of DNA barcode of rbcL sequence. Methods: The fingerprint establishment of Althaeae Roseae Flos was performed on Welchrom Column C18 (300 mm × 4.6 mm, 5 μm) with acetonitrile - 0.1% formic acid solution as mobile phase for gradient elution, with flow rate of 1.0 mL/min, column temperature of 35 ℃, detection wavelength of 365 nm, injection volume of 10 μL. DNA barcode molecular identification method was used for PCR amplification and determination of rbcL sequence. Results: The fingerprints of 11 samples were established, 21 common peaks were obtained, their similarities were calculated, and four components (hyperoside, quercetin, apigenin and kaempferide) were determined. The total length of rbcL sequence of 11 samples was measured, and the G + C content was 44.10%-44.40% and genetic distance (K2P) was 0.001 4. There were 10 ectopic points and the similarity was 99.00%. Conclusion: The two methods are stable and reliable, which can provide basis for the identification and quality control of A. rosea.
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Objective: Based on the Chinese herbal property, the pharmacodynamic evaluation and mechanism discussion of active components were carried out through screening the active components of Chinese patent medicine in the clinical medical insurance catalog. Methods: The most frequently used main herbs were screened through the collection of anti-vitiligo Chinese patent medicine prescriptions, drug properties and material basis. The main compound types were acquired through TCMSP and TCMIP databases. The drug properties were analyzed by admetSAR method to obtain key compounds. The pharmacodynamics were observed by measuring the morphology and melanin content of adult zebrafish and larvae. The safety evaluation was indicated by the survival rate of larvae. RT-PCR was used to reveal the mechanism of the compounds at the transcriptional level. The binding ability of compounds to protein crystal structure was predicted by molecular docking. Results: The most frequently used main herbs were Carthamus tinctorius, Lithospermum erythrorhizon, Tribulus terrestris, Gentianae Radix et Rhizoma., Psoraleae Fructus, and Vernonia anthelmintica. The main compound types through TCMSP and TCMIP database were flavonoids with a total of 81. Based on the druggability and stability, the methoxyflavones kaempferide and isorhamnetin were screened out. Kaempferide (32 μmol/L), isorhamnetin (32 μmol/L) and methoxsalen (25 μmol/L) could promote the regeneration of melanin in zebrafish. Based on the zebrafish embryo model, kaempferide, isorhamnetin and methoxsalen all could accelerate melanogenesis in larvas, and the survival rates of larvas were more than 90% under effective concentration. RT-PCR showed that kaempferide and isorhamnetin upregulated the mRNA levels of MC1R and MITF genes related to melanogenesis. The results of molecular docking between the structures of proteins (MITF, TYR, TYRP1) and kaempferide, isorhamnetin, methoxsalen showed that the binding score of kaempferide or isorhamnetin was higher than that of methoxsalen. Conclusion: Kaempferide and isorhamnetin, the active ingredients in the clinical anti-vitiligo traditional Chinese medicine prescriptions, can promote the melanogenesis in zebrafish by up-regulating the MC1R/MITF signal pathway.
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Periploca forrestii, a traditional medicine commonly used by Miao people, is one of the "three treasures of Miao medicine", which mainly contains various components such as cardiac glycosides, flavonoids, ceramides, terpenoids, phenylpropanoid and volatile oils. It has significant pharmacological effects including cardiotonic, anti-inflammatory, antioxidant, pain-suppressing, and antibacterial activities, and is used to treat rheumatoid arthritis, bruises, stomach pain, dyspepsia, amenorrhea, and dysentery. Relevant domestic and abroad literatures were summarized, and a comprehensive review of the chemical constituents, pharmacological effects, clinical application, quality control and spectrum-effect relationship of Periploca forrestii was conducted, to provide evidences for further investigation of Periploca forrestii Schltr.
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OBJECTIVE@#Kaempferide and 4,2'-dihydroxy-4',5',6'-trimethoxychalcone (DTMC) are two major flavonoids found in Chromolaena odorata Linn. leaf extract. The aim of this study was to elucidate the mechanism by which these two flavonoids exerted their effect on adipogenesis. The inhibitory effect of kaempferide and DTMC on adipocyte differentiation and their mechanisms involving mitotic clonal expansion (MCE) and apoptosis during the early stage of adipogenesis were investigated.@*METHODS@#Confluent 3T3-L1 preadipocytes were induced to differentiate and exposed to the flavonoids during various phases of differentiation. Intracellular lipid accumulation, cell density and expression of the transcription factors peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins α were assessed using AdipoRed, Oil red O and Western blot assays. Effects of both flavonoids on cell proliferation and apoptosis were also determined by carboxyfluorescein diacetate succinimidyl ester and annexin V-fluorescein isothiocyanate/propidium iodide-staining assays, respectively.@*RESULTS@#Kaempferide and DTMC showed significant, concentration-dependent anti-adipogenic activity and effect on cell density in the early phase of adipogenesis. The expression of the transcription factors seemed to be reduced when the treatment was prolonged or in the early phase of adipogenesis. These flavonoids interrupted MCE via inhibition of preadipocyte proliferation and induction of apoptosis. DTMC was nearly three times more potent than kaempferide in inducing apoptosis.@*CONCLUSION@#Kaempferide and DTMC exerted their anti-adipogenic activity through inhibition of MCE, either by suppressing cell proliferation or by inducing apoptosis during the early phase of differentiation.
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Objective To study the quality standard of the imitation ecological planting Anoectochilus roxburghii under forest covering. Methods The identification of medicinal properties, microscopic characteristics, thin-layer chromatography (TLC) and content determination of A. roxburghii cultivated under forest were carried out in this study, and the moisture content, ash and acid-insoluble ash were determined according to Chinese Pharmacopoeia of 2015 edition. Results A. roxburghii cultivated under forest exhibits specific properties in characteristics, microscopic features and TLC results. The average moisture content of A. roxburghii cultivated under forest from five planting bases was 8.69%, the average ash was 11.93%, and the average acid-insoluble ash was 3.27%. Content determination results of the average quality score of quercetin, isorhamnetin and kaempferide were at 0.021 0%,,0.024 7%, and 0.027 3%, respectively. Conclusion The above method is simple, specific and reproducible, which will provide the basis for the quality standard of the imitation ecological planting A. roxburghii under forest covering.
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Objective To develop a method of quantitative analysis of multi-components by single marker( QAMS) for determination of quercetin, kaempferide,isohamnetin in Chuipencao granules,providing method to control the quality of Chuipencao granules.Methods The analysis was performed at 30 ℃,on an Shimadzu C18 column(250 mm ×4.6 mm,5μm),and the mobile phase was methanol-0.4% phosphoric acid solution .The flow rate was 1.0 mL/min, and the injection volume was 10 μL.Quercetin as a reference to establish the relative correction factor kaempferol and between isorhmnetin, the method was evaluated for reproducibility, and the difference between calculated and measured values was compared. Results Quercetin, kaempferol and isorhmnetin respectively 32.36-426.43μg, 11.56-136.87μg, 10.45-155.68μg showed a good linear relationship, and the regression equation was: Y=3625.8X-13658 (R2 =0.9998), Y=2682.9X-10253(R2 =0.9999), Y=3012.2X-11223 (R2 =0.9999).The average recoveries were 99.3%, 99.6%, 98.5% (RSD were less than 1.0%).Determination of 10 batches of Chuipencao granules with QAMS and external standard method, kaempferol and isorhamnetin were measured, and measured values were basically the same.Conclusion The QAMS method is reliable and accurate, which might be used for the quality control of Chuipencao granules.
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Objective: To establish a method of HPLC for thesimultaneous determination of eleven ingredients (tanshinone IIA, puerarin, β-ecdysone, liquiritin, paeoniflorin, spinosin, ferulic acid, kaempferide, emodin, tenuifolin, and 23-acetate alisol B) in Xinnaokang Tablets. Methods: The contents of tanshinone IIA, puerarin, β-ecdysone, liquiritin, paeoniflorin, spinosin, ferulic acid, kaempferide, emodin, tenuifolin, and 23-acetate alisol B were determinedby HPLC. Separation was performed on an InertSustain C18column (250 mm × 4.6 mm, 5 μm); Mobile phase was methanol-acetonitrile (1∶1, A)-0.1% phosphoric acid (B), gradient elution: 0-10.0 min, 80% B; 10.0-20.0 min, 80%-70% B; 20.0-35.0 min, 70%-50% B; and 35.0-50.0 min, 50%-30% B; Flow rate was 0.9 mL/min; Column temperature was 40 ℃; Injection volume was 10 μL. Results: The separations of tanshinone IIA, puerarin, β-ecdysone, liquiritin, paeoniflorin, spinosin, ferulic acid, kaempferide, emodin, tenuifolin, and 23-acetate alisol B were good.The linear ranges were good,28.24-282.42, 15.89-158.90, 20.53-205.26, 4.09-40.94, 12.08-120.76,40.03-404.30, 4.08-40.75, 2.13-21.25, 7.91-79.14, 20.30-202.96, and 4.10-40.96μg/mL for tanshinone IIA, puerarin, β-ecdysone, liquiritin, paeoniflorin, spinosin, ferulic acid, kaempferide, emodin, tenuifolin,and 23-acetate alisol B, respectively, with the correlation r> 0.999 0. The extraction recoveries varied from 98.15% to 101.84% (RSD varied from 0.62% to 1.71%). The contents of tanshinone IIA, puerarin, β-ecdysone, liquiritin, paeoniflorin, spinosin, ferulic acid, kaempferide, emodin, tenuifolin,and 23-acetate alisol Bwere 0.870-0.887, 6.321-6.329, 0.857-0.866, 0.171-0.179, 0.369-0.377, 2.128-2.136, 0.088-0.099, 0.148-0.155, 0.113-0.121,0.371-1.380, and 0.101-0.109 mg/gin six batches of samples, respectively. Conclusion: This method is rapid and has high sensitivity, high accuracy, and good specificity,which can provide the basis for the quality control of Xinnaokang Tablets.
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OBJECTIVE:To establish a method for the determination of 4 flavonoids constituents in Yinqiao capsules. METH-ODS:HPLC method was adopted. The Hypersil ODS C18 column was used with the mobile phase A of methanol-water-acetic acid (10∶88∶2,V/V/V)and B of methanol-water-acetic acid(88∶10∶2,V/V/V)in gradient elution at the flow rate of 1.0 ml/min;the de-tection wavelength was 327 nm,the column temperature was 25 ℃,and the volume was 10 μl. RESULTS:There was a good linear relationship between the amount of quercetin and peak area in the range of 0.050 9-1.018 0 μg(r=0.999 8),kaempferide in the range of 0.050 2-1.004 0 μg(r=0.999 5),isorhamnetin in the range of 0.051 0-1.020 0 μg(r=0.999 4)and rutin in the range of 0.050 4-1.007 0 μg(r=0.999 8). RSDs of precision,stability and repeatability tests were <2%. The average recoveries were 100.09%(RSD=0.93%,n=9),99.83%(RSD=0.75%,n=9),100.51%(RSD=1.17%,n=9) and 101.19%(RSD=1.08%,n=9). CONCLUSIONS:The method is amount specific,stable and reproducible and can be used for the quality control of Yinqiao capsules.
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Objective: To establish a method for determining the contents of quercetin, kaempferide, and isorhamnetin in Anoectochilus roxburghii and to provide scientific method for quality control of A. roxburghii and its related products. Methods: HPLC was used. Using Lanbo-Kromasil-C18 column, with methanol -0.2% phosphoric acid solution (50:50) as mobile phase; The detection wavelength was 360 nm, flow rate was 1.0 mL/min, and column temperature was 30 ℃. Results: Quercetin, kaempferide, and isorhamnetin showed a good linear relationship in the range of 11.0-165.6 (r = 1.0000), 11.4-170.4 (r = 0.9998), and 10.4-156.0 ng (r = 0.9995). The average recoveries of quercetin, kaempferide, and isorhamnetin were 97.69% (RSD = 1.1%, n = 9), 95.09% (RSD = 1.6%, n = 9), and 95.86% (RSD = 1.7%, n = 9). Conclusion: The method is simple, rapid, accurate, reproducible, and can be used as an ideal method for quality control.
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To establish the fingerprint of Alpiniae Oxyphyllae Fructus by UPLC. The chromatographic fingerprint was obtained with ACQUITY UPLC HSS T3 column (100 mm × 2.1 mm, 1.8 μm) and gradient eluted with acetonitrile and aqueous formic acid. The flow rate was 0.5 mL/min, the sample room temperature and the column temperature were maintained at 10℃ and 30℃, respectively. The detection wavelength was set at 254 nm. The injection volume was 2 μ L. The common mode of UPLC fingerprint of Alpiniae Oxyphyllae Fructus was set up firstly. There were 20 common peaks in the fingerprints. Protocatechuic acid, α-hydroxymethyl furfural, oxyphyllenodiol A, teuhetenone A, teuhetenone A, chrysin, kaempferide, tectochrysin, and nootkatone were identified by comparing the retention time and their ultraviolet spectra. The similarities of 15 Alpiniae Oxyphyllae Fructus among 19 batches of Alpiniae Oxyphyllae Fructus were above 0.970. The method is rapid and efficiency. It can be used for the quality evaluation of Alpiniae Oxyphyllae Fructus.
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This study was aimed to establish an HPLC method for the determination of chlorogenic acid, rutin and kaempferide in Solidago decurrens Lour. A Diamonsil C18 column (4.6 mm í 200 mm, 5 μm) was adopted with the mobile phase of acetonitrile-0.2% phosphoric acid. The flow rate was 1.0 mL·min-1. The column temperature was at 25℃. The wavelength of detection was set at 282 nm. The results showed that the calibration curve was linear over the range of 63.2~442.4 μg for chlorogenic acid (r = 0.999 3), 8.1~56.8 μg for rutin (r = 0.999 4) and 10.8~75.7μg for kaempferide (r = 0.999 8). The average recovery of chlorogenic acid was 98.6% (RSD = 1.4%), that of rutin was 99.2% (RSD = 0.8%) and that of kaempferide was 100.3% (RSD = 1.0%). It was concluded that the method was simple, economical and accurate with good reproducibility.
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Objective: To establish a method for quantitative determination of Dipyridamole Injection. Methods: The flavonoids were determined using Agela C18 column (150 mm × 4.6 mm, 5μm), acetonitrile-0.4% phosphoric acid (gradient) as the mobile phase with flow rate 1.0 mL/min, detection wavelength of 360 nm, column temperature of 35℃. The lactones were determined using Agela C18 column (150 mm × 4.6 mm, 5 μm), methanol-tetrahydrofuran and water (10:75) solution (gradient) as the mobile phase with flow rate 0.8 mL/min, with evaporative light scattering detector, and column temperature of 30℃. Results: Respectively taking 10 batches of Dipyridamole Injection, the contents of seven major components such as quercetin, kaempferide, isorhamnetin, ginkgolide A, ginkgolide B, ginkgolide C, and bilobalide were determined as 12.4, 20.8, 5.5, 7.2, 2.6, 4.7, and 102.0 μg/mL. Conclusion: The established method is with good stability, repeatability, and precision, which conforms to the requirements and can be used for the quality control of Dipyridamole Injection.
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Objective: To compare the content of flavonoids from Sophorae Fructus by different processing methods. Methods: HPLC method with Zorbax Eclipse Plus C18 column (250 mm × 4.6 mm, 5 μm) was used in the experiment; Methanol-0.4% acetic acid was used as mobile phase, with gradient elution; Column temperature was set as 30℃; The flow rate was 1.0 mL/min and detection wavelength was 256 nm. Results: Genistin: crude (0.86%) > stir-frying with honey (0.67%) > carbonizing by stir-frying (0.48%); Rutin: crude (3.0%) > stir-frying with honey (2.2%) > carbonizing by stir-frying (0.88%); Sophoricoside: crude (8.08%) > stir-frying with honey (5.73%) > carbonizing by stir-frying (3.58%); Quercetin: crude (0.04%) < stir-frying with honey (0.05%) < carbonizing by stir-frying (0.12%); Genistein: crude (0.06%) < stir-frying with honey (0.08%) < carbonizing by stir-frying (0.21%); Kaempferide: crude (0.01%) < stir-frying with honey (0.02%) < carbonizing by stir-frying (0.54%). Conclusion: Among the flavonoids from Sophorae Fructus after processing, the content of flavonoid glycosides is reduced and the content of flavonoid aglycone is increased simultaneously, which may be related to the different functions of crude Sophorae Fructus, Sophorae Fructus stir-fried with honey, and Sophorae Fructus carbonized by stir-frying pieces.
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ive] To isolate the active constituents with analgesic and antemetic actions from Rhizo-ma Alpiniae Officinarum. [Methods] Polyamide and silica gel column chromatography was used to isolate and extract the analgesic and antemetic constituents. The structure of compounds was identified by ultra violet spectroscopy, infrared spectroscopy, mass spectroscopy and nuclear magnetic resonance spectrosco-py. [ Results ] Galangin and kaempferide are identified as the analgesic and antemetic constituents. [Conclusion] Galangin can be used as the quality control for Rhizoma Alpiniae Officinarum.