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Objective To compare the contents variation of six flavonoids includingdaidzin，glycitin, genistin, daidzein, glycitein and genisteinin black beans, semifinished and finished Sojae Semen Praeparatum．Methods The contents of flavonoids were determined by HPLC，the condition were Diamonsil C18 column (4.6×250 mm, 5 μm) , column temperature 30 ℃, detection wavelength 260 nm, mobile phase 0.2% acetic acid water (A) - methanol (B), gradient elution, flow rate 1.0 ml/min．Results The linearity of this method to determine 6 isoflavones was good (r≥0.9993) within the determination range, and the recovery rate met the requirements. The RSD of precision, repeatability and stability experiment was less than 4%, 3%and 3%. The results of HPLC showed that the contents of six flavonoidsin Sojae Semen Praeparatum increased significantly compared with black beans. And, the contents of six flavonoids in finished Sojae Semen Praeparatum were slightly more than those in semifinished Sojae Semen Praeparatum. Conclusion The HPLC method established in this study could accurately determine the content of 6 isoflavones in Sojae Semen Praeparatum. The content of six isoflavones in black beans could be increased by the fermentation, and the combined isoflavones were transformed into free isoflavones during the fermentation process.
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Objective: To establish a method for the simultaneous content determination of 14 chemical components such as D- amygdalin, puerarin, hesperidin in Fenghan Ganmao Granules (FGG) by UHPLC-UV wavelength switching method, and chemometric analysis was used to analyze the quality differences. Methods: Separation was performed on an Agilent Poroshell 120 EC-C18 (150 mm × 2.1 mm, 2.7 μm) with a gradient elution of acetonitrile and 0.1% phosphoric acid. The content of 14 chemical components in 68 batches of samples from five manufacturers was determined by switching wavelength (210, 254, 310 nm). The radar chart, similarity evaluation, heat map and hierarchical clustering analysis, and principal component analysis (PCA) were used for data analysis. Results: The content of each component was as follows, D-amygdalin 0.063-3.885 mg/g, 3'-hydroxy puerarin 0.012-1.540 mg/g, puerarin 0.036-4.017 mg/g, 3'-methoxy puerarin 0.016-1.837 mg/g, puerarin-6″-O-xyloside 0.004-0.449 mg/g, mirificin 0.021-2.076 mg/g, daidzin 0.010-1.527 mg/g, prim-O-glucosylcimifugin 0.007-0.471 mg/g, 5-O-methylvisammioside 0.062-1.029 mg/g, hesperidin 0.210-8.453 mg/g, rosmarinic acid 0.001-0.237 mg/g, oxypeucedanin hydrate 0.007-0.204 mg/g, glycyrrhizic acid 0.056-1.311 mg/g, oxypeucedanin 0.002-0.042 mg/g, respectively. Chemometric analysis showed that there were some differences among the samples from different manufacturers, and the samples from the same manufacturer were more consistent. Conclusion: The method is simple, reproducible, and specific, which provides a reference method for the overall quality evaluation of FGG.
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Objective: To establish HPLC specific chromatograms of Puerariae Lobatae Radix(PLR) and Puerariae Thomsonii Radix(PTR), and make a distinction about their species and different habitats of PLR by chemical pattern recognition, provide reliable methods for scientific evaluation and effective control of their quality. Method: HPLC was employed to determine the contents of chemical ingredients in 23 batches of PLR and PTR.The similarity analyzed with "Similarity Evaluation System for Chromatographic Fingerprint of Chinese Materia Medica"(version of 2004A), then a common pattern was established.Based on its chemical fingerprint information, the quality of PLR and PTR was comprehensively analyzed by three kinds of chemical pattern recognition methods. Result: In addition to sample S22(from Shaanxi province), the similarities of 23 batches of samples were more than 0.9, which showed that similarity of PLR and PTR was good, this method can not differentiate them.Principal component analysis(PCA) could only identify PLR and PTR, but partial least squares-discriminant analysis(PLS-DA) could distinguish PLR from PTR and the producing areas of PLR with model interpretation of 96.4% and prediction of 74.6%.The result of hierarchical cluster analysis(HCA) was consistent with PLS-DA. Conclusion: Chemical pattern recognition method can make a distinction between PLR and PTR, as well as different habitats of PLR;it is suitable for quality control of their medicinal materials.
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Objective: To determine the components of 3'-hydroxy puerarin, puerarin, daidzin, daidzein, genistein glycosides, and genistein from Pueraria Radix and to study the quality of medicinal materials combining with anti-oxidant activity. Methods: The contents of six main components from Pueraria Radix samples were determined by using UPLC-DAD method, which performed on AgiLent Zorbax SB-C18 (5 mm×2.1 mm, 1.8 μm), acetonitrile and 0.1% acetic acid aqueous solution as mobile phase, column temperature 30 ℃, and the flow rate of 0.4 mL/min. The anti-oxidant activity evaluation of DPPH was accomplished by using UV spectrophotometry. Comprehensive weighted evaluation was based on seven indicators, combined six kinds of ingredients with the IC50 of medicine, and made cluster analysis. Results: Studies had shown that the quality of No. 8, 9, 50, and 51 samples of Pueraria thomsonii was better than others, which collected from Jianyang and Jiangyou. And the quality of No. 19, 20, 29, 30, and 31 samples of cultivated-planting Pueraria omeiensis was poor. The quality of No. 33 ~ 37, 65 ~ 69 samples of Pueraria Radix was better than other places in Sichuan Province, which collected from Pingwu, Beichuan, and Qingchuan. Conclusion: The UPLC wavelength switching technology multicomponent simultaneous determination method is simple and convenient, which has good reproducibility, strong specificity. Comprehensive weighted evaluation method can analyze the quality of medicinal materials better, which provides references for comprehensive evaluation on the main varieties of Pueraria.
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Objective: To establish simultaneous determination method of eight main flavonoids from Puerariae Lobatae Radix (PLR) by using UPLC, and to study the effect of ultrafine grinding technology on the content of flavonoids in PLR for optimizing its production process. Methods: Response surface method was used to investigate the crushing time, sampling capacity, and initial particle size in the superfine pulverizing technology of PLR. Using the particle size distribution (D50, D90), and the content of eight flavonoids (3′-hydroxy puerarin, puerarin, 3′-methoxy puerarin, daidzin, genistin, daidzein, genistein, and formononetin) from PLR powders with various particle sizes (10—40, 40—65, 300 mesh) determined by UPLC, we selected these 10 elements as the response factors to evaluate the ultrafine powders technology for PLR. Results: Eight flavonoids from PLR had a good linear relationship with the linear range of 75.8—242.7, 205.6—658.0, 147.3—417.3, 10.2—163.3, 11.3—182.0, 1.2—18.8, 0.25—4.00, 0.35—5.37 μg/mL, and the average recovery ranged from 98.86%, 99.25%, 99.90%, 100.17%, 100.21%, 101.40%, 100.73%, and 101.42%. In addition, the repeatability, stability, and precision RSD were all less than 3%. UPLC result showed that, compared with the PLR powders with other particle sizes, the ultrafine powder of PLR (300 mesh) had a higher flavonoids contents. Results: of response surface method showed the optimized preparing parameters of PLR ultrafine powder technology as follows: 80 mesh [(180 ± 7.6) μm] of particle size, 247 g of sampling amount, and 26 min of crushing time. Conclusion: Optimization of ultrafine powders process from PLR by response surface method is simple and accurate, which can obtain PLR ultrafine powder with higher content of flavonoids. This process can provide reference for the ultrafine grinding technology of PLR.
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Objective: To establish a method for simultaneously determination of five isoflavones (daidzein, daidzin, puerarin, mirificin, and 6″-O-xylosidepuerarin) in Pueraria Radix by single marker (QAMS), which is feasible and accurate. Methods: Puerarin was taken as internal standard substance to establish the relative correction factor (RCF) for quantitative analysis of multi-components with QAMS. Thus, the contents of mirificin, 6″-O-xylosidepuerarin, daidzin, and daidzein were calculated. The contents in 10 batches of samples were determined by external standard method and QAMS. The scientificalness and feasibility of the methods were evaluated by comparison of the quantitative results between external standard method and QAMS. Results: The reproducibility of RCF was perfect. The results calculated with QAMS were consistent with the results by the external standard method. Conclusion: QAMS is accurate and feasible to evaluate the quality of Pueraria Radix.
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Objective To establish the HPLC fingerprint of Liujing Toutong Tablets (LTT) and multi-wavelength method for determination of the contents of 3’-hydroxy puerarin, puerarin, daidzin, nuezhenide, and daidzein, so as to provide a reference for the quality control. Methods Using the method of HPLC, the fingerprint chromatography of high polarity and low polarity were established, with a mobile phase of acetonitrile-0.1% phosphoric acid and Diamonsil C18 column (250 mm × 4.6 mm, 5 μm). Furtherly, the Diamonsil C18 column was used with a mobile phase of acetonitrile-0.1% acetic acid gradient elution to determine the five contents. Results The fingerprint chromatography of high and low polarities with 11 batches of LTT were established, and the similarities of 11 batches were all over 0.90. The low polarity fingerprint chromatography included 16 mutual peaks, 12 of which belonged to herbs: No. 11, 12, 13 belonged to Angelicae Dahuricae Radix, No. 1, 2, 3, 7 belonged to Puerariae Lobatae Radix, No. 8 belonged to Ligustri Lucidi Fructus, No. 5, 6 belonged to Chuanxiong Rhizoma, No. 15 belonged to Ligustici Rhizoma et Radix and No. 4 belonged to Chuanxiong Rhizoma and Ligustici Rhizoma et Radix. Seven peaks including puerarin (No. 1), 3’-methoxy puerarin (No. 2), daidzin (No. 3), ferulic acid (No. 4), daidzein (No. 7), imperatorin (No. 11), and isoimperatorin (No. 13) were verified by standard compounds. The high polarity fingerprint chromatography included 10 mutual peaks, among them No. 2-9 belonged to Puerariae Lobatae Radix, No. 1, 10 belonged to Ligustri Lucidi Fructus. Five peaks including 3’-hydoxypuerarin (No. 2), puerarin (No. 3), 3’-methoxypuerarin (No. 4), daidzin (No. 7) and specnuezhenide (No. 10) were verified by standard compounds. The five active components were well separated and showed good lineaeity, such as 3’-hydroxy puerarin 71.60-716.00 ng (r = 0.999 1), puerarin 407.78-4 077.80 ng (r = 0.999 4), daidzin 90.72-907.20 ng (r = 0.999 9), nuezhenide 95.80-958.00 ng (r = 0.999 1), daidzein 21.98-219.80 ng (r = 0.999 9). The average recoveries and corresponding RSD values were 101.1% (1.38%), 97.8% (0.72%), 99.1% (0.75%), 97.8% (2.75%), and 98.7% (0.70%). The contents of 3’-hydoxypuerarin, puerarin, daidzin, specnuezhenide and daidzein in the 10 batches of sample were 3.08-3.84 mg/mL, 17.71-21.24 mg/mL, 3.51-4.71 mg/mL, 1.40-5.69 mg/mL, and 0.66-0.86 mg/mL, respectively. Conclusion The HPLC fingerprint combined with multi index determination method can reflect the intrinsic quality of LTT, which is stability, accurate, and reproducible, providing a scientific reference for quality control.
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Objective To develop a quantitative analysis multi-components by single marker (QAMS) method for the quantification of multiple characteristic components, namely, puerarin, daidzin, jatrorrhizine, palmatine, soybean glycol, wogonoside, wogonin, in Gegen Qinlian Pills (GQP) with puerarin as the internal reference. Methods Samples were analyzed by Waters ultra-high efficiency liquid chromatography (UPLC) system, equipped with a reverse phase Acquity BEH C18 chromatographic column, with the mobile phase of methanol-0.1% phosphoric acid solution at a flow rate of 0.3 mL/min. The column temperature and the detection wavelength set at 30 ℃ and 260 nm respectively. Based on puerarin as the internal reference, the relative correction factors (RCFs) with other six characteristic components were calculated, then compared with the external standard method. This result benefits to verify the accuracy and advantages of the established QAMS method. Results Under the linear range of determination, RCFs of daidzin, jatrorrhizine, palmatine, soybean glycol, wogonoside, wogonin with reference to puerarin, were 1.02, 1.07, 1.05, 1.32, 0.62, and 0.90 in GQP, respectively. And the repeatability was good in different experimental conditions. Moreover, there was no significant difference on the quantitative results of seven characteristic components, derived from between external standard method and QAMS method in the 10 batches of GQP samples. The range of seven characteristic components contents in the 10 batches of GQP samples, namely puerarin, daidzin, jatrorrhizine, palmatine, soybean glycol, wogonoside and wogonin, were 8.923-10.746 mg/g, 2.231-2.988 mg/g, 0.825-1.197 mg/g, 1.274-1.522 mg/g, 2.330-2.713 mg/g, 0.836-0.951 mg/g, and 0.901-1.092 mg/g, respectively. Conclusion In present study, a feasible, convenient and accurate QAMS method with puerarin as the internal reference was established. Therefore, it is suitable to quantify multiple characteristic components in GQP and provide a useful approach for the quality control of GQP.
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OBJECTIVE: To establish a UPLC method for simultaneous determination of puerarin, daidzin, daidzine, palmatine, and berberine in HuangdiAnxiao capsules (HDAXC). METHODS: The chromatographic analyses were carried out on an ACQUITY UPLC BEH C18 column (2.1 mm×100 mm, 1.7 μm; Waters Corp., USA) maintained at 30℃. The mobile phase component A was acetonitrile and B was phosphoric acid with gradient elution at 0.2 mL·min-1. The five components were detected by dual wavelength, ie λ1=250 nm, λ2=354 nm. RESULTS: The standard curves of puerarin, daidzin, daidzine, palmatine, and berberine showed good linear relationship in the ranges of 0.022-0.176 μg (r=0.9999), 0.0043-0.0344 μg (r=0.9999), 0.00061-0.0049 μg (r=0.9997), 0.0023-0.0184 μg (r=0.9997), and 0.0086-0.0668 μg (r=0.9996). respectively. The average recoveries were 97.70%, 96.81%, 99.36%, 99.60% and 98.67% with RSDs of less than 2.7% (n=6). CONCLUSION: This method is simple, rapid, accurate, and reproducible, and it can lay basis for the quality evaluation of HDAXC.
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Objective To investigate the protective effects of alpha-lipoic (α-LA) on H9c2 cardiomyocytes under-going hypoxia or hypoxia/reoxygenation(H/R) injury and explore its possible mechanisms. Methods H9c2 car-diomyocytes in hypoxia or H/R injury researches were respectively divided into normal control group, hypoxia or H/R group, hypoxia or H/R + α-LA group (LA group), hypoxia or H/R+α-LA + Daidzin group (Daidzin group) and hypoxia or H/R+α-LA +DMSO group ( DMSO group) . The myocardial cell survival was detected by MTT,the activity of LDH and ALDH2 were respectively analyzed by microtitration and ELISA,the MDA level was measured by TBA. Results Compared to the normal control group, the cell survival rates of hypoxia and H/R group were decreased ( P0. 05 ) . Conclusion α-LA can protect H9 c2 cardiomyocytes from hypoxia or H/R injury by means of upregulating activities of ALDH2 and decreasing hypoxia or H/R induced lipid peroxidation.
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Objective The alm of this study was to determine the solubility and permeability of daldzin and daldzein and the interaction of these two components.Methods With the method inChinese Pharmacopoeia and in situ single-pass intestinal perfusion model we tested the solubility and permeability of daldzin, daldzein and their interaction.Results In pH 7.4 K-R buffer the solubility of daldzin was 6 times than daldzein and both the solubility of these two components were enhanced when they were determined together. In small intestine of rat, the permeability of daldzein was 3 times than daldzin. Daldzin could enhance the permeability of daldzein but the daldzein manifested an opposite trend.Conclusion When compared to daldzin, daldzein owned a lower solubility but a better permeability. When used together, both the solubility and permeability of daldzein would be enhanced. The solubility of daldzin could be enhanced slightly but its permeability would be reduced.
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Objective: To prepare the lyophilized powder of Pueraria flavonoids loaded solid lipid nanoparticles (PF-SLN) and determine the dissolution rate of its four effective components: 3'-hydroxypuerarin, puerarin, daidzin, and daidzein. Methods: PF-SLN was prepared by the high pressure homogenization (HPH) technology. The lyophilized formula contained mannitol as cryoprotectant. The release rates of the four effective components from the PF-SLN lyophilized powder as well as the physical mixture were determined, with artificial gastric juice (pH 1.2) as dissolvent. Results: The technical parameters of PF-SLN preparation optimized by orthogonal test were as follows: The ratio and the dosage of lipid-surfactant were 2:1 and 2.0%, PF dosage was 2.5%, and 150 MPa homogeneity was 15 cycles. The optimal PF-SLN lyophilized powder was loosen with the particle size of (517.1 ± 10.3) nm, polydisperse index of 0.484 ± 0.210, and Zeta potential of (-21.91 ± 2.03) mV, respectively. The in vitro accumulated dissolution rates of PF-SLN lyophilized powder were slower than those of the physical mixture. Conclusion: The method employed to prepare PF-SLN lyophilized powder is feasible. PF-SLN lyophilized powder could delay the in vitro dissolution rate notablely. It might be a novel vehicle potentially for nano-drug delivery system of Pueraria flavonoids.
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Objective: To prepare the lyophilized powder of Pueraria flavonoids nanosuspension (PF-NS) and to determine the dissolution rates of its four effective components (3'-hydroxypuerarin, puerarin, daidzin, and daidzein). Methods: PF-NS was prepared by the high pressure homogenization (HPH) technology. The lyophilized formula contained mannitol as lyoprotectant. The dissolution rates of the four effective components from lyophilized powder of PF-NS as well as the physical mixture were determined, with artificial gastric juice (pH 1.2) as dissolvent. Results: The optimal lyophilized powder of PF-NS was loosed with the particle size of (479.7 ± 14.7) nm, polydisperse index of 0.524 ± 0.220, and Zeta potential of (29.68 ± 3.97) mV, respectively. The in vitro accumulated dissolution rate of lyophilized powder of PF-NS was higher than that of the physical mixture. Conclusion: The method employed to prepare the lyophilized powder in PF-NS is simple and feasible. The lyophilized powder of PF-NS could improve the in vitro dissolution rate notablely. It might be a novel vehicle potentially for nano-drug delivery system of PF.
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Objective To develop a sensitive and specific HPLC method to simultaneously determine the contents of puerarin, daidzin and daidzein in Puerariae Lobatae Radix. Methods The three compounds were obtained by ionic liquid based on dispersive liquid phase microextraction. The determination was carried out on a Phenomenex C18 column (250 mm×4.6 mm, 5μm) with a mixture of methanol-0.2%acetic acid (volume ration 45∶55) as mobile phase at a flow rate of 0.8 mL/min. The UV detective wavelength was 250 nm, and the column temperature was set at 35 ℃. Results The linear response ranged from 6.24×10-6-37.44 μg for puerarin (r=0.999 71), 5.44×10-6-27.20μg for daidzin (r=0.999 85), and 5.60×10-6-28.00μg for daidzein (r=0.999 94), respectively. Conclusion The method is quick, simple and repeatable for simultaneous determination of the contents of puerarin, daidzin and daidzein in Puerariae Lobatae Radix.
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Objective:To establish an HPLC method for the determination of four kinds of isoflavone compounds including daid-zin, tectoridin, daidzein and tectorigenin in the extracts of Pueraria lobata flowers. Methods:The separation was performed on an Agi-lent C18 column(250 mm × 4. 6 mm, 5 μm) using a mobile phase of methanol-acetonitrile-water(2∶1∶2). The flow rate was 1. 0 ml· min-1 ,and the detection wavelength was 264nm. The column temperature was 25℃ and the sample size was 20 μl. Results:The de-tection could be accomplished within 10 minutes with good separation and specificity of four isoflavone compounds with the retention timeof3.4,3.8,5.7and7.2min,respectively. Thelinearrangewas0.784-78.440 μg·ml-1(daidzin),2.000-200.000 μg· ml-1(tectoridin) and 0.800-80.020 μg·ml-1 (daidzein and tectorigenin),and the relative coefficient was 0.999 9, 0.999 8, 0. 999 7 and 0. 999 9, respectively. The average recovery was 100. 54%(RSD=1. 66%,n=6),100. 03%(RSD=1. 00%, n=6), 99. 48%(RSD=1. 76%, n=6) and 100. 92%(RSD=2. 26%, n=6), respectively. Conclusion: The method is simple, rapid and accurate with good repeatability, which can be used in the rapid determination of isoflavone compounds in the flowers of Pueraria loba-ta.
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Isoflavones can be found in grains and leaves of soybean. Currently, these are sold in pharmacies as phytotherapic capsules. Isoflavones have been recommended by doctors, especially for women, due to their ability to relieve menopause symptoms, among other benefits. However, no method exists for the official control of isoflavone content in capsules sold in the Brazilian market. This study aims to develop an appropriate analytical method to determine the total isoflavone content (daidzin, glycitin, and genistin, and their respective aglycone forms) in phytotherapic capsules purchased in pharmacies in Curitiba, Parana State, Brazil, using the technique of high-performance liquid chromatography with UV detection (UV-HPLC). The HPLC system consisted of a quaternary pump, an autosampler, and Waters reversed-phase C18 column (5 μm × 300 mm. Analyses were carried out at 40 °C, using a flow rate of 1.0 mL/min (acetonitrile and acetic acid 0.1%), and detection was performed at 254 nm. The method was validated as required by ANVISA and showed to be reliable for the following parameters: linearity (r² >0.99), selectivity (correlation between 0.99 and 1.00), precision (relative standard derivation <1.59%), accuracy (from 80% to 111.63% intraday and from 80% to 117.88% interday recovery), and robustness.
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Drinking a great amount of alcohol over a long period of time has serious effect on people's health and their families.It has been the chief concern for many experts to find ways to help people to give up alcohol.Radix pueraria is the most typical traditional Chinese herb to help giving up driking alcohol.Some experts had done some deep-seated pharmacological researches on the effect of puerarin and daidzin,the contents distilled from Radix pueraria,on treating alcohol-dependence disease.The present paper is a general survey of recent developments of this research.
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Object A method for the determination of daidzein, genistein, daidzin and genistin in soybean extract by RP HPLC was established Methods Zorbax SB C 18 column (4 6?15 mm, 5 ?m) was used Daidzein and genistein were separated with mobile phase Ⅰ∶methanol acetonitrile 0 1% phosphoric acid solution (30∶8∶62) Daidzin and genistin were separated with mobile phase Ⅱ∶methanol acetonitrie 0 1% phosphoric acid solution (16∶24∶60) The UV detection wavelength was 254 nm Results The average recoveries of daidzein, genistein, daidzin and genistin were 98 7%, 99 5%, 98 4%, 98 6% and the precision (RSD) were 1 4%, 0 9%, 0 9%, 1 7% (n=4) respectively Conclusion: The method was accurate, rapid and with good reproducibility
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AIM: To establish an HPLC method for determing content of puerarin isoflavones in effective parts of Naomaitong Granules.METHODS: Gradient elution with methanol-water-glacial acetic acid was used as the mobile phase.The flow rate was 1.0 mL/min,and the detection wavelength was set at 250 nm.RESULTS: The average recoveries of puerarin,daizein-8-capiosy(1→6) glucoside,3-methoxyl-puerarin,daidzin,daidzein were 100.89%(RSD = 2.1%),100.23%(RSD = 1.06%),101.04%(RSD = 1.92%),99.82%(RSD = 2.02%),102.06%(RSD = 1.34%) respectively.CONCLUSION: The method is simple,accurate and can be used for quality control of the effective parts in Naomaitong Granules.
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Objective: To establish the quality standards for Xueyujiangn Capsules(total icariin daidzin Fructus Foeniculi, etc.).Methods:A method for the assay of daidzin by HPLC was described. Results: The average recovery was 101.75% and RSD was 2.5%. Conclusion: The method is simple. This study provides a method for the quality control of Xueyujiangn Capsules.