Hydroxy Puerarin Research Articles

A comprehensive strategy integrating chemical profiling with deep learning for screening the antithrombin bioactive-chemical quality markers of functional food: Pueraria radix as an example

Pueraria lobata radix (PL) and Puerariae tbatae radix (PT) are nutritious medicinal food homology plants that are widely used in the food and health products industry. Both of them are used as Pueraria radix (PR) for medicine. In this study, the function-related active markers were discovered by chemical profiling coupled with deep learning for assessment of PR. Firstly, IR spectral fingerprints were established. And the IR spectra of PL and PT were different in fingerprint region. HCA, PCA and PLS-DA can distinguish the samples of PL and PT. Furtherly, UPLC-Q-TOF-MS was further applied to identify the main components of PL and PT, and 30 chemical components in PL and 15 in PT were identified. In vitro antithrombin assay was performed to indicate the different batches of PL and PT extracts and they could be distinguished according to the strength of activity. The potential active markers related to antithrombin activity were screened by PCA and PLS-DA. Finally, 3′-hydroxy puerarin, puerarin, 3′-methoxy puerarin, daidzin, and daidzein were screened-out as functional markers applying molecular docking combined with BP-ANN, CP-ANN, SIMCA, and SVMDA. The thrombin-based discovery strategy of bioactive-chemical marker combination was a powerful tool for screening the quality markers for evaluation of PR.

Puerariae lobatae Radix: Progress in Extraction, Separation Methods and Pharmacological Activities Research

Kudzu root (Puerariae lobatae Radix) is the tuberous root of Pueraria lobata, family Leguminosae. Kudzu root contains a variety of beneficial active ingredients such as puerarin, daidzin, daidzein, genistenin, 3′-hydroxy puerarin, β-sitosterol, stigmasterol, arachidic acid, and so on. Modern medical research shows that active ingredients in kudzu root are widely used clinically as raw materials for the treatment of hyperglycemia, non-alcoholic fatty liver disease, myocardial infarction, alcohol addiction, oxidative stress, inflammatory response, and retinal blockage due to their various pharmacological effects such as improving cardiovascular circulation, lowering blood lipids, lowering blood pressure, lowering blood sugar, being antipyretic, being estrogen-like, and relieving alcohol. China has rich resources of kudzu root, and active ingredients are usually extracted before it is made into a preparation, so whether the extraction and separation process is reasonable will directly affect the ease of preparation and the efficacy of the treatment. This paper reviews the process methods for the extraction and separation of active ingredients in kudzu root and its common pharmacological activities. The aim is to provide some references for readers to compare the advantages and disadvantages of various extraction and separation methods as well as understand the active ingredients and pharmacological activities of kudzu root.

Screening and verification of antiviral compounds against HSV-1 using a method based on a plaque inhibition assay

BackgroundHerpes simplex virus type 1 (HSV-1) infection is a common viral disease that mainly causes oral lesions, but can also cause genital lesions in some instances. Current treatments with nucleoside analogs are limited by the emergence of drug resistance. Therefore, novel anti-HSV-1 drugs are urgently needed.MethodsIn this study, we screened a library of 2080 compounds for anti-HSV-1 activity using a plaque formation assay. We selected 11 potential inhibitors of HSV-1 and further evaluated their antiviral effects by plaque reduction assay and real-time polymerase chain reaction (qPCR).ResultsFive compounds, namely ginsenoside Rd, brassinolide, rosamultin, 3’-hydroxy puerarin, and clinafloxacin HCl, showed potent anti-HSV-1 activity and completely suppressed plaque formation at a concentration of 10 µM. Among them, clinafloxacin HCl, a fluoroquinolone antibiotic, exhibited a high selectivity index for HSV-1.ConclusionsOur findings suggest that these five compounds have potential antiviral properties against HSV-1 and may have different mechanisms of action. Further studies are warranted to elucidate the antiviral mechanisms of these compounds and to explore their therapeutic potential for HSV-1 infection.

Application of the selected macroporous resin for the separation and identification of flavonoids from Chinese Radix Pueraria Lobata by HPLC-Q-TOF-MS

A systematic investigation was conducted on the adsorption and desorption properties of macroporous resins including D101, ADS7, HPD600, XAD2 and HPD100 for flavonoids of Radix Pueraria Lobata. Based on the results of the adsorption capacity, desorption ratio, and physical properties of the investigated resins, ADS7 was selected for the purification of flavonoids in this study. On the basis of the correlation coefficients, the experimental results fitted the pseudo-second-order model compared to the pseudo-first model. The separation parameters of the RPL flavonoids were optimised through dynamic adsorption/desorption experiments with a column packed with ADS7. The breakthrough point was found at 30 mL of RPL flavonoids solution, while at 105 mL of 60 % ethanol, the flavonoids were completely desorbed. Furthermore, the total flavonoid content was 3.16 mg/g. HPLC-Q-TOF-MS analysis helped identify five isoflavones, including puerarin, 3′-methoxy puerarin, daidzin, 3′-hydroxy puerarin and daidzein from radix pueraria Lobata.

Rapid detection of multi-indicator components of classical famous formula Zhuru Decoction concentration process based on fusion CNN-LSTM hybrid model with the near-infrared spectrum

Traditional Chinese Medicine (TCM) is a collective term for the medicines of all ethnic groups in China, including Han Chinese and ethnic minority medicines, reflecting the Chinese people's understanding of life, health and disease, and is a system of medicine with a long historical tradition and unique theories and technical methods. Due to the nonlinearity, large hysteresis, and strong interference in the concentration process used in the production of traditional Chinese medicine, it is challenging to use just one conventional control method to achieve the desired control effect. The majority of traditional quality control methods used in processing the samples will result in the loss of the active ingredient and in the detection of impurities. Monitoring of multiple critical quality attributes (CQAs) is crucial for the overall process of the index ingredient. In this study, based on the above background, fast and nondestructive near-infrared spectroscopy (NIRS) technique incorporating convolutional neural network (CNN) and long-short-term memory network (LSTM) was utilized to construct a quantitative model based on the seven characteristic components, puerarin, 3′-hydroxy puerarin, 3′-methoxy puerarin, puerarin apigenin, daidzin, glycyrrhizin and glycyrrhizic acid, during the concentration process. The results showed that the relative mean error (MAPE) values of the different components ranged from 0.7% to 2.8% and R2 from 0.981 to 0.992 based on the proposed CNN-LSTM model. The results showed that the combined model of near-infrared spectroscopy fused with convolutional neural network (CNN) and long-short-term memory network (LSTM) was feasible for accurate and rapid monitoring of the concentration of the classic famous prescription Zhuru Decoction (ZRD) during the decoction process and that the method could provide a reference value for rapid nondestructive testing of traditional Chinese medicine compound formulas.

Advances in Extraction, Purification, and Analysis Techniques of the Main Components of Kudzu Root: A Comprehensive Review.

Kudzu root (Pueraria lobate (Willd.) Ohwi, KR) is an edible plant with rich nutritional and medicinal values. Over the past few decades, an ample variety of biological effects of Pueraria isoflavone have been evaluated. Evidence has shown that Pueraria isoflavone can play an active role in antioxidant, anti-inflammatory, anti-cancer, neuroprotection, and cardiovascular protection. Over 50 isoflavones in kudzu root have been identified, including puerarin, daidzein, daidzin, 3'-hydroxy puerarin, and genistein, each with unambiguous structures. However, the application of these isoflavones in the development of functional food and health food still depends on the extraction, purification and identification technology of Pueraria isoflavone. In recent years, many green and novel extraction, purification, and identification techniques have been developed for the preparation of Pueraria isoflavone. This review provides an updated overview of these techniques, specifically for isoflavones in KR since 2018, and also discusses and compares the advantages and disadvantages of these techniques in depth. The intention is to provide a research basis for the green and efficient extraction, purification, and identification of Pueraria isoflavone and offers investigators a valuable reference for future studies on the KR.

Spatially resolved metabolomics combined with bioactivity analyses to evaluate the pharmacological properties of two Radix Puerariae species

Ethnopharmacological relevanceP. lobata and P. thomsonii are medicinal plants with similar pharmacological functions but different therapeutic effects. A novel method is presented herein to investigate metabolites in terms of their distribution and qualification, quantification is necessary to elucidate the different therapeutic effects of the two Puerariae species. Aim of the studyThe aim of the present study was to perform spatially resolved metabolomics combined with bioactivity analyses to systematically compare the metabolite differences in P. lobata and P. thomsonii by distribution, qualification, quantification, and biological activity to evaluate their pharmacological properties. Materials and methodsAir flow-assisted desorption electrospray ionization–mass spectrometry imaging (AFADESI-MSI) was performed to characterize the differences in the metabolite distributions of P. lobata and P. thomsonii. Further qualitative and quantitative analyses of the differential metabolites were performed using liquid chromatography–mass spectrometry (LC-MS). Biological activities correlated with the differences in the metabolites were validated by MTT assays. ResultsSome metabolites showed complementary distributions of the phloem and xylem in the two species, saccharide, vitamin, and inosine levels were higher in the phloem of P. thomsonii but higher in the xylem of P. lobata. The 3′-hydroxyl puerarin level was higher in the xylem of P. thomsonii but higher in the phloem of P. lobata. Qualitative and quantitative analyses of the metabolites revealed a total of 52 key differential metabolites. MTT assays showed that daidzein, daidzin, puerarin, ononin, genistin, formononetin, 3′-hydroxy puerarin, 3′-methoxy puerarin, mirificin, and 3′-methoxy daidzin exerted protective effects on H9c2 cells against hypoxia/reoxygenation injury. P. lobata extracts exhibited a significantly better protective efficacy than P. thomsonii extracts. ConclusionsIn this study, AFADESI-MSI combined with LC-MS and biological activities comprehensively elucidated metabolite differences in the distribution, qualification, quantification, and pharmacological properties of P. lobata and P. thomsonii. The results of this study could provide a novel strategy for species identification and quality assessment of similar Chinese herbal medicines.

Analysis of Isoflavones in Pueraria by UHPLC-Q-Orbitrap HRMS and Study on α-Glucosidase Inhibitory Activity.

Pueraria is a rich source of bioactive compounds, but there is a lack of comprehensive information concerning its composition. Therefore, a UHPLC-Q-Orbitrap HRMS method was developed to identify and quantify bioactive compounds in pueraria. Twelve isoflavones were quantified, with puerarin being the most abundant, followed by puerarin 6″-O-xyloside, 3'-methoxy puerarin, and 3'-hydroxy puerarin. A further 88 bioactive components in eight categories were also tentatively identified. The 12 isoflavones, except for genistein, exhibited α-glucosidase inhibitory activity. The binding of these compounds to the active site of α-glucosidase was confirmed via molecular docking analysis. These findings provide a basis for identifying pueraria as a promising functional food ingredient.

Investigation of the potential curative effects of Gui-Zhi-Jia-Ge-Gen decoction on wind-cold type of common cold using multidimensional analysis

Ethnopharmacological relevanceGui-Zhi-Jia-Ge-Gen decoction (GJGD) is a classical Chinese medicine prescription that has been widely used in clinical practice for centuries. In recent times, TCM has received considerable attention for its potential efficacy in treating a wind-cold type of common cold. However, the effect of the Gui-Zhi-Jia-Ge-Gen decoction on the wind-cold type of common cold is still not fully understood, which presents challenges for both quality control, research and development. Furthermore, the identification of potential pharmacodynamic ingredients (PPIs) is important for developing quality control procedures for industrial and large-scale production. Aim of the studyThe aim of this study was to investigate the potential curative effect of Gui-Zhi-Jia-Ge-Gen decoction on wind-type of common cold using multidimensional qualitative analysis that combined water-decoction spectrums, in vivo plasma spectrums, and molecular docking to identify key constituents of GJGD. Materials and methodsWater-based GJGDs were formulated according to the clinical usage documented in ancient medical texts. Ultra-high-performance liquid chromatography-quadrupole-time of flight mass spectrometry (UHPLC-Q-TOF-MS) was combined with computer-aided modeling screening to identify GJGD PPIs in rats following oral administration. Molecular docking experiments were carried out to predict the binding affinity of the PPIs to tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin-1β (IL-1β). Finally, the active ingredients of GJGD were further validated through pharmacodynamic experiments by assessing their efficacy in treating a wind-cold type of common cold in rats. ResultsA total of 61 compounds were identified in the GJGD, 8 of which were detected in rat blood samples, providing stronger evidence for PPIs. Molecular docking also confirmed that these 8 compounds had a better affinity for TNF-α, IL-6, and IL-1β. In animal studies, various doses of the GJGD groups and the positive control groups caused significant elevations (P < 0.05) in the levels of white blood cell count and lymphocyte ratio and caused a significant decrease (P < 0.05) in the monocyte ratio and neutrophilic granulocyte ratio compared to the model group. Organ indexes of the GJGD treated groups were higher than the model group (P < 0.05). Significant neutrophil infiltration, hemorrhage, compensatory vacuole, and interstitium proliferation were observed in the lung tissue of the model group. However, the lung tissues of the various dose groups that received GJGD showed a near normal appearance, except for slight thickening, interstitium proliferation, and compensatory vacuole in some areas. The GJGD was found to be effective against a cold-wind type of common cold, which is in accordance with molecular docking studies suggesting that GJGD may be effective against a cold-wind type of common cold. Finally, based on multidimensional analysis, 8 potential compounds in GJGD were identified as PPIs (puerarin, 3′-hydroxy puerarin, 3′- methoxy puerarin, daidzin, cinnamic acid, paeoniflorin, liquiritin, and glycyrrhizic acid). ConclusionThe present study combined water decoction spectral analysis, molecular docking, and in vivo blood plasma spectrum analysis to develop a multidimensional qualitative approach for the development of GJGD and to assess its effectiveness in a wind type of common cold in Sprague Dawley rats. Meanwhile, 8 compounds in the GJGD were identified as PPIs in this study, which may be useful in developing quality standards for complex TCM prescriptions.

A monoclonal antibody-based icELISA for puerarin

Puerarin was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) by periodate oxidation to serve as the immunogen and coating antigen, respectively. BALB/c mice were immunized with puerarin-BSA according to the routine immunization procedure, and the titer and specificity of serum were detected after three immunization. After booster immunization, mouse spleen lymphocytes were fused with mouse myeloma cells, and 24 hybridoma cell lines of the monoclonal antibodies against puerarin were screened by monoclonal antibody screening technique. Ascites was prepared and purified. The cross-reactivity of monoclonal antibody(mAb) M1 with 4'-methoxy puerarin, daidzin, puerarin-6″-O-xyloside, daidzein, mirificin, 3'-methoxy puerarin, and 3'-hydroxy puerarin was 239.84%, 112.18%, 67.89%, 58.28%, 22.37%, 0.40%, and 0.20%, respectively, and those with other analogs such as baicalein and baicalin were all less than 0.10%. The IC_(50) and the working range of the indirect competitive enzyme-linked immunosorbent assay(icELISA) for puerarin were 44.80 ng·mL~(-1) and 8.20-292.30 ng·mL~(-1), respectively. The average recovery was 91.95%-98.20% with an RSD in the range of 0.70%-2.60%. The content of puerarin in different Puerariae Lobatae Radix samples was determined with icELISA and validated by UPLC-MS. The correlation between data obtained from icELISA and UPLC-MS was 0.999 0, indicating that icELISA is suitable for the rapid detection of puerarin in Puerariae Lobatae Radix samples.

Pharmacokinetics of eight active ingredients of Salviae Miltiorrhizae Radix et Rhizoma-Puerariae Lobatae Radix combination in rats

To reveal the rationality of compatibility of Salviae Miltiorrhizae Radix et Rhizoma(SMRR) and Puerariae Lobatae Radix(PLR) from the perspective of pharmacokinetics, this study established a UPLC-MS/MS method for quantitative determination of PLR flavonoids(3'-hydroxy puerarin, puerarin, puerarin 6″-O-xyloside, 3'-methoxy puerarin, puerarin apioside) and salvianolic acids and tanshinones(salvianolic acid B, cryptotanshinone, and tanshinone Ⅱ_A) in plasma of rats. Rats were given SMRR extract, PLR extract, and SMRR-PLR extract by gavage and then plasma was collected at different time. UPLC separation was performed under the following conditions: Eclipse C_(18) column(2.1 mm×50 mm, 1.8 μm), 0.1% formic acid in water(A)-0.1% formic acid in acetonitrile(B) as mobile phase for gradient elution. Conditions for MS are as below: multiple reaction monitoring(MRM), ESI~(+/-). Comprehensive validation of the UPLC-MS/MS method(specifically, from the aspects of calibration curve, precision, accuracy, repeatability, stability, matrix effect, extract recovery) was performed and the result demonstrated that it complied with quantitative analysis requirements for biological samples. Compared with SMRR extract alone or PLR extract alone, SMRR-PLR extract significantly increased the AUC and C_(max) of PLR flavonoids and tanshinones in rat plasma, suggesting that the combination of SMRR and PLR promoted the absorption of the above components. The underlying mechanism needs to be further studied.

Studies on fingerprints and efficacy-related substance of classical prescription Zhuru Decoction

Based on fingerprint and network pharmacology,the whole process quality control of Zhuru Decoction was conducted and efficacy-related substances were predicted.The fingerprints of raw materials,decoction pieces and Zhuru Decoction were established,and 25 common peaks were identified,including 9 common chromatographic peaks of 3'-hydroxy puerarin,puerarin,3'-methoxy puerarin,puerarin,aperioside,daidzin,daidzein,liquiritin,glycyrrhizic acid and 6-gingerol, with similarity all greater than 0.95.The main groups of pharmacodynamic substances can be transferred from raw materials,decoction pieces to Zhuru Decoction step by step,with a clear affiliation relationship.Based on the testability and traceability,the active ingredients were screened,and the network relationship of &quot;component-target-pathway&quot; was constructed and analyzed for the nine chemical components screened by network pharmacology.The enriched pathways included energy metabolism,alcoholism,and smooth muscle contraction and relaxation-related pathways.The nine active components of Zhuru Decoction may achieve the effects of clearing heat, alleviating a hangover, harmonizing stomach and stopping vomiting through these signaling pathways.Based on transitive and traceable properties of the above 9 components as well as their close relationship to the efficacy of Zhuru Decoction,these 9 components can be identified as potential efficacy-related substances and provide basis for the overall quality control of Zhuru Decoction.

A reliable and rapid pharmacokinetic study of pueraria isoflavones using pueraria reference extractive substance in beagle plasma: Application to study of Yufeng Ningxin Tablets

ObjectiveIn order to evaluate the reliability and feasibility of pueraria reference extractive substance (RES) used in biological sample, the pharmacokinetics of 3′‑hydroxy puerarin (3′-HP), puerarin, 3′‑methoxy puerarin (3′-MP), and daidzein-8-C-apiosyl-(1-6)-glucoside (DAG) in beagle plasma following oral administration of Yufeng Ningxin Tablet were quantitated. MethodsA reliable and sensitive high-performance liquid chromatography-tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS) method developed with chromatographic separation was operated on a Merck C18 column, and acetonitrile-5 mmol/L ammonium was used as mobile phase in gradient elution. The plasma samples were deproteinized by acetone, detected by triple quadrupole mass spectrometry with an electrospray ionization interface, and quantified using selected ion monitoring mode. The pharmacokinetic parameters were calculated by Winnonlin 4.1. ResultsThe calibration curves of the reference extractive substance and standard substance methods were linear over the ranges 0.0417–11.3309 µg/mL and 0.0394–10.0000 µg/mL. The intra-day and inter-day precision of the two methods at three concentrations were less than 13.63%, and the average recoveries of 3′-HP, puerarin, 3′-MP, and DAG were more than 70.67%. The RSD of the mean plasma concentrations of the analytes calculated by the two methods was less than 5%, and cos (ϑ) = =1.000. Among the analytes, puerarin showed the highest blood concentration [(940 ± 185) ng/mL] and the longest retention time [(5 ± 1) h] in the dog's bodies. ConclusionPueraria reference extractive substance can be seen as an alternative to the standard substance to overcome the scarcity of standard substance for the analysis of biological samples.

Determination of 9 isoflavonoids in Puerariae Lobatae Radix with quantitative analysis of multi-components by single marker

A method for determination of 9 isoflavones in Puerariae Lobatae Radix was established and the accuracy and feasibility of the method were verified. The relative correction factors of eight isoflavonoids,3'-hydroxy puerarin,puerarinapioside,3'-methoxy puerarin,puerarin 6″-O-xyloside,daidzin,genistin,formononetin and daidzein were determined by HPLC method with puerarin as the internal standard. The contents of 9 isoflavonoids in 11 batches of samples were determined by external standard method and QAMS.The accuracy and feasibility of the methods were evaluated by comparison of the quantitative results between external standard method and QAMS. The reproducibility of the relative correction factors was good under different experimental conditions,and there was no significant difference between the external standard method of the 9 compounds and the content of QAMS method. The results showed that using puerarin as an internal standard to simultaneously determine the 8 isoflavonoids mentioned above is accurate and feasible. Thus,it can be used as quality control of Puerariae Lobatae Radix.

Qualitative and Quantitative Analysis of 24 Components in Jinlianhua Decoction by UPLC-MS/MS.

Jinlianhua Decoction (JD), composed of Flos Trollii, Herba Taraxaci, Folium Isatidis, Radix Puerariae Lobatae, and Folium Perillae in a ratio of 6:15:10:10:6, is a prescription for Fengwen which is a group of febrile diseases due to wind in Chinese medicine. It was originally used for the prevention and treatment of severe acute respiratory syndrome (SARS), and could also be used to treat influenza due to their common pathomechanism. To elucidate the unclear pharmacodynamic basis of JD, the LC-QExactive-MS system was used to qualitatively analyze its main components in this study. As a result, 89 compounds were identified and 24 important ones were selected thereby to further perform the simultaneous quantification in 8 batches of JD samples using LC-QTrap-MS with multiple reaction monitoring (MRM). Based on the qualitative and quantitative results in combination with the bioactivities reported, 16 compounds including orientin, 2″-O-β-l-galactopyranosylorientin, puerarin, trollisin I, rosmarinic acid, 2″-O-(2′″-methylbutanoyl) isoswertisin, daidzin, scutellarin, 3′-methoxy puerarin, vitexin, 3′-hydroxy puerarin, 2″-O-(2′″-methylbutanoyl) vitexin, kaempferol, caffeic acid, 3,4-dimethoxybenzoic acid, and cynaroside were determined as the major components of JD. This study provides a useful combinational method for analyzing the major pharmacodynamic substances of JD and lays a foundation for the quality control research of the decoction.

Green, infrared‐assisted extraction based on statistical modeling for ultra‐high performance liquid chromatography determination of bioactive isoflavones from Puerariae Lobatae

AbstractWe developed a green infrared‐assisted extraction method for the determination of 3′‐hydroxy puerarin, puerarin, daidzin, genistin, and daidzein in Pueraria lobata. After a simultaneous optimization of Box–Behnken design and desirability function, the developed method provided better isoflavone extraction efficiencies with a lower solvent consumption and shorter extraction time than other methods. The optimized extraction used an extraction time of 8 min, solid–liquid ratio of 1:100, and ethanol volume fraction of 60%. Additionally, ultra‐high performance liquid chromatography was used for identification and quantification of the five isoflavones. Our method showed good linearity (r &gt;0.999) over wide concentration ranges. The ranges for the limits of detection and quantitation were 0.03–0.09 μg/mL and 0.14–0.44 μg/mL, respectively, the recovery range was 98.38–103.37%, and the run time was 5.5 min. This approach is efficient, economical, and eco‐friendly, and shows promise for rapid extraction and quantitative analysis of bioactive isoflavones from Pueraria lobata.

Quality research of Puerariae Lobatae Radix from different habitats with UPLC fingerprint and determination of multi-component content

To establish ultra performance liquid chromatography( UPLC) fingerprint of Puerariae Lobatae Radix from different habitats and simultaneously determine the contents of six isoflavonoids. The UPLC fingerprint analysis and content determination were performed on a Waters ACQUITY UPLC BEH C_(18)( 2. 1 mm×50 mm,1. 7 μm) chromatographic column,with acetonitrile-0. 05% formic acid as mobile phase for gradient elution. The detection wavelength was set at 250 nm; the flow rate was 0. 2 mL·min~(-1); the column temperature was 30 ℃ and the injection volume was 2 μL. Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine( TCM) was adopted; principal component analysis( PCA) and discriminant analysis by partial least square method( PLS-DA) in Simca-P software were used to identify the differential components in samples from three habitats. The similarity was over 0. 90 in 29 batches of samples,indicating good consistency of the samples. The samples were clustered into 3 categories by PCA and PLS-DA,and six differential components such as puerarin apioside,daidzin,and isoflavoues aglycone were found. The determination results of 6 isoflavones,including 3'-hydroxy puerarin,puerarin,3'-methoxy puerarin,puerarin apioside,daidzin,and isoflavoues aglycone,showed that the content of the same component and the fluctuation range between different components were all different among different habitats. The total content of 6 isoflavones from different regions was Anhui 11. 21% >Henan 10. 97% >Shannxi 9. 38%. The establishment of UPLC fingerprint combined with simultaneous determination of 6 active components provides a more comprehensive reference for quality control and quality evaluation of Puerariae Lobatae Radix.

Pharmacokinetic characteristics of fourteen main components of Gegen Qinlian Decoction in rats

A specific and selective UPLC-MS/MS method was developed and validated for the simultaneous determination of isoflavonoids(3'-hydroxy puerarin, puerarin, daidzin, daidzein, genistin, genistein), flavonoids (baicalin, baicalein, wogonoside, wogonin, liquiritin)and alkaloids(berberine, jatrorrhizine, palmatine)(14 bioactive compounds) of Gegen Qinlian Decoction(GQD) in plasma. The pharmacokinetics characteristics of 14 bioactive compounds were study after oral administration of GQD at a single dose to rats. Prednisolone was used as the internal standard of liquiritin, and naringin was used as the internal standard of the other thirteen analytes. After the plasma samples were processed by precipitation protein method, the constituents and internal standards were gradient eluted by using a Zorbax SB-18 column with a mobile phase of acetonitrile(A) and 0.1% formic acid aqueous solution(B) using a gradient elution of 0-2.5 min, 15%-30% A; 2.5-3.5 min, 30%-35% A; 3.5-5.0 min, 35%-40% A; 5.0-9.0 min, 40%-60% A; 9.0-11.0 min, 60%-15% A, and the flow rate was 0.4 mL·min⁻¹. The auto sampler was conditioned at 25 °C and the sample injection volume was 5 μL. A mass spectrometry was applied with electrospray ionization (ESI) ion source in the positive and negative ion multiple reaction monitoring(MRM) mode. All pharmacokinetic parameters were processed by non-compartmental analysis with DAS 3.2.2 software. The results showed that the linear correlation coefficient of the 14 components were all greater than 0.99, indicating that the method had good linearity in their respective concentration ranges. Post-preparative stability (25 °C, 24 h), short-term stability(25 °C, 12 h), long-term stability (-20 °C, 7 d), and freeze and thaw stability (3-cycles) of the fourteen constituents were examined to evaluate the stability of methodology. The results of the inner and inter-day relative standard deviations were both less than 10%, indicating legitimate precise and accuracy to the requirement of biological sample analysis. The assay method is proved to be sensitive, accurate and convenient. It can be applied to the pharmacokinetic study of the fourteen analytes. The kinetic parameters of the related drugs were calculated according to the blood concentration of the 14 components. The results showed that the MRT0-t of the isoflavones and flavonoids was 7.5-11.8 h, T1/2z were mainly in 11.0-29.7 h, and the AUC0-t flavonoids were larger than the isoflavones. The MRT0-t of alkaloids were between 4.3-7.2 h, T1/2z were 1.0-5.0 h, AUC0-t were less than flavonoids and isoflavones. The results suggest that flavonoids and isoflavones have a high concentration of blood and long time of action, which are beneficial to the anti-inflammatory and antipyretic effects. The concentration of alkaloids in the body is low and the time of action is short, and it may play its bacteriostasis in the intestinal tract.

Simultaneous determination and pharmacokinetics of fourteen bioactive compounds in rat plasma by LC-ESI-MS/MS following intravenous injection of Gegen-Sanqi compatibility solution.

A high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method was developed and fully validated for simultaneous determination of puerarin, 3'-hydroxy puerarin, 6″-O-xylosyl puerarin, 3'-methoxy puerarin, mirificin, puerarin-7-O-glucoside, daidzin, daidzein, daidzein-7-O-glucoside, ginsenoside-Rd, notoginsenoside-R1, ginsenoside-Re, ginsenoside-Rg1, ginsenoside-Rb1 in rat plasma after intravenous injection of 5mL/kg Gegen-Sanqi compatibility solution (containing Pueraria flavonid 10.8mg/mL and Panax notoginsenosidum 5.4mg/mL). After addition of internal standard (IS) baicalin, the analytes and IS were recovered from rat plasma by protein precipitation using acetonitrile containing 0.1% formic acid. Chromatographic separation was performed on a Capcell pak MG C18 column (3.0mm×75mm, 3.0μm) at 35°C with a flow rate of 0.75mL/min. Mass spectrometry was conducted using multiple reaction monitoring in positive mode. The method was linear for all of the analytes over 1000 times concentration range with correlation coefficients greater than 0.99. The precision and accuracy of the LC-MS/MS assay based on the three analytical quality control (QC) levels were well within the acceptance criteria from FDA guidance for bioanalytical method validation. Method recoveries were higher than 75% and the matrix effects were minimal. All analytes were stable under tested conditions. It was the first time to study the pharmacokinetics of daidzein-7-O-glucoside in rat plasma. To the best of our knowledge, this is the first study for simultaneous determination of so many analytes in rat plasma after intravenous injection of Gegen-Sanqi compatibility solution. It was expected that the present work would provide some helpful references for the apprehension of the mechanism of action and further clinical efficacy studies of Gegen-Sanqi herb-pair.

Brain Pharmacokinetics and the Pharmacological Effects on Striatal Neurotransmitter Levels of Pueraria lobata Isoflavonoids in Rat.

Isoflavonoids are putatively active components of Pueraria lobata and has been demonstrated prominent neuro-protection effect against cerebrovascular disorders, hypertension or Parkinson's disease (PD). However, the molecular basis for the beneficial effect of Pueraria lobata on nervous systems has not been well revealed. The present study aims to assess striatum exposure to main active isoflavonoids and changes of striatal extracellular neurotransmitters levels in rat brain after intravenous administration of Pueraria lobata isoflavonoids extracts (PLF), to further elucidate its' substantial bases for neuro activities. Fifteen rats were divided into 3 groups (five rats in each group) to receive a dose of PLF at 80 or 160 mg/kg or normal saline (vehicle), respectively. An LC-MS/MS method was employed to determine the concentrations of five main isoflavonoids and multiple neurotransmitters in microdialysate from striatal extracellular fluid (ECF) of the rats. The exposed quantities of puerarin (PU), 3′-methoxypuerarin (MPU), daidzein-8-C-apiosyl-(1-6)-glucoside (DAC), and 3′-hydroxypuerarin (HPU) in striatum were dose-dependent. The content of daidzein (DAZ) was too low to be detected in all dialysate samples through the experiment. Optimal dose PLF (80 mg/kg) promoted DA metabolism and inhibited 5-HT metabolism. No obvious change in the level of GLu was determined. The concentration of GABA presented a temporary decline firstly and then a gradual uptrend followed by a further downtrend. Higher dose (160 mg/kg) PLF could enhance the metabolism of both DA and 5-HT, and lower the extracellular level of GLu, without changing GABA concentrations, which might result in alleviation on excitatory toxicity under conditions, such as ischemia. The results infer that different dose of PLF should be chosen to achieve appropriate neurochemical modulation effects under conditions, such as hypertension or ischemia/stroke. These findings may significantly contribute to a better understanding of the neuroprotective effect of Pueraria lobata and provide new insights into its application toward neuro-degenerative diseases in the future.