Isoflavones are widely consumed by people around the world in the form of soy products, dietary supplements and drugs. Many isoflavones or related crude extracts have been reported to exert pain-relief activities, but the mechanism remains unclear. Voltage-gated sodium channels (VGSCs) play important roles in excitability of pain sensing neurons and many of them are important nociceptors. Here, we report that several isoflavones including 3'-methoxydaidzein (3MOD), genistein (GEN) and daidzein (DAI) show abilities to block VGSCs and thus to attenuate chemicals and heat induced acute pain or chronic constriction injury (CCI) induced pain hypersensitivity in mice. Especially, 3MOD shows strong analgesic potential without inducing addiction through inhibiting subtypes Na1.7, Na1.8 and Na1.3 with the IC of 181 ± 14, 397 ± 26, and 505 ± 46 nmol·L, respectively, providing a promising compound or parent structure for the treatment of pain pathologies. This study reveals a pain-alleviating mechanism of dietary isoflavones and may provide a convenient avenue to alleviate pain.
Analgesics ; administration & dosage ; chemistry ; Animals ; Humans ; Isoflavones ; administration & dosage ; chemistry ; Male ; Mice ; Mice, Inbred C57BL ; Pain ; drug therapy ; genetics ; metabolism ; Voltage-Gated Sodium Channel Blockers ; administration & dosage ; Voltage-Gated Sodium Channels ; genetics ; metabolism

Puerarin is a naturally occurring isoflavone C-glycoside,isolated from the root of Pueraria lobata,which has attracted extensive attention in the medical circles because of its various pharmacological effects,such as vasodilation,cardioprotection,neuroprotection,antioxidant,anticancer,anti-inflammation,alleviating pain,promoting bone formation,inhibiting alcohol intake,and attenuating insulin resistance. However,its low oral bioavailability has limited its clinical application. This review gives a comprehensive summary of the researches on physicochemical properties,pharmacokinetics( absorption,distribution,metabolism and excretion,pharmacokinetic parameters) in oral administration,and pharmaceutics research strategies of puerarin in recent years,and the in vivo behavior difference between multicomponent and single component environment was also summarized. The reasons( low water solubility,poor membrane permeability,short half-life,inhibition of P-gp efflux and first-pass metabolic effects of intestinal enzymes,etc.) for low bioavailability were concluded and the idea that multicomponent enviroment would affect the bioavailability was clarified. The aim of this review is to provide literature basis for the development of new dosage forms and new technologies for multivariate compound drug delivery system to improve the bioavailability of oral puerarin,and to propose ways to improve the bioavailability of BCS Ⅳ drugs derived from traditional Chinese medicine by fully enlarging the synergistic effect of multi-components or reasonably using the inhibitory effect between components.
Administration, Oral ; Biopharmaceutics ; Isoflavones ; Pueraria

Buyang Huanwu Decoction (BYHWD) is a well-known traditional Chinese medicine prescription which is used to treat ischaemic stroke and stroke-induced disabilities. However, the exact mechanism underlying BYHWD's amelioration of ischaemic stroke and its effective constituents remain unclear. The present study aimed to identify the effective constituents of BYHWD and to further explore its action mechanisms in the amelioration of ischaemic stroke by testing the activities of 15 absorbable chemical constituents of BYHWD with the same methods under the same conditions. The following actions of these 15 compounds were revealed: 1) Ferulic acid, calycosin, formononetin, astrapterocarpan-3-O-β-D-glucoside, paeonol, calycosin-7-O-β-D-glucoside, astraisoflavan-7-O-β-D-glucoside, ligustrazine, and propyl gallate significantly suppressed concanavalin A (Con A)-induced T lymphocyte proliferation; 2) Propyl gallate, calycosin-7-O-β-D-glucoside, paeonol, and ferulic acid markedly inhibited LPS-induced apoptosis in RAW264.7 cells; 3) Propyl gallate and formononetin significantly inhibited LPS-induced NO release; 4) Hydroxysafflor yellow A and inosine protected PC12 cells against the injuries caused by glutamate; and 5) Formononetin, astragaloside IV, astraisoflavan-7-O-β-D-glucoside, inosine, paeoniflorin, ononin, paeonol, propyl gallate, ligustrazine, and ferulic acid significantly suppressed the constriction of the thoracic aorta induced by KCl in rats. In conclusion, the results from the present study suggest that BYHWD exerts its ischaemic stroke ameliorating activities by modulating multiple targets with multiple components.
Animals ; Apoptosis ; drug effects ; Brain Ischemia ; drug therapy ; physiopathology ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Glucosides ; administration & dosage ; analysis ; Isoflavones ; administration & dosage ; analysis ; Male ; Mice ; Mice, Inbred BALB C ; Monoterpenes ; administration & dosage ; analysis ; PC12 Cells ; RAW 264.7 Cells ; Rats ; Rats, Sprague-Dawley ; Saponins ; administration & dosage ; analysis ; Stroke ; drug therapy ; physiopathology ; Triterpenes ; administration & dosage ; analysis

OBJECTIVE: To investigate whether CaN-NFAT3 pathway mediates the protective effects of aldehyde dehydrogenase (ALDH) 2 in high glucose-treated neonatal rat ventricular myocytes. METHODS: The ventricular myocytes were isolated from the heart of neonatal (within 3 days) SD rats by enzyme digestion and cultured in the presence of 5-Brdu. After reaching confluence, the cultured ventricular myocytes were identified using immunofluorescence assay for -SA protein. The cells were then cultured in either normal (5 mmol/L) or high glucose (30 mmol/L) medium in the presence of ALDH2 agonist Alda-1, ALDH 2 inhibitor Daidzin, or Alda-1 and NFAT3 inhibitor (11R-VIVIT). Fluorescent probe and ELISA were used to detect intracellular Ca concentration and CaN content, respectively; ALDH2, CaN and NFAT3 protein expressions in the cells were detected using Western blotting. RESULTS: Compared with cells cultured in normal glucose, the cells exposed to high glucose showed a significantly decreased expression of ALDH2 protein ( < 0.05) and increased expressions of CaN ( < 0.05) and NFAT3 proteins with also increased intracellular CaN and Ca concentrations ( < 0.01). Alda-1 treatment significantly lowered Ca concentration ( < 0.05), intracellular CaN content ( < 0.01), and CaN and NFAT3 protein expressions ( < 0.05), and increased ALDH2 protein expression ( < 0.05) in high glucose- exposed cells; Daidzin treatment significantly increased Ca concentration ( < 0.01) and intracellular CaN content ( < 0.05) in the exposed cells. Compared with Alda-1 alone, treatment of the high glucose-exposed cells with both Alda-1 and 11R-VIVIT did not produce significant changes in the expression of ALDH2 protein (>0.05) but significantly reduced the expression of NFAT3 protein ( < 0.05). CONCLUSIONS Mitochondrial ALDH2 protects neonatal rat cardiomyocytes against high glucose-induced injury possibly by negatively regulating Ca-CaN-NFAT3 signaling pathway.
Aldehyde Dehydrogenase, Mitochondrial ; antagonists & inhibitors ; metabolism ; Animals ; Animals, Newborn ; Benzamides ; pharmacology ; Benzodioxoles ; pharmacology ; Calcium ; metabolism ; Cells, Cultured ; Culture Media ; Enzyme Inhibitors ; pharmacology ; Glucose ; administration & dosage ; pharmacology ; Isoflavones ; pharmacology ; Mitochondria, Heart ; enzymology ; Myocytes, Cardiac ; drug effects ; metabolism ; NFATC Transcription Factors ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Rats ; Rats, Sprague-Dawley

The present study aimed at investigating the effects of Puerarin (PR), a major isoflavonoid isolated from the Chinese medicinal herb Puerariae radix, on bone metabolism and the underlying mechanism of action. The in vivo assay, female mice were ovariectomized (OVX), and the OVX mice were fed with a diet containing low, middle, and high doses of PR (2, 4, and 8 mg·d(-1), respectively) or 17β-estradiol (E2, 0.03 μg·d(-1)) for 4 weeks. In OVX mice, the uterine weight declined, and intake of PR at any dose did not affect uterine weight, compared with the control. The total femoral bone mineral density (BMD) was significantly reduced by OVX, which was reversed by intake of the diet with PR at any dose, especially at the low dose. In the in vitro assay, RAW264.7 cells were used for studying the direct effect of PR on the formation of osteoclasts. PR reduced the formation of tartrate resistant acid phosphatase (TRAP)-positive multi-nucleated cells in the RAW 264.7 cells induced by receptor activator for nuclear factor-κB Ligand (RANKL). MC3T3-E1 cells were used for studying the effects of PR on the expression of osteoprotegerin (OPG) and RANKL mRNA expression in osteoblasts. The expression of OPG mRNA and RANKL mRNA was detected by RT-PCR on Days of 5, 7, 10, and 12 after PR exposure. PR time-dependently enhanced the expression of OPG mRNA and reduced the expression of RANKL mRNA in MC3T3-E1 cells. In conclusion, our results suggest that PR can effectively prevent bone loss in OVX mice without any hyperplastic effect on the uterus, and the antiosteoporosis activity of PR may be related to its effects on the formation of osteoclasts and the expression of RANKL OPG in osteoblasts.
Animals ; Bone Density ; drug effects ; Drugs, Chinese Herbal ; administration & dosage ; Female ; Femur ; chemistry ; growth & development ; metabolism ; Humans ; Isoflavones ; administration & dosage ; Mice ; Osteoclasts ; drug effects ; metabolism ; Osteoporosis ; genetics ; metabolism ; physiopathology ; prevention & control ; Osteoprotegerin ; genetics ; metabolism ; Ovariectomy ; Pueraria ; chemistry ; RANK Ligand ; genetics ; metabolism

To establish a HPLC method for simultaneously determining plasma concentrations of gastrodin (Gas) and its metabolites hydroxybenzyl alcohol (HBA), puerarin (Pur) and internal standard (IS) p-hydroxyphenylethanol (Tyr) in rats and studying the pharmacokinetic process and interactions of gastrodin and puerarin after single and combined intravenous injection and oral administration. With Tyr as the internal standard, plasma samples were processed with methanol for protein precipitation, supernatant was dried with N2, and residues were re-dissolved with acetonitrile-0.05% phosphoric acid (20: 80). Chromatography was carried out on an Agilent ZORBAX SB-Aq C18 column (4.6 mm x 250 mm, 5 μm), with acetonitrile-0.05% phosphoric acid as the gradient mobile phase for the gradient elution. The UV detector wavelength was set at 221 nm for Gas HBA and IS and 250 nm for Pur. After the single or combined administration of Gas and Pur, their plasma concentrations in rats were detected. WinNonlin 5.2 pharmacokinetic software and SPSS 17. 0 software were used to respectively calculate pharmacokinetic parameters of each group, make a statistical analysis and compare the pharmacokinetic processes of Gas and Pur after the single or combined administration. According to the results, the absolute recoveries between low, media and high concentrations of Gas, HBA and Pur and IS as well as Tyr were more than 77.20%, with a good linearity (r > 0.999 6, n = 5) for Gas, HBA and Pur within concentration ranges of 0.10-101, 0.03-7.58 and 0.05-5.98 mg xL ('1) respectively. The lower limits of quantification for Gas, HBA and Pur were 0.10, 0.03, 0.05 mg x L(-1), respectively. Their in-ra-day and inter-day precisions were less than 12% with the accuracy between 85. 1% -1 10. %. All of the three substances and IS were stable during the whole analysis process. The findings showed significant differences in the main in vivo pharmacokinetic parame-ers in rats (AUC, C.(max) T,½ T.(max) MRT) after the single and combined administration of Gas and Pur. Either after the oral adminis-ration or after the intravenous injection, parameters showed a lower clearance rate ( L) longer mean residence time ( RT) and higher relative bioavailability, especially after the oral administration. Specifically, the relative bioavailability of the combined oral ad-inistration of Pur was 10. 7 times of that of the single administration, while that of Gas was 1. times of that of the single administra-ion. The combined administration of Gas and Pur can promote the absorption, decrease the elimination rate and prolong the mean resi-ence time. The method is simple and accurate and can be applied in the simultaneous determination of plasma concentrations of Gas, HBA and Pur in rats and the pharmacokinetic studies.
Administration, Oral ; Animals ; Benzyl Alcohols ; administration & dosage ; blood ; pharmacokinetics ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; administration & dosage ; analysis ; pharmacokinetics ; Glucosides ; administration & dosage ; blood ; pharmacokinetics ; Isoflavones ; administration & dosage ; blood ; pharmacokinetics ; Male ; Rats ; Rats, Wistar

The present study was designed to determine the effects of puerarin pre-treatment on the pharmacokinetics of the oral anticoagulant agent warfarin and the antiplatelet agent clopidogrel in rats. In the treatment group, rats was gavaged with warfarin or clopidogrel after repeated treatment with puerarin at intraperitoneal doses of 20, 60, or 200 mg·kg(-1) for 7 days, while rats in the control group were administrated only with the same dose warfarin or clopidogrel. Plasma samples were obtained at the prescribed times and analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). The results showed that rats treated with puerarin at all the test doses of 20, 60 and 200 mg·kg(-1) were found to affect the pharmacokinetics of warfarin, but not clopidogrel, suggesting a potential herb-drug interaction between puerarin and warfarin.
Animals ; Anticoagulants ; pharmacokinetics ; Chromatography, Liquid ; Clopidogrel ; Drug Administration Schedule ; Herb-Drug Interactions ; Injections, Intraperitoneal ; Isoflavones ; administration & dosage ; pharmacology ; Male ; Platelet Aggregation Inhibitors ; pharmacokinetics ; Rats ; Rats, Wistar ; Tandem Mass Spectrometry ; Ticlopidine ; analogs & derivatives ; pharmacokinetics ; Vasodilator Agents ; administration & dosage ; pharmacology ; Warfarin ; pharmacokinetics

In order to test the equilibrium solubility of puerarin in different solvents and solubilizer,cilia toxicity and irritation of these excipient, the balance method, toad in the ciliary body toxicity and rat nasal mucosa irritation were used respectively. Results showed that puerarin solubility was 56.44 g x L(-1) in combined solvent of 30% PEG200 and 10% Kolliphor HS 15. With normal saline solution as negative control and sodium deoxycholate as positive control, the effects of 30% PEG200, 30% PEG 400, 10% Kolliphor HS 15 and combination of 30% of PEG200 and 10% Kolliphor HS 15 on toad palate cilium were observed and cilia movement duration was recorded. The results indicated that there was no significant difference in cilia movement duration among 30% PEG200, 10% Kolliphor HS 15 and normal saline group. The rats long-term nasal mucous membrane irritation of 30% PEG 400, 10% Kolliphor HS 15, which had no cilia toxicity, was studied, with normal saline solution as negative control. There were no significant difference revealed on rat nasal mucosa epithelial thickness among 30% PEG 400, 10% Kolliphor HS 15 and normal saline. Above researches showed 30% PEG 400, 10% Kolliphor HS 15 was ideal for solubility of puerarin nasal drops and showed a lower cilia toxicity and irritation, and can be used as the solvent and solubilizer of puerarin nasal drops.
Administration, Intranasal ; methods ; Animals ; Anura ; Cilia ; chemistry ; Female ; Isoflavones ; chemistry ; Male ; Nasal Mucosa ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Sprague-Dawley ; Solubility ; Solvents ; chemistry

Animals ; Hemorheology ; Isoflavones ; administration & dosage ; pharmacology ; Male ; Pulmonary Artery ; physiopathology ; Pulmonary Heart Disease ; blood ; physiopathology ; Rats ; Rats, Sprague-Dawley

AIM: To establish a sensitive and rapid liquid chromatographic-tandem mass spectrometry (LC-MS/MS) method for the quantitative analysis of dehydrated puerarin in rat plasma, and its application for pharmacokinetic studies. METHODS: A plasma sample was pretreated by one-step protein precipitation by the addition of five volumes of methanol. The chromatographic separation was achieved on a Zorbax SB-C18 column (4.6 mm × 150 mm I.D. 5.0 μm, Agilent, USA) at 40 °C at a flow rate of 0.6 mL·min(-1) by an isocratic elution consisting of 10 mmol·L(-1) ammonium acetate in methanol and water containing 0.1% formic acid in a ratio of 20 : 80 (V/V). Detection was performed on a triple quadrupole mass spectrometer in multiple-reaction monitoring (MRM) mode. An atmospheric pressure chemical ionization (APCI) interface in positive ionization mode was used by monitoring the transitions from m/z 399.1→281.0 (dehydrated puerarin) and m/z 271.0→215.0 (internal standard, IS). RESULTS: Calibration curves were linear in the concentration range from 1.50 to 5400 ng·mL(-1), and the lower limit of quantification (LLOQ) was 1.50 ng·mL(-1) in rat plasma. The accuracy and precision values, which were calculated from three different sets of quality control samples analyzed in sextuplicate on three different days, ranged from 95.73% to 103.18%, and from 4.33% to 7.86%, respectively. CONCLUSION The method was successfully applied to assess the pharmacokinetics of dehydrated puerarin after oral administration in rats.
Animals ; Chromatography, High Pressure Liquid ; methods ; Drug Stability ; Drugs, Chinese Herbal ; administration & dosage ; analysis ; metabolism ; pharmacokinetics ; Female ; Isoflavones ; administration & dosage ; blood ; metabolism ; pharmacokinetics ; Male ; Pueraria ; chemistry ; Rats ; Rats, Wistar ; Tandem Mass Spectrometry ; methods