This study aimed to examine the effect and underlying mechanism of Puerariae Lobatae Radix on insulin resistance in db/db mice with type 2 diabetes mellitus(T2DM) based on the analysis of intestinal flora. Fifty db/db mice were randomly divided into a model group(M group), a metformin group(YX group), a high-dose Puerariae Lobatae Radix group(YGG group), a medium-dose Puerariae Lobatae Radix group(YGZ group), and a low-dose Puerariae Lobatae Radix group(YGD group). Another 10 db/m mice were assigned to the normal group(K group). After continuous administration for eight weeks, body weight and blood sugar of mice were measured. Enzyme linked immunosorbent assay(ELISA) was used to detect glycosylated serum protein(GSP) and fasting serum insulin(FINS), and insulin resistance index(HOMA-IR) was calculated. The histopathological changes in the pancreas were observed by HE staining. Tumor necrosis factor(TNF)-α expression in the pancreas was detected using immunohistochemistry. The structural changes in fecal intestinal flora in the K, M, and YGZ groups were detected by 16S rRNA. Western blot was used to detect the expression of farnesoid X receptor(FXR) and takeda G protein-coupled receptor 5(TGR5) in the ileum, cholesterol 7α-hydroxylase(CYP7A1) and sterol 27α-hydroxylase(CYP27A1) in the liver, and G protein-coupled receptors 41(GPR41) and 43(GPR43) in the colon. Compared with the K group, the M group showed increased body weight, blood sugar, serum GSP, fasting blood glucose(FBG), and FINS, increased HOMA-IR, inflammatory infiltration of islet cells, necrosis and degeneration of massive acinar cells, unclear boundary between islet cells and acinar cells, disturbed intestinal flora, and down-regulated FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43. Compared with the M group, the YX, YGG, YGZ, and YGD groups showed decreased body weight, blood sugar, serum GSP, FBG, and FINS, islet cells with intact and clumpy morphology and clear boundary, necrosis of a few acinar cells, and more visible islet cells. The intestinal flora in the YGZ group changed from phylum to genus levels, and the relative abundance of intestinal flora affecting the metabolites of intestinal flora increased. The protein expression of FXR, TGR5, CYP7A1, CYP27A1, GPR41, and GPR43 increased. The results show that Puerariae Lobatae Radix can improve the inflammatory damage of pancreatic islet cells and reduce insulin resistance in db/db mice with T2DM. The mechanism of action may be related to the increase in the abundance of Actinobacteria, Bifidobacterium, and Bacteroides in the intestinal tract and the protein expression related to metabolites of intestinal flora.
Mice ; Animals ; Insulin Resistance ; Blood Glucose/metabolism* ; Diabetes Mellitus, Type 2/genetics* ; Pueraria/chemistry* ; Gastrointestinal Microbiome ; RNA, Ribosomal, 16S ; Body Weight ; Necrosis

The effective components of flavonoids in the "Pueraria lobata-Hovenia dulcis" drug pair have low bioavailability in vivo due to their unstable characteristics. This study used microemulsions with amphoteric carrier properties to solve this problem. The study drew pseudo-ternary phase diagrams through titration compatibility experiments of the oil phase with emulsifiers and co-emulsifiers and screened the prescription composition of blank microemulsions. The study used average particle size and PDI as evaluation indicators, and the central composite design-response surface method(CCD-RSM) was used to optimize the prescription; high-dosage drug-loaded microemulsions were obtained, and their physicochemical properties, appearance, and stability were evaluated. The results showed that when ethyl butyrate was used as the oil phase, polysorbate 80(tween 80) as the surfactant, and anhydrous ethanol as the cosurfactant, the maximum microemulsion area was obtained. When the difference in results was small, K_(m )of 1∶4 was chosen to ensure the safety of the prescription. The prescription composition optimized by the CCD-RSM was ethyl butyrate(16.28%), tween 80(9.59%), and anhydrous ethanol(38.34%). When the dosage reached 3% of the system mass, the total flavonoid microemulsion prepared had a clear and transparent appearance, with average particle size, PDI, and potential of(74.25±1.58)nm, 0.277±0.043, and(-0.08±0.07) mV, respectively. The microemulsion was spherical and evenly distributed under transmission electron microscopy. The centrifugal stability and temperature stability were good, and there was no layering or demulsification phenomenon, which significantly improved the in vitro dissolution of total flavonoids.
Polysorbates/chemistry* ; Flavonoids ; Pueraria ; Surface-Active Agents/chemistry* ; Ethanol ; Emulsions ; Particle Size ; Solubility

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.
Cloning, Molecular ; Genistein ; Glucosyltransferases/metabolism* ; Isoflavones/pharmacology* ; Pueraria/chemistry*

Dangefentong Capsules is a new traditional Chinese medicine preparation for the treatment of diabetic peripheral neuropathy. It is based on the Salviae Miltiorrhizae Radix et Rhizoma-Puerariae Lobatae Radix herb pair with salvianolic acids, tanshinones and pueraria flavonoids as main components. Studying the chemical composition in vivo of Dangefentong Capsules and its metabolites is of great significance for making clear its pharmacodynamic material basis and the action mechanism. The UHPLC-Q/Orbitrap-MS/MS was applied to rapidly analyze the metabolites and metabolic pathways of Dangefentong Capsules in Beagle dogs after gavage. Eclipse plus C_(18) column(2.1 mm×50 mm, 1.8 μm) was used, and gradient elution was performed with 0.1% formic acid aqueous solution(A)-formic acid acetonitrile solution(B). A heated electrospray ion source(HESI) was employed. The scanning mode was set as the positive and negative ion mode, and the mass scanning range was m/z 100-1 000. The plasma, urine and feces samples were collected after male Beagle dogs were administered with Dangefentong Capsules. The prototype components and metabolites were identified by UHPLC-Q/Orbitrap-MS/MS analysis combined with reference substances and references. The results showed that 107 chemical components were identified, including 58 prototype components and 49 metabolites. The identified prototype components included 42 components from Salviae Miltiorrhizae Radix et Rhizoma and 16 components from Puerariae Lobatae Radix. The metabolites consist of 21 and 28 metabolites of Salviae Miltiorrhizae Radix et Rhizoma and Puerariae Lobatae Radix, respectively. They are mainly derived from the methylation, hydroxylation, sulfation and glucuronidation of salvianolic acids, tanshinones and pueraria flavonoids. This research rapi-dly analyzes the chemical components in vivo of Beagle dogs administered with Dangefentong Capsules, laying a basis for illustrating the pharmacodynamic material basis and mechanism of Dangefentong Capsules.
Abietanes ; Acetonitriles ; Alkenes ; Animals ; Capsules ; Chromatography, High Pressure Liquid/methods* ; Dogs ; Drugs, Chinese Herbal/chemistry* ; Flavonoids ; Formates ; Male ; Polyphenols ; Pueraria ; Tandem Mass Spectrometry

Histone lysine-specific demethylase 1(LSD1) has become a promising molecular target for lung cancer therapy. Upon the screening platform for LSD1 activity, some Chinese herbal extracts were screened for LSD1 activity inhibition, and the underlying mechanism was preliminarily investigated at both molecular and cellular levels. The results of LSD1 inhibition showed that Puerariae Lobatae Radix extract can effectively reduce LSD1 expression to elevate the expression of H3 K4 me2 and H3 K9 me2 substrates in H1975 and H1299 cells. Furthermore, Puerariae Lobatae Radix was evaluated for its anti-lung cancer activity. It had a potent inhibitory ability against the proliferation and colony formation of both H1975 and H1299 cells. Flow cytometry and DAPI staining assays indicated that Puerariae Lobatae Radix can induce the apoptosis of lung cancer cells. In addition, it can significantly suppress the migration and reverse the epithelial-mesenchymal transition(EMT) process of lung cancer cells by activating E-cadherin and suppressing the expression of N-cadherin, slug and vimentin. To sum up, Puerariae Lobatae Radix displayed a robust inhibitory activity against lung cancer, and the mechanism may be related to the down-regulation of LSD1 expression to induce the cell apoptosis and suppress the cell migration and EMT process. These findings will provide new insights into the action of Puerariae Lobatae Radix as an anti-lung cancer agent and offer new ideas for the study on the anti-cancer action of Chinese medicine based on the epigenetic modification.
Pueraria/chemistry* ; Histone Demethylases/analysis* ; Plant Roots/chemistry* ; Epithelial-Mesenchymal Transition ; Neoplasms

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.
Animals ; Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal/pharmacokinetics* ; Plant Roots/chemistry* ; Pueraria/chemistry* ; Rats ; Rhizome/chemistry* ; Salvia miltiorrhiza/chemistry* ; Tandem Mass Spectrometry

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.
Chromatography, High Pressure Liquid ; Drugs, Chinese Herbal ; chemistry ; Ecosystem ; Flavonoids ; analysis ; Phytochemicals ; analysis ; Plant Roots ; chemistry ; Pueraria ; chemistry

Puerariae Lobatae Radix is a traditional Chinese medicine,which was first recorded in Shennong Classic of Materia Medica,and was recorded in many ancient books. Its main effect is to relieve muscles to expel heat,produce saliva and promote eruption,invigorate splenic yang and stop diarrhea. CNKI and Wanfang Data were searched in this paper with the words " Pueraria", " puerarin usage" and " puerarin application" as the key words,and it was found that the puerarin usage characteristics were rarely reported.Therefore,the application characteristics of fresh use,crude use and processed use of Puerariae Lobatae Radix in ancient books were summarized in this paper,in order to provide a reference for the modern development of Puerariae Lobatae Radix.
Drugs, Chinese Herbal ; therapeutic use ; Humans ; Medicine, Chinese Traditional ; Plant Roots ; chemistry ; Pueraria ; chemistry

Objective: To explore the effects of Kudzu Root plus Cinnamon Granules (KR+C) on prostatic hyperplasia (PH) in mice. METHODS: Sixty 4-week-old Kunming male mice were randomly divided into six groups: blank control, PH model, high-, medium- and low-dose KR+C, and finasteride control. All the mice except those in the blank control group were subcutaneously injected with testosterone propionate (5 mg / ［kg·d］) at 7 days after surgical castration. The animals of different groups were treated intragastrically with different doses of KR+C, finasteride, and normal saline respectively for 3 weeks and then sacrificed for weighing of the prostate, calculation of the prostatic index, observation of the morphological changes in the prostate after HE staining, determination of the expressions of FGF2, Ki67 and TGF-β1 by immunohistochemistry, detection of 5α-reductase activity by ELISA, and measurement of the apoptosis index of the prostatic cells by TUNEL. RESULTS: Compared with the model controls, the mice of the other groups showed significantly reduced prostatic volume (P <0.05), prostatic index (P <0.05), expressions of FGF2, Ki67 and TGF-β1, and activity of 5 α-reductase (P <0.05), but remarkably increased apoptosis index of the prostatic cells (P <0.05). However, no statistically significant differences were observed in the above parameters between the finasteride control and the three KR+C groups (P>0.05). CONCLUSIONS KR+C can reduce the prostatic volume of PH mice by decreasing the activity of 5α- reductase, inhibiting the expressions of FGF2, Ki67 and TGF-β1, and promoting the apoptosis of prostatic cells.
Animals ; Apoptosis ; Cholestenone 5 alpha-Reductase ; metabolism ; Cinnamomum zeylanicum ; chemistry ; Fibroblast Growth Factor 2 ; metabolism ; Finasteride ; therapeutic use ; In Situ Nick-End Labeling ; Ki-67 Antigen ; metabolism ; Male ; Mice ; Organ Size ; Phytotherapy ; methods ; Plant Roots ; chemistry ; Prostate ; pathology ; Prostatic Hyperplasia ; drug therapy ; metabolism ; pathology ; Pueraria ; chemistry ; Random Allocation ; Testosterone Propionate ; administration & dosage ; Transforming Growth Factor beta1 ; metabolism ; Urological Agents ; therapeutic use

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