Puerarin

Puerarin

Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17β-estradiol (E2) and producing an anti-estrogenic effect. This study was to investigate whether puerarin could suppress the invasion and vascularization of E2-stimulated endometriotic tissue. The endometriotic stromal cells (ESCs) were successfully established and their invasive ability under different treatments was assessed through a Transwell Assay. Simultaneously, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by western blotting. 

The CAM assay indicated that puerarin (10−9 mol/L) also inhibited the angiopoiesis of endometriotic tissue stimulated by the E2 (10−8 mol/L) treatment (P<0.05). Accordingly, immunohistochemistry showed that the accumulation of MMP-9, ICAM-1, and VEGF was reduced whereas that of TIMP-1 increased in the combination treatment group compared with the E2 treatment group. The study demonstrated that puerarin could suppress the tissue invasion by ESCs and the vascularization of ectopic endometrial tissues stimulated by E2, suggesting that puerarin may be a potential drug for the treatment of endometriosis.1

Puerarin Suppresses Invasion and Vascularization of Endometriosis Tissue Stimulated by 17β-Estradiol

Wang D, Liu Y, Han J, Zai D, Ji M, Cheng W, Xu L, Yang L, He M, Ni J, Cai Z, Yu  C. PLoS One. 2011; 6(9): e25011. doi: 10.1371/journal.pone.0025011

Abstract

Puerarin, a phytoestrogen with a weak estrogenic effect, binds to estrogen receptors, thereby competing with 17β-Estradiol (E2) and producing an anti-estrogenic effect. This study was to investigate whether puerarin could suppress the Invasion and Vascularization of E2-Stimulated endometriotic Tissue.

Methodology/Principal Findings: The endometriotic stromal cells (ESCs) were successfully established and their invasive ability under different treatments was assessed through a Transwell Assay. Simultaneously, matrix metallopeptidase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) were detected by western blotting. Vascularization of endometriotic tissues was observed by chicken chorioallantoic membrane (CAM) assay. The staining of MMP-9, intercellular adhesion molecule 1 (ICAM-1), TIMP-1, and vascular endothelial growth factor (VEGF) in grafted endometriotic tissues was examined using immunohistochemistry analysis. The purity of ESCs in isolated cells was >95%, as determined by the fluoroimmunoassay of vimentin. E2 (10−8 mol/L) promoted the invasiveness of ESCs by increasing MMP-9 accumulation and decreasing TIMP-1 accumulation. Interestingly, puerarin (10−9 mol/L) significantly reversed these effects (P < 0.01). The CAM assay indicated that puerarin (10−9 mol/L) also inhibited the angiopoiesis of endometriotic tissue stimulated by the E2 (10−8 mol/L) treatment (P< 0.05). Accordingly, immunohistochemistry showed that the accumulation of MMP-9, ICAM-1, and VEGF was reduced whereas that of TIMP-1 increased in the combination treatment group compared with the E2 treatment group.

Conclusions/Significance: This study demonstrated that puerarin could suppress the tissue invasion by ESCs and the vascularization of ectopic endometrial tissues stimulated by E2, suggesting that puerarin may be a potential drug for the treatment of Endometriosis.

Regulatory mechanism of malignant behavior of endometriosis mediated by puerarin

Yu CQ, Yu J, Han J, Zhou QL, Shen W. J Chin Integr Med (Chin) 2009; 7: 41-47.

To observe the inhibitory effects of puerarin on angiopoiesis of endometriotic tissue, and to explore the regulatory effects of puerarin on tumor-related gene expression of endometriosis.

Methods: The regulatory effects of puerarin on endometriotic angiopoiesis and tumor-related gene expression were observed by using a chicken chorioallantoic membrane model and gene array method.

Results: Chicken chorioallantoic membrane experiment indicated that puerarin obviously inhibited endometriotic vasiculation and angiopoiesis. The area of blood vessels was significantly reduced as compared with the untreated group (P < 0.05). The expressions of oncogenes and genes related to adhesion, invasion, and apoptosis, including ERBB2, ETS2, FOS, S100A4, TEK, TERT, NFKBIA, CDH1, CD44, ITGA6, NCAM1, MMP1, FLT1, AKT1, BCL2L and BIRC5 genes, were obviously higher, while the expressions of the anti-oncogenes, anti-apoptosis genes and anti-invasion genes, including KAI1, KISS1, SERPINB5, TNFRSF25, TNFRSF1A, TNFRSF6 and SERPINB2, were significantly lower in eutopic endometrial tissue from patients with endometriosis than those from endometriosis-free women. The expressions of oncogenes (ERBB2, ETS2, FOS), apoptosis gene (BCL2L1), cyclin-dependent kinases (CDK4, CDC25A), and growth factor and receptors (HGF, FGFR2, TGFBR) were significantly enhanced, while the expressions of the anti-oncogenes (KAI1, SERPINB5), apoptosis genes (BAD and TNF) and cyclin-dependent kinase inhibiting factor (CDKN2A) were obviously reduced in ectopic tissue as compared with those in eutopic tissue from patients with endometriosis. Puerarin significantly enhanced the gene expressions in endometriotic stromal cells, including BAD, BAX, CASP8, CASP9, TNFRSF6, CDKN1B, CDKN2A, IFNA1 and IFNB1, and reduced the gene expressions of FOS, CHEK2, SRC, ITGB5, MMP9, PDGFA and NFKBIA.

Conclusions: The tumor-related gene expression has significant differences in eutopic endometrial tissue between patients with endometriosis and endometriosis-free women, and between ectopic and eutopic tissues from patients with endometriosis. Puerarin can reduce angiopoiesis, regulate tumor-related gene expression and facilitate apoptosis in endometriotic tissue.

Puerariae radix isoflavones and their metabolites inhibit growth and induce apoptosis in breast cancer cells

Lin Y-J, Hou YC, Lin C-H, et al. Biochemical and Biophysical Research Communications. Volume 378, Issue 4, 23 January 2009, Pp 683-8. doi:10.1016/j.bbrc.2008.10.178

Puerariae radix (PR) is a popular natural herb and a traditional food in Asia, which has antithrombotic and anti-allergic properties and stimulates estrogenic activity. In the present study, we investigated the effects of the PR isoflavones puerarin, daidzein, and genistein on the growth of breast cancer cells. Our data revealed that after treatment with PR isoflavones, a dose-dependent inhibition of cell growth occurred in HS578T, MDA-MB-231, and MCF-7 cell lines. Results from cell cycle distribution and apoptosis assays revealed that PR isoflavones induced cell apoptosis through a caspase-3-dependent pathway and mediated cell cycle arrest in the G2/M phase. Furthermore, we observed that the serum metabolites of PR (daidzein sulfates/glucuronides) inhibited proliferation of the breast cancer cells at a 50% cell growth inhibition (GI50) concentration of 2.35 μM. These results indicate that the daidzein constituent of PR can be metabolized to daidzein sulfates or daidzein glucuronides that exhibit anticancer activities. The protein expression levels of the active forms of caspase-9 and Bax in breast cancer cells were significantly increased by treatment with PR metabolites. These metabolites also increased the protein expression levels of p53 and p21. We therefore suggest that PR may act as a chemopreventive and/or chemotherapeutic agent against breast cancer by reducing cell viability and inducing apoptosis.

Puerarin prevents bone loss in ovariectomized mice and inhibits osteoclast formation in vitro

Yuan SY, Sheng T, Liu LQ, Zhang YL, Liu XM, Ma T, Zheng H, Yan Y, Ishimi Y, Wang XX. Chinese Journal of Natural Medicines, Volume 14, Issue 4, April 2016, Pages 265-269.

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 

Induction of apoptosis by puerarin in colon cancer HT-29 cells

Zl & Li Wj. Cancer Letters. Volume 238, Issue 1, Pages 53-60 (8 July 2006)

Puerarin was isolated from Pueraria radix and has beneficial effects on cardiovascular, neurological, and hyperglycemic disorders. The current study showed that puerarin also possessed anti-cancer properties. Methyl thiazolyl tetrazolium assay (MTT) assay revealed a dose-dependent reduction of HT-29 cellular growth in response to puerarin treatment. Apoptosis was observed following treatments with ≥25μM puerarin, as reflected by the appearance of the subdiploid fraction and NDA fragmentations. We then investigated effects of puerarin on expression of apoptosis-associated genes and the results revealed an increase of bax and decreases of c-myc and bcl-2. Finally, puerarin treatment significantly increased the activation of caspase-3, a key executioner of apoptosis. These findings indicate that puerarin may act as a chemopreventive and/or chemotherapeutic agent in colon cancer cells by reducing cell viability and inducing apoptosis.

The Effects of Plant Flavonoids on Mammalian Cells: Implications for Inflammation, Heart Disease, and Cancer

Middleton Jr E, Kandaswami C, & Theoharides TC. Pharmacological Reviews December 1, 2000 vol. 52 no. 4 673-751

Flavonoids are nearly ubiquitous in plants and are recognized as the pigments responsible for the colors of leaves, especially in autumn. They are rich in seeds, citrus fruits, olive oil, tea, and red wine. They are low molecular weight compounds composed of a three-ring structure with various substitutions. This basic structure is shared by tocopherols (vitamin E). Flavonoids can be subdivided according to the presence of an oxy group at position 4, a double bond between carbon atoms 2 and 3, or a hydroxyl group in position 3 of the C (middle) ring. These characteristics appear to also be required for best activity, especially antioxidant and antiproliferative, in the systems studied. The particular hydroxylation pattern of the B ring of the flavonoles increases their activities, especially in inhibition of mast cell secretion. Certain plants and spices containing flavonoids have been used for thousands of years in traditional Eastern medicine. They are usually subdivided according to their substituents into flavanols, anthocyanidins, and flavones, flavanones, and chalcones (See Table).

In spite of the voluminous literature available, however, Western medicine has not yet used flavonoids therapeutically, even though their safety record is exceptional. Suggestions are made where such possibilities may be worth pursuing.

Puerarin promotes osteogenesis and inhibits adipogenesis in vitro

Wang N, Wang Xl, Cheng Wx, et al. Chinese Medicine 2013, 8:17 doi:10.1186/1749-8546-8-17

Puerarin (daidzein 8-C-glucoside) has potential on preventing osteoporosis. This study aims to investigate the effects of puerarin on osteogenesis and adipogenesis in vitro.

Methods: CCK-8 assay, alkaline phosphatase (ALP) activity and Alizarin Red S were used to measure the effects of puerarin on proliferation, osteoblastic differentiation, and mineralization in osteoblast-like MC3T3-E1 cells. The effects of puerarin on adipogenesis were measured by Oil Red O staining and intracellular triglyceride level in preadipocyte 3T3-L1 cells. The mRNA and protein levels of osteogenesis- and adiopogenesis-related factors were detected by qRT-PCR and western blot, respectively. Further, the secreted osteocalcin levels and nuclear translocation of β-catenin were detected by ELISA and immunofluorescence assay, respectively.

Results: As to osteogenesis, puerarin could stimulate proliferation (1 μM, P = 0.012; 10 μM, P = 0.015; 20 μM, P = 0.050), ALP activity (20 μM, P = 0.008) and calcium nodule formation (20 μM, P = 0.011) in a dose-dependent manner. Puerarin (20 μM) promoted osteocalcin secretion (P = 0.004) and the protein expression of both osteopontin (P = 0.001) and osteoprotegerin (P = 0.003). As to adipogenesis, puerarin suppressed adipocytes formation and intracellular triglyceride level (P = 0.001). In addition, puerarin (20 μM) decreased the mRNA and protein levels of CCAAT/enhancer binding protein α (P = 0.001, P = 0.002), proliferator-activated receptor γ (P = 0.005, P = 0.003), and adipocyte lipid-binding protein 4 (P = 0.001, P = 0.001). Moreover, phosphorylation of AKT1-Ser437 (10 μM, P = 0.003; 20 μM, P = 0.007) and GSK-Ser9 (10 μM, P = 0.005; 20 μM, P = 0.003), and the nuclear translocation of β-catenin (10 μM, P = 0.006; 10 μM, P = 0.002) were increased in 3T3-L1 cells treated by puerarin.

Conclusion: Puerarin promoted osteogenesis and inhibited adipogenesis in vivo, and Akt/GSK-3β/β-catenin signaling pathway was involved in the suppression of adipogenesis.

Gut Mucosa

Puerarin group has been shown to significantly lower mucosal MDA and increase mucosal GSH levels after 2 and 8 hr. Puerarin can maintain GSH levels and reduced lipid peroxidation and partly by decreasing Caspase-3 expression.2

Puerarin treatment could alleviate the mucosal histological disruption caused by intestinal ischemia reperfusion injury. In reperfusion, it was shown that iNOS gene expression increased significantly, while serum NO concentration, mucosal iNOS activity increased significantly during the 8 h of reperfusion. In contrast, mucosal iNOS gene expression decreased significantly in puerarin high-treated group.3

Liver Disorders

Puerarin is a major effective ingredient extracted from the traditional Chinese medicine Radix Puerariae (ge gen). Puerarin has antioxidative and antithrombotic effects. Puerarin may help with non-alcoholic fatty liver disease, alcohol-induced adipogenesis, and osteonecrosis. Puerarin can effectively attenuate liver lipid disorder and inflammation by improving the leptin resistance and enhancing the expressions of leptin receptor mRNA and P-JAK2/P-STAT3 proteins.4

Insulin Resistance

The link between obesity and insulin resistance largely accounts for the pathogenesis of metabolic syndrome and diabetes mellitus, in which adipokine expression plays a key role. Puerarin could improve body weight gain, glucose/insulin intolerance and adipokine expression in high-fat diet (HFD)-induced insulin resistant rats, indicating its potential value for the treatment of metabolic syndrome. Assays have demonstrated that, serum levels of leptin and resistin, but not that of adiponectin, were markedly augmented by HFD and retarded by puerarin treatment.5

Gastric Mucosal

Puerarin exerts a significant protective effect on stress-induced gastric mucosal damage by relaxing the vessels, increasing NO level in gastric mucosal, increasing regional gastric mucosal blood flow and inhibiting gastric motility.6

The tumor-related gene expression has significant differences in eutopic endometrial tissue between patients with endometriosis and endometriosis-free women, and between ectopic and eutopic tissues from patients with endometriosis. Puerarin can reduce angiopoiesis, regulate tumor-related gene expression and facilitate apoptosis in endometriotic tissue.7

Diabetes and Cardiovascular Diseases 

Puerarin has been reported to have comprehensive pharmacological action in treatment of diabetes and cardiovascular diseases. There is a complicated interplay among insulin resistance, adipocytes and endothelial dysfunction that links the abnormalities of metabolic syndrome.

Results have shown that puerarin could potentiate insulin-induced preadipocyte differentiation, promote glucose-uptake of adipocytes that have been induced insulin resistance by high glucose, and prevent TNF-a-induced apoptosis and viability loss of endothelial cells.8

The Chinese Pueraria root extract (Pueraria lobata) ameliorates impaired glucose and lipid metabolism in obese mice

Prasain JK, Peng N, Rajbhandari R, Wysse JM. Phytomedicine 2012 doi:10.1016/j.phymed.2012.09.017

The incidence of type 2 diabetes and metabolic disease is rapidly increasing, but effective therapies for their prevention and treatment have been poorly tolerated or minimally effective. In this study, chronic administration of kudzu root extract (8 months, 0.2%, w/w, in diet) decreased baseline fasting plasma glucose (183±14 vs. 148±11mg/dl) and improved glucose and insulin tolerance in C57BL/6J ob/ob mice (1.67±0.17ng/ml [kudzu treated] vs. 2.35±0.63ng/ml [control]), but such treatment did not alter these parameters in lean control mice. Among the mice on the kudzu supplementation, plasma levels of isoflavone metabolites were significantly higher in ob/ob versus lean control mice, and unmetabolized puerarin (11.50±5.63ng/g) was found in adipose tissue only in the treated mice. Together, these data demonstrate that a puerarin containing kudzu diet improves glucose and insulin responsiveness in ob/ob mice, suggesting that puerarin may be a beneficial adjuvant for treating metabolic disease.

References

Wang D, Liu Yh, Han J, et al. Puerarin Suppresses Invasion and Vascularization of Endometriosis Tissue Stimulated by 17β-Estradiol. PLOS one. September 15, 2011 http://dx.doi.org/10.1371/journal.pone.0025011

LIU Jia-xian ,CHEN Jin-he. Effect of puerarin on the expression of iNOS gene in the gut mucosa of intestinal ischemia-reperfusion rats. West China Journal of Pharmaceutical Sciences., 2009-03

LIU Jia-xian,CHEN Jin-he. Protective effect of puerarin against intestinal ischemia-reperfusion injury. Chinese Pharmacological Bulletin, 2005-04

Zheng PY, Ma ZS, Hua YQ, Liu T, Xing LJ, Ji G. Effects of puerarin on expressions of leptin receptor mRNA, phosphorylated Janus kinase 2/phosphorylated signal transducers and activators of transcription 3 proteins in the liver of rats with non-alcoholic fatty liver. Zhong Xi Yi Jie He Xue Bao. 2008 Sep;6(9):921-7. doi: 10.3736/jcim20080909.

Zhang W, Liu C-Q, Wang P-W, Sun S-Y, Su W-J, Zhang H-J, Li X-J and Shu-Yu Yang S-Y. Puerarin improves insulin resistance and modulates adipokine expression in rats fed a high-fat diet. European Journal of Pharmacology. Volume 649, Issues 1-3, 15 December 2010, Pp. 398-402. doi:10.1016/j.ejphar.2010.09.054

Zhongguo, Zhong Yao Za Zhi. 2006 Mar; 31(6).

Yu CQ, Yu J, Han J, Zhou QL, Shen W. J Chin Integr Med. 2009; 7(1): 41-47. Received July 10, 2008; accepted September 12, 2008; published online January 15, 2009. Indexed/abstracted in and full text link-out at PubMed. Journal title in PubMed:

Xu Me, Xiao S-z, Sun Y-h, Zheng X-x, Yang O-y & Chen G. The study of anti-metabolic syndrome effect of puerarin in vitro. Life Sciences. Volume 77, Issue 25, 4 November 2005, Pp. 3183-96.