Biochanin A attenuates spinal cord injury in rats during early stages by inhibiting oxidative stress and inflammasome activation

Previous studies have shown that Biochanin A,a flavonoid compound with estrogenic effects,can serve as a neuroprotective agent in the context of cerebral ischemia/reperfusion injury;howeve r,its effect on spinal cord injury is still unclea r. In this study,a rat model of spinal cord injury was established using the heavy o bject impact method,and the rats were then treated with Biochanin A(40 mg/kg) via intrape ritoneal injection for 14 consecutive days.The res ults showed that Biochanin A effectively alleviated spinal cord neuronal injury and spinal co rd tissue injury,reduced inflammation and oxidative stress in spinal cord neuro ns,and reduced apoptosis and pyroptosis.In addition,Biochanin A inhibited the expression of inflammasome-related proteins(ASC,NLRP3,and GSDMD)and the Toll-like receptor 4/nuclear factor-κB pathway,activated the Nrf2/heme oxygenase 1 signaling pathway,and increased the expression of the autophagy markers LC3 Ⅱ,Beclin-1,and P62.Moreove r,the therapeutic effects of Biochanin A on early post-s pinal cord injury were similar to those of methylprednisolone.These findings suggest that Biochanin A protected neurons in the injured spinal cord through the Toll-like receptor 4/nuclear factor κB and Nrf2/heme oxygenase 1 signaling pathways.These findings suggest that Biochanin A can alleviate post-spinal cord injury at an early stage.

Biochanin A,as the Lrg1/TGF-β/Smad2 pathway blockade,attenuates blood-brain barrier damage after cerebral ischemiareperfusion by modulating leukocyte migration patterns

Background:Biochanin A is an excellent dietary isoflavone that has the concomitant function of both medicine and foodstuff.The attenuation function of biochanin A on blood-brain barrier(BBB)damage induced by cerebral ischemia-reperfusion remains unclear.Methods:C57BL/6 mice were subjected to 1 h middle cerebral artery occlusion(MCAO)followed by 24 h reperfusion.The infarct volume of the brain was stained by TTC,while leakage of the brain was quantitatively stained by Evans blue,and the neurologic deficit score was measured.Microglial-induced morphologic changes were observed via immunofluorescence staining,and rolling and adhering leukocytes in venules were observed via two-photon imaging,while the inner fluorescein isothiocyanate-albumin of venules were compared with those of surrounding interstitial area through venular albumin leakage.Results:The attenuation effect of biochanin A on tight junction injury was compared in ischemia-reperfusion mice or conventional knockdown of leucine-richα2-glycoprotein 1(Lrg1)mice.Biochanin A could ameliorate BBB injury in mice with cerebral ischemiareperfusion in a dose-dependent manner by strengthening the immunostaining volume of occludin,claudin-5,and zonula occludens-1.The amoeba morphologic changes of microglial combined with the elevated expression of Lrg1 could be relieved under the treatment of biochanin A.Biochanin A played a countervailing role on the rolling leukocytes in the vessel,while the leakage of blood vessels was reduced.Biochanin A diminished its functions to further improved attenuation for tight junction injury on conventional Lrg1-knockout mice,as well as the inhibition effects on TGF-β1,and the phosphorylation of suppressor of mothers against decapentaplegic 2(Smad2)/Smad2 via western blot assay.Conclusion:Biochanin A could alleviate tight junction injury induced by cerebral ischemiareperfusion and blocked the Lrg1/TGF-β/Smad2 pathway to modulate leukocyte migration patterns.

Biochanin A and CPe-Ⅲ Peptide Improved Hepatic Infl ammation by Regulating the Hepatic Lipid Metabolic Pathways in Diet-Induced Obese Mice

Biochanin A(BCA) and CPe-Ⅲ peptide, which both exist in chickpea(Cicer arietinum L.), possess significant antihyperlipidemic properties. However, the actualmechanisms ofthose compounds in inhibiting the dysregulation oflipid metabolism and complicated inflammation have not been wellcharacterized. This study investigated the effects ofBCA, CPe-Ⅲ peptide, and combined BCA and CPe-Ⅲ peptide(BC) on the expression ofgenes involved in hepatic lipid and inflammation metabolism. Results demonstrated that BCA, CPe-Ⅲ peptide, and BC significantly attenuated hepatitis and hyperlipidemia by downregulating those genes involved in pro-inflammatory cytokines(TNF-α), hepatic fatty acid(FA) synthesis(ACC1 and FAS), cholesterolmetabolism(SREBP2, HMGCR, and PCSK9), and upregulating key regulators involved in FA oxidation(PPARα and FABP1), lipolysis(ATGL), LDLR, reverse cholesteroltransport(ABCA1, SR-B1, and LXRα), and cholesterolcatabolism(CYP7 A1). Moreover, they also altered the expression oflipid metabolism-related proteins, including SREBP2, PCSK9, LDLR, ABCA1, and CYP7 A1. Finally, these results revealed that the combination treatment ofBCA and CPe-Ⅲ peptide resulted in greater antihyperlipidemic activity compared with individualcompounds.

Effects of biochanin A on Hela cell proliferation, migration and Bcl-2 and Bax gene expression in cervical cancer

Objective:The study is to investigate the regulation of biochanin A on proliferation and migration of cervical cancer cells and its possible molecular mechanism.Methods: Hela cells were cultured in vitro and treated with different concentrations of biochanin A. Cck-8 assay was used to detect the effect of biochanin A on the proliferation of cervical cancer cells, while the Scratch assay was used to detect the effect on cell migration. Besides, the expression of bcl-2 /Bax mRNA was detected by RT-PCR.Results: Biochanin A could inhibit the proliferation and migration of Hela cells in a dose-dependent and time-dependent manner. At 10, 20, 40, 80, 160 mol/L of biochanin A, the inhibition rate of Hela cells increased significantly after 24, 48 h, significantly higher than that of the control group. The expression of pro-apoptotic protein Bax increased, while the expression of anti-apoptotic protein bcl-2 decreased in a dose-dependent manner.Conclusion: hummus A can inhibit the proliferation and migration of Hela cells in cervical cancer, and its mechanism may be related to the down-regulation of bcl-2 and up-regulation of Bax.

NP-15 Effects of Biochanin A on Learning and Memory in Ovariectomized Rats

Objective:To observe the effects of biochanin A on learning and memory of ovariectomized rats and the histopathological changes in uterus and breast tissues,so as to look for a new drug instead of estradiol to prevent or treat neuro-degenerative diseases after menopause.Method:A total of 48 six-month-old female rats were randomly divided into six groups:sham group,model group,17β-estrodiol,Biochanin A high-dose group,middle-dose group and low-dose group.The changes of the spatial learning and memory function were observed through Morris water maze.After the treatment,morphous of the hippocampus were observed after HE stained by microscope.Internal structure of hippocampal neurons was observed under transmission electron microscope.Colorimetry method was used to test SOD and GSH-PX activity and MDA content in the left cerebral homogenate.Western Blot was used to examine the expression of PPAR gamma and MMP-2 in right hippocampus of rats.After the treatment,the histopathological changes in uterus and breast tissues were observed to probe its safety for therapy.Result:Biochanin A could significantly antagonize the decrease of spatial learning and memory induced by OVX.Under the optical microscope and Electron microscopy observation,The number of necrosis nerve cells in Biochanin A treatment groups were significantly reduced.Biochanin A groups could decrease the content of MDA,and increase the activity of GSH-PX,SOD.The expression of PPAR gamma was down regulated and the expression of MMP-2 was up regulated in Biochanin A groups.The number of acini decreased,tissue atrophy in model group,low-dose group and middle-dose group,but the situatiion reserved in high-dose group.Conclusion:Biochanin A could significantly improve memory dysfunction due to lower estrogen levels in rats,the mechanism of which may be related to antioxidative stress.

Effects of biochanin A on lactational performance,nitrogen metabolism,and blood metabolites in dairy cows

Optimizing nitrogen utilization efficiency and mitigating nitrogen losses in cows plays a pivotal role in fostering economic sustainability within contemporary agricultural systems.Biochanin A(BCA),a natural component in red clover,has the potential to improve nitrogen metabolism in dairy cows.The primary objective of this study was to probe the impact of biochanin A supplementation on lactational performance,nitrogen metabolism,and blood metabolites in dairy cows.A complete randomized block design experiment was conducted over 28 d,involving 36 multiparous Holstein cows(comparable milk yield=37.1±2.90 kg,BW=642±70.0 kg,days in milk=92±8.0 d,and parity=2.4±0.50),which were allocated to three treatment groups:the Control group(with 0 g/d BCA),the Low group(with 10 g/d per cow BCA),and the High group(with 40 g/d per cow BCA).Biochanin A supplementation improved the lactational performance of cows by increasing milk yield by 6.3%(P=0.007)and feed efficiency by 12.7%(P=0.009).Total intestinal apparent digestibility was unaffected by BCA supplementation(P>0.05),but microbial nitrogen was increased by 30.0%(P=0.002)for promoting nitrogen utilization efficiency by 20.7%(P=0.004).Milk competent yields(protein,lactose,and non-fat milk solid)were increased with increasing BCA supplementation(P 0.05).BCA did not affect body health of dairy cows.Additionally,none of the plasma endocrine hormones were affected(P>0.05).A total of 95 significantly different metabolites were screened from the plasma metabolites of cows in the BCA-added and non-added groups.After performing an enrichment analysis of the metabolic pathways associated with the different metabolites,six specific pathways were identified:bile acid biosynthesis,aspartate metabolism,pyrimidine metabolism,arginine and proline metabolism,the urea cycle,and ammonia recycling.The inclusion of BCA is suggested to enhance milk yield and modulate nitrogen metabolism by influencing relevant metabolites within the metabolic pathways.

鹰嘴豆芽素A抗HIV-1活性及抑制CD4^+淋巴细胞早期活化作用

目的研究鹰嘴豆芽素A(biochanin A,BioA)对HIV-1活性及CD4+淋巴细胞早期活化作用的影响。方法①以MT-2和H9/HIV-1ⅢB细胞或HIV-1病毒共培养建立HIV-1感染模型,以不同浓度的BioA干预该过程,观察细胞融合情况和测定培养上清中的p24抗原含量变化,以评价BioA的抗HIV-1活性。②以PHA刺激剂诱导人外周血CD4+淋巴细胞活化,利用流式细胞术检测不同浓度BioA对早期活化标志CD69分子的表达影响。结果①BioA在本实验所设浓度下均能减少由HIV-1介导的细胞融合数(EC50=5.1μmol.L-1,SI=39)及p24抗原的表达量(EC50=38μmol.L-1,SI=5.2)。②终浓度5、10、25、50μmol.L-1的BioA对CD4+淋巴细胞早期活化抗原CD69表达具有明显抑制作用(P<0.01),其中50μmol.L-1达到最大抑制率,能使活化率从(60.42±0.52)%降低到(19.70±0.38)%。结论BioA具有抗HIV-1介导的细胞融合和HIV-1复制的活性及抑制CD4+淋巴细胞早期活化作用。

Preparation of National CRMs of Active Compounds from Natural Products

According to the standard of GB/T1500.3-2008 and the guidelines of ISO/REMCO, the preparation of National Certified Reference Materials （CRMs） of active compounds was composed of the following two aspects： preparation and certification. This paper presents an example of the preparation of biochanin A to illustrate the preparation of National CRMs of active compounds from natural products. Firstly, the target compound was extracted and purified from crude materials. Then, its chemical structure was identified by UV spectrum, IR spectrum, ESI-MS, and NMR. The purity of the compound was determined by HPLC with the area normalization method. The certified value assessed by comparing the purity values from six independent qualified laboratories and the value of uncertainty were given. Thermal analysis, residual solvent, homogeneity testing and stability testing was tested in the process.

NP-21 Effects and Mechanisms of Estrogen on Endogenous Growth Hormone in Brain of Ovariectomized Rats

Objective:To observe the effects of estrogen/plant estrogen(Biochanin A,BCA)on learning and memory of ovariectomized rats,so as to explore the possible mechanisms of neuroprotective effect on endogenous growth hormone.Method:A total of 50 six-month-old female rats were randomly divided into five groups:sham group,model group,17β-estrodiol,Biochanin A high-dose group,and low-dose group.The changes of the spatial learning and memory function were observed through Morris water maze and shuttle box.After the treatment,morphological structures of rat neurons were observed by HE staining and electron microscopy.The contents of E2 and GH in serum were determined by ELISA and associated proteins of growth hormone JAK/STAT signaling pathway were examined by western blot.Result:Estrogen/BCA could enhance learning and memory ability of the aging rats,improve the structures of the nerve cells,increase E2,GH and IGF1 levels,improve STAT5 and p-STAT5 protein expression and reduce SOCS2 protein expression.Conclusion:Estrogen/BCA could significantly improve memory dysfunction,the mechanisms may be related to increase secretion of endogenous growth hormone,regulate growth hormone JAK/STAT signaling pathway.