Anti-ageing Effect And Relative Mechanisms Of Isoflavone Metabolites In Caenorhabidities Elegans

The influences of the isoflavone metabolites, including dihydrodaidzein (DHD), dihydrogeni stein (DHG), O-desmethylangolensin (0-Dma), equol (EQ) and5-hydroxy-equol (5-OH-EQ) on the lifespan of Caenorhabidities elegans(C.elegans) were first studied here. The results showed that0.2mmol/L of EQ or5-OH-EQ significantly prolonged the mean lifespan of C.elegans, however, the same concentration of DHD or DHG did not show any effect on the lifespan of C.elegans. To our surprise, the same concentration of O-Dma significantly shortened the mean lifespan of C.elegans. Among all of the tested isoflavone metabolites,5-OH-EQ had the best effect on the longevity of C.elegans. Since5-OH-EQ is a chiral compound, we detected it by using a chiral-stationary phased HPLC. The HPLC elution profile only detected a single peak, indicating that the enantiomeric excess (e.e.) of the metabolite5-OH-EQ is100%. Moreover, the metabolite5-OH-EQ could turn the polarized light to the left side, the metabolite5-OH-EQ used in this research is100%(-)-5-OH-EQ.Then, we compared the effect of different concentrations (0.1mmol/L-1.0mmol/L) of (-)-5-OH-EQ on the longevity of C. elegans. The results showed that (-)-5-OH-EQ could significantly prolong the lifespan of C. elegans when the concentration of (-)-5-OH-EQ was from0.1mmol/L to0.2mmol/L. However, higher concentrations of (-)-5-OH-EQ (≥0.8mmol/L) significantly shortened the lifespan of C. elegans. Since no significant difference was observed in the anti-ageing effect on C. elegans between the two concentrations of (—)-5-OH-EQ, i.e.0.1mmol/L and0.2mmol/L, we chose the concentration of0.1mmol/L for the further study on the mechanisms for the prolonged longevity of C. elegans. Thermotolerance and juglone oxidative assay showed that0.1mmol/L of (—)-5-OH-EQ significantly enhanced the resistance against both thermal and oxidative stress. We concluded that the longevity of C. elegans induced by (-)-5-OH-EQ might be associated with the enhanced capability under adverse circumstances. In addition, the productive capacity of C. elegans was not obviously influenced when being exposed to0.1mmol/L of (—)-5-OH-EQ, the result of which indicated that the life extension effect of (—)-5-OH-EQ on C. elegans was not regulated by the "trade-offs" mechanism between reproductive and lifespan.The quantitative real-time PCR assay showed that the expression of daf-2and tub-1genes in the worms was significantly down-regulated when being exposed to0.1mmol/L of (—)-5-OH-EQ. So the longevity of C. elegans induced by0.1mmol/L of (—)-5-OH-EQ might be dependent on Insulin/IGF-1signaling pathway. In order to ascertain the influence of (—)-5-OH-EQ on the Insulin/IGF-1signaling pathway, the lifespan assay was performed in daf-16(mu86) mutant and daf-2(el370) mutant. The results showed that0.1mmol/L of (—)-5-OH-EQ failed to prolong the lifespan of both daf-16(mu86) mutant and daf-2(el370) mutant, the result of which demonstrated that the prolonged lifespan of C. elegans induced by (—)-5-OH-EQ is indeed through DAF-2/DAF-16Insulin/IGF-1signaling pathway.It is worth noticing here that the expression of some ageing-associated genes, such as skn-1, sod-3and hsp16.2, which are downstream effectors of daf-16, were significantly down-regulated compared to that of the control. It has been reported that only some strong antioxidants, such as vitamin C, down-regulated some ageing-associated genes. The research on superoxide radical-scavenging activity in vitro showed that the antioxidative activity of (—)-5-OH-EQ was much stronger than that of other isoflavone metabolites. Therefore, we proposed that the downregulation of the ageing-associated genes might be associated with the strong antioxidative activity of (—)-5-OH-EQ.Our study first demonstrated that (—)-5-OH-EQ, which is the metabolite of genistein, not only prolonged the lifespan of C. elegans but also significantly enhanced the resistance against thermal and oxidative stress. This is the first report that the prolonged lifespan of C.elegans induced by (—)-5-OH-EQ is through DAF-2/DAF-16Insulin/IGF-1signaling pathway. Studies have confirmed that the Insulin/IGF-1signaling pathway also plays an important regulating role in humans. Therefore, our study provides an important theoretical evidence for the development of (—)-5-OH-EQ as an anti-ageing agent in the future.