Biological Evaluation Of Hesperidin And Genistein As Putative Dietary Factors With Important Health Benefits In Chickens

The ban on using antibiotics as feed additives, antibiotic resistance and the inherent problems of developing new vaccines make a compelling case for developing alternatives for in-feed antibiotics. The alternatives of choice have to be considered under the environmental conditions of the animal. Flavonoids, a group of polyphenolic compounds found mainly in fruits and vegetables, are one of the widely researched alternatives to the antibiotic feed additives in animal nutrition. The results of several in vitro studies indicate that flavonoids could exhibit a variety of potential beneficial effects. The evidence, however, in vivo is conflicting and the real contributions of such compounds to animal performance (including health challenges) and the mechanisms through which they act are still unclear. Biological activity of flavonoids depends on the presence or absence of the glycoside. After ingestion, the flavonoid glycosides are hydrolyzed into the aglycones which are then absorbed, metabolized by liver and intestinal enzymes, distributed to tissue and excreted in urine. Metabolism by the gut microflora is an important factor for flavonoids bioavailability and can be affected by dietary modification. Recently there has been much interest in the isolation of flavonoids degrading bacteria and identification of dietary components that may contribute to flavonoids bioavailability. With these facts in mind, research was conducted to evaluate the in vivo growth and health responses in flavonoids fed broiler chickens while the aim of in vitro study was to isolate flavonoids degrading bacteria from chicken and to examine the effects of dietary components on flavonoids bioavailability.IN VIVO STUDYThe effects of hesperidin and genistein treatments on growth, metabolism and health status were studied in meat type chickens (Gallus gallus). A total of 768 day-old broiler chickens were randomly divided into 8 groups. The birds were fed either a basal diet (control) or the basal diet supplemented with either 2.5 mg hesperidin/kg (HD1),5 mg hesperidin/kg (HD2),10 mg hesperidin/kg (HD3),20 mg hesperidin/kg (HD4) 2.5 mg genistein/kg (GS1),5 mg genistein/kg (GS2) and 10 mg of genistein/kg (GS3) of diet. No significant differences in body weights and feed conversion ratio were observed between control and treated chicks. Glucose showed detectable decrease only on day 42 between control and chicks treated with 2.5 genistein/kg feed (GS1 group). Cholesterol lowering effects were observed by hesperidin (all groups) on day 21 and 2.5 to 10 mg/kg of hesperidin and genistein on day 42. The hepatic expression of type-1 IGF receptor (IGF-1R) was stimulated with high dose of hesperidin (HD4) and low and medium doses of genistein (GS1 and GS2) on 21 days while all doses of hesperidin and high dose of genistein (GS3) elevated IGF-1R gene expression on day 42. Analysis of Endocrine Indices showed that plasma triiodothyronine (T3) mostly remained unaffected while thyroxine (T4) was elevated except that high dose of genistein (GS3) and two last doses of hesperidin (HD3 and HD4) showed non significant effects on day 21 and 42, respectively. Glucagon designated increasing trend in a dose dependent manner for both hesperidin and genistein supplementation on day 42. Oxidative analysis revealed that hesperidin and genistein supplementation generally reduced the plasma malondialdehyde (MDA) concentrations in a dose-related fashion. Hesperidin and genistein also suppressed total antioxidant capacity (TAOC) and superoxide dismutase (SOD) activity on day 21 and 42 except that high dose of genistein (GS3) did not show any significant effect on day 42. Hesperidin, taken as a whole, resulted in marked decline in heat shock protein 70 (HSP70) gene expressions. In case of genistein, hepatic expression of HSP70 decreased in GS1 and GS2 groups while increased in the GS3 group on day 21. In the antiinflammatory parameters investigation, hesperidin significantly suppressed plasma prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) levels on day 42 and hepatic inductible Nitric Oxide Synthase (iNOS) and cyclooxygenase-2 (COX-2) gene expression on day 21. iNOS mRNA level was also down-regulated on day 42 in chickens supplemented with 10 to 20 mg/kg of hesperidin. Age and dose specific effects of genistein were observed for anti-inflammatory parameters. PGE2 increased in high dose (GS3) group on day 21 whereas decreased in GS1 and GS2 groups on day 42. LTB4 also reduced in GS1 and GS2 groups on both experiment days. Hepatic iNOS gene expression augmented with all doses of genistein on day 21 and with high dose (GS3) on day 42. However, iNOS mRNA level was down-regulated with 2.5 to 5 mg genistein/kg feed on day 42. Hepatic COX-2 gene expression increased on day 21 and decreased on day 42 in chickens on GS1 diet while results were vice versa for high dose (GS3) group. Analysis of immunity indices showed significant down-regulation for hepatic interferon-gamma (IFN-γ) gene expression by first two doses of hesperidin (HD1 and HD2) and up regulation for interleukin-10 (IL-10) by first two doses of both hesperidin and genistein on day 21. Hesperidin (all doses) also elevated the IL-10 mRNA level on day 42 while IFN-γgene expression increased only with high doses (HD3 and HD4). In contrast to hesperidin, high dose of genistein (GS3) decreased IFN-γgene expression on day 42. Hepatic interleukin-4 (IL-4) mRNA level did not show detectable differences between controls and chicks treated with both genistein and hesperidin except that it was up-regulated with high dose of hesperidin (HD4). Intestinal intraepithelial lymphocytes (iIELs) increased in hesperidin and genistein treated chickens on both day 21 and 42 depending on the dose applied. Intestinal secretary IgA (sIgA) production, overall, increased with hesperidin treatment and decreased with genistein treatment on day 21 and 42. Hesperidin and genistein supplementation (10 mg/kg diet) decreased the total short chain fatty acids (SCFA) contents in the cecum while DGGE profile remained unaffected. The present study suggests dose and age specific modulation of performance and health indices by hesperidin and genistein in broilers. This work also provides new insights into the mechanisms underlying the effect of these two flavonoids in vivo.IN VITRO STUDYCecal microbiota of chicken was screened for the bacteria involved in the biotransformation of flavonoids. Four anaerobic Lactobacillus like strains designated as MF-01, MF-02 and MF-03 and MF-07 were isolated from the cecum of chicken. MF-01, MF-02 and MF-03 were capable of converting flavonoid diglycosides (hesperidin and rutin) into bioactive aglycones while MF-07 was active in the deglycosylation of the isoflavone genistin and further degradation of aglycone genistein. The degradation rates of flavonoids and influence of different carbon sources, following incubation with isolated strains, were assessed. The role of consortium in the degradation of flavonoids diglycosides and modulation of bioconversion after supplementation with prebiotic (fructooligosaccharide) and organic acids to the fermentation medium was also monitored. Organic acids (lactate, acetate, butyrate or propionate) supplementation suppressed the flavonoids degradation while fructo-oligosaccharide (FOS) appeared to have prebiotic potential by preserving aglycones in vitro. These Lactobacillus microorganisms may lead to a combination of benefits as probiotics as well as that from transformation of flavonoid diglycosides to bioactive aglycones.CONCLUSIONOverall, the findings of this endeavor will be relevant to further understanding in vivo effects of flavonoids in animals, particularly chickens. The dose and age related fashion in the biological activity of hesperidin and genistein may assist in explaining the large variation between animals in the health beneficial effects of flavonoids intake. In addition, the present work may further aid in establishing age specific recommendations for the use of flavonoids in chickens. This is timely considering the current interest in the biodegradation of flavonoids by gut microflora and use of dietary components to modulate flavonoids bioavailability. Furthermore, it could be concluded that use of FOS may help in preserving the deglycosylated metabolites depending upon the subclass of flavonoids.