Effect of white kwao krua Pueraria mirifica extract on reproductive organ development and growth of rice field frog Hoplobatrachus rugulosus

Abstract

To determine effects of Pueraria mirifica (PM) extract which contains high amount of phytoestrogen on reproductive organ development and growth of Rice Field Frogs Hoplobatrachus rugulosus. The tuberous roots of PM used in this study were collected from Huai Hong Khrai Royal Development Study Center, Chiang Mai Province (PM-HHK). Before the PM-HHK was tested in frogs, it was determined the gross morphology and estrogenic activity by vaginal cornification assay in rats compared with those of PM-Wichai3 cultivar, PM Kasetsart University (PM-KU) cultivar, and Stephania venosa. The roots of PM were round or oval shape with the yellow brown and thick bark, and the color of the tuberous meat was white with a number of fibers. The starch granules had diameters of 3.63-4.27 μm. The potency of estrogenic activity of PM-HHK was similar to that of the PM-Wichai3, but was weaker than that of the PM-KU; however, the estrogenic activity of PM-HHK was kept longer than those of PM-Wichai3 and PM-KU. The Stephania venosa did not show any estrogenic activity. The % yield of PM-HHK extracted by 70% ethanol was 17.7%. One hundred fifty 4 weeks old, at complete metamorphosis stage, Rice Field Frogs were selected, divided into 5 groups (30 frogs/group), and treated with 0, 1.77, 17.7, 177 mg/kg BW/day of PM extract (PM-0, PM-10, PM-100 and PM-1000, respectively) or 100 ug/kg BW/day of 17β-estradiol (E₂) by mixing with frog pellets, for 12 weeks. Frogs were individually measured body weights every 2 weeks. Every 4 weeks, frogs were individually measured body lengths and 9 frogs/group were randomly selected and euthanized. After euthanized, blood sample was collected for estrogen (E₂) and testosterone (T) determinations, and livers, kidneys and gonads were weighed and examined the gross morphology and microscopic histology. PM increased body weight and lengths of frogs (p<0.05), but not depended on doses, and showed no toxicological effect on liver and kidney. E₂ could stimulate an increase only in body length, but not on the weight. Determination of the sex ratio of frogs delivered in September, based on the gross morphology, showed the incline to male (88.89/11.11 for male/female). PM treatments significantly increased gonadal weights (p<0.05) and induced a mixed type (ovotestis) of gonad by 55.56%, 59.26% and 74.07% for PM-10, PM-100 and PM-1000, respectively. Once the ovotestis was microscopic examined, many sperms were observed in seminiferous tubules, but not oocytes. This indicated a testis type of gonad. There were no significant differences of serum E₂ levels among all five treatment groups, and the levels were low. Serum T levels of all four PM groups tended to be higher than those of the E₂ group. Thus, the serum E₂/T levels in PM treated groups were highly significantly lower than the E₂ group (p<0.01). From the results of this study, it can conclude that PM should be an alternative herb of growth promoting of Rice Field Frogs. If the treatment is conducted at the complete metamorphosis stage of frogs, it does not induce a sex reversal. However, the different mechanisms of actions on gonadal development between E₂ and PM are proposed and need to be investigated further.