Hepato-renal, cardiac and reproductive effects of phyllanthus amarus leaf extract in streptozotocin-induced diabetic male wistar rats

Abstract

Indigenous knowledge about natural products forms a basis for medicinal plants research. Natural products are useful in traditional applications; hence, a need to conduct robust scientific research to determine their effects and possible toxicity on different biological cells, tissues, organs, and systems. Importantly, it is evident that useful information on the dosage, efficacy and possible beneficial or harmful effects of some medicinal plants on biological models are lacking. In this thesis, we reported on a single-dosage in vitro antioxidant, antidiabetic, skin depigmentation, cytotoxicity and phytoestrogenic potentials of Phyllanthus amarus (PA) extract. Also, we provided a detailed account of the effects of Phyllanthus amarus aqueous extract on the liver, kidney, heart, and testes in diabetes-induced male Wistar rats. Likewise, we carried out in-vitro antioxidant and the anti-tyrosinase evaluation of the extracts using enzyme-linked immunosorbent assay methods. We evaluated the inhibitory action of alpha-glucosidase and alpha-amylase in the aqueous and the methanol extracts. We also performed cytotoxicity and phytoestrogenicity tests on TM4 (Sertoli) and MCF-7 (breast cancer) cell lines using colourimetric MTT assay, cell counts and E-screen assay, respectively. At the same time, the in-vivo assessment of Phyllanthus amarus aqueous extract was done in male rats using fructose: streptozotocin diabetes model, whereby we administered PA (200mg/kgbwt, 400 mg/kgbwt) as well as GLIBEN (glibenclamide) 0.2mgkgbwt per day to the animals for four weeks (28 days). In this study, we noted increased phytochemicals in the methanolic extract. Also, we observed more radical cation scavenging (TEAC) action in the aqueous fraction. Methanolic ORAC results produced a significant (p < 0.0001) result compared to the aqueous extracts. Besides, PA extract produced a significant inhibition in the α-glucosidase activity. The hexane, as well as the aqueous extracts, gave considerable tyrosinase inhibition (p < 0.05) exhibiting half-maximal inhibitory concentrations of 116.08 and 129.25 μg/mL, correspondingly. PA aqueous extract at concentrations between 0.01 and 10 μg/mL produced the highest activity of mitochondrial dehydrogenase. In this instance, the TM4 cell numbers significantly increased versus the untreated control. More so, exposure of Sertoli cells to higher extract concentrations ranging between 100 and 1000 μg/mL resulted in compromised viability. Furthermore, the aqueous extract produced a significant proliferative effect on MCF-7 cells; hence, we confirmed its estrogenic activity. In our in vivo study, we noted that the animals showed persistent bodyweight loss except in the healthy control up until the 21st day of the experiment. The absolute and relative liver weights of untreated diabetic animals versus the healthy controls showed a significant finding (p < 0.0001). Similarly, the absolute and relative testicular weight of untreated diabetic rats and diabetic rats + PA 200mg/kgbwt produced a significant result (p < 0.05). The PA 400mg/kgbwt similarly increased serum insulin compared to the group that received glibenclamide versus untreated diabetic control and healthy control (p > 0.05). PA 200mg/kgbwt significantly lowered serum nitric oxide versus the diabetic controls (p < 0.0001) while GLIBEN 0.2mg/kgbwt significantly reduced serum nitric oxide versus the normal and diabetic controls, PA 200mg/kgbwt as well as PA 400mg/kgbwt (p < 0.0001). PA 200mg/kgbwt, PA 400mg/kgbwt with GLIBEN 0.2mg/kgbwt significantly brought down serum myeloperoxidase (MPO) activity versus the diabetic controls (p < 0.0001). Both PA 200mg/kgbwt, as well as PA 400 mg/kgbwt, reduced H2O2 levels versus diabetic control (p > 0.05). They significantly reduced malondialdehyde (MDA) levels versus diabetic control (p < 0.0001). PA 400mg/kgbwt significantly raised glutathione peroxidase (GPx) activity similarly to the diabetic control (p < 0.0001). PA 200mg/kgbwt significantly brought down serum triglyceride levels correspondingly to the diabetic non-treated animals (p < 0.0001). PA 200mg/kgbwt and PA 400mgkgbwt caused increased liver GPx activity. They reduced the rats’ heart, kidney, and liver lipid peroxidation similarly to glibenclamide versus the untreated diabetic rats, normalised alkaline phosphatase (ALP) with aspartate aminotransferase (AST) activities, respectively versus untreated diabetic rats. In contrast, the PA 200mg/kgbwt normalised the gamma-glutamyl aminotransferase (GGT) activity versus the diabetic untreated diabetic rats. The diabetic groups produced significant hypoproteinemia (p < 0.0001) versus the untreated healthy rats. Moreover, the PA 200mg/kgbwt improved serum testosterone similarly to glibenclamide versus their diabetic control counterparts and similarly raised serum estradiol compared to diabetic control. Nevertheless, these increases were not significant (p > 0.05). It also significantly improved sperm count correspondingly to glibenclamide (p = 0.0033) versus diabetic control and the PA 400mg/kgbwt group. Histological findings suggest that PA 200mg/kgbwt and PA 400mg/kgbwt put a halt to the progressive destruction (necrosis) of the islets of Langerhans, hence its probable ability to stimulate endogenous β-cell proliferation. PA 400mg/kgbwt ameliorated the renal lesions that showed in the diabetic group. This dose of the extract did not have any noticeable protective effect on the hepatocytes. Besides, PA did not ameliorate the lesions observed in the diabetic heart. Moreover, PA 200mg/kgbwt reduced seminiferous tubular diameter and an expanded interstitium, suggestive of its potential to improve reproductive functions through enhanced spermatogenesis. We thus conclude that PA exhibited high antioxidant potentials by being able to inhibit ABTS radical, reduce ferric ion and scavenge oxygen radicals. Both Phyllanthus amarus Hexane and aqueous extracts have a favourable tyrosinase inhibition versus reference kojic acid. Besides, PA extracts possess a significant inhibitory effect on α-glucosidase. PA extract at lower concentrations elicited Sertoli cell proliferation, probably because of the phytoestrogenicity of Phyllanthus amarus extract, brought about via its active principles, namely, phyllanthin and hypophyllanthin, thus, served as an excellent anticancer agent against breast cancer. One could also suggest that aqueous PA extract may improve reproductive functions through increasing spermatogenesis and decreasing testicular free radicals in diabetic rats. Finally, Phyllanthus amarus possesses an ameliorative effect on complications precipitated by diabetes.