Toxicity assessment and effects of Phyllanthus niruri ethanolic extracts on testicular morphology of Sprague Dawley rats as male herbal contraceptive

Phyllanthus niruri is native to Southeast Asia, particularly Malaysia and has been used as traditional preparations for various health benefits. However, previous studies found that P. niruri can negatively affect reproductive functions. Overpopulation of stray animals has recently been a concerning issue within the community. This can lead to transmission of zoonotic and infectious diseases between humans and animals. Despite many approaches being taken to manage this issue, their effectiveness has decreased, and they are highly costly to implement on a large-scale. Hence, a new affordable and effective approach needs to be introduced. Therefore, this study aimed to explore the safety and efficacy of P. niruri extract as a male herbal contraceptive in stray animals. Phyllanthus niruri ethanolic extract was prepared using the cold maceration method in 50% ethanol. The plant extract was then submitted for phytochemical analyses using gas chromatography-mass spectrometry (GC-MS) and liquid chromatography time-of-flight mass spectrometry (LC-TOF-MS/MS) that detected 28 compounds from GC-MS, mainly ketones and esters, while 27 compounds were identified from LC-TOF-MS/MS with mostly phenolics and flavonoids. From the in vitro study, we investigated the cytotoxicity activity of P. niruri ethanolic extract on Vero and primary feline testicular cells (FTC) and DNA apoptotic activity in FTC. The FTC was prepared from feline testes obtained from post-routine castration from local private veterinary clinics, while Vero cells were obtained from the archived cells of the Virology Laboratory, Faculty of Veterinary Medicine Universiti Malaysia Kelantan (FPV UMK). MTT assay was performed to evaluate the cytotoxicity of these plant extracts on FTC and Vero cells. Apoptosis was assessed using a DNA laddering assay on extracted DNA of the treated FTC cells and gel electrophoresis was used to observe the DNA pattern of the treated cells. The 50% cytotoxic concentration (CC50) of the ethanolic extract of P. niruri was determined at the dose of 5 mg/mL. The DNA laddering assay revealed that the ethanolic extract of P. niruri does not induce apoptosis in the FTC. For the in vivo study, acute and 28-day repeated dose oral toxicity were performed to observe the toxicity profile. The animals were divided into two groups (n = 5) and four groups (n = 5) for acute and repeated dose oral toxicity studies, respectively. Whole blood was used for haematological and serum biochemical analyses. Kidneys and liver were extracted for histopathological evaluation. Following that, the antifertility study was carried out with the animals divided into four groups (n = 5). The sperm parameters and changes in the testicular morphology were assessed. Toxicity studies confirmed that the plant is less toxic for doses up to 2000 mg/kg. The antifertility study revealed impairment of sperm parameters with degenerative effect on testicular tissues in experimental groups. However, these antifertility effects were reversible after 14 days of the plant extract withdrawal period. Therefore, these study outcomes indicated that the ethanolic extract of P. niruri has the potential to be developed into a male herbal contraceptive as it is safe and effective for animal use.