Plant materials and extraction
The leaves and roots of Tephrosia vogelii were collected from Hai district, Moshi region; Northern part in Tanzania (Latitude S 03° 15′ 6.4″ and longitude E 37° 14′ 3.8″). The plant species was identified by the plant taxonomist and botanist, Mr. E. John from Tanzania Pesticides Research Institute (TPRI), and the voucher specimen (SH-NM102) was deposited at the Nelson Mandela African Institution of Science and Technology for future reference. The plant materials were air-dried under the shade for 4 weeks then pulverized to powder. The pulverized leaves (0.6 kg) and roots (0.45 kg) were separately soaked in methanol for 48 h followed by filtration to obtain methanolic filtrates. Methanol solvent was completely evaporated using a rotary evaporator under low pressure below 40 °C. The evaporation afforded 12.0 g and 5.0 g of methanolic leaf extracts (TV-L, ME) and methanolic root extracts (TV-R, ME), respectively. Then the extracts were stored at 4 °C prior to biological assays.
Phytochemical screening of secondary metabolites was conducted according to standard procedures as previously described [21,22,23,24].
Test for tannins
The distilled water (1 mL) was added to 0.5 g of extract. Then the mixture was stirred, filtered and few drops of ferric chloride were added to the filtrate. The formation of blue-black precipitates indicated the presence of tannins.
Test for terpenes and steroids
About 0.1 g of the extract was dissolved in 1 mL of chloroform. Then 1 mL of acetic anhydride was added to the mixture. Finally, two drops of concentrated sulfuric acid were added gently to the mixture alongside the test tube. Changes of colour from violet to blue/green indicate the presence of steroids while changes from violet to pink-red indicated the presence of terpenoids.
Test for saponins
About 0.5 g of extract was added to 5 mL of distilled water and shaken well. Then the mixture was gently warmed. Persistent frothing even after warming indicated the presence of saponins.
Test for flavonoids
About 15 mL of distilled water was added to about 0.25 g of plant extracts, and then mixture was filtered. The filtrate, about 10 mL were collected which then divided into two test tubes each containing 5 mL. Then 5 mL of 20% sodium hydroxide was added to 5 mL of the filtrate. In another tube of 5 mL of the filtrate 5 mL of lead acetic solution was added. Formations of yellow colour with either of the reagents added to the filtrate confirmed the presence of flavonoids.
Test for alkaloids
About 0.5 g of the extract was added to 3 mL of 1% aqueous hydrochloric acid and stirred in a steam bath. The mixture was then filtered, and 1 mL of the filtrate was poured into two test tubes, each containing 0.5 mL. Finally, 3 drops of Mayer’s reagent were added to one of the test tubes, and 3 drops of 1% picric acid were added to another test tube. The formation of precipitates with any of the final added two reagents confirmed the presence of alkaloids.
Test for glycosides
About 0.1 g of extract was dissolved in 1 mL of glacial acetic acid containing one drop of ferric chloride solution. Then 1 mL of concentrated sulphuric acid was added to the mixture by pouring alongside the test tube. The brown ring formation indicated the presence of glycosides.
Experimental animals and doses
Animal model experiments were performed in accordance with OECD guidelines 423 and 407 for toxicity assessment [25, 26] and the previous methods with minor modifications [27,28,29,30,31,32]. Fifty-five laboratory albino rats aged 8 weeks were collected from Sokoine University of Agriculture (SUA). All rats were housed in plastic cages and treatments. Acclimatization was for 7 days. They were fed food three times a day; in the morning, afternoon and evening to make sure that they did not starve and die. For easy administration of drugs to rats through oral gavage, water was not provided in the morning to induce thirst. Thus, water was given in the afternoon immediately after the administration of the drugs and during the evening. Extracts doses of 600, 1200, 2000 and 5000 mg/kg body weight were employed [27, 33]. The body weights of the animals were measured using analytical balance before the drug administration regime in order to prepare appropriate doses. Weights of animals were 150 ± 5 g of which the doses were calculated on the average weight of 150 g. The amount of extracts used to prepare concentrations (doses) was established based on the calculation of the “ratio of expected dose multiplied by the weight of rats”. Then, 90, 180, 300 and 750 mg of methanolic leaf and root extracts of T. vogelii were measured regarding rats weights and expected doses. Extracts were dissolved in 1 mL of distilled water to obtain 90, 180, 300 and 750 mg/mL solutions, which were equivalent to doses 600, 1200, 2000 and 5000 mg/kg, respectively, for dosage in rats.
Animal welfare and safety
The Tanzania animal welfare Act  as well as other ethical guidelines for the consideration of animal rights such as the use of few numbers of animals, treatment with or without minimal pain and age of animals, were observed during experimental research [35, 36]. In this study, all rats were safely caged with the intention of protection (rats neither suffocated nor died because of unconducive environments). The sacrificed bodies and organs were sterilized and incinerated after the experiments.
Lethal dose (LD50) assay: groups of animals and treatment
Twenty-seven rats were grouped into nine groups; each group consisted of three rats based to minor modification from previous studies [27, 37]. Animal groups were housed in separate plastic cages for ease observation and treatments. The LD50 test was an in vivo experiment whereby a single dose of the extract(s) administered to laboratory albino rats so as to evaluate doses causing deaths. Both root and leaf methanolic extracts of T. vogelii of doses 600, 1200, 2000, and 5000 mg/kg were administered to rats to determine the LD50 extracts within 72 h. Except for the control group (group 9) which was not treated with extracts, the same dose was administered to all three animals of each group. The leaf methanolic extracts (TV-L, ME) of doses 600, 1200, 2000 and 5000 mg/kg were administered to four groups of rats (groups 1, 2, 3 and 4), respectively. The root methanolic extracts (TV-R, ME) of doses 600, 1200, 2000 and 5000 mg/kg were administered to four groups of rats (groups 5, 6, 7 and 8), respectively. The lethality was conducted followed by sub-acute toxicity assay.
Sub-acute toxicity assay: groups of animals and treatment
The sub-acute toxicity assay was performed through an animal model using laboratory albino rats at SUA. Twenty-eight albino rats were divided into seven experimental groups, and each group had four rats. Each group was housed in an independent plastic cage for conducting treatments. Animal doses were modified from previous studies from Upadhyay and colleagues . The TV-R, ME doses of 600, 1200 and 2000 mg/kg were used to treat groups 1, 2, and 3, respectively. The TV-L, ME doses of 600, 1200 and 2000 mg/kg were used to treat groups 4, 5, and 6, respectively. Group 7 was not treated with extracts as it served as a control group. The dosage regime of the animals with extracts was consecutively conducted once per day for 14 days. The dose administrations were done at noon and made through oral gavaging followed by clinical signs observations. Toxicity (clinical) signs were recorded at the interval of 10 min, 0.5 h, 1 h, 4 h, 8 h and 24 h immediately after oral dose administration. The clinical signs of toxicity such as water intake, mortality, food intake, sedation, convulsion and general behaviour to the treated animals were recorded for 28 days. In each group, two animals were anaesthetized and sacrificed at the day 15th and two animals at the day 29th. Sacrificed animals were conducted in order to see the effects of prolonged administered extracts in 14 days (day 1–14) and when animals stopped receiving doses from day 15–28. Animals were anaesthetized by ketamine/xylazine, 20:1 mg/kg intraperitoneal (20:1 mg/kg IP) injection to avoid pain during dissection. After the anaesthesia reached depth, the animals were sacrificed then kidney and liver organs were removed for histopathological examination. These organs were preferred because they are the target organs involved in the detoxification and excretions of the ingested harmful substances, which are more likely to manifest negative effects in the cells of the organs . The drug effects particularly negative manifestation were studied and collected as histopathological data.
The liver and kidney were collected from the sacrificed and dissected rats then washed with 10% neutral buffered formalin (NBF) solution. The macroscopic examination was carried out using normal eyes before fixation and staining for microscopic examination. The organs were trimmed and processed for paraffin embedding. Then fixation was carried out whereby about 4 μm thick sections of the embedded were prepared and stained with hematoxylin (H) and eosin (E). The stained tissues in slides were then analyzed with the aid of an optical microscope to observe, if any, signs of cell degenerative changes such as inflammation and necrosis evidence. The optical microscopic analyses captured the images with the microscope Motic B1 series and scanned the images through micro-camera Moticam 480 using the Motic Images Plus 2.0 Mult-Language Application Suite Software. The histopathological analyses of hepatic-renal toxicity effects in animals’ organs treated with extracts were established and compared with the control organs of the untreated animals.