Research Paper Effects of Aqueous Bryophyllum pinnatum Leaf Extract on Haematological, Renal and Sperm Indices in Wistar Rats J. S. APRIOKU* AND I. IGBE1 Department of Experimental Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Port Harcourt, PMB 5323, East-West Road, Choba, Rivers State, 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, University of Benin, Benin City, Nigeria Aprioku and Ighodaro: Toxicity Profile of Bryophyllum pinnatum Leaf Extract Bryophyllum pinnatum is a perennial herb, widely used in the treatment of several conditions in folklore medicine. The effects of sub-acute treatment of the aqueous extract of B. pinnatum leaf on haematological, renal and testicular functions were investigated in the Wistar rat. Adult rats were randomly distributed into three groups of six rats each and administered 0, 62.5 or 125 mg/kg of extract daily for 28 days by oral gavage. Blood levels of urea, creatinine and haematological parameters, as well as epididymal sperm parameters were measured. Kidney and testis of animals were removed and organ weight to body weight ratios were determined. Histology of the organs was also analysed. The extract elevated white blood cell count, reduced neutrophil count (P<0.05) without affecting lymphocyte count and packed cell volume, when compared to control. The extract did not alter sperm count, motility, morphology and viability as well as blood urea and creatinine levels. The extract did not alter histology and relative weights of the kidney and testis. It can be concluded that sub-acute administration of the aqueous extract of B. pinnatum did not produce any renal and testicular toxicity, but may depress neutrophil action. Key words: Bryophyllum pinnatum, flavonoids, neutrophil, sub-acute, testis Bryophyllum pinnatum (Lam.), syn. Kalanchoe antiinflammatory[9], antimicrobial[10,11], antitumor[12], pinnata (Lam.), and B. calycinum (Salisb.) is a widely antiulcer[13], insecticidal[14], antidiabetic[15], distributed perennial medicinal herb. It is native to anticonvulsant[16], antioxidant[17], and antihypertensive Madagascar, but has been naturalized in several other properties[18]. Studies have also reported a wide regions, including the temperate regions of Asia, range of active phytochemicals such as alkaloids, Australia, and New Zealand. B. pinnatum is known by triterpenes, glycosides[18], flavonoids[16,19], steroids[9], some common names including, life plant, air plant, bufadienolides[20], lipids and organic acids[21]. These maternity plant, love plant, miracle leaf, cathedral bells, compounds have been considered to be responsible for mother of thousands, leaf of resurrection plant, and Lao the plant’s diverse pharmacological activities. di Sheng gen. The plant is locally called “Never Die” in Although, folklore claims of many herbal remedies are Nigeria, and is very popular in folklore medicine. It has yet to be authenticated scientifically, B. pinnatum has been used for the treatment of a variety of conditions been reasonably studied with justification of most of in tropical America, India, China, Australia and Africa, the claims. This has enhanced the promotion of the use including, rheumatism, body pain, arthritis, heartburn, of B. pinnatum and other plants either as alternatives skin ulcers, peptic ulcer, diabetes mellitus, microbial or to complement orthodox medicines. Additionally, infections, and hypertension[1-6]. In Nigeria, the plant is particularly known for its effective wound healing This is an open access article distributed under the terms of the Creative properties and detachment of the umbilicus of infants. Commons Attribution-NonCommercial-ShareAlike 3.0 License, which Pharmacological studies on B. pinnatum reported allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under several biological activities some of which could the identical terms authenticate the plant’s traditional uses including, Accepted 07 May 2017 [7] [8] immunomodulatory , CNS depressant , analgesic, Revised 07 January 2017 Received 07 July 2016 *Address for correspondence E-mail: [email protected] Indian J Pharm Sci 2017;79(4):521-526 521 Indian Journal of Pharmaceutical Sciences July-August 2017 www.ijpsonline.com cost of orthodox medications and the development administration, the rats were sacrificed by cervical of resistance to most orthodox chemotherapeutic dislocation under deep diethyl ether anaesthesia. Blood agents has resulted in the use of herbal medications samples were collected separately into labelled plain like B. pinnatum leaf as alternatives, especially and EDTA bottles, for measurement of urea, creatinine in underdeveloped countries[22,23]. In many and haematological parameters, respectively. Animals cases, B. pinnatum leaves were soaked in cold water were dissected and sperm was extracted from cauda overnight, boiled in water, squeezed or roasted and the epididymis for sperm analysis. The kidney and testis extracts obtained are used to treat fevers, headache, joint were also removed, weighed and routinely processed pains, arthritis, body pains, asthma, cancers, tonsillitis, for histological evaluation. diarrhoea, cough, and many other conditions[2-4,6]. Blood sample was centrifuged for 15 min at 3000 rpm Aside from the earlier stated factors, B. pinnatum is to separate serum and the serum was stored at −80°. also a popular herbal medicine due to local belief that Serum levels of urea and creatinine were then assayed. natural extracts are free of adverse effects[23,24]. This Creatinine was measured using alkaline picrate makes safety evaluation of frequently used medicinal method[27], and urea was assayed using urease-Berthelot plants imperative. The present study was thus aimed at method[28]. Whole blood collected into EDTA bottle investigating the effects of sub-acute administration of was used to assay packed cell volume (PCV), and total aqueous B. pinnatum leaf extract on haematological, white blood cell (WBC), lymphocyte and neutrophil renal and testicular indices in rats. counts, using standard laboratory techniques[29]. MATERIALS AND METHODS Sperm analysis: Fresh leaves of B. pinnatum were collected from the The method of Amelar et al.[30] was used in collecting botanical garden of the University of Port Harcourt, sperm cells from the epididymis. Briefly, the testis was Port Harcourt. The leaves were air dried, pulverized excised and the caudal epididymis was carefully isolated (400 g) and extracted exhaustively in distilled water for and placed in a petri dish containing 3 ml of sodium 72 h by cold maceration. The filtrate was subsequently bicarbonate (NaHCO3) buffered Tyrodes’ solution. evaporated to obtain the dry extract using a rotary Several (1 mm) incisions were made on it and sperm evaporator. The dry extract was weighed (28 g) and was gently drawn into a plastic transfer pipette and the percentage yield was determined (7%). The extract transferred into 5 ml test tubes and vigorously shaken was then tested for the presence of phytochemicals for homogeneity and dispersal of sperm cells. Sperm using standard methods[25]. was then analysed to determine sperm motility, sperm count, percentage of abnormal sperm cells (sperm Experimental animals: morphology) and percentage of viable sperm cells Eighteen adult male Wistar rats of 12 w of age, (sperm viability) following standard procedures[29]. obtained from the animal house of the University of Histopathological analysis: Port Harcourt, Nigeria were used for the study. The animals were given standard rodent chow and clean The kidney and testicular tissues were fixed in 10% drinking water ad libitum. The animals were kept in buffered formalin. The tissues were embedded in a well-ventilated room with a 12 h light/dark cycle paraffin and tissue sections (5-7 µm) were stained at room temperature. All animal experiments were with haematoxylin and eosin (H and E) and examined approved by the Animal Research Ethics Committee of with light microscope (Nikon Eclipse E400). All the University, in accordance to the guide for care and alterations from the normal structures were registered use of laboratory animals[26]. and histopathological changes between control Experimental design: and experimental rats were noted. The images were photographed with an Olympus Model BX51 The animals were randomly distributed into three microscope at a magnification of 400x. groups of 6 rats per group. The first and second groups were administered 62.5 and 125 mg/kg of aqueous Statistical analysis: B. pinnatum leaf extract by oral gavage (1 ml/kg) The results are presented as mean±SEM for each daily for 28 d, respectively. The animals in the third group. Differences among groups were analysed using group served as control and were given only distilled one-way analysis of variance (ANOVA) followed water (1 ml/kg) daily for 28 d. At the end of extract by Dunette’s multiple comparison test. Data were July-August 2017 Indian Journal of Pharmaceutical Sciences 522 www.ijpsonline.com analysed using GraphPad Prism Version 5 and values TABLE 3: EFFECTS OF SUB-ACUTE were considered significant at P<0.05. ADMINISTRATION OF AQUEOUS B. PINNATUM LEAF EXTRACT ON SPERM PARAMETERS IN RESULTS AND DISCUSSION WISTAR RATS Sperm Sperm Sperm Sperm Dose The doses used in the present study were selected with count motility viability morphology (mg/kg) 6 the specific aim of evaluating the effect of lower (×10 ) (%) (%) (%) Control 63.83±5.02 60.83±3.96 70.25±6.18 9.17±0.83 biologically active doses of the extract. The LD of 50 62.5 58.50±4.77 57.50±3.35 54.78±3.06 6.67±1.05 B. pinnatum (the aqueous extract) has been reported to 125 65.83±2.87 51.67±2.79 62.27±1.78 7.50±1.12 be 1.8 g/kg (i.p.) in rats; while no obvious toxicological Data expressed as mean±SEM, n=6 animals per group signs were observed at 5 g/kg dose administered orally[31]. In many previous animal studies, doses The results indicated that B. pinnatum administration of B. pinnatum used range from 150 mg/kg to as would have no adverse effect on the kidney over the high as 2000 mg/kg.