A chemical and toxicological study of the tuber extracts of Rhoicissus revoilii Planch. (Vitaceae): A Comorian plant used as a pesticide

 Corresponding author: Victor Louis Jeannoda Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution Liscense 4.0. A chemical and toxicological study of the tuber extracts of Rhoicissus revoilii Planch. (Vitaceae): A Comorian plant used as a pesticide Ansufidine Dhoifir 1, 2, Mihajasoa Stella Razanatseheno 1, 2, Maholy Pricille Ratsimiebo 1, 2, Herizo Lalaina Andriamampianina 1, 2, Hanitra Ranjàna Randrianarivo 1, 2, Lovarintsoa Judicaël Randriamampianina 1, 2, Nomenjanahary Lalaina 3, Danielle Aurore Doll Rakoto 1, 2 and Victor Louis Jeannoda 1, 2, * 1 Laboratory of Applied Biochemistry to Medical Sciences (LABASM), Fundamental and Applied Biochemistry Department (DBFA), Faculty of Sciences, University of Antananarivo, P.O. Box 906, Antananarivo 101, Madagascar. 2 Life and Environment Sciences Doctoral School (SVE), University of Antananarivo, P.O. Box 906, Antananarivo 101, Madagascar. 3 Central Pathological Anatomy Laboratory of the University Hospital Centre (CHU) Joseph Ravoahangy-Andrianavalona, BP 4150, Antananarivo 101, Madagascar. World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 Publication history: Received on 07 May 2025; revised on 14 June 2025; accepted on 16 June 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.26.3.2359 Abstract This study aimed to assess the acute toxicity of Rhoicissus revoilii Planch tuber extracts. This plant from the Vitaceae family is used to treat various pathologies and as a pesticide in the Comoros. From dried tuber powder previously treated with hexane, a methanolic extract (ME) was obtained with a yield of 20.33%. Phytochemical screening of the powder revealed the presence of flavonoids, tannins, polyphenols, coumarins, triterpenes and steroids. The toxicity of ME was assessed in mice and cold-blooded animals. In mice, symptoms of intoxication by the intraperitoneal (i.p.) and oral routes mainly included decreased motor activity, muzzle itching, diarrhoea, and increased respiratory rate. The LD50 (24 h) of ME in mice was estimated to be between 21.29 and 22.3 mg/kg body weight (b.w.) by the i.p. route. By the oral route, it was estimated to be between 273 and 275 mg/kg b.w. When administered by these two routes, ME caused dose-dependent histopathological lesions in the liver, lungs and kidneys. The LC50s were estimated to be 7.491 μg/ml, 8.055 μg/ml and 685 μg/ml, respectively, for alvins, legless frog tadpoles and mosquito larvae at stage three of development. Keywords: Rhoicissus revoilii; Vitaceae; Tubers; Toxicity; Pesticide 1. Introduction Rodents represent a significant threat worldwide, causing substantial economic losses and considerable inconvenience to humans. These small mammals, including rats and mice, cause significant damage to various sectors, including agriculture, public health, the food industry and construction [1, 2]. On farms, rodents damage crops by nibbling them, leading to a significant drop in food production and considerable financial losses. Not only do they damage property, they also carry a number of serious infectious diseases, such as leptospirosis, salmonellosis and plague. They often transmit these diseases through their excrement, urine or the parasites they harbour [3] posing a real threat to public health. To deal with this threat, various methods of rodent control have emerged, ranging from traps to chemical products [4]. However, these approaches often pose problems regarding effectiveness, cost, and environmental impact. It is therefore World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1765 crucial to explore new natural and sustainable alternatives for controlling these pests. Certain plants, traditionally used for their repellent properties, could provide a more ecological and less intrusive solution. Rhoicissus revoilii Planch. (Vitaceae), a plant widely grown in the Comoros, is recognised not only for its potential as a natural pesticide (mousicide, rat poison), but also for its therapeutic properties in the Comoros and in East Africa such as Kenya and Tanzania [5, 6, 7, 8]. The main objective of this study was to assess the toxic effects of methanolic extract of Rhoicissus revoilii tubers on various animals. 2. Material and methods 2.1. Materials 2.1.1. The plant (Source: the authors) Figure 1 Rhoicissus revoilii: a) Whole plant; b) Twigs; c) Leaves and fruits; d) Tubers Rhoicissus revoilii Planch., one of the 12 species of the Rhoicissus genus, is a woody climber with tendrils that can reach heights of between one and ten metres. Its trifoliate leaves are dark green shiny and have entire margins. The leaflets are elliptical and asymmetrical and may be hairless or hairy. Its edible fruits are reddish to black in colour, rounded, and two-lobed [8]. Tubers are generally fleshy. They have a rough, brownish to beige skin and white or yellowish flesh. This plant is distributed in East Africa (Ethiopia, Sudan, Democratic Republic of Congo, Zambia, Zimbabwe, Mozambique, South Africa), as well as in Ghana, the Comoros, Saudi Arabia and Yemen [8]. In the Comoros, it is known by the vernacular names Trambamajji, Pumboubissa and Mhamoussi. The plant (Figure 1) was harvested in May in Chaoueni, in the southeast of Anjouan Island, 60 km from Mutsamudu, at the following geographical coordinates: 12°35'61“ S; 44°50'13“ E. World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1766 Identification was carried out at the Malagasy Institute of Applied Research (IMRA) by Rakotonirina Benja. 2.1.2. Animals used in experiments The mice White Mus musculus OF1 mice were used for the experiments. Five-week-old male and female mice weighing an average of 25 ± 2 g were selected. Frog tadpoles Legless frog tadpoles (Ptychadena mascareniensis) were used in the experiments. They were captured in bodies of water around the university campus three days before the test so that they could acclimatise to laboratory conditions. Carp alvins The carp alvins (Cyprinus carpio) were supplied by an approved fish farmer. They ranged in size from 3 to 5 cm and weighed an average of 3 g. They were placed in an aquarium in the laboratory for three days to acclimatise. Mosquito larvae The larvae of the mosquito (Culex quinquefasciatus), in stage 3 of their development, were captured on the day of the test in the stagnant water around the university campus. 2.2. Methods 2.2.1. Preparation of study material The tubers were dug up, washed, sliced into 4 mm-thick pieces and dried for 72 h in a well-ventilated area. The cossettes obtained were then reduced to a powder and stored in airtight jars at room temperature. 2.2.2. Preparation of the extract The exhaustion extraction method was used to extract the plant's toxic principles. The powder was stripped with hexane (at a ratio of 1:10 w/v) until complete discoloration was achieved. To do this, the powder was suspended in the solvent and the mixture was subjected to magnetic stirring at room temperature for 3 h [9, 10]. The dried pomace was then exhausted with methanol following the same extraction procedure. After filtration, the supernatant from each extraction was evaporated at low pressure and 40°C using a rotary evaporator. The dry residue, dissolved in 10 ml of distilled water, constituted the methanolic extract (ME). 2.2.3. Phytochemical screening The reactions used to detect chemical groups in tuber powder and ME were those developed by Marini-Bettolo et al. [11] and Fong et al. [12]. 2.2.4. Toxicological study Study of the effects of ME on mice Two routes of administration were used to assess toxicity in mice: intraperitoneal (i.p.) and oral. For each 25 g b.w., 0.3 ml of extract was administered, after which the animals were observed for 24 h. Mice given a physiological solution (0.9% NaCl) served as negative controls. Both routes of administration were used in acute toxicity tests. These tests included determining symptoms of intoxication, acute toxicity indices, and anatomo-pathological lesions. Acute toxicity indices (LD0, LD50 and LD100) for ME in mice were determined using the methods of Reed and Muench [13] and Boyd [14]. World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1767 2.2.5. Anatomopathological study Any mice that developed symptoms of intoxication were sacrificed. Their organs were then removed and fixed in a 10% formaldehyde solution before being embedded in paraffin. Histological sections of 3 µm were taken and stained using the standard haematoxylin-eosin method [15]. The slides were prepared using a technique developed by Hould [16] and Diebold et al. [17]. They were observed under a light microscope (10x and 40x magnification). Study of the effects on cold-blooded animals The tests carried out on cold-blooded aquatic animals (carp alvins, frog tadpoles and mosquito larvae) followed the protocols described by Razanatseheno et al. [18]. The LC50 (24 h) was determined by testing different concentrations. The results were analysed using ANOVA with GraphPad Prism 7 software. Statistical values were expressed with 95% confidence intervals. 3. Results 3.1. Extraction and purification process The tuber powder extraction process is shown in Figure 2. Figure 2 Summary diagram of the preparation of the ME extract 3.2. Phytochemical screening results The results of the phytochemical screening of the tuber powder and ME are shown in Table 1. Table 1 Phytochemical screening of Rhoicissus revoilii tuber powder and ME Chemical group Detection reaction Tuber powder ME Alcaloïds Mayer test - - Wagner test - - Dragendorff test - - Flavonoids Flavonols + + Flavones + + Flavonones + + Tannins 1% gelatin test + + 10% salted gelatin test + + Polyphenols 10% FeCl3 test + + Leucoanthocyanins Bate-Smith - - World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1768 Coumarins NaOH (hot) + + Triterpenes Liebermann-Burchard test + - Steroids + + Iridoids HCl (hot) - - Saponins Foam test - - +: positive; -: negative Table 1 showed that flavonoids, tannins, polyphenols, coumarins and steroids were present in the tuber powder and in the ME, while triterpenes were only detected in the tuber powder. 3.3. Effects of extracts on mice 3.3.1. Symptoms of intoxication and influence of administration routes The symptoms of intoxication observed included itching around the muzzle, reduced motor activity, diarrhoea, paralysis of the limbs and increased respiratory rate. These symptoms were the same for both routes of administration, with the exception of abdominal contortions and polyuria (i.p. route) and convulsions (oral route). The results depended on the route of administration. At LD100, mice died after 20 min by the i.p. route and after 47 min by the oral route. 3.3.2. Determination of LD50 (24 h) The acute toxicity indices (LD0, LD50 and LD100) of ME in mice via i.p. and oral administration are presented in Table 2. Seven increasing doses ranging from 12.6 mg/kg to 48 mg/kg were used for the i.p. route, and six dose increments ranging from 75 mg/kg to 600 mg/kg were used for the oral route. Table 2 Acute intraperitoneal and oral toxicity of ME in mice Lethal dose (LD) i.p. route (mg/kg) Oral route (mg/kg) LD0 12.6 75 LD50 21.29-22.3 273-275 3.3.3. Anatomopathological lesions in mice After dissecting the animals, the organs were observed macroscopically and then examined under the microscope to detect any lesions or anatomical changes. The effects of ME on organs (brain, lungs, heart, liver, small intestine, kidneys) were studied for the two routes of administration at different doses. A control group of untreated mice was included for comparison. Macroscopic observations No macroscopic changes were observed when the harvested organs were examined. Microscopic observations Lesions observed after 24 h by i.p. route The main lesions caused by 2 doses of ME (12.6 and 15.6 mg/kg b.w.) on each organ are summarised in Table 3 and illustrated in Figure 3. World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1769 Table 3 Lesions caused by 2 doses of ME in mice by i.p. route (24 h) Organs Untreated mice Observed lesions LD0 (12.6 mg/kg) Sub-lethal dose (15.6 mg/kg) Brain Normal aspect Normal structure Normal aspect Lungs Normal aspect - Dilated inter-alveolar blood vessels - Neutrophils in the parenchyma - Neutrophil infiltration (oedema) - Dilated inter-alveolar blood vessels - Presence of alveolar macrophages loaded with haemosiderin - Presence of red blood cells in the alveoli - Haemorrhagic sheets Heart Normal aspect Normal aspect Normal aspect Liver Normal aspect No steatosis or inflammation - Inflammatory infiltrate - Dilated capillaries - Presence of neutrophils - Ruptured sinusoids Small intestine Normal aspect Normal aspect Normal aspect Kidneys Normal aspect - Dilated glomerular capillaries - Presence of neutrophils - Inflammation of the interstitium and tubules - Glomerular infiltrate - Mesangial proliferation Lesions were particularly marked in the lungs (haemorrhagia), kidneys and liver (inflammation), while the brain and small intestine were histologically normal. World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1770 Figure 3 Lesions caused by ME at a dose of 15.6 mg/kg (b.w.) by i.p. route in the lungs, liver and kidneys (a: 10x magnification; b: 40x magnification) 1: Dilated vessels; 2: Haemorrhagic foci; 3: Destroyed alveoli; 4: Neutrophils; 5: Dilated sinusoidal capillary; 6: Dilated centrilobular vein; Lesions observed after 24 h by oral route Two doses of ME corresponding to LD0 (75 mg/kg) and LD100 (600 mg/kg) were administered orally over a 24 h period. Table 4 below shows the histopathological lesions observed following these treatments. World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1771 Table 4 Lesions caused by different doses of ME on mice taken orally (24 h) Organs Untreated mice Observed lesions LD0 (75 mg/kg) LD100 (600 mg/kg) Brain Normal aspect Normal structure Histologically normal Lungs Normal aspect - Dilated interalveolar blood vessels - neutrophils in the parenchyma - neutrophilic infiltrate (oedema) - Dilated blood vessels - Alveolar oedema (lymphocytic) - Dense lymphocytic cellular infiltrate - Congested capillaries and venules - Proliferation of lymphocytes and macrophages - Dilated inter-alveolar blood vessels - Neutrophils in the parenchyma - Haemorrhagic layer Heart Normal aspect Normal aspect Normal aspect Liver Normal aspect Normal aspect - Intracellular lipid accumulation - Hepatocyte necrosis Small intestine Normal aspect Normal aspect Normal aspect Kidneys Normal aspect Normal aspect - Interstitial inflammatory infiltrate - Lymphocytic infiltration between tubular epithelial cells - Interstitial oedema - Dilated glomerular capillaries - Neutrophils These results show that a dose effect was noted. The lesions observed were more severe at 600 mg/kg than at 75 mg/kg. In the lungs, haemorrhagia and proliferation of lymphocytes, macrophages and polymorphs were observed at the highest dose. In the liver, hepatocyte necrosis was also observed at the same dose. In the kidneys, inflammatory infiltrates associated with interstitial oedema were found. At the lowest dose (LD0), the changes were limited to dilatation of the vessels in the lungs. There were no significant effects on other organs, notably the brain, heart and small intestine. Figure 4 Illustrates the different tissue lesions induced by oral administration of ME at 600 mg/kg World Journal of Advanced Research and Reviews, 2025, 26(03), 1764-1777 1772 Figure 4 Lesions caused by ME at a dose of 600 mg/kg (b.w.) orally in the lungs, liver and kidneys (a: 10x magnification; b: 40x magnification) 1: Dilated alveolus; 2: Inflammatory infiltrate; 3: Thickened bronchiolar wall; 4: Dilated blood vessel; 5: Dilated sinusoidal capillaries; 6: Hepatocytic cords; 7: Tubular dilatation