Chemical and toxicological research on extracts from the leaves of Pittosporum senacia Poir. (Pittosporaceae), a medicinal plant endemic to Madagascar

 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. Chemical and toxicological research on extracts from the leaves of Pittosporum senacia Poir. (Pittosporaceae), a medicinal plant endemic to Madagascar Eliane Razafintsalama Vahinalahaja 2, 3, Maholy Pricille Ratsimiebo 1, 2, Herizo Lalaina Andriamampianina 1, 2, Mihajasoa Stella Razanatseheno 1, 2, Lovarintsoa Judicaël Randriamampianina 1, 2, Danielle Aurore Doll Rakoto 1, 2, Hanitra Ranjàna Randrianarivo 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 National Research Centre for Pharmaceutical Application (CNARP), P.O. Box 702, Antananarivo 101, Madagascar. World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 Publication history: Received on 22 June 2025; revised on 29 July 2025; accepted on 01 August 2025 Article DOI: https://doi.org/10.30574/wjarr.2025.27.2.2823 Abstract A toxic activity has been observed in the extracts of Pittosporum senacia leaves, a Pittosporaceae from Madagascar. A bitter crude extract (CE) was obtained by hot aqueous extraction. A purification procedure from CE comprising ethanol precipitation, dialysis and fractionation with n-butanol yielded a partially purified extract (E3). The active ingredients, which had a bitter taste, were thermostable, precipitable by neutral lead acetate, absorbed by activated charcoal, and soluble in water, ethanol, and n-butanol. A phytochemical screening undertaken on E3 revealed the presence of phenolic compounds, unsaturated sterols, triterpenes and saponins. Mice injected with E3 at the lethal dose of 37.5 mg/kg by intraperitoneal (i.p.) route developed symptoms suggesting an attack of the central nervous system. The LD50 was estimated between 26.05 and 27.41 mg/kg of body weight (b.w.). E3 provoked tissue lesions which were mainly characterized by hemorrhages in the heart, lungs and liver, and by vessel congestion in the brain, intestine and kidneys. In vitro, the active principles caused the lysis of sheep red blood cells. CE was also toxic to carp alvins (LC50 = 27.54 μg/ml) and frog tadpoles (LC50 = 28.11 μg/ml). CE inhibited the germination of diverse plant seeds. E3 and CE were active on Staphylococcus aureus with a Minimum Inhibitory Concentration (MIC) of 1.2 mg/ml and a Minimum Bactericidal Concentration (MBC) of 9.6 mg/ml for CE, and a MIC of 5.9 mg/ml and a MBC of 11.9 mg/ml for E3. CE exhibited a bacteriostatic activity (MBC/MIC= 8) and E3 a bactericidal activity (MBC/MIC= 2.01). Keywords: Pittosporum Senacia; Toxicity; Tissue Lesions; Haemolytic Property; Cold-Blooded Animals; Germination Inhibition; Antimicrobial Properties 1. Introduction Medicinal plants are a valuable source of bioactive compounds that have been used in traditional medicine for thousands of years [1]. Madagascar is a hotspot of plant biodiversity, and many of its endemic species are used for therapeutic purposes without prior scientific validation [2]. Among these plants are species of Pittosporum from the Pittosporaceae family. The Pittosporum genus comprises about 160 species growing wild in tropical and subtropical regions [3]. In Madagascar, Pittosporum is represented by 11 species, 9 of which are endemic. World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 60 Several species of Pittosporum are widely used as medicinal plants for the treatment of various diseases. Different Malagasy species are also widely used as medicinal plants. They have anti-inflammatory, antimicrobial and antispasmodic activities [4, 5]. At LABASM, a research programme on Pittosporaceae has been undertaken: Pittosporum verticillatum [6, 7], Pittosporum ochrosiaefolium [8] and other species are currently being studied. This study focused on Pittosporum senacia, another species of Pittosporaceae used in traditional medicine. This choice was motivated by several reasons, including the lack of studies on this plant other than botanical ones, its use in traditional medicine, the wide diversity of biological properties of its congeners around the world, its widespread availability in Madagascar and, above all, the positive results of preliminary toxicity tests on mice using extracts from its leaves and bark. The investigations aimed to characterise the secondary metabolites present in Pittosporum senacia leaf extracts and to assess their potential toxicity through in vitro tests. The objective was to provide a scientific basis for the safe use of this species in a therapeutic context, while contributing to the inventory of Malagasy plant resources with medicinal or toxicological value. 2. Materials and methods 2.1. Plant materials 2.1.1. Pittosporum senacia Pittosporum senacia is a small tree, 5 to 6 m tall (Figure 1). In Madagascar, it grows in coastal rainforests, from sea level up to 300 m altitude, but rarely up to 1,200 m altitude. The plant was harvested in the Andasibe region (142 km from Antananarivo) in January, when it was in its vegetative stage. In this region, it is commonly known as Maimbovitsika and Ambovitsika. (Source: the authors) Figure 1 Pittosporum senacia: the whole plant (a) and leaves (b) 2.1.2. Plant seeds The seeds used came from the National Center for Applied Research for Rural Development (FOFIFA/CENRADERU) (Table 1). World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 61 Table 1 Plants whose seeds were used for germination assays Plant families Liliopsida Magnoliopsida Common name Poaceae Zea mays Maize, corn Oryza sativa Rice Apiaceae Daucus carota Carrot Petroselinum crispum Chinese parsley Brassicaceae Brassica sp Tissam white Amaranthaceae Beta vulgaris subsp. maritima Beetroot Asteraceae Lactuca sativa Lettuce Cucurbitaceae Cucurbita pepo Pumpkin, zucchini Fabaceae Pisum sativum Bean Phaseolus vulgaris Pea Lamiaceae Ocimum basilicum Basil Solanaceae Solanum nigrum Black nightshade Solanum lycopersicum Tomato 2.2. Animals 2.2.1. Mice White Mus musculus OF1 mice from the Pasteur Institute of Madagascar (IPM) breeding farm were used in the experiments. Five-week-old male or female mice weighing 25 ± 2 g were selected. 2.2.2. Tadpoles Legless tadpoles (Ptychadena mascareniensis) were captured on the day of the test in rice fields located around the Ankatso University Campus. 2.2.3. Fishes Carp alvins (Cyprinus carpio) were supplied by approved private fish farmers. Before testing, these alvins must be kept in an aerated aquarium for one week. 2.2.4. Mosquito larvae Mosquito larvae (Culex quinquefasciatus) were collected on the day of testing from stagnant water located on the campus of the University of Antananarivo. 2.2.5. Microbial strains The germs used, including two Gram-positive bacteria, four Gram-negative bacteria and one yeast, were obtained from the Joseph Ravoahangy Andrianavalona Hospital Laboratory (HJRA). They were isolated from human urine or pus (Table 2). World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 62 Table 2 List of germs used Germs Gram Origin Urine Pus Candida albicans x Streptococcus pyogenes + x Staphylococcus aureus + x Klebsiella pneumoniae - x Pseudomonas aeruginosa - x Escherichia coli - x Enterobacter gergoviae - x 2.3. Methods used to prepare the different extracts All purification steps were guided by toxicity tests on mice and homogeneity tests using thin-layer chromatography. 2.3.1. Leaf powder preparation The fresh leaves were dried away from direct sunlight for one week. They were then ground using a Philips Cucina HR1731/6 blender. The resulting fine powder was stored in jars at room temperature. 2.3.2. Hot aqueous extraction The plant powder was suspended in distilled water at a ratio of 1:10 (w/v). The mixture was heated under reflux for 2 h on a hot plate with magnetic stirring, at the boiling point of distilled water. After cooling, the mixture was left to macerate overnight at 4 °C. The macerate was filtered through four layers of gauze. The resulting filtrate was then centrifuged at 16,000 g for 20 min. The pellet was discarded and the supernatant volume was reduced by evaporation using a rotary evaporator at a ratio of 1:1 (1 ml per 1 g of starting material). The resulting solution constituted the crude extract (CE). 2.3.3. Precipitation with ethanol A volume of absolute ethanol was added dropwise to the same volume of extract to be treated. The mixture was magnetically stirred and left to stand at +4 °C for 15 min. The precipitate formed was removed by centrifugation at 12,000 rpm for 15 min. The supernatant was evaporated to dryness and the resulting residue recovered in distilled water. 2.3.4. Dialysis The extract to be treated was introduced into a dialysis membrane with a filtration threshold of 15,000 Da. The counterdialysis liquid used was distilled water, the volume of which was 500 times greater than that of the extract to be dialyzed. This liquid was constantly kept under magnetic stirring. This process was repeated several times. 2.3.5. Activated charcoal treatment Treatment with activated charcoal was carried out using the Jeannoda method [9]. 2.3.6. Precipitation with neutral lead acetate The precipitation method using neutral lead acetate was adapted from the procedure described by Rasoatahina et al. (2024) [10]. 2.3.7. n-Butanol fractionation The same volume of extract and n-butanol were introduced into a separating funnel. After manual stirring, the mixture was left to settle completely. The two phases were collected separately. The aqueous phase was again treated with nbutanol. The two organic phases were combined, and the n-butanol was evaporated after addition of distilled water. World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 63 2.4. Methods used to study effects on animals 2.4.1. Effect on mice Assessment of acute toxicity Acute toxicity in mice was assessed by intraperitoneal (i.p.) injection of 0.3 ml of test extract per 25 g of mouse. For each test, a batch of 3 mice was used. Another batch of 3 mice of the same weight, receiving 0.3 ml of a physiological solution (0.9% NaCl), served as a control. The dose that killed 50% of mice in 24 h or LD50 was determined by calculation and graphical methods [11]. Seven doses of the extract, in geometric progression of reason r =1.113, ranging from 20.03 mg/kg to 37.5 mg/kg b.w., were injected intraperitoneally into 7 batches of 6 mice. A batch of 6 mice injected with 0.3 ml of saline solution served as a control. Histopathological examination Mice that have developed symptoms of intoxication were sacrificed. Hearts, livers, kidneys, brains, lungs and stomachs were rapidly removed and immersed in fixative fluid (BOUIN solution: picric acid 5 ml, formol 40% 20 ml, glacial acetic acid 5 ml). After 48 h, each organ was fragmented into thin 5 mm thick sections using a scalpel. More details of the method used were given in our previous article [10]. 2.4.2. Haemolytic test on sheep red blood cells In the presence of a haemolytic substance, red blood cells were lysed, releasing hemoglobin and turning the supernatant red. Intact red blood cells sedimented. The method used was that described in our previous article [10]. The composition of the medium used for the test is presented in Table 3. Table 3 Composition of the medium for the haemolytic test Well number C+ C - 3 4 5 6 7 8 9 10 11 12 Test extract 1 mg/ml (µl) 0 0 50 25 12.5 6.25 3.125 1.56 0 .78 0.39 0.196 0.097 PBS (µl) 0 50 0 25 37.5 43.75 46.87 48.44 49.22 49.61 49.80 49.90 2% red blood cell suspension (µl) 50 50 50 50 50 50 50 50 50 50 50 50 Distilled water (µl) 50 0 0 0 0 0 0 0 0 0 0 0 Final concentration of extract (mg/ml) 0 0 1 0.5 0.25 0.125 0.065 0.031 0.015 0.007 0.003 0.001 Final volume of the mixture (µl) 100 100 100 100 100 100 100 100 100 100 100 100 C+: Positive control (total haemolysis); C-: Negative control (no haemolysis) 2.4.3. Effect on carp alvins and frog tadpoles Seven alvins or legless tadpoles were selected and placed in crystallisers containing 200 ml of rainwater. Different quantities of the extract to be studied were added in order to obtain different concentrations in geometric progression. The experiment lasted 24 h. The LC₅₀ (24 h) was determined by testing various concentrations of the extract, and the results were analyzed using the graphical method described by Boyd (1966) [12]. 2.4.4. Effect on mosquito larvae Five batches of 20 stage three larvae were used. These larvae were placed in crystallisers containing 200 ml of spring water with various concentrations of the extract to be tested. A further batch placed in water served as a control [13]. 2.5. Methods used to study effects on seed germination For each seed species, two batches of seeds were soaked in tap water for 48 h in darkness at 30°C. The first batch was then germinated on water-soaked cotton wool, while the second was germinated on cotton wool impregnated with the extract at a specific concentration. The results were observed 72 h after soaking. World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 64 2.6. Methods used to study effects on microorganisms’ growth 2.6.1. Antibiogram test All the materials and methods used for antimicrobial assay were detailed in a previous study [14]. The results were interpreted using the scale of Ponce et al. (2003) [15] and Celikel et al. (2008) [16]: bacteria were considered not sensitive for an inhibition zone diameter (IZD) ≤ 8 mm; sensitive for 9 ≤ IZD ≤ 14 mm; very sensitive for 15 ≤ IZD ≤ 19 mm and extremely sensitive for IZD ≥ 20 mm. 2.6.2. MIC and MBC determination The method used was based on the tube dilution technique described in the CLSI guidelines (2012) [17]. It involved the use of Mueller-Hinton broth as the sole medium, without resorting to microplates. Composition of tubes used for MIC determination are presented in Table 4. This approach allowed the determination of the MIC, defined as the lowest concentration showing no visible turbidity, and the MBC identified as the lowest concentration with no bacterial growth after subculturing on Mueller-Hinton agar. The tubes and agar plates were then inspected for turbidity or microbial growth with the unaided eye. The culture was incubated at 37°C for 24 h. Interpretation of the results was based on the standards proposed by Dalmarco et al. (2010) [18]. The extract was considered bactericidal when the MBC/MIC ratio was ≤ 4, and bacteriostatic when the ratio was > 4 [19]. Table 4 Composition of tubes used for MIC determination Test tube number C+ C1 2 3 4 5 6 7 8 9 Inoculum volume (ml) 0 0 1 1 1 1 1 1 1 1 1 Culture medium volume (ml) 2 1 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Extract volume (ml) 0 1 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 Final mixture volume (ml) 2 2 2 2 2 2 2 2 2 2 2 C+: Positive control; C-: Negative control World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 65 3. Results 3.1. Preparation of the various extracts. The various steps used to obtain the different extracts are summarized in Figure 2. Figure 2 Diagram summarizing the extraction and the purification stages of active principles The hot water extraction of 25 g of leaf powder produced a dark brown, bitter-tasting crude extract (CE) with a pH of 6.5 that was toxic to mice. The extraction yield was 28.88%, whereas the purification yield of E3 from CE was 17.31%. The evolution of the homogeneity of the toxic extracts obtained at the different purification stages is shown in Figure 3. Solvent: Butanol/Acetic acid/Water (60/60/20, w/w); Developer: Sulfuric vanillin reagent Figure 3 Thin layer chromatography of extracts obtained at the various purification stages World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 66 3.2. Phytochemical screening Phytochemical screening results of CE and E3 are presented in Table 5. Table 5 Phytochemical screening results of CE and E3 Chemical families Tests Results CE E3 Alkaloids Mayer - - Wagner - - Dragendorff - - Flavonoids Wilstater - - Leucoanthocyanins Bate-Smith - - Tannins Gelatin 1% test - - Salted gelatin test - - Polyphenols Ferric chloride test + + Unsaturated sterols Salkowski + + Triterpenes Liebermann-Burchard + + Steroids - - Anthraquinones Bornträger - - Deoxyoses Keller-Kiliani + - Iridoids - - Saponins Foam test + + +: Positive test; -: Negative test CE contained polyphenols, unsaturated sterols, triterpenes, deoxyoses, and saponins. The E3 fraction contained the same phytochemical compounds with the exception of deoxyoses. 3.3. Effects on animals 3.3.1. Effects on Mice Symptoms of intoxication Intraperitoneal administration of E3 at a dose of 37.5 mg/kg b.w. immediately caused agitation in the mice. After 10 min, they were breathing deeply, with a respiratory rate ranging from 90 to 110 breaths per min., and remained immobile. Thirty min. later, earlobes were turned backwards and became hyperemic. After 50 min, the mice moved only rarely and dragged their hind legs. After 1 h, exophthalmos and cyanosis of the tail appeared. A gradual decrease in respiratory rate was then observed. After 2 h 30 min, clonic convulsions appeared, becoming increasingly severe and leading to the death of the animal. The sublethal dose of 20.03 mg/kg caused the same symptoms as the lethal dose, with the exception of convulsions. The mice remained motionless for several hours and exhibited enophthalmos. A gradual remission was observed after 10 h. LD50 value The LD50 of CE by the i.p. route was estimated at 26.05 mg/kg b.w. and 27.41 mg/kg b.w. by calculation and graphical methods respectively. World Journal of Advanced Research and Reviews, 2025, 27(02), 059-074 67 3.3.2. Histopathology The effects of the E3 at the tissue level were studied at a lethal dose of 37.5 mg/kg of mice by i.p. route. 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