MOLECULAR AND BIOLOGICAL STANDARDISATION OF Alstonia boonei DE WILD. AND Alstonia congensis ENGL. LEAVES AND STEM-BARKS

Abstract

Alstonia boonei (AB) and Alstonia congensis (AC), belonging to the family Apocynaceae are different but closely related species, indigenous to Africa. The two plants possess varied biological activities including antimalarial, antihypertensive and antidiarrhoeal. However, the apparent similarities between the two species could lead to misidentification and inappropriate medicinal utilisation, necessitating the need to establish diagnostic characters for each species for proper identification. This study, was therefore, aimed at evaluating molecular, pharmacognostic, antispasmodic and antidiarrhoeal profiles of the two plants for their pharmacopoeial standardisation. Deoxyribonucleic acid was isolated from nine accessions of AB and AC leaves (ABL and ACL) collected from southwestern Nigeria and amplified, using Internal Transcribed Spacer (ITS) region. Micromorphological diagnostic characters of leaves and stem-barks were studied by light microscopy. Physico-chemical and elemental analysis of the powdered samples were determined using incineration method and Atomic Absorption Spectroscopy, respectively. Aqueous extraction of plant samples was done, extracts were concentrated in vacuo and freeze dried. The freeze-dried extracts were partitioned successively into dichloromethane (DCM), ethyl acetate and aqueous fractions. Antispasmodic activities of the extracts and fractions were evaluated on high-potassium induced and spontaneous contractions on isolated rat ileum. The AB and AC stem-barks (ABSb, ACSb) extracts and their dichloromethane fractions (DCM-ABSb, DCM ACSb) showing high antispasmodic activity were evaluated for in vivo antidiarrhoeal activities in mice (22- 25 g b.w). Loperamide (5mg/kg) was used as standard. Compounds were isolated from the most active fraction (DCM-ABSb) using chromatographic techniques (Column, TLC). Structures of the isolated compounds were elucidated using spectroscopic techniques (NMR and MS) and their antispasmodic activities evaluated. Data were analysed using descriptive statistics, unweighted pair group method with arithmetic mean and one-way ANOVA and Tukey’s multiple comparison at α0.05. The amplification of the ITS region discriminated between the two Alstonia species. The adaxial epidermal cells of both species were polygonal, straight anticlinal walls and leaves were hypostomatic. The vascular bundle of ABL mid-rib was arc-shaped, while that of ACL was V-shaped. Moisture content values of stem bark of AB (6.4±0.06%) was significantly different from that of AC (7.2±0.4%), while the values were not significantly different for the leaves. Elemental analysis revealed that calcium was the most abundant mineral in the leaves of AB (68.2±3.2 mg/g) and AC (65.7 ±1.0 mg/g). The stem-bark extracts of AB and AC were antispasmodic with IC50 of 0.03±0.2, 0.12±0.01 mg/mL and 1.15±0.1, 1.05±0.8 mg/mL on high-potassium induced and spontaneous contractions, respectively. The DCM-ABSb fraction exhibited significant antispasmodic activity (IC50: 0.02±0.05, 0.31±0.02 mg/mL) on high-potassium induced and spontaneous contractions, respectively. The DCM-ABSb (200 mg/kg b.w.) showed significant antidiarrhoeal activity (87.3%) comparable to Loperamide (87.5%). The isolated compounds from DCM-ABSb were β-amyrin and boonein with only boonein exhibiting antispasmodic activities on both high-potassium induced (IC50: 0.09±0.01 µg/mL) and spontaneous (0.29±0.05 µg/mL) contractions. Alstonia boonei and Alstonia congensis were identified as two distinct species. The diagnostic indices of the two plants provided pharmacopoeial standards for their identification. The isolated boonein could serve as a template for the development of antidiarrhoeal drugs