Bioactivity guided isolation of active principles from spondias pinnata of Bangladesh

Abstract

One medicinal plant of Bangladesh, Spondias pinnata (Kurz) (Family: Anacardiaceae) has been investigated for the isolation of its secondary metabolites and evaluation of pharmacological activities. The powdered fruits and leaves of Spondias pinnata were subjected to cold extraction with methanol at room temperature. The concentrated extract of both parts were then partitioned with pet-ether, carbon tetrachloride, chloroform and ethyl acetate by modified Kupchan partition method. The different fractions of crude methanolic extract of Spondias pinnata fruits and leaves were investigated for phytochemical and biological activities. A total of three compounds were isolated form Spondias pinnata. The isolated compounds were identified by extensive analysis of their high resolution 1H NMR spectral data. Successive chromatographic separation and purification of the crude methanol extract of fruits of Spondias pinnata afforded a 7-hydroxy-6-methoxy coumarin (scopoletin) and a β-sitosterol. This is the first report of its occurrence from Spondias pinnata. On the other hand, Vacuum liquid chromatography (VLC) of the crude methanol extract followed by repeated chromatographic separation and purification of fractions of leaves of Spondias pinnata afforded a Stigmasterol. The different partitions of crude methanolic extract of fruit and leaves and SP-FD-7 (Scopoletin) isolated from fruit were evaluated for antioxidant activity by DPPH method. Among them the CCl4 soluble fraction of leaf (CTSFL), ethyl acetate soluble fraction of leaf (EASFL), crude fraction of fruit (CFF), CHCl3 soluble fraction of fruit (CFSFF), petroleum ether soluble fraction of fruit (PESFF) and SP-FD-7 (scopoletin) showed the most significant free radical scavenging activity with IC50 value of 13 µg/ml, 16 µg/ml, 20 µg/ml, 28 µg/ ml 31 µg/ml and 32 µg/ml. A preliminary toxicological investigation of ethyl acetate, chloroform, carbontetrachloride and petroleum ether extract of fruit and leaves and SP-FD-7 (Scopoletin) isolated from fruit of Spondias pinnata were performed by brine shrimp bioassay technique. Among them CCl4 soluble fraction of fruit (CTSFF) and crude fraction of fruit (CFF) showed high level of lethality in brine shrimp lethality assay with same LC50 values of 3.125µg/ml. The crude extracts, different solvent fractions of fruits and leaves; and SP-FD-7 (Scopoletin) isolated from fruit were subjected to antimicrobial investigation by the disc diffusion method. The CHCl3 soluble fraction of fruit (CFSFF), CCl4 soluble fraction of fruit (CTSFF), crude fraction of fruit (CFF), CHCl3 soluble fraction of leaf (CFSFL), petroleum ether soluble fraction of leaf (PESFL) and crude fraction of leaf (CFL) shows good antibacterial activity against all tested bacterial strains at different doses. Petroleum ether soluble fraction of fruit (PESFF), ethyl acetate soluble fraction of fruit (EASFF), ethyl acetate soluble fraction of leaf (EASFL), CCl4 soluble fraction of leaf (CTSFL) shows no activity against any bacterial strains at the dose of 250µg/ disc while ethyl acetate soluble fraction of fruit (EASFF) and CCl4 soluble fraction of leaf (CTSFL) shows a very poor activity against Staphylococcus aureus and Escherichia coli at the dose of 500µg/ disc. The pure compound SP-FD-7 (Scopoletin) isolated from fruit also showed good antibacterial activity against all tested bacterial strains at different doses. The crude extracts of fruits, leafs and pure compound SP-FD-7 (Scopoletin) isolated from fruit were subjected to determine the analgesic activity by acetic acid induced writhing method on mice. The standard group (Diclofenac Na) showed analgesic activity with writhing inhibition of 72% compared to the control group. The crude fraction of fruit (CFF) and crude fraction of leaf (CFL) showed analgesic activity with writhing inhibition of 31% and 22% at 250mg/kg dose respectively and 56% and 44% at 500 mg/kg dose respectively. The pure compound SP-FD-7 (Scopoletin) showed moderate analgesic activity with writhing inhibition of 16% only at 25 mg/kg dose. From this experiment, it is exhibited that the crude fraction of S. pinnata fruit (CFF) have significant (p<0.001) analgesic activity and the activity is dose dependent. For the evaluation of antidiarrhoeal activity with castor oil induced diarrhoea in mice the crude fraction of fruit, leaf and SP-FD-7 (Scopoletin) isolated from fruit were subjected. The standard group showed mean latent period of 1.86 hours compared to the control group 0.66 hrs. The crude fraction of fruit (CFF) and crude fraction of leaf (CFL) showed antidiarrhoeal activity with mean latent period of 0.87 hr, 0.73 hrs at 250 mg/kg dose respectively and 1.24 hr and 0.98 hr at 500 mg/kg dose respectively. Again the crude fraction of fruit (CFF) and crude fraction of leaf (CFL) showed antidiarrhoeal effect on the basis of mean stool count of castor oil induced diarrhoeal episode on mice in the dose of 250mg/kg and 500mg/kg body weight. The standard group showed mean stool count of 4 compared to the control group 14. The crude fraction of fruit (CFF) and crude fraction of leaf (CFL) showed antidiarrhoeal activity with mean stool count of 9 and 10 at 250 mg/kg dose respectively and 6 and 8 at 500 mg/kg dose respectively. The pure compound SP-FD-7 (Scopoletin) showed a moderate antidiarrhoeal activity with mean latent period of 0.76 hours and mean stool count of 11 at 50 mg/kg kg body weight on mice. From this experiment, it may be assumed that crude fraction of fruit (CFF) have most significant antidiarrhoeal activity. From the above findings it may be concluded that fruit of Spondias pinnata has significant antioxidant, cytotoxic, antibacterial, antidiarrhoeal and analgesic potentials. Where as Spondias pinnata leaf has moderate antioxidant, antibacterial, antidiarrhoeal and analgesic potentials. However, further researches are necessary particularly with isolated active compounds from the most active fractions can play a major role in the area of new drug development.