DETERMINATION OF CHEMICAL COMPOSITION AND BIOLOGICAL ACTIVITIES OF TERMINALIA BELERICA, SWERTIA CHIRATA AND ZANTHOXYLUM ARMATUM

Abstract

Three medicinal plants namely; Swertia chirata, Zanthoxylum armatum and Terminalia belerica were selected on the basis of their medicinal importance. We determined the chemical constituents and biological activities due to their medicinal importance. The results of these tests provide a base line for quality control of these herbal plants. Swertia chirata Microscopic examination of powder of aerial parts of S. chirata in chloral hydrate (10%), glycerine (50%) and iodine (5%) revealed the diagnostic features as xylem vessels of stem portion, spiral shape xylem vessel and xylem vessel with annular thickening, raphides, epidermal cells, oil cell, parenchymatous cells with pits, sclerenchymatous cells, fibre, cork cell, sclereid, starch grains, trichome, stone cell and tannin. Preliminary phytochemical analysis of the methanolic extract of S. chirata showed the presence of tannin, alkaloids, sterols, triterpene, diterpene, and carbohydrate. Different specific colours were observed in fluorescence analysis of powder drug of S. chirata. FT–IR spectrum of powder drug of S. chirata revealed the presence of many functional groups such as –OH (alcoholic), C-H, >C=O, benzene ring and C-O-C . In FT–IR spectrum of methanolic extract of S. chirata showed the presence of -OH, C-H, benzene ring and C-O-C groups. TLC of methanolic extract of S. chirata in chloroform -methanol-water (80:20:2) solvent system revealed 10 and 8 spots under UV light of 254 nm and 366 nm respectively. TLC of methanolic extract of S. chirata in ethyl acetate-methanol (100:20) solvent system revealed 10 and 9 spots under UV light of 254 nm and 366 nm respectively. Antibacterial activity of methanolic extract of S. chirata was carried out on eleven gram positive and seventeen gram negative bacteria by agar well method. Gentamicin 10mg disc (Zone of inhibition 12-15mm) was used as a reference drug. It was revealed that six gram negative bacteria (Klebsiella pneumonia, Acinetobacter baumanii, Serratia marcesens, Aeromonas hydrophila, Shigella dysenteriae and Enterobacter aerogenes) and six gram positive bacteria (Corynebacterium diptheriae, Corynebacterium hofmanii, iii Corynebacterium xerosis, Streptococcus fecalis, Streptococcus pyogenes and Streptococcus saprophyticus) were inhibited. Minimum inhibitory concentrations (MIC) of the extract were determined by broth microdilution method. The significant MIC value of extract of S. chirata was 20 mg/ml against Serratia marcesens. Antifungal screening of methanolic extract of S. chirata was carried out on 6 saprophytic, 5 dermatophytic and 6 yeast by agar well method. In this case Griseofulvin (30 mg disc) was used as a reference drug (zone of inhibition 10-12 mm). All saprophytic and dermatophytic were showed resistance by this plant extract except 1 dermatophytic (Microsporum canis) and 5 yeast (Candida albicans, Candida albicans ATCC 0383, Candida galbrata, Candida tropicalis and Candida kruzei). The significant MIC value of S. chirata was 10 mg/ml against Candida tropicalis. The anti-oxidant study was performed by DPPH free radical scavenging method using ascorbic acid as a standard which showed 96.5±0.04 % inhibition of DPPH free radicals. Significant % inhibition of DPPH was observed by S. chirata at concentration 200μg / ml was 70±0.01%. EC50 value of S. chirata extract was 937.5ug/ml. Neuro-pharmacological activities of methanolic extract of S. chirata were studied by open field, memory bench, traction, head dip, rearing test, dark/light and swimming induced depression test. All tests were performed in a calm and peaceful environment at different test doses 500, 300 & 100 mgkg-1 p.o. Diazepam 2mg/kg p.o. used as a standard drug. The test dose 100 mg/kg produce highly significant reduction in motor activity as compare to control in open field activity which showed that S. chirata could possess a neurosedative action in low doses. The test doses 500 & 100 mg/kg of S. chirata showed stimulant and antidepressant activity of this plant in memory bench activity. In head dip test, the methanolic extract of S. chirata at the test dose 500 mg/kg produced highly significant increased in exploratory behavior. In rearing activity all test doses of methanolic extract of S. chirata not produced any prominent results. The test doses 500 and 300 mg/kg produced highly significant muscle stimulant effect as compare to standard drug diazepam 2mg/kg which produced highly significant muscle relaxing effect in traction test. The test dose of 500 mg/kg of S. chirata showed the increase time spent in light that showed anxiolytic effect. In forced swimming test (FST), the test dose 500 mg/kg of methanolic extract of S. chirata significantly increased the duration of mobility in mice suggesting that they have antidepressant effect and stimulant but he test dose 300 mg/kg significantly decreased the duration of mobility in mice that showed S. chirata could possess a neurosedative in low doses. The methanolic extracts of aerial parts of S. chirata at different doses (100, 300 and 500 mg/kg p.o.) were assessed by formalin induced licking/bit behavior in mice. The highly significant % inhibition of lick/bit was noted at the test dose 100 mg/kg and other test doses were produced significant % inhibition after 0-15 min as compared with aspirin. Results from the present study showed that the methanolic extract of aerial parts of S. chirata produced 76.87 % inhibition of writhes after 0-15 min against acetic acid induced writhes at the dose of 500 mg/kg which was highly significant as compare to aspirin produced 84.4% inhibition of writhes. The methanolic extract of aerial parts of S. chirata at the test dose 500 mg/kg has significant anti-inflammatory effects after 1 hr in paw edema induced by formalin. The toxicity of methanolic extract of S. chirata was performed by Brine Shrimp Bioassay at three diferent doses 10, 100 and 1000 μg/ml. The toxic effect was evaluated in terms of death of larvae and etoposide is used as standard drug. The observations showed that this plant extract did not produced any lethality for larvae. Zanthoxylum armatum Microscopic examination of powder of fruit of Zanthoxylum armatum in chloral hydrate (10%), glycerine (50%) and iodine (5%) revealed the diagnostic features as vascular tracheid, mesophyll cell contain sphenoidal crystals, phloem fibre, pitted vessel, sclariform vessel, oil droplet, schizogenous cavities with oil cells, pigmented trichome, lignified fibres, solidified resin, bordered pitted vessel, spiral vessel, group of starch grain, lignified epicarp cells, starch grain with concentric stritations, mesocarp cells, trichome, cell with sphenoidal crystals, simple trichome, tannin, polyhedral cells, prismatic crystals, branched fibres, simple fibre, starch grain with hilum and annular vessels. Preliminary phytochemical analysis of the methanolic extract of Z. armatum revealed the presence of alkaloids, glycosides, flavonoids, tannins, sterols and carbohydrates. Fluorescence analysis of powder drug of Z. armatum revealed different specific colours. Different diagnostic peaks were observed in FT–IR spectrum of powder drug of Z. armatum which represented the presence of many functional groups such as C-H, -OH (alcoholic), >C=C< , >C=O, benzene ring and C-O-C .In FT–IR spectrum of methanolic extract of Z. armatum showed the presence of –OH, benzene ring and C-O-C groups. TLC of methanolic extract of Z. armatum in chloroform -methanolwater (80:20:2) solvent system revealed 9 and 8 spots under UV light of 254 nm and 366 nm respectively. Antibacterial activity of methanolic extract of Z. armatum was carried out on eleven gram positive and seventeen gram negative bacteria by agar well method. Gentamicin 10mg disc (Zone of inhibition 12-15mm) was used as a reference drug. It was revealed that five gram negative (Klebsiella pneumonia, Acinetobacter baumanii, Serratia marcesens, Aeromonas hydrophila and Vibro choleae) and six gram positive bacteria (Corynebacterium diptheriae, Corynebacterium hofmanii, Corynebacterium xerosis, Streptococcus fecalis, Streptococcus pyogenes and Streptococcus saprophyticus) were inhibited. Minimum inhibitory concentration (MIC) of the extract was determined by broth microdilution method. The significant MIC value of Z. armatum was 10 mg/ml against Aeromonas hydrophila. Antifungal screening of methanolic extract of Z. armatum was carried out on 6 saprophytic, 5 dermatophytic and 6 yeast by agar well method. In this case Griseofulvin (30 mg disc) was used as a reference drug (zone of inhibition 10-12 mm). All saprophytic and dermatophytic were showed resistance by this plant extract except 1 dermatophytic (Microsporum canis) and 5 yeast (Candida albicans, Candida albicans ATCC 0383, Candida galbrata, Candida tropicalis and Candida kruzei). The significant MIC value of Z. armatum was 10 mg/ml against Microsporum canis. The anti-oxidant study was performed by DPPH free radical scavenging method using ascorbic acid as a standard which showed 96.5±0.04 % inhibition of DPPH free radicals. Significant % inhibition of DPPH was observed by Z. armatum at concentration 200μg/l was 70±0.02%. EC50 value of Z. armatum extract was 937.5ug/ml. Neuro-pharmacological activities of methanolic extract of Z. armatum were studied by open field, memory bench, traction, head dip, rearing test, dark/light and swimming induced depression test. All tests were performed in a calm and peaceful environment at different test doses 500, 300 & 100 mgkg-1 p.o. Diazepam 2mg/kg p.o. used as a standard drug. The test dose 500 mg/kg produce highly significant reduction in motor activity as compare to control in open field activity which showed that Z. armatum could possess a neurosedative effect. The test doses 500 & 300 mg/kg of Z. armatum showed anxiolytic activity in memory bench activity. In head dip test, the methanolic extract of Z. armatum at the test dose 500 mg/kg produced highly significant decreased in exploratory behavior. In rearing activity at test dose 500 mg/kg of methanolic extract of Z. armatum decreased in exploratory behavior.The test dose 300 mg/kg produced highly significant muscle relaxant effect in traction test.The test dose of 300 mg/kg of Z. armatum showed the increase time spent in light that showed anxiolytic effect. In FST, the test dose 500 mg/kg of methanolic extract of Z. armatum significantly increased the duration of mobility in mice suggesting that they have antidepressant effect and stimulant. The methanolic extracts of Z. armatum at different doses (100, 300 and 500 mg/kg p.o.) were assessed by formalin induced licking/bit behavior in mice. All test doses were not produced any significant results as compared with aspirin. The test doses of 500 and 300 mg/kg of the methanolic extract of Z. armatum produced 68.65% and 64.9% inhibition of writhes after 15-30 min against acetic acid induced writhes which were highly significant as compare to aspirin produced 69.7% inhibition of writhes. The methanolic extract of Z. armatum at the test dose 500 and 100 mg/kg has highly significant anti-inflammatory effects after 30 min in paw edema induced by formalin as compared with aspirin. The toxicity of methanolic extract of Z. armatum was performed by Brine Shrimp Bioassay at three diferent doses 10, 100 and 1000 μg/ml. etoposide was used as standard drug. The observations showed that Z. armatum extract did not produced any lethality for larvae. Terminalia belerica Microscopic examination of powder of fruit of Terminalia belerica in chloral hydrate (10%), glycerine (50%) and iodine (5%) revealed the diagnostic features as spiral vessel, trichome, sieve plate, scalariform vessel, parenchymatous cells, starch grain, rossette shape crystal, stone cell, branched pits through thick cell stone cell, fiber, pitted tracheid, pitted vessel, longitudinal cells, cork cells, starch grain & group of stone cells. Preliminary phytochemical analysis of the methanolic extract of T. belerica revealed the presence of alkaloids, glycosides, flavonoids, tannins, sterols and carbohydrates. viii Fluorescence analysis of powder drug of revealed different specific colours. Different diagnostic peaks were observed in FT–IR spectrum of powder drug of T. belerica which represented the presence of many functional groups such as C -H, >C=O, benzene ring and C-O-C . In FT–IR spectrum of methanolic extract of showed the presence of –OH, C-H, benzene ring and C-O-C groups. TLC of methanolic extract of T. belerica in chloroform -methanol-water (80:20:2) solvent system revealed 6 and 10 spots under UV light of 254 nm and 366 nm respectively. Antibacterial activity of methanolic extract of T. belerica was carried out on eleven gram positive and seventeen gram negative bacteria by agar well method. Gentamicin 10mg disc (Zone of inhibition 12-15mm) was used as a reference drug. It was revealed that six gram negative (Klebsiella pneumonia, Acinetobacter baumanii, Serratia marcesens, Aeromonas hydrophila, Vibro choleae and Shigella dysenteriae) and six gram positive bacteria (Corynebacterium diptheriae, Corynebacterium hofmanii, Corynebacterium xerosis, Streptococcus fecalis, Streptococcus pyogenes and Streptococcus saprophyticus) species were inhibited. Minimum inhibitory concentration (MIC) of methanolic extract of T. belerica was determined by broth microdilution method. The significant MIC value of methanolic extract of T. belerica was 20 mg/ml against Acinetobacter baumanii and Serratia marcesens. Antifungal screening of methanolic extract of T. belerica was carried out on 6 saprophytic, 5 dermatophytic and 6 yeast by agar well method. In this case Griseofulvin (30 mg disc) was used as a reference drug (zone of inhibition 10-12 mm). All saprophytic and dermatophytic were showed resistance by this plant extract. 5 yeasts (Candida albicans, Candida albicans ATCC 0383, Candida galbrata, Candida tropicalis and Candida kruzei) were showed sensitivity against methanolic extract of T. belerica. The significant MIC value of extract of T. belerica was 18 mg/ml against Candida albicans. The anti-oxidant study was performed by DPPH free radical scavenging method using ascorbic acid as a standard which showed 96.5±0.04 % inhibition of DPPH free radicals. Significant % inhibition of DPPH was observed by at concentration 200μg/ml was 55.6±0.02% highly significant EC50 value of this extract was 100ug/ml. ix Neuro-pharmacological activities of methanolic extract of T. belerica were studied by open field, memory bench, traction, head dip, rearing test, dark/light and swimming induced depression test. All tests were performed in a calm and peaceful environment at different test doses 500, 300 & 100 mgkg-1 p.o. Diazepam 2mg/kg p.o. used as a standard drug. The test dose 500 mg/kg produce highly significant reduction in motor activity as compare to control in open field activity which showed that could possess a neurosedative effect. The test dose 500 mg/kg of extract of T. belerica showed anxiolytic activity in memory bench activity. In head dip test, the methanolic extract of T. belerica at the test dose 500 and 300 mg/kg produced highly significant increased in exploratory behavior. In rearing activity at test dose 500 mg/kg of methanolic extract of T. belerica decreased in exploratory behavior. The test dose 500 mg/kg produced significant muscle relaxant effect in traction test. All test doses of T. belerica showed the increase time spent in dark that showed sedative effect. In FST, the test dose 500 mg/kg of methanolic extract of significantly decreased the duration of mobility in mice suggesting that they have anxiolytic effect. Extracts the methanolic of T. belerica at different doses (100, 300 and 500 mg/kg p.o.) were assessed by formalin induced licking/bit behavior in mice. The test dose of 500 mg/kg of methanolic extract of T. belerica produced 52.5% inhibition of licking/bit after 30 min in formalin induced lick/bit response in mice. This was significant inhibition of licking/bit as compare to aspirin which produced 67.6 % inhibition of writhes. All doses of methanolic of T. belerica were produced significant dose dependent % inhibition of writhes as 61.6 %, 59.46 % and 58.92% by 500, 300 and 100 mg/kg p.o. respectively against acetic acid induced writhes as compare to aspirin produced 69.73 % inhibition of writhes in mice. x The methanolic extract of T. belerica at the test dose 500 mg/kg has significant antiinflammatory effects after 1 hr and 3 hr in paw edema induced by formalin as compared with aspirin. The toxicity of methanolic extract of T. belerica was performed by Brine Shrimp Bioassay at three different doses 10, 100 and 1000 μg/ml. etoposide was used as standard drug. The observations showed that extract of T. belerica did not produced any lethality for larvae.