Pharmacognosy

Pharmacognostic Standardization and Anti- Sickling Activities of Bioactive Compounds from Seeds of Picralima Nitida Stapf (Apocynaceae)

Fri, 02 Jun 2017 00:00:00 GMT

Pharmacognostic Standardization and Anti- Sickling Activities of Bioactive Compounds from Seeds of Picralima Nitida Stapf (Apocynaceae) Osuala, Felix Ngozichukwuka This present study was to establish the pharmacognostic standards of the Picralima nitida, to investigate the anti-sickling effects of crude methanol seed extract and fractions of Picralima nitida, to isolate and elucidate the structure of the bioactive constituents. Phytochemical analysis of the extract, fractions and isolates were carried out using standard procedures. Pharmacognostical profile and proximate/numerical standards were also evaluated. The anti-sickling effect was studied using sodium methabisulphite-induced sickling of the HbSS red blood cells obtained from confirmed non-crisis state (steady state) sickle cell patients. The effects of P. nitida on biochemical parameters of HbSS blood were studied. The effect of the extract on the rheology and viscosity of sickle cell blood were studied using various concentrations (2.5, 5 and 10 mg/ml) at 30 minutes intervals through 180 minutes of incubation. Comparison of the anti-sickling effect of methanol seed extract of P. nitida, fractions and isolates on sickled red blood cells was done using a known standard drug, p-hydroxybenzoic acid. The microscopy of the powdered seeds revealed the presence of sclereids, parenchyma and epidermal cells, calcium oxalate crystal and fat globules. Qualitative and quantitative phytochemical analyses of the extract showed the presence of alkaloids (5.84 g), tannins (0.065), flavonoids (7.03 g), glycosides (4.58 g), terpenoids (4.08 g), protein (11.63 g), steroids, resins, reducing sugars, carbohydrates, fats and oils. The analytical standards gave 3.0, 11.5, 3.5, 4.0, 12.5, 20.4 and 8.0 % for moisture content, total ash, acid insoluble ash, water soluble ash, sulphated ash, alcohol soluble and water soluble extractives respectively. The acute toxicity test showed that the extract was safe at dose 3500 mg/kg. The study showed significant (p < 0.05) antisickling activities of the fractions on HbSS blood and were given to be in order CHCl3 ≤ CH2CL2 < EtOAc < Aqueous at the tested concentrations. The Aqueous Fraction showed highest degree of inhibition (85%) and was comparable to P-hydroxybenzoic acid (100%). The negative control (normal saline) showed no change, Compound 1 at 2.5 mg/ml showed the best anti-sickling activity (100% reversal of sickling at 120 min) compared to that of the standard drug p-hydroxybenzoic acid (100 % reversal of sickling at 180 min). The anti-sickling activity of the compounds are in the order CP1 (100 %) > CP3 (95 %) > CP2 (92 %) unsickling at 120 min. However compound 3 and compound 2 at 5 mg/ml showed 100 % reversal of sickling at 180 min.The isolated compounds are Ajmalicine → (19 α)-16, 17-didehydro – 19 – methyl – 2 –oxayohimbin – 16- carboxylic acid methyl ester, was obtained as white powder. Its molecular formula was derived as C21H24O3 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z = 353.1867. (1) And Ajmalicine Oxindole B → (19 α) -19-methyl -2-oxoformosanan -16- carboxylic acid methyl ester, was obtained as light yellow powder. Its molecular formula was derived as C21H24O4 by the high resolution Shimadzu IT-TOF spectrum, showing an [M] + ion at m/z = 369.1815. (2). The results of the study suggest that the methanol extract and fractions of P. nitida possess antisickling effect and justify’s the ethnomedicinal claims

Serum Profiles of Pro-Inflammatory Cytokines and Some Other Biomarkers of Inflammation in Nigerian Tuberculosis Patients Undergoing Standard Chemotherapy at Enugu

Fri, 02 Jun 2017 00:00:00 GMT

Serum Profiles of Pro-Inflammatory Cytokines and Some Other Biomarkers of Inflammation in Nigerian Tuberculosis Patients Undergoing Standard Chemotherapy at Enugu Anusiem, Chikere Atulaegwu Introduction: Tuberculosis (TB) is a communicable disease that has afflicted man for centuries. It is one of the commonest causes of death from infectious diseases in the world. Nigeria is ranked among the 22 countries with the highest TB burden in the world. The discovery of host or pathogen specific biomarkers of TB for diagnosis, treatment, and prognosis has of recent become top research priority globally. The pathogenesis of TB involves cell-mediated immune response against Mycobacterium tuberculosis and different cytokines have been found to be specifically involved in the pathogenesis of pulmonary TB. Cytokines which are a group of regulatory molecules that permit contact-independent communication between the cells of the human body have been considered to be candidate biomarkers for TB. There has been a lot of TB- related research but to the best of our knowledge no prospective study has concurrently and intermittently measured the blood levels of Tumor necrosis factor alpha (TNFα), Interleukin 1 beta (IL-1 β) and Interleukin 6 (IL-6) in Nigerian TB patients from commencement of TB treatment to the end of short course chemotherapy. This study aims at documenting the serum concentrations of these three cytokines (IL-1 β; IL-6; and TNFα) in eligible TB patients using human ELISA kits, from the time of diagnosis to completion of six months short course chemotherapy of TB. This prospective study is expected to yield useful information that would be utilized in the quest for better biomarkers to improve TB management. Methodology: The study was a prospective observational study carried out at the Chest Clinic of the University of Nigeria Teaching Hospital (UNTH) Enugu with the written permission of UNTH Management. The research proposal was approved by the Research Ethics Committee of UNTH Enugu. Freshly diagnosed drug-naive adult TB patients of both genders who met the inclusion criteria were recruited after giving informed consent. Uniform standard WHO - approved treatment was prescribed for and supplied to the patients. Each subject was followed up for six months starting from their respective times of recruitment into the project. Healthy volunteers were also recruited. Blood samples were taken at zero time (i.e. after diagnosis and enrolment into study but before ingestion of TB drugs); at 2 months of treatment; and also at 6months of treatment. Serum concentrations of Interleukin 1 beta (IL-1 β); Interleukin 6 (IL-6); and Tumor necrosis factor alpha (TNFα) were measured using human ELISA kits (e-Bioscience, USA). Erythrocyte Sedimentation Rate (ESR) was measured by Westergren method and Haemoglobin concentration was estimated by the Cyanmethaemoglobin method. Total leukocyte count and differential counts for the percentage counts of Neutrophils, Lymphocytes, Monocytes, Basophils, and Eosinophils were done by routine manual microscopy. Results were analysed using Graphpad prism and SPSS computer soft-wares and displayed as tables, pie, and bar charts.. Results: Forty two tuberculosis patients and thirty one healthy volunteers were able to complete the study. The mean serum concentration of Interleukin 1 beta (IL-1 β) for the TB patients prior to treatment was abnormally raised (30.0 ± 2.0 pg/ml). As treatment progressed there was statistically significant progressive reduction in the mean IL-1 β serum concentration at 2months as well as at 6 months of treatment (22.0±1.1pg/ml and 17.0 ± 1.3 pg/ml respectively). There was a significant difference between the mean IL-1beta obtained after 6months of treatment and for the healthy volunteers (13.0 ± 1.3pg/ml). A similar trend was observed for both Il-6 and TNFα with values obtained at 6months of treatment approximating those of healthy volunteers. TNFα mean serum concentration at zero time was 40.7±2.4pg/ml and 23.1±1.4 pg/ml after 6 months of treatment. For haemoglobin the trend was different. The mean Haemoglobin concentration appreciated from 11.4±0.2g/dl prior to treatment to 11.8±0.2g/dl after 2 months of treatment, and to 13.2±0.1g/dl four months later. Compared to healthy volunteers (14.4±0.2g/dl) the mean haemoglobin concentration of the TB patients was significantly lower at zero time and at 2 months of treatment. The profile of mean Erythrocyte Sedimentation Rate (ESR) documented in the study mirrored those of the cytokines. The mean Monocyte count, Eosinophil count, and Basophil count obtained before commencement of treatment and during the course of treatment all ranged from 0 to 3% and exhibited no significant difference between TB patients, and healthy volunteers on one hand and between zero time values and mean counts made at 2months and 6months of treatment respectively on the other hand. However, both the mean total leukocyte count and the percentage Neutrophil count in the peripheral blood increased during the course of treatment, while lymphocyte count decreased over time during the corresponding time intervals. The mean total leukocyte count was 4785.0 ± 158.0/mm3 at zero time, 4805.0 ± 106/mm3 and 4835.0 ± 143.0/mm3 at six months of treatment. Conclusion: This study shows that in Nigerian adult patients erythrocyte sedimentation rate (ESR) as well as the three cytokines assayed in this study were all very high in the serum of drug-naïve TB patients compared to those of the healthy volunteers but as TB chemotherapy continued over the period of six months there was progressive reduction in the serum levels of both ESR and the 3 cytokines. At the completion of TB treatment, the serum concentrations of the cytokines were comparable to those of the healthy human controls. The serum profiles therefore indicate that these cytokines are good candidate biomarkers for Nigerian TB patients. Larger funded prospective studies would be needed to confirm and elucidate the suitability of these cytokines singly or in combination to serve as diagnostic and/or prognostic host biomarker(s) for tuberculosis.

Evaluation of Hypoglycaemic Effect and Phytochemical Constituents of Methanol Extract of Vernonia Amygdalina Del Fam. (Asteraceae)

Fri, 02 Jun 2017 00:00:00 GMT

Evaluation of Hypoglycaemic Effect and Phytochemical Constituents of Methanol Extract of Vernonia Amygdalina Del Fam. (Asteraceae) Chukwube, Vincent Obisike Leaf of Vernonia amygdalina has been shown to have hypoglycemic activity. This may be as a result of the presence of secondary metabolites as its constituents. The major secondary metabolites present in the leaf are tannins, carbohydrates, reducing sugars, glycosides, steroids and proteins. This work was designed to identify these secondary metabolites responsible for this activity, test the lethality of the constituents and then determine the best solvent for extraction. A 1 kg crushed leaves of the plant Vernonia amygdalina was extracted with methanol using soxhlet apparatus. The dichloromethane /methanol ratio which showed the best TLC separation was fractionated in a column packed with silica gel GF254 and eluted with dichloromethane/methanol in the ratios of 9:1, 8:2, 7:3, 6:1 and 1:1. Fractions that showed similar TLC characteristics on monitoring were then pooled. The fractions were then subjected to antidiabetic studies using Wistar albino rats. Alloxan monohydrate was used to induce hyperglycemic condition in the rats. Acute toxicity test was carried out using Lorke’s method. Both the normal rats and alloxan induced diabetic rats were subjected to 24 hours fasting. Five groups of five rats per group were used. Blood samples were collected from the animal tail vein at fixed time intervals of 3, 6, 9 and 12 hours. The blood glucose levels were then determined using One Touch Glucometer. Percentage reduction of mean blood glucose level was calculated at p≤ 0.05. The percentage yield of methanol extract was 20.00 %. The LD50 showed that the drug is safe at the dose of 5000 mg/kg. The administration of alloxan increased significantly (p<0.05) the blood glucose level of rats. Of all the solvents used, the methylene dichloromethane/methanol extract (7:3) gave the best separation of the extract. The visible bands obtained had RF values of 0.140, 0.288.0, 0.153, 0.510 and 0.598. The finding showed that extract of Vernonia amygdalina have hypoglycemic activity. This justifies the use of the plant in traditional medicine practice. Finally, the result of the chromatographic studies can be a reference for the resolution, isolation and characterization of the constituents.

Pharmacognostic and Antiulcer Properties of Vitex Doniana Sweet (Lamiaceae)

Mon, 24 Oct 2016 00:00:00 GMT

Pharmacognostic and Antiulcer Properties of Vitex Doniana Sweet (Lamiaceae) Anze, Sunday Patrick Gaza Antimicrobial resistance to synthetic pharmaceuticals is a growing concern, which has forced a radical research for alternatives of which natural medicines offer a ready and safe alternative. Vitex doniana is valued for its sweet fruit pulp and young edible leaves. The stem bark is prepared traditionally for various ailments. In this study, the pharmacognostic, antimicrobial and antiulcer properties of Vitex doniana were evaluated. The acute toxicity of the plant was also studied using standard method. Evaluation of the fresh, powdered and anatomical sections of the leaf and stem bark was carried out to determine the macro-morphological,micro-morphological, chemo-microscopic, quantitative microscopical values and phytochemical profiles according to laid down procedures. Cold maceration and concentration of active principles was carried out using 80 % methanol for both leaf and stem bark powder. Fractionation of the crude extracts was done using solvents of increasing polarity: n-hexane, ethyl acetate, butanol and water. Microbiological assay for the crude extracts and fractions (50, 100, 200 and 400 mg/ml) was done in vitro using the agar well diffusion method, with Gentamicin and Fluconazol (2 mg/ml) as standard antibiotics. The test organisms were Bacillus subtilis, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans. Ulcer preventive test was carried out on experimental rats using varying concentrations of the extract and fractions. Gastric lesions were induced using ethanol. Cimetidine was reference drug. The ulcer spots were measured and ulcer index and percentage inhibition was calculated. Macro- and micro-morphology revealed an obovate, digitately compound leaf that were opposite with palmate leaflets. Other features were anomocytic stomata, dorsoventral leaf type and double-walled palisades. There were cicatrices in the spongy mesophyll; spiral vessels between fibres, glandular, multicellular and unicellular trichomes, prisms of calcium oxalate, sclereids, with narrow lumen and thick wall, thick fibres and pitted vessels, starch grains, lignin and proteins.The phytoconstituents included alkaloids, saponins, tannins, flavonoids, carbohydrates and steroids.The yield of extraction was 31.43 (leaf extract) and 8.31 % (stem bark extract). The plant exhibited significant (p < 0.05) antimicrobial activity with an MIC of 50 mg/ml. The stem bark extract and fractions had better antimicrobial activity than the leaf crude extract. Similarly but conversely, ulcer preventive activity was greatest in the leaf extracts (600 mg/kg) which was more effective than Cimetidine (200 mg/kg). Therefore findings of this work shows that Vitex doniana do possesses antimicrobial and antiulcer effect and the pharmacognostic features could be used for proper identification and preparation of a monograph for inclusion in the Pharmacopoeia.