Publications

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    ABSTRACT: Antioxidant potential, lipid peroxidation inhibition ability in brain homogenate and total phenolic and flavonoid contents of ethanol extracts of some leafy vegetables (Vernonia amygdalina, Gongronema latifolium and Ocimum gratissimum) consumed in south-eastern Nigeria were determined. The antioxidant and neuroprotective potentials of the ethanol extracts were assessed by 2, 2-diphenyl-1-picrylhydrazil (DPPH) assay and through determination of thiobarbituric acid (TBA) reactive substances in brain homogenates. Total phenolic and flavonoid contents were quantitatively determined in the extracts spectrophotometrically. Results obtained from the present study showed that G. latifolium and V. amygdalina extracts were effective against hydrogen peroxide-induced lipid peroxidation with threshold inhibitory concentrations (IC50) values of 357.36±15.6µg/ml and 629.5±22.72µg/ml respectively while O. gratissimum achieved only 20% inhibition (IC20) at 785.73±40.23µg/ml. In the DPPH free radical scavenging assay, 50% of DPPH radicals were scavenged at181.5(µg/ml), 153.33(µg/ml), and 189.67(µg/ml) of V. amygdalina, G. latifolium and O. gratissimum extracts concentrations respectively. Free radical scavenging ability of the extracts was in the order G. Latifolium >V. Amygdalina > O. gratissimum. Total phenolic content was 432.79±42.2mg/100g,274.1±37.3mg/100g, and 490.49±58.1mg/100g catechin equivalent, while flavonoid content was 843.09±21.6 mg/100g, 746.34±13.7mg/100g and 1160.98±24.5mg/100g catechin equivalent in V. amygdalina, G. latifolium and O. gratissimum respectively. The inhibition of lipid peroxidation and DPPH radical scavenging followed a logistic dose response pattern.These results indicate that these vegetables possess neuro-protective potentials and free radical scavenging ability. The present data scientifically corroborate with the consumption of these plants as an exogenous sources of antioxidants that could improve nutrition and boost antioxidant defense.
    06/2014;
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    Chinwe Sylvanus Alisi, Paul C. Chikezie
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    ABSTRACT: The present study ascertained air quality (AQ) of chemical stores and level of alterations in erythrocyte oxidative stress biomarkers of storekeepers exposed to laboratory/industrial chemicals in Owerri, Imo State, Nigeria. Erythrocyte percentage methaemoglobin (%MetHb), reduced glutathione (GSH) concentration, thiobarbituric acid reactive substances (TBARS) index and H2O2 induced erythrocyte haemolysis of participants were measured by spectrophotometric methods. The air levels of CO, NH3 and NO2 in the chemical shops were below Environmental Protection Agency (EPA) minimum standards. Particulate matters (PM) of both control and test air samples were within EPA standards. Erythrocyte %MetHb of test participants represented an increase of 61.81% compared to the control participants (p > 0.05), whereas erythrocyte GSH concentration of test participants showed significant difference (p < 0.05). Levels of H2O2 induced erythrocyte haemolysis was significantly (p < 0.05) higher than those of the corresponding control participants. The ratio of erythrocyte TBARS index of the test to the control participants was 1.4:1 approx.; p < 0.05. The present study showed that the gas substances in chemical stores in Owerri compromised AQ with attendant potentials to cause oxidative stress and hazardous consequence to the storekeepers. Keywords: Air quality, Methaemoglobin, Glutathione, Haemolysis, Erythrocyte, Oxidative Stress.
    Free Radicals and Antioxidant. 02/2014; 4(1):67-69.
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    Paul Chikezie, Alisi C.S
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    ABSTRACT: The present study ascertained air quality (AQ) of chemical stores and level of alterations in erythrocyte oxidative stress biomarkers of storekeepers exposed to laboratory/industrial chemicals in Owerri, Imo State, Nigeria. Erythrocyte percentage methaemoglo¬bin (%MetHb), reduced glutathione (GSH) concentration, thiobarbituric acid reactive substances (TBARS) index and H2O2 induced erythrocyte haemolysis of participants were measured by spectrophotometric methods. The air levels of CO, NH3 and NO2 in the chemical shops were below Environmental Protection Agency (EPA) minimum standards. PM of both control and test air samples were within EPA standards. Erythrocyte %MetHb of test participants represented an increase of 61.81% compared to the control participants (p > 0.05), whereas erythrocyte GSH concentration of test participants showed significant difference (p < 0.05). Levels of H2O2 induced erythrocyte haemolysis was significantly (p < 0.05) higher than those of the corresponding control participants. The ratio of erythrocyte TBARS index of the test to the control participants was 1.4:1 approx.; p < 0.05. The present study showed that the gas substances in chemical stores in Owerri compromised AQ with attendant potentials to cause oxidative stress and hazardous consequence to the storekeepers.
    Free Radicals Antioxidants. 01/2014; 3(1):1-3.
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    A.C Ene, C.J.Obika, G.N.Okwu, Chinwe S. Alisi, N.G. Ede
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    ABSTRACT: Picralima nitida plant was tested for in vivo antimalarial activity in Swiss albino mice infected with chloroquine resistant Plasmodium berghei NK 65. Methanol and aqueous extracts of the leaves, stem bark, roots, fruits and seeds of this plant were used for the in vivo study at a dose of 100 mg/kg. Out of the ten extracts screened, the methanol extracts of the root and stem bark showed higher anti-malarial activity compared to the others. There was a statistically significant difference (p<0.05) observed in the level of parasitemia of the infected mice which were treated with the methanol extracts of the roots and stem bark of Picralima nitida. This significant difference was observed when these two extract treated groups were compared with the untreated negative control, the group treated with chloroquine and the other treatment groups. The presence of phytochemicals/ secondary metabolites was also determined in the plant extracts. Steroids, terpenes, flavonoids and alkaloids were present in the methanol extracts of the root and stem of this plant which showed the highest anti-malarial activity. The presence of these secondary metabolites might be responsible for the high anti-malarial activity. These results suggest that some parts of the plant have more anti-malarial activity compared to the other parts of that same plant. The results also suggest that the solvent used in the extract preparation affects the anti-malarial activity of the plant parts.
    Journal of Research in Biochemistry. 04/2013; 1(2):95-105.
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    ABSTRACT: The medicinal values of fresh Raphia hookeri and Elaeis guineensis wines were evaluated. Face-to-face interview questionnaire-based ethno-medical survey on 1000 randomly selected families in some southeastern and southsouthern states in Nigeria on the use of the palm wines as antimicrobial agents, vehicles for antimicrobial agents, galactogogues in postpartum mothers and prophylactic agents against malaria in ethno-medicine were carried out. The presence of bioactive phytochemical and biochemical constituents with reported pharmacological activities were detected and their biochemical modes of action were proposed. In conclusion, the antimicrobial values of the wines are phytochemical and ethanol mediated, their lactogenic effects are saponin-mediated increases in serum prolactin content and their prophylactic effect is by the inhibition of the intra-erythrocytic plasmodial growth.
    Journal of Research in Biology. 09/2012; 2(6):589-595.
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    ABSTRACT: Cell surface hydrophobicity of six phenol-utilizing bacteria isolated from Port Harcourt Petroleum refinery wastewater was assessed via bacterial adhesion to hydrocarbon (BATH), salt aggregation test (SAT) and Congo red binding (CRB) assays. The test organisms exhibited high to moderate hydrophobicity with BATH assay respectively when n-octane and p-xylene were employed. Bacillus sp. RBD, Escherichia coli. OPWW, Corynebacterium sp. DP, Citrobacter sp. RW and Pseudomonas sp. SD showed moderate hydrophobicity in SAT assay. On the other hand, Pseudomonas sp. RWW showed high hydrophobicity in SAT assay. Similar results of moderate hydrophobicity were obtained with CRB except Corynebacterium sp. DP that exhibited high hydrophobicity value of 14.70±1.00µg. The results obtained in this study showed that the isolates are mainly moderately hydrophobic which make them good candidates in the clean up activity of organic pollutants in polluted sites.
    Journal of Reserach in Biology. 06/2012; 2(4):383-391.
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    ABSTRACT: Aqueous filtrates of processed yellow and white garri {fried fermentation products of cassava (Manihot esculenta)} were investigated for their effects on the aggregation of sickle cell trait haemoglobin (HbAS), in vitro. Phytochemicals detected in the two filtrates included tannins, catechins, flavonoids, cyanogenic glycosides, β-carotene and thiocyanates. Biomolecules such as amino acids, monosaccharides and ρ-hydroxybenzoic acid (ρ-HBA) were also present. Saponins and cardiac glycosides were not detected in either of the filtrates. The processed white garri aqueous filtrate did not contain β-carotene. Palm oil did not contain cardiac glycosides. The amino acid with the highest (p<0.05) concentration in the palm oil was aspartic acid (6.29±0.03 g/100 g protein) whereas cysteine and methionine had the lowest (p<0.05) concentrations (0.56±0.08 and 0.57±0.04 g/100 g protein, respectively). Spectrophotometric method was used to investigate the aggregation of haemolysate HbAS molecules treated with sodium metabisulphite (Na 2 S 2 O 5) at λ max = 555 nm for 25 minutes. The present study showed that aqueous filtrate of processed yellow garri exhibited a significantly (p<0.05) higher (82.91%) aggregation inhibitory effect than the aqueous filtrate of processed white garri which had 64.66%.
    Journal of Research in Biochemistry. 06/2012; 1(1):047-052.
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    ABSTRACT: The amino acid profile and the effects of the seed extracts of Sphenostylis sternocarpa, Monodora myristica and Mucuna sloanei were studied based on their ability to inhibit haemoglobin polymerization and improve the Fe 2+ /Fe 3+ ratio of sickle cell erythrocytes. The samples were fractionated into crude aqueous extract (CAE), fat-soluble (FAS), butanol-soluble (BUS) and water-soluble (WAS) fractions. The CAEs of the samples ranked highest in amino acid content in the range of S. sternocarpa (7.12 ± 0.00 g/100g)>M. myristica (6.00 ± 0.15 g/100g)>M. sloanei (3.56 ± 0.21 g/100g). The amino acids identified in appreciable quantities in the seed samples included Phe, Leu, Val, Ile, His, Arg, Tyr, Met, among others. The extracts inhibited polymerization to varying degrees with CAE of both S. sternocarpa and M. myristica, as well as the WAS of M. myristica eliciting significantly (p<0.05) high percent inhibition of polymerization when compared with Phe standard. The extracts improved the Fe 2+ /Fe 3+ ratio of HbSS blood from 1.36% for CAE of M. sloanei to 85.04% for CAE of S. sternocarpa; and from 11.03% for WAS of S. sternocarpa to 36.08% for WAS of M. sloanei. These legumes could, therefore, have immense nutritional and therapeutic importance in the management of sickle cell disease and other related diseases. Keywords: Sickle cell disease; Fe 2+ /Fe 3+ ratio; legumes.
    British Journal of Pharmaceutical Research. 03/2012; 2(2):80-88.
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    ABSTRACT: The synergistic effect of some antisickling nutrients and amino acids was investigated to determine the possibility of combining antisickling agents to achieve enhancement or potentiation. The approach involved the use of amino acids and other nutrients whose antisickling effectiveness have been documented. These were combined and the effect of such combination assessed on some antisickling parameters such as sickle cell hemoglobin polymerization inhibition, sickle cell reversion experiment, Fe 2+ /Fe 3+ ratio of HbSS blood and their action on lactate dehydrogenase (LDH) activity of sickle cell blood. Results from the hemoglobin polymerization assay revealed the relative % inhibition for the following: L-Phe (83.30), L-Serine (76.40), L-Arg(91.70), L-Lys (84.80) and Ascorbate (88.90), but when combined with equimolar/intimate concentrations of other amino acids and vitamins at a final assay concentration of 200µM; results indicated an improvement over the previous result as follows: L-Phe + Vit.C (93.10), Phe+Ser (93.10)and others having the same value. Even the intimate mixtures of Phe+Ser+Arg+Lys+Vit C had the same value of 93.10. For the sickle cell reversion experiment, the time required to revert 50% sickle cell to normal erythrocytes has the sequence: 30s,36s,28s,32s,38s,28s,25s,26s,25s and 26s for Phe, Ser, Arg, Lys, Vit.C , Phe +Ser, Phe+ Arg, Phe+Lys, Phe +Vit.C, Phe +Lys+Vit C+ Ser + Arg respectively. There is a gradation of improvement in the Fe 2+ /Fe 3+ ratio from the single amino acid antisickling agents to the longer polypeptide/ additive combinations which ranged from 17.82 % increase in ratio for L-Phe to 107.29 for Phe+ Ser+ Lys + Arg+Vit C. The effect of the additive principle on serum LDH activity was very outstanding. The antisickling agents were able to reduce the LDH activity from 29.60 % for L-Phe to 45.34 % for the Phe+Ser+Lys+Arg Vit C combination. The synergistic effect of antisickling agents has been the current approach adopted in the design of effective antisickling agents and other conventional drugs used in the treatment of many malignant syndromes similar to sickle cell disease (SCD).as this combination proved more efficacious than the single antisickling agent protocol. A homozygous mutation of the β-globin, a subunit of adult hemoglobin A (HbA), is the proximate cause of sickle cell disease (SCD).Sickle hemoglobin (HbS) shows peculiar biochemical properties which lead to polymerization when deoxygenated. HbS polymerization is associated with a reduction in cell ion and water content (cell dehydration), increased cell density which further accelerates HbS production 1 .Pathophysiological studies have shown that dense dehydrated red cells may play a central role in acute and chronic manifestations of sickle cell disease in which intravascular sickling in capillaries and small vessels lead to vaso-occlusive episodes, resulting in impaired blood flow in a variety of organs and tissues. Vaso-occlusive events in the micro-circulation results from a complex scenario involving interactions between different cell types ,including dense dehydrated sickle cells, reticulocytes, abnormally activated endothelial cells, platelets and plasma factors, such as cytokines and oxidized pro-inflammatory lipids 2,3 . Antisickling agents are nutrients, drugs, phytochemicals and ions which by their actions inhibit the pathphysiological mechanisms
    InstaSci Journal of Pharmaceutical Sciences. 01/2012; 2(1):18-26.
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    ABSTRACT: Honey is a naturally sweet and viscous fluid produced by honeybees (Apis mellifera) from the nectar of flowers. Proline, free amino acid, tannins, phenols and flavonoids content were determined in eight samples of Obudu cattle ranch honeys. Antioxidant content (ascorbic acid equivalent, quercetin equivalents, tocopherol content) and free radical scavenging activity of Obudu ranch honeys were determined by standard methods. The relationship existing between proline and antioxidant content with free radical scavenging activity was assessed by mathematical modelling using Levenberg Marquardt algorithm. Results of our study showed that Obudu ranch honey was of high quality having comparable total free amino acid, proline, phytochemical and antioxidant content with good quality honeys found elsewhere. α-Tocopherol content was 16.50 ± 1.40 μg/100 g, quercetin equivalent antioxidant content (QEAC) was 9.43 ± 0.9 mg/100 g, ascorbic acid equivalent antioxidant content (AEAC) was 18.56 ± 1.78 mg/100 g, while the meaninhibitory concentration (IC50) of the honey samples against the 2,2-diphenyl-1- picrylhydrazyl (DPPH) radicals (RSA: IC50) was evaluated to be 12.74 ± 0.71mg/ml. Proline content, AEAC, QEAC and α-tocopherol were logistically related (r2 = 0.726, 0.971, 0.960 and 0.888 respectively) to the radical scavenging activity. The results obtained from the present study showed that Obudu ranch honeys were of high quality and possessed antioxidant and free radical scavenging property. Since proline content of the tested honey samples were comparable to proline content of unadulterated honey samples elsewhere, the commercial samples may not be adulterated Results showed that traditional usage of the honey for various medicinal usages may be related to its chemical content and radical scavenging property.
    International Journal of Biochemistry Research & Review. 01/2012; 2(4):164-175.
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    Pakistan Journal of Nutrition 01/2012;
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    Chinwe Alisi, Sam Abanobi
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    ABSTRACT: Dehydrogenase activity (DHA) in Gram-positive Staphylococcus aureus isolated from degenerated wound, Gram-negative Salmonella typhi isolated from stool, and Gramnegative Escherichia coli from a high vaginal swab were assayed. Inhibition of dehydrogenase activity of the test organisms by ethanol extract of Euphorbia hyssopifolia, and Euphorbia hirta, were determined and compared to standard antibiotics(Ciprofloxacin and Gentamycin). The total dehydrogenase assay was done using 2, 3, 5-triphenyl tetrazolium chloride (TTC) as the artificial electron acceptor which was reduced to the red-coloured triphenyl-formazan (TPF). Response of the bacterial isolates varied with extract concentration. Dehydrogenase activity was progressively inhibited in a logistic dose-response fashion in the test organism by the extracts and standard drugs. All extract and standards achieved at least 70% inhibition within the tested doses (0-2000μg/ml), except for Euphorbia hirta against Staphylococcus aureus. Threshold inhibitory concentrations (IC50) for Euphorbia hyssopifolia against Staphylococcus aureus, Salmonella typhi and Escherichia coli were 59.92μg/ml, 234.90μg/ml, and 492.46μg/ml respectively, while for Euphorbia hirta IC50 against Salmonella typhi and Escherichia coli was 99.67μg/ml,and 165.90μg/ml with no significant inhibition against Staphylococcus aureus. Inhibition of dehydrogenase activity in the test organism by the extract compared well with the standard antibiotics. Euphorbia hyssopifolia was effective against Grampositive Staphylococcus aureus implicated in delayed wound healing than Gram-negative Salmonella typhi and Escherichia coli implicated in typhoid fever and urinary tract infections respectively, while Euphorbia hirta was effective against Gram-negative organisms implicated in typhoid fever and urinary tract infections, but not effective against Gram-positive Staphylococcus aureus. Secondary plant metabolites found in the extracts may be acting in synergy to bring about their pharmacologic functions and may explain reasons for ethno-medical usage.
    International Journal of TROPICAL DISEASE & Health. 01/2012; 2(2):72-86.
  • American Journal of Drug Discovery and Development. 04/2011;
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    ABSTRACT: The ethanol extract of the leaf of Chromolaena odorata (Linn) was assessed for free-radical-scavenging and antioxidant potentials. Ability of the extract to scavenge reactive intermediates (superoxide ion O2·-, hydrogen peroxide H2O2, nitric oxide NO˙, hydroxyl radical OH˙) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, were used to assess its free radical scavenging potentials. Antioxidant potential was studied by assessing in-vitro inhibition of lipid peroxidation in both the brain (Neuro-protective potentials) and liver homogenates of Fenton-oxidant stressed rabbits. Inhibition of protein oxidation was assessed in-vitro by loss of protein thiol (P-SH), while assessment of the reducing power of the extract was further used to assess antioxidant capacity. Results obtained showed the ability of the extract to scavenge free radicals and reactive intermediates in a dose-response manner. The plant also had good antioxidant capacity. The secondary plant metabolites found earlier in the extract may explain reasons for the bio-efficacy of the plant. These findings are of great importance in view of the availability of the plant and its observed possible diverse applications in medicine and nutrition.
    British Journal of Pharmaceutical Research. 01/2011; 1:141-155.
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    Journal of Research in Biology. 01/2011; 3:209-216.
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    ABSTRACT: Logistic response of antioxidants to lipid peroxide concentration in carbon tetrachloride toxicity in rabbit liver was evaluated. Carbon tetrachloride (CCl4), ethanol extracts of Chromolaena odorata (ETECO), sylimarin (a known hepatoprotective agent) and water, were used to induce variations in the oxidant/antioxidant balance in the test and control animals. This was used as a model to study the delicate balance between the activities and/or the intracellular concentrations of these antioxidants and lipid peroxide. Concentrations of lipid peroxidation product (malondialdehyde) were estimated to access the degree of oxidation of the polyunsaturated fatty acids in the liver tissue. Glutathione (GSH) concentration was estimated to capture the non-enzymatic antioxidant concentration, while glutathione-s-transferase (GST), superoxide dismutase (SOD), and catalase (CAT) activities were assayed in the liver to assess the enzymatic antioxidant activities. Results obtained from this study showed that the concentrations of lipid peroxidation product (malondialdehyde) varied in a logistic fashion with the nonenzymatic antioxidant (glutathione) and the enzymatic antioxidants (glutathione-s-transferase, superoxide dismutase, and catalase). The concentration of the peroxidation product and the concentration/activity of the antioxidants were inversely related, maintaining a highly logistic relationship (R2 = 0.99). The non-enzymatic antioxidant (GSH) concentration and the enzymatic antioxidant (GST, SOD, and CAT) activities were found to be directly related in a sigmoidal manner (R2 = 0.98). These observations indicated that oxidant/antioxidant concentrations and activities in a rabbit liver tissue is tightly related and mathematically associated.
    European Journal of Medicinal Plants. 01/2011; 1:118-129.
  • Alisi, P. Nc, F.I. Buseri, C.S. Alisi
    Journal of Research in Biology. 01/2011; 3:202-208.
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    ABSTRACT: Carbon tetrachloride and its toxic metabolites consistently produce liver injury in many species including man. The hepatoprotective potential of Chromolaena odorata Linn. (C. odorata) was evaluated in male rabbits against carbon tetrachloride-induced liver damage. Carbon tetrachloride intoxicated control (CCl4) and ethanol extract of C. odoratatreated rabbits (ETECO TEST) received a single dose of CCl4 (0.2 ml/kg bw in liquid paraffin 1:1). Pre-treated rabbits received ethanol extract of C. odorata at 400 mg/kg/day in two divided doses of 200 mg/kg in morning and at night for 6 days prior to CCl4 administration. Sylimarin control received 50 mg/kg bw as a replacement for ETECO prior to CCl4 intoxication. Normal animals received only extract in the above stated dose and served as extract controls (ETECO CTRL). Pre-treatment with C. odorata significantly (p<0.05) prevented the elevation of serum aspartate aminotransferase (AST), alanineaminotransferase (ALT), lactate dehydrogenase (LDH), gamma glutamyl transferase ( ץ-GT), total bilirubin and malondialdehyde (MDA) resulting from carbon tetrachloride intoxication. C. odorata extract also significantly (p<0.05) prevented a decrease in serum total protein, albumin, and glutathione (GSH) concentrations. The extract also significantly (p<0.05) prevented a decrease in superoxide dismutase (SOD), catalase (CAT) and glutathione-s-transferase (GST) activities. The presence of secondary plant metabolites like alkaloids, saponins, phenolic compouds, flavonoids and tannins found in C. odorata extract could beresponsible for its hepatoprotective action.
    International Journal of Biochemistry Research & Review. 01/2011; 1:69-81.
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    ABSTRACT: Ethanolic extract of the leaves of Landolphia owariensis were subjected to phytochemical screening and antimicrobial activities on Escherichia coli, Staphylococcus sp. and Proteus sp. using dehydrogenase assay method. The phytochemical screening indicated the presence of alkaloids, flavonoids, tannins and saponins. The bacterial isolates were exposed to different extract concentrations (20 – 2000 mg/ml) of the extract in nutrient broth, and their response was concentration dependent. In all three bacteria, dehydrogenase activity was progressively inhibited at concentrations of about 20 mg/ml, with total inhibition observed with 700 mg/ml for Staphylococcus sp., 1000 mg/ml for Proteus sp. and 1800 mg/ml for E. coli.
    African Journal of Biotechnology (ISSN: 1684-5315) Vol 6 Num 7. 01/2010;
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    ABSTRACT: The leaf ethanolic extract (LEEX) and root ethanolic extract (REEX) of Landolphia owariensis were comparatively subjected to phytochemical and antimicrobial screening. Susceptibility of some clinical isolates ( Staphylococcus spp., Proteus spp. and Escherichia coli ) to the extracts was determined using dehydrogenase assay method. The phytochemical screening indicated the presence of alkaloids, flavonoids, saponins and tannins in both the leaf and root extracts with cyanogenic glycosides present in the root extract only. The response of the bacterial strains to the leaf and root extracts was concentration dependent. Dehydrogenase activity was progressively inhibited by the root extract at a higher concentration range (150 - 2500 μg/ml) than the leaf extract (20 - 2000 μg/ml). LEEX gave an IC50 of 20, 200, and 550 mg/ml against Staphylococcus spp., Proteus spp. and E. coli, respectively, while REEX gave an IC50 of 340, 320, and 1560 mg/ml against the organisms, respectively. The LEEX exhibited higher antibacterial activity than the REEX. Susceptibility of the organisms to the extracts showed: Staphylococcus spp. > Proteus spp. > E. coli. The observed antimicrobial properties could be due to the presence of alkaloid, flavonoids and tannins, which were identified in both extracts. The findings may be of clinical relevance and further substantiates the traditional use of L. owariensis leaves and roots as antimicrobial agents.
    African Journal of Biotechnology (ISSN: 1684-5315) Vol 7 Num 4. 01/2010;

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