cytotoxicity and antitubercular activity of allium sativum and lantana camara against mycobacterial isolates from people living with HIV/AIDS
U Dibua, G Odo, S Udengwu, C Esimone
antitubercular, cytotoxicity, hiv-infected-persons, minimal inhibitory concentration mic
U Dibua, G Odo, S Udengwu, C Esimone. cytotoxicity and antitubercular activity of allium sativum and lantana camara against mycobacterial isolates from people living with HIV/AIDS. The Internet Journal of Infectious Diseases. 2009 Volume 8 Number 1.
The Antitubercular activity of
Tuberculosis (TB), by members of the
Thus, the burden of HIV/AIDS and TB co-infection has prompted this research aimed at screening the leaves of
Materials and Methods
Subject and Location
The subjects used for the survey were volunteers of known HIV status (people living with HIV/AIDS) from the Local Chest Unit and Shanahan Hospital, Nsukka. Sputum samples were collected before mouthwash and analysed within 24h of collection macroscopically to determine parameters of medical importance: colour, whether blood tinged, presence of pus and parasites according to the methods of Cheesbrough13
Cultivation of Organism
Preliminary identification of Mycobacterial isolates was based on the rate of growth, temperature of optimal growth, colony morphology, colony texture and pigment production. Purulent, muco-purulent or cheesy sputa and nasopharyngeal samples were first digested or hydrolyzed with two drops of 40% Potassium hydroxide solution and incubated for 1hr until fluid as described by 14 Hydrolyzed samples were then cultivated on duplicate plates of freshly prepared Lowenstein-Jensen medium (LJ-medium), consisting of 6-8 homogenized hen’s eggs (275ml), 8ml hydrochloric acid, (IN), 153ml salt Glycerol solution, 2.75ml of 2% (w/v) malachite green, and 25000iu penicillin G (Benzyl penicillin), MacConkey, Blood, and Chocolate agar (5-10% CO2), and incubated at both room temperature and at 370C for 24 hours, observed for fast growers and subsequently re-incubated for 3 – 4 weeks. Acid-fast bacilli were then screened for using the Zeihl-Neelsen Staining method 1 for identifying AFB bacilli as described by Ellen
The isolates were further characterized using various biochemical tests: catalase, urease production, nitrate reduction, lipase (Tween 80 hydrolysis), growth in 5% NaCl, growth rate and pigment production as described by 13
Collection and Identification of Plants (Authentication)
The fresh leaf of the plant,
Extraction of Active Components of the Plants
The methanolic extraction of the leaves of
Preparation of Stock solution
A 500 mg quantity of the plant extracts was weighed and dissolved separately in 5ml each of the solvent (20% dimethylsulfuroxide) to a concentration of 100 mg/ml. The stock solution was doubly diluted to a concentration of 100, 50, 25, 12.5, and 6.25mg/ml in each set of tubes.
The Phytochemical analysis described by 16-17 for the determination of the presence of secondary constituents of plants such as alkaloids, flavonoids, tannins, steroids , carbohydrates, reducing sugars, cardiac glycosides, terpenes, fats and oil and saponins were adopted in the study using methanolic extract of L. camara leaves and Allium sativum bulbs
The Disc Diffusion Method Using Industrial Grade Antimycobacterial Agents
The Well-in-agar dilution Method
Antitubercular activity of the plant extracts (500mg/ml) was assayed using the well-in-agar technique described by .18 Sterile plates of the L-J medium were flooded with normal saline culture of the test organisms of turbidity comparable to one (1) McFarland standard. Excess bacterial suspension was drained and the plates allowed drying. Wells of diameter 8mm were bored on the agar plates with a cork borer, and about 0.2ml of each dissolved extracts was introduced into each well and allowed to stand for 2 hrs at room temperature to allow extracts to diffuse into the agar. The plates were then incubated at 370C for 18-21 days. Control experiments were done by dispensing 0.1ml of the test organism dissolved in 0.5ml each of DMSO and distilled water without adding the plant extracts. The Inhibition Zone Diameters (IZDs) were measured with a pair of calipers and a ruler calibrated in millimeters.
Minimum Inhibitory Concentration (MIC) by the Broth dilution Methods
Brine Shrimp Lethality Assay
Phytochemistry of and
The Phytochemistry of
Key: + = positive
- = negative
The antitubercular activity of test plants expressed in their inhibitory zone diameter (IZD) compared with standard antibiotics is presented in Table 2.
The activity profile of test plant extracts on isolated Mycobacterial species expressed in their minimal inhibitory concentration (MIC) is shown in the increasing order:
The cytotoxic effect of the test ethanolic plant extracts on brine shrimps (Table 4) indicated that garlic extracts had LC50 at 49.1 ppm for
Current strategies to overcome the global problem of antitubercular resistance include research in finding new and innovative alternative plant extracts of medicinal valued. The emergence of drug-resistant
Susceptibility test was therefore carried out using some conventional antitubercular drugs, and the ethanolic extract of
The susceptibility of the isolates to garlic extract as shown by the MIC and dose-response effect was remarkable demonstrates its potential for therapeutic uses Garlic contains a stable, effective, and safe organosulfur compound, S-allyl cysteine (SAC major garlic constituent), which is noted for protection against oxidation, free radicals, pollution, cancer, and cardiovascular diseases. SAC has cardiovascular effect: lowers total serum cholesterol and LDL cholesterol hypercholesterolemia by inhibiting the activity of the key enzymes in cholesterol synthesis, ß-hydroxy-ß-methylglutaryl CoA synthetase and reductase, in the liver 27 It’s blood thinning effect was observed by 28-29 who showed that SAC lowered the levels of plasma thromboxane B2, and factor 4 (blood clotting factors) in those with hypercholesterolemia up to 30%; and also decreased platelet aggregation, or blood clotting, induced by the potent clotting agents, collagen and adenosine diphosphate. Inhibition of Vascular Smooth-Muscle Cell (SMC) and Umbilical Endothelial Cell Proliferation was reported by 30. SMC proliferation constitutes an essential aspect in the development of atherosclerosis and of restinosis (narrowing or constriction) of blood vessels subjected to angioplasty. 31 examined the potential role of Aged Garlic Extract as an antioxidant for sickle red blood cells. Unanimously, the patient's count of Heinz bodies decreased from 58.9% to 29.8% during the 4 weeks of the study. These data suggest the significant antioxidant activity of AGE on sickle cell anemia, and may represent a potential therapy to combat complications of the disease. Antioxidative effects of SAC was further reported by 32 who found that SAC could prevent copper, a potent oxidant, from oxidizing LDL cholesterol.33-34 found that SAC decreased the emission of low level chemiluminescence (LLC) initiated by t-butyl hydroperoxide. SAC inhibited LLC emissions 33% at 5 mmol/L and 45% at 10mmol/L. SAC also demonstrated radical and hydrogen peroxide scavenging activities in vivo. SAC demonstrated a scavenging effect on hydrogen peroxide and also inhibited the chain oxidation induced by a hydrophilic radical initiator in another study by. 33 SAC inhibited the emission of low level chemiluminescence and the early formation of TBA-RS (markers of oxidation), whereas water extracts of raw and heat-treated garlic enhanced such emissions35 suggested that SAC has antioxidative efficacy. Attenuating Ischemic Brain Damage. 36 studied the efficacy of SAC as a free radical scavenger and reported that SAC improved (i) motor performance and (ii) memory impairment, and reduced (iii) water contents and (iv) the infarct size. Also, (i) the production of free radicals (alkoxyl radicals) as studied by electron paramagnetic resonance spectroscopy (EPR) was biphasic. Anticancer and Cancer-Preventive Effects of SAC were similarly reported: SAC inhibited the growth of carcinogen-induced tumors of the breast in the studies carried out by 37 :SAC dose-dependently reduced the carcinogen DMBA-DNA adduct formation in the mammary glands, and (0.5 or 2.5 mg/kg diet) supplementation markedly depressed the occurrence of DMBA-DNA adducts in mammary cells by 70 or 80%, respectively. On the other hand,38 found that SAC is an effective inhibitor of N-methylnitrosourea mammary tumors.
The cytotoxic effect of the test ethanolic plant extracts on brine shrimps is apparent in the study: garlic extract was most highly effective, with LC50 at 49.1 ppm for
The antitubercular activity of some plant extracts have been elucidated in this study, and their pharmacokinetics are clear evidence of their possible uses (as natural sources) in solving the global problem of antituberculosis resistant