TOXICITY STUDY

Determination of LD50 range:TOXICITY STUDY:

In screening drugs, determination of LD50 (the dose which has proved to be lethal (causing death) to 50% of the tested group of animals is usually an initial step in the assessment and evaluation of the toxic characteristics of a substance. It is an initial assessment of toxic manifestations (provides information on health hazards likely to arise from short-term exposure to drugs) and is one of the initial screening experiments performed with all compounds. Data from the acute study may: (a) Serve as the basis for classification and labelling; (b) Provide initial information on the mode of toxic action of a substance; (c) Help arrive at a dose of a new compound; (d) Help in dose determination in animal studies; (e) Help determine LD50 values that provide many indices of potential types of drug activity. Determination of median lethal dose of both Methanolic and aqueous extract of Tribulus terrestris: The Herbal preparation, Tribulus terrestris were subjected to toxicity studies according to OECD guide lines. It was observed that the test extract was not mortal for mice even at 10000 mg/kg dose. Hence, 50mg/kg and 100mg/kg of this Plant of each solvent extract dose were selected for further study.

Pharmacological Evaluation of Tribulus terrestris:

Animals:
Studies were carried out using Wistar albino rats of both sexes weighing 175-200g. They were obtained from the Mahaveer enterprises, Hyderabad. All animal experiments strictly complied with the approval of institutional animal ethical committee. The animals were grouped and housed in polyacrylic cages (38cm x 23cm x 10cm) with not more than six animals per cage and maintained under standard laboratory conditions (temp.25c) with dark and light cycle (12/12h). They were allowed free access to standard dry pellet diet and water ad libitum. The rats were acclimatized to laboratory condition for 10 days before commencement of experiment.

Antipyretic activity:
Chemicals: 0.5% CMC, 15% aqueous solution of yeast, Paracetmol
Equipment: Digital Tele thermometer (Hartmann, Germany). Treatment Protocol: The animals were numbered, weighed and then divided into 6 groups with 6 animals in each as follows: Group- I Group- II Group- III Group-IV Group- V Group-VI : : : : : : Control (CMC 10ml/kg. p.o.) Standard (Paracetmol 150mg/kg p.o.) METT (50mg/kg) is suspended with CMC(0.5%) METT (100mg/kg) is suspended with CMC (0.5%) AETT (50mg/kg) is suspended with CMC (0.5%) AETT (100mg/kg) is suspended with CMC(0.5%)

Method Rats were divided into 6 groups consisting of six animals each. The normal body

temperature of each rat was measured rectally at predetermined intervals and recorded. The test was performed in rats by injecting 10 ml/kg, s.c., of 15% aqueous solution of yeast in 0.5% w/v carboxy methyl cellulose solution to induce pyrexia. Rectal temperature of each animal was taken before and 19 h after the yeast injection using digital clinical thermometer (Hartmann, Germany).Only animals displaying a mean basal rectal temperature between 36.8 and 37.4C were selected for the study. Animals that did not show a minimum increase of 0.5 C in temperature 19 h after yeast injections were discarded. Body temperature was measured by inserting a thermostat probe (Yellow Springs Instruments no. 402) 3 cm into the rectum, without removing the animals from their home cages, for 1 min every 30mins upto6 h. The animals were picked up gently and held manually during the

temperature measurements. This procedure was performed at least twice on the day before the experiment to minimize changes secondary to handling. On the day of the experiment, the basal temperature of each animal was determined by four measurements at 30 min intervals, before any injections. The experiments were conducted in a temperature-controlled room (32C). Table5: anti pyretic table

Figure4: METT

Figure5: AETT

DISCUSSION: RESULTS:

ANTI-MICROBIAL ACTIVITIES: Medicinal plants have recently received the attention of the pharmaceutical and scientific communities and various publications have documented the therapeutic value of natural compounds in a bid to validate claims of their biological activity. Attention has been drawn to the antimicrobial activity of plants and their metabolites due to the challenge of growing incidences of drug-resistant pathogens. Some plants have shown the ability to overcome resistance in some organisms and this has led to researchers investigating their mechanisms of action and isolating active compounds. Particular focus is on establishing the effect of the plant extracts in terms of their micro static and microcidal action and the spectrum of organisms affected. This has enabled exploitation of plants for the treatment of microbial infections and in the development of new antimicrobial agents. This requires rigorous research and it is therefore imperative to follow standard methods to authenticate claims of antimicrobial action. The antimicrobial susceptibility test (AST) is an essential technique in many disciplines of science. It is used in pathology to determine resistance of microbial strains to antimicrobials, and in ethno pharmacology research, it is used to determine the efficacy of novel antimicrobials against microorganisms, essentially those of medical importance. The test is the first step towards new anti-infective drug development.

ANTI-BACTERIAL ACTIVITY:Staphylococcus: Staphylococci are Gram-positive cocci occurring in clusters. They can be cultured on normal nutrient mediums both aerobically and anaerobically. The most important species from the viewpoint of human medicine is S.aureus. A number of extracellular enzymes and exotoxins such as coagulase, alphatoxin, leukocidin, exfoliations, enterotoxins, and toxic shock toxin are responsible for the clinical symptoms of infections by this pathogen, which are observed in the three types invasive infections, pure toxicoses, and mixed forms. The antibiotics of choice for therapy of these infections are penicillinase resistant penicillins. Laboratory diagnosis involves identification of the pathogen by means of microscopy and culturing. S.aureus is a frequent pathogen in nosocomial infections and limited outbreaks in hospitals. Hand washing by medical staff is the most important prophylactic measure in hospitals. Coagulase-negative staphylococci are

classic opportunists. S. epidermidis and other species are frequent agents in foreign body infections due to their ability to form biofilms on the surfaces of inert objects. Staphylococci are small spherical cells (1lm) found in grapelike clusters. Staphylococci are non motile, catalase-producing bacteria. The genus Staphylococcus includes over 30 species and subspecies. S.aureus (and E. coli) is among the most frequent causal organisms in human bacterial infection. Escherichia coli: The most important bacterial family in human medicine is the Enterobacteriaceae. This family includes genera and species that cause well-defined diseases with typical clinical symptoms (typhoid fever, dysentery, plague) as well as many opportunists that cause mainly nosocomial infections (urinary tract infections, pneumonias, wound infections, sepsis). Enterobacteriaceae are Gram-negative, usually motile, facultatively anaerobic rod bacteria. The high levels of metabolic activity observed in them are made use of in identification procedures. The species are subdivided into epidemiologically. Significant serovars based on O, H, and K antigens. The most important pathogenicity factors of Enterobacteriaceae are colonizing factors, invasions, endotoxin, and various exotoxins. Enterobacteriaceae are the most significant contributors to intestinal infections, which are among the most frequent diseases of all among the developing world populace. Pseudomonas aeruginosa: Pseudomonas aeruginosa is a common bacterium that can cause disease in animals, including humans. It is found in soil, water, skin flora, and most man-made environments throughout the world. It thrives not only in normal atmospheres but also in hypoxic atmospheres, and has, thus, colonized many natural and artificial environments. It uses a wide range of organic material for food; in animals, the versatility enables the organism to infect damaged tissues or people with reduced immunity. The symptoms of such infections are generalized inflammation and sepsis. If such colonizations occur in critical body organs, such as the lungs, the urinary tract, and the kidneys, the results can be fatal. Because it thrives on most surfaces, this bacterium is also found on and in medical equipment, including catheters, causing crossinfections in hospitals and clinics. It is implicated in hot-tub rash. It is also able to decompose hydrocarbons and has been used to break down tar balls and oil from oil spills.

Procedure: Take the medium and inoculate with the organism and pour it into the petridishes aseptically and allow solidifying. Prepare the stock solution and standard solution containing 100mg/ml. It is prepared from stock solution having 1mg/ml make different dilution of 20, 40, 60, 80 of leaf extract of Tribulus terrestris plant. Then make the bores with the apparatus in the medium filled in petriplates in the triangular corners of the plate. Then transfer the two test solutions of the leaf extract and the standard in to the petriplates. Then incubate for 24 hours for the development of zone of inhibition. Then plot the graph by taking the log conc on x-axis and zone of inhibition on y-axis. Table6: Anti-bacterial activity of different extracts of Tribulus terrestris leaves
Group Standard (Amoxicillin) METT(100g) AETT(100g) E.coli 12.50.52 11.160.71 10.410.51 S.aureus 110.85 10.830.83 9.660.65 P.aeruginosa 10.910.66 10.080.66 8.660.49

Figure6: Anti-bacterial activity of Tribulus terrestris leaves

X-axis shows the Anti-bacterial activity of different extracts on different Organisms Y-axis shows the % of Inhibition

ANTI-FUNGAL ACTIVITY:-

Aspergillus niger: Aspergillus niger is a fungus and one of the most common species of the genus Aspergillus. It causes a disease called black mold on certain fruits and vegetables such as grapes, onions, and peanuts, and is a common contaminant of food. It is ubiquitous in soil and is commonly reported from indoor environments, where its black colonies can be confused with those of Stachybotrys (species of which have also been called "black mould"). Some strains of Aspergillus niger have been reported to produce potent

mycotoxins called ochratoxins, but other sources disagree, claiming this report is based upon misidentification of the fungal species. Recent evidence suggests some true A.niger strains do produce ochratoxin A. Aspergillus niger infection: A rare fungal infection that often causes a black mould to appear on some fruit and vegetables but may also infect humans through inhalation of fungal spores. Fusarium oxisporium: Fusarium oxysporium comprises all the species, varieties and forms recognized by Wollenweber and Rein king [within an infrageneric grouping called section Elegance. While the species, as defined by Snyder and Hansen, has been widely accepted for more than 50 years, more recent work indicates this taxon is actually a genetically heterogeneous polytypic morphospecies whose strains represent some of the most abundant and widespread microbes of the global soil micro flora. These remarkably diverse and adaptable fungi have been found in soils ranging from the Sonoran Desert, to tropical and temperate forests, grasslands and soils of the tundra. F. oxysporium strains are ubiquitous soil inhabitants that have the ability to exist as saprophytes, and degrade lignin and complex carbohydrates associated with soil debris. They are also pervasive plant endophytes that can colonize plant roots and may even protect plants or be the basis of disease suppression. Although the predominant role of these fungi in native soils may be as harmless or even beneficial plant endophytes or soil saprophytes, many strains within the F.oxysporium complex are pathogenic to plants, especially in agricultural settings. Pathogenic strains of F. oxysporium have been studied for more than 100 years. The host range of these fungi is extremely broad, and includes animals, ranging from arthropods to humans, as well as plants, including a range of both gymnosperms and angiosperms. Plant

pathogenic F.oxysporium strains have a broad host range, individual isolates usually cause disease only on a narrow range of plant species. Procedure: Take the medium and inoculate with the organism and pour it into the petridishes aseptically and allow solidifying. Prepare the stock solution and standard solution containing 100mg/ml. It is prepared from stock solution having 1mg/ml make different dilution of 20, 40, 60, and 80 of leaf extracts of Tribulus terrestris plant. Then make the bores with the apparatus in the medium filled in petriplates in the triangular corners of the plate. Then transfer the two test solutions of the leaf extract and the standard in to the petriplates. Then incubate for 24 hours for the development of zone of inhibition. Then plot the graph by taking the log conc. on x-axis and zone of inhibition on y-axis. Table7: Anti-fungal activity of different extracts of Tribulus terrestris leaves
Group Group I Group II Group III A.niger 13.40.51 12.50.52 10.40.79 F.oxisporium 12.830.71 11.830.57 9.250.75

Figure7: Anti-fungal activity of Tribulus terrestris leaves

X-axis shows the Anti-bacterial activity of different extracts on different Organisms Y-axis shows the % of Inhibition