Methadology For Pharmacognosy Students

Collection of plant material: Dried leaves were obtained and authenticated by Dr. ----------------------------------------------. (RRI/ BNG / SMP/ Drug Authentication / 200809 /314) Bangalore.. Pharmacognosy: Morphological examinations: Organoleptic evaluation: Color, Size, Odour and taste were studied. Macro ¬morphological evaluation of leaves or leaflets. Some of characters which are studied include surface appearance and texture, Lamina Structure: Shape of lamina, Margin, Apex, Base, Venation. Microscopic evaluation: Preparation of Crude drug sample for microscopical examination: The microscopic examination of crude drug aims at determination of the chemical of the cell wall along with the determination of the form and chemical nature of the cell contents. Thus it determines the size, shape and relative structure of the different cells tissues in the plant drug. Before taking the TS if samples are dried then it requires softening before preparation for microscopical studies. It may be done by exposing the samples in moist condition (for leaves and flowers) or by boiling in water (for bark, root and wood). Sometimes water soluble components can be removed by soaking in water e.g., starch grain is gelatinized by heating with water. Procedure followed to take the sections of Stem: Small piece of stem was placed in beaker containing water and heated over m a Bunsen flame for few minutes. This softens the hard drug sample, then thin TS was taken (Transverse section was obtained by cutting along the radial plane of a cylindrical portion of the stem). Staining and mounting of sections: The thin sections are placed in a watch glass containing chloral hydrate solution. It was boiled to clear the sections. Then chloral hydrate was removed, phloroglucinol and concentrated hydrochloric acid were added to stain the sections. One thin section was taken out and mounted on a clean glass slide. A drop of glycerin was added and covered with the covered slip. This slide was observed under microscope. Procedure followed to take the powder microscopy leaves: Dried leaves were powdered and sieved to get a fine powder. This fine powder was placed in a watch glass containing chloral hydrate solution, boiled to clear the powder. Then it was stained using phloroglucinol and concentrated hydrochloric acid. After staining, the powder was taken on a clean slide with the solution, then the slide was covered with cover slip and excess solution was wiped with the help of filter paper/tissue paper. This slide was observed under microscope. Procedure followed to take the leaf sections A part of the leaf passing through the midrib was cut. The cut portion of leaf was inserted between the wedge openings of the potato. The portions of leaf protruding the surface of the pith are cut. The vertical sides of the pith are tapered off upwards. Sections are taken by moving the blade back and forth and placed in watch glass

containing water. Thin sections are selected and placed in chloral hydrate and are cleared by boiling. Cleared sections are stained by using phloroglucinol and concentrated hydrochloric acid. One thin section was taken out and mounted on a clean glass slide. A drop of glycerin was added and covered with the cover slip. This slide was observed under microscope and snaps are taken. T Leaf constants: Determination of Stomatal number Stomatal number is the average number of stomata per square mm of the epidermis of the leaf (Table no. 8 page no. 84). Procedure: Piece of leaf (middle part) was cleared by boiling with chloral hydrate solution. The upper and lower epidermis was peeled separately. The peeled epidermis was placed on slide and mounted with glycerin water. Camera lucida and drawing board was arranged for the drawings. With the help of stage micrometer 1 sq. mm was drawn. The prepared slide was placed on the stage, epidermal cells and stomata are traced. The no. of stomata lying in the area of 1 sq. mm are counted including the cell if at least half of its area lying with in the square. Average no. of stomata per sq. mm is calculated by tracing four different fields. Stomatal number is affected by various factors like age of the plant, size of the leaf, environmental conditions etc. Stomatal index is not much affected by these factors. It is relatively constant. Hence it is more significant in the evaluation of a leaf drug. o Determination of Stomatal Index: Stomatal Index: It is the percentage which the numbers of stomata form to the total number of epidermal cells, each stomata being counted as one cell. Stomatal index can be calculated by using following equation. i Stomatal index = S X 100 /E+S Where S= the number of stomata in a given area of leaf; and E = the number of epidermal cells (including trichomes) in the same area of leaf. Procedure The first seven steps are similar as mentioned in the determination of stomatal number. The no. of stomata and the no. of epidermal cells in each field was counted. Stomatal index was calculated using the above formula. Values for upper and lower surface (epidermis) were determined separately. V Determination of palisade ratio: Palisade ratio is the average number of palisade cells beneath one epidermal cell of a leaf. It is determined by counting the palisade cells beneath four continuous epidermal cells. Procedure: A piece of leaf was cleared by boiling in chloral hydrate solution. Camera lucida and drawing board was arranged for the drawings. The four cells of the epidermis are traced off. By focusing down to palisade layer, sufficient cells are traced off to cover the epidermal cells. The no. of palisade cells under the four epidermal cells is counted. (Including the palisade cells in the count when more than half is covered in the area of epidermal cell).

Average no. of cells beneath a single epidermal cell are calculated which gives the palisade ratio. By focusing different part of the leaf, the same was traced and the average was calculated to get the palisade ratio of the leaf. c Determination of vein-¬islet number: A vein islet is the small area of green tissue surrounded by the vein-lets. The vein-islet number is the average number of vein-islets per square mm of a leaf surface. It is determined by counting the number of vein-islets in an area of 4 sq. mm of the central part of the leaf between the midrib and the margin. Procedure: A piece of leaf was cleared by boiling in chloral hydrate solution. Camera Lucida and drawing board was arranged for the drawings. With the help of stage micrometer 1 sq. mm was drawn. The cleared leaf was mounted on the slide and a drop of glycerin water was added then covered with cover slip. The above prepared slide was placed on the stage of the microscope. Veins are traced which are included within the square. The outlines of those islets which overlap two adjacent sides of the square are also traced. The no. of vein-islets in the sq. mm is counted. The islets which are intersected by the sides of square are included on two adjacent sides and excluded on other two sides. The average no. of vein islets from four squares are found, and average no. of vein islets are calculated. v Determination of Vein-let termination number: It is defined as the no. of vein let termination per sq. mm of the leaf midway between midrib of the leaf and its margin. A vein termination is ultimate free termination of vein-let. Procedure: Same as for the determination of the Vein-islet number. The average no. terminations present with in the square was counted from four different get the value for one sq. mm.

surface, the

of vein squares to

Physico chemical parameters: Determination of moisturecontent (Loss on drying ): About 1g Jasminumgrandiflorumpowderwas weighed and placed in Petri plate. Dried in an oven at 105º C for 1 hr. D • Cooled in desiccator, loss in weight was recorded as moisture. Determination of ash values: D Ash values: Residue of the crude drugs after incineration contains mostly inorganic salts known as ash. Used to determine quality and purity of a crude drug. Ash contains inorganic radicals like phosphates, carbonates and silicates of sodium, potassium, magnesium, calcium etc. some times, inorganic variables like calcium oxalate, silica, carbonate content of the crude drug affects ‘total ash’. Such variables are then removed by treating with acid and then acid insoluble ash value is determined. Types of ash values are d Total ash: carbon and organic matter present in the drug is converted to ash at

temperature 450º C. It mostly contains carbonates, phosphates, silicates and s silica. Acid insoluble ash: Total ash may be treated with 2N HCl, which removes many inorganic salts to yield mainly silica in the residue of acid insoluble ash. i Water soluble ash: It is produced by separating the water soluble material from the total ash. In this case most of the water insoluble salts which may contribute in total ash are removed to find water soluble ash content. i Procedure for determination of total ash value: Accurately 2g of course powder was weighed in a tared silica crucible. The drug is incinerated by gradually increasing the heat in a muffle furnace at 450º C for some hours. After complete incineration, it was kept in a desiccator. The weight of ash with silica crucible was noted. Then the total ash was calculated in terms of percentage. c Procedure for determination of acid insoluble ash value: Followed procedure as per the steps mentioned in the procedure for determination of total ash value of a crude drug. Further Using 25 ml. of dilute hydrochloric acid, total ash was washed in to a 100 ml beaker. Beaker was boiled for 5 minutes. Filtered through an ash less filter paper, residue was washed twice with hot water. Filter paper placed in an silica crucible then, incinerated by gradually increasing the heat in a muffle furnace at 450º C for some hours. After complete incineration, it was kept in a desiccator to cool. The weight of acid insoluble ash with silica crucible was noted. Then the acidinsoluble ash was calculated in terms of percentage. a Procedure for determination of water soluble ash value: This is determined in a similar way to acid insoluble ash, using 25 ml. of water in place of HCl. Determination of extractive values: Extractive values: Useful for the evaluation of a crude drug. Gives idea about the nature of the chemical constituents present in a crude drug. Useful for the estimation of chemical constituents, soluble in that particular solvent used for extraction. s Procedure for Determination of alcohol soluble extractive values: About 5 g of powdered was weighed and transferred to a conical flask. 100 ml of the 90 % alcohol was added and closed with the cork. Kept aside for 24 hours with shaking frequently. Filtered, 25 ml of the filtrate was collected and transferred to a weighed, thin porcelain dish. Evaporated to dryness on a water bath and dried completely in an oven at 100º C. Kept in a desiccator to cool, then percentage w/w of extractive with reference to air dried drug was calculated. a Procedure for Determination of water soluble extractive value: Steps are followed similar to those mentioned in the previous procedure Chloroform water was used instead of alcohol. w Extraction procedures The powdered plant material was successively extracted by using Soxhlet extractor.

Lab scale extraction procedure: 14 g of powdered was charged in a ‘thimble’ made of cellulose it was placed in a central compartment of Soxhlet assembly. •200 ml of solvent was placed in a lower compartment and a reflux condenser is attached above the central compartment. Note: Each component of the set up was a separate item of glass ware which was assembled together with an appropriate content, to make a complete apparatus. The solvent in the lower container was heated to boiling. The vapour passes through the sidearm up into the reflux condenser. Here the vapour liquefied and drips into the thimble containing the material to be extracted. The warm solvent percolates through the material and the walls of the thimble and the extracts gradually collect in the central compartment. Once the height of the extract reached the top of the siphon, the entire liquid flowed through this and back to the lower solvent container. The process is than repeated. •The extract collected in the lower vessel, gradually becoming more and more concentrated The vapour rising from the heated extract is pure solvent vapour and so the liquid dripping into the material from the condenser is essentially pure solvent, though derived from the extract. •After complete extraction the lower vessel was removed, solvent recovered and the extract is concentrated and percentage yield was calculated. The solvents were recovered by using simple distillation method. The charged drug from the central compartment was removed, dried, recharged and extracted with benzene. By using Soxhlet extractor exhaustive extraction with a series of solvents of increasing polarity was done. i Solvents used with increasing polarity: Petroleum ether, Benzene, Chloroform, ethanol and finally water. Large scale procedure: Steps are followed similar to those mentioned in the lab scale procedure, using 470 g of powdered drug. The percentage yield at lab scale and pilot scale is given in table no. After extraction phytochemical screening was done and presence of constituents was d determined. Preliminary phytochemical screening: The crude extract obtained by successive extraction from petroleum ether, benzene, chloroform, ethanol and aqueous extraction are subjected to preliminary phytochemical screening. p

Qualitative chemical examination of extracts Q General method followed for screening of the constituents present in the extract

is as follows:-----------------------------------------------------------------------------------------------Detection of carbohydrates: Extracts were dissolved individually in 5ml of distilled water and filtered. The filtrates were used to test the presence of carbohydrates. f Molisch’s test: Filtrates were treated with 2 drops of alcoholic α naphthol solution in a test tube and 2ml concentrated sulphuric acid was added carefully along the sides of the test tubes. Formation of violet ring at the junction indicates the presence of carbohydrates. Benedict’s test: Filtrates were treated with Benedict’s reagent and heated on water bath. Formation of an orange red precipitate indicated the presence of reducing sugars. Fehling’s test: Filtrates were hydrolyzed with dilute hydrochloric acids, neutralized with alkali and heated with Fehlings A and B solutions. Formation of red precipitate indicates the presence of reducing sugars. Detection of alkaloids: Extracts were dissolved individually in dilute hydrochloride acid and filtered. The filtrates were tested carefully with alkaloid reagents. Mayer’s test: Filtrates were treated with Mayers reagent (potassium mercuric iodide), formation of a yellow cream precipitate indicate the presence of alkaloids. Wagner’s test: Filtrates were treated with Wagner’s reagent (iodine in potassium iodide) and observed. Formation of brown/reddish brown precipitate indicates the presence of alkaloids. Dragendroff’s test: Filtrates were treated with Dragendroff’s reagent (solution of potassium bismuth iodide). Formation of red precipitate indicates the presence of alkaloids. Hager’s test: Filtrates were treated with Hager’s reagent (saturated picric acid solution) Formation of yellow colored precipitate indicates the presence of alkaloids. Detection of phytosterols: Salkowski’s test: The extracts were treated with chloroform and filtered separately. The filtrates were treated with few drops of concentrated sulphuric acid, shaken and allowed to stand. If the lower layer turns red, sterols are present. If lower layer turns golden yellow triterpenes are present. Libermann Burchard test: The extracts were treated with chloroform solution and few drops of conc. 1 ml of acetic anhydride solution followed by sulphuric acid. A blue green colour shows the presence of phytosterols. Detection of glycosides: Extracts were hydrolyzed with dilute hydrochloric acid, and the hydrolysate was subjected to glycosides tests. Test for cardiac glycosides: Legals test: To the hydrolysate 1 ml of pyridine and few drops of sodium nitroprusside solution are added and then it is made alkaline with sodium

hydroxide solution. Colour change shows the presence of glycosides. Liebermann’stest:3 ml. of extract with 3 ml. acetic anhydride was heated, cool then added few drops of conc. sulphuric acid. Blue color appears in presence of bufadenolide. Test for deoxysugars (Keller killiani test): To 2 ml. of extract, glacial acetic acid, one drop of 5% FeCl3 and conc. H2SO4 was added. Reddish brown color appears at junction of two liquid layers and upper layer appears bluish green. Test for anthraquinone glycosides: Borntrager’s test: Hydrolysate is treated with chloroform and the chloroform layer is separated. To this, equal quantity of dilute ammonia solution is added. Colour change in the ammoniacal layer shows the presence of O glycosides. Modified Borntrager’s Test: The extracts were treated with ferric chloride solution and heated on boiling water bath for about 5 min. The mixture was cooled and shaken with equal volume of benzene. The benzene layer was separated and treated with half its volume of ammonia solution. The formation of pink or cherry red color in the ammonical layer indicates the presence of C glycoside. Detection of saponins: Froth’s test: The extracts were diluted with distilled water to 20 ml shaken in a graduated cylinder for 15 min. The formation of 1 cm layer of foam indicates the presence of saponins. Detection of phenolic and tannins: Ferric chloride test: The extract was treated with few drops pf neutral ferric chloride solution. The formation of bluish black color indicates the presence of phenolic nucleus. Gelatin test: To the extract, 1% gelatin solution containing sodium chloride was added. The formation of white precipitate indicates the presence of tannins. Test for flavonoids: Lead acetate test: The extracts were treated with few drops of lead acetate solution, formation of yellow precipitate indicates the presence of flavonoids. Alkaline reagent test: The extracts were treated with few drops of sodium hydroxide separately. Formation of intense yellow color, which becomes colorless on addition of few drops of dilute acid, indicates the presence of flavonoids. Shinoda test: The extracts were treated with few fragments of magnesium metal separately, followed by drop wise addition of concentrated hydrochloric acid. The formation of magenta color indicates the presence of flavonoid. Detection of proteins and amino acids: Millons test: The extracts were treated with 2ml of Millons reagent. The formation of white precipitate, which turns to red upon heating, indicates the presence of proteins. Ninhydrin test: To the extracts, 0.25% ninhydrin reagent was added and boiled for few minutes. Formation of blue color indicates presence of amino acid. The results of qualitative chemical analysis of the extracts are tabulated in the Table No. Optimization of TLC solvent system: Different solvents systems were tried for developing a TLC system for identification of constituents in the extract based on the literature survey and keeping in mind the chemical nature of the constituents. The details are given in the below table no.

The following solvents were used for the development of the TLC system: Chloroform Chloroform: Benzene: Ethyl acetate 0.5:0.5:1 Chloroform : methanol (7:3) Chloroform: methanol: Ethyl acetate 0.5:0.5:1 Toluene: acetone: formic acid (6 : 6: 1) Toluene: ethyl acetate: formic acid (5:5:1) Benzene: acetone (1: 0.1) Benzene : chloroform (1: 0.1) 85% Acetonitrile Toluene : Ethyl acetate: Glacial acetic acid (1.4 : 0.4 : 0.2) T Isolation of phyto¬constituents from extracts: The constituents of pet. ether and chloroform extract were isolated by column chromatography and identification and purity determination were done by thin layer chromatographic techniques. Column chromatography The Chloroform extract was subjected to column chromatography using different solvent systems. The fractions collected were further chromatographed, to know the no. of constituents present. Silica gel G as used as stationary phase. Column chromatography was done by using a glass column. The dimension of the column was 5X15 cm in height and 4 cm in diameter. The column was packed with silica gel by wet packing method wherein a padding of cotton was placed at the bottom of the column and then it was filled with eluting solvent of the lowest polarity (pet ether). Then the required amount of stationary phase (silica gel) was poured into the column to form a bed of silica. The extract was then poured on to the bed of silica, a layer of cotton covered it again and more amounts of solvents were poured over it, the column was then eluted gradiently. The general principle applied in column chromatography consisted of following steps: 1.Pre¬column preparation: The precolumn preparation included adsorption of the selected extract/ fraction, charging and saturation of the column. a. Adsorption of the extract: The extract selected for fractionation was adsorbed on stationary phase in ratio 1:1. b. Charging of column: A glass column was selected and rinsed with the solvent. A cotton layer was placed at the bottom and the column was charged with the solvent and stationary phase. The silica gel was used in the ratio (1:2) of the extract to make the gel bed for complete separation. The solvent was eluted up to the level of column bed and the dried extract was charged in the column. Another layer of cotton was placed over the charged matter to prevent the disturbance of the extract bed while pouring the eluting solvent from the top. c.Saturation of the column: The charged column was left for 4 hrs. for complete saturation and removal of air bubbles to make the bed static. s 2. Elution: The charged column was then eluted with different mobile phases with gradual increase in polarity. The fractions collected and the solvent recovered by simple distillation. All the concentrated fractions were subjected to TLC for the identification of the desired bands. i Column Requirements:¬ Stationary phase – silica gel G (60120 mesh) Mobile phase – Pet ether, Benzene, Ethyl acetate, Chloroform, MeOH, EtOH Charged material – Chloroform fraction. Volume of each fraction – 100 ml to 500 ml. Visualization – Iodine, 10% Ethanolic sulphuric acid, anisaldehyde sulfuric acid, ferric chloride reagent, dragendroff reagent. Details of column are tabulated in table no.12.

Procedure: The column was first eluted with 100% pet ether. The polarity of mobile phase was gradually increased with Benzene, Ethyl acetate, MeOH, EtOH. The fraction collected was concentrate. The desired concentrated fractions were screened for phytoconstituents. The desired concentrated and dried fractions were kept in container with suitable label and kept for further use. The details of collected fraction are given in flow chart no. 1 and 2. Purification of compounds: From column Purification of compound I: The fraction 7181 of column (ptether: ethyl acetate 80:20 ), after concentration, it has formed crystals, was buff colored and shown single spot with the mobile phase Toluene: acetone: formic acid (6 : 6: 1) Purification of compound II: The fraction 91101 of column (pet. ether : ethyl acetate 80:20), after concentration, it has formed crystals, remaining solution was decanted. The decanted solution was solidified and it was buff colored and shown single spot with the mobile phase Toluene: acetone: formic acid (6 : 6: 1). Purification of compound III & IV The ethanolic extract gave four spot which mixed and separate the all spot by preparative TLC which yielded white coloured compounds III & IV. Solubility profile of the isolated compounds: The isolated compounds were analyzed for their solubility in different solvents. The concentration used for solubility was 1mg/ml of solvent. Their solubility profile are tabulated in table no.15. The solvents used were pet ether, CHCl3, acetone, methanol and distilled water. Determination of melting points of the isolated compound: Organic chemist not only to identify it, but also to establish its purity uses the melting point of a compound. The melting point indicates purity in two ways. First, the purer the material, the higher its melting point. Second, the purer the material, the narrower its melting point range. Apparatus–Thermonic (liquid paraffin). Sample¬ Compound I, Compound II, Compound III and Compound IV. Procedure– The isolated compounds were subjected to melting point analysis wherein a capillary tube, closed at one end, was taken and a small amount of the sample was placed into it. The capillary tube was then dipped at the closed end into liquid paraffin along with a thermometer. The paraffin was then heated and the temperature at which the sample just started to melt and the temperature at which it completely melted was taken as the melting point temperature range for the sample. It was noted down. The results are given in Table no: 17. UV Spectroscopy of the isolated compound: The UV spectrum of compound I, II, III and IV is given in Page No. 123124. Instrument: UV Visible Spectrometer. Shimadzu Screening of plant extracts for anti-microbial activity: Antibacterial activities of different extracts were studied by the disc diffusion method. Preparation of extracts The plant part was powdered and extracted successively with different solvents by increasing then polarity, in a Soxhlet extractor. The plant extracts were filtered through Whatman No. 1 filter paper into beaker. The filtrates were dried until constant dry weights of each extracts were obtained. The residues were stored at

4°C for further use. Test Organisms The pure cultures of bacteria maintained in the College Microbiology Laboratory were used for the microbiological work. The test organisms were maintained on Nutrient agar medium. The test organisms used for screening are Bacteria, Staphylococcus aureus. Bacillus subtilis. Escherichia coli. Pseudomonas aeruginosa. P Common microorganism: 1. Staphylococcusaureus: Gram positive cocci about 1m in diameter. They are mainly arranged in group like structure, especially when examined in pathological specimen may occur as a single or pair of cells. They are non sporing, non motile, and usually non capsulate when grown on agar for 24hrs. at 37 ° C. Individual colonies are circular, 23mm in diameter with a smooth, shiny surface; colonies appear opaque and are often pigmented (golden yellow, fawn or cream), though a few strains are un pigmented. Staphylococci are salt tolerant and can be selectively isolated from materials such as faeces and food by use of media containing 710% Nacl. Pathogenesis: S.aureus present in nose of 30% of healthy people and may be found on the skin. It causes infection most commonly at sites of lowered host resistance. E.g.: damaged skin or mucous membranes. Infections caused by S.aureus: • Pyrogenic infections: Abscesses, Impetigo, Mastitis, Bacteraemia, Osteomyelitis, Pneumonia Endocarditis. Toxin¬ mediated infections: Penicillinase, Cephallosporins, Aminoglycosides, Tetracyclins, Macrolides, Fluroquinolines, Rifampicin, Trimethoprim, Chloromphenicol F Scalded skin syndrome, Pemphigus neonatorum, Toxic shock syndrome, Food poisoning. Sensitivity to Antibiotics: 2. Bacillus subtilis: Bacillus subtilis, known as the hay bacillus or grass bacillus, is a Gram positive, catalase positive bacterium commonly found in soil. A member of the genus Bacillus ,B.subtilisis rod shaped, and has the ability to form a tough, protective endospore, allowing the organism to tolerate extreme environmental conditions. Pathogenesis: B. subtilis is not considered a human pathogen; it may contaminate food but rarely causes food poisoning. B.subtilis produces the proteolytic enzyme subtilisin. B.subtilis spores can survive the extreme heating that is often used to cook food, and it is responsible for causing ropiness a sticky, stringy consistency caused by bacterial production of long chain polysaccharides in spoiled bread dough. Uses Colonies of B.subtilis grown on a culture dish in a molecular biology laboratory. B.subtilis is used as a soil inoculant in horticulture and agriculture. Enzymes produced by B.subtilis and B.licheniformisare widely used as additives in laundry detergents. Its other uses include the following: a model organism for laboratory studies a strain of B.subtilis formerly known as Bacillusnatto is used in the commercial production of the Japanese delicacy natto as well as the similar Korean food cheonggukjang B.subtilisstra in QST 713 (marketed as QST 713 or Serenade) has a natural

fungicidal activity, and is employed as a biological control agent popular worldwide before the introduction of consumer antibiotics as an immuno stimulatory agent to aid treatment of gastrointestinal and urinary tract diseases. It is still widely used in Western Europe and the Middle East as an alternative medicine can convert explosives into harmless compounds of nitrogen, carbon dioxide, and water plays a role in safe radionuclide waste [e.g. Thorium (IV) and Plutonium (IV)] disposal with the proton binding properties of its surfaces recombinants B.subtilis str. pBE2C1 and B.subtilis str. pBE2C1AB were used in production of poly hydroxy alkanoates (PHA) and that they could use malt waste as carbon source for lower cost of PHA production Used to create amylase enzymes. Although not considered a human pathogen, Bacillus subtilis can contaminate food and cause food poisoning in rare cases a 3. E.coli: Strains of E.coli are usually motile and some, especially those from extra intestinal infections may produce a polysaccharide capsule. They grow well as nonselective media and most strains ferment lactose, producing large red colonies on Maconkey Agar. They grow over a wide range of temperature (15 – 450C) and some strains are more heat resistant than others members of the enterobacteriaceae and may survive 600C for 15 min. Strains of E.coli Entero-pathogenic strains cause acute gastroenteritis in newborns and infants up to 2 years of age. Entero-invasive strain cause diarrhea in older children and adults. E • Entero-toxigenic strain cause diarrhea in adults and infants. Others strains of E.coli which are usually harmless in their normal habitat can cause disease when they gain access to other sites or tissues. Pathogenesis: • UTI, septic infection, bacteremia, meningitis, pulmonary infections, abscesses, skin and wound infection. Treatment: E.coli is susceptible to a wide range of Antibiotics • Doxycycline, Trimethoprim, Norfloxacin, and other Fluroquinones. 4. Pseudomonas aeruginosa P. aeruginosais a Gram negative rod measuring 0.5 to 0.8 m by 1.5 to 3.0 m.. Pseudomonas aeruginosa is a free living bacterium, commonly found in soil and water.. Its metabolism is respiratory and never fermentative, but it will grow in the absence of O2 if NO3 is available as a respiratory electron acceptor. P. aeruginosa strains produce two types of soluble pigments, the fluorescent pigment pyoverdin and the blue pigment pyocyanin. The latter is produced abundantly in media of low iron content and functions in iron metabolism in the bacterium. Pyocyanin (from "pyocyaneus") refers to "blue pus", which is a characteristic of suppurative infections caused by Pseudomonasaeruginosa. Pathogenes is Pseudomona saeruginosa, the disease process begins with some alteration or circumvention of normal host defenses. The pathogenesis of Pseudomonass infections is multifactorial, include septicemia, urinary tract infections, pneumonia, chronic lung infections, endocarditis, dermatitis, and osteochondritis. Endocarditis Respiratory infections. Bacteremia and septicemia. Central nervous system infections Ear infections including external otitis. Eye infections.

Bone and joint infections Urinary tract infections. Gastrointestinal infections Skin and soft tissue infections, including wound infections, pyoderma and S dermatitis . Resistance to Antibiotics Pseudomona saeruginosa is notorious for its resistance to antibiotics and is, therefore, a particularly dangerous and dreaded pathogen. The bacterium is naturally resistant to many antibiotics due to the permeability barrier afforded by its Gram negative outer membrane. Also, its tendency to colonize surfaces in a bio film form makes the cells impervious to therapeutic concentrations antibiotics. Since its natural habitat is the soil, living in association with the bacilli, actinomycetes and molds, it has developed resistance to a variety of their naturally occurring antibiotics. Moreover, Pseudomonas maintains antibiotic resistance plasmids, both R factors and RTFs, and it is able to transfer these genes by means of the bacterial mechanisms of horizontal gene transfer (HGT), mainly transduction and conjugation. • Treatment: Only a few antibiotics are effective against Pseudomonas aeruginosa, including fluoroquinolones, gentamicin and imipenem, and even these antibiotics are not effective against all strains. a Preparation of inoculum Stock cultures were maintained at 4°C on slopes of nutrient agar. Active cultures for experiments were prepared by transferring a loop full of cells from the stock cultures to test tubes of Nutrient broth for bacteria that were incubated without agitation for 24 hrs at 37°C and 25°C respectively. The cultures were diluted with fresh Nutrient broth and Sabouraud dextrose broth. Preparation of Media: Nutrient agar medium (NA) was used for preliminary antibacterial study. The medium was prepared by dissolving the different ingredients in water and autoclaving at 1210C for 15 minutes. Anti-microbial susceptibility test: The disc diffusion method was used to screen the antimicrobial activity. Invitro antimicrobial activity was screened by using Nutrient agar (NA) obtained from Himedia (Mumbai). The NA plates were prepared by pouring 15 ml of molten media into sterile petriplates. The plates were allowed to solidify and 0.1 % inoculum suspension was swabbed uniformly and the inoculum was allowed to dry for 5 minutes. The different extracts were loaded on 5mm sterile disc till saturation. The loaded disc was placed on the surface of medium and the compound was allowed to diffuse for 5 minutes and the plates were kept for incubation at 37°C for 24 hrs. At the end of incubation, inhibition zones formed around the disc were measured with transparent ruler in millimeter. These studies were performed in triplicate by using standard drugs (10 g /disc Penicillin; 60 g /disc g gentamicin;). Anti helmentic activity: The anti helmintic activity was evaluated on adult earthworms (Pheretims posthuma), . Helminths are recognized as a major constraint to livestock production throughout the tropics and elsewhere53. The economic impact of parasitic gastroenteritis (PGE), which is caused by mixed infection with several species of stomach and intestinal round worms, as a production disease in ruminants lies not only in direct losses such as mortality associated with the clinical form of the disease but also indirect insidious losses as a result of weaknesses, loss of appetite, decreased feed efficiency, reduced weight gain and decreased productivity Winrock International (1992) indicated that over $4 billion is lost in animal productivity as a result of animal diseases, with over half of this loss due to internal parasites such as helminthes55 . In an effort to reduce losses due to effects of helminth parasites on livestock industry, approximately

$1.7 billion are spent annually worldwide in control measures . In Nigeria as in many tropical countries, control of helminthosis is mainly by periodic anthelmintic medication. However, as these are expensive and often unavailable to farmers in rural areas, livestock producers have continued to use indigenous plants as dewormers drawing upon centuries of knowledge of herbal medicines. Furthermore, some serious disadvantages of using manufactured drugs have become evident in the western world, such as resistance, food residues and environmental pollution57 . Albendazole is chemically methyl5(propylthio)2benzimidazolecarbamate, C12H15N3O2S. It is a wide58 spectrum anthelmentic drug used for human and animal infections. When administered orally, it is quickly biotransformed into its active intermediate metabolite Albendazole sulphoxide (ABZSO), which is then oxidized to the inactive form of Albendazole sulphone (ABZSO2). Because of their affinity for the parasite âtubulin, both albendazole and ABZSO show anthelmentic activity61. It is also widely used as inhibator for protein synthesis and causes degenerative changes in the intestine and the enveloping membrane and for treatment of cysticercosis has received criticisms60, 61 , their use in the cysticidal treatment of neurocysticercosis has proved efficacious62, 63. Although neurocysticercosis is widespread in underdeveloped countries with an important socioeconomical impact, there is a lack of information about its natural history, treatment and prognosis60, 64 and widely used for treatment and control of helminthes in cattle65, 66 . The method was followed for anthelmintic screening nine groups, each consisting of six earthworms of approximately equal size (7±1) was released into 50ml of desired formulation at room temperature. Each group was treated with one of the following: Vehicle (1%Tween 80 in normal saline), extract (10, 20 and 50mg/ml) in normal saline containing 1% Tween 80. Observations were made for the time taken for paralysis and /or death of individual worms up to four hours of test period. The mean paralysis time and mean lethal time for each extract was recorded .paralysis was said to occur when the worms did not revive even in normal saline. Death was concluded when the worms lost their motility followed with fading away of their body colour. Preparation of Extracts: Known weight of the extract was taken and the emulsion was prepared using Tween 80 and Normal saline solution (8.7 gm in 1000 ml of distill water) to make concentration of 20mg/ml, 40mg/ml, 60mg/ml, 100mg/ml.