The Ash Content Of A Crude Drug Biology Essay

The modify Content Of A Crude do do drugss Biology EssayThe change content of a crude drug is generally interpreted to be the ease remaining after incineration. It ordinarily represents the inorganic salts naturally occurring in the drug and adhering to it, but it may similarly include inorganic amour bring ined for the purpose of adulteration. There is a wide going varies within narrow limits in the case of the same some torso drug. Hence an change determination furnishes a basis for judging the identity and cleanliness of a drug and gives information relative to its adulteration with inorganic matter. Ash standards have been established for a number of official drugs. Usually these standards bum a maximum limit on the summate ash tree or on the sexual exerciseually transmitted disease in meltable ash permitted.The total ash is the correspondence remaining after incineration. The acid in water supply-soluble ash is the part of the total ash which is insoluble in diluted hydrochloric acid.The ash or residue yielded by an organic chemical compound is as a rule, a measure of the amount of inorganic matters present as impurity. In close to cases, the inorganic matter is present in small amounts which argon difficult to remove in the purification process and which are non objectionable if only traces are present. Ash rank are helpful in determining the quality and purity of the crude drugs in powder form. appendages stipulation in Indian pharmacopoeia were used to determine the different ash value such as total ash and acid insoluble ash. numerate ashWeighed accurately ab out(p) 3 gm of air desiccated fine-grained drug was taken in a tarred silica melting pot and incinerated by gradually increasing the temperature to ramp up it dull red until liberal from carbon cooled and heavinessed and and so mensural the contribution of total ash with credit to the air desiccate drug.Acid insoluble ashThe ash obtained as directed below tota l ash above was poached with 25 ml of 2N HCl for 5 minutes. The insoluble matter was calm on ash less(prenominal) distort paper, washed with heatable water light and weighed, thus calculated the per centum of acid insoluble ash with type to the air dried drug.Water soluble ashThe total ash obtained was boiled with 25 ml of water for 5 minutes. The insoluble matter was collected on an ash less slobber paper, washed with baking het water and ignited for 15 minutes at a temperature not exceeding 450C. The weight of insoluble matter was subtracted from the weight of total ash. The difference in weight represents the water soluble ash. The plowshare of water soluble ash calculated with reference to the air dried drug.b. EXTRACTIVE VALUESExtractive value of crude drugs are useful for their paygrade, especially when the constituents of a drug cannot be readily estimated by any other blottos. Further, these determine indicate the temperament of the constituents present in a c rude drug. closing of alcohol soluble elicitive value5 gm of the air-dried brusk powder of Anogeissus latifolia besiege (Roxb.ex.DC) was macerated with superstar C ml of 90% ethanol in a disagreeable flask for 24 hours, thrill frequently during the number one 6 hours and allowing stand for 18hours. Thereafter, it was filtered rapidly taking precautions against the loss of the solvent. Out of that deform, 25 ml of the filtrate was evaporated to temperance in a tarred flat bottomed shallow p all(prenominal), dried at 105C and weighed. The fate of ethanol soluble straighten upive value was calculated with reference to the air- dried drug. The declarations are recorded in the table. finish of water soluble omitive valueWeigh accurately 5 gm of coarsely fine-grained drug and macerate it with 100 ml of put out water in a closed flask for 24 hours, shaking frequently during the first 6 hours and allow to standing for 18 hours. Thereafter, it was filtered rapidly taking precautions against loss of the solvent. Then 25 ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish, dried at 105C and weighed. The percentage of water soluble sublimateive was calculated with reference to the air dried drug. The results are accustomed in the table.c. LOSS ON DRYINGLoss on drying is the loss in weight in percentage w/w determined by means of the procedure given below. It determines the amount of volatile matter of any kind (including water) that can be driven off to a lower place the delineate specified (Desiccators or hot air oven). If the sample is in the form of large crystals, thence slim down the size by quick crushing to a powder.Procedure almost 1.5 gm of powdered drug was weighed accurately in a tarred porcelain dish which was previously dried at 105C in hot air oven to constant weight and then weighed. From the difference in weight, the percentage loss of drying with reference to the air dried magnetic core was calcula ted.d. FLUORESCENCE ANALYSIS Kokate.C.K, 2002 Khandelwal KR 1996.In the near-ultra region of the spectrum (3000-4000A) some of the phytoconstituents show to a great extent than or less brilliant coloration when exposed to radiation. This phenomenon of emitting megascopic wavelengths as a result of being excited by radiation of a different wavelength is cognize as fluorescence. Some quantifys the amount of ultra-violet light normally present with visible light is sufficient to produce the fluorescence, but often a more powerful source of ultra-violet is necessary, e.g. mercury vapour lamp. It is often possible to make use of this phenomenon for the qualitative examination of herbal drugs. A fluorescence characteristic of the powdered leaves of Anogeissus latifolia wall (Roxb.ex.DC) was observe in daylight and UV light. Also the light study was performed on treating the drug powder with different chemical reagents. The discovered results are given in table.e. FOAMING INDEX Diva kar M.C., 1996Foaming baron is mainly performed to determine the saponin content in an aqueous decoction of coif material.Determination of fizzing indicantWeighed accurately about 1g of coarsely powdered drug and transformed to 500ml conical flask containing 100ml of boiling water. Maintained at subdue boiling at 80-90c for about 30min. Cooled and corresponded sufficient water through the filter to make up the leger to 100ml (V1). Cleaned 10 stoppered study tube of uniform dimension were taken and transferred the successive portions of 1,2,3ml up to 10ml and change the volume of the liquid in to each one study tube with water to 10ml.Stoppered the tubes and shaken them in a lengthwise motion for 15 endorsement uniformly and allowed to stand for 15min and measure the height of foam. If the height of the foam in every tube is less than 1cm, the foaming index is less than 100(not significant). present the foam was more than 1cm height after dilution of mark material. If t he height of the foam in every tube is more than 1cm, the foaming index is more than 1000. In this case, 10ml of first decoction of plant material is thrifty and transferred to 100ml volumetric flask (V2) and volume is made to 100ml and followed the same procedure.5.1. 2. PRELIMINARY PHYTOCHEMICAL ANALYSISExtraction of plant material-Petroleum ether educeion-About 400gm of dry coarse leaf powder of the Anogeissus latifolia wall (Roxb.ex.DC) was extracted with petroleum ether 2500ml (40-600c) for 18 hrs by continuous hot percolation rule. It was allowed to cool to 40oC and then filtered apply whatman No.1 filter paper. The filtrate was then difficult in a rotary evaporator and the extract stored at 4C until required. The extract yield (% w/w) from the plant material was recorded.Methanolic extraction-About 400g of air dried coarse powdered material was taken in 1000ml soxhlet apparatus and soaked with petroleum ether for 2 years. At the end of second day the powder was taken o ut and it was dried. subsequently drying it was again packed and extracted by using methyl alcohol (Changshu yangyuan chemicals, China) as solvent, till strain disappeared. The temperature was maintained at 55C-65C. After that extract was concentrated by distillation and solvent was recovered. The final ascendent was evaporated to dryness. The touch, consistency and yield (% w/w) of methanolic extract were noted.S.No.Name of extract twineConsistencyYield% W/W12Methanolic extractPetroleum ether extractBlackish brownBlackish viridityNon Sticky masssticky oily mass16.751.60Table 1. Nature and colour of extract of Anogeissu latifolia wall (Roxb.ex.DC).5.1. 3 CHEMICAL TESTSA) shield for carbohydrates1. Molisch screen out It consists of treating the compounds of a-naphthol and concentrated sulphuric acid along the sides of the sieve tube.Purple colour or reddish violet colour was produced at the junction between twain liquids. (Kokate, C.K et al, 2000)2. Fehlings Test Equal quan tity of Fehlings resultant role A and B is toted. stir up gently, brick red precipitate is obtained.3. Benedicts bear witness To the 5ml of Benedicts reagent, add 8 drops of final result under examination. Mix well, boiling the mixture vigorously for two minutes and then cool. Red precipitate is obtained.4. Barfoeds test To the 5ml of the Barfoeds resolution add 0.5ml of solution under examination, heat to boiling, formation of red precipitate of copper oxide is obtained.B) Test for Alkaloids1. Dragendroffs Test To the extract, add 1ml of Dragendroffs reagent Orange red precipitate is produced.2. Wagners test To the extract add Wagner reagent. Reddish brown precipitate is produced.3. Mayers Test To the extract add 1ml or 2ml of Mayers reagent. Dull white precipitate is produced.4. Hagers Test To the extract add 3ml of Hagers reagent lily-livered Precipitate is produced.C) Test for Steroids and Sterols1. Liebermann Burchard test drop the test sample in 2ml of chloroform in a dry test tube. Now add 10 drops of acetic anhydride and 2 drops of concentrated sulphuric acid. The solution becomes red, then blue and finally bluish yard in colour.2. Salkowski test Dissolve the sample of test solution in chloroform and add lucifer volume of conc. sulphuric acid. Bluish red cherry red and over-embellished color is noted in chloroform bottom, whereas acid assumes marked green fluorescence.D) Test for Glycosides1. Legals test Sample is dissolved in pyridine atomic number 11 nitropruside solution is added to it and made alkaline. Pink red colour is produced.2. Baljet test To the drug sample, sodium picrate solution is added. Yellow to orange colour is produced.3. Borntrager test Add a few ml of dilute sulphuric acid to the test solution. Boil, filter and extract the filtrate with ether or chloroform. Then organic layer is separated to which ammonia is added, tap, red or violet colour is produced in organic layer.4. Killer Killani test Sample is dissolved in ace tic acid containing trace of ferric chloride and transferred to the surface of concentrated sulphuric acid. At the junction of liquid reddish brown color is produced which gradually becomes blue.E) Test for SaponinsFoam test About 1ml of alcoholic sample is diluted one after another with distilled water to 20ml, and shaken in graduated cylinder for 15 minutes.1 cm layer of foam indicates the comportment of saponins.F) Test for FlavonoidsShinoda test To the sample, magnesium turnings and then concentrated hydrochloric acid is added. Red colour is produced.G) Test for Tri-terpenoidsIn the test tube, 2 or 3 granules of tin was added, and dissolved in a 2ml of thionyl chloride solution and test solution is added. Pink colour is produced which indicates the presence of triterpenoids.H) Tests for Tannins and phenoplast CompoundsTo 2-3 ml of extract, add few drops of pursual reagentsa). 5% FeCl3 solution boneheaded blue-black color.b). Lead acetate solution white precipitate.c). Gelat in solution white precipitated). Bromine water decolor of bromine water.e). Acetic acid solution red color solutionf). Dilute iodine solution transient red color.g). Dilute HNO3 reddish to yellow color.I) Test for icy Oils and Fatty acidsa). Spot test pocket-size quantity of the extract is position between two filter papers. Oil stain produced with any extract shows the presence of amend oils and fats in the extracts.b). Saponification testFew drops of 0.5N alcoholic thou hydroxide are added to the extract with few drops of phenolphthalein solution. Later the mixture is modify on water bath for 1-2 hours soap formation indicates the presence of fixed oils and fats in the extracts.J) Test for Gums and Mucilagea). Ruthenium red testSmall quantities of extract are diluted with water and added with ruthenium red solution. A pink colour production shows the presence of gums and mucilage.K) Test for Proteins and Amino acidsBiuret test Add 1 ml of 40% sodium hydroxide and 2 drops of 1 % copper sulphate to the extract, a violet colour indicates the presence of proteins.Ninhydrin test Add 2 drops of freshly prepared 0.2% Ninhydrin reagent to the extract and heat. A blue colour develops indicating the presence of proteins, peptides or amino acids.Xanthoprotein test To the extract, add 20% of sodium hydroxide or ammonia. Orange colour indicates presence of aromatic amino acid.5.1. 4.TOXICOLOGICAL military rankDetermination LD50 value of Anogeissus latifolia (Roxb.ex.DC).wall. catchperrAcute oral Toxicity StudyThe procedure was followed by using OECD guidelines 423 (Acute toxic contour method)Animals bountiful albino rats (Wister strain) of either sex with calculation 150 180gm were used. The animate beings were maintained on the fitted nutritional and environmental tick throughout the experiment. The animals were housed in polypropylene cages with paddy house bed under standard laboratory condition for an acclimation fulfilments of 7 days prior to perfor ming the experiment. The animals had access to laboratory grub and water. The experimental protocols were okay by institutional Animal honorable direction a scripted permission from in house ethical deputation has been taken to learn out (Reference no. JKKMMRF/2010/009) and complete this study.ProcedureTwelve animals (Wister Albino rats, 150-200gm) were selected for studies. The acute toxic class method is a flavour wise procedure with 3 animals of single sex per step. Depending on the death rate and / or moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute perniciousness of the test animals while allowing for acceptable data based scientific conclusion.The method uses defined back breakers (5, 50, 300, 2000 mg / kg body weight) and the results allow a substance to be ranked and classified according to the Globally Harmonized arranging (GHS) for the classification of chemical which cause acute perniciousness.Most of the crud e extracts have got LD50 value more than 2000 mg. /kg of the body weight of animal used. loony toons volume was administered 0.1 ml / 100 gm body weight to the animalby orally after giving the dose the toxic signs were observed within 3-4 hours.Body weight of animals before and after administration, onset of toxicity and signs of toxicity like changes in skin and fur, eyes, and mucous membrane and also respiratory, circulatory, autonomic and central nervous systems and somatomotor activity and behavior pattern, signs of tremors, convulsion, salivation, diarrhoea, lethargy, sleep and stupor was also to be noted, if any , was observed.ObservationNo toxicity or death was observed for these given dose levels, in selected and hard-boiled animals. So the LD 50 of the Anogeissus latifolia wall (Roxb.ex.DC), as per OECD guidelines-423 is greater than 2000mg/kg (LD50 2000mg/kg).Hence, the biological dose was fixed at 200, 400 and 600mg/kg of body weight for the extract.pharmacological EV ALUATION5.2.1 Evaluation of Anti-ulcer Activity-Animals usedAdult albino rats (Wister strain) of either sex with weighing 150 180gm were used. The animals were maintained on the suitable nutritional and environmental condition throughout the experiment. The animals were housed in polypropylene cages with paddy house bedding under standard laboratory condition for an acclimatization periods of 7 days prior to performing the experiment. The animals had access to laboratory chow and water. The experimental protocols were approved by institutional Animal Ethical Committee a written permission from in house ethical committee has been taken to carry out (Reference no. JKKMMRF/2010/009) and complete this study.5.2.2 Experimental procedureEthanol bring on ulcer- antheral albino-Wistar rats were divided in to five roots of half-dozen animals per throng and animals were fasted for 24 hrs prior to the experiment in perforated steel cages to distract coprophagy. Six groups were made as b elow host I animals served as normal controls. assembly II authentic 1% CMC (1.0ml/kg p.o) as vehicle control.Group III received 200mg/kg, p.o methanolic extract of Anogeissus latifolia.Group IV received 400mg/kg, p.o methanolic extract of Anogeissus latifolia.Group V received 100mg/kg, Sucralfate as standardOne hour after the drug treatment the animals were handle with absolute ethanol 5ml/kg to induce ulcers. The animals were sacrificed after 1hrs and persist was opened and percentage inhibition of ulcer was determined. (Mozafar khazaei et al., 2006, capital of Minnesota V. et al 2002, Paul V. et al., 2000)Aspirin induce ulcer-Male albino-Wistar rats were divided in to five groups of six animals per group and animals were fasted for 24 hrs prior to the experiment in perforated steel cages to repeal coprophagy. Six groups were made as belowGroup I animals served as normal controls.Group II received 1% CMC (1.0ml/kg p.o) as vehicle control.Group III received 200mg/kg, p .o methanolic extract of Anogeissus latifolia.Group IV received 400mg/kg, p.o methanolic extract of Anogeissus latifolia.Group V received 100mg/kg, Sucralfate as standardOne hour after the drug treatment the animals were treated with aspirin 200 mg/kg to induce ulcers. The animals were sacrificed after 1hrs and stomach was opened and percentage inhibition of ulcer was determined. (Mozafar khazaei et al., 2006, Paul V. et al 2002, Paul V. et al., 2000)5.2.3 BIOCHEMICAL PARAMETERS-The stomach was carefully excised keeping oesophagus closed and opened along greater curvature and luminal contents were removed. The stomachic contents were collected in a test tube and centrifuged. The gastric contents were analyzed for gastric juice volume, pH, un wantonze and total acidity.5.2.4 Measurement of gastric juice volume and pH-Gastric juice was collected from ethanol induced ulcer rats. The gastric juice thus collected was centrifuged at 3000 rpm for 10 min. The volume of supernatant was measured and expressed as ml/100g body weight. The pH of the supernatant was measured using digital pH meter. (Canmon DC. et al., 1969, Kannappan et al., 2008, Patil K.S. et al., 2008, Paul V. et al., 2000)5.2.5 Determination of free and total acidity-An aliquot of 1.0 ml of gastric juice was pipette out in to a 50 ml conical flask and 2/3 drops of Topfers reagent was added to it and titrated with 0.01N NaOH until all traces of the red colour disappeared and the colour of the solution turned yellowish orange. The volume of 0.01N NaOH was noted which corresponds to free acidity. Then 2/3 drops of phenolphthalein was added and titration was continued until a permanent pink colour was developed. The volume of total alkali consumed was noted which corresponds to total acidity. The free acidity and total acidity was determined using the formula and values are expressed as mEq/l 100g. (Kannappanetal. 2008, Rajkapoor et al., 2002).Acidity = flock of NaOH X Normality of NaOH X 100 (mEq/L p er 100g)0.015.2.6 ulcer index (UI)-The mucosa was flushed with saline and stomach was pinned on frog board. The lesion in glandular portion was examined under a 10x magnifying glass and length was measured using a divider and scale and gastric ulcer was scored. Ulcer index of each animal was calculated by adding the values and their mean values were determined. (Malairajan et al., 2007)0 Normal coloured stomach0.5 Red colouration1 Spot ulceration1.5 Haemorrhagic streak2 ulcers3 Perforations5.2.7 Percentage inhibitionPercentage inhibition was calculated using the following formula. (Malairajan et al., 2007)UI ulcer control UI ulcer treated% inhibition = X 100UI ulcer control5.2. 8. Statistical AnalysisAll the values are expressed as mean S.E.M for groups of six animals each. Analyzed by one way ANOVA and compared by using Tukey- Kramer multiple comparison tests. The values are statistically significant at triad levels, ***p 0.05.5.3. EVALUATION OF DIURETIC ACTIVITYAnimals us edAdult albino rats (Wister strain) of either sex with weighing 150 180gm were used. The animals were maintained on the suitable nutritional and environmental condition throughout the experiment. The animals had access to laboratory chow and water. The experimental protocols were approved by institutional Animal Ethical Committee a written permission from in house ethical committee has been taken to carry out (Reference no. JKKMMRF/2010/009) and complete this study.Experimental procedureThe method of (Lipchitz et.al., 1943) was employed for the evaluation of diuretic activity. The Male Albino-Wistar rats were divided into four groups of six rats in each as mentioned below.Group I received Normal saline (25mg/kg, p.o) as control.Group II received (400mg/kg, p.o) methanolic extract of Anogeissus latifolia.Group III- received (600mg/kg, p.o) methanolic extract of Anogeissus latifolia.Group IV received Furosemide (20mg/kg, p.o) as standard.The animals were fasted and deprived of s ustenance and water for 18hour prior to the experiment. On the day of experiment, the group I animals percentage as control, received normal saline (25ml/kg,p.o), the group II animals received methanolic extract of Anogeissus latifolia wall (Roxb.ex.DC) leaves (400mg/kg,p.o) and group III animals also received methanolic extract (600mg/kg,p.o), the group IV animals received Furosemide (20mg/kg,p.o), respectively, in normal saline. flat after the administration the animals were kept in metabolic cages (three per cage) specially knowing to separate urine and fecal matter and kept at manner temperature of 25 0.5 C throughout the experiment. The total volume of urine was collected at the end of 5hrs after dosing. During this period no water and forage was made available to the animals. The parameters taken for individual rat were body weight before and after test period, total concentration of Na+ , K+ and Cl- in the urine. The Na+ and K+ were measured by brand photometry and Cl- concentration was estimated by titration with silver nitrate (N/50) using three drop of 5% potassium chromate solution as indicator .the results are reported as mean SD, the test of significance (P5.3.1. Statistical abridgmentAll the values are expressed as mean S.E.M for groups of six animals each. Analyzed by one way ANOVA and compared by using Tukey- Kramer multiple comparison tests. The values are statistically significant at three levels, ***p 0.05.5.4 EVALUATION OF ANALGESIC ACTIVITYAnimals usedAdult albino rats (Wister strain) of either sex with weighing 150 180gm were used. The animals were maintained on the suitable nutritional and environmental condition throughout the experiment. The animals were housed in polypropylene cages with paddy house bedding under standard laboratory condition for an acclimatization periods of 7 days prior to performing the experiment. The animals had access to laboratory chow and water. The experimental protocols were approved by institution al Animal Ethical Committee a written permission from in house ethical committee has been taken to carry out (Reference no. JKKMMRF/2010/009) and complete this study.ProceduresEddys hot dental plate methodThe Male Albino-Wistar rats were divided into four groups of six rats in each as mentioned below.Group I received 1% CMC (3ml/kg, p.o) as control.Group II received (400mg/kg, p.o) methanolic extract of Anogeissus latifolia.Group III- received (600mg/kg, p.o) methanolic extract of Anogeissus latifolia.Group IV received pentazocine (5mg/kg, p.o) as standardAnalgesic activity was performed by using Eddys hot plate (Inco, India) maintained at a temperature of 551c. The basal reaction time of all animals towards caloric heat was recorded. The animals which showed forepaw licking or startle response within 6-8 seconds were selected for the study. Male Albino rats were divided into 5 groups having 6 animals each and they were divided into 5 groups having 6 animals each and they wer e fasted overnight during the experiment free access to water. Group first received 1 % CMC (3ml/kg, p.o).Group second, third and fourth received methanolic extract of Anogeissus latifolia (Roxb.ex DC.) wall. Gull perr leaves of dose 400mg/kg and 600mg/kg, orally as a suspension in 1%CMC solution respectively Group five received pentazocine (5mg/kg, p.o) as reference drug .60 mins after the administration of test and reference compounds, the animals in all the six groups were individually exposed to the plate maintained at 55c and observations were recorded for 3 hours. The time taken in seconds for fore paw licking or jumping was taken as reaction time. A cut off period of 15 seconds is observed to avoid damage to the paws. The percentage protection was calculated using the formula,Percentage protection = (T/C-1) -100 where, T is the reaction time of treated group and C the reaction time of control group.