The Ash Content Of A Crude Drug Biology Essay

The Ash Content Of A Crude Drug Biology EssayThe ash tree content of a crude drug is gener all(prenominal)y interpreted to be the equalizer remaining after incineration. It usually represents the inorganic salts naturally occurring in the drug and adhering to it, but it may also include inorganic liaison landed for the purpose of adulteration. There is a considerable difference varies within narrow limits in the case of the same individual drug. Hence an ash 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 formalized drugs. Usually these standards get a maximum limit on the total ash or on the acid non-water- disintegrable ash permitted.The total ash is the residue remaining after incineration. The acid in oil-soluble ash is the part of the total ash which is insoluble in diluted hydrochloric acid.The ash or residue yiel ded by an organic chemical compound is as a rule, a measure of the amount of inorganic matters present as impurity. In most cases, the inorganic matter is present in small amounts which atomic number 18 difficult to remove in the purification process and which are not objectionable if only traces are present. Ash set are helpful in determining the quality and purity of the crude drugs in pulverize form.Procedures given in Indian pharmacopoeia were utilize to determine the different ash value such as total ash and acid insoluble ash.Total ashWeighed accurately about 3 gm of distribute dried powdered drug was taken in a tarred silica crucible and incinerated by step sassy increasing the temperature to make it bleak red until free from carbon cooled and weighted and and then calculated the percentage of total ash with acknowledgment to the activate dried drug.Acid insoluble ashThe ash obtained as directed under total ash above was boiled with 25 ml of 2N HCl for 5 minutes. The insoluble matter was collected on ash less filter paper, washed with hot water ignited and weighed, then calculated the percentage of acid insoluble ash with reference to the line of descent 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 filter paper, washed with hot 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 percentage of water soluble ash calculated with reference to the air dried drug.b. EXTRACTIVE VALUESExtractive values of crude drugs are useful for their evaluation, especially when the constituents of a drug cannot be readily estimated by any other means. Further, these values indicate the nature of the constituents present in a crude drug.Determination of alcohol soluble reciteive value5 gm of the air-dried c oarse powder of Anogeissus latifolia wall (Roxb.ex.DC) was macerated with 100 ml of 90% ethyl alcohol in a closed flaskful for 24 hours, shaking frequently during the first 6 hours and allowing standing(a) for 18hours. Thereafter, it was filtered rapidly taking precautions against the loss of the solvent. Out of that filtrate, 25 ml of the filtrate was evaporated to dryness in a tarred flat bottomed shallow dish, dried at 105C and weighed. The percentage of ethanol soluble pullive value was calculated with reference to the air- dried drug. The results are recorded in the table.Determination of water soluble plagiarizeive valueWeigh accurately 5 gm of coarsely powdered drug and macerate it with 100 ml of chloroform 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 fla t bottomed shallow dish, dried at 105C and weighed. The percentage of water soluble conjure upive was calculated with reference to the air dried drug. The results are given 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 beneath. It determines the amount of volatile matter of any kind (including water) that can be driven off under the condition specified (Desiccators or hot air oven). If the sample is in the form of large crystals, then reduce the surface by quick crushing to a powder.Procedure somewhat 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 substance was calculated.d. FLUORESCENCE ANALYSIS Kokate.C.K, 2002 Khandelwal KR 1996.In the near-ultra region of the spectrum (3000-4000A) some of t he phytoconstituents show more or less brilliant coloration when exposed to radiation. This phenomenon of emitting indubitable wavelengths as a result of being excited by radiation of a different wavelength is known as fluorescence. Some periods the amount of ultra-violet light ordinarily present with visible light is sufficient to produce the fluorescence, but often a more powerful source of ultra-violet is required, e.g. mercury vapour lamp. It is often contingent 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 observed in daylight and UV light. Also the light study was performed on treating the drug powder with different chemical reagents. The observed results are given in table.e. FOAMING INDEX Divakar M.C., 1996Foaming index is primarily performed to determine the saponin content in an aqueous decoction of plant material.Determination of foamin g indexWeighed accurately about 1g of coarsely powdered drug and transformed to 500ml conical flask containing 100ml of boiling water. Maintained at moderate boiling at 80-90c for about 30min. Cooled and added sufficient water through the filter to make up the strength to 100ml (V1). Cleaned 10 stoppered seek tube of uniform dimension were taken and transferred the successive portions of 1,2,3ml up to 10ml and adjusted the volume of the liquid in each(prenominal) test tube with water to 10ml.Stoppered the tubes and shaken them in a lengthwise motion for 15 sec uniformly and allowed to stand for 15min and measure the efflorescence of foam. If the superlative of the foam in every tube is less than 1cm, the foaming index is less than 100(not significant). Here the foam was more than 1cm height after dilution of plant material. If the 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 calculated and transferred to 100ml volumetric flask (V2) and volume is make to 100ml and followed the same procedure.5.1. 2. PRELIMINARY PHYTOCHEMICAL ANALYSISExtraction of plant material-Petroleum ether extraction-About 400gm of dry coarse page number powder of the Anogeissus latifolia wall (Roxb.ex.DC) was extracted with petroleum ether 2500ml (40-600c) for 18 hrs by continuous hot percolation method. It was allowed to cool to 40oC and then filtered using whatman No.1 filter paper. The filtrate was then concentrated 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 days. At the end of insurgent day the powder was taken out and it was dried. After drying it was again packed and extracted by using methanol (Changshu yangyuan chemicals, China) as solvent, t ill colour disappeared. The temperature was maintained at 55C-65C. After that extract was concentrated by distillation and solvent was recovered. The final solution was evaporated to dryness. The colour, consistency and yield (% w/w) of methanolic extract were noted.S.No.Name of extractColourConsistencyYield% W/W12Methanolic extractPetroleum ether extractBlackish brownBlackish atomic number 19Non Sticky masssticky oily mass16.751.60Table 1. Nature and colour of extract of Anogeissu latifolia wall (Roxb.ex.DC).5.1. 3 CHEMICAL TESTSA) Test for carbohydrates1. Molisch Test It consists of treating the compounds of a-naphthol and concentrated sulfuric acid along the sides of the test tube.Purple colour or reddish violet colour was produced at the junction between two liquids. (Kokate, C.K et al, 2000)2. Fehlings Test tolerable quantity of Fehlings solution A and B is added. Heat gently, brick red sharp is obtained.3. Benedicts test To the 5ml of Benedicts reagent, add 8 drops of sol ution under examination. Mix well, boiling the diverseness vigorously for two minutes and then cool. Red precipitate is obtained.4. Barfoeds test To the 5ml of the Barfoeds solution 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 yellow Precipitate is produced.C) Test for Steroids and Sterols1. Liebermann Burchard test Dissolve 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 green in colour.2. Salkowski test Dis solve the sample of test solution in chloroform and add equal volume of conc. sulphuric acid. Bluish red cherry red and purple color is noted in chloroform layer, whereas acid assumes marked green fluorescence.D) Test for Glycosides1. Legals test Sample is turn in pyridine sodium 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 tree colour is produced.3. Borntrager test agree 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, pink, red or violet colour is produced in organic layer.4. Killer Killani test Sample is dissolved in acetic 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 Sapo ninsFoam test About 1ml of alcoholic sample is diluted separately with distilled water to 20ml, and shaken in graduated cylinder for 15 minutes.1 cm layer of foam indicates the presence 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 Phenolic CompoundsTo 2-3 ml of extract, add few drops of following reagentsa). 5% FeCl3 solution deep blue-black color.b). Lead acetate solution white precipitate.c). Gelatin solution white precipitated). atomic number 35 water decolouration 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 Fi xed Oils and Fatty acidsa). Spot test pure quantity of the extract is placed between two filter papers. Oil blur produced with any extract shows the presence of fixed oils and fats in the extracts.b). Saponification testFew drops of 0.5N alcoholic yard hydroxide are added to the extract with few drops of phenolphthalein solution. Later the mixture is heated 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 tag on 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 pres ence 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 EVALUATIONDetermination LD50 value of Anogeissus latifolia (Roxb.ex.DC).wall.GullperrAcute Oral Toxicity StudyThe procedure was followed by using OECD guidelines 423 (Acute toxic class method)AnimalsAdult albino rats (Wister strain) of either sex with advisement 150 180gm were used. The animals were maintained on the suitable nutritional and environmental condition passim the experiment. The animals were housed in polypropylene cages with paddy house bedding under standard research lab condition for an acclimatization periods of 7 days prior to performing the experiment. The animals had annoy to laboratory chow and water. The experimental protocols were approved by institutional Animal Ethical Committee a written permission from in house honest delegation has been taken to carry out (R eference no. JKKMMRF/2010/009) and gross(a) this study.ProcedureTwelve animals (Wister Albino rats, 150-200gm) were selected for studies. The exquisite toxic class method is a step wise procedure with 3 animals of single sex per step. Depending on the mortality and / or moribund status of the animals, on average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test animals while allowing for acceptable data based scientific conclusion.The method uses defined doses (5, 50, 300, 2000 mg / kg bole weight) and the results allow a substance to be ranked and classified according to the Globally Harmonized system (GHS) for the classification of chemical which cause acute toxicity.Most of the crude extracts possess LD50 value more than 2000 mg. /kg of the body weight of animal used.Dose 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 a fter administration, infringement 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 asphyxia was also to be noted, if any , was observed.ObservationNo toxicity or death was observed for these given dose levels, in selected and treated 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 EVALUATION5.2.1 paygrade 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 house d 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 induced ulcer- virile albino-Wistar rats were divide in to five throngs of half-dozen animals per group and animals were fasted for 24 hrs prior to the experiment in perforated steel cages to quash coprophagy. Six groups were made as below assort I animals served as usual 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 100 mg/kg, Sucralfate as standardOne hour after the drug give-and-take the animals were treated with absolute ethanol 5ml/kg to induce ulcers. The animals were sacrificed after 1hrs and bay window was opened and percentage prohibition era of ulcer was determined. (Mozafar khazaei et al., 2006, Paul V. et al 2002, Paul V. et al., 2000)Aspirin induced 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 avoid 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 sa crificed 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 gastric contents were collected in a test tube and centrifuged. The gastric contents were analyzed for gastric juice volume, pH, free and total acidity.5.2.4 Measurement of gastric juice volume and pH-stomachal 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 verbalised 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 pipe tte 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 indissoluble 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 = Volume of NaOH X practiceity of NaOH X 100 (mEq/L per 100g)0.015.2.6 Ulcer index (UI)-The mucous membrane 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 Analysis completely 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 semblance tests. The values are statistically significant at three levels, ***p 0.05.5.3. EVALUATION OF DIURETIC 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 had access to laboratory chow and water. The experimental protocol s 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 food and water for 18hour prior to the experiment. On the day of experiment, the group I animals serving 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. Immediately after the administration the animals were kept in metabolic cages (three per cage) specially designed to separate urine and fecal matter and kept at agency 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 food 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 flame 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 analysisAll the values are expressed as mean S.E.M for groups of s ix 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 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.ProceduresEddys hot plateful methodThe Male Albino-Wistar rats were divided into four groups of six rats in each as men tioned 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 thermal heat was recorded. The animals which showed forepaw licking or jumping rejoinder 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 were 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 s olution 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.