5.2 Collection and authentication of plant material

Transcription

1 5. MATERIALS AND METHODS 5.1 Selection of the plant The medicinal properties of plants have been investigated in the light of recent scientific developments throughout the world, due to their potent pharmacological activities, low toxicity and economic viability. As per the information collected from local vaidhyas and other traditional medicine practitioners, the present study is deals with the evaluatation of hepatoprotective activity of leaves of A. cordifolia, S. veronicaefolia and stem bark of N. arbortristis. 5.2 Collection and authentication of plant material The leaves and stem bark of the selected plant were collected from in and around the local area of village Ingoriya, Ujjain, Madhya Pradesh and were identified and authenticated by Dr. S. K. Billore, (Professor and Director) School of Plant and Environmental Management, Vikram University, Ujjain, Madhya Pradesh. The voucher specimen was deposited at the Department of Pharmacognosy, Mahakal Institue of Pharmaceutical Studies, Ujjain, Madhya Pradesh. (MIPS/N/012/2010) and (MIPS/S/005/2010) 5.3 Preparation of crude drug for extraction The selected plant leaves and stem bark were used for the preparation of the extract. The plants leaves and stem bark were collected and dried under shade and then coarsely powdered with the help of mechanical grinder. The powder Page 94 of 250

2 was passed through sieve No. 40 and stored in an airtight container for the extraction. (Farnsworth et al., 1966) 5.4 Physico-chemical evaluation The dried and stored powder of plant leaves were subjected to standard procedure for the determination of various physicochemical parameters A) Determination of ash values The determination of ash values is meant for detecting low grade products, exhausted drugs and sandy or earthy matter. It can also be utilized as a mean of detecting the chemical constituents by making use of water soluble ash and acid insoluble ash. (Khandelwal, 2004) a) Total ash value Accurately about 3 gm of air dried powder of plants leaves and stem separately were weighed in a tared silica crucible and incinerated at a temperature not exceeding C until free from carbon, cooled and weighed and then the percentage of total ash with reference to the air dried powdered drug was calculated. (Khandelwal, 2004) b) Acid insoluble ash The ashes obtained in the above method were boiled for 5 minutes with 25ml of dilute HCl. The residue was collected on ash less filter paper and washed with hot water, ignited and weighed. The percentage of acid insoluble ash was calculated with reference to the air dried drug. (Khandelwal, 2004) Page 95 of 250

3 c) Water soluble ash The ash obtained in total ash was boiled for 5 minutes with 25 ml of water. The insoluble matter was collected on an ash less filter paper, washed with hot water and ignited to constant weight at a low temperature. The weight of insoluble matter was subtracted from the weight of the ash. The difference in weights represents the water soluble ash. The percentage of water soluble ash with reference to the air dried drug was calculated. (Khandelwal, 2004) B) Determination of extractive values a) Procedure 5 gm of coarsely powdered air dried drug was macerated with 100 ml of solvent (petroleum ether, chloroform, acetone, ethanol and water) in a closed flask for 24 hour, shaking frequently for six hours and allowed to stand for eighteen hours. It was then filtered rapidly taking precaution against loss of alcohol. 25 ml of the filtrate was evaporated to dryness in tared flat bottomed shallow dish, dried at C and weighed. The percentage of alcohol soluble extractive was calculated with reference to the air dried drug. (Khandelwal, 2004) 5.5 Extraction of dried leaves by using various solvents of increasing polarity The collected, cleaned and powdered leaves of A. cordifolia, S. veronicaefolia and stem bark N. arbortristis were used for the extraction purpose. 500 gm of powdered material were evenly packed in the soxhlet apparatus. It were then extracted with various solvents from non polar to polar such as Page 96 of 250

4 petroleum ether, chloroform, acetone and ethanol. The solvents used were purified before use. The extraction method used was continuous hot percolation and carried out with various solvents, for 72 hrs. The aqueous extraction was carried out by cold maceration process. The extracts were concentrated by vacuum distillation to reduce the volume to 1/10; the concentrated extracts were transferred to 100 ml beaker and the remaining solvent were evaporated on a water bath. Then they were cooled and placed in a dessicator to remove the excessive moisture. The dried extracts were packed in airtight containers and used for further studies. (Kokate et al., 2008) 5.6 Preliminary phytochemical studies (Gothoskar et al., 1971; Kokate et al., 2008; Khandelwal, 2004) A) Tests for carbohydrates and glycosides A small quantity of the extracts was dissolved separately in 4 ml of distilled water and filtered. The filtrate was subjected to various tests to detect the presence of Carbohydrates. i) Molisch s test Filtrate was treated with 2-3 drops of 1% alcoholic α- napthol solution and 2 ml of Con sulpuric acid was added along the sides of the test tube. Appearance of brown ring at the junction of two liquids shows the presence of carbohydrates. Another portion of the extract was hydrolysed with hydrochloric acid for few hours on a water bath and the hydrolysate was subjected to Legal s and Borntrager s test to detect the presence of different glycosides. Page 97 of 250

5 ii) Legal s test To the hydrolysate 1 ml of pyridine and few drops of sodium nitropruside solutions were added and then it was made alkaline with sodiumhydroxide solution. Appearance of pink to red colour shows the presence of glycosides. iii) Borntrager s test Hydrolysate was treated with chloroform and then the chloroform layer was separated. To this equal quantity of dilute ammonia solution was added. Ammonia layer acquires pink color, showing the presence of glycosides. B) Test for alkaloids A small potion of the solvent free alcoholic and aqueous extracts were stirred separately with few drops of dilute hydrochloric acid and filtered. The filtrate was tested with various reagents for the presence of alkaloids. i) Dragondorff s test To a small amount of the filtrate, add 1ml of Dragendorff s reagent. Appearance of reddish brown precipitate indicates the presence of alkaloids. ii) Wagner s test To a small amount of filtrate, add 1ml of Wagner s reagent. Appearance of reddish brown precipitate indicates the presence of alkaloids. iii) Mayer s reagent To a small amount of filtrate, add 1ml of Mayer s reagent. Appearance of cream coloured precipitate indicates the presence of alkaloids. Page 98 of 250

6 C) Test for proteins and free amino acids i) Million s test Small quantities of the extracts were dissolved in small quantity of water and treated with Millon s reagent. Appearance of red color shows the presence of proteins and free amino acids. ii) Ninhydrin test Small quantities of the extracts were dissolved in small quantity of water and treated with Ninhydrin reagent. Appearance of violet color shows the presence of proteins and free amino acid. iii) Biuret s test The extracts were dissolved in a small quantity of water and equal volumes of 5% sodium hydroxide solution and 1% copper sulphate solution were added. Appearance of pink or purple color shows the presence of proteins and amino acids. D) Test for phenolic compounds and tannins i)ferric chloride test Small quantities of the extracts were dissolved in water and dilute Ferric chloride solution (5%) was added. Appearance of violet or blue color indicates presence of phenolic compounds and tannins. Page 99 of 250

7 ii) Gelatin test Small quantities of the extracts were dissolved in water and 1% solution of gelatin containing 10% sodium chloride was added. Formation of white precipitate indicates presence of phenolic compounds and tannins. iii) Lead acetate test Small quantities of the extracts were dissolved in water and 10% lead acetate solution was added. Formation of white precipitate indicates presence of phenolic compounds and tannins. E) Test for flavonoids i) Sodium hydroxide test Small quantities of each extracts were dissolved separately in aqueous sodium hydroxide solution. Appearance of yellow to orange indicates presence of flavonoids. ii) Sulphuric acid test To a portion of the extract, add concentreted sulphuric acid. Appearance of yellow orange colour shows the presence of flavonoids. iii) Shinoda s test Small quantities of the extract were dissolved in alcohol, to them piece of magnesium followed by concentreted hydrochloric acid dropwise added and heated. Appearance of magenta color shows the presence of flavonoids Page 100 of 250

8 F) Test for saponins i) Foam Test Place 2ml of the solution of the extract in water in a test tube and shake well. Formation of stable foam (froth) indicates the presence of saponins. G) Test for fixed oils and fats i) Spot test Small quantities of various extracts were separately pressed between two filter papers. Appearance of oil stain on the paper indicates the presence of fixed oils and fats. ii) Saponification test Add few drops of 0.5N alcoholic potassium hydroxide to a small quantity of the extract and heat on a water bath for 1 2 hrs. Formation of soap or partial neutralization of tha alkali shows the presence of fixed oils and fats. H) Test for phytosterols Small quantities of various extracts were dissolved separately in 5ml of water. Then this solution was subjected to the following tests. i) Salkowski test The solution was treated with few drops of concentrated sulphuric acid. Formation of red colour indicates presence of phytosterols. Page 101 of 250

9 ii) Libermann Bucchard s test The solution was treated with few drops of acetic anhydride, boil and cool. Then add conc. Sulphuric acid through the sides of the test tube. Formation of brown ring at the junction of two layers indicates the presence of phytosterols. I) Test for Gums and Mucilage i) Alcohol test A little of the extract is treated with alcohol. If it is not soluble in alcohol it shows presence of gums and mucilage. ii) Precipitation test The extract solution was added to picric acid solution. Formation of yellow precipitate shows the presence of gums and mucilage. 5.7 Pharmacological studies Acute toxicity studies Organization for economic co operation and development (OECD) regulates guideline for oral acute toxicity study. It is an international organization which works with the aim of reducing both the number of animals and the level of pain associated with acute toxicity testing (OECD, 1996) Following are the main type of guideline followed by OECD Guideline 420, fixed dose procedure. ( 5 animals used ) Guideline 423, acute toxic class. ( 3 animals used ) Page 102 of 250

10 Guideline 425, up and. down method. (1 animal used) i) Guideline 423 a) Principle Acute toxic category method is a method for assessing acute oral toxicity that involves the identification of a dose level that causes mortality. This test involves the administration of a simple bolus dose of test substances to fasten healthy young adult rodents by oral gavage, observation for upto 15 days after dosing and recording of body weight and the necropsy of all the animals. In this method pre specified fixed doses of the test substances were used i.e., 5 mg/kg, 50 mg/kg, 300 mg/kg, 2000 mg/kg and 5000 mg/kg. the mortality due to these doses were observed. Generally female animals were used for this study and each dose group should consist of 3 animals. b) Animals Female Wistar albino rats ( g) of approximately the same age, were procured from central drug research institute, Lucknow, and were used for acute toxicity studies. They were housed in polypropylene cages and fed with standard rodent pellet diet (Hindustan Lever Limited, Bangalore) and water ad libitum. The rats were exposed to alternate cycle of 12 hrs of darkness and light each. Before the test, the rats were fasted for at least 12 hrs; the experimental protocols were subjected to the scrutinization of the Institutional animal ethical committee and were cleared by the same. All experiments were performed during morning according to CPCSEA guidelines (CPCSEA, 2003) for care of laboratory animals and the ethical guideline for investigations of experimental pain in Page 103 of 250

11 conscious animals. The standard orogastric cannula was used for oral drug administration in rats. Figure 5.1: Protocol for determining acute toxicity in rats c) Procedure The overnight fasted female rats were weighed and selected. Acetone, ethanol and aqueous extracts were dosed in a stepwise procedure, with the initial dose being selected as the dose expected to produce some signs of toxicity and were observed for a period of two weeks. The toxic doses were selected based on the above chat. Page 104 of 250

12 5.8 Hepatoprotective studies Carbon tetrachloride (CCl 4 ) induced hepatotoxicity a) Principle The drug is metabolized in endoplasmic reticulum and mitochondria with the formation of CCl 3 O -, the reactive oxidative free radical intermediate generated by cytochrome P450, the nascent oxygen O - resulted via lipoperoxidation causes rise in intracellular reactive Fe +2 ions, aldehyde and depletion GSH, and calcium sequestration. Oxidative CCl 3 O -, also by direct covalent interaction induces degeneration of Ca +2 sequestrations. Failure into sequestration results in increased intercellular Ca +2, aggregation by proteolytic enzymes and causes an increase in Fe +2 ions, which in turn by lipid peroxidation precipitates aldehyde cytotoxicity. (Zimmerman,1976) CCl 4 CCl 3 O - + O - b) Test compounds The acetone, ethanol and aqueous extracts of leaves of A. cordifolia, S. veronicaefolia and stem bark N. arbortristis and standard drug silymarin (25 mg/kg, bw) were used. c) Chemicals and reagents Carbon tetrachloride, olive oil and silymarin. Page 105 of 250

13 d) Experimental design Rats of either sex were divided into nine groups of six animals in each group. (n = 6) (Sangameswaran et al., 2008; Balakrishnan et al., 2011) Group I: Received water (5 ml/kg, p.o.) for 9 days once daily, and served as normal control. Group II: Received water (5 ml/kg, p.o.) for 9 days once daily and carbon tetrachloride (1 ml/kg in 50% v/v olive oil, s.c.) on the 7 th day. Group III: Received standard drug silymarin (25 mg/kg, p.o.) for 9 days once daily and carbon tetrachloride (1 ml/kg in 50% v/v olive oil, s.c.) on the 7 th day. Groups IV, V, VI, VII, VIII and IX: Received all extract (500 mg/kg) for 9 days once daily and carbon tetrachloride (1 ml/kg in 50% v/v olive oil, s.c.) on the 7 th day. On the last day, functional parameters i.e., onset of sleep and duration of sleep, morphological parameters i.e., liver weight and liver volume, serum marker enzyme parameters i.e., Serum glutamic pyruvate transaminase (SGPT) (Reitman and Frankel, 1957), Serum glutamic oxaloacetic transaminase (SGOT) (Reitman and Frankel, 1957) and biochemical parameters Alkaline phosphatase (ALP) (Kind and King, 1954) and biochemical parameters i.e. Total bilirubin (Amour et al.,1965) and Total protein (Lowry et al., 1951) were analyzed according to the reported methods. Page 106 of 250

14 5.8.2 Paracetamol induced hepatotoxicity The liver damage associated with paracetamol overdose is due to the formation of a hepatotoxic metabolite. Therapeutic doses of paracetamol are metabolized mostly to sulphate and glucuronide conjugates. The rest is metabolized to a reactive intermediate which is detoxified by conjugation with glutathione. In overdose, the sulphate and glucuronide conjugation pathways are saturated and more drugs is converted to the reactive metabolite. Liver damage can be prevented by providing glutathione like substances, such as acetylcysteine, so that the reactive metabolite can be removed by conjugation and the liver cells are protected. (Grahame smith., 1984) a) Test compounds The acetone, ethanol and aqueous extracts of leaves of A. cordifolia, S. veronicaefolia, stem bark N. arbortristis and standard drug silymarin (25 mg/kg bw p.o.) were used. b) Chemicals and reagents Paracetamol and silymarin. c) Experimental design Rats of either sex were divided into nine groups of six animals in each group. (n = 6) (Haque et al., 2011; Balakrishnan et al., 2011) Group I: Received water (5 ml/kg, p.o.) for 9 days once daily, and served as normal control. Page 107 of 250

15 Group II: Received water (5 ml/kg, p.o.) for 9 days once daily and paracetamol (1 g/kg, p.o.) on the 7 th day. Group III: Received standard drug silymarin (25 mg/kg, p.o.) for 9 days once daily and paracetamol (1 g/kg, p.o.) on the 7th day. Groups IV, V, VI, VII, VIII and IX: Received all extract (500 mg/kg) for 9 days once daily and paracetamol (1 g/kg, p.o.) on the 7th day. On the last day, functional parameters i.e. onset of sleep and duration of sleep, morphological parameters i.e. liver weight and liver volume, serum marker enzyme parameters i.e. Serum glutamic Pyruvate transaminase (SGPT), (Reitman and Frankel, 1957) Serum Glutamic Oxaloacetic Transaminase (SGOT) (Reitman and Frankel, 1957) and Alkaline phosphatase (ALP), (Kind and King, 1954) biochemical parameters i.e. Total bilirubin (Amour et al.,1965) and Total protein (Lowry et al., 1951) were analyzed according to the reported methods Ethanol induced hepatotoxicity Ethanol produces constellation of dose related deleterious effects in the liver. The primary effects are fatty infiltration of the liver, hepatitis, and cirrhosis. Because of its intrinsic toxicity, alcohol can injure the liver in the absence of dietary deficiencies. The accumulation of fat in the liver is an early event and can occur in normal individuals after the ingestion of relatively small amounts of ethanol. This accumulation results from inhibition of both the tricarboxylic acid cycle and oxidation of fat, in part owing to the generation of excess NADH produced by the actions of alcohol dehydrogenase and aldehyde dehydrogenase. Fibrosis, resulting from tissue necrosis and chronic inflammation, is the Page 108 of 250

16 underlying cause of alcoholic cirrhosis. Normal liver tissue is replaced by fibrous tissue. Alcohol can affect directly stellate cells in the liver, causing deposition of collagen around terminal hepatic venules. Chronic alcohol use is associated with transformation of stellate cells into collagen roducing myofibroblast like cells. The histologic hallmark of alcoholic cirrhosis is the formation of Mallory bodies, which are thought to be related to an altered cytokeratin intermediate cytoskeleton. A number of underlying molecular mechanisms have been proposed. (Hardman, 2001) a) Test compounds The acetone, ethanol and aqueous extracts of leaves of A.cordifolia, S. veronicaefolia and stem bark N.arbortristis and standard drug silymarin (25 mg/kg bw/ p.o.) were used. b) Chemicals and reagents Ethyl alcohol and Silymarin. c) Experimental design Rats of either sex were divided into nine groups of six animals (n = 6) in each group. (kapur et al., 1994; Balakrishnan et al., 2011) Group I: Received water (5 ml/kg, p.o.) for 21 days once daily, and served as normal control. Group II: Received water (5 ml/kg, p.o.) for 21 days once daily and 40% ethanol (v/v, 2 ml/l00 g bw, p.o.) for 21 days. Page 109 of 250

17 Group III: Received standard drug silymarin (25 mg/kg, p.o.) for 21 days once daily and 40% ethanol (v/v, 2 ml/l00 g bw, p.o.) for 21 days. Groups IV, V, VI, VII, VIII and IX: Received all extracts (500 mg/kg) 21 days once daily and 40% ethanol (v/v, 2 ml/l00 g bw, p.o.) for 21 days. On the last day, functional parameters i.e. onset of sleep and duration of sleep, morphological parameters i.e. liver weight and liver volume, serum marker enzyme parameters i.e. Serum glutamic Pyruvate transaminase (SGPT), (Reitman and Frankel, 1957) Serum Glutamic Oxaloacetic Transaminase (SGOT) (Reitman and Frankel, 1957) and Alkaline phosphatase (ALP), (Kind and King, 1954) biochemical parameters i.e. Total bilirubin (Amour et al.,1965) and Total protein (Lowry et al., 1951) were analyzed according to the reported methods Rifampicin induced hepatotoxicity Rifampicin is a major drug used for treatment of tuberculosis, but its chronic use is known to cause hepatotoxicity. The mechanism of hepatotoxicity induced by rifampicin is that it competes with bilirubin for transport across the liver cell and conjugated or unconjugated hyperbilirubinaemia can often occur in chronic hepatitis induced by rifampicin. (Gond, 2008) a) Test compounds The acetone, ethanol and aqueous extracts of leaves of A. cordifolia, S. veronicaefolia, stem bark N. arbortristis linn. and standard drug silymarin (25 mg/kg bw p.o.) were used. Page 110 of 250

18 b) Chemicals and reagents Rifampicin (RIF) + Isoniazid (INH) and silymarin. RIF and INH (100mg/kg) solution were prepared separately in sterile distilled water and were administered by i.p. route for 21 days. (Balakrishnan et al., 2010) c) Experimental design The rats were divided into nine groups of 6 animals in each. Group I: Received vehicle water (5ml/kg/p.o) for 21days once daily, and served as normal control. GroupII: Received vehicle water (5ml/kg/p.o) for 21days once daily, and RIF+INH (100mg/kg/i.p.) for 21 days once daily. GroupIII: Received RIF+INH (100mg/kg/i.p.) for 21 days once daily and silymarin for 21 days (25 mg/kg/p.o) once daily. Group IV, V, VI, VII, VIII and IX: : Received RIF+INH (100mg/kg/ i.p.) for 21 days once daily and all extract of selected plant (500mg/kg/p.o), for 21 days once daily. On the last day, functional parameters i.e. onset of sleep and duration of sleep, morphological parameters i.e. liver weight and liver volume, serum marker enzyme parameters i.e. Serum glutamic Pyruvate transaminase (SGPT), (Reitman and Frankel, 1957) Serum Glutamic Oxaloacetic Transaminase (SGOT) (Reitman and Frankel, 1957) and Alkaline phosphatase (ALP), (Kind and King, 1954) biochemical parameters i.e. Total bilirubin (Amour et al.,1965) and Total protein (Lowry et al., 1951) were analyzed according to the Page 111 of 250

19 reported methods. 5.9 Assessment of hepatoprotective effect of selected plant extracts Functional parameters On the last day, thiopentone sodium (40 mg/kg, i.p) was injected and the sleeping time recorded in all the animals have also been used to evaluate the protective effect of drug Physical parameters i) Determination of liver weight Animals were sacrificed and liver were isolated and washed with saline and weights determined by using an electronic balance. The liver weights were expressed with respect to its body weight i.e., gm. ii) Determination of liver volume After recording the weight all the livers were dropped individual in a measuring cylinder containing a fixed volume of distilled water or saline and the volume displaced was recorded. (Ghosh et al., 2007) Biochemical parameters i) Effect of selected plant extracts on serum glutamate pyruvate transamainse (SGPT) a) Intended Use This reagent kit was intended for in vitro quantitative determination of SGPT (ALT) activity in serum/plasma. Page 112 of 250

20 b) Principle SGPT (ALT) catalyzes the transfer of the amino group from L alanine to alpha ketoglutarate to yield pyruvate and L glutamate. Lactate dehydrogenase then converts pyruvate and NADH into lactate and NAD. The conversion on NADH to NAD decreases the absorption at 340 nm. The rate of decrease in absorbance is measured and is proportional to the SGPT activity. GPT (ALT) L alanine + α ketoglutarate L Glutamate + pyruvate LDH Pyruvate + NADH + H + Lactate + NAD + c) Clinical significance Elevation of SGPT (ALT) activity is found in liver and kidney diseases such as infectious or toxic hepatitis, infectious mononucleosis and cirrhosis. Moderate increase is also found in obstructive jaundice, metastatic carcinoma, hepatic congestion and myocardial infarction. SGPT levels may be decreased in patients undergoing long term hemodialysis without supplemental vitamin therapy. d) Sample Fresh, fasting, unhaemolysed clear serum was used as sample. Page 113 of 250

21 e) Reagent composition Reagent 1: Buffer Tris Buffer (ph = 7.5) : 100 mmol/l L Alanine : 500 mmol/l LD : 1200 U/L Reagent 2: substrate α ketoglutarate : 15 mmol/l NADH : 0.18 mmol/l f) Working reagent preparation reagent 1. Add reagent 2 to reagent 1 in 1:4 ratio i.e. 1ml of reagent 2 + 4ml of g) Assay procedure The working reagent was allowed to attain 370 C before performing the test. 1ml of working reagent was mixed with 100 µl of test solution and the absorbance was recorded. Calculation: General formula for converting absorbance change into international units (IU) ofactivity is ALT activity (IU/L) = ( A/min) / kinetic factor Where: A/minute = change in the absorbance per minute, Kinetic factor = 1768 Page 114 of 250

22 i) Normal values SGOT (AST) : 7 21 U/L SGPT (ALT) : 6 21 U/L ii) Effect of selected plant extracts on serum glutamate oxalacetate transminase (SGOT) a) Intended Use This reagent kit was intended for in vitro quantitative determination of SGOT (AST) activity in serum/plasma. b) Principle SGOT (AST) catalyzes the transfer of the amino group from L asparate to alpha ketoglutarate to yield oxaloacetate and L glutamate. Malate dehydrogenase (MDH), then converts oxaloacetate and NADH to malate and NAD. The conversion of NADH to NAD decreases the absorbance at 340 nm, the rate of which is proportional to the SGOT activity. GOT (AST) L Aspartate + α ketoglutarate Oxalacetate+L Glutamate MDH Oxaloacetate + NADH + H + L Malate + NAD + Page 115 of 250

23 c) Clinical significance Organs rich in SGOT (AST) are heart, liver and skeletal muscles. When any of these organs are damaged, the serum GOT level rises in proportion to the severity of damage. In myocardial infarction, SGOT starts increasing by 3 9 hours, peaks on second day returns to normal on 4 th 6 th day. In hepatitis, SGOT peaks usually between 7 12 days and any increase upto 100 times. Increased levels SGOT are also found in mononucleosis, pancreatitis, and trauma of skeletal muscle, renal necrosis and cerebral necrosis. Transaminase is a process in which an amino group is transferred from an amino acid to an alpha ketoacid. It is an important step in the metabolism of amino acids. The enzymes responsible for transamination are called transaminases (now called, amino transferases). Two diagnostically useful transaminases are glutamate oxalacetate transaminase or SGOT and glutamate pyruvate transaminase or SGPT. These enzymes catalyze the following reactions: GOT/AST L aspartate + Oxoglutarate Oxalacetate + Glutamate (or keroglutarate) GPT/ALT L alkaline + Oxoglutarate Pyruvate + Glutamate (or ketoglutarate) Increased serum transaminase activity is seen in liver dysfunction. Greater activity of SGOT (AST) over SGPT (ALT) is typical of myocardial infarction. Page 116 of 250

24 d) Reagent composition: Reagent 1: Buffer Tris Buffer (ph = 7.8) : 80 mmol/l L Aspartate : 240 mmol/l MDH : 600 U/L LD ; 600 U/L Reagent 2: Substrate α ketoglutarate : 12 mmol/l NADH : 0.18 mmol/l e) Working reagent preparation Add reagent 2 to reagent 1 in 1:4 ratio i.e. 1ml of reagent 2 + 4ml of reagent 1. f) Assay procedure The working reagent was allowed to attain 37 0 C before performing the test. 1 ml of workingreagent was mixed with 100 µl of test solution and the absorbance was recorded. Calculation General formula for converting absorbance change into international units (IU) of activity is AST activity (IU/L) = ( A/min) / kinetic factor Where: A/minute = change in the absorbance per minute, Kinetic factor = 1768 Page 117 of 250

25 iii) Effect of selected plant extracts on alkaline phosphatase (ALP) Phosphatases belong to the class of enzymes called hydrolases and they are characterized by their ability to hydrolyse a large variety of organic phosphate with the formation of an alcohol and phosphate ions. Phosphatases of diagnostic significance are alkaline phosphatase and acid phosphatase. These are differentiated by their reaction in alkaline and acidic medium. The ph for measuring the alkaline phosphatase activity is 10 and for acid phosphatase it is 5. a) Principle The substrate, p nitrophenyl phosphate (PNPP) is hydrolysed by ALP to p nitrophenol and phosphoric acid. Some divalent ions like Mg ++ are added to the system which acts as activators. PNPP is colourless in acid or alkaline medium while PNP is yellow in colour in the alkaline medium and colourless in the acid medium. ALP/ACP P Nitrophenyl phosphate + H 2 O p Nitrophenol+ H 3 PO 4 (colorless) (yellow) b) Clinical significance Increased alkaline phosphatase activity may be related to hepatobiliary and bone diseases. Very high alkaline phosphatase activity in serum is seen in patient with bone cancer and marked increase also occurs in obstructive jaundice Page 118 of 250

26 and biliary cirrhosis. Moderate elevations have been noted in case of Hodgkin s disease, congestive heart failure, infective hepatitis and abdominal problems. c) Reagent composition Reagent 1: ALP buffer. AMP : 300 mm Magnesium acetate : 2 mm Zinc sulphate : 0.8 mm Chelators : qs Reagent 2: ALP substrate pnpp : 10 mm stabilizer : qs d) Assay procedure The working reagent was allowed to attain 37 0 C before performing the test. 1ml of working reagent was mixed with 20µl of test solution and the absorbance was recorded. Calculation General formula for converting absorbance change into international units (IU) of activity is ALP activity (IU/L) = A/min / kinetic factor Where: A/minute = change in the absorbance per minute, Kinetic factor = 2712 Page 119 of 250

27 f) Normal value Alkaline Phosphatase (ALP): 20 to 100 U/L. The biochemical parameters were estimated as per the standard procedure prescribed by the manufacturer s instruction manual provided in the kit. (Span and Auto span, India) using Semi Autoanalyser (ARTOS). iv) Estimation of serum bilirubin Bilirubin in serum would only react with diazo regent in the presence of alcohol, after the proteins had been removed by precipitation. Addition of alcohol to the reaction gives positive test for both conjugated and unconjugated bilirubin pigment. The unconjugated bilirubin level is then estimated by subtracting direct bilirubin value from this total value. Normally, 0.25 mg/dl of conjugated bilirubin is present in the blood of an adult. Bilirubin level rises in diseases of hepatocytes, obstruction to biliary excretion into duodenum, in hemolysis and defects of hepatic uptake and conjugation of bilirubin treatment such as Gilbert s disease. a) Intended Use This reagent kit was intended for in vitro quantitative determination of direct and indirect bilirubin from serum/plasma. b) Methodology Jendrassik and grof. Page 120 of 250

28 c) Principle Bilirubin reacts with diazotized sulphanilic acid in acidic medium to form pink colored azobilirubin with absorbance directly proportional to bilirubin concentration. Direct bilirubin, being water soluble directly reacts in acidic medium. However indirect or unconjugated bilirubin is solubilised using a surfactant and then it reacts similar to direct bilirubin.then absorbance was taken at 546 to 630 nm against reagent blank. d) Calculation Total Bilirubin (mg/dl) = Absorbance of test for total bilirubin/absorbance of sample blank for total bilirubin x factor V) Serum protein Liver cells synthesise albumin, fibrinogen, prothrombin, alpha 1 antitrypsin, haeptoglobin, ceruloplasin, transferrin, alpha foetoproteins and acute phase reactant proteins. The blood levels of these plasma proteins are decreased in extensive liver damage. A routinely estimated total protein is in the normal range of 5.5 to 8 gm/dl. Hypoalbuminanemia may occur in liver diseases having significant destruction of hepatocytes. Hyper globulinaemia may be present in chronic inflammatory disorders such as in cirrhosis and chronic hepatitis. (Harshamohan, 2002) Page 121 of 250

29 a) Intended Use This reagent kit was intended for in vitro quantitative determination of total protein from serum/plasma. b) Clinical significance Total protein is useful for monitoring changes in protein levels caused by various disease states, including in chronic liver disease. c) Principle The peptide bonds of protein react with cupric ions in alkaline solution to form coloured chelate, (Modified biuret reaction). The absorbance of which is measured at 578 nm. The biuret reagent contains sodium potassium tartrate, which helps in maintaining the solubility of thethis complex at alkaline ph. The absorbance of the final colour is proportional to the concentration of total protein in the sample. Calculation Total Protein (g/dl) = Absorbance of test / absorbance standard x Histopathological studies (Avadhoot, 1991; Bhanwra, 2000; Mankani, 2005) The animals were sacrificed and the liver of each animal was isolated and was cut into small pieces, preserved and fixed in 10% formalin for two days. Then the liver piece was washed in running water for about 12 hours to remove the formalin and was followed by dehydration with isopropyl alcohol of increasing Page 122 of 250

30 strength (70%, 80% and 90%) for 12 hours each. Then finally dehydration is done using absolute alcohol with about three changes for 12 hours each. Dehydration was performed to remove all traces of water. Further alcohol was removed by using chloroform and chloroform removed by paraffin infiltration. The clearing was done by using chloroform with two changes for 15 to 20 minutes each. After paraffin infiltration the liver pieces were subjected to automatic tissue processing unit. Embedding in paraffin vaccum hard paraffin was melted and the hot paraffin was poured into L shaped blocks. The liver pieces were then dropped into the molten paraffin quickly and allow to cool. Sectioning the blocks were cut using microtome to get sections of thickness of 5 microne. The sections were taken on a micro slide on which egg albumin i.e., sticking substance 5.11 Statistical analysis The results of the study were expressed as mean ± SEM. ANOVA (Gennaro et al., 1995) was used to analyze and compare the data, followed by Dunnet s (Dunnet et al., 1964) test for multiple comparisons.the value of probability less than 5 % (P < 0.05) was considered statistically significant. (Amin et al., 2006) Page 123 of 250