Biochemical studies on galactopietic effect of on crossbred dairy animals

CHAPTER-I
3.0.0. MATERIALS AND METHODS 3.1.0. Place of study: The study was carried out in the Department of Biochemistry, Orissa Veterinary College, OUAT, Bhubaneswar in collaboration with Intas Pharmaceuticals, Ashram Road, Ahmedabad, LBD farm, Khapuria, Cuttack and some private dairy farms in and around Bhubaneswar. 3.1.1. Chemicals: The chemicals used were from SISCO, Qualigen, SRL, Himedia of AR and GR grade and the analytical reagent kits were from Accurex biomedical Pvt. Ltd. and Crest Biosystems, A Division of Clinical Systems But the solvent methanol was of HPLC grade. The glassware and disposable plastic wares used were obtained of Borosil/ Vensil (India) and Tarson (India) respectively. 3.1.2. Instruments: UV-spectrophotometer (Perkin-Elmer), Microwave Digestion System-3000 (Anton Par), Rotary Evaporator (YAMATO), REMI centrifuge, Lyophilizer (CHRIST ALPHA 1-2), UV-Vis Spectrophotometer (Perkin Elmer) and other accessory instruments of the Department and Central Laboratory of University. 3.2.0. Samples: 3.2.1. Plant materials: The plant Asparagus racemosus and its roots were collected from Medicinal Nursery, Patrapada, Bhubaneswar and was identified and classified by the Professor, Department of Botany, OUAT, Bhubaneswar following the description of Saxena and Brahman (1995). The fresh roots were washed properly in tap water and then rinsed in distilled water and dried under shade for 15-20 days. The dried roots were pulverized using a sterile electric blender to obtain a powered form and it was stored in an airtight glass container for preparation of extracts in the study. But the root powder of Leptadenia reiculata was directly procured from Intas Pharmaceuticals Ltd. and was shade dried before use for extract preparation. DP Tripathy, 2011 1

Biochemical studies on galactopietic effect of on crossbred dairy animals

3.2.2. Plant extracts: The root extract was prepared by Multi-wave 3000 (Anton Par) digestion system following the method of (Eskilsson et al., 2000).by taking 2 gm of powdered material and 20 ml of methanol in each vessel. The process of extraction continued for 25 minutes at 800 C temperatures followed by cooling for 15 min. The extracts were filtered through Whatman No.1 filter and exposed to a temperature of 500 C at 200 hpa pressure in rotary evaporator to obtain a concentrated extract to be used for in vitro study. 3.2.3. Commercial galactogogues: Ostovet Forte manufactured by Virbac Animal Health Pvt. Ltd., Mumbai and Calshakti Platina of Intas Pharmaceuticals Ltd., Ahmedabad was supplied by the sponsoror of the project. 3.3.0. In vitro study: The concentrated plant extract was subjected to detect and quantify the concentration of total poly-phenols. 3.3.1. Qualitative detection of polyphenolic constituents: The root extracts were exposed to different chemical tests to know the presence of various polyphenolic compounds before going to quantify them.. (i) Test for tannins FeCl3 test: 0.5 g of powdered sample of each plant was boiled in 20 ml of distilled water in a test tube and then filtered. The filtration was done by a conical flask and filter paper. 0.1% FeCl3 was added to the filtered samples and observed for brownish green or a blue black colouration, which showed the presence of tannins. (ii) Test for flavonoids Shinodas test: A few drops of 1% NH3 solution was added to the aqueous extract of each plant sample in a test tube. A yellow coloration was observed in presence of flavonoid. DP Tripathy, 2011 2

Biochemical studies on galactopietic effect of on crossbred dairy animals

(iii) Test for cardiac glycosides Killer-killanis test: 1 ml of concentrated H2SO4 was prepared in a test tube. 5 ml of aqueous extract from each plant sample was mixed with 2 ml of glacial CH3COOH containing 1 drop of FeCl3.The above mixture was carefully added to the 1 ml of concentrated H2SO4 so that the concentrated H2SO4 was underneath the mixture. Appearance of a brown ring indicated the presence of cardiac glycoside. (iv) Test for saponins Frothing test: 2 g of powdered samples of each plant was boiled together with 20 ml of distilled water in a water bath and filtered.10 ml of the filtered sample was mixed with 5 ml of distilled water in a test tube and shaked vigorously to obtain a stable persistent froth. The frothing was then mixed with 3 drops of olive oil and observed for the formation of emulsion, which indicated the presence of saponins. (v) Test for steroids: Libarman-Burchards test: One ml of the extract was dissolved in 10 ml of chloroform and equal volume of conc. sulphuric acid by adding through side of the test tube. The red upper layer turns and yellow sulphuric acid layer with green fluorescence indicated the presense of steroids. (vi) Tests for phytosterol: The extract was refluxed with solution of alcoholic potassium hydroxide till complete saponification and was diluted and extracted with ether. The ether layer was tested for the presence of phytosterol following after evaporation of the residue. Tha residue was dissolved in few drops of dilute acetic acid and 3 ml of acetic anhydride was added followed by few drops of conc. H2SO4. Appearance of bluishgreen colour showed the presence of phytosterol. (vii) Test for alkaloids: Dragendorff s test was conducted for detection of alkaloids. (viii) Test for Carbohydrates: Molischs test, Barfoeds test and Fehling (reducing sugar) test were conducted (ix) Proteins and Amino acids: Millons, Biuret and Ninhydrin tsts were conducted. 3.3.2. Determination of total phenolics: DP Tripathy, 2011 3

Biochemical studies on galactopietic effect of on crossbred dairy animals

Total phenolics in the root extracts were determined by the method of Singh et al., (2002) and results were expressed as Gallic acid equivalents.

Reagents: 1. 1:10 F. C reagent: 1 ml F. C. reagent was mixed in 9 ml of distilled water. 2. 7.5 % NaCO3: 7.5 gm of NaCO3 dissolved in 100 ml of distilled water. . 3. Gallic acid: 10 mg in 10 ml methanol: water (6:4) Procedure: Samples (0.2 ml) were mixed with 1.0 ml of 10-fold diluted Folin Ciocalteu reagent (F.C Reagent) and 0.8 ml of 7.5% sodium carbonate solution. After standing for 30 min at room temperature the absorbance was measured at 765 nm using a Perkin-Elmer UV-visible spectrophotometer. The estimation of phenolic compounds in the extracts was carried out in triplicate. 0.2 ml of methanol: water (6:4) was taken in blank in place of plant sample. 3.3.3. Determination of flavonoids: The colorimetric method of Chang et al., (2002) was followed for flavonoid determination by using aluminium chloride. The methanolic root extracts (0.5 ml) was separately mixed with 1.5 ml of methanol, 0.1 ml of 10% aluminium chloride, 0.1 ml of 1M potassium acetate and 2.8 ml of distilled water. It was kept at room temperature for 30 minutes; the absorbance of the reaction mixture was measured at 415 nm with a double beam Perkin Elmer UV / Visible spectrophotometer. The calibration curve was plotted by preparing routine solutions at concentrations 12.5 to 100 g / ml in methanol. 3.4.0. In vivo study 3.4.1. Dosage of root extract: The concentrated extract contained approximately of 1 mg/ ml of GAE polyphenols and RE flavonoids in total. Each 100 ml daily dose of Calshakti Platina contains 1g each of Shatavari and Jivanti root which contains approximately 5 mg of total polyphenol and flavonoids. Therefore equivalent concentration of phenolics and flavonoids in 5 ml was selected as the test dosage for in vivo study. DP Tripathy, 2011 4

Biochemical studies on galactopietic effect of on crossbred dairy animals

3.4.2. Grouping of experimental animals: Apparently healthy cross bred milch cows preferably of 2nd and 3rd lactation of Livestock Breeding Farm, Khapuria Cuttack and some private Dairy Farms of in and around Bhubaneswar maintained at normal farm practices were selected for the study. The animals were randomly grouped into the following experimental groups basing on treatment schedule by their identifying numbers. Groups Gr-I Gr-II Description Normal milch cows as control Each cow treated orally with 5 ml root extract of Shatavari containing 5 mg of GAE polyphenols and RE flavonoids in total daily for 60 days Each cow treated orally with 5 ml root extract of Jivanti containing 5 mg of GAE polyphenols and RE flavonoids in total daily for 60 days Each cow treated orally with Calshakti Platina @ 100 ml daily for 60 days Each cow treated orally with Ostovet forte @ 100 ml daily for 60 days The milch cows in Gr-II and III were orally fed with polyphenols and flavonoids of Shatavari and Jivanti roots @ 12 mg/ animal / day for 60 days respectively. In contrast Gr-IV and V animals were received Calshakti Platina and Ostovet Forte @ 100ml / animal / day for 60 days. The animals at Gr-I were kept untreated as control to the test groups of cows. 3.4.2. Sample collection, preparation and preservation: The milk samples were collected in the morning in sterilized sampling vials and stored in the ice box during transportation. The samples were estimated for concentrations of its constituents on the same day within 6-8 hours of collection. The blood samples were collected on 0th, 30th and 60th day of the treatment schedule from jugular vein aseptically in sodium fluoride anticoagulant for estimation of glucose but non haemolysed serum separated from coagulated blood was used for other parameters. The samples collected were transported in an ice box to the laboratory. The collected blood samples were transferred to a wide mouth test tube and were kept in slanting position for about 30 minutes. After retraction of the clot, serum was decanted to a centrifuge tube and was centrifuged at 2000 rpm for 10 DP Tripathy, 2011 5

Gr-III

Gr-IV Gr-V

Biochemical studies on galactopietic effect of on crossbred dairy animals

minutes to separate the cells. The clear serum was transferred to another tube and was stored in the refrigerator at 0- 4C and was used for biochemical estimations. Estimation of enzymes was done on the same day while other biochemical estimations were completed by next day. Sampling for 0 day under Gr-I was made taking the total number of experimental animals into consideration before any treatment schedule. 3.4.3. Estimation Methods: Milk: The density, solid not fat (SNF), fat, protein and lactose of milk samples were estimated by Milky Lab-Milk Analyzer of Astori make. Initially the instrument was calibrated and 30 ml of milk sample taken in a beaker was dipped with the suction probe and respective measures against each parameter were recorded. Blood/ serum: The following biochemical constituents, enzymes and minerals were estimated in the blood/ serum of experimental animals. 3.4.3.1. Biochemical Constituents: The concentration of Glucose in blood and that of total protein, albumin and globulin, Total cholesterol and Blood Urea Nitrogen (BUN) were estimated in serum by using the reagent kits as mentioned in each parameter. Glucose: Blood glucose was estimated by using the reagent kit of Accurex Biomedical Pvt. Ltd. following GOD POD method described by Trinder, (1969). The OD was measured spectrophotometrically at 505 nm wave length to assay the concentration in mg/ dl. Total Protein, Albumin and Globulin: The concentration of total protein by biuret and albumin by BCG method was estimated in serum using the kit of Accurex Biomedical Pvt. Ltd. following the description Gornall, 1949 and Rodkey, 1964 respectively. The ODs of total protein and albumin were recorded colorimetrically at 600 and 550 nm to assay the concentrations in g/ dl respectively. Globulin conc. was determined by subtracting albumin concentration from that of total protein Total Cholesterol: DP Tripathy, 2011 6

Biochemical studies on galactopietic effect of on crossbred dairy animals

The content of total cholesterol was estimated in serum by CHOD/ PAP method using the kit prepared by Accurex Biomedical Pvt. Ltd. following the description Allian et al., (1974). The OD was measured spectro-photometrically at 505 nm wave length against the blank within 60 minutes to assay the concentration in mg/ dl. Urea: The serum urea concentration was estimated in serum by modified method of Berthelot, 1964 as supplied in reagent kit of Accurex Biomedical Pvt. Ltd. The OD was measured spectro-photometrically at 570 nm wave length against the blank within 60 minutes to express the conc. as mg/ dl. 3.4.3.2. Liver specific serum enzymes: The liver specific enzymes such as AST, ALT and ALP were selected for the study to evaluate the effect of polyphenols and flavonoids on liver function and the mechanism of induced/ declined milk production. AST and ALT: The serum AST ALT activities were assayed by UV-Kinetic method of Tietz, 1995 at 340 nm wave length as described in Accurex Biomedical Pvt. Ltd reagent Kit manual and the activities were expressed in IU/ L. ALP: The serum ALP activity was assayed by UV-Kinetic method of Young et al., 1972 at 405 nm wave length as described in Accurex Biomedical Pvt. Ltd reagent Kit manual and the activities were expressed in IU/ L. 3.4.3.3. Serum minerals: The serum macro-minerals of the experimental animals were estimated in both before and after the treatment schedule in each group under study Calcium: It was estimated by OCPC method as described by Gietelman, 1967 as supplied in the reagent kit of Crest Biosystem Pvt. Ltd at 570 nm wave length to expess the result in mg/ dl/. DP Tripathy, 2011 7

Biochemical studies on galactopietic effect of on crossbred dairy animals

Phosphorus: It was estimated by UV End Point method as described by Daly, 1972 at 340 nm wave length following the Kit manual of Accurex Biomedica Pvt. Ltd and the conc. was expressed in mg/ dl. 3.4.4. Statistical analysis The data was subjected to statistical analysis according to Snedecor and Cochran, (1967) particularly analysis of variance (ANOVA) and least significant difference (LSD) using Microsofts MS 2000 Data Analysis package

DP Tripathy, 2011 8