A Test For Carbohydrates Biology Essay

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Standardization of herbal drugs is complex process since biotype; ecotype and genotypic factors are known to affect the level of secondary metabolites of plants.However information on external morphology, anatomy and micrometric data of organized plant material can be suitably used in identification of plant material. In addition, certain physico-chemical parameters such as extractive value, ash values etc can provide rough idea about quality and purity of crude drugs.

5.1.1 Collection and Authentication of plant material

The plant material was collected from the local area of Adgaone of Dist. Nasik, Maharashtra, in the month of September- 2009 at the time of collection, plant including flowers and roots were collected. The plant materialwasobtained from Nasik district (M.S.) and authenticated by Dr. D. A. Patil, reader and the authorized plant identifier of Department of Botany, SSVPS College, North Maharashtra University, Dhule (M.S) India; a specimen is preserved in the college herbarium (KBHSS/PCG/2009/12). During plant material collection, infected parts of plants were carefully separated. The plant material was thoroughly washed with water to remove adhered particles and debris and dried in shade.

5.1.2 Preparation and Storage

The plant parts were placed in open place for 3-4 daysfor drying. Dried material were homogenized and passed through sieve no 40. Powdered drug was stored in airtight container and protected from light for further use.

5.1.3 Morphological study

The external morphology was studied according to reported methods.96 Khandelwal Macroscopic study was performed by using simple microscope. The colour, odour, taste, size and shape of flowers and roots were determined.

5.1.4 Microscopical analysis

The free-hand section of scale of flowers and root were prepared from fresh plant material and finally stained with various staining reagents as per standard procedures. The disaggregation of plant material was performed by reported method. In brief, scales were disaggregated by means of boiling in an aqueous solution of NaOH (5 % w/v) for 5 min. After cooling and washing with water, pieces were treated with an aqueous solution of chromic acid (25 % v/v) for 30 min at room temperature. The section where cleared with chloral hydrates solution, stained with phloroglucinol-hydrochloric acid (1:1) and toludine blue. 97,98 (O'BrieTP,)Kokate C. K., 2008.Powdered drug were used for the observation of power microscopical character. The powdered drug were separately treated with phloroglucinol-hydrochloric acid(1:1)solution, acetic acid and iodine solution to determine the presence of the lignified fibers, Calcium oxalate crystals and starch grains respectively. A series of digital images captured using a Motic Digital microscope fitted with 1/3'' CCD camera imaging accessory and using Motic Images 2000 (1.3 version) analysis software. The micrometric data were generated from average of 30 measurements for each sample and expressed as lower limit mean+SD.99,100(Brain KR, Kokate C.K. 2010..)

5.1.5 Fluorescence analysis: 101, 102(Gokhale S.B.,) Pratt

The fluroscence analysis is carried out on the powdered sample. Dry powdered sample was placed on a slide, treated with several drops of specified reagents and observed within minute under UV lamp.

5.1.6 Behaviour of powder towards specific reagents:103, 104(Sing VK, 2002,Kalaskar)

Behaviour of flower and root powders of T. populanea with different chemical reagents were performed to detect occurrence of phytoconstituents along with colour changes under ordinary daylight by standard method.

5.1.7. Physiochemical constants 105-107 (Khandelwal KR. 2005, Indian Pharmacopoeia. 1996, Mukherjee PK. 2002.)

5.1.7.1. Ash Value

Ash values are indicative to some extent of care taken in collection and preparation of drug for market and of foreign matter content of natural drug. The object of determining ash value of vegetable drugs is to remove all traces of organic matter which may otherwise interfere in analytical determination. On incineration, crude drugs normally leave an ash usually consisting of carbonates, phosphates and silicates of sodium, potassium, calcium and magnesium.

The total, acid-insoluble, water soluble and sulphated ash values were determined according to Indian Pharmacopoeial methods.

5.1.7.1.1. Elemental analysis of Ash 104.

The powdered drug was incinerated in muffle furnance to obtain ash. The ash was treated with 50% hydrochloric acid for 30 minutes and filtered. The filtrate was used for the detection of elements by specific test.

5.1.7.1.2. Test for aluminium: Test solution was treated with dilute ammonia solution observed gelatinous precipitation, soluble in hydrochloric acid, acetic acid and sodium hydroxide solution.

5.1.7.1.3. Test for Copper

a. Test solution treated with hydrogen sulphide produced brownish -black precipitate.

b. Test solution was treated with sodium hydroxide produced light blue precipitation.

5.1.7.1.4. Test for Calcium

a. Test solution treated with ammonium carbonate solution shows white precipitate which after boiling and cooling is insoluble in solution of ammonium sulphide.

b. Test solution treated with potassium chromate observed yellow crystalline precipitate.

5.1.7.1.5. Test for Iron

a. Test solution mixed with dilute hydrochloric acid and potassium permanganate produced faint pink coloration.

b. Test solution mixed with dilute hydrochloric acid and solution of ammonium thiocynate produced blood red coloration.

5.1.7.1.6. Test for Magnesium

a. Test solution shown white precipitation with after boiling with ammonium carbonate.

b. test solution, dilute ammonia and sodium phosphate solution produced white crystalline precipitate.

5.1.7.1.7. Test for Potassium: Test solution and perchloric acid observed white precipitation

5.1.7.1.8. Test for Sodium: Sample moistens with HCl and introduced on platinum wire into the flame of bursen burner shows yellow colour.

5.1.7.2. Moisture Content

Loss on drying is the loss in weight in percent w/w resulting from loss of water and volatile matter of any kind that can be driven off under specific conditions.

5.1.7.3. Extractive value

The alcohol and water soluble extractive values were obtained according to Indian Pharmacopoeial methods. In brief, 5 gm of coarsely powdered and air dried powder was macerated in 100 ml of ethanol (90 % v/v) in a closed container for 24 h. After 24 h, content was filtered rapidly to minimize alcohol loss. Accurately 25 ml of filtrate was evaporated to dryness in a tarred flat bottomed shallow evaporating dish and dried at 1050C and weighed.

Similarly water soluble extractive was determined. In brief, 5 gm of coarsely powdered and air dried sample was macerated in 100 ml of water in a closed container for 24 h, with frequently shaking during the first 6 h and allowed to stand for 18 h. After 24 h, content was filtered rapidly to minimize water loss. Accurately 25 ml of filtrate was evaporated to dryness in a tarred evaporating dish and dried at 1050C and weighed.

5.1.8. Phytochemical Evaluation108 (khadbadi) 98,109 (Kokate, Trease)

50 g of powdered plantmaterialwas packed in white cotton bags and were then extracted successively using different solvents in a Soxhlet apparatus. The solvents used for extraction were petroleum ether (60-80 0C), benzene, chloroform, ethyl acetate, methanol and water.Each time before extracting with the next solvent, the powdered material was dried in an air oven below 500C. The extractive values and preliminary phytochemical studies were carried out on these successive extracts.

In the present study, the qualitative chemical tests were performed on obtained successive extractsto ensure the presence ofphytoconstituents such as alkaloids, glycosides, tannins, flavonoids, terpenoids, steroids, carbohydrates, etc.

5.1.8.1. Test for Alkaloids:

Dragendorff's test: To the extract add Dragendorff's reagent, orange brown precipitate indicates the presence of alkaloids.

Mayer's test: To the extract add Mayer's reagent, cream colored precipitate indicates the presence of alkaloids.

Wagner's test: To the extract add Wagner's reagent, reddish brown precipitate indicates the presence of alkaloids.

Hager's test: To the extract add Hager's reagent, yellow precipitate indicates the presence of alkaloids.

5.1.8.2. Test for Carbohydrates

Molish's test: To the extract add few drops of alcoholic a-naphthol, then add few drops of concentrated sulphuric acid through sides of test tube; purple to violet color ring appeared at the junction of two liquids.

Barfoed's test: 1ml of extract is heated with 1ml of Barfoed's reagent, if red cupric oxide is formed, indicates the presence of monosaccharide. Disaccharides on prolong heating (about 10 min.) may also cause reduction, owing to partial hydrolysis to monosaccharide.

5.1.8.3. Test for Flavonoids

Shinoda test: To the extract add few magnesium turnings and concentrated hydrochloric acid drop wise, pink scarlet, crimson red or occasionally green to blue color appears after few minutes.

Lead acetate test: To small quantity of residue add lead acetate solution yellow colour precipitate is formed indicates the presence of flavonoids.

5.1.8.4. Test for Glycosides

General test:

Test A: Extract 200 mg of drug with 5ml of dilute sulphuric acid by warming on a water bath and filtered. Then neutralize the acid extract with 5% solution of sodium hydroxide. Add 0.1ml of Fehling's solution A and B and heat on a water bath for 5 minutes. First yellow then brick red precipitate is formed indicating the presence of glycosides.

Test B: Extract 200 mg of the drug using 5 ml of water instead of sulphuric acid. After boiling add equal amount of water as used for sodium hydroxide in the above test. Add 0.1 ml Fehling's A and B until alkaline (test with pH paper) and heat on water bath for 2 min. Note the quantity of red precipitate formed.

Compare the quantity of precipitate formed in Test B with that of formed in test A. If the precipitate in Test A is greater than in Test B then glycoside may be present. Since Test B represents the amount of free reducing sugar already present in the crude drug, whereas Test A represents free reducing sugar and those getting after acid hydrolysis of any glycoside in the crude drug.

Legal's test: aqueous or alcoholic extract, add 1 ml pyridine and 1 ml of sodium nitroprusside, pink colour appears indicates the presence of glycosides

Baljet's test: A thick section shows yellow to orange colour with sodium picrate indicates the presence of glycosides.

5.1.8.5. Test for Phenolic compounds (Tannins)

Ferric chloride test: Treat the extract with ferric chloride solution, blue color appears if hydrolysable tannins present and green color appears if condensed tannins present.

Lead acetate test: To small quantity of aqueous extract add lead acetate solution. White colour precipitate is formed indicates the presence of tannins.

5.1.8.6. Test for Proteins:

Biuret test: To the 2 ml extract 2 ml add 4 % NaOH and few drops of 1 % CuSO4 solution Biuret reagent was added, violet color indicates the presence of proteins.

Xanthoproetic test: To the (5 ml) extract, add 1 ml of concentrated H2SO4 and boil, yellow precipitate is formed. After cooling it, add 40% NH4OH solution, orange color is formed.

5.1.8.7. Test for Steroids and Triterpenoids:

Libermann-Burchard test: - Mix the extract with chloroform, add few drops of acetic anhydride boiled and cooled. Then add concentrated sulphuric acid from the side of the test tube, brown ring is formed at the junction of two layers and upper layer turns green which shows presence of steroids and formation of deep red color indicates presence of triterpenoids.

Salkowski test: Treat the extract with few drops of concentrated sulphuric acid red colour at chloroform layer and acid layer shows greenish yellow fluorescence indicates the presence of steroids and formation of yellow coloured lower layer indicates presence of triterpenoids.

5.1.9. Extraction Methodology

5.1.9.1. Extraction of flowers powder

Dried powder of flower was extracted with methanol (4.5 L) for 6 h at room temperature. The methanolic extract of flower (TPF) was combined and evaporated under reduced pressure at 400C to about 50mL. The TPF was partitioned three times with n-butanol saturated with H2O (50 mL each time). Further, n-butanol fraction was partitioned two times with diluted ammonia solution (50 mL each time). Then the ammonia layer was concentrated under reduced pressure at 40°C. The extracts were freezed-dry to yield about 210g.While, the aqueous fraction was treated with chloroform (3 times, 50 mL) to obtain the chloroform fraction (165 g) 110 (He-Long Bai, 2010)

Fig 5.1: Extraction scheme for Thespesia populnea flower powder

5.1.9.2. Extraction of root powder

The dried roots powder (1.5kg) was extracted with methanol (4 L) by using soxhlet method for 7 days at 45°C.Theethanolic extract of root (TPR) was filtered and concentrated. Further, TPR was dissolved in water and extracted with n-hexane for 3hr to obtained n-hexane fraction (240 g).Theaqueous layer treated with carbon tetrachloride (3 times, 100 mL) lower layer of carbone tetrachloride was collected and concentrated (35 g); while, the separated aqueous layer was concentrated using evaporator (110 g).111 (Md.AbdulMuhit, 2010.)

Fig. 5.2: Extraction scheme of root powered of T. populnea Linn.

5.1.10. Chromatographic study

5.1.10.1. Thin layer chromatography (TLC)

TLC study was done with TPF and TPR to find out the probable active compounds present in them. On the precoated silica gel TLC plate, test samples (dissolved in respective solvents) were applied. Then the plate was properly marked on the top for their identification. To avoid insufficient chamber saturation and undesirable edge effect, a smooth sheet of filter paper was placed (in U shape) in rectangular TLC chamber and was allowed to be in the developing solvent. Having been moistened, the paper was then pressed against the walls of the chamber, so that it adhered to the walls. Several solvent systems were tried during the study. The plate was sprayed withanisaldehydesulphuric acid (AS) reagent, Vanillin sulphuric acid (VS) reagent or Libermann- Burched (LB)reagent and heated at 1000C for 5 minutes. The most informative and satisfactory resolution was taken as final solvent system.

5.1.10.1.1 Thin layer chromatography of TPF.112(Cetkovic,).

TLC profile

Stationary phase : Silica Gel 254

Mobile phase :Ethyl acetate: Formic acid: Acetic acid: Water (100:11:11:26)

Saturation time : 20 min.

Detection : under UV light at 366 nm before and spraying with the 2- aminoethyldiphenylborinate/ VS reagent

5.1.10.1.2. Thin layer chromatography of TPR113 (Alam)

TLC profile

Stationary phase : Silica gel 254

Mobile phase : A. Benzene ethyl acetate (9:1)

B. Tolune: Chloroform: Ethyl acetate: Acetic acid

(10:2:1:0.3)

Detection : Mobile phase A=under UV light740nm before and after spraying with AS reagent.

B= under UV light 365nm and spraying with 1% ethanolic ferric chloride.

5.1.11. Quantitative estimation of phytoconstituents from TPF and TPR

5.1.11.1. Estimation of Total Phenolic Content 114, 115 (HarborneAndKhadabadi

Phenols, the aromatic compounds with hydroxyl groups are widespread in plant kingdom. They occur in plant parts. Phenols are said to offer resistance to diseases and pests in plants. Phenols include compounds like tannins, flavonols.

Principle

Total Phenolic content of TPF and TPR was measured by Folin- Ciocalteu reagent method. In this method, phenols react with phosphomolybdic acid in Folin-Ciocalteu reagent in alkaline medium and produce complex (molybdenum blue), which was measured at 760 nm using UV spectrophotometer.

Preparation of reagents:

Folin- Ciocalteau reagent (% v/v)

The reagent was prepared by diluting the 5 ml of the reagent to 5 ml with distilled water to give a stock solution.

Gallic acid solution (%w/v): The solution was prepared by adding 10 mg of gallic acid in 100 ml of water contributing concentration of 100 µg/ml.

Sodium carbonate (%w/v): It was prepared by dissolving 20 gm of sodium carbonate in 100 ml of water.

Preparation of test solution

10 mg of TPF and TPR were dissolved separately in methanol and volume was made up to 100 ml and designed as the stock solution contributing concentration of 100 µg/ml.

Procedure

Various concentrations (20-100 µg/ml) of gallic acid were prepared in triplicate from stock solution and 1ml was transferred in each 25 ml of volumetric flask containing 9 ml of distilled water. Add 5 ml distilled water in each volumetric flask. 1 ml of Folin-Ciocalteau reagent was added to volumetric flasks containing standard and test solution. After 5 min, 10 ml of 7% sodium carbonate solution was added to volumetric flasks containing standard and test solution. Final volume was made up to 25 ml by using distilled water. Blank determination was done by using methanol in place of test or standard solutions. The solutions were mixed thoroughly and absorbance was measured at 725 nm against a reagent blank after incubation for 90 min at room temperature. Total phenolics content of TPF and TPR was measured by the obtained standard curve of gallic acid.

5.1.11.2. Determination of Total Flavonoids Content

Flavonoids in many plants have a wide range of actions. They are anti-inflammatory, anti-oxidant and are especially useful in maintaining healthy circulation. Total flavonoids content was determined by the Aluminium chloride colorimetric assay.

Preparation of reagents:

Sodium nitrate (5%w/v):The solution was prepared by dissolving 5 gm of NaNO2 in 100 ml of distilled water.

Aluminum chloride (10% w/v):The solution was prepared by dissolving 10 gm of aluminium chloride in 100 ml of distilled water.

Sodium hydroxide (1 M): The solution was prepared by adding 0.4 gm of NaOH in 100 ml of distilled water.

Preparation of test solution: 40 mg of TPF and TPR was weighed accurately and dissolved in methanol and volume was made up to 100 ml and designed as the stock solution contributing concentration of 400 µg/ml.

Procedure:

An aliquot (l ml) of standard solution of rutin (20, 40, 60, 80,100 µg/ml) was added to 10 ml volumetric flask containing 4 ml of 5% NaNO2 into it. After 5 minute 0.3 ml of 10% AlCl3 was added. At sixth minute 2 ml of 1 M NaOH was added and the total volume was added and the volume was made up to 10 ml with distilled water. Same dilutions were prepared with the test solution. Blank determination was done by using methanol in place of test or standard solutions.

Mixed well and take absorbance at 358 nm against blank. From the obtained standard curve of rutin the total flavonoids content of TPF and TPR was determined.

5.1.12. Separation and Purification

5.1.12.1. Column chromatography

Column chromatography is isolation and purification technique used extensively by phyto-chemists to obtain pure samples of chemicals from natural sources; either solid or liquid. It is suitable for the physical separation of gram quantities of material. Column chromatography is the solid-liquid type chromatographic technique in which the two phases are solids (stationary phase) and liquid (moving phase). The theory of column chromatography is analogues that of thin layer chromatography. The most common adsorbents are- silica gel and alumina are the same one used in TLC. The sample is dissolved in small quantity of solvent (the eluent) and applied to the top of the column. The eluent, instead of rising by capillary action up a thin layer, flow down through the column filled with the adsorbent. Just, as in TLC there is an equilibrium established between the solute adsorbed on silica gel or alumina and eluting solvent flowing down through the column.

Column packaging: 60 gms of silica gel G (60-120≠) for column chromatography (laboratory grade)was activated in hot air oven at 110°C for 1hr. A specific solvent system for particular phytoconstituents was used to pack silica gel in the glass column. The activated silica was charged in the column, in small portion with gentle tapping after each addition, in order to ensure uniform packing. The small quantity of solvents was allowed to remain at the top of the column (about 4cm). The air bubbles present in the column were removed by the gentle tapping. Crude extract was dissolved in solvent system and adsorbed on silica gel and allowed to dry. This mixture in a powder form was loaded to the top of the column and eluted with specific solvent system. Several fractions were collected and each fraction was evaluated by TLC technique. These fractions were grouped according to their homogeneity, refereed from the TLC analysis.

5.1.12.2. Isolation of phyconstituents from TPF:110,

The n-butanol soluble fraction was subjected to column chromatography with fine silica packing. The column was eluted with isocratic and gradient application of mixture of chloroform-methanol and finally increasing polarity with water. Finally 45 fractions were collected each of 50 ml. All the fractions were monitored by the TLC treating with ammonia vapours reagent. Further, fraction Ba-4(fraction no. 24-31) was eluted with water-methanol mixture to yield 6 sub-fractions. Sub-fraction Ba-4-4 was subjected to preparative thin layer chromatography (Stationary phase- silica gel GF254, mobile phase: Chloroform: Water: methanol (65:25:10), thickness of plates - 0.5 mm) which afforded compound A (450mg).

Column 1

Fig 5.3: Schematic presentation of isolation of compound A from TPF

The chloroform soluble fraction was eluted with isocratic and gradient application of mixture of chloroform-methanol and finally increasing polarity with water. Total24 fractions were collected each of 50 ml. All the fractions were monitored by the TLC treating with ammonia vapours reagent. Further, the fraction SA1 (fraction no.5-11) was eluted with water-methanol mixture. Total 4 sub-fractions (SA1-1, SA1-2, SA1-3 and SA1-4) were collected. Sub-fraction SA1-2 (fraction no.4-6) was subjected to preparative thin layer chromatography (Stationary phase- silica gel GF254, mobile phase: Ethyl acetate: Methanol:water(90: 18: 2), thickness of plates - 0.5 mm) which offer compound B (210 mg).

Column 2

Fig 5.4: Schematic presentation of isolation of compound B from TPF

5.1.12.3. Isolation of phytoconstituents from TPR: 113, 117(Haque)

The previously obtained n-hexane soluble fraction was subjected to column Chromatography packed with fine column silica. The column was then eluted with n-hexane followed by mixtures of n hexane- ethyl acetate then ethyl acetate- methanol and finally with methanol. The polarity was gradually increased by adding increasing proportions of polar solvent. 30 fractions were collected each of 50 mL and grouped. All the fractions were monitored by the TLC spraying with VS/AS reagent. The fraction Hc (fractions no.16-24) was further eluted with n-hexane-chloroform mixture. Again, 15 sub-fractions were collected. Sub-fraction Hc1(sub fractions no.5-10) was subjected to preparative thin layer chromatography using 5% ethyl acetate in toluene and recovered with methanol. (Stationary phase- silica gel GF254, mobile phase: hexane-ethyl acetate, 95:5, thickness of plates - 0.5 mm) The preparative TLC of Sub fraction Hc1 was providedcompound C (15 mg).

Column 3

Fig 5.5: Isolation scheme for compound C from TPR

The carbon tetrachloride soluble fraction was fractionated by column chromatography using n-hexane and with increasing proporation of acetone to provide 15 fractions (25 ml each).After, fraction Cs1 (fraction no.7-9) was eluted with methanol- chloroform mixture, 13 sub fraction was collected .Compound D (92 mg) was obtained by the preparative TLC of Sc1-2 (sub fraction no.5-9). [Stationary phase- silica gel F254, mobile phase-toluene-ethyl acetate (95:5); thickness of plates-0.5 mm]

Column 4

Fig 5.6: Isolation scheme for compound D from TPR

The column chromatography of aqueous fraction was performed with isocratic- gradient elution of ethyl acetate: methanol as mobile phase. 14 fractions (25 ml each) were collected and the solvent was removed under reduce pressure by evaporation in vaccum at 35°C. The dried fractions were suspended in methanol and monitored by TLC method with same solvent system and alcoholic ferric chloride solution (5% w/v) was used as spraying reagent. The fractions with similar pattern were combined and separate compound E (1.25 g) was obtained by preparative TLC.(Stationary phase- silica gel F254, mobile phase toluene: ethyl acetate:methanol (4:3:3); thickness of plates-0.5 mm]

All separated compounds were evaluated by chemical test and Co-TLC.117 pal 2010.

Column 5

Fig 5.7: Isolation Scheme of compound E from TPR

5.1.13. Evaluation of isolated compounds

The all separated compound were monitored through chemical identity test and Co-TLC.

5.1.13.1. Homogeneity of compounds from TPF:

The purified compounds were subjected to testing homogeneity in different mobile phases.

Table 5.1 TLC profile for homogeneity of compound isolated from TPF

Isolated compound

Mobile phases

Visualization

Compound A

EtOAC: FA:AA:H2O(100:11:11:26)

Under UV-366 and sprayed with 2-aminoethyldiphenylborinatereagent

CHCl3: H2O: MeOH(65:23:10)

EtOAC: n-butanol: H2O(5:1:5.5)

Compound B

EtOAC: FA:AA:H2O

(100:11:11:26)

Under UV-366 and sprayed with 2-aminoethyldiphenylborinatereagent

EtOAC: H2O: MeOH (100:2.5: 16.5)

MeOH: H2O (9:1)

5.1.13.2. Homogeneity of compounds from TPR:

The purified compounds were subjected to testing homogeneity in different mobile phases.

Table 5.2: TLC profile for homogeneity of compound isolated from TPR

Isolated compound

Mobile phases

Visualization

Compound C

1. Petroleum ether: Ethyl acetate

(8:2)

Under UV-254 and sprayed with Vanillin- sulphuric acid reagent followed heating at 110°C.

2. n-hexane: Ethyl acetate(9:1)

3. Cyclohexane : ethyl acetate

Compound D

1. Chloroform: Benzene (1:4)

Under UV- 254 and sprayed with Libermann-burched reagent.

2. Chloroform: Methanol : water(30:4:1)

3. n-hexane: acetone(8:2)

Compound E

1. Tolune: Ethyl acetate: Methanol (4:3:3)

Under UV-366nm and spaying with 5% alcoholic ferric chloride.

2. n-butanol: Acetic acid: Water (4:1:5)

3. Ethyl acetate :Methanol

(8:1)

5.1.14. Recognition of Compound A, B, C, D and E

The pure compounds obtained were subjected to spectral analysis like UV, IR, Mass, NMR and melting point. 113, 116, 118, 119, 120(Dyer JR., Kemp W. Mahato,)

5.1.15. Quantitative estimation of phytoconstituents by HPTLC (121Sethi). Refinement in natural product research needs time tested purification and quantization procedure and HPTLC is one of the technology gaining increasing utility in pharmacognosy research. Although the principles of TLC and HPTLC methods are identical, because of the use of kinetically optimized fine-particle layers in HPTLC, separation is faster and more efficient and the results are more reliable and reproducible. In combination with digital scanning profiling, HPTLC also provides accurate and precise RF values and quantitative analysis of sample constituents by in situ scanning densitometry aided by the formation of easily detected derivatives by post-chromatographic chemical reactions as required, as well as a record of the separation in the form of a chromatogram with fractions represented as peaks with defined parameters including absorbance (intensity), RF, height and area. Furthermore, the feature of a pictorial fluorescence image of HPTLC coupled with a digital scanning profile is more and more attractive to herbal analysts for constructing a herbal chromatographic fingerprint by means of HPTLC. The main objective of this study was to evaluate and optimize the HPTLC fingerprint method in standardization of crude drug. These HPTLC fluorescence images coupled with the scanning profiles provided adequate information and parameters for comprehensive identification, assessment and comparison of major active constituent fingerprints in the samples studied to serve as a basis for their use in medicinal preparations for cardiovascular diseases.122(Amanzadeha),

Preparation of standard and sample solutions

Lupeol, β- sitosterol 5mg was accurately weighed into a 10 mL volumetric flask, dissolved in 5 mL chloroform. Gallic acid, rutin and quercetin 5mg were accurately weighed into a 10 mL volumetric flask, dissolved in 5ml methanol and the solution was made up to 10 mL with the same solvent [1mg/mL]. The 50 mg of TPF accurately weighed and dissolved in 5 mL methanol.Whereas,TRP(50 mg) was dissolved in chloroform and methanol, the solutions were filtered through Whatman filter paper No. 42.

Development of HPTLC Technique

The samples were spotted in the form of bands with Camagmicrolitre syringe on a precoated silica gel plates 60F 254 [10 cm X 10 cm with 0.2 mm thickness, E.Merck] using Camaglinomat IV applicator with the band width of 7 mm. The plates weredeveloped in a solvent system in Camag glass twin through chamber previously saturated with the solvent for 20 min. the distance was 8 cm. subsequent to the scanning, TLC plates were air dried and scanning was performed on a Camag TLC Scanner in absorbance at 254, 365 and 740 nm and operated by wincats software 4.03 version.

5.1.15.1. Quantitative estimation of rutin and quarcetin in TPF123 (Sajeeth)

Table 5.3: HPTLC profile for TPF

Parameters

Rutin

Quercetin

Plate

Plate-1

Plate- 2

Stationary phase

Precoated silica gel plates 60F 254

Size of plate

10 cm x 10 cm

Mobile phase

Ethyl acetate: Methanol: Water(8:1:1)

Chloroform: Methanol(8:2)

Spraying reagent

--

--

Scanning after spraying

Scanned at 366 nm

Scanned at 366 nm

Sample application pattern for Plate 1

1. Standard Rutin was applied on Ist track in 2 l (R2) volume.

2. Standard Rutin was applied on IIndtrack in 4 l (R4) volume.

3. Standard Rutin was applied on IIIrd track in 6 l (R6) volume.

4. Standard Rutin was applied on IVth track in 8 l (R8) volume.

5. Standard Rutin was applied on Vthtrack in 10 l (R10) volume.

6. TPF was applied on VIthtrack in 50l (TF) volume.

Sample application pattern for Plate 2

1. Standard Quercetin was applied on Isttrack in 2 l (Q2) volume.

2. Standard Quercetin was applied on IIndtrack in 4 l (Q4) volume.

3. Standard Quercetin was applied on IIIrd track in 6 l (Q6) volume.

4. Standard Quercetin was applied on IVthtrack in 8 l (Q8) volume.

5. Standard Quercetin was applied on IVthtrack in 10 l (Q10) volume.

6. TPF was applied on VIthtrack in 50l (TF) volume

5.1.15.2. Quantitative estimation of lupeol, β-sitosterol and gallic acid in TPR 124(Vaidya,)

Table 5.4 HPTLC profile for TPR

Parameters

Lupeol

β-sitosterol

Gallic acid

Plate no.

Plate 3

Plate 4

Plate 5

Stationary phase

Precoated silica gel plates 60F 254

Size of plate

10 cm x 10 cm

Mobile phase

Benzene: Ethyl acetate (9:1)

Chloroform: Methanol: Formic acid (5.5:3.5:1)

Spraying reagent

Libermann- Burchedreagent

1% ethanolic FeCl3

Scanning after spraying

Scanned at 740nm (after derivatization)

Scanned at 365nm

Sample application pattern for Plate 3

1. Standard lupeol was applied on Isttrack in 2 l (L2) volume.

2. Standard lupeol was applied on IIndtrack in 4 l (L4) volume.

3. Standard lupeol was applied on IIIrd track in 6 l (L6) volume.

4. Standard lupeol was applied on IVth track in 8 l (L8) volume.

5. Standard lupeol was applied on Vth track in 10 l (L10) volume.

6. TPR was applied on VIthtrack in 50l (RL) volume.

Sample application pattern for Plate 4

1. Standard β-sitosterol was applied on Isttrackin 2 l (B2) volume.

2. Standard β-sitosterol was applied on IIndtrack in 4 l (B4) volume.

3. Standard β-sitosterol l was applied on IIIrd track in 6 l (B6) volume.

4. Standard β-sitosterol was applied on IVth track in 8 l (B8) volume.

5. Standard β-sitosterol was applied on Vth track in 10 l (B10) volume.

6. TPR was applied on VIthtrack in 50l (RB) volume.

Sample application pattern for Plate 5

1. Standard Gallic acid was applied on Isttrackin 2 l (G2) volume.

2. Standard Gallic acid was applied on IIndtrack in 4 l (G4) volume.

3. Standard Gallic acid l was applied on IIIrd track in 6 l (G6) volume.

4. Standard Gallic acid was applied on IVth track in 8 l (G8) volume.

5. Standard Gallic acid was applied on Vth track in 10 l (G10) volume.

6. TPR was applied on VIthtrack in 50l (RG) volume.

5.2 PHARMACOLOGICAL INVESTIGATION

5.2.1 Experimental Animals

Albino wistar rats of either sex weighing 150- 250 g and swiss albino mice of either sex weighing 20-30 g were procured from R.V.S. College of Pharmaceutical Sciences, Sulur, Coimbatore. The animals were housed in polypropylene cages and maintained under standard conditions (12 hrs light and dark cycles, at 25 ± 30C and 35-60 % humidity). Standard palletized feed and tap water were provided ad libitum. The study was approved by Institutional Animal Ethical Committee (IAEC) of R.V.S. College of Pharmaceutical Sciences, Sulur, Coimbatore, registered under CPCSEA, India (Registration No.1012/C/06/CPCSEA).

5.2.2 Procurement of chemicals and diagnostic kits

Indomethacin, Pentazocine, Diclofenac, Gemfibrozil, Simvastatin, Freund's advujant, Cholesterol (C75209) (sigma chemicals, USA), Cholic acids (Himedia), Cholesterol, triglyceride, HDL-cholesterol estimating kits (RFCL Pvt. Ltd, Gudgeon, India), Citric acid, Sodium citrate, Dextrose, Thrombin, Heparin (S.D. Fine chemicals, India). Triton WR 1339, Adenosine di-phosphate (sigma chemicals, USA)

5.2.3 Acute Toxicity Test 125(OECD Guidelines)

The procedure was followed by using Organization of Economic Cooperation and Development (OECD) guidelines 423 (Acute Toxic Method). The acute toxic class method is a stepwise procedure with 3 animals of a single sex per step. Depending on the mortality and/or moribund status of the animals, on the average 2-4 steps may be necessary to allow judgment on the acute toxicity of the test substance. This procedure results in the use of a minimal number of animals while allowing for acceptable data based scientific conclusion. The method uses defined doses (5, 50, 300, 2000 mg/kg body weight) and the results allow a substance to be ranked and classified according to the Globally Harmonized System (GHS) for the classification of chemicals which cause acute toxicity.

Experimental procedure

Male wistar rats weighing 150-200 g were used for the study. The starting dose level of Methanolic extract was 2000 mg/kg body weight p.o. As most of the crude extracts posses LD50 value more value more than 2000 mg/kg p.o., the starting dose used was 2000 mg/kg p.o. Dose volume was administered 0.1 ml/10 gm body weight to the rat which were fasted over night with water ad libitum. Food was with held for a further 3-4 hours after administration of methanolic extracts of flower and root and observed for signs of toxicity.

Body weight of the animals before and after termination were noted and any changes in skin and fur, eyes and mucous membranes and also respiratory, circulatory, autonomic and central nervous system and somatomotor activity an behavior pattern were observed, and also sign of tremors, convulsion, salivation, diarrhoea, lethargy, sleep and coma were noted. The onset of toxicity and signs of toxicity were also observed.

5.2.4 Anti-inflammatory activity

5.2.4.1. Acetic acid induce vascular permeability (acute study) 126, 127(Olijade et al., 2000; Whittle et al.,1964)

Procedure

One hour after oral administration of the methanolic extracts TPF and TPR at doses of 100, 200 and 400 mg/kg, mice were injected with 0.25 ml of 0.6% solution acetic acid intraperitoneally. Indomethacin (Indo, 5 mg/kg, p.o.) served as the reference drug, while animals in the control group received CMC suspension (0.5%) at a dose of 1 ml/kg. Immediately after administration, 1 ml/kg of 10% (w/v) Evan's blue was injected intravenously through the tail vain. Thirty minutes after Evan's blue injection, the mice were killed and the viscera exposed. The animals were held by a flap of abdominal wall and the viscera irrigated with distilled water over a petri dish. The exudates was then filtered and made up to 10 ml. The vascular permeability was represented in terms of the absorbance (A610) and % inhibition.

Table 5.5 Groups of TPF and TPR in the evaluation of acetic acid induce vascular permeability

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1ml/ kg, p.o.

% Inhibition

Absorbance (610 nm)

II

Indo

5 mg/kg, p.o.

III

TPF

100 mg/kg, p.o.

IV

TPF

200 mg/kg, p.o.

V

TPF

400 mg/kg, p.o.

VI

TPR

100 mg/kg, p.o.

VII

TPR

200 mg/kg, p.o.

VIII

TPR

400 mg/kg, p.o.

Similar experiment was performed for the evaluation of ethyl acetate extract of flower (FEA) and root (REA) at the dose of 100, 200 and 400mg/kg p.o., similar reference standard was used.

Table 5.6 Groups of FEA and REA in the evaluation of acetic acid induce vascular permeability

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1ml/ kg, p.o

% Inhibition

Absorbance (610 nm)

II

Indo

5 mg/kg, p.o.

III

FEA

100 mg/kg, p.o.

IV

FEA

200 mg/kg, p.o.

V

FEA

400 mg/kg, p.o.

VI

REA

100 mg/kg, p.o.

VII

REA

200 mg/kg, p.o.

VIII

REA

400 mg/kg, p.o.

5.2.4.2. Cotton pellet implants induced granuloma in rats (chronic study) 128, 129, 130 (Winter, mandal, Arumugam).

Procedure

The rats were divided into eight groups with six animals in each group. After shaving off the fur, the animals were anaesthetised. Through a singly needle incision, sterile pre-weighed cotton pellets (20± 0.5 mg) were implanted in the groin and axilla region of each rat. Methanol extracts of flower (TPF) and root (TPR) (100, 200 and 400 mg/kg), Indomethacin (Indo, 10 mg/kg) and CMC suspension (1 ml/kg) were administered orally to the respective group of animals for 7 consecutive days from the day of cotton-pellet implantation. On the eighth day, the animals were anaesthetizedagain; the cotton pellets were removed surgically and made free from extraneous tissues. The moist pellets were weighed and then dried at 600C for 48 h and again weighed. The reduced weights of the cotton pellets observed for the test compounds and anti-inflammatory are compared with the control. This provides a measure to assess the anti-inflammatory effect of the test compounds.

Table 5.7: Groups of TPF and TPR inCotton pellet implants induced granuloma in rats.

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1 ml/ kg, p.o.

Wet weight (mg)

Dry weight (mg)

Granuloma weight (mg)

% Inhibition

II

Indo

5 mg/kg, p.o.

III

TPF

100 mg/kg, p.o.

IV

TPF

200 mg/kg, p.o.

V

TPF

400 mg/kg, p.o.

VI

TPR

100 mg/kg, p.o.

VII

TPR

200 mg/kg, p.o.

VIII

TPR

400 mg/kg, p.o.

Similar experiment was performed for the evaluation of ethyl acetate extract of flower (FEA) and root (REA), similar reference standard was used as follows;

Table 5.8: Groups of FEA and REA inCotton pellet implants induced granuloma in rats.

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1ml/ kg, p.o.

Wet weight (mg)

Dry weight (mg)

Granuloma weight (mg)

% Inhibition

II

Indo

5 mg/kg, p.o.

III

FEA

100 mg/kg, p.o.

IV

FEA

200 mg/kg, p.o.

V

FEA

400 mg/kg, p.o.

VI

REA

100 mg/kg, p.o.

VII

REA

200 mg/kg, p.o.

VIII

REA

400 mg/kg, p.o.

5.2.5 Analgesic activity (131,Selven 2005)

5.2.5.1. Formalin induce nociception in mice (Chemical induction)132, 133 (Hunskaar, Hnatyszyn).

Procedure

In this test 20 µl of 1% formalin was injected into the dorsal surface of the left hind paw of mice 1 h after oral administration of methanolic extract of flower and root at the dose of 100, 200 and 400 mg/kg. Control animals received 1ml/kg of CMC suspension. While,2 mg/kg i.p.Pentazocine (Penta) was given to reference group. Animals were divided into 8 group (n=6). Mice were observed in a chamber with a mirror mounted on three sides to allow an unobstructed view of the paws; time spent licking the injected paw (licking time) was recorded. Animals were observed for the first 5 min post-formalin (early phase) and for 10 min starting at the 20th min post-formalin (late phase).

Table 5.9: Groups of TPF and TPR inFormalin induce nociception in mice

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1ml/ kg, p.o.

Time of licking and biting response during

0-5 min (I phase)

15-30 min (II phase)

II

Penta

2 mg/kg, i.p.

III

TPF

100 mg/kg, p.o.

IV

TPF

200 mg/kg, p.o.

V

TPF

400 mg/kg, p.o.

VI

TPR

100 mg/kg, p.o.

VII

TPR

200 mg/kg, p.o.

VIII

TPR

400 mg/kg, p.o.

Similar experiment was performed for the evaluation of analgesic activity of Ethyl acetate extract of flower (FEA) and root (REA) at the dose of 100, 200 and 400mg/kg p.o., similar reference standard was used.

Table 5.10: Groups of FEA and REA inFormalin induce nociception in mice

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1ml/ kg, p.o.

Time of licking and biting response during

0-5 min (I phase)

15-30 min (II phase)

II

Penta

2 mg/kg, i.p.

III

FEA

100 mg/kg, p.o.

IV

FEA

200 mg/kg, p.o.

V

FEA

400 mg/kg, p.o.

VI

REA

100 mg/kg, p.o.

VII

REA

200 mg/kg, p.o.

VIII

REA

400 mg/kg, p.o.

5.2.3.2. Acetic acid induce writhings in mice (Peripheral analgesia) 134, 135 (Amos,2003,loro)

Procedure

The methanolic extract of flower (TPF) and root (TPR) at doses of 100, 200 or 400 mg/kg was administered orally, to 16 h fasted mice, divided into groups of six animals each. One hour after treatment, the mice were injected intraperitoneally with 0.1 ml/10 gm body weight of acetic acid solution (0.6%) to induce the characteristic writhings. The number of writhings occurring between 5 to 30 min after acetic acid injection was recorded. The response of the extract and Indomethacin (Indo, 5 mg/kg, p.o.) treated groups were compared with those of animals in the control group (CMC suspension, 1 ml/kg).

Table 5.11: Groups of TPF and TPR in Acetic acid induce writhings in mice.

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1ml/ kg, p.o.

Number of writhes during 5 to 30 min

Percent inhibition

II

Indo

5 mg/kg, p.o.

III

TPF

100 mg/kg, p.o.

IV

TPF

200 mg/kg, p.o.

V

TPF

400 mg/kg, p.o.

VI

TPR

100 mg/kg, p.o.

VII

TPR

200 mg/kg, p.o.

VIII

TPR

400 mg/kg, p.o.

Similar experiment was performed for the evaluation of ethyl acetate extract of flower (FEA) and root (REA) at the dose of 100, 200 and 400 mg/kg p.o., similar reference standard was used.

Table 5.12: Groups of FEA and REA inAcetic acid induce writhings in mice.

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1 ml/ kg, p.o.

Number of writhes during 5 to 30 min

Percent inhibition

II

Indo

5 mg/kg, p.o.

III

FEA

100 mg/kg, p.o.

IV

FEA

200 mg/kg, p.o.

V

FEA

400 mg/kg, p.o.

VI

REA

100 mg/kg, p.o.

VII

REA

200 mg/kg, p.o.

VIII

REA

400 mg/kg, p.o.

5.2.5.3 Hot plate method in mice (Thermal induction)

Procedure

Mice that showed nociceptive responses within 20 s when placed on hot plate maintained at 55 ± 0.50C were selected and grouped into eight (n=6). Group I was control and treated with CMC suspension (1 ml/kg); groups III- V and VI-VIII received 100, 200 and 400 mg/kg p.o. of the extract TPF and TPR respectively; while group II received 2 mg/kg i.p. of Pentazocine (Penta). Each mouse was placed singly on the hot plate and the latency to exhibit thermal stimulus were determined before and at 30, 60, 90 and 120 min after treatment. Licking of paws and jumping were the parameters evaluated as the thermal stimulus. 30 sec. was taken as the cut-off time to avoid animal tissue damage. (134Amos et al., 2001; 136Adzu et al., 2003) Each mouse served as its own control. Before treatment, its reaction time was taken as initial reaction time. The mean reaction time for the all groups was pooled to obtain the final control mean reaction time (Tb). This was pooled for the mice in each treatment group and the final test mean value for each treatment group at each measurement was calculated. (137Engidawork et al., 2010) This final test mean value represented the after treatment reaction time (Ta) and was subsequently used to determine the percentage thermal pain stimulus or protection by applying the following formula:

% protection against thermal induction (PATI) = Test mean (Ta) - Control mean (Tb)/ Control mean (Tb)

Table 5.13: Groups of TPF and TPR in Hot plate method in mice.

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1 ml/ kg, p.o.

Pre drug latency

Nociceptive latency at 30, 60, 90 and 120 min

Percent inhibition (% PATI)

II

Penta

2 mg/kg, i.p.

III

TPF

100 mg/kg, p.o.

IV

TPF

200 mg/kg, p.o.

V

TPF

400 mg/kg, p.o.

VI

TPR

100 mg/kg, p.o.

VII

TPR

200 mg/kg, p.o.

VIII

TPR

400 mg/kg, p.o.

Similar experiment was performed for the evaluation of ethyl acetate extract of flower (FEA) and root (REA) at the dose of 100, 200 and 400mg/kg p.o., similar reference standard was used.

Table 5.14: Groups of FEA and REA in Hot plate method in mice.

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1 ml/ kg, p.o.

Pre drug latency

Nociceptive latency at 30, 60, 90 and 120 min

Percent inhibition (% PATI)

II

Penta

2 mg/kg, i.p.

III

FEA

100 mg/kg, p.o.

IV

FEA

200 mg/kg, p.o.

V

FEA

400 mg/kg, p.o.

VI

REA

100 mg/kg, p.o.

VII

REA

200 mg/kg, p.o.

VIII

REA

400 mg/kg, p.o.

5.2.6 Anti-arthritis activity

5,2,6,1 Freund's adjuvant induced arthritis in rat (130Arumugam P. 2008, 138Madav S 1996)

Higher doses of TPF, TPR, FEA and REA showing statistically significant and considerable analgesic and anti-inflammatory activities therefore, TPF, TPR, FEA and REA were selected onlyat the dose of 400 mg/kg for evaluation of antiarthritis activity.

Procedure

The albino wistar rats of either sex (150- 200 g) were divided into six groups (n=6). On day 0, 0.1 ml of freund'scomplete adjuvant (FCA) (Sigma, USA) was injected into the subplantar region of right hind paw of each rat. The negative control group and the positive control group were given 0.2 mlCMC suspension (1 ml/kgp.o.) and Diclofenac sodium (DF) (5 mg/kg, p.o.), respectively and the remaining four groups received the TPF, TPR FEA and REA at doses of 400 mg/kg, p.o., for 21 consecutive days. (139 Ilenti)

Table 5.15: Groups of Freund's adjuvant induced arthritis in rat.

Groups

Treatment

Dose

I

CMC

1 ml/ kg, p.o.

II

DF

5 mg/kg, p.o.

III

TPF

400 mg/kg, p.o.

IV

TPR

400 mg/kg, p.o.

V

FEA

400 mg/kg, p.o.

VI

REA

400 mg/kg, p.o.

Evaluation parameters

1) Primary lesions

The paw volume and ankle diameter of injected paw was measured using digital plethysmometer and digital vernier caliper respectively, on day 0 before administration of adjuvant and at every five days of the entire treatment period ending on day 21. (140 Bani)

2) Photographs

Photographs of injected paws of all animals were taken by using digital camera (Sanyo, Japan) on day 21 after the adjuvant injection.

3) Body weight

Body weights of all the animals were recorded at the interval of 5 days from the day 0 to day 21. (141 Hu)

4) Scoring systems

Following scoring systems were used to access the degree of arthritis. The scores were assigning according to the severity of disease progression. Scores were evaluated at every 3rd day after adjuvant injection for the entire period of drug treatment. (142 laird, , 143 kumar)

A) Arthritis score

Each rat was observed at every 3rd day and scored for severity of swelling and redness of paws & joints. The grading for arthritis was done on 0 to 4 scales where 0 represents least amount of definite swelling and 4 represents maximum swelling. This scoring system involved observations of all four paws of rat & separate score was given for each limb.

B) Flexion pain test

The ankle joint was flexed dorsally until the toe touched the anterior part of the leg. The test was performed 5 times with an inter-test interval of 5 sec and the pain was scored 0, if there was no squeaking and no leg withdrawal; 1, if there was either squeaking or leg withdrawal; 2, if both squeaking and leg withdrawal were present.

C) Mobility score

The mobility of rats whilst they moved freely in a large empty cage were also assessed according to the adaptations of following scale-

0 - Normal, 1 - Limping, 2 - Walks with difficulty, 3 - Paw not touched

D) Stance score

The stance of rats when they placed in large empty cage were assessed according to the following scale -

1 = Paw lifted continuously, 2 = Paw touching but with no weight bearing, 3 = moderate weight bearing on the paw, 4 = Normal

5) Haematological parameters (144 patil, 145 ward)

On the day 21, animals were anesthetized with anaesthetic ether and blood was withdrawn from retro orbital plexus for estimating following haematological parameters.

A) Total leukocyte (WBC) count

Increased values suggest inflammation or infection.

B) Haemoglobin

Decreased values are indicative of anemia.

C) Erythrocyte sedimentation rate

The erythrocyte sedimentation rate (ESR), also called a sedimentation rate, sedrate or Biernacki reaction, is a non-specific measure of inflammation that is commonly used as a medical screening test. ESR was determined by wintrobe method.

D) C- reactive protein (146 deepa)

On the day 21, animals were anesthetized with anaesthetic ether and blood was withdrawn from retro orbital plexus and serum was separated. The semiquantitative determination of c- reactive protien was performed by using commercial CRP kit obtained from Agappe diagnostics Pvt. Ltd., Kerala, India, according to manufacturer's instructions as follows -

1. 50 μL diluted glycine saline buffer was placed on to each of five circles of the slide.

2. Using a 50 μL (0.05 mL) micropipette, 50 μL (0.05 mL) of the serum sample was added to the drop of glycine-saline buffer in the 1st circle.

3. Using the same micropipette, the sample was mixed with saline by aspirating back & forth several times.

4. Aspirated 50 μL (0.05 mL) from the 1st circle and transfered to 2nd circle. Same operation was repeated upto 5th circle. 0.05 mL was aspirated from the 5th circle and discarded.Following dilutions were obtained:

Dilution: 1/2, 1/4, 1/8, 1/16, 1/32

5. Then one drop of CRP-Latex antigen was added to each of above diluted circles and the slide was rocked gently to and fro for 2 min. The agglutination was observed under good source of light.

Calculation:

Concentration of CRP in serum can be calculated as follows,

CRP Concentration (mg/dL) = Sensitivity Ã- Titre (Highest dilution of serum showing agglutination)Where, CRP sensitivity is 0.6 mg/dL

E) Rheumatoid factor

On the day 21, animals were anesthetized with anesthetic ether and blood was withdrawn from retro orbital plexus and serum was separated. The semiquantitative determination of rheumatoid factor was performed by using commercial Rhelax RF kit obtained from Tulip diagnostics Pvt. Ltd., Goa, India, according to manufacturer's instructions as follows -

1. Using isotonic saline solution serial dilutions of the serum sample was prepared as 1:2, 1:4, 1:8, 1:16 and 1:32.

2. Each dilution of the serum sample was pipetted onto separate reaction circle.

3. One drop of Rhelax RF latex reagent was added to each drop of diluted serum sample on the slide. Dropper tip do not touched the liquid on the slide.

4. Using a mixing stick, the sample and latex reagent was mixed uniformly over the entire circle.

Rock the slide gently, back and forth and observed for agglutination macroscopically at two minutes.

Calculations

Agglutination in the highest serum dilution corresponds to the approximate amount of rheumatoid factors in IU/mL present in the test specimen. RF is calculated by using following formula -

RF (IU/mL) = S Ã-D

Where, S = Sensitivity of the reagent, D = Hightest dilution of the serum showing agglutination

NOTE:

Since, the FEA and REA shows lesser extent of considerable pharmacological activities in previous animal models as compared to TPF and TPR; thus, to reduce the sample (animal) size and to avoid redundant results, the further in-vivo studies were continued only with the TPF and TPR.

5.2.7 Anti- hyperlipidemic activity

5.2.7.1 in- vivo study

5.2.7.1.1 High fat diet (HFD) induce hyperlipidemia(hypercholesterolemia) in rats (Chronic study) (147Chattopadhayay,148 devi)

Procedure

Albino wistar rats of either sex (200- 220 gm) were used in the study. The rats were randomly divided into nine groups of six animals each. The groups of animals were prepared in such way that each group had similar body weight distribution. The first group, serving as a normal control group, maintained on a standard pellet diet, and received CMC suspension(1ml/ kg, p.o.). Second group, hyperlipidemic control group maintained on a high fat diet (HFD) composed of cholesterol 4%, cholic acid 1% and 1 ml coconut oil. Group third received a HFD along with reference compound, Simvastatin (Sim, 4 mg/ kg/ day p.o.). Groups 4, 5, and 6 were received a HFD along with the TPF and groups 7, 8 and 9 received HFD along with TPR at the dose of 100, 200 and 400 mg/ kg/ day, p. o. respectively. The experiment was conducted for 30 days and during this period, full access to food and water was provided. Body weights animals of each group were recorded at day 0, day 10, day 20 and day 30.

Table 5.16: Groups of TPF and TPR in High fat diet (HFD) induce hyperlipidemia (hypercholesterolemia) in rats

Groups

Treatment

Dose

Evaluation parameters

I

CMC

1 ml/kg p.o

Biochemical estimation of serum

Total Cholesterol

Triglyceride

HDL-c

LDL-c

VLDL-c

Atherogenic index

Animal body weight

Histopathology

II

HFD

cholesterol 4%, cholic acid 1% and 1 ml coconut oil

III

HFD + Sim

4 mg/kg p.o.

IV

HFD + TPF

100 mg/kg p.o.

V

HFD + TPF

200 mg/kg p.o.

VI

HFD + TPF

400 mg/kg p.o.

VII

HFD + TPR

100 mg/kg p.o.

VIII

HFD + TPR

200 mg/kg p.o.

IX

HFD + TPR

400 mg/kg p.o.

Collection of blood and separation of serum

The animals were anesthetized with anesthetic ether and with the help of small capillary the retro-orbital vein was punctured and 1ml blood was collected into the Ependorf centrifuged tube and it was allowed to clot for 30 minutes. The tubes were kept for the centrifugation (Remi Centrifuge R 24) at 2000 rpm for 10 minutes. The serum was then separated with the help of micropipette into other Ependorf tube and stored at 2-8o C, untill it was used for the estimation of biochemical parameter.

Biochemical estimation

1. Cholesterol determination

CHOD-PAP method (148 Devi et al; 2003) was used to estimate the serum cholesterol.

Principle

Cholesterol esters are hydrolyzed by Cholesterol Esterase (CE) to give free Cholesterol and fatty acid. In subsequent reaction, cholesterol oxidase oxidizes the 3-OH group to free cholesterol to liberate cholest-4-en-3-one and hydrogen peroxide. In presence of peroxidase, hydrogen peroxidase couples with 4- aminoantipyrine and phenol to produce red quinoneimine dye. Absorbance of coloured dye is measured at 505 nm and is proportional to the amount of total cholesterol concentration in the sample.

Assay parameter

Mode : End point

Wavelength : 505 nm

Temperature : 37 oC

Optical path length : 1cm

Blanking : Reagent blank

Sample volume : 10µl

Reagent volume : 1000µl

Incubation time : 10 min. at 370C or 30 min at room temperature

Concentration of standard : 200 mg/dl

Stability of final colour : 1hour

Linearity : 750mg/dl

Units : mg/dl

Procedure

Pipette into tubes marked

Blank

Standard

Test

Serum

-

-

10 µl

Reagent 2

-

10 µl

-

Reagent 1

1000 µl

1000 µl

1000 µl

Mix well; incubate at 370C for 10 minutes or at room temperature (15-300C) for 30 minutes.

Calculation

Total cholesterol (mg/dl) = (Absorbance of test / Absorbance of standard) X 200

2. Triglycerides determination

GPO-PAP method (148 Devi et al; 2003) was used for the estimation of serum triglycerides.

Principle

Triglycerides are hydrolyzed by lipoprotein lipase to produce glycerol and free fatty acid. Further in presence of glycerol kinase, Adenosine triphosphate phosphorylates and glycerol produces glycerol 3-phosphate and adenosine diphosphate.Glycerol 3-phosphate is further oxidized to produce dihydroyacetone phosphate and H2O2 in presence of peroxidase, hydrogen peroxides couples with 4-aminoantipyrine and 4- chlorophenol to produced red quinoneimine dye. Absorbance of coloured dye is measured at 505 nm and is proportional to Triglyceride concentration in the sample.

Assay parameter

Mode : End point

Wavelength : 505 nm

Temperature : 370C

Optical path length : 1cm

Blanking : Reagent blank

Sample volume : 10µl

Reagent volume : 1000µl

Incubation time : 10 min. at 370C or 30 min. at room temperature

Concentration of standard : 200 mg/dl

Stability of final colour : 1hour

Linearity : 1000 mg/dl

Units : mg/dl

Procedure

Pipette into tubes marked

Blank

Standard

Test

Serum

-

-

10 µl

Reagent 2

-

10 µl

-

Reagent 1

1000 µl

1000 µl

1000 µl

Mix well; incubate at 37 oC for 10 minutes or at room temperature (15-30oC) for 30 minutes.

Calculation

Total Triglyceride (mg/dl) = (Absorbance of test/ absorbance of standard) X 200

3. HDL-cholesterol determination

CHOD-PAP method (148 Devi et al; 2003) was used to estimate the serum HDL- cholesterol.

Principle

Low and very low-density lipoproteins are precipitated by a solution containing PEG 6000, leaving behind the high-density lipoproteins in solution. HDL cholesterol is estimated in the supernatant by a series of enzymatic reactions that are initiated by the oxidation of cholesterol to cholestenone by cholesterol oxidase, accompanied by the formation of hydrogen peroxide. In a second reaction catalyzed by peroxidase, 4-aminoantipyrine and phenol react with hydrogen peroxide to form red colouredquinonemine. Absorbance at 505 nm is directly proportional to HDL cholesterol concentration in the specimen.

Serum + precipitating reagent ï‚®Precipitate (

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Coursework Writing Service

Our expert qualified writers can help you get your coursework right first time, every time.

Dissertation Proposal Service

The first step to completing a dissertation is to create a proposal that talks about what you wish to do. Our experts can design suitable methodologies - perfect to help you get started with a dissertation.

Report Writing
Service

Reports for any audience. Perfectly structured, professionally written, and tailored to suit your exact requirements.

Essay Skeleton Answer Service

If you’re just looking for some help to get started on an essay, our outline service provides you with a perfect essay plan.

Marking & Proofreading Service

Not sure if your work is hitting the mark? Struggling to get feedback from your lecturer? Our premium marking service was created just for you - get the feedback you deserve now.

Exam Revision
Service

Exams can be one of the most stressful experiences you’ll ever have! Revision is key, and we’re here to help. With custom created revision notes and exam answers, you’ll never feel underprepared again.