Material And Methods Pharmacognosy General Consideration Biology Essay

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The Plant material of Viscum capitellatum Smith. parasitism on Dendrophthoe falcata which is itself parasitic on M. indica was collected from Amba Ghat, Kolhapur, Western Ghat region of Maharashtra from India in November 2009. The collection are lies [Latitude 16o 58' 0.59"N and Longitude 73° 48' 36.61"E at altitude 1100m]. The plant specimen (Voucher no. 550) was authenticated by Dr. Vinay Raole, Reader, Department of Botany, M.S. University, Baroda, India.

Pharmacognostical Study

Macroscopical Study[68]

It includes the shape, size, colour, texture, surface and odour of the drug in crude or powered form and often sufficient to enable to identify the whole drugs.

Microscopical Study

Histochemistry

It gives the idea about the colour reaction of specific chemical reagent towards plant tissues [68]. Microscopical images are given in Figure no. 2.

Quantitative Microscopy [66-69]

Transverse sections of scale and stems were obtained by means of a microtome and stained with different staining reagents as per standard procedures [66, 70-71]. All observations were performed using Motic Digital Photomicroscope.

Histological study of leaves and stem were performed by reported method [69]. Leaves were boiled in a 5% aqueous solution of NaOH for 5 min while stems were boiled with 10% aqueous solution of NaOH for 10 min. After cooling and washing with water, pieces were treated with a 25% aqueous solution of chromic acid for 30 min at room temperature. Washed pieces of both leaf and stem were pressed in between two slides and slides coves.

Determination of Stomatal Number

The average number of stomata per square millimeter of epidermis is termed the stomatal number.

Determination of Stomatal Index

The percentage proportional to the ultimate divisions of the epidermis of a leaf, which has been converted into stomata, is termed the stomatal index.

SI = S Ã- 100

E + S

Where SI = Stomatal index, S = number of stomata per unit area and E = number of ordinary epidermal cells in the same unit area.

Procedure [68]

Pieces of leaf between margin or midrib was cleared and mounted, and the lower surface examined by means of a microscope with a 4mm objective and an eyepiece containing a 5mm square micrometer disc. Counts were made of the numbers of the epidermal cells and of stomata within a square grid, a cell being counted if at least half of its area lies within the grid. The stomata index was determined for both leaf surfaces. Results pertaining to quantitative microscopical study are given in table no. 8.

Analytical Study

Ash Value[68, 72]

1.1 Total ash

Total ash gives the idea about the residue obtained after ignition. It consist of physiological ash obtain by ignition of plant tissues and non physiological ash obtain by ignition of extraneous matter adhering to the surface of Plant. 2 gm of accurately weighed air dried powdered drug was taken in silica crucible. This silica crucible with drug material was kept in muffle furnace and ignited at temperature 4500C. The material was heated till the white coloured ash and constant weight is obtained. The procedure was performed in triplicate. Result is given in table No. 9.

The total ash was calculated by subtracting the weight of crucible with ash of drug after ignition from weight of crucible with drug powder before ignition. Percentage of total ash was calculated with reference to air-dried drug.

Acid insoluble ash

Acid insoluble ash gives the idea about the presence of inorganic material such as calcium oxalate present in plant material.

The ash obtained in the total ash method was boiled with 25 ml of 2N hydrochloric acid for 5 min. Insoluble matter was collected on ash less filter paper (Whatman paper) and washed with hot water. The material retained on filter paper and along with filter paper, was further ignited and weighed. Percentage of acid insoluble ash was calculated with reference to air dried material. Result is given in table No. 9.

Water soluble ash

The ash obtained from total ash was boiled with 25 ml water for 5 min. All insoluble matter was collected on ash less filter paper, washed with hot water and ignited for 15 min at the temperature not exceeding 4500C.

The percentage of water soluble ash was calculated by subtracting weight of insoluble matter from weight of total ash. The difference between weights represents water soluble ash. Percentage of water soluble ash was calculated with reference to air dried drug. Result is given in table No. 9.

Extractive Value[68, 72]

Extraction by cold maceration

It is the process of extraction of crude drugs with solvents with several daily shakings or stirring at room temperature.1 kg of powdered plant was extracted with 5 lit of methanol by cold maceration method. The extract was concentrated on rotary vacuum evaporator (Roteva Equitron, Mumbai) and further dried in vacuum dryer [73].

Successive extraction by using Soxhlet apparatus

Weighed accurately 200gm of dried, powered crude drug and kept in a filter paper cover which was already placed in thimble. Then the solvent was slowly poured onto it. The solvent from thimble goes to lower round bottom flask via siphon tube due to the siphoning or syphon cycle. Such 2-3 cycles of solvent were performed and then drug powder was kept for 12 hours with solvent for imbibitions. After 12 hours imbibitions, solvent from flask heated to form vapors. Due to heat the solvent from RBF gets converted into its vapors, and then these vapors pass via side tube into the condenser where it gets condensed. This solvent dripped again on to drug material, which was placed in thimble. This process was continued till thimble gets filled with solvent and when level of solvent reaches to syphon tube, pulling of whole solvent into the flask is taken place. All this events repeated several times and drug material gets extracted continuously with fresh solvent. This process was performed for 3 days and when syphon solution showed negative test for phytoconstituents, extraction was completed. Then the heating was stopped and the mixture was collected and cooled. Then this mixture was filtered and concentrated by using rotary flash vacuum evaporator. The extract was dried in vacuum dryer and was stored in freeze. Then this marc obtained after pet ether extraction and subjected again to extraction by following solvents (Table 10) [73].

Moisture content by Loss on Drying[72]

2 g of air powdered drug was placed in a silica crucible. Before that, crucible was cleaned and dried and weight of empty crucible was taken. The powder was spread in a thin uniform layer. The crucible was then placed in the oven at 1050C. The powder was dried for 4 h and cooled in a desiccator to room temperature and weight of the cooled crucible plus powder was noted. Result is given in table no. 9.

Analysis of inorganic constituents (Elemental analysis)

Ash of drug material was prepared and adds 50% v/v HCl or 50% v/v HNO3 to ash. Keep it for 1 hour. Filtered and with the filtrate performed the test as per method reported [74]. The results of analysis of inorganic constituents are given in (Table 11).

Table 3. Elemental Analysis

Sr. no.

Test performed

Observation

Inference

1.

Test for calcium

a) Add dil. NH4OH and saturated ammonium oxalate solution to filtrate.

White ppt of calcium oxalate forms which is soluble in HCl.

Calcium present.

b) Add ammonium carbonate to filtrate.

White ppt which is insoluble in NH4Cl.

Calcium present.

2.

Tests for iron

a) Add 2% potassium ferricyanide to filtrate.

Dark blue coloration.

Iron present.

b) To filtrate, add 5% ammonium thiocyanate.

Blood red color.

Iron present.

c) To filtrate, add dil. HCl and sol. of KMnO4.

Pink color.

Iron present.

3.

Tests for magnesium

a) To filtrate add NaOH.

White ppt.

Magnesium present.

b) To filtrate add (NH4)2CO3.

White ppt, redissolve in NH4Cl.

Magnesium present.

4.

Tests for potassium

a) Add sodium cobalt nitrite to filtrate.

Yellow ppt.

Potassium present.

b) Flame test.

Violet color to flame.

Potassium present.

5.

Tests for sodium

a) Add uranyl zinc acetate to filtrate, shake well.

Yellow crystalline ppt.

Sodium present.

6.

Tests for carbonate

a) Add HgCl2 to filtrate.

Brownish red ppt.

Carbonate present.

b) Add dil. Acid to the filtrate.

Effervescence of CO2

Carbonate present.

c) Add MgSO4 to filtrate.

White ppt.

Carbonate present.

7.

Tests for Sulphate

a) Add BaCl2 to filtrate.

White crystalline ppt

Sulphate present.

b) Add filtrate to lead acetate sol.

White ppt.

Sulphate present.

8.

Tests for phosphate

a) Add HNO3 and ammonium molybdate to filtrate, heat 10 min. cool.

b) Add silver ammonium- nitrate to filtrate

Yellow crystalline ppt.

Light yellow ppt

Phosphate present.

Phosphate present.

9.

Tests for chloride

a) Add AgNO3 to filtrate.

b) To filtrate, add manganese dioxide and H2SO4

White curd ppt, soluble in dil. NH3.

Odour of chlorine

Chloride present.

Chloride present.

10

Tests for nitrate

a) Add water to filtrate, add H2SO4 from side of test tube.

b) Add H2SO4 and copper to filtrate, warm

Brown color at junction of two liquid

Liberation of red fumes

Nitrate present.

Nitrate present.

Determination of Type of Starch Grains

The shape of starch grains present was determined according to the reported method [68]. Size of starch grains were measured with the help of calibrated Photomicroscope using Motic software. Starch grains were identified by staining with Iodine solution. The Motic digital Photomicroscope was calibrated with images obtained with various magnifications (10x, 40x and 100x) by using standard slide in 1.3 software. The images obtained in triplicate and average figures calculated from 20 readings in each parameter (Table no. 12).

Crude Fiber Content [75]

Pre-weighed dried powder material was extracted with Petroleum ether (b.p. 40- 600C) using soxhlet apparatus for 8 h. The marc obtained after extraction was utilized for determination of Crude Fiber Content.

Crude fiber was investigated by acid-base digestion with H2SO4 (1.25%) and of NaOH (1.25%) solution. The marc after extraction was taken into a 500ml beaker and 200ml of boiling H2SO4 added. The content was boiled for 30 minutes, cooled, filtered and the residue washed three times with 50ml of boiling water. The washed residue was further boiled in 200ml of NaOH for 30 minutes. The digest was filtered to obtain residue. This was washed three times with 50ml of boiling water and lastly with 25ml of ethanol.

The washed residue was dried in an oven at 1250C to constant weight and cooled in dessicator. The residue was scraped into a pre-weighed porcelain crucible, weighed, ashed at 5500C for 2 hours, cooled in a dessicator and weighed. Crude fiber content was expressed as percentage loss in weight on ignition. Result is given in table No. 13.

Phyto-chemical Analysis.

Extracts.

Petroleum ether, benzene, chloroform, acetone and methanol extract obtained by successive extraction method and aqueous extract by maceration method [68, 95].

Qualitative analysis

All the extracts were subjected to proximate chemical analysis and its result is given in table no. 14.

. Table 4. Qualitative chemical analysis

Test performed

Observation

Inference

Tests for Acidic compounds:

a) To the test solution add sodium bi-carbonate

b) Test solution treated with warm water and filter. Test the filtrate with litmus paper.

Tests for Alkaloids:

a) Dragendorff's Test: Test solution treated with Dragendorff's reagent (potassium bismuth iodide)

b) Mayer's Test: Test solution treated with Mayer's reagent (Potassium mercuric iodide).

c) Wagner's Test: Test solution treated with Wagner's reagent (Iodine in potassium iodide).

d) Hager's Test: To the test solution add gives with Hager's reagent (Saturated picric acid solution).

e) Tannic acid test: Test solution treated with Tannic acid solution.

f) Picrolonic acid test: Test solution treated with Picrolonic acid.

Test for amino acids:

a) Million's Test: Test solution treated with Million's reagent and heated on a water bath.

b) Ninhydrin Test: Test solution boiled with Ninhydrin reagent.

Test for Carbohydrates:

a) Molisch's Test: To the test solution add with few drops of Molisch's reagent (Alcoholic-naphthol) and 2ml of conc. sulphuric acid is added slowly from the sides of the test tube.

b) Barford's Test: Test solution heated with Barford's reagent on water bath.

c) Selivanoffs test (Test for Ketones): To the test solution add crystals of resorcinol and equal volumes of concentrated hydrochloric acid and heat on a water bath.

d) Test for pentose: To the test solution add equal volumes of hydrochloric acid containing small amount of Phloroglucinol and heat.

e) Osazone formation test: Heat the test solution with the solution of phenyl hydrazine hydrochloride, sodium acetate, and acetic acid.

Test for Flavonoids:

a) Shinoda Test: Test solution treated with fragments of magnesium ribbon and conc. Hydrochloric acid.

b) Alkaline Reagent Test: Test solution treated with sodium hydroxide solution

c) Zinc-Hydrochloride test: Treat test solution with zinc dust and few drops of HCL

Test for glycosides:

General test: Extract 200 mg of drug with 5 ml of dilute sulphuric acid by warming on a water bath, filter it, and neutralize the acid extract with 5 % solution of sodium hydroxide. Add 0.1 ml of Fehling's solution A and B until it becomes alkaline (test with pH paper) and heat on water bath for 2 minutes.

Test B: Repeat Test A procedure by using 5 ml of water instead of dilute sulphuric acid. Note the quantity of red precipitate formed.

Chemical tests for specific glycosides:

Tests for Anthraquinone glycosides:

a) Borntrager's test: Boil the test material with 1ml of sulphuric acid for 5minutes. Filter while hot. Cool the filtrate; shake with equal volume of dichloromethane or chloroform. Separate the lower layer of dichloromethane or chloroform; shake it with half of its volume of dilute ammonia.

b) Modified Borntrager's test: Boil 200 mg of test material with 2ml of sulphuric acid. Treat with 2 ml of 5 % aqueous ferric chloride solution (freshly prepared) for 5 minutes, shake it with equal volume of chloroform and continue the test as above.

c) Test for hydroxy anthraquinones: treat the sample with potassium hydroxide solution.

Tests for cardiac glycosides:

a) Kedde's test: Extract the drug with chloroform, evaporate to dryness. Add one drop of 90 % alcohol and 2 drops of 2 % sodium hydroxide solution.

b) Keller-Killiani Test: (Test for deoxy sugars) Extract the drug with chloroform and evaporate it to dryness. Add 0.4 ml of glacial acetic acid containing ferric chloride, add carefully 0.5 ml of conc. sulphuric acid by the side of test tube.

c) Raymond's number: treat the test solution with hot methanolic alkali.

d) Baljet's Test: The test solution treated with sodium picrate or picric acid.

e) Legal's Test: Test solution treated with pyridine [made alkaline by adding sodium nitroprusside solution].

Tests for coumarins glycosides: Place small amount of sample in test tube and covered it with a filter paper, moistened with dilute sodium hydroxide solution. Placed the covered test tube on water bath for several minutes. Remove the paper and expose it to ultraviolet (UV) light.

Cynogentic glycosides:

Place 200 mg of drug in conical flask and moisten with few drops of water.( Flask should be completely dry because hydrogen cyanide produced will dissolve in the water rather than come off as gas to react with paper) moisten a piece of picric acid paper with 5% aqueous sodium carbonate solution and suspended in neck of flask. Warm gently at about 37oC. Observe the change in color.

Saponin glycosides:

Froth test: Place 2 ml solution of drug in water in a test tube, shake well.

Tests for steroids and triterpenoids:

Liebermann Burchard Test: Treat the extract with few drops of acetic anhydride, boil and cool, add conc. sulphuric acid from the sides of test tube.

Salkowski test: Treat the extract with few drops of conc. sulphuric acid.

c) Sulfur powder test: Add small amount of sulfur powder to the test solution.

Tests for inulin: To the test solution add the solution of -naphthol and sulphuric acid.

Tests for Lignin: Treat the sample with hydrochloric acid and Phloroglucinol.

Tests for Mucilage:

Treat the sample with thionine solution. After 15 min wash with alcohol

Tests for tannins:

a) Ferric-Chloride Test: Treat test solution with few drops of ferric chloride solution.

b) Gelatin test: To the test solution add 1 % gelatin solution containing 10 % sodium chloride.

Tests for proteins:

a) Heat test: Heat the test solution in boiling water bath.

b) Biuret Test: Test solution treated with Biuret reagent (40% sodium hydroxide and dilute copper sulfate solution).

c) Xanthoproteic test: To the test solution, add 1 ml of conc. nitric acid and boil yellow precipitate is formed. After cooling it, add 40 % sodium hydroxide solution.

Test for starch: To the test solution, add weak aqueous iodine solution. Blue color indicates presence of starch, which disappears on heating and reappears on cooling.

Effervescence produces

Litmus paper turns blue

Gives reddish brown colored precipitate

Gives cream colored precipitate

Gives reddish brown colored precipitate

Gives yellow colored precipitate

Gives buff colored precipitate

Gives yellow colored precipitate

White colored precipitate

Gives violet color

Purple to violet ring appears at the junction of two liquids

If red cupric oxide is formed

Rose color is produced

Red color produced.

Yellow crystals formed.

Observe under microscope.

Shows pink scarlet, crimson red or occasionally green to blue color after few minutes.

Shows increase in the intensity of yellow color on addition of few drops of dilute acid.

Shows red color after few minutes.

Red Precipitate formed

compared with precipitate of test A

A rose pink to red color is produced in ammonical layer.

A rose pink to red color is produced in ammonical layer.

Red color produced

Purple color is produced.

Acetic acid layer shows blue colour.

Violet colour produced

Gives yellow to orange color

Gives blood red color

Paper shows green fluorescence.

Reddish purple color

Stable froth (foam) formed

Brown ring is formed at the junction of two layers,

If upper layer turns green

If upper layer turns deep red

Red color at lower layer

Yellow color at lower layer

It sinks at the bottom

Brownish red color formed

Pink color formed

Mucilage turns violet red.

Gives dark blue color

Green color appears

Precipitate formed

Proteins gets coagulated

Gives violet color

Orange color formed

Blue color, which disappears on heating and reappears on cooling

Acidic compounds present

Acidic compounds present

Alkaloids present

Alkaloids present

Alkaloids present

Alkaloids present

Alkaloids present

Alkaloids present

Amino acids present

Amino acids present

Carbohydrates present

Monosaccharides are present.

Carbohydrates present

Carbohydrates present

Carbohydrates present

Flavonoids present

Flavonoids present

Flavonoids present

If the precipitate in Test A is greater than in Test B then glycoside may be present.

Anthraquinone glycosides present

Anthraquinone glycosides present

Hydroxy anthraquinones present

Cardiac glycosides present

Cardiac glycosides present

Cardiac glycosides present

Cardiac glycosides present

Cardiac glycosides present

Coumarins glycosides present

Cynogentic glycosides present

Saponin glycosides

Present

Steroids present

Triterpenoids present

Steroids present

Triterpenoids present

Steroids present

Inulin Present

Lignin Present

Mucilage present

Hydrolysable tannins

Condensed tannins

Tannins present

Proteins present

Proteins present

Proteins present

Starch present

Floroscence Analysis of various extracts

Petroleum ether, Benzene, Chloroform, Acetone, Methanol and Aqueous extracts were screened for fluorescence characteristic. The observation pertaining to their colour in day light and under ultra-violet light were noticed and represented in table. Many substances for example quinine in solution in dilute sulphuric acid when suitably illuminated emit light of a different wavelength or colour from that which falls on them. This emitted light (fluorescence) ceases when the exciting light is removed [68].Results given in Table No. 15.

HPLC Analysis of sample drug.

The chromatographic pattern of plant was obtained as per report with some modifications for which the HPLC conditions are as follows.

Extract: The methanol extract diluted with HPLC grade methanol and filtered through whatman filter paper and used for analysis

Instrument: Shimadzu LC-20AT with UV/visible detector

Stationary Phase: Bonda- pack C-18 column with 250Ã-4mm

Mobile Phase: Methanol (80): Water (20)

Detection wave length: 350 nm

Flow Rate: 2 ml/min.

HPLC Chromatogram is given in Fig. 3 and its retention time is given in Table no. 16

HPTLC Analysis of sample drug.

The chromatographic pattern of plant was obtained as per report with some modifications for which the HPTLC conditions are as follows.

Extract: Methanolic Extract

Instrument: HPTLC (Camag, Switzerland)

Stationary Phase: pre-coated silica gel plates

Mobile Phase: Ethyl acetate: Formic acid: Glacial acetic acid: water (100:05:10:20)

Spraying Reagent: Natural Product Reagent (NP reagent)

Detection: 365 nm.

HPTLC Chromatogram is given in Fig. 4 and its retention time is given in Table no. 17.

Isolation and characterization of chemical principle

Compound I

The methanol extract was dissolved in water and partitioned with ethyl acetate and n- butanol. The ethyl acetate fraction was subjected to column chromatography for isolation of compounds.

Column chromatography: The separation of extract constituents was done by column chromatography. The clean and dried glass column was used. The silica gel for column chromatography (#60-120) was activated at 1100c.The column was filled with silica gel and mobile phase without formation of any air bubbles. The silica gel was then allowed to stabilize in the column. Mixture of two or three compounds was isolated from the ethyl acetate fraction of methanol extract of the plant with following experimental conditions [73].

Height of column: 20 cm

Diameter of column: 3.5 cm.

Stationary phase: Silica gel (#60-120).

Mobile phase: Benzene→ Chloroform → Ethyl acetate→ Methanol with variant Proportions

Elution: Gradient elution.

Fraction quantity: 25 ml

Preparative TLC:

20 X 20 glass plates were coated with the thick layer of silica gel or any other adsorbent material. The plates were then activated at 1100c.The sample-containing mixture of two or more compounds were applied in the form of thin band on the plate. The plate was then developed. The different bands separated on the plate were scratched and recovered with methanol. Purity of dried sample was checked by TLC method. One single compound was isolated with the help of preparative chromatography from fractions 54- 58. The compound is given for spectral analysis. FTIR spectra, Mass spectra and 1HNMR are given in fig. no. 5, 6 and 7 respectively. The spectral data of FTIR and 1HNMR are given in Table no. 18 and 19 respectively. The assumed structure of the compound (Quercetin) is given in Fig. No. 8.

Compound II

Petroleum ether extract obtained is processed for separation of the unsaponifiable and saponifiable matter. Extract is allowed to saponify using alcoholic KOH with reflux and then it is extracted with solvent ether for separation of unsaponifiable matter. The aqueous phase is acidified with concentrated H2SO4 and then again extracted with the solvent ether for separation of the saponifiable matter [73].

Fractionation of unsaponifiable matter

Experimental:

Height of column : 25 cm

Diameter of column : 3.5 cm.

Stationary phase : Silica gel for column chromatography (#60-120).

Mobile phase : Benzene→ Ethyl acetate

Elution : Gradient elution.

Fraction quantity : 30 ml

Fractions No. 24-27 were subjected for thin layer chromatography with following experimental conditions.

Stationary phase : Silica gel H

Mobile phase : Ethyl acetate: Benzene (1: 9)

Detection : Vanilin-sulphuric acid reagent

Identification : Whitish Purple colour

Fraction was concentrated and single band was applied. After plate development; developed band was scraped (Rf. 0.62). After separation of single compound from the silica, it is dried. This sample was further given for spectroscopic analysis. FTIR spectra, Mass spectra and 1HNMR are given in fig. no. 9, 10 and 11 respectively. The spectral data of FTIR and 1HNMR are given in Table no. 20 and 21 respectively. The assumed structure of the compound (Quercetin) is given in Fig. No. 12.

Biochemical Estimations.

a) Estimation of Total carbohydrate content [76]

The estimation of carbohydrate was done using the method acid base digestion.

Principle:

In hot acidic media glucose is converted to hydroxy methyl furfural by dehydration. This forms a green colour product with phenol.

Procedure:

100mg of the aqueous extract was taken and it was hydrolyzed by keeping it on water bath for 3 hours with 5 ml of HCl (2.5N) and cooled at room temperature. Neutralized it with sodium carbonate and volume was made up to 100 ml and from this centrifuge 10 ml of the solution. Then 0.2, 0.4, 0.6, 0.8 and 1ml of working standard was pipetted out into a series of test tube and in separate test tubes 0.1 and 0.2 ml of sample solution was pipetted out and the volume was make up to 1ml with water. The blank was prepared with 1 ml distilled water. Then 1ml phenol solution and 5ml of sulphuric acid (96%) was added to each test tube and shaken well. After 10 min the test tube was placed in water bath at 25-30ï‚°C for 20 min. The absorbance was read at 490 nm. And the amount of total carbohydrate present was calculated in the sample using standard graph. Result pertaining to Total carbohydrate content is given in Table no. 22 and Calibration curve of standard glucose dilutions are given in Fig. No. 13.

Estimation of Bitterness value [77].

The bitterness value of plant material was compared with diluted solution of Quinine hydrochloride.

Preparation of Solutions

Preparation of Quinine hydrochloride solution

The stock solution of 100µg/ml was prepared from which a series of dilutions 42, 44, 46, 48, 50, 52, 54, 56 and 58 µg/ml were prepared.

Preparation of Sample Preparation

Form the stack solution of 1000 µg/ml, 100, 200, 300 and 400µg/ml dilutions were prepared.

Method

Tasted all the dilutions of sample and Quinine sulphate by taking the solution in mouth and swirled it for 30 secs in mouth mainly near to the tongue. After tasting each dilution the mouth wash rinsed thoroughly with drinking water and taken the interval of 10 mins. Until the bitter sensation of previous dilution was no more remain. Then compared the dilution of sample which produced the same bitterness equivalent to the dilution of Quinine sulphate. Then bitterness value was calculated according to following formula.

Bitterness value in units per gram = 2000 Ã- A

B Ã- C

Where A= quantity of Quinine sulphate (mg) having higher bitterness

B= the concentration of stock solution (mg/ml)

C= Volume of sample in ml having higher bitterness

Result pertaining to estimation of bitterness value is given in Table no. 22

Total Phenolic content [78]

The total phenolic content of methanol extract of V. capitellatum Smith. (VCM) was estimated using Folin-Ciocalteu reagent. In this method, the blue colour formed due to the polyphenol was measured at 760 nm using UV spectrophotometer.

Chemicals

Folin- Ciocalteu reagent (Merck Co.)

Gallic acid (Sigma Ltd., USA)

Sodium carbonate (SISCO Research Laboratory Pvt. Ltd., Mumbai, India)

Reagent preparation

Folin-Ciocalteu (phenol) reagent

The reagent was prepared by diluting 1ml with 5ml of distilled water.

Sodium carbonate

15% solution was prepared in distilled water.

Gallic acid solution

The stock solution was prepared by dissolving 1mg gallic acid in 10ml of water from which different concentrations (20-100µg/ml) were prepared.

Sample preparation

Sample solution was prepared by dissolving 10 mg of the extract in 100 ml of methanol to give (100 µg/ml) solution.

Procedure

0.1ml of extract was mixed with the 0.2ml of Folin-Ciocalteu reagent, 2 ml water and 1 ml of sodium carbonate solution, and absorbance was measured at 760 nm after 10 min incubation at 50 0C. The total phenolic was expressed as µg gallic acid equivalent. Result pertaining to Total phenolic content is given in Table no. 22 and Calibration curve of standard gallic acid dilutions are given in Fig. No. 14.

Total Flavonoid Content

Total flavonoid content of VCM was determined using method reported [79].

Chemicals

Al (NO3)3,

Potassium acetate

Quercetin. (Sigma Ltd., USA)

Preparation of reagents

Quercetin solution

The stock solution was prepared by dissolving 1mg quercetin in 10ml of methanol from which different concentrations (20-100µg/ml) were prepared.

Sample preparation

Sample solution was prepared by dissolving 10 mg of the extract in 100 ml of methanol to give (100 µg/ml) solution.

Potassium acetate solution (1 M)

Potassium acetate solution was prepared by dissolving 9.8g of potassium acetate in 100ml of distilled water.

Aluminum nitrate (10% w/w)

Aluminum nitrate solution was prepared by dissolving 10g 0f aluminum nitrate in 100ml of distilled water.

Procedure

0.5 ml of Sample solution, 1.5 ml of ethanol, 0.1 ml of Al (NO3)3, 0.1 ml of CH3COOK and 2.8ml of water were thoroughly mixed and kept at room temperature for 40 min. The absorbance of mixture was measured at 415 nm. Total flavonoid content was calculated according to a standard curve established with quercetin. The total flavonoid was expressed as µg quercetin equivalent. Result pertaining to total flavonoid content is given in Table no. 22 and Calibration curve of standard quercetin dilutions are given in Fig. No. 15.

Total proanthocyanidin content [80].

Total proanthocyanidin content of VCM was determined using method reported [80].

Chemicals

Vanillin (Merck Co.)

Sulphuric acid

Epicatechin (Fluka)

Preparation of reagents

Epicatechin solution

The stock solution was prepared by dissolving 1mg epicatechin in 10ml of methanol from which different concentrations (8-40µg/ml) were prepared.

Sample preparation

Sample solution was prepared by dissolving 10 mg of the extract in 100 ml of methanol to give (100 µg/ml) solution.

Vanillin-H2SO4 reagent

Vanillin-H2SO4 reagent was prepared by dissolving 1g of vanillin in 100ml of 70% H2SO4.

Procedure

1ml of methanol extract (100µg/ml) was mixed with 2 ml of freshly prepared vanillin solution (1% vanillin in 70% of H2SO4) and maintained for 15 min at 200C. The absorbance was measured at 500 nm. Calibration curved was drawn using aqueous solution of epicatechin (8-40 µg/ml) as a reference standard.

All experiments were performed in triplicate and mean values were used for calculation. Spectrophotometric determinations were performed using on Shimadzu UV-2450 spectrophotometer. Result pertaining to total proanthocyanidin content is given in Table no. 22 and Calibration curve of standard epicatechin dilutions are given in Fig. No. 16.

Biological Studies.

Gross behavior and acute toxicity studies for the methanol extract.

The procedure was followed by using OECD guidelines (Organization of Economic Cooperation and Development) 423 (Acute Toxic Method) [81]. 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.

Acute oral toxicity study

Male wistar rats weighing 150-200 g and Swiss albino mices 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 and observed for signs of toxicity. Body weight of the rats 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 also noted.

Pharmacological screening

(1) Analgesic activity

Animals

Swiss Albino mice (20-25 g) were used for study. The animals were housed under standard conditions at temperature of 22 ± 10C, relative humidity of 55 ± 10% and 12 hr light/dark cycles. Animals were fed with standard pellet diet (Amrut, Pranav Agro Industries Ltd., Sangli, India) and water ad libitum. All the experiments performed are approved by the Institutional Animal Ethical Committee.

Grouping of animals

The animals were divided into five groups containing six animals in each group. Group I served as negative control (received normal saline, 5 ml/kg, p.o.), group II served as positive control (received Diclofenac sodium, 5 mg/kg, i.p.) and group III, IV and V were treated with VCM (100, 200, and 400 mg/kg) respectively.

Acetic acid Induced Writhing Test (Chemical Stimulation)

The analgesic activity evaluated using acetic acid induced writhing in mice [82]. In this method, acetic acid was administered intra peritoneally to animals to create the sensation of pain. Diclofenac sodium (i.p.) and methanol extract (VCM, p.o.) were administered to overnight fasted Swiss Albino mice. Test samples and vehicle were given orally 30 minutes before to intraperitoneal giving 0.7% v/v of acetic acid solution (0.1ml/10g) but Diclofenac sodium was given 15 minutes prior to acetic acid injection. Then each animal was observed individually for counting the number of writhing they performing in 5 minutes. This incomplete writhing was considered as half-writhing. Accordingly, two half-writhing were taken as one full writhing. The number of writhings of treated animals was compared with negative control. In this study Diclofenac sodium (5 mg/kg) was used as a reference standard (positive control). Results are given in Table no. 23.

Heat conduction method:

30 min, 1 hour, 2 hour and 3 hour after administration of sample and drug the tail tip of animals was dipped 5 cm into water maintained at 58°C. The response time was noted as the sudden withdrawal of the tail from the hot water. 10 seconds Cut off time was maintained to avoid damage to the tail. The time required for flicking of the tail, was recorded, to assess response to noxious stimulus [83] (Table no. 24).

(2) Anti-arthritic activity

Animals

Wistar male rats (180-220g) were used for study. The animals were housed under standard conditions at temperature of 22 ± 10C, relative humidity of 55 ± 10% and 12 hr light/dark cycles. Animals were fed with standard pellet diet (Amrut, Pranav Agro Industries Ltd., Sangli, India) and water ad libitum. All the experiments performed are approved by the Institutional Animal Ethical Committee.

Grouping of animals

The animals were divided into five groups containing six animals in each group. Group I served as negative control (received normal saline, 5 ml/kg, p.o.), group II served as positive control (received Diclofenac sodium, 5 mg/kg, i.p.) and group III, IV and V were treated with VCM (100, 200, and 400 mg/kg) respectively.

Freund's adjuvant induced arthritis

Effect on Freund's adjuvant induced arthritis was studied according to the method [84]. Extract and reference standard treatment was continued upto 21 days. On first day, animals were injected with 0.05 ml of complete Freund's adjuvant. Paw volume was measured on 3rd, 9th, 15th and 21 day using plethysmometer (Ugo Basile, Italy) (Table25). On day 21st of treatment, blood samples were collected by retro-orbital puncture and investigated for hematological parameters such as hemoglobin content, WBC count, erythrocyte sedimentation rate and platelet count (Table 26). The qualitative analysis of blood samples for presence or absence of rheumatoid factor (IMUNOSTAT, Ranbaxy, India) and C-reactive protein (Rhelax-CRP, Tulip Diagnostics Pvt. Ltd., Goa, India) was studied according to kit manual. Arthritis score was calculated from secondary lesions. Changes in body weight were determined from initial and final readings. Results were given in table 27.

(3) Anti-inflammatory activity

Animals

Wistar male rats (180-220g) were used for study. The animals were housed under standard conditions at temperature of 22 ± 10C, relative humidity of 55 ± 10% and 12 hr light/dark cycles. Animals were fed with standard pellet diet (Amrut, Pranav Agro Industries Ltd., Sangli, India) and water ad libitum. All the experiments performed are approved by the Institutional Animal Ethical Committee.

Grouping of animals

The animals were divided into five groups containing six animals in each group. Group I served as negative control (received normal saline, 5 ml/kg, p.o.), group II served as positive control (received Diclofenac sodium, 5 mg/kg, i.p.) and group III, IV and V were treated with VCM (100, 200, and 400 mg/kg) respectively.

Carageenan-induced rat paw edema

Effect of drug on carrageenan induced acute inflammation was evaluated in rats according to the procedure [85]. After one hour of sample administration, 0.1 ml of 1% carrageenan solution was injected into sub-planter region of left hind paw of each rat. The paw volume was measured using plethysmometer (Ugo Basile, Italy) before and after 30, 60, 120 and 180min injection of carrageenan. Results were given in (Table 28)

Cotton pellet granuloma

Effect of extract in cotton pellet-induced sub-acute inflammation was studied according to the method reported [86] with some modifications. On day first, two sterile cotton pellets (20mg each) soaked in 0.2 ml of distilled water containing penicillin G (0.1 mg) and streptomycin (0.13 mg) were implanted into each groin region of rat. The extract/ reference compound dosing was continued for seven consecutive days. On 8th day the animals were sacrificed by cervical dislocation and pellets together with granuloma were carefully removed, dried in oven at 600C till constant weight. The percentage inhibition was calculated compared to control group. Results were given in (Table 29)

(4) Anti- diabetic activity

Animals

Wistar male rats (180-220g) were used for study. The animals were housed under standard conditions at temperature of 22 ± 10C, relative humidity of 55 ± 10% and 12 hr light/dark cycles. Animals were fed with standard pellet diet (Amrut, Pranav Agro Industries Ltd., Sangli, India) and water ad libitum. All the experiments performed are approved by the Institutional Animal Ethical Committee.

Grouping of Animals

Plant extract VCM and standard drug Glibenclamide (5 mg/kg) and saline were administered with the help of feeding needle. Group I serve as normal control, which received saline for 21 days. Group II to Group VI are diabetic control rats. Group II received Alloxan only and served as negative control. Group III to Group VI (which previously received alloxan) are given a fixed dose of VCM (100 mg/kg and 200 mg/kg, p.o), and standard drug glibenclamide (5mg/kg) for 21 consecutive days.

Induction of Diabetes by Alloxan in Experimental Animals:

Rats were made diabetic by a single intraperitoneal injection of alloxan monohydrate (150 mg/kg). Alloxan was first weighed individually for each animal according to the body weight and then solubilized with 0.2ml saline (154mM NaCl) just prior to injection. Two days after alloxan injection, rats with plasma glucose levels of >140 mg/dl were included in the study. Treatment with plant extracts was started 48 h after alloxan injection [87].

Collection of Blood Sample and Blood Glucose Determination:

Blood samples were drawn from tail tip of rat at weekly intervals till the end of study (i.e., 3 weeks). Fasting blood glucose estimation and body weight measurement were done on day 1, 7, 14 and 21 of the study. Blood glucose estimation can be done by GOD-POD kit using UV spectrophotometer (Shimatzu 1700) Results are given in (Table 30). On day 21, blood was collected from retro-orbital plexus under mild ether anesthesia from overnight fasted rats and fasting blood sugar was estimated. Serum was separated and analyzed for serum cholesterol, serum triglycerides by enzymatic DHBS colorimetric method, serum SGOT, serum SGPT, serum urea and serum insulin was estimated. Results are given in (Table 31).

Oral Glucose Tolerance Test [87]

Rats were divided into six groups containing six animals in each group. All animals fasted before treatment. Group I was kept as vehicle control which received 10ml/kg saline solution, group II received glucose only, group III received VCM 100 mg/kg, group IV received VCM 200 mg/kg, group V received VCM 100 mg/kg, VI received and group VI received reference standard (Glibenclamide 5mg/kg) in a vehicle, respectively. The rats of all groups were loaded with glucose (3 g/kg, p.o.) 30 minutes after drug administration. Blood samples were collected from puncturing the retro orbital sinus just prior to drug administration, and 30, 60, 90, 120 minutes after loading glucose. Serum glucose level was measured immediately by using glucose estimation kit (Nirmal Laboratories Ltd., India.). Results are given in (Table 32).

(5) Cytotoxicity [88]

Animals:

Brine strimp (fishes) of species Artemia salina were used for the assay procedure.

Preparation of stock (Sample):

10mg of the sample extract (VCM) was dissolved in 10ml of methanol to produce 1000µg/ml of the stock solution and further dilutions (5, 10, 25, 50 and 100 µg/ml) were made in distilled water.

Preparation of stock (Standard, caffeine)

1000µg/ml of stock solution was prepared and further diluted (50, 100, 200, and 300µg/ml).

Procedure

In this test, brine shrimp (Artemia salina) eggs were hatched in artificial sea water (40 g/l sea salt). Samples of test drugs for the experiment were prepared in methanol solution generally, which acts as control vehicle. After 48 hr. of incubation, 15 brine shrimps were transferred to each sample vial using pipette and artificial sea water was added to make 5 ml. Sample vials were previously prepared by dissolving specific concentration of test drugs with different dilutions. Concentration of methanol should not exceed than one percent. Survivors were counted after 24 hr and the LC50 values determined using prohibit analysis. Results were given in table 33.Control vial was prepared using methanol only. Three replicated were prepared for each concentration of test drugs.The lethal concentrations of tests resulting in 50 % mortality of the brine shrimp (LC50) and 95% confidence intervals were determine from the 24 hr counts (Table 34).

(6) Anti- oxidant activity

1) DPPH scavenging activity

Preparation of reagent

DPPH solution was prepared by dissolving 33 mg 1, 1-diphenyl -2-picryl-hydrazyl (DPPH) in 1000ml methanol.

Preparation of stock (Sample):

2mg of extract (VCM) was dissolved in 10ml of methanol to give 200 µg/ml solutions.

Preparation of stock of standard (BHA, Ascorbic acid):

1mg of standard drugs was dissolved in 10ml of distilled water to give 100 µg/ml solutions.

Procedure:

The DPPH radical scavenging activity of the methanol extract was using UV spectrophotometer [89-90]. To 1ml of extract (10-100µg/ml) in methanol was added 2.5ml of DPPH (1 mM in ethanol) and methanol to give a final volume of 5ml. The resultant mixture was shaken and incubated at room temperature in the dark for 30 min. At the end the absorbance of mixture was measured at 517 nm using Microtiter Plate Reader (PowerWave XS, Bio-tek, USA). The percent antioxidant activity was calculated by using following formula [65]. Results were given in table 35

% antiradical activity = (A Control. - A sample) / A Control X 100

Where A Control: Control absorbance (DPPH).

A sample: Sample/standard absorbance.

2) Nitric oxide scavenging activity

The nitric oxide radical scavenging activity was evaluated according to reported method [91]. Nitric oxide radicals are generating from sodium nitroprusside solution at physiological pH. (1 ml, 10 mM) Sodium nitroprosside was mixed with (10-100µg/ml) 1ml extract in phosphate buffer (pH 7.4). The mixture was maintained at 250C for 150 min. To 1 ml of this solution, 1ml of Griess reagent (-naphthyl-ethylenediamine dihydrochloride 0.1% in water and sulfanilamide 5% in H3PO4) was added and absorbance was read at 546 nm. The blank reading was taken without extract/standard which was served as control. Results were given in table 36

% Inhibition = (A Control. - A sample) / A Control X 100

Where A Control: Control absorbance.

A sample: Sample/standard absorbance.

3) Anti-lipid peroxidation activity using rat brain homogenate

Table 5. Preparation of reagents:

Sr. no.

Reagent

Preparation

01.

Sodium dodesyl sulphate (SDS) 8.1%w/v

8.1g of SDS was dissolved in distilled water and make up volume up to 100ml.

02.

Acetic acid (20%v/v), pH 3.5

20ml of acetic acid was dissolved in distilled water and make up volume up to 100ml.

03.

Thiobarbituric acid (TBA) 0.8%w/v

0.8g of TBA was dissolved in distilled water and make up volume up to 100ml, heated at 600c to dissolve if necessary.

04.

Tris-HCl buffer ( 20mM, pH 7.4)

50ml of Tris solution (0.02M) was added to 41.4ml of (0.02M) HCl solution and diluted upto 200ml with distilled water.

05.

Ammonium ferric sulphate (100µm)

4.82mg of ammonium ferric sulphate was dissolved in distilled water and make up volume up to 100ml.

Preparation of stock (standard, BHA and ascorbic acid):

10mg of standard drug was dissolved in 100ml of the distilled water to produce 100µg/ml of stock solution.

Preparation of stock (Sample):

25mg of the sample extract (VCM) was dissolved in 100ml of methanol to produce 250µg/ml of the stock solution.

Procedure [92]

To the 100µl of rat brain homogenate 10µl of different concentration (10-100µg/ml) of extract were added. By adding 10µl of 100µM Fe3+ (ammonium ferric sulphate) solution Lipid per oxidation was stimulated and the mixture was maintained for 30min. at 37oC. sodium dodesyl sulphate (SDS) solution was added to stop precipitation. Further oxidation was stopped by adding of 750µl of 20%w/v acetic acid solution. By centrifugation, the precipitated proteins were removed. 500µl of the clear supernatants were heated in glass tubes with an equal volume of 0.8%w/v of Thiobarbituric acid solution at 95 0C for 30min. Samples were cooled and 100µl of each sample was pipetted into 96 well micro titer plate and absorbance was read at 532nm using micro plate reader. Results were given in table 37

4) Determination of reducing power assay

Determination of reducing power assay

Due to the transformation of Fe +3 to Fe +2 in presence of drug/extract the reducing capability can be measured the reported method [93].

Table 6. Preparation of reagents:

Sr. No.

Reagents

Preparation

1)

Phosphate buffer

(pH 7.6).

2.38g disodium hydrogen phosphate + 0.19g potassium hydrogen phosphate + 8g sodium carbonate were dissolved in 1000ml of deionized water.

2)

Potassium ferricyanide

1% (% w/v).

1g of potassium ferricynide was dissolved in 100ml of distilled water.

3)

Trichloroacetic acid (TCA) 10%.

19g of TCA was dissolved in 100ml of distilled water.

4)

Ferric chloride ( 0.1% )

100mg of ferric chloride was dissolved in 100ml of distilled water.

Preparation of stock (Sample):-

2mg of extract (VCM) was dissolved in 10ml of methanol to give 200 µg/ml solutions.

Preparation of stock of standard (BHA, Ascorbic acid):

The stock solution (100µg/ml) was prepared by dissolving 10 mg of standard in 100 ml of distilled water and further diluted to produce 10, 20, 30, 40, 60 and 80 µg/ml.

Procedure

1ml of different concentrations of extract (10-100µg/ml), 2.5ml phosphate buffer (0.25 M) and 2.5 ml potassium ferricyanide (1% w/v) were added. The mixture was maintained at 500C for 20min. After this period 2.5ml of Trichloroacetic acid was added, and then it was centrifuged. 2.5ml of supernatant was taken and to that 2.5ml of distilled water and 0.5ml of ferric chloride solution (0.1% w/v) was added. Then the absorbance was measured at 700 nm. Results were given in table 38

Microbial Screening (Anti-bacterial and Anti- fungal) [96-97]

Extracts for antimicrobial activity

Pet. Ether, benzene, chloroform, acetone, methanol and aqueous extracts were used for studying their antimicrobial activity. In preliminary study, all above extracts were studied for antibacterial and antifungal activity using broth dilution method at the dose of 200µg/ml (Table 7), and extracts which showing 70% inhibition were forwarded for MIC.

Test organisms

The antimicrobial activity was performed using five strains of gram positive bacteria Staphylococcus aureus NCIM 2079, Klebsiella pneumoniae NCIM 2719, Bacillus subtilis NCIM 2250, Bacillus stearothermophilus NCIM 2235, Bacillus pumilus NCIM 2327, five strains of gram negative bacteria Proteus vulgaris NCIM 2813, Salmonella paratyphi NCIM 2479, Shigella sonnei MTCC 2957, Pseudomonas aeruginosa NCIM 2719, Escherichia coli NCIM 2109, and five strains of fungal Fusarium moniliforme NCIM 1099, Candida guilliermondii NCIM 3126, Candida albicans NCIM3471, Aspergillus niger NCIM 545 and Trichoderma viride NMIM 1221. All the strains were procured from the National Chemical Laboratory (NCL), Pune except Shigella sonnei MTCC 2957 (Institute of Microbial Technology, Chandigarh).

Minimum inhibitory concentration (MIC) [98-99]

Requirements: Nutrient broth, incubator and autoclave.

Table 7. Composition of Nutrient broth (Hi- Media, Mumbai, code M002)

Sr. no.

Composition

gm/liter

1.

Peptic digest of animal tissue

5.00

2.

Beef extract

1.50

3.

Yeast extract

1.50

4.

Sodium chloride

5.00

Final pH 4 ± 0.2, take 13 gm/liter

Procedure:

5 ml of sterilized nutrient broth was poured into sterilized test tube. 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 and 350 µg/ml concentrations of different extracts were prepared in 1%DMSO. 0.1ml of above dilutions was added to nutrient broth. 0.1 ml of bacterial/fungal suspension was added to all tubes and the tubes were maintained at 370 C for 18 - 24 hrs. The growth in tube was observed for turbidity using UV spectrophotometer at 625nm and inhibition was determined by absence of growth. MIC was determined by the lowest concentration of sample that prevents the development of turbidity. Results of % inhibition of microbial (Bacteria/ fungi) growth and MIC (MBC/MFC) are given in table 39 and 40 respectively.

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