Evaluation Of Anti-Inflamation & Memory Enhancing Activity For Some Pharmacological Active Extracts

The use of herbal medicines continues to expand rapidly across the world. Many countries now turn towards herbal medicines or herbal products for their health care in national health-care settings. According to WHO, 80% if the rural population in developing countries depend on traditional medicines to meet their primary health care needs (1). Authentication and standardization are prerequisite steps while considering source materials for herbal formulation in any system of medicine (2). In traditional systems of medicine, the drugs are primarily dispensed as water decoction or ethanol extract. Fresh plant parts, juice, or crude powders are a rarity rather than a rule. Thus medicinal plant parts should be authentic and free from harmful materials like pesticides, heavy metals, microbial or radioactive contamination, etc. (3). It is very important that a system of standardization should establish for every plant medicine in the market because the scope for variation in different batches of medicine is enormous. World Health Organization (WHO) encourages, recommends, and promotes traditional / herbal remedies in national health care programmes because these drugs are safe, people have faith in them and easily available at low cost. The WHO is continuously emphasizing to ensure quality control of medicinal plant products by using modern techniques and applying suitable standards (4). India has a rich heritage of traditional medicine constituting with its different components like Ayurveda, Siddha, Unani, Homoeopathy and naturopathy. Traditional health care has been flourishing in this country for many centuries (5). The growing use of botanicals by the public is forcing moves to evaluate the health claims of these agents and to develop standards of quality and manufacture. Various traditional medicine systems, especially Indian system of medicine attracted the global attention due to their long historical clinical use and reliable therapeutic efficacy. Many big pharmaceutical companies are using traditional medicine as an excellent pool for discovering natural bioactive compounds.

MATERIAL & METHODS-

The whole plant parts of Convolvulus pluricaulis Choisy() were purchased from the local Aria and also collected from Govt.Ayurvedic College(Medicinal Garden) Gwalior Madhya Pradesh .

1. 2210763985077298. of total crude extract

Whole plant materials were dried under shade and subjected to coarse powder for extraction process. Accurately weighed quantity of whole plant material was extracted using 95 % ethanol by soxhlet apparatus for 72 h. The ethanolic extracts were dried under the reduced pressure to get crude methanolic extracts. The ethanolic extracts were dried completely under reduced pressure. After drying, the respective extracts were weighed and percentage yield was determined.

1. 2210734273300955. phytochemical tests

Qualitative chemical tests of ethanolic extracts were subjected to various chemical tests to detect various phytoconstituents.

Test for Alkaloids: -

Dragendorff’s test: - To the extract add Dragendorff?s reagent, reddish brown precipitate indicates presence of alkaloids.

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

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

Test for Amino acids:-

Millon’s test: - To the extract add about 2ml of Millions reagent white precipitate indicates presence of amino acids.

Ninhydrine test: - To the extract add Ninhydrine solution, boil, violet colour indicates presence of amino acid.

Test for Flavonoids: -

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

Alkaline reagent test: - To the extract add few drops of sodium hydroxide solution, intense yellow colour is formed which turns to colorless on addition of few drops of dilute acid indicate presence of flavonoids.

Test for Phenolics compounds (Tannins):-

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

Phenazone test: -Add about 0.5 gm of sodium acid phosphate to 5ml of extract warm it and filter. To the filtrate add 2% phenazone solution, bulky precipitate is formed, which is often colored.

Gelatin test: -To the extract add 1% gelatin solution containing 10% sodium chloride. Precipitate is formed.

Test for Proteins: -

Biuret test: - To the extract (2ml) add Biuret reagent (2ml), violet colour indicates presence of proteins.

Hydrolysis test: - Hydrolyze the extract with hydrochloric acid or sulphuric acid. Then carry out the Ninhydrine test for amino acids.

Test for Steroids and Triterpenoids: -

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

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

Test for Carbohydrates: -

Molish’s test: -To the extract add few drops of alcoholic ?-naphthol, then add few drops of concentrated sulphuric acid through sides of test tube, purple to violet colour ring appears at the junction.

Fehling’s test: - Mix 1 ml Fehling?s A and 1 ml Fehling?s B solutions, boil for 1 min. add equal volume of test solution. Heat in boiling water bath for 5-10 min. first a yellow, then brick red precipitate is observed.

Benedict’s test: -Mix equal volume of Benedict?s reagents and test solution in test tube. Heat in boiling water bath for 5 min. solution appears green, yellow or red depending upon amount of reducing sugars present in test solution.

Test for Anthraquinone glycosides: -

Borntrager’s test: - Boil the test material with 1ml of sulphuric acid in a test tube for 5minutes. Filter while hot. Cool the filtrate and shake with equal volume of dichloromethane or chloroform. Separate the lower layer of dichloromethane or chloroform and shake it with half of its volume of dilute ammonia. A rose pink to red colour is produced in the ammonical layer.

Modified Borntrager’s test: - Boil 200mg of the test material with 2ml of dilute sulphuric acid. Treat it with 2ml of 5% aqueous ferric chloride solution (freshly prepared) for 5 minutes, shake it with equal volume of chloroform and continue the test as above. As some plants contain anthracene aglycone in a reduced form, if ferric chloride is used during the extraction, oxidation to Anthraquinone takes place, which shows response to Borntrager?s test.

Test for Coumarin glycosides: -

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

Test for Saponin glycosides: -

Froth formation test: -Place 2ml solution of drug in water in a test tube, shake well, stable froth (foam) is formed.

Test for Cardiac glycosides: -

Keller-killiani test (test for deoxy sugars): -Extract the drug with chloroform and evaporate it to dryness. Add 0.4ml of glacial acetic acid containing trace amount of ferric chloride. Transfer to a small test tube; add carefully 0.5ml of concentrated sulphuric acid by the side of the test tube. Acetic acid layer shows blue colour.

Legal’s test: - Treat the extract with pyridine and add alkaline sodium nitroprusside solution, blood red colour appears.

Baljet’s test: - Treat the extract with picric acid or sodium picrate, orange colour is formed.

1. 1. Activity  

Membrane Stabilizing Activity Of Methanolic Extract

The human red blood cells (HRBC) membrane stabilization had been used as a method to study anti-inflammatory activity. In this method, blood was collected from healthy human volunteers who had not been taken anti-inflammatory drug for 2 weeks prior to the experiments. This blood was mixed with equal volume of sterilized alsever solution (2 ?xtrose, 0.8% sodium citrate, 0.5% citric acid and 0.42%sodium chloride). The blood was centrifuged at 3000 rpm and packed cells were washed with isosaline and a 10% suspension was made with iso saline. Various concentrations of test samples (F1- F6) in concentration of

    protection was calculated using the following formula.

% Protection= Absorbance of control-Absorbance of test×100

                              Absorbance of control

Inhibition Of Protein Denaturation Method

The ethanolic extract of selected plant materials were suspended in distilled water. The following procedure was followed for evaluating the percent of inhibition of protein denaturation: -

Control solution (50 ml) consists of 2ml of Egg albumin (from fresh hen?s egg) and 28 ml of phosphate buffer (PBS, pH 6.4) and 20ml distilled water.

Standard drug (50 ml) consists of 2ml of Egg albumin and 28 ml of phosphate buffer and 20 ml of various concentrations of standard drug Diclofenac sodium (20, 40, 60, 80

& 100 µg/ml).

Test solution (50 ml) consists of 2ml of Egg albumin and 28 ml of phosphate buffer and 20 ml of various concentrations of methanolic extract in a concentration of 100, 200, 300, 400, 500 µg/ml. All of the above reaction mixtures were adjusted to pH 6.4, using a small amount of 1N HCl. The samples were incubated at 37°C for 15 minutes and heated at 70°C for 5 minutes. After cooling, the absorbance of the above solutions was measured using UV- spectrophotometer at 660 nm. The percent inhibition of protein denaturation was calculated using the following formula (57, 58).

Proteinase Inhibitory Activity

The methanolic extract of whole plant materials were suspended in distilled water.

The following procedure was followed for evaluating the Proteinase Inhibitory Activity. Control solution (50 ml) 20 ml consists of 0.06 mg Trypsin, 10 ml 20mM Tris HCl buffer (pH 7.4) and 10 ml distilled water.

Standard drug (50 ml) 20 ml consists of 0.06 mg Trypsin, 10 ml 20mM Tris HCl buffer (pH 7.4) and 10 ml of various concentrations of standard drug Diclofenac sodium. (20, 40, 60, 80

& 100 µg/ml).

Test solution (50 ml) 20 ml consists of 0.06 mg Trypsin, 10 ml 20mM Tris HCl buffer (pH 7.4) and 10 ml of various concentrations of methanolic extract in a concentration of 100, 200, 300, 400, 500 µg/ml. All the reactions were incubated at 37^o C for 5 minutes and then 10 ml of 0.8% (w/v) casein was added. Again the samples were incubated for an additional 20 min and 10 ml of 70% perchloric acid was added to arrest the reaction. Cloudy suspension was centrifuged and the absorbance of the supernatant was read at 210 nm against buffer as blank. The experiment was performed as triplicate. The percent inhibition of proteinase inhibitory activity was calculated (59).

Memory Enhancing Activity Determination of Acetylcholinesterase Inhibitory Activity.

AChE activity was measured using a modified 96-well microplate assay (11) based on Ellman?s method (21). The enzyme hydrolyses the substrate acetylthiocholine resulting in the product thiocholine which reacts with Ellman?s reagent (DTNB) to produce 2- nitrobenzoate-5-mercaptothiocholine and 5-thio-2-nitrobenzoate which can be detected at 412 nm. 50 mM Tris–HCl pH 8.0 was used as a buffer throughout the experiment unless otherwise stated. AChE used in the assay was from electric eel (type VI-S lyophilized powder, 518 U/mg solid, 844 U/mg protein). The enzyme stock solution (518 U/ml) was kept at 280uC. The further enzyme-dilution was done in 0.1% BSA in buffer. DTNB was dissolved in the buffer containing 0.1 M NaCl and 0.02 M MgCl2. ATCI was dissolved in deionized water. In the 96-well plates, 100 ml of 3 mM DTNB, 20 ml of 0.26 U/ml of AChE, and 40 ml of buffer (50 mM tris pH 8.0), 20 ml of extract in various concentrations (25, 50, 100, 250 and 500 mg/ml) dissolved in buffer containing not more than 10% methanol were added to the wells. After mixing, the plate was incubated for 15 min (25uC) and then the absorbance was measured at 412 nm in Tecan infinite 200 microplate reader and the readings were used as blank. The enzymatic reaction was initiated by the addition of 20 ml of 15 mM ATCI and the hydrolysis of acetylthiocholine was monitored by reading the absorbance every 5 min for 20 min. Physostigmine was used as positive control. All the reactions were performed in triplicate. The percentage inhibition was calculated as follows

Percentage Inhibition= (E S)/E ×100   

Where

 E₌ is the activity of the enzyme without extract

 S= is the activity of enzyme with the extract.

RESULTS AND DISCUSSION-

Extractive value determination

Dried whole plant material of Convolvulus pluricaulis Choisy were extracted by ethanol. The percentage yields of all dried extracts were determined by using the following formula.

       
           
       
TableNo.1: Different extracts with their appearance and % yield (in gm)

1. 2210734273300955. phytochemical screening

The preliminary phytochemical analysis revealed that different active constituent present in different extracts such as carbohydrates, proteins, amino acids, fat, oils, steroids, terpenoids, glycosides, alkaloids, tannins and other phenolics compounds.

1. 338898. layer chromatographic studies of bioactive extracts

1. 13133. study has shown the presence of different components present in ethanolic extract of Convolvulus pluricaulis Choisy when the extract were run in specific solvent system. Before reaching to most optimum solvent system a number of systems were employed.

TLC of methanolic extract of Convolvulus pluricaulis Choisy

Membrane Stabilizing Activity of Extracts on Rat Erythrocytes

The ethanolic extract was analyzed by membrane stabilizing property at concentration range of 100-500 µg/ml. Ethanolic extract significantly protect the rat erythrocyte membrane against lysis induced by hypotonic solution. At a concentration of 500 µg/ml, the extract produced 72.60% protection of RBC haemolysis as compared with 73.84% produced by prednisolone.

The ethanolic extract of selected plant materials showed 42.65% inhibition respectively.

The Diclofenac sodium showed 63.89% inhibition against proteinase inhibitory activity.

The ethanolic extracts of selected plant material used in Ayurveda for nervous system disorders were tested for AChE inhibitory activity using Ellman?s colorimetric method in 96- welled microplate. The inhibition curves have been presented in Table 5 representing the % inhibition at 100 mg/ml and IC50 for extracts. Physostigmine was used as the standard AchE inhibitor in this study which showed IC50 of 0.07560.003 mg/ml. IC50 for AchE inhibitory activity was 7763.1 mg/ml respectively. The plant showed very high antioxidant activity also in the DPPH assay.