Collection of Plant Material

    Dostsabha
    By Dostsabha
    1. MATERIALS AND METHODS

    5.1 Collection of Plant Material

    The seeds of Linum usitatissimum was collected from the local market of Indore (M.P.).

    5.2 Identification and Authentification

    The seeds of Linum usitatissimum was identified and authenticated at Department of Botany, Govt. Agriculture College, Indore. The voucher specimen has been deposited at our college for further reference.

    5.3 Preparation of Extract

    The experiments were carried out using air-dried plant materials which were reduced to moderately coarse powder using mechanical grinder. The powdered was passed through sieve no #40 and stored in air-tight containers for further use. The ethanolic extracts of dried powder drug were prepared as follows:

    Preparation of Ethanolic Extract

    The air dried coarse powder of the rhizomes was well packed in soxhlet apparatus and subjected for continuous hot extraction with 70% ethanol. The extract was filtered while hot and the filtrate was distilled under reduced pressure in order to remove solvent completely. The residue was dried and stored in desiccators, used for subsequent experiments. The percentage yield was calculated by using following formula:-

                          

    5.4 Preliminary Phytochemical Studies

    5.4.1 Test for Glycosides

    1. a) Legal Test: - The extract was dissolved in pyridine and sodium nitroprusside was added to the solution to make it alkaline. The formation of pink red to red color showed the presence of glycosides.
    2. b) Baljet Test: - To 1ml of the test extract, 1ml of sodium picrate solution was added and the yellow to orange color revealed the presence of glycosides.
    3. c) Borntrager’s Test: - To 2 ml of dilute Sulphuric acid to 1ml of the extract solution was added. It was boiled and filtered and the filtrate was extracted with choloform. The chloroform layer was treated with 1ml of ammonia. The formation of red color in the ammonical layer showed the presence of anthraquinone glycosides.

    5.4.2 Test for Carbohydrates

    (a) Molisch’s Test:-To 2-3 ml of the extract, few drops of a-napthol solution in alcohol were added, shaken and concentrated sulphuric acid was added through the side of the test tube. Violet colour ring was formed at the junction of the two liquids reveals the presence of Carbohydrates.          

    (b) Fehling’s Test:-To 1ml of the extract, equal quantities of Fehling solution A and B, were added. Upon heating formation of a brick red precipitate indicated the presence of sugars.

    (c)Benedict’s Test:-To 5ml of Benedict’s reagent,  1ml of extract solution was added and boiled for 2 minutes and cooled. Formation of red precipitate showed the presence of reducing sugars.

    5.4.3 Test for Flavonoids

    (a) Shinoda’s Test: - To dry powder or extract, 5 ml 95% ethanol and few drops of conc. HCl were added then 0.5g magnesium turnings were added. Pink color showed the   presence of flavonoids.

    (b) The extract was treated with sodium hydroxide; formation of yellow colour indicated the presence of flavonoids.

    (c)The extract was treated with lead acetate solution, formation of yellow colour precipitate showed presence of flavonoids.

    5.4.4 Test for Saponins

    (a) Foam Test: - Small quantity of alcoholic and aqueous extracts and fractions were taken separately and 20 ml of distilled water was added and shaken in a graduated cylinder for 15 minutes lengthwise. A layer of foam indicated the presence of saponins. (Agarwal et al, 2007).

    5.4.5 Test for Phenolic Compounds and Tannins

    (a) To 2-3ml of alcoholic extract, lead acetate solution was added. Formation of a white precipitate indicated the presence of tannins.

    (b) To 2-3 ml of alcoholic extract, 5% ferric chloride solution was added, formation of a deep blue- black colour product showed the presence of tannins.

    (c) A little quantity of test extract was treated with Potassium ferric cyanide and ammonia solution. A deep red colour indicates the presence of tannins.

    (d) To the test extract, strong Potassium dichromate solution was added; a yellow colour precipitate indicates the presence of tannins (Khandelwal, 2005).

    5.4.6 Test for Triterpenoids

    Noller’s Test- Two or three granules of tin metal was dissolved in 2 ml thionyl chloride and then 1 ml of extract was added to it. Test tube was warmed; the formation of pink colour indicated the presence of triterpenoids.

    5.5 Pharmacological Activity

    Experimental Animals

    Wistar albino rats  of either * * * (150-200g) were used in the study. The animals were housed in polypropylene cages under standard conditions (12 h light; 12 h dark cycle; 25 ± 50 C; 35-60% humidity). They were fed with standard pellet diet and water ad libitum. The experimental protocol was approved by the Institutional Animal Ethical Committee of our institute. ()

    5.5.1 Acute Toxicity Study

    The acute toxicity test aims at establishing the therapeutic index, i.e. the ratio between the pharmacologically effective dose and lethal dose on the same strain and species. Greater is the index; safer is the compound vice versa.

    The acute toxicity study was done according to OECD (Organization of Economic Co-operation and Development) guidelines 420- Fixed Dose Procedure (FDP), as in annex 2D.

    Procedure

    • Healthy adult wistar albino rats of either * * *, starved overnight were divided in to two groups (n=6).
    • Then the defined or fixed dose levels of ethyl alcohol extract of Linum usitatissimum in 1000 mg/kg was given to identify a dose producing evident toxicity.
    • The dose was given by oral route.
    • After giving the dose, the toxicity signs were observed continuously for fourteen days and finally mortality was recorded.
    • Following was observed; body weights of the rats before and after drug administration, onset of toxicity and sign of toxicity like change in skin and fur, eyes and mucous membrane and also respiratory arrest, circulatory, autonomic and central nervous system and somatomotor activity, behavior. (Ghosh M.N et al.,2000, Kulkarni S.K et al., 2002)

     

    Observation: 

    The acute toxicity studies shows that all the extracts were safe up to 1000mg/kg body weight, does 500mg/kg dose was considered as a safe dose. So 2 doses 250mg/kg and 500mg/kg was selected for all In Vivo experiment as maximal dose.

    5.5.2 Carragenan Induced Rat Paw Edema

    The rats were divided into six groups (n=6). Inflammation was induced by injection of 0.1 ml of freshly prepared carragenan (1%) aqueous suspension in normal saline underneath the plantar tissue of the right hind paw of rats. 1 hour prior to carragenan administration groups served test drugs. Group I served as negative control.  Group II served as normal control and received saline solution as a vehicle. Group III served as standard diclofenac sodium 15mg/kg p.o. Group IV and V received test extracts 250 and 500 mg/kg  p.o/ bw. The paw volume was measured initially and then at 1, 2, 3, 4 and 5 h after the carrageenan injection by using digital plethysmometer.

    Experimental Design

     Group I: Negative control served 0.1 ml of carragenan.

    Group II:  Control received vehicle (0.9%w/v Saline) and 0.1 ml of carragenan.

    Group III: Standard received Diclofenac sodium (5 mg/kg bw) and 0.1ml of carragenan

    Group IV: Test Extract (250mg/kg bw) and 0.1 ml of carragenan.

    Group V: Test Extract (500mg/kg bw) and 0.1 ml of carragenan.

    The anti inflammatory effect of Linum usitatissimum extract was calculated by the     following equation: % of inhibition =VC – VT/VC× 100

    Where VC and VT are the paw volume in control rats and treated group of rats respectively. (Mukherjee et al.,2006)

    5.5.3  Histamine Induced Rat Paw Edema

    The rats were divided into six groups (n=6). Inflammation was induced by injection of 0.1 ml of histamine solution underneath the plantar tissue of the right hind paw of rats. 1 hour prior to histamine administration groups served test drugs. Group I served as negative control.  Group II served as normal control and received saline solution as a vehicle. Group III served as standard diclofenac sodium 15mg/kg p.o. Group IV and V received test extracts 250 and 500 mg/kg  p.o/ bw. The paw volume was measured initially and then at 1, 2, 3, 4 and 5 h after the histamine injection by using digital plethysmometer.

    Experimental Design

     Group I: Negative control served 0.1 ml of  histamine solution.

    Group II:  Control received vehicle (0.9%w/v Saline) and 0.1 ml of histamine solution.

    Group III: Standard received Diclofenac sodium (5 mg/kg bw) and 0.1ml of  histamine solution.

    Group IV: Test Extract (250mg/kg bw) and 0.1 ml of histamine solution.

    Group V: Test Extract (500mg/kg bw) and 0.1 ml of histamine solution.

    The anti inflammatory effect of Linum usitatissimum extract was calculated by the     following equation: % of inhibition =VC – VT/VC× 100

    Where VC and VT are the paw volume in control rats and treated group of rats respectively.

    5.5.4 Formalin Induced Rat Paw Edema

    The effects of extracts and indomethacin on the acute phase of inflammation were investigated. Doses of extracts were administered orally once a day for a period of 2 days. An hour after the last dose was administered; 0.2 ml of formalin (1%, w/v) was injected into the rat hind paw. Before formalin injection, the paw volume for each rats were measured separately by means of Plethysmometer. Edema caused by formalin was measured at 3, 6 and 24 h the first day, and measured once per day on the following days until inflammation disappeared. The anti-inflammatory potency of extracts was determined by comparing it with a group in which a 5mg/kg dose of indomethacin was administered orally.

    Experimental Design

     Group I: Negative control served 0.2 ml of formalin solution.

    Group II:  Control received vehicle (0.9%w/v Saline) and 0.2 ml of formalin solution.

    Group III: Standard received Indomethacin (5 mg/kg bw) and 0.2 ml of formalin solution.

    Group IV: Test Extract (250mg/kg bw) and 0.2 ml of formalin solution.

              Group V: Test Extract (500mg/kg bw) and 0.2 ml of formalin solution.

              The anti inflammatory effect of Linum usitatissimum extract was calculated by the

    following equation: % of inhibition =VC – VT/VC× 100

    Where VC and VT are the paw volume in control rats and treated group of rats respectively.

    Statistical Analysis

    The data were expressed as mean±SEM. The statistical significance was determined by using the ANOVA followed by Dunnets t test. Values of P < 0.05 were considered as statistically significant.

     

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