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Phytochemical studies:

• Preliminary Phytochemical studies2, 25
Preliminary test was carryout for the presence/absence of phytoconstituents like Alkaloids, Glycosides, Carbohydrates, Flavonoids, Reducing sugars, Saponins, Sterols, Terpenes, Tannins.

Choice of solvents:-
According to phytochemical studies, methanolic extract contains more chemical constituents than other solvent extract. So Methanol was selected as choice of solvent for extraction of dried leaves of  Polygonum glabrum.

Solvent purification:-
The various solvents there may be admixed with any other substances, so it needs purification before use. Here the solvents are subjected to simple distillation under atmospheric pressure.

A description of method was adopt for performing the tests are summarized below.
1. Alkaloids
(A) With Mayer’s reagent:-

The methanolic extract was treated with few drops of dilute 2N HCl and 0.5ml of Mayer’s reagent, white ppt. obtained

(B)  With Wagner’s reagent:-
The methanolic extract was treated with few drops of dilute 2N HCl and 0.5ml Wagner’s reagent, brown flocculent ppt. was obtained.

(C) With Dragendroff’s reagent:-
The methanolic extract was treated with few drops of dilute 2N HCl and 0.5ml Dragendroff’s reagent ,brown ppt. was obtained.

2. Phenols

(A) With neutral FeCl3:-
The extract was treated with neutral FeCl3, formation of blue, green, or red color, indicates presence of phenols.

(B) With Ferrous sulphate and Sodium potassium Tartarate:-
The reagent was prepared by mixing 0.1%  Ferrous sulphate solution and 0.5% Sodium potassium Tartarate solution. Few drops of reagent was added to a little of the residue, blue, green, violet, or red colouration was produced. Phenols containing two hydroxyl groups to each other give this test.

3. Glycosides

(A). Molisch’s test:-
To a small portion of the extract and 2 drops of 10% solution of α-Napthol in chloroform. Carefully added 1 drop of concentrated H2SO4 from a finely pointed pipette so that it form a separate layer below the water. A violet ring formed at junction of the two layers. On mixing the layers by shaking the test tube in a stream of cold water, a deep purple solution results which on dilution with cold water yields a violet ppt. On shaking and adding a small quantity of suspension to an excess of conc. Ammonia, the color is changed to dull brown.

(B).With Concentrated H2SO4 :-
Extract containing glycosides gives intense colours when treated with cold concentrated H2SO4 .

4. Flavonoids

(A). Shinoda’s test:-
An alcoholic extract of leaves was treated with Magnesium (dust) and concentrated HCl, the appearance of pink tomato colour indicates the presence of flavonoids.
(B).Dissolve sample in 10% HCl and add Zink (dust), pink colour effervescence indicates the presence of flavanones and flavanols.
(C).When treated with neutral lead acetate  gives yellow, orange, red, or brick colour, indicates the presence of flavanoids.
(D).Aques extract dissolved in H2SO4 to give intensely yellow solution.

5. Terpenoids and Steroids
The terpenoids have a common biosynthetic origin. The terpenoids includes
Essentiol oils
Diterpenoids and Gibberlines
Triterpenoids and Steroids
Caretonoids etc.

Lebermann-Burchard reaction
The extract was treated with acetic anhydride and concentrated H2SO4, gives purple red color.

6. Test for Reducing Sugars

(A).Benedict’s test:-
Take 5ml of reagent in test tube, add 8 drops of extract. Placed in boiling water bath for 5min. A green, orange-red ppt. gives a semi quantitative estimation of reducing sugars.
(B). Fehling’s test for Reducing Sugars:-
Take 2ml of extract, added equal volume Fehling A and Fehling B mixture, placed in boiling water bath for 5min. A red ppt. was obtained.

7. Saponins

(A)  Add some water to sample and shake vigorously formation of stable froth with honeycomb structure indicates the presence of saponins.
(B).To an aqueous solution of extract, add solution of lead acetate, formation of white ppt. indicates the presence of saponins.

8. Tannins
A small portion of extract was treated with 5% Ferric Chloride solution. The production of green, to blue color was taken as positive test for tannins.

(B). A creamy ppt. with lead acetate was considered positive test for tannins.

Data for Qualitative Phytochemical studies



1-Dragendroff’s test

2- Wagner’s test

3- Mayer’s test

4- Hager’s test








1- With neutral FeCl3

2- With FeSO4 & Sod. Pot.Tartarate






1- Molisch test

2-With Conc.H2S04






1- Sinoda  test





1-Libermann Burchared test




Reducing Sugar

1-Benedict test

2-Fehling test





1-Saponine test





Pharmacological studies

Animals: Adult Charles Foster albino rats (200±20g), of either sex, were obtained from the Animal house of S.R.L.T, and were randomly distributed into diffrent experimental graph. The rats will be house in group of six in polypropylene cages at an ambient temperature of 250C ± 10Cand45-55%RH, with 12:12 h light/dark cycle. Animal were provided with commercial food pallets and water ad libitum unless stated otherwise. Experiments were conducted between 9.00 and 14.00 h. Animal were acclimatized for at least one week before using them for experiments and exposed only once to every experiment. “Principals of laboratory animal care” (NIH publication number 85-23, revised 1985)28 guidelines were followed.

Drug treatments:
The methanolic extract of Polygonum glabrum were protected from light and stored under refrigeration at 0-40C until its use. Extract was solubilized in distilled waterbefore experimental studies, and administered either orally or intraperitoneally at different dose levels for three consecutive days. Control animals were treated with distilled water 10ml/kg. Standard drugs were used in each set of experiment accordingly and were administered either orally or intraperitoneally to rodents 1h or 30min before experiments for comparison respectively. Experiments were conducted on day 3, 1hr after the last drug administration.

Statistical analysis: The data were expressed as means ±SEM for each treatment group. Statistical significance between groups was performed by the application of analyses of variance ANOVA followed by Dunnett’s test, p values less than 0.05 (p < 0.05) were used as the significant level.

Drug and Chemicals for Screening Methods: The following drug and chemical used and all the reagent and chemicals were of analytical grade.
•         Phenobarbitone (Sigma,St,Louis,USA)(20MG/KG,i.p.) was used as standard anticonvulsant agent.
•         Dopamine (DA),Norepinephrine(NE), Serotonin(5-HT),and 5-hydroxy indole acetic acid (5-HIAA)  was  procure from Sigma,St Louis, USA.
•         n-Butanol(fluorometric grade) use without further purification.
•         n-heptane was wash with one-fifth volume 1N NaOH,then with 1N HCl.
•         Diethyl ether was wash with a saturated solution of FeSO4 to remove accumulated peroxides and subsequently with distilled to remove the FeSO4.
•         Ethylene diamine was redistill and store in a dark bottle in the cold.
•         Acetate buffer (2M,ph 6.8): 2N acetic acid to be adjusted with 2N NaOH to a ph of 6.8.
•         Iodine Solution (0.1N): Iodine (3.175gm) to be dissolve in distilled water containing 12.5gmNaI, diluted to 250 ml, and store in a dark bottle in the cold.
•         Sodium thiosulfate,(0.1N): Na2S2O3.5H2O(6.2gm) to be dissolve in distilled water, make to 250 ml, and  were stored in a dark bottle in the cold.
•         EDTA (10%w/v) Ethylene diamine tetra acetate, disodium salt(10gm) to be  dissolve in distilled water with heading and dilute to 100ml.
•         Alkaline Sulphite/ EDTA Solution: Na2SO3.7H2O(12.6gm) to be dissolve in 25ml of 10% EDTA  and was dilute to 250ml with 5N NaOH.
•         Alkaline ascorbic acid/ethylenediamine solution: Immediately before use, ascorbic acid (200mg) to be dissolve in 2.5ml 0.01n HCl,and was added to 22.5ml 10N NaOH containing 0.5ml redistilled ethylenediamine Since the solution was very viscous, It was necessary to mix it thoroughly.
•         HCl/acetic acid reagent: Concectrated HCl and glacial acetic acid were mixed in a 1:1 ratio.
•         Ascorbic acid solution: immediately before use,100mg ascorbic acid was dissolved in 10ml 0.01N HCl.

Antidepressant activity:
The chemicals /Plant extract which reduces the mental depression which characterized by feelings of intense sadness and despair, mental slowing and loss of concentration, pessimistic worry, lack of pleasure, self-deprecation, and variable agitation or hostilitymay called as Antidepressant Drugs.

Mechanism of Action:- Antidepressants enhance the biological activity of monoamine neurotransmitters in the CNS and that anti-adrenergic compounds may induce depression.

The Antidepressant drugs have effects on cortical, limbic, hypothalamic, and brainstem mechanisms that are of fundamental importance in the regulation of arousal, consciousness, affect, and autonomic functions30.

Apparatus: Graduated glass box (45×20cm) containing 38 cm water



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