Collection Of Plant Material Biology Essay

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Plants were collected in the Tirupathi hills. Healthy plants were chosen. The viral, bacterial or fungal infected plants were discarded. The healthy plants were dried at moderate temperature in the dark for a short period of time and preserved.

6.3 PHARMACOGNOSTICAL STUDY

The plant material when fresh was subjected to macroscopic analysis in order to confirm its identity air dried plant material was finely ground in a suitable mill in to a powder. Extraction was carried out with soxhlet apparatus using solvents pet. Ether and methanol and the extracts were highly concentrated.

6.3.1 EXTRACTION PROCESS43,44

Extraction may be defined as the treatment of the plant or animal tissues with solvent, where by the medicinally active constituents are dissolved and most of the inert matter remains undissolved.

The solvent used for extraction is known as menstrum and the inert insoluble material that remains after extraction is called marc.

The various processes used for extraction are:

Infusion

Decoction

Maceration

Perculation

Digestion

6.3.1a. Continuous hot percolation process or soxhlet extraction

When active constituents of the drug were not freely soluble in the solvent or difficult to be displaced from the cells of the drug, then it becomes necessary to extract the crude drug by the action of hot menstrum for a considerable length of time. The fixed oils from seeds and alkaloids from the drug are extracted by continuous hot percolation process using benzene, chloroform, petroleum ether etc.

Apparatus

The apparatus used for continuous hot percolation process was soxhlet apparatus, which consists of three parts:

A. Flask containing the boiling solvent.

B. Soxhlet extractor in which the drug to be extracted was packed. It had a side tube which carries the vapours of the solvent from the flask to the condenser and a siphon tube which siphon over the extract from soxhlet extractor to the flask.

C. A condenser in which the vapours of the solvent are condensed again into solvent (fig.no.1).

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Procedure

The drug to be extracted was packed in a paper cylinder made from a filter paper and it was placed in the body of sox let extractor. The solvent is placed in the flask.

When solvent was boiled on heating the flask, it gets converted into vapours enter into the condenser through the side tube and get condensed into hot liquid which falls on the column of the drug. When the extractor gets filled with the solvent, the level of siphon tube also raises up to its top. The solvent containing active constituents of the drug in the siphon tube siphon over and run into the flask, thus emptying the body of extractor. This alternation of filling and emptying the body of extractor goes on continuously. The soluble active constituents of the drug remain in the flask while the solvent was repeatedly volatilized. The process of filling and emptying of the extractor was repeated until the drug was exhausted. Normally the process was repeated about 15 times for complete exhaustion of the drug.

6.4 EXTRACTION, ISOLATION AND IDENTIFICATION OF ACTIVE CONSTITUENTS31

The entire plant of Scutia myrtina was dried under shade and then powdered with a mechanical grinder. The powder was passed through sieve number 40 and retained in sieve number 60 and stored in an air tight container for further use. The dried powder material of the plant was defatted with petroleum ether (400 c) for 48 hours in soxhlet apparatus. The defatted plant material thus obtained was further extracted with methanol for 72 hours in the soxhlet. The solvent was removed by distillation under reduced pressure and the resulting semisolid mass was dried in a dessicator. The dried extract were packed in airtight containers and used for studies.

Table no. 1 Percentage Yield of the Plant Extract

Plant name

Part used

Method of extraction

Percentage yield

Pet. Ether (%w/w)

Methanol (%w/w)

Scutia myrtina

Whole plant

Continuous hot percolation

2.75

7.45

Table no. 2 Phytochemical test45

Experiment

Observation

Inference

Test for alkaloids:

Mixed Small portion of extract with few drops of dilute HCI and filter. Filtrate was used to perform the following test to carry out the test for Alkaloids.

A. Dragendroff's Test:

The extract was treated with few ml of Dragendroff's reagent. (Potassium Bismuth Iodide solution).

B. Hager's Test:

The extract was treated with a few ml of Hager's reagent (Saturated Solution of Picric Acid).

C. Wagner's Test:

The extract was treated with few ml of Wagner's reagent (Iodine-Potassium Iodide).

D. Mayer's Test:

The extract was treated with few ml of Mayer's reagent (Potassium Mercuric Iodide solution).

E. Tannic acid test:

To the extract few ml of tannic acid is added.

F. Picrolonic acid test:

To the extract few ml of picrolonic acid is added.

Reddish brown precipitate indicated the presence of alkaloids.

Yellow precipitate indicated the presence of alkaloids.

Reddish brown precipitate indicated the presence of alkaloid.

Cream colour precipitate indicated the presence of alkaloid.

Alkaloids give buff colour precipitate.

Alkaloids give yellow colour precipitate.

Pet. Ether extract -ve

Methanolic extract +ve

Presence of alkaloids

\Presence of alkaloids

Presence of alkaloids

Presence of alkaloids

Presence of alkaloids

Presence of alkaloids

2. Test for Saponins:

A. Foam test:

Dilute 1 gm of extracts with 20 ml distilled water in Graduated cylinder and shaked for 15 minutes.

1cm layer of foam indicated the presence of saponins.

Absence of saponins

3. Test for Glycosides:

The extract was hydrolysed with HCL for few hours on a water bath.

A. Legal's Test:

Dissolve the extract (0.1 gm) in pyridine (2ml) and added sodium nitroprusside solution (2 ml) and made alkaline with sodium hydroxide solution.

B. Raymond Test:

Treat the extract with hot methanolic alkali.

C. Baljets Test:

Treat the extract with picric acid or sodium picrate.

D. Bromine water Test:

Treat the extract with bromine water.

E. Keller-Killiani test:

Treat the extracts with chloroform mix well and evaporated it to dryness. Add 0.4 ml of glacial acetic acid containing trace amount of ferric chloride. Transfer to a small test tube, added carefully 0.5 ml of concentrated sulphuric acid by the side of the test tube.

F. Borntrager's Test: (Anthraquinone Glycosides)

Add a few ml. of dil. Sulphuric acid to 1 ml. of the extract solution. Boil, filter while hot, pipette out the supernatant or filtrate, cool and shake with an equal volume of dichloromethane. Separate the lower dichloromethane layer and shake with half its volume with dilute ammonia.

Pink to red color solution indicated the presence of glycosides.

Violet colour is produced.

Orange colour is produced.

Yellow precipitate was produced.

Acetic acid layer did not show blue colour.

A rose pink to red colour is produced in the ammonical layer.

Pet. Ether extract -ve

Methanolic extract +ve

Presence of glycosides

Presence of glycosides

Presence of glycosides

Presence of glycosides

Absence of digitose sugars.

Presence of anthraquinone glycosides

4. Test for Carbohydrates:

Dissolved small quantity of the extract in distilled water and filter.

A. Molisch's Test:

To the filtrate add 1 ml. of napthol solution, add conc. Sulphuric acid through the sides of the test tube.

B. Fehling's Test:

To 1 ml. of the filtrate add equal quantites of Fehling solution A and B, up on heating.

C. Benedict's Test:

To 1 ml. of the filtrate add 5 ml. of Benedict's Reagent and boil for 2 min and cool.

Purple colour is not produced at the junction of two liquids.

Red precipitate is not produced.

Red precipitate is not produced.

Absence of sugars.

Absence of sugars.

Absence of sugars.

5. Test for tannins:

A. Gelatin Test:

To the extract, 1% gelatin solution containing 10% sodium chloride is added.

B. Ferric chloride Test:

To the extract, ferric chloride is added.

C. Vanillin Hydrochloride Test:

To the extract a few drops of Vanillin Hydrochloride Reagent is added.

White precipitate is produced.

Blue green colour is produced.

Purplish red colour is produced.

Pet. Ether extract -ve

Methanolic extract +ve

Presence of tannins

Presence of tannins

Presence of tannins

6. Test for flavonoids:

A. Shinoda Test (Magnesium HCl reduction test):

To the extract, add few fragments of magnesium ribbon and conc. Sulphuric acid dropwise.

B. Zinc hydrochloride Reduction Test:

To the extract add a mixture of zinc dust and conc. Hydrochloric acid.

C. Alkaline Reagent Test:

To the extract add few drops of sodium hydroxide.

Pink scarlet, crimson red or occasionally green to blue colour appears after few min.

Red colour is produced after few min.

An intense yellow colour which turns to colourless on addition of few drops of dil. acid.

Pet. Ether extract -ve

Methanolic extract +ve

Presence of flavonoids

Presence of flavonoids

Presence of flavonoids

7. Test for sterols and Triterpenoids:

A. Libermann- burchard Test:

1 gm of the extract is dissolved in a few drops of chloroform, 3 ml. of glacial acetic anhydride, 3 ml. of glacial acetic acid were added, warmed and cooled under the tap and drops of conc. Sulphuric acid were added along the sides of the test tube.

B. Salkowski Test:

Dissolve the extract in chloroform and equal volume of conc. Sulphuric acid.

Bluish- green colur was produced.

Bluish red to cherry colour in chloroform layer was produced.

Pet. Ether extract -ve

Methanolic extract +ve

Presence of triterpenoids

Presence of triterpenoids

8. Test for proteins and Amino acids:

A. Biuret Test:

To 2 ml. of the extract add equal volume of 10% sodium hydroxide and 1 drop of 10% CuSO4.

B. Millon's Test:

Test solution with 2 ml. Millon's Reagent (mercuric nitrate in nitric acid containing traces of nitrous acid) and gentle heating.

C. Ninhydrin Test:

To the test extract add Ninhydrin Reagent (Indan 1,2,3 trione hydrate).

Violet colour was not produced.

White to red colour was not produced on gentle heating.

Violet colour is not produced.

Absence of proteins and Amino acids

Absence of proteins and Amino acids

Absence of proteins and Amino acids

9. Test for fixed oils and fats:

A. Spot Test:

Press a small quantity of extract between the filter paper.

B. Saponification Test:

To the extract, add few drops of 0.5 N alc. KOH and a drop of phenopthalein and heat on a water bath for 1-2 hours.

Oil stains on paper was formed.

The formation of soap or partial neutralization of alkali.

Pet. Ether extract +ve

Methanolic extract -ve

Presence of fixed oils and fats

Presence of fixed oils and fats

10. Test for Gums and mucilage:

A. Ruthenium Test:

Ruthenium is used for staining mucilage.

(In 10 ml. of 10% solution of lead acetate, dissolve 0.008 gm of Ruthenium red)

Pink colour is not produced.

Absence of gums and mucilage

The extract was subjected to Preliminary Phytochemical Screening for the presence of different chemical groups. The plant contains phtochemicals such as alkaloids, glycosides, tannins, flavonoids, triterpenoids, fixed oils and fats. The phytochemicals which are present in the plant were indicated in the Table No. 1.

Table No.3 Presence of different plant constituents

plant constitutents

Pet. Ether extract

Methanolic extract

Alkaloids

-ve

+ve

Saponnins

-ve

-ve

Glycosides

-ve

+ve

Carbohydrates

-ve

-ve

Tannins

-ve

+ve

Flavonoids

-ve

+ve

Triterpenoids

-ve

+ve

Proteins and amino acids

-ve

-ve

Fixed oils and fats

+ve

-ve

Mucilage

-ve

-ve

+ve: present, -ve: absent

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