INTRODUCTION:

Medicinal herbs represent a great deal of untapped reservoir of drugs and the active component molecules in future are targeted for lead compounds¹. Couroupita guianensis Aubl. (C. guianensis) belonging to family Lecythidaceae is a deciduous tropical tree 90 feet tall and indigenous to Amazon rainforest. In, India it has been growing for past two to three thousand years at least. The flower are stunning fragrant, waxy aromatic smelling growing directly on the bark. Native Amazonian people used the infusion or teas obtained from leaves, flower and bark of C. guianensis to treat hypertension, tumours, pain and inflammatory processes². In Orissa decoction of flowers has been used to boost the immune system to fight number of disease^{3, 4}. Apart from this, extracts of flower had also been screened for antimicrobial activity⁵, larvicidal activity against vector⁶, cold, intestinal gas formation and stomachache⁷ and immunomodulatory activity⁸. Despite its structural speciality, various part of this tree has been reported to contain oils, ketosteroids, glycosides, isatin, indurubin, couroupitine and phenolic substances with medicinal properties.⁹

Figure No.1 The whole plant and parts of C. guianensis Abul.

Traditional Uses of Couroupita guianensis Abul.

The trees are used to cure cold and stomach ache. Juice made from leaves is used to cure skin diseases and Shamanas of South America have even use tree parts for treating malaria, while the flowers are used to cure cold, intestinal gas formation and stomach ache (Velliangiri Prabhu et al., 2012)¹⁰. The fruit pulp can disinfect wounds and young leaves ease tooth ache. Bark used to treat hypertension, tumors, pains and inflammatory process (Dr. Mahipal Singh, 2014)¹¹.The fresh fruit pulp is used in the preparation of cooling medicinal drink and cure head ache. Leaves are widely used as an analgesic medicine by the rural population worldwide (Geetha et.al, 2005)¹²

MATERIALS AND METHODS:

1. Collection and drying of the plant material:

The Couropita guianesis Abul plant was collected from the Kolhapur district. The collected plant material was washed thoroughly under running tap water, air-dried at room temperature under the shade for 8-10 days. The leaves were separated manually by hand picking. The dried leaves were crushed to the fine powder and stored in tightly sealed polyethylene bags.

2. Authentication of plant species (Lantana camara L. L.):

The plant was authenticated by Prof. D. G. Jagtap, Head of Department of Botany Principal Shri. Vijaysinha Yadav Arts and Science College, Peth Vadgaon, Dist.- Kolhapur.

3. Extraction of plant material:

Leaves of Couropita guianesis Abul. were extracted in a soxhlet extractor, successively with Petroleum ether (60°-80°), Chloroform, and Ethanol (95%) for 24-36 hrs for each solvent Figure No. 2. After extraction with each solvent, the solvent was evaporated and residue was air dried. The residues from each extract were dried and the resultant extracts were stored in an air tight container for further use. The extract was subjected to preliminary phytochemical testing.

Figure No. 2 Soxhlet extraction of Couropita guanesis Abul.

4. Phytochemical investigations of plant

I. Test for Alkaloids

1. Wagner’s Test:

A fraction of extract was treated with 3-5 drops of Wagner’s reagent [1.27g of iodine and 2g of potassium iodide in 100ml of water] and observed for the formation of reddish-brown precipitate (or colouration) which indicates the presence of alkaloids.

2. Mayer’s Test:

Filtrates were treated with Mayer’s reagent (Potassium Mercuric Iodide). Formation of a yellow coloured precipitate indicates the presence of alkaloids.

II. Test for Glycosides:

1. Liebermann’s test:

extract was mixed with each of 2ml of chloroform and 2mL of acetic acid. The mixture was cooled in ice. Carefully concentrated H₂SO₄was added. A colour change from violet to blue to green indicated the presence of steroidal nucleus, i.e., glycine portion of Crude glycoside.

2. Salkowski’s test:

Crude extract was mixed with 2mL of chloroform. Then 2mL of concentrated H₂SO₄ was added carefully and shaken gently. A reddish-brown colour indicated the presence of steroidal ring, i.e., glycone portion of the glycoside.

3. Keller Killiani Test:

Test solution was treated with few drops of glacial acetic acid and Ferric chloride solution and mixed. Concentrated sulphuric acid was added, and observed for the formation of two layers. Lower reddish-brown layer and upper acetic acid layer which turns bluish green would indicate a positive test for glycosides.

III. Test for Phenols:

1. Ferric Chloride Test:

Extracts were treated with 3-4 drops of ferric chloride solution. Formation of bluish black colour indicates the presence of phenols.

IV. Carbohydrates:

1. Benedict's test:

Test solution was mixed with few drops of Benedict's reagent (alkaline solution containing cupric citrate complex) and boiled in water bath, observed for the formation of reddish-brown precipitate to show a positive result for the presence of carbohydrate.

2. Molisch’s Test:

Filtrates were treated with 2 drops of alcoholic α-naphthol solution in a test tube. Formation of the violet ring at the junction indicates the presence of Carbohydrates.

3. Fehling’s Test:

Filtrates were hydrolysed with dil. HCl, neutralized with alkali and heated with Fehling’s A and B solutions. Formation of red precipitate indicates the presence of reducing sugars.

V. Test for Flavonoids:

1. Shinoda test:

Crude extract was mixed with few fragments of magnesium ribbon. Con. HCl was added drop wise. Pink scarlet colour appeared after few minutes which indicated the presence of flavonoids.

2. Alkaline reagent test:

Crude extract was mixed with 2ml of 2% solution of NaOH. An intense yellow colour was formed which turned colourless on addition of few drops of diluted acid which indicated the presence of flavonoids.

VI. Test for Proteins:

1. Biuret Test:

Test solution was treated with equal volume of 10% sodium hydroxide solution and two drops of 1% copper sulphate solution, mixed well and observed for the formation of violet/pink colour. If it is so, presence of proteins was detected.

2. Xanthoproteic Test:

Two ml of extracts were treated with few drops of conc. Nitric acid. Mixed well. Formation of light to dark yellow colour was noted which indicates the presence of proteins.

3. Millon’s Test:

Two ml of crude extract when mixed with 2ml of Millon’s reagent, if a white precipitate appeared which turned red upon gentle heating and disappeared on cooling confirmed the presence of protein.

VII. Test for Saponins:

1) The alcoholic extract was evaporated to dryness, residue was extracted with petroleum ether and acetone. To the insoluble residue after extraction, 5 ml of water was added and shaken well. Formation of stable foam indicates presence of saponins

2) To the alcoholic extract, 3drops of sodium bicarbonate was added and shaken well. Formation of stable foam indicates presence of saponins

VIII. Test for Steroids:

1. Liebermann Burchard test:

Crude extract was mixed with few drops of acetic anhydride, boiled and cooled. Concentrated sulphuric acid was then added from the sides of the test tube and observed for the formation of a brown ring at the junction of two layers. Green coloration of the upper layer indicates a positive test for steroids.

RESULTS AND DISCUSSION:

Couropita guianesis Abul. was successively extracted by using the Soxhlet assembly taking the different solvents such as Petroleum ether, Chloroform and Methanol based on the increasing polarity. All the extracts were evaporated to remove excess of solvent in a water bath. These extracts were then stored in air tight container at cold temperature (approx. 15^oC). These extracts were then used for further chemical test for phytochemical investigation for protein, amino acid, glycoside, alkaloid, phenolic compounds, flavonoids, steroids and tannins etc. the results of phytochemical screening of Couropita guianesis Abul. leaves extracts are mentioned in the following Table No. 01.

Table No. 01

Sr. No.

Test

Petroleum ether

Chloroform

Methanol

Test for Alkaloid

1) Mayer’s reagent

2) Dragondorff’s reagent

3) Wagner’s reagent

4) Hager’s reagent

Test for Glycoside

1) Keller-killiani test

2) Borntrager’s test

3) Ledal’s test

Test for Phenolic

compounds

1) Ferric chloride test

2) Lead acetate test

3) Gelatin test

Test for Carbohydrates

1) Molisch test

2) Fehling’s test

Test for Flavonoids

1) Ammonia test

2) Shinoda/paw test

Test for Proteins and free amino acids

1) Millon’s test

2) Xethoprotein test

3) Biuret test

Test for Saponins

1) The alcoholic extract was evaporated to dryness, residue was extracted with petroleum ether and acetone. To the insoluble residue after extraction, 5 ml of water was added and shaken well.

2) To the alcoholic extract, 3drops of sodium bicarbonate was added and shaken well.

Test for Sterols

1) Salkowski reaction

2) Hersche’s Son’s reaction

Note: + indicates presence and - indicates absence of phytoconstituent

CONCLUSION:

Phytochemicals found present in the Couropita guianesis Abul. indicates their potential as a source of principles that may supply novel medicines. The leaves of Couropita guianesis Abul. petroleum ether extract found to be phytochemicals are present protein, steroids, flavonoids, alkaloids, saponins, phenols. The leaves of Couropita guianesis Abul. chloroform extract found to be phytochemicals are present, protein saponins, alkaloid, phenol’ carbohydrates. The leaves of Couropita guianesis Abul. methanol extract found to be phytochemicals are present alkaloids, glycosides, phenols.

Considering the above facts, isolation, purification and characterization of the phytochemicals found in this species may introduce a future medicine that will change the life of mankind. Furthermore, a detailed study needs to ascertain their antioxidant, anti-inflammatory, anticancer activities.

ACKNOWLEDGMENT:

The authors wish to acknowledge Professor for his inspiration.

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Received on 04.07.2020 Modified on 11.08.2020

Research J. Pharm. and Tech. 2021; 14(8):4161-4164.

DOI: 10.52711/0974-360X.2021.00720