INTRODUCTION:

Phytochemistry is the subject deals with chemicals derived from flora. There are large numbers of phytoconstituent compounds found in plants¹. The chemical substances of the medicinal plants have the capacity of exerting a physiologic action on the human body. The bioactive compounds of plants such as alkaloids, flavonoids, tannins and phenolic compounds were considered to be most important^2,3. The phytochemical research that has been done based on the ethno-pharmacological information forms the valuable approach in the discovery of new anti-infective agents from higher plants⁴.

The secondary metabolites like phenolics and flavonoids from plants have been reported to be potent free radical scavengers. They are found in all parts of plants such as leaves, fruits, seeds, roots and bark⁵. Different phytoconstituents of herbal products are safer than synthetic medicine and beneficial in the treatment of diseases caused by free radicals and it also protect the body by preventing the free radicals to cause tissue injury⁶. Phytochemicals like phenolic compounds when combined with the release of good smelling volatile oils in hot water can boost immunity, increase blood circulation, clear airways, make one feel relaxed and may help recovery in postpartum woman by their antioxidation activities⁷.

Scientific classification:

Common name - Cluster Fig, Indian Fig

Kingdom - Plantae

Class - Monocotyledons

Order - Rosales

Family - Moraceae

Genus - Ficus

Species - racemosa

Fig. 1: Ficus racemosa

Scientific classification:

Common name - Indian oleander

Kingdom - Plantae

Class - Dicotyledons

Order - Gentianales

Family - Apocynaceae

Genus - Nerium

Species - indicum

Fig. 2: Nerium indicum

Scientific classification

Common name - Indian Mulberry

Kingdom - Plantae

Class - Magnoliopsida

Order - Gentianales

Family - Rubiaceae

Genus - Morinda

Species - tinctoria

Fig. 3: Morinda tinctoria

Ficus racemosa (Moraceae) is distributed throughout Bangladesh particularly in evergreen forests and moist localities (Figure 1). Root, bark, leaves and fruit are part of tree used for therapeutic activity. Bark, leaves and unripe fruit are carminative, astringent, stomachic and vermicide⁸. All parts of Ficus racemosa are medicinally important in traditional system of medicine in India and have been used extensively in biliary disorders, jaundice, dysentry, diabetes and diarrhoea and in inflammatory conditions⁹.

Nerium indicum Mill (Family: Apocynaceae) is an ever green plant with a long history of traditional medicinal use in the world, especially tropical and subtropical countries (Figure 2). The plant has been used for treatment of several diseases. The whole plant is believed to have medicinal properties¹⁰.

Morinda tinctoria belongs to the family Rubiaceae and genus Morinda (Figure 3). It is locally known as “Togaru” and is a small tree with immense medicinal properties. In Indian traditional system of medicine, leaves and roots of Morinda tinctoria are used as astringent, deobsterent, emmengogue and to relieve pain in the gout¹¹. It is also used for curing dyspepsia, diarrhoea, ulceration, stomatitis, digestion, wound and fever. The leaf juice is useful as a local application. Root is used to cure inflammation and boils. Unripe fruit is used to cure rheumatism. Ash of the fruit prevents dysentry, vomitting, diarrhoea and cholera¹².

MATERIAL AND METHODS:

Collection of Plant Material:

Ficus racemosa, Morinda tinctoria and Nerium indicum leaves were collected from Coimbatore district, Tamil Nadu. The plants were duly authenticated by Botanical Survey of India, Tamil Nadu Agricultural University, Coimbatore. The collected leaves were cleaned to remove adhering dust particles, washed under running tap water and finally washed with distilled water and gently blotted dry between folds of tissue paper, and used for the further experiments.

Preparation of the Plant Extracts:

Ten gram of fresh leaves of Ficus racemosa, Morinda tinctoria and Nerium indicum were homogenized using motor and pestle with100ml of ethylacetate, ethanol, methanol and aqueous to obtain 20% homogenate. The homogenate was transferred into a 250 ml conical flask, plugged with cotton and wrapped with papers. Then kept on a rotator shaker at 190-220 rpm for 24 hours. Centrifuged at 1000 rpm for 25 minutes. The supernatant was collected and filtered. Filtrate was then concentrated by evaporating. The concentrated extract was then accurately weighed and stored in small vials at -20ºC, for the following studies.

Preliminary Phytochemical Screening of Different Extracts of selected Medicinal Plants:

The fresh leaves of Ficus racemosa, Morinda tinctoria and Nerium indicum were screened for the presence of phytochemicals. The procedures for detection of alkaloids, flavonoids, saponins, phenols¹³, tannins, carbohydrates¹⁴, steroids and terpenoids¹⁵.

i) Detection of Alkaloids:¹³

Small amount of plant extract was dissolved in 5ml of 1% hydrochloric acid on steam bath and filtered. The filtrate is divided into 3 portions to do the following tests.

Dragendroff’s Test:

A portion of filtrate was treated with 6 drops of dragendroff’s reagent and observed for orange precipitate.

Mayer’s Test:

A portion of filtrate was treated with 6 drops of mayer’s reagent. Creamish precipitate indicates the presence of alkaloids.

Wagner’s Test:

A portion of the precipitate was treated with 6 drops of wagner’s reagent. Brownish red precipitate indicates the presence of alkaloids.

ii) Detection of Flavonoids:¹³

Schinoda Test:

A small quantity of plant extract was dissolved in 5ml of ethanol and filtered. Three pieces of magnesium chips were added followed by few drops of conc. hydrochloric acid. Pink or orange colour indicates the presence of flavonoids.

Conc. Sulphuric Acid Test:

A small amount of extract and few drops of conc. sulphuric acid were added. Orange colour indicates the presence of flavonoids.

Sodium Hydroxide Test:

A small quantity of extract was dissolved in water and filtered. To this added 2ml of 10% aqueous sodium hydroxide to produce a yellow colouration. The change in colour from yellow to colourless on addition of dilute hydrochloric acid was observed.

iii) Detection for Saponin:¹³

Frothing Test:

A portion of the extract was added along with 5ml of distilled water in a test tube. A solution was shaken vigorously and observed for stable persistant froth.

iv) Detection of Phenols:¹³

Ferric Chloride Test:

The extract was boiled with distilled water and filtered. To this 2ml of 10% ferric chloride solution was added and observed for the formation of green/blue colour which indicates the presence of phenols.

v)Detection for Tannin:¹⁴

The extract was treated with a few drops of lead solution. The formation of white precipitate indicates the presence of tannin.

vi) Detection of Carbohydrate:¹³

Molisch’s Test:

Added 2 drops of Molisch’s reagent to 2ml of sample in a test tube. Mixed thoroughly and added 2ml of conc. sulphuric acid along the sides of the test tubes. Then were erected the test tubes slowly. The formation of a reddish violet ring at the junction of two liquids indicates the presence of carbohydrate.

vii) Detection for Steroids:¹⁵

Liebermann Burchard Reaction:

A portion of the extract was treated with 10ml chloroform and filtered. To 2ml of filtrate was treated with 2ml of acetic anhydride and conc. sulphuric acid. Blue/green ring indicates the presence of steroids.

viii) Detection of Terpenoids:¹⁵

5ml of extract was mixed with 2ml of chloroform and conc. H₂SO₄(3ml) was carefully added to form a layer. A reddish brown colouration of the interface formed to show positive for the presence of terpenoids.

ix) Detection of Quinones:¹⁵

A small amount of extract treated with conc. HCl and appearance for the formation of yellow colour precipitation indicates the presence of quinines.

x) Detection of Volatile Oils:¹⁴

Shaken 2.0 ml of extract solution with 0.1 ml dilute sodium hydroxide and a small quantity of dilute HCl. Formation of white precipitate indicates the presence of volatile oils.

xi) Detection of Protein:¹³

Bradford’s Test:

To 1 ml of the extract added few drops of Bradford’s reagent (Coomassie Brilliant Blue G 250) and formation of blue colour product indicates the presence of protein.

Total Phenolic Content of Different Extracts of Ficus Racemosa, Morinda Tinctoria and Nerium Indicum :

1g of sample was homogenized using 20ml of 80% ethanol. The homogenate was centrifuged at 10,000rpm for 20 minutes. The supernatant was saved. The residue was extracted with 10ml of 80% ethanol, centrifuged and collected the supernatant and evaporated to dryness. The residue was dissolved in a known volume of distilled water (50ml) and 2.0ml was taken for the experiment. A working standard of 0.5 – 2.5ml catechol solution corresponding to 50 - 250µg of catechol were pipetted out into a series of test tubes. The volume was made upto 2.5ml with water. To all the tubes added 0.5ml of diluted Folin – Ciocalteau reagent. After 3 minutes, 2.0ml of 20% Na₂CO₃ solution was added to each tube and mixed thoroughly. The tubes were placed in a boiling water bath for exactly one minute. Cooled and measured at 650nm against a reagent blank. Constructed a standard graph by plotting the concentration of catechol on X-axis and absorbance on Y-axis. From the graph, the amount of polyphenols present in the sample was estimated and expressed as mg of polyphenols per g of the sample¹⁶.

Statistical Analysis:

The data presented as mean ± SD of three samples in each (n=3) group. Statistical significance p<0.05 protection level was used for comparison.

RESULTS AND DISCUSSION:

All over the world the herbal medicine acts as the representative of the most important fields of traditional medicine. The study on the medicinal plants is essential to promote the proper use of herbal medicine in order to determine their potential as a source for the new drugs⁴. Phytochemical screening is one of the necessary steps to find out the chemical constituents which lead the isolation of compounds¹⁷. Phytochemicals are bioactive non-nutritive compounds found in plants that work with nutrients and dietary fiber to protect against diseases¹⁸. It was observed from Table 1 shows that all the extracts of the selected medicinal plants revealed the presence of carbohydrates, proteins and flavonoids and volatile oil was found to be absent in the aqueous extract. Among the four extracts, the ethanol extract of Ficus racemosa confirmed the presence of phytoconstituents than that of other extracts. It did not shown the presence of quinones and saponins and methanol extract did not show the presence of tannin and saponins. Volatile oils were not found to be present in the ethyl acetate extract. The methanol extract of Morinda tinctoria found to be more phytoconsituents than that of other extracts whereas saponins were also not present in ethanol, methanol, and aqueous extract. The extract of Nerium indicum depicted that more amount of secondary metabolites present in the methanol extract while compared with other extracts. It did not exhibited the presence of saponins, alkaloids, tannins and terpenoids in aqueous extract. Based on the results, the methanol extract of Morinda tinctoria revealed the presence of phytoconstituents than that of other plants. Similar results were reported by Santhi¹⁹, that the aqueous extract of Nerium oleander and Momordica charantia leaves revealed the presence of carbohydrate, cholesterol, proteins, amino acids, alkaloids, flavonoids, tannins, saponins, cardiac glycosides, terpenoids and phlobaotinins followed by ethanol, ethyl acetate, diethyl ether and chloroform. The phytochemical constituents of selected medicinal plants were screened and given in the Table 1.

Table 1: Phytochemical constituents of the different leaves extracts of Ficus racemosa, Morinda tinctoria and Nerium indicum

Test	*Ficus racemosa*				*Morinda tinctoria*
	Ethyl acetate	Ethanol	Methanol	Aqueous	Ethyl acetate	Ethanol	Methanol	Aqueous
Carbohydrate	+	++	+	+	+	+	+	+
Protein	+	++	+	+	+	+	+	+
Alkaloids	+	+	+	-	+	+	++	_
Phenols	+	+++	++	++	+	+	++	+
Flavonoids	+	+++	+	+	+	+	+	+
Tannins	+	+	_	+	+	+	+	_
Quinones	+	-	+	+	+	-	+	+
Saponins	+	+	_	_	+	_	_	_
Terpenoids	+	+++	+	+	++	+	++	_
Steroids	+	++	+	-	++	+	++	+
Volatile oil	-	+	+	-	-	+	+	-

+++- highly presence, ++ moderate presence, + presence, - absence

Table 1: Continue

Test	*Nerium indicum*
	Ethyl acetate	Ethanol	Methanol	Aqueous
Carbohydrate	+	+	+	+
Protein	+	+	+	+
Alkaloids	+	+	+	-
Phenols	+	+	+++	+
Flavonoids	++	++	+++	+
Tannins	++	++	+++	+
Quinones	+	+	+	+
Saponins	_	_	_	_
Terpenoids	+	+	+	-
Steroids	+	+	+	+
Volatile oil	+	+	-	-

Phytochemical screening done by Ashwini ²⁰stated that the fruit powder Coccinia grandis in different organic solvents also revealed the presence of alkaloids, carbohydrates, phytosterols, resins, tannins, flavonoids, proteins and diterpenes. Suriyavathana ²¹also reported that the alcoholic extracts of Desmodium gangeticum and Premna tomentosa wild shows the presence of flavonoid, alkaloid, phenols, tannins and steroids. Tannins are compounds that have the ability to react with protein to form stable water insoluble components^22. .Presence of phytoconstituents in the selected medicinal plants may be responsible for treatment against various diseases.

Total Phenolic Content of Different Extracts of Selected Medicinal Plants:

Phenolic compounds such as flavonoids, phenolic acids and tannins are measured to be chief contributor to the antioxidant capability of plants and various biological activities may be connected to their antioxidant activity. Anti- inflammatory properties of saponins, terpenoids, flavonoids, tannins, steroids and alkaloids were revealed by Orhan²³. The total phenol content of ethyl acetate, ethanol, methanol and aqueous extracts of Ficus racemosa, Morinda tinctoria and Nerium indicum are presented in Figure 5.

Fig. 4: Total phenolic content of Ficus racemosa, Morinda tinctoria and Nerium indicum leaves in different solvents

Values are mean ± SD of three samples in each group

Comparison:

a - Ficus racemosa with Morinda tinctoria;

b - Morinda tinctoria with Nerium indicum;

c - Nerium indicum with Ficus racemosa

* - p<0.05; # – Not significant

The ethanol extract of Ficus racemosa has highest phenolic content (2.35± 0.08 mg/g) compared to all other extract of Ficus racemosa and different solvent extracts of other medicinal plants analysed. The methanol extract of Morinda tinctoria (0.86± 0.01 mg/g) and Nerium indicum (0.35± 0.01 mg/g) was found to contain significant phenolic content than other extracts Mayur²⁴ reported that the methanolic extract of Carpesium abrotanoides L. was found to possess considerable amount of phenols (8.8 mg gallic acid equivalent/g of extract). Sengul²⁵ reported that the total phenolic content of the crude extracts of Viscum album and Crocus sativus had the highest total phenolic content 42.29 mg GAE/g DW respectively. Similar results were reported by Aris²⁶, the total phenolic content of three different extracts of fruits of Ficus deltoidea var angustifolia. It was found that the hexane extract showed the highest phenolic concentration of 259.2 mg/g of GAEs followed by methanol extract (245.2 mg/g of GAEs) and chloroform extract (159.2 mg/g of GAEs).

CONCLUSION:

The present study concluded that most of the biologically active phytochemicals were found the present in the ethanolic extract of Ficus racemosa leaves when compared with other two plants. Thus the Ficus racemosa leaves possesses strong phytochemical constituents which can be used to heal an assortment of diseases. So the various active compounds should be isolated from this leaves, which might be used as therapeutic agents.

CONFLICT OF INTEREST STATEMENT

We declare that we have no conflict of interest.

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Received on 21.09.2016 Modified on 22.10.2016

Research J. Pharm. and Tech 2016; 9(12):2222-2227.

DOI: 10.5958/0974-360X.2016.00449.2