TLC Profiling of Pithecellobium dulce Seed

Sunil H. Ganatra*, Archana M. Ramteke

Department of Chemistry, Institute of Science, R.T. Road, Civil Lines, Nagpur- 440001 (M.S.) India.

ABSTRACT:

Pithecellobium dulce plant belongs to the family Fabaceae and used as an herbal since ancient times. It is abundant in tropical and subtropical Asia. Preliminary phytochemical investigations have been carried out on the seed extract of the plant using n-Hexane, Ethyl acetate, Acetone and Methanol solvents.

Phytochemical analysis reveals the presence of diverse groups of natural products. The major among them are Quinones, Steroids, Flavonoids and Terpenoids. Chemical constituents have shown different R_f values in various extracts of Pithecellobium dulce seeds using thin layer chromatography (TLC). It shows possible combinations of chromatography solvents for separation of phytochemicals.

KEYWORDS: Fabaceae, Pithecellobium dulce, Phytochemical investigations, TLC profiling.

INTRODUCTION:

Natural product plays a vital role in drug design. Nearly 65% of drugs existing in market are originated from natural sources. This is due to diversified chemical compounds with varieties of structures available in natural products. Though varieties of chemical compounds present in individual source, generally one of the chemical is predominating and having drug like properties. The extraction and its further refinement help to procure the individual compound and can be used as an effective drug [1, 2].

Numbers of drugs are already in use as anti-malaria, anti-cancer, anti-inflammatory etc. which are procured from nature. The most important of these bioactive constituents of plants are steroids, terpenoids, phenols, flavonoids, tannins and glycosides. The medicinal properties of a few plants have been documented by number of researchers [3, 4, 5]. Pithecellobium is a genus of about 110 species, mostly native to Asia and tropical America. The taxonomy of this genus has been changed in recent years, and the names of some species are still in debate [6].

The Pithecellobium dulce is known as herbal and widely used in Asian countries. It is must to know the varieties of chemical compounds present in this species along with its drug like properties. The present study is to understand the presence of varieties of natural compounds and TLC profile.

MATERIALS AND METHODS:

Plant material:

Seeds of Pithecellobium dulce have been collected in the summer season from Nagpur region, Maharashtra, India and dried in shade. Roughly 5 kg. of seeds are collected and crushed to form powder. There are no long-term seed storage data for Pithecellobium dulce seeds, but these are typical hard seeded legumes with orthodox storage behaviour. The seeds should be easy to store at low moisture contents (<10%) and low temperatures (any refrigeration) for a number of years [6].

Preparation of extract:

Dried coarse powder of the seeds (30 gm.) was placed into the extractor of a Soxhlet apparatus and subjected to extraction by hot percolation method. The extraction was carried out by using solvents of increasing polarity from n-Hexane, Ethyl acetate, Acetone and Methanol. The extraction was carried out with 250 ml of each solvent for a period of 48 hours. At the end of the extraction the respective extracts were concentrated by evaporation in vacuum oven at 50 °C. .

Protocol for qualitative analysis:

Chemical test were carried out on the different extracts and on the powdered specimen using standard procedure to identify the constituents as described by Mojab et al [7], Harborne [8], Sofowora [9], Trease and Evans [10], and Siddiqui and Ali [11]. Numbers of chemical analysis tests were performed to detect the presence of the active chemical constituents like tannins, saponins, quinones, phenols, steroids, flavonoids, cardiac glycosides, terpenoids. The test details are as follows and their results are depicted in table 1.

Test for Tannins:

In a test tube 1 ml of 5% Ferric chloride is added to the solvent free extract. The presence of tannin is indicated by the formation of bluish black or greenish black precipitate.

Test for Saponins:

The extract was diluted with 20 ml distilled water and was agitated in a graduated cylinder for 15 minutes. The formation of 1cm layer of foam indicates the presence of saponin.

Test for Quinones:

In a test tube 1ml of extract and 1ml of concentrated sulphuric acid (H₂SO₄) was added. Formation of red colour shows the presence of quinone.

Test for Phenols:

In a test tube 1ml of extract 2ml of distilled water was added followed by few drops of 10% ferric chloride (FeCl₃), appearance of blue or green colour indicates presence of phenols.

Test for Steroids:

1ml of extract dissolved in 10 ml chloroform and equal volume of concentrated sulphuric acid (H₂SO₄) added by sides of test tube. The upper layer turns red and sulphuric acid layer shown yellow with green flurosence.This indicated the presence of steroids.

Test for Flavonoids:

In 1ml of extract a few drops of dilute sodium hydroxide (NaOH) was added, an intense yellow colour was produced in the plant extract which becomes colourless on addition of few drops of dilute acid indicates the presence of flavonoid.

Test for Cardiac Glycosides:

In a test tube 5 ml of extract was treated with 2 ml of glacial acetic acid containing a drop of ferric chloride (FeCl₃) solution. This was than underlay with 1ml concentrated sulphuric acid (H₂SO₄). A brown ring of the interface indicates a de-oxy sugar characteristic of cardenolides.

Test for Terpenoids:

In a test tube 5ml of each extract was mixed with 2 ml of chloroform, 3 ml of concentrated sulphuric acid ( H₂SO₄ ) was added to form a layer. A reddish brown precipitate colouration at the interface formed, indicates the presence of terpenoids .

TLC analysis of seed extracts of Pithecellobium dulce:

Each of the aforesaid four extracts was to begin with, checked by thin layer chromatography (TLC) on analytical plates over silica gel- G. For each extracts three different solvent mixtures were used as developing systems. These were Ethyl acetate: Methanol (1:1), Hexane: Methanol (3:2) and Toluene: Ethyl acetate (93:7). In each case, the plates were exposed to iodine vapour and reported spots visualized and noted down.

RESULTS:

Phytochemical analysis performed on prepared extracts of Pithecellobium dulce seeds show positive or negative results as per the test performed and mentioned above. The results are depicted in table 1. It is reported that quinones, steroids, flavonoids and terpenoids are present in all extracts. Tannins and saponins are reported in methanol extract only whereas cardiac glycosides are present in all extracts accept n-Hexane extract. Phenols are present in ethyl acetate and methanol extracts both. From table 1, it is clearly established that the seed extracts of Pithecellobium dulce contains diverse kind of phytochemicals.

Table-1:-Results of the phytochemical tests performed on seed extracts of Pithecellobium dulce.
Phytochemicals	n-Hexane Extract	Ethyl acetate Extract	Acetone Extract	Methanol Extract
Tannins	-	-	-	+
Saponin	-	-	-	+
Quinones	+	+	+	+
Phenols	-	+	-	+
Steroids	+	+	+	+
Flavonoids	+	+	+	+
Cardiac glycosides	-	+	+	+
Terpenoids	+	+	+	+
Note: (+) = Present and (-) = Absent

The observed R_f values are reported in table 2. Extracts from n-hexane and ethyl acetate must contain more numbers of phytochemicals as they reports 3 spots in TLC with Ethyl acetate: Methanol and Toluene: Ethyl acetate solvents mixtures respectively. Whereas TLCs in n-hexane: Ethyl acetate solvents mixture report one spot for all extracts.

Table 2- Showing Rf values of seed extracts of Pithecellobium dulce seeds in various solvent systems
Plant Extracts	TLC Solvent Mixtures
	EA:ME (1:1)	HX:EA (3:2)	Toluene :EA (93:7)
n-Hexane	0.8 0.38 0.07	0.67	0.61
Ethyl acetate	0.7	0.59	0.67 0.38 0.20
Acetone	0.69	0.33	0.7 0.6
Methanol	0.33	0.84	Nil
Note : EA= Ethyl acetate , ME= Methanol , HX= n-Hexane

DISCUSSION:

Qualitative phytochemical screening is an essential step towards discovery of new drugs as it provides the information regarding the presence of a particular primary or secondary metabolite in the plant extract(s) of clinical significance. The presence of any significant bioactive natural product indicates the necessity of separation of the compound from the mixture of compounds through suitable chromatographic techniques.

In the present study, n-hexane extract shows the presence of quinones, steroids, flavonoids, terpenoids. The presence of quinones, phenols, steroids, flavonoids, cardiac glycosides and terpenoids have been found in case of ethyl acetate extract. The acetone extract is having quinones, steroids, flavonoids, cardiac glycosides and terpenoids and in case of methanol extract tannin, saponin, quinones, phenols, steroids, flavonoids, cardiac glycosides and terpenoid have been found. These preliminary phytochemical investigations focus on the importance of separation of the natural compounds from their mixture as they may be used for various clinical practices.

Thin layer chromatographic technique was performed on different solvent systems. Compounds extracted in n-hexane and ethyl acetate have been separated well in TLC using ethyl acetate: methanol (1:1) and toluene: ethyl acetate (93:7) respectively.

CONCLUSION:

Qualitative tests performed on the seed extracts of Pithecellobium dulce indicate the presence of quinones, steroids, flavonoids and terpenoids in major quantity in all the extracts in abundance. Phenols and cardiac glycosides are present in moderate amount and need slightly polar solvent like methanol and ethyl acetate for their separation. Tannins and saponins are also reported in methanol extract of Pithecellobium dulce seeds and their separation is possible using polar solvent.

Various chemical constituents give different R_f values in different solvent systems. It is reported that the difference of R_f values help in identify and elucidate the separated compounds. Extract in methanol shows the presence of nearly all studied phytochemicals, whereas, extracts in n-hexane reports less number of phytochemcial presence. These results may be related with the polarity of these solvents. Methanol is more polar than n-hexane. It can be concluded that polarity helps in extracting more number of phytochemicals in present system.

Pithecellobium dulce seeds extract can be further analysed for the reported phytochemicals to explore the possibility of its use as an herbal medicine with proved results using scientific techniques.

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Received on 01.05.2012

Modified on 25.05.2012

Accepted on 30.05.2012

© A&V Publication all right reserved

Research Journal of Pharmacognosy and Phytochemistry. 4(4): July- August 2012, 220-222