INTRODUCTION:

Samanea saman has long been a source of timber and livestock feed (green forage and pods) for local consumption. Minor medicinal and craft uses also are known. The wood is used for carving items for sale to tourists, and the seeds are strung in garlands¹. The root decoction is used in hot baths for stomach cancer in Venezuela. Rain tree is a folk remedy for colds, diarrhoea, headache, intestinal ailments, and stomachache². There are reports on the phytochemical analysis of the fresh leaves, stem, seed etc of Samanea saman but no reports on the dried fallen leaves of the whole plant. This plant is extensively seen in Coimbatore and there are lots of fallen leaves which are normally burnt or disposed. Hence the present work is aimed at utilizing the fallen parts of the plant and analyze for the presence of organic compounds in it, since plants are a store house of organic compounds.

HPTLC has been used extensively for quantification of compounds in plant extracts because of its simplicity, accuracy, cost effectiveness and rapidly³. Hence in the present study the petroleum ether extract and it’s fractionate have been analyzed by suitable colour tests. The HPTLC finger print of the petroleum extract has been recorded to analyze the presence of constituents in it. One of the fractionates, has been isolated by preparatory TLC and its HPTLC compared with that of extract to know its quantitative presence in the extract.

MATERIALS AND METHOD:

Collection of plant:

Combination of dried fallen leaves, flowers and stems of Samanea saman (Jacq.) Merr. were collected from Coimbatore.

Preparation of extracts:

Solvent extracts (Petroleum ether and ethyl acetate) were prepared by refluxing 940g of the fallen parts of Samanea saman with 1.5 liter of the appropriate solvent. The TLC analysis of the PE, EA, extracts of Samanea saman and the chemically and solvent fractionated portions of the extracts showed presence of a major blue Fluorescence spot at Rf 0.28 in PE: EA solvent system. Since this particular spot gave a characteristic round blue intense fluorescence and showed a prominent spot compared to other spots both in UV and iodine, it was aimed at isolating the compound. The petroleum ether extract was then fractionated with DCM. This fractionate also showed the blue fluorescence spot as a major spot. Hence the DCM fraction of PPE extract was subject to preparatory thin layer chromatographic separation. The band was then separated, washed with DCM first and then alcohol. Solvent evaporation yield 39mg of a white amorphous solid, which is to be further characterized by spectral techniques. In order to establish quantitatively presence of this compound in EA extract a HPTLC fingerprint was carried out.

Phytochemical analysis of extracts of Samanea saman (Jacq).Merr.

The ethyl acetate extract and P.DCM fraction was carried out using standard procedures^4-10 and was test the presence of alkaloids, saponin glycosides, flavonoids, tannins, polyphenolic compounds, quinine, anthroquinone and betacyanin.

Procedure for HPTLC:

Test solution preparation:

The plant extract PEA (SS2) was dissolved in 400µl of dichloromethane and centrifuged at 3000rpm for 2min. This solution was used as test solution for HPTLC analysis. The standard (isolated compound from P.DCM) was dissolved in 200µl of dichloromethane and used for the analysis.

Sample application:

2µl of test solution and standard solution was loaded as 6mm band length in the 3 x 10 Silica gel 60F₂₅₄TLC plate using Hamilton syringe and CAMAG LINOMAT 5 instrument.

Spot development:

The samples loaded plate was kept in TLC twin trough developing chamber (without saturation of Solvent vapor) with respective mobile phase (given mobile phase combination) and the plate was developed in the respective mobile phase up to 90mm.

Photo-documentation:

The developed plate was dried by hot air to evaporate solvents from the plate. The plate was kept in Photo-documentation chamber (CAMAG REPROSTAR 3) and captured the images at White light, UV 254nm and UV366nm.

Scanning:

Before derivatization, the plate was fixed in scanner stage and scanning was done at UV 366nm. The Peak table, Peak display and Peak densitogram were noted.

Analysis details:

Mobile phase: Petroleum ether 60-80° C-ethyl acetate (9: 1).

Detection: Blue coloured fluorescent zones at UV 366nm mode were present in the tracks, it was observed from the chromatogram before derivatization, which confirmed the Presence of compound of interest in the given sample.

RESULTS AND DISCUSSION:

Phytochemical Screening of Samanea Saman (Jacq.)Merr. Extracts and Its Fractionates

The extracts Samanea saman and its fractionates were screened for the presence of phytochemicals using standard colour tests. All the extracts showed the presence of alkaloid, anthraquinone and glycosides. The phytochemical screening results are given in Table 1.

The (EA) extracts showed the presence of alkaloids, flavonoids, saponins, quinine, phenol and glycosides. The ethyl acetate extract additionally showed the presence of saponins. The PE extracts were heated with (10-40ml) HCl for 3h and fractionated with DCM. The DCM portion was again fractionated with hexane (5ml) and this hexane portion was further fractionated with acetone (5ml). The P.DCM fraction showed the presence of alkaloids, flavonoids, quinine, phenol and glycosides.

Our literature survey on the phytochemical screening of Samanea saman revealed the presence of phytosteroids, alkaloids, flavonoids, tannins, glycosides, saponins and steroids. Also the presence of anthraquinone, alkaloids, flavonoids, quinone and carbohydrates in PE and EA extracts of Samanea saman are unreported. Our study revealed the presence of these compounds in the dried fallen parts of Samanea saman. Flavonoids are also shown to inhibit microbes which are resistant to antibiotics by¹¹. New flavonoids are continuously discovered and reported. Flavonoids which recently reported to have antimicrobial activity include quercetin 3'-O-glucoside, rutin¹², coumestrol, genistein and daidzein¹³, morin¹⁴ etc. Saponins are a special class of glycosides which have soapy characteristics¹⁵. It has also been shown that saponins are active antifungal agents¹⁶. Tannins are also known antimicrobial agents. Tannins (commonly referred to as tannic acid) are water-soluble polyphenols that are present in many plant foods. Tannins are water soluble plant polyphenols that precipitate proteins. Tannins have been reported to prevent the development of microorganisms by precipitating microbial protein and making nutritional proteins unavailable for them¹⁶. The growth of many fungi, yeasts, bacteria, and viruses was inhibited by tannins¹⁷. Tannins are reported to have various physiological effects like anti-irritant, antisecretolytic, antiphlogistic, antimicrobial and antiparasitic effects. Phytotherapeutically tannin-containing plants are used to treat nonspecific diarrhoea, inflammations of mouth and throat and slightly injured skins¹⁸. Thus the presence of these phytochemicals in Samanea saman is of significance.

It has been mentioned that antioxidant activity of plants might be due to their phenolic compounds¹⁹. Flavonoids are a group of phenolic compounds with known properties, which include free radical scavenging inhibition of hydrolytic and oxidative enzymes and anti inflammatory action²⁰. The presence of phenols, flavonoids etc in the fallen parts of Samanea saman prompted us to study further their antioxidant activity and its free radical scavenging activity²¹

Table 1: Phytochemical Screening of EA extract and P.DCM fraction

Phytochemicals	EA	P. DCM
Alkaloids	+	+
Flavonoids	+	+
Tannins	+	-
Saponins	+	-
Quinone	+	+
Anthraquinone	+	+
Phenols	+	+
Betacyanin	+	-
Glycosides	+	-

HPTLC Comparison of the Ethylacetate Extract and the Isolated Compound from DCM Fraction:

The isolated compound has been designated SD in the HPTLC profile figure 2 and 3.

HPTLC is an invaluable quantify assessment tool for the evaluation of botanical number. If allows for the analysis of broad number of compounds both officially and cost effectively. Hence this tool was employed for the finger print of NPSS and the compound isolate for DCM fraction figure 1. The HPTLC finger prints of the EA extract showed presence of a major compound figure 2 and additional minor peak corresponding to presence of the isolated compounds.

Figure 1: Chromatogram:

Before derivatization:

Figure 2: Track ethyl acetate extract of PSS Baseline display (Scanned at 366nm)

The HPTLC finger prints of the isolated compound figure 4 showed presence of the isolated compound quantitatively, the peak densitogram display figure 5 shows the amount of compounds SD in the extract. A comparison of the 3D display of HPTLC finger print of EA extract and SD is given in figure 6. The number of peaks obtained, Rf height and area of the peaks are given in Table 2.

Figure 3: Track ethyl acetate extract of PSS – Peak densitogram display

Figure 4: Track isolated compound - Baseline display (Scanned at 366nm)

Figure 5: Track isolated compound – Peak densitogram display

Table 2: HPTLC Peak table

Track	Peak	Rf	Height	Area	Assigned substance
Sample SS2	1	0.13	628.2	11388.7	Ethyl acetate extract (SS2)
Standard SD	1	0.11	488.3	9784.0	Compound (SD)

Figure 6: 3-D Display of Both Tracks

CONCLUSION:

The results of HPTLC reveals that the isolated compound is present as a major fraction in the EA extract of Samanea saman TLC analysis of all the extracts showed the presence of the compound prominently in all fractions. Thus further spectral characteristic of the compound (which is to be carried out as continuation of the present study) would enable to identify the compounds present. Thus the phytochemical screening procedures revealed the presence of important biologically active products in the dried fallen plant parts of Samanea saman, signifying its importance in phytochemistry.

ACKNOWLEDGEMENT:

The authors thank the authorities of the Avinashilingam Deemed University for Women, Coimbatore-43 for having providing the facilities to carry out this research work.

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Received on 06.10.2010 Modified on 22.10.2010

Asian J. Research Chem. 4(2): February 2011; Page 266-269