INTRODUCTION:

Euphoria longan (F- Sapidaceae) commonly known as longan or dragon eye. It is a large handsome evergreen tree and it is native of Southern western India occurs in the forest of Western ghats from Kongan Southwards to Tinnevely hills¹. Leaves are alternate compound are red green. A flower of thin tree is made of small yellow to yellow borne in terminal panicles. Leaves have been used as tonic, brain stimulant, nutrient, stomachic, refrigerant² and for the treatment of amnesia insomnia³anemia palpitations and neurosis.⁴ Due to uniqueness of leaves property in curing of different ailments this part was selected for the study. Hence the present investigation is an attempt in this direction and includes morphological determination, physico chemical constants and preliminary phytochemical screening of Euphoria longan

MATERIAL AND METHODS:

Plant material:–

Euphoria longan was collected in Tinneveli Dist of Tamilnadu. The botanical identify of the plant was confirmed by Dr. V. Chelladurai Research Officer, (Retd) Botany, (C.C.R.A.S. Govt of India) voucher specimen has been deposited at the museum of the Dept. of .Pharmacognosy, K.M.College of Pharmacy. Madurai.

Macroscopic analysis: ⁵

The macroscopy of leaf are 10cm long, equally or unequally pinnate; rachis , rusty-puberulous when young, afterwards glabrous; leaflets 2-5 pairs, reddish when young,coriaceous,6.3-20 by 2.5-5cm.oblong or ovate- lanceolate, more or less oblique, shortly acuminate, glabrous and reticulately veined, base cuneate; main nerves 10-15 pairs, prominent beneath, petiolules 6-12mm.long.

Ethno medical information of Euphoria longan: ^5,6

Fruit - Tonic, brain stimulant, nutrient, stomachic and anthelmintic

Arillus - Anti amnesia, refrigerants, anxiolytic

Seed - Decrease blood pressure

Pulp - Promote hair growth

Color of Euphoria longan

Parts observation

Leaf - green

Bark - yellowish grey

Wood - red

Fruit - red to brown yellow

Flowers - pale yellow to yellow borne

Histochemical and proximate analysis: ^7,8

Histochemical and Physico- chemical parameters of the powdered drug such as ash value, extractive value, loss on drying and crude fiber content were performed according to the method .Extracts were prepared by various solvents by standard methods and percentage of dry extract was calculated in terms of air-dried leaf powder. (Table 1, 2, 3)

Fluorescence characteristics:

When physical and chemical parameters are inadequate as it often happens with the powdered drugs, the plant material may be identified from their adulterants on basis of fluorescence study (Table 4)

Table 1: Histo chemical analysis

Reagent	Test for	Nature of color change	Histological zone
Aniline Hcl	Lignin	Bright yellow	Vascular zone
Phloroglucinol in Hcl	Lignin	Red	Vascular zone
Saffranin 1%	Lignin	Red	Vascular zone
Iodine	Starch	Blue	Phelloderm
Chlor – zinc iodine solution	Cellulose	Blue	Phelloderm
Methylene blue	Mucilage	Deep blue	Absent
Ruthenium red	Mucilage	Deep blue	Absent
Dil. Fecl₃	Tannin	Bluish blank	Epidermal phelloderm
Iodine	Starch	Blue	Phelloderm

Table 2: Proximate analysis

Parameters	Values (%)w/w
Loss on drying	16
Ash Values
A. Total ash	8.72
B. Acid insoluble ash	2.37
C. Water-soluble ash	3.1
D. Sulphated ash	4.1

Table 3: Extractive values

Solvents	Extractive Values w/w (%)
Petroleum ether	2
Benzene	1.8
Chloroform	3.3
Methanol	3.4
Ethanol	3.1
Water	4.2

Behavior of leaf powder with different chemical reagents:

Behavior of leaf of Euphoria longan with different chemical reagents was performed to detect the occurrence of phytoconstituents along with colour changes under ordinary daylight by standard method (Table 5)

Quantitative standards: ^9,10

Total carbohydrate, tannin, saponin, flavonoids, sugar, chlorophlly, crude fiber content in leafs of Euphoria longan was estimated. (Table 6)

Determination of Saponin:

According to the results obtained from positive foaming test and high foaming index of leafs of Euphoria longan study was carried out for the estimation of total saponin content

Estimation of total sapogenin content:

Drug powder was extracted with petroleum ether by refluxing for half hour. Marc obtained, was again refluxed with 90% methanol for half an hour. The methanol extract was distilled off under reduced pressure to obtain semi solid residue. Then the extract was partitioned between distilled water and n-butanol. Aqueous fraction was again partitioned with n-butanol 3times. Combined n-butanol fraction was then evaporated. For hydrolysis of saponin the semi solid mass was then refluxed with 2N HCl for 8 hrs. After cooling the content were partitioned for 3 times with chloroform. Combined chloroform was evaporated and dried to constant weight and total saponin content was calculated.

Fig 1: UV 254nm

Fig 2: UV 366nm

Fig 3 Visible light

Table 4: Fluorescence analysis of leaf powder of Euphoria longan

Treatment	Daylight	UV light 254 nm	UV light 366nm
Powder	Green	Dark Green	Dark Green
Powder treated with distilled water	Green	Dark Green	Blackish green
Powder+CHCL3	Dark Green	Dark Green	Blackish green
Powder +acetone	Yellowish Green	Pale Green	Pale Green
Powder +conc HCL	Pale Green	Yellowish Green	Yellowish Green
Powder +1N HCL	Light Green	Green	Green
Powder +50% HNO3	Pale Green	Pale Green	Pale Green
Powder +50% H2So4	Light Green	Colourless	Pale Green
Powder +1N NaoH in methanol	Buff	Buff Green	Pale Green
Powder +1N NaoH in water	Dark Green	Green	Light Green
Acetic acid	Pale green	Pale green	Dark green
Ammonia solution	Brown	Brownish black	Reddish brown

Table 5: Behavior of powder of Euphoria longan with different chemical reagent

Reagent	Color/ precipitate	Constituents
Powder + Conc H₂So₄	Reddish brown	Steroids present
Powder + Aqs ferric chloride	Black colour	Tannins present
Powder + water	Foam is produced	Saponin present
Powder + Aqs mercuric chloride	Brown colour	Alkaloids present
Powder + Picric acid	Yellow colour	Alkaloids present
Powder + Magnesium Hcl	Black colour	Flavonoids present
Powder+ Aqs silver nitrate	Precipitates	Protein present
Powder + Ammonia solution	Pink colour	Anthraquinone glycosides present
Powder + Aqs. KOH	Pink	Anthraquinone glycosides present

Table 6: Qualitative analysis of Euphoria longan

Components	Results
Tannin content	3 mg/g
Total saponin content	9.2 mg/g
Flavonoid content	2.40 mg/g
Total sugar content	130.31 mg/g
Chlorophyll content	0.741µg/ml
Total sapogenin	3 mg/g
Total crude fibres	7 mg/g
Total protein	90µg/ml

Preliminary phytochemical investigation:^11-14

The qualitative chemical test of various extracts of Euphoria longan was carried out using standard procedure. (Table 7)

Thin Layer Chromatography:^15,16

About 30gms of silica gel – B was weighed out and it was shaken with 100ml of water to form a homogenous suspension. The suspension was poured into a thin layer chromatography applicator which was adjusted to 0.25mm thickness. 20 to 40 Carrier plates (20.5cm) were laid down for air drying.

The plates were kept in the hot air oven at 110°C for one hour to activate the silica gel – G. The plates were stirred in a dry atmosphere and used whenever required. By using the capillary tube the extracts are spotted on the T.L.C plates 2cm above the bottom and in the chromatogram in various solvent systems for different compounds. The spots are developed in solvent system were identified by means of different spraying reagents. (Table 8).

Table 7: Data showing phyto constituents present in different extracts

Test and phytoconstituents	Petroleum ether	Chloroform	Ethanol	Aqueous
Test for Alkaloids Mayer’s reagents Dragendorff’s reagent Hager’s reagent Wagner’s reagent	_	+	+	+
Test for Carbohydrates Molisch’s test Fehlings test Benedicts test Barfords test	-	+	+	-
Test for Glycosides Legal’s test Borntrager’s test	-	+	+	+
Test for Phytosterol Libermann Burchard Test Salkowski Test	- -	+	+	+
Test for fixed oils and fats	_	_	_	_
Test for saponins Foam	-	+	+	+
Test for tannins and phenolic compounds	-	-	+	+
Test For Proteins and Free Amino Acids Millon’s Reagent Ninhydrin Reagent Biuret Test	_	+	+	+
Test for Flavonoids Shinoda’s Test Florescence Test	- -	+	+	+
Test for Lignin	-	-	+	-
Test for Terpenes	-	+	+	-
Test for phlobatannins	-	-	+	+

HPTLC fingerprinting of alcoholic extract:¹⁷

HPTLC instrumentation:

Quantitative and qualitative analysis was performed with the help of HPTLC instrument. The HPTLC system (Camag, Muttenz, Switzerland) consists of

(1) TLC scanner connected with a PC running Win CATS 1.4.3 software under MS Windows

(2) Lincomat V Sample applicator

(3) Photo documentation system Camag

Spotting of samples:

The chromatographic estimation was performed by streaking the extracts in the form of narrow bands of 8mm length on the percoated silica gel 60 F254 aluminium TLC plate,at a constant application rate of 150 µl/s and 10s/µL

was employed with help of Camag 100 µl syringe connected to a Nitrogen tank; using a Camag Linomat V (Camag, Muttenz, Switzerland). The space between three bands was kept 15mm.5µ of 1% concentration solution from each three extracts (Methanol, Choloroform and Petroleum ether) was placed as a spot.

Fig 4:Anisaldehyde H₂SO₄

Plate development and chromatographic conditions:

After spotting the plate, it is subjected to linear ascending development in a solvent system of Toluene: Acetone in the ratio of 9:1 v/v; at Camag Twin Trough glass chamber, which was saturated with the same solvent system at room temperature just 10minutes prior to development.

Fig5: FeCl₃

Fig 6: Libermann-Burchard

Table 9: UV 254nm

Peak	Rf value	Area	Area %
1	0.12	943.5	9.39%
2	0.14	324.5	3.23%
3	0.19	1147.8	11.42%
4	0.57	579.6	5.77%
5	0.68	2347.4	23.35%
6	0.82	4708.2	46.84%

Scanning of plate:

TLC plate was dried in flowing air at room temperature. Densitometric scanning was carried out using Camag TLC Scanner III between wavelength of 200-450nm with a slit dimension of 6.00 x 0.30mm, with scanning speed of 20nm/s, and data resolution was at 100µm/ step. The source lamps for radiation were deuterium and tungsten lamps. The chromatograms were generated using Win CATS evaluation software (Version 1.4.3).

Table 10: UV 366nm

Peak	Rf value	Area	Area %
1	0.12	1251.3	1.62%
2	0.16	781.8	1.01%
3	0.19	8374.8	10.84%
4	0.26	1544.0	2.00%
5	0.37	3520.6	4.56%
6	0.44	711.5	0.92%
7	0.49	1685.1	2.18%
8	0.58	8381.7	10.85%
9	0.70	19608.5	25.39%
10	0.76	8405.3	10.88%
11	0.86	21877.3	28.33%
12	0.93	1088.1	1.41%

Table 11: Visible light

Peak	Rf value	Area	Area %
1	0.12	428.0	4.17%
2	0.14	257.6	2.51%
3	0.19	770.4	7.51%
4	0.49	451.0	4.40%
5	0.59	1009.8	9.84%
6	0.68	3114.3	30.35%
7	0.82	4229.7	41.22%

Table 12: Anisaldehyde H₂SO₄

Peak	Rf value	Area	Area %
1	0.12	95.5	0.75%
2	0.18	1488.2	11.75%
3	0.24	665.4	5.25%
4	0.33	200.6	1.58%
5	0.44	2396.6	18.92%
6	0.52	1115.1	8.80%
7	0.56	957.2	7.56%
8	0.62	1851.5	14.62%
9	0.69	907.5	7.16%
10	0.78	855.6	6.75%
11	0.80	1584.7	12.51%
12	0.88	549.4	4.34%

Table 13: FeCl₃

Peak	Rf value	Area	Area %
1	0.07 Rf	777.0 AU	29.26%
2	0.09 Rf	953.7 AU	35.92%
3	0.14 Rf	425.3 AU	16.02%
4	0.21 Rf	499.3 AU	18.80%

Table 14: Libermann-Burchard

Peak	Rf value	Area	Area %
1	0.06 Rf	475.8 AU	14.25%
2	0.09 Rf	195.3 AU	5.85%
3	0.12 Rf	181.2 AU	5.43%
4	0.19 Rf	429.4 AU	12.86%
5	0.67 Rf	799.2 AU	23.94%
6	0.82 Rf	1257.3 AU	37.66%

Photo documentation of plate:

After the scanning, images of the plate were taken by using different wavelength of lights 254 nm and 366nm with the help of Photo documentation system of Camag.

6 spots with R_f0.12,0.14,0.21,0.52,0.70,0.84 were observed under 254nm ( Fig 1),(Table 9), 12 spots with R_f 0.12,0.16,0.21,0.26,0.34,0.44,0.49,0.58,0.68,0.76,0.84,0.93 were observed under 366nm ( Fig 2), ( (Table 10), 7 spots with R_f0.12,0.14,0.19,0.44,0.59,0.63,0.80 were observed visible light( Fig 3), ( (Table 11) , 12 spots after spraying of anisaldehyde H₂SO₄( Fig 4), ((Table 12) , 4 spots after spraying FeCl₃ ( Fig 5), ( (Table 13) , 6 spots after spraying Libermann – Burchard ( Fig 6), (Table 14)

RESULTS AND DISCUSSIONS:

The present study carried out chemo-microscopy revealed the presence of lignin, tannins, and starch (Table 1). Percentage of moisture content, total ash, acid insoluble ash ,water soluble ash, extractive values was also found Table(2,3) and different chemical compounds such as the glycosides, alkaloids, saponins, tannins among others were detected in various extracts which could make the plant useful for treating different ailments and having a potential of providing useful drugs of human use Table(7). HPTLC technique was used to separate chemical constituents. A qualitative densitometric HPTLC analysis was performed to confirm the presence of glycosides, saponins , tannins.

CONCLUSION:

Euphoria longan were authenticated and physico chemical and phytochemical analysis showed presence of glycosides, tannins saponins, protein, lignin, terpenes, phlobatannins and flavonoids and HPTLC confirmed presence of glycosides, tannins saponins in Euphoria longan leaves.

REFERENCES:

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Received on 30.06.2009 Modified on 25.08.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 31-35

Chemical constituents	Mobile phase	Spraying reagent	Inference
Triterpenoid saponin	n-hexane : ethyl acetate: acetic acid (10:5:1)	Sulphuric acid	Dark pink
Saponin glycoside	Toluene: ethyl acetate: methanol: acetic acid (3:4:3:1)	20% H ₂So₄ in methanol	Dark pink
Phenolic compounds	Toluene: ethyl acetate: acetone (2:4:2)	Sulphuric acid	Yellowish green
Chlorophyll	Petrol :acetone: isopropanol (9:4:0.45)	UV	Yellowish green
Amino acid	n- butanol : acetic acid :water (4:1:5)	Ninhydrin	Purple
Flavonols : Aglycones glycoside	Acetic acid: Con Hcl :water (30:3:10) n-butanol: acetic acid: water (4:1:5)	UV	color
Alkaloid	Methanol: con NH ₄OH (200:3)	Dragondroffs	Orange brown
Cardiac glycoside	n-hexane : ethyl acetate ( 9:6:1:3)	Anisaldehyde in sulphuric acid	Orange
Lignins	Benzene: acetic acid (9:1)	UV	Mauve
Triterpene	Petroleum ether: dicloroethane: acetic acid (2:5:12.5:2)	Chloro sulphonic acid reagent	Dark brown
Phytosterols	Hexane : Diethyl ether (32:1)	Stannic chloride reagent	Orange brown round
Carbohydrate	Butanol : Aceticacid : Water : Ether (9:6:1:3)	Phenol in sulphuric acid	Greenish brown