INTRODUCTION:

The genus Allamanda belongs to family Apocyanaceae comprises of many vatieties like A. blanchetti and A .violacea¹. It is an erect, evergreen, variable shrub; up to 4 meter in height, widely grown in Indian gardens.The plants does not tolerate shade, salty or alkaline soils. They are highly sensitive to frost^1.Allamanda cathartica L. (Apocyanaceae) is commonly known as Angel’s trumpet, Yellow bell¹. It is native to South and Central America and West Indies. It is distributed throughout tropics. It is in many parts of Central, Eastern, and Southern India and in Andaman Islands. Roots, shoots, leaves, flowers, fruits, milky sap and whole plant are used².

Fig. No. 1: Plant of Allamanda cathartica Linn.

DESCRIPTION:^{1, 3}

Leaves:

They are whorled, obviate to oblong lanceolate, 8-12*2.5-4.0cm, wavy and which may be either opposite or whorls of three or four.

Flowers:

Flowers are very large, usually yellow, in few flowered terminal and auxiliary cymes. Corrolla with short tubular base, then suddenly companulate, lobes rounded, contorted to the left, throat with a ring of ciliate scales. Overy one celled with two parietal placentae and many ovules.

Fruits:

These are globus with numerous stout, green prickles, finally two valved.

Seeds:

Capsules are subglobose, 4 to 6 cm in diameter and densely prickled. They contain many tan, flattened, winged seeds.

MATERIALS AND METHODS:

The plant material used in this investigation consists of leaves of Allamanda cathartica Linn (Family: Apocynaceae) which was collected from local market and was authenticated by Nicholas Piramal Pvt. Ltd., Goregaon, Mumbai. Extraction was performed by continuous extraction using Soxhlet extractor. Methanol was the solvent used for extraction.

Preliminary physico-chemical evaluation⁴:

Evaluation of drug means confirmation of its identity and determination of its quality, purity and detection of the nature of adulteration. The evaluation of a crude drug is necessary because of three main reasons: biochemical variation in the drug, deterioration due to treatment and storage and substitution and adulteration. Preliminary Phytochemical screening is a part of chemical evaluation. The qualitative chemical test is useful in detection of adulteration. Air dried leaves were used for quantitative determination of loss on drying, total ash, acid insoluble ash, alcohol soluble extractive values according to standard procedure of Indian Pharmacopoeia (Anonumous, 1996).

Phytochemical investigation⁵:

The plant is biosynthetic laboratory, not only for chemical compounds such as carbohydrates, proteins and lipids, but also for a multitude of compounds like glycosides, alkaloids, volatile oils, tannins etc that exert physiological and therapeutic effects. The compounds that are responsible for medical property of the drug are usually secondary metabolites. A systemic study of a crude drug embraces, through consideration of primary and secondary metabolites. Thus the plant material is subjected to preliminary phytochemical screening for the detection of various plant constituents.²⁴ Phytochemical analysis of extract was conducted following the procedure of Indian Pharmacopoeia (Anonymous, 1996). By this analysis, the presence of several phytochemicals likes alkaloid; flavonoid, tannin, saponin, protein and carbohydrate were tested.

High performance thin layer chromatography (HPTLC):

High Performance Thin Layer Chromatography studies are major advancement of TLC principles. HPTLC studies were carried out to obtain the finger print of methanolic extract of Allamanda cathartica Lin. extract, which offers better quantitative resolution and requires shorter time.

The conditions followed for HPTLC development were as follows:

Conditions of applications:

Instrument used : Linomat IV applicator.

Test plates : Silica gel GF₂₅₄(E-Merk) precoated aluminium plates.

Format : 10 X 10 cm.

Spotting volume : 2µl, 4µl and 10µl.

Band size : 8 mm.

Speed of drying time : 15 sec/µl.

Nitrogen pressure : 2 psi.

Conditions of chromatography:

Solvent system : All analytical grade solvents.

Separation technique : Ascending type.

Development chamber : Twin trough glass chamber.

Distance of migration : 9 cm.

Conditions of scanning:

Instrument: Camag Scanner IV.

Mode : Absorption / reflection with deuterium lamp.

Wavelength: UV-254 nm, 366 nm, 580 nm.

Slit dimension: 6X 0.30 mm.

HPTLC fingerprints of the fractions were obtained using the above chromatographic conditions.

RESULTS AND DISCUSSION:

Preliminary physico-chemical evaluation:⁴

It is necessary that every medicinal plant prior to its use be identified and standardized for its various physicochemical parameters. Air dried leaves were used for quantitative determination of phytochemical values. The ash values, extractive values, percentage yield, color and consistency of methanolic extract of Allamanda cathartica L. were determined five times as per WHO recommendations. (Table No.1, 2)

Table No.5: HPTLC peak area of methanolic extract (10µl) of Allamanda cathartica Linn. at 254 nm.

Peak	Start Rf	Start Ht	Max Rf	Max Ht	Max %	End Rf	End Ht	Area	Area %
1	0.04	28.7	0.06	159.9	12.19	0.07	2.2	1554.0	3.75
2	0.08	7.6	0.09	23.9	1.82	0.11	0.6	266.5	0.64
3	0.18	6.8	0.21	21.0	1.60	0.23	1.6	432.5	1.04
4	0.29	14.0	0.37	302.5	23.05	0.41	27.9	9431.0	22.73
5	0.41	28.0	0.45	134.6	10.26	0.47	55.9	3741.3	9.02
6	0.47	56.0	0.48	66.4	5.06	0.50	50.3	1179.9	2.84
7	0.50	50.7	0.54	118.7	9.04	0.55	100.1	3235.6	7.80
8	0.55	100.5	0.56	123.2	9.39	0.59	13.8	2364.9	5.70
9	0.59	13.8	0.62	44.2	3.37	0.66	3.2	1256.6	3.03
10	0.70	2.9	0.87	205.5	15.66	0.92	100.3	15504.4	37.37
11	0.92	100.3	0.94	112.2	8.56	0.97	1.7	2523.3	6.08

Table No.6: HPTLC peak area of methanolic extract (10µl) of Allamanda cathartica Linn. at 366 nm.

Peak	Start Rf	Start Ht	Max Rf	Max Ht	Max %	End Rf	End Ht	Area	Area %
1	0.09	0.1	0.11	19.5	1.74	0.13	4.6	256.1	0.99
2	0.15	3.8	0.18	56.4	5.05	0.20	22.3	1026.4	3.96
3	0.20	22.3	0.22	41.	3.71	0.24	15.5	747.9	2.89
4	0.24	15.3	0.30	130.6	11.69	0.32	40.2	3347.3	12.93
5	0.32	40.3	0.33	42.8	3.84	0.36	0.9	690.5	2.67
6	0.36	1.0	0.39	315.2	28.22	0.41	162.1	6475.1	25.01
7	0.41	162.2	0.43	283.3	25.37	0.48	54.1	7192.9	27.78
8	0.48	54.9	0.51	73.3	6.56	0.53	1.0	2184.1	8.43
9	0.54	1.3	0.56	38.3	3.43	0.56	36.5	519.8	2.01
10	0.58	38.6	0.60	50.5	4.52	0.64	20.1	1781.9	6.88
11	0.64	19.8	0.68	37.8	3.39	0.71	0.4	1123.7	4.34
12	0.71	0.5	0.74	27.7	2.48	0.77	1.0	547.8	2.12

Table No.7: HPTLC peak area of methanolic extract (10µl) of Allamanda cathartica Linn. at 580 nm.

Peak	Start Rf	Start Ht	Max Rf	Max Ht	Max %	End Rf	End Ht	Area	Area %
1	0.04	1.8	0.05	127.1	5.91	0.07	0.6	1312.9	1.61
2	0.07	0.4	0.08	11.5	0.53	0.09	0.4	86.7	0.11
3	0.10	0.0	0.12	34.4	1.60	0.15	0.2	633.0	0.78
4	0.15	0.1	0.18	149.0	6.93	0.20	0.9	2589.2	3.18
5	0.21	0.0	0.25	64.5	3.00	0.29	7.1	1345.3	1.65
6	0.29	7.3	0.36	247.6	11.51	0.39	110.1	9220.3	11.33
7	0.41	112.5	0.43	153.1	7.11	0.45	101.1	3612.4	4.44
8	0.45	101.2	0.47	184.0	8.55	0.49	137.5	5309.4	6.53
9	0.50	138.8	0.56	366.8	17.05	0.56	362.6	12392.4	15.23
10	0.57	363.0	0.58	383.0	17.80	0.62	163.0	12144.5	14.93
11	0.66	78.7	0.69	128.0	5.95	0.74	70.1	5428.3	6.67
12	0.74	69.9	0.86	302.2	14.05	0.97	7.4	27288.6	33.54

Drug	Total Ash Value	Acid Insoluble Ash	Water Soluble Ash	Sulphated Ash	Water Extractive	Alcohol Extractive
*Allamanda* *cathartica*	9.33%w/w	3.70%w/w	5.25%w/w	6.50%w/w	18.20%w/w	4.88%w/w

Extraction solvent	% Yield	Color	Consistency
Methanol	32.06 %	Greenish Brown	Viscous

Name of the test	Methanolic extract
Carbohydrates	+
Reducing sugar	+
Monosaccharides	+
Pentose sugar	+
Hexose sugar	+
Non reducing polysaccharides	-
Glycosides
Cardiac glycosides	-
Anthraquinone glycosides	-
Saponin glycosides	+
Coumarin glycosides	-
Flavonoids	+
Alkaloids	+
Phenolic compound	+
Steroids	+
Proteins	+
Amino acids	+

Sr. no.	Extract	Mobile phase	Spot observed
1	Methanol	Toluene : Chloroform: Ethanol [8:8:2]	13