INTRODUCTION:

Arisaema leschenaultii Blume. (Araceae) commonly called as Cobra lilly is widely used in the treatment of many aliments. It has widely distributed over the greater part of India on the hills of Assam, Karnataka, Kerala and Tamil Nadu. It has monoecious or dioecious herb. Tubers are globose, about 5 cm diameter roots from the upper side of the tuber stem about 15 cm long, clothed with long mottled sheaths, leaf solitary, petiole stout, 30-60 cm long, pale green.¹ Ariseminone is present in the species of Arisaema.² The phytochemical investigations revealed the presence of carbohydrates, phenolic compounds and tannins, proteins and amino acid and glycosides. It is used in urinary diseases, colitis, eczema, purging, gonorrhea, piles, haemorrhoids, syphilis, roundworms, fistula and sinus etc. Roots are employed as a medicine by the signhalese (Thwaites).¹ This study was aimed at investigating antioxidant potential of tubers of Arisaema leschenaultii Blume. Therefore an effort is made to contribute to establish scientific evidence in this regard.

MATERIALS AND METHODS:

Plant Material:

The tubers of Arisaema leschenaultii Blume. were collected from the various places of Barkatullah University campus and from Balaghat district during the month of September-October and were authenticated by Dr. A. K. Pathak, HOD Department of Pharmacy, Barkatullah University Bhopal. A voucher specimen (BU/4042) has been deposited in the Herbarium of the same department.

Preparation of Extracts:

Alcoholic extract: The collected tubers of plant were dried in shade and powder was exhaustively extracted with alcohol (95 %) in soxhlet apparatus. The ethanolic extract was concentrated in vacuum under reduced pressure using rotary flash evaporator. It was further concentrated and dried in desiccator.

Another 100 g of the coarse powder was extracted exhaustively and successively with various solvents in an increasing order of polarity viz., benzene, chloroform, methanol and water. Each extract was concentrated to a small volume and allowed to dry. After drying, the respective extracts were weighed and percentage extractive values were determined.

Fig.1: Free Radical Scavenging Activity of various groups on DPPH Radical

Phytochemical Screening:

The qualitative chemical investigation of all the extracts was carried out to check the presence of various phytoconstituents. It revealed the presence of carbohydrates, phenolic compounds and tannins, proteins and amino acid and glycosides.

Antioxidant Activity:

Free Radical Scavenging Capacity on DPPH Radical:

For the present study the samples were prepared in different concentrations i.e. 10-100 mcm/mL in AR grade methanol. The samples of above concentrations were mixed with 3 mL of 100 µM of DPPH prepared in AR grade methanol and make up the final volume up to 4 mL with AR grade methanol. The absorbance of the resulting solutions and the blank (with same chemicals except samples) were recorded after 20 min at room temperature, against BHT and ascorbic acid. The disappearance of color was read spectrophotometrically at 517 nm using a JASCO V 530 UV-Visible Spectrophotometer. Radical Scavenging Capacity (RSC) in percent was calculated by following equation:

RSC (%) = 100 x A_blank - A_sample / A_blank

Where

RSC = Radical Scavenging Capacity

A_blank= Absorbance of blank

A_sample= Absorbance of sample

From the obtained RSC values the IC₅₀were calculated, which represents the concentration of the scavenging compound that caused 50 % neutralization.³ The results are shown in Table 1 and Figure 1.

Fig. 2: IC₅₀ values of various groups on DPPH radical

Fig.3: Scavenging of hydroxyl radicals by various groups

Scavenging of Hydroxyl Radicals:

Thiobarbituric Acid Reactive Substances (TBARS) Assay:

All the reagents were dissolved in phosphate buffer, freshly prepared. Reaction mixtures contained deoxyribose (2.8 mM), KH₂PO₄-KOH buffer, pH 7.4 (20 mM), FeCL₃ (0.1 mM), EDTA (0.1 mM), H₂O₂ (1 mM), ascorbate (0.1mM) and drug (variable concentration), in a final volume of 3 mL. The reaction mixture was incubated for 1 h at 37°. After incubating the reaction was stopped by adding 2 mL of ice cold 0.25N HCl containing 15 % trichloroacetic acid, 0.38 % thiobarbituric acid and 0.05 % BHT. Following heating at 80° for 15 min, samples were cooled and centrifuged at 1000 RPM for 15 min; the absorbance of the supernatant was measured at 532 nm. Test compounds were dissolved in 0.05N NaOH and the pH was adjusted to 7.4 with 0.1 N HCl. The absorbance was read against a blank (containing a buffer solution instead of sample) at 532 nm. The absorbance was used for the calculation of the percentage dissolution of 2-deoxy-D- ribose degradation by the sample by using the following formula.

RSC (%) = 100 x A_blank - A_sample / A_blank

BHT was used as a positive control. From the obtained RSC values, the IC₅₀ values were calculated, which represent, the concentration of the scavenging compound that caused 50 % neutralization.⁴ The results are shown in Table 2 and Figure 3.

Table 1: Free Radical Scavenging Activity on DPPH Radical

Conc. Mcg/mL	TE	Su ME	BHT	Conc. Mcg/mL	Asc. acid
10	48.27	34.52	37.86	1	54.99
20	54.55	38.91	41.28	2	61.28
30	60.22	46.28	46.32	3	65.23
40	64.19	51.59	49.69	4	69.05
50	67.98	56.28	53.46	5	74.88
60	73.52	59.22	58.91	6	79.24
70	77.91	62.88	62.38	7	81.54
80	81.23	65.45	66.81	8	84.29
90	84.71	69.10	71.97	9	89.09
100	88.16	71.37	76.29	10	90.46
IC₅₀	26.25	46.57	35.68		16.73

TE – Total Ethanolic, Su ME – Successive Methanolic, BHT – Butylated Hydroxy Toulene, Asc. Acid – Ascorbic acid, IC₅₀ –Inhibition Concentration (the concentration producing 50 % of maximal inhibition)

Fig.4: IC₅₀ values of various groups on hydroxyl radicals

Total Antioxidant Capacity:

100 mcg of extract is added to a mixture of ammonium molybadate (4 mM) and sodium phosphate (28 mM) in 0.6 M H₂SO₄ in total volume of 2 mL in eppendroff tubes and kept at 95° for 90 min and the absorbance will be measured at 695 nm after cooling at room temperature.⁵ The results are shown in Table 3 and Figure 5.

Table 2: Scavenging of Hydroxyl Radicals- TBA Reactive substances

Conc. Mcg/mL	TE	Su ME	BHT	Conc. Mcg/mL	Asc. acid
10	40.21	34.98	27.31	1	50.13
20	46.86	42.87	33.47	2	55.37
30	51.36	48.64	40.01	3	61.10
40	55.97	54.11	45.85	4	67.32
50	61.28	61.19	53.42	5	72.49
60	66.32	67.12	59.97	6	76.58
70	71.08	73.55	65.12	7	80.17
80	75.10	77.22	70.03	8	86.22
90	80.55	80.91	76.00	9	92.36
100	86.74	82.99	83.71	10	98.79
IC₅₀	34.41	35.94	48.00		19.64

RESULTS AND DISCUSSION:

Antioxidant activity of various extracts of Arisaema leschenaultii Blume. were performed by using different invitro models and using Ascorbic acid and BHT as standards. The results are as tabulated in the Table 1, 2 and 3. The total alcoholic extract shown the maximum free radical scavenging capacity as compared to the other extracts. Phytochemical investigations revealed that presence of carbohydrates, phenolic compounds and tannins, proteins and amino acid and glycosides and their presence was substantiated by thin layer chromatographic studies.

Antioxidant studies of different extracts were performed and it was observed that they shows promising antioxidant effects, amongst all, the total alcoholic extract, methanolic extract are active in scavenging the free radicals as compared to the others. The total alcoholic extract exhibited statistically highly significant free radical scavenging capacity on DPPH radical (IC₅₀ value – 26.25 mcg/mL). In hydroxyl radical scavenging and total antioxidant capacity the alcoholic extract showed highly significant results as compared to the standard.

ACKNOWLEDGEMENT:

Authors are greatly thankful to the management of Kamla Nehru College of Pharmacy, Butibori, Nagpur and Department of Pharmacy, Barkatullah University Bhopal for providing free access to their facilities to carry out research work.

Table 3: Total Antioxidant Capacity of Different Extracts of Arisaema leschenaultii Blume.

Test Samples and Standard (Conc. – 100 mcg/mL)	Total Antioxidant Capacity
TE	308
Su ME	289
Su BE	159
Su CH	103
Su Aq	210
BHT	300

TE – Total Ethanolic, Su ME – Successive Methanolic, Su BE – Successive Benzene, Su CH – Successive Chloroform, Su Aq – Successive Aqueous, BHT – Butylated Hydroxy Toulene

Figure 5: Total antioxidant activity of various groups

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2. Rastogi PR and Mehrotra NB. Compendium of Indian Medicinal Plants. Vol V. CDRI and National Institute of Science Communication. New Delhi.

3. Rajkumar DV and Rao MNA. Dehydrozingerone and isoeugenol as inhibitors of lipid peroxidation and as free radical scavengers. Biochem. Pharmacol. 1993; 46(11): 2067- 2072.

4. Raghavan G, Madhavan V, Chandana VR, Shirwaikar A, Rawat AKS and Mehrotra S. Antioxidant potential of Anogeissus latifolia. Biol. Pharm. Bull. 2004; 27(8): 1266- 1269.

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Received on 20.10.2008 Modified on 16.12.2008

Research J. Pharm. and Tech. 2(1): Jan.-Mar. 2009; Page 134-137