INTRODUCTION:

Science might have made advancement in the field of medicine over the years; plants still remain as the primary source of drugs, used in modern medicine for their inexpensiveness, low side effects and compatibility to the human body. Sure, the potential of obtaining new drugs from plant sources is so great that thousands of substances of plant origin are being studied for activity against such enervating foes as heart diseases, respiratory disorders, cancer, and AIDS. In this way, modern medicine will continue to be enriched by the introduction of newer and more potent drugs from plant sources ^[1].With the excessive generation of Reactive Oxygen Species (ROS), the cellular and extracellular macromolecules can undergo oxidative damage, resulting tissue injury in several human chronic diseases including cardiovascular diseases, mutagenesis, cancer, many neurological disorders and the aging process ^{[2, 3]}. In the above situation, antioxidants play an important role in preventing oxidative damage caused by reactive oxygen species (ROS) ^[4].

Many chemotherapeutic drugs have been isolated from plant species or from a natural prototype. More than 50 % of all drugs in clinical trials for anticancer activity were derived from natural sources or are related to them ^[5]. The brine shrimp lethality assay is considered a useful tool for preliminary assessment of toxicity.

Aegle marmelos (common Bangla name, Bael) is a tree belongs to Rutacea family, which grows in most of the areas of Bangladesh, India, and Southeast Asia. Many bioactive compounds have been isolated from the plant ^{[6, 7]} which includes cinnamic acid, aegeline, skimmianine, lupeol, cineole, citral, citronella, cuminaldehyde, eugenol, marmesinin, marmelosin, luvangetin, aurapten, psoralen, marmelide, fagarine, marmin and tannin ^[8-16]. Several previous studies report that different parts of A. marmelos have various therapeutic uses, such as for the treatment of asthma, anemia, fractures, wounds healing, swollen joints, high blood pressure, jaundice, diarrhea ^[17]also for intermittent fever, intestinal ailments, fertility control and treatment after childbirth and fish poison ^[18]. A. marmelos is a traditional herbal medicine for the treatment of diabetes mellitus in folk systems of medicine in India, ^[19] Bangladesh ^[20] and Sri Lanka ^[21]. The unripe dried fruit is used to cure diarrhea and dysentery with its astringent, digestive, stomachic properties.Sweet drink prepared from the fruit pulp give a soothing effect on the patients who have just recovered from bacillary dysentery ^[22]. As many parts of A. marmelos have medicinal values, we herein studied the antioxidative and cytotoxic properties of three different fractions of leaves of A. marmelos in search of new therapeutic uses.

EXPERIMENTAL:

Collection and Identification of Plants

The fresh leaf of the plant Aegle marmelos were collected from the area of Mymensing district during the month of February, 2011. The A. marmelos was taxonomically identified by Bangladesh National Herbarium where a voucher specimen has been deposited having the accession number 35486.

Extraction Procedure

The dried coarse powder samples were dissolved in methanol (800 ml) in a jar and then covered with aluminum foil and shake with mechanical shaker. After that the crude extract was filtered with a sterilized cotton filter. The extract was concentrated by evaporating the solvent under vacuum on a rotary evaporator at 40 °C and stored at 4°C for further use. This procedure was applied similarly to the above mentioned all about dried powdered plant samples. Then fraction of crude extract was performed and prepared methanol (AMM), ethyl acetate (AME) and n-hexane (AMN) extracts.

Chemicals and Drugs

DPPH (1, 1-diphenyl, 2-picrylhydrazyl), was obtained from Sigma Chemical Co. USA. Ascorbic acid was obtained from SD Fine Chem. Ltd., Biosar, India. Naphthyl ethylene diamine dihydrochloride was purchased from Roch-light Ltd., Suffolk, England. Sodium nitroprusside was obtained from Ranbaxy Lab., Mohali. India.

DPPH Radical Scavenging Activity

The free radical scavenging capacity of the extracts was determined using DPPH.^[23]A methanol DPPH solution (0.004% w/v) was mixed with serial dilutions (0 to 500 μg) of extracts and after 10 min the absorbance was read at 515 nm using a spectrophotometer. Ascorbic acid was used as a standard. The inhibition curve was plotted and IC₅₀ values were calculated.

Nitric Oxide Scavenging Capacity Assay

NO radical scavenging was found on the basis of Griess Illosvoy reaction using method followed by Govindarajan et al. ^{[24, 25]} 4 ml of the extracts or standard of different concentration solution was combined with 1 ml (5mM) of Sodium nitroprusside solution and incubated for 2 hours at 30⁰C and then 2 ml solution was withdrawn from the reaction mixture and mix with 1.2 ml of Griess reagent. The absorbance of the solution was measured at 550 nm using a spectrophotometer against the blank solution.

Cytotoxicity Screening

In the bioassay for the bioactive compounds Brine shrimp lethality bioassay is a widely used method.^{[26, 27]} Here simple zoological organism (Artemia salina) was used as a convenient monitor for the screening. The eggs of the brine shrimp were collected from an aquarium shop (Dhaka, Bangladesh) and hatched in artificial seawater (3.8% NaCl solution) for 48 hrs to mature shrimp called nauplii. The cytotoxicity assay was performed on brine shrimp nauplii using Meyer method.^[28] The test samples (extract) were prepared by dissolving them in DMSO (not more than 50 μl in 5 ml solution) and sea water (3.8% NaCl in water) to make concentrations of 10 μg/ml, 25 μg/ml, 50 μg/ml, 250 μg/ml, 500 μg/ml and 1000 μg/ml. A solution of 50 μl DMSO diluted to 5ml was used as a control. Vincristine sulfate was used as positive control. Then matured shrimps were applied to each of all experimental vials and control vial. The number of the nauplii that died after 24 hr was counted. Then % mortality was plotted against respective concentrations used and from the graph LC₅₀ was calculated.

RESULTS:

DPPH Radical Scavenging Activity

The methanol, ethyl acetate, n-hexane extract of leaf of A. marmelos had IC₅₀ values of 6.629µg/ml, 4.383µg/ml and 3.606µg/ml respectively while ascorbic acid has IC₅₀ value of 2.687µg/ml indicating potent candidates of antioxidant compounds (shown in figure 1). As the concentration of the extracts increases the activity was found to increase. In case of the DPPH radical scavenging assay, the n-hexane extract showed better antioxidant activity having IC₅₀value of 3.606 µg/ml and the order of activity is – AMN > AME > AMM.

Figure 1: DPPH scavenging activity of three different extracts of leaf of the A. marmelos and ascorbic acid.

Nitric Oxide Scavenging Capacity Assay

Samples like methanol, ethyl acetate, n-hexane extracts of leaf of A. marmelos showed dose dependent scavenging of nitric oxide (figure 2) with IC₅₀ values of 1.233µg/ml, 5.545µg/ml and 3.050µg/ml respectively while ascorbic acid had IC₅₀ value 3.712µg/ml indicating the presence of potent antioxidant compounds. The order of scavenging activity is – AMM > AMN > AME.

Figure 2: NO scavenging activity of three different extracts of leaf of the A. marmelos and ascorbic acid.

Cytotoxicity Screening

The LC₅₀ values of different fractions of leaf of A. marmelos (in table 1) possess significant bioactivity and it was concentration dependent. The samples of methanol, ethyl acetate, n-hexane extract of leaf had the LC₅₀ value of 4.482µg/ml, 5.278µg/ml and 5.278µg/ml while vincristine sulfate had LC₅₀ value of 3.364µg/ml indicating the presence of potential bioactive substances. The methanol extract of the leaf showed highest cytotoxic activity having LC₅₀value of 4.482µg/ml and the order of cytotoxicity is – AMM > AME = AMN.

Table 1: Cytotoxic potential of three different extracts of leaf of the A. marmelos.

	Conc (μg/ml)	Log C	% mortality	LC₅₀ (μg/ml)
AMM	10	1.000	20	4.482
	25	1.398	90
	50	1.699	100
	250	2.398	100
	500	2.699	100
	1000	3.000	100
AME	10	1.000	30	5.278
	25	1.398	80
	50	1.699	100
	250	2.398	100
	500	2.699	100
	1000	3.000	100
AMN	10	1.000	30	5.278
	25	1.398	80
	50	1.699	100
	250	2.398	100
	500	2.699	100
	1000	3.000	100

DISCUSSION:

Naturally occurred antioxidants, from many plants, foods and beverages can treat various diseases by preventing cellular damage caused by free radicals in the body. ^[29]Antioxidant properties, particularly radical scavenging activities, are very important due to the harmful role of free radicals in foods and biological systems. An antioxidant compound releases an electron which DPPH accepts then decolorized and quantity can be found from the changes in absorbance. Many of the previous studies proposed that ascorbic acid, cysteine, glutathione, flavonoids, tannins, and aromatic amines are reduced and decolorize DPPH by their hydrogen releasing ability. ^[30] NO scavenging capacity of the extracts may help to check the chain of reactions started by the excess generation of Nitric oxide that are damaging to the human body. Many inflammations, cancer and other pathological conditions can be caused by nitric oxide.^[31] This work revealed that the n-hexane fraction needs lowest concentration of 3.606µg/ml to scavenge the DPPH radicals so it is more potent than other extract. The methanolic fraction showed potent antioxidant activity with IC₅₀ value of 1.233µg/ml against NO radicals. This indicates the presence of active antioxidant compounds in the extracts. The brine shrimp lethality bioassay is considered as the primary method of determining cytotoxic action ^{[28, 32]}. The methanolic fraction was more effective than the other two fractions having LC₅₀ of 4.482µg/ml and all fractions showed a concentration dependent cytotoxicity (table- 1). The result obtained from the brine shrimp lethality bioassay of Aegle marmelos can be used as a guide for the isolation of cytotoxic compounds from the different extracts of the leaf of this plant.

CONCLUSION:

The present experiments revealed that the leaves of Aegle marmelos possess potential antioxidant and cytotoxic properties. However, the experiments conducted here are preliminary in nature. Further studies should be carried on for better realizing of the pharmacological activities, mechanism of action and the active compound or compounds responsible for these actions.

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Received on 17.02.2013 Modified on 01.03.2013

Research J. Pharm. and Tech. 6(4): April 2013; Page 384-387