Universal Journal of Pharmacy

Subashini et al. UJP 2013, 02 (01): Page 85-91

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Universal Journal of Pharmacy

Research Article ISSN 2320-303X

Take Research to New Heights

IN VITRO ANTHELMINTIC AND ANTIOXIDANT ACTIVITY OF CRUDE LEAF EXTRACTS OF AEGLE MARMELOS CORREA Gangadhara Angajala, Divya P, Ramya R, Subashini R* Chemistry Research Laboratory, Organic Chemistry Division, School of Advanced Sciences, VIT University, Vellore-632014.

Received 11-01-2013; Revised 20-02-2013; Accepted 26-02-2013

ABSTRACT During the past decades, herbal medicine has emerging as global significance with medicinal and economic implications. Wide spread use of herbs throughout the globe has raised serious concerns over its quality, safety, and efficacy. It is a well known fact that various parts of plants such as leafs, fruits and seeds provide health and nutrition promoting compounds in human diet. Aegle marmelos commonly known as bael is widely used in Indian ayurvedic systems for the treatment of various ailments. In the present work different leaf extracts of Aegle marmelos were evaluated for in vitro anthelmintic studies using albendazole as standard and antioxidant studies were carried out for the leaf extracts using ascorbic acid as standard. The results showed that the aqueous leaf extract of Aegle marmelos with water showed good anthelmintic activity and that of petroleum ether showed enhanced antioxidant activity. Keywords: Aegle marmelos, Anthelmintic, Antioxidant, IC50, Albendazole. Ascorbic acid.

INTRODUCTION In recent times, focus on plant research has increased all over the world to show the immense potential of medicinal plants derived from various traditional systems. There is a widespread belief that the green medicines are harmless and healthier than the synthetic ones1. Many of the drugs commonly used today are of herbal origin mainly because of its safety and efficacy. According to the world health organization it is estimated that at least twenty five percent of the prescription drugs possess at least one active ingredient derived from the plant material2. Pharmaceutical drugs are generally possessing toxicity because of which the users are instructed to take them in restricted doses. They temporarily relieve pain but do not help to improve overall health. More than fifty percent of the drugs people consume are eliminated from the urinary tract via urination while the rest stay lodged in the tissues which cause adverse side effects. By using herbs, the body can easily digest and absorb the necessary things and act as a healing component with less or no side effects. Corresponding Author: Dr.R.Subashini Chemistry Research Laboratory, Vellore:632014. Tamilnadu. India Phone: +919443541305 E-mail: [email protected]

A number of traditional medicinal plants have been in use for the treatment of various diseases3,4. Even though the recovery is slow, the immense therapeutic use of herbs is becoming popular because of its inability to cause side effects. In India, of the 17,000 species of higher plants nearly 7500 are known for its medicinal uses and this is the highest proportion of medicinal plants known for their medical purposes in any country of the world for the existing flora of that respective country5,6. The use of herbs in the treatment of medicinal ailments has been a tradition in India, China etc for so many years, and several diseases can be cured without any side effects7-9. As the pharmacologists are looking forward to develop new drugs from natural sources, development of modern drugs from plants can be emphasized for the control of various diseases. An extensive systemic research has to be carried to explore potential phytoconstituents from medicinal plants for the development of products for their better economic and therapeutic utilization. Aegle marmelos commonly called Bael tree is considered as sacred tree by the Hindus as its leaves are offered to lord shiva during worship. It belongs to the family rutacae and it is medium sized plant originates from India and presently growing all over the country10,11. In India it grows throughout the deciduous forests, outer Himalayan regions, and South Indian plateau with altitudes ranging from 220 to 1250 m and also cultivated for its fruits throughout the India. It is

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Subashini et al. UJP 2013, 02 (01): Page 85-91 moderately sized, slender, aromatic tree, 6.0-7.5 m in height and 90-120 cm in girth. Fruits of Aegle marmelos are 5.0-7.5 cm in diameter, globose, oblong pyriform, rind grey or yellow. Seeds are numerous and arranged in the cells surrounded by slimy transparent mucilage. The unripe fruit is regarded as astringent, digestive and stomachic. The fruit is reputed to be a valuable Ayurvedic medicine for chronic diarrhea and dysentery and said to act as tonic for heart and brain12. The Bael fruit is one of the most nutritious edible fruits, rich in carotenoids, riboflavin and pectin which are used for the preparation of various products like candy, squash, toffee, slab, pulp powder and nectar. Aegle marmelos belongs to monotypic genera of sub family Aurantioideae, tribe Clauseneae and sub tribe Balsamocitrinae and family Rutaceae. It is considered as herbal medicine for the treatment of various ailments. Every part of this plant has its own medicinal property. It is generally used for the treatment of diarrhea, dyspepsia, dysentery, mental diseases, diabetes, jaundice, antifungal, anti-inflammatory, antipyretic, analgesic, antiproliferative, antidislipidemic, cardiotonic, regeneration of damaged pancreas, antiviral, antiulcer, anticancer, heamolytic, larvicidal, hepatoprotective and antibacterial activity asthma, anaemia, healing of wounds, fractures, swollen joints, and also for the management of diabetes13-27. Various chemical constituents like alkaloids, coumarins, steroids are isolated from the different parts of the plant.

www.ujponline.com In the present work in vitro anthelmintic and antioxidant properties of different crude leaf extracts of Aegle marmelos were studied and compared with that of standard. To the best of our knowledge this is the first time the anthelmintic and antioxidant activities of the different extracts of the leaf Aegle marmelos has been reported.

MATERIALS AND METHODS Plant material The Aegle marmelos leaves were collected from Vellore district (Tamilnadu), India during the month of October 2012. The leaves were identiﬁed by Dr. S. Nanthakumar, Prog. Coordinator (Hort.), Tamil Nadu Agriculture University, Krishi Vigyan Kendra, Virinjipuram-632104, Vellore district, Tamilnadu, India. A voucher specimen has been submitted at the office for reference. Extraction and Isolation Fresh green leaves of Aegle marmelos was taken and washed thoroughly with distilled water. The leaves were kept for drying in shade region for 3 days and then finely powdered. A weighed quantity of powdered drug (7g) was taken and extracted with 100 ml petroleum ether (60-80oC) by using soxhlet extractor. The defatted drug was removed and the plant residue was dried by distilling of the solvent in rotary evaporator. To this residue 120 ml methanol was added and extracted for 72 h in soxhlet extractor.

Fig. 1: Isolation of crude extracts from Aegle marmelos leaves


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Subashini et al. UJP 2013, 02 (01): Page 85-91 The extract was filtered using whatman filter paper and the filtrate was evaporated. To the crude plant extract 80 ml water was added and the solution was acidified to pH 2, later it was kept for steam distillation and filtered. To the filtrate 50 ml petroleum ether was added and the two layers namely ether layer, acid layer was separated by using separating funnel. The ether soluble layer was evaporated and the extract was collected (Fig: 1). The acidic aqueous layer containing the alkaloids and plant impurities is made basic with NaOH. The alkaloids were precipitate out and it is filtered and dried. Anthelmintic activity The Anthelmintic activity was carried out as per the procedure given by Ajaiyeoba et al 28. The activity was performed on adult Indian earthworm, pheritima posthuma because of its anatomical and physiological resemblance with that of intestinal parasite round worms of human beings. Indian adult earthworms of equal size were collected from moist soil of puliyanthangal region near sipcot, ranipet, Vellore district, Tamilnadu. Totally four groups each containing different concentrations of crude extracts of Aegle marmelos and approximately equal size Indian earthworms consisting five in each group were used for the study. Test samples of the extract were prepared at the concentrations 5 mg/ml, 10 mg/ml, 15 mg/ml in distilled water. Each group was placed in petridish containing 25 ml of the above test solution. Each group was treated as following: Group-I - Control (Distill water) Group-II - Albendazole (Standard) Group-III- Pet Ether extract of leaves Group-IV- Aqueous extract of leaves Observations were made for the time taken to paralysis and death of individual worms shown in Fig.: 2. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously. Death was concluded when the worms lost their motility followed with fading away of their body colors. All the results were expressed as mean ± SEM of five worms in each group (Table: 1). Statistical analysis Data were analyzed by using one way factorial ANOVA tests followed by t-test on each group. P values were calculated in each case and accordingly interpretation was carried out. Statistical analysis of both paralysis time and death time of the earthworms in case of standard and different concentrations of crude extracts were done and the results are shown in Table:2 and Table:3. Antioxidant activity An antioxidant is a molecule which has the ability to inhibit the oxidation of other molecules. Oxidation reactions can produce free radicals which in turn can start chain reactions and they may have the potential

www.ujponline.com to interact with cellular components like DNA or cell membrane and causes cell death. Free radicals can trap low density lipoprotein in artery wall and begins the formation of plaque. All plants generally produce various secondary metabolites among which phenolics play prominent role in showing good antioxidant activity. The phenolics are generally characterized by the presence of one aromatic ring to which one or more hydroxyl groups are linked. Antioxidants terminate the chain reactions by removing free radical intermediates, and inhibit cell death and other oxidation reactions. Screening of antioxidant activity was carried out by using DPPH radical scavenging assay. DPPH radical scavenging assay The DPPH assay is based on the reduction of DPPH a stable free radical. Radical scavenging activity of compounds against stable DPPH (2,2-diphenyl-2picrylhydrayl hydrate) was determined spetrophotometrically. When Antioxidant react with DPPH, which is a stable free radical becomes paired off in the presence of a hydrogen donor and is reduced to DPPHH as a result the absorption decreases from DPPH radical to DPPHH which is clearly identified by colour change from deep violet to yellow colour measured at 517 nm using UV-Vis light spectrophotometer. A solution of DPPH is mixed with that of a substance that can donate a hydrogen atom, will give rise to the reduced form with the loss of violet colour. The scavenging activity between DPPH radical and antioxidant (RH) is shown in Fig.: 3. DPPH + RH = DPPHH + R Where RH is the reduced form and R is free radical NO2

NO2 . N O2N

NH N

N

NO2

Diphenylpicrylhydrazyl (free radical)

O2N

NO2

Diphenylpicrylhydrazine (non radical)

Fig.: 3. Reduction of DPPH radical to non radical

Antioxidant activity studies using DPPH method The scavenging of the stable DPPH radical model is a widely used method to evaluate antioxidant activities in a relatively very short period of time. Antioxidants cease the free radical reaction by donating hydrogen from the phenolic hydroxyl groups. The addition of different extracts to DPPH solution cause a rapid decrease in optical density at 517 nm which was clearly seen by change in colour from deep violet to light yellow shown in Fig.: 4. Stock solutions were prepared by dissolving 1mg of different plant crude extract in 1ml of ethanol. Then different concentrations of sample 20µg, 50 µg, and 100 µg were dissolved in 3ml ethanol. The solution of DPPH in ethanol was prepared just before UV measurements. 3 ml of sample and 1 ml of DPPH solution were mixed and kept in the dark for 30 min at room temperature and then the decrease in


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Subashini et al. UJP 2013, 02 (01): Page 85 85-91 absorption was measured at 517 nm using UV UV-Vis light spectrophotometer. For the control absorption blank sample containing the same amount of ethanol and DPPH solution was measured. easured. Ascorbic acid was used as standard. The experiment was carried out in triplicate29,30. The IC50 values were calculated for each compound as well as for ascorbic acid as standard which is represented in Table: 4 and graphically represented in Figure: 5. Radical scavenging activity was calculated by following formula: I% (percentage inhibition) = [AB-AA/AB] AA/AB] × 100 Where AB= absorption of blank sample AA= absorption of sample

RESULTS AND DICUSSION Anthelmintic activity The Aqueous and nd Petroleum ether crude leaf extracts (1) of Aegle marmelos were more or less equally potent

www.ujponline.com as far as their anthelmintic activity was concerned. But the aqueous crude extract (2) ( shows better anthelmintic activity than pet ether extract and also comparable with that of standard albendazole. At 15 mg/ml the time of paralysis were observed as 22.56 ± 0.238 min and 12.75 ± 0.641 min for pet ether and aqueous crude extracts which have ha good activity when compared to standard albendazole which was showing 25.32 ± 0.737 min. At 15 mg/ml the time of death for aqueous extract were observed at 35.41 ± 0.478 min which was very closer to standard 33.17 ± 1.014 min. The results obtained from m statistical analysis showed that both pet ether and aqueous crude leaf extracts of Aegle marmelos have significant anthelmintic activity. Particularly statistical analysis of death time of earth worm in case of Pet ether and aqueous leaf extracts have good ood anthelmintic activity (P < 0.005, P < 0.01) in comparison to standard (P < 0.01). 0.01)

Table: 1 Anthelmintic activity of different leaf extracts of Aegle marmelos against Earthworm Group

Treatment

1 2

Control Standard (Albendazole)

3

Pet Ether extract

4.

Aqueous extract

Conc mg/ml Distilled water 5 10 15 5 10 15 5 10 15

Paralysis time (min) Absent 45.23±1.004 38.46±0.456 25.32±0.737 48.52±0.005 34.78±0.524 22.56±0.238 32.47±0.475 25.00±0.002 12.75±0.641

Death Time (min) Absent 66.21±0.324 47.23±0.847 33.17±1.014 69.17±0.324 52.13±0.417 43.85±0.009 63.47±0.427 48.23±0.581 35.41±0.478

Fig.: 2 Anthelmintic activity of Aegle marmelos leaf extracts.

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Subashini et al. UJP 2013, 02 (01): Page 85-91

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Table: 2 Statistical analysis of paralysis time of earth worm in case of standard and crude leaf extracts of Aegle marmelos S.No 1 2 3

Sample (5,10,15) mg/ml Standard Pet ether Aqueous

Df (degree of freedom) 5 5 5

t value

P value

Interpretation

4.874 3.753 2.437

P