INTRODUCTION:

Free radicals are continuously formed in the human body, both by "accidents of chemistry" and for useful metabolic purposes. The harmful effects of free radicals are opposed by antioxidant defenses in the body. As long as a balance is maintained, no unwanted effect will occur. But once this balance is disturbed , it causes oxidative stress, which can lead to cell injury and death. Free radical mechanisms have been implicated in the pathology of several human diseases, including cancer, atherosclerosis, rheumatoid arthritis, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, the pathologies caused by diabetes¹ High frequency of worm infections in the tropics is due to the lack of hygiene, fecal pollution of soil and water, the presence of vectors and the temperature and moisture of the environment. Parasitic infections are a major medical problem throughout the world, especially in developing countries where they prove to be the primary cause of death.

Free Parasitic infections with gastrointestinal nematodes (GINs) remain a major pathological threat in outdoor production systems of various livestock species. Up to now, the control of these parasitic diseases essentially relied on the use of commercial, anthelmintic (AH) drugs. However, resistance to these anthelmintics is now widespread amongst worm populations in sheep and goats across the world ² . Recent results indicate that bioactive, tanniniferous plants represent a valuable option as an alternative to commercial drugs for the control of GINs³.

Borassus flabellifer, the Asian palmyra palm, toddy palm, sugar palm, or Cambodian palm, is native to the Indian subcontinent and Southeast Asia, including India, Bangladesh, Sri Lanka, Cambodia, Laos, Burma, Vietnam, Malaysia and Indonesia⁴. The coconut-like fruits are three-sided when young, becoming rounded or more or less oval. When the fruit is very young, the kernel is hollow, soft as jelly, and translucent like ice, and is accompanied by a watery liquid, sweetish and potable. It has an yellowish brown coloured peel⁵.The active principles reported in literature are gums, saponins, glycosides, carbohydrates, albuminoids, fats, vitamins A, B, and C, steroidal saponins, flabellifer reins, Spirosterol^6,7,8 etc. Different parts of Borassus flabellifer are used in the treatment of gonorrhea, respiratory ailments, hiccups, gastric ailments, styptic for external wounds, diabetes, gangrenous and indolent ulcers, abscesses and laxative⁹. Current work is focused on the antioxidant and anthelmintic activities of methanolic extract of the kernel peels. Peels separated from the kernels and soft jelly like kernel left over after removing the peels are shown in fig1 and 2.

Fig 1:Kernel peels

Fig 2: Soft jelly like kernel

MATERIALS AND METHODS:

Plant material:

The plant material, ie, fruits of Borassus flabellifer were purchased from the nearest fruit market during the month of April-2013, as this was the season when they are available in plenty. They were authenticated in the Botany Department of Siddhartha Arts and Science College, Vijayawada.

Chemicals:

Acetic anhydride (Loba Chemicals), Alkaloidal reagents (Loba Chemicals), Ammonia (Loba chemicals), Ascorbic Acid, Chloroform (Merck Specialties Pvt. Ltd.), 1,1-diphenyl, 2-Picryl hydrazyl (DPPH) from Sigma Aldrich, U.S.A., Ferric chloride, Gum acacia (Loba Chemicals,) Hydrochloric acid (Hi-Pure Chemicals), Lead acetate ( Hi-Pure Chemicals), Methanol (Loba Chemicals), Ninhydrin reagent (Hi-Pure Chemicals), Phosphate buffer (pH6.6), Potassium ferricyanide, Pyridine (Loba Chemicals), Sodium chloride (Loba Chemicals), Sodium hydroxide (SD fine Chemicals), Sodium nitroprusside (Loba Chemicals), Sulphuric acid (Hi-Pure Chemicals), and Tri choloroacetic acid are the chemicals used .

Extraction:

The peels of the kernels of Borassus flabellifer were subjected to sun drying. The peels were spread evenly and allowed to dry for a period of two weeks continuously. The material is dried to such an extent that it can be powdered easily. The dried plant material is subjected to grinding in a mechanical grinder.

The powdered plant material (150g of Borassus flabellifer ) is subjected to successive extraction using different solvents like chloroform, methanol and water. Aqueous extract is obtained using continuous soxhlet extraction. Chloroform and methanol extracts were prepared by maceration. The powder of the peels is subjected to maceration with intermittent shaking. In this process the powder and the solvent are taken in the ratio 1:3 in a conical flask and left for extraction for 6 days. Then the contents are filtered by vacuum filtration. The marc is re-extracted with same solvents (in 1:1 ratio) for 3days. The contents are filtered by vacuum filtration. Both the filtrates are combined and concentrated under reduced pressure and kept in desiccators.

Soxhlet extraction:

After methanol extraction, the solid material (drug powder) is dried and packed in a filter paper and introduced into the thimble which is loaded into the main chamber of the soxhlet extractor. The soxhlet extractor is placed onto a round bottomed flask containing water as the extraction solvent. The soxhlet is then equipped with a condenser and the RB flask was heated on a heating mantel. The solvent vapor travels up a distillation arm and floods into the chamber housing the drug powder. The chamber containing the solid material slowly fills with warm solvent. Some of the desired compound will then dissolve in the warm solvent. When the soxhlet chamber is almost full, the chamber is automatically emptied by a siphon tube, with the solvent running back down to the distillation flask. This cycle was allowed to repeat many times, over eight hours.

Preliminary Phytochemical Screening:

The extracts obtained were weighed and yield was calculated (Table 1)The extracts were abbreviated as follows:

Borassus flabellifer Methanol extract-BFM

Borassus flabellifer Aqueous extract-BFA

Borassus flabellifer Chloroform extract-BFC

Preliminary phytochemical screening was done following standard procedures^10,11. The samples were tested for alkaloids, glycosides, flavonoids etc.

Evaluation of Anthelmintic Activity:

Indian adult earthworms (Pheritima posthuma) were used to study Anthelmintic activity. Earthworms were collected from the water logged areas and then washed with normal saline to remove soil and fecal matter. The earthworms of 5-8 cm in length and 0.2-0.3 cm width, which were active were used in the experimental protocol. They were authenticated in the Department of Zoology, Siddhartha Arts and Science College, Vijayawada.

Experimental Protocol:

Six groups of approximately equal size Indian earth worms consisting of 6 earthworms in each group were used for the study using standard procedure^12,13. Each group was treated in the following manner:

Group	Treatment	Group Code
Group 1	Methanol extract (50mg/ml)	BFM₁
Group 2	Methanol extract (100mg/ml)	BF M₂
Group 3	ALBENDAZOLE (50 mg/ml)	S
Group 4	1% gum acacia	C

Preparation of Test Samples:

Samples for evaluation of Anthelmintic activity were prepared by dissolving 2.5gm of crude extract in 25 ml of 1% gum acacia solution prepared in normal saline. 50 and 100mg/ml conc. were used in the study.

Preparation of Standard:

A standard drug, Albendazole is procured from the nearby medical store. A solution of 50mg/ml conc. is prepared.

Procedure:

The samples were taken in petriplates and labeled. Adult healthy earth worms (n=6) were introduced into them.

Observations were made for the time taken to paralyze and time for death of individual worms. Paralysis was said to occur when the worms do not revive even in normal saline. Death was concluded when worms lost their motility, followed by fading away their body colour.

Evaluation of Anti-Oxidant Activity

· DPPH Radical scavenging activity

· Ferric Reducing power Determination

DPPH radical scavenging activity:

The radical scavenging activity was determined by Szabo et al¹⁴. method with a few modifications. Plant extract (BFM) and standard ascorbic acid solution (STD) of different concentrations viz. 2.5, 5, and 10 µg/ml were added to 3 ml of a 0.004% methanol solution of DPPH. An equal amount of methanol and DPPH served as control. After 30 minutes incubation in the dark, absorbance was recorded at 517 nm, and the percentage inhibiting activity was calculated from [(A0–A1)/A0] ×100, where A0 is the absorbance of the control, and A1 is the absorbance of the extract/standard. The antioxidant activity of the extract was expressed as IC50. The IC50 value was defined as the concentration (in µg/ml) of extracts that inhibits the formation of DPPH radicals by 50%. All the tests were performed in triplicate and the graph was plotted with the average of three observations.

Ferric reducing power determination¹⁵:

Different concentrations of plant extract (BFM) and standard (STD) ascorbic acid solution viz. 20, 40, 60, 80 and 100 µg/ml in 1ml of methanol were mixed with phosphate buffer (2.5 ml, 0.2 M pH 6.6) and potassium ferricyanide [K₃Fe(CN)₆] (2.5 ml, 1%). The mixture was incubated at 50° C for 20 min. A portion (2.5 ml) of tricholoroacetic acid (10%) was added to the mixture, which was then centrifuged at 3,000 g (rpm) for 10 min at room temperature. The upper layer of solution (2.5 ml) was mixed with distilled water (2.5 ml) and ferric chloride (FeCl₃) (0.5 ml, 0.1%). A blue-green colour was obtained in the reaction, whose O.D was measured at 700 nm. An increase in the absorbance of the reaction mixture indicated increased reducing power. All the tests were performed in triplicate and the graph was plotted with the average of three observations.

RESULTS AND DISCUSSION:

Extraction:

Yield was calculated using formula:

Weight of the extract

% Yield = ^{_________________________} X 100

Weight of the plant material

The percentage yield of kernel peel extracts of Borassus flabellifer are given in Table1.The maximum percentage of yield was reported with methanol extract(10.4%), followed by aqueous extract(7.87%) and chloroform extract(1.67%).

Preliminary Phytochemical Screening:

The chloroform extract of kernel peels contains alkaloids, tannins, carbohydrates and phytosterols. The chemical constituents of methanol extract were saponins, tannins, and carbohydrates. Aqueous extract was found to contain fixed oils and carbohydrates. The results are shown in Table 2. Phytochemical screening helps in identifying the chemical constituents of a plant drug. With the knowledge of the chemical constituents, the pharmacological activities can be predicted.

Evaluation of Anthelmintic Activity:

The anthelmintic activity of methanol extracts of Borassus flabellifer was evaluated using Indian earthworms against the standard albendazole. The results of Anthelmintic activity are shown in Table 3 and Fig 3 and 4. Results are expressed as an average of six observations. It was observed that methanol extracts of Borassus flabellifer kernel peels showed significant Anthelmintic activity. Among the samples tested, methanol extract (100mg/ml) showed shortest time of paralysis and death. With the tested dose of 100mg/ml methanol extract, the time required to paralyze the worms was 37+0.30 min and death was recorded at 44+0.45 min. Whereas, the time required to paralyze and kill the worms with standard (Albendazole) was 45+0.45and 55+0.20 min. respectively. The activity of methanol extracts was comparable to that of standard drug (Albendazole) with respect to time taken to paralyze and to kill the worms. The potency of the extracts was inversely proportional to the time taken for paralysis /death of the worms. The control group animals were alive up to 24hrs.

Various mechanisms by which chemotherapeutic agents act as anthelmintic agents are ,by disruption of neuromuscular physiology, blockade of energy metabolism, disrupting reproductive system ¹⁶etc. which is highly efficient in these parasites. The tested extracts may act as anthelmintics by anyone or a combination of these mechanisms. The Anthelmintic activity of the extract may be attributed to the presence of active components such as saponins, carbohydrates and tannins. Moreover, the antheliminitic properties of these compounds are well documented^17-19.

Evaluation of Anti-Oxidant Activity:

DPPH method:

The results of DPPH free radical scavenging activity of methanol extract is shown in Table 4, 5 and fig 5. DPPH radical is a free radical, during reaction with antioxidants it is reduced which can be visualized as a change in the color from purple to yellow^20,21. The tested extracts bleached DPPH indicating their free radical scavenging activity. The standard showed a percentage inhibition of 63% at a dose of 5µg/ml whereas the test at the same dose showed 53% inhibition. This indicates a comparable Antioxidant activity. Further, the antioxidant activity was found to be dose dependant. IC50 values for the standard and methanol extract (BFM) were found to be 3.95 µg/ml and 5.2 µg/ml respectively.

Ferric Reducing Power Assay:

Reduction of Fe(III) is used as an indicator of electron donating activity, which is an important mechanism of phenolic antioxidant action²². In the reducing power assay, the presence of antioxidants in the samples would result in the reduction of Fe3+ to Fe2+ by donating an electron. Amount of Fe2+ complex can then be monitored by measuring the formation of Pearl's Prussian blue at 700 nm. Increasing absorbance at 700 nm indicates an increase in reductive ability and increasing antioxidant activity. The tested extracts exhibited potent reducing ability when compared to the standard. The activity was dose dependant. 100 µg/ml samples showed highest reducing power. The results are shown in Table6 and Fig 6.

Abnormally high levels of free radicals and decreased anti oxidant protection are the main cause for Diabetes, diabetic complications, cancer, aging, obesity, etc²³. Methanol extracts were found to have significant DPPH free radical scavenging activity and Ferric Reducing Power .Tannins , Saponins and carbohydrates are the chief principles identified in the tested extracts. All these compounds are reported^24-26 to possess free radical scavenging and anti oxidant activities. They regulate cellular activities of inflammation related cells, macrophages, lymphocytes, Neutrophils. They also modulate metabolizing enzymes like, Phospholipase A₂, Cyclo Oxygenase (COX), Lipoxygenase (LOX) and Nitric oxide synthase (NOS). Most of the antoxidants also possess anti inflammatory activity which is advantageous in the treatment of inflammation caused by helminth parasites. From the experimental results, we can confirm that the tested extracts play a dual role both as an anthelmintic as well as a promising antioxidant agent.

Table 1: Yield of Extracts

S. No	Extract	Weight in gm	%Yield
1	BFC	2.5	1.67
2	BFM	15.6	10.4
3	BFA	11.8	7.87

Table 2: Preliminary Phytochemical Screening

Phytoconstituent	BFC	BFM	BFA
Alkaloids	+	-	-
Proteins and Amino acids	-	-	-
Fixed oils and fats	-	-	+
Flavonoids	-	-	-
Saponins	-	+	-
Carbohydrates	+	+	+
Phytosterols	+	-	-
Gums and mucilages	-	-	-
Tannins	+	+	-

+ indicates presence - indicates absence

TABLE 3:Anthelmintic Activity

Group	Time of paralysis (min)	Time of death (min)
BFM₁	45± 0.25	52±0.32
BFM₂	37± 0.30	44± 0.45
Albendazole	45± 0.45	55± 0.20
Control	No action	No action

Results given as mean± S.D.

Fig 3:Initial Stage

Fig 4:Final stage

Table4: DPPH Free Radical Scavenging activity of Standard:

S. No	Conc. (µg/ml)	% Scavenging activity	IC50 (µg/ml)
1	2.5	35.0	3.95
2	5.0	63.14
3	10	99.13

Dose response curve

Table 5: DPPH free radical scavenging activity of Borassus flabellifer

S. No	Conc. (µg/ml)	% Scavenging activity	IC50 (µg/ml)
1	2.5	24.11	5.19
2	5.0	53.02
3	10	89.14

Dose response curve

Fig 5: DPPH scavenging activity of Std. and Borassus flabellifer extract

Fig: 6 Reducing power of Std. and Borassus flabellifer:

Table 6: Ferric Reducing Power Determination

S. No	Concentration (µg/ml)	Absorbance of BFM	Absorbance of STD
1	20	0.198	0.242
2	40	0.203	0.287
3	60	0.233	0.287
4	80	0.273	0.310
5	100	0.286	0.362

CONCLUSION:

In the present study methanol extract of the peels of Borassus flabellifer kernels exhibited potential antioxidant activity when compared to the standard drug. These extracts also showed remarkable anthelmintic activity. In developing countries, helminth infestation is one of the main causes of death. The incidence of free radical mediated health problems are also increasing day-by-day. There is a need for more number of natural drugs to be explored to prevent and treat these problems. Nature is a treasure of medicinal plants which need to be discovered. The results discussed in this paper are shown by crude extract of Borassus flabellifer kernels, so, further isolation, purification and characterization of the sample is needed to pin point the chemical entity responsible for the activities reported.

ACKNOWLEDGEMENTS:

The authors thank the management of Siddhartha Academy and the Principal of KVSR. Siddhartha College of Pharmaceutical Sciences for their support.

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Received on 01.09.2014 Modified on 10.09.2014

Res. J. Pharmacognosy & Phytochem. 6(4):Oct. - Dec.2014; Page 181-186