Anthelmintic Activity of the Stem Bark of Juglans regia Linn.

Vishesh Upadhyay, Kambhoja S., Harshaleena K., Veeresh and Dhruva K.

Department of Pharmacognosy, The Oxford College of Pharmacy, Hongasandra, Bangalore- 560068, Karnataka.

ABSTRACT:

Juglans regia Linn. also called as Walnut and Akhrot is a deciduous tree belonging to family Juglandaceae. It is native of Iran, in India it is found throughout the Temperate Himalayas at an altitude of 1000-3000mtrs. In traditional literature, it was found that Juglans regia Linn. stem bark was used as folk medicine as anthelmintic. Thus the extracts of Juglans regia Linn. stem bark were investigated for its anthelmintic activity on adult Indian earthworm, Pheretima posthuma, to substantiate the folkoric claim. The stem bark of Juglans regia Linn. was collected, authenticated, dried, powdered and exhaustively extracted using soxhlet apparatus with different solvents viz. petroleum ether, benzene, chloroform, acetone, methanol, ethanol and distilled water in ascending order of polarity. All the extracts were subjected to anthelmintic activity against Indian earthworm, Pheretima posthuma. Benzene, methanol and ethanol extracts exhibited significant anthelmintic activity as comparable to that of standard drug Piperazine citrate.

KEYWORDS: Juglans regia Linn., anthelmintic activity, Pheretima posthuma, Piperazine citrate.

INTRODUCTION:

The tree Juglans regia Linn. (Juglandaceae) is commonly known as Walnut in English and Akhrot in Hindi. It is a native of Central Asia and found in temperate Himalayas at an altitude of 1000-3000mtrs¹. This tree is reputed to possess varied medicinal properties. In traditional literature it was found that the leaves of Juglans regia Linn. was used as folk medicine as anthelmintic, antibacterial²,aphrodisiac, tonic and the fruits were used as carminative and astringent³. The biological activity viz. antidiabetic⁴, antifungal of the leaf have been reported.

Juglans regia Linn. stem bark contains main chemical constituents viz. β-sitosterol, ascorbic acid, juglone, folic acid, gallic acid, regiolone, and quercitin-3-α-L-arabinoside⁵.

Helminths are recognized as a major constraint to live stock production, crops and affects food production throughout the tropics and elsewhere. Helminths cause considerable problems for human beings and animals. Anthelmintic are used to treat infections with parasitic worms. This includes both flatworms e.g. flukes, tapeworms and roundworms i.e. nematodes. They either kill (vermicides) or expel (vermifuge) infesting helminths⁶. These are of huge importance for human tropical medicine and for veterinary medicine.

There is small repertoire of chemotherapeutic agents available for treatment and the literature review suggests that anthelmintic activity of Juglans regia Linn. hasn’t been comparatively studied and hence in the present study the same was investigated to highlight the importance of the stem bark of Juglans regia Linn. as an anthelmintic drug against adult Indian earthworm, Pheretima posthuma.

MATERIAL AND METHODS:

COLLECTION OF PLANT MATERIAL:

The stem bark of Juglans regia Linn. was collected from the forests of Kullu (H.P.) and authenticated by Dr. K. Madhava Chetty,Asst. Prof. Dept. of Botany, SVU, Tirupathi. (A.P.)

PREPARATION OF EXTRACTS:

The stem bark of Juglans regia Linn. was dried under the sun for two weeks and powdered. The coarse powder (400gm) was subjected to soxhlet extraction with 1.5 ltrs of various solvents viz. petroleum ether, benzene, chloroform, acetone, methanol, ethanol and distilled water in ascending order of polarity. Each time before extraction with next solvent, the marc was air dried and was concentrated by distilling the solvent at low temperature using rotary flash evaporator. The prepared extracts were tested for anthelmintic activity.

PHYTOCHEMICAL SCREENING:

All the extracts were screened for the presence of carbohydrates, cardiac glycosides, flavanoids, steroids and tannins using standard methods⁷.

STUDY OF ANTHELMINTIC ACTIVITY:

The anthelmintic activity was done on adult Indian earthworm, Pheretima posthuma collected and authenticated from GKVK, Bangalore, due to its anatomical and physiological resemblance to the intestine roundworm parasites of human beings.^8,9,10.

Photograph showing anthelminthic activity of different extracts

The earthworms were washed with normal saline to remove all faecal matter and were used for anthelmintic study. The earthworms of 3-6 cm in length and 0.1-0.2 cm in width were used for the experimental protocol.

Emulsions of all the extracts were prepared in normal saline using Tween 80 as vehicle. The final volume of each dilution of the extracts was made up to 20ml.

All drugs and extract solutions were freshly prepared before the start of experiment.

Nine groups of six earthworms of approximately equal size were used for the present study. Different concentrations of extracts were prepared and placed in the petriplates containing earthworms.

Group 1 serves as control, receives only saline + Tween 80.

Group 2 serves as standard, receives standard drug Piperazine citrate of different concentrations viz., 40mg/ml and 60mg/ml.

Group 3 serves as Petroleum ether extract.

Group 4 serves as Benzene extract.

Group 5 serves as Chloroform extract.

Group 6 serves as Acetone extract.

Group 7 serves as Methanol extract.

Group 8 serves as Ethanol extract.

Group 9 serves as Aqueous extract of different concentrations.

Observations were made for the time taken for the paralysis and death of each individual worms. Paralysis was said to occur when the worms lost their mortality followed with fading away of body colour^11,12,13.

All the activity was comparable to the standard drug piperazine citrate (20mg/ml, 40mg/ml).

CHEMICALS USED:

· Piperazine citrate (Standard drug)

· Saline water

· Tween 80

RESULTS AND DISCUSSION:

The preliminary phytochemical screening of the stem bark of Juglans regia Linn. revealed the presence of steroids, tannins, flavanoids and cardiac glycosides.

Results of phytochemical screening are tabulated in table 1.

Anthelminthic activity was done for all the seven extracts viz., petroleum ether, benzene, chloroform, acetone, methanol, ethanol and aqueous extracts.

During anthelminthic study, benzene and methanol extracts exhibited significant activity while acetone and aqueous extracts exhibited moderate activity against the adult Indian earth worm, Pheretima posthuma as compared to the standard drug Piperazine citrate.

Results of anthelminthic activity are tabulated in table 2.

Table 1: Preliminary phytochemical evaluation of the stem bark of Juglans regia Linn.

Phytoconstituents	Petroleum ether Extract	Benzene extract	Chloroform extract	Acetone extract	Methanol extract	Ethanol extract	Aqueous extract
Alkaloids	-	-	-	-	-	+	+
Carbohydrates	-	-	-	-	-	-	+
Cardiac glycosides	-	-	+	+	+	+	-
Flavanoids	-	-	-	+	+	+	+
Steroids	+	+	+	+	+	-	-
Tannins	-	-	-	+	+	+	+

‘+’ : Present; ‘-‘ : Absent

Table 2: Anthelminthic activity of the stem bark of Juglans regia Linn.

GP	TRTMT	PARALYSIS TIME(min)				DEATH TIME(min)
GP	TRTMT	20 mg/ml	40 mg/ml	60 mg/ml	80 mg/ml	20 mg/ml	40 mg/ml	60 mg/ml	80 mg/ml
1^st	Veh	-	-	-	-	-	-	-	-
2^nd	Std	-	43±2.000	33.4± 0.840	-	-	59.4±1.080	55.6±2.100	-
3^rd	P E ext	-	-	-	-	-	-	-	-
4^th	Benz ext	26.5± 2.500	13.3±1.053	13.66±0.420	12.8±0.477	45.66±1.040	34±0.730	29.6±1.053	27.3±0.630
5^th	CF ext	-	-	-	-	-	-	-	-
6^th	Ac ext	76± 3.605	68.8±3.130	59±2.805	54.2±1.681	101.9±3.113	81.6±2.220	73±3.162	70±3.160
7^th	Met ext	54.2±1.796	53.3±2.201	48±2.000	40.3±2.027	74±3.130	73±4.401	70±2.230	67±2.230
8^th	Eth ext	-	-	148± 4.757	134±4.362	-	-	230± 5.420	207.16±.040
9^th	Aq ext	145± 3.162	83± 1.414	76± 0.447	72.83±0.740	167± 2.780	105± 0.694	101± 3.000	87.5± 1.118

GP- Group; TRTMT- Treatment; Veh- Vehicle; Std- Standard; P E ext- Petroleum ether extract; Benz ext- Benzene extract; CF ext- Chloroform extract; Ac ext- Acetone extract; Met ext- Methanol extract; Eth ext- Ethanol extract; Aq ext- Aqueous extract.

It may be concluded that the anthelminthic activity of the stem bark Juglans regia Linn. may be due to the presence of flavanoids, steroids, tannins present in the stem bark Juglans regia Linn.

Further, detailed investigation needs to be underway to determine the exact phytoconstituents and isolate the active principles which are responsible for the anthelminthic activity of the stem bark of Juglans regia Linn.

CONCLUSION:

Thus, the use of Juglans regia Linn. in traditional system of medicine as anthelminthic was justified by this work as it showed significant anthelminthic activity against Indian earthworm, Pheretima posthuma.

The results of the present study indicated a scope for isolating the active principles from the stem bark of Juglans regia Linn. which are responsible for the anthelminthic activity.

ACKNOWLEDGEMENT:

The authors are thankful to the chairman, director and the principal, The oxford college of pharmacy, Bangalore for providing necessary facilities and support to conduct this work and Dr. Madhava Chetty, Asst. Prof., Dept. of Botany, Sri Venkateshwara University, Tirupathi (A.P.) for authenticating the sample.

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Received on 11.10.2010

Accepted on 01.11.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(6): Nov. - Dec. 2010, 465-467