Anthelmintic Activity of Citrus medica L. Peel Extract in Indian Adult Earthworms

 

Kabra A. O.*, Bairagi G. B., Mandade R. J. and Wanare R. S.

Department of Pharmacology, Sudhakarrao Naik Institute of Pharmacy Pusad (M.S). 445204 India

*Corresponding Author E-mail: alk.kabra22@gmail.com

 

ABSTRACT:

Citrus medica L. popularly known as ‘Bara nimbu’ in India belongs to family Rutaceae. The objective of the present work was to evaluate the in-vitro anthelmintic potency of the ethanolic extract of Citrus medica peels using Indian earthworms (Pheretima posthumad). The various concentrations (25-100 mg/ml) of the ethanolic extract were tested in-vitro for anthelmintic potency by determination of time of paralysis and time of death of worm. Piperazine citrate (15mg/ml) used as standard. The result of present study indicates that the Citrus medica L. potentiate to paralyze earthworm and also caused its death after some time. The shortest time of paralysis and time of death was observed at higher dose (100 mg/ml) which was comparable with standard. Thus, the present study demonstrate that the Citrus medica L. as an potent anthelmintic has been confirm as the ethanolic extracts of peels displayed activity against the earthworm used in study.

 

KEYWORDS: Citrus medica, Paralysis, death of earthworm

 


INTRODUCTION:

Helminth infections are among the most widespread infections in humans, distressing a huge population of the world. Although the majority of infections due to helminths are generally restricted to tropical regions and cause enormous hazard to health and contribute to the prevalence of undernourishment, anaemia, eosinophilia and pneumonia1. Parasitic diseases cause ruthless morbidity affecting principally population in endemic areas2. The gastro-intestinal helminthes becomes resistant to currently available anthelmintic drugs therefore there is a foremost problem in treatment of helminthes diseases3. Hence there is an increasing demand towards natural anthelmintic. Citrus medica L., commonly known as citron, is a small tree, having large fruit (20-22.5 cm. long) resembling pineapple in shape.  It is reported that C. medica essential oil showed fungitoxicity against some fungi. C. medica is relevant to treatment of diabetes and alzheimer's disease.4, 5 The Citrus medica L. also have an anti-inflammatory, antihistamine and diuretic action and can cause dilatation of the coronaries. Twenty-seven and twenty nine components were identified in the leaf and peel oils, respectively. Limonene was the major constituent in the oil of leaf and peel.6,7

 

Traditionally, Citrus medica L.claim as anthelmintic but scientifically it is not reveled still thus the present study was design to evaluate the in-vitro anthelmintic activity of ethanolic extract of Citrus medica L. peels.

 

MATERIAL AND METHOD:

Plant: The fresh fruits of Citrus medica L. were collected in the month of October 2010 from its natural habitat at Hadgaon in Nanded region, Maharashtra, India. The plant was authenticated by Dr. Miss. A. Chaturvedi, Post Graduate Teaching Department of Botany, Rashtra Santa Tukadoji Maharaj Nagpur University, Nagpur (Voucher specimen no. 9844).

 

Experimental animals:

All the experiments were carried out in Indian adult earthworms (Pheretima posthumad) collected from moist soil and washed with normal saline to remove all fecal matter were used for anthelmintic activity8 due to its anatomical and physiological resemblance with the intestinal roundworm parasite Ascaris lumbricoids, of human beings.9 Because of easy availability, earthworms have been used widely for the initial evaluation of anthelmintic compounds in vitro.10, 11

 

Materials: Ethanolic extracts of Citrus medica L.peels, Piperazine citrate (GSK. Ltd, Mumbai).

 

Preparation of Extracts of Citrus medica L. peels:

From the collected fruits of Citrus medica L. the peels were removed manually and were dried under shade and undergone crushing in electric blender to form powdered and subjected to extraction by using soxhlet extractor. The percent yield of Peel extract was 20% w/w. The extracts were concentrated by evaporation at room temperature and were used for pharmacological studies.12 Preliminary phytochemical tests of extract was performed by using specific reagents through standard procedures.

 

Administration of Extract:

The suspension of Ethanolic extract of Citrus medica L.  different concentration (25-100mg/ml) were  prepared by using 0.2% v/v of Tween 20 as a suspending agent and final volume was made to 10 ml for respective concentration of\ Citrus medica L. peels. Six groups of approximately equal size worms consisting of six earthworms individually in each group were released into 10 ml of desired concentration of drug and extracts.

 

Administration of Piperazine citrate:

Piperazine citrate (15mg/ml) was used prepared by using 0.2% v/v of Tween 20 as a suspending agent.

 

Experimental Design:

Indian adult earthworms (Pheretima posthumad) collected from moist soil and washed with normal saline to remove all fecal matter were used for anthelmintic activity.

 

Different concentration (25-100mg/ml) of Citrus medica L.ethanolic  extract were prepared by using 0.2% v/v of Tween 20 as a suspending agent and final volume was made to 10 ml for respective concentration of Citrus medica L. A Piperazine citrate (15mg/ml) was used as standard. Six groups of approximately equal size worms consisting of six earthworms individually in each group were released into 10 ml of desired concentration of drug.

 

The anthelmintic assay was carried out as per the method of (Ajaiyeoba et.al, 2001) with minor modification. The animals were divided into ten y group containing six earthworms each different concentration of extracts and standard drug solution were poured in different Petri dishesh. Observations were made for the time taken for paralysis (Paralysis was said to occur when worm did not revive in normal saline) and death (Time for death of worms was recorded after ascertaining that worms neither moved when shaken vigorously nor when shaken vigorously nor when dipped in warm water (500c),followed with their body colors fading away).13

 

Statistical Analysis:

All the results were expressed as Mean ±S.E.M. of six animals in each group. Statistical analysis were performed by one way analysis of variance (ANOVA) followed by student’s t test. At 95% confidence interval, p values < 0.001 were considered significant.14

 

Anthelmintic effect of Citrus medica L. peels in Indian adult earthworms (Pheretima posthumad).

For evaluation of anthelmintic activity Citrus medica L. peels extract, group I and II received normal saline and standard Piperazine citrate while group III, IV, V and VI received  different concentration of  ethanolic extract of Citrus medica L. peels respectively.

 

RESULT AND DISCUSSION:

The qualitative phytochemical investigation of ethanolic extracts of peels of Citrus medica L showed the presence of an array of active chemical constituents including alkaloids, glycosides, terpenoids, Flavonoids and sterols and (Table 1). The results of anthelmintic activity revealed that ethanolic extracts exhibited varying degree of activity against both the worms and caused paralysis followed by death at all tested concentrations. From the above observation made in the Table 2, the extract of Citrus medica L. peels was found to show anthelmintic activity when compared to standard drug. Ethanolic extract of Citrus medica L. peels of highest concentration 100mg/ml showed paralysis at 18.08 min. and death of earthworm at 37.43 min at highest permissible dose which was comparable to standard Piperazine citrate (Table 2) From the above result, it is clear that ethanolic   extract of Citrus medica L. peels have significant anthelmintic activity in dose dependent manner when compared with standard anthelmintic drug. It can be concluded that the active constituents responsible for anthelmintic activity present in the ethanolic extract of peels of Citrus medica L. Further study need to isolate and revealed the active compound contained in the crude extract of Citrus medica L. and to established mechanism(s) of action are required.

 

 

Table 1: Preliminary phytochemical screening of ethanolic extract of C. medica

Tests

Ethanolic extract of Citrus medica L.

Alkaloids

+

Flavonoids

+

Tannins

-

Terpenoids

+

Steroids

+

Glycosides

+

(+) = Present, (–) = Absent

 

 

Table 2: Anthelmintic Potency of Citrus medica L. peels extract

Treatment

Group

Concent ration

(mg/ml)

Time of paralysis (min)

(Mean± S.E.M)

Time of paralysis (min)

(Mean± S.E.M)

Normal Saline

(Control)

 

I

 

-

 

-

 

-

Piperazine citrate

II

15

24.46±0.64

80.69±1.39

Ethanolic extract of  Citrus medica L. peels

III

25

66.33±1.39

86.49±2.23

IV

50

39.79±0.42

60.73±1.02

V

75

32.17±0.62

44.34±0.65

VI

100

18.08±0.22

37.43±0.62

All values represent Mean ± SEM; n=6 in each group. All values are significantly different from reference standard (Piperazine citrate) ***p<0.001. This activity was Concentration dependent. The potency was found to be inversely proportional to the time taken for paralysis and time of death of the worms.

 


 

Figure1-Anthelminthic activity of ethanolic extract of citrus medica L. peels.

 


CONCLUSION:

From the results it concluded that, ethanolic extracts of Citrus medica L. demonstrate to possess dose dependant anthelmintic activity when compared to Piperazine citrate (Figure 1). From results, peels of  Citrus medica L. as an anthelmintic have been confirm as a it displayed activity against the worm used in present study. The potency was found to be inversely proportional to the time taken for paralysis and time of death of the worms. Piperazine citrate acts by increasing chloride ion conductance of worm muscle membrane produces hyperpolarisation and reduced excitability that leads to muscle relaxation and flaccid paralysis. The possible mechanism of the anthelminthics activity of Citrus medica L. cannot be explained on the basis of our present results. However, it may be due to its effect on inhibition of glucose uptake in the parasites and depletion of its glycogen synthesis. Citrus medica L. may also have activated nicotinic cholinergic receptor in the worms resulting in either persistent depolarization or hyperpolarisation.15 The plant may be further explored for its phytochemical profile to recognize the active constituent accountable for anthelmintic activity.

 

REFERENCES:

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7.       Hesperides. A  history of the culture and use of citrus fruits, p.371. John Bale, Sons and Curnow, 1938,  London, England.

8.       Hammond J.A.Fielding D.and Bishop S.C.(1997) : Prospects for plant anthelmints in tropical veterinary medicines, Vet. Res. Coom.,21;213-228.

9.       Vidyarthi RD. A Text Book of Zoology, 14th Edn., S. Chand and Co, New Delhi, 1967.

10.     Sollmann T. Anthelmintics: Their efficiency as tested on earthworms. J. Pharmcol. Exp. Ther. 12: 129-170

11.     Jain ML and Jain SR. Therapeutic utility of Ocimum basilicum var. album. Planta. Med. 1972; 22: 66-70 (1972).

12.     Sharangouda, Patil S.B., Estrogenic activity of ethanolic extract of Citrus medica L. on immature albino rats,Int. Jr. Green Pharm., 2007; 2: 91-94.

13.     Ajaiyeoba EO, Onocha PA and Olarenwaju OT. In vitro anthelmintic properties of Buchholzia coriaceae and Gynandropsis gynandra extracts. Pharma. Biol. 39: 217-220 (2001).

14.     Armitage P. Statistical methods in Medical Research. 1st Edn. Blackwell scientific publication, New York, 1971; 217-220.

15.     Tripathi KD. Essential of Medical Pharmacology. 4th Edn. Jaypee Publication, New Delhi, 2006; 816-817p

 

 

 

 

 

 

Received on 15.01.2011       Modified on 11.02.2011

Accepted on 25.02.2011      © RJPT All right reserved

Research J. Pharm. and Tech. 4(5): May 2011; Page 768-770