Pharmacological Evaluation of Oxalis corniculata Linn for Anthelmintic Activity

 

Santosh B. Dighe1*, B. S. Kuchekar2, Sagar B. Wankhede3

1Department of Pharmacology, Pravara Rural College of Pharmacy, Pravranagar,  Maharashtra, (India)

2Dept. of Pharmaceutical Chemistry, MAEER, Maharashtra Institute of Pharmacy, Pune Maharashtra, (India)

3Department of Pharmaceutical Chemistry, Pad. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pune, Maharashtra, (India)

ABSTRACT:

The present study aimed at the in-vitro comparative study of anthelmintic activity of petroleum ether, ethyl acetate and methanol extracts of whole plant of Oxalis corniculata Linn. using Eisenia foetida at three different concentrations (100, 200, and 400 mg/ml) respectively. The study involved the determination of time of paralysis (P) and time of death (D) of the worms.  At the concentration of 400 mg/ml all the extracts showed very significant activities as compared to the standard drug levamisole (0.55 mg/ml). Each extract with the dose 100, 200, and 400 mg/ml, produced dose-dependent paralysis ranging from loss of motility to loss of response to external stimuli, which gradually progressed to death. In case of methanol extract which was found to be most potent, the time of paralysis and death time was observed as 11.33 and 41.33 min, respectively. In conclusion, the use of O. corniculata as possible anthelmintic is explored  and further studies are suggested to isolate the active principles responsible for the activity.

 

KEYWORDS: Anthelmintic, Eisenia foetida,Oxalis corniculata Linn.,

 

1. INTRODUCTION:

The World Health Organization reveals that over two billion people are suffering from parasitic worm infections.1 It is estimated that by the year 2025, about 57% of the population in developing countries will be influenced.2 The word Helminth is derived from the Greek word helminths meaning worm. Helminth is a broad categorical term referring to various types of parasitic worms that reside in the body.3 The prevalence of parasitic helminths typically displays a negative binomial distribution within an infected population such that relatively few persons carry heavy parasite burdens.

 

Without treatment, those individuals are most likely to become ill and to perpetuate infection within their community.4 Anthelmintics are drugs that may act locally to expel worms from the GIT or systemically to eradicate adult helminths or development forms that invade organs and tissues.5

 

Most of the existing anthelmintics produce side effects such as abdominal pain, loss of appetite, nausea, vomiting, headache and diarrhea.6 Anthelmintics from the natural sources may play a key role in the treatment of these parasite infections.7 Increasing problems of development of resistance in helminths against anthelmintics have led to the proposal of screening medicinal plants for their anthelmintic activity.8 The plant selected for the present studies is O. corniculata  belonging to family Oxalidaceae, very common weed found throughout warmer parts of India.9

This is an annual or perennial plant, the first reproduces by seeds, and in tropical areas it flowers all year. The plant having most diverse 4 genus and consist of about 900 species. The whole plant traditionally used for analgesic, anti-inflammatory and antidysentric properties.10 Soup of Indian sorrel is used in diarrhea.11 Its anti-inflammatory and wound healing property has been reported in 1977 and 2004.12, 13 Also it has antimicrobial and a smooth muscle relaxant property.14,15 O. corniculata has been traditionally used as an anthelmintic. However, anthelmintic activity of O. corniculata has not so far been scientifically proved, so the present study was carried out to assess the anthelmintic activity of O. corniculata against Eisenia foetida by using its different extracts.

 

2. MATERIALS AND METHODS:

Plant material:

The whole plant of O. corniculata was collected from the Loni (Shirdi) in the Ahmednagar district of Maharashtra, India. It was authenticated by Dr. Divakar. Joint Director, Botanical Survey of India, Pune. A voucher specimen (DSOC001) has been deposited in the herbarium section of the Department of Pharmacognosy, PRCOP, Loni, for future reference. The whole plant (1.5 kg) was air dried and pulverized using a mechanical grinder.

 

Preparation of extract:

The whole plant of O. corniculata collected and shade dried and then pulverized in grinder. About 500 gm powdered utilized for extraction was passed through 120-mesh sieve to remove fine powder and coarse powder was used for extraction by using continuous hot Soxhlet extraction.  Different solvents in increasing order of polarity were used like petroleum ether (60-800c), ethyl acetate, and methanol. The extraction was carried out in Soxhlet extractor till all the constituents were extracted. The completion of extraction was indicated by taking sample out of siphon tube on TLC plate and placing it in iodine chamber. Absence of colored spot on plate indicated complete extraction. After completion of extraction, solvent was distilled off and concentrated extract was air-dried. The extract was stored in airtight container. The same procedure was followed during extraction with other solvents.16, 17

 

 

Phytochemical analysis:

Continuous successive soxhlet extraction of powdered whole plant of O. corniculata Linn was carried out using different solvents viz, petroleum ether, and ethyl acetate and methanol. All the three extracts were then subjected to qualitative chemical analysis to detect the chemical constituents present in them. Phytochemical screening of the extracts was carried out using the standard procedure to confirm the presence of carbohydrates and glycosides, phytosterols, phenolic compounds, tannins, flavonoids, proteins and amino acids18. (Table 1)

 

 

Anthelmintic Assay:

The assay was performed on adult earthworm, Eisenia foetida due to its anatomical and physiological resemblance with the intestinal roundworm parasite of human beings. Because of easy availability, earthworms have been used extensively for the preliminary in vitro evaluation of anthelmintics compounds19. 100 ml formulations containing three different concentrations (100, 200, and 400 mg/ml in distilled water), each of Petroleum ether, Ethyl acetate and methanolic extract were prepared and six worms (same type) were placed in it. Time for paralysis was noted when no movement of any sort could be observed except when the worms were shaken vigorously.

 

Time for death of worms were recorded after ascertaining that the worms neither moved when shaken vigorously nor when dipped in warm water (50°C) . Levamisole (0.55 mg/ml) was used as reference standard.

 

Statistical analysis:

Data were analyzed using one way factorial ANOVA tests followed by Dunnett.s t -tests on each group. P values under 0.01 were considered highly significant (shown as **).

 

3. RESULT:

Preliminary phytochemical screening of O. corniculat extract revealed the presence of proteins, saponins, steroids, carbohydrates, alkaloids, tannins, glycosides, flavonoids and phenols (Table 1).

 

Table no. 1: Phytochemical screening of O. corniculata

Test for

Petroleum ether extract

Ethyl acetate extract

Methanol

extract

Carbohydrates

-

-

+

Monosaccharide

-

-

+

Polysaccharide

-

-

+

Flavonoids

-

+

+

Glycosides

-

-

+

Saponin

+

+

+

Alkaloids

+

-

+

Fats and oil

+

-

-

Tannins and Phenol

-

-

+

Proteins and  amino acids

-

+

-

Steroids and Terpenoids

+

+

+

 

Anthelmintic activity:

The anthelmintic activity of methanolic extract of O. corniculata (MEOC) was more potent than Petroleum Ether extract (PEOC) and Ethyl Acetate extract of Oxalis corniculata Linn.(EAOC) was comparable with that of standard drugs Levamisole. Each extract containing concentration of 100, 200, 400 mg/ml, produced dose-dependent paralysis ranging from loss of motility to loss of response to external stimuli, which gradually progressed to death.

 

Table no. 2: Anthelmintic activity of various extract of  O. corniculata

Sr. no.

Test

Concentration (mg/ml)

Time of  Paralysis (min)

Time of Death (min)

1.

Levamisole

0.5

1.23 ± 0.002

4.22 ± 0.05

 

2.

 

PEOC

100

150.16 ± 0.85

255.16 ± 0.66

200

92.33 ± 1.71*

160.16 ± 0.57*

400

28.22 ± 1.57 *

93.55 ± 0.13*

 

3.

 

EAOC

100

120.16 ± 0.15

185.16 ± 0.46

200

63.53 ± 0.71*

100.16 ± 0.47*

400

20.32 ± 1.47 *

73.55 ± 0.33

 

4.

 

MEOC

100

96.83 ± 1.47*

142.66 ± 0.88*

200

31.23 ± 0.93*

70.16 ± 0.79*

400

11.33 ± 0.80**

41.33 ± 0.71**

Where, PEOC = Petroleum extract of Oxalis Corniculata Linn, EAOC = Ethyl acetate extract of Oxalis Corniculata Linn, MEOC = Methanolic extract of Oxalis Corniculata Linn. Values are expressed as mean ± SEM. Values were find out by using ONE way ANOVA followed by Dunnett.s t -test.

 

The Petroleum-ether extracts of dose 100, 200 and 400 mg/ml, produced paralysis within 150.16, 92.33 and 28.22 min respectively. Death was noted within 255.16, 160.16 and 93.55 min respectively.

 

Ethyl acetate extract also showed dose-dependent paralysis at concentration of 100, 200 and 400 mg/ml, paralysis was noted at 120.16, 63.53 and 20.32 min respectively, while death produced at 185.16, 100.16 and 73.55 min respectively.(Fig.1and2)

 

 

Fig 1. Time of Paralysis for various extracts of O. corniculata on Eisenia foetida

 

 

Fig 2. Time of death for various extracts of  O. corniculata  on Eisenia foetida

Each bar is represented as mean ± standard deviation (n = 5).

Group 1 –Standard-1 (Levamisole 0.5 mg/ml), Group 2 to 4 – Pet-ether extract 100, 200 and 400 mg/ml respectively, Group 5 to 7 – Ethyl Acetate extract 100, 200 and 400 mg/ml respectively, Group 8 to 10 – Methanol extract 100, 200 and 400 mg/ml respectively.

 

Methanolic extract at concentration 100, 200 and 400 mg/ml produced paralysis within 96.83, 31.23 and 11.33 min respectively. The death was noted with 100, 200 and 400 mg/ml concentration within142.66, 70.16 and 41.33 min respectively. (Table 2).

 

4. DISCUSSION:

Earthworms are invertebrates composed of many segments. They don’t have bones and move by contracting and relaxing the body segments in sequence. They have the ability to move by ciliary movement. The outer layer of the earthworm is a mucilagenous layer and composed of complex polysaccharides. This layer being slimy enables the earthworm to move freely. Any damage to the mucopolysaccharide membrane will expose the outer layer and this restricts its movement and can cause paralysis. This action may lead to the death of the worm by causing damage to the mucopolysaccharide layer.20 All anthelmintics essentially kill worms by either starving them to death or paralyzing them because worms have no means of storing energy and they must eat almost continuously to meet their metabolic needs. Any disruption in this process results in energy depletion. Interfering with feeding for 24 hours or less is sufficient to kill most adult parasites. Parasites will also die if they become paralyzed and temporarily lose their ability to maintain their position in the gut.21 Preliminary phytochemical screening of O. corniculata extract revealed the presence of proteins, saponins, steroids, carbohydrates, alkaloids, tannins, glycosides, flavonoids and phenols. Table 1 shows the phytochemicals detected in O. corniculata leaf extract.

 

Synthetic phenolic anthelmintics interfere with the energy generation in the helminth parasites by uncoupling the oxidative phosphorylation. Another possible mechanism of action is that they bind to free proteins in the gastrointestinal tract of the host animal or to glycoprotein on the cuticle of the parasite and causes death.22

 

The phytochemicals detected in the extracts are acting as major role to possess medicinal properties. The possible mechanism of action of tannins may be by interfering with energy generation by uncoupling oxidative phosphorylation and interfering with glycoprotein of cell surface23. Alkaloids may act on CNS and caused paralysis of the earthworm.24 The effect would be due to presence of the steroidal alkaloid oligoglycosides which may suppress the transfer of sucrose from the stomach to the small intestine together with its antioxidant effect which is capable of reducing the nitrate generation which could interfere in local homeostasis which is essential for the development of helminthes.25 Levamisole is nicotinic receptor agonists and elicit spastic muscle paralysis due to prolonged activation of the excitatory nicotinic acetylcholine (nACh) receptors on body wall muscle.26

 

5. CONCLUSION:

It could be concluded that the methanolic extract showed most potent anthelmintic activity. The other two extracts i.e. petroleum ether and ethyl acetate extracts, exhibited lesser anthelmintic activity than the methanolic extract. The present study revealed that the anthelmintic activity increases with increasing polarity of solvents. Further studies are required to identify the actual chemical constituents that are present in the crude extracts of this plant which are responsible for anthelmintic activity and to establish the effectiveness and pharmacological rationale for the use of O. corniculata as an anthelmintic drug. Further it can be concluded that active constituents responsible for anthelmintic activity are present in the O. Corniculata

 

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Received on 03.12.2011

Modified on 18.12.2011

Accepted on 24.12.2011                                               

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Research J. Pharmacology and Pharmacodynamics. 4(1):Jan. - Feb., 2012, 1-4