INTRODUCTION:

Helminthiasis is a worldwide and one of the common diseases of all ages. As per WHO more than two billion people suffered from this infestation every year. Development of resistance to most of the commercially available anthelmintics became a severe problem worldwide. So, the indiscriminate use of synthetic anthelmintics can lead to resistance of parasites.¹ Moreover, these drugs are unaffordable, inaccessible or inadequately available to the people of the developing countries.

Herbal drugs have been in use since ancient times for the treatment of parasitic diseases in human and could be of value in preventing the development of resistance.² These factors paved the way to find out the effective herbal formulation for helminthiasis.

In traditional Ayurvedic System of Medicine many formulations are available for the treatment of worm infestation. These traditional preparations are being used for ages without scientific validation. Unique way of prescribing medicines by this system draws attention worldwide for keen research in drugs for reverse pharmacology manner. Panchachurnam, an Ayurvedic polyherbomineral formulation consists of Cassia angustifolia Vahl. leaf, Terminalia chebula Retz. fruit, Zingiber officinale Rosc. rhizome, Foeniculum vulgare Mill. fruit and Saindhava lavana, traditionally used for the management of worm infestation, constipation, haemorrhoids, pain in abdomen, flatulence, assimilatory disorder, rheumatic conditions and in managing all diseases of Kapha Dosha origin. It also improves digestion and ensures timely evacuation of faeces improves functioning of liver and can be used in hyperacidity, heart burn and acidic belching. It provides an antidote for pungent food and promotes the elimination of toxins, supports intestinal immunity and balances tridosha.³ Based on this, the present study was undertaken to investigate the anthelmintic activity of aqueous and ethanolic extracts of Panchachurnam on Indian earth worms (Pheretima posthuma).

MATERIALS AND METHODS:

Collection and identification of plant materials:

The plant materials of Panchachurnam were procured from the local markets of Pithapuram, Samalkot and Peddapuram of East Godavari District, Andhra Pradesh, India. The specimens of the samples were authenticated by Mr. Raghava Rao, Department of Botany, M. R. College, Peddapuram, East Godavari District, Andhra Pradesh, India. Voucher specimens (ACP/LAB/PG-16/17) of the same have been deposited in the museum of Dept. of Pharmacognosy, Aditya College of Pharmacy for future reference.

Preparation of Panchachurnam:

The churna was prepared as per the procedure given in Ayurvedic Formulary of India. All the ingredients were crushed to powder using grinder and were powdered separately and sieve no. #80. In-house Panchachurnam was prepared from these powders by mixing them in one part for each ingredient and passed through sieve no. #60. A physical mixture was made by mixing together in equal proportions to get uniformly blended churna. ^4-6

Preparation of the aqueous extract:

The 100g of Panchachurnam was extracted in water at 50 – 60⁰C in a soxhlet apparatus separately. The extract was concentrated to dryness in a water bath at controlled temperature (50-60⁰C). The dried 95% of the aqueous extract was weighed in a required dose and dissolved in known volume of distilled water, separately for further treatment.

Preparation of ethanolic extract:

The 100g of Panchachurnam was extracted in 95% ethanol at 50–60⁰C in a soxhlet apparatus separately. The extract was concentrated to dryness in a water bath at controlled temperature (50-60⁰C). The dried 95% of the ethanolic extract was weighed in a required dose and dissolved in known volume of distilled water, separately for further treatment.

Preliminary Phytochemical Screening:

Preliminary qualitative phytochemical screening of all the extracts of Panchachurnam was carried by employing standard conventional protocols for the detection of organic constituents by performing chemical tests for alkaloids, glycosides, anthraquinone glycosides, carbohydrates, steroids, terpenoids, flavonoids, tannins, volatile oils, tannins, proteins and fixed oils. Screening of Panchachurnam was also done for the detection of inorganic constituents by performing identification tests for Sodium and Chloride ions.⁷

Drugs, Chemicals and Reagents:

Piperazine citrate (Noel, Mumbai) and Albendazole (Ranbaxy, New Delhi) used as reference standards were purchased from Jagajanani Medical Stores, Peddapuram, East Godavari District, Andhra Pradesh, India. Normal saline was used as control. All chemicals and reagents used throughout the experiment were of analytical grade procured from S.D. Fine-chem., Merck, Fischer scientific and Spectrochem, Mumbai.

Preparation of Standard Solutions and Control:

Piperazine Citrate is taken as standard drug and the concentration of the standard drug was dissolved in 50ml of normal saline solution to get 15mg/ml of solution. Albendazole is taken as standard drug and the concentration of the standard drug was dissolved in 50ml of normal saline solution to get 20mg/ml of solution. Normal saline alone was used as control.

Preparation of test samples:

Samples for experiments were prepared by dissolving extract to obtain a stock solution of 100mg/ml, from the stock solution, different working dilutions were prepared to get concentration range of 15, 25 and 50mg/ml of aqueous extracts (PCAE) and of 15, 25 and 50mg/ml of ethanolic extracts (PCEE) of Panchachurnam.

Experimental worms:

Healthy adult Indian earthworms (Pheritima posthuma) due to its anatomical and physiological resemblance with the intestinal round worm parasites of human beings were used in the present study.They were collected from moist soil of the field and washed with normal water and saline solution to remove soil, adherent material and faecal matter. The Earth worms of 4-8cm in length and 0.2-0.3cm in width were used for all experimental parameters.^8-11

Evaluation of anthelmintic activity:

The evaluation of anthelmintic activity was followed by earlier reported method.^12-14 Three different concentrations of aqueous and ethanolic extract (as given earlier) were prepared and the group of six earthworms which having equal size were released into 10 ml of sample with desired concentration in petridish. Observations were made for the time taken to cause paralysis and death of the individual worms. Mean time for the paralysis in any sort could be observed, except when the worm was shaken vigorously. The worms neither moved when shaken vigorously nor when dipped in warm water (50⁰C). Paralysis is assumed to occur when they do not revive even in saline solution. Death was concluded when the worms lost their motility followed with fading away of their body colour. ^15-16 Potency is inversely proportional to time taken for paralysis and/or death of parasite.

Statistical Analysis:

The data obtained were expressed as mean ± SEM (standard error of the mean). Statistical analysis were performed by one way analysis of variance (ANOVA) followed by Students’‘t’ test. At 95% confidence interval, a p value of <0.05 was considered statistically significant. ¹⁷

RESULTS AND DISCUSSION:

The aqueous (PCAE) and ethanolic (PCEE) extracts of Panchachurnam showed significant anthelmintic activity. Each extract containing 15, 25, and 50mg/ml had produced dose dependent paralysis ranging from loss of motility to loss of response to external stimuli, which eventually progressed to death. The results of anthelmintic activity of Panchachurnam on earthworms Pheritima posthuma were given in Table 1 which was also represented in Figures 1, 2 and 3 respectively.

Table 1 Anthelmintic activity of Aqueous and ethanolic (95%) extracts of Panchachurnam on Indian Earthworms (Pheretima posthuma)

S. No.	Treatment	Concentration (mg/ml)	Time taken for paralysis (Min)	Time taken for death (Min)
1.	Control (Normal Saline)	0.9% NaCl	No paralysis	No death
2.	Piperazine citrate	15	6.10±0.03	8.03±0.98
3.	Albendazole	20	2.50±0.17	3.72±0.62
4.	Aqueous extract of Panchachurnam (PCAE)	15	8.17±0.23	11.13±0.38
5.		25	6.13±0.28	8.68±0.73
6.		50	3.41±0.64	5.20±0.32
7.	Ethanolic extract of Panchachurnam (PCEE)	15	6.93±0.33	8.85±0.65
8.		25	4.79±0.22	6.97±0.24
9.		50	2.80±0.52	4.13±0.41

Results expressed as Mean + SEM from 6 observations, p<0.05 as compared to standard

Figure 1 Control and Anthelmintic activity of Standard Drugs

Figure 2 Anthelmintic activity of Aqueous extracts of Panchachurnam(PCAE)

Figure 3 Anthelmintic activity of Ethanolic extracts of Panchachurnam(PCEE)

The results of the above studies demonstrated that, the aqueous and ethanolic extract of Panchachurnam possess potent anthelmintic activity with varying magnitudes. But the ethanolic extract of Panchachurnam showed highest activity, which is almost equal in effectiveness to the standard Albendazole. The difference in the time taken for induction of paralysis in both Albendazole and Panchachurnam was almost same. Therefore, potency of drug was found to be inversely proportional to the time taken for paralysis/death of worms. The higher concentrations of ethanolic extract produced paralytic effect much earlier and necrotic spots were observed externally on the worms, as compare to aqueous extract. The effect of each extract was compared with albendazole (20mg/ml) and piperazine citrate (15mg/ml) as standard drugs.

The preliminary phytochemical observations of the aqueous and ethanolic extracts of Panchachurnam have shown the occurrence of glycosides, anthraquinone glycosides, carbohydrates, triterpenes, tannins, flavonoids, volatile oils, proteins, fixed oils and resins and also shown the presence of sodium and chloride due to Saindhava Lavana (Rock Salt) which was reported in Table 2 and 3. It indicates that, Panchachurnam is a mixture of all these phytoconstituents and interaction of all these chemicals might be resulted in synergistically enhanced therapeutic efficacy of anthelmintic activity.

Table 2 Phytochemical screening of Panchachurnam

S. No.	Phyto constituents	Panchachurnam
S. No.	Phyto constituents	Aqueous Extract	Ethanolic Extract
1.	Alkaloids	-ve	-ve
2.	Glycosides	+ve	+ve
3.	Anthraquinone Glycosides	+ve	+ve
4.	Carbohydrates	+ve	+ve
5.	Steroids	-ve	-ve
6.	Terpenoids	+ve	+ve
7.	Tannins	+ve	+ve
8.	Flavonoids	+ve	+ve
9.	Volatile Oils	+ve	+ve
10.	Proteins	+ve	+ve
11.	Fixed Oils	+ve	+ve
12.	Resins	+ve	+ve

Table 3 Screening of Panchachurnam for inorganic constituents

S. No.	Inorganic constituents	Panchachurnam
S. No.	Inorganic constituents	Aqueous Extract	Ethanolic Extract
1.	Sodium	+ve	-ve
2.	Chloride	+ve	-ve

CONCLUSION:

The present study reveals that the ethanolic extract of Panchachurnam showed marked anthelmintic activity than the aqueous extract as compared to standard drugs albendazole and piperazine citrate and this formulation will effectively kill the worms and serves as a better anthelmintic preparation. This study would provide the preliminary scientific evidence for the folkloric, ethno-botanical and traditional use of Panchachurnam for destruction of helminthes/parasites and other health benefits. Further, the active constituents responsible for anthelmintic activity can be explored.

ACKNOWLEDGEMENT:

We are grateful to our principal Dr. K. Ravi Shankar and management of Aditya College of Pharmacy, Surampalem for providing us necessary facilities to carry out the research project.

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Received on 12.04.2017 Modified on 01.05.2017

Res. J. Pharmacognosy and Phytochem. 2017; 9(4): 210-214.

DOI: 10.5958/0975-4385.2017.00038.3