Evaluation of Anthelmintic Activity of Aqueous Extract of

Casuarina equisetifolia Inflorescence (IA) and Pollen Grains (Seeds: SA) in Indian Adult Earthworm

 

O. Umamaheswar Rao¹*, M. Chinna Eswaraiah², M. C. Prabhakar³

¹Jawaharlal Nehru Technological University, Kukatpally, Hyderabad - 500085, Telangana, India.

²Anurag Pharmacy College, Ananthagiri, Kodad, Nalgonda District - 508206, Telangana, India.

³Shri Vishnu College of Pharmacy, Bhimavaram - 534202, Andhra Pradesh, India.

*Corresponding Author E-mail: umamaheswarrao.226@gmail.com

Among the most common infections of thedigestive system in human beings are helminth infections. In the developing countries, they pose a large threat to the society. Such parasitic diseases cause severe morbidity, including lymphatic filariasis, onchoserciasis and schistosomiasis. Many traditional plants and herbs are reported to have anthelmintic activity. Casuarina equisetifolia belongs to the family of Casuarinaceae has a long history of traditional use as an anthelmintic. All parts of the plant are a rich source of medicinally useful components eg: coughs, nervous disorders, anticancer, hypoglycemic, antispasmodic, ulcers, diarrhea, gonorrhea, constipation, antibacterial activity, antifungal, etc. The Present study is an attempt to evaluate anthelmintic activity of aqueous extract of Inflorescence (IA) and Pollen grains (Seeds: SA) of Casuarina equisetifolia were investigated on adult Indian earthworms Pheretima posthuma (Pheritimidae) as test worms. The plant extracts at various concentrations i.e. 25mg/mL, 50mg/mLand 100mg/mL were tested in the bioassay. Piperazine citrate (10 mg/mL) was used as a reference standard drug whereas distilled water was used as a control. The bioassay involved the determination of the time of paralysis and time of death control of the worms. The results obtained from this study strongly support the traditional anthelmintic use of Casuarina equisetifolia of aqueous extract of Inflorescence (IA) and Pollen grains (Seeds: SA).

 

 

 

 

INTRODUCTION:

Helminth infections (helminthiasis) are the most common infectionsinhuman beings, affecting a large proportion of the world’s population. The word helminth comes from Greek hélmins, a kind of worm. Most diseases caused by helminths are chronic and debilitating in nature. The diseases originated from parasitic infections causing severe morbidity include lymphatic filariasis, onchocerciasis and schistosomiasis. Globally, an estimated 2 billion people are infected by intestinal nematodes^{1, 2 and 3}. Major risk factors for high levels of intestinal helminthiasis remain poverty, poor environmental sanitation and personal hygiene, lack of potable drinking water, inadequate health care, over-crowding and poor health education. Young children are particularly predisposed to heavy infestations with intestinal parasites due to poor immune systems and habitual play in faecally contaminated soil⁴. The parasitic gastroenteritis is caused by mixed infection with several species of the stomach and intestinal worms, which results in weakness, loss of appetite, decreased feed efficiency, reduced weight gain and decreased productivity.

 

Classification of helminths:

Helminths are divided into three groups based on their body segmentation, namely: Trematodes (flukes), Nematodes (roundworms) and Cestodes (tapeworms). Helminths have multi-cellular bodies and complex life cycles involving maturation in a host organism (Figure 1).

 

As per World Health Organization (WHO), only a few drugs are frequently used in the treatment of these parasite infections. Anthelmintics or antihelminthics are drugs that expel parasitic worms (helminths) from the body, by either stunning or killing them⁵. The evaluations of anthelmintic activity of any drug when carried out in artificial laboratory conditions (in vitro) by using⁶ some samples like Indian earthworm Pheretima posthuma, Ascardia galli, Ascaris lummbricoids, etc. Adult Indian earthworm, Pheretima posthuma has been used as test worm in most of the anthelmintic screenings, as it shows anatomical and physiological resemblance withthe intestinal roundworm parasite of human beings, because of easy availability, earthworms. These in vitro screenings are important as they give abasis for further in vivo studies². Anthelmintic drugs can be classified according to their chemical structure as well as to their action against the specific type of helminths. An ideal anthelmintic must have a wide margin between its toxicity to the worm and its toxic effect on the host. The drug must be effective in one dose. Moreover, these drugs are unaffordable because of their high cost. These factors paved the way for herbal remedies as alternative anthelmintics⁷.

 

Plants are a valuable source of a wide range of secondary metabolites, which are used as pharmaceuticals, agrochemicals, flavors, fragrances, colors, biopesticides and food additives. Natural products possessing medicinal properties present in traditional herbs and medicinal plants have been established to be the reason of the extensive consumption of herbal remedies and healthcare preparations. Medicinal plants are the Nature’s gift to human beings to help them pursue a disease-free healthy life^{8 and 9}. Medicinal plants and derived medicine are widely used in traditional cultures all over the world and they are becoming increasingly popular in modern society as natural alternatives to synthetic chemicals. In the last few decades, there has been an exponential growth in the field of herbal medicine. It is getting popularized in developing and developed countries owing to its natural origin and lesser side effects. At the present juncture, the modern conventional healthcare is burdened with great problems of unsafe medicines, chronic diseases, resistant infections, autoimmune disorders and degenerative disorders of aging, despite great scientific advances. More than 70% of India’s 1.1 billion populations still use these non-allopathic systems of medicine. India possesses almost 8% of the estimated biodiversity of the world with around 0.126% million species. The WHO estimated that approximately 80% of world population relies mainly on traditional medicines, mostly plant drugs in their health care. Today, Ayurveda coexists with themodern system of medicine and is still widely used and practiced. About 30%of the currently used therapeutics is of natural origin¹⁰. Many medicinal plants have been used to treat parasitic infections in man and animals. The plants are known to provide a rich source of botanical anthelmintics¹¹.

 

 

Figure 1: Classification of helminths³

Casuarina equisetifolia is a herbaceous member of the family Casuarinaceaeis a handsome tree with drooping branches, 10-15 m high. It is common along the coast of beaches, rocky coasts, limestone outcroppings, dry hillsides and open forests in both wet and dry zones from sea-level to mid-montane. It is native to South-East Asia, Australia and Polynesia. It is also cultivated as an ornamental, for windbreaks, or as a medicinal plant in some tropical countries in the South Pacific. It contains Ellagic acid, beta-sitosterol, kaempferol and glycosides, quercetin, cupressuflavone, isoquercitrin, several common triterpenoids, trifolin, catechin and epicatechin, cholesterol, stigmasterol, campesterol, cholest-5-en-3-beta-ol derivatives, tannin, proanthocyanidins, juglanin, citrulline and amino acids, afzelin, casuarine, gallicin, catechol derivatives, gentisic acid, hydroquinone, nictoflorin, rutin, trifolin. Phytosterol from the leaves of the plant shows antibacterial activity, hypoglycemic, antifungal, molluscicidal, cytotoxic. Tongans use it to treat coughs, ulcers, stomachaches and constipation. Dysuria and menorrhagia are treated with an infusion of the leaves. Secondary amenorrhoea is treated with a decoction of the leaves. An infusion of the leaves, in Tonga, is used as an emetic to treat throat infections. The plant’s uses in treating throat infections, coughs and stomach-aches are also noted in Fiji and India. In Samoa, an infusion of the leaves is used as a remedy for coughs, asthma and diabetes. Cook Islanders use an infusion of the grated leaves to treat mouth infections and urinary tract infections. In Tahiti, the plant is used to treat nervous disorders, diarrhea, gonorrhoea^{12 and 13} and astringen¹⁴. Pharmacological investigations of bark and wood showed significant anticancer and anthelmintic activities¹⁵. However, no simultaneous anthelmintic activity on the Inflorescence and Pollen grains (Seeds: SA) of Casuarina equisetifolia was scientifically available. The Present study is an attempt to evaluate anthelmintic activity of aqueous extract of Inflorescence (IA) and Pollen grains (Seeds: SA) of Casuarina equisetifolia (Figure 2) were investigated on adult Indian earthworms Pheretima posthuma (Pheritimidae) as test worms.

 

 

Figure 2: Casuarina equisetifolia Inflorescence (IA) and Pollen grains (Seeds: SA)

 

 

MATERIALS AND METHODS:

Materials:

Plant Materials Collection and Preparation of Aqueous Extract of Casuarina equisetifolia Inflorescence (IA) and Pollen grains (Seeds: SA):

The dried inflorescence was collected in bulk from Padmabhushan Dr. B.V. Raju foundation, Vishnupur, Bhimavaram, West Godavari (District), Andhra Pradesh and the Pollen grains (Seeds: SA) were separated from the Inflorescence by using sewing process. The plant material was identified and authenticated on 18/01/2013 by Taxonomist Prof. K. Madhava Shetty, S.V. University, Tirupathi, voucher reference number is 2983. Then the debris and leaves were removed from the collected material and dried in shade. It was coarsely grounded in a mixer. This coarse powder was macerated with distilled water for 24 hrs. The extracts were filtered through muslin cloth then evaporating to dryness under shade to obtain the aqueous extract successively (Chloroform was used as a preservative while drying). This dried extract was collected and was stored in a desiccator for further use. Just before the experiment stars, prepared the dose formulations using distilled water with different concentrations 25 mg/mL, 50 mg/mL, 100 mg/mL and 10 mg/mL for both the extracts Casuarina equisetifolia (inflorescence (IA) and Pollen grains (Seeds: SA)) and Piperazine citrate respectively.

 

Earthworms:

Indian earthworms (Pheretima posthuman) were collected from the water-logged areas of soil in kodad, Nalgonda District - 508206, Telangana, India.The average size of Pheretima posthuma was 5-8 cm. They were washed with water to remove all fecal matters.

 

Method:

Anthelmintic Activity:

The anthelmintic assay was carried out as per the method of Ajaiyeoba et al¹⁶. The assay was performed in vitro using adult earthworm (Pheretima posthuma), owing to its anatomical and physiological resemblance with the intestinal roundworm parasites of human beings for preliminary evaluation of anthelmintic activity ^{17, 18 and 19}. Test samples of aqueous extract of Inflorescence (IA) and Pollen grains (Seeds: SA) of Casuarina equisetifolia were prepared at the concentrations (25mg/mL, 50mg/mL, and 100mg/mL) in distilled water and six earthworms ie.Pheretima posthuma, of approximately equal size (same type) were placed in each 9cm petri dish containing 25 mL of above test solution of extracts. Piperazine citrate 10mg/mL was used as thereference standard and distilled water as control^20-23. All the test solution and standard drug solutions were prepared freshly before starting the experiments. Observations were made, for the time taken for paralysis, it was noted when no movement of any sort could be observed, except when the worms were shaken vigorously. Time for the death of worms were recorded after ascertaining that worms neither moved when shaken vigorously nor when dipped in warm water (50⁰C). Three sets of experiments were done for the statistical significance.

 

RESULTS AND DISCUSSIONS:

In the present study, it was observed that both the aqueous extracts of Casuarina equisetifolia Inflorescence (IA) and Pollen grains (Seeds: SA) have exhibited a positive response to the certain degree of anthelmintic activity. Extracts exhibitedmore potent activity at higher concentration (100 mg/mL) against Pheretima posthuma (earthworm).Evaluation of anthelmintic activity was compared with reference standard Piperazine citrate as shown in Table 1. Piperazine citrate increases chloride ion conductance of worm muscle membrane, producing hyper polarization and reduced excitability that leads to muscle relaxation and flaccid paralysis⁷.

 

Table 1: Anthelmintic Activity of Aqueous Extract of Inflorescence (IA) and Pollen grains (Seeds: SA) of Casuarina equisetifolia

Each Value Represents Mean ± SEM (n=6)

 

 

The effect of aqueous extracts of Inflorescence (IA) and Pollen grains (Seeds: SA) of Casuarina equisetifolia at various concentrations (25mg/mL, 50mg/mL and 100mg/mL) were studied on Pheretima posthuman (Figure 3 to 10)as a preliminary test for anthelmintic activity.

 

 

Figure 3: Control  (Distilled Water)

 

 

Figure 4: Reference Standard

(Piperazine citrate) – 10 mg/mL

 

 

 

Figure 5: Aqueous extract of Inflorescence (IA) - 25 mg/mL

 

Figure 6: Aqueous extract of Inflorescence (IA) - 50 mg/mL

 

Figure 7: Aqueous extract of Inflorescence (IA) - 100 mg/mL

 

 

 

 

Figure 8: Aqueous extract of Pollen grains (Seeds: SA) –

25 mg/mL 

 

 

Figure 9: Aqueous extract of Pollen grains (Seeds: SA) –

50 mg/mL 

 

 

 

Figure 10: Aqueous extract of Pollen grains (Seeds: SA) –100 mg/mL 

 

 

Parameters studied include paralytic time and death time. A Dose-dependent decrease in paralytic time and death time was observed with all the two extracts. Seeds aqueous extract (SA) showed less paralytic time and death time when compared to Inflorescence aqueous extract (IA) [SA > IA]. There was no significant (p˂0.05) difference between SA (100mg/mL) and piperazine (10mg/mL) in paralysis time and death time by one way ANOVA followed by Tukey’s test (Figure 11 and 12).

 

Figure 11: Paralytic Time in different groups *** p < 0.05 vs SA and Piperazine citrate by one way ANOVA	Figure 12:  Death Time in different groups *** p < 0.05 vs SA and Piperazine citrate by one way ANOVA

 

 

 

 

 

 

 

CONCLUSION:

Based on the results obtained from this study, we concluded that the aqueous extracts ofInflorescence (IA) and Pollen grains (Seeds: SA) of Casuarina equisetifoliashowed significant anthelmintic activity on Indian adult earthworms (Pheretima postuma). Therefore, further study may be carried out so that the general people can get actual benefit from this important medicinal plant.

 

ACKNOWLEDGEMENT:

Authors are sincerely thankful to Dr. K. Madhava Chetty, Plant Taxonomist, Department of Botany, Sri Venkateswara University, Tirupati, India for authentification of plant materials.

 

CONFLICT OF INTEREST:

Authors have declared that no conflict of interests exists.

 

REFERENCES:

1.          Sirama V, Kokwaro J, Owuor B, Yusuf A and Kodhiambo M. In-vitro anthelmintic activity of Vernonia amygdalina Del. (asteraceae) roots using adult Haemonchus contortus worms. International Journal of Pharmacological Research. 2015;5(1):1-7.

2.          Ravindra G Mali and Anitha A Mehta. A Review on Anthelmintic Plants. Natural Product Radiance. 2008;7(5):466-475.

3.          Piyush Jain, Seema Singh, Sandeep K Singh, Verma SK, Kharya MD, Sanjeev Solanki. Anthelmintic Potential of Herbal Drugs. International Journal of Research and Development in Pharmacy and Life Sciences. 2013;2(3):412-427.

4.          Uzodimma CE, Ojinnaka NC, Chukwunedum AU and Anthony NI. Prevalence of Intestinal Helminthiasis among Children with Chronic Neurologic Disorders in University of Nigeria Teaching Hospital (UNTH) Ituku-Ozalla. Journal of Neurological Disorders. 2016;4(1):1-6.

5.          Chandan HS, Tapas AR and Sakarkar DM. Anthelmintic activity of extracts of Coriandrum sativum linn. In. International Journal of Phytomedicine. 2011;3(1):36-40.

6.          Murugamani V, Raju L, Baskar Anand Raj V, Manjir Sarmakataki and Girija Sankar G. The New Method Developed for Evaluation of Anthelmintic Activity by Housefly worms and Compared with Conventional Earthworm Method. International Scholarly Research Network Pharmacology. 2012;2012:1-6.

7.          Das R, Mehta DK, and Gupta A. In Vitro Anthelmintic Activity of Leaves of Juglansregia L Against Pheretima posthuma. Scientific reviews and Chemical Communications. 2011;1(1):78-82.

8.          Ali Esmail Al-Snafi. The Pharmacological Importance of Casuarina equisetifolia - An Overview. International Journal of Pharmacological Screening Methods. 2015;5(1):4-9.

9.          Mylene M Uy and Karleen I Garcia. Evaluation of the antioxidant properties of the leaf extracts of Philippine medicinal plants Casuarina equisetifolia Linn, Cyperus brevifolius (Rottb) Hassk, Drymoglossum piloselloides Linn, Ixora chinensis Lam and Piper abbreviatum Opiz. Advances in Agriculture and Botanics – International Journal of the Bioflux Society. 2015;7(2):71-79.

10.       Amit Kumar and Sangh Partap. In Vitro Anthelmintic Activity of Lagenaria siceraria Leaves in Indian Adult Earthworm. Journal of Pharmacognosy and Phytochemistry. 2012;1(4):39-42.

11.       Manas Kumar Pal. Evaluation of Anthelmintic Activity of Leaves of Paederia foetida. International Journal of Pharma and Bio Sciences. 2011;2(1):227-231.

12.       Sriram N. Antidiabetic and Antihyperlipidemic Activity of Bark of Casuarina equisetifolia on Streptozotocin-Induced Diabetic Rats. International Journal of Pharmacy Review and Research. 2011;1(1):4-8.

13.       Nehad M Gumgumjee and Abdulrahaman S Hajar. Antimicrobial efficacy of Casuarina equisetifolia extracts against some pathogenic microorganisms. Journal of Medicinal Plants Research. 2012;6(47):5819-5825.

14.       Aher AN, Pal SC, Patil UK, Yadav SK and Bhattacharya S. Effect of Phenolic Content on Antioxidant Activity of Casuarina equisetifolia. Pharmacologyonline, Newsletter. 2009;1:280-285.

15.       Aher AN, Pal SC, Yadav SK, Patil UK and Bhattacharya S. Antioxidant Activity of Isolated Phytoconstituents from Casuarina equisetifolia Frost (Casuarinaceae). Journal of Plant Sciences. 2009;4(1):15-20.

16.       Ajaiyeoba EO, Onocha PA and Olarenwaju OT. In vitro anthelmintic properties of Buchholzia coriaceae and Gynandropsis gynandra extract. Pharmaceutical Biology. 2001;39(3):217- 220.

17.       Dash GK, Suresh P, Sahu1 SK, Kar DM, Ganapaty S and Panda SB. Evaluation of Evolvulus alsinoides Linn. for anthelmintic and antimicrobial activities. Journal of Natural Remedies. 2002;2(2):182-185.

18.       Shivkar YM and Kumar VL. Anthelmintic Activity of Latex of Calotropis procera. Pharmaceutical Biology. 2003;41(3):263-265.

19.       Vigar Z. Atlas of Medical Parasitology. P. G. Publishing House Singapore. 2nd ed. 1984; pp. 242.

20.       Lal J, Chandra S, Raviprakash V and Sabir M. In vitro anthelmintic action of some indigenous medicinal plants on Ascardia galli worms. Indian Journal of Physiol Pharmacology. 1976;20:64- 68.

21.       Mali RG, Shailaja Mahajan and Patil KS. Anthelmintic activity of root bark of Capparis spinosa. Indian J Nat Prod. 2005;21: 50-51.

22.       Mali RG and Wadekar RR. In Vitro Anthelmintic Activity of Baliospermum montanum Muell. Arg roots. Indian Journal of Pharmaceutical Sciences. 2008;70(1):131-133.

23.       Gbolade AA and Adeyemi AA. Investigation of in vitro anthelmintic activities of Pycnanthus angeolensis and Sphenocentrum jollyanum. Fitoterapia. 2008;79:200-222.

 

 

 

 

 

 

 

 

Received on 07.12.2017         Modified on 25.12.2017

Accepted on 05.01.2018         © AJRC All right reserved