A Comparative Study of Anthelmintic Activity of Hydroalcoholic Extract & Petroleum Ether Extract of Pumpkin Seeds

species include nematodes (roundworms), cestodes (tapeworms), trematodes (flukes), and ancylostomids (hookworms), with nomenclature derived from their morphological features.3 These parasites predominantly colonize the gastrointestinal tract, particularly the stomach and intestines. Clinical manifestations of severe helminthic infections encompass diarrhea,

Helminthiasis, a parasitic infection caused by helminths, poses a significant public health burden globally, particularly in tropical and subtropical regions.4 This condition not only affects human and animal health, including livestock, but also impairs agricultural productivity, thereby influencing global socioeconomic outcomes . Helminths are classified as parasitic worms, and the pharmacological agents employed for their treatment are known as anthelmintic. The primary helminthic infestations, especially abdominal pain, general malaise, and impaired cognitive development. Prolonged those due to hookworms, are associated with chronic blood loss leading to iron- deficiency anemia and intestinal hemorrhage.

Pheretima, a genus of earthworms, has gained widespread acceptance as a model organism in in- vitro anthelmintic research, owing to its anatomical and physiological resemblance to intestinal helminths.6 Pheretima posthuma, a representative species, are long, cylindrical annelids measuring approximately 15–30 cm in length. Typically inhabiting moist, humus-rich soils, these organisms play an essential ecological role in nutrient recycling and soil aeration. Their average lifespan ranges from 3 to 10 years, making them suitable for longitudinal experimental studies.

Phytopharmacological Importance of Pumpkin (Cucurbita spp.)

Pumpkin (Cucurbita spp.), a member of the Cucurbitaceae family, is widely cultivated across tropical and subtropical regions as both a vegetable and a medicinal plant.7 This genus includes several economically and therapeutically

significant species, primarily Cucurbita pepo, Cucurbita maxima, and Cucurbita moschata (Lee et al.). These species are commonly grown throughout the world and have long been recognized for their nutritional and medicinal value. Historically, pumpkins have been used in traditional medicine systems across various countries including China, India, Pakistan, Yugoslavia, Argentina, Mexico, Brazil, and the United States (jia et al.).9 In these cultures, pumpkin seeds in particular have been utilized either alone or in combination with other herbal remedies for the treatment of a wide range of ailments. Pumpkin seeds are rich in bioactive compounds including unsaturated fatty acids, phytoestrogens, and vitamin E isoforms (notably α-tocopherol and γ- tocopherol), which contribute to their pharmaceutical, nutraceutical, and cosmeceutical potential.10 These constituents exhibit a range of therapeutic properties such as antioxidant, anti-aging, anti-inflammatory, and disease- preventive effects. Recent research has increasingly highlighted the anthelmintic potential of pumpkin seeds. Their high content of unsaturated fatty acids and antioxidant vitamins not only contributes to overall health promotion but also plays a role in neutralizing free radicals and combating parasitic infections. The growing body of evidence supporting the medicinal efficacy of Cucurbita seeds has fostered interest in their use as functional foods and in the development of plant-based therapeutic agent.

MATERIAL & METHOD:

Collection of Plant Material

Pumpkin seeds were procured from the local market of Karad. The seeds were cleaned and then ground into a coarse powder using a mechanical grinder to facilitate extraction.

PROCESS OF SOXHELT EXTRACTION:

A comparative study was conducted to evaluate the anthelmintic activity of the hydroalcoholic and petroleum ether extracts of pumpkin (Cucurbita spp.) seeds[11]

The hydroalcoholic solvent system was used to extract polar phytoconstituents such as cucurbitacin, which are known for their anthelmintic properties[12].

 In contrast, petroleum ether, a non-polar solvent, was used to isolate non-pol         ar constituents such as fats and oils.

Initially, 150 g of powdered pumpkin seeds were subjected to extraction using 500 mL of petroleum ether as the solvent. The extraction was carried out over a period of five days at a constant temperature of 55°C. Upon completion, the petroleum ether extract was collected and evaluated for its anthelmintic activity.

The residual marc obtained after petroleum ether extraction was subsequently utilized for hydroalcoholic extraction. A hydroalcoholic solvent system consisting of water and ethanol in a 2:8 ratio (v/v) was employed, with a total volume of 500 mL. This extraction was also performed under identical conditions— maintained at 55°C for five days. After completion, the solvent was evaporated under reduced pressure to obtain the crude hydroalcoholic extract, which was then collected and stored for future analysis.

Experimental Animals:

To evaluate the anthelmintic activity, earthworms were used as the experimental model due to their anatomical and physiological resemblance to human intestinal roundworms. The following standardized procedure was followed to ensure ethical handling, accuracy, and reproducibility of results.

• Selection of Earthworms

The earthworm species selected was Pheretima posthuma. These species are commonly used due to their similarity to parasitic helminths found in humans. ¹³

• Collection and Identification

Earthworms were obtained from local vermiculture units and identified based on their morphological characteristics.

• Cleaning and Acclimatization

Collected earthworms were gently washed with normal saline or distilled water to remove soil and fecal matter. They were then placed in clean containers lined with moist filter paper or sterilized moist soil for 1–2 hours to allow gut clearance.

The worms were maintained at room temperature, avoiding direct sunlight or extreme cold, until use in the experiments.

• Health Check and Grouping

Only healthy, active earthworms of uniform size (6–8 cm in length and 0.3–0.4 cm in diameter) were selected. Weak, injured, or sluggish worms were discarded. The selected worms were randomly divided into experimental groups, including:

1. Control group (Distilled water)

2. Standard group (Albendazole)

3. Test groups (Different concentrations 25,50,75,100 mg/ml)

Each group of worms was placed in separate Petri dishes or beakers containing the respective test or control solution.

Evaluation of Anthelmintic Activity:

The anthelmintic activity was assessed using adult Indian earthworms (Pheretima posthuma) as the test organisms14. The worms were randomly selected and divided into three groups, each consisting of five individuals:

Group I (Control): Treated with distilled water.

Group II (Standard): Treated with Albendazole at concentrations of 50 mg/mL and 100 mg/mL.

Group III (Test): Treated with seed extract (hydroalcoholic or petroleum ether) at concentrations of 25, 50, 75, and 100 mg/ml.

The study focused on two primary parameters:

1. Time to paralysis and

2. Time to death.

• Paralysis was determined when the worms showed no movement in response to a gentle pin prick.

• Death was confirmed by the absence of movement in response to external stimuli and a noticeable fading of the body color[15].

RESULT

The hydroalcoholic pumpkin seed extract produced a significant antihelmintic activity in dose dependent manner as shown in table below:

Table 1: Anthelmintic activity of hydroalcoholic pumpkin seed extract on earthworm

The petroleum ether extract shows anthelmintic activity in dose dependent manner as shown below:

Table 2: Anthelmintic activity of Petroleum ether extract (Oil phase) of pumpkin seeds on earthworm

Table 3: Anthelmintic activity of Standard drug (Albendazole) on earth worm