Therapeutic Potential and Anthelmintic Activity of Bottle Gourd Peel

The helminths are parasitic worms. They are more widespread infectious agents of human beings in the developing world and cause a significant burden of being an infectious disease that surpasses other more established infections, such as malaria and tuberculosis. The prevailing phyla of helminths are two:

1. The nematodes (roundworms), e.g., filarial worms, intestinal worms, and Onchocerca volvulus,
2. Platyhelminthes (flatworms), e.g., tapeworms, schistosomes, and flukes [1].

Today, they disproportionately affect marginalized, low-income, and resource-constrained regions of the world [2]. In this time of rapidly expanding helminth disease and the commitment to finding a drug to control it is essential that resources are allocated in an efficient manner to have the greatest impact. However, many questions remain regarding how best to select a good drug, which is one of the great challenges worldwide for researchers, including the development of synthetic or natural remedies with low side effects [3]. Unfortunately, there are only a limited number of drug classes available for anthelmintic activity in the market, and substantial resistance has been developed in most of the parasitic species. Thus, the synthesis of various anthelminthics with a broad spectrum of action is necessarily important. The drug development may be either synthetic or natural [4]. The activity of any anthelmintic medication is to either deaden or kill the worm and expel it from the body. A portion of the usually accessible medications are albendazole, piperazine, mebendazole, levamisole, and thiabendazole [5,6]. A most popular marketed drug, albendazole, showed good anthelmintic activity either in-vitro or in-vivo in humans primarily against helminth species. However, it is effective against the major intestinal species infecting humans [7].  In spite of this, albendazole shows hepatotoxicity along with hepatic failure in a few cases, as reported in the literature [8]. In comparison with synthetic drugs, natural compounds exhibit a greater tendency to show anthelminthic properties in the treatment of various parasitic diseases. This review focuses on the ability of plant-based natural products in the development of new therapeutic compounds for their anthelminthic activity [9]. Natural compounds in plants have medicinal effects, which are still being explored to derive novel pharmaceuticals [10]. Plant waste, such as leaves, seeds, peels, stems, and roots can be utilized after harvesting [11]. Through this, researchers have found an easy source of antioxidant, antihypertensive, anti-obesity, anticancer, antidiabetic, and antibacterial agents [12]. Bottle gourd is a versatile crop that belongs to the cucurbit family and is mainly found in sub-Saharan Africa (SSA) for human consumption [13].  Bottle gourd (Lagenaria siceraria) shows more genetic inconsistencies and morphological activities compared to other plants in the cucurbit family [14,15]. One of the most common and widespread worm infections is helminthiasis, which is very harmful to the human body [9]. Bottle gourd is mainly used as a nourishing agent, cardio-protective, diuretic, and cardio-tonic [16]. It also possesses both antihepatotoxic activity [17] and antioxidant activity [18].  There are many literature surveys that have revealed that Lagenaria siceraria shows good anthelmintic activity based on the fruit, seed, etc. [19]. Therefore, the present study is carried out based on the bottle gourd peels that perform the anthelmintic activity.

2. METHODS
   1. Drugs and chemicals

The solvents were procured from SD Fine Chem Ltd. and Avra Synthesis Pvt Ltd., Mysore, India. Moreover, all other chemicals used in this study were of analytical grade with high purity.

1. 2. Collection and identification of the plant:

Newly grown bottle gourd was used for the experimental purpose from the area of Mandya district, Karnataka, India. The collected fruits were first washed thoroughly with water to remove dirt and then peeled off the skin manually, and then kept for drying under the sun for 10 days. After complete drying, the entire portions were crushed into a coarse powder with the help of a grinding machine and were stored in an airtight container for further use.

1. 3. Extraction and solvent-solvent partitioning of plant material:

Plant powder was prepared by packaging 20g of Lagenaria siceraria in filter paper and loading it into the thimble, which is placed inside the Soxhlet extractor. Following this, the solvents (250 mL of hexane, dichloromethane, chloroform, ethyl acetate, and water) were added to a round-bottom flask, which was attached to a Soxhlet extractor and condenser on an isomantle. The solvent was heated using the isomantle at a boiling point of hexane (69.1°C), dichloromethane (39.6°C), chloroform (61.2°C), ethyl acetate (77.1°C), and water (100°C). And it started evaporating, passing through the apparatus to the condenser. The condensate dripped then into the reservoir containing the thimble. Once the level of solvent reached the siphon, it was poured back into the flask and the cycle began again. The process should run for about 10 hours. After extraction, the content was concentrated on a water bath for the removal of solvents from samples by using a rotary evaporator apparatus.

1. 4. Preparation of Plant Extracts

Medical plants are useful in both fresh and dry forms. Drying is the most common preservation procedure that occurs post-harvest, which requires prompt execution to uphold quality, prevent contamination, and the deterioration of phytochemical properties. The desiccated plant matter was crushed into small fragments between 2-5 mm using a cylindrical crusher. Approximately 100 g of resinous plant dry powder was placed within a Soxhlet apparatus for solvent extraction, subjected to extraction of solvents, stating sequential extraction from non-polar solvent to polar solvent, i.e., hexane. The extraction samples were preserved at a temperature of 4°C for subsequent analyses. The filtrates were later filtered through Whatman No. 1 filter paper and further concentrated by the use of a rotary evaporator to eliminate the solvents.

Figure 1 – Highlighting the process of study

These concentrated extracts were refrigerated in glass flasks fitted with screw plastic lids, and were appropriately labelled as bottle gourd hexane extract (BH), bottle gourd dichloromethane extract (BD), bottle gourd chloroform extract (BC), bottle gourd ethyl acetate extract (BE), and bottle gourd water extract (BW).

1. 5. Experiment on animals:

An in-depth study was performed to investigate the effective anthelmintic potential of different extracts of bottle gourd peel powder using hexane, dichloromethane, chloroform, ethyl acetate, and water extracts. These extracts have been stringently tested at various levels of concentration, ranging from 10 to 50 mg/mL, with an accurately planned bioassay. This bioassay precisely determined not only the critical time of death of earthworms, roundworms, and tapeworms but also the time period it took for paralysis of worms. It is notable that the reference used for comparison was made by an anthelmintic agent, albendazole.

1. 6. RESULT AND DISCUSSION

In our present study, preliminary examination of various extracts of bottle gourd fruit has revealed its anthelmintic effects. The study revealed that all the fruit extracts had varying levels of positive anthelmintic activity. The method of extraction involved transitioning from non-polar solvents to polar solvents, allowing for the separation of non-polar and polar compounds within the different extracts of bottle gourd fruit, while also examining their respective biological activities. Furthermore, we conducted the experiment to obtain more compounds in polar and non-polar extracts, such as hexane, dichloromethane, ethyl acetate, chloroform, and water. We subjected the extract to thin-layer chromatography using various mobile phases, one of which was hexane: dichloromethane. The hexane extract, in particular, demonstrated greater potency at a lower concentration of 10 mg/mL against A. galli (roundworm). To evaluate the anthelmintic activity, we used a reference (albendazole) as shown in Table 1.

Table 1: Comparative anthelmintic activity of various bottle gourd peel extracts versus albendazole (standard drug).

The hexane extract of bottle gourd represented higher rates of paralysis and mortality of worms, especially at lower concentrations, in comparison to other extracts. It was noteworthy that the ethyl acetate extract displayed significant activity, while the chloroform extract was the least active of all the extracts. The primary mode of action of albendazole on the worms is the inhibition of microtubular function. With a lesser amount of bottle gourd peel extract, it not only exhibits paralysis but also presents the death of the worms.  Albendazole, a most common and widely used broad-spectrum anthelmintic, a standard reference drug in numerous experimental investigations, is associated with a number of adverse effects well documented in human beings. The use of albendazole treatment has been reported to cause gastrointestinal disturbances, which include abdominal pain, nausea, and vomiting, and CNS manifestations, which include headache and dizziness [20,21]. More severe toxicities were observed, particularly with long-term exposure and increased doses, such as hepatotoxicity manifested by increased hepatic transaminases, bone marrow suppression that results in leukopenia or pancytopenia, alopecia, and in rare cases, hypersensitivity reactions [21-23]. Owing to such safety concerns, it is predetermined that the use of albendazole requires strict dose regulation and routine liver function and hematological evaluations. In contrast, natural plant-based extracts under investigation for their anthelmintic effects are more likely to be considered a safer alternative, due to a long history of traditional usage with pronounced pharmacological impact and minimal or no adverse effects at therapeutic concentrations [24,25].

Graph 1 – Representing the time taken for Paralysis (P) in mins (Mean & SEM) in Earthworm, Roundworm, and Tapeworm using various bottle gourd peel extracts against standard (Albendazole).

The conspicuous absence of significant toxicity with regard to these natural extracts highlights the prospects of having safer anthelmintics, especially when used for long-term administration and on more susceptible populations.

Graph 2 – Representing the time taken for Death (D) in mins (Mean & SEM) in Earthworm, Roundworm, and Tapeworm using various bottle gourd peel extracts against standard (Albendazole).