To An In – Vitro Evaluation of The Anthelmintic Activity of Piper Betle on The Pheritima Posthuma Model

The research of new medicinal characteristics of diverse plant species has inspired the attention of the scientists towards the biologically active chemicals since the last couple of decades. This is because the bioactive chemicals are low- or non-toxic and have strong pharmacological activity[1]. The widespread belief that herbal remedies are safer than synthetic medications with serious side effects and the resistance brought on by the careless use of synthetic medications are the primary causes of the growing interest in plant-derived medications[2]. Even though there are several antibacterial drugs accessible in medicine, the improper use and indiscriminate prescription of commercially available antibiotics have led to the development of multidrug resistance in human pathogenic microbes in recent years[3]. From the standpoint of underdeveloped nations today, synthetic medications are not only costly and unable to treat illnesses, but they can also have deadly side effects[4]. As a result, given the rise in the prevalence of newly discovered and reemerging pathogenic diseases, it is imperative to investigate novel antibacterial components with a variety of chemical structures and unique modes of action[5]. to take the place of those that are no longer effective. Additionally, we are aware that higher and aromatic plants have been used in traditional and folklore medicine to prolong the shelf life of goods that exhibit inhibition against yeasts and bacteria[6]. studies have reported that many herbs possess varying degree of antimicrobial activities. Therefore, the natural medicinal plants may be a potent source of new antibacterial agents. A portion of the body infested with parasitic worms such as roundworms (nematodes), tapeworms (cestodes), or flukes (trematodes) is known as helminthiasis[7]. Even though the worms live in the digestive system, they occasionally burrow into the liver and other organs. Anthelmintic medications either eradicate or kill helminthes, or worms, that are causing the infestation. Because of their strong anthelmintic characteristics, medicinal herbs have been studied for scientific advancement worldwide since ancient times[8]. Antihelmintic medications either eradicate or kill helminthes (worm infestations). Because of their strong anthelmintic effects, plants have been studied for their therapeutic qualities for the sake of science worldwide since ancient times[9]. Certain broad spectrum anthelmintics, such as albendazole and piperazine citrate, work effectively against both nematodes and parasitic flatworms. But most medications have a restricted range of effects (like praziquantel), since resistance can emerge quickly[10]. There could be toxicity issues (Akhtar et al., 2000). Finding novel therapeutic plants with broad spectrum anthelmintic action and low toxicity is therefore imperative[11]. The primary sources of reactive oxygen species (ROS) in the forms of superoxide anions, hydroxyl radicals, and hydrogen peroxide are auto- oxidation of lipids and reactive nitrogen species (RNS). The oxidation or nitration processes of lipids, proteins, nucleic acids, and DNAs are likely to be harmed by the production of these extra ROS and RNS by ultraviolet (UV) radiation, smoking, and drug metabolisms. Conversely, these reactive oxygen species are linked to a number of degenerative illnesses, such as cancer, aging, arteriosclerosis, and rheumatism, and they can induce inflammation or lesions on different organs[12]. Naturally occurring phenolic compounds are well known for their capacity to donate hydrogen, chelate metals, reduce, and—most remarkably—capture free radicals and halt chain reactions. According to a report, the hydroxyl groups found in phenolic compounds may play a crucial role in scavenging free radicals and may directly contribute to the antioxidant activity. Plants with high polyphenol content have therefore gained more recognition as natural antioxidants across the globe[13]. The Piperaceae family of vines includes P. betel Linn. (Betel leaf), which is well known as paan in Bangladesh. It is mostly found in tropical and subtropical regions of the world[14]. P. betel leaves have several types of properties, including those that are respiratory depressive, cardiotonic, antitumor, antiulcer, antiplatelet aggregation, antifertility, and antitumor. In addition, it has aphrodisiac, tonic, anthelmintic, stomachic, and carminative properties. According to a number of reports, this plant's leaf has a wide range of advantageous bioactivities, and its extract has a lot of promise for application in the creation of commercial goods[15]. According to numerous reports, this plant's leaf has a wide range of advantageous bioactivities, and its extract has a lot of potential for application in the creation of commercial goods. This served as the foundation for choosing this plant, especially the leaves. Therefore, using an in vitro study paradigm, the evaluation study seeks to establish the total phenolic components of P. betel leaves as well as their antibacterial and anthelmintic activities, all of which may be useful in the development of new, innovative medications[16].

Although there are many antibacterial agents available in the field of medicine, in recent years multidrug resistance has been developed in human pathogenic microorganisms due to indiscriminate prescription and malpractice of commercially available antibiotics[18].

Table No 2[19].

Table No 3

PLANT OF WORK

BIOLOGICAL SOURCE

The betle (piper betle) is a vine belonging to the piperaceae family. which includes both pepper and kava .betel leaf is mostly chewed in asia , and elsewhere in the world by some Asian emigrants, as betel quid or in paan, with areca nut often with added tobacco.

Uses:

1. Antibacterial
2. Anticancer
3. Anti-diabetic
4. Anti-allergic
5. wound healing
6. Anti-inflammatory
7. Gastro protective
8. Anti-hypercholestrolemic [25]

MATERIAL AND METHODS

Collection and preparation of plant material

The leaves of P. betel were collected from Shirdi, Maharashtra in Octomber 2024. The sun dried powdered leaves (500 mg) of P. betel was macerated in 2.5 L of 99.8% methanol (Merck KGaA, Darmstadt, Germany). After 15 days the solution was filtered using filter cloth and Whatman filter paper No. 1. The resulting filtrates were then evaporated in water bath maintained at 45°C to dryness and thus a blackish-green semisolid mass of the extract was obtained (yield 25 g).

Reagents and chemicals

All the solutions, reagents used in this study were of analytical grade.

Evaluation of the anthelmintic activity Collection of worms

The earthworms belonging to species Pheritima posthuma (Annelida), about 3-5 cm in length and 0.1- 0.2 cm in width weighing about 0.8-3.04 g, were collected from the moist soil

of Shirdi , Maharashtra, Sai Krupa Nersery. INDIA.

Reference Drug

For the evaluation of anthelmintic activity of Piper betel, the methanolic extract of leaves of the plant was tested in various doses in each group. Distilled water was used as control.

Albendazole was used as the standard drug for comparative study with methanolic extract.

Anthelmintic assay

The anthelmintic assay was carried out as per the method of Adate et al. with minor modifications. Here the anthelmintic activity was assessed using earthworms because of their anatomical and physiological resemblance with that of the intestinal roundworm parasites of human being . They are widely used as effective tools for anthelmintic study because of their easy availability[20,21] . All of the worms were cleaned and all excrement was removed using regular saline water. Ten milliliters of distilled water were used to weigh the extracts and dissolve them in order to achieve concentrations of 10, 20, 40, 60, and 80 mg/ml. In a petri dish, earthworms were separated into seven groups, each with five worms. After applying the extract to the Petri dishes, the paralysis and death times were ascertained. Periods of paralysis occurred when there was no discernible movement of the worms, with the exception of when they were jolted violently. After determining that the worms did not move when shaken violently or when submerged in heated water (50°C), their body colors gradually faded and the time of death was recorded[22].

Determination of total phenolic content

Total phenolic contents were determined by the Folin-ciocalteau method using Gallic acid as standard. The extract samples (0.5 ml of different dilutions) were mixed with Folin-ciocalteu reagent (2.5 ml, 1:10 diluted with distilled water) for 5 min and aqueous Na2CO3 (2 ml, 7.5 % w/v) was then added. The mixture was incubated for 20 minutes at room temperature. After 20 minutes the absorbance was measured at 760 nm by UV-spectrophotometer. The total phenolic content of the samples were measured using the standard curve prepared from Gallic acid solution with different concentrations (6.25, 12.5, 25, 50, and 100 µg/ml). The phenolic contents of the sample were expressed as mg of GAE (Gallic acid equivalent) / g of the extracts[23].

Statistical analysis

All data are presented as mean±standard deviation (SD) and were analyzed by One-way analysis of variance (ANOVA) (SPSS for windows, version 18.0, IBM corporation, NY, USA). The values were considered significantly different at p<0.05.