Phytochemical screening and antimicrobial activity of plant leaf extract against enteric bacterial pathogens

Punica granatum , a fruit-bearing shrub belonging to the Lythraceae family, offers various benefits to humans, with both its fibers and seeds being valuable. The seeds contain important oil acids such as palmitic, punica, stearic, and oleic acids. In India, P. granatum (pomegranate) leaves have been investigated for their potential antibacterial activity against enteric pathogens. These pathogens are responsible for causing gastrointestinal infections and are often associated with antibiotic resistance. The study aims to explore whether pomegranate leaves possess properties that can combat these enteric pathogens effectively. This research holds promise in identifying a natural and alternative approach to address antibacterial challenges, potentially contributing to the development of novel antimicrobial agents pomegranate is commonly used as a traditional remedy for treating enteric bacterial pathogens. To investigate its antibacterial properties, researchers studied the aqueous extract of P. granatum leaf against enteric pathogens, conducting tests like biochemical examinations and microscopy on the isolates. The phytochemical analysis of P. granatum (pomegranate) leaf aqueous extract revealed the presence of various bioactive compounds. These compounds include amino acids, carbohydrates, alkaloids, steroids, flavonoids, terpenoids, saponins, tannins, and phenols. To evaluate its antimicrobial activity, the researchers used antibiotic discs, including chloramphenicol, vancomycin, and gentamicin, employing the standard Kirby Bauer method. Results showed that vancomycin had the highest zone of inhibition, followed by chloramphenicol and gentamicin, with respective measurements of 20 mm and 19 mm. Chloramphenicol exhibited sensitivity against Escherichia coli (19 mm), while gentamicin showed sensitivity against Salmonella typhi (20 mm), Staphylococcus aureus (19 mm), and E. coli (18 mm). Vancomycin demonstrated sensitivity against S. aureus (21 mm). The aqueous leaf extract displayed significant antibacterial activity at concentrations of 20, 40 mcg, and 60 µ g, showing effective zones of inhibition ranging from 15 mm to 22 mm against Pseudomonas aeruginosa , E. coli , S. aureus, Shigella dysentriae, Proteus vulgaris , and S. typhi . In conclusion, the study established that P. granatum leaf extracts possess remarkable antibacterial properties and contain valuable pharmaceutical bioactive compounds. Due to its efficacy in treating gastrointestinal disorders, it is utilized as a natural remedy in traditional medicine.


INTRODUCTION
Since ancient times, humans have relied on medicinal plants and traditional herbal products for various therapeutic purposes. India, with its rich and diverse flora, has been a hub of such natural remedies (Maghimaa & Palanisamy, 2019;Kabeerdass et al., 2021a;Mathanmohun et al., 2021;Pandeeswari et al., 2021a;Sankareswaran et al., 2021; Phytochemical screening and antimicrobial activity of plant leaf extract against enteric bacterial pathogens 2016; Nashima & Palanisamy, 2016). Among the noteworthy natural power fruits in India is P. granatum, commonly known as Pomegranate, which has been traditionally used for treating various diseases. The fruit is used to prepare juice, as well as other products like jam, jelly, and beverages. Historical records show that ancient Egyptians used extracts from this plant, including its bark and fruits, to cure infections like dysentery and diarrhea. The traditional system of Ayurveda in India has utilized Pomegranate as a medicine for thousands of years (Vakayil et al., 2021a;Kabeerdass et al., 2022a). However, drug resistance has become a significant clinical challenge in treating microbial infections like dysentery and cholera (Kabeerdass et al., 2021b, c;Baburam et al., 2022;Nair et al., 2022;Sharmila et al., 2022;Vakayil et al., 2022a). Despite this, some antibiotics with antibacterial properties remain unexplored in the pharmacological context, supporting their use in traditional anti-diarrheal remedies. Peel and leaf extracts of Pomegranate have shown various pharmacological properties (Fan et al., 2021;Sun et al., 2021;Kabeerdass et al., 2022b, c), and the fruit itself has been reported to possess antiviral properties against influenza, pox, herpes, and HIV-1 virus. Its main components, hydrolyzable pigments like anthocyanins and tannins, offer valuable health benefits to humans, including antibacterial effects. The plant also contains flavonoids like punicalagin, ellagic acid, caffeic acid, and luteolin, among which punicalagin exhibits higher inhibitory effects on the influenza virus. The mature Pomegranate fruit comprises fleshy appendages or seeds covered by a white, membranous, edible layer of the pericarp. P. granatum juice has been found to possess robust antioxidant activity, surpassing that of fruit juices from oranges and grapes. These natural antioxidants contribute to cell rejuvenation and anti-aging effects in humans. Additionally, Pomegranate shows potent anticancer and anti-inflammatory properties against various human malignancies. This research aims to evaluate the antimicrobial and phytochemical properties of P. granatum leaf extract against multidrug-resistant enteric pathogens in vitro. This investigation holds promise for uncovering new potential uses of P. granatum as a natural remedy in combating drugresistant infections.

Preparation of Plant Extract
The leaves were carefully washed, dried in the shade, ground into a powder, and stored in airtight bottles at 4 ºC. To prepare the leaf extract, 50 g of the powder was soaked in 250 mL of water for 8 hours using a soxhlet apparatus at a temperature of 65 ºC. After 8 hours, the leaf extract was collected, concentrated, and stored in an airtight container at 4 ºC for further use (Abirami & Maghimaa, 2019;Vakayil et al., 2019Vakayil et al., , 2020Vakayil et al., , 2021bAbirami et al., 2021).

Phytochemical Analysis
The concentrated aqueous extract was subjected to phytochemical analysis to screen for antimicrobial and phytochemical properties (Sivakumar et al., 2018).

Clinical Sample Collection and Culture Preparation
The diarrheal stool samples were collected in sterile containers from hospitals and laboratories in Salem and transported to the laboratory. The samples were inoculated in MacConkey broth, and after 24 hours, pure cultures were obtained on MacConkey agar plates. The pure cultures were then maintained in nutrient agar slants.

Physiological and Biochemical Analysis
The isolates were identified based on their biochemical and physiological profiles.

Antimicrobial Sensitivity Test
The isolates were subjected to an antibiotic sensitivity test using the disc diffusion method, and the plates were incubated for 24 hours. The zone of inhibition was measured using a plate ruler, and the isolates were categorized as Resistant or Sensitive.

Determination of Antibacterial Activity
The aqueous leaf extracts at different concentrations (20, 40, 60 µg) were evaluated against the isolated pathogens using the agar well diffusion method. The plates were incubated at 370C overnight, and the zone of inhibition (ZOI) was measured. Table 1 displays the presence of various compounds, including amino acids, carbohydrates, alkaloids, steroids, flavonoids, terpenoids, saponins, tannins, and phenols in the aqueous leaf extract of P. granatum. The pathogenic bacteria were isolated from diarrheal stool samples and subjected to biochemical characterization and microscopy. Microscopic examination revealed the presence of both gram-negative and gram-positive bacteria. Biochemical tests, including IMViC, catalase, oxidase, urease, carbohydrate test, and TSI tests, were conducted. The identified pathogens included E. coli, S. aureus, P. aeruginosa, S. dysentriae, S. typhi, and P. vulgaris. Table 2 presents the results of the antibiotic sensitivity test, indicating that Vancomycin had the highest zone of inhibition (21 mm), followed by Chloramphenicol and Gentamicin (20 mm and 19 mm). Chloramphenicol exhibited sensitivity against E. coli (19 mm), while Gentamicin showed sensitivity against E. coli (18 mm), S. aureus (19 mm), and S. typhi (20 mm). Vancomycin was found to be sensitive against S. aureus (21 mm). Table 3 demonstrates the significant antibacterial activity of the aqueous leaf extract at concentrations of 20, 40, and 60 µg, showing an effective zone of inhibition in the range of 15-22 mm against P. aeruginosa, E. coli, S. aureus, S. dysentriae, P. vulgaris, and S. typhi. These findings highlight the potential of the leaf extract of P. granatum as an effective antimicrobial agent against the tested pathogens.

DISCUSSION
There is a growing need for innovative antimicrobial agents derived from plant sources that can offer protection, safety, and cost-effectiveness while addressing antibiotic resistance Pandeeswari et al., 2021b;Sivakumar et al., 2021a, b;Sabarinath et al., 2022a, b) and minimizing the adverse side effects associated with synthetic drugs. Pomegranate (P. granatum) peel has been widely utilized as an antimicrobial agent (Abirami & Maghimaa, 2019), and its antibacterial properties have been extensively studied in the past. Through phytochemical investigation, active inhibitors present in P. granatum leaves have been identified as potent components, making the leaf extract of P. granatum a readily available resource of natural antioxidants (Abirami & Maghimaa, 2019). This finding suggests that the peel extract could serve as an effective antibacterial agent. Such a diverse array of phytochemicals suggests that the P.granatum leaf extract possesses a wide range of potential health benefits. These bioactive constituents are known for their antioxidant, anti-inflammatory, and antimicrobial properties, which make the pomegranate leaf extract a valuable natural resource with potential therapeutic applications. For the scientific community involved in drug discovery and development, the key focus is on identifying novel active constituents responsible for antibacterial activity (Rajkumar et al., 2023a, b). This requires a multidisciplinary approach to recognize and understand the dynamic compounds that contribute to the antimicrobial properties of P. granatum leaf extract. By exploring and harnessing these natural compounds, we may discover valuable solutions to combat bacterial infections in a safer and more sustainable manner.

CONCLUSION
The alarming rise in antibiotic resistance among microorganisms has led to a crucial demand for the development of new antimicrobial compounds. In this context, P. granatum emerges as a vital component, valued for its therapeutic properties in traditional medicine.