Phytochemical screening and antibacterial activities of Momordica charantia and Vernonia amygdalina extracts on some selected enteric isolates

Background: This research focuses on herbal medicine, an ancient healthcare practice, exploring the antibacterial attributes of fresh and dried leaf extracts from Momordica charantia (commonly known as Bitter melon) and Vernonia amygdalina (Bitter leaf). The study specifically investigates their effects on different bacterial strains associated with gastroenteritis. Methods: Four enteric bacterial isolates-Klebsiella pneumoniae , Salmonella typhi , Escherichia coli , and Proteus mirabilis - were obtained from the Medical Laboratory Unit at


Introduction
Plants have been integral to traditional and modern healing practices globally, with a rich history of medicinal use.Herbal medicine, a time-tested approach, relies on complex combinations of plant elements for therapeutic benefits.Identification and quality assessment of phytochemical constituents are crucial to ensure the safety and effectiveness of herbal remedies [1][2][3][4].
Momordica charantia (Figure 1), commonly recognized as bitter melon or balsam pear, is extensively cultivated in regions such as Asia, India, East Africa, and South America.Taxonomically, it belongs to the Kingdom: Plantae, Order: Cucurbitales, Family: Cucurbitaceae, Genus: Momordica and Species: charantia.It has gained recognition for its pharmacological and nutritional advantages and has been traditionally employed to address various health issues, encompassing diabetes, stomach ailments, hypertension, obesity, cancer, inflammation, and infections [10].Recent studies suggest that Momordica charantia exhibits anti-diabetic characteristics, potentially leading to a reduction in blood glucose levels upon consumption [11].A nutritional analysis underscores its abundance in water, protein, lipids, carbohydrates, cellulose, ash, and vitamin C [12].Moreover, it encompasses vitamins, ions, iron, fiber, and lycopene-an antioxidant-in the pulp surrounding the seeds.The bitter element of M. charantia comprises four cucurbitane glycosides, namely momordicosides K and L, and momordicines I and II [12].Various phytochemicals, including polypeptide-p, the steroid glycoside charantin, momordin, oleanolic 3-O-glucuronide, have been identified in both the fruits and seeds of M. charantia.Numerous cucurbitane-type triterpenoids have been isolated from various parts of the plant, including the fruits, seeds, stems, roots, leaves, and vines [10].Given the escalating global interest in plant-based remedies, there is a growing emphasis on exploring the therapeutic potential of herbal treatments with regard to safety, effectiveness, and cost-efficiency [13].Momordica charantia stands out for its diverse biological properties, notably its anti-inflammatory and anti-diabetic effects [12,14].
Vernonia amygdalina (V.amygdalina) is mainly a small shrub with dark green leaves and rough barks which grows majorly in tropical Africa, but is generally spread in many areas of West Africa.It is cultivated as a food supplement in most countries, including Nigeria [15].Because of its bitter flavor, it is generally called a bitter leaf in English, and its local name differs between languages and places.
Vernonia amygdalina is called locally as 'Shuwaka' in Hausa, 'Ewuro' in Yoruba, and Onugbu in Igbo languages [16,17].Vernonia amygdalina (Figure 2) belongs to the family, Asteraceae and is widely reported to have several health benefits [17].Taxonomically, it belongs to Kingdom: Plantae, Order: Asterales, Family: Asteraceae, Genus: Vernonia, and Species: amygdalina.In different parts of the world, V. amygdalina is among the medicinal plants used for treatment.It was reported that the herb is also used for the treatment of diabetes, inflammation, yellow fever, dysentery, constipation, malaria, and stomach aches in Africa and Asia.Moreover, the herb is famous in Africa as a source of food, as a vegetable, and as a culinary herb in soup.Its extracts are found to have lethal effects on human cancer cells of the nasopharynx, effective against amoebic dysentery, and gastrointestinal diseases, and possess antibacterial and anti-parasitic activity [18].Sesquiterpene lactones, flavonoids, and steroid glycosides were found to be the predominant bioactive compounds of the leaves [19].Numerous studies have confirmed the antibacterial activity of Vernonia amygdalina extracts against a range of bacteria [9].
Furthermore, using Vernonia amygdalina as a chewing stick for maintaining oral hygiene has been found to be effective in removing cariogenic microbes and promoting gum healing.The plant exhibits anti-sickling properties, contributes to hemostasis, possesses antibacterial activity, and inhibits plaque formation [20].Water extracts from Vernonia amygdalina leaves have been shown to inhibit fungal growth and eliminate intestinal parasites [21,22].
The significance of investigating the antibacterial properties of Momordica charantia and Vernonia amygdalina becomes particularly crucial in the context of combating bacterial strains associated with gastroenteritis.As antibiotic resistance continues to rise, exploring alternative and complementary therapeutic options from natural sources assumes paramount importance.The potential synergistic effects of phytochemicals in these plants may offer new avenues for developing effective antibacterial agents.This study aims to determine the phytochemicals present and antibacterial activities of these plant extracts against selected enteric isolates, contributing to the ongoing efforts to harness the therapeutic potential of herbal medicine in the face of emerging health challenges.

Source of test isolates
The test isolates, including Escherichia coli, Proteus mirabilis, Salmonella typhi, and Klebsiella pneumoniae, were obtained from the Medical Microbiology and Parasitology laboratory at Babcock University Teaching Hospital (BUTH) in Ilishan-Remo, Ogun State, Nigeria.Before initiating the study, ethical approval was obtained from the Babcock University Health Research Ethics Committee (BUHREC) under registration number BUHREC335/22.

Identification of test isolates
The test isolates underwent sub-culturing from primary agar plates and were subsequently transferred to differential and selective media such as MacConkey agar, Selenite F broth, and Salmonella-Shigella agar.Re-identification and confirmation were carried out using standard biochemical methods and Gram staining, following the protocols outlined by Ochei and Kolhatkar and Cheesbrough [23,24].Source and Authentication of Plant Materials: Fresh leaves of Momordica charantia and Vernonia amygdalina were collected in bulk from a local farm in Igbogbo, Ikorodu, Lagos state, Nigeria.Dr. G.I. Nodza, a botanist from the Department of Botany at the University of Lagos, Lagos state, confirmed the authenticity of the plants.

Extraction of fresh plant materials
The fresh leaves of M. charantia and V. amygdalina were washed with distilled water to remove impurities.Approximately 16 g of each plant material was weighed and homogenized separately in electric blenders with 80 mL of sterile, cold distilled water, hot distilled water, and methanol.This process aimed to achieve a 200 mg/mL concentration, following the method described by Sofowora [25].The resulting homogenate was filtered through sterile cotton wool and Whatman No.1 filter paper into separate containers.The filtrate was then transferred to sterile MacCartney bottles and used immediately for antibacterial susceptibility testing due to the tendency of aqueous extracts to ferment when stored.

Extraction of dried plant materials
The fresh leaves of M. charantia and V. amygdalina were cleaned with distilled water and sun-dried for four consecutive days.The dried leaves were ground into a rough powder using a mortar and pestle, followed by further processing in an electric blender.For extraction, 25 g of the powdered material was soaked in 250 mL of sterile cold distilled water, hot distilled water, and methanol in separate containers.The mixtures were left to stand for 24 hours before filtration, as per the method described by Olayemi and Opaleye [26].
The filtrate underwent filtration using both a mesh and Whatman No.1 filter paper.The resulting liquid was then transferred to a pre-weighed container and subjected to evaporation in a vacuum oven at 45 degrees, yielding a solid residue.The residue was stored in a desiccator until required.In the process of preparing plant extracts, dried leaves from M. charantia and V. amygdalina were utilized.These leaves were employed to generate extracts and reconstituted in distilled water to achieve the desired concentrations.Specifically, 2 g of powdered extract was combined with 10 mL of distilled water, resulting in a 200 mg/mL extract solution.

Phytochemical screening
For phytochemical screening, each leaf extract underwent analysis to identify bioactive components, including alkaloids, terpenoids, flavonoids, steroids, phenols, saponins, and tannins.The screening procedures followed the guidelines outlined by Phillipson [27].Alkaloids test.In the alkaloids test, 1 mg of 10% hydrochloric acid (HCl) was added to 3 mL of the extract, followed by treatment with Meyer's reagent.The presence of alkaloids was indicated by the appearance of precipitation with the reagent.Terpenoids test.For the terpenoids test, 5 mg of the extract was mixed with chloroform (CHCl 2 ) in a test tube, and 3 mL of concentrated sulfuric acid (H 2 SO 4 ) was added carefully to form a distinct layer.Saponins test.To identify saponins, approximately 5 mL of the extract was dissolved in distilled water, vigorously shaken to produce a stable froth, and mixed with three drops of olive oil.The resulting froth was observed.Flavonoids test.For the flavonoids test, a few drops of 1% ammonia solution were added to the extract.Boiled distilled water was added, filtered while hot, and allowed to cool.Subsequently, 5 mL of 20% sodium hydroxide (NaOH) was mixed with an equal volume of the filtrate, and the presence of flavonoids was indicated by the appearance of a yellow solution.Glycosides test.In the glycosides test, 10 mL of 50% concentrated sulfuric acid (H 2 SO 4 ) was added to 1 mL of extract in a boiling test tube.The mixture was heated in boiling water for 5 minutes, and then 10 mL of Fehling's solution was added and boiled.The presence of Submit a manuscript: https://www.tmrjournals.com/mhmdeoxy-sugar, characteristic of cardenolides, was indicated by the formation of a brown ring during the interphase.Sterility check of leaf extracts.The sterility of each extract (both aqueous and methanol extracts) was determined by inoculating 1 mL of each extract on sterile nutrient agar and incubating it at 37 °C for 24 hours, following the procedure outlined by Okunlola et al. [28].The plates were examined for any signs of growth, and the absence of growth indicated sterility.Antibacterial susceptibility testing.Agar Diffusion Test For antibacterial susceptibility testing, the agar diffusion method described by Hudzicki was employed [29].Sterile semi-solid nutrient agar (NA) plates were prepared, and a 1 mL standardized inoculum of the various test isolates was used to flood the plates.After allowing for pre-diffusion, the zones of inhibition were measured in millimeters using a calibrated ruler.Sterile distilled water served as the negative control and Ciprofloxacin was used as the positive control to assess antibacterial activity.The experimental pharmacological protocol is presented in Table 1.Statistical analysis.The statistical analysis involved the application of one-way analysis of variance (ANOVA) and the Turkey-Kramer Multiple Comparisons Test using the Statistical Packages for Social Scientists (SPSS-18.0,version 18.0).

Results
Samples of M. charantia and V. amygdalina plants were sourced from a local farm in Igbogbo, Ikorodu, Lagos state.Authentication of both plant specimens was performed by Dr. G.I. Nodza in the Botany department at the University of Lagos, who assigned them the voucher number LUH9039.A qualitative phytochemical analysis revealed the presence of flavonoids, glycosides, and saponins in both plants, excluding terpenoids.Notably, alkaloids were exclusively identified in V. amygdalina.The antibacterial properties of the plants against specific enteric pathogens were evaluated.The results indicated a lack of inhibitory activity against the test strains, with no observable zones of inhibition for individual or combined solutions.Additionally, when combined with a standard drug, a partial antagonistic effect was observed, resulting in a smaller zone of inhibition compared to the use of the standard drug alone.Concerning bacterial test strains, except for Escherichia coli, the positive control (Ciprofloxacin) demonstrated inhibition zone diameters ranging from 17 mm to 45 mm.The positive control (Cefuroxime) for Escherichia coli exhibited zone diameters ranging from 16 mm to 18 mm.

Discussion
In this study, we investigated the antibacterial properties of extracts derived from both fresh and dried leaves of Momordica charantia (bitter melon) and Vernonia amygdalina (bitter leaf) against specific bacteria associated with gastroenteritis.Previous research has identified these plants as rich sources of various phytochemicals [30,31].The qualitative phytochemical analysis in our study revealed the presence of biologically active secondary metabolites, such as flavonoids, glycosides, and saponins in both plants, with the exception of alkaloids, which were absent in M. charantia Table 2 [10,32,33].These phytochemicals are known for their medicinal significance, displaying various pharmacological, biochemical, and physiological effects, commonly employed in modern medicine [34].Contrary to prior studies, our results indicate that leaf extracts of V. amygdalina and M. charantia did not demonstrate antibacterial activity against the enteric bacterial isolates when tested individually or in combination, despite the presence of essential phytochemicals and the use of different solvents during the extraction process [35][36].In contrast, our outcomes contradict the findings of Zubairu et al., who reported V. amygdalina leaf extract's effectiveness against E. coli and S. typhi [37].
It is also in disagreement with the work of Onifade et al., who reported that the ethyl acetate extract of V. amygdalina (300 mg/mL) inhibited the growth of Pseudomonas aeruginosa, Bacillus subtilis, Klebsiella pneumoniae, Escherichia coli, Proteus mirabilis, Staphylococcus aureus and Staphylococcus epidermidis [38].The most significant inhibitory effect was observed against S. aureus with an inhibition zone of 23.17 ± 0.60 mm.The above protocol shall be adopted for V. amygdalina as well.[36].They also disagree with Ihsan et al.'s report that M. charantia exhibited more significant growth inhibition compared to antibiotics when tested against E. coli, P. aeruginosa, and S. aureus [39].Singh et al., reported a less considerable growth inhibition of M. charantia compared to antibiotics when tested against S. aureus and P. aeruginosa (Table 3-Table 7) [40].The absence of inhibitory activity in the leaf extracts of V. amygdalina and M. charantia from Igbogbo, Ikorodu Local Government Area of Lagos, tested in this study is a noteworthy observation requiring further investigation.The lack of a zone of inhibition, even with the combination of plant extracts, suggests resistance within the bacteria, possibly due to inherent factors such as reduced drug accumulation, up-regulation of target enzymes, altered target sites, decreased target affinity for the drug, or counteraction of the drug's effect [41].

Table 1 Experimental pharmacological protocol
Genetic variations between microbial strains and plant sources may also contribute to this lack of antibacterial activity.Although some medicinal plants, such as Allium sativum (garlic), have shown activity against drug-resistant pathogens, our study did not observe similar effects [42].Furthermore, the absence of critical phytochemicals like terpenoids, known for their bitterness and toxicity against bacteria, may explain our study's lack of antibacterial activity [43].The concentration of these phytochemicals, along with alkaloids, saponins, flavonoids, and glycosides, can vary based on factors like geographical location, plant age, harvest timing, topographical elements, soil nutrient concentrations, extraction methods, and antimicrobial testing protocols [44].Consequently, meticulous scientific protocols should be followed, and additional investigation is essential to comprehend the reasons behind the observed absence of antibacterial activity against the test isolates in this study.
Finally, in this study, we also noted that when the fresh and dried leaf extracts of Momordica charantia and Vernonia amygdalina were combined with a standard drug, smaller mean zone diameters of inhibition were observed compared to the drug tested alone.These findings suggest a potential interaction between the plant extracts and the standard drug.Further investigation is needed to understand these contradictory results and explore the potential interaction between the plant extracts and standard drugs.Additional studies to elucidate the underlying mechanisms and implications of this interaction are very critical.This addition aims to prompt future research in this direction.

Conclusion
Although prior research has highlighted the antibacterial properties of V. amygdalina and M. charantia leaf extracts, emphasizing their substantial alkaloid, saponin, glycoside, and flavonoid content, our present study reveals a divergent outcome.This discovery underscores the imperative for further investigations

Figure 1
Figure 1 Picture of Momordica charantia plant

Figure 2
Figure 2 Vernonia amygdalina of 200 mg/mL of cold water extract of M. charantia 2 It received 0.1 mL of 200 mg/mL of hot water extract of M. charantia 3 It received 0.1 mL of 200 mg/ml of methanolic extract of M. charantia 4 It received Ciprofloxacin (Positive control) 5 It received sterile distilled water (Negative control) . These findings corroborate those of Jia et al., Rashid et al., and Mukherjee et al., except for terpenoids

Table 3 Antibacterial activities of extracts of fresh leaf of Momordica charantia on selected test isolates
Momordica charantia leaf; CAEMCL, cold aqueous extract of Momordica charantia leaf; MEMCL, methanolic extract of Momordica charantia leaf; CMECL, ciprofloxacin and methanolic extract of Momordica charantia leaf; PC, positive control; NC, negative control.

Table 4 Antibacterial activities of extracts of dried leaf of Momordica charantia on selected test isolates
Momordica charantia leaf; CAEMCL, cold aqueous extract of Vernonia amygdalina leaf; MEMCL, methanolic extract of Momordica charantia leaf; CMEMCL, ciprofloxacin and methanolic extract of Momordica charantia leaf; PC, positive control; NC, negative control.

Table 5 Antibacterial activities of extracts of fresh leaf of Vernonia amygdalina on selected test isolates
Vernonia amygdalina leaf; CAEVAL, cold aqueous extracts of Vernonia amygdalina leaf; MEVAL, methanolic extract of Vernonia amygdalina leaf; CMEVAL, ciprofloxacin and methanolic extract of Vernonia amygdalina leaf; PC, positive control; NC, negative control.

Table 7 Antibacterial activities of combined extracts of fresh leaves of Momordica charantia and Vernonia amygdalina on selected test isolates
Momordica charantia and Vernonia amygdalina leaves; CCAEMCVA, combined cold aqueous extract of Momordica charantia and Vernonia amygdalina leaves; CMEMCVA, combined methanolic extract of Momordic acharantia and Vernonia amygdalina leaves; PC, positive control; NC, negative control.