Phytochemical screening of Cocos nucifera And Vernonia amygdalina extract and antibiogram of the mixture (Cocos Nucifera + Vernonia Amygdalina) on Salmonella typhi and Streptococcus pyogenes

doi:10.21203/rs.3.rs-4458581/v2

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Phytochemical screening of Cocos nucifera And Vernonia amygdalina extract and antibiogram of the mixture (Cocos Nucifera + Vernonia Amygdalina) on Salmonella typhi and Streptococcus pyogenes

https://doi.org/10.21203/rs.3.rs-4458581/v2

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Background:Nigeria, a significant player in the global economy, has a rich agricultural heritage that includes diverse crops with potential medicinal properties. Among these, coconut (Cocos nucifera) and bitter leaf (Vernonia amygdalina) are notable for their traditional medicinal uses. This study aimed to evaluate the phytochemical composition and antibacterial efficacy of extracts from these plants, individually and in combination, against Salmonella typhi and Streptococcus pyogenes, addressing the need for alternative antibacterial therapies in the face of rising antibiotic resistance.

Methods: Bitter leaf and coconut were sourced from Bakindogo market, Kaduna, and authenticated by a botanist. Extracts were prepared and subjected to phytochemical screening using standard methods. Antibacterial activity was evaluated through agar disk diffusion to measure zones of inhibition at different extract concentrations. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined for both Salmonella typhi and Streptococcus pyogenes.Top of FormBottom of Form

Results:Phytochemical analysis revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, and carbohydrates in both extracts, with anthraquinones present only in Vernonia amygdalina. The extracts showed significant antibacterial activity, particularly the mixture of Vernonia amygdalina and Cocos nucifera water. The mixture exhibited higher efficacy against Salmonella typhi, with zones of inhibition ranging from 9.5 mm to 17 mm, compared to Streptococcus pyogenes, which showed inhibition zones from 9.0 mm to 13 mm. The MIC for Salmonella typhi was 25 mg/mL, with an MBC of 100 mg/mL, indicating bactericidal properties at higher concentrations. Streptococcus pyogenes showed an MIC of 100mg/ml, but the MBC was not applicable, suggesting bacteriostatic effects.

Conclusions:The study confirms the significant antibacterial potential of Vernonia amygdalina and Cocos nucifera water extracts, particularly against Salmonella typhi. These findings support their traditional use and highlight their potential as alternative antibacterial agents. Further research and pharmaceutical exploration are recommended to fully harness these plants' medicinal properties, particularly for developing new treatments for bacterial infections.

Drug Discovery, Design, & Development

General Microbiology

Vernonia amygdalina

Cocos nucifera

Phytochemical screening

Antibacterial activity

Salmonella typhi

Streptococcus pyogenes

Herbal remedies and Antibiotic resistance

Nigeria stands as a significant player in the global economy, recognized as the largest black nation. With a robust economic growth trajectory, Nigeria's gross domestic product (GDP) soared from $170 billion in 2000 to $451 billion in 2012, albeit unofficial estimations suggest figures closer to $630 billion, inclusive of the informal sector [1][2]. The agricultural sector has consistently been a pivotal contributor to Nigeria's economy, with a noteworthy 26.15% contribution to the GDP in 2017 (NBS, 2019). This sector serves as the cornerstone for economic advancement, development, and poverty alleviation, as emphasized by Sertoglu, Ugural, and Bekun [3], positioning agriculture as the catalyst for sustained economic prosperity.

In Nigeria, agriculture encompasses a diverse array of staple and cash crops, with significant cultivation dedicated to crops such as cassava, yam, maize, sorghum, rice, palm kernel, and coconut, among others [2]. Notably, coconut, revered as the "tree of life," holds immense economic potential, albeit receiving limited attention within Nigeria's agricultural landscape [4]. Originating from Southeast Asia, coconut exhibits remarkable adaptability, thriving across various soil types and climates. Its utility extends beyond traditional consumption, offering a plethora of industrial applications that could bolster Nigeria's industrial development and elevate living standards.

The historical narratives surrounding the origin of coconut are diverse and often steeped in mythology. While Indian mythology attributes the creation of the coconut palm to the sage Vishwamitra, botanical evidence suggests its origin in Papua New Guinea or Malesia, with subsequent dispersal facilitated by natural phenomena and Polynesian voyagers. This dispersion underscores coconut's resilience and capacity for widespread proliferation across diverse geographical regions [5].

Alongside coconut, bitter leaf (Vernonia amygdalina) emerges as a botanical asset in Nigeria's agricultural bounty. This perennial shrub, renowned for its bitter-tasting leaves, harbors extensive medicinal properties, earning it recognition as a staple herb in traditional medicine across West Africa. Indigenous to various ecological zones, bitter leaf thrives in diverse environments, adapting to habitats ranging from forest margins to grasslands.

The morphological characteristics of bitter leaf underscore its resilience and adaptability, with features such as soft woodiness, lanceolate leaves, and bisexual flowers contributing to its ecological versatility. Beyond its ecological significance, bitter leaf holds immense socio-economic value, particularly within Nigerian communities, where it is revered for its medicinal prowess and nutritional benefits a [6].

Amidst the backdrop of Nigeria's agricultural abundance, concerns surrounding food safety and security persist. Bacterial infections continue to pose significant challenges, necessitating innovative approaches to antimicrobial research. The emergence of multidrug-resistant pathogens underscores the urgency for alternative therapeutic interventions, including the exploration of traditional herbal remedies [7][8].

In light of these considerations, this study aims to conduct phytochemical screening of coconut water and bitter leaf extract, evaluating their antibacterial efficacy against Salmonella typhi and Streptococcus spp. By elucidating the phytochemical composition and antibiogram of this botanical mixture, we seek to contribute valuable insights into the potential therapeutic applications of traditional herbal remedies in combating bacterial infections.

In summary, this research endeavors to bridge the gap between traditional botanical knowledge and contemporary biomedical research, offering a pathway towards sustainable healthcare solutions within the context of Nigeria's rich agricultural heritage.

Collection, Identification, and Authentication of the Plants

Bitter leaf and coconut were procured from Bakindogo market in Kaduna. Their identification and authentication were carried out by a botanist from the Department of Biological Science at Kaduna State University. The plants were washed using sterile water and dried at room temperature in the Department of Applied Biology at Kaduna Polytechnic.

Preparation and Testing of the Mixture (Combination) of Bitter Leaf and Coconut Water

The bitter leaf was washed and then squeezed into coconut water. The mixture was left to soak for three days to allow maceration. After soaking for three days, the mixture of bitter leaf and coconut water was periodically shaken to ensure thorough extraction. The macerated mixture was then filtered to separate the micelle from the marc. The micelle was subsequently separated from the menstruum by evaporation using a water bath.

The Mixture Extract Concentration

Various concentrations of the mixture extract were prepared. A stock solution of 100 mg/mL was made by dissolving 1g of the crude extract in 10 mL of sterile distilled water. From this stock solution, different concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, and 12.5 mg/mL) were prepared accordingly.

Phytochemical Screening of Crude Extracts

The presence of bioactive compounds in the methanol extracts was determined by phytochemical tests using standard methods described by [9] and [10].

Test for Carbohydrates (Molisch's Test)

A few drops of Molisch's reagent were added to 2 mL of the extract solution, followed by the addition of 1 mL of concentrated sulfuric acid down the side of the test tube. Changes in coloration at the interface of the two liquids were noted.

Test for Tannins

A few drops of 0.1% ferric chloride solution were added to the extract solutions, and changes were observed.
Additionally, a few drops of lead acetate solution were added to the extract solutions, and changes were observed.

Test for Alkaloids

Meyer's reagent and Two drops of dilute sulfuric acid were added to 1 mL of the extract solution, and observed changes were noted.
Another 1 mL portion of the extract was reacted with Dragendorff's reagent, and changes in the product formed were observed and recorded.
An affirmation test for the presence of alkaloids was performed by adding two drops of Wagner's reagent, and observed changes were recorded.

Test for Saponins

A frothing test was performed by adding 5 mL of de-ionized water to the filtrate of the extracts and shaking vigorously to observe the formation of stable persistent frothing. This frothing was observed for up to 45 minutes.

Test for Flavonoids

Shinoda Test: Filtrates of the extracts (3 mL) were obtained, and four pieces of magnesium fillings were added, followed by a few drops of concentrated hydrochloric acid. Observed color changes were noted.
Sodium Hydroxide Test: 5 mL of 10% sodium hydroxide was added to an equal volume of the extracts. Observed color changes were noted accordingly.

Test for Terpenoids

Liebermann-Burchard Test: Chloroform was added to the extract, mixed with 1 mL of acetic anhydride, and followed by the addition of 1 mL of concentrated sulfuric acid down the wall of the test tube. Observed changes were noted.

Test for Anthraquinones

Bornträger’s Test: 0.5 g of crude extracts was added to 10 ml of chloroform, shaken, and then filtered. Ten ml of the filtrate was reacted with an equal volume of ammonia (10 mL) and shaken gently. Observed changes were recorded.

Characterization and Authentication of Test Organisms

Clinical isolates of Salmonella typhi and Streptococcus spp. were obtained from 44 Army Reference Hospital in Kaduna State, Nigeria.

Storage and Maintenance of Cultures

The cultures were streaked onto Nutrient Agar slants and incubated at 37°C for 48 hours. These cultures were stored in a refrigerator at 4°C for subsequent use. Stock cultures were prepared and kept until needed. Broth cultures were prepared from these organisms.

Preparation and Standardization of Inocula

Nutrient broth was prepared according to the manufacturer’s specifications. The bacterial isolates were tested for sterility on Nutrient Agar, then re-grown in Nutrient broth at 37°C for 24 hours. McFarland's standard method was used to standardize the organisms to 1x10⁴ cfu/mL by preparing four sets of dilution tubes with 9 mL of normal saline and one tube with 4 mL of normal saline, labeled 10⁻¹ to 10⁻⁴. 1 mL of the overnight culture was added to the 10⁻¹ dilution tube, and serial dilutions were prepared to arrive at a dilution of 10⁻⁴ cfu/mL. Dilution with sterile Nutrient broth was done until the optical density matched that of McFarland's standard.

Antimicrobial Susceptibility Test

Nutrient agar was prepared according to the manufacturer’s specifications, and the molten agar was poured into sterile disposable plates and allowed to cool to about 45°C. The plates were swabbed with diluted standardized overnight cultures. Six mm Whatmann No. 1 paper discs, each soaked in the extract concentrations (100 mg/mL, 50 mg/mL, 25 mg/mL, and 12.5 mg/mL), coconut water, and bitter leaf juice, were placed on the agar plates. A standard antibiotic (200 mg/10 mL) was used as a positive control, and sterile distilled water was used as a negative control on a separate plate. Diameters of zones of inhibition were measured after incubating the plates at 25°C for 48 hours. The plates were replicated in triplicate, and the mean zones of inhibition for each organism at each extract concentration were measured using a ruler and recorded.

Determination of Minimum Inhibitory Concentration (MIC)

2 ml of all the four-fold serial dilutions were used to impregnate sterile disks with a diameter of 5 mm. These impregnated disks were used to determine the MIC by placing them on the already streaked isolates of Salmonella typhi and Streptococcus pyogenes and incubated at 37°C for 24 hours. The zone of inhibition was measured using a ruler and recorded according to the concentration incorporated on the disk.

Determination of Minimum Bactericidal Concentration (MBC)

The MBC was determined by sub-culturing the prepared dilutions onto fresh Nutrient agar and further incubating for 24 hours at 37°C. These plates were observed for growth. The plates with the least concentration showing no growth were considered the MBC.

Phytochemical Constituents of Vernonia amygdalina, Cocos nucifera Water, and Their Mixture

Comparative phytochemical screening of Vernonia amygdalina, Cocos nucifera water, and their mixture revealed the presence of alkaloids, flavonoids, tannins, saponins, terpenoids, and carbohydrates in all samples. Anthraquinones were present only in Vernonia amygdalina.

Table 1: Phytochemical Composition of Bioactive Compounds

Phytochemical Components	Cocos nucifera Water	Vernonia amygdalina	Vernonia amygdalina and Cocos nucifera Water Mixture
Alkaloids	Present	Present	Present
Saponins	Present	Present	Present
Tannins	Present	Present	Present
Flavonoids	Present	Present	Present
Terpenoids	Present	Absent	Present
Anthraquinones	Absent	Present	Present
Carbohydrates	Present	Present	Present

Bacterial Susceptibility Standard of Amoxil

Table 2: Susceptibility Standard of Amoxil

R	I	S
≤13	14-16	≥17

R: Resistant I: Intermediate S: Susceptible

≤: less than or equal to ≥: greater than or equal to

Antimicrobial Activity of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes

The antibiogram of the mixture of Vernonia amygdalina and Cocos nucifera water was found to be effective against Salmonella typhi and Streptococcus pyogenes, showing zones of inhibition as summarized in Table 3.

Table 3: Antimicrobial Activity of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes

Test Organisms	Concentration (mg/ml) Zone of Inhibition (mm)				Positive control (500 mg/mL) Amoxicillin	Reaction
	100	50	25	12.5
1st isolate of S. typhi	17±0.1	12.5±0.5	12±0.1	9.5±0.5	18	S
2nd isolate of S. typhi	17±0.1	12.5±0.5	12±0.1	9.5±0.5	18	S
3rd isolate of S. typhi	17±0.1	12.5±0.5	12±0.1	9.5±0.5	18	S
4th isolate of S. typhi	17±0.1	12.5±0.5	12±0.1	9.5±0.5	18	S
1st isolate of S. pyogenes	13±0.1	13±0.1	11±0.5	9±0.1	17	S
2nd isolate of S. pyogenes	13±0.1	13±0.1	11±0.5	9±0.1	17	S
3rd isolate of S. pyogenes	13±0.1	13±0.1	11±0.5	9±0.1	17	S
4th isolate of S. pyogenes	13±0.1	13±0.1	11±0.5	9±0.1	17	S

S: Susceptible

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Mixture on Salmonella typhi and Streptococcus pyogenes

The MIC was determined using the agar disk diffusion and macro tube dilution methods, while the MBC was determined by sub-culturing the prepared dilutions into fresh media. The results in Table 4 show that the mixture of Vernonia amygdalina and Cocos nucifera water has significant antibacterial effects on Salmonella typhi at various concentrations. Streptococcus pyogenes showed partial sensitivity at 100 mg/ml.

Table 4: Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) of the Mixture of Vernonia amygdalina and Cocos nucifera Water on Salmonella typhi and Streptococcus pyogenes

Test Organisms	MIC (mg/ml)	MBC (mg/ml)
1st isolate of S. typhi	25	100
2nd isolate of S. typhi	25	100
3rd isolate of S. typhi	25	100
4th isolate of S. typhi	25	100
1st isolate of S. pyogenes	100	NA
2nd isolate of S. pyogenes	100	NA
3rd isolate of S. pyogenes	100	NA
4th isolate of S. pyogenes	100	NA

NA: Not applicable

Phytochemical screening of Vernonia amygdalina leaf extract and Cocos nucifera water (Table 1) revealed the presence of alkaloids, flavonoids, tannins, anthraquinones, saponins, terpenoids, and carbohydrates, while anthraquinones were present only in Vernonia amygdalina. Studies have shown that alkaloids and flavonoids are key compounds responsible for the antibacterial activities in higher plants [11]. Saponins, tannins, and alkaloids are well-documented plant metabolites known for their antimicrobial properties [12]. The presence of these phytochemicals in the mixture may contribute to its antibacterial activity, supporting their traditional use in treating various bacterial and fungal infections.

The antibacterial susceptibility test showed that all strains of the two clinical isolates, Salmonella typhi and Streptococcus pyogenes (Table 2), were highly susceptible to Amoxicillin (control). This observation aligns with the findings of Kassim et al. [13]. Salmonella typhi exhibited higher sensitivity to Amoxicillin with a zone of inhibition ranging from 18mm to 23mm, compared to Streptococcus pyogenes, which showed a range of 17mm to 20mm.

The mixture of Vernonia amygdalina and Cocos nucifera water showed a broad-spectrum antibacterial activity, with efficacy increasing with higher concentrations. Salmonella typhi strains were highly sensitive to the mixture, with zones of inhibition ranging from 9.5mm to 17mm at concentrations of 12.5 mg/ml to 100 mg/ml. Streptococcus pyogenes strains were less sensitive, showing zones of inhibition from 9.0mm to 13mm at the same concentrations.

The MIC and MBC tests revealed that the mixture had a good antibacterial effect on Salmonella typhi, with MIC and MBC values of 25 mg/ml and 100 mg/ml, respectively. For Streptococcus pyogenes, the MIC was 100 mg/ml, but the MBC was not applicable. These results suggest that the mixture is bacteriostatic at lower concentrations and bactericidal at higher concentrations. This supports the traditional use of large quantities of extracts by traditional practitioners for treating infections [15].

The study investigated the antibacterial activity and minimum inhibitory concentration of the antibiogram mixture of Vernonia amygdalina and Cocos nucifera water. Phytochemical analysis confirmed the presence of alkaloids, flavonoids, tannins, anthraquinones, saponins, terpenoids, and carbohydrates in both plants, with anthraquinones found only in Vernonia amygdalina. The mixture exhibited a high potential to inhibit Salmonella typhi but was less effective against Streptococcus pyogenes, supporting its traditional use in therapy. This study highlights the potential application of these plants as alternative antibacterial agents.

Based on the findings of this research, the following recommendations are made for the development of new antibacterial drugs:

1. Exploration by Pharmaceutical Companies: Pharmaceutical companies should investigate the antibacterial properties of the mixture of Vernonia amygdalina and Cocos nucifera water as a potential source of new antibacterial drugs.

2. Further Studies: Additional research should be conducted on these plants to uncover more of their potential in addressing gastrointestinal infections and other bacterial diseases.

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ETHICS APPROVAL AND CONSENT TO PARTICIPATE: In compliance with local guidelines, the collection of Vernonia amygdalina (Bitter leaf) and Cocos nucifera (Coconut) used in this study followed ethical standards and regulations. The plant material was collected from Bakindogo market in Kaduna, Nigeria. The identification and authentication of the plant were performed by a botanist in the Department of Biological Sciences, Kaduna State University.

Furthermore, it's important to note that the plant material used in this study was cultivated rather than wild. No endangered or protected species were involved in this research.

CONSENT FOR PUBLICATION: Not applicable

AVAILABILITY OF DATA AND MATERIALS: As for the data availability statement, I confirm that all data generated or analyzed during this study are included in the published article. This consists of the phytochemical composition of the Vernonia amygdalina (Bitter leaf), Cocos nucifera (Coconut) water and the combination of the two, antibacterial activity results, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) values.

COMPETING INTERESTS: The authors declare that they have no competing interests

FUNDING: This research was funded by the first author; no any organization or society fund the research.

AUTHORS' CONTRIBUTIONS: EOM carry out the experimental aspect of the research and also contributed to the write up, IAO supervised the experimental aspect and also contribute to the data presentation and the write up, AIU designed the study and also the major contributor in writing the manuscript data presentation and AAD also contributed by supervising the experimental aspect, analyzing the data and the write up.

ACKNOWLEDGEMENTS: Not applicable

The authors declare no competing interests.

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Phytochemical screening of Cocos nucifera And Vernonia amygdalina extract and antibiogram of the mixture (Cocos Nucifera + Vernonia Amygdalina) on Salmonella typhi and Streptococcus pyogenes

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Abstract

BACKGROUND

METHODS

RESULTS

DISCUSSION

CONCLUSION

REFERENCES

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