Antibacterial Activity of Two Bell Pepper Extracts: Capsicum annuum L. and Capsicum frutescens

Abstract

This study was conducted to determine extracts of fruit used as food ingredients and in traditional medicine for antibacterial activities. The effect of Capsicum annuum and Capsicum frutescens methanol and aqueous extracts on selected bacteria (Staphylococcus aureus, Salmonella typhimurium, Vibrio cholerae, Pseudomonas aeruginosa, Escherichia coli, and Shigella dysenteriae) were investigated. Both extracts were found to be effective against Vibrio cholerae, Staphylococcus aureus, and Salmonella typhimurium, while methanol extracts showed the greatest effect. The extract from Capsicum annuum showed a higher antibacterial activity than the one from Capsicum frutescens. The minimal inhibitory concentrations of methanol and aqueous extracts were 0.20 mg mL⁻¹ and 0.25 mg mL⁻¹, respectively. Minimal bactericidal concentrations values of both extracts ranged from 1 to 2.5 mg mL⁻¹. Phytochemical assay revealed the presence of alkaloids, flavonoids, polyphenols, and sterols. Thus, Capsicum fruits may serve as a source of natural bactericidal agents to be used in food and medicinal systems.

Keywords:

• Plant
• Minimum inhibitory concentration
• Pathogen
• Traditional medicine
• Phytochemical compounds
• Minimal bactericidal concentration

INTRODUCTION

Foodborne disease constitutes a public health concern for developed and developing countries.[1,2] Many developing countries use traditional medicine as the primary health care level. Many drugs currently used are economically expensive are not readily available in rural areas. The inconsistent use of these drugs could lead to microbial drug resistance. Medicinal plants contain large varieties of chemical substances which possess important therapeutic properties that can be used in the treatment of human diseases.[1] About 80% of individuals from developed countries use traditional medicine based on medicinal plants.[3]

The traditional Ivorian medicine uses a wide variety of plants to treat gastrointestinal disorders, such as diarrhea; these plants are rarely associated with mortality, but they cause significant morbidity, such as impaired physical and mental development.[4] In rural areas of Côte d'Ivoire, infection is treated with the most accessible medicine, which is traditional medicine. Among the therapeutic elements of this medicine, pepper is almost always cited.[5,6]

Besides the use of Bell pepper as a food ingredient, it is also used in traditional medicine especially treating symptoms such as stomach ache, diarrhoea, dysentery. Most gastroenteritis may be a result of contaminated food.[6,7] Foodborne gastroenteritis affects several million people yearly throughout the world. It is one of the main causes of mortality in infants, children, and the elderly.[8] Incidence of foodborne illnesses is still a major problem, even in developed countries. It has been estimated that 6 to 81 million cases of illnesses and up to 9000 annual deaths were attributed to foodborne pathogens in the USA alone. Salmonella spp, Listeria monocytogenes, Staphylococcus aureus, and Campylobacter jejuni were the main pathogens incriminated in poisoning cases.[9] In Côte d'Ivoire, gastroenteritis is the highest cause of mortality and morbidity. It is, therefore, important to investigate the potential use and antibacterial activities of peppers found in Côte d'Ivoire. To our knowledge, no data in this field is available. The purpose of this study was to investigate Bell pepper fruit (Capsicum) for potential antibacterial activities by bioassay screening.

MATERIALS AND METHODS

Plant Material

The plant materials were Capsicum annuum var Antillais and Capsicum frutescens var Soudanese fruit. These fruits were surveyed and selected to compile a representative list of the Bell pepper fruits mostly used by traditional healers of Côte d'Ivoire for their availability, accessibility, and wide utilization in food. These two Capsicum fruit varieties were identified by the Agency ANADER (National Agency for Rural Development) and confirmed by the national floristic center of the University of Cocody, Abidjan, Côte d'Ivoire. Plants were collected from three major markets in Abidjan: Koumassi, Treichville, and Adjamé. Capsicum annuum selected were fresh, ripened, and firm. Capsicum frutescens selected were dried because they were used in this form.

Test Microorganisms

The following microbial cultures were used in this study: Staphylococcus aureus, American Type Culture Collection (ATCC) 25913, Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella typhimurium ATCC 13311. Shigella dysenteriae and Vibrio cholerae are clinical sources from the National Public Health Laboratory (strains are confirmed by Paris Pasteur Institute), Abidjan, Côte d'Ivoire. All these microorganisms are important foodborne pathogens widely used in screening tests except for Pseudomonas aeruginosa, a nosocomial pathogen recommended by the National Committee for Clinical Laboratory Standards for antibacterial activity screening.[10]

Plant Extracts Preparation

Fresh, unblemished fruits were washed then oven dried for 48 h at 60°C, stored at room temperature, and ground to a fine powder. The aqueous extract was obtained by mixing 10 g of powder in 100 mL of boiling distilled water and then boiled for 15 min. The mixture was filtered through Whatman paper No. 2 (Whatman International, Maidstone, England) then oven evaporated at 50°C until completely dry. The dry extract was re-suspended in distilled water to obtain a final concentration of 100 mg mL⁻¹ of extract.[11]

The methanol extract was prepared by adding 60 mL of methanol to 10 g of fruit powder. The mixture was shaken for 15 min and left at room temperature (22°C ± 2°C). The supernatant was collected. The filtrate was again mixed to 60 mL methanol, shaken, and the supernatant extracted. This extraction process was repeated three times. The extract obtained was then filtered through Whatman paper No. 2. The filtrate was transferred into a vial and oven evaporated at 50°C until completely dry. The dry extract was re-suspended in distilled water to yield a final concentration of 100 mg mL⁻¹ extract.

Inoculums Preparation

Bacteria cultures were grown in Muller-Hinton broth over night then diluted with sterilized Muller-Hinton broth to obtain 0.5 (Marc-Farland) MCF of (Densimat ref. 99535 A version, Biomerieux). The diluted cultures were inoculated in Muller-Hinton agar and allowed to dry for 10 min at 22°C ± 2°C. Excess moisture was removed.

Antibacterial Activity Screening

Disk diffusion method

The disk-diffusion assay used was adapted from Taylor et al.[12] methods. Test disks were prepared by saturating sterilized blotting paper disks in fruits extracts. For methanol negative control, blotting paper disks were dipped into methanol. For positive control, tetracycline paper disks of 0.25 mg mL⁻¹ concentration were used. Disks were transferred onto the surface of Muller-Hinton agar inoculated with each test culture and incubated at 37°C for 18 to 24 h. After the incubation period, the inhibition around each disk was measured.

Well diffusion method

The standard well diffusion assay was used with some modifications.[13] Wells were formed with sterilized Pasteur pipette in Muller-Hinton agar plate inoculated with test organisms. For negative control, 100 μL of methanol was used. For positive control, 100 μL of 0.25 mg mL⁻¹ tetracycline solution was used. Then, 100 μL of each type of pepper fruit extract was aliquoted into the wells; all the plates were left for 3 h at 5°C for pre-diffusion before incubating at 37°C for 18 to 24 h. Results were recorded as presence or absence of the inhibition zone.[14] Inhibition zones (including the diameter of disc or well) were measured, and values above 10 mm were considered as active against bacteria.

Minimal Inhibitory Concentration (MIC) Determination

This assay was performed in Muller-Hinton broth. Serial dilutions of Capsicum extract 0.5, 0.25, 0.2, 0.125, 0.1, 0.0625, 0.05 0.02, 0.0125, and 0.006 mg mL⁻¹ were prepared to determine MIC across 10 dilutions. Two hundred μl of each extract dilution of Capsicum fruits was incorporated in 1800 μL of Mueller-Hinton broth inoculated with each test organism (0.5 McFarland) and homogenized. The bacterial growth controls (positive controls) were made without fruit extracts or commercial antibiotic (tetracycline). Purity control tests were prepared by using MH broth only. The tubes were incubated at 37°C for 18 to 24 h. Bacterial growth was indicated by turbidity. The absence of bacterial growth was interpreted as antibacterial activity. The MIC was taken as the lowest concentration of the extract in broth tube showing no visible bacterial growth.[15,16]

Minimal Bactericidal Concentration (MBC)

The MBC was determined by subculture of Mueller-Hinton broth tubes where no visible bacterial growth (negative tubes) was determined (no visible growth in the MIC assay). Then, 200 μL of each negative tube were incorporated in sterile 1800 μL of Mueller-Hinton broth and homogenized. The tubes were incubated at 37°C during 18 to 24 h. Bacterial growth was indicated by turbidity. The absence of bacterial grown was interpreted as antibacterial activity. The MBC was defined as the lowest concentration (mg L⁻¹) of the extract in a broth tube showing no visible bacterial growth. The MBC was taken as the concentration of the extract that did not show any growth on a new set of agar plates.[16,17] All these tests were repeated three times.

Phytochemical Analysis of the Plant Extract

A small portion of each powder was used for the phytochemical tests.[1,18] Test for tannins: Exactly 1 g of fruit powder was dissolved in 10 mL of distilled water and filtrate (Whatman filter paper No. 1). A blue coloration resulting from the addition of ferric chloride reagent to the filtrate indicated the presence of tannins in the pepper. Alkaloids were tested with exactly 1 g of fruit powder, which was dissolved in 5 mL of 1% HCl on a steam bath. One milliliter of the filtrate was treated with a few drops of Dragendorff's reagent. Turbidity or precipitation was taken as indicative of the presence of alkaloid. For flavonoids, about 0.2 g of fruit powder was dissolved in 2 mL of methanol and heated. A chip of magnesium metal was added to the mixture followed by the addition of a few drops of HCl. The occurrence of red or orange coloration was indicative of flavonoids.

Steroids were tested with about 0.2 g of fruit powder dissolved in 3 mL of chloroform and filtered. Concentrated H₂SO₄ was carefully added to the filtrate to lower layer. A reddish brown color at the interface was taken as positive for the steroid ring. Polyphenols were tested with exactly 1 g of fruit powder dissolved in 10 mL of distilled water or methanol and filtrate (Whatman filter paper No. 1). A blue or black coloration resulting from the addition of 2% ferric chloride reagent to the filtrate indicated the presence of polyphenols.

RESULTS AND DISCUSSION

Results

The Capsicum extracts were effective in inhibiting some of the test cultures and ineffective against others. Though the response was not uniform, all Capsicum extracts showed an activity against some bacterial strains (Staphylococcus aureus, Salmonella typhimurium, and Vibrio cholerae) used in this assay (Tables 1 and 2). Both methanol and aqueous extracts were found to be effective against Staphylococcus aureus, Salmonella typhimurium, and Vibrio cholerae. No inhibition zone was measured for Shigella dysenteriae, Pseudomonas aeruginosa, and E. coli.

Table 1 Antibacterial activity of Capsicum annuum Var Antillais aqueous and methanol extracts using the disk and the wells diffusion assay

	Staphylococcus	Salmonella	Shigella	Pseudomonas		Vibrio
Extracts	aureus	typhimurium	dysenteriae	aeruginosa	E. coli	cholerae
C. annuum Antillais
Aq. disk	11 ± 0^a	11 ± 1.4^a	–	–	–	12 ± 0^a
Met. disk	13 ± 0^a	14 ± 1.4^b	–	–	–	13 ± 0^a
Aq. well	14 ± 1.4^ab	11 ± 1.4^a	–	–	–	16 ± 0^b
Met. well	16 ± 1.4^b	16 ± 0^b	–	–	–	21 ± 1.4^c
Positive control
Disk	22 ± 0^bc	22 ± 0^c	20	–	27	30 ± 1.4^c
Well	24 ± 0^c	24 ± 0^c	20	–	27	30 ± 1.4^c
Negative control
Disk	–	–	–	–	–	–
Well	–	–	–	–	–	–
Negative results indicated by – included all assays in which no zone of inhibition (mm) appeared around the test extract disks. Aq. disk or/wells: aqueous extracts using the disk or wells diffusion assay; Met. disk or/wells: methanol extracts using the disk or wells diffusion assay.
Results with the same letter in the same column indicate no significant difference.

Table 2 Antibacterial activity of Capsicum frutescens Var Soudanais aqueous and methanol extracts using the disc and the wells diffusion assay

	Staphylococcal	Salmonella	Shigella	Pseudomonas		Vibrio
Extracts	aureus	typhimurium	dysenteriae	aeruginosa	E. coli	Cholerae
Capsicum frutescens Soudanais
Aq. disk	11 ± 1.4^a	12 ± 0^a	–	–	–	11 ± 1.4^a
Met. disk	12 ± 1.4^a	12 ± 1.4^a	–	–	–	12 ± 0^a
Aq. well	12 ± 0^a	12 ± 0^a	–	–	–	12 ± 0^a
Met. well	13 ± 0^a	12 ± 1.4^a	–	–	–	16 ± 01^d
Positive control
Disk	24 ± 0^d	23 ± 0^d	20	–	27	30 ± 0^c
Well	24 ± 0^d	23 ± 0^d	20	–	27	30 ± 0^c
Negative control
Disk	–	–	–	–	–	–
Well	–	–	–	–	–	–
Negative results indicated by – included all assays in which no zone of inhibition (mm) appeared around the test extract disks. Aq. disk or/wells: aqueous extracts using the disk or wells diffusion assay; Met. disk or/wells: methanol extracts using the disk or wells diffusion assay.
Results with the same letter in the same column indicate no significant difference.

In antibacterial activity screening, Capsicum annuum Var Antillais inhibition zone ranged from 11 to 14 mm for the disc diffusion assay and from 11 to 21 mm for the well assay, while that of C. frutescens was respectively from 11 to 12 mm and from 12 to 16 mm (Tables 1 and 2). V. cholerae was the most sensitive bacteria. Both extracts gave almost the same inhibition diameter but methanol extract showed a greater antimicrobial activity than the aqueous extract. Capsicum annuum Var Antillais extracts exhibited a stronger antibacterial activity against bacteria tested than C. Frutescens Var Soudanese. The standard well diffusion assay used in this study gave a higher inhibition diameter compared to the disc diffusion method.

The activity profile for MICs of fruit extracts (Table 3) appeared to be the same. The methanol extract showed a much lower MIC (0.2 mg mL⁻¹) for C. annuum and 0.25 mg mL⁻¹ for C. frutescens, indicating a strong antibacterial activity on V. cholerae a Gram negative non Enterobacteriaceae. MIC value for aqueous extract was 0.25 mg mL⁻¹ for both Capsicum species on V. cholerae. For other bacteria, MIC value was 0.5 mg mL⁻¹ for both extracts.

Table 3 Minimum inhibitory concentrations (MIC) of Capsicum annuum and Capsicum frutescens fruits extract

MIC (mg mL^−1)
Extracts	Staphylococcal aureus	Salmonella typhimurium	Shigella dysenteriae	Pseudomonas aeruginosa	E. coli	Vibrio cholerae
C. annuum Antillais
Aq. extr	0.5	0.5	–	–	–	0.25
Met. extr	0.5	0.5	–	–	–	0.20
C. frutescens Soudanais
Aq. extr	0.5	0.5	–	–	–	0.25
Met. extr	0.5	0.5	–	–	–	0.25
Negative control	–	–	–	–	–	–
Positive control	+	+	+	+	+	+
Positive results indicated by + show presence of bacterial growth. Negative results indicated by – show absence of bacterial growth.

The activity profile for minimum bactericidal concentration (MBC) of fruit extracts (Table 4) also appeared to be the same. MBCs values of both extracts ranged from 1 to 2.5 mg mL⁻¹. Both extracts gave almost the same minimum bactericidal concentrations but methanol extract showed a greater bactericidal activity than the aqueous extract. MBCs values were 1 to 2 mg mL⁻¹ on V. cholerae, whereas these values ranged from 2 to 2.5 mg mL⁻¹ against S. aureus and S. typhimurium. The results of phytochemical screening are listed in Table 5. Chemical tests showed the presence of flavonoids, alkaloids, steroids, and polyphenols in both extracts.

Table 4 Minimum bactericidal concentration (MBC) of Capsicum annuum and Capsicum frutescens fruits extracts

CMB (mg mL^−1)
Extracts	Staphylococcal aureus	Salmonella typhimurium	Vibrio cholera
C. annuum Antillais
Aq. extr	2	2	1
Met. extr	2	2	1
C. frutescens Soudanais
Aq. extr	2.5	2.5	2
Met. extr	2	2	1
Negative control	-	-	–
Negative results indicated by – show absence of bacterial growth.

Table 5 Phytochemical active compounds found in Capsicum annuum Var Antillais and Capsicum annuum var Soudanais

Bioactive compounds
	Tannins	Alkaloïds	Flavonoïds	Steroïds	Polyphenols
Capsicum annuumr Antillais	–	+	+	+	+
Capsicum frutescens Soudanais	–	+	+	+	+
–: Absence of compound; +: Presence of compound.

DISCUSSION

Bell pepper, such as Capsicum annuum and Capsicum frutescens, showed antibacterial activities against some foodborne bacteria. Results obtained in this study showed that V. cholerae was the most susceptible bacteria, probably due to the low pH of fruit.[19] Vibrio species can proliferate more readily in alkaline medium; therefore, the effect of acidity of both pepper extracts may have produced an unfavorable pH environment for their survival. These results agreed with those of Careaga et al.[20] These authors evaluated an extract from Capsicum bell pepper type against bacteria and reported varying degrees of inhibition against Salmonella typhimurium inoculated in raw beef meat. Dorantes et al.[21] reported that Capsicum extracts had an inhibitory effect on Salmonella typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus.

The antibacterial activities of pathogenic bacteria observed in this study confirmed results found by Dorantes et al.,[21] which showed that active compounds of Capsicum are soluble in both aqueous and isopropanol solvent extracts and had an antibacterial activity. These results agreed with those of Careaga et al.,[20] and Cichewicz and Thorpe.[22] Careaga et al.[20] recorded aqueous and isopropanol extracts from Capsicum to be effective against Salmonella and Pseudomonas aeruginosa in beef meat. Cichewicz and Thorpe[22] reported aqueous extract from fresh Capsicum species to exhibit varying degrees of inhibition against Bacillus, Salmonella, Streptococcus, and Clostridium sp. Dorantes et al.[5] found isopropanol extracts from Capsicum to exhibit antibacterial activity against Listeria monocytogenes, Staphylococcus aureus, Salmonella typhimurium, and Bacillus cereus. Razavi et al.[23] also showed high antibacterial effect from essential oil of Prangos ferulaceae (L.) Lindl against Bacillus cereus. Both methanol and aqueous extracts were not effective against S. dysenteriae and P. aeruginosa. However, Careaga et al.[20] showed an activity against Pseudomonas.

In this study, Capsicum annuum and Capsicum frutescens exhibited antibacterial activities against some pathogenic bacteria responsible of foodborne disease. Among the bacteria tested, V. cholerae was the most sensitive compared to the four Capsicum extracts. For the methanol extract, Capsicum annuum exhibited a stronger antimicrobial activity against the bacteria tested than Capsicum frutescens. With the disc diffusion method, no significant difference was observed between Capsicum annuum aqueous and methanol extracts inhibition diameter for S. aureus and Vibrio cholerae. However, significant difference was observed for Salmonella typhimurium. With regard to the well diffusion method, there was a significant difference between aqueous and methanol extracts on Vibrio cholerae. The inhibition diameters were, respectively, 16 mm and 21 mm. In all cases, methanol extracts of both plants exhibited higher antibacterial activity than aqueous extracts. This result agreed with that of Sagun et al.[24] who found methanol extracts of all the plants tested showing a higher activity than the other extracts investigated. Since both methanol and aqueous extracts were not effective against S. dysenteriae and P. aeruginosa, these two test organisms were not used for the minimum bactericidal concentration determination. The lowest concentration of extract producing more reduction of bacterial population is 1 mg mL⁻¹. V. cholerae was again the most sensitive bacterium.

In classifying the antibacterial activity as Gram positive or Gram negative, it would generally be expected that a much greater number be active against Gram positive than gram negative bacteria.[25] In the present findings, all extracts showed an activity against Gram positive bacteria (S. aureus). However, the number of bacteria used in screening has been restricted due to the limitation of resources. Results found in this study are in accordance with those of Szabo et al.[26] These authors showed antimicrobial activities of selected spice extracts against Staphylococus auereus (ATCC 25923) and Streptococcus pyogenes (ATCC 49399). Capsicum is a widely used plant in traditional medicine. Its fruits were used as popular household spices. It was found that fresh fruit were more effective than dried ones.[27] These findings support the traditional knowledge of local users and are a preliminary scientific validation for the use of these fruit extracts for their antibacterial activity.

The extract of Capsicum annuum showed encouraging and interesting results against bacterial activities because it is found to be active against Staphylococcus aureus, Salmonella typhimurium, and vibrio cholerae. These bacteria are responsible for stomach ache, diarrhea, and dysentery. Phytochemicals exert antimicrobial activity through different mechanisms; alkaloid, which is one of the largest groups of phytochemical in plants, was found in all Capsicum pepper extracts. One of the most common biological properties of alkaloids is their toxicity against cells of foreign organisms. These activities have been widely studied for their potential use in the elimination and reduction of human cancer cell lines.[28] In addition, alkaloids possess anti-inflammatory, anti-asthmatic, and anaphylactic properties with consequences to alter immunological status in vivo.[29,30] Furthermore, alkaloids have led to the development of powerful pain killer medications.[31]

Flavonoids are also constituents of extracts isolated from Capsicum annuum var Antillais and Capsicum frutescens var Soudanais tested. Flavonoids exhibit a wide range of biological activities that are antimicrobial, anti-inflammatory, anti-angionic, analgesic, anti-allergic effects, cytostatic and antioxidant properties.[32–34] Flavonoids are important for prevention of diseases associated with oxidative damage of membrane, protein, and DNA. Flavonoids in the human diet reduce the risk of various cancers, as well as preventing menopausal symptoms.[32] All these facts support the usefulness of Capsicum fruits in folklore remedies. It is also one of the reasons why these fruits are widely used for the treatment of many diseases among many tribes in Africa. In addition to the antimicrobial activities, flavonoids exhibit antitrypanosomal and antileishmanial activities.[35] Epidemiological studies suggest that the consumption of flavonoids is effective in lowering the risk of coronary heart disease. Furthermore, several flavonoids exhibit antiviral activities.[36] The presence of steroids and polyphenols has earlier been reported having antimicrobial activity. Polyphenols are another group that has exhibited antimicrobial activity.[33,37] Steroids are one of the largest and most diverse groups of secondary metabolites.

CONCLUSION

Extracts isolated from Bell peppers, such as Capsicum annuum and Capsicum frutescens, exhibit antibacterial activities against some bacterial pathogens responsible for foodborne disease. Alkaloids, flavonoids, polyphenols, and sterols were identified as biological active compounds. Thus, Capsicum fruits may serve as a source of natural antibacterial in food and medicinal systems. This article reports understanding of the use of pepper and it is a validation for the use of extracts of Capsicum fruits for antibacterial activity. These results should lend scientific credence to justify the use of pepper as a food ingredient or in traditional medicine. Awareness of communities should be enhanced incorporating the traditional knowledge with scientific findings.

ACKNOWLEDGMENTS

The authors are thankful to the Food Science Department of Abobo-Adjamé University for laboratory facilities, and the National Laboratory of Public Health for providing bacteria, chemical, and technical material support to conduct the research smoothly. The authors also wish to thank the International Journal of Food Properties editor responsible for handling the manuscript.