Antibacterial and Anticandidal Activity of Pomegranate Peel

 Original Article

Antibacterial and Anticandidal Activity of Ethanolic Extracts of Pomegranate (Punica granatum Linn) Peel, Sudan

Saga T. Ahmed¹, Abdelgadir A. Abdelgadir², Bakri Y. M. Nour³,  Asma H. Abuaisha⁴, Elhadi A. Ahmed^1*

 Abstract

Introduction: Pomegranate fruit is grown in Sudan and used in folk medicine to treat various diseases. This study aimed to assess the antimicrobial activity of the ethanol extract against common clinically important bacteria and Candida albicans.

Methods: Pomegranate (Punica granatum Linn) peel was purchased from the local market in Wad Medani, Gezira State. The extraction was carried out by alcoholic maceration and examined against standard strains of Staphylococcus aureus and Klebsiella pneumonae, and clinical isolates of Escherichia coli and Candida albicans. The well-diffusion method was used for antimicrobial testing.

Results: Different concentrations of extracts; 200, 100, 50, 25, 12.5, 6, 3 and 1.5 mg/ml, exhibited strong antibacterial and anticandidal activities. The maximum diameter of inhibition zones was observed against gram negative bacteria; Escherichia coli followed by Klebsiella pneumonae.

Conclusion: The current finding could highlight the Pomegranate fruit as a potential source of new antimicrobial agents.

Keywords: Pomegranate peel, Antibacterial, Anticandidal, Ethanolic extract.

________________________________________________________________________
1Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, University of Gezira, Sudan.
2Department of Pharmacognosy, Faculty of Pharmacy, University of Gezira, Wad Medani, Sudan.
3Department of Medical Parasitology, Faculty of Medical Laboratory Sciences, University of Gezira, Sudan
4Department of Community Medicine, Faculty of Medicine, Almughtaribeen University, Khartoum, Sudan

*Correspondent author: Elhadi A. Ahmed, Department of Medical Microbiology, Faculty of Medical Laboratory Sciences, University of Gezira, Sudan. Email: hadilabone@yahoo.com, hadilabone@uofg.edu.sd

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Introduction: 

Bacterial infections greatly impact human life, especially with the growing resistance of many of these microorganisms to antibiotics. Studies have shown that children’s deaths due to infections are mainly caused by methicillin-resistant Staphylococcus aureus and extended-spectrum beta-lactamase producers of gram-negative bacilli.(1) In recent decades, fungi have become another important cause of diseases, and Candida albicans was considered the most common fungal pathogen.(2) Treatment of fungal diseases is generally difficult due to the presence of a degree of similarity between fungi and human cells and for this reason, there are small numbers of antifungals available.(3) In addition, drug resistance by C. albicans has become a matter of concern.(4,5,6) In the current scenario of the emergence of multiple drug resistance of human pathogens, it is crucial to discover new antimicrobial agents with diverse chemical constituents, novel mechanisms of action against problematic infectious diseases and fewer side effects. Failure in treatment and increased drug resistance encouraged scientists to search for alternative medicinal plants, guided by community practices.(7) Many plants synthesize compounds that have vital and beneficial functions to humans and animals and may have defensive properties against microorganisms.(8)

 Punica granatum Linn belongs to the Punicaceae family and known as Pomegranate, has recently been described as the force fruit of nature and was used in folk medicine to treat many illnesses.(9) The specific components of this plant are multiple, including flavonoids, polyphenols, tannins and organic acids. It also has antioxidant, anti-inflammatory, anticancer and antiviral activities.(10) Methanol is the most common solvent used to extract pomegranate, other solutions used to a lesser degree include water, ethanol and acetone.(11) This study aimed to assess the antimicrobial activity of ethanol extract of Sudanese pomegranate peel against common clinically significant gram-positive and gram-negative bacteria and C. albicans.

Materials and Methods:

Study design 

Experimental laboratory-based study conducted at the Faculty of Medical Laboratory Sciences and Faculty of Pharmacy, University of Gezira, Wad Medani, Sudan.

Microorganisms studied

Laboratory strains of Staphylococcus aureus ATCC (25923) and Klebsiella pneumonae ATCC (13833); as well as clinical isolates of Escherichia coli and Candida albicans identified by Medical Laboratory, University of Gezira were used. Strains were sub-cultured and purified on Nutrient, MacConkey and Sabouraud's dextrose agars.

Plant preparation and extraction

Pomegranate fruit peels were purchased from the Wad Medani City’s local market, Gezira State. The peels were washed thoroughly with distilled water and then shade-dried for 4 days. The dried plant material (79.11 g) was powdered using an electric blender into a coarse powder. The powder was soaked in 500 ml of ethanol (70%) for 7 days with occasional shaking. The extract was then filtered and evaporated using rotary evaporator. The extract was stored in in a refrigerator in an air tight, dark glass bottle (Figure 1). Different concentrations of extract were prepared for testing as follow: 200 mg/ml, 100 mg/ml, 50 mg/ml, 25 mg/ml, 12.5 mg/ml, 6 mg/ml, 3 mg/ml and 1.5 mg/ml.

Figure 1: Process and extraction of pomegranate peel

        1: Pomegranate peel, 2: Dried materials, 3: Dissolved powder, 4: Extracts.

Preparation of McFarland standard turbidity (0.5) for microbial inoculums suspension

Barium sulfate was used to adjust the turbidity of the inoculums. It was prepared by adding a 1% v/v sulfuric acid solution to 1.17% w/v barium chloride solution. The turbidity standard was prepared by adding 0.6 ml of barium chloride to 99.4ml of the sulfuric acid solution.(12)

Antimicrobial sensitivity testing (Well diffusion method)

Bacterial colonies were suspended in sterile normal saline (0.9%) and modified to yield suspension containing about 1010 Colony Forming Units (CFU) (compared with 0.5 McFarland standard turbidity). About 25 ml of nutrient agar medium was poured into the plates and 200 μl of bacterial suspension was added to the agar plates. Plates were gently mixed to achieve equal distribution of bacteria in agar. After the complete solidification of the agar, four circular wells, 8 millimeters in diameter, were cut using a sterile cork borer. Two wells were filled with 200μl of the Ethanolic Extracts of Pomegranate Peel (EEPP/EPPE) while the other wells were filled with 100 μl of 50 μg/ml ceftriaxone as positive control and 100 μl of distilled water as negative control. The plates were left at room temperature for 40 min and then incubated in the upright position overnight at 35-37°C.(13, 14)

The Fungal strain was grown in Sabouraud's dextrose agar for 48 hours, then harvested and washed off with sterile normal saline to produce a suspension match to 0.5 McFarland standards containing approximately 2.5 x 106 CFU/ml. 

Anti-fungal standard 25 μg/ml Nystatin was used as a positive control.

Interpretation of results

Results were stated in diameters of inhibition zones in millimeters using the following standard: < 9 mm zone was considered inactive; 9-12 mm partially active while 13-18 mm was active and > 18 mm was very active. (15, 16)

Results:

Using an agar well-diffusion test, examined extracts showed varying levels of antimicrobial activities against the targeted microorganisms.

Figure2. Well-diffusion test: left, K. pneumoniae at 200 mg/ml EPPE, Right, Escherichia coli at 6mg/ml EPPE.

Figure 2 shows examples of the effectiveness of ethanolic extract on tested microorganisms with different concentrations. Note the large size of Inhibition Zone (IZ) when the highest concentration was used (200 mg/ml) against K. pneumoniae. On the other hand, note the small size of IZ when tested with a concentration of 6 mg/ml against another organism which is E. coli.

E. coli showed the highest susceptibility with a mean zone of inhibition of 34 mm at a 200 mg/ml concentration and 13 mm at a concentration of 1.5 mg/ml as shown in Table (1). This means that the EPPE is effective down to the lowest concentration used.

Table 1. Means of Inhibition Zones of Ethanolic Extract of Pomegranate Peel against E. coli

K. pneumoniae revealed a mean zone of inhibition of 28 mm at the highest concentration used showing effectiveness down to the 0concentration of 3 mg/ml

. (14 mm) as shown in Table (2)

Table 2. Means of Inhibition Zones of Ethanolic Extract of Pomegranate Peel against K. pneumoniae

S. aueeus revealed a mean zone of inhibition of 26 mm at the highest concentration used, which shows effectiveness down to the concentration of 3 mg/ml (14 mm) as shown in Table (3).

Table 3. Means of Inhibition Zones of Ethanolic Extract of Pomegranate Peel against S. aureus

C. albicans showed a mean inhibition zone of 32 mm at the highest concentration, which reveals effectiveness down to the concentration of 3 mg/ml. (Table 4).

Table 4. Means of Inhibition Zones of Ethanolic Extract of Pomegranate Peel against C. albicans

:Discussion

In recent years, drug resistance to human pathogenic bacteria has been commonly reported from all over the world, however, the situation is alarming in developing countries due to indiscriminate use of antibiotics, and therefore alternative antimicrobial strategies are urgently needed such as medicinal plants which could be a good source for a variety of drugs.(17) In this study, the crude ethanolic extract of peel of P. granatum was screened for antimicrobial activity against the common clinically important microorganisms; S. aureus, E. coli, K. pneumoniae and C. albicans using agar well diffusion technique. The most important finding is that the pomegranate extracts possessed broad-spectrum antimicrobial properties against gram-positive and gram-negative bacteria and fungi, and this was also reported by Oraki et al.(18) This unique property distinguished the pomegranate extracts from many other plants which exhibit activity against bacteria or fungi alone. Considering these results pomegranate peel could be experimented for disease treatment such as diarrhea, wound and skin infections, and respiratory illness which previously mentioned microorganisms may cause.

The current study used pomegranate peel ethanol extracts that inhibit the growth of different microorganism types with low concentrations, indicating that the active constituents are mainly water. All examined microorganisms in the study were inhibited by the EPPE at concentrations ranging from 200 to 3 mg/ml. The maximum diameter of inhibition zones was observed against E. coli followed by C. albicans, K. pneumonia and S. aureus. In general, alcoholic extracts of pomegranate peel exert show better activity against microorganisms when compared to other dissolvent such as aqueous and diethyl ether.(19, 20) This finding could help in studying and isolating the possible active substances. Also, another study in Egypt used disc-diffusion technique for testing gram-positive and negative bacteria and yeasts by extracts of P. granatum peel showed positive activity, which supports these findings.(21)

In addition to activity against opportunistic pathogens, primary pathogenic bacteria such as Typhoidal salmonellae and Shigella flexineri from Indian origin were also inhibited by ethanolic extracts of P. granatum peel.(22) Contrasting results on P. granatum peel activity against microorganisms were found by Sangeetha et al.(7), which showed that different extracts including those containing ethanol gave poor antibacterial activity, especially against gram-negative. These differences may in part be due to the different extraction methods employed, potentially the freshness of the fruit used, and variation in environment, soil, season and region of growth.(23)

Pomegranate peel is a rich source of tannins; a high phenolic compound, which many researchers believe is the reason for preventing the growth or killing of microorganisms.(24) Tannins have antimicrobial potential (for example, antiviral, antifungal and antibacterial activities) and it has been suggested that the antimicrobial activity of tannins may be due to the ability of tannin compounds to precipitate proteins, therefore causing leakage of cell membrane of the microorganism, and aiding cell lysis which ultimately leads to cell death, however, the levels of pomegranate peel compounds may vary among pomegranate cultivars which may result in differing levels of bioactivity.(25)‏ Since the exact component that causes the killing or inhibition of the growth of microorganisms has not yet been determined, the constituents of the ethanolic extracts should be considered when conducting identification and isolation studies because of their good results towards bacteria and fungi. This study showed high activity of ethanol extracts of the pomegranate peel part against tested microorganisms, and this is similar to the results of other studies that compared peel and other parts, e.g. seeds, juice, and whole fruits.(26) 

:Conclusion and Perspectives

Ethanolic extract of Punica granatum peel showed strong antimicrobial inhibitory effects and therefore, it could be targeted to isolate and identify promising antimicrobial agents. Furthermore, bio-guided assay fractionation and structure

 .elucidation should be carried out

Acknowledgement 

The authors extend their thanks to the staff of the Department of Medical Microbiology at the Faculty of Medical Laboratory Sciences and Faculty of

. Pharmacy, University of Gezira for their cooperation and technical assistance 

Conflict of interest 

.No conflict of interest to be disclosed

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