{"id":60909,"date":"2024-09-30T10:02:59","date_gmt":"2024-09-30T10:02:59","guid":{"rendered":"https:\/\/biomedpharmajournal.org\/?p=60909"},"modified":"2024-10-22T08:50:41","modified_gmt":"2024-10-22T08:50:41","slug":"evaluation-of-the-antibacterial-effects-of-the-punica-granatum-peels-extracts-against-some-pathogenic-bacteria-an-in-vivo-and-in-vitro-study","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol17no3\/evaluation-of-the-antibacterial-effects-of-the-punica-granatum-peels-extracts-against-some-pathogenic-bacteria-an-in-vivo-and-in-vitro-study\/","title":{"rendered":"Evaluation of the Antibacterial Effects of the Punica granatum Peels Extracts Against some Pathogenic Bacteria: An In vivo and In vitro Study"},"content":{"rendered":"\n

Introduction<\/strong><\/p>\n\n\n\n

Traditional herbal treatments have been used for centuries. Many people in third-world nations rely heavily on herbal treatments as their first line of defense against illness.1,2<\/sup> As bacteria develop resistance to chemical manufactured pharmaceuticals, scientists are looking for safer alternatives, such as herbal medicines and the active components they contain.3,6<\/sup><\/p>\n\n\n\n

Pomegranate, or Punica granatum<\/em> Linn, is a deciduous shrub or small Asian tree (growing to a height of 5\u20138 meters) of the family Punicaceae. It has been farmed for centuries throughout the Asia-Mediterranean area8<\/sup>, Europe, and Africa, although its natural habitat is Iran and northern India. Cancer, heart disease, diabetes mellitus, dental disease, bacterial infection, antibiotic resistance, sun-damaged skin, diarrhoea, bloody diarrhoea, and haemorrhoids are all treated using the bioactive chemicals found in Punica granatum<\/em>. Some forms of sore throat may also be alleviated by using P. granatum<\/em> as a mouth rinse. Flowers, trunk skin, fruits, roots, seeds, and even the plant itself have therapeutic use.7,9<\/sup><\/p>\n\n\n\n

Flowers of the Punica granatum<\/em> are red or reddish in colour, with a diameter of 3.5 to 7 cm, and may be campanulate or cylindrical in shape. You may find both fertilised and unfertilized flowers. The unfertilized flower has tiny, sterile petals that are short in style and short in stamens, and the stigma is located low on the flower, below the anthers. In Iranian alternative medicine, the unfertilized blooms are called “Golnar”.10<\/sup><\/p>\n\n\n\n

Pomegranate tea may be used to heal sore throats and oral irritation, while the skin and roots of the trunk are effective against parasite infections and diarrhoea, including bloody diarrhoea. In ancient Greek medicine, pomegranate was used as a hemostatic agent and to treat diabetic mellitus.11,12<\/sup><\/p>\n\n\n\n

P. granatum’s<\/em> antibacterial activities have only lately been recognized.13,15<\/sup> P. granatum has potent antibacterial activities against Gram-positive and Gram-negative nonoral microorganisms when extracted in ethanol, water, methanol, and acetone.16,17<\/sup> However, only a few research14,17<\/sup> have examined the effectiveness of this plant’s antibacterial activities against oral bacteria.<\/p>\n\n\n\n

Nevertheless, the aforementioned research is quite small and mostly lacks a suitable microbiological technique. Frequently, their procedures lack clarity or provide insufficient explanation. One research on the effect of P. granatum<\/em> flower (petal) water extract on oral microbial pathogens was found after searching the literature, and it mostly dealt with S. sanguinis.(18)<\/sup> On the other hand, Menezes et al.19<\/sup> found that P. granatum<\/em> hydroalcoholic extract was very efficient against biofilm-forming microbes in patients’ mouths.<\/p>\n\n\n\n

This study\nconducted for evaluation of antibacterial effects of Punica granatum<\/em>\nextract against some pathogenic bacteria.<\/p>\n\n\n\n

Materials and Methods<\/strong><\/p>\n\n\n\n

Staphylococcus aureus<\/em> and Escherichia coli<\/em> samples were collected from 130 patients with skin infections in Baghdad, Iraq, between the ages of 15 and 60 over the course of a year. Each and every isolate was positively identified by means of the morphological, cultural, and biochemical assays detailed in reference.14<\/sup><\/p>\n\n\n\n

After air drying and powdering the P. granatum<\/em> peels, 25g were extracted using 500mL of water and ethanol on Soxhlet equipment for 72 hours. The contents of the flasks were cooled, then filtered, concentrated at 40 degrees Celsius to get the crude extract, and then kept at four degrees centigrade in dark vials until use.16<\/sup><\/p>\n\n\n\n

Following the procedures outlined in (17)\nand (18), we tested for the presence of flavonoids, alkaloids, glycosides, tannins,\nas well as steroidal terpenes.<\/p>\n\n\n\n

The efficacy was calculated using well\ndiffusion techniques using Muller Hinton Agar (MHA). The plates were injected\nwith a suspension of the test isolates that had been standardised against\nMcFarland tube 0.5. After drying the plates for 30 minutes at 37o<\/sup>C\nin the incubator, a sterile standard core borer was used to evenly space out\nfive wells, each measuring five millimetres in diameter. To stop the extract\nfrom leaking out from beneath the agar, the well bottoms were sealed with\nsterile molten nutritional agar. The aqueous and ethanolic crude extracts dissolved\nin DMSO served as positive controls, while sterile water and 10% DMSO served as\nnegative controls. Each extract was diluted to a final concentration of 50,\n100, or 200mg\/ml, and 25 ml was added to the appropriate well on the infected\nplate.<\/p>\n\n\n\n

The plates were then incubated for 24 hours at 37 degrees Celsius. A millimeter-calibrated ruler was used to assess the size of the resultant inhibitory zones. The zone of inhibition of the test microorganisms at that dose was calculated as the mean of three measuremens. 19,20<\/sup><\/p>\n\n\n\n

For in vivo<\/em> study, experiment made\nuse of thirty male rats ranging in age from 6 to 8 weeks, weighing 180-220gm.\nAll animals were provided with water and commercial solid food ad libitum<\/em>.\nThese were divided into 3 groups (each group contain 10 animals):<\/p>\n\n\n\n

G1 control negative<\/p>\n\n\n\n

G2 control positive for E. coli<\/em> 1\u00d7108\n<\/sup>CFU\/ml<\/p>\n\n\n\n

G3 oral infection by E. coli<\/em> 1\u00d7108\n<\/sup>CFU\/ml then treated by orally administered 5\u2009mg (using gavage needle) of\nextract (21).<\/p>\n\n\n\n

Numbers of bacteria per gramme of faeces\nwere assessed by collecting faecal samples 0, 1, 2, 3, 4, 5, and 6 days after\nthe bacterial suspensions were provided. Duplicate MacConkey agar plates\n(Difco) were incubated overnight at 37\u00b0C after receiving 100\u2009\u03bcl aliquots of\nfaecal suspensions that had been serially diluted in PBS. using the counting of\ntypical colonies on plates containing 30\u2013300 colonies. <\/p>\n\n\n\n

Statistical\nanalysis was done by using SPSS version 23. <\/p>\n\n\n\n

Results and Discussion<\/strong><\/p>\n\n\n\n

   Clinical isolates of E. coli<\/em> and S.\naureus<\/em> were inhibited by pomegranate extracts at concentration of 200mg\/ml\nas compared with other concentration and this extract concentration was showed non-significant\ndifference with chloramphenicol (P<0.01), as shown in Table 1. <\/p>\n\n\n\n

Table 1: Comparison between different concentrations of extract with chloramphenicol <\/strong><\/p>\n\n\n\n\n\n\n\n
\n

Bacteria<\/strong><\/p>\n<\/td>\n

\n

50 mg\/ml<\/strong><\/p>\n<\/td>\n

\n

100 mg\/ml<\/strong><\/p>\n<\/td>\n

\n

200 mg\/ml<\/strong><\/p>\n<\/td>\n

\n

Chloramphenicol 0.03 mg<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

S. aureus <\/em><\/p>\n<\/td>\n

\n

3.4\u00b10.4<\/p>\n<\/td>\n

\n

4.7\u00b10.9<\/p>\n<\/td>\n

\n

12.5\u00b11.2<\/p>\n<\/td>\n

\n

14.6\u00b12.3<\/p>\n<\/td>\n<\/tr>\n

\n

E. coli <\/em><\/p>\n<\/td>\n

\n

4.2\u00b10.8<\/p>\n<\/td>\n

\n

5.3\u00b10.1<\/p>\n<\/td>\n

\n

13.7\u00b12.4<\/p>\n<\/td>\n

\n

15.2\u00b11.7<\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n\n\n
\"\"<\/td>\n\n

Figure 1: Comparison between different concentrations of extract with chloramphenicol<\/strong><\/p>\n
\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n

Table 2\nshows that there were significant effects of extract treatment on mortality and\nthe quantity of live E. coli bacteria retrieved from faeces. On the first day\nafter infection, ten rats from the G2 and G3 groups were observed to excrete\nviable E. coli bacteria in their faeces. The G2 group’s faeces contained\nbacteria at a concentration of 103-6<\/sup> CFU\u2009g\u22121, while the G3 group’s\nfaeces contained bacteria at a concentration of 102-3 <\/sup>CFU\u2009g\u22121. Also,\nat 6 days after injection, no mice in group G3 had perished, whereas all five\nrats in group G2 had died.<\/p>\n\n\n\n

Table 2: Effects of treatment with pomegranate extract on fecal shedding of\u00a0E. coli<\/em>\u00a0(CFU g\u22121<\/sup>) by rats.<\/strong><\/p>\n\n\n\n\n\n\n\n\n\n\n\n\n\n\n
\n

 <\/p>\n<\/td>\n

\n

Day 0<\/strong><\/p>\n<\/td>\n

\n

Day 1<\/strong><\/p>\n<\/td>\n

\n

Day 2<\/strong><\/p>\n<\/td>\n

\n

Day 3<\/strong><\/p>\n<\/td>\n

\n

Day 4<\/strong><\/p>\n<\/td>\n

\n

Day 5<\/strong><\/p>\n<\/td>\n

\n

Day 6<\/strong><\/p>\n<\/td>\n<\/tr>\n

\n

G2-1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

3 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n<\/tr>\n

\n

G2-2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

2 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

5 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

7 \u00d7 105<\/sup><\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n<\/tr>\n

\n

G2-3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

3 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 105<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 105<\/sup><\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n<\/tr>\n

\n

G2-4<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

7 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 104<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 106<\/sup><\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n<\/tr>\n

\n

G2-5<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

4 \u00d7 105<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 105<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 105<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 106<\/sup><\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n

\n

Death<\/p>\n<\/td>\n<\/tr>\n

\n

G3-1<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

6 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n<\/tr>\n

\n

G3-2<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

2 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 103<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

G3-3<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

2 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

8 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 102<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

G3-4<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

1 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 102<\/sup><\/p>\n<\/td>\n<\/tr>\n

\n

G3-5<\/p>\n<\/td>\n

\n

0<\/p>\n<\/td>\n

\n

4 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

8 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 103<\/sup><\/p>\n<\/td>\n

\n

2 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

3 \u00d7 102<\/sup><\/p>\n<\/td>\n

\n

1 \u00d7 102<\/sup><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n

Pomegranate peel extract was shown to have the highest antibacterial activity 21<\/sup> among the alcoholic extracts of pomegranate seed, fruit, peel, and juice tested against several bacterium strains, including S. aureus<\/em>. Methanolic pomegranate peel extract, as revealed by 18<\/sup>, is more effective against gram-positive bacteria than against gram-negative bacteria. Because of this, methanolic extracts of both sour and sweet pomegranate peels are very effective against S. aureus<\/em> 18<\/sup>. Due to its effective antibacterial activity against S. aureus<\/em> 22<\/sup>, pomegranate peel extract was recommended by 22<\/sup> for use in popular chicken meat items in order to extend their shelf life.<\/p>\n\n\n\n

The content\nof plant extracts varies, which explains why bacteria’s reactions to them vary.\nTo test the antibacterial effects of pomegranate peels against P. aeruginosa<\/em>\nand S. aureus<\/em> (23), (16) extracted them at room temperature using\nseveral polar solvents.<\/p>\n\n\n\n

The minimum inhibitory concentrations (MICs) for active pomegranate extracts against the investigated bacteria, including S. aureus<\/em> and P. aeruginosa<\/em>, were reported by Duman et al. to vary from 40 to >90 g\/ mL (24)<\/sup>. Pomegranate extracts suppress or postpone S. aureus<\/em> development at doses ranging from 0.01 to 1% v\/v 25.<\/sup> <\/p>\n\n\n\n

Antimicrobial properties of pomegranate water, ethanolic, and butanolic extracts against Escherichia coli<\/em>, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus<\/em> have been described in (26,27). Antimicrobial activity (inhibition zone) against S. aureus<\/em> and P. aeruginosa<\/em> was shown to be significantly greater in pomegranate peel fractions than in the control group28.<\/sup><\/p>\n\n\n\n

\u00a0\u00a0 Recently observed that pomegranate fruit extracts exhibit potent antibacterial activity in vitro<\/em> against a wide range of bacteria, including E. coli<\/em>, S. aureus<\/em>, Enterobacter spp<\/em>., Bacillus <\/em>spp., and Micrococcus<\/em> spp. According to research by 29<\/sup>, pomegranate extracts in chloroform, ethanol, and water were very effective against E. coli<\/em> O157:H7.<\/p>\n\n\n\n

Variation in extract composition is a significant contributor to MIC variation 30<\/sup>. Location, harvest time, plant age, growth stage, drying procedure, and extraction method all have a role in determining the extract’s chemical make-up 31,32,33,34.<\/sup><\/p>\n\n\n\n

Many authors revealed an antibacterial effect of pomegranate on E. coli<\/em> in vivo<\/em>, the effects of P. granatum<\/em> peel extract was the primary focus of this investigation. These actions alter plasma membrane permeability, increase cell protein concentrations, and ultimately cause cell death 35<\/sup>. While research on P. granatum<\/em> derivatives’ antibacterial activity has been well-documented 36,37 <\/sup>, the impact on in vivo<\/em> circumstances is less well understood.
<\/p>\n\n\n\n\n\n
\"\"<\/td>\n\n

Figure 2: Gross appearance of S. aureus<\/em><\/strong><\/p>\n
\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n\n\n\n\n
\"\"<\/td>\n\n

Figure 3: Gross appearance of <\/strong>E. coli<\/em><\/strong><\/p>\n
\n

\u00a0<\/p>\n

Click here to view Figure<\/a><\/p>\n<\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

\u00a0<\/p>\n\n\n

Conclusion<\/strong><\/p>\n\n\n\n

In conclusion, pomegranate peel extracts\nexhibit significant antibacterial activity against E. coli<\/em> and S.\naureus<\/em>, particularly at a concentration of 200 mg\/mL, where they perform\ncomparably to chloramphenicol. The in vivo<\/em> study further demonstrated\nthat rats receiving the extract showed reduced bacterial load in feces and\nbetter survival rates compared to the untreated control group. These findings\nunderscore the potential of pomegranate peel extracts as an effective\nantibacterial agent and suggest their utility in controlling bacterial\ninfections and enhancing survival in clinical or experimental settings.<\/p>\n\n\n\n

Acknowledgement<\/strong><\/p>\n\n\n\n

We acknowledge\nthe assistance we received from the members of the National University of\nScience and Technology ( NUST), College of Pharmacy<\/p>\n\n\n\n

Conflict of Interest<\/strong><\/p>\n\n\n\n

The authors do not\nhave any conflict of interest <\/strong><\/p>\n\n\n\n

Funding Sources<\/strong><\/p>\n\n\n\n

The author(s) received no financial support for the research, authorship, and\/or publication of this article <\/strong><\/p>\n\n\n\n

Data Availability Statement<\/strong><\/p>\n\n\n\n

This statement does not apply to this article.<\/p>\n\n\n\n

Ethics Statement<\/strong><\/p>\n\n\n\n

This research did not involve human participants, animal subjects, or any material that requires ethical approval.<\/p>\n\n\n\n

References<\/strong><\/p>\n\n\n\n

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