INTRODUCTION:

Nowadays, the global reliance on medicinal plants as a source of natural plants for treating a wide range of diseases has significantly increased due to the abundant traditional medicine systems found worldwide¹^,². According to the World Health Organisation (WHO), almost 80% of the global population depends on traditional medicine, primarily derived from natural plants, for their main healthcare requirements³. Hence, medicinal plants have been recommended as a rich supplier of phytochemical substances with a variety of biological properties⁴^,⁵.

Phytochemicals in plants exhibit a remarkable diversity, presenting a wide range of health-enhancing attributes, anti-inflammatory, cardiovascular protective, neuroprotective, and anticancer effect⁶^,⁷. Furthermore, medicinal plants comprise numerous essential phytochemical components, including terpenoids, flavonoids, cardiac glycosides, alkaloids, saponins, tannins, and steroids⁸. Among these polyphenol compounds are flavonoids and phenolic acids, which are characterized by aromatic rings and hydroxyl groups⁹^,¹⁰. Extracting these valuable compounds involves several techniques: solvent extraction, supercritical fluid extraction, maceration, and solid-phase extraction to ensure efficient concentration while minimizing unwanted substances that are essential in potential therapeutic, nutritional, and industrial applications¹¹^,¹².

Polyphenols have been shown to have promising results as anticancer medicines in recent years due to their many beneficial properties. They are helpful in cancer treatment because they protect healthy cells from harm while simultaneously killing malignant cells¹²^,¹³. In carcinogenesis, polyphenols regulate growth factor-receptor connections and cell signalling cascades, leading to cell cycle arrest, altered cell survival, and apoptosis¹⁴. Moreover, their pro-oxidative action induces apoptosis, dependent on concentration, target molecules, and conditions of the surrounding environment, while exhibiting anti-oxidative properties¹⁵. Additionally, polyphenols interact differently in healthy and cancerous cells, and they also support the immune system by inhibiting angiogenesis and acting as anti-inflammatory agents. Polyphenols decrease cell adhesiveness and invasiveness in the last stages of cancer, reducing metastatic potential¹⁴^,¹⁶.

Among numerous plant sources, pomegranate (Punica granatum L.) is a remarkable, colorful, and essential medicinal plant¹⁷. Pomegranate is a fruit high in bioactive substances and has a biowaste that can be processed into valuable products⁵^,¹⁸. Nevertheless, the peel constitutes around 60% of its total pomegranate fruit weight and possesses a greater concentration of total phenolic and antioxidant activity content in comparison to the pulp, flower, leaf, and seed. Furthermore, it demonstrates antibacterial and antifungal characteristics^19-21. Moreover, pomegranate botanical parts show potent anticancer properties, offering a perspective for treating chronic conditions like breast, colon, and prostate cancers, skin cancers, and stomach ulcers²².

This study is designed to estimate the phytochemical content of pomegranate peel aqueous and ethanolic extracts as well as their antimicrobial and anticancer effects.

MATERIALS AND METHODS:

Raw materials:

The pomegranate fruits (Punica granatum L.) were purchased from the Horticultural Research Institute, Agriculture Research Center, Giza, Egypt. The pomegranate fruit was cleaned with distilled water, followed by a manual peeling procedure using a sharp knife. the peels had been washed, dried in a 50°C oven, pulverized using a pestle and mortar, a high-speed laboratory mixer, and sieved with a mesh size of 20 to 30 to produce a finely separated powder²³.

Chemicals and reagents:

Gallic acid, the Folin-Ciocalteu reagent, 2.2-diphenyl-1-1picryl-hydrazyl (DPPH), quercetin muller hinton agar, peptone water, and nutrient broth were acquired from Sigma-Aldrich Chime in Stein, Germany. All solvents and chemicals were obtained from MERCK, USA.

Preparation of plant extracts:

Aqueous extract:

100g of powdered pomegranate peel had been soaked twice in 1000ml cold water at room temperature for 24 hours with constant stirring. The extract was filtered using Muslin and Whatman No. 1 filter paper²⁴^,²⁵.

Ethanolic extract:

Three times, 100grams of pomegranate peel powder were macerated in 80% (v/v) ethanol at the room temperature for tow hours, then refrigerated at 4±1˚C for 24hours. Both extracts are condensed using a Stuart Rotary Evaporator (Model RE300) at 40±1°C, freeze-dried (Snijders Scientific model 2040), and The lyophilized preparations were held at 40±1°C until use²⁶^,²⁷.

Determination of total phenolic and flavonoid component:

Total amount of phenolic component contents has been measured by the Folin-Ciocalteau reagent, as described in²⁸. The total amount of flavonoid was assessed utilizing the method published by Jia et al.²⁹.

Fractionation and identification of phenolic and flavonoid components:

The extracts had been separated into fractions and the phenolic and flavonoid components determined using HPLC in accordance with the procedure outlined by (Goupy et al.and Loon et al.). Data analysis utilizing Hewlett Packard Software was utilized to calculate component concentrations based on retention duration and peak area³⁰^,³¹.

Determination of antioxidant activity:

In accordance with Brand-Williams et al.³² colorimetric technique, the antioxidant activity of samples has been measured by ability to scavenge 2, 2'-Diphenyl-1-picrylhydrazyl (DPPH) radicals. Using the Yen and Duh methodology, the percentage of the samples that inhibited the DPPH radical was calculated³³.

Inhibition% = (Ac (0) – AA (t)) / Ac (0) × 100

Where: Ac (0) is the control absorbance at time = 0 min. AA (t) is the antioxidant absorbance at time =1hr.

Antibacterial activity:

In accordance with the protocol described by Patel et al.³⁴. The antimicrobial activity against Escherichia coli and Salmonella typhimurium as gram-negative. Moreover, By determining the inhibition zone's diameter, Staphylococcus aureus and Bacillus subtilis have been assessed as gram-positive bacteria³⁵.

Antifungal activity:

The potato dextrose agar well diffusion assay determined the antifungal efficacy of pomegranate peel aqueous and ethanolic extracts. Every twenty-four hours, the inhibition zone (mm) has been measured to evaluate the antifungal activity against Aspergillus flavus and Candida albicans³⁶.

Cytotoxic and antitumor activities of selected plant extracts:

The cytotoxicity or anticancer efficacy of both ethanolic and aqueous extracts of pomegranate peel was evaluated using the MTT test on two tumor cell lines cell lines of tumors (HT-29 colonic tumor cells and HepG2 liver cancer cells) from the National Cancer Institute Laboratories at Cairo University in Egypt. Spectrophotometric analysis was used to determine the degree of MTT inhibition, and cytotoxicity was reported as the 50% inhibitory concentration (IC₅₀) of various extracts on cell growth. The IC₅₀ values were calculated. (GraphPad Software, S. Diego, CA, USA).

Apoptosis and necrosis cells assay:

The study used the Annexin V-FITC apoptosis detecting kit with two fluorescent channels flow cytometry to assay apoptosis and necrosis cells. ACEA Novo Express™ software (ACEA Biosciences Inc., San Diego, CA, USA) and quadrant analysis were used to count the quantity of FITC and/or PI-positive cells.

Reading (Biological Significance of each phase):

Early apoptosis phase (Q4), Late apoptosis phase (Q2), Necrosis phase (Q1).

Statistical analysis:

In order to analyze the data, SPSS version 22 was employed. Data was displayed as mean ± SD. ANOVA in one direction and an LSD post hoc test were used. Using the Pearson correlation coefficient, the correlations between the variables were determined. Significant P-values were defined as less than 0.05.

RESULTS:

Total phenolic and flavonoid contents of pomegranate peel and its extracts

Table (1) presents the results of an analysis of the total phenolic and flavonoid content of pomegranate peel powder as well as its aqueous and ethanol extracts.

Table 1: Total phenolic and flavonoid contents of pomegranate peel powder and its extract

Samples		Total phenolic (mg/g)	Total flavonoids (mg/g)
Pomegranate peels	Powder	9.93^f± 0.16	2.75^g±0.15
	Ethanol	65.60^a± 0.19	35.32^b±0.22
	Aqueous	33.63^bc±0.20	22.72^d±0.15

The different letters show statistically different means according to LSD post hoc test.

DPPH antioxidant activity of pomegranate peel and extracts:

The results of the current study, which used DPPH to assess the antioxidant activity of pomegranate peel powder and its ethanolic and water extract, are shown in Table (2).

Table 2: DPPH antioxidant activity of pomegranate peel and extracts (%)

Samples

Powder

Ethanol

Aqueous

p-value

Antioxidant activity % (DPPH)

83.22^b± 0.77

95.36^a±

0.60

57.22^c± 0.39

P <0.001

According to the LSD post hoc test, the different letters show that the means are statistically different.

HPLC assay of pomegranate peel and extract phenolic compounds:

HPLC was used to separate and identify the phenolic components in pomegranate peel powder and its extracts, and Table (3) shows the data.

Table 3: HPLC assay of pomegranate peel and extract phenolic compounds

Phenolic compounds mg/100g	Pomegranate peels
Phenolic compounds mg/100g	Powder	Ethanol	Aqueous
Pyrogallol	192.61	238.0	434.7
Gallic acid	21.73	1063.62	124.21
Catechol	2.58	138.42	146.15
4-Aminobenzoic acid	15.27	75.28	53.59
Catechin	41.43	300.4	116.53
Chlorogenic acid	29.81	187.35	107.18
Benzoic acid	20.54	284.89	99.3
Caffeic acid	5.54	23.07	31.42
Ferulic acid	8.67	26.86	22.02
Salicylic acid	11.8	45.88	30.07
Ellagic acid	6.39	815.16	285.37
Coumarin	13.01	34.02	21.94
Caffeine	7.6	56.63	55.83
Protocatechuic acid	1.15	68.01	35.21
p coumaric	7.33	12.31	9.82
Iso-Ferulic acid	10.1	18.49	13.76
α Coumaric	3.25	6.65	4.98
3,4,5-methoxy-cinnamic	13.88	73.59	33.64
Epi-catechine	99.3	120.75	109.57
Total	511.99	3589.38	1735.29

HPLC assay of pomegranate peel and extract flavonoid compounds:

As shown in Table (4), pomegranate peel powder and its extracts' flavonoid components were isolated and identified using HPLC.

Table 4: HPLC assay of pomegranate peel and extract flavonoid compounds

Flavonoid compounds mg/100g	Pomegranate peels
Flavonoid compounds mg/100g	Powder	Ethanol	Aqueous
Apigenin 6-arabinose 8-galactose	5.66	40.98	29.22
Hesperidin	102.69	1807.5	1050.6
Rosmarinic	6.45	39.6	33.54
Rutin	9.34	97.09	70.96
Apigenin 7-glucose	5.11	24.87	19.32
Quercitrin	7.38	46.74	16.06
Naringin	3.78	67.17	30.56
Naringenin	3.34	25.3	13.92
Quercetin	4.68	50.1	18.84
Kampferol 3-2-p-coumaroylglucose	1.34	7.2	4.47
Kampferol	1.64	13.96	9.61
Apigenin	1.12	6.8	4.59
Luteolin 7 glucose	2.64	17.84	11.89
Hespertin	2.47	21.72	16.55
Apignin	2.72	53.72	38.22
Total	160.36	2320.59	1368.35

The antibacterial activity of pomegranate peel extracts:

Four foodborne pathogens were tested to determine the antibacterial effectiveness of ethanolic and aqueous pomegranate peel extracts. Bacillus subtilis, Staphylococcus aureus, Salmonella typhimurium, and Escherichia coli were the bacteria that were tested. Table (5) presents the data.

Table 5: The antibacterial activity of pomegranate peel extracts

Tested microorganisms		Aqueous extract*	Ethanol extract*	Gentamycin*
Tested microorganisms		Zone of growth inhibition (mm)
Gram Positive Bactria	*Staphylococcus aureus* (RCMB 010010 )	14	24	22
Gram Positive Bactria	*Bacillus subtilis* (RCMB 015 (1) NRRL B-543)	11	22	20
Gram Negative Bactria	*Escherichia coli* (RCMB 010052 ATCC 25955)	7	19	15
Gram Negative Bactria	*Salmonella* typhimurium (RCM (RCMB 006 (1) ATCC 14028 )	9	21	18

*The diffusion agar technique was used to conduct the test. Diameter of the well: 6.0 mm (tested with 100 ul)

Positive control was gentamycin for bacteria ( 4 ug/ml)

The ethanol and aqueous extracts were tasted at 10 mg/ml.

The antifungal activity of pomegranate peel extracts:

Pomegranate peel ethanolic and aqueous extracts were tested for their ability to inhibit Aspergillus flavus and Candida albicans. The results were reported as inhibition zone (mm) Table (6).

Table 6: The antifungal activity of pomegranate peel extracts

Tested Microorganisms	Aqueous extract	Ethanol extract	Ketoconazole
Tested Microorganisms	Zone of growth inhibition (mm)
*Aspergillus flavus* (RCMB 002002)	14	18	17
*Candida* *albicans* (RCMB 005003(1)ATCC 10231 )	16	20	19

The diffusion agar technique was utilized for the test. Diameter of the well: 6.0 mm (tested with 100 ul)

Ketoconazole 100 ug/ml as a positive control for fungus

The ethanol and aqueous extracts samples were tasted at a 10 mg/ml concentration.

Cytotoxicity and anticancer activity of selected plant extracts on tumour cell lines:

1. IC ₅₀ of the pomegranate peel powder and their extracts:

IC ₅₀ (extract concentration that kills 50 % of cells) was determined by gradually increasing concentrations (0, 6.1, 12.2, 25, 50, and 100%) of the pomegranate samples to liver and colon cells. The fraction of the surviving cells was determined at each concentration, and the IC₅₀was calculated graphically; the data are displayed in Table (7) .

Table 7: IC₅₀ of the pomegranate peel powder and their ethanolic extract on the liver (HepG2) and colon (HT-29) cells.

Conc ug/ml	Liver cells (HepG2)			Colon cells (HT-29)
Conc ug/ml	Powder	Aqueous	Ethanol	Powder	Aqueous	Ethanol
0	1	1	1	1	1	1
6.1	0.66	0.62	0.51	0.77	0.78	0.59
12.2	0.45	0.43	0.37	0.53	0.51	0.41
25	0.34	0.32	0.25	0.35	0.35	0.30
50	0.28	0.28	0.24	0.25	0.27	0.27
100	0.23	0.23	0.22	0.19	0.22	0.22
IC 50	10.5	9.7	6	15.2	14	9.2

IC₅₀: the concentration of samples that afford a 50% reduction in cell growth (After 72h of incubation).

HT-29: Human Colon cancer cell line.

HepG2: Human liver cancer cell line.

2. Correlation between total phenolic and flavonoid with the calculated IC 50:

Table (8) illustrate the correlation between total phenolic and flavonoid compounds with the IC50 of the different studied plants in liver (HepG2) and colon (HT-29) cells. All correlations are non-significant (p > 0.05).

3. Apoptotic and necrotic cells:

One-way analysis of variance ANOVA demonstrated a highly significant variance in apoptotic and necrotic cells between the aqueous and the ethanolic extract of pomegranate peel, and the data are presented in Table (9) and Figures (1 and 2).

4. Apoptotic/Necrotic ratio:

The apoptotic/ necrotic cell ratio (A/N Ratio) was calculated to determine the aqueous and ethanolic extract of pomegranate peel with the lowest inflammatory potential. For pomegranate peel samples, ANOVA results showed no significant difference between pomegranate peel samples (p <0.001) in Table (9).

Table 9: The apoptotic, necrotic cells and A/N ratio of HC-29 cell line under the effect of pomegranate aqueous and ethanolic extracts.

Pomegranate samples	Apoptotic cells%	Necrotic cells%	A/N Ratio%
Aqueous extracts	42.79^b±1.90	13.06^b±0.78	3.27^a±0.95
Ethanolic extract	47.31^a± 1.30	11.82^a±0.93	4.00^a±0.72
P-value	p <0.001	p <0.001	p <0.001

Data were analyzed with an ANOVA test. The letters show statistically different means according to the LSD post hoc test.

Figure 1: Number and percent of apoptotic (sum Q2 and Q4) and necrotic cells (Q1)under the effect of aqueous extract. Annexin test.

Figure. 2: Number and percent of apoptotic (sum Q2 and Q4) and necrotic cells (Q1) under the effect of ethanolic extract. Annexin test.

DISCUSSION:

Pomegranate peel has a higher phenolic and flavonoid concentration than blossoms, leaves, and seeds¹⁴. Therefore. Data in Table (1) show that the ethanolic extracts of pomegranate peels have the highest concentrations of the total phenols and flavonoids (65.60 mg GAE/g and 35.32 mg QC/g, respectively) in comparison to the aqueous extracts (33.63 mg GAE/g and 22.72mg QC/g). Meanwhile, pomegranate peel powder has the lowest amount of both. The current study is close to those reported by Tabaraki et al.³⁷ who stated that the pomegranate peels ethanolic extract had a significant concentration of total phenolic content, ranging from 49.35 to 86.78mg GAE/g. Furthermore, the finding results were similar to those reported by Elfalleh et al., who observed that the methanolic extracts of various portions of the pomegranate had the highest levels of total polyphenols and flavonoids in comparison to the aqueous extract¹⁴ as well as,³⁸ who mentioned that totalphenolic and flavonoids content were 106.4 and 3.5 mg/g, respectively.The higher concentration of phenolic and flavonoid components in ethanolic extract in comparison to aqueous extract can be due to their increased solubility in organic solvents. Polyphenolic components are polar, efficient, and readily dissolve in polar solvents like aqueous methanol, while they face difficulties in nonpolar solvents like ether³⁹^,⁴⁰.

DPPH is a persistent free radical that has a purple color and absorbs light at 517nm. When it interacts with antioxidants, it undergoes reduction by receiving an electron or hydrogen radical, forming 2,2-diphenyl-1-picrylhydrazine, which is yellow. The sensitivity of this radical allows for detecting low levels of antioxidant compounds in various extracts⁴¹. In the present study, data in Table (2) was shown that there was a wide range in the inhibitory action of pomegranate peel extracts against the DPPH radical. The ethanolic extract exhibited the greatest radical scavenging activity among the extracts studied, followed by water extract (95.36 and 57.22%, respectively). Meanwhile, pomegranate peel powder recorded a moderate power with value (83.22%). These data in agreed with Tabaraki et al, who showed that DPPH radical scavenging activity of methanol extract from pomegranate was greater than that of water extract³⁷ and close to Abdealsiede et al., who found 85.90% antioxidant activity in pomegranate peel ethanolic extract⁴². This is due to the water-extracted free sugars, organic acids, salts, and antioxidant compounds. Ethanol extracts had more phenolic and flavonoid components than water extracts, which helped boost antioxidant activity⁴³.

Data of HPLC assay of pomegranate peel and its extract phenolic compounds, Table (3), show nineteen phenolic compounds were found, and pyrogallol was the main phenolic compound identified in pomegranate peel powder (192.61mg/100g), followed by Epi-catechine and catechin, which had 99.3 and 41.43mg/100g, respectively. Furthermore, chlorogenic, gallic, and benzoic acids were found in moderate amounts, which recorded 29.81, 21.94, and 20.54mg/100g, respectively, while the lowest phenolic acids detected in pomegranate peels powder were catechol and protocatchoic acid (2.58 and 1.15mg/100g, respectively). Concerning the data of pomegranate peel extracts, gallic acid had been the predominant phenolic component detected in the ethanolic extract (1063.62mg/100g). In comparison, pyrogallol was the most prevalent phenolic acid in the aqueous extract, which had 434.7mg/100 g. In contrast, ellagic acid was the second phenolic component identified in ethanolic (815.16mg/100g) and aqueous extract (285.37mg/100g). In addition, α- Coumaric was the lowest phenolic acid in both extracts (6.65 and 4.45 mg/100g, respectively). Furthermore, during the extraction process of pomegranate peels, it's noteworthy that the choice of solvent, such as ethanol and water, significantly influences the levels of phenolic acids obtained. Ethanol extraction yields higher phenolic acid concentrations(3589.38mg/100g) than water extraction (1735.29mg/100g). According to previous work, Middha et al. revealed a few significant phenolic components in pomegranate peel, like ellagic and gallic acids, as well as punicalagin as a main ellagitannin⁴⁴. Moreover, Mansour et al. discovered p-coumaric acid, ellagic acid, gallic acid and caffeic acid in pomegranate peel²⁷^,⁴⁵. While Bouaziz et al. reported that pomegranate leaves, barks, fruits, stems, and roots all contain a variety of bioactive substances, including phenolic compounds such as gallic and ellagic acids, as well as punicalagin, pedunculagin and punicalin. The Folin-Ciocalteu method yielded a higher concentration of total polyphenols than the HPLC approach did⁴⁶. The HPLC method only presented 50 to 60 percent of the amount assessed using the Folin-Ciocalteu method⁴⁷. Generally, it could be observed that the ethanolic extracts had the highest phenolic compound content compared to aqueous extracts and their contents in row materials, which are illustrated before in Table (1), due to the high degree polarity of ethanol, which provides greater extraction capability and can produce a greater number of active constituents and phytochemicals.

Flavonoids are a type of plant pigment that is naturally found in plants. Therefore, In the present study, Fifteen flavonoid components were identified in pomegranate peel powder and extracts, as revealed in Table (4). The flavonoid content of pomegranate peel powder was the lowest (160.36mg/100g) compared to the extract. The predominant flavonoid identified in pomegranate peel powder (102.69mg/100g) was hesperidin. It also contained a moderate content of rutin and quercitrin, with 9.45 and 7.38mg/100g, respectively, while kampferol was the lowest flavonoid compound, with 1.65mg/100g. Regarding the results of pomegranate peel extracts, during the extracting process of pomegranate peels, it's worth noting that the selection of solvents, such as ethanol and water, significantly impacts the levels of flavonoid compounds. Ethanol-based extraction methods typically yield higher concentrations of these flavonoid compounds (2320.59mg/100g) than water-based extractions (1368.35mg/100g). Moreover, the most common flavonoid compound was hesperidin in ethanolic and aqueous extract (1807.5 and 1050.6 mg/100g, respectively). Furthermore, rutin and naringin were the second flavonoid compounds detected with high amounts in an ethanolic extract, with 97.09 and 67.17mg/100g, respectively. In contrast, rutin was the second most important flavonoid compound discovered in the aqueous pomegranate peel extract (70.96 mg/100g). Moreover, the ethanolic extract of pomegranate peel recorded high contents of apignin, quercetin, and quercitrin (53.72, 50.1, 46.74mg/100g, respectively) compared to aqueous extract. In addition, the aqueous extract contained moderate levels of apignin , rosmarinic, naringin and apigenin 6-arabinose 8-galactose (38.22, 33.54, 30.56 and 29.22 mg/100g, respectively). These results are in accordance with those reported by Basiri et al., Rowayshed et al. and Venkitasamy et al., As per the findings of these studies, the primary flavonoids discovered in pomegranate peel were quercetin, catechin, epicatechin, kaempferol, flavan-3-ol, rutin, kaempferol 3-O-glycosideluteolin, , kaempferol 3-O-rhamnoglycoside and luteolin 7-O-glycoside³⁷^,⁴⁸^,⁴⁹.

Pomegranate peel extracts, especially the ethanolic extract, demonstrated strong antibacterial and fungal action. Markedly, it achieves these effects without causing any side effects and offers the benefit of being cost-effective. Also, it is a promising natural alternative for combating microbial infections⁵⁰. In the present study, data in Table (5), The results of aqueous and ethanolic extracts reveal that ethanolic extract demonstrated higher antibacterial activity than aqueous extract and commercial antibiotic gentamycin. Staphylococcus aureus and Bacillus subtilis were the most elevated sensitive bacteria to ethanolic extract of pomegranate peel, which recorded the highest inhibition zones (24 and 22 mm, respectively) compared to the other tested strains, which recorded 21 and 19 mm against Salmonella typhimurium and Escherichia coli, respectively at 10mg/ml. Conversely, The aqueous extract from pomegranate peel showed the least effective antibacterial action against all strains. The findings presented here are consistent with those documented by Dey et al., which indicate that the pomegranate peel extract is an abundant reservoir of antibacterial compounds (tannins and flavonoids) that inhibit the growth of bacteria responsible for contaminated illnesses and food spoilage⁵¹. Also, our results align with the findings those reported by Sami et al.⁵² who indicate that ethanolic pomegranate peel extract is effective against B. subtilis, P. aeruginosa, S. aureus, and K. pneumonia. According to the data, pomegranate peel aqueous and ethanolic extracts are are more effective against gram-positive germs than gram-negative ones, which are resistant due to their outer membrane's peptidoglycan and lipopolysaccharides, which create a strong hydrophilic surface and an effective permeability barrier⁴⁶. Due to its higher polarity, ethanol extracts more active constituents like phenolic and flavonoid compounds with potent antibacterial properties than the aqueous extract against all tested foodborne pathogenic bacteria⁵³. The solubility difference between water and ethanol also affects ability. Water has a high dielectric constant and polar molecules with dipole configurations. Therefore, ethanol dissolves more readily than water⁵⁴.

According to the findings in Table (6), The ethanolic extracts of pomegranate peel demonstrated a much greater antifungal activity against all strains of the disease than did the aqueous extracts and the commercial drug ketoconazole. The ethanolic extract showed the greatest inhibition zone against Candida albicans (20.0 mm), followed by Aspergillus flavus (18.0 mm). However, the pomegranate peel aqueous extract showed the lowest level of inhibition against Aspergillus flavus (14 mm); according to the study of Abdollahzadeh et al. which stated that a methanol extract of pomegranate peel displayed efficiency against C. albicans, A. niger, C. utilis, and S. cerevisae⁵⁵. In addition, Sami et al. found that the ethanolic extracts of pomegranate peel possesses antifungal action against C. albicans, C. pellicullosa, and A. niger⁵². It could be attributed to the ethanolic extract showing more effectiveness against all tested foodborne pathogenic fungi than the aqueous extract due to ethanol's higher polarity, enabling a greater extraction capacity for active constituents like phenolic and flavonoid compounds⁵³.

On the other hand data in Table (7) show that The liver (HepG2) and colon (HT-29) cell lines were most strongly inhibited (IC50) by the ethanolic extract of pomegranate peel (6.0 and 9.2 mg/100g, respectively), followed by the aqueous extracts of pomegranate peel (9.7 and 14.0 mg/100g, respectively), compared to pomegranate peel powder. This indicates that the pomegranate peel ethanolic extract is a safer and more potent anti-cancer agent in accordance with Kasimsetty et al., who stated that the chemopreventive characteristics of ellagitannins, which are derived from pomegranate juice, and their bacterial metabolites, urolithins, urolithins, have been investigated for their capacity to suppress the growth of HT-29 human colon cancer cells. The study reveals that ellagitannins found in pomegranate juice, such as ellagic and punicalagin acid, may also have a role in colon cancer chemoprevention⁵⁶. The data show that the characteristics of the pomegranate peel powder and its ethanolic extract obstruct the proliferation of tested tumor cell lines. The cytotoxic effect on tumor cells could be explained by the high phenolic component content of pomegranate peel powder and its ethanolic extract. Furthermore, polyphenols are well-known antioxidants that can cause DNA damage in the occurrence of metal ions like copper. These effects contribute to their chemopreventive and selective cytotoxicity against cancer cells⁵⁷.

Usually, cell death is either apoptosis or necrosis. Active, programmed cellular disintegration without inflammation is called apoptosis. Necrosis is passive, unintended cell death caused by environmental disturbances and uncontrolled inflammatory cell discharge. Pathologists use necrosis to describe dead tissues or cells and the changes that occur after they die, regardless of prelethal status⁵⁸. In the present study, data in Table (9) and and Figures (1 and 2) have shown that the ethanolic extract caused significant induction of apoptosis (Sum Q2 and Q4) with 47.31%±1.30 compared to the aqueous extract (42.79%±1.90). Meanwhile, ethanolic extract recorded the lowest necrosis cell percentage )Q1), 11.82%± 0.93, compared to the aqueous extract, which recorded 13.06%±0.78. These data agreed with Jeune et al., who showed that when pomegranate extract is combined with genistein, it was more efficient than either agent alone at slowing the development of breast cancer cells and triggering apoptosis⁵⁹. Also, as in study of Nasr et al., which indicate that Pomegranate peels and seed ethanolic extract significantly upregulated pro-apoptotic genes and downregulated anti-apoptotic genes in genes related to apoptosis⁶⁰.Polyphenols may play a crucial role in cancer prevention by controlling oxidative stress in cancer cells, which may explain the correlation between polyphenol concentration and apoptosis and necrotic cells. The impact on signal transduction, redox-sensitive transcription factors, and gene expression influences both cell growth and apoptosis. Polyphenols have a direct impact on the apoptotic process and the expression of regulatory proteins⁶¹^,⁶².

The apoptotic/ necrotic cell ratio (A/N Ratio) in Table (9) showed that ethanol extract of pomegranate peel had the highest ratio (4.00 % ± 0.72), while aqueous showed the lowest ratio (3.27 %± 0.95). A/N Ratio > 1: A higher ratio indicates a prevalence of apoptotic cell death over necrosis. This may suggest that the cells respond to a controlled physiological process or a specific treatment that induces apoptosis⁶³.

CONCLUSION:

Our study demonstrates that pomegranate peel is a great source of polyphenols, such as flavonoids and phenols, which have strong antifungal, antibacterial, and antioxidant properties. The pomegranate peel ethanolic extract exhibits higher levels of these compounds and more potent biological activities than the aqueous extract. Moreover, the pomegranate peel ethanolic extract demonstrates selective cytotoxicity against cancerous cells, inducing apoptosis and necrosis in liver and colon tumor cell lines more effectively and safely compared to the aqueous extract. Therefore, pomegranate peel extract, especially ethanolic extract, has significant potential as a natural alternative for treating chronic diseases such as cancer and bacterial/fungal infections.

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Received on 29.11.2023 Modified on 03.02.2024

Research J. Pharm. and Tech 2024; 17(6):2744-2752.

DOI: 10.52711/0974-360X.2024.00430

Plant	Total phenolic				Total flavonoids
	Liver (HepG2)		Colon (HT-29)		Liver (HepG2)		Colon (HT-29)
	r	P	r	P	r	P	r	P
Pomegra nate	-0.96	0.17	-0.97	0.16	-0.88	0.31	-0.89	0.29