Pomegranate in Cancer Therapy: A Review of Anticancer Potential, Mechanisms and Clinical Challenges in Breast, Colon and Prostate Cancers

ABSTRACT

Pomegranate ( Punica granatum L.) is a plant that has been used in traditional medicine for centuries and that has gain popularity in recent years as an anticancer dietary supplement. For the purpose of this review, a comprehensive literature search was performed to gather articles published between 1993 and 2021 and describing the therapeutic potential of pomegranate fruit extracts, juice, and seed oil on breast, colon, and prostate cancer. The results of the studies highlighted the fact that while the consumption of pomegranate has been associated with a number of beneficial effects on cancer, it still remains unclear which chemicals are responsible for such effects. It is hoped that this review will encourage further investigations on pomegranate in order to identify the compounds (or their metabolites) responsible for biological activity and unravel their mode(s) of action at the molecular level.

Abbreviations

8‐OHdG

8‐hydroxy‐2′‐deoxyguanosine

ADSCs

adipose‐derived stem cells

AR

androgen receptor

BC

breast cancer

DSB

double‐strand break

DSS

dextran sodium sulfate

EA

ellagic acid

EAC

Ehrlich ascites carcinoma

EEPE

ellagitannin‐enriched pomegranate extract

ESC

Ehrlich solid carcinoma

HIF‐1α

hypoxia‐inducible factor‐1α

HR

homologous recombination

IGF‐1

insulin‐like growth factor‐1

IGFBP‐3

insulin‐like growth factor binding protein‐3

JNK

Jun N‐terminal kinases

LPS

lipopolysaccharide

miRNAs

microRNAs

MMP

matrix metalloproteinase

NF‐κB

nuclear factor κB

PBMC

peripheral blood mononuclear cells

PC

prostate cancer

PCNA

proliferating cells nuclear antigen

PE

pomegranate extract

PE‐SLNs

solid lipid nanoparticles with an optimized pomegranate extract

PFE

pomegranate fruit extract

PFJ

pomegranate fruit juice

PI3K

phosphatidylinositol 3‐kinase

PJ

pomegranate juice

PJC

pomegranate juice concentrate

PPE

pomegranate peel extract

PSA

prostate‐specific antigen

PSADT

PSA doubling time

PSE

pomegranate seed extract

RhoA,B,C

GTPases

SDF1α

stromal‐derived growth factor one alpha

SEPFS

standardized extract obtained from the skin of the pomegranate fruit

SHBG

sex hormone binding globulin

SHIP1

SH‐2 containing inositol 5′ polyphosphatase 1

TPT

total pomegranate tannin

TRAP

transgenic rat model

UA

urolithin A

VEGF

vascular endothelial growth factor

WPFE

water‐based extract derived from the pomegranate fruit

1. Introduction

Pomegranate ( Punica granatum L.), of the family Lythraceae, is a deciduous shrub or small tree that has been used in ethnomedicine for centuries (Lansky and Newman 2007). The bark (Granati cortex) from the roots and stems of this plant is reputed for its antimicrobial, antiuremic, anthelminthic, and molluscicidal properties (WHO 2009). The fruit consists of a peel (epicarp) that includes an interior network of membranes, seeds, and arils (Lansky and Newman 2007). The peel is rich in polyphenols, particularly flavonoids and ellagitannins. Flavonoids include flavonols (kaempferol, quercetin, and myricetin) and flavan‐3‐ols (catechin, epicatechin and epigallocatechin‐3‐gallate). Ellagitannins such as punicalagin, punicalin, corilagin, granatin A and B, tellimagrandin, and pedunculagin are prominent constituents. Recent studies have further confirmed the presence of other bioactives such as gallagic acid, ellagic acid glycosides, and various hydrolyzable tannins that contribute to the antioxidant and anticancer activity of the peel (Wong et al. 2021; Salih et al. 2025). The peel also contains pyrrolidine‐type alkaloids (punigratane), lignans (pomegralignan), phenolic acids (caffeic acid, p‐coumaric acid, chlorogenic acid, gallic acid, and ellagic acid) (Amri et al. 2017; Singh et al. 2018), along with polysaccharides, proteins, bioactive peptides, and minerals (Viuda‐Martos et al. 2010; Kaur et al. 2014; Rafiq et al. 2016; Ito et al. 2014). The arils (sarcotesta) consist mainly of water and sugars, as well as pectin, organic acids (ascorbic acid, citric acid, and malic acid) and phenolic compounds (mainly anthocyanins) (Viuda‐Martos et al. 2010; Sreekumar et al. 2014; Elfalleh 2012). Pomegranate juice is obtained either from the arils or the whole fruit (Viuda‐Martos et al. 2010). The juice contains water, sugars (sucrose, fructose, and glucose), and a small amount of pectin. The most prevalent phenolic compounds in the juice are the flavan‐3‐ols, ellagitannins, and ellagic acid derivatives. It is also rich in anthocyanins (i.e., derivatives of delphinidin, cyanidin, and pelargonidin, with high levels of epicatechin‐cyanidin‐3,5‐dihexoside followed by cyanidin‐3,5‐dihexoside, cyanidin‐3,5‐pentoside hexoside, delphinidin‐3‐glucoside, and delphinidin‐dihexoside), hydroxybenzoic acids (gallic acid and ellagic acid), and hydroxycinnamic acids (3‐caffeoylquinic acid, caffeic acid, chlorogenic acid, and p‐coumaric acid) and derivatives (digalloyl‐hexoside 1 and tri‐hexahydroxydiphenic acid hexoside 1) (Viuda‐Martos et al. 2010) (Topalović et al. 2020). Vitamin C, vitamin E, coenzyme Q10, and lipoic acid are present in the juice as well (Viladomiu et al. 2013). The seeds mostly contain an oil, rich in unsaturated acids, with a high amount of punicic acid (9Z,11E,13Z‐octadecatrienoic acid), a conjugated isomer of linolenic acid unique to pomegranate seed oil (Kohno et al. 2004; Viladomiu et al. 2013; Sharma et al. 2017). The oil contains phytoestrogens such as the isoflavones genistein and daidzein, the coumestan coumestrol, as well as the glycosphingolipid N‐palmitoyl cerebroside (Tsuyuki et al. 1981) (Khorsandi and Yazdi 2009). Ellagic acid derivatives (3,3′‐di‐O‐methyl ellagic acid and 3,3,4′‐tri‐O‐methyl ellagic acid), triterpenoids (asiatic acid and betulinic acid), sterols (stigmasterol, sitosterol, cholesterol) and alkaloids have also been reported in the seeds. Three steroids (estrone, testosterone, and estriol) have also been tentatively identified (Elfalleh 2012; Wu and Tian 2017; Wong et al. 2021). The chemical structures of some of the characteristic phenolic compounds found in pomegranate fruit are given in Figure 1.

FIGURE 1.

Chemical structures of the characteristic compounds found in pomegranate fruit. A compound index is provided in the Supporting Information.

In recent years, the consumption of pomegranate has gained in popularity, owing to the potential of its fruit extracts, juice, and seed oil in the treatment and prevention of cancer (Sharma et al. 2017). Preclinical studies have revealed the therapeutic potential of pomegranate fruit against various types of cancers (Wong et al. 2021). This review examines the anticancer potential of pomegranate and its bioactive constituents in breast, colon, and prostate cancers, drawing on evidence from in vitro, in vivo, and, where available, early‐phase clinical studies. Pomegranate‐derived compounds have been shown to inhibit cancer cell proliferation, induce apoptosis, modulate oncogenic gene expression, and reduce inflammation and oxidative stress. These cancer types were selected due to their high global prevalence, shared molecular pathways targeted by pomegranate (such as hormone sensitivity, inflammation, and oxidative stress), and the availability of sufficient preclinical and clinical data to support critical evaluation of translational relevance.

2. Review Methodology

A comprehensive literature search was conducted in PubMed/MEDLINE, ScienceDirect, American Chemical Society, Springer/Kluwer, Wiley Interscience, Google Scholar and SCOPUS for studies reporting recent findings on the anticancer effects of Punica granatum in breast, colon, and prostate cancer. Search terms included “pomegranate” OR “ Punica granatum ” combined with “cancer,” “chemoprevention,” “apoptosis,” “oxidative stress,” “breast cancer,” “colon cancer,” “prostate cancer,” and related MeSH terms. Inclusion criteria were: (i) preclinical and clinical studies on pomegranate and cancer, (ii) peer‐reviewed articles in English, (iii) studies with clear methodologies, (iv) reports on apoptosis, proliferation inhibition, oxidative stress, or inflammation, and (v) those analyzing specific pomegranate bioactive components like punicalagin and ellagic acid. Exclusion criteria were: (i) studies on non‐cancer conditions, (ii) non‐peer‐reviewed literature, (iii) mixed bioactive studies obscuring pomegranate‐specific effects, and (iv) duplicate or methodologically weak studies. Data extraction included: (i) study type, (ii) cancer model, (iii) pomegranate preparation, (iv) bioactive compounds, (v) mechanisms of action, and (vi) key findings. A flowchart outlining the literature selection process is provided to enhance transparency and is summarized in Figure 2. Where reported, effective concentrations of pomegranate extracts or bioactive compounds were noted, along with discussion of factors such as bioavailability and human absorption, which may influence translational relevance. In addition, the potential implications of pomegranate in cancer prevention strategies, future dietary recommendations, and the development of natural product‐based therapeutics are briefly discussed. Results are summarized in tables and figures, highlighting cancer cell line responses, in vivo tumor inhibition, clinical outcomes, and molecular mechanisms.

FIGURE 2.

Flowchart summarizing the literature identification, screening, eligibility assessment and inclusion process for studies on the anticancer effects of Punica granatum in breast, colon and prostate cancers.

3. Therapeutic Potential of Pomegranate on Breast Cancer

3.1. Preclinical Studies of Pomegranate on Breast Cancer

As a leading cancer diagnosis in women, breast cancer (BC) takes up 30% of all new female cancers each year (Łukasiewicz et al. 2021) in the United States (American Cancer Society 2023; Siegel et al. 2023). Fruits and vegetables are potent anti‐carcinogenic agents; it is estimated that more than 20% of all cancers could be eliminated by a proper high in fruits and vegetable diet.

In breast cancer models, ellagitannins such as punicalagin and their gut‐derived metabolites, especially urolithins (urolithin A and B), have been identified as key contributors to the observed anticancer effects(García‐Villalba et al. 2022). These compounds exert pro‐apoptotic, anti‐proliferative, and estrogen‐modulating activities in hormone‐dependent breast cancer cells (García‐Villalba et al. 2022). Urolithin A, in particular, has been shown to inhibit aromatase activity and modulate estrogen receptor signaling, supporting its relevance in estrogen‐sensitive tumors. The bioactivity of these metabolites varies based on individual microbiota composition, which may influence therapeutic outcomes (García‐Villalba et al. 2022). Numerous studies have demonstrated that pomegranate causes an induction of apoptosis and cell cycle arrest, as well as inhibits multiple signaling pathways in vitro and in vivo models of breast cancers (Seeram et al. 2005; Syed et al. 2007; Jurenka 2008; Amin et al. 2009) (Table 1 and Figure 3). Nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NF‐κB) stops apoptosis and encourages proliferation in human breast cancers. Substances that inhibit NF‐κB activation would be effective chemopreventive agents for estrogen receptor‐negative tumors (Biswas et al. 2004). Several downstream carcinogenesis mediators are known to be regulated by NF‐κB, highlighting its importance as a conductor of the cancer process. Previous studies on the topic of pomegranate's anti‐inflammatory properties have shown that it has a down‐regulatory modulative property of pro‐inflammatory eicosanoids, as well as the fact that pomegranate extract, that has been standardized to an ellagitannin content of 37% punicalagin, has a lowering effect on the NF‐κB levels (Khan et al. 2009; Weisburg et al. 2010). Furthermore, pomegranate extracts have been shown to decrease the expression of miR‐155. This decrease coincided with the activation of SHIP‐1, an inositol 5′‐phosphatase regulated by miR‐155, which is a pivotal link in the regulation of the phosphatidylinositol‐3,4,5‐trisphosphate that serves as a secondary messenger in the activation of AKT and nuclear translocation of NF‐κB (Baran et al. 2003; Chao et al. 2010). The anticancer properties of fermented pomegranate juice and seed oil extracts stem from their ability to suppress the expression and activity of NF‐kB, coupled with the ability to decrease the levels of RhoC and RhoA proteins. As a consequence, the metastatic potential of aggressive breast cancer variants is significantly diminished (Khan et al. 2009). Scientific evidence substantiates that the substances derived from pomegranate, specifically ellagitannins, possess the capacity to impede the functionality of the aromatase enzyme and impact the carcinogenic processes of estrogen‐responsive breast cancer (Adams et al. 2010). Estradiol, a form of estrogen, is known to play a role in the development and growth of certain types of breast cancer. Research indicates that estradiol may enhance the likelihood of breast cancer occurrence in postmenopausal women, thereby prompting suggestions that dietary approaches aiming to lower estrogen production could potentially be advantageous in terms of preventing cancer (Thomas et al. 1997). The consumption of pomegranates has the potential to influence tumor characteristics by diminishing their growth and/or invasiveness, as well as inhibiting hormonal‐induced cancer development by diminishing estrogen levels and/or activity. The enzymatic activity of aromatase (17β‐hydroxysteroid dehydrogenase), responsible for converting androgens into estrogens, holds a significant importance in the development of breast cancer. Earlier studies (e.g., Adams et al. 2010) demonstrated that ellagitannin‐derived compounds from pomegranate can inhibit aromatase activity in vitro, potentially reducing estrogen production. More recent work has continued to support pomegranate's anti‐estrogenic effects, including the inhibition of aromatase and 17β‐hydroxysteroid dehydrogenase in breast cancer cell line models (Shabir et al. 2024) (Rauf et al. 2025). However, robust in vivo or clinical data quantifying these enzyme‐inhibitory effects remain limited. Thus, while pomegranate shows promise in modulating enzymes linked to estrogen biosynthesis, further studies are needed to validate its efficacy, potency, and bioavailability under physiological conditions (Rauf et al. 2025) Fermented juice, aqueous pericarp extract, and cold‐pressed and supercritical CO₂‐extracted seed oil of pomegranate all inhibited breast cancer carcinogenesis (Kim et al. 2002). Both the pomegranate seed oil and fermented fruit extracts exhibit a chemo‐preventive activity in mouse mammary organ cultures (Mehta and Lansky 2004). A standardized whole pomegranate fruit extract has shown an inhibiting potential on WA4 (a cell line derived from the mouse mammary tumor virus (MMTV‐Wnt‐1 transgenic mouse) mammary cancer cell proliferation in vitro as well as a cytotoxic activity on the cells in question. These effects stemmed from an arrest of cell cycle progression in the G0/G1 phase, and an increase in caspase‐3 enzyme activity in the presence of the extract (Dai et al. 2010). Moreover, a standardized extract obtained from the skin of the pomegranate fruit (SEPFS), with standardized levels of punicalagins (37%–40%) and free ellagic acid (3.4%), has the potential to impact the DNA repair pathway necessary for the survival of MCF‐7 breast cancer cells. It was demonstrated that the presence of 20 μg/mL of SEPFS for 72 and 96 h, during the incubation with MCF‐7 cells, resulted in a reduction of their growth by approximately 30% and 35%, respectively, whereas 50 mg/mL of SEPFS inhibited cell growth by about 50% and 80% at 72 and 96 h, respectively. SEPFS exhibited inhibitory effects on cancer cell growth, accompanied by arresting the cell cycle at the G2/M phase and inducing apoptosis. DNA microarray analysis revealed that SEPFS downregulated genes associated with mitosis, chromosome organization, RNA processing, DNA replication, and DNA repair, while upregulating genes involved in apoptosis regulation and cell proliferation. SEPFS specifically downregulated vital genes involved in DNA double‐strand break (DSB) repair through homologous recombination (HR), such as MRE11, RAD50, NBS1, RAD51, BRCA1, BRCA2, and BRCC3. This downregulation of HR genes correlated with elevated levels of their predicted microRNAs (miRNAs), including miR‐183 (predicted target RAD50) and miR‐24 (predicted target BRCA1), indicating that SEPFS may influence miRNAs associated with the DNA repair processes. Furthermore, treatment with SEPFS increased the frequency of DSBs, suggesting that it downregulates HR, sensitizing cells to DSBs, growth inhibition, and apoptosis. Given that HR represents a novel target for cancer therapy, the downregulation of HR by SEPFS could potentially be exploited to enhance tumor sensitivity to anticancer drugs. Furthermore, the SEPFS treatment induced apoptosis and caused cell cycle arrest at the G2/M phases (Shirode et al. 2014). Additionally, it was demonstrated that the bioactivity of pomegranate polyphenols is enhanced through their encapsulation, resulting in the inhibition of cancer cell growth in both MCF‐7 and Hs578T breast cancer cell lines (Shirode et al. 2015). Among the tested cells, MCF‐7 breast carcinoma fibroblasts exhibited the highest level of sensitivity to the application of a standardized ethanolic extract obtained from pomegranate peel, which shows promise as a potential agent for anticancer purposes (Keta et al. 2020). Moreover, in MCF‐7 cells, the introduction of 200–300 μg/mL of petroleum ether extract obtained from dried fruit peels for a duration of 48–72 h resulted in an observation of apoptosis and a decrease in proliferation. Particularly, within the specified concentration range, there was an increase in the expression of the pro‐apoptotic gene Bax, while the expression of the anti‐apoptotic gene Bcl‐2 was reduced (Dikmen et al. 2011). In one study, following 48 h of incubation, the acetonic fraction obtained from the pomegranate peel extract demonstrated notable efficacy in inhibiting the proliferation of MCF‐7 cells, as evidenced by the IC₅₀ value of 8.15 μg/mL) (Yassin et al. 2021). The antioxidant and cytotoxic properties of eight distinct pomegranate varieties sourced from Türkiye were assessed in relation to their impact on MCF‐7 breast cancer cells, it was found that all extracts resulted in decreased cell viability of MCF‐7 breast cancer cell lines. The most potent extract contained substantial amounts of anthocyanins such as cyanidin chloride (69.76 ± 8.02 μg/g extract), cyanidin‐3‐O‐glucoside (903.66 ± 101.89 μg/g extract), and punicalagin (992.09 ± 174.53 μg/g extract). This particular extract demonstrated a robust cytotoxic activity against MCF‐7 cells after 24 h of incubation, exhibiting an IC₅₀ value of 49.08 μg/mL (Eroglu Ozkan et al. 2021). Furthermore, an assessment was conducted to evaluate the anticancer efficacy of a water extract obtained from the black pomegranate, and the findings revealed that the extract triggered the cell death pathway in over 70% of the MCF‐7 and BT‐20 cancer cell lines (Khorrami et al. 2019). An inhibitory effect on the proliferation of MCF‐7 breast adenocarcinoma cells, reaching 83.7%, was observed with the administration of a methanolic extract derived from pomegranate peel at a dosage of 5 μg/mL (Modaeinama et al. 2015). The potential of 29 diverse pomegranate accessions to suppress the proliferation of breast cancer cell lines was examined, and the analysis of aqueous peel extracts revealed that certain accessions, including 100‐1, 102‐3, 106‐7, and 127‐28, exhibited considerable efficacy in inhibiting the proliferation of breast cancer cell lines (MCF7 and MDA‐MB‐453). This finding suggests a broad anti‐proliferative activity among these accessions (Orgil et al. 2016). The growth of MCF‐7 cells was regulated and their proliferation was suppressed by a methanolic PE. This inhibitory effect was observed in cells that were stimulated by 27‐hydroxycholesterol, a compound linked to the progression and metastasis of breast cancer tumors (Vini et al. 2016). Pomegranate seed extract (PSE) was evaluated in the MCF‐7 breast cancer cell line and the co‐culture treatment in adipose‐derived stem cells (ADSCs). Apoptosis of the cancer cells and the effect on the cell cycle were monitored. The research showed that PSE in combination with ADSCs expressed higher anti‐proliferative effects on the MCF‐7 breast cancer cell line than ADSCs alone. PSE expressed high cytotoxicity at concentrations higher than 100 μg/mL (33.5% on MCF‐7 and 36.5% cells on ADSCs cells). These effects were also confirmed by Dai et al. (2010). Also, PSE increased the cytotoxicity of ADSCs on MCF‐7 cancer cells. The combination of PSE and ADSCs led to cell cycle arrest and the stimulation of apoptosis, which resulted from a significant increase in Bax genes and caspase‐3 expression and a decline in Bcl‐2 expression. These findings indicated that co‐treatment of ADCSs with PSE reduces breast cancer cell growth via apoptosis and inhibition of angiogenesis via reduction of VEGF expression (Moradi‐Gharibvand et al. 2022).

TABLE 1.

Mechanisms of pomegranate action in breast cancer.

Mechanism of anticancer action	Tested pomegranate concentrations/doses	Type of study	Results/effects	References
Suppression of epithelial‐mesenchymal transition (EMT) and cancer stemness	Water‐based fruit extract (WPFE) (50–500 μg/mL)	In vitro (HMLER, Hs578T)	↓ EMT markers, ↓ Cancer stemness genes, suppression of β‐catenin and ZEB1	Nallanthighal et al. (2017)
Apoptosis	Standardized whole fruit extract (50 mg/mL)	In vitro (WA4—MMTV‐Wnt‐1 transgenic mouse)	↓ Cell cycle progression (G0/G1), ↑ Caspase‐3	Dai et al. (2010)
	Standardized skin extract (SEPFS) (20 μg/mL, 50 mg/mL)	In vitro (MCF‐7)	↓ DNA repair genes (MRE11, RAD50, BRCA1, BRCA2), ↑ Apoptosis, ↑ G2/M cell cycle arrest	Shirode et al. (2014)
	Methanolic pomegranate peel extract (200–300 μg/mL, IC50 = 8.15 μg/mL for MCF‐7)	In vitro (MCF‐7)	↑ Bax, ↓ Bcl‐2, ↑ Caspase‐dependent apoptosis	Dikmen et al. (2011)
	Pomegranate juice concentrate (PJC) (Various doses)	In vitro (MDA breast cancer cells)	↑ Apoptosis via mitochondrial pathway, ↑ Caspase‐3	Habib et al. (2023)
Metastasis inhibition	Fermented juice and seed oil (Standardized ellagitannin content: 37% punicalagin)	In vitro (MDA‐MB‐231, MCF‐7)	↓ NF‐κB activation, ↓ RhoC, ↓ RhoA, ↓ Metastatic potential	Khan et al. (2009)
Anti‐angiogenesis	Pomegranate seed extract (PSE) (100 μg/mL)	In vitro (MCF‐7)	↓ VEGF, ↓ Angiogenesis, ↑ Apoptosis ↓ Proliferation	Moradi‐Gharibvand et al. (2022)
Tumor growth inhibition	Solid lipid nanoparticles with pomegranate extract (PE‐SLNs) (0.5 g transdermal gel)	In vivo (Ehrlich ascites carcinoma (EAC) mice)	↓ Tumor progression by 59.71%, ↑ Tumor necrosis, ↑ Apoptosis	Teaima et al. (2022)
Inhibition of estrogen synthesis and hormonal regulation	Pomegranate juice (PJ) (237 mL/day for 3 weeks)	Clinical study (Postmenopausal women)	↓ Estrone, ↓ Testosterone levels in normal weight women, No significant changes in estradiol or SHBG	Kapoor et al. (2015)

Note: Symbols: ↓—Decrease; ↑—Increase.

Abbreviations: AKT, protein kinase B; Bax, Bcl‐2‐associated X protein; Bcl‐2, B‐cell lymphoma 2; BRCA1, BRCA2, breast cancer susceptibility genes 1 and 2; DSB, double‐strand break; EAC, Ehrlich ascites carcinoma; EMT, epithelial‐mesenchymal transition; G0/G1, G2/M, cell cycle phases; IC₅₀, inhibitory concentration 50%; MCF‐7, Michigan Cancer Foundation‐7; MDA‐MB‐231, highly invasive triple‐negative breast cancer cell line; MDA, metastatic ductal adenocarcinoma; MMTV, mouse mammary tumor virus; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PE‐SLNs, solid lipid nanoparticles with pomegranate extract; PJC, pomegranate juice concentrate; PSE, pomegranate seed extract; SEPFS, standardized extract from pomegranate fruit skin; SHBG, sex hormone‐binding globulin; SHIP‐1, SH2‐containing inositol 5′‐phosphatase 1; VEGF, vascular endothelial growth factor.

FIGURE 3.

Anticancer mechanisms of Punica granatum in breast cancer cells. Pomegranate‐derived bioactive compounds promote apoptosis by increasing Bax expression, reducing Bcl‐2, and activating caspases‐3 and ‐9, leading to mitochondrial damage. Cell cycle arrest occurs through the upregulation of G0/G1 and G2/M checkpoints, inhibiting uncontrolled proliferation. Additionally, pomegranate inhibits epithelial‐mesenchymal transition (EMT) and cancer cell migration and invasion by downregulating β‐catenin, vimentin, ZEB1, RhoA, and RhoC while increasing E‐cadherin levels. Furthermore, pomegranate suppresses NF‐κB activation, reducing inflammation and blocking aromatase, thereby decreasing estrogen biosynthesis and inhibiting hormone‐dependent tumor growth. ↑, Increase; ↓, Decrease; Bax, Bcl‐2‐associated X protein; Bcl‐2, B‐cell lymphoma 2; Caspases‐3, 9, Cysteine‐aspartic proteases 3 and 9; E‐cadherin, Epithelial cadherin; EMT, Epithelial‐mesenchymal transition; G0/G1, G2/M, Cell cycle phases; miR‐155, MicroRNA‐155; NF‐κB, Nuclear factor kappa‐light‐chain‐enhancer of activated B cells; RhoA, RhoC, Ras homolog family member A and C; Vimentin, Intermediate filament protein; ZEB1, Zinc finger E‐box‐binding homeobox 1; β‐catenin, Beta‐catenin.

The cytostatic effect of pomegranate juice concentrate (PJC) was proven by using an MTT assay against MDA (breast cancer) cells. The PJC provokes the death of breast cancer cells via apoptosis (Habib et al. 2023). Furthermore, the extract obtained from the pomegranate fruit exhibited the ability to reduce epithelial to mesenchymal transition and inhibit migration in triple‐negative breast cancer cells (MDA‐MB‐231 cells) through the down‐regulation of nuclear factor kappa light‐chain‐enhancer of activated B cells (Mukherjee et al. 2023). Upon the addition of the water‐based extract derived from the pomegranate fruit (WPFE) to neoplastic mammary epithelial HMLER and breast cancer Hs578T cells, a notable decline in their regenerative capacity was noted. WPFE exerts an influence on the characteristics of breast cancer stem cells and effectively inhibits the process of epithelial‐to‐mesenchymal transition (Nallanthighal et al. 2017).

Pomegranate peel extract (PPE) dulled the migration and invasion of MDA‐MB‐231 cells at a concentration range of 25–1000 μg/mL. In the 500 μg/mL and 1000 μg/mL concentrations, PPE induced apoptosis. PPE suppressed the gene expression of vimentin, ZEB1, and β‐catenin and also increased the expression of E‐cadherin in triple‐negative breast cancer cells. Consequently, PPE presents itself as a hopeful candidate for a drug aimed at diminishing metastasis in triple‐negative breast cancer cells (Bagheri et al. 2018). The cancer cell line MDA‐MB‐231 was used for the investigation of anticancer effects of anthocyanidins (delphinidin, cyanidin, and pelargonidin) present in the pomegranate juice. The efficiencies on the cancer cell growth were as follows: delfinidin > cyanidin > pelargonidin at all three tested concentrations 10, 25 and 50 μM. The same arrangement of anthocyanidins was observed in the analysis of their proapoptotic effects. It was also found that cooper chelation decreased the growth inhibition and apoptosis induced by delphinidin. Namely, neocuproine at the concentration of 50 μM significantly protected MDA‐MB‐231 cells from the effects of delphinidin (50 μM), unlike iron and zinc chelators. Neocuproine and ROS scavengers (thiourea, catalase and superoxide dismutase) significantly inhibited delphinidin‐induced apoptosis indicating that intracellular copper reacts with anthocyanidins and cause DNA damage via the generation of ROS. It was found that delphinidin decreased the migratory capacity of MDA‐MB‐231 cells with less susceptibility to metastasis. The metastatic potential was regained after copper from the cells was chelated by neocuproine in the presence of delphinidin (Farhan et al. 2022). Solid lipid nanoparticles with an optimized pomegranate extract (PE‐SLNs) in a transdermal emulgel were evaluated on mice skin bearing a solid form of Ehrlich ascites carcinoma (EAC). Transdermal gel (0.5 g) was applied twice daily and beside PE‐SLNs, also with free PE, optimized ellagic acid‐SLNs emulgel, and optimized void‐SLNs (without drug) emulgel for comparison. After 6 days of treatment, the progression, and growth of the tumor in all groups decreased, in contrast to the control group. Tumor volume was significantly decreased (p < 0.01) in the group treated with PE‐SLNs in comparison to the negative control group, which didn't receive any drug or external treatment. The maximum average tumor volume in the PE‐SLNs group was 237.42 ± 93.77 mm³ compared to the negative control group where it reached 2.5 times larger volume (589.21 ± 198.52 mm³). The calculated inhibition rate of the tumor value showed the highest inhibition percentage in the group treated with PE‐SLNs (59.71%) in comparison to other groups. The group treated with free PE emulgel exhibited slight inhibition (10.71%). This work suggests and confirms synergistic and/or additive properties of various phytochemical compounds found in pomegranate (Seeram et al. 2006; Hong et al. 2008). It also recommends nanoparticles as biodegradable and biocompatible carriers of ellagitannins. This encapsulation overcomes poor absorption and low bioavailability and gastrointestinal hydrolysis (Shirode et al. 2015). Also, the optimized PE‐SLNs emulgel expressed observable tumor tissue necrosis which was more than 50% per field in Ehrlich solid carcinoma (ESC) tissues. This research presented a promising delivery carrier for PE in the formulation of optimized PE‐SLNs transdermal emulgel to be applied to solid breast carcinoma (Teaima et al. 2022).

3.2. Clinical Studies of Pomegranate on Breast Cancer

The risk of breast cancer is particularly high in post‐menopausal women as levels of estrone (produced from the conversion of adrenal androgens by the aromatase enzyme) and the more biologically‐active estradiol (metabolized from estrone by the 17β‐hydroxysteroid dehydrogenase type 1 enzyme) are increased while the concentration of sex hormone binding globulin (SHBG) is reduced (Bernstein and Ross 1993; Rebecca et al. 2011). Pomegranate juice (PJ) has been reported to inhibit the aromatase enzyme as well as 17β‐hydroxysteroid dehydrogenase type 1 (Kim et al. 2002). The results of the first randomized‐controlled clinical study on the effects of PJ in breast cancer were reported in 2015. In this study, 64 healthy post‐menopausal women drank fresh refrigerated 100% PJ for 3 weeks (118.3 mL in the morning and 118.3 mL in the early evening). The serum levels of estradiol, free estradiol, estrone, testosterone, androstenedione, and SHBG were measured. The results of the study showed that there was no significant reduction, neither in the levels of the sex hormones nor in those of SHBG in the group that consumed PJ compared to the control. A significant reduction in the levels of estrone and testosterone was evident in the serum of normal weight women who consumed the juice compared to the control group (Kapoor et al. 2015). To date, however, clinical evidence directly evaluating pomegranate‐derived interventions in breast cancer patients remains limited. Beyond the endocrine‐biomarker trial in post‐menopausal women, there is a paucity of contemporary, adequately powered randomized studies assessing clinically relevant endpoints (e.g., tumor response, recurrence, or validated surrogate biomarkers) in defined breast cancer populations. Recent synthesis of the clinical literature similarly indicates that human data in breast cancer are scarce and largely restricted to hormonal risk markers, underscoring the need for well‐designed trials using standardized preparations and exposure‐based biomarkers (e.g., circulating/urinary urolithins) to link intake, metabolism, and biological effects (Jang et al. 2024).

4. Therapeutic Effects of Pomegranate on Colon Cancer

4.1. Preclinical Studies of Pomegranate on Colon Cancer: Molecular Mechanisms and Signaling Pathways

4.1.1. In Vitro

The in vitro investigation of anticancer effects of pomegranate and its products on colon cancer cells are quite extensive and numerous (Table 2). These studies usually include the participation of Caco‐2, HT‐29 and HCT116 cell lines, and research with other cancer lines is less reported. Caco‐2 and HT‐29 cells are epithelial cells isolated from human colorectal adenocarcinoma, and HCT116 cell line was isolated from the colon cancer patient, and has a mutation in codon 13 of the ras proto‐oncogene (ATCC 2023). Pomegranate, originated from Egypt, has been studied in several studies on colon cancer lines. Motaal and Shaker (2011) investigated the activity of 50% ethanolic extracts of the peel, pulp, and whole pomegranate fruit on HCT116 cells. Whole fruit extract was the most superior with IC₅₀ of 4.8 ± 0.02 μg/mL, followed by pulp extract (6.1 ± 0.04 μg/mL) and peel extract (9.3 ± 0.06 μg/mL). Although the peel extract contained the highest amount of ellagic acid (1.9% ± 0.01%), it was not the most active in the anticancer activity assay. The best effect was shown by the extract of the whole pomegranate fruit with the concentration of ellagic acid of 0.3% ± 0.05%. Therefore, it is considered that other compounds such as sterols, catechins, epicatechins, anthocyanins, and other flavonoids are also responsible for the activity (Motaal and Shaker 2011). In a further study, an 80% ethanolic extract of the peel expressed less cytotoxicity towards HCT116 cells with IC₅₀ value of 19 μg/mL. The extract was rich in phenolic compounds, primarily in hesperidin (31.9 mg/100 g of the extract), pyrogallol (116.3 mg/100 g), catechin (41.9 mg/100 g) and gallic acid (13.4 mg/100 g) (El‐Feky 2022). Shalaby et al. (2019) studied the ethanolic and aqueous peel extracts, and the juice in a cytotoxic assessment on the same colon cancer cells. The ethanolic peel extract inhibited the cells growth with IC₅₀ of 12.51 ± 1.64 μg/mL, followed by the juice (IC₅₀ = 16.12 ± 1.28 μg/mL) and aqueous extract (IC₅₀ = 19.22 ± 0.13 μg/mL). The tested pomegranate samples were rich in phenolic acids and flavonoids, primarily in pyrogallol and hesperidin in the extracts, and pyrogallol and luteolin‐6‐arabinoside‐8‐glucoside in the juice. Interestingly ellagic acid, which has exhibited excellent cytotoxicity, was totally absent from the ethanolic extract (Shalaby et al. 2019). Moghazy et al. (2019) studied the inhibitory effects of aqueous peel extract on the growth of HCT116 and Caco‐2 cells suggesting the probable mechanism of anticancer effects. The extract, rich in protocatechuic (186.37 μg/mL) and p‐hydroxybenzoic acid (155.08 μg/mL), and rutin (460.50 μg/mL), significantly affected Pre G1 and G2/M phase elevating the cells accumulation, but on the other hand it did not have the effects on the G0‐G1 phase. The anti‐apoptotic activity of the extract showed that the expressions of the pro‐apoptotic genes, caspase‐3 and related p53, were significantly increased while the expressions of the anti‐apoptotic gene (Bcl‐2) was significantly decreased, after the treatment. This aqueous peel extract inhibited the cancer cells growth with IC₅₀ values of 1434 μg/mL for Caco‐2 and 2257 μg/mL for HCT116 cells (Moghazy et al. 2019). Fermented and unfermented pomegranate had cytotoxic effects against HCT116 cells (IC₅₀ = 21.10 ± 1.96 and 24.60 ± 2.60 μg/mL, respectively) attributed to inhibition of the activities of urokinase and histone deacetylase. Pomegranate, in both forms, decreased the level of vascular endothelial growth factor, which is a marker of angiogenesis (210.00 ± 23.50 and 255.88 ± 28.40 pg/mg, respectively, vs. DMSO 610.20 ± 64.30 pg/mg). In addition, they had a potential to inhibit metastasis as measured by reduction in the activity of elastase enzyme (0.80 ± 0.07 and 0.88 ± 0.09 U/mg protein, respectively, vs. DMSO 2.00 ± 0.23/mg protein) (Mahmoud and Ali 2014). Pomegranate fruit, from the United States of America, and its preparations were also studied in vitro. Namely, pomegranate juice, punicalagin, ellagic acid, and a standardized total pomegranate tannin (TPT) extract expressed high antiproliferative and apoptotic activities toward colon cancer cells. TPT extract contained 85% punicalagin, 1.3% ellagic acid hexoside, and in minor quantities other ellagitannins and ellagic acid glycosides. The juice, containing 1.74 mg/mL of punicalagin and 0.14 mg/mL of ellagic acid, was shown to be the most potent antiproliferative agent by inhibiting proliferation from 30% to 100% in the concentrations 12.5–100 μg/mL. Punicalagin, ellagic acid, and TPT inhibited cell growth in a dose‐dependent manner but to a lesser extent compared to juice. Nonmetastatic colon cancer cells, SW480, were sensitive to the investigated polyphenols, whereby ellagic acid inhibited the cell proliferation from 49% to 76%, punicalagin from 1% to 65%, and TPT from 1% to 67%, at the concentration range of 12.5–100 μg/mL. Ellagic acid reduced proliferation from 14% to 35%, punicalagin from 0% to 57% and TPT from 0.02% to 40% in the SW620 metastatic colon cancer cells. HT‐29 cells were sensitive to ellagic acid with the inhibition of 0%–21%, to punicalagin with 1%–55%, and to TPT with 2%‐ 1%. Ellagic acid induced antiproliferative effects in HCT116 cells from 53% to 87%, punicalagin from 0% to 72% and TPT from 13% to 87%. Furthermore, the juice, ellagic acid, punicalagin, and TPT induced apoptosis in HT‐29 cells, at equivalent doses of 100 μg/mL. The apoptosis of HCT116 cells was induced only by the presence of ellagic acid, punicalagin, and TPT. The results indicated the importance of chemical synergism among the compounds in the expression of the biological effects (Seeram et al. 2005).

TABLE 2.

In vitro preclinical studies of pomegranate on colon cancer.

Pomegranate preparation	Tested concentration (IC50)	Colon cancer model	Mechanism of action	References
Whole fruit extract (50% ethanol)	Whole fruit: 4.8 ± 0.02 μg/mL, Pulp: 6.1 ± 0.04 μg/mL, Peel: 9.3 ± 0.06 μg/mL	HCT116 cells	↓ Cell proliferation, ↓ VEGF, ↑ Apoptosis	Motaal and Shaker (2011)
Peel extract (80% ethanol)	19 μg/mL	HCT116 cells	↓ Cell viability, ↓ Proliferation	El‐Feky (2022)
Ethanolic peel extract	12.51 ± 1.64 μg/mL	HT‐29 cells	↓ Cell viability, ↓ Proliferation, ↑ Apoptosis	Shalaby et al. (2019)
Aqueous peel extract	1434 μg/mL (Caco‐2), 2257 μg/mL (HCT116)	Caco‐2, HCT116 cells	↓ Tumor growth, ↓ Inflammatory markers	Moghazy et al. (2019)
Fermented pomegranate extract	21.10 ± 1.96 μg/mL	HCT116 cells	↓ Urokinase activity ↓ Histone deacetylase ↓ VEGF	Mahmoud and Ali (2014)
Pomegranate juice, punicalagin, and TPT extract	Juice: 30%–100% inhibition, TPT: 1%–67% inhibition	HT‐29, SW480, SW620 cells	↓ NF‐κB ↓ COX‐2 ↑ Apoptosis	Seeram et al. (2005)
Methanolic leaf extract	70.56% ± 0.90% inhibition at 250 μg/mL	HT‐29 cells	↓ Cell viability ↑ Caspase‐3	Balamurugan et al. (2021)
Pomegranate seed extract (Nigerian study)	133.77 μg/mL (seeds), 81.056 μg/mL (peel)	HT‐29 cells	↓ Cell viability ↑ Caspase‐3 activation	Mónica et al. (2020)
Pomegranate peel extract (Moroccan study)	127.58–203.24 μg/mL	HT‐29 cells	↓ Cell viability ↓ Tumor growth markers	Eddebbagh et al. (2016)
Bosnian pomegranate peel extract	74.85 ± 2.99 μg/mL (24 h), 40.15 ± 1.25 μg/mL (48 h)	HCT116 cells	↓ Cell viability ↓ Tumor size	Keta et al. (2020)
Pomegranate seed oil extract	63.2 μg/mL	LS174 cells	↓ Tumor proliferation ↑ Apoptosis	Đurđević et al. (2018)
Pomegranate‐derived metal nanoparticles	250 μg/mL (HCT116), ZnO‐nanoparticles: 31.25 μg/mL	HCT116 cells	↑ Apoptosis ↓ Cell viability ↓ Inflammatory markers	Mohamad Sukri et al. (2019)
Hydromethanolic extracts (Mexican pomegranate)	IC50 > 200 μg/mL	LS180 cells	↓ Cell proliferation, moderate cytotoxic effects	Cortez‐Trejo et al. (2022)
Anti‐angiogenic effects of pomegranate extract	753 mg polyphenols/day	Colo 205 cells (angiogenesis model)	↓ Angiogenesis, ↓ VEGF expression, ↓ Tumor weight	Sudha et al. (2021)
Pomegranate peel extract (Italian study)	5–50 μg/mL	LoVo cells	↓ Cell viability, ↑ Apoptosis	Moreira et al. (2017)
Pomegranate extract (various fruit stages)	100 μg/mL (peel, immature and mature fruit extracts)	HCT116 cells	↓ Tumor viability in various fruit stages	Russo et al. (2021)
Punicalagin‐rich pomegranate peel fraction	IC50 = 36.58 ± 4.24 μg/mL	HT‐29 cells	↓ Tumor growth, ↑ Apoptosis via mitochondrial pathway	Chen et al. (2022)
Pomegranate extract in combination with 5‐fluorouracil	2.5 mL/kg/day	HT‐29 cells	↓ Tumor progression, ↑ Sensitivity to chemotherapy	Banerjee et al. (2013)

Note: Symbols: ↓—Decrease; ↑—Increase.

Abbreviations: COX‐2, cyclooxygenase‐2; HCT116, HT‐29, Caco‐2, LS180, colon cancer cell lines; IC₅₀, inhibitory concentration 50%; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; TPT, total pomegranate tannin extract; VEGF, vascular endothelial growth factor.

In further research, it was shown that the pomegranate juice, punicalagin, and TPT inhibited HT‐29 cancer cell proliferation and apoptosis through the modulation of cellular transcription factors and signaling proteins. The juice suppressed TNF‐α induced COX‐2 protein expression by 79% at a concentration of 50 μg/mL. TPT inhibited the protein expression by 55%, and punicalagin by 48% at the same concentration of 50 μg/mL. Pretreatment with the juice completely inhibited AKT activity at 50 μg/mL. In addition, the treatment with juice, at the same concentration, suppressed binding to the NF‐κB response element and phosphorylation of the p65 subunit by 6.4 times. TPT reduced NF‐κB binding 10 times at a concentration of 100 μg/mL, punicalagin 3.6 times at 100 μg/mL, while ellagic acid was inactive (Adams et al. 2006). Balamurugan et al. (2021) studied a methanolic leaf extract, prepared from Indian pomegranate, and determined the maximum inhibition of HT‐29 growth of 70.56% ± 0.90% (for the extract concentration of 250 μg/mL) (Balamurugan et al. 2021). The cancer cell line HT116 was employed for the testing of Indian methanolic pomegranate fruit extract, which was able to inhibit cell growth by 62% at a concentration of 100 μg/mL (Badyal et al. 2016). Mónica et al. (2020) studied Nigerian pomegranate for their cytotoxic potentials revealing that the peel and seeds ethanol, chloroform, and hexane extracts showed inhibitory effect on HT‐29 colon cancer cells. The ethanol extract made of pomegranate seeds and chloroform extract made of peel were superior with IC₅₀ values of 133.77 and 81.056 μg/mL, respectively (Mónica et al. 2020). The methanolic extracts of Moroccan pomegranate peel, leaves, branches, flowers, and corolla expressed antiproliferative effects on HT‐29 cells with IC₅₀ values of 127.58–203.24 μg/mL. The leaf extract was the most potent while peel extract was the weakest in its cytotoxic effect. These effects were in strong correlation with total polyphenolics and flavonoids content (Eddebbagh et al. 2016). Anticancer activity of pomegranate (harvested in Bosnia and Herzegovina) ethanolic peel extract was tested on HCT116 human cancer cell line along with MRC‐5 normal fibroblasts. The extract contained punicalin in the concentration of 182.41 mg/g, followed by punicalagin (50.92 mg/g), gallic acid (6.11 mg/g) and ellagic acid (22.54 mg/g). The test showed that the extract effectively killed the cancer cells with the IC₅₀ values of 74.85 ± 2.99 μg/mL after 24 h, and 40.15 ± 1.25 μg/mL after 48 h. It is of great importance that the extract showed good selectivity, having the effect on cancer cells more than on normal ones. As a response to the treatment of the extract, at the concentration of 16 μg/mL, HCT116 cells showed minor G1 block after 48 h. There were no significant changes detected between treated (16 μg/mL) and untreated cancer cells after 24 h, in the terms of their ability to migrate which was estimated by wound healing assay (Keta et al. 2020). The cell viability of colon cells HT‐29 and HCT116 was determined after the incubation with the 80% methanolic extract prepared from pomegranate seed oil, originated from Italy. The extract (concentration of 0.4 mg/L) inhibited the growth of both cancer cell lines in all tested volumes from 0.12 to 0.60 μL (Costantini et al. 2014).

Russo et al. (2021), conducted an interesting investigation with the extracts prepared from pomegranate fruits. They studied the anticancer actions of acidic hydroalcoholic extracts prepared from mature and immature fruits, mesocarp/aril, and peels. They investigated two forms of immature fruits, ‘baby red’ and ‘baby green’. Extracts (at 100 μg/mL, each) from different maturation stages significantly inhibited HCT116 cancer cell proliferation. The extracts made of peels showed better cytotoxic effects compared to mesocarp/aril extracts (Russo et al. 2021). The strongest anti‐proliferative activities were achieved with the peel extracts from immature pomegranate fruits. ‘Baby red’ pomegranate peel extract expressed the greatest statistically significant anticancer effect. Treatments with peel extracts, made of immature fruits at the concentration of 100 μg/mL, significantly suppressed cell metabolic viability and induced apoptosis by elevating the activity of caspase‐3. The extract of ‘baby green’ immature peels was the richest in polyphenols, with nearly 353 mg polyphenols/g of fresh fruit, followed by the extract of ‘baby red’ immature peels with 296 mg/g of fresh fruit. Punicalagin a and b were the most abundant secondary metabolites in all investigated samples except ‘baby red’ pomegranate mesocarp/arils which contained total gallotannin as the most present compounds (Russo et al. 2021).

Chinese authors studied the anticancer effects of pomegranate peel extracts rich in ellagitannins in HT‐29 human colorectal cancer cells. The punicalagin‐rich fraction was not active after the treatment at the concentration of 80 μg/mL for 24 h. On the other hand, two other fractions, the punicalin and the granatin B + punicalagin fractions reduced cell viability by 33.2% ± 4.4% and 60.1% ± 3.2%, respectively. After 48 h, at the same concentration, all pomegranate peel fractions expressed stronger cell viability reductions. Finally, at 72 h the cell viability reductions were the most superior with the values of 65.3% ± 2.4%, 86.2% ± 2.2%, and 61.9% ± 4.1% for the punicalin‐rich fraction, granatin B + punicalagin fraction, and punicalagin‐rich fraction, respectively. Furthermore, the granatin B + punicalagin fraction exhibited time‐ and dose‐dependent cytotoxicity with the IC₅₀ values of 58.71 ± 4.57 μg/mL after 48 h and 36.58 ± 4.24 μg/mL after 72 h, indicating the best anticancer activity among tested fractions. In addition, the granatin B + punicalagin fraction was the most active in the inhibition of tumor growth in a xenograft mouse tumor model. It is of importance that no pathological changes in the major mouse organs were observed. This pomegranate peel fraction was able to induce apoptosis in the tumors which was indicated by the increased cleavage of caspase 8 and caspase 3. Cell death mechanisms were further investigated, and Hoechst 33342 staining showed that the HT‐29 cells treated with the pomegranate peel fractions, at the concentration of 40 μg/mL for 48 h, exhibited chromatin condensation, karyopyknosis, and apoptotic body formation, which are typical characteristics of apoptosis. The apoptosis induced by the granatin B + punicalagin fraction was further analyzed with annexin V‐FITC/PI double labeling. After 72 h, apoptotic cells treated with in 40, 80, and 160 μg/mL increased to 36.10% ± 1.75%, 76.30% ± 1.72%, and 79.00% ± 1.85%, respectively, compared to the control (7.18% ± 1.85%). JC‐1 staining indicated that the granatin B + punicalagin fraction might trigger ROS‐mediated mitochondrial damage to induce apoptosis further leading to the decrease of MMP. The elevated levels of cleaved caspase 3, 8, and 9 demonstrated that both extrinsic and mitochondria‐mediated apoptotic pathways are involved in the cellular apoptosis. Finally, the analysis of the cell cycle activity showed the extract dose‐dependently arrested the cells in S‐phase and reducing cells in G1‐phase. Moreover, the granatin B + punicalagin fraction sensitized HT‐29 cells to 5‐fluroruracil‐induced cell death and S‐phase cell cycle arrest. The combination of 5‐fluroruracil with the granatin B + punicalagin fraction enhanced the toxicity to HT‐29 cells by increasing cell cycle arrest and NF‐κB signaling, while inhibiting autophagy (Chen et al. 2022). The ethanolic fruit peel extract, originated from Thailand, stimulated Caco‐2 cells proliferation with the ED₅₀ of 4.7 μg/mL. On the other hand, the extract was able to stimulate peripheral blood mononuclear cells (PBMC) proliferation, as well, suggesting that the application of this extract should be used with some caution (Okonogi et al. 2007). Ethanolic extracts made from seeds and husks of pomegranate from Saudi Arabia were highly active against Caco‐2 cells with IC₅₀ values of 45 and 40 μg/mL, respectively (Awad and El‐Awady 2015). On the other hand, Brazilian authors Jardini et al. (2007) revealed that the hydroalcoholic extract of the pomegranate pulp, at a concentration of 200 μg/mL, showed low inhibition of Caco‐2 cells growth of 4.16%, although the absorption of the extract's antioxidant phytocompounds was 63.25% (Jardini et al. 2007).

Caco‐2 cells were used for evaluating the anti‐inflammatory potential of pomegranate extracts. A hydroalcoholic pomegranate (from Morocco) fruit husk extract, rich in punicalagin (87.4 ± 3.8 mg/g) and ellagic acid (23.9 ± 2.5 mg/g), was able to suppress the inflammation in Caco‐2 cells. The cells were pretreated with the extract or punicalagin as a standard compound, at the apical side, simulating the intestinal lumen. Inflammation was induced with lipopolysaccharide and a cocktail of cytokines IL‐1β, TNF‐α and IFN‐γ. It was found that the extract and punicalagin showed anti‐inflammatory effects acting both on the pro‐inflammatory gene transcription and protein levels of IL‐6, IL‐8, and monocyte chemoattractant protein‐1, possibly due to a direct molecular trapping (Hollebeeck et al. 2012). An aqueous pomegranate peel extract, made of waste material of Italian pomegranate juice production, was found to suppress inflammation in Caco‐2 cells and in porcine colonic tissue explants. The inflammations were induced by TNF in the cells and by LPS in the colonic tissue. The expressions of mediators of the inflammatory response, IL‐1α, IL‐6, and CXCL 8 (IL‐8) were significantly decreased by the extract concentration of 5 μg/mL. Chemical composition analysis of the extract determined the presence of α‐ and β‐punicalagin with 146.9 ± 1.465 mg/g and 266.3 ± 1.687 mg/g, and gallic acid, ellagic acid, and granatin B in small quantities (Mastrogiovanni et al. 2019). Other human colon cancer cells, such as LS 180, colo 205, LoVo, LS 174, were used in fewer studies. The hydromethanolic extracts of three pomegranate fruits, from Mexico, of different aryl color (red, pink, and white) were evaluated for their in vitro cytotoxicity. Inhibitory concentrations (IC₅₀) were over 200 μg/mL toward colorectal cancer cells LS 180, and were characterized as moderately active. Among phenolic acids galloyl‐6‐O‐glucoside was the most abundant in all three extracts. D‐(+)‐catechin was a predominant flavonoid in the red‐aryl fruit, and phellatin was the most present in the white‐ and pink‐aryl pomegranate fruit (Cortez‐Trejo et al. 2022). The process of angiogenesis in cancer enables cancer growth and metastasis. The antiangiogenic effect of pomegranate fruit extract (capsule produced in USA containing about 753 mg GAE of polyphenols) against colon cell lines (colo 205) was investigated on the chick chorioallantoic membrane model (CAM). The extract was inoculated to chick embryos at 7th day after cancer cell implantation at the dose of 5–20 μg/mL. The extract significantly reduced the tumor weight, hemoglobin content of tumor mass, and the average blood vessel branch count (Sudha et al. 2021). Water peel extract, made of pomegranate from Israel, exhibited significant inhibition of the growth of colon (LoVo) cancer cells. It is of importance that both doxorubicin‐sensitive and doxorubicin‐resistant cells sublines were sensitive. The inhibitions of the cell growth were 18%–34% and 5%–10%, respectively, especially at higher extract concentrations of 25–75 μg/mL (Moreira et al. 2017). Hydroethanolic peel extracts made of pomegranate from different parts of western Herzegovina, Croatia, expressed antiproliferative effects toward SW620 colorectal adenocarcinoma, metastatic cells. The extracts inhibited 50% of the cell growth with the concentration range of 163–354 μg/mL. The majority of identified phytocompounds in the extracts were tannins (punicalagins, ellagic acid pentose or hexose derivatives, and punicalin) (Peršurić et al. 2020). The seed oil, isolated from Bosnian pomegranate, showed moderate anti‐angiogenic and cytotoxic effects toward LS 174 colon adenocarcinoma cells with an IC₅₀ value of 63.2 μg/mL. The oil contained high quantity of unsaturated fatty acids such as punicic, oleic, and linoleic acids as dominant ones, following by tocopherols and carotenoids. The oil did not show activity against normal MRC‐5, a human diploid lung fibroblast cell line, indicating its safety (Đurđević et al. 2018).

There are also very interesting studies that investigated the anticancer effects of pomegranate‐mediated synthesized metal nanoparticles. The cytotoxicity effects of pomegranate peel water extract showed no activity toward normal colorectal cells CCD112 while cancer cells HCT116 showed more than 50% decrease in viability with the extract concentration of 250 μg/mL. On the other hand, pomegranate/ZnO‐nanoparticles were able to kill both cell lines. The concentration of 31.25 μg/mL of particles expressed cells‐growth reduction of 60%–80% with no specific selection towards the cancerous cells or the normal colonic cells (Mohamad Sukri et al. 2019). Ramya and Narayana Rao (2022) examined the cytotoxicity of Ag and Au nanoparticles made with P. granatum peel water extract. The nanoparticles demonstrated profound effects against HCT116 cells having half‐maximum inhibitory concentrations of 107 and 143 g/mL, respectively (Ramya and Narayana Rao 2022). On the other hand, iron oxide nanoparticles showed no effects (IC₅₀ > 250 μg/mL) against HCT116 cells (Yusefi et al. 2020). Devanesan et al. (2018) examined the cytotoxic effects of Ag nanoparticles made with P. granatum water peel extract toward RKO colon cancer cell line. The cells viabilities were 56% and 61% on 3rd and 5th day, respectively, after the exposure of 12.5 μg of nanoparticles (Devanesan et al. 2018). An interesting investigation by Esther Lydia et al. (2020) showed potent anticancer effect of a functional yoghurt, with added Au nanoparticles made with P. granatum seed oil, on HT‐29 cells with cells viability ranging from 83.3% ± 1.2% to 28.4% ± 1.2% (Esther Lydia et al. 2020).

4.2. In Vivo Studies of Pomergranate on Colon Cancer Animal Models

There are several animal studies with chemically induced colon carcinogenesis which indicated the protective effects of pomegranate (Table 3 and Figure 4). The largest number of studies was performed on the azoxymethane‐induced colon cancer model. The first such study was carried out by Kohno et al. (2004). Azoxymethane was used to develop colonic malignancies in experimental rats in a study of anticancer properties of pomegranate seed oil. The oil contained more than 70% of (9Z,11E,13Z)‐18:3 as conjugated linolenic acids and its effects were compared with that of conjugated linoleic acid. Azoxymethane was administered subcutaneously (20 mg/kg body weight) once a week, for 2 weeks. Before the azoxymethane administration, rats were fed with the pomegranate oil or conjugated linoleic acid for 32 weeks (0.01%, 0.1%, or 1% of the oil or 1% of conjugated linoleic acid). The administration of the pomegranate oil significantly reduced the incidence (for 44%, 38% and 56% in the diet with 0.01%, 0.1%, or 1% of the oil) and multiplicity of colonic adenocarcinomas while conjugated linoleic acid feeding had slightly and insignificantly reducing effects. The dietary administration of the pomegranate oil and conjugated linoleic acid increase the expression of peroxisome proliferator‐activated receptor γ protein in colonic mucosa of azoxymethane‐treated rats. The inhibition of tumors formation by the pomegranate oil was in correlation with an increased content of conjugated linoleic acid ((9Z,11E)‐18:2) in the lipid fraction of rat colonic mucosa and liver (Kohno et al. 2004). Further study investigated pomegranate juice made of the fruits from the USA. The rats received the juice, presented with 20% in a diet, for 13 weeks. Azoxymethane was injected subcutaneously at 16 mg/kg body weight, at 7th and 8th weeks of age. At 17th week of age, the rats were killed and colons were analyzed. Total number of the aberrant crypt foci (proximal + distal colon) in pomegranate‐treated rats was 15.67 ± 1.86 in contrast to untreated group of rats with the number of 171.67 ± 5.6. (Boateng et al. 2007). An aqueous‐methanolic extract of pomegranate (from Egypt) peel was investigated at the doses of 200 and 400 mg/kg. Azoxymethane was administered by s.c. injections at the dose of 15 mg/kg body weight for 12 weeks. Histopathological analysis showed that the treatment with the pomegranate extract improved dysplastic changes of the colonic histoarchitecture. It was found that it significantly, in a dose‐dependent manner, decrease the total number of aberrant crypt foci. Cancer cell proliferation in the colon was also reduced which was shown by proliferating cells nuclear antigen (PCNA). Per os administration of the peel extract led to the reduction of cancer tumor and pathophysiological markers gamma‐glutamyl transpeptidase, carcinoembryonic antigen, alkaline phosphatase and lactate dehydrogenase. Further analysis of the rat colon showed that the expression of pro‐inflammatory proteins and cytokines were significantly reduced (COX‐2, iNOS, TNF‐α and IL‐6) which was confirmed by immunohistochemical analysis (Omara et al. 2011). Waly et al. (2012) investigated suppressing effects of the aqueous extract of pomegranate peel on azoxymethane‐induced carcinogenesis in rat colon. The development of tumors was achieved by intraperitoneal injections of azoxymethane once a week for 2 weeks at a dose of 15 mg/kg bodyweight. This dose of the extract provided 47 mg of polyphenols expressed as GAE/bw. The extract was shown to be an excellent colon protective agent in azoxymethane‐induced cytotoxicity in rat colon. Namely, the administration of the extract to the rats inhibited the aberrant crypt foci development, over three times in comparison to untreated group. Aberrant crypt foci are commonly accepted as the precursors for tumors in the colon. In addition, the extract improved the levels of biochemical indicators of oxidative stress in the colonic tissue such as glutathione transferase, glutathione peroxidase, glutathione reductase, superoxide dismutase, and catalase. In a further study by Waly et al. it was revealed that the same extract prevented chromosomal aberrations decreasing the percentage of bone marrow cells with micronuclei. The antioxidant properties of the extract were demonstrated by its ability to prevent DPPH and ABTS radical formation, as well as the formation of oxidized proteins and malondialdehyde in colon cells. Additionally, the extract was able to decrease the impairment of reduced glutathione/oxidized glutathione (GSH/GSSG) ratio and the activity of myeloperoxidase (Waly et al. 2012). Banerjee et al. (2013) studied the effects of pomegranate juice consumption on azoxymethane‐induced colorectal cancer in rats. The juice, prepared from pomegranates from the USA, was administered for 10 weeks providing 2504.74 mg of polyphenols expressed as gallic acid equivalents/L. Azoxymethane was injected subcutaneously at weeks two and three, at a dose of 15 mg/kg. The treatment with pomegranate juice led to suppressed number of aberrant crypt foci and dysplastic aberrant crypt foci by 29% and 53.5% respectively. The administration of the juice decreased the expression of COX‐2, iNOS, NFκB, VCAM‐1, mRNA, and protein, inhibited phosphorylation of PI3K/AKT and mTOR expression, and increased the expression of miR‐126. After an in vivo study of chemopreventive effects of a pomegranate juice (originated from USA) on azoxymethane‐induced colon cancer in rats, the possible role of miR‐126 was investigated in HT‐29 colon cancer cell lines. The in vitro study confirmed that the juice suppressed colon tumorigenesis targeting, in part, miR‐126‐regulated pathways. An important role of miR‐126 was confirmed using the antagomiR for miR‐126, where the juice reversed its effect on the expression of VCAM‐1, PI3K p85β, and miR‐126 (Banerjee et al. 2013). Bastide et al. (2017) conducted interesting study to examine the protective effects of polyphenols on cured meat promotion of colonic mucin depleted foci in cancer‐induced rats. The authors showed that intake of a cured meat with erythorbate promotes colon carcinogenesis in rats. In addition, they revealed that a pomegranate extract, rich in polyphenolics, could inhibit this effect by limitation of endogenous lipid peroxidation and nitrosation. The extract was administered to the rats, which were fed with the workshop‐made cured meat for 14 days, as well as to the rats for 100 days, which were treated with azoxymethane (20 mg/kg, single dose, i.p.) and fed with cured meat. The concentration of the extract, containing 12% of ellagic acid, in the diet was 0.6% w/w. Both experiments showed the reduced levels of hem, TBARs, percentage of the dead cells in fecal water, and dihydroxynonene mercapturic acid in the urine. Dihydroxynonene mercapturic acid is the main urinary metabolite of 4‐hydroxynonenal, a major end product of endogenous lipid peroxidation. The number of aberrant crypt and mucin‐depleted foci were significantly reduced in the long‐term study. The removal of erythorbate from cured meat led to even more reduction of the foci. The protection which came from the pomegranate extract was associated with the suppression of nitrosyl iron excretion by feces, pointing that this nitroso compound could be a promoter of the process of carcinogenesis (Bastide et al. 2017). The most recent experiment with a similar model of cancer induction tested the juice and peel water extract of pomegranate from Egypt. Azoxymethane solution was applied continuously for 2 weeks at a dose of 20 mg/kg/week. The first group of rats were treated with 5‐fluorouracil (12.5 mg/kg) and two groups were treated with 5‐fluorouracil and the juice (2.5 mL kg/day, by oral gavage) or with 5‐fluorouracil and the extract (1.5 mL kg/day, by oral gavage) for 8 weeks. The solution of 5‐fluorouracil was administered on the first, third and fifth day with a repeat cycle every 4 weeks for 4 months. The results showed that rats treated with juice or aqueous peel extract showed increase in weight gain, food intake, blood hemoglobin, hematocrit, and high‐density lipoprotein cholesterol level, compared to the control. The levels of COX‐2, prostaglandin E2, cytochrome P450 were significantly decreased, as well as total cholesterol, triglycerides, nitric oxide, IL‐1 and TNF‐α compared to the controls. The chemical composition, mineral content and antioxidants of pomegranate peel juice and extract were estimated. The juice and extract were rich in antioxidant phenolics and flavonoids (Abu Al‐Majd et al. 2022). 1,2‐Dimethylhydrazine is a colon specific carcinogen transforming into intermediates azoxymethane and methylazoxymethanol by the liver. These compounds methylate DNA and generate oxidative stress causing aberrant crypts foci (Benguiar et al. 2020). Rat colon carcinogenesis induced by 1,2‐dimethylhydrazine dihydrochloride was significantly reduced after the treatment with a standardized pomegranate fruit extract. The extract was standardized to the major ellagitannins (not less than 30% punicalagin R and punicalagin b) and about 5% of ellagic acid. The results showed that Wnt‐target genes, that is, Wnt5a, FZD8 (frizzled receptor‐8), β‐catenin, TCF/LEF (T cell factor/lymphoid enhancer binding protein), c‐myc, and CCND1 (cyclin D1), were upregulated, while APC (adenomatous polyposis coli) and AXIN1 were downregulated in colonic tissues in the induced cancer group compared with the normal group. The standardized pomegranate extract was able to diminish all the aberrant alterations in the studied genes in cancer colonic tissues. Anticancer effects were also evidenced by the histopathological analysis, and the normalization of survival rate of the rats, inhibition of tumor incidence and a reduction of the level of a serum tumor marker, carcinoembryonic antigen (Sadik and Shaker 2013). Benguiar et al. (2020) showed that pomegranate peel in combination with probiotics could be effective chemopreventive agents in colon carcinogenesis. 1,2‐Dimethylhydrazine, for cancer induction, was administered to the laboratory rats by subcutaneous injection at the dose of 30 mg/kg body weight, for a period of 9 weeks, once every week. Pomegranate peel was given to the rats, alone or in the combination with the probiotics, within high fat diet in the concentration of 2.5%. The results showed that the diets with pomegranate peel significantly reduced total cholesterol, triglycerides, AST and ALT levels in the rat plasma. In addition, the incorporation of pomegranate peel into diets significantly increased ferric reducing ability of plasma levels and reduced the malonaldehyde levels. Finally, the treatment with the pomegranate peel, alone or in combination with probiotics, helped to improve the histological tumor changes of the colon in the rats with induced colon carcinogenesis, compared to controls. Colons of the rats, fed with pomegranate peel only, presented low‐grade dysplasia. Pomegranate peel in combination with probiotics in the diet restored the normal histological appearance of the mucosa with homogenous gland structure, in comparison to untreated group of animals. These treatments significantly reduced the count of pathogenic bacteria, and increased beneficial bacteria (Benguiar et al. 2020). Another study investigated the beneficial effect of a peel methanolic extract made of pomegranate fruit from Egypt, where colon cancer in rats was induced intrarectally using N‐methylnitrosourea. Phenolic components identified in the extract were gallic acid (1.366 mg/mL), protocatechuic acid (0.047 mg/mL), catechin (0.377 mg/mL), rutin (0.136 mg/mL), ellagic acid (4.643 mg/mL), and punicalagin (1.910 mg/mL). N‐methylnitrosourea was administered three times a week in the dose of 2 mg/0.5 mL of water, for 5 weeks. The rats were treated with 5‐fluorouracil (12.5 mg/kg, i.p.) on 1st, 3rd and 5th day with the cycle repeated every 4 weeks, or with the extract (2.25 or 4.5 g/kg, p.o.) administered daily for 6 months. The groups of the rats that received the extract, particularly one treated with higher doses, exhibited a significant reduction of different parameters related to colon cancer induction, with improved histological presentation of the tissue. The extract significantly decreased the plasma levels of TGF‐β and Bcl2, and serum levels of epidermal growth factor, carcinoembryonic antigen, colon cancer specific antigens, and matrix metalloproteinase‐7. Immunohistochemistry revealed a decrease in COX‐2, cyclin D1, and survivin content. The extract was able to downregulate the expression levels of colonic CTNNB1, KRAS, and c‐myc genes in a dose dependent manner. Based on the obtained results the potential mechanism of anticancer effects, which includes pro‐apoptotic, anti‐inflammatory, and antiproliferative activities, might depend on the inhibition of the Wnt/β‐catenin signaling pathway (Ahmed et al. 2017). An ethanolic extract of pomegranate increased the expression of caspase‐3 and induced apoptosis in mouse crypt cells, which was shown in an in vivo study by Kusmardi et al. (2021). The authors treated experimental mice with 2% dextran sodium sulfate (DSS) and pomegranate peel extract in two doses, 240 mg and 480 mg/kg bw/day. The quantity of ellagic acid, determined by HPLC, in the extract was 11.047%. It was shown that the extract in the second dose, in the microscopic observation of immunostaining, increased the caspase‐3 expression compared to the control group of mice. The mean H‐scores obtained for each treatment group were: 213.23 ± 8.32 (DSS group), 243.81 ± 18.69 (normal group), 226.10 ± 12.38 (pomegranate peel extract of 240 mg/kg/day) and 238.84 ± 15.81 (pomegranate peel extract of 480 mg/kg/day)(Kusmardi et al. 2021).

TABLE 3.

In vivo studies of pomegranate on colon cancer.

Pomegranate preparation	Colon cancer model	Doses and treatment duration	Mechanism of action	Effects	References
Pomegranate seed oil	Azoxymethane‐induced rat model	0.01%, 0.1%, or 1% of diet for 32 weeks	↓ Colonic adenocarcinoma incidence, ↑ PPARγ	↓ Tumor incidence by 44%, 38%, 56% depending on dose	Kohno et al. (2004)
Pomegranate juice	Azoxymethane‐induced rat model	20% of diet for 13 weeks	↓ Aberrant crypt foci, ↓ Dysplastic aberrant crypt foci	↓ Aberrant crypt foci by 91%, ↓ Dysplastic aberrant crypt foci	Boateng et al. (2007)
Aqueous‐methanolic pomegranate peel extract	Azoxymethane‐induced rat model	200–400 mg/kg, administered for 12 weeks	↓ Tumor growth, ↓ PCNA, ↓ COX‐2, ↓ TNF‐α, ↓ IL‐6	↓ Tumor progression, ↓ Cell proliferation markers	Omara et al. (2011)
Aqueous pomegranate peel extract	Azoxymethane‐induced rat model	47 mg polyphenols/kg, administered for 2 weeks	↓ Aberrant crypt foci development, ↑ Antioxidant enzyme activity, ↓ Malondialdehyde	↓ Oxidative stress, ↓ DNA damage	Waly et al. (2012)
Pomegranate juice (USA origin)	Azoxymethane‐induced rat model	2504.74 mg polyphenols/L, administered for 10 weeks	↓ COX‐2 ↓ iNOS ↓ NF‐κB ↓ VCAM‐1 ↑ miR‐126	↓ Inflammatory cytokines, ↓ Oncogenic signaling	Banerjee et al. (2013)
Pomegranate extract (polyphenol‐rich)	Azoxymethane and high‐fat diet‐induced rat model	12% ellagic acid in diet (0.6% w/w) for 100 days	↓ Lipid peroxidation, ↓ Nitrosation, ↓ Mucin‐depleted foci	↓ Colon cancer‐promoting markers, ↓ Tumor formation	Bastide et al. (2017)
Pomegranate peel extract (Egyptian study)	N‐methylnitrosourea‐induced colon cancer model	2.25 or 4.5 g/kg body weight, daily for 6 months	↓ TGF‐β, ↓ Bcl‐2, ↓ Cyclin D1, ↓ β‐Catenin, ↓ C‐myc	↓ Tumor size ↓ Cancer proliferation markers	Ahmed et al. (2017)
Pomegranate peel and probiotics	High‐fat diet with 1,2‐dimethylhydrazine induction	2.5% pomegranate peel in high‐fat diet for 9 weeks	↓ Colonic dysplasia, ↓ Pro‐inflammatory markers, ↑ Beneficial gut microbiota	↓ Dysplasia severity, ↓ Tumor risk	Benguiar et al. (2020)
Pomegranate extract (N‐methylnitrosourea model)	N‐methylnitrosourea‐induced colon cancer model	2 mg/0.5 mL, 3 times a week for 5 weeks	↓ Tumor burden, ↓ Carcinoembryonic antigen (CEA), ↓ Matrix metalloproteinase‐7	↓ Tumor growth, ↓ Metastatic potential	Ahmed et al. (2017)
Pomegranate mesocarp decoction	Pirc rat model with mutated Apc gene	151.47 ± 3.42 mg/g ellagitannins in diet for 6 weeks	↓ Mucin‐depleted foci, ↑ Caspase‐3, ↓ PCNA, ↓ Wnt‐target genes	↓ Tumor progression, ↑ Apoptosis	Tortora et al. (2018)

Note: Symbols: ↓: decrease/downregulation. ↑: increase/upregulation.

Abbreviations: % (w/w), percent weight/weight; Apc, adenomatous polyposis coli; Bcl‐2, B‐cell lymphoma 2; CEA, carcinoembryonic antigen; c‐Myc, MYC proto‐oncogene (c‐Myc); COX‐2, cyclooxygenase‐2; DNA, deoxyribonucleic acid; g/kg, grams per kilogram; IL‐6, interleukin‐6; iNOS, inducible nitric oxide synthase; MDA, malondialdehyde; mg/kg, milligrams per kilogram; mg/L, milligrams per liter; miR‐126, microRNA‐126; MMP‐7, matrix metalloproteinase‐7; NF‐κB, nuclear factor kappa B; PCNA, proliferating cell nuclear antigen; Pirc, Polyposis in Rat Colon (rat model carrying a mutant Apc allele); PPARγ, peroxisome proliferator‐activated receptor gamma; TGF‐β, transforming growth factor beta; TNF‐α, tumor necrosis factor alpha; VCAM‐1, vascular cell adhesion molecule 1; Wnt, Wingless/Int‐1 signaling pathway; β‐catenin, beta‐catenin.

FIGURE 4.

Anticancer effects of Punica granatum in colon cancer: insights from in vitro and in vivo studies. In cancer cell lines, pomegranate extract induces cell cycle arrest by increasing G2/M phase regulation and promoting apoptosis through mitochondrial dysfunction, ROS generation, Bax activation, and cyclin‐dependent pathways. It also inhibits angiogenesis by suppressing VEGF and downregulating pro‐inflammatory markers such as IL‐1β, IL‐6, COX‐2, NF‐κB, TNF‐α, and IFN‐γ. Additionally, pomegranate exhibits synergistic effects with chemotherapeutic drugs, enhancing cytotoxicity. In animal models, pomegranate extract administration leads to tumor size and growth reduction, exhibits a chemopreventive effect, reduces aberrant crypt foci, and enhances beneficial gut microbiota. ↑, Increase; ↓, Decrease; Bax, Bcl‐2‐associated X protein; COX‐2, cyclooxygenase‐2; G2/M, cell cycle phase; IFN‐γ, interferon gamma; IL‐1β, interleukin‐1 beta; IL‐6, interleukin‐6; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; ROS, reactive oxygen species; TNF‐α, tumor necrosis factor‐alpha; VEGF, vascular endothelial growth factor.

Pomegranate water extract prepared from leaves, rich in flavonoids, phenols, phytosterols, tannins and carbohydrates, showed strong antigenotoxic effects in an experiment with Swiss albino mice. The protective effect of the extract was observed against cyclophosphamide‐induced DNA damage along with the inhibition of hepatic lipoperoxidation, the increase in reduced glutathione, glutathione S‐transferase, superoxide dismutase, and catalase. Three doses of the leaf extract (200, 400, and 800 mg/kg) were active in antigenotoxic effects estimated by the mouse bone marrow micronucleus test (Dassprakash et al. 2012). The effect of decoction from the mesocarp of Italian‐origin pomegranate was also investigated for its chemopreventive potential in a rat model of colorectal cancer. HPLC analysis confirmed the presence of ellagitannins at a concentration of 151.47 ± 3.42 mg/g, primarily composed of α‐ and β‐punicalagins (67.5 ± 1.24 mg/g), along with ellagic acid and its derivatives (21.7 ± 0.35 mg/g). The decoction was administered for 6 weeks in the diet to the Pirc rats, mutated in Apc, a key‐gene in colorectal cancer. Pirc rats develop polyps and pre‐neoplastic lesions in the colon (mucin‐depleted foci) representing the early step in the development of colon cancer. The number of mucin‐depleted foci were significantly reduced, along with a lower multiplicity, in the group of the rats treated with pomegranate, compared to controls (34 ± 4 vs. 47 ± 3). Additionally, apoptotic index in the foci from treated rats was increased compared to the rats from control groups (2.5 ± 0.2 vs. 1.6 ± 0.2). In order to elucidate the mechanisms of chemopreventive effect of pomegranate decoction, further in vitro assays were performed. The main metabolites of pomegranate urolithin‐A and sodium butyrate, were tested, alone or in combination, on HT‐29 and HCT‐116 colon cancer cells in vitro, and ex vivo in biopsies of normal colon mucosa and cancer colon foci (adenoma) from Pirc rats. The IC₅₀ values for urolithin‐A and sodium butyrate, in the viability test of HT‐29 cells after 72 h, were 43.9 μM and 3 mM, respectively. The results of IC₅₀ for HCT116 cells were 59.2 μM for urolithin‐A, and 0.7 mM for sodium butyrate. It was found that the combination of these two metabolites did not act synergistically but only additively. The effect of the metabolites on proliferation, apoptosis, and inflammation markers was studied in HT‐29 cells. The expression of the proliferation marker PCNA was significantly reduced by sodium butyrate at the concentration of 2.5 mM after 24 h, while urolithin‐A (25 μM) and their combination reduced it slightly but insignificantly, compared to the controls. Further, the expression of caspase‐3, an apoptosis marker, was also increased by the combination of the metabolites, in the same concentrations, during 24 h. The expressions of the inflammatory markers iNOS and COX‐2 in cancer cells were reduced by all the tested compounds after 72 h, but the treatment with their combination was the most active. In a short ex vivo experiment, the combination of urolithin‐A and sodium butyrate inhibited the growth of the cells isolated from normal colon mucosa and adenoma by 75% and 50%, respectively. The effect of the compounds on the proliferation marker PCNA was insignificant. The expressions of the pro‐apoptotic proteins, CASP‐3 and BAK, were increased in both type of colon samples, while COX‐2 protein expression was decreased (Tortora et al. 2018).

4.3. Anticancer Activities of Metabolites of the Pomegranate Compounds in Colon Cancer

In a number of studies, urolithin A, along with other urolithins, was shown to possess strong immunomodulatory and anticancer effects (Rogovskii 2022) (Table 4). It was demonstrated that the P. granatum ellagitannins and their intestinal bacterial metabolites, urolithins, expressed strong effects on the initiation and promotion development stages of colon cancer (Núñez‐Sánchez et al. 2016). Namely, ellagitannins and urolithins inhibited 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin‐induced CYP1‐mediated EROD (ethoxy resorufin‐O‐deethylase) activity in HT‐29 cells. Their IC₅₀ values ranged from 56.7 ± 2.6 μM for urolithin A to 76.3 ± 3.3 μM for urolithin D. The investigated compounds decreased proliferation and clonogenic efficiency of HT‐29 cells in both time‐ and dose‐dependent manners. The HT‐29 cell proliferation was inhibited through cell cycle arrest in the G0/G1 and G2/M stages of the cell cycle, followed by the activation of apoptosis. IC₅₀ of antiproliferation activity for the pomegranate constituents were varied from 123 ± 9.3 μM for hexahydroxydiphenic acid to 378 ± 6.5 μM for urolithin A (Kasimsetty et al. 2010). It was found that exposure of ellagic acid, and urolithin A and B to Caco‐2 cells arrested the cell growth at the S‐ and G2/M‐phases. The changes in the expression levels of MAPK signaling genes such as growth factor receptors (FGFR2, EGFR), oncogenes (K‐Ras, c‐Myc), and tumor suppressors (DUSP6, Fos) and of genes involved in cell cycle (CCNB1, CCNB1IP1) were observed (González‐Sarrías, Espín, et al. 2009). Furthermore, these compounds modulated phase I and II detoxifying enzymes in Caco‐2 cells inducing the expression and activity of enzymes CYP1A1 and UGT1A10, and inhibiting several sulfotransferases (González‐Sarrías, Azorín‐Ortuño, et al. 2009). A pomegranate juice, made of fruits from Iran, and punicalagin were able to inhibit the sulfoconjugation of 1‐naphthol in Caco‐2 human colon carcinoma cells (Saruwatari et al. 2008).

TABLE 4.

Anticancer activities of pomegranate metabolites in colon cancer.

Metabolite	Tested concentration (IC50)	Colon cancer model	Mechanism of action	References
Urolithin A	56.7 ± 2.6 μM	HT‐29 cells	↓ CYP1‐mediated EROD activity, ↓ Cell proliferation, ↓ Clonogenic efficiency, ↑ Cell cycle arrest (G0/G1, G2/M), ↑ Apoptosis	Núñez‐Sánchez et al. (2016)
Urolithin D	76.3 ± 3.3 μM	HT‐29 cells	↓ Cell proliferation ↓ Clonogenic efficiency ↑ Apoptosis	Núñez‐Sánchez et al. (2016)
Ellagic acid	19.0 μg/mL	HCT‐15 cells, Caco‐2	↓ PI3K/AKT, ↑ Bax, ↑ Caspase‐3, ↑ Cytochrome C, ↓ Bcl‐2, ↑ Apoptosis	Umesalma et al. (2015)
Ellagitannins	30 μg/mL	HT‐29, Caco‐2, CCD‐112CoN cells	↓ Cyclins A & B1, ↑ Cyclin E, ↑ Apoptosis (↑caspase‐9 activation)	Larrosa et al. (2006)
Punicalagin	Inhibits sulfoconjugation of 1‐naphthol	Caco‐2 cells	↓ Sulfoconjugation of carcinogens, ↑ Detoxification	Saruwatari et al. (2008)
Hexahydroxydiphenic acid	123 ± 9.3 μM	HT‐29 cells	↓ Cell proliferation, ↓ Wnt signaling, ↑ DNA damage response	Kasimsetty et al. (2010)
Galactomannan polysaccharide	46.3 ± 1 μg/mL	HCT116 cells	↑ Apoptosis, ↑ DNA fragmentation, ↓ Cell viability	Joseph et al. (2013)
Ellagic acid and urolithins (combined)	378 ± 6.5 μM	HCT‐15 cells	↓ Cell cycle progression (S phase, G2/M), ↑ Apoptosis via mitochondrial pathway	Umesalma et al. (2015)
Ellagic acid and punicalagin	Inhibits CYP1‐mediated carcinogen metabolism	Caco‐2 cells	↓ CYP1A1 expression, ↓ Carcinogen bioactivation, ↑ Detoxification	González‐Sarrías, Azorín‐Ortuño, et al. (2009)

Note: Symbols: ↓—Decrease; ↑—Increase.

Abbreviations: AKT, protein kinase B; Bax, Bcl‐2‐associated X protein; Bcl‐2, B‐cell lymphoma 2; CYP1, cytochrome P450 family 1; EROD, ethoxyresorufin‐O‐deethylase; G0/G1, G2/M, S phase, cell cycle phases; IC₅₀, inhibitory concentration 50%; MAPK, mitogen‐activated protein kinase; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PI3K, phosphoinositide 3‐kinase; Wnt, Wingless‐related integration site.

Dietary ellagitannins of pomegranate, punicalagin, and its hydrolysis product, ellagic acid, were tested for their pro‐apoptotic effects on human colon cancer cells Caco‐2 and colon normal cells CCD‐112CoN. Both compounds displayed the same effects on cancer cells with a downregulation of cyclins A and B1 and upregulation of cyclin E, cell‐cycle arrest in S phase, induction of apoptosis via intrinsic pathway (FAS‐independent, caspase 8‐independent) through bcl‐XL downregulation with mitochondrial release of cytochrome c into the cytosol, activation of initiator caspase 9 and effector caspase 3. Punicalagin expressed the effects after its hydrolysis to ellagic acid, which entered into the cells and metabolized to dimethyl derivatives. This study suggests that the anticancer effect of dietary ellagitannins of pomegranate is attributed mainly to their hydrolysis product, ellagic acid, which induced apoptosis via mitochondrial pathway in colon cancer Caco‐2 cells. It is of importance that punicalagin and ellagic acid did not induce apoptosis in normal colon CCD‐112CoN cells (Larrosa et al. 2006). It was found that ellagic acid could induce cell cycle arrest at G2/M phase in HCT‐15 cells. This phenolic compound induced apoptosis which was accompanied by an inactivation of phosphatidylinositol 3‐kinase (PI3K)/Akt pathway. At the same time, it promoted the expression of Bax, caspase‐3, and cytochrome c, and suppressed the activity of Bcl‐2 in HCT‐15 cells with increased annexin V apoptotic cells and DNA fragmentation (Umesalma et al. 2015). Galactomannan polysaccharide, isolated from the fruit peel, showed cytotoxic properties on human colon cancer cell line HCT116 in the wide range from 0.001 to 1000 μg/mL with the IC₅₀ value of 46.3 ± 1 μg/mL after the incubation period of 72 h. A dose‐dependent gradual increase in cytotoxicity was observed, regardless of incubation time. In addition, doxorubicin, a positive control, was about four times weaker with the IC₅₀ value of 189 ± 1.1 μg/mL. Cytotoxicity of galactomannan polysaccharide was achieved through the induction of apoptosis which was evidenced by a decrease in cell number and specific morphological characteristics such as distorted shape, membrane blebbing, and the presence of apoptotic bodies (Joseph et al. 2013). Wnt‐target genes are known to be the most important in human colon carcinogenesis. Sharma et al. (2010) investigated the effects of urolithin‐A, ellagic acid, and ellagitannins‐rich fruit pomegranate extract on Wnt signaling in a human 293 T cells. The fruit extract, with 3.5% of ellagic acid, and > 25% of punicalagin α and punicalagin β, was studied for its effect on Wnt signaling using a luciferase reporter of canonical Wnt pathway‐mediated transcriptional activation. The pomegranate extracts inhibited Wnt signaling (IC₅₀ about 30 μg/mL), along with ellagic acid (IC₅₀ = 19.0 μg/mL) and urolithin‐A (IC₅₀ = 9.0 μg/mL), which both had the prominent effect (Sharma et al. 2010).

4.4. Clinical Studies of Pomegranate on Colon Cancer

Ellagic acid and the ellagitannins from pomegranate, as well as their metabolites (urolithins), have demonstrated protective effects on colon cancer in vitro and in animal models (Núñez‐Sánchez et al. 2015). Ellagic acid, ellagic acid conjugates, gallic acid, and up to 12 different urolithin metabolites were tested for their effects on normal and malignant tissues of colon cancer patients after an intake of pomegranate extracts (PE) at a dose of 900 mg/day for 15 days before surgical resection (Nuñez‐Sánchez et al. 2014). Specific and moderate modulation of various microRNAs in tissues from colon cancer patients can be done after the intake of two plant‐based capsules containing 450 mg of pomegranate extracts per day until surgery. Unfortunately, there is the difficulty in discriminating these effects from those attributed to surgical protocols (Nuñez‐Sánchez et al. 2015). Furthermore, new studies by Nuñez‐Sánchez have shown that colon tissue gene expression changes occurred following oral intake of pomegranate extracts and they were not connected with the metabolite types of the individuals or specific metabolites in the colon tissues gene expression in the human tissues influenced by interindividual variability and experimental procedures (Nuñez‐Sánchez et al. 2017). Another clinical study evaluated the effect of the consumption of pomegranate extracts (900 mg/day) on the levels of lipopolysaccharide‐binding protein, a contributor of endotoxemia, which has been implicated in the onset of colon cancer. The results of the study showed that pomegranate extracts were able to reduce the levels of lipopolysaccharide‐binding protein in patients with newly‐diagnosed colon cancer (González‐Sarrías et al. 2018). Importantly, since these translational studies, few new interventional clinical trials in colorectal cancer have been reported, and available human evidence remains centered on short‐term pre‐surgical supplementation studies evaluating tissue delivery and molecular readouts rather than long‐term clinical outcomes. Accordingly, future research should prioritize multicenter randomized trials with standardized pomegranate formulations, stratification by urolithin metabotype/microbiota features, and clinically meaningful endpoints (e.g., neoplasia recurrence, adjuvant‐treatment tolerance, inflammatory/immune signatures) to better delimit conclusions and inform perspectives.

5. Therapeutic Effects of Pomegranate on Prostate Cancer

5.1. Preclinical Studies of Pomegranate on Prostate Cancer

Numerous studies confirm the antiproliferative and cytotoxic effects of pomegranate ( Punica granatum L.) or its constituents on prostate cancer (PC) cell lines where normal prostate epithelial cells are significantly less affected (Guzmán‐Lorite et al. 2025) (Figure 5). Adaramoye investigated the effect of hydrolyzable ellagitannin punicalagin on two prostate cancer cell lines (PC‐3 and LNCaP) as well as healthy prostate cells (BPH‐1) (Adaramoye et al. 2017). The 10, 50, and 100 μM concentrations inhibited 30%, 60%, and 70% viability of LNCaP in a concentration‐dependent manner, and 28%, 52%, and 55% of PC‐3, while BPH‐1 viability was affected insignificantly. Also, punicalagin (50 and 100 mM) increased cell death in PC‐3 cells where the apoptosis was mediated by the significant expression of caspase‐3 and caspase‐8. LNCaP was affected only with 100 mM and showed insignificant expressions of both enzymes. The mechanisms punicalagin exerted antiproliferative activity in PC cells were apoptosis and anti‐angiogenic effect. The impact of ethyl acetate extracts of pomegranate pericarp polyphenols and fermented juice polyphenols as well as ethanol extract of cold pressed seed oil was investigated on PC‐3, DU 145, and LNCaP prostate cancer cell lines (Albrecht et al. 2004). The effect of the extracts was also evaluated on the normal human prostatic stromal and epithelial cells and human prostatic stromal cells isolated from patients with benign hyperplasia. The most notable effects were seed oil extracts on LNCaP cells, where the lowest concentration required to inhibit prostate cancer cell proliferation by 50% was (IC₅₀ = 20 μg/mL). Pericarp and seed oil extracts suppressed the proliferation of androgen‐independent DU 145 cells with IC₅₀ = 30 μg/mL. The mechanism was 2.3‐fold upregulation of the cyclin‐dependent kinase inhibitor p21 and 0.6‐fold downregulation of proto‐oncogene c‐myc. Fermented juice extract was also most active against DU 145 with IC₅₀ = 45 μg/mL. Overall, all three extracts acted in dosages from 20 to 100 μg/mL on cancer cell lines, while normal and benign hyperplasia cells were inhibited by 50 to 300 μg/mL. It was important to notice that the seed oil extract did not affect the proliferation of normal and benign cells at all. Since the most powerful effect all extracts proved on the DU 145 cell line, different sets of experiments presented that pomegranate extract affected the cell cycle distribution lowering the number of cells in the G1 phase and G2M arrest. Apoptosis was the other mechanism of cell death induced by increased caspase‐3 activity for 5.3 ± 0.9 and 9.4 ± 1.3‐fold in PC‐3 and DU 145 cells, respectively. Extracts also affected gene regulation suggesting the powerful effect that diet and natural compounds could have on cancer prevention and development (Albrecht et al. 2004).

FIGURE 5.

Schematic representation of the proposed anticancer mechanisms of Punica granatum in prostate cancer. The bioactive compounds reduce tumor growth, migration, and invasiveness by inhibiting MMPs and modulating pro‐inflammatory cytokines. They also induce apoptosis through mitochondrial damage, caspase activation, and suppression of antiapoptotic proteins. Additionally, pomegranate impacts cell cycle regulation by promoting G2/M arrest and reduces angiogenesis via VEGF/HIF‐1α inhibition. ↑: increase; ↓: decrease; Bax: Bcl‐2‐associated X protein; Bcl‐2: B‐cell lymphoma 2; c‐Myc: cellular myelocytomatosis oncogene; G1: gap 1 phase of the cell cycle; G2/M: transition between G2 phase and mitosis; G2: gap 2 phase of the cell cycle; HO: heme oxygenase; HUR: human antigen R; ICAM‐1: intercellular adhesion molecule 1; IL‐12: interleukin‐12; IL‐1β: interleukin‐1 beta; IL‐6: interleukin‐6; M: mitosis phase; Mcl‐1: myeloid cell leukemia‐1; MMP‐2: matrix metalloproteinase‐2; MMP‐9: matrix metalloproteinase‐9; p21: cyclin‐dependent kinase inhibitor 1; ROS: reactive oxygen species; S: synthesis phase; SIRT1: sirtuin 1; TIMP1: tissue inhibitor of metalloproteinases‐1; VEGF: vascular endothelial growth factor.

The same cell lines were tested on the activity of pomegranate fruit juice (PFJ) and ellagic acid (EA) for possible mechanisms of apoptosis, while chemopreventive effects on the development and progression of prostate adenocarcinoma were evaluated in a transgenic rat (TRAP) model (Naiki‐Ito et al. 2015). Ellagic acid in physiological pH did not affect any of the cells, yet at pH 6.5 EA inhibited the proliferation of LNCaP cells only up to 35%. The concentrations of 75 and 100 mM EA increased the number of apoptotic cells. Immunoblotting revealed activation of caspase‐3 in LNCaP cells. The Bax was increased while the Bcl‐2 level was slightly decreased. Also, p21 and p27 expressions were decreased and cyclin E increased suggesting that PJF inhibited proliferation through initiation of cycle arrest along with apoptosis. EA also decreased the levels of androgen receptor (AR) and prostate‐specific antigen (PSA) in LNCaP cells in a dose‐dependent manner. The TRAP group treated with PFJ exhibited a significantly lower percentage (p < 0.01) as well as decreased incidence of adenocarcinoma in the lateral prostate compared to the control group (p < 0.0001). Other tests revealed that PFJ suppressed carcinogenesis of the prostate by induction of caspase 3‐dependent apoptosis and in that way inhibited cell growth. These findings suggested that daily intake of PFJ and EA inhibited the early stages of prostate carcinogenesis and that EA is the most likely the active compound that exerts this effect (Naiki‐Ito et al. 2015). Another research of EA investigated anti‐invasive effects on rat prostate cancer cell lines (PLS10) in vitro and androgen‐independent human PC‐3 cancer cells (Pitchakarn et al. 2013). As in the research of Naiki‐Ito et al. (2014), EA did not inhibit the growth of PC‐3 cells. On the other hand, it affected the PLS10 cells with IC₅₀ around 100 μM. Treatment with EA inhibited the invasion of both cell lines. PC‐3 cells were inhibited for 57% and 78% and PLS10 for 39% and 52% in 25 and 50 μM concentrations, respectively. The motility of the cancer cells was also reduced. Inhibition rates for PC3 cells were 41% and 77%, while for PLS10 cells were 13% and 67% in 25 and 50 μM, respectively. This research also established that the invasion of these cell lines does not happen through the regulation of matrix metalloproteinase‐2 (MMP‐2) and MMP‐9 secretion, proteins responsible for tumor growth, angiogenesis, and invasion. EA also significantly inhibited the activity of purified collagenase type IV for 32%, 68%, and 87% in PLS10 cells treated with 25, 50, and 100 μM, respectively indicating that suppression of this enzyme by EA might reduce the invasiveness of prostate cancer cell lines. The impact of pomegranate peel ethanol extract was evaluated on human prostate cancer cell lines DU145, PC3, and mouse prostate cancer cells TRAMP‐C1 (Deng et al. 2017). The main constituents of peel extract were punicalagin and ellagic acid. The extract inhibited the proliferation of both human prostate cancer cell lines in higher doses (50 and 100 μg/mL) after 72 h incubation. Mouse prostate cancer cells were more susceptible to concentration‐dependent inhibition after 24 h incubation. The mechanism of cell growth inhibition in TRAMP‐C1 was mitochondrial apoptotic pathway and an increase in the Bax/Bcl‐2 ratio which manifested as decreased expression of anti‐apoptotic Bcl2 protein and increased expression of caspase‐3 and pro‐apoptotic Bax protein after treatment. Pomegranate peel extract also reduced migration ability and significantly decreased invasion capabilities of TRAMP‐C1 cells via reduced levels of MMP2/MMP9 and increased tissue inhibitors of metalloproteinase 2 (TIMP2) expression. Evidence pointed out that pomegranate extract prepared from skin and arils without seeds and standardized to ellagitannin content (37% punicalagin) exhibited potent apoptotic effects in human prostate cancer cell line LAPC4 (Koyama et al. 2010). This extract inhibited cell proliferation by 20% in 10 μg/mL concentration. The combination of the extract with insulin‐like growth factor binding protein‐3 (IGFBP‐3) (1 μg/mL) suppressed the growth for 30% indicating an additive effect on LACP4 growth inhibition. This co‐treatment had an even more pronounced apoptotic induction of cancer cells than cell growth inhibition. Studying the underlying mechanisms of apoptosis, the same author discovered that the combination of extract and IGFBP‐3 decreased Ser‐473 phosphorylation. On the other hand, the extract reduced phosphorylation of mTOR at Ser2448 and slightly at Ser2481 while IGFBP‐3 slightly increased it. Their combination did not change the mTOR phosphorylation, suggesting their distinct apoptosis induction mechanisms. The extract and IGFBP‐3 increased Jun N‐terminal kinases (JNK) phosphorylation in a dose‐dependent manner, hence their combination did not display the additive outcome. This implies that the pomegranate extract and IGFBP‐3 might activate JNK by the exact mechanism and that its activation by the different factors leads to the induction of distinct targets. It is also established that pomegranate extract may suppress endogenous tumor production of IGF‐I but unlikely that is the only mechanism involved. Apoptotic events in various cancers by the control of the IGF axis may have beneficial curative effects.

Lansky investigated three different pomegranate extracts (fermented juice polyphenols) (in therapeutically active doses), peel polyphenols, and seed oil (in sub‐therapeutic doses) on the proliferation of human prostatic cancer cell line DU 145 as well as the more aggressive PC‐3 cells and its invasion across an in vitro model of metastasis (Matrigel) (Lansky, Harrison, Froom, et al. 2005; Lansky, Harrison, Froom, and Jiang 2005). The results showed that oil (16.5 μg/mL) and peel (6.25 μg/mL) did not affect the cell growth in sub‐therapeutic doses though both acted synergistically with the fermented juice polyphenols with the peak at 25 and 50 μg/mL, respectively. The combination of all three extracts in the total concentration of 3 μg/mL suppressed the invasion of PC‐3 human prostate cancer cells across Matrigel by 99%. The same combination also reduced the expression of phospholipase A2 by 95%, the enzyme linked with prostate cancer cell invasiveness and prostate tumors primarily (Jiang et al. 2002; Krishnan et al. 2009). This research shows the potency of different parts of pomegranate to inhibit the proliferation of prostate cancer cells and a way to treat different cancers or even diseases. Trying to determine which pomegranate compounds are responsible for the antiproliferative activity, Lansky evaluated its four components: ellagic acid, caffeic acid, luteolin, and punicic acid as inhibitors of PC‐3 human prostate cancer cell line and its invasion on artificial membranes (Matrigel) (Lansky, Harrison, Froom, et al. 2005; Lansky, Harrison, Froom, and Jiang 2005). All compounds individually inhibited invasion significantly (p ≤ 0.02), and the best activity than any single agent provided the combination of caffeic acid, luteolin, and punicic acid. These findings suggest that these compounds might express supra‐additive or possibly synergistic effects in inhibiting prostate cancer cell invasion in vitro. Ellagic acid exhibited antiproliferative effects on LNCaP cells by inhibiting mTOR activation and reducing β‐catenin at 25 and 50 μM concentrations (Vanella et al. 2013). The research included an evaluation of EA to induce cell cycle arrest and apoptosis. The anti‐apoptotic proteins: silent information regulator 1 (SIRT1), human antigen R (HuR), and heme oxygenase‐1 (HO‐1) were downregulated by EA, which triggered apoptosis. EA altered the expression of apoptosis‐inducing factor (AIF), leading to a significant rise in ROS levels and caspase‐3 activation. EA treatment increased the percentage of apoptotic cells and enhanced the expression of the tumor suppressor protein p21. The levels of IL‐6 and TGF‐β, both linked to more invasive forms of prostate cancer (Yang et al. 2008; Culig and Puhr 2012), were also decreased by EA treatment. All findings implied that EA could be a new method and a very effective tactic for lowering the incidence of prostate cancer.

Vicinanza examined the effects of EA and urolithin A (UA) on cell growth, cell cycle, and apoptosis in DU‐145 and PC‐3 PC cells and whether EA and UA combinations influenced cell growth (Vicinanza et al. 2013). EA showed better dose‐dependent growth‐inhibitory effects on PC‐3 cells (IC₅₀ = 14.5 ± 1.5 μM), while DU‐145 was strongly inhibited by UA treatments (IC₅₀ = 74.79 ± 2.4 μM). EA caused cell cycle arrest in the S phase linked to reduced cyclin B1 and cyclin D1 levels. UA caused a G2/M arrest and increased cyclin B1. EA triggered apoptosis in both cell lines, while UA had a less noticeable proapoptotic effect only in DU‐145. Combined treatment with low concentrations of EA and UA strongly reduced cell growth (7.5 μM of EA + 15 μM of UA), exhibiting synergism in PC‐3 cells. These data offer information on pomegranate metabolites for the prevention of PC relapse.

Lee investigated possible mechanisms of commercial pomegranate juice on inhibition of human prostate carcinoma cells DU145 (Lee et al. 2012). The most prominent suppression of PFJ happened in concentrations higher than 7.5 mg/mL where 70% of DU145 cells were dead after incubation treatment for 72 h. Normal cells were not affected. Further analysis determined that juice inhibited proliferation by triggering apoptosis in DU 145 cells by DNA fragmentation, mitochondrial pathway, and death receptor signaling. Also, in the highest concentration (12.5 mg/mL) phosphatidylserine translocation in the cell membrane was observed, which is the first sign of apoptosis leading to membrane leakage. 2DE‐based proteomics discovered proteins that were up‐ and downregulated with PJF treatment (valosin containing protein (VCP), human prolidase, α‐enolase (ENO1), β‐actin, proteasome subunit α type 3 (PSMA3), translationally controlled tumor protein (TCTP), mago nashi homolog 2, and ubiquitin‐conjugating enzyme E2N (UBE2N)). High expression of the aforementioned proteins is correlated to metastatic growth, higher invasiveness, antiapoptotic protection of cancer cells, and pure cancer prognosis. The results of the antiproliferative effects of DU145 by PJF are in concordance. Les recorded significant differences in PC‐3 cells at lower doses (31 μg/mL) of lyophilized organic PJ (Les et al. 2015). Cell viability was close to 40% at the highest dose (1000 μg/mL). The antiproliferative effect in PC‐3 cells was dose‐dependent. The authors indicated that other research reported better results of antiproliferative or cytotoxic effects in the same cell lines, but underline the use of different preparation of samples (concentrated and purified extracts) (Malik et al. 2005; Lucci et al. 2015) or even different parts of pomegranate (peel or seed extracts) (Sineh Sepehr et al. 2012). Pomegranate whole seed ethanolic extract, comprised of 72.8% punicic acid, exhibited dose‐dependent antiproliferative activity against hormone‐dependent prostate carcinoma LNCaP, with an IC₅₀ = 8.6 μg/mL (Lucci et al. 2015). This value was 3.4 times lower than the positive control vinblastine (IC₅₀ = 29.3 ± 0.6 μg/mL). Another study analyzed methanolic pomegranate seed extract (PSE) that inhibited the proliferation of 85.37% PC‐3 cells at a concentration of 500 μg/mL. The 20 μg/mL PSE dose exerted the lowest antiproliferative activity (77.8%). Taxol (20 μg/mL) caused 93.1% growth inhibition of PC‐3 cancer cells. Pomegranate peel extract (PPE) caused the highest growth inhibition (84.16% and 84%) of PC‐3 cells in 250 and 500 μg/mL concentrations, respectively (Modaeinama et al. 2015). These doses were comparable and not significantly different from 20 μg/mL Toxol, used as a positive control and expressed 93.1% growth inhibition on PC‐3 cells. Pomegranate seed and peel extracts were assessed for their antiproliferative activity on the PC‐3 cell line (Sineh Sepehr et al. 2012). The peel extract suppressed the growth with the IC₅₀ = 250.21 μg/mL while seed extract did not express significant inhibition. The viabilities of the PC‐3 cells exposed to peel extract at doses 10 and 600 μg/mL were 96.3% ± 7.8% and 24.1% ± 2.5%, respectively. Further analysis confirmed that pomegranate peel extract showed apoptosis of PC‐3 cells in the concentration of 250 μg/mL after 24 h, again showing that pomegranate could be a promising therapeutic agent in PC treatment. Malik indicated that pomegranate fruit extract (PFE) might provide chemo‐preventive and chemo‐therapeutic effects on humans (Malik et al. 2005). The author provided research on PFE (10–100 μg/mL; 48 h) on highly aggressive human prostate cancer PC‐3 cells. Treatment resulted in a dose‐dependent inhibition of cell growth, viability, and induction of cell‐cycle arrest and apoptosis. Apoptosis was associated with alterations in the levels of Bax and Bcl‐2 ratio towards increasing Bax, necrosis, and the dose‐dependent decrease in protein expressions of cyclin D1, D2, and E, as well as cyclin‐dependent kinase 2, 4, and 6. This extract also decreased the expression of prostate‐specific antigen and androgen receptors in CWR22Rv1 cells in a dose‐dependent manner. The highest extract concentration (80 and 100 μg/mL) decreased the AR expressions for 80% and 90%, and PSA protein levels for 38% and 62%, respectively. PFE (0.1 and 0.2%) given orally significantly inhibited the growth of prostate carcinoma CWR22Rv1 tumor xenograft in athymic nude mice as well as PSA secretion. These data can be clinically relevant and lead to the practical implication of pomegranate use. The PE ability to inhibit nuclear factor κB (NF‐κB), one of the most important inflammatory responses related to cancer, was assessed on LNCaP‐AR, LAPC4, CL1, and DU145 prostate cancer cells (Rettig et al. 2008). PE was made of pomegranate skin and standardized to ellagitannins as punicalagin (37%–40%) and ellagic acid (3.4%). The results showed that PE inhibited NF‐κB activity in DU145 cells in a dose‐dependent manner. PE also inhibited NF‐κB activation induced by TNF‐α in DU145 and CL1 cell lines. PE retards the growth and induction of apoptosis in LAPC4 xenografts in castrated mice. This research showed the importance of using pomegranate as the dietary product in the prevention of androgen independence driven by high NF‐κB activity. The same standardized extract (PE) was used in the research of Sartippour (Sartippour et al. 2008). The extract displayed inhibitory activity on LNCaP cells under normoxic (IC₅₀ = 5.7 ± 0.5 μg/mL) and hypoxic (IC₅₀ = 2.0 ± 0.2 μg/mL) conditions. PE (2.5 μg/mL) also reduced the levels of proteins (hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) that under hypoxic conditions, induce angiogenesis). After a 4‐week treatment, the extract exhibited a 5.9‐fold decrease in the size of LAPC4 xenografts in mice, decreased vessel density of tumor, as well as levels and expression of HIF‐1α and VEGF. The pomegranate extract inhibited angiogenesis in tumors and in that way slows the tumor growth. Seeram established that the use of ellagitannin‐enriched pomegranate extract (EEPE) led to the accumulation of ellagitannin metabolites, urolithins, in the prostate, colon, and intestine of the mice (Seeram et al. 2007). The EEPE also inhibited LAPC4 growth of the xenografts in immunodeficient mice. Ellagitannin and its metabolites displayed antiproliferative activity on human PC cells, where urolithins expressed better IC₅₀ values on the investigated cell lines (LNCaP, LNCaP‐AR, DU145, and 22RV1) than EA. This study indicated the treating and chemopreventive role of pomegranate. Pomegranate extract was also studied on metastatic castration‐resistant pancreatic cancer along with docetaxel chemotherapy improvement (Wang and Martins‐Green 2014). The pomegranate extract exerted strong in vitro cytotoxicity in the aggressive phenotypes of advanced cancer cells C4‐2, PC‐3, and ARCaPM with IC₅₀ values of 42, 78, and 161 μg/mL, respectively. Apoptotic induction was associated with increased cleavage of poly (ADP‐ribose) polymerase (PARP) and caspase‐3. The extract significantly reduced protein survivin, hence only modestly affecting Mcl‐1 protein in PC. Both proteins have an anti‐apoptotic effect and strongly correlate with bone metastasis. These findings show that survivin might be a novel molecular target for PC treatment. Treatment with PE along with docetaxel had a synergistic effect regarding toxicity to PC cells. Simultaneous application attenuated survivin levels, activated the apoptosis mechanism, and sensitized PC cells to docetaxel. The administration of PE alone or with docetaxel in athymic nude mice with intratibial C4‐2 xenografts, significantly reduced the PSA levels during the experiment. The extract effectively inhibited survivin, induced apoptosis, hinder C4‐2 tumor growth in the bone, and significantly enhanced the efficacy of docetaxel. Pomegranate juice (1% and 5%) was evaluated for anti‐progression effects on invasive prostate cancer cell lines DU145 and PC‐3 (Wang et al. 2011). The results showed increased cell adhesion by upregulation of genes involved in cell adhesion (E‐cadherin, ICAM‐1 and downregulation of genes involved in cell migration (type I collagen)). PJ significantly lowered the level of secreted pro‐inflammatory cytokines/chemokines such as IL‐6, IL‐12p40, IL‐1b, and RANTES, showing the potential to reduce inflammation and its impact on cancer progression. PJ blocks the ability of the chemokine stromal‐derived growth factor one alpha (SDF1α) to chemoattract cancer cells and prevents metastasis to the bone marrow. Additionally, Wang and associates examined luteolin, ellagic, and punicic acid impact on growth, adhesion, and migration of the same cell lines as in previous research, as well as chemotaxis toward chemokine SDF1α. Luteolin, ellagic, and punicic acid applied individually at 4 or 8 μg/mL significantly inhibited PC3 and DU145 cell migration. A combination of all three compounds (4 or 8 μg/mL) was more efficient in the inhibition of cell migration than any dual combinations. The effect of three compounds combination at 8 μg/mL inhibited SDF1α chemotaxis almost as 5% PJ. Also, the combination (8 μg/mL) enhanced the expression of cell adhesion genes and reduce the expression of genes involved in cell cycle control and cell migration. In addition, they increase several well‐known tumor‐suppression miRNAs, decrease several oncogenic miRNAs, and block the chemokines receptor type 4 (CXCR4)/SDF1α chemotaxis axis. The cancer cell line PC‐3 was used for the investigation of anticancer effects of delphinidin, cyanidin, and pelargonidin from the pomegranate juice. As for the breast cancer line, the effect of the anthocyanidins on PC‐3 cells growth and apoptosis were the strongest for delfinidin, followed by cyanidin and pelargonidin, at all three tested concentrations 10, 25, and 50 μM. It was also found that copper, iron, and zinc chelation could protect PC‐3 cells from the growth‐inhibition by delphinidin. Iron and zinc chelators, on the other hand, did not have any effects on induced apoptosis. As mentioned for breast cancer cells, neocuproine, a copper chelator, and ROS scavengers significantly inhibited delphinidin‐induced apoptosis. The antimetastatic potential of delphinidin was diminished after copper from the cells was chelated by neocuproine (Farhan et al. 2022).

5.2. Clinical Studies of Pomegranate on Prostate Cancer

The first clinical study that evaluated the effect of PJ intake (237 mL per day until disease progression) was carried out on men with PC and rising PSA after surgery or radiotherapy. The study showed a statistically significant effect of PJ on the prolongation of PSA doubling time (PSADT), along with in vitro activity on PC cell proliferation and apoptosis (Pantuck et al. 2006). Pomegranate extract was found to increase PSADT in patients across the range of baseline PSADT values, despite the fact that a shortening of PSADT was noticed in 19.8% of individuals (Paller et al. 2013). Another study showed that pomegranate extract (patients drunk 236.6 mL of the extract per day, equivalent to 1.6 mmol of total polyphenols per day) did not notably prolong PSADT in prostate cancer patients with rising PSA after primary therapy compared to placebo (Pantuck et al. 2015). A phase II randomized control trial revealed that the intake of PJ as an adjunct intervention in men with advanced PC did not result in any significant decrease in PSA levels compared to placebo (Stenner‐Liewen et al. 2013). Interestingly, a double‐blind, placebo‐controlled randomized trial established that supplementing the diet with a polyphenol‐rich food supplement containing a blend of green tea, pomegranate, broccoli, and curcumin had a positive effect on PSA progression in patients with histologically confirmed PC (Thomas et al. 2014). One randomized, placebo‐controlled phase II clinical trial examined the effect of PFE (1000 mg twice daily, for up to 4 weeks) on the levels of serum PSA, the oxidative stress biomarker 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG), and key biomarkers of prostate cancer inflammation, development and progression (NF‐κB expression, pS6 kinase), as well as proliferation (Ki‐67), prior to prostatectomy. The study indicated that low 8‐OHdG levels correlated with the presence of high levels of urolithin A (a metabolite of pomegranate ellagitannins) and it was concluded that PFE may exert a protective role against oxidative DNA damage. However, the extract did not significantly lower 8‐OHdG levels compared to the placebo group. In addition, there was no reduction in the levels of other relevant biomarkers (Freedland et al. 2013). Another study analyzed the expression of proliferation biomarkers (CDKN1A, MKi‐67 and c‐Myc) after the consumption of PJ (200 mL per day for 3 days before surgery) by PC patients. It identified the presence of urolithin A glucuronide, urolithin B glucuronide (traces) and dimethyl ellagic acid, but no obvious changes were observed in the expression of the studied proliferation biomarkers (González‐Sarrías et al. 2010). Additionally, a recent phase II randomized placebo‐controlled trial conducted in men with PC under active surveillance showed there was no difference in plasma IGF‐1 levels, PSA doubling time, and biopsy kinetics after consumption of PFE (1000 mg/day for 12 months). Immunohistochemical analysis revealed that the androgen receptor expression and the levels of 8‐OHdG were reduced following PFE intake. Urolithin A and urolithin A glucuronide were the two metabolites which were the most frequently detected after PFE intake (Jarrard et al. 2021). Notably, the recent clinical literature (post‐2021) remains limited, with few additional published randomized trials of pomegranate preparations in prostate cancer (NCT07161310, NCT02095145). Nevertheless, clinical development continues through registered studies evaluating standardized pomegranate extracts in localized disease settings, including active surveillance and pre‐surgical window‐of‐opportunity designs, as well as trials focusing on ellagitannin‐derived metabolites (urolithins) as mechanistically informed adjuncts.

As shown in Table 5, pomegranate exerts anti‐prostate cancer activity through multiple mechanisms, depending on the preparation used, the experimental model, and the molecular pathways targeted.

TABLE 5.

Mechanisms of pomegranate in prostate cancer.

Mechanism of action	Pomegranate preparation	Prostate cancer model	Molecular pathways affected	References
Apoptosis induction	Punicalagin (10–100 μM)	PC‐3, LNCaP, BPH‐1 cells	↑ Caspase‐3, ↑ Caspase‐8, ↑ Bax/Bcl‐2 ratio	Adaramoye et al. (2017)
Cell cycle arrest	Ethanolic peel extract (50–100 μg/mL)	DU145, PC3, TRAMP‐C1 cells	↓ Cyclin D1, ↓ c‐Myc, ↑ p21 expression, ↑ G2/M arrest	Deng et al. (2017)
Anti‐angiogenesis	Pomegranate juice (1%–5%)	DU145, PC‐3 cells	↓ VEGF, ↓ HIF‐1α, ↓ Tumor vessel density	Wang et al. (2011)
Androgen receptor signaling inhibition	Pomegranate fruit extract (10–100 μg/mL)	CWR22Rv1 cells	↓ Androgen receptor, ↓ PSA, ↓ Cyclin‐dependent kinases	Malik et al. (2005)
Proliferation suppression	Pomegranate skin extract (IC50 = 5.7 ± 0.5 μg/mL)	LNCaP‐AR, LAPC4, CL1, DU145 cells	↓ β‐Catenin, ↓ SIRT1, ↑ ROS, ↓ PCNA	Rettig et al. (2008)
Metastasis and invasion inhibition	Ellagic acid (25–100 μM)	PC‐3, PLS10 cells	↓ MMP2/MMP9, ↓ Cell motility, ↓ Collagenase IV activity	Pitchakarn et al. (2013)
Oxidative stress reduction	Polyphenol‐rich pomegranate extract (1000 mg/day)	Prostate cancer patients under active surveillance	↓ DNA oxidative damage, ↑ Antioxidant enzyme activity	Jarrard et al. (2021)
NF‐κB pathway inhibition	Standardized pomegranate extract (500 mg/day)	LAPC4 xenografts in immunodeficient mice	↓ NF‐κB activation, ↓ Pro‐inflammatory cytokines	Seeram et al. (2007)
Autophagy modulation	Pomegranate extract (in combination with docetaxel)	Metastatic castration‐resistant PC (C4‐2, PC‐3, ARCaPM)	↓ mTOR phosphorylation, ↑ JNK phosphorylation	Wang and Martins‐Green (2014)
Reduction of PSA levels	Pomegranate juice (237 mL/day)	Prostate cancer patients with rising PSA	↑ PSA doubling time, ↓ Tumor progression	Pantuck et al. (2006)
Modulation of inflammatory markers	Pomegranate polyphenol‐rich supplement	Patients with histologically confirmed PC	↓ IL‐6, ↓ TNF‐α, ↓ Inflammatory cytokines	Thomas et al. (2014)

Note: Symbols: ↓—Decrease; ↑—Increase.

Abbreviations: AKT, protein kinase B; AR, androgen receptor; Bax, Bcl‐2‐associated X protein; Bcl‐2, B‐cell lymphoma 2; COX‐2, cyclooxygenase‐2; DU145, PC‐3, LNCaP, prostate cancer cell lines; G0/G1, G2/M, S phase, cell cycle phases; HIF‐1α, hypoxia‐inducible factor‐1 alpha; IC₅₀ , inhibitory concentration 50%; IL‐6, interleukin‐6; NF‐κB, nuclear factor kappa‐light‐chain‐enhancer of activated B cells; PARP, poly (ADP‐ribose) polymerase; PCNA, proliferating cell nuclear antigen; PSA, prostate‐specific antigen; ROS, reactive oxygen species; VEGF, vascular endothelial growth factor.

6. Limitations, Challenges and Clinical Gaps in the Use of Pomegranate for Breast, Colon and Prostate Cancer Treatment

Despite promising preclinical and emerging clinical evidence, several limitations and challenges must be addressed before pomegranate can be fully integrated into cancer prevention and treatment strategies. One major challenge is its low bioavailability, as key polyphenols such as punicalagins and ellagic acid have poor intestinal absorption and are largely metabolized by gut microbiota into urolithins, whose production varies significantly among individuals. This interindividual variability in urolithin metabolism raises concerns about inconsistent therapeutic responses, requiring further research into personalized dosing and microbiome interactions.

Another limitation is the lack of standardization in pomegranate formulations, as studies have used different extracts, juices, seed oils, and purified compounds, making direct comparisons difficult. Establishing quality control measures and standardized formulations with defined bioactive concentrations is essential for reproducibility in clinical trials. A critical gap in research is the lack of large‐scale, randomized clinical trials that assess pomegranate's long‐term effects on tumor progression, recurrence, and survival in breast, colon, and prostate cancer patients. Most existing studies are small, short‐term, and lack placebo controls, making it difficult to draw firm conclusions about clinical efficacy. An additional concern is the translational relevance of in vitro studies using unfeasibly high concentrations of pomegranate extracts or compounds. According to the U.S. National Cancer Institute (NCI), botanical products are considered cytotoxic when the IC₅₀ is below 20 μg/mL or 10 μM after 48–72 h of treatment. Several studies reported IC₅₀ values that exceed this threshold, suggesting limited applicability in a clinical setting due to the unrealistic doses required to replicate these effects in humans. Conversely, studies meeting or exceeding this benchmark are more likely to represent truly promising candidates for therapeutic development. Future preclinical research should prioritize physiologically relevant dosing to ensure meaningful clinical translation. In breast cancer, trials have primarily focused on biomarkers of estrogen metabolism rather than direct clinical outcomes such as tumor regression. In colon cancer, research has largely been limited to gut microbiota modulation and inflammatory markers, with no trials assessing pomegranate's impact on tumor recurrence or overall survival. Prostate cancer research has been more advanced, with Phase II trials suggesting that pomegranate prolongs PSA doubling time, yet the failure of a Phase III trial to meet its primary endpoint raises concerns about patient selection and efficacy variability. Another challenge is the unclear primary mechanism of action, as pomegranate targets multiple pathways, including antioxidant, anti‐inflammatory, pro‐apoptotic, anti‐angiogenic, and hormone‐modulating effects—but lacks a well‐defined molecular target. Unlike conventional therapies such as tamoxifen for breast cancer or androgen deprivation therapy for prostate cancer, pomegranate's broad‐spectrum activity makes it harder to optimize its therapeutic use. Additionally, potential interactions with chemotherapy, radiotherapy, or immunotherapy remain poorly understood, and long‐term high‐dose safety has not been extensively studied. Regulatory and commercial barriers also hinder clinical translation, as pomegranate is categorized as a dietary supplement rather than a pharmaceutical, meaning it does not require FDA or EMA approval for marketing. The absence of rigorous regulatory oversight has led to variability in commercial pomegranate products and potential misleading health claims, which may undermine its credibility as a scientifically validated therapy. To bridge these gaps, future research should focus on improving bioavailability through nanoformulations and probiotic strategies, conducting personalized treatment studies based on urolithin metabotypes, designing large‐scale clinical trials with standardized formulations, and evaluating combination therapies with existing cancer treatments. Addressing these challenges will be essential for establishing pomegranate as a reliable complementary or adjunct therapy in oncology.

7. Conclusion and Future Prospects

Pomegranate has demonstrated promising anticancer effects in breast, colon, and prostate cancers, supported by a growing body of preclinical and emerging clinical evidence. Its bioactive compounds particularly punicalagins, ellagic acid, and urolithins have been shown to induce apoptosis, inhibit proliferation, and modulate oxidative, inflammatory, and hormonal pathways. However, the exact molecular mechanisms remain incompletely defined, and the effectiveness of individual compounds in vivo is still under investigation. Recent studies have expanded the understanding of pomegranate's mechanisms of action, delivery strategies, and clinical potential, highlighting the need to continuously integrate up‐to‐date evidence into future evaluations. Key challenges such as limited bioavailability, interindividual variability in metabolism, and inconsistency in formulations remain major barriers to clinical translation. Further research is required to identify the most therapeutically relevant compounds, optimize delivery systems, and conduct standardized, large‐scale clinical trials. Understanding the influence of gut microbiota on metabolite production, as well as evaluating pomegranate in combination with conventional therapies, may enhance its application in personalized cancer prevention and treatment strategies.

Author Contributions

Dusanka Kitic: investigation, writing – original draft, writing – review and editing, validation, methodology, project administration, data curation, supervision, visualization. Bojana Miladinovic: investigation, writing – original draft, writing – review and editing, validation, methodology, data curation, visualization. Milica Randjelovic: investigation, writing – original draft, writing – review and editing, validation, methodology, data curation, visualization. Agnieszka Szopa: investigation, writing – original draft, writing – review and editing, supervision, validation, methodology, visualization, project administration. Veronique Seidel: investigation, writing – original draft, writing – review and editing, validation, methodology, visualization, supervision, data curation, project administration. Javad Sharifi‐Rad: visualization, writing – review and editing, writing – original draft, investigation, conceptualization, validation, methodology, project administration, data curation, supervision. William N. Setzer: writing – review and editing, validation, methodology, data curation, supervision, investigation, writing – original draft. Daniela Calina: writing – original draft, writing – review and editing, visualization, validation, methodology, investigation, project administration, supervision, data curation.

Funding

This work was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (grant numbers 451‐03‐34/2026‐03/200113).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting Information S1: Compound index for Figure 1.

Data Availability Statement

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.