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POMEGRANATE: Punicalagins and urolithins bioactive products with beneficial health effects

Nowadays, pomegranate punicalagins can be considered as a functional food (FF) due to the amount of published scientific relevant studies.

Report Title

POMEGRANATE: punicalagins and urolithins bioactive products with beneficial health effects

Authors of this report

Quality and Food Safety Group

Department Agro-food Technology

UNIVERSITY MIGUEL HERNÁNDEZ DE ELCHE

Dámaris Girona Ruiz (Senior Technician Nutrition and Dietetics)

Marina Cano Lamadrid (Food Science and Technology Graduate)

Ángel A. Carbonell Barrachina (DOCTOR IN CHEMISTRY) UNIVERSITY PROFESSOR

INDEX

  1. Punicalagins introduction
  2. Pomegranate as a functional food
  3. Chemical composition of pomegranate
    3.1. Beneficial effects of punicalagins
  4. Transformation of punicalagins to urolithins
    4.1. Beneficial effects of urolithins
  5. Products with optimal punicalagin content
  6. Digital blog on pomegranate in different languages
  7. Reference

1. Punicalagins introduction

The aim of this present monograph is to inform readers about the wide range of beneficial properties of the pomegranate fruit (Punica granatum L) based on “scientific” results. It is worth mentioning that punicalagins (one of the most important phenolic compounds in this fruit, responsible for the antioxidant activity) is converting in the human body into urolithins, a bioactive metabolite that presents positive effects on the human organism. Updated information based on different recent scientific articles, assurance of the veracity of the beneficial properties of the pomegranate, is detailed below.

2. Pomegranate as a functional food

Nowadays, pomegranate punicalagins considered as a functional food (FF) due to the amount of published scientific relevant studies. Not only because it provides nutrients but because it also has one or more beneficial effects on different functions of the organism, improving health and human wellbeing. In addition, it has a preventive role by reducing risk factors for certain diseases such as prostate cancer, breast cancer and obesity, among others (Functional Food Guide, FESNAD).

Pomegranate can be considered as a functional food due to its high antioxidant activity. It is mainly attributable to polyphenolic compounds found in different parts of pomegranate tree (bark > flower > leaves > arils > seed) (Bruno, 2016). The majority of the compounds of each of the part of the pomegranate is shown in more detail is Figure 1

Pomegranate as a functional food
Figure 1 – Pomegranate as a functional food

3. Chemical composition of pomegranate

Pomegranate fruit and pomegranate-based products are rich sources of polyphenols, compounds of natural origin that provide healthy effects on the human body due to their antioxidant capacity. Punicalagins is the most outstanding ellagitannin among the hydrolysable tannins found in pomegranate. In addition, it contains ellagic acid and its conjugated glycosides, glycoside and rhamnoside, obtaining hydrolysable tannins (Figure 2) (Ismail et al., 2012). It is worth highlighting that the European Food Safety Agency (EFSA) does not allow health claims for pomegranate product antioxidants, because more clinical trials are needed to establish the connection.

Figure 2: Chemical composition of pomegranate

3.1 Beneficial effects of punicalagins

During last years, several studies on the antioxidant and antiinflammatory capacity of punicalagins have been carrying out, linking these compounds with the prevention of different diseases such as prostate cancer, of breast and obesity, among others (Les et al., 2018) (Figure 3).

Scientific relevant studies showing beneficial properties in both human and animal cells (in both in-vitro and in-vivo assays) have been carried out. For example, an in vitro study on breast cancer cells has recently been published, a decrease in the viability of these cells was observed after treatment with pomegranate extract rich in punicalagins (2.5-25 μL/mL) after 48 h of application (Banerjee et al).

Beneficial effects of punicalagins
Figure 3 – Beneficial effects of punicalagins

Recently, the scientific community has concluded that punicalagins are not directly those compounds which present this antioxidant effect. But, it is well-known that punicalagins act as the precursor of urolithins (González-Sarrías et al., 2017).

4. Transformation of punicalagins to urolithins

High molecular weight polyphenols, such as punicalagins, are not absorbed in the stomach or the small intestine, and reach the colon almost unaltered. Thus, a modulation of the composition of the intestinal microbiota is caused mainly by the population of beneficial microorganisms of the human body (gutmicrobiotaforhealth.com).

Among intestinal microbiota individuals, Gordonibacter and its species urolithinfaciens and pameleae are capable of transforming the punicalagins and the ellagic acid to urolithins (González-Sarrías et al., 2017).

The process begins with the ingestion of punicalagins in the form of concentrated pomegranate juice, and/or peel extracts (Phase I). Once it has been ingested, punicalagins in the presence of hydrochloric acid, HCl (stomach) and the action of the different microorganisms present in the intestine, is transformed into ellagic acid (Figure 4) (Phase II).

Transformation of punicalagins to urolithins
Figure 4 – Transformation of punicalagins to urolithins

Subsequently, different chemical reactions of the catabolic pathway ellagic acid (naturally present in the pomegranate and its derived products, and as a product of the digestion of punicalagins as has been discussed above) degradation occur. Different catabolic intermediates (bioactive metabolites,) called urolithins, are generated (Espin et al., 2013; García-Villalba et al., 2017) (Phase III).

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Can everyone produce urolithins?

Recently, a human in vivo experiment observed that it was evident that not all humans were capable of forming urolithins after the ingestion of high amount of ellagitannins (punicalagins) and/or ellagic acid. As a conclusion, individuals were classified into 3 different phenotypes according to their ability to form these compounds, not depending on their age, sex, BMI (body mass index) and health status (Figure 5). It is important to mention that there is a great variability among individuals with a healthy intestinal microbiota and those presenting an imbalance intestinal microbiota. Therefore, depending on the phenotype the effects on health may be different, whether or not they can have their beneficial role.

Formation of urolithins by different phenotypes
Figure 5 – Formation of urolithins by different phenotypes

Among the three metabolites that can be formed by intestinal microbiota (Figure 5), urolithin A was the most detected in blood and urine, remaining even after 4 days of the pomegranate extracts intake using capsules in one take (1.8 g/dose) (Tomas-Barberan et al., 2014).

4.1 Beneficial effects of urolithins

  1. Recently, an in-vivo study observed a relationship between urolithin A and its ability to inhibit enzymes linked with carbohydrates metabolism such as lipase and α-glucosidase, concentration of 0.032 mg urolithin A/mL and 0.015 mg urolithin A/mL, respectively. As a result, a contribution of decreasing glucose absorption in the digestive tract was noticed. The result was a detriment of diabetes and a prevention of obesity (Les et al., 2018).
  2. It is well-known that prostate cancer is one of the most widespread cancers in the world. Recently, different scientific tests have been carried out to check if the use of urolithins could decrease the proliferative activity of this type of cancer cells. As an example, an in vitro essay with two types of prostate cancer cells (LNCaP and DU-145) was done, observing that the higher concentrations of urolithin A (32.26 μM, 35.7 μM and 45.5 μm), the higher inhibition of the proliferation of this cell type (Stanisławska, et al., 2018).
  3. HDL cholesterol is inversely related to the incidence of atherosclerosis. Polyphenols, including ellagitannins, have been shown to encourage antiatherogenic properties. Urolithin B is formed from these compounds by the intestinal microbiota, and urolithins may be involved in beneficial effects against cardiovascular diseases in vitro. Recently, a study investigated the effect of urolithin B on lipid plaque deposition and reverse cholesterol transport. The conclusion was that urolithin B decreases the deposition of lipid plaque, and urolithin B and urolithin B sulphate induces the inverse transport of cholesterol by influencing the expression of key proteins in this pathway. Urolithin B represents the basis for the development of new drugs for the prevention and treatment of atherosclerosis in humans (Zhao et al., 2019).
Pomegranate as a prevention of different diseases
Pomegranate as a prevention of different diseases

It should be noticed that these beneficial properties are correlated with the phenotypes mentioned above, and can be attributed to those healthy individuals that their intestinal microbiota is able to form urolithins, mainly type A. On the other hand, it is not possible to demonstrate these benefits in humans who are not capable of synthetizing these compounds.

5. Products with optimal punicalagins content

In order to promote the development of urolithins in our body, it is necessary to start from a consumption of products with an optimal content in these polyphenols (punicalagins and ellagic acid). Although more clinical research is needed and additionally the possible benefits depend on what type of phenotype the consumer is, it could be said that an adequate intake of punicalagins would be approximately 200 mg per day. As indicated in the previous paragraphs, the majority of the content of these compounds is found in the peel, and the pomegranate juice from arils has rather lower contents. Approximately 460 mL per day should be consumed to obtain the indicated dose of punicalagins. On the other hand, it is important to mention that the interesting compounds of pomegranate peel during the preparation of infusions (pomegranate tea) could be degraded due to severe heat treatments.

The research group “Quality and Food Safety” of the Department of Agro-Food Technology of the Universidad Miguel Hernández has carried out numerous analyses of pomegranate products since 2007: juices, extracts, concentrates and food supplements marketed in Spain and Europe. Commonly, the content and recommended intake of these products does not provide an adequate amount of punicalagins to be considered as functional foods (Cano-Lamadrid et al., 2017; Cano-Lamadrid et al., 2019). As an exception, standing out for their concentration, among other types of products and brands, the pomegranate powder extract of the brand Granatum Plus used to make its pomegranate capsules Punicalagin Plus and its pomegranate juice concentrated Punicalagin 200. The concentration of punicalagins per gram of product is 308 milligrams, being one of the highest one.

Capsules of pomegranate extract bioactive components

One Punicalagin Plus pomegranate extract capsule contains the bioactive substances present in more than 50 litres of natural pomegranate juice.

The Granatum Punicalagina Plus + food supplement showed a high content of phenolic compounds, mainly attributed to its outstanding punicalagin content; punicalagin represented more than 31% of the net weight of the capsule, with an average value of 231 mg of punicalagin per capsule. The punicalagin content per capsule is clearly higher than that naturally present in the edible parts of the pomegranate, or in the juice made from the fruit.

It should be noted that the scientific literature reports highly variable amounts of punicalagin in pomegranate juice as a consequence of the different extraction techniques. Contributory factors may involve separation or not of the arils prior to pressing and different intensities of pressing.

Previous studies in the field suggest that the average punicalagin content is in the range of 4 mg to 564 mg per litre of natural pomegranate juice (Gil et al. 2000; Fischer et al. 2011; Campillo et al. 2015).

Taking these values as a reference, the intake of a daily capsule of the Granatum Punicalagina Plus +  food supplement would be equivalent to the punicalagin content of between 410 ml and over 50 litres of natural pomegranate juice per day.

REFERENCES

Campillo N, Viñas P, Férez-Melgarejo G, Ochotorena ML, Hernández Córdoba M. Determination ofphenolic acids and hy-drolyzable tannins in pomegranate fruit and beverages by liquid chromatography with diode array detection and time-of-flight mass spectrometry. Food Anal Method. 2014;8:1315-1325.

Fischer UA, Carle R, Kammerer DR (2011) Identification and quantification of phenolic compounds from pomegranate (Punica granatum L.) peel, mesocarp, aril and differently produced juices by HPLCDADESI/MSn. Food Chem, 127:807–821.

Gil MI, Tomás-Barberán FA, Hess-Pierce B, Holcroft DM, Kader AA (2000) Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing. J Agric Food Chem 48:4581–4589

Taking all mentioned above into account, it is recommended that more clinical tests should be conducted. Moreover, the European Food Safety Agency (EFSA) may be focused on the authorization of the health statements mentioned throughout this document. The labelling must always be improved in order to know the origin, the ingredients list, and their nutritional composition, including the composition of certain bioactive compounds such as punicalagins in the case of food supplements.

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6. Digital blog on pomegranate in different languages

After this review of current situation of pomegranate studies, especially about punicalagin as a precursor of urolithins and their healthy properties, it can be said that scientific production has been growing worldwide. There is a blog that compiles the large number of interesting publications that appear in international journals with high scientific rigor: http://www.zumodegranada.com.

Thanks to the University Miguel Hernández de Elche, this blog collects and translate more than 1000 new articles that appear weekly on the Internet. They are published in different languages. It is published all types of information, (i) that which is easily understandable by consumers and (ii) more technical aspects for experts.

Below a series of recent publications on punicalagin and urolithins is shown. The way it is presented is as readers can find it in the aforementioned digital blog, complementing what has been explained along this document:

A Novel Candidate for Prevention and Treatment of Atherosclerosis: Urolithin B Decreases Lipid Plaque Deposition in apoE−/− Mice and Increases Early Stages of Reverse Cholesterol Transport in ox‐LDL Treated Macrophages Cells.

Posted in: Molecular Nutrition Food Research Date:2019

A new candidate for the prevention and treatment of atherosclerosis: urolithin B decreases the deposition of lipid plaque in mice apoE – / – and increases the early stages of reverse cholesterol transport in ox – Macrophagous cells treated with LDL.

HDL cholesterol is inversely related to the incidence of atherosclerosis. Polyphenols including ellagitannins have been shown to exert antiatherogenic properties. Urolithin B is formed from ellagitannins by components of the gut microbiota, and urolithins might be involved in beneficial effects against cardiovascular diseases in vitro. In this study, the influence of urolithin B on several parameters involved in the lipid plaque deposition and the reverse cholesterol transport is investigated.

Pomegranate polyphenols and urolithin A inhibit α-glucosidase, dipeptidyl peptidase-4, lipase, triglyceride accumulation and adipogenesis related genes in 3T3-L1 adipocyte-like cells.

Posted in: Journal of Ethnopharmacology Date:2018

Polyphenols of pomegranate and urolithin A inhibit α-glucosidase, dipeptidil peptidase-4, lipase, triglyceride accumulation and adipogenesis genes in cells similar to adipocytes 3T3-L1

Ethnopharmacological relevance: Pomegranate fruit is considered an antidiabetic medicine in certain systems of traditional medicine. In addition, pomegranate polyphenols are known as powerful antioxidants with beneficial effects such as the reduction of oxidative / inflammatory stress and the increase of protective signalling such as antioxidant enzymes, neurotrophic factors and cytoprotective proteins. Aim of the study: This work evaluates the effects of pomegranate juice, its main polyphenols known as ellagic acid and punicalagin, as well as its main metabolite urolithin A, on physiological and pharmacological targets of metabolic diseases such as obesity and diabetes. Materials and methods: For this purpose, enzyme inhibition bioassays of lipase, α-glucosidase and dipeptidyl peptidase-4 were carried out in cell-free systems. Similarly, adipocytes derived from 3T3-L1 cells were employed to study the effects of ellagic acid, punicalagin and urolithin A on adipocyte differentiation and triglyceride (TG) accumulation. Results: Pomegranate juice, ellagic acid, punicalagin and urolithin A were able to inhibit lipase, α-glucosidase and dipeptidyl peptidase-4. Furthermore, all tested compounds but significantly the metabolite urolithin A displayed anti-adipogenic properties in a dose-dependent manner as they significantly reduced TG accumulation and gene expression related to adipocyte formation such as adiponectin, PPARγ, GLUT4, and FABP4 in 3T3-L1 adipocytes. Conclusion: These results may explain from a molecular perspective the beneficial effects and traditional use of pomegranate in the prevention of metabolic-associated disorders such as obesity, diabetes and related complications.

The effects of urolithins on the response of prostate cancer cells to non-steroidal antiandrogen bicatulamide.

Posted in : Phytomedicine Date:2018

The effects of urolithins on the response of prostate cancer cells to nonsteroidal bicalutamide antiandrogen

Urolithins are bioavailable products of gut microbiota metabolism of ellagitannins. Their biological activity includes anti-cancer effects. The aim of this study was to explore the effects of urolithins on prostate cancer cells and activity of clinically used anti-androgen, bicalutamide.

Cytotoxicity of pomegranate polyphenolics in breast cancer cells in vitro and vivo: potential role of miRNA-27a and miRNA-155 in cell survival and inflammation.

Posted in: Medline Date:2012

Cytotoxicity of pomegranate polyphenolics in in vitro and in vivo breast cancer cells: potential role of Mirna-27a and Mirna-155 in cell survival and inflammation.

Several studies have demonstrated that polyphenolics from pomegranate (Punica granatum L.) are potent inhibitors of cancer cell proliferation and induce apoptosis, cell cycle arrest, and also decrease inflammation in vitro and vivo. There is growing evidence that botanicals exert their cytotoxic and anti-inflammatory activities, at least in part, by decreasing specificity protein (Sp) transcription factors. These are overexpressed in breast tumors and regulate genes important for cancer cell survival and inflammation such as the p65 unit of NF-κB. Moreover, previous studies have shown that Pg extracts decrease inflammation in lung cancer cell lines by inhibiting phosphatidylinositol-3,4,5-trisphosphate (PI3K)-dependent phosphorylation of AKT in vitro and inhibiting the activation of NF-kB in vivo. The objective of this study was to investigate the roles of miR-27a-ZBTB10-Sp and miR-155-SHIP-1-PI3K on the anti-inflammatory and cytotoxic activity of pomegranate extract. Pg extract (2.5-50 μg/ml) inhibited growth of BT-474 and MDA-MB-231 cells but not the non-cancer MCF-10F and MCF-12F cells. Pg extract significantly decreased Sp1, Sp3, and Sp4 as well as miR-27a in BT474 and MDA-MB-231 cells and increased expression of the transcriptional repressor ZBTB10. A significant decrease in Sp proteins and Sp-regulated genes was also observed. Pg extract also induced SHIP-1 expression and this was accompanied by downregulation of miRNA-155 and inhibition of PI3K-dependent phosphorylation of AKT. Similar results were observed in tumors from nude mice bearing BT474 cells as xenografts and treated with Pg extract. The effects of antagomirs and knockdown of SHIP-1 by RNA interference confirmed that the anti-inflammatory and cytotoxic effects of Pg extract were partly due to the disruption of both miR-27a-ZBTB10 and miR-155-SHIP-1. In summary, the anticancer activities of Pg extract in breast cancer cells were due in part to targeting microRNAs155 and 27a. Both pathways play an important role in the proliferative/inflammatory phenotype exhibited by these cell lines.

Biological Significance of Urolithins, the Gut Microbial Ellagic Acid-Derived Metabolites: The Evidence So Far

Posted in: Evidence-Based Complementary and Alternative Medicine Date:2013

Biological significance of urolithins, and metabolites derived from elagic acid from gastrointestinal microbiot

The health benefits attributed to pomegranate have been associated with its high content in polyphenols, particularly ellagitannins. This is also the case for other ellagitannin-containing fruits and nuts including strawberry, raspberry, blackberry, walnuts, and muscadine grapes. The bioavailability of ellagitannins and ellagic acid is however very low. These molecules suffer extensive metabolism by the gut microbiota to produce urolithins that are much better absorbed. Urolithins circulate in plasma as glucuronide and sulfate conjugates at concentrations in the range of 0.2-20 M. It is therefore conceivable that the health effects of ellagitannin-containing products can be associated with these gut-produced urolithins, and thus the evaluation of the biological effects of these metabolites is essential. Recent research, mostly based on in vitro testing, has shown preliminary evidence of the anti-inflammatory, anticarcinogenic, antiglycative, antioxidant, and antimicrobial effects of urolithins, supporting their potential contribution to the health effects attributed to pomegranate and ellagitannin-rich foods. The number of in vivo studies is still limited, but they show preventive effects of urolithins on gut and systemic inflammation that encourage further research. Both in vivo and mechanistic studies are necessary to clarify the health effects of these metabolites. Attention should be paid when designing these mechanistic studies in order to use the physiologically relevant metabolites (urolithins in gut models and their conjugated derivatives in systemic models) at concentrations that can be reached in vivo.

Urolithin A Is a Dietary Microbiota-Derived Human Aryl Hydrocarbon Receptor Antagonist

Posted in: Metabolites Date:2018

Urolithin A is an antagonist of the hydrocarbon of dietary microbiota

Urolithins (e.g., UroA and B) are gut microbiota-derived metabolites of the natural polyphenol ellagic acid. Urolithins are associated with various health benefits, including attenuation of inflammatory signaling, anti-cancer effects and repression of lipid accumulation. The molecular mechanisms underlying the beneficial effects of urolithins remain unclear. We hypothesize that some of the human health benefits of urolithins are mediated through the aryl hydrocarbon receptor (AHR). Utilizing a cell-based reporter system, we tested urolithins for the capacity to modulate AHR activity. Cytochrome P450 1A1 (CYP1A1) mRNA levels were assessed by real-time quantitative polymerase chain reaction. Competitive ligand binding assays were performed to determine whether UroA is a direct ligand for the AHR. Subcellular AHR protein levels were examined utilizing immunoblotting analysis. AHR expression was repressed in Caco-2 cells by siRNA transfection to investigate AHR-dependency. UroA and B were able to antagonize 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced AHR-mediated transcriptional activity. Furthermore, UroA and B attenuated TCDD-mediated stimulation of CYP1A1 mRNA levels. In addition, competitive ligand binding assays characterized UroA as a direct AHR ligand. Consistent with other AHR antagonists, UroA failed to induce AHR retention in the nucleus. AHR is necessary for UroA-mediated attenuation of cytokine-induced interleukin 6 (IL6) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression in Caco-2 cells. Here we identified UroA as the first dietary-derived human selective AHR antagonist produced by the gut microbiota through multi-step metabolism. Furthermore, previously reported anti-inflammatory activity of UroA may at least in part be mediated through AHR.

Improvements in Metabolic Health with Consumption of Ellagic Acid and Subsequent Conversion into Urolithins: Evidence and Mechanisms

Posted in: Advances in Nutrition Date:2016

Improvement of the metabolic health by the consumption of elagic acid as a consequence of the conversion to utolitines: evidence and mechanisms.

Ellagic acid (EA) is a naturally occurring polyphenol found in some fruits and nuts, including berries, pomegranates, grapes, and walnuts. EA has been investigated extensively because of its antiproliferative action in some cancers, along with its anti-inflammatory effects. A growing body of evidence suggests that the intake of EA is effective in attenuating obesity and ameliorating obesity-mediated metabolic complications, such as insulin resistance, type 2 diabetes, nonalcoholic fatty liver disease, and atherosclerosis. In this review, we summarize how intake of EA regulates lipid metabolism in vitro and in vivo, and delineate the potential mechanisms of action of EA on obesity-mediated metabolic complications. We also discuss EA as an epigenetic effector, as well as a modulator of the gut microbiome, suggesting that EA may exert a broader spectrum of health benefits than has been demonstrated to date. Therefore, this review aims to suggest the potential metabolic benefits of consumption of EA-containing fruits and nuts against obesity-associated health conditions.

7. References

THE POMEGRANATE SECRET
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THE POMEGRANATE SECRET
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