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Polyphenols are a class of natural compounds that act as antioxidants, neutralising harmful free radicals that would damage cells and increase the risk of diseases such as cancer, diabetes and heart disease. They also reduce inflammation, which is thought to be at the root of many chronic diseases.We are investigating the photoprotective effects of punicalagin, a type of polyphenolic compound mainly found in pomegranates, against UVA-induced damage in human skin fibroblasts. Punicalagin increases cell viability and reduces the high levels of ROS generated by photooxidative stress through its ability to modulate the Nrf2 transcriptional pathway. Interestingly, activation of the Nrf2 pathway results in an increase in reduced glutathione, NADH, and subsequently protects mitochondrial respiratory capacity. Integrating molecular and imaging approaches, our results demonstrate a potential cytoprotective effect of punicalagin against UVA-induced skin damage through an anti-apoptotic mechanism.

Nanoparticles-based drug delivery system has been a promising approach for the treatment of colorectal cancer (CRC), which can be combined with chemotherapy, targeted therapy and immunotherapy to improve the treatment of CRC. 2'3' cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) is an agonist of the STING signaling pathway activating antitumor immunity. IPI-549 is a small-molecule inhibitor for phosphatidylinositol 3-kinase γ (PI3Kγ), which can induce M1 macrophages polarization to provide pro-inflammatory microenvironment to suppress tumors. Here, we developed a ferric ion-punicalagin network (Fe-PU), which can be not only used as an inducer of ferroptosis, but also serve as a carrier to load cGAMP and IPI-549 to obtain nanohybrid (Fe-PU/CD-IPI). In order to improve the delivery effect and targeted ability to CRC, a cyclic arginine-glycine-aspartic acid peptide linked-bovine serum albumin were utilized to modify Fe-PU/CD-IPI to prepare nanohybrid Fe-PU/CD-IPI@cBSA. The therapeutic effect of various nanohybrids were validated in the mice with spontaneous tumor in the colorectal area and tumor-bearing mice, which lead to the increase of ferroptosis, the activation of STING signaling pathway, and the repolarization of macrophages. Collectively, the cGAMP and IPI-549 co-loaded nanohybrids effectively reshaped the tumor immune microenvironment, and exhibited prominent treatment effect of anti-colorectal cancer in vitro, patient-derived organoids, and in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

ABSTRACT: Poultry production was long plagued by coccidiosis, and the development of alternative therapies will make practical sense. In this work, 2 battery experiments were designed. In battery experiment 1, the best effect of 7 anticoccidial herbs (Sophora japonica Linn, Citrus aurantium L, leaf of Acer palmatum, bark of Magnolia officinalis, fruit peel of Punica granatum L., Eclipta prostrata L., and Piper sarmentosum Roxb.) against Eimeria tenella infection of 21-day-old male Chinese Guangxi yellow-feathered chickens were screened out by clinic indexes (bloody feces scores, cecal lesion scores, oocysts output, relative weight gain rate, and survival rate). According to the results from battery experiment 1 and other literature research, we selected 2 monomers which were extracted from fruit peel of Punica granatum L. for further battery experiment 2 which were similar with battery experiment 1. Clinic results showed that Punicalagin had better anticoccidial effect than Ellagic acid. The anticoccidial mechanism exploration results of Elisa, antioxidant test, and pathological observation showed that Punicalagin reduced the cecal inflammation, improved the expression of immunoglobulin in cecal tissue, improved cecal integrity, and restored its REDOX state. Results of 16S rRNA sequencing analysis showed that Punicalagin also maintained the fecal flora health during E. tenella infection through insignificantly increasing the proportion of Lactobacillus and Faecalibacterium as well as significantly reducing the proportion of pathogenic bacteria, Escherichia–Shigella. RNA-Seq analysis results suggested that Punicalagin may play a role in controlling E. tenella infection by interaction with cytochrome P450 family enzymes. Overall, Punicalagin has promising potential as an alternative therapy for chicken Eimeria tenella infection.

Background: Punicalagin (PG), a component of Punica granatum L., present a significant nutraceutical value in view of its numerous health-promoting properties and abundance. Interest in PG is driven by its complex structure, as the largest molecular weight ellagitannin. Promising results from the consumption of pomegranate juice in clinical trials against several ailments i.e., cardiovascular diseases, diabetes, and prostate cancer have been mostly attributed to its PG content. In recent years, PG has been demonstrated to exhibit excellent pharmacological bioactivities and has been proposed as a safe candidate for chemoprevention. Purpose: To assess the underlying PG's action mechanisms towards chronic diseases with high mortality and morbidity. Methods: Electronic databases including PubMed, ScienceDirect and Scopus were used to compile the literature. Papers describing the biological effects of punicalagin in vitro conditions, experimental animal models, and humans were selected and discussed. In addition, the extraction and isolation procedure with the highest punicalagin yield was included. Results: Among the 1757 primarily selected papers, only a final number of 119 studies were included. The mechanisms of actions ranged from selective induction of apoptosis of cancer cells, autophagy of cancer cells, decrease in oxidative stress, reduction of inflammation via down-regulation of pro-inflammatory factors and up-regulation of anti-inflammatory mediators to maintaining neuronal integrity. This review presents the latest research progress made towards PG role in the prevention and treatment of various chronic diseases and its protective health effects highlighting the need for future research in nutraceuticals. Despite the promising effects of punicalagin, there seems to be a need for human trials to confirm the effects at physiological levels as there can be substantial human inter-individual variability in response to punicalagin metabolism. Further, comparison of the different extraction and isolation methods are presented to highlight the most optimum for PG recovery. Conclusion: This review aims at improving the value-added applications of this unique dietary bioactive phenolic at a commercial scale in view of its role in health and disease management.

Punicalagin, which is derived from pomegranate peel, is reported to exert growth-inhibitory effects against various cancers. However, the underlying mechanisms have not been elucidated. Human papillomavirus (HPV), a major oncovirus, utilizes the host autophagic machinery to support its replication. Here, punicalagin markedly downregulated the levels of the major HPV oncoproteins E6 and E7 in cervical cancer cells through the autophagy-lysosome system. Additionally, punicalagin activated the reactive oxygen species (ROS)-JNK pathway and promoted the phosphorylation of BCL2, which led to the dissociation of BCL2 from BECN1 and the induction of autophagy. Treatment with autophagy and JNK inhibitors or ROS scavengers mitigated the punicalagin-induced degradation of E6 and E7. Moreover, the knockout of ATG5 using the clustered regularly interspaced palindrome repeat/Cas 9 system mitigated the punicalagin-induced downregulation of E6/E7. This indicated that punicalagin-induced degradation of E6 and E7 was dependent on autophagy. The results of in vivo studies demonstrated that punicalagin efficiently inhibits cervical cancer growth. In conclusion, this study elucidated a mechanism of punicalagin-induced autophagic degradation of E6 and E7. It will enable the future applications of punicalagin as a therapeutic for HPV-induced cervical cancer.

Pomegranate extract (PE) and its polyphenols have been reported to show skin protective effects but their cytoprotective effects against methylglyoxal (MGO)-induced DNA damage and cell dysfunctions are unclear. Herein, we evaluated whether PE, punicalagin (PA), ellagic acid (EA), and urolithin A (UA), can alleviate MGO-induced DNA damage in human keratinocytes. PE (50 µg/mL) and PA (50 µM) protected DNA integrity and reduced the formation of MGO-DNA adducts and tailed DNA by 60.2 and 49.7%, respectively, in HaCaT cells. PE and PA reduced MGO-induced cytotoxicity by increasing the cell viability (by 17.5 and 15.0%) and decreasing reactive oxygen species (by 28.3 and 30.0%), respectively. PE and PA also ameliorated MGO-induced cell dysfunction by restoring cell adhesion, migration, and wound healing capacity. Findings from this study provide insights into the skin protective effects of PE and its polyphenols supporting their applications as potential bioactive ingredients for cosmeceuticals.

To develop a more effective and safer drug for the treatment of type 2 diabetes mellitus (T2DM)-induced liver damage, we investigated the protective effects of punicalagin, a major component in pomegranate peel, on the liver of mice with T2DM. After five weeks of punicalagin treatment, blood and liver samples were obtained for the subsequent analyses. Western blotting, reverse transcription polymerase chain reaction, and immunohistochemical staining were performed to determine the expression of endoplasmic reticulum (ER) stress markers in the liver tissue. The results showed that punicalagin alleviated glucose and insulin resistance, increased insulin sensitivity, and reduced serum free fatty acids levels and hepatic steatosis in the mice with T2DM. Furthermore, punicalagin down-regulated the elevated mRNA expression of the ER stress markers eIF2α, GRP78, ATF4, and CHOP in the liver of mice with T2DM. Our results suggest that punicalagin is a potential natural agent for the prevention of T2DM-induced liver damage.

Ultraviolet (UV)-B is an established etiological cause of skin damage. SIRT1, a mammalian SIR2 homolog localized in the nucleus and cytosol, plays a protective role in UVB-induced DNA damage. In this study, we established a method of screening foods and food ingredients, which augment the SIRT1 promoter in HaCaT cells, where we used recombinant HaCaT cells transduced with the vector expressing EGFP under the control of SIRT1 promoter. We identified four SIRT1-augmenting pomegranate-derived polyphenols (ellagic acid, punicalin, punicalagin, and urolithin A). All of these contributed to the proliferation of UVB-irradiated HaCaT cells. Punicalagin and urolithin A removed UVB-induced cyclobutane pyrimidine dimers (CPD) by activating nucleotide excision repair (NER). Both punicalagin and urolithin A upregulated NER-related XPC expression and XPA deacetylation level in a SIRT1-dependent manner. In the present study, we attempted to elucidate the mechanisms underlying the repair of UVB-damaged HaCaT cells by pomegranate-derived polyphenols.

Little is known about the effect of punicalagin extracted from pomegranate or in combination with human chorionic gonadotropin on enhancing the productive and reproductive performance in rabbits that gestate for the first time. The impact of punicalagin alone or in combination with human chorionic gonadotropin on maiden doe body weight, progesterone concentrations, conception rate, gestation rate, litter size, and kit weight was investigated in this study. A completely randomized and balanced experimental design was used to allocate 21 naturally mated maiden does at 6 months of age into the following three treatment groups of 7 does each: Control - intramuscular injection with sterilized water only; Treatment 1 - pre-mating intramuscular injection with punicalagin (100 µg/doe) twice a week and once 3 days post-mating; and Treatment 2 - pre-mating intramuscular injection with punicalagin twice a week and human chorionic gonadotropin (25 IU/doe) once 3 days post-mating. Progesterone was assayed from blood samples taken from the ear marginal vein at mating, post-mating, gestation, and post-kindling phases when all does were weighed. Results indicated significantly positive impacts of punicalagin alone or in combination with human chorionic gonadotropin on all doe reproductive traits, birth, and weaning weights of kits. The study clearly demonstrated an improvement in doe fertility, reproductive performance, and kit survival to weaning. In conclusion, PL and PL+HCG improved maternal body weights and their offspring as well as pregnancy outcomes of young rabbits particularly in the 2nd pregnancy, hence findings of this study could be recommended for improving reproductive health and fertility in maiden rabbit does.

Inflammatory response remains one of the most common and serious complications of disease in clinical studies. It has been established that some molecules found in pomegranate peel polyphenols (PPPs) are related to inflammatory processes; however, the mechanisms are unclear, leaving a significant unmet medical need. The goal of this study was to analyze the anti-inflammatory effects of PPPs in RAW264.7 macrophages and examine the relationship between certain special components such as - punicalagin (PC) and ellagic acid (EA) and systemic inflammation. The results showed that PPPs and their main components PC and EA, could decrease pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) at the mRNA and protein levels and, down-regulate iNOS and COX-2 expression, which in turn reduced NO and PGE2. The molecular mechanism might be associated with the inhibition of MAPKs activation. Additionally, PPPs and PC performed much better than EA. We highlight the key role of food-derived molecules PPPs and its main components in understanding the mechanism of anti-inflammatory.

Pomegranate (Punica granatum) is a heath with abundant hydrolysable tannin in its peel. This study demonstrated that POMx (70% acetone extract of pomegranate peels) significantly reduced interleukin (IL)-1β-induced inducible nitric oxide synthase, cyclooxygenase-2, and matrix metalloproteinase-13 protein expression in primary rat chondrocytes (PRCs). In the type II collagenase-induced osteoarthritis rat model, POMx (150 mg/kg) recovered the weight-bearing ratio changes in experimental animals, suggesting the alleviation of knee-related inflammation and nociception. Therefore, the major compound, punicalagin, was isolated from POMx. Punicalagin also significantly reduced IL-1β-induced inflammatory factors in PRCs and exerted significant antiosteoarthritis effects in the vivo model after 28-d treatment (concentration: 0.50 mg/kg). However, the IC50 values of POMx and punicalagin against prostaglandin E2 production were 83.2 and 36.0 μg/mL, respectively, and the concentration of punicalagin in POMx was 19.1%. Altogether, POMx can be used to develop functional foods for knee-related diseases, and punicalagin can act as an active ingredient.

Abstract: Pomegranates is abundant in polyphenols and is well-known for its antioxidant activity. Punicalagin (PG) is a major polyphenolic compound in the pomegranate peel. In certain conditions, PG can be hydrolyzed to punicallin (PL) and ellagic acid (EA), and PL can be further hydrolyzed to EA. PG, PL, and EA all play important roles in the antioxidant activity of pomegranate peels. This study was conducted to compare the in vitro antioxidant activity and in vivo anti-oxidative stress effects of PG, PL, and EA. For the in vitro test, 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) and superoxide anion ( O2 · ¯ ) scavenging capacities, ferric-reducing antioxidant power (FRAP), and lipid peroxidation (LPO) inhibition capacities of PG, PL, and EA were tested. For the in vivo test, oxidatively stressed mice, which were induced by oxidized fish oil, were administrated PG, PL or EA (10 mg kg−1 d −1) for 21 days. The results showed that the in vitro antioxidant activity trends were EA>PG>PL>Trolox in scavenging DPPH·, PG>PL>EA≈Trolox in scavenging O 2· ¯ , EA>PG≈PL>Trolox in FRAP, and Trolox>PG>EA>PL in LPO inhibition. In the in vivo test, the EA treatment increased the average daily weight gain and total antioxidant capacity (T-AOC) in the plasma (P

Ellagic acid (EA) is a polyphenol that must be released from the non-bioavailable ellagitannins in pomegranates, walnuts or strawberries to be absorbed. To estimate whether EA bioavailability could be improved after consumption of a high free EA amount, we conducted a crossover pharmacokinetic study in healthy volunteers that consumed two pomegranate extracts providing either 130 mg punicalagin+524 mg EA (PE-1) or 279 mg punicalagin+25 mg EA (PE-2). Targeted metabolomics (UPLC-ESI-qTOF-MS/MS) identified plasma free EA but not phase-II conjugates. EA pharmacokinetics showed high interindividual variability. Cmax ranged from 12 to 360 nM (PE-1: 74.8 ± 54.4 nM; PE-2: 64.1 ± 76.8 nM). In vitro digestion supported in vivo results. EA bioavailability was limited by the ellagitannin, pH and protein environment. A higher free EA intake does not enhance its bioavailability but promotes urolithin production. Bioavailability of EA, as the unchanged fraction that reaches the systemic circulation, is not as low as previously thought.

Punicalagin is a functional ingredient in pomegranate juice, which has various beneficial effects for health and prevention of disease. In this study, the anti-teratogenic effect of punicalagin (1 × 10−5 or 1 × 10−4 µM) was investigated in cultured mouse embryos and yolk sac-placentas exposed to nicotine (1 mM), one of the main toxins in cigarette smoke. Nicotine-treated embryos revealed severe anomalies as well as impaired yolk sac vascularization, reduced labyrinth formation, and developmental arrest of blood islands in the chorioallantoic border. Furthermore, nicotine significantly altered the regulations of hypoxia- and vascularization-related genes in yolk sac-placenta and the levels of oxidative stress, apoptosis, and inflammation in both embryos and yolk sac-placentas. However, these detrimental changes were remarkably improved in response to co-treatment with punicalagins. These findings indicate that punicalagin could be crucial to the development of clinical strategies to attenuate nicotine-induced teratogenesis in embryos.

Attention deficit hyperactivity disorder (ADHD) has high incidence rate among children which may be due to excessive monosodium glutamate (MSG) consumption and social isolation (SI)., Aim: We aimed to explore the relationships between MSG, SI, and ADHD development and to evaluate the neuroprotective potential of Punicalagin (PUN)., Methods: Eighty male rat pups randomly distributed into eight groups. Group I is the control, and Group II is socially engaged rats treated with PUN. Groups III to VII were exposed to ADHD-inducing factors: Group III to SI, Group IV to MSG, and Group V to both SI and MSG. Furthermore, Groups VI to VIII were the same Groups III to V but additionally received PUN treatment., Key Findings: Exposure to MSG and/or SI led to pronounced behavioral anomalies, histological changes and indicative of ADHD-like symptoms in rat pups which is accompanied by inhibition of the nuclear factor erythroid 2-related factor 2 (Nrf2)/Heme-oxygenase 1 (HO-1)/Glutathione (GSH) pathway, decline of the brain-derived neurotrophic factor (BDNF) expression and activation of the Toll-like receptor 4 (TLR4)/Nuclear factor kappa B (NF-kB)/NLR Family Pyrin Domain Containing 3 (NLRP3) pathway. This resulted in elevated inflammatory biomarker levels, neuronal apoptosis, and disrupted neurotransmitter equilibrium. Meanwhile, pretreatment with PUN protected against all the previous alterations., Significance: We established compelling associations between MSG consumption, SI, and ADHD progression. Moreover, we proved that PUN is a promising neuroprotective agent against all risk factors of ADHD., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Myocardial infarction (MI) is a life-threatening ischemic disease and is one of the leading causes of morbidity and mortality worldwide. Punicalagin (PU), the major ellagitannin found in pomegranates, is characterized by multiple antioxidant activities. The aim of this study is to assess the protective effects of PU against isoproterenol (ISO)-induced acute myocardial damage and to investigate its underlying vascular mechanisms using rat model. METHODS: Rats were randomly divided into five groups and were treated orally (p.o.) with PU (25 and 50 mg/kg) for 14 days. ISO was administered subcutaneously (S.C.) (85 mg/kg) on the 15th and 16th days to induce Myocardial infarction. Cardiac markers, oxidative stress markers, and inflammatory cytokines levels were determined in the heart tissue. Immunohistochemistry analysis was performed to determine the protein expression pathways of inflammation, apoptosis and oxidative stress (Nuclear factor erythroid 2-related factor 2 (Nrf-2), and heme oxygenase-1 (HO-1) in all the groups. In silico study was carried out to evaluate the molecular interaction of PU with some molecular targets. RESULTS: Our results showed that ISO-induced cardiac tissue injury was evidenced by increased serum creatine kinase-MB (CK-MB), cardiac troponin I (cTnI), and lactate dehydrogenase (LDH), associated with several histopathological changes. ISO also induced an increase of MDA, PCO, NO, and 8-hydroxy-2-deoxyguanosine (8-OHdG), along with a decrease of antioxidant enzyme activities in the myocardial tissues. In addition, an increase of TNF-α, NF-κB, IL-6, IL-1β, iNOS, Nrf2 and (HO-1) was observed. Pre-treatment with PU reduced myocardial infract area, ameliorated histopathological alterations in myocardium, and decreased activities of myocardial injury marker enzymes in ISO-induced rats. In addition, PU remarkably restored ISO-induced elevation of lipid peroxidation and decrease of antioxidants, significantly reduced myocardial pro-inflammatory cytokines concentrations in this animal model. Molecular docking analysis of PU with protein targets showed potent interactions with negative binding energies. In conclusion, PU can protect the myocardium from oxidative injury, inflammatory response, and cell death induced by ISO by upregulating Nrf2/HO-1 signaling and antioxidants., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Fatty acid synthase (FAS) has been recognized as a potential therapeutic target for obesity. In this study, for the first time, the inhibitory effect of pomegranate husk extract, punicalagin and ellagic acid on FAS was investigated. We found them potently inhibiting the activity of FAS with half-inhibitory concentration values (IC50) of 4.1 μg/ml (pomegranate husk extract), 4.2 μg/ml (4.50 μM, punicalagin) and 1.31 μg/ml (4.34 μM, ellagic acid), respectively. Moreover, they all exhibited time-dependent inactivation of FAS. Punicalagin and ellagic acid inhibited FAS with different mechanisms compared to previously reported inhibitors, through inactivating acetyl/malonyl transferase and β-ketoacyl synthase domains, respectively. Additionally, 100 μg/ml pomegranate husk extract, 5.24 μg/ml (5 μM) punicalagin and 4.5 μg/ml (15 μM) ellagic acid effectively reduced lipid accumulation inside FAS over-expressed 3T3-L1 adipocytes. Since FAS plays a key role in the biosynthesis pathway of fatty acid, these findings suggest that pomegranate husk extract, punicalagin and ellagic acid have potential in the prevention and treatment of obesity.

Pomegranates contain a complex mixture of gallotannins, ellagitannins, ellagic acid and anthocyanins. However, label claims on pomegranate supplements (PS) may not correlate with actual content of antioxidants, polyphenols or tannins. Nineteen PS were evaluated for their authenticity by determining ellagitannin composition by RP-HPLC and studying the relationship between total polyphenols as measured by the Folin–Ciocalteau assay and antioxidant capacity by oxygen radical absorbing capacity (ORAC), free radical scavenging properties by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and ferric reducing antioxidant power (FRAP). Only a limited number of pomegranate supplements were authentic. Product labels were inconsistent with polyphenol composition and antioxidant content. A majority of the samples (n = 13) contained disproportionately high amounts of ellagic acid and low or no detectable pomegranate tannins. Only six products had tannin composition that resembled pomegranates (punicalagin, punicalin, ellagitannins and gallotannins). PS-01 (natural pomegranate extract) was the most representative of pomegranate fruit polyphenols with 99% total pomegranate polyphenol and the highest antioxidant capacity across all measures. Correlations between total polyphenols and antioxidant content were high (R2 > 0.87) in products that had polyphenol composition resembling pomegranates. Products that contained high amounts of ellagic acid and low or no detectable pomegranate tannins had poor correlations between total polyphenols and antioxidant content. The results indicate that reliable labeling information, better standardization, improved manufacturing practices and regulation of the market is required to assure consumers of the quality of pomegranate supplements.

Poultry production was long plagued by coccidiosis, and the development of alternative therapies will make practical sense. In this work, 2 battery experiments were designed. In battery experiment 1, the best effect of 7 anticoccidial herbs (Sophora japonica Linn, Citrus aurantium L, leaf of Acer palmatum, bark of Magnolia officinalis, fruit peel of Punica granatum L., Eclipta prostrata L., and Piper sarmentosum Roxb.) against Eimeria tenella infection of 21-day-old male Chinese Guangxi yellow-feathered chickens were screened out by clinic indexes (bloody feces scores, cecal lesion scores, oocysts output, relative weight gain rate, and survival rate). According to the results from battery experiment 1 and other literature research, we selected 2 monomers which were extracted from fruit peel of Punica granatum L. for further battery experiment 2 which were similar with battery experiment 1. Clinic results showed that Punicalagin had better anticoccidial effect than Ellagic acid. The anticoccidial mechanism exploration results of Elisa, antioxidant test, and pathological observation showed that Punicalagin reduced the cecal inflammation, improved the expression of immunoglobulin in cecal tissue, improved cecal integrity, and restored its REDOX state. Results of 16S rRNA sequencing analysis showed that Punicalagin also maintained the fecal flora health during E. tenella infection through insignificantly increasing the proportion of Lactobacillus and Faecalibacterium as well as significantly reducing the proportion of pathogenic bacteria, Escherichia-Shigella. RNA-Seq analysis results suggested that Punicalagin may play a role in controlling E. tenella infection by interaction with cytochrome P450 family enzymes. Overall, Punicalagin has promising potential as an alternative therapy for chicken Eimeria tenella infection., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) helicase NSP13 plays a conserved role in the replication of coronaviruses and has been identified as an ideal target for the development of antiviral drugs against SARS-CoV-2. Here, we identify a novel NSP13 helicase inhibitor punicalagin (PUG) through high-throughput screening. Surface plasmon resonance (SPR)-based analysis and molecular docking calculation reveal that PUG directly binds NSP13 on the interface of domains 1A and 2A, with a K D value of 21.6 nM. Further biochemical and structural analyses suggest that PUG inhibits NSP13 on ATP hydrolysis and prevents it binding to DNA substrates. Finally, the antiviral studies show that PUG effectively suppresses the SARS-CoV-2 replication in A549-ACE2 and Vero cells, with EC 50 values of 347 nM and 196 nM, respectively. Our work demonstrates the potential application of PUG in the treatment of coronavirus disease 2019 (COVID-19) and identifies an allosteric inhibition mechanism for future drug design targeting the viral helicases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Historically, people have been using pomegranate to alleviate many disease conditions. Pomegranate is known for its antiinflammatory, antioxidant, neuroprotective, anticancer, and antibacterial properties. In the current study, we examined effects of 8 dietary phenolics present in pomegranate (DPPs)-cyanidin-3-glucoside, cyanin chloride, delphinidin-3-glucoside, delphinidin-3,5-diglucoside, pelargonidin-3-glucoside, pelargonin chloride, punicalagin, and punicalin-on Escherichia coli ATP synthase and cell growth. DPPs caused complete or near complete (89%-100%) inhibition of wild-type E. coli ATP synthase and partial (5%-64%) inhibition of mutant enzymes αR283D, αE284R, βV265Q, and γT273A. Growth inhibition of wild-type, null, and mutant strains in the presence of DPPs were lower than that of isolated wild-type and mutant ATP synthase. On a molar scale, cyanin chloride was the most potent, and pelargonidin-3-glucoside was the least effective inhibitor of wild-type ATP synthase. Partial inhibition of mutant enzymes confirmed that αR283D, αE284R, βV265Q, and γT273A are essential in the formation of the phytochemical binding site. Our results establish that DPPs are potent inhibitors of wild-type E. coli ATP synthase and that the antimicrobial nature of DPPs can be associated with the binding and inhibition of microbial ATP synthase. Additionally, selective inhibition of microbial ATP synthase by DPPs is a useful method to combat antimicrobial resistance., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Nephrotoxicity is a major complication that limits the therapeutic application of cisplatin (CIS). Oxidative stress and inflammation are implicated in CIS-induced acute kidney injury (AKI) and apoptotic cell death. Punicalagin (PUN), a polyphenol in pomegranate, possesses promising anti-inflammatory and antioxidant activities, and its beneficial effect against CIS-induced AKI has not been fully elucidated. This investigation evaluated the protective effect of PUN against CIS-induced renal oxidative stress, inflammation and cell death. Rats received PUN (25 and 50 mg/kg) for 10 days and a single injection of CIS at day 7. The results showed increased serum urea and creatinine and several histopathological alterations in the kidney of CIS-intoxicated rats. Renal malondialdehyde (MDA) and nitric oxide (NO) were increased, and reduced glutathione, superoxide dismutase and catalase were declined in rats treated with CIS. PUN effectively ameliorated kidney function and attenuated tissue injury induced by CIS, decreased MDA and NO, and enhanced antioxidant defenses. Additionally, PUN downregulated NF-κB p65, iNOS, TNF-α, IL-6 and IL-1β in the kidney of rats that received CIS. Bax and caspase-3 were increased, and Bcl-2 was decreased in the kidney of CIS-intoxicated rats, an effect that was reversed by PUN. PUN upregulated Nrf2 expression in the kidney of CIS-intoxicated rats. In conclusion, PUN prevents CIS-induced AKI in rats by attenuating oxidative stress, inflammatory response and apoptosis, and upregulating Nrf2 and antioxidants., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The novel coronavirus disease (Covid-19) has become a major health threat globally. The interaction of SARS-CoV-2 spike (S) glycoprotein receptor-binding domain (RBD) with ACE2 receptor on host cells was recognized as the first step of virus infection and therefore as one of the primary targets for novel therapeutics. Pomegranate extracts are rich sources of bioactive polyphenols that were already recognized for their beneficial health effects. In this study, both in silico and in vitro methods were employed for evaluation of pomegranate peel extract (PoPEx), their major polyphenols, as well as their major metabolite urolithin A, to attenuate the contact of S-glycoprotein RBD and ACE2. Our results showed that PoPEx, punicalin, punicalagin and urolithin A exerted significant potential to block the S-glycoprotein-ACE2 contact. These in vitro results strongly confirm the in silico predictions and provide a valuable insight in the potential of pomegranate polyphenols for application in SARS-CoV-2 infection., (Copyright © 2021 Elsevier Inc. All rights reserved.)

In this study, a special orthogonal separation method, named as dual-tautomerism separation (DTS), was developed for the purification of tautomeric compounds from complex matrixes. In DTS, isomers of these compounds are individually collected and asymmetrically transformed to the mixtures of isomers. After separating the mixture with an identical method, high-purity compounds can be prepared from the newly generated isomers but impurities remain in another one. To validate the effectiveness, a DTS was developed to prepare punicalagin in gram-scale from pomegranate peel waste. Isomerization kinetic and thermodynamic of punicalagin were accurately assayed by dynamic HPLC built on low-temperature or/and loop-based stop-flow two-dimensional liquid chromatography. After the isomerization based on it, 9.3 g of pomegranate peel extract was firstly separated on C18 column, and F α and F β around α-punicalagin and β-punicalagin were obtained. Then, the proportion of α-punicalagin in F α and F β was optimized to 52.7% and 32.0% based on isomerization kinetics and thermodynamic. With the aid of low-temperature injection, F α and F β were loaded and secondly purified. After waste recycling, totally 3.0 g of punicalagin with the purify of 99.5% was obtained within two days, which would strongly support the resource utilization of pomegranate peel waste. Because only an individual method was employed in the two-step purification, the separation in DTS was fully compatible., Competing Interests: Declaration of Competing Interest The work presented in this report is the subject of pending patents (CN 201910711349.9, CN 201910713229.2, CN 201910711427.5) filed by Xinjiang Technical Institute of Physics and Chemistry, Chinese Academy of Sciences. The authors declared that there are no conflicts of interest to this work and these patents., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The emerging SARS-CoV-2 infection is the cause of the global COVID-19 pandemic. To date, there are limited therapeutic options available to fight this disease. Here we examined the inhibitory abilities of two broad-spectrum antiviral natural products chebulagic acid (CHLA) and punicalagin (PUG) against SARS-CoV-2 viral replication. Both CHLA and PUG reduced virus-induced plaque formation in Vero-E6 monolayer at noncytotoxic concentrations, by targeting the enzymatic activity of viral 3-chymotrypsin-like cysteine protease (3CL pro ) as allosteric regulators. Our study demonstrates the potential use of CHLA and PUG as novel COVID-19 therapies., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Salmonella, a bacterial foodborne pathogen, can contaminate meat, milk, and vegetables. While appropriate measures are available to control Salmonella, the inhibitory phytochemicals from plants are gaining increased attention. Punicalagin, a natural antimicrobial, is one of the main active tannins isolated from Punica granatum L. To obtain a broader understanding of the effect of punicalagin on the cell membranes of Salmonella Typhimurium, the growth curves, extracellular potassium concentration, release of cell constituents, intracellular pH, membrane potential, and morphological features were characterized to elucidate the mechanisms of action. Treatment with punicalagin induced an increase in the extracellular concentrations of potassium and a release of cell constituents. A higher pH gradient, an increase in the intracellular pH, and cell membrane depolarization were observed after punicalagin treatment. Electron microscopy observations showed that the cell membrane structures of Salmonella Typhimurium were damaged by punicalagin. It is concluded that punicalagin inhibits the proliferation of Salmonella Typhimurium and destroys the integrity of the cell membrane, leading to a loss of cell homeostasis. These findings indicate that punicalagin has the potential to be developed as a future alternative to control Salmonella Typhimurium contamination in foods and reduce the risk of salmonellosis., (Copyright ©, International Association for Food Protection.)

Introduction: The world is witnessing a steady rise in the prevalence of gestational diabetes mellitus (GDM), correlated with the current obesity epidemic. Both GDM and obesity negatively impact both the health of women but also that of the next generation. GDM and maternal obesity are associated with increased maternal and fetal inflammation and oxidative stress. A safe and effective intervention that can prevent these pathological features, and reduce the intergenerational burden, is required. Phenolic acids, such as punicalagin and curcumin, possess anti-inflammatory and antioxidant properties. Thus, the aim of this study was to examine the effects of punicalagin and curcumin on pro-inflammatory cytokines and chemokines, and antioxidant expression in an in vitro model of inflammation., Methods: Human placenta, visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) explants were obtained at term elective Caesarean section and stimulated with TNF alpha (TNF)., Results: We found that punicalagin and curcumin significantly supressed TNF-induced pro-inflammatory cytokine (IL1A, IL1B, and IL6) and chemokine (CCL2-4, CXCL1, CXCL5 and CXCL8) expression in human placenta, VAT and SAT. Anti-inflammatory cytokine IL4 and IL13 mRNA expression was also upregulated by punicalagin and curcumin treatment in placenta, VAT and SAT. Punicalagin and curcumin also altered antioxidant (SOD2 and catalase) mRNA expression in placenta, VAT and SAT, with minimal effect on hydrogen peroxide concentrations in tissue lysates., Conclusion: These findings suggest that the phenolic acids punicalagin and curcumin possess potent anti-inflammatory capabilities in in vitro human models of inflammation. Further studies are warranted to determine their suitability as therapeutic interventions for pro-inflammatory gestational complications, including GDM and maternal obesity., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Punicalagin has been found to exert cardiac protective effects against myocardial ischemia/reperfusion (MI/R) injury, although the detailed mechanisms remain largely unknown. This experiment was performed to explore the potential involvement of silent information regulator 1 (SIRT1)-NFE2-related factor 2 (NRF-2)-heme oxygenase-1 (HO-1) pathway in the cardiac protective actions of punicalagin. Sprague-Dawley (SD) rats were subjected to MI/R operation with or without punicalagin treatment (40 mg kg -1 d -1 ). We showed that punicalagin-treated group exhibited enhanced cardiac function, reduced myocardial infarction and decreased cleaved caspase-3 level. Furthermore, myocardial oxidative/nitrosative stress was ameliorated by punicalagin as evidenced by suppressed superoxide generation, gp91 phox and iNOS expressions, NO metabolites as well as myocardial nitrotyrosine level. Additionally, punicalagin decreased myocardial IL-6, TNF-α and the levels of ICAM-1, VCAM-1 and IKK-β expressions as well as IκB-α phosphorylation and NF-κB nuclear translocation. However, these effects were abolished by EX527 (5 mg kg -1 d -1 , a selective SIRT1 inhibitor). We further found that punicalagin dose-dependently enhanced SIRT1 nuclear distribution and NRF-2-HO-1 signaling. While EX527 treatment not only reduced SIRT1 activity, but also reversed the activation of NRF-2-HO-1 pathway. Collectively, these results revealed that punicalagin reduced cardiac oxidative/nitrosative stress and inflammatory response induced by MI/R operation through SIRT1-mediated activation of NRF-2-HO-1 signaling., (Copyright © 2019 Elsevier B.V. All rights reserved.)

N-myristoylation (Myr) is an eukaryotic N-terminal co- or post-translational protein modification in which the enzyme N-myristoyltransferase (NMT) transfers a fatty acid (C14:0) to the N-terminal glycine residues of several cellular key proteins. Depending on the cellular context, NMT may serve as a molecular target in anticancer or anti-infectious therapy, and drugs that inhibit this enzyme may be useful in the treatment of cancer or infectious diseases. As part of an on-going project to identify natural Homo sapiens N-myristoyltransferase 1 inhibitors (HsNMT1), two ellagitannins, punicalagin (1) and isoterchebulin (2), along with eschweilenol C (3) and ellagic acid (4) were isolated from the bark of Terminalia bentzoë (L.) L. f. subsp. bentzoë. Their structures were determined by means of spectroscopic analyses and comparison with literature data. Punicalagin (1) and isoterchebulin (2) showed significant inhibitory activity towards HsNMT1, and also against Plasmodium falciparum NMT (PfNMT) both in vitro and in cellulo, opening alternative paths for new NMT inhibitors development. This is the first report identifying natural products from a botanical source as inhibitors of HsNMT and PfNMT., (Copyright © 2018. Published by Elsevier B.V.)

The intake of fruits has proven to reduce the risk and incidence of cancer worldwide and plays a crucial role in cancer prevention. Pomegranate (Punica granatum), which belongs to the Punicaceae family, is one such plant that contains beneficial nutrients as well as many bioactive components and important phytochemicals that can be attributed to cancer-related therapeutic purposes. Pomegranate possesses antioxidant, anti-inflammatory, anti-proliferative, anti-angiogenic, anti-invasive, and anti-metastatic properties, and induces apoptosis. It also down-regulates various signaling pathways such as NF-κB, PI3K/AKT/mTOR, and Wnt, and down-regulates the expression of genes that are responsible in cancer development, such as anti-apoptotic genes, MMPs, VEGF, c-met, cyclins, Cdks, and pro-inflammatory cytokines. Therefore, inclusion of the fruit in one's diet would assist in a healthy life protected from cancer and also act as an effective chemotherapeutic with no toxic side effects., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Prostate cancer (PCa) is an international health problem and search for its effective treatment is in progress. Punicalagin (PN), polyphenol from pomegranate fruit, is known to exhibit potent anticancer activity in lung, breast and cervical cells. However, there is paucity of information on its effect in PCa. This study evaluated anti-proliferative effects of PN and its effects on extrinsic pathway of apoptosis in PCa cells, and angiogenesis in chicken chorioallantoic membrane (CAM). Antioxidant activities of PN were determined by 2,2-diphenyl-1-picryhydrazyl (DPPH) radical scavenging and inhibition of lipid peroxidation (LPO) methods. PCa (PC-3 and LNCaP) and normal prostate (BPH-1) cells were cultured and treated with PN (10, 50 and 100 μM). Cytotoxicity and viability effects of PN were determined by lactate dehydrogenase (LDH) and XTT assays, respectively. Antiangiogenic effects were measured using CAM assay, while apoptosis was assessed by DNA fragmentation, enrichment factor by Cell Death Detection ELISA kit and expressions of caspases-3 and -8. Results showed that PN (10-200 μM) significantly scavenged DPPH and inhibited LPO in a concentration-dependent manner. Furthermore, PN (10-100 μM) concentration-dependently inhibited viability in PC-3 and LNCaP, while viability in BPH-1 was insignificantly affected. PN had low toxicity on cells in vitro at concentrations tested. Also, PN (100 μM) increased enrichment factor in PC-3 (2.34 ± 0.05) and LNCaP (2.31 ± 0.26) relative to control (1.00 ± 0.00). In addition, PN (50 μM) decreased the network of vessels in CAM, suggesting its anti-angiogenic effect. Moreso, PN increased the expressions of caspases-3 and -8 in PC-3. Overall, PN exerts anti-proliferative activity in PCa cells via induction of apoptosis and anti-angiogenic effect., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Maternal diabetes-induced birth defects remain a significant health problem. Studying the effect of natural compounds with antioxidant properties and minimal toxicities on diabetic embryopathy may lead to the development of new and safe dietary supplements. Punicalagin is a primary polyphenol found in pomegranate juice, which possesses antioxidant, anti-inflammatory and anti-tumorigenic properties, suggesting a protective effect of punicalagin on diabetic embryopathy. Here, we examined whether punicalagin could reduce high glucose-induced neural tube defects (NTDs), and if this rescue occurs through blockage of cellular stress and caspase activation. Embryonic day 8.5 (E8.5) mouse embryos were cultured for 24 or 36 h with normal (5 mM) glucose or high glucose (16.7 mM), in presence or absence of 10 or 20 μM punicalagin. 10 μM punicalagin slightly reduced NTD formation under high glucose conditions; however, 20 μM punicalagin significantly inhibited high glucose-induced NTD formation. Punicalagin suppressed high glucose-induced lipid peroxidation marker 4-hydroxynonenal, nitrotyrosine-modified proteins, and lipid peroxides. Moreover, punicalagin abrogated endoplasmic reticulum stress by inhibiting phosphorylated protein kinase ribonucleic acid (RNA)-like ER kinase (p-PERK), phosphorylated inositol-requiring protein-1α (p-IRE1α), phosphorylated eukaryotic initiation factor 2α (p-eIF2α), C/EBP-homologous protein (CHOP), binding immunoglobulin protein (BiP) and x-box binding protein 1 (XBP1) mRNA splicing. Additionally, punicalagin suppressed high glucose-induced caspase 3 and caspase 8 cleavage. Punicalagin reduces high glucose-induced NTD formation by blocking cellular stress and caspase activation. These observations suggest punicalagin supplements could mitigate the teratogenic effects of hyperglycemia in the developing embryo, and possibly prevent diabetes-induced NTDs., (Copyright © 2015 Elsevier Inc. All rights reserved.)