Comparative transcriptomic and metabolomic profiles reveal fruit peel color variation in two red pomegranate cultivars.

Pomegranate (Punica granatum L.) which belongs to family Lythraceae, is one of the most important fruit crops of many tropical and subtropical regions. A high variability in fruit color is observed among different pomegranate accessions, which arises from the qualitative and quantitative differences in anthocyanins. However, the mechanism of fruit color variation is still not fully elucidated. In the present study, we investigated the red color mutation between a red-skinned pomegranate 'Hongbaoshi' and a purple-red-skinned cultivar 'Moshiliu', by using transcriptomic and metabolomic approaches. A total of 51 anthocyanins were identified from fruit peels, among which 3-glucoside and 3,5-diglucoside of cyanidin (Cy), delphinidin (Dp), and pelargonidin (Pg) were dominant. High proportion of Pg in early stages of 'Hongbaoshi' but high Dp in late stages of 'Moshiliu' were characterized. The unique high levels of Cy and Dp anthocyanins accumulating from early developmental stages accounted for the purple-red phenotype of 'Moshiliu'. Transcriptomic analysis revealed an early down-regulated and late up-regulated of anthocyanin-related structure genes in 'Moshiliu' compared with 'Hongbaoshi'. Alao, ANR was specially expressed in 'Hongbaoshi', with extremely low expression levels in 'Moshiliu'. For transcription factors R2R3-MYB, the profiles demonstrated a much higher transcription levels of three subgroup (SG) 5 MYBs and a sharp decrease in expression of SG6 MYB LOC116202527 in high-anthocyanin 'Moshiliu'. SG4 MYBs exhibited two entirely different patterns, LOC116203744 and LOC116212505 were down-regulated whereas LOC116205515 and LOC116212778 were up-regulated in 'Moshiliu' pomegranate. The results indicate that specific SG members of the MYB family might promote the peel coloration in different manners and play important roles in color mutation in pomegranate.

BELL1 interacts with CRABS CLAW and INNER NO OUTER to regulate ovule and seed development in pomegranate.

Pomegranate (Punica granatum) flowers are classified as bisexual flowers and functional male flowers. Functional male flowers have sterile pistils that show abnormal ovule development. In previous studies, we identified INNER NO OUTER (INO), CRABS CLAW (CRC), and BELL1 (BEL1), which were specifically expressed in bisexual and functional male flowers. However, the functions of ovule identity genes and the mechanism underlying ovule sterility in pomegranate remain unknown. Here, we found that the integument primordia formed and then ceased developing in the ovules of functional male flowers with a vertical diameter of 8.1-13.0 mm. Megaspore mother cells were observed in bisexual flowers when the vertical diameters of flowers were 10.1-13.0 mm, but not in functional male flowers. We analyzed the expression patterns of ovule-related genes in pomegranate ovule sterility and found that PgCRC mRNA was highly expressed at a critical stage of ovule development in bisexual flowers. Ectopic expression of PgCRC and PgINO was sufficient to increase seed number in transgenic lines. PgCRC partially complemented the Arabidopsis (Arabidopsis thaliana) crc mutant, and PgINO successfully rescued the seeds set in the Arabidopsis ino mutant. The results of yeast two-hybrid assays, bimolecular fluorescence complementation assays, and genetic data analyses showed that PgCRC and PgINO directly interact with PgBEL1. Our results also showed that PgCRC and PgINO could not interact directly with MADS-box proteins and that PgBEL1 interacted with SEPALLATA proteins. We report the function of PgCRC and PgINO in ovule and seed development and show that PgCRC and PgINO interact with PgBEL1. Thus, our results provide understanding of the genetic regulatory networks underlying ovule development in pomegranate.

Hepatoprotective effect of methanol fruit extract of Punica granatum L in highly active antiretroviral therapy-induced toxicity in Wistar rats.

Prolonged use of Highly Active Antiretroviral Therapy (HAART) has been linked to toxicity, particularly hepatotoxicity. There are few effective drugs for HAART patients that promote hepatic cell regeneration and prevent liver injury. Therefore, the purpose of this study was to investigate the hepato-protective activity of Methanol fruit extract of Punica granatum (MFEPG) in HAART-administered rats. Thirty rats weighing between 150-200 g were randomly divided into six groups and each group comprised of five rats. Distilled water was given to the rats in group one. Only HAART was given to the rats in group two. MFEPG at doses of 100 and 400 mg/kg was given to the rats in groups three and four. MFEPG dosages of 100 and 400 mg/kg along with HAART were given to the rats in groups five and six, respectively. All treatments were via oral gavage daily for 40 days. Under halothane anesthesia, all rats were sacrificed on day 41. Liver tissues were utilized for lipid peroxidation marker; Malondialdehyde (MDA), antioxidant enzymes; Superoxide dismutase (SOD) and Catalase (CAT) and histological evaluation, while blood samples were examined for biochemical parameters (AST, ALT, ALP, Total cholesterol, Total protein, and Albumin). The HAART-treated group exhibited a significantly higher amount of the lipid peroxidation end product; MDA, and significantly lower levels of antioxidant enzymes; SOD, and CAT. Liver enzymes and total cholesterol were significantly increased with a significant reduction in Total protein and Albumin levels in the HAART-treated group. Conversely, the liver function biomarkers were returned to normal levels in the HAART and MFEPG-treated groups. Histopathological studies revealed that when HAART-exposed rats were treated with MFEPG, both the biochemical and histological results significantly improved. Thus, the antioxidant activity of MFEPG provides protection against HAART-induced liver oxidative damage. More research is needed to determine the safety of using MFEPG in humans.

Prevention of aflatoxin B1 toxicity by pomegranate peel extract and its effects on growth, blood biochemical changes, oxidative stress and histopathological alterations.

Contamination of animal feeds with mycotoxins is one of the most serious issues in the world of animal nutrition. The purpose of this study was to assess the efficacy of pomegranate peel extract (PPE) versus oxytetracycline (OXY) in reducing aflatoxin B1 (AFB1) toxicity in rabbits fed contaminated diets. This experiment was done on 48 weaned NZW, four groups (n = 12). The first (AF) served as the control group feed on an infected diet by AFB1 between 0.02 and 0.03 mg/kg BW; second (AF + OXY) received an infected diet by AFB1 and was treated with OXY 200 mg/kg BW/day; third (AF + PPE) received an infected diet by AFB1 and treated with PPE 130 mg/kg BW/day; fourth (AF + OXY + PPE) received an infected diet by AFB1 and treated with a daily oral dose of 100 mg/kg BW/day of OXY + PPE (65 mg/kg BW/day), the experiment lasted for 8 weeks experiment. The treated group with PPE and its combination with OXY concurrent with AF showed amelioration markedly for liver and kidney function. To summarise, adding PPE and combining it with OXY at a half dose to an AFB1-contaminated diet can help minimise the harmful effects of AFB1 on rabbit performance.

Identification of chemical constituents in pomegranate seeds based on ultra-high-performance supercritical fluid chromatography coupled with quadrupole time-of-flight mass spectrometry.

Pomegranate seeds are a potential source of bioactive compounds. Nonetheless, most pomegranate seeds are discarded in the food processing industry, likely due to the lack of convincing data on their component analysis. METHODS: To reveal the main chemical constituents of pomegranate seeds, a reliable and sensitive method based on ultra-high-performance supercritical fluid chromatography coupled with electrospray ionization and quadrupole time-of-flight mass spectrometry (MS) was developed. A time-dependent MSE data acquisition mode was applied to acquire the mass spectrometric data. The chemical constituents were identified by an automatic retrieval of a traditional Chinese medicine library and relevant literature. RESULTS: A total number of 59 compounds, including fatty acids, sterols, vitamins, cerebrosides, phospholipids, flavonoids, phenylpropanoids, and others, were tentatively identified. Their possible fragmentation pathways and characteristic ions were proposed and elucidated. CONCLUSIONS: The findings of this study, along with the developed methodology, could provide a reference for basic research on the pharmacodynamic substances of pomegranate seeds and shed light on their potential nutritional and therapeutic applications in the future.

Pomegranate sarcotesta lectin (PgTeL) inhibits planktonic growth and disrupts biofilm formed by Cryptococcus neoformans.

AIMS: We investigated the putative fungistatic and fungicidal activities of pomegranate sarcotesta lectin (PgTeL) against Cryptococcus neoformans B3501 (serotype D), specifically the ability of PgTeL to inhibit yeast capsule and biofilm formation in this strain. METHODS AND RESULTS: PgTeL showed a minimum inhibitory concentration of 172.0 µg ml-1, at which it did not exhibit a fungicidal effect. PgTeL concentrations of 4.0-256.0 µg ml-1 reduced biofilm biomass by 31.0%-64.0%. Furthermore, 32.0-256.0 µg ml-1 PgTeL decreased the metabolic activity of the biofilm by 32.0%-93.0%. Scanning electron microscopy images clearly revealed disruption of the biofilm matrix. Moreover, PgTeL disrupted preformed biofilms. At concentrations of 8.0-256.0 µg ml-1, PgTeL reduced metabolic activity in C. neoformans by 36.0%-92.0%. However, PgTeL did not inhibit the ability of B3501 cells to form capsules under stress conditions. CONCLUSIONS: PgTeL inhibited biofilm formation and disrupted preformed biofilms, demonstrating its potential for use as an anticryptococcal agent.

An insight into the neuroprotective effects and molecular targets of pomegranate (Punica granatum) against Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disease that still has no permanent cure. The drugs prescribed in the present days are only for symptomatic relief for the patients. Many studies correlating the reduction in the incidence of AD with the diet consumed have been published. These studies showed that a diet rich in polyphenols is associated with a decrease in the incidence of AD. The present review is focused on the ability of pomegranate and its bioactive components to ameliorate the progression of AD and their ability to exert a neuroprotective effect. Various studies showing the ability of pomegranate in inhibiting enzymes, reducing reactive oxygen species, inhibition of microglial activation, inhibition of tau protein hyperphosphorylation, maintenance of synaptic plasticity, anti-inflammatory activity and its ability to inhibit Beta secretase-1 (BACE-1) has been reviewed in this article. In spite of the lack of studies on humans, there are compelling evidence indicating that pomegranate can reduce various risk factors involved in the causation of AD and thus can be used as a persistent nutraceutical to slow ageing and for providing neuroprotection for the treatment of AD.Highlights An overview of traditional and pharmacological uses of pomegranate (POM).Potential of POM in the treatment of neurodegenerative diseases especially in AD.Insight into the molecular mechanisms of neuroprotective effects of POM in AD.Clinical evaluation studies involving POM and its bioactive components.

PgMYB1 Positively Regulates Anthocyanin Accumulation by Activating PgGSTF6 in Pomegranate.

The peel color of pomegranates is an important exterior quality that determines market value. Anthocyanins are biosynthesized in the cytosol and then transported to the vacuole for storage. However, the molecular mechanism that determines the color variation between red and white pomegranates remains unclear. In this study, we identified an R2R3-MYB protein (PgMYB1) that interacts with the PgGSTF6 promoter and regulates its transcriptional expression, thus promoting the accumulation of anthocyanins in pomegranate. The expression of PgMYB1 and PgGSTF6 was positively correlated with the anthocyanin content in red and white pomegranates. Further investigation showed that the knockdown of PgMYB1 in red pomegranate 'Taishanhong' (TSH), by the virus-induced gene-silencing system, inhibited anthocyanin accumulation. Together, our results indicate that PgMYB1 controls the transport of anthocyanin via PgGSTF6 and thus promotes anthocyanin accumulation in red pomegranates. Our results have a certain reference value for further clarifying the regulation of anthocyanin synthesis and transport in pomegranate fruits.

Pomegranate Seed Oil as a Source of Conjugated Linolenic Acid (CLnA) Has No Effect on Atherosclerosis Development but Improves Lipid Profile and Affects the Expression of Lipid Metabolism Genes in apoE/LDLR-/- Mice.

The aim of this study was to evaluate the anti-atherosclerotic effect of pomegranate seed oil as a source of conjugated linolenic acid (CLnA) (cis-9,trans-11,cis-13; punicic acid) compared to linolenic acid (LnA) and conjugated linoleic acid (CLA) (cis-9,trans-11) in apoE/LDLR-/- mice. In the LONG experiment, 10-week old mice were fed for the 18 weeks. In the SHORT experiment, 18-week old mice were fed for the 10 weeks. Diets were supplied with seed oils equivalent to an amount of 0.5% of studied fatty acids. In the SHORT experiment, plasma TCh and LDL+VLDL cholesterol levels were significantly decreased in animals fed CLnA and CLA compared to the Control. The expression of PPARα in liver was four-fold increased in CLnA group in the SHORT experiment, and as a consequence the expression of its target gene ACO was three-fold increased, whereas the liver's expression of SREBP-1 and FAS were decreased in CLnA mice only in the LONG experiment. Punicic acid and CLA isomers were determined in the adipose tissue and liver in animals receiving pomegranate seed oil. In both experiments, there were no effects on the area of atherosclerotic plaque in aortic roots. However, in the SHORT experiment, the area of atherosclerosis in the entire aorta in the CLA group compared to CLnA and LnA was significantly decreased. In conclusion, CLnA improved the lipid profile and affected the lipid metabolism gene expression, but did not have the impact on the development of atherosclerotic plaque in apoE/LDLR-/- mice.

The effects of pomegranate consumption on inflammatory and oxidative stress biomarkers in adults: a systematic review and meta-analysis.

BACKGROUND: Several studies have shown the effects of pomegranate on oxidative stress and inflammation biomarkers, while some studies showed no effects of pomegranate on these biomarkers. Therefore, we aimed to evaluate the effects of pomegranate consumption on C-reactive protein (CRP), interlukin-6 (IL-6), tumor necrosis factor α (TNF-α), total antioxidant capacity (TAC), and malondialdehyde (MDA) in adults. METHODS: A systematic literature search was performed using databases, including PubMed, Web of Science, and Scopus, up to May 2023 to identify eligible randomized controlled trials (RCTs). Heterogeneity tests of the included trials were performed using the I2 statistic. Random effects models were assessed based on the heterogeneity tests, and pooled data were determined as the weighted mean difference with a 95% confidence interval. RESULTS: Of 3811 records, 33 eligible RCTs were included in the current study. Our meta-analysis of the pooled findings showed that pomegranate consumption significantly reduced CRP (WMD: -0.50 mg/l; 95% CI -0.79 to -0.20; p = 0.001), IL-6 (WMD: -1.24 ng/L 95% CI -1.95 to -0.54; p = 0.001), TNF-α (WMD: -1.96 pg/ml 95%CI -2.75 to -1.18; p < 0.001), and MDA (WMD: -0.34 nmol/ml 95%CI -0.42 to -0.25; p < 0.001). Pooled analysis of 13 trials revealed that pomegranate consumption led to a significant increase in TAC (WMD: 0.26 mmol/L 95%CI 0.03 to 0.49; p = 0.025). CONCLUSION: Overall, the results demonstrated that pomegranate consumption has beneficial effects on oxidative stress and inflammatory biomarkers in adults. Therefore, pomegranate can be consumed as an effective dietary approach to attenuate oxidative stress and inflammation in patients with cardiovascular diseases. PROSPERO REGISTRATION CODE: CRD42023406684.

Is pomegranate husk scald during storage induced by water loss and mediated by ABA signaling?

BACKGROUND: Husk scald (HS) is a physiological disorder limiting the marketability of pomegranate fruit during long-term storage. Herein we propose that HS is triggered by water loss and mediated by ABA signaling. Therefore, pomegranate fruit were stored at three different storage temperatures (3.5, 7 and 11°C) and 96.5% ± 2.3% relative humidity (RH) evaluating weight loss (WL), abscisic acid (ABA), respiration rate (RR), total phenolics (TP), total anthocyanin (TA), antioxidant activity (AA), exocarp electrolyte leakage (EL), malondialdehyde (MDA), color attributes, browning index and visual quality of fruit. RESULTS: HS appeared after 3 months of storage at 11 °C, less at 7°C and non-present at 3.5°C. Incidence of HS occurred along with higher WL, RR, EL, MDA, and ABA content. Conversely, TP, TA and AA decreased significantly. WL increased with water vapor pressure deficit (VPD) at higher temperatures. After 93 days, 11 °C fruit reached ~10% WL while ABA increased to ~150 µg kg-1 . However, 3.5 and 7 °C fruit, reached 5 and 7% WL while ABA was ~31 and 75 µg kg-1 , respectively. CONCLUSION: Herein, we propose a mechanistic model of HS development where water stress induces ABA as a primary signaling molecule that triggers the HS response mediated by reactive oxygen species (ROS). Accumulation of ROS induces phenolic biosynthesis and oxidative stress promotes loss of membrane compartmentalization that induces phenolic degradation. Ultimately, husk scalding becomes visible due to the oxidation of phenolics into brown pigments. © 2022 Society of Chemical Industry.

Freeze dried pomegranate juices of Moroccan fruits: main representative phenolic compounds.

BACKGROUND: The pomegranate (Punica granatum) is an ancient perennial plant species of the Punicaceae family. Its seeds are consumed as food or as juice. Previous studies have noted that pomegranate juice encompasses many active compounds with beneficial effects. The main goals of this work were to study the phenolic components of freeze-dried and reconstituted pomegranate juices obtained from 13 pomegranate genotypes growing in Morocco. RESULTS: We analyzed several pomegranate juices using high-performance liquid chromatography and high-resolution mass spectrometry to determine phenolic compounds. Twenty-seven bio-phenols, belonging to four different classes (phenolic acids, hydrolyzable tannins, anthocyanins, and flavonoids), were identified based on their accurate mass measurements, and quantified. Some encouraging results were obtained. Even though the freeze-drying process introduced a marked degradation of bio-phenols, substantially lowering their levels in the reconstituted fruit juices, these fruit juices were still rich enough in bio-phenols to compete with some fresh fruit juices. The reconstituted juices obtained by rehydration of the lyophilized material still differed enough to enable a statistical classification based on their polyphenol content. A correlation analysis was applied to the polyphenol data to explore correlations and similarities between genotypes. CONCLUSIONS: The results showed that freeze-drying and reconstitution of juices introduced some degradation of the polyphenol content. The overall polyphenolic pattern within the same cultivar, in two different harvesting years, was maintained, however, suggesting the composition stability of the freeze-dried juices produced in this time span. © 2022 Society of Chemical Industry.

Comparative effects of gibberellin A3 , glycine betaine, and Si, Ca, and K fertilizers on physiological disorders and yield of pomegranate cv. Wonderful.

BACKGROUND: Damage from cracking, russeting, and sunscalds causes significant yield losses in pomegranate worldwide and may result from stressful environmental conditions. Although foliar sprays with minerals or growth regulators could be an important orchard management, little is known on the effectiveness of glycine betaine, silicon (Si)-based fertilizers or the response of cv. Wonderful to gibberellin A3 (GA3 ). RESULTS: During a 2-year study, foliar spraying with GA3 at 75 or 150 mg L-1 applied in July substantially reduced cracking, russeting, and sunscald symptoms and increased fruit size, yield, and 100-aril weight, without affecting the % edible portion or % juice, suggesting that arils and skin increased similarly. Nevertheless, they reduced the skin red coloration, especially at the higher dose. GA3 at 75 mg L-1 applied in September resulted in a low number of harvested fruit as a result of delayed maturation. Spraying with glycine betaine at seven repeated times at biweekly intervals starting in July, reduced sunscald symptoms, red coloration, and maturity index only in the year with high damage. Foliar sprays with calcium chloride or Si-based fertilizer containing potassium, applied as in the glycine betaine treatment, did not affect the occurrence of physiological disorders, whereas Si-based fertilizer containing potassium and calcium increased cracking and decreased sunscald only in the year with high damage. CONCLUSION: Spraying with GA3 at 75 mg L-1 in July could have a significant impact on a grower's income by reducing damage from physiological disorders, improving yield with a minimum decrease in red skin coloration. The efficacy of nutrient-related fertilizers and glycine betaine were not constant, and this would be useful to evaluate at earlier application times and under stress conditions. © 2021 Society of Chemical Industry.

Lipidomic Profile and Enzymes Activity in Hepatic Microsomes of Rats in Physiological and Pathological Conditions.

Among the risk factors affecting the development of cancer, nutritional factors occupy a significant place. Pomegranate seed oil (PSO) and bitter melon extract (BME), used for ages in folk medicine, are nowadays used in the prevention of many diseases and as ingredients of dietary supplements. Despite numerous publications on these raw materials or their active substances, their mechanism of action in various pathological states has not been recognized yet, nor has the safety of their simultaneous use been evaluated. The study aimed to assess how dietary supplementation with either PSO, with BME, or both, affects fatty acids' profiles and their metabolism in hepatic microsomes, as well as the activity of selected microsomal enzymes (COX-2 and CYP1B1). Experimental animals (Sprague-Dawley rats) were divided into eight parallel experimental groups, differing in applied dietary modifications (control, PSO, BME and both PSO and BME) and introduction of chemical carcinogen-7,12-dimethylbenz[a]nthracene. Obtained results indicated the pronounced effect of the cancerous process on lipid metabolism and demonstrated the antagonistic effect of applied dietary supplements on the content of individual fatty acids and the activity of CYP1B1 and COX-2. The applied broad analytical approach and chemometric data analysis confirmed that raw materials, for which potential cancer prevention has been previously demonstrated, may differ in effects depending on the coexisting pathological state.

Mitigation of the effect of drought on growth and yield of pomegranates by foliar spraying of different sizes of selenium nanoparticles.

BACKGROUND: Drought is a very important environmental stressor, which has negative effects on the growth of trees, decreasing their yield. The role of different-sized selenium nanoparticles (Se-NPs) in the mitigation of environmental stresses such as drought in crops has not yet been investigated. RESULTS: Trees treated with Se-NPs displayed higher levels of photosynthetic pigments, a better nutrient status, better physical parameters (especially fruit cracking) and chemical parameters, a higher phenolic content, and higher concentrations of osmolytes, antioxidant enzymes, and abscisic acid than untreated trees under drought stress. Foliar spraying of 10 and 50 nm Se-NPs alleviated many of the deleterious effects of drought in pomegranate leaves and fruits and this was achieved by reducing stress-induced lipid peroxidation and H2 O2 content by enhancing the activity of antioxidant enzymes. Furthermore, the 10 nm Se-NPs treatment produced more noticeable effects than the treatment with 50 nm Se-NPs. CONCLUSION: Results confirm the positive effects of nanoparticle spraying, especially the role of 10 nm Se-NPs in the management of negative effects of drought not only for pomegranates but potentially also for other crops. © 2021 Society of Chemical Industry.

Transformation of Microbial Negative Correlations into Positive Correlations by Saccharomyces cerevisiae Inoculation during Pomegranate Wine Fermentation.

The application of starter is a common practice to accelerate and steer the pomegranate wine fermentation process. However, the use of starter needs a better understanding of the effect of the interaction between the starter and native microorganisms during alcoholic fermentation. In this study, high-throughput sequencing combined with metabolite analysis was applied to analyze the effect of commercial Saccharomyces cerevisiae inoculation on the native fungal community interaction and metabolism during pomegranate wine fermentation. Results showed that there were diverse native fungi in pomegranate juice, including Hanseniaspora uvarum, Hanseniaspora valbyensis, S. cerevisiae, Pichia terricola, and Candida diversa Based on ecological network analysis, we found that S. cerevisiae inoculation transformed the negative correlations into positive correlations among the native fungal communities and decreased the Granger causalities between native yeasts and volatile organic compounds. This might lead to decreased contents of isobutanol, isoamylol, octanoic acid, decanoic acid, ethyl laurate, ethyl acetate, ethyl hexadecanoate, phenethyl acetate, and 2-phenylethanol during fermentation. This study combined correlation and causality analysis to gain a more integrated understanding of microbial interaction and the fermentation process. It provided a new strategy to predict certain behaviors between inoculated and selected microorganisms and those coming directly from the fruit.IMPORTANCE Microbial interactions play an important role in flavor metabolism during traditional food and beverage fermentation. However, we understand little about how selected starters influence interactions among native microorganisms. In this study, we found that S. cerevisiae inoculation changed the interactions and metabolisms of native fungal communities during pomegranate wine fermentation. This study not only suggests that starter inoculation should take into account the positive features of starters but also characterizes the microbial interactions established among the starters and the native communities. It may be helpful to select appropriate starter cultures for winemakers to design different styles of wine.

A low cost alternative, using mycorrhiza and organic fertilizer, to optimize the production of foliar bioactive compounds in pomegranates.

AIM: To select the best combination of arbuscular mycorrhizal fungi and efficient vermicompost dose in maximizing the production of leaf metabolites in Punica granatum seedlings. METHODS AND RESULTS: The experimental design was in a 3 × 3 factorial arrangement: three inoculation treatments (inoculated with Gigaspora albida, inoculated with Acaulospora longula and control not inoculated) × 3 doses of vermicompost (0, 5 and 7·5%). After 120 days of inoculation, biomolecules, plant growth parameters and mycorrhizal colonization were evaluated. The combination of 7·5% of vermicompost and A. longula was favourable to the accumulation of leaf phenols, with an increase of 116·11% in relation to the non-inoculated control. The total tannins was optimized/enhanced when G. albida and 7·5% of fertilizer were used, registering an increase of 276·71%. CONCLUSIONS: The application of 7·5% of vermicompost associated with A. longula and G. albida is a low cost alternative to increase the levels of bioactive compounds in pomegranate leaves. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first published report of optimization of bioactive compound production in P. granatum by the combined use of mycorrhiza and vermicompost doses.

A novel non-cytotoxic synthetic peptide, Pug-1, exhibited an antibiofilm effect on Streptococcus mutans adhesion.

The aim of this study was to evaluate the effects of synthetic peptides on the growth and biofilm formation of Streptococcus mutans and their cytotoxicity on a human keratinocyte cell line. Four pomegranate-derived peptides; Pug-1, Pug-2, Pug-3 and Pug-4 were synthesized. Streptococcus mutans (ATCC 25175 and DMST 41283) were used. The antibacterial and anti-adherence activities of the peptides were evaluated. The effect of the peptides on S. mutans glucosyltransferase (Gtf) activity was measured. The peptides' cytotoxicity was evaluated on a HaCaT cell line using an MTT assay. The bactericidal penetration depth of Pug-1 into the biofilm was investigated using confocal laser scanning microscopy (CLSM). Among the four peptides, Pug-1 (200 µg ml-1 ) exhibited antibacterial activity on S. mutans (DMST 41283). All peptides demonstrated anti-adherence activity. Streptococcus mutans Gtf activity was significantly inhibited by Pug-1. Interestingly, Pug-1 peptide was not cytotoxic to the HaCaT cell line. Pug-1 (100-200 µg ml-1 ) exhibited bactericidal penetration at approximately 50-100% of the biofilm depth. Therefore, Pug-1 might be a candidate of anticariogenic agent. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data highlighted that pomegranate-derived antimicrobial peptides (AMPs) inhibit Streptococcus mutans adhesion, virulence-associated genes and enzymes. This in vitro study supports the hypothesis that pomegranate AMPs would be noncytotoxic to human keratinocytes, with a potent anticariogenic effect via an anti-adherence mode-of-action. The present study provides evidence for further investigation of these AMPs as therapeutic and preventive agents for dental caries.

The Effect of Polyphenols on Pomegranate Fruit Susceptibility to Pilidiella granati Provides Insights into Disease Tolerance Mechanisms.

Pilidiella granati, also known as Coniella granati, is the etiological agent of pomegranate fruit dry rot. This fungal pathogen is also well-known as responsible for both plant collar rot and leaf spot. Because of its aggressiveness and the worldwide diffusion of pomegranate crops, the selection of cultivars less susceptible to this pathogen might represent an interesting preventive control measure. In the present investigation, the role of polyphenols in the susceptibility to P. granati of the two royalties-free pomegranate cultivars Wonderful and Mollar de Elche was compared. Pomegranate fruit were artificially inoculated and lesion diameters were monitored. Furthermore, pathogen DNA was quantified at 12-72 h post-inoculation within fruit rind by a real time PCR assay setup herein, and host total RNA was used in expression assays of genes involved in host-pathogen interaction. Similarly, protein extracts were employed to assess the specific activity of enzymes implicated in defense mechanisms. Pomegranate phenolic compounds were evaluated by HPLC-ESI-MS and MS2. All these data highlighted 'Wonderful' as less susceptible to P. granati than 'Mollar de Elche'. In the first cultivar, the fungal growth seemed controlled by the activation of the phenylpropanoid pathway, the production of ROS, and the alteration of fungal cell wall. Furthermore, antifungal compounds seemed to accumulate in 'Wonderful' fruit following inoculation. These data suggest that pomegranate polyphenols have a protective effect against P. granati infection and their content might represent a relevant parameter in the selection of the most suitable cultivars to reduce the economic losses caused by this pathogen.

Pomegranate Peel as Suitable Source of High-Added Value Bioactives: Tailored Functionalized Meat Products.

In the last few years, the consumer's concern with the relationship between health and diet has led to the search of foods with functional properties beyond the nutritional. In this framework, the consumption of pomegranate has increased due to their sensorial attributes and remarkable amounts of bioactive compounds, which generate, at the same time, huge amounts of by-products. A search in the Scopus database for the last 10 years has revealed the rising interest in pomegranate peel (PP), the main residue from this fruit. The meat industry is a food sector that has had to search for new alternatives to substitute the use of synthetic preservatives by new natural additives, to extend the self-life and keep the quality attributes of their processed products. This review sets out the main bioactivities of PP extracts, and their incorporation in meat products is elaborated. PP is a good source of bioactive compounds, including phenolic acids, flavonoids and hydrolyzable tannins, which have beneficial health effects. It can be concluded that the reformulation of meat products with PP extracts is a suitable strategy for enhancing their technological characteristics, in addition to conferring functional properties that make them healthier and potentially more acceptable for the consumer.