Blanching Pre-Treatment Promotes High Yields, Bioactive Compounds, Antioxidants, Enzyme Inactivation and Antibacterial Activity of 'Wonderful' Pomegranate Peel Extracts at Three Different Harvest Maturities.

'Wonderful' pomegranate (Punica granatum L.) peel contains a wide range of phytochemicals including vitamins, dietary fibre, phenolic compounds, and antioxidant properties. Yet, it is often used as animal feed or discarded in landfills, which is not the best eco-friendly way to utilize this phenolic-rich bioresource. Finding novel ways of utilizing pomegranate peel waste could prove a more profitable and eco-friendlier alternative that is far more beneficial to the economy. Adding a blanching pre-treatment step at optimal conditions prior to processing of pomegranate peel aids in the inactivation of quality changing enzymes such as polyphenol oxidase (PPO) and peroxidase (POD), which are accountable for the degradation reactions that cause breakdown of nutrients and phytochemicals. This study aimed to determine the effect of blanching at 80 °C for 3 min on the yield, polyphenol content, antioxidant properties, enzyme inactivation, and antibacterial activity of 'Wonderful' pomegranate peel ethanolic extracts from three different harvest maturities (unripe, ripe, and over ripe), including a comprehensive characterization and quantification using liquid chromatography-mass spectrometry (LC-MS). The blanched unripe peel extracts exhibited the highest total phenolic content, total tannin content, 2,2-diphenyl-1-picryl hydrazyl (DPPH) antioxidant activity, 2,2-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical scavenging activity and ferric ion reducing antioxidant power (FRAP) at 14.0 mg gallic acid equivalent (GAE)/g dry mass (DM), 1.0 mg GAE/g DM, 359.1 µmol Trolox/g DM, 912.2 µmol Trolox/g DM and 802.5 µmol Trolox/g DM, respectively. There was significant (p < 0.05) decrease in PPO and POD activity of all blanched pomegranate peel extracts. The blanched unripe peel extracts had the lowest PPO activity at 0.2 U/g fresh weight (FW), with a 70% PPO inactivation compared to ripe and over ripe harvest, whereas the highest POD inactivation was recorded at 67% in over ripe peel extracts. All blanched peel extracts, irrespective of harvest maturity, had minimum inhibitory concentration (MIC) values at 160 µg/mL against all four bacteria strains tested, which included two Gram-positive bacterial strains (Bacillus subtilis ATCC 6051 and Staphylococcus aureus ATCC 12600) and two Gram-negative bacteria (Escherichia coli 11775 and Klebsiella pneumonia ATCC 13883). A total of 25 metabolites including phenolic acids (4), organic acids (1), flavonoids (4), ellagitannins (13), and other polyphenols (3) in all three pomegranate peel samples were tentatively identified after LC-MS profiling. The blanched unripe peel extracts showed significantly higher punicalin α and ß, ß punicalagin, catechin, epicatechin content at 414 mg/g, and 678 mg/g, 151 mg/g, 229 mg/g, respectively, compared to peel extracts from other harvest maturities. This study provides supportive information for the commercial utilization of pomegranate fruit peel as source of value-added ingredients for the development of novel food, cosmetics, and pharmacological products.

The Implication of Chemotypic Variation on the Anti-Oxidant and Anti-Cancer Activities of Sutherlandia frutescens (L.) R.Br. (Fabaceae) from Different Geographic Locations.

Extracts of Sutherlandia frutescens (cancer bush) exhibit considerable qualitative and quantitative chemical variability depending on their natural wild origins. The purpose of this study was thus to determine bioactivity of extracts from different regions using in vitro antioxidant and anti-cancer assays. Extracts of the species are complex and are predominantly composed of a species-specific set of triterpene saponins (cycloartanol glycosides), the sutherlandiosides, and flavonoids (quercetin and kaempferol glycosides), the sutherlandins. For the Folin-Ciocalteu phenolics test values of 93.311 to 125.330 mg GAE/g DE were obtained. The flavonoids ranged from 54.831 to 66.073 mg CE/g DE using the aluminum chloride assay. Extracts from different sites were also assayed using the 2,2-diphenyl-1-picrylhydrazyl (DPPH•) radical scavenging method and ferric reducing anti-oxidant power (FRAP) methods. This was followed by an in vitro Cell Titer-Glo viability assay of various ecotypes using the DLD-1 colon cancer cell line. All test extracts displayed anti-oxidant activity through the DPPH• radical scavenging mechanism, with IC50 values ranging from 3.171 to 7.707 µg·mL-1. However, the degree of anti-oxidant effects differed on a chemotypic basis with coastal plants from Gansbaai and Pearly Beach (Western Cape) exhibiting superior activity whereas the Victoria West inland group from the Northern Cape, consistently showed the weakest anti-oxidant activity for both the DPPH• and FRAP methods. All extracts showed cytotoxicity on DLD-1 colon cancer cells at the test concentration of 200 µg·mL-1 but Sutherlandia plants from Colesburg (Northern Cape) exhibited the highest anti-cancer activity. These findings confirm that S. frutescens specimens display variability in their bioactive capacities based on their natural location, illustrating the importance of choosing relevant ecotypes for medicinal purposes.

Characterization of immune regulatory molecules B7-H4 and PD-L1 in low and high grade endometrial tumors.

BACKGROUND: The objective of this investigation was to characterize the expression landscape of immune regulatory molecules programmed death-ligand-1 (PD-L1, B7-H1) and B7-H4 in a cohort of endometrial tumors across the spectrum of grade and histology. MATERIALS AND METHODS: With institutional review board approval, 70 endometrial tumors from patients with known clinical outcomes were identified representing a spectrum of grade and histology. Immunohistochemistry (IHC) was performed for PD-L1 and B7-H4 and scored. Microsatellite instability (MSI) status was assessed for endometrioid tumors using the institutional IHC assay for expression of the mismatch repair (MMR) genes, MLH1, MSH2, MSH6 and PMS2. RNA sequencing data from the Cancer Genome Atlas was queried for expression levels of CD274 (PD-L1 protein) and VTCN1 (B7-H4) across molecular subtypes of endometrial carcinoma and were correlated with a T cell infiltration index. RESULTS: We identified 40 low grade endometrioid tumors and a cohort of 30 high grade tumors. PD-L1 expression was observed in both high and low grade endometrial tumors (56% vs 35%, p=0.07). In the low grade tumors, PD-L1 expression was associated with MSI status (p<0.01). The high grade cohort had similar rates of PD-L1 expression compared to low grade MSI tumor (56% and 62% respectively), and both were distinct from low grade MSS tumors (22%, p<0.05). High (3+) B7-H4 positive cells were observed in both high and low grade carcinomas (33% and 31% respectively). RNA profiling data from confirmed highest CD274 expression in POLE and MSI tumors that was linearly correlated with T cell infiltration, while VTCN1 expression appeared consistent across molecular subtypes. CONCLUSIONS: While PD-L1 expression correlated with MSI and high grade tumors, B7-H4 expression was independent of grade, histology and immune cell infiltration. The development and testing of multi-agent therapeutics targeting PD-L1 and B7-H4 may be a novel strategy for endometrial tumors.