Anthocyanin supplementation in humans improves LDL- and HDL-cholesterol concentrations and enhances cellular cholesterol efflux to serum. These benefits may be due to the inhibition of CETP.

Anthocyanins, present in fruits and vegetables as natural colorants, have been well characterized to possess bioactive properties. Anthocyanin components extracted from black rice (Oryza sativa L. indica) separated by gel filtration and identified using LC-MS were cyanidin 3-glucoside and peonidin 3-glucoside. A standardized extract of black rice pigmented fraction (BRE) containing known proportions of cyanidin 3-glucoside and peonidin 3-glucoside exhibited marked antioxidant activities and free radical scavenging capacities in a battery of in vitro model systems. Significant (p < 0.05) prevention of supercoiled DNA strand scission induced by reactive oxygen species (specifically, peroxyl radical and hydroxyl radicals) and suppression of the oxidative modification of human low-density lipoprotein was obtained with BRE. In addition, BRE reduced (p < 0.05) the formation of nitric oxide by suppressing inducible nitric oxide synthase expression in murine macrophage RAW264.7 cells, without introducing cell toxicity. The results of this study show that black rice contains anthocyanin pigments with notable antioxidant and anti-inflammatory properties for potential use in nutraceutical or functional food formulations. Keywords: Black rice; anthocyanin; antioxidant; nitric oxide

BACKGROUND Anthocyanins have been shown to improve endothelial function in animal models. However, whether these compounds have similar beneficial effects in humans is largely unknown. METHODS In a short-term crossover study, 12 hypercholesterolemic individuals were given oral anthocyanins (320 mg) isolated from berries or placebo. Brachial artery flow-mediated dilation (FMD) was assessed before and after the intervention. In a long-term intervention trial (12 weeks), 150 hypercholesterolemic individuals were given anthocyanins (320 mg/day, n = 75) or placebo (n = 75), after which we measured FMD, plasma cGMP, and other serum biomarkers. Another short-term intervention was conducted in the presence of NO-cGMP inhibitors in 6 people and in a rat aortic ring model (n = 8). RESULTS Significant increases of FMD from 8.3% (0.6%) at baseline to 11.0% (0.8%) at 1 h and 10.1% (0.9%) at 2 h were observed after short-term anthocyanin consumption, concomitantly with increases of plasma anthocyanin concentrations (P &lt; 0.05). In the study participants who received long-term anthocyanin intervention, compared with the control group, we observed significant increases in the FMD (28.4% vs 2.2%), cGMP (12.6% vs −1.2%), and HDL-cholesterol concentrations, but decreases in the serum soluble vascular adhesion molecule-1 and LDL cholesterol concentrations (P &lt; 0.05). The changes in the cGMP and HDL cholesterol concentrations positively correlated with FMD in the anthocyanin group (P &lt; 0.05). In the presence of NO-cGMP inhibitors, the effects of anthocyanin on endothelial function were abolished in human participants and in a rat aortic ring model. CONCLUSIONS Anthocyanin supplementation improves endothelium-dependent vasodilation in hypercholesterolemic individuals. This effect involves activation of the NO-cGMP signaling pathway, improvements in the serum lipid profile, and decreased inflammation.

1The COVID-19 pandemic is spreading globally with high disparity in the 2 susceptibility of the disease severity. Identification of the key underlying factors for 3 this disparity is highly warranted. Here we describe constructing a proteomic risk 4 score based on 20 blood proteomic biomarkers which predict the progression to 5 severe COVID-19. We demonstrate that in our own cohort of 990 individuals without 6 infection, this proteomic risk score is positively associated with proinflammatory 7 cytokines mainly among older, but not younger, individuals. We further discovered 8 that a core set of gut microbiota could accurately predict the above proteomic 9 biomarkers among 301 individuals using a machine learning model, and that these gut 10 microbiota features are highly correlated with proinflammatory cytokines in another 11 set of 366 individuals. Fecal metabolomic analysis suggested potential amino 12 acid-related pathways linking gut microbiota to inflammation. This study suggests 13 that gut microbiota may underlie the predisposition of normal individuals to severe : medRxiv preprint ( Figure S1). Gut microbiota data were collected and measured during a follow-up 107 visit of the cohort participants, with a cross-sectional subset of the individuals (n=132) 108 having blood proteomic data at the same time point as the stool collection and another 109 independent prospective subset of the individuals (n=169) having proteomic data at a 110 next follow-up visit ~3 years later than the stool collection. 111 112 Among the cross-sectional subset, using a machine learning-based method: 113 LightGBM and a very conservative and strict tenfold cross-validation strategy, we 114 identified 20 top predictive operational taxonomic units (OTUs), and this subset of 115 core OTUs explained an average 21.5% of the PRS variation (mean out-of-sample 116 R 2 =0.215 across ten cross-validations). The list of these core OTUs along with their 117 taxonomic classification is provided inTable S3. These OTUs were mainly assigned 118 to Bacteroides genus, Streptococcus genus, Lactobacillus genus, Ruminococcaceae 119 family, Lachnospiraceae family and Clostridiales order.120 121To test the verification of the core OTUs, the Pearson correlation analysis showed the 122 coefficient between the core OTUs-predicted PRS and actual PRS reached 0.59 123 (p<0.001), substantially outperforming the predictive capacity of other demographic 124 characteristics and laboratory tests including age, BMI, sex, blood pressure and blood 125 lipids (Pearson's r =0.154, p=0.087) ( Figure 3A). Additionally, we used co-inertia 126 analysis (CIA) to further test co-variance between the 20 identified core OTUs and 20 127 predictive proteomic biomarkers of severe COVID-19, outputting a RV coefficient 128 (ranged from 0 to 1) to quantify the closeness. The results indicated a close 129 association of these OTUs with the proteomic biomarkers (RV=0.12, p<0.05) (Figure 130 S3A). When replicating this analysis stratified by age, significant association was 131 observed...