Foods 2020 , 9 , x FOR PEER REVIEW
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Foods 2020 , 9 , 1147 chromatography tandem mass spectrometry (LC-MS/MS) (Figure S2). Here, we see that positively predicted peptides are distributed across five parent rice proteins. Notably, peptide pep_7XU902 is a subsequence of peptide pep_55AE5D. Again, using differentiated THP-1 macrophages, the anti- inflammatory effect of the individual peptides against LPS induced TNF- α was determined. Following a primary screen, we found that seven peptides tested induced a significant reduction in TNF- α secretion compared to untreated cells while one peptide did not (Figure S3), with the effect of five peptides emerging as highly significant ( p < 0.001; see Figure 3). Similar efficacy to the rice NPN was observed for two of the constituent peptides, where pep_CICEMV and pep_H1REMR respectively produced a 53% and 52% reduction in TNF- α secretion (Figure 3). Further, we found that in all cases the constituent rice NPN peptides selected for testing exhibited more than a 20% reduction in TNF- α secretion (Figure 3). 9of 19 of our selected in vitro predicted peptides in rice NPN was verified by liquid chromatography tandem mass spectrometry (LC-MS / MS) (Figure S2). Here, we see that positively predicted peptides are distributed across five parent rice proteins. Notably, peptide pep_7XU902 is a subsequence of peptide pep_55AE5D. Again, using di ff erentiated THP-1 macrophages, the anti-inflammatory e ff ect of the individual peptides against LPS induced TNF- α was determined. Following a primary screen, we found that seven peptides tested induced a significant reduction in TNF- α secretion compared to untreated cells while one peptide did not (Figure S3), with the e ff ect of five peptides emerging as highly significant ( p < 0.001; see Figure 3). Similar e ffi cacy to the rice NPN was observed for two of the constituent peptides, where pep_CICEMV and pep_H1REMR respectively produced a 53% and 52% reduction in TNF- α secretion (Figure 3). Further, we found that in all cases the constituent rice NPN peptides selected for testing exhibited more than a 20% reduction in TNF- α secretion (Figure 3). Table 1. Sequence and parent protein information for seven positively predicted peptides in rice NPN.
Molecular Weight
Uniprot ID P14323
ID
Sequence
Length
Charge
Table1. Sequence and parent protein information for seven positively predicted peptides in rice NPN.
ID pep_6QEKFQ FYNEGDAPVVAL Sequence pep_ZBRXEN TVFDGVLRPGQL pep_6QEKFQ FYNEGDAPVVAL pep_7XU902 pep_7XU902 pep_CICEMV NGVLRPGQL pep_35E7UW IDGYDTPVEGR pep_CICEMV NGVLRPGQL pep_55AE5D pep_ZBRXEN TVFDGVLRPGQL
12 12 16 14 11
1301.49
0
Length Molecular Weight 1294.37
Charge Uniprot ID Q0E261
− 2 − 1
pep_H1REMR IYGPDTGVDYKDNQMR
1871.97 1642.75 1221.27
P14323 Q0E261 Q0DEV5 P29835 P29835 Q0DEV5 Q0DEV5 P14614 P29835 P14614 P29835 Q0DEV5
12 12
1301.49 1294.37 1871.97 1642.75 1221.27
0
GYYGEQQQQPGMTR
0
− 2 − 1 0 − 2 1 − 2
pep_H1REMR IYGPDTGVDYKDNQMR 16 pep_35E7UW IDGYDTPVEGR GYYGEQQQQPGMTR 14
− 2
9
953.1
1
11
SEEGYYGEQQQQPGMTR
17
1988.05
− 2
9
953.1
pep_55AE5D SEEGYYGEQQQQPGMTR 17
1988.05
Figure 3. Effects of rice NPN constituent peptides on TNF- α secretion from differentiated THP-1 macrophages. Effect of treatment on the secretion of TNF- α in THP-1 differentiated macrophages after treatment with LPS. THP-1 macrophages were treated with predicted peptides (0.5 μ g/mL), or media alone (Untreated) for 24 h before incubation with 100 ng/mL of LPS for 24 h. The secretion of TNF- α was quantified by ELISA. Data presented are the mean ± SEM of at least 3 independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001). 3.3. Supplementation with Rice NPN in an Elderly Cohort Had Beneficial Effects on Physical Activity and Correlated with Markers of Age Associated Inflammation An elderly cohort in otherwise good health was recruited and ingested rice NPN daily over a 12-week period in a double blinded, placebo-controlled trial (Table S1). The primary endpoint for this study was gut discomfort, as measured by a subjective questionnaire, and while no significant changes were observed on that metric following treatment (Table 2), beneficial e ff ects on serum concentrations of TNF- α and HDL were noted, as well as a beneficial e ff ect on glucose uptake. These were associated with increased performance in functional tests such as a chair stand test and short physical performance battery. No serious adverse events reported (Table S1), as such, rice NPN was considered safe to ingest. An elderly cohort in otherwise good health was recruited and ingested rice NPN daily over a 12-week period in a double blinded, placebo-controlled trial (Table S1). The primary endpoint for this study was gut discomfort, as measured by a subjective questionnaire, and while no significant changes were observed on that metric following treatment (Table 2), beneficial effects on serum Figure 3. E ff ects of rice NPN constituent peptides on TNF- secretion from di ff erentiated THP-1 macrophages. E ff ect of treatment on the secretion of TNF- α inTHP-1di ff erentiated macrophages after treatment with LPS. THP-1 macrophages were treated with predicted peptides (0.5 µ g / mL), or media alone (Untreated) for 24 h before incubation with 100 ng / mL of LPS for 24 h. The secretion of TNF- α was quantified by ELISA. Data presented are the mean ± SEM of at least 3 independent experiments (* p < 0.05, ** p < 0.01, *** p < 0.001). 3.3. Supplementation with Rice NPN in an Elderly Cohort Had Beneficial E ff ects on Physical Activity and Correlated with Markers of Age Associated Inflammation
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