Dietary table grape protects against UV photodamage in humans: 1. clinical evaluation

Full text

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Supplemental material
Number of Supplementary Figures: 6
Number of Supplementary Tables: 4
Supplementary Discussion
Supplementary Methods
Supplementary References
List of Supplementary Figures
Supplementary Figure 1. CONSORT Diagram
Supplementary Figure 2. Protocol for MED testing and Skin Biopsy Site
Determination
Supplementary Figure 3. Mass Spectrum
Supplementary Figure 4. MSMS Spectra of the Procyanidin B1 and B2 Ions
Supplementary Figure 5. Decrease in markers of acute UV-induced damage
observed after 14 days of oral CTG administration
Supplementary Figure 6. Nanostring analysis of skin biopsy samples
List of Supplementary Tables
Supplementary Table 1. Inclusion and Exclusion Criteria for Recruitment of
Human Subjects
Supplementary Table 2. Quantitative Analysis of Select Compounds.
Supplementary Table 3. Nutritional Analysis of California Grape Powder.
Supplementary Table 4. Constituents in California Grape Powder
Preparation.
Supplementary Methods

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Supplementary Figure 1
Supplementary Figure 1. CONSORT Diagram
20 healthy human volunteers were recruited for a single-group, open-label intervention
study.

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Supplementary Figure 2
Supplementary Figure 2. Protocol for MED testing and Skin Biopsy Site
Determination
(A) Cohort 1: MED testing was performed on the inner aspect of the forearms. UV
doses given to each of the 6 sites during the MED testing were 113.9, 217.1, 342.6,
500.3, 618.6, and 848.4 J/m2. During visit 2 and visit 4, skin biopsies were taken from
the MED site on the inner forearm and the control site on the ipsilateral hip. (B) Cohort
2: MED testing was performed on the hips. UV doses given to each of the 6 sites during
the MED testing were 40, 80, 120, 160, 200, 240 J/m2. During visit 2, skin biopsies were
taken from the MED site on the hip and the control site on ipsilateral hip (at least 5 cm
away from the MED testing site). During visit 4, skin biopsies were taken from the site
that received the same dose of UV as the MED site on visit 2, as well as the control site
on ipsilateral hip (at least 5 cm away from the MED testing site). UVB, ultraviolet B;
MED, minimal erythema dose.

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Supplementary Figure 3
Supplementary Figure 3. Mass Spectrum
(A) A 3D-representation of positively charged ions from the grape product following
reverse-phase LC-MS analysis. Ions displayed are from m/z 100-1000 over 0-20 min.
(B) A close up of ions with m/z of 579.144 over a period from 10-15 min. The ions are
procyanidins B1 and B2.
A
B

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Supplementary Figure 4
.
Supplementary Figure 4. MSMS Spectra of the Procyanidin B1 and B2 Ions
(A) MSMS of the procyanidin B2 ion eluting at 11.3 min. (B, C and D) MSMS spectra of
the procyanidin B1 ions eluting at 11.7, 12.5 and 13.3 min, respectively. The upper
MSMS spectra (blue) are those observed in the grape powder and the lower ones are
those from the MS-DIAL public positive ion spectral library.
A
C
B
D

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Supplementary Figure 5
Supplementary Figure 5. Decrease in markers of acute UV-induced damage
observed after 14 days of oral CTG administration. Representative microscopic
images of apoptotic cells stained with TUNEL (A, bar=50 μm), CPDs (D, bar=100 μm)
and γ-H2AX (F, bar=100 μm) from cohort 1. Quantification of cells positive for TUNEL
(B), CPDs (E) and γ-H2AX (G). (C) mRNA fold changes for proinflammatory cytokines
from MED site biopsies before and after oral CTG treatment detected by real-time

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polymerase chain reaction. *p < 0.05, mean + SEM. Insets on right correspond to their
color.
Supplementary Figure 6

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Supplementary Figure 6. Nanostring analysis of skin biopsy samples
(A) Gene expression pattern based on k-mean clustering along with their enriched
KEGG terms. (B) Differentially expressed genes between control and UV-exposed skin.
(C) IPA. Differential expression of genes listed are represented as ratios between 4
different biopsy conditions (pre-treatment MED site, pre-treatment control site, post-
treatment MED site and post-treatment control site). (D) Apoptosis panel. Pre-MED,
biopsy taken from MED site before CTG administration; Pre-control, biopsy taken from
control site before CTG administration; Post-MED, biopsy taken from MED site after

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CTG administration; Post-control, biopsy taken from control site after CTG
administration;
Supplementary Table 1. Inclusion and Exclusion Criteria for Recruitment of
Human Subjects

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Inclusion Criteria Exclusion Criteria
- age 19 and older
- able to understand requirements of
the study and risks involved
- able to provide signed consent
- Fitzpatrick sun reactive skin type I-III
- Fitzpatrick sun reactive skin type IV-VI
- a recent history of vitiligo, melasma, and
other disorders of pigmentation with the
exception of postinflammatory
hyperpigmentation
- a known history of photosensitivity
disorders, melanoma or non-melanoma skin
cancers
- tanning bed use
- use of a photosensitizing medication
- women who were lactating, pregnant, or
planning to become pregnant
- recent or anticipated exposure of the UV-
irradiated or control skin sites to the sun
Supplementary Table 2. Quantitative Analysis of Select Compounds

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Compounds Quantity
Catechins
Catechin 33.3 mg/kg +/- 0.58
Epicatechin 14.37 mg/kg +/- 0.28
Anthocyanins
Peonidin 77.4 mg/kg +/- 2.5
Cyanidin 21.6 mg/kg + /- 1
Flavonols
Isorhamnetin 0.37 mg/kg +/- 0.03
Stilbenes
Resveratrol 3.44 mg/kg +/- 0.42
Total Polyphenols (in
gallic acid equivalents)
380 mg/100g
Note: This analysis does not represent the complete phytochemical profile of grapes.
Supplementary Table 3. Nutritional Analysis of California Grape Powder
Nutrient Amount Units

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(per 100 g powder)
Calories 368 kcals
Total Fat, acid hydrolysis 0.31 g
Total Carbohydrate 87.5 g
Protein (N x 6.25) 3.81 g
Beta carotene 0.138 mg
Vitamin A from carotene 230 IU
Vitamin C < 1 mg
Calcium 55.1 mg
Iron 1.88 mg
Sodium < 12.4 mg
Potassium 1170 mg
Thiamin HCl 0.12 mg
Folic Acid 40.6 mcg
Phosphorus 119 mg
Magnesium 39.5 mg
Zinc 0.283 mg
Copper 0.432 mg
Manganese 0.323 mg
Moisture 5.51 g
Ash 2.88 g
Note: nutritional analyses were performed using standard methods of analysis.
References available on request.
Supplementary Table 4. Constituents in California Grape Powder Preparation
Class Compound RT m/z Area Adduct

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(min)
Amino
acid Alanine 4.64 90.056 123,920 [M+H]+
Arginine 4.27 175.11
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4,252,14
1[M+H]+
Glutamine 4.49 147.07
4538,808 [M+H]+
Isoleucine 8.39 132.10
1769,377 [M+H]+
Phenylalanine 9.74 166.08
5839,711 [M+H]+
Proline 5.17 116.06
9
7,445,58
8[M+H]+
Threonine 4.55 120.06
172,924 [M+H]+
Tryptophan 11.26 205.09
6817,183 [M+H]+
Tyrosine 8.33 182.07
9232,016 [M+H]+
Phenolic
sCatechin 13.13 291.08
546,289 [M+H]+
p-Coumaric acid 12.73 165.05
137,422 [M+H]+
Cyanidin-3-
glucoside 15.92 449.10
3263,456 [M]+
Dihydroquercetin 14.27 305.06
150,675 [M+H]+
Diosmetin 12.52 301.07
0206,495 [M+H]+
Epicatechin 12.19 291.08
592,191 [M+H]+
Epicatechin
gallate 14.81 443.09
5110,912 [M+H]+
Ferulic Acid 11.82 195.06
544,547 [M+H]+
trans-p-
Hydroxycinnamic
acid
12.22 X` 140,843 [M+H]+
Icariside B5 13.26 389.21
434,161 [M+H]+
Isorhamnetin 3-
galactoside 16.10 479.11
7150,319 [M+H]+
Luteolin 15.92 287.05
5168,131 [M+H]+
Luteolin b- 15.35 465.10 619,889 [M+H]+

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glucosylgalactosid
e3
Peonidin-3-O-b-D-
glucoside 12.52 463.12
4
3,824,04
3[M+]
Phloracetophenon
e 4'-O-glucoside 13.71 331.09
962,924 [M+H]+
Phloretin 16.42 275.09
134,579 [M+H]+
Procyanidin B1 11.73 579.14
884,121 [M+H]+
Procyanidin B1 12.34 579.14
4150,371 [M+H]+
Procyanidin B1 13.51 579.15
239,435 [M+H]+
Procyanidin B2 11.29 579.14
4175,969 [M+H]+
Quercetin 3-O-
glucuronide 15.35 479.08
0408,559 [M+H]+
Resveratrol 16.07 229.08
664,348 [M+H]+
Rutin 14.68 611.16
562,774 [M+H]+
Tricin 5-glucoside 12.58 493.13
1
2,731,00
6[M+H]+
Indoles 3-Formylindole 11.26 146.06
245,101 [M+H]+
Harmaline 13.16 215.12
3308,361 [M+H]+
Indole 13.96 118.06
438,119 [M+H]+
Indole-3-carbinol 13.99 130.06
376,338 [M+H-
H2O]+
Indolelactic acid 13.99 206.07
9
1,132,17
0[M+H]+
Indoline 9.74 120.08
0296,605 [M+H]+
Other 4-Aminobutanoate 4.49 104.07
059,909 [M+H]+
5'-S-
Methylthioadenosi
ne
10.83 298.09
5
3,162,52
6[M+H]+
Choline 4.78 104.10
5357,870 [M]+
Guanidinoacetate 11.26 118.06
233,874 [M+H]+

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Harmaline 13.16 215.12
3308,361 [M+H]+
N-Fructosyl
isoleucine 8.63 294.15
4
2,889,12
1[M+H]+
N-Fructosyl
phenylalanine 9.83 328.13
7
1,948,77
0[M+H]+
D-Mannosamine 4.18 180.08
5556,332 [M+H]+
Theobromine 10.55 181.06
951,726 [M+H]+
Trigonelline 5.40 138.05
2
1,084,59
8[M+H]+
Supplementary Discussion
Gene expression profile of 800 genes from the Nanostring panel were clustered by k-
mean clustering. The clustered genes were annotated based on significantly enriched
KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway terms (Supplementary
Figure 6, p< 0.05). Five hundred and eighty-four genes were differentially expressed
(219 up-regulated and 239 down-regulated genes) between control samples and MED
samples (Supplementary Figure 6). p53 signaling pathway, apoptosis, mitogen-
activated protein kinase (MAPK) signaling pathway, pathways in cancer, cell cycle,
phosphatidylinositol-3 kinase (PI3K)-Akt signaling pathway, and microRNAs in cancer
were some of the most activated KEGG terms (clusters C, D and E) from the UV-treated
skin site before treatment with CTGs (labeled as Pre-MED). Ingenuity Pathways
Analysis (IPA) indicated that the most activated pathways were involved in diseases or
functions belonging to the following categories: cell proliferation of tumor cell lines,
differentiation of connective tissue cells, mature lymphocytic neoplasm, cell death of
hematopoietic cells, mature B-cell neoplasm, inflammation of organs, repair of DNA, cell
death and apoptosis of hepatic progenitor cells (p-value < 2.89E-39) (Supplementary

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Figure 6). Downregulation of MMP3, implicated in epithelial-mesenchymal transition1,
was seen in the Nanostring apoptosis panel.
Supplementary Methods
Human tissue processing and histology. Paraffin-embedded tissue samples were cut
into 5 μm sections and deparaffinized prior to undergoing staining with hematoxylin and
eosin (Fisher Scientific, Kalamazoo, MI) or analysis with immunohistochemistry or
immunofluorescence. Sections subsequently underwent an antigen retrieval protocol by
heating at 95°C for 30 minutes in a citrate-based (pH=6.0) antigen unmasking solution
(Vector Laboratories, Burlingame, CA). For immunohistochemical staining, which was
used to detect CPDs, slides were incubated in 70 mM NaOH in 70% ethanol for 2
minutes, then neutralized with 100 mM Tris-HCl (pH=7.5) for 1 minute in order to
denature the nuclear DNA. After being washed with phosphate buffered saline (PBS),
sections underwent peroxidase blocking with 0.3% hydrogen peroxide for 10 minutes.
After being washed with PBS again, sections underwent protein blocking with 0.5%
bovine serum albumin (BSA) and 0.5% Casein in PBS (Abcam, Cambridge, MA).
Sections were then incubated in 4°C overnight in anti-Thymine Dimer-HRP mAb
(Kamiya Biomedical Company, Tukwila, WA) in PBS, washed and incubated in a
biotinylated secondary antibody. Sections were developed by incubating 3–5 min with

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diaminobenzidene (DAB) peroxidase substrate solution and counterstained with
hematoxylin prior to being mounted in Permount (Fisher Scientific). For
immunofluorescent staining, which was used to detect γ-H2AX, sections underwent
protein blocking with 2% BSA following antigen retrieval and then incubated in 4°C
overnight in phospho-histone H2A.X (Ser139) (20E3) rabbit mAb (Cell Signaling
Technology, Danvers, MA). Sections were then washed in PBS and incubated in Alexa
Fluor 594-conjugated secondary antibody for 1 hour at room temperature. Slides were
mounted in VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories).
Images were captured using an Olympus EX51 microscope. For each experimental
condition, images were taken for 3 > high-power fields (40x), and each image was
analyzed using Adobe Photoshop (Adobe, San Jose, CA).
TUNEL assay. An In Situ Cell Death Detection Kit (Roche Applied Science, Penzberg,
Germany) was used according to the manufacturer’s protocol. Slides were mounted in
VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories). Images
were captured using an Olympus EX51 microscope. For each experimental condition,
images were taken for 3 >high-power fields (40x), and each image was analyzed using
Adobe Photoshop (Adobe, San Jose, CA).
Mass spectrometry.
Extraction from the grape supplement. The grape supplement (10 mg) was
extracted using 80% ice-cold aqueous methanol (1 ml) and gently vortexed for 30 min.
The extract was centrifuged at 3000xg for 10 min and the supernatant separated from
the pellet and placed in a 1.5 mL micro-centrifuge tube. A portion of the extract (100 µL)
was then dried down by vacuum centrifugation and re-suspended in 0.1% formic acid
(200 µL) for mass spectrometry evaluations.
Global Metabolomics. An aliquot (5 µL) of the resuspended extract was loaded
onto a Nano cHiPLC (6 mm x 200 µm) ChromXP C18-CL 3 µm 120Å reverse-phase
trap cartridge (Eksigent, Concord, Ontario, Canada) at 2 µL/min using an Eksigent
autosampler. After washing the cartridge for 5 min with 0.1% formic acid in double
distilled H2O, the bound metabolites were flushed onto a Nano cHiPLC column (15 cm x
200 µm ID) ChromXP C18-CL 3 µm 120Å (Eksigent, Toronto, Canada) with a 20 min
linear (2-98%) acetonitrile gradient in 0.1% formic acid at 1000 nl/min using an Eksigent
400 NanoLC System (Dublin, CA). The column was washed with 98% acetonitrile-0.1%
formic acid for 5 min and then re-equilibrated with 2% acetonitrile-0.1% formic acid for 5
min. The SCIEX 5600 Triple-Tof mass spectrometer (SCIEX, Concord, Ontario, Canada)
was used to analyze the column eluate. The nanoIonSpray voltage was 2300 V and the
declustering potential was 80 V. Ionspray and curtain gases were set at 10 psi and 25
psi, respectively. The interface heater temperature was 120oC. Eluted metabolites were
subjected to a 1.25 s repeating duty cycle consisting of a 250 ms time-of-flight (TOF)
survey scan from m/z 50-1000 to determine the top twenty most intense ions for MSMS
analysis. High-resolution 50 ms product ion TOF scans of the selected parent ions were
collected over the range from m/z 50-1000 using a rolling collision energy (15-35 V).
The resulting .wiff and .wiffscan data were converted into .mzxml file format using
MSConvert (http://proteowizard.sourceforge.net/download.html) for analysis with
MZmine (version 2.53) 2,3. MZmine enabled generation of a 3D-display of all the
positively charged ions in the grape product, as well as the ions corresponding to

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procyanidin B1 and B2. The .wiff and wiffscan files were also converted into the .abf file
format using the ABF converter (https://www.reifycs.com/AbfConverter/) for analysis
using MS-DIAL (version 4.20).
This program putatively identified compounds that were present in the grape
product and were confirmed by MSMS (Figure S3). These included the procyanidins B1
and B2 (Figure S4). Furthermore, select compounds were quantified with further
analysis (Supplemental Table 2).
Melanin index measurement. The melanin index was measured with a Skin Colorimeter
in a photoprotected site from two volunteers before and after treatment. A DSM II
ColorMeter (Cortex Technology, Hadsund, Denmark) was used to measure the melanin
index. For 2 healthy Caucasian volunteers (Fitzpatrick skin type II and III), the melanin
index was measured on the same site on the inner aspect of the upper arm before and
after CTG treatment.
Nanostring assay. Using a standardized protocol from the UAB Nanostring Laboratory,
RNA was isolated from snap-frozen skin biopsy samples using the PureLink RNA Mini
Kit (Thermo Fisher, Waltham, MA) and quantified with a DeNovix DS-11-
Spectrophotometer (Wilmington, DE). RNA was subsequently concentrated with RNA
Clean & Concentrator-5 (Zymo Research, Irvine, CA) if the RNA concentration was
below 20 ng/µL. 150 ng of RNA was hybridized with codesets and processed on the
Nanostring Prep station. The run was performed on the Nanostring Analysis station.
The Human PanCancer pathways panel was chosen in addition to 31 addition genes
listed.
1. PGE2
2. COX2
3. ODC
4. AURKB
5. CSPG4
6. MITF
7. SOX10
8. ST3GAL5
9. CSAG2
10. GAGE1
11. MAGEA2
12. MAGEA3
13. MAGEC1
14. MAGEC2
15. PRAME
16. IGF2BP3
17. PTPRZ1
18. KIF20A
19. CD2
20. KLRK1
21. HLA-E
22. IFNAR1

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23. ITK
24. CD4
25. LCK
26. IFI27
27. CCR4
28. CTSS
29. CD68
30. IRF2
31. TERT
Nanostring analysis. There were 800 genes with their expression values for nine
replicates of treatment conditions. To investigate the list of differentially expressed
genes (DEGs), DESeq2 with parameters (p-value< 0.05 and log2 fold change |1|) were
employed. The pathway analysis was done by KEGG, ingenuity pathway analysis (IPA)
and reactome with significant parameters.
Statistics. Paired one-tailed t-test analyses were performed using Graphpad Prism
Version 7.0 (GraphPad Software, San Diego, CA). P-values <0.05 were considered
statistically significant. Spearman's rank correlation coefficient was calculated to
examine whether the MED elevation differed significantly based on age. Kruskal-Wallis
test was used to examine whether the MED elevation differed significantly based on
season (spring, summer, autumn and winter).
Power analysis was conducted prior to initiating the study. It indicated that 10
subjects would provide 80% power for statistically significant results for the primary
endpoint (i.e. MED). Furthermore, a post-hoc power analysis was performed again
using the MED data. Using a one-sided t-test at 0.1 significance level and 80% power,
we were able to detect a % change of 74.8% in the MED.
Study approval. Patient consent and protocols observed the Helsinki Guidelines and
were approved by the Institutional Review Board at the University of Alabama at
Birmingham. A written informed consent form was obtained from participants prior to the
study.
Treatment with table grapes. Freeze-dried grape powder was provided by the California
Table Grape Commission. The powder was provided in sealed pouches, each
containing 25 g of freeze-dried grape powder, and was stored at -20° C prior to
consumption. A single serving of fresh grapes (126 g or 0.75 cup) was equivalent to 23
g of the freeze-dried grape powder. Volunteers were instructed to reconstitute the grape
powder by adding approximately 180 mL (3.3 fl. oz=0.41 US cup) of water to the powder
and stirring for a minimum of 30 seconds prior to consumption at home. Each volunteer
took 25 g of grape powder 3 times per day (75 g daily) for 14 days. Patients were given
a drug diary to monitor for compliance.
MED testing and sample collection. Gradually increasing doses of UV were
administered with a broadband UVB light source, Panosol II UVB-204 (SERIAL: 26429,
Lamps: FSX24T12/UVB/HO, 2 AMPS). The output was measured 5 cm away from the
machine with an X-96 Irradiance Meter (Daavlin, Bryan, OH) immediately prior to the

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study. At the pretreatment visit (visit 1) and the post-treatment visit, immediately after
the last dose of CTG (visit 3), six sites (each site, 2x2 inches) were marked and
exposed to incrementally increasing doses of UV. Twenty-four hours later, the sites were
examined for erythema (pre-CTG treatment, visit 2; post-CTG treatment, visit 4).
Different skin sites underwent MED testing for the pre-CTG treatment and post-CTG
treatment exposures. For example, if a subject underwent pre-treatment MED testing on
the right side, he/she underwent post-treatment MED testing on the left side. Six-mm-
punch biopsies were taken from the MED site and the non-sun-exposed control site on
ipsilateral hip. Biopsy samples were bisected. Half was fixed in formalin to be
embedded in paraffin, and the other half was snap frozen in liquid nitrogen.
Supplementary References
1. Radisky DC, Levy DD, Littlepage LE, et al. Rac1b and reactive oxygen species
mediate MMP-3-induced EMT and genomic instability. Nature. 2005;436(7047):123-
127. doi:10.1038/nature03688
2. Pluskal T, Castillo S, Villar-Briones A, Oresic M. MZmine 2: modular framework for
processing, visualizing, and analyzing mass spectrometry-based molecular profile
data. BMC Bioinformatics. 2010;11:395. doi:10.1186/1471-2105-11-395
3. Tsugawa H, Cajka T, Kind T, et al. MS-DIAL: data-independent MS/MS
deconvolution for comprehensive metabolome analysis. Nat Methods.
2015;12(6):523-526. doi:10.1038/nmeth.3393