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Oral ingestion of collagen hydrolysate leads to the transportation of highly concentrated Gly-Pro-Hyp and its hydrolyzed form of Pro-Hyp into the bloodstream and skin. Misato Yazaki, Yukihiko Ito, Masayoshi Yamada, Spyros Goulas, Sachiyuki Teramoto, Masa-aki Nakaya, Shigeo Ohno, and Kohji Yamaguchi J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05679 • Publication Date (Web): 28 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017
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Journal of Agricultural and Food Chemistry
Oral ingestion of collagen hydrolysate leads to the transportation of highly concentrated Gly-Pro-Hyp and its hydrolyzed form of Pro-Hyp into the bloodstream and skin.
Misato Yazaki1,
2 *
, Yukihiko Ito1, Masayoshi Yamada1, Spyros Goulas2,
Sachiyuki Teramoto1, Masa-aki Nakaya2, Shigeo Ohno2 , Kohji Yamaguchi1
1
Research Institute, FANCL Corporation, 12-13 Kamishinano, Totsukaku,
Yokohama, Kanagawa, 244-0806, Japan, 2Department of Molecular Biology, Graduate School of Medical Science, Yokohama City University, 3-9 Fuku-ura, Kanazawaku, Yokohama 236-0004, Japan
Corresponding author *
Misato
Yazaki
(Tel:
+81-45-820-3527,
Fax:
+81-45-820-3526,
email:
[email protected])
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Abstract
2
Collagen hydrolysate is a well-known dietary supplement for the treatment of
3
skin aging, however its mode of action remains unknown. Previous studies have
4
shown that the oral ingestion of collagen hydrolysate leads to elevated levels of
5
collagen-derived peptides in the blood but whether these peptides reach the skin
6
remains
7
collagen-derived peptides after ingestion of high tri-peptide containing collagen
8
hydrolysate in humans. We identified 17 types of collagen-derived peptides
9
transiently, with a particularly enrichment in Gly-Pro-Hyp. This was also
10
observed using an in vivo mouse model in the plasma and skin, albeit with a
11
higher enrichment of Pro-Hyp in the skin. Interestingly, this Pro-Hyp enrichment
12
in the skin was derived from Gly-Pro-Hyp hydrolysis, as the administration of
13
pure Gly-Pro-Hyp peptide led to similar results. Therefore, we propose that
14
functional peptides can be transferred to the skin by dietary supplements of
15
collagen.
unclear.
Here,
we
analyzed
the
plasma
concentration
of
16 17
Kerwords
18
Collagen, Gly-Pro-Hyp, Pro-Hyp, peptide, kinetics, dietary supplement 2 ACS Paragon Plus Environment
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Introduction
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Collagen is the main structural protein of connective tissues in animals.
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Comprising of approximately 30% of all proteins in the body, it is present in
22
fibrous tissues, such as tendons and ligaments, as well as in the cornea,
23
cartilage, bones, skin and blood vessels1. Collagen has a unique triple helix
24
structure with a repeated amino acid sequence of (Gly-X-Y)n, in which X and Y
25
are typically Pro and Hyp. It can be found in various food sources added as a
26
denatured form gelatin or alternatively, manufactured as dietary supplements in
27
an enzymatically hydrolyzed form collagen hydrolysate. Due to the high solubility
28
of collagen hydrolysate, it can be used in drinks and a jelly-like form making it
29
ideal as a food supplement and for cosmeceutical skincare.
30
Over recent years, various beneficial effects have been reported on the
31
consumption of collagen hydrolysates that includes improvements in joint pains2,
32
wound healing
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elasticity and wrinkles of facial skin7 and epidermal barrier function8. Although
34
the mechanism of most of these effects remain unknown, several studies have
35
observed a transient increase in collagen-derived peptides, especially of
36
Pro-Hyp9-11, in the blood
3, 4
, blood pressure5, glucose tolerance6; as well as the moisture,
12-14
and skin
15
after ingestion of collagen hydrolysate. 3
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In dermal fibroblasts, Pro-Hyp has been shown to stimulate cell proliferation,
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increase synthesis of hyaluronic acid
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skin18. Together, this suggests that Pro-Hyp is likely to be a key collagen-derived
40
peptide. However, whether other collagen-derived peptides are beneficial and
41
can be incorporated into the skin remains unclear.
16 17
and accelerate cell migration in the
42
Recent advancements in methods for collagen hydrolysate preparation led to
43
the production of high tri-peptide containing collagen hydrolysate (HTC-col),
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which has high concentrations of tri-peptides comprising of the Gly-X-Y
45
sequence. This has received attention as a potential alternative to traditional
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food supplements of collagen due to reports of HTC-col having inhibitory effects
47
on
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photo-aging properties
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suggests that collagen-derived tri-peptides may also have beneficial effects on
50
the body. Indeed, Gly-Pro-Hyp has been reported to have activity on the central
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nervous system after intraventricular administration 22 and also inhibits dipeptidyl
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peptidase-4 in vitro, which has been implicated in diabetes 23.
atherosclerosis
development in hypercholesterolemic 20
rabbits19,
anti
21
. This
and improvements on the dryness of the skin
53
Previous studies have shown that after ingestion of collagen hydrolysate,
54
Pro-Hyp is the most enriched peptide in the blood 11 and skin 15. In this study we 4 ACS Paragon Plus Environment
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tested using HTC-col, which contains high levels of tri-peptides, whether the
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composition of collagen-derived peptides incorporated into the blood and skin
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are altered.
58 59
Materials and Methods
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Reagents. Two kinds of collagen hydrolysates, HACP-01 and CPAH-5 (provided
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by Jellice, Miyagi, Japan), both made from porcine skin collagen by enzymatic
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hydrolysis, were used. HACP-01 contained mainly tri-peptides (Gly-X-Y) and
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had an average molecular weight 1500 – 1800 Da. This is referred to as High
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tri-peptide containing collagen hydrolysate (HTC-col). Whereas CPAH-5 had an
65
average molecular weight 4500 – 5500 Da and is referred to as Low tri-peptide
66
containing collagen hydrolysate (LTC-col). Synthetic peptides were purchased
67
from Bachem (Bubendort, Switzerland).
68 69
Gel filtration chromatography. Molecular weight of collagen hydrolysate was
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evaluated by gel permeation chromatography HPLC using Gulliver system
71
(JASCO, Tokyo, Japan) equipped with UV-detector operation at 220 nm and the
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column YMC-Pack Diol-60 (500 X 8.0 mmI.D., YMC, Kyoto, Japan). Mobile 5 ACS Paragon Plus Environment
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phase was 80% of 0.1 M Sodium phosphate buffer at pH=7.4, and 20%
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acetonitrile (Wako Chemicals, Osaka, Japan) and the flow rate was at 1.0
75
mL/min.
76 77
Human study. Four healthy men and eight healthy women were recruited to
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participate in this study. The average age of subjects was 31.5 ± 6.5. Exclusion
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criteria included the following: allergies of gelatin foods; liver and kidney and
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gastrointestinal diseases; physical therapy or use of medication; pregnancy or
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during lactation; difficultly to take blood samples; blood donation within a month.
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This study was carried out with sufficient respect for the spirit of the Declaration
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of Helsinki and was approved by institutional review boards of FANCL Co. The
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study was undertaken using a single-blinded and crossover study with no less
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than 7 days of washout period. After overnight fasting for 12h, subjects orally
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ingested collagen hydrolysate HTC-col or LTC-col of dose 300 mg per kg of body
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weight with 20 w/v% water. 12 mL of venous blood was collected in EDTA tubes
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just before and after 15, 30, 60, 120, 300 minutes from oral ingestion, The blood
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plasma was immediately separated by centrifugation and stored at −80 °C until
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analysis. 6 ACS Paragon Plus Environment
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Mouse studies and ethical considerations. All mouse studies were conducted
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under specific pathogen-free conditions. Mice were purchased from SLC
94
(Shizouka, Japan), and acclimatized for a week before experiments and had free
95
access to laboratory standard chow and water. All mice were divided to five per
96
cage randomly. The experiments were performed at around 24 ± 3 °C
97
temperature, with 55 ± 20% humidity, and with 12 h light/dark cycle. All protocols
98
were approved by the animal ethics committee of the FANCL Co.
99 100
Mouse study 1: Five-week old male BALB/c mice were randomly assigned into
101
two groups. Groups were treated with either a low dose (900 mg/kg) or higher
102
dose (1800 mg/kg) of HTC-col. Both groups were fasted for 16h before the oral
103
ingestion of collagen hydrolysate. After anesthetizing mice, blood was taken
104
from the heart, before and after 15, 30, 45, 60, 120, and 360 minutes from oral
105
ingestion. We used five mice for each time point and dose. After blood collection,
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all mice were perfused with PBS to remove all blood and the abdominal and
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dorsal skin was collected to measure of peptide concentrations. The blood
108
plasma and skin were stored at −80 °C until further analysis. 7 ACS Paragon Plus Environment
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109 110
Mouse study 2: Five-week old male BALB/c mice were randomly assigned to
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orally ingest Gly-Pro-Hyp or Pro-Hyp (Bachem) with 0.2 mmol/kg. Blood was
112
taken from the heart before and after 10, 15, 30, 45, 60, and 120 minutes from
113
oral ingestion, and the skin was collected after perfusion. We used five mice at
114
each time point. The blood plasma and skin were stored at −80 °C until further
115
analysis.
116 117
Pretreatment
118
deproteinization of plasma, three volumes of ethanol was added to plasma and
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centrifuged at 400 x g for 10 minutes at 4 °C. Then the supernatant was used for
120
quantification of peptide concentrations by LC-MS/MS.
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Skin: After crushing the skin in liquid nitrogen, samples were sonicated in an
122
equivalent weight of distilled water and centrifuged at 9000 x g for 5 minutes at
123
4 °C. As for the pellet, the same procedure then repeated with 50% ethanol
124
(Wako Chemicals, Osaka, Japan) and subsequently with 100% ethanol. The
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three supernatants were mixed and filtered through a 0.45 µm PVDF filter
126
(Merck Millipore, Massachusetts, USA). This filtrate was then subsequently
of
samples
for
LC-MS/MS.
Blood
plasma:
For
the
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injected into the LC-MS/MS system.
128 129
LC-MS/MS analysis. Peptide concentrations in plasma and the skin were
130
quantified by LC-MS/MS. The LC was performed with ACQUITY UPLC system
131
with the column ACQUITY UPLC HSS T3 (waters, Massachusetts, USA). Binary
132
gradient elution was performed with 0.05%(v/v) heptafluorobutyricacid (Thermo
133
Fisher Scientific, Massachusetts, USA) containing 0.1% formic acid (Wako
134
Chemicals, Osaka, Japan) and acetonitrile. The gradient profile with the
135
following proportions (v/v) of acetonitrile was applied (t (min), %acetonitrile): (0
136
min, 0%), (1 min, 0%), (4 min, 20%), (5min, 30%), (5.2 min, 30%), (5.3 min,
137
100%), (6.3 min, 100%), (6.4 min, 0%), (9.5 min, 0%). The flow rate was 0.4
138
mL/min. Mass spectrometry was performed with electrospray ionization (ESI) in
139
the positive and the multiple-reaction monitoring system was used with ion
140
source temperature 120 °C, desolvation temperature 400 °C, desolvation gas
141
flow 600 L/hr, cone gas flow 50 L/hr and capillary voltage 3 kV.
142 143
Statistical analyses.
144
Statistical analyses were performed using Microsoft Excel 2010 (Microsoft, 9 ACS Paragon Plus Environment
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Washington, USA). The Cmax and AUC of HTC-col group and LTC-col group
146
were compared by paired t-test. p
