Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating

Article abstract of DOI:10.1021/acs.jafc.7b03714

Cyclic dipeptides (2,5-diketopiperazines) are present in a variety of foods and are reported to demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a novel collagen hydrolysate characterized by a high content of X-hydroxyproline (Hyp)-Gly-type tripeptides using ginger protease. In the present study, we found that, through heating, X-Hyp-Gly can be easily converted into Hyp-containing cyclic dipeptides. After heating for 3 h at 85 °C and pH 4.8, Ala-Hyp-Gly was almost completely cyclized to cyclo(Ala-Hyp), in contrast to a slight cyclization of Ala-Hyp. The contents of cyclo(Ala-Hyp) and cyclo(Leu-Hyp) reached 0.5-1% (w/w) each in the ginger-degraded collagen hydrolysate under the heating conditions. Oral administration experiments using mice revealed that cyclo(Ala-Hyp) and cyclo(Leu-Hyp) were absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides, including Pro-Hyp. The high productivity and oral bioavailability of the collagen-specific cyclic dipeptides suggest significant health benefits of the heat-treated ginger-degraded collagen hydrolysate.

Full text of DOI:10.1021/acs.jafc.7b03714

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Article
Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing
Cyclic Dipeptides with High Oral Bioavailability: Efficient Formation
of Cyclo(X-Hyp) from X-Hyp-Gly-Type Tripeptides by Heating
Yuki Taga, Masashi Kusubata, Kiyoko Ogawa-Goto, and Shunji Hattori
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03714 • Publication Date (Web): 07 Oct 2017
Downloaded from http://pubs.acs.org on October 9, 2017
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Page 1 of 33
Journal of Agricultural and Food Chemistry
Identification of Collagen-Derived Hydroxyproline (Hyp)-Containing Cyclic Dipeptides
with High Oral Bioavailability: Efficient Formation of Cyclo(X-Hyp) from X-Hyp-Gly-
Type Tripeptides by Heating
Yuki Taga,* Masashi Kusubata, Kiyoko OgawaꢀGoto, and Shunji Hattori
Nippi Research Institute of Biomatrix, 520ꢀ11 Kuwabara, Toride, Ibaraki 302ꢀ0017, Japan
To whom correspondence should be addressed: Yuki Taga
Nippi Research Institute of Biomatrix, 520ꢀ11 Kuwabara, Toride, Ibaraki 302ꢀ0017, Japan
Tel: +81ꢀ297ꢀ71ꢀ3046; Fax: +81ꢀ297ꢀ71ꢀ3041
Eꢀmail: yꢀtaga@nippiꢀinc.co.jp
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ABSTRACT
Cyclic dipeptides (2,5ꢀdiketopiperazines) are present in a variety of foods and are reported to
demonstrate antioxidant, antidepressant, and other beneficial effects. We recently developed a
novel collagen hydrolysate characterized by a high content of Xꢀhydroxyproline (Hyp)ꢀGlyꢀtype
tripeptides using ginger protease. In the present study, we found that through heating, XꢀHypꢀ
Gly can be easily converted into Hypꢀcontaining cyclic dipeptides. After heating for 3 h at 85°C
and pH 4.8, AlaꢀHypꢀGly was almost completely cyclized to cyclo(AlaꢀHyp) in contrast to a
slight cyclization of AlaꢀHyp. The contents of cyclo(AlaꢀHyp) and cyclo(LeuꢀHyp) reached 0.5–
1% (w/w) each in the gingerꢀdegraded collagen hydrolysate under the heating condition. Oral
administration experiments using mice revealed that cyclo(AlaꢀHyp) and cyclo(LeuꢀHyp) were
absorbed into the blood at markedly higher efficiencies compared to collagenous oligopeptides,
including ProꢀHyp. The high productivity and oral bioavailability of the collagenꢀspecific cyclic
dipeptides suggest significant health benefits of the heatꢀtreated gingerꢀdegraded collagen
hydrolysate.
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Keywords: collagen hydrolysate, cyclic dipeptide, hydroxyproline, ginger, oral bioavailability
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INTRODUCTION
Collagen is an extracellular matrix protein abundant in connective tissues, such as skin, bone,
and tendon. Collagen consists of repeating GlyꢀXaaꢀYaa triplets where Xaa and Yaa are
frequently Pro and 4ꢀhydroxyproline (4ꢀHyp), respectively, forming a stable tripleꢀhelical
structure in the body. Heatꢀdenatured collagen (gelatin) can be extracted from skin, bone, and
fish scales. Oral ingestion of enzymatic hydrolysates of gelatin, referred to as collagen
hydrolysate or gelatin hydrolysate, has been reported to have various beneficial effects, including
reducing joint pain,1, 2 increasing bone density,3, 4 modulating lipid metabolism,5, 6 and lowering
blood pressure.7, 8 Although the mechanism of these effects has not been clarified, the structures
of the collagenꢀderived active ingredients have been determined over the last decade. In 2005,
Sato et al. first identified ProꢀHyp as the most abundant collagenꢀderived peptide in the blood
after oral ingestion of collagen hydrolysate.9 Additionally, after the ingestion, other collagenꢀ
derived Hypꢀcontaining dipeptides and tripeptides, including HypꢀGly and XꢀHypꢀGlyꢀtype
tripeptides, were also found in the blood at significantly high concentrations compared to other
foodꢀderived peptides.9ꢀ13 The presence of Hyp within the peptide sequence confers striking
protease resistance, leading to the high blood levels of the collagenꢀderived dipeptides and
tripeptides.9, 11, 13, 14 Recent studies have revealed that collagenꢀderived oligopeptides, such as
ProꢀHyp and HypꢀGly, have physiological functions, including growth stimulation of skin
fibroblasts,11, 15, 16 promotion of osteoblast and myoblast differentiation,17, 18 and improvement of
skin barrier dysfunction.19
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2,5ꢀDiketopiperazines (cyclic dipeptides, cyclo(XaaꢀYaa)) are found in a wide variety of foods
and beverages, such as bread,20 chicken essence,21 beer,22 coffee,23 cocoa,24 and the distillation
residue of awamori spirits.25 Cyclic dipeptides are mainly formed from peptides and proteins
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during heating and fermentation in food processing and contribute to the taste of foods,
especially bitterness.26 Beneficial effects have been reported for cyclic dipeptides; for example,
cyclo(IleꢀPro), cyclo(PheꢀPro), cyclo(ProꢀVal), and cyclo(LeuꢀPro) generated during the
production of awamori spirits have shown antioxidant activity,25 and cyclo(PheꢀPhe) found in
chicken essence has shown antidepressant and antidementia effects.27 Moreover, cyclic
dipeptides exhibit various other beneficial activities, such as antimicrobial,28ꢀ30 antitumor,31, 32
and antihepatitis activity.33, 34 Cyclic dipeptides can be produced from dipeptides by heating at
high temperature (>100°C) via headꢀtoꢀtail cyclization with dehydration.35ꢀ37 In addition, thermal
formation of cyclic dipeptides also occurs for tripeptides.36ꢀ39 Hayasaka et al. recently reported
that collagenous GlyꢀProꢀYꢀtype tripeptides, including GlyꢀProꢀHyp and GlyꢀProꢀAla, are
converted into cyclo(GlyꢀPro) through heating.39 The generation rate of cyclo(GlyꢀPro) from
GlyꢀProꢀY was much higher than that of GlyꢀPro. However, heating at 95°C for 24 h was needed
to cyclize ~80% of the GlyꢀProꢀYꢀtype tripeptides.
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Several studies have previously reported the presence of Hypꢀcontaining cyclic dipeptides.
Cyclo(PheꢀHyp) was found in culture supernatant of Lactobacillus plantarum,28 and cyclo(Alaꢀ
Hyp), cyclo(LeuꢀHyp), and cyclo(PheꢀHyp) were extracted from cultures of Alternaria
alternata.29 These microorganismꢀderived cyclic dipeptides showed antifungal activity.28, 29
Cyclo(SerꢀHyp), named JBP485, was identified in a hydrolysate of human placenta and
considered to be derived from collagen present in the tissue.40 Previous studies demonstrated that
cyclo(SerꢀHyp) has antihepatitis effects, which were suggested to be associated with antiꢀ
inflammatory, antioxidant, and antiapoptotic activities.33, 34 Moreover, improvement of acute
renal failure41 and promotion of corneal wound healing42 were also reported for cyclo(SerꢀHyp).
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We recently developed a novel collagen hydrolysate using ginger protease that preferentially
cleaves peptide bonds with Pro/Hyp at the P2 position.13 This collagen hydrolysate uniquely
contained substantial amounts of XꢀHypꢀGlyꢀtype tripeptides (approximately 2.5%; w/w). We
demonstrated that oral administration of the gingerꢀdegraded collagen hydrolysate significantly
increased the levels of XꢀHypꢀGly in blood. Thus, it is expected that an oral ingestion of the Xꢀ
HypꢀGlyꢀrich collagen hydrolysate has distinct health benefits. However, during the heat
treatment in the manufacturing process, we observed that XꢀHypꢀGlyꢀtype tripeptides in the
collagen hydrolysate unexpectedly diminished, especially during the sterilization step (data not
reported previously). In the present study, we aimed to identify the products from the heat
treatment of XꢀHypꢀGlyꢀtype tripeptides. In addition, oral bioavailability of the thermal
conversion products was investigated to assess their potential as novel active ingredients in
collagen hydrolysates.
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MATERIALS AND METHODS
Chemicals. Dithiothreitol (DTT),
Lꢀalanine, and glycine were purchased from Wako
Chemicals (Osaka, Japan). Sequencing grade trypsin and sequencing grade chymotrypsin were
purchased from Promega (Madison, WI). Vivaspin 20ꢀ10K was purchased from GE Healthcare
(Piscataway, NJ), and transꢀ4ꢀhydroxyꢀLꢀproline was purchased from SigmaꢀAldrich (St. Louis,
MO). HꢀProꢀHypꢀOH, HꢀHypꢀGlyꢀOH, HꢀGlyꢀAlaꢀHypꢀOH, HꢀGlyꢀProꢀAlaꢀOH, HꢀGlyꢀProꢀ
ArgꢀOH, HꢀGlyꢀProꢀHypꢀOH, HꢀAlaꢀProꢀGlyꢀOH, and cyclo(GlyꢀPro) were purchased from
Bachem (Bubendorf, Switzerland). Other peptides were custom synthesized by AnyGen
(Kwangju, Korea). Ginger rhizomes were purchased from a local supermarket. Gelatin was
prepared from pepsinꢀsolubilized bovine skin collagen as described previously.13
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Ethics Statement. All animal studies were approved by the Experimental Ethical Committee
of Nippi Research Institute of Biomatrix.
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Preparation of Ginger-Degraded Collagen Hydrolysates. Gingerꢀdegraded collagen
hydrolysates for heating experiments or oral administration experiments were prepared as
described previously.13 Briefly, for heating experiments, 50 mg/mL gelatin solution in 2 mM
DTT/100 mM sodium acetate buffer (pH 4.8) was hydrolyzed at 50°C for 16 h with shaking
using 1/10 (w/w) of ginger powder, which was prepared by homogenizing ginger rhizomes in
chilled acetone. After filtration using a 0.8 µm filter, the reactant was stored at −30°C until
analysis. For oral administration experiments, 50mg/mL gelatin solution (pH 4.0 adjusted by
HCl) containing 2 mM DTT was similarly hydrolyzed with ginger powder. The reactant was
filtered to remove the ginger powder, acidified with HCl, filtered through a 0.8 µm filter, and
subjected to ultrafiltration using Vivaspin 20ꢀ10K devices to remove ginger proteases. The flowꢀ
through fraction was lyophilized using a Virtis Genesis 25EL freeze dryer (SP Industries,
Gardiner, NY). After washing with acetone, the collagen hydrolysate was redried using a
centrifugal evaporator CVEꢀ3100 (EYELA, Tokyo, Japan).
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Preparation of Cyclo(Ala-Hyp) and Cyclo(Leu-Hyp). AlaꢀHypꢀGly or LeuꢀHypꢀGly was
heated at 85°C for 8 h in 100 mM sodium acetate buffer (pH 4.8). The heatꢀtreated peptide was
subjected to reversedꢀphase chromatography using a Zorbax Eclipse XDBꢀC18 column (5 µm
particle size, L × I.D. 150 mm × 4.6 mm; Agilent Technologies, Palo Alto, CA) with monitoring
at 220 nm on an Alliance 2690 separation module equipped with a 2487 dual absorbance
detector (Waters, Milford, MA). Separation was performed at a flow rate of 1 mL/min with a
binary gradient as follows: 98% solvent A (0.1% formic acid) for 5 min, linear gradient of 0–
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99% solvent B (100% acetonitrile) for 10 min, and 98% solvent A for 5 min. Fractions of
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cyclo(AlaꢀHyp) at 3.0–3.4 min and cyclo(LeuꢀHyp) at 9.7–9.9 min were collected from heatꢀ
treated AlaꢀHypꢀGly and LeuꢀHypꢀGly, respectively. After drying using the centrifugal
evaporator, the purified cyclic dipeptide was dissolved in distilled water and stored at −30°C.
Heating Experiments of Collagenous Oligopeptides. The gingerꢀdegraded collagen
hydrolysate solution (pH 4.8) was heated at 85°C for 1 or 3 h. Synthetic diꢀ and tripeptides were
incubated at 4–100°C for 1 or 3 h in 100 mM citrateꢀNaOH buffer (pH 2.4), 100 mM sodium
acetate buffer (pH 3.6 and 4.8), 100 mM sodium phosphate buffer (pH 6.0 and 7.2), or 100 mM
sodium carbonate buffer (pH 8.4 and 9.6). The samples were diluted in 1% formic acid for liquid
chromatography (LC)–mass spectrometry (MS) analysis.
Preparation of An Internal Standard Mixture of Collagen-Derived Oligopeptides and
Cyclic Dipeptides. Stable isotopeꢀlabeled collagen (SIꢀcollagen) was first prepared in human
lung fibroblast cultures using stable isotopically heavy Pro, Lys, and Arg as described
previously.12, 13, 43, 44 After denaturation at 60°C for 30 min, SIꢀcollagen was digested using
trypsin and chymotrypsin (1:50 enzyme/substrate ratio, respectively) in 100 mM ammonium
bicarbonate/1 mM CaCl2 at 37°C for 16 h, and further digested with freshly prepared dialyzed
mouse plasma at 37°C for 24 h as described previously.12, 13 After ethanol deproteinization and
drying of the ethanolꢀsoluble fraction using the centrifugal evaporator, the protease digest of SIꢀ
collagen (SIꢀdigest) was heated in 50 mM sodium acetate buffer (pH 4.8) at 85°C for 1 h. The
heatꢀtreated SIꢀdigest was then stored at −30°C.
Oral Administration of Collagen Hydrolysates. The gingerꢀdegraded collagen hydrolysate
was dissolved in 50 mM sodium acetate buffer (pH 4.8) and divided into two aliquots. One
aliquot was heated at 85°C for 3 h to convert XꢀHypꢀGly to cyclo(XꢀHyp), while the other was
excluded from the heat treatment to retain XꢀHypꢀGly. These two hydrolysates were orally
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administered to 3ꢀmonthꢀold male ICR mice (Japan SLC, Shizuoka, Japan), as reported
previously.13 Briefly, blood from the tail vein was collected 1 h after oral administration of 10
mg of the collagen hydrolysates. The mice had been fed a collagen freeꢀdiet, AINꢀ93M (Oriental
Yeast, Tokyo, Japan), from a day before the experiment. Plasma was prepared by centrifugation
and was mixed with heatꢀtreated SIꢀdigest used as an internal standard. After ethanol
deproteinization and drying of the ethanolꢀsoluble fraction, the sample was reconstituted in 0.1%
formic acid for LC–MS analysis. Heatꢀtreated SIꢀdigest was mixed into the calibration standards
as the internal standard.
Oral Administration of Synthetic Peptides. Synthetic peptides were orally administered to
9ꢀmonthꢀold male ICR mice (Japan SLC) as per the method above for the collagen hydrolysates.
Mice were divided into two groups and orally administered peptide mixtures containing 100 µg
each of the following peptides: group 1 [AlaꢀHyp, ProꢀHyp, HypꢀGly, cyclo(AlaꢀHyp),
cyclo(LeuꢀHyp), and cyclo(GlyꢀPro)]; group 2 [GlyꢀAlaꢀHyp, GlyꢀProꢀAla, GlyꢀProꢀHyp, Alaꢀ
HypꢀGly, ProꢀHypꢀGly, and cyclo(AlaꢀHyp)]. Plasma was prepared before administration (0 h)
and at 0.5, 1, 2, 4, and 6 h after administration. LC–MS samples were prepared after the addition
of heatꢀtreated SIꢀdigest as described above. The area under the plasma concentration−time curve
(AUC0−6 h) was calculated using the trapezoidal rule.
LC–MS Analysis. Samples prepared in the peptide heating experiments and oral
administration experiments were analyzed by LC–MS in multiple reaction monitoring (MRM)
mode using a hybrid triple quadrupole/linear ion trap 3200 QTRAP mass spectrometer (AB
Sciex, Foster City, CA) coupled to an Agilent 1200 Series HPLC system (Agilent Technologies).
The samples were loaded onto an Ascentis Express F5 HPLC column (5 µm particle size, L ×
I.D. 250 mm × 4.6 mm; Supelco, Bellefonte, PA) at a flow rate of 400 µL/min and separated by
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a binary gradient as follows: 100% solvent A (10 mM ammonium acetate) for 7.5 min, linear
gradient of 0–75% solvent B (100% acetonitrile) for 12.5 min, 75% solvent B for 5 min, and
100% solvent A for 5 min (for AlaꢀHyp, ProꢀHyp, HypꢀGly, and AlaꢀHypꢀGly in serum
samples); or 100% solvent A (0.1% formic acid) for 7.5 min, linear gradient of 0–90% solvent B
(100% acetonitrile) for 12.5 min, 90% solvent B for 5 min, and 100% solvent A for 5 min (for
other analytes). Analyst 1.6.2 (AB Sciex) was used to perform the data acquisition and analysis.
The MRM transitions of amino acids, oligopeptides, and cyclic dipeptides are shown in Table S1
in the Supporting Information. Capillary voltage was 3 kV, declustering potential was 15 V,
heater gas temperature was 700°C, curtain gas was 15 psi, nebulizer gas was 80 psi, and heater
gas was 80 psi.
Direct Infusion MS Analysis. Before or after heating synthetic AlaꢀHypꢀGly at 85°C for 3 h
in distilled water with a clean glass vial, MS spectra were obtained by direct infusion analysis
using an ultraꢀhigh resolution quadrupole timeꢀofꢀflight (QTOF) mass spectrometer (maXis II,
Bruker Daltonics, Bremen, Germany). The theoretically simulated MS spectrum of cyclo(Alaꢀ
Hyp) was generated by Compass DataAnalysis version 4.3 (Bruker Daltonics).
RESULTS AND DISCUSSION
Heat-Induced Decrease in X-Hyp-Gly. To evaluate thermal stability, we subjected a gingerꢀ
degraded collagen hydrolysate solution, which contains high levels of GlyꢀProꢀY and XꢀHypꢀ
Glyꢀtype tripeptides,13 to heat treatment at 85°C and pH 4.8 for 1 or 3 h. LC–MS analysis
showed that while GlyꢀProꢀYꢀtype tripeptides (initially 78.9 mg/g gelatin in total) only slightly
decreased after 3 h heating (71.7 mg/g) (Fig. 1A), XꢀHypꢀGlyꢀtype tripeptides (initially 16.2
mg/g) dramatically decreased by the heat treatment (6.2 mg/g after 1 h and 1.2 mg/g after 3 h)
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(Fig. 1B). Similarly, synthetic AlaꢀHypꢀGly and LeuꢀHypꢀGly showed 65% and 67% decreases,
respectively, by 1 h of heating at 85°C and pH 4.8 (Fig. 1C). In contrast, other types of
collagenous synthetic peptides, including AlaꢀHyp, ProꢀHyp, HypꢀGly, and GlyꢀAlaꢀHyp, were
thermally stable except for GlyꢀProꢀAla and GlyꢀProꢀHyp, which showed 15% and 7%
decreases, respectively. The slight decreases in GlyꢀProꢀYꢀtype tripeptides are likely due to
thermal conversion into cyclo(GlyꢀPro), as previously reported.39
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The heatꢀinduced significant decrease in XꢀHypꢀGly suggests that this type of collagenꢀderived
tripeptide is thermally degraded into amino acids and/or dipeptides. Thus, we next monitored the
degradation products of AlaꢀHypꢀGly by LC–MS after heating at 85°C and pH 4.8 for 1 or 3 h
(Fig. 1D). Gly was found to be generated concomitantly with the decrease in AlaꢀHypꢀGly.
However, the complement degradation product, AlaꢀHyp, was completely undetectable, as were
other potential degradation products (HypꢀGly, Ala, and Hyp). These observations indicate that
AlaꢀHypꢀGly was converted into an unexpected form by heating.
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Identification of Cyclo(X-Hyp) Converted from X-Hyp-Gly by Heating. We performed
mass spectrometric analysis of heatꢀtreated AlaꢀHypꢀGly using high resolution QTOF–MS to
identify the thermal conversion product. After 3 h heating of AlaꢀHypꢀGly (m/z 260.13, z = 1) in
distilled water, we observed the generation of a large unknown peak “X” (m/z 185.09, z = 1)
(Fig. 2A). From the observed mass and the appearance of Gly shown in Fig. 1D, we predicted
that cyclo(AlaꢀHyp) was formed from AlaꢀHypꢀGly by cyclodehydration that occurred
simultaneously or after cleavage of the peptide bond between Hyp and Gly. Thus, we compared
the measured spectrum of peak X with the simulated spectrum of cyclo(AlaꢀHyp). As shown in
Fig. 2B, the monoisotopic peak of X (m/z 185.0948) was highly matched to that of cyclo(Alaꢀ
Hyp) (m/z 185.0921, C8H12N2O3+H), and the isotopic distribution of peak X was also identical to
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that of the simulated spectrum. These results demonstrate that peak X was cyclo(AlaꢀHyp)
thermally converted from AlaꢀHypꢀGly. The cyclic dipeptide is assumed to be configured as cisꢀ
cyclo(LꢀAlaꢀLꢀHyp) based on the fact that natural constituents of the original tripeptide are Lꢀ
amino acids.26 However, epimerization is possibly occurred by the thermal treatment. NMR
analysis is needed to definitively determine the steric configuration.
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The heatꢀinduced cyclization of AlaꢀHypꢀGly was quantitatively estimated by LC–MS with
authentic cyclo(AlaꢀHyp), which was purified from heatꢀtreated AlaꢀHypꢀGly by reversedꢀphase
chromatography. A substantial amount of cyclo(AlaꢀHyp) was generated by heating at 85°C and
pH 4.8, which was inversely proportional to the decrease in AlaꢀHypꢀGly (Fig. 3A and Fig. S1 in
the Supporting Information). After 3 h of heating, the molar concentration of cyclo(AlaꢀHyp)
reached ~100% of that of the original peptide. In contrast, only a trace amount of cyclo(AlaꢀHyp)
was generated from AlaꢀHyp after heating at 85°C for 3 h (Fig. 3B).
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To define the conditions for the formation of Hypꢀcontaining cyclic dipeptides from XꢀHypꢀ
Glyꢀtype tripeptides, we analyzed three parameters related to the cyclization reaction: peptide
sequence, reaction pH, and reaction temperature. First, we heatꢀtreated various types of XꢀHypꢀ
Gly at 85°C and pH 4.8 for 1 h and observed decreases of more than 50% for all of the peptides
except ProꢀHypꢀGly (Fig. 4A). AlaꢀProꢀGly and LeuꢀProꢀGly were also decreased by the heat
treatment, probably due to cyclo(XꢀPro) formation. Second, we measured the generation of
cyclo(AlaꢀHyp) from AlaꢀHypꢀGly heated at 85°C for 1 h while varying the pH. Cyclization
efficiently occurred at pH 4.8–7.2 (63.0–72.7%), while the reaction was markedly suppressed at
pH 2.4 (8.7%) (Fig. 4B). Finally, we confirmed that the peptide cyclization was highly
dependent on the reaction temperature (0.3–3.3% at 4–55°C, 57.6% at 85°C, and 82.7% at
100°C, under 1 h heating at pH 4.8) (Fig. 4C). These data indicate that we can both promote and
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suppress cyclization of XꢀHypꢀGly (other than ProꢀHypꢀGly) by controlling the process
conditions; high temperature with weakly acidic/neutral pH to produce cyclo(XꢀHyp) and low
temperature with acidic pH to maintain XꢀHypꢀGlyꢀtype tripeptides.
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Rizzi previously described that tripeptides (GlyꢀLeuꢀAla, AlaꢀLeuꢀGly, LeuꢀGlyꢀPhe, and Proꢀ
GlyꢀGly) were converted into cyclic dipeptides consisting of Nꢀterminal dipeptides by heating at
120°C for 0.5 h, showing 7–23% yields.36 The study explained that the cyclodehydration reaction
occurs by attack of the Nꢀterminal amino group on the carbonyl group of the peptide bond
between the internal and Cꢀterminal amino acid residues followed by the loss of the Cꢀterminal
amino acid. However, the reported cyclization rate of the tripeptides used was markedly lower
than that of the dipeptide (ValꢀAla, 94%). In contrast, XꢀHypꢀGly was found to be more easily
converted into cyclo(XꢀHyp) under the relatively moderate heating conditions. We speculate that
the efficient cyclization of XꢀHypꢀGly is attributed to the presence of the imino acid at the
internal position. In addition, the Cꢀterminal Gly is also assumed to be important for the efficient
cyclization based on the comparison with the thermal conversion of GlyꢀProꢀYꢀtype tripeptides
into cyclo(GlyꢀPro) that were previously reported (24 h heating at 95°C for ~80% cyclization).39
However, present data are insufficient to completely support these speculations. Further
experiments are needed to determine the key sequence features for the highly efficient
cyclization of XꢀHypꢀGly.
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Preparation of Stable Isotope-Labeled Internal Standards of Cyclo(X-Hyp). Using LC–
MS, we next investigated whether the Hypꢀcontaining cyclic dipeptides are absorbed into the
blood. A recent study reported simultaneous quantification of 31 kinds of cyclic dipeptides using
LC–MS with good repeatability and linearity for standards of the analytes.45 However, the matrix
effect was suggested to decrease the recovery rate of some of the cyclic dipeptides when spiked
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into tea extracts. Ion suppression (or enhancement) effects in LC–MS caused by coeluting matrix
components sometimes critically impair the quantitative accuracy. This is especially true in
complex biological samples, as reported in our previous study analyzing Hypꢀcontaining
peptides in blood.12 To accurately quantitate the cyclic dipeptides in blood, we utilized a recently
developed internal standard mixture named SIꢀdigest, in which various kinds of collagenꢀderived
oligopeptides, including XꢀHypꢀGly, are stable isotopically labeled.12, 43 We further treated the
SIꢀdigest with heat (85°C and pH 4.8 for 1 h) to generate internal standards of cyclic dipeptides.
Generation of stable isotopeꢀlabeled cyclo(AlaꢀHyp), cyclo(LeuꢀHyp), and also cyclo(GlyꢀPro)
was confirmed as shown in Fig. S2 in the Supporting Information. In the following
experiments, the heatꢀtreated SIꢀdigest was mixed into plasma samples as an internal standard for
LC–MS analysis.
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Absorption of Cyclo(X-Hyp) into Blood after Oral Administration of Ginger-Degraded
Collagen Hydrolysates. The collagen hydrolysate prepared using ginger protease was heated at
85°C and pH 4.8 for 3 h to generate cyclic dipeptides. We confirmed that the contents of
cyclo(AlaꢀHyp) and cyclo(LeuꢀHyp) were increased from 0.7 to 5.2 mg/g and from 1.0 to 9.1
mg/g, respectively, following the heat treatment (Fig. 5A). A heatꢀinduced increase was also
detected for cyclo(GlyꢀPro) (0.6 to 5.7 mg/g), which was likely derived from GlyꢀProꢀYꢀtype
tripeptides, as reported previously.39 The heatꢀtreated and nonꢀtreated (control) collagen
hydrolysates were orally administered to mice, and plasma samples for LC–MS analysis were
obtained 1 h later. After oral administration of the control collagen hydrolysate, only slight
amounts of the cyclic dipeptides were detected in the blood (Fig. 5B). In contrast, all three cyclic
dipeptides displayed markedly higher plasma levels after the administration of the heatꢀtreated
collagen hydrolysate. The plasma concentrations of cyclic dipeptides paralleled their contents in
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the administered collagen hydrolysates. These results demonstrate that orally administered
collagenꢀderived cyclic dipeptides were absorbed into the blood.
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Although we focused on cyclo(AlaꢀHyp) and cyclo(LeuꢀHyp) as representatives of the Hypꢀ
containing cyclic dipeptides in this study, other cyclo(XꢀHyp) were also detected in the gingerꢀ
degraded collagen hydrolysate after heat treatment (data not shown). As shown in Fig. 5A, the
amount of cyclo(AlaꢀHyp) and cyclo(LeuꢀHyp) was comparable or higher than cyclo(GlyꢀPro).
While cyclo(GlyꢀPro) is probably derived from various kinds of GlyꢀProꢀYꢀtype tripeptides, the
Hypꢀcontaining cyclic dipeptides are from their respective precursors (AlaꢀHypꢀGly and Leuꢀ
HypꢀGly), indicating that the total amounts of cyclo(XꢀHyp) in the heatꢀtreated collagen
hydrolysate would be considerably high.
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Efficient Absorption of Cyclo(X-Hyp) Compared to Collagenous Oligopeptides. Further
evaluation of the absorption of the cyclic dipeptides was performed using synthetic peptides.
Mice were orally administered peptide mixtures (100 µg each), which included cyclic dipeptides
and various kinds of collagenous oligopeptides reported to appear in the blood after oral
ingestion of collagen hydrolysate.12, 46 Since some peptides cannot be discriminated after partial
peptide hydrolysis in the gastrointestinal tract and blood (e.g., from AlaꢀHypꢀGly to AlaꢀHyp and
HypꢀGly), we divided the peptides into two experimental groups. After the administration, most
of the peptides were observed to be increased in the plasma, except for AlaꢀHyp, GlyꢀAlaꢀHyp,
and GlyꢀProꢀAla (Fig. 6). Plasma concentrations of the collagenous dipeptides and tripeptides
peaked at 0.5 or 1 h and returned to the basal level at 2 h. Among the oligopeptides, ProꢀHyp
showed the highest maximum plasma concentration (Cmax, 0.118 µg/mL). The AUC0–6h of the
dipeptides and tripeptides ranged from 0.065 to 0.431 µg/mL·h (Table 1). On the other hand,
Hypꢀcontaining cyclic dipeptides showed higher values of Cmax [0.748 and 0.626 µg/mL for
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cyclo(AlaꢀHyp) and 1.495 µg/mL for cyclo(LeuꢀHyp)]. These higher concentrations were
maintained for a relatively longer period of time. At 2 h after the administration, the plasma level
relative to the Cmax was still 43–100%. Due to the characteristic profiles of cyclo(XꢀHyp), the
AUC0–6h of the cyclic dipeptides reached >2 µg/mL·h, which was dramatically higher than that
of the collagenous oligopeptides (Table 1). Similarly, a significantly higher plasma level was
observed for another type of cyclic dipeptide, cyclo(GlyꢀPro). Previous studies by Mizuma et al.
demonstrated that cyclic dipeptides are more efficiently absorbed from the small intestine than
the linear forms due to their high stability to proteolysis and high transportability via peptide
transporters.47, 48 Hydrophobicity is one of the determinants for the recognition of peptides by
peptide transporters.49, 50 Thus, the high hydrophobicity of cyclic dipeptides potentially enhances
their intestinal absorption. These properties of cyclic dipeptides are assumed to contribute to the
high oral bioavailability of cyclo(XꢀHyp). In vitro transport studies using intestinal epithelial
cells (Cacoꢀ2) are now in progress.
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In conclusion, we demonstrate that XꢀHypꢀGlyꢀtype tripeptides are almost completely
converted into Hypꢀcontaining cyclic dipeptides by heating at 85°C and pH 4.8 for 3 h, and that
the cyclic dipeptides are more efficiently absorbed into the blood than conventional collagenous
oligopeptides, including ProꢀHyp. Previous studies have reported that cyclo(SerꢀHyp) found in a
hydrolysate of human placenta has various beneficial effects, including protection against liver
injury,33, 34 improvement of acute renal failure,41 and promotion of corneal wound healing.42
Such beneficial biological activities of cyclo(SerꢀHyp) could provide an indication that other
Hypꢀcontaining cyclic dipeptides might demonstrate beneficial effects as well.
312
313
ABBREVIATIONS USED
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Hyp, hydroxyproline; DTT, dithiothreitol; LC, liquid chromatography; MS, mass spectrometry;
SIꢀcollagen, stable isotopeꢀlabeled collagen; AUC, area under the plasma concentration–time
curve; MRM, multiple reaction monitoring; QTOF, quadrupole timeꢀofꢀflight; Cmax, maximum
plasma concentration
Supporting Information. Figure S1: MRM chromatograms of AlaꢀHypꢀGly and cyclo(Alaꢀ
Hyp). Figure S2: MRM chromatograms of stable isotopeꢀlabeled cyclo(AlaꢀHyp), cyclo(Leuꢀ
Hyp), and cyclo(GlyꢀPro) in SIꢀdigest. Table S1: MRM transitions of amino acids, oligopeptides,
and cyclic dipeptides. This material is available free of charge via the Internet at
http://pubs.acs.org.
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REFERENCES
1. Clark, K. L.; Sebastianelli, W.; Flechsenhar, K. R.; Aukermann, D. F.; Meza, F.; Millard,
R. L.; Deitch, J. R.; Sherbondy, P. S.; Albert, A. 24ꢀWeek study on the use of collagen
hydrolysate as a dietary supplement in athletes with activityꢀrelated joint pain. Curr. Med. Res.
Opin. 2008, 24, 1485ꢀ96.
2.
Schauss, A. G.; Stenehjem, J.; Park, J.; Endres, J. R.; Clewell, A. Effect of the novel low
molecular weight hydrolyzed chicken sternal cartilage extract, BioCell Collagen, on improving
osteoarthritisꢀrelated symptoms: a randomized, doubleꢀblind, placeboꢀcontrolled trial. J. Agric.
Food Chem. 2012, 60, 4096ꢀ101.
3.
Wu, J.; Fujioka, M.; Sugimoto, K.; Mu, G.; Ishimi, Y. Assessment of effectiveness of
oral administration of collagen peptide on bone metabolism in growing and mature rats. J. Bone
Miner. Metab. 2004, 22, 547ꢀ53.
4.
Guillerminet, F.; Beaupied, H.; FabienꢀSoule, V.; Tome, D.; Benhamou, C. L.; Roux, C.;
Blais, A. Hydrolyzed collagen improves bone metabolism and biomechanical parameters in
ovariectomized mice: an in vitro and in vivo study. Bone 2010, 46, 827ꢀ34.
5.
fed a highꢀlipid and highꢀsucrose diet. Food Sci. Technol. Res. 2013, 19, 1149ꢀ1153.
6. Tometsuka, C.; Koyama, Y. I.; Ishijima, T.; Toyoda, T.; Teranishi, M.; Takehana, K.;
Koyama, Y. I.; Kusubata, M. Effects of collagen peptide ingestion on blood lipids in rats
Abe, K.; Nakai, Y. Collagen peptide ingestion alters lipid metabolismꢀrelated gene expression
and the unfolded protein response in mouse liver. Br. J. Nutr. 2017, 117, 1ꢀ11.
7.
Saiga, A.; Iwai, K.; Hayakawa, T.; Takahata, Y.; Kitamura, S.; Nishimura, T.;
Morimatsu, F. Angiotensin Iꢀconverting enzymeꢀinhibitory peptides obtained from chicken
collagen hydrolysate. J. Agric. Food Chem. 2008, 56, 9586ꢀ91.
17
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 33
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
8.
Herregods, G.; Van Camp, J.; Morel, N.; Ghesquiere, B.; Gevaert, K.; Vercruysse, L.;
Dierckx, S.; Quanten, E.; Smagghe, G. Angiotensin Iꢀconverting enzyme inhibitory activity of
gelatin hydrolysates and identification of bioactive peptides. J. Agric. Food Chem. 2011, 59,
552ꢀ8.
9.
Iwai, K.; Hasegawa, T.; Taguchi, Y.; Morimatsu, F.; Sato, K.; Nakamura, Y.; Higashi,
A.; Kido, Y.; Nakabo, Y.; Ohtsuki, K. Identification of foodꢀderived collagen peptides in human
blood after oral ingestion of gelatin hydrolysates. J. Agric. Food Chem. 2005, 53, 6531ꢀ6.
10.
Ohara, H.; Matsumoto, H.; Ito, K.; Iwai, K.; Sato, K. Comparison of quantity and
structures of hydroxyprolineꢀcontaining peptides in human blood after oral ingestion of gelatin
hydrolysates from different sources. J. Agric. Food Chem. 2007, 55, 1532ꢀ5.
11.
Shigemura, Y.; Akaba, S.; Kawashima, E.; Park, E. Y.; Nakamura, Y.; Sato, K.
Identification of a novel foodꢀderived collagen peptide, hydroxyprolylꢀglycine, in human
peripheral blood by preꢀcolumn derivatisation with phenyl isothiocyanate. Food Chem. 2011,
129, 1019ꢀ24.
12.
Taga, Y.; Kusubata, M.; OgawaꢀGoto, K.; Hattori, S. Highly accurate quantification of
hydroxyprolineꢀcontaining peptides in blood using a protease digest of stable isotopeꢀlabeled
collagen. J. Agric. Food Chem. 2014, 62, 12096ꢀ102.
13.
Taga, Y.; Kusubata, M.; OgawaꢀGoto, K.; Hattori, S. Efficient absorption of Xꢀ
hydroxyproline (Hyp)ꢀGly after oral administration of a novel gelatin hydrolysate prepared using
ginger protease. J. Agric. Food Chem. 2016, 64, 2962ꢀ70.
14.
Sontakke, S. B.; Jung, J. H.; Piao, Z.; Chung, H. J. Orally Available Collagen Tripeptide:
Enzymatic Stability, Intestinal Permeability, and Absorption of GlyꢀProꢀHyp and ProꢀHyp. J.
Agric. Food Chem. 2016, 64, 7127ꢀ33.
18
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Page 19 of 33
Journal of Agricultural and Food Chemistry
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
15.
Shigemura, Y.; Iwai, K.; Morimatsu, F.; Iwamoto, T.; Mori, T.; Oda, C.; Taira, T.; Park,
E. Y.; Nakamura, Y.; Sato, K. Effect of Prolylꢀhydroxyproline (ProꢀHyp), a foodꢀderived
collagen peptide in human blood, on growth of fibroblasts from mouse skin. J. Agric. Food
Chem. 2009, 57, 444ꢀ9.
16.
Ohara, H.; Ichikawa, S.; Matsumoto, H.; Akiyama, M.; Fujimoto, N.; Kobayashi, T.;
Tajima, S. Collagenꢀderived dipeptide, prolineꢀhydroxyproline, stimulates cell proliferation and
hyaluronic acid synthesis in cultured human dermal fibroblasts. J. Dermatol. 2010, 37, 330ꢀ8.
17.
Kimira, Y.; Ogura, K.; Taniuchi, Y.; Kataoka, A.; Inoue, N.; Sugihara, F.; Nakatani, S.;
Shimizu, J.; Wada, M.; Mano, H. Collagenꢀderived dipeptide prolylꢀhydroxyproline promotes
differentiation of MC3T3ꢀE1 osteoblastic cells. Biochem. Biophys. Res. Commun. 2014, 453,
498ꢀ501.
18.
Kitakaze, T.; Sakamoto, T.; Kitano, T.; Inoue, N.; Sugihara, F.; Harada, N.; Yamaji, R.
The collagen derived dipeptide hydroxyprolylꢀglycine promotes C2C12 myoblast differentiation
and myotube hypertrophy. Biochem. Biophys. Res. Commun. 2016, 478, 1292ꢀ7.
19.
Shimizu, J.; Asami, N.; Kataoka, A.; Sugihara, F.; Inoue, N.; Kimira, Y.; Wada, M.;
Mano, H. Oral collagenꢀderived dipeptides, prolylꢀhydroxyproline and hydroxyprolylꢀglycine,
ameliorate skin barrier dysfunction and alter gene expression profiles in the skin. Biochem.
Biophys. Res. Commun. 2015, 456, 626ꢀ30.
20.
diketopiperazines in wheat sourdough and bread. J. Agric. Food Chem. 2009, 57, 9563ꢀ8.
21. Chen, Y. H.; Liou, S. E.; Chen, C. C. Twoꢀstep mass spectrometric approach for the
Ryan, L. A.; Dal Bello, F.; Arendt, E. K.; Koehler, P. Detection and quantitation of 2,5ꢀ
identification of diketopiperazines in chicken essence. Eur. Food Res. Technol. 2004, 218, 589ꢀ
597.
19
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 33
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
22.
Gautschi, M.; Schmid, J. P.; Peppard, T. L.; Ryan, T. P.; Tuorto, R. M.; Yang, X.
Chemical characterization of diketopiperazines in beer. J. Agric. Food Chem. 1997, 45, 3183ꢀ
3189.
23.
coffee. J. Agric. Food Chem. 2000, 48, 3528ꢀ32.
24. Stark, T.; Hofmann, T. Structures, sensory activity, and dose/response functions of 2,5ꢀ
Ginz, M.; Engelhardt, U. H. Identification of prolineꢀbased diketopiperazines in roasted
diketopiperazines in roasted cocoa nibs (Theobroma cacao). J. Agric. Food Chem. 2005, 53,
7222ꢀ31.
25.
Takaya, Y.; Furukawa, T.; Miura, S.; Akutagawa, T.; Hotta, Y.; Ishikawa, N.; Niwa, M.
Antioxidant constituents in distillation residue of Awamori spirits. J. Agric. Food Chem. 2007,
55, 75ꢀ9.
26.
and bioactivity. Crit. Rev. Food Sci. Nutr. 2017, 57, 718ꢀ742.
27. Tsuruoka, N.; Beppu, Y.; Koda, H.; Doe, N.; Watanabe, H.; Abe, K. A DKP cyclo(Lꢀ
Borthwick, A. D.; Da Costa, N. C. 2,5ꢀdiketopiperazines in food and beverages: Taste
PheꢀLꢀPhe) found in chicken essence is a dual inhibitor of the serotonin transporter and
acetylcholinesterase. PLoS One 2012, 7, e50824.
28.
Strom, K.; Sjogren, J.; Broberg, A.; Schnurer, J. Lactobacillus plantarum MiLAB 393
produces the antifungal cyclic dipeptides cyclo(LꢀPheꢀLꢀPro) and cyclo(LꢀPheꢀtransꢀ4ꢀOHꢀLꢀ
Pro) and 3ꢀphenyllactic acid. Appl. Environ. Microbiol. 2002, 68, 4322ꢀ7.
29.
Musetti, R.; Polizzotto, R.; Vecchione, A.; Borselli, S.; Zulini, L.; D'Ambrosio, M.; di
Toppi, L. S.; Pertot, I. Antifungal activity of diketopiperazines extracted from Alternaria
alternata against Plasmopara viticola: an ultrastructural study. Micron 2007, 38, 643ꢀ50.
20
ACS Paragon Plus Environment

Page 21 of 33
Journal of Agricultural and Food Chemistry
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
30.
Kumar, N.; Mohandas, C.; Nambisan, B.; Kumar, D. R.; Lankalapalli, R. S. Isolation of
prolineꢀbased cyclic dipeptides from Bacillus sp. N strain associated with rhabditid [corrected]
entomopathogenic nematode and its antimicrobial properties. World J. Microbiol. Biotechnol.
2013, 29, 355ꢀ64.
31.
activity of phenylahistin in vitro and in vivo. Biosci. Biotechnol. Biochem. 1999, 63, 1130ꢀ3.
32. Brauns, S. C.; Milne, P.; Naude, R.; Van de Venter, M. Selected cyclic dipeptides inhibit
Kanoh, K.; Kohno, S.; Katada, J.; Hayashi, Y.; Muramatsu, M.; Uno, I. Antitumor
cancer cell growth and induce apoptosis in HTꢀ29 colon cancer cells. Anticancer Res. 2004, 24,
1713ꢀ9.
33.
Liu, K. X.; Kato, Y.; Kaku, T. I.; Santa, T.; Imai, K.; Yagi, A.; Ishizu, T.; Sugiyama, Y.
Hydroxyprolylserine derivatives JBP923 and JBP485 exhibit the antihepatitis activities after
gastrointestinal absorption in rats. J. Pharmacol. Exp. Ther. 2000, 294, 510ꢀ5.
34.
Yang, T.; Wu, J.; Wang, C.; Liu, Q.; Ma, X.; Peng, J.; Kaku, T.; Liu, K. Protective effect
of JBP485 on concanavalin Aꢀinduced liver injury in mice. J. Pharm. Pharmacol. 2009, 61, 767ꢀ
74.
35.
racemization in dipeptides. Science 1981, 213, 544ꢀ5.
36. Rizzi, G. P. Heatꢀinduced flavor formation from peptides. In Thermal Generation of
Steinberg, S.; Bada, J. L. Diketopiperazine formation during investigations of amino Acid
Aromas; Parliament, T. H., McGorrin, R. J., Ho, C. T., Eds.; American Chemical Society:
Washington, DC, 1989; Vol. 409, pp 172ꢀ181.
37.
Fabbri, D.; Adamiano, A.; Falini, G.; De Marco, R.; Mancini, I. Analytical pyrolysis of
dipeptides containing proline and amino acids with polar side chains. Novel 2, 5ꢀ
21
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 33
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
diketopiperazine markers in the pyrolysates of proteins. J. Anal. Appl. Pyrolysis 2012, 95, 145ꢀ
155.
38.
formation of diketopiperazines. J. Org. Chem. 1983, 48, 2295ꢀ2298.
39. Hayasaka, F.; Yamamoto, S.; Sakai, Y. Production method for cyclic dipeptide derived
from native collagen. Food Sci. Technol. Res. 2016, 22, 477ꢀ483.
40. Yagi, A.; Nagao, M.; Okamura, N.; Ishizu, T.; Itoh, H.; Shida, T. Effect of cyclo (transꢀ4ꢀ
Steinberg, S. M.; Bada, J. L. Peptide decomposition in the neutral pH region via the
LꢀhydroxyprolylꢀLꢀserine) from hydrolysate of human placenta on baby hamster kidneyꢀ21/Cꢀ13
cells. Nat. Med. (Tokyo, Japan) 1998, 52, 156ꢀ159.
41.
Guo, X.; Meng, Q.; Liu, Q.; Wang, C.; Sun, H.; Peng, J.; Ma, X.; Kaku, T.; Liu, K.
JBP485 improves gentamicinꢀinduced acute renal failure by regulating the expression and
function of Oat1 and Oat3 in rats. Toxicol. Appl. Pharmacol. 2013, 271, 285ꢀ95.
42.
promotes corneal epithelial wound healing. Sci. Rep. 2015, 5, 14776.
43. Taga, Y.; Kusubata, M.; OgawaꢀGoto, K.; Hattori, S. Stable isotopeꢀlabeled collagen: a
Nagata, M.; Nakamura, T.; Hata, Y.; Yamaguchi, S.; Kaku, T.; Kinoshita, S. JBP485
novel and versatile tool for quantitative collagen analyses using mass spectrometry. J. Proteome
Res. 2014, 13, 3671ꢀ8.
44.
regulation of prolyl 3ꢀhydroxylation in tendon type I collagen. J. Biol. Chem. 2016, 291, 837ꢀ47.
45. Yamamoto, K.; Hayashi, M.; Murakami, Y.; Araki, Y.; Otsuka, Y.; Kashiwagi, T.;
Taga, Y.; Kusubata, M.; OgawaꢀGoto, K.; Hattori, S. Developmental stageꢀdependent
Shimamura, T.; Ukeda, H. Development of LCꢀMS/MS analysis of cyclic dipeptides and its
application to tea extract. Biosci. Biotechnol. Biochem. 2015, 80, 172ꢀ7.
22
ACS Paragon Plus Environment

Page 23 of 33
Journal of Agricultural and Food Chemistry
459
460
461
462
463
464
465
466
467
468
469
470
471
46.
Yamamoto, S.; Hayasaka, F.; Deguchi, K.; Okudera, T.; Furusawa, T.; Sakai, Y.
Absorption and plasma kinetics of collagen tripeptide after peroral or intraperitoneal
administration in rats. Biosci. Biotechnol. Biochem. 2015, 79, 2026ꢀ33.
47.
glycylphenylalanine: comparison with the linear form. J. Pharm. Pharmacol. 1997, 49, 1067ꢀ71.
48. Mizuma, T.; Masubuchi, S.; Awazu, S. Intestinal absorption of stable cyclic dipeptides by
the oligopeptide transporter in rat. J. Pharm. Pharmacol. 1998, 50, 167ꢀ72.
49. Brandsch, M.; Knutter, I.; Thunecke, F.; Hartrodt, B.; Born, I.; Borner, V.; Hirche, F.;
Mizuma, T.; Masubuchi, S.; Awazu, S. Intestinal absorption of stable cyclic
Fischer, G.; Neubert, K. Decisive structural determinants for the interaction of proline
derivatives with the intestinal H+/peptide symporter. Eur. J. Biochem. 1999, 266, 502ꢀ8.
50.
Tateoka, R.; Abe, H.; Miyauchi, S.; Shuto, S.; Matsuda, A.; Kobayashi, M.; Miyazaki,
K.; Kamo, N. Significance of substrate hydrophobicity for recognition by an oligopeptide
transporter (PEPT1). Bioconjug. Chem. 2001, 12, 485ꢀ92.
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Figure captions
Figure 1. Heating experiments of collagenous oligopeptides. Contents of (A) GlyꢀProꢀYꢀtype
tripeptides and (B) XꢀHypꢀGlyꢀtype tripeptides after heating of gingerꢀdegraded collagen
hydrolysate at 85°C and pH 4.8 for 1 or 3 h. (C) Residual ratio of various types of synthetic
collagenous peptides after heating at 85°C and pH 4.8 for 1 h. (D) Concentrations of AlaꢀHypꢀ
Gly and its potential degradation products after heating of AlaꢀHypꢀGly (10 µg/mL) at 85°C and
pH 4.8 for 1 or 3 h. The data represent the mean ± SD of three separate experiments.
Figure 2. Identification of cyclo(AlaꢀHyp) by mass spectrometric analysis. (A) QTOF–MS
spectra of AlaꢀHypꢀGly (control) and heatꢀtreated AlaꢀHypꢀGly (85°C, 3 h). (B) Measured and
simulated QTOF–MS spectra of cyclo(AlaꢀHyp).
Figure 3. Comparison of heatꢀinduced cyclo(AlaꢀHyp) formation beginning with either Alaꢀ
HypꢀGly or AlaꢀHyp. (A) AlaꢀHypꢀGly (10 nmol/mL) and (B) AlaꢀHyp (10 nmol/mL) were
heated at 85°C and pH 4.8 for 1 or 3 h. The data represent the mean ± SD of three separate
experiments.
Figure 4. Effects of amino acid composition, reaction pH, and reaction temperature on
cyclization of XꢀHypꢀGly. (A) Residual ratios of XꢀHypꢀGly and XꢀProꢀGlyꢀtype tripeptides
heated at 85°C and pH 4.8 for 1 h. (B and C) Conversion ratios for AlaꢀHypꢀGly heated at (B)
85°C for 1 h with varying pH (2.4–9.6), and (C) pH 4.8 for 1 h with varying temperature (4–
100°C). The conversion ratio was calculated according to the concentration of generated
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494
495
496
497
498
499
500
501
502
503
504
505
506
507
508
509
cyclo(AlaꢀHyp) relative to the original concentration of AlaꢀHypꢀGly. The data represent the
mean ± SD of three separate experiments.
Figure 5. Absorption of cyclic dipeptides after oral administration of gingerꢀdegraded collagen
hydrolysates. (A) Contents of cyclo(AlaꢀHyp), cyclo(LeuꢀHyp), and cyclo(GlyꢀPro) in gingerꢀ
degraded collagen hydrolysates with (heat) or without (control) heating at 85°C and pH 4.8 for 3
h. The data represent the mean ± SD of three separate measurements. (B) Plasma concentrations
of cyclo(AlaꢀHyp), cyclo(LeuꢀHyp), and cyclo(GlyꢀPro) after oral administration of 10 mg of the
collagen hydrolysates to ICR mice. The data represent the mean ± SD (n = 3).
Figure 6. Plasma concentrations of collagenous oligopeptides and cyclic dipeptides after oral
administration of the peptides. Synthetic peptide mixtures (100 µg each) were administered to
ICR mice, and plasma concentrations of the peptides were measured. Group 1: AlaꢀHyp, Proꢀ
Hyp, HypꢀGly, cyclo(AlaꢀHyp), cyclo(LeuꢀHyp), and cyclo(GlyꢀPro). Group 2: GlyꢀAlaꢀHyp,
GlyꢀProꢀAla, GlyꢀProꢀHyp, AlaꢀHypꢀGly, ProꢀHypꢀGly, and cyclo(AlaꢀHyp). The data represent
the mean ± SD (n = 5). ND, not detected.
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Tables
Table 1. AUC0–6h of orally administered collagenous oligopeptides and cyclic dipeptides
Peptide
AlaꢀHyp
ProꢀHyp
µg/mL·h
0.070 ± 0.008
0.431 ± 0.089
0.069 ± 0.021
2.350 ± 0.524
3.206 ± 1.128
2.672 ± 0.870
0.196 ± 0.036
0.065 ± 0.015
0.184 ± 0.032
2.036 ± 0.157
Group 1
HypꢀGly
Cyclo(AlaꢀHyp)
Cyclo(LeuꢀHyp)
Cyclo(GlyꢀPro)
GlyꢀProꢀHyp
AlaꢀHypꢀGly
ProꢀHypꢀGly
Cyclo(AlaꢀHyp)
Group 2
The data represent the mean ± SD (n = 5).
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Figure Graphics
Figure 1
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Figure 2
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Figure 3
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