Isolation and Identification of an Antiproliferative Compound from Fructose-Tryptophan Maillard Reaction Products

Article abstract of DOI:10.1021/acs.jafc.6b00157

This study was performed to isolate and identify a compound with antiproliferative activity against human stomach cancer cell lines, from fructose-tryptophan Maillard reaction products (MRPs). The MRPs, prepared from a fructose-tryptophan solution heated at 130 °C for 2 h, were fractionated into five solvent fractions: n-hexane, chloroform, ethyl acetate, butanol, and water. The highest antiproliferative activity was found in the chloroform fraction (85.93% at 200 μg/mL), and the active compound from this chloroform fraction was purified by silica gel column chromatography, TLC, and preparative HPLC. The antiproliferative activity (IC₅₀) of the active compound was 42.24 μg/mL, and the active compound was identified as perlolyrine (C₁₆H₁₀N₂O₂) by ¹H/¹³C NMR, DEPT, HMBC, and LC-ESI-MS. Therefore, this research may be useful in developing perlolyrine as a functional therapeutic agent.

Full text of DOI:10.1021/acs.jafc.6b00157

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Article
Isolation and Identification of an Antiproliferative Compound
from Fructose-tryptophan Maillard Reaction Products
Sang Hoon Lee, Su Jeong Jeong, Gwi Yeong Jang, Min Young Kim, In Guk Hwang, Hyun
Young Kim, Koan Sik Woo, Bang Yeon Hwang, Jin Song, Junsoo Lee, and Heon Sang Jeong
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00157 • Publication Date (Web): 03 Apr 2016
Downloaded from http://pubs.acs.org on April 4, 2016
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Isolation and Identification of an Antiproliferative Compound from Fructose-tryptophan
Maillard Reaction Products
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Sang Hoon Lee1, Su Jeong Jeong1, Gwi Yeong Jang1, Min Young Kim1, In Guk Hwang2, Hyun
Young Kim3, Koan Sik Woo3, Bang Yeon Hwang4, Jin Song2, Junsoo Lee1, and Heon Sang
Jeong1+
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1Department of Food Science and Biotechnology, Chungbuk National University, Cheongju 361-
763, Republic of Korea
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2National Academy of Agricultural Sciences, Rural Development Administration,Wanju 865-851,
Republic of Korea
3National Institute of Crop Science, Rural Development Administration, Wanju 865-851, Republic
of Korea
4College of Pharmacy, Chungbuk National University, Cheongju 361-763, Republic of Korea
Running title: Isolation of antiproliferative compound from MRPs
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* Page proof recipient: Heon Sang Jeong
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Department of Food Science and Biotechnology, Chungbuk National University, 1 Chungdaero,
Seowonꢀgu, Cheongju, Chungbuk 361ꢀ763, Republic of Korea
Phone: +82ꢀ43ꢀ261ꢀ2570, Fax: +82ꢀ43ꢀ271ꢀ4412
Eꢀmail: hsjeong@chungbuk.ac.kr
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ABSTRACT
This study performed to isolate and identify a compound with antiproliferative activity
against human stomach cancer cell lines, from fructoseꢀtryptophan Maillard reaction products
(MRPs). The MRPs, prepared from a fructoseꢀtryptophan solution heated at 130 °C for 2 h, were
fractionated into five solvent fractions; nꢀhexane, chloroform, ethyl acetate, butanol, and water.
The highest antiproliferative activity was found in the chloroform fraction (85.93% at 200 ꢁg/mL),
and the active compound from this chloroform fraction was purified by silica gel column
chromatography, TLC, and prep. HPLC. The antiproliferative activity (IC50) of the active
compound was 42.24 ꢁg/mL and the active compound was identified as perlolyrine (C16H10N2O2)
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by Hꢀ/13CꢀNMR, DEPT, HMBC, and LCꢀESIꢀMS. Therefore, this research may be useful in
developing perlolyrine as a functional therapeutic agent.
Keywords: Maillard reaction, fructoseꢀtryptophan MRPs, antiproliferative effect, perlolyrine
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INTRODUCTION
Thermal processing is commonly used to ensure safety and to extend the shelf life of foods
packaged in glass jars by providing commercial sterility.1 Recently, many studies have reported
that thermal processing may increase the biological activity of various foods, due to chemical
changes that occur during the heat treatment.2─6 These changes include the formation of Maillard
reaction products (MRPs), such as melanoidins.7 The observed phenomenon that food turns brown
during thermal processing or storage is the result of this type of reaction.8
The Maillard reaction (MR) is a class of nonꢀenzymatic browning reactions involving the
interaction of reducing sugars and free amino acids, or amino group of an amino acids, peptides
and proteins.9 Many studies of MR kinetics have focused mainly on the consumption of reactants,
the intensity and chromaticity of MRPs, and the conversion of derivatives and intermediate
MRPs.10,11 Recently, many studies have investigated the physiological activities of the MR and
products of the reaction, especially melanoidins, have been found to possess scavenging free
radicals such as hydroxyl radical, superoxide, and ferrous chelation activity.10,12,13 Various sugarꢀ
amino acid MR model systems produce characteristically different MRPs. Antioxidant and antiꢀ
inflammatory effects of sugar (glucose, fructose, and ribose)ꢀamino acid (lysine and glycine)
MRPs were revealed, and 5ꢀhydroxymethylꢀ2ꢀfurfural (HMF) and 5ꢀhydroxymethylꢀ2ꢀfuroic acid
(HMFA) were identified from glucoseꢀlysine MRPs by Kitts et al.14 The cytotoxicity of ethanol
extracts derived from glucoseꢀlysine and fructoseꢀlysine MRPs in the Cacoꢀ2 cells (a human colon
adenocarcinoma cell line) are reported and cytotoxicity depended on incubation time,
concentration, and sugars.15 Hwang et al.16 reported that, of all the model systems comprising 20
amino acids and sugars (fructose and glucose) evaluated, fructoseꢀtryptophan MRPs displayed the
highest anticancer activity. However, studies to isolate and identify the active compounds among
the MRPs derived from sugars and tryptophan have not yet been conducted.
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Cancer is one of the major causes of worldwide death. All cancers share a common
pathology, wherein abnormal cells display uncontrolled growth and may ultimately spread to other
organs and tissues.17,18 Many studies have been carried out on natural sources including the grains,
fruits, vegetables, algaes, and medicinal herbs, in an attempt to identify compounds with potential
anticancer properties19,20, however, little progress has been made towards isolating such agents
from MRPs. The objective of this present study was to evaluate the antiproliferative activities of
fructoseꢀtryptophan MRPs and to isolate and identify the active compound.
MATERIALS AND METHODS
Chemicals and reagents.
DꢀFructose,
Lꢀtryptophan and 3ꢀ(4,5ꢀdimethylthiazolꢀ2yl)ꢀ2,5ꢀ
diphenylꢀ2Hꢀtetrazolium bromide (MTT) were purchased from Sigma Chemical Co. (St. Louis,
MO, USA). RPMI 1640 mediums and fetal bovine serums (FBSs, from New Zealand) were
purchased from Gibco® of IntrogenTM (Seoul, Korea). Perlolyrine was purchased from American
Custom Chemicals Corporation (API0014476, San Diego, CA, USA). HPLCꢀgrade water and
acetonitrile were purchased from J.T. Baker (Phillipsburg, NJ, USA). Reagents were highest
reagent grade quality.
Preparation of Maillard reaction products (MRPs). Tryptophan (81.68 mg) was weighed
accurately in a 10 mL glass tube and then 4 mL of 0.05 M fructose solution was added to obtain a
0.1 M solution of tryptophan, and the tube was tightly capped. The solution was then heated at 130
°C for 2 h in a heatꢀtreatment apparatus (Jisico, Seoul, Korea), as determined to be optimal
condition in previous studies.16,21 After heated, each reaction mixtures were filtered through a 0.45
ꢁm PVDF membrane filter and were freezeꢀdried (Modulyodꢀ115, Thermo Electron Co., Waltham,
MA, USA). The antiproliferative activity of each sample was determined, and the samples were
used as a source to isolate the active compounds.
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Cell culture and anti-proliferative activity. Human gastric cancer cells (AGS, CRLꢀ1739, P/N
125), lung cancer cells (Aꢀ549, CCLꢀ185, P/N 117), colon cancer cells (HCTꢀ116, CCLꢀ247, P/N
181; SWꢀ620, CCLꢀ227, P/N 124), liver cancer cells (HepG2, HBꢀ8065, P/N 157), and mammary
gland cancer cells (MCFꢀ7, HTBꢀ22, P/N 174) were obtained from the American Type Culture
Collection (ATCC, Cryosite, Lane Cove NSW, Australia). Cells were grown in RPMI 1640 with
10% FBS, 100 U/mL penicillin, and 100 ꢁg/mL streptomycin at 37 °C in 5% CO2 humidified air.
MRPs extract and several fractions (100 ꢁL) were added to 96ꢀwell plates and the cancer cells
were added at a density of 104 cells per 100 ꢁL medium. After incubation for 24 h, the medium
was removed and the cells were incubated with MTT solution (15 ꢁL) at 37 °C for 4 h. The
supernatant was carefully removed, and 100 ꢁL of dimethyl sulfoxide was added to well. The
absorbance was measured at 540 nm by the microplate reader (Infinite® F50 Robotic, Tecan
Trading AG, Zürich, Switzerland). The antiproliferative effects of MRPs evaluated by the
percentage cell viability.
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Purification and identification of antiproliferative substance. Fructoseꢀtryptophan MRPs
(102.12 g) were sequentially partitioned in a separatory funnel using solvents with different
polarity such as nꢀhexane, chloroform, ethyl acetate, waterꢀsaturated nꢀbutanol. All fractions were
concentrated using a rotary vacuum evaporator (Nꢀ100, Eyela, Tokyo, Japan) and dried under a
stream of nitrogen gas at 40 °C. The chloroform fraction (48.90 g) was injected to the openꢀ
column (Pyrex Glass Column, 500 × 35 mm) filled with silica gel (Kiesel gel 60, 70−230 mesh;
Merck, Darmstadt, Germany), and eluted using a mixture of dichloromethane (DCM) and
methanol (MeOH) with different ratio (DCM : MeOH = 40 : 1, 20 : 1, 10 : 1, 5 : 1, 0 : 1, v/v, 500
mL). Eight fractions were collected and dried using the vacuum evaporator and nitrogen gas, and
evaluated the antiproliferative activity. The active fraction S2 (1.71 g) was loaded onto a silica gel
60 F254 glass plates (0.25 mm thickeness, 20 × 20 cm; Merck), and then developed with DCM :
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MeOH = 20 : 1 mixture. The plate was sprayed with 20% sulfuric acid solution (containing in 10%
vanillin/ethanol) for analyze the spot pattern. When the various spots were exposed on the plate,
they were divided into 9 each parts (T1ꢀT9), and then scraped off with a spatula. Methanol (50 mL)
was added to the separated silica gel samples. To extract the material, it was removed from the
silica gel powder by centrifugation and filtration through a 0.2ꢀꢁm PVDF membrane filter, and
then the solvent was evaporated. The active fraction T8 (0.46 g) was further purified by semiꢀ
preparative RPꢀHPLC system (Acme 9000, Younglin Co., Anyang, Korea). The column was used
the Discovery® C18 column (250 × 10 mm, 5 ꢁm, Supelco, Bellefonte, PA, USA) and mobile
phase was 60% acetonitrile (flow rate: 3.0 mL/min) and detector was UV/visible detector at 254
nm. The structure of the active compound P2 (0.042 g, 0.04% of FꢀT MRP) was identified using
analytical HPLC, LCꢀMS and NMR. HPLC chromatogram and UV spectrum were analyzed using
an HPLCꢀPDA system (Acme 9000, Younglin). The column was used Mightysil® RPꢀ18 GP
column (250 × 4.6 mm, 5 ꢁm, Kanto chemical Co., Tokyo, Japan) at a flow rate of 0.6 mL/min and
detection at 200−400 nm using photodiode array detector (PDA). The molecular weight of active
compound measured using the LCQ Fleet LCꢀMS (Thermo Scientific, San Jose, CA, USA)
equipped with electrospray ion source (ESI). Briefly, active compound (1 mg/mL) was subjected
into the ESI and eluted at a flow rate of 0.25 mL/min and a mass scan range was from 200 m/z to
1,000 m/z. MS conditions in positive mode were a spray voltage of 5.0 kV, a capillary temperature
of 275 °C, a capillary voltage of 35 V, a tube lens offset of 120 V, a multipole 0 offset of −4.89 V,
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a multipole 1 offset of −12.5 V, a sheath gas flow (N2) and 10 A.U. The H nuclear magnetic
resonance (NMR, 900 MHz), 13C nuclear magnetic resonance (NMR, 125 MHz), Distortionless
Enhancement by Polarization transfer (DEPT) and Heteronuclear Multiple Bond Correlation
(HMBC) were recorded on a spectrometer (Avance 500; Bruker, Madison, WI, USA) using
CD3OD as a solvent.
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Statistical analysis. The results were presented as the mean ± standard deviation. Significance
of differences was analyzed using oneꢀway analysis of variance (ANOVA) and independent tꢀtest
followed by Duncan’s and Student’s test, respectively, at a level of P<0.05. All analysis was
performed using SPSS version 12.0 for Windows (SPSS Institute, Chicago, IL, USA).
RESULTS AND DISCUSSION
Antiproliferative effects of fructose-tryptophan MRPs. The antiproliferative activities of
fructoseꢀtryptophan MRPs were evaluated on various human cancer cell lines, including human
gastric cancer cell line (AGS), lung cancer (Aꢀ549), colon cancer (HCTꢀ116, SWꢀ620), liver
cancer (HepG2) and mammary cancer (MCFꢀ7). Cells were treated with different concentrations of
fructoseꢀtryptophan MRPs (200ꢀ800 ꢁg/mL) for 24 h. The IC50 values ranged from 525 to 1,650
ꢁg/mL (Figure 1). The AGS cells showed the highest susceptibility (IC50 = 525 ꢁg/mL), followed
by HCTꢀ116 cells (IC50 = 752 ꢁg/mL) and SWꢀ620 cells (IC50 = 753 ꢁg/mL). Many studies have
reported the beneficial effects of MRPs, including their antioxidant properties10,12,13, however, very
little research has been investigated on their anticancer activity. Jing and Kitts14 demonstrated that
the cytotoxicity in Cacoꢀ2 cell was seen the ethanolic extracts derived from glucoseꢀlysine and
fructoseꢀlysine MRPs and high molecular weight polymer of MRP possesses higher toxicity in
comparison with low molecular weight MRP. Wang et al.22 reported that MRPs changes in cell
morphology and inhibit RNA synthesis, however no inhibit DNA and protein synthesis. Hwang et
al.2 reported that MRPs derived from fructoseꢀtyrosine and fructoseꢀtryptophan showed higher
antiproliferative activity than other nonꢀpolar amino acid MRPs and described the isolation of 2,4ꢀ
bis(pꢀhydroxyphenyl)ꢀ2ꢀbutenal, which was identified to have antiproliferative activity on HCTꢀ
116 human colon cancer cells, from fructoseꢀtyrosine MRPs.
Purification and identification of an antiproliferative substance. Since the fructoseꢀ
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tryptophan MRPs showed a strong antiproliferative effect on the AGS human gastric cancer cell
lines (Figure 1), we attempted to isolate an antiproliferative compound from fructoseꢀtryptophan
MRPs heated 130 °C for 2 h. The MRPs were purified using a series of solvent partitions; nꢀ
hexane, chloroform, ethyl acetate, water saturated nꢀbutanol and water. The highest
antiproliferative effects were observed in chloroform fraction at concentrations ranging from 25–
200 ꢁg/mL, followed by the ethyl acetate, nꢀhexane, water and water saturated nꢀbutanol fraction
(Figgure 2). At a concentration of 200 ꢁg/mL, the chloroform fraction displayed a cytotoxicity of
85.93%. The chloroform fraction was subjected to silica gel column chromatography and the
separated products were eluted with increasing concentrations of MeOH in DCM. Eight subꢀ
fractions were collected and investigated for antiproliferative effects. The highest antiproliferative
activity was observed in the S2 fraction (Figure 3). This fraction displayed a cytotoxicity of 84.87%
at 100 ꢁg/mL and 91.54% at 200 ꢁg/mL. Each fraction was loaded onto a preparative TLC plate,
which was then developed with DCM:MeOH (20:1) solvent. Nine subꢀfractions were obtained,
and the antiproliferative activity of each fraction was measured at different concentrations (20–80
ꢁg/mL). The T8 fraction displayed the highest antiproliferative activity with 87.37% cytotoxicity
at 80 ꢁg/mL (Figure 4). The T8 fraction was further separated to 3 subꢀfractions using preparative
HPLC, and the antiproliferative activity of each fraction was evaluated. The P2 fraction showed
the highest antiproliferative effect (IC50 = 42.24 ꢁg/mL, Figure 5). The preparative HPLC
chromatogram of the T8 fraction is shown in Figure 6(a). The analytical HPLC chromatogram and
UV spectrum of the pure active compound (P2 fraction) isolated by preparative HPLC are shown
in Figure 6(b) and (c). The isolated and purified antiproliferative compound was analyzed by UV,
LCꢀMS, 1Hꢀ and 13CꢀNMR, DEPT, and HMBC. The chemical structure of the compound is shown
in Figure 6(d). The isolated compound was identified as perlolyrine (C16H10N2O2): UV max
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(MeOH) nm: 239, 293, 369 and 382; FABꢀMS: m/z 263 [M–H]–; H NMR (900 MHz, CD3OD):
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8.30 (1H, d, J=5.4), 8.03 (1H, d, J=5.4), 8.20 (1H, d, J=8.1), 7.30 (2H, br t, J=7.2), 7.59 (2H, br t,
J=7.2), 7.72 (1H, d, J=8.1), 7.23 (1H, d, J=3.2), 6.60 (1H, d, J=3.2), 4.77 (s); 13C NMR (125 MHz,
CD3OD): 143.16 (Cꢀ1), 138.82 (Cꢀ3), 134.58 (Cꢀ13), 132.70 (Cꢀ10), 132.26 (Cꢀ12), 130.08 (Cꢀ6),
122.68 (Cꢀ5), 122.28 (Cꢀ11), 115.07 (Cꢀ4), 111.06 (Cꢀ8). There are no significant difference of
antiproliferative activity between P2 fraction and perlolyrine with various concentrations (P>0.05,
Figure 7). The purities of perlolyrine isolated in our own laboratory and purchased from ACCC
were similar to 94.8% and 96.0%, respectively. The strongly fluorescent βꢀcarboline alkaloid
perlolyrine, also known as "yellow substance", was first isolated from Lolium perenne L.23, and
was later found to occur in several other plants such as Panax gingseng24, Tribulus terrestris L.25,
Cyclocarya paliurus26, and Ligusticum wallichii27, as well as in soy sauce and Japanese sake
(Gassner, Brossi, Bembenek, & Abell, 1988).16,28 Several studies concerning the physiological
effect of perlolyrine have been reported. Knoch and Baltes29 described the formation of perlolyrine
after roasting tryptophan with sugars. Tryptamine, formed by the thermolytic decarboxylation of
tryptophan and 5ꢀhydroxymethylꢀ2ꢀfuraldehyde (5ꢀHMF), is one of the intermediate products of
the Maillard reaction, and it forms tetrahydroperlolyrine that is then oxidized to yield perlolyrine.
In previous studies, MRPs derived from sugars and amino acids, and from fructoseꢀtyrosine and
fructoseꢀtryptophan in particular, were shown to have strong antiproliferative effects on human
colon cancer cells. Hwang et al.30 identified the 2,4ꢀbis(pꢀhydroxyphenyl)ꢀ2ꢀbutenal from fructoseꢀ
tyrosine MRPs as being bioactive. In this study, we isolated perlolyrine from fructoseꢀtryptophan
MRPs, which was identified as a compound with antiproliferative activity against tumor cells. Liu
et al.27 demonstrated a protective effect of perlolyrine on injured vascular endothelial cells, and
Tang et al.31 reported an antithrombotic effect of perlolyrine and its analogues on platelet
aggregation. Recently, Li et al.26 reported that isolates prepared from soy sauce could induce
quinone reductase (a biomarker for phase II enzyme) in murine hepatoma cells. However, the
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antiproliferative activity of perlolyrine isolated from fructoseꢀtryptophan MRPs has not yet been
reported. Therefore, this research will be important in the development of perlolyrine as a potential
therapeutic agent.
AUTHOR INFORMATION
Corresponding Author
Professor Dr. Heon Sang Jeong, Eꢀmail: hsjeong@chungbuk.ac.kr, Phone: +82 43 261 2570, Fax:
+82 43 271 4412. Department of Food Science and Biotechnology, Chungbuk National University,
1 Chungdaero, Seowonꢀgu, Cheongju, Chungbuk 361ꢀ763, Republic of Korea
Notes
The authors declare no competing financial interest.
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Figure 1. Antiproliferative effects of fructoseꢀtryptophan Maillard reaction products on various
cancer cell lines. AGS: human gastric cancer cell line, Aꢀ549: human lung cancer cell line,
HCTꢀ116 and SWꢀ620: human colon cancer cell line, HepG2: human liver cancer cell line,
MCFꢀ7: human mammary gland cancer cell line. Different capital and small letters on the
error bars indicate a significant difference (P<0.05) among different cancer cell lines and
concentrations, respectively.
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Figure 2. Antiproliferative effects of solvent fractions of fructoseꢀtryptophan Maillard reaction
products on human gastric cancer cell line (AGS). Different capital and small letters on
the error bars indicate a significant difference (P<0.05) among different solvent fractions
and concentrations, respectively.
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Figure 3. Antiproliferative effects of silica gel subꢀfractions from chloroform fraction on human
gastric cancer cell line (AGS). Different capital and small letters on the error bars indicate
a significant difference (P<0.05) among different subꢀfractions of chloroform and
concentrations, respectively.
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Figure 4. Antiproliferative effects of preparative TLC fractions from S2 fraction on human gastric
cancer cell line (AGS). Different capital and small letters on the error bars indicate a
significant difference (P<0.05) among different subꢀfractions of S2 fraction and
concentrations, respectively.
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Figure 5. Antiproliferative effects of preparative HPLC subꢀfractions from T8 fraction on human
gastric cancer cell line (AGS). Different capital and small letters on the error bars indicate
a significant difference (P<0.05) among different subꢀfractions of T8 fraction and
concentrations, respectively.
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Figure 6. Preparative HPLC chromatogram (a), analytical HPLC chromatogram (b), UV spectrum
(c) of P2 fraction from T8 fraction and structure of antiproliferative compound isolated
from fructoseꢀtryptophan Maillard reaction products (d).
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Figure 7. Antiproliferative effects of P2 fraction and perlolyrine on human gastric cancer cell line
(AGS). The ns means no significant difference (P>0.05) between P2 fraction and
perlolyrine by Student’s tꢀtest.
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TOC graphic
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