Rambut Jagung 2

Subscriber access provided by Fudan University
Article
Corn Silk Extract and Its Bioactive Peptide Ameliorated Lipopolysaccharide-
Induced Inflammation in Mice via Nuclear Factor-#B Signaling Pathway
Tin-Yun Ho, Chia-Cheng Li, Hsin-Yi Lo, Feng-Yuan Chen, and Chien-Yun Hsiang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03327 • Publication Date (Web): 08 Jan 2017
Downloaded from http://pubs.acs.org on January 11, 2017
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical

Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.
Page 1 of 47 Journal of Agricultural and Food Chemistry
1 Corn Silk Extract and Its Bioactive Peptide Ameliorated
2 Lipopolysaccharide-Induced Inflammation in Mice via Nuclear Factor-κB
3 Signaling Pathway
5 Tin-Yun Ho,†,‡,a, Chia-Cheng Li,†,a, Hsin-Yi Lo,†, Feng-Yuan Chen,† and Chien-Yun
6 Hsiang*,§
†
7 Graduate Institute of Chinese Medicine, China Medical University, Taichung 40402,
8 Taiwan
‡
9 Department of Health and Nutrition Biotechnology, Asia University, Taichung,
10 Taiwan
§
11 Department of Microbiology, China Medical University, Taichung, Taiwan
12
*
13 Corresponding author. Department of Microbiology, China Medical University, 91
14 Hsueh-Shih Road, Taichung 40402, Taiwan. Tel.: +886 4 22053366 x 2163. Fax:
15 +886 4 22053764. E-mail address: cyhsiang@mail.cmu.edu.tw
16
a
17 These authors contributed equally to this work.
18
19 Short title: Anti-inflammatory effects of corn silk and its bioactive peptide
20 1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry Page 2 of 47
21 ABSTRACT
22 Bioactive peptides derived from foods have shown beneficial anti-inflammatory
23 potential. Inhibitory κB kinase-β (IKKβ) plays a crucial role on the activation of
24 nuclear factor-κB (NF-κB), a transcription factor involved in inflammation. Here we
25 applied proteomic and bioinformatics approaches to identify anti-inflammatory
26 peptides that target IKKβ from corn silk. Corn silk extract significantly suppressed
27 lipopolysaccharide (LPS)-induced NF-κB activities (1.7±0.2 fold vs. 3.0±0.6 fold,
28 p<0.05) in cells. Trypsin hydrolysate of corn silk also suppressed LPS-induced
29 NF-κB activities (1.1±0.3 fold vs. 3.3±0.5 fold, p<0.01). In addition, both corn silk
30 extract and trypsin hydrolysate significantly inhibited the LPS-induced
31 interleukin-1β (IL-1β) production by 58.3±4.5% and 55.1±7.4%, respectively. A
32 novel peptide FK2, docked into the ATP-binding pocket of IKKβ, was further
33 identified from trypsin hydrolysis of corn silk. FK2 inhibited IKKβ activities, IκB
34 phosphorylation, and subsequent NF-κB activation (2.3±0.4 fold vs. 5.5±0.4 fold,
35 p<0.001). Moreover, FK2 significantly reduced NF-κB-driven luminescent signals
36 in organs by 5 to 11 fold, and suppressed LPS-induced NF-κB activities and IL-β
37 productions in tissues. In conclusion, our findings indicated that corn silk displayed
38 anti-inflammatory abilities. In addition, we first identified an anti-inflammatory
39 peptide FK2 from corn silk. Moreover, the anti-inflammatory effect of FK2 might be

40 through IKKβ-NF-κB signaling pathways.
41
42 KEYWORDS: corn silk, peptide, inflammation, nuclear factor-κB, inhibitory κB
43 kinase-β
44

45 INTRODUCTION
46 Inflammation is a protective response of tissues to numerous harmful stimuli, such
47 as pathogens, injury, or irritants. Upon stimulation, inhibitory κB kinase (IKK) is
48 activated and enhances the nuclear translocation of nuclear factor-κB (NF-κB),
49 leading to the expression of proinflammatory cytokines, such as interleukin-1β
50 (IL-1β) and tumor necrosis factor-α (TNF-α). While proinflammatory cytokines are
51 released from cells, they attract the infiltration of immune cells and further result in
52 the inflammatory process.1 Despite its protective response, inflammation is also
53 strongly associated with many pathological conditions, including atherosclerosis,
54 autoimmune diseases, cancers, cardiovascular diseases, diabetes, obesity, and
55 neurological diseases.2 Dexamethasone and non-steroidal anti-inflammatory drugs,
56 such as aspirin or ibuprofen, are commonly used for the amelioration of
57 inflammation. However, long-term use of these drugs can cause several adverse
58 effects, including gastric erosions or kidney damage.3 Therefore, discovery of better
59 anti-inflammatory agents from natural products has been gaining a significant
60 importance now.
61 Protein or peptide drugs have been the most rapidly growing segments of the
62 prescription drug market in the last few years because of higher potency, higher
63 specificity, lower toxicity, less accumulation in organs, and fewer drug-drug

64 interactions.4 Bioactive peptides derived from various foods have shown some
65 beneficial anti-inflammatory potential.5,6 For example, tripeptides VPP and IPP
66 derived from bacterial fermentation of casein and VPY derived from enzymatic
67 hydrolysis of soy protein decrease the production of cytokines and improve the
68 inflammatory bowel disease in mice.7,8 VPP and IPP also prevent the atherosclerotic
69 changes through reducing the expression of pro-inflammatory genes in
70 apolipoprotein E-knockout atherosclerosis model.9 Lunasin, a 43-amino-acid-residue
71 peptide from enzymatic hydrolysis of soy protein, ameliorates murine
72 ovalbumin-induced asthma by reducing the cytokine expression and inflammatory
73 infiltration.10,11 Tripeptide IRW derived from thermolysin and pepsin hydrolysis of
74 egg white protein ovotransferrin reduces TNF-α-induced expression of adhesion
75 molecules in aorta and mesenteric arteries in spontaneously hypertensive rats.12,13
76 Tripeptide PAY in salmon pectoral fin byproduct pepsin hydrolysate and bioactive
77 peptides RQRK and VIK in pepsin-pancreatin hydrolysates from commercially
78 available soy products suppress the protein expression of inducible nitric oxide
79 synthase (iNOS) and cyclooxygenase-2 (COX-2) and attenuate the production of
80 pro-inflammatory cytokines in lipopolysaccharide (LPS)-treated murine
81 macrophages.14,15 These results indicate that bioactive peptide from food is a
82 promising approach for anti-inflammatory drug discovery.

83 Corn or maize (Zea mays L.) is one of the most important cereal crops in the
84 world. In 2016, the global production of corn is about 830 million metric tons.16
85 Corn silk is the yellowish thread from the stigmas of corn fruits. Although corn silk
86 is a waste material from corn cultivation, it has been used for the treatment of
87 various illnesses in traditional medicine in many countries. For example, corn silk
88 has been applied for the treatment of metabolic disorders, such as diabetes, obesity,
89 edema, hyperlipidemia, and hypertension. It displays antioxidant, anti-fatigue, and
90 anti-depressant activities. It has been used for the treatment of inflammatory
91 diseases, such as cystitis, urinary infections, nephritis, as well as prostatitis.17
92 Pretreatment with corn silk extract reduces carrageenin-induced pleurisy exudate,
93 number of leukocytes, oxidative stress, and O2- levels at the inflammatory site, also
94 suggesting that corn silk extract may be a potent treatment for inflammatory
95 diseases.18 Corn silk consists of various constituents, including proteins, flavonoids,
96 polysaccharides, saponins, alkaloids, and steroids.19,20 Anti-inflammatory activities
97 of flavonoids, polysaccharide, and essential coil in corn silk have been reported
98 previously. However, the presence of bioactive peptides with anti-inflammatory
99 potentials in corn silk is still unclear.
100 To address this question, we applied proteomic and bioinformatics approaches to
101 identify anti-inflammatory peptides from protein-rich extract of corn silk. The

102 anti-inflammatory activities and mechanisms of peptides were further evaluated by
103 luciferase assay, Western blot analysis, ex vivo NF-κB-driven bioluminescent
104 imaging, and immunohistochemical staining (IHC). IKKβ plays a dominant role in
105 the proinflammatory signal-induced phosphorylation of inhibitory κB (IκB) and is
106 also the target of anti-inflammatory drugs, such as aspirin and salicylate.21,22
107 Therefore, we applied IKKβ as a target to identify the peptides with
108 anti-inflammatory potentials in corn silk. Our data showed that a novel bioactive
109 peptide FK2, a 9-amino-acid-residue peptide derived from trypsin hydrolysis of corn
110 silk extract, ameliorated LPS-induced inflammation in mice. Moreover, the
111 anti-inflammatory effect of FK2 might be through IKKβ-NF-κB signaling pathways.
112
113
114 MATERIALS AND METHODS
115 Chemicals. All chemicals, except indicated, were purchased from Sigma-Aldrich
116 (St. Louis, MO). Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine
117 serum (FBS) were obtained from Life Technologies (Carlsbad, CA). D-Luciferin was
118 purchased from Xenogen (Hopkinton, MA). OptEIATM Set for mouse IL-1β was
119 purchased from BD Pharmingen (San Diego, CA). TPCA-1 was purchased from
120 abcam (Cambridge, MA). IKKβ kinase was purchased from Promega (Madison, WI).

121 Rabbit polyclonal antibodies against IκB-α, phosphorylated IκB-α, and β-actin, and
122 peroxidase-conjugated anti-rabbit polyclonal antibody were purchase from Cell
123 Signaling (Beverly, MA). Mouse monoclonal antibody against p65 was purchased
124 from Chemicon (Temecula, CA). Rabbit polyclonal antibody against IL-1β was
125 purchased from Santa Cruz (Dallas, TX).
126
127 Preparation of Aqueous Extract and Trypsin Hydrolysate of Corn Silk. Fresh
128 corn silk (Stigma Maydis) was obtained from local market in Taichung, Taiwan. The
129 aqueous extract of corn silk was prepared by mixing 10 g of fresh corn silk with 30
130 ml of distilled water at 40C with shaking. Twenty-four hours later, the supernatant
131 was collected and digested by trypsin according to Trypsin Gold manual (Promega,
132 Madison, WI). Corn silk extract and trypsin hydrolysate were stored at -200C for
133 further analysis.
134
135 Preparation of FK2 Peptide. FK2 peptide was synthesized by PeptideSynTM
136 technology (LifeTein, Somerset, NJ). The purity and amino acid sequences of FK2
137 were verified by high-performance liquid chromatography and mass spectrometry
138 (MS), respectively. The purity of FK2 used in this study was ≥95%.
139

140 Luciferase Assay and Cell Viability Assay. Recombinant HepG2/NF-κB cells,
141 which carried the luciferase genes driven by NF-κB, were cultured in DMEM
142 supplemented with 10% FBS and incubated at 370C with 5% CO2.23 Cells (2×105
143 cells/well) were seeded in a 96-well plate and incubated at 370C for 24 h. LPS (from
144 Escherichia coli O55:B5; 100 ng/ml) and various amounts of corn silk extract or
145 peptide were then added to cells and incubated at 370C for another 24 h. Luciferase
146 assay was performed as described previously.23 Relative NF-κB activity was
147 calculated by dividing the relative luciferase units (RLUs) of extract- or
148 peptide-treated cells by the RLUs of solvent-treated cells. Cell viability was judged
149 by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide colorimetric
150 assay.
151
152 Animal Experiments. Mouse experiments were conducted under ethics approval
153 from China Medical University Animal Care and Use Committee (Permit No.
154 2016-034). Female BALB/c mice (4-5-week-old) were obtained from National
155 Laboratory Animal Center (Taipei, Taiwan). Transgenic mice, which carried
156 NF-κB-driven luciferase genes, were constructed previously.24-26 Mice were
157 maintained under a 12 h day - 12 h night cycle and had free access to water and
158 food.

159 BALB/c mice were challenged intraperitoneally with 1 mg/kg LPS and then
160 orally given with various amounts of corn silk extract or peptide. Four hours later,
161 mice were sacrificed, and sera and organs were collected. The amount of IL-1β in
162 serum was quantified according to OptEIATM Set.
163
164 2-Dimensional Electrophoresis (2-DE) and Liquid Chromatography Coupled
165 with Tandem MS (LC-MS/MS) Analysis. The composition of corn silk extract was
166 analyzed by sequential wavelength scanning with a spectrophotometer (Beckman
167 Coulter, Fullerton, CA). 2-DE was performed as described previously,27,28 Briefly,
168 corn silk extract was precipitated by mixing with 10% trichloroacetic acid in
169 acetone/20 mM dithiothreitol and then resuspended in rehydration buffer (9.8 M
170 urea, 4% CHAPS, 40 mM dithiothreitol, 0.2% biolytes). Protein samples (200 µg),
171 quantified according to the Bradford method, were applied to IPG strips (7 cm, pH
172 3-10). Isoelectric focusing was performed using a Protean IEF Cell (Bio-Rad,
173 Hercules, CA). Focused IPG strips were then separated by sodium dodecyl
174 sulfate-polyacrylamide gel (SDS-PAGE) and protein spots on the gels were then
175 visualized by Coomassie Brilliant Blue R-250. For LC-MS/MS analysis, protein
176 spots were excised from stained gels, digested by trypsin, and identified using an
177 Ultimate capillary LC system (LC Packings, Amsterdam, The Netherlands) coupled
10

178 to a QSTARXL quadrupole-time-of-flight mass spectrometer (Applied
179 Biosystem/MDS Sciex, Foster City, CA). MS/MS data were matched against green
180 plants (Viridiplantae) taxonomy using the MASCOT search program
181 (http://www.matrixscience.com). Probable protein candidates were determined by
182 the average theoretical molecular weight, isoelectric point (pI), and sequence
183 coverage.
184
185 Docking Calculations. Docking analysis was performed by Swiss-Dock software
186 (http://www.swissdock.ch/).29 The crystal structure of human IKKβ asymmetric
187 dimer (PDB ID: 4KIK) was obtained from protein data bank (http://www.rcsb.org/).
188 Chain A of IKKβ was chosen as a target using the target selection tab in
189 SWISS-Dock. The structures of peptides were generated using MarvinSketch
190 (https://www.chemaxon.com) and saved as a mol2 format. We added hydrogen
191 atoms using Structure/Add/Explicit Hydrogens function in MarvinSketch and further
192 make sure that peptides had correct topology using Structure/Clean 3D/Cleaning
193 Method/Fine with Hydrogenize function in MarvinSketch. The mol2 files were
194 uploaded using the ligand selection tab in SWISS-Dock. The clusters of binding
195 modes and binding affinity were evaluated by the simple fitness and full fitness
196 score. Simple fitness ignores the solvent effect, while full fitness score accounts for
11

197 the solvation free energy. All docking structure figures were prepared by UCSF
198 Chimera (https://www.cgl.ucsf.edu/chimera/).
199
200 IKKβ Activity Assay. IKKβ activity was measured using ADP-Glo™ kinase
201 assay (Promega, Madison, WI). Briefly, a mixture containing 20 ng IKKβ, 1 µg
202 IKKtid peptide, 50 µM ATP, and various amounts of TPCA-1, corn silk extract, or
203 FK2 was incubated at room temperature for 1 h. ADP was then detected using
204 ADP-Glo™ reagent and measured by a luminometer. Relative IKKβ activity was
205 calculated by dividing the RLUs of reaction containing compounds by the RLUs of
206 reaction containing solvent.
207
208 Western Blot Analysis. Recombinant HepG2/NF-κB cells were cultured in
209 25-cm2 flasks, pretreated with various amounts of corn silk or FK2 for 30 min, and
210 then treated with 100 ng/ml LPS. Thirty minutes later, cells were collected using a
211 cell scraper and lyzed with RIPA buffer (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.5%
212 sodium deoxycholate, 0.1% SDS, 150 mM sodium chloride, 2 mM EDTA, 50 mM
213 sodium fluoride). Protein samples (50 µg) were separated by 10% SDS-PAGE and
214 transferred electrophoretically to nitrocellulose membranes. Membranes were then
215 probed with anti-IκB-α, anti-phosphorylated IκB-α, or anti-β-actin antibody. The
12

216 bound antibody was detected with peroxidase-conjugated anti-rabbit antibody
217 followed by chemiluminescence (Phototope®-HRP Western detection kit, New
218 England Biolabs, Ipswich, MA) and exposed by autoradiography. Bands on the
219 X-ray films were measured using Gel-Pro® Analyzer (Media Cybernetics, Inc.,
220 Silver Spring, MD).
221
222 Ex vivo Bioluminescent Imaging. Female transgenic mice (5-6-week-old) were
223 challenged intraperitoneally with 1 mg/kg LPS and then orally given with 1 mg/kg
224 FK2 peptide. Four hours later, mice were injected intraperitoneally with 150 mg/kg
225 D-luciferin and sacrificed 5 min later. The organs were then removed and imaged for
226 1 min by IVIS Imaging System (Xenogen, Hopkinton, MA).24-26 The intensity of
227 signal was quantified as the sum of all detected photon counts per second in the
228 region of interest.
229
230 IHC. Parafilm-embedded small intestines were cut into 5-µm-thick sections and
231 incubated with anti-p65 and anti-IL-1β antibodies (1:200 dilution) overnight at 40C.
232 Sections were then incubated with biotinylated secondary antibody (Zymed
233 Laboratories, Carlsbad, CA) at room temperature, incubated with avidin-biotin
234 complex reagent, and stained with 3,3'-diaminobenzidine (Histostain-Plus, Zymed
13

235 Laboratories, Carlsbad, CA). IL-1β-positive area was measured using ImageJ
236 (Media Cybernetics, Bethesda, MD). The expression of IL-1β was calculated as
237 (area occupied with brown color/area of whole tissue) × 100. The number of
238 p65-positive cells was counted by counting 100 cells in each view. The proportion of
239 p65-positive cell (%) was calculated as (the number of brown nuclei/the total
240 number of nuclei) × 100.
241
242 Statistical Analysis. Data were presented as mean ± standard error. Data were
243 analyzed by one-way ANOVA and post hoc Bonferroni test using SPSS Statistics
244 version 20 (IBM, Armonk, NY). A p-value of less than 0.05 was considered as
245 statistically significant.
246
247
248 RESULTS
249 Corn Silk Extract Suppressed LPS-Induced NF-κB Activities In Vitro and
250 Reduced LPS-Induced IL-1β Production In Vivo. To analyze the
251 anti-inflammatory effects of corn silk extract, we first treated cells with LPS and
252 various amounts of corn silk extract, and monitored NF-κB activities by luciferase
253 assay. Recombinant HepG2/NF-κB cells were used to report the LPS-induced
14

254 NF-κB activities in this study. Hepatocytes are responsive to LPS stimulation
255 because of the presence of toll-like receptor 4 on hepatocytes.30 Therefore, we first
256 evaluated the anti-inflammatory activities of corn silk in cells and then analyzed the
257 anti-inflammatory effects in mice with LPS-induced systemic inflammation. As
258 shown in Figure 1A, LPS stimulated the NF-κB activity by approximately 3-fold,
259 compared with mock. However, corn silk extract significantly inhibited
260 LPS-induced NF-κB activities (1.7±0.2 fold vs. 3.0±0.6 fold, p<0.05) in a
261 dose-dependent manner. Trypsin hydrolysate of corn silk also suppressed
262 LPS-induced NF-κB activities (1.1±0.3 fold vs. 3.3±0.5 fold, p<0.01). And the
263 suppression displayed a dose-dependent manner. No visible cytotoxic effect was
264 observed during treatment. Then, we challenged mice with LPS and treated mice
265 with various amounts of corn silk extract. The amount of pro-inflammatory cytokine
266 IL-β in sera was quantified. As shown in Figure 1B, LPS stimulated the production
267 of IL-β in mice, while both corn silk extract and trypsin hydrolysate at 1 g/kg
268 significantly inhibited the LPS-induced IL-1β production by 58.3±4.5% and
269 55.1±7.4%, respectively. Moreover, the inhibitory effects of corn silk extract
270 displayed a dose-dependent manner. These data suggested that corn silk extract and
271 trypsin hydrolysate of corn silk inhibited LPS-induced NF-κB activities in vitro and
272 ameliorated LPS-induced acute inflammation in mice via the suppression of IL-β
15

273 production.
274
275 Identification of Protein Constituents in Corn Silk Extract. To analyze the
276 chemical composition of corn silk extract, we scanned corn silk extract with
277 sequential wavelength ranging from 250 to 800 nm. As shown in Figure S1, a
278 maximal absorbance was observed at 280 nm, suggesting that proteins might be the
279 major constituents in corn silk extract. In addition, the recovery of corn silk extract
280 was 21.65 mg dry weight of extract/10 g fresh corn silk and the amount of protein in
281 corn silk extract was 16.22 mg/10 g fresh corn silk, also suggesting that the corn silk
282 extract was a protein-rich extract.
283 We further analyzed the protein constituents of corn silk extract by 2-DE and
284 LC-MS/MS. 2-DE analysis showed that there were seven significant spots and some
285 small smears in the gel (Figure 2A). The protein spots were then excised from the
286 gel, digested by trypsin, and identified by LC-MS/MS. The identified protein spots
287 of corn silk extract are summarized in Table 1. Fructokinase-1 accounted for 6.6% of
288 corn silk extract, followed by beta-1,3-glucanase precursor (5.2%), chitinase (5.1%),
289 glycine-rich RNA-binding protein 2 (2.8%), putative beta 4 proteasome subunit
290 (1.5%), chitinase chem5 precursor (1.3%), and trypsin inhibitor precursor (1.3%).
291
16

292 Identification Bioactive Peptides with Anti-Inflammatory Potentials in Corn
293 Silk Extract. NF-κB plays a crucial role in the regulation of inflammation, while the
294 upstream IKK is essential for NF-κB activation.1 Corn silk extract was able to
295 inhibit LPS-induced NF-κB activities in cells in this study. We wondered whether
296 there were bioactive peptides interacting with NF-κB signaling transduction pathway.
297 Docking analysis was therefore performed to evaluate the interaction between corn
298 silk peptides and IKKβ. IKKβ plays a crucial role on the activation of NF-κB, a
299 nuclear transcription factor involved in inflammation. In addition, anti-inflammatory
300 properties of aspirin and salicylate are mediated in part by their specific inhibition of
301 IKKβ. Therefore, IKKβ seems to be a rational target for anti-inflammatory drug
302 development. IKKβ is a 677-amino-acid polypeptide that contains an N-terminal
303 kinase domain (residues 15-308), a leucine zipper region (residues 458-479), and a
304 helix-loop-helix region (residues 603-642).21 Fructokinase-1 was the most abundant
305 polypeptide in the corn silk extract in this study. After trypsin digestion, 11 peptides
306 ranging from 6 to 20 amino acid residues in length were generated (Figure 2B).
307 However, only three peptides FK1 (GSISLIAEP), FK2 (TMKLLLVTL), and FK3
308 (AGALLSYDP) of fructokinase-1 docked into the ATP-binding pocket in the
309 N-terminal kinase domain of IKKβ (Figure 2C). TPCA-1, a selective inhibitor of
310 IKKβ, was used as a reference compound for docking analysis. As shown in Figure
17

311 2C and Table 2, TPCA-1, as expected, bound to the ATP-binding site (Lys-44) of
312 IKKβ with the binding energy of -4167 kcal/mol. The binding energies between corn
313 silk peptides and IKKβ were slightly higher than that of TPCA-1. However, FK2
314 was the only one peptide that might interact with the catalytic site (Asp-145) and
315 block the ATP-binding pocket of IKKβ. These findings suggested that FK2 was a
316 potent anti-inflammatory bioactive peptide that interacted with IKKβ and
317 consequently inhibited NF-κB activities.
318
319 FK2 Suppressed LPS-Induced Acute Inflammation In Vivo via
320 Downregulation of NF-κB Pathway.
321 To analyze whether FK2 interacted with IKKβ, inhibited the phosphorylation of
322 IκB-α, and consequently inhibited the activation of NF-κB, we first performed IKKβ
323 activity assay in the presence of various amounts of FK2. As shown in Figure 3A,
324 TPCA-1, the IKKβ inhibitor, suppressed IKKβ activities in a dose-dependent
325 manner and 90% of IKKβ activity was abolished by 100 µM TPCA-1. FK2 and corn
326 silk extract also inhibited IKKβ activities. FK2 and corn silk extract suppressed 50%
327 and 70% of IKKβ activities at 100 µM FK2 and 25 mg/ml corn silk extract,
328 respectively, and the inhibition displayed a biologic dose response. Therefore, these
329 data suggested that FK2 might interact with IKKβ and affect the activity of IKKβ.
18

330 Upon the activation of IKKβ, IKKβ stimulates the phosphorylation of IκB-α and, in
331 turn, induces the translocation of NF-κB to the nucleus. We therefore performed
332 Western blot and luciferase assay to elucidate the effects of FK2 on the
333 phosphorylation of IκB-α and the activation of NF-κB. Western blot shows that LPS
334 stimulated the phosphorylation of IκB-α, while FK2 reduced the amount of
335 phosphorylated IκB-α induced by LPS (Figure 3B). Corn silk extract also displayed
336 the inhibition on the phosphorylation of IκB-α. Further luciferase assay shows that
337 LPS activated NF-κB activities by 6-fold, compared with mock (Figure 3C).
338 However, LPS-induced NF-κB activities were significantly suppressed by FK2
339 (2.3±0.4 fold vs. 5.5±0.4 fold, p<0.001) in a dose-dependent manner. These findings
340 suggested that FK2 interacted with IKKβ, affected IκB-α phosphorylation, and, in
341 turn, suppressed NF-κB activation in cells.
342 We further analyzed whether FK2 inhibited NF-κB activities and suppressed
343 LPS-induced inflammation in mice. Mice were challenged intraperitoneally with
344 LPS and given orally with various amounts of FK2. The concentration of IL-1β was
345 increased after LPS administration, while LPS-induced IL-1β production was
346 significantly inhibited by FK2 (Figure 3D). Moreover, the inhibition displayed a
347 dose-dependent manner. These data suggested that oral administration of FK2
348 suppressed LPS-induced inflammation in mice. Transgenic mice were further
19

349 challenged intraperitoneally with LPS and given orally with 1 mg/kg FK2, the
350 maximal effective dosage of FK2 used in BALB/c mice. Ex vivo imaging showed
351 that the NF-κB-dependent luminescent signals were induced by LPS in various
352 organs (Figure 4). However, oral administration of FK2 significantly suppressed
353 LPS-induced luminescent intensities in organs, except liver. The maximal
354 suppression was observed in small intestine, followed by brain, spleen, heart,
355 stomach, lung, and kidney. Pathological examination showed no visible changes in
356 small intestine after a 4-h challenge of LPS (Figure 5). IHC analysis was performed
357 using antibodies against p65 nuclear localization sequence and IL-1β. As shown in
358 Figure 5, the expression of IL-1β and the number of p65-positive cells in the
359 intestine were significantly increased by LPS, compared with mock. However, oral
360 administration of FK2 significantly decreased the expression of IL-1β and the
361 number of p65-positive cells in intestine. These findings suggested that FK2
362 ameliorated LPS-induced inflammation in mice. Moreover, the anti-inflammatory
363 activity of FK2 might be related with the IKKβ-NF-κB signaling transduction.
364
365
366 DISCUSSION
367 Corn silk has been consumed as a therapeutic remedy for a long time. The aqueous
20

368 extract of corn silk has been used for patients with urinary tract disorders in many
369 countries.17 In traditional Chinese medicine, the aqueous extract of corn silk has also
370 been used to alleviate fever (inflammation). The ethanolic extract of corn silk
371 exhibits anti-inflammatory activities by the inhibition of TNF-α and LPS-induced
372 cell adhesion and intracellular adhesion molecule-1 (ICAM-1) production in
373 endothelial cells.31 The aqueous extract of corn silk ameliorates carrageenin-induced
374 pleurisy in rat model via the reduction of cytokine production, oxidative stress, and
375 complement C3 protein level. It also blocks inflammation-related events, such as
376 ICAM-1 and iNOS production, by NF-κB activity.18 In this study, we found that
377 protein-rich aqueous extract of corn silk inhibited LPS-induced NF-κB activation in
378 cells and inhibited LPS-induced cytokine productions in LPS-induced systemic
379 inflammation models. These studies provided scientific evidences for the
380 ethnopharmacological usage of corn silk on inflammation.
381 The nutritional compositions of corn silk contain 40% crude fiber, 17.6% crude
382 protein, 9.65% moisture, 3.91% ash, and 0.29% crude fat.32 Some bioactive
383 constituents, such as flavonoids, saponins, alkaloids, polysaccharides and essential
384 oils, have been identified from corn silk. For example, maysin
385 (rhamnosyl-6-C-(4-ketofucosyl)-5,7,3',4'tetrahydroxyflavone) is a major flavonoid
386 in corn silk. Maysin inhibits the growth of androgen-independent human prostate
21

387 cancer cells via the stimulation of mitochondria-dependent apoptotic cell death.33 It
388 displays neuroprotective effects against oxidative stress-induced apoptotic death of
389 human neuroblastoma cells through its anti-oxidative action.34 It also exhibits
390 immunomodulatory activities by activation of macrophages through up-regulation of
391 the immune response-related signaling pathways.35 Polysaccharides from corn silk
392 regulate the level of blood glucose, improve the movement of gastrointestine, and
393 lead to the loss of body weight.36,37 Moreover, corn silk polysaccharides inhibit the
394 tumor growth, extend the survival time, and increase the production of serum IL-2,
395 IL-6 and TNF-α in mice bearing H22 hepatocellular carcinoma, suggesting that the
396 antitumor activity of corn silk polysaccharides might be at least in part due to
397 immune function activation.38
398 Protein constituents involved in physiologic process, metabolism, or development
399 of corn silk have been identified.39 However, bioactive proteins or peptides with
400 pharmacological potentials have not been discovered from corn silk so far. Here we
401 applied proteomic approaches and found that aqueous extract of corn silk contained
402 seven significant protein spots. Most protein constituents, such as frucotokinase-1,
403 beta-1,3-glucanase and chitinase, are involved in carbohydrate metabolism.
404 Fructokinase-1, the major protein constituent in the corn silk extract, specifically
405 catalyzes the transfer of a phosphate group from ATP to fructose. It may play an
22

406 important role in maintaining the flux of carbon towards starch formation in
407 endosperm. It may also be involved in a sugar-sensing pathway.40 After trypsin
408 digestion of corn silk extract, 11 peptides of fructokinase-1 were identified by
409 LC-MS/MS. We further found that a 9-amino-acid-residue peptide FK2 exhibited
410 anti-inflammatory potentials in mice by NF-κB-driven bioluminescent imaging.
411 NF-κB bioluminescent imaging has been used to assess the biocompatibility of
412 biomaterials and the anti-inflammatory potentials of herbs or compounds.23-26,41 Here
413 we found that oral administration of FK2 significantly suppressed LPS-induced
414 NF-κB activations in most organs, such as brain, heart, lung, spleen stomach,
415 intestine, and kidney. These findings suggested that FK2 might be a potent
416 broad-spectrum anti-inflammatory peptide in the corn silk. Nevertheless, how the
417 FK2 was absorbed via an intragastric route, distributed in the body, and excreted
418 from the body remained to be demonstrated experimentally. In addition, trypsin
419 usually cleaves peptide bonds at the C-terminal side of lysine or arginine residues,
420 except when followed by proline.42 In comparison of trypsin from bovine and
421 porcine, bovine trypsin produces a higher number of peptides containing missed
422 cleavages, whereas porcine trypsin produces more semitryptic peptides. In addition,
423 differences in protein affinity into the substrate binding pockets are related to
424 variable digestion kinetics patterns of cleavage sites.43 Therefore, we speculated that
23

425 there were missed cleavages around the sequences of FK2, resulting in the FK2
426 sequence starting from Thr-224.
427 IKK plays a crucial role in the NF-κB signaling transduction pathway. IKK
428 complex consists of two kinases (IKKα and IKKβ) and a regulatory subunit (NF-κB
429 essential modulator or NEMO). IKKα and IKKβ share similar structural
430 characteristics. Both contain an N-terminal kinase domain, a leucine zipper region
431 that allows homo- or hetero-dimerization of the kinases, a helix-loop-helix region
432 that functions in modulating IKK activity, and a NEMO-binding domain that
433 interacts with NEMO. In comparison with IKKα, IKKβ contains an ubiquitin-like
434 domain between kinase domain and leucine zipper region. In addition, IKKβ plays a
435 dominant role in the proinflammatory signal-induced phosphorylation of IκB, while
436 IKKα is dispensable for this function.21 Moreover, anti-inflammatory properties of
437 aspirin and salicylate are mediated in part by their specific inhibition of IKKβ.22
438 These finding suggest that IKKβ is a rational target for anti-inflammatory drug
439 development. Lys-44 and Asp-145 in N-terminal kinase domain of IKKβ are key
440 amino acid residues involved in ATP binding and catalysis, respectively.21 So far,
441 various IKKβ inhibitors, like thiol-reactive inhibitors, ATP analogues and allosteric
442 inhibitors of IKKβ, have been identified. For example, 1-dehydro-[10]-gingerdione
443 from ginger functions as a thiol-reactive inhibitor of IKKβ that inhibits cytoplasmic
24

444 IKKβ-catalyzed IκB-α phosphorylation, disrupts NF-κB activation, and further
445 suppresses the expression of NF-κB-regulated genes, such as iNOS, COX-2, and
446 IL-6.44 Withaferin A from the Ayurvedic plant Withania somnifera, exerts its
447 anti-inflammatory effects by targeting the crucial Cys-179 residue located in the
448 catalytic site of IKKβ.45 Gamabufotalin, a major bufadienolide of Chansu, has been
449 used for cancer therapy. Yu et al (2014) found that gamabufotalin inhibits the
450 phosphorylation of IKKβ via targeting the ATP-binding site, abrogates NF-κB
451 binding and p300 recruitment to COX-2 promoter, and therefore suppresses COX-2
452 activity.46 Staurosporine, an alkaloid isolating from Streptomyces staurosporeus, and
453 quercetin, a flavonoid occurring in many fruits and vegetables, display
454 anti-inflammatory responses. Several reports indicate that staurosporine is an ATP
455 competitive inhibitor of IKKβ and quercetin shows a mixed inhibition mechanism
456 towards ATP. Furthermore, Avila et al (2009) showed that the inhibitory activities of
457 staurosporine and quercetin toward IKKβ may be explained based principally on
458 hydrogen-bonding interactions and ATP binding-site occupancy by these
459 compounds.47 In this study, we found that FK2 occupied the ATP-binding pocket and
460 formed hydrogen bonds with Gly-27, Asp-145, and Lys-147 in the kinase domain of
461 IKKβ. Therefore, we speculated that FK2 exhibited an IKKβ-inhibitory ability via
462 hydrogen-bonding interactions and ATP-binding site occupancy in IKKβ.
25

463 In conclusion, our findings indicated that both protein-rich aqueous extract of
464 corn silk and trypsin hydrolysate of corn silk displayed anti-inflammatory abilities in
465 vitro and in vivo. Moreover, by proteomic and docking analysis, we first identified a
466 bioactive peptide FK2 with anti-inflammatory potentials from trypsin hydrolysate of
467 corn silk extract. FK2 suppressed LPS-induced NF-κB activation and
468 proinflammatory cytokine production in mice. In addition, the anti-inflammatory
469 potentials of FK2 might through the interaction of IKKβ. Although both corn silk
470 extract and FK2 interacted with IKKβ, inhibited the phosphorylation of IκB-α, and
471 suppressed the activation of NF-κB, it was difficult to judge which one displayed a
472 better anti-inflammatory activity because corn silk extract consisted of several kinds
473 of constituents while FK2 was a synthetic peptide. Subchronic toxicity study showed
474 that corn silk has no adverse effects in rats orally giving with corn silk extract for
475 consecutive 90 days.32 Our findings suggested that oral administration of corn silk
476 ameliorated acute systemic inflammation induced by LPS in mice. Therefore,
477 consumption of corn silk extract might have some beneficial effects against acute
478 inflammation in ethnopharmacological usage. The long-term study needs to be
479 performed to prove the effects of corn silk on chronic inflammation.
480
481
26

482 FUNDING SOURCES
483 This work was supported by grants from Ministry of Science and Technology
484 (MOST104-2320-B-039-018-MY3, MOST104-2325-B-039-004, and
485 MOST105-2320-B-039-017-MY3), China Medical University (CMU104-H-01 and
486 CMU104-H-02), and CMU under the Aim for Top University Plan of the Ministry of
487 Education, Taiwan.
488
489 NOTES
490 The authors declare no competing financial interest.
491
492 ABBREVIATIONS USED
493 2-DE, 2-dimensional electrophoresis; COX-2, cyclooxygenase-2; DMEM,
494 Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; IHC,
495 Immunohistochemical staining; IκB, inhibitory κB; IKK, inhibitory κB kinase;
496 IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; LC-MS/MS, liquid
497 chromatography coupled with tandem mass spectrometry; LPS, lipopolysaccharide;
498 NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; pI, isoelectric point;
499 RLU, relative luciferase units; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide
500 gel; TNF-α, tumor necrosis factor-α
501 27

502 REFERENCES
503 (1) Vallabhapurapu, S.; Karin, M. Regulation and function of NF-κB
504 transcription factors in the immune system. Annu. Rev. Immunol. 2009, 27, 693-733.
505 (2) Zhao, Y.; Forst, C. V.; Sayegh, C. E.; Wang, I. M.; Yang, X.; Zhang, B.
506 Molecular and genetic inflammation networks in major human diseases. Mol.
507 Biosyst. 2016, 12, 2318-2341.
508 (3) White, T. J.; Arakelian, A.; Rho, J. P. Counting the costs of drug-related
509 adverse events. PharmacoEconomics 1999, 15, 445-458.
510 (4) Fosgerau, K.; Hoffmann, T. Peptide therapeutics: current status and future
511 directions. Drug Discov. Today 2015, 20, 122‐128.
512 (5) Chakrabarti, S.; Jahandideh, F.; Wu, J. Food-derived bioactive peptides on
513 inflammation and oxidative stress. Biomed. Res. Int. 2014, 2014, 608979.
514 (6) Majumder, K.; Mine, Y.; Wu, J. The potential of food protein-derived
515 anti-inflammatory peptides against various chronic inflammatory diseases. J. Sci.
516 Food Agric. 2016, 96, 2303-2311.
517 (7) Chatterton, D. E.; Nguyen, D. N.; Bering, S. B.; Sangild P. T.
518 Anti-inflammatory mechanisms of bioactive milk proteins in the intestine of
519 newborns. Int. J. Biochem. Cell Biol. 2013, 45, 1730-1747.
28

520 (8) Kovacs-Nolan, J.; Zhang, H.; Ibuki, M.; Nakamori, T.; Yoshiura, K.; Turner,
521 P. V.; Matsui, T.; Mine, Y. The PepT1-transportable soy tripeptide VPY reduces
522 intestinal inflammation. Biochim. Biophys. Acta 2012, 1820, 1753-1763.
523 (9) Nakamura, T.; Hirota, T.; Mizushima, K.; Ohki, K.; Naito, Y.; Yamamoto, N.;
524 Yoshikawa, T. Milk-derived peptides, Val-Pro-Pro and Ile-Pro-Pro, attenuate
525 atherosclerosis development in apolipoprotein E-deficient mice: a preliminary study.
526 J. Med. Food 2013, 16, 396-403.
527 (10) Yang, X.; Zhu, J.; Tung, C. Y.; Gardiner, G.; Wang, Q.; Chang, H. C.; Zhao,
528 B. Lunasin alleviates allergic airway inflammation while increases antigen-specific
529 Tregs. PloS ONE 2015, 10, e0115330.
530 (11) Alaswad, A. A.; Krishnan, H. B. Immunological investigation for the
531 presence of lunasin, a chemopreventive soybean peptide, in the seeds of diverse
532 plants. J. Agric. Food Chem. 2016, 64, 2901-2909.
533 (12) Huang, W.; Chakrabarti, S.; Majumder, K.; Jiang, Y.; Davidge, S. T.; Wu, J.
534 Egg-derived peptide IRW inhibits TNF-α-induced inflammatory response and
535 oxidative stress in endothelial cells. J. Agric. Food Chem. 2010, 58, 10840-10846.
536 (13) Majumder, K.; Chakrabarti, S.; Davidge, S. T.; Wu, J. Structure and activity
537 study of egg protein ovotransferrin derived peptides (IRW and IQW) on endothelial
29

538 inflammatory response and oxidative stress. J. Agric. Food Chem. 2013, 61,
539 2120-2129.
540 (14) Dia, V. P.; Bringe, N. A.; de Mejia, E. G. Peptides in pepsin-pancreatin
541 hydrolysates from commercially available soy products that inhibit
542 lipopolysaccharide-induced inflammation in macrophages. Food Chem. 2014, 152,
543 423-431.
544 (15) Ahn, C. B.; Cho, Y. S.; Je, J. Y. Purification and anti-inflammatory action of
545 tripeptide from salmon pectoral fin byproduct protein hydrolysate. Food Chem. 2015,
546 168, 151-156.
547 (16) USDA. FAS Grain: World Markets and Trade.
548 http://www.fas.usda.gov/data/grain-world-markets-and-trade.
549 (17) Hasanudin, K., Hashim, P., Mustafa, S.. Corn silk (Stigma maydis) in
550 healthcare: a phytochemical and pharmacological review. Molecules 2012, 17,
551 9697-9715.
552 (18) Wang, G. Q.; Xu, T.; Bu, X. M.; Liu, B. Y. Anti-inflammation effects of
553 corn silk in a rat model of carrageenin-induced pleurisy. Inflammation 2012, 35,
554 822-827.
555 (19) Kuhnen, S., Bernardi Ogliari, J., Dias, P. F.; da Silva Santos, M.; Ferreira, A.
556 G.; Bonham, C. C.; Wood, K. V.; Maraschin, M. Metabolic fingerprint of Brazilian
30

557 maize landraces silk (stigma/styles) using NMR spectroscopy and chemometric
558 methods. J. Agric. Food Chem. 2010, 58, 2194-2200.
559 (20) El-Ghorab, A.; El-Massry, K. F.; Shibamoto, T. Chemical composition of
560 the volatile extract and antioxidant activities of the volatile and nonvolatile extracts
561 of Egyptian corn silk (Zea mays L.). J. Agric. Food Chem. 2007, 55, 9124-9127.
562 (21) Liu, S.; Misquitta, Y. R.; Olland, A.; Johnson, M. A.; Kelleher, K. S.; Kriz,
563 R.; Lin, L. L.; Stahl, M.; Mosyak, L. Crystal structure of a human IκB kinase β
564 asymmetric dimer. J. Biol. Chem. 2013, 288, 22758-22767.
565 (22) Yin, M. J.; Yamamoto, Y.; Gaynor, R. B. The anti-inflammatory agents
566 aspirin and salicylate inhibit the activity of IκB kinase-β. Nature 1998, 396, 77-80.
567 (23) Hsiang, C. Y.; Cheng, H. M.; Lo, H. Y.; Li, C. C.; Chou, P. C.; Lee, Y. C.;
568 Ho, T. Y. Ginger and zingerone ameliorate lipopolysaccharide-induced acute
569 systemic inflammation in mice, assessed by nuclear factor-κB bioluminescent
570 imaging. J. Agric. Food Chem. 2015, 63, 6051-6058.
571 (24) Li, C.C.; Hsiang, C.Y.; Lo, H.Y.; Pai, F.T.; Wu, S.L.; Ho, T.Y. Genipin
572 inhibits lipopolysaccharide-induced acute systemic inflammation in mice as
573 evidenced by nuclear factor-κB bioluminescent imaging-guided transcriptomic
574 analysis. Food Chem. Toxicol. 2012, 50, 2978-2986.
31

575 (25) Hsiang, C. Y.; Hseu, Y. C.; Chang, Y. C.; Kumar, K. J. S.; Ho, T. Y.; Yang,
576 H. L. Toona sinensis and its major bioactive compound gallic acid inhibit
577 LPS-induced inflammation in nuclear factor-κB transgenic mice as evaluated by in
578 vivo bioluminescence imaging. Food Chem. 2013, 136, 426-434.
579 (26) Hsiang, C. Y.; Lo, H. Y.; Huang, H. C.; Li, C. C.; Wu, S. L.; Ho, T. Y.
580 Ginger extract and zingerone ameliorated trinitrobenzene sulfonic acid-induced
581 colitis in mice via modulation of nuclear factor-κB activity and interleukin-1β
582 signaling pathway. Food Chem. 2013, 136, 170-177.
583 (27) Lo, H. Y.; Ho, T. Y.; Lin, C.; Li, C. C.; Hsiang, C. Y. Momordica charantia
584 and its novel polypeptide regulate glucose homeostasis in mice via binding to insulin
585 receptor. J. Agric. Food Chem. 2013, 61, 2461-2468.
586 (28) Lo, H. Y.; Ho, T. Y.; Li, C. C.; Chen, J. C.; Liu, J. J.; Hsiang, C. Y. A novel
587 insulin receptor-binding protein from Momordica charantia enhances glucose
588 uptake and glucose clearance in vitro and in vivo through triggering insulin receptor
589 signaling pathway. J. Agric. Food Chem. 2014, 62, 8952-8961.
590 (29) Grosdidier, A.; Zoete, V.; Michielin, O. SwissDock, a protein-small
591 molecule docking web service based on EADock DSS. Nucleic Acids Res. 2011, 9,
592 270-277.
32

593 (30) Liu, S.; Gallo, D. J.; Green, A. M.; Williams, D. L.; Gong, X.; Shapiro, R.
594 A.; Gambotto, A. A.; Humphris, E. L.; Vodovotz, Y.; Billiar, T. R. Role of toll-like
595 receptors in changes in gene expression and NF-κB activation in mouse hepatocytes
596 stimulated with lipopolysaccharide. Infect. Immun. 2002, 70, 3433-3442.
597 (31) Habtemariam, S. Extract of corn silk (stigma of Zea mays) inhibits the
598 tumour necrosis factor-α- and bacterial lipopolysaccharide-induced cell adhesion
599 and ICAM-1 expression. Planta Med. 1998, 64, 314-318.
600 (32) Wang, C.; Zhang, T.; Liu, J.; Lu, S.; Zhang, C.; Wang, E.; Wang, Z.; Zhang,
601 Y.; Liu J. Subchronic toxicity study of corn silk with rats. J. Ethnopharmacol. 2011,
602 137, 36-43.
603 (33) Lee, J.; Lee, S.; Kim, S. L.; Choi, J. W.; Seo, J. Y.; Choi, D. J.; Park, Y. I.
604 Corn silk maysin induces apoptotic cell death in PC-3 prostate cancer cells via
605 mitochondria-dependent pathway. Life Sci. 2014, 119, 47-55.
606 (34) Choi, D. J.; Kim, S. L.; Choi, J. W.; Park, Y. I. Neuroprotective effects of
607 corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC
608 cells. Life Sci. 2014, 109, 57-64.
609 (35) Lee, J.; Kim, S. L.; Lee, S.; Chung, M. J.; Park, Y. I. Immunostimulating
610 activity of maysin isolated from corn silk in murine RAW 264.7 macrophages. BMB
611 Rep. 2014, 47, 382-387.
33

612 (36) Du, J.; Xu, Q. T.; Gao, X. H. Effects of stigma maydis polysaccharide on
613 gastrointestinal movement. J. Traditional Chin. Med. 2007, 32, 1203-1206.
614 (37) Zhao, W. Z.; Yin, Y. G.; Yu, Z. P.; Liu, J. B.; Chen, F. Comparison of
615 anti-diabetic effects of polysaccharides from corn silk on normal and hyperglycemia
616 rats. Int. J. Biol. Macromol. 2012, 50, 1133-1137.
617 (38) Yang, J.; Li, X.; Xue, Y.; Wang, N.; Liu, W. Anti-hepatoma activity and
618 mechanism of corn silk polysaccharides in H22 tumor-bearing mice. Int. J. Biol.
619 Macromol. 2014, 64, 276-280.
620 (39) Ma, Z.; Qin, Y.; Wang, Y.; Zhao, X.; Zhang, F.; Tang, J.; Fu, Z. Proteomic
621 analysis of silk viability in maize inbred lines and their corresponding hybrids. PLoS
622 One 2015, 10, e0144050.
623 (40) Zhang, S.; Nichols, S. E.; Dong, J. G. Cloning and characterization of two
624 fructokinases from maize. Plant Sci. 2003, 165, 1051-1058.
625 (41) Ho, T. Y.; Chen, Y. S.; Hsiang, C. Y. Noninvasive nuclear factor-κB
626 bioluminescence imaging for the assessment of host-biomaterial interaction in
627 transgenic mice. Biomaterials 2007, 28, 4370-4377.
628 (42) Siepen, J. A.; Keevil, E. J.; Knight, D.; Hubbard, S. J. Prediction of missed
629 cleavage sites in tryptic peptides aids protein identification in proteomics. J.
630 Proteome Res. 2007, 6, 399-408.
34

631 (43) Walmsley, S. J.; Rudnick, P. A.; Liang, Y.; Dong, Q.; Stein, S. E.;
632 Nesvizhskii, A. I. Comprehensive analysis of protein digestion using six trypsins
633 reveals the origin of trypsin as a significant source of variability in proteomics. J.
634 Proteome Res. 2013, 12, 5666-5680.
635 (44) Lee, H. Y.; Park, S. H.; Lee, M.; Kim, H. J.; Ryu, S. Y.; Kim, N. D.; Hwang,
636 B. Y.; Hong, J. T.; Han, S. B.; Kim, Y. 1-Dehydro-[10]-gingerdione from ginger
637 inhibits IKKβ activity for NF-κB activation and suppresses NF-κB-regulated
638 expression of inflammatory genes. Br. J. Pharmacol. 2012, 167, 128-140.
639 (45) Heyninck, K.; Lahtela-Kakkonen, M.; Van der Veken, P.; Haegeman, G.;
640 Vanden Berghe, W. Withaferin A inhibits NF-κB activation by targeting cysteine
641 179 in IKKβ. Biochem. Pharmacol. 2014, 91, 501-509.
642 (46) Yu, Z.; Guo, W.; Ma, X.; Zhang, B.; Dong, P.; Huang, L.; Wang, X.; Wang,
643 C.; Huo, X.; Yu, W.; Yi, C.; Xiao, Y.; Yang, W.; Qin, Y.; Yuan, Y.; Meng, S.; Liu,
644 Q.; Deng, W. Gamabufotalin, a bufadienolide compound from toad venom,
645 suppresses COX-2 expression through targeting IKKβ/NF-κB signaling pathway in
646 lung cancer cells. Mol. Cancer. 2014, 13, 203.
647 (47) Avila, C. M.; Romeiro, N. C.; Sant'Anna, C. M.; Barreiro, E. J.; Fraga, C. A.
648 Structural insights into IKKβ inhibition by natural products staurosporine and
649 quercetin. Bioorg. Med. Chem. Lett. 2009, 19, 6907-6910.
35

650 FIGURE CAPTIONS
651 Figure 1. Anti-inflammatory effects of corn silk extract and trypsin hydrolysate in
652 vitro and in vivo. (A) Luciferase assay. NF-κB recombinant cells were treated
653 without or with various amounts of corn silk extract (no digestion) or trypsin
654 hydrolysate (trypsin digestion), and then challenged with 100 ng/ml LPS. Luciferase
655 activity and cell viability were determined at 24 h. Bar represents relative NF-κB
656 activity, which is present in comparison to RLU of solvent-treated cells. Line
657 represents cell viability during treatment. Values are mean ± standard error of
658 triplicate assays. (B) IL-1β production. Mice were orally given various amounts of
659 corn silk extract corn silk extract (no digestion) or 1 g/kg trypsin hydrolysate
660 (trypsin digestion), and intraperitoneally injected with 1 mg/kg LPS. Four hours later,
661 sera were collected and the amount of IL-1β in sera was measured using OptEIATM
662 set. Relative IL-1β amount was calculated by dividing the IL-1β amount of mice
663 given with LPS or corn silk by the IL-1β amount of mice given with solvent. Values
###
664 are mean ± standard error (n = 5/group). p <0.001, compared with mock. *p <
665 0.05, **p < 0.01, compared with LPS.
666
667 Figure 2. Protein identification of corn silk extract and docking analysis of
668 fructokinase-1 peptide fragment. (A) 2-DE of corn silk extract. Protein was
36

669 separated on a pH 3-10 strip followed by SDS-PAGE on a 15% polyacrylamide gel.
670 Proteins were visualized by Coomassie Brilliant Blue R-250. Protein size markers
671 (in kDa) are shown at the left. Protein spots analyzed by LC-MS/MS are indicated
672 by red circles. Photos are representative images of three independent experiments.
673 (B) Fructokinase-1. Amino acid sequences of peptides generated from trypsin
674 digestion of fruntokinase-1 are shown in red. Peptides used for docking analysis are
675 underlined and indicated. (C) Docking analysis. IKKβ is shown in a ribbon
676 presentation. TPCA-1, FK1, FK2, and FK3 are shown in a stick format. Hydrogen
677 bonds are displayed as yellow dashed lines. The side chains of active amino acid
678 residues (Lys-44 and Asp-145) are shown in a stick format.
679
680 Figure 3. Anti-inflammatory effects of FK2. (A) IKKβ activity assay. A mixture
681 containing IKKβ, ATP, IKKtid peptide substrate, and various amounts of TPCA-1,
682 corn silk extract, or FK2 was incubated at room temperature for 1 h. ADP was then
683 detected using ADP-Glo™ reagent and measured by a luminometer. Relative IKKβ
684 activity was calculated by dividing the RLU of reaction containing compounds by
685 the RLU of reaction containing solvent. Values are mean ± standard error. (B)
686 Western blot analysis. Cells were pretreated with various amounts of corn silk
687 extract or FK2 for 30 min and then treated with 100 ng/ml LPS for 30 min. IκB-α
37

688 and phosphorylated IκB-α were detected by Western blot and visualized by
689 chemiluminescence. Band intensity with respect to mock is shown at the bottom.
690 Photos are representative images. (C) In vitro. NF-κB recombinant cells were treated
691 without or with various amounts of FK2 and then treated with 100 ng/ml LPS. Bars
692 represent relative NF-κB activity, which is presented in comparison to RLU of
693 solvent-treated cells. Line represents cell viability during treatment. Values are mean
694 ± standard error of triplicate assays. (D) In vivo. Mice were orally given various
695 amounts of FK2 and intraperitoneally injected with 1 mg/kg LPS. Four hours later,
696 the amount of IL-1β in sera was measured using OptEIATM set. Values are mean ±
### ** ***
697 standard error (n = 5/group). p <0.001, compared with mock. p < 0.01, p<
698 0.001, compared with LPS.
699
700 Figure 4. NF-κB-dependent luminescence in organs. (A) Ex vivo imaging.
701 Transgenic mice were intraperitoneally administered 1 mg/kg LPS and orally given
702 with 1 mg/kg FK2. Four hours later, mice were injected intraperitoneally with
703 D-luciferin and sacrificed 5 min later. Organs were then excised rapidly and
704 subjected to imaging. The color overlay on the image represents the photons per
705 second emitted from organs, as indicated by the color scale. Photos are
706 representative images (n = 5/group). (B) Quantification of photon emissions from
38

###
707 organs. Values are mean ± standard error. p < 0.001, compared with mock. **p <
708 0.01, ***p < 0.001, compared with LPS.
709
710 Figure 5. Histological examination and IHC analysis of small intestine. Mice were
711 orally given with 1 mg/kg FK2 and intraperitoneally injected with 1 mg/kg LPS.
712 Four hours later, small intestines were excised, and sections were stained with
713 hematoxylin and eosin (H&E) (40× magnification) or by IHC using antibody against
714 p65 or IL-1β. For IHC, upper pictures show a low magnification (40×), while lower
715 pictures show a magnification (100×) of the rectangle in upper pictures. Photos are
716 representative images (n=5/group). Quantification of p65-positive cell or
717 IL-1β-positive area (%) is shown at the bottom. Values are mean ± standard error
718 (n=5/group). #p < 0.05, ###

p < 0.001, compared with mock. *p < 0.05, ***
p < 0.001,
719 compared with LPS.
39

Table 1 Corn silk proteins identified by LC-MS/MS
Molecular
Spot no. Protein ID Accession no.a pI Scoreb Amount (%)c
weight (Da)
1 Chitinase chem5 precursor NP_001266396 30442 4.1 74 1.34
2 Trypsin inhibitor precursor NP_001104908 20136 4.6 120

1.29
3 Fructokinase-1 NP_001105210 34691 5.0 248
6.62
4 Putative beta 4 proteasome subunit NP_001105922 23244 5.4 40
1.54
5 Beta-1,3-glucanase precursor ADL60383 36068 9.4 379
5.23
6 Chitinase ACX37090 29973 8.6 605
5.05
7 Glycine-rich RNA-binding protein 2 ACG40707 15646 9 73
2.77
a
Protein accession number according to NCBI protein database.
b
MASCOT MS/MS ion score obtained for individual peptide sequences. The significant threshold is ~37.
c
The integrated density of each spot was analyzed by Gel-Pro Analyzer.
40

Table 2 Interaction parameters between ligands and IKKβ phosphokinase domain.
Ligand Formula or sequence Simple fitness Full fitness Hydrogen bond Distance
(kcal/mol) (kcal/mol)
TPCA-1 C12H10FN3O3 -19.8 -4167 Lys-44 1.99 Å
FK1 GSISLIAEP 54.6 -3911 Leu-21 3.07 Å
FK2 TMKLLLVTL 38.1 -3877 Gly-27 2.51 Å
Asp-145 2.15 Å
Lys-147 2.07 Å
FK3 AGALLSYDP 52.7 -3930 Arg-57 2.49 Å
Tyr-169 1.96 Å
Tyr-199 2.11 Å
41

Figure 1
(A) No digestion Trypsin digestion

No digestion Trypsin digestion
4 ## 120
##
3.5
Relative NF-κB activity
100
3
Cell viability (%)

80
2.5 * *
*
**
2 * * 60
**
1.5 **
40
1
20
0.5
0 0
Mock LPS 1 10 25 50 100 250 500
Corn silk (µg/ml)
(B)
35
30 ###
Relative IL-1β amount
25
20
15 ** **
**
10
0
Mock LPS 10 100 1000 1000
Corn silk (mg/kg)
No digestion Trypsin
digestion
42

Figure 2
(A) (B)
pH 3 --------------------------------------> pH 10 MAAGRELVVSFGEMLIDFVPTVAGVSLAEAPAFLKAPGGA 40
kDa
200
116 PANVAIAVSRLGGGAAFVGKLGDDEFGRMLAAILRDNGVD 80
97
66 DGGVVFDSGARTALAFVTLRADGEREFMFYRNPSADMLLT 120
45
3
1 5 ADELNVELIKRAAVFHYGSISLIAEPCRTAHLRAMEIAKE 160
31
FK1
6 AGALLSYDPNLREALWPSREEARTQILSIWDQADIVKVSE 200
21 2 4 FK3
14 7 VELEFLTGIDSVEDDVVMKLWRPTMKLLLVTLGDQGCKYY 240
FK2
7 ARDFHGAVPSFKVQQVDTTGAGDAFVGALLQRIVKDPSSL 280
QDEKKLVESIKFANACGAITTTKKGAIPSLPTEAEVLQLI 320
EKA 323
(C)
TPCA-1 FK1 FK2 FK3
43

Figure 3
(A) (C)
(B) (D)
Corn silk (mg/ml) FK2 (µM)
100 250 500 1 10 100
LPS －＋＋＋＋＋＋＋
p-IκB-α
1.0 1.9 2.0 1.8 1.3 1.7 2.0 0.9
IκB-α
1.0 0.9 1.1 1.2 1.2 1.1 1.2 1.2
β-actin
44

Figure 4
(A)
Brain Heart Lung Liver Spleen Stomach Kidney Intestine
Mock
LPS
LPS/FK2
(B)
40 ###
Mock
35
LPS
FK2
30
Relative NF-κB fold
25
###
20 ###
###
15 ###
###
10
*** *** ***
**
** ** **
5
0
Brain Heart Lung Liver Spleen Stomach Kidney Intestine
45

Figure 5
Mock LPS LPS/FK2
H&E
p65
7.6±1.1% 25.2±0.8%### 5.2±0.4%***
IL-1β
5.1±1.0% 14.2±4.5%# 8.9±1.9%*
46

Table of Contents Graphics
Identification of protein constituents FK2 docked with IKKβ
3 5
1
6
2 4 7
FK2 suppressed LPS-induced IL-1β production and NF-κB activity
Mock LPS LPS/FK2

Rambut Jagung 2

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Rambut Jagung 2

Uploaded by

Copyright:

Available Formats

Subscriber access provided by Fudan University

Journal of Agricultural and Food Chemistry is published by the American Chemical

1 Corn Silk Extract and Its Bioactive Peptide Ameliorated

2 Lipopolysaccharide-Induced Inflammation in Mice via Nuclear Factor-κB

15 +886 4 22053764. E-mail address: cyhsiang@mail.cmu.edu.tw

ACS Paragon Plus Environment

22 Bioactive peptides derived from foods have shown beneficial anti-inflammatory

23 potential. Inhibitory κB kinase-β (IKKβ) plays a crucial role on the activation of

24 nuclear factor-κB (NF-κB), a transcription factor involved in inflammation. Here we

25 applied proteomic and bioinformatics approaches to identify anti-inflammatory

27 lipopolysaccharide (LPS)-induced NF-κB activities (1.7±0.2 fold vs. 3.0±0.6 fold,

28 p<0.05) in cells. Trypsin hydrolysate of corn silk also suppressed LPS-induced

30 extract and trypsin hydrolysate significantly inhibited the LPS-induced

31 interleukin-1β (IL-1β) production by 58.3±4.5% and 55.1±7.4%, respectively. A

35 p<0.001). Moreover, FK2 significantly reduced NF-κB-driven luminescent signals

36 in organs by 5 to 11 fold, and suppressed LPS-induced NF-κB activities and IL-β

38 anti-inflammatory abilities. In addition, we first identified an anti-inflammatory

ACS Paragon Plus Environment

40 through IKKβ-NF-κB signaling pathways.

42 KEYWORDS: corn silk, peptide, inflammation, nuclear factor-κB, inhibitory κB

ACS Paragon Plus Environment

46 Inflammation is a protective response of tissues to numerous harmful stimuli, such

47 as pathogens, injury, or irritants. Upon stimulation, inhibitory κB kinase (IKK) is

48 activated and enhances the nuclear translocation of nuclear factor-κB (NF-κB),

49 leading to the expression of proinflammatory cytokines, such as interleukin-1β

52 the inflammatory process.1 Despite its protective response, inflammation is also

53 strongly associated with many pathological conditions, including atherosclerosis,

54 autoimmune diseases, cancers, cardiovascular diseases, diabetes, obesity, and

55 neurological diseases.2 Dexamethasone and non-steroidal anti-inflammatory drugs,

56 such as aspirin or ibuprofen, are commonly used for the amelioration of

58 effects, including gastric erosions or kidney damage.3 Therefore, discovery of better

59 anti-inflammatory agents from natural products has been gaining a significant

63 specificity, lower toxicity, less accumulation in organs, and fewer drug-drug

ACS Paragon Plus Environment

65 beneficial anti-inflammatory potential.5,6 For example, tripeptides VPP and IPP

69 changes through reducing the expression of pro-inflammatory genes in

70 apolipoprotein E-knockout atherosclerosis model.9 Lunasin, a 43-amino-acid-residue

71 peptide from enzymatic hydrolysis of soy protein, ameliorates murine

72 ovalbumin-induced asthma by reducing the cytokine expression and inflammatory

73 infiltration.10,11 Tripeptide IRW derived from thermolysin and pepsin hydrolysis of

74 egg white protein ovotransferrin reduces TNF-α-induced expression of adhesion

75 molecules in aorta and mesenteric arteries in spontaneously hypertensive rats.12,13

77 peptides RQRK and VIK in pepsin-pancreatin hydrolysates from commercially

79 synthase (iNOS) and cyclooxygenase-2 (COX-2) and attenuate the production of

80 pro-inflammatory cytokines in lipopolysaccharide (LPS)-treated murine

81 macrophages.14,15 These results indicate that bioactive peptide from food is a

82 promising approach for anti-inflammatory drug discovery.

ACS Paragon Plus Environment

89 edema, hyperlipidemia, and hypertension. It displays antioxidant, anti-fatigue, and

90 anti-depressant activities. It has been used for the treatment of inflammatory

91 diseases, such as cystitis, urinary infections, nephritis, as well as prostatitis.17

92 Pretreatment with corn silk extract reduces carrageenin-induced pleurisy exudate,

95 diseases.18 Corn silk consists of various constituents, including proteins, flavonoids,

96 polysaccharides, saponins, alkaloids, and steroids.19,20 Anti-inflammatory activities

98 previously. However, the presence of bioactive peptides with anti-inflammatory

99 potentials in corn silk is still unclear.

100 To address this question, we applied proteomic and bioinformatics approaches to

ACS Paragon Plus Environment

102 anti-inflammatory activities and mechanisms of peptides were further evaluated by

103 luciferase assay, Western blot analysis, ex vivo NF-κB-driven bioluminescent

105 the proinflammatory signal-induced phosphorylation of inhibitory κB (IκB) and is

107 Therefore, we applied IKKβ as a target to identify the peptides with

110 silk extract, ameliorated LPS-induced inflammation in mice. Moreover, the