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Rambut Jagung 2
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
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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:
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
21 ABSTRACT
26 peptides that target IKKβ from corn silk. Corn silk extract significantly suppressed
29 NF-κB activities (1.1±0.3 fold vs. 3.3±0.5 fold, p<0.01). In addition, both corn silk
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,
37 productions in tissues. In conclusion, our findings indicated that corn silk displayed
39 peptide FK2 from corn silk. Moreover, the anti-inflammatory effect of FK2 might be
41
43 kinase-β
44
45 INTRODUCTION
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
57 inflammation. However, long-term use of these drugs can cause several adverse
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
64 interactions.4 Bioactive peptides derived from various foods have shown some
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
76 Tripeptide PAY in salmon pectoral fin byproduct pepsin hydrolysate and bioactive
78 available soy products suppress the protein expression of inducible nitric oxide
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,
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
97 of flavonoids, polysaccharide, and essential coil in corn silk have been reported
101 identify anti-inflammatory peptides from protein-rich extract of corn silk. The
104 imaging, and immunohistochemical staining (IHC). IKKβ plays a dominant role in
106 also the target of anti-inflammatory drugs, such as aspirin and salicylate.21,22
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
112
113
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
123 Signaling (Beverly, MA). Mouse monoclonal antibody against p65 was purchased
124 from Chemicon (Temecula, CA). Rabbit polyclonal antibody against IL-1β was
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
134
136 technology (LifeTein, Somerset, NJ). The purity and amino acid sequences of FK2
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
148 peptide-treated cells by the RLUs of solvent-treated cells. Cell viability was judged
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
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
163
165 with Tandem MS (LC-MS/MS) Analysis. The composition of corn silk extract was
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
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
179 Biosystem/MDS Sciex, Foster City, CA). MS/MS data were matched against green
182 the average theoretical molecular weight, isoelectric point (pI), and sequence
183 coverage.
184
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
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
199
200 IKKβ Activity Assay. IKKβ activity was measured using ADP-Glo™ kinase
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
207
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%
213 sodium fluoride). Protein samples (50 µg) were separated by 10% SDS-PAGE and
12
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.,
221
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
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
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
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
246
247
248 RESULTS
249 Corn Silk Extract Suppressed LPS-Induced NF-κB Activities In Vitro and
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
256 evaluated the anti-inflammatory activities of corn silk in cells and then analyzed the
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
262 LPS-induced NF-κB activities (1.1±0.3 fold vs. 3.3±0.5 fold, p<0.01). And the
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
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
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
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%),
290 (1.5%), chitinase chem5 precursor (1.3%), and trypsin inhibitor precursor (1.3%).
291
16
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
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
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
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
318
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,
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).
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
342 We further analyzed whether FK2 inhibited NF-κB activities and suppressed
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
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
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
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
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
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
379 inflammation models. These studies provided scientific evidences for the
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
384 oils, have been identified from corn silk. For example, maysin
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
389 human neuroblastoma cells through its anti-oxidative action.34 It also exhibits
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
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,
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
411 NF-κB bioluminescent imaging has been used to assess the biocompatibility of
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
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
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
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
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
443 from ginger functions as a thiol-reactive inhibitor of IKKβ that inhibits cytoplasmic
24
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
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
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
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
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
477 consumption of corn silk extract might have some beneficial effects against acute
480
481
26
483 This work was supported by grants from Ministry of Science and Technology
486 CMU104-H-02), and CMU under the Aim for Top University Plan of the Ministry of
488
489 NOTES
491
494 Dulbecco’s modified Eagle’s medium; FBS, fetal bovine serum; IHC,
496 IL-1β, interleukin-1β; iNOS, inducible nitric oxide synthase; LC-MS/MS, liquid
498 NEMO, NF-κB essential modulator; NF-κB, nuclear factor-κB; pI, isoelectric point;
501 27
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588 uptake and glucose clearance in vitro and in vivo through triggering insulin receptor
591 molecule docking web service based on EADock DSS. Nucleic Acids Res. 2011, 9,
592 270-277.
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593 (30) Liu, S.; Gallo, D. J.; Green, A. M.; Williams, D. L.; Gong, X.; Shapiro, R.
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595 receptors in changes in gene expression and NF-κB activation in mouse hepatocytes
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601 Y.; Liu J. Subchronic toxicity study of corn silk with rats. J. Ethnopharmacol. 2011,
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607 corn silk maysin via inhibition of H2O2-induced apoptotic cell death in SK-N-MC
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610 activity of maysin isolated from corn silk in murine RAW 264.7 macrophages. BMB
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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
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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
35
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
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 <
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
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
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
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<
699
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
38
###
707 organs. Values are mean ± standard error. p < 0.001, compared with mock. **p <
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
717 IL-1β-positive area (%) is shown at the bottom. Values are mean ± standard error
39
Molecular
Spot no. Protein ID Accession no.a pI Scoreb Amount (%)c
weight (Da)
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
Ligand Formula or sequence Simple fitness Full fitness Hydrogen bond Distance
(kcal/mol) (kcal/mol)
Asp-145 2.15 Å
Lys-147 2.07 Å
Tyr-169 1.96 Å
Tyr-199 2.11 Å
41
Figure 1
100
3
0 0
Mock LPS 1 10 25 50 100 250 500
(B)
35
30 ###
Relative IL-1β amount
25
20
15 ** **
**
10
0
Mock LPS 10 100 1000 1000
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)
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
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
IL-1β
46
3 5
1
6
2 4 7