New Rice-Derived Short Peptide Potently Alleviated Hyperuricemia

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Bioactive Constituents, Metabolites, and Functions

A new rice-derived short peptide potently alleviated hyperuricemia induced by potassium oxonate in rats Naixin Liu, Ying Wang, Meifeng Yang, Wenxin Bian, Lin Zeng, Saige Yin, Ziqian Xiong, Yan Hu, Siyuan Wang, Buliang Meng, Jun Sun, and Xinwang Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b05879 • Publication Date (Web): 18 Dec 2018 Downloaded from http://pubs.acs.org on December 19, 2018

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Journal of Agricultural and Food Chemistry

A new rice-derived short peptide potently alleviated hyperuricemia induced by potassium oxonate in rats Naixin Liu†, ‡, Ying Wang†, §, Meifeng Yang†, ‡, Wenxin Bian‡, Lin Zeng∥, Saige Yin‡, Ziqian Xiong‡, Yan Hu‡, Siyuan Wang§, Buliang Meng*, ‡, Jun Sun*, ‡, Xinwang Yang*, ‡

‡Department

of Anatomy and Histology & Embryology, Faculty of Basic Medical

Science, Kunming Medical University, Kunming 650500, Yunnan, China. §Ethic

Drug Screening & Pharmacology Center, Key Laboratory of

Chemistry in Ethnic Medicine Resource, State Ethnic Affairs Commission & Ministry of Education, Yunnan MinZu University, Kunming 650500, Yunnan, China. ∥Public

Technical Service Center, Kunming Institute of Zoology, Chinese

Academy of Science, Kunming 650223, Yunnan, China.

1

ACS Paragon Plus Environment


1 2

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Abstract Gout that caused by hyperuricemia affects human health seriously and

3

more efficient drugs are urgently required clinically. In this study, a novel

4

peptide named RDP1 (AAAAGAKAR, 785.91 Da) was identified from the

5

extract of shelled fruits of Oryza Sativa. Our results demonstrated that RDP1

6

(the minimum effective concentration is 10 µg/kg) could significantly reduce

7

the serum uric acid & creatinine and alleviate hyperuricemic nephropathy in

8

rats by intragastric administration. RDP1 inhibited xanthine oxidase, which

9

also was verified at the animal level. Results from molecular docking indicated

10

that RDP1 can inhibit uric acid formation by occupying the binding site of

11

xanthine oxidase to xanthine. Besides, RDP1 showed no toxicity on rats and

12

was stable in several temperatures, demonstrated its advantages of

13

transportation. This research was the first discovery of anti-hyperuricemic

14

peptide from the shelled fruits of O. Sativa and provided a new candidate for

15

the development of hypouricemic drugs.

16 17

Keywords: peptide, Oryza Sativa, hyperuricemia, renoprotection.

18 19 20 21 22 2


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23

Introduction

24

Hyperuricemia is a symptom caused by excessive production and/or low

25

excretion of uric acid1, 2. In humans, uric acid is mainly derived from xanthine in

26

the liver, in which xanthine oxidase (XO) is the key enzyme involved in its

27

production3. Persistent hyperuricemia may increase the prevalence of gout,

28

which can lead to acute arthritis, gout stone, interstitial nephritis, severe joint

29

deformities and dysfunction. Recent studies have shown that hyperuricemia is

30

also closely related to some chronic metabolic diseases4, 5, and can significantly

31

increase the risk of hypertension, diabetes, kidney and cardiovascular

32

diseases6-9, which may make hyperuricemia become the major point of tertiary

33

prevention for gout. Clinical drugs for hyperuricemia can target at 1) the

34

inhibition of uric acid production, such as allopurinol, febuxostat and

35

topiroxostat and so on; 2) the promotion of uric acid excretion, such as

36

probenecid, benzbromarone and so on10. The inhibition of uric acid production

37

is particularly critical for anti-hyperuricemic treatments. Unfortunately, 25 % to

38

50 % of patients with hyperuricemia fail to show positive responses to

39

medications for gout because of the drug contraindications or serious side

40

effects11. For example, allopurinol is highly susceptible to cross react with other

41

drugs and may cause gastrointestinal symptoms, rashes, Stephen Johnson's

42

syndrome and allopurinol hypersensitivity syndrome12-14. Febuxostat may

43

cause cardiovascular problems and is costly16-18. Both probenecid and

44

benzbromarone may increase the crystallization of uric acid in the kidney, while 3



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45

benzbromarone may also exert hepatotoxicity19. Therefore, it is urgent to

46

explore or develop less untoward reaction and more economical new anti-

47

hyperuricemic, potent drug candidates.

48

Substances that have been reported to have anti-hyperuricemic activity

49

are small molecule compounds majorly, difficult to produce and store20-25. In

50

recent decades, many studies have shown that short bioactive peptides

51

containing 5 to 10 amino acid residues are more easily absorbed and often

52

more readily to exert significant beneficial effects, such as regulating

53

hypertension, hypertriglyceridemia and hypercholesterolemia26. Short peptides

54

often possess high activity, stability and specificity27. In addition, their bulk

55

production can be easy and economical. Therefore, the small peptides aroused

56

great research attention. Today there are already in use clinical polypeptides

57

such as exenatide, insulin and ziconotide28, 29. Meanwhile, a large number of

58

biopeptides with other activities also have been found, such as antimicrobial

59

peptides, analgesic peptides and so on30,

60

discovery of peptides with anti-hyperuricemic activity is still in infancy27, 32, 33.

31.

However, the research and

61

In this research, a novel peptide RDP1 was identified from the extract of

62

shelled fruits of local O. Sativa from Yunnan, China. Our results revealed that

63

RDP1 exert potent anti-hyperuricemic activity, which showed its potential value

64

in the development of anti-gout drugs and health food.

65 66 4


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Materials and methods

68

Sample preparation and animal care

69

O. Sativa was collected from Yunnan, China (shown as Supplement Fig.

70

1). Fruits of O. Sativa were shelled and then rice was obtained. The extract of

71

rice was obtained as following procedures, briefly, rice was soaked in

72

deionized water for 12 h at 4 °C, then supernatants were filtered by a filter

73

paper and centrifuged at 1,2000 g for 20 min at 4 °C, and then lyophilized and

74

stored at -80 °C until use.

75

Sprague Dawley male rats (150 ± 20 g) were commercially obtained from

76

the Hunan Slack Jingda Laboratory Animal Co., Ltd. (Hunan, China). Rats

77

were housed under room temperature (22 ± 2 °C), with free access to food

78

and water. Animal handling was in accordance with the Provision and General

79

Recommendation of Chinese Experimental Animals Administration

80

Legislation. All animal care and handling procedures were conducted in

81

accordance with the requirements of the Ethics Committee of Kunming

82

Medical University (KMMU20180012).

83

Purification procedures

84

The purification of peptide was performed as our previous report with some

85

modifications34. Briefly, the sample was purified by Sephadex G-50 (1.5 × 31

86

cm, superfine, GE Healthcare, Sweden) gel filtration column. The pre-

87

equilibrium used a 25 mM Tris-HCl buffer containing 0.1 M NaCl (pH 7.8) and

88

elution was achieved with the same buffer at a flow rate of 0.3 mL/min. An 5



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89

automatic fractionation collector (BSA-30A, HuXi Company, Shanghai, China)

90

was used to collected samples in tubes every 10 min and their absorbance at

91

280 nm was detected (Fig. 1A). The fraction was merged and then injected to

92

a C18 HPLC column (Hypersil BDS C18, 4.0 × 300 mm, Elite, China) with an

93

injection volume of 1 mL, which was pre-balanced with ultra-pure grade water

94

containing 0.1 % (v/v) trifluoroacetic acid (TFA). The elution was achieved by a

95

linear gradient (0-40 % ACN, 40 min, as shown in Fig. 1B) of acetonitrile (ACN)

96

containing 0.1 % (v/v) TFA at a flow rate of 1 mL/min and monitored at 220 nm.

97

The peak indicated by an arrow in Fig. 1B was collected and purified by a

98

second round of HPLC under same conditions as procedures mentioned above

99

(Fig. 1C).

100

The determination of peptide primary structure

101

The molecular mass of the sample was detected by mass spectrometry.

102

Briefly, 1 μL sample was mixed with 1 μL α-cyano-4-hydroxycinnimic acid (5

103

mg/mL, dissolved in 50 % ACN, 0.1 % TFA) and spotted on sample plate for

104

crystallization. The crystallized sample was analyzed by AutoFlex Speed

105

MALDI TOF/TOF mass spectrometer on positive mode. In order to determine

106

the amino acid sequence, sample was dissolved in 25 mM NH4HCO3, reduced

107

by dithiothreitol at 37 °C for 1 h and blocked by iodoacetamide for 30 min.

108

Then the sample was mixed with α-cyano-4-hydroxycinnimic acid and

109

analyzed by tandem mass spectrometry on the same equipment.

110

The artificial synthesis of peptide 6


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111


The RDP1 (AAAAGAKAR) with purity of > 95 % was synthesized and

112

provided commercially by Wuhan Bioyeargene Biotechnology Co., Ltd. (Wuhan,

113

China).

114

Hemolytic activity assay

115

Hemolysis activity was referenced to the experiment before and some

116

modifications were made35. Briefly, human red blood cells were washed three

117

times with saline. Different doses of RDP1 (10 µg/mL, 100 µg/mL, 1 mg/mL)

118

were incubated with red blood cells for 30 min at 37 °C and then centrifuged for

119

4 min, 4000 g at room temperature (22 ± 2 °C). The absorbance of the

120

supernatant was measured at 540 nm. 1% Triton X-100 was used to determine

121

the maximum hemolysis.

122

Acute toxicity assay

123

The acute toxicity test was carried out according to the previous report36.

124

Different concentrations of RDP1 (10 µg/kg, 100 µg/kg and 1 mg/kg) and equal

125

amount of saline were injected into rats by intraperitoneal injection, respectively,

126

then the mortality, toxicity and behavioral changes of rats within 24 h were

127

observed and recorded.

128

Anti-hyperuricemic assays in vivo

129

Animal assays were performed according to methods described in

130

previous research22. Briefly, Rats were randomly divided into six groups, the

131

control group, the model group, the allopurinol group (the positive control) and

132

three RDP1 groups (10 µg/kg, 100 µg/kg, 1 mg/kg). The control group was 7



Page 8 of 39

133

administrated 1 mL saline per day. Other groups were administrated 450

134

mg/kg potassium oxonate (POX, Dalian Meilun Biological Technology Co.,

135

Ltd, Dalian, Liaoning, China) and 100 mg/kg adenine (Dalian Meilun Biological

136

Technology Co., Ltd, Dalian, Liaoning, China) per day. RDP1 or allopurinol

137

was administrated to rats by intragastric administration 1 h after the treatment

138

of POX and adenine. The control and model group were treated with saline;

139

the allopurinol group was treated with allopurinol (10 mg/kg, Dalian Meilun

140

Biological Technology Co., Ltd, Dalian, Liaoning, China). RDP1 groups were

141

treated with different concentrations of RDP1 (10 µg/kg, 100 µg/kg and 1

142

mg/kg). POX and adenine were dissolved in saline, and rats were treated

143

daily with POX and adenine or saline (control) for 7 days by intragastric

144

administration.

145

In order to compare anti-hyperuricemic activity of the crude extract of

146

shelled fruits of O. Sativa and RDP1, rice (1 mg/kg) and RDP1 (1 mg/kg) was

147

administrated to rats by intragastric administration 1 h after the treatment of

148

POX and adenine.

149

Blood and kidney samples were obtained on the seventh day after the last

150

administration of RDP1, allopurinol or saline. The blood of rats was centrifuged

151

at 6000 g at room temperature (22 ± 2 °C) for 5 min to obtain the serum. The

152

serum level of uric acid and creatinine were measured with uric acid and

153

creatinine kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, Jiangsu,

154

China), and all operations were performed as follows: 8


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155

For the detection of serum uric acid, the blank sample (0.2 mL distilled water

156

was mixed sufficiently with 2 mL tungstate protein precipitator), standard

157

sample (0.2 mL 50 mg/L uric acid solution was mixed sufficiently with 2 mL

158

tungstate protein precipitator) and test sample (0.2 mL rat blood was mixed

159

sufficiently with 2 mL tungstate protein precipitator) were prepared. After 10 min,

160

these samples were centrifuged at 3000 g, 4 °C for 5 min and the supernatants

161

were kept. Next, 1.6 mL supernatant, 500 µL CUT reagent and 500 µL

162

phosphotungstic acid were mixed for 10 min and the absorbance values at 690

163

nm were detected. The concentration of serum of uric acid was calculated as

164

follows: Test ― Blank

165

Serum of uric acid (mg/L) = 50 ×

166

For the detection of serum creatine, the test sample (0.2 mL rat blood was

167

mixed sufficiently with 2 mL tungstate protein precipitator) was mixed and

168

centrifuged at 3500 g, 4 °C for 10 min, then took the supernatant. The blank

169

sample (1.6 mL distilled water was mixed sufficiently with 500 µL picric acid

170

solution and 500 µL 0.75 M NaOH solution), standard sample (1.6 mL 50 µM

171

creatine solution was mixed sufficiently with 500 µL picric acid solution and 500

172

µL 0.75 M NaOH solution) and test sample (1.6 mL supernatant was mixed

173

sufficiently with 500 µL picric acid solution and 500 µL 0.75 M NaOH solution)

174

were prepared. Next, they were incubated at 37 °C for 10 min and the

175

absorbance values at 510 nm were detected. The concentration of serum of

176

creatinine was calculated as follows:

Standard ― Blank

9



177 178

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Test ― Blank

Serum of creatinine (µM) = 50 × 11 × Standard ― Blank Histopathological Examination

179

The HE staining was performed according previous report35. Kidney tissues

180

of rats were fixed in 4 % formalin for 24 to 48 h, then dehydrated with gradient

181

ethanol (75 % for 12 h, 85 % for 12 h, 95 % and 100 % for 2 h, respectively).

182

Renal tissues were embedded in paraffin and sliced at a thickness of 5 µm and

183

operated the HE staining. The treated sections were visualized under a light

184

microscopy (Zeiss, Germany) at 200 × magnifications.

185

Measurement of inhibitory effects against XO in vivo and vitro

186

The xanthine oxidase inhibition assay was carried out according to the

187

previous method with slightly modifications 27. A 50 mM Tris-HCL buffer with a

188

pH of 8 was prepared, and the following samples and medicines were dissolved

189

in this buffer. 2 mM xanthine (Dalian Meilun Biological Technology Co., Ltd,

190

Dalian, Liaoning, China) solution and 0.52 mU/mL XO (Dalian Meilun Biological

191

Technology Co., Ltd, Dalian, Liaoning, China) solution were prepared. The

192

xanthine solution (128 µL), XO solution (16 µL), sample solution (32 µL, RDP1

193

or serum of rats) and buffer solution (928 µL) were mixed and incubated at

194

37 °C for 15 min. 48 µL 1M HCL was added to terminate the reaction and

195

absorbance at 292 nm was measured. Allopurinol and buffer were used as

196

positive control and negative control, respectively. The inhibitory activity is

197

calculated as follows:

198

XO inhibition rate (%) = 100 % ×

Negtive Control ― Sample Negtive Control

10


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199


Molecular docking

200

Molecular docking of the RDP1−XO complex was conducted to explore the

201

potential binding details37. The molecular docking experiments were conducted

202

by the MGL tools 1.5.6 with AutoDock vina 4.0. The X-ray crystal structure of

203

XO in complex with indole-3-aldehyde (PDB ID: 3NVZ) was downloaded from

204

the RCSB Protein Data Bank (http://www.rcsb.org/pdb). The 3D structures of

205

RDP1 was predicted by the PEP-FOLD3 server (http://bioserv.rpbs.univ-paris-

206

diderot.fr/services/PEP-FOLD3/). All the water molecules in XO were excluded

207

and polar hydrogen atoms were added before assigning Gasteiger charges to

208

the macromolecule file for the preparation of docking simulation. A grid box with

209

dimensions of 50 Å × 50 Å ×50 Å was defined to enclose the predicted binding

210

site with a certain grid spacing of 1.0 Å. Afterwards, the docking simulations

211

were carried out with the default vina parameters. Finally, all the docking

212

models were sorted by the estimated affinity value and the one with the lowest

213

value (highest affinity) was selected as its most favorable binding mode.

214

Stability of RDP1

215

The stability of RDP1 was tested as previously described, with some

216

modifying35. In the detection of stability in plasma, 100 µL human plasma and

217

100 µL RDP1 (10 µg/mL) were mixed and incubated at 37 °C, then the content

218

of RDP1 was detected every 5 min until the peptide degraded completely. 100

219

µL urea (8 M), 60 µL trichloroacetic acid (1 g/mL) were added in the mixture to

220

terminate the reaction, then centrifuged them at 1,2000 g for 30 min and the 11



Page 12 of 39

221

supernatant was obtained. In iterative freezing and thawing stability test, 1 mL

222

RDP1 (10 µg/mL) was placed in liquid nitrogen for 5 min, then the sample was

223

taken out and thawed at 37 °C for 5 min. This operation was repeated for

224

different times, and then content of RDP1 was detected. The stability of RDP1

225

at 4 °C, 37 °C and 60 °C were also checked. Briefly, RDP1 (10 µg/mL) was

226

incubated at 4 °C, 37 °C and 60 °C for different days, samples were taken at

227

the settled time points and then centrifuged at 1,2000 g for 20 min, the

228

supernatant was obtained.

229

The samples were injected to a C18 HPLC column (Hypersil BDS C18, 4.0

230

× 300 mm, Elite, China) with an injection volume of 1 mL, which was pre-

231

balanced with ultra-pure grade water containing 0.1 % (v/v) TFA. The elution

232

was achieved by a linear gradient (0-30 % ACN, 30 min) of ACN containing 0.1

233

% (v/v) TFA at a flow rate of 1 mL/min and monitored at 220 nm. The residual

234

quantity of RDP1 was determined and quantified from the area of peak (the

235

elution time of RDP1) absorbance at 220 nm.

236 237

Results and Discussion

238

Purification and the primary structure of RDP1.

239

Extracts from shelled fruits of O. Sativa were purified by Sephadex G-50

240

gel filtration column and fractioned into several parts. Samples collected from

241

the peak indicated in Fig. 1A by an arrow was further separated and purified

242

by RP-HPLC. As shown in Fig. 1B, more than 40 peaks were obtained and 12


Page 13 of 39


243

one of them (indicated by an arrow in Fig. 1B) was further purified by HPLC,

244

and a peak with elution time of 14.4 min was obtained (Fig. 1C). Then this

245

sample was analyzed by mass spectrum.

246

As displayed in Fig.2A, a main peak with m/z of 785.91 was detected,

247

indicated both the molecular weight and purity of this sample. Tandem mass

248

spectrometry analysis was employed to elucidate the sequence of this sample

249

and ‘AAAAGAKAR’ was confirmed (Fig. 2B). The results from Blastp search in

250

the NCBI database revealed that this peptide showed no obvious sequence

251

similarity with other peptides. Therefore, it was considered to be a new bioactive

252

peptide and named as RDP1 (Rice-Derived-Peptide-1). Its chemical structure

253

was shown in Fig. 2C. The theoretical molecular mass, as calculated at

254

http://web.expasy.org/compute_pi/, was 785.90 Da, which was fit well with the

255

observed

256

posttranslational modification of RDP1.

molecular

mass

(785.91

Da),

indicating

there

was

no

257

Up to day only a few anti-hyperuricemic peptides have been identified,

258

including ‘YLDNY’ and ‘SPPYWPY’ from shark cartilage water extract, some

259

dipeptides from milk protein, ‘WPPKN’ and ‘ADIYTE’ from walnut protein

260

hydrolysate27, 32, 33. Based on our knowledge, RDP1 was the first discovered

261

peptide with anti-hyperuricemic activity from rice, and the second known plant-

262

derived anti-hyperuricemic peptide. Compared with those reported anti-

263

hyperuricemic peptides (‘YLDNY’ and ‘SPPYWPY’, ‘WPPKN’ and ‘ADIYTE’),

264

RDP1 (AAAAGAKAR) displayed the longest sequence. It was worth noting that, 13



Page 14 of 39

265

RDP1 contained six alanine residues out of its own nine amino acid residues,

266

as contrary, in sequences of other known anti-hyperuricemic peptides, there

267

was no such phenomenon in which one kind of the amino acid occupied such

268

a high proportion.

269

RDP1 showed neither hemolytic activity against human blood cells nor

270

acute toxicity against rats.

271

RDP1 had no hemolytic activity at the highest concentration of 1 mg/mL

272

(as shown in Table 1). In the acute toxicity test, rats were injected with RDP1

273

intraperitoneally at a single dose of 10 µg/kg, 100 µg/kg and 1 mg/kg, no dead

274

individual was observed in 24 h (as shown in Table 2) and the general

275

condition was not changed (data not shown). The results confirmed RDP1

276

revealed no adverse side effects on neither the rats nor human red cells.

277

RDP1 significantly decreased serum levels of uric acid & creatinine and

278

alleviated kidney injury in hyperuricemic rats.

279

When the concentration of uric acid in the blood increases, or pH value of

280

body changes, the supersaturated uric acid will precipitate into uric acid

281

crystals. The crystals deposited in joints and various tissues can cause joint

282

pain, visceral damage and so on10. In this study, in order to determine the

283

anti-hyperuricemic effect of RDP1 in vivo, serum levels of uric acid and

284

creatinine in hyperuricemic rats were measured. As shown in Fig. 3A, the

285

serum level of uric acid was 14.52 ± 0.49 mg/L (n = 5) in the control group, as

286

contrary, the serum uric acid of the model group was 49.24 ± 1.96 mg/L (n = 14


Page 15 of 39


287

5), suggesting that the administration of POX and adenine induce

288

hyperuricemia in rats. In the allopurinol group, the level of serum uric acid was

289

30.74 ± 1.25 mg/L (n = 5), which significantly alleviated the hyperuricemia

290

induced by POX and adenine. In the RDP1 groups (10 µg/kg, 100 µg/kg and 1

291

mg/kg), as illustrated in Fig. 3A, concentrations of serum uric acid were 32.99

292

± 0.98 mg/kg, 28.52 ± 1.14 mg/kg and 24.74 ± 1.21 mg/kg, respectively (n =

293

5), thus, conclusion came to that, RDP1 showed obvious capacity in lowering

294

the serum uric acid, and the activity was concentration-dependent. It was also

295

worth mentioning that, RDP1 (100 µg/kg and 1 mg/kg) demonstrated a more

296

potent anti-hyperuricemic ability than that of allopurinol (positive control) (10

297

mg/kg), which exhibited the potential of RDP1 to develop into an anti-

298

hyperuricemic drug candidate. In consideration of that POX was used to

299

inhibit the degradation of urate by urate oxidase. Therefore, the decrease of

300

serum urate levels after the intragastric administration of RDP1 may be

301

caused not only by the inhibition of XO, but also by the loss of inhibition of

302

urate oxidase.

303

Uric acid nephropathy is due to the accumulation of uric acid crystals in renal

304

tissues which may cause recurrent inflammation. In clinical, serum level of

305

creatinine is a sensitive indicator for renal injury10,

306

serum creatinine concentration of the control group was 56.81 ± 4.43 µM (n =

307

5), while that of the model group was 135.75 ± 9.77 µM (n = 5). Compared with

308

the control group, rats in the model group exhibited a significant increase in the

38.

As shown in Fig. 3B,

15



Page 16 of 39

309

serum creatinine (P < 0.0001), suggesting that the obviously renal injury

310

occurred to rats treated with POX and adenine. The serum creatinine

311

concentration of the allopurinol group (10 mg/kg) was 58.51 ± 6.68 µM (n = 5),

312

and serum creatinine concentrations of the RDP1 groups (10 µg/kg, 100 µg/kg,

313

1 mg/kg) were 83.02 ± 12.71 µM, 71.17 ± 16.92 µM and 63.90 ± 8.45 µM,

314

respectively (n = 5). It was also observed that the creatinine-lowering ability of

315

RDP1 (1mg/kg) was close to that of allopurinol (10 mg/kg) and the activity was

316

concentration-dependent. In order to further verify the protective effect of RDP1

317

on renal injury induced by hyperuricemia, the HE staining was also performed.

318

As depicted in Fig. 4, the renal pathological changes in the model group were

319

marked by the disappearance of brush margin and the atrophy of renal tubules.

320

The renal pathological changes in the RDP1 group and allopurinol group were

321

significantly alleviated, and the effect was similar. All these results suggested

322

that RDP1 exert significant anti-hyperuricemic and renal protective activity, and

323

was significantly effective by intragastric administration.

324

Among other known anti-hyperuricemic peptides, ‘YLDNY’ and ‘SPPYWPY’

325

were treated at higher concentrations of 5 mg/kg, 15 mg/kg, 50 mg/kg, and

326

‘SPPYWPY’ only exhibited anti-hyperuricemic activity by intraperitoneal

327

injection; ‘WPPKN’ and ‘ADIYTE’ showed anti-hyperuricemic activity at a much

328

higher concentration of 300 mg/kg27, 32, 33. Thus, compared with them, RDP1

329

demonstrated the reducing uric acid activity at lower concentrations (10 µg/kg,

330

100 µg/kg and 1 mg/kg). The remarkable biological activity of RDP1 provided 16


Page 17 of 39


331

solid evidence for its potential to be develop as a new drug candidate to treat

332

against gout. However, another crucial issue which should be considered is that

333

adenine can be oxidized to 2, 8-dihydroxyadenine by XO and thus may cause

334

adenine nephropathy. In this connection, the therapeutic effect of RDP1 is

335

apparent because it can attenuate not only hyperuricemic nephropathy, but also

336

adenine nephropathy, and indeed, it may alleviate at least one of two renal

337

diseases.

338

In addition, the anti-hyperuricemic activity of extracts of rice from O. Sativa

339

and RDP1 were also determined and compared. As displayed in Fig. S2A, the

340

uric acid-lowering activity of rice was only half of that of RDP1, besides, as

341

shown in Fig.1A and B, the purification procedures of RDP1 revealed a poor

342

content in the extract of shelled fruits of O. Sativa, these results suggested that

343

the content of RDP1 in shelled fruits of O. Sativa was not predominant. However,

344

the creatinine-lowering activity of RDP1 was lower than the extract of rice from

345

O. Sativa (Fig. S2B). Therefore, it could be hypothesized that there is a

346

synergistic effect between the RDP1 and the other compounds existing in the

347

extracts of shelled fruits of O. Sativa with substantial renal-protective ability to

348

RDP1. Anyway, these results also indicated that the shelled fruits of O. Sativa,

349

rice, can be directly used as a health product for gout and uric acid nephropathy.

350

RDP1 inhibited XO activity both in vivo and in vitro.

351

In the purine metabolic pathway of higher mammals, XO converts

352

hypoxanthine to xanthine and then to uric acid39. Therefore, XO is an important 17



Page 18 of 39

353

target against gout40. To further explore the underlying molecular mechanism

354

of RDP1 involving in the reduction of uric acid levels, we explored its inhibitory

355

effect on XO in vivo and in vitro. As shown in Fig. 5A, the direct interaction of

356

RDP1 with XO was detected in vitro, and RDP1 inhibited XO concentration-

357

dependently. At the concentration of 1 mg/kg, the inhibitory activity of RDP1

358

was 3/4 out of allopurinol (1 mg/mL) (n = 5). From above experimental results,

359

we assumed that RDP1 have the potential of binding and interacting with XO,

360

which was further confirmed by molecular docking.

361

In vivo, the serum of rats treated with 10 µg/kg RDP1 showed no XO-

362

inhibitory activity, whereas, 100 µg/kg and 1 mg/kg RDP1 exhibited the

363

inhibitory activity against XO (n = 5). The XO inhibitory activity of RDP1 (1 mg/kg)

364

was higher than that in allopurinol (10 mg/kg) (Fig. 5B). As the previous report

365

mentioned, proteins and peptides can be further degraded into smaller peptides

366

or amino acids during digestion and absorption and thus their biological

367

activities were affected27. Therefore, RDP1 might be broken down or digested

368

into shorter peptides after digestion and absorption through the gastrointestinal

369

tract, and the ability to inhibit XO was enhanced accordingly.

370

Results from molecular docking revealed RDP1 occupied the binding

371

site of XO to its substrate xanthine.

372

In order to further explore the interaction between RDP1 and XO,

373

molecular docking was performed. As shown in Fig. 6A and B, RDP1 mainly

374

consisted of hydrophilic residues, which form electrostatic interaction and 18


Page 19 of 39


375

hydrogen bonding with the surrounding residues, and with almost no

376

hydrophobic accumulation. The XO structure (PDB ID: 3nvz) was submitted to

377

the CavityPlus server, and 10 possible cavities were predicted, then five

378

possible binding sites were obtained according to the location of cavity in the

379

XO structure (Table S1). Autodock-vina software was used to carry out

380

peptide-protein docking in the above 5 binding sites respectively. The

381

combination of RDP1 and site1 was most likely with the affinity of -10.2 (Table

382

S2). Therefore, the following analysis were about the docking results of site1

383

and RDP1. As shown in Fig. 6C and D, the amino N of Ala1 in RDP1 forms a

384

hydrogen bond with Asn19 amide O on XO at a distance of 3.3 Å; the carboxyl

385

group on Arg9 in RDP1 formed a hydrogen bond with the side chains of

386

Arg32, Arg598 and Glu676 carboxylic groups, and the heavy atom distances

387

are 3.0 Å, 3.3 Å and 3.1 Å, respectively; the amino side chains on Arg9

388

formed a hydrogen bond with the carboxyl groups of Asp21 and Glu232 on

389

XO with the heavy atom distances of 3 Å and 3.3 Å respectively. In

390

conclusion, RDP1 occupied the Mo domain, which is one of a drug active

391

cavity of XO, and Glu232, as one of the binding sites of xanthine (substrate)-

392

XO (enzyme) interaction, plays an important role in this reaction41. Therefore,

393

it could be speculated that RDP1 inhibited the interaction between XO and

394

xanthine, thus reducing the production of uric acid.

395

RDP1 was rapidly degraded in plasma, but stable in some other cases.

396

As shown in Fig. 7A, the stability of RDP1 in the plasma environment was 19



Page 20 of 39

397

first detected. 20 min after incubation with plasma, RDP1 was completely

398

degraded, and the half-life was about 4.6 min (calculated by GraphPad Prism

399

software) (n = 3). However, in animal experiments, the level of serum uric acid

400

in rats treated with RDP1 for more than 1 h showed a significantly reducing

401

compared with the model group, which demonstrated the anti-hyperuricemic

402

activity of RDP1 still maintain after 1 h in vivo. Therefore, we speculated that

403

RDP1 is transferred into shorter peptides by enzymolysislytic effect in vivo, and

404

its biological activity is not affected or even enhanced.

405

In order to explore the characteristics of RDP1 in storage and

406

transportation, its stability under various conditions was tested. In repeated

407

freeze-thaw test, after repeated freezing and thawing for ten times, the content

408

of RDP1 was remained about 80 % (Fig. 7B) (n = 3). As shown in Fig. 7B, the

409

content of RDP1 at 4°C and 37°C showed a stable characteristic even in 10

410

day. At 60 °C in 10 day, RDP1 didn’t completely degrade and maintained about

411

50 % (n = 3). The excellent stability, which can be maintained for a long time at

412

4 °C and 37 °C and can be maintained for a time at 60 °C, demonstrated the

413

advantages of transportation and preservation of RDP1.

414

In conclusion, RDP1 (AAAAGAKAR, 785.91 Da) originated from the extract

415

of shelled fruits of O. Sativa, displayed little hemolytic or acute toxin effects.

416

RDP1 exhibited significant ability to reduce the serum level of uric acid and

417

alleviate hyperuricemic nephropathy with a minimum effective concentration of

418

10 µg/kg. RDP1 could control hyperuricemia by directly inhibiting the XO. These 20


Page 21 of 39


419

results suggested the potential of RDP1 as an anti-hyperuricemic drug, and

420

indicated that the local rice of O. Sativa can be developed as a new generation

421

of anti-gout health food.

422 423

Author information

424

*Corresponding

425

Dr. Xinwang Yang: [email protected]

426

Dr. Jun Sun: [email protected]

427

Dr. Buliang Meng: [email protected]

428

Author Contributions

429

†Naixin

Author

Liu, Ying Wang and Meifeng Yang contributed equally to this work.

430 431

Notes

432

The authors declare no competing financial interest.

433 434 435

Acknowledgements This work was supported by the Chinese National Natural Science

436

Foundation (81760648, 31670776 and 31460571), Yunnan Applied Basic

437

Research Project Foundation (2017FB035) and Yunnan Applied Basic

438

Research Project-Kunming Medical University Union Foundation (2018FE001

439

(-161))

440 21



441

Page 22 of 39

Supporting Information

442

Fig. S1, plants, fruits and rice from O. Sativa collected from Yunnan, China;

443

Fig. S2, anti-hyperuricemic activity of crude extracts of shelled fruits of O. Sativa

444

and RDP1; Table S1, docking parameters of XO; Table 2, affinity value of 5

445

sites of RDP1 and XO.

446 447

References

448

1. McCarty, D. J., A historical note: Leeuwenhoek's description of crystals

449

from a gouty tophus. Arthritis Rheum 1970, 13, 414-8.

450

2. Dalbeth, N.; Bardin, T.; Doherty, M.; Liote, F.; Richette, P.; Saag, K. G.;

451

So, A. K.; Stamp, L. K.; Choi, H. K.; Terkeltaub, R., Discordant American

452

College of Physicians and international rheumatology guidelines for gout

453

management: consensus statement of the Gout, Hyperuricemia and Crystal-

454

Associated Disease Network (G-CAN). Nat Rev Rheumatol 2017, 13, 561-

455

568.

456

3. Li, H.; Zhao, M.; Su, G.; Lin, L.; Wang, Y., Effect of Soy Sauce on Serum

457

Uric Acid Levels in Hyperuricemic Rats and Identification of Flazin as a Potent

458

Xanthine Oxidase Inhibitor. J Agric Food Chem 2016, 64, 4725-34.

459

4. Hu, J.; Wu, H.; Wang, D.; Yang, Z.; Zhuang, L.; Yang, N.; Dong, J.,

460

Weicao capsule ameliorates renal injury through increasing autophagy and

461

NLRP3 degradation in UAN rats. Int J Biochem Cell Biol 2018, 96, 1-8.

462

5. Niskanen, L. K.; Laaksonen, D. E.; Nyyssonen, K.; Alfthan, G.; Lakka, H. 22


Page 23 of 39


463

M.; Lakka, T. A.; Salonen, J. T., Uric acid level as a risk factor for

464

cardiovascular and all-cause mortality in middle-aged men: a prospective

465

cohort study. Arch Intern Med 2004, 164, 1546-51.

466

6. Kokichi, A., Recent decreasing trends of exposure to

467

PCDDs/PCDFs/dioxin-like PCBs in general populations, and associations with

468

diabetes, metabolic syndrome, and gout/hyperuricemia. J Med Invest 2018,

469

65, 151-161.

470

7. Cheng, D.; Du, R.; Wu, X. Y.; Lin, L.; Peng, K.; Ma, L. N.; Xu, Y.; Xu, M.;

471

Chen, Y. H.; Bi, Y. F.; Wang, W. Q.; Dai, M.; Lu, J. L., Serum Uric Acid is

472

Associated with the Predicted Risk of Prevalent Cardiovascular Disease in a

473

Community-dwelling Population without Diabetes. Biomed Environ Sci 2018,

474

31, 106-114.

475

8. Kawada, T., Hyperuricaemia and type 2 diabetes mellitus. Clin Exp

476

Pharmacol Physiol 2018, 45, 870.

477

9. Mene, P.; Punzo, G., Uric acid: bystander or culprit in hypertension and

478

progressive renal disease? J Hypertens 2008, 26, 2085-92.

479

10. Pascart, T.; Liote, F., Gout: state of the art after a decade of

480

developments. Rheumatology (Oxford) 2018.

481

11. Pascart, T.; Richette, P., Investigational drugs for hyperuricemia, an

482

update on recent developments. Expert Opin Investig Drugs 2018, 27, 437-

483

444.

484

12. Kim, S. C.; Neogi, T.; Kang, E. H.; Liu, J.; Desai, R. J.; Zhang, M.; 23



Page 24 of 39

485

Solomon, D. H., Cardiovascular Risks of Probenecid Versus Allopurinol in

486

Older Patients With Gout. J Am Coll Cardiol 2018, 71, 994-1004.

487

13. Alemzadeh-Ansari, M. J.; Hosseini, S. K.; Talasaz, A. H.; Mohammadi, M.;

488

Tokaldani, M. L.; Jalali, A.; Pourhosseini, H., Effect of High-Dose Allopurinol

489

Pretreatment on Cardiac Biomarkers of Patients Undergoing Elective

490

Percutaneous Coronary Intervention: A Randomized Clinical Trial. Am J Ther

491

2017, 24, e723-e729.

492

14. Ansari-Ramandi, M. M.; Maleki, M.; Alizadehasl, A.; Amin, A.; Taghavi, S.;

493

Alemzadeh-Ansari, M. J.; Kazem Moussavi, A.; Naderi, N., Safety and effect

494

of high dose allopurinol in patients with severe left ventricular systolic

495

dysfunction. J Cardiovasc Thorac Res 2017, 9, 102-107.

496

15. Garritsen, F. M.; van der Schaft, J.; de Graaf, M.; Hijnen, D. J.; Bruijnzeel-

497

Koomen, C. A. F.; van den Broek, M. P. H.; De Bruin-Weller, M. S., Allopurinol

498

Co-prescription Improves the Outcome of Azathioprine Treatment in Chronic

499

Eczema. Acta Derm Venereol 2018, 98, 373-375.

500

16. Kwak, C. H.; Sohn, M.; Han, N.; Cho, Y. S.; Kim, Y. S.; Oh, J. M.,

501

Effectiveness of febuxostat in patients with allopurinol-refractory

502

hyperuricemic chronic kidney disease. Int J Clin Pharmacol Ther 2018, 56,

503

321-327.

504

17. Mukri, M. N. A.; Kong, W. Y.; Mustafar, R.; Shaharir, S. S.; Shah, S. A.;

505

Abdul Gafor, A. H.; Mohd, R.; Abdul Cader, R.; Kamaruzaman, L., Role of

506

febuxostat in retarding progression of diabetic kidney disease with 24


Page 25 of 39


507

asymptomatic hyperuricemia: A 6-months open-label, randomized controlled

508

trial. EXCLI J 2018, 17, 563-575.

509

18. Ruggeri, M.; Basile, M.; Drago, C.; Rolli, F. R.; Cicchetti, A., Cost-

510

Effectiveness Analysis of Lesinurad/Allopurinol Versus Febuxostat for the

511

Management of Gout/Hyperuricemia in Italy. Pharmacoeconomics 2018, 36,

512

625-636.

513

19. Lee, M. H.; Graham, G. G.; Williams, K. M.; Day, R. O., A benefit-risk

514

assessment of benzbromarone in the treatment of gout. Was its withdrawal

515

from the market in the best interest of patients? Drug Saf 2008, 31, 643-65.

516

20. Les, F.; Prieto, J. M.; Arbones-Mainar, J. M.; Valero, M. S.; Lopez, V.,

517

Bioactive properties of commercialised pomegranate (Punica granatum) juice:

518

antioxidant, antiproliferative and enzyme inhibiting activities. Food Funct 2015,

519

6, 2049-57.

520

21. Zhang, Z. C.; Su, G. H.; Luo, C. L.; Pang, Y. L.; Wang, L.; Li, X.; Wen, J.

521

H.; Zhang, J. L., Effects of anthocyanins from purple sweet potato (Ipomoea

522

batatas L. cultivar Eshu No. 8) on the serum uric acid level and xanthine

523

oxidase activity in hyperuricemic mice. Food Funct 2015, 6, 3045-55.

524

22. Chen, L.; Lan, Z., Polydatin attenuates potassium oxonate-induced

525

hyperuricemia and kidney inflammation by inhibiting NF-kappaB/NLRP3

526

inflammasome activation via the AMPK/SIRT1 pathway. Food Funct 2017, 8,

527

1785-1792.

528

23. Wang, M. X.; Zhao, X. J.; Chen, T. Y.; Liu, Y. L.; Jiao, R. Q.; Zhang, J. H.; 25



Page 26 of 39

529

Ma, C.; Liu, J. H.; Pan, Y.; Kong, L. D., Nuciferine Alleviates Renal Injury by

530

Inhibiting Inflammatory Responses in Fructose-Fed Rats. J Agric Food Chem

531

2016.

532

24. Honda, S.; Masuda, T., Identification of Pyrogallol in the Ethyl Acetate-

533

Soluble Part of Coffee as the Main Contributor to Its Xanthine Oxidase

534

Inhibitory Activity. J Agric Food Chem 2016.

535

25. Tung, Y. T.; Hsu, C. A.; Chen, C. S.; Yang, S. C.; Huang, C. C.; Chang, S.

536

T., Phytochemicals from Acacia confusa heartwood extracts reduce serum

537

uric acid levels in oxonate-induced mice: their potential use as xanthine

538

oxidase inhibitors. J Agric Food Chem 2010, 58, 9936-41.

539

26. Sachdeva, S.; Lobo, S.; Goswami, T., What is the future of noninvasive

540

routes for protein- and peptide-based drugs? Ther Deliv 2016, 7, 355-7.

541

27. Murota, I.; Taguchi, S.; Sato, N.; Park, E. Y.; Nakamura, Y.; Sato, K.,

542

Identification of antihyperuricemic peptides in the proteolytic digest of shark

543

cartilage water extract using in vivo activity-guided fractionation. J Agric Food

544

Chem 2014, 62, 2392-7.

545

28. Soudry-Kochavi, L.; Naraykin, N.; Di Paola, R.; Gugliandolo, E.; Peritore,

546

A.; Cuzzocrea, S.; Ziv, E.; Nassar, T.; Benita, S., Pharmacodynamical Effects

547

of Orally Administered Exenatide Nanoparticles Embedded in Gastro-resistant

548

Microparticles. Eur J Pharm Biopharm 2018.

549

29. Hamad, M. K.; He, K.; Abdulrazeq, H. F.; Mustafa, A. M.; Luceri, R.;

550

Kamal, N.; Ali, M.; Nakhla, J.; Herzallah, M. M.; Mammis, A., Potential Uses of 26


Page 27 of 39


551

Isolated Toxin Peptides in Neuropathic Pain Relief: A Literature Review.

552

World Neurosurg 2018, 113, 333-347 e5.

553

30. Wang, Y.; Li, X.; Yang, M.; Wu, C.; Zou, Z.; Tang, J.; Yang, X., Centipede

554

venom peptide SsmTX-I with two intramolecular disulfide bonds shows

555

analgesic activities in animal models. J Pept Sci 2017, 23, 384-391.

556

31. Yang, X.; Lee, W. H.; Zhang, Y., Extremely abundant antimicrobial

557

peptides existed in the skins of nine kinds of Chinese odorous frogs. J

558

Proteome Res 2012, 11, 306-19.

559

32. Li, Q.; Kang, X.; Shi, C.; Li, Y.; Majumder, K.; Ning, Z.; Ren, J.,

560

Moderation of hyperuricemia in rats via consuming walnut protein hydrolysate

561

diet and identification of new antihyperuricemic peptides. Food Funct 2018, 9,

562

107-116.

563

33. Nongonierma, A. B.; Fitzgerald, R. J., Tryptophan-containing milk protein-

564

derived dipeptides inhibit xanthine oxidase. Peptides 2012, 37, 263-72.

565

34. Li, X.; Wang, Y.; Zou, Z.; Yang, M.; Wu, C.; Su, Y.; Tang, J.; Yang, X.,

566

OM-LV20, a novel peptide from odorous frog skin, accelerates wound healing

567

in vitro and in vivo. Chem Biol Drug Des 2018, 91, 126-136.

568

35. Bian, W.; Meng, B.; Li, X.; Wang, S.; Cao, X.; Liu, N.; Yang, M.; Tang, J.;

569

Wang, Y.; Yang, X., OA-GL21, a novel bioactive peptide from Odorrana

570

andersonii, accelerated the healing of skin wounds. Biosci Rep 2018, 38.

571

36. Cao, X.; Wang, Y.; Wu, C.; Li, X.; Fu, Z.; Yang, M.; Bian, W.; Wang, S.;

572

Song, Y.; Tang, J.; Yang, X., Cathelicidin-OA1, a novel antioxidant peptide 27



Page 28 of 39

573

identified from an amphibian, accelerates skin wound healing. Scientific

574

reports 2018, 8, 943.

575

37. Trott, O.; Olson, A. J., AutoDock Vina: improving the speed and accuracy

576

of docking with a new scoring function, efficient optimization, and

577

multithreading. J Comput Chem 2010, 31, 455-61.

578

38. Bellomo, R.; Ronco, C.; Kellum, J. A.; Mehta, R. L.; Palevsky, P.; Acute

579

Dialysis Quality Initiative, w., Acute renal failure - definition, outcome

580

measures, animal models, fluid therapy and information technology needs: the

581

Second International Consensus Conference of the Acute Dialysis Quality

582

Initiative (ADQI) Group. Crit Care 2004, 8, R204-12.

583

39. Blau, N.; Erlandsen, H., The metabolic and molecular bases of

584

tetrahydrobiopterin-responsive phenylalanine hydroxylase deficiency. Mol

585

Genet Metab 2004, 82, 101-11.

586

40. Elion, G. B., The purine path to chemotherapy. Science 1989, 244, 41-7.

587

41. Truglio, J. J.; Theis, K.; Leimkuhler, S.; Rappa, R.; Rajagopalan, K. V.;

588

Kisker, C., Crystal structures of the active and alloxanthine-inhibited forms of

589

xanthine dehydrogenase from Rhodobacter capsulatus. Structure 2002, 10,

590

115-25.

591 592

Figure legends:

593

Figure 1. Peptide purification procedures.

594

The extracts of shelled fruits of O. Sativa were separated by a Sephadex G28


Page 29 of 39


595

50 column and the samples indicated by an arrow in Fig. 1A was collected and

596

purified by a HPLC procedure, the sample indicated by an arrow in Fig. 1B was

597

further purified by another round of HPLC procedure. Finally, a peptide was

598

purified, which was indicated by an arrow in Fig. 1C.

599

Figure 2. Primary structure of RDP1.

600

A. The observed molecular weight of native RDP1.

601

B. Sequence of RDP1. The complete sequence of RDP1 was determined as

602

‘AAAAGAKAR’ by tender mass analysis.

603

C. Chemical structure of RDP1. The structure was manually produced by

604

ChemDraw software.

605

Figure 3. RDP1 significantly reduced the serum level of uric acid and

606

creatinine of hyperuricemic rats.

607

In anti-hyperuricemic assays in vivo, RDP1 (1 mg/kg) showed more potent

608

activity than allopurinol (10 mg/kg) (n = 5) in uric acid reducing (showed in Fig.

609

3A), moreover, as shown in Fig. 3B, RDP1 (1 mg/kg) showed similar renal

610

protection activity with allopurinol (10 mg/kg) (n = 5).

611

*P < 0.05, **P < 0.01, and ***P < 0.0001 indicate significantly different from

612

the control (Student’s t tests).

613

Figure 4. RDP1 alleviated renal injury on hyperuricemic rats.

614

Rats were randomly divided into six groups: the control group, the model

615

group, the allopurinol group (10 mg/kg) and three RDP1 groups (10 µg/kg,

616

100 µg/kg, 1 mg/kg). The renal tissues were collected on the seventh day 29



Page 30 of 39

617

after the last administration of RDP1, allopurinol or saline, and were operated

618

with the HE staining. The samples were observed at 200 × magnifications.

619

Figure 5. RDP1 inhibited the activity of XO in vivo and in vitro.

620

As shown in Fig. 5A, in vitro, RDP1’s (1 mg/kg) inhibiting activity against XO

621

was a quarter of that of allopurinol (1 mg/kg) (n = 5). In vivo, RDP1 (1 mg/kg)

622

revealed a higher inhibitory activity against XO than that of allopurinol (10

623

mg/kg) (n = 5) (Fig. 5B).

624

*P < 0.05, **P < 0.01, and ***P < 0.0001 indicate significantly different from

625

the control (Student’s t tests).

626

Figure 6. Molecular docking

627

The molecular docking results of RDP1 to XO revealed the chief amino acid

628

residues of RDP1 and XO in the active site and the ligand were represented

629

with sticks. The yellow dashed lines stood for hydrogen bonds. In Fig. 6, the

630

global (Fig.6 A) and local (Fig.6 B) visual angle of interaction between RDP1

631

and XO were showed, besides, the global (Fig.6 C) and local (Fig.6 D) view of

632

residues interacting with RDP1 and XO were showed.

633

Figure 7. Stability of RDP1.

634

The stability of RDP1 in the plasma environment was showed in Fig. 7A,

635

RDP1 was completely degraded in 20 min, and the half-life was about 4.626

636

min (calculated by GraphPad Prism software) (n = 3). In freeze-thaw assay, the

637

content of RDP1 was remained about 80 % in ten times (n = 3) (Fig. 7B) and

638

Fig. 7B also showed the stability of RDP1 at 4 °C, 37 °C and 60 °C in 10 days 30


Page 31 of 39

639


(n = 3).

Tables Table 1. RDP1 showed no hemolytic activity. Group

Hemolytic ratio (%)

Triton

100.0

Saline

4.5 ± 0.1

10 µg/mL RDP1

5.0 ± 0.6

100 µg/mL RDP1

4.9 ± 0.1

1 mg/mL RDP1

5.4 ± 0.2

Table 2. RDP1 showed no acute toxicity activity. Number of rats Group

male

female Mortality rate (%)

Negative control (saline)

3

3

0

10 µg/kg RDP1

3

3

0

100 µg/kg RDP1

3

3

0

1 mg/kg RDP1

3

3

0

Experimental group

31



Figure 1. Peptide purification procedures. The extracts of shelled fruits of O. Sativa were separated by a Sephadex G-50 column and the samples indicated by an arrow in Fig. 1A was collected and purified by a HPLC procedure, the sample indicated by an arrow in Fig. 1B was further purified by another round of HPLC procedure. Finally, a peptide was purified, which was indicated by an arrow in Fig. 1C. 84x122mm (300 x 300 DPI)


Page 32 of 39

Page 33 of 39


Figure 2. Primary structure of RDP1. A. The observed molecular weight of native RDP1. B. Sequence of RDP1. The complete sequence of RDP1 was determined as ‘AAAAGAKAR’ by tender mass analysis. C. Chemical structure of RDP1. The structure was manually produced by ChemDraw software. 82x106mm (300 x 300 DPI)



Figure 3. RDP1 significantly reduced the serum level of uric acid and creatinine of hyperuricemic rats. In anti-hyperuricemic assays in vivo, RDP1 (1 mg/kg) showed more potent activity than allopurinol (10 mg/kg) (n = 5) in uric acid reducing (showed in Fig. 3A), moreover, as shown in Fig. 3B, RDP1 (1 mg/kg) showed similar renal protection activity with allopurinol (10 mg/kg) (n = 5). *P < 0.05, **P < 0.01, and ***P < 0.0001 indicate significantly different from the control (Student’s t tests). 84x177mm (300 x 300 DPI)


Page 34 of 39

Page 35 of 39


Figure 4. RDP1 alleviated renal injury on hyperuricemic rats. Rats were randomly divided into six groups: the control group, the model group, the allopurinol group (10 mg/kg) and three RDP1 groups (10 µg/kg, 100 µg/kg, 1 mg/kg). The renal tissues were collected on the seventh day after the last administration of RDP1, allopurinol or saline, and were operated with the HE staining. The samples were observed at 200 × magnifications. 82x109mm (300 x 300 DPI)



Figure 5. RDP1 inhibited the activity of XO in vivo and in vitro. As shown in Fig. 5A, in vitro, RDP1’s (1 mg/kg) inhibiting activity against XO was a quarter of that of allopurinol (1 mg/kg) (n = 5). In vivo, RDP1 (1 mg/kg) revealed a higher inhibitory activity against XO than that of allopurinol (10 mg/kg) (n = 5) (Fig. 5B). *P < 0.05, **P < 0.01, and ***P < 0.0001 indicate significantly different from the control (Student’s t tests). 84x163mm (300 x 300 DPI)


Page 36 of 39

Page 37 of 39


Figure 6. Molecular docking The molecular docking results of RDP1 to XO revealed the chief amino acid residues of RDP1 and XO in the active site and the ligand were represented with sticks. The yellow dashed lines stood for hydrogen bonds. In Fig. 6, the global (Fig.6 A) and local (Fig.6 B) visual angle of interaction between RDP1 and XO were showed, besides, the global (Fig.6 C) and local (Fig.6 D) view of residues interacting with RDP1 and XO were showed. 177x114mm (300 x 300 DPI)



Figure 7. Stability of RDP1. The stability of RDP1 in the plasma environment was showed in Fig. 7A, RDP1 was completely degraded in 20 min, and the half-life was about 4.626 min (calculated by GraphPad Prism software) (n = 3). In freezethaw assay, the content of RDP1 was remained about 80 % in ten times (n = 3) (Fig. 7B) and Fig. 7B also showed the stability of RDP1 at 4 °C, 37 °C and 60 °C in 10 days (n = 3). 84x128mm (300 x 300 DPI)


Page 38 of 39

Page 39 of 39


84x70mm (300 x 300 DPI)


New Rice-Derived Short Peptide Potently Alleviated Hyperuricemia

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