Eritadenine from Edible Mushrooms Inhibits Activity of Angiotensin

Subscriber access provided by Washington University | Libraries

Article

Eritadenine from Edible Mushrooms Inhibits Activity of Angiotensin Converting Enzyme in vitro Sadia Afrin, Md. Abdur Rakib, Boh Hyun Kim, Jeong Ok Kim, and Yeong Lae Ha J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05869 • Publication Date (Web): 03 Mar 2016 Downloaded from http://pubs.acs.org on March 7, 2016

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 27

Journal of Agricultural and Food Chemistry

1

Eritadenine from Edible Mushrooms Inhibits Activity of

2

Angiotensin Converting Enzyme in vitro

3 4

Sadia Afrin1, Md. Abdur Rakib1, Boh Hyun Kim1, Jeong Ok Kim2,

5

and Yeong Lae Ha1†

6 7

1

8

Agriculture & Life Science, Gyeongsang National University, Jinju 660-

9

701, Republic of Korea, 2HKBiotech., Co. Ltd., Jinju 660-884, Republic of

10

Division of Applied Life Sciences, Graduate School, and Institute of

Korea

11 12

*

13

Graduate School, Gyeongsang National University, Jinju 660-701,

14

Republic of Korea

15

E-mail: [email protected]; Tel: +82-55-772-1964; Fax: +82-55-772-1969

Correspondence to: Yeong Lae Ha, Division of Applied Life Science,

16 17

Afrin1, S. and Rakib1 A. equally contributed to this study

18 19 20

1

ACS Paragon Plus Environment


21

ABSTRACT

22

The inhibition of angiotensin converting enzyme (ACE) activity was

23

determined in vitro by mushroom-derived eritadenine (EA), which was

24

analyzed in 11 principle Korean edible mushrooms. EA inhibited ACE

25

activity with 0.091 µM IC50, whereas the IC50 of captopril (CP), which is a

26

reference compound, was 0.025 µM. Kinetic measurements of ACE

27

reaction in the substrate of hippuryl-L-histidyl-L-leucine (HHL) with or

28

without EA revealed that Vmax (0.0465 O.D/ 30 min) was unchanged, but

29

the Km increased from 2.063 mM to 3.887 mM, indicating that EA

30

competes with HHL for the active site. When EA was analyzed by HPLC,

31

Lentinus edodes with soft cap contained the highest amount EA (642.8

32

mg%); however, Phellinus linteus with hard cap contained a least amount

33

of EA (9.4 mg%). These results indicate that EA was a strong competitive

34

inhibitor for ACE, and edible mushrooms with soft caps contained a

35

significant amount of EA.

36 37

KEYWORDS: angiotensin converting enzyme (ACE), competitive

38

inhibition, eritadenine, IC50 value, edible mushrooms

39 40

2


Page 2 of 27

Page 3 of 27


41

INTRODUCTION

42

Mushrooms are macrofungi, which are the spore-bearing fruiting bodies

43

that may grow above or below ground on the soil. They are consumed in

44

many countries and are used as therapeutic foods to prevent several

45

diseases such as hypercholesterolemia, cancer and hypertension 1. More

46

than 1,100 mushroom species are commonly eaten and collected from at

47

least eighty five countries in the world 2. In Korea, there are approximately

48

300 edible species of mushrooms, but only 20 species are commonly

49

consumed 3. These edible mushrooms contain many secondary metabolites

50

4

51

. Eritadenine [2(R), 3(R)-dihydroxy-4-(9-adenyl)-butanoic acid, 253

52

daltons; EA] is one of these secondary metabolites (Figure 1, A), which is

53

a purine alkaloid 5. EA was firstly isolated from the shiitake mushroom,

54

Lentinus edodes 6, and later from Agaricus bisporus also known as bottom

55

mushroom 7. This compound is an inhibitor of S-adenosyl-L-homocysteine

56

hydrolase and has hypocholesterolemic activity in animals 8-10. However,

57

L. edodes mushroom containing EA have been used to prevent or treat

58

hypertension in human 1, 11.

59 60

The renin–angiotensin system (RAS) is a major clinical target for the treatment of hypertension 12. RAS-mediated hypertension is initiated when

3



61

renin stimulates the conversion of angiotensinogen to angiotensin I. Then,

62

ACE stimulates the conversion of Angiotensin I to Angiotensin II, which

63

is a potent vaso-active peptide that causes blood vessels to constrict,

64

resulting in increased blood pressure 13. Inhibition of this ACE activity is

65

considered as a useful therapy method that may yield major hypotension

66

benefits. Some ACE inhibitory drugs with small molecular weights

67

currently used for antihypertensive treatment are captopril (CP, 217

68

daltons; Figure 1, B), ramipril (416 daltons) and enalapril (376 daltons) 14.

69

Hence, it is of significance to investigate whether EA acts as an ACE

70

inhibitor.

71

The objective of the present study is to examine ACE inhibitory

72

activity of EA and to quantify EA in 11 principle edible mushrooms in

73

Korea. To achieve this objective, an ACE inhibition assay was performed

74

in vitro using EA, with reference to CP. The HPLC-PDA-MS/MS and

75

analytical HPLC systems were applied for identification and quantification

76

of EA in mushroom samples, respectively.

77 78 79 80

4


Page 4 of 27

Page 5 of 27

81 82


MATERIALS AND METHODS Materials. EA derived from L. edodes was obtained from Santa Cruz

83

Biotechnology (Dallas, TX, USA). Eleven principle fresh edible

84

mushrooms were obtained from the Department of Mushroom Culture,

85

Gyeongnam Agricultural Research & Extension Services (Jinju, Rep. of

86

Korea: Table 2). ACE from rabbit lung (≥ 2 units per mg protein),

87

hippuryl-L-histidyl-L-leucine (HHL), O-phthaldialdehyde (OPA), caffeic

88

acid (CA), trifluoroacetic acid (TFA), and sodium borate decahydrate were

89

purchased from Sigma-Aldrich (Saint Louis, MO, USA). Acetonitrile and

90

methanol (all HPLC grades) were purchased from Sigma-Aldrich. All

91

other reagents used were analytical grade.

92

ACE Inhibition Assay. The inhibition of ACE activity by various

93

concentration of EA and CP as well as their IC50 were measured using

94

spectrophotometric method with HHL as substrate as described by Chang

95

et al. 15 with modification. Briefly, a 20 mM sodium borate buffer

96

containing 0.3 M NaCl (pH 8.3) was used for preparation of EA, CP, ACE

97

and substrate HHL solutions. The ACE-catalyzed reaction was performed

98

for 30 min at 37 °C in test tubes of the following compositions: 100 µL

99

EA or CP, 100 µL ACE solution (40 mU/ mL), and 100 µL HHL (15 mM)

100

solutions (A1); 100 µL EA or CP solution, and 200 µL borate buffer (A2);

5



101

100 µL borate buffer, 100 µL ACE solution, and 100 µL HHL solution

102

(A3); and 300 µL borate buffer (A4). The enzymatic reaction was stopped

103

by adding 3 mL alkaline solution of OPA solution (pH 12.0). The

104

absorbance of each reaction was measured at 390 nm using a Beckman

105

DU-640 (Brea, CA, USA), after incubation for 20 min at 25 °C. Inhibition of

106

ACE by EA or CP was calculated using the following equation: Inhibition

107

(%) = [1 - (A1 - A2) / (A3 - A4)] x 100. The IC50 value of ACE activity

108

was calculated by the following equation: IC50 = (50-b)/m derived from

109

linear regression graph of ACE activation, where b is the intercept and m

110

is the slope of the equation.

111

Determination of kinetic parameters of ACE inhibition. Kinetic

112

parameters of Vmax and Km values were determined according to the

113

Michaelis-Menten kinetic model 16. The reaction rate for the formation of

114

L-histidyl-L-leucine from HHL by ACE (40 mU/ mL) was determined by

115

above mentioned method with EA (0.091 µM) or CP (0.00625 µM) and

116

without EA or CP to get the saturation curves, and then plotted against

117

HHL concentrations (0.94, 1.85, 3.75, 7.50, 15 mM). Lineweaver-Burk

118

plot was derived using the saturation curves to determine the type of

119

inhibition. Kinetic parameters (Km and Vmax) were calculated using MS

120

ExcelTM .

6


Page 6 of 27

Page 7 of 27

121


Purification and quantification of EA. Fresh mushroom sample (10

122

g) in a centrifuge test tube (250 ml) containing 10 mg CA (internal

123

standard) was homogenized in 60% ethanol (100 ml) with a homogenizer

124

(IKA, Ultra-Turrax T25). The homogenate was centrifuged with an Ilsin

125

ultracentrifuge (Seoul, Republic of Korea) for 15 min at 7,000 rpm. These

126

homogenization and centrifugation steps were repeated twice. For each

127

repeated centrifugation step, the supernatant was removed and replaced

128

with a new batch of 60% ethanol. The combined 60% ethanolic extract

129

was filtered through filter paper (Whatman number 2) and concentrated to

130

5 mL volume using a vacuum evaporator (EYELA, Rotary evaporator N-

131

1000, Shanghai, China) at 60 °C. This concentrate then underwent HPLC

132

analysis for EA quantification after filtering through a 0.2 µM membrane

133

filter (Toyo Roshi Kaisha, Ltd., Tokyo, Japan). EA content was expressed

134

as a dry weight base.

135

The EA concentration was analyzed using a reversed-phase HPLC

136

system equipped with a Agilent Zorbax SB-C18 column (4.6 x 250 mm, 5

137

µm) and gradient system as described by Enman et al.5 with slight

138

modification. Solution A was 0.03% TFA in distilled water, while solution

139

B was 0.03% TFA in acetonitrile. The B solution (3%) at 0 min was

140

linearly increased to 20% for 5 min and then linearly decreased to 3% for

7



Page 8 of 27

141

10 min, followed by extension of 3% flow for additional 5 min (20

142

min/run). EA was detected at 270 nm using a Beckman DU-640 with a

143

flow rate of 1 mL/ min. CA was used as an internal standard for the

144

quantification of EA as described previously by Ha et al,

145

obtain the response factor (RF), a mixture of authentic EA and CA

146

compounds were subjected to the extraction procedure and reversed-

147

phased HPLC analysis. The RF was calculated as follows: RF = (area

148

CA/weight CA) x (weight EA/area EA), where CA refers to internal

149

standard. Using the RF, the amount of EA in the sample was calculated by

150

mg/100 g dried weight (mg% EA/ dried weight) = [(area EA/area CA) x

151

weight CA (mg)]/g sample (dried weight) x 100 x RF (0.85).

152

Identification of EA by HPLC-MS/MS. EA in L. edodes mushroom

153

extracts was identified by HPLC-PDA-MS/MS system (Thermo scientific,

154

Waltham, MA, USA) equipped with an Agilent Zorbax SB-C18 column

155

(4.6 x 250 mm, 5 µm). Mobile phase was a gradient system with distilled

156

water and acetonitrile containing 0.03% TFA as described in HPLC

157

analysis. Analytical condition was as followed: spray voltage, 3,800V;

158

vaporizer temperature, 350℃; capillary temperature, 380℃; ionization

159

mode, ESI positive; and scan type, full scan (100 -1,000 M+) mode by

160

detection at UV 270 nm using PDA detector.

161 162

17

. Briefly, to

Statistical analysis. Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed using one-way analysis 8


Page 9 of 27


163

of variance (ANOVA), followed by Duncan’s multiple range test for

164

comparison of means using SPSS for Window’s version 11 (SPSS, Inc.,

165

USA). Values with p < 0.05 were considered to be statistically significant.

166 167 168

RESULTS Antihypertensive behaviors of EA were measured using an ACE

169

inhibitory assay system with reference to CP, which is a potent inhibitor of

170

ACE 18. The A and B panels of Figure 2 show ACE activities of EA and

171

CP at different concentrations, respectively. The IC50 value for ACE

172

inhibition by EA was found to be at a concentration of 0.091 µM, whereas

173

the IC50 value for ACE inhibition by CP was 0.025 µM (Table 1). This

174

indicated that EA is a strong antihypertensive agent.

175

Furthermore, we determined the action mode of EA in ACE inhibition

176

at various HHL substrate concentrations (Figure 3 and Table 1). The Km

177

and Vmax values of the ACE reaction were found to be 2.063 mM and

178

0.0465 O.D/ 30 min, respectively. Meanwhile, when EA was present in the

179

reaction of ACE, the Km value increased to 3.887 mM, but Vmax did not

180

change, results indicate that EA acted as a competitive inhibitor against

181

substrate HHL, similar to the effect of CP on ACE (Vmax, 0.0448 O.D/ 30

182

min, and Km, 6.549 mM). These results indicate that EA likely binds to

9



183 184

the active site of ACE similar to known mechanism of CP18-19. It is of significance to quantify the EA content in edible mushrooms,

185

since levels of EA are only reported in a few types of mushrooms, such as

186

L. edodes 6 and A. bisporus 7, Hence, we extracted L. edodes with 60%

187

ethanol (v/v) containing CA to identify EA by HPLC-PDA-MS/MS system

188

(Figure 4). A peak (4.28 min of retention time) in L. edodes sample was

189

identified as EA with 254 (M+) (Figure 4, panel A), with reference to M+

190

(254) of authentic EA (Figure 4, panel B). Similarly, a peak (8.93 min of

191

retention time) was identified as CA with 181 (M+) (Figure 4).

192

Eleven principle Korean edible mushrooms were extracted with 60%

193

ethanol (v/v) containing CA to quantify EA content by reversed-phase

194

HPLC system using CA as an internal standard. In Figure 5, A panel shows

195

a typical HPLC chromatogram of L. edodes sample analysis, and B panel

196

shows the HPLC chromatogram of EA and CA. The EA contents (mg%

197

dry weight) of the 11 edible mushrooms are shown in Table 2. L. edodes

198

with soft cap contained 642.8 mg%, which was the highest amount of EA.

199

A significant amount of EA was found in mushrooms with soft caps:

200

Flammulina velutipes (427.1 mg%); Hericium erinaceus (409.7 mg%);

201

Agaricus blazei (403.2 mg%); A. bisporus (375.5 mg%); and Cordyceps

202

militaris (367.3 mg%), whereas lower level of EA was detected in

10


Page 10 of 27

Page 11 of 27


203

Ganoderma lucidum and Phellinus linteus with hard caps, 21 mg% and 9.4

204

mg%, respectively. According to these results, mushrooms with soft caps

205

contained significant amount of EA, but lower level of EA was present in

206

mushrooms with hard caps.

207 208 209

DISCUSSION The present study clearly demonstrated that EA is a strong inhibitor of

210

ACE in vitro through competitive inhibition with an IC50 of 0.091 µM.

211

This inhibitory efficacy resembled that of CP (IC50 of 0.025 µM), which is

212

a potent competitive inhibitor of ACE19. Principle Korean edible

213

mushrooms with soft caps contained a significant amount of EA, but those

214

with hard caps contained a least amount.

215

Many agents, like CP, were identified as competitive inhibitors of

216

ACE19. EA, which was originally isolated from L. edodes 6, was found to

217

be a strong competitive inhibitor of ACE, as shown in Table 1 and Figure

218

3. More interestingly, this is the first report of EA as a strong competitive

219

inhibitor for ACE with similar efficacy to that of CP, which is a reference

220

compound.

221

The kinetic analysis provides insight into the capability of EA to block

222

or reduce ACE activity, along with the quantity of inhibitor required either

11



223

for the reaction to continue or for ACE activity to be inhibited 19- 22.

224

Lineweaver-Burk plots for ACE reactions in various concentrations of

225

HHL with EA (0.091 µM) or without EA revealed that Vmax (0.0465 O.D/

226

30 min) did not change, but Km increased from 2.063 mM to 3.887 mM

227

HHL, indicating competitive inhibition (Table 1 and Figure 3). There is no

228

data to directly compare the kinetic data for the ACE reaction with EA in

229

the literature, but kinetic data of EA, including IC50 values, were

230

comparable to those of CP, which is a reference compound, in the present

231

study (Table 1) and in the literature 23. Therefore, these results indicate that

232

EA is a strong ACE inhibitor, which may bind competitively with HHL at

233

the active site of ACE.

234

EA was analyzed only from L. edodes and A. bisporus so far6,7. No

235

other edible mushrooms were analyzed for EA due to a lack of interest in

236

EA as a nutraceutical or pharmaceutical agent, except for as a

237

hypocholestrolemic agent8-9. Meanwhile, the present study revealed a

238

novel biological function of EA as a potent inhibitor of ACE to attenuate

239

hypertension, and hence, EA will acquire more interest as a nutraceutical

240

or pharmaceutical compound, and might be quantified in edible

241

mushrooms other than L. edodes and A. bisporus. In the present study, EA

242

was determined from 11 different edible mushrooms ranging from 642.8

12


Page 12 of 27

Page 13 of 27


243

mg% EA in L. edodes to 9.4 mg% in P. linteus dry matter (Table 2). The

244

amount of EA present in L. edodes after methanol extract has been

245

reported at levels of 60-70 mg% dry weight in the caps and 30-40 mg% in

246

the stems 7, and 73 mg% in shitake mycelium 24. Saito et al. 7 and Enman

247

et al.5 reported the content of EA in dry matter of whole L. edodes

248

mushrooms to be 40-70 and 317-633 mg%, respectively. The amount

249

(642.8 mg%) of EA contained in the whole mushroom of L. edodes in the

250

present report was found to be similar to that (317-633 mg%) of reported

251

values by Enman et al. 5 Similarly, A. bisporus contained 375.5 mg% in

252

dry matter, a value higher than that of an earlier report 7.These results may

253

be attributed to different growth environments of the given mushrooms.

254

Interestingly, mushrooms with soft caps contained a significant amount of

255

EA, while mushrooms with hard caps contained less EA. This requires

256

further clarification.

257

It is of significance to note the analytical condition of EA in

258

mushroom samples. We found that EA was soluble in less than 70%

259

ethanol (v/v), and other materials from the mushroom samples, such as

260

carbohydrates and proteins, were easily removed by precipitation in 60%

261

ethanol (v/v). Hence, 60% ethanol was chosen for the extraction and

262

purification of EA from mushroom samples in the present study. More

13



263

importantly, fresh mushrooms were used as samples for analysis, since

264

they were easy to grind and the extraction in 60% ethanol (v/v) was easy

265

to perform. The concentration of TFA in mobile phases was critical to

266

better separation of EA, and the best concentration was found to be 0.03%

267

TFA5. Finally, EA and CA have different chromophores with UV

268

absorption profile: EA, λmax at 261 nm in distilled water; and CA, a broad

269

spectrum from 265 to 310 nm in distilled water; hence, HPLC analysis for

270

EA was performed at 270 nm.

271

In conclusion, this study firstly reported that EA is a potent

272

competitive inhibitor of ACE with an IC50 of 0.091 µM. The principle

273

Korean edible mushrooms contained a significant amount of EA ranging

274

from 642.8 to 9.4 mg% in L. edodes and P. linteus dry matter, respectively.

275

These results suggest that EA and EA–containing edible mushrooms could

276

be useful for the treatment of high blood pressure in humans. Further

277

research is needed to clarify the antihypertensive action of EA in vivo.

278 279

Acknowledgements

280

This study was partly supported by the grant from Basic Research

281

Program through the National Research Foundation of Korea (2013-

282

R1A1A2011587) funded by the Ministry of Science, ICT and Future

14


Page 14 of 27

Page 15 of 27


283

Planning, Republic of Korea, and the grant (C0219166) from the Small

284

and Medium Business Administration, Republic of Korea.

15



285

References

286

(1) Manzi, P.; Aguzzi, A.; Pizzoferrato, L. Nutritional value of mushrooms

287 288 289 290 291 292

widely consumed in Italy. Food Chem. 2001, 73, 321-325. (2) Boa, E. Wild edible fungi: a global overview of their use and importance to people. Roma: FAO 2004, 17. (3) Heo, H. Y. The Biodiversity and protected area of Korea; Ministry of Environment (Korea). 2007, 45. (4) Barros, L.; Cruz, T.; Baptista, P.; Estevinho, L. M.; Ferreira, I. C. Wild

293

and commercial mushrooms as source of nutrients and nutraceuticals.

294

Food Chem. Toxicol. 2008, 46, 2742-7.

295

(5) Enman, J.; Rova, U.; Berglund, K. A. Quantification of the bioactive

296

compound eritadenine in selected strains of shiitake mushroom

297

(Lentinus edodes). J. Agric. Food Chem. 2007, 55, 1177-80.

298

(6) Chibata, I.; Okumura, K.; Takeyama, S.; Kotera, K. Lentinacin: a new

299

hypocholesterolemic substance in Lentinus edodes. Experientia. 1969,

300

25, 1237-8.

301

(7) Saito, M.; Yasumoto, T.; Kaneda, T. Quantitative analyses of

302

eritadenine in shiitake mushroom and other edible fungi. J. Jpn.Soc.

303

Food Nutr. 1975, 28, 503-513.

304

(8) Fukada, S. –I.; Setoue, M.; Morita, T.; Sugiyma, K. Dietary

305

eritadenine suppresses guanidinoacetic acid-induced hyperhomo-

306

cysteinemia in rats. J. Nut. 2006, 136, 2797-2802.

307

(9) Sekiya, A.; Fukada, S. -I.; Morita, T.; Kawagishi, H.; Sugiyama, K.

308

Suppression of methionine-induced hyperhomocysteinemia by dietary

309

eritadenine in rats. Biosci. Biotech. Bioch. 2006, 70, 1987-1991.

310 311

(10) Yamada, T.; Komoto, J.; Lou, K.; Ueki, A.; Hua, D. H.; Sugiyama, K.; Takata, Y.; Ogawa, H.; Takusagawa, F. Structure and function of 16


Page 16 of 27

Page 17 of 27


312

eritadenine and its 3-deaza analogues: Potent inhibitors of S-

313

adenosylhomocysteine hydrolase and hypocholesterolemic agents.

314

Biochem. Pharmacol. 2007, 73, 981-989.

315

(11) Bisen, P. S.; Baghei, R. K.; Sanodiya, B. S.; Thakur, G. S.; Prasad, G.

316

B. K. S. Lentinus edodes: A macrofungus with pharmacological

317

activities. Curr. Med. Chem. 2010, 17, 2419-2430.

318

(12) Ma, T. K.; Kam, K. K.; Yan, B. P.; Lam, Y. Y. Renin-angiotensin-

319

aldosterone system blockade for cardiovascular diseases: current

320

status. Br. J. Pharmacol. 2010, 160, 1273-92.

321

(13) Hammoud, R. A.; Vaccari, C. S.; Nagamia, S. H.;

Khan, B. V.

322

Regulation of the renin–angiotensin system in coronary

323

atherosclerosis: a review of the literature. Vasc. Health Risk Manag.

324

2007, 3, 937-45.

325

(14) Chang, C. –H.; Lin, J. –W.; Caffrey, J. L.; Wu, L. –C.; Lai, M. –S.

326

Different angiotensin-converting enzyme inhibitors and the

327

associations with overall and cause-specific mortalities in patients

328

with hypertension. Am. J. Hypertens. 2105, 28, 823-830.

329

(15) Chang, B. W.; Chen, R. L.; Huang, I. J.; Chang, H. C. Assays for

330

angiotensin converting enzyme inhibitory activity. Anal. Biochem.

331

2001, 291, 84-8.

332

(16) Ojeda, D.; Jimenez-Ferrer, E.; Zamilpa, A.; Herrera-Arellano, A.;

333

Tortoriello, J.; Alvarez, L. Inhibition of angiotensin convertin enzyme

334

(ACE) activity by the anthocyanins delphinidin- and cyanidin-3-O-

335

sambubiosides from Hibiscus sabdariffa. J. Ethnopharmacol. 2010,

336

127, 7-10.

337 338

(17) Ha, Y. L.; Grimm N. K.; Pariza, M. W. Newly recognized anticarcinogenic fatty acids: identification and quantification in 17



339 340 341 342 343

natural and processed cheeses. J. Agric. Food Chem. 1989, 37, 75-81. (18) Jurca, T.; Vicaş, L. Complexes of the ACE-inhibitor captopril. Farmacia 2010, 58, 198-202. (19) Bryan, J. From snake venom to ACE inhibitor-The discovery and rise of captopril. Pharm. J. 2009, 282, 455.

344

(20) Yahaya, N. F. M.; Rahman, M. A.; Abdullah, N. Therapeutic potential

345

of mushrooms in preventing and ameliorating hypertension. Trends

346

Food Sci. Tech. 2014, 39, 104-115.

347

(21) Girgih, A. T.; Udenigwe, C. C.; Li, H.; Adebiyi, A. P.; Aluko, R. E.

348

Kinetics of enzyme inhibition and antihypertensive effects of hemp

349

seed (Cannabis sativa L.) protein hydrolysates. J. Am. Oil Chem. Soc.

350

2011, 88, 1767-1774.

351

(22) Onuh, J. O.; Girgih, A. T.; Malomo, S. A.; Aluko, R. E.; Aliani, M.

352

Kinetics of in vitro renin and angiotensin converting enzyme

353

inhibition by chicken skin protein hydrolysates and their blood

354

pressure lowering effects in spontaneously hypertensive rats. J. Funct.

355

Foods 2015, 14, 133-143.

356 357 358

(23) Cushman, D.W.; Ondetti, M. A. Design of angiotensin converting enzyme inhibitors. Nat. Med. 1999, 5, 1110-1112. (24) Lelik, L. V. G.; Lefler, J.; Hegoczky, J.; Nagy-Gasztonyi, M.;

359

Vereczkey, G. Production of the mycelium of shiitake (Lentinus

360

edodes) mushroom and investigation of its bioactive compounds. Acta

361

Aliment. 1997, 26, 271-277.

362

18


Page 18 of 27

Page 19 of 27

363


Figure Captions

364 365

Figure 1. Chemical structures of EA (A) and CP (B).

366

Figure 2. Inhibition of ACE activity by EA (A panel) and CP (B panel) in

367

vitro. IC50 value of EA and CP was calculated to be 0.091 and 0.025 µM,

368

respectively. Each value is expressed as a mean (n = 3). SD is less than 5%

369

of mean value.

370

Figure 3. Lineweaver–Burk plots for the inhibition of ACE by EA (0.091

371

µM) and CP (0.00625µM), of which the CP concentration was diluted to 4

372

times IC50 value (0.025 µM) to draw on the same plot with EA. Each value

373

is expressed as a mean (n = 3). SD is less than 5% of mean value.

374

Figure 4. MS/MS spectra of L. edodes sample (A panel) and authentic EA

375

and CA (B panel) by LC-PDA-MS/MS. Peak identification: EA, 4.28 min

376

(M+, 254); and CA. 8.93 min (M+, 181).

377

Figure 5. Typical reverse-phased HPLC chromatograms of L. edodes

378

sample (A panel) and authentic EA and CA compounds (B panel). Peak

379

identification: EA, 3.1 min; and CA, 11.8 min.

380 381 382

19



Page 20 of 27

383 384

Table 1. ACE inhibition and kinetic parameters by EA kinetic parameterb) compound

IC50 (µM)a) Vmax (O.D/ 30 min)

Km (mM)

no inhibitor

-

0.0465

2.063

eritadenine

0.091 ± 0.009

0.0465

3.887

captopril

0.025 ± 0.015

0.0448

6.549

385

a)

IC50 (µM) was calculated from Figure 2

386

b)

Kinetic parameter was calculated from Figure 3

387 388

20


Page 21 of 27

389


Table 2. Amount of EA in some edible mushrooms in Korea EA contentb) Moisture a)

scientific name

trivial name

(mg%. dry weight) (%)

Lentinus edodes

Pyogo

642.8 ± 23.2a

82.0

Flammulina velutipes

Paengeui

427.1 ± 12.1 b

89.0

Hericium erinaceus

Norugongdengeui

409.7 ± 10.3 c

88.5

Agaricus blazei

Sinryoung

403.2 ± 20.5 c

88.0

Agaricus bisporus

Yangsongeui

375.5 ± 15.7 cd

91.0

Cordyceps militaris

Dongchunghacho

367.3 ± 13.9 cd

66.2

Pleurotus eryngii

Saesongeui

335.8 ± 16.2 e

82.7

Tricholoma matsutake

Songeui

280.1 ± 15.9 f

88.0

Pleurotus ostreatus

Neutari

212.1 ± 15.8 g

89.3

Ganoderma lucidum

Yeongji

21.0 ± 0.6 h

16.1

Phellinus linteus

Sangwhang

9.4 ± 0.3 h

12.5

390

a)

Trivial name is Korean name of given mushroom.

391

b)

EA content was calculated by internal standard method using a CF (0.85)

392 393 394

and was expressed as mg% (mg per 100 g, dry weight). c)

Mean ± SD (n =3). Means with different superscript small letters are significantly different at p

Eritadenine from Edible Mushrooms Inhibits Activity of Angiotensin

Recommend Documents