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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

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Journal of Agricultural and Food Chemistry

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Eritadenine from Edible Mushrooms Inhibits Activity of

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Angiotensin Converting Enzyme in vitro

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

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and Yeong Lae Ha1†

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

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

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

Korea

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*

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

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Republic of Korea

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

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

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

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ACS Paragon Plus Environment


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ABSTRACT

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The inhibition of angiotensin converting enzyme (ACE) activity was

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

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analyzed in 11 principle Korean edible mushrooms. EA inhibited ACE

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

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reference compound, was 0.025 µM. Kinetic measurements of ACE

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

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

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

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

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

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

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

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

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significant amount of EA.

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KEYWORDS: angiotensin converting enzyme (ACE), competitive

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inhibition, eritadenine, IC50 value, edible mushrooms

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INTRODUCTION

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

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

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many countries and are used as therapeutic foods to prevent several

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diseases such as hypercholesterolemia, cancer and hypertension 1. More

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

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

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

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consumed 3. These edible mushrooms contain many secondary metabolites

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4

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

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

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

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

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

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

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

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hypertension in human 1, 11.

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

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

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

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

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

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considered as a useful therapy method that may yield major hypotension

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benefits. Some ACE inhibitory drugs with small molecular weights

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currently used for antihypertensive treatment are captopril (CP, 217

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

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

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inhibitor.

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The objective of the present study is to examine ACE inhibitory

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

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

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

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analytical HPLC systems were applied for identification and quantification

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of EA in mushroom samples, respectively.

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

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

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mushrooms were obtained from the Department of Mushroom Culture,

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

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

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

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

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

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

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other reagents used were analytical grade.

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ACE Inhibition Assay. The inhibition of ACE activity by various

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

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spectrophotometric method with HHL as substrate as described by Chang

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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is the slope of the equation.

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Determination of kinetic parameters of ACE inhibition. Kinetic

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parameters of Vmax and Km values were determined according to the

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

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

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

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

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

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

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

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ExcelTM .

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Purification and quantification of EA. Fresh mushroom sample (10

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

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

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

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

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homogenization and centrifugation steps were repeated twice. For each

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repeated centrifugation step, the supernatant was removed and replaced

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

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

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

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

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analysis for EA quantification after filtering through a 0.2 µM membrane

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

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as a dry weight base.

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The EA concentration was analyzed using a reversed-phase HPLC

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

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

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

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

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

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

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

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

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quantification of EA as described previously by Ha et al,

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

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compounds were subjected to the extraction procedure and reversed-

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

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

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

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

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

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

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

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

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

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water and acetonitrile containing 0.03% TFA as described in HPLC

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

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

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

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detection at UV 270 nm using PDA detector.

161 162

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. Briefly, to

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


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

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

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

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RESULTS Antihypertensive behaviors of EA were measured using an ACE

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

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

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

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

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

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indicated that EA is a strong antihypertensive agent.

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

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

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

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

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

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

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

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

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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,

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

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

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

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

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

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

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

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Eleven principle Korean edible mushrooms were extracted with 60%

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

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

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

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

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

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

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

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

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

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

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

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

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

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mushrooms with hard caps.

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

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

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

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a potent competitive inhibitor of ACE19. Principle Korean edible

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

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with hard caps contained a least amount.

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

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

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

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

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

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compound.

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

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

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

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Lineweaver-Burk plots for ACE reactions in various concentrations of

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

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

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

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

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

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

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

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

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the active site of ACE.

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

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

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

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

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

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

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or pharmaceutical compound, and might be quantified in edible

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

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was determined from 11 different edible mushrooms ranging from 642.8

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

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

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

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

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

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

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

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

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

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

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be attributed to different growth environments of the given mushrooms.

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Interestingly, mushrooms with soft caps contained a significant amount of

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

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further clarification.

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

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

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

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

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

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

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

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

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

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

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

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

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

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EA was performed at 270 nm.

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

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

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Korean edible mushrooms contained a significant amount of EA ranging

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

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

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

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

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Acknowledgements

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This study was partly supported by the grant from Basic Research

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Program through the National Research Foundation of Korea (2013-

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

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

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and Medium Business Administration, Republic of Korea.

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Figure Captions

364 365

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

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

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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.

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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

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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

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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 ... - ACS Publications

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