Characterization of selenium-enriched mycelia of Catathelasma ventricosum and their antihyperglycemic and antioxidant properties.

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Characterization of selenium-enriched mycelia of Catathelasma ventricosum and their antihyperglycemic and antioxidant properties Yuntao Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5050316 • Publication Date (Web): 22 Dec 2014 Downloaded from http://pubs.acs.org on December 27, 2014

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Characterization of selenium-enriched mycelia of Catathelasma ventricosum and their antihyperglycemic and antioxidant properties

Journal: Manuscript ID: Manuscript Type: Date Submitted by the Author: Complete List of Authors:

Journal of Agricultural and Food Chemistry jf-2014-050316.R2 Article 22-Dec-2014 Liu, Yuntao; Sichuan Agricultural University, College of Food Science; Sichuan Agricultural University, Animal Nutrition Institute Li, Caiming; Jiangnan university, Luo, Xiaohu; Jiangnan university, Han, Guoquan; Sichuan Agricultural University, Xu, Shude; Guangdong VTR Bio-tech Co., Ltd, Niu, Fuge; Jiangnan university, Hu, Xinjie; Sichuan Agricultural University, ; Jiangnan university, Wu, Hejun; Sichuan Agricultural University, Zhang, Huimin; Yangzhou University,

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Title: Characterization of selenium-enriched mycelia of Catathelasma ventricosum and their antihyperglycemic and antioxidant properties ⊥

Authors: Yuntao Liu,*,†, Caiming Li,‡ Xiaohu Luo,‡ Guoquan Han,† Shude Xu,§ Fuge Niu,‡ Xinjie Hu,†,‡ Hejun Wu,† Huimin Zhang# Affiliations: College of Food Science, Sichuan Agricultural University, Yaan 625014, China. ⊥ Animal Nutrition Institute, Sichuan Agricultural University, Chengdu 611130, China. ‡ State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China. § Guangdong VTR Bio-tech Co., Ltd, Zhuhai 519060, China. # College of Animal Science and Technology, Yangzhou University, Yangzhou 225009, China †

Running title: Characterization of selenium-enriched mycelia Figures: 1 Tables: 4 Word count: 7006 *Correspondence: Yuntao Liu, PhD College of Food Science Sichuan Agricultural University 46 Xinkang Road, Yaan, Sichuan 625014, China Tel: 86-835-2882212 Fax: 86-835-2882311 Email: [email protected]

1



1

ABSTRACT

2

This is the first report concerning the selenium enrichment of Catathelasma ventricosum

3

mycelia. The selenium-containing proteins present in selenium-enriched mycelia (Se-MC)

4

were identified using size-exclusion chromatography-inductively coupled plasma-mass

5

spectrometry (SEC-ICP-MS). The selenium-containing amino acids liberated by hydrolysis of

6

these proteins were identified using anion exchange-ICP-MS. Se-MC was found to contain

7

selenoproteins with molecular weights ranging from 1.7 to 60.5 kDa. The main

8

selenium-containing amino acids within them were selenomethionine and selenocysteine.

9

Furthermore, Se-MC possessed excellent antihyperglycemic and antioxidant properties.

10

Se-MC normalized biochemical parameters like insulin level, blood glucose level, body

11

weight, and antioxidant enzyme activity in streptozocin-induced diabetic mice. It also

12

inhibited the α-amylase and α-glucosidase activities present in in vitro gastric and intestinal

13

models. In conclusion, Se-MC has the potential to serve as a dietary supplement of selenium,

14

an antioxidant, or an ingredient for the formulation of nutraceuticals.

15

Keywords: Mushroom, Antihyperglycemic activity, Antioxidant activity, Chemical

16

composition, Selenium speciation

2


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INTRODUCTION

18 19

Diabetes mellitus, a chronic metabolic disease caused by insulin deficiency or

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dysfunction, is characterized by high blood glucose levels.1 Hyperglycemia has been shown to

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increase oxidative stress through the overproduction of reactive oxygen species.2 These

22

oxygen radicals cause lipid oxidation and play a role in the development of diabetic

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complications. Oxidative stress is thought to link diverse mechanisms for the pathogenesis of

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diabetic complications.3 Although several hypoglycemic drugs are available to treat diabetes

25

mellitus, many of them have serious side effects.4 Relatively low-cost management of

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hyperglycemia with minimal clinical side effects remains a challenge for the medical system.5

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Phytochemicals from relatively low-cost bioresources have relatively few side effects.

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Exploiting this resource offers a new approach for the treatment of various diseases, including

29

diabetes.6

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Catathelasma ventricosum, an edible fungus, is a delicious source of valuable nutrition.

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Its fruiting body provides an excellent source of nutrients because of its high protein,

32

polysaccharide, and mineral content, as well as its low content of toxic metals, fat and energy.

33

A previous report has demonstrated that the fruiting body of wild C. ventricosum has

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antihyperglycemic and antioxidative effects.7 Selenium (Se), a trace element important for

35

human health, is a component of several selenoproteins that preform essential biological

36

functions, including glutathione peroxidase (GSH-Px) and selenoprotein P. The Se-dependent

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enzyme GSH-Px reduces oxidized glutathione and minimizes lipid peroxidation by catalyzing

38

the reduction of both lipid peroxides and hydrogen peroxide. Selenoprotein P, an extracellular 3



39

antioxidant present in the vascular endothelium, lowers the level of peroxinitrile (ONOO−), a

40

reactive nitrogen specie.8 Se deficiency has been associated with several diseases, including

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hepatocyte damage, cardiovascular disease and cancers.9-11 Se supplementation using

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microorganisms may be an excellent solution to the problem of Se deficiency because organic

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Se compounds are generally believed to be better and safer dietary supplements than

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inorganic Se compounds. A variety of Se-enriched food supplements have been

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commercialized, including garlic, yeast and lactic acid bacteria.12

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Submerged basidiomycete culture is currently the preferred technique for producing

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stable, safe mycelia and mushroom biometabolites. Importantly, submerged culture is less

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time consuming than mushroom cultivation and leads to the production of products with

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mushroom metabolite contents that are more consistent than those of products consisting of

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fruiting bodies.1 Furthermore, based on our preliminary experiment, the capacity of C.

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ventricosum for selenium enrichment is greater than that of some famous mushrooms, like

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Lentinula edodes, Pleurotus ostreatus, Ganoderma lucidum, Coriolus versicolor, Tremella

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fuciformis, etc (data not show). In order to assess the suitability of Se-enriched mycelia from

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C. ventricosum (Se-MC) as a candidate for diabetes treatment, the chemical composition and

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Se speciation in Se-MC was determined. The antihyperglycemic and antioxidant properties of

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Se-MC were also investigated.

57 58

MATERIALS AND METHODS

59 60

Chemicals. Sodium selenite and sodium selenate were obtained from Alfa Aesar (Ward Hill, 4


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MA, US). Working solutions for the determination of total selenium were prepared daily by

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appropriate dilution of a 1 mg/mL Se (IV) standard solution (Merck, Darmstadt, Germany).

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Glibenclamide, Streptozocin (STZ), 4-N-trophenyl-α-D-glu-copyranoside (PNPG),

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selenocysteine (SeCys), selenomethionine (SeMet) and amino acid standards were purchased

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from Sigma-Aldrich Co. LLC (St. Louis, MO, US). Reagent kits for the determination of

66

catalase (CAT), maleic dialdehyde (MDA), superoxide dismutase (SOD), glutathione

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peroxidase (GSH-Px) and insulin were obtained from the Jianchen Bioengineering Institute

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(Nanjing, Jiangsu, China). The SXT Blood Glucose Monitoring System and Test Strip were

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from Sinocare Co., Ltd. (Changsha, Hunan, China). All other materials were of the highest

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grade commercially avaialbe and obtained from Sinopharm Chemical Reagent Co. Ltd.

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(Shanghai, China).

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Se-enriched fermentation C. ventricosum mycelia. The seed of C. ventricosum was

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purchased from Mianyang Edible Fungi Research Institute (Mianyang, Sichuan, China) and

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maintained on synthetic potato dextrose agar (PDA).

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C. ventricosum was initially grown on PDA at 22 oC for 4 days. Aliquots (2.5 mL) of this

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4-day-old liquid culture were then transferred to two hundred 250 mL flasks. Each flask

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contained 47.5 ml of Se-enriched fermentation culture medium (medium components were

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based on our previous reports13) and incubated at 22 oC on a shake platform at 100 rpm for 15

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days. The harvest mycelia are referred to as Se-MC. C. ventricosum was also incubated in an

81

otherwise identical fermentation culture medium without Na2SeO3 as a control. This control

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material is referred to as MC. 5



83 84

In vitro simulation of gastrointestinal digestion. Gastrointestinal digestion was simulated

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using a method based upon a published procedure that mimicks the physiological events

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taking place in the upper gastrointestinal tract (stomach and small intestine).14 Three-gram

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samples of powdered C. ventricosum mycelia (Se-MC and MC were freeze-dried and ground

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to a fine powder (40 mesh)) were mixed with 18 mL of saline (140 mM NaCl, 5 mM KCl and

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150 µM butylated hydroxytoluene (BHT)), acidified to pH 2 with 0.1 M HCl, and then mixed

90

with 500 µL pepsin solution. After incubation at 37 oC for 1 h in a shaking water bath, 0.1 M

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NaHCO3 was added to increase the pH to 6.9. At this point, 2.5 mL pancreatin-bile solution

92

was added. After incubation at 37 oC for 2 h in a shaking water bath, the simulated

93

gastrointestinal digestion (GI) extract was centrifuged, and then the supernatant was dried

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under nitrogen gas. The dried powder was used for the in-vitro assay. The samples of Se-MC

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and MC were treated in triplicate.

96 97

Chemical composition. Proximate analysis. The crude protein, crude fat, moisture, ash, and

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total phenolic contents of Se-MC and MC were determined in triplicate using the Association

99

of Official Analytical Chemists (AOAC) procedures (1995).15 The determination of

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carbohydrate content (g) and energy content (KJ) were performed based on the methods of

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Liu et al. (2012).7

102 103

Amino acid analysis. Extracts were prepared according to the method of Ribeiro et al.

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(2008).16 Amino acid analysis was performed using our previously published methods7, with 6


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slight modifications. Briefly, the amino acid composition was determined using a

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reversed-phase high-performance liquid chromatography (HPLC, Agilent 1100) system

107

equipped with a 250 × 4.6 mm, 5 µm ODS HYPERSIL column (Agilent) (Santa Clara, CA,

108

US). The analysis was conducted using gradient elution at 40 °C and a flow rate of 1 mL/min.

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The mobile phase gradient was formed from buffer A (8 g sodium acetate dissolved in 1000

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mL deionized water plus 225 µL triethylamine and 5 mL tetrahydrofuran; pH adjusted to 7.2 ±

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0.05 with acetic acid) and buffer B (8 g sodium acetate dissolved in 400 mL deionized water

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plus 800 mL acetonitrile and 800 mL methanol; pH adjusted to 7.2 ± 0.05 with acetic acid).

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A/B ratios were 92: 8, 40: 60, 0: 100, 0: 100, 0: 100 and 92: 8, at run times of 0, 27.5, 31.5, 32,

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34 and 35.5 min, respectively. Samples were derivatized with o-phthaldialdehyde (OPA,

115

Sigma) (St. Louis, MO, US) prior to HPLC; the detection wavelength was set to 338 nm, or

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262 nm for proline. Stock solutions of individual amino acids, except for tryptophan, were

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prepared in 0.1 M HCl. Tryptophan solutions were prepared in water. The concentrations of

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the individual amino acids were approximate 1 mM. The samples of Se-MC and MC were

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analyzed in triplicate.

120 121

The content of Se and its speciation. Determination of total Se. Dried samples

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(approximately 0.1 g) were digested with a mixture containing 0.5 mL of H2O2 (30%) and 2

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mL of HNO3 (65%). After 24 h, the solutions were digested using a microwave system. A

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mineralization program was performed based on the method of Wrobel et al. (2004).17 After

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digestion, the final solutions were diluted to a total volume of 25 mL with distilled water. The

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standard solutions and reagent blanks were digested using the same procedure. The samples 7



127 128

of Se-MC and MC were treated in triplicate. The total selenium concentration was determined using inductively coupled plasma mass

129

spectrometetry (ICP-MS, Agilent 7500ce) (Tokyo, Japan). Isotopes 77Se, 78Se, 80Se and 82Se

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were monitored. The instrument settings were: forward power, 1590 W; carrier gas flow rate,

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1.2 L/min; plasma gas flow rate, 15 L/min; and dwell time, 0.3 s per isotope. All samples

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were analyzed in triplicate.18

133 134

Investigation of selenoproteins in mycelia. Mycelia samples (1 g) were added to 100 mL of

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buffer containing 20 mM Tris-HCl (pH 7.5) and 150 mM NaCl. The mixtures were extracted

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with magnetic stirring at 37 oC for 24 h. After centrifugation (8000 rpm for 10 min), 1 mL of

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supernatant was transferred to 5 mL acetone. This mixture was extracted with magnetic

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stirring at 4 oC for 24 h, and then placed at 5 oC for 24 h. After centrifugation (8000 rpm for

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10 min), the supernatant was discarded and the protein precipitate was dried under nitrogen

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gas. The samples of Se-MC and MC were treated in triplicate.

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The dry precipitate was re-solubilized with 1 mL of mobile phase buffer for analysis by

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size exclusion chromatography (SEC)-ICP-MS. Chromatographic separations were performed

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using an Agilent 1100 liquid chromatograph (Palo Alto, CA, US) equipped with a Superdex

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peptide 10-300 GL (300 mm × 10 mm × 15 µm) column. The mobile phase was composed of

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20 mM Tris-HCl, 150 mM NaCl (pH 7.5), the injection volume was 50 µL, and the flow rate

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was 0.5 mL/min.18 The SEC column was calibrated using a standard mixture of albumin (66

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kDa), myoglobin (16 kDa), cytochrome c (12.5 kDa), methylcobalamin (1.34 kDa) and (Gly)6

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(0.36 kDa). All samples were analyzed in triplicate. 8


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Se speciation in the mycelia. Enzymatic hydrolysis was performed to identify the species that

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possess covalent bonds between Se and proteins or peptides.19 In this method, the precipitated

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selenoproteins were re-solubilized with 1 mL of a solution containing 20 mM Tris-HCl (pH

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7.5), 150 mM NaCl, and 0.03 g of trypsin. After incubation at 50 oC for 24 h in a shaking

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water bath, 0.03 g of proteinase K was added to the mixture described above, and the

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resulting solution was kept in a shaking water bath at 50 oC for an additional 24 h. After

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centrifugation (8000 rpm for 10 min) and filtration (0.45 m polyvinylidene fluoride filter), the

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supernatant was passed through 5 kDa molecular weight cutoff filters (Ultrafree-0.5,

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Millipore) to remove high molecular weight proteins from the extracts before the analysis of

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the Se-containing species. The samples were treated in triplicate.

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Se species were analyzed by anion exchange-HPLC-ICP-MS and identified by

161

comparing their retention times with those of available standards (sodium selenite, sodium

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selenite, SeCys and SeMet).20 Chromatographic separations were performed using an Agilent

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1100 liquid chromatograph (Palo Alto, CA, US) equipped with a Hamilton PRP-X100 (250

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mm × 4.1 mm × 10 µm) column. The mobile phase consisted of 5 mM ammonium citrate in

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2% methanol (pH 5.2), the flow rate was 1 mL/min, and the injection volume was 50 µL. All

166

samples were analyzed in triplicate.18

167 168

Antihyperglycemic assays in vitro. α-Glucosidase inhibitory activity. Inhibition of

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α-glucosidase activity was assayed using the method of Palanisamy et al. (2011), with minor

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modifications.3 Various dilutions of the GI extracts described above were mixed with 9



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α-glucosidase (0.4 IU) dissolved in 2 mL phosphate buffer (0.1 M, pH 6.8) containing 50 µL

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glutathione (1 mg/mL) and 50 µL PNPG (0.1 M). These reaction mixtures were incubated at

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37 °C for 15 min. The reactions were stopped with 10 mL of 0.1 M sodium carbonate, and the

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extent of reaction was determined by measuring the absorbance at 400 nm using a UV-visible

175

spectrophotometer. Acarbose was used as the positive control, while the absence of sample

176

was used as a negative control. The percentage inhibition was calculated as: % inhibition = (1

177

– absorbance of sample/absorbance of negative control) × 100. The α-glucosidase inhibitory

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activity was expressed as an EC50 value (amount of sample causing 50% inhibition). All

179

samples were analyzed in triplicate.

180 181

α-Amylase inhibitory activity. Porcine pancreatic α-amylase (1 IU) was dissolved in 0.5 mL of

182

buffer (20 mM phosphate, pH 6.9) containing various dilutions of the GI extracts described

183

above. These mixtures were incubated at 37 °C for 15 min. Each reaction mixture was

184

subsequently added to 0.5 mL of soluble starch solution (1.5%) and incubated at 37 °C for 5

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min. Each reaction was stopped at this point using 1.0 mL of 3,5-dinitrosalicylic acid reagent.

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The samples were then incubated in a boiling water bath for 10 min, cooled to room

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temperature, and diluted with distilled water. The absorbance was measured at 520 nm.

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Acarbose was used as the positive control and reactions lacking sample were used as a

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negative control. The percentage inhibition was calculated as: % inhibition = (1 – absorbance

190

of sample/absorbance of negative control) × 100. The α-amylase inhibitory activity was

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expressed as an EC50 value (amount of sample causing 50% inhibition). All samples were

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analyzed in triplicate. 10


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Animals. The male ICR mice (body weight 18 ± 2 g) used in this study were purchased from

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SLAC Laboratory Animal Company (Shanghai, China). The mice were acclimatized for at

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least one week before starting the experiment. Before and during the experiment, the mice

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were housed in a segregated air-conditioned room at 25 oC with a 12 h light and dark cycle.

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Mice were provided a basal diet (Shanghai Laboratory Animal Company) and free access to

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drinking water. This experiment was approved by the Institutional Animal Ethics Committee

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of Jiangnan University (Wuxi, China). All experimental animals were overseen and approved

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by the Animal Care and Use Committee of our Institute before and during experiments.

202 203

Establishment of a diabetic mouse model. Diabetes was induced in mice by the

204

intraperitoneal (i.p.) injection of a freshly prepared solution of STZ at a dose of 150 mg/kg

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body weight.21 Mice displaying polydipsia and polyuria after 72 h were chosen for

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measurement of their fasting glucose levels using blood glucose test strips. Hyperglycemia

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mice (blood glucose level > 16.8 mmol/L) were used as the diabetic mice for further studies.

208 209

Experimental design. Based on a preliminary experiment (data not shown), the STZ-induced

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diabetic mice were randomly divided into five groups (6 mice per group). Group I: normal

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mice treated with saline solution as control; Group II: STZ-induced diabetic mice treated with

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saline solution as diabetic control; Group III: STZ-induced diabetic mice receiving oral

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glibenclamide (20 mg/kg/d) as a positive control; Group IV: STZ-induced diabetic mice

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receiving oral MC at a dose of 500 mg/kg/d; Group V: STZ-induced diabetic mice receiving 11



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oral Se-MC at a dose of 500 mg/kg/d. After 30 days of treatment, the animals were fasted

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overnight and sacrificed by cervical dislocation. Their blood glucose levels and body weight

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were measured. Blood collected from their eyes was immediately centrifuged for 5 min to

218

obtain serum (stored at –70 oC). Kidney and liver tissues were excised from the animals,

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washed with saline solution, and stored at –70 oC.

220 221

Biochemical analysis. Blood glucose levels were determined with blood glucose test strips

222

using blood samples taken from the tail veins of overnight fasted mice. The serum insulin

223

concentration was determined using commercially available kits according to the

224

manufacturer’s instructions. Liver and kidney homogenates were immediately assayed for

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SOD, CAT, GSH-Px, and MDA activities using the procedures described in the instructions

226

contained in their corresponding assay kits.

227 228

Statistical analyses. All of the assays described above were carried out in triplicate. The

229

results are expressed as the mean ± SD. Data were analyzed using one-way analysis of

230

variance (ANOVA) followed by Tukey's test. Statistical analyses were performed using SPSS

231

software, version 16.0. Differences leading to values of p < 0.05 were considered statistically

232

significant.

233 234

RESULTS

235 236

Chemical composition. The proximate compositions of the investigated mushrooms are 12


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shown in Table 1. The order of the contents (g/100 g dry weight) was total carbohydrate >

238

crude protein > ash > crude fat > total phenols. These data demonstrate that Se-MC contained

239

slightly more protein and ash than the control MC, and slightly less total carbohydrate. The

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crude fat and total phenol contents of Se-MC were about half or less of those of the MC

241

control. The total energy content of the Se-MC was slightly lower than that of the MC control.

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The percentage composition of 17 amino acids was investigated in the mycelia of C.

243

ventricosum; the results are presented in Table 2. Se-MC and MC were found to contain 13

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and 12 amino acids, respectively. Se-MC contained seven of the essential amino acids,

245

lacking only isoleucine. The ratios of the essential amino acids to non-essential amino acids

246

were 0.87 and 0.80 in Se-MC and MC respectively.

247 248

The content of Se and its speciation. The total Se contents of Se-MC and the MC control

249

were determined by digesting samples and quantifying the Se by ICP- MS. The total Se

250

content of Se-MC was 867.11 µg/g of dry mycelia. The mycelial yield was about 12.64 g dry

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mycelia per L of culture. The conversion of organic Se was approximate 11%. No Se was

252

detected in the MC control.

253

The SEC-ICP-MS chromatograms of the Se-MC extracts are presented in Fig. 1(a). The

254

molecular weights (MW) of the three main Se-containing fractions were about 60.5, 11.2, and

255

1.7 kDa. Based on the description of Kapolna et al. (2007)18, the fractions with high

256

molecular weights (HMW; 60.5 and 11.2 kDa) represent Se-containing proteins, those with

257

the low molecular weight (LMW) of 1.7 kDa correspond to Se-containing peptides, while the

258

molecular weights of selenoamino acids and inorganic selenium are less than 1.7 kDa. 13



259

A chromatogram of the Se species standards, obtained using anion

260

exchange-HPLC-ICP-MS, is displayed in Fig. 1(b). The species were identified by comparing

261

their retention times to those of available standards. The selenium species profile of the

262

Se-MC extracts is shown in Fig. 1(c). Selenomethionine (SeMet) was the predominant

263

selenium species in the Se-MC extracts, comprising approximately 68% of the total selenium

264

content. Other selenium species identified in Se-MC samples included selenocysteine (SeCys,

265

6%), selenite (Se-IV, 2%) and selenate (Se-VI, 5%).

266 267

Antihyperglycemic activity in vitro. The antihyperglycemic properties of Se-MC and MC

268

were evaluated using two assays that measured the inhibition of α-glucosidase and α-amylase

269

activities, respectively. Based on the results, the highest α-glucosidase inhibitory activity

270

(EC50 = 8.3 µg/mL) was found in Se-MC, the highest α-amylase inhibitory activity (EC50 =

271

19.4 µg/mL) was found in MC, while the α-glucosidase and α-amylase inhibitory activities of

272

acarbose (positive control) were 15.9 and 26.7 µg/mL, respectively.

273 274

Effects of Se-MC and MC on blood glucose, body weight, and insulin in STZ-induced

275

diabetic mice. Mice in the diabetic control group (group II) exhibited a severe decrease in

276

insulin level and body weight and a significant increase in blood glucose level, compared with

277

mice in the normal group (group I; p < 0.05) (Table 3). After administration of Se-MC, MC or

278

glibenclamide (positive control) for 30 days, the levels of blood glucose in groups III, IV and

279

V were much lower than that in group II. On the other hand, the insulin levels and body

280

weights of the mice in groups III, IV and V showed a significant increase compared with 14


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those of the mice in group II.

282 283

Effects of Se-MC and MC on MDA and antioxidant enzymes in kidney and liver of

284

STZ-induced diabetic mice. The MDA levels in the kidney and liver of untreated

285

STZ-induced diabetic mice were significantly increased (p < 0.05), whereas the activities of

286

antioxidant enzymes in the kidney and liver of untreated STZ-induced diabetic mice were

287

significantly decreased (p < 0.05) compared with those of the mice in group I (Table 4). After

288

administration of Se-MC or MC for 30 days, the MDA levels present in the mice in groups IV

289

and V were significantly decreased (p < 0.05), while, SOD, GSH-Px, and CAT activity were

290

significantly increased (p < 0.05) in the kidney and liver, compared with those of the mice in

291

group II and III.

292 293

DISCUSSION

294 295

The moisture content of mushrooms ranges from 86–94 g/100g fresh weight. The

296

moisture contents of Se-MC and MC were 93.03 and 93.55 g/100g fresh weight, respectively,

297

which is higher than that of the fruiting body of C. ventricosum.7 It seems that liquid culture

298

conditions can increase the moisture content of C. ventricosum. Kalač (2009) indicated that

299

low dry matter and lipid contents result in mushrooms with low energy values.22

300

The concentration order of the components in the proximate analyses of Se-MC and MC

301

is consistent with that revealed in our previous report.7 This indicates that the physical state of

302

the culture medium (liquid state or solid state) may have a limited effect on the chemical 15



303 304

composition of C. ventricosum. The concentration of protein in Se-MC (31.22% of dry matter) is very high compared

305

with those of most plants and mushrooms. The results are in consistent with the conclusions

306

of Belitz and Grosch (1999)23: Se concentrations are positively correlated with protein content

307

in food. Moreover, Pappa et al. (2006)24 indicated that the Se concentration is negatively

308

correlated with food fat content. The concentration of crude fat in Se-MC (2.17% of dry

309

matter) is very low compared with those of most plants and mushrooms.22 In a word, C.

310

ventricosum is an ideal candidate for Se enrichment.

311

The content of crude protein in Se-MC, on the other hand, was 30% higher than that in

312

MC. It seems the presence of Se in the culture medium can affect protein metabolism in C.

313

ventricosum. One possibility is that Se can replace sulfur in amino acids like

314

selenomethionine (SeMet) and selenocysteine (SeCys), due to their physicochemical

315

similarity, which may stimulate the synthesis of protein (especially Met and Cys). The results

316

of the amino acid analysis also support this assumption. The concentration of methionine

317

(Met) and cystine (Cys) in Se-MC was much higher than that in MC. Moreover, Dumont et al.

318

(2006)25 indicated that most plants lack the ability to accumulate large amounts of Se (they

319

rarely exceed 100 µg/g dry weight). However, some mushrooms have been recognized as Se

320

accumulators. These mushrooms can take up a large amount of Se (> 1000 µg/g dry weight)

321

without exhibiting any negative effects because they increase their intracellular concentrations

322

of SeCys and SeMet, which are normally incorporated into proteins.8,25

323

Although selenium is beneficial to normal biological function when supplied in

324

appropriate quantities, excessive selenium intake can cause toxicosis.26 The essential and 16


Page 16 of 33

Page 17 of 33


325

toxic levels of Se are similar. The tolerable upper Se intake level for adults is set at 400

326

µg/day, while the recommended dietary allowance for Se is 55 µg/day for both men and

327

women.27 Therefore, determination of the total Se content of Se-MC is of great significance.

328

The total Se content of Se-MC (867.11 µg/g of dry mycelia) is higher than some other

329

Se-enriched plants and mushrooms, like Allium schoenoprasum (1.07 µg/g), Lentinula edodes

330

(770.7 µg/g) and Cordyceps militaris (42 µg/g).18,19,28

331

The bioavailability of Se depends strongly on the chemical form of the Se that is

332

ingested. Organic Se compounds are generally more bioavailable than inorganic Se

333

compounds. 8 Ferri et al. (2007)29 indicated there is a lack of information about the chemical

334

forms of selenium in foods. This lack is mainly the result of the difficulties inherent in their

335

identification. The application of different hyphenated techniques (HPLC-MS,

336

HPLC-ICP-MS etc.) has begun to change this situation. In this study, we employed

337

HPLC-ICP-MS to analyze the Se species. Previous reports have identified selenate, selenite,

338

SeMet and SeCys as the primary selenocompounds in mushrooms.19,28 These reports agree

339

well with the results of this study. In addition, the ratio of organic Se to inorganic Se in

340

Se-MC is 74:7, which is higher than the ratio seen in some Se-enriched foods.20,28

341

Two in vitro assays were carried out to evaluate the antihyperglycemic activity of a

342

simulated gastrointestinal digestion extract of Se-MC and MC: an α-glucosidase inhibition

343

assay and an α-amylase inhibition assay. α-Glucosidase, located in the brush-border surface

344

membrane of intestinal cells, is a key enzyme of carbohydrate digestion. α-Amylase catalyzes

345

the hydrolysis of the α-1,4-glucosidic linkages in glycogen, starch, and other oligosaccharides.

346

Inhibition of the α-glucosidase and α-amylase activities in the human digestive tract can 17



347

effectively control diabetes by diminishing the absorption of glucose derived from the

348

degradation of starch by these enzymes.30 There are two main concepts to explain the

349

interaction mechanism between α-glucosidase/α-amylase and their inhibitor: (1) The inhibitor

350

inhibits α-glucosidase/α-amylase activity by hydrolyzing them;31 (2) α-glucosidase/α-amylase

351

and their inhibitor combine to form a soluble complex, which inactivates the enzymes.32

352

Yasuda (2003)33 indicated α-glucosidase/α-amylase inhibitor is not a competitive inhibitor

353

that competes with α-glucosidase/α-amylase for starch, disaccharides or various

354

oligosaccharides. However, the inhibitor will combine with α-glucosidase/α-amylase directly

355

and change their conformations, inactivating them. Acarbose, an inhibitor of a-glucosidase

356

and α-amylase used in the treatment of diabetes mellitus, was chosen as the positive control.3

357

The inhibitory activities of Se-MC and MC were significant greater than those of acarbose (p

358

< 0.05), which indicates that Se-MC and MC possess excellent antihyperglycemic activity.

359

Inhibition of intestinal α-glucosidase and α-amylase activities is an important

360

antihyperglycemic mechanism of Se-MC and MC. However, the antihyperglycemic activities

361

of Se-MC and MC are very similar. It seems that adding Se into the culture medium does not

362

have has much of an influence on the α-glucosidase and α-amylase inhibitory activities of C.

363

ventricosum mycelium.

364

Animal models in which diabetes mellitus is induced by the administration of STZ or

365

other chemicals have been reported.21 The advantages of STZ are its greater stability and

366

relative lack of extrapancreatic toxicity. In the present study, the diabetic mouse model was

367

established by intraperitoneal injection of STZ, all of the STZ-induced diabetic mice

368

displayed polydipsia and polyuria. 18


Page 18 of 33

Page 19 of 33

369


Stapleton (2000)34 reported that selenium has been found to have insulin-mimetic

370

activities in vitro and in vivo. Based on the results presented here, Se-MC possesses better

371

antihyperglycemic properties than MC. The levels of blood glucose and insulin in the mice in

372

groups III and V were very close (p > 0.05), although they didn't recover completely

373

(compared with the normal group, p < 0.05). However, the body weight of the mice in groups

374

IV and V was significantly lower than that of the mice in the positive control (group III) and

375

normal mice (group I). It seems that both Se-MC and MC lack a strong ability to increase

376

body weight in STZ-induced diabetic mice. Chen and Ianuzzo (1982)35 suggested that the

377

decrease in body weight observed in diabetic mice might be the result of protein wasting

378

caused by the inability to use carbohydrates as an energy source. Zhao et al. (2011)36 reported

379

that administration of Opuntia dillenii polysaccharides significantly increased the body

380

weight of STZ-induced diabetic mice.

381

Diabetes mellitus is associated with the generation of reactive oxygen species that cause

382

oxidative damage to the liver and kidney.37,38 Major antioxidant enzymes, including GSH-Px

383

and SOD, are the first line of defense against the reactive oxygen species generated during

384

oxidative stress in vivo.39,40 In the present study, a significant increase in MDA levels and a

385

significant decrease in GSH-Px, SOD and CAT activity were found in kidney and liver of

386

mice with STZ-induced diabetes (Group II; compared with mice in Group I), which suggests

387

that severe oxidative stress occurred in liver and kidney tissues. However, after treatment with

388

Se-MC (mice in group IV) or MC (mice in group V), MDA levels were significantly

389

decreased (p < 0.05) and GSH-Px, SOD, CAT activity were significantly increased (p < 0.05)

390

in liver and kidney, compared with those of mice in group II. In particular, except MDA levels 19



391

in kidney, activities of GSH-Px, SOD, CAT and MDA in group V almost returned to their

392

normal level, which indicated Se-MC possess a strong effect on increasing the synthesis of

393

these antioxidant enzymes. MDA, the end product of lipid peroxidation, is used as an index of

394

cellular damage and cytotoxicity. The MDA generated by high levels of free radicals can

395

damage proteins and inactivate membrane-bound enzymes.41 Chen et al. (2013)39 indicated

396

decreasing MDA levels in diabetic rats may be associated with a drug's potential organ

397

protective effect through improved glycometabolism and lipid metabolism. Therefore, it can

398

be inferred that Se-MC probably exhibit its antihyperglycemic property by increasing these

399

antioxidant enzymes in diabetic mice. These enzymes protect tissues from peroxidative

400

damage and maintain their function, improving the sensitivity and response of target cells to

401

insulin. Simultaneously, after the administration of glibenclamide, there was no difference

402

among these biochemical parameters in group III compared with group II, which reveals that

403

glibenclamide does not have an obvious ability to stimulate the synthesis of these antioxidant

404

enzymes. This observation is consistent with those of a previous study, which found that

405

glibenclamide is not capable of modulating antioxidant enzymes and diminishing

406

STZ-induced damage to the liver, which is the main organ to synthesize antioxidant

407

enzymes.13

408

In conclusion, the selenoproteins in Se-MC possess a wide-range of molecular weights

409

and SeMet and SeCys were identified as the main selenium compounds bound to these

410

proteins. Moreover, Se-MC was found to be an effective antihyperglycemic and antioxidant.

411

These results demonstrate that the mycelia of Se-enriched C. ventricosum can be used as a

412

source of nutrients because of their high protein and carbohydrate content and their low fat 20


Page 20 of 33

Page 21 of 33


413

and energy content. Mushrooms can also be uesd as a natural source of Se supplements for

414

both animals and humans, especially in Se-deficient areas. Future studies may show that the

415

Se-enriched mycelium of C. ventricosum is a source of new antidiabetic drugs,

416

chemopreventive preparations, or food supplements.

417 418

ABBREVIATIONS USED: Catalase (CAT), glutathione peroxidase (GSH-Px), high

419

performance liquid chromatography (HPLC), maleic dialdehyde (MDA), selenium-enriched

420

mycelia (Se-MC), superoxide dismutase (SOD), size-exclusion chromatography-inductively

421

coupled plasma-mass spectrometry (SEC-ICP-MS).

422 423

ACKNOWLEDGEMENT

424 425

This work was supported by Scientific Research Foundation of Sichuan Agricultural

426

University (No. 06021400). YL designed the study, conducted the experiments and prepared

427

the manuscript. CL and XLcontributed to data interpretation and manuscript preparation. GH

428

performed statistical analysis. SX, FN, XH, HW and HZcontributed to data interpretation. All

429

authors have no conflicts of interest to declare.

430 431

REFERENCES

432 433 434

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metabolites: a critical review. Anal. Bioanal. Chem. 2006, 385, 1304– 1323.

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26. Rayman, M. P.; Infante, H. G.; Sargent, M. Food-chain selenium and human health:

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28. Dong, J. Z.; Ding, J.; Yu, P. Z.; Lei, C.; Zheng, X. J.; Wang, Y. Composition and

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32. Doi T.; Yoshimatsu, H.;Katsuragi, I.; Kurokawa, M.; Takahashi, H.;Motoshio, A.; Sakata,

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T. alpha-amylase inhibitor increases plasma 3-hydroxybutyric acid in food-restricted rats.

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Experientia 1995, 51, 585–588.

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33. Yasuda, K.; Shimowada, K.; Uno, M.; Odaka, H.; Adachi, T.; Shihara, N.; Suzuki, N.;

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effects of voglibose, a potent intestinal alpha-glucosidase inhibitor, in spontaneous

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diabetic GK rats. Diabetes Res. Clin. Pract. 2003, 59, 113–22.

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34. Stapleton, S. R. Selenium: An insulin mimetic. Cell Mol. Life Sci. 2000, 57, 1874–1879.

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35. Chen, V.; Ianuzzo, C. D. Dosage effect of streptozotocin on rat tissue enzyme activities

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and glycogen concentration. Can. J. Physiol. Pharmacol. 1982, 60, 1251–1256.

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36. Zhao, L.Y.; Lan, Q. J.; Huang, Z. C.; Ouyang, L. J.; Zeng, F. H. Antidiabetic effect of a

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661–668.

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37. Kakkar, R.; Mantha, S. V.; Radhi, J.; Prasad, K.; Kalra, J. Increased oxidative stress in rat

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liver and pancreas during progression of streptozotocin induced diabetes. Clin. Sci. 1998,

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38. Mohamed, A. K.; Bierhaus, A.; Schiekofer, S. The role of oxidative stress and NF-kappaB activation in late diabetic complications. Biofactors 1999, 10,175–179. 39. Chen, X. M.; Tang, J.; Xie, W. Y.; Wang, J. J.; Jin, J.; Ren, J.; Jin, L. Q.; Lu, J. X.

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Protective effect of the polysaccharide from Ophiopogon japonicus on

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body. Indian J. Exp. Biol. 2002, 40, 1233–1239. 41. Bagchi, D.; Bagchi, M.; Hassoun, E. A.; Stohs, S. J. In vitro and in vivo generation of

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pesticides. Toxicology 1995, 104, 129–140.

549 550

FIGURE CAPTIONS

551

Fig. 1. HPLC-ICP–MS chromatograms of the mycelia of Se-enriched C. ventricosum. (a):

552

protein fractions of Se-MC were analyzed by SEC-ICP-MS; (b): chromatogram of Se species

553

standard obtained by anion-exchange ICP-MS; (c): chromatogram of Se species profile in

554

Se-MC obtained by anion-exchange ICP-MS

27



Page 28 of 33

TABLE Table 1. Moisture (g/100 g of fresh weight), macronutrients (g/100 g of dry weight) and total energy (kJ/100 g of dry weight) of C. ventricosum. C. ventricosum Selenium-enriched mycelia Mycelia a

Components Moisture a

93.03±3.67 93.55±2.07

Ash

Crude protein

Crude fat

Total carbohydrates

Total energy

Total phenols

10.64±1.48 9.29±1.17

31.22±2.59 21.42±5.03

2.17±0.34 6.19±0.29

55.97±4.94 63.10±7.39

1562.52±87.42 1665.87±102.53

0.44±0.04 0.83±0.08

Each value is expressed as the mean ± SD (n =3).

28


Page 29 of 33


Table 2. Free amino acid composition (% weight) of C. ventricosum. Amino acidsa Valb Thrb Metb Leub Ileb Trpb Pheb Lysb Gly His Tyr Asp Ser Cys Arg Ala Glu

C. ventricosum Selenium-enriched mycelia (Se-CM)

Mycelia (CM)

13.59±0.63c 1.45±0.49 16.37±0.51 1.07±0.05 ND 4.19±0.63 2.73±0.34 7.11±0.39 1.06±0.08 10.52±0.96 ND ND 8.74±1.02 9.74±0.18 ND 18.62±2.74 4.81±0.43

12.14±0.53 7.92±0.82 4.06±0.31 NDd ND 8.63±0.77 5.19±0.49 6.48±0.67 1.53±0.08 12.15±0.75 ND ND 17.53±1.24 1.46±0.16 ND 12.18±0.21 10.73±1.11

a

Valine (Val); Threonine (Thr); Methionine (Met); Leucine (Leu); Isoleucine (Ile); Tryptophan (Trp); Phenylalanine (Phe); Lysine (Lys); Glycine (Gly); Histidine (His); Tyrosine (Tyr); Aspartic acid (Asp); Serine (Ser); Cystine (Cys); Arginine (Arg); Alanine (Ala); Glutamic acid (Glu); b Essential amino acids; c Each value is expressed as mean ± SD (n =3); d ND: not detected.

29



Page 30 of 33

Table 3. Effects of selenium-enriched mycelia and mycelia on body weight, blood glucose and insulin in diabetic mice. Groupe Insulin level (µIU/ml) Blood glucose (mmol/L) Body weight (g) e

Group I (control)

Group II (diabetic mice)

Group III (positive control)

Group IV (diabetic mice + MC)

Group V (diabetic mice + Se-MC)

28.7 ± 1.7c 5.8 ± 0.6a 35.3 ± 1.5d

8.3 ± 0.8a 26.3 ± 3.1d 25.8 ± 3.2a

19.1 ± 2.0b 11.5 ± 1.1b 32.5 ± 2.4c

11.2 ± 0.7a 17.7 ± 1.4c 28.7 ± 1.4b

18.3 ± 1.2b 13.1 ± 0.9b 28.2 ± 1.7b

Each value is expressed as the mean ± SD (n = 6). Means with different letters within a row are significantly different (p < 0.05).

30


Page 31 of 33


Table 4. Effect of selenium-enriched mycelia and mycelia on antioxidants in liver and kidney of diabetic mice. Groupe Liver GSH-Px (U/mg protein) SOD (U/mg protein) CAT (U/mg protein) MDA (nmol/mg protein) Kidney GSH-Px (U/mg protein) SOD (U/mg protein) CAT (U/mg protein) MDA (nmol/mg protein) e

Group I (control)

Group II (diabetic mice)

Group III (positive control)

Group IV (diabetic mice + CM)

Group V (diabetic mice + Se-CM)

877.1 ± 74.1d 478.1 ± 39.5c 194.4 ± 20.1c 4.2 ± 0.8a

413.5 ± 32.3a 277.3 ± 14.6a 41.7 ± 2.6a 9.0 ± 0.7c

442.0 ± 29.7a 234.9 ± 10.7a 49.9 ± 2.8a 8.5 ± 0.8c

593.6 ± 30.1b 299.7 ± 19.4b 92.7 ± 10.7b 6.2 ± 1.1b

797.4 ± 59.6c 424.5 ± 21.7c 167.4 ± 17.3c 4.7 ± 0.7a

564.2 ± 34.9c 277.3 ± 14.9c 83.8 ± 7.5c 3.1 ± 0.5a

277.4 ± 17.7a 104.4 ± 8.5a 37.5 ± 3.7a 7.3 ± 0.4d

263.7 ± 20.4a 115.8 ± 11.0a 42.1 ± 3.5a 7.3 ± 0.6d

400.3 ± 35.2b 197.6 ± 20.4b 66.3 ± 4.7b 4.9 ± 0.2c

584.5 ± 67.6c 283.7 ± 17.7c 89.1 ± 9.5c 3.8 ± 0.7b

Each value is expressed as the mean ± SD (n = 6). Means with different letters within a row are significantly different (p < 0.05); glutathione

peroxidase (GSH-Px); superoxide dismutase (SOD); Catalase (CAT) maleic dialdehyde (MDA).

31



FIGURE Fig. 1.

32


Page 32 of 33

Page 33 of 33


Graphic for table of contents:

33


Extraction of polysaccharides from Phellinus nigricans mycelia and their antioxidant activities in vitro.

Synthetic anthocyanidins and their antioxidant properties.

Optimization of Mycelia Selenium Polysaccharide Extraction from Agrocybe cylindracea SL-02 and Assessment of their Antioxidant and Anti-Ageing Activities.

Production of bioactive compounds by actinomycetes and their antioxidant properties.

Does antioxidant properties of the main component of essential oil reflect its antioxidant properties? The comparison of antioxidant properties of essential oils and their main components.

Phytochemical Characterization of Chinese Bayberry (Myrica rubra Sieb. et Zucc.) of 17 Cultivars and Their Antioxidant Properties.

Antioxidant and immunological activity in vitro of polysaccharides from Phellinus nigricans mycelia.

Hypoglycemic, antihyperglycemic, and antioxidant effects of the edible plant Anoda cristata.

Antioxidant activity and glucose diffusion relationship of traditional medicinal antihyperglycemic plant extracts.

low sporing mutants.

The physicochemical properties of geraniin, a potential antihyperglycemic agent.

Isolation, characterization, and antitumor activity of a novel heteroglycan from cultured mycelia of Cordyceps sinensis.

Synthesis of hydroxycinnamoyl β-D-xylopyranosides and evaluation of their antioxidant properties.

Chemical characterization and evaluation of antioxidant properties of açaí fruits (Euterpe oleraceae Mart.) during ripening.

Topical analgesic, anti-inflammatory and antioxidant properties of Oxybaphus nyctagineus: phytochemical characterization of active fractions.

Production and Characterization of Antioxidant Properties of Exopolysaccharide(s) from Peanibacillus mucilaginosus TKU032.

naphthalene-containing azaphenothiazines and their potent in vitro antioxidant properties.

The extraction of different proteins in selenium enriched peanuts and their antioxidant properties.

Antiglycation and Antioxidant Properties of Momordica charantia.

Physicochemical and antioxidant properties of black garlic.

Aflatoxin is degraded by heated and unheated mycelia, filtrates of homogenized mycelia and filtrates of broth cultures of Aspergillus parasiticus.

Antihyperglycemic, antihyperlipidemic and antioxidant effects of ethanol and aqueous extracts of Cyclocarya paliurus leaves in type 2 diabetic rats.

Characterization and antioxidant properties of six Algerian propolis extracts: ethyl acetate extracts inhibit myeloperoxidase activity.

Preliminary characterization, antioxidant properties and production of chrysolaminarin from marine diatom Odontella aurita.