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REVISED 3-22-11

International Journal ofMedicinal Mushrooms

416

Antihypoxic Activities of the Golden Chanterelle Mush-room, Cantharellus cibarius (Higher

Dr. Ebrahimzadeh:

Mohammad Ali Ebrahimzadeh, Department of Medici-nal Chemistry, School of Pharmacy, 18th km. Bolvar khazar, Mazandaran University of Medical Sciences, Sari, 48189, Iran;

International Journal of Medicinal Mushrooms, 16(4): 339–344 (2014)

3391045-4403/14/$35.00 © 2014 Begell House, Inc. www.begellhouse.com

Antihypoxic Activities of the Golden Chanterelle Mushroom, Cantharellus cibarius (Higher Basidiomycetes)Masoumeh Khalili, Mohammad Ali Ebrahimzadeh,* Farnoosh Omrani, & Mohammad Karami

Pharmaceutical Sciences Research Center, School of Pharmacy, Mazandaran University of Medical Sciences, Sari, Iran

*Address all correspondence to: Mohammad Ali Ebrahimzadeh, Department of Medicinal Chemistry, School of Pharmacy, 18th km. Bolvar khazar, Mazandaran University of Medical Sciences, Sari, 48189, Iran; Tel.: +98 151 3543081-3; Fax: +98 151 3543084; zadeh20@yahoo.com.

ABSTRACT: Cantharellus cibarius is an edible mushroom with worldwide distribution. Because of its good radical scavenging and strong iron-chelating activity, this mushroom was nominated for assay of antihypoxic activity. Protective effects of Chanterelle extract against hypoxia-induced lethality in mice were evaluated by 3 experimental models of hypoxia: asphyctic, hemic, and circulatory. Antihypoxic activity was especially pronounced in the hemic model. The effect was dose dependent. C. cibarius extract (600 mg kg-1) kept the mice alive for 10.07 ± 1.18 min. It significantly (P < 0.0001) and dose dependently prolonged survival time compared to control group (7.00 ± 0.63 min). Extract at 300 mg kg-1 prolonged survival time to 9.94 ± 0.87 min, which was statistically significant (P < 0.0001) compared to control group. In circulatory model, C. cibarius extract (600 mg kg-1) was effective. It prolonged latency for death significantly with regard to the control group (15.18 ± 4.21 vs. 9.84 ± 0.75 min; P < 0.001). At 300 mg kg-1, the extract also prolonged survival time (13.57 ± 0.87 min), and this effect was also statistically significant compared to the control group (P < 0.01). Extract showed no activity in the asphyctic model. Mice in the control group died of hypoxia in 28.20 ± 3.27 min. Extract (600 mg kg-1) prolonged latency for death, but this activity was not statistically significant (P > 0.05). Phenytoin prolonged latency for death to 55.00 ± 6.05 min (P < 0.0001).

KEY WORDS: medicinal and edible mushrooms, antihypoxia, Cantharellus cibarius, circulatory hypoxia, hemic hypoxia

ABBREVIATIONS: NaNO2, sodium nitrite; ROS, reactive oxygen species.

I. INTRODUCTION

In recent years, an increase in the consumption of wild edible and medicinal mushrooms has been ob-served, even in the developed world. Wild-growing mushrooms have been a popular delicacy in many countries, mainly in central and eastern Europe,1 be-cause they add flavor and texture to food. The gold-en chanterelle mushroom, Cantharellus cibarius Fr. (Cantharelaceae, Cantharellales, higher Basidiomy-cetes), is widely viewed as among the most desirable of edible mushrooms. Chanterelle has been reported in North America, Europe, Africa, Central America, and Asia,2 including Iran.3 It is highly appreciated for its wonderful fruity, apricot-like aroma and is particularly prized for cooking throughout Europe.4 In addition to the flavor and smell, its texture is also

nice. The chanterelle is of great economic interest for the northeastern region of Iran. We recently re-ported the antioxidant activity of C. cibarius. Previ-ous studies of the C. cibarius concerned its contents of minerals, amino acids,5 vitamin D,6 ash, protein, fat, fiber, ascorbic acid,7 and trace elements.8 In general, phenolic compounds (such as caffeic acid, p-coumaric acid, and rutin)4 and organic acids are known to contribute to the organoleptic character-istics of fruits and vegetables.9 The C. cibarius fruit body has been shown to protect against various dis-eases through its antioxidant activity. Several fatty acids, phenolic compounds, organic acids, carot-enoids, and steroids from this mushroom have been shown to exhibit antioxidant and anti-inflammatory properties.10 The C. cibarius has been found to show various biological properties such as antimicrobial

International Journal of Medicinal Mushrooms

Khalili et al.340

activity,11 insecticidal properties,12 scavenging of lipid peroxidation,13 antioxidant activity, pain kill-er properties, and antiaging and anticancer activi-ties.3,14,15

The imbalance between low oxygen supply and oxygen demands determines organ hypoxia. It occurs especially in heart diseases, ischemia, bleeding, heart attack, and immature newborns, causing numerous deleterious effects and finally resulting in tissue destruction and death.16,17 Hy-poxia mediates the production of nitric oxide.16,17 Therefore, scavenging of nitric oxide may offer a possibility for neutralizing hypoxia. Hypoxia in-duces the production of reactive oxygen species (ROS) as well.18 It seems antioxidants are able to scavenge ROS and therefore are able to exhibit an antihypoxic property. In addition, the presence of redox-active metals (copper, iron) increases the amount of ROS. Therefore, chelating agents may offer some benefit in the prevention and treatment of hypoxia.

There is increasing interest in the use of natu-ral antioxidants instead of chemical ones. Studying local plant foods is of particular interest because they often contain large amounts of bioactive com-pounds.19,20 Their nutritional and medicinal effects must be documented and supported with scientif-ic studies. Because of the phenol and flavonoids content and the good nitric oxide scavenging and strong iron-chelating activities of C. cibarius,3 it was chosen for antihypoxic activity assay. Antihy-poxic activity was investigated using 3 different hypoxic models: asphyctic, hemic, and circulatory hypoxia. To the best of our knowledge, nothing about the antihypoxic activity of C. cibarius has been reported to date.

II. MATERIALS AND METHODS

A. Mushroom Material

C. cibarius was obtained throughout the fall of 2012 from indigenous people who collect edible mushrooms from forests in Sari, Iran. The sample was authenticated by Dr. Bahman Eslami, and the voucher specimen was deposited in the herbarium

at the Sari School of Pharmacy. The macroscopic descriptions of size, shape, color, texture, and odor were noted. Color of the carpophore, shape of the cap and stipe, color of the flesh and latex, smell, and habitat also were noted. The mushroom sam-ples were transported to the laboratory. Samples were dried at 45°C for 36 hours then at room tem-perature for 4 days.

B. Preparation of Mushroom Extract

Dried mushroom (100 g) was extracted by percola-tion using methanol (400 mL) for 24 hours at room temperature. The extract then was separated from the sample residue by filtration through Whatman No.1 filter paper. This procedure was repeated thrice. Extract was filtered and concentrated under reduced pressure at 40°C using a rotary evapora-tor until a crude solid extract was obtained, which then was freeze-dried for complete removal of the solvent (yield, 9.1%).

C. Chemicals

Sodium fluoride and sodium nitrite were purchased from Merck (Germany).

D. Animals

Groups of 10 male Swiss albino mice (20 ± 2 g) were randomly housed in polypropylene cages at an ambient temperature (25 ± 1°C and 45–55% rel-ative humidity), with a 12-hour light/12-hour dark cycle (lights on at 7 a.m.). The animals had free access to standard pellet and water and ad libitum. Experiments were conducted between 8:00 a.m. and 2:00 p.m.. All the experimental procedures were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The Institutional Ani-mal Ethical Committee of Mazandaran University of Medical Sciences also approved the experimen-tal protocol.

Volume 16, Number 4, 2014

Antihypoxic Activities of Golden Chanterelle 341

E. Maximum Nonfatal Dose

Extract (2 g kg-1) was injected to a group of 4 mice to determine the maximum nonfatal dose. After 48 hours, mortality identified the maximum nonfatal dose.21

F. Asphyctic Hypoxia

Mice received single intraperitoneal injections of 300 and 600 mg kg-1 doses of extract and a 50 mg kg-1 dose of phenytoin 30 minutes before they were subjected to hypoxia. Another control group was treated with normal saline.22Hypoxia was induced putting animals individually in a tightly closed 300-mL glass container, which was placed in an aquarium under water (25°C). The animals had convulsions and died from hypoxia. The latencies for death were recorded. The animals died approx-imately 2 minutes following convulsions.

G. Hemic Hypoxia

Mice (n = 24) were divided into 3 groups, each containing 8 mice. The control group was treated with normal saline. Thirty minutes after intraperi-toneal administration of 300 and 600 mg kg-1 doses of extract, sodium nitrite (NaNO2; 360 mg kg–1) was applied intraperitoneally to mice, and anti-hypoxic activity was estimated as the latent time (minutes) to evidence of hypoxia.21,23

H. Circulatory Hypoxia

Mice (n = 24) were divided into 3 groups, each containing 8 mice. The control group was treated with normal saline. Thirty minutes after intraperi-toneal administration of 300 and 600 mg kg-1 doses of extract, sodium fluoride (150 mg kg–1) was ap-plied intraperitoneally to mice, and antihypoxic ac-tivity was estimated as the latent time (minutes) to evidence of hypoxia.21,23

I. Statistical Analysis

All the experimental results were centered using 3 parallel measurements of the mean ± standard

deviation. Analysis of variance was performed. Newman-Keuls multiple comparison test was used to determine the differences in means. All P values <0.05 were considered significant.

III. RESULTS AND DISCUSSION

A. The Maximum Nonfatal Doses

The maximum nonfatal dose for extract was at least 2 g kg-1. At these doses no mortality was ob-served after 48 hours.

B. Antihypoxic Activity

Brain hypoxia affects the central nervous system, leading to many physiological alterations such as mental retardation and motor impairment.24 Hy-poxia causes oxidative stress, which produces strong deleterious effects on cells because of dam-age to proteins, lipids, and DNA molecules.25 ROS were demonstrated to be involved in a large num-ber of normal physiologic and pathological pro-cesses. The increased amount of ROS in hypoxia is the result of increased reduction equivalents in the mitochondrial electron transport system caused by the lower level of oxygen. In general, many extracts produced from plants have potent antioxidant and scavenging activities. Statistically significant antihypoxic activities of some doses of extract were established in the experimental models of hemic and circulatory hypoxia in mice. The results of antihypoxic activities are shown in Figs. 1–3. During investigations of NaNO2 on brain metabolism, Gibson et al.26 demonstrated a close relationship between oxidative metabolism and cholinergic function. In our study, chemical hypoxia was induced by the intraperitoneal injec-tion of NaNO2 (360 mg kg-1), which reduces the oxygen-carrying capacity of the blood by convert-ing hemoglobin to methemoglobin. This lethal dose was injected 30 minutes after treatment with the extract. Immediately after the NaNO2 injection, the animals were placed in small cages, and the time between the injection of NaNO2 and cessation of respiration was recorded.27 The results obtained

Running head OK?

International Journal of Medicinal Mushrooms

Khalili et al.342

from the hemic hypoxia model are shown in Fig. 1. C. cibarius extract at 600 mg kg-1 is capable of keeping the mice alive for 10.07 ± 1.18 minutes. It significantly (P < 0.0001) and dose-dependently prolonged survival time compared with the control group (7.00 ± 0.63 minutes). C. cibarius extract at 300 mg kg-1 prolonged survival time to 9.94 ± 0.87

minutes. This effect was statistically significant (P < 0.0001) compared with the control group. The results of circulatory hypoxia are shown in Fig. 2. The effect of the extract was dose dependent. Ex-tract at 600 mg kg-1 was effective. It significantly prolonged latency for death among treated mice (15.18 ± 4.21 minutes) compared with the control group (9.84 ± 0.75 minutes; P < 0.001). At 300 mg kg-1, the extract prolonged survival time (13.57 ± 0.87 minutes). This effect also was statistically significant compared with the control group (P < 0.01). There are data in the literature showing that administration of sodium fluoride, a substance that induces circulatory hypoxia, increases blood his-tamine content and decreases the oxygen-carrying capacity of the blood.

Extract showed no activity in the asphytic model (Fig. 3). Mice in the control group died of hypoxia in 28.20 ± 3.27 minutes. C. cibarius ex-tract at 600 mg kg-1 prolonged latency of death to 31.15 ± 2.14 minutes, but this activity was not sta-

tistically significant (P > 0.05). Extract at 300 mg kg-1 kept mice alive for 30.54 ± 3.89 minutes, but this increase was not statistically significant com-pared with the control (P > 0.05). Phenytoin pro-longed latency for death to 55.00 ± 6.05 minutes (P < 0.0001).

A significant protective effect of Ginkgo bilo-ba, which contains flavonoids, against other forms

FIG 1: Antihypoxic activity of Cantharellus cibarius ex-tract in hemic hypoxia. Data are mean ± standard devia-tion (n = 8). P < 0.0001 compared to control.

<AU: Sentence OK as edited? “It significantly prolonged la-tency for death among treated mice (15.18 ± 4.21 minutes) compared with the control group (9.84 ± 0.75 minutes; P < 0.001).”>

Control

300

600

0

5

10

15

Concentration (mg / kg)

Surv

ival

tim

e (m

inut

e)

Control

300

600

0

5

10

15

20

25

Concentration (mg / kg)

Surv

ival

tim

e (m

inut

e)

FIG 2: Antihypoxic activity of Cantharellus cibarius ex-tract in circulatory hypoxia. Data are mean ± standard deviation (n = 8). P < 0.001 for 600 mg kg-1and P < 0.01 for 300 mg kg-1 compared to control.

Phenyto

in

Normal

salin

e30

060

00

20

40

60

80

Concentration (mg / kg)

Surv

ival

tim

e (m

inut

e)

FIG 3: Antihypoxic activities of Cantharellus cibarius ex-tract in asphyctic hypoxia in mice. Data are expressed as mean ± standard deviation (n = 8). P < 0.0001 for phenytoin compared to control.

<AU: Sentence OK as edited? “There are data in the literature showing that administra-tion of so-dium fluoride, a substance that induces circula-tory hypoxia, increases blood histamine content and decreases the oxygen-carry-ing capacity of the blood.”>

Volume 16, Number 4, 2014

Antihypoxic Activities of Golden Chanterelle 343

of hypoxia, such as hypobaric hypoxia, has been reported.28 Our results may be supported by other data from the literature showing that flavonoids in-crease cerebral blood flow and possess antihypoxic activity.29 The mechanism of this protective action seems to be in part a result of the antioxidant activ-ity of phenolic acids.30,31

IV. CONCLUSIONS

C. cibarius extract is valuable source of antihypox-ic activity. Extract showed a very good protective effect against hypoxia in some models. In particu-lar, it produced a significant and dose-dependent antihypoxic effect in the hemic and circulatory hypoxia models. Pharmacological effects may be attributable to the presence of polyphenols in the extract. Further investigation of individual com-pounds is required.

ACKNOWLEDGMENTS

This research was supported by a grant from the research council of Medical Sciences University of Mazandaran, Iran. The authors thank Dr. Bahman Eslami for authenticating the mushroom’s scien-tific name.

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