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Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Physico-chemical properties and cytotoxic potential of Cordyceps sinensis metabolites a
a
a
a
Eun-Jeong Lee , Ka-Hee Jang , Seon-Young Im , Yoon-Kyung Lee , bcd
Muhammad Farooq
e
, Rozbeh Farhoudi & Dong-Jin Lee
a
a
Department of Crop Science and Biotechnology, Dankook University, Cheonan, Republic of Korea b
Department of Agronomy, University of Agriculture, Faisalabad, Pakistan
Click for updates
c
The UWA Institute of Agriculture, The University of Western Australia, Crawley, WA6009, Australia d
College of Food and Agricultural Sciences, King Saud University, Riyadh11451, Saudi Arabia e
Department of Agronomy and Plant Breeding, Islamic Azad University, Shoushtar Branch, Shoushtar, Iran Published online: 19 Aug 2014.
To cite this article: Eun-Jeong Lee, Ka-Hee Jang, Seon-Young Im, Yoon-Kyung Lee, Muhammad Farooq, Rozbeh Farhoudi & Dong-Jin Lee (2015) Physico-chemical properties and cytotoxic potential of Cordyceps sinensis metabolites, Natural Product Research: Formerly Natural Product Letters, 29:5, 455-459, DOI: 10.1080/14786419.2014.948438 To link to this article: http://dx.doi.org/10.1080/14786419.2014.948438
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
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Natural Product Research, 2015 Vol. 29, No. 5, 455–459, http://dx.doi.org/10.1080/14786419.2014.948438
SHORT COMMUNICATION Physico-chemical properties and cytotoxic potential of Cordyceps sinensis metabolites Eun-Jeong Leea, Ka-Hee Janga, Seon-Young Ima, Yoon-Kyung Leea, Muhammad Farooqbcd*, Rozbeh Farhoudie and Dong-Jin Leea* a
Department of Crop Science and Biotechnology, Dankook University, Cheonan, Republic of Korea; Department of Agronomy, University of Agriculture, Faisalabad, Pakistan; cThe UWA Institute of Agriculture, The University of Western Australia, Crawley, WA 6009, Australia; dCollege of Food and Agricultural Sciences, King Saud University, Riyadh 11451, Saudi Arabia; eDepartment of Agronomy and Plant Breeding, Islamic Azad University, Shoushtar Branch, Shoushtar, Iran
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b
(Received 9 May 2014; final version received 22 July 2014) This study was conducted to estimate the antioxidant activities, biochemical properties and biological activities of one of the entomopathogenic fungi, Cordyceps sinensis. Analysis of fungal metabolites indicated that the most abundant free sugar was glucose; the highest component of organic acids was citric acid from 10-day culture medium and the glutamate was the predominant amino acid observed from 3-day culture medium. Maximum total polyphenols and flavonoids were detected in the 15-day culture medium. For cytotoxicity test, three cancer cell lines, HepG2 (liver), MCF-7 (breast) and A549 (lung) were used. The IC50 values of the highest toxicity of HepG2 cell lines were observed from 10-day cultured medium, whereas the highest toxicity of MCF-7 and A549 was observed on 5-day cultured medium. This is the first study reporting on the strong antioxidant and cytotoxic potential of C. sinensis. Culture medium of C. sinensis may thus be used as an effective antioxidant and anticancer treatment of natural origin. Keywords: Cordyceps sp; free sugar; organic acid; amino acid; mineral; antioxidant; cytotoxicity
1. Introduction Since prehistoric times, natural substances including plants and their extracts have been used for their healing properties. Synthetic compounds are derived from plants quite often. The use of valuable natural resources for medicinal purposes was possible through the analysis of specific physicochemical properties in the source. The reported functional properties such as antioxidant and anticancer activities are connected with certain target functions, and serve as a foundation in developing treatment for various diseases (Kunzek et al. 2002). Cordyceps sp. is an entomopathogenic fungus, which parasites on caterpillar larvae of Lepidoptera. It was found naturally in the mountains of Tibet and China, and has been used as ancient Chinese medicine since the 1700s. Cordyceps is known to affect multiple body systems, such as the immune system, and kidney and liver function (Holliday & Cleaver 2008). Several investigations have been conducted to develop advanced biomaterials and biologically active substances from Cordyceps sp. (Roberts 1989; Kwon et al. 2000). For instance, Wei (2003) identified mannitol, nucleosides, ergosterol and aminophenol as major biologically active substances in Cordyceps sinensis. Some other researchers reported the role of
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
456
E.-J. Lee et al.
C. sinensis metabolites in antitumour effects, organ transplantation and the prevention of kidney, liver and heart diseases (Shi 2005). Nonetheless, time scale collection and isolation of biologically active substances from C. sinensis metabolites have been rarely reported. Moreover, antioxidant and anticancer potentials of C. sinensis metabolites are not known. In this experiment, C. sinensis was evaluated as a natural biologically active substance. Each culture medium based on different culture period was subjected to component analysis, and efficacy for antioxidant and cytotoxic activities, to provide the basic information for its commercial use and applications.
Downloaded by [Selcuk Universitesi] at 10:20 10 January 2015
2. Results and discussion 2.1. Physicochemical properties Glucose, maltose and fructose were identified as free sugars in C. sinensis culture medium (Table 1). The glucose contents gradually decreased as the culture period went on. The maltose contents demonstrated an increasing trend from day 3 to day 10, but the value suddenly dropped on day 15. In this study, the glucose content decreased continuously; considering the quantity, glucose was the major free sugar component of the culture medium (Table 1). Fructose was in minimum fraction amongst the free sugars. Moreover, contents of free sugars varied with change in the duration of culturing (Table 1). Fungi-derived simple sugars and polysaccharides have attracted a great deal of attention because of their significant pharmacological activities (Zhong et al. 2009). For instance, sugars in Cordyceps sp. possess strong protective effect against chronic renal failure caused by fulgerising kidneys (Wang et al. 2010). Six different organic acids in the culture medium were identified as citric acid, malic acid, formic acid, lactic acid, acetic acid and succinic acid (Table 1). The content of citric acid on day 10 was the maximum amongst all. Other than the citric acid, highest concentrations of malic acid, formic acid, lactic acid, acetic acid and succinic acid were noted on the day 3 compared with other culture periods (Table 1).
2.2. Antioxidant activity assay Analysis of total polyphenols and flavonoids, on each culture period, indicated that the highest contents, both of polyphenol and flavonoids, in the medium were turned out to the day 15 (Table 2). According to Lee et al. (2009) total polyphenols are more in the culture medium than the mycelia. However, culturing conditions such as temperature, time and period for polyphenol extraction need to be optimised. A simple method has been developed to determine the antioxidant activity by utilising the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Originally purple coloured free radical turns to yellow as the odd electron of DPPH radical becomes paired with a hydrogen from a free radical-scavenging antioxidant to form the reduced DPPH-H. The antioxidant activity increased as the culture periods lasted from day 3 till day 15 (Table 2). Compared with the IC50 value of ascorbic acid, which is one of the synthetic antioxidants, a standard, the antioxidant activity of C. sinensis culture medium was not remarkably high. Superoxide dismutase (SOD) is an essential enzyme that catalyses the elimination of superoxide radical anion (O2 2 ) to hydrogen peroxide (H2O2) and O2, and thus protects cells from the damage induced by free radicals. SOD activity is related to various diseases such as Alzheimer and cardiovascular diseases (Balin 1982). The SOD activity of C. sinensis culture medium increased as the culture period went on from day 3 to the day 15 (Table 2). Although, the SOD activity of culture medium was lower than that of ascorbic acid, the value is still considered to be high enough for using the culture medium in natural antioxidant development.
Maltose
0.4 ^ 0.03 0.8 ^ 0.06 1.3 ^ 0.04 0.9 ^ 0.03
Glucose
15.7 ^ 0.6 12.9 ^ 0.6 10.0 ^ 0.7 8.2 ^ 0.4
0.3 ^ 0.03 0.2 ^ 0.04 0.1 ^ 0.03 0.2 ^ 0.06
Fructose 279.2 ^ 2.3 264.7 ^ 1.1 309.2 ^ 2.0 233.6 ^ 2.1
Citric acid
6.27 ^ 0.88b 6.07 ^ 0.58b 6.24 ^ 0.32b 7.04 ^ 0.94a –
1.08 ^ 0.07b 1.03 ^ 0.07b 1.01 ^ 0.04b 1.11 ^ 0.07a –
Total flavonoids (ppm CE/DW) 341.14 ^ 4.4d 233.72 ^ 7.9c 137.43 ^ 3.1b 107.59 ^ 3.9ab 64.02 ^ 1.3a
DPPH free radical-scavenging ability (mg mL21)
27.9 ^ 1.0 24.6 ^ 1.3 24.3 ^ 0.7 25.9 ^ 1.0
Malic acid 7.5 ^ 0.3 5.8 ^ 0.6 2.8 ^ 0.3 3.8 ^ 0.1
Lactic acid
107.77 ^ 6.68b 103.04 ^ 6.42ab 98.27 ^ 0.62ab 95.51 ^ 3.77ab 49.11 ^ 1.35a
Superoxide dismutase activity (mg mL21)
11.8 ^ 0.7 3.4 ^ 0.6 2.4 ^ 0.4 1.4 ^ 0.4
Formic acid
Organic acid (mg L21)
7.0 ^ 0.7 0.7 ^ 0.1 0 0
Succinic acid
86.89 ^ 3.0d 78.53 ^ 2.9c 71.03 ^ 0.8b 66.03 ^ 2.8a 81.02 ^ 0.8c
ABTS free radical-scavenging ability (mg mL21)
5.6 ^ 0.4 2.6 ^ 0.2 1.8 ^ 0.1 1.2 ^ 0.1
Acetic acid
Notes: Means with same letters, within a column, differ significantly at p , 0.05 by Duncan’s multiple range test. GAE, gallic acid equivalent; CE, catechin equivalent; results are expressed as the mean ^ SD. a Ascorbic acid was used as standard antioxidant.
3 5 10 15 Ascorbic acida
Culture Total polyphenols (ppm duration (days) GAE/DW)
Table 2. Antioxidant activities in C. sinensis cultured for different durations.
3 5 10 15
Culture duration (days)
Free sugars (g L21)
Table 1. Free sugars and organic acid composition of C. sinensis cultured for different durations.
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As the experimental results for antioxidant activity may vary with the bioassays used, ABTS free radical-scavenging ability was tested to re-examine the antioxidant activity of the culture medium and to confirm it. ABTS free radical-scavenging ability was measured in values of IC50; the activity increased as the culture period went on day 15 . day 10 . day 5 . day 3 (Table 2). The comparison of scavenging ability of culture medium with ascorbic acid indicated that the scavenging ability of the culture medium at all culture periods, except for day 3, was higher than the ascorbic acid (Table 2). This indicates a great scope to use the C. sinensis culture medium as a natural source of antioxidants.
2.3. Cytotoxic activity assay The C. sinensis culture medium was applied to several cancer cells to monitor the cytotoxic activity. The cancer cells used in the experiment are A549 (human lung adenocarcinoma epithelial cell lien), HepG2 (human hepatocellular liver carcinoma cell line) and MCF-7 (human breast adenocarcinoma cell line), and the cytotoxic effect was measured in IC50 values. Maximum cytotoxic activity of C. sinensis culture medium on A549 cell lines was observed on day 5, whereas maximum cytotoxic activity for HepG2 cell lines was observed on day 10. However, the highest cytotoxic activity of C. sinensis culture medium for MCF-7 cell lines was observed on day 5 (Table 3). The mild oxidative stress in a cell is only related to the growth and survival of the cell, but severe oxidative stress not only suppresses the growth of the cell, but also toxifies the cell and greatly affects its survival. It can be inferred from the lower level of antioxidant enzyme of cancer cell than that of primary cell, as the antioxidants play a key role in cancer treatment (Halliwell 2007). In this experiment, high cytotoxic activity of C. sinensis culture medium was observed on day 5 for HepG2 cell lines and MCF-7 cell lines, while 10 days for A549 cell lines. However, the cytotoxic activity of the culture medium was generally lower than the control, doxorubicin. C. sinensis possesses great potential for its use in pharmaceutical industry. For example, C. sinensis possesses a strong protective effect against chronic renal failure caused by fulgerising kidneys (Wang et al. 2010). Moreover, it is beneficial for heart rhythm disturbances, such as cardiac arrhythmias and chronic heart failure (Zhu et al. 1998). C. sinensis is also quite helpful for respiratory patients, for example in a double-blind placebo-controlled study with 30 elderly volunteers, Cordyceps improved the maximum amount of oxygen the people were able to assimilate (Xiao et al. 1999). Some in vivo and in vitro effects of C. sinensis indicated its potential role as an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males (Halpern & Miller 2002).
Table 3. Comparison of cytotoxic activities on HepG2, MCF-7 and A549 cell lines of C. sinensis. Cytotoxic activities (IC50; mg mL21) Culture periods (days) 3 5 10 15 Doxorubicina
A549
HepG2
MCF-7
333.1 ^ 6.3e 235.6 ^ 12.6b 287.7 ^ 8.5d 268.0 ^ 14.2c 47.1 ^ 0.1a
522.5 ^ 5.5c 474.2 ^ 21.7c 84.7 ^ 2.8b 595.3 ^ 61.9d 78.5 ^ 0.3a
406.70 ^ 4.6c 208.23 ^ 8.7b 466.19 ^ 10.9c 1413.52 ^ 7.9d 41.23 ^ 0.6a
Note: Means with same letters, within a column, differ significantly at p , 0.05 by Duncan’s multiple-range test. Results are expressed as the mean ^ SD. a Doxorubicin was used as standard substance for cytotoxicity assay.
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3. Conclusion This is the first study reporting strong antioxidant and cytotoxic potential of C. sinensis. Culture medium of C. sinensis may thus be used as an effective antioxidant and for anticancer treatment of natural origin. However, further studies are required to optimise the conditions, such as temperature, time and period, for the extraction of bioactive substances. Supplementary material Experimental details relating to this article are available online. Funding
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This study was supported by the research [grant number PJ0089802013] from the Rural Development Administration (RDA) of South Korea.
References Balin AK. 1982. Testing the free radical theory of aging. In: Adelman RR, Roth GS, editors. Testing the theories of aging. Boca Raton, FL: CRC Press; p. 137–182. Halliwell B. 2007. Dietary polyphenols: good, bad, or indifferent for our health? Cardiovasc Res. 73:341–347. Halpern GM, Miller A. 2002. Medicinal mushrooms: ancient remedies for modern ailments. New York: M. Evans & Company. Holliday J, Cleaver M. 2008. Medicinal value of the caterpillar fungi species of the genus Cordyceps (Fr.) link (Ascomycetes). A review. Int J Med Mushrooms. 10:219–234. Kunzek H, Muller S, Vetter S. 2002. The significance of physicochemical properties of plant cell wall materials for the development of innovative food products. Eur Food Res Technol. 214:361– 376. Kwon HC, Moon HI, Choi SH, Lee JO, Cho SY, Jung IY, Kim SY, Lee KR. 2000. Cytotoxic constituents of Bombycis corpus. Yakhak Hoeji. 43:169–172. Lee KM, Hee NS, Suk SH, Hong YJ, Gill LK, Hwan BY. 2009. Total phenol contents and DPPH radical scavenging activity of entomopathogenic fungi. Korean J Appl Entomol. 48:377–383. Roberts DW. 1989. World picture of biological control of fungi. Mem Inst Owaldo Cruz Rio de Janeiro. 84:89–100. Shi YX. 2005. Update on researches of pharmacological effects of Cordyceps. Chin Pharm. 14:72–74. Wang Y, Yin H, Lv X, Wang Y, Gao H, Wang M. 2010. Protection of chronic renal failure by a polysaccharide from Cordyceps sinensis. Fitoterapia. 81:397–402. Wei L. 2003. Research advanced in functional composition content of Cordyceps. West Chin J Pharm Sci. 18:359–360. Xiao Y, Huang XZ, Chen G, Wang MB, Zhu JS, Cooper CB. 1999. Increased aerobic capacity in healthy elderly humans given fermented Cordyceps Cs-4: a placebo controlled trial. In: Annual Meeting American College of Sports Medicine, Seattle, WA, June 2–5, 1999; p. 774. Zhong S, Pan H, Fan L, Lv G, Wu Y, Parmeswaran B, Pandey A, Soccol CR. 2009. Advances in research of polysaccharides in Cordyceps species. Food Technol Biotechnol. 47:304– 312. Zhu JS, Halpern GM, Jones K. 1998. The scientific rediscovery of a precious ancient Chinese herbal regimen: Cordyceps sinensis. Part II. J Altern Complement Med. 4:429–457.
Natural Product Research: Formerly Natural Product Letters Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/gnpl20
Physico-chemical properties and cytotoxic potential of Cordyceps sinensis metabolites a
a
a
a
Eun-Jeong Lee , Ka-Hee Jang , Seon-Young Im , Yoon-Kyung Lee , bcd
Muhammad Farooq
e
, Rozbeh Farhoudi & Dong-Jin Lee
a
a
Department of Crop Science and Biotechnology, Dankook University, Cheonan, Republic of Korea b
Department of Agronomy, University of Agriculture, Faisalabad, Pakistan
Click for updates
c
The UWA Institute of Agriculture, The University of Western Australia, Crawley, WA6009, Australia d
College of Food and Agricultural Sciences, King Saud University, Riyadh11451, Saudi Arabia e
Department of Agronomy and Plant Breeding, Islamic Azad University, Shoushtar Branch, Shoushtar, Iran Published online: 19 Aug 2014.
To cite this article: Eun-Jeong Lee, Ka-Hee Jang, Seon-Young Im, Yoon-Kyung Lee, Muhammad Farooq, Rozbeh Farhoudi & Dong-Jin Lee (2015) Physico-chemical properties and cytotoxic potential of Cordyceps sinensis metabolites, Natural Product Research: Formerly Natural Product Letters, 29:5, 455-459, DOI: 10.1080/14786419.2014.948438 To link to this article: http://dx.doi.org/10.1080/14786419.2014.948438
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content.
Downloaded by [Selcuk Universitesi] at 10:20 10 January 2015
This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/termsand-conditions
Natural Product Research, 2015 Vol. 29, No. 5, 455–459, http://dx.doi.org/10.1080/14786419.2014.948438
SHORT COMMUNICATION Physico-chemical properties and cytotoxic potential of Cordyceps sinensis metabolites Eun-Jeong Leea, Ka-Hee Janga, Seon-Young Ima, Yoon-Kyung Leea, Muhammad Farooqbcd*, Rozbeh Farhoudie and Dong-Jin Leea* a
Department of Crop Science and Biotechnology, Dankook University, Cheonan, Republic of Korea; Department of Agronomy, University of Agriculture, Faisalabad, Pakistan; cThe UWA Institute of Agriculture, The University of Western Australia, Crawley, WA 6009, Australia; dCollege of Food and Agricultural Sciences, King Saud University, Riyadh 11451, Saudi Arabia; eDepartment of Agronomy and Plant Breeding, Islamic Azad University, Shoushtar Branch, Shoushtar, Iran
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b
(Received 9 May 2014; final version received 22 July 2014) This study was conducted to estimate the antioxidant activities, biochemical properties and biological activities of one of the entomopathogenic fungi, Cordyceps sinensis. Analysis of fungal metabolites indicated that the most abundant free sugar was glucose; the highest component of organic acids was citric acid from 10-day culture medium and the glutamate was the predominant amino acid observed from 3-day culture medium. Maximum total polyphenols and flavonoids were detected in the 15-day culture medium. For cytotoxicity test, three cancer cell lines, HepG2 (liver), MCF-7 (breast) and A549 (lung) were used. The IC50 values of the highest toxicity of HepG2 cell lines were observed from 10-day cultured medium, whereas the highest toxicity of MCF-7 and A549 was observed on 5-day cultured medium. This is the first study reporting on the strong antioxidant and cytotoxic potential of C. sinensis. Culture medium of C. sinensis may thus be used as an effective antioxidant and anticancer treatment of natural origin. Keywords: Cordyceps sp; free sugar; organic acid; amino acid; mineral; antioxidant; cytotoxicity
1. Introduction Since prehistoric times, natural substances including plants and their extracts have been used for their healing properties. Synthetic compounds are derived from plants quite often. The use of valuable natural resources for medicinal purposes was possible through the analysis of specific physicochemical properties in the source. The reported functional properties such as antioxidant and anticancer activities are connected with certain target functions, and serve as a foundation in developing treatment for various diseases (Kunzek et al. 2002). Cordyceps sp. is an entomopathogenic fungus, which parasites on caterpillar larvae of Lepidoptera. It was found naturally in the mountains of Tibet and China, and has been used as ancient Chinese medicine since the 1700s. Cordyceps is known to affect multiple body systems, such as the immune system, and kidney and liver function (Holliday & Cleaver 2008). Several investigations have been conducted to develop advanced biomaterials and biologically active substances from Cordyceps sp. (Roberts 1989; Kwon et al. 2000). For instance, Wei (2003) identified mannitol, nucleosides, ergosterol and aminophenol as major biologically active substances in Cordyceps sinensis. Some other researchers reported the role of
*Corresponding authors. Email: [email protected]; [email protected] q 2014 Taylor & Francis
456
E.-J. Lee et al.
C. sinensis metabolites in antitumour effects, organ transplantation and the prevention of kidney, liver and heart diseases (Shi 2005). Nonetheless, time scale collection and isolation of biologically active substances from C. sinensis metabolites have been rarely reported. Moreover, antioxidant and anticancer potentials of C. sinensis metabolites are not known. In this experiment, C. sinensis was evaluated as a natural biologically active substance. Each culture medium based on different culture period was subjected to component analysis, and efficacy for antioxidant and cytotoxic activities, to provide the basic information for its commercial use and applications.
Downloaded by [Selcuk Universitesi] at 10:20 10 January 2015
2. Results and discussion 2.1. Physicochemical properties Glucose, maltose and fructose were identified as free sugars in C. sinensis culture medium (Table 1). The glucose contents gradually decreased as the culture period went on. The maltose contents demonstrated an increasing trend from day 3 to day 10, but the value suddenly dropped on day 15. In this study, the glucose content decreased continuously; considering the quantity, glucose was the major free sugar component of the culture medium (Table 1). Fructose was in minimum fraction amongst the free sugars. Moreover, contents of free sugars varied with change in the duration of culturing (Table 1). Fungi-derived simple sugars and polysaccharides have attracted a great deal of attention because of their significant pharmacological activities (Zhong et al. 2009). For instance, sugars in Cordyceps sp. possess strong protective effect against chronic renal failure caused by fulgerising kidneys (Wang et al. 2010). Six different organic acids in the culture medium were identified as citric acid, malic acid, formic acid, lactic acid, acetic acid and succinic acid (Table 1). The content of citric acid on day 10 was the maximum amongst all. Other than the citric acid, highest concentrations of malic acid, formic acid, lactic acid, acetic acid and succinic acid were noted on the day 3 compared with other culture periods (Table 1).
2.2. Antioxidant activity assay Analysis of total polyphenols and flavonoids, on each culture period, indicated that the highest contents, both of polyphenol and flavonoids, in the medium were turned out to the day 15 (Table 2). According to Lee et al. (2009) total polyphenols are more in the culture medium than the mycelia. However, culturing conditions such as temperature, time and period for polyphenol extraction need to be optimised. A simple method has been developed to determine the antioxidant activity by utilising the stable 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. Originally purple coloured free radical turns to yellow as the odd electron of DPPH radical becomes paired with a hydrogen from a free radical-scavenging antioxidant to form the reduced DPPH-H. The antioxidant activity increased as the culture periods lasted from day 3 till day 15 (Table 2). Compared with the IC50 value of ascorbic acid, which is one of the synthetic antioxidants, a standard, the antioxidant activity of C. sinensis culture medium was not remarkably high. Superoxide dismutase (SOD) is an essential enzyme that catalyses the elimination of superoxide radical anion (O2 2 ) to hydrogen peroxide (H2O2) and O2, and thus protects cells from the damage induced by free radicals. SOD activity is related to various diseases such as Alzheimer and cardiovascular diseases (Balin 1982). The SOD activity of C. sinensis culture medium increased as the culture period went on from day 3 to the day 15 (Table 2). Although, the SOD activity of culture medium was lower than that of ascorbic acid, the value is still considered to be high enough for using the culture medium in natural antioxidant development.
Maltose
0.4 ^ 0.03 0.8 ^ 0.06 1.3 ^ 0.04 0.9 ^ 0.03
Glucose
15.7 ^ 0.6 12.9 ^ 0.6 10.0 ^ 0.7 8.2 ^ 0.4
0.3 ^ 0.03 0.2 ^ 0.04 0.1 ^ 0.03 0.2 ^ 0.06
Fructose 279.2 ^ 2.3 264.7 ^ 1.1 309.2 ^ 2.0 233.6 ^ 2.1
Citric acid
6.27 ^ 0.88b 6.07 ^ 0.58b 6.24 ^ 0.32b 7.04 ^ 0.94a –
1.08 ^ 0.07b 1.03 ^ 0.07b 1.01 ^ 0.04b 1.11 ^ 0.07a –
Total flavonoids (ppm CE/DW) 341.14 ^ 4.4d 233.72 ^ 7.9c 137.43 ^ 3.1b 107.59 ^ 3.9ab 64.02 ^ 1.3a
DPPH free radical-scavenging ability (mg mL21)
27.9 ^ 1.0 24.6 ^ 1.3 24.3 ^ 0.7 25.9 ^ 1.0
Malic acid 7.5 ^ 0.3 5.8 ^ 0.6 2.8 ^ 0.3 3.8 ^ 0.1
Lactic acid
107.77 ^ 6.68b 103.04 ^ 6.42ab 98.27 ^ 0.62ab 95.51 ^ 3.77ab 49.11 ^ 1.35a
Superoxide dismutase activity (mg mL21)
11.8 ^ 0.7 3.4 ^ 0.6 2.4 ^ 0.4 1.4 ^ 0.4
Formic acid
Organic acid (mg L21)
7.0 ^ 0.7 0.7 ^ 0.1 0 0
Succinic acid
86.89 ^ 3.0d 78.53 ^ 2.9c 71.03 ^ 0.8b 66.03 ^ 2.8a 81.02 ^ 0.8c
ABTS free radical-scavenging ability (mg mL21)
5.6 ^ 0.4 2.6 ^ 0.2 1.8 ^ 0.1 1.2 ^ 0.1
Acetic acid
Notes: Means with same letters, within a column, differ significantly at p , 0.05 by Duncan’s multiple range test. GAE, gallic acid equivalent; CE, catechin equivalent; results are expressed as the mean ^ SD. a Ascorbic acid was used as standard antioxidant.
3 5 10 15 Ascorbic acida
Culture Total polyphenols (ppm duration (days) GAE/DW)
Table 2. Antioxidant activities in C. sinensis cultured for different durations.
3 5 10 15
Culture duration (days)
Free sugars (g L21)
Table 1. Free sugars and organic acid composition of C. sinensis cultured for different durations.
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As the experimental results for antioxidant activity may vary with the bioassays used, ABTS free radical-scavenging ability was tested to re-examine the antioxidant activity of the culture medium and to confirm it. ABTS free radical-scavenging ability was measured in values of IC50; the activity increased as the culture period went on day 15 . day 10 . day 5 . day 3 (Table 2). The comparison of scavenging ability of culture medium with ascorbic acid indicated that the scavenging ability of the culture medium at all culture periods, except for day 3, was higher than the ascorbic acid (Table 2). This indicates a great scope to use the C. sinensis culture medium as a natural source of antioxidants.
2.3. Cytotoxic activity assay The C. sinensis culture medium was applied to several cancer cells to monitor the cytotoxic activity. The cancer cells used in the experiment are A549 (human lung adenocarcinoma epithelial cell lien), HepG2 (human hepatocellular liver carcinoma cell line) and MCF-7 (human breast adenocarcinoma cell line), and the cytotoxic effect was measured in IC50 values. Maximum cytotoxic activity of C. sinensis culture medium on A549 cell lines was observed on day 5, whereas maximum cytotoxic activity for HepG2 cell lines was observed on day 10. However, the highest cytotoxic activity of C. sinensis culture medium for MCF-7 cell lines was observed on day 5 (Table 3). The mild oxidative stress in a cell is only related to the growth and survival of the cell, but severe oxidative stress not only suppresses the growth of the cell, but also toxifies the cell and greatly affects its survival. It can be inferred from the lower level of antioxidant enzyme of cancer cell than that of primary cell, as the antioxidants play a key role in cancer treatment (Halliwell 2007). In this experiment, high cytotoxic activity of C. sinensis culture medium was observed on day 5 for HepG2 cell lines and MCF-7 cell lines, while 10 days for A549 cell lines. However, the cytotoxic activity of the culture medium was generally lower than the control, doxorubicin. C. sinensis possesses great potential for its use in pharmaceutical industry. For example, C. sinensis possesses a strong protective effect against chronic renal failure caused by fulgerising kidneys (Wang et al. 2010). Moreover, it is beneficial for heart rhythm disturbances, such as cardiac arrhythmias and chronic heart failure (Zhu et al. 1998). C. sinensis is also quite helpful for respiratory patients, for example in a double-blind placebo-controlled study with 30 elderly volunteers, Cordyceps improved the maximum amount of oxygen the people were able to assimilate (Xiao et al. 1999). Some in vivo and in vitro effects of C. sinensis indicated its potential role as an alternative medicine for the treatment of some reproductive problems caused by insufficient testosterone levels in human males (Halpern & Miller 2002).
Table 3. Comparison of cytotoxic activities on HepG2, MCF-7 and A549 cell lines of C. sinensis. Cytotoxic activities (IC50; mg mL21) Culture periods (days) 3 5 10 15 Doxorubicina
A549
HepG2
MCF-7
333.1 ^ 6.3e 235.6 ^ 12.6b 287.7 ^ 8.5d 268.0 ^ 14.2c 47.1 ^ 0.1a
522.5 ^ 5.5c 474.2 ^ 21.7c 84.7 ^ 2.8b 595.3 ^ 61.9d 78.5 ^ 0.3a
406.70 ^ 4.6c 208.23 ^ 8.7b 466.19 ^ 10.9c 1413.52 ^ 7.9d 41.23 ^ 0.6a
Note: Means with same letters, within a column, differ significantly at p , 0.05 by Duncan’s multiple-range test. Results are expressed as the mean ^ SD. a Doxorubicin was used as standard substance for cytotoxicity assay.
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3. Conclusion This is the first study reporting strong antioxidant and cytotoxic potential of C. sinensis. Culture medium of C. sinensis may thus be used as an effective antioxidant and for anticancer treatment of natural origin. However, further studies are required to optimise the conditions, such as temperature, time and period, for the extraction of bioactive substances. Supplementary material Experimental details relating to this article are available online. Funding
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This study was supported by the research [grant number PJ0089802013] from the Rural Development Administration (RDA) of South Korea.
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