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Determination of essential and toxic elements in Cordyceps kyushuensis Kawam by inductively coupled plasma mass spectrometry
Journal of Pharmaceutical and Biomedical Analysis 72 (2013) 172–176
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Determination of essential and toxic elements in Cordyceps kyushuensis Kawam by inductively coupled plasma mass spectrometry Guoying Zhang a,b , Yanxin Zhao c , Fengjun Liu d , Jianya Ling a,∗ , Jianqiang Lin a,∗ , Changkai Zhang a a
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China Shandong University of Traditional Chinese Medicine, Jinan 250014, China Jinan Central Hospital, Jinan 250013, China d Qianfoshan Hospital, Jinan 250014, China b c
a r t i c l e
i n f o
Article history: Received 9 April 2012 Received in revised form 5 August 2012 Accepted 7 August 2012 Available online 16 August 2012 Keywords: Cordyceps kyushuensis Kawam ICP-MS Microwave digestion Multi-element analysis Food composition
a b s t r a c t In this study, a total of 20 elements (essential, non-essential and toxic): lithium (Li), sodium (Na), potassium (K), gallium (Ga), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), vanadium (V), chromium (Cr), nickel (Ni), cobalt (Co), molybdenum (Mo), selenium (Se), barium (Ba), tin (Sn), arsenic (As), lead (Pb) cadmium (Cd) and mercury (Hg) in natural and cultured Cordyceps kyushuensis have been determined by means of inductively coupled plasma mass spectrometry (ICP-MS). Cultured stroma, natural stroma and natural worm were digested by microwave-assisted method before analysis. The proposed ICP-MS method was validated by analyzing a certified reference material (CRM) GBW10015 (spinach). The results of one-way analysis of variance (ANOVA) revealed that the element concentrations in the three kinds of samples were significantly different (p < 0.05). Except for Mg, Zn, Cu, the values of other elemental contents were the highest in the stroma of natural C. kyushuensis. In comparison with the worm, the concentrations of determined elements in wild stroma were higher. The remarkable difference of elemental contents between cultured and natural stroma may be caused by distinct growing environment. This finding highlighted the usefulness of ICP-MS elemental analysis and enhanced the value of C. kyushuensis as a candidate for nourishing food based on its composition. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The fungi of genus Cordyceps belonging to Clavicipitaceae, Ascomycotina [1], have received significant attention from medical and pharmacological researchers. Cordyceps was commonly used as a traditional Chinese medicine for the treatment of fatigue, night sweating, hyperglycemia, hyperlipidemia, asthenia after severe illness, respiratory disease, renal dysfunction, arrhythmias, other heart and liver diseases. The pharmacological actions of Cordyceps such as anti-oxidation [2], potentiating the immune system [3] and anti-tumor activities [4] have been demonstrated. Recently, Cordyceps has become more and more attractive as dietary food and source of medicine in some countries [5–7]. Cordyceps kyushuensis Kawam, is a unique species of Cordyceps, which parasitizes on the larvae of Clanis bilineata walker. Some pharmacologically active substances, such as cordycepin and adenosine have been found in both stroma and worm [8]. However
∗ Corresponding authors. Tel.: +86 531 88364427; fax: +86 531 88567250. E-mail addresses: [email protected] (J. Ling), [email protected] (J. Lin). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.08.007
no research has been devoted to the elemental composition of C. kyushuensis to date. Many analytical methods, such as graphite furnace atomic absorption spectrometry (GF-AAS), inductively coupled plasma atomic emission spectroscopy (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS) have been used to investigate the element concentrations of all kinds of biological samples, foodstuffs, medicinal materials, and other samples. GF-AAS is sensitive enough to detect trace elements, but as a mono-elemental technique, it is slow and tedious when a large number of elements needed to be analyzed. Even though ICP-AES is a multi-elemental technique, but the disadvantage of insufficient sensitivity made it little bit in trace elements detection. ICP-MS, employed in this study, has been widely used as a technique for its significant advantages, such as simultaneous multi-element measurement capability and very low detection limits [9]. Moreover, it offers a wider linear dynamic range which allows the determination of major and trace elements in the same sample [10]. The aim of this study was to apply the validated ICP-MS method to determine the content of 20 essential, non-essential and toxic elements (Na, K, Ca, Mg, Cu, Fe, Zn, V, Se, Cr, Co, Mo, Mn, Ni, Ba, Sn, Pb, Cd, As and Hg) in cultured C. kyushuensis stroma, as well as natural stroma and worm of this fungus.
G. Zhang et al. / Journal of Pharmaceutical and Biomedical Analysis 72 (2013) 172–176 Table 1 Operating parameters for ICP-MS. Operating parameter
Value
Type of nebulizer Nebulizer Ar flow rate Auxiliary Ar flow rate Plasma Ar flow rate Analog stage voltage Lens voltage Pulse stage voltage Sample uptake rate ICP RF power Radiofrequency generator Spray chamber Sample cone Skimmer cone Type of torch Scanning mode Dwell time Integration time Number of readings per replicate Number of replicate
Cross-flow 0.86 L min−1 1.2 L min−1 15 L min−1 −1900 V 5.7 V 900 V 1.0 L min−1 1100 W 40 MHz Ryton® , double-pass Nickel, 1.1 mm orifice diameter Nickel, 0.9 mm orifice diameter Standard quartz torch Peak hopping 55 ms 1000 ms 1 5
2. Materials and methods 2.1. Instrumentation, equipment and reagents A microwave oven (Milestone Ethos A Digestion/Extraction Microwave Labstation, Sorisole, Italy) equipped with twelve 100 mL TFM Teflon vessels, with an energy output of 900 W, was employed to digest the samples. The maximum digestion temperature and pressure were 300 ◦ C and 3.45 MPa, respectively. A quadrupole ELAN DRC-e inductively coupled plasma mass spectrometer (Perkin-Elmer SCIEX, Concord, Ont., Canada) was used; a gem tipped cross-flow nebulizer interfaced with a (Ryton) Scott double pass spray chamber, using a 2.0 mm id alumina injector tube, comprised the sample introduction system. ICP conditions were selected that maximized the ion signals of the elements studied while reducing the background to a minimum. Optimized with a multi-element calibration stock solution (10 g/L) containing Ba, Be, Ce, Co, In, Mg, Pb, Rh, and U (obtained from Perkin-Elmer, Concord, Ont., Canada), the operating parameters of the ICP-MS for the analysis were summarized in Table 1. The content of each trace element was obtained from the average value of five replicates of one pooled sample. Rh mono-element standard solution from PerkinElmer (Norwalk, CT, USA) for internal standard was employed. High purity de-ionized water with a resistivity of 18.2 M cm−1 was obtained using a Synergy water purification system with SynergyPak purification cartridges (Billerica, MA, USA). Concentrated nitric acid (65%, Merck, Germany) used for sample mineralization was high-grade purity reagent, and hydrogen peroxide (30%, Merck) was analytical reagent. The working standard solutions used to establish calibration curves were prepared at the concentration from 25, 50, 75, 100, 125, 250, 500, 750 and 1000 g/mL with the 1000 g/mL each single-element stock standard solution (Traceable to National Institute of Standards and Technology Reference Materials) diluted with de-ionized water immediately before analysis. All prepared solutions were stored in polyethylene volumetric flasks and contain 1% (v/v) HNO3 . All microwave vessels and plastic materials were washed with de-ionized water three times, then they were immersed in 15% HNO3 solution for 24 h. Finally they were thoroughly rinsed with ultrapure water and dried before use.
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standard plant sample (GBW10015, spinach) was purchased from National Standard Center of China. Natural C. kyushuensis was collected from Mount Meng, Shandong province of China in August 2011, and identified by Professor Yinglan Guo (Institute of Microbiology, Chinese Academy of Science, China). Cultured fruiting body of C. kyushuensis was cultivated in our laboratory. The anamorph strain, originally isolated from fresh specimen, was confirmed by means of both morphological and molecular methods and kept on potato dextrose agar (PDA) slant culture at 4 ◦ C. Mycelial fragments were suspended in 5 mL of sterile deionized water and used as inocula in 250 mL flasks containing 95 mL PDA broth for pre-cultures. Flasks were shaken at 150 rpm for 6 days at 25 ◦ C. Subsequently, the husked rice medium was inoculated with the liquid seed culture. After culturing at 25 ◦ C for about 15 days with the supplement of moisture and illumination, the fungus proliferated all over the medium and then was aged for about 60 days. 2.3. Sample preparation Natural C. kyushuensis sample, divided into stroma and worm parts, were washed with ultrapure water and oven-dried at 60 ◦ C to constant weight together with cultured stroma. Then all samples were homogenized with a micro plant crusher and screened with a 200 mesh sieve. As an excellent technology can reduce contamination and improve efficiency of mineralization, microwave-assisted digestion was employed to decompose the samples at a temperature of 200 ◦ C for ramping 10 min and holding 20 min. Several mixtures of nitric acid and hydrogen peroxide were attempted for mineralization of samples, such as 5 mL HNO3 + 2 mL H2 O2 (procedure A), 7 mL HNO3 + 1 mL H2 O2 (procedure B), 9 mL HNO3 + 2 mL H2 O2 (procedure C), 10 mL HNO3 + 2 mL H2 O2 (procedure D). Approximately 0.2 g of each sample and CRM were accurately weighed and transferred to the Teflon digestion vessels, and then the corresponding volume of concentrated HNO3 and 30% (v/v) H2 O2 were added. Each sample was prepared in triplicated. Sample blank was used in accordance with the mineralization procedure. After microwave digestion, the solutions were evaporated to a small volume at the temperature not higher than 60 ◦ C. Then the concentrated solutions were transferred to 50 mL volumetric flasks, and 0.5 mL HNO3 was added before adjusting the final volume to 50 mL with high purity de-ionized water. Rh was added to a final concentration of 10 g/L for its evaluation as internal standard. In order to avoid the possible interferences caused by sample matrix and oxidizing materials, the isotope masses shown in Table 1 were selected for simultaneous monitoring. The spectral interferences were checked under the standard mode ICP-MS (DRC OFF) in this experiment. 2.4. Validation of the method The accuracy and precision of the proposed method were assessed by analyzing the trace elements in CRM (spinach). Linearity of each analytical element was expressed as the related coefficients (R) of specification curves and the minimum acceptable value of R was 0.9995. According to the recommendations of IUPAC, the limit of detection (LOD) was determined as three times the standard deviation of ten consecutive measurements of sample blank solutions prepared by the same microwave procedure as the samples. 2.5. Statistical analysis
2.2. Samples Routinely used as the certified reference material (CRM) for fruits, vegetable, staple and non-staple foodstuff, a Chinese national
One-way analysis of variance (ANOVA) was employed to perform statistical analysis of the results. It was conducted using the statistical analysis packages of SPSS version 16.0
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for windows. The level of statistical significance was set at P-value < 0.05. 3. Results and discussion 3.1. Sample digestion procedure Sample digestion is one of the most important steps for chemical analysis. Because complete decomposition without contamination or loss of elements is the prerequisites for reproducible and accurate analysis, several mineralization methods, such as dry ashing method, wet digestion procedure, and microwave-assisted digestion mode [11], have been employed to decompose biological, environmental and other various samples. A long period of time is needed to incinerate samples in a crucible furnace by dry ashing method. The use of concentrated acids and careful monitoring of digestion for varying periods are needed in wet digestion procedure. Both methods have shortcomings of time-consuming and possible volatilization loss of some elements. An alternative method is microwave-assisted digestion coupled with closed vessels, which has better recovery of volatile elements, more reproducible procedures and a better working environment [12]. Several combinations of nitric acid with hydrogen peroxide were investigated in this paper. This was an attractive procedure because the study had been aimed at avoiding the use of HCl, HClO4 , and HF in the digestion of the fungi of genus Cordyceps. HCl may introduce additional interference elements into ICP-MS system [13], Toxic HF is found to depress the fluorescence signal [14], and explosive HClO4 is expensive and dangerous [15,16]. From the experimental results, great quantity of black precipitate remained in the resultant solutions at the end of program A and B, respectively. There was still a small amount of white residue revealed after program C. The Cordyceps samples were found to be thoroughly decomposed after performing program D. Therefore program D of microwave-assisted digestion had been applied to all analysis. The final results also shown that the microwave digestion procedure gave the best agreement between measured elemental mass fractions and certified values. 3.2. Selection of isotopes Measurements of trace elements by ICP-MS method usually suffer from interferences caused by isotopes, sample matrices, plasma working gas, double-charge ions, and some polyatomic ions. The absence of polyatomic interferences was checked when required, by measuring several isotopes of the elements and checking the isotopic ratio. Specific isotopes should be carefully considered and monitored according to the sensibility of the element and/or possible isobaric and polyatomic interferences. Through the results of our previous determination and other researchers’ reports, the isotopes measured were shown in Table 2. 52 Cr and 59 Co were chosen to avoid the potential polyatomic interferences caused by 40 Ar, 12 C,43 Ar and 16 O, through adopted microwave digestion procedure, whereas double-charge ions and oxide interferences could be generally eliminated by means of selecting appropriate instrumental working parameters. 111 Cd was selected to be determined rather than 114 Cd because of the isobaric interference of tin (114 Sn) [17]. Due to the severe polyatomic isobaric interferences in 56 Fe [18], which cannot be used with a simple quadrupole, 57 Fe had to be measured and the sensitivity was lower. Similarly, in the determination of nickel, 60 Ni was used to avert interferences with 23 Na35 Cl or 58 Fe when 58 Ni was analyzed [19].
3.3. Method validation As a part of the quality assurance–quality control protocol, the CRM (GBW10015) was analyzed to determine the accuracy (%RE), precision (%RSD) and LOD of the proposed method. The results shown in Table 2 were generally in good agreement with the certified concentrations in CRM, indicating validity of the method employed in this work. The accuracy for all of the detected elements was less than 10%. The analytical precision expressed as %RSD, for Na, Cd, Co, Cu, Ni, Mn and Ba, was routinely between 5 and 10%, for other elements, were less than 5%. Except for Se, Sn and Hg, the percentage of relative error (%RE) was the lowest for Na and the highest for Cu, and the percentage of relative standard deviation (%RSD) was minimum for Cr and maximum for Ba. The correlation coefficients (R) of calibration curves were at least 0.9998, indicating good linear relationships throughout the concentration range analyzed. The method detection limits obtained for the selected elements were also shown in Table 2, which were determined as 3 SD of the 20 consecutive measurements of the reagent blanks multiplied by the dilution factor used for sample preparation (250 mg of sample/50 mL). In the absence of polyatomic interferences, the level of a selected isotope for a given element had a great influence on the detection limits of the particular element. The greater the abundance of the isotope, the lower the detection limit. 3.4. Concentration of trace elements in natural and cultured C. kyushuensis In this study, the elements determined were 20 subdivided into essential (macro and micro) and non-essential or toxic. Among the essential elements estimated in the present work we found the macro elements (Na, K, Ca and Mg) and microelements (Zn, Fe, Cu, Mn, V, Cr, Ni, Co, Mo, and Se); along the nonessential elements and the toxic elements Ba, Sn, As, Cd, Pb and Hg. The results are presented in Table 3. Trace elements contained in three different samples were considerably different at the 95% confidence level according to the results of one-way analysis of variance (ANOVA). For macro element, in this study, Na (1420 g/g) and K (946 g/g) in wild stroma exhibited the highest levels and were significantly different from the other two samples. While the maximum content of Mg (986 g/g) was found in the cultured stroma, which is considerably higher than those of wild stroma and worm. For Ca, there is a similar content in the cultured stroma (547 g/g) and natural stroma (453 g/g), and a relatively lower concentration (238 g/g) was found in natural worm. Potassium and magnesium are essential electrolytes for maintaining normal fluid balance in cells and a delicate balance of these two elements is reported to prevent an increase in blood pressure and to maintain normal cardiac rhythm. Calcium is known to be involved in muscle contraction and relaxation, blood clotting, proper nerve function and body immune defenses [20,21]. The biological roles of K, Mg and Ca are essential for disease prevention and control [22]. In addition to the four macro elements analyzed in this study, 16 trace elements were also studied. Trace elements, in minute quantities, are essential for the normal health and function of humans. They can be divided into three groups of essential microelements, possible essential microelements and potentially toxic microelements or non-essential microelements. Trace elements, whether essential or non-essential, can cause morphological abnormalities, reduce growth and increase mortality and mutagenic effects, above their critical threshold concentrations [23]. Essential microelements, including Fe, Zn, Cu, Mn, Cr, Mo, Co, Se, Ni and V were determined in this work. The result showed that the concentrations of Fe, Cu, Mn, V and Cr in natural stroma are between
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Table 2 Accuracy, precision and LOD validated by certified reference material with Rh (10 g/L) as internal standard. Element
Isotopes (m/z)
Certified value (g/g)
Found value (g/g)a
%RE
%RSD
Detection limit (ng/g)
Na K Ca Mg Fe Zn Mn Pb Ba Cu Cr Ni V Mo Cd As Co Se Sn Hg
23
15,000 24,900 6600 5520 540 35.3 41 11.10 9.00 8.90 1.40 0.92 0.87 0.47 0.15 0.23 0.22 0.09 0.06 0.02
15,238 23,860 6248 5630 556 32.9 39 11.32 8.37 8.10 1.43 0.89 0.82 0.45 0.14 0.25 0.21 0.09 0.06 0.02
1.59 −4.18 −5.33 +1.99 +2.96 −6.80 −4.88 +1.98 −7.00 −9.00 +2.14 −3.26 −5.75 −4.26 −6.67 +8.70 −4.55 0 0 0
5.60 3.48 4.24 2.54 3.67 4.46 6.38 3.85 8.71 7.74 2.12 5.47 4.71 2.15 8.46 4.45 7.82 0 0 0
80 90 100 80 70 30 50 40 70 20 10 7 5 5 4 5 5 3 7 1
a
Na 39 K 43 Ca 24 Mg 56 Fe 66 Zn 55 Mn 208 Pb 137 Ba 63 Cu 53 Cr 60 Ni 51 V 98 Mo 111 Cd 75 As 59 Co 82 Se 118 Sn 202 Hg
Results were mean ± standard deviation obtained from five replicates of one pooled sample.
18.62 and 114.72 g/g, which is higher than those of cultured strom and natural worm, except that Zn is higher in the cultured strom (84.81 ppm) than in the natural stroma (65.25 ppm), and the concentration of Cu is broadly similar in cultured strom (14.50 g/g) and natural stroma (13.18 g/g). For Ni, Co, Mo and Se, the content of wild stroma is between 0.74 and 3.23 g/g. While the cultured stroma (0.46–2.86 g/g) and natural worm (0.20–1.35 g/g) showed a lower content for the same elements. Fe, Cu, Mn and Zn are essential elements in enzyme metabolism. They have immunomodulatory functions and thus influence the susceptibility to the course and the outcome of a variety of viral infections [24]. The importance of iron in maintaining good health and well being has long been recognized by nutritionists [25]. Co, as a component of the vitamin B12 complex, is an essential trace element of human body, which can significantly stimulate the production of blood and effectively cure the macrocytic anemia [19]. The essential trace element molybdenum is bound to a required for the physiological function of some enzymes, such as xanthine oxidize, aldehyde oxidase, sulfite oxidase, nitrate reductase, and so on. Metallic vanadium is probably an essential trace element, which most common valence states are V3+ , V4+ , and V5+ . The pentavalent state (VO3 − ) is the most common form in extracellular fluid whereas the quadrivalent form (VO2+ ) predominates in intracellular [26]. Although the concentration of V in most foods is low, food is the main source of this trace element for the general population. Non-essential elements such as Ba and Sn were also detected in this study. The element Ba is rich not only in natural stroma (8.12 g/g), but also in the cultured stroma (1.89 g/g) and natural
worm (6.60 g/g), while the concentration of Sn is only between 0.15 and 0.76 g/g in all the three samples. Although there are no reported physiological effects of Ba and Sn at low concentrations, their accumulation in the body system could be detrimental to health. Under normal conditions, the element barium has no remarkable trophism or toxic action to the human body. However, some barium salts are usually toxic to human beings. The amount of an essential nutrient considered adequate for requirements of humans is termed the dietary reference intake (DRI) [27]. Ingestion limits of barium have not been established because of this element has no DRI or radiological component. Potentially toxic microelements, including Pb, Cd, Hg and As are strictly limited at ultra-low levels in food and herbal medicine because of their potent toxicity. Harmful elements, such as Cd and Hg, were not detected in the cultured stroma. Their levels are also very low in both the natural stroma and the natural worm. Pb and As, the content is low in the three samples. For heavy metals, the highest levels in Table 3 were less than the maximum levels according to Chinese Pharmacopeia (2010). The contents of Cu, Hg, Pb, As and Cd are limited to 20, 0.2, 5.0, 2.0, 0.3 g/g separately. Cd, Pb, Hg and As have no known physiological function yet reported. They are reported to be toxic and should be considered as a high risk factor to public health in general [28]. Cd has an adverse effect on brain metabolism and has other severe effects such as prostate cancer, kidney, liver, lungs and bone damage. Children are particularly at risk from Pb consumption, both before and after birth [20]. However, the potential of some elements to be harmful or beneficial is highly dependent upon their speciation. For example, the toxicity of arsenic is tremendously different in various
Table 3 Concentration of essential and toxic elements in dry samples. Sample
Concentration of element (g/g)a Na
Cultured stroma 923 ± 19.22 1420 ± 14.13 Natural stroma 876 ± 3.67 Natural worm
Cultured stroma Natural stroma Natural worm a
K 530 ± 18.00 946 ± 13.41 724 ± 2.45
Ca 547 ± 13.72 453 ± 3.40 238 ± 2.21
Mg
Zn
Fe
Cu
986 ± 8.30 84.81 ± 0.03 68.23 ± 0.21 14.50 ± 0.04 280 ± 4.48 65.24 ± 0.05 114.72 ± 1.26 13.18 ± 0.18 117 ± 2.34 27.43 ± 0.04 39.20 ± 1.17 3.56 ± 0.96
Ni
Co
Mo
Se
2.86 ± 1.12 3.23 ± 0.97 1.35 ± 0.46
0.46 ± 0.01 2.77 ± 0.07 0.92 ± 0.02
0.61 ± 0.03 0.74 ± 0.05 0.20 ± 0.03
0.98 ± 0.01 1.89 ± 1.04 2.73 ± 0.02 8.12 ± 2.48 0.42 ± 0.01 6.60 ± 0.91
Ba
Sn
As
0.28 ± 0.33 0.76 ± 0.03 0.15 ± 0.01
0.69 ± 0.11 1.32 ± 0.17 0.38 ± 0.04
Results were mean ± standard deviation obtained from five replicates of one pooled sample. ND: not detected.
Mn
V
Cr
3.12 ± 0.12 34.21 ± 0.09 13.20 ± 0.18
1.94 ± 0.10 20.97 ± 0.70 5.27 ± 0.11
3.21 ± 0.49 18.62 ± 1.65 2.55 ± 0.29
Pb
Cd
Hg
ND 1.70 × 10−2 0. 30 × 10−2
ND 0. 40 × 10−2 0. 10 × 10−2
0.42 ± 0.11 1.09 ± 0.41 0.51 ± 0.22
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compounds. Inorganic compounds such as arsenite and arsenate are known to be highly toxic, while organics are generally considered to be non-toxic. Chromium has been well known to be helpful in treatment of diabetes mellitus and hypercholesterolemia. The trivalent chromium (Cr3+ ) is essential while the hexavalent (Cr6+ ) is highly toxic to human and has the ability to traverse biological membranes [29]. In order to know the speciation of the toxic elements in C. kyushuensis, further studies need to be performed with the same sample. Except for Mg, Zn and Cu, which concentration in cultured stroma was maximum, the contents of all other determined trace elements were highest in wild stroma. And there is a distinct difference between the concentrations of cultured stroma and natural stroma. The situation is the same between any other two parts, which may indicate the effect of biological accumulation possessed by stroma of this fungus. Different contents of mineral elements between wild stroma and cultured stroma may be caused by the distinct growing environments (temperature, humidity, pH, illumination climate, and organic matter content of artificial medium, natural soil, and wild worm). This indicated that the element contents of cultured C. kyushuensis might be improved by optimizing the composition of artificial medium. The trace element contents in three samples may depend on the selective uptake, deposition of elements in tissues, and the ability of cultured and wild C. kyushuensis to absorb elements from the substrate.
[4] [5]
[6]
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4. Conclusions In this work, 20 elements (essential, non-essential and toxic) in cultured and wild (stroma and worm) C. kyushuensis have been determined by means of ICP-MS. According to the results, the element concentrations in the three samples were significantly different (p < 0.05). The values of elemental contents were the highest in the stroma of natural C. kyushuensis except for Mg, Zn and Cu. The discrepancy of element content between cultured and natural C. kyushuensis samples might be resulted from different growing conditions, such as temperature, humidity, pH, quantity of illumination, element level of soil and culture medium, and so on [30]. The data obtained in this research will be valuable for complementing the composition knowledge of C. kyushuensis.
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This research work was supported in part by the National Natural Science Foundation of China (No. 30770041 and 30970012), the China Postdoctoral Science Foundation funded project, the Shandong Provincial Foundation for Science (No. 2007GG2NS02056), and the Foundation of State Key Laboratory of Microbial Technology (No. M2010-02). The authors appreciate their help in earnest. References
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Journal of Pharmaceutical and Biomedical Analysis journal homepage: www.elsevier.com/locate/jpba
Determination of essential and toxic elements in Cordyceps kyushuensis Kawam by inductively coupled plasma mass spectrometry Guoying Zhang a,b , Yanxin Zhao c , Fengjun Liu d , Jianya Ling a,∗ , Jianqiang Lin a,∗ , Changkai Zhang a a
State Key Laboratory of Microbial Technology, Shandong University, Jinan 250100, China Shandong University of Traditional Chinese Medicine, Jinan 250014, China Jinan Central Hospital, Jinan 250013, China d Qianfoshan Hospital, Jinan 250014, China b c
a r t i c l e
i n f o
Article history: Received 9 April 2012 Received in revised form 5 August 2012 Accepted 7 August 2012 Available online 16 August 2012 Keywords: Cordyceps kyushuensis Kawam ICP-MS Microwave digestion Multi-element analysis Food composition
a b s t r a c t In this study, a total of 20 elements (essential, non-essential and toxic): lithium (Li), sodium (Na), potassium (K), gallium (Ga), magnesium (Mg), zinc (Zn), iron (Fe), copper (Cu), manganese (Mn), vanadium (V), chromium (Cr), nickel (Ni), cobalt (Co), molybdenum (Mo), selenium (Se), barium (Ba), tin (Sn), arsenic (As), lead (Pb) cadmium (Cd) and mercury (Hg) in natural and cultured Cordyceps kyushuensis have been determined by means of inductively coupled plasma mass spectrometry (ICP-MS). Cultured stroma, natural stroma and natural worm were digested by microwave-assisted method before analysis. The proposed ICP-MS method was validated by analyzing a certified reference material (CRM) GBW10015 (spinach). The results of one-way analysis of variance (ANOVA) revealed that the element concentrations in the three kinds of samples were significantly different (p < 0.05). Except for Mg, Zn, Cu, the values of other elemental contents were the highest in the stroma of natural C. kyushuensis. In comparison with the worm, the concentrations of determined elements in wild stroma were higher. The remarkable difference of elemental contents between cultured and natural stroma may be caused by distinct growing environment. This finding highlighted the usefulness of ICP-MS elemental analysis and enhanced the value of C. kyushuensis as a candidate for nourishing food based on its composition. © 2012 Elsevier B.V. All rights reserved.
1. Introduction The fungi of genus Cordyceps belonging to Clavicipitaceae, Ascomycotina [1], have received significant attention from medical and pharmacological researchers. Cordyceps was commonly used as a traditional Chinese medicine for the treatment of fatigue, night sweating, hyperglycemia, hyperlipidemia, asthenia after severe illness, respiratory disease, renal dysfunction, arrhythmias, other heart and liver diseases. The pharmacological actions of Cordyceps such as anti-oxidation [2], potentiating the immune system [3] and anti-tumor activities [4] have been demonstrated. Recently, Cordyceps has become more and more attractive as dietary food and source of medicine in some countries [5–7]. Cordyceps kyushuensis Kawam, is a unique species of Cordyceps, which parasitizes on the larvae of Clanis bilineata walker. Some pharmacologically active substances, such as cordycepin and adenosine have been found in both stroma and worm [8]. However
∗ Corresponding authors. Tel.: +86 531 88364427; fax: +86 531 88567250. E-mail addresses: [email protected] (J. Ling), [email protected] (J. Lin). 0731-7085/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jpba.2012.08.007
no research has been devoted to the elemental composition of C. kyushuensis to date. Many analytical methods, such as graphite furnace atomic absorption spectrometry (GF-AAS), inductively coupled plasma atomic emission spectroscopy (ICP-AES), and inductively coupled plasma mass spectrometry (ICP-MS) have been used to investigate the element concentrations of all kinds of biological samples, foodstuffs, medicinal materials, and other samples. GF-AAS is sensitive enough to detect trace elements, but as a mono-elemental technique, it is slow and tedious when a large number of elements needed to be analyzed. Even though ICP-AES is a multi-elemental technique, but the disadvantage of insufficient sensitivity made it little bit in trace elements detection. ICP-MS, employed in this study, has been widely used as a technique for its significant advantages, such as simultaneous multi-element measurement capability and very low detection limits [9]. Moreover, it offers a wider linear dynamic range which allows the determination of major and trace elements in the same sample [10]. The aim of this study was to apply the validated ICP-MS method to determine the content of 20 essential, non-essential and toxic elements (Na, K, Ca, Mg, Cu, Fe, Zn, V, Se, Cr, Co, Mo, Mn, Ni, Ba, Sn, Pb, Cd, As and Hg) in cultured C. kyushuensis stroma, as well as natural stroma and worm of this fungus.
G. Zhang et al. / Journal of Pharmaceutical and Biomedical Analysis 72 (2013) 172–176 Table 1 Operating parameters for ICP-MS. Operating parameter
Value
Type of nebulizer Nebulizer Ar flow rate Auxiliary Ar flow rate Plasma Ar flow rate Analog stage voltage Lens voltage Pulse stage voltage Sample uptake rate ICP RF power Radiofrequency generator Spray chamber Sample cone Skimmer cone Type of torch Scanning mode Dwell time Integration time Number of readings per replicate Number of replicate
Cross-flow 0.86 L min−1 1.2 L min−1 15 L min−1 −1900 V 5.7 V 900 V 1.0 L min−1 1100 W 40 MHz Ryton® , double-pass Nickel, 1.1 mm orifice diameter Nickel, 0.9 mm orifice diameter Standard quartz torch Peak hopping 55 ms 1000 ms 1 5
2. Materials and methods 2.1. Instrumentation, equipment and reagents A microwave oven (Milestone Ethos A Digestion/Extraction Microwave Labstation, Sorisole, Italy) equipped with twelve 100 mL TFM Teflon vessels, with an energy output of 900 W, was employed to digest the samples. The maximum digestion temperature and pressure were 300 ◦ C and 3.45 MPa, respectively. A quadrupole ELAN DRC-e inductively coupled plasma mass spectrometer (Perkin-Elmer SCIEX, Concord, Ont., Canada) was used; a gem tipped cross-flow nebulizer interfaced with a (Ryton) Scott double pass spray chamber, using a 2.0 mm id alumina injector tube, comprised the sample introduction system. ICP conditions were selected that maximized the ion signals of the elements studied while reducing the background to a minimum. Optimized with a multi-element calibration stock solution (10 g/L) containing Ba, Be, Ce, Co, In, Mg, Pb, Rh, and U (obtained from Perkin-Elmer, Concord, Ont., Canada), the operating parameters of the ICP-MS for the analysis were summarized in Table 1. The content of each trace element was obtained from the average value of five replicates of one pooled sample. Rh mono-element standard solution from PerkinElmer (Norwalk, CT, USA) for internal standard was employed. High purity de-ionized water with a resistivity of 18.2 M cm−1 was obtained using a Synergy water purification system with SynergyPak purification cartridges (Billerica, MA, USA). Concentrated nitric acid (65%, Merck, Germany) used for sample mineralization was high-grade purity reagent, and hydrogen peroxide (30%, Merck) was analytical reagent. The working standard solutions used to establish calibration curves were prepared at the concentration from 25, 50, 75, 100, 125, 250, 500, 750 and 1000 g/mL with the 1000 g/mL each single-element stock standard solution (Traceable to National Institute of Standards and Technology Reference Materials) diluted with de-ionized water immediately before analysis. All prepared solutions were stored in polyethylene volumetric flasks and contain 1% (v/v) HNO3 . All microwave vessels and plastic materials were washed with de-ionized water three times, then they were immersed in 15% HNO3 solution for 24 h. Finally they were thoroughly rinsed with ultrapure water and dried before use.
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standard plant sample (GBW10015, spinach) was purchased from National Standard Center of China. Natural C. kyushuensis was collected from Mount Meng, Shandong province of China in August 2011, and identified by Professor Yinglan Guo (Institute of Microbiology, Chinese Academy of Science, China). Cultured fruiting body of C. kyushuensis was cultivated in our laboratory. The anamorph strain, originally isolated from fresh specimen, was confirmed by means of both morphological and molecular methods and kept on potato dextrose agar (PDA) slant culture at 4 ◦ C. Mycelial fragments were suspended in 5 mL of sterile deionized water and used as inocula in 250 mL flasks containing 95 mL PDA broth for pre-cultures. Flasks were shaken at 150 rpm for 6 days at 25 ◦ C. Subsequently, the husked rice medium was inoculated with the liquid seed culture. After culturing at 25 ◦ C for about 15 days with the supplement of moisture and illumination, the fungus proliferated all over the medium and then was aged for about 60 days. 2.3. Sample preparation Natural C. kyushuensis sample, divided into stroma and worm parts, were washed with ultrapure water and oven-dried at 60 ◦ C to constant weight together with cultured stroma. Then all samples were homogenized with a micro plant crusher and screened with a 200 mesh sieve. As an excellent technology can reduce contamination and improve efficiency of mineralization, microwave-assisted digestion was employed to decompose the samples at a temperature of 200 ◦ C for ramping 10 min and holding 20 min. Several mixtures of nitric acid and hydrogen peroxide were attempted for mineralization of samples, such as 5 mL HNO3 + 2 mL H2 O2 (procedure A), 7 mL HNO3 + 1 mL H2 O2 (procedure B), 9 mL HNO3 + 2 mL H2 O2 (procedure C), 10 mL HNO3 + 2 mL H2 O2 (procedure D). Approximately 0.2 g of each sample and CRM were accurately weighed and transferred to the Teflon digestion vessels, and then the corresponding volume of concentrated HNO3 and 30% (v/v) H2 O2 were added. Each sample was prepared in triplicated. Sample blank was used in accordance with the mineralization procedure. After microwave digestion, the solutions were evaporated to a small volume at the temperature not higher than 60 ◦ C. Then the concentrated solutions were transferred to 50 mL volumetric flasks, and 0.5 mL HNO3 was added before adjusting the final volume to 50 mL with high purity de-ionized water. Rh was added to a final concentration of 10 g/L for its evaluation as internal standard. In order to avoid the possible interferences caused by sample matrix and oxidizing materials, the isotope masses shown in Table 1 were selected for simultaneous monitoring. The spectral interferences were checked under the standard mode ICP-MS (DRC OFF) in this experiment. 2.4. Validation of the method The accuracy and precision of the proposed method were assessed by analyzing the trace elements in CRM (spinach). Linearity of each analytical element was expressed as the related coefficients (R) of specification curves and the minimum acceptable value of R was 0.9995. According to the recommendations of IUPAC, the limit of detection (LOD) was determined as three times the standard deviation of ten consecutive measurements of sample blank solutions prepared by the same microwave procedure as the samples. 2.5. Statistical analysis
2.2. Samples Routinely used as the certified reference material (CRM) for fruits, vegetable, staple and non-staple foodstuff, a Chinese national
One-way analysis of variance (ANOVA) was employed to perform statistical analysis of the results. It was conducted using the statistical analysis packages of SPSS version 16.0
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for windows. The level of statistical significance was set at P-value < 0.05. 3. Results and discussion 3.1. Sample digestion procedure Sample digestion is one of the most important steps for chemical analysis. Because complete decomposition without contamination or loss of elements is the prerequisites for reproducible and accurate analysis, several mineralization methods, such as dry ashing method, wet digestion procedure, and microwave-assisted digestion mode [11], have been employed to decompose biological, environmental and other various samples. A long period of time is needed to incinerate samples in a crucible furnace by dry ashing method. The use of concentrated acids and careful monitoring of digestion for varying periods are needed in wet digestion procedure. Both methods have shortcomings of time-consuming and possible volatilization loss of some elements. An alternative method is microwave-assisted digestion coupled with closed vessels, which has better recovery of volatile elements, more reproducible procedures and a better working environment [12]. Several combinations of nitric acid with hydrogen peroxide were investigated in this paper. This was an attractive procedure because the study had been aimed at avoiding the use of HCl, HClO4 , and HF in the digestion of the fungi of genus Cordyceps. HCl may introduce additional interference elements into ICP-MS system [13], Toxic HF is found to depress the fluorescence signal [14], and explosive HClO4 is expensive and dangerous [15,16]. From the experimental results, great quantity of black precipitate remained in the resultant solutions at the end of program A and B, respectively. There was still a small amount of white residue revealed after program C. The Cordyceps samples were found to be thoroughly decomposed after performing program D. Therefore program D of microwave-assisted digestion had been applied to all analysis. The final results also shown that the microwave digestion procedure gave the best agreement between measured elemental mass fractions and certified values. 3.2. Selection of isotopes Measurements of trace elements by ICP-MS method usually suffer from interferences caused by isotopes, sample matrices, plasma working gas, double-charge ions, and some polyatomic ions. The absence of polyatomic interferences was checked when required, by measuring several isotopes of the elements and checking the isotopic ratio. Specific isotopes should be carefully considered and monitored according to the sensibility of the element and/or possible isobaric and polyatomic interferences. Through the results of our previous determination and other researchers’ reports, the isotopes measured were shown in Table 2. 52 Cr and 59 Co were chosen to avoid the potential polyatomic interferences caused by 40 Ar, 12 C,43 Ar and 16 O, through adopted microwave digestion procedure, whereas double-charge ions and oxide interferences could be generally eliminated by means of selecting appropriate instrumental working parameters. 111 Cd was selected to be determined rather than 114 Cd because of the isobaric interference of tin (114 Sn) [17]. Due to the severe polyatomic isobaric interferences in 56 Fe [18], which cannot be used with a simple quadrupole, 57 Fe had to be measured and the sensitivity was lower. Similarly, in the determination of nickel, 60 Ni was used to avert interferences with 23 Na35 Cl or 58 Fe when 58 Ni was analyzed [19].
3.3. Method validation As a part of the quality assurance–quality control protocol, the CRM (GBW10015) was analyzed to determine the accuracy (%RE), precision (%RSD) and LOD of the proposed method. The results shown in Table 2 were generally in good agreement with the certified concentrations in CRM, indicating validity of the method employed in this work. The accuracy for all of the detected elements was less than 10%. The analytical precision expressed as %RSD, for Na, Cd, Co, Cu, Ni, Mn and Ba, was routinely between 5 and 10%, for other elements, were less than 5%. Except for Se, Sn and Hg, the percentage of relative error (%RE) was the lowest for Na and the highest for Cu, and the percentage of relative standard deviation (%RSD) was minimum for Cr and maximum for Ba. The correlation coefficients (R) of calibration curves were at least 0.9998, indicating good linear relationships throughout the concentration range analyzed. The method detection limits obtained for the selected elements were also shown in Table 2, which were determined as 3 SD of the 20 consecutive measurements of the reagent blanks multiplied by the dilution factor used for sample preparation (250 mg of sample/50 mL). In the absence of polyatomic interferences, the level of a selected isotope for a given element had a great influence on the detection limits of the particular element. The greater the abundance of the isotope, the lower the detection limit. 3.4. Concentration of trace elements in natural and cultured C. kyushuensis In this study, the elements determined were 20 subdivided into essential (macro and micro) and non-essential or toxic. Among the essential elements estimated in the present work we found the macro elements (Na, K, Ca and Mg) and microelements (Zn, Fe, Cu, Mn, V, Cr, Ni, Co, Mo, and Se); along the nonessential elements and the toxic elements Ba, Sn, As, Cd, Pb and Hg. The results are presented in Table 3. Trace elements contained in three different samples were considerably different at the 95% confidence level according to the results of one-way analysis of variance (ANOVA). For macro element, in this study, Na (1420 g/g) and K (946 g/g) in wild stroma exhibited the highest levels and were significantly different from the other two samples. While the maximum content of Mg (986 g/g) was found in the cultured stroma, which is considerably higher than those of wild stroma and worm. For Ca, there is a similar content in the cultured stroma (547 g/g) and natural stroma (453 g/g), and a relatively lower concentration (238 g/g) was found in natural worm. Potassium and magnesium are essential electrolytes for maintaining normal fluid balance in cells and a delicate balance of these two elements is reported to prevent an increase in blood pressure and to maintain normal cardiac rhythm. Calcium is known to be involved in muscle contraction and relaxation, blood clotting, proper nerve function and body immune defenses [20,21]. The biological roles of K, Mg and Ca are essential for disease prevention and control [22]. In addition to the four macro elements analyzed in this study, 16 trace elements were also studied. Trace elements, in minute quantities, are essential for the normal health and function of humans. They can be divided into three groups of essential microelements, possible essential microelements and potentially toxic microelements or non-essential microelements. Trace elements, whether essential or non-essential, can cause morphological abnormalities, reduce growth and increase mortality and mutagenic effects, above their critical threshold concentrations [23]. Essential microelements, including Fe, Zn, Cu, Mn, Cr, Mo, Co, Se, Ni and V were determined in this work. The result showed that the concentrations of Fe, Cu, Mn, V and Cr in natural stroma are between
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175
Table 2 Accuracy, precision and LOD validated by certified reference material with Rh (10 g/L) as internal standard. Element
Isotopes (m/z)
Certified value (g/g)
Found value (g/g)a
%RE
%RSD
Detection limit (ng/g)
Na K Ca Mg Fe Zn Mn Pb Ba Cu Cr Ni V Mo Cd As Co Se Sn Hg
23
15,000 24,900 6600 5520 540 35.3 41 11.10 9.00 8.90 1.40 0.92 0.87 0.47 0.15 0.23 0.22 0.09 0.06 0.02
15,238 23,860 6248 5630 556 32.9 39 11.32 8.37 8.10 1.43 0.89 0.82 0.45 0.14 0.25 0.21 0.09 0.06 0.02
1.59 −4.18 −5.33 +1.99 +2.96 −6.80 −4.88 +1.98 −7.00 −9.00 +2.14 −3.26 −5.75 −4.26 −6.67 +8.70 −4.55 0 0 0
5.60 3.48 4.24 2.54 3.67 4.46 6.38 3.85 8.71 7.74 2.12 5.47 4.71 2.15 8.46 4.45 7.82 0 0 0
80 90 100 80 70 30 50 40 70 20 10 7 5 5 4 5 5 3 7 1
a
Na 39 K 43 Ca 24 Mg 56 Fe 66 Zn 55 Mn 208 Pb 137 Ba 63 Cu 53 Cr 60 Ni 51 V 98 Mo 111 Cd 75 As 59 Co 82 Se 118 Sn 202 Hg
Results were mean ± standard deviation obtained from five replicates of one pooled sample.
18.62 and 114.72 g/g, which is higher than those of cultured strom and natural worm, except that Zn is higher in the cultured strom (84.81 ppm) than in the natural stroma (65.25 ppm), and the concentration of Cu is broadly similar in cultured strom (14.50 g/g) and natural stroma (13.18 g/g). For Ni, Co, Mo and Se, the content of wild stroma is between 0.74 and 3.23 g/g. While the cultured stroma (0.46–2.86 g/g) and natural worm (0.20–1.35 g/g) showed a lower content for the same elements. Fe, Cu, Mn and Zn are essential elements in enzyme metabolism. They have immunomodulatory functions and thus influence the susceptibility to the course and the outcome of a variety of viral infections [24]. The importance of iron in maintaining good health and well being has long been recognized by nutritionists [25]. Co, as a component of the vitamin B12 complex, is an essential trace element of human body, which can significantly stimulate the production of blood and effectively cure the macrocytic anemia [19]. The essential trace element molybdenum is bound to a required for the physiological function of some enzymes, such as xanthine oxidize, aldehyde oxidase, sulfite oxidase, nitrate reductase, and so on. Metallic vanadium is probably an essential trace element, which most common valence states are V3+ , V4+ , and V5+ . The pentavalent state (VO3 − ) is the most common form in extracellular fluid whereas the quadrivalent form (VO2+ ) predominates in intracellular [26]. Although the concentration of V in most foods is low, food is the main source of this trace element for the general population. Non-essential elements such as Ba and Sn were also detected in this study. The element Ba is rich not only in natural stroma (8.12 g/g), but also in the cultured stroma (1.89 g/g) and natural
worm (6.60 g/g), while the concentration of Sn is only between 0.15 and 0.76 g/g in all the three samples. Although there are no reported physiological effects of Ba and Sn at low concentrations, their accumulation in the body system could be detrimental to health. Under normal conditions, the element barium has no remarkable trophism or toxic action to the human body. However, some barium salts are usually toxic to human beings. The amount of an essential nutrient considered adequate for requirements of humans is termed the dietary reference intake (DRI) [27]. Ingestion limits of barium have not been established because of this element has no DRI or radiological component. Potentially toxic microelements, including Pb, Cd, Hg and As are strictly limited at ultra-low levels in food and herbal medicine because of their potent toxicity. Harmful elements, such as Cd and Hg, were not detected in the cultured stroma. Their levels are also very low in both the natural stroma and the natural worm. Pb and As, the content is low in the three samples. For heavy metals, the highest levels in Table 3 were less than the maximum levels according to Chinese Pharmacopeia (2010). The contents of Cu, Hg, Pb, As and Cd are limited to 20, 0.2, 5.0, 2.0, 0.3 g/g separately. Cd, Pb, Hg and As have no known physiological function yet reported. They are reported to be toxic and should be considered as a high risk factor to public health in general [28]. Cd has an adverse effect on brain metabolism and has other severe effects such as prostate cancer, kidney, liver, lungs and bone damage. Children are particularly at risk from Pb consumption, both before and after birth [20]. However, the potential of some elements to be harmful or beneficial is highly dependent upon their speciation. For example, the toxicity of arsenic is tremendously different in various
Table 3 Concentration of essential and toxic elements in dry samples. Sample
Concentration of element (g/g)a Na
Cultured stroma 923 ± 19.22 1420 ± 14.13 Natural stroma 876 ± 3.67 Natural worm
Cultured stroma Natural stroma Natural worm a
K 530 ± 18.00 946 ± 13.41 724 ± 2.45
Ca 547 ± 13.72 453 ± 3.40 238 ± 2.21
Mg
Zn
Fe
Cu
986 ± 8.30 84.81 ± 0.03 68.23 ± 0.21 14.50 ± 0.04 280 ± 4.48 65.24 ± 0.05 114.72 ± 1.26 13.18 ± 0.18 117 ± 2.34 27.43 ± 0.04 39.20 ± 1.17 3.56 ± 0.96
Ni
Co
Mo
Se
2.86 ± 1.12 3.23 ± 0.97 1.35 ± 0.46
0.46 ± 0.01 2.77 ± 0.07 0.92 ± 0.02
0.61 ± 0.03 0.74 ± 0.05 0.20 ± 0.03
0.98 ± 0.01 1.89 ± 1.04 2.73 ± 0.02 8.12 ± 2.48 0.42 ± 0.01 6.60 ± 0.91
Ba
Sn
As
0.28 ± 0.33 0.76 ± 0.03 0.15 ± 0.01
0.69 ± 0.11 1.32 ± 0.17 0.38 ± 0.04
Results were mean ± standard deviation obtained from five replicates of one pooled sample. ND: not detected.
Mn
V
Cr
3.12 ± 0.12 34.21 ± 0.09 13.20 ± 0.18
1.94 ± 0.10 20.97 ± 0.70 5.27 ± 0.11
3.21 ± 0.49 18.62 ± 1.65 2.55 ± 0.29
Pb
Cd
Hg
ND 1.70 × 10−2 0. 30 × 10−2
ND 0. 40 × 10−2 0. 10 × 10−2
0.42 ± 0.11 1.09 ± 0.41 0.51 ± 0.22
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compounds. Inorganic compounds such as arsenite and arsenate are known to be highly toxic, while organics are generally considered to be non-toxic. Chromium has been well known to be helpful in treatment of diabetes mellitus and hypercholesterolemia. The trivalent chromium (Cr3+ ) is essential while the hexavalent (Cr6+ ) is highly toxic to human and has the ability to traverse biological membranes [29]. In order to know the speciation of the toxic elements in C. kyushuensis, further studies need to be performed with the same sample. Except for Mg, Zn and Cu, which concentration in cultured stroma was maximum, the contents of all other determined trace elements were highest in wild stroma. And there is a distinct difference between the concentrations of cultured stroma and natural stroma. The situation is the same between any other two parts, which may indicate the effect of biological accumulation possessed by stroma of this fungus. Different contents of mineral elements between wild stroma and cultured stroma may be caused by the distinct growing environments (temperature, humidity, pH, illumination climate, and organic matter content of artificial medium, natural soil, and wild worm). This indicated that the element contents of cultured C. kyushuensis might be improved by optimizing the composition of artificial medium. The trace element contents in three samples may depend on the selective uptake, deposition of elements in tissues, and the ability of cultured and wild C. kyushuensis to absorb elements from the substrate.
[4] [5]
[6]
[7]
[8]
[9] [10] [11]
[12]
[13]
[14]
[15]
4. Conclusions In this work, 20 elements (essential, non-essential and toxic) in cultured and wild (stroma and worm) C. kyushuensis have been determined by means of ICP-MS. According to the results, the element concentrations in the three samples were significantly different (p < 0.05). The values of elemental contents were the highest in the stroma of natural C. kyushuensis except for Mg, Zn and Cu. The discrepancy of element content between cultured and natural C. kyushuensis samples might be resulted from different growing conditions, such as temperature, humidity, pH, quantity of illumination, element level of soil and culture medium, and so on [30]. The data obtained in this research will be valuable for complementing the composition knowledge of C. kyushuensis.
[16]
[17]
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[19]
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Acknowledgements [23]
This research work was supported in part by the National Natural Science Foundation of China (No. 30770041 and 30970012), the China Postdoctoral Science Foundation funded project, the Shandong Provincial Foundation for Science (No. 2007GG2NS02056), and the Foundation of State Key Laboratory of Microbial Technology (No. M2010-02). The authors appreciate their help in earnest. References
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