Molecules 2014, 19(5), 6123-6141; doi:10.3390/molecules19056123

Article
Distribution of Nucleosides in Populations of Cordyceps cicadae
Wen-Bo Zeng 1, Hong Yu 1,*, Feng Ge 2, Jun-Yuan Yang 1, Zi-Hong Chen 1, Yuan-Bing Wang 1, Yong-Dong Dai 1 and Alison Adams 3
1
Yunnan Herbal Laboratory, Institute of Herb Biotic Resources, Yunnan University, Kunming 650091, Yunnan, China; E-Mails: zengwenboherb@163.com (W.-B.Z.); icecream6973@sina.com (J.-Y.Y.); czh78@tom.com (Z.-H.C.); wangyb001@126.com (Y.-B.W.); daidiy555@gmail.com (Y.-D.D.)
2
Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming 650500, Yunnan, China; E-Mail: gefeng79@aliyun.com
3
Department of Biological Sciences, College of Engineering, Forestry and Natural Science, Northern Arizona University, Flagstaff, AZ 86011-5640, USA; E-Mail: Alison.Adams@nau.edu
*
Author to whom correspondence should be addressed; E-Mail: herbfish@163.com or hongyu@ynu.edu.cn; Tel.: +86-137-006-766-33; Fax: +86-871-650-346-55.
Received: 15 January 2014; in revised form: 25 April 2014 / Accepted: 5 May 2014 /
Published: 14 May 2014

Abstract

: A rapid HPLC method had been developed and used for the simultaneous determination of 10 nucleosides (uracil, uridine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenine, adenosine, 2'-deoxyadenosine and cordycepin) in 10 populations of Cordyceps cicadae, in order to compare four populations of Ophicordyceps sinensis and one population of Cordyceps militaris. Statistical analysis system (SAS) 8.1 was used to analyze the nucleoside data. The pattern of nucleoside distribution was analyzed in the sampled populations of C. cicadae, O. sinensis and C. militaris, using descriptive statistical analysis, nested analysis and Q cluster analysis. The total amount of the 10 nucleosides in coremium was 1,463.89–5,678.21 µg/g in 10 populations of C. cicadae, 1,369.80–3,941.64 µg/g in sclerotium. The average contents of the 10 analytes were 4,392.37 µg/g and 3,016.06 µg/g in coremium and sclerotium, respectively. The coefficient of variation (CV) of nucleosides ranged from 8.36% to 112.36% in coremium of C. cicadae, and from 10.77% to 155.87% in sclerotium of C. cicadae. The CV of the nucleosides was wide within C. cicadae populations. The nested variation analysis by the nine nucleosides’ distribution indicated that about 42.29% of the nucleoside variability in coremium was attributable to the differentiation among populations, and the remaining 57.71% resided in the populations. It was also shown that about 28.94% of the variation in sclerotium was expressed between populations, while most of the variation (71.06%) corresponded to the populations.
Keywords:
Cordyceps cicadae; nucleosides; distribution

1. Introduction

Cordyceps cicadae X. Q. Shing (Figure 1), named “Chan Hua”, belongs to the genus Cordyceps (family Clavicipitaceae, Ascomycotina), and its anamorph is Isaria cicadae Miq [1], which is a major parasitic fungus growing on the nymph of Cicada flammata Distant, Platypleura kaempferi Fabricius, Crytotympana pustulata Fabricious [2], Platylomia pieli Kato [3] and Oncotympana maculatieollis Motsch (Figure 2). Cordyceps cicadae has been used as a Traditional Chinese Medicine and food for about 1,500 years in China [2], much longer than Ophicordyceps sinensis (Berk.) G. H. Sung, J. M. Sung [4,5].

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Figure 1. Cordyceps cicadae used for this study.

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Figure 1. Cordyceps cicadae used for this study.
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Figure 2. The nymph and adult of Oncotympana maculatieollis, as one host of C. cicadae, collected from Kunming in Yunnan (Pop CCKSG).

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Figure 2. The nymph and adult of Oncotympana maculatieollis, as one host of C. cicadae, collected from Kunming in Yunnan (Pop CCKSG).
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Furthermore, C. cicadae has been used as a substitute for O. sinensis. Its putative active functions include: (1) treatment of childhood convulsions; (2) antitumor activity [6,7]; (3) analgesic activity and sedative function [3,8]; (4) amelioration of renal function [9]; (5) anti-fatigue effects [10]; (6) immunomodulatory effects [7].

Cordyceps cicadae is a cosmopolitan species in many regions of the World, and its habitat demands are less strict than those of O. sinensis. The distribution of C. cicadae had been surveyed in China (Table 1). It has also been recorded in South Asia, Europe, North America [11] and Jeju Island in South Korea [12,13].

Table Table 1. Distribution of C. cicadae in China.

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Table 1. Distribution of C. cicadae in China.
ProvinceLocation
YunnanMojiang, Fengyang [13] Lanping, Weixi, Xianggelila, Zhaotong and Kunming
SichuanMount Emei, Qingcheng mountain and Qingyun mountain [13] and Xiangcheng
GuizhouFanjing mountain, Libo karst geopark, Guiyang forest park and Huaxi [13,14]
JiangsuYixing
GuangxiLeye [12]
HainanWuzhi mountain [12]
FujianWushan in Fuzhou [13]
ShanghaiTianma mountain [15]
ZhejiangHangzhou [16]
Guangdong [11]a
Hunan [11]
Hubei [11]

a No details.

According to Traditional Chinese Medicine, C. cicadae had been considered as a drug similar to O. sinensis, with its effective composition of amino acids, polysaccharides, and mannitol being similar to those of O. sinensis [17]. Several components, such as nucleosides, polysaccharides, ergosterol and mannitol, had been used as markers for quality control of Cordyceps and its products [4]. The following chemical constituents have been isolated from C. cicadae: polysaccharides [18,19], galactomannan [20], adenosine, uridine, inosine, guanosine [21], ISP-1(myriocin) [22,23] and ergosterol peroxide [24].

Previous studies showed that the most important bioactive constituents in O. sinensis and its analogs were soluble nucleosides. Since cordycepin (3'-deoxyadenosine) with antitumor activity was isolated from cultured C. militaris in 1950 [25], nucleosides in Cordyceps have become a focus of research. To date, more than ten nucleosides were detected or isolated from this group, such as adenine, adenosine, 2'-deoxyadenosine, 3'-deoxyadenosine, uracil, uridine, 2'-deoxyuridine, guanine, cytosine, guanosine, hypoxanthine, inosine, thymine and thymidine [4,26,27]. Adenosine plays a key role in the pharmacological effects, as it depressed the excitability of CNS neurons and inhibited the release of various presynaptic neurotransmitters [28,29], and adenosine has been used as a marker for quality control of O. sinensis in the Chinese Pharmacopoeia [30]. Inosine, the major biochemical metabolite resulting from oxidative deamination of adenosine, stimulated axon growth in vitro and the adult central nervous system [31]. Cordycepin, one of the main compounds found in C. militaris, had also shown multiple pharmacological activities [32,33,34]. However, whether or not natural and cultured O. sinensis contain cordycepin is still controversial [27,35,36,37]. In addition, nucleosides were reported to play a role in growth and differentiation of the gastrointestinal tract, as well as to play a role in the maintenance of the immune response [38,39]. So far, several methods, including HPLC [27,35,36,37,40,41], LC–MS [26,36,42,43], CE–MS [44], CE [45], CEC [46], ultra-performance liquid chromatography (UPLC) [47], and CZE [48], had been established to determine nucleosides in O. sinensis and related species.

Cordyceps cicadae is recorded as one of the most valued Traditional Chinese Medicines [17]. It consists of the dried fungus Isaria cicadae growing on the nymphs of cicadas. The fruiting body (coremium) and the nymph (sclerotium) of C. cicadae have been applied together in Traditional Chinese Medicine and food. Up to now, the nucleosides of coremium and sclerotium have not been determined in populations of C. cicadae. In this study, a simple and convenient HPLC method was used to analyze the nucleosides in coremium and sclerotium of C. cicadae populations, comparing with those of O. sinensis and C. militaris. This method included a system of 10 nucleosides, i.e., uracil, uridine, 2'-deoxyuridine, inosine, guanosine, adenine, thymidine, adenosine, 2'-deoxyadenosine and cordycepin (3'-deoxyadenosine). The nucleoside distribution patterns in populations of C. cicadae were revealed, and these compounds could become as useful markers for the authentication and quality control of C. cicadae.

2. Results and Discussions

Statistical analysis system (SAS) 8.1 was used to analyze the contents of nucleosides in C. cicadae, O. sinensis and C. militaris. Descriptive statistical analysis, nested analysis and Q cluster analysis (average cluster) of the data are presented in this paper.

2.1. Descriptive Statistical Analysis

The mean content and coefficient of variation (CV) of 10 nucleosides in coremium and sclerotium of C. cicadae, O. sinensis and C. militaris are shown in Table 2 and Table 3. In coremium, the content of uracil was 279.84–444.47 µg/g, the CV was 22.23%–40.87%, and the average content was 344.60 µg/g. The content of uridine was 363.30–1,928.73 µg/g, the CV was 12.93%–81.78%, the average content of uridine was 1,468.78 µg/g. The content of 2'-deoxyuridine was 49.89–350.41 µg/g, the CV was 33.31%–95.22%, the average content was 171.24 µg/g. The content of inosine was 79.65–1,166.62 µg/g, the CV was 10.33%–82.36%, the average content was 456.03 µg/g. The content of guanosine was 351.44–1,483.06 µg/g, the CV was 8.36%–58.24%, the average content was 1,016.53 µg/g. The content of adenine was 33.63–166.17 µg/g, the CV was 32.36%–66.30%, the average content was 70.17 µg/g. The content of thymidine was 17.73–83.81 µg/g, the CV was 29.20%–91.67%, the average content was 38.96 µg/g. The content of adenosine was 201.54–1,153.78 µg/g, the CV was 10.33%–56.48%, the average content was 797.92 µg/g. The content of 2'-deoxyadenosine was 13.01–54.62 µg/g, the CV was 25.13%–112.36%, the average content was 28.13 µg/g.

Table Table 2. The content of 10 nucleosides in 10 populations of C. cicadae, four populations of O. sinensis and one population of C. militairis.

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Table 2. The content of 10 nucleosides in 10 populations of C. cicadae, four populations of O. sinensis and one population of C. militairis.
PopPositionContents (mean (µg/g)/CV (%))Total amount (µg/g)
UracilUridine2'-DeoxyuridineInosineGuanosineAdenineThymidineAdenosine2'-deoxyadenosineCordycepin
CCKSGcoremium444.47/33.731577.49/19.00350.41/95.221166.62/10.33733.03/42.09166.17/32.3683.81/29.20837.00/39.9354.62/25.13b5413.62
sclerotium315.63/62.24774.72/41.3257.24/49.62198.44/77.06314.61/48.9999.15/34.9575.02/39.66510.99/74.4052.94/26.472398.75
CCLHHcoremium334.54/27.491587.46/12.93119.21/40.45291.28/54.311330.38/12.1472.39/32.7043.05/34.95857.36/21.1332.81/28.254668.47
sclerotium149.32/26.571353.86/36.2362.34/81.9298.06/44.89720.48/34.9277.91/71.95138.32/126.94742.77/36.52120.84/155.873463.90
CCLHQcoremium371.92/38.411560.08/29.22299.71/70.19490.96/65.311003.10/37.9867.06/44.3033.06/53.97723.85/29.6415.61/66.454565.36
sclerotium266.09/29.501221.69/17.3085.03/33.91152.94/33.56648.18/18.8162.09/43.6043.49/84.05575.99/16.4222.74/47.993078.23
CCLHXcoremium351.85/22.231365.09/14.30100.01/41.75267.39/20.71867.27/8.3658.80/32.4928.27/75.87811.56/10.3325.17/42.683875.42
sclerotium243.02/25.841224.83/17.0448.13/62.7095.47/16.39626.64/10.7755.06/39.0868.09/108.80629.61/11.2934.13/98.293024.97
CCLSHcoremium279.84/40.871676.29/36.0788.49/61.11456.96/65.691091.44/40.4866.65/57.1121.56/55.73727.60/39.9314.31/53.314423.14
sclerotium237.62/20.181387.93/20.3153.99/48.53181.70/43.04614.19/12.7376.20/29.1345.70/44.16585.88/26.6625.17/41.583208.38
CCLTDcoremium305.86/23.191278.23/21.5394.33/33.31409.24/58.69751.05/17.9143.21/41.1727.58/64.09749.17/21.5035.72/46.073694.39
sclerotium204.83/21.191117.27/22.6940.83/39.27159.18/52.50661.92/11.2343.13/54.5552.58/73.60678.05/18.4844.48/48.673002.28
CCLTLcoremium289.52/37.701460.48/21.09169.42/62.02418.79/82.361207.12/39.5851.70/39.7617.73/91.67923.07/21.9313.01/53.704550.85
sclerotium191.12/28.901159.95/28.5763.93/53.4796.83/33.11608.91/16.3161.13/100.8459.21/73.47564.37/15.2532.67/57.022838.13
CCLZDcoremium404.82/23.431928.73/19.57263.10/84.10587.61/66.121347.46/36.6975.06/66.3045.65/60.17994.31/27.9531.48/47.225678.21
sclerotium261.29/23.351587.68/26.0980.34/50.33179.66/42.85802.61/14.5760.07/43.5791.87/74.81715.34/13.0055.68/43.893834.54
CCWYJcoremium334.14/38.451890.68/35.48177.81/85.93391.80/61.141483.06/36.7867.02/43.2449.37/40.091153.78/37.7342.67/39.455590.34
sclerotium207.19/25.521516.08/28.4240.94/81.69105.91/43.88859.40/25.0852.03/40.70101.56/62.21961.26/23.6297.28/67.093941.64
CCYTScoremium329.00/39.83363.30/81.7849.89/78.9679.65/72.59351.44/58.2433.63/44.8039.51/51.17201.54/56.4815.94/112.361463.89
sclerotium187.25/32.74520.61/19.8223.97/44.9574.61/42.69260.00/30.7433.93/32.3129.55/77.86230.20/35.149.68/89.861369.80
OSDQIstroma353.39/45.172276.38/23.946.67/138.48155.27/22.061599.02/9.48178.67/65.9163.85/15.681685.50/8.5047.01/18.416365.76
sclerotium213.93/60.961432.24/22.9016.76/136.97430.95/9.82864.00/44.4091.23/25.40124.32/19.25675.15/70.5849.26/25.253897.84
OSLTAstroma55.31/24.311572.67/13.23141.80/50.781176.66/10.4679.75/24.1124.03/44.751388.17/11.9546.75/22.654485.14
sclerotium48.18/43.551468.93/19.52615.71/20.66978.12/4.5362.08/29.1169.50/24.76451.90/47.6852.18/33.813746.60
OSMNIstroma170.45/47.781387.75/7.9496.01/40.871481.03/8.16108.00/17.0052.05/29.991619.77/12.7044.54/32.964959.60
sclerotium116.40/15.011255.39±/5.47399.60/12.431070.80/3.2799.19/24.49147.95/12.60752.39/8.9258.51/17.553900.23
OSNBEstroma149.76/11.152765.61/2.927.70/19.07580.85/17.062130.34/3.59113.59/21.6758.34/26.802544.76/4.0246.77/24.738397.72
sclerotium127.95/15.591599.98/6.539.92/27.352073.63/18.431131.88/4.76154.86/11.56177.09/9.041081.60/8.63106.28/17.616463.19
CMSMBstroma319.18/19.511900.92/11.025.01/24.8885.08/20.141215.38/16.31313.75/20.1269.41/14.691613.28/13.5158.18/19.15659.29/19.116239.49
sclerotium332.93/20.041743.60/13.8712.77/18.48189.93/13.141075.99/16.58264.18/20.1768.87/16.741655.93/12.3776.98/15.844173.57/13.819594.75
Table Table 3. The average content (µg/g) and CV (%) of 10 nucleosides in C. cicadae, O. sinensis and C. militairis.

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Table 3. The average content (µg/g) and CV (%) of 10 nucleosides in C. cicadae, O. sinensis and C. militairis.
SpeciesPositionMean content (µg/g)/CV (%)Total amount (µg/g)
UracilUridine2'-DeoxyuridineInosineGuanosineAdenineThymidineAdenosine2'-deoxyadenosineCordycepin
C. cicadae
(n c = 10)
coremium344.60/34.661468.78/38.58171.24/104.35456.03/79.551016.53/46.9270.17/65.9938.96/66.95797.92/42.2128.13/64.84b4392.37
sclerotium226.34/40.181186.46/37.0555.68/63.15134.28/60.32611.69/36.7962.07/59.8570.54/106.70619.45/40.6449.56/142.543016.06
O. sinensis
(n = 4)
stroma182.23/75.882000.60/31.483.59/157.80243.48/86.321596.76/23.20120.00/56.3749.57/40.121809.55/26.1446.27/23.076052.06
sclerotium126.61/68.281439.14/16.906.67/192.88879.97/83.681011.20/20.51101.84/38.79129.71/34.04740.26/45.6266.56/41.414501.96
C. militaris
(n = 1)
stroma319.18/19.511900.92/11.025.01/24.8885.08/20.141215.38/16.31313.75/20.1269.41/14.691613.28/13.5158.18/19.15659.29/19.116239.49
sclerotium332.93/20.041743.60/13.8712.77/18.48189.93/13.141075.99/16.58264.18/20.1768.87/16.741655.93/12.3776.98/15.844173.57/13.819594.75

b Not detected; c Number of population.

In sclerotium, the content of uracil was 149.32–315.63 µg/g, the CV was 20.18%–62.24%, the average content was 226.34 µg/g. The content of uridine was 520.61–1587.68 µg/g, the CV was 17.04%–41.32%, the average content of uridine was 1,186.46 µg/g. The content of 2'-deoxyuridine was 23.97–85.03 µg/g, the CV was 33.91%–81.92%, the average content was 55.68 µg/g. The content of inosine was 74.61–198.44 µg/g, the CV was 16.39%–77.06%, the average content was 134.28 µg/g. The content of guanosine was 260.00–859.40 µg/g, the CV was 10.77%–48.99%, the average content was 611.69 µg/g. The content of adenine was 33.93–99.15 µg/g, the CV was 29.13%–100.84%, the average content was 62.07 µg/g. The content of thymidine was 29.55–138.32 µg/g, the CV was 39.66%–126.94%, the average content was 70.54 µg/g. The content of adenosine was 230.20–961.26 µg/g, the CV was 11.29%–74.40%, the average content was 619.45 µg/g. The content of 2'-deoxyadenosine was 9.68–120.84 µg/g, the CV was 26.47%–155.87%, the average content was 49.56 µg/g.

Analysis of the nucleosides revealed obvious differences between coremium and sclerotium in populations of C. cicadae. The contents of uracil, uridine, 2'-deoxyuridine, inosine, guanosine, adenine and adenosine in coremium were higher than those in sclerotium. The coefficient of variation in coremium was 34.66%–104.35%, and the CV in sclerotium was 36.79%–142.54%, with a great variation of nucleosides content in populations of C. cicadae. The wide variation of nucleosides in C. cicadae populations may mainly be derived from the genetic differences of the C. cicadae population, being affected by different location, geography, climate, maturation of the C. cicadae. Furthermore, Li et al. reported that after storage of O. sinensis at 75% relative humidity and 40 °C for 10 days, the contents of uridine, guanosine and adenosine in natural O. sinensis were markedly increased about one to four fold [49], implying that the storage conditions might be another factor affecting the variation of nucleosides in C. cicadae.

Nucleosides were believed to be the active components in Cordyceps-like fungi [50], indeed, Cordyceps-like fungi contained a higher concentration of nucleosides [51], and some unique nucleosides, such as cordycepin, 2'-deoxyuridine and 2'-deoxyadenosine were detected in Cordyceps-like fungus [26,27,45,47,51,52,53], which could be used as markers for distinguishing Cordyceps-like fungi from their counterfeits.

Cordycepin in natural O. sinensis was found in very low amounts [36,46,54], about several tens of micrograms per gram [55]. However, in this study, cordycepin was not detected in C. cicadae and O. sinensis, and cordycepin in C. militaris was high, up to 659.29 µg/g in stroma and 4173.57 µg/g in sclerotium, in accordance with the reports of Guo et al., and Yang and Li [36,37]. 2'-Deoxyadenosine was detected in C. cicadae, i.e., 28.13 µg/g in coremium and 49.56 µg/g in sclerotium. Cordycepin and 2'-deoxyadenosine are isomers of each other, and there are a lot of reports about the pharmacological activities of cordycepin [32,33,34], while the pharmacological activities of 2'-deoxyadenosine in Cordyceps-like fungi are worth studying further.

Li et al. reported that the levels of adenosine, guanosine and uridine were very similar in stroma and sclerotium of O. sinensis [54]. The average content of nucleosides of 10 populations of C. cicadae, four populations of O. sinensis and one population of C. militaris are shown in Table 3. Several nucleosides such as uracil, uridine, guanosine, adenine and adenosine in coremium were higher than those in sclerotium of C. cicadae. On the contrary, the content of thymidine and 2'-deoxyadenosine in coremium were lower than those in sclerotium of C. cicadae. The average content of inosine in coremium (456.03 µg/g) was 3-fold higher than that in sclerotium (134.28 µg/g) of C. cicadae.

Hsu et al. reported that the content of adenosine in stroma was approximately 6-fold higher than that in sclerotium of O. sinensis [56]. However, the content of adenosine in coremium was approximately 1.5 times that in sclerotium of C. cicadae. The total content of the 10 nucleosides in coremium was approximately 1.5 times that in sclerotium in C. cicadae. On the contrary, the content of the 10 analytes in stroma was approximately 1.5 times that in sclerotium of C. militaris. The distribution of the 10 nucleosides in C. cicadae was similar to that in O. sinensis, being different from the distribution pattern of the 10 analytes in C. militaris.

2.2. Nested Analysis

Nested analysis was used to analyze the uracil, uridine, 2'-deoxyuridine, inosine, guanosine, adenine, thymidine, adenosine and 2'-deoxyadenosine in C. cicadae, investigating the percent of total variance of the nine analytes between populations and individuals.

The result of nested variation analysis by the nine nucleosides’ distributions is shown in Table 4. It was indicated that about 42.29% of the variation in coremium was attributed to the differentiation among populations, and the remaining 57.71% was resided among individuals within populations. It was also showed that about 28.94% of the variation in sclerotium was expressed between populations, while most of the variation 71.06% was resided among individuals within populations.

Table Table 4. Nested analysis of nine nucleosides in coremium and sclerotium of C. cicadae.

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Table 4. Nested analysis of nine nucleosides in coremium and sclerotium of C. cicadae.
PositionAnalytePercent of total variance (%)Percent of population variance (%)Percent of individual variance (%)F ValuePr > F
coremiumuracil100.009.1390.872.000.0477
uridine100.0052.4547.5512.03<0.0001
2'-deoxyuridine100.0023.8376.174.130.0002
inosine100.0054.4845.5212.97<0.0001
guanosine100.0045.2554.759.27<0.0001
adenine100.0053.5746.4312.54<0.0001
thymidine100.0044.8255.189.12<0.0001
adenosine100.0046.6953.319.76<0.0001
2'-deoxyadenosine100.0050.3949.6111.16<0.0001
Mean100.0042.2957.71
sclerotiumuracil100.0019.2580.753.380.0013
uridine100.0047.5252.4810.06<0.0001
2'-deoxyuridine100.0019.1980.813.370.0013
inosine100.0022.0477.963.830.0004
guanosine100.0063.7036.3018.55<0.0001
adenine100.0016.4883.522.970.0038
thymidine100.009.4590.552.040.0432
adenosine100.0047.4452.5610.03<0.0001
2'-deoxyadenosine100.0015.3884.622.820.0058
Mean100.0028.9471.06

2.3. Q Cluster Analysis

O. sinensis, one of the most precious Traditional Chinese Medicines grows in a very restricted habitat, and is usually found in the soil of prairies or fir forests at an altitude from 3,500 to 5,000 m, mainly in provinces like Sichuan, Qinghai, Yunnan, Tibet and Gansu in China. In Nepal, Bhutan and India, O. sinensis is collected as well. In China, this fungus is usually called “Dong Chong Xia Cao”. O. sinensis has been used for the treatment of hyperglycemia, respiratory and liver diseases, renal dysfunction, renal failure and has antioxidant properties [57,58]. It was initially recorded in Ben-Cao-Bei-Yao by Wang Ang in 1694. Because of its scarcity in nature and high price, some studies have been carried out in order to find substitutes for O. sinensis [49,59,60]. C. militaris have been used as the main substitute for O. sinensis [50,61], and Traditional Chinese Medicine considers C. cicadae to be a drug similar to O. sinensis, as these two species have similar active components and medicinal value [17], however, little scientific information about the proximate composition and bioactive ingredients of C. cicadae and O. sinensis is available. Q cluster analysis (average linkage) was used to analyze the 10 nucleosides in C. cicadae, O. sinensis and C. militaris (Figure 3 and Figure 4).

Molecules 19 06123 g003 200
Figure 3. Q-Cluster of 10 nucleosides assayed in coremium (stroma) of 10 populations of C. cicadae, four populations of O. sinensis, and one population of C. militaris, using the average linkage method.

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Figure 3. Q-Cluster of 10 nucleosides assayed in coremium (stroma) of 10 populations of C. cicadae, four populations of O. sinensis, and one population of C. militaris, using the average linkage method.
Molecules 19 06123 g003 1024

Figure 3 shows the 15 populations of C. cicadae, O. sinensis and C. militaris separated into three branches; clade A includes populatation CCKSG, clade C includes population CMSMB, and clade B includes all other populations. The populations of C. cicadae collected at Lanping and Weixi county clustered as one subclade, which showed the geological differences. In Figure 4, 15 populations of C. cicadae, O. sinensis and C. militaris also separate into three branches; clade D includes 10 populations of C. cicadae and three populations of O. sinensis, clade E includes population OSNBE, and clade F includes population CMSMB.

Molecules 19 06123 g004 200
Figure 4. Q-Cluster of 10 nucleosides assayed in sclerotium of 10 populations of C. cicadae, four populations of O. sinensis, and one population of C. militaris by the average linkage method.

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Figure 4. Q-Cluster of 10 nucleosides assayed in sclerotium of 10 populations of C. cicadae, four populations of O. sinensis, and one population of C. militaris by the average linkage method.
Molecules 19 06123 g004 1024

The four populations of O. sinensis could not be separated into a single clade, indicating that the nucleosides in O. sinensis had no obvious differences from those of C. cicadae. The average clusters based on the average content of uracil, uridine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenine, adenosine, 2'-deoxyadenosine and cordycepin had been constructed, showing that C. cicadae should be a better substitute for O. sinensis than C. militaris.

3. Experimental

3.1. Sample Preparation

The details of the sources of C. cicadae, O. sinensis and C. militaris, are shown in Table 5. The samples, divided into the fruiting body (coremium or stroma) and the nymph or caterpillar (sclerotium), were dried at 50 C and ground into powder. These were separately weighed into a 5 mL volumetric flask, 20% methanol was added to the flask to about 90% of its volume, and after sonication for 90 min, the mixture was diluted to the mark with 20% methanol. After centrifugation at 25 C for 10 min at 4,000 rpm/min, sample solutions were passed through a 0.45 μm membrane filter. Duplicate analytical samples were prepared for each sample. The HPLC chromatograms of C. cicadae and mixed standards are shown in Figure 5.

Table Table 5. Localities of the 10 populations of C. cicadae, four populations of O. sinensis and one population of C. militaris.

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Table 5. Localities of the 10 populations of C. cicadae, four populations of O. sinensis and one population of C. militaris.
SpeciesNO. of populationsSamlpe sizeLocus of extractionLocality
C. cicadaeCCKSG10CoremiumGelezicun, Shuanglong township, Kunming City, Yunnan
10Sclerotium
CCLHH10CoremiumHedongqingcun, Hexi township, Lanping county, Yunan
10Sclerotium
CCLHQ10CoremiumQidenglongcun, Hexi township, Lanping county, Yunan
10Sclerotium
CCLHX10CoremiumXiaqingtoucun, Hexi township, Lanping county, Yunan
10Sclerotium
CCLSH10CoremiumHuilongcun, Shideng township, Lanping county, Yunan
10Sclerotium
CCLTD10CoremiumDeqingcun, Tongdian township, Lanping county, Yunan
10Sclerotium
CCLTL10CoremiumLianqiaoshacun, Tongdian township, Lanping county, Yunan
10Sclerotium
CCLZD10CoremiumDatujicun, Zhongpai township, Langping county, Yunnan
10Sclerotium
CCWYJ10CoremiumJuxiangcun, Yongchun township, Weixi county, Yunan
10Sclerotium
CCYTS10CoremiumSanzhou Mountain, Taihua town, Yixing city, Jiangsu
10Sclerotium
O.sinensisOSDQI5StromaDeqin, county, Yunnan
5Sclerotium
OSMNI5StromaManicun, Lengda township, Jiacha county, Tibet
5Sclerotium
OSLTA5StromaLitang county, Sichuan
5Sclerotium
OSNBE5StromaNepal
5Sclerotium
C.militarisCMSMB5StromaBaiyi township, Songming county, Yunnan
5Sclerotium
Molecules 19 06123 g005 200
Figure 5. HPLC chromatograms of (A) C. cicadae (Pop CCLTL); (B) mixed standards; 1: uracil; 2: uridine; 3: 2'-deoxyuridine; 4: inosine; 5: guanosine; 6: adenine; 7: thymidine; 8: adenosine; 9: 2'-deoxyadenosine; 10: cordycepin (3'-deoxyadenosine).

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Figure 5. HPLC chromatograms of (A) C. cicadae (Pop CCLTL); (B) mixed standards; 1: uracil; 2: uridine; 3: 2'-deoxyuridine; 4: inosine; 5: guanosine; 6: adenine; 7: thymidine; 8: adenosine; 9: 2'-deoxyadenosine; 10: cordycepin (3'-deoxyadenosine).
Molecules 19 06123 g005 1024

3.2. Chemicals and Reagents

HPLC-grade methanol was obtained from Merck KGaA (Darmstadt, Germany). Water was purified using a Millipore Simplicity system (Billerica, MA, USA). Uracil, uridine, 2'-deoxyuridine, inosine, guanosine, thymidine, adenine, adenosine, 2'-deoxyadenosine and cordycepin (purity ≥ 98.0%) were purchased from Sigma (St. Louis, MO, USA).

3.3. Liquid Chromatography Conditions

HPLC was conducted on a Dionex liquid chromatograph system (DIONEX, Sunnyvale, CA, USA) equipped with a LPG-3400A quaternary pump and a PDA-3000 photodiode array detector. The sample extracts were separated and analyzed using a Waters Symmetry® C18 column (250 mm, 4.6 mm, 5μm) at 30 °C. The mobile phase consisted of 10% solvent A (methanol) and 90% solvent B (water). The flow rate was 1.0 mL·min−1. The detecting wavelength was set between 190 and 380 nm, and the chromatographic peaks were measured at a wavelength of 260 nm for the detection of nucleosides.

3.4. Method Validation

3.4.1. Calibration Curves

Stock solutions were prepared by dissolving the standards in 20% methanol to give 1–2 mg/mL for uracil, uridine, 2'-deoxyuridine, inosine, guanosine, adenine, thymidine, adenosine, 2'-deoxyadenosine and cordycepin respectively. Further dilution with 20% methanol was performed to prepare the standard solutions for calibration curves. At least six concentrations of the solution were analyzed in triplicate, and then the calibration curves were constructed by plotting the peak areas versus the concentration of each analyte. The results were shown in Table 6.

Table Table 6. Linear regression data, LOD, and LOQ of 10 nucleosides at 260 nm.

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Table 6. Linear regression data, LOD, and LOQ of 10 nucleosides at 260 nm.
Analyteλ max (nm)Linear Regression Equationr2Test Range (μg/mL)LOD (μg/mL)LOQ (μg/mL)
uracil260.1y = 0.0145x − 0.00760.99998.00–240.000.0060.018
uridine263.1y = 0.0233x + 0.0080.99998.00–240.000.0080.024
2'–deoxyuridine263.2y = 0.0234x − 0.00160.99997.00–140.000.0080.024
inosine249.8y = 0.0368x − 0.04790.99957.60–380.000.0150.045
guanosine254.2y = 0.023x + 0.0290.99978.00–400.000.0080.024
adenine261.5y = 0.0108x0.99998.00–400.000.0040.012
thymidine268.2y = 0.0315x + 0.00090.99998.60–172.000.0120.036
adenosine261.0y = 0.0178x − 0.00090.99998.40–420.000.0060.018
2'–deoxyadenosine261.1y = 0.0171x + 0.00250.99998.40–420.000.0060.018
cordycepin261.2y = 0.0189x − 0.00050.99998.40–420.000.0070.021

3.4.2. Limits of Detection and Quantification

The stock solution containing ten reference compounds was diluted to a series of appropriate concentrations with the same solvent, and an aliquot of the diluted solutions were injected into HPLC for analysis. The limits of detection (LOD) and quantification (LOQ) under the present chromatographic conditions were determined at a signal-to-noise ratio (S/N) of about 3 and 10, respectively. The LOD and LOQ data for each compound investigated were shown in Table 6. The identification of investigated compounds was carried out by comparison of their retention times and UV spectra with those obtained injecting standards in the same conditions or by spiking Cordyceps samples with stock standard solutions.

3.4.3. Reproducibility and Accuracy

Reproducibility and accuracy were determined for 10 standard samples at a certain concentration, which was described in Table 7. The intra-day coefficients of variation for the 10 analytes were 0.65%–2.49%. The inter-day coefficients of variation for the 10 analytes were 1.08%–2.12%. The accuracy (%) of the method was expressed as the mean deviation of all repetitions from the nominal value. The intra-day accuracy for the 10 analytes was 98.98%–101.38%. The inter-day accuracy for the 10 analytes was 98.88%–101.53%.

Table Table 7. Reproducibility and accuracy analysis of 10 nucleosides (n = 5).

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Table 7. Reproducibility and accuracy analysis of 10 nucleosides (n = 5).
AnalyteNominal Concentration (µg/mL)Assay Value (mean ± SD) (µg/mL)Coefficient of Variation (%)Accuracy (%)
intra-day d
uracil40.0039.84 ± 0.501.2699.60
uridine40.0039.65 ± 0.882.2299.13
2'-deoxyuridine70.0070.71 ± 1.371.94101.01
inosine38.0037.65 ± 0.802.1299.08
guanosine40.0040.55 ± 1.012.49101.38
adenine40.0039.59 ± 0.972.4598.98
thymidine86.0085.32 ± 1.151.3599.21
adenosine42.0041.65 ± 0.270.6599.17
2'-deoxyadenosine40.0039.77 ± 0.621.5699.43
cordycepin42.0042.39 ± 0.541.27100.93
Inter-day d
uracil40.0039.71 ± 0.481.2199.28
uridine40.0039.55 ± 0.731.8598.88
2'-deoxyuridine70.0070.82 ± 1.251.77101.17
inosine38.0037.62 ± 0.741.9799.00
guanosine40.0040.61 ± 0.932.29101.53
adenine40.0039.63 ± 0.681.7299.08
thymidine86.0085.17 ± 1.271.4999.03
adenosine42.0041.57 ± 0.451.0898.98
2'-deoxyadenosine40.0039.62 ± 0.842.1299.05
cordycepin42.0042.49 ± 0.691.62101.17

d The sample was analyzed five times within one day (intra-day) and over two consecutive days (inter-day).

3.4.4. Extraction Recoveries

Recoveries and reproducibility of the proposed methods for target compounds were calculated using the C. cicadae (population CCLTL) mixture sample as a representative. The extraction recovery was performed by adding a known amount of individual standards into a 0.50 g of C. cicadae sample. Three replicates were performed for the test. The mixture was extracted and analyzed using the method mentioned above. Table 8 shows the recoveries of 10 nucleosides.

Table Table 8. Recoveries for the assay of 10 nucleosides in C. cicadae (n = 3).

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Table 8. Recoveries for the assay of 10 nucleosides in C. cicadae (n = 3).
AnalyteOriginal (µg)Spiked Amount (µg)Found e (mean ± SD) (µg)Recovery f (%)Coefficient of Variation (%)
uracil145.35140.00282.08 ± 4.8998.851.73
uridine714.12700.001398.28 ± 11.6098.880.83
2'-deoxyuridine63.8560.00126.87 ± 2.36102.441.86
inosine140.62140.00276.66 ± 2.1998.590.79
guanosine415.96400.00806.39 ± 3.9998.830.50
thymidine39.6140.0080.55 ± 1.28101.181.59
adenine31.9630.0061.05 ± 0.7098.531.14
adenosine375.96370.00736.62 ± 4.8198.750.65
2'-deoxyadenosine25.3130.0054.20 ± 0.7697.991.40
cordyceping150.00152.12 ± 0.78101.410.51

e The data were present as an average of three determinations; f Recovery (%) = 100 × ((amount found − original amount)/amount spiked); g Not detected.

3.5. Statistical Analysis

The data were statistically analyzed using the Statistical Analysis System (SAS) 8.1 software.

4. Conclusions

Simple and convenient HPLC methods for the determination of the content of nucleosides in C. cicadae populations were described. The method might be used for fast determination of the nucleosides in Cordyceps materials.

Chemical constituents of natural crude drugs, including C. cicadae occurring in Nature, are affected by location, geography, climate and microenvironment. The variance of nucleosides was large in natural C. cicadae, and might be derived from genetic differences. The genetic differentiation of C. cicadae populations by DALP and EST-SSR will be discussed in future papers.

The use of C. cicadae as a Traditional Chinese Medicine and tonic food has been appreciated for more than 1,500 years, and it has been used as a substitute for O. sinensis. The content and distribution of nucleosides in C. cicadae were similar to those in O. sinensis, and the medicinal effectiveness of C. cicadae was also similar to that of O. sinensis. Furthermore, the habitat demands of C. cicadae are less strict than those of O. sinensis, and its resource distribution and reserves were much larger than those of O. sinensis. The price of C. cicadae was about 2,000 yuan per kilogram in 2013, which was 1/100 of that of O. sinensis. It was suggested that C. cicadae should be used as substitute for O. sinensis.

Acknowledgments

This work was supported by the Specialized Research Fund for the Doctoral Program of Higher Education (20125301110001) and Yunnan Natural Science Foundation of China (2008CC019).

Author Contributions

Wen-Bo Zeng, Hong Yu and Feng Ge designed research; Wen-Bo Zeng, Jun-Yuan Yang, Zi-Hong Chen, Yuan-Bing Wang and Yong-Dong Dai performed experiments and analyzed the data; Wen-Bo Zeng, Hong Yu, Yuan-Bing Wang and Yong-Dong Dai collected the Cordyceps materials, Wen-Bo Zeng, Hong Yu and Alison Adams wrote the paper. All authors read and approved the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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