Cordyceps is a fungus that lives on certain caterpillars in the high mountain regions of China. Most cordyceps supplements are made in a lab.
Cordyceps might improve immunity by stimulating cells and specific chemicals in the immune system. It might also help fight cancer cells and shrink tumor size, particularly with lung or skin cancers. Natural cordyceps is hard to get and might be expensive.
People most commonly use cordyceps for athletic performance, kidney disorders, liver problems, and sexual problems, but there is no good scientific evidence to support these uses.
Arsenic Species in Cordyceps sinensis and Its Potential Health Risks
Yaolei Li1, Yue Liu2, Xiao Han3, Hongyu Jin1* and Shuangcheng Ma1*
1National Institutes for Food and Drug Control, Beijing, China
2School of Chinese Pharmacy, Beijing University of Chinese Medicine, Beijing, China
3Department of Pharmacy, Beihua University, Jilin, China
High arsenic residues make Cordyceps sinensis a concern in China. Arsenic toxicity is related to its species. Many studies have evaluated the toxicity of total arsenic, but few have studied its species. In this study, the species of arsenic in C. sinensis and its potential health risk were investigated. SEC-HPLC-ICP-MS was used to analysis of arsenic in C. sinensis and unknown arsenic (uAs) was discovered. Additionally, arsenic in C. sinensis was mainly found in alkali-soluble proteins. The trend of arsenic transformation indicated that unknown arsenic in C. sinensis may be converted into free inorganic arsenic, which enhanced toxicity. The result of risk assessment indicated that there were potential health risks of uAs. Hereon, we proposed recommendations for the use of C. sinensis and regulatory recommendations for arsenic standards. This study contributed to the toxicity reveal, safety evaluation, and risk assessment of arsenic in C. sinensis.
Cordyceps sinensis, which distributes mainly in alpine regions in Qinghai, Tibet, Sichuan, Yunnan, Gansu, and Guizhou of China (Li et al., 2006; Yang et al., 2009), is a traditional Chinese medicine (TCM) with a long history. It is parasitic in the larvae of the bat moth, making the larvae body rigid, and forms a rod-shaped sub-seat in the head of the worm in summer (Yue et al., 2008; Huang et al., 2018). It’s expensive due to its unique growth environment (Dawn et al., 2012) and important medical value. Compendium of materia medica records its role of protecting the lungs and kidneys, stopping bleeding and removing blood stasis (Li and Tsim, 2004). Annals of Sichuan also records that it works the same as ginseng (Kang et al., 2013). It is included in the 2015 edition of the Chinese Pharmacopoeia (Chinese Pharmacopoeia Committee, 2015).
High arsenic residues and exorbitant price make C. sinensis a concern in China. In 2016, the National Medical Products Administration (NMPA) issued that the total arsenic (tAs) was founded to be 4.4–9.9 mg kg-1 in C. sinensis and its related products. Meanwhile, the consumption indicated that the long-term use may cause health risks (National Medical Products Administration, 2016). This has caused widespread concern of social media and consumers. They have no consensus about arsenic in C. sinensis. Some view tAs accumulates in human body and is harmful (Lu et al., 2017), however, other view arsenic is harmless because its level in C. sinensis is negligible (China Science Daily, 2016). Thus, it is necessary to explore whether arsenic in C. sinensis is safe to humans.
The toxicity of arsenic is closely related to its species (Liu Q et al., 2018), different species have different toxicities. Combined with its chemical forms and toxicological characteristics, arsenic is mainly divided into inorganic arsenic (iAs) and organic arsenic (oAs). Inorganic arsenic has been listed as Group 1 carcinogen by International Agency for Research into Cancer (IARC) (Sharma and Sohn, 2009). The LD50 values of iAs including As(III) and As(V) in rat are 14 and 20 mg kg-1, respectively (Moe et al., 2016). Organic arsenic such as arsenocholine and arsenosugar are considered to be nontoxic (Liu et al., 2013). The evaluation of arsenic with tAs in TCM has many shortcomings (Zhu et al., 2013). For instance, most of the marine drugs mainly contain arsenosugar, and it’s unreasonable to evaluate the toxicity of arsenic by tAs. Thus, it is necessary to accurately study the arsenic species in C. sinensis.
Currently, the Chinese Pharmacopoeia (edition 2015) contains detection methods for six arsenic species (Chinese Pharmacopoeia Committee, 2015). Other national standards such as USP, EP, etc. do not include. There are many research methods and progress (Zhao et al., 2006; Monika and Danuta, 2016; Werner et al., 2018), and most of them concern the detection of small-molecule arsenic compounds in TCM, however only few studies on macromolecular compounds such as arsenosugar, arsenic-binding protein, and other unknown arsenic species. Many studies have evaluated the toxicity of tAs, but few have studied its species (Moreda-Piñeiro et al., 2011; Cao et al., 2015; Zhou et al., 2017; Lu et al., 2017; Sun et al., 2017). Before this work, we had focused on establishing a method to analysis arsenic speciation and test 34 batches of C. sinensis using 10% nitric acid (v/v) combined with microwave extraction. This condition was intense enough to make all species convert to iAs and maximized the health risks of arsenic in C. sinensis. The results showed that there were only iAs in C. sinensis, and As(III) was more abundant than As(V) (Zuo et al., 2018). However, according to the past research, most of the arsenic in biomimetic extraction was not detected (Cao et al., 2015). The administration of C. sinensis involves consumption after boiling and cooling, with almost all of it taken into the human digestive system. Hence, considering that it may cause human health and safety risks, it’s very meaningful to study arsenic speciation in C. sinensis.
In this study, we first extracted the arsenic species in C. sinensis by simulating gastric juice and nitric acid with different proportions. The transformation trend of arsenic species in C. sinensis was predicted and the distribution of arsenic was investigated. We discovered the unknown arsenic species by establishing SEC-HPLC-ICP-MS. Furthermore, we conducted a health risk assessment to describe its potential health risks. Finally, the medicinal and regulatory recommendations for C. sinensis were provided. Our study provides a valuable guide to the toxicological risk of arsenic in C. sinensis for consumers.
Materials and Methods
Seventeen batches of wild C. sinensis were collected in this study. These samples came from some production areas in China—Qinghai, Tibet, Gansu, Yunnan, Sichuan, and other regions. The production area, latitude, and other information of the sample are shown in Figure 1 and Supplementary Table 1. All the residual soil in the sample was carefully washed with ultrapure water. After drying at room temperature, the sample was pulverized and passed through a sieve of 0.3 mm to obtain powder. All samples were authenticated by Mr. Shuai Kang, who was an associate researcher on the identification of medicinal materials in National Institutes for Food and Drug Control (NIFDC). All the samples have stored in traditional Chinese drugs museum and where it belongs to NIFDC.
Figure 1 Distribution of sampling sites in China.
Reagents and Materials
Nitric acid (HNO3, 65.0%) was of ultrapure quality (Merck, Munchen, Germany). Analytical-grade ammonium carbonate ([NH4]2CO3), sodium hydroxide (NaOH), sodium chloride (NaCl), ethanol (C2H5OH), acetone (CH3COCH3), n-hexane (C6H14), chloroform (CHCl3), and hydrochloric acid (HCl) were all purchased from Beijing Chemical Reagent Co. (Beijing, China). Tris was purchased from Roche Diagnostics Gmbh (Berlin, Germany). Purified pepsin was purchased from Sigma-Aldrich (Sigma-Aldrich, USA). Deionized water (18.2 MΩ) was produced using a Millipore ultrapure water system (Millipore, Bedford, USA). The total arsenic standard solution (100.0 µg ml-1), dimethyl arsenic (DMA), monomethyl arsenic (MMA), arsenate [As(V)], arsenobetaine (AsB), and arsenocholine (AsC) standard solution were purchased from National Standard Material Research Center (Beijing, China). The arsenate [As(III)] standard solution was purchased from the National Institute of Standards and Technology (Gaithersburg, USA). Standard working solutions of AsB, As(III), DMA, AsC, MMA, and As(V) were prepared by diluting stock solutions immediately before use. The internal standard solution containing germanium (m/z = 74, 100.0 µg ml-1) was purchased from Agilent (Agilent Technologies, Folsom, CA, USA).
Total Arsenic Determination by ICP-MS
For total arsenic analysis, all samples were digested using a MARS 5 microwave digestion system (CEM, USA). The digestion method listed in the Pharmacopoeia of the People’s Republic of China (Chinese Pharmacopoeia Committee, 2015). Approximately 0.2 g of the sample was weighed into a PTFE digestion tube, and then 8 ml of HNO3 was added in sequence. The microwave digestion program was as follows: heating for 3 min to 120°C and holding for 3 min, heating for 2 min to 150°C and holding for 3 min, heating for 2 min to 200°C and holding for 12 min. After removing the excess nitric acid, the digested solutions were allowed to cool to room temperature. The solution was then transferred to a polyethene flask and diluted with deionized water to 50 ml. All samples were filtrated through hydrophilic microporous membrane filters (0.45 µm, Nylon 66, Jinteng, China) before determination by ICP-MS (Agilent 7700X, Agilent Technologies Co., USA). Standard working solutions of total arsenic (0–50 µg L-1) were prepared by diluting of stock solutions immediately before use. Take germanium as internal standard, and simultaneously enter the sample and internal standard.
Arsenic Speciation Analysis by HPLC-ICP-MS and SEC-HPLC-ICP-MS
Speciation analysis of the arsenic species in the extracts was conducted by directly coupling high-performance liquid chromatography (HPLC, Agilent 1200 HPLC Pump, USA) with ICP-MS. The separation of six arsenic species, namely, AsB, As(III), DMA, AsC, MMA, and As(V), was performed using an anion exchange column (DioncxIon Pac™ AS7 Analytical column, 250×4.6 mm, USA) with a gradient mobile phase of [NH4]2CO3. In this study, we used a previously validated test method (Zuo et al., 2018). The gradient elution using [NH4]2CO3 and water solutions A (100 mmol L-1) and B (deionized water) was carried out (Zuo et al., 2018). The step-gradient program was as follows: 10% A linearly increasing to 50% A from 0 to 3 min, linearly increasing to 100% A from 3.0 to 4.0 min, remaining at 100% A from 4.0 to 11 min, linearly reduce to 10% A from 11 to 13 min, re-equilibration to the initial concentration of 10% A and 90% B from 13 to 17 min and remaining until completion of the separation run. The flow rate of the mobile phase was set to 0.5 ml min-1, and the injection volume was 10 µl. The size exclusion chromatography (SEC) condition was performed using a gel column (Shodex PROTEIN KW-802.5, Japan) with an Isocratic mobile phase of Tris-HCl (30 mmol L-1, pH = 7.5, prepare 30 mmol L-1 Tris first, then adjust the pH to 7.5 with hydrochloric acid), the flow rate of the mobile phase was set to 1.0 ml min-1, and the injection volume was 20 µl. The analysis time of SEC-HPLC-ICP-MS was 20 min.
The ICP-MS instrument was tuned and optimized for m/z 75 at the beginning of the experiment every day. A standard torch was used with a plasma gas flow rate of 15 L min-1, a carrier gas flow rate of 1.0 L min-1 and a makeup gas flow rate of 0.25 L min-1. The plasma RF power was 1,550 W in the experiment. The signal at m/z 75 for arsenic was monitored and collected in the time-resolved analysis mode (TRA). The integration time for arsenic at m/z 75 was 0.3 s. The six stock solutions were prepared by appropriate dilutions of corresponding standards in doubly deionized water. Working solutions (0–500 µg L-1) for arsenic species were prepared daily by diluting the stock solution (1,000 µg L-1). All solutions were stored at 4°C until analysis.
Extraction of Bioaccessible Arsenic
0.25 g of each sample powder was accurately weighed and placed into a 50 ml centrifuge tube, followed by the addition of 10 ml of simulated gastric juice. The mixture was extracted by shaking in a water bath at 37°C for 12 h. After cooling, the extract was filtered through hydrophilic microporous membrane filters (0.45 µm) and subsequently analyzed. Briefly, the simulated gastric juice was prepared using 10 g of purified pepsin (Sigma-Aldrich, USA) and 16.4 ml of diluted nitric acid (take 10.5 ml of concentrated nitric acid diluted to 100 ml with water) diluted to 1,000 ml with deionized water.
Exploring the Trend of Arsenic Transformation
After weighing accurately 0.3 g of the sample powder, 10 ml of 0, 1, 2, 3, 5, 8, 10, 13, and 15% (v/v) nitric acid solution was added. The mixture was extracted by a microwave rapid extraction system (EXPLORER, CEM, USA) at 70°C for 10 min. After cooling to room temperature, all samples were filtered through 0.45 µm membrane filters before determination. A blank solution was prepared using the same method.
Investigate the Different Distribution of Arsenic in C. Sinensis
Extraction of Arsenic From Different Proteins
In this experiment, water-soluble protein, salt-soluble protein, alkali-soluble protein, and alcohol-soluble protein in C. sinensis were extracted. First, 0.3 g samples were accurately weighed, followed by extraction in 10 ml of water, 5% NaCl, 0.08 mol L-1of NaOH and 70% ethanol solution, respectively. After 12 h at room temperature, ultrasonic extraction was performed three times for 20 min each time. The sample was then centrifuged at 5,000 r min-1 for 10 min, and the supernatant was combined. Next, ice-cold acetone was slowly added to the supernatant to increase the concentration to 80%. The protein was precipitated in a refrigerator at 4°C for 12 h and then was centrifuged at 5,000 r min-1 for 15 min to separate the supernatant. Finally, the organic solvent in the four proteins was blown off with nitrogen to obtain the purified protein (Liu et al., 2015). These proteins were dissolved in 5 ml of 30 mmol ml-1 Tris-HCl buffer (pH = 7.5) (Liu et al., 2015), and filtered through hydrophilic microporous membrane filters (0.45 µm). Furthermore, the obtained supernatant was concentrated to 5 ml and used for subsequent analysis.
Extraction of Arsenic From Crude Water-Soluble Polysaccharides
The polysaccharide was prepared by water extraction and alcohol precipitation. First, it weighed 0.5 g sample and immersed in 10 ml of water for 12 h. The mixture was extracted by microwave extraction at 85°C and filtered. The filter residue was repeatedly extracted three times. All filtrates were concentrated at 80°C to 1/4 of the original volume. Three volumes of 95% ethanol were added to the filtrate for 24 h, which was centrifuged at 3,000 r min-1 for 40 min, and the precipitate was dissolved in 5 ml of hot water. The protein was removed by the Sevag method (chloroform: n-butanol = 4:1, v/v) until no precipitation in the organic phase to obtain a water-soluble crude polysaccharide solution. Protein residue was dissolved in 5 ml of 30 mmol ml-1 Tris-HCl buffer (pH = 7.5), and filtered through hydrophilic microporous membrane filters (0.45 µm). Furthermore, the obtained supernatant was concentrated to 5 ml and subsequently analyzed.
Extraction of Arsenic From Lipids
After accurately weighing 0.3 g of the sample powder, 30 ml of n-hexane was added and followed by microwave extraction three times at room temperature. Next, the sample was centrifuged at 5,000 r min-1 for 10 min, and the supernatants were combined and condensed to 10 ml.
Analysis, Quality Assurance, and Quality Control
The standard reference material citrus leaf was used during the total arsenic measurement by ICP-MS. The tAs concentration in the citrus leaf was 1.2 mg kg-1, which agreed well with the certified values (1.1 ± 0.2 mg kg-1). For the speciation analysis, the spiked recoveries for different arsenic species were in the range of 85–118%, with an RSD < 10% (n = 6). The six arsenic species showed a good linear relationship, and the acceptance criterion coefficients of linear regression (R2) were ≥ 0.9990. These results met the quality requirements for metal analysis.
Health Risk Assessment
To assess the health risks of arsenic in C. sinensis, the risk assessment method used the following formula (USEPA, 1998).
In the formula, EDI (µg/BWkg day) is the estimated daily intake of arsenic, C (mg kg-1) is the concentration of arsenic, IR is the C. sinensis intake for an adult from the Chinese Pharmacopoeia 2015 (g person-1 day-1) (Chinese Pharmacopoeia Committee, 2015), and the maximum daily dose was 9 g in Chinese Pharmacopoeia, which was used in this equation to provide the “worst-case” scenario. BW (kg) is the average body weight and was considered to be 60 kg in this study (USEPA, 2013).
HQ is the hazard quotient of arsenic. If the HQ value is above 1, toxic risk exists, with an increasing possibility as the value increases. RfD is the oral reference dose (µg kg−1 day−1), and its value of arsenic is 0.3 µg kg−1 day−1 (USEPA, 2015). EF is the exposure frequency (365 d year-1). ED is the exposure duration (70 years). AT is the average exposure time (25,550 days) (Liu et al., 2013; Liu L et al., 2018).
The cancer risk (CR) associated with iAs exposure for C. sinensis consumers was calculated according to the following equation:
where SF (BWkg day µg-1) is the cancer slope factor set by USEPA only for iAs (USEPA, 2015; Liu L et al., 2018). The SF value for iAs was 1.5×10-3 BWkg day µg-1.
Bioaccessible Arsenic in Cordyceps Sinensis
After treated samples by simulated gastric juice, the extracts were detected by HPLC-ICP-MS, and tAs was determined by ICP-MS. According to the measurement results (Figure 2), the tAs in 17 batches of C. sinensis ranged from 4.7 to 15.5 mg kg-1, with an average content of 9.5 mg kg-1, which was consistent with some previous studies (Liu et al., 2016; Zhou et al., 2017; Li et al., 2019). The bAs content ranged from 2.0 to 10.1 mg kg-1, and the average content was 5.0 mg kg-1. There were little As(III) and As(V) detected in this experiment. The typical chromatographic are shown in Figure 3. As(III) content ranged from 0.05 to 0.2 mg kg-1, and the average content was 0.1 mg kg-1. The As(V) content ranged from 0.07 to 0.3 mg kg-1, the average content was 0.2 mg kg-1, and the total amount of iAs was only 0.4 mg kg-1. Further, there were huge differences among tAs, bAs, and iAs (Figure 4). The bAs accounted for 52% of tAs, while iAs accounted for 4%. It should be noted that only 8% of free iAs in the bAs was detected by HPLC-ICP-MS. Many chromatographic conditions were tried in this study and did not detect other arsenic species. Thus, its need to further verify whether there is unknown arsenic species present in C. sinensis.