Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Determination of Arsenic Species in Ophiocordyceps sinensis from Major Habitats in China by HPLC-ICP-MS and the Edible Hazard Assessment

Determination of Arsenic Species in Ophiocordyceps sinensis from Major Habitats in China by... molecules Article Determination of Arsenic Species in Ophiocordyceps sinensis from Major Habitats in China by HPLC-ICP-MS and the Edible Hazard Assessment 1 , † 2 , † 1 1 Lian-Xian Guo , Gui-Wei Zhang , Jia-Ting Wang , Yue-Ping Zhong and 1 , Zhi-Gang Huang * Dongguan Key Laboratory of Environmental Medicine, School of Public Health, Guangdong Medical University, Dongguan 523808, China; [email protected] (L.-X.G.); [email protected] (J.-T.W.); [email protected] (Y.-P.Z.) Shenzhen Academy of Metrology and Quality Inspection, Shenzhen 518000, China; [email protected] * Correspondence: [email protected]; Tel.: +86-0769-22896573 † These authors contributed equally to this work. Received: 2 April 2018; Accepted: 24 April 2018; Published: 26 April 2018 Abstract: This study sought to determine the concentration and distribution of arsenic (As) species in Ophiocordyceps sinensis (O. sinensis), and to assess its edible hazard for long term consumption. The total arsenic concentrations, measured through inductively coupled plasma mass spectrometry (ICP-MS), ranged from 4.00 mg/kg to 5.25 mg/kg. As determined by HPLC-ICP-MS, the most V V V III V concerning arsenic species—AsB, MMA , DMA , As , and As —were either not detected (MMA V V III and DMA ) or were detected as minor As species (AsB: 1.4–2.9%; As : 1.3–3.2%, and As : 4.1–6.0%). III The major components were a cluster of unknown organic As (uAs) compounds with As , which accounted for 91.7–94.0% of the As content. Based on the H O test and the chromatography 2 2 behavior, it can be inferred that, the uAs might not be toxic organic As. Estimated daily intake (EDI), hazard quotient (HQ), and cancer risk (CR) caused by the total As content; the sum of inorganic As (iAs) and uAs, namely i+uAs; and iAs exposure from long term O. sinensis consumption were calculated and evaluated through equations from the US Environmental Protection Agency and the uncertainties were analyzed by Monte-Carlo Simulation (MCS). EDI and EDI are total As i+uAs approximately ten times more than EDI ; HQ and HQ > 1 while HQ < 1; and CR iAs As i+u As i As total total As 4 4 and CR > 1  10 while CR < 1  10 . Thus, if the uAs is non-toxic, there is no particular i+uAs iAs risk to local consumers and the carcinogenic risk is acceptable for consumption of O. sinensis because the concentration of toxic iAs is very low. Keywords: Ophiocordyceps sinensis; arsenic speciation; risk assessment; HPLC-ICP-MS 1. Introduction Ophiocordyceps sinensis (O. sinensis), a mysterious entomogenous fungus distributed on the Qinghai–Tibet Plateau, is popularly referred to as winter-worm-summer-grass (Dong Chong Xia Cao in Chinese) [1,2] (Figure 1). O. sinensis has been utilized in China (Figure 2A) and surrounding countries for more than 2000 years as a rare functional food to promote health and to treat diverse chronic diseases. In 1993, O. sinensis became famous worldwide because Chinese female athletes broke several world records in running events at the National Games and the meritorious performances were later attributed (at least in part) to the consumption of this fungus [3]. Subsequently, modern pharmaceutical research has shown that its predominant functions are anti-tumor, anti-inflammatory, nephroprotective, Molecules 2018, 23, 1012; doi:10.3390/molecules23051012 www.mdpi.com/journal/molecules Molecules 2018, 23, 1012 2 of 14 antioxidant, antihyperglycemic, anti-apoptosis, immunoregulatory, and hepatoprotective [4,5]. Accordingly, these pronounced medicinal functions have resulted in a large demand for wild O. sinensis and have also increased the local prosperity of economically poor production areas around the Molecules 2018, 23, x FOR PEER REVIEW 2 of 14 Qinghai–Tibet Plateau and in adjacent countries. Some households earn as much as two-thirds of economically poor production areas around the Qinghai–Tibet Plateau and in adjacent countries. their income from the collection of O. sinensis (Figure 2B), and pastoralists are earning an income of a Some households earn as much as two-thirds of their income from the collection of O. sinensis (Figure size unrecorded in their history [6,7]. However, concerns about human health have arisen since the 2B), and pastoralists are earning an income of a size unrecorded in their history [6,7]. However, CFDA (China Food and Drug Administration) revealed in 2016 that excessive Arsenic (As) content concerns about human health have arisen since the CFDA (China Food and Drug Administration) (total As: 4.4–9.0 mg/kg [8]) was detected in O. sinensis, a level five times the reference value of revealed in 2016 that excessive Arsenic (As) content (total As: 4.4–9.0 mg/kg [8]) was detected in O. 1 mg/kg for total As in functional foods (GB16740-2014) [9]. Subsequently, the CFDA ordered that all sinensis, a level five times the reference value of 1 mg/kg for total As in functional foods (GB16740- the pilot work on O. sinensis as a functional food be discontinued on 26 February 2016 [10]. Moreover, 2014) [9]. Subsequently, the CFDA ordered that all the pilot work on O. sinensis as a functional food numerous media outlets had arbitrarily declared that O. sinensis is a poison instead of a functional be discontinued on 26 February 2016 [10]. Moreover, numerous media outlets had arbitrarily declared food. This assertion has caused a great uproar in the health food market and has seriously affected the that O. sinensis is a poison instead of a functional food. This assertion has caused a great uproar in O. sinensis-dependent economic chain [11]. the health food market and has seriously affected the O. sinensis-dependent economic chain [11]. Figure 1. Life history of Ophiocordyceps sinensis (O. sinensis). (a) The eggs of the host Thitarodes insect, Figure 1. Life history of Ophiocordyceps sinensis (O. sinensis). (a) The eggs of the host Thitarodes insect, which are scattered on the grassland, incubate; (b) The host larvae safely reside in the soil throughout which are scattered on the grassland, incubate; (b) The host larvae safely reside in the soil throughout the long-lasting larval stage; (c) The ascospores erupt from mature fruiting bodies of O. sinensis; (d) the long-lasting larval stage; (c) The ascospores erupt from mature fruiting bodies of O. sinensis; The 4–5th instar larvae may be infected by the infective conidia of the O. sinensis fungus in the soil; (d) The 4–5th instar larvae may be infected by the infective conidia of the O. sinensis fungus in the soil; (e) The caterpillar-shaped sclerotium (winter-worm) is formed; (f) The stroma germinates out of the (e) The caterpillar-shaped sclerotium (winter-worm) is formed; (f) The stroma germinates out of the head capsule and the mature O. sinensis (summer-grass-winter-worm) is formed. head capsule and the mature O. sinensis (summer-grass-winter-worm) is formed. Arsenic can be present in both organic and inorganic forms. The toxicity of As is dependent on Ш V its chemical form, with inorganic species (iAs), such as arsenite (As ) and arsenate (As ), being the Arsenic can be present in both organic and inorganic forms. The toxicity of As is dependent on its III V most toxic [12]. Organic species are the metabolic products of iAs, such as monomethylarsonic acid chemical form, with inorganic species (iAs), such as arsenite (As ) and arsenate (As ), being the most V V (MMA ) and dimethylarsenic acid (DMA ), and are much less toxic than iAs to humans. toxic [12]. Organic species are the metabolic products of iAs, such as monomethylarsonic acid (MMA ) Additionally, some other organic As complexes (arsenocholine, arsenobetaine, various arsenosugars and dimethylarsenic acid (DMA ), and are much less toxic than iAs to humans. Additionally, some and arsenolipids) are generally considered nontoxic [13], according to previous studies on As toxicity. other organic As complexes (arsenocholine, arsenobetaine, various arsenosugars and arsenolipids) are However, recently, experimental results have documented the presence of trivalent intermediates, generally considered nontoxic [13], according to previous studies on As toxicity. However, recently, Ш Ш monomethylarsonous acid (MMA ) and dimethylarsinous acid (DMA ) in the urine of humans experimental results have documented the presence of trivalent intermediates, monomethylarsonous exposed to drinking water containing high levels of inorganic As [14]. These trivalent intermediates III III acid (MMA ) and dimethylarsinous acid (DMA ) in the urine of humans exposed to drinking are structurally different from the pentavalent compounds and are more reactive and more water containing high levels of inorganic As [14]. These trivalent intermediates are structurally carcinogenic [15–17]. More recently, the subsequent metabolic products of DMA , sulfur-containing different from the pentavalent compounds and are more reactive and more carcinogenic [15–17]. intermediary metabolites (dimethylmonothioarsinicacid, DMMTA )[18], and several As containing hydrocarbons (AsHC 332, AsHC 360 and AsHC 444) were shown to high toxic effects on organisms [19]. The different toxicities of As species reinforce the importance of distinguishing its chemical Molecules 2018, 23, 1012 3 of 14 III More recently, the subsequent metabolic products of DMA , sulfur-containing intermediary metabolites (dimethylmonothioarsinicacid, DMMTA ) [18], and several As containing hydrocarbons (AsHC 332, AsHC 360 and AsHC 444) were shown to high toxic effects on organisms [19]. The different toxicities of As species reinforce the importance of distinguishing its chemical form, as the total amount Molecules 2018, 23, x FOR PEER REVIEW 3 of 14 of As does not provide enough information about the toxicity of the analyzed sample. Therefore, it is incorrect to consider O. sinensis is toxic according to its total As content. Unlike previous reported form, as the total amount of As does not provide enough information about the toxicity of the arsenic accumulated mushrooms, which germinated on plant sourced media, O. sinensis only lives analyzed sample. Therefore, it is incorrect to consider O. sinensis is toxic according to its total As in Thitarodes larva of 4–5th instar. Inspired by the significantly higher organic As proportion in content. Unlike previous reported arsenic accumulated mushrooms, which germinated on plant animal-sourced traditional Chinese medicine (TCM) compared to plant-sourced TCM [20], some sourced media, O. sinensis only lives in Thitarodes larva of 4–5th instar. Inspired by the significantly scholars have proposed that due to larva-fungus complexity (Figure 2C,D), O. sinensis might contain higher organic As proportion in animal-sourced traditional Chinese medicine (TCM) compared to a lar plge ant- pr sou oprortion ced TCM of [20 organic ], some As. scRecently holars ha ,ve resear propos chers ed tha have t dpr ue to la ovided rva- evidence fungus compl for the exispeculation ty (Figure III V that 2C iAs ,D),species O. sinensis wer might co e minor ntain (As a large and proportion of As ) or below organ the ilevel c As. Recently, of detection reseafter archers h examining ave provided the As Ш V evidence for the speculation that iAs species were minor (As and As ) or below the level of detection speciation in O. sinensis through high-performance liquid chromatography-hydride generation-atomic after examining the As speciation in O. sinensis through high-performance liquid chromatography- fluorescence spectrometry (HPLC-HG-AFS). In addition, they found that the largest proportion of hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). In addition, they found that As was composed of an unknown organic As (uAs) species [21]. Because of the shortcomings of V V the largest proportion of As was composed of an unknown organic As (uAs) species [21]. Because of conventional HPLC-HG-AFS, in that research, they only discriminated four As species (MMA , DMA , V the shortcomings of III conventional HPLC-HG-AFS, in that research, they only discriminated four As As and As ) in O. sinensis samples [21]. Non-forming hydride species, such as AsB, important in V V V Ш species (MMA , DMA , As and As ) in O. sinensis samples [21]. Non-forming hydride species, such biota samples, cannot be evaluated using this approach [22]. as AsB, important in biota samples, cannot be evaluated using this approach [22]. Figure 2. The producing area of Ophiocordyceps sinensis in China and sampling details of this study. Figure 2. The producing area of Ophiocordyceps sinensis in China and sampling details of this study. (A) Schematic map illustrating the sampling sites in the Qinghai–Tibetan Plateau and its adjacent (A) Schematic map illustrating the sampling sites in the Qinghai–Tibetan Plateau and its adjacent high-altitude areas. Litang (LT), Naqu (NQ), and Yushu (YS) were chosen as the sampling sites. high-altitude areas. Litang (LT), Naqu (NQ), and Yushu (YS) were chosen as the sampling sites. (B) (B) The grassland of the O. sinensis habitat. Pastoralists are encamped there to collect it. (C) The stroma The grassland of the O. sinensis habitat. Pastoralists are encamped there to collect it. (C) The stroma (a) of O. sinensis emerged out of the ground. (D) O. sinensis in the soil, the yellow arrows pointing out (a) of O. sinensis emerged out of the ground. (D) O. sinensis in the soil, the yellow arrows pointing out its sclerotium (b) and stroma (c). its sclerotium (b) and stroma (c). To further clarify the As speciation in O. sinensis and oral intake hazards in longtime consumption, the present study examined the total As content, including the most concerning As Ш V V V species (As , As MMA , DMA , and AsB), in O. sinensis using the most commonly applied techniques: inductively coupled plasma mass spectrometry (ICP-MS) and anion exchange high- Molecules 2018, 23, 1012 4 of 14 To further clarify the As speciation in O. sinensis and oral intake hazards in longtime consumption, III the present study examined the total As content, including the most concerning As species (As , V V V As MMA , DMA , and AsB), in O. sinensis using the most commonly applied techniques: inductively coupled plasma mass spectrometry (ICP-MS) and anion exchange high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC-ICP-MS) [23,24]. Due to the extraordinary prices of the O. sinensis and chronic injury effects to organisms due to Arsenic consumption, a hazard assessment via experimental animal trials would be costly. Thus, in this study, we chose the most widely accepted model (Environment Protection Agency, EPA) of evaluation for health risks [25,26] to evaluate the potential non-carcinogenic risk and the probability of excess lifetime cancer risk due to As exposure from O. sinensis consumption. Additionally, Monte-Carlo Simulation (MCS) were employed to analyze the uncertainty. The health risk posed by O. sinensis consumption from exposure to total As, iAs, and i+uAs (iAs and uAs together) were comparatively estimated in this study. These contributions might be useful in efforts to revive the local O. sinensis-dependent economy in the Qinghai–Tibet Plateau and surrounding countries by providing a comprehensive edible hazard evaluation of O. sinensis. 2. Results and Discussion 2.1. Analytical Performances of the Proposed Method Extraction efficiency for As speciation analysis was assessed based on a comparison between the total As concentration in 0.15 mol/L HNO extracts and concentrated HNO extracts using a 3 3 microwave digestion system (see Section 3.2). The concentrations of the total As in the extracts through different extraction methods were quantitively consistent, with an extraction efficiency of 92.3–104% (the ratio of total As concentration in 0.15 mol/L HNO extracts to that in concentrated HNO extracts). 3 3 This efficiency indicates that most As species were thoroughly extracted through the current method (see Section 3.2). The analytical performances of the proposed ICP-MS, for total As content analysis, and HPLC-ICP-MS, for As species analysis, methods were validated by determining the linearity, limits of detection (LOD), and limits of quantification (LOQ) as shown in Table 1. The linear correlation 1 1 coefficients, in the range of 0.5–500 g L (for total As) and 0.2–300 g L (for As species), were V III V V greater than 0.9997. LOD of total As, AsB, DMA , As , MMA , and As were 2.3, 1.1, 1.3, 1.0, 2.2, 1 1 and 1.1 g kg , respectively, And the LOQ were found to be in the range of 3.0–6.9 g kg for the total As content and the five arsenic species. The recoveries of the total As content were in the range 92.3106.6% and the relative standard deviations (RSDs) were in the range 2.4~4.6%. The recoveries and RSDs of arsenic species were studied by spiking three concentration levels of the five arsenic species into the O. sinensis samples. The recoveries of arsenic species were in the range 86.3~111.7% and the RSDs were in the range 0.8~6.6%, as shown in Table 2. The analysis results of certified reference materials (CRM) using the same method were in good agreement with their certified values and the recoveries were 97.5–101% for total As analysis and 110.8% for iAs analysis as shown in Table 3. Table 1. Analytical performance of inductively coupled plasma mass spectrometry (ICP-MS) for total arsenic content and HPLC-ICP-MS for arsenic species. Analytes Linear Range (g/L) Linear Equation R LOD (g/kg) LOQ (g/kg) Total As 0.5–500 y = 2731.03x + 5.5567 0.9998 2.3 6.9 AsB 0.2–300 y = 18,867.9x + 2319.6 0.9999 1.1 3.3 DMA 0.2–300 y = 19,212.4x + 4065.2 0.9999 1.3 4.0 III As 0.2–300 y = 17,090.6x + 3289.1 0.9997 1.0 3.0 0.5–300 y = 18,373.6x + 196.8 1.0000 2.2 6.6 MMA As 0.2–300 y = 18,504.6x + 8247.2 1.0000 1.1 3.3 Molecules 2018, 23, 1012 5 of 14 Table 2. Recovery and precision of the methods. Background Value Added Measured Value Recovery RSD Analytes (mg/kg) (g/L) (g/L) (%) (%, n = 6) 5.00 14.8~15.3 92.3~99.4 4.6 10.0 20.4~21.5 94.7~102.3 3.8 Total As 0.51 50.0 60.7~64.3 95.8~106.6 2.4 2.00 2.09~2.26 91.7~100.1 4.2 10.0 9.45~9.87 91.9~96.2 1.9 AsB 0.010 50.0 42.0~47.0 83.6~93.4 5.0 2.00 1.73~1.98 86.5~99.0 5.7 10.0 9.20~9.55 92.0~95.5 1.5 ND DMA 50.0 50.4~52.6 100.9~105.2 1.7 2.00 45.3~45.6 86.3~99.2 5.5 III 10.0 52.8~53.7 88.8~98.0 4.3 1.70 As 50.0 96.8~99.2 106.9~111.7 2.0 2.00 2.07~2.19 99.4~105.7 2.7 0.0031 10.0 10.6~10.8 105.0~107.2 0.85 MMA 50.0 47.2~48.6 94.2~97.1 1.2 2.00 4.81~5.08 86.6~100.0 6.6 0.12 10.0 12.8~13.0 97.4~99.4 0.80 As 50.0 49.0~50.2 91.9~94.4 1.1 Table 3. National standard reference materials values (mg/kg, mean  standard deviation) and determined values for total As and inorganic arsenic (iAs) content (n = 5). Certified Value Determined Value Recovery Sample Type Reference Materials (mg/kg) (mg/kg) (%) Green Chinese onion GBW10049 0.52  0.11 0.507  0.08 97.5 Pork liver GBW10051 1.4  0.3 1.42  0.15 101.4 Yellow-fin tuna GBW08573 5.08  0.39 4.98  0.11 98.0 0.16  0.02 (total As) 0.165  0.012 103.1 Rice GBW100358 0.13  0.02 (iAs) 0.144  0.006 110.8 2.2. Total Arsenic Concentration and Arsenic Species in O. sinensis Samples The total As content in O. sinensis ranged from 4.00 mg/kg to 5.25 mg/kg of dry mass (Table 4), which is consistent with that in the previous studies [21] that caused uneasiness and fear in consumers. The Chinese government, through the National Health and Family Planning Commission of the People’s Republic of China, established a reference value of 1 mg/kg for total As in functional foods (GB16740-2014) [9]. Thus, the total As content in O. sinensis exceeds the limit of As in functional foods in China (Figure 3). These data demonstrate that there is an urgent need to determine As speciation in O. sinensis samples, because the total amount of As does not provide sufficient toxicological information. III In Figure 4B, the large peak area indicates that As , which is generally considered most toxic, might be the major As species in O. sinensis, however, an H O test proved that most of the As is not oxidized 2 2 V III to As (Figure 4C) and hence the major overlapped peak cannot be the toxic As . Thus, according to current chromatography conditions, an H O test is necessary to minimize misidentification. The iAs, 2 2 calculated according to the As content in Figure 4C, is relatively abundant compared to the total As content, in the range 6.0% to 8.3% (the amount of As in Figure 4C), but is small compared with the amount of organic As. Organic As species were predominant, and the percentage of total As content V V ranged from 91.7% to 94.0%. The two potential toxic organic As species, MMA and DMA , were V V found to be negligible. Because the initial organic metabolites of iAs, MMA , and DMA were trace in O. sinensis sample, it can be inferred that they were transformed into other organic arsenic metabolites in the organism such as AsB (which was also detected as a minority compound in these samples, ranging from 1.4% to 2.9%) and various unknown organic As species (uAs, a cluster of unknown compounds, with a retention time from 3.2 to 4 min, representing 89.0% to 92.3% of the of the total As content, Molecules Molecules Molecules Molecules 2018 2018 2018 2018, , , , 23 23 23 23, x FO , x FO , x FO , x FOR P R P R P R PEER EER EER EER R R R RE E E EVIEW VIEW VIEW VIEW 6 of 6 of 6 of 6 of 14 14 14 14 Molecules 2018, 23, 1012 6 of 14 Molecules 2018, 23, x FOR PEER REVIEW 6 of 14 Molecules 2018, 23, x FOR PEER REVIEW 6 of 14 8 8 8 89 9 9 9.0 .0 .0 .0% to 92 % to 92 % to 92 % to 92.3% of .3% of .3% of .3% of the of the tota the of the tota the of the tota the of the totallll As content, Fi As content, Fi As content, Fi As content, Figu gu gu gure re re re 4C). B 4C). B 4C). B 4C). Ba a a ased on sed on sed on sed on previous studies, comparison of previous studies, comparison of previous studies, comparison of previous studies, comparison of the retenti the retenti the retenti the retentio o o on ti n ti n ti n time beha me beha me beha me behavi vi vi vior of or of or of or of st st st sta a a an n n nda da da dards wi rds wi rds wi rds with tha th tha th tha th thattt t of the extra of the extra of the extra of the extrac c c cts ts ts ts upon cha upon cha upon cha upon chan n n nging pH of ging pH of ging pH of ging pH of the the the the mobi mobi mobi mobile le le le 89.0% to 92.3% of the of the total As content, Figure 4C). Based on previous studies, comparison of Figure 4C). Based on previous studies, comparison of the retention time behavior of standards with 89.0% to 92.3% of the of the total As content, Figure 4C). Based on previous studies, comparison of the retention time behavior of standards with that of the extracts upon changing pH of the mobile p p p ph h h hase c ase c ase c ase ca a a an n n n asc asc asc asce e e ert rt rt rtain t ain t ain t ain th h h he p e p e p e pr r r re e e es s s sence of ence of ence of ence of an an an an e e e ex x x xp p p pe e e ect ct ct cted co ed co ed co ed compound or giv mpound or giv mpound or giv mpound or give e e e useful useful useful useful in in in information on th formation on th formation on th formation on the nature e nature e nature e nature that the retenti of the extracts on time beha upon changing vior of stapH ndards wi of the th tha mobile t of the extra phase can c ascert ts upon cha ain thenpr ging pH of esence of the an expected mobile phase can ascertain the presence of an expected compound or give useful information on the nature of unknown of unknown of unknown of unknown c c c co o o ompounds [27]. In this mpounds [27]. In this mpounds [27]. In this mpounds [27]. In this stu stu stu stud d d dy, de y, de y, de y, despite th spite th spite th spite the lack of stan e lack of stan e lack of stan e lack of standa da da dards f rds f rds f rds fo o o or the r the r the r the conf conf conf confi i i ir r r rm m m ma a a ati ti ti tion on on on of of of of uAs, uAs, uAs, uAs, compound phase can or asc give ertain t useful he prinformation esence of an eon xpethe cted co natur mpound or giv e of unknown e useful compounds information on th [27]. In this e nature study, Ш Ш Ш Ш of unknown compounds [27]. In this study, despite the lack of standards for the confirmaIII tion of uAs, based on the overlap w based on the overlap w based on the overlap w based on the overlap w of unknown compounds [27]. In this iiiith As th As th As th As , or , or , or , organ gan gan gan stu iiiic As c As c As c As dy, de(oA (oA (oA (oA spite th s ss s) s ) s ) s ) sp p p p e lack of stan ecies wh ecies wh ecies wh ecies which ich ich ich da have low r have low r have low r have low r rds for the conf e e e etttte e e ent nt nt nt irion unde ion unde ion unde ion unde mation ofr anion r anion r anion r anion uAs, despite the lack of standards for the confirmation of uAs, based on the overlap with As , organic As based on the overlap with AsШ, organic As (oAs) species which have low retention under anion exchange exchange exchange exchange based on the overlap w HP HP HP HPLC LC LC LC-IC -IC -IC -ICP P P P-M -M -M -MS c S c S c S c ith As h h h hrom rom rom romaaa a, or tttto o o ogr gr gr gr gan am am am am ic As s, s s, s s, s s, su u u uch ch (oA ch ch as as as as s) sar ar ar ar pecies wh senol senol senol senoliiiip p p piiiids ds ds ds ich an an an an have low r d AsHC d AsHC d AsHC d AsHCes w s w s w s w tenth h h hion unde ich were ich were ich were ich were r anion us us us usua ua ua uall ll ll ll y y y y (oAs) species which have low retention under anion exchange HPLC-ICP-MS chromatograms, such as exchange HPLC-ICP-MS chromatograms, such as arsenolipids and AsHCs which were usually exchange HPLC-ICP-MS chromatograms, such as arsenolipids and AsHCs which were usually analy analy analy analyz z z zed ed ed ed using rever using rever using rever using revers ss sed ed ed ed-phase -phase -phase -phase HP HP HP HPLC-ICP-MS, c LC-ICP-MS, c LC-ICP-MS, c LC-ICP-MS, ca a a an n n n be be be be excluded excluded excluded excluded [28]. In [28]. In [28]. In [28]. In addi addi addi addittttiiiion, accor on, accor on, accor on, accord d d din in in ing g g g tttto o o o it it it its s s s arsenolipids and AsHCs which were usually analyzed using reversed-phase HPLC-ICP-MS, can be analyzed using reversed-phase HPLC-ICP-MS, can be excluded [28]. In addition, according to its analyzed using reversed-phase HPLC-ICP-MS, can be excluded [28]. In addition, according to its st st st stabil abil abil abilit it it ity un y un y un y unde de de der t r t r t r th h h he H e H e H e H2 2 2 2O O O O2 2 2 2 trea trea trea treatment, unprotected tri tment, unprotected tri tment, unprotected tri tment, unprotected triv v v va a a al ll lent oAs sp ent oAs sp ent oAs sp ent oAs spec ec ec ecies which c ies which c ies which c ies which ca a a an n n n be oxidized be oxidized be oxidized be oxidized, such as , such as , such as , such as excluded [28]. In addition, according to its stability under the H O treatment, unprotected trivalent 2 2 stability under the H2O2 treatment, unprotected trivalent oAs species which can be oxidized, such as Ш Ш Ш Ш Ш Ш Ш Ш V V V V stability under the H2O2 treatment, unprotected tri III valent oAs sp III ecies which can be oxidized, such as V DMA DMA DMA DMA , MM , MM , MM , MMA A A A , , , , and t and t and t and th h h hio io io io- - - -o o o organo rgano rgano rganoa a a ars rs rs rseni eni eni enic c c c (DM (DM (DM (DMM M M MTA TA TA TA ) ) ) ),,,, can can can can also be also be also be also be e e e ex x x xclude clude clude cluded d d d. Th . Th . Th . Thu u u us s s s, d , d , d , du u u ue to e to e to e to its c its c its c its ch h h hemical emical emical emical oAs species which can be oxidized, such as DMA , MMA , and thio-organoarsenic (DMMTA ), can Ш Ш V DMAШ, MMAШ, and thio-organoarsenic (DMMTA V), can also be excluded. Thus, due to its chemical DMA , MMA , and thio-organoarsenic (DMMTA ), can also be excluded. Thus, due to its chemical char char char characterist acterist acterist acteristic ic ic ics, the u s, the u s, the u s, the uA A A As s s s in in in in O. sine O. sine O. sine O. sinensis nsis nsis nsis ca ca ca cannot be the nnot be the nnot be the nnot be the toxi toxi toxi toxic oAs whi c oAs whi c oAs whi c oAs whic c c ch h h h ha ha ha have be ve be ve be ve been d en d en d en diiiis ss sc c c cover over over overe e e ed d d d s s s so o o o fa fa fa farrr r also be excluded. Thus, due to its chemical characteristics, the uAs in O. sinensis cannot be the toxic characteristics, the uAs in O. sinensis cannot be the toxic oAs which have been discovered so far characteristics, the uAs in O. sinensis cannot be the toxic oAs which have been discovered so far (Fi (Fi (Fi (Fig g g gure ure ure ure 5) 5) 5) 5). T . T . T . Th h h he chrom e chrom e chrom e chroma a a atttto o o ograp grap grap graphy hy hy hy b b b be e e eh h h ha a a av v v viiiior or or or indic indic indic indica a a atttte e e es ss s t t t th h h hat at at at it it it it m m m miiiigh gh gh ghtttt b b b be e e e an an an an arsen arsen arsen arseno o o osug sug sug suga a a arrr r((((s) s) s) s), w , w , w , wh h h hich ich ich ich are are are are oAs which have been discovered so far (Figure 5). The chromatography behavior indicates that it (Figure 5). The chromatography behavior indicates that it might be an arsenosugar(s), which are (Figure 5). The chromatography behavior indicates that it might be an arsenosugar(s), which are ШШ Ш frequently r frequently r frequently r frequently re e e eported to be ported to be ported to be ported to be co-elu co-elu co-elu co-eluted w ted w ted w ted wiiiith DMA th DMA th DMA th DMA,,,, AsB AsB AsB AsB,,,, MM MM MM MMA, A, A, A, an an an and d d d As As As As under under under under anion anion anion anion exch exch exch exchang ang ang ange e e e HP HP HP HPLC- LC- LC- LC- might be an arsenosugar(s), which are frequently reported to be co-eluted with DMA, AsB, MMA, frequently reported to be co-eluted with DMA, AsB, MMA, and AsШ under anion exchange HPLC- frequently reported to be co-eluted with DMA, AsB, MMA, and As under anion exchange HPLC- III IC IC IC ICP-MS P-MS P-MS P-MS chro chro chro chrom m m ma a a at t t to o o ogram gram gram grams [ s [ s [ s [2 2 2 29] 9] 9] 9].... and As under anion exchange HPLC-ICP-MS chromatograms [29]. ICP-MS chromatograms [29]. ICP-MS chromatograms [29]. Figure 3. The concentration (mg/kg dry weight) of total As and As speciation detected in O. sinensis. Figure 3. The concentration (mg/kg dry weight) of total As and As speciation detected in O. sinensis. Figure 3. Figure 3. Figure 3. Figure 3. The concentration (mg/k The concentration (mg/k The concentration (mg/k The concentration (mg/kg g g g dry weight) of dry weight) of dry weight) of dry weight) of total As and As total As and As total As and As total As and As specia specia specia speciat t t ti ii ion detected on detected on detected on detected in in in in O. O. O. O. sinensis sinensis sinensis sinensis. . . . Figure 3. The concentration (mg/kg dry weight) of total As and As speciation detected in O. sinensis. Ш V Inorganic As (█ AsШ and █ As V) are shown in red and yellow sections, and organic As (█ AsB and III Ш Ш V V V Inorganic As (█ As Ш Ш and █ As V V ) are shown in red and yellow sections, and organic As (█ AsB and In In Inor In Inorg org org org ganic a a a an n n niiiic A c A c A c A As s s s s ( ( ( ( (█ █ █ █ As As As As As and and and and and █ █ █ █ As As As As As ) are ) are ) are ) are ) are show show show show shown n in re n in re n in re n in re in red d and d and d and d and and y y y y yellow e e e ellow llow llow llow sec sec sec sec sections, t tt tions ions ions ions, a , a , a , a and n n n nd org d org d org d org organic a a a anic nic nic nic As As As As As ( (( ( (█ █ █ █ A A AsB A As s s sB and B and B and B and and █ uAs) is shown in dark and light gray sections. █ uAs) is shown in dark and light gray sections. █ █ █ █ uAs) u u u uA A A As) i s) i s) i s) i is s s s s shown show show show shown in dark n in dark n in dark n in dark in dark and and and and and li li li li light g g g gh h h ht g t g t g t g gray r r r ray ay ay ay sectio sectio sectio sectio sections. ns. ns. ns. ns. Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard samples V Ш V samples of AsB, DMA V, AsШ, MMA, and As V, at 10 ppb of each arsenic species. (B) The extracts of V III V samples of AsB, DMA , As , MMA, and As , at 10 ppb of each arsenic species. (B) The extracts of of AsB, DMA , As , MMA, and As , at 10 ppb of each arsenic species. (B) The extracts of sample Figure 4. Figure 4. Figure 4. Chromatograms Chromatograms Chromatograms obtai obtai obtain n ned ed ed i i in n n qu qu quan an antification tification tification by HPLC-IC by HPLC-IC by HPLC-ICP P P-M -M -MS. ( S. ( S. (A A A) A mi ) A mi ) A mix of s x of s x of stttand and andaaard rd rd Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard III NQ1. The As and unknown V V V V Ш ШШ Ш organic As peaks V V V V overlap. (C) Oxidation products of the extracts of NQ1. sam sam samp p ples of As les of As les of AsB, B, B, DMA DMA DMA , As , As , As , MMA , MMA , MMA, an , an , and A d A d As s s , at , at , at 10 ppb 10 ppb 10 ppb of of of each arseni each arseni each arsenic c c sp sp specie ecie ecies. s. s. ( ( (B B B) The ) The ) The e e ex x xtracts of tracts of tracts of samples of AsB, DMA , As , MMA, and As , at 10 ppb of each arsenic species. (B) The extracts of III V Any As is transformed into As when H O is added to the extracts. 2 2 Molecules 2018, 23, x FOR PEER REVIEW 7 of 14 sample NQ1. The As and unknown organic As peaks overlap. (C) Oxidation products of the extracts Molecules 2018, 23, 1012 7 of 14 Ш V of NQ1. Any As is transformed into As when H2O2 is added to the extracts. Figure 5. Inference on the toxicity of the unknown As detected in O. sinensis. (a) 1 mL of H O was Figure 5. Inference on the toxicity of the unknown As detected in O. sinensis. (a) 1 mL of H2O2 was 2 2 added to the extracts, and the unknown As could not be oxidized (Figure 4C). Thus, the unknown added to the extracts, and the unknown As could not be oxidized (Figure 4C). Thus, the unknown As III III V Ш Ш V As cannot be the toxic MMA , DMA , or DMMTA which can be oxidized under treatment with cannot be the toxic MMA , DMA , or DMMTA which can be oxidized under treatment with H2O2. III H O . (b) The unknown peak presents a similar retention time with As , indicating that it is not a low 2(b) The unknown peak prese 2 nts a similar retention time with As , indicating that it is not a low retention component, such as an As hydrocarbon (AsHC). retention component, such as an As hydrocarbon (AsHC). Table 4. Concentration of total arsenic and arsenic species in Ophiocordyceps sinensis. Table 4. Concentration of total arsenic and arsenic species in Ophiocordyceps sinensis. b Ш V b AsB uAs III As VAs iAs oAs Total As Sample AsB uAs As As iAs oAs Total As V V V V Sample Name DMA MMA DMA MMA Name mg/kg (%) mg/kg (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg NQ1 0.10 (2.1%) nd nd 4.34 (91.2%) 0.22 (4.6%) 0.10 (2.1%) 0.32 (6.7%) 4.44 (93.3%) 4.76 NQ1 0.10 (2.1%) nd nd 4.34 (91.2%) 0.22 (4.6%) 0.10 (2.1%) 0.32 (6.7%) 4.44 (93.3%) 4.76 NQ2 0.09 (1.8%) nd nd 4.59 (91.8%) 0.21 (4.2%) 0.11 (2.2%) 0.32 (6.4%) 4.68 (93.6%) 5.00 NQ2 0.09 (1.8%) nd nd 4.59 (91.8%) 0.21 (4.2%) 0.11 (2.2%) 0.32 (6.4%) 4.68 (93.6%) 5.00 NQ3 0.12 (2.3%) nd nd 4.81(91.6%) 0.25 (4.8%) 0.07 (1.3%) 0.32 (6.1%) 4.93 (93.9%) 5.25 NQ4 0.08 (2.0%) nd nd 3.69 (90.0%) 0.23 (5.6%) 0.10 (2.4%) 0.33 (8.0%) 3.77 (92.0%) 4.10 NQ3 0.12 (2.3%) nd nd 4.81(91.6%) 0.25 (4.8%) 0.07 (1.3%) 0.32 (6.1%) 4.93 (93.9%) 5.25 LT1 0.07 (1.7%) nd nd 3.69 (90.2%) 0.20 (4.9%) 0.13 (3.2%) 0.33 (8.1%) 3.76 (91.9%) 4.09 NQ4 0.08 (2.0%) nd nd 3.69 (90.0%) 0.23 (5.6%) 0.10 (2.4%) 0.33 (8.0%) 3.77 (92.0%) 4.10 LT2 0.09 (2.2%) nd nd 3.72 (89.6%) 0.22 (5.3%) 0.12 (2.9%) 0.34 (8.2%) 3.81 (91.8%) 4.15 LT3 0.11 (2.8%) nd nd 3.56 (89.0%) 0.24 (6.0%) 0.09 (2.3%) 0.33 (8.3%) 3.67 (91.7%) 4.00 LT1 0.07 (1.7%) nd nd 3.69 (90.2%) 0.20 (4.9%) 0.13 (3.2%) 0.33 (8.1%) 3.76 (91.9%) 4.09 LT4 0.09 (1.7%) nd nd 4.75 (92.2%) 0.21 (4.1%) 0.10 (1.9%) 0.31 (6.0%) 4.84 (94.0%) 5.15 LT2 0.09 (2.2%) nd nd 3.72 (89.6%) 0.22 (5.3%) 0.12 (2.9%) 0.34 (8.2%) 3.81 (91.8%) 4.15 YS1 0.12 (2.6%) nd nd 4.19 (89.3%) 0.27 (5.8%) 0.11 (2.3%) 0.38 (8.1%) 4.31 (91.9%) 4.69 YS2 0.08 (1.9%) nd nd 3.80 (90.5%) 0.24 (5.7%) 0.08 (1.9%) 0.32 (7.6%) 3.88 (92.4%) 4.20 LT3 0.11 (2.8%) nd nd 3.56 (89.0%) 0.24 (6.0%) 0.09 (2.3%) 0.33 (8.3%) 3.67 (91.7%) 4.00 YS3 0.07 (1.4%) nd nd 4.69 (92.3%) 0.25 (4.9%) 0.07 (1.4%) 0.32 (6.3%) 4.76 (93.7%) 5.08 LT4 0.09 (1.7%) nd nd 4.75 (92.2%) 0.21 (4.1%) 0.10 (1.9%) 0.31 (6.0%) 4.84 (94.0%) 5.15 YS4 0.15 (2.9%) nd nd 4.65 (90.5%) 0.22 (4.3%) 0.12 (2.3%) 0.34 (6.6%) 4.8 (93.4%) 5.14 0.09 (1.9%) nd nd 4.21 (90.9%) 0.23 (5.0%) 0.10 (2.2%) 0.33 (7.1%) 4.3 (92.9%) 4.63 AVR YS1 0.12 (2.6%) nd nd 4.19 (89.3%) 0.27 (5.8%) 0.11 (2.3%) 0.38 (8.1%) 4.31 (91.9%) 4.69 Concentrations are presented as the average value of three measurements with a relative standard deviation YS2 0.08 (1.9%) nd nd 3.80 (90.5%) 0.24 (5.7%) 0.08 (1.9%) 0.32 (7.6%) 3.88 (92.4%) 4.20 (RSD) of less than 8% (the ranges of RSD values were as follows, RSD : 2.1~5.6%, RSD : 1.8~6.1%, RSD : AsB DMA V As III YS3 0.07 (1.4%) nd nd 4.69 (92.3%) 0.25 (4.9%) 0.07 (1.4%) 0.32 (6.3%) 4.76 (93.7%) 5.08 2.0~6.7%, RSD : 1.4~5.2%, and RSD : 1.1~7.3%; AsB, MMA, DMA, uAs, AsIII, AsV, iAs, oAs, and Total As MMA V As V wereYS4 the abbreviat 0.15 (2 ion .9%) of arsenobetaine, nd nd monomethylarsonic 4.65 (90.5%) 0.22 (4 acid, .3%) dimethylarsenic 0.12 (2.3%) acid, 0.34 (6 unknown .6%) 4.8 (93 organic .4%) arsenic, 5.14 arsenite, and arsenate, inorganic arsenic (total), organic arsenic (total), and total arsenic, respectively. not detected; AVR 0.09 (1.9%) nd nd 4.21 (90.9%) 0.23 (5.0%) 0.10 (2.2%) 0.33 (7.1%) 4.3 (92.9%) 4.63 average value among all the samples. Concentrations are presented as the average value of three measurements with a relative standard deviation (RSD) of less than 8% (the ranges of RSD values were as follows, RSDAsB: 2.1~5.6%, RSDDMAV: 2.3. Hazard Risk Assessment of Long-Term O. sinensis Consumption 1.8~6.1%, RSDAsШ: 2.0~6.7%, RSDMMAV: 1.4~5.2%, and RSDAsV: 1.1%~7.3; AsB, MMA, DMA, uAs, AsⅢ, Because the toxicity of As species differs [17], it is especially important to determine the chemical AsⅤ, iAs, oAs, and Total As were the abbreviation of arsenobetaine, monomethylarsonic acid, form of As in O. sinensis samples, and a health risk assessment should focus on toxic As species dimethylarsenic acid, unknown organic arsenic, arsenite, and arsenate, inorganic arsenic (total), c d because of their carcinogenic potential rather than the total As content. Based on the As speciation organic arsenic (total), and total arsenic, respectively. not detected; average value among all the analysis sam method ples. of the latest Chinese national standard to determine As species in functional food (GB 16740-2014), in this work iAs and AsB were minor As components. Additionally, MMA and 2.3. Hazard Risk Assessment of Long-Term O. sinensis Consumption DMA were negligible, leaving the majority of As content as unknown organic As. Till the end of the 1990s, iAs was assumed as the toxic actor among all the As species, and oAs was assumed Because the toxicity of As species differs [17], it is especially important to determine the chemical less toxic or non-toxic. Thus, according to conventional opinion on the toxicity of As species, form of As in O. sinensis samples, and a health risk assessment should focus on toxic As species the negligible abundances of toxic As in O. sinensis show that the oral intake hazard might be lower because of their carcinogenic potential rather than the total As content. Based on the As speciation than in iAs-accumulated mushrooms, such as Laccaria amethystea [30], Collybia butyracea [31] and other analysis method of the latest Chinese national standard to determine As species in functional food mushrooms [32]. However, this assumption was questioned with the development of analytic methods. (GB 16740-2014), in this work iAs and AsB were minor As components. Additionally, MMA and Arsenic undergoes rapid and complicated metabolism in organisms, and several organic As species, DMA were negligible, leaving the majority of As content as unknown organic As. Till the end of the III III V discovered as intermediate metabolites (MMA , DMA , DMMTA ) in organisms or discovered in 1990s, iAs was assumed as the toxic actor among all the As species, and oAs was assumed less toxic animal-sourced foods, were found to be strongly toxic (AsHCs) [15–19]. In this study, although the or non-toxic. Thus, according to conventional opinion on the toxicity of As species, the negligible V V traditionally considered toxic As (iAs, DMA , MMA ) in O. sinensis is minor, the large amount of Molecules 2018, 23, 1012 8 of 14 organic As cannot be accurately determined for its speciation and thus its toxicity cannot be arbitrarily evaluated. In order to comprehensively assess long-term O. sinensis consumption, based on the intake of total As, i+uAs (considering uAs is toxic, as iAs), and iAs (consider uAs is non-toxic, as AsB) the average values of the estimated daily intake (EDI), hazard quotient (HQ), and cancer risk (CR) were calculated according to Equations (1)–(3), respectively. According to the intake of total As, the average of HQ = 2.6 and CR = 1.2  10 . total As total As A Monte-Carlo simulation was used to model the uncertainties of the EDI, HQ, and CR. After 10,000 iterations of each simulation, EDI, HQ, and CR were derived, and the distribution patterns are illustrated in Figure 6. In terms of the health risk associated with total As exposure, the average EDI (Figure 6A) from O. sinensis consumption is 8.00  10 mg/(kgday), the HQ > 1 total As total As (Figure 6B), and the mean, median, 5th percentile, and 95th percentile values of CR are total As 3 3 4 3 1.23  10 , 1.20  10 , 8.14  10 , and 1.78  10 , respectively (Figure 6A–C). According to the average value and uncertainty analysis of EDI, HQ, and CR, O. sinensis would not be recommended for long-time consumption, as assessed by the total As content (HQ > 1 and CR > 1  10 ). total As total As However, as we discussed before, applying total As content in the hazard risk assessment is unfair; the hazard risk assessment should be based on the concentration of toxic As species. However, the majority of the As species in O. sinensis were unknown using the current five arsenic standards, thus the toxicity is also unknown. Hence, we assess the edible hazard according to two postulated III situations. First, if the uAs is as toxic as As , based on the intake of i+uAs, HQ = 2.62 and i+uAs CR = 1.2  10 . In terms of the health risk associated with i+uAs exposure after the Monte-Carlo i+uAs simulation, the mean, median, 5th percentile, and 95th percentile values of CR are 1.18  10 , i+u As 3 3 3 1.18  10 , 1.00  10 , and 1.36  10 , respectively (Figure 6D–F). These results indicate a similar edible hazard as that predicted by the total As analysis. Second, if the uAs is non-toxic as AsB and AsC, the relative abundance of the toxic iAs is only 10% of the total As content, and, and correspondingly, 5 5 the average value (EDI = 5.95  10 mg/(kgday), HQ = 0.19 < 1, and CR = 8.7  10 < iAs iAs iAs 1  10 ) and uncertainty analysis of EDI, HQ, and CR were much lower and below the hazardous thresholds. The mean, median, 5th percentile, and 95th percentile values of CR were 8.93  10 , toxic As 5 5 4 8.66  10 , 5.83  10 , and 1.29  10 , respectively (Figure 6G–I). The CR value exceeded the threshold value (1  10 ) at the 72.7th percentile. Thus, if the uAs is non-toxic, the long-term consumption of O. sinensis is relatively safe according to the hazard analysis on iAs and the reputation of O. sinensis should be rehabilitated because of its non-toxicity. As we discussed in Section 2.2, the uAs is might be an arsenosugar, which are generally considered to be non-toxic or have low toxicity [29]. Thus, according to the current recognition on the toxicity of organic As species, the uAs might be non-toxic, and accordingly the long consumption of O. sinensis might be safe in terms of As speciation analysis. However, determination of the structure and toxicity of the uAs should be carried out to provide strong evidence for our inference. Although As has become notorious because some As species cause acute toxicity, chronic toxicity, and cancer via environmental exposure at low doses, its medicinal properties have been a focus in ancient China and Greece for over 3000 years [13]. Until the late 1940s, diseases such as psoriasis and syphilis were frequently treated with As [33]. More recently, inorganic arsenic has also shown significant activity in curing acute promyelocytic leukemia (APL) [13]. Inorganic arsenic has shown activity against other hematologic and solid organ malignancies but is also associated with serious toxicities, especially when applied at higher doses. For decades, scientists have attempted to seek ideal As compounds that are nontoxic and have medicinal potential. Few efforts have been successful, V V and the medical use of the most concerning forms of organic As (such as MMA , DMA , and AsB) has not been proven yet [33]. The recent development of orally bio-available organic arsenic derivatives (OAD) offering improved toxicity profiles and better efficacy may expand the application of arsenic compounds in hematologic malignancies and solid tumors [33,34]. O. sinensis has been massively reported to have predominant efficiency in anti-tumorigenesis, and most researchers consider this the result of chemical constituents such as cordycepin, adenosine, the exopolysaccharide Molecules 2018, 23, 1012 9 of 14 fraction, cordyglucans, and monosaccharide saponins [5]. From our newly obtained data, inorganic As comprised only 7% of the total As content, much lower than that (90%) of unknown organic As. Inspired by the above-mentioned progress in the medicinal use of organic arsenic derivatives (OAD) [33,34], we infer that the unknown As might play certain important roles in anti-tumorigenesis or other functional uses of O. sinensis. Therefore, combining the risk hazard analysis and potential functional speculation, As might be considered functional in O. sinensis instead of poisonous. Further research to provide sufficient evidence on the above speculation including structural identification, Molecules 2018, 23, x FOR PEER REVIEW 9 of 14 toxicology, and pharmacology assessments, especially animal testing, should be implicated in future. Figure Figure 6. 6. Estim Estimated ated distribution distribution patterns patterns and and d descriptive escriptive st statisti atistics cs of of ( (A A,,D D,,G G)) estimated estimated daily intake daily intake ((EDI EDI ), ), ( (B B ,E ,E ,H ,H )) hazar hazard quotient ( d quotient (HQ HQ ), ), an and d ( (C C ,F ,F ,I ,I )) cancer ri cancer risk sk( (CR CR ). ). Where in Where in ((A A– –C C)) values w values wer ere derived e derived accor according to ding to th thee total As concentration in total As concentration in O. O. si sinensis nensis;; in in ( (D D –– F F )) values were values were deriv derive ed d according to the according to the i+uAs i+uAs conc concentration entration (the su (the sum of m of iAs and iAs and uAs) u inAO. s) in sinensis O. sinensis ; and in; and in ( (G–I) values G–I wer ) values w e derived ere derived according to according the iAs concentration to the iAs concentra in O. sinensis tion in . O. sinensis. Although As has become notorious because some As species cause acute toxicity, chronic toxicity, and cancer via environmental exposure at low doses, its medicinal properties have been a focus in ancient China and Greece for over 3000 years [13]. Until the late 1940s, diseases such as psoriasis and syphilis were frequently treated with As [33]. More recently, inorganic arsenic has also shown significant activity in curing acute promyelocytic leukemia (APL) [13]. Inorganic arsenic has shown activity against other hematologic and solid organ malignancies but is also associated with serious toxicities, especially when applied at higher doses. For decades, scientists have attempted to seek ideal As compounds that are nontoxic and have medicinal potential. Few efforts have been V V successful, and the medical use of the most concerning forms of organic As (such as MMA , DMA , and AsB) has not been proven yet [33]. The recent development of orally bio-available organic arsenic derivatives (OAD) offering improved toxicity profiles and better efficacy may expand the application of arsenic compounds in hematologic malignancies and solid tumors [33,34]. O. sinensis has been massively reported to have predominant efficiency in anti-tumorigenesis, and most researchers Molecules 2018, 23, 1012 10 of 14 3. Materials and Methods 3.1. Reagents Deionized water obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used for the preparation of all solutions. Anhydrous sodium acetate, potassium nitrate, sodium dihydrogen phosphate, and disodium ethylenediaminetetraacetate (EDTA) were used for the preparation of mobile phases. H O (31%) and concentrated HNO (69%) were used for sample 2 2 3 digestion and extraction. Standards for the determination of both total As content and As speciation were prepared by diluting or compounding the standard stock solution sourced from the National Institute of Metrology (Beijing, China). The former was diluted to 10 mg As/L from the stock solution (As ) with certified concentration of 1000 mg As/L, and the latter was prepared with III V V V As (0.233 mol/g), As (1.011 mol/g), MMA (0.355 mol/g), DMA (0.706 mol/g), and AsB (0.518 mol/g). All stock solutions were kept at 4 C, and further dilutions for analysis were prepared daily. National Standard reference materials: GBW10049 (green Chinese onion, Institute of Geophysical and Geochemical Exploration, Langfang, China), GBW10051 (pork liver, Institute of Geophysical and Geochemical Exploration, Langfang, China), GBW08573 (yellow-fin tuna, Second Institute of Oceanography, Huangzhou, China) and GBW(E)100358 (rice, National Analysis Center for Ion and Steel, Beijing, China) were used as certified reference materials (CRMs) in this study. 3.2. Sample Collection and Preparation Three typical production areas (Litang, Naqu, and Yushu) were chosen as the sampling sites (Figure 2), and twenty O. sinensis samples of similar size (approximately 0.3 g per sample) were collected at each site in May 2017. All the samples were mechanically cleaned of soil, rinsed with deionized water and freeze-dried to reach a constant weight. To gather enough material for subsequent As analysis, each of the five O. sinensis samples was combined into a batch sample. Therefore, the original twenty O. sinensis taken at each sampling site were organized into 4 sample batches (referred to as NQ1–4, LT1–4 and YS1–4). These sample batches were individually pulverized using a mortar and pestle to reduce the particle size. Then, the fine powder was passed through a sieve with a pore size of 0.25 mm and stored in sealable plastic bags at 4 C until analysis. For total As content analysis, digestion of approximately 500 mg of sample powder was conducted in a microwave digestion system using concentrated HNO . The operating program of the microwave system was as follows: the samples were heated to 120 C in 5 min and held at 120 C for 5 min in the first step, heated to 150 C in 5 min and held at 150 C for 5 min in the second step, heated to 170 C in 5 min and held at 170 C for 5 min in the third step, and heated to 190 C in 5 min and held at 190 C for 20 min in the fourth step. Finally, the samples were cooled to room temperature. For As speciation analysis, approximately 1 g of sample powder was diluted with 20 mL 0.15 mol/L HNO in a 50 mL polyethylene centrifuge tube and then incubated in a 90 C water bath for 12 h. All the digestion products were centrifuged, filtered, and stored at 4 C before total As and As speciation analysis. 3.3. Total Arsenic Analysis The total As content of each sample was measured using ICP-MS (Agilent 7800, Santa Clara, CA, USA). The supernatant was first diluted with water up to 25 mL and then subjected to ICP-MS. The operating parameters of the equipment were as follows: radio frequency (RF) power, 1550 W; carrier gas, 1.05 L/min; collision mode, Helium (HE) flow 4.2 mL/min; plasma gas flow rate = 15 L/min; auxiliary gas flow rate = 0.1 L/min; and selected isotope = m/z 75. Samples were quantified using an external calibration curve from As standards (calibration points: 5, 10, 50, 100, and 200 ppm). Triplicate analyses were performed for each sample. For quality control purposes, the standards used for the calibration curve were run before and after each sample series. The corresponding digestion blanks (one for each sample digestion series) were also measured. Molecules 2018, 23, 1012 11 of 14 The total As in the CRMs (GBW10049, GBW10051, GBW08573 and GBW100358) were determined according the same methods. 3.4. Arsenic Speciation Analysis The separation and determination of As species were performed according to our previous III V V V HPLC-ICP-MS method [35]. Five As species (As , As , MMA , DMA , AsB) were separated by an Agilent 1260 HPLC system (Agilent, USA). The chromatograph was equipped with a standard autosampler, IonPac AG19 guard column (4  50 mm), and an IonPacAS19 separation column (4  250 mm). The main chromatography conditions used for HPLC were as follows: a mobile phase of 10 mmol/L anhydrous sodium acetate; 3 mmol/L potassium nitrate; 10 mmol/L sodium dihydrogen phosphate; 0.2 mmol/L disodium ethylenediaminetetraacetate buffer; a flow rate at 1.0 mL/min; a column temperature of room temperature; and an injection volume with a 50 L sample. The separated As species were examined by ICP-MS (as described previously) and identified by comparison of retention times with standards (Figure 4A). External calibration curves (calibration V V III V points: 0, 2.5, 5, 10, 50, and 100 ppb) were used to quantify MMA , DMA , As , As , and AB according to the corresponding standards. Triplicate analyses were performed for each sample. Extraction blanks were also analyzed by HPLC-ICP-MS in each work session. To check the extraction and digestion efficiency for the As species analysis, the As species extraction was completely measured using ICP-MS according to 3.3. Then the measured total As concentration in the As species extraction which was digested by 0.15 mol/L HNO , was compared with the total As digested by concentrated HNO to calculate the extraction efficiency. The iAs species in one CRM (GBW100358) were determined according the same methods. III Due to the complexity of the metrics of the O. sinensis sample, the As peak could not be separated from the other unknown peaks (Figure 4B); therefore, 1 mL of H O was added to the supernatant 2 2 III V before analysis. This operation completely oxidizes As into As (Figure 4C), without conversion of other arsenic species into new As compounds, according to the comparison of the number and the area of the corresponding peaks in Figure 4B and C. The inorganic As content was identified and quantified as As using external standards. The total organic As amounts were quantified according to the delta value between the total As and the inorganic As content. Thus, the content of the unknown As species was quantified according to the delta value between the organic As and the sum of the V V three determined As species (MMA , DMA , and AsB). 3.5. Health Risk Assessment The health risks for consumers with exposure to total toxic As (the sum of inorganic As, DMA, and MMA ), total As content, i+uAs, and iAs in O. sinensis were evaluated. According to the model of evaluation for health risks (Environment Protection Agency, EPA) [25,26], the estimated daily intake of As from O. sinensis consumption was calculated according to Equation (1): EDI = C  IR BW, (1) As where EDI (mg/kgday) is the estimated daily intake of As, C (mg/kg) is the concentration of total As As or toxic As in O. sinensis (dry weight), IR (0.01 kg/day) is the daily ingestion rate of O. sinensis [36], and BW is the average body weight (58.7 kg) [37] of the consumer. Risk characterization was quantified according to the potential non-carcinogenic risks of As exposure from O. sinensis ingestion and was reflected by the hazard quotient (HQ). The HQ value was calculated using equation (2). If HQ < 1, there might be no concern for non-carcinogenic effects. Otherwise, if the HQ exceeds 1, there might be serious concern for non-carcinogenic effects [25]. HQ = (EDI R f D)  (EF  ED AT) (2) Molecules 2018, 23, 1012 12 of 14 The probability of the excess lifetime cancer risk (CR) due to As exposure for O. sinensis consumption was estimated according to Equation (3). According to the US EPA, the incremental probability of cancer risk for consumers over a lifetime is characterized by CR with an acceptable 6 4 6 range of 1  10 –1  10 . A CR less than 1.0  10 indicates no obvious concern for cancer risk. However, a CR greater than 1.0  10 indicates an obvious potential cancer risk [25]. CR = EDI  SF  (EF  ED AT) (3) In Equations (2) and (3), RfD is the oral reference dose for As, EF (days/year) is the exposure frequency, ED (years) is the exposure duration, AT (days) is the average exposure time to As (usually AT is equal to the average life expectancy), and SF is the slope factor of As through oral intake. In this study, RfD, SF, ED, EF, and AT were determined to be 3  10 mg/(kgday) [26], 1.5 mg/(kgday) [26], 70 years, 365 days/year, and 25,550 days [37], respectively. To assess the long-term health risks of O. sinensis to arsenic exposure, the uncertainty of EDI, HQ, and CR were modeled through a Monte-Carlo simulation conducted with CrystalBall Software (V. 11.1.2.0.00, Oracle, Redwood Shores, CA, USA). The parameter distributions used in the model are presented in Table A1. 4. Patents A new method for risk assessment of Ophiocordyceps sinensis. Patent No. 201711087936.2. Document No. 2017110900896410. Author Contributions: L.-X.G. and Z.-G.H. conceived and designed the experiments; G.-W.Z. and J.-T.W. performed the experiments; Y.-P.Z. and L.-X.G. analyzed the data; Z.-G.H. contributed analysis tools; L.-X.G. wrote the paper. Acknowledgments: This work was jointly supported by the Excellent College Teachers Training Program of Guangdong Province, China (No. YQ2015084), the Natural Science Foundation of China (No. 81303155), and the Guangdong Provincial Science and Technology Plan Project (No. 2015A020208009). Conflicts of Interest: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Abbreviations O. sinensis Ophiocordyceps sinensis As Arsenic III As Arsenite As Arsenate DMA Dimethylarsinic acid MMA Methylarsonic acid AsB Arsenobetaine DMMTA Thio-organoarsenic uAs Unknown organic As oAs Organic arsenic iAs Inorganic arsenic i+uAs Sum of iAs and uAs LOQ Limits of quantification LOD Limits of detection EDI Estimated Daily Intake HQ Hazard Quotient CR Cancer Risk High-Performance Liquid Chromatography–Inductively Coupled Plasma Mass HPLC-ICP-MS Spectrometry Molecules 2018, 23, 1012 13 of 14 Appendix A Table A1. Input parameters for Monte-Carlo simulation. Parameters Units Distribution Adult Body weight, BW BW kg Lognormal (Mean = 58.7, SD = 12.0) Daily ingestion rate, IR kg/day Lognormal (Mean = 0.01, SD = 0.0143) Total As concentration, C g/kg Lognormal (Mean = 4.63, SD = 0.42) total As i+uAs concentration, C g/kg Lognormal (Mean = 4.53, SD = 0.41) i+uAs iAs concentration, C g/kg Lognormal (Mean = 0.34, SD = 0.03) iAs References 1. Dong, C.H.; Wen-Jia, L.I. Cordyceps industry in China: Current status, challenges and perspectives–Jinhu declaration for Cordyceps industry development. Mycosystema 2016, 35, 1–15. [CrossRef] 2. Guo, L.X.; Hong, Y.H. Fungus-larva relation in the formation of Cordyceps sinensis as revealed by stable carbon isotope analysis. Sci. Rep. 2017, 7, 7789. [CrossRef] [PubMed] 3. Martel, J.; Ko, Y.F. Myths and realities surrounding the mysterious caterpillar fungus. Trends Biotechnol. 2017, 35, 1017. [CrossRef] [PubMed] 4. Qi, W.; Lei, W. Pharmacological study progress of the Cordyceps sinensis. Global Tradit. Chin. Med. 2014, 7, 227–232. 5. Liu, Y.; Wang, J. The chemical constituents and pharmacological actions of Cordyceps sinensis. Evid. Based Complement. Altern. Med. 2015, 2015, 575063. 6. Caplins, L.; Halvorson, S. Collecting Ophiocordyceps sinensis: An emerging livelihood strategy in the garhwal, indian himalaya. Mt. Sci. 2017, 14, 390–402. [CrossRef] 7. Winkler, D. Yartsa gunbu (Cordyceps sinensis) and the fungal commodification of Tibet’s rural economy. Econ. Bot. 2008, 62, 291–305. [CrossRef] 8. Consumption Tips on Cordyceps sinensis Products. Available online: http://samr.cfda.gov.cn/WS01/CL1986/ 144020.html (accessed on 4 February 2016). 9. The Common Standards of Functional Food; GB16740-2014; National Health and Family Planning Commission of the People’s Republic of China: Beijing, China, 2014. 10. Notice on the Suspension of All the Pilot Work on Cordyceps sinensis for Health Food. Available online: http://samr.cfda.gov.cn/WS01/CL0847/146100.html (accessed on 4 March 2016). 11. Wang, H.; Shan, Y. The utilization and analysis of present market situation for Cordyceps sinensis. Res. Pract. Chin. Med. 2016, 30, 83–86. 12. Tsuda, T.; Babazono, A. Inorganic arsenic: A dangerous enigma for mankind. Appl. Organomet. Chem. 2010, 6, 309–322. [CrossRef] 13. Khairul, I.; Wang, Q.; Naranmandura, H. Metabolism, toxicity and anticancer activities of arsenic compounds. Oncotarget 2017, 8, 23905–23926. [CrossRef] [PubMed] 14. Cohen, S.M.; Arnold, L.L.; Eldan, M.; Lewis, A.S.; Beck, B.D. Methylated arsenicals: The implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit. Rev. Toxicol. 2006, 36, 99–133. [CrossRef] [PubMed] 15. Styblo, M.; Razo, L.M.D. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch. Toxicol. 2000, 74, 289. [CrossRef] [PubMed] 16. Huang, C.; Ke, Q. Molecular mechanisms of arsenic carcinogenesis. Mol. Cell. Biochem. 2004, 255, 57–66. [CrossRef] [PubMed] 17. Hughes, M.F. Arsenic toxicity and potential mechanisms of action. Toxicol. Lett. 2002, 133, 1–16. [CrossRef] 18. Hua, N.; Carew, M.W. Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells. Chem. Res. Toxicol. 2011, 24, 1586–1596. 19. Meyer, S.; Matissek, M.; Müller, S.M.; Taleshi, M.S.; Ebert, F.; Francesconi, K.A.; Schwerdtle, T. In vitro toxicological characterisation of three arsenic-containing hydrocarbons. Metallomics 2014, 6, 1023–1033. [CrossRef] [PubMed] 20. Hao, C.; Wang, G. Arsenic speciation analysis of 15 traditional Chinese medicines. Instrum. Anal. 2009, 28, 918–921. Molecules 2018, 23, 1012 14 of 14 21. Cao, X.; Wang, J. Analysis of arsenic speciation in Cordyceps sinensis in Tibet by HPLC-HG-AFS. Chin. Tradit. Patent Med. 2015, 37, 1985–1989. 22. Gómezariza, J.L.; Sánchezrodas, D. A comparison between ICP-MS and AFS detection for arsenic speciation in environmental samples. Talanta 2000, 51, 257. [CrossRef] 23. Popp, M.; Hann, S. Environmental application of elemental speciation analysis based on liquid or gas chromatography hyphenated to inductively coupled plasma mass spectrometry—A review. Anal. Chim. Acta 2010, 668, 114–129. [CrossRef] [PubMed] 24. Sankararamakrishnan, N.; Mishra, S. A Comprehensive Review on Various Analytical Methods for the Determination of Inorganic and Organic Arsenic in Environmental Samples. In Environmental Contaminants; Springer: Singapore, 2018. 25. U.S. EPA. Risk Assessment Guidance for Superfund (RAGS). In Human Health Evaluation Manual (Part A); EPA Publication: Washington, DC, USA, 1989. 26. U.S. EPA. Integrated Risk Information System (IRIS). In Arsenic, Inorganic (CASRN 7440-38-2)-748; EPA Publication: Washington, DC, USA, 2009. 27. Francesconi, K.A.; Goessler, W.; Panutrakul, S.; Irgolic, K.J. A novel arsenic containing riboside (arsenosugar) in three species of gastropod. Sci. Total Environ. 1998, 221, 139–148. [CrossRef] 28. Sele, V.; Sloth, J.J.; Holmelid, B.; Valdersnes, S.; Skov, K.; Amlund, H. Arsenic-containing fatty acids and hydrocarbons in marine oils—Determination using reversed-phase HPLC-ICP-MS and HPLC-qTOF-MS. Talanta 2014, 121, 89–96. [CrossRef] [PubMed] 29. Niegel, C.; Matysik, F.M. Analytical methods for the determination of arsenosugars—A review of recent trends and developments. Anal. Chim. Acta 2010, 657, 83. [CrossRef] [PubMed] 30. Larsen, E.; Hansen, M. Speciation and health risk considerations of arsenic in the edible mushroom Laccaria amethystina collected from contaminated and uncontaminated locations. Appl. Organomet. Chem. 1998, 12, 285–291. [CrossRef] 31. Kuehnelt, D.; Goessler, W. Arsenic compounds in terrestrial organisms I: Collybiamaculata, Collybiabutyracea and Amanita muscaria from arsenic smelter sites in Austria. Appl. Organomet. Chem. 1997, 11, 289–296. [CrossRef] 32. Chen, S.; Guo, Q. Determination of arsenic species in Edible mushrooms by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. Food Anal. Methods 2017, 10, 740–748. [CrossRef] 33. Quintás-Cardama, A.; Verstovsek, S. Chemical and clinical development of darinaparsin, a novel organic arsenic derivative. Anti-Cancer Agents Med. Chem. 2008, 8, 904. [CrossRef] 34. Kuntze, A.M.; Braz, O. Development of an organic arsenic derivative as a therapy for leukaemia. Spies Int. Symp. Opt. Sci. 2003, 2003, 347–355. 35. Tang, L.; Zhang, L.L. Arsenic speciation of dried Lentinus edodes by HPLC-ICP-MS. Food Ind. 2015, 36, 275–277. 36. Feng-Lei, X.U.; Wen-Ying, H. Clinical efficacy of Cordyceps sinensis for chronic kidney diseases: A systematic review. Chin. J. Evid. Based Med. 2006, 6, 804–808. 37. Ma, L.; Yang, Z.; Kong, Q.; Wang, L. Extraction and determination of arsenic species in leafy vegetables: Method development and application. Food Chem. 2017, 217, 524–530. [CrossRef] [PubMed] Sample Availability: Samples of the compounds AsB, DMA, MMA, AsIII, AsV and uAs are available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Molecules Multidisciplinary Digital Publishing Institute

Determination of Arsenic Species in Ophiocordyceps sinensis from Major Habitats in China by HPLC-ICP-MS and the Edible Hazard Assessment

Download PDF
14 pages

Loading next page...
 
/lp/multidisciplinary-digital-publishing-institute/determination-of-arsenic-species-in-ophiocordyceps-sinensis-from-major-wWU3omgQBO

References (38)

Publisher
Multidisciplinary Digital Publishing Institute
Copyright
© 1996-2019 MDPI (Basel, Switzerland) unless otherwise stated
ISSN
1420-3049
DOI
10.3390/molecules23051012
pmid
29701658
Publisher site
See Article on Publisher Site

Abstract

molecules Article Determination of Arsenic Species in Ophiocordyceps sinensis from Major Habitats in China by HPLC-ICP-MS and the Edible Hazard Assessment 1 , † 2 , † 1 1 Lian-Xian Guo , Gui-Wei Zhang , Jia-Ting Wang , Yue-Ping Zhong and 1 , Zhi-Gang Huang * Dongguan Key Laboratory of Environmental Medicine, School of Public Health, Guangdong Medical University, Dongguan 523808, China; [email protected] (L.-X.G.); [email protected] (J.-T.W.); [email protected] (Y.-P.Z.) Shenzhen Academy of Metrology and Quality Inspection, Shenzhen 518000, China; [email protected] * Correspondence: [email protected]; Tel.: +86-0769-22896573 † These authors contributed equally to this work. Received: 2 April 2018; Accepted: 24 April 2018; Published: 26 April 2018 Abstract: This study sought to determine the concentration and distribution of arsenic (As) species in Ophiocordyceps sinensis (O. sinensis), and to assess its edible hazard for long term consumption. The total arsenic concentrations, measured through inductively coupled plasma mass spectrometry (ICP-MS), ranged from 4.00 mg/kg to 5.25 mg/kg. As determined by HPLC-ICP-MS, the most V V V III V concerning arsenic species—AsB, MMA , DMA , As , and As —were either not detected (MMA V V III and DMA ) or were detected as minor As species (AsB: 1.4–2.9%; As : 1.3–3.2%, and As : 4.1–6.0%). III The major components were a cluster of unknown organic As (uAs) compounds with As , which accounted for 91.7–94.0% of the As content. Based on the H O test and the chromatography 2 2 behavior, it can be inferred that, the uAs might not be toxic organic As. Estimated daily intake (EDI), hazard quotient (HQ), and cancer risk (CR) caused by the total As content; the sum of inorganic As (iAs) and uAs, namely i+uAs; and iAs exposure from long term O. sinensis consumption were calculated and evaluated through equations from the US Environmental Protection Agency and the uncertainties were analyzed by Monte-Carlo Simulation (MCS). EDI and EDI are total As i+uAs approximately ten times more than EDI ; HQ and HQ > 1 while HQ < 1; and CR iAs As i+u As i As total total As 4 4 and CR > 1  10 while CR < 1  10 . Thus, if the uAs is non-toxic, there is no particular i+uAs iAs risk to local consumers and the carcinogenic risk is acceptable for consumption of O. sinensis because the concentration of toxic iAs is very low. Keywords: Ophiocordyceps sinensis; arsenic speciation; risk assessment; HPLC-ICP-MS 1. Introduction Ophiocordyceps sinensis (O. sinensis), a mysterious entomogenous fungus distributed on the Qinghai–Tibet Plateau, is popularly referred to as winter-worm-summer-grass (Dong Chong Xia Cao in Chinese) [1,2] (Figure 1). O. sinensis has been utilized in China (Figure 2A) and surrounding countries for more than 2000 years as a rare functional food to promote health and to treat diverse chronic diseases. In 1993, O. sinensis became famous worldwide because Chinese female athletes broke several world records in running events at the National Games and the meritorious performances were later attributed (at least in part) to the consumption of this fungus [3]. Subsequently, modern pharmaceutical research has shown that its predominant functions are anti-tumor, anti-inflammatory, nephroprotective, Molecules 2018, 23, 1012; doi:10.3390/molecules23051012 www.mdpi.com/journal/molecules Molecules 2018, 23, 1012 2 of 14 antioxidant, antihyperglycemic, anti-apoptosis, immunoregulatory, and hepatoprotective [4,5]. Accordingly, these pronounced medicinal functions have resulted in a large demand for wild O. sinensis and have also increased the local prosperity of economically poor production areas around the Molecules 2018, 23, x FOR PEER REVIEW 2 of 14 Qinghai–Tibet Plateau and in adjacent countries. Some households earn as much as two-thirds of economically poor production areas around the Qinghai–Tibet Plateau and in adjacent countries. their income from the collection of O. sinensis (Figure 2B), and pastoralists are earning an income of a Some households earn as much as two-thirds of their income from the collection of O. sinensis (Figure size unrecorded in their history [6,7]. However, concerns about human health have arisen since the 2B), and pastoralists are earning an income of a size unrecorded in their history [6,7]. However, CFDA (China Food and Drug Administration) revealed in 2016 that excessive Arsenic (As) content concerns about human health have arisen since the CFDA (China Food and Drug Administration) (total As: 4.4–9.0 mg/kg [8]) was detected in O. sinensis, a level five times the reference value of revealed in 2016 that excessive Arsenic (As) content (total As: 4.4–9.0 mg/kg [8]) was detected in O. 1 mg/kg for total As in functional foods (GB16740-2014) [9]. Subsequently, the CFDA ordered that all sinensis, a level five times the reference value of 1 mg/kg for total As in functional foods (GB16740- the pilot work on O. sinensis as a functional food be discontinued on 26 February 2016 [10]. Moreover, 2014) [9]. Subsequently, the CFDA ordered that all the pilot work on O. sinensis as a functional food numerous media outlets had arbitrarily declared that O. sinensis is a poison instead of a functional be discontinued on 26 February 2016 [10]. Moreover, numerous media outlets had arbitrarily declared food. This assertion has caused a great uproar in the health food market and has seriously affected the that O. sinensis is a poison instead of a functional food. This assertion has caused a great uproar in O. sinensis-dependent economic chain [11]. the health food market and has seriously affected the O. sinensis-dependent economic chain [11]. Figure 1. Life history of Ophiocordyceps sinensis (O. sinensis). (a) The eggs of the host Thitarodes insect, Figure 1. Life history of Ophiocordyceps sinensis (O. sinensis). (a) The eggs of the host Thitarodes insect, which are scattered on the grassland, incubate; (b) The host larvae safely reside in the soil throughout which are scattered on the grassland, incubate; (b) The host larvae safely reside in the soil throughout the long-lasting larval stage; (c) The ascospores erupt from mature fruiting bodies of O. sinensis; (d) the long-lasting larval stage; (c) The ascospores erupt from mature fruiting bodies of O. sinensis; The 4–5th instar larvae may be infected by the infective conidia of the O. sinensis fungus in the soil; (d) The 4–5th instar larvae may be infected by the infective conidia of the O. sinensis fungus in the soil; (e) The caterpillar-shaped sclerotium (winter-worm) is formed; (f) The stroma germinates out of the (e) The caterpillar-shaped sclerotium (winter-worm) is formed; (f) The stroma germinates out of the head capsule and the mature O. sinensis (summer-grass-winter-worm) is formed. head capsule and the mature O. sinensis (summer-grass-winter-worm) is formed. Arsenic can be present in both organic and inorganic forms. The toxicity of As is dependent on Ш V its chemical form, with inorganic species (iAs), such as arsenite (As ) and arsenate (As ), being the Arsenic can be present in both organic and inorganic forms. The toxicity of As is dependent on its III V most toxic [12]. Organic species are the metabolic products of iAs, such as monomethylarsonic acid chemical form, with inorganic species (iAs), such as arsenite (As ) and arsenate (As ), being the most V V (MMA ) and dimethylarsenic acid (DMA ), and are much less toxic than iAs to humans. toxic [12]. Organic species are the metabolic products of iAs, such as monomethylarsonic acid (MMA ) Additionally, some other organic As complexes (arsenocholine, arsenobetaine, various arsenosugars and dimethylarsenic acid (DMA ), and are much less toxic than iAs to humans. Additionally, some and arsenolipids) are generally considered nontoxic [13], according to previous studies on As toxicity. other organic As complexes (arsenocholine, arsenobetaine, various arsenosugars and arsenolipids) are However, recently, experimental results have documented the presence of trivalent intermediates, generally considered nontoxic [13], according to previous studies on As toxicity. However, recently, Ш Ш monomethylarsonous acid (MMA ) and dimethylarsinous acid (DMA ) in the urine of humans experimental results have documented the presence of trivalent intermediates, monomethylarsonous exposed to drinking water containing high levels of inorganic As [14]. These trivalent intermediates III III acid (MMA ) and dimethylarsinous acid (DMA ) in the urine of humans exposed to drinking are structurally different from the pentavalent compounds and are more reactive and more water containing high levels of inorganic As [14]. These trivalent intermediates are structurally carcinogenic [15–17]. More recently, the subsequent metabolic products of DMA , sulfur-containing different from the pentavalent compounds and are more reactive and more carcinogenic [15–17]. intermediary metabolites (dimethylmonothioarsinicacid, DMMTA )[18], and several As containing hydrocarbons (AsHC 332, AsHC 360 and AsHC 444) were shown to high toxic effects on organisms [19]. The different toxicities of As species reinforce the importance of distinguishing its chemical Molecules 2018, 23, 1012 3 of 14 III More recently, the subsequent metabolic products of DMA , sulfur-containing intermediary metabolites (dimethylmonothioarsinicacid, DMMTA ) [18], and several As containing hydrocarbons (AsHC 332, AsHC 360 and AsHC 444) were shown to high toxic effects on organisms [19]. The different toxicities of As species reinforce the importance of distinguishing its chemical form, as the total amount Molecules 2018, 23, x FOR PEER REVIEW 3 of 14 of As does not provide enough information about the toxicity of the analyzed sample. Therefore, it is incorrect to consider O. sinensis is toxic according to its total As content. Unlike previous reported form, as the total amount of As does not provide enough information about the toxicity of the arsenic accumulated mushrooms, which germinated on plant sourced media, O. sinensis only lives analyzed sample. Therefore, it is incorrect to consider O. sinensis is toxic according to its total As in Thitarodes larva of 4–5th instar. Inspired by the significantly higher organic As proportion in content. Unlike previous reported arsenic accumulated mushrooms, which germinated on plant animal-sourced traditional Chinese medicine (TCM) compared to plant-sourced TCM [20], some sourced media, O. sinensis only lives in Thitarodes larva of 4–5th instar. Inspired by the significantly scholars have proposed that due to larva-fungus complexity (Figure 2C,D), O. sinensis might contain higher organic As proportion in animal-sourced traditional Chinese medicine (TCM) compared to a lar plge ant- pr sou oprortion ced TCM of [20 organic ], some As. scRecently holars ha ,ve resear propos chers ed tha have t dpr ue to la ovided rva- evidence fungus compl for the exispeculation ty (Figure III V that 2C iAs ,D),species O. sinensis wer might co e minor ntain (As a large and proportion of As ) or below organ the ilevel c As. Recently, of detection reseafter archers h examining ave provided the As Ш V evidence for the speculation that iAs species were minor (As and As ) or below the level of detection speciation in O. sinensis through high-performance liquid chromatography-hydride generation-atomic after examining the As speciation in O. sinensis through high-performance liquid chromatography- fluorescence spectrometry (HPLC-HG-AFS). In addition, they found that the largest proportion of hydride generation-atomic fluorescence spectrometry (HPLC-HG-AFS). In addition, they found that As was composed of an unknown organic As (uAs) species [21]. Because of the shortcomings of V V the largest proportion of As was composed of an unknown organic As (uAs) species [21]. Because of conventional HPLC-HG-AFS, in that research, they only discriminated four As species (MMA , DMA , V the shortcomings of III conventional HPLC-HG-AFS, in that research, they only discriminated four As As and As ) in O. sinensis samples [21]. Non-forming hydride species, such as AsB, important in V V V Ш species (MMA , DMA , As and As ) in O. sinensis samples [21]. Non-forming hydride species, such biota samples, cannot be evaluated using this approach [22]. as AsB, important in biota samples, cannot be evaluated using this approach [22]. Figure 2. The producing area of Ophiocordyceps sinensis in China and sampling details of this study. Figure 2. The producing area of Ophiocordyceps sinensis in China and sampling details of this study. (A) Schematic map illustrating the sampling sites in the Qinghai–Tibetan Plateau and its adjacent (A) Schematic map illustrating the sampling sites in the Qinghai–Tibetan Plateau and its adjacent high-altitude areas. Litang (LT), Naqu (NQ), and Yushu (YS) were chosen as the sampling sites. high-altitude areas. Litang (LT), Naqu (NQ), and Yushu (YS) were chosen as the sampling sites. (B) (B) The grassland of the O. sinensis habitat. Pastoralists are encamped there to collect it. (C) The stroma The grassland of the O. sinensis habitat. Pastoralists are encamped there to collect it. (C) The stroma (a) of O. sinensis emerged out of the ground. (D) O. sinensis in the soil, the yellow arrows pointing out (a) of O. sinensis emerged out of the ground. (D) O. sinensis in the soil, the yellow arrows pointing out its sclerotium (b) and stroma (c). its sclerotium (b) and stroma (c). To further clarify the As speciation in O. sinensis and oral intake hazards in longtime consumption, the present study examined the total As content, including the most concerning As Ш V V V species (As , As MMA , DMA , and AsB), in O. sinensis using the most commonly applied techniques: inductively coupled plasma mass spectrometry (ICP-MS) and anion exchange high- Molecules 2018, 23, 1012 4 of 14 To further clarify the As speciation in O. sinensis and oral intake hazards in longtime consumption, III the present study examined the total As content, including the most concerning As species (As , V V V As MMA , DMA , and AsB), in O. sinensis using the most commonly applied techniques: inductively coupled plasma mass spectrometry (ICP-MS) and anion exchange high-performance liquid chromatography–inductively coupled plasma mass spectrometry (HPLC-ICP-MS) [23,24]. Due to the extraordinary prices of the O. sinensis and chronic injury effects to organisms due to Arsenic consumption, a hazard assessment via experimental animal trials would be costly. Thus, in this study, we chose the most widely accepted model (Environment Protection Agency, EPA) of evaluation for health risks [25,26] to evaluate the potential non-carcinogenic risk and the probability of excess lifetime cancer risk due to As exposure from O. sinensis consumption. Additionally, Monte-Carlo Simulation (MCS) were employed to analyze the uncertainty. The health risk posed by O. sinensis consumption from exposure to total As, iAs, and i+uAs (iAs and uAs together) were comparatively estimated in this study. These contributions might be useful in efforts to revive the local O. sinensis-dependent economy in the Qinghai–Tibet Plateau and surrounding countries by providing a comprehensive edible hazard evaluation of O. sinensis. 2. Results and Discussion 2.1. Analytical Performances of the Proposed Method Extraction efficiency for As speciation analysis was assessed based on a comparison between the total As concentration in 0.15 mol/L HNO extracts and concentrated HNO extracts using a 3 3 microwave digestion system (see Section 3.2). The concentrations of the total As in the extracts through different extraction methods were quantitively consistent, with an extraction efficiency of 92.3–104% (the ratio of total As concentration in 0.15 mol/L HNO extracts to that in concentrated HNO extracts). 3 3 This efficiency indicates that most As species were thoroughly extracted through the current method (see Section 3.2). The analytical performances of the proposed ICP-MS, for total As content analysis, and HPLC-ICP-MS, for As species analysis, methods were validated by determining the linearity, limits of detection (LOD), and limits of quantification (LOQ) as shown in Table 1. The linear correlation 1 1 coefficients, in the range of 0.5–500 g L (for total As) and 0.2–300 g L (for As species), were V III V V greater than 0.9997. LOD of total As, AsB, DMA , As , MMA , and As were 2.3, 1.1, 1.3, 1.0, 2.2, 1 1 and 1.1 g kg , respectively, And the LOQ were found to be in the range of 3.0–6.9 g kg for the total As content and the five arsenic species. The recoveries of the total As content were in the range 92.3106.6% and the relative standard deviations (RSDs) were in the range 2.4~4.6%. The recoveries and RSDs of arsenic species were studied by spiking three concentration levels of the five arsenic species into the O. sinensis samples. The recoveries of arsenic species were in the range 86.3~111.7% and the RSDs were in the range 0.8~6.6%, as shown in Table 2. The analysis results of certified reference materials (CRM) using the same method were in good agreement with their certified values and the recoveries were 97.5–101% for total As analysis and 110.8% for iAs analysis as shown in Table 3. Table 1. Analytical performance of inductively coupled plasma mass spectrometry (ICP-MS) for total arsenic content and HPLC-ICP-MS for arsenic species. Analytes Linear Range (g/L) Linear Equation R LOD (g/kg) LOQ (g/kg) Total As 0.5–500 y = 2731.03x + 5.5567 0.9998 2.3 6.9 AsB 0.2–300 y = 18,867.9x + 2319.6 0.9999 1.1 3.3 DMA 0.2–300 y = 19,212.4x + 4065.2 0.9999 1.3 4.0 III As 0.2–300 y = 17,090.6x + 3289.1 0.9997 1.0 3.0 0.5–300 y = 18,373.6x + 196.8 1.0000 2.2 6.6 MMA As 0.2–300 y = 18,504.6x + 8247.2 1.0000 1.1 3.3 Molecules 2018, 23, 1012 5 of 14 Table 2. Recovery and precision of the methods. Background Value Added Measured Value Recovery RSD Analytes (mg/kg) (g/L) (g/L) (%) (%, n = 6) 5.00 14.8~15.3 92.3~99.4 4.6 10.0 20.4~21.5 94.7~102.3 3.8 Total As 0.51 50.0 60.7~64.3 95.8~106.6 2.4 2.00 2.09~2.26 91.7~100.1 4.2 10.0 9.45~9.87 91.9~96.2 1.9 AsB 0.010 50.0 42.0~47.0 83.6~93.4 5.0 2.00 1.73~1.98 86.5~99.0 5.7 10.0 9.20~9.55 92.0~95.5 1.5 ND DMA 50.0 50.4~52.6 100.9~105.2 1.7 2.00 45.3~45.6 86.3~99.2 5.5 III 10.0 52.8~53.7 88.8~98.0 4.3 1.70 As 50.0 96.8~99.2 106.9~111.7 2.0 2.00 2.07~2.19 99.4~105.7 2.7 0.0031 10.0 10.6~10.8 105.0~107.2 0.85 MMA 50.0 47.2~48.6 94.2~97.1 1.2 2.00 4.81~5.08 86.6~100.0 6.6 0.12 10.0 12.8~13.0 97.4~99.4 0.80 As 50.0 49.0~50.2 91.9~94.4 1.1 Table 3. National standard reference materials values (mg/kg, mean  standard deviation) and determined values for total As and inorganic arsenic (iAs) content (n = 5). Certified Value Determined Value Recovery Sample Type Reference Materials (mg/kg) (mg/kg) (%) Green Chinese onion GBW10049 0.52  0.11 0.507  0.08 97.5 Pork liver GBW10051 1.4  0.3 1.42  0.15 101.4 Yellow-fin tuna GBW08573 5.08  0.39 4.98  0.11 98.0 0.16  0.02 (total As) 0.165  0.012 103.1 Rice GBW100358 0.13  0.02 (iAs) 0.144  0.006 110.8 2.2. Total Arsenic Concentration and Arsenic Species in O. sinensis Samples The total As content in O. sinensis ranged from 4.00 mg/kg to 5.25 mg/kg of dry mass (Table 4), which is consistent with that in the previous studies [21] that caused uneasiness and fear in consumers. The Chinese government, through the National Health and Family Planning Commission of the People’s Republic of China, established a reference value of 1 mg/kg for total As in functional foods (GB16740-2014) [9]. Thus, the total As content in O. sinensis exceeds the limit of As in functional foods in China (Figure 3). These data demonstrate that there is an urgent need to determine As speciation in O. sinensis samples, because the total amount of As does not provide sufficient toxicological information. III In Figure 4B, the large peak area indicates that As , which is generally considered most toxic, might be the major As species in O. sinensis, however, an H O test proved that most of the As is not oxidized 2 2 V III to As (Figure 4C) and hence the major overlapped peak cannot be the toxic As . Thus, according to current chromatography conditions, an H O test is necessary to minimize misidentification. The iAs, 2 2 calculated according to the As content in Figure 4C, is relatively abundant compared to the total As content, in the range 6.0% to 8.3% (the amount of As in Figure 4C), but is small compared with the amount of organic As. Organic As species were predominant, and the percentage of total As content V V ranged from 91.7% to 94.0%. The two potential toxic organic As species, MMA and DMA , were V V found to be negligible. Because the initial organic metabolites of iAs, MMA , and DMA were trace in O. sinensis sample, it can be inferred that they were transformed into other organic arsenic metabolites in the organism such as AsB (which was also detected as a minority compound in these samples, ranging from 1.4% to 2.9%) and various unknown organic As species (uAs, a cluster of unknown compounds, with a retention time from 3.2 to 4 min, representing 89.0% to 92.3% of the of the total As content, Molecules Molecules Molecules Molecules 2018 2018 2018 2018, , , , 23 23 23 23, x FO , x FO , x FO , x FOR P R P R P R PEER EER EER EER R R R RE E E EVIEW VIEW VIEW VIEW 6 of 6 of 6 of 6 of 14 14 14 14 Molecules 2018, 23, 1012 6 of 14 Molecules 2018, 23, x FOR PEER REVIEW 6 of 14 Molecules 2018, 23, x FOR PEER REVIEW 6 of 14 8 8 8 89 9 9 9.0 .0 .0 .0% to 92 % to 92 % to 92 % to 92.3% of .3% of .3% of .3% of the of the tota the of the tota the of the tota the of the totallll As content, Fi As content, Fi As content, Fi As content, Figu gu gu gure re re re 4C). B 4C). B 4C). B 4C). Ba a a ased on sed on sed on sed on previous studies, comparison of previous studies, comparison of previous studies, comparison of previous studies, comparison of the retenti the retenti the retenti the retentio o o on ti n ti n ti n time beha me beha me beha me behavi vi vi vior of or of or of or of st st st sta a a an n n nda da da dards wi rds wi rds wi rds with tha th tha th tha th thattt t of the extra of the extra of the extra of the extrac c c cts ts ts ts upon cha upon cha upon cha upon chan n n nging pH of ging pH of ging pH of ging pH of the the the the mobi mobi mobi mobile le le le 89.0% to 92.3% of the of the total As content, Figure 4C). Based on previous studies, comparison of Figure 4C). Based on previous studies, comparison of the retention time behavior of standards with 89.0% to 92.3% of the of the total As content, Figure 4C). Based on previous studies, comparison of the retention time behavior of standards with that of the extracts upon changing pH of the mobile p p p ph h h hase c ase c ase c ase ca a a an n n n asc asc asc asce e e ert rt rt rtain t ain t ain t ain th h h he p e p e p e pr r r re e e es s s sence of ence of ence of ence of an an an an e e e ex x x xp p p pe e e ect ct ct cted co ed co ed co ed compound or giv mpound or giv mpound or giv mpound or give e e e useful useful useful useful in in in information on th formation on th formation on th formation on the nature e nature e nature e nature that the retenti of the extracts on time beha upon changing vior of stapH ndards wi of the th tha mobile t of the extra phase can c ascert ts upon cha ain thenpr ging pH of esence of the an expected mobile phase can ascertain the presence of an expected compound or give useful information on the nature of unknown of unknown of unknown of unknown c c c co o o ompounds [27]. In this mpounds [27]. In this mpounds [27]. In this mpounds [27]. In this stu stu stu stud d d dy, de y, de y, de y, despite th spite th spite th spite the lack of stan e lack of stan e lack of stan e lack of standa da da dards f rds f rds f rds fo o o or the r the r the r the conf conf conf confi i i ir r r rm m m ma a a ati ti ti tion on on on of of of of uAs, uAs, uAs, uAs, compound phase can or asc give ertain t useful he prinformation esence of an eon xpethe cted co natur mpound or giv e of unknown e useful compounds information on th [27]. In this e nature study, Ш Ш Ш Ш of unknown compounds [27]. In this study, despite the lack of standards for the confirmaIII tion of uAs, based on the overlap w based on the overlap w based on the overlap w based on the overlap w of unknown compounds [27]. In this iiiith As th As th As th As , or , or , or , organ gan gan gan stu iiiic As c As c As c As dy, de(oA (oA (oA (oA spite th s ss s) s ) s ) s ) sp p p p e lack of stan ecies wh ecies wh ecies wh ecies which ich ich ich da have low r have low r have low r have low r rds for the conf e e e etttte e e ent nt nt nt irion unde ion unde ion unde ion unde mation ofr anion r anion r anion r anion uAs, despite the lack of standards for the confirmation of uAs, based on the overlap with As , organic As based on the overlap with AsШ, organic As (oAs) species which have low retention under anion exchange exchange exchange exchange based on the overlap w HP HP HP HPLC LC LC LC-IC -IC -IC -ICP P P P-M -M -M -MS c S c S c S c ith As h h h hrom rom rom romaaa a, or tttto o o ogr gr gr gr gan am am am am ic As s, s s, s s, s s, su u u uch ch (oA ch ch as as as as s) sar ar ar ar pecies wh senol senol senol senoliiiip p p piiiids ds ds ds ich an an an an have low r d AsHC d AsHC d AsHC d AsHCes w s w s w s w tenth h h hion unde ich were ich were ich were ich were r anion us us us usua ua ua uall ll ll ll y y y y (oAs) species which have low retention under anion exchange HPLC-ICP-MS chromatograms, such as exchange HPLC-ICP-MS chromatograms, such as arsenolipids and AsHCs which were usually exchange HPLC-ICP-MS chromatograms, such as arsenolipids and AsHCs which were usually analy analy analy analyz z z zed ed ed ed using rever using rever using rever using revers ss sed ed ed ed-phase -phase -phase -phase HP HP HP HPLC-ICP-MS, c LC-ICP-MS, c LC-ICP-MS, c LC-ICP-MS, ca a a an n n n be be be be excluded excluded excluded excluded [28]. In [28]. In [28]. In [28]. In addi addi addi addittttiiiion, accor on, accor on, accor on, accord d d din in in ing g g g tttto o o o it it it its s s s arsenolipids and AsHCs which were usually analyzed using reversed-phase HPLC-ICP-MS, can be analyzed using reversed-phase HPLC-ICP-MS, can be excluded [28]. In addition, according to its analyzed using reversed-phase HPLC-ICP-MS, can be excluded [28]. In addition, according to its st st st stabil abil abil abilit it it ity un y un y un y unde de de der t r t r t r th h h he H e H e H e H2 2 2 2O O O O2 2 2 2 trea trea trea treatment, unprotected tri tment, unprotected tri tment, unprotected tri tment, unprotected triv v v va a a al ll lent oAs sp ent oAs sp ent oAs sp ent oAs spec ec ec ecies which c ies which c ies which c ies which ca a a an n n n be oxidized be oxidized be oxidized be oxidized, such as , such as , such as , such as excluded [28]. In addition, according to its stability under the H O treatment, unprotected trivalent 2 2 stability under the H2O2 treatment, unprotected trivalent oAs species which can be oxidized, such as Ш Ш Ш Ш Ш Ш Ш Ш V V V V stability under the H2O2 treatment, unprotected tri III valent oAs sp III ecies which can be oxidized, such as V DMA DMA DMA DMA , MM , MM , MM , MMA A A A , , , , and t and t and t and th h h hio io io io- - - -o o o organo rgano rgano rganoa a a ars rs rs rseni eni eni enic c c c (DM (DM (DM (DMM M M MTA TA TA TA ) ) ) ),,,, can can can can also be also be also be also be e e e ex x x xclude clude clude cluded d d d. Th . Th . Th . Thu u u us s s s, d , d , d , du u u ue to e to e to e to its c its c its c its ch h h hemical emical emical emical oAs species which can be oxidized, such as DMA , MMA , and thio-organoarsenic (DMMTA ), can Ш Ш V DMAШ, MMAШ, and thio-organoarsenic (DMMTA V), can also be excluded. Thus, due to its chemical DMA , MMA , and thio-organoarsenic (DMMTA ), can also be excluded. Thus, due to its chemical char char char characterist acterist acterist acteristic ic ic ics, the u s, the u s, the u s, the uA A A As s s s in in in in O. sine O. sine O. sine O. sinensis nsis nsis nsis ca ca ca cannot be the nnot be the nnot be the nnot be the toxi toxi toxi toxic oAs whi c oAs whi c oAs whi c oAs whic c c ch h h h ha ha ha have be ve be ve be ve been d en d en d en diiiis ss sc c c cover over over overe e e ed d d d s s s so o o o fa fa fa farrr r also be excluded. Thus, due to its chemical characteristics, the uAs in O. sinensis cannot be the toxic characteristics, the uAs in O. sinensis cannot be the toxic oAs which have been discovered so far characteristics, the uAs in O. sinensis cannot be the toxic oAs which have been discovered so far (Fi (Fi (Fi (Fig g g gure ure ure ure 5) 5) 5) 5). T . T . T . Th h h he chrom e chrom e chrom e chroma a a atttto o o ograp grap grap graphy hy hy hy b b b be e e eh h h ha a a av v v viiiior or or or indic indic indic indica a a atttte e e es ss s t t t th h h hat at at at it it it it m m m miiiigh gh gh ghtttt b b b be e e e an an an an arsen arsen arsen arseno o o osug sug sug suga a a arrr r((((s) s) s) s), w , w , w , wh h h hich ich ich ich are are are are oAs which have been discovered so far (Figure 5). The chromatography behavior indicates that it (Figure 5). The chromatography behavior indicates that it might be an arsenosugar(s), which are (Figure 5). The chromatography behavior indicates that it might be an arsenosugar(s), which are ШШ Ш frequently r frequently r frequently r frequently re e e eported to be ported to be ported to be ported to be co-elu co-elu co-elu co-eluted w ted w ted w ted wiiiith DMA th DMA th DMA th DMA,,,, AsB AsB AsB AsB,,,, MM MM MM MMA, A, A, A, an an an and d d d As As As As under under under under anion anion anion anion exch exch exch exchang ang ang ange e e e HP HP HP HPLC- LC- LC- LC- might be an arsenosugar(s), which are frequently reported to be co-eluted with DMA, AsB, MMA, frequently reported to be co-eluted with DMA, AsB, MMA, and AsШ under anion exchange HPLC- frequently reported to be co-eluted with DMA, AsB, MMA, and As under anion exchange HPLC- III IC IC IC ICP-MS P-MS P-MS P-MS chro chro chro chrom m m ma a a at t t to o o ogram gram gram grams [ s [ s [ s [2 2 2 29] 9] 9] 9].... and As under anion exchange HPLC-ICP-MS chromatograms [29]. ICP-MS chromatograms [29]. ICP-MS chromatograms [29]. Figure 3. The concentration (mg/kg dry weight) of total As and As speciation detected in O. sinensis. Figure 3. The concentration (mg/kg dry weight) of total As and As speciation detected in O. sinensis. Figure 3. Figure 3. Figure 3. Figure 3. The concentration (mg/k The concentration (mg/k The concentration (mg/k The concentration (mg/kg g g g dry weight) of dry weight) of dry weight) of dry weight) of total As and As total As and As total As and As total As and As specia specia specia speciat t t ti ii ion detected on detected on detected on detected in in in in O. O. O. O. sinensis sinensis sinensis sinensis. . . . Figure 3. The concentration (mg/kg dry weight) of total As and As speciation detected in O. sinensis. Ш V Inorganic As (█ AsШ and █ As V) are shown in red and yellow sections, and organic As (█ AsB and III Ш Ш V V V Inorganic As (█ As Ш Ш and █ As V V ) are shown in red and yellow sections, and organic As (█ AsB and In In Inor In Inorg org org org ganic a a a an n n niiiic A c A c A c A As s s s s ( ( ( ( (█ █ █ █ As As As As As and and and and and █ █ █ █ As As As As As ) are ) are ) are ) are ) are show show show show shown n in re n in re n in re n in re in red d and d and d and d and and y y y y yellow e e e ellow llow llow llow sec sec sec sec sections, t tt tions ions ions ions, a , a , a , a and n n n nd org d org d org d org organic a a a anic nic nic nic As As As As As ( (( ( (█ █ █ █ A A AsB A As s s sB and B and B and B and and █ uAs) is shown in dark and light gray sections. █ uAs) is shown in dark and light gray sections. █ █ █ █ uAs) u u u uA A A As) i s) i s) i s) i is s s s s shown show show show shown in dark n in dark n in dark n in dark in dark and and and and and li li li li light g g g gh h h ht g t g t g t g gray r r r ray ay ay ay sectio sectio sectio sectio sections. ns. ns. ns. ns. Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard samples V Ш V samples of AsB, DMA V, AsШ, MMA, and As V, at 10 ppb of each arsenic species. (B) The extracts of V III V samples of AsB, DMA , As , MMA, and As , at 10 ppb of each arsenic species. (B) The extracts of of AsB, DMA , As , MMA, and As , at 10 ppb of each arsenic species. (B) The extracts of sample Figure 4. Figure 4. Figure 4. Chromatograms Chromatograms Chromatograms obtai obtai obtain n ned ed ed i i in n n qu qu quan an antification tification tification by HPLC-IC by HPLC-IC by HPLC-ICP P P-M -M -MS. ( S. ( S. (A A A) A mi ) A mi ) A mix of s x of s x of stttand and andaaard rd rd Figure 4. Chromatograms obtained in quantification by HPLC-ICP-MS. (A) A mix of standard III NQ1. The As and unknown V V V V Ш ШШ Ш organic As peaks V V V V overlap. (C) Oxidation products of the extracts of NQ1. sam sam samp p ples of As les of As les of AsB, B, B, DMA DMA DMA , As , As , As , MMA , MMA , MMA, an , an , and A d A d As s s , at , at , at 10 ppb 10 ppb 10 ppb of of of each arseni each arseni each arsenic c c sp sp specie ecie ecies. s. s. ( ( (B B B) The ) The ) The e e ex x xtracts of tracts of tracts of samples of AsB, DMA , As , MMA, and As , at 10 ppb of each arsenic species. (B) The extracts of III V Any As is transformed into As when H O is added to the extracts. 2 2 Molecules 2018, 23, x FOR PEER REVIEW 7 of 14 sample NQ1. The As and unknown organic As peaks overlap. (C) Oxidation products of the extracts Molecules 2018, 23, 1012 7 of 14 Ш V of NQ1. Any As is transformed into As when H2O2 is added to the extracts. Figure 5. Inference on the toxicity of the unknown As detected in O. sinensis. (a) 1 mL of H O was Figure 5. Inference on the toxicity of the unknown As detected in O. sinensis. (a) 1 mL of H2O2 was 2 2 added to the extracts, and the unknown As could not be oxidized (Figure 4C). Thus, the unknown added to the extracts, and the unknown As could not be oxidized (Figure 4C). Thus, the unknown As III III V Ш Ш V As cannot be the toxic MMA , DMA , or DMMTA which can be oxidized under treatment with cannot be the toxic MMA , DMA , or DMMTA which can be oxidized under treatment with H2O2. III H O . (b) The unknown peak presents a similar retention time with As , indicating that it is not a low 2(b) The unknown peak prese 2 nts a similar retention time with As , indicating that it is not a low retention component, such as an As hydrocarbon (AsHC). retention component, such as an As hydrocarbon (AsHC). Table 4. Concentration of total arsenic and arsenic species in Ophiocordyceps sinensis. Table 4. Concentration of total arsenic and arsenic species in Ophiocordyceps sinensis. b Ш V b AsB uAs III As VAs iAs oAs Total As Sample AsB uAs As As iAs oAs Total As V V V V Sample Name DMA MMA DMA MMA Name mg/kg (%) mg/kg (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg (%) (%) mg/kg mg/kg NQ1 0.10 (2.1%) nd nd 4.34 (91.2%) 0.22 (4.6%) 0.10 (2.1%) 0.32 (6.7%) 4.44 (93.3%) 4.76 NQ1 0.10 (2.1%) nd nd 4.34 (91.2%) 0.22 (4.6%) 0.10 (2.1%) 0.32 (6.7%) 4.44 (93.3%) 4.76 NQ2 0.09 (1.8%) nd nd 4.59 (91.8%) 0.21 (4.2%) 0.11 (2.2%) 0.32 (6.4%) 4.68 (93.6%) 5.00 NQ2 0.09 (1.8%) nd nd 4.59 (91.8%) 0.21 (4.2%) 0.11 (2.2%) 0.32 (6.4%) 4.68 (93.6%) 5.00 NQ3 0.12 (2.3%) nd nd 4.81(91.6%) 0.25 (4.8%) 0.07 (1.3%) 0.32 (6.1%) 4.93 (93.9%) 5.25 NQ4 0.08 (2.0%) nd nd 3.69 (90.0%) 0.23 (5.6%) 0.10 (2.4%) 0.33 (8.0%) 3.77 (92.0%) 4.10 NQ3 0.12 (2.3%) nd nd 4.81(91.6%) 0.25 (4.8%) 0.07 (1.3%) 0.32 (6.1%) 4.93 (93.9%) 5.25 LT1 0.07 (1.7%) nd nd 3.69 (90.2%) 0.20 (4.9%) 0.13 (3.2%) 0.33 (8.1%) 3.76 (91.9%) 4.09 NQ4 0.08 (2.0%) nd nd 3.69 (90.0%) 0.23 (5.6%) 0.10 (2.4%) 0.33 (8.0%) 3.77 (92.0%) 4.10 LT2 0.09 (2.2%) nd nd 3.72 (89.6%) 0.22 (5.3%) 0.12 (2.9%) 0.34 (8.2%) 3.81 (91.8%) 4.15 LT3 0.11 (2.8%) nd nd 3.56 (89.0%) 0.24 (6.0%) 0.09 (2.3%) 0.33 (8.3%) 3.67 (91.7%) 4.00 LT1 0.07 (1.7%) nd nd 3.69 (90.2%) 0.20 (4.9%) 0.13 (3.2%) 0.33 (8.1%) 3.76 (91.9%) 4.09 LT4 0.09 (1.7%) nd nd 4.75 (92.2%) 0.21 (4.1%) 0.10 (1.9%) 0.31 (6.0%) 4.84 (94.0%) 5.15 LT2 0.09 (2.2%) nd nd 3.72 (89.6%) 0.22 (5.3%) 0.12 (2.9%) 0.34 (8.2%) 3.81 (91.8%) 4.15 YS1 0.12 (2.6%) nd nd 4.19 (89.3%) 0.27 (5.8%) 0.11 (2.3%) 0.38 (8.1%) 4.31 (91.9%) 4.69 YS2 0.08 (1.9%) nd nd 3.80 (90.5%) 0.24 (5.7%) 0.08 (1.9%) 0.32 (7.6%) 3.88 (92.4%) 4.20 LT3 0.11 (2.8%) nd nd 3.56 (89.0%) 0.24 (6.0%) 0.09 (2.3%) 0.33 (8.3%) 3.67 (91.7%) 4.00 YS3 0.07 (1.4%) nd nd 4.69 (92.3%) 0.25 (4.9%) 0.07 (1.4%) 0.32 (6.3%) 4.76 (93.7%) 5.08 LT4 0.09 (1.7%) nd nd 4.75 (92.2%) 0.21 (4.1%) 0.10 (1.9%) 0.31 (6.0%) 4.84 (94.0%) 5.15 YS4 0.15 (2.9%) nd nd 4.65 (90.5%) 0.22 (4.3%) 0.12 (2.3%) 0.34 (6.6%) 4.8 (93.4%) 5.14 0.09 (1.9%) nd nd 4.21 (90.9%) 0.23 (5.0%) 0.10 (2.2%) 0.33 (7.1%) 4.3 (92.9%) 4.63 AVR YS1 0.12 (2.6%) nd nd 4.19 (89.3%) 0.27 (5.8%) 0.11 (2.3%) 0.38 (8.1%) 4.31 (91.9%) 4.69 Concentrations are presented as the average value of three measurements with a relative standard deviation YS2 0.08 (1.9%) nd nd 3.80 (90.5%) 0.24 (5.7%) 0.08 (1.9%) 0.32 (7.6%) 3.88 (92.4%) 4.20 (RSD) of less than 8% (the ranges of RSD values were as follows, RSD : 2.1~5.6%, RSD : 1.8~6.1%, RSD : AsB DMA V As III YS3 0.07 (1.4%) nd nd 4.69 (92.3%) 0.25 (4.9%) 0.07 (1.4%) 0.32 (6.3%) 4.76 (93.7%) 5.08 2.0~6.7%, RSD : 1.4~5.2%, and RSD : 1.1~7.3%; AsB, MMA, DMA, uAs, AsIII, AsV, iAs, oAs, and Total As MMA V As V wereYS4 the abbreviat 0.15 (2 ion .9%) of arsenobetaine, nd nd monomethylarsonic 4.65 (90.5%) 0.22 (4 acid, .3%) dimethylarsenic 0.12 (2.3%) acid, 0.34 (6 unknown .6%) 4.8 (93 organic .4%) arsenic, 5.14 arsenite, and arsenate, inorganic arsenic (total), organic arsenic (total), and total arsenic, respectively. not detected; AVR 0.09 (1.9%) nd nd 4.21 (90.9%) 0.23 (5.0%) 0.10 (2.2%) 0.33 (7.1%) 4.3 (92.9%) 4.63 average value among all the samples. Concentrations are presented as the average value of three measurements with a relative standard deviation (RSD) of less than 8% (the ranges of RSD values were as follows, RSDAsB: 2.1~5.6%, RSDDMAV: 2.3. Hazard Risk Assessment of Long-Term O. sinensis Consumption 1.8~6.1%, RSDAsШ: 2.0~6.7%, RSDMMAV: 1.4~5.2%, and RSDAsV: 1.1%~7.3; AsB, MMA, DMA, uAs, AsⅢ, Because the toxicity of As species differs [17], it is especially important to determine the chemical AsⅤ, iAs, oAs, and Total As were the abbreviation of arsenobetaine, monomethylarsonic acid, form of As in O. sinensis samples, and a health risk assessment should focus on toxic As species dimethylarsenic acid, unknown organic arsenic, arsenite, and arsenate, inorganic arsenic (total), c d because of their carcinogenic potential rather than the total As content. Based on the As speciation organic arsenic (total), and total arsenic, respectively. not detected; average value among all the analysis sam method ples. of the latest Chinese national standard to determine As species in functional food (GB 16740-2014), in this work iAs and AsB were minor As components. Additionally, MMA and 2.3. Hazard Risk Assessment of Long-Term O. sinensis Consumption DMA were negligible, leaving the majority of As content as unknown organic As. Till the end of the 1990s, iAs was assumed as the toxic actor among all the As species, and oAs was assumed Because the toxicity of As species differs [17], it is especially important to determine the chemical less toxic or non-toxic. Thus, according to conventional opinion on the toxicity of As species, form of As in O. sinensis samples, and a health risk assessment should focus on toxic As species the negligible abundances of toxic As in O. sinensis show that the oral intake hazard might be lower because of their carcinogenic potential rather than the total As content. Based on the As speciation than in iAs-accumulated mushrooms, such as Laccaria amethystea [30], Collybia butyracea [31] and other analysis method of the latest Chinese national standard to determine As species in functional food mushrooms [32]. However, this assumption was questioned with the development of analytic methods. (GB 16740-2014), in this work iAs and AsB were minor As components. Additionally, MMA and Arsenic undergoes rapid and complicated metabolism in organisms, and several organic As species, DMA were negligible, leaving the majority of As content as unknown organic As. Till the end of the III III V discovered as intermediate metabolites (MMA , DMA , DMMTA ) in organisms or discovered in 1990s, iAs was assumed as the toxic actor among all the As species, and oAs was assumed less toxic animal-sourced foods, were found to be strongly toxic (AsHCs) [15–19]. In this study, although the or non-toxic. Thus, according to conventional opinion on the toxicity of As species, the negligible V V traditionally considered toxic As (iAs, DMA , MMA ) in O. sinensis is minor, the large amount of Molecules 2018, 23, 1012 8 of 14 organic As cannot be accurately determined for its speciation and thus its toxicity cannot be arbitrarily evaluated. In order to comprehensively assess long-term O. sinensis consumption, based on the intake of total As, i+uAs (considering uAs is toxic, as iAs), and iAs (consider uAs is non-toxic, as AsB) the average values of the estimated daily intake (EDI), hazard quotient (HQ), and cancer risk (CR) were calculated according to Equations (1)–(3), respectively. According to the intake of total As, the average of HQ = 2.6 and CR = 1.2  10 . total As total As A Monte-Carlo simulation was used to model the uncertainties of the EDI, HQ, and CR. After 10,000 iterations of each simulation, EDI, HQ, and CR were derived, and the distribution patterns are illustrated in Figure 6. In terms of the health risk associated with total As exposure, the average EDI (Figure 6A) from O. sinensis consumption is 8.00  10 mg/(kgday), the HQ > 1 total As total As (Figure 6B), and the mean, median, 5th percentile, and 95th percentile values of CR are total As 3 3 4 3 1.23  10 , 1.20  10 , 8.14  10 , and 1.78  10 , respectively (Figure 6A–C). According to the average value and uncertainty analysis of EDI, HQ, and CR, O. sinensis would not be recommended for long-time consumption, as assessed by the total As content (HQ > 1 and CR > 1  10 ). total As total As However, as we discussed before, applying total As content in the hazard risk assessment is unfair; the hazard risk assessment should be based on the concentration of toxic As species. However, the majority of the As species in O. sinensis were unknown using the current five arsenic standards, thus the toxicity is also unknown. Hence, we assess the edible hazard according to two postulated III situations. First, if the uAs is as toxic as As , based on the intake of i+uAs, HQ = 2.62 and i+uAs CR = 1.2  10 . In terms of the health risk associated with i+uAs exposure after the Monte-Carlo i+uAs simulation, the mean, median, 5th percentile, and 95th percentile values of CR are 1.18  10 , i+u As 3 3 3 1.18  10 , 1.00  10 , and 1.36  10 , respectively (Figure 6D–F). These results indicate a similar edible hazard as that predicted by the total As analysis. Second, if the uAs is non-toxic as AsB and AsC, the relative abundance of the toxic iAs is only 10% of the total As content, and, and correspondingly, 5 5 the average value (EDI = 5.95  10 mg/(kgday), HQ = 0.19 < 1, and CR = 8.7  10 < iAs iAs iAs 1  10 ) and uncertainty analysis of EDI, HQ, and CR were much lower and below the hazardous thresholds. The mean, median, 5th percentile, and 95th percentile values of CR were 8.93  10 , toxic As 5 5 4 8.66  10 , 5.83  10 , and 1.29  10 , respectively (Figure 6G–I). The CR value exceeded the threshold value (1  10 ) at the 72.7th percentile. Thus, if the uAs is non-toxic, the long-term consumption of O. sinensis is relatively safe according to the hazard analysis on iAs and the reputation of O. sinensis should be rehabilitated because of its non-toxicity. As we discussed in Section 2.2, the uAs is might be an arsenosugar, which are generally considered to be non-toxic or have low toxicity [29]. Thus, according to the current recognition on the toxicity of organic As species, the uAs might be non-toxic, and accordingly the long consumption of O. sinensis might be safe in terms of As speciation analysis. However, determination of the structure and toxicity of the uAs should be carried out to provide strong evidence for our inference. Although As has become notorious because some As species cause acute toxicity, chronic toxicity, and cancer via environmental exposure at low doses, its medicinal properties have been a focus in ancient China and Greece for over 3000 years [13]. Until the late 1940s, diseases such as psoriasis and syphilis were frequently treated with As [33]. More recently, inorganic arsenic has also shown significant activity in curing acute promyelocytic leukemia (APL) [13]. Inorganic arsenic has shown activity against other hematologic and solid organ malignancies but is also associated with serious toxicities, especially when applied at higher doses. For decades, scientists have attempted to seek ideal As compounds that are nontoxic and have medicinal potential. Few efforts have been successful, V V and the medical use of the most concerning forms of organic As (such as MMA , DMA , and AsB) has not been proven yet [33]. The recent development of orally bio-available organic arsenic derivatives (OAD) offering improved toxicity profiles and better efficacy may expand the application of arsenic compounds in hematologic malignancies and solid tumors [33,34]. O. sinensis has been massively reported to have predominant efficiency in anti-tumorigenesis, and most researchers consider this the result of chemical constituents such as cordycepin, adenosine, the exopolysaccharide Molecules 2018, 23, 1012 9 of 14 fraction, cordyglucans, and monosaccharide saponins [5]. From our newly obtained data, inorganic As comprised only 7% of the total As content, much lower than that (90%) of unknown organic As. Inspired by the above-mentioned progress in the medicinal use of organic arsenic derivatives (OAD) [33,34], we infer that the unknown As might play certain important roles in anti-tumorigenesis or other functional uses of O. sinensis. Therefore, combining the risk hazard analysis and potential functional speculation, As might be considered functional in O. sinensis instead of poisonous. Further research to provide sufficient evidence on the above speculation including structural identification, Molecules 2018, 23, x FOR PEER REVIEW 9 of 14 toxicology, and pharmacology assessments, especially animal testing, should be implicated in future. Figure Figure 6. 6. Estim Estimated ated distribution distribution patterns patterns and and d descriptive escriptive st statisti atistics cs of of ( (A A,,D D,,G G)) estimated estimated daily intake daily intake ((EDI EDI ), ), ( (B B ,E ,E ,H ,H )) hazar hazard quotient ( d quotient (HQ HQ ), ), an and d ( (C C ,F ,F ,I ,I )) cancer ri cancer risk sk( (CR CR ). ). Where in Where in ((A A– –C C)) values w values wer ere derived e derived accor according to ding to th thee total As concentration in total As concentration in O. O. si sinensis nensis;; in in ( (D D –– F F )) values were values were deriv derive ed d according to the according to the i+uAs i+uAs conc concentration entration (the su (the sum of m of iAs and iAs and uAs) u inAO. s) in sinensis O. sinensis ; and in; and in ( (G–I) values G–I wer ) values w e derived ere derived according to according the iAs concentration to the iAs concentra in O. sinensis tion in . O. sinensis. Although As has become notorious because some As species cause acute toxicity, chronic toxicity, and cancer via environmental exposure at low doses, its medicinal properties have been a focus in ancient China and Greece for over 3000 years [13]. Until the late 1940s, diseases such as psoriasis and syphilis were frequently treated with As [33]. More recently, inorganic arsenic has also shown significant activity in curing acute promyelocytic leukemia (APL) [13]. Inorganic arsenic has shown activity against other hematologic and solid organ malignancies but is also associated with serious toxicities, especially when applied at higher doses. For decades, scientists have attempted to seek ideal As compounds that are nontoxic and have medicinal potential. Few efforts have been V V successful, and the medical use of the most concerning forms of organic As (such as MMA , DMA , and AsB) has not been proven yet [33]. The recent development of orally bio-available organic arsenic derivatives (OAD) offering improved toxicity profiles and better efficacy may expand the application of arsenic compounds in hematologic malignancies and solid tumors [33,34]. O. sinensis has been massively reported to have predominant efficiency in anti-tumorigenesis, and most researchers Molecules 2018, 23, 1012 10 of 14 3. Materials and Methods 3.1. Reagents Deionized water obtained from a Milli-Q water purification system (Millipore, Bedford, MA, USA) was used for the preparation of all solutions. Anhydrous sodium acetate, potassium nitrate, sodium dihydrogen phosphate, and disodium ethylenediaminetetraacetate (EDTA) were used for the preparation of mobile phases. H O (31%) and concentrated HNO (69%) were used for sample 2 2 3 digestion and extraction. Standards for the determination of both total As content and As speciation were prepared by diluting or compounding the standard stock solution sourced from the National Institute of Metrology (Beijing, China). The former was diluted to 10 mg As/L from the stock solution (As ) with certified concentration of 1000 mg As/L, and the latter was prepared with III V V V As (0.233 mol/g), As (1.011 mol/g), MMA (0.355 mol/g), DMA (0.706 mol/g), and AsB (0.518 mol/g). All stock solutions were kept at 4 C, and further dilutions for analysis were prepared daily. National Standard reference materials: GBW10049 (green Chinese onion, Institute of Geophysical and Geochemical Exploration, Langfang, China), GBW10051 (pork liver, Institute of Geophysical and Geochemical Exploration, Langfang, China), GBW08573 (yellow-fin tuna, Second Institute of Oceanography, Huangzhou, China) and GBW(E)100358 (rice, National Analysis Center for Ion and Steel, Beijing, China) were used as certified reference materials (CRMs) in this study. 3.2. Sample Collection and Preparation Three typical production areas (Litang, Naqu, and Yushu) were chosen as the sampling sites (Figure 2), and twenty O. sinensis samples of similar size (approximately 0.3 g per sample) were collected at each site in May 2017. All the samples were mechanically cleaned of soil, rinsed with deionized water and freeze-dried to reach a constant weight. To gather enough material for subsequent As analysis, each of the five O. sinensis samples was combined into a batch sample. Therefore, the original twenty O. sinensis taken at each sampling site were organized into 4 sample batches (referred to as NQ1–4, LT1–4 and YS1–4). These sample batches were individually pulverized using a mortar and pestle to reduce the particle size. Then, the fine powder was passed through a sieve with a pore size of 0.25 mm and stored in sealable plastic bags at 4 C until analysis. For total As content analysis, digestion of approximately 500 mg of sample powder was conducted in a microwave digestion system using concentrated HNO . The operating program of the microwave system was as follows: the samples were heated to 120 C in 5 min and held at 120 C for 5 min in the first step, heated to 150 C in 5 min and held at 150 C for 5 min in the second step, heated to 170 C in 5 min and held at 170 C for 5 min in the third step, and heated to 190 C in 5 min and held at 190 C for 20 min in the fourth step. Finally, the samples were cooled to room temperature. For As speciation analysis, approximately 1 g of sample powder was diluted with 20 mL 0.15 mol/L HNO in a 50 mL polyethylene centrifuge tube and then incubated in a 90 C water bath for 12 h. All the digestion products were centrifuged, filtered, and stored at 4 C before total As and As speciation analysis. 3.3. Total Arsenic Analysis The total As content of each sample was measured using ICP-MS (Agilent 7800, Santa Clara, CA, USA). The supernatant was first diluted with water up to 25 mL and then subjected to ICP-MS. The operating parameters of the equipment were as follows: radio frequency (RF) power, 1550 W; carrier gas, 1.05 L/min; collision mode, Helium (HE) flow 4.2 mL/min; plasma gas flow rate = 15 L/min; auxiliary gas flow rate = 0.1 L/min; and selected isotope = m/z 75. Samples were quantified using an external calibration curve from As standards (calibration points: 5, 10, 50, 100, and 200 ppm). Triplicate analyses were performed for each sample. For quality control purposes, the standards used for the calibration curve were run before and after each sample series. The corresponding digestion blanks (one for each sample digestion series) were also measured. Molecules 2018, 23, 1012 11 of 14 The total As in the CRMs (GBW10049, GBW10051, GBW08573 and GBW100358) were determined according the same methods. 3.4. Arsenic Speciation Analysis The separation and determination of As species were performed according to our previous III V V V HPLC-ICP-MS method [35]. Five As species (As , As , MMA , DMA , AsB) were separated by an Agilent 1260 HPLC system (Agilent, USA). The chromatograph was equipped with a standard autosampler, IonPac AG19 guard column (4  50 mm), and an IonPacAS19 separation column (4  250 mm). The main chromatography conditions used for HPLC were as follows: a mobile phase of 10 mmol/L anhydrous sodium acetate; 3 mmol/L potassium nitrate; 10 mmol/L sodium dihydrogen phosphate; 0.2 mmol/L disodium ethylenediaminetetraacetate buffer; a flow rate at 1.0 mL/min; a column temperature of room temperature; and an injection volume with a 50 L sample. The separated As species were examined by ICP-MS (as described previously) and identified by comparison of retention times with standards (Figure 4A). External calibration curves (calibration V V III V points: 0, 2.5, 5, 10, 50, and 100 ppb) were used to quantify MMA , DMA , As , As , and AB according to the corresponding standards. Triplicate analyses were performed for each sample. Extraction blanks were also analyzed by HPLC-ICP-MS in each work session. To check the extraction and digestion efficiency for the As species analysis, the As species extraction was completely measured using ICP-MS according to 3.3. Then the measured total As concentration in the As species extraction which was digested by 0.15 mol/L HNO , was compared with the total As digested by concentrated HNO to calculate the extraction efficiency. The iAs species in one CRM (GBW100358) were determined according the same methods. III Due to the complexity of the metrics of the O. sinensis sample, the As peak could not be separated from the other unknown peaks (Figure 4B); therefore, 1 mL of H O was added to the supernatant 2 2 III V before analysis. This operation completely oxidizes As into As (Figure 4C), without conversion of other arsenic species into new As compounds, according to the comparison of the number and the area of the corresponding peaks in Figure 4B and C. The inorganic As content was identified and quantified as As using external standards. The total organic As amounts were quantified according to the delta value between the total As and the inorganic As content. Thus, the content of the unknown As species was quantified according to the delta value between the organic As and the sum of the V V three determined As species (MMA , DMA , and AsB). 3.5. Health Risk Assessment The health risks for consumers with exposure to total toxic As (the sum of inorganic As, DMA, and MMA ), total As content, i+uAs, and iAs in O. sinensis were evaluated. According to the model of evaluation for health risks (Environment Protection Agency, EPA) [25,26], the estimated daily intake of As from O. sinensis consumption was calculated according to Equation (1): EDI = C  IR BW, (1) As where EDI (mg/kgday) is the estimated daily intake of As, C (mg/kg) is the concentration of total As As or toxic As in O. sinensis (dry weight), IR (0.01 kg/day) is the daily ingestion rate of O. sinensis [36], and BW is the average body weight (58.7 kg) [37] of the consumer. Risk characterization was quantified according to the potential non-carcinogenic risks of As exposure from O. sinensis ingestion and was reflected by the hazard quotient (HQ). The HQ value was calculated using equation (2). If HQ < 1, there might be no concern for non-carcinogenic effects. Otherwise, if the HQ exceeds 1, there might be serious concern for non-carcinogenic effects [25]. HQ = (EDI R f D)  (EF  ED AT) (2) Molecules 2018, 23, 1012 12 of 14 The probability of the excess lifetime cancer risk (CR) due to As exposure for O. sinensis consumption was estimated according to Equation (3). According to the US EPA, the incremental probability of cancer risk for consumers over a lifetime is characterized by CR with an acceptable 6 4 6 range of 1  10 –1  10 . A CR less than 1.0  10 indicates no obvious concern for cancer risk. However, a CR greater than 1.0  10 indicates an obvious potential cancer risk [25]. CR = EDI  SF  (EF  ED AT) (3) In Equations (2) and (3), RfD is the oral reference dose for As, EF (days/year) is the exposure frequency, ED (years) is the exposure duration, AT (days) is the average exposure time to As (usually AT is equal to the average life expectancy), and SF is the slope factor of As through oral intake. In this study, RfD, SF, ED, EF, and AT were determined to be 3  10 mg/(kgday) [26], 1.5 mg/(kgday) [26], 70 years, 365 days/year, and 25,550 days [37], respectively. To assess the long-term health risks of O. sinensis to arsenic exposure, the uncertainty of EDI, HQ, and CR were modeled through a Monte-Carlo simulation conducted with CrystalBall Software (V. 11.1.2.0.00, Oracle, Redwood Shores, CA, USA). The parameter distributions used in the model are presented in Table A1. 4. Patents A new method for risk assessment of Ophiocordyceps sinensis. Patent No. 201711087936.2. Document No. 2017110900896410. Author Contributions: L.-X.G. and Z.-G.H. conceived and designed the experiments; G.-W.Z. and J.-T.W. performed the experiments; Y.-P.Z. and L.-X.G. analyzed the data; Z.-G.H. contributed analysis tools; L.-X.G. wrote the paper. Acknowledgments: This work was jointly supported by the Excellent College Teachers Training Program of Guangdong Province, China (No. YQ2015084), the Natural Science Foundation of China (No. 81303155), and the Guangdong Provincial Science and Technology Plan Project (No. 2015A020208009). Conflicts of Interest: The authors declare no conflict of interest. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Abbreviations O. sinensis Ophiocordyceps sinensis As Arsenic III As Arsenite As Arsenate DMA Dimethylarsinic acid MMA Methylarsonic acid AsB Arsenobetaine DMMTA Thio-organoarsenic uAs Unknown organic As oAs Organic arsenic iAs Inorganic arsenic i+uAs Sum of iAs and uAs LOQ Limits of quantification LOD Limits of detection EDI Estimated Daily Intake HQ Hazard Quotient CR Cancer Risk High-Performance Liquid Chromatography–Inductively Coupled Plasma Mass HPLC-ICP-MS Spectrometry Molecules 2018, 23, 1012 13 of 14 Appendix A Table A1. Input parameters for Monte-Carlo simulation. Parameters Units Distribution Adult Body weight, BW BW kg Lognormal (Mean = 58.7, SD = 12.0) Daily ingestion rate, IR kg/day Lognormal (Mean = 0.01, SD = 0.0143) Total As concentration, C g/kg Lognormal (Mean = 4.63, SD = 0.42) total As i+uAs concentration, C g/kg Lognormal (Mean = 4.53, SD = 0.41) i+uAs iAs concentration, C g/kg Lognormal (Mean = 0.34, SD = 0.03) iAs References 1. Dong, C.H.; Wen-Jia, L.I. Cordyceps industry in China: Current status, challenges and perspectives–Jinhu declaration for Cordyceps industry development. Mycosystema 2016, 35, 1–15. [CrossRef] 2. Guo, L.X.; Hong, Y.H. Fungus-larva relation in the formation of Cordyceps sinensis as revealed by stable carbon isotope analysis. Sci. Rep. 2017, 7, 7789. [CrossRef] [PubMed] 3. Martel, J.; Ko, Y.F. Myths and realities surrounding the mysterious caterpillar fungus. Trends Biotechnol. 2017, 35, 1017. [CrossRef] [PubMed] 4. Qi, W.; Lei, W. Pharmacological study progress of the Cordyceps sinensis. Global Tradit. Chin. Med. 2014, 7, 227–232. 5. Liu, Y.; Wang, J. The chemical constituents and pharmacological actions of Cordyceps sinensis. Evid. Based Complement. Altern. Med. 2015, 2015, 575063. 6. Caplins, L.; Halvorson, S. Collecting Ophiocordyceps sinensis: An emerging livelihood strategy in the garhwal, indian himalaya. Mt. Sci. 2017, 14, 390–402. [CrossRef] 7. Winkler, D. Yartsa gunbu (Cordyceps sinensis) and the fungal commodification of Tibet’s rural economy. Econ. Bot. 2008, 62, 291–305. [CrossRef] 8. Consumption Tips on Cordyceps sinensis Products. Available online: http://samr.cfda.gov.cn/WS01/CL1986/ 144020.html (accessed on 4 February 2016). 9. The Common Standards of Functional Food; GB16740-2014; National Health and Family Planning Commission of the People’s Republic of China: Beijing, China, 2014. 10. Notice on the Suspension of All the Pilot Work on Cordyceps sinensis for Health Food. Available online: http://samr.cfda.gov.cn/WS01/CL0847/146100.html (accessed on 4 March 2016). 11. Wang, H.; Shan, Y. The utilization and analysis of present market situation for Cordyceps sinensis. Res. Pract. Chin. Med. 2016, 30, 83–86. 12. Tsuda, T.; Babazono, A. Inorganic arsenic: A dangerous enigma for mankind. Appl. Organomet. Chem. 2010, 6, 309–322. [CrossRef] 13. Khairul, I.; Wang, Q.; Naranmandura, H. Metabolism, toxicity and anticancer activities of arsenic compounds. Oncotarget 2017, 8, 23905–23926. [CrossRef] [PubMed] 14. Cohen, S.M.; Arnold, L.L.; Eldan, M.; Lewis, A.S.; Beck, B.D. Methylated arsenicals: The implications of metabolism and carcinogenicity studies in rodents to human risk assessment. Crit. Rev. Toxicol. 2006, 36, 99–133. [CrossRef] [PubMed] 15. Styblo, M.; Razo, L.M.D. Comparative toxicity of trivalent and pentavalent inorganic and methylated arsenicals in rat and human cells. Arch. Toxicol. 2000, 74, 289. [CrossRef] [PubMed] 16. Huang, C.; Ke, Q. Molecular mechanisms of arsenic carcinogenesis. Mol. Cell. Biochem. 2004, 255, 57–66. [CrossRef] [PubMed] 17. Hughes, M.F. Arsenic toxicity and potential mechanisms of action. Toxicol. Lett. 2002, 133, 1–16. [CrossRef] 18. Hua, N.; Carew, M.W. Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells. Chem. Res. Toxicol. 2011, 24, 1586–1596. 19. Meyer, S.; Matissek, M.; Müller, S.M.; Taleshi, M.S.; Ebert, F.; Francesconi, K.A.; Schwerdtle, T. In vitro toxicological characterisation of three arsenic-containing hydrocarbons. Metallomics 2014, 6, 1023–1033. [CrossRef] [PubMed] 20. Hao, C.; Wang, G. Arsenic speciation analysis of 15 traditional Chinese medicines. Instrum. Anal. 2009, 28, 918–921. Molecules 2018, 23, 1012 14 of 14 21. Cao, X.; Wang, J. Analysis of arsenic speciation in Cordyceps sinensis in Tibet by HPLC-HG-AFS. Chin. Tradit. Patent Med. 2015, 37, 1985–1989. 22. Gómezariza, J.L.; Sánchezrodas, D. A comparison between ICP-MS and AFS detection for arsenic speciation in environmental samples. Talanta 2000, 51, 257. [CrossRef] 23. Popp, M.; Hann, S. Environmental application of elemental speciation analysis based on liquid or gas chromatography hyphenated to inductively coupled plasma mass spectrometry—A review. Anal. Chim. Acta 2010, 668, 114–129. [CrossRef] [PubMed] 24. Sankararamakrishnan, N.; Mishra, S. A Comprehensive Review on Various Analytical Methods for the Determination of Inorganic and Organic Arsenic in Environmental Samples. In Environmental Contaminants; Springer: Singapore, 2018. 25. U.S. EPA. Risk Assessment Guidance for Superfund (RAGS). In Human Health Evaluation Manual (Part A); EPA Publication: Washington, DC, USA, 1989. 26. U.S. EPA. Integrated Risk Information System (IRIS). In Arsenic, Inorganic (CASRN 7440-38-2)-748; EPA Publication: Washington, DC, USA, 2009. 27. Francesconi, K.A.; Goessler, W.; Panutrakul, S.; Irgolic, K.J. A novel arsenic containing riboside (arsenosugar) in three species of gastropod. Sci. Total Environ. 1998, 221, 139–148. [CrossRef] 28. Sele, V.; Sloth, J.J.; Holmelid, B.; Valdersnes, S.; Skov, K.; Amlund, H. Arsenic-containing fatty acids and hydrocarbons in marine oils—Determination using reversed-phase HPLC-ICP-MS and HPLC-qTOF-MS. Talanta 2014, 121, 89–96. [CrossRef] [PubMed] 29. Niegel, C.; Matysik, F.M. Analytical methods for the determination of arsenosugars—A review of recent trends and developments. Anal. Chim. Acta 2010, 657, 83. [CrossRef] [PubMed] 30. Larsen, E.; Hansen, M. Speciation and health risk considerations of arsenic in the edible mushroom Laccaria amethystina collected from contaminated and uncontaminated locations. Appl. Organomet. Chem. 1998, 12, 285–291. [CrossRef] 31. Kuehnelt, D.; Goessler, W. Arsenic compounds in terrestrial organisms I: Collybiamaculata, Collybiabutyracea and Amanita muscaria from arsenic smelter sites in Austria. Appl. Organomet. Chem. 1997, 11, 289–296. [CrossRef] 32. Chen, S.; Guo, Q. Determination of arsenic species in Edible mushrooms by high-performance liquid chromatography coupled to inductively coupled plasma mass spectrometry. Food Anal. Methods 2017, 10, 740–748. [CrossRef] 33. Quintás-Cardama, A.; Verstovsek, S. Chemical and clinical development of darinaparsin, a novel organic arsenic derivative. Anti-Cancer Agents Med. Chem. 2008, 8, 904. [CrossRef] 34. Kuntze, A.M.; Braz, O. Development of an organic arsenic derivative as a therapy for leukaemia. Spies Int. Symp. Opt. Sci. 2003, 2003, 347–355. 35. Tang, L.; Zhang, L.L. Arsenic speciation of dried Lentinus edodes by HPLC-ICP-MS. Food Ind. 2015, 36, 275–277. 36. Feng-Lei, X.U.; Wen-Ying, H. Clinical efficacy of Cordyceps sinensis for chronic kidney diseases: A systematic review. Chin. J. Evid. Based Med. 2006, 6, 804–808. 37. Ma, L.; Yang, Z.; Kong, Q.; Wang, L. Extraction and determination of arsenic species in leafy vegetables: Method development and application. Food Chem. 2017, 217, 524–530. [CrossRef] [PubMed] Sample Availability: Samples of the compounds AsB, DMA, MMA, AsIII, AsV and uAs are available from the authors. © 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Journal

Molecules – Multidisciplinary Digital Publishing Institute

Published: Apr 26, 2018

There are no references for this article.