Cordyceps militaris
Preliminary Information
Cordecyps militaris is an entomopathogenic fungus, known for over 4000 years, and is a leading pioneer in the Green Pharmacy Revolution. "Cordyceps" is traditionally and conventionally understood as the dried fungus of C. militaris and sinensis (both species are myco-chemically and pharmacologically similiar and can be used interchangably) that commonly grows out caterpillar larvae and other entomology. These larvae and other entomologies include about 20 species of five genera, Hepialus, Hepialiscas, Napialus, Forkalus and Bipectilus, which are classified as Hepialidae. Among them, Hepialus armoricanus, H. obilfurcus, H. baimaensis and H. renzhiensis are predominant hosts of C. sinensis.124 The parasitic complex of the fungus and the caterpillar is found in the soil of the prairie at an elevation of 3500–5000 meters in the northwestern part of China. It is for this geographically exclusive reason the natural Cordyceps is a significantly rare species in the current marketplace.
It is of great interest and would be of great benefit to hummanity for researchers to aim at developing techniques for isolating strains of C. sinensis. Particularly, fermentable strains are of significant interest because fermentation allows more medicinal activity to be made from smaller amounts of the mushroom. Fermented sodas and tonics of C. militaris and sinensis have been clinically proven to be incredibly therapeutic, prophylactic, and be potent vehicles of the mushrooms diverse medicinal and therapeutic devices.
General Background
C. militaris and sinensis, mycopharmaceuticals and plant medicine in general is a prime example of the "Back to the Pavillion theory". This theory suggests that nowadays, people are willing to wear cotton clothes instead of synthetic/polyester ones, eat vegetables and organic foods rather than processed mass produced gorceries, and in the same manner, willing to take herbal/natural medicines rather than the synthetic drugs.
Because of this cycling back to our roots (or mycelium I could say) we are re-discovering the reason we humans were able to persevere so well over the millinea. It wasn't becasue a technoloigcal age of information and innovation of industrial advancments allowing us to live more lavish and opulent lifestyles. It was moreso our direct connection to nature and our richly organic and diverse natural lives which sustained and fertilized the spirit of manking to its indomnitable and unconquerable state. A state which is greatly lacking in the modern era.
This being said, C. militaris is extremely popular among TCM as a Dietetic Chinese drug, also known as ‘edible Chinese drugs’, ‘dietetic material medicine’ or ‘medicinal food’, although the word ‘drug’ could be misinterpreted in the West. Its clear Hippocrates reciveved his medicinal initiation from more ancient sources who have been practicing with food as medicine for thosuands of years before him. What else did these ancients know and understand? What other wisdom do we lack that we must re-learn from our common ancestry?
Currently, there is explosive research into Cordecyps within the field of pharmacognosy. Cordecyps represent a largely untapped reservoir of rare and species unique bioactive chemicals that we already know are serving as miriculous medicines, we (westerners) have simply yet to name the 'active constitents' responsible for the medicinal effects. This art of naming is hard (headed) sciences way of coming to understand the nature and efficacy and potential applications of this unique medicine. While we are FAR from a comprehensive analysis of the Cordyceps myco-chemical profile, we have still come a far way already. It is worth noting that internal constituents and phenotypes are equally as reliant on the environmental conditions for an organisms expression, as is the genetic makeup itself. It is known that most flora only are expressing about 20% of their available genetic code, leaving about 80% unactualized genetic potential, which means under slightly different environmental conditions, the same genetic code could potentially produce a nearly completely different mushroomwith totally different chemical constituents. This same law applies to the majority of the flroa and fauna, and certainly humzn beings (although, this is another discussion). All things consdiered, even with our limited knowledge and conceptually limited modes of analysis, again, we still have been able to peer into the mystery of the mushrooms to shine light on their wondrous ability to heal us.
Below is a brief overveiw and condensation of much of the current scientific literature describing the principle medicinal constituents and pharmacologic actions of Cordyceps militaris and sinensis, and it is followed by a nutritional analysis. I call this, MYCOPHARMACY.
Mycopharmacy
There have been many empirically found medicinal and therpeutic effects of this mushroom which include pro-sexual, anti-inflammatory, anti-oxidant/anti-aging, anti-tumour/anti-cancer/anti-leukemic, anti-proliferative, anti-metastatic, immunomodulatory, anti-microbial, anti-bacterial, anti-viral, anti-fungal, anti-protozoal, insecticidal, larvicidal, anti-fibrotic, steroidogenic, hypoglacaemic, hypolipidaemic, anti-angiogenetic, anti-diabetic, anti-HIV, anti-malarial, anti-fatigue, neuroprotective, liver-protective, reno-protective as well as pneumo-protective. There is antitumour effect, hepatoprotection and inflammatory effects, and antioxidant, nephroprotective, and antiapoptotic properties. It also has the unique funtion of delaying senility. It is commonly used in China to replenish the kidney and soothe the lung, for the treatment of fatigue, night sweating, hyposexuality, hyperglycaemia, hyperlipidaemia, asthenia after severe illness, respiratory disease, renal dysfunction and renal failure, arrhythmias and other heart disease and liver disease. A large amount of experimental evidence has shown that fungal polysaccharides have a wide range of bioactivities including antitumor [52], anti-influenza virus [53], immunopotentiation [54], hypoglycemic [55], hypocholesterolemic [56], and antioxidant effects [51].
Cordecypin
The main mechanism of cordycepin is to inhibit purine biosynthesis pathway. Cordycepin also induces RNA or DNA chain termination and interacts with receptors for mTOR which has an important role in the regulation of protein synthesis. Cordycepin is converted into 5’mono-, di- and tri-phosphates and can inhibit the activity of ribose-phosphate, pyrophosphokinase and 5-phosphoribosyl-1-pyrophosphate amidotransferase enzyme which play important role in biosynthesis of purines.
The structure of cordycepin is similar to the adenosine, a cellular nucleoside. Therefore it can act as a nucleoside analogue. Some other key active components inlcude (such as polysaccharides, ergosterol, mannitol, homocitrullyl aminoadenosine, cordycepic acid).
Cordycepin is a category of compounds that exhibits significant therapeutic potential and has many intracellular targets, including nucleic acid, apoptosis, and cell cycle. Tuli et al. researched the variety of molecular mechanisms that mediate the pharmacological effects of cordycepin. Besides, they deem that cordycepin can participate in various molecular processes in cells because of its similarity with adenosine [7]. Wang et al. investigated the effects of cordycepin in prevention of focal cerebral ischemic/reperfusion (IR) injury and suggested that cordycepin has a neuroprotective effect in the ischemic brain, which is due to the inhibition of inflammation and increase of antioxidants activity related to lesion pathogenesis [23, 24]. So cordycepin could be an attractive therapeutic candidate with oral activity against I/R-associated heart diseases such as myocardial infarction [25]. Besides, cordycepin showed the obvious analgesic effect.
Cordycepin is a potent anti-inflammatory and analgesic medicine. In addition, cordycepin stimulated the release of some cytokines of resting PBMCs and influenced proliferation of PBMCs and transcription factors in THP-1 cell line. Accordingly, cordycepin can intensively regulate the functions of human immune system. ]. Cordycepin is a broad spectrum biocidal compound possessing not only antitumor activity but also antibacteria, antivirus, and insecticidal activities [2].
To date, six nucleobases (Figure 3), including cytosine, uracil, thymine, adenine, guanine, and hypoxanthine, were determined in natural and cultured Cordyceps sinensis. The overall content of nucleosides is much higher in cultured Cordyceps sinensis than in natural ones by comparison.
Cordecypic acid
Cordycepic acid is also a principal bioactive compound of C. militaris. The structure was identified first as 1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid. Later, this compound was crystallized and identified as d-mannitol. It is used in the anti-aging nutritional supplement and also for its strong pro-sexual activity.
Pro-Sexual Activity of Cordycepic acid
Traditional medicine has used Cordyceps species for improving the sexual function. Wen-Hung Lin et al., have shown the spermatogenic effect of C. militaris. diet supplemented with C. militaris mycelium to subfertile boars for 2 months. The sperm production was increased significantly in boars fed with diet contained C. militaris extract in both quality of fertile sperm and the quantity of semen volume and total sperm number. They also detected the cordycepin in plasma in boars supplemented with C. militaris. More interesting, an amount of motile sperm cells and sperm morphology were also enhanced significantly.
Polysaccharides
Cordyceps sinensis contains a great deal of polysaccharides, which can be in the range of 3–8% of the total weight. Poly saccharides are organic sugar molecules containing carbon, hydrogen, and oxygen, also known as carbohydrates (that is, Carbo- for carbon, hydr- for hydrogen, and -ate for oxygen). A large amount of experimental evidence has shown that fungal polysaccharides have a wide range of bioactivities including antitumor [52], anti-influenza virus [53], immunopotentiation [54], hypoglycemic [55], hypocholesterolemic [56], and antioxidant effects [51].
Cordycepic acid is a phosphorylated monosaccharide (D-mannitol) which carries an array of activity beyond the pro-sexual. It is namely an anti-aging molecule which protects afainst genetic degradation, it carries anti cancer effects, is anti oxidizing and anti inflammatory of course. But the key thing i believe is its ability to maturally modulate the immun responses By provoking innate responses from the immunology, it strengthens innate immunity rather than supplying foreign devices to do the bodies work for it. This exercising the endowed healing system is of great benefit for long lasting health and wellness, opposed to temporary short term relief often generated by other specialized metabolites, although this is likewise highly useful.
In general, polysaccharide activity is typically more complex and has more bioactivity when a compound itself is more conplex structurally, alementally, and through other physical traits. For example, many other studies have suggested that the pharmacological activity of the polysaccharide was correlated with its characteristics. In many cases of medicinal mushrooms, polysaccharides' milecular weight was the determining factor of the potency of the antitumor activity [57]. In other words, Sasaki et al. confirmed that the fungi polysaccharide's antitumor activity is related to the molecular weight, and the fungi polysaccharide has antitumor activity if its molecular weight is greater than 16000.
the pharmacological activity of the polysaccharide was correlated with its characteristics such as molecular weight. It has been reported that polyglucans with higher molecular weight (10–1000 kDa) tend to have greater water solubility and therefore have a more effective antitumour activity. The anticancer effects of fungal polysaccharides arise from the enhancement of the body's immune system rather than direct cytocidal effects.62
Crude protein and amino acids
Kwan showed C. militaris has a high ratio of the essential amino acids, such as lysine (101.2 mg/g), methionine (62.7 mg/g), and acidic amino acids of glutamic acid (57.5 mg/g) and aspartic acid (43.9 mg/g), a low of tyrosine content (4.7 mg/g) [14
Previous papers have reported that the content of crude protein was in the range 29.1–33%.27,33,66 The protein was composed of 18 amino acids, including aspartic acid, threonine, serine, glutamate, proline, glycine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, cystine, cysteine and tryptophan. The content of amino acids after hydrolysis is mostly reported as 20–25%, the lowest being 5.53%, the highest being 39.22%. The highest contents are glutamate, arginine and aspartic acid, and the major pharmacological components are arginine, glutamate, tryptophan and tyrosine.67 The content of amino acids in the commercial preparation of Cordyceps is significantly higher than in the mycelia of C. sinensis, which is similar to the content in the fruit body of C. sinensis.
Protection of heart and antihypertensive effects
Xu et al.108 reported that the alcoholic extracts had a protective effect on myocardial injury induced by adriamycin in rats. The mechanism was suggested to be that mannitol, amino acids and polysaccharides in the extracts played an important role; all the substances had the effect of nourishing the myocardium and enhancing its anti-injury capacity.108 With the same experimental methods, Gu et al.109 reported that Lungfit (C. sinensis compounds) could be beneficial for myocardial ischaemia/reperfusion injury as well. However, both alcohol and water extracts had obviously protective effects on the rat isolated heart.
Nutritional Information
Another study on a Cordyceps species analyzed 20 different elemental minerals subdivided into essential (macro and micro) and non-essential/toxic from two different asscessions, a wild type, and a cultivated type. The table is pictured below and a description of the results taken directly from the literature are described directly below that.
"Among the essential elements estimated in the present work we found the macro elements (Na, K, Ca and Mg) and microelements (Zn, Fe, Cu, Mn, V, Cr, Ni, Co, Mo, and Se); along the nonessential elements and the toxic elements Ba, Sn, As, Cd, Pb and Hg. The results are presented in Table 3. Trace elements contained in three different samples were considerably different at the 95% confidence level according to the results of one-way analysis of variance (ANOVA).
For macro element, in this study, Na (1420g/g) and K (946g/g) in wild stroma exhibited the highest levels and were significantly different from the other two samples. While the max- imum content of Mg (986 g/g) was found in the cultured stroma, which is considerably higher than those of wild stroma and worm. For Ca, there is a similar content in the cultured stroma (547 g/g) and natural stroma (453 g/g), and a relatively lower concentration (238 g/g) was found in natural worm.
Potassium and magnesium are essential electrolytes for main- taining normal fluid balance in cells and a delicate balance of these two elements is reported to prevent an increase in blood pres- sure and to maintain normal cardiac rhythm. Calcium is known to be involved in muscle contraction and relaxation, blood clotting, proper nerve function and body immune defenses [20,21]. The bio- logical roles of K, Mg and Ca are essential for disease prevention and control [22].
In addition to the four macro elements analyzed in this study, 16 trace elements were also studied. Trace elements, in minute quan- tities, are essential for the normal health and function of humans. They can be divided into three groups of essential microelements, possible essential microelements and potentially toxic microele- ments or non-essential microelements. Trace elements, whether essential or non-essential, can cause morphological abnormalities, reduce growth and increase mortality and mutagenic effects, above their critical threshold concentrations [23].
Essential microelements, including Fe, Zn, Cu, Mn, Cr, Mo, Co, Se, Ni and V were determined in this work. The result showed that the concentrations of Fe, Cu, Mn, V and Cr in natural stroma are between 18.62 and 114.72 g/g, which is higher than those of cultured strom and natural worm, except that Zn is higher in the cultured strom (84.81 ppm) than in the natural stroma (65.25 ppm), and the con- centration of Cu is broadly similar in cultured strom (14.50 g/g) and natural stroma (13.18 g/g). For Ni, Co, Mo and Se, the content of wild stroma is between 0.74 and 3.23g/g. While the cul- tured stroma (0.46–2.86 g/g) and natural worm (0.20–1.35 g/g) showed a lower content for the same elements.
Fe, Cu, Mn and Zn are essential elements in enzyme metabolism. They have immunomodulatory functions and thus influence the susceptibility to the course and the outcome of a variety of viral infections [24]. The importance of iron in maintaining good health and well being has long been recognized by nutritionists [25]. Co, as a component of the vitamin B12 complex, is an essential trace element of human body, which can significantly stimulate the pro- duction of blood and effectively cure the macrocytic anemia [19]. The essential trace element molybdenum is bound to a required for the physiological function of some enzymes, such as xanthine oxidize, aldehyde oxidase, sulfite oxidase, nitrate reductase, and so on. Metallic vanadium is probably an essential trace element, which most common valence states are V3+ , V4+ , and V5+ . The pentavalent state (VO3 − ) is the most common form in extracel- lular fluid whereas the quadrivalent form (VO2+) predominates in intracellular [26]. Although the concentration of V in most foods is low, food is the main source of this trace element for the general population.
Non-essential elements such as Ba and Sn were also detected in this study. The element Ba is rich not only in natural stroma (8.12 g/g), but also in the cultured stroma (1.89 g/g) and natural worm (6.60 g/g), while the concentration of Sn is only between 0.15 and 0.76g/g in all the three samples. Although there are no reported physiological effects of Ba and Sn at low concentra- tions, their accumulation in the body system could be detrimental to health. Under normal conditions, the element barium has no remarkable trophism or toxic action to the human body. However, some barium salts are usually toxic to human beings. The amount of an essential nutrient considered adequate for requirements of humans is termed the dietary reference intake (DRI) [27]. Ingestion limits of barium have not been established because of this element has no DRI or radiological component.
Potentially toxic microelements, including Pb, Cd, Hg and As are strictly limited at ultra-low levels in food and herbal medicine because of their potent toxicity. Harmful elements, such as Cd and Hg, were not detected in the cultured stroma. Their levels are also very low in both the natural stroma and the natural worm. Pb and As, the content is low in the three samples. For heavy metals, the highest levels in Table 3 were less than the maximum levels accord- ing to Chinese Pharmacopeia (2010). The contents of Cu, Hg, Pb, As and Cd are limited to 20, 0.2, 5.0, 2.0, 0.3 g/g separately.
Cd, Pb, Hg and As have no known physiological function yet reported. They are reported to be toxic and should be considered as a high risk factor to public health in general [28]. Cd has an adverse effect on brain metabolism and has other severe effects such as prostate cancer, kidney, liver, lungs and bone damage. Chil- dren are particularly at risk from Pb consumption, both before and after birth [20]. However, the potential of some elements to be harmful or beneficial is highly dependent upon their speciation. For example, the toxicity of arsenic is tremendously different in various
A further study by Wei et al. on C. sinesis analyzed 24 mineral elements, the abstract and conclusion of the report will be copied below, followed by the graphics.
Abstract
Cordyceps sinensis (C. sinensis) is a natural product that has diverse nutritional and medicinal values. Since the availability of natural C. sinensis becomes limited its authentication and quality control is of high significance. Herein we report on profiling of metals in C. sinensis by using inductively coupled plasma mass spectrometry (ICP-MS). The analysis reveals that C. sinensis contains a wide array of essential elements, including P, Mg, Zn, Cu, Fe, etc. Toxic metals detected are Cd, Pb, and As. In all five samples analyzed Pb contents are below 2.0 ppm. Arsenic level in C. sinensis caterpillar is significantly higher than that in its mycelium and varies from 3.0 to 32 ppm likely due to soil contamination. It‘s for the first time demonstrated in this work that clustering analysis on the proposed metal profiles consisting of 24 elements is very useful to identify “abnormal” C. sinensis samples, thus adding another dimension to the effective means for authentication and quality assessment of this highly demanded previous natural product.
4. Conclusions
226 Profiling of metals in C. sinensis, a precious natural product by using an ICP-MS method has been performed for the first time. The profiling reveals
228 that C. sinensis contains a wide array of essential elements. Among them P, Mg, Fe, and Zn are present at high levels. It also contains toxic metals,
230 including Pb and As. While Pb level is below the limit of 5 ppm set for Pb in herbal medicines according to Pharmacopeia it is found that As level in
232 caterpillar samples can be as high as 32 ppm likely due to soil contamination. Clusterin
analysis on the proposed metal profiles consisting of 24 elements
234 proves effective for identifying “abnormal” C. sinensis samples.
Mineral Analysis Conclusions
In this work, 20 elements (essential, non-essential and toxic) in cultured and wild (stroma and worm) C. kyushuensis have been determined by means of ICP-MS. According to the results, the element concentrations in the three samples were significantly dif- ferent (p < 0.05). The values of elemental contents were the highest in the stroma of natural C. kyushuensis except for Mg, Zn and Cu. The discrepancy of element content between cultured and natural C. kyushuensis samples might be resulted from different growing conditions, such as temperature, humidity, pH, quantity of illumi- nation, element level of soil and culture medium, and so on [30]. The data obtained in this research will be valuable for complementing the composition knowledge of C. kyushuensis.
Mycopharmacy - Dietetic Medicine - Extracts and Nutrition
The most bioactive components extracted from Cordyceps, such as cordycepin, cordycepic acid and CP, which have anti-aging and sleep regulating effects, have been authorized as new medicinal resources. Many of these medicines are being prepared as polysaccharide enriched fractions of C. sinensis and militaris, which have greatly overlapping chemical profiles, pharmacological actions, and historical applications.
These products have many functions that are mainly focused on the following aspects: enhancing physique, anti-aging, protecting the heart, improving sleep, increasing appetite, increasing immunity, etc.
Extraction Protocol †
Extraction protocol was designed based on the work of Wang et al. who optimized an Ultrasonic-Assisted Extraction of Cordycepin from Cordyceps militaris using orthogonal experimental design. Factors examined included ethanolic percentage, extraction time, extraction temperature, liquid to solid ratio and ultrasonnic frequency (kHz). Cordycepin is the principle active component of Cordyceps medicinal mushroom and is therefore the principle target of the soul extraction. Here in alchemistry we use the term 'soul' interchangably with 'specialized metabolites' where the soul is representative of the total profile of specialized metaboilites within the mushroom, not just the singluar principle component. Secondary targets include cordycepic acid, polysaccharides, steroids, fatty acids, proteins. Last but certainly not least, this extraction focused on a mineral retrieval from the 'body' of the mushroom material. Minerals represent essential nutritional value to human beings and are exclusively found in spagyric medicines. The only other way to get potent mineral nutritional satiation would be to eat the mushroom or food stuffs, which we highly encourage! 60 min extraction duration, 60% ABV, and 60ºC temperature were all targeted in the soxhlet extraction. Duration began when full saturation was achieved and the first flush of the soxhlet apparatus was initiated. At the one hour mark, this fraction was separated and the solvent was replaced with DI water and extracted at the same temperature until the solvent ran clear. When solvent was clear, this fraction was removed and solvent was replaced with 95%ABV and extracted further until the solvent ran clear. Finally, three fractions were separated, a Cordycepin fraction, a water fraction, and a 95% ethanol fraction. The cordycepin fraction remained separate while the other two were evaporated of solvent in a dehydrator and the residues were collected and combined together to form a powder. We further took the plant body to undergoe spagyric extraction, calcining the remaining material at 550ºF, dissolving the remaining white ashes into DI water, filtering and crystallizing the salts from the aqueous solution. The mineral salts were then pulverized and combined into the 50% ABV cordycepin fraction, and refluxed for 4 hours until total dissolution was achieved. The resulting product was bottled and labeled and stored in a refrigerator at ~35ºF.
Citations
Das, Shonkor Kumar, et al. "Medicinal uses of the mushroom Cordyceps militaris: current state and prospects." Fitoterapia81.8 (2010): 961-968.\
Liu Y, Wang J, Wang W, Zhang H, Zhang X, Han C. The Chemical Constituents and Pharmacological Actions of Cordyceps sinensis. Evid Based Complement Alternat Med. 2015;2015:575063. doi: 10.1155/2015/575063. Epub 2015 Apr 16. PMID: 25960753; PMCID: PMC4415478.
Zhou, Xuanwei, et al. "Cordyceps fungi: natural products, pharmacological functions and developmental products." Journal of Pharmacy and Pharmacology 61.3 (2009): 279-291.
The American Journal of Chinese MedicineVol. 35, No. 04, pp. 559-574 (2007)No AccessGanodermataceae: Natural Products and Their Related Pharmacological FunctionsXuanwei Zhou, Juan Lin, Yizhou Yin, Jingya Zhao, Xiaofen Sun, and Kexuan Tang https://doi.org/10.1142/S0192415X07005065
Mizuno, Takashi. "Medicinal effects and utilization of Cordyceps (Fr.) Link (Ascomycetes) and Isaria
TY - https://www.researchgate.net/journal/Journal-of-Pharmacy-and-Nutrition-Sciences-1927-5951 Bui Thanh, Tung, Lan, Do, Lan, Pham, Nha, Pham 2016/12/02 Cordyceps militaris (L.) Link: Chemical Bioactive Compounds and Pharmacological Activities VL - 610.6000/1927-5951.2016.06.04.4 Journal of Pharmacy and Nutrition Science. DOI:10.6000/1927-5951.2016.06.04.4
Zhang, Guoying, et al. "Determination of essential and toxic elements in Cordyceps kyushuensis Kawam by inductively coupled plasma mass spectrometry." Journal of Pharmaceutical and Biomedical Analysis 72 (2013): 172-176.
Journal of Proteomics Volume 181, 15 June 2018, Pages 24-35
A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis. Author links open overlay panelXu Zhang a, Qun Liu a, Wei Zhou a, Ping Li a, Raphael N. Alolga a, Lian-Wen Qi a b, Xiaojian Yin aaState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinabClinical Metabolomics Center, China Pharmaceutical University, Nanjing 211198, China. Received 11 January 2018, Revised 27 March 2018, Accepted 27 March 2018, Available online 30 March 2018, Version of Record 5 May 2018.
Vannucci L, Krizan J, Sima P, Stakheev D, Caja F, Rajsiglova L, Horak V, Saieh M. Immunostimulatory properties and antitumor activities of glucans (Review). Int J Oncol. 2013 Aug;43(2):357-64. doi: 10.3892/ijo.2013.1974. Epub 2013 Jun 5. PMID: 23739801; PMCID: PMC3775562.
Wei, X., Hu, H., Zheng, B., Arslan, Z., Huang, H.-C., Mao, W., & Liu, Y.-M. (2017). Profiling metals in Cordyceps sinensis by using inductively coupled plasma mass spectrometry. Analytical Methods, 9(4), 724–728.doi:10.1039/c6ay02524b
Preliminary Information
Cordecyps militaris is an entomopathogenic fungus, known for over 4000 years, and is a leading pioneer in the Green Pharmacy Revolution. "Cordyceps" is traditionally and conventionally understood as the dried fungus of C. militaris and sinensis (both species are myco-chemically and pharmacologically similiar and can be used interchangably) that commonly grows out caterpillar larvae and other entomology. These larvae and other entomologies include about 20 species of five genera, Hepialus, Hepialiscas, Napialus, Forkalus and Bipectilus, which are classified as Hepialidae. Among them, Hepialus armoricanus, H. obilfurcus, H. baimaensis and H. renzhiensis are predominant hosts of C. sinensis.124 The parasitic complex of the fungus and the caterpillar is found in the soil of the prairie at an elevation of 3500–5000 meters in the northwestern part of China. It is for this geographically exclusive reason the natural Cordyceps is a significantly rare species in the current marketplace.
It is of great interest and would be of great benefit to hummanity for researchers to aim at developing techniques for isolating strains of C. sinensis. Particularly, fermentable strains are of significant interest because fermentation allows more medicinal activity to be made from smaller amounts of the mushroom. Fermented sodas and tonics of C. militaris and sinensis have been clinically proven to be incredibly therapeutic, prophylactic, and be potent vehicles of the mushrooms diverse medicinal and therapeutic devices.
General Background
C. militaris and sinensis, mycopharmaceuticals and plant medicine in general is a prime example of the "Back to the Pavillion theory". This theory suggests that nowadays, people are willing to wear cotton clothes instead of synthetic/polyester ones, eat vegetables and organic foods rather than processed mass produced gorceries, and in the same manner, willing to take herbal/natural medicines rather than the synthetic drugs.
Because of this cycling back to our roots (or mycelium I could say) we are re-discovering the reason we humans were able to persevere so well over the millinea. It wasn't becasue a technoloigcal age of information and innovation of industrial advancments allowing us to live more lavish and opulent lifestyles. It was moreso our direct connection to nature and our richly organic and diverse natural lives which sustained and fertilized the spirit of manking to its indomnitable and unconquerable state. A state which is greatly lacking in the modern era.
This being said, C. militaris is extremely popular among TCM as a Dietetic Chinese drug, also known as ‘edible Chinese drugs’, ‘dietetic material medicine’ or ‘medicinal food’, although the word ‘drug’ could be misinterpreted in the West. Its clear Hippocrates reciveved his medicinal initiation from more ancient sources who have been practicing with food as medicine for thosuands of years before him. What else did these ancients know and understand? What other wisdom do we lack that we must re-learn from our common ancestry?
Currently, there is explosive research into Cordecyps within the field of pharmacognosy. Cordecyps represent a largely untapped reservoir of rare and species unique bioactive chemicals that we already know are serving as miriculous medicines, we (westerners) have simply yet to name the 'active constitents' responsible for the medicinal effects. This art of naming is hard (headed) sciences way of coming to understand the nature and efficacy and potential applications of this unique medicine. While we are FAR from a comprehensive analysis of the Cordyceps myco-chemical profile, we have still come a far way already. It is worth noting that internal constituents and phenotypes are equally as reliant on the environmental conditions for an organisms expression, as is the genetic makeup itself. It is known that most flora only are expressing about 20% of their available genetic code, leaving about 80% unactualized genetic potential, which means under slightly different environmental conditions, the same genetic code could potentially produce a nearly completely different mushroomwith totally different chemical constituents. This same law applies to the majority of the flroa and fauna, and certainly humzn beings (although, this is another discussion). All things consdiered, even with our limited knowledge and conceptually limited modes of analysis, again, we still have been able to peer into the mystery of the mushrooms to shine light on their wondrous ability to heal us.
Below is a brief overveiw and condensation of much of the current scientific literature describing the principle medicinal constituents and pharmacologic actions of Cordyceps militaris and sinensis, and it is followed by a nutritional analysis. I call this, MYCOPHARMACY.
Mycopharmacy
There have been many empirically found medicinal and therpeutic effects of this mushroom which include pro-sexual, anti-inflammatory, anti-oxidant/anti-aging, anti-tumour/anti-cancer/anti-leukemic, anti-proliferative, anti-metastatic, immunomodulatory, anti-microbial, anti-bacterial, anti-viral, anti-fungal, anti-protozoal, insecticidal, larvicidal, anti-fibrotic, steroidogenic, hypoglacaemic, hypolipidaemic, anti-angiogenetic, anti-diabetic, anti-HIV, anti-malarial, anti-fatigue, neuroprotective, liver-protective, reno-protective as well as pneumo-protective. There is antitumour effect, hepatoprotection and inflammatory effects, and antioxidant, nephroprotective, and antiapoptotic properties. It also has the unique funtion of delaying senility. It is commonly used in China to replenish the kidney and soothe the lung, for the treatment of fatigue, night sweating, hyposexuality, hyperglycaemia, hyperlipidaemia, asthenia after severe illness, respiratory disease, renal dysfunction and renal failure, arrhythmias and other heart disease and liver disease. A large amount of experimental evidence has shown that fungal polysaccharides have a wide range of bioactivities including antitumor [52], anti-influenza virus [53], immunopotentiation [54], hypoglycemic [55], hypocholesterolemic [56], and antioxidant effects [51].
Cordecypin
The main mechanism of cordycepin is to inhibit purine biosynthesis pathway. Cordycepin also induces RNA or DNA chain termination and interacts with receptors for mTOR which has an important role in the regulation of protein synthesis. Cordycepin is converted into 5’mono-, di- and tri-phosphates and can inhibit the activity of ribose-phosphate, pyrophosphokinase and 5-phosphoribosyl-1-pyrophosphate amidotransferase enzyme which play important role in biosynthesis of purines.
The structure of cordycepin is similar to the adenosine, a cellular nucleoside. Therefore it can act as a nucleoside analogue. Some other key active components inlcude (such as polysaccharides, ergosterol, mannitol, homocitrullyl aminoadenosine, cordycepic acid).
Cordycepin is a category of compounds that exhibits significant therapeutic potential and has many intracellular targets, including nucleic acid, apoptosis, and cell cycle. Tuli et al. researched the variety of molecular mechanisms that mediate the pharmacological effects of cordycepin. Besides, they deem that cordycepin can participate in various molecular processes in cells because of its similarity with adenosine [7]. Wang et al. investigated the effects of cordycepin in prevention of focal cerebral ischemic/reperfusion (IR) injury and suggested that cordycepin has a neuroprotective effect in the ischemic brain, which is due to the inhibition of inflammation and increase of antioxidants activity related to lesion pathogenesis [23, 24]. So cordycepin could be an attractive therapeutic candidate with oral activity against I/R-associated heart diseases such as myocardial infarction [25]. Besides, cordycepin showed the obvious analgesic effect.
Cordycepin is a potent anti-inflammatory and analgesic medicine. In addition, cordycepin stimulated the release of some cytokines of resting PBMCs and influenced proliferation of PBMCs and transcription factors in THP-1 cell line. Accordingly, cordycepin can intensively regulate the functions of human immune system. ]. Cordycepin is a broad spectrum biocidal compound possessing not only antitumor activity but also antibacteria, antivirus, and insecticidal activities [2].
To date, six nucleobases (Figure 3), including cytosine, uracil, thymine, adenine, guanine, and hypoxanthine, were determined in natural and cultured Cordyceps sinensis. The overall content of nucleosides is much higher in cultured Cordyceps sinensis than in natural ones by comparison.
Cordecypic acid
Cordycepic acid is also a principal bioactive compound of C. militaris. The structure was identified first as 1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid. Later, this compound was crystallized and identified as d-mannitol. It is used in the anti-aging nutritional supplement and also for its strong pro-sexual activity.
Pro-Sexual Activity of Cordycepic acid
Traditional medicine has used Cordyceps species for improving the sexual function. Wen-Hung Lin et al., have shown the spermatogenic effect of C. militaris. diet supplemented with C. militaris mycelium to subfertile boars for 2 months. The sperm production was increased significantly in boars fed with diet contained C. militaris extract in both quality of fertile sperm and the quantity of semen volume and total sperm number. They also detected the cordycepin in plasma in boars supplemented with C. militaris. More interesting, an amount of motile sperm cells and sperm morphology were also enhanced significantly.
Polysaccharides
Cordyceps sinensis contains a great deal of polysaccharides, which can be in the range of 3–8% of the total weight. Poly saccharides are organic sugar molecules containing carbon, hydrogen, and oxygen, also known as carbohydrates (that is, Carbo- for carbon, hydr- for hydrogen, and -ate for oxygen). A large amount of experimental evidence has shown that fungal polysaccharides have a wide range of bioactivities including antitumor [52], anti-influenza virus [53], immunopotentiation [54], hypoglycemic [55], hypocholesterolemic [56], and antioxidant effects [51].
Cordycepic acid is a phosphorylated monosaccharide (D-mannitol) which carries an array of activity beyond the pro-sexual. It is namely an anti-aging molecule which protects afainst genetic degradation, it carries anti cancer effects, is anti oxidizing and anti inflammatory of course. But the key thing i believe is its ability to maturally modulate the immun responses By provoking innate responses from the immunology, it strengthens innate immunity rather than supplying foreign devices to do the bodies work for it. This exercising the endowed healing system is of great benefit for long lasting health and wellness, opposed to temporary short term relief often generated by other specialized metabolites, although this is likewise highly useful.
In general, polysaccharide activity is typically more complex and has more bioactivity when a compound itself is more conplex structurally, alementally, and through other physical traits. For example, many other studies have suggested that the pharmacological activity of the polysaccharide was correlated with its characteristics. In many cases of medicinal mushrooms, polysaccharides' milecular weight was the determining factor of the potency of the antitumor activity [57]. In other words, Sasaki et al. confirmed that the fungi polysaccharide's antitumor activity is related to the molecular weight, and the fungi polysaccharide has antitumor activity if its molecular weight is greater than 16000.
the pharmacological activity of the polysaccharide was correlated with its characteristics such as molecular weight. It has been reported that polyglucans with higher molecular weight (10–1000 kDa) tend to have greater water solubility and therefore have a more effective antitumour activity. The anticancer effects of fungal polysaccharides arise from the enhancement of the body's immune system rather than direct cytocidal effects.62
Crude protein and amino acids
Kwan showed C. militaris has a high ratio of the essential amino acids, such as lysine (101.2 mg/g), methionine (62.7 mg/g), and acidic amino acids of glutamic acid (57.5 mg/g) and aspartic acid (43.9 mg/g), a low of tyrosine content (4.7 mg/g) [14
Previous papers have reported that the content of crude protein was in the range 29.1–33%.27,33,66 The protein was composed of 18 amino acids, including aspartic acid, threonine, serine, glutamate, proline, glycine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine, histidine, cystine, cysteine and tryptophan. The content of amino acids after hydrolysis is mostly reported as 20–25%, the lowest being 5.53%, the highest being 39.22%. The highest contents are glutamate, arginine and aspartic acid, and the major pharmacological components are arginine, glutamate, tryptophan and tyrosine.67 The content of amino acids in the commercial preparation of Cordyceps is significantly higher than in the mycelia of C. sinensis, which is similar to the content in the fruit body of C. sinensis.
Protection of heart and antihypertensive effects
Xu et al.108 reported that the alcoholic extracts had a protective effect on myocardial injury induced by adriamycin in rats. The mechanism was suggested to be that mannitol, amino acids and polysaccharides in the extracts played an important role; all the substances had the effect of nourishing the myocardium and enhancing its anti-injury capacity.108 With the same experimental methods, Gu et al.109 reported that Lungfit (C. sinensis compounds) could be beneficial for myocardial ischaemia/reperfusion injury as well. However, both alcohol and water extracts had obviously protective effects on the rat isolated heart.
Nutritional Information
Another study on a Cordyceps species analyzed 20 different elemental minerals subdivided into essential (macro and micro) and non-essential/toxic from two different asscessions, a wild type, and a cultivated type. The table is pictured below and a description of the results taken directly from the literature are described directly below that.
"Among the essential elements estimated in the present work we found the macro elements (Na, K, Ca and Mg) and microelements (Zn, Fe, Cu, Mn, V, Cr, Ni, Co, Mo, and Se); along the nonessential elements and the toxic elements Ba, Sn, As, Cd, Pb and Hg. The results are presented in Table 3. Trace elements contained in three different samples were considerably different at the 95% confidence level according to the results of one-way analysis of variance (ANOVA).
For macro element, in this study, Na (1420g/g) and K (946g/g) in wild stroma exhibited the highest levels and were significantly different from the other two samples. While the max- imum content of Mg (986 g/g) was found in the cultured stroma, which is considerably higher than those of wild stroma and worm. For Ca, there is a similar content in the cultured stroma (547 g/g) and natural stroma (453 g/g), and a relatively lower concentration (238 g/g) was found in natural worm.
Potassium and magnesium are essential electrolytes for main- taining normal fluid balance in cells and a delicate balance of these two elements is reported to prevent an increase in blood pres- sure and to maintain normal cardiac rhythm. Calcium is known to be involved in muscle contraction and relaxation, blood clotting, proper nerve function and body immune defenses [20,21]. The bio- logical roles of K, Mg and Ca are essential for disease prevention and control [22].
In addition to the four macro elements analyzed in this study, 16 trace elements were also studied. Trace elements, in minute quan- tities, are essential for the normal health and function of humans. They can be divided into three groups of essential microelements, possible essential microelements and potentially toxic microele- ments or non-essential microelements. Trace elements, whether essential or non-essential, can cause morphological abnormalities, reduce growth and increase mortality and mutagenic effects, above their critical threshold concentrations [23].
Essential microelements, including Fe, Zn, Cu, Mn, Cr, Mo, Co, Se, Ni and V were determined in this work. The result showed that the concentrations of Fe, Cu, Mn, V and Cr in natural stroma are between 18.62 and 114.72 g/g, which is higher than those of cultured strom and natural worm, except that Zn is higher in the cultured strom (84.81 ppm) than in the natural stroma (65.25 ppm), and the con- centration of Cu is broadly similar in cultured strom (14.50 g/g) and natural stroma (13.18 g/g). For Ni, Co, Mo and Se, the content of wild stroma is between 0.74 and 3.23g/g. While the cul- tured stroma (0.46–2.86 g/g) and natural worm (0.20–1.35 g/g) showed a lower content for the same elements.
Fe, Cu, Mn and Zn are essential elements in enzyme metabolism. They have immunomodulatory functions and thus influence the susceptibility to the course and the outcome of a variety of viral infections [24]. The importance of iron in maintaining good health and well being has long been recognized by nutritionists [25]. Co, as a component of the vitamin B12 complex, is an essential trace element of human body, which can significantly stimulate the pro- duction of blood and effectively cure the macrocytic anemia [19]. The essential trace element molybdenum is bound to a required for the physiological function of some enzymes, such as xanthine oxidize, aldehyde oxidase, sulfite oxidase, nitrate reductase, and so on. Metallic vanadium is probably an essential trace element, which most common valence states are V3+ , V4+ , and V5+ . The pentavalent state (VO3 − ) is the most common form in extracel- lular fluid whereas the quadrivalent form (VO2+) predominates in intracellular [26]. Although the concentration of V in most foods is low, food is the main source of this trace element for the general population.
Non-essential elements such as Ba and Sn were also detected in this study. The element Ba is rich not only in natural stroma (8.12 g/g), but also in the cultured stroma (1.89 g/g) and natural worm (6.60 g/g), while the concentration of Sn is only between 0.15 and 0.76g/g in all the three samples. Although there are no reported physiological effects of Ba and Sn at low concentra- tions, their accumulation in the body system could be detrimental to health. Under normal conditions, the element barium has no remarkable trophism or toxic action to the human body. However, some barium salts are usually toxic to human beings. The amount of an essential nutrient considered adequate for requirements of humans is termed the dietary reference intake (DRI) [27]. Ingestion limits of barium have not been established because of this element has no DRI or radiological component.
Potentially toxic microelements, including Pb, Cd, Hg and As are strictly limited at ultra-low levels in food and herbal medicine because of their potent toxicity. Harmful elements, such as Cd and Hg, were not detected in the cultured stroma. Their levels are also very low in both the natural stroma and the natural worm. Pb and As, the content is low in the three samples. For heavy metals, the highest levels in Table 3 were less than the maximum levels accord- ing to Chinese Pharmacopeia (2010). The contents of Cu, Hg, Pb, As and Cd are limited to 20, 0.2, 5.0, 2.0, 0.3 g/g separately.
Cd, Pb, Hg and As have no known physiological function yet reported. They are reported to be toxic and should be considered as a high risk factor to public health in general [28]. Cd has an adverse effect on brain metabolism and has other severe effects such as prostate cancer, kidney, liver, lungs and bone damage. Chil- dren are particularly at risk from Pb consumption, both before and after birth [20]. However, the potential of some elements to be harmful or beneficial is highly dependent upon their speciation. For example, the toxicity of arsenic is tremendously different in various
A further study by Wei et al. on C. sinesis analyzed 24 mineral elements, the abstract and conclusion of the report will be copied below, followed by the graphics.
Abstract
Cordyceps sinensis (C. sinensis) is a natural product that has diverse nutritional and medicinal values. Since the availability of natural C. sinensis becomes limited its authentication and quality control is of high significance. Herein we report on profiling of metals in C. sinensis by using inductively coupled plasma mass spectrometry (ICP-MS). The analysis reveals that C. sinensis contains a wide array of essential elements, including P, Mg, Zn, Cu, Fe, etc. Toxic metals detected are Cd, Pb, and As. In all five samples analyzed Pb contents are below 2.0 ppm. Arsenic level in C. sinensis caterpillar is significantly higher than that in its mycelium and varies from 3.0 to 32 ppm likely due to soil contamination. It‘s for the first time demonstrated in this work that clustering analysis on the proposed metal profiles consisting of 24 elements is very useful to identify “abnormal” C. sinensis samples, thus adding another dimension to the effective means for authentication and quality assessment of this highly demanded previous natural product.
4. Conclusions
226 Profiling of metals in C. sinensis, a precious natural product by using an ICP-MS method has been performed for the first time. The profiling reveals
228 that C. sinensis contains a wide array of essential elements. Among them P, Mg, Fe, and Zn are present at high levels. It also contains toxic metals,
230 including Pb and As. While Pb level is below the limit of 5 ppm set for Pb in herbal medicines according to Pharmacopeia it is found that As level in
232 caterpillar samples can be as high as 32 ppm likely due to soil contamination. Clusterin
analysis on the proposed metal profiles consisting of 24 elements
234 proves effective for identifying “abnormal” C. sinensis samples.
Mineral Analysis Conclusions
In this work, 20 elements (essential, non-essential and toxic) in cultured and wild (stroma and worm) C. kyushuensis have been determined by means of ICP-MS. According to the results, the element concentrations in the three samples were significantly dif- ferent (p < 0.05). The values of elemental contents were the highest in the stroma of natural C. kyushuensis except for Mg, Zn and Cu. The discrepancy of element content between cultured and natural C. kyushuensis samples might be resulted from different growing conditions, such as temperature, humidity, pH, quantity of illumi- nation, element level of soil and culture medium, and so on [30]. The data obtained in this research will be valuable for complementing the composition knowledge of C. kyushuensis.
Mycopharmacy - Dietetic Medicine - Extracts and Nutrition
The most bioactive components extracted from Cordyceps, such as cordycepin, cordycepic acid and CP, which have anti-aging and sleep regulating effects, have been authorized as new medicinal resources. Many of these medicines are being prepared as polysaccharide enriched fractions of C. sinensis and militaris, which have greatly overlapping chemical profiles, pharmacological actions, and historical applications.
These products have many functions that are mainly focused on the following aspects: enhancing physique, anti-aging, protecting the heart, improving sleep, increasing appetite, increasing immunity, etc.
Extraction Protocol †
Extraction protocol was designed based on the work of Wang et al. who optimized an Ultrasonic-Assisted Extraction of Cordycepin from Cordyceps militaris using orthogonal experimental design. Factors examined included ethanolic percentage, extraction time, extraction temperature, liquid to solid ratio and ultrasonnic frequency (kHz). Cordycepin is the principle active component of Cordyceps medicinal mushroom and is therefore the principle target of the soul extraction. Here in alchemistry we use the term 'soul' interchangably with 'specialized metabolites' where the soul is representative of the total profile of specialized metaboilites within the mushroom, not just the singluar principle component. Secondary targets include cordycepic acid, polysaccharides, steroids, fatty acids, proteins. Last but certainly not least, this extraction focused on a mineral retrieval from the 'body' of the mushroom material. Minerals represent essential nutritional value to human beings and are exclusively found in spagyric medicines. The only other way to get potent mineral nutritional satiation would be to eat the mushroom or food stuffs, which we highly encourage! 60 min extraction duration, 60% ABV, and 60ºC temperature were all targeted in the soxhlet extraction. Duration began when full saturation was achieved and the first flush of the soxhlet apparatus was initiated. At the one hour mark, this fraction was separated and the solvent was replaced with DI water and extracted at the same temperature until the solvent ran clear. When solvent was clear, this fraction was removed and solvent was replaced with 95%ABV and extracted further until the solvent ran clear. Finally, three fractions were separated, a Cordycepin fraction, a water fraction, and a 95% ethanol fraction. The cordycepin fraction remained separate while the other two were evaporated of solvent in a dehydrator and the residues were collected and combined together to form a powder. We further took the plant body to undergoe spagyric extraction, calcining the remaining material at 550ºF, dissolving the remaining white ashes into DI water, filtering and crystallizing the salts from the aqueous solution. The mineral salts were then pulverized and combined into the 50% ABV cordycepin fraction, and refluxed for 4 hours until total dissolution was achieved. The resulting product was bottled and labeled and stored in a refrigerator at ~35ºF.
Citations
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Liu Y, Wang J, Wang W, Zhang H, Zhang X, Han C. The Chemical Constituents and Pharmacological Actions of Cordyceps sinensis. Evid Based Complement Alternat Med. 2015;2015:575063. doi: 10.1155/2015/575063. Epub 2015 Apr 16. PMID: 25960753; PMCID: PMC4415478.
Zhou, Xuanwei, et al. "Cordyceps fungi: natural products, pharmacological functions and developmental products." Journal of Pharmacy and Pharmacology 61.3 (2009): 279-291.
The American Journal of Chinese MedicineVol. 35, No. 04, pp. 559-574 (2007)No AccessGanodermataceae: Natural Products and Their Related Pharmacological FunctionsXuanwei Zhou, Juan Lin, Yizhou Yin, Jingya Zhao, Xiaofen Sun, and Kexuan Tang https://doi.org/10.1142/S0192415X07005065
Mizuno, Takashi. "Medicinal effects and utilization of Cordyceps (Fr.) Link (Ascomycetes) and Isaria
TY - https://www.researchgate.net/journal/Journal-of-Pharmacy-and-Nutrition-Sciences-1927-5951 Bui Thanh, Tung, Lan, Do, Lan, Pham, Nha, Pham 2016/12/02 Cordyceps militaris (L.) Link: Chemical Bioactive Compounds and Pharmacological Activities VL - 610.6000/1927-5951.2016.06.04.4 Journal of Pharmacy and Nutrition Science. DOI:10.6000/1927-5951.2016.06.04.4
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Journal of Proteomics Volume 181, 15 June 2018, Pages 24-35
A comparative proteomic characterization and nutritional assessment of naturally- and artificially-cultivated Cordyceps sinensis. Author links open overlay panelXu Zhang a, Qun Liu a, Wei Zhou a, Ping Li a, Raphael N. Alolga a, Lian-Wen Qi a b, Xiaojian Yin aaState Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, ChinabClinical Metabolomics Center, China Pharmaceutical University, Nanjing 211198, China. Received 11 January 2018, Revised 27 March 2018, Accepted 27 March 2018, Available online 30 March 2018, Version of Record 5 May 2018.
Vannucci L, Krizan J, Sima P, Stakheev D, Caja F, Rajsiglova L, Horak V, Saieh M. Immunostimulatory properties and antitumor activities of glucans (Review). Int J Oncol. 2013 Aug;43(2):357-64. doi: 10.3892/ijo.2013.1974. Epub 2013 Jun 5. PMID: 23739801; PMCID: PMC3775562.
Wei, X., Hu, H., Zheng, B., Arslan, Z., Huang, H.-C., Mao, W., & Liu, Y.-M. (2017). Profiling metals in Cordyceps sinensis by using inductively coupled plasma mass spectrometry. Analytical Methods, 9(4), 724–728.doi:10.1039/c6ay02524b
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