Cordyceps. cicadae (C. cicadae) also known as cicadae flower, Chanhua or Sandwhe, belongs to the family Clavicipitaceae and the genus Cordyceps, which strictly parasitize on the cicada nymph or the larva of Cicada flammate, Platypleura kaempferi, Cryptotympana pustulata and Patylomia pieli. The host larva were consumed as nutrition and became a tightly packed mass of mycelium, then formed the flower bud-shaped stroma from the mouth, head or bottom of the cicada larva. It is a kind of wonderful biological complex of fungus and larva. The anamorph of C. cicadae is called as Isaria cidadae that was originally described by Miquel in 1838, after which many scientific names such as Isaria basili, Sphaeria sinclairi and Paecilomyces cicadae were developed [1].

Distribution of C. cicadae

C. cicadae is usually distributed in the tropical and subtropical region with temperature ranging from 18-24? and relative humidity of >80%. C. cicadae is usually grow vertically on sunny slopes at an altitude of 700-950 m. In China, they are most frequently seen in Fujian, Zhejiang, Yunnan, Sichuan and Jiangsu province or river valley of the Yunnan-Tibet Plateau [2-4]. In Japan, C. cicadae are mainly distributed in mountain and forest region at low altitude, south of Fukushima. In South Korea, C. cicadae are found on Jeju Island. In Taiwan, the fruiting body of C. cicadae is present in bamboo forest of northern Taiwan Mountain. Furthermore, they are also seen in Thailand, Southeast Asia, North America and Europe [5].

Record of C. cicadae in ancient traditional chinese medicines works

C. cicadae are one of the most valued traditional Chinese medicines (TCM) and have been used for about 1,600 years in China. It was first mentioned in Lei’s Treatise on Preparing Drugs (Lei Gong Pao Zhi Lun) written by Xiao Lei in the Liu Song Period of Northern and Southern Dynasties [6]. Its morphology was first described in Tu-Ching Pents’ao compiled by Song Su in the Song Dynasty that horn-like protuberances occurred on the heads of Cicada in mountain. Shi-Zhen Li in the Ming Dynasty clarified in Compendium of Materia Medica that C. cicadae exhibits activities of dispelling wind and heat, relieving convulsion, improving eyesight, removing cloudiness of eyes, and promoting eruption. Shi-Zhen Li also mentioned that C. cicadae were primarily used in treatment of infantile convulsions and morbid night crying of babies, palpitation and malaria. Ancient TCM works emphasized the therapeutic effect of C. cicadae for various eye diseases. For example, Prescriptions of the Bureau of Taiping People’s Welfare Pharmacy (Taiping Huimin Heji Ju Fang) written by Cheng Chen during the Song Dynasty stated that Superb C. cicadae Powder is specifically for treatment of acute conjunctivitis, chronic blepharitis, chronic dacryocystitis and pterygium.

Bioactivity

Modern pharmacological studies have indicated that C. cicadae exhibit a variety of biological functions (Table 1). Shi-Zhen Li claimed in Compendium of Materia Medica that C. cicadae have equivalent effect as cicada slough, so we also listed cicada slough in clinical application of C. cicadae (Tables 2 and 3). C. cicadae have been used to recede nephelium of eyeball and improving vision for a long time. The ideal efficacy of C. cicadae in eye treatments may be attributed to the antibiotics produced to fight fungal and bacterial infection, or specific active constituents. It is of great significance to explore bioactive components targeting vision improvement and its underlying mechanisms. There are two patent pending on “Preparation and application of active ingredients and its drug combinations of C. cicadae” in Taiwan (pending number 104112814) and China (pending number 201510303766.1). Another great commercial potential of C. cicadae application lies in prevention of chronic renal failure. Wang et al. [7] showed that C. cicadae obviously improved renal tubular function in chronic renal failure patients with the renal tubular dysfunction [7]. Studies have also found that active ingredients from C. cicadae exhibit renal protection. For example, Zhu et al. [8] reported that 12.5 μg/ml ergosterol peroxides from C. cicadae could ameliorate TGF-β1-induced renal fibroblast proliferation and fibrtonectin expression, thus combating progression of renal fibrosis [8]. Another example is that Peng et al. [9] demonstrated the protective effect of HEA on renal ischemia-reperfusion injury in mice [9]. There are many mushrooms possessing functions of lowering blood glucose levels and immunomodulation, but protection capacity on kidney of those mushrooms are rarely reported. In Taiwan, the numbers of patients undergoing hemodialysis have leaped to the first place worldwide. C. cicadae that had the equivalent pharmacological effect with C. sinensis might act as a better alternative, offering potential kidney-specific nutritional and health benefits.

Table 1: Physiological Activity of C. cicadae.

Table 2: Physiological Activity and Clinical Application of C. cicadae Powder.

Table 3: Food Safety Evaluation Tests of C. cicadae.

Potentially bioactive constituents of C. cicadae

Myriocin (ISP-1): Fujita et al [48] isolated and identified myriocin from fermented liquid of C. cicadae [48]. Its derivate FTY720 was developed and transferred to Novartis (Basel, Switzerland) for approval as a treatment for multiple sclerosis in USA and Europe, and obtained approval in USA in 2010 [49]. Pharmacological activities of FTY720 include immune adjustment [50], atherosclerosis alleviation [51] and anti-fungus [52].

Nucleosides: Adenosine: Adenosine is a metabolite of adenine nucleotides, which is an FDA approved drug in the USA and widely used for arrhythmia management [53]. The highest content of adenosine (1.90 mg/g) is seen in C. cicadae mycelium, which is 4 times higher (0.48 mg/g) than that in C. sinensis [54]. The content of adenosine in C. cicadae fruiting body is 3.437 mg/g [39]. Pharmacological studies showed that adenosine has beneficial effects on central nervous system [55], cardiovascular disorders [56], anti-platelet aggregation, radiation resistance and anti-tumor effect [57].

Cordycepin: Cordycepin is the first active ingredient of nucleosides isolated from fungi, displaying notable cytotoxicity. Li et al. [58] found that cordycepin concentration in C. cicadae mycelia was 2.77 mg/g, substantially higher than that in C. sinensis and equivalent to those in Cordyceps militaris (C. militaris) [58]. Cordycepin concentration in C. cicadae fruiting body is 0.736 mg/g [39]. Cordycepin exhibits biological activities such as anti-tumor [59], anti-microbial [60], anti- HIV effects [61], immunomodulation and endocrine modulation [62] as well as lowered serum cholesterol and lipoprotein levels. At present, cordycepin has been manufactured into drugs mainly intended for leukemia treatment, which have been under clinical trials.

N6-(2-hydroxyethyl) adenosine (HEA): Endogenous nucleosides and their analogues may participate in pain modulation [63,64]. The level of HEA in C. cicadae fruiting body was 7.025 mg/g [39]. HEA is the pivotal quality indicator for C. cicadae. HEA can bind to ?-receptors, thus exerting analgesic effect by curbing neurotransmitter release. The mechanism underlying pain regulation of HEA is different from that of opioid analgesics, so there is no dependence following treatment of HEA and is safer for clinical application. The great commercial potential in cancer treatment is worthy expecting [65]. Other researches revealed that HEA might be involved in calcium ion and myocardial contraction, which was beneficial to cardiovascular disorders [66]. In addition, its protective effect on renal ischemia- reperfusion injury in mice was proved [67].

Ergosterol and its Peroxides: Ergosterol and its peroxides are isolated from C. cicadae mycelium. Ergosterol possesses anti-oxidative capacity and is a precursor of vitamin D2 [68]. Ergosterol peroxides have many bioactivities such as anti-tumor, anti-inflammatory, anti-viral and anti-atherosclerosis activities. It can also inhibit T cell activation and proliferation induced by PHA activation and works as an immunosuppressor [69]. Zhu et al. [8] reported that ergosterol peroxides (12.5 μg/ml) from C. cicadae ameliorated TGF-ß1-induced renal fibroblast proliferation and fibrtonectin expression, thus combating progression of renal fibrosis [8].

Cyclic Heptapeptide: Duarte et al. [70] obtained 5 cyclic heptapeptides from C. cicadae, including bassintin, bassintin A, beauvericin, beauvericin A and beauvericin B [70]. Bassintin and bassintin A exhibit significant inhibitory effect on platelet aggregation induced by adenosine diphosphate (ADP) in rabbit (IC₅₀=1.4 × 10-4 mol/l) [71]. Pharmacological studies showed that beauvericins could against convulsion and tumor and had effect on arrhythmia and sedation [72]. Wang et al. [73] revealed that beauvericin J, beauvericin and beauvericin A from C. cicadae suppressed multiple drug-resistance hepatocellular carcinomas Hep G2/ADM cells, with an IC₅₀ of 2.40- 2.93 μM that far lower than 71.8 μM for doxorubicin [73].

Polysaccharide: Polysaccharide has distinct bio-activities sharing with other TCM, such as anti-tumor, anti-bacteria, anti-virus, antiradiation and anti-aging. Polysaccharides are also used for beautifying and moisture-holding of skin [74] and lowered blood glucose levels [25]. Recently, its immune-modulatory effect becomes the hotspot. C. cicadae polysaccharides significantly enhanced host’s immunity by enhancing phagocytic activity of reticuloendothelial cells in mice [75]. Takano et al. [1] also found that C. cicadae polysaccharide promoted Th1 immune response and up-regulated IL-2 and IFN-γ expression in Pyle collection of lymph nodes in rats [1]. No toxicity resulting from polysaccharide was found in animal toxicity test. Kim et al. [76] showed that polysaccharides derived from C. cicadae mycelium promoted maturity of dendritic cells, thus inducing anti-tumor immune responses [76].

Artificial cultivation of C. cicadae

Because of long-term excessive collection of wild C. cicadae, the supply of wild C. cicadae fails to meet the market demands. Artificially cultivated C. cicadae is an ideal substitute in developing healthcare products, by which the time cost and bacterial contamination rates can be reduced and bioactive components can be manipulated (Figure 1). Different cultivation techniques offer markedly different production of bioactive metabolites. Therefore, distinct bioactive constituents can be obtained by different cultivation methods to meet different aspects of healthcare demands.

Figure 1: Wild and artificially cultivated C. cicadae. (A) Wild C. cicadae. The parasitic nymphs of C. cicadae are with pale or pale yellow bodies of about 3 cm long and 1-1.5 cm wide. The head of the nymphs were clavate and roughened, displaying white at the front. The fine mycelia or fruiting body growing within the nymph body are referred to as coremium, presenting oval shape. The stroma will develop on the nymph body and extruded from the soil. The stroma usually has 3-4 branches in antler-like, cylindrical or rod-like shape, which form flower bud-shaped stroma. (B) Solid-state fermentation of C. cicadae. (C) Liquid fermentation of C. cicadae. After several days of inoculation, the color of the fermented fluid was turned purple.

Fruiting body production based on wild culture matrix

Chai et al. [77] established a novel solid-state fermentation method, by which fruit body with yellow pointed end could occur within 30 days [77]. The Lab of Bioengineering Center of Grape King Bio Ltd also successfully cultivated fruiting body of C. cicadae from brown ricebased culture matrix in 2011, from cultivation to harvesting fruiting bodies within about one month period [6]. In 2014, professor Jian- Yi Wu of Dayeh University also reported the cultivation method for substantial fruiting body by grains-based culture matrix [78].

Cultivation based on infected nymphs

Hu et al. [79] made the silkworm larva and silkworm chrysalis infected with Paecilomyces cicadae, and found that the infection rates of the two hosts were higher than 70%, with higher infection rate (90%-100%) seen in the silkworm chrysalis [79]. In 2009, South Korean scholar Jeong successfully inoculated C. cicadae mycelia onto the silkworm and developed fruiting bodies, on which high-dose oral toxicity test was carried out afterwards. The hosts of C. cicadae are not as specific as that of C. sinensis, indicating more easy availability in terms of mass cultivation and development.

Liquid fermentation of mycelium

Solid-stage cultivation ties up a great deal of manpower, and the remaining matrix may affect the quality of produced C. cicadae. Therefore, many institutions are flocking to liquid-state fermentation of C. cicadae mycelium. Hsu et al. [80] yielded mass production of C. cicadae mycelium using a variety of carbon source, nitrogen source and inorganic salts combined with liquid fermentation technique, offering obvious advantages of less pollution and short production cycle [80]. Furthermore, the functional component contents and physiological activity of produced C. cicadae mycelium was not inferior to that of wild C. cicadae mycelium.

Safety of C. cicadae

Safety evaluation of the artificially cultivated C. cicadae is an imperative process in development of C. cicadae-related products due to variance in bioactive components resulting from diverse cultivation procedures.

Reports on mistaken ingestion of wild C. cicadae

There are many reports on mistaken ingestion of C. cicadae in China. People are likely to mistakenly eat C. cicadae and be poisoned [81]. Harvested wild C. cicadae are mud-covered and can easily turn mouldy if not being fully cleaned. The reason of poisoning might be attributed to molds growth under the humid storage conditions.

Food safety evaluation tests

Jeong et al. [82] showed that rats were orally administered with fruiting bodies powder of the Paecilomyces sinclairii in concentration of 5% for 13 weeks had no significant effect on serum levels of BUN and creatinine [82]. In the acute toxicity test, Chen et al. [83] demonstrated that mice were orally administrated with extracts of C. cicadae or Paecilomyces cicadae (60 g/kg) had no significant effect on mortality 72 h following treatment, with a LD₅₀ of 14.2 ± 2.1 g/kg and 12.5 ± 2.1 g/ kg, respectively [83]. Song et al. [84] found that the maximum tolerable dose of C. cicadae was 80 g/kg in acute toxicity test, which was 444 times higher than that the clinically recommended dose [84]. In the subacute toxicity test, Kwack et al. [85] revealed that mice were orally administered with extracts of C. cicadae or Paecilomyces cicadae for 14 days had no significant effect on mortality, with a LD₅₀ of 5 g/kg [85]. There were no other abnormalities except for decreased thymus weight presented in male mice. Other research further confirmed that a 28- day consecutive oral administration of extracts of Paecilomyces cicadae at dose of 9 g/kg caused no toxicity-related lesions in rats. No toxic effect was observed in the acute toxicity test (piglet) [86], 3 different test systems of genotoxicity test [80], a 90-day oral toxicity test (rats) [87], as well as teratogenicity test (data unpublished) of C. cicadae mycelium produced using artificial fermentation method.

C. cicadae possesses multiple pharmacological activities that offers several featured advantages such as low toxicity, low price and easy availability of raw materials from artificial cultivation. The gene mutation rate of C. cicadae is only 1%, which is greatly lower than 10% seen in the C. sinensis, indicating that C. cicadae has relatively stable genetic structures and expected to be easily cultivated [88]. Because of the convenient cultivation technique for C. cicadae, a broad line of functional products and supplements can be manufactured based on fermented mycelia, incorporating healthcare foods, cosmetics, biological agriculture, and pharmaceuticals. At present, researches on C. cicadae are limited to preliminary medicinal chemistry and pharmacology studies. Further exploration on bioactive metabolites of C. cicadae is expected to expand its application scope and better exert its healthcare efficacy. Through advanced extraction and purification technique, the bioactive components of the C. cicadae can be used to develop botanical new drug. Development of C. cicadae on vision and kidney healthcare is a worth expecting project.