Evaluation of an Epitypified Ophiocordyceps formosana (Cordyceps s.l.) for Its Pharmacological Potential

The substantial merit of Cordyceps s.l. spp. in terms of medicinal benefits is largely appreciated. Nevertheless, only few studies have characterized and examined the clinical complications of the use of health tonics containing these species. Here, we epitypified C. formosana isolates that were collected and characterized as Ophiocordyceps formosana based on morphological characteristics, molecular phylogenetic analyses, and metabolite profiling. Thus, we renamed and transferred C. formosana to the new protologue Ophiocordyceps formosana (Kobayasi & Shimizu) Wang, Tsai, Tzean & Shen comb. nov. Additionally, the pharmacological potential of O. formosana was evaluated based on the hot-water extract from its mycelium. The relative amounts of the known bioactive ingredients that are unique to Cordyceps s.l. species in O. formosana were found to be similar to the amounts in O. sinensis and C. militaris, indicating the potential applicability of O. formosana for pharmacological uses. Additionally, we found that O. formosana exhibited antioxidation activities in vitro and in vivo that were similar to those of O. sinensis and C. militaris. Furthermore, O. formosana also displayed conspicuously effective antitumor activity compared with the tested Cordyceps s.l. species. Intrinsically, O. formosana exhibited less toxicity than the other Cordyceps species. Together, our data suggest that the metabolites of O. formosana may play active roles in complementary medicine.


Introduction
The entomopathogenic fungus Cordyceps s.l. (sensu lato) species, including Ophiocordyceps sinensis and Cordyceps militaris, are among the prestigious traditional Chinese medicines and have a long history of use as tonics and folk medicines that can be applied to cancer and diabetes treatments and used as antioxidants among other uses [1]. Rather than the safety of the use of Cordyceps s.l. (sensu lato) species, much attention has been focused on their high value due to their pharmacological and therapeutic benefits in Cordyceps militaris mycelia and fruiting bodies were obtained from Mucho Biotechnology Inc. as described previously [23]. The mycelia and fruiting bodies of O. sinensis were purchased from Tongrentang Co., Ltd., Beijing, China.

Micromorphological Characteristic Examination.
Microscopic examinations were performed via cryosectioning and examination under a compound light microscope (Olympus, Japan). The samples were fixed with 4% formaldehyde in PBS for one day with one change of solution. After the samples were mounted in optimal cutting temperature (OCT) compound, cryosectioning was performed with a LEICA CM3050-S cryostat (Leica Biosystems, Germany) to obtain sections ranging in thickness from 5 m to 10 m. The sections were examined and photographed under an Olympus BH2 light microscope (Olympus, Japan) equipped with a Canon-ds126 camera.

Genomic DNA Extraction from Cordyceps spp.
The genomic DNA extraction from Cordyceps spp. was performed according to a modified version of the protocol of Doyle [24]. Briefly, either the fungal samples were directly cut down from the stroma and stalks of Cordyceps or mycelia were harvested from culture plates. The collected samples were lyophilized and ground to powder with a pestle with 500 L of hot CTAB buffer (2% CTAB, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris at pH 8.0, and 2% PVP-40) in combination with 3 L of fresh -mercaptoethanol. After incubation at 60 ∘ C for 20 minutes, 500 L of chloroform : isoamyl alcohol (CI; 24 : 1) was added and mixed gently for 10 minutes prior to centrifugation at 13,200 g for 2 minutes. The supernatant was carefully transferred to a new Eppendorf tube following the administration of a 0.6x volume of isopropanol. Next, the mixture was allowed to incubate at −20 ∘ C for 30 minutes to  The DNA was harvested by centrifugation at 13,200 g for 30 minutes along with a single 75% cold  ethanol wash, drying with a vacuum (LABCONCO, USA), and resuspension in 50 L of ddH 2 O.

DNA Amplification and
Sequencing. PCR was employed for the amplification of specific DNA fragments as described below. In general, the PCR reaction mixtures contained 2.5 L of 10x reaction buffer, 1 L of 10 M of each specific primer (see Supporting Table 1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/189891), 0.5 L of 10 mM dNTPs, 2 L of template DNA, and 0.3 L of 1 U Taq polymerase in a total volume of 25 L. The RPB1 sequences were amplified using the forward primer cRPB1 and the reverse primer RPB1c-r [25] according to the following steps: denaturation at 95 ∘ C for 5 minutes; 40 cycles of 95 ∘ C for 1 minute, 55 ∘ C for 1 minute, and 72 ∘ C for 1 minute; and an additional extension step at 72 ∘ C for 10 minutes. The RPB2 sequences were amplified with the forward primer fRPB2-5f and the reverse primer fRPB2-7cr [26] according to the following steps: denaturation at 95 ∘ C for 5 minutes; 40 cycles of 95 ∘ C for 50 seconds, 55 ∘ C for 1 minute, and 72 ∘ C for 90 seconds; and an additional extension step at 72 ∘ C for 10 minutes. The EF1-sequences were amplified using the forward primer EF1-2218R and the reverse primer EF1-983F [27] according to the following steps: denaturation at 94 ∘ C for 5 minutes; 40 cycles of 94 ∘ C for 1 minute, 46 ∘ C for 30 seconds, and 72 ∘ C for 2 minutes; and an additional extension step at 72 ∘ C for 7 minutes. The PCR products were electrophoresed and eluted for sequencing using an ABI 3730XL system (Applied Biosystems, MA, USA). The obtained sequences were then assembled and trimmed using a ContigExpress of vector NTI.

Phylogenetic
Analysis. The nucleotide sequences of three genes, that is, the largest and second largest subunits of RNA polymerase II (RPB1, RPB2) and translation elongation factor 1 (EF1-), were either obtained from our sequenced PCR products of the O. formosana isolates, retrieved from the NCBI database following the criteria described by Sung et al. [21], or acquired by whole genome ortholog BlastNs of C. militaris CM01. A total of 51 species/isolates were included in this study (Supporting Table 3). The nucleotide sequences of these three genes were concatenated, aligned, and tested in models using MEGA 6.0 [28]. The best model according to the AIC and BIC values was the GTR+I+G model. The aligned sequences were analyzed using CIPRES portal-based Raxml blackbox (RAxML 7.2.7) [29] for maximum likelihood (ML) tree searching with the default settings, an estimation of the gamma distribution, invariable sites, and Bayesian inference of the phylogeny with MrBayes 3.2.3 with the parameters obtained from the model test. The resulting phylogenetic trees were saved and plotted using Adobe Illustrator CS3.

Cordyceps Extract Preparation and HPLC Analysis.
All of the desiccated Cordyceps samples that were subjected to HPLC (HITACHI, D-2000 HSM, Japan) analyses for de novo secondary metabolites were pulverized with a homogenizer at 1,300 rpm and subsequently stored in a humidity-controlled desiccator at room temperature. The preparation of the Cordyceps extracts was performed according to the standard operation procedure described below. Briefly, 1 g of Cordyceps powder was dissolved with deionized water at a solid-to-solvent ratio of 1 : 40 by vortexing for 30 seconds in a 50 mL falcon tube (BD Biosciences, USA). Next, the extraction was performed with 50 ∘ C hot water reflux for 2 hours (including the first 30 minutes of ultrasonication). After 20 min of centrifugation at 4,000 rpm (approximately 3,200 g), the supernatant was collected and filtered with a 0.22 m filter. The resulting extracts were directly subjected to HPLC analyses or stored at −20 ∘ C.
High-performance liquid chromatography (HPLC) was performed on RP-18 column (150252 Purospher STAR RP-18 endcapped (5 m) LiChroCART 250-4, Merck) at a flow rate of 1 mL/min. The composition of the mobile phase was 20% methanol/H 2 O. Three of the indicated components, D-mannitol, adenosine, and cordycepin, were detected at 260 nm using a DAD detector and the corresponding retention times of the individual standard compounds in the same HPLC conditions. Quantitation was preformed according to the established standard curve of each compound.

Cell
Culture and MTT Assay. The MTT assay was used to measure the abilities of the Cordyceps extracts to inhibit human cancer cell proliferation. Approximately 5 × 10 3 cells were seeded onto each well of a 96-well plate containing 200 L of culture medium (per well) 24 hours before treatment. After exposure to various concentrations of the indicated extracts for 96 hours, the cells were incubated with 20 L of MTT (Thiazolyl Blue Tetrazolium Bromide, Sigma, USA) solution for 4 hours. Cell proliferation was measured by the absorption at 570 nm using a spectrophotometer (SpectraMax 190 UV-Vis Microplate, Molecular Devices, USA). The measurements were performed in triplicate. The percentages of viable cells were calculated as (sample OD 570 nm/Mock OD 570 nm) × 100 (%) [30].

DPPH Antioxidation
Assay. The scavenging of DPPH radicals was estimated by vigorously mixing equal amounts of the Cordyceps extract and DPPH solution (0.1 mM in methanol). The mixture was then incubated at room temperature for 50 minutes before measurement at 517 nm using a spectrophotometer (SpectraMax 190 UV-Vis Microplate, Molecular Devices, USA). The scavenging activity was determined by comparing the blank (100%) that contained only DPPH and solvent. The scavenging efficiency percentages were calculated as (sample OD 517 nm/Mock OD 517 nm) × 100 (%) [31].

Cell-Based ROS Scavenging
Assay. Approximately 5 × 10 4 CHO-K1 cells were seeded in each well of a 12-well plate and exposed to the indicated Cordyceps extracts for 1 hour after 16 hours of serum starvation. Here, the 10 mM Nacetylcysteine treatment served as a positive control. After H 2 O 2 (1800 M) challenge for 5 minutes, the cells were incubated with fluorescent probes (CM-H2DCFDA, Sigma, USA) for 30 minutes in a 37 ∘ C incubator (light was excluded).
Finally, the cells were trypsinized and subjected to ROS quantification via the detection of the DCF levels, which corresponded to the relative ROS amounts at 525 nm using a flow cytometer (BD FACSCanto, USA).

Tumor
Xenografts. An in vivo tumorigenesis assay was conducted as previously described [32]. Briefly, 5 × 10 6 MDA-MB-231 breast cancer cells were inoculated into the left thighs of 10-week-old nude mice (BALB/cAnN.Cg-Foxn1 nu /CrlNarl) via subcutaneous injections. The oral gavage treatments with O. formosana extract began daily immediately after 10 days when the tumor growth was palpable. The mice were divided into three groups: two groups were treated with O. formosana water extracts at concentrations of 1x (25 mg/mL, 100 L/day) and 5x (125 mg/mL, 100 L/day), and a control group was treated with PBS. The tumor sizes were measured every four days and calculated with the formula tumor size = 0.4 × length × width 2 according to previous reports [33]. Statistical strategy was based on Ku's study [34]. After 26 days of O. formosana water extract treatment, the tumors in the mice were photographed.
2.11. Statistical Analyses. All data are expressed as the means ± the SEMs based on at least three independent experiments. The statistical analyses were performed using one-way analyses of variance (ANOVA) followed by Tukey's tests, and significance was measured as indicated.

Collection and Morphological Features of Ophiocordyceps formosana.
We collected Cordyceps spp. from varied potential niches in Taiwan to assess their potential medicinal values and applications. In Taoyuan Figure 1.
Obviously, the ascostromata arose from the heads or abdomens of the infected insect hosts (here, darkling beetle larva of the Tenebrionoidea superfamily), and the stalks exhibited long-cylindrical shapes of 10-30 mm × 0.5-2 mm in size that were orange (6A7) in color and bore short hairs but lacked membranous sheaths. The oblong ascostromata heads were 4-6 mm × 1-4 mm in size and largely consisted of pseudoparenchymatous epidermal tissues. Moreover, the perithecia were embedded within the stromata and were brownish orange (7C8) and ovoid shaped (360-480 × 240-320 m) with short necks, with ostioles of 60 m in width. The perithecial wall was 20 m thick underneath with approximately 40 m-thick epidermal tissue. Furthermore, the asci displayed long-cylindrical shapes with attenuated bases (6.5-7.9 m × 160-240 m) and thick dome-shaped apices (3.9-5.3 × 3.2-4.6 m) along with narrow slits. The asci contained eight ascospores and hyaline, were cylindrical and filamentous, and were often fragmented into more than 10-20 cylindrical and truncated partspores (2.6-3.0 × 6.5-7.3 m) at maturity. Colonies grown on potato dextrose agar (PDA) were orange (5A7) to white in color and pulvinate to umbonate in shape and exhibited velutinous to floccose mycelia that were whitish light-orange to pink in color and exudation droplets. The anamorphic state was similar to Hirsutella type, and the conidiogenous cells were monophialidic, hyaline, and elongated ampuliform-shaped (1.5-2.3 × 8.6-17.0 m). The conidia were hyaline and cylindrical (1.6-2.3 × 3.6-6.9 m in size).  Table 2). The ITS sequences were not utilized due to time and reagent limitations. However, the multigene analysis results of the three genes were sufficient to indicate that our collected specimens were identical to Cordyceps formosana TNM F13893 as verified by the molecular markers.

Molecular Identification of
Indeed, the phylogenetic trees based on the maximum likelihood method and Bayesian principles resulted in two identical trees with different support strengths. A maximum likelihood bootstrap tree is shown in Figure 2, and the posterior probability ML bootstrap support values are denoted after the slashes. The topologies of Cordyceps s.l. in the three newly created families, Ophiocordycipitaceae, Cordycipitaceae, and Clavicipitaceae s.s., were largely congruent with the results of Sung et al. [21].    (Figure 3(a)) was reminiscent of those of O. sinensis and C. militaris, which indicated that the biochemical properties of O. formosana were similar to those of other well-known Cordyceps species. Additionally, we compared three predominate metabolites that were unique in Cordyceps spp., cordycepin, adenosine, and D-mannitol. As shown in Figure 3(  Collectively, we epitypified O. formosana based on detailed studies of its morphological, molecular, and biochemical features.

The Ophiocordyceps formosana Water Extract Displayed
Antioxidation Activity. Ophiocordyceps formosana displayed biochemical features that were analogous to those of other valuable medicinal Cordyceps spp. s.l. We furthermore examined its biological and pharmacological properties. The nature of the antioxidant activity of the hot water extract from O. formosana was evaluated based on DPPH assay and cell-based ROS scavenging bioactivity. As shown in  (Figure 4(a)) that was even comparable with that of the extract derived from the O. sinensis fruiting body (Figure 4(b)). Coincidently, an in vivo cellbased ROS scavenging assay using CHO-K1 cells revealed that O. formosana exhibited a nonstatistically significantly greater antioxidant effect than the mycelial extracts (Figure 4(c)). Notably, the extract from the O. formosana mycelia was also comparable to the extracts derived from the O. sinensis and C. militaris fruiting bodies (Figure 4(d)). Our data imply that  O. formosana may serve as an alternative and/or substitute for tonic and medicinal uses of O. sinensis and C. militaris.

The Ophiocordyceps formosana Water Extract Exhibits
Anticancer Activity. To determine the anticancer potential of O. formosana, the hot water extracts from the mycelia and fruiting bodies of O. formosana, O. sinensis, and C. militaris were tested against various human cancer cell lines, including A549 lung cancer, MDA-MB-231 breast cancer, Huh7 liver cancer, and HL-60 leukemia cells, at different dosages. As shown in Figure 5(a), the O. formosana extract exerted anticancer activities IC 50 s of 1.0 mg/mL for the A549 cells, 0.53 mg/mL for the MDA-MB-231 cells, 0.44 mg/mL for the Huh7 cells, and 0.19 mg/mL for the HL-60 cells. Apparently, the O. formosana extract exhibited more effective cancer cell suppression activities than the extracts from the O. sinensis and C. militaris mycelia that were even comparable to those of the extract derived from the O. sinensis fruiting bodies in all tested cancer cell lines (Table 1). Strikingly, the O. formosana extract yielded the lowest toxicities (maintaining a survival rate greater than 90%) to the tested normal cell lines, which included HCEC normal human corneal epithelial cells and MCF10A normal human breast epithelial cells, among all of the other Cordyceps extracts as shown in Figure 5(b).

In Vivo Anticancer Activity of the Ophiocordyceps formosana Water Extracts.
To further validate the antitumor activities of the extracts from O. formosana, we conducted an in vivo antitumorigenesis assay using tumor xenograft mouse models. Briefly, nude mice were first implemented with the human MDA-MB-231 breast cancer cells and then treated with the water extracts of O. formosana at low (2.5 mg/day) and high doses (12.5 mg/day) daily by oral gavages (Figure 6). The results showed that the treated groups exhibited decreases in tumor size compared to the nontreated group in a dose-dependent manner. Our study highlights the potential future applications of O. formosana in tonics and medicines.

Discussion
Ophiocordyceps formosana was originally collected from Xitao, Nantou County, Taiwan, and described and named Cordyceps formosana in the subgenus Eucordyceps by Kobayasi [17,22]. Nevertheless, Ophiocordyceps formosana did not receive much attention until a revisit based on a new collection found in the Guanwu National Forest Recreation Area of Hsinchu County, Taiwan [35]. Consistent with our discovery, the indigenous Ophiocordyceps formosana seemed to prefer to inhabit Tenebrionoidea larva in areas of temperate broadleaf forests at approximately 1500-1800 m in altitude (Figure 1(h)). More recently, O. formosana was also found in Huangshan, Anhui Providence of China, by Li et al., which extended the distribution of O. formosana beyond Taiwan [36].
Morphologically, our collected samples were nearly almost identical to the holotype of Cordyceps formosana described by Kobayasi [22] and Li et al. [36], which therefore justified the identification of the entomopathogen discovered in this study as C. formosana (Figure 1). However, the morphology and the genotype of C. formosana had not been clearly defined and linked. To address this issue, the nucleotide sequences of the RPB1, RPB2, and EF1housekeeping genes that were obtained and sequenced from our fungal samples were aligned and compared with the documented allied sequences of the reported C. formosana TNM F13893 to further validate the identities of both fungal specimens. The reason that we used RPB1, RPB2, and EF1-to infer the species phylogeny was based on a hypothesis that essential genes are more likely to be subjected to the same selection forces, and convergent evolution in amino acid sequences is less likely to occur; this hypothesis has also been supported in several previous studies of the phylogenies of the Cordyceps relatives [20,21]. Other approaches involving the use of a neutral locus, such ITS, should be appropriate for inferring species phylogeny; however, ITS sequences change too fast and are therefore unsuitable for inferring the phylogenies of distantly related species. Indeed, we failed to obtain the ITS sequence of C. formosana using the "so-called" conserved primer sequences. Therefore, in this study, we were dealing with distantly related species, so these three genes were selected to infer the species phylogeny. Interestingly, the phylogenetic tree-based maximum likelihood and Bayesian inference phylogenetic methods both revealed the same topology, although, due to restrictions in computer capacity, we were unable to rule out the possibility that the trees were trapped in local optima. However, we replicated two runs of the BI analyses, and the results were identical, which supports our hypothesis that Ophiocordyceps formosana resides in Ophiocordycipitaceae. Due to the clear evidence from not only the morphological characteristics but also the molecular linkages, we epitypified C. formosana to O. formosana. Moreover, the anamorphic state of Hirsutella type in culture was also consistent with the description from Li et al. [36]. According to the definition of Ophiocordyceps proposed by Kobayasi, the anamorphs of this genus consist of four genera: Hirsutella, Hymenostilbe, Paraisaria, and Syngliocladium types [22]. The results of the phylogenetic investigation further support our suggestion for combining and transferring C. formosana to the new protologue Ophiocordyceps formosana (Kobayasi & Shimizu) Wang, Tsai, Tzean & Shen comb. nov. (see Supporting Text 1 for details), although the comparisons of several phylogenetically related Ophiocordyceps spp. indicated that O. formosana apparently displays a brighter yellow color of its stromata that distinguishes it from other species in this clade ( Figure 1). For example, O. ravenelii exhibits superficially distributed perithecia and dark brown-colored clavate stromata [37]. The stromata of O. nigrella are also dark brown and clavate, but the perithecia are embedded in mycelial tissues [38], whereas O. konnoana exhibits yellow, clavate stromata and superficial perithecia [39]. However, O. heteropoda and O. gracilis exhibit embedded perithecia and capitate stromata [40,41], which indicates that these two species may be comparatively less related to O. formosana. Interestingly, the clade that was phylogenetically allied to O. formosana and included O. ravenelii, O. nigrella, and O. konnoana appears to exhibit a similar host preference, that is, Coleoptera  larvae. In contrast, O. heteropoda and O. gracilis parasitize Auchenorrhyncha (cicada) nymphs and Lepidopteran larvae, respectively. Indeed, regarding phytopathogens, linkages between host ranges and evolution have been suggested [42]. Although it is currently recognized as belonging to Cordyceps s.l., the strict host specificities of some of the clades, such as Elaphocordyceps, may be due to recent host jumping [21,43] that might have given rise to strong driving forces for an evolutionarily genetic drift. Moreover, this clade also shares morphological features of the stromata; however, this seems to be only a symplesiomorphic character within Ophiocordyceps spp. [43]. The adverse effects of oxidative stress on human health have attracted considerable interest. Indeed, the exposure of organisms or cells to excessive amounts of reactive oxygen species (ROS) results in striking homeostatic imbalances [44]. Eventually, high levels of ROS are capable of inducing cellular and DNA damage, triggering cell apoptosis, and contributing to deleterious complications, such as aging, high blood pressure, inflammatory responses, and even cancer. Two Cordyceps spp., that is, O. sinensis and C. militaris, are commonly used to ameliorate conditions associated with aging [45] and several malignant complications, including lung disorders [46], diabetes [47], kidney failure [48], and cancer [23]. It is believed that the antioxidation activities derived from different preparations or fractions of Cordyceps are effective in therapeutics [49]. Using the in vitro DPPH assay and the cell-based anti-ROS scavenging assay (Figure 4), the extract from the mycelia of O. formosana was found to exhibit prominent antioxidative activities that exceeded those of the mycelia of C. militaris and O. sinensis and were even comparable to those of the fruiting bodies of O. sinensis. Hence, our evidence-based results suggest the value of the complementary and alternative medicine O. formosana for oxidation-related complications.
In recent years, increasing amounts of evidence have indicated that Cordyceps spp. are competent as alternative antitumor medicines and adjuvants for chemo-and radiotherapy in the treatment of various cancers [50]. Notably, the hot water extract from the mycelia of O. formosana exhibits superior antitumor activities against various cancer cell lines via induction of cancer apoptosis (unpublished data), including breast, lung, and liver cancers and leukemia, compared to extracts from the mycelia of O. sinensis and C. militaris ( Figure 5(a)). Moreover, the O. formosana mycelial extract even exhibited an anticancer efficacy that was comparable to that of the extract from the O. sinensis fruiting bodies (Table 1); therefore, benefits are available for many markets because a massive supply is achievable. Intriguingly, the relatively lower toxicity of O. formosana to normal cells renders O. formosana as more competent as an alternative medicine for the treatment of cancer ( Figure 5(b)). In support of this idea, we have observed no apparent deleterious effects on any of the dissected tissues/organs or in the in vivo biochemistries of mice that have been orally treated with O. formosana for 14 successive days at concentrations of up to 2 g/kg (unpublished data).

Conclusions
In conclusion, we established an evidence-based alternative indigenous medicinal Cordyceps s.l. named Ophiocordyceps formosana that exhibits pharmacological potential as an anticancer and antioxidative stress-related complication agent. With the support of the HPLC analyses, the chemical profile indicated that key bioactive ingredients, including D-mannitol, adenosine, and cordycepin, were present in Ophiocordyceps formosana at amounts that were comparable or even exceeded those in other known traditional medicinal Cordyceps species. In addition to its high level of pharmacological efficacy, O. formosana also exhibited lower toxicity than the documented valuable Cordyceps species O. sinensis and C. militaris. Together, these findings support the notion that this epitypified Ophiocordyceps formosana (Cordyceps s.l.) can serve as an alternative medicinal fungus.