Cordycepin Content Research Articles

Dynamic Analysis of Nucleosides and Carbohydrates during Developmental Stages of Cordyceps militaris in Silkworm (Bombyxmori).

Background: Cultured Cordyceps militaris is very popular. Objective: To gain dynamic insight into activity markers in fruiting body of Cordyceps militaris (C. militaris) in Bombyxmori (B. mori), also named silkworm. Methods: The development stages of samples at 3, 9, 12, 19, 27, and 33 days after inoculation (DAI) were collected. HPLC coupled with diode array detection and evaporative light-scattering detection method (HPLC-DAD-ELSD) was used to determine eight makers, including six nucleosides and two carbohydrates from the samples. Results: C. militaris cultured 33 DAI with fifth star silkworm larva could accumulate higher levels of cordycepin (13.43 mg/g) than the highest reported cordycepin (8.57 g/L). The contents of cordycepin, adenosine, and trehalose were gradually increased with the formation of C. militaris fruiting bodies on silkworm larva, while mannitol was decreased. The change of guanosine was similar to uracil. Conclusions: Results suggested that mannitol could be accumulated in a short period during mycelium growth and could metabolize and transform into energy store and trehalose during fruit body formation. The inosine in the insect was completely utilized and transformed. The synergistic formation of cordycepin and adenosine or differences in metabolized pathways are a great possibility according to the same trend. Highlights: This research offered some reference to further find a certain regularity or metabolic mechanism.

Cordycepin suppresses cell proliferation and migration by targeting CLEC2 in human gastric cancer cells via Akt signaling pathway

PurposeGastric cancer is a common malignancy worldwide, and is associated with high morbidity and mortality rates. Cordycepin is a 3′-deoxyadenosine drug with significant anti-cancer effects. The aim of this study was to determine the molecular mechanisms underlying cordycepin action on gastric cancer cell proliferation and migration. MethodsThe human gastric cancer cell lines MGC-803 and HGC-27 were treated with different concentrations of cordycepin (25 μM, 50 μM, 100 μM and 5 μM, 25 μM and 50 μM) for 48 h. Cell proliferation was assessed by MTT and colony formation assays, and in vitro migration by the wound healing and transwell assays. In addition, Flow Cytometry was used to detect the cell cycle and apoptosis. RT-PCR and Western blotting were used to evaluate the expression levels of key factors. ResultsCordycepin significantly inhibited gastric cancer cell proliferation and migration in a dose-dependent manner, in addition to inducing apoptosis and arresting the cell cycle at the G2 phase. Mechanistically, cordycepin targeted the PI3K/Akt signaling pathway by significantly altering the expression levels/activation of several key mediators, and upregulated the anti-metastatic factor CLEC2. ConclusionCordycepin inhibited the proliferation and migration of gastric cancer cells by upregulating CLEC2 via the Akt signaling pathway.

Experimental and In Silico Analysis of Cordycepin and its Derivatives as Endometrial Cancer Treatment.

The aim of this study was to investigate the inhibition effects of cordycepin and its derivatives on endometrial cancer cell growth. Cytotoxicity MTT assays, clonogenic assays, and flow cytometry were used to observe the effects on apoptosis and regulation of the cell cycle of Ishikawa cells under various concentrations of cordycepin, cisplatin, and combinations of the two. Validated in silico docking simulations were performed on 31 cordycepin derivatives against adenosine deaminase (ADA) to predict their binding affinities and hence their potential tendency to be metabolized by ADA. Cordycepin has a significant dose-dependent inhibitory effect on cell proliferation. The combination of cordycepin and cisplatin produced greater inhibition effects than did cordycepin alone. Apoptosis investigations confirmed the ability of cordycepin to induce the apoptosis of Ishikawa cells. The in silico results indicate that compound MRS5698 is least metabolized by ADA and has acceptable drug likeness and safety profiles. This is the first study to confirm the cytotoxic effects of cordycepin on endometrial cancer cells. This study also identified cordycepin derivatives with promising pharmacological and pharmacokinetic properties for further investigation in the development of new treatments for endometrial cancer.

Enrichment of cordycepin for cosmeceutical applications: culture systems and strategies.

Cordyceps spp. is the herbal medication initially used in China and has been reported as the unique resource of cordycepin. Cordycepin exhibits many health benefits, including anti-photoaging and anti-pigmentation; therefore, it potentially is a bioactive ingredient of cosmetic products. In order to enrich cordycepin content in Cordyceps, two artificial cultivation procedures, which are solid-state fermentation and liquid culture, were developed and optimized. The aim of this review is to illustrate cordycepin biosynthesis pathway in Cordyceps, and its bioactivity for cosmeceutical applications, as well as comparing the two different cultivation procedures. The basic model of artificial cultivation of Cordyceps is introduced; meanwhile, the potential application of modern biotechnology to the artificial cultivation is also discussed. This review should be of interest to the readers for the development of cordycepin bioproduction in order to be applied in cosmeceutical industry and some other uses.

Effect of Culture Time on the Bioactive Components in the Fruit Bodies of Caterpillar Mushroom, Cordyceps militaris CM-H0810 (Ascomycetes).

Cordyceps militaris are widely cultivated in China for an important raw material for health foods. CM-H0810 is a C. militaris strain used in the production of C. militaris in Shanghai, the surrounding areas of Shanghai, and Guangdong province in China. We evaluated the effect of culture time on the bioactive components in the fruit bodies of C. militaris CM-H0810 to provide scientific references for production of C. militaris fruit bodies with good quality. The results showed that the polysaccharide contents increased gradually during 35-45 d, but it declined with the prolongation of culture time. The highest polysaccharide content was 3.46% at 45 d. With the prolongation of culture time the cordycepin content gradually increased; the highest cordycepin content was 3.57 μg/mg at 60 d, which increased 321% compared to that at 35 d. Contrary to cordycepin, the adenosine content declined gradually, with the highest content of 1.86 μg/mg at 35 d and the lowest content of 1.48 μg/mg at 60 d. Our study indicates that it is necessary to select suitable harvest times in view of different compounds that are desirable to obtain in high quantities.

Enhancing cordycepin production in liquid static cultivation of Cordyceps militaris by adding vegetable oils as the secondary carbon source

This study evaluated different vegetable oils as the second carbon source in liquid static culture of Cordyceps militaris in terms of mycelial growth and cordycepin production. The maximum mycelial concentration and cordycepin production were observed under cottonseed oil and peanut oil induction, respectively. In the condition of adding 20 g/L of peanut oil at Day 0, the final concentration of cordycepin reached to the highest, about 5.29 g/L, which was about 3.17 times higher than that of the control. The qRT-PCR and enzyme activity analysis confirmed that addition of peanut oil up-regulated the expression of the genes encoding glucose-6-phosphate dehydrogenase and isocitrate lyase, as well as the genes in the cordycepin biosynthesis pathway, cns1 and cns2, during the cultivation in C. militaris.

갈색거저리 동충하초의 Streptococcus thermophilus KCCM 3782 유산균 발효에 따른 물리화학적 특성과 산화방지 활성 비교

This study aimed to provide basic evidence regarding the development of materials by analyzing the physicochemical properties and antioxidant activities of Streptococcus thermophilus KCCM 3782 strain-fermented Cordyceps militaris grown on Tenebrio molitor. Among the solvents tested, DPPH radical scavenging activity was the highest in the hot water-extracted sample after 30 min of extraction. Moisture content decreased, whereas crude protein and fat content increased, after lactic acid bacteria-mediated fermentation. Sodium, magnesium, calcium, and zinc contents increased, with potassium levels attaining the maximum value, whereas free amino acid content decreased after the fermentation. Among Hunter’s color values, a value increased to 66.7→149.92 after fermentation, whereas the L and b values decreased to 15.79→-15.75 and 54.45→0.01, respectively. Cordycepin content assay increased from 7.02 mg% to 8.66 mg%. The DPPH free radical scavenging activity of the fermented product was 91.92 EDA%, which was higher than that of the extract (84.69 EDA%). The ABTS free radical scavenging and superoxide dismutase (SOD) activities were also higher in the fermented products.

Development of Cordyceps militaris under different monochromatic lights

Light is an essential environmental factor for the stroma's production, accumulation of bioactive substances and pigmentation in Cordyceps militaris. In this study, different LED monochromatic lights (blue, green, red) were used for C. militaris cultivation. Effects of various monochromatic lights on bio-efficiency of stroma, biomass, and content of cordycepin were evaluated. Furthermore, expression of the Cmwc-1 gene in different light conditions was investigated.

Effect of strain separated parts, solid-state substrates and light condition on yield and bioactive compounds of Cordyceps militaris fruiting bodies

ABSTRACTCordyceps militaris is widely cultivated as a functional food and traditional medicine in China and Southeast Asia. In this study, C. militaris hybrid strains were isolated from different parts of hybrid fruiting body cultivated through mating-based sexual reproduction of ascospores. We investigated the effect of strains, solid-state substrates and light condition on production of yield and contents of bioactive compounds (cordycepin and adenosine) using HPLC in fruiting bodies. The results indicated that yield and production of bioactive compounds exist distinct differences on strain separated part, solid-substrates and light conditions. The middle strain obtained the highest yield on wheat substrate. The yield was higher on wheat substrate, the concentration of cordycepin and adenosine in fruiting bodies were higher on rice substrate. The red light is beneficial to high yield, and white light is conducive to the accumulation of cordycepin in fruiting bodies, the light condition has little effect on the adenosine content.

교배형 분자마커를 이용한 신품종 밀리타리스 동충하초 ‘도원홍초 2호’의 품종 특성

The mushroom species Cordyceps militaris has been studied and cultivated as a medicinal mushroom due to its multiple valuable biological and pharmaceutical activities. For breeding new strains of C. militaris, multiplex PCR assays were performed using primers specific for its mating type genes, MAT1-1 and MAT1-2. Mating types and mating status were confirmed, as evidenced by DNA bands at 233-bp and 191-bp for MAT1-1 and MAT1-2 respectively. The novel 'Dowonhongcho 2ho’ was developed through mating; they were found to possess high-quality fruiting bodies when grown in artificial media. The stromata of the new strain were club-shaped, with a bright orange-red color, and measured 7.1 cm in length. They had an average cordycepin content of 0.33%. Compared to 'Dowonhongcho,' the new strain had a 7% higher yield, as well as firm fruiting bodies. The optimum temperature for mycelial growth was 20~25°C, and the optimum temperature for stroma development was 18~22 °C. The fruiting bodies developed after 49.1 days from inoculation. The use of mating type molecular markers improved the breeding efficiency of the new strain 'Dowonhongcho 2ho.’ Thus, they may be valuable for artificial cultivation and industrial-scale production of C. militaris with excellent characteristics.

Transcriptome analysis of Paecilomyces hepiali at different growth stages and culture additives to reveal putative genes in cordycepin biosynthesis

Paecilomyces hepialid (PH) is an endoparasitic fungus of Cordyceps sinensis (CS) and has become a substitute for CS due to their similar pharmacological activities. Because the market demand for CS is difficult to satisfy, and cordycepin, the effective compound of CS, is difficult to industrially produce, we produced 5 samples of PH by culturing for different durations and adding different additives to the culture broth, and detected their cordycepin content with UPLC ESI MS/MS. Then we grouped these cultures into five transcriptome comparisons containing 3 time variable groups and 2 additive variable groups. We used next-generation (NG) sequencing methods to acquire transcriptomic information and investigated the response of gene expression to the additives and the influence of different growth stages. This work will contribute to a better understanding of purine metabolism in PH, and possibly in other Cordyceps species. It will provide a useful resource to further advance transcriptomics studies in Cordyceps species.

Genome shuffling improved the nucleosides production in Cordyceps kyushuensis

Genome shuffling was first applied to improve the production of nucleosides in Cordyceps kyushuensis. Six improved strains were selected for genome shuffling by UV and HNO2 mutagenesis. Ten improved genome shuffling strains with good genetic stability were obtained, among which, the production of cordycepin in R6 was 9.624 times higher than that of the ancestor. While in R18 and R19, the yield of cordycepin, adenosine, guanosine and uridine were all increased greatly compared with the ancestor. Based on the four phenotypes of the content of cordycepin, adenosine, guanosine and uridine, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were applied to infer the relationships between genome shuffling strains and mutants.

Cordycepin induces apoptosis of human acute monocytic leukemia cells via downregulation of the ERK/Akt signaling pathway.

The aim of the present study was to examine the apoptotic effect of cordycepin (COR) on human THP-1 acute monocytic leukemia cells. THP-1 cells were exposed to different concentrations of COR for 24, 48, 72 or 96 h. The cell viability and apoptotic rate were analyzed. The gene expression of Akt1, Akt2, Akt3, B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X protein (Bax) were assessed by reverse-transcription quantitative PCR. Western blot analysis was used to detect the protein levels of phosphorylated (p)-Akt, p-extracellular signal-regulated kinase (ERK) and cleaved caspase-3. It was found that the viability of THP-1 cells was inhibited by COR in a dose- and time-dependent manner. After treatment with 200 µM COR for 24 h, the percentage of apoptotic cells was significantly increased. COR also downregulated the levels of Bcl-2, Akt1, Akt2 and Akt3, and elevated the expression of Bax. The protein levels of p-Akt and p-ERK were suppressed and cleaved caspase-3 was increased after treatment of COR. In conclusion, COR was found to induce apoptosis of THP-1 acute monocytic leukemia cells through downregulation of ERK/Akt signaling.

Characterization of Newly Bred Cordyceps militaris Strains for Higher Production of Cordycepin through HPLC and URP-PCR Analysis.

Cordyceps militaris, a member of Ascomycota, a mushroom referred to as caterpillar Dongchung-ha-cho, is commercially valuable because of its high content of bioactive substances, including cordycepin, and its potential for artificial cultivation. Cordycepin (3'-deoxyadenosine) is highly associated with the pharmacological effects of C. militaris. C. militaris is heterothallic in that two mating-type loci, idiomorph MAT1-1 and MAT1-2, exist discretely in two different spores. In this study, nine C. militaris strains were mated with each other to prepare newly bred strains that produced a larger amount of cordycepin than the parent strains. Nine strains of C. militaris were identified by comparing the internal transcribed spacer sequence, and a total of 12 single spores were isolated from the nine strains of C. militaris. After the MAT idiomorph was confirmed by PCR, 36 mating combinations were performed with six single spores with MAT1-1 and the others with MAT1-2. Eight mating combinations were successfully mated, producing stroma with perithecia. Cordycepin content analysis of all strains by high-performance liquid chromatography revealed that the KASP4-bred strain produced the maximum cordycepin among all strains, regardless of the medium and stroma parts. Finally, universal rice primer-PCR was performed to demonstrate that the bred strains were genetically different from the parental strains and new C. militaris strains. These results may be related to the recombination of genes during mating. The newly produced strains can be used to meet the industrial demand for cordycepin. In addition, breeding through mating suggests the possibility of producing numerous cordycepin-producing C. militaris strains.

Cordycepin negatively modulates lipopolysaccharide-induced cytokine production by up-regulation of heme oxygenase-1

AimsThe present study is to investigate the effect of cordycepin on the expression of heme oxygenase-1 (HO-1) in lipopolysaccharide (LPS)-activated microphages, as well as its mechanism of action. MethodsMouse RAW264.7 cells were treated with different concentrations of cordycepin for 0–16h. Western blotting was used to determine the expression of HO-1 and the phosphorylation of c-Src and the p47phox subunit of NADPH oxidase. Intracellular reactive oxygen species (ROS) level was determined using H2DCFDA as fluorescent probe. Laser-scanning confocal microscopy was used to visualize the nuclear translocation of NF-E2-related factor 2 (Nrf2). Enzyme-linked immunosorbent assay was performed to measure the inhibitory effect of cordycepin on LPS-induced secretion of tumor necrosis factor-α and interleukin-6. ResultsCordycepin induced the phosphorylation of c-Src and p47phox subunit of NADPH oxidase in RAW264.7 cells. Cordycepin increased the secretion of ROS by activating NADPH oxidase. In addition, cordycepin enhanced the expression of HO-1 in RAW264.7 cells in both dose- and time-dependent manners. Of note, elevated HO-1 expression induced by cordycepin treatment was regulated by c-Src/NADPH oxidase/ROS pathway. HO-1 expression induced by cordycepin was dependent on the activation of Nrf2, which was regulated by c-Src/NADPH oxidase/ROS. Cordycepin reduced LPS-induced secretion of proinflammatory cytokines through up-regulation of HO-1. ConclusionThe present study demonstrates that cordycepin induces the expression of HO-1 in RAW264.7 cells via c-Src/NADPH oxidase/ROS/Nrf2 pathway, and plays an anti-inflammatory role by inhibiting the secretion of cytokines from macrophages.

Development of High Cordycepin-Producing Cordyceps militaris Strains

Cordyceps militaris, known as Dong-Chong-Xia-Cao, produces the most cordycepin among Cordyceps species and can be cultured artificially. For these reasons, C. militaris is widely used as herb or functional food in the East Asia. In this study, we developed a new strain of C. militaris that produces higher cordycepin content than parent strains through mating-based sexual reproduction. Twenty parent strains were collected and identified as C. militaris based on internal trasncrived spacer and rDNA sequences. Seven single spores of MAT 1-1 idiomorph and five single spores of MAT 1-2 idiomorph were isolated from 12 parent strains. When 35 combinations were mated on the brown rice medium with the isolated single spores, eight combinations formed a stroma with a normal perithecia and confirmed mated strains. High pressure liquid chromatography analysis showed that mated strain KSP8 produced the most cordycepin in all the media among all the tested strains. This result showed due to genetic recombination occurring during the sexual reproduction of C. militaris. The development of C. militaris strain with increased cordycepin content by this approach can help not only to generate new C. militaris strains, but also to contribute to the health food or medicine industry.

동충하초균주로 발효한 감초의 주요성분 함량 변화 및 NO 생성 억제 효과

Traditional Korean fermented herbal plants are potential sources of new food that promote health, but they are still produced by yeast, fungi or bacteria fermentation. In the present work, mushroom (Paecilomyces tenuipes and Cordyceps militaris) fungal dongchunghacho were used to fermented Glycyrrhiza uralensis Fischer (licorice) or mixed with pupa. The pupa were tested as solid substrates for the production of corcycepin, liquiritin, and liquiritigenin. The fermented substrates were analyzed the content of cordycepin, liquiritin, liquiritigenin, and glycirrhizin productivity and inhibitory activity of NO. The cordycepin content of 70% EtOH extract from the fermented mixture of licorice and 50% pupa with C. militaris increased maximum at 33 times. Pupa was very excellent for the production of cordycepin. The liquiritin content was decreased in all the assays inoculated with P. tenuipes and C. militaris dongchunghachos. The liquiritigenin content was higher when fermented with P. tenuipes than C. militaris. The addition of pupa significantly reduced the liquiritin content and glycyrrhizin production. As a result, the liquiritigenin content increased in fermented P. tenuipes and C. militaris, and liquiritin and glycyrrhizin decreased. The inhibition of NO production in the different ethanolic extracts fermented with licorice and pupa was also significantly increased and higher than that of a nonfermented extract in higher polar solvent extracts. The contents of cordycepin and biological active compounds were altered in accordance with the concentration of pupa and fungi. This study provides basic data for use in developing dongchunghacho fungi as a functional food resource.

The Quality Improvement of Solid-State Fermentation with Cordyceps militaris by UVB Irradiation.

Cordyceps militaris, a medicinal and edible mushroom, was used to ferment buckwheat and embryo rice by solid-state fermentation (SSF). Our aim was to investigate the effect of ultraviolet B (UVB) light irradiation on the content of vitamin D2 and biologically effective components, and antioxidant properties of buckwheat and embryo rice in SSF with C. militaris. Irradiated samples of buckwheat and embryo rice fermented by C. militaris had significantly increased vitamin D2 content, from 0-0.3 to 1.18-16.79 μg/g, while the increase in fresh embryo rice fermented by C. militaris was up to 16.79 μg/g. The content of adenosine, cordycepin and polysaccharide in irradiated dry samples fermented by C. militaris was 0.08 to 11.15 mg/g, higher than that of the irradiated fresh samples fermented by C. militaris (0.07-8.40 mg/g). Samples fermented by C. militaris had lower EC50 values and higher content of antioxidants than did unfermented samples. When the solid-state fermented sample was irradiated with UVB light, the content of biologically effective and antioxidant components and antioxidant property of sample decreased. However, it still contained enough of these biologically effective and antioxidant components.

Determination of cordycepin content of <i>Cordyceps militaris</i> recombinant rice by high performance liquid chromatography

Purpose: To assess the suitability of high performance liquid chromatography (HPLC) for the determination of cordycepin content of Cordyceps militaris recombinant riceMethods: Cordyceps militaris recombinant rice was made by mixing brown rice with artificial Chinese caterpillar fungus culture medium powder using twin-screw extrusion technology. Cordycepin content was determined by reversed-phase HPLC with water:acetonitrile (95:5, v/v) as mobile phase, detection wavelength of 260 nm, and flow rate of 1.0 mL/min.Results: Cordycepin contents showed good linearity in the range of 1 - 50.0 μg/mL (r2 = 0.9996), and while recovery ranged from 103.2 to 109.9 %. Relative standard deviation (RSD), precision and repeatability RSD was 2.38, 0.76 and 1.46 %, respectively.Conclusion: The HPLC method is simple, fast, accurate and reproducible. It is suitable for determination of cordycepin content of artificial Chinese caterpillar fungus culture medium and brown rice recombinant rice.Keywords: Recombinant rice, Cordycepin, Chinese caterpillar fungus, Aweto, Cordyceps militaris

The effects of cordycepin on the cell proliferation, migration and apoptosis in human lung cancer cell lines A549 and NCI-H460.

Our study aimed to evaluate the effect of cordycepin on human lung cancer cell lines A549 and NCI-H460. Human lung cancer A549 cells and NCI-H460 cells were treated with different concentrations of cordycepin for different times. Cells incubated without cordycepin were defined as a control. The cell proliferation, migration and apoptosis were, respectively, determined by MTT assay, transwell migration assay and flow cytometry. Additionally, the expression levels of related proteins associated with cell cycle, epithelial-mesenchymal transition (EMT) and apoptosis were examined. The survival rate of A549 cells and NCI-H460 cells treated with cordycepin significantly decreased compared with untreated cells in a concentration-dependent manner, while the apoptosis rate increased. The migration number of cells treated with cordycepin significantly decreased as the increase in concentration. qRT-PCR and Western blot analysis showed that the aberrant expression of related molecules associated with cell cycle, migration and apoptosis was observed in the lung cancer cells, such as cyclin B, cyclin E, MMP-9, caspase-3 and Bcl-2. Cordycepin may exert inhibitory effects on the development of human lung cancer via inhibiting cell proliferation, suppressing migration and inducing apoptosis, suggesting that cordycepin may have therapeutic potential for the treatment of this disease.