본 연구는 동충하초 추출물을 PC12 및 BV2 세포에서 인지능력 개선에 대한 효능을 평가하고자 하였다. 동충하초 추출물을 증류수로 추출하여 PC12 및 BV2 세포로 MTT 분석을 통해 세포 생존율을 확인하고 L-glutamate로 유도한 PC12 세포를 통해 세포 보호 효능과 아세틸콜린 함량 및 아세틸콜린에스테아제 활성을 평가하였다. 또한, LPS로 유도한 BV2 세포를 통해 nitric oxide (NO) 및 prostaglandin E2 (PGE2) 생성량 등의 항염증 효능을 측정하고 western blot 분석을 통해 NK-ĸB, p38, JNK, caspase-3 등의 단백질 발현량을 확인하였다. 동충하초 추출물은 200 ㎍/㎖ 농도를 제외하고 1, 10, 100 (㎍/㎖) 농도에서 세포 독성이 나타나지 않았다. L-glutamate로 유도한 PC12 세포에서 유의성있게 세포 보호 효능과 아세틸콜린 함량의 증가, 아세틸콜린에스테아제 활성 감소가 나타났다. 또한, 동충하초 추출물은 NO 및 PGE2 생성량과 NK-ĸB, p38, JNK, caspase-3 등의 단백질 발현을 억제하였다. 이와 같은 결과는 동충하초 추출물이 인지능력에 대한 예방 및 개선 효능이 있음을 나타낸다. 따라서 동충하초 추출물은 인지능력 개선을 위한 새로운 천연 소재로 활용될 수 있다.

발아대두 동충하초의 유산균 발효를 위하여 발아대두와 동충하초가 함유된 배지에서 생육이 우수한 유산균주를 김치로부터 분리하여 동정한 결과 Lactobacillus plantarum KCB001로 명명하였다. 선정된 유산균을 이용하여 발아대두 동충하초의 고상발효조건을 최적화한 결과, 동충하초와 발아대두의 혼합비는 4:1, 가수량은 40%(v/v), 종배양액의 첨가량은 20%(v/w), 최적 발효온도와 시간은 각각 37oC와 72시간으로 확인되었다. 유산균발효에 의해 총 폴리페놀함 량과 DPPH 소거능이 증가하였으며, 특히 동충하초의 지표 물질인 cordycepin 함량은 발효에 의해 24% 증가함으로써 발효에 의해 발아대두 동충하초의 기능성이 증가함을 확인하였다.

Through data mining of the Cordyceps militaris genome, a lectin-encoding gene, CMLec3, was identified. In this study, the CMLec3 sequence was analyzed using bioinformatics approaches, and the gene was heterologously expressed in Escherichia coli BL21 cells. The biological activity of the product was examined. In addition, CMLec3 gene expression levels were assessed. The results showed that the CMLec3 protein contained a lectin domain structure and was successfully expressed. The CMLec3 protein partly inhibited HeLa cell proliferation. CMLec3 exhibited the highest gene expression in the primordium at a level 5.19 times that of the mycelium and 1.35 times that of the fruiting body. This suggests that the gene may be related to fruiting body development.

A entomopathogenic ascomycetous fungus, Cordyceps militaris is distributed world wide and cultivated as a medicinal mushroom having many valuable biological and pharmaceutical activities. In the breeding of new C. militaris mushroom, single ascospores were isolated and tested their characteristics such as mycelial growth, mycelial density, fruiting body formation ability, the production of perithecia. Also two opposite mating types were assayed using two sets of primers specific for C. militaris, which were amplified a 191-bp fragment for MAT1-1 and 233-bp fragment for MAT1-1. F1 hybrids resulted in well-developed perithecial fruiting bodies and their crossings were confirmed by the PCR assays. New hybrids were produced showing high quality fruiting bodies in artificial media. Mycelial growth of new strain 'Dowonhongcho 2’ was better on SDAY and at 15-25°C when it was compared with that of 'Yedang 3'. The stromata of new strain were club-shaped and bright orange-red color. Its length was 7.1 cm and the cordycepin content was 0.33% on average. In comparison with 'Yedang 3', the new strain had a yield that was 7% higher and it produced fruiting bodies which were firmer. Days of fruiting body were similar with 50 days from inoculation. This cultivar may serve as a valuable one for artificial cultivation and industrial-scale production of C. militaris.

For heaths and vitality Cordyceps sinensis has been widely used as an important medicinal mushroom. C. militaris is popular for its substitute and can be cultivated artificially. Its new strains have been improved by crossing single spore strains. A bipolar heterothallic fungus, C. militaris has two strains of compatible mating types and they can be differentiated by crossing, fruiting body formation ability and the production of perithecia. Each isolate was determined to be monokaryotic or dikaryotic on the basis of perithecial formation by fruiting tests. This process was also carried out by molecular markers for the detection of two opposite mating types or F1 hybrids using two sets of primers specific for C. militaris amplifying a 191-bp fragment for MAT1-2 and 233-bp fragment for MAT1-1. Therefore the crossing of two compatible mating types resulted in well-developed perithecial fruiting bodies and their crossings were confirmed by the PCR assays for the rapid and specific detection of both MAT1-1 and MAT1-2. This results showed that molecular markers provide an efficient alternative for identification of hybridal status or mating types in the breeding program for artificial cultivation and industrial-scale production of C. militaris.

Light is a necessary environmental factor for stroma formation and development of Cordyceps militaris, a well-known edible and medicinal fungus. In this study, photo morphogenesis and the blue-light receptor gene were studied using five representative strains of C. militaris. The results suggest that light was essential for colony pigmentation and could promote conidia production. Cmwc-1 gene inactivation results in thicker aerial hyphae, disordered fruit body development, a significant reduction in conidial formation, and carotenoid and cordycepin production. These characteristics were restored when the ΔCmwc-1 strains were hybridized with wild-type strains of the opposite mating type. A genomewide expression analysis revealed that there were 1042 light responsive genes in the wild-type strain and only 458 in the ΔCmwc-1 strain. Among five putative photoreceptors identified, Vivid, cryptochrome-1, and cyclobutane pyrimidine dimer photolyase are strongly induced by light in a Cmwc-1- dependent manner, while phytochrome and cryptochrome-2 were not induced. The transcription factors involved in the fungal light reaction were mainly of the Zn2Cys6 type. CmWC-1 regulates adenylosuccinate synthase, an important enzyme for adenosine de novo synthesis, which could explain the reduction in cordycepin production. Some G proteincoupled receptors that control fungal fruit body formation and the sexual cycle were regulated by CmWC-1, and the cAMP pathway involved in light signal transduction in N. crassa was not critical for the photoreaction in the fungus here. A transcriptional analysis indicated that steroid biosynthesis was more active in the ΔCmwc-1 strain, suggesting that CmWC-1 might switch the vegetative growth state to primordia differentiation by suppressing the expression of related genes.

This study was conducted to in vitro antioxidant and anti-inflammatory activities of EtOH extracts of Cordyceps militaris (CM). Antioxidant potential, total phenolic and flavonoid contents of CM EtOH extracts were determined by Folin-Ciocalteu method and the aluminum chloride colorimetric method, respectively. Antioxidant activity of CM extracts was measured by following some well-established methods for free radical scavenging such as 2,2-diphenyl-picrylhydrazyl hydrate and 1,2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid). Moreover, Anti-inflammatory activity of CM extracts was determined by measuring the inhibition of nitric oxide (NO) production in lipopolysaccharide/interferon-γ-activated RAW264.7 macrophage-like cells. In addition, cytotoxicity of CM extracts against macrophages was determined by MTT assay. Our results showed that total phenolic content was 19.7 mg gallic acid/g extract. Total flavonoid content was 5.0 mg Naringin/g. Its antioxidant activity was assessed by IC50 value and the values are 338.8 μg/ml (DPPH radical scavenging), 35.4 μg/ml (ABTS radical scavenging). In addition, CM extracts attenuated NO production through the reduction of cellular inducible NO synthase protein expressions. Using MTT assay on indicate that CM extracts showed no toxicity. In conclusion, these results provide important evidence that CM extracts can potentially be used to antioxidant and anti-inflammatory agent.

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.

Cordyceps species are important mushrooms traditionally used for heaths and vitality. C. sinensis has been widely used as a medicinal mushroom and C. militaris is popular for its substitute. C. militaris can be cultivated artificially and new strains has been improved by crossing single spore strains. As a bipolar heterothallic fungus C. militaris has two strains of compatible mating types and they can be differentiated by crossing, fruiting body formation ability and the production of perithecia. However this process is very laborious and time consuming to carry out. In this study, molecular markers were designed and used for the detection of two opposite mating types. Two mating types were assayed using two sets of primers specific for C. militaris, which were amplified a 191-bp fragment for MAT1-2 and 233-bp fragment for MAT1-1. After crossing of two compatible mating types F1 hybrids resulted in well-developed perithecial fruiting bodies and their crossings were confirmed by the multiplex PCR assays for the rapid and specific detection of both MAT1-1 and MAT1-2. This results may serve as a efficient process in the breeding program for artificial cultivation and industrial-scale production of C. militaris.

Single ascospores were isolated and tested their characteristics such as mycelial growth, mycelial density, fruiting body formation ability, the production of perithecia in the breeding of new Cordyceps militaris mushroom. Among them selected isolates were crossed and hybrids were produced showing high quality fruiting bodies in artificial media. New strain 'Dowonhongcho’ was better on SDAY and at 10~25°C when it was compared with that of 'Yedang 3' in mycelial growth. The stromata of new strain were club-shaped and bright orange-red color. Its length was 6.1 cm and the cordycepin content was 0.34% on average. In comparison with 'Yedang 3', the new strain had a yield that was 9% higher and it produced fruiting bodies which were firmer. The optimum temperature for mycelial growth was 22~25°C and the optimum temperature for stroma development was 18~22°C. Days of fruiting body were similar with 45 days from inoculation. This cultivar may serve as a valuable one for artificial cultivation and industrial-scale production of C. militaris.

Cordyceps militaris is being studied and cultivated as a medicinal mushroom having many valuable biological andpharmaceutical activities. In the breeding of new C. militaris mushroom, single ascospores were isolated and examined theirmycelial growth, mycelial density, and production of stroma and perithecia. Among them selected isolates were crossed andhybrids were produced showing high quality fruiting bodies on artificial media. Mycelial growth rate of new strain‘Dowonhongcho’ was higher than that of better on ‘Yedang 3’ on SDAY at 10-25oC. The stromata of new strain were club-shaped and bright orange-red. Its height was 6.1 cm and the cordycepin content was 0.34% on average. The new strainshowed 9% higher yield than ‘Yedang 3’ with producing firmer fruit bodies. The optimum temperature for mycelial growthwas 22~25oC and the optimum temperature for stroma development was 18~22oC. Fruiting bodies were began to produce45 days later after inoculation. This new cultivar may serve as a valuable one for artificial cultivation and industrial-scaleproduction of C. militaris.

Cordyceps species, including C. sinensis, C. militaris, C. pruinosa and C. bassiana and C. staphylindaecola are prized traditional medicinal materials. From many Cordyceps species, Cordyceps militaris is well known and is ethopharmacologically known in Far East Asian countries such as Korea, China, and Japan. Continuous scientific studies have found that these preparations are capable of exhibiting numerous pharmacological activities as anti-oxidative, anti-viral, anti-cancer, anti-fibrotic, antiinflammatory, anti-nociceptive, anti-angiogenic, anti-diabetic, and anti-obesity drugs. Many species of Cordyceps (more than 600 types) have not yet been evaluated for their pharmacological activities. A preliminary study indicated that the biological activity varied between species and even between strains of the same species. But the chemical and functional properties and constituents of Cordyceps militaris have been studies extensively. The chemical constituents of Cordyceps militaris include cordycepin (3'-deoxyadenosine) and its derivatives, ergosterol, polysaccharides, a glycoprotein and peptides containing aminoisobutyric acid. They include anti-tumour, anti-metastatic, mmunomodulatory, antioxidant, anti-inflammatory, insecticidal, antimicrobial, hypolipidaemic, hypoglycaemic, anti-ageing, neuroprotective and renoprotective effects. Cordycepin is reported to possess many pharmacological activities including immunological stimulating, anti-cancer, anti-virus, insecticidal, antibacterial and anti-infection activities. Polysaccharide accounts for the anti-inflammatory, antioxidant, antitumour, anti-metastatic, immunomodulatory, hypoglycaemic, steroidogenic and hypolipidaemic effects. Ergosterol exhibits anti-tumour and immunomodulatory activity. The aim of this presentation is to review the chemical constituents and pharmacological actions of Cordyceps militaris.

일반성분 중 조회분 함량은 버섯이 균사체 보다 높은 조회분 함량을 보였다. 조단백질은 모든 시험구에서 19% 내외로 나타났고, 조지방 함량은 동충하초 균사체에서 가장 높았으며, 조섬유는 팽이버섯에서 가장 높게 나타났다. 가용성물질소물은 팽이버섯균사체와 동충하초균사체에서 60% 이상의 함량을 나타내었다. 각 시료별 총단백질과 총당의 구성 비율은 팽이버섯과 팽이버섯 균사체의 단백다당류의 구성이 다름을 확인하였다. 시료별 핵산관련물질 함량은 팽이버섯에서 5'-GMP, 5'-XMP, 5'-IMP순으로 높게 나타났고, 팽이버섯 균사체는 5'-XMP, 5'-GMP, 5'-IMP, 동충하초 균사체는 5'-XMP, 5'-IMP, 5'-GMP순으로 높게 나타났다. 모든시료에서 각각 17종의 아미노산이 검출되었다. 각 시료의 총 아미노산 함량을 살펴보면 팽이버섯 18,980 mg%, 팽이버섯 균사체 17,604 mg%, 동충하초 균사체 18,429 mg%로 나타났고 모든 시료에서 glutamic acid 함량이 가장 높았다. 유리아미노산 함량도 구성아미노산과 유사하게 팽이버섯, 팽이버섯 균사체, 동충하초 균사체 순으로 높게 나타났다. 본 실험결과 팽이버섯, 팽이버섯 균사체 및 동충하초 균사체는 핵산관련 물질과 아미노산 함량이 높게 나타나, 아미노산 함유 식품 개발에 적합한 자원으로 활용도가 기대된다.

Cordyceps militaris is a entomopathogenic ascomycetous fungus and cultivated as a medicinal mushroom having many valuable biological and pharmaceutical activities. It was cultivated on five different grain media which were compared with silkworm pupa medium for the artificial fruiting body production. C/N ratio of pupa and bean media was low at 5.9 and 5.8 respectively, which was attribute to low nitrogen contents compared to others. The various media had a significant effect on mycelial growth, cordycepin contents, fruiting body production. The mycelial growth were greatest at barley and wheat media but least at pupa medium. The cordycepin contents were highest at bean and pupa media with 2.9 and 2.6 mg/g, respectively. This was followed by wheat, corn, barley and brown rice media. The yield of fruiting body was greatest at barley and wheat media but the yield and days to harvest were least at silkworm pupa medium.

The effects of environmental and nutritional requirement such as temperature, pH, different media, carbon, nitrogen, and carbon-to-nitrogen ratio on the mycelial growth of Cordyceps militaris strain ‘Yedang 3’ were studied. The optimum temperature and pH for the growth of mycelium were 20-25oC and pH 6-7, respectively. Out of ten media tested, mushroom complete media (MCM) was the best medium for fast mycelial growth, and Sabouraud’s dextrose agar yeast extract (SDAY), malt extract yeast extract agar (YMA) also were favored. The color and shape of colonies varied in different media. The best carbon sources for mycelial growth were fructose, mannitol, and sucrose, whereas the best nitrogen sources were tryptone and peptone. However, mycelia grew slowly in inorganic nitrogen compounds such as NH4Cl, (NH4)2SO4, NH4NO3, and NaNO3. The optimum C:N ratio observed on the culture media was 30-40 range. These results provided basic information on cultural characteristics of vegetative growth and might be useful for spawn production in Cordyceps militaris.