D01:10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis the enhancement of applied basic research by modern Study and development of substitutes of the DCXC using technology,the establishment of nature reserve for alpine modern biotechnology meadow,the strict control of grazing capacity,and the Nowadays,the rare wild DCXC has been in danger because of development of artificial grassland.Besides.the related laws and regulations are also essential (Yang et al.,2002). unrestrained exploitation,and an effective approach to protect them is to study and develop the substitutes by means of bioengineering techniques,such as cell engineering,chromo- Application and prospect of biotechnology in the some engineering,cell fusion and so on.The new develop- research and development of the DCXC fungi ment of alternatives can have a high proportion of natural Cultivation,species selection and identification of authentic medicinal ingredients as does the wild DCXC.The Chinese DCXC fungus medicine ingredients from the DCXC are mainly secondary metabolites,and now they can be efficiently produced The technology of DNA molecular markers can be used to through fungus cultivation and fermentation technology.To identify and breed the DCXC.To distinguish the DCXC and increase the output of metabolites,we can add suitable its adulterants is an essential precondition for scientific inducer and precursor in the fermentation process of research of the DCXC in respect of pharmacognosy,prepar- Cordyceps fungus.Fermentation technology is widely used ation science,chemistry and pharmacodynamics,as well as in the production of fungal drugs(Zhou et al.,2005).With the quality assessment and clinical application.In general,the help of fermentation engineering,we can cultivate the conidia identification of the DCXC is mainly based on the origin, of the DCXC fungus with the same active ingredients as the characters,microscopic characteristics,physicochemical DCXC,or develop the relatives of the DCXC like properties and ultraviolet absorption spectrum and a relatively C.malitaris,which can serve as a substitute of the DCXC objective criterion of quality identification has been set up (Su et al.,2008).Furthermore,the transgenic and cell culture uepn d according to biological taxonomy (origin identification), technologies,which are increasingly mature,can highly cytohistology (microscopic identification)and chemistry express and produce the active ingredients similar to the (physicochemical differentiation)(Li,2004;Yang Duan, ones from the DCXC.The H.sinensis or the other endophytic 2012).DNA is the most stable genetic information and, fungi from the DCXC are suitable to be the expression tissues,organs and even cells in an individual organism have systems of fungi.Thus,transgenic technology is very helpful the same genetic information.Based on the relationship to efficiently express exogenous genes and obtain new between the DNA and pharmaceutical ingredient,we can medicinal fungal resources with the active ingredients. develop and protect better crude drugs as well as seek new To some extent,the transgenic technology is an important resources.The technology of RAPD and RFLP are usually pathway to optimize the quality of the DCXC cultivated and employed to identify different species and determine the resolve the problem of the related resource shortage. molecular properties of authentic drug by building DNA finger print.Furthermore,both of them are also used to make specific probes for detecting the corresponding drugs,which Acknowledgements will lay a foundation for the quality standardization of TCM. We would like to thank Dr.Wenxia Song (Department of Cell Biology and Molecular Genetics,University of Maryland College Park)for the help to provide an easy learning and Cultivation of high-quality larva and fungus of the DCXC working environment. using modern biotechnology 日 The application of biotechnology to the production of the Declaration of interest DCXC chiefly lies in preventing disease and pests,optimizing quality,reducing pollution of pesticide and heavy metal.The This research is financially supported by the Lhasa Science disease damage of the host insect is mainly caused by fungi and Technology Bureau (Project No.YZ201110),Shanghai such as Metarhizium anisopliae,Paecilomyces,Beauveria and Science and Technology Committee and Tibet Shengniu Paecilamyces.The pest damage is due to nematodes,mites Biotechnology Co.Ltd. and mosquitoes as a disease medium.To ensure the survival rate of the larvae,it is very necessary to regularly disinfect the References food and growing space of the host larvae (Liu et al.,2007; Berkeley MJ.(1843).On some entomogenous Sphaeriae.London J Bot, Zeng Chen,2001). 2.205-11. At present,the major limitation to artificial cultivation of Boesi A,Cardi F.(2009).Cordyceps sinensis medicinal fungus: the DCXC is the survival rate of the host larvae artificially traditional use among Tibetan people,harvesting techniques,and modern uses.Herbal Gram.83.52-61. bred.Thus,to increase the survival rate of the host larvae,the Bian D,Li Chun,Yang XM,Li L.(2008).Analysis of the situation of technology of feeding larva should be optimized and mean- grassland degradation and it's mechanism of the alpine pastoral area while the host larvae with high quality need to be introduced in northwestern Tibet.J Nat Resour,23.254-62.(In Chinese). Chen CY,Feng QG.(1992).Studies on industrial submerged fermen- and selected.Modern biotechnology can be used to select the tation of Cordyceps sinensis.Chin Tradit Herbal Drugs.23,409-11. host larvae of high quality,and transgenic technology also (In Chinese). enables the larvae to acquire the insect-resistant and antiviral Chen HF,Wang LY,Han HX.(2004).Fauna sinical insecta.Beijing: abilities.If so,it is very likely to reduce the use of pesticides in Science Press. artificial environments and ensure the DCXC natural and safe, Chen M,Dai RQ.(1995).Comparison of esterases isoenzymes,total proteins and free proteins in the fermentation mycelia of Cordyceps which will improve the marketing and export of the DCXC. sinensis.Chin J Chin Mater Med,20.585-6.(In Chinese). RI G H T S L I N Kthe enhancement of applied basic research by modern technology, the establishment of nature reserve for alpine meadow, the strict control of grazing capacity, and the development of artificial grassland. Besides, the related laws and regulations are also essential (Yang et al., 2002). Application and prospect of biotechnology in the research and development of the DCXC fungi Cultivation, species selection and identification of authentic DCXC fungus The technology of DNA molecular markers can be used to identify and breed the DCXC. To distinguish the DCXC and its adulterants is an essential precondition for scientific research of the DCXC in respect of pharmacognosy, prepar￾ation science, chemistry and pharmacodynamics, as well as quality assessment and clinical application. In general, the identification of the DCXC is mainly based on the origin, characters, microscopic characteristics, physicochemical properties and ultraviolet absorption spectrum and a relatively objective criterion of quality identification has been set up according to biological taxonomy (origin identification), cytohistology (microscopic identification) and chemistry (physicochemical differentiation) (Li, 2004; Yang & Duan, 2012). DNA is the most stable genetic information and, tissues, organs and even cells in an individual organism have the same genetic information. Based on the relationship between the DNA and pharmaceutical ingredient, we can develop and protect better crude drugs as well as seek new resources. The technology of RAPD and RFLP are usually employed to identify different species and determine the molecular properties of authentic drug by building DNA finger print. Furthermore, both of them are also used to make specific probes for detecting the corresponding drugs, which will lay a foundation for the quality standardization of TCM. Cultivation of high-quality larva and fungus of the DCXC using modern biotechnology The application of biotechnology to the production of the DCXC chiefly lies in preventing disease and pests, optimizing quality, reducing pollution of pesticide and heavy metal. The disease damage of the host insect is mainly caused by fungi such as Metarhizium anisopliae, Paecilomyces, Beauveria and Paecilamyces. The pest damage is due to nematodes, mites and mosquitoes as a disease medium. To ensure the survival rate of the larvae, it is very necessary to regularly disinfect the food and growing space of the host larvae (Liu et al., 2007; Zeng & Chen, 2001). At present, the major limitation to artificial cultivation of the DCXC is the survival rate of the host larvae artificially bred. Thus, to increase the survival rate of the host larvae, the technology of feeding larva should be optimized and mean￾while the host larvae with high quality need to be introduced and selected. Modern biotechnology can be used to select the host larvae of high quality, and transgenic technology also enables the larvae to acquire the insect-resistant and antiviral abilities. If so, it is very likely to reduce the use of pesticides in artificial environments and ensure the DCXC natural and safe, which will improve the marketing and export of the DCXC. Study and development of substitutes of the DCXC using modern biotechnology Nowadays, the rare wild DCXC has been in danger because of unrestrained exploitation, and an effective approach to protect them is to study and develop the substitutes by means of bioengineering techniques, such as cell engineering, chromo￾some engineering, cell fusion and so on. The new develop￾ment of alternatives can have a high proportion of natural medicinal ingredients as does the wild DCXC. The Chinese medicine ingredients from the DCXC are mainly secondary metabolites, and now they can be efficiently produced through fungus cultivation and fermentation technology. To increase the output of metabolites, we can add suitable inducer and precursor in the fermentation process of Cordyceps fungus. Fermentation technology is widely used in the production of fungal drugs (Zhou et al., 2005). With the help of fermentation engineering, we can cultivate the conidia of the DCXC fungus with the same active ingredients as the DCXC, or develop the relatives of the DCXC like C. malitaris, which can serve as a substitute of the DCXC (Su et al., 2008). Furthermore, the transgenic and cell culture technologies, which are increasingly mature, can highly express and produce the active ingredients similar to the ones from the DCXC. The H. sinensis or the other endophytic fungi from the DCXC are suitable to be the expression systems of fungi. Thus, transgenic technology is very helpful to efficiently express exogenous genes and obtain new medicinal fungal resources with the active ingredients. To some extent, the transgenic technology is an important pathway to optimize the quality of the DCXC cultivated and resolve the problem of the related resource shortage. Acknowledgements We would like to thank Dr. Wenxia Song (Department of Cell Biology and Molecular Genetics, University of Maryland College Park) for the help to provide an easy learning and working environment. Declaration of interest This research is financially supported by the Lhasa Science and Technology Bureau (Project No. YZ201110), Shanghai Science and Technology Committee and Tibet Shengniu Biotechnology Co. Ltd. References Berkeley MJ. (1843). On some entomogenous Sphaeriae. London J Bot, 2, 205–11. Boesi A, Cardi F. (2009). Cordyceps sinensis medicinal fungus: traditional use among Tibetan people, harvesting techniques, and modern uses. Herbal Gram, 83, 52–61. Bian D, Li Chun, Yang XM, Li L. (2008). Analysis of the situation of grassland degradation and it’s mechanism of the alpine pastoral area in northwestern Tibet. J Nat Resour, 23, 254–62. (In Chinese). Chen CY, Feng QG. (1992). Studies on industrial submerged fermen￾tation of Cordyceps sinensis. Chin Tradit Herbal Drugs, 23, 409–11. (In Chinese). Chen HF, Wang LY, Han HX. (2004). Fauna sinical insecta. Beijing: Science Press. Chen M, Dai RQ. (1995). Comparison of esterases isoenzymes, total proteins and free proteins in the fermentation mycelia of Cordyceps sinensis. Chin J Chin Mater Med, 20, 585–6. (In Chinese). DOI: 10.3109/07388551.2013.791245 Advances in research of Ophiocordyceps sinensis 9 Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Fudan University on 07/22/13 For personal use only