Production of paclitaxel and the related taxanes by cell suspension cultures of Taxus species

Medicinal plants are the most promising source for the development of drugs, and many types of active ingredients from the plant resources have been studied in order to clarify the relationship between the chemical structure and the activity. However, it is not easy to develop drugs from those active compounds, and in many cases, the supply of active compounds can have some problems: 1) limited quantity of active compounds in plant; 2) low plant growth rate; 3) the limited localization of active ingredients in the specific organs; and 4) from the perspective of the conservation of natural resources. Therefore, the stable supply of the compounds commercially is very difficult and contains risk hedge. Plant cell culture is an attractive technology to solve these problems by securing the stable supply of the active compounds without damage to the natural plant resources. Recently, an efficient production process of anticancer drug paclitaxel by Taxus cell suspension cultures was constructed. The established Taxus cell lines produced paclitaxel and related taxanes by specific external stimuli, such as methyl jasmonate. The time-course analysis revealed that there are two regulatory steps existing in the paclitaxel biosynthesis: the taxane-ring formation step that is up-regulated by MeJA, and the acylation step at the C-13 position. By applying the data from the two-stage culture and the high-density culture, a large-scale culture process was developed with a stable paclitaxel production in the range of 140-295 mg L(-1), reaching 295 mg L(-1) at maximum.     

Development of in vitro techniques for the important medicinal plant Veratrum californicum

VERATRUM CALIFORNICUM (Liliaceae) is an important monocotyledonous medicinal plant which is the only source of the anticancer compound cyclopamine. An IN VITRO culture system for somatic embryogenesis and green plant regeneration of VERATRUM CALIFORNICUM was developed. Embryogenic calli were induced from mature embryos on induction medium. Five basal media supplemented with different growth regulators were evaluated for embryogenic callus induction, modified MS medium with 4 mg/L picloram showing the best result for embryogenic callus production. Fine suspension cell lines were established by employing friable embryogenic calli as starting material and AA medium and L2 medium as culture media. The suspension cell lines cultured in AA medium with 4 mg/L NAA appeared to be fresh yellow and fast growing. The suspension cells were cryopreserved successfully and recovered at a high rate. Green plants were regenerated from embryogenic calli maintained on solid medium with 73 % regeneration ability (green plants/100 calli) in 27-month-old culture. The IN VITRO plantlets contained the steroid alkaloids cyclopamine and veratramine. This IN VITRO system will form the basis for metabolic engineering of VERATRUM cells in the context of biotechnological production of pharmaceutically important secondary metabolites. DMSO:dimethyl sulfoxide fw:fresh weight NAA:naphthaleneacetic acid 2,4-D:2,4-dichlorophenoxyacetic acid picloram:4-amino-3,5,6-trichloro-2-pyridinecarboxylic acid dicamba:3,6-dichloro-2-methoxybenzoic acid.     

Using natural products for drug discovery: the impact of the genomics era

Introduction: Evolutionarily selected over billions of years for their interactions with biomolecules, natural products have been and continue to be a major source of pharmaceuticals. In the 1990s, pharmaceutical companies scaled down their natural product discovery programs in favor of synthetic chemical libraries due to major challenges such as high rediscovery rates, challenging isolation, and low production titers. Propelled by advances in DNA sequencing and synthetic biology technologies, insights into microbial secondary metabolism provided have inspired a number of strategies to address these challenges.

Areas covered: This review highlights the importance of genomics and metagenomics in natural product discovery, and provides an overview of the technical and conceptual advances that offer unprecedented access to molecules encoded by biosynthetic gene clusters.

Expert opinion: Genomics and metagenomics revealed nature’s remarkable biosynthetic potential and her vast chemical inventory that we can now prioritize and systematically mine for novel chemical scaffolds with desirable bioactivities. Coupled with synthetic biology and genome engineering technologies, significant progress has been made in identifying and predicting the chemical output of biosynthetic gene clusters, as well as in optimizing cluster expression in native and heterologous host systems for the production of pharmaceutically relevant metabolites and their derivatives.     

Bio-inspired plant leaf skeleton based three dimensional scaffold for three dimensional cell culture

Large quantities of natural fibers are available in the plant biomass that can be utilized for various purposes including three dimensional cell culture and tissue engineering due to their biocompatibility, ecofriendly, easy availability and cost effective. Especially, leaf skeletons (venation architecture) have a complex hierarchical architecture with novel properties. In this present invention describes about developing three dimensional scaffold from plant leaf skeletons for three dimensional cell culture. Plant leaf skeleton is prepared by simple and rapid method which using sodium hydroxide pretreatment under pressurized condition at 120°C for 1 h. The prepared plant skeleton has microporous surface topography. Also, the plant leaf skeleton is mainly composed by hemicellulose, cellulose and lignin. The microscopic analysis clearly indicates that human mesenchymal stem cells (hMSCs) attached and proliferated on plant leaf skeleton. Interestingly, cell density of hMSCs is increased on plant leaf skeleton by incubation time-dependent manner. Our study confirmed that sodium hydroxide via surface modified Ficus religiosa leaf skeleton enhances the attachment and proliferation of the human mesenchymal stem cells because of their biocompatibility and porous nature. The chemical nature and venation architecture of leaf skeleton has facilitated nutrient and oxygen absorption to promote cell-cell interaction, long term cell culture, and possible scope for induction of cell differentiation. Thus, leaf venation architecture can be applied as three dimensional (3D) cell-culture platform for multi-layer cell culture, cell-based assay model, high-throughput drug screening, cell-replacement therapy, wound healing and substitute for skin. Moreover, this scaffold could also be well-suited to co-culture screening strategies and stem cell differentiation for tissue engineering. Our present invention highlighted agro-wastes as precursor for novel scaffold materials to construct the 3D cell culture platform.     

药用植物细胞悬浮培养体系生物转化的研究进展

细胞悬浮培养体系生物转化获得目标产物已成为获取天然活性产物的重要途径。利用不同药用植物的细胞悬浮培养体系及不同的反应类型对先导活性化合物进行生物转化,可以获得新的活性化合物或提高活性化合物的产率。本文综述了近年来药用植物细胞悬浮培养体系类型及生物转化的新进展,并对活性成分的结构修饰与资源利用进行探讨。     

The production of secondary metabolites by plant cells cultivated in bioreactors1

Plant cell cultures are a potential source of pharmaceutically important plant metabolites. In the past few years a lot of success has been achieved in the field of the cultivation of plant cells on a large scale. Special bioreactor systems, such as airlift or drum-type fermentors have been devised for the mass cultivation of plant cells. Classical stirred-tank bioreactors (up to 75000 l volume) have also been used for the cultivation of plant cells and the production of important plant metabolites. Plant cells proliferate much slower than microbial cells. In consequence, the time taken to grow a plant cell suspension from the shake-flask level (300 ml) to the production scale (20,000 l) takes about 3 to 6 weeks. Nevertheless, the cost analyses available show that the production of valuable chemicals by a suitable plant cell culture process could be commercially viable. Plant cells have been grown in batch and repeated batch culture, and single- stage as well as two-stage processes have been developed for the production of secondary metabolites by fermentor-cultured plant cells. There are, however, only few cell culture processes that have attracted industrial attention as yet. Biological rather than technological problems are the main obstacles to a more common use of fermentor-cultured plant cells in industry.     

桔梗悬浮培养对细胞天麻素的生物转化

天麻素即对羟基甲基苯 β Ｄ 吡喃葡糖苷 (4 ｈｙｄｒｏｘｙｍｅｔｈｙｌｐｈｅｎｙｌ β Ｄ ｇｌｕｃｏｐｙｒａｎｏｓｉｄｅ) ,为兰科植物天麻 (ＧａｓｔｒｏｄｉａｅｌａｔａＢｌ .)的主要活性成分[1] ,有镇静、抗惊厥、抗炎、镇痛及增强机体免疫功能等作用。用植物细胞悬浮培养物对外源底物进行生物转化从而对其结构进行修饰 ,以获得更有意义的产物的报道[2 ,3 ] 很多 ,也是当今研究的热点。生物转化(ｂｉｏｔｒａｎｓｆｏｒｍａｔｉｏｎ) ,也称生物催化 (ｂｉｏｃａｔａｌｙｓｉｓ) ,是利用植物离体培养细胞或器官、动物、微生物及细…     

长春花组织培养研究进展

长春花含有100多种吲哚类生物碱,具有抗肿瘤、降血压等多种生物活性,有较高的药用价值,但是含量偏低。长春花组织培养可从提高繁殖系数、调控次生代谢产物的累积等来提高长春花生物碱类成分含量。本文就外植体和培养基的选择、药用成分累积的影响因素、悬浮细胞培养应用、影响毛状根因素和基因工程技术应用等方面,综述了长春花组织培养的主要研究进展,为进一步开发利用长春花药物资源提供参考。     

Glycosyltransferases, important tools for drug design

An increasing appreciation of carbohydrates as components of natural products has uncovered new opportunities in carbohydrate-based drug design. Glycosylated natural products produced by microorganisms contain a variety of different sugars. Usually the biosynthetic pathways to deoxysugars start from a monosacchride-1-phosphate which is converted to a NDP-hexose by a nucleotidyltransferase. Modification of this intermediate by different enzymes (e.g. dehydratases, epimerases, aminotransferases) yields the final sugar. In contrast to microorganisms, plant products mostly contain glucose, galactose, rhamnose and xylose as structural elements. In all organisms the nucleotide-activated sugar is attached to an aglycon by a glycosyltransferrase (GT). As no single universal GT has been uncovered yet, accomplishing the generation of novel glycosylated compounds requires a deep understanding of the function of glycosyltransferases (GTs) and its specificity. In this review we will present important drugs that contain sugar components. We will give an overview about the existing natural product GTs and we will discuss the structural features of GTs. Through specific examples within different compound classes, we will highlight recent examples of metabolic and combinatorial engineering approaches successfully applied to the production of novel glycosylated compounds.     

Accumulation and Biotransformation of Chromenes and Benzofurans in a Cell Suspension Culture of Ageratina adenophora

A cell suspension culture of AGERATINA ADENOPHORA was shown to yield several novel chromene and benzofuran derivatives in minute amounts that were different to the compounds found in seedlings of the same species. The structure elucidation of the new compounds is described. When two of the seedling chromenes (demethoxyencecalin and demethylencecalin) were fed to the cell suspension culture, one biotransformation product each was obtained in high yields (80%) that originated from a hydroxylation at one of the geminal methyl groups of the chromene heterocycle. These products accumulated largely in the growth media even though the presence of cells was necessary for the biotransformations to occur. When the third seedling chromene (encecalin) was fed to the cell auspension culture, no significant biotransformation was noted but several of the benzofurans present as cell culture metabolites showed a significantly increased accumulation in the growth media of the treated cultures. This increased accumulation of benzofurans was found to be inducible also by adding yeast extract to the cell culture. The metabolism of chromenes and bezofurans in the cell suspension culture is discussed.     

Biologically active chitosan systems for tissue engineering and regenerative medicine

Biodegradable polymeric scaffolds are widely used as a temporary extracellular matrix in tissue engineering and regenerative medicine. By physical adsorption of biomolecules on scaffold surface, physical entrapment of biomolecules in polymer microspheres or hydrogels, and chemical immobilization of oligopeptides or proteins on biomaterials, biologically active biomaterials and scaffolds can be derived. These bioactive systems show great potential in tissue engineering in rendering bioactivity and/or specificity to scaffolds. This review highlights some of the biologically active chitosan systems for tissue engineering application and the associated strategies to develop such bioactive chitosan systems.     

Preparation of botanical samples for biomedical research

Plants are chemical storehouses, a fact which has driven countless multidisciplinary quests for bioactive compounds. As the very first step of botanical research, the whole desire is to find "hit" plants with specific bioactivities. It is logical to use some strategies that can maximize the chances of finding these "hits" with limited time and resources. In addition to selecting the right plants for screening, how the plant extracts are prepared can also influence the bioactivity screening outcomes. An extract from the same plant material can be quite different in chemical composition having different preparations. Because of the complex mixture nature of plant extracts, it is possible artifact activities may be observed. Thus confirmatory activity tests are often necessary to warrant the next laborious isolation step. A bioassay directed isolation approach may be the most efficient in identifying the bioactive compounds because of the narrowed focus at each isolation step, but a phytochemistry isolation approach is appropriate to characterize a purified bioactive extract. In fact, these two approaches can be taken intermittently whenever efficiency can be improved. Finally, use of the identified active compounds is now broader. In addition to determining a lead compound to continue a drug development path, there is an increasing interest in support for the use of botanical extracts as botanical drugs. Instead of dropping the extract after extracting the lead compound, the natural analogues representing the purified extract now have a chance to become leading compounds in the pursuit of novel therapies for metabolic syndrome and other diseases.     

Screening of chemicals for human bioaccumulative potential with a physiologically based toxicokinetic model

Human bioaccumulative potential is an important element in the risk assessment of chemicals. Due to the high number of synthetic chemicals, there exists the need to develop prioritisation strategies. The purpose of this study was to develop a predictive tool for human bioaccumulation risk assessment that incorporates not only the chemical properties of the compounds, but also the processes that tend to decrease the concentration of the compound such as metabolisation. We used a generic physiologically based toxicokinetic model that based on in vitro human liver metabolism data, minimal renal excretion and a constant exposure was able to assess the bioaccumulative potential of a chemical. The approach has been analysed using literature data on well-known bioaccumulative compounds and liver metabolism data from the ECVAM database and a subset of the ToxCast phase I chemical library—in total 94 compounds covering pharmaceuticals, plant protection products and industrial chemicals. Our results provide further evidence that partitioning properties do not allow for a reliable screening criteria for human chemical hazard. Our model, based on a 100% intestinal absorption assumption, suggests that metabolic clearance, plasma protein-binding properties and renal excretion are the main factors in determining whether bioaccumulation will occur and its amount. It is essential that in vitro metabolic clearance tests with metabolic competent cell lines as well as plasma protein-binding assays be performed for suspected bioaccumulative compounds.     

Impacts of diversification of cytochrome P450 on plant metabolism

Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines.     

Synthetic TLR4-active glycolipids as vaccine adjuvants and stand-alone immunotherapeutics

The design of vaccine adjuvants and stand-alone immunotherapeutics has historically been a mix of alchemy and accident partly because of the complex nature of the molecular mechanisms involved in immune system function. The recent discovery of pattern recognition receptors and toll-like receptors (TLRs) in particular on cells of the immune system has shown the important role that stimulation of these cell receptors by microbial products plays in both innate and adaptive immune responses. Considerable effort has been directed at developing pharmaceutically acceptable mimetics of many TLR-active natural products, including the main cell-surface component of Gram-negative bacteria: lipopolysaccharide (LPS). LPS and its active principle, lipid A, are potent stimulators of host defense systems via their interaction with TLR4. However, the profound pyrogenicity and lethal toxicity of LPS and lipid A have precluded their medicinal use. Structure/activity investigations on natural S. minnesota R595 lipid A and its derivatives have led to the development of a novel class of synthetic lipid A mimetics known as aminoalkyl glucosaminide phosphates (AGPs). This review discusses the evolution of the AGPs and related TLR4-active glycolipids with emphasis on structure/activity relationships in the AGP series and pre-clinical/clinical development of selected AGPs, including the potent vaccine adjuvant RC-529.     

Marine-derived anticancer agents in clinical trials

Anticancer agents may be derived either from the isolation of an active lead compound occurring spontaneously in nature or by novel chemical synthesis in the laboratory. There are examples of successful drugs being derived from both sources, which have had a profound impact on the natural history of various types of cancer. The treatment of lymphomas and acute leukaemias with the use of combination chemotherapy, including anthracyclines and vinca alkaloids, are examples of the contribution of nature. In contrast, agents such as 5-fluorouracil, methotrexate and more recently, the humanised anti-CD20 antibody rituximab and the tyrosine kinase inhibitor imatinib are examples of synthetic compounds, which were designed with a clear rationale, that are routinely used in patients with solid tumours and haematological malignancies. Until recently, the tradition in natural product-derived anticancer drug development was to rely almost exclusively on the screening of terrestrial sources (plant extracts and fermentation products) for their cytotoxic properties. Although C-nucleosides obtained from Caribbean sponge were the initial inspiration for the synthesis of antiviral substituted nucleosides and the successful anticancer agent citarabine, active against leukaemias and lymphomas, the contribution of marine compounds as a source of anticancer agents was modest. In recent years, the improvements in the technology of deep-sea collection and aquaculture added to the growing recognition of the tremendous biodiversity present in the marine world, and has contributed to the growing interest of exploring the oceans as a potential source of new anticancer candidates. This is reflected in the number of marine-derived compounds undergoing preclinical and early clinical development. In this paper, the authors discuss the available literature on anticancer agents that have reached clinical trials, such as didemnin B, aplidine, dolastatin-10, bryostatin-1 and ecteinascidin-743 (ET-743, trabectedin), as well as other promising compounds still undergoing tests in the laboratory.     

Marine-derived anticancer agents in clinical trials

Anticancer agents may be derived either from the isolation of an active lead compound occurring spontaneously in nature or by novel chemical synthesis in the laboratory. There are examples of successful drugs being derived from both sources, which have had a profound impact on the natural history of various types of cancer. The treatment of lymphomas and acute leukaemias with the use of combination chemotherapy, including anthracyclines and vinca alkaloids, are examples of the contribution of nature. In contrast, agents such as 5-fluorouracil, methotrexate and more recently, the humanised anti-CD20 antibody rituximab and the tyrosine kinase inhibitor imatinib are examples of synthetic compounds, which were designed with a clear rationale, that are routinely used in patients with solid tumours and haematological malignancies. Until recently, the tradition in natural product-derived anticancer drug development was to rely almost exclusively on the screening of terrestrial sources (plant extracts and fermentation products) for their cytotoxic properties. Although C-nucleosides obtained from Caribbean sponge were the initial inspiration for the synthesis of antiviral substituted nucleosides and the successful anticancer agent citarabine, active against leukaemias and lymphomas, the contribution of marine compounds as a source of anticancer agents was modest. In recent years, the improvements in the technology of deep-sea collection and aquaculture added to the growing recognition of the tremendous biodiversity present in the marine world, and has contributed to the growing interest of exploring the oceans as a potential source of new anticancer candidates. This is reflected in the number of marine-derived compounds undergoing preclinical and early clinical development. In this paper, the authors discuss the available literature on anticancer agents that have reached clinical trials, such as didemnin B, aplidine, dolastatin-10, bryostatin-1 and ecteinascidin-743 (ET-743, trabectedin), as well as other promising compounds still undergoing tests in the laboratory.     

天然产物促进药物发现（英文）

Natural products or natural product derived drugs comprised 32% of small molecule approved drugs between 1981 and 2010.In the same period of time,16% of small molecule approved drugs were synthetic or natural mimics based on the study of pharmacophores related to natural products.Indisputably,natural products provide diverse structural diversity and intricate carboskeletal frameworks.As it is believed that nature has evolved optimized biologically active compounds-the secondary metabolites-to ensure survival of the species that produce them,natural products are perceived by some to be more′drug-like′than totally synthetic compounds.As such,natural products may provide us with the′best′lead compounds yet for drug discovery,giving rise to natural product inspired drug design.This talk will provide an overview of some of my research in this area.Specifically,I will outline the challenges and some of the lessons learnt in this quest to develop natural products as leads to potential drugs.