Earticle

Functional cosmetics in Korea are defined as the products which have anti-wrinkle, and/or whitening, and/or sun-screen effects. Recently, various efforts are being made to develop functional cosmetic ingredients from natural resources. In our laboratory, we are searching for the natural products having anti-aging or whiteningactivities from plants growing in Jeju Island, Korea. We have screened anti-tyrosinase, anti-elastase and DPPH radical scavenging activities using the ethanol extracts of plants collected in Jeju. From the activity tests, some plant species such as Distylium racemosum, Quercus glauca, Maakia fauriei, Cornus kousa, Sasa quelpaertensis were chosen for the further phytochemical study to find out bioactive components. In this presentation, we will disclose our results on the biological activities as well as isolation and identification of some natural products of interest.

Skin wrinkles typically appear as a result of aging processes. Wrinkling in skin is caused by habitual facial expressions, aging, sun damage, smoking, poor hydration, and various other factors. Although wrinkle can signify wisdom, most people nowadays would rather not have them. Many products and procedures promise to reduce wrinkles. Some do little or nothing (like the products that claim they reduce "the appearance of fine lines," which means that they don't reduce the lines themselves). Others can achieve a fair amount of success. Jeju island is the most beautiful place in Korea. There are approximately 2000 plant species. We were tried to find new anti wrinkle agents derived from the plants. We found a few materials originated from the plants have got anti wrinkle effects in vitro and/or in vivo. Asiaticoside: A saponin component isolated from Centella asiatica, has been shown to induce type I collagen synthesis in human dermal fibroblast cells, We have been characterized the action mechanism of Asiaticoside in molecular level. We also evaluated the effects of a preparation containing asiaticoside on the periorbital wrinkles of a group of human volunteers. I this study, with 12 weeks of treatment with the Asiaticoside cream, most of the periorbital wrinkles were attenuated to some degree. Emodin: Matrix metalloproteinases(MMPs) are the proteases in the degradation of the extracellular matrix. In this study, we found that Emodin, an anthraquinone which has been isolated from the rhizome of Rheum palmatum, significantly inhibited TNFalpha-induced MMP-1 expression. These findings suggest that Emodin could be of therapeutic value in the prevention of MMP-1-related aging process containing wrinkle formation.

The organic cosmetics started around 2000 with “Wellbeing” concerning for better qualified life style and many products having concept for “Natural” and “Eco-Friendly” had been released on the market following with Welling boom. Nowadays natural is not a main concept of cosmetics any more, it is just required basic factor. The more advanced marketing concept of cosmetics is “Organic” which is including quality of materials, environment and pureness. The organic cosmetics have been known for customers for several years, but unfortunately, the word “Organic” was prohibited for using by government because there was no definition and regulation. But organic cosmetic certification body named ECO-CERT from France had definition for organic cosmetics starting around 2003. Ecocert is doing inspection and certification for manufacturer with very high qualified verification system, so their certified organic cosmetics are getting well known for EU, USA and others. When we think about the Jeju island, it has good image for clean, pure and beautiful. So it is the right time for Jeju to pay attention for organic cosmetics certified by Ecocert with their unique and special local products.

L-Ascorbic acid (vitamin C), a representative water soluble vitamin, has a variety of biological, pharmaceutical and dermatological functions; it promotes collagen biosynthesis, provides photoprotection, causes melanin reduction, scavenges free radical, and enhances the immunity (anti-virus effect), etc. These functions are closely related to the well-known antioxidant properties of this compound. Vitamin C, however, is very unstable to air, moisture, light, heat, metal ions, oxygen, and base. Therefore, the applications of vitamin C in the fields of cosmetics, dermatologicals, and pharmaceuticals are limited despite of its useful functions. Therefore in this experimental study, we determined the effects of polyhydric alcohols, chelating agents, the ionic shielding effects and the various water-soluble polymers on L-ascorbic acid stability. Then we prepared polymethyl methacrylate (PMMA) microcapsules using a polyol in oil in polyol (P/O/P) emulsion solvent evaporation method as a means of stabilization of L-ascorbic acid. Furthermore, in order to enhance the encapsulation efficiencies of L-ascorbic acid, L-ascorbic acid pretreated with cationic polymer to form an ionic complex, and the ionic complex was applied to PMMA microcapsules. The morphology of the microcapsule was evaluated using a microscope, which showed a spherical shape with several sub-micron microstructures inside of the microcapsule. The pretreatment of L-ascorbic acid to form an ionic complex in inner polyol phase resulted in a slight increase in the encapsulation efficiencies. In order to determine influence of encapsulation to the L-ascorbic acid stability in aqueous solution, free Lascorbic acid and the encapsulated L-ascorbic acid were incubated at 42°C. When the L-ascorbic acid encapsulated by PMMA microcapsule, the residual contents showed 70% or more retention at 42°C 42 days after. However, the free L-ascorbic acid stored at 42°C, residual contents of the quercetin gradually decreased to about 3% during 42 days. In conclusion, this study suggests that the polyol in oil in polyol (P/O/P) emulsion solvent evaporation method can be applied to the encapsulation of L-ascorbic acid. And the PMMA microcapsule system was devised to achieve improvement of Lascorbic acid stability thus holding a potential for future applications.

Hyperpigmentation mechanisms including UVB-melanosis have been recently progressed in terms of paracrine cytokine regulation between melanocytes and keratinocytes/fibroblasts as well as of endothelins or stem cell factor-induced intracellular signalings leading to gene expression of melanogenic molecules including tyrosinase, endothelin B receptor or melanosomal protein Pmel17. According to these mechanisms, several novel depigmenting agents have recently been developed. However, post-translational processing of melanogenesis subsequent to gene and protein expression of melanogenic molecules including tyrosinase and Pmel 17 is poorly understood and there is few depigmenting agents developed based on the above strategy. Previously we found that the recovery process of melanization following glycosylation inhibition of B-16 melanoma cells which induces complete loss of melanization in melanosome due to the interrupted transfer of tyrosinases compartmentalized in coated vesicles is a good tool for searching post-translational regulation of melanogenesis. In this study, using the pigmentation recovery process of glycosylation-inhibited B-16 melanoma cells and western blotting analysis, we determined whether tyrosinases are originally present in immature melanosome or is transferred from Golgi area to melanosomes. In glycosylation-inhibited B-16 melanoma cells, tyrosinases are not localized in melanosome-rich fraction but in cytosol fraction, indicating that tyrosinases are transferred from Gogi area to melanosomes. The addition of reduced glutathione into the recovery process abolished the recovered translocation of tyrosinases from Gogi-endoplasmic reticulum-lysosome(GERL) to melanosomes despite no glycosylation inhibition potential of reduced glutathione, resulting in the continuous complete loss of melanization. These findings suggest that reduced glutathione serves as a translocation inhibitor for tyrosinases but not as a tyrosinase inhibitor. To assess whether glycosylation inhibitors have a potential to inhibit epidermal hyperpigmentation stimulated by melanogenic cytokines released from UVB-exposed keratinocytes, we used guinea pig skin organ culture or three-dimensional cultures consisting of human keratinocytes and human melanocytes to examine the inhibitory effect of glycosylation inhibitors on epidermal pigmentation. In both models, glycosylation inhibitors markedly abolished visibly increased pigmentation without affecting increased melanocyte population. The present study suggests that glycosylation inhibitors or reduced gluthathione can serve as novel depigmenting agents with unique strategy such as intracellular translocation inhibition if they can penetrate into the epidermis at effective concentrations when topically applied.

Microphthalmia associated transcription factor (Mitf) is a key regulatory transcriptional factor of pigmentation-related genes including tyrosinase. Previously, we reported the construction of a protein chip to detect binding of Mitf protein and E-box DNA. To discover the small molecule inhibitors of Mitf-DNA binding, by using the protein chip, we screened 27 chemicals which were selected from a pharmacophore data base by virtual screening. Among them, compound #18 was found to show the most potent inhibitory activity against Mitf-DNA binding. To confirm its inhibitory activity against Mitf-DNA binding, an electrophoretic mobility shift assay (EMSA) was performed. The depigmenting activity of compound #18 was confirmed by cellular melanin assay, RT-PCR, and Western blot. These results demonstrated that compound #18 is the first reported small molecule inhibitor of MITF-DNA binding with depigmenting activity and that the Mitf protein chip is a powerful HTS tool for the discovery of depigmenting agents based on inhibition of Mitf-DNA binding.

Although many hypo-pigmenting agents are currently available, the demand for novel whitening agents is increasing, in part due to the weak effectiveness and unwanted side effects of currently available compounds. To screen for novel hypo-pigmenting agents, many methodologies such as cell culture and enzymatic assays are routinely used. However, these models have disadvantages in terms of physiological and economic relevance. In this study, we validated zebrafish as a whole-animal model for phenotype-based screening of melanogenic inhibitors or stimulators. We used both the well-known melanogenic inhibitors (PTU, arbutin, kojic acid, MBT) and newly developed small molecule compounds (haginin, YT16i). All the tested compounds produced inhibitory effects on the pigmentation of zebrafish, most likely due to their inhibitory potential on tyrosinase activity. In simultaneous in vivo toxicity tests, a newly developed melanogenic inhibitor YT16i showed massive abnormalities in terms of deformed morphologies and cardiac function. Together, these results provide a rationale in screening and evaluating the putative melanogenic regulatory compounds. We suggest that the zebrafish system is a novel alternative to mammalian models, with several advantages including the rapidity, cost-effectiveness, and physiological relevance.

Labisia pumila has been used for many generations by the Malay women as a post-partum medicine. The herb Kacip Fatima has long been regarded as the "queen" of Malaysian herbs. But its skin efficacy has not been studied yet. In this study, we have thus focused on finding anti-aging and anti-melanogenesis efficacy of the Labisia pumila extract. Extracts of Labisia pumila were analyzed for their antioxidant activities by DPPH (2,2-diphenyl-1-picrylhydrazyl) methods and found it has strong antioxidant activity. Treatment with the Labisia pumila extract enhanced collagen synthesis and inhibited the production of pro-inflammatory cytokines. Labisia pumila extract protects cell damage induced by UVB. Collagen synthesis of human dermal fibroblasts treated with Labisia pumila is increased dose dependent manner. Labisia pumila extract showed the strongest anti-melanogenesis activity. Treatment with the Labisia extract inhibits α -melanocyte stimulating hormone induced melanin synthesis in B16F1 melanoma cells. However the Labisia extract did not inhibit mushroom tyrosinase activity in vitro. Instead internal tyrosinase activity was reduced significantly by the treatment of the Labisia extract, which suggested that Labisia extract suppressed the expression of tyrosinase gene. The results show that the Labisia pumila extract has strong radical scavenging activity, anti-inflammatory activity and also restores collagen production by the UVB irradiated dermal cells. It also has strong inhibitory activity in melanogenesis. Taken together, the Labisia pumila extract has a good potential as anti-aging and whitening agent for cosmetic ingredient.

Atopic dermatitis (AD) is an itchy inflammatory dermatitis with a chronic course with remissions and exacerbations. AD occurs primarily in children, but persisting cases in adulthood as well as adult late-onset AD occur. Usually the personal and/or family history of other atopic conditions, such as asthma and allergic rhinitis, are positive. It is estimated that about 20% of the population of the industrialized countries has an atopic constitution, and between 5 and 20% suffers at least during some period of their life from AD. Treatment of AD is largely confined to the application of anti-inflammatory drugs such as corticosteroids; calcineurin inhibitors are frequently prescribed as steroid sparing agents. The integrity of the skin barrier, in particular the composition of the stratum corneum, appears to be of great importance for the development and progression of skin lesions in patients with AD. Optimal barrier function requires the presence of sufficient extracellular lipids to form a competent lamellar bilayer system. The barrier abnormality in AD correlates with a global reduction in these stratum corneum (SC) lipids, along with a selective reduction in 1 of the 3 key species, the ceramide fraction. Yet current water-in-oil emollients and moisturizers, though considered mainstays in the symptomatic management of AD, neither address nor correct the underlying SC lipid abnormality. Moreover, improperly formulated emollients and moisturizers can aggravate permeability barrier abnormalities, and therefore, in theory, they could both sustain preexisting disease and exacerbate previously quiescent dermatoses. One of the vital functions of the stratum corneum, the outermost layer of the skin, is known to provide a barrier against water loss through the skin. The effective barrier function of stratum corneum has been attributed to a highly ordered structure of lipid bilayers observed in the intercellular space of stratum corneum. In this study, we investigated the barrier function of a lamellar structure which is similar to the lipid bilayers in the skin. To prepare lamellar structures which are made up pseudo ceramide PC104, stearic acid and cholesterol, the thermotropic properties and the structural characteristics of ceramide PC104/stearic acid/cholesterol were studied by differential scanning calorimetry(DSC) and X-ray diffraction. A mixture of ceramide PC104, stearic acid and cholesterol was in a stable α-form having a lamellar structure. Also, stearic acid was found to be able to stabilize the lamellar structure of ceramide PC104. Moisturizers are widely recommended and used in AD, and when their use is encouraged by nursing assistants, they greatly reduce topical steroid usage. Many studies showed that “skin physiological lipid mixture” is effective for accelerating barrier recovery. But there are some requirements to accelerate barrier recovery with the inclusion of lipid mixture to moisturizing product. It is well documented that the adequate molar ratio of lipids is important for barrier recovery. Unless a substantial dose is provide, the lipid mixture cannot achieve barrier recovery for AD. And the free fatty acid component should comprise predominantly which are of sufficient length to interact within the lamellar membrane system. We developed pseudoceramide (PC-104) dominant, physiologic lamella structure base (DermaONTM) from above rationale and assessed the efficacy of the product with it on barrierdisrupted and Atopic skins.

The whole cell bioconversion of daidzein were examined for ortho-hydroxylation using Streptomyces avermitilis MA-4680 and Nocardia species screened from stock cultures. The major enzymes responsible for the monohydroxylation step are cytochrome P450s. The cytochrome P450s belong to a family of ubiquitous heme containing monooxygenases and catalyze diverse reactions, such as hydroxylation, epoxidation, reduction, oxidation, Odemethylation, desulfonation, peroxidation, deamination and dehalogenation reactions. Among the various reactions of cytochrome P450, hydroxylation with high regio- and stereoselectivity is of special interest for biotechnological applications. To identify monooxygenases which play a key role for the ortho-hydroxylation, 33 and dozens of cytochrome P450s from Streptomyces avermitilis MA-4680 and Nocardia species were cloned and examined, respectively, all the genes were constructed in E. coli BL21(DE3) host system and each P450s was co-expressed with redox proteins, camA (putidaredoxin reductase) and camB (putidaredoxin) from Pseudomonas putida, to produce ortho-specific monohydroxylated daidzein(trihydroxyisoflavonoid). The major hydroxylated products of daidzein were 7,3’,4’-trihydroxyisoflavone, 6,7,4’-trihydroxyisoflavone and 7,8,4’- trihydroxyisoflavone which were mono-hydroxylated at ortho position of hydroxy group of daidzein. The target P450s which showed the highest activity among the tested P450s were prepared for the templates of the in-vitro evolution.

국내 화장품 시장은 이미 성숙 및 포화 수준에 도달하고 있어 앞으로 세계시장에서의 경쟁력을 향상시켜야 할 필요성이 절실한 상황이며, 우리나라 고유의 국산 화장품 브랜드의 개발과 수출 전략 마련의 필요성이 대두되고 있다. 그러나 우리나라 화장품 제조에 사용되는 원료의 약 80% 이상은 해외 수입을 통해 이루어지고 있는 실정이다. 화장품 원료 수입품 가운데 수입실적이 높은 원료 및 제품들은 각국이 특허 등의 방법을 통해 산업차원에서 보호하고 있기 때문에 국내에서 이를 수입할 경우 완제품으로 수입하거나 고가의 로열티를 지불해야 하는 실정이다. 이에 더해 수입화장품의 성장세가 지속되고 있는데, 이를 극복하기 위한 방안으로 내세우는 것이 바로 국내 한방화장품으로 인식되고 있다. 한방화장품은 수입에만 의존하던 화장품 원료를 국산으로 대체할 수 있고 시대적 트랜드인 웰빙에 부합하면서 소비자의 수요가 지속적으로 늘어나고 있어 국내 화장품 산업 발전 도모와 국제시장에서의 수출 교두보를 마련할 수 있을 것으로 기대된다. 이미 한방 화장품은 시장에서 좋은 반응을 얻고 있으나 차별화, 세계화를 위해서는 중장기적인 계획을 가지고 한방 화장품의 특징을 과학적이고 객관적으로 입증할 수 있도록 하는 연구를 활성화시키는 것이 무엇보다 중요하다. 또 우리나라만의 특색이 있는 한방 화장품 개발로 서양의 허브나 다른 천연추출물을 이용한 화장품과의 차별화가 되지 않으면 한방 화장품이란 의미도 점점 퇴색되어지리라 생각된다. 지금이야말로 우리 한방 화장품을 고유 브랜드로 정착시키고 세계화하기 위한 필요 요인들을 점검할 때이다. 누룩은 우리의 전통술 제조상 가장 특징적이며 독특한 효 소 및 발효 미생물원으로서 다양한 미생물과 자연중의 세균, 효모, 곰팡이 등이 부착되어 충분히 번식함으로서 amylase, glycosidase, protease 등의 효소 생성을 유도할 수 있어 이들 복합효소를 이용하면 한방 원료가 갖는 toxin을 완화내지는 제거할 수 있으며 폴리페놀 배당체를 비배당체 형태로 생물전환하여 활성화시킬 수 있다. 누룩곰팡이는 발효공업에서 매우 중요한 곰팡이이며 된장, 간장, 단술, 청주 등 우리의 전통 식품과 깊은 관련을 갖는다. 누룩곰팡이는 1980년대부터 이용되어온 대표적인 미백 소재인 코지산(kojic acid)을 생산하는 균으로 코지산의 미백효과와 누룩곰팡이가 분비하는 글루코시다제를 이용해 한방재료의 폴리페놀을 활성화하여 얻은 발효액으로 한방발효 화장품을 개발하게 된다면 피부미백과 주름에 뛰어난 기능성 소재를 개발할 수 있을 것으로 판단된다. 본 연구에서는 누룩 곰팡이를 이용해 고기능성 성분을 추출 및 생산할 수 있는 한방재료로 감초와 천마를 선정하였다. 현재 국내에서 기능성화장품 원료로 주로 사용되는 감초는 Glycyrrhiza glabra로부터 추출한 유용성감초추출물로 주요 활성성분은 glabridin으로 알려져 있다. 그러나 본 연구에서는 한방 감초로 Glycyrrhiza uralensis를 선정하였으며 이로부터 고농도 liquiritigenin을 함유한 발효액 내지는 효능성분을 추출정제한 고함량 제품을 얻어 고기능성미백제와 주름개선제로 개발하고자 하였다. 이처럼 Glycyrrhiza uralensis로부터 다량의 liquiritigenin을 얻기 위해서는 누룩곰팡이와 같이 폴리페놀배당체에 대해 강력한 글루코시다제 활성을 갖는 효소를 필요로 한다. Glycyrrhiza glaba가 최근까지도 기능성 화장품 제조에 '감초'와 같이 사용되어 왔지만 수종에 따라 유효성분 및 효능에 차이가 있다는 사실에 대한 인식의 부족으로 liquiritigenin 배당체를 이용한 기능성 화장품이 개발된 사례가 없는 실정이다. 본 연구자들은 ibroblast cell을 이용한 어세이 결과 누룩발효를 진행한 감초추출발효액이 발효전의 감초추출액과 비교하여 콜라겐 생성능이 증가되었으며, 발효의 진행일수별로 liquiritigenin의 함량이 증가함을 확인하였다. 이것은 감초에 다량 함유되어 있는 성분들이 누룩발효과정 중에 일어나는 강력한 효소작용(Aspegillus niger에 의한 glycosidase 활성)에 의해 보다 강한 활성을 가진 식물성 여성호르몬의 일종인 liquiritigenin으로 전환되어 생성되는 것으로 해석할 수 있었다. 향후 Glycyrrhiza uralensis로부터 전구체 성분을 효과적으로 추출하고, 누룩곰팡이를 생물전환반응의 최적화 연구를 통해 다량의 liquiritigenin을 함유한 고농도 발효액 및 정제물을 제조하여, 이를 한방발효 화장품의 주름개선용 소재로 개발하고자 안정성과 안전성, 유효성 등의 임상시험을 수행하고자 한다. 천마(天麻, Gastrodia elata)는 예로부터 정풍초(定風草)라는 이름으로 불리기도 하는데, 이것은 중풍을 치료하는 약초란 뜻이며, 산삼에 버금가는 약초로 알려져 왔다. 천마의 경우 1990대 중반에 천마의 인공재배기술이 개발되고, 고소득 작목으로 각광을 받아 98년도 생산량이 286톤까지 증가하였다. 식약청에서 식품원료로서의 사용이 허용된 2000년도를 기점으로 2001년도부터 급격하게 생산량이 증가하는 추세를 나타내어 2004년도의 생산량이 404 톤으로 이제는 과잉생산을 염려해야 할 시기이다. 현재 시중에 유통되고 있는 천마제품은 향후 소득대체 작목으로서 천마공급이 증가하는 추세임을 감안해 볼 때 보다 다양한 형태의 천마 가공제품 개발 및 천마제품의 품질 고급화를 위한 연구가 더욱더 절실히 요구되고 있다. 그러므로 천마의 가공제품 개발과 더불어 1차 상품인 천마를 기능성 한방화장품으로 소재화 함으로서 부가가치를 높이는 것 역시 매우 필요한 일로 사료된다. 당사는 Tyrosinase inhibition assay와 Melanocyte를 이용한 미백효능 시험에서 누룩발효를 진행한 천마추출발효액이 발효전의 천마추출액과 비교하여 그 효능이 증가되었으며, 발효전에는 관찰되지 않는 4-hydroxybenzyl alcohol(4-HBA)의 피크가 발효후에는 현격히 증가함을 HPLC assay로 직접 확인할 수 있었다. 향후 연구에서는 생물전환반응이 일어나는 기작을 연구하고, 발효공정의 최적화 연구를 통해 생산수율을 극대화하며, 하이드로퀴논과 유사한 분자구조 물질인 4-HBA의 안정성과 안전성, 유효성을 임상시험을 통해 평가함으로써 4-HBA를 다량 함유한 고농도 발효추출물 및 정제물을 한방 미백 원료로 개발하고자 한다. 한방화장품에 대한 관심 집중으로 현재 한방 화장품 시장에 진출한 업체수는 30여개를 넘어섰고 설화수, 후, 수려한, 백옥생, 십장생, 산심, 수향진 등 브랜드수만 120여개에 달한다. 현재 관련 업계는 한방화장품을 장기적인 신성장 동력으로 삼고 독자적인 기술력을 확보하겠다는 전략이다. 또한 해외시장 진출을 위한 포석으로 삼을 계획이며 FTA 등 시장 개방 시대에 세계와 경쟁할 수 있는 유일한 경쟁력으로 삼고 더욱 특화시키겠다는 의지를 보이고 있다. 이처럼 한반화장품시장의 장기화가 지속될 것으로 보는 현시점에서 보다 차별화된 한방원료의 개발이 절실하다고 판단되며 이에 대한 방안으로 본 연구에서는 누룩 발효가 갖는 우리의 전통적인 이미지를 한방소재와 접목시킴으로서 한방화장품의 한류브랜드를 보존․정착시키고 누룩곰팡이가 갖는 효소전환반응을 이용해 한방소재의 기능성을 보다 과학적으로 향상시키고자 한다. 본 연구는 앞으로 고기능성 한방화장품 개발을 위한 전환점을 마련하는 계기가 될 수 있을 것으로 보이며 또한, 한방발효화장품이 국내시장에서의 차별화된 컨셉으로 성장할 수 있는 교두보를 마련하는 것은 물론 국제시장에서 경쟁력을 확보하는데 기여할 수 있을 것으로 판단된다.

Cell based efficacy tests can be a promising counterproposal against in vivo tests with animals. Efficacy tests with the human dermal fibroblast (HDF) were applied for screening of akin anti-ageing agents from various natural resources. Very reliable dose and effect consequences could be assessed with the proteome expression profiling. Especially for the skin anti-ageing effects, analysis of five different functional protein groups and their pattern profiling could result reliable efficacies of various agents from natural products. Multiple analysis for skin anti-ageing effects was also possible by proteome expression profiling.

Thermosensitive polymers with a lower critical solution temperature (LCST) near or below body temperature are of great interest for biomedical applications such as local drug delivery and body temperature sensitive drug release1. The advantages of thermosensitive polymers for drug delivery materials have been explained in terms of local delivery and controlled release of drugs2. We have studied biodegradable thermosensitive poly(organophosphazenes) bearing α-amino-ω- methyl-poly(ethylene glycol) (AMPEG), some hydrophobic amino acid esters, and a depsipeptide ethyl ester (ethyl-2-(O-glycyl)glycolate) as a hydrolysis sensitive moiety have been synthesized. Most of the poly(organophosphazenes) synthesized showed sol-gel transition properties in an aqueous solution. The aqueous solutions of the present polymers were injected subcutaneously into rats, which resulted in immediate gelation. The biocompatibility testing of the polymers has showed the minimum or mild tissue reaction in rats. The anticancer agent, doxorubicin, loaded easily in the polymers at low temperature was released over a month from the polymer gels in a controlled rate. These gels will be expected to be useful for local delivery of protein and anticancer drugs. In this study, we discuss thermosensitive properties and application of these poly(organophosphazenes) as drug carriers.

Tumor necrosis factor(TNF)-related apoptosis-inducing ligand(TRAIL) is an attractive anticancer therapeutic due to the tumor cell specificity. However its rapid inactivation, low stability and solubility, and severe hepatotoxicity on systemic delivery have been considered as critical obstacles for clinical applications. Chemical PEGylation is a known means to increase stability and to influence pharmacokinetic properties of a protein therapeutic. In this study, we applied the N-terminal specific PEGylation technology on active trimeric TRAIL for enhanced pharmaceutical characteristics with improved tumor therapeutic efficacy. The PEG-TRAIL showed improved physiological stability and pharmacokinetic profile than unmodified TRAIL with minimal activity loss. The improved pharmacological characteristics resulted in the enhanced therapeutic potentials on inoculated tumor animal models without detectable side effects. Furthermore, the in vivo tumor targeting capacity might offer the possibility of the PEG-TRAIL as new tumor target therapeutics.

In recent years, advances in medical imaging technology have been emphasized along with new advances in the field of cellular and molecular biology. Emerging field of medical imaging, i.e. Molecular Imaging, allows not a traditional image of anatomical changes, but a biological characterization of disease state at molecular or cellular lever. With combining molecular imaging technology and drug delivery system, a novel design of drug screening or new approaches in drug development can be designed with animal models. For example, polymer nanoparticles bearing near-infrared fluorescent can be utilized in determining optimized therapeutic dosages or the frequencies of drug administration. Nanoparticles also can be utilized as molecular probes for visualization of therapeutic efficacy in small animals. Design of novel polymeric nanoparticles with high specificity in cellular or molecular responses, application of molecular imaging probes as well as delivery carriers for therapeutic agent will be presented in current study.

Recent progress in polymer nanotechnology has been directed to the application of biodegradable nanoassemblies based on natural proteins and polysaccharides as drug delivery systems, due to their biodegradation, sustained-release properties, sub-cellular size and stability. Recombinant human gelatin-based nanoparticles were prepared by a simple coacervation method, which are suitable for the hydrophilic protein drug delivery. In addition, a hydrophobically modified glycol chitosan (HGC) was prepared by a conjugation of glycol chitosan with 5β-cholanic acid. In aqueous phases, the HGC conjugates formed nanoparticles. Their hydrophobic cores were surrounded with a hydrophilic outer domain. The inner domain can serve as a nano-reservoir for a variety of hydrophobic anticancer drugs. The characteristics of nanoparticles were investigated by various physicochemical techniques to examine the possibility as drug carriers. Although recent progress in biotechnology enables production of various protein drugs for therapeutic purposes, several challenges still remain, including enzymatic susceptibility, stability during storage, and efficacy after administration into the body. Promising approaches to overcome these limitations is to use nanoparticular drug delivery systems or in situ forming gels to achieve the controlled protein drug release, systemically or locally, over a long period of time. The controlled local drug delivery is aimed to facilitate high site-specific concentrations of therapeutic agents with prolonged retention at lower doses with reduced systemic toxicity. In situ gels can solve the problems associated with most widely used biodegradable microsphere formulations, such as the drug loss caused by low encapsulation efficiencies of microspheres, the use of harsh organic solvent and manufacturing costs increases by several processes. Definitely, the parenteral delivery including in-situ polymer gels is currently most demanding and suitable for the successful development of recombinant therapeutic proteins by stabilizing and controlling the release rate of sensitive proteins.

Hepatitis B virus (HBV) is one of the main pathogens responsible for hepatitis and hepatocellular carcinoma. Human plasma-derived Hepatitis B immune globulin (HBIG) is being used for prophylactic and liver transplantation currently. However, it may be necessary to replace a HBIG with a recombinant one because of limited availability of human plasma with high anti-HBs antibody titer and possible contamination of human pathogens. To meet these requirements, a Chinese hamster ovary (CHO) cell line, HB-C7A, was established which produces a fully human IgG1 that binds HBV. The HB-C7A recognizes the conformational “a” determinant of HBsAg and binds HBV particle more efficiently than the Hepabig (a plasma-derived HBIG from Green Cross Corp., Yongin, Korea). The virus neutralizing efficacy of this antibody was proved using HBV-naïve chimpanzees. The HB-C7A exhibits ∼3500 units/mg of antibody and its affinity (Ka) is estimated ∼7 fold higher than that of Hepabig. The HB-C7A binds to HBVinfected human liver tissue but does not bind to normal human tissues. Currently it is in clinical phase I. In addition the possibility of chronic hepatitis B treatment by this antibody was investigated. This HB-C7A has several advantages compared to the Hepabig such as activity, safety and availability.

CJ는 1964년 MSG사업을 시작으로 50여년 동안 식품 첨가물과 동물사료용 아미노산 등을 생산, 공급하며 세계 시장을 주도하여 왔다. CJ는 세계 최고 수준의 발효기술과 대량생산 경험, 글로벌 사업 노하우, 원가 경쟁력 등 탁월한 역량을 발휘하여, 라이신은 세계 2위의 M/S, 핵산은 2007년 말 세계 시장점유율 1위를 기록하여 2008년 산업자원부 선정 세계 일류상품에 선정되었다. 라이신은 동물에서 가장 부족하기 쉬운 필수 아미노산으로서 2007년 시장규모가 약 16억$로 향후에도 시장이 계속 확대될 전망이며 CJ는 뛰어난 기술과 원가경쟁력을 바탕으로 지속적으로 수율과 생산성을 개선하여 세계 1위의 라이신 기업으로 도약할 계획에 있다. 라이신 시장은 원료 수급을 위한 입지 경쟁력과 기술경쟁력이 핵심 경쟁력으로서, 바이오연구소에서는 신균주 개발 및 발효 공정 최적화로 원가절감에 기여하고 있으며, 특히 오믹스 및 대사공학을 활용하여 고생산성/고수율 균주를 지속적으로 개발함과 동시에 발효 공정 최적화를 통한 현장 기술이전을 매년 실시하고 있다. 또한 IMP와 GMP 등 핵산의 경우, 신균주 개발과 함께 신규 전환효소 발굴 및 개량을 통한 혁신적 생물전환공정을 개발 중에 있다. 향후 CJ 바이오연구소는 향후 현 사업 기술경쟁력 1위를 달성함과 동시에 아미노산 확장 및 유도체 개발, 친환경 BT제품 개발로 글로벌 바이오산업을 주도하는 연구소를 지향하고 있다.

Vitamin B2 (B2) plays an important role in the organisms because it is the precursor of flavin mononucleotide and flavin adenine dinucleotid, which function as coenzymes for a wide variety of enzymes in the intermediate metabolism. Thus, B2 have become one of the important fermentation products in biotechnology industry. The B2 has been produced from fungi (Ashbya gossypii, Aspergillus terreus, & Eremothecium ashybii), yeasts (Candida famata or guilliermondii, Mycobacterium pheli, Saccharomyces, Hansenula, & Pichia etc), and bacteria (Clostridium acetobutylicum & Bacillus subtilis). The starting material in B2 biosynthesis is guanosine triposphate (GTP), which is converted to B2 through six enzymatic reactions. Though B. subtillus, C. famate, and A. gossypii operate via different pathways until GTP, they follow the same pathway from GTP to B2. The GTP fluxes originate from three sources, serine, threonine and glyoxylates cycles. Our company investigated various vegetable oils as the sole carbon source using A. gossypii for effective B2 production. Among various vegetable oils, soybean oil was the best one for effective B2 production. Especially, When lauric acid, myristic acid, palmitic acid, stearic acid or arachidic acid was used, the cell concentrations were similar to those without the addition of each fatty acid. However, in the case of the medium containing high linolenic acid, the cell concentration was markedly decreased. With the addition of linoleic acid or oleic acid, the B2 production was about 2.0-3.0 fold higher than that obtained without linoleic acid or oleic acid. Among the nitrogen sources, yeast extract and gelatin appeared to be the most important sources for the production of B2. After B2 fermentation, the decantation was carried out for separating B2 and supernatant. There were a lot of free amino acids, peptide, and protein in the supernatant. Our company is investigating the supernatant for the use of microorganism media, fertilizer, and agrochemical etc.

대한민국 고려홍삼만으로 6,000억의 획기적 매출을 탄생시킨 정관장의 도전역사와 그 노하우는 ? 한국인삼공사는 1999년 KT&G로부터 분리후 홀로서기 10년에 접어들고 있다. 1999년 독립 당시 1,400억이었던 매출규모가 2008년 말 기준 6,000억 초과달성을 눈앞에 두면서 ‘99년 매출 대비 4배 이상의 성장을 하고 있다. 전세계 인삼시장규모를 약 3조원정도로 추정하여 볼 때 개별 기업에서 탄생시킨 동 매출액은 인삼종주국인 한국의 위상정립과 더불어 국내기업의 자랑일 수 밖에 없다. 한국인삼공사의 위와 같은 지속적 성장은 항상 소비자입장에서 고객 건강과 행복을 최우선으로 하는 기업문화에 의하여 만들어 졌다고 할 수 있다. “정관장 고려홍삼(Korean Red Ginseng)”에서 가장 중요한 것은 고객의 토탈건강(Total Health)을 중심으로한 “안심성”과 “효능성” 이라 할 수 있으며,이에 대한 체계적이고 과학적인 연구개발이 바로 믿 음 정관장의 오늘을 탄생시킨 장본인이라 판단된다. 더불어 토양부터 재배, 수확, 가공에 이르기 까지 각 단계별로 예방적 관점(문제점의 예측 보강 및 후속 단계로 진행 방지)의 안전성분석/평가를 통한 보다 좋은 제품생산, 유통제품을 중심으로한 다양한 복용개선효과 입증, 새로운 시장 도전과 창출을 위한 신제품 개발, 신뢰도 향상을 위한 전문유통망확장, 브랜드에 대한 국민 친근성, 소비자에 대한 편안한 서비스등이 바로 세계속의 정관장을 그 주인공으로 만든 것이다.

첨단생명공학산업은 21세기 과학기술과 산업의 성장을 주도할 핵심 산업으로 선진 각 국에서는 첨단생명공학기술을 이용한 생물의약품의 신약개발 경쟁력 선점을 위하여 국가차원에서 과감한 투자와 정책적인 지원을 집중하고 있다. 이는 생물의약품 분야가 신약개발 채산성이 저하되고 있는 기존의 합성의약품에 비하여 R&D risk가 비교적 적고(합성의약품의 약 40%의 개발비용 소요), 특히 IT, NT 등과 접목하여 다양한 학문분야와 함께 발전하면서 기술혁신이 이루어지는 등 폭넓은 시너지 효과에 의한 경제적 파급효과가 대단히 큰 산업분야이기 때문이다. 우리나라의 경우도 1983년 생명공학육성법이 제정된 이후 「제1차 생명공학육성기본계획(1994～2006)」에 따라 생명공학에 대한 국가차원의 체계적인 연구개발 육성에 착수하여 기술적인 인프라를 구축하였고, 2007년부터 향후 10년간은 생명공학분야 세계7위의 기술 강국 진입을 목표로 「제2차 생명공학육성기본계획(2007～2016)」을 확정하여 생명공학의약품 실용화를 위한 로드맵을 추진하고 있는 상황이다. 생물의약품(생명공학의약품, 바이오의약품)으로 탄생되기 위해서는 최종적으로 그 제품의 안전성과 유효성이 입증되어야 하고, 식품의약품안전청의 허가 절차가 필수적인 바, 생물의약품 허가과정, 관련 법규 등 전반적인 허가절차 및 주요 정책방향등을 소개하도록 하겠다.

Biologics Bureau is responsible for ensuring the safety & efficacy of vaccines, blood products, biotechnology derived products, human stem cell therapies, gene therapies, allergenic materials and anti-toxins Biologics, in contrast to drugs that are chemically synthesized, are derived from living sources (such as humans, animals, and microorganisms). Most biologics are complex mixtures that are not easily identified or characterized, and many biologics are manufactured using biotechnology. Biological products often represent the cutting-edge of biomedical research and, in time, may offer the most effective means to treat a variety of medical illnesses and conditions that presently have no other treatments available. The objectives of the nonclinical safety studies are to define pharmacological and toxicological effects not only prior to initiation of human studies but throughout clinical development. Toxicity studies are expected to be performed in compliance with good laboratory practice(GLP). Conventional approaches to toxicity testing of pharmaceutical may not be appropriate for biologics due to the unique and diverse structural and biological properties of the latter that may include species specificity, immunogenicity and unpredicted pleiotropic activities. A sponsor shall submit an IND to KFDA if the sponsor intends to conduct a clinical investigation with investigational biologics. Our primary objectives in reviewing an IND are, in all phases of investigation, to assure the safety and right of subjects, and, in phase 2 and 3, to help assure that the quality of the scientific evaluation of drugs is adequate to permit an evaluation of biologics' effectiveness and safety. Therefore, although our review of phase 1 submissions will focus on assessing the safety of phase 1 investigations, our review of phase 2 and 3 submissions will also include assessment of scientific quality of clinical investigations and likelihood that the investigations will yield data capable of meeting statutory standards for marketing approval. Current regulation, Guidance on IND application(KFDA Administrative Notice 2008-32, 2008.6.18), contains the general requirements for IND' s content and format. In case of biologics, the amount and depth of CMC information depends on the phase of the investigation. All the clinical investigations should be complied with Enforcement provision 29 and Good Clinical Practice(KFDA Administrative Notice 2008-39, 2008.6.27).

Biologics often represent the cutting-edge of biomedical research and may offer the most effective means to treat a variety of medical illnesses and conditions that presently have no other treatments available. Biologics such as vaccines, plasma-derived products, recombinant products, monoclonal antibodies, cell and gene therapy products are derived from living organisms, so they are quite different from chemically synthesized drugs. Most biologics are not easily identified or characterized and cannot be completely defined in physical and/or chemical terms alone. Because of characteristics of biologics, there is an increased risk of the introduction of adventitious agents. Biologics tend to be heat sensitive and susceptible to microbial contamination. Therefore, it is necessary to use aseptic principles from initial manufacturing steps. It is also important to make consistent products during manufacturing process and should be guaranteed the quality, safety, and efficacy during the validity period.

The term 'generic' drug is used for chemical drug that are structurally and therapeutically equivalent to an originator product and a regulatory pathway for its approval has been established. However a variety of terms such as 'similar biological medicinal products(biosimilars)', 'follow-on protein products', 'biogenerics', and 'subsequent entry biologics' have been used by different country or region about biological product. And the approach established for small molecule generic product is not appropriate for development, evaluation and licensing of subsequent entry biological product. Because biological product is large and highly complex molecule that are difficult to characterized. Also structure of the biological product is generally very sensitive to various production parameters such as media, temperature, scale so that it is unlikely that one manufacturer will be able to reproduce biological product manufactured by another company. On the other hand, biological products have been important to treatment of many life-threatening and chronic diseases such as cancer, chronic renal failure, diabetes mellitus. However, there has been a problem with limiting their access to patients because of relatively high cost. Recently the expiration of patents and data protection for the first major group of innovative biological products that were licensed based on full registration dossier is ushering in an era of subsequent entry biological products. For this reason, subsequent entry biological products are increasingly widen their range and are needed overall regulation and guideline for their evaluation. Therefore KFDA go forward with preparing the guideline for subsequent entry biological products and harmonize international guideline with it.

Molecular chaperones are proteins that help the folding of other proteins, usually through cycles of binding and release, without forming part of their final native structure. The functional importance of molecular chaperones such as the calnexin, calreticulin, ERp57, and protein disulphide isomerase that are engaged in protein folding is summarized here. The relevance of the molecular chaperones in the context of Chinese hamster ovary cell engineering to enhance the cell specific productivity (qp)1-2, in apoptosis engineering3 and protein quality control4 is discussed in detail with examples.

Lowering culture temperature has become popular in a CHO cell culture for improving recombinant protein production and quality. Many reports showed that a reduction in culture temperature (28°C-32°C) resulted in growth arrest, metabolism decrease and improved cell viability with variable effects on recombinant protein productivity. Most published studies revealed lowering culture temperature enhanced specific productivity1, qp, while a few demonstrated no increase in qp at low culture temperature2. Recently, it has been shown that enhancing effect of low culture temperature on qp depends on clones3. Therefore, the selection of clones displaying enhanced qp at low culture temperature is critical for improvement of recombinant protein production. In addition to enhanced qp, lowering culture temperature is known to be advantageous for improving protein quality4 and reducing protein aggregation5. Owing to its various beneficial effects, the application of low culture temperature to a CHO cell culture will be more attractive.

Most of insect cells have a simple N-glycosylation process and consequently, paucimannosidic or simple core glycans are predominant. Previously, we also revealed that paucimannosidic N-glycan structures are dominant in Drosophila S2 cells. It has been proposed that β-N-acetylglucosaminidase (GlcNAcase), a hexosaminidase that exists in Golgi membrane and cuts off a terminal N-acetylglucosamine (GlcNAc), might be a factor for simple N-glycosylation in several insects and their derived cells. In the present work, we investigated substantial suppression effects of GlcNAcase on Nglycan patterns in Drosophila S2 cells using two suppression strategies; RNA interference (RNAi) that is a mRNA-level method and specific chemical inhibitor, 2-acetamido-1,2-dideoxynojirimycin (2-ADN), that is a protein-level method. Compared to the original N-glycan sample from human erythropoietin (hEPO)-secreting stably transfected S2 cells, we found that improved N-glycan structures were clearly shown to have a terminal GlcNAc and/or galactose through high-performance liquid chromatography (HPLC) and matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analyses. Therefore, we proved that GlcNAcase is a possible major factor for formation of paucimannosidic core N-glycans in Drosophila S2 cells. These data suggest that complex glycoproteins might be possible in the engineered Drosophila S2 cells by suppression of GlcNAcase in N-glycosylation pathway.

Research and development of globular seamless capsules at Morishita Jintan Co., Ltd. has been performed for 35 years using an original dropping method. The application of the capsules with living microorganisms varies from DDS (Drug Delivery System) to as far as bioreactors. 1. Background of research and development Bifidobacterium longum has been one of the well known probiotics for benefit and efficacy to human intestinal condition. As B. longum is affected unfavorably by gastric acid, it was difficult to deliver significant numbers of B. longum alive to the intestines via the stomach. Delivery of living B. longum to the intestines has long been anticipated for improvements of human health. 2. Application for enteric DDS The concept of triple-layer capsule was developed to offer acid-resistant and pH-dependent disintegrated functions to its layers for protection of B. longum from the gastric acid and the release of viable B. longum in the intestines. The encapsulation of freeze-dried, viable B. longum JBL-01 was realized using a concentric triple nozzle. When tested in human volunteer subjects, the oral administration of the resultant capsule containing B. longum JBL-01 significantly relieved constipation and diarrhea. The capsules with living B. longum JBL-01 have been commercialized as supplements and drugs, and currently become some of the leading products of Morishita Jintan Co., Ltd. Recently, it was demonstrated in hemodialysis patients that the oral administration of this capsule decreased the blood serum levels of uremic toxins (phosphate, homocysteine and indoxyl sulfate). 3. Application for immobilized microorganisms Culture liquid was also encapsulated using the triple nozzle, and cell growth was observed in the capsule in a medium, i.e. incubation inside the capsule. Using a porous synthetic resin, entrapment of living microorganisms has successfully been practiced. This plastic capsule, with stable shell, can immobilize microorganisms to make itself function as a mini-bioreactor. As the density of microorganisms become very high in the capsules, the capsules can potentially process the fermentation to produce such as bio-ethanol, lactic acid or fine chemicals much economically than conventional method.

Bioethanol experiments using three forms of waste from sugarcane production process has been conducted by utilizing pre treatments and enzimatic hydrolisis and fermentation in an SSF bioreactor. The solid form of waste (i.e. bagasse), the liquid waste (i.e. molasse and the sludge (i.e. Blotong) from a sugarcabe industry in Malang, East Java were firstly characterised to determine the lignin, cellulose, carbohydrate, fibre and ash composition. Combination of pre treatment with white rot fungi and steaming were done to degrade the linocellulotic bound in bagasse. A 3000 ml Simultaneous Saccharification and Fermentation (SSF) bioreactor converted the raw material into bioethanol. No pre treatment was needed for molasse. Most of blotong contain ash and only slight carbohydrate, i.e. 5-7 %, rendering very difficult process to produce ethanol and the yield was hardly detected although combination of cellulase and cellubiase were used. The highest ethanol production was from molasses with around 81.3-87.9% yield. Modest ethanol yield was from bagasse 12.0%. However, combination of pretreatments and enzymes increased the ethanol yield to 36.4%. The increasing of ethanol production in cellulase-cellubiase hydrolysis confirmed that beside glucose, cellubiose was also produced in partial hydrolysis of cellulose. Afterwards, cellubiose was converted to glucose by cellubiase. This fact was indicated by the increase in glucose content about 16.2%. And then glucose was converted to ethanol simultaneously with S. cerevisiae. Furthermore, combination of cellulase-cellubiase-xylanase enzymes showed more ethanol yield. The reaction includes hydrolysis of cellulose to glucose and cellubiose, hydrolysis of xylan to xylose and hydrolysis of cellubiose to glucose. These reactions were indicated by the weight loss of cellulose and hemicellulose of bagasse after SSF process from 50% and 20% to 22% and 10%.

Monoclonal antibodies (mAb) have recently proven to be excellent biopharmaceutical agents. Generation of hybridomas from antigen-stimulated B cells has been a key technology for obtaining mAbs; however, it is a laborious and time-consuming process, and there are sometimes difficulties in acquiring mAbs for molecules conserved between species because of immunological tolerance. Thus, it is of great importance to develop in vitro technologies for generating useful Abs as drug candidates. We have been challenging to develop a novel in vitro antibody generation system using a chicken B cell line DT40, which displays Abs and mutates Ig genes during culture, thereby generating a useful Ab library for screening mAbs. First, we have generated an engineered cell line DT40-SW whose mutation machinery can be reversibly switched on and off. The Abgeneration system using DT40-SW was found to be useful as follows: (1) mAbs for various model antigens including antoantigens can be obtained from DT40-SW Ablibrary that is free from immunological tolerance; (2) the switching device of the mutation machinery enables to fix desirable Ig mutants by stopping mutation; (3) by repeated culture and sorting of clones bearing higher affinity for target antigens, affinity maturation can be mimicked in vitro. We have also genetically improved DT40-SW cells for mutation efficiency and Abproduction. The Ab generation system will be applicable for obtaining valuable Abs such as anti-tumor Abs.