Determination toxic effects of Hystrix Brachyura Bezoar extracts using cancer cell lines and embryo zebrafish (Danio rerio) models and identification of active principles through GC-MS analysis.

ETHNOPHARMACOLOGICAL RELEVANCE: Porcupine bezoar (PB) is used as folk medicine for various medical conditions including cancer treatment in Malaysia. However, its toxicity profile has never been thoroughly ascertained to confirm its safe nature as an efficacious traditional medicine in the treatment of cancer as well as other ailments. AIM OF THE STUDY: This study was aimed to reveal three different PBs' aqueous extracts(viz. PB-A, PB-B, PB-C) chemical constituent's profile using GC-MS analysis, anticancer property on A375, HeLa and MCF7 cancer cells, toxicity profile on zebrafish embryo morphology, EC50, LC50 and teratogenicity index. MATERIALS AND METHODS: PBs' extracts characterization was performed through GC-MS analysis, in vitro anticancer effect was carried out on A375, HeLa and MCF7 cancer cell lines and finally and toxicity properties on three different PBs aqueous extracts (viz. PB-A, PB-B, PB-C) were determined using zebrafish embryo model. RESULTS: The GC-MS analysis revealed 10 similar compounds in all PBs' extracts. Dilauryl thiodipropionate was found to be a major compound in all PBs' extracts followed by tetradecanoic acid. An in vitro anticancer study revealed PB extracts exerted median inhibition concentration (IC50) <50 µg/mL, on cancer cells viz. A375, HeLa and MCF7 with no significant toxicity on normal cells viz. NHDF cells. In vivo toxicity of PBs extracts found affecting tail detachment, hatching, craniofacial, brain morphology, soft tissues, edema, spinal, somites, notochord and cardiovascular system (brachycardia, disruption of blood circulation) deformities. The LC50 and EC50 demonstrated PB extracts effect as dose and time dependent with median concentration <150.0 µg/mL. Additionally, teratogenicity index (TI) viz. >1.0 revealed teratogenic property for PB extracts. CONCLUSIONS: The findings revealed that all three PBs aqueous extracts possessed anticancer activity and exhibited significant toxicological effects on zebrafish embryos with high teratogenicity index. Hence, its use as an anticancer agent requires further investigation and medical attentions to determine its safe dose.

Association between Bioactive Molecules in Breast Milk and Type 1 Diabetes Mellitus.

The association between breastfeeding and type 1 diabetes mellitus (T1DM) is controversial. However, several recent studies have established a link between these two factors, necessitating a need to review this subject to raise public awareness. Current research indicates that breast milk contains a variety of bioactive substances including immunoglobulins, oligosaccharides, insulin, lactoferrin, lysozyme, cytokines, epidermal growth factors, leukocytes, nucleotides, beneficial bacteria and vitamins. Such substances strengthen the breastfeeding infant's immune system, both directly, by increasing gut microbiota diversity and attacking harmful bacteria and pro-inflammatory molecules, and indirectly, by increasing thymus performance. Accordingly, a lack of or inadequate breastfeeding may predispose infants to several autoimmune disorders, including T1DM. Nursing mothers and caregivers are therefore advised to follow optimal breastfeeding practices prior to introducing complementary foods.

A Review of Bioactive Factors in Human Breastmilk: A Focus on Prematurity.

Preterm birth is an increasing worldwide problem. Prematurity is the second most common cause of death in children under 5 years of age. It is associated with a higher risk of several pathologies in the perinatal period and adulthood. Maternal milk, a complex fluid with several bioactive factors, is the best option for the newborn. Its dynamic composition is influenced by diverse factors such as maternal age, lactation period, and health status. The aim of the present review is to summarize the current knowledge regarding some bioactive factors present in breastmilk, namely antioxidants, growth factors, adipokines, and cytokines, paying specific attention to prematurity. The revised literature reveals that the highest levels of these bioactive factors are found in the colostrum and they decrease along the lactation period; bioactive factors are found in higher levels in preterm as compared to full-term milk, they are lacking in formula milk, and decreased in donated milk. However, there are still some gaps and inconclusive data, and further research in this field is needed. Given the fact that many preterm mothers are unable to complete breastfeeding, new information could be important to develop infant supplements that best match preterm human milk.

Small molecules and extrinsic factors promoting differentiation of stem cells into insulin-producing cells.

In the modern world, type-2 diabetes mellitus has become a leading public healthcare problem, due to major risks of morbidity and mortality. Prevalence has increased significantly in recent decades. Treatment involves oral hypoglycemic agents or insulin replacement therapy. Development is ongoing for cell-based diabetes therapies using stem cells with the potential to differentiate into insulin-producing cells (IPCs): embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and stem cells from adult pancreas, liver, central nervous system, bone marrow and adipose tissue. Successful induction of iPSCs, however, depends on the quantity and quality of available stem cells and the development of adapted protocols determining the environment of extrinsic factors and involvement of small molecules. Validating such new cell therapies must be founded on this experimental rationale.

Chemical Profile, In Vitro Enzyme Inhibitory, and Antioxidant Properties of Stereum Species (Agaricomycetes) from Turkey.

Recently, mushroom species have been the focus of researchers' interest because of several bioactivities. The aim of this study was to investigate the chemical profile and biological activities of various extracts of two Stereum species (S. rugosum and S. sanguinolentum). Antioxidant activity was tested using ß-carotene-linoleic acid, DPPH scavenging, ABTS·+ scavenging, cupric-reducing antioxidant capacity (CUPRAC), and metal chelating assays. The extracts were also tested for their enzyme inhibitory activities against acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). HPLC-DAD was applied for the analysis of phenolic compounds, and fatty acid compositions were determined using GC and GC-MS. When fumaric acid and catechin hydrate were found as the most abundant phenolic compounds in both Stereum species, oleic acid and palmitic acid were identified as major fatty acids. Both of the studied Stereum methanol extracts were determined as the most active in ß-carotene-linoleic acid, DPPR, ABTS·+, and CUPRAC assays; the n-hexane extracts were found to be most active in metal chelating and AChE inhibitory activity assays. In addition, the methanol extract of S. sanguinolentum (IC50: 34.26 ± 0.31 µg/mL) showed higher ABTS·+ scavenging activity than α-tocopherol (IC50: 38.51 ± 0.54 µg/mL). The acetone extracts were found as potent inhibitors against BChE. These results suggest that Stereum species could be an antioxidant source and cholinesterase agent in pharmaceutic, food, and cosmetics industries.

Phytochemical composition, antioxidant and antiproliferative activities and effects on nuclear DNA of ethanolic extract from an Italian mycelial isolate of Ganoderma lucidum.

ETHNOPHARMACOLOGICAL RELEVANCE: Ganoderma lucidum (Curtis) P. Karst. (also known as Linghzhi and Reishi) is the most appreciated and revered medicinal mushroom across many Asian countries, but its properties have also attracted interest in Western countries. Indeed, in the West, it is now commercially available as a dietary supplement in preparations mainly made from spores, fruiting bodies and mycelia. It is employed in both nutraceutical and pharmacological formulations either for its immuno-modulating anti-inflammatory properties or as an effective adjuvant therapy in the treatment of several chronic diseases as well as in cancer treatment. AIM OF THE STUDY: The aim of this investigation was to show the phytochemical composition and antioxidant and antiproliferative activities of an ethanolic extract from an Italian mycelial isolate of Ganoderma lucidum and to assess its effects on nuclear DNA. MATERIALS AND METHODS: LC/ESI-MS and tandem mass spectrometry MSMS were used to obtain structural identification of ethanolic G. lucidum extract constituents. Antioxidant activities were determined by the DPPH method, chelating effect on Fe2+ and lipoxygenase inhibition while cytotoxic activities using the MTT assay. Effects on nuclear DNA were evaluated using the DNA nicking assay in a cell-free system and the fast halo assay performed on oxidatively injured human U937 cells; apoptosis induction was investigated using the non-denaturing fast halo assay and DNA laddering detection. RESULTS: This extract was rich in several bioactive compounds, mainly phenolic and triterpenic acids. It showed antioxidant activity and protective effects in oxidatively injured DNA in cell-free analyses and antiproliferative, genotoxic, and proapoptotic effects in the cell model. CONCLUSIONS: Italian G. lucidum mycelium isolate appears to be a source of various natural compounds that may have applications as chemopreventive agents or functional foods.

Chemical, Physical, and Biological Factors Shape Littoral Invertebrate Community Structure in Coal-Mining End-Pit Lakes.

Aquatic invertebrates form the base of the consumer food web in lakes. In coal-mining end-pit lakes, invertebrates are exposed to an environment with potentially challenging physical and chemical features. We hypothesized that the physical and chemical features of end-pit lakes reduce critical littoral habitat and thus reduce invertebrate diversity, thereby limiting the potential for these lakes to be naturalized. We used a multivariate approach using principle component analysis and redundancy analysis to study relationships between invertebrate community structure, habitat features, and water quality in five end-pit lakes and five natural lakes in the Rocky Mountain foothills of west-central Alberta, Canada. Results show a significantly different invertebrate community structure was present in end-pit lakes as compared with reference lakes in the same region, which could be accounted for by water hardness, conductivity, slope of the littoral zone, and phosphorus concentrations. Habitat diversity in end-pit lakes was also limited, cover provided by macrophytes was scarce, and basin slopes were significantly steeper in pit lakes. Although water chemistry is currently the strongest influencing factor on the invertebrate community, physical challenges of habitat homogeneity and steep slopes in the littoral zones were identified as major drivers of invertebrate community structure. The addition of floating wetlands to the littoral zone of existing pit lakes can add habitat complexity without the need for large-scale alterations to basing morphology, while impermeable capping of waste-rock and the inclusion of littoral habitat in the planning process of new pit lakes can improve the success of integrating new pit lakes into the landscape.

Edible bird's nest modulate intracellular molecular pathways of influenza A virus infected cells.

BACKGROUND: Edible Bird's Nest (EBN) as a popular traditional Chinese medicine is believed to have health enhancing and antiviral activities against influenza A virus (IAV); however, the molecular mechanism behind therapeutic effects of EBN is not well characterized. METHODS: In this study, EBNs that underwent different enzymatic preparation were tested against IAV infected cells. 50% cytotoxic concentration (CC50) and 50% inhibitory concentration (IC50) of the EBNs against IAV strain A/Puerto Rico/8/1934(H1N1) were determined by HA and MTT assays. Subsequently, the sialic acid content of the used EBNs were analyzed by fluorometric HPLC. Western Blotting and immunofluorescent staining were used to investigate the effects of EBNs on early endosomal trafficking and autophagy process of influenza virus. RESULTS: This study showed that post inoculations of EBNs after enzymatic preparations have the highest efficacy to inhibit IAV. While CC50 of the tested EBNs ranged from 27.5-32 mg/ml, the IC50 of these compounds ranged between 2.5-4.9 mg/ml. EBNs could inhibit IAV as efficient as commercial antiviral agents, such as amantadine and oseltamivir with different mechanisms of action against IAV. The antiviral activity of these EBNs correlated with the content of N-acetyl neuraminic acid. EBNs could affect early endosomal trafficking of the virus by reducing Rab5 and RhoA GTPase proteins and also reoriented actin cytoskeleton of IAV infected cells. In addition, for the first time this study showed that EBNs can inhibit intracellular autophagy process of IAV life cycle as evidenced by reduction of LC3-II and increasing of lysosomal degradation. CONCLUSIONS: The results procured in this study support the potential of EBNs as supplementary medication or alternative to antiviral agents to inhibit influenza infections. Evidently, EBNs can be a promising antiviral agent; however, these natural compounds should be screened for their metabolites prior to usage as therapeutic approach.

Productivity, Physicochemical Changes, and Antioxidant Activity of Shiitake Culinary-Medicinal Mushroom Lentinus edodes (Agaricomycetes) Cultivated on Lignocellulosic Residues.

The effects of substrate and strain on productivity, physicochemical characteristics, and compounds with antioxidant activity were evaluated in basidiomes of the shiitake mushroom, Lentinus edodes. Strains IE-245 and IE-256 and the substrates oak wood shavings (OW), sorghum stubble (SS), and sugar cane bagasse (SC) were used. Productivity was evaluated by measuring biological efficiency (BE), production rate (PR), and yield. Total sugars, total soluble solids, pH, titratable acidity, color parameters, total phenolics, flavonoids, ascorbic acid, and antioxidant activity of the basidiomes were measured. BE, PR and yield were higher with the combination IE-256/SS, at 103.71%, 1.32%, and 34.57%, respectively. The largest amount of total sugars (17.61 mg glucose · g-1 dry weight) was found with combination IE-256/SS. Variation was observed in basidiome color; the lowest luminosity (L*) value (darkest color) was found in the IE-256 strain on the OW substrate (L* = 30.45), whereas that of the IE-245 strain on the SC substrate was the lightest in color (L* = 57.00). The largest amounts of total phenolics were recorded in the IE-256 strain on the OW (6.50 mg gallic acid equivalents [GAE] · g-1 dry weight) and the SS substrates (5.85 mg GAE · g-1 dry weight). The best antioxidant activity was obtained with IE-256-0.80, 0.65, and 0.59 µmol Trolox equivalents · g dry weight-1-on the OW, SC, and SS substrates, respectively. Based on the values of BE, PR, and yield, IE-256/SS was the most productive. Substrate and strain, and their interactions, influenced the physicochemical characteristics of the basidiomes and the amounts of compounds with antioxidant activity they contained.

Available sustainable alternatives replace endangered animal horn based on their proteomic analysis and bio-effect evaluation.

The use of endangered animal products in traditional Chinese medicine (TCM) and other ethno-medicines is culturally widespread across many regions of Asia. In the present study, traditional efficacies of seven types of animal horn including antipyretic, sedative and procoagulant activities were evaluated. Shotgun proteomic analysis was performed on material from horns following separation into soluble and insoluble fractions. Over 200 proteins were identified in each sample using nano LC-MS/MS, and these were classified according to their molecular function and cellular component using principal component analysis (PCA). The results indicated that seven horns showed antipyretic, sedative and procoagulant effect. Proteomic analysis showed that YH and WBH were similar to RH in terms of protein profile, and GH was similar to SAH. In addition, YH and GH were similar to RH in their cellular component classification profile. PCA based on the composition of keratin and keratin-associated proteins showed that constituents of WBH and GH were similar to RH and SAH, respectively. This is the first analysis of the protein content of animal horns used in TCM, and it is effective to substitute the horn of endangered animals with sustainable alternatives from domestic animals.

Chemical screening and identification of high cordycepin containing cultured isolate(s) of medicinal Chinese caterpillar mushroom, Ophiocordyceps sinensis (Berk.) G.H. Sung et al.

Forty isolates of Ophiocordyceps sinensis collected from Himalayan alpine meadows of Uttarakhand, India, and cultivated on Jhangora (Echinochloa crusgalli) grains were screened to identify the isolate(s) of high cordycepin content. The cultured mycelia were extracted with 50% methanol-chloroform and analyzed by HPTLC using chloroform:methanol (6:1 v/v) as mobile phase and densitometry scanning at 263 nm. Cordycepin varied from 0.002% to 0.029% was detected in twenty-one isolates. Compared to natural O. sinensis (0.004%, 0.006%), cordycepin was determined to be enhanced in twelve cultured samples.

Comparative study of wild edible mushrooms as sources of antioxidants.

The purpose of the study was to explore sixteen of the most popular edible species of wild-growing mushrooms as potential sources of antioxidants. Among the mushrooms tested, the highest total polyphenol contents, exceeding 100 mg/100 g fresh mass, were found in five mushrooms: Boletus chrysenteron, B. edulis, Leccinum scabrum, L. aurantiacum, and Macrolepiota procera. Antioxidant activity was measured with the FRAP, TEAC, DPPH scavenging ability and ferrous ions chelating ability assays. Results of the study show that wild mushrooms vary according to their antioxidant properties. The highest FRAP potentials, exceeding 1 mmol/100 g, were found in five species ofBoletales: Boletus edulis, B. chrysenteron, Leccinum scabrum, L. aurantiacum, and Suillus grevillei. TEAC values were from 1.07 to 4.01 mmol/100 g fresh mass. High TEAC values (>2.3 mmol/100 g) were found in Leccinum scabrum, L. aurantiacum, Macrolepiota procera, Boletus chrysenteron, and B. edulis. The DPPH radical scavenging effectiveness of mushroom extracts, expressed as EC50 values, was in range 2.91-13.86 mg/mL. Scavenging ability was the highest for B. edulis and B. chrysenteron. The metal chelating ability of mushroom extracts expressed as ECso values of chelating ability on ferrous ions were from 8.02 mg/mL in Cantharellus cibarius to 12.10 mg/mL in Suillus luteus. Among the mushrooms tested, Boletus chrysenteron and B. edulis were characterized by high scores of polyphenol contents and antioxidant activity in the FRAP, TEAC, and DPPH assays. These results place these culinary species of wild-growing mushrooms among products with considerable antioxidant potential.

The effect of royal sun agaricus, Agaricus brasiliensis S. Wasser et al., extract on methyl methanesulfonate caused genotoxicity in Drosophila melanogaster.

The effect of culinary-medicinal Royal Sun Agaricus (Agaricus brasiliensis) hot water extract on methyl methane sulfonate (MMS) induced mutagenicity/genotoxity in Drosophila melanogaster was studied using a quick and broadly applicable in vivo assay, i.e., the wing somatic mutation and recombination test. We used 2nd instar larvae, trans-heterozygous for the third chromosome recessive markers, i.e., multiple wing hairs (mvh) and flare-3 [flr (3)], and fed them for 24 h with the aqueous extract of A. brasiliensis. For antigenotoxicity studies a 24-h pretreatment with the extract was done, followed by a 48-h treatment of the then 3rd instar larvae with MMS. The frequency of mutations of the wing blade changes (i.e., of the number of wing spots of different sizes) induced in somatic cells was determined as a parameter of genetic changes of the wing imaginal discs. The results showed that A. brasiliensis extract did not cause any genotoxic or mutagenic effects. No antigenotoxic and/or protective effect against the induction of mutations by MMS was observed. Instead, a possible enhanced mitotic recombination frequency by MMS was seen after pretreatment of the larvae with A. brasiliensis extract. Possible mechanisms of action are discussed.

Antiproliferative effect of methanolic extraction of tualang honey on human keloid fibroblasts.

BACKGROUND: Keloid is a type of scar which extends beyond the boundaries of the original wound. It can spread to the surrounding skin by invasion. The use of Tualang honey is a possible approach for keloid treatment. The objective of this study was to determine the antiproliferative effect of methanolic extraction of Tualang honey to primary human keloid fibroblasts and to identify the volatile compounds in methanol extraction of Tualang honey. METHODS: Crude Tualang honey was extracted with methanol and then dried using rota vapor to remove remaining methanol from honey. Normal and keloid fibroblasts were verified and treated with the extracted honey. Cell proliferation was tested with [3-(4,5-dimethylthiazol-2-yi)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] (MTS) assay. Extraction of Tualang honey using methanol was carried out and the extracted samples were analysed using gas chromatography-mass spectrometry (GC-MS). The result was analysed using SPSS and tested with Kruskal-Wallis and Mann-Whitney tests. RESULTS: Methanolic extraction of honey has positive anti proliferative effect on keloid fibroblasts in a dose-dependent manner. The presence of fatty acids such as palmitic acid, stearic acid, oleic acid, linoleic acid and octadecanoic acid may contribute to the anti-proliferative effect in keloid fibroblasts. CONCLUSIONS: The methanolic honey extraction has an antiproliferative effect on keloid fibroblasts and a range of volatile compounds has been identified from Tualang honey. The antiproliferative effect of keloid fibroblasts towards Tualang honey may involve cell signaling pathway. Identifying other volatile compounds from different organic solvents should be carried out in future.

[Active ingredients and efficacies of Ganoderma lucidum cultivated on non-medicinal parts of Chinese medicinal herbs].

OBJECTIVE: Ganoderma lucidum was cultivated on non-medicinal parts of Salvia miltiorrhiza, Chrysanthemum morifolium, Ptatycodgn grandlfiorum, as all are Chinese traditional herbal medicines. We studied the changes of active ingredients and efficacies of the Ganoderma lucidum fruit bodies. METHODS: The agronomic characters, polysaccharide and terpene contents, acute toxicity and efficacy of Ganoderma lucidum grown on the non-medicinal part of the three materials were compared with that grown on the ordinary formula group (OF. G) which was composed of corn cob, cotton seed shell. RESULTS: Biological conversion efficiencies of the Ganoderma lucidum fruit body using non-medicinal parts were higher than that of using the ordinary formula group (OF. G), though growth periods became longer; Contents of active ingredients were all improved except that the terpene content of the Salvia miltiorrhiza group was decreased. Both polysaccharide and terpene from the Chrysanthemum morifolium group were the highest, contents of which were respectively 2.47% and 0.79%; Acute toxicity test showed that Ganoderma lucidum fruit bodies were all with low toxicities. Mice maximum tolerance dose were 100 g/kg weight. In hemolysin test and sleeping promotion test, the Chrysanthemum morifolium group showed better effect than the ordinary formula group (OF. G). In anti-fatigue test, only the ordinary formula group (OF. G) proved to be more effective. CONCLUSION: It's feasible to cultivate Ganoderma lucidum and active ingredients and efficacies of Ganoderma lucidum have been changed using the non-medicinal parts of Chinese medicinal herbs.

Metabolite profiles for Antrodia cinnamomea fruiting bodies harvested at different culture ages and from different wood substrates.

Antrodia cinnamomea is a precious edible fungus endemic to Taiwan that has long been used as a folk remedy for health promotion and for treating various diseases. In this study, an index of 13 representative metabolites from the ethanol extract of A. cinnamomea fruiting body was established for use in quality evaluation. Most of the index compounds selected, particularly the ergostane-type triterpenoids and polyacetylenes, possess good anti-inflammation activity. A comparison of the metabolite profiles of different ethanol extracts from A. cinnamomea strains showed silmilar metabolites when the strains were grown on the original host wood (Cinnamomum kanehirai) and harvested after the same culture time period (9 months). Furthermore, the amounts of typical ergostane-type triterpenoids in A. cinnamomea increased with culture age. Culture substrates also influenced metabolite synthesis; with the same culture age, A. cinnamomea grown on the original host wood produced a richer array of metabolites than A. cinnamomea cultured on other wood species. We conclude that analysis of a fixed group of compounds including triterpenoids, benzolics, and polyacetylenes constitutes a suitable, reliable system to evaluate the quality of ethanol extract from A. cinnamomea fruiting bodies. The evaluation system established in this study may provide a platform for analysis of the products of A. cinnamomea.

[Determination of biological activity of extract from hirudo by N-benzoyl-L-arginine ethyl ester].

As a potent anticoagulant, leech a traditional Chinese medicine, has become increasing topics. Hirudin, which is the primary effective component in leech, is a specific and efficient inhibitor of thrombin, mainly used in prevention and treatment of thrombus on the clinic practice. However, there is still no accurate and convenient method reported about the determination of it's biological activity. This paper reported a method for the determination of the biological activity the of extract from hirudo. The extra thrombin, which was not inhibited by hirudin in the extract from hirudo, reacted with N-benzoyl-L-arginine ethyl ester and was determined. The biological activity of the hirudo extract was determined, indirectly. The linear of calibration curve and accuracy were both perfect, the method was accurate and reliable.

Chemical diversity of biologically active metabolites in the sclerotia of Inonotus obliquus and submerged culture strategies for up-regulating their production.

Inonotus obliquus (Fr.) Pilat is a white rot fungus belonging to the family Hymenochaetaceae in the Basidiomycota. In nature, this fungus rarely forms a fruiting body but usually an irregular shape of sclerotial conk called 'Chaga'. Characteristically, I. obliquus produces massive melanins released to the surface of Chaga. As early as in the sixteenth century, Chaga was used as an effective folk medicine in Russia and Northern Europe to treat several human malicious tumors and other diseases in the absence of any unacceptable toxic side effects. Chemical investigations show that I. obliquus produces a diverse range of secondary metabolites including phenolic compounds, melanins, and lanostane-type triterpenoids. Among these are the active components for antioxidant, antitumoral, and antiviral activities and for improving human immunity against infection of pathogenic microbes. Geographically, however, this fungus is restricted to very cold habitats and grows very slowly, suggesting that Chaga is not a reliable source of these bioactive compounds. Attempts for culturing this fungus axenically all resulted in a reduced production of bioactive metabolites. This review examines the current progress in the discovery of chemical diversity of Chaga and their biological activities and the strategies to modulate the expression of desired pathways to diversify and up-regulate the production of bioactive metabolites by the fungus grown in submerged cultures for possible drug discovery.

Development of evaluation system for bioactive substances using human artificial chromosome-mediated osteocalcin gene expression.

Bioactive substances in daily food and supplements are expected to prevent various lifestyle-related diseases. Recently, many evaluation systems for bioactive substances were developed with cell lines integrated with green fluorescence protein (GFP) reporter gene. To evaluate osteogensis activity in functional food, we developed a novel cell line that reports osteocalcin gene expression using the human artificial chromosome (HAC) vector. HAC vectors are able to avoid various problems in usual plasmid vector such as difficulty in control of transgene copy number. HAC is transmitted to cells as an independent chromosome from host chromosomes, and expresses transgenes depending on host cell circumstances. We established Chinese hamster ovary cell lines that carried GFP gene regulated by osteocalcin gene promoter on the HAC. Expression of GFP was responded to vitamin D(3) [1alpha,25(OH)(2)D(3)]. Furthermore, we constructed HAC vector bearing tandem repeats of reporter gene unit, to enhance intensity of gene expression. GFP expression in these reporter cells is related to the copy number of reporter gene units. Using the evaluation system for bioactive substances, we could show osteogenic activity in some fish oils.