Potency of Phlebia species of white rot fungi for the aerobic degradation, transformation and mineralization of lindane.

The widespread use of the organochlorine insecticide lindane in the world has caused serious environmental problems. The main purpose of this paper is to investigate the potency of several Phlebia species of white rot fungi to degrade, transform and mineralize lindane, and to provide the feasibility of using white rot fungi for bioremediation at contaminated sites. Based on tolerance experiment results, Phlebia brevispora and Phlebia lindtneri had the highest tolerance to lindane and were screened by degradation tests. After 25 days of incubation, P. brevispora and P. lindtneri degraded 87.2 and 73.3% of lindane in low nitrogen medium and 75.8 and 64.9% of lindane in high nitrogen medium, respectively. Several unreported hydroxylation metabolites, including monohydroxylated, dehydroxylated, and trihydroxylated products, were detected and identified by GC/MS as metabolites of lindane. More than 10% of [14C] lindane was mineralized to 14CO2 by two fungi after 60 days of incubation, and the mineralization was slightly promoted by the addition of glucose. Additionally, the degradation of lindane and the formation of metabolites were efficiently inhibited by piperonyl butoxide, demonstrating that cytochrome P450 enzymes are involved in the fungal transformation of lindane. The present study showed that P. brevispora and P. lindtneri were efficient degraders of lindane; hence, they can be applied in the bioremediation process of lindane-contaminated sites.

Molecular breeding of lignin-degrading brown-rot fungus Gloeophyllum trabeum by homologous expression of laccase gene.

The basidiomycete Gloeophyllum trabeum KU-41 can degrade Japanese cedar wood efficiently. To construct a strain better suited for biofuel production from Japanese cedar wood, we developed a gene transformation system for G. trabeum KU-41 using the hygromycin phosphotransferase-encoding gene (hpt) as a marker. The endogenous laccase candidate gene (Gtlcc3) was fused with the promoter of the G. trabeum glyceraldehyde-3-phosphate dehydrogenase-encoding gene and co-transformed with the hpt-bearing pAH marker plasmid. We obtained 44 co-transformants, and identified co-transformant L#61, which showed the highest laccase activity among all the transformants. Moreover, strain L#61 was able to degrade lignin in Japanese cedar wood-containing medium, in contrast to wild-type G. trabeum KU-41 and to a typical white-rot fungus Phanerochaete chrysosporium. By using strain L#61, direct ethanol production from Japanese cedar wood was improved compared to wild type. To our knowledge, this study is the first report of the molecular breeding of lignin-degrading brown-rot fungus and direct ethanol production from softwoods by co-transformation with laccase overproduction constructs.

Melanin biosynthesis inhibitory activity of a compound isolated from young green barley (Hordeum vulgare L.) in B16 melanoma cells.

In the course to find compounds that inhibit melanin biosynthesis (i.e., whitening agents), we evaluated the effects of the methanol-soluble fraction (i.e., the water-soluble portion of methanol extracts-CHP20P-MeOH eluted fraction) from young green barley leaves on melanin production in B16 melanoma cells. Activity-guided fractionation led to an isolate called tricin (compound 1) as an inhibitory compound of melanin production in B16 melanoma cells. Furthermore, tricin analogs such as tricetin, tricetin trimethyl ether, luteolin, and apigenin were used for analyzing the structure-activity relationships (SAR) of 5,7-dihydroxyflavones studies. Tricin demonstrated stronger inhibitory activity compared to three other compounds. The results suggest that a hydroxyl group at the C-4' position and methoxy groups at the C-3',5' positions of the tricin skeleton may have important roles in this inhibitory activity in B16 melanoma cells. Our results suggest that tricin inhibits melanin biosynthesis with higher efficacy than arbutin, and it could be used as a whitening agent.

Hypotensive effects and angiotensin-converting enzyme inhibitory peptides of reishi (Ganoderma lingzhi) auto-digested extract.

Reishi (Ganoderma lingzhi) has been used as a traditional medicine for millennia. However, relatively little is known about this mushroom's proteins and their bioactivities. In this study, we used reishi's own proteases to hydrolyze its protein and obtained auto-digested reishi (ADR) extract. The extract was subjected to in vitro assays and administered to spontaneous hypertensive rats (SHRs) to determine its potential for use as a hypotensive medication. Bioassay-guided fractionation and de novo sequencing were used for identifying the active compounds. After 4 h administration of ADR, the systolic pressure of SHRs significantly decreased to 34.3 mmHg (19.5% change) and the effect was maintained up to 8 h of administration, with the decrease reaching as low as 26.8 mmHg (15% reduction-compare to base line a decrease of 26.8 mmHg is less than a decrease of 34.3 mmHg so it should give a smaller % reduction). Eleven peptides were identified and four of them showed potent inhibition against ACE with IC50 values ranging from 73.1 µM to 162.7 µM. The results showed that ADR could be a good source of hypotensive peptides that could be used for antihypertensive medication or incorporation into functional foods.

Identification of novel glycosyl hydrolases with cellulolytic activity against crystalline cellulose from metagenomic libraries constructed from bacterial enrichment cultures.

To obtain cellulases that are capable of degrading crystalline cellulose and cedar wood, metagenomic libraries were constructed from raw soil sample which was covered to pile of cedar wood sawdust or from its enrichment cultures. The efficiency of screening of metagenomic library was improved more than 3 times by repeating enrichment cultivation using crystalline cellulose as a carbon source, compared with the library constructed from raw soil. Four cellulase genes were obtained from the metagenomic libraries that were constructed from the total genome extracted from an enrichment culture that used crystalline cellulose as a carbon source. A cellulase gene and a xylanase gene were obtained from the enrichment culture that used unbleached kraft pulp as a carbon source. The culture supernatants of Escherichia coli expressing three clones that were derived from the enrichment culture that used crystalline cellulose showed activity against crystalline cellulose. In addition, these three enzyme solutions generated a reducing sugar from cedar wood powder. From these results, the construction of a metagenomic library from cultures that were repetition enriched using crystalline cellulose demonstrated that this technique is a powerful tool for obtaining cellulases that have activity toward crystalline cellulose.

Effect of chemical factors on integrated fungal fermentation of sugarcane bagasse for ethanol production by a white-rot fungus, Phlebia sp. MG-60.

Bioethanol production through integrated fungal fermentation (IFF), involving a unified process for biological delignification with consolidated biological processing by the white-rot fungus Phlebia sp. MG-60, was applied to sugarcane bagasse. Initial moisture content of the bagasse was found to affect biological delignification by MG-60, and 75% moisture content was suitable for selective lignin degradation and subsequent ethanol production. Additives, such as basal media, organic compounds, or minerals, also affected biological delignification of bagasse by MG-60. Basal medium addition improved both delignification and ethanol production. Some inorganic chemical factors, such as Fe(2+), Mn(2+), or Cu(2+), reduced bagasse carbohydrate degradation by MG-60 during delignifying incubations and resulted in increased ethanol production. The present results indicated that suitable culture conditions could significantly improve IFF efficiency.

Biodegradation of heptachlor and heptachlor epoxide-contaminated soils by white-rot fungal inocula.

The ability of certain white-rot fungi (WRF) inocula to transform heptachlor and heptachlor epoxide and its application in artificially contaminated soil were investigated. Fungal inoculum of Pleurotus ostreatus eliminated approximately 89 % of heptachlor after 28 days of incubation, and chlordene was detected as the primary metabolite. The fungal inoculum of Pleurotus ostreatus had the highest ability to degrade heptachlor epoxide; approximately 32 % were degraded after 28 days of incubation, and heptachlor diol was detected as the metabolite product. Because Pleurotus ostreatus transformed heptachlor into a less toxic metabolite and could also effectively degrade heptachlor epoxide, it was then selected to be applied to artificially contaminated soil. The spent mushroom waste (SMW) of Pleurotus ostreatus degraded heptachlor and heptachlor epoxide by approximately 91 and 26 %, respectively, over 28 days. This finding indicated that Pleurotus ostreatus SMW could be used to bioremediate heptachlor- and heptachlor epoxide-contaminated environments.

Wild Mushrooms in Nepal: Some Potential Candidates as Antioxidant and ACE-Inhibition Sources.

Twenty-nine mushrooms collected in the mountainous areas of Nepal were analyzed for antioxidant activity by different methods, including Folin-Ciocalteu, ORAC, ABTS, and DPPH assays. Intracellular H2O2-scavenging activity was also performed on HaCaT cells. The results showed that phenolic compounds are the main antioxidant of the mushrooms. Among studied samples, Inonotus andersonii, and Phellinus gilvus exhibited very high antioxidant activity with the phenolic contents up to 310.8 and 258.7 mg GAE/g extracts, respectively. The H2O2-scavenging assay on cells also revealed the potential of these mushrooms in the prevention of oxidative stress. In term of ACE-inhibition, results showed that Phlebia tremellosa would be a novel and promising candidate for antihypertensive studies. This mushroom exhibited even higher in vitro ACE-inhibition activity than Ganoderma lingzhi, with the IC50 values of the two mushrooms being 32 µ g/mL and 2 µ g/mL, respectively. This is the first time biological activities of mushrooms collected in Nepal were reported. Information from this study should be a valuable reference for future studies on antioxidant and ACE-inhibitory activities of mushrooms.

Inhibitory effects of the leaves of loquat (Eriobotrya japonica) on bone mineral density loss in ovariectomized mice and osteoclast differentiation.

The loquat, Eriobotrya japonica Lindl. (Rosaceae), is a small tree native to Japan and China that is widely cultivated for its succulent fruit. Its leaves are used as an ingredient of a tasty tea called "Biwa cha" in Japanese. The anti-osteoporosis effects of the leaves of loquat in vitro and in vivo have been investigated. After 15 days of feeding normal diet or diet supplemented with 5% loquat leaves, the body weight, viscera weights, and bone mineral density (BMD) of both groups of eight ovariectomized (OVX) mice were compared. The result showed that the loss of BMD in loquat-fed mice was significantly prevented in three parts of the body, especially in the trabecular bone of the head (P < 0.05), abdomen (P < 0.01), and lumbar (P < 0.05) compared to the control group. No hypertrophy in the uterus by the loquat leaves diet was observed. The effect of the extract (447.25 g) prepared from the dried leaves of loquat (2.36 kg) was further studied on RANKL-induced osteoclast differentiation and cell viability. The extract suppressed the differentiation of osteoclasts under 50, 125, 250, and 500 µg/mL. Through bioactivity-guided fractionation, ursolic acid (1) was isolated and inhibited osteoclast differentiation under 4 and 10 µg/mL. It was concluded that loquat leaves possess the potential to suppress ovariectomy-induced bone mineral density deterioration.

Rational design and synthesis of topoisomerase I and II inhibitors based on oleanolic acid moiety for new anti-cancer drugs.

Semisynthetic reactions were conducted on oleanolic acid, a common plant-derived oleanane-type triterpene. Ten rationally designed derivatives of oleanolic acid were synthesized based on docking studies and tested for their topoisomerase I and IIα inhibitory activity. Semisynthetic reactions targeted C-3, C-12, C-13, and C-17. Nine of the synthesized compounds were identified as new compounds. The structures of these compounds were confirmed by spectroscopic methods (1D, 2D NMR and MS). Five oleanolic acid analogues (S2, S3, S5, S7 and S9) showed higher activity than camptothecin (CPT) in the topoisomerase I DNA relaxation assay. Four oleanolic acid analogues (S2, S3, S5 and S6) showed higher activity than etoposide in a topoisomerase II assay. The results indicated that the C12-C13 double bond of the oleanolic acid skeleton is important for the inhibitory activity against both types of topoisomerases, while insertion of a longer chain at either position 3 or 17 increases the activity against topoisomerases by various degrees. Some of the synthesized compounds act as dual inhibitors for both topoisomerase I and IIα.

Expression of a manganese peroxidase isozyme 2 transgene in the ethanologenic white rot fungus Phlebia sp. strain MG-60.

BACKGROUND: The white-rot fungus Phlebia sp. strain MG-60 was proposed as a candidate for integrated fungal fermentation process (IFFP), which unifies aerobic delignification and semi-aerobic consolidated biological processing by a single microorganism based on its ability to efficiently degrade lignin and ferment the sugars from cellulose. To improve IFFP, the development of a molecular breeding method for strain MG-60 is necessary. The purpose of this study is to establish the transformation method for the strain MG-60 and to obtain the over-expressing transformants of lignin-degrading enzyme, manganese peroxidase. FINDINGS: In the present study, the expression vector regulated by Phlebia brevispora glyceraldehyde-3-phosphate dehydrogenase promoter and terminator was constructed. A polyethylene glycol transformation method for the ethanol-fermenting white-rot fungus Phlebia sp. MG-60 was established with high transformation efficiency, and the manganese peroxidase isozyme 2 gene (MGmnp2) transformants were obtained, showing higher MnP activity than control transformants. MGmnp2 transformants showed higher selective lignin degradation on Quercus wood powder. CONCLUSIONS: This first report of MG-60 transformation provides a useful methodology for widely accessible to interested researches. These results indicate the possibility of metabolic engineering of strain MG-60 for improving IFFP.

Corncobs as a potential source of functional chemicals.

Phytochemical examination of corncob extracts led to the isolation of a new lignan identified as 7,7'-dihydroxy-3'-O-demethyl-4-methoxymatairesinol, together with seven known compounds, identified as ß-sitosterol, ß-sitosteryl-ß-D-glucoside, 6ß-hydroxy-campest-4-en-3-one, 5α,8α-epidioxyergosta-6,22-dien-3ß-ol, tricin, kaempferol and p-coumaric acid. The isolated compounds were identified by one and two-dimensional NMR spectroscopies and mass spectrometry.

Structure-activity relationships of lanostane-type triterpenoids from Ganoderma lingzhi as α-glucosidase inhibitors.

A series of lanostane-type triterpenoids, identified as ganoderma alcohols and ganoderma acids, were isolated from the fruiting body of Ganoderma lingzhi. Some of these compounds were confirmed as active inhibitors of the in vitro human recombinant aldose reductase. This paper aims to explain the structural requirement for α-glucosidase inhibition. Our structure-activity studies of ganoderma alcohols showed that the OH substituent at C-3 and the double-bond moiety at C-24 and C-25 are necessary to increase α-glucosidase inhibitory activity. The structure-activity relationships of ganoderma acids revealed that the OH substituent at C-11 is an important feature and that the carboxylic group in the side chain is essential for the recognition of α-glucosidase inhibitory activity. Moreover, the double-bond moiety at C-20 and C-22 in the side chain and the OH substituent at C-3 of ganoderma acids improve α-glucosidase inhibitory activity. These results provide an approach with which to consider the structural requirements of lanostane-type triterpenoids from G. lingzhi. An understanding of these requirements is considered necessary in order to improve a new type of α-glucosidase inhibitor.

Comparative biological study of roots, stems, leaves, and seeds of Angelica shikokiana Makino.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica shikokiana has been used as a health food for its anticancer, anti-inflammatory, antibacterial, antiallergic, and blood vessel dilating effects in Japan. It can also be used to prevent and treat hepatitis, diabetes, hyperlipidemia, and arteriosclerosis. AIM OF THE STUDY: The present study was designed to compare the biological activities such as melanin synthesis inhibitory, anti-allergy, anti-lipase, anti-bacterial, anti-oxidant, and neuroprotective activities of different parts of the plant that may justify the use of this plant in folk medicine. MATERIAL AND METHODS: The roots, stems, leaves and, seeds of Angelica shikokiana were separately extracted with water and ethanol. Each extract was examined for melanin synthesis inhibitory and anti-allergy activity on B16-melanoma and RBL-2H3 cells using IgE and A23187 as a stimulant for ß-hexosaminidase release, respectively. We also evaluated the inhibition of two enzymes, lipase and acetylcholine esterase, and of the bacterial growth of two species, Escherichia coli and Staphylococcus aureaus. The anti-oxidant activity was determined using oxygen radical anti-oxidant capacity, ORAC assay and its relation to the phenolic content was estimated using the Folin-Ciocalteu method. Besides, the protective effect of the extracts against H2O2-induced oxidative stress in mouse neuroblastoma, Neuro-2A cells was investigated. RESULTS: The most active extract exhibiting melanin synthesis inhibition (63%) and at the same time with low cytotoxicity (15%) was the ethanol extract of roots at 20 µg/ml, followed by the ethanol extract of stems (57% inhibition, 5% cytotoxicity). On the other hand, the highest inhibitions of ß-hexosaminidase release were recorded for the ethanol extract of leaves with IC50 value of 6.89 µg/ml followed by the water extract of the seeds and leaves with IC50 value of 78.32 and 88.44 µg/ml, respectively. For anti-lipase assay, ethanol extracts of the stems and roots showed the strongest inhibition with IC50 values of 204.06 and 216.24 µg/ml, respectively. None of the examined extracts showed any activity against Escherichia coli. while the ethanol extract of the roots and stems showed moderate inhibition for Staphylococcus aureus with minimum inhibitory concentration of 400 µg/ml. Ethanol extract of the roots showed only 30% inhibition of acetylcholine esterase enzyme. The results of anti-oxidant, phenolic content and protective effect against H2O2-induced cytotoxicity assays showed highly correlated data. Ethanol extract of the stems (ORAC value of 1.08 µmol Trolox/mg and phenolic content 44.25 µg GAE/mg) increased the cell viability of H2O2-treated Neuro-2A cells by 28%.

Putative cationic cell-wall-bound peroxidase homologues in Arabidopsis, AtPrx2, AtPrx25, and AtPrx71, are involved in lignification.

The final step of lignin biosynthesis, which is catalyzed by a plant peroxidase, is the oxidative coupling of the monolignols to growing lignin polymers. Cationic cell-wall-bound peroxidase (CWPO-C) from poplar callus is a unique enzyme that has oxidative activity for both monolignols and synthetic lignin polymers. This study shows that putative CWPO-C homologues in Arabidopsis , AtPrx2, AtPrx25, and AtPrx71, are involved in lignin biosynthesis. Analysis of stem tissue using the acetyl bromide method and derivatization followed by the reductive cleavage method revealed a significant decrease in the total lignin content of ATPRX2 and ATPRX25 deficient mutants and altered lignin structures in ATPRX2, ATPRX25, and ATPRX71 deficient mutants. Among Arabidopsis peroxidases, AtPrx2 and AtPrx25 conserve a tyrosine residue on the protein surface, and this tyrosine may act as a substrate oxidation site as in the case of CWPO-C. AtPrx71 has the highest amino acid identity with CWPO-C. The results suggest a role for CWPO-C and CWPO-C-like peroxidases in the lignification of vascular plant cell walls.

Target proteins of ganoderic acid DM provides clues to various pharmacological mechanisms.

Ganoderma fungus (Ganodermataceae) is a multifunctional medicinal mushroom and has been traditionally used for the treatment of various types of disease. Ganoderic acid DM (1) is a representative triterpenoid isolated from G. lingzhi and exhibits various biological activities. However, a universal starting point that triggers multiple signaling pathways and results in multifunctionality of 1 is unknown. Here we demonstrate the important clues regarding the mechanisms underlying multi-medicinal action of 1. We examined structure-activity relationships between 1 and its analogs and found that the carbonyl group at C-3 was essential for cytotoxicity. Subsequently, we used 1-conjugated magnetic beads as a probe and identified tubulin as a specific 1-binding protein. Furthermore, 1 showed a similar Kd to that of vinblastine and also affected assembly of tubulin polymers. This study revealed multiple biological activities of 1 and may contribute to the design and development of new tubulin-inhibiting agents.

Volatiles emitted from the roots of Vetiveria zizanioides suppress the decline in attention during a visual display terminal task.

The perennial grass Vetiveria zizanioides (vetiver) is mainly cultivated for its fragrant essential oil. Although the components of the oil and their biological activities have been studied extensively, the effect of the volatiles emitted from the roots of V. zizanioides on humans has so far remained unexplored. We investigated the effects of volatile compounds emitted from the cut roots of V. zizanioides (1.0 g, low-dose conditions; 30 g, high-dose conditions) on individuals during a visual display terminal task. Participants who breathed the volatile compounds emitted under low-dose conditions showed faster reaction times and stimulation of sympathetic nerve activity as measured by electrocardiography. These effects were not observed under high-dose conditions. The total amounnt of volatiles emitted during the experiment was 0.25 µg under low-dose conditions and 1.35 µg under high-dose conditions. These findings indicate that volatile compounds emitted from the roots of V. zizanioides under low-dose conditions may have helped subjects to maintain performance in visual discrimination tasks while maintaining high sympathetic nerve system activity.

Osteoclastogenesis inhibitory effect of ergosterol peroxide isolated from Pleurotus eryngii.

Ergosterol peroxide was isolated from the ethanol extract of Pleurotus eryngii as an inhibitor of osteoclast differentiation. This compound showed an inhibitory effect in a dose-dependent manner and an inhibition rate of up to 62% with low cytotoxicity, even at a concentration as low as 1.0 microg/mL.

The production of ß-glucosidases by Fusarium proliferatum NBRC109045 isolated from Vietnamese forest.

Fusarium proliferatum NBRC109045 is a filamentous fungus isolated from Vietnamese forest due to high production of ß-glucosidases. Production of the enzyme was studied on varied carbon source based mediums. The highest activity was obtained in medium containing 1% corn stover + 1% wheat bran (3.31 ± 0.14 U/ml). It is interesting to note that glucose (0.69 ± 0.02 U/ml) gave higher activity and just followed by cellobiose among the di- and mono-saccharides, which is generally regarded as a universal repressor of hydrolases. We improved the zymogram method to prove that in response to various carbon sources, F. proliferatum could express various ß-glucosidases. One of the ß-glucosidases produced by F. proliferatum growing in corn stover + wheat bran based medium was partially purified and proved to have high catalytic ability.