Lobothallia alphoplaca exhibited highest FRAP value. Methanolic extract of Xanthoparmelia stenophylla showed the highest ABTS radical scavenging capacity. The n-hexane extract of Rhizoplaca chrysoleuca exhibited highest DPPH radical scavenging capacity. Highest antioxidant capacity in terms of β-carotene linoleic acid bleaching property was observed in the water extract of Xanthoria elegans. Similarly, Melanelia disjuncta water extract showed highest NO scavenging capacity. Among n-hexane, methanol and water extracts of all lichens, the methanolic extract of Xanthoparmelia mexicana showed highest total proanthocyanidin, flavonoid and polyphenol content. From cytotoxic assay, it was observed that the methanolic extracts of L. alphoplaca and M. disjuncta were exhibiting high cytotoxic effects against cancer cell growth. Similarly, the water extract of Dermatocarpon vellereum, Umbilicaria vellea, X. elegans and M. disjuncta and the methanolic extract of M. disjuncta and X. stenophylla were found to possess high antioxidant capacities and were non-toxic and may be used as natural antioxidants for stress related problems. Our studies go on to prove that the unique trans-Himalayan lichens are a hitherto untapped bioresource with immense potential for discovery of new chemical entities, and this biodiversity needs to be tapped sustainably.Fourteen saxicolous lichens from trans-Himalayan Ladakh region were identified by morpho-anatomical and chemical characteristics. The n-hexane, methanol and water extracts of the lichens were evaluated for their antioxidant capacities. The lichen extracts showing high antioxidant capacities and rich phenolic content were further investigated to determine their cytotoxic activity on human HepG2 and RKO carcinoma cell lines. The ferric reducing antioxidant power (FRAP), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH) and nitric oxide (NO) radical scavenging capacities and β-carotene-linoleic acid bleaching property exhibited analogous results where the lichen extracts showed high antioxidant action. The lichen extracts were also found to possess good amount of total proanthocyanidin, flavonoid and polyphenol. The methanolic extract of  Reactive oxygen species (ROS) react swiftly with free radicals to become radicals themselves thereby starting free radical chain reaction. Superoxide anion (Oviz. butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), tertbutylhydroquinone (TBHQ) and propyl gallate (PG) have been reported to exert toxic effects in vitro studies on medicinal plants and vegetables strongly supports the fact that phytoconstituents with antioxidant capacity are capable of exerting protective effects against oxidative stress in biological systems Antioxidants are compounds which can impede the oxidation process by reacting with free radicals, chelating catalytic metals and scavenging oxygen in biological systems Lichens, the unanimously disseminated organisms occurring in the most adverse and varied geo-climatic circumstances ranging from the ice-capped poles to the tropics and from the plains to the highest mountains on earth and substrates, encompass the most inimitable group of organisms in nature, growing simultaneously in a close successful symbiotic alliance of two distinct organisms, an exhabitant fungus (the mycobiont) and the photoautotrophic partner algae (the phycobiont) viz. Umbilicariaceae , Teloschistaceae  and Parmeliaceae  are used as food ingredients and for medicinal purposes in different parts of the world The Indian Himalayan region is well known for its varied characteristic ecosystems endowed with rich floristic and faunal wealth. Plants of the Himalayan region are widely used in traditional medical systems both as prophylactics and therapeutics for high altitude maladies and a number of natural products and botanical supplements have been developed from our institutes, which are useful to combat these problems. These products have been reported to possess high nutritional and antioxidant properties. Extensive research work was carried out by the previous investigators to explore the medicinal and aromatic plants of trans-Himalaya viz. Dermatocarpon vellereum, Umbilicaria vellea, Rhizoplaca chrysoleuca, Rhizoplaca melanophthalma, Pleopsidium flavum, Xanthoparmelia mexicana, Acarospora badiofusca, Xanthoria elegans, Lecanora frustulosa, Lobothallia alphoplaca, Physconia muscigena, Melanelia disjuncta, Xanthoparmelia stenophylla and Peccania coralloides from trans-Himalayan cold desert of Ladakh have not been reported till date. For this rationale, the present investigation was desinged to estimate the in vitro antioxidant capacities and phenolic profile of n-hexane, methanol and water extracts of the aforementioned fourteen saxicolous lichens from trans-Himalaya. The lichen extracts showing high antioxidant capacities and rich phenolic content were further investigated to determine their cytotoxic activity on human hepatocellular carcinoma HepG2 and colon carcinoma RKO cells.However, the medicinal and therapeutic effects with respect to antioxidant capacities, cytotoxic activity and phenolic composition of different extracts of saxicolous lichens s-triazine (TPTZ), ferrous sulfate (FeSO4.7H2O), aluminium chloride (AlCl3), sodium acetate (C2H3NaO2), sodium carbonate (Na2CO3), potassium persulfate (K2S2O8), potassium chloride (KCl), ferric chloride (FeCl3), sodium nitroprusside {Na2[Fe(CN)5NO].2H2O}, sulfanilic acid (C6H7NO3S), butylated hydroxytoluene (BHT), ascorbic acid, gallic acid, quercetin and catechin were purchased from Sigma-Aldrich . RPMI medium 1640, penicillin, streptomycin, trypsin-EDTA, sulphorhodamine B, trichloroacetic acid, acetic acid, Tris base and all other chemicals used for the cytotoxicity assay were of analytical grade and also purchased from Sigma-Aldrich . Folin Ciocalteu's phenol reagent, vanillin, silica gel precoated aluminium TLC plates (20×20 cm), hydrochloric acid, sulphuric acid, methanol, n-hexane, chloroform, ethanol, toluene, dioxane, acetic acid, diethyl ether, formic acid and sodium carbonate were purchased from Merck Chemical Supplies . All the other chemicals used including solvents were of analytical grade.1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,4,6-tripyridyl-All necessary permits were obtained for the described field studies and lichen collection. The permit was issued by Dr. B. Balaji (IFS), Divisional Forest Officer, Leh Forest Division, Jammu & Kashmir, India.viz. Dermatocarpon vellereum (Zschacke), Umbilicaria vellea (L.) Ach. em. Frey, Rhizoplaca chrysoleuca (Sm.) Zopf, Rhizoplaca melanophthalma (DC) Leuck. & Poelt, Pleopsidium flavum (Bell.) Korb., Xanthoparmelia mexicana (Gyeln.) Hale, Acarospora badiofusca (Nyl.) Th. Fr., Xanthoria elegans (Links.) Th. Fr., Lecanora frustulosa (Dick.) Ach., Lobothallia alphoplaca (Wahlenb. ex Ach.), Physconia muscigena (Ach.) Poelt., Melanelia disjuncta (Essl.), Xanthoparmelia stenophylla (Ach.) Ahti & Hawksw. and Peccania coralloides  Massal. were collected from the northward slope of hill , Indus valley, Leh-Ladakh, J&K, India, in the peak winter period  of January, 2012. Umbilicaria vellea was collected from the location near Chang-La Top , Changthang valley, Leh-Ladakh, J&K, India in May, 2012. Lichens were carefully collected using scalpel and forceps following standard procedure. Then dust, soil, and rock debris were removed by using brushes, forceps and needles, shade dried to a constant weight  and lichen samples were kept at room temperature until extraction in the sterile petriplate .Amongst the fourteen lichen specimens thirteen lichens viz. solvent system A (toluene: dioxane: acetic acid:: 180: 60: 8), solvent system B (hexane: diethyl ether: formic acid:: 130: 100: 20) and solvent system C (toluene: acetic acid:: 200: 30) were used as mobile phase. TLC was performed in accordance with the previous reports for lichen identification Lichens were identified morpho-anatomically using a stereomicroscope, light microscope, and chemically with the help of color reactions, UV light and standardized thin-layer chromatography (TLC) viz. n-hexane, methanol and water by vortexing for 10 min, and then centrifugation at 10,000 rpm for 15 min. This cycle of extraction for each lichen sample was at 10°C and repeated two times as each solvent added freshly to ensure complete extraction Lichen specimens were air dried at room temperature and ground separately to powder. Finely ground dry thalli of all lichen species (170.4 mg to 330 mg depending upon their abundance) were sequentially extracted in 1 ml of solvents s-triazine (TPTZ) solution and 1 part of 20 mM FeCl3.6H2O]. The content was incubated in the dark for 30 min. The increase in absorbance with the formation of colored product (ferrous tripyridyltriazine complex) was measured at 593 nm. The antioxidant capacity of the lichen extract was determined based on a calibration curve plotted using FeSO4.7H2O at a concentration ranging between 0.125 and 2 mM. The calibration equation for FeSO4.7H2O was y = 0.464×−0.0114, R2 = 0.9996, where x is the concentration of FeSO4.7H2O mM/l and y is the absorbance at 593 nm. Results were expressed in mM Fe (II)/g of extract.Ferric reducing antioxidant power (FRAP) assay was performed as suggested by previous investigators •+ radicals. Working solution was prepared freshly before each assay by diluting the stock solution by mixing of stock solution to 50% methanol for an initial absorbance of about 0.700±0.02 at 745 nm, with temperature control set at 30°C. Free radical scavenging activity was assessed by mixing 30 µl of different fractions (different concentration in respective solvents) with 300 µl of ABTS working standard. The decrease in absorbance was measured after 6 min at 30°C. Quercetin and ascorbic acid were used as positive control. The scavenging activity was estimated based on the percentage of ABTS radicals scavenged by the following formula:The ABTS assay was performed according to the previously established protocol 0 was absorption of control, AS was absorption of tested extracts and standards.Where, A50) for scavengers  was calculated 50 value was determined by plotting the scavenging capacity against the logarithm of sample concentration.The half maximal inhibitory concentration  2 = 0.9975, where x was the absorbance and y was the CAE. Extract samples were evaluated at a final concentration of 100 µg/ml.Determination of proanthocyanidin was performed according to the method of Sun et al. (1998) 3 in ethanol solution were allowed to react. The reaction mixture was incubated for 1 h at room temperature and the absorbance was measured at 420 nm. Total flavonoid content was calculated as quercetin equivalent (QAE) in mg/g of extract by using the following equation based on the calibration curve: y = 0.0235x−0.1803, R2 = 0.9994, where x was the absorbance and y was the QAE at a final concentration of 100 µg/ml.Total flavonoid was analyzed with the method of Ordonz et al. (2006) 2O, and incubated at room temperature for 3 min. After that 100 µl of sodium carbonate  was added to the reaction mixture. The content was incubated at room temperature in the dark for 30 min and absorbance was measured at 765 nm. Estimation of total polyphenol content was calculated as gallic acid equivalent (GAE) in mg/g of extract on the basis of calibration curve of gallic acid, y = 0.0104× −0.0589, R2 = 0.9978, where x was absorbance and y was GAE at a final concentration of 100 µg/ml.Total polyphenol content (TPC) in extract was determined by Folin Ciocalteu's colorimetric method as described by Gao et al. (2000) 2 and 95–100% humidified air atmosphere.Human hepatocellular carcinoma (HepG2) and colon carcinoma (RKO) cell lines acquired in liquid nitrogen (−180°C) were procured from American Type Culture Collection (ATCC), USA. Cells were maintained in RPMI medium 1640  at 37°C in 5% CO2 and 95–100% humidified air atmosphere . Microscopic examination was performed (10X eyepiece and 10X objective lenses) using inverted phase contrast microscope . The changes in cellular morphology following different treatments were acquired at different time intervals .Logarithmically growing cells were seeded in 96 flat-well plate,  and incubated for 24 h to have a partial monolayer. The extracts of lichens were dissolved in RPMI medium 1640 and cells were treated with different concentration  of extracts. Thereafter, cells were incubated at 37°C in a 5% CO2, in humidified incubator (95–100% humidity), the SRB assay was performed. At both points of time, the medium was removed and the cells were fixed with trichloroacetic acid  at 4°C for 1 h, stained for 30 min with SRB  dissolved in 1% acetic acid for 30 min and washed four times with 1% acetic acid. The protein-bound dye was made solubilised with 10 mmol/L tris base, pH 10.5 and the absorbance was recorded at 565 nm using spectrophotometer .The Sulphorhodamine B (SRB assay) was performed for evaluating cellular growth and colorimetric determination of cytotoxicity of lichen extracts was performed The percentage growth (PG) at each extract concentration level was calculated as:50, µg/ml) was the concentration where {(Tt−T0)/(C−T0)} ×100 = 50. Total growth inhibition concentration  was the concentration where {(Tt −T0)/(C−T0)} ×100 = 0 = PG. Lethal concentration that kills 50% of the cells  was the concentration level where {(Tt−T0)/T0} ×100 = −50. These parameters were determined following established reports 0 =  absorbance of the extract treated cells just after time zero, and Tt =  absorbance of the extract treated cells after 72 h, C =  absorbance of control (cells untreated with extract) after 72 h.The 50% growth inhibition concentration  using statistical analysis with SPSS 17.0  version. Analysis of variance (ANOVA) in a completely randomised design, Duncan's multiple range test and Pearson's correlation coefficients were performed to compare the data. Post hoc analysis was performed using Neuman Keuls Test, and values with viz. Lobothallia alphoplaca, Rhizoplaca melanophthalma and Xanthoparmelia mexicana were reported to the area near the northward slope of hill of Indus valley for the first time as per our previously reported work In the present study lichen specimens were collected from Chang-La Top of Changthang valley and northward slope of hill of Indus valley, Ladakh, India. The collected lichens specimens were identified using standardized analytical techniques, significant lichen identification keys and monographs p<0.05) FRAP values in comparison with the water and n-hexane extracts. Among all extracts, methanol extract of L. alphoplaca exhibited highest FRAP value (1673.32±94.83 µM Fe (II)/g extract). The maximum FRAP values in n-hexane and water extracts were observed in X. elegans (136.55±12.91 µM Fe (II)/g extract) and P. coralloides (335.19±12.92 µM Fe (II)/g extract), respectively. The positive standards viz., ascorbic acid (9851.60±752.15 µM Fe (II)/g extract) and BHT (3160.14±251.72 µM Fe (II)/g extract) were found to exhibit significantly higher FRAP values than the lichen extracts . The maximum ABTS radical scavenging capacity in n-hexane and water extracts was rendered by A. badiofusca (IC50 6.55±0.10 mg/ml) and U. vellea (IC50 3.45±0.11 mg/ml). ABTS radical scavenging capacity of methanol extracts was significantly higher (p<0.05) in comparison with water and n-hexane extracts for all the lichen species. Although X. elegans water extract (IC50 5.08±0.10 mg/ml) showed significantly higher (p<0.05) radical scavenging capacity than the methanol and n-hexane extracts. The n-hexane extract of most of the lichen species under investigation exhibited significantly lower (p<0.05) antioxidant capacity when compared with the methanol and water extracts. However, the n-hexane extracts of A. badiofusca (IC50 6.55±0.10 mg/ml), L. alphoplaca (IC50 9.52±0.13 mg/ml) and P. coralloides (IC50 14.84±0.09 mg/ml) showed significantly higher (p<0.05) antioxidant capacity in comparison with the corresponding water extracts. Quercetin (IC50 0.33±0.03 mg/ml) and ascorbic acid (IC50 0.18±0.01 mg/ml) were found to produce significantly higher (p<0.05) radical scavenging potential compared to the lichen extracts  of the fourteen lichen species in different solvent extracts has been depicted in extracts .viz. quercetin and ascorbic acid were compared through their ability to scavenge DPPH radical. The DPPH radical scavenging capacity of the lichen extracts and the positive controls increased in a dose dependent manner at concentration of 0.1–0.5 mg/ml. The half maximal inhibitory/scavenging concentration (IC50 or RSa50) values were determined and have been depicted in 50 indicates higher DPPH radical scavenging capacity. The rate of DPPH radical scavenging capacity was found to depend on lichen species and extracting solvent. For most of the lichen species, DPPH radical scavenging capacity of n-hexane extract was significantly higher (p<0.05) in comparison with methanol and water extract. The radical scavenging capacity of n-hexane extracts of D. vellereum (IC50 1.24±0.10 mg/ml), U. vellea (IC50 1.23±0.10 mg/ml), R. chrysoleuca (IC50 1.02±0.02 mg/ml), A. badiofusca (IC50 2.48±0.10 mg/ml), X. elegans (IC50 3.37±0.10 mg/ml), L. frustulosa (IC50 3.04±0.03 mg/ml), M. disjuncta (IC50 3.65±0.10 mg/ml) and X. stenophylla (IC50 5.08±0.02 mg/ml) increased significantly (p<0.05) when compared with the methanol and water extracts. However, the water extract of R. melanophthalma (IC50 1.65±0.09 mg/ml), X. mexicana (IC50 1.97±0.08 mg/ml) and P. coralloides (IC50 5.95±0.05 mg/ml) showed significant increase (p<0.05) in radical scavenging ability than n-hexane and methanol extract. In addition, the methanol extract of L. alphoplaca (IC50 2.29±0.01 mg/ml), P. muscigena (IC50 5.59±0.10 mg/ml) and P. flavum (IC50 1.09±0.01 mg/ml) were found to produce significantly higher (p<0.05) radical scavenging capacity when compared with the corresponding n-hexane and water extracts. The n-hexane extract of R. chrysoleuca exhibited the highest radical scavenging capacity (IC50 1.02±0.02 mg/ml) among all lichen extracts under our study. The maximum DPPH radical scavenging capacity in methanol and water extracts was retained by P. flavum (IC50 1.09±0.01 mg/ml) and D. vellereum (IC50 1.59±0.11 mg/ml), respectively. Quercetin (IC50 0.08±0.02 mg/ml) and ascorbic acid (IC50 0.05±0.01 mg/ml) were found to produce significantly higher (p<0.05) DPPH radical scavenging effect compared to the lichen extracts . The highest antioxidant capacity for n-hexane and methanol extracts was determined in X. stenophylla (IC50 0.33±0.11 mg/ml) and M. disjuncta (IC50 0.18±0.08 mg/ml) respectively. The methanol and water extracts of majority of the lichen species showed significantly higher (p<0.05) antioxidant capacity in comparison with the n-hexane extract. The methanol extracts of D. vellereum (IC50 1.01±0.11 mg/ml), R. chrysoleuca (IC50 0.52±0.11 mg/ml), P. muscigena (IC50 0.22±0.08 mg/ml), M. disjuncta (IC50 0.18±0.08 mg/ml), P. coralloides (IC50 0.22±0.11 mg/ml), X. stenophylla (IC50 0.22±0.10 mg/ml) and A. badiofusca (IC50 0.32±0.10 mg/ml) were found to produce significantly higher (p<0.05) antioxidant capacity when compared with the corresponding n-hexane and water extracts. In addition, the water extracts of X. mexicana (IC50 0.14±0.08 mg/ml), L. frustulosa (IC50 0.20±0.10 mg/ml), L. alphoplaca (IC50 0.28±0.09 mg/ml), U. vellea (IC50 0.50±0.10 mg/ml), R. melanophthalma (IC50 0.53±0.10 mg/ml) and X. elegans (IC50 0.08±0.01 mg/ml) showed significantly higher (p<0.05) antioxidant capacity in comparison with the corresponding methanol and n-hexane extracts. However, P. flavum n-hexane extract (IC50 0.45±0.10 mg/ml) showed significantly higher (p<0.05) antioxidant capacity than the corresponding methanol and water extracts. BHT was used as positive control and it showed significantly higher (p<0.05) antioxidant capacity (IC50 0.04±0.01 mg/ml) in comparison with all lichen extracts (The β-carotene-linoleic acid bleaching capacity of different solvent extracts of fourteen lichen species was expressed as scavenging capacity (%). All the lichen extracts and positive control BHT showed β-carotene-linoleic bleaching activity in a dose dependent manner at concentration of 0.1-0.5 mg/ml and it was found to vary with extracting solvent and lichen species . The IC5 and BHT . In the extracts .50 or RSa50 values of lichen extracts and BHT were calculated and have been depicted in M. disjuncta (IC50 0.75±0.09 mg/ml) water extract showed the highest nitric oxide radical scavenging capacity among all lichen extracts under investigation. For n-hexane, methanol and water extracts, maximum radical scavenging capacity was detected in R. chrysoleuca (IC50 1.39±0.11 mg/ml), L. alphoplaca (IC50 1.75±0.09 mg/ml) and M. disjuncta (IC50 0.75±0.09 mg/ml) respectively. Majority of the lichen species were found to produce significantly higher (p<0.05) nitric oxide radical scavenging capacity in their n-hexane and and water extracts in comparison with corresponding methanol extracts. The n-hexane extracts of D. vellereum (IC50 6.29±0.10 mg/ml), U. vellea (IC50 2.11±0.11 mg/ml), R. chrysoleuca (IC50 1.39±0.11 mg/ml), R. melanophthalma (IC50 3.31±0.10 mg/ml), X. elegans (IC50 2.75±0.08 mg/ml), L. frustulosa (IC50 3.33±0.09 mg/ml) and P. muscigena (IC50 2.61±0.09 mg/ml) were found to exhibit significantly higher (p<0.05) radical scavenging capacity in comparison with the corresponding water and methanol extracts. In addition, the water extracts of P. coralloides (IC50 1.46±0.13 mg/ml), X. stenophylla (IC50 3.26±0.07 mg/ml), M. disjuncta (IC50 0.75±0.09 mg/ml), A. badiofusca (IC50 2.82±0.10 mg/ml) and P. flavum (IC50 1.01±0.11 mg/ml) showed significantly increased (p<0.05) radical scavenging capacity in comparison with the corresponding n-hexane and methanol extracts. However, the methanol extracts of X. mexicana (IC50 4.14±0.09 mg/ml) and L. alphoplaca (IC50 1.75±0.09 mg/ml) were found to produce significantly higher (p<0.05) radical scavenging capacity in comparison with the corresponding n-hexane and water extracts. BHT showed significantly higher (p<0.05) nitric oxide radical scavenging capacity (IC50 0.15±0.04 mg/ml) in comparison with all lichen extracts (The scavenging capacity (%) of the different solvent extracts of fourteen lichen species against nitric oxide released by sodium nitroprusside was studied and the result has been depicted in extracts .p<0.05) TPAC in comparison with the corresponding n-hexane and water extracts. Methanol extract of X. mexicana showed highest TPAC (4218.59±5.21 mg CAE/100 g) among all lichen extracts. The maximum TPAC in the n-hexane and water extracts was found in M. disjuncta (178.52±1.56 mg CAE/100 g) and P. coralloides (1071.82±0.64 mg CAE/100 g) respectively (The TPAC of the different lichen extracts was expressed as mg CAE/100 g dry weight (dw) of extract. The methanol extracts of all lichen species were found to contain significantly higher (ectively .p<0.05) TFC in comparison with the corresponding n-hexane and water extracts. The methanol extract of X. mexicana showed highest TFC (3607.56±4.18 mg QAE/100 g) among all lichen extracts. The maximum TFC in n-hexane and water extracts was determined in L. frustulosa (202.19±30.97 mg QAE/100 g) and X. mexicana  respectively  TPC in comparison with the corresponding n-hexane and water extracts. The methanol extract of X. mexicana showed highest TPC (5439.93±61.45 mg GAE/100 g) among all lichen extracts. The maximum TPC in n-hexane and water extracts was determined in R. chrysoleuca (255.70±10.58 mg GAE/100 g) and P. coralloides (604.07±21.67 mg GAE/100 g) respectively  and all concentrations were non-toxic below this level. L10M and L12M crossed the GI50 at 275 µg/ml (% growth 21.28±1.94) and 281 µg/ml (% growth 24.61±1.87) and were found to be toxic. LW1, L10M and L12M were non-toxic upto 222.22 µg/ml. LW8 was very toxic at 333.33 µg/ml where it approached almost near to GI90 and crossed GI50 at 189 µg/ml. L13M and LM5 were extremely toxic for HepG2 cells as they crossed the GI50 at 41 µg/ml and 191 µg/ml, TGI at 69 µg/ml and 266 µg/ml, and LC50 at 190 µg/ml and 324 µg/ml respectively. They showed −66.31±4.52% and −58.19±4.54% growth at 333.33 µg/ml. All these results have been depicted in In HepG2 cells, extract LW2 and LW12 showed no toxicity as they remained far above from GIectively . LW1 was50 at 289 and 330 µg/ml. The percent growth recorded with the treatment of LW8 and L10M extracts at highest concentration 333.33 µg/ml was 14.77±1.87 and 46.25±3.53 respectively. L12M was toxic as it crossed GI50 at 213 µg/ml, cells showed 23.58±2.15% growth at 333.33 µg/ml and it was non-toxic upto148.15 µg/ml concentration. LM5 and L13M were toxic as they approached the LC99 at 333.33 µg/ml and showed −99.21±0.88% and −99.28±0.38% growth respectively. LM5 crossed the LC50, TGI and GI50 at 282, 229 and 187 µg/ml respectively. L13M crossed the LC50, TGI and GI50 at 121, 76 and 46 µg/ml respectively. All these results have been depicted in In case of RKO cells, LW1, LW2 and LW12 were non-toxic at all concentration levels. However, LW8 and L10M crossed GIIn morphological visualization after 24, 48 and 72 h, extract LW1, LW2 and LW12 were non toxic at all concentration levels whereas L13M was highly toxic as it showed toxicity up to 98.77 µg/ml for HepG2 cell line and up to 65.84 µg/ml for RKO cell line. Extract LW8, L5M and L12M were non toxic up to 222.22, 148.15 and 148.45 µg/ml, respectively, for both cell lines. L10M was found to be non toxic up to 222.22 µg/ml for HepG2 cell line and up to 148.15 µg/ml for RKO cell line. In HepG2 cell line, extract LM5 showed dead cells at 333.33 µg/ml after 24, 48 and 72 h; and 50% dead cells at 222.22 µg/ml after 24 h. L12M showed 50% cell death at 333.33 µg/ml after 24 and 48 h. L13M showed dead cells at 222.22 and 333.33 µg/ml after 24 h, at 98.77 to 333.33 µg/ml after 48 h and at 148.15 to 333.33 µg/ml after 72 h. In RKO cells, L13M showed dead cells at 222.22 and 333.33 µg/ml after 24 h, at 98.77 to 333.33 µg/ml after 48 h and at 148.15 to 222.22 µg/ml after 72 h. LM5 showed dead cells at 333.33 µg/ml after 48 and 72 h. All results related to the morphological analysis have been depicted in viz. antitumour, antibacterial, antifungal, antiviral, antiinflammatory and antioxidant capacities Lichens, one of the prominent life-form in the trans-Himalayan Ladakh region contain natural antioxidants which provide protection from the damage caused by the climatic conditions of high altitude environments and support their adaptability in extreme environmental conditions like extreme habitats , low temperature, drought, prolonged winter, high solar irradiance and ultraviolet radiations. Numerous extracellular deposits in lichen thallus may contribute to the activation of antioxidant defence system in these conditions. In addition, diverse secondary metabolites are synthesized by lichens that are unique and found rarely in other plants in vitro and in vivo suggest probable explanations for structure-activity relationships. In general, the antioxidant capacity is known to be associated with the species, extraction solvent, method of extraction, temperature, conditions of the test systems, composition of the extract and hydrophobic or hydrophilic nature of the antioxidants. In our result, a wide range of antioxidant properties in different lichen extracts were observed that may be attributed to presence of diverse antioxidant compounds with different polarity in these lichen species. The bioactive antioxidants in the lichen extracts showed their diverse antioxidative effects in different antioxidant assays depending upon their individual redox reactions that may differ from each other by different magnitude of electrostatic interactions. In agreement with previous findings, the present study also revealed that the lichen extracts are the complex mixture of different compounds with distinct antioxidant capacities The antioxidant capacities of plant extracts have been ascribed to various mechanisms of actions such as deterrence of chain initiation, radical scavenging activity, binding of transition metal ion catalysts, increasing endogenous status of antioxidant enzymes etc to prevent oxidative damage Previous reports on lichen diversity in the Antarctic regions revealed that lichens are well adapted for extreme climatic conditions and they can tolerate long periods of drought, severe cold and arid atmosphere. They have developed protective mechanisms to evade damage caused by high radiation and freezing temperatures Researchers pay keen attention on the biological roles of lichen secondary substances that are reported to have biological activities such as antitumour, antibacterial, antifungal, antiviral, antiinflammatory and antioxidant activities Biologically active phytochemicals like polyphenols, flavonoids, flavonols, proanthocyanidins, alkaloids, terpenoids, carotenoids etc. are derived from plant sources and natural products which have promising health promoting properties and protective effects against chronic diseases while acting in combination It is well established that the antioxidant capacity of plant is associated with the phenolic compounds in vitro antioxidant capacity assays obtained by the available chemical tests are not sufficient to prove the medicinal and therapeutic potential of these natural resources unless they are supported by the scientific data with the cytoprotective effects of these compounds in cell culture system. Therefore, the antioxidant capacity and cytotoxic effect of a given compound should be studied directly in a cellular model as it provides the optimal environment for possible interaction of the intracellular components with the compound of interest that would be missed in a chemical assay system. Moreover, we must be well aware of the safety of these lichen extracts because of the fact that many natural antioxidant may act as pro-oxidant based on the dose and on the ambient redox conditions In recent time a good number of research investigations were carried out to establish the positive biological properties of lichen extracts and their metabolites in different assays like antimicrobial 50 at 333.33 µg/ml, whereas it was non-toxic to RKO cells as there was 82.68±3.78% growth at the same concentration. The 222.22 µg/ml concentration of LW8 was toxic for HepG2 cells as there was 37.24±3.55% growth at this level whereas RKO cells was non-toxic at the same concentration level with 82.2±4.21% growth. The lichen X. elegans (LW8) are edible as medicine 50 values against both types of carcinoma cells, whereas LW1, LW2, LW8 and LW12 can be used for further trial in search of potent radioprotector at the safe and non-toxic concentration levels considered under the present study. Hence, from the results of cytotoxic effects of lichen extracts in carcinoma cells we observed that the methanol extract of L. alphoplaca and M. disjuncta were exhibiting high cytotoxic effects against the cancer cell growth and these extracts hold immense potential for use as anticancer agents. However identification of potential bioactive compound needs to be done in different extracts. Similarly, the water extract of D. vellereum, U. vellea, X. elegans and M. disjuncta and the methanol extract of M. disjuncta and X. stenophylla showed high antioxidant capacities and were found to be non-toxic and may be used as natural antioxidants for stress related problems.In comparison to RKO cells, HepG2 cells were more susceptible to LW1, LW2, LW8 and LW12 and the RKO cells were mostly susceptible to the remaining extracts –5. LW1 w50 value <100 µg/ml 50 of L13M was well below 100 µg/ml and that of LM5 was above 100 µg/ml, but at 333.33 µg/ml concentration both crude extracts showed almost equal percent of deaths of cells. All selected lichen extracts exhibited cytotoxic effect with increased cell death whereas reduction of cytotoxicity was observed with reduced extract concentration which finds support to the previous investigations The results of cytotoxicity assay revealed that the selected lichen extracts used in the present study exert cytotoxicity and antiproliferative action on HepG2 and RKO cell lines , Table 7U. vellea and X. elegans extracts against cellular damage by environmental stressors and survival potential of these lichens under extreme environmental condition was reported previously Our results corroborate the previous investigations where cytotoxic and anticancer action of lichen extracts were explored in different lichen species from different parts of the world In the present study, fourteen saxicolous lichens from trans-Himalayan cold desert of Ladakh have been identified with their morpho-anatomical and chemical compositions and the antioxidant and free radical scavenging capacities of these lichen extratcts were reported for the first time. This study revealed that these lichen species have broad spectrum free radical scavenging effect and high antioxidant capacity. Moreover, the antioxidant capacity was found to be associated with the species and the polarity of solvent used for extraction. Our studies clearly showed that the high altitude saxicolous lichens can be an interesting source of new antioxidative substrates with the potential to be used for scavenging various types of free radicals by different scavenging mechanisms. The lichen extracts were found to contain considerable amount of phenolic compounds which were responsible for their high antioxidant and free radical scavenging ability and could be used as natural source of antioxidants to ameliorate oxidative stress related disorders. Among all extracts, eight lichen extracts having high antioxidant capacity and phenolic compounds were further investigated for cytotoxic action on human carcinoma cell lines and these extracts were found to exhibit anticancer and radioprotective effects that would be of great interest for further chemical investigations and could lead to the discovery of novel cytotoxic anticancer molecules from lichens of the trans-Himalayan region. With these present primary findings, further studies are also required to ascertain the biological activities of the lichen extracts in animal models.


Background. There is evidence that Tregs are important to prevent allergic diseases like asthma but limited literature exists on role of TH17 cells in allergic diseases. Methods. Fifty children with asthma and respiratory allergy (study group) and twenty healthy children (control group) were recruited in this study. Total IgE levels and pulmonary function tests were assessed. The expression of Tregs and cytokines was determined by flow cytometry. Results. The average level of total IgE in study group (316.8 ± 189.8 IU/mL) was significantly higher than controls . The frequency of TH17 cells and culture supernatant level of IL-17 in study group (12.09 ± 8.67 pg/mL) was significantly higher than control group . Alternatively, the frequency of FOXP3 level was significantly lower in study group [(49.00 ± 13.47)%] than in control group [(95.91 ± 2.63)%] and CD4+CD25+FOXP3+ to CD4+CD25+ ratio was also significantly decreased in study group [(6.33 ± 2.18)%] compared to control group [(38.61 ± 11.04)%]. The total serum IgE level is negatively correlated with FOXP3 level . The FOXP3 expression is negatively correlated with the IL-17 levels  and IL-4 levels . Conclusions. Imbalance in TH17/Tregs, elevated IL-17, and IL-4 response and downregulation of FOXP3 were associated with allergic asthma. H2 immune response, is a chronic inflammatory disorder, affecting children worldwide [H2 cytokines play a critical role in amplifying asthma [H1 cytokines prevent this allergic inflammation [H1 and TH2 cells. Some studies have suggested that other T cell subsets like TH17 and Treg also play a role in regulating asthma [H1, TH2, TH17, and allergen specific IgE. They have also been found to suppress basophils, eosinophils, and mast cells but induce levels of specific IgG4 [high) present on the surface of T cells and the expression of FOXP3 required for the generation and maintenance of their suppressive activity [+CD25+ T cells and is considered as a master switch for development and function of natural Treg cells [+CD25high Tregs are impaired in pediatric asthma [+ T cells, termed as TH17, produces IL-17 [H17 cells are now considered the key mediator in development of asthma [H17 cells enhance both neutrophilic and eosinophilic airway inflammation in mouse model of asthma [H17 cells play a key role in filling the gap between TH1 and TH2 by secreting IL-17A and IL-17F and also contributing to immunity against certain extracellular bacteria and fungi [+ T cells and also from monocytes, mast cells, macrophages, and neutrophils [H1 and TH2 cells as well as TH17 differentiation are suppressed by Tregs [H17 cells in vitro [+CD25highFOXP3+ Tregs and TH17 cells play an important role in mediating asthma.Asthma, characterized by Torldwide . It is nammation , 4. In rg asthma . Treg ceg asthma  by supprfic IgG4 . Naturalactivity , 8, 9. Feg cells –13. Receeg cells  or via aeg cells , 16. Somes IL-17 . TH17 cef asthma . TH17 cef asthma , 21. TH1nd fungi . IL-17, trophils , 24, hastrophils . TH1 andby Tregs . Howeverin vitro , 29. RecHypothesis. The null hypothesis states that T regulatory cells do not play any role in bronchial asthma. We hypothesize that T regulatory cells play a protective role in asthma. T regulatory cells, which regulate the balance between TH1 and TH2 cells, are downregulated in cases of asthma and allergy.Fifty children with asthma (study group) and twenty healthy children (control group) who were matched for age (in months) (control (88.86 ± 38.67); study group, (85.95 ± 35.55)) attended the Advanced Pediatric Centre in Post Graduate Institute of Medical Education and Research (PGIMER), Chandigarh, and were diagnosed as asthma and were recruited in this study with their informed consent. The sera of age and sex matched nonallergic patients were taken as controls. The Ethics Committee of PGIMER approved this study (Micro/2006/754/8th May 2006).H1, TH2, and TH17 expression and Treg cells.The diagnosis of asthma was made by clinical history, physical examination, FEV1 measurement, positive response to bronchodilators, positive skin prick test, and elevated total IgE. The asthma of all patients was under control with inhaled corticosteroids. Blood samples were collected for evaluation of TThe total IgE of allergic patients was measured using PATHOZYME immunoglobulin E OD 417 kit. The absorbance was measured at 450 nm after addition of tetramethyl benzidine hydrochloride (TMB) substrate and dilute hydrochloric acid. The concentration of IgE is directly proportional to the color intensity of the test samples. This test was calibrated to WHO 2nd International Reference Preparation 75/502 (1981).Five milliliters of heparinized blood was obtained from 20 healthy subjects and 50 asthmatic patients. For cytokine analysis, plasma was isolated from peripheral blood and stored at −80°C until it was used. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized blood sample by density gradient centrifugation (250 g for 20 minutes at room temperature) using Histopaque .γ), Th2 , and Th17 (IL-17) were assessed using BD CBA flex set. Tests were performed according to manufacturer's instructions . The analysis was carried out using flow cytometry  with FACS Diva Software).The serum levels of cytokines Th1  of the cells  and intracellular phenotyping (FOXP3) were performed by staining, paraformaldehyde fixation, and permeabilization according to the manufacturer's instructions . PBMCs were determined using forward and side scatter properties based on size and granularity by FACSCanto  with FACS Diva Software. The following mAbs (BD biosciences) were used: APC antihuman CD4, PE-Cy antihuman CD25, and PE antihuman FOXP3. To correct nonspecific binding, matched isotype controls were used.For analysis of Treg cells, the buffy coat (lymphocytes and monocytes) was separated. The cell pellet washed with PBS  was centrifuged at 200 g for 15 minutes. PBMCs were cultured in a petri dish containing 5% COr rank correlation coefficients were used to evaluate relationship between variables. When assessing the flow cytometric data, Student's t-test was used. P values <0.05 were considered statistically significant.Data were analyzed with SPSS  and Graphpad prism . The mean values and their internal differentiation with standard deviations were calculated. The spearman's  t-test was applied between study group and control group for total IgE level. The average level of total IgE in study group  was significantly higher than in control group  (P < 0.0001)   . FEV1 (% 0.0001) . Total s 0.0004) .γ, and IL-17 in sera were expressed as mean ± standard deviation. The average level of IL-17 expression in study group (12.09 ± 8.67 pg/mL) was significantly higher than the corresponding values in control group  (γ were significantly lower in study group (12.08 ± 8.67 pg/mL) compared to control group  (P = 0.51); IL-4: 21.88 ± 10.35 pg/mL versus 19.79 ± 12.38 pg/mL (P = 0.47); IL-6: 18.17 ± 10.49 pg/mL versus 15.11 ± 9.79 pg/mL (P = 0.08); IL-10: 22.82 ± 19.16 pg/mL versus 18.62 ± 5.31 pg/mL (P = 0.35); IL-12: 17.58 ± 9.27 pg/mL versus 16.94 ± 11.00 (P = 0.52); IL-13: 34.55 ± 17.51 pg/mL versus 29.39 ± 10.12 pg/mL (P = 0.40))   but valu= 0.009) . No sign= 0.40)) . t-test was done to analyze IL-17 in control and study group. The results depict a significant difference between the two groups (P < 0.0001). Similarly, there was also significant difference between study group and control group for IFN-γ (P = 0.009).The Student's+CD25+ cells for both control (n = 20) (n = 50) (P < 0.0001)   and stud(n = 50) . The per 0.0001) .+CD25+FOXP3+ to CD4+CD25+ ratio, which was significantly decreased in study group ((6.33 ± 2.18)%) compared to control group ((38.61 ± 11.04)%, P < 0.0001)  .+CD25high and total IgE level  (There was a significant negative correlation between %FOXP3/CD4 0.0001) .r = −0.5631, P < 0.0001)  (FOXP3 percentage and IL-17 level had a significant inverse correlation with each other ( 0.0001) . There w 0.0460) .H17 and Treg cells. It is universally accepted that total IgE level is directly correlated with allergy and asthma. In our study, the average level of total IgE was significantly higher in children with bronchial asthma compared to healthy subjects. On the basis of available studies, we had hypothesized that Treg cells would be associated with lower levels of allergy markers such as IgE and TH2 cytokines. Most studies of Treg activity come from immunotherapy studies in allergic diseases [H17/Treg cytokine profile in study group. Studies have suggested that asthma is associated with chronic and recurrent inflammation [H2 cells are associated directly with inflammation whereas TH17 cells behave primarily as proinflammatory markers [H17 cells. The interaction between TH17 cells and Tregs in various inflammatory diseases needs to be further defined [H17 phenotype. Thus, there is a correlation of increase in IL-17 levels in patients with asthma coupled with a significant decrease in transcription factor FOXP3 Treg level when compared to controls.The present work demonstrates the relationship between Tdiseases . FOXP3 tdiseases , 11, 31.ammation . TH2 cel markers . Studies defined . The kno defined –40. Howe defined –43. Thes defined –46. Anot defined . It has  defined . In patiH17 with Treg response in the milieu of TH2 activity. These results show that there is a correlation between FOXP3 and IL-17 level and also a functional imbalance in TH17/Treg in children with asthma. In this study, we demonstrated that IL-17 and FOXP3 are reciprocally interconnected with each other. It has already been shown that CD4+CD25+FOXP3+ play a protective role in autoimmune disease [+CD25high T cells was variable, which is already reported in previous studies [+CD25+ T cells and is expressed by these cells [We could not find a study in children with asthma reporting the relationship of T disease . We foun studies , 50. FOXse cells , 11, 31.H17 and Tregs associated with asthma, which may play a potential role in development of asthma. Our study also shows inverse correlation between IL-17 and FOXP3. Future studies are needed to clarify these findings.In conclusion, the present study demonstrates that there is an imbalance between T


To evaluate the clinical features, treatment results, prognostic factors and late toxicities of nasopharyngeal carcinoma in children and adolescents.Between January 1990 and January 2011, 158 NPC patients younger than 20 years old were treated in our institution, and the patient’s clinical characteristics, treatment modalities, outcomes and prognostic factors were retrospectively analyzed.p = 0.01, stage IV and stage II-III were 77.1% and 90%, p = 0.04, respectively. In multivariate analysis, T4 (p = 0.02) and stage IV (p = 0.04) were the independent adverse prognostic factors for OS. Significant reduction in trismus  and G2 xerostomia  was observed in patients treated by IMRT.There were 9 (5.7%) patients in stage II, 60 (38.0%) in stage III and 89 (56.3%) in stage IV according to the UICC2002 staging system. Neck mass (32.3%), headache (21.5%) and nasal obstruction (15.2%) were the most common chief complaints. With a median follow-up time of 62.5 months (range 2.0-225.0 months), the 5-year overall survival (OS) rate, local-regional control (LRC) rate and distant metastasis-free survival (DMFS) rate were 82.6%, 94.9% and 76.4%, respectively. There were 43 (27.2%) patients failed during the follow up, with seven local-regional recurrences and 38 distant metastases. In univariate analysis, the 5-year OS of T4 and T1-3 were 75% and 87.9%, Most childhood and adolescence nasopharyngeal carcinoma patients were locally advanced diseases at first diagnosed. The treatment results of radiotherapy, with or without chemotherapy, are excellent in our institution. Reducing distant metastasis with new strategies and late toxicities with intensity-modulated radiotherapy are the future directions for the treatment of adolescent nasopharyngeal carcinoma. Nasopharyngeal carcinoma (NPC) is the most commonly diagnosed head and neck malignancy in China and Southeast Asian countries, but children and adolescent nasopharyngeal carcinoma is very rare worldwide. The incidence among children and adolescents varies greatly among different regions and races, accounts for 0.1-2.3% of all NPCs in our nation . StandarBetween January 1, 1990 and January 31, 2011, 168 pathologically confirmed NPC patients under 20 years old were treated in our institution. Ten patients were excluded due to their undefined stage or lost follow-up shortly after treatment. The median age was 16 years (range 8–20 years). All patients received thorough physical examinations, biopsies of the nasopharynx, dental care, general status evaluations, blood counts, chest X-rays, as well as neck and abdomen ultrasonography. Indirect mirrors  and fiberoptic nasopharyngoscopes  were used to examine the nasopharynx. All patients were restaged according to the UICC 2002 staging system. 153 patients (96.8%) were staged with either CT, MRI or both, among them, 76 patients (48.1%) had bone scans before treatment. X-rays and clinical examinations were used in the other five as staging tools.All patients received radical external beam radiotherapy, there were 24 (15.2%) patients received neoadjuvant chemotherapy, 43 (27.2%) patients in stage III/IV received concurrent chemo-radiotherapy and 20 (12.7%) patients with residue primary lesions after radiotherapy received adjuvant chemotherapy.There were 103 (65.2%) patients received conventional 2D radiotherapy using 6 MV photons combined with 6–12 MeV electrons. The technique and dose used were as follows: first, a facial-cervical field  to a total dose of 36 Gy, followed by a smaller facial-cervical field  or a preauricular field to avoid excessive exposure of the brainstem and the spinal cord, to a total dose of 70–72 Gy to the nasopharyngeal region. The irradiation dose to the neck lymph node drainage region was as follows: 50 Gy was delivered to the lower neck and supraclavicular region and 60 Gy to the upper neck when cervical nodes were negative. 60 Gy were applied to the entire neck and supraclavicular region when cervical nodes were positive, then 70–72 Gy to the positive lymph nodes, with the division of the upper and lower neck set at the lower edge of the cricoid bone. After 70–72 Gy of irradiation, an additional of 10–21 Gy was boosted to the residue tumor with a shrinking-field external beam, brachytherapy or stereotactic radiosurgery technique. The residual lymph nodes were removed by surgical excision or regional neck dissection after 2–3 months observation.There were 55 (34.8%) patients received intensity-modulated radiation therapy (IMRT) technique. The entire neck IMRT technique was used to cover the primary lesion, nodal disease and entire neck including the supraclavicular region. The prescription dose for T1 and T2 primary lesions (GTVp) was 70 Gy in 33 fractions at 2.12 Gy per fraction, while 74 Gy at 2.24 Gy per fraction was applied to T3 or T4 diseases and involved retropharyngeal nodes with a maximum diameter > 1.5 cm, and all positive lymph nodes (GTVnd) received 70 Gy at 2.12 Gy per fraction. The elective radiation dose of 60 Gy at 1.82 Gy per fraction encompassed the high-risk regions including the uninvolved skull base, parapharyngeal spaces, the posterior third of the nasal cavity and high-risk nodal levels. If there were no positive neck nodes in the neck, 50–54 Gy was delivered to the bilateral lower neck and supraclavicular region using a two-phase IMRT plan, with the phase 1 IMRT plan (28 treatment fractions) covering the primary lesion, positive nodes, the high-risk region and the lower neck/supraclavicular region. The phase 2 IMRT plan (5 treatment fractions) covered only the primary lesion, positive nodes and high-risk regions. The dose limited to the major organs at risk were as follows: the brain stem with a 3 mm margin, Dmax < 54 Gy; spinal cord with a 5 mm margin, Dmax < 40 Gy; the optic nerve, chiasm and temporal lobe, Dmax < 54 Gy; and the parotid gland, V30-35 < 50%.For patients who received neoadjuvant chemotherapy, the target volumes depended on the pre-chemo involved regions.2/week regimen (median cycles: 7), 9 patients received cisplatin 80–100 mg/m2/q3w regimen (median cycles: 3), and adjuvant chemotherapy were PF-based regimen for 2–6 cycles.Seventy (44.3%) patients received chemotherapy, with 24, 43 and 20 patients treated by neoadjuvant, concurrent and adjuvant chemotherapy, respectively. Neoadjuvant chemotherapy consisted of cisplatin and a 5-FU based regimen(PF regimen) every 3 weeks for 1–2 cycles. In concurrent chemotherapy, 34 patients received a cisplatin 30 mg/mAll patients were evaluated weekly during radiation therapy, with a required follow-up after they completed radiotherapy: one month after the completion of radiotherapy, every three months in the first two years, every six months from the second to fifth years, and annually thereafter. Late radiation effects were evaluated and scored according to the Radiation Therapy Oncology Group/European Organization for Research and Treatment of Cancer late effects scale.The statistic was performed by SPSS 19.0 software. Kaplan-Meier method was used for calculating the survival, chi-square test and Cox regression analysis were used to detect statistically significant differences among the late toxicities and potential prognostic factors between the different groups.This study has been approved by ethics committee of cancer hospital, Chinese academy of Medical Sciences.Among 158 patients eligible for analysis, there were 119 male and 39 female, with median age of 16 years old (range 8–20 years), 9 patients in stage II, 60 in stage III and 89 in stage IV, respectively. 139 (88%) patients were non-keratinizing diseases. The median history time was 4.8 months (range 0.2-60.0 months). Neck mass (32.3%), headache (21.5%) and nasal obstruction (15.2%) were the most common chief complaints. There were 36 patients (22.8%) observed cranial nerve palsy, the grigeminal nerve (V) and abducent nerve (VI) were the most commonly involved, with incidence of 7.6% in V1 (12), 12.0% in V2 (19), 10.1% in V3 (16) and 6.3% in VI repectively  and stage IV (p = 0.04) were the independent adverse prognostic factors for OS. No significant prognostic factors were found for LRC and DMFS in either univariate or multivariate analysis.The median follow-up time was 62.5 months (range 2.0-225.0 months), with 13.3% patients lost follow-up. The 5-year OS, LRC and DMFS were 82.6%, 94.9% and 76.4%, respectively  failed during follow up, with 7 in local-regional, 38 in distant metastasis and 2 in both. The median failure times were 15 months (3–39 months) and 5 months (1–38 months) for local-regional recurrence and distant metastasis. The most common site of metastasis was bone ; other sites included lung (n = 7), liver (n = 1) and distant lymph nodes (n = 3). The causes of death were primary disease in 24, second malignancy in 2 and unknown in 4.v 3.6%, p = 0.03) and G2 xerostomia  was observed in those patients treated by IMRT.For those patients who had detail medical records, the most common late toxicities were xerostomia, neck fibrosis, and hearing loss, with incidence of 94.8% (55/58), 91.2% (52/57) and 52.6% 30/57) respectively. Hypothyroidism was reported in 50.8% (30/59) patients. Severe late sequela included blindness (2 patients), deafness (2 patients), bone necrosis (2 ptatients) and nasopharynx hemorrhage (1 patient). Due to a lack of sufficient information, radiation encephalopathy could not be fully documented (see detail in Table 0/57 respThere were six patients (3.8%) developed second primary neoplasm in the radiation field after 38–123 months of radiotherapy Table .Table 4v 62% ± 0.8% (p < 0.001) [The incidence of NPC varies widely among different regions and races, and is most often seen among patients in their fifth or sixth decade in endemic countries. However, there was a bimodal incidence graphs in sporadic regions such as North America and Mediterranean basin countries, a second minor early peak was observed at 10–20 years of age, with incidence of 2-18% . Huang e< 0.001) .3 increase in primary tumor volume. Although the recommended dose for the primary site ranges from 59.4 Gy to 66 Gy (1.8-2.0 Gy per fraction) in the literature, it seems that a higher dose provided better outcomes; Ozyar et al. [p = 0.01) in multivariate analysis, while other authors reported that a 64-80Gy or higher dose was needed [v 28%). Laskar et al. [p = 0.03) and G2 xerostomia (p = 0.02) because of IMRT allows for the delivery of high doses to the target area while sparing the surrounding critical structures and offers superior target coverage compared with conventional radiotherapy and 3D-CRT in improving therapeutic ratios. Using new techniques, such as IMRT, to improve local control and protect normal tissue will be a key focus in future daily practice.In our univariate and multivariate analysis, T4 was the adverse prognostic factor. T4 patients usually have a larger tumor volume than those with T1-3 and a higher tumor burden needs a higher dose to control. Sze et al.  found ths needed . In conts needed  reporteds needed ,10. In os needed ,14. Woldr et al.  reportedMany studies focused on using neoadjuvant chemotherapy combined with lower dose radiotherapy to reduce the radiation related severe late toxicities. In NPC-91-GPOH study and NPC-2003-GPOH/DCOG study , after cIn the present series of young NPC patients, distant metastasis remained the major pattern of failure ,12,20,whMost childhood and adolescence nasopharyngeal patients had local advanced diseases at first diagnosed. The treatment results of radiotherapy with or without chemotherapy are excellent in our institution. Reducing distant metastasis with new strategies and late toxicities with intensity-modulated radiotherapy will be the future directions for the treatment of children and adolescent nasopharyngeal carcinoma. Further, response-adapted RT is worth further evaluation to minimize late toxicities.


R-hydroxy-10E,12Z,15Z-octadecatrienoic acid (9R-HOTE) from α-linolenic acid (ALA) in perilla seed oil (PO) hydrolyzate was achieved using purified recombinant 9R-lipoxygenase (9R-LOX) from Nostoc sp. SAG 25.82. The specific activity of the enzyme followed the order linoleic acid (LA) > ALA > γ-linolenic acid (GLA). A total of 75% fatty acids (ALA and LA) were used as a substrate for 9R-LOX from commercial PO by hydrolysis of Candida rugosa lipase. The optimal reaction conditions for the production of 9R-HOTE from ALA using 9R-LOX were pH 8.5, 15°C, 5% (v/v) acetone, 0.2% (w/v) Tween 80, 40 g/L ALA, and 1 g/L enzyme. Under these conditions, 9R-LOX produced 37.6 g/L 9R-HOTE from 40 g/L ALA for 1 h, with a conversion yield of 94% and a productivity of 37.6 g/L/h; and the enzyme produced 34 g/L 9R-HOTE from 40 g/L ALA in PO hydrolyzate for 1 h, with a conversion yields of 85% and a productivity of 34 g/L/h. The enzyme also converted 9R-hydroxy-10E,12Z-octadecadienoic acid (9R-HODE) from 40 g/L LA for 1.0 h, with a conversion yield of 95% and a productivity of 38.4 g/L. This is the highest productivity of HFA from both ALA and ALA-rich vegetable oil using LOX ever reported. Therefore, our result suggests an efficient method for the production of 9R-HFAs from LA and ALA in vegetable oil using recombinant LOX in biotechnology.Hydroxy fatty acids (HFAs) derived from omega-3 polyunsaturated fatty acids have been known as versatile bioactive molecules. However, its practical production from omega-3 or omega-3 rich oil has not been well established. In the present study, the stereo-selective enzymatic production of 9 Z,4Z)-pentadienes to form hydroperoxy fatty acids (HPFAs) that reduced to hydroxy fatty acids (HFAs) [Lipoxygenase (LOX) catalyzes the regio- and stereo-selective hydroperoxidation of free polyunsaturated fatty acids (PUFAs) possessing one or more 1Z,4Z-pents (HFAs) , 3. Extes (HFAs) –7.To date, LOX has mainly been applied to the production of HPFAs, which are utilized to produce green note volatiles combined with hydroperoxy lyase . Due to n-3) is an essential fatty acid that requires dietary uptake as it is not synthesized by the human body due to the absence of endogenous desaturases such as Δ5- and Δ6-desaturases. Perilla (Perilla frutescens) seed oil (PO) is known to contain approximately 60% ALA [E,12Z,15Z- or 13-hydroxy-9Z,11E,15Z-octadecatrienoic acid (9- or 13-HOTE), have been known as antifungal [R-LOX from Nostoc sp. showed a wide range of substrate specificity for PUFAs that form HPFAs [Nostoc 9R-LOX can be applied to produce HFA from ALA in omega-3 rich vegetable oil.α-Linolenic acid  were purchased from Nu-Check Prep  and Sigma , respectively. 9-Hydroperoxy-trioctadecatrienoic acid (9-HPOTE) and 9S-HOTE standards were purchased from Cayman Chemicals . 9R-HOTE standard was prepared by the previously reported procedure [n-hexane, and methanol, were high-performance liquid chromatography (HPLC) grade and ethanol and ethyl acetate for extraction were reagent grade. These solvents were supplied from Duksan Chemical . Commercial PO was obtained from a local market. Luria-Bertani (LB) medium was purchased from Fermentas . Ampicillin, boric acid, and isopropyl-β-d-thiogalactopyranoside (IPTG) were purchased from USB . Lipases from recombinant Aspergillus oryzae (AOL), Candida rugosa (CRL), Pseudomonas cepacia (PCL), Pseudomonas fluorescense (PFL), Rhizomucor meihei (RML), and Thermomyces lanoginosus (TLL) were obtained from Sigma.High-quality (>99%) ALA and linoleic acid  in pET-15b (9R-lox/pET-15b) was used for expression in E. coli [E. coli ER2566 cells by electroporation and selected on LB agar containing 50 μg/ml ampicillin. A single colony of E. coli cells containing the 9R-lox/pET-15b plasmid were inoculated into 3 ml of liquid LB medium and cultivated at 37°C with shaking at 220 rpm overnight. This culture was transferred into a 2,000 ml flask containing 500 ml of LB medium and 50 μg/ml ampicillin at 37°C with shaking at 200 rpm. When the optical density of the culture reached 0.6 at 600 nm, expression of the 9R-lox gene was induced by adding isopropyl-β-D-thiogalactopyranoside with a final concentration of 0.1 mM, and the culture was further incubated for 16 h at 16°C with shaking at 150 rpm.The constructed plasmid harboring  E. coli . This plThe cultured cells were harvested by centrifugation at 6000×g for 20 min at 4°C and washed twice with 0.85% (w/v) NaCl. The cells were disrupted by sonication on ice for 2 min in 50 mM Tris-HCl buffer (pH 8.0) containing 300 mM NaCl and 1 mg/ml lysozyme. The unbroken cells and debris were removed by centrifugation at 13000×g for 20 min at 4°C, and the obtained supernatant was used as a crude extract. Purification was carried out using a fast protein liquid chromatography system (Bio-Rad) in a cold room at 4°C. The crude extract was applied to a His-Trap affinity chromatography column  equilibrated with 50 mM Tris-HCl buffer (pH 8.0). The bound enzyme was then eluted at 4°C with the same buffer containing 250 mM imidazole at a flow rate of 1 ml/min. The active fractions were collected and dialyzed at 4°C for 16 h against 50 mM Tris-HCl buffer (pH 8.5). The resulting solution was used as the purified enzyme.−1 cm−1 was used to calculate the activity of the enzyme for 9-HPODE or 9-HPOTE [234 < 1.0. One unit (U) of LOX activity is defined as the amount of enzyme catalyzing the formation of 1 μmol of product per min. The rate of the reaction was compared to that without the addition of enzyme as a control.LOX activity was determined by measuring conjugated diene formation at 234 nm using a Beckman Coulter DU-700 spectrophotometer . An extinction coefficient of 25,000 M 9-HPOTE , 25. UnlR-LOX activity for the production of 9R-HOTE from ALA, the pH values were varied from 7.0 to 10.0 using 50 mM Tris-HCl buffer (pH 7.0–9.0) and 0.2 M sodium borate buffer (pH 9.0–10.0) at a constant temperature of 25°C, while the temperatures were varied from 10 to 50°C at a constant pH of 8.0. The effect of temperature on enzyme stability was monitored for 4 h by incubating the enzyme solution at different temperatures from 5 to 50°C in 50 mM Tris-HCl buffer (pH 8.5). Samples were withdrawn at time intervals and then assayed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA and 0.05 U ml−1 enzyme at 15°C for 5 min. The half-life of the enzyme was calculated using Sigma Plot 10.0 software .To investigate the effects of pH and temperature on 9R-HOTE from ALA. Tween 80 concentration was varied from 0.005% to 1% (w/v) to determine the optimum concentration of the detergent. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA, 0.1 U/ml enzyme, 5% (v/v) acetone, and Tween 80 at 15°C for 10 min.Several organic solvents at concentrations of 4% and 6% (v/v), including methanol, ethanol, butanol, isopropanol, heptanol, octanol, acetone, ethyl acetate, dichloromethane, hexane, and dimethyl sulfoxide (DMSO) were tested for selecting the optimum solvent. The effect of solvent concentration on LOX activity was evaluated by varying the concentration of acetone from 1% to 10% (v/v). The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA, 0.1 U/ml enzyme, and acetone at 15°C for 10 min. Several detergents, including Tween 20, Tween 40, Tween 80, Span 20, Span 60, Span 80, Triton-X 100, Brij 58, and sodium dodecyl sulfate (SDS), were tested to find the suitable detergent for the maximum production of 92 using a Clark-type electrode  with a high sensitivity membrane and oxygen monitor (YSI Model 5300A) in a thermostatic oxygen cell with a cell volume of 1.5 ml at 25°C [2/N2 atmosphere. The reactions were initiated by the addition of 1−5 μL concentrated enzyme, corresponding to 0.1−2 U/mg specific activity, via a gastight syringe. Nitrogen was used to adjust to different O2 concentrations. Enzyme activity was determined from the initial rate of oxygen uptake and consumption, as reported previously [Determination of all kinetic measurements was obtained by monitoring the time-dependent consumption of O at 25°C , 26. Theeviously , 26. LA,R-HOTE from ALA by recombinant 9R-LOX were determined by varying the enzyme concentration from 0.05 to 2 mg/ml at a constant ALA concentration of 10 g/L and varying the substrate concentration from 5 to 50 g/L at a constant enzyme concentration of 0.5 mg/ ml. The reactions were performed in 50 mM Tris-HCl buffer (pH 8.5) containing different concentration of ALA and enzyme at 15°C for 20 min.The optimal concentrations of enzyme and substrate for the increased production of 9R-HOTE from ALA by recombinant 9R-LOX was performed in 50 mM Tris-HCl buffer (pH 8.5) containing 40 g/L ALA, 5% (v/v) acetone, and 0.2% (w/v) Tween 80 at 15°C for 80 min. 9R-HODE production from LA was carried out under the same conditions as the production of 9R-HOTE from ALA.The production of 9R-HOTE. The time course reactions for the production of 9R-HOTE from ALA by 9R-LOX were carried out in a 500 ml baffled flask in 80 ml of 50 mM Tris-HCl buffer (pH 8.5) consisting of PO hydrolyzate, which contained 40 g/L ALA, 10 g/L LA, 15.3 g/L of other fatty acids, 5% (v/v) acetone, 0.2% (v/v) Tween 80, and 1 g/L purified 9R-LOX at 15°C for 100 min, with agitation at 250 rpm and oxygen supplementation for efficient oxygenation. After the reactions, 9R-HOTE and 9R-HODE were obtained from 9R-HPODE and 9R-HPOTE by the addition of 8 ml of 1 M NaBH4, and an equal reaction volume of ethyl acetate. The ethyl acetate in the reaction mixture was removed using a rotary evaporator under reduced pressure, and the concentrations of 9R-HODE and 9R-HOTE in the reaction solutions were determined.PO with a high content of ALA was hydrolyzed using AOL, CRL, PCL, PFL, RML, and TLL. The reaction was carried out in 9 ml of 50 mM Tris-HCl buffer (pH 7.5) containing 10 g/L PO, 1 g/L CRL (700 U/mg), and 0.02% (w/v) Tween 80 at 37°C with shaking at 250 rpm for 2 h. The reaction was stopped by boiling the reaction mixture at 100°C for 50 min. The sample was kept at −20°C for analyzing the concentrations of fatty acids. The samples were extracted twice using ethyl acetate and dried under reduced pressure. The dried samples were collected and used for the production of 9R-HOTE and 9R-HODE standards were prepared by a preparative HPLC after solvent extraction, as described previously [9eviously . The pur3 and 0.05 ml of tridecanoic acid  as an internal standard in a screw-cap Pyrex tube (16×150 mm). The mixture was incubated at 55°C in a water bath for 1 h. The reactant was cooled and then 2 ml of saturated NaOCH3 solution and 3 ml of hexane were added in the same reaction tube for extraction. The hexane layer containing the FAME was taken into a gas chromatography (GC) vial. The vial was capped and placed at −20°C until GC analysis. A GC-flame ionization detector (FID) (Agilent 6890N) equipped with a DB-23 column  was used for analysis. The chromatographic conditions were as follows: injector temperature, 250°C; oven temperature, started at 80°C, increased to 180°C at 25°C min−1, increased to 250°C at 4°C/min, and held for 5 min; column temperature, maintained at 160°C for 1 min, and then raised to 250°C at a rate of 4°C/min. Identification of compounds was confirmed by comparison with authenticated reference standards for PO . The concentrations of fatty acids were determined using the peak of the internal standard as a reference value and the standard curves of authentic compounds.The composition of total fatty acids from PO hydrolyzate was determined by fatty acid methyl ester (FAME) analysis, as described previously . Briefly9R-LOX products, including 9-HPOTE, 9-HOTE, 9-HPODE, 9-HODE, 13-HOTE, and 13-HODE, were analyzed by specific measurement at 234 nm using an reversed-phase HPLC system  equipped with a UV detector and a Nucleosil C18 column  with a guard cartridge. The column was run and eluted with a gradient of solvent A  and solvent B  as described [n-hexane/2-propanol/acetic acid  [R-LOX were determined using linear calibration curves and correlating the peak areas to the concentrations of standards.Substrates, ALA and LA were analyzed by non-specific measurement at 202 nm, while ) using an electrospray ionization (ESI) interface. Two hundred microliters obtained from the HPLC fraction was concentrated under N2 stream to complete dryness and dissolved in 20 μl ethanol containing 0.1% acetic acid. The flow rate was 250 μl min−1 and a gradient of 0.1% formic acid in acetonitrile was used. The operation parameters were as follows: capillary temperature, 275°C; ion source voltage, 5 kV; nebulizer gas, 40 psi nitrogen; capillary voltage, 46 V in positive ionization mode and 15 V in negative ionization mode; average scan time, 0.01 min; average time to change polarity, 0.02 min; and collision energy (CE), approximately 35% abundance of the precursor ion.Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) analysis of HFAs was carried out using a ThermoFinnigan LCQ Deca XP plus ion trap mass spectrometer  with a Synergi fusion-RP 80Å column  was carried out using Tukey’s method with a significance level of p < 0.05 using SigmaPlot 10.0 . Data were reported as mean ± standard deviation (SD) for triplicate treatments.R-LOX from Nostoc sp. SAG 25.82 was purified as a soluble protein from crude extract by His-Trap affinity chromatography. The molecular mass of the purified enzyme analyzed by SDS-PAGE was approximately 52 kDa (web.expasy.org/compute_pi/). Based on the molecular masses of the reference proteins, the native enzyme may exist as a trimer with a molecular mass of 156 kDa, as determined by gel filtration chromatography . Among the solvents tested, acetone was the most effective solvents for LOX activity . The maximum enzyme activity occurred at 5% (v/v) acetone . Tween 20, Tween 40, and Tween 80 at these concentrations showed 1.2- to 1.6-fold higher activity than that of control . The maximum enzyme activity occurred at 0.2% (v/v) Tween 80 (R-LOX than that without the addition of solvent and detergent.The effect of organic solvent on the activity of 9activity . The eff acetone . The eff control . Tween 8Tween 80 . SolventKm), turnover numbers (kcat), and catalytic efficiencies (kcat/Km) of Nostoc sp. 9R-LOX were investigated using PUFAs, including LA, ALA, and GLA  acetone and 0.2% (w/v) Tween 80 at pH 8.5 and 15°C for 5 min. Under these conditions, the specific activity and Michaelis−Menten constants  acetone and 0.2% (w/v) Tween 80 at pH 8.5 and 15°C. The maximum production of 9R-HOTE was observed at 1.0 g/L enzyme  acetone and 0.2% (w/v) Tween 80 at pH 8.5 and 15°C  acetone, and 0.02% (w/v) Tween 80 with agitation speed of 250 rpm and oxygen at 20 ml of working volume in a 250 ml-baffled flask. Under the optimal conditions, the production of 9R-HOTE and 9R-HODE from PO hydrolyzate containing ALA and LA were performed using 9R-LOX. The enzyme produced 36.5 g/L of 9R-HOTE and 9.5 g/L of 9R-HODE from 40 g/L of ALA and 10 g/L of LA in PO hydrolyzate for 100 min. 9R-HOTE production from ALA in PO hydrolyzate was increased to 34 g/L at 60 min and plateaued at 35 g/L at 80 min. The conversion yield and volumetric productivity for 9R-HOTE from ALA in PO hydrolyzate were 85% and 34 g/L/h, respectively. 9R-HODE production from LA in PO hydrolyzate was more rapid than that from ALA and plateaued at 20 min with a conversion yield of 95%  and docosahexanoic acid , the important precursors of human signaling cascades [The substrate specificity of bacterial LOXs toward omega-3 PUFAs including ALA based on kinetic parameters is also noteworthy properties. The kinetic parameters of bacterial LOXs can be determined by not only an Ofrom ALA , 30, 31.for PUFA . Therefois study . 9R-ConFbacteria , compariS-HPODE from LA by the crude extract or the partially purified enzyme conversion, immobilization, biphasic reaction [S-HPODE or 9S-HODE from LA using crude extract or by immobilization [S-HPODE and 13S-HPOTE, respectively. SLOX produced 5.7 g/L 13S-HPODE from 6 g/L LA in TLH with a conversion yield of 96% [E,12Z,15Z-form of 13S-HPOTE from 78.5 g/L ALA in FOH with a conversion yield of 62.8% and a productivity of 31.4 g/L/h [Gaeumannomyces graminis tritici LOX secreted in Trichoderma reesei showed a conversion yield of 40% and a productivity of 3.6 g/L/h for 10E,12Z-13S-HPOTE production [Pseudomonas aeruginosa that was cloned and secreted in Trichoderma reesei showed mixed production of 9S- and 13S-HPODE from LA with a low yield [Burkholderia thailandensis LOX showed 10.4 g/L/h of productivity with a molar conversion yield of 98.5% [The production of HPFAs and HFAs is summarized in reaction –38. Crudlization , 40. A fd of 96% . SLOX pr.4 g/L/h . Fungal oduction . A LOX fow yield . Recentlof 98.5% .R-LOX from Nostoc sp. produced 9R-HOTE with a higher conversion yield and productivity from ALA in PO hydrolyzate as well as reagent-grade ALA than those of other LOXs for PUFAs , 10 mM LA, and 0.25 U ml(TIF)Click here for additional data file.S2 Figa Effect of pH. The reactions were performed by varying the pH from 7.0 to 10.0 using 50 mM Tris-HCl buffer (pH 7.0–9.0) (filled circle) and 50 mM sodium borate buffer (pH 9.0–10.0) (empty circle) containing 0.25 mM ALA and 0.05 U/mL enzyme at a constant temperature of 25°C for 5 min. b Effect of temperature. The reactions were performed by varying the temperature from 10 to 50°C in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA and 0.05 U ml−1 enzyme for 5 min. c The effect of temperature on 9R-LOX stability. Experiments were carried out after incubation at temperatures ranging from 4 to 45°C for 4 h. Samples were withdrawn at time intervals and then assayed in 50 mM Tris-HCl buffer (pH 8.5) containing 0.25 mM ALA and 0.05 U ml−1 enzyme at 15°C for 5 min.The data represent the means of three separate experiments, and error bars represent the standard deviation. (TIF)Click here for additional data file.S3 Figa Reacted with LA. b Reacted with ALA. c Reacted with GLA.(TIF)Click here for additional data file.S4 Fig−1 lipase, and 0.1 g−1 L−1 of PO at 30°C for 30 min. Recombinant AOL, CRL, TLL, RML, PCL, and PFL were used for the hydrolysis of PO.The reactions were performed in 50 mM Tris-HCl buffer (pH 7.5), 1 U ml(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Lentinus edodes, commercial obtained in daily life, had recently become more attractive in physiological research. Zinc was now considered as a major element in assuring the correct functioning of an organism and essential for maintaining coordination of the major homeostatic networks. To investigate the bioconversion of zinc and the physiological effects of their complex (MZPS), the present studies were processed.In the modern society, aging had been a major problem. People may rely on many medicines to delay it. However, lots of medicines were chemosynthetic, and they would do a bad side-effect on human body. Microbial sources could be used as a potential means of producing natural antioxidants. Lentinus edodes SD-08 were extracted by hot water leaching and purified by DEAE-52 cellulose anion-exchange column chromatography separately. The zinc content was determined by flame atomic absorption spectrometry. The evaluation of monosaccharide compositions and proportions used gas chromatogram. The analysis of molecular weight used HPGPC chromatogram. The typical structure of polysaccharide was evaluated by IR spectrum. The antioxidant activities in vitro measured through reducing power, the scavenging effects on hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. The anti-aging activities in vivo measured through the total antioxidant capacity (T-AOC), GSH peroxide (GSH-Px), superoxide dismutase (SOD) and the contents of malondialdehyde (MDA).Mycelia polysaccharides (MPS) and mycelia zinc polysaccharides (MZPS) of Lentinus edodes SD-08 were extracted and purified by DEAE-52 cellulose anion-exchange column chromatography separately, and four fractions  were obtained. In addition, MPS composing of rhamnose, arabinose and mannose (molar proportion = 1.75:1.00:3.02) and MZPS containing rhamnose, arabinose, mannose and glucose (molar proportion = 7.19:2.26:1.00:8.39) were investigated by gas chromatography. Infrared spectrum analysis indicated that there were C-H, C=O and -CH2 bonds in MPS and MZPS. MPS also had the typical absorption of -NH3+, -NH2 and -COOH. Compared with MPS, MZPS showed in vitro positive rising of reducing power and certain scavenging effects on hydroxyl radical and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals. MZPS were found to upregulate in vivo the anti-aging activities of total antioxidant capacity (T-AOC), GSH peroxide (GSH-Px), superoxide dismutase (SOD), and decrease the contents of malondialdehyde (MDA).MPS and MZPS of in vivo and antioxidant activities in vitro, and the molecular constituents, chemical bonds and functional groups of MZPS were superior to MPS, suggesting that the MZPS of L. edodes SD-08 could be used as a potential natural antioxidant.MZPS effectively showed potential anti-aging activities  Lentinus edodes, as its nutritional value and medical application, has been the second most popular edible mushroom [L. edodes, because of its antioxidant activity, immunomodulatory and antitumor effects [Mushrooms play an important role in diet and have recently become attractive as sources for the development of drugs, so many researchers paid much attention on them ,2. In thmushroom ,4. Lenti effects -7. AlthoZinc (Zn) is one of the most important trace elements in human body and now considered as a major element in assuring the correct functioning of an organism. Zinc is essential for maintaining coordination of the major homeostatic networks, i.e., the nervous, neuroendocrine and immune systems, adds support to a new theoretical approach to explain aging processes  during tLentinus edodes SD-08 was cultured in liquid media using ZnSO4 as zinc supplement. The mycelia polysaccharide (MPS) and mycelia zinc polysaccharide (MZPS) of L. edodes SD-08 were separately purified, and four fractions  were obtained by DEAE-52 cellulose anion-exchange column chromatography. Molecular constituents, chemical bonds and functional groups of MPS or MZPS were investigated. The in vivo anti-aging capacities and in vitro antioxidant activities of MZPS were evaluated.In this paper, L. edodes SD-08 was from our laboratory at 4°C and maintained on synthetic potato dextrose agar (PDA) slants. The liquid fermentation technology was used to produce L. edodes SD-08 mycelia. Each 1 L erlenmeyer flask, containing 500 mL of liquid medium , was inoculated with a 1 cm2 mycelial block of L. edodes SD-08 from the solid media. The liquid culture was grown at 25°C with a shaking of 160 r/min for 14 d.1,1-dipheny-l,2-picrylhydrazyl (DPPH) was from Sigma Chemicals Company . DEAE-52 cellulose anion-exchange was from Whatman Chemicals Company (UK). All other chemicals used in this experiment were analytical reagent grade and purchased from local chemical suppliers in China.Three times of distilled water were added to a beaker with mycelia powder 100 g, and the mixture was sonicated for 600 s using sonifier cell disrupter . The homogenate was heated at 85°C for 3 h and centrifuged at 3000 r/min for 10 min. The supernatant was collected and the above process was repeated three times. Four times of alcohol  was mixed with the supernatant and kept at –4°C for 12 h. After centrifuged at 3000 r/min for 20 min, the precipitation (MPS or MZPS) was lyophilized. The content of MPS or MZPS was determined by phenol–sulfuric acid method, using glucose as standard .MPS and MZPS were separately fractionated by DEAE-52 cellulose anion-exchange column (2.5 × 65 cm). When polysaccharides were poured into the DEAE-52 cellulose, DEAE-52 could combine the polysaccharides which could be eluted by the increasing intensity of NaCl , and only strong enough ionic force could elute the different fragments . The maj3  were added to the mixture for terminating the reaction, and then centrifuged at 1200 r/min for 10 min. After incubating at 25°C for 15 min, the absorbances of the samples were measured at 700 nm.The reducing power of polysaccharides was measured estimated according to the method of Oyaizu  with a s2O2, 1.0 mL of 9 mM FeSO4, and 1 mL of 9 mM salicylic ethanol solution. After incubating at 37°C for 30 min, the absorbances of polysaccharides were then measured at 510 nm using UV-4802 double-beam UV-spectrophotometer. The hydroxyl radical scavenging activity was expressed as:Hydroxyl radical scavenging activity was measured according to the method of Winterbourn and Sutton . One mil0 is the absorbance of the blank and A1 is the absorbance of the sample.Where AThe experimental of DPPH scavenging activity of MPS and MZPS were according to the method of Liu and Zhao . The reac is the absorbance of 2 mL DPPH and 2 mL absolute ethanol, Ai and Aj is the absorbance of 2 mL sample and 2 mL DPPH or absolute ethanol.Where A3 and 2 mL HClO4 and let the mixture standing overnight. The mixture was boiled until the cooking liquor clear. After digestion of the samples, the mixture was diluted to 25 mL. The zinc content was measured by using flame atomic absorptions spectrometric method .MPS and MZPS (0.1 g) were weighed severally, and then added 5 mL HNOMonosaccharide composition was determined by gas chromatography (GC)  equipped with a capillary column of Rtx-1 (30 mm × 0.25 mm × 0.25 μm) using the method in the literature  with sliThe molecular weight and homogeneity of MZPS were determined by high performance liquid gel permeation chromatography (HPGPC) that was operated with a HPLC system  equipped with a SHODEX SB-806HQ column (8.0 mm × 300 mm) and a refractive index detector. The injection volume was 100 μL. NaCl aqueous solution (0.2M) was used as mobile phase at a flow rate of 0.5 mL/min, and the column temperature was maintained at 35°C. A series of standard dextrans with known Mw (molecular weights) values were used to make the calibration curve, and the regression equation of logMw against elution time (ET) was as follows: logMw = –0.3429ET + 11.975, R2 = 0.9991. Molecular weights was analysed by Agilent GPC software.-1 [The IR spectrum of the polysaccharides was carried out using a Fourier transform infrared spectrophotometer  equipped with OPUS 3.1 software. The components of MPS or MZPS were ground with KBr powder and then pressed into pellets for FTIR measurement at the frequency range of 4000–500 cm-1 .Forty-two male mice (Kunming strain), weighing 20 ± 2 g were provided by Center for Animal Testing of Shandong Lukang Drugs Group Ltd.  and housed in stainless steel cages under controlled conditions  with free access to standard food. After a 3 day acclimatization period, all animals were randomly divided into six groups (7 in each group). Model group (MG) was intraperitoneal injected with 0.2 mL D-galactose . Blank group (BG) was gavaged with 0.2 mL distilled water. Low MPS (LMPS) and high MPS (HMPS) were perfused 200 and 800 mg/kg body weight of MPS, respectively. Low MZPS (LMZPS) and high MZPS (HMZPS) were perfused 200 and 800 mg/kg body weight of MZPS, respectively. LMPS, HMPS, LMZPS and HMZPS were intraperitoneal injected with 0.2 mL D-galactose  simultaneously. The mice were allowed to have free access to water and food for 30 d. The experiments were performed under the guidance of Committee of Shandong Agricultural University.At the end of the experiment, all animals were sacrificed under ether anesthesia, and blood samples were taken from the retrobulbar vein with a vacutainer and anticoagulated by heparin (stored at –80°C). The hearts, livers, spleens, and kidneys were rapidly removed, weighed, and homogenized  immediately in 0.2 M phosphate buffer , respectively. The homogenates were centrifuged (6000 r/min) at 4°C for 20 min and the supernatants were stored at –20°C for the evaluation of the activities of the enzyme and nonenzyme total antioxidant capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and the content of malonaldehyde (MDA).The T-AOC and SOD of serum and liver were analyzed using commercial kits .3, 0.15 mM NADPH, 1 mM GSH, and 2.4 U/mL of glutathione reductase (GR). The reaction was initiated by adding 0.15 mM H2O2. The rate of nicotinamide adenine dinucleotide phosphate (NADPH) consumption was recorded at 340 nm. The activity of GSH-Px was expressed as mM of NADPH oxidized per minute per milligram of tissue or micromoles per minute per milliliter of blood.The activity of GSH-Px was measured as described by Flohé and Günzler . The reaThe content of MDA was measured according to the method of Zhao et al.  with a s0 is the absorbance at 532 nm, A1 is the absorbance at 600 nm, and A2 is the absorbance at 450 nm.Where AAll experiments were carried out in triplicates and results were recorded as means ± standard deviation (S.D.). P < 0.05 was considered to be statistically significant. Determination of parameters of MPS and MZPS were processed and analyzed using SPSS 16.0.The results of MPS and MZPS purification were shown in Figure P. baumii [A. bisporus [L. edodes SD-08 had the higher antioxidant capacities in vitro.As shown in Table . baumii  and 0.38bisporus , indicatP. nebrodensis [C. militaris [, 38.10% of T. matsutake [P. ostreatus [L. edodes SD-08, especially MZPS-1, significantly affects the scavenging of hydroxyl radical.As shown in Table rodensis , 28.30% ilitaris , 38.10% atsutake , and 0.7streatus , respectC. militaris [C. sinensis [It can be seen from Table ilitaris  and 17.8sinensis . The DPPThe content of zinc in MPS and MZPS were analyzed. The zinc content in MZPS (2.95 ± 0.05 mg/g) was markedly higher than that in MPS (0.62 ± 0.03 mg/g) (P < 0.01).Gas chromatographic figure of mixed standard monosaccharides, MPS and MZPS contained many peaks as shown in Figure 5, 7.14 × 102 and 7.56 × 105 Da, respectively. The Mw/Mn value of MZPS was 168.19 and the Mz/Mw value was 6.30.HPGPC chromatogram Figure  indicate-1 and 3417.35 cm-1 characteristic of hydroxyl groups and a weak C-H band at around 2925.17 cm-1 and 2922.30 cm-1. The relatively strong absorption peak at 1638.03 cm-1 and 1633.81 cm-1 (around 1600-1650 cm-1) indicated the existing of C=O, the peaks at 578.03 cm-1 and 575.98 cm-1 suggestted the presence of -CH2 [-1, 1079.34 cm-1, 1023.96 cm-1 and 1152.30 cm-1, 1079.35 cm-1, 1026.52 cm-1, respectively. In addition, MPS had an absorption peak at 1618.54 cm-1 characteristic of -NH3+ and -NH2, showing that MPS was a protein-bound polysaccharide. The absorption peak at 1416.65 cm-1 indicated that the characteristic of -COOH in MPS. The absorption peaks at 2852.22 cm-1 and 1407.18 cm-1 indicated that MZPS was an aliphatic polysaccharide and had a C-H band.Infrared spectrum of the MPS and MZPS, as shown in Figure In our present work, we built the senile model using D-galactose. Since free-radical-induced lipid peroxidation has been associated with a number of diseases , free-raThe T-AOC activities of the mice in MZPS were significantly lower than those of the mice in MPS. The T-AOC activities of HMZPS reached 25.01 ± 0.01 U/mg, 26.04 ± 0.01 U/mg, 15.23 ± 0.10 U/mg and 20.39 ± 0.01 U/mL in heart, liver, kidney and blood, which were 16.59 U/mg, 13 U/mg, 6.86 U/mg and 11.42 U/mL higher than that of LMZPS, respectively. Moreover, The T-AOC activities of HMZPS in heart, liver, kidney and blood were 5.72 U/mg, 0.51 U/mg, 0.29 U/mg and 1.76 U/mL higher than that of HMPS, respectively. The results indicated that HMZPS had the highest activity among the four groups .Significant increases in GSH-Px activities were observed between MZPS and MPS. The GSH-Px activities of HMZPS were increased by 0.07 U/mg, 0.17 U/mg, 0.07 U/mg and 0.03 U/mL than that of LMZPS, and by 0.01 U/mg, 0.10 U/mg, 0.04 U/mg and 0.02 U/mL than that of HMPS, respectively. The results indicated that the MZPS had the higher activities of GSH-Px.Results showed that the SOD activities rose in a dose-dependent manner. The SOD activities of HMZPS reached 633.11 ± 0.69 U/mg, 302.25 ± 1.40 U/mg, 14.24 ± 0.42 U/mg and 290.28 ± 6.03 U/mL in heart, liver, kidney and blood, which were increased by 234.89 U/mg, 59.14 U/mg, 6.1 U/mg and 177.04 U/mL than that of LMZPS. Compared to HMPS, the SOD activities of HMZPS increased 64.56 U/mg, 39.17 U/mg, 1.05 U/mg and 21.20 U/mL, respectively.The MDA contents of HMZPS reached 1.21 ± 0.01 nmol/g, 0.43 ± 0.01 nmol/g, 0.04 ± 0.10 nmol/g and 1.03 ± 0.01 nmol/mL in heart, liver, kidney and blood, which were 1.56 nmol/g, 2.55 nmol/g, 0.34 nmol/g and 1.50 nmol/mL  lower than that of LMZPS, respectively, and 0.13 nmol/g 0.11 nmol/g, 0.02 nmol/g and 0.22 nmol/mL lower than that of HMPS, respectively. The results indicated that MZPS had ability of lowering lipid contents in mice tissue.5 Da. Li et al. [L. edodes fruiting bodies was composed of arabinose, galactose, glucose, xylose, and mannose with a molar ratio of 0.72:3.01:33.77:1.34:3, and the Mw was 1.06 × 106 Da [L. edodes.In the present work, the preliminary chemical characterization of MZPS was determined. The results indicated that the major component of MZPS was glucose, and the Mw of MZPS was 1.20 × 10i et al.  also fouin vitro of MPS and MZPS and their four fractions. Free radicals are known to be the major cause of various chronic and degenerative diseases, including aging, coronary heart disease, inflammation, stroke, diabetes mellitus and cancer [We measured various antioxidant activities d cancer . The antd cancer . Reductad cancer . Hydroxyd cancer , they ard cancer . DPPH isd cancer . When DPd cancer . It has in vivo of MPS and MZPS. In this work, markedly decreased T-AOC, GSH-Px and SOD activity, increased MDA levels were found in the Model group. The MPS and MZPS could significantly counteract the increased oxidative stress by promoting the activities of enzymatic and non-enzymatic antioxidants and reducing the level of lipid peroxidative product after fed to the mice. These experimental data indicated that the MPS and MZPS have antioxidant activity and a potential for decreasing risk of aging.We also measured various anti-aging effects in vivo and antioxidant activities in vitro, and the molecular constituents, chemical bonds and functional groups of MZPS were different from that of MPS, suggesting that the MZPS of L. edodes SD-08 could be used as a potentially natural antioxidant. However, the relationship of structure and activity of MZPS and its possible mechanisms were the areas for future studies.MZPS effectively showed anti-aging activities


Flavobacterium enshiense DK69T is a Gram-negative, aerobic, rod-shaped, non-motile and non-flagellated bacterium that belongs to the family Flavobacteriaceae in the phylum Bacteroidetes. The high quality draft genome of strain DK69T was obtained and has a 3,375,260 bp genome size with a G + C content of 37.7 mol % and 2848 protein coding genes. In addition, we sequenced five more genomes of Flavobacterium type strains and performed a comparative genomic analysis among 12 Flavobacterium genomes. The results show some specific genes within the fish pathogenic Flavobacterium strains which provide information for further analysis the pathogenicity. Flavobacterium enshiense DK69T (= CCTCC AB2011144T = TKCTC 23775) is a type strain that belongs to the genus Flavobacterium of the family Flavobacteriaceae [Flavobacterium were identified and widely distributed in soil, fresh water, marine water, sediment, microbial mat, and glaciers [Flavobacterium strains are fish pathogens including Flavobacterium columnareTATCC 49512 causing columnaris disease [Flavobacterium psychrophilum JIP02/86T causing cold-water disease [Flavobacterium branchiophilum FL-15T causing bacterial gill disease [Flavobacterium strains are Gram-negative, non-spore-forming, yellow-pigmented, rod-shaped, aerobic and with a low DNA G + C content (30–41 mol %) [Flavobacterium strains contained iso-C15:0 as the major fatty acid, phosphatidylethanolamine as the major polar lipid and menaquinone-6 as the major respiratory quinone [The common characters of 1 mol %) –12. The  quinone –12.Flavobacterium species, we sequenced six Flavobacterium strains including F. enshiense DK69T [,Flavobacterium beibuense F44-8T [Flavobacterium cauense R2A-7T [Flavobacterium rivuli WB 3.3-2T [Flavobacterium subsaxonicum WB 4.1-42T [Flavobacterium suncheonense GH29-5T [Flavobacterium genomes in the NCBI, Flavobacterium indicum GPTSA100-9T [Flavobacterium frigoris PS1T [Flavobacterium sp. F52 [Flavobacterium columnareTATCC 49512, Flavobacterium psychrophilum JIP02/86T and Flavobacterium branchiophilum FL-15T. Here, we present the description of the non-contiguous finished genomic sequencing of F. enshiense DK69T and the comparative genome analysis of the 12 Flavobacterium genomes.In order to provide genome information of se DK69T ,Flavobace F44-8T , Flavobae R2A-7T , FlavobaB 3.3-2T , Flavoba 4.1-42T  and FlavSA100-9T , Flavobaris PS1T , Flavoba sp. F52 , FlavobaF. enshiense DK69T is a Gram-negative, strictly aerobic, yellow-pigmented rod shaped bacterium isolated from soil collected at a pharmaceutical company in Enshi, Hubei province, China. The total soil C, N, P, S and Fe concentrations were 39.83, 3.34, 0.68, 0.36, 33.80 g kg−1, respectively, and the pH was 6.97 [T was clustered within a branch containing other species in the genus Flavobacterium . The quantity of DNA was measured by the NanoDrop Spectrophotometer to ensure that the DNA concentration is greater than 20 ng/μl, then 5 μg of DNA was sent to Majorbio  for sequencing.F. enshiense DK69T. TruSeq DNA Sample Preparation Kits are used to prepare DNA libraries with insert sizes of 300–500 bp for single, paired-end, and multiplexed sequencing. The protocol used 1 μg of DNA sheared by either sonication or nebulization [F. enshiense DK69T generated 8,329,997 x 2 reads totaling 1,682,659,394 bp data with an average coverage of 498.4 x. Then SOAPdenovo v1.05 [The Illumina Hiseq2000 with the Paired-End library strategy was used to determine the whole-genome sequence of lization . The gen+. SignalP [The annotation of the genomic sequences was completed using the NCBI Prokaryotic Genome Annotation Pipeline which was combined using Best-placed reference protein set and the gene caller GeneMarkS SignalP  and SOSU SignalP  were use SignalP . The Gen SignalP  and the  SignalP  BLASTP sF. enshiense DK69T was found to have a total length of 3,375,260 bp, a G + C content of 1,273,385 bp (37.7 mol %) and 74 contigs. From a total of 3,054 genes predicted, 2,848 genes are protein-coding genes, 50 are RNA genes, 57.9 % are assigned with putative functions and the remaining are annotated as hypothetical proteins or proteins of unknown functions. The distribution of genes into COGs functional categories is shown in Table The genome statistics are provided in Table F. enshiense DK69T  The genome based phylogenetic analysis results is in agreement with the 16S rRNA gene based one; (2) The genomic data are correlated with some phenotypes of strain DK69T; (3) Compared to the three fish pathogenic Flavobacteriumstrains, no pathogenic related genes was detected in the environmental strain DK69T which indicated its non-pathogenicity; and (4) Some specific genes were found within the three fish pathogenic Flavobacterium strains which provides information for further analysis the pathogenicity.The genomic results of