Patent Application: US-59874408-A

Abstract:
the present invention relates to butenolide compounds having cytoprotection such as antioxidant , anti - inflammatory and / or antifungal properties , and which are derived from the marine fungus aureobasidium .

Description:
the following examples are given by way of illustration of the present invention and should not be considered to limit the scope of the present invention . the optimized protocol for the production of the gamma - alkyl butenolides involves culturing the marine aureobasidium sp . strain , aqp1639 in a carbohydrate enriched media ( e . g . 24 g potato dextrose medium in 1 l natural seawater ) prepared with natural seawater . production of the gamma - alkyl butenolides by aqp1639 also requires agitated planktonic suspension cultivation with adequate oxygen supply , and alkalisation of the ethyl acetate extract from crude natural product . the yield of the gamma - alkyl butenolides , 5 - hexylfuran - 2 ( 5h )- one ( named p1639c ; fig1 ), produced under non - optimised culture conditions is approximately 10 ˜ 15 mg / l . aqp1639 was pre - cultured in potato dextrose agar ( pda , oxoid ) prepared with natural sea water . the growth medium was autoclaved at 121 ° c . for 15 min before plates were made . aqp1639 was inoculated on a pda seawater plate and cultivated for two to three weeks until darkly pigmented arthroconidia became obvious . the pre - grown colonies were used to inoculate potato dextrose broth ( pdb , oxoid ) which was also prepared using natural seawater from the same source , followed by shaken flask cultivation at 30 ° c . at a speed of 220 rpm for 20 days . the cultivation was carried out until a visible black biofilm was established at the air / liquid interface on the flask wall , which usually took three to four weeks . dark oil could be seen at air / liquid interface at this stage . the ethyl acetate extract of the culture supernatant , which showed antimicrobial activity , was dried and then alkalinised using 0 . 5m naoh water solution in room temperature ( 20 ° c .˜ 24 ° c .) until the oil - like material dissolved in water completely . meoh was then added to the alkalinised solution and mixed thoroughly , followed by hexane extraction . the hexane extract was then evaporated down completely and again reconstituted in 2 ml hexane . the hexane reconstitute was then loaded to silica sep - pak ® cartridge to carry out antifungal activity guided fractionation using 100 % hexane , 90 % hexane : 10 % etoac and 80 % hexane : 20 % etoac . the active fractions were pooled and reconstituted in etoac , then further purified using c18 - hplc and isocratic 70 % meoh : 30 % h 2 o . the active fractions were pooled and the structure was characterised using nmr spectroscopy and mass spectrometry . p1639c showed a lrms of m / z 191 . 2 ( m + na ) + . careful analysis of the nmr data ( table 2 ) suggested a molecular formula of c 9 h 14 o 2 . with an unsaturation number of three suggested the presence of one ring in the system . correlations from cosy and hmbc ( fig3 ) yielded a known butenolide - type , 5 - hexylfuran - 2 ( 5h )- one ( mukku , 2000 ) compound . this compound produced a pleasant fragrance similar to coconut - oil , and is an analogue of the known 2 , 6 - dimethyl - 5 - oxo - heptanoic acid , a widely used flavour in food stuffs , alcoholic beverages , perfumery and pharmaceutical products ( sinha , 2004 ). optical rotation measurements was recorded using a perkin elmer , model 343 polarimeter at 589 nm . an antioxidant assay was carried out using an antioxidant reporter cell line to determine if p1639c upregulated the protective anti - oxidant gene battery which is under control of the anti - oxidant response element ( are ). the cell line are32 was incubated with eight concentrations of p1639c and the positive control tert - butylhydroquinone ( tbhq ) for 24 hours , and the luciferase activity measured ( luciferase assay system , promega ). p1639c enhanced induction of are - driven luciferase activity up to 18 - fold ( at a concentration of 30 μm ) ( fig2 b ) compared with normal cells without any oxidative induction . thus , the butenolide p1639c , produced by aqp1639 , showed potent antioxidant elicitation activity . the are comparison was also carried out between p1639c ( 5 - hexyl - 2 ( 5h )- furanone ), 4 - decanolide ( 5 - hexyl - 2 ( 3h )- furanone ) and 2 ( 5h )- furanone . as can be seen in fig8 compound p1639c showed an elicitation of are - driven gene expression up to 18 - fold by treatment with 30 μm of p1639c . however , 4 - decanolide or 2 ( 5h )- furanone did not show significant anti - oxidant elicitation effects using are - driven gene expression methods . the basis of the assay is to assess the ability of different concentrations ( 100 - 2000 μm ) of a test compound to compete with a standard concentration of spin trap ( tempone - h ; 50 μm ) for hydroxyl or superoxide radicals . to our knowledge , this is the first assay that descriminates between scavenging capabilities for different oxygen - centred radicals . in the absence of antioxidant , tempone - h reacts with oxygen - centred radicals to generate a spin signal at a rate that is determined by the concentration of the radical generating compound ( menadione for . oh and pyrogallol for superoxide ). inclusion of an antioxidant with an equivalent rate constant for reaction with either oh or superoxide at the same concentration as tempone - h ( 50 μm ) will effectively compete for radicals and will diminish the signal at a given timepoint by ˜ 50 %. by monitoring the effect of a variety of different concentrations of test compound against a set concentration of tempone - h , a concentration - effect curve can be established , from which an ic 50 ( concentration required to reduce the signal by 50 % of control ) can be derived for each test compound . test compounds can then be compared to a known agent ( in this case , ascorbic acid ) and can be ranked according to scavenging capabilities for each of the radicals in question (. oh and o 2 − ). the lower the ic 50 , the more potent the antioxidant . incubation ( 60 min , 37 ° c .) of tempone - h ( 50 μm ) with menadione ( 150 μm ; oh generator ) or pyrogallol ( 150 μm o 2 − generator ) caused a time - dependent increase in epr signal ( fig1 a and b ). inclusion of ascorbic acid ( 50 - 300 μm ) in the incubation mixture caused a concentration - dependent reduction in signal development ( fig1 ); 50 % reduction in area under the curve was calculated to be ˜ 70 μm ( ic 50 ) for . oh and 130 μm for o 2 − . equivalent experiments with test compound p1639c caused a less dramatic reduction in epr signal ( with a calculated ic 50 of ˜ 900 μm for oh experiments and ˜ 725 μm for o 2 − . compound p1639c has direct antioxidant effects against both . oh and o 2 − ( to similar degrees ) the potency of this agent is lower than that of ascorbic acid against both radical species (˜ 1 order of magnitude ). candida albicans and malassezia furfur were used to carry out the antifungal assay in potato dextrose agar ( pda ) media containing 0 . 2 % yeast extract . the minimum inhibitory concentration ( mic ) was recorded ( table 1 ). p1639c showed antifungal activity against c . albicans at an mic of 1 - 1 . 5 μg / ml and against m . furfur at 0 . 8 μg / ml . the comparison of antifungal activity using various butenolide compounds suggested that the length of the gamma side chain has significant effect on the anti - fungal activity . results indicated that the longer the side chain , the better antifungal activity it possesses . however , considering the water solubility of the lactone compounds , a gamma side chain with carbon number between c5 and c8 is preferable . in regard to the antifungal activity , the double bond between c2 and c3 or c3 and c4 did not show significant effect on the activity . anti - inflammatory assay was carried out based on the nfκb expression and ikkβ activity . nfκb expression was tested using princess ® nina nfκb assay kit . p1639c and other related lactone compounds were supplemented to a mammalian cell culture in which nfκb has been stimulated by tnf - α . the assay was also coupled with an in vitro cell toxicity assay . ikkβ was tested using 5 - 20 mu of ikkβ , which was diluted in 50 mm tris ( ph 7 . 5 ), 0 . 1 mm egta , 1 mg / ml bsa , 0 . 1 %, b - mercaptoethanol . the kinase is assayed against substrate peptide ( lddrhdsgldsmkdeey ) in a final volume of 25 . 5 μl containing 50 mm tris ( ph 7 . 5 ), 0 . 1 mm egta , 0 . 1 %, b - mercaptoethanol , 300 μm substrate peptide , 10 mm magnesium acetate and 0 . 005 mm [ 33p - g - atp ] ( 500 - 1000 cpm / pmole ) and incubated for 30 mins at room temperature . assays are stopped by addition of 50 of 0 . 5m ( 3 %) orthophosphoric acid . the phosphorylated peptide was harvested on a p81 filterplate and the phosphorylation level was measured by scintillation counts . p1639c was supplemented to the mammalian cell culture with a series of concentration at 0 . 001 mm , 0 . 002 mm , 0 . 005 mm , 0 . 01 mm , 0 . 02 mm , 0 . 05 mm , 0 . 1 mm , 0 . 2 mm and 0 . 5 mm . seap provided in the princess nina kit was used as a stimulatory control to show normal inflammatory response in cells , whereas bay11 - 7082 at a final concentration of 5 μm was used as the positive control of inhibited nf - κb activity . the cell with tnf - α was used as the negative control . the cell response to the supplemented compounds was measured by the 450 nm emission ( 360 nm excitation ) after addition of inactivation buffer and mup solution . cell viability was carried out by incubation of the cells with resazurin and measuring the absorption at 600 nm against a reference measurement of above 650 nm . lactone compounds sharing similar structure with p1639c were also examined for the anti - inflammatory activity . the comparison of single and double bonds with length of side chains were made between these lactones ( fig7 a & amp ; b ). in addition , ikkβ phosphorylation activity decreased to 64 %± 2 % when 50 μm p1639c was present supporting the anti - inflammatory activity of p1639c . the toxicology assay was carried out using an in vitro human hepatocytes model where human hepatocytes were exposed to varying concentrations of compound p1639c as well as tamoxifen and chlorpromazine for three hours . the depletion rate of intracellular atp was used as the parameter to measure the level of toxicity observed by each of the compounds . both tamoxifen and chlorpromazine elicited a moderate decreasing rate of intracellular atp level , which suggested a moderate level of toxicity . however , significant atp depletion was not observed in human hepatocytes after exposure to compound p1639c , which suggested that the compound has low toxicity in human hepatocytes ( fig6 a - c ). thus the applicant has found that in particular , the alkalinisation of the ethyl acetate extract from the aqp1639 culture supernatant generates a series of gamma - alkyl butenolides , in particular , a potent antifungal compound 5 - hexylfuran - 2 ( 5h )- one , which also possesses an intense coconut fragrance , and strong antioxidant effects with low toxicity in human heptocyte toxicological models . braun , d ., n . pauli , et al . 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