inflammation is an important signal for human body , which indicates that something is wrong . when body tissues are damaged regardless it is due to biological , physical or chemical factors , macrophages around the damaged tissues will be activated to eliminate the foreign objects . at the same time , they will also release some factors to activate other immuno - defense systems , such as , nitric oxide , tumor necrosis factor , interleukin , granulocyte - monocyte colony - stimulating factor , granulocyte colony - stimulating factor , and monocyte colony - stimulating factor . the concentration of the above factors was elevated in inflamed tissue . in 1975 carswell and colleague reported that by injecting mice with bacterial lps , a tumor - killing factor could be detected in the serum , and they called it tumor necrosis factor ( tnf ). later shalaby ( 1985 ) named the tnf produced by macrophage tnf - α , and the lymphotoxin that produced by t lymphocyte as tnf - β . tnf - α is produced by monocyte and macrophage , and bacterial lps served as a strong stimulant . bacterial endotoxin frequently caused severe ailment in digestive system , and bacterial lps is a major endotoxin . animal studies indicate that lps will delay gastric emptying and it is related to the induction of the immune responses . when body was stimulated by lps , it would induce the formation of tnf - α , il - 1β , il - 6 , and other factors to participate defense and repair process . however , too much these factors will also cause undesirable effect on our body , for example , too much tnf - α will cause organ prostration , toxic shock or even death . it has been shown that the addition of tnf - α antibody can effectively prevent the onset of detrimental shock syndrome caused by endotoxin . researches indicated that the formation of tnf - α , il - 1β , and il - 6 were related . lps will induce the synthesis of tnf - α which is then induced the formation of il - 1β , and then the later will induce the formation of il - 6 . metabolism of arachidonic acid by the type - 2 cyclooxygenase ( cox - 2 ) pathway produces prostaglandins and thromboxanes . different kinds of prostaglandin are produced by different type of cell . for example , monocytes and marcophages produce large quantities of pge2 and pgf2 ; neutrophils produce moderate amounts of pge2 ; mast cells produce pgd2 . prostaglandins have diverse physiological effects , including increased vascular permeability , increased vascular dilation , and induction of neutrophil chemotaxis associated with inflammation responses . from the inhibitive activities on the release of tnf - α , il - 1β , il - 6 and prostaglandin and the expression of cox - 2 as described in following examples , it is demonstrated that the present bromelain preparation exhibits a significantly improved effct in anti - inflammation . the other aspects and features of the invention will become apparent in the descriptions of following examples . these examples are given for illustration of the invention and are not intended to be limiting thereof . starch and oragnic acid were mixed at 2 : 1 by weight , the mixture was heated at 80 ° c . for 5 min with stirring . the stirred mixture was irradiated with electron beam of 15 kgy for 3 seconds to form an organic network polymer . the mixture of organic network polymer was neutralized with appropriate amount of nahco 3 to about ph 7 . the neutralized mixture was added same weight of bromelain at 30 ° c . then stirred for 3 min . the mixture was subjected the second irradiation with electron beam of 15 kgy for 3 seconds to embed bromelain into the organic network polymer and obtained the bromelain preparation . the bromelain preparation was tested for residual activity in acidic envirronment of ph 3 , 4 , 5 , 6 , and 7 at 30 ° c . for 2 hours . the remaining activities at ph 3 , 4 , 5 , 6 and 7 were 82 %, 86 %, 92 %, 95 %, and 96 %, compared to the residual activities of uncoated bromelain of less than 50 % after treated at ph 3 and 4 at 30 ° c . for 2 hours . male sd rats , weight about 250 g each , were purchased from biolasco taiwan co ., ltd . rats were kept under 23 ° c ., with 12 hours day / night cycle , and fed with regular diet . drinking water was pre - treated with reverse osmotic technique . five sets of rats were treated differently , and each set contain 10 rats . in the controls set , rats were held under regular diet through the experiment . in lipopolysaccharide ( lps ) treated set , rats were held under regular diet and then escherichia coli lps ( 2 . 5 mg / kg ) was injected into the abdominal cavity of each rat . in the remaining 3 sets of rat , before the injection of lps , their diets were supplemented with different rates of the improved bromelain preparation ( 10 mg / kg , 50 mg / kg or 100 mg / kg ) for 7 days . after lps injection , rats were kept on a 24h - fast . blood were then drawn from celiac vein of each rat before and after stress treatment for immunological study . the data were analyzed using one - way analysis of variance ( anova ). the concentrations of interleukin - 1 ( il - 1 ), interleukin - 6 ( il - 6 ) and tumor necrosis factor - α ( tnf - α ) were assayed using elisa . as shown in fig1 to 3 , feeding of the improved bromelain preparation could effectively reduced the serum contain of tnf - α , il - 1β , and il - 6 when the rats were challenged with bacterial lps . the reduction of serum tnf - α , il - 1β , and il - 6 concentration by the improved bromelain preparation is dose - dependent . in the serum immunoassay of il - 1β ( fig1 ), feeding of low dose ( 10 mg / kg ) of the improved bromelain preparation reduced il - 1β content in serum from lps - challenged rats by 31 %, comparing to the lps group . similarly at low dose , the concentration of serum il - 6 and tnf - α were reduced by 41 % and 40 % respectively , comparing to the lps group ( see fig2 and 3 ). feeding of medium and high dose ( 50 and 100 mg / kg ) of the improved bromelain preparation exhibited significant anti - inflammation effects in treated animals , as shown in the following table 1 . we used microglia bv - 2 cells as model to investigate the cytotoxicity and inhibitive effects in prostaglandin pge 2 release and cox - 2 expression of the improved bromelain preparation . bv - 2 cell line was maintained in deme supplemented with 10 % fbs and antibiotics at 37 ° c . under 5 % co 2 . confluant cultures were passed by trypsinization . for experiments , cells were washed twice with warm dmem ( without phenol red ), then treated in serum - free medium . in all experiments , cells were treated with the bromelain preparation in 1 × pbs ( phosphate - buffered saline ). cytotoxicity was determined by measuring the release of lactate dehydrogenase ( ldh ). bv - 2 cells were preincubated in 24 - well plates at a density of 5 × 10 5 cells per well for 24 hours , then washed with phosphate - buffered saline ( pbs ). bv - 2 cells with various concentration of the bromelain preparation were treated with lps for 24 hours and the supernatant was used to assay ldh activity . the reaction was initiated by mixing 0 . 1 ml of cell free supernatant with potassium phosphate buffer containing nadh and sodium pyruvate in a final volume of 0 . 2 ml to 96 - well plate . the rate of absorbance value was read at 490 / 630 nm on an automated spectramax 340 microtiter plate reader . data were expressed as the mean percent viable cell vs . lps control . the result was shown in fig4 . from the result of ldh release assay , it was demonstrated that the present bromelain preparation has no significant cytotoxicity to bv - 2 cells , and meams that it is harmless to bv - 2 cells . prostaglandin pge 2 release by lps - stimulated bv - 2 cell with various concentrations of the bromelain preparation plus 1 μg / ml lps after 24 hours treatment was measured by elisa immunoassay kit ( r & amp ; d system , minneapolis , usa ). the linear range of the assay was from 10 to 1000 μg / ml . bv - 2 cell suspensions were diluted or concentrated to achieve values that fall within the linear ranges of the assays . the pge 2 values were read at 450 / 570 nm on an automated spectramax 340 microtiter plate reader . data were expressed as the mean percent viable cell compared to the control . as shown in fig5 , treatment with 10 μg / ml of the bromelain preparation reduced the release of pge 2 by lps - stimulated bv - 2 cells by 27 %, compared to the lps control group . treatments with 50 μg / ml and 100 μg / ml of the bromelain preparation significantly reduced the release of pge 2 by lps - stimulated bv - 2 cells by 50 % and 80 % respectively , compared to the lps control group . for detecting the effects of the bromelain preparation on cyclooxgenase - 2 ( cox - 2 ) activity and expression in lps - stimulated bv - 2 cells , total rna was purified from bv - 2 cell with various concentrations of the bromelain preparation plus 1 μg / ml lps after 4 hours incubation and using trizol ( gibco brl ) following the protocol recommended by the manufacturer . cox - 2 expression ( real - time rt - pcr ) was analysed by real time quantitative rt - pcr assay . total rna ( 0 . 5 μg ) was reverse transcribed with random primers with m - mlv reverse transcriptase , in the presence of rnase out ™ ( invitroben , usa ). one hundred nanograms reverse transcribed rna was primed with specific oligonucleotides for cox - 2 : pcr was accomplished with abi prism 7000 deetection system ( applied biosystems , usa ). the pcr product was visualized by electrophoresis in 2 % agarose gel , staining with ethidium bromide . verification of specific genes was established by their predicted size under uv light . the result was shown in fig6 . it suggested that the bromelain preparation supressed the expression of cox - 2 and further inhibited the biosynthesis and release of prostaglandin and inflammation - related cytokines such as il - 1 , il - 6 , tnf - α and the like , acting as one kind of nsaid ( nonsteroidal anti - inflammatory drug ). following the prostaglandin pge 2 release assay described in example 2 , the anti - inflammation effect of the bromelain preparation was compared with seven marketing products at same dosage . as shown in fig7 , the bromelain preparation of the invention ( cometrue ) reduced the pge 2 release by 50 %. comparing to the current marketing bromelain products , only product b and product e exhibited the reducing effects in pge 2 release by 44 % and 18 % respectively , and the other product showed no such inhibition effects in pge 2 release by lps - stimulated bv - 2 cells . the present bromelain preparation indeed exhibits improved anti - inflammation effects than the prior marketing bromelain products .
the present invention relates to improved preparation of bromelain in which the bromelain is coated with an organic network polymer constructed by cross - linkage between organic acids and polysaccharides . the present invention also relates to a pharmaceutical composition comprising the present bromelain preparation for treating inflammation , alleviating pains , and / or enhancing immuno - defense in a subject who needs such treatment .