Patent Application: US-201314423547-A

Abstract:
the present invention relates to a microparticle containing essential palmarosa and lemongrass oils for controlling enteric diseased caused by microorganisms . more specifically , the microparticle according to the present invention can be used for controlling enteric infections caused by pathogenic bacteria in humans and animals , and can be used as an alternative to the administration of antibiotics for that purpose . the microparticle can be used in the veterinary and pharmaceutical fields and can be administered orally .

Description:
the present invention relates to a microparticle comprising the following components : the microparticle may contain the ratios of 1 : 3 , 1 : 1 or 3 : 1 of essential oils of cymbopogon citratus and cymbopogon martinii , preferably 3 : 1 . the essential oils of cymbopogon citratus and cymbopogon martinii can be obtained by hydrodistillation , by steam distillation or other processes that allow obtaining it . the wall material can be selected from the group of arabic gum , maltodextrin , chitosan , cashew tree gum , among others or a mixture thereof , preferably maltodextrin . the microparticle coating can be obtained by any method known in the art , which allows oils micro - encapsulation , for example , by rodrigues ( 2004 ). the emulsifier may be selected from the group of tween 80 , 60 , or 20 , soy lecithin , among others , which is preferably tween 80 . the microparticle has variable size between 100 and 1000 μm and spherical shape . the objective of its application is to prevent infections of the gastrointestinal tract caused by pathogenic bacteria in humans and animals . the microparticles may be administered in its original form or in vehicles pharmaceutically and / or veterinary acceptable in the form of tablets , emulsion , suspension , gel capsule , among others . additionally , the microparticles of the present invention can be mixed with water , food , animal feed or any other for oral administration . for the preparation of microparticles was performed a process known in the art . the wall material was dissolved in distilled water with a stir plate . the oils and a emulsifier agent were added to the preparation and homogenized . after homogenization , the formulation was passed through the mini spray dryer . the operating conditions for microencapsulation were optimized by varying the inlet temperature of 180 ° c .± 5 ° c ., the ratio of oil ( 20 % compared to maltodextrin ) and a total of solids of 30 %. the carrier gas used was n 2 . antimicrobial activity of cymbopogon spp essential oil for e . coli in different seasons the antimicrobial activity of cymbopogon citratus and cymbopogon martinii oils for different e . coli isolates as well as for the standard strain was evaluated by broth microdilution assay , in accordance with the m7 - a6 standard ( clsi , 2003 ). in a sterile 96 - well microplate were deposited 100 μl of luria broth ( e . coli ), whereas the column 12 was used for the control of microorganism and culture medium sterility . to the lane 1 — line a were added 50 μl of essential oil solution of known concentration , and further were added 50 μl of medium , these ones related to the control of the samples sterility . then , 100 μl of the same materials were added in line b , the content of the homogenized wells with medium and transferred to the wells of the next line ( c ), repeating this procedure until the line h , so as to obtain a concentration decreasing of the material . 100 ml of residue were discarded . then , 100 μl of microorganism inoculum , which turbidity was compared to the 0 . 5 mcfarland scale and diluted to a final concentration of 10 4 cells / ml , were added . the plates were sealed with parafilm ® and incubated for 24 hours at 36 ° c . alter the incubation period , 50 μl of a solution of triphenyl tetrazolium chloride — ttc ( 0 . 5 %) were added and the plates were reincubated for 3 h . the mic was defined as the lowest sample concentration , capable of preventing the appearance of the red color , given to the medium when the cells have respiratory activity , that is , when there was growth of the microorganism . it was also included in the assay the gentamicin sulfate antibiotic control , as well as a control to confirm the sterility of the culture medium and the microorganism growth . the activity of c . citratus and c . martinii oils was also evaluated from the mixture thereof ( the present invention ) in different ratios , i . e ., 75 : 25 , 25 : 75 and 50 : 50 ( w / w ). the mic results ( minimum inhibitory concentration — mg / ml ) are shown in table 1 . according to the results we can state that the species c . martinii ( palmarosa ) proved to be the most active against different strains of e . coli isolated from porcines with diarrhea , with wide spectrum of action , with best activities between autumn and winter . however , the mixture of the c . citratus and c . martinii oils in the ratio 25 : 75 ( 1 : 3 ) or 75 : 25 ( 3 : 1 ) incurred even better activities . in vitro cytotoxicity of the cymbopogon citratus and cymbopogon martinii essential oils of the present invention the in vitro cytotoxicity of essential oils ( present invention ) were evaluated against two normal cell lines ( vero — african green monkey kidney epithelial cells and hacat — immortal human keratinocytes ) and against a panel of human tumor cells . based on the results , growth rate graphics from the sample concentration for each of the tested strain were generated . three effective concentrations called gi 50 ( growth inhibition , the concentration required to disrupt 50 % cell growth ), tgi ( total growth inhibition , the concentration required for 0 % of cell growth ) and lc 50 ( lethal concentration , concentration required for 50 % cell death ) could be calculated by nonlinear regression , sigmoidal , using 8 . 0 origin software . the test results obtained are shown in fig1 ( present invention ) and table 2 . in this model , one way to infer the growth rate is by spectrophotometric reading of the absorbance of cellular proteins stained with sulforhodamine b ( srb ), an anionic dye staining bright pink . this dye can bind to protein terminations of basic amino acids in living cells fixed with trichloroacetic acid ( tca ), thus being an independent assay of cell metabolism and allowing a sensitive measurement of protein linearly with the number of cells in culture . the srb assay is considerably fast , simple and shows sensitivity comparable to those fluorescent assays and upper to the assays that use visible dyes , even at low cell concentrations ( 1000 to 2500 cells per chamber ) ( skehan et al , 1990 ). * mean tgi : arithmetic mean of the values for tgi tumor lines . parameters for evaluation of activity on tumor cell lines : inactive ( mean tgi & gt ; 50 μg / ml ), weak activity ( 15 μg / ml & lt ; mean tgi & lt ; 50 μg / ml ), moderate activity ( 6 . 25 μg / ml & lt ; mean tgi & lt ; 15 μg / ml ) and potent activity ( mean tgi & lt ; 6 . 25 μg / ml ) ( fouche et al . 2008 ) this test allows us to evaluate the antitumor activity through exposure of human tumor cells in exponential growth phase at different concentrations of the sample and whether that exposure led to an interruption in the growth rate without cell death ( cytotoxic activity ) or caused the death cell ( cytocidal activity ). to measure the activity , an effective concentration called tgi ( total growth inhibition , concentration required for 0 % cell growth occurs ) ( shoemaker , 2006 ) was calculated . chemotherapy doxorubicin was used as positive control . in addition to a standard of comparison , the main purpose of using this control was to verify that all employed cell lines kept the profile of response to chemotherapy . this is because with successive passages necessary for the maintenance of cell culture , there is the possibility of changing the lineage in culture , and this mutation could be detected by the change in response against doxorubicin . furthermore , in order to minimize the occurrence of mutations , the cell lines are perpetuated only by up to 20 passages , after which they are replaced by new cells of the same strain that were maintained frozen . with respect to the normal strains , the present invention exhibited antiproliferative activity of weak to moderate on the vero line ( tgi 84 . 2 μg / ml ) for c . martinii and c . citratus ( table 2 ), suggesting a small cytotoxic action on kidney cells . however , the analysis of the in vivo tests showed that the in vitro effect did not translate into toxicity in animals evaluated . otherwise , the present invention did not interfere with the human keratinocytes cell line growth ( hacat ), with tgi values & gt ; 250 μg / ml at the maximum tested concentration ( table 2 ). as the action on human tumor cell lines , the present invention was tested inactive , since the tgi was above the limit set for activity ( 50 μg / ml ). the object of the present invention , the microparticle of 3 : 1 cymbopogon citratus and cymbopogon martinii essential oils was administered in a single dose of 5000 mg / kg to five animals , namely : pet 2 . 1 ( preliminary test ) and animals 2 . 2 to 2 . 5 ( main test ). five additional animals ( identified as 3 . 1 to 3 . 5 ) did not received any oral treatment , being considered as a second control group (“ satellite ”) of the experiment . twenty - four hours after administration of the present invention oral dose of 5000 mg / kg , piloerection was observed in all experimental animals , the appearance being normalized by 48 hours after start of the test . in the subsequent days , the individual clinical assessment of experimental animals , including appearance ( general condition of the animal , and skin and fur changes ), body weight evolution , clinical signs ( heart and respiratory rate ), spontaneous behavior ( hyperactivity / lethargy ) and response to stimuli ( changing reflections ) showed no changes in behavior and mood , motor activity , muscle tone , reflexes , central and autonomic nervous system activity . throughout the trial period , it was possible to smell the characteristic odor of the essential oils exhaled by animals . at the end of the trial , at the time of euthanasia , all the animals were healthy . table 3 shows the corresponding values of the body weight evolution per animal during the experimental period of the animals treated with the present invention and the animals receiving no treatment (“ satellite ”). fig2 shows the animal body weight gain , taking into account the difference of the individual weights at the beginning and the end of the acute toxicity test . when compared with the body weight gain data of wistar / uni mice , males , kept in the same environmental conditions and fed with the same feed (“ satellite ” group — fig3 ), the experimental animals showed weight gain compatible with normality . the statistical analysis ( duncan test ) in weight gain between the group treated with the present invention and the “ satellite ” control revealed no significant difference , suggesting the non - toxicity of the present invention . at the end of the experimental period ( 14 days ), necropsy of all the animals and histopathological analysis was performed and revealed no evidence suggestive of toxicity . using the ghs chemicals classification system , the microparticles of the present invention were classified as nontoxic after a single dose ( ld 50 greater than 5000 mg / kg ). according to the results of the acute oral toxicity test in rodents , the present invention showed no systemic toxic effects that could be shown in adult wistar / uni rats after single oral administration at a dose of 5000 mg / kg . following the ghs (“ globally harmonized system of classification and labelling of chemicals ”) classification for acute toxicity tests , the microparticles of the present invention were classified as nontoxic after a single dose ( ld 50 greater than 5000 mg / kg ). experimental animals treated with the present invention with a single dose of 5000 mg / kg showed no systemic toxic effects that could be shown , with a body weight gain consistent with the same age (“ satellite ” group ). the uniformity in weight gain between the experimental groups was statistically analyzed by duncan &# 39 ; s test ( p & gt ; 0 . 05 ), corroborates this assertion . after euthanasia , no macroscopic changes suggestive of toxicity in vital organs were detected . performance evaluation and score of diarrhea in nursery phase in piglets fed with the present invention in the preferred embodiment the present invention ( ratio 3 : 1 ) was evaluated on performance and score of diarrhea in piglets in the nursery phase ( treatment cb006 - s ). for this evaluation , 72 piglets of both sexes with starting age of 28 days and terminal of 63 days . 4 piglets were housed in each pen ( two males and two females ) which formed an experimental unit . the analyzed variables were : animal performance , diarrhea score , and percentage of piglets delivered . the study periods were : i — from 28 to 35 ; ii — from 28 to 49 ; and iii from 28 to 63 days old . the treatments were : 1 )— negative control — basal diet , 2 )— antibiotic — basal diet plus growth promoter msd 08 63 g / ton of feed ( 5 ppm ), 3 )— test — basal diet plus cb006 - s 2 . 000 g / ton of feed . the performance results of the piglets during the first period ( 8 to 35 days ) are shown in table 4 for different groups . table 5 initial weight , final weight , average daily gain ( gmdp ), average daily feed intake ( cmdr ) and feed conversion ( ca ) averages of piglets for period 2 of the 28 to 49 days old . initial final gmdp cmdr ca 28 group weight weight 28 to 49 28 to 49 to 49 1 7 . 56 13 . 31b 0 . 274b 0 . 371b 1 . 94a 2 7 . 54 15 . 12a 0 . 360a 0 . 539a 1 . 51a 3 7 . 48 14 . 16ab 0 . 318ab 0 . 537a 1 . 74a cv % 2 . 64 9 . 47 19 . 64 14 . 81 6 . 28 averages followed by the same letter in the columns do not differ by tukey test ( p & gt ; 0 . 05 ). performance results for the gmdp and final weight showed significant difference ( p & gt ; 0 . 05 ) in favor of group 2 , even though group 3 was not statistically different from groups 1 and 2 ; however , the cmdr was significantly lower for group 1 when compared to the other groups , and between groups 2 and 3 there was no significant difference . there was no significant difference about feed conversion between the tested groups . the performance results of the piglets during period 3 ( from 28 to 63 days ) are shown in table 6 for the different groups . the results showed that the final weight was higher in group 3 , while group 2 did not differ statistically from groups 1 and 3 ; the same results were observed for gmdp . for cmdr , groups 2 and 3 did not differ statistically between themselves , but were higher than group 1 . for feed conversion parameter there was no difference between groups in this period . there was no statistical difference between the groups in relation with the diarrhea incidence and the percentage of piglets delivered to units of growth and termination ( table 7 ) was not influenced ( p & lt ; 0 , 05 ) by experimental treatments , noting that in all groups the percentage of delivered animals was within the desired standards ( 80 - 85 %) for a pig farming system adopted by agribusinesses . in the experimental conditions , the additive cb006 - s showed similar results to antibiotic msd08 and superior to control in piglets &# 39 ; performance , in the period of 28 - 63 days of age showing that it can be used in diets for weanling piglets as possible substitute to antibiotics used in this phase . no animal showed signs of intoxication after ingestion of the tested product . hur , j . ; lee , j . h . comparative evaluation of a vaccine candidate expressing enterotoxigenico escherichia coli ( etec ) adhesins for collibacilosis with a commercial vaccine using a pig model . vaccine , v . 30 , p . 3829 - 3833 , 2012 . wu , s . ; zhang , f . ; huang , z . ; liu , h . ; xie , c . ; zhang , j . ; thacker , p . a . ; qiao , s . effects of the antimicrobial peptide cecropin ad on performance and intestinal health in weaned piglets challenged with escherichia coli . peptides , v . 35 , p . 225 - 230 , 2012 . hayat , a . m . ; tribble , dr . ; sanders , j . w . ; faix , d . j . ; shiau , d . ; armstrong , a . w . ; riddle , m . s . knowledge , attitudes , and 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