Patent Application: US-84517107-A

Abstract:
the present invention provides a nanomicell for a skin , and the nanomicell includes an oil substance , an extract of a angelica radix , an extract of a lithospermum radix , and a phospholipid layer . the extract of a angelica radix is formed by extracting the angelica radix with the oil substance , and the extract of a lithospermum radix is formed by extracting the lithospermum radix with the oil substance . the extract of the angelica radix and the extract of the lithospermum radix are packaged within the phospholipid layer to form a plurality of micells having a diameter of nano - level .

Description:
the invention is described more specifically with reference to the following embodiments . it is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for the purpose of illustration and description only ; it is not intended to be exhaustive or to be limited to the precise form disclosed . the lithospermum radix used in the following experiments is arnebia euchroma , and the angelica radix is the dry roots of angelica sinensis ( oliv .) diels . the preparing method of different shiunko nanomicells will be introduced first , and then the creativeness of the shiunko nanomicells will be showed by different function testing experiments . the materials in s group include : lithospermum radix 90 gm , angelica radix 90 gm , and sesame oil 300 gm . the materials in group 0 includes : lithospermum radix 90 gm , angelica radix 90 gm , and olive oil 300 gm , and the materials in group m includes : lithospermum radix 90 gm , angelica radix 90 gm , and mineral oil 300 gm . the preparing method of shiunkos include steps of : first , purifying the angelica radix by eliminating the impurities , and then soaking the angelica radix in different oil substances as listed above . after 24 hours , the oil substances containing angelica radix are heated to a temperature between 130 ° c . to 140 ° c . until angelica radix has a scorched surface , and then adding lithospermum radix to the oil substances and keeping in the same temperature for 15 minutes . second , when the oil substances having a red color , heating is stopped , and then angelica radix and lithospermum radix are removed from the respective oil substances . finally , the oil substance and the extract of angelica radix and lithospermum radix therein are filtered with 4 layers of sterile swabs respectively , and then the respective filtrates are stored in different glass bottles . the materials include : the shiuriko oils sampled from the group s , the group o and the group m respectively , and further includes glycerin , phospholipids and pure water . the steps of the preparing methods include : first , adding the phospholipids into the samples s , o , and m respectively , and stirring uniformly to get respective mixtures . second , the respective mixtures above are added into the glycerin respectively and stirred , and then the pure water is also added into . after high speed stirred in homogenizer ( 6000 rpm , 3 minutes ), the respective mixtures are homogenized in high pressure homogenizer , and filtered with 0 . 1 g / m filter membrane . finally , the condensed solutions of a sesame shiunko nanomicell ( group s ), olive shiunko nanomicell ( group 0 ), and mineral shiunko nanomicell ( group m ) having a red color are collected respectively . the shiunko nanomicell gels could be made by mixing 100 ml of respective shiunko nanomicells mentioned above with 100 gm of transparent gel ( lubrajel dv ), and then stored in bottles . drops of sesame shiuriko nanomicell ( ssn ), olive shiunko nanomicell ( osn ), and mineral shiuriko nanomicell ( msn ) are sampled respectively , and diluted in 100 ml of pure water to form diluents respectively . after stirring homogeneously , drops of the respective diluents are sampled and analyzed by a particle size analyzer ( beckman coulter ™ n5 ). the results are showed in table 1 . please also refer to fig1 , which is a polygon illustrating the variation in particle size of the different nanomicells after different storage times according to the embodiment of the present invention . as fig1 shows , ssn , osn and msn all have a particle size below 100 nm , which are stable even stored under the room temperature for 150 days . the bacteria tested in the present invention include pseudomonas aeruginosa , escherichia coli , enterococcus faecalis , proteus mirabilis and acinetobacter baumannii , which are usually discovered in wounds of scalds and burns . the bacteria tested in the present invention are showed in table 2 . 10 μl of respective bacteria fluids of above - mentioned bacteria , of which the total number of bacteria are 10 4 to 10 5 , are sampled and mixed with 1 ml of tryptic soy broth containing no drugs ( a ), silver sulfadiazine 1 % cream ( b ), traditional shiunko ( c ), ssn , osn , and msn respectively , and stirred homogeneously in respective bottles . after 0 minute , 8 hours and 24 hours of culture , 10 μl of culture medium are sampled from the respective bottles , and plated on horse blood agar to subculture in 35 ° c . for 24 hours . the growing conditions are recorded and the colony forming units are calculated . the calculated results of respective bacteria are showed in table 3 to table 7 . please also refer to fig2 ( a )( b ), which are diagrams showing the bacterial culture results of pseudomonas aeruginosa after 0 , 8 , and 24 hours of culture with no drugs ( control group )( a ), silver sulfadiazine 1 % cream ( b ), traditional shiunko ( c ), ssn ( d ), osn ( e ) or msn ( f ) respectively according to the embodiment of the present invention . accordingly , the nanomicells provided according to the preferred embodiment of the present invention are actually have the ability in inhibition of pseudomonas aeruginosa growing , while pseudomonas aeruginosa is the commonest bacteria existed in wounds of burns and scalds and can easily induce bacteremia that has a high fatality rate . the detail results are introduced as follows : the calculating results of colony forming units of pseudomonas aeruginosa are showed in table 3 . as the table shows , first , in silver sulfadiazine 1 % cream treating group , after 0 , 8 , and 24 hours of culture , the colony forming units are more than 10 5 , 0 , and 0 respectively , which shows the silver sulfadiazine 1 % cream has a great ability in inhibition of pseudomonas aeruginosa . second , in the traditional shiunko treating group , the colony forming units calculated show that the traditional shiunko has no ability in inhibition of pseudomonas aeruginosa . third , in the ssn treating group , after 0 , 8 , and 24 hours of culture , the colony forming units are more than 10 5 , 5 and 0 respectively , which shows the ssn has a good ability in inhibition of pseudomonas aeruginosa . fourth , in the osn treating group , after 0 , 8 , and 24 hours of culture , the colony forming units are more than 10 5 , 10 and 0 respectively , which shows the ssn has a good ability in inhibition of pseudomonas aeruginosa . fifth , in the msn treating group , after 0 , 8 , and 24 hours of culture , the colony forming units are more than 10 5 , 26 and 0 respectively , which shows the ssn has a good ability in inhibition of pseudomonas aeruginosa . the calculating results of colony forming units of escherichia coli are showed in table 4 . as the table shows , first , the silver sulfadiazine 1 % cream has a great ability in inhibition of pseudomonas aeruginosa . second , the traditional shiunko has no ability in inhibition of escherichia coli . third , the ssn , osn , and msn have no ability in inhibition of escherichia coli . the calculating results of colony forming units of enterococcus faecalis are showed in table 5 . as the table shows , first , the silver sulfadiazine 1 % cream has a great ability in inhibition of enterococcus faecalis . second , the traditional shiunko has no ability in inhibition of enterococcus faecalis . third , the ssn , osn , and msn have no ability in inhibition of enterococcus faecalis . the calculating results of colony forming units of proteus mirabilis are showed in table 6 . as the table shows , first , the silver sulfadiazine 1 % cream has a great ability in inhibition of proteus mirabilis . second , the traditional shiuriko has no ability in inhibition of proteus mirabilis . third , the ssn , osn , and msn have no ability in inhibition of proteus mirabilis . the calculating results of colony forming units of acinetobacter baumannii are showed in table 7 . as the table shows , first , the silver sulfadiazine 1 % cream has a great ability in inhibition of acinetobacter baumannii . second , the traditional shiunko has no ability in inhibition of acinetobacter baumannii . third , the ssn , osn , and msn have a bad ability in inhibition of p acinetobacter baumannii . function test iii : evaluation of treating effects in burns and scalds injury the experimental animals are twelve rabbits ( male , new zealand rabbit ), and the experiment is implemented in the lab of experimental animal of kaohsiung medical university . first , the experimental rabbits were washed with non - antibacterials soaps , and the dorsal fur was shaved off to expose the skin . second , the rabbits are anaesthetized with ketamine 40 mg / kg i . m . third , the twelve rabbits are divided into 6 groups ( rabbits a , b are group 1 ; rabbits c , d are group 2 ; rabbits e , f are group 3 ; rabbits g , h are group 4 ; rabbits i , j are group 5 ; rabbits k , l are group 6 ,) and the predetermined injury regions of each rabbits are the same . fourth , after heated in hot water having a temperature of 95 ° c . for 5 minutes , a iron column having a diameter of 2 cm is used to contact the exposed skin to produce 6 wounds respectively . after removing the iron and cooling the injury regions in room temperature for 10 minutes , the injury regions are treated with the different ointments or gels ( the control group , the silver sulfadiazine 1 % cream , the traditional shiunko , the ssn gel , the osn gel , and the msn gel ) clockwise in each group , and finally the transparent dressings ( tegaderm ™) are put on each wounds respectively . fifth , after recovering from anesthetization , the experimental rabbits are raised in normal condition . the ointments ( or gels ) and the dressings are replaced daily , and the wounds are recorded by photograph too . sixth , from the time of 31 days after the injury , the ointments ( or gels ) and the dressings are replaced and the wounds are recorded every two days until all the rabbits heal . seventh , after each treating , the rabbits are forced to wear special neck masks designed for preventing the rabbits licking the wounds . eighth , the rabbits are sacrificed respectively at the time of 5 , 10 , 15 , 20 , 25 and 30 days after the injury , and the skin of the wound is took off by scissors and soaked in 10 % formalin . the tissue pathological variations of the skin are diagnosed under the direction of the pathological medicine specialist . ninth , the 36 wounds of the other six rabbits are treated until healing , and the cutaneous wound regeneration and repair are observed and recorded . the rabbits are sacrificed , and the wound tissues are sampled 3 cm × 3 cm followed by soaking in 10 % fommalin . after dehydration , defatting and embedding , 3 ˜ 6 μm wound tissue sections are prepared by microtome and fixed on slides . after stained with hematoxylin & amp ; eosin , the wound tissue sections are observed by light microscope . further , the tissue pathological variation are evaluated and scored depend on eight histomorphologic features of hyperkeratosis , epidermal hyperplasia , hair follicles , apocrine glands , smooth muscles , fibroplasia , vascular proliferation and collagen orientation respectively ( adam j . singer et al ., 2000 ). the cutaneous wound regeneration and repair are scored by the following standard : 5 points : the scabs have came off , and the wounds have healed , and the healing area is flat ; 4 points : the scabs have came off , and the wounds have healed , and the healing area is raised ; 3 points : the scabs have came off , and the wounds have not healed ; 2 points : the scabs have formed over the wounds , and there is no exudate ; 1 point : the scabs have formed over the wounds partially , and there is exudate sting . the cutaneous wound regeneration and repair are scored at the time of 31 and 37 days after the injury , and the more points scored , the more well healing effects are evaluated . the cutaneous wound regeneration and repair scores are showed in tables 8 and 9 . please also refer to fig3 and 4 , which are diagrams showing the wounds of the experimental rabbits at the time of 31 and 37 days after the burn injury and continuing treatment of no drugs ( a ), silver sulfadiazine 1 % cream ( b ), traditional shiunko ( c ), ssn ( d ), osn ( e ), and msn ( f ) respectively according to the embodiment of the present invention , no matter at 31 or 37 days , the scores of the wounds treated with ssn , osn and msn are better than other groups . further , the cutaneous wound regenerating and repairing abilities of osn and msn are a bit better than ssm based on the scores of the time of 37 days , and osm has a better ability in reducing the scar tissues . on the other hand , the traditional shiunko is prepared based on the wax , which has the disadvantages of hard - to - clean , being painful when replacing the dressings , and being disagreeable to the sight because of the residue having a red - purple color . however , all the disadvantages are overcomed by replacing the dressings with ssn , osn or msn gels . please refer to table 11 , which is a histomorphologic scale of the rabbit i at the time of day 5 after the injury . the scores are from 0 point of worst condition to 10 points of almost normal skin . the wound tissue sections are evaluated at the time of the days 5 , 10 , 15 , 20 , 25 and 30 after the injury , and the final scores are showed in table 10 . please refer to table 10 and also fig5 ( a )-( f ), which are diagrams showing wound tissue sections in lm 20 × after stained with hematoxylin & amp ; eosin at the time of 20 days after the burn injury and successive treatment of no drugs ( a ), silver sulfadiazine 1 % cream ( b ), traditional shiunko ( c ), ssn ( d ), osn ( e ), and msn ( f ) respectively according to the embodiment of the present invention . as the table 10 and the figures show , first , there are five dressings ( control group , silver sulfadiazine 1 % cream , traditional shiunko ointment , ssn gel , msn gel ) having greater scores at the time of day 5 than those at the times of days 10 , 15 , 20 , 25 and 30 . maybe it is because that the blood circulation of the wound tissues has not yet been cut off after burn injury , and hence the most wound tissues still have normal histomorphology . second , based on the total scores , it is clear that the three different nanomicell gels have a better ability in wound repairing and healing than the control group , silver sulfadiazine 1 % cream and traditional shiunko . further , the msn gel has the best ability in wound repairing and healing than the osn and the ssn gels , wherein the osn gel is better than the ssn gel . based on the mention above , the ssn , the osn and the msn provided by the present invention , have a particle size below 100 nm even stored under the room temperature for 150 days , which shows the great stability . further , the shiunko nanomicell also shows the great ability in inhibition of pseudomonas aeruginosa . as to the ability of wound repairing and healing , the shiunko nanomicell obviously has the better ability than the traditional shiunko prepared with wax and the silver sulfadiazine 1 % cream . in addition , the shiunko nanomicell is easy to be cleaned , and will not leave residues , which are all the benefits of the present invention . from the evaluated results of the burn and scald model of rabbits , it shows that the osn and the msn gels have the better ability in wounds healing , however the osn has a better ability in reducing scar . the evaluated results of wound tissue sections shows that the msn has the greatest ability in healing than the osn and ssn gels , and further the osn is better than the ssn . however , no matter msn , osn , or ssn gel , is better than the control group , silver sulfadiazine 1 % cream or traditional shiunko . accordingly , the shiunko nanomicell gels provided by the present invention can be applied in burn and scald wounds to improve the healing and reduce the scar tissue , and further applied in relating skin diseases . additional advantages and modifications will readily occur to those skilled in the art . therefore , the invention in its broader aspects is not limited to the specific details and representative embodiments shown and described herein . accordingly , various modifications may be made without departing from the spirit or scope of the general inventive concept as defined by the appended claims and their equivalents . 1 . adam j . singer , henry c . thode jr ., steve a . mcclain . development of a histomorphologic scale to quantify cutaneous scars after burns . acad emerg med . 2000 , 7 ( 10 ): 1083 - 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