1(1)32-36 CONTACT&:&Sabrina&Resky&Pratiwi&&&&&&&sabrinareskyp7@gmail.com3 ©3International3Journal3of3Applied3Biology3 323 Abstract& Earthworms3 (Lumbricus) rubellus)3 extract3 is3 known3 to3 contain3 bioactive3 protein3 as3 antibacterial3 compounds.One3 of3 the3 disadvantages3 of3 polar3 compounds3is3slow3penetration3into3the3skin3layers3which3can3be3solved3by3 formulating3it3in3the3form3of3ethosomal3drug3delivery3system.3The3aims3of3 this3research3was3to3find3out3information3about3ethanol3concentration3that3 provide3the3highest3entrapment3 efficiency3of3 the3ethosome.3Earthworms3 powder3 was3 macerated3 using3 50%3 ethanol3 for3 33 days.3 The3 extract3 was33 formulated3into3ethosome3with3variation3of3ethanol3concentration3that3are3 20%,330%,340%,3and350%,3respectively.3The3measurement3of3entrapment3 efficiency3 was3 conducted3 by3 measuring3 the3 amount3 of3 active3 protein3 of3 earthworms3 extract3 that3 was3 entrapped3 in3 ethosome3 vesicles3 using3 spectrophotometer3 UVNVisible.3 The3 results3 showed3 that3 the3 entrapment3 efficiency3was3directly3proportional3to3ethanol3concentration3in3formula.33 & ISSN3:32580N24103 eISSN3:32580N21193 3 3 3 A&Study&on&Entrapment&Efficiency&of&Earthworms&(Lumbricus)rubellus)&& Extract&in&the&Ethosomal&Drug&Delivery&System) 3 Sabrina&Resky&Pratiwi,&Emilia&Utomo,&Hardyanti,Nur&Ainiah,&Dini&Rusdayanti&Putri&&&Sartini& 3 Faculty3of3Pharmacy,3Hasanuddin3University,3Makassar,3Indonesia3 3 3 & & & & & & & & & & & & Introduction& Earthworms3(Lumbricus)rubellus)L.)3had3been3used3as3China3traditional3medicine3and3 empirically3used3by3people3to3treat3thypoid3fever3caused3by3Salmonella)thyposa3and3acne3vulgaris3 caused3by3Propionibacterium)acnes3(Immanita,32012;3Sun,32015).3This3treatment3was3supported3 by3previous3studies3which3showed3that3earthworms3has3antibacterial3and3antioxidant3activity3 (Aldarraji,3 2013;3 Mathur,3 2011).3 Earthworm3 extract3 in3 concentration3 7.5%3 could3 inhibit3 the3 growth3 of3 Propionibacterium) acnes) and3 treat3 inflammation3 due3 to3 acne3 (Asmawati,3 2016).3 Earthworm3 L.) rubellus3 was3 made3 in3 a3 solid3 dosage3 form3 proved3 to3 have3 broad3 spectrum3 antimicrobial3against3GramNpositive3bacteria3S.)aureus,3GramNnegative3bacteria3E.)coli,3and3fungi3 C.)albicans3(Damayanti3et)al.,32008).3Besides,3earthworms3also3contained3phenolic3compound3 which3has3antioxidant3activity3(Aldarraji,32013).3 The3disadvantages3of3earthworms3extract3are3having3bad3smell3and3slow3absorption3into3 skin3 layer3 caused3 by3 polar3 bioactive3 compounds.3 Previous3 research3 has3 conducted3 the3 formulation3of3earthworm3extract3in3the3gel3preparation,3but3it3wasnot3able3to3cover3the3smell3 up3(Asmawati,32016).3Another3formula3containing3earthworm3extract,3maltodextrin,3distilled3 &&&&&&&&OPEN&ACCESS& & & &&&&&&&&&&International&Journal&of&Applied&Biology& Keyword3 Ethosome,3 ethanol3concentration,3 entrapment3efficiency,3 Lumbricus)rubellus.3 3 Article&History3 Received3183June320173 Accepted3193August3201733 3 International Journal of Applied Biology is licensed under a Creative Commons Attribution 4.0 International License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 3 International Journal of Applied Biology, p-ISSN : 2580-2410 e-ISSN : 2580-2119. Journal homepage : http://journal.unhas.ac.id/index.php/ijoab International)Journal)of)Applied)Biology,)Vol.)1,)Issue)1,)2017) 3 333 water3 (23 :3 53 :3 50)3 was3 made3 by3 encapsulating3 extract3 in3 maltodextrin3 and3 tested3 for3 its3 antibacterial3activity3(Istiqomah,32012).33 The3 ethosome3 is3 a3 carrier3 of3 a3 kind3 of3 soft3 vesicle3 contained3 phospholipid,3 high3 concentrations3 of3 alcohol,3 and3 water3 (Tiwari,3 20103 ;3 Patel,3 2013).3 Ethosomal3 drug3 delivery3 system3has3many3advantages3such3as3good3delivery3of3protein3molecules,3containing3non3toxic3 materials,3 increasing3drug3permeation3into3skin3 layers,3and3convenient3to3use.(Razavi,32015;3 Sujatha,32014).3Good3penetration3of3ethosome3is3caused3by3two3mechanisms.3Firstly3ethanol3 takes3a3role3as3penetration3enhancer3which3is3penetrate3into3intercelluler3lipid,3increase3lipid3 fluidity3of3cell3membrane,3and3decrease3multilayer3lipid3of3cell3membrane.3Secondly,3ethosome3 increases3skin3permeability3so3that3it3is3easily3permeated3into3inner3layer3of3skin3and3release3the3 drug3(Parashar,32013).3 Entrapment3efficiency3is3the3most3important3thing3in3characterization3study3of3ethosome3 formulation.3It3indicates3the3amount3of3drug3or3bioactive3compounds3which3is3entrapped3in3the3 vesicles.3 It3 will3 absolutely3 affect3 the3 amount3 of3 drug3 that3 reaches3 the3 target3 of3 therapy3 (Barupal,2010).3In3order3to3find3out3the3highest3entrapment3efficiency3of3earthworms3extract,3 the3measurement3of3entrapment3efficiency3was3done3to3four3ethosome3formula3with3variation3 of3ethanol3concentration3(20%,330%,340%3and350%).3The3aims3of3this3research3was3to3find3out3 information3about3ethanol3concentration3that3provide3the3highest3entrapment3efficiency3of3the3 ethosome.33 & Materials&and&Methods& Earthworm&Preparation& Dried3earthworms3(Lumbricus)rubellus)L.)3that3were3used3in3this3research3obtained3from3 Makassar,3South3Sulawesi,3Indonesia.33 3 Extraction&of&Earthworms& Earthworms3extraction3was3done3by3using3maceration3method.3A3total3of35003g3dried3 earthworms3was3macerated3using350%3solvent3ethanol3for333days.3The3solvent3was3evaporated3 using3rotary3evaporator3and3continued3with3lyophilization3to3obtain3viscous3extract.3 3 Measurement&of&Total&Protein&Content&of&Earthworms&Extract& & A3total3amount3of3503mg3extract3was3dissolved3in3distilled3water3to3103ml3(530003ppm).3 Taken32003µl3of3the3solution3and3then3added3with3reagent3C,3 incubated3for3103minutes3and3 reagen3 D,3 incubate3 for3 203 minutes.3 Distilled3 water3 was3 added3 up3 to3 53 ml3 (2003 ppm).3 The3 absorbances3were3measured3using3spectrophotometer3UVNVisible3at37023nm.3 & Preparation&of&Ethosomal&Dispersion& The3composition3of3ethosome3formula3of3earthworms3extract3is3shown3in3Table31.3 Table&1.&Composition&ofðosome&formula&of&earthworms&extract& Compositions& Amount& F1& F2& F3& F4& Earthworms3extract3 0.53%3 0.53%3 0.53%3 0.53%3 Soya3lecithin3 2%3 2%3 2%3 2%3 96%Ethanol33 20%3 30%3 40%3 50%3 Propyleneglycol3 10%3 10%3 10%3 10%3 Distilled3water3up3to3 100%3 100%3 3100%3 3100%3 International)Journal)of)Applied)Biology,)Vol.)1,)Issue)1,)2017) 3 343 The3ethosome3was3made3using3cold3method.3Earthworms3extract3was3dissolved3in3ethanol3and3 homogenized3using3magnetic3stirrer3for353minutes3at37503rpm,340oC.3After3that,3soya3letichin3was3 dispersed3into3the3mixture.3After353minutes,3propyleneglycol3was3added3into3the3mixture3and3 homogenized3for353minutes.3Distilled3water3at330oC3was3added3at3last.3The3size3of3ethosome3 vesicles3was3minimized3by3sonicating3for3153minutes.3 3 Determination&of&Entrapment&Efficiency& & An3amount3of313ml3of3ethosome3was3added3to3effendorf3tube,3then3centrifugated3for323 hours3at313.0003rpm,34oC.3Taken3250µl3of3the3supernatant,3then3was3added3with3reagen3C,3 incubated3for3103minutes3and3reagen3D,3incubated3for3203minutes.3Distilled3water3was3added3up3 to3 53 ml.3 The3 absorbance3 was3 measured3 using3 spectrophotometer3 UVNVisible.3 Percentage3 of3 entrapment3efficiency3was3calculated3using3the3formula3below:3 !! = #$%&'(%)*'(+#,,*--+&.#$%&'(%)(%(#/-+&. 0100%& & Results&and&Discussion&& Entrapment3efficiency3is3an3important3thing3in3the3characterization3study3in3ethosomal3 formulation.3It3indicates3the3amount3of3drug3entrapped3in3the3prepared3vesicles3(Verma,32011).3 Because3of3that,3it3is3expected3that3the3higher3the3entrapment3efficiency,3the3higher3amount3of3 drug3that3reaches3target3of3therapy.3In3this3study,3there3are3four3concentration3of3ethanol3that3 was3used3that3are3F13(20%),3F23(30%),3F33(40%),3and3F43(50%).3 The3results3showed3the3percentage3of3entrapment3efficiency3of3F1,3F2,3F3,3and3F43that3 are335.56%,330.45%,359.16%,3and372.58%,3respectively3as3shown3in3Figure31.3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 & & Figure&1.&Entrapment&Efficiency&of&Ethosome&of&Earthworms&Extract& & & The3 results3 indicated3 that3 the3 entrapment3 efficiency3 was3 affected3 by3 ethanol3 concentration3in3formula.3The3highest3entrapment3efficiency3was372.58%3given3by3F43which3 containing350%3of3ethanol.3This3is3likely3due3to3the3solubility3of3extract3in3the3ethosomal3core3was3 higher3in3F43than3other3formulas.3In3this3study,3the3entrapment3efficiency3of3the3formulation3 escalated3along3with3the3increasing3of3alcohol3concentrations.3Alcohol3is3a3natural3enhancer,3 which3has3the3property3to3alter3the3skin3permeability.3However,3transdermal3permeability3of3 35.559 30.447 59.163 72.583 0 10 20 30 40 50 60 70 80 90 F1 F2 F3 F4 En tr ap m en t&E ff ic ie nc y& (% ) Treatment International)Journal)of)Applied)Biology,)Vol.)1,)Issue)1,)2017) 3 353 ethosomal3formulations3was3found3to3be3higher3compared3to3hydroalcoholic3drug3solution3which3 indicates3that3alcohol3is3not3the3only3one3contributor3to3increase3the3skin3permeability.33 Several3studies3have3investigated3the3possible3mechanism3of3enhancing3skin3permeability3 by3lipid3vesicular3system.3Vesicles3can3interact3with3the3stratum3corneum3lipids3and3alter3the3 permeability,3 which3 facilitates3 penetration3 of3 drug3 across3 stratum3 corneum.3 Enhanced3 permeation3of3drug3with3ethosomal3formulations3could3be3caused3by3the3combination3effect3of3 alcohol3and3lipid3vesicular3system3(Chourasia,2011).3According3to3Vijayan3(2015),3entrapment3 efficiency3was3affected3by3molar3ratio3of3lecithin3and3ethanol.3In3addition,3in3the3formulation3of3 serratiopeptidase3enzyme3ethosome,3the3highest3entrapment3efficiency3was375.37%3given3by3 formula3containing3lecithin35%3(w/w)3:3ethanol3203ml,3propyleneglycol3103ml,3active3compound3 0.053 mg3 (Vijayan,3 2015).3 Another3 previous3 research3 showed3 that3 there3 are3 two3 factors3 influencing3the3entrapment3efficiency3that3are3Soya3Phosphatidyl3Choline3(SPC)3and3ethanol3 concentration.3The3amount3of3these3materials3influenced3the3entrapment3of3ketoprofen3inside3 lipid3vesicles3in3a3positive3way.3It3has3been3said3the3entrapment3efficacy3of375N80%3was3provided3 by3 formula3 containing3 30N40%3 of3 alcholo3 and3 2,5N3%3 of3 SPC.3 While3 the3 lower3 efficacy3 was3 performed3by3formula3containing3333320N25%3of3alcohol3and31N2%3of3SPC3(Chourasia,2011).33 In3addition,3research3about3aceclofenac3ethosome3has3shown3that3the3highest3and3the3 lowest3 entrapment3 efficiency3 was3 given3 by3 formula3 containing3 30%3 and3 50%3 of3 ethano3 respectively.3There3was3increase3in3percent3entrapment3efficiency3was3observed3with3an3increase3 of3 ethanol3 concentration,3 but3 when3 ethanol3 concentration3 exceeded3 30%,3 a3 decrease3 in3 entrapment3efficiency3was3observed.3If3ethanol3concentration3increased3above330%3resulting3 into3leakage3of3drug3from3fluidized3bilayer3of3vesicles3(Barupal,32010).3The3obtained3results3of3 this3research3was3in3line3with3previous3mentioned3research3which3indicated3that3the3higher3the3 concentration3of3ethanol,3the3higher3its3entrapment3efficiency.3Besides,3based3on3mechanism3of3 drug3entrapment3in3ethosome,3hydrophilic3drugs3are3entrapped3in3the3aqueous3core3of3lipid3 carrier3 while3 lipophilic3 drugs3 are3 retained3 in3 the3 nonpolar3 chain3 (Chourasia,3 2011).3 So,3 earthworms3extract3which3is3hydrophilic3was3entrapped3in3the3core3of3ehtosomes3vesicle.3 3 Conclusion&& 3 The3highest3percentage3of3entrapment3efficiency3of3earthworms3extract3ethosome3was3 72.58%3 presented3 by3 formula3 containing3 earthworms3 extract3 5%,3 soya3 lecithin3 2%,3 propyleneglycol3 10%,3 and3 ethanol3 50%.3 The3 entrapment3 efficiency3 of3 the3 formulations3 was3 observed3to3increase3along3with3the3increasing3concentration3of3alcohol.33 3 Acknowledgements& 3 This3 research3 was3 supported3 by3 The3 Ministry3 of3 Research,3 Technology3 and3 Higher3 Education3of3Republic3of3Indonesia3for3the3Research3Grant.3 & References& Aldarraji,3 Q.M.,3 Halimoon,3 N.3 and3 Majid,3 N.M.2013.3 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