Cod liver oil (CLO) has long been used as medicine or as a functional food. The CLO is a potential source of vitamin D, vitamin A, and omega fatty acids (eicosapentaenoic acid/EPA and docosahexaenoic acid/DHA). Self-nanoemulsifying drug delivery system (SNEDDS) can enhance dissolution, absorption, and bioavailability of hydrophilic and lipophilic substances for oral administration. The objective of this study was to develop a SNEDDS of CLO with good physical characteristics and stability. The optimization formula was carried out using various ratios of oil, surfactant, and cosurfactant. The physical properties of SNEDDS were determined by transmittance percentage, dispersibility, robustness, thermodynamics stability (heating-cooling cycle, centrifugation, and freeze-thaw cycle), and globule size distribution. The optimum formula of CLO-SNEDDS was obtained at a ratio of surfactant and cosurfactant 2 1 and a comparison of oil and surfactant mixtures 1 6. The CLO-SNEDDS meets the requirement of percent transmittance (97.90 0.85), dispersibility (grade A), and stability based on robustness and thermodynamic stability tests. Diluted SNEDDS has an average globule size of 125 nm with a polydispersity index (PDI) of 0.515. CLO-SNEDDS preparation has good physical characteristics and stability.

Cod liver oil (CLO)is a food supplement extracted from the cod liver. Cod liver oil has long beenused as medicine or as a functional food. Cod liver oil is a potential sourceof vitamin D, vitamin A, and omega fatty acids (eicosapentaenoic acid/EPA anddocosahexaenoic acid/DHA)1. Cod liver oil canprevent heart disease by increasing the elasticity of blood vessels, loweringblood pressure, reducing lipid level, and increasing the HDL level2-4. Cod liver oil cansupport the treatment of type II diabetes mellitus (T2DM) by increasing insulinsensitivity5. Other studies haveshown that CLO has cytotoxic, neuroprotective, hepatoprotective, anti-ulcer, andanti-inflammatory activities. Cod liver oil can also help brain development forchildren's growth6,7.

In oral use, CLO isgenerally developed as an Emulsion-based delivery system. In the oil in a wateremulsion system, CLO is in a dispersed form that is coated or protected bywater8,9. The emulsion formof CLO is further modified into a nanoemulsion form10,11. The advantage ofthe nanoemulsion system is has a small globule size (nano-scale) and increasingits absorption and bioavailability12. However, the studythat develops cod liver oil into another form of nanoemulsion system: Theself-nanoemulsifying drug delivery system (SNEDDS), has not been carried out.

In this study, CLOwas developed in SNEDDS. The SNEDDS is a preconcentrated or anhydrous form ofnanoemulsion, a mixture of oils, surfactants, and cosurfactants13. This system isexpected to self-emulsify quickly in the aqueous stomach system and producesnano globules with a size of 20-200 nm. The advantages of SNEDDS overnanoemulsion are enhancing the physical and chemical stability of theformulation and the ability to fill them into unit dosage forms, such assoft/hard capsules, which improves their commercial viability and patientcompliance14.

Several studies havebeen carried out to develop SNEDDS for lipophilic active compounds. The resultsshow that SNEDDS can increase the absorption and bioavailability of activecompounds in oral delivery15,16. This study aimed todevelop SNEDDS of CLO for oral delivery. Physical and stability evaluationswere carried out to the preparations.

The materials used in this studywere COD (Lancida Hebar Technology), Cremophor®RH-40 (BASF), polyethyleneglycol (PEG) 400 (Bratachem), and monopotassium phosphate/KH2PO4 (Merck). Themain instruments used in this study were gas chromatography-mass spectrometry/GC-MS(Shimadzu GCMS-QP2010 Ultra), magnetic stirrer (Thermolyne S131120-33Q),particle size analyzer/PSA (Beckman Coulter), spectrophotometer UV-Vis(Shimadzu UV mini-1240), sonicator (Branson), analytical balance (MettlerToledo AL204), dan vortex mixer (Thermo Scientific).

The research was carried out inseveral stages of the method as shown in Figure 1.

Figure 1. Research flow chart

Miscibility study of oil and surfactant/co-surfactant

The test was carried out by themiscibility study between CLO and surfactant/cosurfactant. The surfactants usedwere Cremophor®RH-40 and Tween 80; the cosurfactant used was propylene glycol,Transcutol®, and PEG 400. Miscibility studies were performed by preparing 1 mLof the CLO in a test tube then added with surfactant/cosurfactant until a clearmixture was obtained. Stirring and observation were provided every timeaddition of 1 mL of surfactant/cosurfactant17.

Optimization of surfactant and cosurfactant ratio for CLO-SNEDDS

At this stage, the SNEDDS formulawas optimized by varying the concentration of surfactants and cosurfactants ata ratio of oil and Smix (surfactant + cosurfactant) was 1 : 9. Comparison ofsurfactant and cosurfactant used was 3 : 1; 2 : 1; and 3 : 2. The SNEDDSprepared by mixing oil, surfactant, and co-surfactant at a temperature of 40°C,then stirred until homogeneous. The CLO-SNEDDS preparations were characterizedby the percent transmittance test18.

Physical evaluation of CLO-SNEDDS

Percent transmittance – As much as 1 mL of each SNEDDSpreparation was diluted up to 100 mL using distilled water. Measurement wascarried out at a wavelength of 650 nm using a UV-Vis spectrophotometer.Distilled water was used as blank19.

Dispersibility test – A dispersibility test wasperformed using a type II dissolution apparatus. As much as 1 mL of CLO-SNEDDSwas added to the 250 mL of distilled water and stirred at 37±0.5ºC at 50 RPM.Visual observation was carried out according to the emulsification grade, asshown in Table I17,20.

Table I. Grade of dispersibility

Robustness to dilution test – Robustness tests were carried outby diluting 1 mL of each CLO-SNEDDS with 100 mL of distilled water, 0.1 N HCl,and phosphate buffer pH 6.8. The mixtures were stirred using a magnetic stirrerand then stored for 24 hours to observe any physical change of system,including phase separation and precipitation21,22.

Determination of droplet size andpolydispersity index – Droplet size and polydispersity index were determined using a PSA.The test was carried out on the diluted CLO-SNEDDS. A total of 100 μL ofCLO-SNEDDS was diluted with 50 mL of distilled water23.

Thermodynamic stability study – The stability was evaluated by centrifugation, aheating-cooling cycle, and a freeze-thaw cycle. Centrifugation tests werecarried out at 3,500 RPM for 30 minutes. At heating cooling tests, CLO-SNEDDSwere stored at two different temperatures (45 and 4ºC). The tests were carriedout in three cycles with storage at each temperature for not less than 48hours. The freeze-thaw tests were carried out at two different temperatures:-21 and 25ºC, conducted for three cycles with storage at each temperature for notless than 48 hours24.

The SNEDDS was known to be one ofthe nano-based drug delivery systems suitable for the delivery of hydrophilicor lipophilic compounds. The CLO delivery in SNEDDS was expected to increaseits dissolution, absorption, and bioavailability. The SNEDDS was prepared bymixing oil, surfactants, and cosurfactants without adding water, so SNEDDS wasalso known as a preconcentrated or anhydrous form of nanoemulsion13. The SNEDDS would form ananoemulsion system spontaneously by gastrointestinal (GI) peristaltic.Miscibility of oil with surfactants and cosurfactants was important for theeffectiveness of SNEDDS formation. The result showed that CLO has goodmiscibility property with Cremophor®RH-40 and PEG 400. Surfactants andcosurfactants in the nanoemulsion were used to form a good and flexibleinterfacial film and decrease surface tension value to almost zero and supportthe spontaneous formation of nano globules25, as shown in Table II.

Table II. Resultof CLO miscibility study

The first step of the optimizationformula was to determine the optimum ratio of the surfactant and thecosurfactant. Surfactants and cosurfactants used were Cremophor®RH-40 and PEG400. The percent transmittance test was used as the initial screening method toselect the optimum ratio of surfactants and cosurfactants. The result showedthat formula F2 (2 : 1) had a percent transmittance value closest to 100%, asshown in Table III. Therefore, the 2 : 1 ratio ofsurfactant and cosurfactant was used for the following research step.

Table III. Optimization result of ratiosurfactant and cosurfactant

The next step was carried out todetermine the optimum ratio of oil and smix using the percent transmittancetest as an initial screening. The result showed that F2A, F2B, F2C, F2D, andF2E meet the percent transmittance requirement for SNEDDS (>90%), as shownin Table IV26. Therefore, the five formulas ofCLO-SNEDDS were continued for further evaluation.

Table IV. Optimizationresult of ratio oil and smix

Dispersibility tests, robustness tests, and thermodynamic stabilitytests were carried out on F2A, F2B, F2C, F2D, and F2E, as shown in Table V. The objective of dispersibility tests was todetermine the ability of SNEDDS to disperse entirely and quickly when subjectedto dilution under mild agitation. The test was carried out by visualobservation, and then the grade was determined according to Table I. The SNEDDS preparations must exhibit grade A andB characteristics on the dispersibility test. The CLO-SNEDDS (F2A-F2E) couldquickly produce a clear emulsion (nanoemulsion) system when diluted in water sothat it was categorized as grade A in the dispersibility test. The ability ofSNEDDS to form nanoemulsion spontaneously when diluted in water occurs due tothe presence of surfactants and cosurfactants that were capable of forming aninterfacial layer in the nano globules system27.

The purpose of the robustness test was to determine the system'sstability after the SNEDDS preparation is diluted in three types of solventswith different pH values, like in the digestive tract conditions. The solventsused were distilled water medium, HCl 0.1 N (stomach condition), and aphosphate buffer pH 6.8 (small intestine condition). The results showed thatCLO-SNEDDS was able to form a stable system after dilution, using the threetypes of solvents, characterized by the absence of phase separation andprecipitation28.

The subsequent study was the thermodynamic stability test, carried outby three types of tests (centrifugation, heating cooling, and freeze-thaw). Theresults showed that CLO-SNEDDS (F2A, F2B, F2C, and F2D) had good stabilitymarked by not occurring phase separation and sedimentation. This indicates thatthe CLO-SNEDDS (F2A, F2B, F2C, and F2D) had good kinetic and thermodynamicstability. The F2E formula shows instability in thermodynamic testing, whichcould occur because the number of surfactants and cosurfactants wasinsufficient to form a stable nanoemulsion system29.

Based on the dispersibility, robustness, and thermodynamic test, itcould be concluded that CLO-SNEDDS (F2A, F2B, F2C, and F2D) had good physicalcharacteristics. Formula F2D with a ratio of oil and smix 1 : 6 was chosen asthe final formula (Figure 2). The formula was chosen because itstill produces SNEDDS with good physical characteristics, with the least amountof smix. This was expected can minimize the risk of negative effects ofsurfactants and cosurfactants for oral administration.

Table V. Dispersibility,robustness, and thermodynamic stability tests results

The F2D formula was furtherevaluated by determining an average of globules size and polydispersity index(PDI) of the nanoemulsion system. The results of globule size analysis using aparticle size analyzer showed that CLO-SNEDDS (F2D) was able to form a nanoemulsionsystem after dilution with a globule size of 125 nm and a PDI value of 0.515.These results followed the globule size requirements for SNEDDS (20-200 nm) andPDI of <0.730.

Figure 2. The CLO-SNEDDS Formula F2D

The CLO-SNEDDS preparation with the ratio of asurfactant and cosurfactant (2 : 1) and a ratio of oil and smix (1 : 6) hadgood physical characteristics based on %transmittance, dispersibility,robustness, and thermodynamic stability studies. The CLO-SNEDDS (F2D)preparation was able to produce a nanoemulsion system after dilution with aglobule size of 125 nm and a PDI of 0.515.

We would like tothank the research Centre of Universitas Islam Bandung for funding thisresearch.

Sani Ega Priani: Conceptualization, supervisor, methodology, datacuration, formal analysis, writing original draft. Desti Puspa Rahayu:Methodology, investigation, formal analysis, administration. Indra TopikMaulana: Conceptualization, data curation, supervisor.

All data are available from the authors.

The authors declare no conflict of interest.

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