Encapsulation Of β-Carotene And Curcumin In Liposome From Soybean Lecithin And L-α-Phosphatidylcholine Using SC-CO2 With The Assistance Of Ultrasonication

The newest method to maintain better bioavailability of antioxidant compounds in the human digestive process is encapsulation using liposomes. The liposomes used were lecithin from soybean seeds and L-α-Phosphatidylcholine. Furthermore, curcumin and β-carotene were encapsulated in soy lecithin liposomes using SC-CO2 with 100% ethanol, aquadest 50% v/v ethanol and 100% aquadest at 50°C, 60°C, and 70°C and pressure 15 MPa, 20 MPa and 25 MPa by ultrasonication for 60 minutes. This research was conducted to obtain liposomes from curcumin and β-carotene encapsulation, analyse the effect of lecithin from soybean seeds on the formation of encapsulation in liposomes, analyse the effect of operating conditions on the encapsulation process on the size of curcumin and β-carotene in liposomes and determine the size characteristics of liposomes. This was analysed using a UV-Vis Spectrophotometer and Particle Size Analyzer (PSA) with the Dynamic Light Scattering method and also using the Scanning Electron Microscope (SEM) method. The results of the PSA test showed that the increase in operating temperature and pressure resulted in an increase in liposome particle size. UV-Vis analysis proved that the content of curcumin and β-carotene in liposomes from L-α-Phosphatidylcholine had better results. It was found that using SC-CO2+Sonicator had higher concentrations of curcumin and β-carotene with the condition that the higher the temperature, the higher the curcumin content. The use of liposomes has increased the stability of encapsulated suspensions and prevented flocculation. SEM analysis proved that the encapsulation of curcumin with L-α-Phosphatidylcholine has the smallest particle size distribution of 2 micro meters so that it can be categorized as multilamellar vesicles (MLV).


Introduction
Supercritical Carbon Dioxide (SC-CO2) is a fairly good alternative used as a green solvent to replace toxic organic solvents.CO2 is easily separated, has a low critical temperature, low operating temperature which is suitable for processing and encapsulating compounds that cannot withstand high heat [1][2][3][4][5][6].SC-CO2 is non-polar but its solubility to polar phospholipids and water is relatively low, so it is necessary to mix ethanol with SC-CO2 [7][8][9].In recent years, a method for preparing liposomes using SC-CO 2 as solvent, without using ethanol, has been investigated by introducing ultrasonication [10].Various nutraceutical components have been successfully encapsulated in liposomes using a supercritical liquid-based method, but the method used still involves organic solvents.Trucillo et al. [11] minimized the number of organic solvents in the product by optimizing the extended gas-to-liquid ratio.The use of ethanol solvent is needed when SC-CO2 as an alternative solvent that causes 1239 (2023) 012021 IOP Publishing doi:10.1088/1755-1315/1239/1/012021 2 contamination problems from the liposome solution.In recent years, a method for preparing liposomes using SC-CO2 as solvent, without using ethanol, has been investigated by introducing ultrasonication but the results obtained are less than optimal due to the high pressure used which can cause leakage in the vessel [12].
Liposomes are small spherical artificial vesicles that can be made from natural, non-toxic cholesterol and phospholipids.Liposomes are widely used as carriers of various molecules in the cosmetic and pharmaceutical industries.The use of liposome encapsulation to grow delivery systems that can trap 3 unstable compounds such as antioxidant compounds and protect their function [13].Liposomes/vesicles can encapsulate aqueous hydrophilic and hydrophobic drugs within the bilayer and are often classified based on the size and number of bilayers: small uni lamellar vesicles (SUV), large uni-lamellar vesicles (LUV), and multilamellar vesicles (MLV) [14].SUVs and LUVs exhibit diameters in the range of 20-100 and 100-1000 nm, respectively [15,16] and are frequently used as drug carriers [17].
The best encapsulation process is using liposomes because they can provide stability and are able to maintain functional components for nutraceutical, pharmaceutical, cosmetic, and medical applications, and various nutraceutical components have been encapsulated in liposomes using SC-CO2 [18][19][20].The encapsulation process often uses commercial liposomes as encapsulation media, but they are quite expensive, so it is necessary to find alternative sources of liposomes that are cheaper.Lecithin consists of glycolipids, triglycerides, and phospholipids (phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol).Lecithin is classified in the United States by the Food and Drug Administration as products that are generally recognized as safe for human consumption [23].The phospholipid content of lecithin comes from the phosphate fraction extracted from vegetables such as soybeans, sunflowers, rice beans, and rapeseed seeds [24].Lecithin extracted from soybeans is more economical, safer, and stable from a production point of view [25,26].Lecithin from soybeans has a much higher amount of phospholipid components when compared to other vegetable sources [24][25][26][27].Soy lecithin is more stable, less polyunsaturated fatty acids, safer, widely available in pure and impure forms, and less expensive for laboratory and commercial production of liposomes [28].
β-Carotene is a natural pigment or natural antioxidant compound found in vegetables and fruits such as carrots, pumpkin, and sweet potatoes [29].β-Carotene consumption is very important to balance daily intake and meet the needs of antioxidants in the human body [30].In the human body, β-carotene is converted to vitamin A and exhibits antioxidant activity so that it can ward off free radicals that can damage body tissues and increase the risk of exposure to cancer and other diseases [31].β-Carotene is hydrophobic because of its low absorption ability in the human body and is sensitive to heat and light so it is easily oxidized due to its poor bioavailability.Therefore, β-Carotene must be encapsulated to improve bioavailability, prevent oxidation and degradation of the membrane barrier function [32,33].
Curcumin (diferuloylmethane) is an active compound found in Curcuma Xanthorriza and turmeric in the form of polyphenols with the chemical formula C21H20O6.Curcumin, which is a natural yellow pigment, can be isolated from turmeric [34,35] is easily degraded by pH factors and organic solvents, and is relatively stable in the human body.Curcumin has potential as an anti-inflammatory [36], antitumor, DNA-carcinogen inhibitor, and antioxidant [37].WHO states that the acceptable daily intake of curcumin as a food additive ranges from 0-3 mg/kg.Its therapeutic use has also been investigated and is known to have potential anticancer use [38,39].Curcumin is available in several forms including capsules, tablets, ointments, energy drinks, soaps, and cosmetics.Curcuminoids have been approved by the US Food and Drug Administration (FDA) as "Generally Recognized as Safe" (GRAS) [40].
Many researchers have conducted research related to encapsulation using liposomes regarding the use of solvents and also the type of liposomes used.The encapsulation process will use a type of liposome from soybeans resulting from subcritical extraction and also use pure phosphatidylcholine from soybeans.This was done to analysis the effect of extracted soybean lecithin on encapsulation results because lecithin extracted from subcritical is more economical than pure phosphatidylcholine from soybeans.The solvent used will also be compared between using organic solvents and also without using organic solvents.This is to reduce the use of organic solvents because later the results of this encapsulation will be used as a conductor of antioxidant compounds for consumption in the human body.The aims of The aims of this research is to get the best analysis results from the encapsulation process of antioxidant compounds using the type of liposome and the type of solvent in terms of size, stability, and content of antioxidant compounds that can be encapsulated.The encapsulation of these compounds using liposomes with soy lecithin and L-α-Phosphatidylcholine with a mixture of SC-CO2, distilled water, and ethanol as solvents was used to minimize the use of toxic organic solvents in liposomes.A vessel containing the solute, SC-CO2, and ultrasonicated 1239 (2023) 012021 IOP Publishing doi:10.1088/1755-1315/1239/1/0120213 with ultrasonic probe sonicator to encapsulate β-Carotene and curcumin in liposomes.To reach the desired operating temperature, the vessel is immersed in an acrylic chamber filled with water.

Samples and chemicals
Soybean lecithin produced from subcritical water extraction and L-α-Phosphatidylcholine (soybean ≥99%, lyophilized powder) were used as an encapsulant or liposome without further modification.β-Carotene and Curcumin were purchased from Sigma-Aldrich Co. (St.Louis, MO, USA) and used as antioxidant compound for encapsulated, Ethanol (C2H5OH, 99,5%) were purchased from Wako Pure Chemical Industries Inc. (Tokyo, Japan), Distilled water, Carbon dioxide (CO2) and Nitrogen were supplied by Samator, Surabaya, Indonesia.

Liposome preparation and experimental procedure
Figure 1 provides an illustration related to the schematic of the apparatus used to produce encapsulation of curcumin and β-Carotene liposomes with SC-CO 2 with the help of a probe sonicator for the ultrasonication process.Lecithin (6 mg) and curcumin (4 mg) were filled with 45 mL of co-solvent (ethanol, 50% ethanol and 50% distilled water, 100% distilled water) in a pressure-resistant stainless-steel vessel (SS-316; inner diameter 45 mm; outer diameter 80 mm; length 100 mm).The sealed vessel was placed in a water bath made from acrylic material and heated in the range of 50-70 0 C. SC-CO2 is pumped in the range of 15-25 MPa [27][28][29], and the vessel was ultrasonically irradiated 45 kHz and 600 W (input) for ∼60 min (Sonic Vibra Cell VCX 500 Sonicator & 13mm Probe, 53 Church Hill Rd.Newtown) SC-CO2 was pumped using Jas.coPU 4386 Semi Preparative CO2 Pump with a certain pressure to obtain liposomal dispersion with 7,5 mL/minute of CO2 gas flowrate.To release the pressure, the back-pressure regulator was stretched, and then the vessel was removed from the water bath and its cover is slowly opened for 15 minutes so there are no water bubbles coming out from vessel.The same method was used to produce liposomes from Lα-Phosphatidylcholine for curcumin and β-Carotene.Each measurement was carried out under various pressure and temperature and whether liposomes were used.The results will be quoted as the mean ± standard deviation.

β-carotene and curcumin concentration analysis
Analysis using a UV-Vis Genesys 10S spectrophotometer from Thermo Scientific aims to determine how much curcumin and β-Carotene are encapsulated and to determine the stability of the encapsulated antioxidant compounds.The sample is exposed to a monochromatic beam of light.Some of the light beam is absorbed and the rest will be received by the detector.The detector will then calculate the light received and know the light absorbed by the sample.The amount of light absorbed is proportional to the concentration of the substance contained in the sample so that the concentration of the substance in the sample will be known quantitatively.The UV-Vis Spectrophotometer was chosen as the analytical method because the analytical method developed on the UV-Vis Spectrophotometer is simple, can be validated, and is accurate.This method was successfully validated and can easily be used for routine quality control analysis of curcumin [41].

Particle size analysis
The particle size obtained from the encapsulation results was determined by the Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Worcestershire, UK) using the dynamic light scattering (DLS) mechanism.The research sample was diluted 5 times with distilled water and measured at 25 0

Characterization analysis
Particles formed after the encapsulation process were analyzed using the Scanning Electron Microscopy test to determine the morphology of the particles formed and the diameter of the particles produced.From the SEM results, an image of particle morphology will be obtained.From the figure it can be measured particle diameter so that particle distribution and particle morphology can be known.The test equipment used is a product of the FEI inspect S50 type

Result and Discussion
3.1.Effects of temperature, pressure, and solvent on particle size distributions of liposome-encapsulated βcarotene and curcumin Figure 2 shows the standard deviation size distribution of the encapsulated nanosuspension particles obtained at each pressure and temperature in the range of 15-25 MPa and 50-70 0 C. It is shown in the graph that the higher the temperature and pressure used, the resulting standard deviation in the mean diameter is narrower indicating that the particle distribution is narrower in the resulting particle diameter size range.The experimental results show that there is a single peak diameter over the particle size distribution.The average size of the encapsulated nanosuspension particles was in the range of 70-150 nm with most of them having diameters of 100-300 nm.The largest nanosuspension particle size was produced from using pure distilled water as a co-solvent.Even distilled water still has an influence on the encapsulation process with curcumin because of the ability of curcumin to dissolve in water.The use of liposome influences the formation of nanosuspension particles as shown in Figure 3 which shows a smaller particle size compared to without using liposome.Increasing temperature can disrupt the soybean phosphatidylcholine molecules thereby increasing their mobility and flexibility in aqueous solutions [42], from 50 to 70 0 C at the constant pressure (15 MPa) as shown in The use of various CO2 pressures showed that the solubility of liposome with low solvent was not enough to dissolve soybean phosphatidylcholine molecules and form nanosuspensions sizes that were not much different, but the highest peak intensity was obtained for a pressure of 25 MPa.Meanwhile, increasing the pressure from 15 to 25 MPa at the same temperature (60 0 C) was able to form smaller diameter nanosuspension particles as shown in Figure 5 capable of increasing dispersion and causing the formation of smaller nanosuspension particles [3][4][5]43].Zhao et al. [3][4][5] carried out experiments and found that in addition to the smaller particle diameter, a narrower particle size distribution would be obtained at higher pressures when the nanosuspension particles formed with the liposome from soybean lecithin were produced with SC-CO2 at pressure and temperature are in the range of 6-30 MPa and 40-65 0 C, respectively.Meanwhile, increasing the pressure from 15 to 25 MPa at the same temperature (60 0 C) was able to form smaller diameter nanosuspension particles as shown in Figure 6 capable of increasing dispersion and causing the formation of smaller nanosuspension particles [3][4][5]43].Zhao et al. [3][4][5] carried out experiments and found that in addition to the smaller particle diameter, a narrower particle size distribution would be obtained at higher pressures when the nanosuspension particles formed with the liposome from soybean lecithin were produced with SC-CO2 at pressure and temperature are in the range of 6-30 MPa and 40-65 0 C, respectively.Figure 6 shows the comparison of nano suspended particles using different solvents, where the use of ethanol solvents produces a much smaller particle size compared to the two solvents.

Effects of temperature, pressure, and solvent on amounts of liposome-encapsulated β-carotene and curcumin
Nanoparticles and particle distribution determine the success of a compound encapsulation process using liposomes.However, the concentration of β-carotene and curcumin and the level of stability of the nanoparticles in suspension are also important things to note. Figure 8 showed the concentration of curcumin in suspension which was able to be dispersed in L-α-phosphatidylcholine and solvents using SC-CO2 + Ultrasonication where the results showed that increasing the pressure used caused curcumin levels to rise, this was due to an increase in pressure in the water-CO2 system at temperatures up to 100 0 C so that the solubility of CO2 in water to equilibrium dissociation can encourage the formation of CO2 derivative molecules.

Figure 8 Concentration of compounds in encapsulated suspension using L-α-Phosphatidylcholine with various operating conditions
An increased anhydrous CO2 molecule can lead to the formation of liposomes through the interaction of fatty acyl chains.The hydrophobic part of the liposome will interact either with curcumin or β-Carotene, so that it is able to envelop these antioxidant compounds [44].Like the solubility of CO2 in water, the solubility of curcumin and carotene CO2 also increases with increasing pressure at constant temperature due to an increase in CO2 solubility so that it can affect the interaction between carotene or curcumin molecules and soy phosphatidylcholine content.Carotene encapsulation efficiency increases under increased pressure [45].As shown Figure 9 the concentration of curcumin and β-carotene encapsulated by liposomes did not increase with increasing temperature from 50 to 70°C at constant pressure (15 MPa).

Figure 9 Concentration of compounds in encapsulated suspension using soy lecithin with various operating conditions
Increasing temperature can disrupt the process of increasing molecular mobility and flexibility caused by Van der Waals interactions between soy phosphatidylcholine molecules so that it can reduce the solubility of CO2 in water and the formation of CO2 derivative molecules that can affect liposome production through interactions between fatty acyl chains in the soy phosphatidylcholine structure [19].In addition, although an increase in temperature negatively affects the solubility of CO2 with carotene due to a decrease in the density of CO2, it will allow the vapor pressure of the solute to increase, thereby increasing solubility.Due to such complex interrelated phenomena, increasing the temperature from 50 to 70 0 C at 15 MPa did not significantly affect the encapsulation efficiency of curcumin and β-carotene [46].
The use of various liposome variations also determines the formation of nanosuspension particles and also the concentration of compounds that can be dispersed in solvents as shown in Figure 10 where the concentration of curcumin which is able to be encapsulated by lecithin has a lower concentration compared to L-αphosphatidylcholine because the lecithin used is a product of extraction that allows the presence of other compounds and lipids that are not needed to be extracted so that the encapsulation process occurs imperfectly.

Figure 10 Concentration of compounds in encapsulated suspension using liposome variations at constant operating conditions
Figure 11 showed that the concentration of curcumin in the encapsulation without using liposome on day 30 decreased significantly and was not stable.This is because encapsulation does not occur perfectly due to the presence of hydrophobic chains in compounds that do not bind to anyone and the poor solubility of curcumin and beta carotene in distilled water.

Liposome characterizations
Liposomes using L-α-Phosphatidylcholine were observed by Scanning Electron Microscopy (SEM).Examples of SEM images of these liposomes using L-α-Phosphatidylcholine, curcumin and β-carotene in ethanol and distilled water are reported in Figure 13 (a), (b) which show that the liposomes are spherical in shape and have a smooth surface on their surfaces.Size analysis performed on SEM images confirmed the average liposome diameter to be between 100-300 nm when L-α-Phosphatidylcholine is loaded in the inner core.shows the SEM results for the encapsulation results of carotene compounds in liposomes.From the figure it can be seen that the curcumin encapsulated product has a smaller size and more distribution compared to the carotene encapsulated product.The results of curcumin encapsulation during the SEM test have particle sizes of 2-5 micrometers while the results of curcumin encapsulation during the SEM test have particle sizes of 3-100 micrometers so that it can be said that the results of curcumin encapsulation in L-α-Phosphatidylcholine liposomes are categorized as multilamellar vesicles [15,16].

Conclusion
Each liposome encapsulation was loaded from antioxidant compounds β-carotene and curcumin which were produced from lecithin and L-α -Phosphatidylcholine in aqueous media with SC-CO2 and ultrasonication.Batch experiments were carried out by applying ultrasonication for 60 minutes in the range of 50-70•C and 10-25 MPa.The experimental results show that there is a single peak diameter over the particle size distribution.The average size of the encapsulated nanosuspension particles is in the range of 70-150 nm with most of them having a diameter of 100-300 nm.UV-Vis spectra showed that the nanosuspension was more effective and able to bind antioxidant compounds as indicated by the use of a mixture of 50% ethanol and 50% aquadest as a solvent using liposomes in the form of L-α-Phosphatidylcholine and had good results.Stable results even preventing flocculation up to 30 days.

Figure 2
Figure 2 Standard deviations for variations in operating conditions, compounds, and solvents used with the L-α -phosphatidylcholine liposome.

Figure 3
Figure 3 Particle size distribution of the encapsulated nanosuspension with liposome variations.

Figure 5
Figure 5 Particle size distribution of the encapsulated nanosuspension with pressure variations at 60 0 C

Figure 6 7 Figure 7
Figure 6 Particle size distribution of the encapsulated nanosuspension with solvent variations.This is due to the ability of β-Carotene and curcumin to dissolve easily in organic solvents.The use of antioxidant compounds curcumin and β-Carotene can also affect the formation of nanosuspension particles due to

Figure 11
Figure 11 Concentration of compounds in encapsulated suspension using liposome variations at constant operating conditionsFrom Figure12it can be seen that the resulting encapsulation solution uses antioxidant compounds in the form of curcumin and β-carotene.

Figure 13 (
a) shows the SEM results for the encapsulation of curcumin compounds in liposomes.

Figure 13 (Figure 13 (Figure 13 (
Figure 13(a) Concentration of compounds in encapsulated suspension using liposome variations at constant operating conditions