INTRODUCTION:

Currently, therapy for various diseases, for instance, cancer, diabetes, and heart disease is less effective due to the limitations of drugs in reaching the target site of action. This case can be caused by inappropriate selection of the drug delivery system. Many diseases occur due to improper lifestyle and daily nutritional intake, resulting in inflammation and cell damage. To overcome this problem, many people have applied treatment using various antioxidants to maintain health. However, some antioxidants provide low stability against heat, oxygen, and light¹. Capsicum annum containing capsanthin is a source of natural antioxidants originating from plants. Capsanthin as a part of the fat-soluble carotenoid group is responsible for the red color in Capsicum annum. Capsanthin as an antioxidant shows high antioxidant activity² due to the presence of a conjugated keto group and multiple double bonds in the molecular structure, resulting in many therapeutic effects such as anticancer^3,4, anti-inflammatory⁵ and anti-obesity^6–8. Previous studies with population-based cohorts showed that consumption of Capsicum annuum significantly reduced mortality from diseases such as ischemic heart and respiratory diseases as well as cancer⁹. Another study such as in Mediterranean populations showed that the consumption of chili peppers (Capsicum annuum) resulted in lower mortality related to cardiovascular and cancer diseases ^10,11.

Capsanthin as a part of carotenoid provides lipophilic properties exhibiting protective activity in lipoproteins and cell membranes through quenching singlet oxygen and peroxyl radicals thereby producing a protective effect against possible inflammation and oxidative stress ¹². In addition, antioxidants such as capsanthin can act as cancer chemoprevention¹³. However, the low stability and lipophilic nature of capsanthin results in low oral bioavailability^14,15. Therefore, it is a promising strategy to develop nanoparticles as drug carriers to enhance the therapeutic effects of capsanthin. The use of nanotechnology, for instance, nanoparticles can overcome the problem of low oral bioavailability due to the low solubility and instability of drugs^16–18including the herbal medicine^19,20. This research aimed to develop a lipid-based nanocarrier, namely a Nanostructured lipid carrier (NLC) for capsanthin delivery.

NLC as a biodegradable and biocompatible lipid nanocarrier consists of solid and liquid lipid present in a lipid matrix in the core of the nanoparticle. Lipid nanoparticles are promising carriers to enhance the drugs oral bioavailability²¹. The size of nanoparticles in the range of 10 – 400nm indicates great capability as drug delivery systems. The small size of particles leads to a higher surface area resulting in the enhancement of drug therapeutics effects and reduce the toxic effects²². Solid lipids that can be used are glycerides, steroids, or fatty acids, while liquid lipids used are oleic acid, olive oil, and other vegetable oils.^23–25. In this study, the solvent injection method was chosen for the manufacture of NLC. This method provides several advantages including the simple method of preparation, the minimum energy used, and the simple equipment needed in the preparation. In addition, it is fast to handle during the preparation of NLC^26–28. NLC can load lipophilic drugs in the NLC lipid core matrix in dissolved form resulting in a higher amount of drug absorption. The choice of NLC materials or components needs to be considered due to the type of lipid, surfactant, and cosolvent chosen can have an impact on its ability to dissolve the active substance^29,30. Previous study showed that NLC can improve intestinal lymphatic drug transport leading to the enhancement of drug absorption³¹. Based on its various advantages, NLC can be a smart lipid-based nanocarrier for drug delivery and is a promising nanocarrier to be developed.

Within this research, the development of capsanthin-loaded NLC formulation using various liquid lipids was performed. The identification of capsanthin as an active ingredient such as Fourier-transform infrared spectroscopy (FTIR) and thin-layer chromatography (TLC) was conducted. In addition, the measurement of particle size, polydispersity index, and zeta potential of capsanthin-loaded NLC was carried out to identify the properties of this formulation. Furthermore, the selected capsanthin-loaded NLC formulation determined the morphology, loading capacity as well as release study.

MATERIALS AND METHODS:

Materials:

Capsanthin standard (> 95% purity) was purchased from BOC Sciences, USA. Capsanthin extract from red chili peppers was purchased from Qin Health Industry (Shaanxi) Co., Ltd. Oils including wheat germ and almond oil were obtained from TSbali, Indonesia. Linoleic acid, Tween 20, and Span 60 were acquired from Nitrakimia, Yogyakarta. Glyceryl monostearate (GMS) was purchased from Multi Jaya Kimia, Indonesia.

Methods:

Identification of Capsanthin Using FTIR:

The capsanthin extract was identified for its capsanthin content using FTIR (BRUKER) to confirm the functional group of capsanthin. As a reference, the capsanthin standard was used in this measurement. A small amount of sample powder, both standard and capsanthin extract, was measured at wave numbers of 4000–500 cm^-1. The spectra obtained were observed for similarities in the functional groups of capsanthin standard and extract.

Identification of Capsanthin Using TLC:

Another capsanthin identification performed in this study was the TLC analysis. The capsanthin standard and extract were dissolved in ethanol followed by spotting on a 10 x 20cm TLC plate with a silica gel as an absorbent. The plate containing the sample spots was inserted into a chamber that had previously been saturated with a mobile phase consisting of acetone: chloroform: and ethanol (2:1:1) followed by elution. Afterward, the plate was dried and placed in a closed chamber that had been saturated with iodine. Furthermore, capsanthin spots originating from standard and extracts were detected using a UV lamp at 254nm.

Formulation Optimization of Capsanthin-Loaded NLC:

Preparation of capsanthin-loaded NLC was carried out based on the formulation composition as shown in Table 1. Modification of the solvent injection method was used for the preparation of NLC²⁸. Briefly, solid lipid was melted with the addition of ethanol at a temperature of 60°C, followed by the addition of liquid lipid and lipophilic surfactant namely Span 60. Subsequently, a solution of capsanthin extract in ethanol was added to the mixture after cooling down to room temperature. The mixture was stirred using a magnetic stirrer at 1000 rpm for 1 hour. Thereafter, the water phase containing water-soluble surfactant namely Tween 20 was added dropwise to the lipid mixture followed by stirring at the same temperature for 1hour. Afterward, centrifugation at 3500rpm at 4°C for 20minutes was carried out and the supernatant was collected followed by evaporating the ethanol in the fume hood in a dark condition overnight. At the end of the process, lyophilization was conducted to obtain the product that will be used for further experiments.

Characterization of capsanthin-loaded NLC:

Measurement of particle size, polydispersity index, and zeta potential:

The properties of capsanthin-loaded NLC including particle size, polydispersity index, and zeta potential were characterized through measurements using a Zetasizer (Malvern). The samples were dispersed in 20 mM phosphate buffer saline (PBS) pH 7 followed by stirring on a magnetic stirrer at a speed of 300rpm and a temperature of 37^oC before being measured.

Analysis using transmission electron microscopy (TEM):

To describe the morphology of the capsanthin-loaded NLC, analysis using TEM (JEOL JEM-1400 electron microscope) was carried out. The sample dispersed in water was dropped onto the grid and allowed to dry, followed by the addition of 10µL of 2% uranyl acetate and left to stand until absorbed into the sample. Afterward, the samples were left to dry and continued with observation using TEM.

Quantification of loading capacity (LC) of capsanthin in capsanthin-loaded NLC:

A certain amount of capsanthin-loaded NLC (Mp) was dissolved completely in an adequate amount of ethanol (Ve). The concentration of capsanthin (Cc) was measured using the spectrophotometer UV/VIS (Shimadzu) at a wavelength of 476nm. The loading capacity was calculated using the following equation 1.

LC (%) = (Cc x Ve)/Mp x 100%(1)³²

Release study of capsanthin-loaded NLC:

A capsanthin release study from capsanthin extract and capsanthin-loaded NLC was started by dispersing samples at an equal amount of capsanthin in 5mL of 20 mM phosphate buffer saline (PBS) pH 7. Afterward, the dispersion was filled into a dialysis membrane tubing (MWCO 14000 Dalton) followed by inserting the dialysis membrane into the basket and filling the chamber in the dissolution tester with 400mL of 20mM PBS pH 7 as a medium. The basket was rotated at 50 rpm and 4.0ml of release medium was collected at predetermined time points at 0, 10, 15, 30, and 60 minutes, 2, 3, 4, 5, 6, 7, and 8hours. The new release medium at the same temperature and volume was added to the system after sampling. The concentration of capsanthin in the collected samples was determined using a spectrophotometer UV/Vis at a wavelength of 476 nm. The release study was also carried out using a different medium namely HCl 0,1 M containing saline with the same protocol above^32,33.

Statistical data analysis:

To compare the influence of various liquid lipids used in the characteristics of capsanthin-loaded NLC one-way ANOVA followed by Tukey HSD test was performed. For the impact of capsanthin incorporation into NLC on release properties, an independent sample t-test was applied. The program software, namely SPSS 17, was used to perform statistical analysis.

RESULTS AND DISCUSSION:

Identification of capsanthin using FTIR and Thin Layer Chromatography (TLC):

NLC is a potential carrier for the delivery of various active compounds due to the ability of NLC to enhance the therapeutic effects of active ingredients ^27,34, such as capsanthin as performed in this research. For the preparation of NLC, red chili pepper extract was used as a source of capsanthin. Consequently, identification of capsanthin was carried out to confirm the presence of capsanthin, including the analysis using FTIR to ensure that the functional groups were the same as the standard as depicted in the molecular structure of capsanthin as shown in Figure 1.

Figure 1. Molecular structure of capsanthin

Based on the FTIR results as indicated in Figure 2, functional groups can be detected in both capsanthin including standard (Figure 2 A) and extract (Figure 2 B) providing the same functional groups at the same wavenumber. The broad spectrum around 3400-3300 cm^-1 indicates the presence of OH originating from capsanthin. In addition, the presence of conjugated ketone (–C=0) at 1650-1630 cm^-1, CH- stretching at a wavenumber of 2970-2880 cm^-1, and the -CH3 bending band at 1380-1370 cm^–1 are evidence of the presence of capsanthin in the extract.

Figure 2. FTIR spectra of capsanthin standard (A) and capsanthin extract (B) indicate similar functional groups including a broad spectrum of -OH stretching band at a wavenumber of 3400-3300 cm^-1(a), -CH- stretching at a wavenumber of 2970-2880 cm^-1 (b), a typical of conjugated ketone (–C=0) at 1650-1630 cm^-1(c), and -CH3 bending band at 1380-1370 cm^–1(d).

Furthermore, to ensure the presence of capsanthin in the extract used, TLC analysis was also conducted in this research. The results as exhibited in Figure 3, showed that the capsanthin standard produced a spot with an Rf of 0.78, the same as the Rf of capsanthin in the extract. Therefore, based on the FTIR and TLC results, it can be confirmed that the extract used contains capsanthin as an active substance.

Figure 3. TLC of capsanthin standard (1) and capsanthin extract with three replications (2,3,4).

Formulation optimization and characterization of capsanthin-loaded NLC:

Capsanthin was incorporated into NLC with the addition of solid lipid, liquid lipid, and surfactant. Formulation optimization was carried out through the preparation of various NLC formulations including the different liquid lipids used as shown in Table 1. The selected capsanthin-loaded NLC formulation was obtained based on the measurement of the NLC properties including the particle size and zeta potential. Particle size and zeta potential are nanocarrier characteristics describing the carrier's ability to interact with the target locations in the body, for instance, the intestinal membrane^35,36. Particle size as one of the characteristics of nanocarriers not only affects the stability of the carrier system during storage but also has an impact on increasing the therapeutic effect. The smaller the particle size, the higher its ability to reach the intestinal membrane. Particles with a size of more than 300 nm will have difficulty penetrating mucus covering the intestinal membrane. Tween 20 and Span 60, as hydrophilic and hydrophobic surfactants, respectively, act as NLC components playing an important role in maintaining the particle size of NLC. The characteristics and concentration of surfactants influence the properties of lipid nanoparticles, not the particle size but also the stability of the particles by preventing phase separation³⁷. However, the particle size is not only influenced by the concentration and type of surfactant but also by the chemical composition of the nanoparticles, and processing or preparation methods such as temperature and pH³⁸.

Based on particle size measurements as shown in Figure 4, Formulation 1 (F1) indicates a significantly smaller size than the other formulations. The difference in size of each formulation is likely caused by differences in liquid oil content in the formulations between F1, F2, and F3. Different liquid oils affect the lipid core formed in the NLC because of the differences in fatty acids contained in each liquid lipid.

Figure 4. Characterization of capsanthin-loaded NLC formulations (F1, F2, F3) including particle size (blue bars) and polydispersity index (red circles). Data are presented as mean ± SD (n = 3).

Figure 5. Zeta potential of capsanthin-loaded NLC formulations (F1, F2, F3). Data are presented as mean ± SD (n = 3).

Zeta potential is another factor that influences not only the stability of nanocarriers but also the ability of nanocarriers to interact with membranes ³⁹, especially intestinal membranes. The more positive the zeta potential, the greater the possibility of strong interaction with the intestinal mucous membrane. This occurs due to the presence of sialic acid and sulphonic acid in the intestinal mucosa having negative charges^40–42. Therefore, the zeta potential of the nanocarrier needs to be taken into account as it impacts the amount of drug that can be transported across the membrane to reach the systemic circulation. In this study, the zeta potential of F1, as shown in Figure 5 indicates a significantly less negative value compared to F2 and F3, namely around -15 mV, which leads to the low possibility of being repelled by the negative charges of mucous membranes. Based on the particle size and zeta potential data, the formulation F1 as shown in Figure 6 was selected for further experiments including loading capacity quantification, TEM analysis, and release study.

Figure 6. Capsanthin-loaded NLC of selected formulation F1

The loading capacity (LC) of F1 was 22±4%, and it is categorized as a high drug loading in the nanocarrier preparation⁴³. LC describes the drug concentration in the nanocarrier, where the higher the LC, the higher the amount of drug that can be incorporated in the carrier, thus it is effective as a drug delivery. The drug loading depends on the solubility of the drug in the lipid carrier which is affected by the molecular structure of the drug⁴⁴. In addition, the morphology of NLC containing capsanthin can be described using TEM as shown in Figure 7 as spherical particles with nanometer size. This result is in line with the F1 size measurement where the particle size is below 400nm.

Figure 7. The transmission electron microscopy (TEM) of capsanthin-loaded NLC of selected formulation (F1)

Release studies of capsanthin:

Release studies of capsanthin from extract and capsanthin-loaded NLC were performed using two types of media including HCl containing saline and PBS with pH 1 and 7, respectively. The use of two media HCl and PBS describes the condition in the gastric and intestinal, respectively. Based on the results of the release studies as exhibited in Figure 8, the capsanthin detected in the PBS medium of the NLC sample experienced a delay showing a lag time of about 30 minutes, whereas this condition did not occur in an acidic medium. Since capsanthin does not contain either acidic or basic groups, its solubility in water is not affected by pH. Therefore, the difference in lag time was determined by the integrity of the surfactant layer covering the NLC matrix. The lag time indicated that the drug molecules diffused slowly in the NLC matrix of lipids covered by the surfactant layer. The difference in lag time of release between using acidic and alkali media indicated that surfactant membrane integrity was affected by the pH of the medium. This is basic information to develop pH-responsive structured carriers in further research.

Figure 8. Release study of capsanthin-loaded NLC of selected formulation (F1) in 0.1 M HCl and 20 mM PBS pH 7. Data are presented as mean ± SD (n = 3).

Based on the result in Figure 8., the release of capsanthin from NLC as a carrier is slower than capsanthin from extract in both media, namely HCl and PBS. In the first 2 hours of the experiment, almost 100 % of capsanthin from unloaded capsanthin was released into the medium, while approximately 50% of the capsanthin from the NLC was detected in the medium. This means that capsanthin was protected in the NLC, thus it is in accordance with the purpose of the nanocarrier design, namely to incorporate capsanthin into the NLC.

CONCLUSION:

Within this research, NLC was developed as a carrier for capsanthin. The selected NLC formulation namely capsanthin-loaded NLC using wheat germ oil (F1) as a liquid lipid provided various properties including the particle size, zeta potential, and loading capacity supporting as a drug delivery system. The morphology of capsanthin-loaded NLC could be confirmed with the TEM analysis. In addition, the release study indicated that capsanthin could be incorporated into the NLC marked by a significantly slower capsanthin release compared to unloaded capsanthin. Therefore, NLC can be used as a promising capsanthin carrier.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors would like to thank LPPM UAD for supporting this research through an international collaborative research grant.

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Received on 03.02.2024 Revised on 28.09.2024

Accepted on 01.02.2025 Published on 01.07.2025

Available online from July 05, 2025

Research J. Pharmacy and Technology. 2025;18(7):2941-2947.

DOI: 10.52711/0974-360X.2025.00421

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Formulations	Solid lipid GMS (mg)	Liquid lipid Wheat germ oil (mg)	Liquid lipid Linoleic acid (mg)	Liquid lipid Almond oil (mg)	Surfactant (Tween 20) (mg)	Surfactant (Span 60) (mg)	Capsanthin extract (mg)
F 1	120	60			120	60	100
F 2	120		60		120	60	100
F 3	120			60	120	60	100