Food Technology and Biotechnology

Materials

Curcumin (95%) was purchased from Sciphar Natural Products Co. Ltd. (Shaanxi, PR China). Octyl and decyl glycerate (ODO) was obtained from Henan Zhengtong Food Technology Co. Ltd. (Henan, PR China). Tween 80 and Tween 60 were provided by Guangzhou Runhua Chemical Co. Ltd. (Guangzhou, PR China). Phosphatidylcholine 60 (PC 60) was produced by Aikang Fine Chemical Co. Ltd. (Shanghai, PR China). Hydrophilic fumed silica (Aerosil 380) was provided by Evonik Degussa (Essen, Germany). Povidone K30 (PVP (polyvinylpyrrolidone) K30) was purchased from Star-Tech & JRS Specialty Products Co. Ltd. (Chongqing, PR China). Carboxymethylcellulose sodium (CMC-Na) was obtained from Yuhe Food Additive Co. Ltd. (Zhengzhou, China). All the ingredients were of food grade.

The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical was provided by Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), and 1,1,3,3-tetraethoxypropane (97%) was purchased from Shanghai Macklin Biochemical Co. Ltd. (Shanghai, PR China). Phosphotungstic acid, potassium bromide (KBr), trichloroacetic acid (TCA) and 2-thiobarbituric acid (TBA) (≥98.5%) were from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, PR China). Pepsin, pancreatin and bile extract were from Sigma-Aldrich, Merck (St. Louis, MO, USA). DMEM, fetal bovine serum (FBS) and penicillin-streptomycin were purchased from Cellmax Cell Technology Co. Ltd. (Beijing, PR China). All other chemicals used were of analytical grade without further purification.

Preparation of curcumin nanoemulsion

Several mass fractions of Tween 80 (2.0–5.0%), Tween 60 (2.0–5.0%) and PC 60 (2.0–5.0%) were dissolved in 10% ODO at 70 °C, followed by the addition of 0.3% curcumin raw material. PVP K30 0.6–1.2% was added to the oil phase after curcumin was completely dissolved. Then, Milli-Q water (Arium pro Basic, Sartorius, Goettingen, Germany) (73.5–83.1%) was added to the oil mixture as the continuous phase. The mixture was stirred for 10 min to form oil in water (o/w) emulsion.

Optimisation of curcumin nanoemulsion

The composition of curcumin nanoemulsion was optimized by orthogonal experiment design with 5 factors (A-E) at 4 levels respectively: A=temperature (25, 40, 55 and 70 °C) of the preparation, and the mass fractions of B=Tween 80 (2, 3, 4 and 5%), C=Tween 60 (2, 3, 4 and 5%), D=PC 60 (2, 3, 4 and 5%) and E=PVP K30 (0.6, 0.8, 1.0 and 1.2%). Emulsion stability index (ESI) was chosen as evaluation index (25). The emulsion was stored at room temperature for 48 h after preparation, and then the data were measured to calculate the ESI as follows:
where h_C is the height of creamed phase, h_E is the total height of the emulsion and h_S is the height of sedimentation phase.

Preparation of curcumin silica-lipid hybrid microparticles

According to the method of Tan et al. (22), the microparticles were prepared using a two-step process: the production of silica-stabilised nanoemulsion and vacuum drying. Curcumin nanoemulsion was prepared as described in the section Preparation of curcumin nanoemulsion. The silica was added into o/w nanoemulsion in different silica-to-lipid ratio by stirring constantly. The silica-stabilised emulsion was then vacuum-dried (DZF-6090; Jinghong, Shanghai, PR China) under a pressure of 100 Pa at 50 °C to form solid powder. Drying lasted 4 h. Different silica-to-lipid mass ratios (1:1, 1:2 and 1:3) were chosen to explore the effect of different additions on the formulation.

Morphological analysis

Loading of curcumin

The loading of curcumin in nanoemulsion and microparticles was measured by UV/Vis spectrophotometer (755B; Jinghua, Shanghai, PR China) at the wavelength of 425 nm. A mass of 0.1 g nanoemulsion or microparticles was mixed with 9.9 g ethanol to extract the curcumin from the formulations. The ethanol extractions of curcumin were diluted 100 times and centrifuged (TGL-16; Xiangyi) at 5595×g for 10 min before measurement. Curcumin loading was calculated by the following formula:

In vitro antioxidant activity

DPPH scavenging activity

A volume of 100 μL of nanoemulsion and silica-lipid hybrid microparticles in ethanol (100 μL of ethanol as blank control group) with various mass fractions of curcumin were added to 3.9 mL DPPH ethanol solution, and then mixed evenly. The reaction was performed at ambient temperature in the dark. After 30 min of reaction, the absorbance of the mixture was measured at 517 nm by UV/Vis spectrophotometer (755B; Jinghua, Shanghai, PR China). The antioxidant activity was calculated as follows:

Anti-lipid peroxidation effects

Different concentrations of curcumin nanoemulsion and silica-lipid hybrid microparticles in Milli-Q water (Arium pro Basic, Sartorius) were prepared. A volume of 0.5 mL of the sample was mixed with 1 mL of 1% (m/V) soybean phospholipid solution (0.5 mL of Milli-Q water as blank), 1 mL of 0.2 M (pH=7.4) phosphate buffer solution (PBS) and 1 mL of 5 mM FeCl₂ in the tubes. The mixtures were incubated at 37 °C for 60 min in the dark. Afterwards, 3 mL of TBA-TCA-HCl (containing 0.025 M thiobarbituric acid (TBA), 0.92 M TCA and 0.58 M HCl) were added to the mixtures to stop the reaction. The tubes were placed at 95 °C for 15 min then cooled in an ice bath. The supernatant was collected after centrifugation (TGL-16; Xiangyi) at 685×g for 10 min and the absorbance was measured at 532 nm. Thiobarbituric acid reactive substances (TBARS) inhibition rate was calculated as follows:

In vitro simulation of digestion

A two-step experiment was performed to simulate the gastric and small intestinal digestion of curcumin nanoemulsion and silica-lipid hybrid microparticles. Unformulated curcumin was suspended in 0.5% (m/V) CMC-Na solution as a control group (26). The experiment was done according to Huang et al. (27) with some modifications.

To test simulated gastric digestion, simulated gastric fluid (SGF) was prepared by adding 7 mL HCl (36.5%) and 2 g NaCl to 1 L Milli-Q water (Arium pro Basic, Sartorius), and then adjusting the pH to 1.2 with 1.0 M HCl. A volume of 10 mL of nanoemulsion diluent, aqueous dispersion of microparticles and curcumin CMC-Na suspension containing 1 mg/mL of curcumin was mixed with 10 mL of SGF, respectively. The mixture was incubated at 37 °C for 2 h with agitation at 100 rpm (EMS-30; Qianyue, Shanghai, PR China) after 32 mg of pepsin were added to it.

To test simulated intestinal digestion, 0.05 M potassium dihydrogen phosphate solution served as simulated intestinal fluid (SIF). Curcumin encapsulated in silica nanoparticles stabilised Pickering emulsion during storage and simulated digestion. The pH of the gastric phase was adjusted to 7.0 after simulated gastric digestion. Later, 10 mL of SIF containing 47.6 mg of pancreatin and 51.6 mg of bile extract were added to the mixture and incubated at 37 °C. At this point, the pH-stat titration served to maintain the pH of digestive juice at 7.0 by adding 0.1 M NaOH dropwise. The volume of NaOH was recorded at different time points during 2 h. The released free fatty acids (FFA) were calculated according to the following equation (11):

where V_NaOH is the total volume of NaOH (mL) used to neutralize the released FFA, c_NaOH=0.1 M is the concentration of NaOH in the burette, M_lipid is the molecular mass of the oil in the formula (g/mol), m_lipid is the total mass (g) of lipid in the digestive juice. The particle size of the supernatant of the mixture was measured after each phase.

The in vitro bioaccessibility of curcumin was measured after simulated digestion. The digestive solution was centrifuged (TGL-16; Xiangyi) at 5595×g for 10 min and separated into two parts: one was the supernatant containing the mixed micelles, the other was the opaque sediment phase. The amount of curcumin in the supernatant was determined and the bioaccessibility was calculated as follows:

Cell toxicity evaluation

L929 murine fibroblast cell line, purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, PR China) was chosen for cell studies. Cells were cultured in DMEM medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a 5% CO₂ incubator.

For the assessment of cell viability, the cells were plated in 96-well plates (100 μL/well) at a density of 10⁴ cells/mL and cultured for 12 h (blank group was only given an equal volume of cell culture medium without cells). The curcumin nanoemulsion, blank nanoemulsion, silica-lipid hybrid microparticles and blank particles were diluted to various concentrations (4, 8, 12, 20, 30, 45, 60, 80, 100 and 150 μg/mL) with culture medium and then added to 96-well plates (Goyoobiotech, Nanjing, PR China) (100 μL/well). Control and blank groups received 100 μL cell culture medium. The medium was removed and the cells were washed with PBS after 24 h of treatment. Then, 200 μL cell counting kit-8 (CCK-8; Goyoobiotech) solution (10% in culture medium) were added to the wells for further incubation for 1.5 h, and then the absorbance (A) was recorded by a microplate reader (K3; Thermo Scientific) at 450 nm. The cell viability was calculated as follows:

Statistical analysis

All the experiments were done in triplicate. Results were expressed as mean value and standard deviation (mean±S.D.). The results were analyzed by ANOVA and t-test. Data were considered significant at p<0.05.

Results of orthogonal experiment

Orthogonal experiment is a method to analyze the correlations of variables at different levels by an orthogonal table and statistical analysis, which can be used for the optimization of multiple formulae (28, 29). The stability of o/w emulsion is closely related to many factors such as the preparation conditions and formulation composition. An orthogonal experiment was designed to optimize the formulation of curcumin nanoemulsion by testing the effects of preparation temperature and emulsifier mass fraction on the ESI. The results and analysis of orthogonal experiment are shown in Table 1. The sequence of effects on ESI to evaluate the preparation was: A (temperature)>D (PC 60)>E (PVP K30)>C (Tween 60)>B (Tween 80), which indicated that the temperature had significant effect on the stability of the formula and Tween 80 had little influence on the ESI of the o/w emulsion. Comprehensively considering the performance of each level, the optimum preparation conditions were A₄B₄C₃D₂E₁. Based on the results, the optimal conditions for the preparation of nanoemulsion were ODO 10%, curcumin 0.3%, Tween 80 5%, Tween 60 4%, PC 60 3%, PVP K30 0.6% and Milli-Q water 77.1%, and preparation temperature 70 °C.

Curcumin silica-lipid hybrid microparticle formation

Microparticles with different silica-to-lipid mass ratios (1:1, 1:2 and 1:3) were prepared by vacuum drying. The sample with silica-to-lipid mass ratio 1:3 appeared as a thick, oily mixture after vacuum drying and there was no powder product formed. The sample with 1:2 ratio resulted in yellow agglomerated mass with poor powder mobility. The microparticles with silica-to-lipid mass ratio 1:1 formed dry and free flowing powder which was up to the standard.

Physicochemical characterization of nanoemulsion and microparticles

Table 2 shows the particle size (redispersed particle size) and PDI of curcumin microparticles. The particle size of the nanoemulsion and microparticles was further analyzed by TEM. The hydrophobic tails of emulsifier molecules attach to hydrophobic core formed by the oil phase, while the hydrophilic heads protrude into aqueous phase, forming small spheroid particles dispersed in the aqueous phase. The spherical structure of nanoemulsion droplets is observable in Fig. 1a. The dynamic light scattering test showed that the particle size of nanoemulsion was (40.5±2.4) nm, which was substantiated by the diameter of particles in the TEM. PDI of nanoemulsion and microparticles was (0.16±0.02) and (0.26±0.02), respectively. The particle size and PDI of nanoemulsion were significantly smaller ((177.2±3.3) nm) than of microparticles. This could be ascribed to the oil droplet interfaces absorbed by tiny silica particles, leading to an increase in the particle size of the redispersed microparticles (30). The SEM images of Aerosil 380 and microparticles are shown in Fig. 1b and Fig. 1c, respectively. In a previous study (31), Aerosil 380 consisted of 7-nm primary particles, which indicates a porous internal structure with pore sizes ranging from 25 to 100 nm. The SEM image (Fig. 1b) showed a loose, porous and nearly spherical shape which gave the particles highly specific surface area and strong adsorption ability. Nevertheless, the microparticles (Fig. 1c) lost these features and had a rough surface, with microparticles formed when the oil phase was adsorbed onto the porous silica matrices. The results were consistent with the findings of Simovic et al. (21) where droplets were uniformly distributed and absorbed onto the surface of silica instead of forming a continuous film. In other study, Tan et al. (22) proposed that there could be redistribution and self-assembly of the Aerosil 380 particles from the continuous phase to the droplet surface and the inner wick during dehydration. The curcumin contents of nanoemulsion and microparticles were (0.30±0.02) and (0.67±0.02) %, respectively, indicating that curcumin loading increased after solidification.

Fig. 1

Transmission electron microscopic image of: a) curcumin nanoemulsion, and scanning electron microscopic images of: b) hydrophilic fumed sillica (Aerosil 380), and c) curcumin silica-lipid hybrid microparticles

XRD spectra

Crystalline or amorphous phase of curcumin in the silica-lipid hybrid microparticles was investigated by XRD analysis. The XRD spectra of curcumin, Aerosil 380, physical mixture of curcumin and Aerosil 380 and curcurmin microparticles are shown in Fig. 2a. Several sharp peaks in the diffractogram of the physical mixture of curcumin and Aerosil 380 indicated that curcumin existed in crystalline form. In contrast, the XRD spectrum of microparticles was similar to that of Aerosil 380 particles and there was no characteristic peak observed. The results suggested that curcumin was in an XRD-amorphous state in the microparticles.

Fig. 2

The spectra of: a) X-ray diffraction of curcumin, Aerosil 380, physical mixture of curcumin and Aerosil 380, and curcumin silica-lipid hybrid microparticles (Cur-SLH), and b) FTIR of curcumin, blank silica-lipid hybrid microparticles (Blank SLH), physical mixture of curcumin and blank SLH, and Cur-SLH microparticles

FTIR spectra

FTIR was chosen to investigate the interactions between curcumin and excipients in the solid state. The FTIR spectra of curcumin, blank silica-lipid hybrid microparticles, physical mixture of curcumin and curcumin silica-lipid hybrid microparticles are shown in Fig. 2b. A characteristic peak observed at 3508 cm^–1 in the spectra of curcumin powder corresponded to the O–H stretching vibration, which was different from the broad peak of amorphous curcumin in the study of Li et al. (32). This phenomenon suggested a different molecular environment of hydroxyl groups between crystalline curcumin and amorphous curcumin. The peak at 1627 cm^–1 was related to the C=O group and the band at 1509 cm^–1 indicated the presence of C=C group. Meanwhile, these characteristic peaks still existed in the spectra of physical mixture, indicating no interaction between the curcumin and blank microparticles. However, the characteristic peaks disappeared in curcumin microparticles and the spectrum was similar to that of blank microparticles. Therefore, the results indicate that curcumin was incorporated in Aerosil 380 and remained in dissolved state in the microparticles (33, 34).

In vitro antioxidant activity of curcumin nanoemulsion and microparticles

DPPH free radical scavenging assay

DPPH is a stable radical which has been extensively applied in the analysis of antioxidant activity. Generally, DPPH signals decrease when the odd electron of DPPH radical becomes paired with hydrogen from a free radical scavenging antioxidant to form the reduced DPPH-H (35). In this study, the DPPH assay was used to explore the free radical scavenging activity of curcumin nanoemulsion and microparticles. According to the results in Fig. 3a, the nanoemulsion and microparticles showed a higher DPPH scavenging activity than curcumin ethanol solution. The DPPH scavenging activity of nanoemulsion was 87.98 and of microparticles 87.06%, which was higher than of control (81.17%) when the concentration was 1 mg/mL. It can be postulated that the nanoparticles protected curcumin from degradation and thus increased its antioxidant activity. In addition, the radical scavenging activity of microparticles was slightly lower than that of nanoemulsion when the concentration was lower than 1 mg/mL. Sari et al. (36) concluded that the encapsulation of silica not only reduces the degradation of curcumin but may also preserve its antioxidant activity. Hence, this result indicates that the free radical scavenging activity of curcumin was not affected by the encapsulation.

Fig. 3

Results of: a) DPPH scavenging activity of curcumin ethanol solution (Cur-EtOH), curcumin nanoemulsion (Cur-NE) and curcumin silica-lipid hybrid microparticles (Cur-SLH). Results are shown as mean value±S.D. (N=3), and b) TBARS inhibition by Cur-EtOH, Cur-NE and Cur-SLH. Results are shown as mean value±S.D. (N=3)

Anti-lipid peroxidation

Lipid peroxidation is often responsible for biological damage, particularly the biological membrane of the brain or liver (37). Generally, malondialdehyde is one of the major products of lipid peroxidation, whose levels can be spectrophotometrically determined by forming a complex with the 2-thiobarbituric acid called TBA reactive substances (TBARS). The TBARS inhibition by curcumin nanoemulsion, silica-lipid hybrid microparticles and curcumin ethanol solution is shown in Fig. 3b. It was observed that the anti-lipid peroxidation capacities of nanoemulsion and microparticles increased in a dose-dependent manner, and the inhibition rate was over 50% at the concentration of 100 μg/mL. However, the TBARS inhibition by microparticles was slightly lower than of curcumin ethanol solution, which can be explained as the result of the slow release of curcumin from Aerosil 380.

Results of in vitro simulation of digestion study

A two-step experiment was used to investigate the simulated digestion of curcumin nanoemulsion and silica-lipid hybrid microparticles. The particle sizes of digestive juice were measured after each digestion phase by DLS technique. The changes in the particle size are observable in Fig. 4a. Nanoemulsion and microparticles before digestion had mean particle size of (40.5±2.4) and (177.2±3.3) nm, respectively. After gastric digestion, there was not much change in the particle size of the two samples. This result indicates that the structures of the two samples remained stable and the oil droplets were not destroyed during gastric digestion. However, after 2 h of intestinal digestion, the particle sizes of nanoemulsion and microparticles increased significantly to (100.0±2.3) and (822.0±4.7) nm, respectively. The augmentation of particle sizes suggested that droplets were destabilized in the process of simulated intestinal digestion. Previous research showed that bile salts can take the place of surfactant molecules from the oil-water interface, facilitate the binding of pancreatic enzymes to the oil-water interfacial layer and promote the lipid digestion, leading to the destabilization of emulsion (38, 39). The complex reactions that occurred in the intestine resulted in the formation of mixed micelles with the ability to transport the encapsulated components to the surface of the enterocytes and enhance the absorption of nutrients (40).

Fig. 4

Results show: a) particle size of curcumin nanoemulsion (Cur--NE) and curcumin silica-lipid hybrid microparticles (Cur-SLH) before digestion in simulated gastric fluid (SGF) and simulated intestinal fluid (SIF). Results are shown as mean value±S.D. (N=3). Different symbols mean significant difference (p≤0.01), b) cumulative FFA released by Cur-NE and Cur-SLH during the simulated gastric digestion. Results are shown as mean value±S.D. (N=3), and c) bioaccessibility of curcumin from Cur-CMC-Na suspension (as a control group), Cur--NE and Cur-SLH after the simulated digestion. Results are shown as mean value±S.D. (N=3). ***Significantly different (p≤0.01)

During the intestinal digestion, oil droplets are broken down into free fatty acids and monoglycerides so the bioactive molecules can be released (41). In this study, the degree of lipid digestion from the nanoemulsion and microparticles was measured by instilling 0.1 M NaOH into digestive juice to neutralize free fatty acids. As shown in Fig. 4b, there was a rapid increase in the rate of free fatty acid release from nanoemulsion and microparticles in the first 30 min, followed by a more gradual increase from 30 to 120 min. However, there were some differences in the rate and extent of digestion between nanoemulsion and microparticles. The initial digestion rate of nanoemulsion was faster than that of microparticles, which can be attributed to the difference in the droplet size. A number of studies have found that the release rate of free fatty acids increases as the lipid droplet size decreases (42). Due to large specific surface area of small droplets, their contact with lipase molecule increased (43). Moreover, the final amount of free fatty acids released from the microparticles was lower than from nanoemulsion, which indicated that the microparticles can inhibit the release of oil droplets. The possible reason was that the oil-water interface was adsorbed by silica particles which led to the inhibition of oil release (30).

Bioaccessibility is defined as the fraction of the active ingredient which is solubilised from the food during simulated gastrointestinal digestion, so it is considered as an estimable method of oral bioavailability (44). The bioaccessibility of the samples was measured at the end of simulated intestinal digestion. As shown in Fig. 4c, there was a significant increase in bioaccessibility of nanoemulsion and microparticles compared to curcumin CMC-Na suspension, indicating that nanocarriers can improve the bioavailability of curcumin. However, it was obvious from the results that the bioaccessibility of nanoemulsion was higher than that of microparticles. Similarly, the bioaccessibility of the free fatty acids increased with the increase of their release. The difference in curcumin bioaccessibility between nanoemulsion and microparticles may be due to two reasons. Lower release of free fatty acids resulted in more undigested lipids, which entrap curcumin. Furthermore, there were not enough mixed micelles to dissolve curcumin (45).

Storage stability of curcumin nanoemulsion and microparticles

Stability is an important factor in examining the quality of the system. In this study, the retention ratios of curcumin in nanoemulsion, silica-lipid hybrid microparticles and ethanol solution was measured during 6 weeks of storage at different temperatures to investigate the chemical stability of the three samples. Fig. 5a shows that the retention ratios of curcumin in the nanoemulsion did not change significantly during storage at 4 and 25 °C, and changed slightly at 40 °C. On the whole, the nanoemulsion maintained good chemical stability during 6 weeks of storage. The same result was also obtained with microparticles (Fig. 5b). However, the samples stored in light behaved differently from those in the dark (Fig. 5c). During 6 weeks of storage, the curcumin retention ratios of nanoemulsion declined to 31.20%, which suggested the formulation was unstable when exposed to light and should be stored in the dark. The retention ratio of microparticles also decreased and remained at 73.23%. Even so, it was still clearly higher than that of nanoemulsion. This observation confirmed that solid preparations protected curcumin better when exposed to light. The possible reason was that the curcumin was encapsulated in the pores of Aerosil 380, which could reduce the influence of light on curcumin (45). Table 2 shows the dispersed particle size and appearance of curcumin microparticles during storage. The dispersed particle size and appearance have not changed significantly, which indicates a good physical stability.

Fig. 5

Retention ratios of curcumin in: a) curcumin nanoemulsion (Cur-NE), b) curcumin silica-lipid hybrid microparticles (Cur-SLH) during storage for 6 weeks at 4, 25 and 40 °C in the dark, and c) curcumin in ethanol solution (Cur-EtOH), Cur-NE and Cur-SLH during storage for 6 weeks at 25 °C under light. Results are shown as mean value±S.D. (N=3)

Cell toxicity evaluation of curcumin nanoemulsion and microparticles

Viability of L929 murine fibroblast cells was measured by CCK-8 assay and the results are in Fig. 6. The preliminary cell studies demonstrated that the blank carriers do not have negative effect on the cell viability, which indicated high safety of the excipients used in the formula. Curcumin nanocarriers deterred the cell viability in a dose-dependent manner. The cell viability was over 80% when the concentration of nanocarriers was less than 45 μg/mL. The viability dropped to less than 50% when L929 murine fibroblast cells were treated with 150 μg/mL nanocarriers. The results indicate that curcumin at a certain concentration had evident cytotoxicity. In addition, the same concentration of curcumin microparticles was less cytotoxic than nanoemulsion when the concentration was higher than 30 μg/mL, which was probably due to the incomplete release of curcumin in the solid preparation.

Fig. 6

Cell viability of curcumin nanoemulsion (Cur-NE), blank nanoemulsion (Blank-NE), curcumin silica-lipid hybrid microparticles (Cur--SLH) and blank silica-lipid hybrid microparticles (Blank-SLH). Results are shown as mean value±S.D. (N=3)