Construction, characterization and bioactivity evaluation of curcumin nanocrystals with extremely high solubility and dispersion prepared by ultrasound-assisted method

Graphical abstract

Abstract

Curcumin (Cur) as a natural pigment and biological component, can be widely used in food and beverages. However, the water insolubility of Cur significantly limits its applications. In this study, we prepared a series of nanocrystals via ultrasound-assisted method to improve the solubility and availability of Cur. The results showed artemisia sphaerocephala krasch polysaccharide (ASKP), gum arabic (GA) and wheat protein (WP) were outstanding stabilizers for nanocryatals except traditional agent, poloxamer 188 (F68). The obtained curcumin nanocrystals (Cur-NC) displayed a rod-shaped, crystal- and nanosized structure, and extremely high loading capacity (more over 80 %, w/w). Compared with raw powder, Cur-NC greatly improved the water solubility and dispersibility, and the slow and complete release of Cur of Cur-NC also endowed them excellent antioxidant capacities even at 10 μg/mL. Importantly, as functional factor additive in beverages (e.g. water and emulsion), Cur-NC could increase the content of Cur to at least 600 μg/mL and retain a good stability. Overall, we provided an effective improvement method for the liposoluble active molecules (e.g. Cur) based on the nanocrystals, which not only tremendously enhanced its water solubility, but also strengthened its bioactivity. Notably, our findings broadened the application of water-insoluble compounds.

1. Introduction

Curcumin (Cur) is a natural polyphenol compound extracted from curcuma longa.

It is commonly used as a natural color in food and beverages due to its relatively intense yellow color [1]. Besides, the multiple biological activities, such as antioxidant, anti-inflammatory and antibacterial, also make it as a functional factor widely used in functional foods and beverages [2]. However, Cur is rapidly degraded in neutral and alkaline environments, strong light and heat conditions, affecting its storage stability. Importantly, as a liposoluble component (logP = 3.29), the water insolubility (11 ng/mL) makes it difficult to be dispersed, released in water environment and absorbed by organism, which weakens its bioavailability and activity, and greatly limits its application in food and beverages [3].

It has been reported that approximately 70 % of bioactive compounds researched in laboratory are insoluble in water according to the statistics [4]. There are many emerging encapsulation and delivery technologies have emerged to ameliorate the difficulties in solubility, such as cyclodextrin inclusion complexes [5], liposomes [6], nanoemulsions [7], etc. Among them, nanocrystallization is in the spotlight due to its extremely high loading capacity (LC). Nanocrystals are nano-sized colloidal dispersed system with 100 ∼ 1000 nm particle size and contain bioactive compound and stabilizers only. The stabilizer content is very low or even no, thus, nanocrystals show extremely high LC and biocompatibility. Besides, due to the small particle size and large specific surface area, nanocrystals could improve the solubility and dissolution rate, prolong the action time based on the surface adhesion in the mucus layer, thereby promoting the absorption in the gastrointestinal tract and improving the bioavailability of bioactive compound [8], [9].

In general, there are three main technologies for nanocrystals preparation, including top-down, bottom-up and the combined method. Top-down approach is crushing particles from larger to nano size by physical–mechanical method, such as ultrasound and media grinding method [10]. On the contrary, bottom-up method makes particles grow slowly from small to large based on the supersaturated solution precipitation, for example, supercritical anti-solvent technology and solvent evaporation method, etc. [11]. However, top-down approach always needs special equipments, high energy consumption and requires operating experience, and the removal of organic solvents as well as uncontrollable particle size are the drawbacks of bottom-up method. Combined techniques are the mixture of bottom-up and top-down method, which can significantly make up for the shortcomings of the above two methods. The ultrasound-assisted approach belongs to the combined techniques, in which the particles based on the anti-solvent precipitation are further broken under the ultrasound technique. Ultrasound-assisted is one of the promising methods for the preparation of poorly water-soluble actives, where the stable nanocrystals with high solubility can be controlled by adjusting the technical parameter, such as amplitude level and sonication time, etc., which is convenient for industrial production [12].

Nanocrystals are thermodynamically unstable systems due to their high surface area and Gibbs free energy, and often manifested as sedimentation, aggregation, particle growth and crystalline transformation [13]. Particle size is a key factor affecting the stability of nanocrystals, as evidenced by their unique size-dependent surface energy, and the large specific surface area of nanocrystals allows them to have high surface free energy, which causes them to aggregate. In order to prevent their instability, some appropriate kinds and amount of stabilizers are often added into the nanocrystals. The stabilizers not only adsorb on the particle surface to provide activation energy for inhibiting particles aggregation and Ostwald ripening, but also maintain the stability of the nanocrystals by increasing the viscosity and avoiding mutual collisions between particles [14]. These stabilizers mainly stabilize nanocrystals through electrostatic repulsion, certain spatial site resistance, etc. [15], [16]. However, the traditional stabilizers of nanocrystals widely used in the market are surfactants and synthetic polymers (such as sodium dodecyl sulfate, Tween 80, microcrystalline cellulose, etc.), which belong to the pharmaceutical excipients and exist certain potentially toxic, limiting the application of nanocrystals in other fields, like functional foods and beverages.

Therefore, in this study, the curcumin nanocrystals (Cur-NC) prepared with ultrasound-assisted approach and stabilized by novel stabilizers (such as water-soluble proteins and polysaccharides) were carried out. The formulation process and prescription of series Cur-NC were optimized in terms of LC and particle size. After that, the morphology, crystallinity as well as other properties of the obtained Cur-NC were systematically characterized. In addition, the cumulative release, anti-oxidation activity and cytotoxicity of Cur-NC were also evaluated. Importantly, the thermodynamic stability, re-dispersibility, especially the additivity in beverages of the obtained nanocrystals were focused on. The results showed the feasibility of polysaccharides and proteins as stabilizers of Cur-NC expect polymers, and also provided novel and efficient approach for the liposoluble bioactive compounds application.

2. Materials and methods

2.1. Materials

The Cur was purchased from Shanghai Eon Chemical Technology Co., Ltd. (Shanghai, China). Acetonitrile was acquired from Tianjin Comio Chemical Reagent Co., Ltd. (Tianjin, China). Dimethyl sulfoxide (DMSO) was obtained from Tianjin Fuyu Fine Chemical Co., Ltd. (Tianjin, China). Poloxamer 188 (F68), Hydroxypropyl Methyl Cellulose (HPMC) and Polyvinyl pyrrolidone (PVP) were provided by BASF Europe. Polyvinyl Alcohol (PVA) was purchased from Shin-Etsu. (Shin-Etsu, Japan). Artemisia Sphaerocephala Krasch Polysaccharide (ASKP) was provided by Wuhan Xinrunbao Biotechnology Co., Ltd. (Wuhan, China). Gum Arabic (GA) was obtained from Tianjin Tianli Chemical Reagent Co., Ltd. (Tianjin, China). Chitosan (CS) was supplied by Yuanye Biotechnology Co., Ltd. (Shanghai, China). Prululan Polysaccharide (PU) was supplied by Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Whey protein isolation (WPI), Hydrolyzed Wheat Protein (WP), Sodium caseinate (SC) and Soy protein isolate (SPI) were derived from by Xi'an Binghe Biotechnology Co., Ltd. (Xi'an, China).

2.2. Preparation of the Cur-NC

Cur was firstly dissolved in DMSO to form a Cur solution with a concentration of 10 mg/mL. Then, 900 μL of the Cur solution was added to 9 mL deionized water (containing certain content stabilizer) at a slow and uniform rate, mixed and treated by Ultrasonic Cell Crusher (on for 3 s and off for 2 s, Xinzhi Biotechnology Co, Ningbo, China) at set time and power. After that, the DMSO was removed by dialysis (molecular weight 8000–14,000 Da), and the obtaining sample was further treated by ultrasound at the same power for 15 min. To obtain preferred nanocrystals, the ultrasonic power (200 W, 300 W and 400 W) and ultrasonic time (5 min, 10 min and 15 min) were optimizated. Further, the kind and concentration of stabilizers were also selected.

2.3. The loading capacity (LC) and encapsulation efficiency (EE) of the Cur-NC

The Cur-NC was centrifuged at 1000 r/min for 10 min to remove large Cur particles. Then 200 μL of supernatant was added to 2 mL DMSO and mixed thoroughly, and then the concentration of Cur in the Cur-NC was measured by UV spectrophotometer (General Instrument Co, Shanghai, China) at 425 nm and mass of Cur was calculated (m₁). The EE was calculated by the following equation:

$$\text{Encapsulation}\text{efficiency}=\frac{{\text{m}}_1}{{\text{m}}_2}\times 100\%$$

m₂ is theoretical mass of Cur.

In addition, the freshly prepared Cur-NC was frozen at −20 °C for 12 h and transferred to a vacuum freeze dryer (10 N/A, Scientz biotechnology Co, Ningbo, China) and lyophilizated at −50 °C for 24 h. Lyophilized powders were weighed for mass (m₃) and measured concentration of Cur in lyophilized powder as well as counted mass of Cur (m₄).

$$\text{Loading}\text{capacity}=\frac{{\text{m}}_4}{{\text{m}}_3}\times 100\%$$

2.4. Particle size, zeta potential and polydispersity index (PDI) of Cur-NC

The freshly prepared Cur-NC were diluted with distilled water to get concentration of Cur around 200 μg/mL. Then, the particle size, zeta potential and PDI of the diluted Cur-NC were measured by dynamic light scattering technique (Anton Paar GmbH, Shanghai, China).

2.5. X-ray diffraction analysis (XRD)

The XRD patterns of the lyophilized nanocrystals were obtained by an XRD (Bruker, Germany) with a CuKa radiation generated at 40 kV and 40 mA. The scanning region of diffraction angle (2θ) was ranged from 5° to 40° with a scanning speed of 5°/min.

2.6. Scanning electron microscopy (SEM)

The morphology of the lyophilized nanocrystals were further observed by a SEM (FEI, America). The lyophilized powders were fixed on the aluminum stake with a conductive tape, and sprayed with gold. The images were finally obtained at an acceleration voltage of 30 kV. The length and diameter of particles in SEM was counted by Image J software.

2.7. In vitro cumulative release

Briefly, 1 mL of the Cur-NC (100 μg/mL) were added into the dialysis bags (molecular weight 8000–14,000 Da) and sealed at both ends. Then, the bags were placed in PBS (10 mL, pH 7.4) buffer containing 2 % (w/v) Tween-80 at 37 °C and stirred (Sedaris Laboratory and Analytical Instruments Manufacturing Factory, Tianjin, China) at a rate of 100 rpm. This release medium mimics intestinal fluid, where 2 % Tween 80 primarily promotes Cur solubilization. At the set time point, 1 mL of the sample was removed from the release medium, and 1 mL of the fresh medium at the same temperature was supplemented immediately. The Cur raw powder dispersed in water (RP) and DMSO (Solution) were used as control. The absorbance of the Cur from different groups was determined. The release amount of the Cur could be calculated by substituting it into the standard curve of the Cur.

$$\text{Cumulative}\text{release}\left(\%\right)=\frac{\left({\text{C}}_1+{\text{C}}_2+{\text{C}}_3+\dots \dots +{\text{C}}_{\text{n}-1}\right)\times \text{V}+{\text{C}}_{\text{n}}\times {\text{V}}_{\text{total}}}{{\text{m}}_{\text{theory}}}\times 100\%$$

where C₁, C₂, C₃……C_n is the mass concentration of Cur at the 1st, 2nd, 3rd….….. n time points (µg/mL) and m_theoryis the theory of Cur-NC and V_total is the volume of release medium (10 mL).

2.8. Ferric cyanide (Fe³⁺) reducing antioxidant power

Briefly, 2.5 mL of the Cur-NC was mixed with 2.5 mL of 1 % (w/v) potassium ferricyanide, incubated at 50 °C for 20 min, and then cooled to room temperature. After that, the sample was acidified with trichloroacetic acid (2.5 mL, 10 %, w/v) and centrifuged at 1500 g for 10 min. The obtaining supernatant of 2.5 mL was mixed with equal volume of distilled water, reacted with 2.5 mL of 0.1 % (w/v) Fecl₃ solution for 10 min, and then measured by a UV spectrophotometer (TU-1810 PC, Pu-Analysis General Instrument Co, Beijing, China) at 700 nm. All samples were prepared away from light throughout the experiment.

2.9. Cytotoxicity of the Cur-NC

Caco-2 cells were evenly distributed in 96-well plates and incubated in complete culture medium for 48 h. When the cell density reached about 60 %, the cell medium was removed and the cells were washed by PBS. Following, the complete culture medium containing different Cur-NC (10 μg/mL) were added and incubated with cells at 37 °C for 24 h. Then, the incubation medium was removed and the cells were washed with PBS. After 100 μL of MTT (500 μg/mL) solution was added, the cells were further incubated at 37 °C for 4 h. After that, the DMSO was added to each well and shaken for 10 min on a plate shaker (Kirin Bell Instrument Manufacturing Co., Haimen, China). The absorbance of the resulting solution was measured by the UV at 490 nm and calculate the cell viability.

2.10. The storage stability of Cur-NC

The Cur-NC samples were stored at a glass bottle at 4 °C away from light. The particle size, PDI and photographs were measured on days 0, 3, 7, 14 and 30.

2.11. The redispersibility of Cur-NC

Lyophilized powder and samples dispersed in water were photographed. Oil-in-water emulsions consisting of 0.5 % (w/w) WPI as stabilizer and 10 % (v/v) MCT as oil phase were prepared under condition of high-speed shear (15000 rpm, 4 min, Ultra Turrax, Germany) and probe ultrasound (500 W, 3 min). 600 μg lyophilized powders and fresh Cur-NC suspension were dispersed in prepared emulsion and then the particle size, PDI and stability of these samples were determined.

2.12. Statistical analysis

All experiments were repeated three times, SPSS (version 26.0, SPSS Inc., Chicago, IL, USA) was used for one-way of variance (ANOVA) and Duncan’s multiple comparisons. The data were expressed as mean ± standard deviation. The results were analyzed and plotted using GraphPad Prism software. The significance between groups was set as p < 0.05.

3. Results and discussion

3.1. Optimization of the preparation process of the Cur-NC

3.1.1. Ultrasonic technology

Loading capacity (LC) is the percentage of the bioactive substance in the total delivery system, higher LC means that fewer additives are added, thus reducing the potential toxicity associated with the use of additives [17]. Besides, higher LC was one of the important advantages of nanocrystals, so it was also selected as the index of the optimization of the preparation process of Cur-NC here. Ultrasound-assisted is mainly based on the shock wave generated by cavitation effect to break the agglomerates, thus reducing the particle size [18]. The ultrasonic power and time were critical parameters for ultrasonic output and we optimized them first. As shown in Fig. 1A, when the ultrasound time increased from 5 to 15 min, the LC were all around 80 % with little difference between each other. The longer the ultrasound time, the greater the energy consumption required, it was obvious that 5 min was the optimal ultrasound time. Similarly, it also showed no significant difference in LC with the ultrasonic power increased from 200 W to 400 W, while the series of Cur-NC prepared under 300 W had the smallest particle size (∼300 nm) and the PDI were around 0.2 (Fig. 1B). It was reported that smaller particle size could improve the solubility and dissolution rate of the bioactive substance and have greater surface adhesion, which could enhance the retention time in the gastrointestinal tract and improve the absorption and bioavailability of bioactive substance [19]. Therefore, the ultrasonic condition of 300 W and 5 min were selected in the follow-up experiment. In general, the increase of ultrasonic power and time would reduce the particle size although the reduction is not infinite [20]. However, in our results the particle size did not decrease with the increase of ultrasonic time and power, which may be due to the fact that the system temperature were increased with the strengthening of ultrasonic condition and then the Brownian motion were enhanced, thus the re-aggregation of nanocrystals occurred [21].

Fig. 1.

(A) The LC and (B) particle size detected by dynamic light scattering (DLS) of Cur-NC prepared with different ultrasonic powers and times. Different lowercase letters indicate significant differences (p < 0.05).

3.1.2. The stabilizers of the Cur-NC

In order to ameliorate the unstable phenomena of nanocrystals (such as settling, aggregation and flocculation) in a liquid environment, various stabilizers are always introduced [22]. Here, four common polymers, F68 (poloxamer 188, NC_F68), PVA (polyvinyl alcohol, NC_PVA), HPMC (hydroxypropyl methyl cellulose, NC_HPMC) and PVP (polyvinyl pyrrolidone, NC_PVP) were selected as stabilizers. Fig. 2A displayed that the LC of the obtained Cur-NC were almost 80 ∼ 90 %, in which the NC_HPMC had the lowest LC. The NC_F68 and NC_PVA showed nano-size in the DLS detection (Fig. 2B), while the NC_F68 had higher LC, Encapsulation efficiency (EE, Fig. S1) and smaller size than NC_PVA and thus was be chosen ultimately. F68 has certain hydrophilic and lipophilic properties, which can form a thicker hydration layer around the particles, spatially preventing the particles from approaching each other and maintaining the stability of the Cur-NC. In addition, the amphiphilic ability also can enhance the wettability and dissolution performance of nanocrystals [23]. HPMC could be dispersed in water and attach to the surface of nanocrystals through hydrogen bonding, while the viscosity of HPMC will gradually decrease with time, resulting in instability of nanocrystals [24]. PVP had high affinity and then could adsorb on the nanocrystals surface, preventing inter-nanoparticle aggregation by forming spatial site resistance [25]. The small molecular weight and viscosity may be the reason of larger particle size of NC_PVP. Besides, as shown in Fig. 2C, NC_F68 with different concentrations of F68 exhibited differentiated LC. 5 % and10% were more excellent in LC while 10 % of F68 had higher EE and stability than 5 % (Fig. S2), and thus 10 % F68 was selected. Similarly, Germini G [26] et al. also found that the amounts of stabilizer affected the LC, and less amount of stabilizer would inadequately cover the particle surface while more ones cause aggregation and the decrease of LC.

Fig. 2.

(A) The LC and (B) particle size of Cur-NC prepared with different kinds of polymer stabilizers. (C) The LC of Cur-NC prepared with different concentrations (w/w) of F68. Different lowercase letters indicate significant differences (p < 0.05).

Compared to the synthetic excipients, there are various natural biomacromolecules (e.g. proteins and polysaccharides) with good biocompatible and biodegradable, and can act as stabilizer in the nano-system through thickening, forming electrostatic effect and steric hindrance etc [15]. As shown in Fig. 3A, the nanocrystals with four polysaccharides, GA (gum arabic, NC_GA), ASKP (artemisia sphaerocephala krasch polysaccharide, NC_ASKP), CS (chitosan, NC_CS) and PU (pullulan polysaccharide, NC_PU) showed high LC and indicated the feasibility of polysaccharide as stabilizers. But in the subsequent storage process, there were obvious precipitate appeared in NC_CS and NC_Pu. CS is an ionic surfactant and can prevent nanoparticle aggregation through electrostatic repulsion and spatial site resistance effects [27]. The relatively harsh dissolution condition (ice-acetic acid) may be the main reason for the instability of NC_CS. GA has good hydrophilicity and lipophilicity and prevents particle aggregation mainly by forming a thick protective film around the encapsulated active ingredient core and acting as an emulsifier [28]. ASKP possesses good film-forming property and can also be used as a thickening agent, which makes the intermolecular Brownian motion smaller and thus increases the stability of Cur-NC [29]. Fig. 3B and 3C demonstrated that the LC of Cur-NC at 5 % and 10 % concentration were preeminent both in NC_GA and NC_ASKP. The Cur-NC prepared with10% stabilizer showed better reproducibility for NC_GA and EE (Fig. S3) for NC_ASKP, so 10 % GA and ASKP were chosen as the final concentration.

Fig. 3.

The LC of Cur-NC prepared with different kinds of polysaccharide stabilizers (A) and different concentrations (w/w) of GA (B) and ASKP (C). Different lowercase letters indicate significant differences (p < 0.05).

Food proteins always have foaming and emulsifying abilities based on their amphiphilic property [30]. Therefore, we also investigated their potential as stabilizers for nanocrystals. As presented in Fig. 4A, the LC of Cur-NC prepared with WP (hydrolyzed wheat protein, NC_WP), WPI (whey protein isolation, NC_WPI), SPI (soy protein isolate, NC_SPI), and SC (sodium caseinate, NC_SC) were approximately 80 % and did not have significant differences between each other. Further, NC_SPI and NC_WPI precipitated visibly under the storage and the EE of NC_SC (Fig. S4) was the lowest one. WPI and SC are amphiphilic proteins which can be quickly adsorbed to the surface of nanocrystals and stabilize them. The stability of nanocrystals is related to the isoelectric point pH (4.5–5.5) of WPI and isoelectric point 4.6 of SC, the electrostatic repulsion between nanoparticles is weakened near the isoelectric point of proteins, which destabilizes the system. The pH of the system is approximated to the isoelectric point of WPI at around 5, so the system is unstable [30]. SPI is mainly composed of globular proteins with molecular weight between 200 and 300 kDa, and because of its large molecular weight, it can be easily shed on the surface of nanocrystals, thus making the system unstable [31], [32]. So WP was selected as the optimal protein stabilizers here. WP has good emulsification, foaming, and foam stability [33]. Qin Ma [34] et al. also concluded that protein as a stabilizer is related to protein molecular weight and isoelectric point, similar to our results. Besides, as shown in Fig. 4B, 10 % and 25 % concentration of WP had higher LC, but 10 % concentration was proved as the optimal concentration due to the better reproducibility.

Fig. 4.

The LC of Cur-NC prepared with different kinds of protein stabilizers (A) and different concentrations (w/w) of WP (B). Different lowercase letters indicate significant differences (p < 0.05).

3.2. Characterization of the obtained Cur-NC

3.2.1. Size distribution, zeta potential and loading capacity

LC and PDI were the abbreviations of loading capacity and polydispersity, respectively. NC_NO was Cur-NC without stabilizer.

As shown in Table 1, the particle sizes of Cur-NC were all almost 300 nm except the one without stabilizer (NC_NO, 397 nm) and all the PDI of Cur-NC were less than 0.2, predicting a better dispersion of the Cur-NC in water and improving dissolution rate as well as bioavailability of Cur [35]. Besides, with the appropriate surface charges (negative zeta potentials), Cur-NC would show good stabilities. Meanwhile, the LC of Cur-NC fluctuated 75 % and 90 % (w/w), presenting bright advantage over other delivery systems [37].

Table 1.

Characterization of the Cur-NC prepared with different kinds of stabilizers.

	NCNO	NCF68	NCGA	NCASKP	NCWP
Particle Size (nm)	397.37 ± 12.1	274.46 ± 2.00	300.34 ± 17.10	282.34 ± 8.24	295.56 ± 19.52
PDI	0.16 ± 0.03	0.18 ± 0.01	0.10 ± 0.05	0.15 ± 0.09	0.20 ± 0.03
Zeta potential (mV)	−22.00 ± 1.30	−18.00 ± 0.30	−18.00 ± 0.40	−20.00 ± 0.50	−17.00 ± 0.50
LC (%)	—	88.26 ± 2.20	82.46 ± 3.40	77.16 ± 4.50	78.50 ± 3.10

3.2.2. X-ray diffraction

Crystallization is an important feature of nanocrystals and the crystalline states of series Cur-NC were detected by XRD. As shown in Fig. 5, there were sharp peaks appeared in all Cur samples, indicating the crystal states of raw powder (RP) and Cur-NC. The peaks of RP were dense and numerous, showing a highly crystalline state, which was consistent with the previous report [36]. Cur-NC demonstrated a reduced crystalline state based on the sparse and few peaks. Compared with RP, Cur-NC lost some peaks, such as 8°, 16°, 17.5°, and 24°, which may be caused by the re-formation of crystals under the ultrasound assisted preparation. Besides, there was basically no difference on the crystal peaks between Cur-NC with/without stabilizers, probably due to the fact that stabilizers existed in the Cur-NC systems mainly act to prevent the aggregation of particles rather than the formation of crystals [37].

Fig. 5.

XRD detection of the obtained Cur-NC prepared with different kinds of stabilizers. RP: raw powder.

3.2.3. Morphology

The morphology of Cur-NC were observed by SEM. As shown in Fig. 6A, RP showed irregular shapes with micron size, while the Cur-NC prepared with different kinds of stabilizers were all nonosized and showed uniform rod shapes. After further statistical calculation, it was found that NC_F68, NC_GA and NC_WP were all around 700 nm in length and 150 nm in diameter, and NC_ASKP showed slight smaller size (540 nm and 100 nm, respectively (Fig. 6B). It demonstrates that ultrasound-assisted significantly reduced the particle size of Cur-NC. Notably, there were obvious difference between the results detected by DLS (∼300 nm) and SEM, which was mainly due to the different principles of the two measurements and the former calculated the particle size through simulating the particle as a regular spherical shape.

Fig. 6.

(A) SEM images of Cur-NC prepared with different kinds of stabilizers. (B) Statistics of SEM images based on at least 50 particles.

3.3. In vitro cumulative release

Limited by solubility and particle size, water-insoluble active substances are often difficult to dissolve in biological body fluids, which greatly restricts their activities [38]. Here, the in vitro release of Cur-NC in PBS 7.4 (simulation of small intestine) were evaluated. As shown in Fig. 7A, the release of the Cur solution (in DMSO) was fastest and essentially complete (90 %) within 12 h, contributed by their free molecular state. In contrast, RP just released 10 % even though 7 days owing to its poor solubility in the water. Moderately, it showed the release of four Cur-NC were around 40–60 %, which were more complete compared to the RP while slower to the solution group, and this also manifested that Cur-NC could ensure free molecules released from the particles slowly and sustainably due to small particle size, high solubility and good water dispersibility. Therefore, the physiological activity would be maximized based on the suitable release of Cur-NC. Fig. 7B revealed that the release of NC_ASKP and NC_GA were higher than NC_F68 and NC_WP. In the environment of PBS 7.4, GA and ASKP were easily detached from the Cur-NC, and GA had good hydrophilicity and lipophilicity, which perhaps jointly lead to the fastest and highest release of NC_ASKP and NC_GA. The lowest release rate of NC_F68 was probably due to the fact that F68 could form a certain spatial potential resistance and thick hydration layer around the particles, which made the particles stable and prevented F68 shed from the particles [28].

Fig. 7.

(A) In vitro cumulative release curves of Cur-NC. (B) Significance analysis of the cumulative release within 7 days. Different lowercase letters indicate significant differences (p < 0.05).

3.4. Antioxidant capacity

The imbalance between free radical production and burst will produce oxidative stress, and active free radicals will attack cell membranes and react with serum anti-protease, causing damage to our body. Antioxidants can scavenge excess free radicals in the body and reduce the damage to the organism [39]. As an antioxidant, we explored the antioxidant capacity of Cur by ferric cyanide (Fe³⁺) reducing antioxidant power assay. As shown in Fig. 8A, the absorbance of Cur-NC were higher than that of the RP, indicating the better antioxidant property of Cur-NC. Meanwhile, Fig. 8B demonstrated that the cell viability of Caco-2 cells were all above 90 % when the concentration of Cur was 10 μg/mL (the concentration used as antioxidant), indicating that Cur had good biocompatibility. In general, the biological activity of NC were obviously stronger than that of power, but there were no significant difference between different nanocrystals. According to the Noyes-Whitney equation, the smaller the particle size, the larger the specific surface area, the faster the dissolution rate of particles [9]. This endowed the Cur-NC faster the dissolution rate and more free molecules dissolved in liquid (Fig. 7), and thus played a stronger biological activity than raw power.

Fig. 8.

The Ferric-reducing ability (A) and the cell viability (B) of Cur-NC. Different lowercase letters indicate significant differences (p < 0.05).

3.5. Storage stability

The stability of nanocrystals plays an important role in the transport and storage of bioactive substances [40]. Therefore, we also evaluated the stability of the Cur-NC via the observation of appearance and determination of particle size during one month storage. As shown in Fig. 9A, four Cur-NCs had translucent yellow appearance and were uniformly dispersed in water, which were quite different from the obvious stratification and precipitation in RP, indicating that nanocrystals could improve the solubility and dispersibility of water insoluble bioactive compounds. There were no obvious settlement appeared in Cur-NC except the increase of clarification during the storage, demonstrating the excellent stability of Cur-NC. The increased clarification of nanocrystals may be because of the reversible flocculation, notably, it could be re-dispersed evenly via waggling, stirring or ultrasound. Meanwhile, Fig. 9B exhibited that the particle size of Cur-NC prepared with stabilizers were lower than that of NC_NO. Notably, there were no significant changes of particle size and PDI of each Cur-NC, indicating that Cur-NCs were stable at 4 °C for one month, which was consist with the result of Fig. 9A.

Fig. 9.

(A) The appearances and (B) particle size and PDI of Cur-NC during 30-days storage at 4 °C, the bar chart represents the particle size and the solid circle represents the PDI.

3.6. The additivity in beverages

The solid state of bioactive substances show better stability and more convenience as a precursor for additives, tablets and capsules, compared to the liquid state [41]. As shown in Fig. 10A, Cur-NC powders after lyophilization were fluffier than RP, in which Cur-NC with stabilizers were superior to NC_NO. After re-dispersed these powders in water (∼600 μg/mL, significantly enhanced compared to RP (∼11 ng/mL)), respectively, it presented different phenomenon. The Cur-NC solution with stabilizers all displayed homogeneous while NC_NO had weak stratification, and RP sank completely (Fig. 10B). The particle size of Cur-NC re-dispersion solutions were further measured and it showed that the particle size and PDI were slightly larger than that of corresponding fresh Cur-NC (Fig. 10C). On the one hand, this phenomenon may be due to the bridge caused by the hydrogen bond of curcumin. Besides, the process of lyophilization contains removing inter-particle moisture, freezing stress and the drying stress, which leads to the collapse and aggregation of nanocrystals [42]. All in all, Cur-NC in aqueous solutions had the good re-dispersibility of, especially Cur-NC with stabilizers. Qimeng Wang [43] et al. loaded Cur into a nanocarrier made of partially hydrolyzed α-lactalbumin (α-lac) self-assembly, thus improving the redispersibility of Cur, similar to our experimental results.

Fig. 10.

(A) The lyophilized powder appearances of the lyophilized Cur-NC. (B) The re-dispersibility and (C) particle size and PDI of fresh Cur-NC and lyophilized powers of Cur-NC in water. Concentration of Cur is 600 μg/mL.

Besides, emulsions (e.g milk) are also used commonly as carriers for functional beverages in addition to aqueous solutions. Therefore, we prepared three emulsions (blank emulsion, Emu), Emu with fresh Cur-NC solution (Emu_FNC) and Emu with lyophilized Cur-NC powder (Emu_LNC) to evaluate the redispersibility of Cur-NC in emulsions here. As shown in Fig. 11A, Emu, Emu_FNC and Emu_LNC had a single-peak distribution, indicating the uniform droplets size. Compared to Emu, Emu_FNC and Emu_LNC showed smaller size (Fig. 11B, 400 nm vs 600 nm), which may be due to the small particle sizes of Cur-NC. The appearance of emulsions in Fig. 11C displayed that Emu_LNC and Emu_FNC had uniform milky yellow color, and there were no obvious delamination and sedimentation during the 7-days storage, showing a good stability. These data demonstrated that Cur-NC could be an excellent additive state for functional beverages. In addition, based on the reference of Cur, other insoluble active substances (e.g., resveratrol and β-carotene) could also be prepared into nanocrystals and lyophilized powders and thus be added into beverages as powerful additives for functional beverages.

Fig. 11.

(A) The droplet distribution (B) size and PDI (the bar chart represents the particle size and the solid circle represents the PDI) and (C) appearance of Emu_LNC, Emu_FNC and Emu. Concentration of Cur is 600 μg/mL. Different lowercase letters indicate significant differences (p < 0.05).

4. Conclusion

In this work, we successfully prepared various Cur-NC with high loading capacity (more than 80 %) via ultrasound-assisted method to improve the water solubility and availability of Cur. These Cur-NC were stabilized with little amounts of F68, GA, ASKP and WP, respectively, and showed rod-shape and nanosize structures. The uniform particle size (about 300 nm) and zeta potential (∼−20 mV) endowed Cur-NC good stability during 30-days storage at 4 °C. Notably, the high water solubility and appropriate release of Cur-NC greatly improved the biological activity of Cur. Coupled with superior dispersibility, the Cur-NC greatly broadened their application as an additive or delivery system. Overall, this study demonstrated the feasibility of polysaccharides and proteins as stabilizers of Cur-NC, and also provided novel and efficient approach for improving the bioactivities of the liposoluble compounds.

CRediT authorship contribution statement

Dan Yang: Formal analysis. Lili Wang: Writing – original draft, Methodology, Data curation, Conceptualization. Linxuan Zhang: Data curation. Mengqi Wang: Data curation. Dan Li: Formal analysis. Ning Liu: Data curation. Dechun Liu: Funding acquisition, Data curation. Mouming Zhao: Supervision. Xiaolin Yao: Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article: