Highly bioavailable curcumin preparation with a co‐grinding and solvent‐free process

Abstract Curcumin (Cur.) is a natural product isolated from the rhizome of Curcuma longa, with a variety of biological and pharmacological activities in food and pharmaceutical products. However, curcumin's poor solubility in water greatly limits its bioavailability and clinical applications. In this study, co‐grinding curcumin with food additives produced a mixture, which was evaluated for the solubility in water, dissolution, material morphology, in vivo bioavailability, cell uptake and entry mechanism. We tested 9 food additives in total and found that poloxamers performed the best. The 2 co‐grinding mixtures Cur./Kolliphor® P407 and Cur./Kolliphor® P188 with high drug loading at 65.5% significantly improved the curcumin aqueous solubility, subsequently increased its intestinal epithelial cell uptake and oral bioavailability. The relative bioavailabilities for the 2 co‐grinding mixtures were 309% and 163%, respectively, compared with curcumin API. Co‐grinding process has a broad application prospect and is suitable for industrial production.

Although these methods can improve the OB of curcumin to some extent, their wider application is hindered by some disadvantages exposed in commercial production, such as complicated prescription and sophisticated process, high cost price, and low drug-loading capacities. For example, when completely dissolved in microemulsion/ nanoemulsion/SEDDS formulations, the amount of curcumin is limited by its solubility in surfactant-oil mixtures. Being incorporated in soft gelatin capsules, the final cost of microemulsion/nanoemulsion/ SEDDS preparations will raise. With further solidifying of these formulations, the concentration of curcumin in such preparations decreases even more, resulting in larger volume in these solid dosage forms (including tablets or capsules) to provide a therapeutic dose, thereby reducing patient compliance. Furthermore, some frequently used excipients have irritation to gastric mucosa (such as some absorption promoters and a large number of surfactants). Similarly, the preparation of curcumin polymeric micelle, liposomes, nanoparticles, and solid dispersions usually involves the utilization of organic solvents and drying process, such as spray-drying or freeze-drying with excipients, which leads to increase in production cost and commercial price (Madhavi & Kagan, 2014). Some of the steps are also environmentally unfriendly. Therefore, further research and improvement are required.
Poly (ethylene oxide)-poly (propylene oxide) amphiphilic block copolymers (Poloxamers) serve well as pharmaceutical excipients because of their highly tunable association properties, low toxicity, and ability to functionalize (Bodratti & Alexandridis, 2018). Poloxamer 407 and Poloxamer 188 are the most widely used excipients in the field of pharmaceutical preparations. They usually served as matrix, solvent, stabilizer, emulsifier, absorption promoter, solid dispersion carrier, etc., to control drug release, improve drug stability, increase the solubility of insoluble drugs, and enhance drug bioavailability. So they are considered as ideal carriers for hydrophobic drugs including curcumin.
Here, we report a new and simple co-grinding process via solvent-free approach to improve the OB of curcumin with high drug loading at 65.5%. For the first time, poloxamer with good safety is employed as the excipient to mix and co-grind with curcumin to obtain a solid curcumin raw material. Compared with curcumin in specific dosage forms, this preparation can be used for the further processing of various dosage forms such as tablets, capsules, granules, or pills with a broader application prospect. What's more, being simple and cost-effective, the prescription process is more suitable for industrial production.

| Establishment of HPLC analytical method for curcumin in vitro
An appropriate amount of curcumin standard was weighed and dissolved in methanol to prepare curcumin methanol solution with a concentration of 50 mg/ml. Zero was corrected with methanol, UV scanning was performed within the wavelength range of 200-800 nm, and the maximum absorption wavelength was selected as the detection wavelength.
By exploring different mobile phases and ratios, curcumin was required to be separated from methoxycurcumin and dimethoxycurcumin at baseline, and the peak time was less than 25 min (Syed et al., 2015).
A proper amount of curcumin API was weighed and dissolved in methanol to prepare mother liquor with a concentration of 1 mg/ml, which was successively diluted with methanol to different concentrations ranging from 500 μg/ml to 0.122 μg/ml. The sample was injected to HPLC according to the chromatographic conditions explored above, and the peak time and peak area were recorded, respectively. The standard curve was drawn with the concentration as the horizontal coordinate and the peak area as the vertical coordinate.

| Determination of solubility
The solubility of curcumin was studied in different solvents, including ultrapure water, 0.1 M hydrochloric acid, and phosphate buffer (PBS) with a pH of 6.8. Excessive curcumin API was added to 10 ml centrifuge tubes containing different solvents and were oscillated for 72 hr at 100 rpm in a constant temperature shock box at 37°C.
The supernatant was removed and analyzed by HPLC after highspeed centrifugation at 13,000 rpm for 10 min.

| Determination of oil-water partition coefficient
The absorption, distribution, metabolism, and excretion (ADME) of the drug in the body are closely related to its water solubility and lipid solubility. Therefore, the oil-water partition coefficient (P) of curcumin was measured, that is, the ratio of the drug concentration in the oil phase and water phase. In this experiment, the octanolwater system was adopted. By measuring the drug concentration c 1 in octanol saturated with water and c 2 in water saturated with octanol (the two phases have the same volume), formula p = c 1 /c 2 was used to calculate the oil-water partition coefficient.
Specifically, measure 10.00 ml water-saturated octanol solution of curcumin API in a 50 ml centrifuge tube with a pipette accurately, and then measure 10.00 ml water saturated with octanol.
After being sealed, the centrifugal tube was oscillated for 72 hr at 100 rpm in a constant temperature shock box at 37°C. Finally, the oil phase and water phase were separated, the oil phase was diluted with methanol and analyzed by HPLC while the water phase was analyzed directly by HPLC.

| Solubilization effect of different excipients
The solubilization effect of 9 excipients (the mass ratio of curcumin API and excipients is 1:1) was investigated to screen excipients with superior solubilization effect for the next experiments.
Different excipients include PVP, Chitosan, Kolliphor ® P407, PEG 6,000, Phospholipid, Kolliphor ® HS15, Kolliphor ® ELP, Soluplus ® , and Kolliphor ® P188 were weighted into separate 2 ml test tubes, and equal mass of curcumin API was added. Two small steel balls were added to each tube, which was placed in a grinder for grinding at a frequency of 60 Hz for 2 min (Automatic Grinding Instrument (JXFSTPRP24, Shanghai Jingxin Industrial Development Co., Ltd.)).
The drug-loading capacity (DL) is calculated according to the formula DL = m Curcuminoids / (m Curcuminoids + m Excipient ) *100%, m, mass. 70 mg of each ground powder was placed in a 10-ml centrifuge tube with 7 ml ultrapure water in it, respectively. The centrifuge tube was oscillated for 72 hr at 100 rpm in a constant temperature shock box at 37°C. The upper liquid was centrifuged at 13,000 rpm for 10 min, and the supernatant was taken for HPLC analysis.

| Scanning electron microscope (SEM)
Scanning electron microscope can obtain more abundant characteristic information of sample surfaces by scanning the sample with electron beams. SEM was used to observe the surface characteristics of the curcumin API and its mixture after grinding with excipients, so as to see whether there is a difference in physical morphology between them.

| Dissolution test in vitro
Two kinds of excipients with superior solubilization effect under 2.6 were selected for the dissolution test. Superior solubilization means the solubility of curcumin is above 10 μg/ml. The dissolution medium was 900 ml of a 0.5% sodium dodecyl sulfate (SDS) solution. The rotation speed is 75 rpm, and the temperature is 37°C. 20 mg of Soluplus ® curcumin co-grinding mixture, Kolliphor ® P188 curcumin co-grinding mixture, Kolliphor ® P407 curcumin co-grinding mixture, and 10 mg curcumin API were put into different dissolution tanks, which were sampled 3 ml in the same position with a sampling needle for 5 min, 10 min, 20 min, 40 min, 1 hr, 1.5 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, and immediately supplemented dissolution cylinder with isothermal and isovolumetric media (0.5% SDS). The removed samples were filtered by 0.22μm microporous filter membrane and analyzed by HPLC.

| Cell culture
Caco-2 cells were cultured in DMEM high-glucose culture solution (containing 10% fetal bovine serum, 1% penicillin-streptomycin mixed solution, 1% nonessential amino acid) and incubated in an incubator at 37°C and 5% CO 2 . The solution was changed every two days and digested with 0.25% EDTA-trypsin when the growth was over 80%. HT-29 cells and Raji-B cells were cultured in R1640 culture solution (containing 10% fetal bovine serum and 1% mixed solution of penicillin-streptomycin) and incubated in an incubator at 37°C and 5% CO 2 . The solution was changed every two days. When the growth was over 80%, they were passaged at 1:5.

| Cell tight junction test (ZO-1 cell immunofluorescence test)
Caco-2 cells at logarithmic growth stage were seeded in confocal dishes at a density of 2 × 10 5 cells/ml. The culture solution was changed every other day during the first week and daily from the second week. After 14 days of culture under the condition of 37°C and 5% CO 2 , the culture solution was removed and the Caco-2 cells were washed with PBS for three times, and 1 ml of the drugcontaining culture solution was added there to have a curcumin concentration of 20 μg/ml (drug in group 2: curcumin; drug in group 3: curcumin and chitosan). After 4 hr of co-incubation, the drug-containing solution was removed, Caco-2 cells were washed with PBS for three times, and 4% paraformaldehyde was used to fix the cells for 20 min. After the fixation was completed, PBS was used to wash cells twice for 5 min each time. The immunostaining blocking solution was added and blocked for 60 min. After blocking, PBS was used to wash cells twice for 5 min each time. The ZO-1 primary antibody was added and incubated at 37°C for one hour. Washed with PBS 3 times for 5 min each time. The goat antirabbit secondary antibody was added and incubated at 37°C for one hour, and washed with PBS 3 times for 5 min each time. 1 ml of ready-to-use DAPI was added to each well, and after incubation for 8 min, PBS was used to wash cells 3 times. Cells were observed under a confocal microscope.

| Cell uptake (qualitative)
Caco-2 cells at logarithmic growth stage were seeded in confocal dishes at a density of 4 × 10 4 cells/ml. The culture solution was changed every other day during the first week and daily from the second week. After 14 days of culture under the condition of 37°C and 5% CO 2 , the culture solution was removed, and cells were cultured for 4 hr with a drug-containing serum-free solution (curcumin concentration: 20 μg/ml). After three times of washing with PBS in dark, the fluorescence of intracellular curcumin in different groups was observed and photographed under a confocal microscope. The stronger the fluorescence was, the more the uptake was indicated (Kunwar et al., 2008).

| Cell uptake (quantitative) and cell entry mechanism study
Confocal microscope can visually observe the relative amount of curcumin ingested by cells, but it cannot accurately show the difference of curcumin ingested by different groups of cells within 4 hr, so quantitative study on the intake of curcumin is needed. To calculate the cell uptake rate of drugs, flow cytometry with high sensitivity was used to determine the fluorescence value of curcumin for quantitative determination.
Here, we used a three-cell model: Caco-2 cells and HT-29 cells at logarithmic growth stage were seeded in 6-well plates at densities of 7 × 10 4 cells/ml and 3 × 10 4 cells/ml, respectively. The culture solution was changed every other day during the first week and daily from the second week. The culture lasted for 14 days. In the last 3 days, Raji-B cells at logarithmic growth stage were inoculated at the density of 1 × 10 4 cells/ml. After 14 days, the culture solution was removed and the drug-containing serum-free solution (curcumin concentration: 20 μg/ml) was added into plates and cultured cells for 4 hr. After that, the cells were removed in dark, washed with PBS for three times, and digested with 0.25% EDTAtrypsin. The serum-containing culture solution was added to terminate the digestion. The cells were centrifuged at 1,200 rpm for 5 min, washed with PBS for two times, and then resuspended and sieved. Using the characteristics of curcumin autofluorescence, the cell uptake rate of curcumin was quantitatively determined by  To explore cell entry mechanism, after 14 days' culture, the culture solution was removed and the inhibitor was added for half an hour (sodium azide concentration was 1 mg/ml, ketoconazole concentration was 10 μg/ml, and verapamil concentration was 300 μg/ ml), and then, the inhibitor was removed, and curcumin-containing serum-free solution (curcumin concentration: 20 μg/ml) was added.
The following steps including cell digesting, washing, and detection by flow cytometry are the same as above.

| Establishment of HPLC analytical method for curcumin in blood samples
The nitrendipine was used as an internal standard to quantify curcumin in plasma. By exploring different mobile phases and ratios, curcumin was required to be separated from methoxycurcumin and dimethoxycurcumin at baseline, and the peak time was less than 25 min.

| Pharmacokinetics study
The male Sprague Dawley rats were divided into 4 groups (n = 3).
Group 1 was administered orally 50 mg/kg body weight (BW) curcumin solution (preparation method: curcumin and P407 with a mass ratio of 1:9 were completely dissolved in ethanol. Rotary evaporation under reduced pressure was used to remove ethanol for later use, and saline was added to obtain a clear and transparent solution before gavage administration). Group 2 received 75 mg/kg BW grinding mixture of curcumin and P407 dissolved in saline, and group 3 received 75 mg/kg BW grinding mixture of curcumin and P188 dissolved in saline. Group 4 was administrated orally 50 mg/kg BW curcumin API dissolved in saline. In summary, all rats were given 50 mg/kg BW of curcumin in different preparations.
After administration, a 0.5 ml blood sample was collected into tubes containing heparin sodium from infraorbital venous plexus in 15 min, 30 min, 45 min, 1 hr, 2 hr, 3 hr, 4 hr, 5 hr, 6 hr, and 9 hr intervals. After centrifugation at 4,000 rpm for 5 min, 200 μL of plasma was taken and added to 100 μL nitrendipine dissolved in ethyl acetate solution (250 μg/ml), and then, 1 ml of ethyl acetate was added and mixed in a vortex for 2 min. The supernatant (organic layer contained curcumin and nitrendipine) was obtained by centrifugation at 12,000 rpm for 5 min. After evaporating the ethyl acetate, 100 μL of methanol was used to redissolve it. The standard curve solution was treated using the same method.

| Curcumin HPLC analytical method
The optimized condition of the curcumin HPLC analytical method for plasma sample analysis. The liquid chromatography diagram is shown in Figure 1.

| Solubility and oil-water partition coefficient of curcumin
The solubility of curcumin in water, PBS, and 0.1mol/L hydrochloric acid was 1.622, 0.310, and 0.675 μg/ml, respectively. The water solubility of curcumin was proved to be extremely poor.
The solubility of curcumin was 0.103 μg/ml in octanol-saturated water and 1,487.0 μg/ml in water-saturated octanol. p = 14,504.67, so the calculated oil-water partition coefficient of curcumin log P was 4.16. The results showed that curcumin had strong lipophilic property and poor hydrophilicity. It suggests that improving the solubility of curcumin may be a key factor in improving the oral absorption and bioavailability of curcumin.

| Solubilization effect of different excipients
When directly mixed together without grinding process, the complex of curcumin and excipients may lack stability due to nonuniformity in their density, particle size and surface properties, especially in transit or after long-term storage. Now, the process of mixing and grinding curcumin with excipients, on the one hand, averages their differences in particle size and reduces the possibility of material layering. On the other hand, the two are brought into full contact by grinding, so that the surface static of curcumin can be decreased by the surface activity of excipients, which is also beneficial to maintaining the stability of the mixture. Besides, the close combination of excipients and curcumin can form a locally water-soluble microenvironment around curcumin, improving the solubility of curcumin and facilitating dissolution during the digestion process. The dissolution experiment in vitro (Fig. S1) showed that the release of curcumin in co-grinding mixture (micronized) is faster than which without grinding (directly mixing, nonmicronized), and the dissolution difference was particularly obvious in the first hour. The rapid dissolution of curcumin brought by the grinding process was beneficial to its absorption in vivo. A preliminary study showed that the homogeneous material with good dispersion can be obtained by controlling certain parameters as follows: 2 small steel balls, frequency of 60 Hz, and a time of 2 min.
The solubility changes of curcumin after co-grinding with different excipients were shown in Table 1

| Scanning electron microscope (SEM)
SEM showed that after grinding curcumin with P407, Soluplus, and P188, the surface morphology did not change significantly, indicating that no solid dispersions and other preparation forms were formed, which was beneficial to the long-term stability of the mixture of curcumin and excipients. As displayed in Figure 2, surface morphology is irregular particle aggregation.

| Dissolution test in vitro
The results of the dissolution experiment of curcumin API, cur./ Soluplus, cur./P407, and cur./P188 after grinding were shown in Figure 3. At the first 5 sampling points (5 min, 10 min, 20 min, 40 min, and 1h), the dissolution of curcumin in cur./Soluplus co-grinding mixture, cur./P407 co-grinding mixture, and cur./P188 co-grinding mixture was significantly accelerated due to the presence of excipients.
In group cur./P188 co-grinding mixture, the dissolution of curcumin reached over 80% at 10 min and over 90% at 20 min. Based on the

F I G U R E 3
The release of curcumin from Soluplus ® curcumin co-grinding mixture, Kolliphor ® P188 curcumin co-grinding mixture, Kolliphor ® P407 curcumin co-grinding mixture, and curcumin in 900 ml 0.5% sodium dodecyl sulfate (SDS) solution at 75 rpm and 37°C during 6 hr TA B L E 2 Cumulative release rate of curcumin (Cur.) from different co-grinding mixture in the first 40 min (%) above results, we chose P407 and P188 groups for subsequent experiments ( Figure 3 and Table 2).

| Cell tight junction test (ZO-1 cell immunofluorescence test)
It has been reported that chitosan can open tight connections between small intestinal epithelial cells and promote drug absorption (Thanou et al., 2001;Yeh et al., 2011). Our experimental results also confirmed the role of chitosan in opening tight junctions in Caco-2 cells, which was beneficial for the absorption of curcumin (Figure 4).
There are two ways for the material to pass through the small intes-

| Cell uptake and cell entry mechanism study
Curcumin itself can emit green fluorescence under excitation. Under the same fluorescence parameters, we observe the intensity of green fluorescence to reflect the uptake of curcumin by Caco-2 cells.
It can be seen in Figure  The effect of chitosan on cell tight junctions. Caco-2 cells were co-incubated with 20 μg/ml curcumin and chitosan at 37°C for 4 hr and dyed before being observed under confocal microscope. The nucleus was stained blue by DAPI, and cell tight junctions were stained red by ZO-1 primary antibody followed by goat anti-rabbit secondary antibody. Bar indicates 100 μm experiments also confirm that curcumin uptake increased by 22.89, 3.79, and 11.11 times, respectively, after the addition of p-gp-specific inhibitor verapamil (300 μg/ml), energy-dependent transport inhibitor sodium azide (1 mg/ml), and cyp3a4-specific inhibitor ketoconazole (10 μg/ml), indicating that the uptake of curcumin was influenced by p-gp and cyp3a4 enzyme.
And in order to explore the degree of increase in curcumin solubilization (the solubilization limit is completely soluted) in its absorption in vivo, we also added an experimental group of which curcumin was completely dissolved.

| Pharmacokinetics study of curcumin
The curcumin level in plasma isolated from the infraorbital venous plexus of rats was recorded in Figure 7, and their pharmacokinetic parameters were calculated in Table 3 (Gupta et al., 2015), which was more beneficial to the uptake of curcumin. However, the better solubilization F I G U R E 5 Uptake of curcumin in Caco-2 cells. The cells were exposed to Cur., Cur./P407, Cur./P407/cs., Cur./P188, and Cur./P188/cs. co-grinding mixture at 37°C for 4 hr, respectively, and then being visualized by confocal microscopy. Images are at 60×; bar indicates 100 μm F I G U R E 7 The concentration of curcumin in plasma collected from the infraorbital venous plexus of rats (n = 3) orally administered the curcumin formulations (a. Cur, b. Cur./P188 = 2/1 co-grinding mixture, c. Cur./P407 = 2/1 co-grinding mixture, and d. Cur./P407 = 1/9 dissolved in physiological saline). Values were expressed as mean ± SD effect of P407 in rats prevailed, leading to a greater increase in bioavailability. It is worth noting that even if curcumin were completely dissolved, its relative bioavailability would be only 618%.

| CON CLUS ION
In this study, partial mechanisms of curcumin ingested by intestinal epithelial cells were described, and curcumin formulations were investigated (excipients and curcumin were mixed and co-grinded to make a mixture). Kolliphor ® P407 with excellent performance was screened, which significantly improved the solubility and dissolution of curcumin, increased intestinal epithelial cells uptake, enhanced oral bioavailability, and increased cytotoxicity to mammary carcinoma 4T1 (Fig S2,   S3). The preparation has high drug loading, good safety, and flexible form, which can also be further used in combination with metabolic inhibitors such as piperine and further processed into various dosage forms appropriate for different populations. Being simple and costeffective, the prescription process is suitable for industrial production.
In addition, our results showed that the relative bioavailability would be only 618% even if curcumin was completely dissolved. To further improve the oral bioavailability of curcumin, more consideration should be given to protecting curcumin from complex biochemical environments in the gastrointestinal tract such as pH, ions, and metabolic enzymes. This research may lay a good foundation for further development of subsequent curcumin oral preparations.

| E THI C AL RE VIE W
This study was approved by the ethics committee of Zhejiang University.

INFORMED CONS ENT
Written informed consent was obtained from all study participants.

ACK N OWLED G M ENTS
This study was supported by the National Key R&D Program of China (No. 2017YFE0102200). We thank Dr. Zhen Zhao for her linguistic assistance during the preparation of this manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.