Curcumol (purity ≥99%) was purchased from the National Institutes for Food and Drug Control (Beijing, China), and curzerenone (purity >98%) was obtained from Weikeqi Bio-Technology Co., Ltd. (Chengdu, China). HPLC-grade methanol and acetonitrile were purchased from Merck KGaA (Darmstadt, Germany). Formic acid, ethanol, and Kolliphor HS15 were purchased from Sigma-Aldrich Corp. (St. Louis, MO, United States). Milli-Q water (18.2 MΩ cm) was obtained from a Purelab OptionS7 ultra-pure water system (ELGA LabWater, High Wycombe, United Kingdom).

Male Sprague Dawley (SD) rats (age 6 weeks) were purchased from Zhejiang Vital River Laboratory Animal Technologies Co., Ltd. in this study. (Zhejiang, China). Animal experiments were approved by the Animal Experiment Committee of the Center for Drug Safety Evaluation and Research of Zhejiang University. All animal use and studies complied with all relevant ethical regulations and were approved by the Institutional Animal Care and Use Committee (IACUC) at Zhejiang University (permit number: IACUC-s22-019). The animals were acclimatized in a laboratory environment for 7 days before the experiment. They are kept in plastic cages for 24 h, given free pellet food and water, and put on a 12-h light/dark cycle.

The UPLC system is equipped with an ACQUITY UPLC I-Class (Waters Corp., Milford, MA, United States) and an ACQUITY UPLC BEH C8 (2.1 mm × 100 mm, 1.7 µm, Waters Corp.) for separation. The mobile phase (A) was water containing 0.1% formic acid, and the mobile phase (B) was acetonitrile containing 0.1% formic acid. The gradient elution procedure was as follows; 0.00–1.00 min: 10%B→97%B, 1.00–2.30 min:97%B, 2.30–2.31 min: 97%B→10%B, 2.31–3.00 min: 10%B with a constant flow rate of 0.400 mL/min. At this flow rate, the column was subjected to pressures approaching 9,000–10,000 psi. The injection volume was 20.0 µl, and the autosampler and column temperature were maintained at 4°C and 40°C, respectively.

The mass spectrometry system consisted of a Xevo TQS triple quadrupole MS/MS (Waters Corp.) equipped with an ESI source. Multiple reaction monitoring (MRM) was selected to quantify curcumol in positive ion mode. Curcumol m/z 237.19→135.20 and IS m/z 231.19→89.13 were chosen as the MS transitions for quantification. The full scan precursor ion spectra of Curcumol (A) and product ion spectra (B) are shown in Figure 2. The optimized parameters of the instrument were obtained as follows: capillary voltage, 3.00 kV; ion source temperature, 150°C; desolvation gas temperature, 500°C; collision gas flow rate, 0.15 mL/min; desolvation gas flow rate, 1000 L/h. Data were processed using UNIFI v1.9.3 software (Waters Corp.).

The appropriate amount of each standard substance was dissolved in methanol: water (50:50, V/V) to obtain the concentration of curcumol and IS at 1 mg/mL, respectively. Calibration curves were prepared at concentrations of 10, 20, 50, 100, 200, 500, 1,000, and 2000 ng/mL for the plasma; 5, 10, 50, 100, 200, 500, 1,000, and 2000 ng/mL for the brain, heart, liver, spleen, lung, kidney; 10, 20, 50, 100, 500, 1,000, 2000 and 5,000 ng/mL for the small intestine, and colon. The QC samples were prepared at concentrations of 30, 400, and 1,600 ng/mL for the plasma; 15, 160, and 1,600 ng/mL for the heart, liver, spleen, lung, and kidney; 30, 400, and 4,000 ng/mL for the small intestine, and colon.

About 0.20 mL blood samples were collected and centrifuged (5417R centrifuge, Eppendorf AG, Hamburg, Germany) for 10 min at 3,000 rpm to obtain the plasma. Then, 50 µl of plasma was mixed with 150 µl of acetonitrile (containing internal standard solution at a concentration of 10 ng/mL) and centrifuged at 12,000 rpm for 5 min after a vortex using a multi-tube vortex mixer (Allsheng, Hangzhou, China) for 5 min 100 µl of supernatant was taken for UHPLC-MS/MS analysis. The whole tissues were collected and washed with 0.9% normal saline, and the tissue samples (0.3 g) were homogenized in 0.9% normal saline (1: 3, w/v). Then, 75 μl of tissue homogenate mixed with 150 µl of acetonitrile (containing internal standard solution at a concentration of 10 ng/mL) was treated in the same manner as the plasma sample.

For the pharmacokinetics study, twenty Sprague-Dawley rats were randomly divided into four groups (n = 5). After weighing, single doses of 10 mg/kg, 40 mg/kg, and 80 mg/kg of curcumol were given via intragastric administration (i.g.) for three groups of rats, and the rest group of rats was given a single dose of 2.0 mg/kg via intravenous administration (i.v.). 5% ethanol and 5% Kolliphor HS15 saline were added to form a uniform emulsion for immediate administration. Rat blood was collected from the tail vein into heparinized tubes at 0 (pre-dose), 0.25, 0.50, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0, and 24 h after i.g. Dosing and at 0 (pre-dose), 0.083, 0.25, 0.50, 1.0, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24 h after i.v. Dosing. After centrifugation at 3,000 rpm for 10 min at 4°C, plasma were immediately collected and stored at −80°C until analysis. See “2.5 sample preparation” for the subsequent plasma sample processing. The pharmacokinetic parameters of the study were obtained by pharmacokinetic software DAS 3.0 with non-compartment analysis.

In the distribution experiment, thirty SD rats were orally administered 40 mg/kg curcumol; five rats were sacrificed at 0.5, 1, 2, 4, 8, and 24 h. The tissues were collected, including the heart, liver, spleen, lung, kidney, brain, small intestine, and colon. The tissues were collected rapidly and rinsed thoroughly with normal saline (0.9%). All the tissue samples were wiped dry with filter paper and then stored at −80°C. See “2.5 sample preparation” for the subsequent tissue sample treatment and analysis.

Curcumol plasma protein binding in rat plasma was performed in vitro. Rat plasma was spiked with curcumol working solutions to make plasma samples at three different concentrations (100, 300, and 600 ng/mL). The bound and unbound fractions of curcumol were separated from rat plasma samples by rapid equilibrium dialysis through the RED Device Inserts (Thermo Scientific, United States). Spiked plasma samples in duplicates (300 μl) were placed in the sample chamber with 550 μl dialysis buffer placed into the buffer chamber. After incubating for 120 min at 37°C, 50 µl plasma and 50 µl buffer samples were taken from the sample chamber and the buffer chamber, respectively. Then pieces mixed with 150 µl of acetonitrile (containing internal standard solution at a concentration of 10 ng/mL) were treated and centrifuged at 12,000 rpm for 5 min after a vortex using a multi-tube vortex mixer (Allsheng, Hangzhou, China) for 5min. Supernatant was taken for UHPLC-MS/MS analysis.

To determine the concentration of curcumol in plasma and tissues, we established standard curves for each matrix from corresponding blank tissue and spiking different concentrations of curcumol. Representative chromatograms of curcumol in plasma and tissues of the blank, curcumol spiked with IS, and oral groups are shown in Figure 3. The retention time of curcumol and IS were 1.87 and 1.85 min, respectively, and the total analysis time was in 3 minutes. There was no significant interfering peak observed.

The calibration curves showed good linear linearity at the concentration ranges of curcumol in rat plasma and tissue homogenates (Table 1). Quality control (QC) samples at three concentration levels were determined to evaluate the precision and accuracy. The RSDs of precision ranged from 0.9% to 12.3% for the intra-day and 1.7%–10.7% for the inter-day, and the accuracy was within ±15.0% for both the intra- and inter-day (Table 2). The extraction recoveries at three levels for curcumol were more than 89.0%, and the matrix effect ranged from 90.5% to 113.5% in plasma and tissue homogenates in Table 3.

The stability data of curcumol in rat plasma and tissues are listed in Table 4. The results showed that curcumol was stable for 12 h at room temperature and stored in the autosampler for 12 h. After three freeze-thaw cycles and 30 days at −80°C, no significant degradation was observed.

The validated method for quantifying curcumol was applied to pharmacokinetic studies in rats. The mean plasma concentration-time curves and the main pharmacokinetic parameters of curcumol are shown in Figure 4 and Table 5, respectively. After oral administration of 10, 40, and 80 mg/kg curcumol, the T_max was less than 1.0 h, indicating a rapid absorption of curcumol in rats. Quick elimination of curcumol was observed in the plasma with half-lives ranging from 3 to 5 h. Figure 5 shows that the increasing oral dose increased the C_max and AUC. After an intravenous injection of 2.0 mg/kg curcumol, the pharmacokinetic results showed that AUC_0−t and AUC_0−∞ were 196.15 ± 40.15 and 218.09 ± 42.97 ng/mL·h. The C_max was 233.80 ± 53.73 ng/mL, and the T_1/2 was 1.25 ± 0.42 h. The apparent V_d of curcumol was 17.23 ± 7.24 L/kg, and the CL was 9.45 ± 1.78 L/h/kg. These results indicated a quick elimination of curcumol in plasma and a certain extent of tissue distribution. The absolute oral bioavailability was calculated by comparing the AUCs of curcumol after oral and intravenous administration according to Equation: F (%) = (AUC_ig × Dose_iv)/(AUC_iv × Dose_ig)×100, the absolute bioavailability of curcumol ranges from 9.2% to 13.1% at the three oral doses.

To further reveal the characteristics of curcumol in vivo and potential target organs, the quantitative method in rat tissues was used to investigate the distribution of curcumol in rat tissues after 40 mg/kg curcumol oral administration. According to the pharmacokinetic study results, six time points of 0.5, 1.0, 2.0, 4.0, 8.0, and 24 h were selected for tissue distribution in this experiment, including the distribution phase, equilibrium phase, and elimination phase. The concentrations of the brain, heart, liver, spleen, kidney, lung, small intestine, and colon within 24 h were shown in Figure 6. In most tissues, the highest concentration of curcumol can be observed within 0.5–1 h, suggesting a rapidly and widely distributed. After 8 h, the tissue concentration decreased obviously with no long-term accumulation. As shown in Figure 6, the Tmax of curcumol in the intestine and colon was 0.5 and 2 h, with the Cmax of 6.86 ± 0.65 and 6.37 ± 1.42 μg/g respectively, which were much higher than that in other tissues.

A study on plasma protein binding rate was carried out to reveal the distribution of curcumol in blood. We studied protein binding rates at concentrations ranging from 100–600 ng/mL, containing the concentration 126.36 and 237.60 ng/mL, which were the C_max of curcumol at the effective dose 40 and 80 mg/kg. The rapid equilibrium dialysis system separated bound and unbound drugs from spiked plasma. Calculate the percentage of the curcumol bound as follows: Unbound (%) = (conc. In buffer chamber/conc. In plasma chamber) × 100%; Bound (%) = 100% - Unbound (%). Plasma protein binding of curcumol was found to be high in rat plasma, with 85.6%, 91.4%, and 93.4% at 100, 300, and 600 ng/mL, respectively.