Curcumin Is Primarily Distributed in Lung, Spleen and Liver, Metabolized to Glucuronide and Sulfate, and Excreted through Bile and Urine by Using an Amorphous Curcumin Formulation with High Absorbability

Abstract

Curcumin (CUR), a polyphenol, is a promising compound for use in functional foods owing to various biological properties. However, the kinetics of CUR remains unclear because CUR has extremely low water solubility and absorbability. Here, we tried to elucidate the distribution, metabolism, and excretion of CUR by using amorphous CUR, a novel formulation that has dramatically improved water solubility and absorbability. When amorphous CUR was orally administered, CUR was predominantly distributed in the lungs, spleen, and liver, with low levels of accumulation over 24 h. Moreover, most of the CUR metabolites were observed to be glucuronide and sulfate conjugates. Furthermore, CUR was found to be excreted not only in bile but also in urine. Taken together, we have systematically demonstrated the kinetics of CUR by using a highly absorbable CUR formulation. In order to develop functional foods with high quality, it is important to not only evaluate the function and toxicity of CUR but also to correctly understand its kinetics, such as absorption, distribution, accumulation, metabolism, and excretion.

INTRODUCTION

It is said that the world will soon have more older people than children.¹⁾ An aging global population, owing to increased life expectancy, is expected to be accompanied by an overall decline in the QOL and an increase in medical expenses. For example, individuals 65 years of age or older make up 29.1% of the population of Japan as of 2023, resulting in medical expenses reaching a record high.²⁾ Therefore, strategies to maintain health, prevent diseases, and promote recovery from diseases are required to ensure a long and healthy life, improve the overall health of the population, and reduce medical expenses. Moreover, the treatment of diseases should not be the only strategy to ensure the health of the population because drug discovery has become increasingly difficult in recent years. Therefore, it is essential to prevent people from contracting diseases. In this regard, functional foods are expected as a preventive method that can be easily implemented and followed for a long time because they can be consumed on a daily basis.³⁾

Among functional foods, curcumin (CUR), which is the main component of turmeric, has been reported to have various benefits, such as high antioxidant content⁴⁾ as well as anticancer⁵⁾ and anti-inflammatory properties.⁶⁾ CUR has also been reported to contribute to the improvement of memory function.⁷⁾ Therefore, CUR is expected to have preventive effects against various diseases. Thus, some formulations have been developed that improve upon the issues of CUR, which is extremely insoluble in water⁸⁾ and is hardly absorbed in the body.⁹⁾ On the other hand, the safety of CUR when absorbed in higher amounts is not clear. To develop the CUR-based products that are highly safe and efficacious, it is important to understand not only the biological response to CUR but also its kinetics, such as absorption, distribution, metabolism, and excretion. For example, water-insoluble functional food ingredients cannot be absorbed in the body and, therefore, cannot induce any biological response.¹⁰⁾ In addition, no direct function or toxicity is expressed in the tissues where a functional food ingredient is not distributed. Furthermore, elucidating the metabolism and the routes of excretion will lead to an understanding of the functions and toxicity of a functional food ingredient.

However, the kinetics of CUR after gastrointestinal absorption have largely been unknown. In this regard, we have developed a novel amorphous CUR formulation with high absorbability and a blood triglyceride-lowering effect.¹¹⁾

Here, we aim to elucidate (1) tissue distribution and accumulation, (2) metabolism profile, and (3) route of excretion, after gastrointestinal absorption, using the novel amorphous CUR formulation that has high water solubility and absorbability.

MATERIALS AND METHODS

Reagents and Samples

Standard CUR >98% (Nagara Science, Gifu, Japan), emodin (Tokyo Chemical Industry, Tokyo, Japan), CUR-d₆, CUR-glucuronide (Toronto Research Chemicals, Toronto, ON, Canada), CUR powder (AVT Natural Products Limited, India), polyvinylpyrrolidone K30 (PVP) (BASF Japan, Tokyo, Japan), polyglycerol fatty acid esters (PGFEs) (San-Ei Gen FFI, Osaka Japan), and a commercial formulation of CUR (Theracurmin®) (Theravalues, Tokyo, Japan) were purchased as indicated. A β-glucuronidase solution from Helix pomatia, biochemistry grade isoflurane, LC/MS grade acetonitrile, formic acid, and methanol were purchased from Wako Pure Chemical Corporation (Osaka, Japan).

Preparation of Amorphous CUR Formulations

CUR powder, PVP, and PGFEs were blended in a ratio of 16.3 : 48.7 : 35 (w/w). The physical mixtures were processed using a twin screw extruder (Process 11 Twin Screw Extruder, Thermo Fisher Scientific, Germany) at a screw speed of 100 rpm, between 100 and 200°C. All extrudates were milled and sieved through a 30-mesh screen.

Dissolution Test

The water solubility of each CUR formulation was analyzed using a dissolution tester (Miyamoto Riken Kogyo, Osaka, Japan). Briefly, 900 mL of phosphate-buffered saline at pH 6.8, as per the Japanese Pharmacopoeia (JP) 2nd fluid, was stirred at 50 r.p.m., at 37°C, and the CUR formulations (10 mg/100 mL CUR) were added to the JP 2nd fluid. The JP 2nd fluid containing CUR was collected after 30, 60, 120, and 360 min and filtered through a 0.2-μm membrane filter. Dissolved CUR concentrations were determined using HPLC under the following conditions: an L-column ODS (4.6 × 250 mm, 5 μm, Chemicals Evaluation and Research Institute, Tokyo, Japan) was used for chromatographic separation with a column temperature of 40°C, with a mobile phase comprised of 50% acetonitrile containing 0.1% phosphoric acid, with a flow rate of 1.0 mL/min. The CUR peak was detected at 420 nm absorption using a UV–vis detector (Agilent Technologies, CA, U.S.A.). Each solubility was evaluated with 0.04 μg/mL as the lower limit of quantitation.

X-Ray Diffraction

The X-ray diffraction (XRD) profile of each CUR formulation was obtained using a diffractometer (Smart Lab, Rigaku Co., Ltd., Tokyo, Japan) with Cu Kα radiation, an X-ray tube voltage of 40 kV, and an X-ray tube current of 30 mA. The diffractograms were recorded in the 2θ range from 5 to 40° at a scan rate of 1°/min.

Animals

Sprague–Dawley rats (male, 7 weeks old), BALB/c mice (male, 7 weeks old), and ICR mice (male, 7 weeks old) were purchased from Japan SLC, Inc. (Shizuoka, Japan). The animals were kept under a 12-h light/dark cycle (lights on at 08:00, lights off at 20:00) at 23°C with ad libitum access to food and water. All animals used in the experiments were euthanized by inhalation of isoflurane. All experimental protocols were performed under conditions approved by the animal research committee of Osaka University, Japan.

Absorption Analysis

Each CUR formulation (100 mg CUR/kg) was orally administered to rats. Blood samples from experimental rats were collected at 0, 0.5, 1, 2, 4, 8, and 24 h after administration. The collected blood was centrifuged at 3000 × g for 15 min at 4°C to prepare plasma samples. β-Glucuronidase in acetate buffer (pH 4) was added to the plasma and incubated for 60 min at 37°C. After incubation, 200 ng/mL emodin, serving as an internal standard, was added to the mixture. After deproteinization using acetonitrile, the samples were vortexed, and centrifuged at 10000 × g for 5 min. The resulting supernatant was dried under nitrogen and redissolved in 100 μL methanol. The CUR concentrations in the solutions were analyzed using LC-tandem mass spectrometry (LC/MS/MS) under the following conditions.

Analysis of Tissue Distribution and Accumulation

BALB/c mice were provided with water containing 1 mg CUR/mL of an amorphous CUR formulation or a commercial CUR formulation. After 3 months or after stopping the treatment for 24 h, samples from their heart, lungs, spleen, testis, brain, kidney, and liver were harvested and stored at –80°C until analysis. From each organ, 1 g of tissue was weighed and homogenized after adding 4 mL of 1% formic acid methanol solution. Then, 500 μL of homogenate was collected and centrifuged at 10000 × g for 5 min at 4°C. The supernatant was dried under nitrogen and redissolved in 200 μL of 80% methanol and then centrifuged at 10000 × g for 5 min at 22°C. The supernatant was filtered through a 0.45-μm membrane filter and used as the solution for LC/MS/MS analyses.

Evaluation of Metabolites

Amorphous CUR formulation or commercial CUR formulation (100 mg CUR/kg) or water as a control was orally administered to ICR mice. Peripheral blood samples from experimental ICR mice were collected from the jugular vein at 5, 10, 30, and 60 min after administration. To collect portal vein blood, mice were anesthetized with isoflurane and their abdomens were incised. Portal vein blood was collected at 5, 10, 30, and 60 min after administration. After each collection, the mice were euthanized. The collected blood was centrifuged at 3000 × g for 15 min at 4°C to prepare plasma samples.

For CUR analysis in plasma, 50 μL of plasma sample was mixed with 10 μL of acetate buffer (pH 4), 25 μL of 200 ng/mL emodin as an internal standard, and 500 μL of acetonitrile. The solution was centrifuged at 10000 × g for 5 min at 22°C after mixing. The supernatant was dried under nitrogen and redissolved in 200 μL of 80% methanol and then used as the solution for LC-MS/MS analyses.

For CUR glucuronide analysis in plasma, 50 μL of plasma sample was mixed with 450 μL of ultrapure water and 250 μL of acetonitrile. The solution was centrifuged at 10000 × g for 5 min at 22°C after mixing. The supernatant was dried under nitrogen and redissolved in 200 μL of 10% methanol and then centrifuged at 10000 × g for 5 min at 22°C. The supernatant was filtered through a 0.45-μm membrane filter and used for LC-MS/MS analyses.

Evaluation of Excretion

Amorphous CUR formulation or commercial CUR formulation (100 mg CUR/kg) was orally administered to rats. Urine and fecal samples from experimental rats were collected at 0, 1, 2, 4, 9, 12, 24, 36, and 48, and at 0, 12, 24, 36, and 48 h, respectively, after administration.

For urine pretreatment, 100 μL of urine was taken and 10 μL of acetate buffer (pH 4) was added. Only if enzymatic treatment was carried out, 25 μL of β-glucuronidase was added to the urine sample and incubated at 37°C for 1 h. Then, 50 μL of 200 ng/mL CUR-d₆, as an internal standard, and 500 μL of ethyl acetate–methanol (95 : 5, v/v) solution were added to the mixture. The solution was centrifuged at 10000 × g for 5 min at 4°C after mixing. The supernatant was dried under nitrogen and redissolved in 200 μL of 80% methanol and centrifuged at 10000 × g for 5 min at 22°C. The supernatant was filtered through a 0.45-μm membrane filter and used for LC/MS/MS analyses.

For feces pretreatment, the collected feces samples were homogenized by adding water in twice the amount of the feces, and 10 μL of acetate buffer (pH 4) was added to this. Only if enzymatic treatment was carried out, 25 μL of β-glucuronidase was added to the feces sample and incubated at 37°C for 1 h. Then, 50 μL of 200 ng/mL CUR-d₆, as an internal standard, and 500 μL of ethyl acetate–methanol (95:5, v/v) solution was added to the mixture. The solution was centrifuged at 10000 × g for 5 min at 4°C after mixing. The supernatant was dried under nitrogen and redissolved in 200 μL of 80% methanol and centrifuged at 10000 × g for 5 min at 22 °C. The supernatant was filtered through a 0.45-μm membrane filter and used as the solution for LC/MS/MS analyses.

LC/MS/MS Conditions for Analysis of CUR

LC/MS/MS analysis was performed using a Waters Acquity ultra-performance liquid chromatography (UPLC) system coupled to a Quattro Premier XE mass spectrometer (Waters, Milford, MA, U.S.A.) equipped with an electrospray interface (ESI). Chromatographic separation was performed using an Acquity UPLC® BEH C18 column (1.7 μm, 100 × 2.1 mm i.d.) from Waters. The column was maintained at 40°C. The mobile phase consisted of solvent A (ultrapure water with 0.1% formic acid) and solvent B (acetonitrile). The flow rate was 0.3 mL/min throughout the whole separation. The LC/MS elution program was similar to that of HPLC elution program, which is indicated in Table 1. The mass spectrometer was operated in the positive ESI mode. MS parameters for the analysis were as follows: ESI source block and desolvation temperatures, 120 and 350°C, respectively. The selected reaction monitoring transitions for determining CUR, emodin, CUR-d₆, and CUR-glucuronide were m/z 369.17 > 176.9, m/z 269.01 > 244.9, m/z 375.51 > 180.11, m/z 545.45 > 117.00, respectively.

Table 1. Gradient Profile Applied in LC/MS/MS Method

(1)		(2)		(3)		(4)
Time (min)	% B	Time (min)	% B	Time (min)	% B	Time (min)	% B
0	50	0	35	0	0	0	10
4	95	8	95	8	95	8	95
7	95	11	95	11	95	11	95
7.1	50	11.1	35	11.1	0	11.1	10
10	50	14	35	14	0	14	10

(1) Analysis of CUR concentration in tissue and blood, (2) analysis of CUR glucuronide in blood, (3) analysis of CUR metabolites in blood, and (4) analysis of CUR and CUR glucuronide concentration in urine and feces.

RESULTS

Physical Properties, Water Solubility, and Absorbability Evaluation in Amorphous CUR Formulation

To analyze the physical properties of our amorphous CUR, the crystallinity of the amorphous CUR formulation was compared to unformulated CUR powder and commercial CUR formulation, which is reported as a water solubility-improved formulation. XRD analysis showed that many peaks derived from CUR crystals were observed in CUR powder and commercial CUR. On the other hand, the peaks disappeared in amorphous CUR. The data suggested that the amorphous CUR formulation we have devised was amorphous (Fig. 1a).

Fig. 1. Evaluation of Crystallinity, Dissolution, and Absorption of Each CUR Formulation

Various profiles of amorphous CUR, commercial CUR, or CUR powder were evaluated. (a) X-ray diffraction profiles of each CUR formulation were performed using a diffractometer (Smart Lab, Rigaku Co., Ltd., Tokyo, Japan). The diffractograms were recorded in the 2θ range from 5 to 40° at a scan rate of 1°/min. (b) Dissolution profiles of each CUR formulation were evaluated. Each CUR formulation (10 mg of CUR) was added to 100 mL of phosphate buffer (pH 6.8) at 37°C. The dissolution process was monitored for 6 h using a dissolution tester. The dissolved concentration of CUR was quantified using high-performance liquid chromatography. (c) The absorbability of each CUR formulation was evaluated. Each CUR formulation (100 mg CUR/kg) was orally administered to rats. CUR was extracted from blood collected at 0, 0.5, 1, 2, 4, 8, and 24 h and quantified using LC/MS/MS. Data represent the mean ± standard error.

Next, the water solubility of the amorphous CUR formulation was compared to unformulated CUR powder and commercial CUR formulation. The dissolution test of each CUR formulation showed that the water solubility of amorphous CUR was much higher than that of commercial CUR and CUR powder (Fig. 1b). The data suggested that the amorphous state of each formulation was also verified by its water solubility.

Finally, the absorbability of the amorphous CUR formulation was compared to unformulated CUR powder and commercial CUR formulation. CUR concentration analysis in the plasma showed that the area under the blood concentration–time curve (AUC) in the CUR powder-treated rat was only 489 ng/mL·h. Moreover, the AUC in the commercial CUR formulation-treated rat was 4754 g/mL·h. In contrast, the AUC in the amorphous CUR formulation-treated rat was 30344 ng/mL·h (Fig. 1c). These data suggest that the amorphous CUR might be absorbed 6.4 or 62.1 times more than the commercial CUR or the CUR powder, respectively, although it will be necessary to calculate the bio-availability by analyzing the AUC in intravenous injection.

Taken together, the amorphous CUR formulation we have devised is more water soluble due to amorphization, so the absorption is also improved. Therefore, the amorphous CUR formulation is suitable for analyzing the kinetics of CUR after absorption.

Tissue Distribution and Accumulation Analysis of CUR Absorbed in the Gastrointestinal Tract

In order to evaluate the kinetics of CUR absorbed in the gastrointestinal tract, we analyzed the tissue distribution of CUR in BALB/c mice after providing them with water containing amorphous CUR or the commercial CUR for 3 months. In the group that received commercial CUR, the presence of CUR was hardly detected in any tissue. On the other hand, in the group that received amorphous CUR, it was observed that CUR was predominantly distributed in the liver, lungs, and spleen (Fig. 2a). The data suggested that administration of amorphous CUR, which has high absorbability, led to an understanding of the tissue distribution of CUR. Next, in order to evaluate the accumulation of CUR distributed in each tissue, we analyzed the amount of CUR distributed in each tissue after stopping CUR intake for 24 h. LC/MS/MS analysis showed that almost no CUR was present in any tissue in the groups that received either commercial CUR or amorphous CUR (Fig. 2b). These findings suggested that the distributed CUR is less likely to accumulate in each tissue and may be metabolized and excreted within 24 h.

Fig. 2. Tissue Distribution of CUR after Amorphous CUR or Commercial CUR Were Orally Administered

(a) After male BALB/c mice were provided with water containing 1 mg CUR/mL of amorphous CUR or commercial CUR for 3 months, CUR concentration in each tissue (brain, heart, kidney, liver, lungs, spleen, and testis) was determined using LC/MS/MS. (b) After stopping the CUR intake for 24 h from (a), CUR concentration in each tissue was determined. Data represent the mean ± standard error of the mean (n = 3).

Taken together, a high potential for safety can be inferred from the low accumulation of CUR in the distributed tissues, although the functional expression may be transient.

Identification of CUR Metabolites and Analysis of the Metabolic Processes

Given the low level of tissue accumulation of CUR, it was inferred that the CUR was rapidly metabolized. CUR-derived metabolites contained in the blood were analyzed 1 h after a single administration of the amorphous CUR to ICR mice. LC/MS/MS analysis showed that no peak was observed in the control group. Peaks with retention times of 3.5, 3.9, 4.6, 5.1, and 5.8 min were detected in the group that received amorphous CUR (Figs. 3a, 3f). Thus, a precursor ion that produces fragment ions with an m/z of 134, which are characteristic of CUR, was next scanned. Precursor ion scan analysis showed that precursor ions with m/z of 623.4, 447.5, 543.5, and 367 were detected in only the group that received amorphous CUR. Furthermore, the retention time of each precursor ion matched that of peaks in Fig. 2f (Figs. 3g–3j). Considering the molecular weight (MW) of these compounds, CUR (MW, 368) was estimated to be metabolized to CUR glucuronide/sulfate (MW, 625), CUR sulfate (MW, 449), and CUR glucuronide (MW, 545). CUR has already been reported to be metabolized to CUR glucuronide¹²⁾; therefore, these findings can be considered reliable.

Fig. 3. The Chromatograms of ICR Mice Plasma after Oral Administration of Amorphous CUR

(a–e) Water (control) or (f–j) amorphous CUR was orally administered to the mice (100 mg CUR/kg, p.o.). After 1 h, blood was collected from the mice. The peaks were detected only in the group that received amorphous CUR, comprehensively monitored by UV 420 nm (a and f). A precursor ion that produces fragment ions of m/z 134, which are characteristic of CUR, was scanned (b and g: m/z 623.4; c and h: m/z 447.5; d and i: m/z 543.5; e and j: m/z 367).

Next, in order to evaluate the metabolic process, we quantitatively analyzed the amount of CUR and CUR glucuronide, as a representative metabolite, in peripheral blood after single oral administration of the amorphous CUR in ICR mice. LC/MS/MS analysis showed that CUR was hardly detected in peripheral blood. On the other hand, CUR glucuronide was present at significantly detectable levels from 5 min after administration. Finally, the ratio of AUC0–60 min of CUR and AUC0–60 min of CUR glucuronide was approximately 3:97 (Fig. 4a). The data suggested that CUR was metabolized before exposure to peripheral blood because CUR was hardly detected during the early phase after administration, but substantial levels of CUR glucuronide were detected. We also focused on the portal vein blood that connects the small intestine to the liver and receives no first-pass effect in the liver. Quantitative analysis of CUR and CUR glucuronide in the portal vein blood showed that CUR glucuronide was present at significantly higher levels than CUR, similar to the profile observed in peripheral blood (Fig. 4b). The data suggested that a large amount of CUR had already been metabolized in the small intestine.

Fig. 4. CUR Concentration–Time Profiles in Peripheral Blood and Portal Blood of ICR Mice after Oral Administration of Amorphous CUR

Amorphous CUR (100 mg CUR/kg) was orally administered to ICR mice. At 0, 5, 10, 30, and 60 min after administration, peripheral blood and portal blood samples were collected from the mice. CUR and CUR glucuronide concentrations in (a) peripheral blood and (b) portal blood were determined using LC/MS/MS. Data represent the mean ± standard error of the mean (n = 3).

Evaluation of CUR Excretion

Bile acid excretion and urinary excretion were assumed as the CUR excretion routes. Thus, after a single oral administration of the amorphous CUR or the commercial CUR to rats, we collected feces and urine at each time point. In order to compare the amount of CUR and CUR glucuronide in feces and urine samples, which have many foreign substances, we focused on the quantitative method with/without glucuronidase treatment. After CUR glucuronide was converted to CUR using glucuronidase treatment, the total amount of CUR and CUR glucuronide was determined by quantifying the amount of CUR by LC/MS/MS. Moreover, the amount of CUR itself was determined by quantifying the amount of CUR without glucuronidase treatment. Consequently, the amount of CUR glucuronide was estimated by subtracting the amount of CUR from the total amount of CUR and CUR glucuronide.

The analysis of the group that received commercial CUR showed that CUR and CUR plus CUR glucuronide were present at negligible levels in urine. In contrast, both CUR and CUR plus CUR glucuronide were present at high concentrations in feces (Figs. 5a, 5c), as previously reported.¹³⁾ In Fig. 5a, the amount of CUR was higher than the amount of CUR plus CUR glucuronide, but the difference was not statistically significant. Therefore, the data suggest that most of the CUR excreted in the feces was not in the form of CUR glucuronide, but CUR itself, with a fecal excretion rate of 56%. Considering the low absorbability of the commercial CUR, most of the CUR was excreted without being absorbed, rather than excreted through bile acids after absorption.

Fig. 5. Excretion Profile of CUR and CUR Glucuronide after Oral Administration of Amorphous CUR or Commercial CUR

Amorphous CUR or commercial CUR (100 mg CUR/kg) was orally administered to rats. Feces and urine samples were collected at 0, 12, 24, 36, and 48, and at 0, 1, 2, 4, 9, 12, 24, 36, and 48 h, respectively. CUR and CUR glucuronide concentrations in feces of the group that received (a) commercial CUR and (b) amorphous CUR, and in urine of the group that received (c) commercial CUR and (d) amorphous CUR were determined using LC/MS/MS. Data represent the mean ± S.E.M. (n = 3). Significance was determined by t-test (*p < 0.05 vs. CUR).

On the other hand, the analysis in the group that received amorphous CUR showed that the presence of both CUR glucuronide and CUR alone was detected in urine, with excretion rates of 0.1% each. Moreover, the levels of CUR plus CUR glucuronide were substantially higher than those of CUR alone (Fig. 5d). The data suggested that both CUR glucuronide and CUR were excreted from the urine, indicating that CUR is well absorbed in the body. Furthermore, the levels of CUR plus CUR glucuronide were equal to or greater than the levels of CUR alone in feces, with the fecal excretion rates of each being approximately 6%. In addition, the levels of CUR plus CUR glucuronide in the group that received the amorphous CUR were lower than those in the groups that received commercial CUR (Fig. 5b). These data suggested that not only CUR but also CUR glucuronide was excreted in the feces after absorption in the group that received amorphous CUR, unlike the commercial CUR, which was not absorbed and was instead excreted in the feces.

Taken together, these findings indicated that CUR was metabolized to CUR glucuronide after absorption and subsequently excreted in the bile. Moreover, the data primarily suggests that CUR is also excreted in the urine as CUR and CUR glucuronides, although the amount was very small.

DISCUSSION

In this study, we evaluated (1) tissue distribution and accumulation, (2) metabolism profile, and (3) route of excretion, after gastrointestinal absorption, using the amorphous CUR with high water solubility and absorbability.¹¹⁾

In the distribution analysis, CUR was detected in various kinds of tissues, such as the lungs, spleen, and liver, when amorphous CUR was administered to mice (Fig. 2a). It has been previously reported that CUR after intravenous administration was distributed in the lungs, spleen, and liver,¹⁴⁾ which is similar to the findings in our study. It has been reported that CUR is distributed in several tissues as it is highly hydrophobic and passively diffuses into tissues.¹⁵⁾ Therefore, CUR could be predominantly distributed in the tissues that receive high blood flow, such as the lungs, which are responsible for the exchange of oxygen in the blood, the spleen, which has hematopoietic and blood storage functions, and the liver, which plays a central role in metabolism. Moreover, it was also observed in this study that the amount of CUR distributed in the lungs and spleen was greater than that in the liver, although, in general, most of the absorbed compounds are mainly distributed from the portal vein to the liver. In this regard, it was reported that the expression of glucuronyl transferase, the enzyme for metabolizing CUR to the glucuronide conjugates, was lower in the lungs and spleen than in the liver.¹⁶⁾ Therefore, although CUR was distributed in the liver, the CUR distributed in the liver was metabolized faster than that in the lung and spleen. As a result, the amount of CUR could be smaller in the liver than in the lung and spleen.

In the metabolism analysis, CUR was mainly metabolized to CUR glucuronide, as was previously reported.¹⁷⁾ In addition, it is known that CUR glucuronide is converted to CUR by glucuronidase derived from the intestinal microflora, which then undergoes enterohepatic cycling.¹⁸⁾ In this study, CUR glucuronide was identified as one of the major metabolites of CUR, which could have partly undergone enterohepatic cycling, because the amount of CUR in portal vein blood at 60 min was higher than that at 30 min (Fig. 4b). Taken together, the data are consistent with previous reports, indicating the reliability of the current findings.

In the excretion analysis, most of the CUR in the group that received commercial CUR was excreted without being absorbed, rather than excreted through bile acids after absorption. On the other hand, when CUR was well absorbed from the gastrointestinal tract in the group that received amorphous CUR, it was observed that CUR was excreted not only in bile but also in urine as CUR and CUR glucuronide. Some researchers have reported that CUR was mostly excreted into the feces and very little was excreted through urine.¹⁹⁾ Although CUR metabolites were not analyzed in these reports, it was considered that CUR, which has low absorbability, was directly excreted through feces, similar to the results observed in the group that received commercial CUR in this study. Taken together, when excretion is analyzed after oral administration, it is necessary to understand the degree of absorption using the metabolites as one of the promising indicators.

CONCLUSION

This study provided new insights into the kinetics of CUR under the following global conditions. It has been reported that CUR after oral administration is distributed in various kinds of tissues, mainly metabolized to CUR glucuronide, and mostly excreted through feces. However, the kinetics of CUR were not completely understood when using of less absorbent CUR formulations.

In this study, using amorphous CUR with improved absorbability, we showed that CUR absorbed after oral administration was mainly distributed in the lungs, spleen and liver, with low levels of accumulation. It was metabolized to not only the glucuronide but also the sulfate forms, and excreted not only through bile but also through urine (Fig. 6).

Fig. 6. Schematic Illustration of the CUR Kinetics after CUR Formulations with Different Absorbability Were Orally Administrated

(a) When commercial CUR with low absorbability was orally administered, CUR was (1) hardly distributed in various tissues and (2) hardly excreted in the urine. Consequently, orally administered CUR was directly excreted in the feces as CUR. (b) When amorphous CUR with high absorbability was orally administered, it was found that CUR was predominantly distributed in the lungs, spleen, and liver with low levels of accumulation, metabolized to both the glucuronide and the sulfate conjugates, and excreted not only through bile but also through urine.

Consequently, in future analyses of CUR function and toxicity, not only CUR absorption but also the distribution, accumulation, metabolism, and excretion data shown in this study should be considered.

Acknowledgments

We appreciate the useful advice and incisive comments from Dr. Y. Goda, Dr. H. Nabeshi (National Institute of Health Sciences), and Dr. H. Akiyama (Hoshi University and National Institute of Health Sciences).

This research was supported, in part, by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 17H04724 to K.N.), by the Health Labor Sciences Research Grant from the Ministry of Health, Labor, and Welfare of Japan (No. H30-syokuhin-wakate-002 to K.N.), and by Pharmaceutical Research Grants from Takeda Science Foundation (No. 2024067194 to K.N.). Moreover, this research was partially supported by the Research Support Project for Life Science and Drug Discovery (Basis for Supporting Innovative Drug Discovery and Life Science Research [BINDS]) from AMED under Grant Numbers 23ama121052 and JP23ama121054.

Conflict of Interest

The results in this research were partly collected using the collaborative research fee from San-Ei Gen F.F.I., Inc.

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