Administration of cyclodextrin-ferulic acid complex significantly increases the plasma concentration of the intact form of ferulic acid in mice

Abstract

We previously demonstrated that dietary ferulic acid (FA) (10 g FA/kg diet) significantly suppressed white adipose tissue (WAT) deposits and body weight gain via induction of beige adipocyte formation in mice. However, a lower dose of FA (2 g FA/kg diet) did not significantly suppress fat deposits or induce beige adipocyte formation due to low bioavailability. In the present study, we found that administration of α-cyclodextrin-FA complex (CD-FA) to mice significantly elevated the FA concentration in plasma and inguinal WAT. In particular, CD-FA administration significantly increased the intact form of FA in plasma, suggesting that it can be expected to enhance the induction of beige adipocyte formation at a lower dose. However, a CD-FA-supplemented diet (12.4 g CD-FA/kg, including 2.0 g FA/kg) did not significantly induce beige adipocyte formation. To reduce the required functional dose of FA, the further development of bioavailable FA formulations should be investigated.

Introduction

Ferulic acid (FA) is one of the phenolic acids in wheat grains and is also found in the form of γ-oryzanol in rice bran, which contains rice hulls, seed skins, and germ. Dietary FA has been reported to significantly reduce body weight gain in genetically obese mice and suppress fat accumulation in white adipose tissue (WAT) and the liver (Wang et al., 2019).

The formation of beige adipocytes is induced in WAT, whereupon they release excess energy in the form of heat via a process mediated by uncoupling protein 1 (UCP1), as in brown adipose tissue (Giralt and Villarroya, 2013). Based on the literature, we hypothesized that the suppressive effect of FA intake on body fat accumulation might be due to the induction of beige adipocyte formation as well as inguinal white adipose tissue (iWAT)-specific thermogenesis. Indeed, we demonstrated that dietary FA-mediated suppression of fat deposits is associated with the induction of beige adipocyte formation and thermogenesis in iWAT (Tanaka and Tsuda, 2023).

To induce beige adipocyte formation in mice, the mean daily intake of FA was calculated to be approximately 1.6 g/kg body weight/day (10 g FA/kg diet, 4 weeks) in our previous study (Tanaka and Tsuda, 2023). The human equivalent dose was calculated to be about 768 mg FA for a 60-kg human (Nair and Jacob, 2016), indicating the need for a high functional dose of FA for beige adipocyte induction. For example, because 100 g of brown rice contains 41.8 mg of FA (Nishizawa et al., 1998), about 1.8 kg of brown rice must be consumed to induce beige adipocyte formation. This is mainly because FA is poorly soluble in water and has low oral bioavailability (Li et al., 2011). To reduce the functional dose of FA required to induce beige adipocyte formation, it is necessary to develop highly bioavailable FA formulations.

In our previous study, highly water-soluble and bioavailable α-monoglucosyl hesperidin (αGH) was synthesized from hesperidin (which has low water solubility and poor oral bioavailability) via transglucosylation, and αGH intake significantly reduced body fat accumulation in mice via beige adipocyte formation (Nishikawa et al., 2019). However, dietary hesperidin did not reduce body fat deposits or the induction of beige adipocyte formation (Nishikawa et al., 2019). The data suggest that improvements in the water solubility and bioavailability of FA might enable the use of FA as a supplement with anti-obesity properties and expand the variety of FA applications in the food industry. Of the possible formulation methods, food-derived factors encapsulated by cyclodextrin (CD) can improve bioavailability. Among CDs, α-CD is highly water soluble, and encapsulation of FA with α-CD would improve its water solubility and bioavailability.

Accordingly, the present study was conducted to examine whether α-CD-FA complex (CD-FA) significantly increases the concentration of FA in mouse plasma and iWAT. In addition, we examined the effect of CD-FA intake on the induction of beige adipocyte formation at a lower dose than in our previous work (Tanaka and Tsuda, 2023).

Materials and Methods

Chemicals  FA (No. H0267; purity > 98 %) was purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). CD-FA (Lot No. FA-A201201) was kindly provided by CycloChem Bio Co., Ltd. (Kobe, Japan); the CD-FA contained 16.1 % FA. Anti-UCP1 (ab209483) antibody was purchased from Abcam (Tokyo, Japan). Glyceraldehyde-3-phosphate dehydrogenase antibody (016-25523) was obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan).

Animal experiments  The design of the animal experiments was approved by the Animal Experiment Committee of Chubu University, and their guidelines were followed for the maintenance of all mice used in this work (Permission No. 202110016).

FA concentration in plasma and iWAT after single administration of FA or CD-FA to mice  Male C57BL/6J mice (8 weeks old, n = 40) were obtained from Japan SLC (Hamamatsu, Japan). To wash out the effect of the non-purified laboratory diet, the mice were allowed free access to water and a semi-purified AIN-93G diet. After 7 days, mice were divided into two groups (FA group, n = 4 per indicated time point; CD-FA group, n = 4 per indicated time point), and food was withheld for 24 h, after which FA (70 μmol FA/kg body weight) (Zhao et al., 2003) or CD-FA (84 mg CD-FA/kg body weight, including 70 μmol FA/kg body weight) dissolved in saline was orally administered to mice by direct stomach intubation. Blood samples were collected under anesthesia (isoflurane) using a syringe containing heparin at 15, 30, 60, 120, or 240 min after administration and plasma was removed by centrifugation and stored at -80 °C until use. iWAT was also removed, immediately frozen in liquid nitrogen, and stored at −80 °C until use.

FA levels in plasma and iWAT were determined according to previous reports (Tanaka and Tsuda, 2023; Zhao et al., 2003). To measure the intact form of FA in plasma, methanol was added to the plasma to remove protein. After centrifugation, the supernatant was carefully evaporated to dryness in vacuo. The dried extract was dissolved using 20 % acetonitrile in water containing 10 mM phosphoric acid, and the FA concentration was analyzed by high-performance liquid chromatography (HPLC) using 10 mM phosphoric acid in 20 % acetonitrile as the solvent and an ODS column (Luna C18 (2), 4.6 × 150 mm; Phenomenex, Torrance, CA). UV was detected at a maximum absorption wavelength of 320 nm, which is specific for FA (Tanaka and Tsuda, 2023; Zhao et al., 2003). An aliquot of plasma was enzymatically hydrolyzed (β-glucuronidase/sulfatase type HP-2; Sigma-Aldrich, St. Louis, MO) and methanol was added to remove protein. After centrifugation and concentration of the supernatant, the level of total FA (intact and the β-glucuronide/sulfate-conjugated form) was analyzed as described above.

To determine the FA level in iWAT, the tissue was homogenized in 0.1 M acetate buffer (pH 5.0), and the homogenate was prepared for determination of the intact form of FA or total FA (intact and glucuronide/sulfate-conjugated form) with or without enzymatic hydrolyzation by β-glucuronidase/sulfatase (Tanaka and Tsuda, 2023; Zhao et al., 2003). After the treatment, methanol was added to remove protein. After centrifugation and concentration of the supernatant, the FA level was analyzed by HPLC as described above. Preliminary experiments indicated adequate recovery (88 %) compared with authentic FA in plasma and iWAT homogenate.

Effects of dietary FA or CD-FA intake on the induction of beige adipocyte formation in mice  Male C57BL/6J mice (4 weeks old) were obtained from Japan SLC. After 7 days, the animals were allocated to one of three groups for 4 weeks: a control diet group (fed a partially modified AIN-93G diet) (Tanaka and Tsuda, 2023), a 0.2-FA group (control diet supplemented with 2 g FA/kg diet), and a 0.2-CD-FA group (control diet supplemented with 12.4 g CD-FA/kg, including 2 g FA/kg). After 4 weeks of feeding, iWAT was isolated and the level of UCP1 protein in iWAT was determined as in our previous work (Tanaka and Tsuda, 2023). The protocol of the immunoblot analysis is shown in the Supplemental Materials. The FA dose (2 g FA/kg diet) was based on our previous study to show that the supplementation level did not affect the induction of UCP1 expression and suppression of fat deposits (Tanaka and Tsuda, 2023).

Statistical analysis  Differences between the means of two groups were analyzed by Student’s t-test (Figs. 1 and 2). Data were analyzed by the Tukey–Kramer test for comparison with the control for the expression level of UCP1 protein (Table S1 and Fig. S1). All data are expressed as the means ± SEM. P values < 0.05 were considered statistically significant.

Fig. 1

Plasma concentrations of total (intact and glucuronide/sulfate-conjugated form) FA (A) and the intact form of FA (B) in mice after single administration of FA or CD-FA. The area under the curve of the total (C) or intact (D) form from 0 to 120 min after FA or CD-FA administration. (E) The intact form/total form ratio based on the area under the curve in the FA or CD-FA group. Data are presented as means ± SEM (n = 4). ^*Significantly different at P < 0.05 compared with the FA group at that time point.

Fig. 2

iWAT concentrations of total (intact and glucuronide/sulfate-conjugated form) FA (A) and the intact form of FA (B) in mice after single administration of FA or CD-FA. The area under the curve of the total (C) or intact (D) form from 0 to 240 min after FA or CD-FA administration. (E) The intact form/total form ratio based on the area under the curve in the FA or CD-FA group. Data are presented as means ± SEM (n = 4). ^*Significantly different at P < 0.05 compared with the FA group at that time point.

Results and Discussion

In this study, both FA and CD-FA were dissolved in saline to precisely compare their bioavailability. Although CD-FA is water soluble and can be dissolved in saline, the use of organic solvents or surfactants can break CD-FA complexation and is therefore not appropriate. FA can be dissolved in organic solvents or water supplemented with surfactant. However, forcing FA to dissolve in such a manner can significantly affect its bioavailability, making it difficult to perform an accurate comparison of bioavailability between FA and CD-FA.

The concentrations of the total (intact + β-glucuronidase/sulfatase-conjugated form) and intact forms of FA reached a maximum 15 min after administration (total form: FA group, 17.7 ± 1.7 μmol/L; CD-FA group, 47.7 ± 4.1 μmol/L; intact form: FA group, 7.6 ±1.0 μmol/L; CD-FA group, 29.1 ± 3.0 μmol/L) and began to fall thereafter in both groups (Fig. 1A and B). At 15 min after administration, the concentrations of both the intact and total forms of FA were significantly higher in the CD-FA group than in the FA group. Fig. 1C and D show the area under the curve of the total and intact forms from 0 to 120 min after FA or CD-FA administration. The levels of both the total and intact forms were significantly higher in the CD-FA group than in the FA group. Furthermore, based on the area under the curve, CD-FA significantly increased the intact form/total form ratio in plasma compared with FA (Fig. 1E). These results indicate that CD-FA administration significantly increases the intact form of FA in the blood. An increase in the concentration of the intact form of FA may be expected to enhance its bioactivity.

In iWAT, a significant increase in the FA concentration was observed in the CD-FA group compared with the FA group 15 min after FA or CD-FA administration (Fig. 2A and B), but there was no significant difference in the area under the curve from 0 to 240 min after FA or CD-FA administration or in the intact form as a percentage of the total (Fig. 2C, D and E).

Because the administration of CD-FA significantly increased the concentration of FA in plasma, CD-FA intake is expected to suppress body fat accumulation and induce beige adipocyte formation at the low dose of FA (2 g FA/kg diet), which did not significantly suppress fat deposits or induce beige adipocyte formation in our previous study (Tanaka and Tsuda, 2023). To compare the ability of CD-FA to induce beige adipocyte formation with our previous study (Tanaka and Tsuda, 2023), which was involved intake of an FA-supplemented diet, rather than direct stomach intubation, we examined whether intake of a CD-FA-supplemented diet (12.4 g CD-FA/kg, including 2 g FA/kg) suppressed body fat accumulation and significantly induced beige adipocyte formation. Neither iWAT, epididymal WAT, and interscapular brown adipose tissue weights nor body weight gain differed among the three groups (Table S1). In addition, no difference was seen in the protein expression of UCP1 among the three groups (Fig. S1). These results suggest that 0.2 CD-FA did not significantly suppress body fat deposits or induce beige adipocyte formation.

As shown in Fig. 1, the plasma concentrations of the total and intact forms of FA after CD-FA administration were significantly elevated at 15 min compared with the group administered FA and the ratio of the intact form of FA was also significantly increased in the CD-FA group. However, no significant suppression of fat deposits or induction of beige adipocyte formation was observed in the 0.2-CD-FA group, suggesting that the improved bioavailability identified in the CD-FA group is transient and that maintenance of a sustained FA concentration may be necessary for FA-mediated induction of beige adipocyte formation. In a report on the improved bioavailability of CD-curcumin complex, the decrease in the blood concentration of curcumin was gradual (Li et al., 2018). It remains unclear why the increase in the blood concentration of FA after CD-FA administration is transient. Several studies have reported that curcumin formulations using liposomes or an amorphization technique can be efficiently absorbed by the bodies of rodents and humans (Storka et al., 2015; Sunagawa et., al., 2021). Such techniques can maintain a sustained increase in plasma concentration after FA administration.

In conclusion, we found that oral administration of CD-FA significantly increased the concentration of the intact form of FA in plasma compared with FA. However, a CD-FA-supplemented diet (12.4 g CD-FA/kg, including 2 g FA/kg) did not significantly suppress body fat deposits or induce beige adipocyte formation. To reduce the required functional dose of FA needed to induce beige adipocyte formation, other highly bioavailable FA formulations need to be developed.

Acknowledgements  This study was supported in part by Grants-in-Aid for Scientific Research from the Japan Society for Promotion of Science (JSPS KAKENHI) (Nos. 23K05083 and 21H04863 to Takanori Tsuda) and The Public Foundation of Elizabeth Arnold—Fuji (Takanori Tsuda). We thank Naomi Tagami (Chubu University) for her technical assistance.

Conflict of interest  There are no conflicts of interest to declare.

Supplemental materials.

Immunoblot analysis.  The tissue samples were homogenized, centrifuged and the total protein concentrations of the obtained supernatant were determined using a Protein Assay System (Bio-Rad, Richmond, CA, USA) with bovine γ-globulin employed as a standard. Aliquots of the supernatant were treated with Laemmli sample buffer for 5 min at 100 °C. The samples were then loaded onto an SDS-PAGE system. The resulting gel was transblotted onto a PVDF membrane, which was blocked with 5% skim milk for 1 h at room temperature. After a washing with 20 mM Tris-HCl-buffered saline containing 0.05 % (w/v) Tween 20 (TTBS), the membrane sheets were reacted with antibodies (anti-UCP1 or anti-glyceraldehyde-3-phosphate dehydrogenase) for 16 h at 4 °C. After a washing with TTBS, the membranes were reacted with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibodies (Cell Signaling Technology, Tokyo, Japan) for 1 h at room temperature. After a washing, immunoreactivity was visualized using the ECL reagent (Thermo Fisher Scientific, Yokohama, Japan), and the relative signal intensity was evaluated with iBright CL1500 Imaging System (Thermo Fisher Scientific). The protein levels of UCP1 are expressed as relative fold changes compared to the level of the control group (=1) after normalization to glyceraldehyde-3-phosphate dehydrogenase protein expression.

Table S1. Body weight, food intake, and relative adipose tissue weights in mice fed Control, 0.2-FA, or 0.2-CD-FA diet for 4 weeks.¹

	Control	0.2-FA	0.2-CD-FA
Initial body weight, g	18.1 ± 0.3	18.1 ± 0.4	18.1 ± 0.5
Final body weight, g	25.2 ± 0.5	25.2 ± 0.6	25.2 ± 0.5
Food intake, g/(4 weeks ■ mouse)	102.9 ± 7.6	102.6 ± 7.1	105.4 ± 7.7
eWAT2, g/100 g body	1.54 ± 0.08	1.60 ± 0.13	1.35 ± 0.10
iWAT, g/100 g body	1.37 ± 0.07	1.38 ± 0.11	1.20 ± 0.07
Interscapular BAT3, g/100 g body	0.47 ± 0.02	0.46 ± 0.02	0.43 ± 0.03

¹  Values are presented as means ± SEM (n = 10).

²  eWAT, epidydimal WAT

³  BAT, brown adipose tissue

Fig. S1

Immunoblot analysis of UCP1 and GAPDH in iWAT in the control, 0.2-FA, and 0.2-CD-FA groups at 4 weeks. The protein levels are expressed as relative fold changes compared to the level of the control group (=1) after normalization to glyceraldehyde-3-phosphate dehydrogenase protein expression. Data are presented as means ± SEM (n = 10).

Abbreviations

CD

cyclodextrin

FA

ferulic acid

HPLC

high-performance liquid chromatography

iWAT

inguinal white adipose tissue

UCP1

uncoupling protein 1

WAT

white adipose tissue

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