# Analytical evaluation of fatty acid esters of hydroxy fatty acid quantity in fermented brown rice and rice bran (FRBA)

Ayu Watanabe, Laurence Balas, Daisuke Saigusa, Jiro Ogura, Thierry Durand, Nariyasu Mano, Hiroaki Yamaguchi

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Ayu Watanabe, Laurence Balas, Daisuke Saigusa, Jiro Ogura, Thierry Durand, et al.. Analytical evaluation of fatty acid esters of hydroxy fatty acid quantity in fermented brown rice and rice bran (FRBA). Food Chemistry Advances, 2022, 1, pp.100040. 10.1016/j.focha.2022.100040. hal-04813776

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# Analytical evaluation of fatty acid esters of hydroxy fatty acid quantity in fermented brown rice and rice bran (FRBA)

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Ayu Watanabe<sup>a,1</sup>, Laurence Balas<sup>b,1</sup>, Daisuke Saigusa<sup>c,d</sup>, Jiro Ogura<sup>e,f</sup>, Thierry Durand<sup>b</sup>, Nariyasu Mano<sup>a,e</sup>, Hiroaki Yamaguchi<sup>a,e,f,*</sup>

## ARTICLE INFO

### Keywords:

Branched saturated fatty acid esters of
hydroxyl fatty acids (FAHFA)

Fermented brown rice and rice bran (FBRA)

Anti-inflammatory effect

Liquid chromatography-tandem mass
spectrometry (LC-MS/MS)

## ABSTRACT

Fermented brown rice and rice bran with *Aspergillus oryzae* (FBRA) is a processed food known to have anti-inflammatory and anti-tumorigenic effects. Moreover, suppressive effects on spontaneous type-1 diabetes have recently been reported in mice. The determination of the active ingredients in FBRA is crucial to explain its health benefits. We investigated the presence of branched saturated fatty acid esters of hydroxyl fatty acids (FAHFAs) in rice bran and FBRA by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using commercially available and custom-synthesized standards. FAHFAs are novel bioactive lipids that exhibit anti-diabetic and anti-inflammatory effects. A possible involvement in cancer prevention has also been suggested. The FAHFA profiles differed between rice bran and FBRA both qualitatively and quantitatively. Interestingly, in both samples, the quantity of branched saturated FAHFAs was much higher than that previously reported for other cereals, vegetables and fruits. Therefore, given the beneficial effects of FAHFAs, regular consumption of FRBA may prevent metabolic disorders, insulin resistance, and chronic inflammatory disorders.

## 1. Introduction

Fermented brown rice and rice bran with *Aspergillus oryzae* (FBRA) is manufactured by fermenting a mixture of brown rice and rice bran with *Aspergillus oryzae* to improve its digestibility. FBRA exhibits many physiological effects, including anti-tumorigenic (Katayama et al., 2002; Kuno et al., 2006, 2015, 2016, 2018; Long et al., 2007; Mori, 2019; Onuma et al., 2015; Phutthaphadoong et al., 2010) and anti-inflammatory effects (Onuma et al., 2015; Phutthaphadoong et al., 2010; Umeyama et al., 2021). Recently, in mice, after consumption of FBRA, preventive effect on spontaneous type-1 diabetes has been reported (Kataoka, Nemoto, Sakurai, Yasutomo & Shikanai, 2021). FBRA has been reported to contain functional components, such as dipeptides (Tanaka et al., 2017), ergothioneine (Horie et al., 2020), ferulic acid (Ogawa et al., 2017), spermidine (Horie et al., 2019), and steryl glucosides (Murai et al., 2020). Dipeptides found in fermentation products

have been reported to exhibit angiotensin-converting enzyme (ACE) inhibition activity (Pripp, Isaksson, Stepaniak & Sørhaug, 2004). Recently, Horie et al. (2020) showed that the L-ergothioneine content, which has powerful antioxidant and cytoprotective properties, was higher in fermented rice bran (176 µg/g) than in fermented white rice (59.8 µg/g) and brown rice (85.5 µg/g). L-Ergothioneine in rice bran was enriched 140 times after fermentation for 45 h. Ferulic acid is a component of the rice cell wall and has been shown to have excellent antioxidant properties. Ogawa et al. (2017) found that fermentation significantly increased the content of phenolic acids such as ferulic acid, sinapic acid, caffeic acid, syringic acid, and protocatechuic acid, which are known antioxidants. Spermidine is a polyamine known to prevent age-related diseases. The spermidine content in rice bran increased by 158% after fermentation (Horie et al., 2019). Murai et al. (2020) analyzed steryl glucosides in FBRA samples and revealed that the content of acylated steryl glucosides in FBRA increased 1.5-fold during the fermentation process. Acy-

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<footnote><sup>a</sup> Faculty of Pharmaceutical Sciences, Tohoku University, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan</footnote>

<footnote><sup>b</sup> IBMM, Univ Montpellier, CNRS, ENSCM, Montpellier, France</footnote>

<footnote><sup>c</sup> Faculty of Pharma-Science, Teikyo University, 2-11-1 Kaga, Itabashi-ku, Tokyo, 173-8605, Japan</footnote>

<footnote><sup>d</sup> Tohoku University Tohoku Medical Megabank Organization, 2-1 Seiryo-machi, Aoba-ku, Sendai 980-8573, Japan</footnote>

<footnote><sup>e</sup> Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryo-machi, Aoba-ku, Sendai, 980-8574, Japan</footnote>

<footnote><sup>f</sup> Yamagata University Graduate School of Medicine/Department of Pharmacy, Yamagata University Hospital, 2-2-2 Iida-nishi, Yamagata, 990-9585, Japan</footnote>

<footnote>* Corresponding author at: Yamagata University Graduate School of Medicine/Department of Pharmacy, Yamagata University Hospital, 2-2-2 Iida-nishi, Yamagata, 990-9585, Japan.</footnote>

<footnote>E-mail address: hiroaki.yamaguchi@med.id.yamagatau.ac.jp (H. Yamaguchi).</footnote>

<footnote n="1">¹ Contributed equally.</footnote>
lated steryl glucosides are known to exhibit cholesterol-lowering and anti-diabetic effects (Ito, Nakashima & Matsuoka, 2015; Usuki et al., 2011). Detailed understanding of the bioactive ingredients in FBRA will further clarify the health benefits of FBRA intake.

To date, no information is available on the lipid profile of FBRA. Many lipids have bioactive properties. We focused on the branched fatty acid esters of hydroxyl fatty acids (FAHFAs), which are novel bioactive lipokines (Brejchova et al., 2020) that exhibit anti-diabetic and anti-inflammatory effects. Studies have also reported inverse correlation between the levels of specific FAHFAs and some cancers (Liu et al., 2020; Rodríguez et al., 2019; Zhu et al., 2017). Initially discovered in white adipocytes (Aryal et al., 2021; Yore et al., 2014), FAHFAs have also been detected in several vegetables, fruits, and meat (Benlebna et al., 2021b; Liberati-Čizmek et al., 2019; Pham et al., 2019; Yore et al., 2014; Zhu, Yan, Zhang, Xiao & Feng, 2018). FAHFAs are produced by the esterification of the carboxyl group of fatty acids with the hydroxyl group of hydroxy fatty acids (Yore et al., 2014). The major FAHFAs comprise a combination of palmitic acid, stearic acid, oleic acid (OA), or palmitoleic acid (PO) and their corresponding hydroxy fatty acids (hydroxy palmitic acid; HPA, hydroxy stearic acid; HSA, hydroxy oleic acid; HOA, and hydroxy palmitoleic acid; HPO, respectively). There are several FAHFA families, including the palmitic acid ester of hydroxy palmitic acid (PAHPA), oleic acid ester of hydroxy palmitic acid (OAHPA), palmitic acid ester of hydroxy oleic acid (PAHOA), oleic acid ester of hydroxy oleic acid (OAHOA), palmitic acid ester of hydroxy stearic acid (PAHSA), oleic acid ester of hydroxy stearic acid (OAHSA), palmitoleic acid ester of hydroxy stearic acid (POHSA), and stearic acid ester of hydroxy stearic acid (SAHSA) (Brejchova et al., 2020; Yore et al., 2014; Zhu et al., 2018). Moreover, many isomers are present in each family because of the diverse positions of the branched esters of hydroxy fatty acids and the configuration (R or S) of the hydroxyl group (Brezinova et al., 2018; Nelson et al., 2017). The nomenclature for FAHFAs is illustrated in the Materials and Methods section (Fig. 1).

Various physiological actions of FAHFAs are known. For example, 5- and 9-PAHSA enhance the secretion of glucagon-like peptide-1 (GLP-1) and transfer glucose transporter 4 (GLUT4) to the cell membrane, thereby increasing the uptake of sugar from the blood (Yore et al., 2014). However, these effects have not been studied in other families. Moreover, 9-PAHSA alleviates obesity-related inflammation by inhibiting the secretion of tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-12 via the G-protein-coupled receptor 120 (GPR120) (Smith & Kahn, 2016).

The 5-, 9-, 10-, and 12/13-PAHSA levels in the serum and adipose tissue of insulin-resistant individuals have been reported to be lower than those of insulin-sensitive individuals (Yore et al., 2014). Therefore, a decrease in the FAHFA levels in the body may increase the risk of developing diabetes. Since 9- and 13-PAHSA and 12- and 13-SAHS levels are reduced in the serum of breast cancer patients compared with those of healthy individuals (Zhu et al., 2018), their association with cancer has also attracted attention. Moreover, the oral administration of 5- and 9-PAHSA for three days in mouse models of colitis, a chronic inflammatory disease, prevents disease-related inflammation and damage (Lee et al., 2016).

We recently reported the novel biological effects of 9-PAHPA and 9-OAHPA (Benlebna et al., 2020a; 2020b, 2021a). The long-term high intake of 9-PAHPA or 9-OAHPA increases basal metabolism and insulin sensitivity in healthy mice (Benlebna et al., 2020a). In contrast, liver homeostasis is disrupted under these conditions. Similarly, in obesogenic diet-fed mice, the long-term intake of 9-PAHPA or 9-OAHPA modulates basal metabolism and has an insulin-sensitizing effect (Benlebna et al., 2021a). However, no major adverse effects on liver metabolism and homeostasis were observed in the obesogenic diet-fed mice. Moreover, the influence of 9-PAHPA and 9-OAHPA on the C2C12 myoblasts and skeletal muscle phenotype in mice was examined. These molecules inhibited C2C12 myoblast proliferation and in-

creased the expression of oxidative myosin in the skeletal muscle of mice (Benlebna et al., 2020b).

In this study, we investigated the presence of FAHFAs in FBRA, for the first time, as these recently discovered lipids may be correlated with FBRA biological properties. A comparison with the FAHFA content in unfermented rice bran is also discussed.

## 2. Materials and methods

The nomenclature for FAHFAs is illustrated in Fig. 1.

### 2.1. Commercially available materials

5-POHSA, 9-POHSA, 10-POHSA, 12-POHSA, 13-POHSA, 5-PAHSA, 9-PAHSA, 10-PAHSA, 12-PAHSA, 13-PAHSA, 5-OAHS, 9-OAHS, 12-OAHS, 13-OAHS, 5-SAHSA, 9-SAHSA, 10-SAHSA, 12-SAHSA, 13-SAHSA, 5-OAHS-d₁₇, 12-OAHS-d₃₁, 9-PAHSA<sup>13</sup>C₄, 12-PAHS-d₃₁, and 5-POHSA-d₁₄ were purchased from Cayman Chemical Co. (Ann Arbor, MI). All other solvents and reagents used were of the highest purity available.

2.2 Chemical synthesis of 5-PAHPA, 7-PAHPA, 9-PAHPA, 5-OAHPA, 7-OAHPA, 9-OAHPA, 7-PAHSA, (Z)-10-OAHC18, 7-OAHS, and 10-OAHS

These non-commercial FAHFAs were synthesized using a previously reported procedure (Balas et al., 2016). A summary of the general procedure used for these chemical syntheses is given below.

**General procedures for the synthesis of non-commercially available FAHFAs used in this study.** The syntheses involve the following steps: silylation of the primary alcohol group of the commercial starting material (alkene or alkyne derivatives), epoxidation of a terminal olefin, $BF_3$-mediated epoxide ring-opening by lithium acetylide, Steglich esterification with a fatty acid, fluoride-mediated TBS-deprotection, and Jones oxidation. These seven steps are outlined in Fig. S1. The detailed experimental conditions for performing these seven chemical reactions were reported by Balas et al. in 2016. (Balas et al., 2016).

POHSA, palmitoleic acid ester of hydroxy stearic acid; OAHPA, oleic acid ester of hydroxy palmitic acid; PAHPA, palmitic acid ester of hydroxy palmitic acid; OAHS, oleic acid ester of hydroxy stearic acid; PAHSA, palmitic acid ester of hydroxy stearic acid; SAHSA, stearic acid ester of hydroxy stearic acid; 12(Z)-10-OAHC18, 10-oleic acid ester of 10-hydroxy-12(Z)-octadecenoic acid.

### 2.3. Chromatographic and mass spectrometric conditions

Chromatographic separation was performed using an ACQUITY UPLC® H-Class Bio System (Waters, Milford, MA, USA). A SunShell C30 analytical column (2.6 × 150 mm, 2 µm, ChromaNik Technologies Inc., Osaka, Japan) was used. The column temperature was maintained at 45 °C. The mobile phase comprised water/methanol/1-butanol/acetic acid (8:88:4:0.1, v/v/v/v), at a flow rate of 0.4 mL/min. The overall run time was 70 min. Mass spectrometry was performed on a Xevo™ TQ-S tandem quadrupole mass spectrometer (Waters, Milford, MA, USA) using the negative ionization electrospray technique. The capillary voltage was set to -3 kV, and the desolvation temperature was 650 °C. Selected reaction monitoring (SRM) transitions were analyzed and are summarized in Table 1. The data were acquired and analyzed using Masslynx™ software (Waters, Milford, MA, USA).

### 2.4. Preparation of synthetic standard solutions for lc-ms/ms analyses

Standard stock solutions were prepared in methanol (5-POHSA, 9-POHSA, 10-POHSA, 12-POHSA, 13-POHSA, 5-PAHSA, 9-PAHSA, 10-PAHSA, 12-PAHSA, 13-PAHSA, 5-OAHS, 9-OAHS, 12-OAHS, 13-OAHS, 5-SAHSA, 9-SAHSA, 10-SAHSA, 12-SAHSA, and 13-SAHSA: 200 µg/mL; 12(Z)-10-OAHC18 and 7-OAHS: 100 µg/mL; and 7-PAHPA, 9-PAHPA, 7-OAHPA, 9-OAHPA, 7-PAHSA, and 10-OAHS:
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Chemical structures and nomenclature of FAHFAs measured in this study.</figcaption></figure>

90 µg/mL). The stock solutions were diluted to 10 µg/mL in methanol, mixed, and evaporated to dryness under a nitrogen stream. The dried residue was re-dissolved in methanol in varying concentrations (0.01, 0.02, 0.05, 0.1, 0.2, and 0.5 µg/mL). Standard stock solutions of 5-OAHSA-d<sub>17</sub>, 12-OAHSA-d<sub>31</sub>, 9-PAHSA<sup>13</sup>C<sub>4</sub>, 12-PAHSA-d<sub>31</sub>, and 5-POHSA-d<sub>31</sub> (20 µg/mL) were prepared in methanol, then diluted to 1 µg/mL in methanol and stored at -80 °C.

## 2.5. FBRA and rice bran samples

The FBRA sample used in this study was produced by the fermentation of brown rice and rice bran with Aspergillus oryzae (Koken Co. Ltd., Tobetsu, Hokkaido, Japan) according to an established procedure (Horie et al., 2019; Tanaka et al., 2017). Rice bran was manufactured in a factory in Hokkaido, Japan. FBRA and rice bran were powdered and stored at -20 °C prior to analysis.

## 2.6. Evaluation of extraction recovery and matrix effect

To investigate whether the matrix constituents in FBRA and rice bran interfered with the assay for the evaluation of specificity and selectivity, extracts containing stable isotope-labeled FAHFAs were prepared

and analyzed. Ten milligrams of FBRA powder or rice bran powder was washed three times with ultrapure water (1 mL). Sample extraction was performed using methanol as follows: liquid-liquid-extraction (LLE) using *tert*-butyl methyl ether (TBME), or solid-phase extraction (SPE) using a Bond Elut C18 cartridge (100 mg/1 mL) (Agilent, Mississauga, ON, CA, USA), or a combination of LLE and SPE. The extraction recoveries of the stable isotope-labeled FAHFAs from FBRA and rice bran were evaluated by comparing the peak areas obtained from samples spiked with stable isotope-labeled FAHFAs (10 ng each) before extraction with those obtained from samples to which stable isotope-labeled FAHFAs were added after extraction. The matrix effect on the stable isotope-labeled FAHFAs by the matrix components in FBRA and rice bran was evaluated by comparing the peak areas of an extract from FBRA and rice bran to which stable isotope-labeled FAHFAs were added after extraction with those of the mobile phase to which the same quantity of stable isotope-labeled FAHFAs was added.

## 2.7. Sample analysis

FBRA powder or rice bran powder (10 mg) was washed three times with ultrapure water (1 mL). Sample extraction was performed by the addition of methanol (1 mL) to the washed FBRA or rice bran. The tube
<table><caption>**Table 1**
SRM parameters for determination of FAHFAs.</caption><thead><tr><th>Analyte</th><th>SRM transition</th><th>Cone voltage (V)</th><th>Collision energy (V)</th></tr></thead><tbody><tr><td>5-PAHPA</td><td>m/z 509.4 → m/z 255.2</td><td>58</td><td>14</td></tr><tr><td>7-PAHPA</td><td></td><td></td><td></td></tr><tr><td>9-PAHPA</td><td></td><td></td><td></td></tr><tr><td>5-OAHPA</td><td>m/z 535.4 → m/z 271.2</td><td>58</td><td>18</td></tr><tr><td>7-OAHPA</td><td></td><td></td><td></td></tr><tr><td>9-OAHPA</td><td></td><td></td><td></td></tr><tr><td>5-POHSA</td><td>m/z 535.4 → m/z 299.3</td><td>58</td><td>20</td></tr><tr><td>9-POHSA</td><td></td><td></td><td></td></tr><tr><td>10-POHSA</td><td></td><td></td><td></td></tr><tr><td>12-POHSA</td><td></td><td></td><td></td></tr><tr><td>13-POHSA</td><td></td><td></td><td></td></tr><tr><td>5-PAHSA</td><td>m/z 537.4 → m/z 255.2</td><td>58</td><td>16</td></tr><tr><td>7-PAHSA</td><td></td><td></td><td></td></tr><tr><td>9-PAHSA</td><td></td><td></td><td></td></tr><tr><td>10-PAHSA</td><td></td><td></td><td></td></tr><tr><td>12-PAHSA</td><td></td><td></td><td></td></tr><tr><td>13-PAHSA</td><td></td><td></td><td></td></tr><tr><td>12(Z)-10-OAHC18</td><td>m/z 561.4 → m/z 281.2</td><td>58</td><td>24</td></tr><tr><td>5-OAHS</td><td>m/z 563.4 → m/z 281.3</td><td>58</td><td>24</td></tr><tr><td>7-OAHS</td><td></td><td></td><td></td></tr><tr><td>9-OAHS</td><td></td><td></td><td></td></tr><tr><td>10-OAHS</td><td></td><td></td><td></td></tr><tr><td>12-OAHS</td><td></td><td></td><td></td></tr><tr><td>13-OAHS</td><td></td><td></td><td></td></tr><tr><td>5-SAHS</td><td>m/z 565.4 → m/z 283.2</td><td>75</td><td>16</td></tr><tr><td>9-SAHS</td><td></td><td></td><td></td></tr><tr><td>10-SAHS</td><td></td><td></td><td></td></tr><tr><td>12-SAHS</td><td></td><td></td><td></td></tr><tr><td>13-SAHS</td><td></td><td></td><td></td></tr><tr><td>5-OAHS-d<sub>17</sub></td><td>m/z 580.6 → m/z 298.4</td><td>58</td><td>58</td></tr><tr><td>12-OAHS-d<sub>31</sub></td><td>m/z 580.8 → m/z 298.5</td><td>68</td><td>28</td></tr><tr><td>9-PAHSA<sup>13</sup>C<sub>4</sub></td><td>m/z 541.6 → m/z 259.3</td><td>84</td><td>26</td></tr><tr><td>12-PAHSA-d<sub>31</sub></td><td>m/z 568.9 → m/z 286.5</td><td>2</td><td>32</td></tr><tr><td>5-POHSA-d<sub>14</sub></td><td>m/z 549.7 → m/z 267.3</td><td>30</td><td>20</td></tr></tbody></table>

The structures of these FAHFAs are depicted in Fig. 1.

was shaken for 1 h and centrifuged at 15,000 × g for 10 min at room temperature. The methanol extract (supernatant) was purified via LLE using TBME. The upper ether layer was transferred to a different tube and evaporated to dryness. The dried residue was re-dissolved in the mobile phase (100 µL). Five microliters of the solution was injected into the HPLC column. Calibration curves were created for FAHFA concentrations using the standard addition method by adding 0, 1, 2, 5, 10, 20, and 50 ng FAHFAs to the FBRA and rice bran samples.

# 3. Results and discussion

## 3.1. Method development

We first developed a LC-MS/MS method for the simultaneous quantification of branched saturated FAHFAs in FBRA using 34 synthetic FAHFA standards. Thus, besides some commercially available standards, including stable isotope-labeled standards, we also used some custom-synthesized FAHFAs to obtain a detailed FAHFA profile in this fermented functional food.

The negative ion mass spectra (Q1) of the FAHFAs indicated the presence of deprotonated molecules ([M-H]⁻) as the predominant ions for each compound (data not shown). The product ion mass spectrum of each analyte, ion pairs selected for SRM, and parameter settings are listed in Table 1. Several isomeric pairs exhibited similar fragmentation patterns in the product ion mass spectra. Therefore, it was necessary to separate these isomeric pairs using chromatography.

To determine the optimal HPLC conditions, we performed experiments using different combinations of mobile phase eluents (acetonitrile, alcohol, and/or other solvents), buffers (acetic acid, formic acid, or ammonium), analytical columns (C18 or C30 reversed phase), and flow rates. Except for the commercial 12/13-regioisomer mixtures, all the regioisomers within a specific FAHFA family with simi-

lar fragmentation patterns were successfully separated (Fig. 2) by performing isocratic elution with water/methanol/1-butanol/acetic acid (8:88:4:0.1, v/v/v/v) using a C30 reverse phase column as described in Section 2.

To optimize the pretreatment method, the extraction recovery and matrix effect were evaluated using five stable isotope-labeled branched saturated FAHFAs (5-OAHS-d<sub>17</sub>, 12-OAHS-d<sub>31</sub>, 9-PAHSA<sup>13</sup>C<sub>4</sub>, 12-PAHSA-d<sub>31</sub>, and 5-POHSA-d<sub>31</sub>). We evaluated the LLE by TBME and SPE using a Bond Elut C18 cartridge; the results are illustrated in Fig. 3. For the FBRA sample, the extraction recoveries obtained from LLE, SPE, and LLE plus SPE were 79.2–98.9%, 79.4–85.7%, and 34.9–42.3%, respectively. The matrix effect observed was 30.3–155.3%, 39.8–160.7%, and 46.0–172.6%, respectively. For the rice bran samples, the extraction recoveries from LLE, SPE, LLE plus SPE were 63.5–84.1%, 67.9–80.0%, and 9.5–14.0%, respectively. The matrix effect was 64.6–151.7%, 56.2–95.6%, and 107.4–146.0%, respectively. These results indicate that the extraction recoveries and matrix effects from both samples depend on both the pretreatment method and properties of the analytes. In particular, the matrix components in FBRA and rice bran could be strongly affected by the ionization of the analytes. Based on these results, we decided to extract samples with LLE and determine the FAHFA concentrations using the standard addition method. The standard addition method is very effective in mitigating the influence of the matrix and is widely used in the quantitative analysis of foods by LC-MS (Mochizuki, 2011). The number of powdered samples used in our study was not as limited as that of biological samples, such as blood or tissues.

Extracted samples from FBRA (A–C) and rice bran (D–F) were obtained (open column, extraction recovery; solid column, matrix effect). Samples were pretreated with LLE (A, D), SPE (B, E), or LLE plus SPE (C, F). Each column represents mean ± SD (n = 3).

Calibration curves were constructed by spiking samples with seven different analyte concentrations. The calibration data, including the
<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Representative SRM chromatograms of FAHFAs. 500 pg of each analyte was injected.</figcaption></figure>
<figure><img src="image_6.png" /><figcaption>Fig. 3. Extraction recovery and matrix effect.</figcaption></figure>

<table><caption>**Table 2**
Calibration curves for FAHFAs.</caption><thead><tr><th rowspan="2">Analyte</th><th colspan="3">FBRA</th><th colspan="3">Rice bran</th></tr><tr><th>Slope</th><th>Intercept</th><th>r</th><th>Slope</th><th>Intercept</th><th>r</th></tr></thead><tbody><tr><td>5-PAHPA</td><td>1.57×10<sup>4</sup></td><td>-1.16×10<sup>3</sup></td><td>0.999</td><td>8.70×10<sup>3</sup></td><td>3.09×10<sup>4</sup></td><td>0.998</td></tr><tr><td>7-PAHPA</td><td>2.14×10<sup>4</sup></td><td>8.93×10<sup>4</sup></td><td>0.999</td><td>2.68×10<sup>4</sup></td><td>3.84×10<sup>5</sup></td><td>0.981</td></tr><tr><td>9-PAHPA</td><td>1.04×10<sup>4</sup></td><td>1.24×10<sup>5</sup></td><td>0.988</td><td>5.03×10<sup>3</sup></td><td>2.23×10<sup>5</sup></td><td>0.952</td></tr><tr><td>5-OAHPA</td><td>1.50×10<sup>4</sup></td><td>1.04×10<sup>6</sup></td><td>0.853</td><td>-6.29×10<sup>2</sup></td><td>2.58×10<sup>6</sup></td><td>-0.040</td></tr><tr><td>7-OAHPA</td><td>1.20×10<sup>3</sup></td><td>1.17×10<sup>4</sup></td><td>0.989</td><td>6.38×10<sup>2</sup></td><td>3.18×10<sup>4</sup></td><td>0.937</td></tr><tr><td>9-OAHPA</td><td>3.57×10<sup>3</sup></td><td>3.70×10<sup>4</sup></td><td>0.992</td><td>1.61×10<sup>3</sup></td><td>2.89×10<sup>4</sup></td><td>0.977</td></tr><tr><td>5-POHSA</td><td>3.02×10<sup>2</sup></td><td>-3.11×10<sup>2</sup></td><td>0.996</td><td>177×10<sup>2</sup></td><td>3.24×10<sup>2</sup></td><td>0.996</td></tr><tr><td>9-POHSA</td><td>2.96×10<sup>3</sup></td><td>-1.86×10<sup>3</sup></td><td>0.998</td><td>2.18×10<sup>3</sup></td><td>5.98×10<sup>2</sup></td><td>0.999</td></tr><tr><td>10-POHSA</td><td>2.56×10<sup>3</sup></td><td>-6.90×10<sup>2</sup></td><td>1.000</td><td>2.68×10<sup>3</sup></td><td>-2.62×10<sup>3</sup></td><td>0.997</td></tr><tr><td>12/13-POHSA</td><td>6.47×10<sup>3</sup></td><td>-6.23×10<sup>3</sup></td><td>0.999</td><td>4.35×10<sup>3</sup></td><td>-2.04×10<sup>3</sup></td><td>0.999</td></tr><tr><td>5-PAHSA</td><td>8.02×10<sup>3</sup></td><td>2.85×10<sup>3</sup></td><td>0.999</td><td>6.29×10<sup>3</sup></td><td>5.21×10<sup>3</sup></td><td>0.999</td></tr><tr><td>7-PAHSA</td><td>1.30×10<sup>4</sup></td><td>3.42×10<sup>3</sup></td><td>0.999</td><td>1.00×10<sup>4</sup></td><td>1.55×10<sup>4</sup></td><td>0.999</td></tr><tr><td>9-PAHSA</td><td>3.90×10<sup>3</sup></td><td>4.07×10<sup>4</sup></td><td>0.990</td><td>2.48×10<sup>3</sup></td><td>8.35×10<sup>4</sup></td><td>0.958</td></tr><tr><td>10-PAHSA</td><td>8.54×10<sup>3</sup></td><td>3.33×10<sup>4</sup></td><td>0.997</td><td>5.34×10<sup>3</sup></td><td>7.44×10<sup>4</sup></td><td>0.984</td></tr><tr><td>12/13-PAHSA</td><td>2.76×10<sup>4</sup></td><td>1.24×10<sup>5</sup></td><td>0.997</td><td>1.60×10<sup>4</sup></td><td>2.20×10<sup>5</sup></td><td>0.981</td></tr><tr><td>12(Z)-10-OAHC18</td><td>2.45×10<sup>4</sup></td><td>5.68×10<sup>4</sup></td><td>0.974</td><td>2.07×10<sup>5</sup></td><td>3.43×10<sup>7</sup></td><td>0.795</td></tr><tr><td>5-OAHS</td><td>9.17×10<sup>4</sup></td><td>2.79×10<sup>5</sup></td><td>0.981</td><td>6.55×10<sup>3</sup></td><td>5.28×10<sup>5</sup></td><td>0.942</td></tr><tr><td>7-OAHS</td><td>1.93×10<sup>4</sup></td><td>1.93×10<sup>4</sup></td><td>0.999</td><td>1.89×10<sup>4</sup></td><td>3.45×10<sup>4</sup></td><td>0.999</td></tr><tr><td>9-OAHS</td><td>2.28×10<sup>4</sup></td><td>2.59×10<sup>5</sup></td><td>0.992</td><td>1.21×10<sup>4</sup></td><td>4.21×10<sup>5</sup></td><td>0.946</td></tr><tr><td>10-OAHS</td><td>2.89×10<sup>4</sup></td><td>2.71×10<sup>5</sup></td><td>0.990</td><td>1.40×10<sup>4</sup></td><td>3.43×10<sup>5</sup></td><td>0.963</td></tr><tr><td>12/13-OAHS</td><td>6.55×10<sup>4</sup></td><td>9.94×10<sup>5</sup></td><td>0.985</td><td>2.72×10<sup>4</sup></td><td>7.31×10<sup>5</sup></td><td>0.952</td></tr><tr><td>5-SAHSA</td><td>1.80×10<sup>3</sup></td><td>-1.60×10<sup>3</sup></td><td>0.998</td><td>1.33×10<sup>3</sup></td><td>2.83×10<sup>2</sup></td><td>0.996</td></tr><tr><td>9-SAHSA</td><td>4.50×10<sup>3</sup></td><td>-7.81×10<sup>2</sup></td><td>0.998</td><td>3.64×10<sup>3</sup></td><td>5.84×10<sup>3</sup></td><td>0.998</td></tr><tr><td>10-SAHSA</td><td>4.64×10<sup>3</sup></td><td>-4.17×10<sup>2</sup></td><td>0.997</td><td>3.60×10<sup>3</sup></td><td>5.78×10<sup>3</sup></td><td>0.999</td></tr><tr><td>12/13-SAHSA</td><td>1.07×10<sup>4</sup></td><td>8.56×10<sup>2</sup></td><td>0.998</td><td>7.45×10<sup>3</sup></td><td>1.76×10<sup>4</sup></td><td>0.999</td></tr></tbody></table>

The structures of these FAHFAs are illustrated in Fig. 1.

slope, intercept, and correlation coefficient, are summarized in Table 2. The calibration curves for FAHFAs, except for 5-OAHPA in both FBRA and rice bran samples and 12(Z)-10-OAHC18 in the rice bran sample, were linear (r>0.9). One possible reason for the lack of linearity in these samples (5-OAHPA in FBRA and rice bran samples and 12(Z)-10-OAHC18 in the rice bran sample) could be the presence of interfering compounds in the original sample.

## 3.2. Quantification of FAHFAs in *fra* and *rice bran*

For the first time, we detected branched saturated FAHFAs in FBRA and unfermented rice bran. Several families of branched FAHFAs and several regioisomers in each family were identified. The quantities of

FAHFAs are summarized in Table 3. Rice bran contains a 1.8 to 6 times higher quantity of FAHFAs than that contained in FBRA. The major FAHFAs in rice bran were 12(Z)-10-OAHC18 (approximately 70 times more concentrated than in FBRA) and 5-OAHS, whereas FBRA samples were mainly rich in 5-OAHPA and 5-OAHS. Previous reports have shown that FAHFAs are present in various plant foods (Benlebna et al., 2021b; Liberati-Čizmek et al., 2019; Yore et al., 2014). Our results demonstrated that FBRA and rice bran contain a higher quantity of FAHFAs than those in the previously studied foods (Kokotou, 2020; Liberati-Čizmek et al., 2019; Yore et al., 2014). The reason for the lower content of FAHFAs in the FBRA sample compared to that in the unfermented rice bran may be that FAHFAs were decomposed, conjugated to other species, and/or oxidized during the fermentation process.
<table><caption>**Table 3**
Quantity of FAHFAs in FBRA and rice bran.</caption><thead><tr><th>Analyte</th><th>FBRA</th><th>Rice bran</th></tr></thead><tbody><tr><td>5-PAHPA</td><td>N.D.</td><td>0.355</td></tr><tr><td>7-PAHPA</td><td>0.417</td><td>1.43</td></tr><tr><td>9-PAHPA</td><td>1.24</td><td>4.43</td></tr><tr><td>5-OAHPA</td><td>6.94</td><td>N.D.</td></tr><tr><td>7-OAHPA</td><td>0.978</td><td>4.99</td></tr><tr><td>9-OAHPA</td><td>1.04</td><td>1.79</td></tr><tr><td>5-POHSA</td><td>N.D.</td><td>0.183</td></tr><tr><td>9-POHSA</td><td>N.D.</td><td>0.0274</td></tr><tr><td>10-POHSA</td><td>N.D.</td><td>N.D.</td></tr><tr><td>12/13-POHSA</td><td>N.D.</td><td>N.D.</td></tr><tr><td>5-PAHSA</td><td>0.0356</td><td>0.083</td></tr><tr><td>7-PAHSA</td><td>0.0263</td><td>0.155</td></tr><tr><td>9-PAHSA</td><td>1.05</td><td>3.37</td></tr><tr><td>10-PAHSA</td><td>0.390</td><td>1.39</td></tr><tr><td>12/13-PAHSA</td><td>0.451</td><td>1.37</td></tr><tr><td>12(Z)-10-OAHC18</td><td>0.232</td><td>16.6</td></tr><tr><td>5-OAHS</td><td>3.04</td><td>8.05</td></tr><tr><td>7-OAHS</td><td>0.100</td><td>0.182</td></tr><tr><td>9-OAHS</td><td>1.14</td><td>3.48</td></tr><tr><td>10-OAHS</td><td>0.936</td><td>2.45</td></tr><tr><td>12/13-OAHS</td><td>1.52</td><td>2.69</td></tr><tr><td>5-SAHSA</td><td>N.D.</td><td>0.021</td></tr><tr><td>9-SAHSA</td><td>N.D.</td><td>0.160</td></tr><tr><td>10-SAHSA</td><td>N.D.</td><td>0.161</td></tr><tr><td>12/13-SAHSA</td><td>N.D.</td><td>0.237</td></tr></tbody></table>

FAHFAs contents are expressed as µg/g.

N.D., not detected.

The structures of these FAHFAs are depicted in Fig. 1.

Liberati-Čizmek et al. (2019) reported that SAHSA was the most abundant FAHFA in garlic and clementines. In our study, this family of FAHFAs was observed in low quantities. The main FAHFAs in FBRA are the esters of OA. This may be because the fatty acid composition of our samples was mainly OA. Furthermore, it has been reported that OA is abundant in rice bran (Nakamura et al., 2017).

Liberati-Čizmek et al. (2019) quantified a mixture of OAHPA and POHSA because their method could not distinguish between these FAHFAs. The chromatographic behaviors of OAHS and POHSA are very similar, and they are very difficult to separate, thus, the SRM transitions $m/z$ 535.5→$m/z$ 281.2/253.2 for OAHPA and $m/z$ 535.5→$m/z$ 253.2/281.2 for POHSA were applied. In this study, we separated OAHPA and POHSA using mass spectrometry by selecting transitions that do not interfere with each other ($m/z$ 535.4→$m/z$ 271.2 for OAHPA and $m/z$ 535.4→$m/z$ 299.3 for POHSA). The retention time for both 9-OAHPA and 9-POHSA was 17.1 min, thereby, increasing the difficulty of separation using chromatography.

Moreover, we detected 12(Z)-10-OAHC18 in plants and food for the first time. 12(Z)-10-OAHC18 is an ester of OA and 10-hydroxy-12(Z)-octadecenoic acid (HYA). HYA is a metabolite of linolenic acid produced by the gut microbiota and has been reported to exhibit anti-diabetic, triglyceride-lowering, and pylori-infection-preventing effects (Matsui et al., 2017; Miyamoto et al., 2019; Nanthirudjanar et al., 2015). Although there are no reports on the physiological effects of 12(Z)-10-OAHC18, this FAHFA may play a key role in the health benefits of rice bran and FBRA.

Notably, 5-PAHSA, one of the most studied FAHFAs to date, exhibited anti-diabetic and anti-inflammatory effects and was detected at low doses in both the rice samples investigated in this study. 9-PAHSA, which was analyzed similarly to 5-PAHSA, occurred in the highest abundance among the PAHSA family investigated. Recently, Aryal et al. (2021) reported the biological effects of POHSAs, OAH-SAs, and SAHSAs. Many FAHFAs potentiate glucose-stimulated insulin secretion, activate G-protein coupled receptor 40, inhibit LPS-induced chemokine and cytokine gene expression and secretion, and phagocytose immune cells (Aryal et al., 2021). We detected several OAHSA re-gioisomers, but none in the POHSA and SAHSA families, and in the FBRA

sample. Notably, 5- and 9-OAHS, which exhibited insulin-stimulated glucose uptake and anti-inflammatory effects, were abundant in the FBRA sample, suggesting that they may be directly involved in the bio-logical effects of this functional food.

On the whole, this study showed that the traditional Asian food FRBA is an exceptionally rich source of FAHFAs, some recently discovered lipokines that regulate human health. Further investigations are required to elucidate the role and biological relevance of the branched saturated FAHFAs in these samples.

## 4. Conclusions

This paper revealed the presence of some recently uncovered bioactive lipids, called FAHFAs, in FBRA (a traditional Asian manufactured functional food) and in unfermented rice bran unprecedentedly. The levels of FAHFAs we detected in FBRA and rice bran were high compared to the FAHFA levels in other vegetables reported so far. The two rice samples showed very different FAHFA profiles. PAHPA, OAHPA, PAHSA, OAHS, and 12(Z)-10-OAHC18 were detected in FBRA and rice bran samples, whereas POHSA and SAHSA were detected only in the rice bran samples. Our results not only provide a better knowledge of the lipid components in a functional food, but also suggest that regular consumption of FRBA in our diet may help us to maintain good health. Our contemporary societies have changed their eating habits resulting in a high prevalence of obesity, diabetes and chronic inflammatory diseases, mainly in Western countries. FBRA is a traditional food in Asian countries that is losing its notoriety/popularity. FAHFAs exhibit anti-inflammatory and anti-diabetic effects and may have protective and preventive effects against some cancers. By showing that FBRA is an enriched source of FAHFAs, mainly 5-OAHPA which is not detected in the unfermented precursor rice bran, our study suggests that the consumption of this traditional food may bring many human health benefits. Further research is needed to elucidate the biological properties of all the detected FAHFAs and correlate the health benefits of FBRA. However, this study shows attractive and promising perspectives since the industrial production of FBRA (which could be optimized by lipid monitoring with the FAHFA analysis method presented in this paper) and the commercialization of FBRA should promote its use, and regular consumption could contribute to the prevention of diabetes and chronic inflammatory diseases, especially in Western countries.

### CRediT authorship contribution statement

**Ayu Watanabe:** Formal analysis, Investigation, Methodology, Writing-Reviewing and Editing, Visualization. **Laurence Balas:** Resources, Syntheses of FAHFAs, Writing-Reviewing and Editing. **Daisuke Saigusa:** Writing-Reviewing and Editing. **Jiro Ogura:** Writing-Reviewing and Editing. **Thierry Durand:** Resources, Writing-Reviewing and Editing. **Nariyasu Mano:** Methodology, Writing-Reviewing and Editing, Supervision. **Hiroaki Yamaguchi:** Conceptualization, Data Curation, Methodology, Project Administration, Writing-Original Draft Preparation.

### Funding

This work was supported in part by the Tohoku Medical Megabank Project of MEXT, Japan Agency for Medical Research and Development (AMED; grant numbers JP20km0105001 and JP20km0105002).

### Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this study.
## Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.focha.2022.100040.

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