Eubacterium limosum JCM6421^T influences quercetin production from isorhamnetin and short-chain fatty acid production from various sugars

Abstract

Isorhamnetin (3′-methylquercetin) is one of the major metabolites of quercetin. We hypothesized that Eubacterium limosum JCM6421^T transforms isorhamnetin back to quercetin and the type of sugar affects short-chain fatty acid production by E. limosum JCM6421^T. Anaerobic broth (0.2 mL) containing this bacterium was added to the isorhamnetin solution and anaerobically incubated for 8 and 24 h. Additionally, E. limosum JCM6421^T was anaerobically incubated with various 2 µL-sugar solutions for 24 h and short-chain fatty acid production by this bacterium was measured. Quercetin was produced by anaerobic incubation of isorhamnetin with E. limosum JCM6421^T. Its production increased with an increase in incubation time from 8 to 24 h. Mannitol was the ideal sugar for butyric and acetic acid production by E. limosum JCM6421^T among the investigated sugars. We discovered that E. limosum JCM6421^T produces quercetin from isorhamnetin.

Introduction

Quercetin is one of the major flavonoids found in many plant foods. Several beneficial effects of quercetin have been reported; for example, a quercetin diet decreases hepatic fat accumulation (Kobori et al., 2011; Wang et al., 2014) and blood pressure in hypertensive subjects (Edwards et al., 2007). Furthermore, quercetin has been reported to prevent cancer (Gibellini et al., 2011; Turner et al., 2009) and possess anti-allergic effects (Mlcek et al., 2016). Therefore, quercetin is an important food component. However, once quercetin is absorbed from the intestine, some of it is converted into its methylated derivative, isorhamnetin, in the liver and then released with unmetabolized quercetin in the gut via bile (Ueno et al., 1983). The chemical structures of quercetin and isorhamnetin are shown in Fig. 1. A large amount of isorhamnetin is present in the digestive tract (Matsukawa et al., 2009) and plasma (Manach et al., 1997) after intake of a diet containing quercetin or quercetin glycosides. Some intestinal bacteria can degrade quercetin (Keppler et al., 2006; Zhang et al., 2014), however, there are few reports on the intestinal bacteria that can transform isorhamnetin back to quercetin. The physiological function of quercetin appears to be greater than that of isorhamnetin (Bandaruk et al., 2012; Yamamoto et al., 1999). Therefore, intestinal bacteria that can demethylate isorhamnetin to quercetin are important since they increase the bioavailability of quercetin. Eubacterium limosum produces genistein from biochanin A (genistein 4′-methyl ether) and daidzein from formononetin (7-hydroxy-4′-methoxyisoflavone), suggesting that it is capable of O-demethylation of several compounds (Hur et al., 2000). Therefore, this bacterium is expected to demethylate isorhamnetin to produce quercetin.

Fig. 1.

Chemical structure of (a) isorhamnetin and (b) quercetin.

Short-chain fatty acids produced by intestinal microbiota have been suggested to affect disease etiology and relate to the systemic effects of diet. Short-chain fatty acids are involved in immune regulation and intestinal epithelial barrier maintenance (Cong et al., 2022). Butyrate is one of the major short-chain fatty acids.

E. limosum is a major component of the human intestinal microbiota and an active producer of butyrate (Jeong et al., 2015), which is thought to provide many health benefits in the intestine.

E. limosum has also been reported to decrease IL-10 and IL-13 release from colonic biopsies of healthy controls compared with that of post-infectious irritable bowel syndrome patients (Sundin et al., 2015). This suggests that the bacterium can act against low-grade inflammation. The levels of E. limosum and its relatives are more than ten-fold higher in centenarians (Biagi et al., 2010). E. limosum may contribute to the health of centenarians with intestinal inflammation by its anti-inflammatory properties (Kanauchi et al., 2006), and by its metabolic activity to convert various isoflavonoids (Hur et al., 2000) in the gut.

E. limosum appears to be an important intestinal bacterium. The present study hypothesized that E. limosum could transform isorhamnetin back to quercetin, isorhamnetin affects short-chain fatty acid production by E. limosum, and some sugars enhance butyrate production by E. limosum.

Materials and Methods

Materials    Isorhamnetin and quercetin were purchased from Extrasynthese (Genay, France). E. limosum JCM6421^T was obtained from the Japan Collection of Microorganisms, RIKEN BioResource Research Center (Ibaraki, Japan).

In vitro qualitative incubation of E. limosum JCM6421^T with isorhamnetin    E. limosum JCM6421^T was anaerobically incubated on glucose blood liver (BL) agar (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) plates under a CO₂ atmosphere generated using the AnaeroPack system (Mitsubishi Gas Chemical Company Inc., Tokyo, Japan) at 37 °C for 24 h. E. limosum colonies on the BL agar medium were harvested with an inoculating loop and suspended in anaerobic broth (final concentration of 2.6 × 10⁸ CFU / mL), which was prepared from the semi-solid Gifu anaerobic medium (GAM; Nissui Pharmaceutical Co., Ltd, Tokyo, Japan). To prepare the isorhamnetin solution, 10 mg of isorhamnetin was dissolved in 1 mL of a dimethyl sulfoxide and N, N-dimethylformamide mixture (1 : 1). Then, 1 µL (10 µg) and 2 µL (20 µg) of the isorhamnetin solution were each transferred to 0.2 mL of the anaerobic medium with suspended E. limosum and incubated anaerobically under a CO₂ atmosphere generated using the AnaeroPack system at 37 °C for 8 and 24 h. After incubation, 800 µL of a mixture of acetic acid and methanol (100 : 5) was added to the culture medium, vortexed vigorously for 2 min, and centrifuged at 12 000 × g for 10 min at 4 °C. The supernatant was then subjected to highperformance liquid chromatography (HPLC) analysis. Three samples were prepared for each test.

Analysis of quercetin and isorhamnetin    To detect quercetin and isorhamnetin, a photodiode array detector (MD-2018; JASCO Co., Ltd., Tokyo, Japan) was used to monitor the spectral data from 200 to 400 nm for each peak. Quercetin and isorhamnetin were used as standard samples and were quantified using spectral data at 254 nm. The mobile phase comprised a mixture of methanol, acetic acid, and water (45 : 5 : 50 by volume). The HPLC analysis was conducted under the following conditions: column temperature, 40 °C; flow rate, 1 mL / min; column, Capcell pak C-18 UG 120 (4.6 × 250 mm, Osaka Soda Co., Ltd., Osaka, Japan).

In vitro incubation of E. limosum JCM6421^T with isorhamnetin or sugars for analysis of short-chain fatty acids    The culture method for short-chain fatty acid analysis following in vitro incubation of E. limosum JCM6421^T with isorhamnetin is identical to the method described above. For short-chain fatty acid analysis following in vitro incubation of E. limosum JCM6421^T with sugars, the culture method was the same as above except for the concentration of bacteria and additives to the medium. The final concentration of E. limosum JCM6421^T was 3.2 × 10⁸ CFU/mL. For the cultivation, 2 µL of a sugar solution (10% sugar: glucose, fructose, mannose, mannitol, xylose, or xylitol in sterilized water) and 2 µL of sterilized water as the control was individually transferred to 0.2 mL of the previously prepared anaerobic medium with suspended E. limosum and incubated anaerobically under a CO₂ atmosphere generated using the AnaeroPack system at 37 °C for 24 h. After incubation, 300 µL of distilled water was added for analysis of short-chain fatty acids and the short-chain fatty acid profile was evaluated. Three samples were prepared for each test.

Evaluation of short-chain fatty acid profile    After incubation of the culture broth, 300 µL of distilled water was added, vortexed vigorously for 2 min, and centrifuged at 12 000 × g for 5 min at 4 °C. The supernatants were analyzed using a short-chain fatty acid analysis kit (YMC Co. Ltd., Kyoto, Japan), as previously described by Tamura et al. (2020). This kit converts the carboxyl group of a short-chain fatty acid to 2-nitrophenylhydrazine, which can be detected in the ultraviolet and visible regions with high sensitivity. For HPLC analysis, 20 µL of each preparation was injected into a 250 × 6.0 mm YMC-Pack FA (YMC). To detect the hydrazides in short-chain fatty acids, a photodiode array detector was used to monitor the spectral data from 200 to 400 nm for each peak. Acetic, propionic, and butyric acids were used as standards. Crotonic acid was used as the internal standard. Spectral data at 254 nm were used to quantify the hydrazide content of short-chain fatty acids. The mobile phase consisted of a mixture of acetonitrile, methanol, and water (30 : 16 : 54 by volume, pH 4.5). The HPLC analysis was conducted under the following conditions: column temperature, 50 °C; flow rate, 0.9 mL / min.

Statistical analysis    Data are expressed as the mean ± standard error (SE). All data were analyzed using Sigma Plot 13 (Systat Software, Inc., San Jose, CA, USA) using a one-way analysis of variance, and Tukey's test was used for multiple comparisons or using a t-test analysis. Statistical significance was set at p < 0.05.

Results and Discussion

In vitro qualitative incubation of E. limosum JCM6421^T with isorhamnetin    E. limosum was found to transform isorhamnetin to quercetin regardless of the concentration of isorhamnetin used in this study (Fig. 2). Quercetin production from isorhamnetin increased as the incubation time increased from 8 to 24 h. Isorhamnetin concentration was significantly lower in both the 8 and 24 h culture media compared to the 0 h incubation broth. The ratios of quercetin to isorhamnetin in the culture media are shown in Fig. 3. This ratio was significantly greater in the 24 h-10 µg culture medium than in the 8 h-10 µg and 8 h-20 µg culture media. The 20 µg isorhamnetin concentration is too high for E. limosum to perform the demethylation reaction and substrate inhibition may have occurred in this bacterium. As a result, quercetin production may have been higher at the 10 µg isorhamnetin concentration.

Fig. 2.

Concentrations of quercetin and isorhamnetin in the culture media supplemented with 1 µL (10 µg) and 2 µL (20 µg) of isorhamnetin and incubated with Eubacterium limosum JCM6421^T for 0 h (0 h-1 µL and 0 h-2 µL), 8 h (8 h-1 µL and 8 h-2 µL), and 24 h (24 h-1 µL and 24 h-2 µL). Values are expressed as the mean ± SE (n = 3). ** indicates significant difference from the 0 h-incubation broth (p < 0.01). * indicates significant difference from the 0 h-incubation broth (p < 0.05).

Fig. 3.

The ratio of quercetin to isorhamnetin concentrations in the culture media supplemented with 1 µL (10 µg) and 2 µL (20 µg) of isorhamnetin and incubated with Eubacterium limosum JCM6421^T for 8 h and 24 h. Values are expressed as the mean ± SE (n = 3). a: p < 0.05 as compared to 8 h-1 µL, b: p < 0.05 as compared to 8 h-2 µL.

Betaine is fermented to N, N-dimethylglycine by E. limosum under anaerobic incubation with CO₂ (Müller et al., 1981). E. limosum converts isoxanthohumol in hops to the phytoestrogen 8-prenylnaringenin in vitro—the methyl group of isoxanthohumol is demethylated to produce 8-prenylnaringenin (Possemiers et al., 2008). Isorhamnetin (quercetin-3'-methyl ether) is a flavonoid with a methyl group, and E. limosum likely demethylates the methyl group in isorhamnetin to produce quercetin. Secoisolariciresinol is a type of polyphenol with methyl groups. It has been reported that strains of Peptostreptococcus productus and E. limosum demethylated secoisolariciresinol (Clavel et al., 2006). In contrast, a number of functional or phylogenetic relatives of E. limosum and P. productus did not demethylate secoisolariciresinol (Clavel et al., 2006). The demethylation activity of E. limosum is unique and the activity appears to be conserved within this species.

The isorhamnetin concentration was significantly lower in the isorhamnetin 8 h and 24 h culture medium than in the isorhamnetin 0 h culture medium. On the other hand, quercetin was detected in the 8 and 24 h culture medium. Our results suggest that E. limosum produces quercetin from isorhamnetin. The quercetin concentration was higher in the 24 h than the 8 h incubation broth. For E. limosum to produce high amounts of quercetin via anaerobic incubation, a longer incubation period may be necessary.

The higher ratio of quercetin to isorhamnetin in the 24 h-10 µg culture medium indicates that 10 µg isorhamnetin in 0.2 mL of culture medium for 24 h was the ideal condition for the production of quercetin from isorhamnetin in our study. In contrast, the 24 h-20 µg culture medium is unsuitable for quercetin production, as indicated by its high concentration of isorhamnetin.

Quercetin is reported to possess more biological activities than isorhamnetin (Bandaruk et al., 2012; Yamamoto et al., 1999) and is considered a useful functional component. However, when quercetin is ingested, it undergoes methylation in the body, and thus results in a large amount of methylated-quercetin or isorhamnetin being present in the digestive tract (Matsukawa et al., 2009). It has been reported that rats fed a diet containing quercetin glycoside showed cecal pools of monomethylated quercetin (isorhamnetin and tamarixetin) of about 40 µmol (Matsukawa et al., 2009). In our experiment, the isorhamnetin concentration in the culture media was between 157.5 and 313.7 µmol/L. Thus, the isorhamnetin concentrations we used seem to be sufficiently high for predicting isorhamnetin metabolism in the gut.

In summary, intestinal bacteria such as E. limosum play an important role in improving the functionality of quercetin by transforming isorhamnetin back to quercetin in the gut.

E. limosum ameliorates 2.0% dextran sodium sulfate-induced colonic inflammation in mice (Kanauchi et al., 2006). Moreover, the levels of E. limosum and its relatives are increased by more than ten-fold in centenarians (Biagi et al., 2010). We conclude that E. limosum produces bioactive quercetin from isorhamnetin, and the ability to convert isorhamnetin to quercetin in the gastrointestinal tract is another important role of this bacterium. It has been reported that in an in situ rat intestinal perfusion test using 50 µmol/L quercetin-glucoside, the plasma isorhamnetin concentration was about one-fifth of the quercetin concentration (Arts et al., 2004). If about 50% of isorhamnetin is converted to quercetin by the action of E. limosum in the digestive tract, an increase in bioavailability of about 10% is expected. E. limosum may increase the bioavailability of quercetin by producing quercetin from isorhamnetin in the gut and enhance the functionality of diets containing quercetin. The physiological significance of E. limosum in the in vivo bioavailability of quercetin is a crucial area for future research.

In vitro incubation of E. limosum JCM6421^T with isorhamnetin for short-chain fatty acid analysis    Short-chain fatty acid concentrations in the culture media with different isorhamnetin concentrations are shown in Fig. 4. No significant differences were observed in acetic and butyric acid concentrations between the 8 and 24 h isorhamnetin culture media and 8 and 24 h control culture media. The average propionic acid concentration in the 8 h-10 µg isorhamnetin culture medium was higher than that in the 8 h-20 µg isorhamnetin broth. The average propionic acid concentration in the 24 h-10 µg control culture medium was higher than that in the 24 h-10 µg and 24 h-20 µg isorhamnetin broths. These results suggest that isorhamnetin might have an inhibitory effect on propionic acid production.

Fig. 4.

Concentrations of acetic acid (Fig. 4-a), propionic acid (Fig. 4-b), butyric acid (Fig. 4-c) in the culture media supplemented with 1 µL (10 µg) and 2 µL (20 µg) of isorhamnetin and incubated with Eubacterium limosum JCM6421^T for 8 h (Is 8 h-1 µL, Is 8 h-2 µL) and 24 h (Is 24 h-1 µL, Is 24 h-2 µL). Concentrations of short-chain fatty acids in the culture medium supplemented with 1 µL and 2 µL of a 1:1 mixture of dimethyl sulfoxide and N, N-dimethylformamide and incubated with Eubacterium limosum JCM6421^T for 8 h (Cont 8 h-1 µL and Cont 8 h-2 µL) and 24 h (Cont 24 h-1 µL and Cont 24 h-2 µL). The different superscripted letters indicate significant difference (p < 0.05).

In vitro incubation of E. limosum JCM6421^T with sugars for short-chain fatty acid analysis    The short-chain fatty acid concentrations in the culture media containing various sugars are shown in Fig. 5. Mannitol was the most effective sugar for enhancing acetic and butyric acid production by E. limosum among the examined sugars. Mannitol induced significantly higher acetic acid production than mannose. After mannitol, fructose increased butyrate production by E. limosum to the next highest level. The addition of xylose to the medium resulted in the lowest butyrate production observed. In a study examining the energy sources required for the growth of E. limosum, fructose, glucose, and mannitol were found to be excellent energy sources, whereas xylose, mannose, and xylitol supported little or no growth (Genthner et al., 1981). Both fructose and glucose are excellent energy sources for E. limosum growth; however, the levels of acetic and butyric acids produced by E. limosum in the medium containing mannitol were significantly higher than those in the media containing other carbohydrates. Mannitol may be an ideal carbohydrate for short-chain fatty acid production by E. limosum. Aloe vera whole leaf extract supplementation to pure E. limosum cultures enhanced butyric acid production at 24 and 48 h (Pogribna et al., 2008). Based on this and our present results, we infer that some food components may enhance butyric acid production in E. limosum. Recently, many studies have focused on the beneficial effects of butyric acid production by intestinal bacteria (Coppola et al., 2021; Zhang et al., 2021). Mannitol may be one of the candidate sugars for increasing butyric acid production by E. limosum.

Fig. 5.

Concentrations of acetic acid (Fig. 5-a), propionic acid (Fig. 5-b), butyric acid (Fig. 5-c) in the culture media supplemented with 2 µL of fructose, glucose, mannitol, mannose, xylose, xylitol, and water (control) and incubated with Eubacterium limosum JCM6421^T for 24 h. The different superscripted letters indicate significant difference (p < 0.05).

In conclusion, we discovered that E. limosum JCM6421^T produces quercetin from isorhamnetin. E. limosum may increase the bioavailability of quercetin by transforming isorhamnetin back to quercetin in the gut and thus enhance the functionality of diets containing quercetin. Mannitol was the ideal sugar for butyric and acetic acid production by E. limosum JCM6421^T among the investigated sugars.

Acknowledgements    This study was financially supported by a Grant-in-Aid for Scientific Research (C) (21K05480) from the Japan Society for the Promotion of Science.

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

References

•  Arts,  I.C.,  Sesink,  A.L.,  Faassen-Peters,  M.,  Hollman,  P.C. (2004). The type of sugar moiety is a major determinant of the small intestinal uptake and subsequent biliary excretion of dietary quercetin glycosides. Br. J. Nutr., 91, 841-847.
•  Bandaruk,  Y.,  Mukai,  R.,  Kawamura,  T.,  Nemoto,  H., and  Terao,  J. (2012). Evaluation of the inhibitory effects of quercetin-related flavonoids and tea catechins on the monoamine oxidase-A reaction in mouse brain mitochondria. J. Agric. Food. Chem., 60, 10270-10277.
•  Biagi,  E.,  Nylund,  L.,  Candela,  M.,  Ostan,  R.,  Bucci,  L.,  Pini,  E.,  Nikkïla,  J.,  Monti,  D.,  Satokari,  R.,  Franceschi,  C.,  Brigidi,  P., and  De Vos,  W. (2010). Through ageing, and beyond: gut microbiota and inflammatory status in seniors and centenarians. PLoS One, 5, e10667.
•  Clavel,  T.,  Henderson,  G.,  Engst,  W.,  Doré,  J.,  Blaut,  M. (2006) Phylogeny of human intestinal bacteria that activate the dietary lignan secoisolariciresinol diglucoside. FEMS Microbiol. Ecol. 55, 471-478.
•  Cong,  J.,  Zhou,  P.,  Zhang,  R. (2022) Intestinal microbiota-derived short chain fatty acids in host health and disease. Nutrients 14., 1977. doi: 10.3390/nu14091977.
•  Coppola,  S.,  Avagliano,  C.,  Calignano,  A., and  Berni Canani,  R. (2021). The protective role of butyrate against obesity and obesity-related diseases. Molecules, 26, 682., doi: 10.3390/molecules26030682.
•  Edwards,  R.L.,  Lyon,  T.,  Litwin,  S.E.,  Rabovsky,  A.,  Symons,  J.D., and  Jalili,  T. (2007). Quercetin reduces blood pressure in hypertensive subjects. J. Nutr., 137, 2405-2411.
•  Genthner,  B.R.,  Davis,  C.L., and  Bryant,  M.P. (1981). Features of rumen and sewage sludge strains of Eubacterium limosum, a methanol- and H₂-CO₂-utilizing species. Appl. Environ. Microbiol., 42, 12-19.
•  Gibellini,  L.,  Pinti,  M.,  Nasi,  M.,  Montagna,  J.P.,  De Biasi,  S.,  Roat,  E.,  Bertoncelli,  L.,  Cooper,  E.L., and  Cossarizza,  A. (2011). Quercetin and cancer chemoprevention. Evid. Based. Complement. Alternat. Med., 2011, 591356.
•  Hur,  H., and  Rafii,  F. (2000). Biotransformation of the isoflavonoids biochanin A, formononetin, and glycitein by Eubacterium limosum. FEMS Microbiol. Lett., 192, 21-25.
•  Jeong,  J.,  Bertsch,  J.,  Hess,  V.,  Choi,  S.,  Choi,  I.G.,  Chang,  I.S., and  Müller,  V. (2015). Energy conservation model based on genomic and experimental analyses of a carbon monoxide-utilizing, butyrate-forming acetogen, Eubacterium limosum KIST612. Appl. Environ. Microbiol., 81, 4782-4790.
•  Kanauchi,  O.,  Fukuda,  M.,  Matsumoto,  Y.,  Ishii,  S.,  Ozawa,  T.,  Shimizu,  M.,  Mitsuyama,  K., and  Andoh,  A. (2006). Eubacterium limosum ameliorates experimental colitis and metabolite of microbe attenuates colonic inflammatory action with increase of mucosal integrity. World. J. Gastroenterol., 12, 1071-1077.
•  Keppler,  K.,  Hein,  E.M., and  Humpf,  H.U. (2006). Metabolism of quercetin and rutin by the pig caecal microflora prepared by freeze-preservation. Mol. Nutr. Food Res., 50, 686-695.
•  Kobori,  M.,  Masumoto,  S.,  Akimoto,  Y., and  Oike,  H. (2011). Chronic dietary intake of quercetin alleviates hepatic fat accumulation associated with consumption of a Western-style diet in C57/BL6J mice. Mol. Nutr. Food Res., 55, 530-540.
•  Manach,  C.,  Morand,  C.,  Demigné,  C.,  Texier,  O.,  Régérat,  F., and  Rémésy,  C. (1997). Bioavailability of rutin and quercetin in rats. FEBS Lett., 409, 12-16.
•  Matsukawa,  N.,  Matsumoto,  M.,  Shinoki,  A.,  Hagio,  M.,  Inoue,  R., and  Hara,  H. (2009). Nondigestible saccharides suppress the bacterial degradation of quercetin aglycone in the large intestine and enhance the bioavailability of quercetin glucoside in rats. J. Agric. Food Chem., 57, 9462-9468.
•  Mlcek,  J.,  Jurikova,  T.,  Skrovankova,  S., and  Sochor,  J. (2016). Quercetin and its anti-allergic immune response. Molecules, 21, 623.
•  Müller,  E.,  Fahlbusch,  K.,  Walther,  R., and  Gottschalk,  G. (1981). Formation of N,N-dimethylglycine, acetic acid, and butyric acid from betaine by Eubacterium limosum. Appl. Environ. Microbiol., 42, 439-445.
•  Pogribna,  M.,  Freeman,  J.P.,  Paine,  D., and  Boudreau,  M.D. (2008). Effect of Aloe vera whole leaf extract on short chain fatty acids production by Bacteroides fragilis, Bifidobacterium infantis and Eubacterium limosum. Lett. Appl. Microbiol., 46, 575-580.
•  Possemiers,  S.,  Rabot,  S.,  Espín,  J.C.,  Bruneau,  A.,  Philippe,  C.,  González-Sarrías,  A.,  Heyerick,  A.,  Tomás-Barberán,  F.A.,  De Keukeleire,  D., and  Verstraete,  W. (2008). Eubacterium limosum activates isoxanthohumol from hops (Humulus lupulus L.) into the potent phytoestrogen 8-prenylnaringenin in vitro and in rat intestine. J. Nutr., 138, 1310-1316.
•  Sundin,  J.,  Rangel,  I.,  Repsilber,  D., and  Brummer,  R.J. (2015). Cytokine response after stimulation with key commensal bacteria differ in post-infectious irritable bowel syndrome (PI-IBS) patients compared to healthy controls. PLoS One, 10, e0134836.
•  Tamura,  M.,  Hori,  S.,  Inose,  A., and  Kobori,  M. (2020). Effects of γ-polyglutamic acid on blood glucose and caecal short chain fatty acids in adult male mice Food. Nutr. Sci., 11, 8-22.
•  Turner,  N.D.,  Paulhill,  K.J.,  Warren,  C.A.,  Davidson,  L.A.,  Chapkin,  R.S.,  Lupton,  J.R.,  Carroll,  R.J., and  Wang,  N. (2009). Quercetin suppresses early colon carcinogenesis partly through inhibition of inflammatory mediators. Acta. Hortic., 841, 237-242.
•  Ueno,  I.,  Nakano,  N., and  Hirono,  I. (1983). Metabolic fate of [₁₄C] quercetin in the ACI rat. Jpn. J. Exp. Med., 53, 41-50.
•  Wang,  S.,  Moustaid-Moussa,  N.,  Chen,  L.,  Mo,  H.,  Shastri,  A.,  Su,  R.,  Bapat,  P.,  Kwun,  I., and  Shen,  C.L. (2014). Novel insights of dietary polyphenols and obesity. J. Nutr. Biochem., 25, 1-18.
•  Yamamoto,  N.,  Moon,  J.H.,  Tsushida,  T.,  Nagao,  A., and  Terao,  J. (1999). Inhibitory effect of quercetin metabolites and their related derivatives on copper ion-induced lipid peroxidation in human low-density lipoprotein. Arch. Biochem. Biophys., 372, 347-354.
•  Zhang,  Z.,  Peng,  X.,  Li,  S.,  Zhang,  N.,  Wang,  Y., and  Wei,  H. (2014). Isolation and identification of quercetin degrading bacteria from human fecal microbes. PLoS One, 9, e90531.
•  Zhang,  M.,  Wang,  Y.,  Zhao,  X.,  Liu,  C.,  Wang,  B., and  Zhou,  J. (2021). Mechanistic basis and preliminary practice of butyric acid and butyrate sodium to mitigate gut inflammatory diseases: a comprehensive review. Nutr. Res., 95, 1-18.