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. 2022 Nov 9;32(3):371–379. doi: 10.1007/s10068-022-01198-6

The effect of fermented rice germ extracts on the inhibition of glucose uptake in the gastrointestinal tract in vitro and in vivo

Ye Ji Hyun 1, Soo-yeon Park 1, Ji Yeon Kim 1,2,✉
PMCID: PMC9905455  PMID: 36778085

Abstract

This study aimed to evaluate the effect of fermented rice germ extracts on the inhibition of glucose uptake in the gastrointestinal (GI) tract. Samples were prepared by extracting rice germ fermented with Lactobacillus plantarum with 30% ethanol (RG_30E) or 50% ethanol (RG_50E). Ferulic acid was determined as the active component in the samples. RG_30E significantly inhibited glucose uptake and mRNA expression of GLUT2 and SGLT1 to a larger extent than RG_50E in Caco-2 cells. A single oral administration was performed on C57BL/6 mice to confirm which substrate (glucose, sucrose, or maltose) the sample inhibited absorption of, improving postprandial blood glucose elevation. As a result, RG_30E resulted in significantly lower blood glucose levels and AUC after glucose and sucrose administration. Therefore, fermented rice germ extracted with 30% ethanol regulates glucose uptake through glucose transporters and can be expected to alleviate postprandial hyperglycemia in the GI tract.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-022-01198-6.

Keywords: Fermented rice germ, Glucose uptake, Glucose transporter, Gastrointestinal tract, Postprandial hyperglycemia

Introduction

Diabetes has become a major health problem worldwide. Diabetes is a metabolic disorder with a variety of causes characterized by chronic hyperglycemia due to defects in insulin secretion and metabolism (Vinayagam et al., 2016). If hyperglycemia is maintained for a long period, the function of the beta cells of the pancreas is impaired, resulting in impaired insulin synthesis and secretion (Schalkwijk and Stehouwer, 2005). This leads to more hyperglycemia. Studies have shown that diabetic patients have higher intestinal glucose transporter expression than healthy people (Dyer et al., 2002). Therefore, in diabetic patients, more glucose is absorbed in a shorter period of time, and blood glucose rises rapidly after postprandial glycemia. Mitigating the intestinal glucose transporter and glucose absorption is effective in suppressing and delaying diabetes and associated complications (Shamloo et al., 2018).

Carbohydrates taken from food are digested by amylase and glycosidases in the intestine to produce monosaccharides. Monosaccharides are absorbed from epithelial cells of the intestine by glucose transporters and provide glucose throughout the body (Shimizu et al., 2000). Glucose transporters play an important role in energy metabolism and regulation of glucose transport across cell membranes. Glucose in the intestine is transported primarily by two glucose transporters, glucose transporter 2 (GLUT2) and sodium-dependent glucose transporter 1 (SGLT1) (Manzano and Williamson, 2010). In the intestine, GLUT2 is expressed primarily in the basolateral membrane and transports glucose into the blood. SGLT1 is expressed in the apical membrane of intestinal epithelial cells and employs Na+/K+-ATPase (Li et al., 2020).

Caco-2 cells are a human colon cell line derived from colon cancer and are widely used in vitro to confirm glucose transport kinetics and glucose metabolism in the intestine. These cells have basolateral membranes, brush borders with microvilli, and tight junctions. Since Caco-2 cells have a phenotype similar to that of actual intestinal cells and abundantly express both GLUT2 and SGLT1, it is effective to confirm changes in glucose uptake under active transport or facilitated transport conditions (Johnston et al., 2005; Zheng et al., 2012).

Rice (Oryza sativa L.) contains various bioactive components. Ferulic acid, γ-oryzanol, GABA, and tocopherols are the main phytochemicals contained in rice germ. Some bioactive ingredients in rice germ from fermentation were found to have a beneficial effect on health because fermentation increases the nutritional value of food (McGovern et al., 2004; Tanaka et al., 2017). The total polyphenol content was significantly higher in rice embryos after fermentation than before fermentation. It is assumed that water-soluble polyphenol compounds are produced by microbial fermentation (Song and Lee, 2017). In particular, extraction is a process that can increase the amount of phenolic acid contained in fermented rice germs and increase antioxidant activity (Moongngarm et al., 2012; Perez-Ternero et al., 2017; Shin et al., 2019).

Ferulic acid (FA) is a phenolic acid and a phytochemical substance present in fruits, vegetables, and various plants. FA is a substance that is expected to treat diabetes because it increases the activity of antioxidant enzymes and prevents cell damage caused by diabetes (Dyer et al., 2002; Schalkwijk and Stehouwer, 2005). According to a recent study, FA decreased the expression of the GLUT2 gene in the liver tissue of high-fat and fructose-induced type 2 diabetic mice (Shamloo et al., 2018). In addition, in a diabetes model, FA showed antidiabetic potential by inhibiting the activity of α-glucosidase in the intestine (Adisakwattana et al., 2009). Previous studies have shown through the oral glucose tolerance test (OGTT) that FA helps lower blood glucose levels (Narasimhan et al., 2015a; Wang et al., 2015). However, although research on glucose uptake is actively being conducted, studies on the absorption of disaccharides containing glucose molecules such as sucrose and maltose are insufficient. The purpose of this study was to investigate the inhibition of glucose uptake by GLUT2 and SGLT1 in fermented rice germ extracts using a small intestine Caco-2 cell model and the absorption of glucose, sucrose, and maltose after a single administration of fermented rice germ extracts in C57BL/6 mice.

Materials and methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), DMEM low glucose, fetal bovine serum (FBS), penicillin–streptomycin, and Dulbecco’s phosphate buffered saline (DPBS) were purchased from Biowest (Nuaille’, Cholet, France). Bovine calf serum (BS), sodium pyruvate (100 mM), and MEM nonessential amino acids (NEAAs) were obtained from Gibco (Rockville, MD, USA). Ferulic acid, ethanol, ethyl acetate, methanol, acetonitrile, α-glucosidase, and phosphate buffer were purchased from Sigma–Aldrich (St. Louis, MO, USA). OPA reagent, borate buffer, and FMOC reagent were purchased from Agilent Technologies (Santa Clara, USA).

Preparation of samples

Rice germ was supplied by Cheongwon Saengmyeong Agricultural Cooperative Federation Rice Association Corporate. Lactobacillus plantarum KACC 15357 was used as a fermentation strain, and it was purchased from the Korean collection for type culture (KCTC, Korea). L. plantarum was cultured in MRS medium at 37 °C for 24 h. The culture medium was homogenized at 5,000 rpm for 10 min and then sterilized at 95 °C for 15 min to be used for fermentation. 3% (w/v) of the L. plantarum culture medium was inoculated onto a rice fermentation medium composed of 15% (w/v) rice germ and 15% (w/v) glucose. Fermented rice germ was produced by anaerobic fermentation for 24 h in a 37 °C incubator. Using 30% ethanol (Duksan, Seoul, Korea) and 50% ethanol, the fermented rice germ was extracted at 60 °C for 6 h. Vacuum extraction and concentration were performed with Cosmos 660 (Gyeongseo E&P, Incheon, Korea). The samples differ in the ratio of extracted ethanol. The samples extracted with 30% ethanol and 50% ethanol were expressed as RG_30E and RG_50E, respectively.

Quantification of compounds by HPLC analysis

The phenolic compounds were analyzed using an Ultimate 3000 HPLC system (Thermo Dionex, USA) equipped with a Youngjinbiochrom Inno C-18 column (250 mm × 4.6 mm, 5 μm). The mobile phases were 0.1% trifluoroacetic acid in distilled water (DW) (v/v) (A) and acetonitrile (B) under the following gradient conditions: 0 min, 10% B; 0–25 min, 10–60% B; 25–30 min, 60–100% B; 30–35 min, 100–100% B; 35–36 min, 100–10% B; 36–40 min, 10–10% B. The flow rate was 0.8 mL/min, and the injection volume was 10 µL. Wavelengths of 254 nm (4-hydroxybenzoic acid and vanillic acid) and 340 nm (ferulic acid) were used to detect phenolic compounds.

The GABA was analyzed using an Agilent LC system (Agilent Technologies, Santa Clara, USA) equipped with a Capcellpak UG120 C-18 column (250 mm × 4.6 mm, 5 μm) and photodiode array detector. The mobile phases were 40 mM monosodium phosphate (pH 7.8) (A) and acetonitrile/methanol/DW (45:45:10, v/v/v) (B) under the following gradient conditions: 0 min, 5% B; 0–31 min, 5–56% B; 31–33 min, 56–56% B; 33–34 min, 56–100% B; 34–38 min, 100–100% B. The flow rate was 1.5 mL/min, and the temperature was 40 °C. A wavelength of 338 nm was used to detect GABA.

The γ-oryzanol content of the fermented rice germ was analyzed using a slight modification of a method described in Huang and Ng (Huang and Ng, 2011). The analysis was carried out using an HPLC 1100 series system (Agilent Technologies, Santa Clara, USA) equipped with a GL Science Inertsil SIL 100 Å column (250 mm × 4.6 mm, 5 μm). The mobile phase was hexane/isopropanol/ethyl acetate/acetic acid (97.6:0.8:0.8:0.8, v/v/v/v), and the flow rate of isocratic elution was 1.0 mL/min. The injection volume was 10 µL, and the UV detector was set at a wavelength of 325 nm.

Tocopherols were analyzed using the Shiseido SI-2 series HPLC system (Shiseido, Tokyo, Japan) equipped with an Osaka soda Capcell Pak C18 MG S5 column (250 mm × 4.6 mm, 5 μm) by following the slightly modified method of Panfili et al. (Panfili et al., 2003). The mobile phase was composed of methanol/water/butanol (92:4:4, v/v/v) at a flow rate of 1.0 mL/min. The injection volume was 20 µL, and the detector was set at a wavelength of 292 nm.

α-Glucosidase inhibitory activity

α-Glucosidase activity was measured by a slight modification of the method reported by Jung et al. (Jung et al., 2007) and Laoufi et al. (Laoufi et al., 2017). An α-glucosidase enzyme solution was adjusted to 0.2 U/mL with phosphate buffer (0.1 M, pH 7.5). Then, 20 µL of the α-glucosidase enzyme solution and 20 µL of the phosphate buffer were mixed with 20 µL of samples or acarbose (Sigma, St Louis, Missouri, USA). The mixture was preincubated at 37 °C for 10 min. Then, 40 µL of 4-nitrophenyl-α-d-glucopyranoside (PNPG) was added and incubated at 37 °C for 40 min. To stop the reaction, 300 µL of 0.1 M sodium carbonate was added, and the absorbance was measured at 400 nm using a spectrometer (BioTek Instruments, Inc., Winooski, VT, USA).

Cell culture

Caco-2 cells were purchased from Korea Cell Line Bank (Seoul, Korea) and are a human intestinal epithelial cell line. Caco-2 cells were cultured in DMEM containing 10% FBS, 1% penicillin–streptomycin, and 1% NEAA at 37 °C in an atmosphere of 5% CO2.

Glucose uptake assay

Caco-2 cells were incubated for 48 h by seeding 2 × 105 cells/mL into 6-well plates with a pore size of 0.4 μm (Corning Costar Corp., NY, USA) containing 10% FBS and 1% penicillin–streptomycin. After washing with DPBS, the medium was replaced with glucose-free DMEM. Then, 10 mg/mL glucose and samples (RG_30E, RG_50E, and FA) were added and incubated for 48 h. A glucose (GO) assay kit (Sigma, St Louis, Missouri, USA) was used to measure the amount of glucose in the supernatant. The control was not treated with either glucose or rice germ, and G10 was treated with only 10 mg/mL glucose. All sample groups were treated with 10 mg/mL glucose and each sample.

RNA extraction and qRT–PCR

Total RNA was isolated from cultured Caco-2 cells using TRIzol solution (Life Technologies, Rockville, MD, USA) following the manufacturer’s protocol. TRIzol and chloroform were added to Caco-2 cells and centrifuged at 12,000 rpm for 20 min. The supernatant was separated, and an equal amount of isopropanol was added. cDNA was synthesized by reverse transcription using a Transcriptor First Strand cDNA Synthesis Kit (Life Technologies, Rockville, MD, USA). The relative mRNA expression levels were quantified by the comparison 2−ΔΔCT method. The primers were as follows: GLUT2 forward 5′-CCCTGTCTGTATCCAGCTTTG-3′ and reverse 5′-TGTTTGCTACTAACATGGCTTTG-3′; SGLT1 forward 5′-CTGGCAGGCCGAAGTATG-3′ and reverse 5′-CCACTTCCAATGTTACTAGCAAAG − 3′; GAPDH forward 5′-AGCCACATCGCTCAGACAC-3′ and reverse 5′-GCCCAATACGACCAAATCC-3′.

Animals

Sixty-nine 6-week-old male C57BL/6 mice were purchased from Hanabio (Ansan, Gyeonggi-do, Korea). Experimental animals were quarantined at the Southeast Medi-Chem Institute (SEMI; Animal Facility Registration Certificate: No. 412) and acclimated for 1 week. All mice were housed at 26.8 ± 0.5 °C temperature and 48.4 ± 1.7% humidity with lights on from 07:00 to 19:00. During the period of acclimatization and experimentation, the AIN-93G diet, which is a normal diet for experimental animals, was supplied. The diet and drinking water were freely accessed, and three to four animals were bred per cage. The individual body weight was measured, and the mice were divided into nine groups (n = 7/group) with equal average body weights between each group: G (glucose + DW); G30E (glucose + RG_30E); G50E (glucose + RG_50E); S (sucrose + DW); S30E (sucrose + RG_30E); S50E (sucrose + RG_50E); M (maltose + DW); M30E (maltose + RG_30E); and M50E (maltose + RG_50E). This study was conducted in accordance with the policies and regulations of SEMI’s Animal Experimental Ethics Committee (SEMI-20-007).

Oral glucose, sucrose, and maltose tolerance tests

All mice were fasted overnight, blood was collected from the tail vein, and blood glucose was measured (0 min). Sample solution (200 mg/kg BW) was administered as a single oral dose, and immediately, 0.3 mL of 2 g/kg BW substrate solution (glucose, sucrose, or maltose) was administered to each mouse. The control groups (G, glucose; S, sucrose; M, maltose) of each group were administered the same volume of DW. Blood glucose was measured in blood samples from the tail vein at 15, 30, 45, 60, 90, and 120 min. The area under the curve (AUC) was calculated using the trapezoid method (Purves, 1992).

Statistical analysis

All results are expressed as the mean ± standard error (SE). Data were statistically analyzed using SAS 9.4 (SAS Institute, Cary, NC, USA). Student’s t-test was used to determine the level of significance of the content of compounds. Analysis of variance (ANOVA) was performed followed by Duncan’s multiple range test, and p < 0.05 was defined as statistically significant. Significant differences between each group are indicated with different letters.

Results and discussion

The content of compounds in fermented rice germ extracts

The contents of phenolic compounds, GABA, γ-oryzanol, and tocopherols in the fermented rice germ extracts are shown in Table 1. The chromatograms of the analyzed standards and samples are shown in Supplementary Fig. 1A–F. The data are presented as mg per 100 g of the RG_30E and RG_50E powders. The content of all compounds was higher in RG_30E than in RG_50E. The phenolic acids contained in fermented rice germ extracts were ferulic acid, 4-hydroxybenzoic acid, and vanillic acid. The contents of phenolic compounds contained in fermented rice germ extracted with 30% ethanol were 8.18 ± 0.07 mg/100 g, 2.01 ± 0.13 mg/100 g, and 2.96 ± 0.13 mg/100 g, respectively. The contents of phenolic compounds in fermented rice germ extracted with 50% ethanol were 7.71 ± 0.05 mg/100 g, 1.93 ± 0.01 mg/100 g, and 2.10 ± 0.09 mg/100 g, respectively. RG_30E contained significantly higher amounts of ferulic acid and vanillic acid than RG_50E (p < 0.05). RG_30E contained 929.00 ± 7.00 mg/100 g GABA, and RG_50E contained 912.67 ± 1.53 mg/100 g GABA. RG_30E also had a significantly higher content of GABA than RG_50E (p < 0.05). γ-Oryzanol, α-tocopherol and γ-tocopherol were 180.89 ± 1.03 mg/100 g, 4.98 ± 0.29 mg/100 g, and 1.44 ± 0.12 mg/100 g for RG_30E and 167.75 ± 8.90 mg/100 g, 4.69 ± 0.52 mg/100 g, and 1.35 ± 0.01 mg/100 g for RG_50E, respectively. Bioactive compounds such as phenolic compounds, GABA, γ-oryzanol, and tocopherols are known to be the most abundant in rice bran, including rice germ (Moongngarm et al., 2012; Perez-Ternero et al., 2017; Shin et al., 2019). In particular, the results demonstrated that the contents of ferulic acid, vanillic acid, and GABA in RG_30E were significantly higher than those in RG_50E (p < 0.05).

Table 1.

Content of compounds in fermented rice germ extracts

Group Content of compounds (mg/100 g)
Ferulic acid 4-Hydroxybenzoic acid Vanillic acid GABA γ- Oryzanol α-Tocopherol γ- Tocopherol
RG_30E 8.18 ± 0.07* 2.01 ± 0.13 2.96 ± 0.13* 929.00 ± 7.00* 180.89 ± 1.03 4.98 ± 0.29 1.44 ± 0.12
RG_50E 7.71 ± 0.05 1.93 ± 0.01 2.10 ± 0.09 912.67 ± 1.53 167.75 ± 8.90 4.69 ± 0.52 1.35 ± 0.01
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The values are mean ± standard error (SE). RG_30E, fermented rice germ extracted with 30% ethanol; RG_50E, fermented rice germ extracted with 50% ethanol. Asterisks indicate significant differences between the two groups at the p < 0.05 level using student’s t-tests

*p-value < 0.05

Fermented rice contains more bioactive compounds than non-fermented raw rice, such as ferulic acid (Nisa et al., 2019). Ferulic acid is a prominent phenolic compound during the fermentation process (Schmidt et al., 2014). Ferulic acid is an active component that can have antidiabetic potential due to its high antioxidant activity and involvement in glucose mechanisms such as glucose transporter expression and glucose load in the GI tract (Narasimhan et al., 2015a; 2015b; Srinivasan et al., 2007; Vinayagam et al., 2016; Wang et al., 2015). Furthermore, it has been reported that γ-oryzanol, a bioactive phytochemical abundant in rice, is a mixture of ferulic acid esters of triterpene alcohols and sterols and is mainly hydrolyzed into ferulic acid in the body (Kokumai et al., 2019). It was confirmed that the γ-oryzanol content of the fermented samples decreased continuously during fermentation, but the ferulic acid concentration increased (Sirilun et al., 2015). Hence, this study focused on the significant difference in ferulic acid between fermented samples.

Inhibition of α-glucosidase activity

The α-glucosidase activity of the samples was confirmed compared with acarbose, which is known as an inhibitor of α-glucosidase activity. Acarbose showed 53.93 ± 0.45% α-glucosidase activity inhibition at 8 mg/mL. RG_30E and RG_50E inhibited α-glucosidase activity by 18.39 ± 1.55% and 18.13 ± 1.62%, respectively, at a concentration of 100 mg/mL (Table 2).

Table 2.

The effect of fermented rice germ extracts on α-glucosidase activity inhibition

Group
RG_30E RG_50E Acarbose
Concentration (mg/mL) 100 100 8
α-glucosidase activity inhibition (%) 18.39 ± 1.55b 18.13 ± 1.62b 53.93 ± 0.45a
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The values are mean ± standard error (SE). RG_30E, fermented rice germ extracted with 30% ethanol; RG_50E, fermented rice germ extracted with 50% ethanol. Data with different letters are significantly different at p < 0.05 followed by Duncan’s multiple range test

The inhibition of α-glucosidase activity was measured to determine the effect of α-glucosidase in fermented rice germ on reducing postprandial hyperglycemia. Low α-glucosidase activity delays the rise of blood glucose by inhibiting the degradation of oligosaccharides into monosaccharides, which are uptake forms. Acarbose is known to inhibit the increase in blood glucose by inhibiting α-glucosidase activity. However, acarbose has a side effect that causes GI disorders due to the fermentation of carbohydrates that are not absorbed by the intestine. Therefore, this study confirmed whether fermented rice germ extracts could inhibit the increase in postprandial blood glucose by suppressing α-glucosidase activity and replacing acarbose (Adisakwattana et al., 2009; Bischoff, 1995; Kelley et al., 1998). According to the results, compared to 8 mg/mL acarbose, the samples had a weaker inhibitory effect on α-glucosidase activity and did not directly inhibit glucose uptake. Thus, this study confirmed the metabolism of glucose uptake other than α-glucosidase, which fermented rice germ extracts suppress.

Inhibition of glucose uptake

The inhibition of glucose uptake in Caco-2 cells is shown in Fig. 1. Compared to G10, RG_30E retained a greater amount of glucose than RG_50E. In particular, it was confirmed that the higher the concentration of both RG_30E and RG_50E, the more effectively glucose uptake was inhibited. Glucose uptake decreased significantly after FA treatment by 5.37% compared to G10 (p < 0.05). Compared to G10, the amount of glucose remaining in the medium of cells treated with RG_30E at 250 µg/mL and 500 µg/mL increased by 8.63% and 11.21%, respectively (p < 0.05). With RG_50E treatment at 250 µg/mL and 500 µg/mL, glucose uptake was downregulated by approximately 5.65% and 8.91%, respectively (p < 0.05).

Fig. 1.

Fig. 1

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The effect of fermented rice germ extracts on glucose uptake in Caco-2 cells. CON without glucose and sample, RG_30E fermented rice germ extracted with 30% ethanol, RG_50E fermented rice germ extracted with 50% ethanol, FA ferulic acid. Data with different letters are significantly different at p < 0.05 followed by Duncan’s multiple range test

One effective way to manage diabetes is to control postprandial blood glucose concentrations by inhibiting glucose uptake in the intestine (Goto et al., 2012; Kim et al., 2011). This result indicates that fermented rice germ extracted with 30% ethanol regulates intestinal glucose uptake, a potentially important factor in diabetes management.

The effect of rice germ on the expression of glucose transporter-related mRNA

The effect of rice germ on the expression of glucose transporter-related mRNA is shown in Fig. 2. With FA treatment, the mRNA levels of GLUT2 and SGLT1 were significantly downregulated by 79.89% and 66.18%, respectively (p < 0.05). RG_30E significantly decreased the concentration-dependent gene expression of GLUT2 and SGLT1 (p < 0.05). The GLUT2 expression levels after RG_30E treatment significantly decreased by 37.46% and 53.02% at 250 µg/mL and 500 µg/mL, respectively, and the expression levels of SGLT1 also decreased significantly by 39.26% and 59.2% at 250 µg/mL and 500 µg/mL, respectively (p < 0.05). RG_30E resulted in lower gene expression than RG_50E. However, GLUT2 significantly decreased by 38.29% (at 250 µg/mL concentration) and 28.44% (at 500 µg/mL concentration) compared to G10 (p < 0.05). The expression of SGLT1 mRNA decreased, but not significantly (p < 0.05).

Fig. 2.

Fig. 2

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The effect of fermented rice germ extracts on the expression of glucose transporter-related mRNA in Caco-2 cells. Relative mRNA expression indicated by fold-change compared to the control treated with only 10 mg/mL glucose. CON without glucose and sample, RG_30E fermented rice germ extracted with 30% ethanol, RG_50E fermented rice germ extracted with 50% ethanol, FA ferulic acid. A GLUT2 mRNA expression; B SGLT1 mRNA expression. Data with different letters are significantly different at p < 0.05 followed by Duncan’s multiple range test

Few studies have shown that fermented rice germ prevents hyperglycemia by inhibiting glucose uptake in the GI tract. However, recent studies have shown that ferulic acid can inhibit glucose uptake by regulating the expression of GLUT2 (Narasimhan et al., 2015b). Since fermented rice germ extracts contain a relatively large amount of ferulic acid, the effect of glucose metabolism by the glucose transporter was confirmed in the fermented rice germ extracts. The glucose transporters involved in glucose uptake in the GI tract are GLUT2 and SGLT1. GLUT2 is located on the basolateral surface of enterocytes and is involved in the transfer of glucose from the intestine to the circulatory system. SGLT1 is located across the apical membrane of enterocytes and contributes to glucose absorption (Alzaid et al., 2013). There is evidence that the combination of GLUT2 and SGLT1 affects glucose uptake through the apical membrane of intestinal cells (Kellett et al., 2008). These in vitro results showed that fermented rice germ extracted with 30% ethanol has the potential to lower postprandial blood glucose levels by inhibiting glucose uptake by affecting glucose transporters in the GI tract.

Quantification of oral glucose, sucrose, and maltose tolerance tests (OGTT, OSTT, and OMTT)

Based on the in vitro results, a single administration of fermented rice germ extracts (200 mg/kg BW) to C57BL/6 mice and administration of a substrate (2 g/kg BW) demonstrated an acute inhibitory effect on the increase in blood glucose levels by the substrates (glucose, sucrose, maltose). The effect of fermented rice germ extracts on blood glucose levels was confirmed by an OGTT, oral sucrose tolerance test (OSTT), and oral maltose tolerance test (OMTT) in C57BL/6 mice. After administering glucose, sucrose, or maltose and a single administration of RG_30E or RG_50E, the blood glucose level reached its peak in 15 min and then returned to fasting levels. Compared to the other groups, the group intaking RG_30E dropped blood glucose levels more rapidly in the OGTT, OSTT, and OMTT (Fig. 3). Figure 4 shows the glucose AUC values. In the OGTT and OMTT, the AUC value decreased by 6.68% and 7.29% in the G + RG_30E and M + RG_30E groups, respectively, compared to the control of glucose and maltose groups (p < 0.05). In addition, OSTT indicated a decrease in both S + RG_30E and S + RG_50E groups, but only the S + RG_30E group significantly decreased by 7.53% compared to only sucrose group (p < 0.05). In experiments with oral administration of glucose and sucrose, but not maltose, the AUC value was significantly reduced by RG_30E, as shown in Fig. 4 (p < 0.05).

Fig. 3.

Fig. 3

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The effect of fermented rice germ extracts on substrate (glucose, sucrose, maltose)-loaded C57BL/6 mice. A Blood glucose level after oral administration of glucose for 120 min; B Blood glucose level after oral administration of sucrose for 120 min; C Blood glucose level after oral administration of maltose for 120 min. After the solution of rice germ extracts was administered to C57BL/6 mice, the substrate solution (2 g/kg BW) was orally administered. The substrates and samples were loaded for 120 min, and blood glucose was measured at each time point. G only glucose, G + RG_30E glucose with RG_30E, G + RG_50E glucose with RG_50E, S only sucrose, S + RG_30E sucrose with RG_30E, S + RG_50E sucrose with RG_50E, M only maltose, M + RG_30E maltose with RG_30E, M + RG_50E maltose with RG_50E. Data with different letters are significantly different at p < 0.05 followed by Duncan’s multiple range test

Fig. 4.

Fig. 4

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The effect of rice germ extracts on AUC by oral substrate loading test in C57BL/6 mice. A Oral glucose tolerance test (OGTT); B oral sucrose tolerance test (OSTT); C oral maltose tolerance test (OMTT). After the solution of rice germ extracts was administered to C57BL/6 mice, the substrate solution (2 g/kg BW) was orally administered. The substrates and samples were loaded for 120 min, and blood glucose was measured at each time point. G only glucose, G + RG_30E glucose with RG_30E, G + RG_50E glucose with RG_50E, S only sucrose, S + RG_30E sucrose with RG_30E, S + RG_50E sucrose with RG_50E, M only maltose, M + RG_30E maltose with RG_30E, M + RG_50E maltose with RG_50E. Data with different letters are significantly different at p < 0.05 followed by Duncan’s multiple range test

Oral tolerance tests were used to determine the potential mechanism of action in diabetes treatment and focused on a variety of glucosidase enzymes for certain types of α-glycoside bonds and the transport of glucose (Solares-Pascasio et al., 2021). Our results indicated that fermented rice germ extracts have a poor ability to inhibit glucose production by breaking the bonds between glucose molecules. On the other hand, it was suggested that it helped suppress the rise of blood glucose by inhibiting the absorption of glucose, a monosaccharide, and the production of glucose and fructose from sucrose. Sucrose absorption is regulated after hydrolysis by the apical membrane uptake rate of glucose and fructose (Hurtado and Waasdorp, 2018). Therefore, the extend our previous observations by showing that the hypoglycaemic effects of fermented rice germ extract are in part mediated via regulation of hepatic glycolysis and gluconeogenesis in db/db mice. Our research previously shown that fermented rice germ extract reduce blood glucose concentrations of type 2 diabetes mice improving hyperglycemia and hepatic glucose metabolism (Hyun et al., 2021).

However, there are several limitations to this study. First, the mechanism by which the enzyme inhibits glucose uptake in the intestine was not identified. The inhibition of α-glucosidase activity was measured, and the fermented rice germ extracts did not affect enzyme activity as much as acarbose. Therefore, it was confirmed that fermented rice germ extracts regulate glucose uptake by affecting glucose transporters in Caco-2 cells. Subsequent studies will need to confirm that fermented rice germ extracts have the potential to help control the rise of blood glucose by promoting the activity of enzymes associated with glucose absorption. Second, the fermented rice germ extracts were administered orally as a single dose to confirm the loading of various sugars containing glucose molecules. However, no study has been conducted on the inhibition of postprandial blood glucose elevation after long-term ingestion of fermented rice germ extracts. Long-term consumption of fermented rice germ extracts is expected to improve postprandial hyperglycemia and promote antidiabetic effects.

In conclusion, this study has demonstrated the effect on the uptake of glucose in the GI tract by extracting fermented rice germ with 30% (RG_30E) or 50% (RG_50E) ethanol. RG_30E suppressed glucose uptake and GLUT2 and SGLT1 expression in Caco-2 cells compared with RG_50E. Blood glucose was measured for 120 min after a single oral administration of fermented rice germ extracts in C57BL/6 mice and glucose, sucrose, or maltose administration. RG_30E inhibited the absorption of all three substrates, and in particular, the absorption of glucose and sucrose significantly decreased (p < 0.05). Therefore, fermented rice germ extract helps improve postprandial hyperglycemia by inhibiting glucose uptake in the GI tract.

Supplementary Information

Below is the link to the electronic supplementary material.

10068_2022_1198_MOESM1_ESM.docx (626.1KB, docx)

Supplementary material 1 (DOCX 626.1 kb)

Acknowledgements

This work was supported by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Agro and Livestock Products Safety·Flow Management Technology Development Program, funded by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) (119072-1). We appreciate the Cheongwon Saengmyeong Agricultural Cooperative Federation Rice association corporation for supporting the fermented rice germ extract samples.

Declarations

Conflict of interest

The authors have no financial conflicts of interest to declare.

Footnotes

Publisher’s Note

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Contributor Information

Ye Ji Hyun, Email: yj0706hyun@naver.com.

Soo-yeon Park, Email: Sooyeon.park@seoultech.ac.kr.

Ji Yeon Kim, Email: jiyeonk@seoultech.ac.kr.

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Supplementary material 1 (DOCX 626.1 kb)

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