Jeju Citrus (Citrus unshiu) Leaf Extract and Hesperidin Inhibit Small Intestinal α-Glucosidase Activities In Vitro and Postprandial Hyperglycemia in Animal Model

Abstract

Citrus fruits are widely distributed in East Asia, and tea made from citrus peels has demonstrated health benefits, such as a reduction in fever, inflammation, and high blood pressure. However, citrus leaves have not been evaluated extensively for their possible health benefits. In this study, the α-glucosidase-inhibitory activity of Jeju citrus hot-water (CW) and ethyl alcohol (CE) extracts, along with hesperidin (HP) (a bioactive compound in citrus leaf extracts), was investigated, and furthermore, their effect on postprandial blood glucose reduction in an animal model was determined. The hesperidin contents of CW and CE were 15.80 ± 0.18 and 39.17 ± 0.07 mg/g-extract, respectively. Hesperidin inhibited α-glucosidase (IC₅₀, 4.39), sucrase (0.50), and CE (2.62) and demonstrated higher α-glucosidase inhibitory activity when compared to CW (4.99 mg/mL). When using an SD rat model, during sucrose and starch loading tests with CE (p < 0.01) and HP (p < 0.01), a significant postprandial blood glucose reduction effect was observed when compared to the control. The maximum blood glucose levels (Cmax) of the CE administration group decreased by about 15% (from 229.3 ± 14.5 to 194.0 ± 7.4, p < 0.01) and 11% (from 225.1 ± 13.8 to 201.1 ± 7.2 hr·mg/dL, p < 0.05) in the sucrose and starch loading tests, respectively. Our findings suggest that citrus leaf extracts standardized to hesperidin may reduce postprandial blood glucose levels through the observed inhibitory effect against sucrase, which results in delayed carbohydrate absorption. Our findings provide a biochemical rationale for further evaluating the benefits of citrus leaves.

Keywords: Citrus unshiu leaf; diabetes; inhibition; postprandial hyperglycemia; α-glucosidases.

Figure 1

Chemical structure of hesperidin.

Figure 2

HPLC profiles of citrus leaf extracts (standard solution (a), hot-water extract (b), and ethyl alcohol extract (c)). 1. Rutin; 2. Neoeriocitrin; 3. Narirutin; 4. Rhoifolin; 5. Naringin; 6. Hesperidin; 7. Neohesperidin; 8. Neoponcirin; 9. Poncirin; 10. Naringenin; 11. Hesperetin; 12. Isosinensetin; 13. Sinensetin; 14. 4,5,7-Trimethoxy flavon; 15. Nobiletin; 16. 4,5,6,7-Tetramethoxy flavon; 17. Tangeretin; 18. 5-Demethyl nobiletin; and 19. Gardenin B.

Figure 2

HPLC profiles of citrus leaf extracts (standard solution (a), hot-water extract (b), and ethyl alcohol extract (c)). 1. Rutin; 2. Neoeriocitrin; 3. Narirutin; 4. Rhoifolin; 5. Naringin; 6. Hesperidin; 7. Neohesperidin; 8. Neoponcirin; 9. Poncirin; 10. Naringenin; 11. Hesperetin; 12. Isosinensetin; 13. Sinensetin; 14. 4,5,7-Trimethoxy flavon; 15. Nobiletin; 16. 4,5,6,7-Tetramethoxy flavon; 17. Tangeretin; 18. 5-Demethyl nobiletin; and 19. Gardenin B.

Figure 3

Dose-dependent changes in SD rat small intestinal α-glucosidase-inhibitory activity (% inhibition) of GO2KA1 (GO), Jeju citrus leaf hot-water extract (CW), Jeju citrus leaf ethyl alcohol extract (CE), and hesperidin (HP). Different corresponding letters indicate significant differences at p < 0.05 by Duncan’s test. ^a–d First letter indicates differences among different samples, and ^A–C second one indicates differences among different concentrations of same samples.

Figure 4

Dose-dependent changes in SD rat small intestinal sucrase-inhibitory activity (% inhibition) of GO2KA1 (GO), Jeju citrus leaf hot-water extract (CW), Jeju citrus leaf ethyl alcohol extract (CE), and hesperidin (HP). Different corresponding letters indicate significant differences at p < 0.05 by Duncan’s test. ^a–c First letter indicates differences among different samples, and ^A–C second one indicates differences among different concentrations of same samples.

Figure 5

Dose-dependent changes in SD rat small intestinal maltase-inhibitory activity (% inhibition) of GO2KA1 (GO), Jeju citrus leaf hot-water extract (CW), Jeju citrus leaf ethyl alcohol extract (CE), and hesperidin (HP). Different corresponding letters indicate significant differences at p < 0.05 by Duncan’s test. ^a–c First letter indicates differences among different samples, and ^A–D second one indicates differences among different concentrations of same samples.

Figure 6

Dose-dependent changes in SD rat small intestinal glucoamylase-inhibitory activity (% inhibition) of GO2KA1 (GO), Jeju citrus leaf hot-water extract (CW), Jeju citrus leaf ethyl alcohol extract (CE), and hesperidin (HP). Different corresponding letters indicate significant differences at p < 0.05 by Duncan’s test. ^a–c First letter indicates differences among different samples, and ^A–D second one indicates differences among different concentrations of same samples.

Figure 7

Dose-dependent anti-hyperglycemic effect of ethyl alcohol extracts of citrus leaves (CE) in sucrose loading test. After fasting for 24 h, 5-week-old male SD rats were orally administered sucrose solution (2.0 g/kg-body weight (b.w.)) with or without samples (CE 0.1 g/kg-b.w., CE 0.5 g/kg-b.w., and positive control: GO2KA1 0.5 g/kg-b.w.). Each point represents mean ± standard deviation (n = 10). ** p < 0.01 and *** p < 0.001 compared to different samples at the same concentration by unpaired Student’s t-test.

Figure 8

The dose-dependent anti-hyperglycemic effect of hesperidin (HP) on sucrose loading test results. After fasting for 24 h, 5-week-old male SD rats were orally administered a sucrose solution (2.0 g/kg-body weight (b.w.)) with or without the test samples (HP 0.1 g/kg-b.w. and HP 0.5 g/kg-b.w.). Each point represents mean ± standard deviation (n = 10). *** p < 0.001 compared to different samples at the same concentration by unpaired Student’s t-test.

Figure 9

The dose-dependent anti-hyperglycemic effect of ethyl alcohol extracts of citrus leaves (CE) on starch loading test results. After fasting for 24 h, 5-week-old male SD rats were orally administered a starch solution (2.0 g/kg-body weight (b.w.)) with or without samples (CE 0.1 g/kg-b.w., CE 0.5 g/kg-b.w., and positive control: GO2KA1 0.5 g/kg-b.w.). Each point represents mean ± standard deviation (n = 10). * p < 0.05 and *** p < 0.001 compared to different samples at the same concentration by unpaired Student’s t-test.

Figure 10

The dose-dependent anti-hyperglycemic effect of hesperidin (HP) on starch loading test results. After fasting for 24 h, 5-week-old male SD rats were orally administered a starch solution (2.0 g/kg-body weight (b.w.)) with or without samples (HP 0.1 g/kg-b.w. and HP 0.5 g/kg-b.w.). Each point represents mean ± standard deviation (n = 10). *** p < 0.001 compared to different samples at the same concentration by unpaired Student’s t-test.