Effect of herbal extracts on glucose absorption
Blood glucose levels peaked at 30 min after ingesting 5 g/kg of starch under fasting conditions (Fig. 1). C. limetta extract slightly reduced the hyperglycemic peak (0.12-fold), while B. media and I. sonorae decreased (p < 0.05) postprandial glucose levels by 0.34-0.36-fold. This hints a mild postprandial antihyperglycemic activity. In vitro tests demonstrated that I. sonorae and B. media had the highest inhibitory activity α-amylase (25.4 and 20.5%, respectively) as compared to C. limetta (5.3%) (Table 1). Acarbose exerted even greater inhibitory activity (88.1%). For α-glucosidase, C. limetta and B. media showed similar low inhibition (5.1 and 4.4%, respectively), while I. sonorae lacked this effect.
Table 1
In vitro inhibitory activity of I. sonorae, C. limetta, and B. media extracts against carbohydrates digestive enzymes.
Herbal extracts | α-Amylase inhibition | α-Glucosidase inhibition |
I. sonorae | 25.4 ± 0.3b | ND |
C. limetta | 5.3 ± 0.5 | 5.1 ± 0.5b |
B. media | 20.5 ± 2.0c | 4.4 ± 0.4b |
Acarbose | 88.1 ± 6.3a | 81.9 ± 6.6a |
Values are expressed as maximum percentage of inhibition (%). Data are expressed as mean values ± standard deviation (n = 3). Means within a column followed by the same letter are not significantly different (p < 0.05) by Tukey’s test. ND: not detected.
Effect of herbal extracts on glucose uptake in 3T3-L1 adipocyte cells
All herbal extracts increased (p < 0.05) glucose uptake as compared to the control cells (Fig. 2). C. limetta and I. sonorae led to lower extracellular glucose levels (0.22-0.38-fold) as compared to the control group. Remarkably, B. media significantly reduced extracellular glucose levels (0.68-0.76-fold) at both concentrations, comparable to effect of insulin at the highest B. media concentration (1.0 mg/mL). B. media extract induced significant overexpression of Glut4, Irs1, and Pi3k genes (2.27–2.85, 1.98–2.60, and 2.51-3.19-fold, respectively) as compared to the control cells, showcasing a dose-response relationship (Fig. 3).
Polyphenol profile of herbal extracts
A comprehensive analysis of the polyphenol profiles was conducted (Table 2). I. sonorae had 31 identified phenolic compounds, including eight hydroxybenzoic acids, twelve hydroxycinnamic acids, three flavanones, six flavonols, and two hydroxycoumarins.
C. limetta roots extract had 39 phenolic compounds, including eleven hydroxybenzoic acids, twenty hydroxycinnamic acids, one flavanol, four flavanones, two flavonols, and one hydroxycoumarin. B. media contained 28 identified polyphenols, including twelve hydroxybenzoic acids, ten hydroxycinnamic acids, one flavanol, four flavanones, three flavonols, and two hydroxycoumarins. Multivariate analyses linked phenolic compounds to the previously described antidiabetic potential. I. sonorae and B. media extracts showed the highest in vitro α-amylase inhibitory activity (Table 1), which was linked mainly to hydroxybenzoic acid hexoside (PA_5), feruloylquinic acid (PA_24), and benzoic acid (PA_10) (Fig. 4). Accordingly, these compounds were most abundant in these extracts (Table 2).
Table 2
Polyphenol profile of I. sonorae, C. limetta, and B. media extracts by UPLC-QTOF MSE.
Code | Polyphenols | Rt (min) | Molecular Formula | Expected mass (Da) | Observed mass (Da) | Mass error (ppm) | I. sonorae | C. limetta | B. media |
| Hydroxybenzoic acids | | | | | | | | |
PA_1 | Hydroxybenzoic acid isomer I | 1.22 | C7H6O3 | 138.0317 | 138.0312 | -3.3747 | 1293.3 | 1772.8 | 620.4 |
PA_2 | Protocatechuic acid hexoside | 1.63 | C13H16O9 | 316.0794 | 316.0790 | -1.4622 | 21512.3 | 347697.0 | 5984.6 |
PA_3 | Vanillic acid† | 1.70 | C8H8O4 | 168.0423 | 168.0417 | -3.2769 | ND | 26039.5 | 51512.0 |
PA_4 | Dihydroxybenzoic acid isomer I | 1.94 | C7H6O4 | 154.0266 | 154.0266 | -0.1933 | 668.5 | 446.8 | 20571.0 |
PA_5 | Hydroxybenzoic acid hexoside | 2.20 | C13H16O8 | 300.0845 | 300.0834 | -3.6896 | 3536.4 | 487.7 | 3027.3 |
PA_6 | Gallic acid ethyl ester | 2.36 | C9H10O5 | 198.0528 | 198.0523 | -2.5838 | ND | 96098.1 | 20600.6 |
PA_7 | Hydroxybenzoic acid isomer II | 3.13 | C7H6O3 | 138.0317 | 138.0315 | -1.4297 | 1614.8 | 966.4 | 87775.5 |
PA_8 | Protocatechuic acid† | 3.32 | C7H6O4 | 154.0266 | 154.0259 | -4.5348 | 853.3 | 444.1 | 1993.8 |
PA_9 | Dihydroxybenzoic acid isomer II | 4.21 | C7H6O4 | 154.0266 | 154.0259 | -4.7078 | ND | ND | 506.4 |
PA_10 | Benzoic acid | 4.27 | C7H6O2 | 122.0358 | 122.0368 | 0.3424 | 2721.0 | 829.1 | 1300.0 |
PA_11 | Syringic acid† | 4.48 | C9H10O5 | 198.0528 | 198.0523 | -2.5853 | ND | 16507.5 | 11358.3 |
PA_12 | Hydroxybenzoic acid isomer III | 6.67 | C7H6O3 | 138.0317 | 138.0316 | -0.3344 | 489.8 | 3217.6 | 1763.7 |
| Hydroxycinnamic acids | | | | | | | | |
PA_13 | Caffeoyl tartaric acid | 1.08 | C13H12O9 | 312.0481 | 312.0475 | -1.9704 | ND | 938.1 | ND |
PA_14 | Cinnamic acid† | 1.45 | C9H8O2 | 148.0524 | 148.0519 | -3.6684 | 3911.7 | ND | ND |
PA_15 | Caffeoylquinic acid isomer I | 2.57 | C16H18O9 | 354.0951 | 354.0954 | 0.8135 | 6010.1 | 450.2 | ND |
PA_16 | Caffeic acid ethyl ester | 3.52 | C11H12O4 | 208.0736 | 208.0731 | -2.1574 | ND | 2734.5 | ND |
PA_17 | Coumaroylquinic acid isomer I | 3.68 | C16H18O8 | 338.1002 | 338.1005 | 1.0962 | ND | 747.8 | ND |
PA_18 | Ferulic acid† | 3.68 | C10H10O4 | 194.0579 | 194.0576 | -1.8077 | ND | 2984.7 | ND |
PA_19 | Ferulic acid hexoside | 3.69 | C16H20O9 | 356.1107 | 356.1107 | -0.1713 | ND | 8275.5 | 394.7 |
PA_20 | Coumaroyl hexose | 4.09 | C15H18O8 | 326.1002 | 326.0996 | -1.7866 | 1237.7 | 1070.8 | ND |
PA_21 | Caffeoylquinic acid isomer II | 4.17 | C16H18O9 | 354.0951 | 354.0943 | -2.0983 | 1451.5 | 999.5 | 10313.2 |
PA_22 | Coumaroyl glycolic acid | 4.45 | C11H10O5 | 222.0528 | 222.0523 | -2.4526 | ND | 879.9 | 5151.3 |
PA_23 | Ellagic acid hexoside | 4.73 | C20H16O13 | 464.0591 | 464.0602 | 2.3819 | ND | 690.2 | ND |
PA_24 | Feruloylquinic acid | 5.12 | C17H20O9 | 368.1107 | 368.1103 | -1.1118 | 1715.0 | ND | 1050.0 |
PA_25 | Coumaric acid† | 5.18 | C9H8O3 | 164.0473 | 164.0472 | -0.8242 | 613.7 | 1563.4 | 780.1 |
PA_26 | Sinapoylquinic acid isomer I | 5.23 | C18H22O10 | 398.1213 | 398.1200 | -3.3438 | ND | 446.0 | ND |
PA_27 | Ellagic acid pentoside | 5.32 | C19H14O12 | 434.0485 | 434.0492 | 1.4832 | ND | 2870.2 | ND |
PA_28 | Coumaroylquinic acid isomer II | 5.34 | C16H18O8 | 338.1002 | 338.0999 | -0.7703 | ND | 502.8 | 369.2 |
PA_29 | Ellagic acid† | 5.63 | C14H6O8 | 302.0063 | 302.0067 | 1.2807 | ND | 1194.1 | 1477.4 |
PA_30 | Isoferulic acid | 5.73 | C10H10O4 | 194.0579 | 194.0576 | -1.8164 | 539.7 | 4217.5 | 2327.3 |
PA_31 | Dicaffeoylquinic acid isomer I | 6.29 | C25H24O12 | 516.1268 | 516.1257 | -2.0080 | 72712.9 | 2850.5 | ND |
PA_32 | Sinapoylquinic acid isomer II | 6.36 | C18H22O10 | 398.1213 | 398.1231 | 4.6479 | ND | 1554.8 | 1459.7 |
PA_33 | Dicaffeoylquinic acid isomer II | 6.39 | C25H24O12 | 516.1268 | 516.1260 | -1.5250 | 28039.8 | 1634.2 | 2519.1 |
PA_34 | Caffeoylquinic acid isomer III | 6.40 | C16H18O9 | 354.0951 | 354.0947 | -0.9726 | 4404.7 | ND | ND |
PA_35 | Dicaffeoylquinic acid isomer III | 6.73 | C25H24O12 | 516.1268 | 516.1272 | 0.8322 | 75354.8 | 2355.7 | ND |
PA_36 | Rosmarinic acid† | 6.82 | C18H16O8 | 360.0845 | 360.0841 | -1.1864 | 586.0 | ND | 1831112.9 |
| Flavanols | | | | | | | | |
F_1 | (Epi)-catechin hexose | 5.20 | C21H24O11 | 452.1319 | 452.1328 | 2.0200 | ND | 1731.3 | 678.3 |
| Flavanones | | | | | | | | |
F_2 | Neoeriocitrin | 4.50 | C27H32O15 | 596.1741 | 596.1736 | -0.8151 | 679.8 | 802.3 | ND |
F_3 | Narirutin | 6.37 | C27H32O14 | 580.1792 | 580.1788 | -0.7378 | ND | ND | 5223.6 |
F_4 | Naringin† | 6.57 | C27H32O14 | 580.1792 | 580.1796 | 0.6403 | ND | 190414.1 | ND |
F_5 | Hesperidin† | 6.76 | C28H34O15 | 610.1898 | 610.1892 | -0.9442 | ND | 662410.3 | 1141.0 |
F_6 | Eriodictyol | 7.73 | C15H12O6 | 288.0634 | 288.0636 | 0.5717 | 191.1 | ND | ND |
F_7 | Naringin malonate | 8.03 | C36H44O22 | 828.2324 | 828.2318 | -0.7540 | ND | ND | 788.0 |
F_8 | Naringenin† | 8.68 | C15H12O5 | 272.0685 | 272.0686 | 0.3929 | 816.7 | 724.7 | 917.0 |
| Flavonols | | | | | | | | |
F_9 | Myricetin rutinoside | 5.17 | C27H30O17 | 626.1483 | 626.1494 | 1.6816 | 358.1 | ND | ND |
F_10 | Rhamnetin hexoside | 5.25 | C22H22O12 | 478.0747 | 478.0757 | 2.1079 | 391.5 | ND | ND |
F_11 | Myricetin hexoside | 5.27 | C21H20O13 | 480.0904 | 480.0905 | 0.1751 | 2583.4 | 3125.6 | ND |
F_12 | Kaempferol sophoroside | 5.72 | C27H30O16 | 610.1534 | 610.1535 | 0.1290 | ND | ND | 5046.0 |
F_13 | Kaempferol hexoside-rhamnoside | 5.86 | C27H30O15 | 594.1585 | 594.1605 | 3.4310 | ND | ND | 1449.4 |
F_14 | Quercetin hexoside | 5.90 | C21H20O12 | 464.0955 | 464.0953 | -0.4036 | 16498.3 | 735.0 | ND |
F_15 | Quercetin malonyl-hexoside | 6.18 | C24H22O15 | 550.0959 | 550.0948 | -2.0132 | 246.8 | ND | ND |
F_16 | Kaempferol malonyl-hexoside | 6.75 | C24H22O14 | 534.1010 | 534.0992 | -3.2217 | 402.2 | ND | ND |
F_17 | Quercetin† | 7.90 | C15H10O7 | 302.0427 | 302.0422 | -1.5475 | ND | ND | 431.4 |
| Furanocoumarins | | | | | | | | |
C_1 | Psoralen | 6.92 | C11H6O3 | 186.0317 | 186.0320 | 1.3986 | ND | ND | 29060.3 |
| Hydroxycoumarins | | | | | | | | |
C_2 | Esculin | 3.20 | C15H16O9 | 340.0794 | 340.0786 | -2.5525 | 1690.6 | 1427.8 | ND |
C_3 | Esculetin | 4.06 | C9H6O4 | 178.0266 | 178.0260 | -3.3713 | 403.6 | ND | 26338.5 |
C_4 | Hydroxycoumarin | 6.82 | C9H6O3 | 162.0317 | 162.0318 | 0.7271 | ND | ND | 112663.3 |
Results are expressed as arbitrary units. Data are showed as mean values (n = 3). Rt: retention time; ND: not detected; PA: phenolic acids; F: flavonoids; C: coumarins. †Identification confirmed with commercial standards.
Eighteen polyphenols were associated with reduced glucose uptake (Fig. 5): dihydroxybenzoic acid isomer I (PA_4), hydroxybenzoic acid isomer II (PA_7), rosmarinic acid (PA_36), protocatechuic acid (PA_8), dihydroxybenzoic acid isomer II (PA_9), caffeoylquinic acid isomer II (PA_21), hydroxybenzoic acid isomer I (PA_3), ellagic acid (PA_29), quercetin (F_17), naringin malonate (F_7), kaempferol hexoside-rhamnoside (F_13), narirutin (F_3), kaempferol sophoroside (F_12), naringenin (F_8), esculetin (C_3), hydroxycoumarin (C_4), and psoralen (C_1). These polyphenols were found in greater amount in B. media (Table 2), which exerted the greatest effect on glucose uptake in adipocyte cells (Fig. 2). Additionally, eighteen polyphenols were linked to Glut4, Irs1, and Pi3k gene overexpression. Seventeen of these were also associated with an increase in glucose uptake of B. media (Fig. 5B), while sinapoylquinic acid isomer II (PA_32) was linked solely to gene overexpression in the insulin cascade pathway, not increased glucose uptake.