In vitro and in silico Analysis of PTP1B and α-Glucosidase Inhibitory Activities of Isoflavonoids Isolated from Belamcanda chinensis Roots

Introduction

Type 2 diabetes (T2D), also known as non-insulin-dependent diabetes mellitus, is currently the most prevalent form of diabetes and the number of people affected by this pathology is continuously increasing worldwide. The International Diabetes Federation (IDF) estimates that 536.6 million people are living with diabetes (diagnosed or undiagnosed) in 2021, and this number is projected to increase by 46%, reaching 783.2 million by 2045.¹ T2D is characterized by insulin resistance—possibly due to diminished post-receptor insulin signaling. T2D is characterized by insulin resistance—possibly due to diminished post-receptor insulin signaling, which typically begins several years before hyperglycemia.² α-Glucosidase and protein tyrosine phosphatase 1B (PTP1B) are well-known as the promising target enzymes for the treatment of T2D.³ α-Glucosidase is an enzyme that catalyzes glucose release from the nonreducing end of dietary carbohydrates, thereby elevating the blood glucose level. Therefore, inhibition of α-glucosidase is one of the well-established therapeutic approaches for controlling postprandial hyperglycemia.³ While protein tyrosine phosphatase 1B (PTP1B) plays an important role in the negative regulation of insulin signal transduction pathway and has been validated as a novel therapeutic strategy for the treatment of type 2 diabetes. Inhibition of PTP1B can improve insulin sensitivity, suggesting that the PTP1B inhibitors are conducive to T2D treatment.⁴ It has been recognized that natural phytochemicals with dual inhibition of α-glucosidase and PTP1B enzymes effectively suppress hyperglycemia due to their synergistic effect and significantly contribute to the enhancement of insulin sensitivity.^3,5

Belamcanda chinensis (L.) DC (syn. Iris domestica and known as "sa kan" in Korea) belongs to the Iridaceae family and is widely distributed in East and Southeast Asia and North America.⁶ With pharmacological effects such as reducing throat swelling, clearing heat, and detoxifying, this plant is used in traditional medicines to treat sore throat, cough, bronchitis, and chronic tracheitis.^7,8 Previous phytochemical investigations on B. chinensis reported iridal-type triterpenoids, isoflavonoids, flavonoids, benzoquinones, and steroids.^6–10 Various pharmacological activities have been reported from the components and extracts of B. chinensis such as anti-osteoclastogenic, anti-inflammatory, antidiabetic, antioxidant, antitumor, hepatoprotective, antimutagenic, neuroprotective, antibacterial, and neuroprotective activities.^6-10 In our previous study, six sucrosephenylpropanoid esters, five iridal-type triterpenoids, twelve isoflavonoids, and four flavonoids were isolated from B. chinensis roots.¹⁰ Among them, one sucrosephenylpropanoid ester (belamchinoside A) and one isoflavonoid (irilin D) exhibited strong effects on RANKL-induced osteoclast formation using RAW 264.7 cells and BMMs in a concentration-dependent manner.¹⁰ In this present study, we report the isolation and NMR characterization of twelve isoflavonoids and four flavonoids from the B. chinensis roots, as well as the investigation of their inhibitory ability against α-glucosidase and PTP1B enzymes in vivo and in silico.

Experimental

Chemicals and reagents – Protein tyrosine phosphatase 1B (PTP1B, human recombinant) and dithiothreitol (DTT) were purchased from Biomol® International LP (Plymouth Meeting, PA, USA) and Bio-Rad Laboratories (Hercules, CA, USA), respectively. Yeast α-glucosidase, p-nitrophenyl phosphate (p-NPP), p-nitrophenylα-D-glucopyranoside (p-NPG), acarbose, ethylene diamine tetraacetic acid (EDTA), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).

Plant material – The plant sample (B.chinensis roots) was collected at the Medicinal Plants Garden of Daegu Catholic University, Republic of Korea, in May 2024, and authenticated by Professor Byung Sun Min, College of Pharmacy, Daegu Catholic University, Republic of Korea. The voucher specimen (CUD-24052) was stored at the Laboratory of Pharmacognosy in the College of Pharmacy, Daegu Catholic University, Republic of Korea.

Extraction and isolation – The dried roots (1.5 kg) of B. chinensis were extracted four times with methanol (MeOH, 5 L for 3 hours each) at room temperature and filtered. The filtrate was evaporated under reduced pressure to obtain the MeOH extract (305 g), which was suspended in distilled water and successively partitioned with n-hexane, CH₂Cl₂, and EtOAc, respectively. The CH₂Cl₂ fraction (73 g) was separated into ten fractions (MC1-MC10) using column chromatography (CC), silica gel (CH₂Cl₂:MeOH; 50:1 → 0:1, v/v). Fraction MC7 (11 g) was subjected to silica gel column chromatography (CC) and eluted with a solvent system of n-hexane-acetone (5:1, v/v) to yield compounds 2 (0.8 g) and 1 (135 mg). Fraction MC6 (358 mg) was chromatographed on a Sephadex LH-20 column (MeOH-H₂O, 2:1, v/v) to yield compounds 3 (6 mg) and 4 (5 mg). Fraction MC5 (326 mg) was further purified using an RP-18 silica gel column (MeOH-H₂O, 2:1, v/v) to yield compounds 6 (6 mg) and 5 (4 mg). Fraction MC8 (5.2 g) was separated using an RP-18 silica gel column (MeOH-H₂O, 2:1, v/v) to yield seven sub-fractions (MC8.1–MC8.7). Sub-fractions MC8.3 (216 mg) was subjected to Sephadex LH-20 CC (MeOH-H₂O, 1.5:1, v/v) to yield compound 7 (5 mg). The EtOAc fraction (34 g) was chromatographed on a silica gel column (CH₂Cl₂-MeOH, 15:1 to 0:1, v/v) to yield five fractions (E1–E5). Fraction E1 (4 g) was separated using a silica gel column (n-hexane-CH₂Cl₂-MeOH, 3:5:0.3, v/v/v) to give compounds 9 (5 mg) and 12 (7 mg). Fraction E2 (209 mg) was purified using a Sephadex LH-20 CC eluted with a mixture of MeOH-H₂O (2:1, v/v) to give 10 (6 mg). Fraction E3 (3.9 g) was subjected to a silica gel column (CH₂Cl₂-MeOH, 30:1, v/v) to give compounds 11 (5 mg) and 8 (7 mg). Fraction E4 (5.6 g) was chromatographed on a silica gel column (CH₂Cl₂-MeOH, 20:1, v/v) to yield four fractions (E4.1-E4.4). Compounds 16 (5.5 mg) and 15 (7 mg) were isolated from subfraction E4.3 (358 mg) by RP-18 silica gel CC (MeOH-H₂O, 2:1, v/v). Fraction E5 (368 mg) was purified using a RP-18 silica gel column (MeOH-H₂O, 3:1, v/v). to yield compounds 13 (7 mg) and 14 (5 mg).

3,5,4'-trihydroxy-7,5'-dimethoxyflavanone (5) – Yellow amorphous powder; ¹H-NMR (400 MHz, methanol-d₄): δ 7.14 (1H, d, J = 2.0 Hz, H-6ʹ), 7.01 (1H, dd, J = 8.0, 2.0 Hz, H-2ʹ), 6.87 (1H, d, J = 8.0 Hz, H-3ʹ), 6.11 (1H, s, H-8), 6.08 (1H, s, H-6), 5.05 (1H, d, J = 11.2 Hz, H-2), 4.64 (1H, d, J = 11.2 Hz, H-3), 3.91 (3H, s, -OCH₃-7), 3.84 (3H, s, -OCH₃-5ʹ); ¹³C-NMR (100 MHz, methanol-d₄) δ (ppm): 198.9 (C-4), 169.8 (C-7), 165.0 (C-5), 164.3 (C-8a), 148.9 (C-4ʹ), 148.4 (C-5ʹ), 129.7 (C-1ʹ), 122.2 (C-2ʹ), 115.9 (C-3ʹ), 112.5 (C-6ʹ), 102.6 (C-4a), 96.0 (C-6), 95.1 (C-8), 85.3 (C-2), 73.7 (C-3), 56.5 (-OCH₃-5ʹ), 56.4 (-OCH₃-7).

3,5,3'-trihydroxy-7,4',5'-trimethoxyflavanone (6) – Yellow amorphous powder; ¹H-NMR (400 MHz, methanol-d₄): δ 6.74 (1H, s, H-6ʹ), 6.73 (1H, s, H-2ʹ), 6.13 (1H, s, H-8), 6.11 (1H, s, H-6), 5.03 (1H, d, J = 11.2 Hz, H-2), 4.60 (1H, d, J = 11.2 Hz, H-3), 3.90 (3H, s, -OCH₃-5ʹ), 3.86 (3H, s, -OCH₃-7), 3.85 (3H, s, -OCH₃-4ʹ); ¹³C-NMR (100 MHz, methanol-d₄): 198.7 (C-4), 169.8 (C-7), 165.0 (C-5), 164.7 (C-8a), 154.5 (C-3ʹ), 151.1 (C-5ʹ), 138.1 (C-4ʹ), 134.1 (C-1ʹ), 109.9 (C-2ʹ), 104.5 (C-6ʹ), 102.6 (C-4a), 96.1 (C-6), 95.1 (C-8), 85.2 (C-2), 73.7 (C-3), 60.9 (-OCH₃-5ʹ), 56.5 (-OCH₃-7), 56.4 (-OCH₃-4ʹ).

Noririsflorentin (7) – Yellow amorphous powder; ¹H-NMR (400 MHz, methanol-d₄): δ 8.05 (1H, s, H-2), 6.79 (2H, s, H-2ʹ and 6ʹ), 6.73 (1H, s, H-8), 6.10 (2H, s, H-9), 4.05 (3H, s, -OCH₃-4ʹ), 3.87 (6H, s, -OCH₃-3ʹ and 5ʹ); ¹³C-NMR (100 MHz, methanol-d₄), see Table 1.

Table 1.

13C-NMR data of isoflavonoids 1–4 and 7–10

Irilin D (8) – Yellow amorphous powder; ¹H-NMR (400 MHz, acetone-d₆): δ 13.2 (1H, s, OH-5), 8.13 (1H, s, H-2), 7.14 (1H, d, J = 2.0 Hz, H-6ʹ), 6.94 (1H, dd, J = 8.0, 2.0 Hz, H-2ʹ), 6.88 (1H, d, J = 8.0 Hz, H-3ʹ), 6.47 (1H, s, H-8), 3.86 (3H, s, -OCH₃-6); ¹³C-NMR (100 MHz, methanol-d₄), see Table 1.

Dichotomitin (9) – Yellow amorphous powder; ¹H-NMR (400 MHz, methanol-d₄): δ 8.47 (1H, s, H-2), 6.87 (1H, s, H-8), 6.72 (1H, s, H-2ʹ), 6.67 (1H, s, H-6ʹ), 6.17 (2H, s, H-9), 3.79 (3H, s, -OCH₃-5ʹ), 3.69 (3H, s, -OCH₃-4ʹ); ¹³C-NMR (100 MHz, methanol-d₄), see Table 1.

Genistein (10) – Yellow amorphous powder; ¹H-NMR (400 MHz, methanol-d₄): δ 8.06 (1H, s, H-2), 7.39 (2H, d, J = 8.0 Hz, H-2ʹ and 6ʹ), 6.87 (2H, d, J = 8.0 Hz, H-3ʹ and 5ʹ), 6.35 (1H, s, H-8), 6.23 (1H, s, H-6); ¹³C-NMR (100 MHz, methanol-d₄), see Table 1.

Hispidulin (11) – Yellow amorphous powder; ¹H-NMR (400 MHz, DMSO-d₆): δ 7.92 (2H, d, J = 8.8 Hz, H-2ʹ, 6ʹ), 6.92 (2H, d, J = 8.8 Hz, H-3ʹ, 5ʹ), 6.76 (1H, s, H-3), 6.58 (1H, s, H-8), 3.74 (3H, s, -OCH₃-6); ¹³C-NMR (100 MHz, DMSO-d₆): δ 182.1 (C-4), 163.8 (C-4ʹ), 161.2 (C-2), 157.4 (C-7), 152.8 (C-5), 152.4 (C-8a), 131.4 (C-6), 128.5 (C-2ʹ and 6ʹ), 121.2 (C-1ʹ), 116.0 (C-3ʹ and 5ʹ), 104.1 (C-4a), 102.4 (C-3), 94.3 (C-8), 60.0 (-OCH₃-6).

Kanzakiflavone-2 (12) – Yellow amorphous powder; ¹H-NMR (400 MHz, DMSO-d₆): δ 7.94 (2H, d, J = 8.4 Hz, H-2ʹ and 6ʹ), 6.94 (2H, d, J = 8.4 Hz, H-3ʹ and 5ʹ), 6.91 (1H, s, H-3), 6.85 (1H, s, H-8), 6.16 (2H, s, -OCH₂O-9); ¹³C-NMR (100 MHz, DMSO-d₆): δ 182.5 (C-4), 164.1 (C-4ʹ), 161.3 (C-2), 153.7 (C-7), 152.5 (C-8a), 141.1 (C-5), 129.4 (C-6), 128.5 (C-2ʹ and 6ʹ), 120.9 (C-1ʹ), 116.0 (C-3ʹ and 5ʹ), 106.6 (C-4a), 102.7 (C-3), 102.7 (-OCH₂O-9), 89.7 (C-8).

Tectoridin (13) – Yellow amorphous powder; ¹H-NMR (400 MHz, DMSO-d₆): δ 8.37 (1H, s, H-2), 7.34 (2H, d, J = 8.4 Hz, H-2ʹ and 6ʹ), 6.77 (2H, d, J = 8.4 Hz, H-3ʹ and 5ʹ), 6.82 (1H, s, H-8), 3.70 (3H, s, -OCH₃-6), 5.05 (1H, d, J = 7.2 Hz, H-1ʹʹ), 3.65, 3.42 (each 1H, m, H2-6ʹʹ), 3.58 (1H, m, H-3ʹʹ), 3.42 (1H, m, H-5ʹʹ), 3.26 (1H, m, H-2ʹʹ), 3.14 (1H, m, H-4ʹʹ); ¹³C NMR (100 MHz, DMSO-d₆): δ 180.8 (C-4), 157.5 (C-4ʹ), 156.6 (C-7), 154.7 (C-2), 152.9 (C-5), 152.5 (C-8a), 132.4 (C-6), 130.2 (C-2ʹ and 6ʹ), 122.1 (C-1ʹ), 121.1 (C-3), 115.1 (C-3ʹ and 5ʹ), 106.5 (C-4a), 100.1 (C-1ʹʹ), 94.0 (C-8), 77.3 (C-5ʹʹ), 76.7 (C-3ʹʹ), 73.1 (C-2ʹʹ), 69.7 (C-4ʹʹ), 60.7 (CH₂-6ʹʹ), 60.3 (-OCH₃-6).

Iridin (14) – Yellow amorphous powder; ¹H-NMR (400 MHz, acetone-d₆): δ 8.29 (1H, s, H-2), 6.79 (1H, d, J = 2.0 Hz, H-2ʹ), 6.78 (1H, d, J = 2.0 Hz, H-6ʹ), 6.85 (1H, s, H-8), 3. 85 (3H, s, -OCH₃-5ʹ), 3.81 (3H, s, -OCH₃-6), 3.78 (3H, s, -OCH₃-4ʹ), 5.18 (1H, d, J = 7.2 Hz, H-1ʹʹ), 3.43–3.92 (sugar protons); ¹³C-NMR (100 MHz, acetone-d₆): 181.8 (C-4), 157.9 (C-7), 155.6 (C-5), 154.7 (C-8a), 153.8 (C-2), 153.7 (C-5ʹ), 151.1 (C-3ʹ), 137.4 (C-4ʹ), 134.4 (C-6), 127.4 (C-1ʹ), 123.7 (C-3), 110.6 (C-2ʹ), 107.9 (C-4a), 105.8 (C-6ʹ), 101.6 (C-1ʹʹ), 95.0 (C-8), 78.1 (C-5ʹʹ), 78.0 (C-3ʹʹ), 74.5 (C-2ʹʹ), 71.7 (C-4ʹʹ), 62.5 (CH₂-6ʹʹ), 60.8 (-OCH₃-4ʹ), 60.7 (-OCH₃-6), 56.3 (3H, s, -OCH₃-5ʹ).

6ʹʹ-O-p-hydroxybenzoyliridin (15) – Yellow amorphous powder; ¹H-NMR (400 MHz, acetone-d₆): δ 8.17 (1H, s, H-2), 7.94 (2H, d, J = 8.8 Hz, H-2ʹʹʹ and 6ʹʹʹ), 6.94 (2H, d, J = 8.4 Hz, H-3ʹʹʹ and 5ʹʹʹ), 6.84 (1H, s, H-8), 6.80 (1H, d, J = 2.0 Hz, H-2ʹ), 6.80 (1H, d, J = 2.0 Hz, H-6ʹ), 5.27 (1H, d, J = 7.2 Hz, H-1ʹʹ), 4.76 (1H, dd, J = 12.0, 2.0 Hz, H-6ʹʹa), 4.38 (1H, dd, J = 12.0, 8.0 Hz, H-6ʹʹb), 4.04 (1H, m, H-5ʹʹ), 3.88 (3H, s, -OCH₃-5ʹ), 3.82 (3H, s, -OCH₃-4ʹ), 3.81 (3H, s, -OCH₃-6), 3.67 (1H, m, H-3ʹʹ), 3.66 (1H, m, H-2ʹʹ), 3.53 (1H, m, H-4ʹʹ); ¹³C-NMR (100 MHz, acetone-d₆): δ 181.8 (C-4), 166.3 (C-7ʹʹʹ), 162.7 (C-4ʹʹʹ), 157.7 (C-7), 155.2 (C-2), 154.8 (C-5), 153.9 (C-5ʹ), 153.6 (C-8a), 151.0 (C-3ʹ), 137.4 (C-4ʹ), 134.1 (C-6), 132.6 (C-2ʹʹʹ and 6ʹʹʹ), 127.4 (C-1ʹ), 123.7 (C-1ʹʹʹ), 122.3 (C-3), 116.0 (C-3ʹʹʹ and 5ʹʹʹ), 110.7 (C-2ʹ), 108.0 (C-4a), 105.9 (C-6ʹ), 101.4 (C-1ʹʹ), 94.9 (C-8), 77.9 (C-5ʹʹ), 75.3 (C-3ʹʹ), 74.4 (C-2ʹʹ), 71.5 (C-4ʹʹ), 64.6 (CH₂-6ʹʹ), 60.8 (-OCH₃-4ʹ), 60.7 (-OCH₃-6), 56.3 (3H, s, -OCH₃-5ʹ).

6ʹʹ-O-vanilloyliridin (16) – Yellow amorphous powder; ¹H-NMR (400 MHz, methanol-d₄): δ 7.83 (1H, s, H-2), 7.53 (1H, dd, J = 8.4, 2.0 Hz, H-6ʹʹʹ), 7.47 (1H, d, J = 2.0 Hz, H-2ʹʹʹ), 6.78 (1H, d, J = 8.4 Hz, H-5ʹʹʹ), 6.70 (2H, s, H-2ʹ and 6ʹ), 6.57 (1H, s, H-8), 5.08 (1H, d, J = 7.2 Hz, H-1ʹʹ), 4.66 (1H, dd, J = 12.0, 2.0 Hz, H-6ʹʹa), 4.45 (1H, dd, J = 12.0, 8.0 Hz, H-6ʹʹb), 3.87 (3H, s, -OCH₃-5ʹ), 3.83 (3H, s, -OCH₃-4ʹ), 3.79 (3H, s, -OCH₃-6), 3.77 (3H, s, -OCH₃-3ʹʹʹ), 3.37–3.62 (4H, m, H-2ʹʹ, 3ʹʹ, 4ʹʹ and 5ʹʹ); ¹³C-NMR (100 MHz, methanol-d₄): δ 182.3 (C-4), 166.7 (C-7ʹʹʹ), 152.8 (C-4ʹʹʹ), 157.6 (C-7), 155.7 (C-2), 154.6 (C-5), 154.5 (C-5ʹ), 154.1 (C-8a), 151.5 (C-3ʹ), 137.9 (C-4ʹ), 134.1 (C-6), 122.4 (C-6ʹʹʹ), 115.9 (C-6ʹʹʹ), 127.7 (C-1ʹ), 124.3 (C-1ʹʹʹ), 122.3 (C-3), 113.8 (C-2ʹʹʹ), 148.6 (C-3ʹʹʹ), 111.2 (C-2ʹ), 108.3 (C-4a), 106.1 (C-6ʹ), 101.4 (C-1ʹʹ), 95.3 (C-8), 77.8 (C-5ʹʹ), 75.8 (C-3ʹʹ), 74.6 (C-2ʹʹ), 72.1 (C-4ʹʹ), 64.9 (CH₂-6ʹʹ), 61.4 (-OCH₃-4ʹ), 61.1 (-OCH₃-6), 56.6 (3H, s, -OCH₃-5ʹ), 56.3 (3H, s, -OCH₃-3ʹʹʹ).

PTP1B and α-glucosidase inhibitory assays – The inhibition assays against recombinant human protein tyrosine phosphatase 1B and α-glucosidase enzymes were conducted with p-nitrophenyl phosphate (p-NPP) and p-nitrophenyl α-D-glucopyranoside (p-NPG) as the substrates, respectively, according to our previously published protocols.^5,11

Enzyme kinetic analysis with PTP1B and α-glucosidase – The kinetic mechanisms of the active compound (irilin D, 8) in PTP1B and glucosidase inhibition were investigated using complementary Lineweaver–Burk and Dixon plots.^5,11 The inhibition of irilin D (8) against PTP1B and α-glucosidase enzymes was monitored at different substrate concentrations (2, 1, and 0.5 mM of p-NPP for PTP1B and 2.5, 1.25, and 0.625 mM of p-NPG for α-glucosidase) on Dixon plots (single reciprocal plots). In addition, Lineweaver-Burk plots for the inhibition of PTP1B and α-glucosidase of irilin D (8) were obtained in the presence of its various concentrations (0, 21.5, 38.2, and 55.5 μM for PTP1B and 0, 32.5, 44.0, and 60.5 μM for α-glucosidase). The kinetic results were analyzed using the SigmaPlot 12.0 software (SPCC Inc., Chicago IL, U.S.A). The kinetic data were graphically presented by the Lineweaver-Burk and Dixon plots, from which the type of enzyme inhibition was determined. Kinetic constant values (K_i) were assessed using Dixon plots.

Molecular docking simulation in PTP1B and α-glucosidase inhibition – Molecular docking simulations of irilin D (8) against PTP1B and α-glucosidase enzymes were performed using AutoDock 4.2 software, according to our previously published protocol.^5,11 The complex structure of PTP1B (PDB ID of 1T49) and its selective allosteric inhibitor 3-(3,5-dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide (compound A), together with isomaltase (PDB ID: 3A4A) were obtained from the RCSB Protein Data Bank (https://www.rcsb.org/). Isomaltase, with the same organism and highly similar to α-glucosidase, was used as a substitute in this study. The 3D structure of (Z)-3-butylidenephthalide (BIP) was obtained from the PubChem Compound (NCBI) with compound CID of 5352899. The results were analyzed using Discovery Studio and PyMOL.

Statistical analysis – Statistical analyses were performed by one-way analysis of variance (ANOVA), followed by the Fisher least significant difference test. p<0.05 was considered statistically significant. The results are presented as the mean±standard error of the mean (SEM).

Results and Discussion

The structure of isolated compounds were identified as irigenin (1),¹² irisflorentin (2),¹² iristectorigenin A (3),¹³ irilone (4),¹⁴ 3,5,4'-trihydroxy-7,5'-dimethoxyflavanone (5),¹⁵ 3,5,3'-trihydroxy-7,4',5'-trimethoxyflavanone (6),¹⁶ noririsflorentin (7),¹⁷ irilin D (8),¹⁸ dichotomitin (9),¹⁹ genistein (10),¹⁹ hispidulin (11),²⁰ kanzakiflavone-2 (12),²¹ tectoridin (13),¹⁹ iridin (14),¹⁹ 6ʹʹ-O-p-hydroxybenzoyliridin (15),²² and 6ʹʹ-O-vanilloyliridin (16)²² (Fig. 1) based on the analysis of their observed and reported spectroscopic data.

Fig. 1.

Chemical structures of compounds 1–16

The anti-diabetic potential of the B. chinensis compounds (1-16) was investigated via their inhibition against PTP1B and α-glucosidase, and the results are expressed as IC₅₀ values (Table 2). Whereby, three isoflavonoids including irilone (4, IC₅₀ = 37.95 µM), irilin D (8, IC₅₀ = 31.86 µM), and genistein (10, IC₅₀ = 24.64 µM) showed moderate inhibition against PTP1B in comparison to the positive control, ursolic acid (IC₅₀ = 5.99 µM). Interestingly, these three compounds displayed potent α-glucosidase inhibitory activity with corresponding IC₅₀ values of 76.92, 44.22, and 14.71 µM, significantly lower than the IC₅₀ value (216.30 µM) of the positive control, acarbose. The remaining compounds showed weak or no activity. Similar structure-activity relationships were observed in PTP1B and α-glucosidase assays for the isoflavonoids 1-4 and 7-10. The structures of these isoflavonoids are similar, except for the absence of the methoxy substituents in the B ring of active compounds irilone (4), irilin D (8), and genistein (10). This interesting finding suggested that the presence of the methoxy substituents in the B ring of isoflavonoids might have negatively affected the PTP1B and α-glucosidase inhibitory activities of the isoflavonoids. These results are consistent with the previous reports about the PTP1B and α-glucosidase inhibitory activities of genistein (10) and its derivatives.^23,24 To the best of our knowledge, this is the first investigation on the inhibitory activity of irilin D (8) against PTP1B and α-glucosidase enzymes. Therefore, this compound was selected for the kinetic and molecular docking studies.

Table 2.

Inhibitory activities of compounds 1-16 against PTP1B and α-glucosidase

To determine the PTP1B and α-glucosidase inhibition mechanisms of irilin D (8), two kinetic methods were used, the Lineweaver-Burk plot and Dixon plot. The inhibition mechanisms of each enzymatic inhibition at various concentrations of irilin D (8) were evaluated by monitoring the effects of various substrate concentrations via the Dixon plot, and the effects of various concentrations of irilin D (8) were evaluated via the Lineweaver-Burk plot. In the Lineweaver-Burk plot, the plotted lines for irilin D (8) that crossed the same point on the x-intercept, represented the noncompetitive inhibition of irilin D (8) against both PTP1B and α-glucosidase. The inhibition constants (K_i) were calculated by interpretation of the Dixon plots, where the value of the x-axis means - K_i. As a result, the inhibition constants of irilin D (8) against PTP1B and α-glucosidase were determined as 30.83 and 44.22 µM, respectively.

Fig. 2.

Lineweaver-Burk and Dixon plots for the inhibition of PTP1B [(A) and (B)] and α-glucosidase [(C) and (D)] enzymes by irilin D (8), respectively.

The interaction and binding modes of irilin D (8) with PTP1B were investigated via the simulation and comparison with the reference ligand, compound A (an allosteric inhibitor), using AutoDock 4.2 software. According to the simulation results shown in Fig. 3, both the reference ligand and irilin D (8) were stably positioned into the same pocket (allosteric site) of PTP1B with negative binding energies of −11.16 and −7.92 kcal/mol, respectively. With −7.92 kcal/mol of binding energy, the corresponding ligand interactions of irilin D (8) at the allosteric site of PTP1B were four hydrogen bonding interactions between its ketone and hydroxyl groups and three interacting residues: Asn193, Ala189, and Lys197. Van der Waals interactions were observed between irilin D (8) and PTP1B residues Glu276, Phe194, Glu200, and Gly277, which may help this compound further be stabilized in the active site of PTP1B. In addition, irilin D (8) and compound A shared some similar binding residues, including the allosteric residue Asn193 via H-bond interaction; Glu200 via van der Waals interaction; and Phe196, Ala189, Phe280, and Leu192 via hydrophobic interactions. The molecular docking result indicated the good binding ability of irilin D (8) to the allosteric site of PTP1B, confirming its noncompetitive type of PTP1B inhibition.

Fig. 3.

Molecular docking related PTP1B inhibition by compound A (red stick) and 8 (cyan stick) (A). 2D diagram of PTP1B inhibition by 8 (B) and compound A (C) at the PTP1B allosteric site. The figure was generated using PyMOL and Discovery Studio Visualizer.

Based on molecular docking studies using AutoDock 4.2 software, binding site-directed inhibition of α-glucosidase by irilin D (8) was predicted, in which (Z)-3-butylidenephthalide (BIP) was selected as the standard ligand for validating the simulation results. The results are shown in Fig. 4 and Table 3, which include the binding energy, interacting residues with the type of interactions including H-bond, van der Waals, and hydrophobic interactions. The ligand-enzyme complexes of irilin D (8)/or BIP were posed stably in the same pocket (allosteric site) of the α-glucosidase enzyme with negative binding energies of −8.23 and −6.86 kcal/mol, respectively. The α-glucosidase-irilin D (8) inhibitor complex contained five hydrogen bonds with three interacting residues Cys342, Glu296, and Asn259. In addition, van der Waals interactions with Lys16, Thr340, Asp341, Trp343, Ser298, Thr290, Trp15, Lys13, His295, and Ile272 residues stabilized the α-glucosidase-irilin D (8) complex. One hydrophobic interaction (π-σ) was observed between irilin D (8) and the Ala292 residue. As listed in Table 3, irilin D (8) and BIP shared many similar binding residues including Glu296 via H-bond interaction; Lys16, Asp341, Trp343, Ser298, and Thr290 via van der Waals interactions; and Leu292 via hydrophobic interaction. Taken together, the findings of kinetic analysis and molecular docking simulation showed that the α-glucosidase inhibitory activity of irilin D (8) was promising. Interestingly, the α-glucosidase inhibition of irilin D (8) in silico was recently recognized and reported via its molecular docking simulation with the 3TOP receptor (a part of α-glucosidase enzyme in human gut).²⁵

Fig. 4.

Molecular docking related α-glucosidase inhibition by BIP (yellow stick) and 8 (blue stick) (a). 2D diagram of α-glucosidase inhibition by 8 (b) and BIP (c) at the allosteric site of α-glucosidase. The figure was generated using PyMOL and Discovery Studio Visualizer.

Table 3.

Binding site residues and docking scores of irilin D (8) and reference compounds in PTP1B and α-glucosidase

In conclusion, our present study reports the isolation and investigation of the anti-diabetic activity of sixteen B. chinensis compounds (twelve isoflavonoids and four flavonoids) via their inhibitory ability against α-glucosidase and PTP1B enzymes in vivo and in silico. The results showed that two isoflavonoids (irilin D and genistein) act as dual α-glucosidase and PTP1B inhibitors. The first kinetic investigation of irilin D indicated its noncompetitive inhibition type for both of these two enzymes. In addition, molecular docking simulations of irilin D and the reference ligands showed negative binding energies and similar proximity to residues in the PTP1B and α-glucosidase binding pocket, which means they are closely connected and strongly binding with the active enzyme site. Thereby suggesting the potential of B. chinensis isoflavonoids in the prevention and treatment of type-2 diabetes.

Acknowledgments

This research was supported by the National Research Foundation of Korea (NRF) of Korea (NRF-2021R1A2C2011940) and Gyeongsangbukdo (RISE).

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