Evaluation of the Enzyme Inhibitory and Antioxidant Activities of Entada spiralis Stem Bark and Isolation of the Active Constituents
Abstract
:1. Introduction
2. Results and Discussion
2.1. Extraction and Fractionation
2.2. Total Phenolic Contents (TPC)
2.3. Total Flavonoid Contents (TFC)
2.4. In Vitro Antioxidant Activity
2.4.1. Dot-Blot Staining Assay Result
2.4.2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay
2.4.3. 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Assay
2.5. In Vitro Digestive Enzymes Inhibitory Activity
2.5.1. In Vitro α-Amylase Inhibitory Assay
2.5.2. In Vitro α-Glucosidase Inhibitory Assay
2.6. Identification of the Isolated Compounds
3. Materials and Methods
3.1. General Information
3.2. E. spiralis Stem Bark Preparation and Extraction
3.3. Fractionation of the Active Extract
3.4. Estimation OF Total Phenolic Content (TPC)
3.5. Estimation of Total Flavonoid Contents (TFC)
3.6. In Vitro Antioxidant Activity Assay
3.6.1. Dot-Blot Staining Assay
3.6.2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) Assay
3.6.3. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) Assay
3.7. In Vitro Digestive Enzymes Inhibitory Activity
3.7.1. In Vitro α-Amylase Assay Inhibitory Assay
3.7.2. In Vitro α-Glucosidase Inhibitory Assay
3.8. Active Principle Isolation through Column Chromatography
3.9. Isolation Using Chromatotron
3.10. Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are not available from the authors. |
Extract | TPC (µg GAE/mg dw) | TFC (µg QUE/mg dw) |
---|---|---|
Methanol Extract | 42.5 ± 8.59 e | 28.94 ± 2.93 e |
Chloroform Extract | 28.3 ± 1.38 d | 12.73 ± 1.93 d |
Petroleum Ether Extract | 2.6 ± 0.95 b | 0.84 ± 0.24 b |
F1 | 10.71 ± 1.43 c | 6.88 ± 1.20 c |
F2 | 8.16 ± 3.18 c | 5.12 ± 1.93 c |
F3 | 2.04 ± 3.91 b | 0.51 ± 0.99 b |
F4 | 0.79 ± 1.23 a | −468 ± 21.55 a |
Sample | IC50 (µg/mL) | |||
---|---|---|---|---|
DPPH Assay | ABTS Assay | α-Amylase | α-Glucosidase | |
Methanol extract | 42.67 ± 4.10 C | 37.39 ± 0.05 C | 98.15 ± 6.04 C | 20.63 ± 0.44 A |
Chloroform extract | 472.83 ± 11.20 F | 90.84 ± 3.12 D | 405.29 ± 7.36 F | 74.96 ± 24.77 C |
Petroleum Ether extract | 1050.08 ± 23.21 G | 625.16 ± 10.58 F | -47.60 ± 9.25 G | 172.93 ±1.77 F |
F1 | 37.09 ± 0.88 C | 19.92 ± 1.11 A | 312.14 ± 8.78 E | 24.17 ± 1.24 B |
F2 | 30.18 ± 1.91 B | 30.50 ± 0.21 B | 13.19 ± 0.18 B | 28.15 ± 2.25 B |
F3 | 78.52 ± 5.21 D | 29.45 ± 4.12 B | 215.86± 16.62 D | 123.18 ± 2.05 D |
F4 | 326.63 ± 13.93 E | 303.75 ± 12.29 E | −67.45 ± 8.74 H | 143.76 ±21.43 E |
Qc | 29.82 ± 3.73 A | - | - | 4.85 ± 1.05 |
Ac | 24.67 ± 0.45 A | 16.74± 1.76 A | - | - |
Acarbose | - | - | 0.85 ± 0.19 | - |
Tx | - | 15.23 ± 2.15 A |
Sample | IC50 (µmol/L) | |||
---|---|---|---|---|
DPPH Assay | ABTS Assay | α-Amylase | α-Glucosidase | |
FEQ-2 | 5.0 × 10−2 | 1.0 × 10−2 | ND | 5.3 × 10−1 |
FEQ-3 | 3.9 × 10−2 | 7.0 × 10−3 | 3.3 × 10−1 | 7.7 × 10−2 |
FEQ-4 | 1.9 × 10−1 | 1.0 × 10−1 | 1.4 × 100 | 2.2 × 10−1 |
FEQ-5 | 3.6 × 10−2 | 7.5 × 10−3 | 5.1 × 10−1 | 3.8 × 10−2 |
FEQ-7 | 4.3 × 10−2 | 6.4 × 10−3 | ND | 3.5 × 10−1 |
FEQ-8 | 4.0 × 10−2 | 3.9 ×10−3 | ND | 3.4 × 10−1 |
Qc | 9.9 × 10−2 | − | − | 1.6 × 10−2 |
Ac | 1.4 × 10−1 | 9.5 × 10−2 | − | − |
Acarbose | − | − | 1.3 × 10−3 | − |
Tx | − | 6.1 × 10−2 |
Position | δH (ppm), m J (Hz) | δC (ppm) Type |
---|---|---|
1 | - | 127.06, C |
2 | 7.10 1H, dd (6.8, 1.4) | 109.32, CH |
3 | 6.95 d, (6.8) | 146.78, CH |
4 | - | 147.92, C |
5 | - | 114.72, C |
6 | 7.06 1H, d (1.5) | 123.04, CH |
7 | 7.60 1H, d (15.9) | 144.65, CH |
8 | 6.33 1H d (15.9) | 115.67, CH |
1′ | 4.20 2H, t (6.8) | 64.64, OCH2 |
2′ | 1.63–1.73, 2H, m | 31.93, CH2 |
3′ | 1.63–1.73, 2H, m | 28.87, CH2 |
4′ | 1.35–1.43, 2H, m | 29.71, CH2 |
5′ | 1.35–1.43, 2H, m | 26.06, CH2 |
6′ | 0.90 3H, t (7.14) | 14.11, CH3 |
OCH3 | 3.95 3H, s | 55.93, OCH3 |
C=O | - | 167.42 |
O-H | 5.96 1H, s | - |
Compound | MP (°C) | UV (λmax MeOH) | FT-IR (cm−1) | MF | Compound’s Name |
---|---|---|---|---|---|
FEQ-3 | 278–280 | 214, 281 | 3291, 2924, 1257, 1613 | C15H10O6 | Kaempferol |
FEQ-4 | 170–172 | 225, 256 | 3260, 2938, 1606, 1250 | C18H17O7 | Pachypodol |
FEQ-7 | 174–178 | 236, 280 | 3325, 2832, 1647, 1448 | C15H15O6 | (+)-Catechin |
FEQ-8 | 241–244 | 280, 238 | 3325, 2883, 1633, 1448 | C15H15O6 | (−)-Epicatechin |
Position | FEQ-3 | FEQ-4 | FEQ-7 | FEQ-8 | ||||
---|---|---|---|---|---|---|---|---|
δH, m J (Hz) | δC | δH, m J (Hz) | δC | δH, m J (Hz) | δC | δH, m, J (Hz) | δC | |
2 | - | 146.7 | - | 156.5 | 4.49 (d) (J = 7.5) | 81.5 | 4.71 (m) | 78.5 |
3 | - | 135.7 | 3.81 (s) | 138.7 | 3.84 (dd) (J = 13.2, 7.6) | 66.8 | 3.82 (m) | 65.4 |
4 | - | 176.6 | - | 178.8 | 2.38(m) 2.68 (dd) (J = 6.0, 5.3) | 28.3 | 2.49 (dd) (J = 16.4, 4.4), 2.66 (m) | 28.7 |
5 | - | 161.1 | - | 161.3 | - | 156.6 | - | 157.0 |
6 | 6.21 (d) (J = 2.0) | 97.9 | 6.33 (d) (J = 2.2) | 97.7 | 5.90 (d) (J = 2.0) | 95.6 | 5.90 (d) (J = 1.9) | 95.6 |
7 | - | 164.3 | 3.89 (s) | 165.8 | - | 156.9 | - | 156.7 |
8 | 6.42 (d) (J = 2.04) | 93.1 | 6.55 (d) (J = 2.2) | 91.9 | 5.70 (d) (J = 2.1) | 94.3 | 5.73 (d) (J = 1.9) | 94.6 |
9 | - | 156.9 | -- | 156.9 | - | 155.8 | - | 156.2 |
10 | - | 103.1 | - | 105.9 | - | 99.5 | - | 99.0 |
1′ | - | 122.4 | - | 122.7 | - | 131.1 | - | 131.1 |
2′ | 8.12 (dd) (J = 2.0, 7.0) | 129.3 | 7.65 (m) | 110.9 | 6.70 (d) (J = 2.0) | 115.0 | 6.69 (m) | 115.4 |
3′ | 6.94 (dd) (J= 2.0, 9.0) | 115.0 | 3.97 (s) | 146.1 | - | 145.3 | 144.9 | |
4′ | - | 159.2 | 150.3 | - | 145.3 | - | 145.0 | |
5′ | 6.94 (dd) (J= 2.0, 9.0) | 115.0 | 7.03 (d) (J = 8.4) | 114.9 | 6.73 (d) (J = 1.8) | 115.5 | 6.90 (s) | 115.2 |
6′ | 8.12 (dd) (J = 2.0, 7.0) | 129.3 | 7.65 (m) | 120.9 | 6.60 (dd) (J = 8.1, 1.9) | 118.9 | 6.69 (m) | 118.4 |
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Roheem, F.O.; Mat Soad, S.Z.; Ahmed, Q.U.; Ali Shah, S.A.; Latip, J.; Zakaria, Z.A. Evaluation of the Enzyme Inhibitory and Antioxidant Activities of Entada spiralis Stem Bark and Isolation of the Active Constituents. Molecules 2019, 24, 1006. https://doi.org/10.3390/molecules24061006
Roheem FO, Mat Soad SZ, Ahmed QU, Ali Shah SA, Latip J, Zakaria ZA. Evaluation of the Enzyme Inhibitory and Antioxidant Activities of Entada spiralis Stem Bark and Isolation of the Active Constituents. Molecules. 2019; 24(6):1006. https://doi.org/10.3390/molecules24061006
Chicago/Turabian StyleRoheem, Fatimah Opeyemi, Siti Zaiton Mat Soad, Qamar Uddin Ahmed, Syed Adnan Ali Shah, Jalifah Latip, and Zainul Amiruddin Zakaria. 2019. "Evaluation of the Enzyme Inhibitory and Antioxidant Activities of Entada spiralis Stem Bark and Isolation of the Active Constituents" Molecules 24, no. 6: 1006. https://doi.org/10.3390/molecules24061006