Analysis of Antioxidant Constituents from Ilex rotunda and Evaluation of Their Blood–Brain Barrier Permeability
Abstract
:1. Introduction
2. Materials and Methods
2.1. Plant Materials
2.2. Preparation of Extracts
2.3. DPPH Radical Scavenging Assay
2.4. ABTS Radical Scavenging Assay
2.5. Fractionation and Separation of Marker Compounds 1–8
2.6. Method Validation
2.6.1. Detection Wavelength
2.6.2. Preparation of Calibration Standard Solution
2.6.3. Chromatographic and Separation Conditions
2.6.4. Mass Conditions
2.7. HPLC-DPPH Method and ELISA Assay
2.8. Parallel Artificial Membrane Permeability Assay for the Blood–Brain Barrier (PAMPA-BBB)
2.9. Statistical Analysis
3. Results
3.1. Screened DPPH and ABTS Activities Guided Extraction and Solvent Selection
3.2. Antioxidant Activities of Fractions
3.3. Isolation and Identification of Marker Compounds 1–8
3.4. Method Validation of Marker Compounds (1–8) from I. rotunda
3.4.1. Optimization of HPLC Condition
3.4.2. Method Validation of Quantitative Analysis
Linearity, LODs, and LOQs
Precision, Accuracy, and Recovery
Quantification of Marker Compounds in I. rotunda
3.5. Screening of Antioxidants by HPLC-DPPH Method and ELISA Assay
3.6. Screening of Brain-Penetrable Antioxidants by PAMPA-BBB Method
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Marker Compound | Concentration Range (µg/mL) | a Regression Equation | b Correlation Coefficient (R2) | c LOD (µg/mL) | d LOQ (µg/mL) |
---|---|---|---|---|---|
syringin (1) | 6.25 ~ 200 | y = 11,125x + 16,180 | 0.9999 | 0.18 | 0.55 |
chlorogenic acid (2) | 12.5 ~ 400 | y = 2977.6x + 10,941 | 0.9993 | 0.45 | 1.38 |
rutin (3) | 6.25 ~ 200 | y = 10,785x + 18,732 | 0.9998 | 0.13 | 0.42 |
rotundarpenoside B (4) | 12.5 ~ 400 | y = 4358.5x + 4988.8 | 0.9999 | 0.26 | 0.81 |
3,4-dicaffeoylquinic acid (5) | 12.5 ~ 400 | y = 3462.4x − 8499.8 | 0.9995 | 0.65 | 1.98 |
3,5-dicaffeoylquinic acid (6) | 25 ~ 800 | y = 4918.7x − 13,540 | 0.9998 | 0.38 | 1.18 |
4,5-dicaffeoylquinic acid (7) | 25 ~ 800 | y = 5893x − 25,506 | 0.9993 | 0.27 | 0.78 |
3,4,5-tricaffeoylquinic acid (8) | 12.5 ~ 400 | y = 2617.1x + 916.7 | 0.9999 | 0.58 | 1.76 |
Marker Compound | Concentration Range (µg/mL) | a Recovery (%) | b Precision (RSD %) | |
---|---|---|---|---|
Intraday | Interday | |||
syringin (1) | 40 | 96.60 | 0.40 | 2.48 |
100 | 97.60 | |||
200 | 95.30 | |||
chlorogenic acid (2) | 64 | 104.62 | 1.15 | 3.65 |
160 | 102.73 | |||
400 | 104.53 | |||
rutin (3) | 40 | 104.03 | 0.46 | 2.53 |
100 | 102.10 | |||
200 | 100.47 | |||
rotundarpenoside B (4) | 64 | 103.92 | 0.85 | 3.27 |
160 | 99.78 | |||
400 | 102.58 | |||
3,4-dicaffeoylquinic acid (5) | 64 | 104.7 | 0.51 | 2.81 |
160 | 97.26 | |||
400 | 100.21 | |||
3,5-dicaffeoylquinic acid (6) | 128 | 99.93 | 0.47 | 2.70 |
320 | 98.18 | |||
800 | 99.09 | |||
4,5-dicaffeoylquinic acid (7) | 128 | 99.44 | 0.68 | 3.04 |
320 | 97.88 | |||
800 | 98.79 | |||
3,4,5-tricaffeoylquinic acid (8) | 64 | 100.89 | 0.47 | 2.74 |
160 | 100.32 | |||
400 | 103.6 |
Marker Compounds | a Reduction of the Peak Area (%) | EC50 Values (μM) |
---|---|---|
syringin (1) | 6.98 ± 0.44 | – |
chlorogenic acid (2) | 60.51 ± 0.31 | 35.50 ± 0.38 |
rutin (3) | 21.45 ± 0.90 | – |
rotundarpenoside B (4) | 18.99 ± 0.90 | – |
3,4-dicaffeoylquinic acid (5) | 45.24 ± 0.67 | – |
3,5-dicaffeoylquinic acid (6) | 58.88 ± 0.44 | 10.88 ± 0.04 |
4,5-dicaffeoylquinic acid (7) | 83.67 ± 0.19 | 13.84 ± 0.24 |
3,4,5-tricaffeoylquinic acid (8) | 67.25 ± 1.00 | 10.89 ± 0.14 |
Ascorbic acid * | – | 22.56 ± 0.77 |
Marker Compounds | BBB Permeability log Pe (cm/s) | Cross BBB Potential a |
---|---|---|
syringin (1) | b n.d. | - |
chlorogenic acid (2) | n.d. | - |
rutin (3) | n.d. | - |
rotundarpenoside B (4) | n.d. | - |
3,4-dicaffeoylquinic acid (5) | n.d. | - |
3,5-dicaffeoylquinic acid (6) | n.d. | - |
4,5-dicaffeoylquinic acid (7) | −5.85 ± 0.01 | + |
3,4,5-tricaffeoylquinic acid (8) | n.d. | - |
c coumarin | −4.54 ± 0.01 | ++ |
d caffeic acid | −9.08 ± 0.01 | - |
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Kim, C.-K.; Ahn, J.; Yu, J.; Le, D.; Han, S.; Lee, M. Analysis of Antioxidant Constituents from Ilex rotunda and Evaluation of Their Blood–Brain Barrier Permeability. Antioxidants 2022, 11, 1989. https://doi.org/10.3390/antiox11101989
Kim C-K, Ahn J, Yu J, Le D, Han S, Lee M. Analysis of Antioxidant Constituents from Ilex rotunda and Evaluation of Their Blood–Brain Barrier Permeability. Antioxidants. 2022; 11(10):1989. https://doi.org/10.3390/antiox11101989
Chicago/Turabian StyleKim, Chang-Kwon, Jeongjun Ahn, Jayeon Yu, DucDat Le, Sanghee Han, and Mina Lee. 2022. "Analysis of Antioxidant Constituents from Ilex rotunda and Evaluation of Their Blood–Brain Barrier Permeability" Antioxidants 11, no. 10: 1989. https://doi.org/10.3390/antiox11101989