GC-MS/MS Quantification of EGFR Inhibitors, β-Sitosterol, Betulinic Acid, (+) Eriodictyol, (+) Epipinoresinol, and Secoisolariciresinol, in Crude Extract and Ethyl Acetate Fraction of Thonningia sanguinea
Abstract
:1. Introduction
2. Results and Discussion
2.1. Validation Parameters of Analytical Method Using GC-MS/MS
2.1.1. Linearity
2.1.2. Limit of Detection and Limit of Quantification
2.1.3. Precision and Accuracy
2.1.4. Specificity and Sensitivity
2.2. Application of Validated Analytical Method
2.3. Molecular Docking Studies of Isolated Compounds toward EGFR
2.4. Compounds (+) Eriodictyol and Secoisolariciresinol Inhibited EGFR
3. Materials and Methods
3.1. Plant Material and Collection Process
3.2. Extraction, Fractionation, and Isolation of Pure Compounds
3.2.1. Extraction Process and Fractionation Procedure
3.2.2. Isolation of Pure Compounds
3.3. Gas Chromatography Coupled to Tandem Mass Spectrometry (GC-MS/MS)
3.3.1. Calibration Graph of Isolated Compounds (1–5)
3.3.2. GC-MS/MS Analysis Conditioning
3.3.3. Plant Sample Assay
3.4. Molecular Docking Studies
3.5. EGFR Protein Kinase (PK) Inhibition
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Parameter | β-Sitosterol | Betulinic Acid | Eriodictyol | Epipinoresinol | Secoisolariciresinol |
---|---|---|---|---|---|
Concentration range (μg/mL) | 7.0–12.0 | 7.0–12.0 | 7.0–12.0 | 7.0–12.0 | 7.0–12.0 |
Correlation coefficient (R2) | 0.9851 | 0.9928 | 0.9773 | 0.9937 | 0.9960 |
LOD (μg/mL) | 0.08 | 0.06 | 0.08 | 0.12 | 0.11 |
LOQ (μg/mL) | 0.26 | 0.18 | 0.25 | 0.4 | 0.38 |
Slope (b) | 2071.8 | 4704.4 | 996.82 | 1223.2 | 738.37 |
Intercept (a) | 74,876 | 13,942 | 68,478 | 4446.2 | 25,573 |
Analytes | Added Conc. (μg/mL) | Intra-Day | Inter-Day | ||
---|---|---|---|---|---|
Recovery% ± SD | RSD | Recovery% ± SD | RSD | ||
β-Sitosterol | 7.0 | 99.21 ±1.01 | 1.02 | 99.56 ± 1.11 | 1.11 |
10.0 | 100.25 ±1.21 | 1.21 | 100.35 ±1.35 | 1.35 | |
12.0 | 101.26 ± 0.89 | 0.88 | 99.95 ±1.45 | 1.45 | |
Betulinic acid | 7.0 | 100.67 ± 1.09 | 1.08 | 101.11 ±1.21 | 1.20 |
10.0 | 98.90 ± 1.21 | 1.22 | 100.91 ± 1.12 | 1.11 | |
12.0 | 99.45 ± 1.45 | 1.46 | 99.15 ± 1.32 | 1.33 | |
Eriodictyol | 7.0 | 100.13 ± 1.33 | 1.32 | 99.93 ± 1.14 | 1.14 |
10.0 | 99.93 ± 0.85 | 0.85 | 98.56 ± 1.15 | 1.17 | |
12.0 | 99.87 ± 0.77 | 0.77 | 99.96 ± 1.10 | 1.10 | |
Epipinoresinol | 7.0 | 101.00 ± 0.87 | 0.86 | 101.10 ± 0.99 | 0.98 |
10.0 | 101.73 ± 0.92 | 0.90 | 102.31 ± 0.98 | 0.96 | |
12.0 | 99.64 ± 0.87 | 0.87 | 100.45 ± 0.87 | 0.87 | |
Secoisolariciresinol | 7.0 | 99.32 ±1.07 | 1.08 | 99.89 ± 1.08 | 1.08 |
10.0 | 99.77 ±1.14 | 1.14 | 100.95 ± 0.98 | 0.97 | |
12.0 | 101.45 ± 1.01 | 1.00 | 102.31± 1.34 | 1.31 |
Samples | Average Conc. (μg/mL) | ||||
---|---|---|---|---|---|
β-Sitosterol | Betulinic Acid | Eriodictyol | Epipinoresinol | Secoisolariciresinol | |
Methanol crude extract | 21.1 ± 0.56 | 0.88 ± 0.06 | 22.74 ± 0.71 | 11.0 ± 0.33 | 7.22 ± 0.21 |
Ethyl acetate fraction | 5.5 ± 0.12 | 0.86 ± 0.05 | 38.9 ±0.89 | 23.8 ±0.67 | 24.08 ±1.02 |
Compound | Binding Affinity (Kcal/mol) | Type of Interaction | Bond Length (A°) | Interaction Moiety Involved | Amino Acid |
---|---|---|---|---|---|
Co-crystallized ligand | −11.26 | H-acceptor | 1.70 | -N- | Met 769 |
(+) Eriodictyol | −19.93 | H-acceptor | 2.34 | -OH | Met 769 |
Secoisolariciresinol | −16.63 | H-acceptor | 1.77 | -OH | Met 769 |
Compound | Percentage of Autophosphorylation Inhibition at Conc. [10 µM] | IC50 nM * |
---|---|---|
(+) Eriodictyol | 80.34 ± 2.64 | 76.16 ± 1.66 |
Secoisolariciresinol | 79.62 ± 2.16 | 80.26 ± 1.67 |
Erlotinib | 83.89 ± 2.03 | 78.65 ± 1.85 |
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Elhady, S.S.; Ibrahim, E.A.; Goda, M.S.; Nafie, M.S.; Samir, H.; Diri, R.M.; Alahdal, A.M.; Thomford, A.K.; El Gindy, A.; Hadad, G.M.; et al. GC-MS/MS Quantification of EGFR Inhibitors, β-Sitosterol, Betulinic Acid, (+) Eriodictyol, (+) Epipinoresinol, and Secoisolariciresinol, in Crude Extract and Ethyl Acetate Fraction of Thonningia sanguinea. Molecules 2022, 27, 4109. https://doi.org/10.3390/molecules27134109
Elhady SS, Ibrahim EA, Goda MS, Nafie MS, Samir H, Diri RM, Alahdal AM, Thomford AK, El Gindy A, Hadad GM, et al. GC-MS/MS Quantification of EGFR Inhibitors, β-Sitosterol, Betulinic Acid, (+) Eriodictyol, (+) Epipinoresinol, and Secoisolariciresinol, in Crude Extract and Ethyl Acetate Fraction of Thonningia sanguinea. Molecules. 2022; 27(13):4109. https://doi.org/10.3390/molecules27134109
Chicago/Turabian StyleElhady, Sameh S., Elsayed A. Ibrahim, Marwa S. Goda, Mohamed S. Nafie, Hanan Samir, Reem M. Diri, Abdulrahman M. Alahdal, Ama Kyeraa Thomford, Alaa El Gindy, Ghada M. Hadad, and et al. 2022. "GC-MS/MS Quantification of EGFR Inhibitors, β-Sitosterol, Betulinic Acid, (+) Eriodictyol, (+) Epipinoresinol, and Secoisolariciresinol, in Crude Extract and Ethyl Acetate Fraction of Thonningia sanguinea" Molecules 27, no. 13: 4109. https://doi.org/10.3390/molecules27134109