Quality Related Safety Evaluation of a South African Traditional Formulation (PHELA®) as Novel Anti-Biofilm Candidate
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Procurement of Plant Material
2.3. Extraction of the Plant Material
2.4. UPLC-MS Analysis of PHELA® Extract
2.5. RP-HPLC Analysis of PHELA® Extract
2.6. Safety Assessment of PHELA® Extract
2.6.1. MTT Cytotoxicity Assay
2.6.2. CYP450 Enzyme Inhibition Study
2.7. Antibacterial and Anti-Biofilm Activity of PHELA® Extract
2.7.1. MIC Determination by Broth Dilution Method Using the 96-Well Plate
2.7.2. Determination of MBC
2.7.3. Growth Curves Analysis
2.7.4. Inhibition of Biofilm Formation
2.7.5. AFM Assisted Characterization of the Effects of PHELA® Extract on S. aureus Biofilm
3. Statistical Analysis
4. Results
4.1. UPLC-MS Analysis of PHELA® Extract
4.2. RP-HPLC Analysis of PHELA® Extract
4.3. Safety Assessment of PHELA® Extract
4.4. Antibacterial and Anti-Biofilm Activity of PHELA® Extract
Determination of MIC and MBC
4.5. Growth Curve Analysis
4.6. Effect of PHELA® on Formation of Biofilm
5. Discussion
6. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
EPS | Extracellular polymeric substances |
MHB | Muller Hinton Broth |
ATCC | American Type Culture Collection |
PHAE | Hydro-alcoholic extract of PHELA® |
RP-HPLC | Reverse Phase- High Performance Liquid Chromatography |
PDA | Photodiode Array |
CYP | Cytochrome |
MIC | Minimum Inhibitory Concentration |
MBC | Minimum Bactericidal Concentration |
AFM | Atomic Force Microscopy |
Sq | Root Mean Square |
Spk | Reduced peak height |
Svk | Reduced valley depth |
Sa | Roughness Average |
Ssk | Surface skewness |
Ska | Coefficient of kurtosis |
Sdr | Surface Area Ratio |
Sz | % Maximum height |
r2 | correlation coefficient |
Rt | Retention time |
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Rt (min) | Compound Name | Chemical Formula | Acquired [M-H]− m/z Ratio | Theoretical [M-H]− m/z Ratio | Calculated Accurate Mass (Da) | Mass Error (ppm) | References |
---|---|---|---|---|---|---|---|
0.6 | 6-0-α-D-glucopyranosyl-D-fructose | C12H22O11 | 341.1089 | 341.1084 | 342.1162 | 1.5 | [32] |
6.7 | Quercetin-3-O-alpha-l-rhamnopyranosyl(1-2)-beta-D-glucopyranoside-7-O-alpha-l-rhamnopyranoside | C34H44O19 | 755.2406 | 755.2399 | 756.2476 | 0.9 | [31,33] |
6.8 | Hesperidin methyl chalcone | C29H36O15 | 623.1974 | 623.1976 | 624.2054 | −0.3 | [34] |
7.9 | Martynoside | C31H40O15 | 651.2285 | 651.2289 | 652.2366 | −0.6 | [35] |
13.4 | Carnosol | C20H26O4 | 331.1906 | 331.1909 | 330.1831 | −0.9 | [36] |
Rt(min) | Compound name | Chemical formula | Acquired [M+H]+ m/z | Theoretical [M+H]+ m/z | Calculated accurate mass (Da) | Mass error (ppm) | References |
10.18 | Isoliquiritin | C21H22O9 | 419.1349 | 419.1342 | 418.1263 | 1.7 | [37] |
11.4 | Emodin | C15H9O5 | 269.0448 | 269.0450 | 268.0371 | −0.7 | [38] |
Name of the Plant | CYP3A4 (mg/mL) ± SEM | CYP2D6 (mg/mL) ± SEM |
---|---|---|
PHELA® formulation | 0.2317 ± 2.14 | 0.2817 ± 3.65 |
Positive control | Ketoconazole | Fluoxetine |
0.0643 ± 1.12 | 0.0739 ± 1.69 |
Gram-Positive Bacteria | MIC (mg/mL) | MBC (mg/mL) |
---|---|---|
S. aureus | 0.3125 ± 0.019 | <1.00 |
B. substilis | 0.356 ± 0.007 | >1.00 |
Gram-negative bacteria | ||
P. auregenosa | 0.625 ± 0.017 | >1.25 |
E. coli | 0.512 ± 0.011 | >1.5 |
Statistical Parameters | Control | Standard | PHELA® Extract |
---|---|---|---|
Root Mean Square (Sq) | 9.165 nm | 4.344 nm | 3.523 nm |
Reduced peak height (Spk) | 36 nm | 14.12 nm | 17.51 nm |
Reduced valley depth (Svk) | 28.56 nm | 14.75 nm | 10.42 nm |
Roughness Average (Sa) | 7.30 nm | 3.52 nm | 2.81 nm |
Surface skewness (Ssk) | 0.19 | 0.14 | 0.4 |
Coefficient of kurtosis (Ska) | 0.07 | 0.19 | 0.27 |
Surface Area Ratio (Sdr) % | 5.98 | 1.75 | 1.90 |
Maximum height (Sz) | 64.56 nm | 28.87 nm | 27.93 nm |
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Das, B.; Kar, A.; Bhowmik, R.; Karmakar, S.; Tripathy, S.; Matsabisa, M.G.; Mukherjee, P.K. Quality Related Safety Evaluation of a South African Traditional Formulation (PHELA®) as Novel Anti-Biofilm Candidate. Molecules 2022, 27, 1219. https://doi.org/10.3390/molecules27041219
Das B, Kar A, Bhowmik R, Karmakar S, Tripathy S, Matsabisa MG, Mukherjee PK. Quality Related Safety Evaluation of a South African Traditional Formulation (PHELA®) as Novel Anti-Biofilm Candidate. Molecules. 2022; 27(4):1219. https://doi.org/10.3390/molecules27041219
Chicago/Turabian StyleDas, Bhaskar, Amit Kar, Rudranil Bhowmik, Sanmoy Karmakar, Satyajit Tripathy, Motlalepula G. Matsabisa, and Pulok Kumar Mukherjee. 2022. "Quality Related Safety Evaluation of a South African Traditional Formulation (PHELA®) as Novel Anti-Biofilm Candidate" Molecules 27, no. 4: 1219. https://doi.org/10.3390/molecules27041219