Neuroprotective Effect of CR-777, a Glutathione Derivative of Withaferin A, Obtained through the Bioconversion of Withania somnifera (L.) Dunal Extract by the Fungus Beauveria bassiana
Abstract
:1. Introduction
2. Results
2.1. Production, Isolation and Structural Elucidation of the Bioconversion Products Produced through the Bioconversion of Withania somnifera Extract by Beauveria bassiana ATCC 7159
2.2. Stereochemistry of C-5 and C-6 in CR-591 (1) and CR-777 (2)
2.3. Mechanism of Formation of CR-591 (1) and CR-777 (2)
2.4. Impact on Parkinson’s Mimetics
3. Discussion
4. Materials and Methods
4.1. Analytical and Structure Elucidation Methods of Target Molecules
4.2. Plant Extraction Procedure
4.3. Cultivation of B. bassiana
4.4. Fermentation of the Plant Extract
4.5. Fractionation of Extract and Purification of Target Compounds
4.6. Isolation of Withaferin A
4.7. Compliance with the Rules for Laboratory Animal Use
4.8. Culture of Mesencephalic Neurons for MPP+ Injury
4.9. Culture of Mesencephalic Neurons for α-Synuclein Injury
4.10. CR-777 Exposure
- MPP+ injury: On day 6 of culture, the medium was removed and fresh medium was added, without or with MPP+ (Sigma Aldrich), at 4 µM diluted in control medium with or without CR-777 for 48 h.
- 6-OHDA injury: On day 6 of culture, the medium was removed and fresh medium was added, without or with 6-OHDA (Sigma Aldrich), at 20 µM diluted in control medium with or without CR-777 for 48 h.
- α-Synuclein injury: On day 7 of culture, the medium was removed and fresh medium was added, without or with alpha synuclein (rPeptide), at 250 nM diluted in control medium with or without CR-777 for 24 h.
4.11. Immunostaining: TH Positive Neron (Dopaminergic Neurons)
4.12. Immunostaining: Overepression of α-Syuclein (Dopaminergic Neurons)
- Monoclonal anti-tyrosine hydroxylase (TH, Sigma) antibody produced in mice at a dilution of 1/10,000 in PBS containing 1% FCS and 0.1% saponin for 2 h at room temperature. This antibody was revealed with Alexa Fluor 488 goat anti-mouse IgG (Molecular Probe) at the dilution of 1/800 in PBS containing 1% FCS and 0.1% saponin for 1 h at room temperature.
- Monoclonal anti-alpha-synuclein (Cell Signaling) antibody produced in rabbit at a dilution of 1/200 in PBS containing 1% FCS and 0.1% saponin for 2 h at room temperature. This antibody was revealed with Alexa Fluor 568 goat anti-rabbit IgG (Molecular Probe) at the dilution of 1/800 in PBS containing 1% FCS and 0.1% saponin for 1 h at room temperature.
- Immunostaining: MAP-2 tau phosphor Ser214, Thr212 (cortical neurons)
- Mouse monoclonal antibody anti phosphoT (phospho Thr212/Ser214; AT100) at a dilution of 1/400 in PBS containing 1% fetal calf serum and 0.1% saponin.
- Chicken polyclonal antibody anti microtubule-associated-protein 2 (MAP-2) at a dilution of 1/1000 in PBS containing 1% fetal calf serum and 0.1% saponin (this antibody stains specifically cell bodies and neurites, allowing study of neuronal cell survival and neurite network).
4.13. Analysis
4.14. Statistical Analysis
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Sample Availability: Samples of the compounds 1 and 2 are available from the authors. |
Position | δC | δH (mult; J in Hz) | HMBC |
---|---|---|---|
1 | 204.1 | - | |
2 | 127.5 | 5.89 (1H; dd; 10.2; 2.0) | C-4, C-10 |
3 | 148.8 | 6.58 (1H; dd; 10.2, 1.9) | C-1, C-5 |
4 | 67.3 | 4.89 (1H; m) | C-1, C-2, C-3, C-6 |
5 | 81.4 | - | |
6 | 52.4 | 3.02 (1H; d; 12.3) | C-4, C-5, C-7, C-10, Cys (35,3) |
7 | 38.7 | 2.19 (1H; m), 1.55 (1H; m) | C-6, C-8, C-9, C-14 |
8 | 36.7 | 1.67 (1H; m) | |
9 | 47.0 | 1.39 (1H; m) | |
10 | 59.3 | - | |
11 | 28.4 | 1.81 (1H; m), 1.42 (1H; m) | |
12 | 40.5 | 1.14 (2H; m) | |
13 | 44.4 | - | |
14 | 56.3 | 1.18 (1H; m) | C-15 |
15 | 25.2 | 1.70 (1H; m), 1.27 (1H; m) | C-14 |
16 | 24.3 | 1.39 (1H; m), 0.90 (1H; m) | C-13 |
17 | 53.1 | 1.23 (1H; m) | |
18 | 12.4 | 0.75 (3H; s) | C-12, C-13, C14, C-17 |
19 | 10.5 | 1.25 (3H; s) | C-1, C-5, C-9, C-10 |
20 | 40.3 | 1.94 (1H; m) | |
21 | 13.6 | 0.99 (3H; d; 6.8) | C-17, C-20, C-22 |
22 | 80.1 | 4.44 (1H; d; 13.4) | C-20, C-24 |
23 | 30.8 | 2.52 (1H; d; 17.3), 2.16 (1H; m) | C-22, C-24, C-25 |
24 | 157.8 | - | |
25 | 126.4 | - | |
26 | 168.5 | - | |
27 | 56.5 | 4.34 (2H; m) | C-24, C-25, C-26 |
28 | 20.3 | 2.09 (3H; s) | C-23, C-24, C-25 |
L-Cys | 35.3 | 3.13 (1H; dd; 14.3, 4.7), 2.95 (1H; dd; 14.3, 6.6) | C-6, Cys (55,7) |
55.7 | 3.70 (1H; m) | Cys (35,3), Cys (172,8) |
Position | δC | δH (mult; J in Hz) | HMBC |
---|---|---|---|
1 | 204.2 | - | |
2 | 127.5 | 5.89 (1H; dd; 10.2; 2.0) | C-4, C-10 |
3 | 148.6 | 6.57 (1H; dd; 10.2, 1.9) | C-1, C-5 |
4 | 67.3 | 4.90 (1H; m) | C-1, C-2, C-3, C-6 |
5 | 81.3 | - | |
6 | 52.7 | 3.16 (1H; d; 12.3) | C-4, C-5, C-7, Cys (36.3) |
7 | 38.5 | 2.15 (1H; m), 1.51 (1H; m) | C-6, C-8, C-9, C-14 |
8 | 36.6 | 1.66 (1H; m) | |
9 | 47.0 | 1.39 (1H; m) | |
10 | 59.2 | - | |
11 | 28.5 | 1.81 (1H; m), 1.40 (1H; m) | |
12 | 40.3 | 1.95 (1H; m) | |
13 | 44.3 | - | |
14 | 56.3 | 1.18 (1H; m) | C-15 |
15 | 25.2 | 1.70 (1H; m), 1.29 (1H; m) | C-14 |
16 | 24.2 | 1.38 (1H; m), 0.89 (1H; m) | C-13 |
17 | 53.1 | 1.23 (1H; m) | |
18 | 12.3 | 0.75 (3H; s) | C-12, C-13, C14, C-17 |
19 | 10.3 | 1.25 (3H; s) | C-1, C-5, C-9, C-10 |
20 | 40.3 | 1.94 (1H; m) | |
21 | 13.6 | 0.99 (1H; d; 6.8) | C-17, C-20, C-22 |
22 | 80.1 | 4.44 (1H; d; 13.4) | |
23 | 30.8 | 2.52 (1H; d; 17.3), 2.14 (1H; m) | C-22, C-24, C-25 |
24 | 157.8 | - | |
25 | 126.4 | - | |
26 | 168.5 | - | |
27 | 56.5 | 4.34 (2H; m) | C-24, C-25, C-26 |
28 | 20.2 | 2.09 (3H; s) | C-23, C-24, C-25 |
L-Cys | 36.3 | 3.08 (1H; dd; 13.7, 5.7), 2.81 (1H; dd; 13.7, 7.6) | C-6, Cys (55.0), Cys (172.9) |
55.0 | 4.59 (1H; t; 6.) | Cys (36.3), Cys (172.9), Glu (175.2) | |
172.9 | - | ||
L-Glu | 27.9 | 2.13 (2H; m) | Glu (55.5), Glu (175.2) |
33.2 | 2.54 (2H; m) | Glu (55.5), Glu (175.2) | |
55.5 | 3.67 (1H; m) | Glu (27.9), Glu (33.2), Glu (173.9) | |
173.9 | - | ||
175.2 | - | ||
L-Gly | 42.9 | 3.87 (2H, m) | Cys (172.9), Gly (173.9) |
172.9 | - | ||
173.9 | - |
Inhibitor | Activity |
---|---|
Farnesylthiosalicylic acid (FTS) | Inhibits the Ras/Raf pathway by disrupting Ras at the membrane. |
BEZ-235 | Dual PI3K/mTOR inhibitor |
T0070907 | Selective PPAR-γ inhibitor |
Nitro-L-arginine methyl ester (L-NAME) | Inhibitor of nitric oxide synthase (NOS). |
ABT-199 | Inhibitor of BLC2 |
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Rabhi, C.; Arcile, G.; Le Goff, G.; Da Costa Noble, C.; Ouazzani, J. Neuroprotective Effect of CR-777, a Glutathione Derivative of Withaferin A, Obtained through the Bioconversion of Withania somnifera (L.) Dunal Extract by the Fungus Beauveria bassiana. Molecules 2019, 24, 4599. https://doi.org/10.3390/molecules24244599
Rabhi C, Arcile G, Le Goff G, Da Costa Noble C, Ouazzani J. Neuroprotective Effect of CR-777, a Glutathione Derivative of Withaferin A, Obtained through the Bioconversion of Withania somnifera (L.) Dunal Extract by the Fungus Beauveria bassiana. Molecules. 2019; 24(24):4599. https://doi.org/10.3390/molecules24244599
Chicago/Turabian StyleRabhi, Chérif, Guillaume Arcile, Géraldine Le Goff, Christian Da Costa Noble, and Jamal Ouazzani. 2019. "Neuroprotective Effect of CR-777, a Glutathione Derivative of Withaferin A, Obtained through the Bioconversion of Withania somnifera (L.) Dunal Extract by the Fungus Beauveria bassiana" Molecules 24, no. 24: 4599. https://doi.org/10.3390/molecules24244599