Background
Methods
Plant material
Preparation of crude extract
Preparation of alkaloid-enriched fractions
Test for the presence of steroidal alkaloids
Cellular assays
Cell maintenance
Cytotoxicity evaluation
P-glycoprotein inhibition
Isolation of active compound
Bioassay-guided fractionation
Isolation of active constituents by column and solid phase extraction chromatography
UPLC-TOF-MS fingerprinting of the crude extract and alkaloid-enriched fractions
Bioactivity
Synergistic potential evaluation of the aqueous alkaloid-enriched fraction and active isolated compound in combination with doxorubicin
Statistics
Results
Isolation and structural elucidation of steroidal alkaloids
Solamargine (19) (deuterated chloroform and methanol) | Compound 1 (2015) (deuterated methanol) | ||||
---|---|---|---|---|---|
Carbon no (type) | C13-NMR | H-1-NMR (J in Hz) | C13-NMR | H-1-NMR (J in Hz) | |
Aglycone | C-1 (CH2) | 37.9 | 38.70 | ||
C-2 (CH2) | 30.5 | 31.13 | |||
C-3 (CH) | 79 | 79.50 | |||
C-4 (CH2) | 40.4 | 41.19 | |||
C-5 (C) | 141.1 | 142.07 | |||
C-6 (CH) | 122.2 | 5.37 (d, J 4.2) | 122.78 | 5.38 (d, J 4.6) | |
C-7 (CH2) | 32.5 | 32.94 | |||
C-8 (CH) | 32.1 | 31.81 | |||
C-9 (CH) | 50.7 | 51.90 | |||
C-10 (C) | 37.5 | 38.20 | |||
C-11 (CH2) | 21.4 | 22.15 | |||
C-12 (CH2) | 38.9 | 39.67 | |||
C-13 (C) | 41.1 | 41.77 | |||
C-14 (CH) | 57.1 | 57.90 | |||
C-15 (CH2) | 30.1 | 30.90 | |||
C-16 (CH) | 79.7 | 4.33 (m) | 80.45 | 4.34 (m) | |
C-17 (CH) | 63.2 | 64.21 | |||
C-18 (CH3) | 16.8 | 0.84 (s) | 17.00 | 0.83 (s) | |
C-19 (CH3) | 19.6 | 1.05 (s) | 19.91 | 1.05 (s) | |
C-20 (CH) | 42 | 42.85 | |||
C-21 (CH3) | 15.4 | 0.97 (d, J 7.0) | 15.60 | 0.97 (d, J 7.2) | |
C-22 (C) | 98.9 | 99.60 | |||
C-23 (CH2) | 34.4 | 33.36 | |||
C-24 (CH2) | 32.7 | 33.14 | |||
C-25 (CH) | 31.3 | 31.81 | |||
C-26 (CH2) | 47.8 | 2.6 (m, br) | 48.47 | 2.53 (m) | |
C-27 (CH3) | 19.6 | 0.87 (d, J 6.0) | 19.98 | 0.85 (d, J 6.4) | |
Glucose (glu) | C-1′ (CH) | 99.9 | 4.48 (d, J 7.8) | 100.63 | 4.50 (d, J 7.6) |
C-2′ (CH) | 79.5 | 80.23 | |||
C-3’(CH) | 77.3 | 78.19 | |||
C-4′ (CH) | 75.7 | 76.73 | |||
C-5′ (CH) | 78.6 | 79.50 | |||
C-6′ (CH2) | 61.4 | 62.12 | |||
Rhamnose A (rha A) | C-1″ (CH) | 102.4 | 4.87 (s) | 103.16 | 4.84 (s) |
C-2″ (CH) | 71.8 | 72.60 | |||
C-3″ (CH) | 71.5 | 72.35 | |||
C-4″ (CH) | 73.3 | 74.10 | |||
C-5″ (CH) | 69.1 | 69.94 | |||
C-6″ (CH3) | 17.7 | 1.29 (m) | 18.11 | 1.26 (m) | |
Rhamnose B (rha B) | C-1″‘(CH) | 101.5 | 5.23 (s) | 102.44 | 5.21 (s) |
C-2″‘(CH) | 71.6 | 72.52 | |||
C-3″‘(CH) | 71.3 | 72.35 | |||
C-4″‘(CH) | 73 | 73.89 | |||
C-5″‘(CH) | 70.1 | 70.85 | |||
C-6″‘(CH3) | 17.6 | 1.29 (m) | 18.0. | 1.26 (m) |
Cytotoxicity
IC50 ± SEM | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
Cancerous cell lines | Non-cancerous cell lines | |||||||||||
Extract/fractiona | A2780 | Caco-2 | DU-145 | HepG2 | MCF-7 | MDA-MB-231 | SH-SY5Y | SK-Br3 | 3 T3-L1 | C2C12 | EA.hy.926 | SC-1 |
Crude 24 ha | 34.73 ± 1.07 | 36.46 ± 1.20 | 74.33 ± 1.14 | 9.44 ± 1.11 | 44.35 ± 1.06 | 26.87* | 19.10 ± 1.07 | 33.25 ± 1.10 | 37.24 ± 1.34 | 93.41 ± 1.08 | 3.88 ± 1.07 | 28.73 ± 1.09 |
Crude 72 ha | 32.88* | 24.40 ± 1.13 | 47.11 ± 1.18 | 7.04 ± 1.08 | 27.94 ± 1.11 | 24.00* | 12.97 ± 1.07 | 20.11 ± 1.08 | 19.55 ± 1.23 | 91.18 ± 1.13 | 2.79 ± 1.55 | 23.07 ± 1.10 |
Aqueous 24 ha | 31.45* | 50.37 ± 1.26 | 46.53 ± 1.21 | 40.02 ± 1.08 | 31.91 ± 1.06 | 28.05* | 29.11 ± * | 29.24* | 21.95 ± 1.50 | > 100 | 16.45 ± 1.06 | 19.89 ± 1.09 |
Aqueous 72 ha | 29.81* | 25.21 ± 1.12 | 34.08* | 20.66 ± 1.10 | 25.49 ± 1.13 | 26.11* | 17.21 ± 1.15 | 18.81 ± 1.14 | 12.89 ± 1.23 | 62.00 ± 1.22 | 9.35 ± 1.08 | 21.10 ± 1.08 |
Solamargine 72 hb | X | X | X | X | X | X | 15.62 ± 1.45 | 18.59 ± 1.13 | X | 20.25 ± 1.08 | 8.30 ± 1.12 | X |
P-gp inhibitor | ||||||||||||
Verapamil 24 hb | 93.48 ± 1.05 | > 100 | > 100 | 18.72 ± 1.08 | > 100 | > 100 | > 100 | > 100 | > 100 | > 100 | 31.55 ± 1.12 | > 100 |
Verapamil 72 hb | 65.75 ± 1.06 | > 100 | > 100 | 8.34 ± 1.08 | 46.76 ± 1.14 | 47.5 ± 1.09 | > 100 | 41.92 ± 1.09 | 79.22 ± 1.21 | > 100 | 5.69 ± 1.11 | 60.01 ± 1.16 |
P-glycoprotein inhibition
Synergistic potential of the aqueous alkaloid-enriched fraction and solamargine with doxorubicin
Cell type | Cancerous | Non-cancerous | ||||||
---|---|---|---|---|---|---|---|---|
Cell line | SH-SY5Y | SK-Br3 | C2C12 | EA.hy926 | ||||
Compound/Fraction | Solamargine | Aqueous alkaloid-enriched fraction | Solamargine | Aqueous alkaloid-enriched fraction | Solamargine | Aqueous alkaloid-enriched fraction | Solamargine | Aqueous alkaloid-enriched fraction |
IC50 of (A) [IC50 sample + doxorubicin (μM or μg/mL)] | 3.4 | 7.39 | 4.3 | 11.18 | 30.77 | 15.7 | 5.5 | 2.62 |
IC50 of (B) [IC50 doxorubicin + sample (nM)] | 16.27 | 24.22 | 40.83 | 47.48 | 149.9 | 140.7 | 213.4 | 220 |
IC50 (A) [Solamargine (μM) or aqueous alone (μg/mL)] | 15.62 | 26.5 | 18.59 | 19.7 | 20.25 | 13.7 | 8.3 | 7.3 |
IC50 (B) [doxorubicin alone (nM)] | 56.6 | 56.6 | 94.1 | 94.1 | 148.5 | 148.5 | 381.6 | 381.6 |
FIC (A) | 0.22 | 0.28 | 0.23 | 0.57 | 1.52 | 1.15 | 0.66 | 0.36 |
FIC (B) | 0.29 | 0.43 | 0.43 | 0.5 | 1.01 | 0.95 | 0.56 | 0.58 |
FIX value | 0.51 | 0.71 | 0.66 | 1.07 | 2.53 | 2.1 | 1.22 | 0.94 |
Result | Additive effect | Additive effect | Additive effect | Indifferent effect | Antagonistic effect | Antagonistic effect | Indifferent effect | Additive effect |