A Combination of Alkaloids and Triterpenes of Alstonia scholaris (Linn.) R. Br. Leaves Enhances Immunomodulatory Activity in C57BL/6 Mice and Induces Apoptosis in the A549 Cell Line
Abstract
:1. Introduction
2. Results and Discussion
2.1. Characterization of the Chemical Compounds by HPLC and LC/MS/MS
No | Classification | Compound identity | TR (min) | (+)ESI-MS m/z | UVmax (nm) | Ion fragments | Formula |
---|---|---|---|---|---|---|---|
1 | Alkaloids | Scholaricine | 11.65 | 357 | 336, 298, 285, 266 | 339, 325, 311, 198 | C20H24N2O4 |
2 | 19-Epi-scholaricine | 11.84 | 357 | 360, 311, 293, 286 | 339, 325, 311, 198 | C20H24N2O4 | |
3 | Sarpagine | 12.57 | 311 | 364, 256 | 294, 278, 253, 226, 186 | C19H22N2O2 | |
4 | N4-Demthylechitamine | 12.85 | 371 | 297, 266 | 353, 339, 256, 189 | C21H26N2O4 | |
5 | Echitamidine | 15.83 | 386 | 329, 304, 294, 262 | 3369, 355, 271, 211 | C22H29N2O4 | |
6 | Strictamine | 18.30 | 323 | 365, 261, 239 | 295, 263, 216, 156 | C20H22N2O2 | |
7 | Akuammidine | 19.64 | 353 | 279, 251 | 307, 293, 230, 202 | C21H24N2O3 | |
8 | Vallesamine | 20.63 | 341 | 362, 258, 240 | 323, 309, 267, 108 | C20H24N2O3 | |
9 | Picrinine | 22.49 | 339 | 336, 257, 239 | 307, 265, 107 | C20H22N2O3 | |
10 | Picralinal | 28.31 | 367 | 365, 259, 240 | 337, 324, 280, 260 | C21H22N2O4 | |
11 | Triterpenoids | Cylicodiscic acid | 4.77 | 473 | 223, 203 | 455, 441, 423, 232 | C30H48O4 |
12 | Betulinic acid | 5.54 | 457 | 210 | 441, 429, 411, 220 | C30H48O3 | |
13 | Betulin | 6.62 | 443 | 208 | 425, 413, 395, 204 | C30H50O2 | |
14 | Oleanolic acid | 13.07 | 457 | 205 | 439, 427, 409, 381, 259, 217 | C30H48O3 | |
15 | Ursolic acid | 13.86 | 457 | 205 | 439, 427, 409, 381, 259, 217 | C30H48O3 | |
16 | Cycloeucalenol | 16.17 | 423 | 200 | 408, 385, 367,315, 289, 213 | C30H48O | |
17 | α-amyrin acetate | 18.94 | 468 | 225 | 435, 419, 401, 381 | C32H52O2 |
2.2. The Inhibition of Alkaloids, Triterpenes and Their Combination on Cell Proliferation in A549 Cells
2.3. The Inhibition of Alkaloids, Triterpenes and Their Combination on Tumor Growth in C57BL/6 Mice
2.4. The Combination of Alkaloids and Triterpenes Enhances Thymus and Spleen Indexes in Tumor-Bearing C57BL/6 Mice
2.5. The Effect of Alkaloids, Triterpenes and Their Combination on TNF-α and IL-6 Level Increases
Groups | Dose (raw material/kg/d) | TNF-α (pg/mL) | IL-6 (pg/mL) |
---|---|---|---|
Blank | − | 46.33 ± 1.98 | 34.56 ± 3.76 |
CTX | 20 mg/kg/d | 40.35 ± 2.36 $$ | 28.44 ± 4.59 $$ |
Alkaloids | 7.5 g | 53.73 ± 3.62 | 42.66 ± 2.84 |
15 g | 59.57 ± 2.93 # | 47.16 ± 2.78 # | |
30 g | 62.16 ± 2.54 ## | 51.64 ± 1.77 ## | |
Triterpenes | 7.5 g | 52.44 ± 2.18 | 41.75 ± 2.67 |
15 g | 58.39 ± 1.15 # | 48.95 ± 3.00 # | |
30 g | 62.05 ± 1.69 ## | 51.41 ± 1.68 ## | |
Alkaloids and triterpenes | 7.5 g | 57.33 ± 2.30 # | 46.97 ± 2.75 |
15 g | 61.70 ± 1.42 *## | 52.69 ± 3.02 *## | |
30 g | 65.38 ± 1.95 **## | 56.83 ± 4.25 **## |
2.6. Annexin-FITC and PI Double Staining for Apoptosis Analysis
2.7. Cell Cycle Analysis
Groups | Dose (raw material/mL) | G0/G1 (%) | S (%) | G2/M (%) |
---|---|---|---|---|
Blank control | − | 65.1 ± 2.6 | 26.5 ± 4.4 | 8.3± 1.4 |
Cisplatin | 10 μg/mL | 49.8 ± 1.9 $$ | 39.2 ± 3.8 $$ | 11.1 ± 2.8$ |
Alkaloids | 1 mg | 63.8 ± 2.4 | 27.4 ± 2.9 | 8.8 ± 1.5 |
2 mg | 58.9 ± 3.3 | 33.3 ± 3.6 | 7.8 ± 1.7 | |
3 mg | 52.2 ± 2.8 | 41.7 ± 2.5 | 6.4 ± 2.2 | |
Triterpenes | 1 mg | 62.2 ± 1.4 | 25.4 ± 4.6 | 12.4 ± 1.6 |
2 mg | 61.0 ± 2.7 | 30.6 ± 3.9 | 8.4 ± 1.2 | |
3 mg | 56.3 ± 2.9 | 34.4 ± 2.1 | 8.3 ± 0.8 | |
Alkaloids and triterpenes | 1 mg | 56.6 ± 4.7 *# | 29.3 ± 5.2 | 14.1 ± 3.1 |
2 mg | 48.1 ± 3.2 **## | 42.2 ± 3.7 **## | 9.7 ± 1.8 | |
3 mg | 44.6 ± 1.3 **## | 48.9 ± 4.3 *## | 6. 5 ± 2.4 ## |
2.8. Apoptosis-Related Protein Expressions
3. Experimental
3.1. Materials
3.2. Cell Culture
3.3. HPLC Analysis
3.4. LC/MS/MS Analysis
3.5. MTT Assay for Cell Viability
3.6. Lewis Tumor-Bearing C57BL/6 Mice
3.7. ELISA Assay for IL-6 and TNF-α in Serum
3.8. Annexin-V/PI Double-Staining Assay and Cell Cycle
3.9. Western Blot Assay
3.10. Statistical Analysis
4. Conclusions
Acknowledgments
Conflicts of Interest
References
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Feng, L.; Chen, Y.; Yuan, L.; Liu, X.; Gu, J.-F.; Zhang, M.-H.; Wang, Y. A Combination of Alkaloids and Triterpenes of Alstonia scholaris (Linn.) R. Br. Leaves Enhances Immunomodulatory Activity in C57BL/6 Mice and Induces Apoptosis in the A549 Cell Line. Molecules 2013, 18, 13920-13939. https://doi.org/10.3390/molecules181113920
Feng L, Chen Y, Yuan L, Liu X, Gu J-F, Zhang M-H, Wang Y. A Combination of Alkaloids and Triterpenes of Alstonia scholaris (Linn.) R. Br. Leaves Enhances Immunomodulatory Activity in C57BL/6 Mice and Induces Apoptosis in the A549 Cell Line. Molecules. 2013; 18(11):13920-13939. https://doi.org/10.3390/molecules181113920
Chicago/Turabian StyleFeng, Liang, Yan Chen, Ling Yuan, Xuan Liu, Jun-Fei Gu, Ming-Hua Zhang, and Ying Wang. 2013. "A Combination of Alkaloids and Triterpenes of Alstonia scholaris (Linn.) R. Br. Leaves Enhances Immunomodulatory Activity in C57BL/6 Mice and Induces Apoptosis in the A549 Cell Line" Molecules 18, no. 11: 13920-13939. https://doi.org/10.3390/molecules181113920