Table 1.
Hydroponic culture conditions.
Fig 1.
(A) HPLC analyses of alcoholic extracts of Ashwagandha roots (1) and leaves (2) raised at distant places, Ibaraki (K) and Tokushima (A and B) and Punjab, India (P). Purified Withaferin A (Wi-A) and Withanone (Wi-N) were used as standards. (B) Withanolide yield and ratio of Wi-N/Wi-A in extracts from root and leaves of Ashwagandha grown at distant places are shown. (C) Representative comparative cytotoxicity assay for root and leaf extracts of Ashwagandha. Cytotoxicity as determined by viability assay (short term cytotoxicity) and phase contrast images of cells in plates for colony forming assays (long term cytotoxicity) are shown.
Fig 2.
(A) Hydroponic cultivation system for Ashwagandha and (B) hydroponically grown leaves are shown. (C) Quantitative analysis of the effect of cultivation medium on plant characteristics showing no significance effect on the indicated attributes. (D) Cultivation in medium with 4 units caused hypertrophic roots, and addition of 4 units of NaCl caused decrease in root volume. (E) Effect of UV and temperature stress on hydroponically cultivated Ashwagandha. Exposure to UV-A during night caused leaf curling. Cultivation temperature 37/22 caused thick and dark green leaves, and 42/22 (light/dark) was lethal. (F) Cytotoxic assays of extracts (10 μg/ml) prepared from dried hydroponic leaves (100 mg/ml) cultivated under UV and temperature stress did not show any significant difference.
Fig 3.
(A) Representative HPLC analyses of bioactives (Withanone and Withaferin A) in hydroponically grown Ashwagandha under different media and light conditions are shown. Leaves cultivated on land were used as controls. (B) Cytotoxicity assays of extracts (10 μg/ml) prepared from hydroponically grown Ashwagandha leaves (100 mg/ml). High Withanone content corresponded to selective toxicity to cancer cells.
Fig 4.
(A) HPLC analysis of roots (A-1) and leaves (A-2) from Ashwagandha plants cultivated under different light conditions. Quantitation of Withanone and Withaferin A in root and leaves of Ashwagandha cultivated under different light conditions. (B) Cytotoxicity of Ashwagandha root and leaf extracts (derived from 1 mg/ml of root or leaf powder) to human cancer (U2OS) cells showing higher cytotoxicity of the latter. (C) Cytotoxicity of leaf extracts (derived from 0.5 mg/ml leaf powder) from plants raised under different light conditions to human osteosarcoma.
Fig 5.
(A) Cyclodextrin-assisted water extractions of Ashwagandha leaves were performed and the level of Withanone and Withaferin A was determined by HPLC analysis. As shown, CD-assisted water extractions yield significant enrichment of Withanone and Withaferin A. (B) Residual gamma-CD precipitate analyzed by HPLC showed high ratio (17:1) of Withanone:Withaferin A. (C) In vitro cytotoxicity assay revealed that the CD extract of land raised Ashwagandha leaves (B2) was toxic to cancer as well as normal cells. CD-B1 (root) extract that possessed low content of Withanolides did not show activity. Gamma-CD residual precipitates (DM) that contained high level of Withanone showed higher cytotoxicity to cancer cells and were mild to normal cells. Leaves from hydroponically grown plants under the treatment of red (S-C2) and blue (S-A2) lights showed selective toxicity to cancer cells. (D) IC50 of each of the extracts obtained from several independent experiments is shown. DM extracts showed higher toxicity to the cancer than to the normal cells. (E) In vivo tumor formation assays in nude mice revealed that the gamma CD could enhance the anticancer potential in water extracts of Ashwagandha leaves.