1. Aim or purpose
The aim of this study was to evaluate the cancer chemopreventive properties and expand the knowledge about the
therapeutic potential of soxhlet extract (HIHEsox) of Hemidesmus indicus (Indian sarsaparilla, commonly used in
Ayurvedic medicine) on an acute lymphoblastic leukaemia cell line (CCRF-CEM).
2. Materials and methods
Chemical characterisation
Soxhlet extraction was performed in order to extract non-volatile and semivolatile organic compounds from dried H.
indicus hydroalcoholic extract with 1:1 mixture of CHCl3 and ethanol.
HIHEsox was subjected to RP-HPLC-DAD-MS analysis to identify and quantify its main phytomarkers.
Chemopreventive and antitumor properties
The proapoptotic and cytostatic effects were analysed using Annexin V and propidium iodide, respectively. To study the
mechanisms involved in the cellular effects of HIHEsox, poly ADP-ribose polymerase polymerase (PARP), cyclin E, and
p21 expression, caspases 3 and 8 activity, and mitochondrial potential were evaluated by flow cytometry. The
antigenotoxic potential was investigated studying the ability of HIHEsox to inhibit the phosphorylation of H2AX induced
by different genotoxicants (H2O2, etoposide, doxorubicin and camptothecin).
3. Results
Chemical characterisation
2-hydroxy-4-methoxybenzaldehyde resulted the main component (>77%), followed by 3-hydroxy-4-
methoxybenzaldehyde, 2-hydroxy-4-methoxybenzoic acid, 4-hydroxybenzaldehyde, and 4-hydroxy-3-methoxybenzoic
acid; protocatechuic and syringic acids were identified but under the limit of quantitation.
Chemopreventive and antitumor properties
HIHEsox induced a significant proapoptotic activity through a reduction in the mitochondrial transmembrane potential
(45.39% of cells with decreased mitochondrial potential versus 7.65% in untreated cells at 25 µg/mL treatment), the
increased activity of caspase 8 and caspase 3 (2.36- and 6.62-fold increase, respectively, at 25 µg/mL), and the upregulation
of cleaved PARP. Furthermore, HIHE soxhlet induced a cell-cycle block in S phase starting from the lowest
tested concentrations (from 16% of untreated cells to a maximum of 21% for treated cells) and supported by a modulation
of cyclin E. p21 was not affected by HIHE soxhlet.
Due to the lack of genotoxic activity of the extract in preliminary experiments (no significant increase in phosphorylation
of H2AX was observed at any tested concentration and at any time point), the evaluation of the antigenotoxicity potential
of HIHEsox was performed. We observed a significant inhibition of the genotoxicity of etoposide, doxorubicin, and
H2O2-mediated genotoxicity. A slight protective effect was recorded against the genotoxicity of camptothecin.
4. Conclusions
These data shed a light on the wide spectrum of therapeutic potential against cancer, as well as on the in vitro cancer
chemopreventive activity of HIHEsox. It induces apoptosis and cell-cycle inhibition in human leukaemia cells, and
exhibits genoprotective activity against mutagens acting through a different DNA damage mechanism. These data add to
the growing body

4. Keywords:
Chemopreventive, Cytostatic, Antigenotoxic, Hemidesmus indicus, Ayurveda

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