Cell Growth Inhibition of Saponin XII from Dipsacus japonicus Miq. on Acute Myeloid Leukemia Cells

In previous studies, we isolated the known compound saponin XII from the roots of Dipsacus japonicus Miq. Here, we show that this compound reduced the number of acute myeloid leukemia OCI-AML3 cells as evaluated by a hemocytometer. Flow cytometry analyses demonstrated that the reported activity was associated with a significant increase of apoptosis and of cells in the G0/G1 phase of the cell cycle, with a decrease of cells in the S and G2/M phases. Thus, the inhibition of cell growth in OCI-AML3 cells was due to antiproliferative and pro-apoptotic effects. Interestingly, the bioactivity of saponin XII exerted its effect at a concentration as low as 1 µg/mL.


Introduction
Plants are a valuable source of therapeutically useful compounds, and herbal medicine is the most practiced form of traditional medicine, defined by the World Health Organization (WHO) as "knowledge, skills, and practices based on theories, beliefs, and experiences of an indigenous culture used to maintain health and prevent, diagnose, or treat physical and mental illness" [1]. The value of traditional medicinal plants is not only due to their pharmacological properties but also to the belief that these "natural" remedies are very low in toxicity [1]. However, the beneficial properties of medicinal plants, along with the compounds isolated from them, require globally accepted high standard of manufacturing and biological validation through biotechnological methods [1].
Among many other herbal medicines, the genus Dipsacus is widely used in traditional remedies around the world, and is diffused in Europe, Africa and Asia with about 20 species belonging to its

Isolation and Identification of Saponin XII
The chemical structure of purified compound 1 was analyzed by NMR, and further identified by NMR spectroscopy as saponin XII. Dipsacus japonicus extraction and separation were modified from a previously described method [9].
HR-ESI-MS of 1 gave a pseudo-molecular sodium adduct ion peak at m/z 1567.7136 ([M + Na] + , calcd. 1567.7144), corresponding to a molecular formula of C 71 H 116 O 36 (M = 1544). The 1 H and 13 C NMR spectra of 1 (in CD 3 OD) exhibited six methyl singlets, δ H (0.73, 0.83, 0.93, 0.96, 1.00, 1.12, each 3H), and for the analogous carbons at δ C (14.0, 16.1, 17.4, 26.0, 33.0 and 23.6, C24-27; 29-30 respectively). The aglycon was identified as hederagenin by the comparison of the 1 H, 13 C NMR and DEPT data obtained in 2D NMR experiments with literature values [13]. The chemical shift of C-3 (δ C 78.6) and upfield chemical shift of C-28 (δ C 176.5) in the 13 C NMR spectra (in pyridine-d 5 ) implied that 1 was a bisdesmosidic glycoside, with sugar chains at C-3 and C-28. Sugar aglycon and sugar-sugar linkages were confirmed by the HMBC correlations. Based on spectroscopic analyses, including HR-ESI MS, 1D and 2D NMR experiments, as well as comparison with those reported data, the structure of 1 was determined unambiguously as 3-O-[β-d-glucopyranosyl(1→4)][α-l-rhamnopyranosyl(1→3)]emphβ-d-glucopyranosyl(1→3)-α-L-rhamnopyranosyl(1→2)-α-arabinopyranosyl hederagenin 28-O-β-dglucopyranosyl(1→6)-β-d-glucopyranoside (saponin XII), as shown in Figure 1 [9,16]. µg/mL) significantly increased apoptotic cell death, although there was no significant difference between these two groups. Since apoptosis is, by definition, a caspase-dependent process [19], experiments to see if the executioner caspase-3 was activated [20] were performed and, as shown, caspase-3 was activated upon treatment with both 1 and 2 µg/mL of saponin XII. In the cell cycle analysis, saponin XII, added for 24 or 48 h, significantly increased the percentage of cells in the G0/G1 phase and decreased the percentage of cells in the S phase ( Figure 4) with an MEC of 1 µg/mL. The reason for the missing analysis after 72 h of treatment with the higher concentration (2 µg/mL) of saponin XII is that no living cells were recovered.  In conclusion, this is the first report on the bioassay of saponin XII. The results indicate that this compound inhibits the cell growth of OCI-AML cells by an increase of apoptosis-a known effect of saponins that is exerted through caspase 3/8/9 activation [13,21]. This effect is shown in particular by Dipsacus saponins as previous studies demonstrate that Dipsacus saponins decrease apoptosis by increasing the anti-apoptotic molecule Bcl-2, decreasing the expression of pro-apoptotic molecules Bax, caspase-9 and caspase-8 [22], and by decreasing proliferation. Due to a limited amount of the studied compound, it was not possible to perform studies on structure-activity relationships. Hopefully, additional bioactive studies will clarify this interesting effect on acute myeloid leukemia cell lines and examine its potential therapeutic use.

Biological Activity of Isolated Saponin XII
Saponin XII significantly decreased the number of OCI-AML3 cells at concentrations of 1 and 2 µg/mL (0.648, 1.295 µM) (Figure 2). At these concentrations, the effect of cell number reduction reached its plateau, whereas higher concentrations were toxic to the cells as evidenced by the excessive number of dead cells and by the unreadable cell cycle profile. The decrease in cell number could have been due to apoptotic induction, inhibition of proliferation or both. To evaluate these processes, we stained cell nuclei with propidium iodide (PI) and performed flow cytometry analysis after the exclusion of necrotic cells by forward light scatter (FSC), without the need for annexin V staining [18] to investigate apoptosis and cell cycle of treated vs. untreated cells. As shown in Figure 3, saponin XII (2 and 1 µg/mL) significantly increased apoptotic cell death, although there was no significant difference between these two groups. Since apoptosis is, by definition, a caspase-dependent process [19], experiments to see if the executioner caspase-3 was activated [20] were performed and, as shown, caspase-3 was activated upon treatment with both 1 and 2 µg/mL of saponin XII. In the cell cycle analysis, saponin XII, added for 24 or 48 h, significantly increased the percentage of cells in the G 0 /G 1 phase and decreased the percentage of cells in the S phase ( Figure 4) with an MEC of 1 µg/mL. The reason for the missing analysis after 72 h of treatment with the higher concentration (2 µg/mL) of saponin XII is that no living cells were recovered.
Molecules 2020, 25, x FOR PEER REVIEW 5 of 9 collected by centrifuge and washed in PBS (phosphate-buffered saline). DNA was stained by incubating the cells in PBS containing 50 µg/mL PI for 30 min at 4 °C. This allows direct DNA staining in PI hypotonic solution without the requirement of RNase treatment as the RNA is removed by hypotonic shock [18]. Fluorescence was measured and analyzed by flow cytometry using Coulter Epics XL-MCL equipment (Beckman Coulter Inc., Brea, CA, USA) [23]. Apoptosis data were reported on a logarithmic scale (FL3). For cell cycle analyses, apoptotic cells were gated out and data were reported on a linear scale (FL2). Doublet discrimination was done with the Coulter Epics XL-MCLTM Flow Cytometer SYSTEM IITM Software, which can detect >90% cellular doublet in cells ≥7 µm, using peak vs. integral discrimination. A peak fluorescence signal was assigned to AUX to measure peak vs. integral fluorescence.

Statistical Analysis
Statistical significance was determined using the Mann-Whitney U test as specified in the figure legends. Differences were considered statistically significant according to the following criteria: * p < 0.05; ** p < 0.01; *** p < 0.001.    In conclusion, this is the first report on the bioassay of saponin XII. The results indicate that this compound inhibits the cell growth of OCI-AML cells by an increase of apoptosis-a known effect of saponins that is exerted through caspase 3/8/9 activation [13,21]. This effect is shown in particular by Dipsacus saponins as previous studies demonstrate that Dipsacus saponins decrease apoptosis by increasing the anti-apoptotic molecule Bcl-2, decreasing the expression of pro-apoptotic molecules Bax, caspase-9 and caspase-8 [22], and by decreasing proliferation. Due to a limited amount of the studied compound, it was not possible to perform studies on structure-activity relationships. Hopefully, additional bioactive studies will clarify this interesting effect on acute myeloid leukemia cell lines and examine its potential therapeutic use. The purity of compound 1 was estimated to be greater than 95% by integration in 1 H and 13 C NMR.

Extraction and Purification of Saponin XII
The ground and dried roots of DJ (1 kg) were extracted three times with 90% aqueous MeOH at room temperature. MeOH was evaporated in vacuo and the aq. solution was partitioned with n-hexane followed by EtOAc and n-BuOH (each three times), giving three extracts, respectively. The n-BuOH extract was separated on silica gel using  [9,16], compound 1 was confirmed to be saponin XII. The purity of saponin XII was determined by HPLC ( Figure S2).

OCI-AML Culture Conditions
We used subtype 3 of the OCI-AML cell line, a generous gift from Prof. Maria Paola Martelli (section of hematology, Department of Medicine, University of Perugia). OCI-AML3 cells were maintained in RPMI 1640 medium with 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 • C in 5% CO 2 . The cell line was obtained from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Lower Saxony, Germany), kept at logarithmic growth and cultured in 24-well plates to assess the number and morphology of cells. Cultures, kept at 2 × 10 5 cells/mL, were treated with different concentrations of dimethylsulfoxide (DMSO) (the higher concentration utilized of DMSO was 0.6 µL/mL). The test compounds at the final concentrations of 0, 5, 1 and 2 µg/mL and cells were harvested after 24, 48 or 72 h depending on the different types of experiments.

Analysis of Cell Number, Apoptotic Cell Death and Cell Cycle Progression
Cells were counted manually using a hemocytometer. Cell viability and cell cycle progression were analyzed by flow cytometry to determine the DNA content of cell nuclei stained with propidium iodide (PI) after the exclusion of necrotic cells by forward light scatter (FSC). Briefly, cells were collected by centrifuge and washed in PBS (phosphate-buffered saline). DNA was stained by incubating the cells in PBS containing 50 µg/mL PI for 30 min at 4 • C. This allows direct DNA staining in PI hypotonic solution without the requirement of RNase treatment as the RNA is removed by hypotonic shock [18]. Fluorescence was measured and analyzed by flow cytometry using Coulter Epics XL-MCL equipment (Beckman Coulter Inc., Brea, CA, USA) [23]. Apoptosis data were reported on a logarithmic scale (FL3). For cell cycle analyses, apoptotic cells were gated out and data were reported on a linear scale (FL2). Doublet discrimination was done with the Coulter Epics XL-MCLTM Flow Cytometer SYSTEM IITM Software, which can detect >90% cellular doublet in cells ≥7 µm, using peak vs. integral discrimination. A peak fluorescence signal was assigned to AUX to measure peak vs. integral fluorescence.

Statistical Analysis
Statistical significance was determined using the Mann-Whitney U test as specified in the figure legends. Differences were considered statistically significant according to the following criteria: * p < 0.05; ** p < 0.01; *** p < 0.001.