Two new glycosides isolated from Sapindus mukorossi fruits: effects on cell apoptosis and caspase-3 activation in human lung carcinoma cells

Two new glycosides (1, 2) and two saponins (3, 4) were isolated from the fruits of Sapindus mukorossi Gaertn. The two glycosides were designated as sapindoside G (1) and 4′′,4′′′′′-O-diacetylmukurozioside IIa (2). All four compounds exhibited inhibitory effects against A549 human lung adenocarcinoma cells with inhibition rates up to 69.2–83.3% at a concentration of 100 μg/mL. Flow cytometric analysis revealed that compounds 1–4 could suppress A549 cell growth by promoting cell apoptosis, which was related to the activation of caspase-3. Graphical abstract


Introduction
The genus Sapindus belongs to the Sapindaceae family, which consists of 2000 species . Sapindus mukorossi Gaertn, commonly known as Wu-huan-zi, is prevalent in southern China and has been used in the treatment of asthma, dermatological disorders and hepatic disorders (Sharma et al. 2011). According to the literature, there are several phytochemicals present in the pericarp, seeds, leaves, roots, stems and galls. The major constituents in the fruit are saponins (10.0-11.5%) and sugars (10%) (Verma 2012). The glycosides isolated from S. mukorossi are mainly sesquiterpene oligoglycosides and triterpenoidal saponins of hederagenin, dammarane and tirucullane (Upadhyay & Singh 2012). These glycosides have antimicrobial (Ibrahim et al. 2006), cytotoxic (Chen et al. 2010), molluscicidal (Huang et al. 2003;Upadhyay & Singh 2011), insecticidal (Rahman et al. 2007), fungicidal (Supradip et al. 2010) and hepatoprotective (Peng et al. 2014) properties. In this study, we isolated and characterized two new glycosides and evaluated the in vitro antiproliferative activity of all compounds against A549 cells.
By comparing the physical and spectroscopic data with those reported in the literature (Chirva et al. 1970;Nakayamak et al. 1986), compounds 3 and 4 were identified as Hishoushisaponin Ee and Sapindoside A, respectively.

Cell cycle and cell apoptosis
To further confirm the induction of cell apoptosis, cells were stained with annexin V/PI for flow cytometry. As shown in Figure S5, the early apoptotic rates of A549 cells were 52.4% (1), 4.9% (2), 52.4% (3) and 56.6% (4). Compared to untreated A549 cells, the percentage of late apoptotic cells increased by 1.8% (1), 3.3% (2), 13.9% (3) and 8.1% (4) following 24-h treatment. Compound 2 had a weaker effect on A549 cells apoptosis than the other three compounds. Late apoptosis was induced in the test concentration of compound 2. Therefore, the inhibition of A549 cell growth by glycosides is attributed to an induction in cell apoptosis. Compound 2 has different inhibitory mechanisms than compounds 1, 3 or 4. For cell cycle analysis, the percentages of cells in G0/G1, S and G2/M phases were determined by flow cytometry. The effect of compounds 1-4 on the cell cycle distribution of A549 cells is shown ( Figure S6). As a result, the compounds had little influence in the A549 cell cycle arrest.

Induction of apoptosis by activating caspase-3
Caspase family proteins play crucial roles in cell apoptosis. To assess whether compounds 1-4 activate the caspase-dependent cell death pathway, we studied the activation of caspase-3 using colorigenic tetrapeptide substrates such as Ac-DEVD-pNA, which is selective for caspase-3 enzymatic activities. Compared to the control group, compound-treated groups had higher absorbance measurements at 405 nm. After a 2-h treatment with compound 1, caspase-3 activity increased from 1.44 ± 0.26 μM pNA/2 h of protein to 4.33 ± 0.48 μM pNA/2 h of protein in a concentration-dependent manner ( Figure S7). The activity of the enzyme increased in the presence 6.25-75 μg/mL of compounds 2-4 and decreased in the presence of 100 μg/mL of compounds 2-4. These results indicated that compounds 1-4 from S. mukorossi activated caspase-3. A high caspase-3 activity was observed during the early apoptotic stage. The highest caspase-3 enzymatic activities obtained were 4.33 ± 0.48 μM pNA/2 h (with 100 μg/mL of 1), 5.09 ± 0.17 μM pNA/2 h (with 75 μg/mL of 3) and 3.47 ± 0.40 μM pNA/2 h (with 75 μg/mL of 4). Compound 2 had a lower induction of caspase-3 activity, consistent with the apoptotic rate described in the flow cytometry results.

Conclusions
Two new glycosides (1, 2) and two saponins (3, 4) were isolated from the fruits of S. mukorossi Gaertn. Compounds 1, 3 and 4 showed inhibition of A549 cell growth in a dose-dependent manner by promoting cell apoptosis via activation of caspase-3. Studies are in progress to elucidate the possible mechanism of action of these glycosides.