Antimicrobial Activity, Growth Inhibition of Human Tumour Cell Lines, and Phytochemical Characterization of the Hydromethanolic Extract Obtained from Sapindus saponaria L. Aerial Parts

The hydromethanolic extract of Sapindus saponaria L. aerial parts was investigated for antimicrobial activity (against several Gram-positive and Gram-negative bacteria and fungi) and capacity to inhibit the growth of different human tumor cell lines as also nontumor liver cells. The evaluated extract was further characterized in terms of phytochemicals using UV, 1H-NMR, 13C-NMR, and MS spectroscopic tools. The extract has shown a significant antimicrobial activity on all tested bacterial and fungal species. The best activity was achieved against Bacillus cereus and Staphylococcus aureus among bacteria and against all three Penicillium species tested. It also revealed cytotoxicity against human colon (HCT-15), cervical (HeLa), breast (MCF-7), and lung (NCI-H460) carcinoma cell lines, with HeLa being the most susceptible tumor cell line. The extract was not toxic for nontumor liver cells. Chromatographic separation of the extract resulted in the isolation and identification of stigmasterol, oleanolic acid, luteolin, luteolin 8-C-β-glucoside (orientin), luteolin 6-C-β-glucoside (isoorientin), luteolin 7-O-β-glucuronide, and rutin. The results of the present findings may be useful for the discovery of novel antitumor and antimicrobial agents from plant origin.


Introduction
Plants are considered as one of the main sources of biologically active compounds. In spite of the recent domination of the synthetic chemistry as a method to discover and produce drugs, the potential of bioactive plants or their extracts to provide new and novel products for disease treatment and prevention is still enormous [1].
The discovery of antibiotics has decreased the spread and severity of a wide variety of diseases. However, and as a result of their uncontrolled use, the efficiency of many antibiotics is being threatened by the emergence of microbial resistance to existing chemotherapeutic agents [2]. While bioactive natural compounds have been isolated mainly from cultivable microbial strains, untapped biologically active metabolites of different resources including plants remain to be investigated to alleviate or help responding to current health care situations [3]. Plant-derived natural products represent an attractive source of antimicrobial agents since they are natural, have manageable side effects, and are available at affordable prices [4]. Furthermore, those natural products may have different mechanisms from conventional drugs and could be of clinical importance in health care improvement [5].
Cancer is characterized by uncontrolled growth and spread of abnormal cells [6]. For many years, scientists were searching for chemically synthesized compounds with For the extract preparation, 1.2 kg of air dried powder of S. saponaria aerial parts was extracted with methanol : water 80 : 20 (v/v) at room temperature several times until exhaustion. The extract was concentrated under reduced pressure to give 68 g of crude extract.

Evaluation of Antimicrobial Activity of the Hydromethanolic Extract
Antibacterial Activity. . Cells were routinely maintained as adherent cell cultures in RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 2 mM glutamine (MCF-7, NCI-H460, and HCT-15) or in DMEM supplemented with 10% FBS, 2 mM glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin (HeLa and HepG2 cells), at 37 ∘ C, in a humidified air incubator containing 5% CO 2 . Each cell line was plated at an appropriate density (7.5 × 10 3 cells/well for MCF-7, NCI-H460, and HCT-15 or 1.0 × 10 4 cells/well for HeLa and HepG2) in 96-well plates and allowed to be attached for 24 h. Sulforhodamine B assay was performed according to a procedure previously described by the authors [17]. Briefly, cells were then treated for 48 h with various extract concentrations. Following this incubation period, the adherent cells were fixed by adding 10% cold trichloroacetic acid (TCA, 100 L) and incubated for 60 min at 4 ∘ C. Plates were then washed with deionized water and dried; sulforhodamine B solution (0.1% in 1% acetic acid, 100 L) was then added to each plate well and incubated for 30 min at room temperature. Unbound SRB was removed by washing with 1% acetic acid. Plates were air dried, the bound SRB was solubilised with 10 mM Tris (200 L) and the absorbance was measured at 540 nm in the microplate reader mentioned above. For hepatotoxicity evaluation, a cell culture was prepared from a freshly harvested porcine liver obtained from a local slaughter house, according to a procedure established by the authors [17]; it was designed as PLP2. Cultivation of the cells was continued with direct monitoring every two to three days using a phase contrast microscope. Before confluence was reached, cells were subcultured and plated in 96-well plates at a density of 1.0 × 10 4 cells/well and cultivated in DMEM medium with 10% FBS, 100 U/mL penicillin, and 100 g/mL streptomycin. Ellipticine was used as positive control (0.24-65.2 g/mL).
Three independent experiments were performed in triplicate, and the results were expressed as mean values ± standard deviation (SD).

Results and Discussion
3.1. Antimicrobial Activity. The results of antibacterial activity of hydromethanolic extract of S. saponaria are presented in Table 1 and  species tested than both mycotics. Also, the tested extract exhibited higher fungicidal activity against P. ochrochloron and P. verrucosum than ketoconazole. According to the existing literature, the investigation of Sapindus saponaria extracts is limited, but there is more information about isolated compounds. Extracts from the dried pericarp of S. saponaria fruits were investigated for their antifungal activity against clinical isolates of yeasts Candida albicans and . non-albicans from vaginal secretions of women with vulvovaginal candidiasis. From all tested extracts the n-BuOH and one of its fractions showed strong activity against all Candida isolates tested [10]. The acetylated saponin 3-b-O-[a-L-rhamnopyranosyl-(1-3)-b-Dglucopyranosyl] hederagenin was detected by Lemos et al. [18] who observed antimicrobial activity against Pseudomonas aeruginosa, Bacillus subtilis, and Cryptococcus neoformans. Triterpenoid saponins, with hederagenin or oleanolic acid as aglycone, have been found to possess antifungal activity against C. glabrata, C. albicans, Trichosporon beigelii, Penicillium avelaneum UC-4376, Pyricularia oryzae, Cryptococcus neoformans, Coccidioides immitis, and Saccharomyces cerevisiae as well as against the dermatophytes Microsporum canis and Trichophyton mentagrophytes [19,20]. Triterpene acids (ursolic and oleanolic) isolated from Miconia species showed a significant antibacterial activity against some bacterial strains [21]. Luteolin showed selectively antibacterial activity against MRSA and methicillin-sensitive S. aureus strains with MIC, 3.9 to 15.6 and 62.5 to 125 g/mL, respectively [22]. Also luteolin 7--glucosides possess antibacterial activity [23], and rutin has been shown to exhibit antimicrobial activity [24].
From previous results it could be noticed that all flavonoid aglycones (myricetin, quercetin, and luteolin) showed better antibacterial and antifungal activities than other flavonoid glycosides [25,26]. Luteolin exhibited relatively higher activities than the other compounds and was more effective against fungi than against bacteria [27,28]. These authors presented that certain features relating to flavonoid structure and antimicrobial activity can be identified. The active flavonoids were polyhydroxylated (myricetin, datiscetin, quercetin, luteolin, and kaempferol), except for flavone which does not contain any hydroxyl group. These active flavonoids have in common the obligatory C-4 keto group and hydroxyl group substitutions at C-3, C-5, and C-7 and have at least one hydroxyl group on ring B. These observations indicate that the more hydrophilic flavonols or flavones are better inhibitors than less hydrophilic ones. These observations suggest that the hydroxyl group at C-3 is required for activity. This requirement, however, is not necessary for the flavone skeleton, as in the case of luteolin. Antimicrobial activity of total extract (although low compared to others) may be due to the presence of some aglycones, unstable flavonoid glycosides, or some other bioactive secondary metabolites. This is in agreement with the literature that plant extracts generally contain flavonoids in glycosidic form. This may be the reason why the plant extract did not produce as marked inhibition as some fractionated extracts or as many of the pure compounds [29].

Growth Inhibition of Human Tumor Cell Lines.
The hydromethanolic extract of S. saponaria revealed cytotoxicity against human colon, cervical, breast, and lung carcinoma cell lines (  tested tumor cell lines. The extract up to 800 g/mL did not show capacity to inhibit the growth of HepG2 cell line (hepatocellular carcinoma). Moreover, the hydromethanolic extract of S. saponaria, up to 800 g/mL, was not toxic for nontumor liver cells. Ellipticine was used as positive control, but the comparison with the extract results should be avoided because it is an individual/purified compound and not a mixture like the extract (in the crude extract the concentration of each individual bioactive compound is certainly much lower and in the range of the pure compound). Furthermore,  There is no valuable data about previous investigation of this plant extracts. Some of the compounds isolated from this extract were already tested for some biological activity. Flavonoids are generally regarded to have a wide range of pharmacological activity (antioxidant, anti-inflammatory, antimicrobial, antiviral, and antitumor); among these compounds, apigenin and luteolin have been confirmed to have antitumor activity [22]. Stigmasterol, a constituent isolated from Bacopa monnieri Linn aerial parts, showed therapeutic efficacy against Ehrlich ascites carcinoma in mice [30]. Oleanolic acid is a pentacyclic triterpenoid widely distributed in nature and possessing various important bioactivities, such as antitumor and hepatoprotection [31].
The findings revealed that the antimicrobial and antitumor properties of the hydromethanolic extract of S. saponaria provide preliminary scientific validation for the traditional medicinal use of this plant as a potential phytotherapeutic agent in certain diseases and for the control of bacteria and fungi in the environment. However, the extracts and active compound isolated from S. saponaria should be further studied in animal models in order to evaluate their in vitro efficacy and toxicity.   light that changed to violet colour on spraying with vanillin sulphuric and heating in an oven at 110 ∘ C for 5 min. NMR spectral data has shown signals very close to that reported by Yinusa et al. [32] for stigmasterol. Compound (2) was identified as oleanolic acid: white amorphous powder.  (4) and (5) showed deep purple spots under UV light which changed to yellow with ammonia vapor indicating that they are flavones with free 5-OH and 4 -OH [35]. Complete acid hydrolysis of the two compounds revealed that no sugars were detected meaning that they remained without change in addition to the appearance of an additional spot on the chromatogram (Wessely-Moser rearrangement between C-6 and C-8), which may be due to acid isomerization [36], indicating that the compounds are mono-C-glycosides. Thus, the compounds were subjected to ferric chloride degradation and cochromatographed with authentic sugars samples, where glucose was detected. UV spectral data and NMR signals for the two compounds are very similar to the ones reported for orientin and isoorientin, respectively [37,38].