Mammillaria Species—Polyphenols Studies and Anti-Cancer, Anti-Oxidant, and Anti-Bacterial Activities

Discovering new natural resources of polyphenols is the aim of many recent studies in the field of natural product research. This study tentatively investigated the polyphenols profile of the stems of seven Mammillaria species (M. rhodantha, M. spinosissima, M. hahniana, M. crucigera, M. candida, M. albilanata, and M. muehlenpfordtii) using high performance liquid chromatography with DAD detector (HPLC-DAD) method. Furthermore, the anti-cancer, anti-oxidant, and anti-bacterial potentials of these extracts as well as major identified phenols were explored. The HPLC-DAD study confirmed the availability of six phenolic acids, including gentisic acid, chlorogenic acid, caffeic acid, protocatechuic acid, sinapic acid, and p-hydroxybenzoic acid. The dominant compounds were: gentisic acid in M. rhodantha and M. spinosissima; chlorogenic acid in M. muehlenpfordtii, M. crucigera, and M. rhodantha; and caffeic acid in M. rhodantha, M. crucigera, and M. spinosissima. Stems of Mammillaria sp. showed antiproliferative effects against HeLa, MCF-7, and Jurkat cells. In HeLa and MCF-7 cells, the best antiproliferative activities were found in the treatments with M. rhodantha, M. spinosissima, and M. muehlenpfordtii. The apoptotic assay of M. rhodantha, M. spinosissima, and M. muehlenpfordtii showed accumulation of necrotic cells in the early and late apoptotic phase. M. rhodantha, M. spinosissima, and M. muehlenpfordtii showed the highest anti-oxidant activities using 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene bleaching, and ferric reducing anti-oxidant power (FRAP) assays. M. rhodantha was the best source of antioxidants. Mammillaria sp. showed moderate anti-bacterial effects against bacteria and the highest effects were found using the extracts of M. rhodantha, M. spinosissima, M. crucigera and M. muehlenpfordtii against most bacteria. The anti-bacterial activities were attributed to other phenolic compounds (e.g., chlorogenic acid) than gentisic acid, which was not active against most bacteria. Mammillaria sp. could be considered to be an important natural source of phenolic acids with anti-cancer, anti-bacterial, and anti-oxidant activities.


Anti-Cancer Effects
Stems of Mammillaria sp. showed antiproliferative effects against selected cancer cells as shown in Table 2. The highest activities were against HeLa, MCF-7and Jurkat cells. In HeLa and MCF-7 cells, the best antiproliferative activities were found in the treatments with M. rhodantha, M. spinosissima,

Anti-Cancer Effects
Stems of Mammillaria sp. showed antiproliferative effects against selected cancer cells as shown in Table 2. The highest activities were against HeLa, MCF-7and Jurkat cells. In HeLa and MCF-7 cells, the best antiproliferative activities were found in the treatments with M. rhodantha, M. spinosissima, and M. muehlenpfordtii. The only anti-cancer activity against HT-29 was found in the extracts of M. rhodantha and M. spinosissima. The best anti-cancer effects against Jurkat were found in the treatments of M. rhodantha and M. spinosissima. Gentisic acid showed comparable activities to those of M. rhodantha and M. spinosissima. The apoptotic assay of M. rhodantha, M. spinosissima, and M. muehlenpfordtii showed accumulation of necrotic cells in the early and late apoptotic ( Figure 2).

Anti-oxidant Effects
Mammillaria sp. showed obvious anti-oxidant effects by several methods (Table 3). M. rhodantha, M. spinosissima, and M. muehlenpfordtii showed the highest anti-oxidant activities using DPPH, β-carotene bleaching, and ferric reducing anti-oxidant power (FRAP) assays compared to other species. M. rhodantha was best the source of antioxidants as revealed by lowest IC 50 . Furthermore, butylated hydroxytoluene (BHT) and Trolox showed higher activities than all stem extracts.
Values with different letters within a column indicates significant differences (p = 0.05). TEAC: Trolox equivalents anti-oxidant.

Anti-Bacterial Activities
Mammillaria sp. showed moderate anti-bacterial effects against all studied bacteria: Bacillus cereus, Listeria monocytogenes, Escherichia coli, Micrococcus flavus, Staphylococcus aureus, and Pseudomonas aeruginosa ( Table 4). The highest effects were found using the extracts of M. rhodantha, M. spinosissima, M. crucigera, and M. muehlenpfordtii against most bacteria. Other Mammillaria sp. showed much lower anti-bacterial effects. The gentisic acid did not show activities against most bacteria. The chlorogenic acid showed high anti-bacterial effects which is comparable to antibiotics.

Discussion
The seven Mammillaria species-M. rhodantha, M. spinosissima, M. hahniana, M. crucigera, M. candida, M. albilanata, and M. muehlenpfordtii-under HPLC-DAD qualitative and quantitative study showed that six phenols were present in all the species, which are gentisic acid, chlorogenic acid, caffeic acid, protocatechuic acid, sinapic acid, and p-hydroxybenzoic acid. This is the first report on the tentative phenolic composition of the stems of this genus. Indeed, further analyses with more chromatographic techniques would be recommended, for proper confirmation of detected compounds. Previous investigation revealed that the pollen of Mammillaria heyderi Sensu Lato contained quercetin, herbacetin glycoside derivatives, and kaempferol [22]. Under our study we did not detect quercetin or kaempferol in the studied steam extracts. The major phenol in M. rhodantha and M. spinosissima was gentisic acid. Gentisic acid was discovered in a few species such as Momordica charantia leaves (12.75 mg g −1 ) [23] and Vaccinium oxyccocos fruit (0.3 mg 100 g −1 ) [24] and is rare in general. Gentisic acid is a strong anti-oxidant and has important health benefits [25,26]. Chlorogenic acid is the ester of caffeic acid and is much more common than gentisic acid. Chlorogenic acid was isolated from different plants species such as Etlingera elatior leaves, coffee, and fruit [27] as well as medicinal plants [17]. The chlorogenic acid is known for strong its anti-oxidant, anti-cancer, anti-inflammatory, and antiviral properties, and modulates the anti-oxidant enzyme activities [28,29]. Protocatechuic acid, sinapic acid and p-hydroxybenzoic acid were relatively low but they are considered to be important anti-oxidant and anti-cancer compounds [30][31][32].
Mammillaria species showed antiproliferative effects against cancer cells. Most activities were against HeLa and MCF-7 cells using M. rhodantha, M. spinosissima, and M. muehlenpfordtii stem extracts. The three species are rich in specific phenolic compounds such gentisic acid, which is dominant in M. rhodantha and M. spinosissima, and chlorogenic acid, which is dominant in M. muehlenpfordtii. Gentisic acid showed comparable antiproliferative and cytotoxic activities to those of M. rhodantha and M. spinosissima. Gentisic acid is a quinonoid phenolic acid that is important for cancer prevention and treatment. These phenols activate the brain chemotherapeutics that help in the reduction of brain tumors [33] and has anti-oxidant activities related to the control of HCT-116 cancer cells [34]. In M. muehlenpfordtii there were strong antiproliferative activities against different cancer cells, which is explained by the activity of the major phenol found, which is chlorogenic acid. This phenol has anti-cancer activities on liver, colorectal, and laryngeal cancer. It influences the expression of specific genes such as GSK-3β and APC (up-regulation) and β-catenin (down-regulation) which promote the apoptosis of tumor cells [35]. The apoptotic activity of protocatechuic acid has been reported on human gastric carcinoma [36]. Sinapic and caffeic acids have been related to anti-cancer activities. Sinapic acid is considered to be a hydroxycinnamic acid derivative which up-and down-regulate specific genes in PC-3 and LNCaP prostate cancer cells [37]. Caffeic acid has anti-cancer activities against different cancer cells, such as human cervical cancer [38].
Mammillaria sp. had anti-oxidant activities as found in M. rhodantha, M. spinosissima, and M. muehlenpfordtii extracts. M. rhodantha was the best source of antioxidants in this study because it was rich in the strong antioxidants of gentisic acid, chlorogenic acid, caffeic acid, and protocatechoic acid. The four of these phenols have been associated with anti-oxidant activities as revealed in several studies [17,38,39]. Previous reports have found that gentisic acid provided protection to human erythrocytes against gamma radiation [25]. In another study, gentisic acid and protochatechuic acid imparted oxidative stability in sardine oil [32]. M. spinosissima is rich in gentisic acid and caffeic acid and both are strong antioxidants. M. muehlenpfordtii is rich in chlorogenic acid, which is responsible for anti-oxidant activities in this species. Previous investigation showed that chlorogenic acid can mitigate oxidative stresses in vitro and in vivo [39]. It was obvious that these dominant phenols were responsible for anti-oxidant activities.
Mammillaria sp. showed anti-bacterial effects against bacteria, and the highest effects were found in the extracts of M. rhodantha, M. spinosissima, M. crucigera and M. muehlenpfordtii against most bacteria. The gentisic acid did not show activities against most bacteria. Gentisic acid showed weak or no activity against E. coli, P. aeruginosa, and S. aureus in the previous study [40]. The anti-bacterial activities of M. rhodantha is mainly attributed to other major phenols including chlorogenic acid, caffeic acid, and protocatechoic acid. Chlorogenic acid has anti-bacterial activities against bacteria such as E. coli [41] as found in the current study. It increases plasma membrane permeability, causing loss of barrier function [42]. Caffeic acid has antimicrobial activities against several microorganisms including S. aureus [43]. In a previous investigation, protocatechoic acid showed strong anti-bacterial activities against E. coli, L. monocytogenes, S. aureus, and B. cereus [44]. Furthermore, it has anti-oxidant, anti-inflammatory, and anti-cancer activities [30]. It was obvious that the anti-bacterial activities of the extracts of M. rhodantha, M. spinosissima, M. crucigera, and M. muehlenpfordtii against most bacteria was attributed to the chlorogenic composition as well as other major phenols.

Plant Material and Preparation
The

Anti-Cancer Activities
Antiproliferative and cytotoxic effects of stem extracts were tested on MCF-7, HeLa, Jurkat, and HT-29, as well as HEK-293 (human normal cells) [1,2,16]. Extracts were solubilized in DMSO (1%). Vinblastine sulfate and taxol were considered positive controls. A microplate reader (Thermo, Waltham, MA, USA) was used at a 570 nm wavelength. The percentage of activity inhibition was calculated in triplicate: % Inhibition = (Abs. 570 nm control-Abs. 570 nm sample)/Abs. 570 nm control × 100. Furthermore, IC 50 values were obtained by plotting the percentage of cell viability against extract concentration and expressed in µg/mL. The IC 30 and IC 50 were determined in the apoptotic cell population (flow cytometry, FAC Scan, Becton Dickinson, Iowa, USA) following [1,15,16]. Control cells were untreated with extracts or methanol standards.

Anti-Oxidant Activity
DPPH, β-carotene bleaching [48] and FRAP [2,49] assays were used. For DPPH, the wavelength of 517 nm was used to determine the absorbance. During the β-carotene bleaching assay, the wavelength of 470 nm was used. Samples amount required to scavenged 50% of the DPPH/β-carotene bleaching solutions (IC 50 in µg/mL) was determined by plotting the inhibition percent against extract concentration. The butylated hydroxytoluene (BHT) was incorporated as control. The FRAP reagent was prepared as described in previous studies (e.g., [43]) using tripyridyl triazine (TPTZ, Sigma-Aldrich, Berlin, Germany). Aliquots (100 µL) of bark extracts or Trolox (Sigma-Aldrich, Berlin, Germany) were added to FRAP reagent (3 mL), mixed, incubated for half an hour at 37 • C and the absorbance was measured at 593 nm. Aqueous solutions of known serial concentrations of Trolox (0-0.5 Mmol/L) were used for the calibration. Two sets of triplicate replications were conducted for all experiments.  [17,50]. Microtiter plates (96-well) containing a serial concentration of the stem extracts (0.05-200 mg mL −1 ) in each well mixed with bacterial inoculum (1.0 × 10 4 CFU per well) in 100 µL tryptic soy broth were incubated at 37 • C for 24 h in a rotary shaker. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of the stem extract that exhibited no visible growth using a binocular microscope and was determined following the incubation period of the microtiter plates. The minimum bactericide concentration (MBC), which was defined as the lowest concentration that caused no visible growth and indicated the killing of 99.5% of the inoculum, was determined using serial sub-culturing of the stem extract or standard. The optical density was determined at a wavelength of 655 nm. A positive control was used (streptomycin, 0.01-10 mg/mL), as well as a negative one (DMSO, 1%). The optical density was determined at a wavelength of 655 nm. A positive control was used (streptomycin, 0.01-10 mg/mL), as well as a negative one (DMSO, 1%).

Statistical Analyses
The SPSS (Version 21.0) was used to determine least significance difference (LSD). The standard deviation (SD) of means of three replicates was used.

Conclusions
This is the first report exploring seven Mammillaria species giving information about phenolic acid composition, as well as anti-cancer, anti-oxidant, and anti-bacterial activities. In all studied species, gentisic, chlorogenic, caffeic, protocatechuic, sinapic, and p-hydroxybenzoic acids were estimated qualitatively and quantitatively. The dominant compounds were gentisic acid in M. rhodantha and