Anticancer and Antibacterial Activity of Gallic Acid from Caesalpinia mimosoides Lamk

Cancer and diarrheal diseases are major causes of mortality in developing countries (WHO, 2012). There are limitations to the standard treatments for cancer, especially when treating patients in the advanced stages of the disease (Khan et al., 2002; Thongprasert, 2005). Bacterial foodborne illnesses are also a common problem worldwide, including those caused by Salmonella spp. and Plesiomonas shigelloides. Many antibiotics have been extensively used, leading to increases in drug-resistant bacteria (Collignon 2009; Abatcha et al., 2014). Therefore, alternative therapeutic agents with anticancer and antibacterial activities are needed. Bioactive compounds from plant phytochemicals could be a potential source for effective drugs as well as food supplements and nutraceuticals. Caesalpinia mimosoides Lamk (C. mimosoides Lamk) is an Asian vegetable; the young shoots and leaves are consumed, indicating that plant compounds could be safely used as therapeutics. Extensive experimental works over the past decade have identified beneficial effects of C. mimosoides Lamk related to its antimicrobial activities (Chanwitheesuk et al., 2007; Daduang et al., 2011), antioxidant activities


Introduction
Cancer and diarrheal diseases are major causes of mortality in developing countries (WHO, 2012). There are limitations to the standard treatments for cancer, especially when treating patients in the advanced stages of the disease (Khan et al., 2002;Thongprasert, 2005). Bacterial foodborne illnesses are also a common problem worldwide, including those caused by Salmonella spp. and Plesiomonas shigelloides. Many antibiotics have been extensively used, leading to increases in drug-resistant bacteria (Collignon 2009;Abatcha et al., 2014). Therefore, alternative therapeutic agents with anticancer and antibacterial activities are needed. Bioactive compounds from plant phytochemicals could be a potential source for effective drugs as well as food supplements and nutraceuticals. Caesalpinia mimosoides Lamk (C. mimosoides Lamk) is an Asian vegetable; the young shoots and leaves are consumed, indicating that plant compounds could be safely used as therapeutics. Extensive experimental works over the past decade have identified beneficial effects of C. mimosoides Lamk related to its antimicrobial activities (Chanwitheesuk et al., 2007;Daduang et al., 2011), antioxidant activities RESEARCH ARTICLE

Chemicals and reagents
Hexane, ethyl acetate, and methanol were purchased from S.C. Science Co., Ltd. (Thailand). Silica gel 60 and thin layer silica gel 60 F254 were purchased from Merck (Germany). Dulbecco's modified Eagle's medium-high glucose (DMEM-HG), fetal bovine serum, penicillinstreptomycin, and trypsin-EDTA were obtained from Gibco BRL (Grand Island, NY). Catechin, quercetin, ferulic acid, rutin, caffeine, gallic acid and neutral red were obtained from Sigma-Aldrich Co. LLC. The other chemicals used were all reagent grade.

Plant material and extraction
Shoot and leaves of C. mimosoides Lamk were collected from the Udon Thani province in January 2014. The taxonomic identity was confirmed by Prof. Dr. Arunrat Chaveerach, Department of Biology, Faculty of Science, Khon Kaen University. Samples were washed with tap water and air-dried at 50ºC. C. mimosoides Lamk powder (0.95 kg) was obtained by crushing the plants with an electric blender. Then, it was extracted by incubation in 4 liters of ethyl acetate (EtOAc) at room temperature for 24 hr. This process was repeated for 24 hr, and the solvents were combined. Removal of the solvent from the extract was performed under reduced pressure by a rotary evaporator (BUCHI, Model R240). Finally, 29.61 g of crude EtOAc extract was stored in desiccators for further purification.

Isolation of crude ethyl acetate extract
The crude EtOAc extract was separated by silica gel column chromatography. Silica gel (silica gel F254 size 0.063 mm) was packed into a glass column (8 cm x 60 cm) and 29.61 g of crude EtOAc extract was applied to the column. The phytochemical compounds were separated by gradually increasing the polarity of the eluting solvents: hexane, hexane:EtOAc, EtOAc, EtOAc:methanol (MeOH) and MeOH, respectively. Two column volumes of each eluate were collected and characterized by thin layer chromatography (TLC). TLC was carried out by spotting the sample on a silica gel 60 F254 precoated alumina sheet (Merck, Cat. No. MC1057190001) and using the same mobile phase system as described above. Spots were visualized under ultraviolet (UV) light at 254 and 365 nm. The chemical reactivity of these fractions was also tested by oxidizing with p-anisaldehyde, and 5 fractions, named E1-E5, showed positive results. The 30.10 mg of the selected fraction E4 was re-separated on a silica gel column and eluted with the same eluting system described above. The subfraction number E4.4.5 was obtained and recrystallized with EtOAc for characterization.

Characterization by high performance liquid chromatography (HPLC)
High performance liquid chromatography (HPLC) was used into characterize the compounds from fraction E4.4.5. Twenty microliters of the tested fraction was injected on a C18-Symmetry column, size 15 cm x 3.0 mm, 5 µm (Waters). The compounds were separated using a gradient elution of MeOH and 0.5% phosphoric acid. The gradient was run with 5%, 70% and 90% MeOH from 0-17 min, 17-18 min and 18-20 min, respectively, at a flow rate of 1.5 ml/min. Gallic acid, catechin, caffeine, ferulic acid, rutin, and quercetin were used as standard compounds by measuring the absorbance at 270 nm (Waters, UV 2479). The peaks were analyzed by the Clarity program (Waters).
A white solid obtained from fraction E4.4.5 was further characterized by 1 H and 13 C nuclear magnetic resonance spectroscopy (NMR) (Varian NMR-400 MHz Spectrometer).

Cancer cell cultures
Human cholangiocarcinoma (CCA) cell lines M213 and M214 were obtained from Prof. Banchob Sripa, Department of Pathology, Faculty of Medicine, Khon Kaen University. They were cultured in RPMI 1640 medium supplemented with 10% FBS and 1% penicillinstreptomycin (Sigma-Aldrich) at 37°C in a CO 2 incubator (95% relative humidity, 5% CO 2 ). Cell lines were trypsinized with 1X trypsin-EDTA when they reached confluence.
Cell viability and IC 50 determination A neutral red (NR) assay modified from Borenfreund and Puerner (1984) was used to test for cell viability. Briefly, cells were detached from the culture flask using 1X trypsin-EDTA. Then, RPMI supplemented with 10% FBS was added. The cells were counted using a hemocytometer, and approximately 6,000 cells were seeded per well in a 96-well plate. The cells were cultured in complete RPMI 1640 medium at 37°C in a CO 2 incubator for 24 hr. All the cell lines were treated with varying concentrations (30,50,70,90,110, 120 150 and 170 µM) of nGA and incubated for an additional 24 hr. NR was added to the test cells for 3 hr. Cell viability was detected at 540 nm using an RT-2100c microplate reader (Rayto, China). In parallel, cGA was also tested to compare cell growth inhibition to nGA.

Morphological changes
The morphological changes of M213 and M214 cells treated with IC 50 values of cGA for 24 hr were observed by staining with 5 mg/ml of 4',6-diamidino-2-phenylindole (DAPI) for 10 min and analyzing under a fluorescence microscope.

Antimicrobial activity test
The gallic acid from C. mimosoides Lamk was then evaluated for its antibacterial activity. The 2 human pathogenic bacterial strains tested were Salmonella spp. (serogroup B) and Plesiomonas shigelloides. They were obtained from the Department of Clinical Microbiology at the Faculty of Associated Medical Sciences, Khon Kaen University.
The minimum inhibition of concentration (MIC) of nGA and cGA was investigated using the disc diffusion method (Bauer et al.,1966). For each species, 0.5 McFarland inoculums were swabbed onto nutrient agar plates; then, 20 µL nGA and cGA at concentrations of 10-140 mM was added to the 6 mm diameter sterile paper discs on dried plates and placed on the test plate. After incubation at 37°C for 16-24 hr, the diameters of the clear zones that appeared were measured; 10% dimethylsulfoxide (DMSO) was the negative control, and 10 µg ampicillin was the positive control. The lowest concentration of the tested compound that showed no visible growth was reported as the MIC. All experiments were conducted in triplicate. The percent inhibition was calculated as shown in the following formula.
%Inhibition= (zone of sample test-zone of negative control) (zone of positive control) x 100

Scanning electron microscopy (SEM)
SEM was performed following the method described in Uawonggul et al. (2007), with slight modifications.
Asian Pacific Journal of Cancer Prevention, Vol 17, 2016 1343 DOI:http://dx.doi.org/10.7314/APJCP.2016.17.3.1341 Anticancer and Antibacterial Activity of Gallic Acid from Caesalpinia mimosoides Lamk Briefly, bacterial strains were cultured in nutrient broth and harvested at the logarithmic phase of growth, washed twice with phosphate buffered saline (pH 7.0), and re-suspended to yield a final concentration of 10 6 CFU/ml. Aliquots of the bacterial suspension (100 µL) were individually incubated with the previously determined MIC of cGA at 110 mM for Plesiomonas shigelloides and 117 mM for Salmonella spp. at 37°C for 1 and 2 hr, respectively. The treated bacteria (150 µL) were fixed with equal volumes of 2.5% glutaraldehyde (Sigma-Aldrich Co. LLC) in a 0.1 M phosphate buffer, pH 7.2, for 2 hr and pipetted onto 0.2 µM polycarbonate membrane filters (Whatman, Germany) for 5 min. The materials were dehydrated in graded ethanol concentrations (30, 50, 70 and 90%) for 15 min each and then coated with gold palladium by a sputter coater (Polaron, SC7620, England) and examined with a scanning electron microscope (LEO Electron Microscopy Ltd., LEO1450VP, England) operated at 12 to 20 kV. For the negative control, all procedures were performed in a similar manner, except that the bacterial cells were incubated with a PBS buffer instead of cGA.

Growth inhibition of CCA cells by gallic acid from C. mimosoides Lamk
The effects of nGA compared to cGA on 2 cell lines were tested. The results showed that the IC 50 value of nGA was similar to cGA in all cells tested (Table 1). The IC 50 values of nGA when treating M213 and M214 were 120 and 124 µM, respectively. The IC 50 values of cGA when treating M213 and M214 were 119 and 147 µM, respectively. Treatment of all cells with nGA and cGA resulted in similar marked suppression of cell proliferation in a dose-dependent manner. The morphological alterations of M213 and M214 cells treated with cGA were observed with a fluorescence microscope. In the fixed DAPI-stained cells, smaller and more brightly stained nuclei were observed, indicating cell death by apoptosis (Figure 3).

Antibacterial activities of gallic acid from C. mimosoides Lamk against foodborne bacteria
The MIC of nGA for the bacterial species Salmonella spp. (serogroup B) and Plesiomonas shigelloides was    (Table 2). SEM analysis showed that the bacterial membrane was wrinkled following treatment with 120 mM cGA to Salmonella spp. (serogroup B) ( Figure 4A) and 110 mM cGA to Plesiomonas shigelloides ( Figure 4B) for 1 and 2 hr, respectively.
M213 and M214 cells were treated with cGA to assess its anticancer activity. The fixed cells were DAPI stained, and smaller, brighter nuclei were observed with a fluorescence microscope, indicating that the cells had undergone apoptosis (Figure 3). This result is similar to those of several studies reporting that gallic acid is an anticancer agent that significantly inhibits cell proliferation and induces apoptosis. Faried et al. (2007) reported that gallic acid induced apoptosis in esophageal cancer cells (TE-2) by upregulating the pro-apoptotic protein Bax and downregulating the anti-apoptotic proteins Bcl-2 and Xiap and the Akt/mTOR survival pathway. You et al. (2010) found similar results using gallic acid and HeLa cervical cancer cells. Chandramohan et al., (2012) reported that gallic acid induced apoptosis in a chronic myeloid leukemia cell line (K562) through the death receptor and mitochondrial-mediated pathways via inhibition of BCR/ ABL kinase, NF-κB and COX-2. Daduang et al. (2015) found that caspases 3/7, 8, and 9 were significantly more active in HeLa cells treated with gallic acid, indicating the induction of apoptosis via intrinsic and extrinsic pathways. However, SiHa and C33A cells only showed increased caspase 8 levels, revealing that gallic acid induced apoptosis via the intrinsic pathway in these cell lines. Moreover, Rattanata et al. (2015) used Fourier transform infrared spectroscopy (FTIR) analysis and found a lipid signal (2948, 2835, 2915, and 2848 cm-1) that was a strong marker of apoptosis in cholangiocarcinoma cells (M213) treated with gallic acid.
The antibacterial activity of nGA against 2 gramnegative pathogenic bacterial strains was compared to treatment with cGA. The MIC of gallic acid from these two different sources was the same, suggesting that nGA affected the bacterial cell membrane in the same manner as cGA. The primary action of phenolic acids on the cell membrane has been supported by several published works. Ramos-Nino et al. (1996) found that the antilisterial activity of phenolic acids was dependent on the lipophilicity and degree of ionization of the molecules. Other groups also found the phenolic acid activity against Listeria monocytogenes to be dependent on pH, which is consistent with the passive diffusion of phenolic acids through the cell membrane (Kouassi and Shelef, 1998;Wen et al., 2003). Moreno et al. (2006) reported that phenolic compounds can penetrate a cell due to changes in its membrane permeability. Altogether, the results indicate that most of the phenolic acids (particularly those from C. mimosoides Lamk) alter bacterial size and shape, and this was observed in both gram-negative and gram-positive bacteria (Daduang et al, 2011).
In conclusion, this study demonstrates the bioactivity of extracts from C. mimosoides Lamk. We successfully isolated an active substance, gallic acid, using an ethyl acetate extraction. The nGA exhibited both anticancer activity and antimicrobial activities with similar IC 50 and MIC values to cGA. Our results suggest that gallic acid may be a potential anticancer and antibacterial agent; however, in depth in vivo studies are needed to elucidate the exact mechanism.