Antimicrobial and Antiproliferative Activities of Chia ( Salvia hispanica L.) Seeds

: The genus Salvia L. (Lamiaceae) has been traditionally used for the treatment of various illnesses since ancient times. Salvia hispanica L., commonly known as Chia, is an annual herbaceous plant which was one of the most significant crops for pre-Columbian civilizations (Aztec and Maya) in America. Nutritional potential and beneficial effects of Chia seeds on human health have been previously reported. Therefore, this study aims to investigate anti(myco)bacterial, antifungal, and antiproliferative activities of Chia seeds. Ethanol extract of Chia seeds were tested against Staphylococcus aureus (ATCC 25925), Bacillus subtilis (ATCC 6633), Escherichia coli (ATCC 25923), Acinetobacter baumannii (ATCC 02026), Aeromonas hydrophila (ATCC 95080), Candida albicans (ATCC 14053), Candida tropicalis (ATCC 1369), and Candida glabrata (ATCC 15126) using broth microdilution method. Antimycobacterial activity was performed against Mycobacterium tuberculosis H37Rv using resazurin microtiter plate method. Ampicillin, Ethambutol, Isoniazid, and Fluconazole were chosen as reference drugs. Antiproliferative effect of the various concentrations (200, 100, 50, and 25 μg/mL) of ethanol extract was tested against A549 human lung cancer cell lines using MTT method. Ethanol extract was found to be more effective against A. baumannii (MIC: 62.5 µg/mL) than reference drug Ampicillin (MIC: 125 µg/mL). There was a correlation between increased doses and antiproliferative activity of extract against A549 human lung cancer cell lines ( p <0.05).


INTRODUCTION
Intake of dietary phytochemicals has been played important roles in the prevention of various illnesses including cancer, inflammatory, and cardiovascular diseases. Due to their medicinal properties plants and their metabolites are also used in different industries [1]. Salvia L. (sage) is the most species-rich genus of the family Lamiaceae (mint family) with approximately 1000 species [2]. It has been reported since ancient times that Salvia species have been traditionally used in the treatment of tuberculosis, bronchitis, and microbial infections [3]. Some species of the genus have been used worldwide on account of their beneficial effects on human health and nutritional properties [4]. Salvia hispanica L., commonly named as Chia, is an annual herbaceous plant which is native to northern Guatemala and southern Mexico and is also cultivated in some countries including Mexico, Bolivia, Australia, Argentina, Colombia, Peru, and Guatemala [4]. In the recent years, Chia seeds which were one of the most significant crops for pre-Columbian civilizations (Aztec and Maya) in America [4,5], have been used in the food, animal feed, medical, cosmetics, and pharmaceutical industries [6]. Chia seeds have important roles as nutritional supplement and functional food. Moreover, seeds contain no toxic components and gluten, thus making Chia seeds a safe ingredient also gluten free diets [4].
According to the literature, antiproliferative activity of Chia seeds was studied against some cancer cell lines [7,8]; however, we didn't reach any available literature on antiproliferative effect of Chia seeds against A549 human lung cancer cell lines. Additionally, antimicrobial activity of Chia seeds has been investigated in few studies [9,10]. But some factors such as geographical origin and extraction procedure were changed composition of bioactive compounds in seeds. The consumption of Chia seeds has been increasing over the years due to their health benefits and uses in cooking [4]. Therefore, in this study, we aimed to investigate in vitro anti(myco)bacterial, antifungal, and antiproliferative activities of Bolivian Chia seeds.

Plant Material and Extraction Procedure
Commercially available Chia seeds from Bolivia (2019 harvest) were purchased from a local market. Powdered seeds were extracted twice with ethanol (20 mL solvent per 1 g seed; 96%) by stirring overnight at room temperature then filtered using Whatman Grade No.1 filter paper. Solvent was evaporated via a vacuum evaporator (Heidolph Instruments, Germany) and obtained extract was kept in the dark at 4 °C.

Antimicrobial Activity
Gram

Antibacterial Activity
Antibacterial activity was studied using a broth microdilution method [11]. Ampicillin was used as a reference drug. Sample was dissolved in DMSO for preparing initial concentration (2000 μg/mL). The mixture was used for having stock solution which was diluted in Mueller-Hinton broth. Further dilutions of reference drug and extract were prepared (1000-0.9 μg/mL). Standard strain working suspensions were made in sterile tubes. Turbidity adjusted to match McFarland standard No: 0.5. Further dilutions (1:20) of suspensions were prepared in distilled water and added to each plate (10 µL). Thus each plate's bacterial concentration was adjusted to 5x10 5 CFU/mL. Effect of DMSO was tested. The minimal inhibitory concentration (MIC) values were determined in duplicate tests.

Antimycobacterial Activity
Antimycobacterial activity was tested using the resazurin microtiter assay [11]. Isoniazid and Ethambutol were used as reference drugs. Resazurin reagent was prepared using Resazurin sodium salt powder. Middlebrook 7H9 broth containing 0.1% casitone, 0.5% glycerol, and 10% oleic acid-albumin-dextrose-catalase and 7H9-S medium were used for preparing culture medium. A resazurin working solution (0.01% (w/v)) made in distilled water and stock solutions (1000 μg/mL) of extract and reference drugs prepared in DMSO were filtered through 0.22 μm membrane filter (Ministar, Goettingen, Germany). A two-fold dilution series were performed using 7H9-S medium (100 μL) in a 96-well microtiter plate. 0.12-250 μg/mL concentration ranges were detected. A growth control and a sterility control were added to each plate. The bacterial inoculum was prepared in a tube which was containing 7H9-S medium (5 mL) via resuspending a loopful of Lowenstein-Jensen culture medium. During 2 min the tube was mixed then waited to allow sediment. After supernatant was added in sterile tube, the turbidity adjusted to match McFarland standard No: 1. 7H9-S medium was used to prepare dilutions (1:20) of these suspensions. Plates were inoculated with diluted suspension (100 μL) then put into plastic bags. After incubation period (37 °C, 7 days) Resazurin working solution (30 μL) was added to each well then plates were incubated (37 °C, 24 h) and results were visually recorded. The lowest concentration that prevents complete color change of resazurin from blue to pink was determined as MIC value. Experiments were done in duplicate.

Antifungal Activity
Antifungal activity was studied using a broth microdilution method of NCCLS [12]'s standard document (M27-A2) with minor modifications [11]. RPMI 1640 medium which buffered to pH 7.0 with 0.165 M 3-(N-morpholino) propanesulfonic acid was used. Fluconazole was used as a reference drug. Working suspensions of standard strains were made as a 1:100 dilution followed by a 1:20 dilution of the stock suspensions using RPMI 1640 medium. Stock solutions (1000 μg/mL) of extract and reference drug dissolved in DMSO were filtered through membrane filters. Two-fold dilution series were added in a 96-well microtiter plate using RPMI 1640 medium (100 μL). 250-0.12 μg/mL concentration ranges were tested. A growth control and a sterility control were added to each plate. 100 μL of working inoculum suspension was added to each plate and plates were incubated (48 h, 35 °C). MIC values were visually determined in duplicate tests.

Antiproliferative Activity
Determination of cell viability was studied by MTT method. A549 human lung cancer cell lines were procured from ATCC (American Type Culture Collection, VA, USA). DMEM which was supplemented with FCS (10%) was used for cell cultivation. Cells were kept in suitable culture conditions (95% air; 5% CO2; 37 °C). After reaching 70-80% confluency cells were detached with Trypsin-EDTA solution (3.0 mL) and settled to 96-well plates (10 4 cells per well). After 24 h, various concentrations (200, 100, 50, and 25 μg/mL) of ethanol extract dissolved in DMSO were applied and cells were incubated (24 h). Cells treated with growth medium containing no FCS were used as a control. After incubation, supernatants were replaced with MTT (1 mg/mL) dissolved in growth medium then incubated (37 °C) until purple precipitate was visually detected. The supernatants were removed; cells which absorbed MTT were dissolved in DMSO. Plates were detected using a spectrophotometer (Epoch, Winooski, USA) at a 550 nm. Effect of DMSO was tested. Experiments were done in four replicates [13].

Statistical Analysis
SPSS 25.0 (IBM, NY, USA) was used for statistical analyses. The data are provided as the mean ± SD. Kruskal Wallis H and one-way analysis of variance (ANOVA) with Tukey's post hoc test were used. P values < 0.05 were considered as significant.

RESULTS and DISCUSSION
In the present study antimicrobial and antiproliferative activities of ethanol extract (yield 20.67% (w/w)) of Chia seeds were evaluated. Antimicrobial activity results are provided in Table 1. When compared to reference drug Ampicillin (MIC: 125 µg/mL) seed extract had greater activity against A. baumannii (MIC: 62.5 µg/mL). Extract showed antimycobacterial activity against M. tuberculosis H37Rv (MIC: 62.5 µg/mL); however, the efficiency of the extract was not found as strong as Isoniazid and Ethambutol (MIC values: 0.97 µg/mL and 1.95 µg/mL, respectively). Extract showed the highest antifungal activity against C. glabrata; but result was not found as high as Fluconazole (MIC values: 31.25 µg/mL and 3.90 µg/mL, respectively). Antimicrobial effect of Chia seeds was investigated against several microorganisms including E. coli, A. baumanii, S. aureus, and C. albicans; and aqueous and aqueous-ethanol extracts exhibited antimicrobial activity against E. coli [10]. Despite these results, protein hydrolysates of Chia seeds were not showed antimicrobial activity against E. coli, Salmonella typhi, Shigella flexneri, Klebsiella pneumoniae, S. aureus, B. subtilis, and Streptococcus agalactiae [9]. In our study, seeds exhibited antibacterial activity against both tested gramnegative and gram-positive bacterial strains. According to the literature, composition and concentration of bioactive compounds in Chia seeds vary depending on some factors like geographical origin, climatic conditions, agricultural practices, and extraction procedures [4]. These factors also change the effectiveness of the tested extract. This may explain different results in our study and in previous studies. Due to their hydrophilic cell wall structure which contains lipopolysaccharides inhibits accumulation of hydrophobic oils and extracts, and penetrations of these substances through the target cell membrane, gram-negative bacteria are more resistant against natural components than gram-positive bacteria [14]. When compared to Ampicillin, seeds showed greater activity against gram-negative nosocomial pathogen A.
baumannii which is one of the important healthcare problems worldwide because of its ability to gain resistance to all classes of antimicrobial agents used against it [15]. According to the our results, Chia seeds might be promising sources in the development of novel therapeutic agents against infections caused by A. baumannii.
Antiproliferative activity results are shown in Table 2. Significantly lower cell viability levels were observed in 100 μg/mL concentration applied group than control group (0.963 ± 0.036 and 1.092 ± 0.012, respectively) and in 200 μg/mL concentration applied group than control and DMSO groups (0.936 ± 0.036; 1.092 ± 0.012; and 1.085 ± 0.009, respectively) (p <0.05). However, there were no significantly differences found between the other groups (p >0.05). Chia oil reduced tumor growth, metastasis, and cell mitosis in neoplastic tissue and increased apoptosis. However; in Walker 256 model Chia flour supplementation didn't prevent tumor bearing effects [8]. Effects of mucilage compounds on some cancer lines (HeLa, HCT-15, HCT-116, MCF7, MDA-MB-231, MCF7/Vin, MCF7/Vin + , MCF7/Vincells, Vero, and HepG2 cells) were evaluated and significant inhibition on proliferation of MCF7, HeLa, and HepG2 cells with low toxicity were determined [7]. However, we didn't reach any available literature on antiproliferative effect of Chia seeds against A549 human lung cancer cell lines. The lung cancer which is the most common type of malignant tumors with high mortality is expected to cause over than 3 million deaths for the year 2035 [16]. A549 human lung cancer cell lines have been widely studied for cancer research since 1976 [17]. In our study, ethanol extract of Chia seeds were found to be more effective against A549 human lung cancer cell lines at a dose of 200 µg/mL and there was a correlation between increased doses and activity.

CONCLUSION
The consumption of Chia seeds has been increasing over the years and its health benefits related to chronic diseases like cardiovascular diseases, obesity, cancer, and diabetes. In the current study, we investigated some biological properties of Bolivian Chia seeds. Chia seeds might be promising sources in the development of novel therapeutic agents against A. baumannii. Antiproliferative effect of Chia seeds against A549 human lung cancer cell lines was determined for the first time. In the future, this study may be the basis for further studies on the effects of seeds against lung cancer and in addition to its nutritional potential this might be a new topic to support Chia consumption.

Declaration of Conflicting Interests and Ethics
The authors declare no conflict of interest. This research study complies with research publishing ethics. The scientific and legal responsibility for manuscripts published in IJSM belongs to the author(s).