|Year : 2014 | Volume
| Issue : 1 | Page : 45-49
Lack of in vitro anticancer and antimicrobial activities in Suaeda maritima (seablite) crude extracts
Department of Applied Sciences, Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok 10300, Thailand
|Date of Web Publication||16-Jul-2014|
Faculty of Science and Technology, Suan Sunandha Rajabhat University, Bangkok 10300
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Aims: The aim was to determine antimicrobial and anticancer activities for water and ethanol extracts of leaves and shooting parts of seablite. Materials and Methods: Anticancer activity tests of seablite extracts were performed by resazurin microplate assay (REMA) and tested with 3 cell lines including KB cell line, epidermoid carcinoma of the oral cavity (American Type Culture Collection [ATCC] CCL-17), MCF-7 cell line, breast adenocarcinoma (ATCC HTB-22) and NCI-H187 cell line, and small cell lung carcinoma (ATCC CRL-5804). Antimicrobial activities of seablite water and ethanol extracts against herpes simplex virus type-I (HSV-1) and Mycobacterium tuberculosis H 37 Ra strain were tested according by green fluorescent protein-based assay; Candida albicans (ATCC 90028) and Bacillus cereus (ATCC 11778 or TISTR 687) were tested according by REMA; and Plasmodium falciparum, K1 strain was tested according by micro-culture radioisotope techniques. Results: Both extracts were not possessed anticancer activity to KB-oral cavity cancer, MCF7-breast cancer and NCI-H187-small lung cancer cell lines at maximum concentration = 50 μg/mL. In the same way to anticancer activity assays, both extracts were not inhibited HSV-1, P. falciparum K1 strain, M. tuberculosis H 37 Ra strain and B. cereus at the same concentration. Conclusions: Ethanol and water extracts of leaves and shooting parts of seablite had no significant anticancer and antimicrobial activities.
Keywords: Anticancer, antimicrobial, seablite, Suaeda maritima
|How to cite this article:|
Sudjaroen Y. Lack of in vitro anticancer and antimicrobial activities in Suaeda maritima (seablite) crude extracts. J Pharm Negative Results 2014;5:45-9
|How to cite this URL:|
Sudjaroen Y. Lack of in vitro anticancer and antimicrobial activities in Suaeda maritima (seablite) crude extracts. J Pharm Negative Results [serial online] 2014 [cited 2018 Jan 24];5:45-9. Available from: http://www.pnrjournal.com/text.asp?2014/5/1/45/136796
| Introduction|| |
Seablite grows in alkaline soil, mostly found in swamp area or mangrove forest. Seablite's young leaves are both cooked and eaten fresh, but sometimes local people scald them to dilute saltiness.  Seablite leaves are part of households in Samut Songkram. Not only in Thailand, are seablites also popular in the Southern part of India as pickled seablite in vinegar. Seablite can be cooked in curry, and be the food of farm animals. ,
Sudjaroen  had determined nutritive values of seablites, which have high calcium, beta-carotene and fiber with have low carbohydrate and fat. Thus, it was very interesting to be introduced as healthy organic cuisine or be modified for other usage. Besides, seablites leaves can prevent hepatitis , and can be antiviral. ,, Its biological activity is related to having triterpenoids and sterols.
Patra et al.  were determined the antioxidant activity of seablite's leaf and stem extracted by organic solvent (acetone, ethanol and methanol) and water. Later, all extracts were determined total antioxidant capacity, total phenolic content, ascorbic acid content, 2, 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, metal chelating, nitric oxide scavenging, and reducing power. Furthermore, the extracts were possessed antimicrobial activity against Gram-positive and Gram-negative pathogenic bacteria by agar well diffusion and micro dilution technique to find minimal inhibiting concentration (MIC) and minimal bactericidal concentration (MBC) of seablite extracts in each solvent. The finding was seablite extract from leaves and stems in all solvent can perform 70-92% of antioxidant from total antioxidant capacity, total phenolic content, and DPPH radical scavenging. From other ways to test the antioxidant of seablite, the result was also satisfying. The result after testing the antimicrobial activities showed that seablite acetone extract was inhibited four types of pathogenic bacteria: Vibrio cholera, Staphylococcus epidermidis, Bacillus subtilis and Escherichia More Details coli. However, seablite ethanol extract was inhibit only three types of pathogenic bacteria including Pseudomonas aeruginosa, S. epidermidis and E. coli.
Nonetheless, the bioactivity of seablite from literature in this research was studied on seablite from India. Thai seablite may or may not have the same activities with Indian seablite. Furthermore, there were few more in vitro antimicrobial activities that need to be tested, e.g. antituberculosis, antimalarial (Plasmodium falciparum), antiviral (herpes simplex type-I), antifungal (Candida albicans), antibacterial (Bacillus cereus) and in vitro screening for anticancer activity test against cell lines also interesting. Beneficial biological activities of edible parts needs to be claim and confirm their activities are corresponding to previous studies.
| Materials and methods|| |
In this study, Ampawa Agricultural Office, Samut Songkram, Thailand provided help to collect the sample seablite. The air dried seablite was grinded and kept for screening biological test. The sample must be dried by hot air and ground. Bring 121.47 g of ground seablite for continuous extraction, then, extract with ethanol and water using Soxhlet apparatus. Finally, get the solvent evaporated through rotary evaporation apparatus under vacuum. The extract could also be dissolved in dimethyl sulfoxide (DMSO) and be the test to the anticancer and antimicrobial activities onward.
Total phenolic content
In this step, 0.1 mL of 1 mg sample extract was input into the test tube, mixing with 4.6 mL distilled water and 1 mL of Folin-Ciocalteu reagent. After that, the extract was left inside the room in room temperature for 3 min. Next, 3 mL of 2% Na 2 CO 3 (w/v) was filled into the tube, and shaken with the speed of 150 rpm for 2 h. Later, the extract was measured to find out the light absorbance at 760 nm by comparing with the gallic acid at the intensity of 1, 0.875, 0.75, 0.625, 0.5, 0.375, 0.25, and 0.125 mg/mL. The total phenolic content was calculated into mg of gallic acid per g of the extract  .
Anticancer activity test
Three cell lines, including KB cell line, epidermoid carcinoma of the oral cavity (American Type Culture Collection [ATCC] CCL-17), MCF-7 cell line, breast adenocarcinoma (ATCC HTB-22) and NCI-H187, small cell lung carcinoma (ATCC CRL-5804) were used in this study. The resazurin microplate assay (REMA) developed by O'Brien et al.  was performed for anticancer test. In brief, the cells were cultured in proper condition and diluted by culture medium at 2.2 × 10 4 cells/mL for KB cell and 3.3 × 10 4 cells/mL for MCF-7 and NCI-H187 cell line. The next step was to add the 5% DMSO 50 μL into cell suspension 45 μL in the 384-well plates. Then, the extract was incubated at 37°C in the incubator which contained 5% of CO 2 . After incubation (KB and MCF-7 took 3 days, NCI-H187 took 5 days), 12.5 μL resazurin in 62.5 μg/mL was added. The incubation was continued for 4 h, then measured fluorescence signal by SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) at excitation and emission wavelength of 530 and 590 nm, respectively. Dose response curve could be done in the 6 th test. 3-fold serial intensity dilution and the intensity of the cell-restraint extract 50% (IC 50 ) could be calculated by SOFTMax Pro software (Molecular Devices, USA). Ellipticine, doxorubicin and tamoxifen was used as a positive control. 0.5% DMSO and water were used as a negative control.
Antimicrobial activity test
Anti-herpes simplex virus type-I test
Before the test of antivirus activity, there should be the cytotoxicity test conducted first to make sure that the extract is noncytotoxic. The antivirus test was conducted by green fluorescent protein (GFP)-based assay.  The extracts diluted by 10% DMSO at 10 μL/well were added into 96-well plate. Next, added GFP-expressing Vero cell suspension 1 × 10 5 cells/mL mixed with herpes simplex virus type-I (HSV-1) (ATCC VR260) 5 × 10 5 PFU/mL for 190 μL/well. Then the sample was incubated at 37°C by incubator which has 5% of CO 2 for 4 days. After that, fluorescence signal was measured by SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) at excitation and emission wavelength 485 and 535 nm, respectively (bottom-reading mode). Fluorescence signal from the 4 th day of incubation will be deducted on the 1 st day (day = 0) of incubation. The IC 50 was calculated by SOFTMax Pro software (Molecular Devices, USA) from testing 6 levels of 2-fold serial dilution extracts. Acyclovir was used as a positive control and 0.5% DMSO was used as a negative control.
Anti-Candida albicans test
The test was performed by taking C. albicans yeast (ATCC 90028) to culture on potato dextrose agar plate at 30°C for 3 days. After that, 3-5 colony of yeast were taken to culture in shaking flask that had RPMI-1640 medium until the density was 5 × 10 5 CFU/mL. Next, the yeast cell suspension was brought to be tested in antiyeast activity by REMA.  45 μ of cell suspension and 5 μL extracts from each density diluted by 0.5% DMSO were added into 384-well plate. The plate was cultured at 37°C for 4 days, then 10 μL/well of 62.5 μg/mL resazurin solvent were added and incubated for another 30 min. After that, fluorescence signal was measured by SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) at excitation and emission wavelength 530 and 590 nm, respectively. The IC 50 was calculated by SOFTMax Pro software (Molecular Devices, USA) from testing 6 levels of 2-fold serial dilution extracts. Amphotericin B was used as a positive control and 0.5% DMSO was used as a negative control.
Antimalarial activity test
Plasmodium falciparum (K1, multi drug resistant strain) was cultured in the test tube (in vitro) developed by Trager and Jensen method.  It was cultured by RPMI 1640 medium which had 20 mM of N-2-hydroxyethylpiperazine-N'- 2-ethanesulfonic acid, 32 mM of NaHCO 3 and 10% of heat inactivated human serum with 3% of erythrocyte mixed together. Then, it was incubated at 37°C by 3% CO 2 in CO 2 incubator. The culture medium and erythrocyte were changed every day during the test.
The evaluation of in vitro antimalarial test was performed by micro-culture radioisotope techniques.  200 μl mixture which has 1.5% of erythrocyte infected by 1% of malaria (1% parasitemia). In early ring stage, it was mixed with 25 μl of medium that mixed with sample extract in each density distilled by 1% DMSO (the total was 0.1% DMSO). After that, the sample was incubated for 24 h. After that, 25 μL of [ 3 H] hypoxanthine (Amersham, USA) would be added into medium (0.5 μCi) in each plate, and incubated again for another 24 h. Radioactive labeled on hypoxanthine indicates the growth of cell. Top Count microplate scintillation counter (Packard International Inc., USA) was used to find out the radioactive volume. 50% inhibition concentration (IC 50 ) could tell that the cell development was reduced to 50%. This experiment used 1 and 10 μg/mL extract to prevent P. falciparum to calculated the IC 50 . Dihydroartemisinine and 0.1% DMSO was used as positive control and negative control, respectively.
Green fluorescent protein expressing Mycobacterium tuberculosis H 37 Ra strain (H 37 Ra gfp) culture was developed by Changsen et al.  and Collins et al.  H 37 Ra gfp was cultured on plate 7H10 agar consisting of kanamycin 30 μg/mL. The incubation at 37°C was 4 weeks long. After that, the single colony of the cell was taken to culture on 7H9 broth which had 0.2% of glycerol v/v, 0.1% of casitone w/v, 0.05% of tween 80 v/v, 10% of middle brook oleic albumin dextrose catalase enrichment solution (BD Biosciences) v/v and 30 μg/mL of kanamycin. All substances were incubated at 37°C in 200 rpm shaker incubator until the 550 nm optical density was around 0.5-1. For batch cultivation, 1/10 of the ingredient above was taken to incubate at the same condition. Then the cells were cleansed and suspended by PBS buffer, and then were sonicated 8 times (15 s/time). The cultures were divided into tubes, and kept at −80°C for 2-3 months before experiment session.
During test session, the cells were tested their density in 384-well plate at around 1 × 10 5 CFU/mL/well. The tests took 4 times (quadruplicate), or within 4 wells/test. Each testing plate was contained 5 μL of 0.5% DMSO (diluted by serial dilution) and 45 μL cell suspension. The plate was incubated at 37°C for 10 days. To find out the fluorescence signal, I used SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) with excitation and emission wavelength 485 and 535 nm, respectively (bottom-reading mode). The fluorescence signal on 10 th incubation day were deducted on = 0 of incubation. It could be calculated in MIC by rifampicin, ofloxacin, streptomycin, isoniazid and ethambutol as positive control and 0.5% DMSO as negative control.
Antibacterial activity test against Bacillus cereus
Bacillus cereus ATCC 11778 (TISTR 687) in this research was given by ATCC. It was cultured by streaking the virus on to medium called Tryptic Soy Agar. The culturing lasted 1-day incubation at 37°C. Next, the single colony were cultured in Tryptic Soy Broth (TSB) for 5 mL, and incubated at 37°C in 200 rpm shaker incubator for 30 min. (OD 600 ~ 0.1) Activated B. cereus culture were diluted for 200 times. The 20 mL TSB medium were incubated at 37°C in 200 rpm shaker incubator for another 3 h (OD 600 ~ 0.4-0.5). After that, the test to find out antimicrobial activity continued by adding 7.5 μL extract, 25 μL of resazurin 0.25 mM and bacterial cell suspension until the final volume. Also, 75 μL (~15,000 cells) in Mueller Hinton broth was put onto 384-well plate. The sample was taken to incubate at 37°C for 2 h, following by measuring signal by SpectraMax M5 multidetection microplate reader (Molecular Devices, USA) at excitation and emission wavelength of 530 and 590 nm.  Vancomycin was used as positive control and 0.5% DMSO used as negative control.
| Results|| |
It was found that seablite extracted with water (SW) contained crude extract more than the seablite extracted with ethanol (SE) at yield = 19.65 and 9.34% respectively, which was related with the total phenolic content of SW, which was higher than that of SE at 14.47 and 6.93 mg of gallic acid equivalent/g extract. Water and ethanol extracts were not possessed anticancer activity to KB-oral cavity cancer, MCF7-breast cancer and NCI-H187-small lung cancer cell lines at maximum concentration = 50 μg/mL [Table 1]. In the same way to anticancer activities, both extracts were not significantly inhibit HSV-1, C. albicans, P. falciparum, K1 strain, M. tuberculosis H 37 Ra strain and B. cereus at maximum concentration of test (50 μg/mL) [Table 2].
|Table 1: Cytotoxic effect of SE and SW against KB, MCF7 and NCI-H187 cell linesd |
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|Table 2: Antimicrobial of SE and SW against HSV-1, Mycobacterium tuberculosis, Candida albicans, Bacillus cereus and Plasmodium falciparumf|
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| Discussion|| |
Our recent study was reported that water and ethanol extracts of seablite contain phenolic compounds with antioxidant activity by scavenging DPPH and 2, 2-azinobis (3-ethyl-benzothiazoline-6-sulfonic acid) radicals.  Polyphenolic compounds may the major bioactive components in seablite, which are responsible for antioxidation and antiproliferation. Natural antioxidants have been proved to inhibit tumor growth selectively, because of different redox status between normal cells and cancer cell.  Moreover, seablite contains antioxidant and might contain phenolic substances that can inhibit pathogens as well.
The three flavonol glycosides including, quercetin 3-O-α- l-rhamnopyranosyl- β-d-galactopyranoside-7-O-β-d-glucopyranosyl-glucopyranoside, kaempferol-3-O-α-l-rhamnopyranosyl-β-d-galactopyranoside-7-O-β-d- glucopyranosyl-glucopyranoside, and kaempferol 3-O-α-l-rhamnopyranosyl-β-d-galactopyranoside-7-O-(2″″'- O-trans-feruloyl)-β-d-glucopyranosyl-β-d-glucopyranoside, known compounds, namely, quercetin and kaempferol, methyl cis, trans-ferulate, and methyl trans-ferulate, which were isolated from 70% seablite methanol extract. All of isolated compounds were showed cytotoxic activity against the human tumor cell lines MCF7, HCT116, and HepG2.  However, our results were contrasted by lack of anticancer activities of seablite extracts against KB, MCF7 and NCI-H187 cell lines. Especially, MCF7 cell line was presented different results, may due to variation of plant, method of extraction, concentration, and solubility of extract and type of assay.
In case of antimicrobial activities, this study was showed negative results of seablite extracts along with various pathogens including, HSV-1, C. albicans, P. falciparum, K1 strain, M. tuberculosis H 37 Ra strain and B. cereus at preferable concentration (50 μg/mL). When compared this finding with previous study  which reported that ethanol extract of seablite were inhibited three types of bacteria including P. aeruginosa, S. epidermidis and E. coli and the MIC and MBC of all seablite extract were around 2.5-5.0 mg/mL, which could be implied that seablite may has activity when increase the concentration of seablite extract from μg/ml to mg/mg level. However, at this concentration (mg/ml) was difficult to dissolving in all in vitro assays (in presented of DMSO) and it mean that both seablite extracts were not possessed anticancer and antimicrobial activities at preferable concentration. The 50 μg/mL of plant extract was the common maximum concentration used to "cut-off" for the significant of biological activity, which was referred by the National Centre for Genetic Engineering and Biotechnology, Thailand. This study will be helpful to avoid any study repeated in this direction in the future.
| Conclusion|| |
Ethanol and water extracts of leaves and shooting parts of seablite had no significant anticancer activities against to KB, MCF7, and NCI-H187 cell lines, which tested resazurin by microplate assay. Both extracts had no significant antimicrobial activities against to HSV-1 and M. tuberculosis H 37 Ra strain were tested according by GFP-based assay; C. albicans (ATCC 90028) and B. cereus (ATCC 11778 or TISTR 687) were tested according by REMA; and P. falciparum, K1 strain was tested according by micro-culture radioisotope techniques.
| Acknowledgments|| |
The researcher would like to express their gratitude to the Office of Higher Education, Ministry of Education, Thailand, and Research and Development Institute of Suan Sunandha Rajabhat University, Bangkok, Thailand for the funding support. I am grateful to Faculty of Science and Technology, Saun Sunandha Rajabhat University, National Center for Genetic Engineering and Biotechnology for research facility support. I would like to sincerely thank all staffs of Microbiology Laboratory Section, Division of Clinical Pathology, National Cancer Institute, Bangkok for support antimicrobial tests.
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[Table 1], [Table 2]