Anti-microbial and Anti-cancer Properties of Echinocystic Acid Extracted from Luffa cylindrica
Received Date: Jan 18, 2018 / Accepted Date: Feb 05, 2018 / Published Date: Feb 12, 2018
Triterpenoids are the most ubiquitous non-steroidal secondary metabolites in terrestrial and marine flora and fauna occurring in the free form as well as in the forms of ether, ester and glycosides. The diverse structural types of triterpenoids with useful biological activities, identification, biosynthesis for feed industry increases interest in triterpenoids. The widely occurring plant Luffa cylindrica was investigated for active triterpenoid sapogenins. The triterpenoid sapogenins were isolated by solvent extraction followed by chromatography and were defined by newer spectroscopic methods. These triterpenoid sapogenins from Luffa cylindrica were isolated and characterized. The anti-microbial and anti-cancer properties of echinocystic acid will be discussed.
Keywords: Luffa cylindrica; Anti-microbial activity; Anti-cancer activity; Disc Diffusion assay; MTT assay
Triterpene of diverse structural types are widely distributed in terrestrial and marine sediments, prokaryotes as well as eukaryotes. The triterpenoids have a range of unique and potentially useable biological effects. The most important triterpenoid structures are oleanane, ursane, lupane and dammarane-euphane triterpenoids on biological point of view. Luffa cylindrica (Cucurbitaceae) is a traditionally important medicinal plant. It is widely distributed in tropical and subtropical areas throughout India. It is used in clinical problems relating to child birth. The chemical constituents such as sapogenins, saponins and proteins of Luffa cylindrica used as potentially effective clinical agent in health care in many communities reported [1-4]. The saponins and sapogenins isolated from the plant have the same oleanane type skeleton. The two major sapogenins of the plants are mainly oleanolic acid (1) and echinocystic acid (2) . Its seed and fruits are used as a vegetable either prepared like squash or eaten like cucumbers. Khajuria et. al  reported the immunomodulatory effects of two oleanane sapogenins, oleanolic acid (1) and echinocystic acid (2) in mice isolated from Luffa cylindrica. In this study, the anti-microbial and anti-cancer properties of oleanane triterpenoid sapogenin, echinocystic acid (2) will be discussed.
Materials and Methods
Seeds of Luffa cylindrica were collected from Jammu, India and identified in the Botany Department of Regional Research of Laboratory (Formerly RRL, CSIR-IIIM), Jammu. The voucher specimen is deposited in the herbarium of the institute. Dulbecco’s Modified Eagle’s Medium (DMEM), Roswell Park Memorial Institute (RPMI)-1640 culture media, fetal bovine serum (FBS), penicillin-streptomycin antibiotic solution, phytohemagglutinin (PHA-M) and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from HiMedia (Mumbai, India). Heparin and trypan blue were purchased from SRL (India); Histopaque, were procured from Sigma-Aldrich (St Louis, MO, USA).
The methanolic extract (60 g) of the defatted powdered seeds of L. cylindrica was hydrolysed with aqueous MeOH-HCl under reflux for 4 h. The acid hydrolysate was separated into acidic and neutral fractions by treatment with a saturated solution of NaHCO3. The acidic fractions were separately subjected to column chromatography on silica gel using n-Hexane-CHCl3 in various ratios 9:1, 3:1, 1:1 and CHCl3- MeOH (24:1) and similar fractions so obtained were further purified by preparative TLC (CHCl3-Pyridine-H2O 9:0.5:0.5) followed by crystallization. Thus, two sapogenins, 1 (950 mg) and 2 (200 mg) were obtained. The two sapogenols were identified by IR, NMR and ESIMS. The two polar sapogenins are oleanolic acid (1) mp. 308°C-310°C and echinocystic acid (2), mp. 300°C-305°C. The echinocystic acid (2) was dissolved in non-toxic organic solvent dimethyl sulphoxide (DMSO) and stored at 4°C until use.
The standard bacterial strains used as test organisms were obtained from Microbial Type Culture Collection (MTCC) from Institute of Microbial Technology, Chandigarh, India. They are belonged to the Gram Positive and Gram-negative category. The Gram-positive food borne pathogens are Bacillus subtilis (MTCC121), Staphylococcus aureus (MTCC96) and Listeria monocytogenes (MTCC657). Escherichia coli (MTCC1667) and Pseudomonas aeruginosa (MTCC741) are Gram negative bacteria. Salmonella typhimurium (MTCC98) is most predominating food spoilage human pathogenic Gram-negative bacteria.
Anti-bacterial activity screening
The Anti-bacterial screening of echinocystic acid (2) was determined by Disc Diffusion method of Bauer et al.  against some Gram positive and Gram negative pathogenic bacterial strains. This study was performed using 2500 μg/ml, 250 μg/ml, 25 μg/ml and 2.5 μg/ml of echinocystic acid (2). The inhibition zones were obtained at 250 μg/ml and values were found to be at one fourth of 250 μg/ml of oleanolic acid (1). Ciprofloxacin was used as positive controls.
Anti-microbial mode of action of Echinocystic acid (1) on Gram positive and Gram negative bacterial cells by counting colony forming units (CFUs)
Echinocystic acid (2) once applied to actively growing liquid culture (exponential phase of growth) of Listeria monocytogenes and Salmonella typhimurium and their colony forming units (CFU) were counted for next 24 h along with untreated control.
The echinocystic acid (2) at its MIC was treated on actively growing culture (8 h grown) and allowed to grow further till 24 hrs. In every two hours interval aliquot of culture (both treated and untreated) was withdrawn aseptically diluted serially with sterilized distilled water and plated into nutrient agar medium, incubated at 37°C for another 24 h. The colonies appeared were counted per ml of culture broth [7-11].
Antifungal activity screening
The antifungal screening of echinocystic acid (2) was determined by same Disc Diffusion method of Bauer et al.  against Candida albicans MTCC3018. Fluconazole was used as standard.
MCF7 and MDA-MB-231 breast cancer cell lines were procured from the National Centre for Cell Science (Pune, India). Cell culture was done according to the description of Banerjee et al. . Briefly, cells were grown in DMEM, supplemented with 1% penicillin– streptomycin and 10% (v/v) FBS solution at 37°C in a humidified CO2 incubator (5% CO2) (Thermo Fisher Scientific, Waltham, MA, USA).
Cells (1 × 104) were seeded per well of a 96-well plate and incubated in standard culture condition for 24 h. After that, complete DMEM media was removed and cells were replenished with incomplete media (without FBS) and then treated with different concentrations of echinocystic acid (2). Both negative and positive controls were used along with the treated groups. Treated cells were incubated at 37°C in the presence of 5% humidified CO2 for 48 h, and the proliferation rates were estimated by MTT assay at 595 nm using an ELISA reader (Bio Rad, USA). The percentage of viable cells was calculated taking viability of untreated cells as 100%.
Isolation of human peripheral blood mononuclear cells; culture and MTT assay
Fresh blood was collected from five healthy non-smokers, nonalcoholic male donors (21–25 years of age) with written informed consents by venipuncture into heparinised falcon tubes. Blood was collected by pathologists employed at the university hospital, under the supervision of medical doctors. All studies were performed complying fully with the approved “Ethical Guidelines for Biomedical Research on Human Subjects” formulated by the Indian Council of Medical Research, India. The work was reviewed and approved by the Institutional Ethics Committee for Human Research of Visva-Bharati University. Human peripheral blood mono nuclear cells (HPBMC) were isolated according to the method of Bøyum  with minor modification. Blood was layered over equal amount of Histopaque and centrifuged at 1000 × g for 30 min. The buffy coat was aspirated into 3-5 ml PBS and centrifuged at 1000 × g for 10 min, and the washing process was repeated thrice. The pellet was resuspended in RPMI-1640 media (1 × 106 cells/ml) and viability was checked using the trypan blue dye exclusion method.
Cell viability was found to be >95%, which indicated successful isolation. Isolated HPBLs (0.5 ml) were stimulated by PHA and cultured in 5 ml of RPMI-1640 media with 10% FBS along with antibiotics. After 24 h, cells were treated for 48 h with respective IC50 doses of echinocystic acid (2) in MCF7 and MDA MB 231 cell lines and proliferation rates were estimated by MTT assay at 595 nm using a ELISA reader (Bio Rad, USA). Percentage of viable cells was calculated taking viability of untreated cells as 100%.
Results were expressed as mean ± standard error (SE). Statistical analysis of the data was performed by paired t-test, and p<0.05 were considered significant.
Results And Discussion
In the Disc Diffusion study, the anti-microbial activity of echinocystic acid (2) was evaluated against all the Gram-positive Bacillus subtilis, Listeria monocytogenes , Staphylococcus aureus and Gram-negative Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa bacteria (Table 1) [8-11]. It showed significant anti-microbial potential against the Gram-positive Bacillus subtilis, Listeria monocytogenes , Staphylococcus aureus and Gram-negative Salmonella typhimurium bacteria (Figure 1). The echinocystic acid (2) could kill all the tested bacteria at 250 μg/ml and maximum zones of inhibition were obtained using highest concentration (2500 μg/ml) suggests that Echinocystic acid (2) killed the Gram-positive food borne pathogens Bacillus subtilis, Listeria monocytogenes , human pathogenic Staphylococcus aureus and one Gram-negative Salmonella typhimurium bacterium. MIC of echinocystic acid (2) obtained against all the tested bacteria was 62.2 μg/ ml. A clear cut bacteriostatic pattern of growth for both the strains, the Gram-positive Listeria monocytogenes and Gram-negative Salmonella typhimurium was found in the anti-microbial mode of action study (Table 2). Figures 2 and 3 clearly indicated static type of mode of action upon treatment with echinocystic acid (2) at its MIC. Echinocystic acid (2) has moderate anticandidal activity against Candida albicans (Table 3). With regard to the structure-activity relationship it was suggested that both the hydroxyl and carboxyl groups are important for their Anti-bacterial activity of echinocystic acid (2). MTT assay results showed that echinocystic acid (2) inhibited the cell viability of human breast cancer cell lines (Figure 4). Both MCF7 and MDA-MB 231 cell lines showed dose dependent inhibition of cell growth after treatment with echinocystic acid (2). Significant inhibitory effect was observed at the dose of 60 μM. Calculated IC50 value for MCF7 cell line was 41.72 μM and for MDA-MB 231 cell line was 48.17 μM. Isolated HPBMCs were treated with both these IC50 for 48 h. 96.29% of cell growth inhibition was observed at 41.72 μM which was 84.34% at the dose of 48.17 μM. This shows that echinocystic acid (2) is not cytotoxic to normal cells.
|Bacterial strains||Echinocystic acid (2)|
|Bacillus subtilisMTCC 121 Gram (+) ve||+++|
|Listeria monocytogenesMTCC 657Gram (+) ve||+++|
|Staphylococcus aureusMTCC 96Gram (+) ve||+++|
|Escherichia coliMTCC 1667 Gram (-) ve||-|
|Salmonella typhimuriumMTCC 98 Gram (-) ve||+++|
|Pseudomonas aeruginosaMTCC 741 Gram (-) ve||-|
|+++, inhibition zone diameter more than 20 mm; -, no inhibition|
Table 1: Anti-bacterial screening of Echinocystic acid (2) against six Gram-positive and Gram-negative bacteria.
|Log CFU count at different hours||Control
|Treated on actively growing culture (8 hrs culture)||Control
|Treated on actively growing culture (8 hrs culture)|
Table 2: Mode of action of Echinocystic acid (2) on Gram positive and Gram negative bacterial cells by counting colony forming units (CFUs).
|Fungal species||Echinocystic acid (2)||Echinocystic acid (2)||Fluconazole|
|MIC100 and MIC80: Concentration of echinocystic acid (2) that caused 100% and 80% reduction of the growth control respectively.|
Table 3: Minimum inhibitory concentration (MIC) in μg/ml of Echinocystic acid (2).
Due to its potential anti-microbial properties echinocystic acid (2) can be considered as natural preservatives in food processing industry to reduce the microbial growth in processed food products. Echinocystic acid has potential anti-cancer activity on human breast cancer cell lines and it can be used in human health care. We are pursuing further studies of anti-microbial and anti-cancer activities of the two major oleanane sapogenins, oleanolic acid (1) and echinocystic acid (2) of the plant to provide a basis for discussion of their activity in relation to their chemical structures. This is the first report of anti-microbial and anti-cancer properties of echinocystic acid of Luffa cylindrica.
Financial support from CSIR, New Delhi is gratefully acknowledged.
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Citation: Garai S, Ghosh R, Bandopadhyay PP, Mondal NC, Chattopadhyay A (2018) Anti-microbial and Anti-cancer Properties of Echinocystic Acid Extracted from Luffa cylindrica. J Food Process Technol 9:717. DOI: 10.4172/2157-7110.1000717
Copyright: ©2018 Garai S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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