Isolation of methyl gamma linolenate from spirulina platensis using flash chromatography and its apoptosis inducing effect
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Isolation of methyl gamma linolenate from Spirulina platensis using flash chromatography and its apoptosis inducing effect against human lung carcinoma A- 549 cell lines.
Gamma linolenic acid is an important omega-6 polyunsaturated fatty acid (PUFA) of medicinal interest was isolated from microalgae Spirulina platensis using flash chromatography system (Isolera system) as its methyl ester. The isolated methyl gamma linolenate was characterized by IR, 1H NMR, 13C NMR and mass spectral analysis and the data were consistent with the structure.
The percentage yield of isolated methyl gamma linolenate is found to be 71 % w/w, which is a very good yield in comparison to other conventional methods. It was subjected to in-vitro cytotoxic screening on A-549 lung cancer cell lines using SRB assay and result was compared with standard rutin.
It may be concluded that the Flash chromatography system plays a major role in improving the yield for theisolation of methyl gamma linoleate from Spirulina platensis and the isolated molecule is a potent cytotoxicagent towards human lung carcinoma cell lines, however it may be further taken up for an extensive study.
KeywordsSpirulina platensis Lung cancer Gamma linolenic acid (GLA) Cytotoxicity Flash chromatography
Gamma linolenic acid
Fatty acid methyl esters
Methyl, gamma linolenate
Spirulina is a commercial well known micro algae, which contains various antioxidants, especially phycocyanin. Apart from being sold as a nutraceutical, Spirulina is incorporated as a functional ingredient in food products and beverages. Spirulina refers to the dried biomass of Arthrospira platensis, belonging to the family Phormidiaceae, an oxygenic photosynthetic bacterium found worldwide in fresh and marine waters . This algae represents an important staple diet in humans and has been used as a source of protein and vitamin supplement in humans without any significant side-effects. Apart from the high (up to 70 %) content of protein, it also contains vitamins, especially B12 and provitamin A (β-carotenes) and minerals especially iron. It is also rich in phenolic acids, tocopherols and gamma linolenic acid (GLA) [2, 3].
Many commercial methods are available for producing gamma linolenic acid (GLA) concentrates include winterization, fractional distillation, urea-inclusion, high performance liquid chromatography and argentated silica gel chromatography [4, 5, 6, 7]. Even though GLA concentrates mentioned above are sufficient for most applications; however pharmaceutical applications of GLA require higher concentrations of GLA, often in excess of 90 % but many of these methods did not produce the satisfied output . Hence new method for producing higher concentrations of gamma linolenic acid and its ester forms from Spirulina platensis are urgently needed. Lung cancer is one of the leading causes of cancer deaths (15 %) in the world and is by far the most common cancer in the western world. Globally lung cancer is responsible for more deaths annually than those due to breast cancer, colorectal cancer, and prostate cancer combined together [9, 10, 11]. As per the National Institute of Health Analysis (NIH) report, the medical expenditure for the diagnosis and treatment of cancer could be as high as $ 207 billion, out of which $ 12 billion could be only for the treatment of lung cancer .
A number of investigations have demonstrated that, a variety of modified fatty acid analogues are promising molecules in cancer prevention and have potential in the treatment of cancer [13, 14]. Some studies have shown that GLA can slow or stop the growth of some types of cancer cells in tissue cultures in the laboratory. The same kinds of studies suggest that GLA may help some cancer drugs to work better [15, 16, 17, 18]. However, there is very little evidence as yet that GLA work to prevent or treat cancers in humans. Human studies are under way to evaluate the role of GLA and other essential fatty acids on the growth of cancer. As existing anticancer drugs of synthetic origin are having more adverse effects, the developments of safe compounds are urgently needed. In recent years, more importance has been given to nutraceuticals/functional foods because they can provide physiological benefits additional to nutritional and energetic, as for instance antihypertensive and anticancer activities. Since the gamma linolenic acid has come under the category of Nutraceuticals/functional foods, its exploration in the treatment of lung cancer may be considered as safe. Considering all these facts, the present research is aimed to produce the high concentrates of methyl gamma linolenate from Spirulina platensis using flash chromatography as a molecular target towards treatment of lung cancer and the isolated product is characterized by using HPTLC, NMR and mass spectra. The characterized molecule is subjected to in-vitro cytotoxic studies on human lung carcinoma A-549 cell lines.
All chemicals used were purchased from Fluka chemicals. Their purity was checked by GC. All solvents were purified by distillation using Rotavap (Buchi R120) and if necessary residual water was removed. The components of solvents and elements are given in volume ratios of the components. Methyl gamma linolenate was isolated and purified by using Isolera Flash chromatography System (Biotage INC) and identified using different spectral and HPTLC techniques. The standard methyl gamma linolenate and routine were purchased from Sigma-Aldrich uses thin layer chromatography. The 1H NMR and 13C NMR were recorded on Bruker DRX-300 (300 MHz FT-NMR) using deuterated chloroform as solvent and TMS as internal standard. The mass spectra of compounds were recorded on JEOL GCMATE II GC-MS.
Fresh cultures of Spirulina platensis were obtained from Antenna Research Foundation Pvt Ltd., “Madurai, Tamilnadu, India”. The cell paste was lyophilized and stored at −20 °C for further use.
General procedure for isolation of methyl gamma linolenate
The Isolera system is a high-performance flash chromatography system, consisting of the following integrated components:
Isolera flash chromatography system
A touch screen display, which is a solvent-resistant, color LCD screen with a resolution of 800 × 600 pixels. It serves both as a display and as the system’s input device via on-screen touch controls. A fraction collector, which collects fractions into a wide variety of collection racks and vessels. A pump module, which directs liquid flow through the system. A default flow rate is specified for each cartridge but, if desired, the flow rate can be changed. If the flow rate is increased, the system will start the run at the default flow rate and then regulate towards the flow rate defined in the method. The system regulates on both flow rate and pressure. If 90 % of the maximum allowed pressure is reached before the defined flow rate, the flow rate at 90 % pressure will be used. An internal detector, which provides the system with information on the light absorbance of the solvents and samples passing through the detector flow cell. The different fractions can be collected through a automated fraction collector based on the Rf the compound of interest can be identified and pooled together after performing thin layer chromatography analysis.
Preparation of fatty acid methyl esters (FAME)
Freeze dried biomass of Spirulina platensis (50 g) was extracted by reflux for 4 h using a mixture of methanol and acetyl chloride (95:5, 800 ml). The extract obtained was diluted with water and extracted thrice with equal volume of n-hexane containing 0.01 % butylated hydroxyl toluene. The combined n-hexane layer was evaporated to get the fatty acid methyl ester (FAME).
Enrichment of FAME for methyl gamma linolenate (GLA-ME)
The FAME obtained was subjected to urea complexation as described previously to remove the saturated fatty acids from the polyunsaturated fatty acids. For the urea complexation, methanol (9 ml) and urea (3 g) were added to FAME. The mixture was heated to get a clear solution. It was cooled at room temperature and stored at 0 °C overnight. Then it was filtered to remove the crystals settled at the bottom. The filtrate was extracted with n- hexane containing 0.01 % butylated hydroxyl toluene. The n-hexane fraction was evaluated for the presence of GLA methyl ester by HPTLC.
Quantification of GLA-ME in enriched GLA fraction by HPTLC
Preparation of sample solution
Whole enriched GLA-ME fraction (122.5 mg) was dissolved in 10 ml of n-hexane. This solution was used for application on to the TLC plate.
Preparation of standard solution
Standard GLA-ME was prepared by dissolving 27 mg of GLA-ME and in 10 ml of methanol in a volumetric flask.
Stationary phase : Silica gel 60 F 254
Mobile phase : Hexane: Acetone (6:4)
Mobile phase volume : 10 ml
Band length : 6 mm
Application rate : 10 s/μl
Development chamber : Camag twin trough development chamber (10 × 10 cm)
Development distance : 6 cm from the application position
Scanner : Camag TLC scanner III
Detection wavelength : 235 nm
Slit dimension : 4.00 × 0.30 mm
Scanning speed : 20 mm/s
Data resolution : 100 μm/step
Measurement mode : Absorption
Peak area of GLA-ME standard = 1379.2
Peak area of GLA-ME in sample = 3605.6
Separation of GLA-ME using flash chromatography system
Characterization of GLA-ME
FAB-MS: 292 (M, 100 %): 254.70 [+(CH2)4CH = CHCH2CH = CH (CH2)4COOCH3] m/z + 1, 178.82 [CH3(CH2)4CH = CHCH2CH = CHCH2CH = CH+],151.69 [CH3(CH2)4CH = CHCH2CH = CHCH2CH+] m/z + 1, 121.90 [CH3(CH2)4CH = CHCH2CH+] m/z-3. .
1H NMR (300 Mhz,CDCl3 δH); 5.5 (m, 6H, olefinic C6, C7, C9,C10,C12,C13), 3.7 (s, 3H, CH3 ester C19), 2.6 (t, 4H, CH2 between double bonds C8,C11), 2.3 (t, 2H, CH2 adjacent to C = O, C17), 2.1 (m, 4H, CH2 adjacent to double bond C5,C14), 1.7 (m, 8H, C3, C4, C15 and C16) 1.3 (m, 2H, CH2 next to CH3 C2), 0.9 (t, 3H, CH3 terminal C1) ppm
13CNMR (300MHZ,CDCl3 δC): 14.14- C1, 22.53- C2, 24.55 – C8, 24.75 - C11, 25.60 – C16, 26.85 – C5, 27.18 - C14, 29.21- C15, 29.23 – C4, 31.72- C17, 33.83- C3, 51.51- C19, 127.56 – C7, C12, 129.55 – C9, C10, 130.41 – C6, C13, 174.82 – C18.
Cell lines and culture medium
A-549 cell cultures used in the experiments were procured from National Centre for Cell Sciences, Pune, India. A-549 cells were grown in Earl’s Minimal Essential Medium supplemented with 2 mmol L-glutamine, 10 % Fetal Bovine Serum, Penicillin (100 μg/ml), streptomycin (100 μg/ml) and amphotericin B (5 μg/ml) and the cells were maintained at 37 °C in a humidified atmosphere with 5 % CO2 and subculture twice a week.
In-vitro apoptosis inducing effect
Results and discussion
Determination of cytotoxicity by SRB method
% Growth Inhibition
The novel method has been developed for the isolation of GLA-ME by using a flash chromatography system which improved the percentage yield up to three folds (71 %) in comparison with the conventional methods (18-20 %). The cytotoxicity shown by GLA-ME may be due to the induction of apoptosis of tumor cells by augmenting free radical generation only in the tumor cells but not normal cells. It is evidenced by the research work carried out by Undurti et al. . They also reported that induction of apoptosis of tumor cells by GLA is due to its action at the gene/oncogene level and by altering BCl-2 expression. Hence, it may be concluded the cytotoxicity shown by GLA-ME may be due to induction of apoptosis effect. However, a detailed study of this mechanism is in progress.
Methyl gamma linolenate was isolated from the Spirulina platensis by flash chromatography system and the percentage yield is found to be 71 %. It may be concluded that the Flash chromatography system plays a major role in improving the yield for the isolation of methyl gamma linoleate from Spirulina platensis. Since the process consumes very less time compared to conventional isolation, many chemists searching for new technologies for the production of plant constituents of medicinal interest can adopt this method. The isolated molecule is a potent cytotoxic agent towards human lung carcinoma cell lines, it may be further taken up for extensive study.
The authors greatly acknowledge for financial grant under the Department of Science and Technology (DST) (SR/SO/HS-0288/2012) Government of India also acknowledges JSS University and JSS College of Pharmacy for providing the necessary facilities.
- 2.Fedekar FM, Abde WK, Hoda SN. Production and nutritive value of Spirulina platensis in reduced cost media. Egypt J Agric Res. 2012;38:51–7.Google Scholar
- 7.Guerrero JL, Madrid PC, Juarez RN. Isolation of some PUFA from edible oils by argentated silica gel chromatography. Grasasy y Aceties. 2003;54:116–21.Google Scholar
- 8.Jubie S, Dhanabal SP, Afzal Azam M, Muruganantham N, Kalirajan R, Elango K. Synthesis and characterization of some novel fatty acid analogues: A preliminary investigation on their activity against human lung carcinoma cell line. Lipids Health Dis. 2013;12:45–51.CrossRefPubMedPubMedCentralGoogle Scholar
- 19.Jubie S, Gayathri R, Srividya AR, Kalirajan R, Prabitha P, Sankar S, et al. Synthesis and characterization of some novel quinoxaline-2, 3-dione derivatives: a preliminary investigation on their activity against a human epithelial carcinoma cell line. Lett Drug Design Discov. 2011;8:317–20.CrossRefGoogle Scholar
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