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Authors: Kumar, Naresh
Keywords: Hydroxycinnamic Acids
Natural Source of Antioxidants
L-Tyrosine Through Shikimate Pathway
Issue Date: May-2015
Publisher: Dept. of Biotechnology iit Roorkee
Abstract: Hydroxycinnamic acids are natural source of antioxidants; arise from the metabolism of Lphenylalanine and L-tyrosine through shikimate pathway in plants. The hydroxycinnamic acids are the largest class of phenolic compounds, represented by p-coumaric, caffeic and ferulic acids. They have been consistently associated with the reduced risk of cardiovascular disease, cancer and other chronic diseases. The p-coumaric acid is a ubiquitous plant metabolite known to exhibit useful biological activities viz., antioxidant, anti-inflammatory, antiplatelet, anti-melanogenic; inhibits melanin synthesis in B16F10 cells without affecting CREB phosphorylation or tyrosinase protein production. The caffeic acid is a polar compound with a strong chelating capacity towards metals, and coffee is the primary source of it in the human diet. However, it can also be found in other food sources. Like other phenolics, caffeic acid also exhibits strong antioxidant activity. Ferulic acid is an abundant phenolic phytochemical, known to enhance the stability of cytochrome c, inhibits the apoptosis induced by cytochrome c, increases the IgE binding to peanut allergens and many more. During its formation, phenylalanine and tyrosine are converted into cinnamic and p-coumaric acid with the help of phenylalanine ammonia lyase and tyrosine ammonia lyase, respectively. The p-coumaric gets converted into ferulic by hydroxylation and methylation reaction. Oxidation and methylation of ferulic acid and other aromatic compounds give di- and trihydroxy derivatives of cinnamic acid, which takes part in the lignin formation together with ferulic acid. In 1925, it was chemically synthesized and its structure was confirmed by spectroscopic techniques, depicted the presence of an unsaturated side chain and existence of two isomeric forms (cis and trans). The resonance stabilized phenoxy radical accounts for the effective antioxidant activity of ferulic acid. It catalyzes the stable phenoxy radical formation upon vi absorption of ultra-violet light, which gives the strength to ferulic acid for terminating free radical chain reactions. Isolation of the hydroxycinnamic acids from Parthenium hysterophorus L. followed by their structural characterization using elemental analysis, FT-IR, NMR, ESI-MS and single X-ray crystallography had been carried out. Molecular structures of these acids were confirmed by single crystal X-ray diffractrometer, which showed that the p-coumaric acid crystallized in the monoclinic crystal system with space group P 21/c, while caffeic and ferulic acids were crystallized in the monoclinic crystal system with space group P 21/n. The thermograms of the acids clearly indicate that all three acids are stable up to 100ºC but at higher temperatures, curves shows irregular pattern with one stage thermal decomposition during thermal analysis. The molecular geometry, harmonic vibrational frequencies and structural parameters were computed at DFT/B3LYP/6-311G** basis set by Gaussian 09. The comparisons between experimental and simulated data of spectroscopic analysis and geometrical parameters were accomplished for their statistical validation by curve fitting analysis and found statistically close to each other with the values of correlation coefficient for bond lengths and bond angles 0.985, 0.992, 0.984, 0.975, and 0.913, 0.933 in p-coumaric, caffeic and ferulic acids, respectively. The HOMO and LUMO analysis were also carried out to find out the charge transfer within the molecule. Motivated by the broad spectrum of biological activities shown by ferulic acid, its derivatives and the compounds possessing amide moiety, we have developed a strategy towards the synthesis of a number of novel derivatives by the structural modifications of ferulic acid. The design, synthesis and characterization of four series of mono and bis- amide derivatives (IIIa-IIIo, Va-Vg, VIIa-VIIg and IXa-IXe) of ferulic acid had been carried out by microwave irradiation under solvent free reaction conditions. The previously extracted ferulic acid together with the vii different categories of primary amines were taken in equimolar concentration in a petri dish, mixed carefully and subjected to microwave irradiation at 300-450W power for 3-10 min. The structural studies of all the newly synthesized compounds had been performed by FT-IR, NMR, mass spectroscopy and elemental analysis for C, H, and N. The 1H-NMR was taken after keeping all of those for ten days and six months at room temperature did not show any change, confirmed that the compounds are stable in solid as well in liquid phase. These results were also supported by their thermal studies. Further, all the synthesized compounds have been screened for their in vitro cytotoxicity and free radical scavenging activity by MTT against five different human cancer cell lines, i.e. breast (MDA-MB-231 and MCF-7), cervical (HeLa), lung (A549), and liver (HepG2) as well as one normal stem cell line at 10μM concentration and DPPH assay, respectively. The results of MTT assay showed that 21 compounds exhibited good activity against all types of cancer cell lines and very less or negligible activity alongside normal stem cells. These 21 compounds were further studied for their IC50 value and found that the compounds Va-Vg (containing acridine moiety) exhibited the best IC50 (07.11-11.08 μM). The compound Vb was found to be the most promising against breast (MCF-7; IC50 = 07.49 μM and MDA-MB-231; IC50 = 07.28 μM), Vd against lung (A549; IC50 = 07.11μM) and liver (HepG2; IC50 = 08.32 μM) and Ve against cervical (HeLa; IC50 = 07.14 μM) cancer cell line. Data obtained free radical scavenging activity revealed that nine compounds (IIIf, IIIl, IIIo, VIIe and IXa-IXe) showed good antioxidant activity (EC50; 18.37 to 25.44μM) as compared to L-ascorbic acid (EC50; 20.14μM), thirteen compounds showed poor activity (EC50 values more than 35μM), and twelve compounds exhibited moderate activity (EC50; 26.89-34.81μM). viii The theoretical calculations were also performed in two manners; Firstly, the quantum chemical calculations of all the compounds for their geometry optimization and computation of structural parameters based on DFT with a hybrid function B3LYP at 6-311G** basis set. To evaluate the behavior in terms of energy of synthesized compounds, calculations were done both in solvent and gasseus phase. The values of energies (HOMO and LUMO), electro-negativity (χ), chemical hardness (η), chemical softness (S), electronic chemical potential and electrophilic index (ω) were also calculated using Koopman’s theorem for closed shell molecule. The optimized geometry showed the positive harmonic vibrational frequencies, indicates that optimized geometries attained the global minimum state on the potential energy surface. Secondly, the 3DQSAR (quantitative structure activity relationship) studies for anticancer and antioxidant activities were also carried out by using CoMFA (comparative molecular field analysis). In the work, six (6) different models were generated (5 for anticancer and 1 for antioxidant) and analyzed. The statistical, contour map, steric and electrostatic effects analysis were carried out to understand the structure–activity relationship of ferulic acid derivatives. Commenting on the statistical results, we inferred that 3D-QSAR values lie statistically closed to the values obtained from experimental results for in vitro anticancer and antioxidant activity, and outcome for all the cases to be worthy in respect to their statistical significance for correlation.
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