ANTI-OXIDANT EVALUATION AND MOLECULAR DOCKING STUDIES OF PHYTOCOMPOUNDFROM MADHUCALONGIFOLIAAS POTENTIAL THYMIDYLATE SYNTHASE INHIBITOR

77  ANTI-OXIDANT EVALUATION AND MOLECULAR DOCKING STUDIES OF PHYTOCOMPOUNDFROM MADHUCALONGIFOLIAAS POTENTIAL THYMIDYLATE SYNTHASE INHIBITOR Karpagam T, Varalakshmi B, Shanmuga Priya,Sugunabai J, Gomathi S, BharathiV, Priyadarshini, Jannathul Firdous Department of Biochemistry, Shrimati Indira Gandhi College, Tiruchirappalli, India. Department of Biochemistry, SeethalakshmiRamaswamy College, Tiruchirappalli, Tamil Nadu, India. 3 Biological and Bioinformatics Research Centre, Trichy, Tamil Nadu, India. Cluster for Integrative Physiology and Molecular Medicine (CIPMM), Faculty of Medicine, Royal College of Medicine Perak Universiti Kuala Lumpur, No.3, Jalan Greentown, 30450, Ipoh, Perak, Malaysia. *Corresponding Author: Jannathul Firdous Running Title: Anti-oxidant Evaluation and Molecular docking studies of Madhucalongifolia ABSTRACT Best alternative for cancer treatment is medicinal plants with numerous pharmacological properties which is used in many countries around the world. The present study was focussedto implement docking analysis of some phytocompounds present in Madhucalongifolia for anticancer action on thymidylate synthase to analyse potency of phytocompound. Madhucalongifolia leaves were dried and powdered. The powder was extracted with ethanol and water. In order to know the antioxidant potential of plant extract, phytochemical analysis followed by DPPH scavanging assay was done. The highest antioxidant activity was observed in ethanolic extract and therefore, this extract was chosen for further stuides. The phytocompounds were functionally analysed by FTIR and GC-MS analysis. The GC-MS analysis determines the existance of various compounds in Madhucalongifoliaethanolicextracts. 5,5',8,8'-Tetrahydroxy-3,3'-dimethyl-2,2'-binaphthalene1,1',4,4'-tetrone (C22H14O8) was one of the compound used for docking studies. Binding energy valueshowedthe synthesized compound selectivity towards ATP-binding pocket of Thymidylate synthase, the enzyme target in cancer chemotherapy. The computational methodology such as molecular docking analysis is efficient in finding effective drugs made of natural origin against these diseases. Its evident that Madhucalongifolia contains various phytocomponents and considered as a plant of medicinal value against cancer.


INTRODUCTION
Oxidative stress results out of increased free radicals are responsible for the development of various life threatening diseases including cancer. Haemorrhagic shock, arthritis, atherogenesis, Alzheimer disease, Parkinson's disease and some gastrointestinal disorders are the diseases resulting from free radicals 1 . Deleterious effects of free radicals such as oxidative damage of living cells are prevented by antioxidants, both exogenous or endogenous. This free radical scavengers can be synthetic and natural. Butylatedhydroxyanisole (BHA), Butylatedhydroxytoluene (BHT), tert-butylhydroquinone (TBHQ) and propyl gallate (PG) are the synthetic antioxidants induces toxicity during long time usage 2 . Now, many in-depth studies are carried out in searching natural antioxidants from herbal sources. Cancer is an abnormal growth of cells with potential speed in spreading to otherbodyparts. Cancer can affect different types of organ such as digestive, nervous, and circulatory systems, where hormones are released abnormally even to untargeted organ results in affecting the normal body function 3 .Eventough there are many medicinal treatment available to treat cancer, they are not effective in treating the disease to normal due to its high systemic toxicity and drug resistance. The final outcome may therefore not success. There is a continuous search of new herbal cures for cancer with enhancing efficacy and less toxicity to normal tissues 4 . In human cells, Thymidylate synthase (TS, EC 2.1.1.45), only source of enzyme for deoxythymidyne-monophosphate (dTMP) synthesis and this dTMP is an important precursor for DNA synthesis. When this enzyme is inhibited, replication process of DNA will be terminated and apoptosis occurs in fast dividing cells. This process is known as thyminelesscell death 5 . The molecular knowledge of such biological pathwaynotea unique option of mechanism usingthymidylate synthase inhibitors to treat cancer whereespeciallythymidylate synthase is involved. In cancer chemotherapy, thymidylate synthase enzyme is inhibited using 5-Fluorouracil (5-FU) 6 . This synthetic drug when used to treat cancer, results in the continuous exposure to Thymidylate synthase inhibitors on the site of tumour causing an increase in levels of intracellular Thymidylate synthase activity which may lead to resistance. The understanding of TS inhibition and its interaction with action of anticancer agents can explore new strategies with an effective TS inhibition without any resistance 7 . Madhucalongifolia comes from Sapotaceaefamily. Its pharmacological functions are well known for treating phlegm, inflammation, fractures, insect bite, diarrhoea, chronic tonsilitis, fever and rheumatism. This plant was reported to contain phytochemicals such as cardiac glycosides, proteins, starch, anthraquinone glycosides, phenolic compounds, tannins, terpenoids and saponins.These phytochemical compounds are responsible for their wound healing, antimicrobial, antioxidant, anti-inflammatory and anticancer activities. 8 Madhuca longifolia bark showed significant antioxidant potential and rule out lipid peroxidation when extracted with ethanol in a study was reported. Madhuca species also exhibits potent anti-inflammatory activity when extracted with crude alkaloid, ethanol and even saponin mixture in various studies. This reported action is due to the mechanism of phytochemicals present in Madhuca inhibits the synthesis of prostaglandin and it reduces the intercellular cell adhesion molecule-1 expression 9 . In considering all these views, the present studywasdone to perform antioxidant potential of M. Indicaethanolic extract. The scavenging effect and in-silico docking analysis of the phytocompounds present in Moringaoleifera for anticancer activity were evaluated.

MATERIALS AND METHODS Preparation of plant extract
Madhucalongifoliawas identified and authenticated at Rapinat Herbarium, St. Joseph College, Tiruchirappalli, Tamilnadu.Madhucalongifolialeaves were washed thrice in distilled waterand dried at room temperature for about two to three weeks. The dried leaves were coarsely powdered using mechanical blender and stored in an airtight container for futher use. Based on the sucessive solvent extraction method, Madhucalongifoliapowderwere successively extracted with different solvents viz., ethanol and waterusing a Soxhlet apparatus. The extraction was carried out for 18 h with the selected solvents with a ratio 1:4 w/v, based on their polarity viz., ethanol and aqueous 10 .

Phytochemical Studies
The ethanol and aqueous extracts of leaves obtained by successive solvent extraction were subjected to various phytochemical analysis to detect the presence of various phytochemicals. Test for alkaloids, saponins, tannins, phenolic compound, flavanoids, steroids and triterpenoids were done according to the method reported in previous study 11 . DPPH radical scavenging activity DPPH radical scavenging activity was used to test the antioxidant properties of any plant extract using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical 12 . Briefly, methanolic DPPH solution (0.1 mM) was prepared and added to plant extract in ratio 1:3, at different concentrations (100-200 μg/ml). The absorbance value was noted using UV-visible Spectrophotometer at 517nm after 20 mins. Ascorbic acid was used as standard for comparison. Value in percentage was calculated according to the formula, which showed the DPPH inhibition of free radicals with the formula: %RSA = 100 × [A c -A s ]/A c Where A c, control absorbance and A s , test sampleabsorbance.

FTIR and GC-MS Analysis
Fourier transform infrared (FTIR) spectrometry was carried out to identify the chemical compound responsible for Madhucalongifoliaextract antioxidant potential. FTIR measures the chemical bond vibration related with their functional groups and creates a spectrum. By attaining IR spectra of particular plant extract, it is possible to identify the concrete structure of certain plant secondary metabolites 13 . The compounds exist in Madhucalongifoliaethanolic extract was identified by GC-MS analysis. The principle of GC-MS associates an electron ionization system with them to use high energy carrying electrons (70 eV). The equipment has TR 5-MS capillary standard non-polar column, dimension: 30Mts, ID: 0.25 mm, Film thickness: 0.25μm. The carrier gas used is Helium with injection volume about 1 μl. The constituents present in Madhucalongifoliaextractwas identified by comparing the data available in the library (NIST and Willey) where the data was previously obtained from GC-MS instrument and the results obtained was tabulated 14 .

Molecular docking studies
Docking studies was performed to get into the binding mode of the compounds. Here, the enzyme 3D structure (PDB ID: 1HVY) was restored and the study was conducted using Autodock version 4.2.6. Active constituent from Madhucalongifoliawas restored in form of 3D structure and compound structure was generated as ligand. The protein and ligandbinding were checked in the docking test. The compound was docked into the active site of binding pocket of Thymidylate synthase through positional root-mean-square deviation (RMSD) and results with less than 2A o was considered as best stabilizing energy form.

RESULTS AND DISCUSSION Phytochemical analysis
The preliminary phytochemical test was carried out inMadhucalongifolialeaves extractasphytochemicals are direct medicinal agents. The phytochemical analysis was carried out in ethanolic and water extracts. Qualitative analysis of two different extractsconfirmed the existence of alkaloid, flavonoid, tannin, terpenoid, quinine, saponin, phenols and coumarins as shown in Figure 2. The quinine was completely absent in both. The saponins and terpenoidswere found to present only in ethanolic extract. Flavonoids are antioxidant agents with multiple biological activities including anti-carcinogenic, anti inflammatory, antibacterial, anti-allergic, antiviral and radioprotective effects 15 . Tannins usually scavenge the hydroxyl radical toxins by directly react with it and forms tannin-protein complex. This complex act as a potential free radical scavenger in preventing the diseases 16 .Saponin act as a mild detergent and in medicine, it is used in hypercholesterolaemia, hyperglycaemia, cancer treatments 17 .   Table 2. So the etanolic extract of Madhucalongifoliahave maximum antioxidant activity than aqueous extract. Phenolic compounds from different natural sources function as both primary and secondary antioxidants by different mechanisms. The existance of phytochemicals such as phenolics, tannins and coumarins in both the plant extracts may be a contributing factor for their potential free radical scavenging activity. The mechanism is that they exhibit antioxidant potential by their hydroxyl groups which function as hydrogen donor 18 .   The peak for 1087 corresponds to C-N stretching linked to Aliphatic amines and the peak at 680 corresponds to C-Br Stretch related to alkyl halides. Fourier transform infrared (FTIR) spectrometry measures the bonds within its structural groups and creates a spectrum. This is smilar as biochemical/metabolic fingerprint assessed by FTIR for the given sample. Through FTIR, we can detect the changes in primary and secondary metabolites of any given sample 19 .   GC-MS spectrum in addition to FTIR spectrum results, confirmed the existence of components using different retention times.. The mass spectrometer identify the compounds structure and during analysis, the large compound fragmented into small compounds which appeared as peaks at different m/z ratios. These mass spectra fingerprint of compound can be identified from the data library 20 Table  3 and 4. Through the grid map, the interaction between proteins and ligands through binding pocket was determined. The grid map was used with 60 points in each x, y, and z direction, equally spaced at 0.375A°. Docking by Lamarckian genetic algorithm was studied with population size of 150; random starting position and conformation. The results of the molecular docking analysis indicate that the compounds were more selective towards the ATP-binding pocket of Thymidylate synthase (1HVY). Lowest Binding Energy (LBE) and hydrogen bond interaction results during docking was shown in Table 4. The binding energy was between -9.38 and -5. dimethyl-2,2'-binaphthalene-1,1',4,4'-tetrone from Madhucalongifolia showed hydrogen bonds with less distance CONCLUSION Antioxidant activity ofMadhucalongifoliaethanolicextractwas confirmed by the presence of major phytochemicals such as flavonoids and tannins. In DPPH scavanging activity, significant difference between ethanol and aqueous plant extracts was observed and ethanol extracts exhibited excellent antioxidant activity . Therefore, for further study, ethanolicextract was selected.From the results of FTIR and GC-MS analysis, it could be concluded that Madhucalongifoliacontains various bio-active compounds. Evaluation of pharmacological activity of various compounds are needed in future to know as it may help further to establish the application of isolated compound in treatment of various diseases and provide more assurance in application of such isolated compounds. From the docking analysis of the results of pharmacological studies, it was concluded that the synthesized compound accommodated in ATP-binding pocket of Thymidylate synthase. Financial support and sponsorship: Nil Conflict of Interests: There are no conflicts of interest.