In Silico Analysis of Phytochemicals From Coffea arabica Against Haemophilus influenzae Causing Conjunctivitis ABSTRACT: Phytochemicals are secondary metabolites obtained from plants through various metabolic pathways . It has been reported that Coffea arabica plant extract is used to cure conjunctivitis. The plant extract contains different phytochemicals. Conjunctivitis is caused by Haemophilus influenzae. One of the key enzymes involved in its biochemical pathway is shikimate dehydrogenase. The molecular docking of the phytochemicals with the enzyme was studied using Biovia Discovery Studio. The strength of the interaction was evaluated based on -CDocker energy and -CDocker interaction energy. High positive values for both the parameters indicated that out of different phytochemicals ,caffeine and cholorogenic acid can effectively deactivate the shikimate dehydrogenase enzyme thereby interrupting the life cycle of Haemophilus influenza. KEY WORDS: Phytochemical, Biovia, Discovery studio, Coffea arabica,Haemophilus influenzae. 1. INTRODUCTION: The faster and developed life styles affects the health of an individual. Now a day’s lifestyles are an important factor for health. According to WHO 60% health factor directly related with way of living [1]. Millions of people follow unhealthy lifestyle like unhealthy diet, smoking, alcohol consuming, drug abuse, stress and so on. Hence, they are encountered with illness, disability and problems like joint pain, cardiovascular disease, hypertension, overweight, skin lesions, low vision and low hearing problems in early age. A little change in way of living can prevent these diseases from annexing our society [2]. Vagbhatta's Astangahrdayam gives information about how plants are used for curing disease [3].Medicinal plants are the major constituents of indigenous natural medicine, modern medicine, food supplements, bioactive principles, pharmaceutical intermediates and synthetic drugs [4].Medicinal plants are used for the preparation of drugs from Sumerian clay slab from Nagpur approximately 5000 years old. It comprised 12 recipes for drug preparation referring to over 250 various plants, some of them belong to group alkaloid such as poppy, henbane, and mandrake.In ancient period there was not sufficient information about particular plant for curing specific disease. In time , the reason for the usage of specific medicinal plants for treatment of certain diseases were being discovered, thus the medicinal plants usage gradually abandoned the empiric framework [5].Phytochemicals are biologically avtive compounds that are produced by plants and are non nutritive in nature. Phytochemicals act as natural defence mechanism in their host plants. Phytochemicals are also called as phytonutrients and secondary metabolites produced by plants. Plant based medicinal constituent can be derived from any part of plant like bark ,leaves, flower,roots,fruits and seeds [6]. Coffee belongs to family Rubiaceae [7].Coffee leaf extract is used to cure disease like conjunctivitis [8] Coffee is known to contain phytochemicals like Cafestol, triglycerides,Caffeine,Cholorogenic acid [9]. Coffea benghalensis flowers are used to treat conjunctivitis in West Bengal .Coffea fragrans flowers are used to treat eye ailments too in the district of Assam [10].There is high possibility that some of the phytochemicals might play a major role in curing conjunctivitis. However, there is no report identifying the specific phytochemical responsible to cure conjunctivitis. Phenolic compounds in coffee leaves provides important biological functions, among them chlorogenic acid exhibits anti-diabetic and anti- lipidemic properties through regulation of the activation pathway of the cellular energy homeostasis enzyme AMPK [11]. Haemophilus influenzae is generally known to cause conjunctivitis, which belongs to genus Haemophilus. They are gram negative , coccobacillary, facultatively anaerobic bacteria . Conjuctivitis is a common bacterial disease that affects the nose and the throat. Haemophilus influenzae bacteria typically live in nasopharynx [12]. The present study focuses on the identification of the phytochemical of Coffea arabica responsible to cure conjunctivitis caused by Haemophilus influenza through in silico analysis. 2. MATERIALS AND METHOD: 2.1 Software used: Discovery studio module of Biovia software (Dassault Systemes of France) was used for analysis. The software utilizes machine learning techniques to predict the level of molecular interaction. Biovia is the scientific brand of Dassault Systems that is focused on the Virtual biosphere and Materials. Biovia is committed to enhancing and speeding innovation, improving productivity. Biovia is used for reducing cost and accelerating product development. The Discovery Studio module of Biovia is used for identifying molecular interaction and perform molecular docking of phytochemicals from plant extract that act as a ligand and form strong covalent bond with bacterial protein to successfully inhibit microbe. 2.2 METHODOLOGY: 2.2.1 List of phytochemicals : Plants secret phytochemicals as secondary metabolites to protect them from predators that may include bacteria, viruses, fungi etc. When these plants or their parts are consumed by humans these phytochemicals fight off threats to health. Some phytochemicals have been used as poisons and others as traditional medicine. Published works showed that Coffea arabica contains cafestol, caffeine, chlorogenic acid and triglycerides. It has already been established that Coffea arabica plant belonging to Rubiaceae family has potential to help controlling conjunctivitis. This work is focused on identification of the particular phytochemical responsible for inhibiting and controlling of conjunctivitis. 2.2.2 Enzyme found in Haemophilus influenzae: Various metabolic cycles have been seen in the bacterial life cycle for its survival. These metabolic cycles are regulated by different enzymes. Brenda enzyme database was used to identify and list different enzymes found in Haemophilus influenza . It has been found that shikimate dehydrogenase enzyme (EC 1.1.1.25 and protein database code 1NPY) is involved in phenylalanine, tyrosine and tryptophan biosynthesis (KEGG) and is very crucial for survival of this particular microbe. 2.2.3Molecular Docking: Molecular docking method has been used to identify the phytochemical from the plant extract , that act as a ligand and form a strong covalent bond with the bacterial protein to successfully inhibit the microbe. The Discovery studio module of Biovia software was used for identifying molecular interaction and performs molecular docking. In this process first the sdf files for the phytochemicals found in the Coffea arabica plant were downloaded from the website (https://pubchem.ncbi.nlm.nih.gov). The protein database code of the shikimate dehydrogenase enzyme was identified from the website (www.rcsb.org). The active site of the enzyme was identified via “receptor cavity” protocol found under "receptor-ligand interaction" menu. Molecular docking was done using the CDocker protocol of Biovia softwere under “receptor- ligand interaction”. The enzyme molecule was treated as the receptor molecule and the phytochemical was treated as the ligand. The “-CDOCKER_ENERGY” and “- CDOCKER_INTERACTION_ENERGY” were used as indicator for the quality of molecular docking. The high positive value of those indicators presented a good interaction between the ligand and the receptor. Thus, the interactions with high values might indicate the major phytochemical responsible for curing the disease. 3. RESULTS AND DISCUSSION: Figure1. shows the active site of the Shikimate dehydrogenase enzyme. It appears as light green colour. CDOCK is a molecular dynamics (MD) simulated-annealing-based algorithm. It is a grid-based molecular docking method and optimized for accuracy. The ligand conformations were obtained by Molecular Dynamic methods. Figure 1. Active Site of Shikimate Dehydrogenase Enzyme -CDOCKER energy was calculated based on the internal ligand strain energy and receptor-ligand interaction energy. -CDOCKER interaction signifies the energy of the nonbonded interaction that exists between the protein and the ligand. The criteria for best interaction was chosen based on a) high positive value of -CDOCKER energy and b)small difference between -CDOCKER energy and -CDOCKER interaction energy [13]. Table 1 shows that shikimate dehydrogenase - caffeine interaction has the highest positive value of -CDOCKER energy (14.444) and minimum value of the difference (8.0708) between - C DOCKER interaction energy and - C DOCKER energy followed by chlorogenic acid. Thus the results indicated that caffeine and chlorogenic acid can effectively deactivate the shikimate dehydrogenase enzyme thereby interrupting the biological cycle of H. influenzae. Higher positive values for caffeine indicated that it was the most active ingredient against H. influenzae. On the other hand, cafestol can deactivate the enzyme to a small extent (negative -CDocker energy but positive -CDocker interaction energy). Triglycerides cannot interact with shikimate dehydrogenase enzyme. Thus, the key phytochemicals preventing conjunctivitis caused by H. influenzae are caffeine and cholorogenic acid. Table 1. Results of CDocking of Phytochemicals with Shikimate Dehydrogenase (receptor) SL LIGAND - C DOCKER - C DOCKER DIFFERENCE NO ENERGY INTERACTION BETWEEN – ENERGY CDOCKER INTERACTION ENERGY AND – CDOCKER ENERGY 1 Caffeine 14.444 22.5148 8.0708 2 Chlorogenic