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Volume: 3: Issue-2: April-June-2014 Copyrights@2014 ISSN: 2278-0246 Received: 12th May-2014 Revised: 27th May-2014 Accepted: 28th May-2014

Coden: IJAPBS www.ijapbs.com Research article

IN-VITRO AND IN-SILICO SCREENING OF ANGIOTENSIN CONVERTING ENZYME ACTIVATOR; A SOLUTION FOR HYPOTENSION

Sarina P Kabade1, Savinay Kumar J.C1, Harish Kumar C V1, SurendraBabu K.C1, Abhinava J V1, Guruprasad R2

1Department of PG Studies and Research in Biotechnology, Government Science College, Bangalore-560001.

2DurgaFemto Technologies and Research, Chamarajpet, Bangalore-560018.

ABSTRACT: The angiotensin converting enzyme (ACE) plays a vital role in regulating the blood pressure. The activity of ACE was screened between the eye and the heart of the zebrafish, by using hippuryl-L-histidyl-L-leusin as a substrate. Among these zebrafish eyes shows maximum activity. The screening of ACE activator is done from different plant methanol extract such as Salviniamolesta, Murrayakoenigii, Lantana camara & Eucalyptus oblique. Among these plants Lantanacamar a shows maximum ACE activity. The GC-MS analysis of Lantana camara detects 21 different compounds whereas cis-9-Octadecenal (19.8%), Squalene (19%), Hexadecanoic acid (17%) are in high concentration. All 21 compounds were subjected to the docking studies with an ACE enzyme using the software auto dock. Among these compounds Stigmast-5-en-ololeat shows maximum affinity towards the enzyme and this compound can be used as a potent ACE activator. Key words: ACE activator, Hypotension, Zebrafish, Salviniamolesta, Murrayakoenigii, Lantana camara, Eucalyptus oblique, GC-MS, Stigmast-5-en-ololeat, Auto Dock INTRODUCTION The cardiovascular homeostasis is the important phenomenon in regulating the heart. The renin-angiotensin system is one of the well known regulating systems for the cardiovascular homeostasis. In this angiotensin II plays a vital role in regulating the sodium flux and thus regulating the blood pressure [1, 2, 3, 4]. The ACE is a zinc-containing dicarboxypeptidy peptidase enzyme, which is involved in conversion of peptide angiotension I to angiotensin II. Thus the regulation of angiotension II will completely depend on the ACE activity [5, 6,7, 8]. The ACE was discovered in mid-1950’s and the ACE inhibitors are widely used for hypertension [9, 10,11]. In humans as well as in other animals the ACE was founded in Blood serum [12, 13], lungs [14], kidney [15], brain [16], and other tissues [17]. In in-vitro experiments the activity of ACE was measured using radioimmunoassay [18] and spectrophotometric assay [19]. The chemicals such as Z-Pro-Phe-His-Leu[20],Hip-His- Leu [21], Hip -Gly-Gly, Phe-Phe -Gly [22, 23] and Z-Phe(NO2)-Gly- Gly [24] were used as a substrate for the ACE assay. Today hypotension is one of the common diseases found in elder peoples [25, 26, 27, 28]. The use of ACE inhibitors is effectively decreasing the blood pressure, thus the ACE activator will increase the blood pressure [29, 30, 31]. Insilico drug designing is one of the modern accepted methods used for drug designing [32, 33]. In this investigation the in silico method is used in finding the ACE activator, which can be used as a solution for hypotension. MATERIALS AND METHODS Isolation of ACE The different parts of the zebra fish were dissected and homogenized with 10 volumes of 10mM phosphate buffer at 40C. The homogenate was centrifuged at 5000g for 10min at 40C. To the supernatant 20 volumes of 10mM buffer were added and centrifuged at 5000g for 10 min. The supernatant was dialyzed and stored at 40C, which serves as an enzyme source.

International Journal of Analytical, Pharmaceutical and Biomedical Sciences Page: 40 Available online at www.ijapbs.com

Guru Prasad et al IJAPBS ISSN: 2278-0246 Assay of ACE The assay was carried out by modifying the assay has done by Makoto Hayakari et al [34].This assay is done by a 0.5ml reaction mixture containing 300µmol NaCl, 3mM HHL in 40µmol phosphate buffer at pH 8.3. The mixture was incubated for 30 min at room temperature. The reaction mixture was terminated in boiling water bath for 10min. To the terminated mixture 3ml of 0.2M phosphate buffer of pH 8.3 and 1.5ml of 3% 2,4,6-trichloro-s-triazine(TT) in dioxane was added. The mixture is stirred vigorously until turbidity solution becomes transparent and centrifuged at 1000g for 10min. To supernatant the absorbance at 382nm was taken and enzyme activity was calculated. The above procedure was carried out for different sources and enzyme activity was calculated. Preparation of plant extract Soxhelet apparatus was used to prepare methanol extracts of Salviniamolesta, Murrayakoenigii, Lantana camara & Eucalyptus oblique. Screening of ACE activator To the enzyme reaction mixture the different plant extracts were added in the concentration of 5µg/ml before incubation. Further procedure is carried out as above mentioned in the ACE assay and enzyme activity is calculated. GC-MS analysis The GC-MS analysis of methanol extract of Lantanacamara carried out to determine the compounds present in it. The GC-MS was run with a column oven temperature of 600C and injection temperature of 2500C with split mode of injection and liner velocity flow control. The pressure applied for GC is 57.4kpa which gives the column flow of 1.00ml/min and linear velocity of 36.5 cm/Sec, with a purge flow of 3.0ml/min and split ratio is 10.0. The ion source temperature was set at 2000C and the interface temperature is 3000C, with 2.00min solvent cut time. The mass spectra taken with intervals of 0.50Sec, with scan range of 40- 600 m/z with a scan speed of 1250. The total time has taken is 34.00 min and FTD detector is used for detection. Docking studies The structures of the compounds obtained from the methanol extract Lantanacamara was drawn by using Chem Sketch and are converted to pdb by using Open Babel software. These were subjected to docking studies with the crystal ACE enzyme (PDB108A). The complete docking studies were done by using Auto Dock Vina software [35]. RESULT Enzyme activity of eye and heart The ACE activity of zebra fish eye and heart was shown in the figure 1.

Fig 1: the enzyme activity of heart and eye were presented as mean ±SD.


Guru Prasad et al IJAPBS ISSN: 2278-0246 Effect of plant extracts on enzyme activity The different plant extract shows varied effect on enzyme activity and the detailed result was shown in figure 2 Fig 2: Effects of different plants source on enzyme activity was presented as mean ± SD. Among this

Lantanacamara shows maximum activity. GC-MS analysis The methanol extract of Lantanacamara was subjected to GC-MS analysis and it shows 21 different compounds. The complete details of GC-MS chromatogram was shown in figure 3. The list of 21compounds along with the Retention time and the area was shown in the table 1.

Fig 3: The GC-MS chromatogram showing peaks at different time intervals.


Guru Prasad et al IJAPBS ISSN: 2278-0246

Table 1: List of compounds identified in methanol extract of Lantana camara during GC-MS analysis.

Peak R. Time Area Area% Name1 6.126 368490 0.72 Melamine2 7.112 427678 0.83 2,3-Dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one3 7.373 861290 1.68 Octanoic acid4 8.275 1424808 2.78 5-Oxymethylfurfurole5 11.057 2161305 4.22 Caryophyllene6 11.496 963621 1.88 Alpha.-Humulene7 12.019 612669 1.20 (+)-Cycloisosativene8 12.294 319390 0.62 Delta.-Cadinene9 12.565 441992 0.86 Dodecanoic acid10 13.570 413359 0.81 2-methyl-4-(2,6,6-trimethyl-cyclohex-1-enyl)-but-2-en-1-ol11 13.734 159253 0.31 (+)-Aromadendrene12 14.838 661695 1.29 Tetradecanoic acid13 15.690 2598880 5.07 Neophytadiene14 16.943 8709808 17.00 Hexadecanoic acid15 18.409 1679953 3.28 trans-Phytol16 18.685 10142781 19.80 cis-9-Octadecenal17 18.833 3191193 6.23 Octadecanoic acid18 24.147 9731916 19.00 Supraene19 26.820 956067 0.87 Stigmast-5-en-3-ol, oleat20 26.953 3467593 6.77 Alpha-tocopheryl-beta-d-mannosid21 29.589 1927935 3.76 gamma.-Sitosterol

Docking Studies The 21 compounds obtained from GC-MS shows different binding affinity towards ACE. Among these Stigmast-5-en-3- ol, oleat (-11.0kcal/mol) shows maximum affinity towards the ACE. The structure of the Stigmast-5-en-3-ol, oleat was shown in the figure 4. The binding of Stigmast-5 -en-3-ol, oleatto ACE was shown in the figure 5. The docking result of Stigmast-5-en-3-ol, oleat to ACE table was shown in the table 2.

Fig. 4: 3D structure of Stigmast-5-en-3-ol, oleat


Guru Prasad et al IJAPBS ISSN: 2278-0246

Fig 5: Docking of crystal ACE protein with Stigmast-5-en-3-ol, oleat ligand.

Table 2: Represents the docking result of Stigmast-5-en-3-ol, oleat to crystal ACE.

Mode affinity(kcal/mol) dist from rmsdl.b best mode rmsdu.b.

1 -11.0 0.000 0.0002 -10.8 32.638 40.3213 -10.4 42.205 45.4854 -10.3 3.899 7.0155 -10.3 3.295 4.7156 -10.2 6.827 10.3957 -10.0 32.912 39.9938 -10.0 48.689 51.4489 -9.9 4.182 8.582

DISCUSSION Among the eye and heart, the zebra fish eye shows the maximum activity (fig. 1). Due to its maximum activity eye is used as a source of the ACE. The four plants were subjected to determine the ACE activity, among these methanol extracts of Lantana camara was shown to be maximum activity. Due to its maximum activity of Lantana camara extract, it was subjected to the GC-MS analysis. From GC-MS analysis 21 compounds were identified (fig. 2). Among these cis- 9-Octadecenal (19.8%), Squalene (19%), Hexadecanoic acid (17%) having maximum concentration.(table 1) All 21 compounds were subjected to the docking study with crystal ACE, among this Stigmast-5 -en-3-ol, oleat shows maximum affinity towards the enzyme (fig. 4). Thus Stigmast-5-en-3-ol, oleat can be used as a potent ACE activator, which helps in controlling the hypotension. CONCLUSION The treatment of hypotension is necessary to avoid cardiovascular risks. The ACE activator is one of the solutions for avoiding the hypotension. This investigation successfully screened a potent ACE activator from the Lantanacamaraextract. The Stigmast-5-en-ol, oleat from the Lantana camaracan be used as a solution for the hypotension.

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