2016-03-17T01:09:07+05:302016-03-17T01:09:02+05:302016-03-17T01:09:07+05:30Acrobat PDFMaker 11 for Worduuid:5e77db86-69a2-48fb-a880-490d219de421uuid:ad0e60dd-3f2c-4225-9078-200dc63227aa9xmlANTIOXIDANT ASSAY OF C-2-HYDROXYPHENYLCALIX[4]RESORCINARENE USING DPPH METHODValued Acer CustomerAdobe PDF Library 11.0D:20160314011642Acer
International Journal of ChemTech Research CODEN (USA): IJCRGG ISSN: 0974-4290 Vol.9, No.02 pp 278-283, 2016
Antioxidant Assay of C-2-Hydroxyphenylcalix[4] Resorcinarene using DPPH Method Santi Nur Handayani, Mustofadan Respati Tri Swasono1,2, Jumina12, 1 University of Gadjah Mada, Yogyakarta 55281, Indonesia 1Department of Chemistry, Faculty of Mathematics and Natural Sciences 2Department of Chemistry, of Mathematics and Natural Sciences, University of enderal Soedirman Purwokerto, Dr. Suparno street, Karangwangkal, Purwokerto, 53123 Indonesia University of Gadjah Mada Yogyakarta, Indonesia 2Pharmacology and Therapy Unit, Faculty of Medicine, Abstract: C-2-Hydroxyphenylcalix[4]resorcinarene can be synthesized via condensation of 2-hydroxybenzaldehyde and resorcinol with acid catalyst. Structural elucidation of products was performed using FT-IR spectrophotometer, GC-MS, 1H NMR and 13C NMR. The product of condensation was subjected to antioxidant assays using DPPH (1-1-diphenyl-2-picrylhydrazyl) method. Reaction of 2-hydroxybenzaldehyde, HCl and ethanol was performed by refluxing the mixture for 24 hours. The aromatic electrophilic substitution-cyclization 2-hydroxybenzaldehyde and resorcinol in presence of HCl gave C-2-hydroxyphenylcalix[4] resorcinarena as yellow solid in 93.14 % yield with m.p > 368.8 oC. The product was analyzed with FT-IR, 1H NMR and 13C NMR. It has strong antioxidant activity in DPPH methods with ES50 77.4322 ppm. Keywords : antioxidant assay, 2-hydroxyphenylcalix[4]resorcinarene, DPPH method. 1. Introduction Excessive sun exposure may cause the process of aging. This will trigger formation of free radicals that will cause autoxidation reactions in the body, especially in the lipid layer of the cell membrane1. Free radicals are known to have high activities to trigger a chain reaction in the cell. Autoxidation process will continue to produce hydroperoxides which may react further to form aldehydes, ketones, alcohols and hydrocarbons2. This may damage cells and cause various diseases, such as inflammation, cardiovascular disease, atherosclerosis, cancer, Alzheimer's, Parkinson's, diabetes etc1. The radical activity can be inhibited by the action of antioxidants3,4. Antioxidants are chemical compounds that function as inhibitors of oxidation reaction. Antioxidants inhibit oxidation by donating one or more electrons to free radicals, so the free radicals activities can be suppressed5. Antioxidants also inhibit the formation of reactive oxygen species (ROS), such as superoxide, O2•-, radical hydroxyl, OH•, peroxy, ROO•, alkoxyl, RO•, and hidroperoxcyl, HO2• 6,1. Sunscreens which also function as antioxidants might be prepare from compounds having conjugated double bonds likes aromatic or phenolic group as active materials. Calixarenes, groups of synthetic oligomer compounds containing an aromatic ring in a cyclic series connected by methylene or methyne bridges are suitable for this purpose. The structure of calix[4] resorcinarene allows to be modified to have properties as antiradical, antioxidant as well as sunscreen agents. Hasbullah7. reported p-methoxyphenylcalix[4]resorcinarene as an antioxidant with free radicals reduction of 67.30%. Antioxidants and antiradical activities of resorcinarene tetranitroxide are found 100 times more effective than resorcinol in reducing free radicals as an antioxidant with free radicals reduction of 67.30%. Antioxidants and antiradical activities of resorcinarene tetranitroxide are found 100 times more effective than resorcinol in reducing free radicals as an antioxidant with free radicals reduction of 67.30%. Antioxidants and antiradical activities of resorcinarene tetranitroxide are found 100 times more effective than resorcinol in reducing free radicals 2. Experimental 1.1 Chemicals Chemicals used in this research were salicylaldehyde, resorcinol, hydrochloric acid (HCl 37%), ethanol, aquadest, acetone, hexane, methanol, DMSO, and DPPH ((1,1-diphenyl-2-picrilhidrazyl). All chemicals except aquadest were purchased from E. Merck. Aquadest was obtained from Laboratory of General Chemistry, UGM. 1.2 Equipments Equipments used in this research were laboratory glassware, Büchner funnel, Buchi evaporator R-124, melting-point apparatus (Electrothermal 9100), Camac UV-Cabinet II, analytical mass balance (Mettler AT200), infra red spectrophotometer (IR, Shimadzu-Prestige 21), proton nuclear magnetic resonance spectrometer ( 1H-NMR, JEOL JNM-MY60 and JEOL MY-500 MHz), carbon nuclear magnetic resonance spectrometer (13C-NMR, JEOL MY-500 MHz), and UV-Vis spectrophotometer (Type 722). 2.3 Procedures 2.3.1 Synthesis of C-2-hydroxyphenylcalix[4]resorcinarene Into 100 mL three-necked flask equipped with water condenser, 1.1 g (10 mmol) of resorcinol and 1.22 g (10 mmol) of salicylaldehyde were dissolved in 100 mL of ethanol. Then, concentrated hydrochloric acid (1 mL) was added into the solution. The mixture was refluxed for 24 hours and monitored by TLC (acetone:hexane = 7:1) and allowed to cool to room temperature. The solid was collected by vacuum filtration with Büchner funnel, washed using the mixture of water and ethanol (1:1) and dried. The C-hydroxyphenylcalix[4]resorcinarene was obtained orange solid in 93.14 % yield with m.p. > 368.8 oC. . IR (KBr pellet) wave number in cm-1 : 3479.29 (-OH), 1612.49 and 1427.2 (C=C aromatic), 1427.9 (C-H bridge and methylen), 2931.80 (Csp3-H), 1426 and 1072.42 (C-OC). 1H NMR (DMSO) δ in ppm from TMS: 5.8418 and 5.9492 (H bridge), 6.0595 -6.7288 ppm (Ar-H), and 8.1336-8.4124 ppm (-OH). 13C NMR (DMSO) δ in ppm from TMS: 27 (-C-H bridge) and 100- 135 ppm 101.3641 (Ar-C). 2.3.2 Antioxidant test A stock solution of DPPH was prepared by dissolving 0.4 mg of DPPH in 1L methanol and the solution was kept in the dark at 4 oC [9]. A stock solution of the C-2-hydroxyphenylcalix[4]resorcinarene was prepared at 12.5, 25, 50, 100, and 200 ppm in DMSO respectively. 500µL from the stock solution of the compound was added to the 2 mL of DPPH 0,05 mM. The mixture was shaken well and kept in dark at room temperature for 2 hour. The absorbance of the mixture was measured at 517 nm by using spectrophotometer. The percent inhibition of radical scavenging ability was calculated as : % inhibition = x 100 % Linear regresion regresion y = ax + b made concentration as absis (x axis) and % inhibition as ordinat y. The 50% inhibition (ES5010) of antioxidant activity was calculated as the concentrations of samples that inhibited 50% of scavenging activity of DPPH radicals activity under these conditions . 4. Results and Discussion 4.1. Synthesis of C-2-hydroxyphenylcalix[4]resorcinarene 2-hydroxybenzaldehyde is a benzaldehyde derivative and can be condensation with resorcinol as raw materials in the synthesized of calix[4]resorcinarene. In order to obtain it, 2-hydroxybenzaldehyde and resorcinol (1:1) was refluxed in ethanol in the presence of hydrochloric acid catalyst. The reaction was carried out for 24 hours. The reaction gave orange solid in 93.14 % yield with m.p > 368,8 hours. The reaction gave orange solid in 93.14 % yield with m.p > 368,8 hours. The reaction gave orange solid in 93.14 % yield with m.p > 368,8 The FT IR spectrum (Figure.1) of calix showed characteristics absorption at 3479,29 cm-1 indicating the presence of hydroxyl (-OH) group. The most important evidence indicated that the reaction had taken place was the disappearance of strong aldehyde carbonyl absorption of the reactant. Figure 1. IR spectrum of the condensation between 2-hidroxybenzaldehyde and resorcinol Figure 2. 1H-NMR spectrum of the condensation between 2-hidroxybenzaldehyde and resorcinol Figure 2. 13C NMR spectrum product of cyclisation 2-hydroxyphenylcalix[4]resorcinarene 1H NMR spectrum of the synthesized product (Figure 2) showed that were 3 proton groups with different chemical environment. They were aromatic and hydroxyl groups. First group (A) consisted of signal at δ 5.8418 and 5.9492 ppm with total integration of 4 protons belonged to proton of methyne bridge. The product condensation reaction shown a mixture more conformer calyx[4]resorcinarene in cone, partial cone or the other (12). Signal A with ratio of integration δ 5.8418 and 5.9492 ppm 1.1146 : 0.577 are shown partial cone (C4v) conformer and crown (C2v) with comparation: 1:2. Second group (B) consisted of signal multiplet ( 24 H, δ 6.0595 -6.7288 ppm) came from the resonance of aryl protons with the detail 16 protons from residue of 2-hydroxybenzaldehyde and 8 protons residue of resorcinol. The last signals (C, 8.1336-8.4124 ppm ) represented 8 hydroxyl protons.
etanolOHHOH+OHHOOHHHOHOHOHOHHOHOHHOH+OHHOOH2HHO- H2OOHHOHHOOHHOOHHOHOHHOOHHOOHOHHOHOOHOHHOOHOHHOHO2-hydroxyphenylcaliks[4]resorcinarene C2h : C4v = 2 :1 Figure 3. Mechanism reaction of 2-hydroxyphenylcalix[4]resorcinarene Based on 13C NMR spectrum (Figure 3), there were several peaks representating the carbons existed on the product. Spectrum 13C NMR analysis was also conducted to support the previous analyses. Peaks at 27 ppm ppm indicated methylen bridge carbons in compound. Peaks at 100 ppm shown carbons in aromatic between carbons of bonds with OH groups. Peaks at region 115- 135 ppm indicated the presence of aromatic carbons (12 C). This reaction condensation and cyclization between 2-hydroxybenzaldehyde and resorcinol yielded 2-hydroxyphenylcalix[4]resorcinarene. 4.2 Antioxidant assays of product cyclisation with DPPH methods Antioxidant property of C-2-ethoxyphenylcalix[4]resorcinarene was calculated with decrease in absorbance 1,1-diphenyl-2-picrylhydrazyl (DPPH). Calix[4]resorcinarene can donated proton to the non radical from DPPH is highly antioxidant agent. Percent inhibition can calculated % inhibition = x 100 %. The electron scavenging 50 (ES50) was calculated with intrapolation concentration (axis x) versus % inhibition (axis y) curve ( Figure 4 and Tabel 1) )represent 77.4322 ppm. 2-hydroxyphenylcalix[4]resorcinarene has a medium antioxidant property, because it can stabilize the radical DPPH to reaction with the hidroxy phenol groups produce in molecul stabilization. Table 1. Concentration of 2-hydroxyphenylcalix[4]resorcinarene versus % inhibition
Concentration (ppm)
Absorbans
% Inhibition
Y= mx + C
ES 50 (ppm)
12.5
0.87
22.043
y = 0.3096x + 26.027
77.4322
25.0
0.822
26.344
R² = 0.8498
50.0
0.516
53.763
100.0
0.376
66.308
200.0
0.205
81.631
Kontrol
1.116
Figure 4. Curve concentration of 2-hydroxyphenylcalix[4]resorcinarene vs % inhibition Nihlati et al. [11] sugested a standard activities antioxidant of degree a group of compound based activites values of that : - ES50 < 50 µg/mL = very strong - ES50 50- 100 µg/mL = strong - ES50 101- 150 µg/mL = moderate - ES50 > 150 µg/mL = weak Conclusion 2-Hydroxyphenilcalix[4]resorcinarene was synthesize in a good yield (93.14 %) and displayed strong antioxidant activities (ES 50 77.4322 ppm) with DPPH methods.References
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