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Volume 1803
International Symposium on Applied Chemistr y (ISAC) 2016
Tangerang Banten, Indonesia
3–5 October 2016
Editor
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ISBN 978-0-7354-1471-6 ISSN 0094-243X
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Melville, New York, 2017 AIP Conference Proceedings
To learn more about AIP Conference Proceedings visit http://proceedings.aip.org
Conference collection Conference collection
International Symposium on Applied Chemistr y (ISAC) 2016
Tangerang Banten, Indonesia
3–5 October 2016
Editor
Silvester Tursiloadi
Indonesian Institute of Science, Tangerang Selatan, Indonesia
Sponsoring Organization
Himpunan Kimia Indonesia (Indonesian Chemical Society)
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Volume 1803
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AIP Conference Proceedings, Volume 1803
International Symposium on Applied Chemistry (ISAC) 2016 Table of Contents
Preface: International Symposium on Applied Chemistry (ISAC) 010001
Organizing Committee: International Symposium on Applied Chemistry (ISAC) 010002
Evaluation of lignin-based black liquor decolorization by Trametes versicolor U 80
Feni Amriani, Ajeng Arum Sari, R. Irni Fitria A., Haznan Abimanyu, and Sanro Tachibana 020001 High-loading-substrate enzymatic hydrolysis of palm plantation waste followed
by unsterilized-mixed-culture fermentation for bio-ethanol production
Teuku Beuna Bardant, Ina Winarni, and Hadid Sukmana 020002
Decolorization of black liquor from bioethanol G2 production using iron oxide coating sands
Vera Barlianti, Eka Triwahyuni, Joko Waluyo, and Ajeng Arum Sari 020003 Effect of coagulant/ occulant dosage and pH to water recovery of black liquor wastewater
in bioethanol production from oil palm empty fruit bunch using response surface methodology
Dian Burhani, Anissa Winarni, and Ajeng Arum Sari 020004
Chemical characterization of lignin from kraft pulping black liquor of Acacia mangium Euis Hermiati, Lucky Risanto, M. Adly Rahandi Lubis, Raden Permana Budi Laksana,
and Aniva Rizkia Dewi 020005
Lipid extraction of wet BLT0404 microalgae for biofuel application
Dieni Mansur, Muhammad Arifuddin Fitriady, Dwi Susilaningsih, Sabar Pangihutan Simanungkalit,
and Egi Agustian 020006
Degradation of black liquor from bioethanol process using coagulation and Fenton-like methods
Muryanto Muryanto, Ajeng Arum Sari, and Haznan Abimanyu 020007
Statistical analysis of NaOH pretreatment effects on sweet sorghum bagasse characteristics
Ary Mauliva Hada Putri, Eka Tri Wahyuni, and Yanni Sudiyani 020008
Bio-oil production from palm fronds by fast pyrolysis process in uidized bed reactor
Nino Rinaldi, Sabar P. Simanungkalit, and Kiky Corneliasari S. 020009 Degradation of palm oil empty fruit bunch (EFB) into bio-oil in sub-and supercritical solvents
Rakhman Sarwono and Eka Dian Pus tasari 020010
Hydrothermal pretreatment of palm oil empty fruit bunch
Sabar Pangihutan Simanungkalit, Dieni Mansur, Boby Nurhakim, Astrid Agustin, Nino Rinaldi,
Muryanto, and Muhammad Ariffudin Fitriady 020011
Reducing sugar production of sweet sorghum bagasse kraft pulp
Nissa Nurfajrin Solihat, Triyani Fajriutami, Deddy Triyono Nugroho Adi, Widya Fatriasari,
and Euis Hermiati 020012
Utilizing thermophilic microbe in lignocelluloses based bioethanol production: Review
Sriharti, Wawan Agustina, Lia Ratnawati, Tau k Rahman, and Takiyah Salim 020013 Kinetic studies of cellulose enzymatic hydrolysis from pretreated corn cob
Jeannie Stevanie, Irvan Kartawiria, and Haznan Abimanyu 020014
Optimization pretreatment condition of sweet sorghum bagasse for production of second generation bioethanol
Yanni Sudiyani, Joko Waluyo, Eka Triwahyuni, Dian Burhani, Muryanto, Prasetyo Primandaru,
Andika Putra Riandy, and Novia Sumardi 020015
Immobilization of Saccharomyces cerevisiae using Ca-alginate for bioethanol production from empty fruit bunch of oil palm
Joko Waluyo, Dian Burhani, Nurul Hikmah, and Yanni Sudiyani 020016
Bioactivities examination of Cinchona leaves ethanol extracts
Nina Artanti, Linar Z. Udin, M. Hana , Jamilah, Ida Rahmi Kurniasih, Gian Primahana, Yulia Anita,
Andini Sundowo, and Yoice Sri Kandace 020017
Characteristic of fermented spinach (Amaranthus spp.) polyphenol by kombucha culture for antioxidant compound
Aspiyanto, Agustine Susilowati, Jeti M. Iskandar, Hakiki Melanie, Yati Maryati,
and Puspa D. Lotulung 020018
Molecular identi cation of marine symbiont bacteria of gastropods from the waters of the Krakal coast Yogyakarta and its potential as a Multi-Drug Resistant (MDR) antibacterial agent
Muhammad Syaifudien Bahry, Delianis Pringgenies, and Agus Trianto 020019 Antioxidant activities of phenolic compounds isolated from the leaves of Macaranga allorobinsonii
Whitmore
Akhmad Darmawan, Sofa Fajriah, Megawati, Indah D. Dewijanti, Sofna Banjarnahor, Tri Yuliani,
Sri Hartati, Tjandrawati Mozef, Ruslan Effendi, and Greesty F. Swandiny 020020 Phytochemical analysis and antioxidant capacity of Lycopodium clavatum Linn. from Lake Sebu,
South Cotabato, Philippines
Angem L. Descallar, Maria Pamela S. Nuñez, Maria Luisa N. Cabrera, Tres Tinna B. Martin,
Christine Dawn G. Obemio, and Rhumer S. Lañojan 020021
Enhancement of antioxidant activity, α-glucosidase and α-amylase inhibitory activities by spontaneous and bacterial monoculture fermentation of Indonesian black grape juices
Andri Frediansyah, Rifa Nurhayati, and Fitrio Romadhoni 020022
Chemical characteristic and functional properties of arenga starch-taro (Colocasia esculanta L.) our noodle with turmeric extracts addition
Ervika Rahayu N. H., Dini Ariani, Miftakhussolikhah, Maharani P. E., and Yudi P. 020023 Effects of culture medium compositions on antidiabetic activity and anticancer activity
of marine endophitic bacteria isolated from sponge
Faiza Maryani, Hani Mulyani, Nina Artanti, Linar Zalinar Udin, Rizna Triana Dewi,
Muhammad Hana , and Tutik Murniasih 020024
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Characteristic of phenolic compound and antioxidant activity of fermented broccoli (Brassica oleracea L. ssp.) beverage by lactic acid bacteria (LAB)
Yati Maryati, Agustine Susilowati, Hakiki Melanie, and Puspa D. Lotulung 020025
Rubrofusarin from Aspergillus niger GTS01-4 and its biological activity
Megawati, Rizna Triana Dewi, Hanny Mulyani, Faiza Maryani, Puspa Dewi N. Lotullung,
and Minarti 020026
Comparative anthelmintic activity investigation of selected ethno-medicinal weeds
Kirstin Rhys S. Pueblos, Mark Bajalla, Dixie Pacheco, Sheila Ganot, Daisy Paig, Radyn Tapales,
Jeanne Lagare, and Mark Tristan J. Quimque 020027
Potency of microfiltration membrane process in purifying broccoli (Brassica oleracea L.) fermented by lactic acid bacteria (LAB) as functional food
Agustine Susilowati, Aspiyanto, Yati Maryati, Hakiki Melanie, and Puspa D. Lotulung 020028
Extraction of coffee silverskin to convert waste into a source of antioxidant
Patrick Tangguh and Samuel P. Kusumocahyo 020029
Preliminary phytochemical screening and alpha-glucosidase inhibitory activity of Philippine taro (Colocasia esculenta (L.) Schott var. PSB-VG #9)
Richemae Grace R. Lebosada and Ivy L. Librando 020030
Enzymatic production of DFA III from fresh dahlia tubers as raw material
Thelma A. Budiwati, D. Ratnaningrum, and S. Pudjiraharti 020031
Steam distillation extraction of ginger essential oil: Study of the effect of steam ow rate and time process
Muhammad Arifuddin Fitriady, Anny Sulaswatty, Egi Agustian, Salahuddin,
and Deska Prayoga Fauzi Aditama 020032
The utilization of patin sh head for instant stock paste
Gabriela Mawi Hartanto, Della Rahmawati, and Maria Gunawan Puteri 020033
Fermented inulin hydrolysate by Bi dobacterium breve as cholesterol binder in functional food application
Hakiki Melanie, Agustine Susilowati, and Yati Maryati 020034
Cooking characterization of Coleus tuberosus noodle in various arenga starch substitution
Miftakhussolikhah, Dini Ariani, Mukhamad Angwar, and Jeremia Kevin M. M. 020035
Colour and pH changes of tempe during extended fermentation
D. Muzdalifah, Z. A. Athaillah, W. Nugrahani, and A. F. Devi 020036
Application of lemon peel essential oil with edible coating agent to prolong shelf life of tofu and strawberry
Della Rahmawati, Mega Chandra, Stefanus Santoso, and Maria Gunawan Puteri 020037
The effect of temperature and extraction period of time on the chemicals content of emprit ginger ethanol extract (Zingiber of cinale var. Rubrum)
Diah Ratnaningrum, Een Sri Endah, and Sri Pudjiraharti 020038
Development of more friendly food packaging materials base on polypropylene through blending with polylacticacid
Achmad Hana Setiawan and Fauzan Aulia 020039
Developing a prebiotic yogurt enriched by red bean powder: Microbiological, physi-cochemical and sensory aspect
Fitri Setiyoningrum, Gunawan Priadi, and Fi A ati 020040
Recovery of fermented inulin fiber by lactic acid bacteria (LAB) from inulin hydrolysate using fungi inulinase enzymes of Scopulariopsis sp.-CBS1 and class of Deuteromycetes-CBS4 as cholesterol binder
Agustine Susilowati, Hakiki Melanie, Yati Maryati, and Aspiyanto 020041 Screening of the presence organophosphates and organochlorines pesticide residues in vegetables
and fruits using gas chromatography-mass spectrometry
Dillani Putri, Nurhani Aryana, Yosi Aristiawan, and Dyah Styarini 020042 Emulsion polymerization of polystyrene-co-acrylic acid with Cu2O incorporation
Sri Fahmiati, Sri Budi Harmami, Yenny Meliana, and Agus Haryono 020043 Synthesis of PP-g-MA as compatibilizer for PP/PLA biocomposites: Thermal, mechanical and
biodegradability properties
Muhammad Ghozali and El Nur Rohmah 020044
Interaction between vegetable oil based plasticizer molecules and polyvinyl chloride, and their plasticization effect
Agus Haryono, Evi Triwulandari, and Pingping Jiang 020045
Esteri cation free fatty acid in palm fatty acid distillate using sulfonated rice husk ash catalyst
Arif Hidayat and Bachrun Sutrisno 020046
Hydrogenation of artemisinin to dihydroartemisinin over heterogeneous metal catalysts
Anis Kristiani, Ralentri Pertiwi, and Indri Badria Adilina 020047
Formation of six-membered rings via alkyne insertion into four-membered rings
Takanori Matsuda, Norio Miura, and Takeshi Matsumoto 020048
Copper (II) ion adsorption from aqueous solution onto fatty hydroxamic acid - Immobilized zeolyte
Muhsinun, Purwantiningsih Sugita, and Henny Purwaningsih 020049
Synthesis of Ni supported by CaO from Ni(0)L complexes (L=dihydrazine, bisethylenediamine)
Sabarmin Perangin-angin, Nimpan Bangun, Adil Ginting, and Nabila Karina Putri 020050 Optimization and adsorption kinetic studies of aqueous manganese ion removal using chitin
extracted from shells of edible Philippine crabs
Mark Tristan J. Quimque, Marvin C. Jimenez, Meg Ina S. Acas, Danrelle Keth L. Indoc,
Enjelyn C. Gomez, and Jenny Syl D. Tabuñag 020051
CP_Prelim_pages_8.5x11_Blue.indd 6 09-01-2017 21:14:49
Nano porous alkaline earth metal silicates as free fatty acid adsorbents from Crude Palm Oil (CPO)
Indra Masmur, Seri Bima Sembiring, Nimpan Bangun, Jamaran Kaban, and Nabila Karina Putri 020052 In uence of reaction condition on viscosity of polyurethane modi ed epoxy based on glycerol
monooleate
Evi Triwulandari, Mohammad Kemilau Ramadhan, and Muhammad Ghozali 020053 Catalytic activity of titania zirconia mixed oxide catalyst for dimerization eugenol
S. Tursiloadi, A. Kristiani, S. N. Aisyiyah Jenie, and J. A. Laksmono 020054 Computational calculation of acetalization of benzaldehyde using acid catalysts (HCl) with
computational method (Ab-Initio)
Muhammad Yusuf and Deby Elfrinasti Br Sitepu 020055
Gravimetric dilution of calibration gas mixtures (CO2, CO, and CH4 in He balance): Toward their uncertainty estimation
Harry Budiman, Muhammad Rizky Mulyana, and Oman Zuas 020056
Glycerol conversion into value added chemicals over bimetallic catalysts in supercritical carbon dioxide
Luth ana N. Hidayati, Sudiyarmanto, and Indri B. Adilina 020057
Bracketing method with certi ed reference materials for high precision and accuracy determination of trace cadmium in drinking water by Inductively Coupled Plasma - Mass Spectrometry
Rosi Ketrin, Eka Mardika Handayani, and Isna Komalasari 020058
Identi cation of di(ethylhexyl) phthalate as impurity in the analysis by using chromatography gas tandem mass spectrometry
Eka Dian Pus tasari, Hendris Hendarsyah, Salahuddin, and Novita Ariani 020059 Optimization of squalene extraction from Palm Fatty Acid Distillate (PFAD) in multistage process
Leah Sibuyo, Diah Widiputri, and Evita Legowo 020060
Optimization of squalene produced from crude palm oil waste
Irda Wandira, Evita H. Legowo, and Diah I. Widiputri 020061
Development of PVA-alginate as a matrix for enzymatic decolorization of textile dye in bioreactor system
Dede Heri Yuli Yanto, Syifa Zahara, Raden Permana Budi Laksana, Sita Heris Anita,
Maulida Oktaviani, and Fahriya Puspita Sari 020062
PREFACE: International Symposium on Applied Chemistry (ISAC)
We welcome you to the Second International Symposium on Applied Chemistry (ISAC) held October 2-3, 2016 at Indonesia Convention Exhibition (ICE), BSD City, Indonesia. As a premier conference in the field of chemistry by carrying the theme, "Chemistry for Better Life", ISAC 2016 provides a highly competitive forum for reporting the latest developments in the research and application of Applied Chemistry. We are pleased to present the proceedings of the conference as its published record.
ISAC is the 2nd international scientific conference organized by Indonesian Chemical Society (HKI) and supported by Indonesian Institute of Sciences (LIPI). It will facilitates sharing of ideas and recent research experiences and its application in chemical related fields, as well as to promote international cooperation among participants. It covers the areas of organic chemistry, inorganic chemistry, analytical chemistry, food chemistry material chemistry, environmental chemistry, green chemistry, medicinal chemistry, chemical biology, electrochemistry and physical chemistry.
The conference program represents the efforts of many people. We want to express our gratitude to the members of the Advisory Board, Scientifics and Editorial Board, Organizing Comitee, and the external reviewers for their hard work in reviewing submissions. We also thank the invited speakers, Dr. Oaki Yuya (Keio University, Japan), Prof. Nico Voelcker (University of South Australia, Australia), Prof. Dr. Arief Budiman (Universitas Gadjah Mada, Indonesia), Dr.
Muhammad Abdul Kadir Martoprawiro (Chairman of Indonesian Chemical Society), Prof. Dr.
Swapandeep Singh Chimni (University Amritsar, India), Dr. Mark A.T. Blaskovich (University of Queensland, Australia), Dr. Hyunjoo Lee (Korea Institute of Science and Technology, Korea) and Dr. Jungho Jae (Korea Institute of Science and Technology, Korea), for sharing their insights with us. Finally, the conference would not be possible without the excellent papers contributed by authors. We thank all the authors for their contributions and their participation in ISAC 2016!
We hope that this program will further stimulate research in Applied Chemistry and provide practitioners with better techniques and knowledge. We feel honored and privileged to serve the best recent developments in the field of Applied Chemistry to you through this exciting program.
Prof. Dr. Silvester Tursiloadi, M.Eng.
Chair Person of Comitee and Coordinator of S&E Board Research Center for Chemistry
Indonesian Institute of Sciences – LIPI
ORGANIZING COMMITTEE Advisory Board:
·
Prof. Dr. Ir. Iskandar Zulkarnaen (Chairman of Indonesian Institute of Sciences)
·
Dr. Laksana Tri Handoko (Deputy Chairman for Engineering Sciences of the Indonesian Institute of Sciences)
·
Dr. Muhamad Abdul kadir Martoprawiro (Chairman of Indonesian Chemical Society)
·
Dr. Agus Haryono (Director of the Center for Chemistry- Indonesian Institute of Sciences)
Scientific and Editorial (S&E) Board:·
Prof. Dr. Silvester Tursiloadi (Coordinator of S&E board, Research Center for Chemistry, LIPI, Indonesia)
·
Prof. Volkan Degirmenci (University of Warwick, United Kingdom)
·
Prof. Dr. Shogo Shimadzu (Chiba University, Japan)
·
Prof. Nico Voelcker (University of South Australia, Australia)
·
Prof. Reinout Heijungs (Leiden University, Netherland)
·
Prof. Dr. Dong Jin Suh (Korean Institute of Science and Technology, South Korea)
·
Prof. Dato' Dr. Ir. Abdul Wahab Mohammad (CESPRO, University Kebangsaan Malaysia)
·
Prof. Dr. Khalid M. Khan (ICCBS, University of Karachi, Pakistan)
·
Prof. David Lennon (University of Glasgow, United Kingdom)
·
Prof. Dr. Arief Budiman (Gadjah Mada University, Indonesia)
·
Prof. Muhammad Hanafi (Research Center for Chemistry, LIPI, Indonesia, Indonesia)
·
Prof. Habibah Wahab (Malaysia Institute of Pharmaceutical and Nutraceutical, Malaysia)
·
Prof. Yanni Sudiyani (Research Center for Chemistry, LIPI, Indonesia)
·
Prof. Dr. Subyakto (Research Center for Biomaterials, LIPI, Indonesia)
·
Dr. Oaki Yuya (Keio University, Japan)
·
Dr. Habil Andreas Martin (Leibniz Institut fuer Katalyse LIKAT, Germany)
·
Dr. Ahmad Hanafi Setiawan (Research Center for Chemistry, LIPI, Indonesia, Indonesia)
·
Dr. Yuni Krisyuningsih Krisnandi (University of Indonesia)
·
Dr. Rosi Ketrin (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. Tjandrawati Mozef (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. Jarnuzi Gunlazuardi (University of Indonesia)
·
Dipl. Ing. Haznan Abimanyu, Ph.D (Indonesian Chemical Society)
·
Dr. Edi Iswanto Wiloso (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. Oman Zuas, M.Sc (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. Wahyu Dwianto, M.Agr. (Research Center for Biomaterials, LIPI, Indonesia)
·
Dr. Oman Zuas, M.Sc (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. rer.nat. Neni Sintawardani (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. Yenny Meliana (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. Dini Mansur (Research Center for Chemistry, LIPI, Indonesia)
·
Dr. Fransiska Herkrismastuti (Research Center for Chemistry, LIPI, Indonesia)
Organizing Committee:
·
Chairperson : Prof. Dr. Silvester Tursiloadi, M.Eng
·
Co-Chairperson : T. Beuna Bardant, M.Sc
·
General secretary : A. Rahman Setiawan, M.Eng
·
1st Secretary :Sujarwo Suryaputra, S.Si
·
2nd General secretary :Eka Dian, S.Si
·
Program coordinator : Dr. Heru Susanto, M.Sc
Characterization and antioxidant activity of gallic acid derivative
Krissan Malinda, Hery Sutanto, and Akhmad Darmawan
Citation: AIP Conference Proceedings 1904, 020030 (2017);
View online: https://doi.org/10.1063/1.5011887
View Table of Contents: http://aip.scitation.org/toc/apc/1904/1 Published by the American Institute of Physics
Characterization and Antioxidant Activity of Gallic Acid Derivative
Krissan Malinda
1,a), Hery Sutanto
1, and Akhmad Darmawan
21Department of Chemical Engineering, Faculty of Life Sciences and Technology, Swiss German University, Tangerang Selatan, Banten, Indonesia.
2Research Center for Chemistry, Indonesian Institute of Sciences, Kawasan PUSPIPTEK Serpong, Tangerang Selatan, Banten, Indonesia.
a)Corresponding author: [email protected]
Abstract. Peroxidase enzyme was used to catalyze the dimerization process of gallic acid. The structure of the dimerization product was characterized by 1H NMR and LC-MS-MS. The mechanism of gallic acid dimerization was also discussed. It was proposed that ellagic acid was formed through an oxidative coupling mechanism that lead to the formation of a C-C bond and followed by an intramolecular Fischer esterification mechanism that lead to the formation of two C-O bonds. Moreover, the antioxidant activity of gallic acid and ellagic acid were also studied. Gallic acid and ellagic acid exhibited the DPPH radical scavenging activity with IC50 values of 13.2 M and 15.9 M, respectively.
INTRODUCTION
Ellagic acid is a condensed dimer of gallic acid, which is also the prevalent phenolic acid in plant tannins. With the molecular weight m/z 302.2, ellagic acid is classified as a polyphenolic compound present in some natural sources, including pomegranate, strawberries, blackberries, and raspberries [1]. Ellagic acid was found to be an effective antioxidant after subjecting it in different in vitro antioxidant activity and radical scavenging assays.
Compared to standard antioxidant compounds such as BHA and BHT, results showed that ellagic acid has a higher antioxidant activity in DPPH free radical scavenging activity assay, superoxide radical scavenging assay, and Fe3+
reducing powerassay [2].
Noticing this potential, there has been increasing attention to the use of gallic acid as a basis for drug development. However, since extraction method was considered to have a great dependency on nature and uncertain quantities of phytochemicals on each fruit [3], various ways were done to synthesize ellagic acid chemically, including through the dimerization process. Dimerization is the process of combining two smaller (identical) molecules into a larger molecule. To catalyze the reaction, it was claimed in the US Patent 5541091A that peroxidase enzyme can be used in the dimerization of an aromatic compound [4].
An earlier study on the dimerization of gallic acid indicated that peroxidase enzyme can be used to catalyze the dimerization process of gallic acid via an oxidative coupling mechanism. The Liquid Chromatography – Mass Spectrometry (LC-MS) analysis showed the presence of the product with a molecular weight which is similar to ellagic acid [5].
In this study, the dimerization process of gallic acid as well as product characterization were examined. In addition, the antioxidant activity of ellagic acid was also evaluated in comparison to gallic acid.
MATERIALS AND METHODS Materials
Gallic acid monohydrate, Folin Ciocalteu’s phenolic reagent, sodium carbonate, methanol, buffer pH 7, hydrogen peroxide 30%, and ethyl acetate were obtained from Merck, Germany. Peroxidase enzyme from horseradish with activity of ≥ 250 units/ mg and ellagic acid were obtained from Sigma-Aldrich, USA. Free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) was obtained from Sigma-Aldrich, Germany.
Methods
Dimerization of Gallic Acid
In 2.5 ml of methanol, 100 mg of gallic acid was diluted (the initial concentration of gallic acid is equal to 0.235 M). As much as 15 mg of peroxidase enzyme in 20 ml buffer (pH 7) solution and 4 ml of 30% hydrogen peroxide were added. The mixture was stirred for 9 hours in 37°C [5].
The reaction product was separated using separatory funnel, and ethyl acetate was used as the solvent to captivate the desired product, which was the gallic acid dimer. After the addition of the solvent, two layers were formed. The upper (organic) layer was collected, while the bottom layer was rinsed repeatedly until two phases were no longer formed. The upper phase was brought to a rotary evaporator, where the solvent evaporates and leaves a concentrated product.
Structure Elucidation
The process of determining the chemical structure of the dimerization product was conducted by using 1H Nuclear Magnetic Resonance Spectroscopy (Jeol JNM ECA-500, Japan). The confirmation of the molecular weight was done by using Liquid Chromatography – Tandem Mass Spectrometry (Waters Corporation UPLC Acquity 1, USA). The analysis results were compared with an appropriate reference, which is the ellagic acid obtained from Sigma-Aldrich, USA.
In vitro Antioxidant Activity.
The DPPH (1.1-diphenyl-2-picryl-hydrazyl) scavenging assay was performed based on the method used by Mishra, et al., (2012) with slight modification [6]. The objective of this method was to investigate the scavenging activity of the sample (gallic acid and ellagic acid) towards free radical DPPH.
The DPPH solution was prepared. To make a 2536 M of DPPH (Mr = 394.32 g/mol) grandparent solution, 10 mg DPPH powder was diluted in 10 ml of methanol. From the grandparent solution, DPPH parent solution of 253.6 M was made. Both grandparent and parent solution were stored in an amber glass bottle covered in aluminum foil at 4°C. As a note, DPPH solution that was going to be used in the procedure described below was the one that has a concentration of 253.6 M (the one that was referred as the parent solution).
In brief, the sample with various concentrations was prepared in methanol solution. In the test tubes, 1.5 ml of the sample with various concentrations was mixed with 1.5 ml of 253.6 μM DPPH solution in methanol. Control solution was made by replacing the proportion of the sample with 1.5 ml of methanol. The test tube for blank solution was filled with 3 ml of methanol. All test tubes were brought to vortex shaker and incubated at room temperature and in a dark chamber for 30 minutes. The absorbance of each sample was analyzed using UV-Vis spectrophotometer (λ = 517 nm). The percentage scavenging of DPPH radical was calculated using the equation: [1 - (B/A)] x 100%; whereas A is absorbance of the control solution and B is absorbance of the sample solutions.
For each sample, a graph of DPPH radical scavenging activity (in %) versus sample concentration (in μM) was made in order to calculate the IC50 (the half maximal inhibitory concentration) of each sample. In this study, the parameter IC50 was used to estimate the amount of antioxidant necessary to decrease the initial DPPH by 50%.
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RESULTS AND DISCUSSIONS Dimerization of Gallic Acid
The mixture of gallic acid solution and peroxidase enzyme solution produced a light brown color. Upon the addition of H2O2, the color of the solution changed rapidly to dark brown. After 9 hours of incubation, the color of the solution turned into yellow. The color changes indicated that gallic acid has been transformed into a new substance.
The reaction product was brought into a separatory funnel to be separated by using ethyl acetate. Ethyl acetate was used as the solvent due to its efficiency in obtaining extracts enriched in ellagic acid [7]. Moreover, gallic acid has a low solubility in ethyl acetate [8]. This fact supports the use of ethyl acetate as a solvent to separate the dimerization product [Compound III] from gallic acid.
Compound III 1H NMR (500 MHz in CD3OD): δ 7.4868 (s, 2H, H-3/3’). ESI-MS: [M+H]+ m/z 303.01088.
During the dimerization process of gallic acid, the catalytic cycle of horseradish peroxidase (HRP) enzyme was started upon the addition of hydrogen peroxide to the solution of gallic acid and peroxidase enzyme. Figure 1 shows the catalytic cycle of horseradish peroxidase enzyme with gallic acid.
FIGURE 1. Catalytic Cycle of Horseradish Peroxidase Enzyme with Gallic Acid as Reduction Substrate; (a) the reaction between hydrogen peroxide and HRP-Fe(III) at its resting state; (b) The reaction lead to the formation of water and compound I which was known as an Fe(IV) oxoferryl centre; (c) the reaction of gallic acid, as a reducing
substrate, with compound I. The reaction resulted in the formation of gallic acid radical and compound II; (d) the reaction of compound II with other gallic acid resulted in the recovery of HRP-Fe(III) and another gallic acid radical
The catalytic cycle of horseradish peroxidase enzyme marked the first and second step of the radical dimerization reaction, which was chain initiation and chain propagation. At the end of the propagation step, gallic acid radicals were formed. As free radicals were not stable, they were undergoing delocalization to stabilize themselves.
Moreover, during the catalytic cycle, there were two different possible hydrogen positions that could be attacked by compound I and II; namely the hydroxyl group in the meta and para position. Figure 2 and 3 presents the radical stabilization of gallic acid radical against attacks on hydrogen at meta and para substituted – OH Group, respectively.
FIGURE 2. Resonance Stabilization of Gallic Acid Radical Against Attacks at Meta Substituted – OH Group
FIGURE 3. Resonance Stabilization of Gallic Acid Radical Against Attacks at Para Substituted – OH Group The final step of the radical dimerization reaction was termination. From many possible gallic acid radicals that may form (as shown in Figure 2 and 3), not all of them would be so easily form connections with each other due to the stability of the radicals formed and the steric factors.
An interesting phenomenon was observed when the gallic acid radical (b) initiated a chain termination with the same gallic acid radical (b), as shown in Figure 4. The compound shows an equilibrium of keto and enol form.
James (2010) stated that “aldehydes and ketones are somewhat lycanthropic (having an altered behavior) chemical
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species”. It means that they can form equilibrium between isomers, but not resonance [9]. For aldehydes and ketones, the keto form is mostly considered as the most stable. However, due to aromatic stabilization, the enol form is greatly favored in phenols. The chain termination mechanism also marked the formation of a new C-C bond.
FIGURE 4. Chain Termination of Gallic Acid Radicals
Apparently, there was a possibility for the compound (in enol form) to undergo an intramolecular Fischer esterification mechanism as displayed in Figure 5.
FIGURE 5. Gallic Acid Intramolecular Fischer Esterification Mechanism
During the mechanism, a nucleophilic (a molecule that has a tendency of donating electrons to create a new bond) oxygen atom from the hydroxyl group attacked the carbonyl carbon atom, forming an oxonium (oxygen cation with three bonds) ion. Proton transfer from the oxonium ion to the adjacent hydroxyl group gave a tetrahedral intermediate and a new oxonium ion. The loss of water from this oxonium ion gave the ellagic acid and water [10].
Therefore, during the dimerization process of gallic acid, the formation of ellagic acid was associated with the formation of three bonds. One C-C bond was formed during the chain termination of two gallic acid radicals.
Meanwhile, two C-O bonds were formed during the intramolecular Fischer esterification mechanism, in which water was removed. Figure 6 shows the molecular structure of ellagic acid.
FIGURE 6. Molecular Structure of Ellagic Acid with C Atoms Numbering
H+ H+ H+
Structure Elucidation
To characterize Compound III, the number and types of hydrogen was analyzed by 1H NMR spectroscopy. As a reference, the standard ellagic acid were also subjected to 1H NMR spectroscopy.
Compound III 1H NMR (CD3OD, 500MHz): δ 7.4868 (s, 2H, H-3/3’). The 1H NMR spectrum of Compound III shows a singlet signal at δ 7.4868 for two hydrogens of the aromatic ring, since it is recognized that hydrogens bonded to a substituted benzene ring have signals that appear in the region δ 6.5-8.5 [11].
Due to the spectrum similarity with the standard ellagic acid that was also examined, the interpretation of Compound III by 1H NMR spectroscopy indicates the presence of ellagic acid. The 1H NMR spectrum of the standard ellagic acid shows a singlet signal at δ 7.4583 for two hydrogens of the aromatic ring [1H NMR (DMSO, 500MHz): δ 7.4583 (s, 2H, H-3/3’)]. Moreover, a literature review also reported that the characterization of ellagic acid was marked by the presence of a singlet signal at δ 7.45 for two hydrogens of the aromatic ring [1H NMR (DMSO-d6) ppm: δ 7.45 (s, 2H, ArH) [12].
In order to identify the molecular mass of the components present in the sample, the Compound III was subjected to LC-MS-MS. As a reference, a standard ellagic acid was also observed. The solvent used was a gradient of acetonitrile and water.
The mass spectrum of peaks at retention time of 2.76 min indicates the presence of Compound III at high intensity by molecular ion [M+H]+ m/z 303.01088. This compound was predicted to be ellagic acid. The identification of the compound as ellagic acid was made according to the suitability of the retention time and the mass spectrum of the compound to the standard ellagic acid that was also examined.
The chromatogram of the standard ellagic acid was observed in negative ion mode. There were two observed peaks with the retention time of 2.49 and 2.79 min. From the mass spectrum of peaks at retention time of 2.49 min, ellagic acid was detected at high intensity by molecular ion [M–H]− m/z 301.00125. Identification of peaks at retention time of 2.79 min also shows the presence of ellagic acid by molecular ion [M–H]− m/z 300.99770. The result indicated that the two ions were considered to originate from the same compound, which is ellagic acid.
Therefore, based on NMR and ESI-MS measurements, it can be confirmed that Compound III is ellagic acid.
In vitro Antioxidant Activity
The antioxidant activity of the samples (gallic acid and ellagic acid) were determined using the DPPH radical scavenging assay. The assay determines the antioxidant activity based on the ability of the sample to neutralize or inhibit the DPPH free radical. Gallic acid and ellagic acid possessed an antioxidant activity with IC50 values of 13.2 M and 15.9 M, respectively. The result of this experiment showed that based on DPPH radical scavenging assay, ellagic acid possessed a lower antioxidant activity compared to gallic acid.
By means of Density Functional Theory (DFT), Saqib, et al., (2015) mentioned that, even though the numbers of hydroxyl substituents in a compound are crucial, the antioxidant activity is mainly depending on the bond dissociation enthalpy (BDE) value of each substituent.
Among some possible reaction pathways, the mechanism of DPPH free radical scavenging assay is based on the hydrogen atom transfer (HAT) from an antioxidant to free radical DPPH. During the reaction, DPPH free radical is reduced by receiving a hydrogen atom from antioxidants. The HAT mechanism is governed by the O-H bond dissociation enthalpy (BDE). Low value of BDE indicates that the hydrogen atoms are more easily separated and act as a hydrogen donor during the reaction with DPPH. Therefore, from a theoretical perspective, low BDE values are identical with high antioxidant capacity [14]. By using DFT theory, the BDE values in gallic acid free radicals are in the range of 72.79 kcal/mol to 99.46 kcal/mol. Otherwise, the BDE values in ellagic acid free radicals are in the range of 77.08 kcal/ mol to 85.01 kcal/ mol [13, 14].
Additionally, Badhani, et al., (2015) stated that gallic acid possesses strong antioxidant activities among various polyphenols. The research declared that the defining factor of the antioxidant activity of gallic acid is the three hydroxyl groups that are bonded to its aromatic ring. Arranged in the ortho position with each other, these hydroxyl groups form an intramolecular hydrogen bond that not only affects the antioxidant activity of gallic acid, but also stabilizes the antioxidant radicals that are formed. The carboxylic group bonded to carbon number 1 is also believed to have a valuable impact to the antioxidant abilities of gallic acid. Compared to pyrogallol, whose molecular structure differs only in carboxylic group, gallic acid showed a higher antioxidant activity [15].
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CONCLUSIONS
The dimerization process of gallic acid using peroxidase enzyme as the catalyst was successfully performed.
ESI-MS analysis showed the presence of Compound III by molecular ion [M+H]+ m/z 303.01088 at the retention time of 2.76 min, which is similar to ellagic acid. 1H NMR spectroscopy result also indicated the presence of ellagic acid [1H NMR (CD3OD, 500MHz): δ 7.4868 (s, 2H, H-3/3’)]. These results proved that Compound III is ellagic acid
Gallic acid and ellagic acid showed their IC50 value of 13.2 M and 15.9 M, respectively. Therefore, it can be concluded that based on the DPPH radical scavenging assay, gallic acid is a better antioxidant than ellagic acid.
ACKNOWLEDGEMENTS
The authors would like to express gratitude to the Research Center for Chemistry, Indonesian Institute of Sciences for providing the materials, equipment, and all of the analytical needs for this research. We also acknowledge Swiss German University, along with the lecturers and colleagues for the support, both financially and emotionally.
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