Post on 24-Feb-2018
Parte
"Synthesis of derivatives of Anthraquinone 2-ethylquinizarin and
evaluate antioxidant activity and electrochemical properties of
2-ethyIquinizarin"
Chapter 5
Introduction to Anthraquinones
Chapter 5
5.1 Introduction
Literature survey revealed that many of the reported compounds isolated from
various parts of the plants belonging to genus Artocarpus contained mainly, three six
membered rings that are linearly fused. In addition, most of the compounds
encompass carbonyl group and hydroxyl group in their molecular structures. Hence it
was contemplated to synthesize similar type of molecules with the aim to get the
enhanced activity with minimal side effects.
Anthracene is a polynuclear hydrocarbon in which three benzene rings are fused
in linear fashion. Its derivative anthraquinone encloses two carbonyl groups in the
central benzene ring. Therefore it was envisaged to synthesize anthraquinone bearing
hydroxy groups on flanked benzene rings and subject the synthesized compounds for
biological investigation.
Hence it was thought that it will be more appropriate and necessary to give brief
introduction about both synthetic and naturally occurring anthraquinones, giving
emphasis on their biological importance and methods of synthesis.
Quinones are a unique class of organic compounds identified by the presence of a
cyclic diketone structure. Anthraquinones constitute a large and diverse subgroup
within the quinone superfamily. Anthraquinones are a group of functionally diverse
aromatic compounds structurally related to anthracene, also known as 9,10-
anthraquinone, 9,10-anthracenedione, anthradione and anthracene-9,10-quinone (1)
with parent structure as shown.
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Anthraquinones are found as pigments in many plants and composite
microorganisms. Synthetic anthraquinones can be prepared by using various methods.
Its derivatives are widely used as raw materials for the manufacture of different kinds
of dyes, as catalyst in manufacturing paper, in manufacturing hydrogen peroxide, as
bird repellent on seeds, etc. Some of the derivatives find their application in
pharmaceutical and cosmetic industries.
5.2 Natural Anthraquinones
Naturally occurring anthraquinones are found in plants, fungi and lichens. Natural
anthraquinones in higher plants are formed by two main biosynthetic pathways, by
polyketide pathway and chorismate / 5-succinylbenzoic acid pathway. The latter
pathway occurs in the Rubiaecae^ family in synthesizing anthraquinones. Most of the
anthraquinones in which only one ring is substituted is often found in families
Rubiaceae, Bignoniaceae, and Verbenaceae. But do not appear to occur in fungi, and
the families Polygonaceae, Rhamnaceae, Caesalpinoideae or Liliaceae(A\oQ) where
the anthraquinones are formed via polyketide pathway. Polyketide pathway
(acetate/malonate pathway) is commonly found in microorganisms and insects ' .
Anthraquinones found in various plants, fungi and lichen species exhibit broad
spectrum of biological activities. Anthraquinones isolated from family
Caesalpiniaceae^ are used as laxatives, in treatment of asthma, tumors, ulcers, skin
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diseases such as pruritis, eczema and itching. Kim^ et al. has reported the antifungal
activity of anthraquinones isolated from seeds of Cassia tora of Leguminoseae family
against phytopathogenic fungi. Anthraquinones with antiplasmodial activity from the
roots of Rennellia elliptica Korth of family Rubiaceae is reported by Osman^ et al.
Many naturally occurring anthraquinones, isolated from plants and
microorganisms, are studied for their structural and biological importance. Rhubarb is
a species of plant in the family Polygonaceace, which has been used in traditional
Chinese medicine since ancient times and still being used in various herbal
preparations. The most abundant anthraquinone of rhubarb, Emodin (2) was capable
of inhibiting cellular proliferation, induction of apoptosis (process of programmed cell
death), and prevention of metastasis (spread of cancer from one organ or part to
another non-adjacent organ or part). Aloe-emodin (3) an other major component of
rhubarb found to have antitumor properties. Rhein (4) is also a major rhubarb
anthraquinone, which was found to effectively inhibit the intake of glucose in tumor
cells, causing changes in membrane -associated functions and led to cell death, this
study of rhubarb was reported by Huang et al. Also He Z H et al. have reported the
anti-angiogenic effects of rhubarb and its anthraquinones.
OH O OH O
Srinivas et al. have studied molecular mechanism action of Emodin (2) ie, its
transition from laxative ingredient to an antitumor agent^. Aloe-emodin (3) is also
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present in sap or leaves of Aloe vera and the leaves of senna, and has a stimulant
laxative action. Aloe emodin (3) is a new type of anticancer agent with selective
activity against neuroectodermal tumors' . Rhein (4) is commonly found as a
glycoside such as rhein-8-glucoside or glucorhein. Rhein has also been reported as an
effective antirheumatic drug, inhibited superoxide anion production from human
neutrophils", anti-proliferative effect'^ of (4) on human adenocarcinoma cells has also
been studied.
OH O
Damnacanthal (5) is an anthraquinone isolated from the roots of Morinda
citrifolia. It has been used in traditional medicine in treating human cancers such as
lungs, colon and leukemia. It is also found to exhibit antitumorigenic'^ activity in
human colorectal cancer cells. Damnacanthal (5) and nordamnacanthal (6) are
naturally occurring anthraquinones and are widely present in Morinda species. They
both have some unique and biological properties. They are found to be cytotoxic
towards the MCF-7(breast carcinoma) and CEM -SS(T.lymphoblastic leukemia) cell
lines'"* and are found to display antiviral and antimicrobial activities.
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O O ^
O OH
Alitheen et al. have evaluated the cytotoxicity study of (5) and (6) on human
leukemia cell lines(HL-60) and mouse myelomonocyte leukemia(WEHI-3B)cell
lines'^ The immunomodulatory effect of (5) was evaluated by using lymphocytes
proliferation assay on mice thymocytes and human peripheral blood mononuclear
cells(PBMC)'^
.17 Parameswaran et al. have reported the isolation and structural elucidation of the
following four anthraquinone derivatives from a fungus, Eurotium sp, isolated from
the leaves of the mangrove plant Porteresia coarctata. They are physcion (7),
fluoroglaucin (8), alaterinin (9) and catenarin (10). These compounds have been
shown to be associated with antibacterial, antioxidant and cytotoxic properties.
OH O
O R
(7) = R'=R^ = H, R = Me
(8) = R' =H, R2 = OH, R = Me
(9) = R' = 0H, R2 = R ^ = H
(10) = R'=R^ = H, R2 = 0 H
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Two new fiangal anthraquinones 1-0-methyl ethers (11) and (12) of Physcion and
Emodin, respectively, along with Emodin anthrone (13) were isolated for the first
1 R
time from, Basidiomycotina by Melvyn Gill et al. On the basis of spectral study,
they could arrive at the structure of these compounds.
OR^ O OR^
(11) = R ' = H ,R^ = Me ,R^ = Me
(12) = R' = H , R2 = Me, R = H
OH O
(13)
Natural anthraquinones produced from liquid cultures of Fusarium oxysporum,
were isolated from the roots of citrus trees, which were affected by root rot disease.
These natural anthraquinones were used as natural dyes for dyeing of wool fabrics .
Similarly, many derivatives of anthraquinones have been isolated from plants,
ftingi, lichens and insects, which have been evaluated for their biological activities.
5.3 Synthetic anthraquinones
Synthetic anthraquinones are prepared commercially by oxidation of anthracene
or condensation of benzene and phthalic anhydride, followed by dehydration of the
condensation product. Many anthraquinones have been synthesized and used in textile
industry as dyes, paints, pharmaceutical agents, in rubber and paper industry, etc. In
pharmaceutical industry it is used as laxatives, antitumor agents, antimicrobial drugs
and anticancer drugs in chemotherapy of various types of cancers. Because of their
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vast applications in various fields, synthesis of anthraquinones and their derivatives of
better quality and in good yields is a challenge for organic chemists.
The following methods are commonly used for synthesis of anthraquinones.
a. Friedel crafts reaction of benzene and phthalic anhydride in the presence of
anhydrous aluminium chloride. The resulting o-benzoylbenzoic acid then
undergoes cyclization forming anthraquinone^*^"^ .
b. Diels Alder reaction of naphthaquinone and butadiene followed by oxidative
dehydrogenation will also produce 9,10 anthraquinone. Many substituted
aza '* and diaza^^ anthraquinones are synthesized by this method.
c. 9, 10 Anthraquinone is obtained industrially by the oxidation of anthracene.
The above methods are modified by using different catalysts, reagents, and by
varying reaction durations, to synthesize various substituted anthraquinones
with better yields, possessing potential pharmacological properties.
Methodologies to synthesize various substituted anthraquinones are discussed in
detail in chapter 6 of this thesis.
5.3.1 Pharmacological activities of synthetic anthraquinones
There are many substituted anthraquinones that have been synthesized and studied
for their various pharmacological properties. Among them few are discussed in the
following paragraphs.
Ametantrone (14) and mitoxantrone (15) are structurally similar antitumor drugs
of anthraquinone class. Mitoxantrone is developed by a rational modification of the
parent 1,4-dehydroxy analogue, ametantrone. Relationship between their
pharmacological activity and their ability to condense nucleic acids have been studied
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Chapter 5
and found that pharmacological effects of these antitumor drugs could involve
condensation of nucleic acids^^, primarily of RNA in nucleoli.
Skladanowski^^ et al. reported that (14) and (15), induce covalent DNA cross
links in HeLa S3 cells(type of immortal cell line used in scientific research) at
concentrations 5-10)iM. The study suggested that this DNA cross linking is associated
with the cytotoxic and antitumour activity of these compounds.
R O HN' ,NH
^OH
R O HN, ~NH
,0H
(14) R=H , (15) R = OH
Also, the anthraquinone antineoplastic agents (14) and (15) are found to be
potential inhibitors of basal and drug stimulated lipid peroxidation in variety of
subcellular systems . They are also found to inhibit hydrogen peroxide dependent
9Q
fatty acid peroxidation which consists of initiation and propagation reactions .
Rufigallol (16) belonging to the anthraquinone class is used as antimalarial *^ drug
in combination with another antimalarial structurally similar compound, exifone.
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Anthraquinones are known to exhibit anticancer activity against various types of
cancers. 9,10-Anthraquinone monoalkylaminoalkylhydrazones were synthesized by
Antonini et al. The hydrazones were converted to hydrochlorides and tested for their
cytotoxicity activity against L1210 murine leukemia cells and the study proved that
two derivatives exhibited moderate activity against the cells.
5.3.2 Synthetic anthraquinones in textile industry
Anthraquinones play major role in manufacturing various kinds of dye and paints.
Anthraquinone based dyes are the most resistant to degradation due to their fiised
aromatic structures, which retain colour for long periods of time. These dyes
constitute the second largest class of textile dyes after azo dyes and are used
extensively in the textile industry due to their wide variety of colour shades, ease of
application and minimal energy comsumption ' . Synthetic dyes are often derived
from 9,10- anthraquinone, alizarin (17), 1-nitro anthraquinone (18), anthraquinone-1-
suphonic acid (19).
(18) R - NO,, (19) R =S03H
,34 New-2-aminopyridine based acid anthraquinone dyes have been synthesized, by
condensing 2-aminopyridine and bromamine acid, to obtain 1-amino-4-(2-
aminopyridinyl)anthraquinone-2-sulphonic acid (20) which was diazotized and
coupled with different naphthalene based coupling components(R) like gamma acid.
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m-amino benzoyl K-acid, N-methyl J acid, sulfogamma acid etc. which resulted in
the formation of new derivatives of acid anthraquinone dye (21). The dyeing
performance of newly synthesized dyes were studied on nylon, wool and silk fibres
and found that all the dyes gave good light fastness on each kind of fibre. Their
antimicrobial screening revealed that all these acid anthraquinone dyes are inactive
against bacteria Pseudomonas Sp., Bacillus subtilis, Ceretium and Escherichia coli at
100 |ag/ml and 200 p.g/ml concentration compared to Penicillin, Ampicillin and
Amoxicillin.
SO3H SO3H
(20) (21)
R = Naphthalene based coupling components.
Anthraquinone dyes with carboxylic acid as anchoring group are designed and
synthesized as sensitizers for dye-sensitized solar cells (DSSCs) and the study showed
that these anthraquinone dyes had low performance on DSSC applications^^.
Anthraquinone based dyes are very resistant to degradation due to their fused
aromatic structures, low biodegradability and thus remain coloured for longer time in
waste water. The increased use of this class of dyes has led to the necessity to know
not only the possibility of removal of colour of these dyes by electrochemical
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methods but also to understand their electrochemical behavior. Reactive blue 4(RB4)
(22) dye is an anthraquinone dye, extensively used in dyeing. Cameiro et al. has
investigated the electrochemical reduction for the removal of RB4 dye from the
aqeous solution using reticulated glassy carbon electrode. The results of the study
concluded that 60% of colour removal was achieved after three hours of RB4 dye
electrolysis at acidic and neutral conditions and only 37% at alkaline conditions.
(22)
Christian et aP'' have synthesized series of dyes by nucleophilic aromatic
substitution reactions followed by methacrylation. l,4-bis(4-((2-
methacryloxyethyl)oxy) phenylamino) anthraquinone (23), is one of such kind which
is blue in colour. These dyes can be successfully used in medical applications, such as
in manufacturing iris implants.
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Due to their complex behavior in solutions, anthraquinones are considered to be a
promising redox-active group for electrochemical applications. Most of the
electrochemical studies of anthraquinone and its derivatives are conducted in organic
phases due to their low solubility in aqueous media. Cyclic voltammetry was used by
Haque et al. * to study the electrochemical behavior of water soluble anthraquinone
derivative, the sodium salt of anthraquinone -2- sulfonic acid (24) in aqueous solution
at glassy carbon electrode, and also in the presence of a cationic surfactant
cetyltrimethylammonium bromide (CTAB). The results revealed that the current
potential behavior of anthraquinones depends on the concentration of CTAB and
micellization(A micelle is an aggregate of surfactant molecules dispersed in a liquid
colloid, the forming of micelles is known as micellization) had a profound effect on
the electrochemical behavior of anthraquinones.
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Chapter 5
Present work
In the present work, an anthraquinone, 2-ethylquinizarin is synthesized, starting
from easily available hydroquinone, using Clemmensen reduction and Friedel-Crafts
reaction. Structural elucidation of the newly synthesized anthraquinone is carried out
using IR, ^H NMR, COSY, DEPT, ' C NMR, and mass spectral data.
Antioxidant activity of 2- ethylquinizarin was evaluated by Ferric reducing
antioxidant power (FRAP), and Free radical scavenging (DPPH) methods. Also the
antioxidant activity of 2-ethylquinizarin is estimated by electrochemical studies using
cyclic voltammetry.
The work carried out is organized and presented as follows.
Chapter 6: Synthesis of derivatives of anthraquinone.
Chapter 7: Antioxidant activity of 2-ethylquinizarin.
Chapter 8: Electrochemical studies of 2-ethylquinizarin using cyclic voltammetry.
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Chapter 5
References
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2. PM Dey, JB Harbome. Plant Biochemistry, 1997, Academic press, 413.
3. K Nakanishi, T Goto, S Ito, S Natori, S Nozoe. Natural Product Chemistry, Vol.3, 1993 Kodansha Scientific Books, 457.
4. Hemen Dave, Lalita Ledwani. Indian J. Nat. Prod. Resour., 2012: 3(3): 291.
5. YM Kim, CH Lee, HG Kim, HS Lee. J. Agric .Food Chem., 2004: 52(20): 6096.
6. Puteh Osman, Nor Hadiani Ismail, Rohaya Ahmad, Norizan Ahmat, Khalijah Awang, Faridahanim Mohd Jaafar. Molecules, 2010: 15: 7218.
7. Q Huang, G Lu, HM Shen, MC Chung, CN Ong. Med Res. Rev., 2007: 27(5): 609.
8. ZH He, MF He, SC Ma, PP But. J. Ethnopharmacol, 2009: 121(2): 313.
9. G Srinivas, S Babykutty, PP Sathiadevan, P Srinivas. Med. Res. Rev., 2007: 27(5): 591.
10. Teresa Pecere, M Vittoria Gazzola, Carla Mucignat, Cristina Parolin, Francesca Dalla Vecchia, Andrea Cavaggioni, Giuseppe Basso, Alberto Diaspro, Benedetto Salvato, Modesto Carli, Giorgio Palu. Cancer Res., 2000: 60: 2800.
11. M Mian, S Brunelleschi, S Tarli, A Rubino, D Benetti, R Fantozzi, L Zilletti. J. Pharm Pharmacol, 1987: 39(10): 845.
12. G Aviello, I Rowland, CI Gill, AM Acquaviva, F Capasso, M McCann, R Capasso, AA Izzo, F Borrelli. J. Cell. Mol .Med, 2010: 14 (7): 2006.
13. T Nualsanit, P Rojanapanthu, W Gritsanapan, SH Lee, D Lawson, SJ Back. J.Nutr.Biochem., 2012: 23(8): 915.
14. AM Ali, NH Ismail, MM Mackeen, LS Yazan, SM Mohamed, AS Ho, NH Lajis. Pharm.Biol, 2000: 38(4): 298.
15. NB Alitheen, AR Mashitoh, SK Yeap, M Shuhaimi, A Abdul Manaf, L Nordin. International Food Research Journal, 2010: 17: 711.
16. NB Alitheen, AA Manaf, SK Yeap, M Shuhaimi, L Nordin, AR Mashitoh. Pharm. Biol, 2010: 48(4): 446.
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17. PS Parameswaran, Dnyaneshwar Gawas, Supriya Tilvi, Chandrakant G.Naik, Proceedings ofMBR 2004 National seminar on New frontiers in Marine Bioscience Research, 2004: 3.
18. Melvyn gill, Peter M.Morgan, ARKIVOC, 2001: 7: 145.
19. FANagia, RSR EL-Mohamedy. Dyes and Pigments., 2007: 75 (3): 550.
20. C Friedel, JM Crafts. Compt. Rend., 1877: 84: 1392 & 1450.
21. CC Price. Org. React., 1946: 3:1.
22. JK Groves. Chem. Soc. Rev., 1972: 1: 73.
23. H Heaney. Comp. Org. Syn., 1991: 2: 733.
24. Mohamed Chigr, Houda Pillion, Anny Rougny. Tetrahedron Lett, 1998: 29(46): 5913.
25. C Gesto, E de la Cuesta, C Avendano. Tetrahedron, 1989: 45(14): 4477.
26. Jan Kapuscinski, Zbigniew Darzynkiewicz. Proc. Nati. Acad. Sci. USA., 1986: 83: 6302.
27. A Skladanowski, J Konopa. Br. J. Cancer., 2000: 82(7): 1300.
28. ED Kharasch, RF Novak. J. Pharmacol. Exp. Ther., 1983: 226: 500.
29. ED Kharasch, RF Novak. J. Biol. Chem., 1985: 260(19): 10645.
30. RW Winter, KA Cornell, LL Johnson, M Ignatushchenko, DJ Hinrichs, MK RiscoQ. Antimicrob. Agents Chemother., 1996: 40(6): 1408.
31.1 Antonini, P Polucci, D Cola, G Palmieri, S Martelli, M Bontemps-Gracz . Farmaco., 1993: 48(12): 1641.
32. Klaus Hunger. Industrial Dyes: Chemistry, Properties, Applications. 2003: Wiley -VCH, 35.
33. Navin B Patel, Ashok L Patel. Asian J. Chem., 2009: 21(6): 4435.
34. Navin B Patel, Ashok L Patel. Indian J. Chem., 2009: 48B: 705.
35. Chaoyan Li, Xichuan Yang, Ruikui Chen, Jingxi Pan Haining Tian, Hongjun Zhu, Xiuna Wang, Anders Hagfeldt, Licheng Sun. Sol. Energ. Mat. Sol .C, 2007: 91(19): 1863.
36. Patricia A Cameiro, Nivaldo Boralle, Nelson R Stradiotto, Maysa Furlan, Maria Valnice B Zanoni. J. Braz. Chem. Soc, 2004: 15(4): 587.
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37. Christian DUendorf, Susanne Katharina Kreth, Soo Whan, Helmut Ritter. Beilstein. J. Org.Chem., 2013: 9: 453.
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Chapter 6
Synthesis of derivatives of anthraquinone
Chapter 6
6.1 Methodologies in synthesizing substituted anthraquinones
The importance of anthraquinone and its derivatives has been already discussed in
Chapter 5. There are several methods available in literature for synthesizing
substituted anthraquinones. Some of them have been discussed in the following
paragraphs:
• Oxidation of anthracene
Oxidation of anthracene produces anthraquinone. Ammonium cerium(IV)- nitrate
has been used to oxidize anthracene to anthraquinone (Scheme-6.1)'. In this method
anthracene in THF was stirred in presence of ammonium cerium(IV)-nitrate.
Scheme-6.1
Ce(IV)(NH,)2(NO0
Rodriguez et al. used acetic acid with air in presence of nitric acid in selective
oxidation of anthracene to get better quality anthraquinone in good yields. This
oxidation reaction of anthracene was favoured by addition of one mol of nitric acid
per mol of substrate.
• Diels Alder reaction of naphthoquinone and butadiene
Diels Alder reaction of naphthoquinone and butadiene followed by oxidative
dehydrogenation to produce 9,10-anthraquinone. This is one of the industrial methods
used to synthesize anthraquinones''. This method involves catalytic gas phase
oxidation of naphthalene into 1,4-naphthoquinone which on reacting with 1,3-
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Chapter 6
butadiene (Scheme-6.2) forms 1, 4, 4a, 9a-tetrahydroanthraquinone(THA). Oxidative
dehydrogenation of THA using DMSO, gives 9,10-anthraquinone.
Scheme-6.2
O
+0.
(VA)
+
o H H
Oxidative
dehydrogenation 1
DMSO
• Friedel Crafts reaction of benzene and ptittialic anhydride
Friedel Crafts reaction of benzene and phthalic anhydride in the presence of
anhydrous aluminium chloride also gives anthraquinone in good yields, hi this
method benzene and phthalic anhydride reacts in presence of anhydrous aluminium
chloride. The resulting o-benzoylbenzoic acid then undergoes cyclization, forming
anthraquinone. This method is very useful in the synthesis of substituted
anthraquinones in high yield"*" . Since the present work involved synthesis of
anthraquinones using Friedel Crafts reaction, the following paragraphs gives us brief
introduction to Friedel Crafts reaction used to synthesize substituted anthraquinones.
Friedel Crafts reactions of benzene and different substituted benzenes, in the
presence of molten mixture of AlCh-NaCl (2:1) have been used to synthesize various
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Chapter 6
anthraquinones*"'°. To overcome the lack of regioselectivity and improve the yields,
1119 1 "
some modifications ' were made in carrying out Friedel Crafts reaction. Singh et
al. have reported the synthesis of anthraquinones by replacing traditional reagent,
molten mixture of AlC^-NaCl, by eco friendly, non-corosive, cheaper and reusable
montmoriUonite clays. Phthalic anhydride and substituted benzenes were added to the
activated montmoriUonite clay (heated at 125-130'*C for 24 hr). The use of
montmoriUonite clay also avoids the hydrolysis of methoxy group in the benzene ring.
The Scheme-6.3 depicts the reaction.
Scheme-6.3
^ ^ < OR
o +
MontmoriUonite Clay
125-130°C
R
AlClj-NaCl melt.
R — H, CHj
2-Aminoanthraquinone (1) is used as an intermediate in the synthesis of
anthraquinone dyes, which finds applications in automotive paints, high quality paints
and enamels, plastics, rubber, printing inks and textile industry. Various methods to
synthesize 2-aminoanthraquinone, are collected by Gouda et al.'"*, one of the methods
is reported in Scheme-6.4'^. In this experimental protocol the reaction between
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Chapter 6
phthalic anhydride and nitrobenzene in the presence of AICI3 and concentrated
methane sulphonic acid produced 2-nitroanthraquinone directly. This on reduction
with SO2 / NO2 group / mole nitrocompound in 30%-60% H2SO4 at pH < 3 and 80-
180°C in the presence of HI, FeS04.7H20, CUSO4.5H2O, SnCb , or TiCh as catalyst
resulted in formation of 2-aminoanthraquinone (1).
Scheme 6.4
,NOc
0 + V ^
AlCL/MeSO,H - »
heat
.16 Dhananjayan et al. have synthesized substituted anthraquinones by a single step
Friedel Crafts acylation reaction between phthalic anhydride and substituted benzenes
using AlCls-NaCl mixture as shown in (Scheme-6.5).
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Chapter 6
Scheme-6.5
R
R
R
R
AlCl3-NaCl
O R O R
R' - H, OH, R = OH, R = OH, R = H, OH, CH3, R = H, OH.
The antifilarial properties of these synthetic anthraquinone derivatives was carried
out which showed activity against microfilaria, as well as adult male and female
worms of Brugia malayi.
Recently, Madje et al. have used Alum ( KA1(S04)2.12H20)'^ as catalyst in the
synthesis of anthraquinone derivatives starting from phthalic anhydride and
substituted benzenes (Scheme- 6.6) in good to excellent yields (70-96%), using water
as a solvent at ambient temperature. This efficient one-pot s)/nthesis of anthraquinone
has been catalyzed by alum.
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Chapter 6
Scheme-6.6
O
/Ri ^^^^' Alum( 25 mol%), Hp , rt
K^
R,, Rj = OH, CH3, Br, CI, NOj
Ri
O
Madje et al'^, have also used Boron sulphonic acid B(HS04)3 as catalyst to carry
out the reaction of phthalic anhydride and substituted benzenes to synthesize
anthraquinone derivatives under solvent free condition in good to excellent yields
(70-96%), the reaction was complete in 60-120 min. Therefore this method was
considered to be much faster and ecofriendly. The study proved that B(HS04)3 can be
used as an efficient solid catalyst for the synthesis of anthraquinone derivatives.
Literature survey about importance and synthesis of substituted anthraquinones,
prompted us to take up the work of synthesis of 2-ethylquinizarin (2-EQ) via Friedel
Crafts reaction and estimate the antioxidant activity of 2-EQ by biochemical and
electrochemical studies.
6.2 Present work
Present work involves the synthesis of 2-EQ, starting from easily available
hydroquinone, which was converted to 2-EQ by series of reactions that include Fries
rearrangement, Clemmensen reduction and Friedel Crafts reaction. The Scheme-6.7
represents the sequence of reactions involved in the synthesis of 2-EQ.
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Chapter 6
Scheme-6.7
2CH3COCI
conc.H^SO,,
0 OH
OH 0
AlClj-NaCl-melt
AICI3 -NaCl melt
3.5 hrs
Zn-Hg/HCl
The sequence of reactions shown in Scheme-6.7 is carried out in following steps.
Step 1. Preparation of hydroquinone diacetate (2) from hydroquinone (1)
Step 2. Fries rearrangement of hydroquinone diacetate to get
quinacetophenone (3)
Step 3. Clemmensen reduction of quinacetophenone to ethyl hydroquinone (4)
Step 4. Synthesis of 2-ethylqunizarin (5) by Friedel Crafts reaction
Step 1. Preparation of hydroquinone diacetate (2) from hydroquinone
To synthesize substituted anthraquinones by Friedel Crafts reaction, normally
substituted benzenes and (or) substituted phthalic anhydrides are used. Similar
methodology has been adapted in the present investigation. Thus the required
hydroquinone diacetate (2) was obtained by acetylation of hydroquinone (1).
Acetylation of (1) was accomplished by reacting it with acetyl chloride in presence of
few drops of concentrated sulphuric acid.
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Chapter 6
OH
2CH3COCI
conc.H2S04
OH
1
OCOCH3
OCOCH3
2
The product (2) did not answer the neutral FeCb test, indicating the absence of
free phenolic hydroxyl groups which was present in the precursor (1). Further
confirmation for formation of (2) was obtained by recording IR spectrum. IR
spectrum of (2) did not have the broad band which was seen in the IR spectrum of its
precursor (1). The mixed melting point of this compound with known sample did not
show any depression.
Step 2. Fries rearrangement of hydroquinone diacetate to get
quinacetophenone (3)
To achieve the synthesis of desired quinacetophenone (3) from diacetate (2), Fries
rearrangement was sought to be the most appropriate approach. Generally anhydrous
aluminium chloride is used as catalyst in such reactions'^. However in order to get
better yields and to decrease the reaction time, instead of using AICI3 alone, molten
mixture^" of AICI3 and NaCl was used. The reaction was completed within 10 min,
with better yield when compared with the yield of product obtained by using only
anhydrous aluminium chloride.
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Chapter 6
OCOCH3 OH O
AlCl3-NaCl-melt
OCOCH3
2
OH
3
Formation of (3) was confirmed by recording its ' H N M R spectrum (Figure-6.1),
which exhibited the peak as a singlet at 6 2.6 due to acetyl protons. Peaks at 8 8.1 and
8 II.7 were attributed to the two hydroxyl protons, and the aromatic protons exhibited
multiplet in the aromatic region between 8 6.75 to 7.3.
Step 3. Clemmensen reduction of quinacetophenone to ethyl hydroquinone (4)
Quinacetophenone (3) and phthalic anhydride were refluxed in presence of AICI3
for eight hr on an oil bath at 160*'C, expecting the formation of anthraquinone, 2-
acetyl quinizarin. But the reaction did not proceed as expected even after modifying
the reaction conditions like increasing the reflux time and using molten mixture of
AlCb-NaCl.
OH O
160-180°C
AlCL-NaCl melt ^
OH
3
O OH Expected 2-acetylquinizarin.
Therefore it was thought of to convert acetyl group to ethyl group and then carry
out the Friedel Crafts reaction. In this regard it was planned to convert the carbonyl
group of quinacetophenone to alkyl group by Clemmensen reduction^' of
Page 126
Chapter 6
quinacetophenone in the presence of zinc amalgam and HCl to get ethyl
hydroquinone^^'^^
OH O
OH
3
Zn-Hg / HCl
Clemmensen reduction
Conversion of quinacetophenone (3) to ethyl hydroquinone (4) was confirmed by
recording the ' H NMR spectrum (Figure-6.2) of (4) which exhibited a triplet at 5 1.1
indicating the presence of methyl protons and a quartet at 5 2.55 due to -CH2 protons.
The presence of two hydroxyl groups was confirmed by two peaks at 5 7.44 and 6
7.49. Three aromatic protons appeared as multiplet at 5 6.4 to 6.65.
Page 127
Chapter 6
Mechanism of Clemmensen reduction of quinacetophenone (3) to ethyl
hydroquinone (4)
OH :0 ^* 9H rP"^" ^^ ^^
HCI
Step 4. Synthesis of 2-ethyIqunizarin by Friedel Crafts reaction
Synthesis of 2-alkylquinizarins were reported by Marschalk et al. '* by reducing
quinizarin with alkaline dithionite, leucoquinizarin was obtained which reacted with
aldehydes to form 2-alkylquinizarins. Friedel Crafts reaction used to synthesize
anthraquinones has been modified to get better yields and to decrease reaction time. In
the present work the synthesis of 2-EQ by reacting phthalic anhydride and ethyl
hydroquinone was carried out in presence of molten mixture of AlCl3-NaCl ^°' ' ^ in
the ratio(5 : 1). For every mole of substrate four moles of AICI3 was used, and AICI3:
NaCl mixture in the ratio(5 : 1) was used, as this combination of molten mixture
along with the reactants refluxed for about 1 hr in oil bath gave excellent yield of 2-
EQ.
Page 128
Chapter 6
O
160-180°C »
AlCl3-NaCl(5:I)
The formation of compound 2-EQ, has been confirmed by recording its IR, ' H
NMR, '^C NMR, COSY, DEPT and mass spectra. IR spectrum (Figure-6.3a) of (5)
exhibited absorption bands at 1624 cm"' and 1584 cm"' accounting for two carbonyl
groups and two broad bands at 3689 cm"' and 2967 cm"' due to presence of two
hydroxyl groups as the molecule is unsymmetrical. ' H NMR spectrum (Figure-6.3b)
and (Figure-6.3c) of (5) exhibited triplet at 6 1.3 due to methyl protons and a quartet
at 5 2.8 due to two protons of methylene group of the ethyl group. Singlet at 6 7.18 is
attributed to the proton at C-3, as there are no protons on the adjacent carbon atom.
The multiplet between 5 7.82 and 8.5 is due to protons on the aromatic ring at position
C-6 and C-7, and multiplet at downfield 6 8.35 is due to the aromatic protons at
position C-5 and C-8 on the aromatic ring. The two hydroxyl protons gave two (D2O)
exchangeable peaks at 5 13.0 and 13.42.
' H - ' H COSY (Figure 6.3d) spectrum of 2-EQ exhibits distinct contours on the
diagonal, by extending vertical and horizontal lines from the contour at 5 1.3 (due to
methyl protons) on the diagonal to the off diagonal peaks at 5 2.8 (due to methylene
protons) on both the axes indicates that methyl protons are coupled with methylene
protons. Similarly the horizontal and vertical lines extended from contour on the
diagonal at 5 7.82 (Ar-H, at C-6 and C-7) to the off diagonal peak at 5 8.35 (Ar-H, C-
5 and C-8), on both the axes implies that the protons at C-6 and C-7 are coupled with
protons at C-5 and C-8 of aromatic ring. Also contour on the diagonal at 6 7.18 (Ar-H
Page 129
Chapter 6
at C-3) do not show any off diagonal peaks, as this proton is not coupled with any
other protons.
' C NMR spectrum (Figure-6.3e) of (5) exhibited two peaks at 6 186 and 6 187
due to two carbonyl carbon atoms. Peaks at 5 157 and 5 158 due to carbons
possessing hydroxyl groups, peaks at 5 146, 134.6, 134.5, 134.3, 134.2, 134,133.6,
127.2, 127, 126.8 are attributed to ten aromatic carbons, peaks at 5 23.2 and 5 12.8
are due to methylene and methyl carbon respectively of ethyl group in 2-EQ. C
DEPT spectrum was recorded at angle 135^, which shows an inverted peak at 6 25,
which can be attributed to the -CH2 carbon in the ethyl group of 2-EQ, while carbon
of methyl group gives rise to a peak at 5 12.8 (Figure-6.3f)- Finally the structure was
confirmed by its mass spectrum (Figure-6.3g) which exhibited the molecular ion peak
m/z 267 corresponding to its molecular formula C16H12O4.
Page 130
Chapter 6
Mechanism for formation of 2-ethylquinizarin (Friedel Crafts reaction)
o
0 : + AlCI, O-AICL
0 - O H
O OH
CI3AIO
Page 131
Chapter 6
•a s s o a S o
o
s s •^ u <u
a
I
u 9
Page 134
Chapter 6
Characterization data of the synthesized compounds (2-5) are presented in
Table-6.1.
Table-6.1: Characterization data of compounds (2-5)
Compounds
2
3
4
5
Molecular formula C10H10O4
CgHgOs
CgHioOi
C16H12O4
MP( "C)
122
202-204
113
168
Yield( %)
75
89
83
78
6.4 Experimental
6.4.1 Synthesis of hydroquinone dicaetate (2) from hydroquinone (1)
A drop of cone, sulphuric acid was added to a mixture of hydroquinone(55 g , 0.5
moles) and acetyl chloride(95.5 ml, 1.3 moles) taken in a round bottomed flask fitted
with a calcium chloride guard tube. The mixture was swirled gently for few min. A
white solid of hydroquinone diacetate 2 was removed using cruhed ice (500 g). This
was filtered, washed with ice cold water and dried. It was recrystallized from ethanol.
6.4.2 Fries rearrangement of hydroquinone diacetate to get quinacetophenone (3)
Two necked round bottomed flask containing melt of anhydrous aluminium
chloride(72.5 g, 0.5 moles) and sodium chloride(13.5 g, 0.2 moles), with one neck
fitted with a condenser and calcium chloride guard tube, and other neck is tightly
closed, is heated on a oil bath at temperature of 160^C. Hydroquinone diacetate (15.5
g, 12.5 moles) was added in portions through the second neck, with vigorous stirring.
Page 141
Chapter 6
Immediately after addition of hydroquinone diacetate, anhydrous condition is
maintained and the temperature of the mixture was raised to IQS^C and the melt was
stirred for 9 min. The reaction mixture was cooled and hydrolyzed with a
concentrated HCl (75 ml, 2 moles) in water (l.OL), and left overnight at room
temperature. The quinacetophenone (3) formed was filtered, washed with water and
dried, ft was recrystallised from aqueous ethanol.
6.4.3 Clemmensen reduction of quinacetophenone to get ethyl hydroquinone (4)
A mixture of zinc dust (30 g, 0.46 moles), mercuric chloride (6 g, 0.02 moles),
concentrated hydrochloric acid (10 ml, 37%, 0.3 moles) and water (50 ml) was stirred
for 5 min. The aqueous solution was decanted, and resulting amalgam was used for
the reaction.
In a two necked flask fitted with reflux condenser and dropping funnel, mixture of
quinacetophenone (13 g, 0.08 moles) in water (35 ml), toluene (26 ml, 0.2 moles),
acetic acid (13 ml, 0.2 moles) and zinc amalgam was taken. To this concentrated
hydrochloric acid (65 ml, 2 moles) was added drop wise slowly through the dropping
fiinnel and the mixture was refluxed for 45 hr with constant stirring on a magnetic
stirrer. Then the mixture was transferred to a separating funnel, the aqueous layer is
separated and extracted with ether (20 ml x 3) and combined with organic layer. Ether
was evaporated to get colorless prisms of 2-ethylhydroquinone (4). It was
recrystallised from carbon tetrachloride.
Page 142
Chapter 6
6.4.4 Synthesis of 2-ethylquinizarin (5) by Friedei Crafts reaction
To a molten mixture of anhydrous aluminium chloride (3.99 g, 0.03 moles) and
sodium chloride (0.79 g, 0.01 mole) a freshly prepared mixture of phthalic anhydride
(1.48 g, 0.01 mole) and 2-ethylhydroquinone (1.38 g, 0.01 mole) was added in small
lots. The mixture was maintained at 160^C for 30 min and then was refluxed at 180V
for 3.5 hr. The reaction mixture was cooled and treated with ice. Then the red
precipitate of 2-ethylquinizarin was filtered and washed with water and dried. It was
recrystallised from n-hexane as spongy orange red needles.
Page 143
Chapter 6
References
1. Tse-Lok Ho, Tse-Wai Hall, CM Wong. Synthesis. 1973: 1973(4): 206.
2. Francisco Rodriguez, Ma Dolores Blanco, Luis F Adrados, Jose C Bruillo, Julio F Tijero. Tetrahedron Lett., 1989: 30(18): 2417.
3. Elena G Zhizhina, Klavdiy I Matvee, Vladimlen V Russkikh. Chemistry for Sustainable Development, 2004: 12: 47.
4. RM Christie. Colour Chemistry, 2001: The Royal Society of Chemistry, l" Edn, Cambridge, 85.
5. Ved P Aggarwala, R Gopal, Sumat P Garg. J.Org.Chem., 1973 : 38(6): 1247.
6. Knut Danielsen. Acta Chem. Scand, 1996: 50: 954.
7. Muhammad Nadeem Akhtar, Seema Zareen , Swee Keong Yeap , Wan Yong Ho, Kong Mun Lo, Aurangazeb Hasan, Noorjahan Banu Alitheen. Molecules, 2013: 18: 10042.
8. Z Horii, H Hakusui, T Momose. Chem. Pharm. Bull., 1968: 16: 1262.
9. Z Horii, T Momose, Y Tamura. Chem. Pharm. Bull, 1965: 13: 797.
10. BR Dhruba, VB Patil, AV Rama Rao. Indian J. Chem., 1976: 14B: 622.
11. U Hofmann. Angew. Chem. Int. Ed Engl, 1968: 7(9): 681
12. Nirada Devi, Mausumi Ganguly. Indian J.Chem., 2008: 47B: 153.
13. Ram Singh, Geetanjali. /. Serb. Chem. Soc, 2005: 70 (7): 937.
14. Moustafa Ahmed Gouda, Moged Ahmed Berghot, Alaa Shoeib, Khaled M Elattar, Abd El-Galil Mohamed Khalil. Turk. J. Chem., 2010: 34: 651.
15. H Naeimi, R Namdari. Dyes and Pigments, 2009: 81: 259.
16. Mugunthu R Dhananjeyan, Youli P Milev, Michael A Kron, Muraleedharan G Nair. J. Med Chem., 2005: 48 (8): 2822.
17. BR Madje, Kiran F Shelke, Suryakant B Sapkal, Gopal K Kakade, Murlidhar S Shingare. Green.Chem.Lett.Rev., 2010: 3(4): 269.
18. BR Madje, MB Ubale, JV Bharad, MS Shingare. Bulletin of the Catalysis Society of India, 2011: 9: 19.
19. GC Amin, NM Shah. J. Indian Chem. Soc, 1948: 25: 377.
20. DB Bruce, AJS Sorrie, RH Thomson. J.Chem.Soc, 1953: 2403.
Page 144
Chapter 6
21. E Clemmensen. Chem.Ber., 1914: 47: 681.
22. TB Johnson, WW Hodge. J. Amer .Chem .Soc., 1913: 35: 1014.
23. L Elmore Martin. J. Amer. Chem. Soc, 1936: 58: 1440.
24. C Marschalk, F Koenig, N Ouroussoff. Bull.soc .Chim. Fr., 1936: 1545.
25. H Raudnitz, G Laube. Ber., 1929: 62: 509.
26. Peter Wasserscheid, Thomas Velton. Ionic Liquids in Synthesis, 2008: Vol.1: WILEYVCH2"''Edn:303.
Page 145
Chapter 7
' ^ ^ ^ ^ * ' T
4
x. • J .
^
Antioxidant activity of 2-ethylquinizarin
Chapter 7
7.1 Introduction
Oxidation is a chemical reaction, during which transfer of electrons or hydrogen
takes place from a substance to an oxidizing agent. Oxidation reactions can produce
free radicals, in turn these radicals can start chain reactions. When these chain
reactions take place in a cell it can cause cell damage or even death. Antioxidants can
terminate these chain reactions by removing the free radical intermediates, and thus
inhibit other oxidation reactions. Therefore, antioxidants are the molecules that inhibit
oxidation reaction of other molecules. Although oxidation reactions are crucial for
life, they can also be damaging. Cell damage' caused by free radicals appears to be a
major contributor to aging and to degenerative diseases of aging such as cancer,
cardiovascular disease, cataracts, immune system decline, and brain dysfiinction .
Overall, free radicals have been implicated in the pathogenesis of at least 50
diseases '"*.
Reactive oxygen species (ROS) is a term which includes all highly reactive
oxygen containing molecules, including free radicals, hydroxyl radical, the superoxide
anion radical, hydrogen peroxide, singlet oxygen, nitric oxide radical, hypochlorite
radical, and various lipid peroxides. All these are capable of reacting with membrane
lipids, nucleic acids, proteins, enzymes, and other small molecules, resulting in
cellular damage. To protect the cells and organs of the body against these reactive
oxygen species, humans have evolved a highly sophisticated and complex antioxidant
protection system which involves a variety of compounds, both endogeneous and
exogeneous in origin, that functions interactively and synergistically to neutralize free
radicals^. These compounds include,
Page 147
Chapter 7
• Nutrient derived antioxidants such as, ascorbic acid, tocopherols, tocotrienols,
carotenoids and other low molecular weight compounds like glutathione and
lipoic acid.
• Antioxidant enzymes, e.g., superoxide dismutase, glutathione peroxidase, and
glutathione reductase.
• Metal binding proteins, such as ferritin, lactoferrin, albumin, and
ceruloplasmin that
"sequester free iron and copper ions that are capable of catalyzing oxidative
reactions.
• Antioxidant phytonutrients like flavonoids present in wide variety of fruits,
vegetables, green tea extracts, etc.
Insufficient levels of antioxidants, or inhibition of the antioxidant enzymes, cause
oxidative stress and may damage or kill cells. As oxidative stress appears to be an
important cause for many human diseases , the use of antioxidants in pharmacology is
intensively studied particularly in treatments of hypertension and neurodegenerative
diseases.
Antioxidants are widely used in dietary supplements , as preservatives in food
packaging units and skin care formulations^. In industry, antioxidants are used as
stabilizers in fiiels and lubricants to prevent oxidation, and also in gasoline to prevent
polymerization that leads to the formation of engine fouling residues'^. They are
widely used to prevent oxidative degradation of polymers such as rubbers, plastics
and adhesives that causes a loss of strength and flexibility in these materials".
Due to their high demand, work on both natural and artificial antioxidants has
attracted many scientists around the world, who are now trying to evaluate antioxidant
Page 148
Chapter 7
activity of the natural products they extract from plant and animal source, or the new
synthetic compounds that they synthesize. Chromatography, electrochemical
techniques and spectroscopy are used as tools to evaluate antioxidant activity of a
substance.
7.2 Spectroscopic methods used to determine antioxidant activity of compounds
1 0
Following are the few spectroscopic techniques which are commonly used to
determine antioxidant activity of compounds in most of the research labs.
Ferric reducing antioxidant power assay'^
The reagent used in this method is TPTZ (2,4,6-tripyridyl-s-triazine) in acetate
buffer 300mM per litre, pH 3.6 and FeCl3.6H20. At low pH, reduction of ferric
tripyridyl triazine (Fe III TPTZ) complex to ferrous form (which has an intense blue
colour) can be monitored by measuring the change in absorption at 593 nm. The
reaction is non specific, in that any half reaction that has lower redox potential, under
reaction conditions, than that of ferric ferrous half reaction, will drive the ferrous (Fe
III to Fe II) ion formation. The change in absorbance is therefore, directly related to
the combined or "total" reducing power of the electron donating antioxidants present
in the reaction mixture. Trolox and ascorbic acid are used as standards in this method.
DPPH (2,2 diplienyl 1-picryl iiydrazyi) or Free radical scavenging metiiod'"'
In this method DPPH (2,2-diphenyl-1-picryl hydrazyl) is the reagent used to
analyse the antioxidant activity of the compound. DPPH* is a free radical with an
unpaired valence electron, and molecules do not dimerize in either its solid state or in
solution. In the solution of DPPH dissolved in methanol, the odd electron in the
DPPH* free radical results in the formation of purple colour, with an absorption band
Page 149
Chapter 7
with a maxima at 519 nm. The colour turns from purple to yellow as the molar
absorptivity of the DPPH* radical at 519 nm reduces from 9660 to 1640 when the odd
electron present in DPPH* radical becomes paired with a hydrogen from antioxidant
being studied to form the reduced DPPH-H. The resulting decolorization is
stoichiometric with respect to number of electrons captured. Butylated hydroxyl
anisole or ascorbic acid can be used as standard.
Oxygen radical absorbance capacity (ORAC) method'^
This method measures the oxidative degradation of the fluorescent molecule
(either fluorescein or beta-phycoerythrin) against free radical generators such as
AAPH(2,2'-azobis(2-amidino-propane) dihydrochloride). Fluoroscein is used as
fluorescent probe. The loss of fluorescence of fluorescein indicates the extent of
damage from its reaction with the peroxyl radical. Antioxidants present in the analyte
are considered to protect the fluorescent molecule from the oxidative degeneration.
The degree of protection of these fluorescent molecules is measured using a
fluorometer. The degeneration of fluorescence of fluorescein is measured, as the
presence of the antioxidant slows down the fluorescence decay. The standard used
here is trolox.
The hydroxyl radical antioxidant capacity ( HORAC) assay'^
This method is based on, the oxidation reaction of fluorescein by hydroxyl
radicals via a classic hydrogen atom transfer mechanism. Free hydroxyl radicals are
produced by making use of hydrogen peroxide. These free radicals are used to
suppress the fluorescence of fluorescein over time. But in the presence of
antioxidants, these hydroxyl radicals formed are blocked from reacting with the
fluorescein, until all of the antioxidant activity of the antioxidant is completely
Page 150
Chapter 7
exhausted. Then the remaining hydroxyl radicals react with the fluorescein and
quench its fluorescence. The area under the fluorescence decay curve is used to
quantify the total hydroxyl radical antioxidant activity in the sample and is compared
with the standard curve obtained using different concentrations of gallic acid, which is
used as standard.
The 2, 2'-azino-bis(3-ethyIbenzothiazoline-6-suphonic acid (ABTS) assay'''
This method employs ABTS, which is oxidised by addition of sodium persulphate
or manganese dioxide, giving rise to the radical cation (ABTS'+) which absorbs at
743 nm, giving a bluish-green colour. In the presence of an electron donating
antioxidant, the (ABTS'+) radical is neutralized by addition of one electron, resulting
in the decolourisation of the solution. The spectrophotometric method based on the
absorbance diminution of ABTS cation radical was applied to determine the
antioxidant content in the given analyte. Trolox is considered to be the best standard
antioxidant for this method.
Total peroxy radical trapping antioxidant parameter (TRAP) assay'^
The reagent used to generate peroxy radicals here is AAPH(2,2'-azobis(2-
amidino-propane) dihydrochloride). The luminol-enhanced chemiluminescence (CL)
is used to follow up the peroxyl radical. The CL signal is driven by the production of
luminol derived radicals from thermal decomposition of AAPH. In the presence of
antioxidants, which neutralizes the peroxy radicals produced, the CL signal is affected
due to less number of peroxy radicals. The TRAP value is determined from the
duration of the time in which the antioxidant sample quenched the CL signal due to
the presence of antioxidants in it. CL signal is measured in the presence of known
amount of the standard antioxidant trolox, and compared with that of the analyte.
Page 151
Chapter 7
Potassium ferricyanide reducing antioxidant power (PFRAP) method'^
In this method, the reducing ability of antioxidants / antioxidant extracts is related
to the absorbance increase. The compounds possessing antioxidant capacity reacts
with potassium ferricyanide to form potassium ferrocyanide, which reacts with ferric
trichloride to yield ferric ferrocyanide, a blue coloured complex with a maximum
absorbance at 700 nm^ .
Some of the other spectrometric methods which are also used in analyzing
antioxidant activity of analytes are the lipid peroxidation inhibiton method^' and the
cupric reducing antioxidant power assay^ .
Electrochemical methods^^ are also applied to study the antioxidant content and
antioxidant capacity of the analytes. Cyclic voltammetry and biamperometry are the
most frequently used methods.
Using the above mentioned available methods to analyse the antioxidant activity
of natural extracts, synthetic compounds, biofluids, etc., various researchers around
the world are studying the antioxidant capacities or the antioxidant contents present in
the new compounds that have been extracted from natural sources, as well as the
compounds that are synthesized.
Study of antioxidant activities of some of the natural extracts and synthetic
compounds includes the freeze dried citrus fruit peels "*, guava fruit , bovine milk'^,
solvent extracts of Eichhornia crassipes^'^, plant foods, beverages and oils consumed
in Italy , phenolic compounds , human fluids , flavonoids and phenolic acids ,
TO "? 1 " 9
synthetic chalcones , synthetic diarylamines , synthetic indole derivatives ,
Page 152
Chapter 7
quinazolinone derivatives^^, anthraquinones and anthrones "*, anthraquinones and
flavonoids from flower of Reynoutria sachalinensis , etc.
Literature survey revealed that much work has not been carried out in evaluation
of antioxidant activity of synthetic anthraquinones, and there is lot of scope for
studying the antioxidant properties of anthraquinones, in this regard it was planned to
carry out the present work.
7.3 Present work
The present work involves the analysis of antioxidant activity of 2-ethylquinizarin
(2-EQ) by ferric reducing antioxidant power assay and free radical scavenging
(DPPH) method. Anthraquinone 2-EQ was synthesized as shown in Scheme-6.1 in
chapter 6 of this thesis. The structure of the compound 2-EQ was established using,
IR, ' H NMR, , ' H - ' H C O S Y , ^^C NMR, '^C DEPT and mass spectral data, which
confirms the assigned structure for 2-EQ (1).
The antioxidant activity of 2-EQ was analyzed by following two methods.
Page 153
Chapter 7
7.3.1 Potassium Ferricyanide Reducing Antioxidant Power (PFRAP) metliod
Procedure 36
The PFRAP assay of different concentrations of 2-EQ was carried out as follows.
Various concentrations of sample (10, 50 and lOO^g) were mixed with 2.5 ml of
200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide.
The mixture was incubated at 50°C for 20 min. Next, 2.5 ml of 10% trichloroacetic
acid (w/v) were added, 5 ml of above solution was mixed with 5 ml of distilled water
and 1 ml of 0.1% of ferric chloride. The absorbance was measured
spectrophotometrically at 700 nm. Butylated hydroxy anisole (BHA) was used as
standard antioxidant.
Results are tabulated as shown in Table-7.1.
TabIe-7.1: Absorbance measured in presence of 2-EQ and BHA at 700 nm
Sample
(in ^g)
10
50
100
2EQ
0.016
0.057
0.107
BHA
0.11
0.315
0.551
Page 154
Chapter 7
n fi -U.D
g 0.5 -c o O 0 . 4 -
re S 0.3 -(0
| 0 . 2 -
** 0.1 -n -U n 1 1
10 50
Concentration (ug)
100
- • - 2 E Q
-A-BHA
Flgure-7.1: Comparison of Potassium ferricyanide reducing antioxidant power
of 2-EQ with that of Butylated hydroxy anisole (BHA)
7.3.2 Free radical scavenging activity (DPPH) method
Procedure 37
Different concentrations (10, 50 and 100|ig) of sample in dimethyl sulphoxide and
butylated hydroxy anisole (BHA) were taken in a series of test tubes. The volume was
adjusted to 500|il by adding methanol. Five milliliters of a 0.1 mM methanolic
solution of l,l-diphenyl-2-picryl hydrazyl (DPPH) was added to these tubes and
shaken vigorously. A control without the test compound, but with an equivalent
amount of methanol was maintained. The tubes were allowed to stand at room
temperature for 20 min. The absorbance of the samples was measured at 519 nm.
Radical scavenging activity was calculated using the following formula.
% free radical scavenging activity -• Absorbancecontrol~ Absorbance2EQ
Absorbance control xlOO
Page 155
Chapter 7
Results of percentage free radical scavenging activity is presented in Table-7.2.
Table -7.2: Percentage free radical scavenging activity
Sample
10
50
100
2EQ
0.36
4.98
6.35
BHA
14.80
50.47
71.34
80
70
60
50
O) c a> >
_ •> 40
.y « 30
5 20 0) 0) 10
2EQ Samples
BHA
10 ug
Figure-7.2: Comparision of free radical scavenging activity of 2-EQ with that of
Butylated hydroxy! anisole
7.3.3 Calculation of IC50 value
The half maximal inhibitory concentration, denoted as IC50 is defined as the
measure of the effectiveness of a compound in inhibiting a biochemical or biological
reaction. It is the half maximal (50%) inhibitory concentration (IC) of a compound
Page 156
Chapter 7
(50% IC or IC50), which represents the concentration of a compound or a drug that is
required for 50% inhibition in vitro.
Percentage free radical scavenging activities are plotted against the concentration
of the compounds used to get linear regression, IC50 then estimated using the slope ie,
Y=ax + b
IC50 is calculated using the equation = (0.5 - b) /a.
Figure-7.3: Comparison of effect of 2-EQ and BHA on DPPH free
radical scavenging activity
The IC50 value of 2-EQ was found to be 1.03 |ig /ml, where as IC50 of the
reference standard BHA was 19.35 |ag/ml.
Page 157
Chapter 7
Conclusion
In FRAP method, we can see that, the absorbance values of the solution
containing 2-ethylquinizarin is less, which means there were few antioxidant
molecules to react with potassium ferricyanide to form, potassium ferrocyanide.
And also in DPPH method, formation of DPPH-H complex was very less in the
presence of 2-EQ. Therefore the results of above studies reveal that 2-ethylquinizarin
exhibits very mild antioxidant activity when compared to that of butylayed hydroxy
anisole. Antioxidant activity of 2-ethylquinizarin, may be improved by substituting
different functional groups on the aromatic ring.
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Chapter 7
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15. Milan Ciz, Hana Cizova, Petko Denev, Maria Kratchanova, Anton Slavov, Antonin Lojek. Food Control, 2010: 21: 518.
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17. J Chen, H Lindmark-Mansson, L Gorton, B Akesson. Int. Dairy J., 2003: 13: 927.
18. Pallavi Saxena, Alka Arora, Samiran Dey, Yogendra Malhotra, K Nagarajan, Pranjal Kr Singh. Journal of Drug Delivery & Therapeutics, 2011: 1(1): 36.
19. Jiangfei Meng, Yulin Fang, Ang Zhang, Shuxia Chen, Tengfei Xu, Zhangcheng Ren, Guomin Han, Jinchuan Liu, Hua Li, Zhenwen Zhang, Hua Wang. Food Research International, 2011: 44(9): 2830.
Page 159
Chapter 7
20. M Oyaizu. Japan Journal Nutrition. 1986: 44: 307.
21. J Zhang, RA Stanley, LD Melton. Mol. Nutr. Food .Res., 2006: 50(8): 714.
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24. G Kroyer. Z Ernahrungswiss., 1986: 25(1): 63.
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26. N Pellegrini, M Serafini, B Colombi, D Del Rio, S Salvatore, M Bianchi, F Brighenti. J.Nutr., 2003: 133(9): 2812.
27. M Lopez, F Martinez, C Del Valle, M Ferrit, R Luque. Talanta., 2003: 60: 609.
28. D Koracevic, G Koracevic, V Djordjevic, S Andrejevic, V Cosic. J.Clin. Pathol, 2001: 54: 356.
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31. Diana Pinto-Basto, Joao P Silva, Maria -Joao RP Queriroz, Antonio J Moreno, Olga P Coutinho. Mitochondrion, 2009: 9(1): 17.
32. Sibel Suzen. Top Heterocycl Chem., 2007: 11: 145.
33. S Rajasekaran, GopalKrishna Rao, PN Sanjay Pai, Gurupreet Singh Sodhi. J. Chem. Pharm. Res., 2010: 2(1): 482.
34. Gow-Chin-Yen, Pin-Der Duh, Da-Yon Chuang. Food Chemistry, 2000: 70(4): 437.
35. X Zhang, PT Thuong, W Jin, ND Su, DE Sok, K Bae, SS Kang. Arch .Pharm. Res., 2005: 28(1): 22.
36. JCM Barreira, ICFR Ferreira, MBPP Oliveira, JA Pereira. Food Chem. Toxicol, 2008: 4(6): 2230.
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Page 160
Chapter 8
Coirmtl
111 a^ n.
tf(ftn^
Vollat*
Electrochemical Studies of 2-ethylquinizarin using
Cyclic Voltammetry
Chapter 8
8.1 Introduction
Cyclic Voltammetry (CV) is an important electrochemical technique that can be
used to study the electrochemical properties of a redox species present in the
solution'. CV was first reported in 1938 and described theoretically in 1948 by
Randies and Sevick. This technique allows us to study the redox reactions of the
electroactive species present in the solution. Voltammetric measurement is carried out
using an electrochemical cell made up of three electrodes namely the working
electrode, reference electrode, and the counter electrode, immersed in a solution
containing the redox species and an excess of a nonreactive electrolj'te called the
supporting electrolyte, using a solvent which has a high dielectric constant to enable
the electrolyte to dissolve, so that the passage of current is made possible. The
working electrode, in which the electrochemical phenomena (oxidation or reduction)
being investigated is usually made up of inert metals such as gold, platinum or glassy
carbon. The reference electrodes, like Ag/AgCl or Cu-Cu(II) or saturated calomel
electrode Hg/Hg2Cl2 etc. are used to maintain the constant potential. The auxiliary
electrode or the counter electrode is usually, which is often a platinum wire that
serves to conduct electricity from the signal source through the cell to the other
electrodes. These three electrodes are immersed in a solution containing the redox
species, typically in a very low concentration (10" M), and an excess of supporting
electrolyte, which is an electrochemically inert salt like tetra butylammonium
perchlorate (TBAP) is added. The cyclic voltammetric setup and instrumentation is as
shown in the Figure- 8.1a and 8.1b.
Page 162
Chapter 8
Electrodes
Reforenea Auxiliar)- CA«.'A«CI) ViVittltlg (Pi*ir«) .^ •Jt,Aii«-C)
j Moveable t Nitrogimcr
Hcfiun Inki
OUg* Cdl
Electrode GEomctrics
Lsod
Figure-8.1a: Cyclic voltammetric setup
Figure-8.1b: Instrumentation of Cyclic voltammetry
In cyclic voltammetry the potential of a stationary working electrode, in an
unstirred solution is scanned linearly using a triangular potential waveform as showTi
in the Figure- 8.2.
Page 163
Chapter 8
- Cycle l-\
initial Forward Scan Switcliiits
Figure-8.2: Cyclic voltammetry waveform
Depending upon the information required single or multiple cycles are carried out.
In a cyclic voltammetry experiment during the potential sweep, the potentiostat
measures the current between the working and reference electrodes resulting from the
potential applied. This current is plotted versus the applied voltage to get a cyclic
voltammogram as in Figure-8.3. The cyclic voltammogram is a complicated, time
dependent function of a large number of physical and chemical parameters.
0.9 0.7 0.5 Poten&il (V vs Ag/AgCI)
0.3
Figure-8.3: Typical cyclic voltammogram showing oxidation and reduction peaks
In Cyclic Voltammogram the Ipc is the cathodic peak current the corresponding
peak potential is called the cathodic peak potential denoted by Epc is reached when all
the substrate at the surface of the electrode is reduced. When the voltage is decreased,
the reverse oxidation process occurs this results in the anodic peak current Ipa, and the
corresponding peak potential, called the anodic peak potential denoted by Epa is
Page 164
Chapter 8 ^
reached when all of the substrate on the surface of the electrode has been oxidized in
a typical reversible reaction.
The half wave potential Ei/2(Equation.l) can be obtained from a voltammogram
by calculating the average value of the anodic and cathodic peaks.
Epa+Epc Ei/2= (1)
E1/2 is the half-wave potential, at this point the concentrations of the reduced and
oxidized species are equal, and in this context the concentration refers to
concentration of a given species on the electrode, but not in the bulk solution.
Recently cyclic voltammetry has become a widely used electrochemical
technique to study the qualitative information about various chemical processes such
as the thermodynamics of redox reactions, to determine the presence of intermediates
in oxidation-reduction reactions, the kinetics of heterogenous electron transfer
reactions, the kinetics of coupled reactions, the electron stoichiometry of a system and
redox potential which is used as a identification tool to determine hydrogen bonding
and antioxidant activity of a compound.
Literature survey reveals that the antioxidant compounds can act as reducing
agents and in solutions they are oxidized at the inert electrodes. Based on this study,
much work has been carried out in this field " , and has reported the relationship
between electrochemical behavior exhibited by the antioxidant compounds and their
antioxidant capacity, and it was concluded that 'the low oxidation potential' of a
compound corresponds to its 'high antioxidant power'.
Page 165
Chapter 8 _ _ _ ^
Here is the brief account on applications of cyclic voltammetry in different fields.
It has been reported that the CV is a strong tool to investigate not only the
homogeneous catalysis, but also heterogeneous catalysis. The study fiirther proved
that the homogeneous catalysis can be used in Nitric oxide (NO) removal in the
environment, which involves the reduction and decomposition of NO and also CV is
a powerful means to select catalysts for NOx removal^.
Gold nanoparticles(AuNPs) are widely used as carriers for drugs such as
paclitaxel , to detect the location of tumor^, as radiotherapy dose enhancer'* , and
synthesis of these AuNPs in various sizes can be achieved by fast scan CV".
Technique of CV is employed to investigate the enhanced catalytic current generated
by bio-film modified anodes of Geobacter sulfurreducens strain DLl vs variant strain
KN400 (new strain of geobacter sulphurreducens), which generates approximately 2-8
fold greater current than strain DLl depending upon the electrode material, enabling
comparative electrochemical analysis to study the mechanism of current generation'^.
CV is finding its application widely in the field of study of antioxidant activities
of various food products like green tea, apple vinegar, citrus fruits, ascorbic acid,
crude extracts and pure compounds derived from natural sources, etc. The cyclic
voltammograms recorded for such compounds provide information about the
antioxidant capacity of a particular compound or a crude extract that is being
analysed^. CV is used to study the electrochemical behavior of anthocyanins and
anthocyanidins, so as to evaluate their antioxidant properties'^. Similarly CV study
has been carried out to evaluate total antioxidant capacity of plasma'* which
concluded that CV experiments that are time and cost effective can be used as simple
and relatively reliable method for assessment of body antioxidant status.
Page 166
Chapter 8 ^
CV studies of carbon steel corrosion in chloride-formation solution, effect of some
inorganic salts containing anions like sulphide, sulphate, and bicarbonate anions on
the pitting corrosion behavior of carbon steel has also been reported'^.
Based on the above observations it is evident that CV can be used as powerful
electrochemical technique in studying antioxidant capacity of food products and
biological fluids, corrosion behavior of metals, the reaction kinetics, synthesis and
electrochemical behavior of gold, silver'^ and PbS nanoparticles,'^ etc. hispired by
such interesting applications of CV, and to apply the principle and procedures to carry
out electrochemical studies on the organic compound of our interest, the present work
was taken up in our lab.
Oxidation reactions are very important process that takes place in all living
organisms, these oxidation reactions produce free radicals, which initiates the chain
reactions. The chain reactions taking place in the cell can cause cell damage'^ and
even cell death, which leads to many diseases like heart diseases, types of cancers,
stroke, premature aging etc. So to control these chain reactions and lower the risk of
cellular damage, body has a defense system of antioxidants, such as the enzymes
catalase, superoxide dismutase, and various peroxidases which react with the free
radicals and hence protecting cells'^. Inhibition and insufficient levels of such
enzymes leads to cell damage, therefore it is necessary to use antioxidant supplements
to protect our body from diseases caused by cell damage.
Antioxidants are abundantly found in natural sources like fruits and vegetables.
Beta carotene, Lutien, Lycopene, Selenium, Vitamin C, etc. are some of the examples
for this class of antioxidants. Many chemists are working on synthesizing these
molecules that can act as potential antioxidants with minimal or no side effects on the
Page 167
Chapter 8
human body. Antioxidants are not only used in the field of medicine, but also in
cosmetic and food processing industries to increase the shelf lives of their products.
Therefore analyzing the antioxidant capacity of natural as well as synthetic
compounds helps in identifying more compounds to function as antioxidants.
Many techniques such as spectrophotometric methods ' , chemiluminescence ,
fluorescence' ^ and electron spin resonance spectroscopy^'' have been used to evaluate
the antioxidant activities of plant extracts, fruit juices, biological fluids, pure
compounds isolated from natural sources, synthetic compounds, etc.
In the present work, the electrochemical technique such as cyclic voltammetry is
used to study the electrochemical behavior of 2-ethylquinizarin(2-EQ). The
electrochemical behavior was studied using glassy carbon electrode as a working
electrode at different scan rates. The effect of concentrations of the analyte at different
pH values, and in the presence of different electrolytes was also studied.
8.2 Materials and methods
8.2.1 Chemicals and reagents
The compound 2-EQ was synthesized, as described in chapter 6. Solvent DMSO
of analytical grade was used, appropriate amount of pure compound was dissolved in
DMSO to get 5mM standard solution of 2-EQ. Tetra butylammonium perchlorate
(TBAP) was used as supporting electrolyte. Deionized water was used throughout the
experiments.
8.2.2 Preparation of Britten Robinson Buffer
The Britton-Robinson buffer solutions were prepared as per the procedure in
literature^ ' ^. 0.01 M acetic acid, 0.01 M phosphoric acid and 0.01 M boric acid
Page 168
Chapter 8
solutions were prepared separately in 100 ml volumetric flasks and then the solutions
of identical volume ratio of three acids were mixed together and titrated to the desired
pHwithO.OlMNaOH.
8.2.3 Cyclic voltammetry Cell
Cyclic voltammetric experiments were performed using three electrode system
consisting of the glassy carbon electrode as working electrode, saturated calomel as a
reference electrode and a platinum counter electrode are immersed in solution of
analyte, along with the supporting electrolyte taken in a cell as shown in Figure-8.4.
Since the response of the compound being studied at the electrode surface depends on
how the electrode has been prepared prior to running the experiment, the working
electrode is polished and rinsed before starting the experiment.
Figure-8.4: The electrochemical cell
Page 169
Chapter 8
8.2.4 Cleaning of the working electrodes
Prior to the experiment, the GCE was polished in an aqueous suspension of 0.05
|im alumina (BAS) on an alumina polishing pad to get mirror surface, and then rinsed
with double distilled water in an ultrasonic bath for few minutes. This cleaning
procedure was applied always before electrochemical measurements.
8.3 Experimental Procedure
About 10 ml of supporting electrolyte in DMSO was taken in an electrochemical
cell, the solution was stirred well for a minute using the magnetic stirrer. Cyclic
voltammetric measurements for the analyte 2-ethylquinizarin, were run from -1.1 to -
0.2V/S, at different scan rates, different concentrations of the analyte, in the presence
of different electrolytes and at different pH values of the solution containing 2-EQ.
8.3.1 Effect of different scan rates on electrochemical behavior of 2-EQ
Effects of scan rate on the peak current of 2-EQ were studied at GCE. For each of
the CV runs made, the scan rate was varied as 25, 50, 75, 100 upto 300 mV/s at room
temperature. From the parameters obtained from the cyclic voltammograms of 2-
ethylquinizarin at different scan rates, it is well evident that the anodic current and
oxidation potential increases with increasing sweep rate. The anodic peak current (Ipa)
increase linearly with the square root of the scan rate (Inset of Figure-8.5, correlation
coefficient R = 0.9956) which is typical of a diffusion controlled electrochemical
process .
Page 170
Chapter 8
Table-8.1: Effect of different scan rates on the peak current and potentials of
2-EQ in DMSO at O.lmV/s
Diff. Scan rate (mV) 25
50
75
100
125
150
175
200
225
250
275
300
Epa (V)
0.8549
0.8878
0.9044
0.9223
0.9361
0.9499
0.9499
0.9584
0.9693
0.9734
0.9726
1.0010
Apa
(mA)
0.0023
0.0030
0.0035
0.0041
0.0051
0.0056
0.0059
0.0064
0.0067
0.0073
0.0082
0.0084
Page 171
Chapter 8
0.0100
0.0075
0.0025-
0.0000
— I 1 1 1 1 1 —
0.45 0.60 0.75 0.90
EN vs SCE
1.05
Figure -8.5: Cyclic voltammograms at different scan rates (a-1) 25, 50, 75,100, 125,150,175,200,225,250,275 and 300mV/s in a solution containing 5mM 2-EQ. Inset: The calibration curve, plot of Ip/(scanrate)''^
8.3.2 Effect of different electrolytes on electrochemical behavior of 2-EQ
Effects of different electrolytes like, NaCl, KCl, LiCl, HgzCli, K2S04and K2CO3
on the peak current at the GCE is studied. For the 5ml of DMSO, 200|aL of analyte
(5mM of 2-EQ), and appropriate amount of electrolytes were added, and CV
readings were recorded for every electrolyte at the constant scan rate O.lmV/s, CV
profiles of 2-EQ change with the different type of electrolytes. Among all the
electrolytes studied here, the redox reaction of 2-EQ was found to be more for NaCl,
where as peak potentials and the redox peak current of 2-EQ was found to be less for
KCl. This could be explained on the basis of size of the cation present in the
Page 172
Chapter 8 ^
electrolyte used. Since the cations predominate in the electrical double layer at these
potentials and increase in size of cation leads to decrease in peak current making
reaction more difficult, resulting in shift of peak potentials towards more negative
values ' as presented in Table-8.2.
Table- 8.2: Effect of different electrolytes on the peak current and potentials of 2-EQ in DMSO at O.lmV/s
Different
electrolytes
GCE
HgaCb
K2CO3
K2SO4
KCl
LiCl
NaCl
Epa
(V)
0.2123
0.9030
0.9030
0.8892
0.7593
0.7951
0.8267
Epc
(V)
0.2399
0.2424
0.2399
0.3146
0.2926
0.2813
^ Epa + Epc
(V)
0.5744
0.5727
0.5645
0.5369
0.5438
0.5540
(mA)
9.0695E"^
0.001957
0.002637
0.002616
0.002331
0.002657
0.003578
(mA)
-8.0240 E"
-8.2200 E "
-0.001094
-4.9268 E-
-9.2394 E-"
-8.2200 E"
Thus among the electrolytes NaCl, KCl, and Hg2Cl2, the size of Na^ cation was
found to be smaller, hence the higher value of redox peak current for 2-EQ observed
as shown in Figure-8.6.
Page 173
Chapter 8
0.0045 -
0.0030 -
<
10.0015-
0.0000 -
-0.0015-
a. GCE b. Hg^CI c. K^CO^ d.K.SO, e.Kbl f. LiCI g. NaCI
1 ' 1 " ' ' •""r"-"-" i •
h >
\ ' "I ' 1 0.0 0.2 0.4 0.6 0.8
EA^ VS SCE 1.0
Figure-8.6 : Cyclic voltammograms at different electrolytes (a-g) GCE, HgiCh, K2CO3, K2S04,KC1, LiCl, NaCI in a solution containing 200^L of 5mM of 2-EQ at scan rate O.lmV/s
8.3.3 Effect of pH on redox peak currents and peak potentials of 2-EQ
The effect of pH on the GCE, was investigated under constant concentration of 2-
EQ (5mM) at constant scan rate of O.lmVs"' in the range of pH values 2-10, using
Britton-Robinson buffer. pH of the electrolyte solution has significant influence on
the redox reaction of 2EQ at GCE. Results of peak potentials and peak currents are
shown in Table-8.3 Both peak current and peak potential were found to be shifted
towards the lower values with increase in pH. This implies that the oxidation of 2-EQ
becomes increasingly slow in the basic pH values^^. Figure-8.7 and Figure-8.8 shows
the cyclic voltammograms recorded for 5mM of 2-EQ with the GCE at the different
values of pH 6-9 and pH 2-5 respectively.
Page 174
Chapter 8
Table-8.3: Effect of different pH values on the peak current and potentials of 2-EQ in DMSO at O.lmV/s
Different
pHs
PH = 2
PH = 3
PH = 4
PH = 5
PH = 6
PH = 7
PH = 8
PH = 9
PH=10
Epa
(V)
1.0000
0.7597
0.8196
0.6203
0.7037
0.6203
0.5570
0.5125
0.3498
Epc
(V)
0.5408
0.5757
0.4231
0
0.2853
0.2091
0.2221
0.2594
-0.7557
I? Epa+Epc
(V)
0.7704
0.6677
0.6213
0.3101
0.4945
0.4147
0.3895
0.3859
-0.2029
• •pa
(mA)
0.0048
0.0029
0.0030
0.0081
0.0036
0.0026
0.0023
0.0018
0.0320
ipc
(mA)
-6.6848E-^
-7.9683E-^
-9.8033E"'
-0.0011
-9.1370E-^
-5.7345E"'
-4.8115E-^
-4.8825E-^
-0.0069
Page 175
Chapter 8
<
E
0.005
0.000
-0.005-
-0.010
0.005
0.000
-0.005
-0.010-j
pH6
pH 8
pH7
pH9
— I — I — I — ' — I — • — I — ' — I — ' — I — I — I — I — I — 1 — I — • — I —
-1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0
EA/ vs SCE
Figure-8.7: Cyclic Voltammograms for 2-ethylquinizarin at pH values 6 - 9
0.005
0.000-
•0.005
< E 0.005
0.000
-0.005
pH2
pH4
pH3
pHS
- 0 . 0 1 0 -1 1 1 1 1 1 • 1 1 ) 1 1 I I r—] 1 1 1 p
-1.0 -0.5 0.0 0.5 1.0 -1.0 -0.5 0.0 0.5 1.0
EA/ vs SCE
Figure- 8.8: Cyclic Voltammograms for 2-ethylquinizarin at pH values 2 - 5
Page 176
Chapter 8 ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^
8.3.4 Effect of concentration on the peak currents and peak potentials of 2-EQ
The effect of concentration on the peak current of 2-EQ at different concentrations
and constant scan rate of O.lmV/s using TBAP as supporting electrolyte has been
investigated. Initially the voltammogram of the oxygen reduction was recorded in the
supporting electrolyte, TBAP in DMSO(lOml) without the analyte 2-EQ to obtain
the original limiting value of the oxygen current (/or), which corresponds to the
oxygen solubility in this electrolyte. Then the supporting electrolyte was bubbled for
3-5 min with nitrogen to remove oxygen from the electrolyte. The voltammogram of
the supporting electrolyte without oxygen was scanned to obtain the residual current
value (/res). Later different concentrations of antioxidant 2-EQ (5Mm of 2-EQ was
dissolved in 10ml of DMSO) were added to the renewed portion of the supporting
electrolyte and under the same conditions, the proportional decrease of the oxygen
current corresponding to the concentrations of the added antioxidant 2-EQ at constant
potential were recorded as shown in TabIe-8.4.
Page 177
Chapter 8
Table- 8.4: Cyclic voltammetric parameters of different concentrations of 2-EQ at scan rate of O.lmVs'*
Concentration
of 2-EQ
(liM)
O^M
15nM
20 nM
25|aM
30nM
35nM
40^M
45 iM
50|aM
55|aM
Epa
(V)
-0.6933
-0.6850
-0.6933
-0.6898
-0.6894
-0.6931
-0.6898
-0.6976
-0.6976
-0.6996
Epc
(V)
-0.5957
-0.6002
-0.6055
-0.6041
-0.6082
-0.6087
-0.6084
-0.6123
-0.6118
T? Epa+Epc Ei/2= 2
(V)
-0.3466
-0.6403
-0.6475
-0.6476
-0.6467
-0.6506
-0.6492
-0.6530
-0.6549
-0.6557
(mA)
0.0061
0.0037
0.0030
0.0025
0.0021
0.0016
0.0013
0.0010
6.9444E"^
6.8914E"'*
Ape
(mA)
-0.0058
-0.0072
-0.0083
-0.0095
-0.0105
-0.0112
-0.0123
-0.0131
-0.0142
Page 178
Chapter 8
0.010^
0.005 -
^ 0.000-
E
— -0.005 -
-0.010-
-0.015-
-1,
o^M
200 400 600 C of 2EQ/MM
1 ' 1 n—'—I—'—I—r •1.0 -0.8 -0.6 -0.4 -0.2
E/VvsSCE
Figure-8.9: Cyclic voltammograms for different concentrations of 2EQ, ie 0(iM, ISiiM, 20MM, 25^M, 30MM, 35MM, 40|iiM, 45^M, SO^iM, 55^M of 2EQ (Inset: decrease in peak currents with increasing concentration of 2-EQ)
From the Figure-8.9 it can be inferred that after addition of the analyte 2-EQ, the
anodic peak current decreases. This depletion in anodic peak current is due to the
antioxidant substrate reacting with the superoxide O2 and decreasing its concentration
at the electrode surface. The quantity of current decreased was directly proportional to
the radical scavenging capacity of antioxidant-2EQ. This behavior of 2-EQ is similar
to the behavior of standard antioxidants ascorbic acid and uric acid^\
As a result of our investigations the curve of the relative change of the oxygen
reduction current density[ j /(jor - jres)\ against antioxidant 2-EQ concentration in the
bulk of the solution at the GCE in supporting electrolyte were plotted as shown in
Figure-8.10. Also coefficient of antioxidant activity K, the relative strength of
antioxidants to scavenge the O2' was determined as the slope of the curve as shown in
Page 179
Chapter 8
Figure-8.10, which was found to be 0.025, where j is the peak current at the
particular concentration of 2-EQ.
0.6-
0.5-
0.4-V)
•• s 0.3 -• ^ ^
~~" • ^ ^
0.2-
0.1 -
•
1
0.8
• \
• V
N. •
, . 1 1 1 1 1 1 1.2 1.6 2.0 2.4
Cone, of 2 - E Q / m M
Figure-8.10: Curve of the relative change of the oxygen reduction current density against different concentrations of antioxidant 2-EQ
Antioxidant activity coefficient K can be calculated by using equation (2)*
A/-K
{jor -jres)^C Equation. (2)
Aj = Change in the anodic peak current density with the addition of investigating
analyte.
AC = Change in the concentration of analyte.
jor = Limiting current density in the presence of oxygen, without the analyte in the
solution.
jres = Residual current density without oxygen in the solution.
Page 180
Chapter 8
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