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.

Page 101

Chapter 5

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

Page 102

Chapter 5

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

Page 103

Chapter 5

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.

Page 104

Chapter 5

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

Page 105

Chapter 5

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

Page 106

Chapter 5

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

Page 107

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.

Page 108

Chapter 5

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.

Page 109

Chapter 5

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

Page 110

Chapter 5

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.

Page 111

Chapter 5

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.

Page 112

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.

Page 113

Chapter 5

References

1. Ying-Shan Han, Robert Van der Heijden, Robert Verpoorte. Plant cell. Tissue and Organ Culture, 2001: 67: 201.

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.

Page 114

Chapter 5

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.

Page 115

Chapter 5

37. Christian DUendorf, Susanne Katharina Kreth, Soo Whan, Helmut Ritter. Beilstein. J. Org.Chem., 2013: 9: 453.

38. M Anamul Haque, M Muhibur Rahman, M Abu Bin Hasan Susan. J. Solution C/?ew., 2011: 40(5): 861.

Page 116

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-

Page 118

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

Page 119

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

Page 120

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).

Page 121

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.

Page 122

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.

Page 123

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.

Page 124

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.

Page 125

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.

Page 158

Chapter 7

References

1. Dr. Mark Pervical. Clinical Nutrition Insights, NUT031 1/96 Rev. 10/98, 1998 Advanced Nutrition Publications, 1.

2. Helmut Sies, Wilhelm Stahl, Alfred R Sundquist. Ann. NY Acad ScL, 1992: 669: 7.

3. L Langseth. Antioxidant Vitamins Newsletter., 1993: 4: 3.

4. B Halliwell. Lancet, 1994: 344: 721.

5. RA Jacob. Nutr. Res., 1995: 15(5): 755.

6. FJ Kelly. J. Int. Fed Clin. Chem., 1998: 10(1): 21.

7. Barrie Cassileth, Ian Yarrett .The Asco. Post, 2012: 3(15): 96.

8. C Andre, I Castanheira, JM Cruz, P Paseiro, A Sanches-Silva. Trends in Food Science and Technology, 2010: 21(5): 229.

9. I Bogdan Allemann, L Baumann, Skin Therapy Lett., 2008: 13(7): 5.

10. GS Hammond, CE Boozer, CE Hamilton, JN Sen. J.Am.Chem.Soc, 1955: 77(12): 3233.

11. Boyong Xue, Kenichi Ogata, Akinori Toyota. Polym. Degrad. Stabil, 2008 93(2): 347.

12. Aurelia Magdalena Pisoschi, Gheorghe Petre Negulescu. Biochem. and Anal. Biochem., 2011: 1: 106

13. FF Benzie, JJ Strain. Method Enzymol, 1999: 299: 15.

14. Om P Sharma, Tej K Bhat. Food Chemistry., 2009: 113: 1202

15. Milan Ciz, Hana Cizova, Petko Denev, Maria Kratchanova, Anton Slavov, Antonin Lojek. Food Control, 2010: 21: 518.

16. Titus A M Msagati. The Chemistry of Food Additives and Preservatives, 2013, John Wiley & Sons, Ltd, l" edn. West Sussex, 17.

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.

22. R Apak, K Guclu, M Ozyurek, B Bektasoglu, M Bener. Methods Mol. Biol, 2010: 594:215.

23. Jiri Sochor, Jiri Dobes, Olga Krystofova, Branislav Ruttkay-Nedecky, Petr Babula, Miroslav Pohanka, Tunde Jurikova, Ondrej Zitka, Vojtech Adam, Borivoj Klejdus, Rene Kizek. Int. J. Electrochem.Sci., 2013: 8: 8464.

24. G Kroyer. Z Ernahrungswiss., 1986: 25(1): 63.

25. Lim Yau Yan, Lim Theng Teng, Tee Jing Jhi. Sunway Academic Journal, 2006: 3: 9.

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.

29. CA Rice-Evans, NJ Miller, G Paganga. Free Radic. Biol Med, 1996: 20(7): 933.

30. RJ Anto, K Sukumaran, G Kuttan, MN Rao, V Subbaraju, R Kuttan. Cancer Lett., 1995 : 97(1): 33.

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.

37. S Kumar, D Kumar, Manjusha, K Saroha, N Singh, B Vashishta. Acta. Pharm., 2008:58:215.

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

References

1. Heinze, Jurgen. Angew.Chem. Int.Ed.Engl, 1984: 23( 11): 831.

2. AJ Blasco, MC Gonzalez, A Escarpa. Anal Chim Acta., 2004: 511:71.

3. AJ Blasco, MC Rogerio, MC Gonzalez, A Escarpa. Anal. Chim Acta., 2005: 539:

237.

4. S Chevion, MA Roberts, M Chevion. Free Radic. Biol. Med., 2000: 28: 860.

5. MS Cosio, S Buratti, S Mannino, S Benedetti. Food Chem., 2006: 97: 725. 6. EI Korotkova, YA Karbainov, AV Shevchuk. J .Electroanal. Chem, 2002: 518:

56.

7. Junjiang Zhu, Zhen Zhao, Dehai Xiao, Jing Li, Xiangguang Yang. Electrochem.Commun., 2005: 7(1): 58.

8. Jacob D Gibson, Bishnu P Khanal, Eugene R Zubarev. J.Am. Chem.Soc, 2007: 129: 11653.

9. Qian Ximei. Nat. Biotechnol, 2008: 26: 1

10. Hainfeld, James. Phys.Med.Biol, 2004: 49: N309 .

11. Mohammad Etesami, Norita Mohamed. Int. J. Electrochem. Sci., 2011: 6: 4676.

12. Sarah M Strycharz, Anthony P Malanoski, Rachel M Srinder, Hana Yi, Derek R Lovley, Leonard M Tender. Energy Environ.Sci., 2011: 4: 896 .

13. Andreia A de Lima, Eliana M Sussuchi, Wagner F De Giovani. Croat. Chem. Acta., 2007: 80(1): 29.

14. J Psotova, J Zahalkova, J Hrbac, V Simanek, J Barek. Biomed. Pap. Med. Fac. Univ. Palacky Olomouc. Czech. Repub., 2001: 145(2): 81.

15. MA Deyab, ST Keera Egypt. J.Pet., 2012: 21(1): 31.

16. Marcella Giovanni, Martin Pumera. Electroanal, 2012: 24(3): 615.

17. Yu Jun Yang, Lun Ying He, Qin Fa Zhang. Electrochem. Commun., 2005: 7(4): 361.

18. Dr. Mark Pervical. Clinical Nutrition Insights, NUT03I 1/96 Rev. 10/98, 1998: Advanced Nutrition Publications, 1.

19. B Halliwell. Nutr.Rev., 1994: 52: 253.

Page 182

Chapter 8

20. C Sanchez-Moreno, JA Larrueri, F Saura-Calixto. J. Sci. Food Agri., 1998: 76: 270.

21. A Cano, J Hernandez-Ruiz, F Garcia-Canovas, M Acosta MB Amao. Phytochem. Anal.,l99S: 9: 196.

22. AP Arzamasceva, EI Shkarina, TV Maksimova. Chemico. Pharm. J. (Russia)., 1999: 11: 17.

23. K Krumova, P Oleynik, P Karam, G Cosa. J.Org.Chem., 2009: 4(10): 3641.

24. Teresita S Martin, Hiroe Kikuzaki, Masashi Hisamoto, Nobuji Nakatani. J.Am.Oil Chem.Soc, 2000: 77(6): 667.

25. HTS Britton. Hydrogen Ions, 3"* edn Chapman and Hall, 1942: 1.

26. HTS Britton, RA Robinson. J. Chem. Soc, 1931: 1456.

27. Simic Aleksandra, Dragan Manojlovic, Dejan segan, Marija Todorovic. Molecules, 2007: 12: 2327.

28. GS Suresh, LK Ravindranath. Bull.electrochem., 2000: 16(11): 515.

29. RRarsons. J. Electroanal.Chem., 1969: 21: 35.

30. Andreea Lungu, Daniela Bala, Teodora Staicu, Constantin Mihailciuc. Rev.Roum.Chim., 2012: 57(7-8): 707.

31. R Manjunath. Ph.D thesis entitled "Development of Layer-by-Layer self Assembled thin films for Chemical and Biochemical Sensors", Kuvempu University, 2013: 182.

Page 183