Journal of Pharmacognosy and Phytochemistry 2015; 3(6): 122-124

E-ISSN: 2278-4136
P-ISSN: 2349-8234 Comparative study of antioxidant activity of polyphenols
JPP 2015; 3(6): 122-124
Received: 02-01-2015
isolated from frozen and fresh leaves of Trachyspermum
Accepted: 18-02-2015 ammi (Ajwain)
Mazahir Raza
Chief Chemist
Mazahir Raza, Anshul K. Shukla, Tatheer Fatima, Shaista Ali
R&D Centre, AMA Herbal
Abstract
Laboratories (P) Ltd, Lucknow,
Trachyspermum ammi, Ajwain is a rich in vitamins and minerals, it is also concentrated in health-
India 226017.
promoting phytonutrients such as carotenoids (beta-carotene, and lutein) and flavonoids to provide
powerful antioxidant protection. The aim of this was to determine the effect on total antioxidant activity
Anshul K. Shukla
in fresh and frozen ajwain leaves. The extract of fresh and frozen ajwain leaves was subjected to in vitro
Chemist, R&D Centre, AMA
free radical scavenging DPPH assay. The results obtained were analyzed. Antioxidant activity was
Herbal Laboratories (P) Ltd,
exhibited significantly higher in frozen ajwain leaves than fresh one. Lutein content was also found
Lucknow, India 226017.
higher in frozen Ajwain leaves in comparison with fresh Ajwain. Hence the antioxidant activity of ajwain
is highly correlated to the concentration of polyphenols and freezing condition.
Tatheer Fatima
Asst. Chemist
Keywords: Ajwain, Antioxidant activity, Radical scavenging activity, Polyphenols Lutein. Carotenoids
R&D Centre, AMA Herbal
Laboratories (P) Ltd, Lucknow,
India 226017. 1. Introduction
Leaves of Ajwain, Trachyspermum ammi is used as green vegetable for salad has high
Shaista Ali nutrition values. The dark green leaves contain many valuable nutrients, especially the
Research Scholar antioxidant carotenoids, lutein and zeaxanthin. To get availability of ajwain leaves throughout
Department of Botany, the year supply of frozen ajwain has become popular now a days. It is most important to check
Maharshi university, Lucknow how freezing effects the antioxidant property of ajwain leaves.
226008 Poly phenols mainly lutein is reducing agent and belong to the group of antioxidants (Sies
1997) [13]. The important role of lutein is to neutralize the free radicals or reactive oxygen
species such as the hydroxyl radical (·OH), the superoxide anion (O2·−) and others (Bergamini
et al. 2004; Dean et al. 1997; Stoian et al. 1996) [2, 4, 15]. Lutein exert the antioxidant actions
based on their singlet oxygen quenching properties and ability to trap peroxyl radicals (Paiva
and Russell 1999; Stahl and Sies 1996) [10, 14]. The first one depends on the number of
conjugated double bonds of the molecule and is influenced to a lesser extent by cyclic or
acyclic carotenoid groups (Paiva and Russell 1999; Krinsky 1998) [10, 6]. Also, cyclic
substituents as an end group of polyene hydrocarbon chain (in the structure of carotenoids, as
well as xanthophylls like lutein), influence the antioxidant activity of the mentioned
compounds.
There are several methods for assays have been introduced for the measurement of the total
antioxidant activity of pure compounds (Re et al. 1999; Rice-Evans et al. 1996; Miller and
Rice-Evans 1994; Miller et al. 1996; Kono et al. 1995) [11, 9, 12, 8, 12, 5]. Each method relates to
the generation of a different radical, acting through a variety of mechanisms and the
measurement of a range of end points at a fixed time point or over a range (Miller et al. 1996;
Kono et al. 1995; Arnao et al. 1990) [9, 12, 5, 1] Two types of approach have been taken into
consideration. One of them is the inhibition assays; in that, the extent of scavenging by
hydrogen or electron donation of a preformed free radical is the marker of antioxidant activity,
as well as assays involving the presence of antioxidant system during the generation of the
radical (Re et al. 1999) [11]. These assays have been based on different principles and
experimental conditions. In general, DPPH· (1, 1-diphenyl-2-picrylhydrazyl) and ABTS (2, 2′-
azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) radical cations are the basis of the
Correspondence: spectrophotometric methods that have been applied to the measurement of the total antioxidant
Mazahir Raza activity of solutions of pure substances. The total antioxidant activity can be expressed by the
Chief Chemist ferric reducing antioxidant power assay (FRAP), oxygen radical absorbance capacity (ORAC),
R&D Centre, AMA Herbal superoxide radical scavenging activity or Trolox equivalent antioxidant activity (Bertoncelj et
Laboratories (P) Ltd, Lucknow, al. 2007) [3]. The aim of this study was to evaluate the radical scavenging activity of
India 226017.
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samples of extracts fresh ajwain and frozen ajwain. The darkness for 30 min. After 30 min, absorbance at 515 nm was
antioxidant activity of extracts from ajwain is highly correlated measured, and blank was prepared by the use of methanol.
to the concentration of polyphenols including phenolic acids, Because one of the most important constituents of ajwain
flavonoids as well as carotenoids. For that reason, the leaves extract is lutein, we decided to measure antioxidant
concentrations of these compounds in analysed extracts were activity of this compound. The antioxidant activity of lutein
determined. (Roth, Karlsruhe, Germany) solutions in the concentration
range of 10 to 100 μg/mL of substance was evaluated. The
2. Materials and Methods antioxidant activity was expressed in radical scavenging
2.1 Materials, Chemicals and Reagents activity (RSA). RSA was measured by using equation.
Fresh and frozen ajwain leaves were obtained from a local
marketplace at Lucknow, 1, 1-Diphenyl-2-picrylhydrazyl RSA= (ADPPH – A) X100%
(DPPH), methanol, acetonitrile, Folin–Ciocalteu reagent, ADPPH
phosphomolybdate, phosphotungstate, gallic acid, lutein, ethyl
acetate were all of analytical grade. 2.4 Determination of Total Phenolic Contents in Ajwain
leaves extracts
2.2 Preparation of extracts of Ajwain Leaves According to references (Wettasinghe and Shahidi 1999; Mélo
For separation of polyphenols from ajwain leaves, et al. 2006) [17, 7]. The total phenolic content was measured
Supercritical fluid extractor SFE 7680 T (Hewlett Packard, spectrophotometrically at 760 nm using the Folin–Ciocalteu
Avondale, PA, USA) was applied. Polyphenols and other reagent. This reagent is a mixture of phosphomolybdate and
compounds were extracted from ajwain with supercritical fluid phosphotungstate used for the colorimetric assay of
and liquid CO2. The solvent modified with methanol (5%) was polyphenols. Standards of gallic acid in the concentration
applied at the pressures of 100, 175 and 258 bar. Additionally, range of 0.1 to 1.3 mg/mL were also measured. The results
extraction at 258 bar was made without organic modifier. were expressed as gallic acid equivalents (GAE) of dry matter.
From every step of extraction, four fractions were obtained (1-
mL volume of methanolic extract). Particular supercritical 2.5 Quantitative Analysis of Ajwain Extracts and Lutein
fluid extraction (SFE) conditions include the following: by HPLC–UV-VIS
extraction time, 30 min; supercritical fluid, carbon dioxide The ajwain leaves extracts were analysed using an Agilent
(purity 99.99%) with organic modifier (5% methanol); fluid model 1100 liquid chromatographic system consisting of a
temperature, 50 °C; fluid pressures, 100, 175 and 258 bar; quaternary pump and a UV-VIS detector (Agilent
temperature of extraction, 39 °C; receiver temperature, 65 °C Technologies, Waldbronn, Germany). Compounds were
and trap, a stainless steel tube. For every extraction, 0.2 g of separated on a Discovery C18 column (150×4.6 mm, 5 μm)
dried ajwain leaves was taken and frozen ajwain leaves was from Supelco (Bellefonte, PA, USA). Methanol: water
previously kept at room temperature for 3 h. (80:20v/v) was used as eluent A, while ethyl acetate was used
as eluent B. The mobile phase was delivered at 1.0 mL/min in
2.3 DPPH Radical-Scavenging Assay of ajwain extracts a gradient mode. For the calibration procedure, standard
and Lutein Solutions mixtures of lutein in methanol were prepared. The
The inhibition of free radical scavenging activity of ajwain concentration of lutein was in the range of 5.0–100.0 μg/mL.
leaves extracts was measured by spectrophotometer using The linearity of the detector response was determined by the
DPPH [W. Brand-Williams, M. E. Cuvelier and C. Berset square correlation coefficients of the calibration curves
1995] [16]. Three millilitres of DPPH solution (4 mg of pure generated by three repeated injections of standard solutions at
DPPH dissolved in 10 mL of acetonitrile and 190 mL of six concentration levels (5.0–100.0 μg/mL). We defined a
methanol) was mixed with 100 mL of extract and stored in the signal to noise ratio of 3 as a limit of detection

Table 1: Calibration data used for the determination of lutein in ajwain extracts by HPLC–UV-VIS

RSD
Concentration Retention R2 LOD LOQ
Calibration For peak
Method estimated range (μg/mL, time (μg/mL, (μg/mL, (μg/mL,
curve areas
injected) tR (min) injected) injected) injected)
(%)
HPLC–UV-Vis 5.0–100.0 8.208 y040.189x−18.907 0.9992 0.2 0.6 5.5

(LOD) and a signal to noise ratio of 9 as a limit of leaves. The RSA value obtained for frozen ajwain leaves were
quantification (LOQ). All calibration data for lutein, including 30.3%. Meanwhile, the RSA obtained for fresh ajwain did not
retention time (tR), calibration equations using peak area, exceed 19.8 %. Particular results obtained during experiments
linearity with correlation coefficient (R2) of the calibration concerning the determination of RSA for fresh and frozen
curves, LOD and LOQ; precision data as a relative standard ajwain leaves are presented in Table 2.
deviation (RSD) estimated for peak areas are presented in Lutein, which is a xanthophyll and belongs to the carotenoid
Table 1. Standard solutions and plant extracts were analysed in group. Radical scavenging activity was evaluated for lutein
triplicate. solutions in the concentration value range of 10 to 100 μg/mL
of substance. The lower concentration of lutein in the solution
3. Results and Discussion gave a RSA value of 6.2%, but for ten times higher
In the presented study it was found that we obtained higher concentration value of lutein (100 μg/mL), obtained values of
results of RSA for frozen ajwain leaves than for fresh ajwain RSA did not exceed 10.0%. Obtained results show that lutein
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as a component of ajwain leaves extracts takes an insignificant measuring naringin using 2, 2′-azinobis (3-
part in the total antioxidant activity of these plant extracts. The ethylbenzothiazoline-6-sulfonic acid). Anal Biochem
total content of polyphenols in ajwain samples varied from 1990; 185:335–338.
1.823 to 4.946 mg/g of dry matter. 2. Bergamini CM, Gambetti S, Cervellati C. Oxygen,
For the determination of lutein, the quantitative analysis of reactiveoxygen species and tissue damage. Curr Pharm
ajwain leaves extracts was performed by the use of the Des 2004; 10:1611–1626.
external standard method. Data obtained for the standards in 3. Bertoncelj J, Doberšek U, Jamnik M, Golob T. Evaluation
concentration values that range from 5.0 to 100.0 μg/mL gave of the phenolic content, antioxidant activity and colour of
a calibration curve, which was used for the determination of Slovenian, honey. Food Chem 2007; 105(2):822–828.
lutein in real samples (extracts from fresh and frozen ajwain 4. Dean RT, Fu S, Stocker R, Davies MJ. Biochemistry and
leaves). Regression coefficients of the calibration curves 0.999 pathology of radical-mediated protein oxidation. Biochem
for UV-VIS detection were obtained. LOD and LOQ for UV- J 1997; 324:1–18.
VIS detection were 0.2 μg/mL and 0.6 μg/mL, respectively. 5. Kono Y, Shibata H, Kodama Y, Sawa Y. The suppression
Since the values for LOD and LOQ are low in relation to the of the N-nitrosating reaction by chlorogenic acid.
usual content of lutein in ajwain leaves extracts, we concluded Biochem J 1995; 312:947–953.
that this method can be used for quantitative analysis. RSD 6. Krinsky NI. Overview of lycopene, carotenoids, and
values estimated for the peak areas of the lutein standards did disease prevention. Proc Soc Exp Biol Med 1998; 218:95-
not exceed 5.5%. 97.
The results after analysis of extracts from fresh and sample of 7. Mélo EA, Lima VLAG De, Maciel MIS, Caetano ACS,
frozen ajwain leaves shown the highest concentration values of Leal FLL. Polyphenol, ascorbic acid and total carotenoid
lutein 0.88 mg/g for frozen ajwain and 0.83 mg/g for fresh contents n common fruits and vegetables. Braz J Food
ajwain. Polyphenol, and lutein concentrations in samples of Technol 2006; 9(2):89–94.
green ajwain leaves (calculated for dry matter of sample, mean 8. Miller NJ, Rice-Evans CA. Total antioxidant status in
of triplicate analysis) plasma and body fluids. Methods Enzymol (1994);
234:279–293.
Sample Concentration of Concentration of 9. Miller NJ, Castelluccio C, Tijburg L, Rice-Evans CA. The
Ajwain polyphenolic acids (mg lutein (mg/g dry antioxidant properties of thioflavines and their gallate
leaves GAE/g dry matter) matter) esters— radical scavengers or metal chelator? FEBS Lett
Fresh 1.791±0.040 0.722±0.047 1996; 392:40–44.
Frozen 4.312±0.098 0.871±0.049 10. Paiva SAR, Russell RM. β-Carotene and other carotenoids
as antioxidants. J Am Coll Nutr 1999; 18(5):426–433.
The detection of the amount of polyphenols and carotenoids 11. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M,
must be related to their radical scavenging activity values, as Rice-Evans C Antioxidant activity applying an improved
this assay shows the potential health benefit of these ABTS cation radical decolorization assay. Free Radic Biol
compounds, which is very often independent of their absolute Med 1999; 26:1231–1237.
quantity. The RSA value is useful in the choice of the plant 12. Rice-Evans CA, Miller NJ, Paganga G. Structure-
genotype and also provides information on the optimal antioxidant activity relationships of flavonoids and
preparation way, which is the most important step to be phenolic acids. Free Radic Biol Med 1996; 20:933–956
checked before freezing the vegetables to maintain the 13. Sies H Oxidative stress: oxidants and antioxidants. Exp
antioxidant activity of polyphenols and carotenoids. Physiol 1997; 82:291–295.
14. Stahl W, Sies H Lycopene. A biologically important
4. Conclusions carotenoid for humans? Arch Biochem Biophys 1996;
This study confirms that the freezing of ajwain leaves is not 336:1–9.
the only way of food storage, but freezing also affects the 15. Stoian I, Oros A, Moldoveanu E. Apoptosis and free
release of carotenoids from plants. The low temperature radicals: mini review. Biochem Mol Med 1996; 59:93–97.
influences the destruction of the walls of the plant cells. 16. Brand-Williams W, Cuvelier ME, Berset C. “Use of Free
Antioxidant activity of extracts from ajwain leaves is Radical Method to Evaluate Antioxidant Activity,”
correlated to the concentration of carotenoids and polyphenols. Lebensmittel Wissenschaft und Technologie 1995;
The high level of the content of polyphenol acids and 28(1):25-30.
flavonoids in ajwain leaves influences the high antioxidant 17. Wettasinghe M, Shahidi F. Evening primrose meal: a
activity. source of natural antioxidants and scavenger of hydrogen
peroxide and oxygen-derived free radicals. J Agric Food
5. References Chem 1999; 47:1801–1812.
1. Arnao MB, Casas JL, del Rio JA, Acosta M, Garcia-
Canovas F. An enzymatic colorimetric method for

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