ЖУРНАЛ АНАЛИТИЧЕСКОЙ ХИМИИ, 2015, том 70, № 2, с. 196-200
ANTIOXIDANT ACTIVITY OF ESSENTIAL OIL OF Coriandrum sativum AND STANDARDIZATION OF HPTLC METHOD FOR THE ESTIMATION
OF MAJOR PHYTOMARKERS © 2015 K. Singh, R. Rani, P. Bansal, S. Medhe, M. M. Srivastava1
Department of Chemistry, Dayalbagh Educational Institute Dayalbagh, Agra-282110, UP, India 1E-mail: firstname.lastname@example.org Received 02.11.2012; in final form 06.08.2013
The essential oils of seed and whole plant of Coriandrum sativum were examined for their in vitro antioxidant activities which were found to be higher compared to different extracts of this plant. A simple, precise and specific HPTLC method has been developed for the determination of important phytomarkers of essential oils. The proposed method was validated in terms of linearity, precision, accuracy, limits of detection, and quantification.
Keywords: essential oil, C. sativum, antioxidant activity, HPTLC, phytomarker.
The demand of essential oils in the developed countries is increasing day by day as more and more spicy snacks are being introduced with standardized taste and fragrance by the fast food chains. The prolonged cooking of spice results in the loss of its nutritive and fragrance values; therefore, essential oils are used for cooking these days. They offer smoother texture in very small volume than their ground counterparts. One drop of essential oil is the equivalent of 1/4 tsp of the ground version . Essential oils are especially suitable as they can be used very conveniently (without any handling of the raw spice) and produce a standardized effect on taste. In addition to taste and aroma, the chemical composition of essential oils also provides valuable physical therapeutic benefits, as they have been shown to contain the concentrated phytomarkers . Among various medicinal uses, several bioactive constituents present in essential oils are found to exhibit antioxidant efficacy . The role of antioxidants in the prevention of degenerative diseases has been well documented [4, 5]. Interestingly, synthetic anti-oxidants such as butylated-hydroxyanisole, butylated-hydroxyltoluene, terbutyl-hydroquinone and propyl gallate, although are effective in the protection of un-saturated fats and oils, are discouraged because of their association with several demerits such as liver swelling, non-selective killing of the cells and carcinogenicity, and consequently their use has been restricted . Thus, the search for novel natural antioxidants has gained much focus in research [7, 8]. Natural antioxi-dants are assumed to be safe since they occur in plant foods and are seen more desirable than their synthetic
counterparts. Bioactive polyphenols, especially bioflavonoids, are very interesting as antioxidants because of their natural origin and ability to act as efficient free radical scavengers .
Furthermore, the development of instrumental methods for the identification and quantification of individual components in food and beverages has become extremely important for establishing their quality and genuineness. High performance thin layer chromatography (HPTLC) is recently introduced technique for the analysis of food products without chemical treatment of the samples that has the several advantages such as simplicity, speed, reproducibility and cost effectiveness . It is an offline technique: the subsequent steps are relatively independent, allowing parallel treatment of multiple samples during chromatography, derivatization and detection. Unlike other methods, HPTLC produces visible chromato-grams in which the complex information about the entire sample is available at a glance .
The plant, Coriandrum sativum belonging to family Umbelliferae, commonly known as Dhania, is highly reputed ayurvedic medicinal plant used as digestive stimulant, anti-inflammatory, antimicrobial, hypolipidemic, anti-mutagenic anti-carcinogenic . The present piece of work is aimed to monitor the antioxidant activity of essential oil of C. sativum and standardization of HPTLC method for the estimation of its important phytomarkers (linalool and geranyl acetate). The proposed HPTLC method is validated according to ICH guidelines based on linearity, accu-
racy in terms of recovery percentage, precision, limit of detection and quantification .
Reagents. The plant material (seeds and plant) of C. sativum was obtained from AG MARK shop, Bich-puri, Agra. All chemicals used including the solvents were of analytical grade. 2,2-Diphenyl-1-picrylhydra-zyl (DPPH), ascorbic acid, a-tocopherol, linalool and geranyl acetate, toluene and ethyl acetate were obtained from sigma Chemical Co., USA. HPTLC plates (silica gel 60 F254, 20 x 10 cm) were purchased from E. Merck (Darmstadt, Germany).
Sample preparation and analysis. Coriander plant (500 g) and seeds (300 g) were grounded in a blender and subjected to hydro distillation using a Clevenger glass apparatus for 4 h, separately. The oil samples were stored in vials in the refrigerator, after drying them over anhydrous sodium sulfate.
DPPH assay. Free radical scavenging capacity of seed and plant essential oils of C. sativum was determined using DPPH radical method . DPPH is a stable free radical with purple color (absorbing at 517 nm). Radicals were scavenged by antioxidant addition; solution becomes yellow because of the formation of 2,2'-diphenyl-1-picrylhydrazine. The reaction mixture containing dilution series of sample was incubated with DPPH solution in methyl alcohol. The content was mixed vigorously and allowed to stand for 30 min at room temperature. The extent of discoloration indicates the amount of DPPH scavenged. The ab-sorbance was measured at 517 nm. Ascorbic acid and a-
Table 1. IC50 values (^g/mL) of essential oils of C. sativum
Analyte DPPH assay Fenton's assay
C. seed oil 47.2 42.3
C. plant oil 35.4 31.1
a-Tocopherol 28.2 26.3
Ascorbic acid 21.8 20.0
tocopherol were used as standard antioxidants. All the tests were performed in triplicate and the percent inhibition was calculated using the formula: [(C - T)] x 100, where C is the absorbance of the control and T for the test sample
Fenton assay. The hydroxyl radical scavenging activity of test samples was evaluated according to the Fenton reaction. In Fenton's assay  the reaction mixture containing different dilution series of essential oils was incubated with deoxyribose, H2O2, FeCl3, EDTA, and ascorbic acid in phosphate buffer at pH 7.4. The reaction was terminated by 1 mL of thiobar-bituric acid (1%, w/v) which resulted in the formation of thiobarbituric acid reactive substance. The resulting OH radicals are measured in terms of their ability to degrade deoxyribose sugar into the fragments that react with thiobarbituric acid to form a pink chromogen. The IC50 value is the concentration of the sample required to scavenge 50% OH free radical. The percentage inhibition of hydroxyl radical was calculated using the formula reported earlier.
100 90 80 70 .2 60
л й НЧ
50 40 30 20 10 0
I I Coriander seed oil I I Coriander plant oil [ Tocopterol ( ] Ascorbic acid
10 20 30 40 Concentration, p.g/mL
50 45 40 35 30 25 20 15 10
C. seed oil C. plant oil Tocopterol Ascorbic
Fig. 1. Percentage inhibition of DPPH radicals (a), and concentration dependency of essential oils of C. sativum seed and plant against reference a-tocopherol and ascorbic acid and IC50 values (each value is mean ± SD, n = 3,p < 0.05) (b).
SINGH и др.
100 90 80 70
2 50 й
I Coriander seed oil - Coriander plant oil
I I Ascorbic acid
40 30 20 10
45 40 35
* 25 й
'is 20 ^
8 15 й
10 20 30 40 Concentration, p.g/mL
C. seed oil C. plant oilTocopterol Ascorbic
Fig. 2. Percentage inhibition of OH radicals (a), and concentration dependency of essential oils of C. sativum seed and plant against reference a-tocopherol and ascorbic acid and IC50 values (each value is mean ± SD, n = 3,p < 0.05) (b).
Fig. 3. Image for HPTLC fingerprinting of essential oils of C. sativum.
Insrumentation. Standard solutions of linalool and geranyl acetate were freshly prepared by dissolving them separately (100 ^g/mL) in toluene. Essential oils were separately mixed with toluene and sonicated for 10 min for proper mixing and then loaded on the HPTLC plates for the analysis. HPTLC system (Ca-mag, Muttanz, Switzerland) consisted of a TLC scanner controlled by WinCATS software. It consist of an auto sampler Linomat V using 100 ^L syringe, connected to a nitrogen cylinder; a UV scanner and visu-alizer. Each HPTLC plate contains different tracks of samples and standards under following conditions: band width 8 mm; distance between bands 12.5 mm; number of tracks 15; application volume of standards
0.5—3.5 ng; gas ow rate 10 ^L/s. UV scanner was set for the maximum light optimization with the following settings: slit dimension, 6.00 mm x 0.30 mm, micro; scanning speed, 20 mm/s; data resolution, 100 ^m/step. Regression analysis and statistical data were automatically generated by the WinCATS software .
Operating procedures. All experimental data were expressed in percent inhibition with respect to the control. The percentage inhibition was used to determine IC50 values. The IC50 value was calculated using probit analysis. All experimental data are given as mean ± SD. Statistical analysis was carried out using
80 70 60 50 40 30 20 10
250 300 350 400 450 500 550 600 [nm] 700
200 150 100 50 0
-0.14 0.06 0.26 0.46 0.66 0.86 1.06
Fig. 4. Chromatographic profile of standards linalool (a)
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