1. Location of harvesting plot
ON STANDARDIZATION OF ANTIOXIDANT ACTIVITY MEASUREMENTS WITH MEDICINAL PLANT EXTRACTS M. Orłowska1, K. Pytlakowska1, M. Bartoszek1, J. Polak1, T. Kowalska1, M. Waksmundzka-Hajnos2, M. Sajewicz1 1Institute of Chemistry, University of Silesia, 9 Szkolna Street, Katowice, Poland 2Department of Inorganic Chemistry, Medical University of Lublin, 4 A. Chodźki Street, Lublin, Poland  
1. Introduction
The Thymus genus embraces ca. 350 different species of aromatic plants from the mint (Lamiaceae) family. Chemical composition of plants belonging to the Thymus genus is extremely rich. The most important groups of biologically active compounds characteristic of this genus are phenolic acids, flavonoids, terpenoids, and essential oils. Owing to an abundance of these compounds, the thyme species have found applications in medicine, cosmetics, and pharmacy. The most appreciated are their antioxidant, antibacterial and anti-inflammatory properties [1,2].
Polyphenolics is a group of compounds which includes phenolic acids, flavonoids, and natural pigments, which all are produced in different parts of the plants. The main sources of polyphenols in human diet are such natural products, as fruits, vegetables, cereals, drinks (infusions, tea, and coffee), wines, and spices. The amounts of the phenolics determined in plants depends on multiple factors such, as the extraction time and temperature, polarity of extracting solvents, etc. [3,4].
Antioxidants are the compounds able to neutralize the oxygen and nitrogen free radicals present in a given system. Participating in numerous metabolic processes, they exert positive effect on various different physiological functions. Owing to that, antioxidants exert prophylactic, or even curative effect and they mainly appear in edible parts of the plants [5].
3. Experimental
Plant material investigated in this study originates from Botanical Garden of the Maria Curie - Sklodowska University in Lublin. The eighteen thyme (Thymus L.) species were collected in July, 2012, and dried under adequate conditions.
Preliminary extraction in Soxhlet apparatus (to remove ballast compounds)
Extraction of phenolic compounds by Accelerated Solvent Extraction under an elevated pressure and at the optimized working parameters (extraction temperature, 130oC; methanol : water, 27 : 73 (v/v) [6] as eluent)
2. Aim
The aim of present work was to compare the total antioxidant properties for examined thyme species (Tab. 1.) determined with the use of DPPH by different analytical techniques. Obtained results were presented as mg applied reference substance per 1 g dry herbal matter.
Extracts
“Dot blot” test
UV-Vis spectrophotometry
Tab.1. List of the investigated thyme species and location of the harvesting plot within Botanical Garden and comparison of results obtained with applied measurement techniques.
Electron paramagnetic
resonance spectrometry (EPR)
Thyme species
Location of harvesting plot
EPR
UV-Vis
“dot blot”
mg TEAC/1g dry herbal matter
mg AA / 1g dry herbal matter
mg GA / 1g dry herbal matter
T. vulgaris
Commodity
85.60
28.33
8.77
25.97
T. serpyllum
Arboretum
74.13
24.73
7.56
32.54
T. pulegioides subsp. pulegioides
Alpinarium
133.00
39.25
12.44
45.26
T. kosteleckyanus
Mediterranean
77.07
27.62
8.53
44.38
T. citriodorus ‘golden dwarf’ leaves
Systematic
33.61
17.68
5.19
25.53
T. marschallianus
74.54
19.85
5.92
30.79
T. degenii
106.55
29.15
9.05
41.75
T. pseudolanuginosus
97.88
30.47
9.49
33.86
T. pannnonicus
99.43
24.87
7.61
36.49
T. austriacus
94.47
22.18
6.71
37.36
T. praecox ‘pygmaeus’
58.53
17.04
4.98
20.70
T. tiflisiensis
88.31
24.09
7.35
36.05
T. pulegioides subsp. pulegioides
74.47
27.99
8.66
29.47
T. citriodorus ‘golden dwarf’ rhizome
56.61
12.24
3.37
11.94
T. serpyllum ‘aureus’
133.38
38.93
12.33
49.64
T. serpyllum ‘albus’
137.53
48.03
15.39
60.16
77.75
22.59
6.84
31.23
T. praecox subsp. arcticus
74.78
18.79
5.57
35.17
Euclidean distance
Method 3 Method 2 Method 1
Sorted thyme species
Fig.1. Dendrogram of eighteen investigated thyme extracts based on their antioxidant properties measured by the following three methods: method 1, EPR; method 2, UV-Vis; and method 3, the “dot blot” test. Dendrogram is augmented with the heat map representing the auto-scaled values of antioxidantpotential .
4. Conclusions
[1] Applied measurement techniques (EPR, UV-Vis spectrophotometry and “dot blot” test) enabled determination of total antioxidant activity for all examined thyme species.
[2] Results obtained with use of applied measurement techniques showed that T. pulegioides subsp. pulegioides alpinarium, T. serpyllum ‘aureus’ arboretum and T. serpyllum ‘albus’ commodity species (Tab. 1, red colour) characterize with the highest total antioxidant activity. The lowest total antioxidant activity was observed for T. citriodorus ‘golden dwarf’ leaves systematic, T. praecox ‘pygmaeus’ alpinarium and T. citriodorus ‘golden dwarf’ rhizome systematic (Tab. 1, blue colour).
5. References
[1] Oh S.Y., Ko J.W., Jeong S.Y., Hong J.: Journal of Chromatography A, 2008, 1205,
[2] Rubió L., Serra A., Macià A., Borràs X., Romero M.P., Motilva M.J.: Journal of Chromatography B, 2012, 905, 75–84.
[3] Borosa B., Jakabováa S., Dörnyeia Á., Horváthd G., Pluháre Z., Kilár F., Felingera A.: Journal of Chromatography A, 2010, 1217, 7972–7980.
[4] Costa P., Gonçalves S., Valentão P., Andrade P.B., Coelho N., Romano A.: Food Chemistry, 2012, 135, 1253–1260.
[5] L. Czerwiecki, Rocznik - Państwowy Zakład Higieny, 3 (2009)
[6] M. Orłowska, D. Staszek, I. Stanimirova, M. Waksmundzka-Hajnos, M. Sajewicz T. Kowalska,
Journal of AOAC International – in press
Acknowledgements
One author (MO) acknowledges the financial support of the DoktoRIS project, co-financed by the European Union within the European Social Found.