Miglena Valyova,Journal Alexander of Chemical Tashev, Technology Stanimir Stoyanov, and Metallurgy, Stanislava 51, 3,Yordanova, 2016, 271-274 Yordanka Ganeva In vitro FREE-RADICAL SCAVENGING ACTIVITY OF Aegopodium podagraria L. AND Orlaya grandiflora (L.) Hoffm. (Apiaceae) Miglena Valyova1, Alexander Tashev1, Stanimir Stoyanov2, Stanislava Yordanova2, Yordanka Ganeva2 1 Faculty of Ecology and Landscape Architecture Received 30 June 2015 University of Forestry, 10 Kliment Ohridski Accepted 26 February 2016 1756 Sofia, Bulgaria 2 Faculty of Chemistry and Pharmacy Sofia University St. Kl.Ohridski 1 James Bourchier, 1164 Sofia, Bulgaria E-mail: [email protected] ABSTRACT The radical scavenging activity of extracts from Aegopodium podagraria L. and Orlaya grandiflora (L.) Hoffm. aerial parts was determined for the first time using 1,1-diphenyl-2-picrylhydrazyl free radical (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) in vitro assays. The IC50 values obtained measured the corresponding antioxidant potential. The ethanol extract of A. podagraria L. exhibited the highest antioxidant potential in both assays, -1 -1 DPPH (IC50 = 66.135±1.6 µg ml ) and ABTS (IC50= 73.9±8.7 µg ml ). Chloroform and ethyl acetate extracts of the plants investigated possessed a very weak radical scavenging activity. The findings of the present study suggest that ethanol extracts of A. podagraria L. and O. grandiflora (L.) Hoffm. can be used as a natural antioxidant source preventing free radicals provocated diseases. Keywords: Aegopodium podagraria L., Orlaya grandiflora (L.) Hoffm., radical scavenging activity. INTRODUCTION elder belongs to the family Apiaceae growing in Central, North and Southeastern Europe, Caucasus, Southwestern Reactive oxygen species (ROS) such as singlet Asia, Siberia. The leaves of the plant are used as a spring oxygen, superoxide, peroxy radical, hydroxyl radicals vegetable like spinach [9]. A. podagraria L. is used in and peroxynitryl radical are generated in the course of popular medicine for the treatment of gout, arthritis, uri- the usual biochemical reactions in the body. Excess ROS nary disorders, cystitis, haemorroids, nervousness, pains, there can lead to degenerative or pathological processes, inflammations [3, 4]. The extracts of A. podagraria L. such as cancers, Alzheimer’s disease, neurodegenera- are evaluated for antibacterial activity [5, 6]. Literature tive disorders, atherosclerosis, coronary heart diseases, survey reveals the presence of polyacetylene falcarin- cataracts and aging [1, 2]. diol having a COX-1 activity, hydroxycinnamic acids, Many plants contain antioxidant compounds which flavonoids, coumarins, essential oil components [4, 7-9]. protect cells against the damaging effects of ROS. There- Orlaya grandiflora (L.) Hoffm. (White Lace Flower, fore, the search for antioxidants from natural sources has family Apiaceae) is an annual herbaceous plant spread in received much attention and these naturally occurring South, Central and Western Europe, the Mediterranean, antioxidants can be formulated to give nutraceuticals, Caucasus, Central and Southwestern Asia. The species which can help to prevent oxidative damage. is a weed and ornamental plant [10]. O. grandiflora (L.) Aegopodium podagraria L. commonly called ground Hoffm. is completely unknown in the phytopharmaco- 271 Journal of Chemical Technology and Metallurgy, 51, 3, 2016 logical literature. The decoctions of aerial parts from ebinthus, Quercus pubescens, Acer monspessulanum, O. grandiflora (L.) Hoffm possess laxative effect [11]. Paliurus spina-christi, Ulmus minor etc.; 41º39’59.3’’ It is investigated for insecticidal and growth inhibition N; 25º49’54.6’’ E; elev. 440 m. a. s. l. activity against Spodoptera littoralis larvae [12]. The Voucher specimens were deposited at the Herbarium chemical composition of O. grandiflora (L.) Hoffm. of the Institute of Biodiversity and Ecosystem Research is insufficiently studied. Previous studies report the (IBER) at the Bulgarian Academy of Sciences (Sofia, identification of quercetin, quercimeritrin, kaempferol Bulgaria). 3- glucoronide [13, 14]. Literature data shows no information on the appli- Preparation of the extracts cation of A. podagraria L. and Orlaya grandiflora (L.) The extraction method used was as follows: the Hoffm. as a source of natural antioxidants. Therefore air-dried plant material of A. podagraria L. (35 g) and the aim of this study is to investigate these plants using Orlaya grandiflora (L.) Hoffm. (50 g) was extracted different extracting solvents and to determine their radi- twice (500 ml for each material) using different solvents cal scavenging activity in order to search for a powerful (chloroform, ethanol and ethyl acetate), correspondingly. non-toxic antioxidant source. The extraction mixtures were refluxed in a water bath at 50°C-55°C for 3 h and the samples were filtered at EXPERIMENTAL atmospheric pressure. The six extracts obtained were concentrated to oil products under reduced pressure Chemicals at 40°C using a rotary evaporator. The stock solutions 1,1-diphenyl-2-picrylhydrazyl (DPPH), a-tocoph- used for analysis were of a concentration of 1 mg ml-1. erol, (±)-6-hydroxy-2,5,7,8-tetramethylchromane- 2-carboxylic acid (Trolox), 2,2’-azinobis(3-ethylbenzo- DPPH radical scavenging activity thiazolyne-6-sulfonic acid) diammonium salt (ABTS), The free radical scavenging activity of the investi- potassium persulfate were purchased from Sigma gated extracts was assayed using a stable DPPH radical Chemical Co (St. Louis, MO, USA). The other chemicals following a standard method with slight modifications and solvents used in this study were of analytical grade. [15, 16]. The DPPH• solution in methanol (0.1 mM) was prepared daily, and 0.8 ml of this solution was Plant material mixed with 2.4 ml of methanolic solutions of the plant Plant material - Bulgaria, A. podagraria L. and O. extracts. The samples were allowed to incubate for 10 grandiflora (L.) Hoffm. min at a room temperature, and then the absorbance A. podagraria L. - SOM 168717; Alexander Tashev, decrease was measured at 517 nm using Unicam UV Vitosha, above “Simeonovo” district, in an open area 500 spectrophotometer (Thermo Spectronic, UK). The close to a forest of Carpinus betulus, 42º36‘45.7’’ N; radical scavenging activity was calculated using the 23º19‘35.9’’ E; elev. 908 m. a. s. l. following formula: A. podagraria L. - SOM 168718; Alexander Tashev, Vitosha, above “Simeonovo” district,, in an open area % Inhibition = [(AB – AE)/ AB] x 100 (1) close to a forest of Carpinus betulus, Corylus avellana etc.; 42º36’45.9’’ N; 23º19’35.9’’ E; elev. 908 m. a. s. l. where AB was the absorbance of the blank sample, while O. grandiflora (L.) Hoffm. - SOM 168568; 168569; AE was the absorbance of the plant extract. α-tocopherol Alexander Tashev, Eastern Rhodope mountains, „Kru- was used as a reference compound. movgrad” Forestry, above the road from Krumovgrad ABTS radical scavenging activity to Kovil village; 41º29‘48.4‘‘ N; 25º 39‘09.7‘‘ E; elev. The free radical scavenging potential of A. podag- 359 m. a. s. l. raria L. and O. grandiflora (L.) Hoffm. extracts was also O. grandiflora (L.) Hoffm. - SOM 168604; 168605; studied using ABTS radical cation decolorization assay Alexander Tashev, Eastern Rhodope mountains, near [17]. ABTS was dissolved in distilled water to reach 7 the town of Madjarovo, on a slope above the road mM concentration. ABTS radical cation (ABTS+•) was Madjarovo-Harmanli under bushes of Pistacia ter- produced through the reaction of ABTS solution with 272 Miglena Valyova, Alexander Tashev, Stanimir Stoyanov, Stanislava Yordanova, Yordanka Ganeva 2.45 mM potassium persulfate. Then the mixture was podagraria L. displays the highest antioxidant activity -1 kept for 12 h in the dark at a room temperature. For the (EC50 = 73.9±8.7 μg ml ). study, the ABTS+• solution was diluted with methanol The ethanol extract of O. grandiflora (L.) Hoffm. -1 to give an absorbance of 0.7 ± 0.01 at 734 nm. Plant shows a high activity (IC50 = 87.0 ± 6.1 μg ml ) ). The extracts (1 ml) reacted with 2 ml of the ABTS solution chloroform and ethyl acetate extracts of the studied and the absorbance was taken at 734 nm after 1 min. The plants exhibit insignificant radical scavenging capacity. ABTS scavenging capacity of the extracts was compared to that of Trolox. All solutions were used on the day of CONCLUSIONS preparation, and all measurements were carried out in triplicate. The percentage of inhibition of ABTS+• was The present work reveals that the ethanol extracts of calculated using the formula presented above (eq. 1). O. grandiflora (L.) Hoffm. and A. podagraria L. exhibit a high antioxidant activity in both DPPH and ABTS RESULTS AND DISCUSSION scavenging assays. These in vitro assays indicate that the plant extracts are a significant source of a natural DPPH radical scavenging activity antioxidant, which might be helpful in preventing the DPPH radical scavenging assay is one of the most progress of various oxidative damages. However, the widely used method for screening of antioxidant activity components responsible for the antioxidative activity of plant extracts. The DPPH radical scavenging capacity are currently unclear. Therefore, further investigations in this study was reported after 10 min reaction time for are required to isolate and identify the antioxidant com- all samples investigated. IC50 value is the parameter used pounds present in the plant extracts. to measure the antioxidant activity of the extracts evalu- ated. The free-radical scavenging activity of chloroform, Acknowledgements ethanol and ethyl acetate extracts from A. podagraria L. The authors are grateful to the University of For- и O. grandiflora (L.) Hoffm. measured by DPPH assay is estry, Sofia, Bulgaria, for the financial support (Contract reported for the first time. The effect of the investigated number 133/14.03.2012). samples on DPPH radicals has been checked at various concentrations: from 15 μg ml-1 to 125 μg ml-1 and from REFERENCES 45 μg ml-1 to 240 μg ml-1, respectively. The results show that the ethanol extract of A. podagraria L. exhibits the 1. I.