Chemometric Analysis of Selected Medicinal Plants from Serbia

Received for publication, October 12, 2014 Accepted, June 06, 2015

BILJANA ARSIC1, DANIJELA KOSTIC1, SASA RANDJELOVIC1, BLAGA RADOVANOVIC1, SLAVICA SUNARIC2, SLAVICA ILIC3 1 Faculty of Sciences and Mathematics, The University of Nis, Visegradska 33, Nis, Serbia; 2 Faculty of Medicine, The University of Nis, Bulevar Dr Zorana Djindjica 81, Nis, Serbia; 3 Faculty of Technology, The University of Nis, Bulevar Oslobodjenja 124, Leskovac, Serbia Corresponding author: Biljana Arsic, e-mail: [email protected]

Abstract The contents of total phenols, total flavonoids and toxic metals (Fe, Zn, Cu, and Mn) were investigated in plants Delphinidum consolida L., Origanum vulgare L., Calendula officinalis L., Cichorium intybus L., Prunus spinosa L., Crataegus oxyacantha L., and Papavear rhoeas L. The highest content of total phenols was determined in 50 % ethanol extracts of oregano, and the lowest in the ethanol extract of hawthorn. Among metals, the highest quantity was recorded for iron; meanwhile, the content of zinc, copper and manganese were considerably lower. The lowest transfer of toxic metals was shown in aqueous extracts, and the highest in ethanol extracts. Principal component analysis was used as a statistical tool for experimental sets of obtained values for total phenols and toxic metals. Performed statistics with Pearson correlation matrix on plant samples based on the content of metals showed that there was a moderate correlation between quantity of iron and copper (0.606), and low correlation between iron and manganese (0.401) and copper and manganese (0.206).

Keywords: correlation, PCA analysis, total phenols, total flavonoids, toxic metals.

1. Introduction The modern way of life increases the need for vegetables and fruits (J. A. VINSON & al. [1]). Some toxic metals can be present in food only in certain limits (Cu, Ni, Mn, Fe, Cr, V, Mo) (U. S. EPA [2], FDA [3]). It is well known that wild plants are richer in polyphenol compounds than grown species. Therefore, we investigated the following plants: Delphinidum consolida L., Origanum vulgare L., Calendula officinalis L., Cichorium intybus L., Prunus spinosa L., Crataegus oxyacantha L. and Papavear rhoeas L. (F. SHAHIDI and M. NACZK [4], A. ZARGARI [5], P. H. LIST and L. HORHAMMER [6], B. BORKOWSKI & al. [7]). Cichorium intybus L. is known in Serbian folk medicine as an anti-diabetic because of the presence of inulin as a reserve food. Cichorium intybus L. has been used to treat skin disorders, such as gout, because of its anti-hepatotoxic activity (B. AHMED & al. [8], R. ZAFAR and S. M. ALI [9]). Calendula officinalis L. possesses anti-bacterial and bactericidal effect; it is used for the treatment of wounds, psoriasis, etc. (V. SLAVKOVSKA & al. [10]). Calendula officinalis L. (flower) is known for its anti-inflammatory and anti-cancer properties (A. R. BILIA & al. [11]). Origanum vulgare L. has anti-spasmodic, bronchodilating, and

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BILJANA ARSIC, DANIJELA KOSTIC, SASA RANDJELOVIC, BLAGA RADOVANOVIC, SLAVICA SUNARIC, SLAVICA ILIC diuretic effect (J. H. A. IETSWAART [12]). Delphinium consolida L. is used to treat intestinal worms, fluid retention, poor appetite, and trouble in sleeping (insomnia). It is also used as a sedative to cause relaxation (J. D. OLSEN & al. [13]). Crataegus leavigata L. (hawthorn) is used in the treatment of atherosclerosis, relieves symptoms of angina pectoris and cardiac arrhythmias, heart and menopausal problems (WHO [14]). Prunus spinosa L. (blackthorn) is used to treat skin problems, soothes stomach cramps, etc. (K. BROWICS [15]). Papaver rhoeas L. is claimed to exhibit sedative, narcotic, and emollient effects (A. ZARGARI [16]). Regarding applied statistical analysis tool, we used principal component analysis (PCA) in order to obtain the initial solution reducing the original dataset. Thus obtained linear solution is then linearly transformed, according to some criterion chosen by the researcher, getting the solution which is easy for the interpretation (J. D. HOREL [17]). PCA expresses the data as a linear combination of its basis vectors. It is a non-parametric analysis (J. SHLENS [18]). The correlation between a component and a variable gives information, and the correlation is called a loading. The squared loadings are easier to interpret than the loadings (J. D. HOREL [17]). Pearson’s correlation is a normalization method where standard deviation is in use for scaling the data between -1 and +1 (M. C. JANZEN & al. [19]). The earliest works in principal component analysis can be found in Karl Pearson (1901) (P. JOLICOEUR and J. E. MOSIMANN [20]). PCA in chemistry was firstly introduced by Malinowski in 1960s as a principal factor analysis (S. WOLD & al. [21]) and after 1970 a large number of chemical applications have been published (F. MALINOWSKI and D. HOWERY [22], Y. BOUTON & al. [23], M. FRAU & al. [24]). The aim of our study was to determine the quantity of phenols, flavonoids and selected toxic metals, and to perform grouping of samples using PCA on the basis of the content of phenols and selected metals.

2. Materials and Methods 1. Reagents All used reagents were of the analytical purity (Merck, Germany). The working solutions were prepared immediately before the analysis from the basic solution with 1000 mg l-1 for all metals. High purity Milli-Q water was used for the preparation of standard solutions. The glassware and polyethylene containers used for analysis were washed with tap water, then soaked overnight in 6 M HNO3 solution and rinsed several times with ultra-pure water to eliminate absorbance due to detergent. Folin-Ciocalteu’s phenol reagent and sodium carbonate were purchased from Merck Chemical Suppliers (Darmstadt, Germany).

2. Apparatus Atomic absorption measurements were made using Varian SpectraAA 10 with background correction and hollow cathode lamps. Air-acetylene flame was used for determination of all elements. The standard procedure described by Association of Official Analytical Chemists (AOAC) was followed for the preparation of samples for the analysis of heavy metals (AOAC [25]). Accurately weighed (2 g) sample was transferred into a silica crucible and kept in a muffle furnace for ashing at 450 ˚C for 3 h and then 5 ml of 6 M hydrochloric acid were added to the crucible. Care was taken to ensure that all the ash came into contact with acid.

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Further, the crucible containing acid solution was kept on a hot plate and digested to obtain a clean solution. The final residue was dissolved in 0.1 M nitric acid and made up to 50 ml. Working standard solutions were prepared by diluting the stock solution with 0.1 M nitric acid for checking the linearity.

3. Preparation of herbal extracts Dried material (flowers or fruits or leaves) of plant species was ground in a blender. Samples of 1 g of each species were weighed from the ground and homogenized herb was extracted with solvents listed in Table 1. The extraction was carried out for 15 min three times with 30, 20, and 20 ml of solvent, respectively. The selected solvents were: ethanol, ethanol- water: 50-50, v/v (%), and water. The extraction was performed in an ultrasonic bath. The extracts were filtered through a Buchner funnel and filter paper (blue collar) (CHMLAB, Spain), transferred into a volumetric flask (100 ml) and the same solvent was added to the mark.

4. Determination of the total phenolics Total phenol contents in extracts were determined by the modified Folin-Ciocalteu method (V. L. SINGLETON and J. A. ROSSI [26]). An aliquot of the extracts (1 ml) was mixed with 0.5 ml of Folin-Ciocalteu reagent and 1.5 ml of sodium carbonate solution (20 %). Tubes were vortexed for 15 s and allowed to stand at 40 0C for 30 min in order to develop colour. Total phenol content was expressed as mg g-1 of gallic acid equivalent (GAE).

5. Determination of the total flavonoids Flavons and flavonols with aluminium chloride form the complex with intensive absorption at 415 nm, while flavanons have weak absorption at this wavelength. Flavanons (naringenin and hesperetin) with 2,4-dinitrophenylhydrazine form phenyl hydrazones with strong absorption at 495 nm. These characteristics of flavonoids were used for the determination of total flavonoids content using two complementary colorimetric methods: aluminium chloride colorimetric method (R. WOISKY and A. SALATINO [27], F. POURMORAD & al. [28]) and 2,4-dinitrophenylhydrazine colorimetric method (M. NAGY and D. GRANCAI [29]). The total content is expressed as mg CE (catechin equivalent)/g or QE (quercetin equivalent)/g of the sample.

6. Analysis of correlations among the evaluated parameters Principal component analysis was used as a statistical tool. It is used with the aim to evaluate the dataset, reducing its dimension and conserving most of the statistical information. The analysis was performed using data analysis and statistical application available for Microsoft Excel® (XLSTAT 2013.4.07, Addinsoft SARL, Paris, France).

3. Results and discussion

1. Content of metals and total phenols Table 1 contains the data on total phenols, flavonoids and metals content in plants and their extracts.

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Table 1. Content of total phenols, total flavonoids and metals in different plant extracts

Total Total phenols Fe Zn Cu Mn Plant Extract flavonoids (mg GAE g-1) (µg g-1) (µg g-1) (µg g-1) (µg g-1) (mg QE g-1)

Plant - 423.32±8.4 31.80±0.6 35.50±0.7 20.61±0.4

L. Water 19.9±0.4 11.45±0.0 - 2.20±0.0 - -

Ethanol 13.1±1.5 9.53±0.02 78±1.56 3.50±0.0 22.10±0.4 16.4±0.3 rhoeas Papavear Ethanol 50 % 14.3±0.2 9.07±0.07 / 7.60±0.5 5.30±0.1

Plant - 172.16±3.4 50.80±1.0 9.97±0.1 16.02±0.3 L. Water 24.8±0.6 0.76±0.03 29±0.5 5.60±0.1 3.26±0.1 2.70±0.0 Ethanol 2.1±0.1 0.56±0.02 118±2.3 20.10±0.4 2.85±0.1 7.20±0.1 Crataegus Crataegus

oxicantha Ethanol 50 % 19.3±0.4 0.55±0.02 - 35.70±0.7 0.52±0.1 -

Plant - 261.6±5.2 62.2±1.2 8.9±0.1 9.1±0.1 L Water 12.1±0.1 0.42±0.01 - 12.7±0.2 - - Ethanol 15.3±0.1 0.70±0.11 91±1.8 7.1±0.1 2.3±0.2 2.1±0.0 Prunus Prunus spinosa spinosa Ethanol 50 % 20.9±0.7 1.24±0.09 - 5.2±0.1 - - Plant - 402±8 32.4±0.6 20.5±0.4 49.4±1.0

L. Water 8.6±0.2 0.59±0.01 - - 1.1±0.08 - Ethanol 5.3±0.8 0.2±0.0 68.0±0.6 28.8±0.6 8.5±0.2 7.2±0.1 intybus intybus Cichorium Cichorium Ethanol 50% 4.8±0.0 0.32±0.01 29.0±0.6 3.7±0.1 3.2±0.1 4.8±0.1 Plant - 137±3 18.1±0.4 12.8±0.3 24.4±0.5 L. Water 45.1±0.0 0.12±0.01 16.0±0.3 4.5±0.1 1.5±0.05 5.4±0.1 Ethanol 31.9±1.1 0.09±0.01 76.0±1.6 5.3±0.1 2.4±0.07 13.7±0.3 Calendula Calendula

officinalis Ethanol 50 % 29.8±0.7 0.17±0.01 17.0±0.3 4.4±0.1 1.1±0.0 3.6±0.1 Plant - 152±3 49.6±1.0 23.9±0.5 21.8±0.4 L. L. Water 74.3±1.2 2.06±0.04 - 29.1±0.6 4.5±0.1 - Ethanol 23.6±0.4 0.97±0.01 113±2 22.3±0.4 3.8±0.1 16.3±0.3 Origanum Origanum vulgare vulgare Ethanol 50 % 75.2±0.6 1.97±0.02 - 22.0±0.4 0.4±0.1 -

Plant - 286±6 39.8±0.8 23.1±0.5 13.8±0.3 L. Water 24.2±0.4 1.69±0.02 14.0±0.3 11.0±0.2 1.6±0.06 - Ethanol 23.9±1.8 1.08±0.01 176±4 14.0±0.3 18.8±0.4 3.3±0.1 Delphinium Delphinium consolida consolida Ethanol 50 % 18.3±0.4 1.32±0.02 - 12.2±0.2 3.6±0.1 -

The total phenol content in ethanol extracts varies from 2.1 to 31.9 mg GAE g-1, in 50 % ethanol extracts from 4.8 to 75.2 mg GAE g-1 of the investigated sample and in aqueous extracts from 8.6 to 74.3 mg GAE g-1 of the investigated sample. The highest content of total phenols was shown in 50 % ethanol extracts of oregano, and the lowest in the ethanol extract of hawthorn (2.1 mg GAE g-1 of the investigated sample). Aqueous extracts have higher content of total phenols comparing to 50 % ethanol extracts and ethanol, with the exception of blackthorn. The plant order in decreasing content of total phenols in aqueous extract: Origanum vulgare L. > Calendula officinalis L. > Crataegus oxyacantha L. > Delphinium consolida L. > Prunus spinosa L. > Papavear rhoeas L. > Cichorium intybus L. The content of phenols determines the pharmacological characteristics of the plant and in the case of medicinal plants it goes from 0.23 to 2.85 mg GAE g-1 of the fresh sample (W. ZHENG and S. Y. WANG [30]), while the concentration of phenols in cultivated plants goes from 0.26 to 17.51 mg GAE g-1 of the fresh sample. On the basis of the literature data, the highest content of phenolic compounds contains the cultivated plant from genus Origanum, around 20 mg GAE g-1 of the fresh sample. On the basis of this fact, we can conclude that poppy belongs to plant group with relatively high content of phenolic compounds.

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The content of total phenols in the ethanol extract of dry fruit of hawthorn was 35.4±2.48 mg GAE g-1 of dry weight by Tadic et al. (M. V. TADIC & al. [31]). Bernatoniene et al. analyzed the content of total phenols and flavonoids in aqueous and ethanol ((v/v), 1:4) extracts of dry fruits of C. monogyna, and got values for total phenols 24.4±0.9 g GAE/100 g, i.e. for flavonoids 1.29±0.07 g QE/100 g for ethanol extracts. These values are very similar to our results (J. BERNATONIENE & al. [32]). Fraternale et al. determined in their study that the content of total phenols in sloe (Prunus spinosa L.) was 83.5±2.5 mg GAE g-1 of dry matter (D. FRATERNALE & al. [33]), which is significantly higher than our values. In this study dry material was analyzed, and in our fresh fruits were extracted. A. FILIPIAK & al. [34] investigated different parts of the plant Cichorium intybus L. in order to determine the content of phenolic compounds, flavonoids and phenolic acids. They performed HPLC analysis of ethanol extracts of Cichorium intybus L. The average contents of investigated flavonoids and phenolic acids were 0.98-4.12 mg g-1, i. e. 2.13-14.78 mg g-1, respectively, for different extracts. The content of phenolic compounds in ethanol extracts of Cichorium intybus L. goes from 7.64 to 32.78 %. Mehmood et al. investigated the extracts of chicory with methanol, chloroform, ethyl acetate, n-butanol and n-hexane. Results show that the contents of total phenols are the highest in the methanol extract 285±4.92 mg GAE/100 g of dry plant material, which is significantly lower than our results (N. MEHMOOD & al. [35]). Cetkovic et al. investigated the content of phenolic compounds in the flower of marigold (Calendula officinalis L.). Results show that the content of total phenols in methanol solution of marigold is 15.12 mg equivalent chlorogenic acid g-1 of the sample, while the content of total flavonoids is 5.13 mg rutin equivalent g-1 of the sample (G. S. CETKOVIC & al. [36]). The content is expressed in different equivalents and the extraction was performed using the process of maceration with ethanol for 24 h in the ratio dry matter : solvent = 1:50, and the dry residue was extracted using solvents of different polarities, and extracts were analyzed using thin layer chromatography. Literature data for spices where Origanum vulgare L. belongs show that it contains total phenols from 0.26 to 17.51 mg GAE g-1 of dry matter (W. ZHENG and S. Y. WANG [30]). Kaurinovic et al. analyzed the extracts of oregano in ether, chloroform, ethyl acetate, n- butanol and water. The content of total phenols varied from 4.72 to 14.13 mg GAE g1 material (B. KAURINOVIC & al. [37]). The highest content of total phenols was in the ethyl acetate extract. Extract was prepared by the extraction with methanol for 24 h, and then the extract was evaporated to dryness, and then the extraction was performed using other solvents and there the content of total phenols and flavonoids was determined. Obtained values are different from ours as a consequence of different extraction procedure and application of solvents of different polarities. Chrpova et al. determined in the aqueous extract of oregano that the content of total phenols (91.4 mg GAE g-1) is negligibly lower than in our aqueous extract (D. CHRPOVA & al. [38]). The content of flavonoids in P. rhoeas goes from 7.90 to 11.45 mg g-1 of the fresh sample. It was observed that the aqueous solution contains higher quantities of total flavonoids compared to the rest of the extracts. The most important flavonoids found in hawthorn are quercetin, hyperoside, vitexin, vitexin-4’-L-rhamnoside, vitexin-4’-L-rhamno-D-glucoside, vitexin-7-di-D-glucoside, rutin, quercetin-3-rhamno-galactoside (A. Y. LEUNG and S. FOSTER [39]). Cardiotonic effect of hawthorn extracts comes from hyperoside and vitexin primarily (H. R. T. AMMON & al. Romanian Biotechnological Letters, Vol. 21, No. 1, 2016 11119

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[40]). There are data on the determination of flavonoids using thin layer chromatography and UV spectrophotometry (A. DEUTSCHES [41]). It was shown that leaves and flowers of hawthorn contain flavonoids (rutin, hyperoside, vitexin, vitexin-2”-O-rhamnoside and acetylvitexin-2”-O-rhamnoside) from 0.1 to 2 % (WHO [42]). Mihajlovic et al. identified in the extracts of flowers and leaves using thin layer chromatography, flavonoids vitexin and hyperoside (D. MIHAJLOVIC & al. [43]). Gudzic et al. analyzed the extracts of flowers of white hawthorn and got that it contains quercetin (B. GUDZIC & al. [44]). Bernatoniene et al. analyzed the content of total phenols and flavonoids in aqueous and ethanol ((v/v), 1:4) extracts of dry fruits of C. monogyna, and got the value for total phenols 24.4±0.9 g GAE/100 g i. e. for flavonoids 1.29±0.07 g QE/100 g for ethanol extracts which is very similar to our results (J. BERNATONIENE & al. [32]). The content of total flavonoids in investigated extracts of hawthorn is very low, and goes from 0.56 to 0.99 mg QE g-1 of fresh sample. It was observed that aqueous extracts contain higher quantities of total flavonoids compared to ethanol extracts. Tadic et al. found that ethanol extracts contain total flavonoids from 0.14 to 0.18 mg rutin equivalent g-1 of dry sample (M. V. TADIC & al. [31]). These values for total flavonoids are lower compared to our results. It can be a consequence of outdoor factors on the flavonoid synthesis, different extraction procedure and different equivalent showing the content of flavonoids in the sample. Quantitative investigations show that the content of flavonoids in flowers of the population of sloe in Poland is significant-around 2.7 % in the form of aglycone and 3.8 % in the form of glycoside (M. OLSZEWSKA and M. WOLBIS [45]). Investigations in the population of this plant in Romania show that it contains 1.2 % of flavonoids in the form of the aglycone (M. TAMAS [46]). In flowers, flavonoids are usually present in the form of monoglycoside, mainly kaempferol and quercetin-3-O-arabinoside. In leaves, glycosides are present, mainly kaempferol 3,7-O-dirhamnoside (M. OLSZEWSKA & al. [47]). The structures of these compounds are determined using chemical and instrumental methods (UV, IR, H-NMR, C-NMR, MS). Flavonoids content in flowers of the population of sloe in Romania revealed that 1.16 % of total flavonoids aglycons represent quercetin. The content of total flavonoids in the investigated extracts is very low. It goes from 0.419 to 1.31 mg QE g-1 of the fresh sample. It was observed that ethanol (50 %) extract contains higher quantities of total flavonoids than other extracts. Uzelac et al. determined the content of flavonoids in the sloe fruit (from 0.436 to 0.656 mg GAE kg-1 of fresh fruit). Extraction was performed using 80 % ethanol (V. UZELAC & al. [48]), which agrees with our results. The content of total flavonoids in the investigated extracts of chicory is very low, and goes from 0.23 to 0.59 mg QE g-1 of dry weight. Mehmood et al. analyzed chicory extracts using methanol, chloroform, ethyl acetate, n-butanol and n-hexanes. The highest content of total flavonoids was in the methanol extract-1.50 mg CE g-1 of dry plant material (N. MEHMOOD & al. [35]), which is higher than our result. The content of total flavonoids in investigated samples of marigold is very low, and it goes from 0.097 to 0.17 mg QE g-1 of dry weight. The content of total flavonoids in methanol extract of marigold is 5.13 mg rutin equivalents g-1 of the sample (G. S. CETKOVIC & al. [36]). These results are significantly higher compared to our results; the contents are expressed using different equivalents, and the extraction process was performed by maceration during 24 h. Kleinubing et al. analyzed the content of heavy metals and flavonoids in aqueous and ethanol extracts. Content of flavonoids was 0.66-2.33 mg QE g-1 in ethanol extract (S. KLEINUBING & al. [49]). Obtained values are in accordance to our results.

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The content of total flavonoids in the investigated extracts of oregano is very low, and it goes from 0.97 to 2.11 mg g-1 of dry weight of the sample. Kaurinovic et al. analyzed the extracts of oregano in ether, chloroform, ethyl acetate, n-butanol and water. The content of total flavonoids was from 9.37 to 28.54 mg rutin equivalent g-1 of dry material (B. KAURINOVIC & al. [50]). The highest content of flavonoids was in the ethyl acetate extract. The content of total flavonoids in the investigated extracts of larkspur is very low, and it goes from 1.085 to 1.69 mg QE g-1 of dry sample. Among metals, the highest quantity was recorded of iron; meanwhile, the contents of zinc, copper and manganese were considerably lower. The iron content was varied from 137.53 to 423.32 g g-1 of plant mass. The highest content of iron was shown in poppy, and the lowest in marigold. The iron content in investigated extract was considerably lower than iron content in plant material. The iron content in ethanol extracts varies from 76.0 to 176 g g-1, and in 50 % ethanol extracts from 0.0 to 29.0 g g-1 and in aqueous extracts from 0.0 to 29.0 g g-1 of the investigated sample. The highest iron content was in ethanol extracts and the plants order in decreasing values of iron is: Delphinium consolida L. > Crataegus oxyacantha L. > Origanum vulgare L. > Prunus spinosa L. > Papavear rhoeas L. > Calendula officinalis L. > Cichorium intybus L. The highest zinc content was in berries of blackthorn- 62.20 g g-1, and the lowest in marigold 18.15 g g-1. The zinc content in extracts was considerably lower. Zinc content in ethanol extract varies from 2.8 to 3.5 g g-1, in 50 % ethanol extract from 3.7 to 35.7 g g-1 and in aqueous extract from 0.0 to 29.1 g g-1 of the investigated sample. The highest content of zinc is in 50 % ethanol extracts, and the plants order in descending zinc values is: Crataegus oxyacantha L. > Origanum vulgare L. > Cichorium intybus L. > Delphinium consolida L. > Prunus spinosa L. > Papavear rhoeas L. > Calendula officinalis L. > Cichorium intybus L. The highest copper content was detected in dry poppy material-35.5 g g-1, and the lowest in blackthorn-8.91 g g-1. Copper content in ethanol extracts varies from 2.3 to 22.1 g g-1, in 50 % ethanol extracts from 0 to 5.3 g g-1, and in aqueous extracts from 0.0 to 4.5 g g-1 of the investigated sample. Plant order in descending values of copper in ethanol extracts is: Papavear rhoeas L.>Delphinium consolida L.> Cichorium intybus L. > Origanum vulgare L. > Crataegus oxyacantha L. > Calendula officinalis L. > Prunus spinosa L. The content of manganese is the highest in dry material of chicory 49.39 g g-1, and the lowest in hawthorn 9.15 g g-1. Manganese content is the highest in ethanol plant extracts and varies from 2.1 to 16.4 g g-1 of the investigated sample. The manganese content varies from 0.0 to 5.4 g g-1 in aqueous extracts of 50 % ethanol. The plants order in descending manganese values in ethanol extracts is: Papavear rhoeas L. > Origanum vulgare L. > Calendula officinalis L. > Crataegus oxyacantha L. > Cichorium intybus L. > Delphinium consolida L. > Prunus spinosa L.

2. PCA Analysis Performed statistics with Pearson correlation matrix on plant samples based on the content of metals shows that there is a moderate correlation between quantity of iron and copper (0.606), and low correlation between iron and manganese (0.401) and copper and manganese (0.206). Eigenvalues for the first and the second factor are higher (2.122 and 1.007, respectively) compared to values for the third and the fourth factor (0.628 and 0.243, respectively). So, the Romanian Biotechnological Letters, Vol. 21, No. 1, 2016 11121

BILJANA ARSIC, DANIJELA KOSTIC, SASA RANDJELOVIC, BLAGA RADOVANOVIC, SLAVICA SUNARIC, SLAVICA ILIC first two factors must be used to explain the obtained variabilities (53.06 %, 25.17 %, 15.69 % and 6.08 %) (Figure 1). Some values for factors (F1, F2, F3 and F4) are positive and some of them are negative. Observation plot based on the content of metals is represented in Figure 2. From Figure 2, it is visible that the highest content of iron is in Cichorium intybus L. and Papaver rhoeas L. Also, information about the content of zinc is visible and suggests that it is the highest in Prunus spinosa L. On the other hand, the performed statistics with Pearson correlation matrix on plant samples based on total phenols content shows that there is a strong correlation between quantity of total phenols in aqueous and water/ethanol extracts (r=0.948), and a moderate correlation between quantity of total phenols in aqueous and ethanol extracts (r=0.596) and low correlation between quantity of total phenols in ethanol and ethanol/water extracts (r=0.496). Eigenvalues for the first and the second factor are higher (2.381 and 0.575, respectively) compared to the value for the third factor (0.044), which means that the first and second factor explain the obtained variabilities (79.36 % and 19.18 %, respectively). In this case, values for eigenvectors are positive for factor 1 (F1); for factor F2, there are some positive and some negative values (positive for the quantity of total phenols in ethanol extract). Observation plot based on the content of total phenols is represented on Figure 3. From Figure 3, it can be concluded that the highest quantity of total phenols among the investigated plants is present in aqueous extracts of Origanum vulgare L.

Figure 1. Plot shows the importance of factors and values of cumulative variabilities

Figure 2. Principal component score plot (F1 and F2) of the studied plant samples based on the contents of toxic metals

Figure 3. Principal component score plot (F1 and F2) of the studied plant samples based on the content of total phenols.

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4. Conclusions Determinations of the total phenols, total flavonoids and toxic metals contents were performed in plants Delphinidum consolida L., Origanum vulgare L., Calendula officinalis L., Cichorium intybus L., Prunus spinosa L., Crataegus oxyacantha L., and Papavear rhoeas L.. The highest content of total phenols was found in 50 % ethanol extracts of oregano. Among toxic metals, the highest quantity was shown of iron. The quantities of toxic metals in the investigated plants were low, making them suitable for use.

Acknowledgments Authors want to thank for the financial support to Ministry of Science, Education and Technological Development of Republic of Serbia (Project No. 174 007).

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