130 Journal homepage: www.fia.usv.ro/fiajournal Journal of Faculty of Food Engineering, Ştefan cel Mare University of Suceava, Romania Volume XVIII, Issue 2 - 2019, pag. 130 - 135 STUDY OF VOLOVATIC (CARDUUS GLAUCINUS) ACTIVE PRINCIPLES FOR POTENTIAL USE AS A MEDICINAL PLANT Ofelia ARVINTE 1 , *Liliana NOROCEL 1 , Sonia AMARIEI 1 1Faculty of Food Engineering, Stefan cel MareUniversity, Suceava, Romania, liliana.norocel@fia.usv.ro, *Corresponding author Received 15 May 2019, accepted 28 June 2019 Abstract: The aim of this study is to establish the antioxidant capacity and phenolic content of one of the most popular medicinal plants in Bucovina area of Romania, named Volovatic or mountain Volovatic (Carduus glaucinus), a plant of the Asteraceae family, Carduus genus, defloratus Carduus species, Carduus glaucinus (glaucus) subspecies, less studied but widely used in the area of origin. It was determined Volovatic antioxidant potential, respectively polyphenol content and antioxidant activity, contents of metals and metalloids from the plant in order to assess a possible toxic effect. Keywords: volovatic, polyphenol content, DPPH, heavy metals 1. Introduction Medicinal plants constitute one of the main sources of dietary supplements, phytochemicals and pro-vitamins very important in maintaining good health. Most medicinal plants are used in the form of teas. The role of herbal tea in disease prevention and cure is due mostly to antioxidant properties of their constituents [1]. Although knowledge and literature about herbs are very vast, resources are not exhausted. In the mountainous area of Suceava County there are still plants used for therapeutic purposes by the native population, plants that are not at all or sporadically occurring references related to their therapeutic properties, respectively the content in active principles. In the Dorna area, Volovatic (Carduus glaucinus) flowers are used from ancestral herds to the preparation of liver tea [2, 3]. Tea is bitter, which confirms the existence of bitter substances that might be bitter or not. Carduus glaucinus is a perennial herbaceous plant in which renewal buds appear under the layer of dead leaves of basal rosette [4]. Belongs to the genus thistle, although no spikes. Taxonomic classification and botanical characterization of the Volovatic is the following: Kingdom – plantae, Phylum – spermatophyta, Class - dicotoledonatae, Order -asteridae/ compositae, Family- asteraceae, Gender-carduus, Species - carduus glucinus/carduus defloratus. The stem is simple, high 30-50 cm, up to 80 cm, without leaves and covered with bristles. The leaves are lanceolate to top undivided. They form a basal rosette, and if they are arranged on the stem, they alternate. The upper part of the leaves is milky; the bottom is covered with hairs [5]. The tubular flowers, purple, hermaphrodite form a chapel surrounded by sculptors. It blooms from June (July in the mountain area) until September. Flowers are pollinated by insects. The fruit is an achene clear ovoid with a white doll made of hairs [5]. Finally, in place of the flowers, the crook is made up of a tusk of white hairs like silver. Under each puff there is a nacelle, and inside a chicken of the plant. The wind http://www.fia.usv.ro/fiajournal mailto:liliana.norocel@fia.usv.ro Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 2 – 2019 Ofelia ARVINTE, Liliana NOROCEL, Sonia AMARIEI, Study of volovatic (Carduus Glaucinus) active principles for potential use as a medicinal plant, Food and Environment Safety, Volume XVIII, Issue 2 – 2019, pag. 130 – 135 131 is blowing, the puffs are loosening, the sun lounges are stretched, and the baby is wrapped in the diapers [6]. These are an alpine species, which appear from the boreal to the subalpine. These love light, are not pretentious with regard to temperature, hears well the dryness and acidity of the mountain coast [7]. Although the sub-mountain hills and the coasts of the Dornelor Depression are full of blooming shrimps in the summer, Volovatic is a rare plant. It is mentioned in "The Flora and Vegetation of Moldova" as appearing in Suceava County and rarely in Neamt County [2]. It is not mentioned in the flora of other mountain areas (Retezat, Ciucaş, Bucegi) [8-10]. Other species of carduus (thistles) appear, such as: c. candicans, c. kerneri, c. viridis, c. achantoides, c. personata, în Bucegi Mountains [9]. Perhaps because Volovatic is a little widespread plant on the territory of Romania, and also quite rarely encountered in the world, there are few studies that remind it. Besides the fact that the inflorescences of this plant are used by the local population to prepare the tea for the regeneration and proper functioning of the liver, in the Carduus genus of which it is part, we find a number of representatives recognized for their sanogenic effects but which do not grow in the mountainous area of Suceava County: Carduus marianus, Carduus benedicta, Carduus nutans, Silybum marianum, Armurariu [11]. In view of the likely local ancestral use of volovace in combating indigestion/liver problems and the fact that many of its close relatives have demonstrated various beneficial actions on the liver, it is very necessary and challenging to research and find the active principles it contains as well their applicability to phytotherapy. Taking into account these premises, the antioxidant potential of the volovate, namely the content of polyphenols and the antioxidant activity, as well as the mineral content, was determined. Particularly the presence of heavy metals and their content was compared with the maximum admissible limits. 2. Materials and methods Materials Harvested plants were used from the commune of Coşna, Suceava County, from two neighboring areas, located at 47,378558 latitude and 25,171808 long, stereo GPS coordinates 70 x = 513111,239m, y = 653287,138 m respectively 47,375816 and 25,175646 long, stereo coordinates 70: x = 513349,851 m, y = 6256978,629m and at about 860m altitude. For the determinations, aerial parts of plants were harvested during the flowering and naturally dried, shade, and fresh roots, autumn harvested at the end of the vegetation period. Reagents. All reagents were of analytical grade and were purchased from Sigma Aldrich (Germany). Methods The moisture (W) was determined by weighing at the analytical balance of samples from all parts of the plant, dried in the oven until the mass remained constant using ZRD-A5055 oven (Zhicheng, China). W% = (msample – mdried sample)*100/msample where: msample - sample mass before drying mdried sample- sample mass after drying The ash was obtained by calcining at 8000C the samples from all parts of the plant using a Nabertherm LE 2/11/R6 Muffle Furnace. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 2 – 2019 Ofelia ARVINTE, Liliana NOROCEL, Sonia AMARIEI, Study of volovatic (Carduus Glaucinus) active principles for potential use as a medicinal plant, Food and Environment Safety, Volume XVIII, Issue 2 – 2019, pag. 130 – 135 132 Determination of antioxidant capacity was achieved by the DPPH (2.2 -diphenyl- 1-picrylhydrazyl) method. The methanolic solution of DPPH has a maximum absorption at the wavelength of 517 nm due to the unpaired electron. In the presence of an antioxidant, the electron is mated to form the discoloured DPPH-H form, the discoloration being directly proportional to the number of electrons captured. The determinations were carried out for the aqueous extract: 5 g sample to 200 ml water, 30 min boiled extract and alcohol extract: 1 g sample to 50 ml ethanol obtained in an ultrasonic bath at 40°C and 25 KHz frequency from the aerial parts of the plant: leaves, flowers, stem. The samples of 0.5 ml were mixed with 2.5 ml of DPPH solution, 6 x 10-5 M. The absorbance was measured with 3600 Schimadzu UV-VIS-NIR Spectro- photometer. The percent inhibition of free radicals, calculated by comparison with DPPH, was calculated using the formula: I% = A0 – Ap/A0 x100 (1) where: A0 = the absorbance of the reference substance, AP = absorbance of the analysed sample. Determination of the polyphenol content was achieved by the Folin-Ciocalteu method. As aromatic chemical compounds as several hydroxyl groups inserted on the aromatic ring, the polyphenols can be oxidized by the Folin Ciocalteu (Fc) reagent, with a blue colour formation, with a maximum absorption of 750 nm. The Folin - Ciocalteu Index is specific only for phenolic compounds with reducing properties. Total phenolic content was determined with Folin Ciocalteu method, using gallic acid as a standard. The results were expressed as g of gallic acid equivalents (GAE)/L on a dry weight basis [16]. For analysis 2 mL of extract, 1 mL of Folin Ciocalteu reagent and 8 mL of 7.5% Na2CO3 were mixed for 5 min and stored in the dark for 30 min then it was measured the absorbance at 750 nm [17]. Determination of the mineral content in different samples was performed using a mass spectrometer with inductively coupled plasma (ICP-MS Agilent Technologies 7500 Series) Digestion of the samples was performed with concentrated nitric acid and hydrochloric acid, using double deionized water obtained with a Water purification system Thermofisher. Concentration (C) of heavy metals in samples obtained is expressed in µg/g sample and is calculated from the formula [2]: C m V a  (2) where: a - concentration value measured, [ppb]; V - volume of acid that dissolved sample [ml] m - mass of mineralized sample [g]. 3. Results and discussion The ash content shows (Table 1) the mineral richness of the plant, and its determination was used for their elemental analysis. Table 1. Water and ash content the various anatomical parts of the medicinal plant Carduus glaucinus Samples Moisture % Ash % Dried leaves 6.25 8.54 Dried flowers 6.05 3.85 Dried strain 6.38 2.24 Fresh root 30.58 5.24 It is noticeable that the highest mineral content was found in the leaves, followed by root, flowers and plant stem. Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 2 – 2019 Ofelia ARVINTE, Liliana NOROCEL, Sonia AMARIEI, Study of volovatic (Carduus Glaucinus) active principles for potential use as a medicinal plant, Food and Environment Safety, Volume XVIII, Issue 2 – 2019, pag. 130 – 135 133 Table 2. Metals and metalloids content of the various anatomical parts of the medicinal plant Carduus glaucinus, in μg/g. Tea 1 (Leaves) Tea 2 (Flowers) Li 0.366 9.129 Na 30.853 656.689 Mg 32.361 675.819 Al 0 13.035 Ca 305.613 2789.826 Ti 59.375 553.567 V 0.065 3.958 Cr 1.649 39.300 Mn 1.928 55.729 Fe II 0.116 3.545 Fe III 0 0.907 Co 0 1.219 Ni 0.142 10.130 Cu 0.100 9.102 Zn 0.014 1.687 Ga 0 0.104 As 0.072 0.350 Se 0.083 1.101 Sr 0 136.398 Mo 57.546 519.011 Pd 0 0.013 Ag 0 0.014 Cd 0.630 0.735 In 4.019 0.527 Sn 0 6.117 Sb 0.105 0.193 Te 0 2.065 Cs 0.278 4.432 Ba 0.270 4.360 Pt 0 0 Au 0 0.657 Hg 0.059 0.056 Tl 0.010 0.144 Pb 0 0 U 0 0 Table 2 shows the high Mg and Ca content of the plant flowers, followed by those of the root and then of the stem, the smallest values being in the leaves. Fe2+, Se and Zn are high in flowers and roots. In terms of heavy metals, Pb is absent in the entire plant, and Hg and Cd to be well below the maximum limit of 10mg/kg for children. [18]. Tea 3( Strain) Tea 4 (Root) Li 5.097 2.982 Na 290.811 302.707 Mg 185.585 247.688 Al 294.393 1019.286 Ca 2021.654 2749.519 Ti 360.831 822.616 V 1.831 13.717 Cr 19.751 2.744 Mn 25.737 200.609 Fe II 1.979 8.144 Fe III 0.417 1.782 Co 0.571 1.697 Ni 4.769 8.384 Cu 4.572 6.818 Zn 1.095 1.493 Ga 0.081 0.248 As 0.387 0.492 Se 1.015 4.179 Sr 157.211 4674.264 Mo 597.403 4542.413 Pd 0.010 0.039 Ag 7.393 0.019 Cd 0.763 0.010 In 0.786 0.276 Sn 7.394 3.884 Sb 0.178 0.143 Te 0.428 1.338 Cs 9.218 10.729 Ba 8.839 13.805 Pt 0 0 Au 0 0 Hg 0.032 0.046 Tl 0.083 0.121 Pb 0 0 U 37.234 0 From the analysis of the results it can be concluded that, from the point of view of the elemental composition, the flowers are Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 2 – 2019 Ofelia ARVINTE, Liliana NOROCEL, Sonia AMARIEI, Study of volovatic (Carduus Glaucinus) active principles for potential use as a medicinal plant, Food and Environment Safety, Volume XVIII, Issue 2 – 2019, pag. 130 – 135 134 most suitable for use in the form of tea. Cadmium, arsenic, mercury and antimony were within the safety limits and lead was below the detection limits. Table 3. Antioxidant capacity of the various anatomical parts of the medicinal plant Carduus glaucinus (I %) Samples Aqueous extract Alcoholic extract Leaves 61.348 2.033 Flowers 89.947 20.444 Strain 78.652 30.497 It can be seen that for all parts of the plant, the aqueous extract is much richer in antioxidants, the biggest difference being observed in the case of the leaves. Table 4. Polyphenol content of the various anatomical parts of the medicinal plant Carduus glaucinus Samples Aqueous extract Alcoholic extract Dried leaves 8.526 g/L 0.865 g/L Dried flowers 9.107 g/L 1.009 g/L Dried strain 11.116 g/L 0.755 g/L In according with the previous result, the concentration of polyphenols is much higher in the aqueous extract than in the alcohol, being the highest in the strain. In the alcoholic extract the highest amount of polyphenols is found in the flowers. 4. Conclusion Volovatic (Carduus glaucinus) is a rich source of antioxidants and phenolic compounds and can protect the human body from the action of free radicals. The determined metals and metalloids show that the traditional medicinal plant is safe to use. Along with the other plants of the Cardus species, tea from Carduus glaucinus can be used for the regeneration and proper functioning of the liver especially in the treatment and control of liver disease. 5. References [1]. D. IVANAOVA, D. GEROVA, T. CHERVENKOV, T.YANKOVA, Polyphenols and antioxidant capacity of Bulgarian medicinal plants, Journal of Ethnopharmacology, Volume 96, Issues 1–2, 4 January, Pages 145-150 (2005). [2]. CHIFU TOADER, MÂNZU CIPRIAN, ZAMFIRESCU OANA, Flora and vegetation of Moldova (Romania), Alexandru Ioan Cuza University Publishing House Iaşi, (2006). [3]. https://it.wikipedia.org/Carduus_defloratus; [4]. https://dexonline.ro/definiţie/hemicriptofita [5]. MITITELU D., VIŢALARIU GH., CHIFU T., ŞTEFAN N., DĂSCĂLESCU D., HOREANU CL., – Flora of Calimani Mountains, (in Romanian) An. Şt. Univ. „Al. I. Cuza” Iaşi, 32, s. II a., Biol.: 28-30 (1986); [6]. SIMIONESCU ION, Flora of Romania, Editura Albatros, (in Romanian) (1973) [7]. AESCHIMANN D., LAUBER K., MOSER D. M., THEURILLAT J.-P., – Alpin Flora. Atlas des 4500 plantes vasculaires des Alpes, vol. I-III, Belin, Paris, p. 622-623 vol. I, 560-561, 576-577 vol.II (2004); [8]. NYARADI E. I. Flora and vegetation of Retezat Mountain, (in Romanian), Publishing House of the Romanian People's Republic (1958). [9]. BELDIE ALEXANDRU, Flora and vegetation of Bucegi Mountain, Publishing House of the Academy of the Socialist Republic of Romania, (in Romanian) (1967). [10]. CIUCĂ MARIA, Flora and vegetation of grassland from Ciucaş Mountain, Publishing House of the Academy of the Socialist Republic of Romania, (in Romanian), (1967). [11]. BUHNER, STEPHEN HARROD, Plants for liver disease Publishing House of Guides, Bucureşti (2006). [12]. RABEA PARVEEN, SANJULA BABOOTA, JAVED ALI, ALKA AHUJA, SURUCHI S. VASUDEV, AND SAYEED AHMAD, Effects of Silymarin Nanoemulsion against Carbon Tetrachlorideinduced Hepatic Food and Environment Safety - Journal of Faculty of Food Engineering, Ştefan cel Mare University - Suceava Volume XVIII, Issue 2 – 2019 Ofelia ARVINTE, Liliana NOROCEL, Sonia AMARIEI, Study of volovatic (Carduus Glaucinus) active principles for potential use as a medicinal plant, Food and Environment Safety, Volume XVIII, Issue 2 – 2019, pag. 130 – 135 135 Damage, Arch Pharm Res Vol 34, No 5, 767-774, (2011); [13]. GOINA, T., CONSTANTINESCU E, CIULEI I et al., Pharmacognosy, Didactic and Pedagogical Publishing House, Bucharest, (in Romanian), (1967). [14]. ISTUDOR VIORICA, Pharmacognosy, phytochemistry of phytotherapy, vol I, Medical Publishing House, Bucharest, (in Romanian), 20 (1998). [15]. PÂRVU C. The Universe of Plants - Little Enciclopedia, Enciclopedia Publishing House Bucharest, (in Romanian), (1991). [16]. SU, X., DUAN, J., JIANG, Y., DUAN, X., & CHEN, F., Polyphenolic profile and antioxidant activities of oolong tea infusion under various steeping conditions. International journal of molecular sciences, 8(12), 1196-1205. (2007). [17]. SRIPAKDEE, T., SRIWICHA, A., JANSAM, N., MAHACHAI, R., & CHANTHAI, S. Determination of total phenolics and ascorbic acid related to an antioxidant activity and thermal stability of the Mao fruit juice. International Food Research Journal, 22(2), (2015). [18]. AMARIEI, S., GUTT, G., OROIAN, M., Study on Toxic Metal Levels in Food Supplements. Revista de chimie, 68(6), 1298-1301 (2017). [19]. ALEKSANDRA STANOJKOVI -SEBIC, RADMILA PIVIC, DRAGANA JOSIC, ZORAN DINIC, ALEKSANDAR STANOJKOVIC, Heavy Metals Content in Selected Medicinal Plants Commonly Used as Components for Herbal Formulations, Journal of Agricultural Sciences, 21 317-325, (2015). 1. Introduction 4. Conclusion