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Chemical Composition and Antioxidant Activity of Leaves of Mycorrhized

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Chemical Composition and Antioxidant Activity of Leaves of Mycorrhized Chemical composition and antioxidant activity of leaves of mycorrhized sea- buckthorn (Hippophae rhamnoides L.) Anna Jaroszewska1, and Wioletta Biel2* ABSTRACT INTRODUCTION Natural products, either as extracts or as pure compounds, The leaves of sea-buckthorn (Hippophae rhamnoides L.) give limitless possibilities for the discovery of new drugs and can be a rich source of nutrients and biologically active improving food quality, also thanks to their wide availability. substances. Their levels depend on growing conditions, Sea-buckthorn (Hippophae rhamnoides L.) contains a series of agricultural technology and climate. The studies however, compounds including, among others, carotenoids, tocopherols, mainly focus on nutritious value of fruit and seeds and sterols, flavonoids, lipids, ascorbic acid, and tannins. These there is shortage of the information regarding to buckthorn compounds are of interest not only from the chemical point leaves. The aim of the experiment was to determine the of view, but also because many of them exhibit biological and effect of symbiotic mycorrhizal fungi on the chemical therapeutic activities, including antioxidant, antitumor, hepato- composition and antioxidant activity of sea-buckthorn protective, and immunomodulatory properties (Saikia and leaves. The study was conducted in 2014 and 2015 at the Handique, 2013). Evaluation of the nutritional value of sea- Experimental Station in Lipnik, Poland. Mycorrhization buckthorn usually concerns its fruit and seeds, while the leaves improved the nutritional value of leaves of sea-buckthorn are rather neglected. However, some studies confirm that also its by increasing levels of total protein (3%), N free extract leaves are a rich source of compounds with anti-inflammatory (1%), Ca (9%), Na (16%), Fe (18%), Cr (34%), and (Padwad et al., 2006) and antibacterial properties (Upadhyay partially elevating antioxidant activity by increasing the et al., 2011). They contain large amounts of crude protein, concentration of polyphenols (7%). Leaves of ‘Habego’ relatively high crude fat, and are a good source of macroelements had a higher nutritional value, containing more total (Jaroszewska et al., 2016). Therefore, sea-buckthorn leaves may protein (4%), crude fat (7%), crude fiber (30%), and all be an excellent source of biologically active phytochemicals in fractions of dietary fiber. ‘Hergo’ had more beneficial both medicine and human nutrition. levels of minerals (P-27%, K-67%, Mg-4%, Na-30%, Zn- The arbuscular mycorrhizal symbiosis is recognized for its 8%, and Cr-21%), polyphenols (51%), total flavonoids multiple positive effects on plant growth and for its important (35%), carotenoids (10%), and L-ascorbic acid (8%), as contribution to the maintenance of soil quality. In spite of these well as higher antioxidant activity (40%). The results of benefits to agriculture, the realization of the full potential of our study partly confirmed the earlier scientific reports on this symbiosis has not yet been reached (Karagiannidis et al., the impact of mycorrhiza on the chemical composition of 2012). During the establishment of the arbuscular mycorrhizal plants. However, it is not possible to compare our results symbiosis, a range of chemical and biological parameters is with data on berry plants, including sea-buckthorn, due to affected in plants, including the pattern of secondary compounds the lack of information in the literature. (Karagiannidis et al., 2012). Previous studies confirm that arbuscular mycorrhizal fungi (AMF) affects the content of Key words: Antioxidants, chemical composition, fiber phenols (Hazzoumi et al., 2015). Higher content of flavonoids fractions, mycorrhiza, vitamins. has been demonstrated in mycorrhizal roots of alfalfa (Medicago sativa L.) (Larose et al., 2002). The AMF root colonization factor leads to significantly increased concentrations of most of the phenolics in purple coneflower (Araim et al., 2009) and in 1 West Pomeranian University of Technology in Szczecin, Department strawberry fruits (Castellanos-Morales et al., 2010). of Agronomy, Papieża Pawła VI 3, 71-434 Szczecin, Poland. 2West Pomeranian University of Technology in Szczecin, Department Currently, there is little information on the chemical of Pig Breeding, Animal Nutrition and Food, Judyma 10, 71-460 composition and antioxidant properties of sea-buckthorn Szczecin, Poland. *Corresponding author ([email protected]). (Hippophae rhamnoides L.) leaves. There is also a lack of data Received: 13 December 2016. concerning the potential impact of symbiotic mycorrhizal fungi on Accepted: 23 February 2017. the quality of berry plants, including sea-buckthorn. Therefore, in doi:10.4067/S0718-58392017000200155 this study we conducted a field experiment to evaluate the impact of symbiotic mycorrhizal fungi on the chemical composition RESEARCH RESEARCH CHILEAN JOURNAL OF AGRICULTURALCHILEAN JOURNAL RESEARCH OF 77(2) AGRICULTURAL APRIL-JUNE RESEARCH 2017 77(2) APRIL-JUNE 2017 155 and antioxidant activity of leaves of two varieties of sea- (NDF) was conducted on an ash-free basis and buckthorn. included sodium dodecyl sulfate (NaC12H 25SO4) (822050, Merck, Kenilworth, Nueva Jersey, USA). MATERIALS AND METHODS Determination of acid detergent fiber (ADF) included hexadecyltrimethylammonium bromide (C16H 33) The study was conducted in 2014 and 2015 at the N(CH3)3Br (Merck 102342), while acid detergent lignin Experimental Station in Lipnik (53°20’35” N, 14°58’10” (ADL) was determined by hydrolysis of ADF sample in E, 7 m a.s.l.), Poland. The soil on which the experiment 72% sulfuric acid (H2SO4). Hemicellulose (HCEL) was was carried out belonged to typical rusty soils (Polish calculated as the difference between NDF and ADF, while Soil Classification, 2011), classified as Haplic Cambisol cellulose (CEL) as the difference between ADF and ADL. according to IUSS Working Group WRB (2015). At the The material for the macro-components concentration Ap level, the soil has a slightly acidic loamy sand. The analyses was subjected to mineralization in concentrated level of humus is formed from clay sands. The analysis sulfuric acid (H2SO4) and perchloric acid (HClO4), of soil minerals showed high P levels and moderate Mg whilst the material for micro-components concentration and K levels. The experiment was designed according to a analyses was digested in a mixture of nitric acid (HNO3) completely randomized method in five replicates (one shrub and perchloric acid (HClO4). The concentration of P was = one repeat). Shrubs were planted in 4 × 3 m spacing. The determined by the Egner-Riehm colorimetric method, with size of single plot was 12 m2. The total number of plots in ammonium molybdate, at wavelength 660 nm, by using a the experiment was 20. The subject of the study included Specol 221 apparatus (866287, Carl Zeiss Jena, Germany). 2 and 3 yr old shrubs, female varieties Habego and Hergo. An atomic absorption spectrometer (ASA) (iCE 3000 Mycorrhization was conducted with ectomicorrhizal Series, Thermo Fisher Scientific, Waltham, Massachusetts, mycelium, which is symbiotic for plants of the olive family. USA) was used to determine K, Na, and Ca – by means The isolate was obtained from the natural ecosystems in of emulsion flame spectroscopy, and Mg, Zn, Fe, Pb, Cr Croatia. It contains symbiotic mycorrhizal fungi (Glomus and Cu – by means of absorption flame spectroscopy. The spp., Gigaspora spp., Pochonia spp., Lecanicillium spp.), nitrate content was determined by potentiometry. Prior and the root bacteria (Bacillus spp.) A dose of 15 mL was to assay Ca content, K and Mg trials were appropriately applied in two places the root zone of plants in the first diluted. Other mineral compounds were determined in year of experiment. The mycelium contained an addition of concentrated samples. hydrogel (ensuring humidity essential for the initial fungi For total polyphenols, total flavonoids, and antioxidant development). Leaves for the analysis were collected during activity determination, methanol extracts were prepared harvest from shoots without fruiting. Leaves were collected according to Kumaran and Karunakaran (2007). The total from the outside of the bush, at half of their height. The phenolic content of plant extracts was determined using 3 leaves were taken from the 1 yr old shoots without any Folin-Ciocalteu reagent (Yu et al., 2002); 0.1 cm plant 3 signs of aging or mechanical damage. extract was mixed with 0.5 cm Folin-Ciocalteu reagent and 3 The experiment factors were mycorrhiza (half of the 1.5 cm 20% sodium carbonate. The mixture was shaken 3 bushes from each variety were subject to mycorrhization, thoroughly and made up to 10 cm using distilled water. The and half were used as a control group) and variety (Habego mixture was allowed to stand for 2 h. Then the absorbance and Hergo). at 765 nm was determined. These data were used to All determinations were expressed on a dry weight estimate phenolic content using a standard curve obtained (DW) basis. Dry matter, crude protein, ether extract, crude from various concentrations of gallic acid. Total polyphenol fiber, crude ash and N free extract (NFE) were determined content in samples was calculated as the amount of gallic -1 by AOAC (2012). Dry matter was evaluated by drying acid equivalent (GAE) in mg kg sample DW. at 105 °C to constant weight; ether extract by Soxhlet The total flavonoids content was determined by the extraction with diethyl ether; crude ash by incineration method of Kumaran and Karunakaran (2007) using 3 in
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