buckwheat intercrops as influenced by fertilization

Accepted Manuscript Antioxidant capacity and polyphenols in buckwheat seeds from fenugreek /buckwheat intercrops as influenced by fertilization

Aliyeh Salehi, Sina Fallah, Hans-Peter Kaul, Karin Zitterl-Eglseer PII:

S0733-5210(18)30165-6

DOI:

10.1016/j.jcs.2018.06.004

Reference:

YJCRS 2584

To appear in:

Journal of Cereal Science

Received Date:

22 February 2018

Accepted Date:

08 June 2018

Please cite this article as: Aliyeh Salehi, Sina Fallah, Hans-Peter Kaul, Karin Zitterl-Eglseer, Antioxidant capacity and polyphenols in buckwheat seeds from fenugreek/buckwheat intercrops as influenced by fertilization, Journal of Cereal Science (2018), doi: 10.1016/j.jcs.2018.06.004

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ACCEPTED MANUSCRIPT 1

Antioxidant capacity and polyphenols in buckwheat seeds from fenugreek/buckwheat

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intercrops as influenced by fertilization

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Aliyeh Salehi*1,2, Sina Fallah1, Hans-Peter Kaul2, Karin Zitterl-Eglseer 3

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*corresponding

Faculty of Agriculture, Shahrekord University, Shahrekord, Iran

Division of Agronomy, Department of Crop Sciences, University of Natural Resources and Life Sciences, Vienna (BOKU), Konrad Lorenz-Straße 24, 3430 Tulln, Austria Institute of Animal Nutrition and Functional Plant Compounds, Department for Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Veterinärplatz 1, 1210 Vienna, Austria author: [email protected] ; Tel: 00(43)68120585956

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Abstract

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A two-year field experiment was conducted to investigate the effects of different intercropping ratios and

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fertilizer types on antioxidant activity, total phenolic content (TPC), total flavonoids content (TFC) and

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content of main flavonoids of buckwheat seeds (Fagopyrum esculentum Moench). The treatments consisted

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of sole cropping of buckwheat (B), fenugreek (F), and three intercropping ratios (F:B = 1:2, 1:1 and 2:1)

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under three fertilizer types: chemical fertilizer (CF), integrated fertilizer (IF) and broiler litter (BL). The

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buckwheat seeds grown in intercropping had higher antioxidant activity measured by DPPH (20.2% in 2014

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and 29.6 in 2015) and FRAP (16.9% in 2014 and 29.9% in 2015) assay, TPC (13.6% in both years), TFC

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(22.9% in 2014 and 11.3% in 2015), flavonoids such as rutin (12.4% in 2014 and 10.8% in 2015), vitexin

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(10.4% in 2014 and 14.2% in 2015), isovitexin (18.1% in 2014 and 7.8% in 2015), orientin (7.3% in 2014

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and 7.07% in 2015), isoorientin (6.6% in 2014 and 9.6% in 2015) and hyperoside (25.1% in 2014 and 27.4%

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in 2015) than with sole cropping. The intercropping ratio of F:B (2:1) was the most suitable for promoting

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the tested antioxidant activity and bioactive compounds. Overall, the IF and BL showed significant benefits

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compared to CF for all detected compounds in both sole and intercrops.

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Keywords: flavonoids, fagopyrum esculentum Moench, organic manure

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1. Introduction

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Interest in organic and medicinal products and the use of these sources as food are increasing in

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the world (Pacheco et al., 2017). One of the most important functional foods and traditional

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medicine worldwide is common buckwheat (Fagopyrum esculentum Moench) (Lee et al., 2016). It

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is a versatile crop with different agricultural purposes (Žvikas et al., 2016) grown in Europe, USA,

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Brazil, Canada, Australia, South Africa and Asia (Kiprovski et al., 2015).

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In recent years, common buckwheat is gaining interest in the development of new food products

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due to health-promoting, biofunctional properties, gluten-freeness and its high nutritional value,

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specifically based on proteins with a high biological value, essential amino acids, natural

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antioxidants, high levels of flavonoids (rutin, hyperoside, vitexin, isovitexin, orientin, isoorientin,

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catechin and epicatechin gallate) and dietary fiber, vitamins and minerals (Kiprovski et al., 2015;

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Žvikas et al., 2016; Dziadek et al., 2016, Lee et al., 2016).

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The fruit of buckwheat is a triangular achene. The hull (pericarp, fruit coat), the outer layer of

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the achene, has a hard fibrous structure that is usually dark brown or black in colour. (Steadman et

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al., 2001). The hull is a waste products and only groats as raw or processed into flour can be source

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of functional products such as pasta, bread, beer and many others in the form of buckwheat or

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fortified buckwheat products. On the other hand, the extent of variation in the content of rutin and

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flavone C-glucosides in the hull may be useful for researchers working on the resistance of

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buckwheat lines against pathogens and diseases whilst extent of variation in the flavonoid

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composition in groats should be important from the nutritional point of view (Zielińska et al.,

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2012).

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Rutin is a polyphenolic compound that is extensively distributed in plants and buckwheat is a

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good source of dietary rutin among grain crops. It has been shown that flavonoids have a wide

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spectrum of positive biological effects on health such as anti-inflammatory, anti-prostaglandin and

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antioxidant activities, support the relaxation of cardiac muscles, protect ascorbic acid and low3

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density lipoproteins against oxidation, and have anti-thrombosis, antibacterial and antiallergic

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effects (Afanas’ev et al., 2001; Lee et al., 2016;). Studies demonstrated that buckwheat is rich in

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many rare compounds with positive effects to some chronic diseases and it has been shown that the

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consumption of buckwheat is related to a broad range of biological and healthy effects which

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phenolic compounds are assumed to be responsible for these benefits (Giménez-Bastida and

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Zielinski, 2015). Also, Zielińska and Zieliński (2009) indicated that buckwheat seeds (both hull and

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kernel) are sources of antioxidant activities which are important ingredients of food products.

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Numerous of the health benefits of buckwheat have been attributed to its very high antioxidant

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activity, resulting in a low incidence of cancers, cardiovascular diseases and also age-related

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degenerative processes. Several scientific studies have shown that the levels of different minerals

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and bioactive phytochemicals depend on crop management and environmental conditions (Kalinova

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and Vrchotova, 2011). Therefore, to achieve organic food products and improve the composition

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and seed quality; organic farming (Žvikas et al., 2016) and intercropping with legumes (Elsheikh et

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al., 2009) can be implemented. Buckwheat is mainly grown for seed production, because of

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significant medical and nutritional benefits (Kiprovski et al., 2015).

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Intercropping is attracting increasing interest and is widespread practice in low-input crop

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production systems in the world. Generally, it has been shown to achieve higher quality and

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quantity of production than with sole cropping (Weisanya et al., 2015; Salehi et al., 2017).

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Reasonable intercropping cultivation with legumes could increase productivity and quality of seeds

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due to enhanced utilization of resources, i.e. water, space, time, radiation and an increased plant

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macro- and micronutrients supply resulting partly from biological nitrogen fixation in legume-

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rhizobia symbiosis and its consequent improvement of soil fertility (Salehi et al., 2017, 2018).

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Weisanya et al. (2015) reported that few studies have been implemented on intercropping of

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medicinal plants with legumes, and it has been shown that intercropping systems may affect

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production, qualitative aspects and chemical composition of plant extracts.

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Also fertilizer can affect the phytochemical status of food. Chemical fertilizers are expensive,

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while application of mineral fertilizers only, in the long-term, has resulted in significantly negative

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environmental impacts such as soil acidification and therefore progressive decline of soil fertility.

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Nutrient supply, however, is an important factor in sustainable or organic agriculture that improves

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both bioactive compounds content and productivity of crops, thereby increasing the medicinal and

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aromatic plant quality (Costa et al., 2013). In these systems, constant search for alternatives to

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replace the use of chemical fertilizers occurs (Pacheco et al., 2017). The successful application of

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some alternative organic fertilizers have been shown with broiler litter in intercropped fenugreek-

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buckwheat system (Salehi et al., 2017, 2018).

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Organic fertilizers are a readily available organic source for supply with essential plant nutrients

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and adequate nourishment, due to improvement of the chemical, physical and biological

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characteristics of the soil and also replenishing of organic matter (Elsheikh et al., 2009). Some

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studies reported that mineral fertilizers decrease antioxidant levels while organic fertilizers improve

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antioxidant activity in plants (Faller and Fialho, 2010).

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So far many studies considered the efficient utilization of the available resources by legume/non-

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legume or medicinal plant intercropping systems on the mineral content and bioactive compounds

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composition. But to the best of our knowledge, information on bioactive compounds composition in

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seed obtained from fenugreek-buckwheat intercrops grown under application of organic and

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inorganic fertilization have not been reported to date.

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Therefore we hypothesized that application of organic fertilizer can improve the content of

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bioactive compounds and seed quality in both sole and buckwheat intercrops compared to chemical

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fertilizer. The aims of our study were to analyse: (i) antioxidant activity i.e., DPPH and FRAP

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assay, (ii) total phenolic content (TPC), (iii) total flavonoids content (TFC) and main flavonoid

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compounds content i.e., rutin, vitexin, isovitexin, orientin, isoorientin and hyperoside of buckwheat

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seeds from intercrops compared to sole crops as affected by intercropping ratio and fertilizer type.

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2. Materials and methods 5

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2.1.

Experimental plan

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A field experiment was established at the research farm of Shahrekord University (32°21´ N,

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50°49´ E; 2050 m a.s.l.), Iran, in the consecutive years 2014 and 2015. The experimental site is

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characterized by average yearly temperatures of 10.5 °C and amounts of rainfall of 280 mm. The

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soil texture is clay loam and the soil type is a fine, carbonatic, mesic Calcixerept.

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2.2. Experimental design and treatments

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A two-factorial experiment in randomized complete block design was conducted with three

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replications in 2014 and 2015. The first factor was cropping system with 5 levels, i.e. sole cropping

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of buckwheat (B), sole cropping of fenugreek (F), and three substitutive row intercropping ratios,

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i.e. F:B = 1:2 (one row of fenugreek + two rows of buckwheat), 1:1 (one row of fenugreek + one

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row of buckwheat) and 2:1 (two rows of fenugreek + one row of buckwheat). The second factor

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was N fertilizer type with 3 levels, i.e. chemical fertilizer (CF), broiler litter (BL) and integrated

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fertilizer (IF = 50% CF + 50% BL).

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2.3. Experimental set-up, fertilization rates and management

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Individual experimental plots had an area of 7.5 m2 (2.5 × 3 m). The amount of nitrogen applied

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was 80 and 60 kg N ha-1 for fenugreek and buckwheat, respectively, according to local farmers’

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practices. Application of 10 and 7.5 Mg ha-1 of broiler litter provided 80 and 60 kg N ha−1,

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respectively, assuming 50% mineralization of broiler litter N during the first cropping season.

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Intercrops received equivalent fertilizer amounts according their species composition. Broiler litter

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was applied by hand before sowing. In the chemical fertilizer treatments urea was used, with one-

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third of the total rate applied at planting and two-thirds at 30 days after planting. Phosphorus, as

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triple superphosphate, and micronutrients such as Fe, Mn, Cu, and Zn were applied to the urea-

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fertilized plots at a rate equivalent to the total amounts added by the broiler litter treatments in order

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to compensate for the nutrient inputs of these elements with the organic fertilizer. In the integrated

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fertilizer treatment we applied 50% of chemical fertilizer and 50% of broiler litter each according to

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the indicated procedures.

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Seeds of buckwheat were provided by a German commercial source (Rudloff Feldsaaten

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GmbH). Fenugreek and buckwheat were sown by hand simultaneously on May 29, 2014 and May

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23, 2015 at 120 and 50 plants m-2 at a depth of 2-4 and 1-2 cm, respectively, both in sole and

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intercropped plots. In each planting hole we sowed three seeds of fenugreek or buckwheat, and to

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obtain required plant densities, fenugreek and buckwheat seedlings were thinned at the 3-4 leaf

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stage. Irrigation was done at 6-day intervals. More information can be obtained from Salehi et al.

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(2017, 2018).

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2.4.

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Plants were harvested manually as samples of 20 buckwheat plants by cutting on the soil surface at

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full maturity on September 6, 2014 and September 1, 2015. Thereafter, seeds were separated from

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plants and oven-dried at 70˚C for 48 h to obtain a constant weight.

Seed harvest

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For determination of bioactive compounds content, seed subsamples of buckwheat were

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transferred to the Institute of Animal Nutrition and Functional Plant Compounds, Department for

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Farm Animals and Veterinary Public Health, University of Veterinary Medicine Vienna, Austria.

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2.5.

Characteristics evaluated

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2.5.1.

Reagents and chemicals

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Acetonitrile HPLC grade (Chem-Lab NV, Belgium); TPTZ (2,4,6-tripyridyl-s-triazin, > 99%)

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(Fluka); FeCl3.6H2O, Folin-Ciocalteu reagent (Merck); acetic acid p.a., AlCl3.6H2O, caffeic acid (>

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99%), ethanol (96%) p.a., HCl, hyperoside (ROTICHROM®, > 99%), isovitexin (ROTICHROM®,

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> 99%), methanol p.a., NaCH3COOH.3H2O, Na2CO3, NaOH, NaNO2, vitexin (ROTICHROM®, >

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99%) were purchased from Roth; DPPH: 2,2-Diphenyl-1-Picrylhydrazyl (> 99%), isoorientin

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(HPLC-grade, > 98%), orientin (> 97%), rutin (> 99%), trolox: (±)-6-Hydroxy-2,5,7,8-tetra-

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methylchromane-2-carboxylic acid (> 98%) were from Sigma-Aldrich.

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2.5.2. Extraction 7

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For determination of antioxidant activity (measured by DPPH and FRAP assay), total phenolic

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content, total flavonoids content and main flavonoid compounds content, 400 mg of milled dried

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buckwheat seeds were weighted into a test tube and extracted with 4 ml of 80% ethanol in an

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ultrasonic bath (Sonorex RK 156H, Bandelin) for 30 min. Then they were filtered through folded

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filters (Whatman) and the extracts transferred to sealed vials. All extracts were kept at -20 °C prior

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to further analysis.

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2.5.3. Determination of antioxidant activity

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2.5.3.1. DPPH assay

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The DPPH (2,2-Diphenyl-1-Picrylhydrazyl) assay was performed as described by Brand-

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Williams et al. (1995) with some modifications. In a 96-well microplate, 100 µl of DPPH solution

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(3.8 mg DPPH in 25 ml MeOH) was mixed with 15 µl of each seed sample extract and 85 µl

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MeOH. Then, microplate was covered with parafilm and incubated in dark at room temperature for

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30 min. The decrease of absorbance was estimated at λ= 490 nm using a microplate reader

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(iMarkTM, Biorad). Trolox was used to generate a calibration curve (8 different concentration

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points, each concentration point represented the mean of four measurements). Each experimental

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sample was measured four times. Trolox was used to produce a calibration curve. The antioxidant

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activity for each seed sample was expressed as mg of trolox equivalents (TE) per g of dry weight

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(DW) of seed.

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2.5.3.2. FRAP assay

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The FRAP (Fe+++-Reduction, Ferric reducing antioxidant power) assay was carried out as described

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by Benzie and Strain (1996), with some modifications. FRAP reagent was prepared by mixing 10

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mM/l TPTZ in 40 mM/l HCl, 20 mM/l FeCl3.6H2O, and 300 mM/l acetate buffer (pH 3.6) using the

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ratio 1:1:10 (v/v/v). Then 180 µl of FRAP reagent was mixed with 9 µl seed sample extract and

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15µl distilled water in 96-well plates. Finally, mixtures were incubated in dark for 5 min at room

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temperature. The absorbance of resulting solution was then measured at λ= 490 nm using a 8

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microplate reader. Trolox was used to generate a calibration curve (8 different concentration points,

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each concentration point represented the mean of four measurements). Each experimental sample

215

was measured four times. The antioxidant activity for each sample was expressed as mg of trolox

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equivalents (TE) per g of dry weight (DW) of seed.

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2.5.4. Determination of total phenolic content (TPC)

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Total phenolic content was determined by Folin-Ciocalteu-Reagent (FCR) method according to

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Singleton and Rossi (1965) with some modifications using a microplate reader. In an alkaline

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milieu the phenolic compounds react with FCR resulting in a blue colour, which can be used for

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photometric measurement. Briefly, 5 µl of FCR was mixed with 10 µl of each seed sample extract

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and 100 µl of distilled water (AD) in 96-well plates. Mixtures were shaken for 3 minutes and then

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10 µl of Na2CO3 (35 g in 100 ml AD) and 125 µl AD were added. Finally, microplate was covered

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with parafilm and incubated at room temperature in dark for 60 min. Absorbance of samples was

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recorded at λ= 750 nm. Caffeic acid was used to generate a calibration curve (8 different

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concentration points, each concentration point represented the mean of four measurements). Each

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experimental sample was measured four times. The total phenolic content for each sample was

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expressed as mg of caffeic acid equivalents per g of dry weight (DW) of seed.

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2.5.5. Determination of total flavonoids content (TFC)

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Determination of total flavonoids content was performed according to Leontowicz et al. (2003)

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with some modifications. Briefly, to each 40 µl buckwheat seed sample extract 100 µl Aqua dest.

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(AD), 15 µl 2.5% NaNO2-solution and 15 µl 10% AlCl3.6H2O-solution were added in 96-well

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plates. Mixtures were shaken for 5 min on the IKA MTS4 and after adding 50µl 1M NaOH to each

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sample again the plate was shaken for 5 min. Finally, absorbance of resulting solution was recorded

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at λ= 490 nm using a microplate reader. Rutin was used to generate a calibration curve (8 different

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concentration points, each concentration point represented the mean of four measurements). Each

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experimental sample was measured four times. Rutin was used to generate a calibration curve. The 9

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total flavonoids content for each sample was expressed as mg of rutin equivalents per g of dry

239

weight (DW) of seed.

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2.5.6. Quantitative determination of flavonoids by high-performance liquid chromatography with

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photodiode array detection (HPLC-PDA)

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The chromatographic separation was performed on a Shimadzu Nexera HPLC system

243

(Shimadzu) which consisted of pump (LC-20ADXR), degasser (DGU-20A5R), column oven

244

(CTO-20AC), controller (CBM-20A), autosampler (SIL-20AXR), photodiode array detector (SPD-

245

M20A) and software LabSolution. The ethanolic extracts of all buckwheat seed samples were

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filtered through membrane filters (Rotilabo® nylon, pore size 0.20 µm, Roth) before injection. All

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chromatographic analyses were performed on a WatersXBridge Shield column RP-18, 150 x

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4.6 mm; 3.5 µm (Waters) in combination with an appropriate guard column (security guard

249

cartridge, RP-18, 4 x 3 mm, 5 µm, Phenomenex).

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The mobile phase consisted of 2% acetic acid in water (v/v) (solvent A) and acetonitrile (solvent

251

B). A linear gradient of the solvent B was applied from 6-17% for 28 min, and from 17-20% for 15

252

min, followed by an isocratic wash out phase of 100% B for 5 min before returning to the initial

253

conditions. The flow rate was 1 ml min-1, injection volume: 10 µl. The column temperature was set

254

at 25°C, and the components were detected at 354 nm. The identification of the main phenolic

255

compounds was based on the retention times (orientin 22.8 min, isoorientin 23.7 min, vitexin 25.5

256

min, isovitexin 29.5 min, rutin 32.4 min, hyperoside 33.3 min) and their UV-spectral data in

257

comparison with authentically commercially available reference substances (Fig. 1).

258

Details about validation of the HPLC method: To determine the selectivity of the method, the

259

chromatograms of the reference substances (rutin, vitexin, isovitexin, orientin, isoorientin and

260

hyperoside) in 80% ethanol were compared to the chromatograms of the ethanolic extracts of

261

buckwheat. Possible interference by coeluent components was excluded by comparison of UV

262

spectral data by PDA of each relevant substance with data of reference substances. For calibration

263

curves rutin, vitexin, isovitexin, orientin, isoorientin and hyperoside were prepared in methanol 10

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(80%) in duplicate at five concentrations in the range of 5-200 µg/ml for each compound. Recovery

265

(%) was calculated analysing four samples with known amounts of reference substances. In order to

266

evaluate precision the repeatability was analysed using six replicate quality control samples. These

267

were prepared in the same way as the calibration samples. Quantification of the substances in the

268

buckwheat samples was performed using the external standard method. The content of each

269

flavonoid in the 80% ethanolic extract of each seed sample was expressed in mg per gram of dry

270

weight (DW) of seed.

271

2.6.

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An analysis of variance (PROC ANOVA) for two factorial experiments was performed on data

273

from each year considering intercropping ratio as the first factor and fertilizer type as the second

274

factor by using SAS version 9.2. Means were separated by least significant differences (LSD),

275

when the F-test indicated factorial effects on the significance level of p<0.05. MSTAT-C was used

276

to calculate least significant differences (LSD) and letters were used for comparing means of

277

different treatments. The significance of the relationships between antioxidant activity (measured

278

by DPPH and FRAP) and total phenolic, total flavonoids and main flavonoid compounds content

279

was determined using Pearson correlation coefficients.

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3. Results and discussion

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3.1. Antioxidant activity

282

3.1.1. DPPH assay

Statistical analysis

283

In our study, the antioxidant activity was estimated using two different in vitro antioxidant

284

assays (DPPH and FRAP). For the DPPH assay, the reaction between DPPH radicals and

285

antioxidant is obtained through electron transfer mechanisms and hydrogen-atom absorption

286

(Huang and et al., 2005). In both years, a significant interaction effect of intercropping and fertilizer

287

type was found for the antioxidant activity (DPPH) of buckwheat seeds (Table 1 and Table 2). In

288

this study, DPPH in seed samples varied from 2.68 to 6.28 mg TE/g DW (2014) and 3.00 to 6.35 11

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mg TE/g DW (2015). Zielińska et al. (2012) reported antioxidant capacity determined by DPPH

290

assay of ripe buckwheat seeds of 2.15 mg TE/g DW.

291

In the seeds grown in intercropped plots, the DPPH level was higher on average by 20.2%

292

(2014) and 29.6% (2015) compared to the sole buckwheat plots (Table 2). Our finding indicate that

293

in the intercropped plots, the DPPH level was highest in the F:B (2:1) intercrops on average with

294

5.00 (2014) and 5.66 (2015) mg TE/g DW (Table 2). In both years, DPPH assay was in intercrops

295

and sole plots higher with IF and BL than with CF fertilizer. In sole plots, the DPPH level was

296

significantly higher with application of IF with 4.58 (2014) and 4.38 (2015) mg TE/g DW.

297

According to results by Chlopicka et al. (2012), buckwheat flour had the higher antioxidant activity

298

(DPPH) of 2.20 mg TE/g DW than for flour of quinoa, wheat and amaranth.

299

In the intercropped plots, the DPPH level was significantly higher with application of both IF

300

and BL on average by 84.9% (2014) and 34.1% (2015) than for CF (Table 2). Overall, the

301

maximum DPPH levels in the buckwheat seeds were found in F:B (2:1) plots fertilized with IF with

302

6.28 (2014) and 6.34 (2015) mg TE/g DW.

303

3.1.2. FRAP assay

304

The FRAP assay is based on electron transfer mechanism without the involvement of free

305

radicals (Huang et al., 2005). A significant interaction effect of intercropping and fertilizer type was

306

observed for FRAP in the seeds of buckwheat in both years (Table 1 and Table 2). The FRAP in

307

seed samples varied from 4.31 to 6.60 mg TE/g DW (2014) and 3.45 to 7.26 mg TE/g DW (2015).

308

In a comparison of the antioxidant activity (FRAP) of buckwheat, quinoa, wheat and amaranth

309

flour, buckwheat showed the highest level at 2.15 mg TE/g DW (Chlopicka et al., 2012). The

310

higher levels in our study indicate, that in the whole seeds more compounds with antioxidant

311

activity are available than in the flour.

312

The results of this study indicated that the seeds grown in the intercropping plots had higher

313

FRAP value on average by 16.9% (2014) and 29.9% (2015) than for sole crops. In the intercropping 12

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plots, the greatest antioxidant activity measured by FRAP was found in the F:B (2:1) plots with

315

5.90 (2014) and 6.67 (2015) mg TE/g DW. Dziadek et al. (2016) reported the antioxidant activity in

316

buckwheat whole seeds was on average 11.32 mg TE/g DW.

317

The seeds grown in sole buckwheat fertilized with both IF and BL treatments had higher FRAP

318

on average by 16.4% (2014) and 29.9% (2015) than for CF treatments (Table 2). In the

319

intercropped plots, IF and BL treatments had the higher antioxidant activity (FRAP) on average by

320

23.2% (2014) and 23.6% (2015). In agreement with our results, Jouzi et al. (2017) have

321

demonstrated that the higher concentration of antioxidant activity and health and nutrition benefits

322

were found in the organic foods.

323

In our study the highest and the lowest antioxidant activity (DPPH and FRAP) was found with

324

application of organic fertilizers (IF and BL) and chemical fertilizer, respectively. Similarly, other

325

researchers have shown that organic fertilizers improve while mineral fertilizers reduce antioxidant

326

levels (Faller and Fialho, 2010).

327

We obtained the highest antioxidant activity (DPPH and FRAP) in the F:B (2:1) intercrops

328

fertilized with IF and BL, which also had the higher content of total phenolic and total flavonoid

329

compounds (Table 2). Lee et al. (2016) also found the highest antioxidant activity in tartary

330

buckwheat groats compared to the tartary hull, common hull and common groat, which had the

331

higher phenolic content compounds.

332

3.2. Total phenolic content (TPC)

333

There was a significant interaction effect of intercropping ratio and fertilizer type on the total

334

phenolic content in both years (Table 1 and Table 2). The total phenolic content in buckwheat seeds

335

varied from 2.65 to 4.30 mg CA/g DW (2014) and 2.63 to 4.26 mg CA/g DW (2015). Lee et al.

336

(2016) reported that the total free phenolic content in buckwheat ranged from 4.40 to 20.9 mg/g. A

337

similar level of 3.5 mg total phenolic/g of grain in acetone or aqueous methanol extracts was found

338

in barley (Verardo et al., 2008).

13

ACCEPTED MANUSCRIPT 339

Our data showed that a higher TPC occurred in seeds grown in the intercropping plots on

340

average by 13.6% (both years) than in seeds grown in sole crops. On the other hand, when either IF

341

or BL were applied, the TPC in sole crops was higher by 24.5% (2014) and 21.4% (2015) than with

342

CF, while in the intercropped plots, the TPC was significantly higher by 12.8% (2014) and 18.1%

343

(2015) on average in IF and BL treatments than with CF (Table 2).

344

In the present study, the total phenolic content increased in the seeds grown with the lowest

345

share of buckwheat (F:B (2:1)) on average with 3.87 (2014) and 3.83 (2015) mg CA/g DW (Table

346

2). Some studies report elevated phenolic compounds content in organic products, while others

347

have shown similar or lower phenolic compounds content in organic products (Vinković Vrček et

348

al., 2011; Huber et al., 2011). In our study, intercropping combined with integrated fertilizer or

349

broiler litter contributed to the more efficient utilization of resources (Salehi et al., 2017, 2018).

350

In general, the quantity and behavior of nitrogen in the soil influence the synthesis of

351

compounds. The similar observation was reported about tomato by Zhang et al. (2016), who

352

indicated that different levels of organic fertilizers and nitrogen fertilizer can improve the synthesis

353

of phenolic compounds and increase the phenolic content

354

3.3. Total Flavonoid content (TFC).

355

The total flavonoid content in buckwheat seeds was significantly affected by the interaction

356

effect of intercropping and fertilizer type in both years (Table 1 and Table 2). The TFC of

357

buckwheat seeds ranged from 4.77 to 11.4 mg RU/g DW in 2014 and from 4.94 to 11.9 mg RU/g

358

DW in 2015 (Table 2). Lee et al. (2016) found total flavonoid contents in buckwheat from 0.31 to

359

12.2 mg/g. Other researchers showed that the highest total flavonoid content was found in common

360

buckwheat flowers at 203.6 mg RE/g DW and the lowest was observed in the ripe seeds at 5.78 mg

361

RE/g DW (Zielińska et al., 2012). Kiprovski et al. (2015) showed that buckwheat cultivars Bosna 1

362

and Bosna 2 accumulated the higher rutin content and the highest total flavonoids content as well

363

(3.7 and 3.8 mg/g DW) compared to other cultivars.

14

ACCEPTED MANUSCRIPT 364

Our finding show that in the seeds grown in intercrops, TFC increased on average by 22.9%

365

(2014) and 11.3% (2015) compared to sole crops with best results for a lower share F:B (2:1) of

366

buckwheat in intercrops (Table 2). In sole and intercropped systems, the effect was more

367

pronounced with integrated fertilizer and broiler litter than with chemical fertilizer in both years.

368

Similarly, other researchers confirm that a moderate reduction of inorganic fertilizer can improve

369

plant flavonoid synthesis and accumulation (Zhang et al., 2016).

370

We found that the seeds grown in sole buckwheat fertilized with both IF and BL had a higher

371

TFC on average by 46.6% (2014) and 18.6% (2015) than with CF fertilizer. In the seeds grown in

372

the intercrops, when broiler litter was applied, TFC was higher by 55.9% (2014) and 70.4% (2015)

373

than with chemical fertilizer (Table 2). The highest total flavonoid content was obtained in F:B

374

(2:1) with 11.4 (2014) and 11.9 (2015) mg RU/g DW by application of broiler litter (Table 2).

375

Overall, our results corroborate with a study by Zhang et al., (2016), who reported that combined

376

application of organic fertilizer can increase content of flavonoids in tomato.

377

3.4. Flavonoids compounds content

378

Validation of HPLC method: No interfering peaks were observed at the expected retention times

379

indicating a high selectivity of the method (Fig 1). Linearity was obtained in concentration ranges

380

of 5-200 µg/ml for all of the six compounds (R2 > 0.9995). The determination of precision showed

381

< 5.0 % RSD reproducibility. Recovery was found at 97.8-99.1% for the analysed substances.

382

3.4.1. Rutin

383

The rutin content in seeds was significantly affected by the interaction effect of intercropping

384

and fertilizer type in both years (Table 1 and Figs. 2 A, B). The rutin content of buckwheat seeds

385

varied from 0.75 to 1.26 mg/g DW in 2014 and from 0.77 to 1.16 mg/g DW in 2015 (Fig. 2 A, B).

386

Kalinova and Vrchotova (2011) reported rutin content in buckwheat groats ranging from 0.05 to

387

0.09 mg/g DW.

15

ACCEPTED MANUSCRIPT 388

In this study, in seeds grown in the intercropped plots, the rutin content was on average by

389

12.4% (2014) and 10.8% (2015) higher compared to the sole plots. In sole buckwheat plots, when

390

both IF and BL were applied, the rutin content was enhanced on average by 12.8% (2014) and

391

10.8% (2015) compared to the CF treatment (Fig. 2 A, B). Other researchers also showed that, the

392

average rutin content in organically produced buckwheat groats was higher than in the

393

conventionally grown groats (Kalinova and Vrchotova, 2011).

394

The maximum rutin content of our buckwheat seeds was found in F:B (2:1) of 1.28 (2014) and

395

1.16 (2015) mg/g DW with IF, in F:B (1:2) of 1.08 (2014) and 1.00 (2015) mg/g DW with BL and

396

in F:B (1:1) of 0.92 (both years) mg/g DW with IF, respectively (Fig. 2 A, B). Advantages of

397

intercropping buckwheat with legumes under application of integrated fertilizer and broiler litter on

398

content of flavonoids can be attributed to a more efficient utilization of resources such as nitrogen

399

and phosphorus. This is in agreement with Wu et al. (2008), who reported that the main affecting

400

factors of the rutin content in the leaves of Eucomnia ulmoides are the level of organic matter and

401

total phosphorus in the soil.

402

3.4.2. Vitexin and Isovitexin

403

According to ANOVA results there were significant interactions of intercropping ratio and

404

fertilizer type for vitexin and isovitexin content of buckwheat seeds in both years (Table 1 and Fig.

405

2 C, D). The vitexin content in buckwheat seeds varied from 1.20 to 1.56 mg/g DW (2014) and 1.16

406

to 1.58 mg/g DW (2015). Overall, the highest vitexin content was obtained in seeds grown in the

407

intercropping plots on average by 10.4% (2014) and 14.2% (2015) more than for seeds grown in

408

sole plots. It increased with increasing share of fenugreek (F:B (2:1)) in intercrops in both years

409

(Fig. 2 C, D). The seeds grown in sole buckwheat fertilized with IF had higher vitexin content on

410

average by 14.7% (2014) and 14.8% (2015) than for both CF and BL fertilizers. The seeds grown in

411

the organically (IF and BL) fertilized intercrops had greatest vitexin content on average by 12.2%

16

ACCEPTED MANUSCRIPT 412

(2014) and 10.9% (2015) higher than for CF fertilizer (Fig. 2 C, D). This finding confirms that

413

application of fertilizer can influence phytochemical status of foods (Salama et al., 2015).

414

In the current study the isovitexin content of buckwheat seeds varied from 0.02 to 0.04 mg/g DW

415

in both years (Fig. 2 E, F). In the intercropped plots, the isovitexin content was on average by

416

18.1% (2014) and 7.8% (2015) higher than with sole buckwheat. Overall, in the seeds grown in the

417

intercrops, the highest isovitexin content was obtained at F:B (2:1) (on average with 0.04 mg/g DW

418

in both years) followed by F:B (1:1) (on average with 0.03 mg/g DW in both years). In 2014, the

419

sole buckwheat plots had significantly higher isovitexin content when IF was applied, on average

420

by 14.3% compared to the CF and BL fertilizers. In 2015 the highest isovitexin content of

421

buckwheat seeds was observed in IF and BL fertilized sole plots on average by 34.8% above that in

422

CF fertilized plots (Fig. 2 E, F). The intercropped plots fertilized with IF and BL had higher

423

isovitexin content on average by 15.9% (2014) and 15.3% (2015) than with CF, but there were no

424

significant differences in isovitexin content between fertilizers (CF, IF, BL) at F:B (2:1) plots in

425

both years (Fig. 2 E, F).

426

Intercropped buckwheat seeds under integrated fertilizer and broiler litter increased the content

427

of vitexin and isovitexin in both years. It can be attributed to positive effect of organic fertilizers

428

and intercropping advantages due to the enhancement plant nutritional status. These positive effects

429

of organic fertilizers were also found by Pavla and Pokluda (2008).

430

3.4.3. Orientin and Isoorientin

431

The ANOVA analysis showed significant interaction effects of intercropping ratio and fertilizer

432

type on orientin and isoorientin content of buckwheat seeds in both years (Table 1 and Fig. 2 A, B).

433

The orientin content in seed samples varied from 0.07 to 0.10 mg/g DW (2014) and 0.08 to 0.10

434

mg/g DW (2015). The seeds grown in the intercropping plots had higher orientin content on

435

average by 7.36% (2014) and 7.07% (2015) than for sole plots, and it increased with increasing

436

share of fenugreek in intercrops. The highest orientin content was observed after application of BL 17

ACCEPTED MANUSCRIPT 437

(0.10 mg/g DW, in both years) and IF (0.09 mg/g DW in both years) at F:B (2:1) followed by IF

438

(0.09 mg/g DW, in both years) at F:B (1:1), while the lowest orientin content was found in sole and

439

intercropped buckwheat seeds that were grown with chemical fertilizer of 0.08 mg/g DW in 2014

440

and 2015 (Fig. 3 A, B).

441

Isoorientin for buckwheat seeds in our observations ranged from 0.12 to 0.15 mg/g DW in 2014

442

and 0.12 to 0.17 mg/g DW in 2015 (Fig. 3 C, D). The highest isoorientin content of buckwheat

443

seeds was found in F:B (2:1) at 0.15 (2014) and 0.17 (2015) mg/g DW with broiler litter. The

444

chemical fertilizer produced the lowest isoorientin content in seed on average with 0.12 mg/g DW

445

in both years (Fig. 3 C, D). There were no significant differences in isoorientin content in the sole

446

buckwheat plots and in F:B (1:2) intercropped plots (Fig. 3 C, D).

447

3.4.4. Hyperoside

448

The hyperoside content in seeds was significantly affected by the interaction effect of

449

intercropping and fertilizer type in both years (Table 1 and Fig. 3 E, F). The hyperoside content in

450

buckwheat seeds ranged from 0.23 to 0.38 mg/g DW (2014) and 0.21 to 0.39 mg/g DW (2015).

451

Overall, the highest hyperoside content was observed in seeds grown in intercropping on average

452

by 25.1% (2014) and 27.4% (2015) higher than for seeds grown in sole plots. It increased with

453

increasing share of fenugreek (F:B (2:1)) in intercrops in both years (Fig. 3 E, F). The seeds grown

454

in sole buckwheat fertilized with both IF and BL had higher hyperoside content on average by

455

24.1% (2014) and 31.2% (2015) than with chemical fertilizer. In our study, the seeds grown in the

456

organically (IF or BL) fertilized intercrops had highest hyperoside content on average by 32.1%

457

(2014) and 19.8% (2015) higher than for CF fertilizer (Fig. 3 E, F).

458

The nitrogen and phosphorus content in the soil are possible factors that could influence the

459

flavonoid compounds. Therefore, we assume that the soil nitrogen and phosphorus level in both

460

sole and intercropping system with application of organic fertilizers (IF and BL) could contribute to

461

the direct improvement of flavonoid compounds content in the buckwheat seeds (vitexin, isovitein, 18

ACCEPTED MANUSCRIPT 462

orientin, isoorientin and hyperoside). Kalinova and Vrchotova (2011) concluded that the nitrogen

463

level could contribute to the direct improvement of the flavonol content in vegetative plant tissues,

464

but the increase of the rutin content in the generative parts of buckwheat was not directly dependent

465

on the soil nitrogen level. Thus phosphorus may have played a major role in current experiments. It

466

has been reported that phosphorus plays an important role in the biosynthesis of some compounds

467

and may affect the quality (Sell, 2003).

468

3.5. Correlation among antioxidant activity, total phenolic, total flavonoids and main flavonoid

469

compounds content

470

It is known, that a high antioxidant activity is caused by a high content of phenolics. Flavonoids

471

have phenolic moieties too. Therefore, positive correlations between antioxidant activity and

472

phenolics as well as flavonoids were expected.

473

The correlations among antioxidant activity (measured by DPPH and FRAP), total phenolic,

474

total flavonoids and main flavonoid compounds content were tested by means of Pearson

475

correlation coefficient. As shown in Table 3, for the DPPH assay significant and positive

476

correlations were observed with total phenolics and total flavonoids in 2014 (r= 0.71 and r= 0.72)

477

and in 2015 (r= 0.80 and r= 0.70, respectively). Our findings show that antioxidant activity

478

measured by DPPH was positively correlated with rutin, vitexin, isovitexin, orientin, isoorientin and

479

hyperoside (Table 3). Lee et al. (2016) found significant and positive correlations among

480

antioxidant activity (DPPH), total flavonoids and flavonoid compounds (rutin, vitexin, isovitexin

481

and epicatechin gallate), too.

482

Pearson correlation results in Table 3 indicate that antioxidant activity measured by FRAP

483

significantly correlated with total phenolics and total flavonoids in 2014 (r= 0.62 and r= 0.52,

484

respectively) and in 2015 (r= 0.76 and r= 0.65, respectively). As reported in Table 3, the antioxidant

485

activity measured by FRAP was positively and significantly correlated with rutin, vitexin,

486

isovitexin, orientin, isoorientin and hyperoside. Lee et al. (2016) found no significant correlations 19

ACCEPTED MANUSCRIPT 487

among FRAP assay, total flavonoids and flavonoid compounds (rutin, vitexin, isovitexin and

488

epicatechin gallate) in common buckwheat, but a significant and positive correlation between total

489

phenolic content and antioxidant activity (FRAP) of buckwheat grains were previously described by

490

Hodzic et al. (2009). Results of assays evaluating antioxidative activity of substances can differ

491

because of the different principles of the methods. In the DPPH assay the chemically stable radical

492

DPPH is reduced and discoloured as a consequence in the presence of antioxidative substances. In

493

the FRAP assay antioxidative substances reduce Fe3+ ions. The resulting Fe2+ ions form a blue

494

complex with TPTZ, which can be photometrically analyzed.

495 496

4. Conclusions

497

The present study highlights that the application of both broiler litter (BL) alone and in

498

combination with chemical fertilizer as integrated fertilizer (IF) is significantly more effective in

499

promoting the antioxidant activity measured by DPPH and FRAP assay, total phenolic content, total

500

flavonoids content and flavonoids compounds content (rutin, vitexin, isovitexin, orientin,

501

isoorientin and hyperoside) of buckwheat seeds compared to the chemical fertilizer (CF)

502

application. This was valid for both sole buckwheat and intercropped with fenugreek. For

503

fenugreek-buckwheat intercrops, the F:B (2:1) ratio appears to be optimum for highest antioxidant

504

activity (DPPH and FRAP) and all of the bioactive compounds measured in this study. Generally,

505

these results are of great interest for buckwheat producers, especially in semiarid areas. Buckwheat

506

seeds grown in intercropping with fenugreek under integrated fertilizer or broiler litter application

507

achieve high concentrations of phenolic compounds, making them are an important source of

508

functional and medicinal food products with great advantages for health and nutrition. Further work

509

on other medicinal plants containing phenolic compounds is recommended to confirm the

510

enhancement of the content of phenolic compounds resulting in a higher antioxidant activity by

511

organic fertilizers and intercropping methods. 20

ACCEPTED MANUSCRIPT 512

Acknowledgements

513

We are grateful to Shahrekord University for financial support of the experiments as well as to

514

the University of Veterinary Medicine Vienna, for their kind support by providing laboratory

515

equipment and materials used in the study, as well as Martin Finsterböck for technical assistance,

516

and also to the University of Natural Resources and Life Sciences, Vienna (BOKU) for hosting A.

517

Salehi during a research scholarship.

518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 21

ACCEPTED MANUSCRIPT 533

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630

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631 632 633 634 635 25

ACCEPTED MANUSCRIPT 636

Figure captions

637

Figure 1. HPLC chromatograms of flavonoid compounds extracted from a sample of buckwheat

638

seeds as affected by intercropping ratio × fertilizer type interaction in 2014 and 2015. The

639

identified peaks are: orientin (1), isoorientin (2), vitexin (3), isovitexin (4), rutin (5) and hyperoside

640

(6).

641 642

Figure 2. Rutin content (A and B), vitexin content (C and D) and isovitexin content (E and F) of

643

buckwheat seeds as affected by intercropping ratio × fertilizer type interaction in 2014 and 2015.

644 645

Figure 3. Orientin content (A and B), isoorientin content (C and D) and hyperoside content (E and

646

F) of buckwheat seeds as affected by intercropping ratio × fertilizer type interaction in 2014 and

647

2015.

26

ACCEPTED MANUSCRIPT

Fig. 1. HPLC chromatograms of flavonoid compounds extracted from a sample of buckwheat seeds as affected by intercropping ratio × fertilizer type interaction in 2014 (A) and 2015 (B). The identified peaks are: orientin (1), isoorientin (2), vitexin (3), isovitexin (4), rutin (5) and hyperoside (6).

1

ACCEPTED MANUSCRIPT 2014

2015

Fig. 2. Rutin content (A and B), vitexin content (C and D) and isovitexin content (E and F) of buckwheat seeds as affected by intercropping ratio × fertilizer type interaction in 2014 and 2015. Different letters indicate significant differences at P < 0.05 by LSD. See Table 2 for treatments.

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ACCEPTED MANUSCRIPT 2014

2015

Fig. 3. Orientin content (A and B), isoorientin content (C and D) and hyperoside content (E and F) of buckwheat seeds as affected by intercropping ratio × fertilizer type interaction in 2014 and 2015. Different letters indicate significant differences at P < 0.05 by LSD. See Table 2 for treatments.

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ACCEPTED MANUSCRIPT Highlights



Organic fertilizers enhanced antioxidant activity and bioactive compounds of buckwheat seeds.



Organic fertilizers had positive effects on flavonoid compounds of buckwheat seeds.

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Intercropping system promoted tested antioxidant activity of common buckwheat.

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Buckwheat seeds grown in intercropping had higher bioactive compounds.

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Total phenolic, total flavonoids and flavonoids compounds were highly correlated with antioxidant activity.

Table 1 ANOVA results (F values) for antioxidant activity measured by DPPH and FRAP assay, total isoorientin and hyperoside of buckwheat seeds in 2014 and 2015. Total Total Sources of variation df DPPH FRAP Rutin phenolic flavonoids Replication 2 0.34ns 0.13ns 0.01ns 0.45ns 0.02ns 2014 *** *** *** *** Intercropping ratio (IR) 3 3.81 1.96 1.15 14.02 0.08*** *** *** *** *** Fertilizer type (Fert) 2 19.33 4.30 1.02 30.24 0.14*** ** * ** * IR×Fert 6 1.29 0.49 0.09 2.03 0.03* Error 22 0.18 0.18 0.01 0.60 0.01 CV (%) 9.58 7.93 3.32 10.62 10.85

2015

Replication 2 0.17ns 0.13ns 0.01ns 0.44ns *** *** *** Intercropping ratio (IR) 3 7.59 6.92 1.34 13.57*** *** *** *** Fertilizer type (Fert) 2 6.42 8.46 1.25 35.59*** IR×Fert 6 0.83** 0.43* 0.14** 1.84* Error 22 0.09 0.16 0.03 0.72 CV (%) 6.65 7.10 5.33ns 11.24 ns not significant. *, **, *** Significant effect at P < 0.05, 0.01, 0.001, respectively.

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0.003ns 0.04*** 0.09*** 0.01* 0.003 6.17

phenolic, total flavonoids, rutin, vitexin, isovitexin, orientin, Vitexin

Isovitexin

Orientin

Isoorientin Hyperoside

0.01ns 0.06*** 0.09*** 0.01* 0.004 4.87

0.00001ns 0.0001*** 0.0001*** 0.00001* 0.000005 7.62

0.00002ns 0.0002*** 0.0005*** 0.00005* 0.00002 5.22

0.00001ns 0.0005*** 0.0004*** 0.0001** 0.00003 4.22

0.001ns 0.01*** 0.02*** 0.002* 0.001 10.33

0.01ns 0.09*** 0.08** 0.01* 0.006 5.75

0.000004ns 0.00005ns 0.00004*** 0.0002** 0.0001*** 0.0003*** 0.00001* 0.0001* 0.000004 0.00002 6.14 5.67

0.00003ns 0.001** 0.001*** 0.0002*** 0.00003 4.03

0.02ns 0.01*** 0.01*** 0.0013* 0.0005 7.45

Table 2 Antioxidant activity measured by DPPH and FRAP, total phenolic content (TPC) and total flavonoids content (TFC) of buckwheat seeds as affected by intercropping ratio × fertilizer type interaction in 2014 and 2015. Sole B F:B (1:2) F:B (1:1) F:B (2:1) 2014 2015 2014 2015 2014 2015 2014 2015 CF 2.94f 3.00e 3.00f 3.32de 2.68f 3.50de 3.10f 4.86c IF 4.58cd 4.38c 5.37b 5.74b 3.82e 3.68d 6.20a 6.34a DPPH (mg TE/g DW) de d a b bc c b BL 3.91 3.67 6.27 5.39 5.03 4.39 5.75a 5.77b FRAP

TPC

(mg TE/g DW)

CF IF BL

4.31f 4.77def 5.26cd

3.45g 5.12f 5.43ef

5.19cde 5.77bc 5.64bc

5.67def 6.25bcd 6.51bcd

4.50ef 6.22ab 5.32cd

4.12g 5.46ef 6.55abc

4.84def 6.60a 6.25ab

5.93cde 6.82ab 7.26a

(mg CA/g DW)

CF IF BL

2.65h 3.35de 3.24ef

2.63g 3.38cd 2.99ef

3.13fg 3.46cd 3.82b

2.96ef 3.34cd 3.80b

2.99g 3.24ef 3.27ef

2.73fg 3.23cde 3.14de

3.56c 3.76b 4.30a

3.43c 3.79b 4.26a

CF 4.77g 6.20efg 5.93efg 4.94g 5.60fg 5.33fg 6.94de 6.87de ef de de de def def c TFC (mg RU/g DW) IF 6.89d 6.79 6.93 7.54 6.37 6.58 8.51 9.29bc de cd b b cd bcd a BL 7.09 7.91 9.97 9.35 7.40 7.96 11.44 11.89a CF, IF, BL, F:B (1:2), F:B (1:1) and F:B (2:1) are chemical fertilizer, integrated fertilizer, broiler litter, one row of fenugreek + two rows of buckwheat, one row of fenugreek + one row of buckwheat, and two rows of fenugreek + one row of buckwheat, respectively. Different letters indicate significant differences at P < 0.05 by LSD.

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Table 3 Pearson’s correlation coefficients (r) among antioxidant activity (measured by DPPH and FRAP), total phenolic, total flavonoid and flavonoid compounds in buckwheat seeds in 2014 and 2015 (n= 36). Antioxidant activity DPPH FRAP 2014 2015 2014 2015 Total phenolic 0.71*** 0.80*** 0.62*** 0.76*** Total flavonoid 0.72*** 0.70** 0.52** 0.65*** Flavonoid compounds Rutin 0.73*** 0.76** 0.69*** 0.66*** *** ** *** Vitexin 0.62 0.57 0.53 0.58*** ** ** ** Isovitexin 0.53 0.60 0.52 0.69*** *** ** *** Orientin 0.66 0.50 0.66 0.52** Isoorientin 0.55** 0.53** 0.62** 0.60*** ** ** *** Hyperoside 0.67 0.68 0.68 0.81*** ** and *** Significant effect at P < 0.01 and 0.001.

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