The influence of shading on the yield and quality of southern sweet-grass (Hierochloë australis (Schrad.) Roem. & Schult.

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1 The influence of shading on the yield and quality of southern sweet-grass (Hierochloë australis (Schrad.) Roem. & Schult.) raw material WIESŁAW PODYMA *1, KATARZYNA BĄCZEK 2, MIROSŁAW ANGIELCZYK 2, JAROSŁAW L. PRZYBYŁ 2, ZENON WĘGLARZ 2 1* Ministry of Agriculture and Rural Development The Department of Plant Breeding and Protection Wspólna 30, Warsaw, Poland 2 Warsaw University of Life Sciences SGGW Faculty of Horticulture and Landscape Architecture Department of Vegetable and Medicinal Plants Nowoursynowska Warsaw, Poland * corresponding author: phone: , wieslaw.podyma@minrol.gov.pl Summary Southern sweet-grass (Poaceae) rarely occurs in Polish coniferous or mixed forests. Leaves of this plant, rich in coumarin compounds, are mainly used as a flavouring raw material in alcohol, tobacco and cosmetic industry. The aim of the study was to investigate the influence of shading on the yield and quality of southern sweet-grass. The experiment was established at the certificated organic field of Department of Vegetable and Medicinal Plants. The sunlight was reduced up to 50 and 70% by using shading nets. The object of experiment was two-year-old plants grown on the alluvial soil. Leaves were harvested two times: in mid-may and in the last week of August (regrowth). The highest yield of leaves was observed at 50% of shading. The shaded plants were characterized by higher content of coumarin and 3,4-dihydrocoumarin and lower content of bergapten in comparison to non-shaded ones. In the leaves five phenolic acids and two flavonoids were identified. Key words: plant shading, yield of leaves, accumulation of biologically active compounds

2 The influence of shading on the yield and quality of southern sweet-grass (Hierochloë australis (Schrad.) Roem. & Schult.) raw material INTRODUCTION Southern sweet-grass is a tuft grass occurring in coniferous or mixed forests [1-3]. Main active compounds of this plant are coumarins, particularly coumarin. They reveal antifungicidal and antitumor activities, increase blood-thinning and flow in the veins but decreases capillary permeability. When used at high doses for a long period coumarins can be toxic [4-6]. Extracts of the plant leaves are used in alcohol, tobacco and cosmetic industry as a flavouring. Coumarin compounds are responsible for the sweet and cherry flower-like odour of the grass and coumarin is regarded to be the strongest one [7, 8]. High and growing demand for the leaves of the southern sweet-grass makes this species endangered in natural habitats and for this reason it is under legal protection in Poland [9]. The aim of undertaken study was to investigate the influence of plant shading on the yield and content of coumarin and phenolic compounds in the leaves of this plant cultivated with organic farming methods. 15 MATERIALS AND METHODS The experiment was carried out in 2007 and 2008, at the certificated organic field of Department of Vegetable and Medicinal Plants on the medium-heavy alluvial soil enriched by manure compost and highmoor peat. The objects of experiment were two-year-old plants planted out 40 x 40 cm in spring 2005 and The experiment was established by the method of randomized blocks in 4 replication with 10 plants on each plot. The sunlight was reduced up to 50 and 70% by using shading nets. Plants without shading were used as a control. The leaves were collected two times during vegetation period. First cut was carried out in the mid of May when the leaves were fully developed, second in August (regrowth). After harvest leaves were dried at 35 C. One gram of grounded raw material was extracted with 100 ml of methanol in Büchi B-811 Extraction System. After solvent evaporation, the residue was dissolved in 10 ml of methanol, filtered through a Supelco IsoDisc PTFE 25 mm 0.45 μm filter, and subjected to HPLC for determination of coumaric compounds, flavonoids and polyphenolic acids. The analysis was carried out using the Shimadzu chromatograph with SPD-M10A VP DAD detector. Phenomenex Luna C18 (2) 5 μm mm column was used. Gradient elution of 10% ACN (mobile phase A) and 55% ACN (mobile phase B) (Lab- Scan) in water adjusted to ph 3.0, flow rate 1 ml min -1, and temperature 30ºC were applied. Peaks were identified by comparison of retention time and spectral data with adequate parameters of standards purchased from ChromaDex. Quantification was based on the peak area at 254 nm (coumarin, 3,4-dihydrocoumarin, bergapten, rutin, elagic and o-coumaric acids), 264 nm (astragalin) and 330 nm (chlorogenic, rosmarinic and ferulic acids). The results presented in tables 1, 2 and 3 are mean values from two years. Vol. 56 No

3 P. Jakowienko, B. Wójcik-Stopczyńska 16 The results were analysed statistically with Stargraphics Plus, version 4.1. The differences between the means were compared with the Tukey test. The yield of leaves (g plant -1 ) harvest shading degree Table 1. control (non-shaded plants) 50% 70% mean fresh mass (g plant -1 ) air dry mass (g plant -1 ) 1 st cut * 2 nd cut total b c a 1 st cut * 2 nd cut total b c 8.55 a Values marked with the same letter in rows do not differ at α=0.05 Values marked with * differ at α=0.05 Table 2. Coumarin compounds in the air dried leaves (mg 100g -1 ) shading degree coumarin compounds harvest control (non-shaded 50% 70% mean plants) 1 st cut a b c coumarin 3,4 dihydrocoumarin bergapten 2 nd cut b c a * mean a ab b 1 st cut a a b * 2 nd cut a b ab mean a b c 1 st cut c b a nd cut b b a * mean c b a Values marked with the same letter in rows do not differ at α=0.05 Values marked with * differ at α=0.05

4 The influence of shading on the yield and quality of southern sweet-grass (Hierochloë australis (Schrad.) Roem. & Schult.) raw material 17 Phenolic compounds in the air-dried leaves (mg 100g -1 ) phenolic compounds harvest control (non - shaded plants) shading degree Table 3. 50% 70% mean astragalin rutin chlorogenic acid elagic acid ferulic acid o-coumaric acid rosmarinic acid 1 st cut 3.49 a 6.38 b 5.99 b 5.29* 2 nd cut 2.73 a 4.30 b 5.65 c 4.23 mean 3.11 a 5.34 b 5.82 b 1 st cut a b b nd cut a b a 41.01* mean a b a 1 st cut 9.85 a b b 11.06* 2 nd cut 7.03 a b b 9.27 mean 8.44 a b b 1 st cut 5.95 a 7.43 b 7.30 b nd cut 9.29 a c b 13.39* mean 7.62 a c b 1 st cut 0.36 a 1.52 b 0.39 a nd cut 1.06 a 2.23 b 1.47 a 1.59* mean 0.71 a 1.87 b 0.93 a 1 st cut 0.19 a 0.27 b 0.19 a nd cut 0.22 a 0.46 b 0.17 a 0.28* mean 0.20 a 0.36 b 0.18 a 1 st cut 0.70 a 3.49 c 1.42 b nd cut 2.81 b 4.44 c 1.17 a 2.81* mean 1.75 a 3.96 b 1.29 a Values marked with the same letter in rows do not differ at α=0.05 Values marked with * differ at α=0.05 RESULTS AND DISSCUSSION Southern sweet-grass is one of the wild growing medicinal plants being under partial legal protection in Poland [9]. Excessive collecting resulted in a total deterioration of many natural sites of this species [1, 10]. The most effective way to preserve this plant in natural environment seems to be its introduction into cultivation. Such trials have already been undertaken in Department of Vegetable and Medicinal Plants [10]. The results presented in this paper seem to be very promising. The highest cumulative yield of leaves (1 st and 2 nd cut) was obtained at 50% shading (48.04 g of fresh mass per plant) in comparison to control and Vol. 56 No

5 P. Jakowienko, B. Wójcik-Stopczyńska 18 70% shading (32.81 and g of fresh mass per plant, respectively, see tab. 1). The highest content of coumarin and 3,4-dihydrocoumarin was observed in the plants grown under 70% shading, in contrary to bergapten which was accumulated in the highest amount in non-shaded plants (tab. 2). The accumulation of detected phenolic compounds (astragalin, rutin and chlorogenic, elagic, ferulic, o-coumaric, rosmarinic acids) appeared to be higher in raw materials originated from plants grown under 50% shading (tab. 3). The results of the experiment clearly indicate that cultivation of southern sweet-grass is possible and very effective. However, relatively high yield is possible only by using shading. Taking into consideration the content of active compounds, specially the coumarins, the quality of raw materials from organic cultivation was even better than from natural sites [11]. CONCLUSIONS Plant shading resulted in increasing the yield of leaves by about 50%. Plant shading increased the content of coumarin and 3,4-dihydrocoumarin and decreased the content of bergapten in the leaves. In the leaves 5 phenolic acids and two flavonoids were identified. The results of this investigation indicate the possibility to obtain relatively high yield and quality of southern sweet-grass leaves in the system of organic cultivation. REFERENCES 1. Gawłowska J, Sulma T, Wierzchowska-Renke K. Turówka wonna (Hierochloë odorata) i turówka leśna (Hierochloë australis) zasoby i zagrożenia. Chrońmy Przyr Ojcz 1989; 5(6): Ćwikliński E, Głowacki Z. Nowe stanowiska rzadszych gatunków w dolinie dolnego Bugu. Zesz Nauk Wyższa Szkoła Rolniczo-Pedagogiczna w Siedlcach, Seria: Nauki Przyr 1990; 19: Świejkowski L. Rośliny lecznicze i przemysłowe. Warszawa 1990: Cisowski W. Biologiczne właściwości kumaryn. I. Działanie na rośliny oraz właściwości farmakologiczne i przeciwbakteryjne. Herba Pol 1983; 3(4): Lake BG. Coumarin metabolism, toxicity and carcinogenicity: relevance for human risk assessment. Food Chem. Toxicol 1999; 37: Kohlmünzer S. Farmakognozja. Wyd 5. Warszawa 2000: Wierzchowska-Renke K. Uwagi o suszeniu turówki wonnej (Hierochloë odorata Wahlb.) i turówki leśnej (Hierochloë australis Roem. et Schult.). Ziel Biul Inf 1972; 1: Sulma T, Wierzchowska-Renke K. Badanie ziela turówki (żubrówki) Herba Hierochloë. III. Ocena handlowego surowca Herba Hierochloë z plantacji na Żuławach. Acta Pol Pharm 1974; 2: Polakowski B. Rośliny chronione. Atlas. Warszawa 1995: Węglarz Z, Geszprych A, Angielczyk M, Pawełczak A. Wstępne badania nad plonowaniem i wewnątrzgatunkową zmiennością chemiczną turówki leśnej (Hierochloë australis (Schrad.) Roem. et Schult.). Zesz Probl Post Nauk Roln 2004; 497: Maciejuk A. Wpływ intensywności światła i terminu zbioru ziela na rozwój, plonowanie i skład chemiczny turówki leśnej (Hierochloë australis (Schrad.) Roem. et Schult.). Praca magisterska wykonana w Katedrze Roślin Warzywnych i Leczniczych Szkoły Głównej Gospodarstwa Wiejskiego. Warszawa 2002.

6 The influence of shading on the yield and quality of southern sweet-grass (Hierochloë australis (Schrad.) Roem. & Schult.) raw material WPŁYW ZACIENIANIA NA PLON I JAKOŚĆ SUROWCA TURÓWKI LEŚNEJ (HIEROCHLOË AUSTRALIS (SCHRAD.) ROEM. & SCHULT.) 19 WIESŁAW PODYMA* 1, KATARZYNA BĄCZEK 2, MIROSŁAW ANGIELCZYK 2, JAROSŁAW L. PRZYBYŁ 2, ZENON WĘGLARZ 2 1* Ministerstwo Rolnictwa i Rozwoju Wsi Departament Hodowli i Ochrony Roślin ul. Wspólna Warszawa 2 Szkoła Główna Gospodarstwa Wiejskiego w Warszawie Wydział Ogrodnictwa i Architektury Krajobrazu Katedra Roślin Warzywnych i Leczniczych ul. Nowoursynowska Warszawa *autor, do którego należy kierować korespondencję: tel.: , wieslaw.podyma@minrol.gov.pl Streszczenie Turówka leśna (Poaceae) występuje rzadko na terenie Polski. Rośnie głównie w lasach iglastych i mieszanych. Liście tej rośliny, bogate w kumaryny, stosowane są przede wszystkim jako surowiec aromatyzujący w przemyśle alkoholowym, tytoniowym i kosmetycznym. Celem niniejszej pracy było określenie wpływu cieniowania na plon i jakość liści tej rośliny. Doświadczenie założono na certyfikowanym, ekologicznym Polu Doświadczalnym Katedry Roślin Warzywnych i Leczniczych. Dostępność światła słonecznego modyfikowano poprzez cieniowanie roślin siatką raszlową (50 i 70% zacienienia). Obiektem badań były rośliny dwuletnie rosnące na podłożu składającym się z mieszanki mady rzecznej, kompostu z obornika i torfu wysokiego. Liście turówki ścinano dwukrotnie: w połowie maja oraz w ostatnim tygodniu sierpnia (odrost). Najwyższy plon liści wytworzyły rośliny rosnące przy pięćdziesięcioprocentowym zacienieniu. Rośliny cieniowane charakteryzowały się również wyższą zawartością kumaryny i 3,4-dihydrokumaryny oraz niższą zawartością bergaptenu w porównaniu z roślinami nie cieniowanymi (kontrola). W liściach zidentyfikowano pięć kwasów polifenolowych i dwa flawonoidy. Słowa kluczowe: cieniowanie roślin, plon liści, zawartość związków biologicznie aktywnych Vol. 56 No