Variations in the Chemical Profile and Biological Activities of Licorice (Glycyrrhiza Glabra L.), As Influenced by Harvest Times
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Acta Physiol Plant (2013) 35:1337–1349 DOI 10.1007/s11738-012-1174-9 ORIGINAL PAPER Variations in the chemical profile and biological activities of licorice (Glycyrrhiza glabra L.), as influenced by harvest times Jose´ Cheel • Lenka Tu˚mova´ • Carlos Areche • Pierre Van Antwerpen • Jean Ne`ve • Karim Zouaoui-Boudjeltia • Aurelio San Martin • Ivan Vokrˇa´l • Vladimı´r Wso´l • Jarmila Neugebauerova´ Received: 2 August 2012 / Revised: 27 November 2012 / Accepted: 30 November 2012 / Published online: 18 December 2012 Ó Franciszek Go´rski Institute of Plant Physiology, Polish Academy of Sciences, Krako´w 2012 Abstract This study investigates the variations in the of 0.88–11.38 %, 1.86–10.03 %, 1.80–18.40 % and 5.53– chemical profile, free radical scavenging, antioxidant and 16.31 %, respectively. These fluctuations correlated posi- gastroprotective activities of licorice extracts (LE) from tively with changes in the antioxidant and free radical plants harvested during the months of February to scavenging activities of licorice. In general, the samples November. Correlations between biological properties and from May and November showed the most favorable free the chemical composition of LE were also investigated. radical scavenging and antioxidant effects, whereas The results showed that the total contents of phenols, the best gastroprotective effect was in May. Liquiritin and flavonoids and tannins in LE varied at different harvest glycyrrhizin, the major constituents in the February and times. Liquiritin and glycyrrhizin, the major components May LE, appeared to contribute to the superoxide radical of LE, varied in the range of 28.65–62.80 and scavenging and gastroprotective effects. Glabridin and 41.84–114.33 mg g-1, respectively. The relative propor- glabrene, the compounds with the highest relative propor- tion of glycyrrhizin derivative (3), glabridin (4), glabrene tion in the November LE, accounted for the antioxidant and (5) and liquiritigenin derivative (6), varied in the range DPPH scavenging activities of licorice. It is concluded that the chemical profile of licorice quantitatively varied at different harvest times and these fluctuations determined Communicated by M. H. Walter . changes in its bioactivities. These data could pave the way Electronic supplementary material The online version of this to optimize harvesting protocols for licorice in relation article (doi:10.1007/s11738-012-1174-9) contains supplementary with its health-promoting properties. material, which is available to authorized users. J. Cheel Á L. Tu˚mova´ K. Zouaoui-Boudjeltia Department of Pharmacognosy, Faculty of Pharmacy, Laboratory of Experimental Medicine, CHU Charleroi, Charles University in Prague, Heyrovske´ho 1203, Universite Libre de Bruxelles, 6110 Montigny-le-Tilleul, 500 05 Hradec Kra´love´, Czech Republic Belgium J. Cheel (&) I. Vokrˇa´l Department of Research and Development, Department of Pharmacology and Toxicology, Contipro Biotech s.r.o, Dolnı´ Dobroucˇ 401, Faculty of Pharmacy, Charles University, 561 02 Dolnı´ Dobroucˇ, Czech Republic Heyrovske´ho 1203, 500 05 Hradec Kra´love´, Czech Republic e-mail: [email protected] V. Wso´l C. Areche Á A. San Martin Department of Biochemical Sciences, Departamento de Quı´mica, Facultad de Ciencias, Faculty of Pharmacy, Charles University, Heyrovske´ho 1203, Universidad de Chile, Casilla 653, Santiago, Chile 500 05 Hradec Kra´love´, Czech Republic P. Van Antwerpen Á J. Ne`ve J. Neugebauerova´ Laboratory of Pharmaceutical Chemistry and Analytical Faculty of Horticulture, Mendel University in Brno, Platform of the Faculty of Pharmacy, Universite Libre de Valticka´ 337, 691 44 Lednice, Czech Republic Bruxelles, 1050 Brussels, Belgium 123 1338 Acta Physiol Plant (2013) 35:1337–1349 Keywords Licorice Á Harvest times Á Antioxidant activity Á licorice collected at different harvest times during a 1-year HPLC Á Flavonoids period. Introduction Materials and methods Licorice, the dry roots of Glycyrrhiza glabra L. (Fabaceae), Chemicals is considered one of the oldest and most widely used herbs around the world (Isbrucker and Burdock 2006). Glycyr- N-Methylphenazonium methyl sulfate (PMS), b-nicotin- rhiza glabra is a perennial herb native to the Mediterranean amide adenine dinucleotide reduced (NADH), nitrotetrazo- region, and the Middle East that is now widely cultivated lium blue chloride (NBT), 2,2-diphenyl-1-picryl-hydrazyl throughout Europe (Nassiri-Asl et al. 2007). Licorice has (DPPH), hide powder, linoleic acid, polyoxyethylene sor- been traditionally used in herbal medicines for its emolli- bitan monopalmitate (Tween-40) and standards (glycyr- ent, antitussive and gastroprotective properties (Sabbioni rhizin and quercetin) were obtained from Sigma-Aldrich et al. 2005). Glycyrrhizin and flavonoids, such as liquiritin, (Steinheim, Germany). Standard liquiritin was purchased isoliquiritin and their aglycones, have been reported as the from Wuhan Sunrise Technology Development Co., Ltd. major constituents of licorice and they are perceived as the (Hong Kong, China) and b-carotene was from Koch-light active principles responsible for its pharmacological effi- Laboratories Ltd. (England). Folin–Ciocalteu phenol cacy (Zhang and Ye 2009). Modern pharmacological reagent, aluminum chloride hexahydrate, formalin, abso- studies have shown that licorice possesses a variety of bio- lute ethanol and sodium carbonate were from Merck activities, including antiinflammatory, antioxidant, immu- (Darmstadt, Germany). HPLC grade solvents were also nostimulating, antiviral, antiulcerogenic, anticarcinogenic from Merck. Ultrapure water from the Milli-Q RG system and hepatoprotective activities (Nassiri-Asl and Hossein- (Millipore, Molsheim, France) was used, and lansoprazole, zadeh 2008). Besides medicinal usage, the licorice extract captopril and flufenamic acid were purchased from Sigma has GRAS (generally regarded as safe) status as a flavoring Chemical Co. (USA). and sweetening agent for tobaccos, chewing gums, candies, toothpaste, and beverages (Isbrucker and Burdock 2006). Animals The growing use of licorice extracts as active ingredients in cosmetic and personal care products is also worth noting. Male Swiss albino mice were purchased from the Instituto de Recently, the demand for licorice has been increasing, Salud Pu´blica de Chile, Santiago. The mice weighing 30 ± while the availability of wild licorice has declined 3 g were used and they were fed on the certified Champion (Yamamoto and Tani 2006). The promotion of licorice diet with free access to water under standard conditions of cultivation as an additional and stable source of the 12-h dark–light period, and 22 ± 2 °C room temperature. medicinal plant requires additional studies to determine the influence of enviromental factors on its chemical content Sample collection and preparation of extracts and biological properties. The harvesting of licorice root occurs in the autumn of its third or fourth year of growth The thickened roots of 4-year-old cultivated Glycyrrhiza (Isbrucker and Burdock 2006), which is mainly dependent glabra L. were collected in the middle of February, May, on the total yield of the plant rather than on its chemical August and November of 2008 in the Botanical Garden of composition. Botanicals have been reported to have vari- the Faculty of Horticulture, Mendel University of Agricul- able chemical compositions due to variations in soil types ture and Forestry, Czech Republic at 164 m above sea level. and climates as well as to harvest times or growing periods Three samples of G. glabra from each harvest time were (Incerti et al. 2009; Yesil-Celiktas et al. 2007;C¸ yrak et al. used. The genetic resource was identified with the code 2007). The influence of harvest times and their associated 0001. The mean values for maximum and minimum tem- enviromental factors on the chemical and biological con- perature (°C) for the months of February, May, August and ditions of licorice have been largely unexplored, except for November 2008 were 7.3 °C/-0.8 °C; 20.5 °C/9.5 °C; a few studies revealing seasonal variation in glycyrrhizin 25.7 °C/14.1 °C and 9 °C/4.1 °C, respectively. The mean and isoliquiritin contents (Hayashi et al. 1998), and the values of global and UV radiation for February, May, influence of UV radiation on glycyrrhizin biosynthesis August and November of 2008 were 6.73 and 39.57; 20.21 (Afreen et al. 2005). and 247.09; 17.77 and 250.97; and 3.38 and 22.09 MJ m-2, The present study is aimed to investigate the variation in respectively (data provided by the Solar and Ozone the chemical composition and free radical scavenging, Observatory of the Czech Hydrometeorological Institute). antioxidant and gastroprotective activities of extracts of The plant material was dried at 40 °C in an oven and then 123 Acta Physiol Plant (2013) 35:1337–1349 1339 ground to fine powders (mesh size 20). A precisely weighed 2.1 9 100 mm), at 40 °C under a flow rate of amount (1.50 g) of the powder was extracted under reflux 175 ll min-1. The solvent gradient was similar to those for with 150 ml of 80 % methanol at 60 °C for 6 h. Extractions HPLC–DAD analyses. Experiments were performed in were carried out in triplicate and materials obtained were negative ion mode. The range for scanning was 200–600 filtered over Whatman No. 1 paper and methanol was then and the capillary temperature was set at 350 °C. High spray removed under reduced pressure by a rotary evaporator at voltage was set at 4,500 V. Nitrogen was used as both 35 °C. The resulting aqueous extract was lyophilized and sheath and auxiliary gas at a flow rate of 25 arbitrary units the extraction yield was calculated based on the dry weight (approximately 0.56 l min-1) for sheath gas and 5 arbitrary of the licorice. These extracts (LE) were assessed for their units (approximately 1.5 l min-1) for auxiliary gas. MS/ chemical profile and biological activities. MS were carried out using helium as the target gas, and the collision energy was set at 50–60 %. Identifications were Determination of total content of phenols and tannins achieved on the basis of the ion molecular mass, MS/MS, and UV–Visible spectra.