Comparison of phenolic compounds content in indeciduous Quercus species

Department of Environmental Science, Konkuk University, Seoul 143-701, Korea. 2 Department of Ethnobotany and Social Medicine, Sikkim University, Gangtok737 102, Sikkim, India. 3 Southern Forest Research Center, Korea Forest Research Institute, Jinju 660-300, Korea. 4 Forest Practice Research Center, Korea Forest Research Institute, Pocheon 487-821, Korea. 5 Institute of Life and Environment, Konkuk University, Seoul 143-701, Korea. 6 Suppro Plant Environment Research Center, AT Center, Seoul 137-787, Korea. 7 Department of Applied Bioscience, Konkuk University, Seoul 143-701, Korea.


INTRODUCTION
Leaves of indeciduous Quercus species have been used in Korean folk medicine for dysentery, diarrhea, hemorrhagia, dermatitis, and exclusion of extravasated blood (Kim et al., 2008).The species Quercus salicina Blume, which is distributed in the southern part of Korea and in Japan, has especially been used for antiinflammatory, diuretic and litholytic remedies (Elliott, 2001;Walters et al., 2001;Redwane et al., 2002;Goun et al., 2002).
aging, cancer and various diseases in humans such as heart disease and cataracts (Pietta, 2000;Katalinic et al., 2006;Halliwell and Gutteridge, 1998).Phenolics are secondary plant metabolites, mainly synthesized by the pentose phosphate pathway and skimate and phenylpropanoid pathways (Randhir and Shetty, 2005).Phenolic compounds have shown antioxidant effects on lipid oxidation (Kaarina et al., 2004;Heinonen et al., 1998;Hopia et al., 1999;Kähkönen and Heinonen, 2003) and a wide range of anti-allergenic, anti-atherogenic, antiinflammatory, anti-microbial, anti-thrombotic and cardioprotective effects (Wei et al., 1990;Pietta, 2000;Middleton et al., 2000).Phenolic compounds have been associated with the health benefits of edible plants, and could be a major determining factor on antioxidant capacity (Balasundram et al., 2006).In addition, phenolic compounds contain an aromatic ring bearing hydroxyl substituent.Phenolic acids and flavonoids are the major class of phenolic compounds, widely distributed in plants (Cai et al., 2004).In phenolic compounds, phenolic acids consist of two kinds of subgroups of hydroxybenzoic and hydroxycinnamic acids (Bravo, 1998).Flavonoids constitute the largest group of plant phenols and naturally generating phenolic compounds (Harbone et al., 1999) and have the basic skeleton of diphenylpropanes.
Numerous quantitative studies on phenolic compounds have been conducted with various methods (Häkkinen et al., 1998;Kawaii et al., 1999;Christopher et al., 2006).In this study, high performance liquid chromatography (HPLC) was used for the determination of phenolic acids and flavonoids in Quercus species used in Korean folk medicine.It is of interest to investigate the functional components of Quercus species traditionally used in Korean folk medicine.The aim of this study was to determine the concentrations of phenolic compounds in Quercus species as medicinal plants.In particular, we determined the concentration of phenolic compounds in Q. salicina Blume leaves and shoots, and in the leaves of four other Quercus species.

Plant description
The indeciduous Quercus species studied were Q. salicina Blume, Quercus acuta Thunberg, Quercus phillyraeoides A. Gray, Quercus glauca Thunberg and Quercus myrsinaefolia Blume, which were collected from southern coast and southern Island of Korea.The collection of shoots and leaves from this tree species was conducted from May 10, 2009to May 15, 2009.In particular, those from Q. salicina Blume were collected from an experimental forest in the Warm-Temperate Research Center of the Korea Forest Research Institute in Jeju Island.Other species were collected from an experimental forest in the Southern Forest Center of Korea.

Preparation of samples
Leaves from five Quercus species were freeze-dried and then ground.Sample extractions were prepared as per the method described by Kim et al. (2006).Two grams of ground samples were extracted in 10 ml of acetonitrile (ACN) and 2 ml of 0.1 N hydrochloric acid, and stirred for 2 h at room temperature.The extract was filtered through a No. 42 Whatman filter paper (125 mm Dia x 100 circles, England) and concentrated by a vacuum evaporator.Residues were re-dissolved in 10 ml of 80% aqueous methanol (HPLC grade, J.T. Baker, USA) and filtered through a 0.45 µM syringe filter (TITAN, Nylon; Sun Sri, USA) and then transferred to 2-ml vials.These samples were used for phenolic compounds analyses.

Statistical analyses
Statistical analyses were conducted using the general linear model procedure (GLM) of the SAS program (SAS Institute, Inc., 2000).The experimental design was a completely randomized design with three replicates.Means separation was performed using the least significant difference (LSD) test at the 0.05 probability level.

Comparison of phenolic compounds contents between the leaf and shoot of Q. salicina Blume
The total concentrations of phenolic compounds in Q. salicina Blume showed differences between leaf and shoot (Figure 1).The average total phenolic compounds in Q. salicina Blume leaves and shoots were 21081.98 µg g -1 .The total amount of phenols in leaves and shoots were 25702.13µg g -1 and 16461.82µg g -1 respectively.Thus, the amount of phenolic compounds in leaves was about 1.56 times than shoots.Among the individual phenolic compounds, gentisic acid was the highest, especially in leaves (9587.50µg g -1 ).Also, the leaves had higher chlorogenic acid (6081.11µg g -1 ) and pyrogallol (3708.30µg g -1 ) content than other substances.The vanillic acid and m-coumaric acid were only detected in leaves.However, (+)-catechin (576.43 µg g -1 ) was only detected in the Q. salicina Blume shoot.
The proportions of phenolic acids and flavonoids are shown in Figure 2. The proportion of phenolic acids among the Q. salicina Blume leaves and shoots varied from 87.86 to 94.08%.The flavonoid proportion in shoots (12.14%) was higher than leaves (5.92%).), Q. phillyraeoides A. Gray (16988.82µg g -1 ), and Q. glauca Thunberg (16003.35µg g -1 ) had higher than average concentration total phenolic compounds (Table 2, Figure 4).The total of phenolic compounds concentration in the leaves of the five Quercus species leaves ranged from 900.91 µg g -1 in Q. myrsinaefolia Blume to 25702.13 µg g -1 in Q. salicina Blume.The highest total phenolic compounds content was found in Q. salicina Blume, which contained 25702.13µg g -1 .The plant with the second highest content was Q. phillyraeoides A. Gray, with 16988.82µg g -1

Comparison of phenolic compounds contents between leaves of the five
. Moreover, Q. myrsinaefolia Blume (900.91 µg g -1 ) showed the lowest concentrations of total phenolic compounds.
The proportions of phenolic acids and flavonoids in Quercus species are shown in Figure 8.The proportion of phenolic acids among the five species of Quercus leaf varied from 57.75 to 94.56%, and flavonoids varied from 5.44 and 42.25%.In particular, Q. acuta Thunberg (94.56%) and Q. salicina Blume (94.08%) had high phenolic acid content, while Q. phillyraeoides A. Gray contained a high percentage of flavonoids (42.25%).
Their activities are related to the number of hydroxyl functional groups in their structures (Middleton et al., 2000;Balasundram et al., 2006).Q. salicina Blume has been used as a folk medicine for various diseases, with  proven effects.
A previous study reported the antioxidative compounds of Q. salicina Blume shoot (Kim et al., 2008), but there have been no reports on the different functional substances such as phenolic acids and flavonoids.In this study, the total concentrations of phenolic compounds varied in leaf and shoot extract of Q. salicina Blume.Among 31 kinds of phenolic compounds, the shoots had higher content of   gallic acid and other 10 phenolic compounds (homogentisic acid, catechin, caffeic acid, vanillin, rutin, hesperedin, ο-coumaric acid, myricetin, quercetin and formononetin) than leaves.On the other hand, Q. salicina Blume leaves were higher in 20 other phenolic compounds.
Thus, this study demonstrated differences in phenolic compounds content in different parts of plants.Several studies have also showed the presence of various phenolic compounds in different tissues of the leaf (Jahne et al., 1993;Weissenbock et al., 1986).Variation in phenolic compounds at different stages of maturity have been reported in Quercus species (Makkar et al., 1991).Moreover, seasonal variation of phenolic compounds composition of Quercus species has also been reported by Brossa et al. (2009).
In addition, we compared the amounts of phenolic compounds in leaf extracts from five Quercus species.The quantitative analyses of 31 phenolic compounds were done using HPLC.In this analyses, Q. salicina Blume leaves showed higher concentration of phenolic compounds than other Quercus species.Three Quercus species (Q.salicina Blume, Q. acuta Thunberg and Q. phillyraeoides A. Gray) showed higher gentisic acid than chlorogenic acid content.Q. salicina Blume leaf had the highest concentration of both gentisic acid and chlorogenic acid, while gentisic acid was not detected in Q. myrsinaefolia Blume, and chlorogenic acid was not detected in Q. glauca Thunberg.On the other hand, Q. glauca Thunberg contained the highest levels of vanillic acid, syringic acid, (+)-catechin, quercetin, and hesperedin.
In summary, the Q. salicina Blume leaf contained the highest concentration of phenolic compounds.Phenolic compounds have important anticancer and anti-aging properties.
In this study, we found differences between Quercus species in the amounts of different phenolic compounds.Overall, Quercus species are useful sources of antioxidants.Future studies are needed for the determination of various Quercus germplasms as useful folk medicines.

Figure 2 .
Figure 2. Proportions of phenolic acids and flavonoids in Quercus salicina Blume leaf and shoot.

Figure 5 .
Figure 5.Comparison of 20 phenolic acids contents in leaves of Quercus salicina Blume.

Figure 6 .
Figure 6.Comparison of 11 flavonoids contents in leaves of Quercus salicina Blume.

Table 1 .
Calibration curve equation for the 31 phenolic compounds standards.

Table 2 .
Phenolic compounds content of the Quercus salicina Blume leaf and shoot.

Table 2 .
Cont.Figure 1.Comparison of total phenolic compounds in Quercus salicina Blume leaf and shoot.

Table 3 .
Phenolic compounds content of the Quercus species.