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Chemical classification changes of phenylpropane compounds related to harvest time during leaf development of Three Acacia Species

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https://www.eduzhai.net International Journal of Plant Research 2014, 4(3): 72-83 DOI: 10.5923/j.plant.20140403.02 The Chemotaxonomic Changes in Phenylpropanoids Compounds Related to the Harvest Time during Leaves Development in Three Phlomis sp Heidar Ali Malmir Department of Biology, University of Bu Ali Sina, Hamedan, Iran Abstract In this study, changes in the levels of none flavonoids, flavonoids and radical scavenging capacity (RSC) during development stages of three phlomis sp leaves were investigated. The total phenolic acid (TPA) and total flavonoids contents (TF) were significantly influenced by the species, harvests and species × harvests interaction (p≤0.05). The individual phenolic acid (cinnamic, p-coumaric and caffeic) decreased in mature leaves, while complex phenolic (benzoic, salicylic, gallic, rosmarinic and lignanglucoside) increased showed significantly variable synthesize trends (p≤0.05). The chrysoeriol p-coumaroylglucoside, chrysoeriol, luteolin 7-o-glucuronide, chrysoeriol 7-o-glucuronide, and chrysoeriol 7-o-glucoside that belong to the group of flavonoids had an opposite synthesize curve compared to naringenin, luteolin, luteolin 7-o-glucoside (p≤0.05). This study revealed that individual flavonoids and phenolic could be used as quantity descriptors for monitoring the synthesize variations and chemotaxonomic differences of flavonoids in phlomis leaves and may be useful in determining the optimal harvest time at which TPA and TF reaches a maximum level in harvesting time. Keywords Synthesize alterations, Seasonal changes, Chemotaxonomic, Phlomis 1. Introduction Phlomis is one of the Labiatae species and the genus Phlomis is composed of about 100 species, of which 14 species are described in flora of Iranica, among them 10 are endemic [12]. The phenolic compounds have their origin in the general phenylpropanoid metabolism. These compounds are a group of aromatic secondary metabolites ubiquitously distributed in plants, leaves, fruits, and vegetables. The phenolic compounds belong to two main groups, flavonoids (anthocyanins, flavanols, luteolin, andflavonols) and non-flavonoid (phenolic acids, phenolic alcohols, stilbenes, and their derivatives). Non-flavonoid may vary in structure due to difference in number and position of the hydroxyl groups on the aromatic ring. In many flavonoids, anthocyanins are the largest group of water soluble pigments [7]. Some of the biological effects of flavonoids compounds may be related to the presence of hydroxyl groups on the aromatic ring. The flavonoids compounds especially the luteolin, auroside and luteolin-7-glucopyranoside acid participate in cell protection against the harmful action of ROS [14]. In common phlomis, Phlomis tuberosa, the iridoids, phenylethanoidglycosides and verbascoside are * Corresponding author: malmir1@basu.ac.ir (Heidar Ali Malmir) Published online at https://www.eduzhai.net Copyright © 2014 Scientific & Academic Publishing. All Rights Reserved considered as the major flavonoids compounds [2]. Kabouche et al. (2005) [6] reported from P. crinita three known flavonoids namely, luteolin, luteolin 7-β-D-glucopyranoside and chrysoeriol as the predominant compounds, But small amounts of non-flavonoid (chlorogenic acid and ferulic acid) were found in P. crinita only in small amounts. The non-flavonoid, a wide variety of caffeic acid derivatives, including acteoside, chlorogenic acid, salicylic acid and lignanglucoside from some Phlomis sp have been identified [1]. The protein precipitation capacity of non-flavonoid has been suggested as an important factor reducing the suitability of plants for herbivore and among non-flavonoid compound gallic acid, chlorogenic acid, Vanillic acid, and Ferulic acid are of great interest due to its chemical reactivity. However, along with other measures of biological activities of flavonoids and non-flavonoid (phenolic acid), this capacity varies depending on the chemical structure of the compound [8]. Prestos et al. (2006) [15] reported that phlomis sp have one of the highest contents of RSC among all herbal plant. Mainly oxygen free radicals, reactive oxygen species (ROS) produced in response to environmental stresses such as salinity, drought, high light intensity or mineral nutrient deficiency. Lopez et al. (2010) [9] reported that, phlomis leaves have been used in the pharmaceutical and cosmetic industries. The leaves in phlomis sp are easily available and in amounts are considered a source of healthcare compounds, and have been intensively used in traditional medicine for International Journal of Plant Research 2014, 4(3): 72-83 73 treatment of venous insufficiency and for its antidiarrheic and antihelmintic depurative and astringent properties. Under field conditions, the flavonoids and non-flavonoids composition of plant leaves varies considerably with seasonal, genetic, and climatic factors [1, 10]. Although many studies have revealed that the beneficial health effects of phlomis are due to the several flavonoids. Researches on the exact composition components in phlomis have still not been fully characterized. Furthermore, the changes in contents of the phenolic and felavonoids according to the harvest times of three phlomis sp leaves, especially (Phlomislanceolata, phlomisaucheri and phlomiscaucasica) was not studied. The objectives of this research project were to (1) identify and quantify major flavonoids and phenolic acid compounds present in the three species by HPLC-PDA. (2) Evaluate and compare the antioxidant activities of various extracts from three species. (3) To gain knowledge of the factors that regulate the flavonoids and phenolic acid composition of the leaves according to the harvest times. 2. Materials and Methods The data collection was carried out in two habitats in Khangormaz and Alvand Mountains (36°46′N, 48°34′E). This area of jurisdiction is the province of the Hamedan state of IRAN. Phlomis lanceolata and phlomis aucheri were collected in the Khangormaz Mountains and phlomis caucasica was collected in the Alvand Mountains. The plant was selected random and three replicates for each harvest were prepared to give 12 plants. Plant leaves were collected from 25 Jun 2010, until 25 Aug 2010, in 10-day intervals. The air-dried plant materials (100g) from each species were ground to a fine powder in a mechanical grinder with a 2 mm diameter mesh and then successively extracted with petroleum ether and methanol in a soxhlet apparatus for 10 h. After filtration of each solvent, the organic phases were independently concentrated under a vacuum by evaporating to dryness. The methanol extracts were stored at 40 ◦C for further analysis. The extraction efficiency (%) of methanol extracts is shown in Table 3. Each sample was extracted in three replicate. The water content and dry weight (DW) of leaves were measured from the samples for chemical analyses. Table 1. Results of repeated measures two-way ANOVA for ten phenolic acid and nine flavonoids compounds of three phlomis species on seven harvests stage and the interaction species × harvests stage Phenolic acid species harvests stagespecies × harvests stage TPA marinic acid Lignanglucoside Galic acid Chlorogenic acid Caffeic acid p-coumaric acid Ferulic acid Benzoic acid Salisilic acid Cinamic acid CV Flavonoids TFC Luteolin p-coumaroylglucoside Chrysoeriol p-coumaroylglucoside Chrysoeriol p-coumaroylglucoside Chrysoeriol p-coumaroylglucoside Luteolin 7-o-glucoside Luteolin 7-o-glucoronide naringenin luteolin chrysoeriol Total errors CV F P 11.6 *** 8.4 * 14.6 *** 16.7 *** 5.5 NS 21.5 *** 8.5 ** 6.7 * 19.6 *** 7.2 * 5.8 * 12.6 9.3 29.7 *** 10.5 ** 15.6 *** 19.5 *** 13.6 ** 6.2 ** 15.4 NS 30.7 *** 17.9 *** 5.4 NS 37.5 21.5 F P F P 22.7 *** 10.5 *** 4.7 ns 2.6 ns 45.8 *** 14.8 *** 13.7 ** 6.3 * 3.6 ns 2.6 ns 36.8 *** 15.4 *** 6.4 * 5.8 * 3.2 ns 3.5 ns 8.5 ** 6.8 * 8.11 ** 7.11 ** 3.7 ns 3.7 ns 25.5 11.8 15.3 5.7 67.9 *** 17.3 *** 5.7 * 6.2 ** 23.7 *** 9.3 ** 6.7 ** 6.11 ** 16.3 *** 5.8 ** 3.8 ns 4.7 * 7.3 ** 3.6 * 12.5 *** 8.6 ** 5.2 * 4.2 * 7.7 ** 3.7 ns 45.7 12.6 16.6 9.8 Not significant NS, statically significant difference at P value bellow 0.05 ***, statically significant difference at P- value bellow 0.01 *** 74 Heidar Ali Malmir: The Chemotaxonomic Changes in Phenylpropanoids Compounds Related to the Harvest Time during Leaves Development in Three Phlomis sp Table 2. Synthesis variation of individual phenolic and flavonoids compounds in three Phlomis sp leaves. Each mean is an average of 3× 7 leaves Individual phenolic TPA(mg GAE/G) Rosmarinic acid Lignanglucoside Galic acid Chlorogenic acid Caffeic acid p-coumaric acid Ferulic acid Benzoic acid Salisilic acid Cinamic acid TF (mg EE/g) Luteolin p coumaroylglucoside cherisoeriol p- coumaroylglucoside cherisoeriol 7- o-glucoronide cherisoeriol p-o- lglucoside Luteolin 7-o glucoside luteolin 7- o-glucoronide naringenin luteolin chrysoeriol P.caucasica 39.62b 10.74c 7.97b 0.54b 12.43b 1.35b 0.13a 4.66c 6.22a 8.3c 3.45b 45.5a 2.15b 1.3a 0.75a 6.97a 1.99a 1.31a 8.92a 4.26a 7.71b P. P. lanceolata 43.2b 7.13b 7.09b 0.34a 11.14b 0.67a 0.15b 0.65b 10.24c 4.53b 0.17a 53.98b 0.69a 2.89b 2.56c 8.01b 2.19a 1.34a 12.73b 4.33a 11.73c PP.aucheri 32.16a 5.64a 3.97a 0.26a 6.81a 0.53a 0.1a 0.14a 8.61b 1.65a 0.15a 52.17b 3.26c 1.37a 1.11b 5.76a 3.02b 1.26a 16.04c 4.9b 2.78a Means in rows with different letters that are shown under the number are significantly different according to the Duncan multiple range test, P < 0.05. Table 3. Analytical characteristics of the calibration graphs, Retention times (Rt), correlation coefficient (R), UV absorbance maxima and intercept of y the phenolic and flavonoids compounds Individual phenolic TPA(mg GAE/G) Equation R2 Rt Y=0.011x+0.015 -0.76 98.8 uvλmax 320 intercept -4.5 Rosmarinic acid Y=0.022x+0.012 -0..64 37.7 300-320 -2.4 Lignanglucoside Y=0.016x+0.076 0.73 38.4 290sh-320 1.67 Galic acid Y=0.013x+0.025 0.75 9.7 2700.32 Chlorogenic acid Y=0.026x+0.067 0.68 21.5 3251.21 Caffeic acid Y=0.016x+0.027 -0.68 16.3 35 - 0.82 p-coumaric acid Y=0.021x+0.037 - 0.78 17.5 234sh-310 -1.05 Ferulic acid Y=0.058x+0.045 -0.42 9.1 325 2.06 Benzoic acid Y=0.053x-0.02 0.73 33.6 250-280sh 1.23 Salisilic acid Y=0.078x+0.05 0.69 36.6 250-278sh 1.8 Cinamic acid Y=0.039x+0.073 0.54 65.3 255sh-3201.64 TF (mg EE/g) Y=0.068x+0.029 0.83 68.4 250-278sh 2.6 Luteolin p coumaroylglucoside Y=0.043x+0.018 0.72 19.5 255sh-269, 316 1.03 cherisoeriol p- coumaroylglucoside Y=0.057x+0.071 0.84 22.4 255sh-269, 320 1.17 cherisoeriol 7- o-glucoronide Y=0.048x+0.077 0.78 22.7 252-265sh, 348 0.84 cherisoeriol7-o- lglucoside Y=0.039x+0.028 0.72 22.4 252-267sh, 3481.8 Luteolin 7-o glucoside Y=0.031x+0.048 -0.52 19.3 255-265sh, 3482.07 luteolin 7- o-glucoronide Y=0.062x+0.084 0.73 19.8 255-267sh, 348 0.92 naringenin Y=0.046x+0.057 -0.84 32.9 288-330sh 1.46 luteolin Y=0.094x-0.043 -0.65 19.3 360 -2.07 chrysoeriol Y=0.096x-0.072 0.69 22.8 251-265sh, 347 1.37 International Journal of Plant Research 2014, 4(3): 72-83 75 2.1. The Standard Experiment The following standards were used for quantification of phenolic and flavonoids compounds: Caffeic, ferulic and ascorbic acids (purity >99.0% each), 2,2-diphenyl-1-picrylh ydrazyl radical (DPPH*, «90.0%) and 2,2'-azinobis(3-ethylb enzothiazolin-6-sulfonate) diammonium salts (ABTS, R»98.0%) were purchased from Sigma-Aldrich Chem. Comp. (USA); gallic acid monohydrate (>98.0%), Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid, a hydrophilic derivative of tocopherol, purum, >99%, for HPLC), Folin-Ciocalteu reagent (FC reagent) and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ, puriss, >99.0%) were obtained from FlukaChemie (Buchs, Switzerland). syringic acid (4-hydroxy-3,5-dimetoxybenzoic) and 1,4-naphthoqui none were obtained from Merck (Darmstadt, Germany), chlorogenic acid (5-caffeoylquinic acid), ellagic acid and myricetin (3,30,40,5,50,7-hexahydroxyflavone) were from Sigma (St. Louis, USA), vanillic acid was from Fluka (Buchs, Switzerland), (+)-catechin was from Roth (Karlsruhe, Germany) and juglone (5-hydroxy-1,4-naphthoquinone) was from Aldrich (Milwaukee, USA). Standards were prepared in methanol as stock solutions at concentration 5 mg/l, except chlorogenic acid were at 2 mg/l and myricetin at 1 mg/l. Diluting stock standards were made at the level commonly found in leaves for precisely determination phenolic compounds contents in analysed samples. Butylate dihydroxytoluene dissolved in methanol and used in extraction solution, was obtained from Sigma (St. Louis, USA). Acetonitrile and methanol used as elutants in HPLC system were of HPLC grade and were purchased from Merck (Darmstadt, Germany). Water used for sample preparation and analyses was bidestillated, purified with a Milli-Q water purification system (Millipore, Bedford, USA). 2.2. Chromatographic Analysis HPLC The flavonoids and phenolic acids were determined by using the high-performance liquid chromatography (HPLC-PAD) device; Varian LC system (Palo Alto, USA), equipped with prostar 230 Delivery Module, Prostar 330 PDA (Photodiode Array detector) [16]. The separation of flavonoids and phenolic acids was performed on Omnispher C18 column (250 mm × 4.6 mm, 5 µm, Varian, Palo Alto, USA) equipped with Chromsepher C18 guard-column (1 cm × 3 mm, Varian, Palo Alto, USA). The mobile phase A was 0.1% aqueous phosphoric acid, mobile phase B was 100% methanol of HPLC purity. Elution conditions were 5-80% B, 0-30 min; 80% B, 30-33 min and 80-95% B, 33-35 min, with the flow rate of 0.8 mL min-1. Performance conditions were column temperature 25°C, injected volume 20 and wavelength range from 220 to 600 nm. (Table 3). The total phenolics were expressed as milligrams of gallic acid equivalents (GAE) per gram of extract. The calibration equation for gallic acid was y = 0.011x + 0.015(R2 = 0.989) (Table 3). The total flavonoid content was expressed as grams of epicatechin equivalents (EE) per gram of extract. The calibration equation for epicatechin was y = 0.068x + 0.029(R2= 0.986) (Table 3). 2.3. Determination of Radical Scavenging Capacity (RSC) The RSC of the stable 1,1-diphenyl-2-picrylhy-drazyl (DPPH) free radical was determined by the method described by Socha et al (2009) [18]. Briefly, the reaction medium contained 2 ml of 100 pM DPPH' violet solution in ethanol and 2 ml of plant extract (or water for the control). The reaction mixture was incubated in the dark for 15 min and the absorbance was recorded at 517 nm. The assay was carried out in triplicate. The decrease in absorbance on addition of test samples was used to calculate the antiradical activity, as expressed by the inhibition percentage (%IP) of DPPH radical, following the equation: %IP = [(Ac - As)/Ac] × 100 Where Ac and As are the absorbencies of the control and of the test sample after 15 min, respectively. From a plot of concentration against %IP, a linear regression analysis was performed to determine the IC50 (extract concentration resulting in a 50% inhibition) value for each sample. mg (GAG) g-1 extract mg (EE) g -1 extract P.caucasica 70 P. lanceolata 60 50 40 30 20 10 0 10 20 30 40 50 Harvest (day) P. aucheri 60 70 p.caucasica p. lanceolata p. aucheri 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 Harvest (day) (a) (b) Figure 1. Synthesize variation in the contents of TPA (1a) and TF (1b) contents in leaves of Phlomis sp 76 Heidar Ali Malmir: The Chemotaxonomic Changes in Phenylpropanoids Compounds Related to the Harvest Time during Leaves Development in Three Phlomis sp 2.4. Statistical Analysis The regression equations were used to improve the estimation of the regression line by giving more influence to data that had higher phenolic and flavonoids (Table 3). Prior to either linear or non-linear regression analysis, the mean and variance of the dependent variable were calculated for each species. 3. Results 3.1. Variations of Individual Phenolic Compounds The analyses of the HPLC of phenolic fraction in the leaves during development in the species P. caucasica, P. aucheri and P. lanceolata indicate major differences. The used method (see Table 3) exhibited a strictly linear dependence between absorbance and concentration of all standards (gallic, ferulic and caffeic acids, catechin, Trolox, phenol and iron sulphate) and possible interfering substances (ascorbic acid, glucose, fructose and saccharose). Ferullic acid, Fig. 2g: In P. caucasica, it increased from the first to the end harvest. In P. lanceolata, on the contrary, the statistically significant peak was measure at the beginning in July and afterwards it significantly decreased (Table 1 and 2; p≤0.05). In P. aucheri there was no significant difference in ferulic acid contents between first and end harvests (p≤ 0.05). Rosmarinic acid, Fig. 2a: In P. aucheri, the rosmarinic acid was increasing continuously from Juan to August. In P. caucasica and P. lanceolata they increased from the first to the two harvests, afterwards it significantly decreased (p≤0.05). The highest rosmarinic acid was exhibited by the P. caucasica. The P. lanceolata and P. aucheri showed the lowest rosmarinic acid content (Table 2; p≤0.05). P-coumaric acid, Fig. 2f: The highest content of p-coumaric acid was measured in first harvests in the species P. aucheri. In the case of the species P. caucasica and P. lanceolata, the level of p-coumaric acid in Juan was higher than in august (Table 2; p≤0.05). Gallic acid, Fig. 2c: In P. aucheri there was no significant difference in gallic acid contents between the first harvest and the end harvest. In the case of the species P. caucasica and P. lanceolata the contents of gallic acid were significantly increased from the first harvests to the end harvests (Table; p≤0.05). The gallic acid was significantly lower for the P. aucheri than for the other species, while no differences in gallicacid were found among P. lanceolata and P. caucasica (Table 2; p≤0.05). Salicylic acid, Fig. 2i: In P. aucheri and P. lanceolata the salicylic acid was increasing continuously from Juan to August while in P. caucasica, the leaves contained significantly more salicylic acid on the four harvests than on the others. The salicylic acid was significantly higher for the P. aucheri than for the two species, while differences in salicylic acid were found among P. lanceolata and P. caucasica (p≤0.05; Table 2). Cinnamic acid, Fig. 2j: In three species, the cinnamic acid was increasing continuously from the first to the four harvests, afterwards it decreased. In P. aucheri and P. lanceolata there was no significant difference in cinnamic acid contents between the firstand the end harvests. The cinnamic acid was significantly higher for the P. caucasica than for the two species, while no differences in cinnamic acid were found among P. lanceolata and P. aucheri (Table 2; p≤0.05). Lignanglucoside, Fig. 2b: In three species, the lignanglucoside increased from the first to the end harvest. The lignanglucoside was significantly higher for the P. caucasica and P. lanceolata than for the P. aucheri (Table 2; p≤0.05). Benzoic acid, Fig. 2h: In benzoic acid, the pattern of synthesize was similar in three species. The benzoic acid was significantly higher in the leaves of P. lanceolata than P. caucasica and P. aucheri (Table 2; p≤0.05). In the case of benzoic acid P. aucheri and P. caucasica showed significant differences among harvests (Table 1 and 2; p≤0.05). It was the lowest in first harvest and then it increased to the three harvests, afterwards it significantly decreased. In P. lanceolata there was no significant difference in benzoic acid contents between the first and the end harvests. µ g-1mg µ g -1mg p.caucasica p.lanceolata 14 12 10 8 6 4 2 0 10 20 30 40 50 Harvest (Day) (a) p.aucheri 60 70 p.caucasica 14 p.lanceolata p.aucheri 12 10 8 6 4 2 0 10 20 30 40 50 60 70 Harvest (Day) (b) International Journal of Plant Research 2014, 4(3): 72-83 77 µ g -1mg 14 p.caucasica p. lanceolata 12 10 8 6 4 2 0 10 20 30 40 50 Harvest (Day) (c) p.aucheri 60 70 µ g -1 mg p. caucasia" p.lanceolata 25 20 15 10 5 0 10 20 30 40 50 Harvest (Day) (d) p.aucheri 60 70 µg -1mg p. caucasica 2.5 p. lanceolata p. aucheri 2 1.5 1 0.5 0 10 20 30 40 50 60 70 Harvests (Day) (e) P. caucasica 8 7 6 5 4 3 2 1 0 10 20 P. lanceolata 30 40 50 Harvest (Day) (g) P. aucheri 60 70 µg -1mg µ g -1mg P. caucasica P. lanceolata 0.35 0.3 0.25 0.2 0.15 0.1 0.05 0 10 20 30 40 50 Harvest (Day) P. aucheri 60 70 (f) P. aucheri 16 14 12 10 8 6 4 2 0 10 20 P. lanceolata P. caucasica 30 40 50 60 70 Harvest (day) (h) µ g -1mg µ g -1mg P. caucasica 12 10 8 6 4 2 0 10 20 P. lanceolata 30 40 50 P. aucheri 60 70 µ g -1mg P.caucasica 5 P. lanceolata 4 3 2 1 0 10 20 30 40 50 60 P. aucheri 70 Harvest (Day) Harvest (Day) (i) (j) Figure 2. Seasonal changes in individual phenolic acid concentration and harvests stages in phlomis sp (according to regression equations shown in Table 3). Each mean is an average of three leaves. Vertical bar represents ± S.E.2a: rosmarinic acid, 2b: lignanglucoside acid, 2c: galic acid, 2d: chlorogenic acid, 2e: caffeic acid, 2f: p-coumaric acid, 2g: ferulic acid, 2h: benzoic acid, 2i: salicylic acid and 2j: cinnamic acid (µg/mg of DW) in leaves of P. lanceolata, P. aucheri and P. caucasica on seven harvests 78 Heidar Ali Malmir: The Chemotaxonomic Changes in Phenylpropanoids Compounds Related to the Harvest Time during Leaves Development in Three Phlomis sp Chlorogenic acid, Fig. 2d: the P. caucasica and P. lanceolata species showed higher chlorogenic acid than in P. aucheri (Table 2; p≤ 0.05). In P. aucheri no differences between these harvests were recorded. In the case of chlorogenic acid P. lanceolata showed significant differences among harvests. It was the lowest in two harvests and then it increased to the five harvests, whereas in species P. caucasica the highest chlorogenic acid level was determined on the two harvests, afterwards it significantly decreased (Table 2; p≤ 0.05). Caffeic acid, Fig. 2e: The highest content of caffeic acid was measured in middle July in the three species, afterwards it significantly decreased. The caffeic acid was significantly higher for the P. caucasica than for the other species, while no differences in caffeic acid were found among P. lanceolata and P. aucheri (Table 2; p≤ 0.05). 3.2. Variations of Individual Flavonoids Compounds The compounds detected and quantified during the HPLC are presented in Table 2. The method of extraction and the HPLC programmers were more suitable for flavonoid. Absorbance of the peaks at the λmax of each flavonoid was used as a measure of the relative amounts (see Table 3). Luteolin p-coumaroylglucoside, Fig.3a: The content of the luteolin p-coumaroylglucoside was three fold larger in the P. aucheri than the content of it in the P. caucasica and even two fold larger compared to the amount of it in the P. lanceolata (Table 2; p≤ 0.05). In three species the pattern of synthesize variation in luteolin p-coumaroylglucoside was similar. In P. lanceolata it reached statistically significantly the highest level in the four harvests. In P. aucheri and P. caucasica the highest content luteolin p-coumaroylglucoside was measured in the five harvests, afterwards it significantly decreased. Chrysoeriol, Fig. 3i: The chrysoeriol increased from the first to the three harvest in P. lanceolata, afterwards it significantly decreased. While in P. aucheri and P. caucasica it increased from the first to the three harvests and remained high till the end of harvests. The content of the chrysoeriol was 1.5 fold larger in the P. lanceolata than the content of chrysoeriol in the P. caucasica and even three fold larger compared to the amount of chrysoeriol in the P. aucheri (Table 2; p≤ 0.05). Luteolin 7-o-glucoside, Fig. 3e: In luteolin 7-o-glucoside, the pattern of synthesize was similar in three species. In three species, it increased from the beginning of the first harvests until three harvests. The luteolin 7-o-glucoside was significantly higher for the P. aucheri than for the P. lanceolata and P. caucasica (Table 2; p≤ 0.05). While no differences in luteolin-7-o-glucosidewere found among P. lanceolata and P. caucasica. µg -1mg P.caucasica 5 4 3 2 1 0 10 20 P. lanceolata 30 40 50 Harvest (Day) (a) P. aucheri 60 70 P. caucasica 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 10 20 P. lanceolata 30 40 50 Harvest (Day) (c) P. aucheri 60 70 µg-1mg µ g -1mg P. caucasica 5 4 3 2 1 0 10 20 P.lanceolata 30 40 50 Harvest (Day) (b) P. aucheri 60 70 P. caucasica 12 P.lanceolata 10 8 6 4 2 0 10 20 30 40 50 Harvest (Day) P.aucheri 60 70 (d) µ g g-1mg International Journal of Plant Research 2014, 4(3): 72-83 79 µ g -1mg µ g -1mg P. cauvasica 5 4.5 4 3.5 3 2.5 2 1.5 1 0.5 0 10 20 P. lanceolata 30 40 50 Harvest (Day) (e) P. aucheri 60 70 P. aucheri 30 P. lanceolata P. caucasica 25 20 15 10 5 0 10 20 30 40 50 60 70 Harvest (Day) (f) µg -1mg µg -1mg P.caucasica 2.5 P. lanceolata 2 1.5 1 0.5 0 10 20 30 40 50 Harvest (day) (g) P.aucheri 60 70 P. caucasica 9 8 7 6 5 4 3 2 1 0 10 20 P. lanceolata 30 40 50 Harvest (Day) (h) P. aucheri 60 70 P. caucasica 20 P. lanceolata P. aucheri µ g -1mg 15 10 5 0 10 20 30 40 50 60 70 Harvest (day) (i) Figure 3. Seasonal changes in individual flavonoids concentration and harvests stages in phlomis sp (according to regression equations shown in Table 3). Each mean is an average of three leaves. Vertical bar represents ± S.E.3a: luteolin-p-coumaroylglucoside, 3b: chrysoeriol-p-coumaroylglucoside, 3c: chrysoeriol-7-o-glucuronide, 3d: chrysoeriol-7-o-glucoside, 3e: luteolin-7-o-glucoside, 3f: luteolin-7-o-glucuronide, 3g: naringenin, 3h: luteolin and 3i: chrysoeriol (µgmg-1 of DW) in leaves of P. lanceolata, P. aucheri and P. caucasica on seven harvests Naringenin, Fig. 3g: In naringenin, the pattern of synthesize was similar in three species. The naringenin increased from the first to the four harvests in P. lanceolata, afterwards it significantly decreased while in P. caucasica and P. aucheri it increased from the first to the three harvests, afterwards it significantly decreased. The naringenin was significantly higher for the P. lanceolata than for the P. aucheri and P. caucasica (Table 2; p≤0.05). Luteolin, Fig. 3h: In luteolin, the pattern of synthesize was similar in three species. The luteolin increased from the first to the three harvests in P. lanceolata and P. caucasica, afterwards it significantly decreased. In P. aucheri the highest content luteolin was measured in four harvests, afterwards it significantly decreased (Table 2; p≤0.05). Lteolin 7-o-glucoronid, Fig. 3f: The luteolin 7-o-glucoronid showed very similar pattern of synthesize with regard to the species. In P. caucasica and P. lanceolata the highest content luteolin 7-o-glucoronid was measured in three harvests, afterwards it decreased. In P. aucheri the highest content luteolin 7-o-glucoronid was measured in four harvests, afterwards it significantly decreased. Chrysoeriol 7-o-glucoside, Fig. 3d: In chrysoeriol 7-o-glucoside, the pattern of synthesize was different in three species. In P. caucasica it increased from the beginning of 80 Heidar Ali Malmir: The Chemotaxonomic Changes in Phenylpropanoids Compounds Related to the Harvest Time during Leaves Development in Three Phlomis sp the season until seven harvests. The chrysoeriol 7-o-glucoside was significantly higher for the P. lanceolata than for the P. aucheri and p. caucasica (Table 2; p≤0.05). In P. aucheri and P. lanceolata the highest content chrysoeriol 7-o-glucoside was measured in two and three harvests, afterwards it decreased. Chrysoeriol p-coumaroylglucoside, Fig.3b: the chrysoeriol p-coumaroylglucoside showed very similar pattern of synthesize with regard to the species. In P. lanceolata and P. aucheri they increased from the beginning of the season until four harvests, than remained high till the end of harvest. The content of the chrysoeriol p-coumaroylglucoside was 3 fold larger in the P. aucheri than the content of it in the P. caucasica and P. lanceolata (Table 2; p≤0.05). Chrysoeriol7-o-glucoronide, Fig. 3c: the content of the chrysoeriol 7-o-glucoronide was 2.5 fold larger in the P. lanceolata than the content of it in the P. caucasica and P. aucheri (Table 2; p≤0.05). In P. aucheri and P. caucasica the highest content chrysoeriol 7-o-glucoronide was measured in four harvests, afterwards it decreased. In P. lanceolata it increased from the beginning of the season until seven harvests. 4. Discussion In the present study, the data from the quantitative analyses of the methanol extracts from P. caucasica, P. lanceolata and P. aucheri leaves using HPLC (calibration graphs) is presented in table 3. In this study, the variations in environmental conditions were observed among habitats. This study revealed that individual flavonoids and phenolic could be used as quantity descriptors for monitoring the synthesize variations and chemotaxonomic differences of flavonoids and phenolic in phlomis leaves. The ratio (individual flavonoids)/ (total flavonoids) have been suggested as a quantity index for measuring the differences in flavonoids levels in FW phlomis leaves across the phlomis leaves development. In the khangormaz habitate the quantity index have almost differences leaf flavonoid profiles with the absence of the simple flavonoid (luteolin, naringein and chrysoeriol) found in P. aucheri and P. lanceolata respectively (Table 2 and 5). However, at alvand habitat level identical were found in that the distribution of the different flavonoid classes. Mechanisms that induce different synthetic in phlomis leaves may include all of the following environmental conditions, such as habitat and temperature, which vary markedly across leaves development [5, 13]. Thus, P. aucheri is unique in producing luteolin, whereas P. lanceolata contain chrysoeriol and P. caucasica differs from two species in the presence naringein. The first chemical group within the species contains phenolic acid which are characterised by an accumulation of (rosmarinic, chlorogenic. lignanglucoside, benzoic, salicylic) acid. There are large individual differences among the phenolic acid profiles of the species in these sections, but no overall distinction in (ferulic, p-coumaric, caffeic, galic) acid patterns among the three species. The second group corresponds to those flavonoids assigned to the species, which accumulate three luteolin, Chrysoeriol and naringein. Luteolin and Chrysoeriol are the most common type of flavonoid in the three species, and therefore are very informative about relationships within this species. The P. lanceolata showed the highest chrysoeriol and naringinin per leaves and the P. aucheri showed the highest luteolin compound per leaves (Table 2; p≤ 0.05). The accumulation of chrysoeriol varies strongly with the developmental state of the leaves, is a result of a balance between biosynthesis, and further it converted to other chrysoeriol compounds [6]. The chrysoeriol compounds, which are the main compounds in P. lanceolata leaves, did not correlate with TF (Table 5). The results of multiple regressions showed that increase in TF in P. lanceollata over the seven harvests had a positive effect on the chrisoeriol, chrisoeriol 7-o- glucoronide, chrisoeriol 7-o- glucoside and chrisoeriol p-coumaroylglucoside content, whereas the naringenin and luteolin compound had a slightly negative effect (Table 5). High TF contents during the end harvest of the P. aucheri leaves could be related to an increased the luteolin compounds and are slightly correlated to the individual luteolin content in the leaves (Table 1 and 5). Apart from this coarse control at the first step on the pathway luteolin activity, it seems probable that subsidiary control mechanisms operate other key stages of luteolin metabolism. These reactions are the four and five step in the phenylpropanoid pathway and ultimately lead to multiple classes of flavonoids natural products [7, 13]. A significant positive correlation (r=0.92; Table 4) was observed between the RSC and the TF of phlomis leaves. The higher RSC in five harvests was relating to the higher TF content that increased with a decrease in free phenolic. Results showed that during the early harvests stage, the average TPA were predominant (Table 2; p< 0.05) with 55.3µg/mg of FW against 65.6 µg/mg of FW for the average TF. However, during the other harvests stages, the average TF became dominant (Table 2; p< 0.05). These results are agreement with those of Ross et al., (2009) [16] who reported that flavonoids were predominant during the flowering stage of bean. The Phenolic acids may vary in structure due to difference in number and position of the hydroxyl groups on the aromatic ring. Variations of the amounts of different phenolic and flavonoids compounds during the seven harvests stages is given in table 1, 2 and 3. Their contents were statistically significantly influenced by the harvest time and the species (Table 1; p≤ 0.05). Different phenolic and flavonoids groups could be a possible explanation of the verified differences between three species. The P. aucheri leaves grown in Alvand mountain had significantly lower (rosmarinic, ferulic, p-coumaric, caffeic, chlorogenic, lignanglucoside) acid contents than P. caucasica and P. lanceolata (Table 2; p≤ 0.05). The P. caucasica leaves grown in khangormaz mountain had significantly higher (rosmarinic, Gallic, ferulic, caffeic, chlorogenic, International Journal of Plant Research 2014, 4(3): 72-83 81 lignanglucoside) acid content than P. lanceollata (Table 2; p≤ 0.05). The phenolic acid profiles observed on the HPLC showed very little variation among three species. Moreover, there was surprisingly little variation in major phenolic acid profiles among species belonging to the same habitat (khangormaz). In khangormaz habitat the differences between the two species were not significant. The P. lanceolata leaves grown in khangormaz mountain had significantly higher (Chrysoeriol -p-coumaroylglucoside, Chrysoeriol, naringenin, chrysoeriol -7-o-glucuronide, chrysoeriol-7-o-glucoside, p-coumaric acid and benzoic acid) contents than P. caucasica and P. aucheri (Table 2; p≤ 0.05). The P. aucheri leaves grown in Alvand mountain had significantly higher (luteolin, luteolin-p-coumaroylglucoside, luteolin-7-o-glucuronide, luteolin-7-o-glucoside) content than P. caucasica and P. lanceolata (Table 2; p≤ 0.05). The results are consistent with those of other researcher [1, 3, 9 and 19]. The flavonoid profiles observed on the HPLC showed very high variation among three species. Moreover, there was surprisingly high variation in major flavonoid profiles among species belonging to the same habitats. The research results show that each phenolic acid and flavonoids determined in Phlomis sp leaves has its own curve of synthesize fluctuations of concentrations (Fig. 2a, 2b, 2c, 2d, 2e, 2f, 2g, 2h and 2i and Fig 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h and 3i). These trends are in the same range as reported by Amaral et al., (2004) and Stampar et al., (2006) [1, 19]. In Phlomis sp leaves, the average TPA in the phlomis sp leaves from the three species studied showed the same trend during development (Fig 1a). The P .lanceolata had significantly higher TPA (43.2 µg/mg FW) than P. caucasica and P. auchri (39.6 and 32.1 µg/mg FW, respectively (Table 2; p≤ 0.05). In three species the highest content TPA was measured in the two and three harvests, afterwards it significantly decreased (Fig 1a; p≤ 0.05). The decreased level of TPA was the results of the level rosmarinic, caffeic and p-coumaric acid were decreased, while the contents salicylic, lignanglucoside and gallic acid were increased (Fig. 2a, 2e, 2f, 2i, 2b and 2c). This agrees with previous findings in Phlomis lychnitis and in other plants [9, 11]. The results of multiple regressions showed that decrease in (rosmarinic, caffeic, p-coumaric and benzoic) acid over the seven harvests had a negative effect on the TPA content (Fig. 2a, 2e, 2f and 2h), whereas (salicylic, gallic and lignanglucoside) acid had a slightly positive effect (Fig. 2i, 2c, 2b; Table 5, p≤ 0.05). The phenolic compounds present in three phlomis sp leaves, such as chlorogenic acid, cinnamic acid and ferulic acid which showed no trend over the developmental stage, but differences were noted when these contents of the three species were compared (Fig. 2d, 2j and 2g; Table 5; p≤ 0.05). These differences can be used to distinguish one species from the other, indicating a distinct individual phenolic metabolism in the leaves of the three species. We suggest using different behavior on phenolic and flavonoids compounds in order to differentiate leaves in Phlomis sp. Individual Phenolic production and its consumption during leaves developmental have been more pronounced in this experiment [16, 17]. From the physiological point of view, these results suggest that the early growth stage could be characterized by the high ferulic, benzoic, rosmarinic, p-coumaric, caffeic, naringenin, luteolin, luteolin-7-o-glucoside and high growth period of leaves. This reactions are the first step in the phenylpropanoid pathway and ultimately leads to multiple classes of flavonoids natural products, such as salicylic, cinnamic, gallic, lignanglucoside, luteolin-p-coumaroylglu coside, chrysoeriol-p-coumaroylglucoside, chrysoeriol, luteolin-7-o-glucuronide, lignanglucoside, rosmarinicand chlorogenic acid. After this stage, the leaves must reduce its growth and prepare itself to the development stage. In fact, it could be postulated that during maturation stage of leaves, the leaves decreased ferulic, benzoic, rosmarinic, p-coumaric, caffeic, naringenin, luteolin, luteolin7-o-gluco side and provide to prepare itself to the lignifications process in order to slow down its growth. Indeed, many studies reported that lignin is a polymer synthesized from soluble phenolic acid compounds of phenyl propane type [15]. The reactions in leaves due to individual phenolic acid oxidation may have an enzymatic and non-enzymatic [4, 13]. The synthesize pattern showing a positive correlation between leaves FW and the concentration of ferulic, benzoic, rosmarinic, p-coumaric, caffeic, naringenin, luteolin, luteolin-7-o-glucoside was also found in three Phlomis sp, and a negative correlation between leaves FW and the concentration of salicylic, lignanglucoside and gallic acid was also found in three Phlomis sp (Table 5). Calis et al. 2005, [2] also see the association between the decrease of TPA in the Phlomis tuberose leaves in June and the rapid development of the flower at the time when most of the nutrients and photo assimilates are employed in flower growth. The flavonoids groups were significantly influenced by the species, harvest and their interaction (Table 1 and 2; p≤ 0.05). At the first harvests, at the stage of the leaves low development, the leaves contained 33.53 µg/mg of TF of FW. In the end of July, the content of TFC was highest 62.70 µg/mg of FW (Table 2). This agrees with previous findings in phlomis leaves and in other plants [17]. In the end of July, the leaves started to lignified, and at the same time, leaves growth was in the phase of full extensions, three flavonoids chrisoeriol-7-o-glucoronide, luteolin-7-o-glucoronide and chrisoeriol-p-coumaroylglucoside content significantly increased (Fig. 3c, 3f and 3b), while the concentrations luteolin, naringenin and luteolin-7-o-glucoside were decreased (Fig. 3h, 3g, and 3e;Table 2 and 4; p≤ 0.05). The individual flavonoids showed very different pattern of synthesize with regard to the species (Fig 3a, 3b, 3c, 3d, 3e, 3f, 3g, 3h and 3i). The synthesize differences on chrysoeriol compound and luteolin compound at harvesting time were observed between species. Since P. lanceolata showed the higher value of chrysoeriol compound in comparison to P. caucasica and P. aucheri (Table 2; p≤ 0.05). The flavonoids production and its accumulation during leaves developmental have been more pronounced in plants (Fig

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