seasonal dynamics of constitutive levels of phenolic components lead to alterations of antioxidant capacities in acer truncatum leaves

Accepted Manuscript Original article Seasonal dynamics of constitutive levels of phenolic components lead to alterations of antioxidant capacities in Acer truncatum leaves Lingguang Yang, Peipei Yin, Ke Li, Hang Fan, Qiang Xue, Xiang Li, Liwei Sun, Yujun Liu PII: DOI: Reference: S1878-5352(17)30019-9 http://dx.doi.org/10.1016/j.arabjc.2017.01.009 ARABJC 2047 To appear in: Arabian Journal of Chemistry Received Date: Revised Date: Accepted Date: October 2016 12 January 2017 14 January 2017 Please cite this article as: L Yang, P Yin, K Li, H Fan, Q Xue, X Li, L Sun, Y Liu, Seasonal dynamics of constitutive levels of phenolic components lead to alterations of antioxidant capacities in Acer truncatum leaves, Arabian Journal of Chemistry (2017), doi: http://dx.doi.org/10.1016/j.arabjc.2017.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Seasonal dynamics of constitutive levels of phenolic components lead to alterations of antioxidant capacities in Acer truncatum leaves Lingguang Yang, Peipei Yin, Ke Li, Hang Fan, Qiang Xue, Xiang Li, Liwei Sun*, and Yujun Liu* National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China Corresponding authors’ e-mail addresses: Liwei Sun: lsun2013@bjfu.edu.cn Yujun Liu: yjliubio@bjfu.edu.cn Yujun Liu will handle correspondence at all stages of refereeing and publication, also post-publi cation Phone number: +86-13521148866 Complete postal address: National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University Qinghuadonglu No 35, Haidian District, Beijing 100083, China Other authors’ e-mail addresses: Lingguang Yang: yanglingguangxdjqz@163.com Peipei Yin: happy62889@126.com Ke Li: like17931@163.com Hang Fan: fwqh1990@163.com Qiang Xue: 1183930244@qq.com Xiang Li: 18289743335@163.com All the authors, including co-authors, corresponding authors, shared the same affiliation and address: National Engineering Laboratory for Tree Breeding, College of Biological Sciences and Biotechnology, Beijing Forestry University Qinghuadonglu No 35, Haidian District, Beijing 100083, China Seasonal dynamics of constitutive levels of phenolic components lead to alterations of antioxidant capacities in Acer truncatum leaves Abstract ： Acer truncatum leaves (ATL) have long been used in China as a substitutional tea with health benefits However, little is known about the antioxidant capacities as well as its bioactive components The present study was to investigate constitutive phenolic compositions and evaluate antioxidant activities of ATL through its whole growing season The results showed that all seasonal ATL samples contained remarkable phenols, tannins and flavonoids, and exhibited high antioxidant capacities Constitutive levels of total phenols, tannins and flavonoids, and three individual phenolic compounds, namely 1,2,3,4,6-penta-O-galloyl-β-D-glucose (PGG), methyl gallate (MG), and quercetin-3-O-L-rhamnoside (QR), and DPPH• and ABTS+• scavenging capacities were all significantly higher in both the beginning (S4) and ending (S11) months than those in the middle months (S5-S10), resulting in a consistent seasonal saddle-shaped pattern Since S4 and S11 showed significant differences in oxygen radical absorbance capacity (ORAC) values but not in total flavonoids, and there was no significant correlation between ORAC and total flavonoids, it can be inferred that total flavonoids contributed less to the ORAC values than total phenols and tannins did Correlation analyses further revealed that total phenols, tannins and flavonoids were the main contributors to antioxidant ability, and PGG, MG and QR were found to be the key components of phenols (including tannins and flavonoids) in ATL Gallic acid was first discovered in ATL, and high levels of PGG and QR (both higher than 1g/100g d.w in April) were corroborated through HPLC analysis Impressive antioxidant abilities of S4 and S11 were further demonstrated by cellular antioxidant assay, and flavonoids showed stronger antioxidant capacity in the cellular level than other phenolic compositions In conclusion, seasonal dynamics of constitutive levels of phenolic components lead to alterations of antioxidant capacities in ATL, and November and April were the ideal harvesting time with highest levels of phenols and antioxidant capacity Key words: Acer truncatum leaves (ATL), phenolic compounds, antioxidant capacity, HPLC, seasonal dynamics, harvesting time Introduction Significance of antioxidant phytochemicals in maintenance of overall human health and alleviation of risks with cardiovascular disease and cancer gains growing interest with emergence of the concept of functional foods (Abergel, 2002) Phenolic components including simple phenols, flavonoids, anthocyanins and tannins are accepted as the primary antioxidants among complex phytochemicals to deliver antioxidant ability and protection against the attack of harmful reactive oxygen species (ROS) (Antolovich et al., 2002; Matkowski and Wołniak, 2005; Sarikurkcu et al., 2009) Phytochemical antioxidants like phenolic components are thus widely used in many fields such as food, cosmetic and pharmaceutical industries (Sindhi et al., 2013) Thus it has become a trend to seek novel, low-cost and safe antioxidant resources from plants Acer truncatum is an abundant and widespread species native in China, Korea and Japan, and is also found in Europe and Northern America (Guo et al., 2014; More and White, 2003) In China, A truncatum is planted as an ecological and commercial tree, the seed kernel in its samara is extracted to produce superior oil rich in nervonic acid (Wang et al., 2006), and it has been officially admitted as edible oil by the Ministry of Health of the People's Republic of China On the other hand, A truncatum leaves (ATL), traditionally used as a substitutional tea, were authenticated to inhibit activity of fatty acid synthase (Zhao et al., 2014) and exhibit considerable antioxidation (Ma et al., 2005a) and cytotoxic effect on CAES-17, BGC-823, MCF-7, and BEL-7402 cancer cell lines (Zhao et al., 2006) and was also demonstrated to have a strong antibacterial activity on bacterial β-oxoacyl-(acyl carrier protein) reductase (FabG) (Zhang et al., 2008) A few phytochemical investigations on ATL showed that it contained substantial contents of flavonoids and tannins (Ma et al., 2005a), and lots of individual flavonoids and tannins were also determined in the leaves of other Acer species (Bi et al., 2016; Royer, Diouf, Stevanovic, 2011; Zhang et al., 2016) However, no investigation has been done on seasonal dynamics of phenolic components and/or their correlation with antioxidant capacities in ATL It was reported that the content of phenolic compositions in plants varies among organs, tissues and between different seasons (Duda et al., 2015; Kim et al., 2013; Pacifico et al., 2015), which might impose impacts on the antioxidant capacity Therefore, total phenols, flavonoids and tannins might be used as the parameters for quality assessment of antioxidant 1,2,3,4,6-penta-O-galloyl-β-D-glucose potentials (PGG) in (Zhao ATL In et al., addition, 2007), quercetin-3-O-L-rhamnoside (QR) (Ma et al., 2005b) and methyl gallate (MG) (Ma et al., 2005a) have been identified in the partially purified extract of ATL These three ingredients are well known for their bioactive functions such as antioxidant, antitumor and anti-obesity, respectively (Asnaashari et al., 2014; Cincin et al., 2014; Lee et al., 2013; Mohan et al., 2013), thus could be also used as indexes to evaluate ATL’s quality High Performance Liquid Chromatography (HPLC) fingerprinting is highly suitable for both qualitative and quantitative control of various herbs and has been widely utilized in evaluating both food and medicinal resources such as Chrysanthemum indicum flower (He et al., 2015), Rosa flower (Riffault et al., 2014), ginkgo leaf (van Beek and Montoro, 2009), green tea (Alaerts et al., 2012), etc Therefore, HPLC was employed in the present work to analyze phenolic compounds in ATL Although there are various methods being available for evaluating antioxidant capacity, they are based on different principles and mechanisms of action, and present some limitations on reflecting the real antioxidant potential when applied solely (Nimse and Pal, 2015) Therefore, it is of great importance for assessing antioxidant potential with different methods in order to achieve a comprehensive understanding Among these methods, DPPH and ABTS are based on electron transfer reaction, oxygen radical absorbance capacity (ORAC) follows the principle of hydrogen atom transfer, and all these three methods are chemical antioxidant assays On the other hand, cellular antioxidant assay (CAA) is a cell-based assay that can be used for analyzing the intracellular antioxidant ability of a sample Accordingly, three chemical antioxidant assays were chosen to estimate the tendency of seasonal antioxidant capacity in ATL, and the cell-based assay was chosen to confirm the selected ATL with optimal phenol contents and chemical antioxidant capacities Altogether, the overall characteristics of phenolic components in ATL are not yet available, and little is known about the seasonal dynamics of major bioactive components and antioxidant capacities, as well as the optimal harvesting time for ATL Those key information is required to be clarified before ATL could be more widely developed as an antioxidant resource The objective of the present study was to evaluate the seasonal dynamics of total phenols, flavonoids and tannins, and major individual compounds presented in ATL, together with its seasonal antioxidant variation, thus elucidating the correlation between phenolic components and antioxidant capacities in ATL Materials and methods 2.1 Reagents, chemicals, and collection and extraction of samples All standards for HPLC were purchased from National Institutes for Food and Drug Control (China), Folin-Ciocalteu 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic reagents, acid 2,2’-diphenyl-1-picrylhydrazyl (Trolox), (DPPH), 2,2’-azobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH), and fluorescein were purchased from Sigma Chemical (USA) HPLC-grade acetonitrile and formic acid were purchased from Tedia Company (USA) Ultra-pure water was prepared using a Milli-Q50 SP Reagent Water System (Millipore Corporation, USA) Other reagents (analytical grade) were purchased from Sinopharm Chemical Reagent Co Ltd (China) Twenty Acer truncatum Bunge trees, authenticated by associate professor Dr Z Liu, were randomly selected surrounding the approximate center point (GPS coordinate, N 40°00'57.21'', E 116°19'43.36'') within a rough area of 300 m2 and an altitude range from 39 to 41 m in Bajia Outskirts Park, Beijing, China As shown in Table 1, ATL was collected at every 20th from April through November and correspondingly marked as S4-S11 (note: S11 was the fallen leaves on the top layer of the ground) Five leaves from each direction, namely east, west, north, and south (totally 20 leaves), were collected at the same height from each selected tree, and those from 20 trees (400 leaves) were combined as one sample in a single month Sampling of leaves covered their whole growth and development period After gentle cleaning, collected leaves were put into an oven set firstly at 105°C for 15 de-enzyming then 80°C for 24 h drying Dried leaves were ground and passed a 250  250 μm2 sieve, then the powder was stored at -20°C for subsequent extraction Table Phenophases at sampling dates of Acer truncatum leaves in 2015 No S4 S5 S6 S7 S8 S9 S10 S11 Sampling dates 20th, April 20th, May 20th, June 20th, July 20th, August 20th, September 20th, October 20th, November phenological stages leaf expansion; blooming intensive leaf growth; samara formation Leaf maturity leaf coloration; samara maturation full defoliation To prepare an extract, 30 ml of 80% methanol was added to 3.000 g ATL powder, then the mixture was sonicated in a 240-W water bath for 30 at room temperature with occasional stirring The sonicated mixture was filtrated by a 0.22-μm filter to obtain the supernatant The extraction process was repeated twice more with the residue, and three supernatants were poured together and diluted to 90 ml with 80% methanol for further experiments 2.2 Measurements of total phenols, tannins and flavonoids 2.2.1 Total phenols Assay of total phenols was performed according to the Folin–Ciocalteu method (Singleton, Orthofer, Lamuela-Raventos, 1999) with some modifications In brief, 40 μL of 25% Folin–Ciocalteu solution was added to a 96-well plate, followed by addition of 20 μL of standards (10-400 mg/L gallic acid, R2 = 0.998), samples or blank (MilliQ water) to designated wells After blending, 140 μL of 700 mM Na 2CO3 solution was added to each well and the plate was shaken for at 250 rpm The microplate was then incubated in dark at 40 °C for 30 min, followed by absorbance measurement at 765 nm with a microplate reader (Bio-Rad xMark™ Microplate Absorbance Spectrophotometer, USA) Results were expressed as mg gallic acid equivalent (GAE)/100 g d.w of ATL powder 2.2.2 Total tannins Total tannins were determined based on phosphomolybdium tungstic acid-casein reaction (Zhao et al., 2011) Briefly, 10 mL of ATL extract solution was blended with antioxidant capacities between S4 and S11 [Insert Figure here] As shown in Figure 4, both PGG (373.27-1226.92 mg/100 g d.w.) and QR (274.401090.33) exhibited considerable levels as they both exceeded g/100 g d.w in April, both their highest level MG (93.58-683.77) showed to be lower than PGG and QR, yet much higher than GA (25.79-220.44) The sum of peak areas of PGG, QR and MG accounted for more than 20% the total peak areas in profiles of any ATL sample, and 0.97-3.04% on the basis of mg/100 g d.w of plant material As shown in the top of Figure 4, GA and MG are simple phenols and share the basic phenolic structure: a hydroxyl bonding directly to an aromatic ring, PGG belongs to hydrolysable tannins with a glucose ring linked by five gallic acids, and QR is a flavonol rhamnoside Together with the high contents of MG, PGG and QR in ATL, therefore, we recognized these three as the representative components in the ATL, and thus they were reasonably considered as main constituents contributing to the phenols (including tannins and flavonoids) and antioxidant ability of ATL Furthermore, the seasonal dynamic of PGG, MG and QR shared a same pattern, with their contents higher in S4 and S11 while lower in S5-S10, which was similar to the saddle-shaped pattern exhibited in the previous results The contents of PGG (1090.33 mg/100 g d.w.), QR (1226.92), and GA (220.44) were the highest in S4, while slightly lower in S11, and the content of MG was the highest in S11 (683.77) On the contrary, seasonal dynamic of GA was relatively steady, and its level maintained to be the lowest among such four compounds throughout the whole season This confirmed our recognition 25 that PGG, QR and MG played important roles in ATL, and the preferable levels of phenols (including tannins and flavonoids) and antioxidant abilities in S4 and S11 were further corroborated through analysis of those three major compounds by HPLC [Insert Figure here] 3.4 Correlation analysis Correlations among phenolic components (total phenols, flavonoids and tannins), antioxidant capacities (DPPH, ABTS and ORAC), and four identified phenolic compounds (QR, PGG, MG and GA) were evaluated by SPSS regression analyses As shown in Table 3, there is extremely significant correlation among total phenols, flavonoids and tannins (0.919 < R2 < 0.996, P < 0.01), confirming that both flavonoids and tannins are major components of phenols Among three antioxidant assays, DPPH shows extremely significant correlation with ABTS (R2 = 0.968, P < 0.01), while ORAC exhibits no significant correlation with either DPPH or ABTS (0.514 < R2 < 0.657, P > 0.05) This may be attributed to different principles between ORAC and DPPH or ABTS Among the identified four phenolic compounds, PGG shows extremely significant correlation with MG (R2 = 0.948, P < 0.01) and significant correlation with QR (R2 = 0.786, P < 0.05), and QR is significantly correlated with GA (R2 = 0.769, P < 0.05) Furthermore, both PGG and MG exhibit extremely significant correlation with total phenols and total tannins (0.876 < R2 < 0.970, P < 0.01), and significant correlation with total flavonoids (0.800 < R2 < 0.807, P < 0.05), while QR, a flavonoid, has 26 extremely significant connection with total flavonoids (R2 = 0.893, P < 0.01) and significant correlation with total phenols (R2 = 0.748, P < 0.05) Because of its relative low contents, GA exhibits no significant correlation with the total phenols, tannins or flavonoids These findings further demonstrated that PGG, MG and QR were the main contributors to phenolic composition in ATL with the PGG and MG exhibiting the leading roles in total tannins as well as total phenols and QR exhibiting the leading role in total flavonoids In addition, total phenols, flavonoids and tannins are all extremely significantly correlated with DPPH and ABTS (0.891 < R2 < 0.988, P < 0.01), validating that phenol compositions are the decisive contributors to their antioxidant capacities Meanwhile, there is significant correlation between ORAC and total phenols and tannins, while no significant correlation between ORAC and total flavonoids This fact proves our previous hypothesis that total flavonoids contributed less to the ORAC values than total phenols and tannins did Lastly, both PGG and MG exhibit extremely significant correlation with DPPH and ABTS (0.909 < R2 < 0.951, P < 0.01), and QR is significantly correlated with DPPH and ABTS (R2 = 0.768, 0.822, P < 0.05) Interestingly, in correlation analysis between ORAC and MG (a simple phenol), PGG (a tannin) and QR (a flavonoid), R2 of QR (R2 = 0.376, P > 0.05) is found to be the lowest in comparison with those of MG (R2 = 0.623, P > 0.05) and PGG (R2 = 0.438, P > 0.05) These results are consistent with that R2 of total flavonoids is the lowest in the correlation between ORAC and total phenols, tannins and flavonoids Thus this agreement further validates that total 27 flavonoids made fewer contributions to ORAC value Table Correlation analyses among phenolic compositions (total phenols, total flavonoids, total tannins), antioxidant capacities (DPPH, ABTS, ORAC), and four identified phenolic compounds (QR, PGG, MG and GA) TP TF TT DPPH ABTS TP TF 919** TT ** 879** ** ** 937** ** ** 968** * 514 657 DPPH ABTS ORAC QR PGG MG GA 996 947 ** 988 * 742 * 748 ** 880 ** 958 288 ORAC QR PGG MG GA 891 947 632 ** 893 * 800 * 807 514 973 754 692 ** 876 ** 970 221 * 768 ** 931 ** 951 363 * 376 ** 438 786* ** 623 684 948** 484 259 822 909 948 412 -.145 * 769 *Significant correlation at P