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Seasonal dynamics of constitutive levels of phenolic components lead to alterations of antioxidant capacities in Acer truncatum leaves
⁎Corresponding authors. lsun2013@bjfu.edu.cn (Liwei Sun), yjliubio@bjfu.edu.cn (Yujun Liu)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
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 and 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 1 g/100 g 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.
Keywords
Acer truncatum leaves (ATL)
Phenolic compounds
Antioxidant capacity
HPLC
Seasonal dynamics
Harvesting time
1 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 such as 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 were 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 et al., 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 potentials in ATL. In addition, 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) (Zhao et al., 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), and thus could be also used as indexes to evaluate ATL’s quality. High Performance Liquid Chromatography (HPLC) fingerprinting is highly suitable for both qualitative control 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), and green tea (Alaerts et al., 2012). 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. This 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.
2 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), and Folin-Ciocalteu reagents, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2′-diphenyl-1-picrylhydrazyl (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 min de-enzyming and then 80 °C for 24 h drying. Dried leaves were ground and passed a 250 × 250 μm2 sieve, and then the powder was stored at −20 °C for subsequent extraction.
No.
Sampling dates
Phenological stages
S4
20th, April
Leaf expansion; blooming
S5
20th, May
Intensive leaf growth; samara formation
S6
20th, June
Leaf maturity
S7
20th, July
S8
20th, August
S9
20th, September
S10
20th, October
Leaf coloration; samara maturation
S11
20th, November
Full defoliation
To prepare an extract, 30 ml of 80% methanol was added to 3.000 g ATL powder, and then, the mixture was sonicated in a 240-W water bath for 30 min 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 et al., 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 Na2CO3 solution was added to each well and the plate was shaken for 5 min 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 4 g casein and kept shaking at 200 rpm for 3 h at room temperature. The mixture was subjected to a 0.22-μm filter to obtain the supernatant, which was defined as sample after Casein-Precipitating Reaction. Another 10 mL of ATL extract solution untreated with casein was defined as sample before Casein-Precipitating Reaction. The following steps were the same as those for determination of total phenols. A standard calibration curve of gallic acid (0–400 mg/L, R2 = 0.996) was plotted. Total tannins were calculated as the difference of total phenols between sample before and after Casein-Precipitating Reaction.
2.2.3 Total flavonoids
Total flavonoids were estimated by a modified aluminum chloride colorimetric assay (Dewanto et al., 2002). Briefly, 120 μL of standards (10–100 mg/L rutin), samples or blank was added to designated wells of a 96-well microplate, followed by addition of 8 μL of 50 mg/mL NaNO2 to each well. After mixing and 6 min incubation, 8 μL of 100 mg/mL AlCl3 was added to each well and pipetted up and down for 10 times and the plate was stood for 5 min at room temperature, and then, 100 μL of 40 mg/mL NaOH was added. The reaction mixture in each well was then pipetted up and down for 10 times again. After 30 min incubation at room temperature, the microplate reader was used to examine absorbance at 410 nm. A standard calibration curve of rutin (0–100 mg/L, R2 = 0.999) was plotted, and results were expressed as mg rutin equivalent (RTE)/100 g d.w. of ATL powder.
2.3 Determination of antioxidant capacity
2.3.1 DPPH• scavenging activity
DPPH• scavenging activity was determined by the method reported by Brand-Williams et al. (1995) with slight modifications. In brief, 10 μL of standards, samples or blank was added to designated well of a 96-well microplate, followed by addition of 40 μL of 1 mM freshly prepared DPPH solution to each well. Subsequently, 190 μL of methanol was added to each well, and the plate was then stood in an orbital shaker setting at 200 rpm for 1 min. After 30 min incubation in dark at room temperature, absorbance was recorded at 517 nm using the microplate reader. A standard calibration curve of Trolox (0–400 mg/L, R2 = 0.999) was plotted, and results were expressed as μmol Trolox equivalent (TE)/100 g d.w. of ATL powder.
2.3.2 ABTS+• scavenging activity
ABTS+• scavenging capacities of samples were evaluated by a previous method (Re et al., 1999) with some modifications. ABTS+• was generated by the reaction of a 7-mM ABTS aqueous solution with 2.4 mM aqueous solution of K2S2O8 in equivalents, and the mixture was incubated in dark at room temperature for 12–16 h. Subsequently, the ABTS solution was diluted with methanol to an absorbance of 0.70 ± 0.02 at 734 nm measured using the microplate reader to generate an ABTS working solution. After the working solution was prepared, 5 μL of standards, samples or blank was added to corresponding wells in a 96-well microplate, followed by addition of 200 μL ABTS working solution to each well. After 5 min incubation in dark at 30 °C, the absorbance was measured also at 734 nm. A standard calibration curve of Trolox (0–800 mg/L, R2 = 0.999) was plotted, and the results were expressed as μmol Trolox equivalent (TE)/100 g d.w. of ATL powder.
2.3.3 ORAC
ORAC assay was performed with all reagents prepared in a 75-mM phosphate buffer (pH7.4) according to a report by Ou et al. (2001), and direct light was not allowed during the whole process. Briefly, 25-μL of standards (Trolox 5–50 μM, R2 = 0.997), samples or blank (75 mM phosphate buffer) was mixed with 75 μL fluorescein (0.20 μM) in wells of a 96-well microplate. After 5 min shaking at 250 rpm, the plate was incubated in a 37 °C-prewarmed oven for 15 min. Subsequently, 100 μL of 37 °C-prewarmed AAPH was immediately added to each well with a 12-channel multipipet to initiate the reaction. The fluorescence was recorded every 1.5 min with a multi-functional fluorescence detector (Tecan infinite M200, Swiss) at 37 °C for 75 min with an excitation at 530 nm and emission at 485 nm. The net Areas under Curve (AUC) of samples and standards were calculated by subtracting the AUC of the blank. Results were calculated by comparing the net AUC of each sample with that of the standard. ORAC values were calculated as μmol Trolox equivalents (TE)/100 g d.w. of ATL powder.
2.3.4 CAA
Human hepatocellular carcinoma (HepG2) cell-based antioxidant CAA assay was performed by using the method reported by Wolfe and Liu (2007) with slight modifications. Briefly, HepG2 cells were seeded in wells of a 96-well microplate at a density of 5 × 104/well and grown in 100 μL DMEM supplemented with 10% FBS, 4 mM l-glutamine and 1% penicillin–streptomycin. After attachment for 24 h at 37 °C in 5% CO2, the medium was removed and subjected to PBS wash for three times, and the HepG2 cells were treated with 100 μL ATL extract at different concentrations (30–80 μg solid/mL) plus 25 μM DCFH-DA dissolved in antioxidant treatment medium (DMEM with 4 mM L-glutamine and 10 mM Hepes) for 1 h. The medium was then removed and 100 μL AAPH (600 μM) dissolved in oxidant treatment medium (HBSS with 10 mM Hepes) was added to each well, and the 96-well microplate was monitored with the multi-functional fluorescence detector every 5 min for 60 min at 538 nm and 485 nm for excitation and emission, respectively. The control wells contained the cells treated with DCFH-DA and AAPH and the blank wells contained the cells treated with DCFH-DA and HBSS but without AAPH. EC50 values were calculated and CAA values were expressed as μmol quercetin equivalent (QEE)/100 g d.w. of ATL powder (R2 = 0.999).
2.4 HPLC analyses
HPLC analyses were performed using a Shimadzu HPLC system (Shimadzu, Japan) equipped with two LC-10AT VP pumps, a SPDM20A ultraviolet detector, and a SIL-20AC TH autosampler controlled by an analytical software (LC Solution-Release 1.23SP1). The chromatographic separation was performed on a reversed phase column (Diamonsil C18 5 μm 250 × 4.6 mm i.d., Dikma, China) with a column temperature set at 30 °C. Two solvents were applied for elution: water containing 0.4% (v:v) formic acid (A) and acetonitrile (B). A linear gradient program was adopted in HPLC separation: 0–10 min, 10% B; 10–12 min, 10–18% B; 12–33 min, 18% B; 33–35 min, 18–15% B; 35–40 min, 15% B; 40–42 min, 15–18% B; 42–60 min, 18% B; 60–100 min, 18–85% B; 100–102 min, 85–100% B; 102–115 min, 100% B. The flow rate was set at 1.0 mL/min, and the injection volume was 10 μL. Detection wavelength was set at 275 nm to monitor all polyphenols simultaneously. For each running, the column was equilibrated for 10 min under initial conditions.
2.5 Correlation analysis
Data were expressed as mean ± SD, and EC50 was calculated by regression analysis. The statistical significance (t-test: two-sample equal variance, using two-tailed distribution) was determined by using SPSS software (Version 22.0, SPSS Inc., Chicago, IL, USA). P values <0.05 were set as significant.
3 Results and discussion
3.1 Effects of seasonal variation on total phenols, flavonoids and tannins in ATL
3.1.1 Total phenols
To determine the total phenols of seasonal ATL, Folin-Ciocalteu method was applied. As shown in Fig. 1A, seasonal dynamic of total phenols was characterized in a saddle-shaped pattern, with the contents being significantly higher in samples of both the beginning (S4) and ending (S11) months than those in the middle months (S5–S10). For S4 and S11, the difference was also significant, with the content of S11 being 1.34 times higher than that of S4. For the middle months, the contents from May to October showed a general ascending trend as indicated by the dash line, and there was no significant difference between S6 and S8 as well as S9 and S10. It should be noted that the phenol content of S10 (the maximum in the middle months) was only 1.35 times, but the content of S11 (the maximum during all the seasons) was 2.64 times higher than that of S5 (the minimum in the middle months). The overall rank order was as follows: S11 > S4 > S10 > S9 > S8 > S6 > S7 > S5.
Seasonal changes of total phenols, tannins and flavonoids in ATL. Gallic acid was used as the positive control for assays of total phenols and total tannins, while rutin was for total flavonoids. The absorbance was determined at 765 nm for total phenols (A) and tannins (B), and 410 nm for total flavonoids (C). The results were presented as mean ± SD of three independent experiments (n = 3) and expressed as μmol standards/100 g d.w. Different letters mean significant difference (P < 0.05). Dash lines were presented based on linear regression analysis.
Among 36 plant species commonly consumed in Spain as tea or beverage resources (Jimenez-Zamora et al., 2016), total phenols of S11 (8996.17 mg GAE/100 g d.w.) in the present study exceed those of 35 species, including black tea (1882.50 mg GAE/100 g d.w.; for comparison, this value as well as following values has been converted into unit the same as our data.), red tea (2385.00) and Ginkgo biloba beverage (705.00), and just a little below that of green tea (10402.50). S4 (6724.34) was superior to 33, and even S5, exhibiting the minimum total phenols in our ATL samples, preceded 31 of the 36 plant species. Kuppusamy et al. (2016) investigated total phenols of 25 plant (tree) wastes, which are the potential sources of function food including substitutional tea. Fruit peel of Quercus robur, the optimum among these 25 wastes, exhibited a dramatic content of phenols (6670 mg GAE/100 g). Nevertheless, both S4 and S11 in ATL exhibited a higher phenol content comparing with the fruit peel of Q. robur. Even S5 was still at the forefront of the 25 plant materials. Furthermore, Jin et al. (2016) found that total phenols of 110 herbal teas were within the range of 20.00–7242.50 mg GAE/100 g and those of eight green teas were in the range of 3742.50–4680.00. S11 showed a higher phenolic content compared with those 110 herbal teas while S4 also ranked at the top in the herbal list, and both S11 and S4 exhibited total phenols higher than all these eight green teas. Therefore, it can be concluded that total phenols in our seasonal ATL (3407.33–8996.17) were considerably high among the main tea or substitutional tea products, with S11 and S4 being the maximum, suggesting that ATL should be a worthwhile candidate for phenol products.
3.1.2 Total tannins
Total tannins of the ATL were analyzed by a casein-precipitating reaction. Fig. 1B shows that seasonal dynamic of total tannins was almost the same with that of total phenols, except for a bit more dramatic rising trend from May to October as indicated by the slope of the dash line and a reverse order of the final two samples. The order was as follows: S11 > S4 > S10 > S9 > S8 > S6 > S5 > S7, namely, the lowest content of total tannins occurred in S7 instead of S5 for total phenols (see Fig. 1A).
Bizuayehu et al. (2016) analyzed total tannins of eight teas commonly used in Ethiopian, and found that green tea possessed the highest total tannins (745 mg tannic acid equivalent/100 g d.w.; for comparison, this value as well as following values has been converted into unit the same as our data.). Our results showed that total tannins of all seasonal ATL (1961.86–6775.13 mg GAE/100 g d.w.) were dramatically higher than those of green tea. Some deviation may occur as the variation of standards in two researches. Some deviation may occur as the variation of standards in two researches, but the results were reliable even if tannic acid was twice functional than gallic acid (mg/mg) in the total tannins assay. Furthermore, S11 still exhibited even higher level of total tannins (6775.13) comparing with that in Castanea mollissima bur (5130.00), a tannins-rich resource reported by Zhao et al. (2011).
3.1.3 Total flavonoids
Fig. 1C shows total flavonoids of the ATL analyzed by an aluminum chloride colorimetric assay. Similar to total phenols and tannins, both S4 and S11 still exhibited significantly higher flavonoids than those in the middle months (S5–S10). No significant difference, however, was observed between S4 and S11. In the middle months, total flavonoids presented in a rising trend generally mild when comparing with those of total phenols and tannins. The detailed rank order was as follows: S11 > S4 > S6 > S7 > S8 > S9 > S10 > S5, with some changes in S5-S10 from those of the total phenols and tannins (see Fig. 1A and B).
Lee et al. (2016) and Bizuayehu et al. (2016) determined total flavonoids in green tea with the same method as ours and obtained identical results (2320 and 2340 mg catechin equivalent/100 g d.w., respectively). Therefore, it could be concluded that total flavonoids in ATL (5621.306–9451.282 mg RTE/100 g d.w.) were much higher and quite noticeable when compared with the main tea plants even if catechin was twice functional than rutin (mg/mg) in the total flavonoids assay.
By calculation based on gallic acid equivalent, total tannins contributed to more than 50% of total phenols in all seasonal ATL (54.84–75.31%), with the largest proportion in S11. Thus it can be concluded that tannins were one of the major components in the ATL. In addition, as total flavonoids in rutin equivalent accounted for more than 5.5% of ATL in dry weight (5.62–9.45%), it can be conferred that ATL contained noticeable flavonoids. Furthermore, it is worthwhile to note that total flavonoids of S4 showed no significant difference with those of S11 while total phenols and tannins of S11 were significantly higher than those of S4. Thus conclusion can be drawn that the extra total phenols in S11 was composited by phenols other than flavonoids, to be specific, might be mainly by tannins. Overall, ATL contained considerable phenols, tannins and flavonoids comparing with other tea or substitutional tea resources. S11 and S4 shared the highest contents of these three phenolic components.
3.2 Effects of seasonal variation on antioxidant capacities in ATL
3.2.1 DPPH• and ABTS+• scavenging activities
DPPH• antioxidant assay was applied to examine the free radical scavenging activities of seasonal ATL, and Trolox (0–800 mg/L) was used as the standard. As shown in Fig. 2A, seasonal dynamic of antioxidant capacities against DPPH• fitted the saddle-shaped pattern described above (see Fig. 1). Namely, the antioxidant capacity was significant stronger in both S4 and S11 than those in S5–S10, and the capacity of S11 was significantly stronger than that of S4. DPPH values from May to September also presented a general increasing trend as indicated by the dash line. An exception was found in October as there was a decline occurred from S9 to S10. The detailed rank order was as follows: S11 > S4 > S9 > S8 > S7 > S10 > S6 > S5.
Seasonal changes of antioxidant capacities in ATL. Trolox was used as the standard for all the three assays. The absorbance was determined at 517 nm for DPPH (A), and 734 nm for ABTS (B), while the fluorescence for ORAC (C) was recorded every 1.5 min for 75 min with an excitation at 530 nm and emission at 485 nm. Data were calculated as the radical scavenging activity (RSA) (%) for both DPPH and ABTS, and as the net Area under Curve (AUC) for ORAC. Results were presented as mean ± SD of three independent experiments (n = 3) and expressed as μmol trolox/100 g d.w. Different letters mean significant difference (P < 0.05). Dash lines were presented based on regression analysis.
Free radical scavenging ability of seasonal ATL was also determined by ABTS+• antioxidant assay, and Trolox (0–800 mg/L) was also used as the standard. Fig. 2B shows that both S4 and S11 still presented stronger ABTS+• antioxidant capacity than those in S5–S10, and an increasing trend also occurred from S5 to S9 (also see the dash line) as that in DPPH• antioxidant capacity, which was in agreement with those in the total phenols and tannins. Overall, these findings may suggest that total phenols and tannins may play a major role in scavenging of DPPH• and ABTS+•. In addition, there was no significant difference among the period from May to July. The detailed rank order was as follows: S11 > S4 > S9 > S10 > S8 > S6 > S7 > S5, being quite similar with that of DPPH. The similarity in these two assays demonstrated that antioxidant values of ATL were reliable and rigorous. Furthermore, the consistent saddle-shaped pattern occurred in phenolic components (total phenols, tannins and flavonoids) and antioxidant capacity (DPPH and ABTS) may suggest that total phenols, tannins and flavonoids play a major role in scavenging ability of free radicals.
Comparing with 36 plant species used as beverages mentioned above (Jimenez-Zamora et al., 2016), S11 ranked the third, just below green tea (184800.00 μmol TE/100 g d.w.; for comparison, this value as well as following values has been converted into unit the same as our data.) and lemon balm (81150.00) in the DPPH assay, and S4 ranked the fourth and presented a favorable result as well. With respect to ABTS, S11 and S4 ranked the fourth and fifth, respectively. Jin et al. (2016) examined antioxidant capacities of 110 herbal teas and eight green teas with DPPH and ABTS. Compared with the eight green teas, both S4 and S11 exhibited even stronger antioxidant capacity than their strongest, Xinyang Maojian green tea (55302.50 for DPPH and 59100.00 for ABTS). And compared with those herbal teas, S11 still ranked the first and exceeded all herbal teas in DPPH (0.00–68357.50) and ABTS (152.50–71780.00), and S4 was also at the top of the herbal teas’ list.
3.2.2 Oxygen radical absorbance capacity
The ORAC assay, a well-recognized method for assessing total antioxidant activity in vitro, can reflect more closely real antioxidant capacity as peroxyl radical is the predominant free radical in the biological systems (Prior et al., 2003). As shown in Fig. 2C, ORAC exhibited a biphasic rising pattern rather than the saddle-shaped pattern found in DPPH and ABTS. The ORAC value increased from S4 (75605.05 μmol TE/100 g d.w.) and reached the first peak at S6 (86539.45), then gradually decreased to the minimum at S8 (64321.41), and followed by a constant rising to the second peak at S11 (111522.45). The overall rank order was S11 > S6 > S10 > S5 > S4 > S9 > S7 > S8, being distinguished from those of DPPH and ABTS, as well as total phenols, tannins and flavonoids. This might be caused by the difference in measurement principle between ORAC and DPPH or ABTS. It should be noted that the antioxidant capacity of S4 (75605.05 μmol TE/100 g d.w.) ranked the fifth in ORAC instead of the second in DPPH, ABTS, the total phenols, tannins and flavonoids. Such enormous variation may be related to certain components in S4 showing weaker antioxidant capacity in ORAC than that in the DPPH and ABTS. Interestingly, the huge variation in ORAC values existed between S4 and S11 while there was no significant difference in total flavonoids between them. Thus we hypothesized that flavonoids in ATL might make fewer contributions to ORAC values than other phenolic components, including tannins, did.
Cao et al. (1996) reported ORAC values of 22 common vegetables, together with green tea and black tea. By comparing our data with their data obtained using the same AAPH radical generator, it is clear that our S11 (111522.45 μmol TE/100 g d.w.) showed an even higher ORAC value than all those 22 common vegetables as well as black tea (92,700; for comparison, this value as well as following values has been converted into unit the same as our data.) and green tea (81,400), and other ATL samples (64321.41–86539.45 μmol TE/100 g d.w.) also showed considerable values. When compared with green teas reported by Sharpe et al. (2016), however, the ORAC value of S11 was only higher than the average value calculated from 24 varieties of the first-brew green teas (102,000 μmol TE/100 g), but lower than that of green teas with six-brews being pooled together (214,000 μmol TE/100 g). The variation of ORAC values for green tea in these two articles may be caused by the adjustment of ORAC protocol by Sharpe et al. (2016), including PH, concentration of fluorescein and AAPH. Thus it is not hard to explain why the ORAC value of ATL in the current study was higher than that of green tea in the former article (Cao et al., 1996) but lower than that in the latter (Sharpe et al., 2016). We recognized that the ORAC protocol used in the current study was more closed to the protocol reported by Cao et al. (1996) than Sharpe et al. (2016), and thus, our results were comparable to the former one. Therefore, conclusion can be drawn that the ORAC values of all seasonal ATL were proven to be comparatively high, and S11 was even higher comparing with those main tea resources.
3.2.3 Cellular antioxidant assay
Comparing to chemical antioxidant activity assays, cell culture models provide a tool that closely reflects reactions in real biological systems (Wolfe and Liu, 2007). Being the optimal seasonal samples in above experiments, S11 and S4 were chosen to conduct the CAA in HepG2 cell lines for further validation. ATL extract used in this experiment (15–150 mg/L) was under the non-cytotoxic level to HepG2 cells. According to results in Table 2, EC50 values of quercetin (5.72 μM) and kaempferol (6.53) were both similar with those (quercetin, 5.92; kaempferol, 7.85) reported by Wolfe and Liu (2007). In addition, CAA value of kaempferol (76.63 μmol of QEE/100 μmol) in the present study was approximately the same with that (75.30) reported by them. All these results indicated that our CAA values were reliable. Slight differences in both EC50 and CAA values of kaempferol may be due to different free radicals used (AAPH rather than ABAP) and growth condition of HepG2 cells (Liu and Huang, 2015).
Compound
EC50a
CAAb
Quercetin
5.72 ± 0.84
Kaempferol
6.53 ± 0.71
76.63 ± 14.75
S11 solid
28.29 ± 1.27
19.66a ± 0.38
S4 solid
30.08 ± 0.935
18.44a ± 0.75
EC50 values of S4 and S11 were calculated to be 30.08 and 28.29 μg/mL, respectively, and these corresponded with their CAA values of 18.44 and 19.66 μmol QEE/100 mg d.w. Both EC50 and CAA values indicated that S11 showed slightly higher cellular antioxidant capacity than S4 but with no significant difference (P > 0.05). This approximation between S11 and S4 may be interpreted by the fact that flavonoids showed stronger antioxidant capacity in this cellular antioxidant assay (Wolfe and Liu, 2007), and S11 exhibited only slightly higher total flavonoids than those of S4 with also no significant difference (see Fig. 1C). Comparing with some tea or beverage plants, CAA values of both S11 and S4 were significantly higher than those of the beverage resources such as black tea (16.40 μmol QEE/100 mg d.w.) (Liu and Huang, 2015), Chinese hawthorn (678.00–1200.00 μmol QEE/100 g d.w.) (Wen et al., 2015), Yerba mate (31.01–39.30) (Boaventura et al., 2015), and adlays (0.53–9.87) (Wang et al., 2016). These indicate that S11 and S4 of ATL can deliver a relative stronger antioxidant effect by protection from oxidant attack to cell membrane, and thus stabilize oxidant homeostasis in human liver cells.
3.3 The effect of seasonal variation on bioactive compositions in ATL by HPLC profile
Fig. 3A shows HPLC chromatograms of four standards that were well-separated with each other, and they all exhibited with different contents in the overlapped profiles of seasonal samples (S4–S11; Fig. 3B), with PGG, QR and MG being already found (Ma et al., 2005a,b; Zhao et al., 2007) but GA, a well-known simple phenol, being demonstrated for the first time in ATL. As shown in Fig. 3B, the majority of peaks in eight ATL samples’ HPLC profiles mainly occurred within 60 min. Furthermore, it was not hard to figure out that intensities of seven peaks labeled with ‘∗∗’ were obviously higher in both S4 and S11 than those in S5–S10. These compounds may partially explain why S4 and S11 possessed higher phenols, tannins and flavonoids as well as stronger antioxidant capacities than those in S5–S10. Moreover, there were nine peaks labeled with ‘∗’ being higher in either S4 or S11 than those in S5–S10, and these, while may also contribute to the superior performance in S4 or S11, might further corroborate the difference of phenols and antioxidant capacities between S4 and S11.
HPLC profiles of four standards and eight seasonal ATL samples. ATL samples were applied to a C18 RP-HPLC column and elution was achieved by a gradient at a flow rate of 1.0 mL/min and was monitored at 275 nm. The mixed standards (A) and liquid extract of seasonal samples (B; S4-S11 from the top to the bottom) were subjected to HPLC to perform composition analysis. Peaks exhibiting higher intensity in S4 and S11 than those in S5–S10 of the middle months were marked with ‘**’, and peaks being higher either in S4 or in S11 than those in the middle months were labeled with ‘*’. Gallic acid, methyl gallate, 1,2,3,4,6-penta-O-galloyl-β-d-glucose, and quercetin-3-O-l-rhamnoside were labeled with their corresponding abbreviation.
As shown in Fig. 4, both PGG (373.27–1226.92 mg/100 g d.w.) and QR (274.40–1090.33) exhibited considerable levels as they both exceeded 1 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% of 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 Fig. 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 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.
Contents of quercetin-3-O-l-rhamnoside (QR), 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG), methyl gallate (MG) and gallic acid (GA) in the eight seasonal samples of ATL. Data were calculated by comparing the identified peak areas of QR (♢), PGG (○), MG (△) and GA (□) shown in Fig. 3B with those of the corresponding standards shown in Fig. 3A. The results were presented as mean ± SD of three independent experiments (n = 3) and expressed as μmol standards/100 g.
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). TP: total phenols; TF: total flavonoids; TT: total tannins; QR: quercetin-3-O-l-rhamnoside; PGG: 1,2,3,4,6-penta-O-galloyl-β-d-glucose; MG: methyl gallate; GA: gallic acid.
TP
TF
TT
DPPH
ABTS
ORAC
QR
PGG
MG
GA
TP
1
TF
.919**
1
TT
.996**
.879**
1
DPPH
.947**
.891**
.937**
1
ABTS
.988**
.947**
.973**
.968**
1
ORAC
.742*
.632
.754*
.514
.657
1
QR
.748*
.893**
.692
.768*
.822*
.376
1
PGG
.880**
.800*
.876**
.931**
.909**
.438
.786*
1
MG
.958**
.807*
.970**
.951**
.948**
.623
.684
.948**
1
GA
.288
.514
.221
.363
.412
−.145
.769*
.484
.259
1
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 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 there is 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 flavonoids made fewer contributions to ORAC value.
4 Conclusion
Overall, all seasonal ATL samples contained considerable phenols, flavonoids and tannins, and exhibited high antioxidant capacity. The seasonal dynamics of total phenols, tannins and flavonoids, and PGG, MG and QR, as well as DPPH and ABTS, with ORAC as an exception, all exhibited a consistent and characterized saddle-shaped pattern. Furthermore, results of correlation analysis corroborated that all above phenol constituents as well as three individual compounds were significantly correlated with antioxidant capacities determined by DPPH and ABTS with also an exception in ORAC. Thus PGG, MG and QR were proved to be the key components of phenols (including tannins and flavonoids), and all these phenolic constitutions were displayed to be the main antioxidant contributors in ATL. In addition, we are the first to demonstrate that gallic acid existed in ATL, and high levels of PGG and QR (both higher than 1 g/100 g d.w. in April) were corroborated through HPLC analysis. Since S4 and S11 showed significant differences in 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. Impressive cellular antioxidant abilities of S4 and S11 were further corroborated through CAA, and flavonoids showed stronger antioxidant capacity in the cellular antioxidant assay than other phenolic compositions. Taken together, November (S11) and April (S4) were confirmed to be the ideal harvesting time for their high levels of phenols and antioxidant ability. In respect of flavor and texture matter, ATL in April (tender leaf) was thus recommended to be developed as substitutional tea, and ATL in November (deciduous leaf) may be more suitable as phenols and/or antioxidant resource for industrial exploration. Our original findings provided baseline data for wide and safe development of ATL as an antioxidant and/or phenolic resource, and gave innovative insights into the utilization of deciduous leaves. Nevertheless, more specific identification of phenol compounds in the ATL, together with confirmation of the key compounds contributing to the antioxidant activity requires to be further studied to provide the point-to-point information for the exploration of ATL.
Author contributions
Lingguang Yang, Peipei Yin, Liwei Sun and Yujun Liu designed the study, carried out the research and drafted the manuscript. Ke Li, Hang Fan, Qiang Xue and Xiang Li participated in the experiments. Yujun Liu and Liwei Sun provided facilities and reviewed the manuscript. All authors read and approved the final manuscript.
Conflicts of interest
The authors declare no conflict of interest.
Acknowledgments
This work was supported by the Fundamental Research Funds for the Central Universities (BLX2013025), and some material used in this study was kindly offered by Shandong Yongchuntang Company.
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