The aim of this study was to determine the composition and content of phenolic compounds in the ethanol extracts of apple leaves and to evaluate the antioxidant activity of these extracts. The total phenolic content was determined spectrophotometrically, as well as the total flavonoid content in the ethanol extracts of apple leaves and the antioxidant activity of these extracts, by the ABTS, DPPH, and FRAP assays. The highest amount of phenolic compounds and flavonoids as well as the highest antioxidant activity was determined in the ethanol extracts obtained from the apple leaves of the cv. Aldas. The analysis by the HPLC method revealed that phloridzin was a predominant component in the ethanol extracts of the apple leaves of all cultivars investigated. The following quercetin glycosides were identified and quantified in the ethanol extracts of apple leaves: hyperoside, isoquercitrin, avicularin, rutin, and quercitrin. Quercitrin was the major compound among quercetin glycosides.
Studies on the chemical biodiversity of plants are recognized as being relevant and are carried out with the aim of enriching the assortment of raw medicinal plant materials and to evaluate their potential application to the demands of practical medicine. A search for plants accumulating phenolic compounds, which have recently been considered as an object of many scientific studies, is especially promising. It is of importance to assess the composition and content of phenolic compounds in plant vegetative organs, to determine the patterns of their accumulation and identify new, promising sources of plant phenolic compounds.
The domestic apple (
Phenolic compounds, acting as natural antioxidants, scavenge free radicals and inhibit their production, stimulate the synthesis of antioxidant enzymes, and thus prevent oxidative stress resulting in damage to the structural molecules of the body [
Therefore, the aim of this study was to determine the composition and content of phenolic compounds in the ethanol extracts of apple leaves harvested from the cultivars Aldas, Auksis, Ligol, and Lodel grown under Lithuanian climatic conditions, to assess the variation of phenolic compounds and to compare their antioxidant activity in the apple leaf samples obtained from different cultivars.
The following apple cultivars were included into this study: Auksis (early winter cv., bred in Lithuania) and Ligol (winter cv., bred in Poland) that are two main cultivars in commercial apple orchards as well as Aldas (early winter cv., bred in Lithuania) and Lodel (winter cv., bred in Poland), two cultivars resistant to apple scab (
All the solvents, reagents, and standards used were of analytical grade. Acetonitrile and acetic acid were obtained from Sigma-Aldrich GmbH (Buchs, Switzerland) and ethanol from Stumbras AB (Kaunas, Lithuania). Hyperoside, rutin, quercitrin, phloridzin, phloretin, caffeic acid, and chlorogenic acid standards were purchased from Extrasynthese (Genay, France), (+)-catechin and (–)-epicatechin from Fluka (Buchs, Switzerland) and avicularin and isoquercitrin from Chromadex (Santa Ana, USA). 1,1-Diphenyl-2-picrylhydrazyl (DPPH•) radical, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), potassium persulfate, sodium acetate trihydrate, iron (III) chloride hexahydrate, and 2,4,6-tripyridyl-s-triazine (TPTZ) were obtained from Sigma-Aldrich (Steinheim, Germany). Folin-Ciocalteu reagent, gallic acid monohydrate, sodium carbonate, aluminum chloride hexahydrate, and hexamethylenetetramine were purchased from Sigma-Aldrich GmbH (Buchs, Switzerland). Deionized water, produced by the Crystal E high-performance liquid chromatography (HPLC, Adrona SIA, Riga, Latvia) water purification system, was used.
An amount of 0.25 g of lyophilized apple leaf powder (exact weight) was weighed, added to 10 mL of ethanol (70%, v/v), and extracted in a Sonorex Digital 10 P ultrasonic bath (Bandelin Electronic GmbH & Co. KG, Berlin, Germany) for 40 minutes at 60°C. The extract obtained was centrifuged for 7 minutes at 6000 rpm with a Hermle Z206A centrifuge (Denville Scientific Inc., USA). The extract was collected and filtered through a membrane filter with a pore size of 0.22
All the spectrophotometric measurements were carried out with a Genesys-10 UV/Vis spectrophotometer (Thermo Spectronic, Rochester, USA). The total phenolic content (mg GAE/g DW) in the ethanol extracts of apple leaves was determined by the Folin-Ciocalteu method [
Qualitative and quantitative analysis of phenolic compounds was performed according to the
The identification of the chromatographic peaks was achieved by comparing the retention times and spectral characteristics (
All the experiments were carried out in triplicate. Means and standard deviations were calculated with the SPSS 20.0 software (Chicago, USA). A single factor analysis of variance (ANOVA) along with the post hoc Tukey test was employed for statistical analysis. The Kolmogorov-Smirnov test was applied to examine the normality of distribution. To verify the hypothesis about the equality of variances, Levene’s test was employed. The correlation was evaluated by Pearson analysis. Differences at
In order to determine the patterns of the accumulation of biologically active compounds in plants, it is important to identify their composition and content in separate plant organs. The secondary metabolites of plant metabolism, phenolic compounds, have been detected in apple leaves [
In this study, the total amount of phenolic compounds in the ethanol extracts of apple leaves varied from 98.81 ± 1.51 mg GAE/g DW (cv. Auksis) to 163.35 ± 4.36 mg GAE/g DW (cv. Aldas) (Table
The total amounts of phenolic compounds (TP) and flavonoids (TFd) in the ethanol extracts of apple leaves of different cultivars.
cv. | TP (mg GAE/g DW)a | TFd (mg RE/g DW)a |
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Aldas |
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Auksis |
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Ligol |
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Lodel |
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bBy ANOVA test.
Studies investigating the total phenolic content in the leaf samples of other plants belonging to the
The biological effects of many plant raw materials depend on flavonoids; therefore, studies on the variation in their content are important and relevant. This study determined the total flavonoids content in the ethanol extracts of apple leaves, which ranged from 21.59 ± 0.52 mg RE/g DW (cv. Auksis) to 45.02 ± 0.90 mg RE/g DW (cv. Aldas) (Table
The data on the patterns of variation in the total phenolic and flavonoid contents of apple leaves are scarce. Therefore, this study provides new knowledge about total phenolics and flavonoids content in the apple leaves of the cultivars grown under Lithuanian climatic conditions, allows the comparison of the results obtained with those of other studies, and is valuable to carrying out a search for promising, biologically active substance-accumulating plant raw materials.
After studying the total phenolics and flavonoids content of apple leaves harvested from different cultivars grown under Lithuanian climatic conditions, it is important to examine and assess the antioxidant activity in the extracts of apple leaves. The results obtained during studies will be useful for the selection of apple cultivars in order to provide a consumer with products rich in antioxidants, will be useful for the assessment and standardization of quality of plant raw materials and their products, and will allow predicting an antioxidant effect of apple leaves
Herbal extracts are multicomponent matrices with antioxidant activity determined by the set of different mechanism reactions, so antioxidant effect cannot be adequately tested using only one method [
The antioxidant activity of ethanol extracts obtained from apple leaves and determined by using DPPH, ABTS, and FRAP assays. Values are means and errors bars indicate standard deviations (
It has been previously reported that antioxidant capacity determined by
The analysis of the antioxidant activity in apple leaves revealed differences in the antioxidant activity among the ethanol extracts of the apple leaves of the cultivars investigated (Figure
In order to determine the relationship between the antioxidant activities of the ethanol extracts of apple leaves assessed by the DPPH, ABTS, and FRAP assays and total phenolic as well as flavonoid contents in these extracts, the correlation analysis was carried out. There was a strong positive correlation between total phenolic as well as flavonoid contents and the antioxidant activity assessed by all the methods (
The phenolic compounds of various groups, (+)-catechin, chlorogenic acid, caffeic acid, (–)-epicatechin, rutin, hyperoside, isoquercitrin, avicularin, quercitrin, phloretin, and phloridzin, in the analyzed ethanol extracts obtained from the apple leaves of the cultivars Aldas, Auksis, Ligol, and Lodel were identified by the HPLC method. The values of resolution (Rs > 2) were achieved in all the samples of extracts. The applied HPLC method allowed for an effective separation of quercetin glycosides: the separation of rutin and hyperoside in ethanol extracts of lyophilized apple leaf samples with the resolution of 2.63; the separation of hyperoside and isoquercitrin with the resolution of 3.28; and the separation of avicularin and quercitrin with the resolution of 2.94. The chromatograms of ethanol extracts of apple leaf samples obtained from all the cultivars investigated are identical regarding the number of analytes and retention time. The example of chromatogram of the ethanol extract of the apple leaf sample (cv. Aldas) is shown in Figure
Chromatogram of ethanol extract of apple leaf sample (cv. Aldas) investigated (
The apple leaves of the cv. Aldas contained the highest total amount of the quercetin glycosides identified and quantified. It was 1.7 times higher than their lowest amount found in the apple leaves of the cv. Ligol (Table
Content of quercetin glycosides in ethanol extracts obtained from the apple leaves of cultivars grown in Lithuania.
Compound | Content of quercetin glycosides, mg/g (expressed for absolute dry weight)a | |||
---|---|---|---|---|
Aldas | Auksis | Ligol | Lodel | |
Hyperoside | 8.95 ± 0.35a | 5.67 ± 0.20b | 4.59 ± 0.17c | 7.03 ± 0.20d |
Isoquercitrin | 3.48 ± 0.12a | 2.79 ± 0.11b | 1.84 ± 0.07c | 2.40 ± 0.08d |
Rutin | 0.33 ± 0.01a | 0.75 ± 0.03b | 0.67 ± 0.02c | 0.54 ± 0.02d |
Avicularin | 2.48 ± 0.10a | 2.82 ± 0.10b | 2.09 ± 0.07c | 2.51 ± 0.08d |
Quercitrin | 13.36 ± 0.51a | 10.29 ± 0.48b | 7.77 ± 0.27c | 12.31 ± 0.59d |
Rutin was the minor component among all the quercetin glycosides quantified in all ethanol extracts of the apple leaf samples analyzed. The results obtained are confirmed by previously published data on the variation in the composition and content of quercetin glycosides in apple leaves [
When comparing quercetin glycoside content and composition variation in apple fruits and leaves grown in Lithuanian climatic conditions, it was found that apple leaves have higher amounts of these compounds. For example, cv. Aldas, cv. Auksis, and cv. Ligol apple fruits had hyperoside amounts from 0.05 mg/g (cv. Auksis) to 0.19 mg/g (cv. Aldas) [
All the quercetin glycosides identified and quantified in the ethanol extracts of apple leaf samples can be ranked in the following ascending order by their content: rutin < isoquercitrin< avicularin < hyperoside < quercitrin. This order was characteristic of the apple leaves of the cultivars Auksis, Ligol, and Lodel. In the apple leaves of the cv. Aldas, the amount of isoquercitrin was higher than that of avicularin.
Phloridzin and phloretin, which belong to the dihydrochalcone class, were identified in the ethanol extracts of apple leaf samples. Phloridzin was the major phenolic compound in the apple leaves. It accounted for 76.9% to 84.2% of all phenolic compounds identified and quantified in the extracts of apple leaves by the HPLC method. The results obtained confirm literature data that phloridzin is a predominant component of phenolic compounds in apple leaves [
Phloridzin has a wide spectrum of biological effects, inhibits the growth of cancer cells [
The apple leaves of the cv. Ligol contained the highest concentration of phloridzin. The identified amounts of phloretin were lower than those of phloridzin (Table
Content of dihydrochalcones, phenolic acids, and catechins in ethanol extracts obtained from the apple leaves of cultivars grown in Lithuania.
Compound | Content of dihydrochalcones, phenolic acids, and catechins, mg/g (expressed for absolute dry weight)a | |||
---|---|---|---|---|
Aldas | Auksis | Ligol | Lodel | |
Phloridzin | 106.01 ± 4.23a | 108.9 ± 4.32b | 114.43 ± 4.72c | 109.51 ± 4.62d |
Phloretin | 1.81 ± 0.07a | 1.52 ± 0.06b | 2.40 ± 0.09c | 1.40 ± 0.06d |
Chlorogenic acid | 0.48 ± 0.02a | 1.38 ± 0.06b | 1.12 ± 0.05c | 0.86 ± 0.03d |
Caffeic acid | 0.26 ± 0.02a | 0.23 ± 0.03b | 0.15 ± 0.01c | 0.14 ± 0.01d |
(+)-Catechin | 0.05 ± 0.01a | 0.27 ± 0.02b | 0.09 ± 0.01c | 0.17 ± 0.01d |
(−)-Epicatechin | 0.72 ± 0.02a | 0.38 ± 0.02b | 0.76 ± 0.02c | 0.39 ± 0.01d |
The highest total amount of phenolic acids identified and quantified by the HPLC method (1.61 ± 0.07 mg/g) was found in the apple leaves of the cv. Auksis (Table
The chemical composition of catechins (monomeric flavan-3-ols) in ethanol extracts obtained from the apple leaves of the cultivars Aldas, Auksis, Ligol, and Lodel was studied. The highest and lowest total amounts of the catechins identified were found in the apple leaves of the cultivars Ligol and Lodel, respectively (0.85 ± 0.04 mg/g versus 0.56 ± 0.02 mg/g) (Table
The HPLC analysis of ethanol extracts obtained from apple leaves revealed that phloridzin was the major compound in the samples investigated, and its amounts were considerably higher than those of other phenolic compounds. Quercitrin was a predominant component among quercetin glycosides. The results of the HPLC analysis show that apple leaves are a valuable, natural source of dihydrochalcones and quercetin glycosides. This encourages further research on this plant as a raw material for use in pharmacy.
In conclusion, the results of this study will provide new knowledge about the composition and content of phenolic compounds in apple leaves and the antioxidant activity of their extracts, which will give a wide range of possibilities to employ these plants as the source of phenolic compounds. The highest total amounts of phenolic compounds and flavonoids were determined in the apple leaves of the cv. Aldas (163.35 ± 4.36 mg GAE/g DW and 45.02 ± 0.90 mg RE/g DW, resp.). Phloridzin was the major compound in the ethanol extracts of apple leaves of all the cultivars investigated. The apple leaves of the cv. Ligol had the highest amount of phloridzin (114.43 ± 4.72 mg/g DW). Quercitrin was the predominant component among the quercetin glycosides identified and quantified in ethanol extracts, and its amount in the apple leaves of different cultivars ranged from 7.77 to 13.36 mg/g DW.
The preliminary
The results reported in this study prompt further research on the chemical composition and biological effect of apple leaves by evaluating the antioxidant activity of individual phenolic compounds
A single factor analysis of variance
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
Cultivar
2,2-diphenyl-1-picrylhydrazyl
Dry weight
Ferric reducing antioxidant power
Gallic acid equivalent
High-performance liquid chromatography
Resolution
Trolox equivalent
Total amount of flavonoids
Total amount of phenolic compounds
2,4,6-tripyridyl-s-triazine
Vitamin C equivalent.
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by the Foundation of the Lithuanian University of Health Sciences and a Grant from the Research Council of Lithuania (no. SVE-02/2011).