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The effect of solvents on recovery of polyphenols from the pink Fuji apple skin.


Phenolic acids and flavonoids are secondary metabolites found in most of the plants. These compounds protect plants against ultraviolet radiation, pathogens, and herbivores [1]. The daily intake of flavonoids by humans can range from 50 mg to 1 g, depending on the geographic regions and consumption of fruits and vegetables and their products [2]. Most of the protective effects of phenolics in biological systems are ascribed to their antioxidant abilities, capacity to transfer electrons, quenching of free radicals and chelating abilities [3], activate of [antioxidant enzymes, reduce alpha-tocopherol radicals and inhibit oxidases [4]]. There has been considerable interest in finding the naturally occurring antioxidants to replace synthetic food antioxidants such as butylated hydroxyl toluene. Several studies have been carried out to analyze the antioxidant potential of a variety of herbs [5, 6]. Among the different parts of plants studied, the leaves are reported to have highest antioxidant properties [7, 8, 9] and the most active principle among the phytochemicals is the phenolic fraction [10,11]. The phenolics have in vivo antioxidant activities and have been used as natural antioxidants in food [12,13].

Apple is one of the attractive and delicious fruits towards the consumers due to its appearance and attributes of firmness, taste, and health benefits. Flavonoids, contribute to the colour of the fruits and are widely believed to possess antioxidative, antimicrobial, antimutagenic and anticarcinogenic properties [14, 15, 16]. Epidemiological studies have shown an inverse relationship between the intake of fruits, vegetables and beverages rich in flavonoids and the incidence of coronary heart disease, but the relationship with cancer is not clear [17]. Apple is one of the main sources of flavonoid intake in the European diet, after onion and tea [15]. Flavonols such as quercetin 3-O-glycosides, monomeric and oligomeric flavan-3-ols such as catechin, epicatechin and procyanidins and anthocyanins such as cyanidin 3-O-glycosides are the major flavonoid presence in apple. These fruits also contain considerable amounts of hydroxycinnamic acid derivatives, which are mainly represented by chlorogenic acid [18,19]. Flavonoids and chlorogenic acid contribute to the quality aspects of apples and the red colour of the fruits is primarily due to the flavonoids cyanidin-3-O -galactoside located in the vacuoles of epidermal cells of peel [20,21], and the browning occurring in processed apple such as juices and ciders is mainly due to oxidation of chlorogenic acid by oxidative enzymes [15]. The biosynthesis of flavonoids in apple, as in other plant tissues, includes precursors from both the shikimate and the acetate-malonate pathways via several enzymatic steps [22, 23]. Flavonoids are generally present in plant tissues as glycosides. In apple, the predominant sugar involved in glycosylation is galactose. Other sugars involved are glucose, rhamnose, xylose, arabinose and the disaccharide rutinose. Unlike other flavonoids, flavan-3-ols are generally found in the free rather than in the glycosylated forms. The different flavonoid classes are predominantly located in the skin [24, 25]. Thus, it is concluded that culture and growing conditions have limited effects on the polyphenol profiles of the cortex and peel of apple fruits but did not discuss effects on actual concentrations [25]. There is considerable qualitative information on the development and environmental regulation of anthocyanin biosynthesis in apples [26, 27], but quantitative information on the amplitude of variation and hence the potential for control is almost lacking. For other flavonoids even the variation in content due to parietal and environmental factors is poorly studied.

The main objective of the present study was to observe the influence of different solvent extraction systems on three main polymethoxylated flavones in the skin of apple fruit, such as sinensetin (SEN), rutin (RU), 3'-hydroxy-5, 6, 7, 4'-tetramethoxyflavone (TMF) and the major phenolic acid, rosmarinic acid (RA).


Chemicals and reagents

Standard samples of SEN, RU, TMF and RA were purchased from Indofine Chemical Co. (Hillsborough, NJ, USA). Solvents used for chromatographic separation were methanol, acetone, chloroform (GC grade) and water (GC grade) obtained from Merck (Darmstadt, Germany). Membrane filter (0.45-um pore size) from Millipore was used for filtration of the mobile phase and the samples. The other chemicals used have analytical or GC grade.

Sample collection

Different types of apple are available in Bangladesh markets all round the year. Fuji (Malus domestica Borkh) is one of the most popular apple cultivars grown in India. In this study, this variety was used. Apples were collected from different local markets of Bangladesh during the period of November 2008. The skin of this variety is initially yellowish green, but turns red when exposed to the sun's radiation. Ripe apples without visual symptoms of damage are usually harvested from both the outer and inner part of the tree canopy, resulting in apples with various levels of flavonoids in the peel. The colour of the apple ranged from pale green to dark red. After harvesting, the fruits were stored at 2 [degrees]C.

Extraction of Polyphenol

The fruits were cut out as a plug of skin with a cork borer (17mm diameter) followed by frozen in liquid nitrogen. Measured amount of skin (200 mg with the thickness of 1mm) was separated from the flesh under frozen condition, cut into six pieces with a scalpel and rapidly transferred to a tube containing 1mL of 0.01M HCl in methanol. The sample was sonicated for 30 min in an ultrasonic bath (CTU150, Coax Teknik, Lynge, Denmark) followed by 30 min shaking (1400 rpm) in a CM-9 Mixer (SARSTEDT, Numbrecht, Germany). The extract was transferred to another tube before re-extraction of the fruit materials with the same procedure as described above. The combined extract (2 mL) was filtered through a 0.45 um Millex HA filter (Millipore, Molsheim, France) prior to GC-MS analysis. The entire procedure was carried out at 4[degrees]C and shaded from incident light.

Preparation of samples for GC-MS analyses

One gram of the powder material of fruit peel was extracted successively with water, methanol, acetone, chloroform, 50% aqueous methanol and 70% aqueous acetone for 2, 4, 6, and 8 hours, respectively, at 40 [degrees]C in a water bath with continuous shaking. The extracts were subjected to qualitative and quantitative GC-MS analysis. The extracts were completely evaporated by K-D evaporator. Each of the extracts was diluted with methanol followed by filtration through 0.45 um membrane filters (Molsheim, France) prior to GC-MS analysis.

Identification and quantification of the samples by GC-MS

The GC-MS analysis of the crude methanol extract of the samples was performed using a Varian GC-MS (Model Varian CP 3800, USA) equipped with a VF-5 fused silica capillary column (30m x 0.25 i. d., film thickness 0.25 m, Varian, USA). An electron ionization system with the ionization energy of 70 eV was used for the detection o f GC-MS. Helium was used as carrier gas with constant flow rate of 1 ml/min. Injector and mass transfer line temperature were set at 250 and 300 [degrees]C, respectively, whereas the oven temperature was programmed from 50 to 200 at 8 [degrees]C/min, followed by isothermal elution for 20 min and finally raised to 300 [degrees]C at 10 [degrees]C/min. Dilute samples (1/100, v/v, in methanol) of 0.21 were injected manually in the split less mode. Identification of the crude methanol extract was based on GC retention time on VF-5 capillary column and computerized matching of mass spectra with standards (Mainlab, Replib and Tutorial data of GC-MS systems). The following reference compounds were used as markers: [Sinensetin (SEN), Rutin (RU), Tetramethoxyfavone (TMF) and Rosmarinic acid (RA)]. The markers were accurately weighed and dissolved in methanol to produce a series of concentrations. Standard calibration curves were established by plotting the peak areas against different concentrations of the reference compounds (varying from 5.0 to 1000 ng on column for SEN, RA and TMF and 10 to 1000 ng for RU). The external standard method was used for quantification of the markers in the apple extract.

The system suitability of the method was evaluated by the intra- and inter-day precision and accuracy of replicates. The accuracy was evaluated through recovery studies by adding known amounts of the standard solution to the extract. Controls from al samples were prepared and analyzed. The recovery experiment was performed at three different standard concentrations.

Statistical analyses

The experimental results were expresed as the means of triplicate measurements. Correlations were obtained by Pearson correlation coefficient in bivariate correlations. Means were compared by Tukey-HSD and L SD (l east significant differences).


The fruits that were selected for this investigation were similar in sizes and al the results calculated based on dry weight basis.

Concentrations of the markers in methanol extracts of apple peel

The GC-MS method applied was a modification o f what was reported [21] for the analysis of flavones present in apples. In the present study, a programmed method was used for simultaneous assay of the authentic markers in which the chemical structures were shown in Figure 1. All standards were determined in a single GC-MS run. The standards were resolved and eluted at 5 .35, 1 3.08, 1 7.52 and 25.35 min, with respect to RA, TMF, SEN and RU (Figure 2). The markers (5, 50, 500, 750 and 1000 ng on column for SEN, RA and TMF and 10, 100, 500, 750 and 1000 ng for RU) showed a good linearity in the range from 5.0 to 1000 ng in the calibration curves that were obtained by GC-MS analysis. Al reference markers were present in the chromatographic profiles of the samples from various locations when the sample solution was analyzed by GC-MS (Figure 3). The peaks of RA, TMS, SEN and RU were confirmed by comparison of their retention times with reference standards (Indofine Chemical C o., Hillsborough, NJ USA)




To asses the precision of these methods, standard solutions of RA, TMS, RU and SEN were determined six times on the same d ay and over a six-day period. The results showed a good precision, ranging from 5 to 100 [pounds sterling]g/ml (Table 1). The accuracy of the method was evaluated through recovery studies. The recovery experiments were performed at three concentrations (5, 50 and 100 ng) of the standard added to sample solutions, in which the marker content had been determined, using a sample from Dhaka. The results for the recoveries of RA, RU, TMS and SEN were in the range of 96[??] 103%. The limit of detection (LOD) of the GC-MS method, established at signals three times that of the noise for SEN, RU, TMF and RA, was 2.0, 2.5, 2.0 and 2.0 ng, respectively.

The GC-MS procedure was applied to determine the markers presence in the pee Significant amounts of the markers (with a wide range) (Table 2) were present in the samples. The variation may be ascribed to environmental conditions and variation in sample sourcing. Although the fresh and similar size of fruits were collected for the present investigation, the chemical composition of the peel could be affected by the age and soil fertility levels [15, 26]. The values obtained for the markers appear to fall within the range reported for the markers in peel [29, 30, 31]. However, the overall levels of the marker concentration were considerably higher in sample. RA was the main component found in peel where the concentrations ranging from 0.072 to 1.6530%o of total dry skin weight. Concentrations of TMF, SEN and RU ranged from 0.002 to 0.29%%, 0.433 to 0.029%% and 0.010 to 0.146%%, respectively (Table 2).

Flavonoids and phenolic acids have been implicated as natural antioxidants in plants, fruits and vegetables. Lipophilic flavones and caffeic acid derivatives were identified in the skin of apple and quantified by GC-MS. Caffeic acid derivatives, rosmarinic acid and lipophilic flavones were predominant phenolics in the methanol extract (Table 2) and their antioxidant properties have been well documented [6,20].

The GC-MS procedure was applied to identify as well as quantify the presence of the markers in apple peel that was extract with different solvents. The quantitative analysis was performed using external standard technique. As shown in Table 2, al the various solvent systems showed a wide range in the concentration of the markers. The amount of rosmarinic acid, the principal polyphenol of peel and the most polar compound among the markers was high in aqueous 70% acetone extract and these amount was significantly different form other solvent systems those were extracted for 2, 4 and 6 hours, respectively, (Table 2). Presence of IRA, in different solvent systems were found in the increasing order of aqueous 70% acetone > aqueous 5 0% methanol > 100%% methanol > water > 1 00% acetone >chloroform.


It was observed that the chloroform extracts contained the highest amount of RU and SEN when the peel was extracted 4 and 6 hours, respectively. There was a significant difference of the amount of SEN and RU in chloroform extracts compared to water, methanol, aqueous 50% methanol, aqueous 70% acetone and acetone used as solvents. Higher concentration of TMF was observed 4 to 6 hours extraction with acetone as well a s aqueous 7 0% acetone but the amount was not significantly different as compared to water and methanol extracts. Polymethoxylated flavones, the principal flavonoid aglycones, present in apple peel are exclusive with a methoxy group at C-5 a structural feature rare in flavonoids. The polarity of the basic ring structure of the polymethoxylated flavones used as markers is quite similar, although the addition of hydroxyl group(s) to ring B in TMF and rings A and B in RU may result in substantial increase in the polarity of the molecules (Fig. 1). The presence of the methoxy groups, five in SEN and four in TMF, renders them lipophilic hence more soluble in lipohilic solvents. Among the various solvents used in the present study for the extraction, chloroform has the lowest polarity, so the proportion of lipopholic markers should be high but the polar marker, RA, was not detected in the chloroform extracts by the study.


It was concluded that 4 to 6 hours of extraction using chloroform efflux of dehydrated apple skin provided complete recovery of SEN and RU. Similarly, 4 to 6 hours extraction with acetone and aqueous 70% acetone under reflux was optimum for recovery of TMF. The recovery of RA was high when apple skin was treated with water for 8 hours at 40[degrees]C. The optimum extraction conditions with different solvent followed by GC-MS method developed in the present study can be used to analyze the polymethoxylated flavones and phenolic acids in apple skin as well as other botanical resources.


The authors are grateful to Sohela Akhtar, Head, Chemistry Division, Atomic Energy Centre, Dhaka, Bangladesh for her constant encouragement and helpful suggestions. They are also grateful to Mr. Zahidul I slam and Mr. Abbas Ali for their help to preparation the apple samples. The study was supported by a University Grant Commision, Dhaka, Bangladesh.




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Amzad H.M.* (1), Salehuddin S.M. (2), Mizanur R.S.M. (3) and M.J. Kabir (2)

Amzad Hossain * Corresponding author e-mail: and/or

(1) Biotechnology Research Institute, University Sabah Malaysia, Locked Bag No. 2073, 88999 Kotakinabalu, Sabah, Malaysia

(2) Chemistry Division, Atomic Energy Centre, GPO Box 164, Ramna, Dhaka-1000, Bangladesh

(3) Department of Chemistry, University of Dhaka, Dhaka-1000, Bangladesh
Table 1: Analytical characteristics of calibration curves
for the marker phenolics

                       Ret.         Range          Regression
Constituent            Time (min)   (ng/ml)        Equation

Sinensetin (SEN)       17.52        2-1000         0.1917x-0.3250

Rutin                  25.35        2.5-1000       0.0698x-0.0243

tetramethoxyflavone    13.08        2 [??] 1 000   0.1950x+0.3032

Rosmarinic acid (RA)   5.35         2-1000         0.2619x+0.214

                                                (%) (SDa)
                                                (n=5, 10ng/ml)

Constituent            Limit (mg)   [R.sup.2]   Interday   Intraday

Sinensetin (SEN)       2.0          0.9997      0.78       0.37

Rutin                  2.0          0.9994      0.83       0.42

tetramethoxyflavone    2.5          0.9998      1.52       1.21

Rosmarinic acid (RA)   2.0          0.9995      0.91       0.83

(a) SD = standard deviation.

Table 2: Isolation of polyphenol with different solvent systems (a)

Percent concentrations of marker polyphenol in the pink skin apple
fruit collected from different markets of Bangladesh between 1th
November to 30th November 2008a

         Time                          50%
Marker   (hrs)   Water      Methanol   Methanol

RA       2       0.540 c    0.541 c    1.030 b
         4       0.661 c    0.648 c    0.978 b
         6       0.643 c    0.678 c    0.938 b
         8       0.799 b    0.871 b    0.868 b
TMF      2       0.006 ab   0.017 a    0.003 b
         4       0.010 a    0.010 a    0.003 b
         6       0.009 ab   0.009 a    0.003 b
         8       0.009 a    0.008a     0.002 b
SEN      2       0.030 c    0.067 b    0.029 c
         4       0.033 c    0.080 b    0.030 c
         6       0.037 c    0.074 b    0.031 c
         8       0.030 c    0.079 b    0.031 c
RU       2       0.027 b    0.099 b    0.030 c
         4       0.016 b    0.113 b    0.021 c
         6       0.018 a    0.086 b    0.015 c
         8       0.013 a    0.114 a    0.010 c

         Time              70%
Marker   (hrs)   Acetone   Acetone   Chloroform

RA       2       0.035 d   1.325 a   ND
         4       0.048 d   1.503 a   ND
         6       0.106 d   1.653 a   ND
         8       0.072 c   1.520 a   ND
TMF      2       0.011 a   0.010 a   0.005 ab
         4       0.011 a   0.011 a   0.006 ab
         6       0.011 a   0.012a    0.005 ab
         8       0.011 a   0.010 a   0.004 b
SEN      2       0.098 b   0.087 b   0.365 a
         4       0.097 b   0.090 b   0.433 a
         6       0.093 b   0.097 b   0.407 a
         8       0.110 b   0.097 b   0.344 a
RU       2       0.088 b   0.098 b   0.124 a
         4       0.094 b   0.081 b   0.146 a
         6       0.126 a   0.068 b   0.140 a
         8       0.146 a   0.129 a   0.120 a

(a) Mean within column with different letters indicate significantly
different values (P < 0 .05). This experiments were performed in
November 2008.
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Article Details
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Author:Amzad, H.M.; Salehuddin, S.M.; Mizanur, R.S.M.; Kabir, M.J.
Publication:African Journal of Food, Agriculture, Nutrition and Development
Article Type:Report
Geographic Code:9BANG
Date:May 1, 2010
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