Polyphenol Content in Figs (Ficus carica L.): Effect of Sun-Drying

Abstract

In order to investigate the effect of sun-drying on the health-related constituents as well as bioaccessibility of figs; total phenolics, flavonoids, proanthocyanidins, anthocyanins, antioxidant capacity, and major phenolic compounds were determined for two commercial figs (Sarilop and Bursa siyahi) with different color (yellow and purple). In addition, release of phytochemical was studied by simulating of in vitro gastrointestinal digestion. In both varieties, rutin and cyanidin-3-rutinoside were confirmed as the major flavonol and anthocyanin, respectively. For both varieties, analyses of total phenolics, anthocyanins and antioxidant activity revealed lower levels after sun-drying. On the other hand, 75 and 71% higher total flavonoid and proanthocyanidin contents were observed for yellow figs.

Keywords:

• Fig
• Ficus carica L.
• Drying
• Polyphenols
• Antioxidant
• Bioaccessibility

INTRODUCTION

The common fig (Ficus carica L.), a deciduous tree belonging to the Moraceae family, is one of the earliest cultivated fruit trees and an important crop worldwide for both fresh and dry consumption.^[1] Figs are source of vitamins, minerals, dietary fibers, and amino acids.^[2] Furthermore, they are also one of the most abundant fruits in the Mediterranean diet, which has been reported to promote health and quality of life.^[3] Fig skin color varies from green to dark purple. Figs can be consumed as a whole fruit, but they are often peeled; the pulp is consumed and the skin is discarded.^[1] Fresh fruits naturally have a short post-harvest life of seven to ten days; however, with a combination of a cooler and a CO₂-enriched atmosphere, fruit can be preserved for up to two to four weeks. Figs are also very popular as dried fruit, since drying increases their shelf life.^[2] Dried figs can be stored for six to eight months; therefore drying can ensure proper preservation of figs.^[4] Drying of exotic tropical fruits such as durian, guava, jackfruit, mango, and figs have been reported to preserve the overall nutritional qualities and retain the concentration of bioactive compounds.^[5]

Phenolic compounds are common plant secondary metabolites which not only play physiological roles in plants but also have favorable effects for human health, because they can act as antioxidants by donating a hydrogen atom or an electron to other compounds, scavenging free radicals, and quenching singlet oxygen.^[6] Figs are sources of phenolic compounds. Indeed, red wine and tea, two well-publicized sources of phenolic compounds contain lower levels of phenolics than figs.^[7] The phenolic content of fig is usually influenced not only by the cultivar, but also varies significantly from one fruit part to another.^[2]

In recent years, several data have been generated on the polyphenol constituents in a variety of food materials, including figs. Phenolics, flavonoids, anthocyanins, and related total antioxidant activities based on chemical extraction have typically been measured using methanol or methanol/water mixtures.^[1–^3,8,9] The nature of extractable phytochemicals, their stability, and their antioxidant activity depend on many factors; including the food matrix, pH, temperature, presence of inhibitors or enhancers of absorption, presence of enzymes, and other related factors.^[10] Nevertheless, to obtain any influence in a specific tissue or organ these bioactive compounds must be bioavailable, i.e., effectively absorbed from the gut into the circulation and transferred to the appropriate location within the body while still maintaining their bioactivity.^[11] It has been proven that the measurement of bioaccessibility by in vitro models can be well correlated with conclusions from human studies and animal models.^[10]

To the best of the authors knowledge, there are only a few studies investigating the effect of sun-drying on polyphenols of figs. However, no previous study evaluated the effect of sun-drying on polyphenols for the above mentioned varieties (Sarilop and Bursa siyahi). In addition, no studies have been reported on simulation of in vitro gastrointestinal (GI) digestion of fig fruit. Given the above, the aim of this study was: (a) to examine the influence of sun-drying on total phenolics, flavonoids, proanthocyanidins, anthocyanins, and antioxidant capacity as well as major phenolic compounds of Sarilop and Bursa siyahi fig varieties, and (b) to evaluate the total phenolics, flavonoids, anthocyanins, and antioxidant activity of these figs at different phases of simulated GI digestion, using an in vitro model.

MATERIALS AND METHODS

Plant Material

Two different varieties (Sarilop; yellow and Bursa siyahi; purple) of fresh fig fruits were collected from orchards in the Aydin and Bursa provinces, respectively. The fig fruits were harvested at the optimal ripening time in September 2011. For each variety, three repetitions were carried out with 50 to 60 fruits per repetition. One quarter of each repetition were used for drying. For the drying procedure, fruits were uniformly distributed on sample trays in a single layer to be exposed to the sunlight at a height of 1 m above the ground and were placed indoors at night. Drying took eight days and during this period, the average day temperature ranged between 31 to 34°C. The average sun-light intensity has been recorded as ˜350 cal/cm²-day.^[12] Immediately after all samples were transferred to laboratory, four fractions were prepared for each variety including skin, pulp, whole fruit, and dried fruit. All fractions were ground to a fine powder in liquid nitrogen using a pre-cooled grinder (IKA A11, Germany), and stored at –80°C before analysis. Moisture content was determined following the guidelines of the official TS 1129-ISO 1026 method.^[13] For each sample, ca 2 g was placed on pre-weighed aluminum pans and weighed. The pans were kept in vacuum oven (Gallenkamp, UK) at 70°C until the equilibrium moisture content was reached (6 h). All samples were analyzed in triplicate and average values were calculated.

Preparation of Extracts

Three independent extractions for each fraction were carried out as described previously by Capanoglu et al.^[14] with slight modifications. 2 ± 0.01 g of each sample was extracted with 5 ml of 75% aqueous-methanol containing 0.1% (v/v) formic acid in a cooled ultrasonic bath (Azakli, Turkey) for 15 min. The treated samples were centrifuged (Hettich Zentrifugen Universal 32R, UK) for 10 min at 2700 xg and the supernatant was collected. Another 5 ml 75% aqueous-methanol containing 0.1% (v/v) formic acid was added to the pellet and this extraction procedure was repeated two more times. All three supernatants were combined and adjusted to a final volume of 15 mL. Prepared extracts were stored at –20°C until analysis. Samples of each extraction were analyzed in triplicate.

Determination of total phenolic content (TP)

The TP of extracts was determined using Folin-Ciocalteu reagent according to the method modified from Velioglu et al.^[15] using gallic acid as a standard. The TP of extracts was expressed on a dry weight (DW) basis as mg of gallic acid equivalent (GAE) per 100 g DW of sample.

Determination of total flavonoid content (TF)

The TF was measured colorimetrically as described by Kim et al.^[16] The TF of extracts was determined by a (+)-catechin standard curve and expressed as mg of (+)-catechin equivalent (CE) per 100 g of DW of sample.

Determination of total proanthocyanidin content (TPA)

The TPA was determined by UV spectrophotometry method based on acid hydrolysis and color formation.^[17] The TPA of extracts was expressed as mg of cyanidin (CYD) equivalent per 100 g of DW of sample.

Determination of total anthocyanin content (TA)

The TA content was determined according to the pH differential method.^[18] The TA of extracts was expressed as mg of cyanidin-3-glycoside (C3G) equivalent per 100 g of DW of sample.

Determination of total antioxidant capacity (TAC)

The TAC was estimated by four different assays. The 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazil (DPPH), ferric reducing antioxidant power (FRAP) and cupric ion reducing antioxidant capacity (CUPRAC) assays were performed according to Miller and Rice-Evans,^[19] Karuman and Karunakaran,^[20] Benzie and Strain,^[21] and Apak et al.,^[22] respectively. In all assays, Trolox was used as a standard and results were expressed in terms of mg of Trolox equivalent (TE) per 100 g DW of sample.

HPLC Analysis of Major Phenolic Compounds

Major phenolic compounds were determined following the method of Capanoglu et al.^[14] Extracts were filtered through a 0.45-μm membrane filter and analyzed using a Waters W600 HPLC system with PDA (Waters 996) and fluorescence (Waters 2475) detectors. A Luna 3 μ C18 150 × 4.60 mm column (Phenomenex, Torrance, CA, USA) was used. The mobile phase consisted of solvent A, Milli-Q water with 0.1% (v/v) TFA and solvent B, acetonitrile with 0.1% (v/v) TFA. A linear gradient was used as follows: at 0 min, 95% solvent A and 5% solvent B; at 45 min, 65% solvent A and 35% solvent B; at 47 min, 25% solvent A and 75% solvent B; and at 54 min returning to initial conditions. The flow rate was 1 mL/min. Detection was done at 280, 312, 360, and 512 nm. Identification was based on the retention times and characteristic UV spectra and quantification was done by external standard curves. All analyses were performed in triplicate.

GI Digestion Simulation

In order to simulate the in vivo GI digestion conditions, and to determine the amount of free soluble polyphenols potentially available for further uptake, the procedure adapted from McDougall et al.^[11] was followed. To follow the release of phytochemicals from fig matrices, at different stages of digestion, aliquots from gastric and intestinal digesta were analyzed in terms of total phenolic, flavonoid, and anthocyanin contents. Antioxidant activity was also determined.

Statistical Analysis

Data were collected from three independent extractions for each fraction and reported as mean ± SD. For multiple comparisons, data were subjected to statistical analysis using SPSS software (version 16.0 for Windows, SPSS Inc.) for the analysis of variance (ANOVA). General Lineer Model (GLM) was applied to measure the differences between the two varieties. Duncan’s new multiple range test was used to analyze differences between treatments (p < 0.05). The correlation coefficients (R²) for spectrophotometric assays were calculated using the Microsoft Office Excel 2007 software (Microsoft Corporation, Red-mond, WA).

RESULTS

Moisture Content

The moisture content of the samples is reported in Table 1. The moisture content from fresh to dry fruit was decreased by 76% for yellow and 39% for purple figs, respectively. After drying, purple figs had higher moisture content compared to yellow figs. Purple fig skins were thicker compared to yellow fig skins, which may have prevented water loss.

TABLE 1 Moisture content and total phenolic, flavonoid, proanthocyanidin, anthocyanin, antioxidant capacity values of fig skin, fig pulp, whole and dried fruits^a

	Yellow figs				Purple figs
Analyses	Fruit skin	Fruit pulp	Whole fruit	Dried fruit	Fruit skin	Fruit pulp	Whole fruit	Dried fruit
Moisture (%)	74.2 ± 0.6 a	74 ± 2 a	72.6 ± 0.6 a	17.3 ± 0.2 b	78.8 ± 0.9 b	81.3 ± 0.5 a	81 ± 1 a	50 ± 2 c
TP^b (mg of GAE/100 g)	351 ± 36 a	167 ± 16 b	211 ± 38 b	193 ± 16 b	930 ± 91 a	351 ± 86 c	493 ± 59 b	417 ± 33 bc
TF^c (mg of CE/100 g)	63 ± 3 a	7 ± 2 c	8 ± 3 c	14 ± 3 b	234 ± 20 a	44 ± 13 b	66 ± 5 b	52 ± 4 b
TPA^d (mg of CYD/100 g)	6.6 ± 0.6 b	7.2 ± 0.8 b	7 ± 1 b	12.0 ± 0.5 a	220 ± 43 a	34 ± 2 bc	62 ± 13 b	16 ± 5 c
TA^e (mg of C3G/100 g)	0.8 ± 0.1 c	5.9 ± 0.5 a	4.6 ± 0.3 b	0.1 ± 0.0 d	196 ± 7 a	38 ± 1 c	83 ± 9 b	14.5 ± 0.3 d
ABTS (mg of TE100 g)	446 ± 37 a	226 ± 13 b	255 ± 11 b	222 ± 12 b	2169 ± 181 a	780 ± 194 bc	1066 ± 97 b	739 ± 62 c
DPPH (mg of TE/100 g)	148 ± 11 a	86 ± 7 c	110 ± 8 b	47 ± 2 d	627 ± 57 a	177 ± 32 b	241 ± 14 b	104 ± 14 c
FRAP (mg of TE/100 g)	167 ± 21 a	89 ± 5 b	96 ± 12 b	39 ± 3 c	986 ± 147 a	203 ± 52 bc	273 ± 25 b	140 ± 39 c
CUPRAC (mg of TE/100 g)	637 ± 91 a	202 ± 27 b	278 ± 27 b	202 ± 22 b	2616 ± 304 a	666 ± 206 b	1025 ± 115 b	776 ± 70 b

^a Data represent average values ± standard deviation of three independent samples;

All contents are expressed per 100 g DW;

Different letters in the rows within each cultivar represent statistically significant differences (p < 0.05);

^b TP: Total phenolic content, ^c TF: Total flavonoid content, ^d TPA: Total proanthocyanidin content, ^e TA: Total anthocyanin content.

TP

TP of all fractions is expressed on a DW basis in Table 1. Results showed that purple figs were richer in phenolic material compared to yellow figs. Similarly, skin had higher phenolic content than pulp. In fact, the highest TP content among all fractions was observed in purple fig skin. In addition, after drying TP was decreased by 8 and 15% in yellow and purple figs, respectively. There was no significant difference between the fresh and dried fruits of both yellow and purple figs (p < 0.05). Caliskan and Polat^[3] and Bucic-Kojic et al.^[17] measured similar TP values in figs compared to our purple fig results. Lower results of TP (56.0 to 74.9 mg GAE/100 g fresh weight [FW]) have been reported in fresh fig fruits by Solomon et al.,^[1] Pande and Akoh,^[23] and Slatnar et al.^[4] “Cuello Dama,” the only fig fruit variety analyzed in both dry and fresh conditions by Vallejo et al.,^[7] resulted in a similar loss (15%) in purple figs as a result of drying. In comparison to pears in which a decrease by 94% in TP was observed after sun-drying,^[24] this analysis of figs showed a higher recovery of phenolics. On the other hand, Al-Farsi et al.^[25] reported that the levels of TP were higher in dried dates compared to fresh ones.

TF

TF of two fig varieties was measured colorimetrically (Table 1) and found to be significantly higher in purple figs compared to yellow figs, with most flavonoids being located in the fruit skin. Also, the flavonoid content in the pulp of purple figs was significantly higher than that of yellow figs. The decrease in TF as a result of drying was found to be 21% in purple figs while an increase of 75% was observed in yellow figs after drying. For purple figs, there was no significant difference between the TF of fresh and dried fruits. However, the TF of dried fruits of yellow figs was significantly higher than for fresh fruits (p < 0.05). In this study, higher TFs were found compared to those reported by Bucic-Kojic et al.^[17] On the other hand, Marinova et al.^[26] determined higher TF results (20.2 mg CE/100 g FW) for fresh figs. These differences might be the result of agroecological differences as well as the difference in extraction method used.

TPA

The pattern of variation in TPA (Table 1) was generally similar to that observed in TF, with a maximum content in purple fig skin. Moreover, purple figs had a ten times higher TPA when compared to yellow figs. Also, the proanthocyanidins in purple fig pulp were higher than for yellow fig pulp. Furthermore, drying resulted in a significant decrease of 74% in TPA in purple figs whereas a statistically significant increase of 71% was observed for yellow figs (p < 0.05).

TA

Of two varities, purple figs showed the highest anthocyanin content, with most of the anthocyanins accumulated in the fruit skin (Table 1). In pulp, TA was significantly higher in purple figs compared to yellow figs. The highest loss as a result of drying was observed in TA (83% in purple figs, 98% in yellow figs) which was statistically significant for both varieties (p < 0.05). Results for purple figs were found to be ten-fold higher than those reported by Solomon et al.^[1] who examined the same fig variety. After the drying of strawberry, apple, and peach, higher amounts of total anthocyanins were reported by Rababah et al.^[27] In contrast, these results indicated that drying has a negative effect on the anthocyanin content in figs.

TAC

TAC was measured using four different methods (ABTS, DPPH, FRAP, CUPRAC), which show different trends (Table 1). In agreement with the above results, TAC was found to be higher in purple. For both varieties, skin was the major contributing tissue to the TAC, having ˜2–5 fold higher capacity than pulp. According to all four analyses carried out, after drying TAC was decreased by 24–57 and 13–57% in purple and yellow variety, respectively. On the basis of the CUPRAC method, for both varieties TAC was not significantly different in dried fruit compared to fresh fruit. In contrast, according to the DPPH and FRAP methods, TAC decreased significantly after drying. As judged by the ABTS method, the TAC value did not change significantly for yellow figs, while it decreased significantly for purple figs as a result of drying (p < 0.05). The reduction in TAC after drying has also been reported for other fruits. Sun-drying caused a significant loss (ranging from 30 to 43%) of antioxidant activity in date varieties.^[25] In contrast, in some studies either an increase or no change in TAC was observed after drying. Piga et al.^[28] reported an increase in antioxidant activity in plums after drying.

Correlation between Spectrophotometric Assays

TP and TF of fig fruits showed a linear relationship with a high correlation coefficient of R² = 0.943 (Table 2). Among all four TAC assays, the highest correlation was demonstrated between TP and CUPRAC (R² = 0.969), followed by TF and CUPRAC (R² = 0.967), TPA and FRAP (R² = 0.965), and TA and DPPH (R² = 0.936). These results imply that flavonoids and other phenolics were the major contributors to the antioxidant capacity of the investigated fig fruits.

TABLE 2 The correlation coefficients (R²) for spectrophotometric assays

	TP^a	TF^b	TPA^c	TA^d	ABTS	DPPH	FRAP	CUPRAC
TP^a	−	0.9432	0.8603	0.8751	0.9636	0.9055	0.9060	0.9690
TF^b	0.9432	−	0.9090	0.8606	0.9147	0.9444	0.9612	0.9670
TPA^c	0.8603	0.9090	−	0.9308	0.9003	0.9551	0.9649	0.9281
TA^d	0.8751	0.8606	0.9308	−	0.9259	0.9362	0.9179	0.8977
ABTS	0.9636	0.9147	0.9003	0.9259	−	0.9244	0.9182	0.9680
DPPH	0.9055	0.9444	0.9551	0.9362	0.9244	−	0.9853	0.9501
FRAP	0.9060	0.9612	0.9649	0.9179	0.9182	0.9853	−	0.9570
CUPRAC	0.9690	0.9670	0.9281	0.8977	0.9680	0.9501	0.9570	−

^a TP: Total phenolic content, ^b TF: Total flavonoid content, ^c TPA: Total proanthocyanidin content, ^d TA: Total anthocyanin content.

Major Phenolic Compounds

The major phenolic compounds present in the fruit fractions are reported in Table 3. Up to 14 phenolic compounds were detected in analyzed fig samples, belonging to five groups of phenolics including phenolic acids, flavonols, flavons, flavan-3-ols, and anthocyanins. For yellow figs, the predominant phenolic compound was rutin (Fig. 1a) whereas cyanidin-3-rutinoside (C3R) was detected at the highest level for purple figs (Fig. 1b).

FIGURE 1 (A) HPLC chromatograms (PDA, recorded at 360 nm) of extracts of whole yellow fruit (upper panel) and dried yellow fruit (lower panel). (B) HPLC chromatograms (PDA, recorded at 512 nm) of whole purple fruit (upper panel) and dried purple fruit (lower panel). Numbers refer to the major phenolic compounds identified: (1) apigenin; (2) rutin; (3) Q3G; (4) C3G; (5) C3R.

Of the phenolic acids, chlorogenic acid, ellagic acid, gallic acid, and p-coumaric acid were determined. Results showed that purple figs were richer in phenolic acids compared to yellow figs. For both varieties, skins had higher levels of chlorogenic acid, ellagic acid, and p-coumaric acid than pulp, whereas the gallic acid content was higher in pulp compared to skin. Moreover, after drying, chlorogenic acid and p-coumaric acid contents of both varieties decreased (58 and 59% for Sarilop; 87 and 34% for Bursa siyahi, respectively). In contrast, drying caused an increase of 67% in gallic acid for both yellow and purple figs. Ellagic acid remained unchanged for yellow figs, whereas a 50% increase was observed for purple figs as a result of drying. Gallic acid and p-coumaric acid contents of fresh and dried fruits of yellow figs were found to be significantly different (p < 0.05). According to the results of Slatnar et al.,^[4] sun-drying reduced the amount of chlorogenic acid for the fig samples obtained in September, which is also valid in this case. Although, previously no researchers have reported the presence of p-coumaric acid in fig fruit, it was detected in considerable amounts in both varieties. On the other hand, according to Pande and Akoh,^[23] fig leaves do contain high amounts of p-coumaric acid (5.9 mg/100 g FW). Next to the phenolic acids determined in this study, Oliveira et al.^[6] also identified 5-O-caffeoylquinic acid and ferulic acid in figs.

TABLE 3 Major phenolic compounds of fig skin, fig pulp, whole, and dried fruits^a

	Yellow figs				Purple figs
	Fruit skin	Fruit pulp	Whole fruit	Dried fruit	Fruit skin	Fruit pulp	Whole fruit	Dried fruit
Chlorogenic acid	12 ± 2 a	0.5 ± 0.2 b	1.9 ± 0.8 b	0.8 ± 0.3 b	21 ± 7 a	6 ± 2 b	8 ± 2 b	1.1 ± 0.5 b
Ellagic acid	0.3 ± 0.1 a	0.2 ± 0.0 a	0.2 ± 0.0 a	0.2 ± 0.0 a	0.7 ± 0.1 a	0.1 ± 0.0 c	0.2 ± 0.1 bc	0.3 ± 0.1 b
Gallic acid	0.2 ± 0.0 b	0.3 ± 0.1 a	0.3 ± 0.0 a	0.1 ± 0.0 c	0.3 ± 0.0 a	0.5 ± 0.2 a	0.3 ± 0.0 a	0.5 ± 0.1 a
p-Coumaric acid	5.1 ± 0.4 a	2.2 ± 0.3 c	2.7 ± 0.1 b	1.1 ± 0.2 d	18 ± 4 a	3.2 ± 0.2 b	4.7 ± 0.8 b	3.1 ± 0.8 b
Kaempferol-rutinoside	1.4 ± 0.4 a	nd^b	0.3 ± 0.1 b	0.2 ± 0.0 b	4 ± 1 a	0.1 ± 0.1 b	0.3 ± 0.1 b	0.9 ± 0.1 b
Quercetin-3-glucoside	1.9 ± 0.5 a	0.1 ± 0.0 b	0.3 ± 0.1 b	0.2 ± 0.0 b	9.4 ± 0.9 a	0.2 ± 0.1 b	1.2 ± 0.7 b	0.7 ± 0.2 b
Quercetin derivative 1	1.7 ± 0.1 a	0.1 ± 0.0 c	0.4 ± 0.1 b	0.2 ± 0.1 bc	7 ± 1 a	0.5 ± 0.1 b	1.1 ± 0.1 b	0.2 ± 0.0 b
Quercetin derivative 2	3.7 ± 0.7 a	0.1 ± 0.1 b	0.6 ± 0.2 b	0.3 ± 0.1 b	18 ± 3 a	0.1 ± 0.0 b	2 ± 1 b	0.6 ± 0.2 b
Rutin	86 ± 21 a	2 ± 1 b	12 ± 3 b	9 ± 2 b	180 ± 34 a	0.9 ± 0.1 b	13 ± 1 b	15 ± 5 b
Apigenin	3.5 ± 0.5 a	0.2 ± 0.1 b	0.6 ± 0.1 b	0.5 ± 0.1 b	12.7 ± 0.5 a	0.3 ± 0.0 d	1.9 ± 0.1 c	3.2 ± 0.4 b
Catechin	8 ± 3 a	2.1 ± 0.5 b	2.3 ± 0.3 b	1.5 ± 0.1 b	6 ± 1 c	27 ± 2 a	13 ± 4 b	2.5 ± 0.7 c
Epicatechin	2.7 ± 0.3 a	0.9 ± 0.1 b	1.1 ± 0.0 b	0.6 ± 0.1 c	29 ± 6 a	4.8 ± 0.9 b	6.9 ± 0.7 b	2.2 ± 0.5 b
Cyanidin-3-glucoside	nd	0.1 ± 0.0 a	0.1 ± 0.0 a	nd	23 ± 6 a	2.8 ± 0.4 bc	6 ± 1 b	0.1 ± 0.0 c
Cyanidin-3-rutinoside	0.1 ± 0.0 b	2.2 ± 0.4 a	2.1 ± 0.1 a	nd	180 ± 31 a	18 ± 4 bc	36 ± 6 b	1.5 ± 0.6 c

^a Data represent average quantities ± standard deviation (determined by HPLC-PDA or HPLC-fluorescence detection) of three independent samples;

All contents are expressed per 100 g DW;

Different letters in the rows within each cultivar represent statistically significant differences (p < 0.05);

^b nd: not detected.

The following flavonol compounds were determined: kaempferol-rutinoside, quercetin-3-glucoside (Q3G), quercetin derivative 1, quercetin derivative 2 (derivatives were calculated in terms of Q3G), and rutin. In terms of flavonol content, Q3G was significantly higher in purple figs compared to yellow figs. For both varieties, rutin was the major flavonol identified, mostly located in the fruit skin. Kaempferol-rutinoside was not detected in the pulp of yellow figs, but was observed in the skin. The drying process caused a decrease in all flavonols of yellow figs (24% for rutin (Fig. 1a), 33% both for kaempferol-rutinoside and Q3G (Fig. 1a), and 50% for quercetin derivatives). For purple figs, the loss in Q3G and quercetin derivatives 1 and 2 as a result of drying was found to be 42, 82, and 71%, respectively. Drying resulted in 16 and 200% increases in rutin and kaempferol-rutinoside, respectively. Both this study and that of Slatnar et al.^[4] report an increase in rutin and Q3G as a result of sun-drying. In addition to the flavonols that we have identified in thid study, Vallejo et al.^[7] also determined quercetin-acetylglucoside in fig skins as well as in dried figs. This compound may be one of the unidentified quercetin derivatives observed in this study.

From the group of flavones only apigenin was detected. For both varieties, skin was the major contributing tissue to apigenin content. Peak 1 from Fig. 1a shows apigenin in extracts from whole and dried fruits of yellow figs. From the figure, although it seems like drying increased the apigenin content in yellow figs, it is actually reduced by 17% on DW basis but this was not statistically significant (p < 0.05). On the other hand, for purple figs, the apigenin content was increased significantly (68%) as a result of drying (p < 0.05). Very recently, the presence of flavones, namely luteolin 6C-hexose-8C-pentose and apigenin-rutinoside, was detected in fig skin.^[7] Similarly in the present study, apigenin was determined in all fruit parts including the skin, pulp, whole, and dried fruits.

Both (+)-catechin and (-)-epicatechin from the group of flavan-3-ols were detected. Results showed that purple figs contained higher amounts of flavan-3-ols compared to yellow figs. For both varieties, catechin was the major flavan-3-ol identified, mostly located in the fruit skin for yellow figs and in the pulp of purple figs. For yellow figs, drying resulted in 35 and 45% lower levels of catechin and epicatechin, while the losses for purple figs were 45 and 68%, respectively. After drying, catechin contents of purple figs and epicatechin contents of yellow figs were found to be significantly different (p < 0.05). Slatnar et al.^[4] reported an increase in both catechin and epicatechin contents in figs as a result of sun-drying. On the other hand, according to this study, flavan-3-ols resulted in lower levels after sun-drying. Previous results of a study on sun-dried pear^[24] showed that as a result of drying, both catechin and epicatechin decreased (91 to 96%), which is in agreement with these results. According to Slatnar et al.,^[4] procyanidins were better preserved by the drying process than hydroxycinnamic acids.

Cyanidin-3-glucoside (C3G) and C3R were identified from the group of anthocyanins. For purple figs, skin was the major contributing tissue to anthocyanin content compared to the pulp, having eight and ten fold higher levels of C3G and C3R, respectively. On the other hand, C3G was not detected in the skin of yellow fig as expected from its light color. Peak 4 and 5 from Fig. 1b shows C3G and C3R in extracts from whole and dried fruits of purple figs, respectively. As shown in the figure, drying caused a significant decrease for both anthocyanins (98% for C3G and 96% for C3R). According to the literature, C3R, accounting for 80% of the TA, is the main anthocyanin in figs, followed by C3G,^[9] which is in agreement with the data reported in the present study. In addition to C3R and C3G, Duenas et al.^[9] also reported the presence of several other anthocyanins in figs including cyanidin-3,5-diglucoside, pelargonidin-3-glucoside and peonidin-3-glucoside. The results indicated that drying has a negative effect on each anthocyanin, as has been reported by others.^[4,7]

In vitro GI Digestion

The impact of in vitro GI digestion on TP is shown in Fig. 2. For both fig varieties, no differences in TP were evident when comparing levels before and after pepsin digestion. When the amount after intestinal digestion was analyzed, 22 and 32% of the compounds were present in the dialyzed fraction (IN) for yellow and purple figs, respectively (Fig. 3), whereas the rest remained in the non-dialyzed fraction (OUT). For both varieties, IN values were higher in skin fractions compared to pulp. A decrease in IN value of 28% as a result of drying was found for purple figs; while an increase of 30% was observed for yellow figs. For all fractions (skin, pulp, whole, and dried fruit) in both varieties, IN values were significantly lower compared to the initial TP values (p < 0.05).

FIGURE 2 Total phenolic content of fig skin, fig pulp, whole and dried fruits, expressed as GAE per 100 g DW. The terms represent; Initial: as initially determined from fruit matrix using 75% aqueous-methanol containing 0.1% formic acid; Post Gastric: phenolics remaining after gastric digestion; IN: dialyzed fraction after intestinal digestion; OUT: non-dialyzed fraction after intestinal digestion. Data represent average values ± standard deviation of three independent samples. Different letters for each fruit fraction represent statistically significant differences (p < 0.05).

FIGURE 3 Recovery of TP, TF, TAC, and TA in fig skin, fig pulp, whole and dried fruits after in vitro intestinal digestion, dialyzed fractions of the intestinal digestion (IN), expressed as percentage. Data represent average values ± standard deviation of three independent samples. Different letters for each fruit fraction represent statistically significant differences (p < 0.05).

During the in vitro simulation of GI digestion, for both fruit varieties, an increase in TF was observed after pepsin digestion (data not shown). The dialyzed flavonoid fraction represented 17 and 32% of the initial TF values for Sarilop and Bursa siyahi, respectively (Fig. 3). Similar to TP, IN values were higher in skin compared to pulp. For purple figs, the loss in IN value as a result of drying was found to be 67%. However, drying resulted with 181% higher TF values for the IN fraction of yellow figs.

In order to determine the effect of in vitro GI digestion on TAC, CUPRAC assay was performed, because among all four TAC assays the highest correlation was observed between either TP or TF and CUPRAC assays (Table 2). For both fruit varieties, an increase in TAC was observed after pepsin digestion (data not shown). The compounds present in the IN for yellow and purple figs was 80 and 86%, respectively (Fig. 3). Similar to TP and TF, IN values were lower in pulp compared to skin. Drying caused a decrease in IN values for both varieties (17 and 30% for yellow and purple figs, respectively).

To identify the influence of simulated in vitro GI digestion on TA, only purple fig samples were analyzed. The dialyzed anthocyanin fraction (Fig. 3) of the whole fruit represented only nine percent of the initial TA value. Similar to the results mentioned above, the IN value was higher in the skin compared to the pulp. The decrease in IN value as a result of drying was found to be 75%. During the in vitro simulation of GI digestion, an increase in TA of purple figs was observed after gastric digestion. The reason for this situation could be the effect of pH which was observed to be pH = 1.7 after the pepsin digestion, considerably lower than the pH of fresh fruit (pH = 5), rendering an increase of the flavylium cation in the solution. Also, a statistically significant decrease in anthocyanin content was observed in the INs of purple figs (p < 0.05). This decrease could partly be explained by the transformation of the flavylium cation to the colorless chalcone at pH 7. Nevertheless, it is important to take into consideration that due to the high pH, the flavylium form would not be the predominant form in the human body after intestinal digestion. The reason for the high loss of anthocyanins may be related to that they are metabolized to some non-colored forms, oxidized, or degraded into other chemicals, which may not be detected under the present conditions.^[29] The results of this study are compatible with other studies in which a low bioaccessibility of anthocyanins is described.^[10,11,29] As shown in Fig. 3, for all fruit fractions except for yellow fig skin, percentage recovery of TP and TF was not significantly different. Recovery of TAC was significantly higher for all samples, and except for dried purple figs, TA was significantly lower compared to TP, TF, and TAC.

DISCUSSION

Drying of fruits is a very ancient practice for food preservation and is still widely in use today. In the literature, there are several reports on the effects of drying on phenolic compounds of various fruits including figs.^{[4,24,25,27,28]} The findings in this study were generally compatible with the existing literature. However, some differences were observed which might be attributed to differences in variety and growing conditions. The present study represents the first published data describing the changes in polyphenols and antioxidant capacity of purple colored figs as a result of sun-drying. In addition, the bioaccessibility of phenolics, flavonoids and anthocyanins, as well as the antioxidant capacity of figs during in vitro GI digestion have also investigated for the first time. Overall the results demonstrate the complexity of the biochemical composition of figs, how this is modified upon fruit processing and the potential relevance of these changes to fruit quality and issues relating to bioaccessibility of antioxidants upon ingestion.

According to the results, TP was found to be insufficient to reflect the changes in phenolics during drying process and, therefore, is not recommended for determining changes in phenolic compounds during fig fruit drying as a single method. Due to the lack of specificity of this method for phenolic compounds, the presence of other reducing compounds reduces the accuracy of the assay. Therefore, to obtain more accurate results, HPLC analysis of individual phenolic compounds should also be performed as was done here.

Drying, in general, is considered as unfavorable due to the possibility of inducing oxidative decomposition either enzymatically or by thermal degradation of polyphenols. However, one of the major findings of this study was an increase both in TF and TPA of yellow figs after sun-drying. Previously, various studies demonstrated an increase in total antioxidant activity and phenolic levels after exposure to several pre- and post-harvest stresses including heat stress in several fruits and vegetables such as strawberries and tomatoes.^[30,31] The increase in the amount of TF as a result of drying can be explained by higher extractability of compounds from the samples as a result of break down in cell walls and/or release from sequestration.^[32] Another explaination of this increase may be Maillard reaction products (MRPs), which can be formed as a consequence of heat treatment or prolonged storage and which generally, exhibit strong antioxidant properties.^[33] Still, this increase in TF is conflicting since HPLC analysis results did not show an increase in any individual flavonoids of yellow figs. Even more interestingly, although the TF of purple figs decreased after sun-drying, some of the major individual flavonoids including rutin, apigenin, and kaempferol-rutinoside were found to increase. Since, HPLC analysis of individual flavonoids is known to give more accurate results, TF measurements might be affected by the presence of other compounds such as vitamins. It is evident that the broad methodologies reported here demonstrate clear drying effects on flavonoids however, for those specifically interested in these metabolites it is recommended to use more comprehensive methods such as Liquid Chromatography-Mass Spectrometry (LC-MS).

Polyphenols act as antioxidants by scavenging free radicals or limiting their formation.^[34,35] Several analytical methods have been developed to measure the TAC. In this study, TAC was measured using four alternative assays which showed different trends. In terms of reflecting the changes as a result of drying, the best correlated methods were DPPH and FRAP assays. The measurement of antioxidant activities, especially in the case of multifunctional or complex multiphase systems, cannot be evaluated satisfactorily by a single method. The principles of the methods vary greatly depending on the radical that is generated or the time of reaction. Even the methods based on the same principle such as ABTS and DPPH can show several important differences in their response to antioxidants. Therefore, it is highly recommended to apply several test procedures to evaluate antioxidant activities to obtain the full picture. In addition, to obtain more reliable results, improved methods such as radical initiator and probe combination^[36] or Flow Injection Analysis- 2,2-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid (FIA-ABTS) method should be applied.^[37]

HPLC analysis of individual phenolic compounds allowed determination of the detailed alteration of each compound as a result of drying. For yellow figs, the predominant phenolic compound was rutin, whereas C3R was detected at the highest level in purple figs. All individual compounds decreased in yellow figs on drying, while for purple figs compounds including ellagic acid, gallic acid, rutin, apigenin, and kaempferol-rutinoside increased. The highest loss after drying was observed in the anthocyanins. The decrease in TA of both yellow and purple figs were similar to the results obtained by HPLC analysis of individual anthocyanins.

There is still a need for more information to fully understand the mechanism behind the drying process. Different drying methods show very different trends. For instance, while sun-drying increased the TP of apricots in one study,^[38] in another study a lower TP was determined in air dried apricots.^[39] Thus, in order to clarify the effect of drying on fig polyphenols, a single drying method is not sufficient. Analysis based on drying kinetics would also provide useful information in order to clarify the effect of drying.

Besides investigating the antioxidant capacity of food materials, it is also important to evaluate the bioavailability of the health associated compounds present. This will provide valuable data for elucidating the true biological relevance of these compounds in the context of nutrition and human health. For this reason, as a part of this study, an in vitro GI digestion study was performed to determine the bioaccessibility of polyphenols from skins, pulps, whole, and dried fruits of two fig varieties.

Bioavailability differs greatly from one polyphenol to another, and for some compounds it depends on dietary source. The absorption of phenolic compounds is considered to be low, not exceeding a plasma concentration of 10 μM. This low absorption may be partially characterized with the chemical structures of different polyphenols that determine their gut absorption. It is believed that the absorption of polyphenols occurs through passive diffusion across the membranes of the gut epithelial cells. In addition, most polyphenols exist in food in the form of esters, glycosides or polymers that cannot be absorbed in their native form. Only aglycones and some glucosides can be absorbed in the small intestine.^[40]

While studies regarding the bioavailability of polyphenols require in vivo experiments, in vitro methods are also useful to determine their stability under GI conditions. In fact, despite their limitations, such as typically constituting only a static model of digestion, it has been shown that the evaluation of bioaccessibility by in vitro models can be well correlated with results from human studies and animal models.^[10] In vitro digestion and dialysis methods for simulating the GI digestion are being extensively used since they are rapid, safe, and do not have the same ethical restrictions as in vivo methods.^[41] The effect of in vitro GI digestion on the stability of polyphenols has already been tested in foods such as pomegranate juice,^[29] raspberry,^[11] chockberry,^[42] apple,^[10] and mulberry.^[41] However, no previous study evaluated the effect of in vitro GI digestion on fig fruit polyphenols. Furthermore, in vitro GI digestion model can be considered as a novel method and should further be studied.

Comparing in vitro studies is difficult since differences may arise from the raw material as well as the in vitro digestion method used. Dialysis is a complicated process affected by different factors such as, volume and composition of the buffer used, sugar concentrations in the sample, or ability of certain molecules to bind with the membrane. All of these parameters can affect the dialyzation of a specific compound, which cannot be truly correlated with absorption of the compound in vivo or to levels of this compound in serum.^[42]

It has been clearly demonstrated that processing has a significant effect on the availability of polyphenols for absorption by the human body. This indicates that disruption of the food matrix and thermal treatments during processing could be major factors affecting the bioaccessibility of the antioxidative compounds. Therefore, in addition to in vitro studies, clinical studies investigating the bioavailability of those compounds would provide valuable data for elucidating the effect of food processing on health-associated compounds in food materials.

In conclusion, this work has focused on the effect of sun-drying on polyphenol content, antioxidant activity and bioaccessibility of figs. Although, the results obtained with the present model of simulated in vitro GI digestion cannot directly predict the human in vivo conditions, it is suggested that this model is helpful for investigating the food matrix and enzymes impacting polyphenol bioavailability. In further studies, it would be interesting to focus on carotenoid bioavailability of fig fruit, perhaps coupled together with cellular models such as Caco-2.

ACKNOWLEDGMENT

The authors would like to thank Dr. Robert D. Hall from Plant Research International (PRI), Wageningen UR for discussions and his comments on the manuscript.

FUNDING

This study was financially supported by the EU 7th Frame ATHENA Project (FP7-KBBE-2009-3-245121-ATHENA).