Inﬂ uence of extraction solvents on the polyphenol contents, compositions, and antioxidant capacities of ﬁ g ( Ficus carica L.) seeds

: Fig seeds are considered to be signifi cantly responsible for the bioactive compounds of fi g. In this study, the effects of six different solvents (100% acetone, 100% methanol, 100% ethanol, 50% (v/v) aqueous acetone, 50% (v/v) aqueous methanol and 50% (v/v) aqueous ethanol) with changing polarities on the polyphenol contents and antioxidant capacities of fi g seed extracts were investigated. Total polyphenol contents (TPCs), total fl avonoid contents (TFCs), antioxidant capacities (DPPH and FRAP assays) and polyphenol compositions of the extracts were evaluated. The results indicated that fi g seeds extracted by 50% (v/v) aqueous methanol exhibited the highest TPC (714 mg GAE/kg DM), TFC (312 mg (+)-CE/kg DM), DPPH (41.6%) and FRAP (8504 mg FeSO 4 /kg DM) values. Also, same extract had the maximum values of chlorogenic acid (131.9 mg/kg DM), (-)-epicatechin (166.4 mg/kg DM) and rutin (50.7 mg/kg DM) ( p <0.05). The extractability of syringic acid was determined to be highest with 50% aqueous methanol (8.03 mg/kg DM) and 50% aqueous ethanol (8.13 mg/kg DM) ( p >0.05). The psoralen extractability was highest in 50% aqueous acetone (53.0 mg/kg DM) and 50% aqueous ethanol (54.0 mg/kg DM) ( p >0.05). High correlations among TPCs, TFCs, antioxidant capacities and individual polyphenols of fi g seed extracts were also observed.


INTRODUCTION
Fresh and dried fruits of the fig tree (Ficus carica L., family Moraceae) are important parts for the consumers worldwide (Rtibi et al. 2018, Rodríguez-Solana et al. 2018 . Fig fruits contain carbohydrates, amino acids, minerals, vitamins, low amounts of lipids, phytosterol, organic acids, and polyphenols (Nadeem & Zeb 2018). Besides being delicious, they have been consumed for centuries because of their antioxidant, antiherpes simplex virus (HSV), hypoglycemic, antidiabetic, anti-hyperlipidemic, hepato-protective and immune sensitive properties (Nadeem & Zeb 2018, Rodríguez-Solana et al. 2018. These health promoting properties are mainly attributed to the fact that fi g fruits contain high amounts of polyphenols with antioxidant properties that can prevent oxidative stress-related diseases (Bahrin et al. 2018).
Fig fruit consists of a fl eshy hollow receptacle with tiny pedicellate druplets called fi g seeds (Mars 2003). Each fig fruit has small whitish seeds with numbers ranging from 30 to 1600 (Badgujar et al. 2014). In this complex matrix, fi g seeds are thought to contribute signifi cantly to the nutrient content and health-promoting effects of fruit. Therefore, it is very important to reveal the polyphenol contents and antioxidant capacities of fi g seeds.
One of the most important factors affecting the extraction efficiency of polyphenols and their associated health benefits is the extraction solvent used (Ngo et al. 2017). Polar solvents are often utilized for recovering polyphenols from plant materials (Do et al. 2014). The use of organic solvents such as ethanol, methanol, acetone and diethyl ether or their aqueous mixtures is generally preferred for this purpose (Wijekoon et al. 2011, Do et al. 2014. Aqueous acetone has been generally determined to be good for the extraction of higher molecular weight flavanols, whereas methanol is found more effective for the extraction of lower molecular weight polyphenols (Do et al. 2014). Ethanol, which is known to be safe for human consumption, is thought to be a good solvent for polyphenol extraction (Do et al. 2014). The nature of the bioactive compounds is appeared to vary depending on the plant material. Therefore, in general, it is very difficult to suggest a suitable extraction solvent for every plant materials (Wijekoon et al. 2011). According to previous studies, the most suitable extraction solvent that can be used to determine the polyphenols and antioxidant capacities of various plant materials has been found as 50% (v/v)

Samples
Fresh fruits of the Sarilop variety were purchased from local market in Izmir (Turkey) at the beginning of July 2018. Fig seeds were extracted after chopping the fresh fruits into a water bath. The seeds washed several times with water and allowed to dry at room temperature for one week. All the materials were kept at 4°C prior to analyses. Before extraction, fig seeds were crushed with a coffee grinder to make them homogeneous.

Extraction process
Solid-liquid extraction system was used for the extraction of antioxidant compounds from fig seeds. Six different solvents including 100% acetone, 100% methanol, 100% ethanol, 50% (v/v) aqueous acetone, 50% (v/v) aqueous methanol and 50% (v/v) aqueous ethanol were utilized in the extractions. The sample was extracted in these solvents using a fixed solid/liquid ratio of 1:5 (w/v), for example 3 g of sample by mixing with 15 mL of solvent. The mixture was then stirred at 50°C for 90 min in a shaking water bath (Memmert WB10, Schwabach, Germany). It was centrifuged (10,000 rpm, 4°C) for 20 min and filtered using a 0.45 μm PTFE membrane filter (Sartorius, Germany). The volume of aliquot extract was completed to 15 mL. Liquid extracts were immediately analyzed for the determination of polyphenol content and antioxidant capacities of fig seeds.

Spectrophotometric determination of polyphenols and antioxidant capacity
Total polyphenol content (TPC) of the extracts was quantified by a spectrophotometric method (Xu & Chang 2007) using Folin-Ciocalteu reagent. The amount of total polyphenols were expressed as mg gallic acid equivalents (GAE) per kg of dry matter (DM) (y = 0.0021x + 0.0367). Total flavonoid content (TFC) of the extracts was determined according to the aluminum chloride colorimetric method described by Heimler et al. (2005) and calibrated against (+)-catechin as the reference standard. The TFC were evaluated as mg (+)-catechin equivalents ((+)-CE) per kg of DM (y = 0.0013x + 0.0059). The capacity of the extracts to scavenge the 2,2-diphenyl-1picrylhydrazyl (DPPH) radical was assessed according to a modified method described by Chu et al. (2000) and Cheung et al. (2003). 6-Hydroxy-2,5,7,8-tetramethylchromane-2carboxylic acid (trolox) was used as a reference antioxidant and the concentration was 15 μM. It was expressed as percentage inhibition of DPPH (%). The ferric reducing ability power (FRAP) of the extracts was determined following the modified methods described by Guo et al. (2003) and Xu et al. (2004). Butylated hydroxytoluene (BHT) was used as a reference antioxidant and the concentration was 10 mM. The results were expressed as mg reduced iron equivalents (FeSO 4 ) per kg DM (y = 0.0626x + 0.0163).

Determination of individual polyphenols using HPLC-DAD detection
The method described by Çam et al. (2014) was adapted with slight modifications for the qualitative and quantitative determinations of individual polyphenols. The polyphenol standards were purchased from Sigma Aldrich Co. (St. Louis, MO, USA). The contents of individual polyphenols in the extracts were measured using a Agilent 1200 LC system (Agilent Technologies, Palo Alto, CA, USA) using DAD detection at 272 nm for gallic acid, 275 nm for (-)-epicatechin, 279 nm for chlorogenic acid, syringic acid and psoralen and 356 nm for rutin, on a Hichrom C 18 column (250 mm × 4.6 mm i.d. with 5 m particle diameter, Hichrom Ltd., Reading, Berkshire, UK) which was maintained at 40°C. The mobile phases were composed of A and B solvent systems; solvent A was water/acetic acid (98:2, v/v); solvent B was 100% methanol. An used elution gradient was as follows: a linear gradient from 95% A to 50% A for 10 min, from 50% A to 30% A for 5 min. The flow rate was 1 mL/min and the injection volume was 20 L. Quantitative determinations were carried out using the external standard method (y = 0.5522x + 15.9797 for gallic acid, y = 0.0783x + 3.1251 for (-)-epicatechin, y = 0.1666x + 4.1505 for chlorogenic acid, y = 0.4325x + 13.0645 for syringic acid, y = 0.3481x + 6.4216 for psoralen and y = 0.2212x + 5.0548 for rutin). Qualitative determinations were performed by comparing the retention times and spectra of the samples and standards, as well as the use of standard addition method. The HPLC method used was validated by determining LOD, LOQ, and recovery values.

Statistical analysis
The results were given as mean value ± standard deviation for triplicate determinations. ANOVA and Duncan multiple range test were used to determine the differences between values by SPSS ver.20.0 (SPSS Inc., Chicago, USA) at a significance level of p<0.05. Also, Pearson correlation test was used to determine the correlation among variables.

Total polyphenol contents, total flavonoid contents and antioxidant capacities of the fig seed extracts
In this study, the polyphenols and antioxidant compounds of fig seeds were extracted with various solvents having different polarities. As indicated in Figure 1a, b, total polyphenol and flavonoid contents varied in the different extracts (p<0.05). TPC increased in the following order: 100% acetone<100% ethanol<100% methanol<50% (v/v) aqueous acetone<50% (v/v) aqueous ethanol<50% (v/v) aqueous methanol (p<0.05). TFCs of the extracts were in the order of 100% acetone=100% ethanol<100%  2015) found that the extracts of sunflower disc florets revealed the highest TPC values were 90% methanol and 50% ethanol extracts as well as 50% methanol extract, supported the findings of this study. The study reported by Wijekoon et al. (2011), which the 50% methanol extract of bunga kantan inflorescence had one of the highest percent inhibition of DPPH. Similar results have been recorded in the present study.

Polyphenol compositions of fig seed extracts
Individual polyphenols in the fig seed extracts were determined qualitatively and quantitatively by using the calibration curves plotted with six different standards in the concentration range of 0.834 -83.34 mg/L (R 2 >0.99) (Figure 2.). The LOD, LOQ and recovery values for each polyphenol are given in Table I The polarities of polyphenols in the food matrix may be different from each other. This   Table II In addition, no statistically significant difference was observed among the absolute solvents used for syringic acid extraction (p>0.05). The lowest amount of psoralen was also found in 100% methanol extract of fig seeds (p<0.05). These findings were consistent with the results of TPC and TFC and also showed that aqueous solvents, in particular 50% (v/v) aqueous methanol, were more successful than the other solvents used to extract polyphenols from fig seeds. These results correlated with Butsat & Siriamornpun (2016) who reported that the aqueous methanol extract of Amomum chinense C. leaves had high contents of catechin, rutin and chlorogenic acid compared to other aqueous solvents.

Correlation analysis among polyphenols and antioxidant capacities
Correlation analyses were conducted among polyphenols and antioxidant capacities of fig seed extracts (Table III) ions. Moreover, the correlation between DPPH and TPC is higher than that of FRAP because Psoralen was not correlated with FRAP values of extracts, implying that this compound was not responsible for the ferric-reducing antioxidant power of the tested extracts. Several studies in literature have also been clearly stated a close relationship between the polyphenol content and the antioxidant capacity (Xu & Chang 2007, Ye et al. 2015, Ghasemzadeh et al. 2015.

CONCLUSIONS
The results obtained from this study showed that fig seeds were found to be a natural source of bioactive compounds and the important parts of polyphenols and antioxidant compounds came from fresh figs. It was also found that extraction with solvents of different polarities affected the TPC, TFC, individual polyphenol content and antioxidant capacity of fig seed extract. The extractability of bioactive compounds in fig seeds was increased by addition of water to organic solvents. This proved that medium polar solvents (aqueous solution of methanol, ethanol, acetone, chloroform etc.) were more effective than solvents with low polarity (absolute organic solvents) in the extraction of