In vitro antioxidant activity of Ficus carica L. latex from 18 different cultivars

As synthetic antioxidants that are widely used in foods are known to cause detrimental health effects, studies on natural additives as potential antioxidants are becoming increasingly important. In this work, the total phenolic content (TPC) and antioxidant activity of Ficus carica Linn latex from 18 cultivars were investigated. The TPC of latex was calculated using the Folin–Ciocalteu assay. 1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and ferric ion reducing antioxidant power (FRAP) were used for antioxidant activity assessment. The bioactive compounds from F. carica latex were extracted via maceration and ultrasound-assisted extraction (UAE) with 75% ethanol as solvent. Under the same extraction conditions, the latex of cultivar ‘White Genoa’ showed the highest antioxidant activity of 65.91% ± 1.73% and 61.07% ± 1.65% in DPPH, 98.96% ± 1.06% and 83.04% ± 2.16% in ABTS, and 27.08 ± 0.34 and 24.94 ± 0.84 mg TE/g latex in FRAP assay via maceration and UAE, respectively. The TPC of ‘White Genoa’ was 315.26 ± 6.14 and 298.52 ± 9.20 µg GAE/mL via the two extraction methods, respectively. The overall results of this work showed that F. carica latex is a potential natural source of antioxidants. This finding is useful for further advancements in the fields of food supplements, food additives and drug synthesis in the future.

www.nature.com/scientificreports/ in other parts of F. carica 14 . Oliveira et al. analysed the latex of F. carica and identified 38 bioactive compounds by using gas chromatography mass spectrometry. Seven of the bioactive compounds are phytosterols, 13 are free amino acids, and 18 are fatty acids. They identified phytosterols, such as β-sitosterol, lupeol, α-and β-amyrin, betulol and lanosterol, and amino acids, such as leucine, phenylalanine, tryptophan, histidine, alanine, glutamine, glycine, serine, ornithine, lysine, asparagine, tyrosine and cysteine 15 . Although many researchers have successfully determined the antioxidant activity and total phenolic content (TPC) of crude extracts from F. carica leaves, fruits and bark, limited reports on the antioxidant activity and TPC of F. carica latex are available. Moreover, the data of published reports are only for few cultivars, and the methods used are complex. The different methods of extraction include maceration extraction 16 , microwave-assisted extraction [17][18][19] and supercritical fluid extraction [20][21][22] . Most of these approaches, however, are time consuming and require comparatively more solvents than others and are not economically viable given their high cost 23 . However, as a better alternative to these methods, ultrasound-assisted extraction (UAE) is more efficient; requires relatively less solvents; and has good reproducibility, rapid extraction time, low temperature and easy scaling up for application in industries [24][25][26] . This process breaks down the cell walls, enables the cell content to be washed out and has high efficiency for isolating antioxidant and phenolic compounds 27,28 . Maceration is also a simple, convenient and less costly extraction process in terms of instrumentation 29 . Therefore, this method is more appropriate than others for both small and medium-sized enterprises in developing countries 30 .
Numerous in vitro assays are used to determine the antioxidant activity of biological samples. Comparing one assay with another is hard, and evaluating the antioxidant activity using a single antioxidant test method only is not possible because different methods measure antioxidant activity from different angles 31,32 . Amongst the various in vitro methods, 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay is more simple, rapid and inexpensive, whilst the 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical assay is appropriate for both hydrophilic and lipophilic samples 31 . In this study, the TPC and antioxidant activities of fig latex from 18 different cultivars were evaluated. The Folin-Ciocalteu (FC) assay for TPC and three different in vitro assays, such as DPPH, ABTS and ferric ion reducing antioxidant power (FRAP), were used to determine the antioxidant activities of the samples. Two different extraction methods, such as maceration and UAE, were used. Initially, the solvent effect and the effect of solvent-to-latex ratio were also studied to select the proper solvent for extraction in this study.

Materials and methods
Chemicals and reagents. All chemicals were analytical reagent grade and used without further purification. All chemicals with their chemical formulas, manufacturers and purity are listed in Table 1. Milli-Q water was used to prepare standard materials and reactant solutions and perform extraction.
Equipment. The equipment used in this study and their manufacturers with model number are listed in Table 2. An orbital shaker, Thermoline ultrasonic bath, centrifuge machine and steam distillatory were used for extraction. A UV-Vis spectrophotometer was used to determine the antioxidant capacities of the samples.
Latex sample collection and preparation. The Figure 1 shows the green leaves from the 18 cultivars of F. carica.
Extraction of crude sample from F. carica latex. Various methods are used to extract antioxidant compounds from plant materials. In this study, modified maceration extraction with continuous shaking and UAE were used with the same conditions. The extraction conditions were selected on the basis of the primary screening and optimisation of this study mentioned in our previous work 23 . For maceration extraction, the samples were extracted using an incubator shaker at 200 rpm and 35 °C. Ultrasonication was conducted by using a Thermoline ultrasonic bath at 35 °C. F. carica latex (1 g) from the cultivar 'Wuhan' was kept in two different 25 mL capped long glass vial, and 10 mL of 75% ethanol was added in each vial. Then, the mixtures were transferred into the shaker and ultrasonic bath for maceration and ultrasonication for 30 min. After extraction, the samples were centrifuged at 4,000 rpm for 10 min by using a laboratory centrifuge machine. The supernatant liquids were filtered and used to determine TPC and antioxidant activity and to perform other analyses. The same extraction process was repeated for the cultivars 'White Genoa' , 'Masui Effect of solvent. The solvent effects were investigated via maceration extraction for the cultivar 'White Genoa' before the final extraction of all cultivars. From the primary screening data of this study, 'White Genoa' extract obtained via maceration showed the highest TPC and antioxidant activity. Therefore, 'White Genoa' was used to investigate the effects of solvent and latex-to-solvent ratio on extraction. Initially, different types of solvents, such as 100% methanol, 100% ethanol, 75% ethanol, 100% ethyl acetate and 100% n-hexane, were used with the same extraction condition to investigate the solvent type. 'White Genoa' was extracted via maceration, and its TPC and antioxidant activity were determined via DPPH assay to study the effects of solvent. The effect of latex-to-solvent ratio was investigated with four different ratios, such as 1:1, 1:5, 1:10 and 1:15 g/mL (w/v). F. carica latex (1 g) from 'White Genoa' was extracted using different amounts of 75% ethanol, such as 1, 5, 10 and www.nature.com/scientificreports/ 15 mL. The result was validated using the same experiment on 'B110' as the second highest active cultivar, and the results are shown in the supplementary data (Tables S1 and S2).

Determination of TPC.
The TPC of leaves of F. carica was analysed using FC reagent with some modifications 23,33 . The FC reagent was used as the oxidising agent. Standard gallic acid or plant extract (100 μL) was mixed with 3.25 mL of 12 times pre-diluted FC reagent. After proper mixing, the samples were allowed to stand for 7 min; then 750 µL of 20% Na 2 CO 3 was added to the solution and kept for 2 h in incubation in the dark. Finally, absorbance was recorded at 760 nm on the basis of a colorimetric redox reaction from a standard curve (y = 0.0033x + 0.0471, R 2 = 0.9951) and using standard gallic acid solution of 31.25-500 µg/mL. The data are shown as μg gallic acid equivalent/mL sample. Each sample was measured as triplicate.
Determination of antioxidant activity. In this study, three different scavenging assays were used to determine the antioxidant activity from F. carica latex. DPPH, ABTS and FRAP assays were used with the same latex samples.
DPPH free radical scavenging assay. Antiradical activity was determined spectrophotometrically using a UVvisible spectrophotometer by monitoring the disappearance of DPPH · at 520 nm in accordance with a previously described procedure with some modifications. The reaction mixtures in the sample consisted of 100 μL of supernatant and 3.9 mL of 0.1 mM DPPH · dissolved in ethanol. The samples were incubated for 30 min at room temperature. Every sample was measured in triplicate. Ethanol was used as blank, and the sample without antioxidant was used as control. Trolox equivalent antioxidant capacity (TEAC) was calculated by preparing a standard Trolox curve (y = -0.0008x + 0.4956, R 2 = 0.9998) from 31.25 µg/mL to 1.0 mg/mL of a standard Trolox solution. The outcomes were presented as mg Trolox equivalent (TE)/g sample. Each experiment was carried out in triplicate. The DPPH activity was expressed as a percentage of inhibition and calculated using Eq. (1) 34 : where A B = absorbance of control sample (t = 0 h) and A S = absorbance of a tested sample after the reaction (t = 1 h).
ABTS radical scavenging assay. The ABTS radical scavenging assay was calculated on the basis of the method of Gorinstein 35 with some modifications. Firstly, the radical solution was prepared by mixing stock solutions, such as 7 mM aqueous solution of ABTS and 2.45 mM potassium persulfate (K 2 S 2 O 8 ) solution at a ratio of 1:1 36 . The mixture was kept for 12-16 h in dark conditions at room temperature. Then, the fresh working solution was prepared for each bioassay by diluting 1 mL of ABTS radical solution with the required amount of ethanol to obtain the absorbance of 0.700 ± 0.02 units at 745 nm. Afterwards, 100 μL of different extracts or different standard Trolox solutions were added to 3.9 mL of an ABTS + solution. The absorbance was measured immediately at 745 nm after 6 min incubation at room temperature. Aqueous ethanol (75%) and Trolox were used as blank and positive control, respectively. TEAC was calculated by preparing a Trolox curve for ABTS assay (the standard curve equation: y = − 0.0009x + 0.4836, R 2 = 0.9978 from 31.25 µg/mL to 500 µg/mL), and the results were presented as μg TE/mL sample. The percentages of inhibition of ABTS was calculated using Eq. (1).
FRAP assay. The FRAP of fig latex was determined using the potassium ferricyanide-ferric chloride method described by Oyaizu 37 with some modifications. The ethanolic extracts (100 μL aliquots) of F. carica latex were added to 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferricyanide (1%). After 20 min of incubation at 50 °C of the mixtures, 2.5 mL of trichloroacetic acid (10%) was added. From the mixture, 2.5 mL was taken and again mixed with 2.5 mL of water and 0.5 mL of 1% FeCl 3

Results
Effect of solvent type and solvent-to-latex ratio. To obtain better activity of natural extracts, it is very essential to select proper solvents and solvent ratios. Therefore, the solvent type and solvent-to-sample ratio were investigated before extraction as mentioned previously 23 . Methanol and ethanol are the main polar solvents used for extracting antioxidants and TPC from plant materials. Table 3 shows the effect of different solvents on the DPPH and TPC of F. carica latex.
(1)  [38][39][40][41] . The activity of 75% ethanol (DPPH, 63.76%; TPC, 298.15 µg GAE/mL) was higher than that of 100% ethanol. The effect of latex-to-solvent ratio was also studied with different latex-to-solvent ratios (1/1, 1/5, 1/10 and 1/15 g/mL) over optimum condition (30 °C, 35 min and 75% ethanol). The outcomes are shown in Table 4. The antioxidant activity increased with the increase of solvent up to 5 mL and then decreased as the amount of solvent increased. Antioxidant activity for 5 and 10 mL solvent were nearly the same. The TPC decreased with the increase of solvent up to 15 mL.    DPPH free radical scavenging activity. The DPPH scavenging activities of the latex of 18 F. carica cultivars were evaluated at the same extraction conditions (30 °C extraction temperature, 35 min extraction time and 75% ethanol as extraction solvent), and the results are presented in Fig. 3a and b. For both extraction methods, the DPPH antioxidant activity was analysed and expressed as percentage inhibition and TEAC. The percentage of DPPH activity for maceration extraction ranged from 20.82% ± 1.54 to 64.93% ± 2.00% and 110.75 ± 9.92 µg to 394.17 ± 12.82 µg TE/mL for percentage of inhibition and TEAC, respectively. The activities of the extracts obtained via UAE ranged from 18.16% ± 1.07 to 58.22% ± 1.78% and 93.67 ± 6.88 µg to 351.08 ± 11.41 µg TE/ mL, respectively. Amongst the 18 cultivars, 'Qing Pi' showed the lowest antioxidant activity (20.82% ± 1.54% and 110.75 ± 9.92 µg TE/mL), whereas 'White Genoa' showed the highest activity (64.93% ± 2.00% and 394.17 ± 12.82 µg TE/mL) via maceration. 'B110′ showed the second highest DPPH activity, (61.38% ± 1.75% and 367.00 ± 11.25 µg TE/mL). The cultivar ' Alma' showed the third highest antioxidant activity (59.97% ± 2.15%  ABTS + radical scavenging activity of F. carica latex. The results of the ABTS radical scavenging assay were expressed as the percentage of inhibition and TEAC similar to DPPH and shown in Fig. 4a

cluster analysis of cultivars of F. carica. Hierarchical cluster analysis is used to classify the F. carica
cultivars on the basis of their TPC and antioxidant activities. Ward's method was used to create the dendrogram, and the similarity between cultivars according to their activities was measured using Euclidean distance. Cultivars with higher TPC and antioxidant activities, as indicated by the DPPH, ABTS and FRAP assays, were placed in the same cluster, whereas cultivars with lower antioxidant and TPC activities were placed in a different cluster. On the basis of antioxidant activity and TPC, three main clusters were obtained at the Euclidean distance of 20.0, including cluster (I), cluster (II) and cluster (III) (Fig. 6). The cultivars in cluster (I) showed the lowest TPC and antioxidant activity. Two cultivars, namely, 'Wuhan' and 'Qing Pi' , were included in this cluster. The second cluster (II), which was the second lowest active cluster based on TPC and antioxidant activity,  correlation analysis of F. carica latex. Significant correlations were obtained amongst the antioxidant activities and TPC via different assays. Figure 7 shows the correlation amongst DPPH, ABTS and FRAP assays of F. carica latex extracts obtained via maceration and UAE. A positive relationship exists between the maceration DPPH and ultrasonic DPPH (r = 0.92). This result indicates a 92% possibility that the same bioactive compounds or the same factors attributed to maceration and UAE influenced the DPPH activity. The DPPH and ABTS activities of extracts obtained via maceration showed a positive correlation with the highest r-value (r = 0.95). So, there have 95% possibility of same reasons, same mechanisms or same bioactive compounds influence the antioxidant activity of F. carica latex with DPPH and ABTS assays via maceration. The FRAP assay results indicated a strong positive relationship between maceration and UAE (r = 0.93). However, a very weak but positive correlation was detected amongst FRAP, DPPH and ABTS assays. So, there has a less similarity of mechanism or the compounds which influence the mechanism of FRAP assay compared to the DPPH and ABTS assay.
Microscopic studies. The structures of leaf anatomy from the leaf shoot of 'White Genoa' cultivar was studied under a compound microscope. After chopping, the leaf shoot was cleaned with ethanol, chloroform and acetic acid mixture (60:30:10 v/v) followed by deionised water. Then, the anatomical segment (500 μm) was analysed under the microscope at different projections. Figure 8 shows the transverse section of fig leaf shoot from the 'White Genoa' cultivar at 10 × and 20 × projections. The veins inside the lamina are visible to the naked eye. All areal parts of the shoot and the simple hairy granular trichomes can be seen (Fig. 8). The cross-section of petiole showed a number of xylem vessels inside of the fibre, and piths are present in the centre. Small pores can be seen inside of the pith vessels, cortex and fibre of F. carica leaf shoot, which may contain the latex (Fig. 8c and  d).

Discussion
The biological activity of F. carica latex depends on the solvent. A diluted solvent can better extract antioxidants and polyphenols from plants compared with a pure solvent 23,42,43 . Ethanolic extracts of Psidium guajava L. have the highest activity amongst chloroform, petroleum ether and water extracts 44 . The bioactive compounds from 70% ethanolic extracts of Moringa oleifera show better activity than those of others 30 . The maximum TPC value of the 70% ethanolic extract obtained via maceration was 5.35 g GAE/100 g of powder. Polar solvents are more effective than non-polar solvents in extracting bioactive compounds from plant materials 45 . The current study indicated that non-polar solvents, such as n-hexane, and less polar solvents, such as ethyl acetate (polarity index 4.4), showed low capability for extracting bioactive compounds from F. carica latex. The activities of the extracts using 1, 5 and 10 mL solvents were significantly different. A high latex-to-solvent ratio increases the rate of diffusion, which improves the solvent-based extraction. It also increases the rates of leaching that allows solvents to come into contact with bioactive compounds. Therefore, 1:10 (g/mL) of solvent ratio was chosen as the optimum ratio to maximise the speed of mass transfer 23,46,47 . Also, a high solvent ratio helps to maximise the extraction rate, minimise the use of latex and increase the percent of yield. www.nature.com/scientificreports/ The TPC and antioxidant activity of the extracts from F. carica latex are mainly due to the presence of different active compounds. The highest result for specific cultivars may be due to the presence of more bioactive compounds than other cultivars. Maceration extraction was better than UAE for obtaining extracts from F. carica latex. In the case of F. carica latex, soaking and shaking help increase the amount of antioxidants and bioactive compounds obtained from the latex 30,44,45,48 . UAE was conducted by ultrasound but without shaking. The solvents used in soaking and shaking for maceration extraction also play a vital role 49 . During maceration, the tissues of F. carica latex are disintegrated first by shaking and heating. Finally, the desired bioactive compounds were diffused from the cell sap to the solvent and showed higher activity than UAE. However, most of the bioactive compounds found inside the cells cannot permeate the cell walls. Most of the water-soluble components with low molecular weights generally diffuse out of the cell when the tissue is treated, and its osmotic control is disrupted. An example is continuous shaking or heating to 60 °C. Even when an osmotic barrier is absent, the diffusion from tissues is often slow, especially with large molecules, such as proteins or gums [50][51][52] . Thus, the tissues of F. www.nature.com/scientificreports/ carica latex were disintegrated first by shaking and heating. Finally, the desired bioactive compounds were diffused from the cell sap to the solvent and showed high activity. Maceration may also work for extracting other non-antioxidant and polyphenolic compounds, which cannot be done via UAE. Data showed that the difference between maceration and UAE was higher for TEAC than the percentage of inhibition. This result is due to the high range of values for TEAC. The SDs amongst the three replicates of the same cultivars are also significant for TEAC but not for the percentage of inhibition. 'White Genoa' showed the highest TPC and antioxidant activity, which might be due to the presence of more bioactive polyphenolic compounds than the other cultivars. Moreover, non-antioxidant compounds may also affect the antioxidant activity of F. carica latex 53,54 . The latex of 'White Genoa' is stickier and more viscous than those of other cultivars. Thus, the latex of this cultivar is more concentrated than those of others. The latex concentration may also affect the antioxidant activity of different cultivars. However, to the best our knowledge, information about the relationship between the physical properties of plant latex and antioxidant activity has not been reported yet.
Data from the FRAP assay indicated that the antioxidant capacities of the samples extracted via maceration and UAE were not significant for all cultivars. Thus, the antioxidant components in proportion to various cultivars cannot be isolated via FRAP assay. The antioxidants present in the F. carica latex exhibited reducing power by reducing Fe 3 to Fe 2 . The values of the three different assays used to measure the antioxidant activity of F. carica latex varied. These differences are attributed to the varying reaction mechanisms of the assays. Moreover, the antioxidants from extracts have different abilities to mitigate peroxyl radicals and to reduce the ABTS + , DPPH free radical and ferric ion 55,56 . This phenomenon may be also due to their different properties, such as molecular size. ABTS radical is formed initially, whilst DPPH radical is a stabilised radical itself. They may also have different affinities against the compounds present in the sample. However, a positive correlation exists amongst the assays due to their similar redox reaction 57 .
Some authors also have reported that maceration extraction is more effective than other methods. Ethanolic extracts of Psidium guajava L. obtained via maceration showed the highest yield of phytoconstituents 44 . The antioxidant activity of methanolic extracts from Garcinia atroviridis obtained via maceration showed good results with a minimum EC 50 value of 9.32 and 5.32 μg/mL for DPPH and ABTS assay, respectively 45 . The extracts of Cosmos caudatus 48 and M. oleifera 30 obtained via maceration exhibited the highest activity compared with others. The 70% ethanolic extract of M. oleifera obtained using maceration showed a minimum EC 50 value of 62.94 μg/ mL with DPPH and 51.50 mmol FeSO 4 eqv/100 g of extract with FRAP assay. The DPPH values for squeezing, decoction and percolation were 367.32, 123.44 and 95.94 μg/mL, respectively. Maceration was used to extract the F. carica latex 11 from Tunisian caprifig.
From the cluster analysis of 18 F. carica cultivars, the cultivar 'White Genoa' had the best antioxidant effect and can be used as a natural source of TPC and antioxidants. Correlation analysis revealed that DPPH and ABTS can be used to evaluate the antioxidant activity of F. carica extracts. Previously, Ajmol et al. 58

conclusion
The extract from the 'White Genoa' latex obtained via maceration showed the highest antioxidant activity and TPC compared with that obtained via UAE. The latex of 'B110' and ' Alma' also showed good activities compared with 'White Genoa' . Although 'White Genoa' showed the highest antioxidant activity and TPC, 'B110' and ' Alma' are also potential sources of TPC and natural antioxidants. The latex of these three F. carica L. cultivars could be a potential source of natural antioxidants and polyphenols. The estimation of total cost to isolate the antioxidant compounds from the latex of cultivar 'White Genoa' of F. carica commercially will help in the proper selection of technology for real-life applications. Developing a cost-effective natural extract with an efficacy similar to or better than that of the current F. carica cultivars could draw a substantial market share. The latex of F. carica cultivars with the highest activity can be subjected to in vitro and in vivo studies to consider their modes of action as a antioxidant. Also, these cultivars can be potential candidates for further phytochemical and pharmacological studies. However, further research should be carried out to determine the effects of the physical properties of fig latex (viscosity and water content), season, cultivation condition (fertiliser application and watering) and soil properties (physical and chemical properties) on the antioxidant activity of the reported fig cultivars.