Effect of oven and freeze drying on antioxidant activity, total phenolic and total flavonoid contents of fig (Ficus carica L.) leaves

Elshaafi, I.M., Musa, K.H. and Abdullah Sani, N. Department of Biological Sciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia Faculty of Agriculture and Veterinary Medicine, Qassim University, Buraydah, Qassim, Saudi Arabia Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600, UKM Bangi, Selangor, Malaysia


Introduction
In the past few years, farmers in Malaysia successfully started importing and growing different fig (Ficus carica L.) cultivars, where the main products sold are fruits and leaves. Their leaves have been used as a tea and claimed to have medicinal properties. Previous studies of antioxidant activity of fig leaves were conducted (Trifunschi and Ardelean, 2013;Ahmad et al., 2013;Allahyari et al., 2014), but no research has been directed on the impact of drying process on their antioxidant, total phenolic (TPC) and total flavonoid (TFC) activities.
The benefits of fig leaves are associated with the secondary metabolites made by these plants. The use of plants as an origin of antioxidants remains substantial for the reason that they are consumed to heal illnesses (Oliveira et al., 2006;Lim and Murtijaya, 2007;Hossain et al., 2010). Moreover, the secondary metabolites of plant work as antioxidants have been proved to fight cardiovascular diseases, cancer and diabetes (Chan et al., 2009;Shahinuzzaman et al., 2019). The protective properties fighting these illnesses remain possibly implemented by the occurrence of several functional secondary metabolites, like polyphenols, vitamins and minerals (Asami et al. 2003;Chang et al., 2006;Roy et al., 2007;Sagrin and Chong, 2013). Polyphenols from various plant sources include an excessive diversity of bioactive compounds, such as flavonoids (anthocyanins, flavonols, flavanols, flavanones) and numerous classes of non-flavonoids such as (phenolic acids, stilbenes and other molecules) (Panche et al., 2016;Tan et al., 2018).
Drying is mainly a process of water removing and reducing the content of moisture, intended to prevent enzymatic and microbial activities, subsequently protecting the crop for lengthening shelf life and phytochemical effectiveness. Furthermore, it also reduces the volume and weight of the crop with significant positive consequences to improve reduction of final product cost, such as storage and shipping (Calixto, 2000;Chan et al., 2009;Tan et al., 2013).
Nevertheless, to the best of our knowledge, there is no literature on the effect of drying of Ficus carica L. leaves on the antioxidant, TPC and TFC. Among the most used drying technique is oven drying which is easy to handle, available and relatively cheap to operate. While freeze drying is not as reachable as oven drying, it is assumed to be more efficient medicinally to other drying methods.
Since antioxidants are delicate to air, heat and light, an appropriate drying process procedure must be optimized for each type of plant's leaves depending on their structure physically and chemically. Therefore, the aim of this study was to evaluate the effectiveness of oven and freeze-drying processes on the TPC, TFC and antioxidant activities of selected fig leaves cultivars. leaves' cultivars namely Brown Turkey Masuri 6 (BTM 6), Masui Dauphine Jumbo (MD-J) and Taiwan Golden Fish Jumbo (TGF-J). The leaves' samples were transported back to the food science laboratory in the Faculty of Science and Technology, Universiti Kebangsaan Malaysia. to be processed accordingly. The samples were divided to two groups; the first as fresh and the second were handled with two drying methods: non-thermal drying using a freeze dryer (Labconco, Benchtop Freeze Dry, USA) and thermal drying using an oven at different temperatures (40 o C, 50 o C and 60 o C) (Memmert Gmbh + CO. KG, Germany) for 48 hrs, then ground with a food grinder (Philips, China) to produce a fine powder. About 0.1 g each of F. carica L. leave samples were weighed and 10 mL aqueous 50% acetone (VWR International, France) was added. All extracted samples were centrifuged using Eppendorf centrifuge (5810 R, Eppendorf, Germany) for 10 mins at 2180 × g and then filtered with 0.22 µm PTFE syringe filter (Osaka Chemical, China).

Determination of moisture content
Moisture content was determined after 48 hrs of oven drying by Association of Official Analytical Chemists (AOAC) (Association of Social Analytical Chemist, 1990) methods.

Determination of total phenolic content (TPC)
The determination of TPC was conducted based on Singleton and Rossi (1965) with modification based on the method of Aminah and Permatasari (2013). The calculation of results was based on the equation generated by the gallic acid standard curve. The result was expressed as milligrams of gallic acid equivalents per 100 g of dry sample (mg GAE/100 g DW).

Determination of total flavonoid content (TFC)
TFC content was determined following the method of Benzie and Strain (1996) with modification based on the method of Bakar et al. (2009). The calculation of results was based on the equation generated by quercetin standard curve. Results were expressed as milligrams of quercetin equivalents (QE) per 100 g of dry sample (mg QE/100 g DW).

Radical-scavenging activity (DPPH)
DPPH determination was carried out according to the method of Musa et al. (2011). The calculation of results was based on the equation generated by Trolox standard curve. Results were expressed as milligrams of Trolox equivalents per 100 g of dry sample (mg TE/100 g DW).

Radical-scavenging activity (ABTS)
ABTS determination was carried out according to the method of van den Berg et al. (1999). The calculation of results was based on the equation generated by Trolox standard curve. Results were expressed as milligrams of Trolox equivalents per 100 g of dry sample (mg TE/100 g DW).

Ferric reducing/ antioxidant power (FRAP)
FRAP determination was carried out according to the method of Benzie and Strain (1999) with modification based on the method of Abdullah Sani et al. (2018). The calculation of results was based on the equation generated by Trolox standard curve. Results were expressed as milligrams of Trolox equivalents per 100 g of dry sample (mg TE/100 g DW). CUPRAC determination was carried out according to the method of Apak et al. (2008). The calculation of results was based on the equation generated by Trolox standard curve. Results were expressed as milligrams of Trolox equivalents per 100 g of dry sample (mg TE/100 g DW).

Statistical analysis
Data were analyzed using MINITAB® (version 17.1.0, USA). One-way ANOVA with Fisher test at p<0.05 was carried out to test significant differences between levels of treatment. Principal component analysis (PCA) was performed using XLstate software (Addinsoft, version 2016.02, France). Pearson's correlation analyses were performed to determine the relationship between antioxidant, TPC and TFC activities.

Moisture content
The  Table 1.

Effect of drying on the total phenolic content (TPC)
The TPC of the three fig leaves' cultivars were affected by drying processes. As in Table 1, oven drying at 40 o C showed the highest TPC for the three cultivars, where BTM 6 revealed significantly (p<0.05) the highest activity (1,125 mg GAE/100 g DW), followed by TGF-J (1,056 mg GAE/100 g DW) and MD-J (993 mg GAE/100 g DW). Significantly there were no differences among freeze drying and oven drying at (50 o C and 60 o C) results of the three fig cultivars, where the activities were ranging from 591 to 655 mg GAE/100 g DW. In contrast, fresh leaves showed significantly (p<0.05) the lowest TPC and ranged between 187 to 240 mg GAE/100 g DW.

Effect of drying on the total flavonoid content (TFC)
The TFC of the three fig leaves' cultivars were affected by drying process. As in Table 1

Effect of drying on radical-scavenging activity (DPPH)
The radical-scavenging activity by DPPH assay of three fig leaves' cultivars was affected by drying processes (Table 1) respectively. There were no significant (p<0.05) differences between freeze drying for all the three cultivars tested. Fresh samples revealed significantly (p<0.05) the lowest activity and ranged between 303 to 168 mg TE/100 g DW.

Effect of drying on radical-scavenging activity (ABTS)
The radical-scavenging activity by ABTS assay of three fig leaves' cultivars was affected by the drying process (Table 1)

Effect of drying on ferric reducing/ antioxidant power (FRAP)
The antioxidant activity by FRAP assay of the three fig leaves' cultivars was affected by drying process (Table 1)

Effect of drying on cupric reducing antioxidant capacity (CUPRAC)
The antioxidant activity by CUPRAC assay of the three fig leaves' cultivars was affected by drying processes (Table 1)

Correlation analysis
The correlations between antioxidant, TPC and TFC activities with regard to the different drying processes were studied (Table 2)

Principal component analysis (PCA)
The PCA of fig leaves achieved by various drying process is shown in Figure 1. Fig leaves' cultivars showed the same trend. For fig cultivar BTM 6 leaves, the PC1 vs. PC2 biplot accounted for 99.21% of the total variance (PC1 = 97.46%, PC2 = 1.75%). According to Figure 1 (A), fresh leaves grouped on the left of the graph, freeze dried and oven dried leaves at 50 o C and 60 o C were grouped in the middle of the graph. While oven drying at 40 o C clustered on the right side of this figure. This distribution revealed that different drying processes present significant differences (p<0.05) in the antioxidant, TPC and TFC activities. Oven drying at 40 o C grouped together with antioxidant, TPC and TFC assays. Based on grouping, TPC and ABTS were formed into one group, similarly, with TFC, DPPH and FRAP, while CUPRAC stands alone. As for fig cultivar TGF-J leaves, the PC1 vs PC2 biplot accounted for 94.75% of total variance (PC1 = 94.74%, PC2 = 3.99%) (Figure 1 (B)) and for fig cultivar MD-J leaves, the PC1 vs. PC2 biplot accounted for 97.09% of total variance (PC1 = 97.09%, PC2 = 2.01%) (Figure 1 (C)).

Discussion
The stabilization of plant leaves by drying comprises fluctuation in the plant matter which could alter the combinations of chemical structure in the dry matter and the extractability (Mediani et al., 2014;Pham et al., 2015). Hence, in this study the antioxidant, TPC and TFC activities of fig leaves dried with various drying processes were assessed. Higher oven drying temperature showed reduction in the activities. Moreover, the reduction of antioxidant levels in the fig leaves correlated with the TPC and TFC activities (  Table 2. Pearson's correlation coefficients between TPC, TFC and antioxidant activities x under influence of different drying process (n = 3) .y X Total phenolic content (TPC), total flavonoid content (TFC), radical-scavenging activity (DPPH), radical-scavenging activity (ABTS), ferric-reducing antioxidant power (FRAP) and cupric reducing antioxidant capacity (CUPRAC), y Replication, * Significant level at p<0.05.
above are due to deterioration of the phenolic and flavonoid compounds. Consequently, drying process is crucial in the production of fig leaves which preserve their antioxidant activities and levels of polyphenolic compounds. Larrauri et al. (1998) suggested that thermal degradation was the main reason of significant decline of antioxidant activity with drying at high temperatures up to 100 o C, which agrees with the current findings. Katsube et al. (2003) for the purpose of drying of mulberry leaves, temperatures between 40 -110°C were used. Wiriya et al. (2009) dried chilies at temperatures between 50 -70°C and Rodríguez et al. (2016) dried Aristotelia berries at 40 -80°C. According to the abovementioned references, when drying temperatures used lower than 60°C, this involves a lengthier drying period especially with air drying, causing a reduction in phenolic content in the presence of oxygen which causes oxidation. While using temperatures at 60°C and above lower the phenolic content because of thermal degradation. Comparing other researchers' results with the current findings using lower temperature at 40 o C for 48 hrs in the oven confirmed that the drying method and temperature is chosen were crucial and should be studied for each plant types and parts.
It suggests that the drying process affects the antioxidant activities, TPC and TFC. Thermal drying at 40 o C showed the highest correlation among TPC, TFC and antioxidant activities, while thermal drying at 50 o C showed the least positive or negative correlations. Thermal drying at 60 o C and freeze drying showed a similar trend, while fresh fig leaves showed mostly negative correlations especially between TPC and TFC.
The strong negative relationship proposed that different drying processes might affect the behaviour of the antioxidant activities, TPC and TPC. The negative correlation found between ABTS and DPPH has been reported previously, revealing that some of the secondary metabolites with ABTS activity may not show radical scavenging capacity by DPPH assay (Wang et al., 1998;Thoo et al., 2010).

Conclusion
It was found that oven drying at 40 o C showed the highest antioxidant activities, TPC and TFC. In order to improve the drying processes, the temperature of drying has to be as low as possible without reducing the fig product quality.