Characterization of Bioactive Ligands with Antioxidant Properties of Kiwifruit and Persimmon Cultivars Using In Vitro and in Silico Studies

: The current study attempted to understand the interaction proﬁles of phytoconstituents in new and traditionally used fruit cultivars with human serum albumin (HSA) in the context of predicting the biological role under in vivo conditions. Therefore, polyphenols, ﬂavonoids, ﬂavanols, tannins, vitamin C, secondary metabolites and their antioxidant capacities of organic kiwifruit Actinidia ( A .) eriantha cv. Bidan (AEB) and A. arguta cv. Cheongsan (AAC), as new cultivars grown in Korea, and widely consumed A . deliciosa cv. Hayward (ADH) and Diospyros kaki Thunb. cv. Fuyu (DKF) were determined and compared. All investigated fruits showed relatively high antioxidant capacities. To complement the bioactivity of these fruits, the binding properties between extracted polyphenols and HSA were determined by 3D-ﬂuorescence spectroscopy and docking studies. The most bioactive was AEB with the highest percentage of binding, following by AAC, ADH and DKF. Our study for the ﬁrst time unveils the di ﬀ erential binding properties of kiwifruit and persimmon phytoconstituents with HSA. Although cultivars possess virtually the same phytoconstituents, presence of one unique compound signiﬁcantly alters the binding properties of HSA. The results of ﬂuorescence quenching and molecular docking showed that these fruits possess multiple properties, which have a great potential to be used in industry with emphasis on the formulation of functional foods and medicinal applications.


Chemicals and Reagents
The chemicals were bought from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) and Fluka Chemie GmbH, Buchs, Switzerland.

Plant Material
Three batches of organic kiwifruits, including Actinidia (A.) deliciosa cv. Hayward (ADH), A. eriantha cv. Bidan (AEB), A. arguta Cheongsan (AAC) and one batch of Diospyros kaki Thunb. cv. Fuyu (DKF) were collected in different commercial orchards from Boseong and Muan counties, Jeonnam and The samples were washed with tap water and dried. The fruits were fractionated into edible fraction (pulps), peels and seeds. Only for DKF 5-8 seeds were separated from pulps. Their edible parts were prepared manually without using steel knives. The peeled fruits (pulps) were weighed, chopped and homogenized in liquid nitrogen in a high-speed blender (Silex professional model, Hamilton Beach, Virginia, USA). A weighed portion (50-100 g) was then lyophilized for 48 h (Virtis model 10-324, Midland, Canada), and the dry weight was determined. The samples were ground to pass through a 60-mesh sieve and stored at −20 °C until the bioactive substances were analyzed.

Determination of Firmness, Total Soluble Solids (TSS), pH, Total Acidity (TA), and Dry Matter
The fruits were analyzed for firmness by measuring penetration force in kilograms using a fruit-firmness tester (Model KM, Fruit Test Tech, Tokyo, Japan). After peeling, the tester penetrates (punches) the flesh with hand pressing. The mean values of the firmness were expressed in Newtons (N): 1 N is 9.8 kg. The peeled fruits were homogenized and filtered through cheesecloth in order to obtain a clear juice for determination of TSS (Brix), pH, TA, and dry matter. The TSS was measured using a digital refractometer (Atago Co. Ltd., Tokyo, Japan), pH was checked with a pH meter (Model 8100, ETI Co. Ltd., Worthing, West Sussex, England). The TA was measured in 4 mL of juice, diluted to 20 mL of distilled water and titrated with 0.1 N NaOH. The TA was expressed as a percentage of citric acid. After peeling, dry matter (%) was calculated by the ratio of initial fresh weigh/dry weight [3,19,23,24].

Sample Extraction
The lyophilized samples of kiwifruit and persimmon cultivars were extracted with deionized water (pH 3) during 60 min at 25 o C. For mass spectrometry (MS) measurements the samples were extracted with ethanol: water mixture (8/2, v/v) during 60 min at 25°C. The proportion of sample to the solvent was 1/10w/v. The extracts were filtered in a Buchner funnel. After removal of the solvent in a rotary evaporator and the aqueous solution were freeze-dried [25,26]. The samples were washed with tap water and dried. The fruits were fractionated into edible fraction (pulps), peels and seeds. Only for DKF 5-8 seeds were separated from pulps. Their edible parts were prepared manually without using steel knives. The peeled fruits (pulps) were weighed, chopped and homogenized in liquid nitrogen in a high-speed blender (Silex professional model, Hamilton Beach, Virginia, USA). A weighed portion (50-100 g) was then lyophilized for 48 h (Virtis model 10-324, Midland, ON, Canada), and the dry weight was determined. The samples were ground to pass through a 60-mesh sieve and stored at −20 • C until the bioactive substances were analyzed. The fruits were analyzed for firmness by measuring penetration force in kilograms using a fruit-firmness tester (Model KM, Fruit Test Tech, Tokyo, Japan). After peeling, the tester penetrates (punches) the flesh with hand pressing. The mean values of the firmness were expressed in Newtons (N): 1 N is 9.8 kg. The peeled fruits were homogenized and filtered through cheesecloth in order to obtain a clear juice for determination of TSS (Brix), pH, TA, and dry matter. The TSS was measured using a digital refractometer (Atago Co., Ltd., Tokyo, Japan), pH was checked with a pH meter (Model 8100, ETI Co., Ltd., Worthing, West Sussex, UK). The TA was measured in 4 mL of juice, diluted to 20 mL of distilled water and titrated with 0.1 N NaOH. The TA was expressed as a percentage of citric acid. After peeling, dry matter (%) was calculated by the ratio of initial fresh weigh/dry weight [3,19,23,24].

Sample Extraction
The lyophilized samples of kiwifruit and persimmon cultivars were extracted with deionized water (pH 3) during 60 min at 25 • C. For mass spectrometry (MS) measurements the samples were extracted with ethanol: water mixture (8/2, v/v) during 60 min at 25 • C. The proportion of sample to the solvent was 1/10w/v. The extracts were filtered in a Buchner funnel. After removal of the solvent in a rotary evaporator and the aqueous solution were freeze-dried [25,26].

Total Contents (T) of Phenolics (TPs), Flavonoids (TFAs), Flavanols (TFLs), Tannins (TNs), Vitamin C (VC) and Main Secondary Metabolites
The TPs content was determined by Folin-Ciocalteu colorimetric method [27], where 0.25 mL of phenolic extract solution (edible fraction) or standard was mixed with 1 mL of Folin-Ciocalteu reagent. In the next step 0.75 mL of 20% sodium carbonate was added and incubated for 6 min in a water bath at 45 • C. The absorbance of the resulting mixture was measured at 750 nm. Quantification of TPs in the samples was performed using a standard curve prepared with gallic acid, and the values were expressed as mg of gallic acid equivalent (GAE) per g dry weight (DW).
The TFA content [28] was measured in the mixture of 0.5 mL of the extract with 2.25 mL of distilled water, followed by the addition of 0.15 mL of 5% (w/v) NaNO 2 solution. After 6 min, 0.3 mL of a 10% AlCl 3 ·6H 2 O solution was added. The reaction was allowed to stand for another 5 min before addition of 1.0 mL of 1 M NaOH. The mixture was mixed well by vortexing, and the absorbance was measured immediately at 510 nm.
TFLs were estimated using the p-dimethylaminocinnamaldehyde (DMACA) method, where 0.2 mL of fruit extract was introduced into a 1.5-mL Eppendorf tube, and 1 mL of DMACA solution was added. The mixture was vortexed and allowed to react at room temperature for 10 min. The absorbance at 640 nm was then read against a blank prepared similarly without DMACA. The presence of flavanols on the nuclei subsequent staining with the DMACA reagent resulted in an intense blue coloration in fruit extract [29].
TNs were estimated by spectrophotometric measurements of 0.5 mL fruit extract, where 3 mL of a 4% methanol vanillin solution and 1.5 mL of concentrated hydrochloric acid were added [30]. The mixture was allowed to stand for 15 min. The absorption of samples and blank against water was measured at 500 nm.
A catechin standard was used for the elaboration of the analytical curve and the results of TFAs and TNs contents were expressed as mg catechin equivalent (CE) per g DW. TFLs content was calculated as µg CE per g DW. VC (mg Asc per g DW) was evaluated in fruit extracts, where 100 mg of freeze-dried fruit sample was extracted with 5 mL water. Then cupric reducing antioxidant capacity (CUPRAC) method was conducted and formed bis (Nc)-copper (I) chelate was determined spectrophotometrically at 450 nm [31].
The ethanolic extracts were submitted to MS analysis for main secondary metabolites determination and were processed exactly as described previously [1,32]. A mass spectrometer, a TSQ Quantum Access Max (Thermo Fisher Scientific, Basel, Switzerland) was used. All samples were analyzed by direct infusion in the mass spectrometer by electrospray ionization (ESI) in negative mode, full scan analysis, range of 100-900 m/z. For optimization of the acquisition parameters and for identity confirmation, only a part of standards was employed, not for all compounds that were found in the investigated samples.

Antioxidant Capacities
Four complementary assays were used for determination of antioxidant capacities: 2, 2-Azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diamonium salt (ABTS +· ) was generated by the interaction of ABTS (7 mM) and K 2 S 2 O 8 (2.45 mM). The mixture was kept in the dark at room temperature for 12-16 h before use. This solution was diluted until the absorbance reached 0.7 at 734 nm and equilibrated at 30 • C. After addition of 1.0 mL of diluted ABTS +· solution to 10 µL of extract or Trolox standards, the absorbance reading was taken 1 min after initial mixing and up to 6 min. The percentage decrease of the absorbance was calculated and plotted as a function of the concentration of the extracts and Trolox for the standard reference data [33]. and persimmon extract samples as the appropriate reagent blank. The absorbance was measured at 595 nm after 30 min [34].
Cupric reducing antioxidant capacity (CUPRAC) is utilizing the copper (II)-neocuproine [Cu (II)-Nc] reagent as the chromogenic oxidizing agent. To the mixture of 1 mL of [Cu (II)-Nc] and NH 4 Ac buffer solution, acidified and non acidified extracts of fruits (or standard) solution (x, in mL) and H 2 O ((1.1-x) mL) were added to make the final volume of 4.1 mL. The absorbance at 450 nm was recorded against a reagent blank [35].
The units for all antioxidant capacities were µM TE (Trolox equivalent) per g DW. The absorbances of all investigated resulted mixtures were measured on Hewlett-Packard, model 8452A spectrophotometer.

Fluorometric Measurements
Two (2D-FL) and three dimensional (3D-FL) fluorescence measurements for all kiwifruit and persimmon extracts at a concentration of 0.01 mg/mL were recorded on a model FP-6500, Jasco spectrofluorometer, serial N261332, Tokyo, Japan, equipped with 1.0 cm quartz cells and a thermostat bath. The 2D-FL measurements were taken at emission wavelengths from 310 to 500 nm and at excitation of 295 nm. The 3D-FL spectra were collected with subsequent scanning emission spectra from 200 to 400 nm at 1.0 nm increments by varying the excitation wavelength from 200 to 500 nm at 10 nm increments. For comparison of the obtained results gallic acid and epicatechin was used [1,10,32]. The solutions for the reaction were in the following concentrations: 1.0 × 10 −5 mol/L HSA; 0.05 mol/L Tris-HCl buffer with 0.1 mol/L NaCl, pH 7.4.

Protein and Ligand Preparation for Molecular Docking Studies
The potential interaction of ligands from the different varieties of kiwifruit and persimmon with HSA was investigated using Autodock docking program version 4.2 [37]. The crystal structure of the HSA was obtained from the RCSB Protein Data Bank (PDB code: 1H9Z). Prior to docking studies, the ligands were downloaded as 2D structure (.sdf format) from PubChem database and converted to 3D structure (.pdb format) (Supplementary Table S1). The receptor protein HSA was prepared by removing the water molecules, adding hydrogen atoms and by assigning partial charges based on the CHARMM force field. The ligand structures were minimized by applying MMFF94 Force Field and other parameters were set to their default. Furthermore, the binding cavity region of the receptor was set to spherical cut-off of 8 Å for non-bonding interactions. Finally, the docking protocol was applied to the processed protein and ligand structures. The resulting best poses were extracted and evaluated using the scoring algorithm and visualized through BIOVIA Discovery Studio 4.5 software (Dassault Systemes BIOVIA Corporate, San Deigo, CA, USA) [38].

Statistical Analysis
All obtained data were calculated on the basis of statistical analysis of Duncan's multiple range test. Values are means ± SD per gram dry weight (DW) of 25 measurements, representing commercial maturity status of fruits and their replicates. Five replications of five extracts from each cultivar were performed. To determine the statistical significance as 95% interval of reliability, ANOVA, one-way analysis on variance, was used.

Biologically Active Compounds and Secondary Metabolites in Investigated Fruits
The contents of total phenolics (TPs), flavonoids (TFAs), flavanols (TFLs), tannins (TNs) and vitamin C (VC) are presented in Table 1. The main active compounds in the fruits were polyphenols and vitamin C and their values were ranging from 32.37 ± 1.34 to 4.31 ± 0.21 mg GAE/g DW and from 36.51 ± 1.65 to 2.31 ± 0.23 mg AA/g DW, respectively (Table 1), showing the highest values for kiwifruit cultivar 'Bidan'. The obtained results differ from previously presented data [1,5,32,39], because of different conditions of extraction, including various solvents, temperature and time of the process. In the present research, as mentioned above, the extraction of bioactive substances was performed in water (pH 3), because for polyphenols most extractions are carried out under acidic conditions. The polyphenols are more stable in low pH, and the acidic condition helps polyphenols to stay neutral [25,26]. The aqueous extracts are important for the further use in the interaction of polyphenols with HSA, because such reactions appeared in human metabolism under similar conditions [40]. The present results (Table 1) are in line with other reports [20], evaluating that among various ripe kiwifruit grown in South Korea, 'Bidan' had the highest total phenolics and antioxidant capacity, and the lowest total flavonoids; whereas, another cultivar 'Chiak' had the highest level of total flavonoids, but the lowest antioxidant capacity. It was reported that the raw 'Hayward' kiwifruit had a VC content of 0.55 mg/g fresh weight (FW) [11]. A. arguta [20,41] showed lower results of VC of about 1.5-2.0 mg AA/g FW and TPs of 656-1400 mg GAE/kg FW than the obtained values in Table 1, but the present data well agreed with similar characterizations of the same fruits by other investigators [42]. The evaluation of 62 consumed fruits [18] showed that persimmon with 112.09 mg GAE/100 g FW was the seventh most beneficial fruit in terms of antioxidant and phenolic properties, a result that agrees well with the present results.

Antioxidant Capacities
Evaluation of the antioxidant capacities by 2, 2-Azino-bis (3-ethyl-benzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ferric reducing antioxidant power (FRAP), cupric ion reducing capability (CUPRAC) and 1, 1-diphenyl-2-picrylhydrazyl scavenging radical (DPPH) assays showed the highest values by CUPRAC between 103.81 ± 5.43 and 19.61 ± 0.93 µMTE/g DW for all kiwifruit cultivars and persimmon ( Table 1). The results calculated by ABTS, FRAP, CUPRAC and DPPH which connected with different kiwifruit cultivars are in agreement with several reports. So, the IC50 values of ABTS radical cation scavenging activities showed that A. arguta determined to be 1.26 mg/mL in comparison with A. deliciosa cv. Hayward of 22.72 mg/mL, presenting the strongest antioxidant activities among all tested kiwifruit cultivars, such as A. chinensis, A. polygama and A. macrosperma [6,20]. The estimated results of AAC (Table 1) showed that the ABTS values were 2.4 times greater than ADH. The antioxidant capacities of kiwifruit flour 'Hayward' in vitro by DPPH and FRAP assays were 20 and 25 µmol TE/g DW, respectively, and the free phenolic content was 14.57 mg GAE/g DW [7]. These results are superior to those presented in Table 1, where the DPPH and FRAP values were 12.45 and 10.28 µmol TE/g DW, respectively, and TPs were 6.27 mg GAE/g DW. These values exhibit good correlation between the polyphenols and their antioxidant capacities, showing that the cited results [7] were 1.6, 2.43 and 2.32 times higher for DPPH, FRAP and TPs, respectively, than the presented ones in Table 1. The values of persimmon 'Fuyu' showed TPs of 4.31 mg GAE/g DW, antioxidant capacities (µmol TE/g DW) by ABTS, FRAP and DPPH methods-16.48, 9.03 and 9.58, respectively, and VC of 2.31 mg AA/g DW (Table 1). These results can be compared with cv. Vanilla, in which total polyphenols were estimated of 1 mg GAE/g FW, antioxidant capacities by ABTS-5 µmol TE/g FW, FRAP-6 µmol TE/g FW, and DPPH-0.3 mg GAE/g FW. VC content was in the range of 0.1-0.2 mg/g FW [44]. Two innovative products (kaki and kiwi) were characterized by their bioactivity [8]. The total phenolics have ranged from 2.1 mg GAE/g DW (kiwi) to 8.7 mg GAE/g DW (kaki), while dried fruit antioxidant capacity was from 23.09 µmol Fe 2+ /g DW to 137.5 µmol Fe 2+ /g DW, as was shown in previous results [17]. The most important phytochemical class including apple, kiwi, and kaki dried fruits (from 74.6% to 93.3%), as it was demonstrated in other reports were phenolics [26]. The evaluation of bioactive substances of presently investigated fruits, including polyphenols, tannins, flavanols, flavonoids, vitamin C and antioxidant capacities (Table 1) well agreed with the data, discussed in recent investigations [39,45,46]. A positive and highly significant correlation between the contents of total phenols and radical scavenging capacities against all used radicals, and especially against ABTS and DPPH in persimmons, are in line with described data [21].

Binding Properties of Bioactive Compounds of Investigated Fruits with Serum Protein Measured by Fluorescence
The results of changing in the fluorescence intensity during interaction of polyphenols with HSA are shown in Tables 1 and 2 and Figure 2. For the standards were chosen gallic acid and epicatechin. The quenching of polyphenols extracted from persimmon 'Fuyu' and kiwifruit 'Hayward' were in the same range and were comparable with gallic acid and among other kiwifruit cultivars were the lowest. As it was described previously [1,2,5], the quenching of the protein depends on the amount of the fruit extract added to the reaction, then fluorescence intensity decreases, by the shifting in the emission value. The change of the fluorescence intensity after interaction with HSA was compared with HSA before interaction, and the peaks a and b were measured, and the binding properties were calculated. The binding properties measured by 2D-FL (Table 2) differ from the values estimated by 3D-FL (Table 1, Figure 2). In 3D-FL the main changes were found according to the decrease of fluorescence intensity mostly in peak a. The results showed that the binding properties of 'Bidan' were greater than other investigated fruits and estimated of about 51%, according to the changes in the fluorescence intensity of peak a (Table 1).  Figure 2). The high binding properties of the investigated fruits can be explained by their antioxidant capacities. The antioxidant capacities of 'Bidan' by DPPH and ABTS assays were 4.2 times greater than for 'Hayward' and the binding properties of 'Bidan' were also greater than for 'Hayward' by 2.8 times (Table 1). These results are in full agreement with the correlation analysis which demonstrated that the phenolic and flavonoid contents are responsible for increasing the scavenging activities of DPPH and ABTS, where protocatechuic and chlorogenic acids were the predominant phenolic acids in kiwifruit pulp, as well as gallic acid, epicatechin, and catechin [47]. The fluorescence results indicate that there was a static quenching mechanism in the interactions of gallic acid (GA) with BSA [48] and support the data obtained in Table 2, where the binding properties of gallic acid were slightly higher that for kiwifruit 'Hayward'. Epicatechin showed as well relatively high binding properties which were closed to the ability of kiwifruit A. Arguta (Table 2). These results are consistent with others [49], where the interaction of copper complexed with (-)-epigallocatechin-3-gallate (EGCG) and bovine serum albumin (BSA) was investigated using fluorescence. The fluorescence quenching efficiency of BSA by EGCG was enhanced after the formation of the complex of EGCG with copper. The EGCG-Cu complex exhibited a higher apparent binding affinity to BSA compared with EGCG alone. The binding affinities to HSA were ranked in the order that EGCG showed the highest value between the investigated compounds [40]. The synergetic effect of pure standards in comparison with the mixture of different antioxidants in the investigated fruits showed higher reactivity than the pure substances themselves [13,14,39]. Molecular docking supported the obtained results.  Table 1 (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).
Binding properties of 'Hayward' and 'Fuyu' were comparable and showed 18% and 14%, respectively (Table 1, Figure 2). The high binding properties of the investigated fruits can be explained by their antioxidant capacities. The antioxidant capacities of 'Bidan' by DPPH and ABTS assays were 4.2 times greater than for 'Hayward' and the binding properties of 'Bidan' were also greater than for 'Hayward' by 2.8 times (Table 1). These results are in full agreement with the correlation analysis which demonstrated that the phenolic and flavonoid contents are responsible for increasing the scavenging activities of DPPH and ABTS, where protocatechuic and chlorogenic acids were the predominant phenolic acids in kiwifruit pulp, as well as gallic acid, epicatechin, and catechin [47]. The fluorescence results indicate that there was a static quenching mechanism in the interactions of gallic acid (GA) with BSA [48] and support the data obtained in Table 2, where the binding properties of gallic acid were slightly higher that for kiwifruit 'Hayward'. Epicatechin showed as well relatively high binding properties which were closed to the ability of kiwifruit A. Arguta (Table 2). These results are consistent with others [49], where the interaction of copper complexed with (-)-epigallocatechin-3-gallate (EGCG) and bovine serum albumin (BSA) was investigated using fluorescence. The fluorescence quenching efficiency of BSA by EGCG was enhanced after the formation of the complex of EGCG with copper. The EGCG-Cu complex exhibited a higher apparent binding affinity to BSA compared with EGCG alone. The binding affinities to HSA were ranked in the order that EGCG showed the highest value between the investigated compounds [40]. The synergetic effect of pure standards in comparison with the mixture of different antioxidants in the investigated fruits showed higher reactivity than the pure substances themselves [13,14,39]. Molecular docking supported the obtained results.

Docking Studies
Fluorescence studies revealed the binding properties of kiwifruit and persimmon water extracts with HSA. To delineate the mechanisms at a molecular level, 15 compounds identified from kiwifruit and persimmon extracts were analyzed through docking studies. Ligands exhibiting high binding affinity towards HSA are shown in Figure 3. Caffeic acid, quinic acid, catechol, syringic acid,  Table 1 (for interpretation of the references to color in this figure legend, the reader is referred to the web version of this article).

Docking Studies
Fluorescence studies revealed the binding properties of kiwifruit and persimmon water extracts with HSA. To delineate the mechanisms at a molecular level, 15 compounds identified from kiwifruit and persimmon extracts were analyzed through docking studies. Ligands exhibiting high binding affinity towards HSA are shown in Figure 3. Caffeic acid, quinic acid, catechol, syringic acid, hesperidin and quercetin were identified to be the major ingredients [1,5,39] that are common among the three varieties of kiwifruit ('Hayward', 'Bidan' and 'Cheongsan') used in the current study (Table 3). From the interaction analysis, hesperidin and quercetin were identified as the top scorers among the compounds screened from the kiwifruit. Hesperidin was found to have a docking and binding energy score of −168.71 and −140.3 kcal/mol, respectively. Whereas, quercetin exhibited the highest dock score of −163 and binding energy score of −116.8 kcal/mol. Hesperidin (Figure 4) formed five conventional hydrogen bond interactions with three residues (Leu115, Arg117 and Leu185). hesperidin and quercetin were identified to be the major ingredients [1,5,39] that are common among the three varieties of kiwifruit ('Hayward', 'Bidan' and 'Cheongsan') used in the current study (Table 3). From the interaction analysis, hesperidin and quercetin were identified as the top scorers among the compounds screened from the kiwifruit. Hesperidin was found to have a docking and binding energy score of −168.71 and−140.3 kcal/mol, respectively. Whereas, quercetin exhibited the highest dock score of −163 and binding energy score of −116.8 kcal/mol. Hesperidin (Figure 4) formed five conventional hydrogen bond interactions with three residues (Leu115, Arg117 and Leu185).    In addition, a carbon-hydrogen bond interaction with Pro113 and a pi-donor hydrogen bond interaction with Tyr161 and Gly189 were exhibited. Other key interactions include pi-sigma (Ile142), pi-alkyl and alkyl interactions (Tyr138, Pro118 and Leu115). Quercetin formed three hydrogen bond interactions with Leu135 and Arg117 and one carbon-hydrogen bond and pi-donor hydrogen bond interaction with Tyr138. It formed van der Waals interaction with Leu135 and other stabilizing interactions such as pi-pi stacked (Tyr161), amide-pi stacked (Tyr161) and pi-alkyl interactions (Ala158, Ile142 and Leu182). Kaempferol and rutin are the major compounds reported only in In addition, a carbon-hydrogen bond interaction with Pro113 and a pi-donor hydrogen bond interaction with Tyr161 and Gly189 were exhibited. Other key interactions include pi-sigma (Ile142), pi-alkyl and alkyl interactions (Tyr138, Pro118 and Leu115). Quercetin formed three hydrogen bond interactions with Leu135 and Arg117 and one carbon-hydrogen bond and pi-donor hydrogen bond interaction with Tyr138. It formed van der Waals interaction with Leu135 and other stabilizing interactions such as pi-pi stacked (Tyr161), amide-pi stacked (Tyr161) and pi-alkyl interactions (Ala158, Ile142 and Leu182). Kaempferol and rutin are the major compounds reported only in 'Hayward' and 'Bidan' and not in Actinidia arguta. Kaempferol and rutin have a dock score of −138.14 and −82.84 and binding energy of −106.3 and −78.9, respectively. Among the ligands, rutin has the highest hydrogen bond energy of −16.9 kcal/mol on interactions with the receptor protein HSA. The highest interactions are evident from six hydrogen bonds from Tyr161, Lys190, Pro113 and Glu141 and other carbon-hydrogen and pi-donor hydrogen bond interactions from Arg186, Ser193 and Leu115. Besides, it forms pi-alkyl (Arg186 and Leu115) and pi-sigma interactions with HSA (Leu115). Protocatechuic acid (3, 4-dihydroxybenzoic acid) was identified only in 'Bidan' cultivar and has shown −90.03 as dock score and −69.0 kcal/mol as binding energy. It formed one conventional hydrogen bond interaction with Leu154, pi-donor hydrogen bond with Tyr161 and van der Waals interaction with Leu139. The other scaffold stabilizing interactions such as pi-pi stacked (Tyr138), amide-pi stacked (Tyr138) and pi-alkyl (Ala158) were also observed.
From persimmon 'Fuyu' extract six compounds were identified such as gallic, vanillic and caffeic acids, epicatechin, kaempferol, and quercetin (Table 4). Among them, caffeic acid, kaempferol, and quercetin are reported for kiwifruit varieties. Quercetin and kaempferol have achieved high binding affinity with HSA with the dock score of −163 and −138.14, respectively [4,5]. The ligand epicatechin has a dock score of −138.14, binding energy of −106.3 kcal/mol, van der Waals energy of −102.8 and the highest H-bond energy of −3.4, which might have favored the interaction with HSA (Table 4). Comparing the docking results, it was observed that the 'Bidan' cultivar among the kiwifruit has the maximum binding affinity with HSA and it is in good agreement with the fluorescence experiments where it showed the highest percentage of binding (51%) with HSA. All the kiwifruit cultivars (used in the current study) have hesperidin and quercetin ligands, which were predicted with high docking and binding energy values among all the ligands. Although, 'Bidan' shares similar ligands with 'Cheongsan', it possesses the distinct ligand named protocatechuic acid, which might have played a significant role in enhancing the binding affinity of the ligands to HSA receptor. Hence, 'Bidan' cultivar might have shown the highest binding in the fluorescence experiments. Interestingly, kaempferol and rutin are absent in 'Cheongsan'. However, 'Hayward' and 'Fuyu' have shown the least interaction with HSA in fluorescence results. HSA, a globular protein of 585 amino acids, comprises three homologous domains: I (5-197 residues), II (198-382 residues) and III (495-585 residues). Each domain has been further subdivided in to subdomain A and B (IA, IB, IIA, IIB, IIIA and IIIB). HSA has been reported to bind to a wide range of ligands at multiple sites with different binding affinities.
From our docking analysis, it is apparent that all the ligands identified from kiwifruit and persimmon extracts have binding pockets in domain I of HSA.
These results were in accordance with other investigations. Molecular docking showed that (-)-epigallocatechin-3-gallate (EGCG) mainly bound to subdomain IIA and IIIA Results of competitive binding experiments confirmed that the location of EGCG binding in BSA was site I [40,49]. Molecular docking analysis highlighted that GA binds at Sudlow site I of BSA and binding of GA at site I of BSA is stable [48]. Remarkably, domain I has been reported to bind hemin and fatty acids, and thus the site has been represented as a major drug binding pocket. The major residues involved in binding are Tyr138, Tyr161, Arg114, His146, Lys190, Ile142, Arg117 and Leu182. Among these Tyr138 and Tyr161 are the crucial residues involved in drug recognition [50,51]. Our results are consistent with these earlier reports wherein the identified ligands were found to have interactions with these major residues. Thus, from our docking studies it is envisaged that the presence of more potential ingredients in the 'Bidan' cultivar might have led to multiple binding pockets in HSA and thereby it might have enhanced the binding affinity towards HSA. Kiwifruit has also been reported for preventing platelet aggregation and has shown an effect on Angiotensin converting enzyme (ACE) [52]. Quercetin identified as one among the top hits has been reported with effective antiviral activity against herpes viruses and Dengue virus [53]. Similarly, kaempferol, quercetin and hesperidin were reported as promising drug candidates to prevent enterovirus 71 replication [54]. Thus, bioactive flavonoids identified and investigated in the present study with potential binding ability with HSA have several health benefits to the human system both as an immune booster as well as in terms of antibacterial and antiviral activities [55].

Conclusions
The exotic fruits such as kiwifruit and persimmon have been investigated previously and suggested to be used as food supplementation, especially for patients with cardiovascular risk factors. Spectrophotometric and fluorometric methods and molecular docking were applied for characterization of several kiwifruit cultivars and persimmon. Antioxidant capacities directly correlated with the amount of polyphenols and the quenching properties of extracted fruit polyphenols and human protein albumin. In the present study several bioactive ligands with antioxidant properties were identified from kiwifruit and persimmon by molecular docking. Quercetin, kaempferol, quercetin, epicatechin and hesperidin as bioactive flavonoids, possess antibacterial and antiviral activities. These bioactive ligands are also present in several fruits and vegetables. Based on the obtained results of secondary metabolites, we have selected only the major hit compounds with high abundance. Docking was performed for the selected (highly abundant) compounds and reported with the binding affinity. Docking studies are in good agreement with the experimental results. Since the main objective of the study was to report that the investigated fruits can serve as a promising functional food with medicinal properties, we validated this hypothesis with robust in silico docking approaches. Therefore, consuming such fruits and vegetables as such or as a complex mixture of phytochemicals can provide beneficial effects to human health by protecting against several infections. In addition to nutritional and antioxidant properties, kiwifruit and persimmon have potential role in pharmacological applications.