Determination of phenolic compounds in Diospyros lotus by RP-HPLC-DAD and evaluation of antioxidant and cytotoxic properties

This research aimed to extract and profile phenolic compounds from seed and fruit of Diospyros lotus, evaluate and compare the antioxidants and cytotoxic potential of the extracts. Seven phenolic acids and four other flavonoids extracts were subsequently obtained following different extraction methods. The extracts were evaporated, lyophilized, and prepared for RP-HPLC-DAD, MTT assay, and antioxidant evaluation by the ferrous ion chelating, ferric ion reducing power, FRAP, DPPH•, •OH and NO• scavenging methods. The RP-HPLC-DAD revealed an epigallocatechin, a flavonoid, and fourteen phenolic acids. Phenolic compounds with the highest concentrations are epigallocatechin (1460.80 ± 10.74 µg/gDW), gallic acid (1167.11 ± 4.04 µg/gDW) and t-cinnamic acid (244.45 ± 3.61 µg/gDW). The bound phenolic acids from acid hydrolysis-1 (BPAH-1) extract of the fruit sample exhibit the highest DPPH• activity, reducing power, and FRAP. The flavonol extract of the fruit sample unveils the highest Fe2+ chelating effect. The BPAH-1 and BPAH-2 extracts of the seed reveal the highest •OH and NO• activities, respectively. Seed sample gave the highest total phenolic/flavonoid/tannin contents. The cytotoxicity of the extracts on HepG2 cell lines > HeLa cell lines and the effects are more pronounced 48 h after treatments. Specifically, flavan-3-ol methanolic and BPAH-2 extracts of the seed, and flavan-3-ol methanolic and BPAH-1 extracts of the fruit show good cytotoxic effect against HepG2 cell lines (IC50 < 37 µg/mL). This research unveils the potential of D. lotus seed and fruit as possible antioxidant and anticancer agent. The antioxidant and cytotoxicity properties observed from the samples might be due to the phenolic compounds determined. Graphical abstract Graphical abstract

In this study, we extracted phenolic compounds from D. lotus seed and fruit using several solvent systems. Four flavonoid extracts (flavone, flavonol, flavan-3-ol methanolic and flavan-3-ol acidic), three free phenolic acids extracts (FPA, BHPA and AHPA) and four bound phenolic acids extracts (BPBH-1, BPAH-2, BPAH-1 and BPBH-2) were obtained. Phenolic compounds in the extracts were determined by means of RP-HPLC-DAD. The different extracts were evaluated for antioxidant and cytotoxic properties. Antioxidant evaluation was made through ferrous ion chelating ability, ferric ion reducing power, FRAP, DPPH • , • OH and NO • scavenging methods. Cytotoxicity evaluation against cancer cell lines was made by MTT assay. The novelty of the study is that, to the best of our knowledge, this is the first study that comprehensively extracted and profiled phenolic compounds in seeds and fruits of D. lotus and evaluated antioxidant capacities and cytotoxic potential of the phenolic extracts against HeLa and HepG2 cell lines.

Samples collection and preparation
The fruit samples of the mature D. lotus (Fig. 1) were collected from Bizim Yöresel, Ordu/Turkiye and washed thoroughly to remove any dust and impurities and then air-dried. Thereafter, seeds were removed from the fruit pods. Both the seeds and fruit pods were then shaded to dry for some days and lyophilized until constant weight. The lyophilized sample was ground into powder using a mill grinder.

Soxhlet extraction
To remove liposoluble substances from the plant parts, certain amounts of the milled powders were defatted with n-hexane for 4 h using Soxhlet equipment. The defatted brans were allowed to dry at room temperature and kept at 4 °C for further analysis.

Flavone extraction
Two (2 g) of the defatted bran was extracted with 150 mL diethyl ether on a thermo-shaker incubator (25 °C) for 20 min and filtered. The residue was again re-extracted 2 more times with 75 mL diethyl ether for 10 min, and the three filtrates were combined and evaporated in a fume hood [21].

Flavan-3-ol extraction
The defatted bran (2 g) was extracted three times with 40 mL of absolute methanol in an ultrasonic bath (60 °C) for 2 h, centrifuged at 5000 rpm (Hettich universal 320 R) for 10 min and filtered. The filtrates were pooled and labelled flavan-3-ol methanolic extract [23]. In acid hydrolysis, the dried pellets were firstly weighed and then extracted with 100 mL 2.5 M HCI-methanol (4:1, v/v) in a water bath for 2 h at 100 °C, centrifuged (5000 rpm) for 10 min and filtered. The filtrate was subjected to liquid-liquid extraction (LLE) three times with 40 mL ether and four times with 40 mL ethyl acetate. The ether layer was concentrated (up to approximately 2 mL) to dryness under vacuum in a fume hood. The ethyl acetate layer was evaporated with a rotavapor at reduced pressure for up to approximately 2 mL aliquot. The two aliquots of ether and ethyl acetate were combined and labelled flavan-3-ol acidic extract. The flavone, flavonol and flavan-3-ol methanolic and acidic extracts, were concentrated to dryness with a rotavapor and lyophilized. The extraction yields of each extract were eventually determined. Thereafter, lyophilized extracts were kept in deep freezer till further analysis.

Phenolic acids extraction
The defatted sample (6 g) was extracted twice with 120 mL 80% methanol on a magnetic mixer at room temperature for 1 h, centrifuged (5000 rpm) for 10 min and filtered. The residues were allowed to dry before being subjected to assays related to bound phenolic acids. The two filtrates were combined, evaporated with a rotavapor at reduced pressure, freeze-dried and lyophilized. The lyophilized crude methanolic extract (CME) was further extended for analysis to obtained extractable phenolic acids [24].

Extractable phenolic acids
The CME of the two samples were liquefied in 12 mL of acidified water (pH 2.0 with HCl) and subjected to LLE three times with 30 mL ether. The three ether layers were combined, concentrated dryness under vacuum in a fume hood and labelled free phenolic acids (FPA) extract. The aqueous part was neutralized (pH 7.0) with 2 M NaOH, dried and lyophilized. The lyophilized extract was liquefied detection was achieved with a G1315B diode array detector. The column temperature was set at 25 °C and the injection volume was 20 µL.

Chromatographic analysis of flavone
The solvent phase consisted of A: 5% aqueous formic acid, v/v, and B: HPLC grade methanol. A volumetric flow rate of 1 mL/min was set for a total run time of 35 min. The sample was eluted with the following gradient: 0-5 min 50% A, 5-30 min 40% A and 30-35 min 20% A before returning to the initial conditions. The chromatograms were recorded at 350 nm [21].

Chromatographic analysis of flavonol
The solvent system consisted of A: 0.5% aqueous orthophosphoric acid, v/v and B: HPLC grade methanol. A flow rate of 1 mL/min was set for 30 min. The sample was eluted with the following gradient: 0-10 min 60% A, 10-21 min 40% A, 21-23 min 40% A and 23-30 min 60% A. The chromatogram peaks were monitored at 254 nm for isoquercetin and quercitrin, and at 370 nm for rutin, kaempferol, isorhamnetin, myricetin and quercetin [22].

Chromatographic analysis of phenolic acids
The solvent system is comprised of HPLC grade acetonitrile (A) and 2% (v/v) aqueous acetic acid solution (B). A flow rate of 1 mL/min was set for 60 min. The sample was eluted with the following gradient: 0-30 min 100% B, 30-50 min 85% B, 50-55 min 50% B and 55-60 min 0% B. Chromatographic peaks were observed at 280 and 320 nm for the benzoic acid and cinnamic acid derivatives, respectively [24].

Qualitative and quantitative analysis of phenolic compounds
Flavonoids and phenolic acids were recognized from their retention time and observing similarities between the spectral features of their peaks and those of available standards. Quantitation was achieved from the calibration plots in 12 mL 2 M NaOH, stirred at room temperature for 4 h, changed to pH 2.0, subjected to LLE as stated earlier, and the resultant was labelled alkaline-hydrolysable phenolic acids (BHPA) extract. The aqueous layer obtained after BHPA extraction was immediately treated with 12 mL 6 M HCl, boiled at 95 °C for 20 min, subjected to LLE, and labelled acid-hydrolysable phenolic acids (AHPA) extract. The FPA, BHPA and AHPA extracts were freeze-dried and lyophilized. The extraction yields were eventually determined.

Bound phenolic acids extraction
The dried residue obtained following initial methanolic (80%) extraction was divided into two parts and subjected to hydrolysis to extract hydrolysable phenolic acids in the bran that were not extracted by the 80% methanol. The hydrolysis was carried out using two different protocols. Protocol I started with alkaline hydrolysis, in which one part of the residue was extracted with 60 mL 2 M NaOH at room temperature for 4 h, centrifuged (5000 rpm), filtered, and the supernatant was labelled bound phenolic acids from basic hydrolysis-1 (BPBH-1) extract. The pellet was subjected to acid hydrolysis with 6 M HCl at 95 °C for 1 h, centrifuged (5000 rpm), filtered, and labelled bound phenolic acids from acid hydrolysis-2 (BPAH-2) extract. In protocol 2, acid hydrolysis was made first then followed by alkaline hydrolysis, and two other extracts (BPAH-1 and BPBH-2) were obtained. The four extracts, BPBH-1, BPAH-2, BPAH-1 and BPBH-2, were acidified to pH 2.0 and subjected to LLE three times with 60 mL ether. The ether phases were combined, concentrated dryness under vacuum in a fume hood, freeze-dried and lyophilized. The extraction yields of the four extracts were eventually determined.

Total polyphenols extraction
A total of 2 g of the defatted bran was extracted with 160 mL aqueous methanol (methanol: water, 3:1, v/v) in a Soxhlet apparatus for 4 h, evaporated with a rotavapor at reduced pressure. Thereupon, the extract was concentrated under vacuum in a fume hood, freeze-dried and lyophilized. The extraction yield was then determined.

RP-HPLC-DAD analysis
The lyophilized flavonoids and phenolic acids extracts were reconstituted in methanol to 1000 µg/mL for HPLC analysis and antioxidant activity determination. The RP-HPLC-DAD analysis were performed using an Agilent technologies 1100 series. The separation was accomplished by a C18 reverse-phase column (inner diameter: 4.6 mm, length: 150 mm, particle size: 5 μm). The chromatograms where A 0 = absorbance of blank; A 1 = absorbance of sample.

Hydroxyl radical scavenging assay
The deoxyribose assay was employed to evaluate the extract • OH scavenging ability as described by Halliwell et al. [29].

Nitric oxide scavenging assay
The NO • scavenging ability of the extract was determined by using an SNP generating NO • system [30]. The Griess reagent was obtained by mixing an equal volume of 2% (w/v) sulphanilamide prepared with 4% H 3 PO 4 solution, and 0.2% (w/v) NEDA·2HCl solution. Extract (200 µL) at different concentrations were combined with 500 µL 10 mM SNP solution and 1175 µL PBS buffer 20 mM, and incubated in light condition at 25 °C for 150 min. Subsequently, 500 µL of an upper layer of solution was pipetted and diluted with 500 µL of Griess reagent. The absorbance of the chromophore produced during diazotization process of nitrite with sulphanilamide and subsequent coupling with the NEDA·2HCl was taken at 542 nm. Vitamin C was used as a positive control.

Ferrous ion chelating assay
The binding of Fe 2+ by the extract was measured following the Dinis et al. [31] method. Extract (200 µL) at different concentrations were mixed with 20 µL 1 mM FeCl 2 and 740 ddH 2 O and incubated in the dark at room temperature for 30 min. The reaction was initiated on the addition of 40 µL 5 mM ferrozine and re-incubated. A blank reading without an extract or standard was also prepared. After the reaction mixture had been equilibrated for 10 min, the absorbance was taken at 562 nm. The EDTA was used as a positive control. The percentage of inhibition of Fe 2+ -ferrozine complex was obtained using Eq. 1 and the IC 50 was eventually obtained.
acquired by plotting peak areas versus the concentrations of standard solutions. The standards calibration curves of the phenolic compounds are shown in Table S1 in supplementary file.

Total phenolic content
The total phenolic content (TPC) was evaluated following the Folin-Ciocalteu method [25]. Extract (30 µL), were combined with ddH 2 O (282.5 µL) and FCR (62.5 µL), and allowed to react at room temperature for 6 min. Thereafter, 345 µL of Na 2 CO 3 (7%, w/v) was added and incubated at room temperature in the dark condition for 2 h, and the absorbance was recorded at 760 nm. The results are presented in mg gallic acid equivalent per gram dry weight (mg GAE/g DW ).

Total flavonoid content
The total flavonoid content (TFC) was evaluated following the aluminium chloride method [26]. Extract (200 µL), ddH 2 O (1120 µL), 10% (w/v) AlCl 3 (40 µL) and 1 M sodium acetate (40 µL) were mixed, incubated away from light at room temperature for 30 min, and the absorbance was taken at 415 nm. The results are expressed in mg quercetin equivalents per gram dry weight (mg QE/g DW ).

Total tannin content
The total tannin content (TTC) was evaluated following the vanillin/HCl method [27]. To 100 µL of extract, 600 µL 4% (v/v) vanillin solution and 300 µL concentrated HCl were added, incubated in the dark condition at room temperature for 20 min, and the absorbance was measure at 500 nm. The results are expressed in mg tannic acid equivalents per gram dry weight (mg TAE/g DW ).

DPPH radical scavenging assay
Each extract (200 µL) at various concentrations were mixed with 550 µL methanol and 250 µL 1 mM DPPH • reagent, incubated in the dark condition at room temperature for duration of 30 min, and the absorbance was recorded at 517 nm. A blank reading without an extract or standard was also prepared [28]. Vitamin C was used as a positive control. The DPPH • , • OH and NO • scavenging activity of the extracts was measured using Eq. 1 and the IC 50 values were subsequently determined.

Statistical analysis
Each assay was done thrice from the same extract. All values are expressed in mean ± S.D. The data obtained were analysed by student's t-test and one-way analysis of variance (ANOVA) test using IBM SPSS Statistics 25. The oneway ANOVA is followed by a post hoc analysis for multiple comparisons. Tukey-Kramer's multiple comparisons test was used to determine the significant differences. A p < 0.05 was considered statistically significant.

Extractions yields
Flavonoids and phenolic acids were extracted from the two plant samples using different reagents. The results of extractions yields are presented percentage (%) and are shown in Table 1. Except for BHPA and BPAH-2 extractions, D. lotus fruit showed higher yields in the remaining extractions.

Phenolic acids profile
Phenolic acids are the second most abundant plant's polyphenols. They are described as aromatic acid compounds consisting of a phenolic ring and carboxylic group (-COOH) attached to the C 6 -C 1 bridge. Phenolic acids have medicinal properties and evidence of their role in disease prevention are studied. They can act as therapeutic agents against oxidants, cancer, inflammatory agents, bacteria, and fungi [35].

Ferric ion to ferrous ion reducing power assay
Ferric ion reducing ability of the extracts was investigated according to Oyaizu [32] method. Extracts (200 µL) were mixed with 500 µL 0.2 M phosphate buffer (pH 6.6) and 500 µL 1% (w/v) K 3 Fe(CN) 6 , and incubated at 50 °C for 20 min. Thereafter, 0.5 mL 10% (w/v) TCA solution was added and centrifuged (3000 rpm) for 10 min. A portion (500 µL) of an upper layer was mixed with 0.5 mL ddH 2 O and 0.1 mL 0.1% (w/v) FeCl 3 . Absorbance was recorded at 700 nm and results are expressed in mg vitamin C equivalent (VCE) per g DW .

Ferric reducing antioxidant power (FRAP) assay
FRAP solution was prepared by combining 25 mL 300 mM acetate buffer (pH 3.6), 2.5 mL 10 mM TPTZ and 2.5 mL 20 mM FeCl 3 solutions, and then warmed at 37 °C for 10 min before use [33]. Extracts (75 µL) were added to 1425 µL FRAP solution and incubated in the dark condition at 37 °C for 30 min. Reading of the intense colored product [Fe 2+ -(TPTZ) 2 ] 2+ was taken at 593 nm and results are expressed in mg VCE per g DW .

MTT assay
The MTT assay was used to test the cytotoxicity effects of the extracts against HeLa and HepG2 cell lines. The assay was carried out in accordance with the Mosmann [34] method and adapted as follows. Cell lines at a density of 1.5 × 10 5 cells/mL per well were seeded in flat-bottom microplates (96-well) and allowed to adhere in a humidified incubator at 37 °C with 5% CO 2 . After incubation, the culture medium was carefully removed and then treated with the 20-80 µg/mL concentrations of the extract prepared in 10% (v/v) dimethyl sulfoxide and incubated for 24 or 48 h at 37 °C in a 5% CO 2 incubator. The control cells were treated with only 10% dimethyl sulfoxide. After incubation, 20 µL 5 mg/mL of MTT solution was added per well and the plates were further incubated for 3.5 h. Next, medium was carefully pipette and discarded and the formazan crystals were solubilized by adding 100 µL of dimethyl sulfoxide per well and the absorbance was measured using the microplate reader at 570 nm. The percentage inhibition (I) was obtained using Eq. 1 and the IC 50 values were determined by plotting percentage inhibition against a corresponding concentration of the extracts. A low value of IC 50 implies greater cytotoxicity activity. and Kim et al. [24] in which they opined that some phenolic acids occur as conjugates with carbohydrates, fatty acids, or proteins. Acidic and basic hydrolyses are usually employed to break down the ester bond [24]. Phenolic acids can bind to the plant's cell walls and help them to defend themselves against invading pathogens [37]. The bound phenolics can also protect the plants against biotic and abiotic stresses, freezing tolerance, drought resistance [7]. Moreover, the esters bond formed by the bound phenolic acids in conjugation with other molecules can help to protect the cells against oxidation caused by free radical species, such as ROS [37]. Table 3 shows the profile of individual phenolic acids determined in the different extracts of D. lotus fruit. Five benzoic acid derivatives (4-hydroxybenzoic, gallic, protocatechuic, syringic and vanillic acids), along with five cinnamic acid derivatives (chlorogenic, ferulic, sinapic, o-coumaric and sinapic, p-coumaric and t-cinnamic acids, were determined and quantified. The chromatograms of the seven extracts of D. lotus seed are presented in supplementary file (Figs. S1 -S7). Some of the phenolic acids (gallic, protocatechuic, caffeic, p-coumaric and t-cinnamic) determined in this study were also identified from the fruit and seed of D. lotus in another study carried out in Trabzon/Turkiye [15]. This suggest that Turkish persimmon is reach with the stated phenolic acids. Different extracts of D. lotus seed contained different phenolic acid profiles. Few phenolic acids were determined in the CME; three as FPAs, two as BHPAs and four as AHPAs. The individual bound phenolic acids were higher than the extractable phenolic acids determined in the CME. Four were identified in BPAH-1 and BPBH-1, six in BPAH-2 and eight in BPBH-2 fractions. The most abundant phenolic acids determined in D. lotus seed were four benzoic acid derivatives (vanillic, 4-hydroxybenzoic, protocatechuic and gallic acids), and two cinnamic acid derivatives (o-coumaric and t-cinnamic acids). These phenolic acids were determined in three or more extracts. In terms of quantity, gallic acid determined in FPA (94.10 ± 1.67 µg/g DW ), BHPA (27.13 ± 0.61 µg/g DW ) and BPAH-1 (233.34 ± 3.84 µg/g DW ) extracts; t-cinnamic acid determined in AHPA (244.45 ± 3.61 µg/g DW ), BPAH-2 (89.40 ± 1.35 µg/g DW ) and BPBH-2 (95.27 ± 1.62 µg/g DW ) extracts; and protocatechuic acid determined in BPBH-1 compounds, including phenolic acids. Therefore, it can also be used to manufacture bioactive compounds with desired qualities in the cosmetic, food, and nutritional industries.

Flavonoids profile
Flavonoids are the most diverse groups of polyphenols, incorporating two or more aromatic rings, with one or more OH groups connected by a carbon bridge [38,39]. The current study determined only one flavonoid, an epigallocatechin, in significant amount in the flavan-3-ol acidic extract of both seed and fruit. The chromatogram peaks in the other fractions do not possess spectral characteristics that are like the available standard, hence not determined. The results are shown in Table 4 and the chromatograms are presented in supplementary file (Figs. S14 -S15). The biological properties of flavonoids are accredited to their configurational structure, the position of functional groups, and the total number of hydroxyl groups attached to the structure [40]. Epigallocatechin has multiple pharmacological effects and has been used in the treatments of cancer, oral diseases, CVD and NDD [41]. Flavonoids have shown anticancer effect by acting as pro-oxidants in cancer cells and help in activating apoptotic pathways. The effect is attributed to the presence of phenolic hydroxyl groups [42].
t-cinnamic acids), were recognized and quantified by RP-HPLC. These phenolic acids were determined in six extracts (i.e., FPA, BHPA, BPAH-1, BPAH-2, BPBH-1, BPBH-2). The chromatograms of the six extracts of D. lotus fruit are presented in supplementary file (Figs. S8 -S13). Some of the phenolic acids (gallic, protocatechuic, t-cinnamic, syringic and ferulic acids) determined in this study were also identified in D. lotus fruit from recent studies in Turkiye and China [15,17,18]. The chromatogram peaks in the AHPA fraction do not possess spectral characteristics that are like the available standard, hence not determined. The bound phenolic acids were quantified in significant concentrations than the extractable phenolic acids determined in the CME. D. lotus fruit is rich in gallic, protocatechuic, vanillic, ferulic and o-coumaric acids; they were detected in relatively high amounts in three or more extracts. Except for the BPBH-2 extract, gallic acid appeared as the major component of the remaining five extracts, namely FPA, BHPA, BPAH-1, BPAH-2 and BPBH-1. Edible fruits like persimmon (D. lotus) can synthesize variety of phenolic

Total polyphenolic content
The total polyphenol content of D. lotus seed and fruit extracts are shown in Table 5. The TPC was evaluated by the Folin-Ciocalteu method which is based on the transfer of electrons from a phenolic compound that is energetically oxidized in an alkaline medium to phosphomolybdic acid (a strong acid and oxidant compound present in FCR) [25]. The TFC was determined using the AlCl 3 . In this assay, the AlCl 3 in the reaction mixture will bind with flavone and flavonol group in either the C 4 keto group, C 3 or C 5 OH group, or the ortho-dihydroxyl group in the A-and B-ring group of flavonoids and generate a stable complex [43]. Measurement of the extent of complex formation would indicate the flavonoids contents in the extract. TPC, TFC -CH = CHCOOH group of the hydroxycinnamic acid derivatives, and -CH 2 COOH group of the hydroxyphenyl acetic acid derivatives has the weakest electron-donating potential. An electron-donating group can reduce the dissociation energy of the phenolic OH bond and then intensify its proton donation ability, free radical scavenging capability, and chelate formation.

• OH Scavenging Activity
The • OH scavenging assay is a widely used method to detoxify • OH, a highly reactive member of ROS. • OH, is a strong oxidants and very unstable entities that are generated in a wide range of environments by Fenton reaction. The • OH have been found to attack and damage biological structure and initiates lipid peroxidation [51,52]. Phenolic compounds, for example phenolic acids and flavonoids, often show protective effects against oxidation of biological structure via • OH scavenging property [51]. Data obtained in this study showed that the • OH scavenging ability of the extracts increase in a concentration-dependent manner. As it can be seen from Table 6 all the extracts exhibited good • OH scavenging activities (IC 50 = 9.28 ± 0.13-16.48 ± 0.12 µg/ mL). Specifically, the seed BPAH-1 extract shows the highest • OH scavenging activity. This is closely followed by the flavonol extract of the fruit, flavan-3-ol methanolic and BHPA extracts of the seed and fruit (IC 50 = 10.08 ± 0.16-10.36 ± 0.16 µg/mL). The statistical analysis on the mean values of these five extracts revealed no significant difference among determinations (p > 0.05). The current study revealed a better result in comparison with earlier reported study in China [46]. Phenolic acids, 4-hydroxybenzoic, gallic, protocatechuic and p-coumaric determined were detected in significant amounts in the seed BPAH-1 extract, and they could be responsible for the exceptional • OH scavenging activity observed in the extract. In addition, these phenolic acids have multiple OH groups in their structures, they can scavenge • OH through proton donation.

NO • scavenging activity
NO • is an extremely unstable species, it readily combines with molecular O 2 to generate stable compounds, such as nitrate (NO 3 -) and nitrite (NO 2 − ). The Griess assay is usually employed for the measurement of NO • production in living systems. The chemistry of the Griess test is based on the principle that at physiological pH, SNP in aqueous solution spontaneously produced NO • , which will subsequently be combined with molecular O 2 to generate NO 2 − that can be measured by Griess reagent [53,54]. Any scavenger of NO • in the reaction mixture compete with O 2 leading to the reduction of NO 2 − generation. The result derived in

In vitro antioxidant activities
The antioxidant activities of flavone, flavonol, flavan-3-ol methanolic, flavan-3-ol acidic, and seven phenolic acids extracts of D. lotus seed and fruit are presented in Table 6. The antioxidant properties of phenolic compounds originate from their properties of proton donation, and scavenging of radicals such as DPPH • , • OH and NO • . In antioxidant assays, the lower the IC 50 value of DPPH • , • OH and NO • scavenging, and ferrous ion chelating assays, the higher the antioxidant potential. Also, the higher the value obtained from ferric ion reducing power and FRAP assays, the higher the antioxidant capacity of the extract. The ability to scavenge free radicals is an essential metric for assessing natural compounds' antioxidant capability [47]. Different letters in the same column denote significant differences at p < 0.05.

DPPH • scavenging activity
DPPH • scavenging assay is a widely used stable free radical scavenging method to evaluate the antioxidant capacity of bioactive compounds and food extracts [46]. The DPPH • scavenging capacity of a phenolic extract is attributed to its proton donating ability. In this assay, hydrogen is donated by an antioxidant compound to a free stable DPPH • and converted into DPPH-H. In doing so, the DPPH • reagent colour is reduced. The decrease in absorbance reflects the extent of the DPPH • scavenging power of the antioxidant [48]. In this study, it was found that the DPPH • scavenging activities of all the extracts increased with increasing concentration ( Table 6). The flavan-3-ol methanolic and FPA extracts of the seed, flavan-3-ol acidic, BPAH-1 and BPAH-2 extracts of the seed and fruit show the largest DPPH • scavenging capacity (IC 50 = 45.42 ± 0.68-50.71 ± 0.51 µg/mL). The statistical analysis on the mean values of these extracts revealed no significant difference among determinations (p > 0.05). These extracts yield better DPPH • scavenging capacities in comparison with previously reported works [20,46,49,50]. Some phenolic acids determined by RP-HPLC in these extracts: seed FPA extract (gallic, protocatechuic and rosmarinic), seed BPAH-1 extract (4-hydroxybenzoic, gallic, protocatechuic and p-coumaric), seed and fruit BPAH-2 extracts (hydroxybenzoic, gallic, protocatechuic, vanillic, o-coumaric and t-cinnamic), fruit BPAH-1 extract (gallic and chlorogenic); could be responsible for the strong DPPH • scavenging activity. These activities might be related to the electron-donating potential of the carboxylic acid groups attached to the phenyl groups of these benzoic and cinnamic acids derivatives. According to Chen et al. [8], the -COOH group of the hydroxybenzoic acid derivatives has the strongest electron-donating ability, followed by the the concentration of the extracts. As can be seen in Table 6, flavonol extract of the fruit exhibit the highest Fe 2+ chelating power (IC 50 = 62.32 ± 1.08 µg/mL). This followed by the seed BPBH-1 extract (IC 50 = 71.55 ± 0.95 µg/mL), fruit FPA extract (IC 50 = 74.27 ± 0.92 µg/mL), and seed FPA extract (IC 50 = 78.09 ± 1.08 µg/mL). Nevertheless, flavone extract from the fruit, and flavan-3-ol acidic, AHPA and BPAH-2 extracts from the seed were the worst Fe 2+ chelators (IC 50 > 200 µg/mL). Some of the extracts of the current exhibited higher Fe 2+ chelation than the previous studies [49,59]. In contrast, Moghaddam and colleagues [20] found higher potent chelating activity in D. lotus seed extract collected from Iran (IC 50 = 42 ± 2.54 µg/mL). The higher potent activity observed by the researchers could be due to the different in the sample geographical locations, extraction time and solvent used for the extraction in comparison with the current study. The study used more polar solvent (distilled water) for the extraction. The results of the current study suggest that D. lotus seed and fruit have chelating activity and therefore capture Fe 2+ first before ferrozine thus disrupting the formation of radical species and these chelating properties are related to the phenolic compound determined. Some of the phenolic compounds with possible Fe 2+ chelating capability determined in the extracts that showed high chelating power are: hydroxybenzoic, gallic, protocatechuic, rosmarinic, sinapic, vanillic, ferulic and o-coumaric.
Interestingly, the chromatogram peak observed in the fruit flavonol extract that exhibited the highest Fe 2+ chelating power (IC 50 = 62.32 ± 1.08 µg/mL) does not correspond to our available standard hence no flavonol compounds were determined. In the reaction mixture of ferrous ion chelating assay, Fe 2+ react with ferrozine to form Fe 2+ -ferrozine complex. But, in the presence of chelating molecule, the complex formation is disrupted. Thus, the agent captures ferrous ion first before ferrozine, which in turn decrease the formation of radical species [60]. The Fe 2+ can bind to the phenolic structures at several coordination sites and therefore be chelated. The possible coordination sites of Fe 2+ to the flavonoids structures include a) in-between 5-OH and 4-carbonyl group, b) in-between 3-OH and 4-carbonyl group, c) in-between 3',4'-OH group in B ring. The possible coordination sites of Fe 2+ to the phenolic acids structures include (a) in-between 3-methoxy and 4-OH groups (b) in-between 3-OH and 4-OH groups. It is worth mentioning that chelating agents are effective as secondary antioxidants because they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion [61].

Ferric ion to ferrous ion reducing power
Ferric ion reducing power assay method is based on the principle that antioxidants, which have reduction potential, this study presented the D. lotus as a potent scavenger of NO • generated through the Griess system. Different seed and fruit extracts of D. lotus exhibited different potent NO • scavenging activity. Like the DPPH • and • OH scavenging assays, the scavenging activity of D. lotus extracts on NO • increases with an increase in the concentration of the extract. The seed BPAH-2 extract shows an exceptional NO • scavenging activity (IC 50 = 24.16 ± 0.15 µg/mL). It is closely followed by the flavan-3-ol acidic extracts obtained from seed (IC 50 = 34.91 ± 0.49 µg/mL) and fruit (IC 50 = 37.35 ± 0.54 µg/mL). D. lotus extracts of the current study revealed more promising results than those determined in two Iranian studies [20,49]. The NO • scavenging activity of phenolic compounds could be related to the methoxy and the phenolic groups attached to their structures [30]. NO • has double-edged sword functions, it serves as a key cell signalling molecule and it has a significant role in homeostasis, neurotransmission, immune defences, vasodilation and can inhibit cancer growth [3]. However, overproduction of NO • in living cells can lead to nitrosative stress that could cause nitrosylation reactions that can affect the structure of proteins and so inhibit their normal function [55]. Furthermore, excess generation of NO • and other nitrogen species in biological systems is known to cause inflammation, liver and kidney injury, cancer, and other pathological conditions [56]. Scavenging NO • could help to arrest the chain of reactions started by overproduction of NO • which are injurious to human health. The D. lotus of the current study have shown the property to curb the deleterious effects of NO • generation in the human body and may be of considerable interest in preventing the ill effects. As a result, the seed or fruit of the plant may be useful in the production of bioactive compounds that may be utilized to reduce/inhibit the quantity of NO • produced in living cells.

Ferrous ion chelating power
Transition metal ion, Fe 2+ can donate a single electron to several compounds, and therefore generate radical species [57]. Fe 2+ plays a significant role as catalysts of oxidative processes, that will lead to the decomposition of H 2 O 2 and formation of • OH by Fenton reaction [52]. Fe 2+ chelation is crucial in avoiding ROS and RNS generation that can cause oxidative damage to several compounds including lipids, carbohydrates, membranes, proteins, lipoproteins, DNA and RNA [5]. Also, the chelation of Fe 2+ is of great significance and would provide an effective therapeutic approach in the management of NDD, CVD, cancer and diabetes that is caused by the radical species [58]. Taking this into account, the current study evaluated the Fe 2+ chelation ability of D. lotus extracts. The chelating powers of all the extracts were found to increase with an increase in ferric ion by binding to TPTZ, therefore protecting cells from the detrimental effects of ferric ion.

In vitro cytotoxic activities
The in vitro cytotoxic activity of different extracts of D. lotus seed and fruit on HeLa and HepG2 cell lines were evaluated by MTT assay. The assay is based on the ability of the cellular mitochondrial dehydrogenase enzyme to reduce the yellow MTT into purple formazan crystals that is quantitatively measured at 570 nm [65]. The effect of the extracts at different concentrations on HeLa and HepG2 cell lines were tested for 24 and 48 h. As it can be seen from Fig. 2 The cytotoxicity effect of the extracts on HepG2 cell lines was also investigated for 24 and 48 h (Fig. 3). Most of the extracts show a good cytotoxic effect against HepG2 cell line (IC 50 < 37 µg/mL). Precisely, seed flavan-3-ol methanolic extract (IC 50 = 33.89 ± 1.69 µg/mL) and seed BPAH-2 extract (IC 50 = 32.46 ± 1.53 µg/mL), and fruit flavan-3-ol methanolic extract (IC 50 = 33.62 ± 1.81 µg/mL) and fruit BPAH-1 extract (IC 50 = 36.43 ± 1.85 µg/mL) were obtained. The good activities exhibited by these extracts could be as a result of the phenolic compounds identified by RP-HPLC-DAD, such as epigallocatechin, gallic and t-cinnamic acids. Results obtained in this study is similar with previous study [68]. Yue et al. [68] reported significant anticancer effect, 40.98% mortality rate of D. lotus extract on HEGP2 cell lines. Furthermore, the current study exhibits an excellent cytotoxic effect on HepG2 cell lines 48 h after treatments in comparison with the previous work [66]. The researchers react with potassium ferricyanide to generate potassium ferrocyanide, which then reacts with ferric chloride to generate intense coloured Prussian blue complex that possess a strong absorbency at 700 nm [62]. The change in absorbance at λ max of 700 nm is strongly linked to the reduction potential of the electron-donating compound present in the reaction mixture. Higher absorbance of the reaction mixture indicate greater Fe 3+ to Fe 2+ transformation ability, thus, higher reducing power of a compound. Table 6 shows the Fe 3+ reducing power of different extracts of D. lotus ranging in concentrations from 86.19 ± 0.61 to 1011.22 ± 3.33 mg VCE per g DW . Fruit BPAH-1 extract is the best reductant with the mean reducing power of Fe 3+ to Fe 2+ as 1011.22 ± 3.33 mg VCE per g DW . Likewise, flavan-3-ol methanolic, flavonol and flavone extracts from fruit show the worst Fe 3+ to Fe 2+ transformation ability. Statistical analysis on the mean values of these three extracts revealed no significant difference among determinations (p > 0.05). All the extracts in this study exhibit significantly higher Fe 3+ to Fe 2+ reducing power than those obtained in other studies [46,49]. Ferric ion to ferrous ion by a reducing agent or an antioxidant compound has been used as an indicator of the electrondonating capacity of such compound [62,63]. The reducing ability of any compound could signify its potential antioxidant property. The reducing ability of D. lotus seed and fruit may originate from phenolic compounds present in their extracts which can donate electrons to the Fe 3+ and reduce it Fe 2+ ion that can comfortably be captured through ferrous ion-chelating, which in turn decrease the formation of radical species and eventually ameliorate the progression of diseases caused by the radical species. Hence, the D. lotus employed in the present research might be exploited as a natural antioxidant source with ion reduction properties.

FRAP
The FRAP is a colorimetric assay based on the capacity of antioxidants to reduce the [Fe 3+ -(TPTZ) 2 ] 3+ complex to the [Fe 2+ -(TPTZ) 2 ] 2+ form at low pH. The end-product [Fe 2+ -(TPTZ) 2 ] 2+ has an intense blue colour and can be observed by measuring the change in absorbance at 593 nm [64]. The intense color gave higher absorbance, and the increase of absorbance is directly associated with the [Fe 3+ -(TPTZ) 2 ] 3+ -[Fe 2+ -(TPTZ) 2 ] 2+ reducing/transformation ability of the electron-donating compound inside the reaction mixture [62]. The FRAP test is simple, robust, and it does not need extensive skills or equipment. The reducing power of the extracts using the FRAP method varies between 26.74 ± 0.16-856.79 ± 2.87 mg VCE per g DW . Like the other Fe 3+ to Fe 2+ reducing assay, FRAP value increases with an increase in absorbance. The current study findings revealed that D. lotus seed and fruit had the ability to reduce

Conclusion
Turkiye is a rich source of edible plants, such as D. lotus, that plant can be found wild in various regions of the country. Different parts of D. lotus can be utilized to produce different natural products that have biological significance. Extracts of them were used over the years in traditional treated HepG2 cell lines with the methanolic extracts of various parts of Theobroma cacao for 48 h and obtained the following IC 50 : root (237.3 µg/mL), husk (396.0 µg/mL), cherelle (427.3 µg/mL), unfermented shell (464.3 µg/mL), leaf (493.3 µg/mL), bark (828.3 µg/mL) and pith (951.0 µg/ mL).