Distribution of polyphenolic compounds, antioxidant potential, and free amino acids in Ziziphus fruits extract; a study for determining the influence of wider geography

Abstract Ziziphus fruits have attracted much attention within the field of medicine due to their high potential against central nervous system disorders. Abundance of secondary metabolites and their composition is key to the pharmaceutical potential and commercial qualities of plants. The in vitro antioxidant activities of Ziziphus nummularia (Burm. f.) and Ziziphus oxyphylla Edgew fruit extract were analyzed using 2,2‐diphenil‐1‐pycrilhydrazyl (DPPH) and 2,2′‐azino‐bis (3‐ethylbenzothiazoline)‐6‐sulfonic acid (ABTS) free radical scavenging assay methods. Phenolic profiles were explored using high‐performance liquid chromatography‐diode array detector (HPLC‐DAD). The result revealed high concentration of polyphenols and their antioxidant potential. In Z. nummularia, the total phenolic content (TPC) (80.270 ± 0.422 μg/ml), DPPH (62.03 ± 0.98 μg/ml), ABTS (66.32 ± 0.73 μg/ml), and TFC (90.683 ± 0.274 μg/ml) were recorded. However, in Z. oxyphylla, DPPH and ABTS values were 60.66 ± 0.56 μg/ml and 61.55 ± 0.77 μg/ml, respectively, indicative of the impacts of climate and soil nutrients. The overall screening of phytochemicals revealed that both the Ziziphus species contain diverse bioactive compounds, including spinacetine‐3‐O‐(2 feruloyl glucopyranosyl)‐glucopyranoside, kaempferol‐3‐O‐glucoside‐7‐O‐glucoside, and caffeic acid; p‐hydroxybenzoyl hexose, p‐coumaric acid, salicylic acid, and ellagic acid pentoxide. Additionally, the highest concentrated amino acid noted was of Lue 0.19 g/100 g with 596.00 retention time (RT), followed by Thr>Ale>Isl>Phya>Val in Z. nummularia. Similarly, the highest concentration of Lue amino acid was recorded as 0.18/100 g with 564.52 RT followed by Pr>Thr>Ale>Lue>Isl>Phya>Val in all genotypes of Z. oxyphylla. Reporting of polyphenols rich and stable species along with identification of favorable regions of cultivation for amino acid, polyphenols, and higher antioxidant potential may lead the way for the identification of elite clones of the species as well as may result in new drug discovery.


| INTRODUC TI ON
Plants deliver a wide range of supplementary metabolites, among which phenolic compounds have gained significant attention because of their pharmacological potential and putative health benefits (Macheix et al., 2005). Secondary metabolites and bioactive compounds of plants play a key role in maintaining plants in their natural environments (Theapparat et al., 2019;Duan et al., 2019).
Regulation of gene expression in response to environmental stresses results in up-or downregulation of the levels of the metabolites, and thus, amounts of the secondary metabolites in plants play a significant role in handling stress (Macheix et al., 2005). Furthermore, plant origin polyphenols are easily accessible and they are important antioxidants for all living beings and are being used by humans to control oxidative stresses and cardiovascular and chronic diseases (Theapparat et al., 2019;Duan et al., 2019). Accumulation of free radicals may disrupt biologically important molecules and may trigger impulses. However, polyphenols decrease the excess amounts of free radicals' generation inside the cells. These oxidative processes may lower the immunological activity and increase the chances of diabetes, infectious diseases, rheumatoid, arthritis, respiratory disorders, atherosclerosis, and a series of destructive processes due to aging, Alzheimer's disease, and schizophrenia (Mukherjee et al., 2007).
The genus Ziziphus is cosmopolitan in distribution and consists of about 100 species of deciduous or evergreen trees and shrubs (Chen et al., 2017;Razi et al., 2013). The genus is believed to have originated in China and South Asia and is represented widely in tropical and subtropical regions of the world including Pakistan (Razi et al., 2013). The genus Ziziphus has been widely used in folk and alternative medicines for treating different diseases (Adzu et al., 2001;Abdel-Zaher et al., 2005;Nisar et al., 2007;De Omena et al., 2007;Al-Reza et al., 2009;Bahadur et al., 2020;Ashfaq et al., 2019).
In Pakistan, Ziziphus is represented by six species; Z. rugosa, Z. mauritiana, Z. nummularia, Z. spina-christi, Z. oxyphylla, and Z. jujuba (Kaleem et al., 2014;Qaiser and Nazimudin, 1984). However, two species, the Z. nummularia and Z. oxyphylla, are native and widely distributed in Districts Dir and Swat regions of Pakistan. Deep rooting and large carbohydrate reserves in its roots contribute to the strong regeneration potential of the species. The fruits of Z. nummularia are good sources of mineral and contain vitamin C and sugars, which contribute to cooling, astringent, appetizer, stomachic, they cure mucous, and increase biliousness effects, and dried fruits of this plant contain alkaloids, saponins, and triterpenoids (Jabeen et al., 2009).
Similarly, Z. oxyphylla is a large shrub to a medium-sized glabrous tree with small curved and unpaired spine along with oval-shape edible fruits. Furthermore, Z. oxyphylla plant parts such as root, leaves, stem, and mostly fruits are used in folklore and traditional medicines for treating jaundice, diabetes, hypertension, as well as in gas troubles (Jan et al., 2009;Sher, 2011). All plant parts, that is, leaf, root, stem, and bark of both species, possess medicinally important properties (Dahiru & Obidoa, 2008;Dahiru et al., 2006).
High diversity of kingdom Plantae is presents in their phytochemicals too, and these phytochemicals are excellent sources of a variety of industrial and commercial applications (Khoddami et al., 2013).
Indeed, several phytochemicals have been isolated from a plant that is extremely important for their pharmaceutical applications, and provide an opportunity for novel drug discovery (Zeb 2015a).
Polyphenolic profile of plants can be complemented as an efficient markers system along with other morphological, biochemical, or molecular markers (Macheix et al., 2005). Free radicals inside both plant and animal cells are detrimental and they affect cell division and lead to deficiency of immunological system, risk of developing cancer, diabetes, infectious diseases, rheumatoid diseases, arthritis, respiratory diseases, atherosclerosis, and a series of destructive processes due to aging, Alzheimer's disease, and schizophrenia (Temple, 2000).
Phenolic and flavonoids are secondary metabolites that include anthocyanins, hydroxycinnamic acids, and flavonoids, which play a key role in nutrition, health-promoting, and commercial properties of plants (Zeb, 2015a(Zeb, , 2015b. In addition, it has a wide array of important functions in plants, structural support, and water transport (Cáceres et al., 2014;Lattanzio et al., 2008). Thus, plant parts rich in phenolic or flavonoids could serve as excellent resources to cure diseases linked to oxidative stresses. However, isolation and quantification of phenolic compounds have always posed challenges (Šola et al., 2018).
Several methods are available for the determination of phenolic compounds, that is, gas chromatography (GC), high-performance liquid chromatography (HPLC), capillary (GC), mass spectrometry (MS), etc. (Angerosa et al., 1996). Among these options, coupling of HPLC-MS with atmospheric pressure ionization techniques, electrospray ionization (ESI), time of flight (TOF) (García-Villalba et al., 2010;Lozano-Sánchez et al., 2013) are powerful and suitable tools for the precise identification of natural products in crude plant extracts (Lozano-Sánchez et al., 2013). Furthermore, the combination of both techniques LC-NMR offers a robust and powerful separation technique of liquid chromatography with the most informationrich spectroscopic technique (NMR) providing structure elucidation (Christophoridou et al., 2005). Over recent years, several chromatographic methods have evolved for the identification and isolation of phenolic compounds from the plant's extracts (Khoddami et al., 2013). HPLC-diode array detection (DAD) is one the most important and main methods which is used very broadly for the identification of phenolic compounds (Tomas-Barberan et al., 2001).
To facilitate selection of wild species that are locally acceptable and that provide benefits such as food and medicine, the main K E Y W O R D S antioxidative effects, ecological regions, free amino acid, functional food, phenolic compounds, Ziziphus objectives of this study were as follows: (a) to identify polyphenols in these Ziziphus species through HPLC-DAD, (b) to determine the total phenolic and flavonoid contents of the fruit extract, and (c) to compare the antioxidant potential and free amino acid contents of Ziziphus fruit extract collected from different geographical locations.

| Plant collection
The fruits of Ziziphus species were collected from different areas of Districts swat and Dir (L), Khyber Pakhtunkhwa, Pakistan ( Table 1).
The plants were identified using the flora of Pakistan and verified using the online resource www.thepl antli st.org. The voucher specimens (NU-001-HUP to NU-040-HUP) were deposited at the Herbarium of Hazara University Mansehra, Pakistan.

| Soil analysis
During 2016, a total of 40 soil samples (three replicates each) were collected randomly from eight regions of the study area (Table 1).
Samples were collected from 30 to 35 cm depth, transferred into clean polyethylene bags, and were analyzed in the Soil Lab at the Agricultural Research Institute (North) Mingora Swat, KP, Pakistan. The soil samples were air-dried, converted into powder form by hand, passed through a sieve <2.00 mm, and stored in a polyethylene bag until they were ready for analysis. Briefly, a 0.5 g dried powdered soil sample was taken into a 50-ml conical flask, and 15 ml of aqua-regia (nitric acid [HNO 3 ]), sulfuric acid (H 2 SO 4 ), and perchloric acid (HCLO 4 ) were added to it in the ratio of 5:1:1.
Samples were kept overnight and then gently heated on a hot plate at 80°C until a transparent extract was obtained. The extracts were evaluated for different compounds and their concentration was determined on an atomic absorption spectrophotometer, and soil samples were determined by the diluted hydrochloric acid (HCL) method using azomethine-H for different color measurements as described in (20) at 420 nm on a spectrophotometer.
For soil moisture analysis (%), the difference between dry and wet samples was used.

| Determination of total phenolic content (TPC)
The total phenolic content (TPC) of Z. nummularia and Z. oxyphylla was determined using the Folin-Ciocalteu (FC) method (Zeb, 2015a(Zeb, , 2015b, with minor modifications. A volume of 100 µl diluted extract was taken in a test tube and 500 µl of distilled water and 100 µl of FC reagent were added, mixed, and allowed to remain for 6 min. Then, 1000 µl of 7% sodium carbonate and 500 µl of distilled water were added. After 90 min, absorbance was measured at 765 nm using a UV-Spectrophotometer (Shimadzu). The gallic acid standard curve was obtained using dilutions (31.05, 62.5, 125, 250, 500, and 1000 µg/ml) for measuring the TPC, which was expressed as mg of gallic acid equivalent per gram (mg GAE/g) of dry sample. The total phenolic activity was estimated using a standard curve and was prepared with the absorbance noted of different concentrations of the standard, the % Soil moistures = Wet weight of dry soil (g)∕wet weight (g) * 100 Note: All 40 genotypes were collected from these 08 sites of the two districts.

TA B L E 1 Location and distribution of
Ziziphus two species in KP, Pakistan range of latitude, longitude, and altitude formula being y = 0.0342x + 20.0301 (R 2 = .6959), and TPC was determined and expressed as mg of gallic acid equivalent per gram (mg GAE/g) of dry sample.

| Total flavonoid contents (TFC)
The total flavonoid content (TFC) was investigated using the aluminum chloride colorimetry method described by Kim et al. (2002).
Quercetin was used as a standard and TFC was determined in milligram of quercetin equivalent (mg QE/g). The calibration curve for quercetin was obtained using different dilutions (31.05, 62.5, 125, 250, 500, and 1000 µg/ml) prepared in methanol. A total volume of 100 µl of each of these dilutions was taken and mixed with 500 µl of distilled water. Then, 100 µl of 5% sodium nitrate was mixed and allowed to remain for 6 min. Thereafter, 150 µl of 10% aluminum chloride solution was added and allowed to remain for 5 min. Finally, 200 µl of 1 M sodium hydroxide was added and absorbance was recorded at 510 nm using UV-Spectrophotometer.
TFC contents was estimated based on the standard curve which was y = 0.0182x + 9.9939 (R 2 = .9516), in mg QE/g. All results were recorded from triplicate samples.

| DPPH radical scavenging assay
The technique of Brand-Williams et al. (1995) was followed for 2, 2diphenyl-1-picryl-hydrazyl-hydrate (DPPH) assay: 2 mg DPPH was dissolved in 100 ml methanol, while for the stock solution of samples, a concentration of 1 mg/ml was mixed in methanol and diluted to concentrations of 1000, 500, 250, 125, 62.5, and 31.5 μg/ml. For each sample of 0.1 ml, a diluted solution was mixed with 3 ml of DPPH solution in methanol and incubated for 30 min at 23°C, and then the solution absorbance was measured at 517 nm. Ascorbic acid were used as a positive control. All concentration data were observed in triplicates and the data are presented as mean ± SE; the data were calculated by the following formula:

| ABTS free radical scavenging assay
Scavenging activity of methanolic fruit extracts of both species was assessed against 2, 2-azinobis (3-ethylbenzthiazoline)-6-sulfonic acidic (ABTS), using standard assay (Re et al., 1999). A solution of ABTS (7 mM) and potassium persulfate (2.45 mM) was prepared and kept overnight in dark to deliver free radicals and the absorbance of ABTS arrangement was changed following 0.7 at 745 nm by the expansion of 50% methanol. At this point, 300 μl of samples was taken and 3 ml of ABTS solution was added to it and absorbance was estimated at 745 nm for 6 min. For positive control, ascorbic acid was used. The information was recorded in triplicate and percent ABTS free radicals scavenging potential was calculated as follows:

| Estimation of IC 50 values
The median inhibitory concentration, that is, IC 50 values of DPPH and ABTS, was calculated for all test samples through the MS Excel program and origin Pro 7.5 software.

| HPLC-DAD analyses of phenolic compounds
Polyphenolic contents were extracted from samples by using the already-reported method (Zeb & Ullah, 2016). The pericarp was peeled, and shade dried for 20 days and ground into fine powder.
About 2 kg of powder was macerated in 80% methanol and 20% distilled water and strongly shaken. The mixture was filtered after 14 days and centrifuged for 10 min at 6000 rpm. The filtrate was subjected to a rotary evaporator for 2 h and solidified (Zeb & Ullah, 2016). For the separation of the phenolic compound using an Agilent 1260, the Infinity HPLC system consists of a degasser, auto-sampler, quaternary, and diode array detector (DAD). The separation of the phenolic compound was carried out with the help of Agilent Rapid Resolution Zorbax Eclipse plus C18 (4.6 × 100 mm, 3.5 μm) column, which was maintained at 25°C. The gradient system consists of (10:2:88) solvent A (methanol: acetic acid: deionized water) (Zeb, 2015a(Zeb, , 2015b. The elution program was begun with 100% A at 0 min, 85% A at 5 min, 50% A at 20 min, 30% A at 25 min, and 100% B from 30 to 40 min. The flow rate was 1 ml/min. The chromatograms were acquired utilizing 280 nm for analysis of phenolic contents. The spectra were recorded from 190 to 450 nm. The available literature was used for the identification of compounds, its retention times, and UV absorption spectra.

| Estimation of free amino acid
Mature and fresh fruits selected for amino acid were analyzed by the methods of acid-base titration and sulfuric acid fluorenone colorimetry (Luo et al., 2009;Wang et al., 2016). One gram of dry fruit was used for the determination of total amino acid by using HPLC (Agilent1200): 6 mol/L HCL (300 ml HCL and 300 water), 0.1 mol/L (8.3 ml HCL and 1 liter water), and Na 2 CO 3 /Na2HCO 3 (0.53 g Na 2 CO 3 and 0.42 g Na2HCO 3 [pH;9]) were dissolved in 10 ml distilled water, and 3 g poison was added to 10 ml CH 3 CN, and 2.5 g CH 3 COONa (1.5 triethylamine and 1170 μl CH 3 COOH) was added to 1 liter water, after which nylon filter paper was used. Ten milliliter (6 mol/L HCL) was added and placed in the oven for 24 h up to 110°C, before the oven process closes the test tube through the airborn pump (Butan China) then place it into an electric thermostatic blast oven for 24 h. 2 ml liquid was taken and centrifuged (8000 rpm) for 6 min; the and dried by using nitrogen evaporator system and heating system for up to 90°C, then 0.1 mol/L HCL (2 ml) was added %inhibition = Control Absorbance − Sample absorbance∕Control absorbance * 100 % ABTS scavenging activity = Control absorbance − sample absorbance absorbance Control × 100 and kept into the water bath for 90 min on 90°C, and 50 μl (10%) CH 3 COOH and 550 μl water added than, filtered through a nylon filter paper for the HPLC analyses to identify the total amino acid in Ziziphus. Proline was used as a stander concentration of 1, 0.5, 0.4, 0.3, 0.2, and 0.1 mg/ml.

| Statistical analysis
All data were taken in triplicates and mean ± SE values were ana-

| Bioactive compounds in Ziziphus species collected from different areas
Different phenolic compounds are naturally plant-occurring phytochemicals and are highly important, as they possess antioxidant potential to control oxidative activities/oxidative rancidity of food (Guo et al., 2020). Flavonoids are the largest groups of phenolic compounds, namely flavanols, flavones, isoflavones, flavones, and chalcones, which have shown different activities such as antioxidant, anticarcinogenic, and cardioprotective assays (Khan et al., 2011).
The current soil analysis included physical and chemical abilities of the selected soil samples profiling, soil pH, and nutrient cycles between plant species and soil, which are most important in determining the area's soil properties. In the current analysis, pH of the soil ranged from 7.4 to 8.0, which shows that there is not much variation in the pH values of different collected soil samples. The soil analysis (% clay, silt, sand, soil moisture, %N, Na, Mg, and %K) was determined (Table S1). The soil moisture and clay ranged from 34% to 91% and 8.8 to 18%, and the maximum nitrogen (N) and  (Table S1). Our result confirmed that the Blueberries (46.56 mg GAE/g dry extract), cranberries (22.13 mg GAE/g dry extract), and gooseberries (5.37 mg GAE/g dry extract) (Deng et al., 2015).   (Ozcan, 2006).

| Antioxidant potential of Ziziphus species
The radical scavenging activity of plant extracts contributes to the presence of different phenolic compounds as well as to the relationship among all these compounds (Barros et al., 2010;Miguel et al., 2009). Here, not only the amount of phenolic and flavonoid content was estimated but also their antioxidant potential was assessed using ABTS and DPPH assays.  (Table S2 and Figure S1). A substantial antioxidant activ-

| Pearson's correlation analysis
Pearson's correlation analysis (r 2 ) among TPC, TFC, DPPH, and ABTS, soil nutrients, and dry weight is summarized in Table 3. The

| Polyphenols identification by HPLC-DAD
Fruits are rich sources of bioactive constituents such as antioxidants which can neutralize lipid free radicals or inhibit the accumulation of free radicals, and thus suppress the accumulation of volatile products from hydroperoxides: for example, aldehydes and ketones, which give unpleasant odors and flavors that may cause rancidity to lipid-containing foods (Leal et al., 2008;Santos et al., 2014). They are also known to exhibit antiviral, antibacterial, antiallergenic, and antiinflammatory activities, as well as reduce the risk of heart disease, cancer, and diabetes (Leal et al., 2008;Santos et al., 2014).   (Table 4), while the Z. oxyphylla has some differences, which as collected from two different areas District Swat, the data are characterized in Table 1.  (Table 5).
Similarly, identification and composition of polyphenolic compounds were done in Z. oxyphylla collected from Swat (Kotlai and Sogalai) and Dir (L) (Checkdara hill and Gull muqam regions samples using HPLC-DAD (Table 5)).
The general classification was noted using Shi et al. (2003).
The phenolic compounds in different plants may be divided into two categories, phenolic acid and flavonoids, which were noted as cinnamic acids (caffeic acid, quercetin 3-rutinoside, quercetin-

3-galactoside, and kaempferol-3-O-glucoside-7-O-glucoside) and
benzoic acids (p-hydroxybenzoic, protocatechuic, vanillic, and gallic acids) (Ozcan, 2006). Even though these results were obtained from different plants which were similar we recorded in the current result and identified the correlation with our polyphenols. Z. nummularia collected from both districts had very closely related compounds and also contained a high number of phenols, that is, caffeic acid, quercetin 3-rutinoside, quercetin-3-galactoside, kaempferol-3-O-glucoside-7-O-glucoside, and quercetin derivative presented in Table 6. A similar result was mentioned in Santos et al. (2014) andJan et al. (2018). Brassica family has also been identified by a different phenolic which is like our results, that is, quercetin-3-O-triglucoside . Quercetin derivative and quercetin are found in protecting the cells from different cytotoxicities and hydrogen peroxide. A similar observation was recorded by Zeb (2015a).
Thus, Ziziphus species have large amounts of quercetin and their derivatives which help in antioxidant activities (Lu et al., 2011;Mohammed et al., 2013;Pontis et al., 2014;Sohaib et al., 2015;Zeb, 2015aZeb, , 2015b. Many variations were recorded in the composition of the compounds among the Ziziphus species the chromatogram were represented in (Figure 3). To the best of our abilities, the current investigation is the first report of its kind. These metabolites

TA B L E 3 (Continued)
F I G U R E 2 HPLC-DAD chromatograms of methanolic extracts from the fruits of (a and b) Z. nummualaria and (c and d) Z. oxyphylla. Retention time and peak area are shown in Table 6 exposure to environmental factors such as air, light, and tempera-  of phenolic compounds was observed in Z. nummulara, which was 49.5 ± 1.3 and 32.6 ± 1.8 mg/g, respectively. In the samples which were collected from Swat, the compounds ranged from 44.6 ± 1.1 TA B L E 4 Identification and composition of polyphenols compounds in Z. nummularia collected from swat district (Barikot and Seghram) and district Dir (Ghoraghat and Gullabad) regions samples using HPLC-DAD

No.
Phenolic compound

Dir (Ghoraghat and Gullabad)
Swat (Barikot and Seghram) and 30.9 ± 0.8 mg/100 g. While lowest polyphenol compound concentrations were noted in Z. oxyphylla collected from Swat region with a high concentration range of 7.91 ± 0.1 mg/100 g, the lowest range was 0.287 ± 0.01 (Ouerghemmi et al., 2016;Santos et al., 2014;Zeb, 2015aZeb, , 2015b.  (Table 6). abetic, and anti-inflammatory (Tang et al., 2007;Chen et al., 2004;Hwang et al., 2006;Madlener et al., 2007), and were changes in the expression of hundreds of genes in response to temperatures are followed by increases in the levels of hundreds of metabolites, some of which are known to have protective effects against the damaging effects of different stresses (Tang et al., 2007).

| Estimation of amino acid in Ziziphus species
Free amino acids in plant foods have a dual role in diets. They represent a source of nitrogen and nutritionally essential amino acids such as Lys, Met, and Thr. They can also participate in reactions to form browning products. One browning product, acrylamide, formed from free Asn and glucose during food processing, is potentially toxic (Friedman and Levin, 2008;Wang et al., 2016). Here, we describe the distribution of free amino acids, and their variations in two Ziziphus species as enlisted in Table 7 and Figure 3.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
This article does not contain any studies involving animal's trails performed by any of the authors. Furthermore, this article does not contain any studies involving human participants performed by any of the authors.

DATA AVA I L A B I L I T Y S TAT E M E N T
The original contributions presented in the study are included in the article/Supplementary Material, and further inquiries can be directed to the corresponding author/s.