Biochemical profile, antioxidant capacity and allelopathic effects from five Ziziphyus spina-christi (L.) provenances growing wild in Saudi Arabia

The aim of this work was to evaluate phenolic profile, antioxidant capacity and allelopathic effects of six Ziziphyus spina-christi provenances in Saudi Arabia. Total phenols, flavonoids, tannins and antioxidant activity were evaluated. Total phenols, condensed tannins and total flavonoids were present at levels of 62.29 mg GAE/g DM, 16.69 mg CE/g DM and 15.45 μg mg QE/g DM, respectively. The high antioxidant activity (0.15 mg/ml) was noted in Mecca Road provenance. The highest germination, shoots and radicle lengths of tested species were observed in At-taif provenance. Z. spina-christi leaf extracts may be suggested in foods and pharmaceutical industries. Leaf extracts could also provide a natural herbicide with a positive impact on the environment. Provenance of Jizan, Northern Borders and Mecca Road were shown to be particularly effective in antioxidant capacity, while Provenance of Riyadh and Northern Borders shown to have the best allelopathic activity.


Introduction
Ziziphyus spina-christi (Z. spina-christi), an evergreen tree, had been credited with many medicinal fields of applications, including immunity and nutrition (Jiang et al., 2007). The fruit of Z. spina-christi, as known as 'Nbeg', tasted like a mixture of dates and apples and was usually eaten fresh or dried (Bukar et al., 2015). Many glycoproteins, proteoglycans and glycopeptides had been identified from the Z. spina-christi honey (Seif Eldin et al., 2015). In addition, the allelopathy activity, an important biological process in ecology, had been widely employed to protect crops and replace herbicides, fungicides and insecticides being harmful to belongs to the family of Rhamnaceae with about 85 species and Ziziphyus is the most common plants of economic importance in Saudi Arabia and are the major floral sources of high-valued expensive honeys Alqarni, 2015). Z. spina-christi can grow either as a tree or as a shrub. The leaves are short, the flowers are pedunculated and the yellow or red fruits are edible . Ziziphyus jujuba, a plant locally grown in Saudi Arabia and used extensively for its health benefits. The medicinal properties of Ziziphyus tree, also known as Nabq or Sidr, were also recognized and have been in use in Pharaonic carpentry, diet, and medicine (El-Ansary et al., 2018). Z. spina-christi is a medicinal plant and its leaf extract (peptide and cyclopeptide alkaloids) has neuroprotective and therapeutic roles against pentylenetetrazol convulsant effect (Waggas et al., 2010). The Quran mentions the sidr tree twice and accordingly it is highly respected by the Muslims throughout the Middle East (Farooqi, 1997). It is traditionally used in the Muslim countries to wash the bodies of dead Muslims prior to burial, with water in which Ziziphyus jujuba leaves had been soaked (Dafni et al., 2005. In Saudi Arabia, it is used for the treatment of many diseases like wounds, ulcers, etc. Mizrahi et al. (2002) reported that Ziziphyus species are cultivated in hot and arid regions. Some pharmacological screening studies indicated that Z. spina-christi leaves appear to be a safe alternative to lower the blood glucose level. Honeys originating from Sidr (Ziziphyus spina-christi L.) trees in Saudi Arabia exhibited substantial antimicrobial activity against pathogenic (Owayss et al., 2020). Leaves of Ziziphyus jujuba contain a compound known as Ziziphyus which demonstrates medicinal properties and is commonly used in traditional medicine. Among the medicinal properties of Ziziphyus jujube are sedative properties, anticancer, antioxidants, antimicrobial and anti-inflammatory effects (Li et al., 2005;Abdel-Zaher et al., 2014). In Saudi Arabia, Ziziphyus spina-christi is one of the most popular species of Ziziphyus. It is a wild and cultivated plant distributed in the Middle East, Pakistan and in the North and East of Africa (Alansi et al., 2016).
In this study, total phenolic, total flavonoid, condensed tannin contents and antioxidant activity of Ziziphyus spina-christi methanolic leaf extracts harvested from six Saudi Arabia provenances were evaluated and compared. In vitro allelopathic activities of aqueous extracts of the leaves were also tested on Trigonella foenum-graecum and Lens culinaris seeds.

Materials and Methods
Plant material Leaves of Z. spina-christi were sampled from At-taif, Riyadh, Jizan, Northern Borders, Mecca Road, Jeddah ( Figure 1). The plant species were collected (200-250 g per species) during the vegetative phase. The harvested provenances of Z. spina-christi were authenticated and named using existing literature (Cope, 1985;Chaudhary and Akram, 1987;Mighaid, 1996;Collenette, 1999;Chaudhary, 1999Chaudhary, 1999Chaudhary, , 2000Al-Hassan, 2006). The harvested plant samples were processed (powdered at around 100 mesh) then dried in the shade at room temperature for two weeks) and finally stored in in plastic bags in the dark until chemical analysis.

Preparation of the plant extracts
The aerial part of each provenance was finely powdered and used for the different biochemical assays. The solvent is used in order to obtain plant extracts solutions (1 g / 10 ml), which were macerated for 24h in shaking conditions (50 rpm) and used to assay the total content of polyphenols, the total content of flavonoids and the total antioxidant activity (DPPH test). Then extracts were pooled and concentrated under vacuum (Hammi et al., 2015). Total polyphenol content Total phenolics were determined with Folin-Ciocalteu assay as per the Singleton et al. (1999) method which was slightly modified by Deawanto et al. (2002). The results are obtained using a UV/Vis Jenway-6300 spectrophotometer (United Kingdom) under 760 nm. The total phenolics content was expressed as milligrams of Gallic acid equivalents per gram of dry matter (mg GAE/g DM) through the calibration curve of Gallic acid.
The sample was analysed in three replications (Rouz et al., 2015).

Total flavonoid contents
Total flavonoid contents were determined using the Aluminium trichloride method (Rice-Evans et al. 1996;Popova et al., 2004;). 1 ml of AlCl3 (2%) was added to 1 ml of plant extract. The volume was adjusted to 25 ml with methanol and thoroughly mixed. The absorption was measured after 40 min by a Shimadzu UV-160 (Tokyo, Japan) spectrophotometer at 420 nm. Total flavonoids were expressed in milligrams quercetin equivalents per gram of dry matter (mg QE/g DM) through the calibration curve of quercetin. All measurements were repeated three times for accuracy and reliability (Elaloui et al., 2016).

Condensed tannin contents
Condensed tannin levels were assayed following the Earp et al. (1981) method. 1 ml of vanillin (1%) mixed with 4 ml of HCl were added to 200 µl of leaf extracts and incubated 20 min in obscurity. Catechin was used as a standard (0-1250 µg/ml) and the results were expressed as microgram catechin equivalent per gram dry weight (µg CE/g DM). All measurements were performed in three replications. After agitation, the absorbance was read at 500 nm using a Jenway 6100 spectrophotometer.

Antioxidant activity
In test tubes 2.36 mg of DPPH, previously dissolved in 100 ml of ethanol, was mixed and incubated in obscurity. Different concentrations (0.75; 0.5; 0.25; 0.125 µg/ml) were prepared from 1 mg/ml of each leaf extracts. The control sample was done using ethanol and DPPH. The absorbance was measured at 490 nm after incubation for 30 min in dark. Measurements for each experiment were done in triplicate. Antioxidant activity expressed as inhibitory effect of the DPPH radical was calculated using this formula: The percentage of inhibition = [(A0 -Ac) / A0] × 100 Where A0 was the absorbance of the control and Ac was the absorbance of the plant extract/ standard. The IC50 value, the concentration (in μg/ml) of the compound required to scavenge DPPH radical by 50, were determined graphically by the linear regression (Basuny et al., 2013).
Allelopathic activity of Ziziphyus spina-christi leaf aqueous extracts Extracts were obtained by macerating from the Ziziphyus spina-christi provenances leaf extracts as various concentrations: 5 g, 20 g, 40 g, 60 g and 100 g powdered plant in 1 litre of distilled water for 24 h. The solutions were filtered through double layers of muslin cloth followed by filter paper. Trigonella foenumgraecum and Lens culinaris seeds were sterilized with 0.3% calcium hypochlorite, rinsed in distilled water, and dried on filter paper in the laboratory at room temperature for 7 days. For germination, two layers of filter papers were placed in 90-mm-diameter glass petri dishes. Twenty seeds were placed in each petri dish, followed by 02 mL of plant extract. The control groups were each given 2 ml of deionized water and leave at room temperature (25 °C). The Petri dishes were sealed with plastic wrap to prevent the loss of moisture and avoid contamination. Starting from the first day after the experiment began, germinated seeds were counted and removed daily. The experimental design was carried out as a randomized complete block (RCB) with 5 replications. The germination percentage (GP) was calculated. For growth bioassays, the seeds of Trigonella foenum-graecum and Lens culinaris were germinated on filter paper in the dark at room temperature (25 °C) and a photoperiod of 16 hours. Fifteen germinated seeds were transferred to petri dishes, which were filed with 25 g of sterilized quartz sand, and 02 mL of tested extract was added. In addition, control sample was added to the experiment without any treatment. Shoot (SL) and root lengths (RL) of seedlings were measured 15 days after treatment (DAT).

Statistical analysis
A descriptive analysis was performed to describe the entire results within each kind of test. Concerning the biochemical composition analysis and antioxidant activity a one-way analysis of variance (ANOVA oneway) followed by Duncan test was performed to test possible significant differences among mean values from different provenances. The level of significance was set at P<0.05 for all analyses. Statistical analyses were performed using PSS v.23 software.

Analysis of secondary metabolites and antioxidant properties
Total polyphenol, total flavonoid and tannins contents Total polyphenol levels varied from 23.65 mg GAE/g DM of the At-taif provenance to 62.29 mg GAE/g DM of the Mecca Road provenance (Table 1). The Jizan provenance has an important total polyphenol level (50.63 GAE/g DM). The relative values of total flavonoids (Table 1) oscillated between 6.35 (At-taif) and 15.45 mg QE/g DM (Jeddah). The Riyadh provenance has an important total polyphenol level (12.68 QE g/g DM). As we see in these results, the tree of Ziziphyus spina-christi in At-taif was the most stressed tree provenance. Leaf extracts of Jizan Z. spina-christi found to contain higher amounts of tannins (16.69 mg CE/g DM) as compared to provenances of At-taif (12.36 mg CE/g DM), Riyadh (9.25 mg CE/g DM) Northern Borders (11.26 mg CE/g DM) Mecca Road (7.16 mg CE/g DM) and Jeddah (14.39 mg CE/g DM).
Antioxidant activity The antioxidant activity ranged between 0.15 mg/mL and 0.06 mg/mL, respectively ( Table 2). The provenance of At-taif, Riyadh and Jeddah provenances have a lower value of IC50 compared with Jizan, Northern Borders and Mecca Road provenances.  Effects of aqueous Z. spina-christi leaf extracts on L. culinaris and T. foenum-graecum Effects on seed germination T. foenum-graecum seeds were more sensitive than those of L. culinaris seeds after treatment by aqueous leaf extracts with concentrations of 60 and 100 g/l (Tables 3 and 4). All extract of provenances of Ziziphyus have a negative effect on germination of T. foenum-graecum and L. culinaris seeds. The extract of Riyadh and Northern Borders provenance have a very remarkable influence on the germination of T. foenum-graecum (3 and 4% at 100 g/l respectively) and the germination of L. culinaris (9% and 8% respectively at 100 g/l).

Effects on root and shoot lengths
Root lengths are very sensitive to aqueous Z. spina-christi leaf extracts (Tables 3 and 4). Compared with other provenances, the two provenances which have a great effect on the root length of Trigonella foenumgraecum L. are those of Riyadh (10 and 16 mm at 20 and 100 g / l respectively) and Northern Borders (15 and 2 mm at 60 and 100 g / l respectively). These two provenances also have the greatest effect on the root length of Lens culinaris L.: 13 and 9 mm at 60 and 100 g / l respectively for Riyadh provenances and 14 and 8 mm at 60 and 100 g / l for Northern Borders provenances (Tables 3 and 4). Shoot lengths are very sensitive to aqueous Z. spina-christi leaf extracts (Tables 3 and 4). Riyadh Northern Borders provenance have a very negative effect on seedling shoots of Trigonella foenum-graecum L. compared with other provenances with 23 and 8 mm at provenance have a very negative effect on seedling shoots of Lens culinaris L. compared with other provenances with 15 and 8 mm at 60 and 100 g / l respectively and 24 and 13 mm at 60 and 100 g / l respectively (Tables 3  and 4).

Analysis of secondary metabolites and antioxidant properties
The total polyphenol content varies from one species to another. The leaves of the Ziziphyus of Mecca Road presented the highest total polyphenol (62.29 mg GAE/g DM) while Ziziphyus of At-Taif is the lowest one (23.65 mg GAE/g DM). The variations are probably due to the genetic characteristics of the species and by the environmental conditions in which the plants are found. These results corroborate with those obtained by Bettaieb et al. (2012), Hadacek (2002), Miliauskas et al. (2004), Ebrahimi et al. (2008), Andarwulan et al. (2010). Similar results were reported by Rouz et al. (2015). Our results were higher than those obtained for the Ziziphyus spina-christi Tunisian provenances (Elaloui et al., 2016a;2016b) where trees are from a young age than ours. This variability the total polyphenol levels could reflect the influence of the geographic conditions (humidity, temperature, altitude), the soil-growth conditions, and the origin and the period of leaves harvest (Brito et al., 2015). The methanol was found to be the efficient solvent to extract Ziziphyus leaves. This idea was also confirmed by Medini et al. (2014). Indeed, the increase in the biosynthesis and accumulation of phenolic compounds occurs frequently in plant tissue in response to biotic and abiotic stresses. These compounds are involved in the defence against reactive oxygen species (ROS), which are inevitably produced when the aerobic metabolism or photosynthetic is weakened by environmental stress. Genetic factors and growth conditions of the plant can also play an important role in the formation of secondary metabolites, including phenolic acids (Islam et al., 2003;Hashempour et al., 2010). Other factors especially the extraction method and the quantification method may also influence the estimate of the content of total phenols (Lee et al., 2003). The polyphenols played an important role in disease resistance by exerting free-radical scavenging, modulation of enzymatic activity and alteration of signal transduction pathways (Elaloui et al., 2016a). This study revealed a strong influence of the localities on the constituent of the leaf's extracts. This composition could also be influenced by the extraction time and the ratio of aqueous methanol (Elaloui et al., 2016c). As well, this variability could be explained by many factors including the origin, the period of harvest, the age and the stage of plant development. Other environmental factors (temperature, altitude, sunshine, animal aggression and diseases) could also influenced this variability (Elaloui et al., 2017). The amount of flavonoids of Z. spina-christi significant varied (P <0.01) between 6.35 (At-taif provenance) and 15.45 mg QE/g DM for Jeddah provenance. With the same method, Z. spina-christi leaves for our provenance had more important flavonoid levels than those obtained for Z. joazeiro (Elaloui et al., 2016a;2016b;Adzu and Haruna, 2007). Similar results were reported by Rouz et al. (2015). It has been proven that the contents of total phenols and flavonoids are high. The difference in the phenolic content including flavonoids described in the literature can be attributed to several factors such as the extraction method and the quantification method. Moreover, variables such as temperature, humidity and light intensity, added to the effects of the biota and the physiochemical structure of the soil can affect not only the production of secondary metabolites but also the chemical structure of substances (Reigosa et al., 2013). It has been proved that the levels of total phenols and flavonoids were high when the environment conditions of the plant were not adequate. In this case, the plant promoted the synthesis of secondary metabolites in order to adapt and survive Tim and Lamb (2005). A few papers reported the amount of phenolics in some of plants; recently, in a review about the biochemical composition of different parts of Z. lotus, Azize (2016) reported around 7 mg/g of phenolics to be present in the leaves, almost 5 times less than the amount measured in Tlili et al. (2019). Moreover, in the same review, the tannin content observed in the leaves was the same that Tlili et al. (2019) measured (around 3.0 mg/g). Conversely, the content of phenolics in the leaves of T. hirsuta and R. tripartitum, including flavonoids, and the DPPH values found by Akrout et al. (2011) andItidel et al. (2013) were in line with those measured by Tlili et al. (2019). Habib et al. (2014), studied the phenolic compounds in some types of honeys including Z. spina-christi L. Sidr honey which was found to contain the highest amount of total phenolics. Z. spina-christi L. samples were obtained from two different regions in Yemen. Ours results were in consistence with those obtained in Z. mauritiana (IC50 = 0.11 mg/mL) (Ashraf et al., 2015). Ours studies showed that the antioxidant activity augmented by the increase in total phenol levels. Adzu and Haruna (2007) also confirmed this idea. For Ziziphyus lotus (L.) Tlili et al. (2019) found the same results as we found in our study. When Alghazeer et al. (2012) studied the antioxidant activity of some plants growing in Libya, he found two times higher the amount of polyphenols in Hyoscyamus albus than we found in this study. The flavonoids in honey make it a good source of antioxidants (Bader et al., 2020).
Effects of aqueous Z. spina-christi leaf extracts on L. culinaris and T. foenum-graecum Effects on seed germination For both target species (L. culinaris and T. foenum-graecum), the highest germination was obtained for the control and inhibition of germination, which increases by elevating the concentration of aqueous extracts.
The concentration of 100 g/l gives the lowest germination for both species with a respective L. culinaris and T. foenum-graecum of 8 at 19% and 2 at 12%. Similar results were reported by Rouz et al. (2015). Inhibitory effect on germination and seedling development were obtained especially at higher concentrations (60 and 100 g/l).
Similar findings were also recorded by many researchers (Arora et al., 2015;Prasad et al., 2016;Turker et al., 2008). The degree of inhibition was largely dependent on the concentration of the aqueous extracts . These results confirm the herbicidal properties of this medicinal specie of Z. spina-christi containing natural molecules like juglone and artemisin that affect membrane permeability, water balance and osmotic regulation (Sanchez-Moreiras et al., 2008).

Effects on root and shoot lengths
Ours results showed that interaction between level of concentration of aqueous extracts and species decrease significantly the roots growth. Similar results were reported by Rouz et al. (2015). With the concentrations of 100 g/l the length of the roots of L. culinaris varied between 8.16 and 18.15 mm and the shoot varied between 8.15 and 21.15 mm. With same concentration the length of the roots of T. foenumgraecum varied between 4.36 and 16.84 mm and the shoot varied between 6.45 and 19.35 mm. The root lengths were more inhibited than shoot lengths. These results are in agreement with those of Patil and Kore (2016).
These results show that the aqueous extracts of the six provenances of Ziziphyus studied have allelopathic potential and significantly inhibit the germination and growth of T. foenum-graecum and L. culinaris. These excerpts are provided with a potent cytotoxic activity. Root growth is more sensitive to different concentrations of aqueous extracts of six provenances of this medicinal specie than germination.  concluded that the aqueous extracts of allelopathic plants generally act on root growth rather than growth of the hypocotyl. This was explained by the fall, which made the roots in contact with the first allelochemicals inhibit cell division in meristematic tissues (Ashrafi et al., 2008). Mitosis is the most important event in the development of the plant and many enzymes and regulatory factors of these phenomena are affected by allelochemicals via a chromosome aberration (Turker et al., 2008). Thus, the aqueous extract can affect the absorption of water by the seed and delay the mobilization of reserves (Kato-Noguchi and Macias, 2006). Even if the seed happens to germinate, we recorded a blocking root growth and / or the aerial part of plants. Tucker et al. (2008) reports that the growth phenomenon is more sensitive to the phytotoxic allelochemicals than germination. So, the exact identity of the compounds responsible for this apparent phytotoxic activity, and quantified with certainty needs to be known. Overall, the observed results could form the basis for further investigations of the biological properties of aqueous extracts of six provenances of this medicinal specie. In addition, the use of these aqueous extracts as a biopesticide is a promising path towards recovery of this fraction.

Conclusions
This study demonstrated that allelopathic potential of aqueous extracts of six provenances of medicinal specie (Ziziphyus) and invasive grass by influencing either the germination or growth are the two phenomena that target both species. This indicates that these extracts are rich in natural bioactive substances, suggesting that their importance in the development of natural bioherbicides as an alternative to minimize the release of synthetic non-degradable molecules in crop production. Further studies are needed to understand the mode of action of these allelochemicals. Also, a separation, purification and identification of bioactive molecules that could potentially be used as herbicides or as plant growth regulators would be need to be recorded for further study. Allelopathy, used as herbicides, had an economic benefit to farmers and also reduced negative impacts on the environment and on the health of the human being. In conclusion, all the data confirm the importance of the Saudi Arabia local vegetation as a potential source of various bioactive phytochemical compounds; the investigation is based on the need for different biological agents from natural sources with potent activity and lesser side effects as substitutes for chemical therapeutics.

Authors' Contributions
Both authors read and approved the final manuscript.