Phenolic profile, antioxidant capacity of five Ziziphus spina-christi (L.) Willd provenances and their allelopathic effects on Trigonella foenum-graecum L. and Lens culinaris L. seeds

Abstract The aim of this work was to evaluate some secondary metabolites, antioxidant activity of methanolic leaf extracts of five Ziziphus spina-christi provenances (INRGREF, Tozeur, Degueche, Nafta and Kebelli) and their allelopathic effects on Trigonella foenum-graecum and Lens culinaris. Leaves were collected during 2013 and 2014. Total phenols, flavonoids, tannins and antioxidant activity were evaluated using the Folin ciocalteux, Aluminum trichloride, vanillin and scavenging activity on 22-diphenyl-1-picrylhydrazyl (DPPH) radical methods, respectively. Total phenols, tannins and flavonoids were present, at levels of 57.41 mg GAE/g DW, 31.98 mg RE/g DW and 14.68 μg CE/g DW, respectively. The high antioxidant activity (0.086 μg/mL) was noted in kebelli provenance (2013). The highest germination, plumule and radicle lengths of tested species were observed in INRGREF 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.


Introduction
Ziziphus 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 indentified 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 the environment. The leaf extracts of Z. spina-christi presented allelopathic effects on the growth and the development of other plants or micro-organisms (Bhadoria et al. 2011). The Tunisian people used these leaves in traditional medicines (as infusion, cataplasm for abscesses and furuncles) without knowing their bioactive compounds. However, a great attention was especially accorded to multiple biological activities and medicinal uses of Z. jujuba and Z. lotus that become of great interest to both researchers and consumers (Elaloui et al. 2014(Elaloui et al. , 2016a(Elaloui et al. , 2016bGhazghazi et al. 2014).
In this study, total phenolic, flavonoid, condensed tannin contents and antioxidant activity of Z. spina-christi methanolic leaf extracts harvested from five Tunisian provenances during two years (2013 and 2014) were evaluated and compared. In vitro allelopathic activities of aqueous extracts of the leaves were also tested on fenugreek and lentil seeds.

Total phenol, flavonoid and tannin contents
Total phenol levels varied from 48.07 to 57.41 mg GAE/g DW (Figure 1(a)). The relative amounts of flavonoids (Figure 1(b)) oscillated between 6.60 (Tozeur) and 14.68 mg RE/g DW (Nafta). The highest rate was shown in 2013. As we see in these results, Nafta was the most stressed provenance. These results were lower (2 mg RE/g DW) than those obtained for the same provenances, but the trees were older than ours (Elaloui et al. 2016b). This variability 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). With the same method, Z. spina-christi leaves had more important flavonoid levels than those obtained (7.37 mg/g DW) for Z. joazeiro (Adzu & Haruna 2007). The methanolic extracts of INRGREF provenance (Figure 1(c)) contained the highest level of condensed tannin (31.98 mg CE/g DW) compared to Tozeur provenance (25.6 mg CE/g DW) during the same year (2014). Leaf extracts of Z. spina-christi found to contain higher amounts of tannins (9.54 mg CE/g DW) as compared to Peltophorum pterocarpum extracts (Muthukumaran et al. 2016).
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 harvest year on the constituent of the leaves extracts. This composition could also be influenced by the extraction time and the ratio of aqueous methanol (Elaloui et al. 2016b).

Antioxidant activity
The antioxidant activity ranged between 0.108 μg/mL (in 2014) and 0.086 μg/mL (in 2013), respectively ( Figure 2S). These results were in consistence with those obtained in Z. mauritiana (IC50 = 0.11 mg/mL) (Ashraf et al. 2015). Other studies showed that the antioxidant activity augmented by the increase in total phenol levels. Adzu and Haruna (2007) also confirmed this idea. Notes. 1. the data are the mean values of three measurements ± sd (standard deviation). 2. the confidence intervals were calculated at the threshold of 5%.

Effects on seed germination
T. foenum-graecum seeds were more sensitive than those of L. culinaris (Table S1), especially after treatment by leaves collected in 2014.

Conclusion
This study should be conducted with the same plants on the field. The compounds of phenolic extracts, responsible for the inhibition of germination and the seedlings development, must be identified and characterised. 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.