Biosynthesis of zinc oxide nanoparticles using Phoenix dactylifera and their effect on biomass and phytochemical compounds in Juniperus procera

Biosynthesized nanoparticles have played vital role recently, as suggested to be alternative to physical and chemical methods. In this study, biosynthesis of zinc oxide nanoparticles (ZnO NPs) were carried out using leaf extracts of Phoenix dactylifera L. and Zinc nitrate. The effect of ZnO nanoparticles on biomass and biochemical parameters was investigated. Biosynthesized ZnO nanostructure was characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), UV–visible spectrophotometer and Fourier transform infrared spectroscopy (FTIR). Which resulted in spherical shape with size ranging between 16 to 35 nm of Biosynthesized ZnO nanoparticles and UV absorption beak at 370.5 nm with clear peaks of functional groups. The impact of different concentrations (0.0 mg/L, 80 mg/L and 160 mg/L) of biosynthesized ZnO nanoparticles on biomass and bioactive compounds production of Juniperus procera in vitro was investigated. The results showed that, biosynthesized ZnO NPs (80 mg/L and 160 mg/L) concentrations were boosted the growth of J. Procera with significantly compared to non-treated plants in vitro. The highest concentration (160 mg/L) of ZnO NPs was enhanced the growth of plant at beginning period, one month later shoots became yellow and callus turned to be brownish. Moreover, the influence of ZnO NPs on phytochemical compounds in callus of Juniperus procera was examined using GC–MS analysis. The differences among treatments were recoded. Overall, zinc oxide nanoparticles substantially improved the growth of shoots and callus with increasing of biochemical parameters such as chlorophyll a, total phenolic and flavonoids contents, besides the total protein and, SOD, CAT and APX activity. ZnO NPs might be induced some phytochemical compounds as well as inhibit.

www.nature.com/scientificreports/ Superoxide dismutase (SOD, EC 1.15.1.1) activity was estimated using the method of Marklund and Marklund 32 . The reaction mixture has contained 1 mL of 0.25 mM pyrogallol, 1.9 mL of 0.1 M sodium phosphate buffer (pH 7.4), and 100 μL of enzyme extract. The absorbance was measured at 420 nm. The SOD activity (U g − 1 protein) was defined as the amount of enzyme needed for 50% inhibition of pyrogallol oxidation.
The catalase (CAT, EC 1.11.1.6) activity was estimated by measuring the absorbance at 240 nm, as per the method described by 33 . 1 mL of 0.059 M H2O2 in 0.1 M sodium phosphate buffer (pH 7.4), 1.9 mL of distilled water, and 100 μL of enzyme extract. The CAT activity was expressed as unit g − 1 of protein.
Proline measurement. Proline was extracted following the method described by 35 . Liquid nitrogen has been used to grind fresh sample (0.5 g) and the product was extracted in 10 ml of 3% aqueous sulfo-salicylic acid. The mix was centrifuged and 2 ml of supernatant was added to 2 mL ninhydrin plus 2 mL glacial acetic acid. The mixture was boiled at 100 °C for 1 h, then the reaction was stopped by transferring the tubes to an ice bath for 5 min. Subsequently, 6 ml of toluene was added, mixed vigorously for 15 s and the absorbance of the upper phase was read at 520 nm. The proline content was expressed in μg/g fresh weight.
Estimation of the total flavonoids. Estimation of the total flavonoids in the callus of J. procera extracts was carried out using the method describe by 36 . 0.5 mL of methanol extract, a volume of 0.5 mL of 2% AlCl3 water solution was added. After 24 h at room temperature, the absorbance was measured at 420 nm. A calibration curve was constructed, using quercetin (50-0400 µg/ml) as standard. Total flavonoid contents were expressed as quercetin (mg/g.dry wt.) using the following equation based on the calibration curve (y = 0.0014x + 0.0595).
Estimation of total phenolic content. The total phenolic content of the callus of J. procera extract was determined by using Folin-Ciocalteu reagent following method described by Ainsworth 37 . Gallic acid was used as a reference standard calibration curve. A volume of 0.5 mL of the plant extract (100 µg/mL) was mixed with 2 mL of the Folin-Ciocalteu reagent (diluted 1:10 with de-ionized water) and were neutralized with 4 mL of sodium carbonate solution (7.5%, w/v). The reaction mixture was incubated at room temperature for 30 min. The absorbance of the resulting blue color was measured at 765 nm using UV-VIS spectrophotometer (SHI-MADZU, UV − 1800). The total phenolic contents were determined from the linear equation of a standard curve prepared with Gallic acid. The content of total phenolic compounds expressed as mg/g gallic acid equivalent (GAE) of dry extract.
Preparation of callus extracts for GC-MS analysis. 50 mg of callus of J. procera were lyophilized before grinding. Then, has been extracted in 2.0 ml of methanol of 99.98% using Tissue Layser LT (Qiagen.) Voltage 24VDC/ power 40 VA for 2 h at 25 °C. The organic and aqueous phases were separated by centrifugation at 5000 rpm for 15 min. Then, supernatant was filtered using 0.45 µm nylon syringe before injected into GC-MS analysis.

Statistical analysis.
All experiments were done in triplicate and the results were reported in the figures and tables are the average of three replicate ± standard deviations. The statistical software SPSS (version 20) one-way ANOVA was used for evaluating statistical significance and at (P < 0.05).
Legal statement. The collection of plant materials which are used in this study complies with relevant institutional, national, and international guidelines and legislation. Seeds and seedlings of the date palm and African pencil cedar were collected and provided by Botany and Microbiology Department (Garden and Herbarium Unit), College of Science, King Saud University (KSU) with full permission to collect plant materials by accepting the terms and conditions of national and international standards.

Results and discussion
Biosynthesis and characterization of ZnO nanoparticles. The synthesis of biosynthesized ZnO NPs was carried out by taking 2:2 (v/v) of leaves extract of and zinc nitrate solution to obtain a mixture solution in a round-bottom flask, and incubated with constant stirring (100 rpm) at 40 °C for 24 h. The color of the reaction mixture was changed to yellow after 30 min of incubation time. The changing of color during the incubation time is the first sign of ZnO NPs formation. Then, the obtained biosynthesized ZnO Powder was submitted to various analytical techniques for characterization and to ascertain their shape, size and functionalization. The biosynthesized ZNO powder was dissolved in Milli-Q water to detected the UV-Visible spectra by using SHI-MADZU SPECTROPHOTOMETER (UV-1800) in the range of 200-800 nm. The UV-Visible analysis showed that an absorption peak at 370.5 nm (Fig. 1a). No another major peak shifts had been observed during reactions and optimization. According to 38 the range of UV spectrum for ZnO NPs was 368 nm. Moreover 39 , has been stated that, UV spectrum for ZnO NPs was observed at 375 nm. Additionally, many researchers reported that, UV Spectra of green zinc oxide nanoparticles is fluctuated between 360-380 nm. The shape and size of biosynthesized ZnO nanostructures was investigated using transmission electron microscope (TEM). TEM image of ZnO nanoparticles showed that, the particle size is ranged from 17 to 36 nm (Fig. 1b). XRD spectra of the biosynthesized ZnO powder was detected by X-ray diffractor, which resulted in different crystal planes such as Procera significantly compared to nontreated plants (Fig. 2a,b). While, among ZnO NPs concentrations, the treatment of 80 mg/L biosynthesized ZnO NPs had the best biomass fresh weight (2.3 g) compared to 160 mg/L treatment (1.5 g) (Fig. 2a,b). Obviously, the addition of biosynthesized ZnO NPs to the plants media has improved regeneration of shoots and callus formation substantially. This result supported by physiological characterization which revealed that, ZnO NPs were increased the amount of chlorophyll a (Fig. 2c) and total protein content ( Fig. 3 a) significantly compared to the control. In case of Chl b no significant result was recorded among different treatments (Fig. 2d).These findings were in accordance with 41 who stated that, the presence of ZnO NPs was incorporated into plant hormone such auxin and improved plants growth. Chlorophyll an increased with significant result under ZnO NPs at 80 mg/L compared to control. Whereas at level (160 mg/L) was decreased significantly compared to control (Fig. 2c).  www.nature.com/scientificreports/ May be due to toxic level of ZnO NPs, while no significant differences were recorded in case of chlorophyll b. The highest concentration (160 mg/L) of Biosynthesized ZnO NPs was enhanced growth of Plants at beginning, one month later the color of callus turned into brownish and shoots were changed to yellowish (Fig. 2a). Might be due high dose of zinc oxide nanoparticle, its seemed to be toxic. Although, zinc is an essential mineral at higher concentrations this metal is toxic mentioned by 42 . Moreover, ZnO NPs at high concentration inhibited the expression of genes involved in chlorophyll synthesis and photosystem structure 43 . Our findings in agreement with 44 who has been reported that, plants treated with ZnO NPs nanoparticles showed significant growth compared to the control. Moreover, it was reported that, ZnO NPs play a major role in the increase in biomass, nutrients in wheat 45 . So far, ZnO NPs with low concentrations, it stimulated the callus growth and pointed out the nanoparticles role in regeneration, decontamination, organogenesis, callus induction and activated a protein that has a vital role in growth recounted by 14,15 . In turn, zinc oxide nanoparticles have potential to enhancement the growth and yield of crops 46,47 . A few studies have been focused on phytotoxicity and toxicological effect of ZnO NPs on plants. In general, studies with NPs indicated a certain degree of phytotoxicity, especially at high concentrations 48 . Exposure plants to NPs have induced reactive nitrogen species 49 . Moreover, the highly concentration of ZnO NPs in the rhizosphere solution and root surface could potentially impact the ryegrass growth stated by 50 . Recent studies have shown that plant growth, development and physiology are significantly affected by nanoparticles. Finally, the lowest values of plants biomass were recorded in non-treated plant (Fig. 2a,b).
The impact of ZnO NPs on Protein contents and enzymes activity of callus of J. procera in vitro. Total protein contents. The total protein of callus of J. procera was determined using Nano drop. The results showed that, there were significant differences in total protein contents among callus treated with different concentrations of ZnO NPs. While, among the ZnO NPs treatments, the highest concentration of biosynthesized ZnO NPs (160 mg/L) has given the highest level of total protein ( Fig. 3a and Table 1). In accordance, ZnO nanoparticles have increased protein content in tomato even under salt stress, stated by 41 . Moreover 51 who has been reported that, ZnO nanoparticles have a positive effect on protein content of callus of Nicotiana tabacum. ZnO NPs cause a great effect on expression of some genes encoding certain proteins, it could be caused turn on or turn off the expression of some genes reported by 52 . Also, it is suggested that ZnO nanoparticles might be provide the plants with bio-available Zn ion at cellular level. www.nature.com/scientificreports/ Superoxide dismutase. The influence of ZnO nanoparticles on SOD activity in callus of J. procera under different treatments was investigated as an important scavenger for reactive oxygen species (ROS). SOD activity was induced by highest levels (160 mg) of ZnO NPs in the medium compared to untreated control significantly ( Fig. 3b and Table 1). It has been stated that, zinc oxide nanoparticles was increased the activity of SOD in Punica granatum callus 53 which is in agreement with our findings in this study. This may be due to The regulations in SOD in response to stress which might be caused by nanoparticles, SOD is well-known as powerful ROS Scavenger.
Catalase. The effect of ZnO NPs on CAT activity in callus of J. procera exposed to different levels of ZnO NPs was evaluated. The result indicates that; the activity of CAT was stimulated with the increased levels of ZnO NPs significantly compared non-treated-control. On the other hand, callus was significantly affected by ZnO nanoparticles. Additionally, the result showed that CAT activity in the case of NPs represented strong correlations with ZnO NPs (Fig. 3c and Table 1). In this context 53 , who has been reported that, zinc oxide nanoparticles were increased the CAT activity in callus of pomegranate.  Table 1. Effect of ZnO NPs on total protein, enzymes activity, proline, flavonoids and total phenolic contents of J. procera. The data are presented the average of total protein, enzymes activity, proline, total flavonoids and total phenolic contents ± standard deviation (SD). a,b,c Means within the same column with different superscripts differ significantly (P < 0.05).  Fig. 3d and Table 1). APX activity was induced by zinc oxide nanoparticle and has strong association with ZnO NPs concentrations reported by 53 .

ZnO NPs treatments
Proline. Proline (Pro) accumulation is physiological response in many plants to a wide range of abiotic and biotic stresses and can be a reactive oxygen species scavenger 54 . The examined of pro in this study showed that, the ZnO NPs have increased the level of pro in callus of J. procera significantly compared to non-treated plants.
The increasing of the pro has strongly correlation with the increasing of nanoparticles ( Fig. 4 and Table 1). ZnO nanoparticles might be induced plant to form proline. It has been reported that, the addition of ZnO NPs to plants have increased proline contents significantly 55 .
Total flavonoids. Flavonoids are secondary metabolites with antioxidant activity, the potency of which depends on the number and position of free OH groups 56 . Flavonoids have many biological activities such as the treatment of asthma, bronchitis and cardiovascular disease, the improvement of peripheral blood flow and reduction of cerebral insufficiency 57 . Here, in this study, the estimation of total flavonoid content of callus extract of J. procera was carried out by using Uv-spectrophotometer and quercetin as calibration curve (y = 0.0014x + 0.0595, R2 = 9839) ( Fig. 5a and Table 1). The results showed that, biosynthesized zinc oxide nanoparticles have increased the total flavonoids in treated callus of J. procera significantly compared to non-treated callus (Fig. 5b). ZnO nanoparticles could be cause stress to the plants which lead to accumulation of flavonoids to act as scavengers.
Higher content of flavonoids and phenol was observed in ZnO NPs as compare to crude extract was reported by 58 .
Total phenolic contents. Phenols are excellent oxygen radical scavengers because the electron reduction potential of the phenolic radical is lower than the electron reduction potential of oxygen radicals 59,60 , and also because phenoxyl radicals are generally less reactive than oxygen radicals 61 . Therefore, phenolic compounds can scavenge reactive oxygen intermediates without promoting further oxidative reactions 59 . The total phenolic content in callus of J. procera was determined using Uv-spectrophotometer and Gallic acid was used as a reference  www.nature.com/scientificreports/ standard calibration curve (Fig. 6a). This result indicates that, ZnO NPs nanoparticles at highest level have induced the formation of phenolic content significantly compared control ( Fig. 6b and Table 1). During the stress caused by heavy metals, phenolic compounds act as metal chelators and accumulated. Hence, increases in antioxidant activity of plants exposed to NPs, is mainly due to the increase in phenolic compounds, which are ROS scavengers 62 .
The effect of biosynthesized ZnO NPs on bioactive compounds production. In nature, plants produce bioactive compounds as a protection mechanism against abiotic and biotic stress, and attraction or signaling. Phytochemicals have been used by human as condiments, flavorings and for treating health disorders and preventing diseases including epidemics. The availability of some phytochemicals constituents from its current natural sources are limited. Hence, alters and inducers agents are needed to increase the productivity of bioactive compounds or even to generate new ones. As in nature, plants produce these products as repose to stress. Therefore, plants have been exposed to different concentrations of biosynthesized zinc oxide nanoparticles. The effect of ZnO NPs on bioactive compounds in callus of J. procera was investigated. The differences have been observed among the treatments. The main phytochemical compound in methanol extract of callus of J. procera under different ZnO nanoparticles concentrations is ferruginol (Table 2    www.nature.com/scientificreports/ significantly affected. Nanoparticles could be promoting formation of some phytochemicals as well as inhibit others. Moreover, plants develop resistance to metal stress by altering phytochemicals accumulation and certain antioxidant enzymes to counteract oxidative damages of cellular components and biomolecules caused by highly reactive free radicals 63 . The effect of nanoparticles on plant secondary metabolism still obscure 16 . Therefore, it is a priority to understand the impact of nanoparticles on secondary metabolites which might be helped in production process and it can be used as promotors.

Conclusion
In summary, it could be concluded that, the addition of biosynthesized ZnO nanoparticles to the media of plants in vitro had played vital role in biomass production. ZnO nanoparticles had greater and more-responsive effect on the J. procera shoots and calli growth and physiological indices compared to non-treated plants. Which resulted in higher growth of callus, shoots, chlorophyll, total protein content, total phenols and flavonoids contents and enzymes activity. Obviously, this study showed that, ZnO nanoparticles simultaneously induced  www.nature.com/scientificreports/ growth promoting or factor can cause oxidative stress effects in the plant cells depends on concentrations of ZnO nanoparticles. However, our knowledge of the influence of nanoparticles on living systems is mostly inconsistent to date. The increasing number of studies on nanoparticles has been focused on nanoparticles bio-availability with respect to their concentration. Moreover, the influence of nano material is widely investigated under in vivo systems, as elicitors accompanied by biotic or abiotic conditions. But the effect of nanoparticles on bioactive compounds production in vitro is requested to be developed and validated. Therefore, we suggested that, the effect of nanoparticle on bioactive compounds should be to elucidated. The result of current study could be concluded that, the nature of nanoparticle-derived Zn ions might have had a more significant effect when other factors are taken into consideration, such as doses, particle size, particle and concentration and growth media. Further, the study which is designed and presented in this paper can be extended to involve quantification of bioactive compounds and biosynthesis pathways of the important phytochemical compounds can be tested under ZnO nanoparticles treatment, which might be lead to better understanding of the effect of nanoparticles on antioxidant systems of plants.

Data availability
The data used or analyzed during the present study are available from the corresponding author/KSU.