Effect of the Green Synthesized rGO and Mg/rGO Nanocomposites Using High Molecular Weight Polyphenols of Rosa Canina Fruit and Investigation of Their Effects on Phytochemical Assay and Toxicity in Organ Culture of Mentha Longifolia

Reduced graphene oxide (rGO) and Mg/rGO nanocomposites (NCs) were prepared by an eco-friendly technique using high molecular weight polyphenols of Rosa canina fruit. Physicochemical properties and cytotoxicity to Mentha longifolia in vitro cultures of these nanomaterials were examined by using XRD, FESEM, EDX, FTIR, DLS/Zeta potential, UV–Visible and GC-MS techniques. The characterization techniques conrmed the synthesis of rGO and Mg/rGO NCs with particle sizes less than 20 nm (based on FESEM). In accordance to the biological measurements, rGO showed in vitro cytotoxicity to M. longifolia shoot cultures. Mg/rGO NCs showed no signicant difference in the growth parameters except for a decrease in the soot number at the concentrations of 50 and 150 mg/L and a decrease in the length of the tallest root at the concentrations of 100 and 150 mg/L, however, eciently improved the photosynthetic pigment contents. The phytochemical assay depicted that the total content of volatile compounds was increased in the treated cultures with 25, 50, and 100 mg/L of rGO and Mg/rGO NCs in comparison to the control. Generally, the more oxygenated and hydrocarbon sesquiterpenes was observed in the cultures treated with 25 and 100 mg/L of rGO and 25 and 50 mg/L of Mg/rGO NCs.


Introduction
Because of useful physicochemical properties, low cost, and availability for mass production, graphenebased nanomaterials (NMs) have been found to be promising agents for various applications in electronics, energy storage, catalysis, drug delivery, cancer imaging, and photothermal therapy (Fan et  . It has been revealed that these NMs also show a high antimicrobial ability . Besides, they have been considered as basic units for fabricating functional nanocomposites (NCs), incorporated with polymers, metals, metal oxides, and organic crystals for a range of utilization (Huang et al., 2012). Among them, reduced graphene oxide (rGO), as an important derivative of graphene family, has been suggested to be a favorite candidate for the immobilization of metal nanoparticles (NPs) ( Various chemical and thermal reduction methods has been reported for the successfully synthesis of rGO and their NCs with metal nanoparticles (Chen et al., 2012). However, encounter problems such as high cost and use of toxic reducing agents (for instance hydrazine, di-methylhydrazine, hydroquinone, sodium borohydride etc.) have steered researchers to focus on the alternative synthesize methods, particularly for NMs with bio-related applications (De Silva et al., 2017). Accordingly, green synthetic approaches using some plant extracts such as Euphorbia wallichii (Atarod et al., 2015), grape (Upadhyay et al., 2015) and Hibiscus sabdariffa (Nazari et al., 2018) have been applied for the reduction of GO and GO-based NCs in order to overcome the above mentioned problems. Rosa canina L. (Rosaseae family) is a known medicinal plant with various traditional uses. It has been reported that the fruit extracts of this plant exhibit anti-in ammatory, antioxidant, and antiproliferative activities (Lattanzio et al., 2011;Tumbas et al., 2012). According to the phytochemical studies, the aqueous extract of R. canina fruit is rich of high molecular weight polyphenols, lignins and tannins (Roman et al., 2013). These bioactive compounds can act as reducing and stabilizing agents for NMs preparation (Nazari et al., 2020;Veisi et al., 2016).
Latest investigations have revealed that NMs have signi cant effects on plant growth, development, and physiology. Several phytotoxicity studies demonstrated that GO could be transported and accumulated in plant tissues and consequently affected their physiological responses (Anjum et al., 2013;Zhao et al., 2015). Nonetheless, their effect on plant metabolism is still unknown. Metabolomics analyses can be used to reveal the mechanisms of toxicity of NMs in plants. Indeed, metabolic alterations re ect biological processes in a direct way (Hu et al., 2014). So far, some metabolomics-based studies have been provided valuable information about the changes in metabolites and biochemical pathways in response to various environmental stressors (Shulaev et al., 2008;Wu et al., 2017). Beside toxic effects, NMs-mediated changes in the metabolism could also be bene cial. It has been suggested that some NMs can be used as elicitors to enhance the production of desired secondary metabolites such as artemisinin and diosgenin (Marslin et al., 2017). Also, nano-biofertilizers intended to improve crop production and plant nutrition (Subramanian et al., 2015). Therefore, investigations on the changes in primary and secondary metabolism in response to NMs stress are crucial to tackle the adverse effects or take advantage of the positive impacts such as manipulating the production of valuable compounds.
Mentha longifolia (Lamiaceae) is an aromatic medicinal plant traditionally used as medicine for the treatment of lots of diseases and disorders. Several pharmacological properties of M. longifolia including antibacterial, anti-in ammatory, and antioxidant activities can be associated with the bioactive metabolites such as avonoids, phenolic acids, terpenoids, and essential oils (Asghari et al., 2018). Monoterpenes such as pulegone, menthone, isomenthone, menthol, 1,8-cineole, borneol, and piperitenone have been reported as the main volatile compounds of this plant (Mikaili et al., 2013). In this work, the main aim was to study the metabolic response of M. longifolia to rGO and Mg/rGO NCs treatments. Accordingly, the rGO and Mg/rGO were synthesized by using aquatic fruit extract of R. canina as a reducing and stabilizing agents. As-prepared rGO and Mg/rGO NCs were characterized by various techniques such as Uv-Vis spectroscopy, XRD, FESEM, EDX, FT-IR, and DLS. Subsequently, the growth characteristic and photosynthetic pigments contents of M. longifolia in vitro cultures were measured under the synthesized NMs stress. Finally, the changes in metabolite pro les were analyzed using GC-MSbased metabolomics.

Materials And Methods
Preparation of rGO A modi ed Hummer synthesize method was applied for the preparation of GO (Wang et al., 2009). Initially, 5 g of graphite powder and 2.5 g of NaNO 3 were stirred in 115 mL of concentrated H 2 SO 4 at the temperature of 0 °C. Subsequently, 15 g of KMnO 4 was slowly added to the mixture under constant stirring at 35 °C. The mixture's temperature was kept at 35 °C for 30 min. Then, 230 mL of deionized water was added and followed by the addition of 700 mL of hot deionized water and 12.5 mL of 30% H 2 O 2 solution after 15 min. After washing with dilute HCl solution and deionized water, the obtained sediment was dried at 50 °C for 3 h.
For the preparation of rGO, 50 mg of GO was dispersed in 90 mL of deionized water using an ultrasonic bath sonicator for approximately 30 min. The obtained suspension was mixed with 15 mL of R. canina fruit extract prepared according to the method described previously (Nazari et al., 2020) and was subjected to re ux. As-synthesized rGO was dried after washing with deionized water.
Preparation of Mg/rGO NCs using both heating (H) and microwave (M) irradiation methods For the green synthesis of Mg/rGO NCs, 0.053 g of the synthesized GO was mixed in 4 mL of deionized water and placed in an ultrasonic bath for 2 h. Then, 0.045 g of Mg(NO 3 ) 2 .6H 2 O 2 was mixed in 6 mL of deionized water and gradually added to the stable GO dispersion. The obtained mixture was put in an ultrasonic bath for 3 h and re uxed for 20 h at 45 °C. Subsequently, 15 mL of R. canina extract was added to the reaction mixture and re uxed for 24 h at 90 °C. Finally, the obtained product was collected and washed several times with deionized water and dried at 50 °C for 3 h.
In an alternative method, Mg/rGO NCs was also prepared using M method. In this direct, the abovementioned method (H) was used except that the mixture was placed under microwave irradiation at 900 W during 7 minutes instead of re ux for 24 h at 90 °C . Finally, the obtained product was collected and washed several times with deionized water and dried at 50 °C for 3 h.

In vitro cultures and treatments
Surface sterilization of M. longifolia seeds was down using 70% ethanol (3 min) and 20% sodium hypochlorite (15 min) followed by repeated washes with sterile distilled water. Then, the seeds were germinated on solidi ed Murashige and Skoog (MS) medium (0.8% agar, pH 5.6-5.8) at 25 ± 1 °C under continuous light for 21 days. Subsequently, shoot cultures were initiated by transferring stem explants with 2-3 nodes to solid MS medium containing 0.8% agar, 3% sucrose, and different concentrations of rGO and Mg/rGO NCs (prepared by CH method) (0, 25, 50, 100, and 150 mg/L) with pH 5.6-5.8 and kept at under 24 h light photoperiod at 24 ± 1 ˚C for 28 days.

Measurement of growth characteristic
In order to investigate the effect of rGO and Mg/rGO NCs on growth characteristic of M. longifolia shoot cultures, the nal fresh weight (FW), dry weight (DW, dried at 35 o C for 24 h), the number of nodes, leaves, shoots, and roots, the length of the smallest shoot and root were measured.

Quanti cation of photosynthetic pigments
Harvested shoots were homogenized in dimethyl sulfoxide solvent and centrifuged at 5900 J for 15 min. The absorbance of the supernatant was determined at 480, 649, and 665 nm. Chlorophylls and carotenoids contents were calculated according to the equations described by (Wellburn, 1994):

GC-Mass analysis
Extraction of volatile compounds was carried out using the method reported by (Barazani et al., 1999). Brie y, 200 mg of plantlets of treated (except for the cultures exposed to 150 mg/L of rGO and 150 mg/L Mg/rGO NCs) and untreated shoot cultures were extracted by n-hexane solvent (7 mL) and subjected to GC-MS analysis (Agilent MS 5973 N) with a HP 5MS column (30 m, 0.25 mm, 0.25 µm lm thickness). The injection volume and injector temperatures were 1 µl and 150 °C, respectively. The temperature program was used as follows: 70 °C for 3 min, 10 °C/min to 120 °C, 120 °C for 2 min, 10 °C/min to 150°C , 150 °C for 2 min, 7 °C/min to 240 °C, 240 °C for 5 min. The carrier gas was helium with a ow rate of 1 mL/min. The standard of n-alkanes containing n and n+1 carbons (Sigma) was used to calculate the retention indices (RI) using a generalized equation (Van den Dool and Kratz, 1963). Further identi cation was made by the comparison of the mass spectra of compounds with the NIST standard reference database.

Statistical Analysis
All the experiments consisted of three replicates with 8 explants per treatment. Statistical signi cant differences between groups were determined using analysis of variance (ANOVA) test with the statistical software SPSS19. Statistical signi cance was considered at p values less than 0.05.

Results And Discussion
Nanomaterial characteristics In the present study, rGO and Mg/rGO NCs were synthesized using R. canina fruit extract. In order to determine the properties of the desired NMs, rst of all, the reduction of GO to rGO and synthesis of Mg/rGO NCs were monitored using UV-Vis spectroscopy. The UV-Vis spectrum of the synthesized rGO showed absorption peaks in the range of 270 to 300 nm (Fig. 1A). The peak at λ max 281 nm con rmed the formation of rGO. Furthermore, the absorption at wavelength 254-285 nm was obtained for Mg/rGO NCs indicating the formation of Mg NPs (Fig. 1B, C). In addition, XRD analysis of rGO (Fig. 1D) (Fig. 1E, F).
On the other hand, the particle size and morphology of rGO, Mg/rGO NCs were investigated using FESEM ( The hydrodynamic diameter of the rGO and the NCs was measured in 25 o C (Fig, 3 A-  In FT-IR spectra of all samples (Fig. 3D-G), the typical peak at 3000-3500 cm −1 corresponding to the O-H and N-H groups (Rezaei et al., 2019). Thus, the presence of fruit polyphenols like A-type proanthocyanidin as capping agents in the matrix of rGO and Mg/rGO NCs was con rmed. Fig. 4 shows the suggested mechanism for the stabilizing effects of the R. canina fruit extract. As previously was pointed out it is rich of several secondary methabolites such as polyphenols and tannins (Roman et al., 2013). According to the suggested mechanism, the hydroxyl groups of A-type proanthocyanidin can chelated with the magnesium cations in medium. In addition, A-type proanthocyanidin caused van der Waals bonding with epoxy and carboxyl groups of graphene oxide surface and reduced them relatively ( Only some of the stomata were identi ed as closed (Fig. 5C). Similarly, it has been reported that 500 mg/L of CNTs caused the formation of the elongated, irregularly shaped cells and epidermis swelling in red spinach. Some stomata of the leaves exposed to CNTs were closed (Begum et al., 2011). According to the reports, stomata closure is one of the most common plant response to the environmental stresses in order to prevent water losses which leads to decreased CO 2 concentration and formation of the reduced forms of electron acceptors in the photosynthetic electron transport chain. Subsequently, this phenomenon can result in production of free radicals and oxidative damages (Mohammadi et al., 2016).

Growth properties
The impact of rGO NPs and Mg/rGO NCs on the growth parameters of M. longifolia is presented in Table  2. The number of nodes and leaves were observed to be less in the treated cultures with rGO, but no  (Table 2, Fig. 5 D-F). In addition, no signi cant differences were found in the fresh and dry weight of the cultures exposed to both rGO and Mg/rGO NCs.
According to the growth measurements, the potential toxicity of rGO on M. longifolia plant was approved.
The negative impact of graphene-based materials on the growth of several plants such as cabbage, tomato, red spinach, and rapeseed has also earlier demonstrated (Begum et al., 2011;Cheng et al., 2016). Mg, as an important nutrient, is the structural component of chlorophyll and many co-enzymes involved in several biological activities; it is necessary for plant growth and development and has a key role in plant defense system in stress conditions. Therefore, it seems that the direct availability of Mg in the culture medium released from Mg/rGO NCs is probably required to improve the plant tolerance to rGO stress. On the other hand, it has been reported that Mg-based NPs exhibit positive effects on the photosynthetic activity and growth in plants (Raliya et al., 2014;Rathore and Tarafdar, 2015). Findings suggest that the nano-biofertilizers not only can supply the desired nutrients for the plant but also help in the facile uptake and transport of available nutrients resulting in enhanced crop growth and yield (Benzon et al., 2015). Therefore, there is need for a deeper and more systematic assessment to investigate the ability of Mg/rGO NCs as Mg nano-fertilizer in plants.

Photosynthetic Pigment Composition
The results in Table 3 suggest that the content of chlorophyll a, b and carotenoids were sensitive to rGO and Mg/rGO NCs exposure in M. longifolia in vitro cultures. Spectrophotometric analysis showed that treated cultures with rGO revealed a signi cant decrease in chlorophyll and carotenoid contents, except for the cultures exposed to 100 mg/L of rGO, which showed no signi cant difference in the content of

Phytochemical assay and metabolic pro le
Metabolomics is one of the most important approaches for evaluating the plant response to different stress conditions. Among the multiple analytical techniques, GC-MS is a fully developed method for plant metabolite pro ling which analyzes several chemical compounds including sugars, organic acids, amino acids, sugar alcohols, aromatic amines and fatty acids (Shulaev et al., 2008). Therefore, in the present study, the changes in the chemical compounds induced by rGO and Mg/rGO NCs were detected after 28 days of exposure in M. longifolia shoot cultures. The overall chemical pro les of the n-hexane extracts, the identity, and the percentage content of the components are brie y presented in Table 4 and their chemical class distribution are also reported in Table 5 Oxygenated monoterpenes, including L-carvone, thymol, o-Cymen-5-ol, carvacrol, and neryl propanoate, were found to be the major metabolites in approximately all treatments except for 25 mg/L of rGO. However, monoterpene hydrocarbons, including α-pinene, β-myrcene, α-thujene, α-phellandrene, βphellandrene and sabinene, were only observed in the treatment of 25 mg/L of Mg/rGO NCs; β-pinene was observed in the treatments of 25 and 50 mg/L of Mg/rGO NCs. Besides, bis-(2-ethylhexyl) phthalate was identified in high concentrations under the NMs stress with the exception of 100 mg/L of rGO. High levels of oxygenated sesquiterpenes (for example α-bisabolol and α-cadinol) and sesquiterpene hydrocarbons (for example germacrene D, α-copaene, trans-β-farnesene, and δ-cadinene) were determined in the treatments of 25 mg/L of rGO (8.03%) and Mg/rGO NCs (15.37%), respectively. The most abundant oxygenated triterpene was friedelan-3-one constituted 30.13% of the extract of the cultures treated with 50 mg/L of rGO. GC-MS analysis of the extracts showed the abundance of oxygenated terpenoids in the treated cultures with rGO. It has been demonstrated that the difference in chemical structure and saturation of terpenoids are the key factors in uencing the potential biological activity of the essential oils. oxygenated terpenoids are described to have the higher antimicrobial and antioxidant activities than the non-oxygenated ones (Kumar et al., 2011). Therefore, it seems that the elevation of these bioactive compounds might exhibit protective role against the stress caused by the NMs. Consequently, considering the medicinal importance of bioactive compounds, application of the effective concentrations of rGO and Mg/rGO NCs as an elicitor can be a suitable approach to manipulate the content and type of the secondary metabolites.

Conclusion
The current study proposed the green synthesis of rGO and Mg/rGO NCs and presented new insights on their impacts on M. longifolia growth and metabolism. The applied characterization techniques indicated the preparation of rGO and Mg/rGO NCs via an eco-friendly chemical reduction method. Besides, cytotoxicity results evidenced the adverse effects of rGO on the biosynthesis of photosynthetic pigments and the growth of M. longifolia in vitro cultures. In contrast, Mg/rGO NCs showed to be quite biocompatible compared with rGO. Both rGO and Mg/rGO NCs caused a gradual increase in total volatile compounds content at the concentrations ranging from 25 to 100 mg/L. Generally, the more oxygenated and hydrocarbon sesquiterpenes was observed in the cultures treated with 25 and 100 mg/L of rGO and 25 and 50 mg/L of Mg/rGO NCs. Accordingly, detailed studies are necessary to investigate the ability of Mg/rGO NCs as Mg nano-biofertilizer to sustain high crop yield or better-quality of medicinal plants.

Declarations
Data availability Data is available by request to the corresponding author.
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Competing interest
The authors have no con icts of interest to declare that are relevant to the content of this article.     The proposed mechanism for stabilizing stage of the Mg/rGO NCs using A-type proanthocyanidin as a polyphenol in the aqueous extract of R. canina fruit extract.