Induction of Catharanthus roseus Secondary Metabolites When Calotropis procera Was Used as Bio-Stimulant

Available information associated with Calotropis procera posted its phytotoxic effect as bio-herbicide scarce works studied its stimulatory/nutritive effect. A pot experiment was performed to assess the validity of using Calotropis procera (C. procera) leaves extract as a bio-stimulant for the growth and quality of a medicinal plant Catharanthus roseus (C. roseus) evaluated by some physio-biochemical indices. Different types of C. procera leaves extracts (CLEs) (methanolic, cold water and autoclaved water extracts) were delivered by two different modes of application. The results revealed that application of CLEs as irrigation or foliar spraying caused a stimulation effect on C. roseus plant. Root and shoot length, dry and fresh weight were significantly improved due to CLEs applications. C. roseus bioactive molecules such as anthocyanins, phenolics, flavonoids, alkaloids, ascorbic acid, reduced glutathione and α-tocopherol were abundance increased significantly with CLEs applications. Reactive oxygen species (ROS) decreased explaining the involvement of CLEs in induction of antioxidant enzymes catalase, ascorbate peroxidase, polyphenol oxidase, guaiacol peroxidase and glutathione-S-transferase for modifying cell oxidative status witnessed by lower lipid peroxidation that kept below the untreated plants’ baseline reflected the improvement of growth and quality rather than phytotoxic effect. The promotion of wholesome-promoting secondary metabolites by CLEs was closely correlated to elevated phenylalanineammonialyase activity. The comparable efficient effect induced by all treatments might be judged by the relation between C. procera phytochemicals and C. roseus metabolism (donor-receiver relation). It is concluded that application of CLEs can be a promising approach for improving the yield and quality of plants despite using polluting fertilizers. The current investigation may provide a matrix for coming studies to seek illustration of numerous plants’ response to C. procera extracts.


Introduction
Within the agricultural and horticultural industry, high yield and high-quality crops are of major commercial and economic importance. The demands for increased yield and improved quality can be met through improvements in crop genotypes by selection, breeding, and genetic engineering and by improvement of the crop growth environment through irrigation, fertilization, and the use of plant protection products. Chemical growth regulators are also one of the tools used to improve both crop growth and quality. However, the extensive and irrational use of these agro-chemicals as inevitable practice for plant production posed undesirable environmental impacts. An alternate strategy to reduce the dependence on synthetic growth regulators and encourage the use of botanical sources is considered safe for the environment and helps in achieving global food security sustainably

Plant Materials and Tissue Collection
C. procera leaves were collected from the Agriculture Faculty Campus, University of Al-Azhar, Assiut, Egypt. The Flora of Egypt [16] was used for authentication of the plant. For phytochemical profile analysis four biological replicates from leaves were sampled from one-year-old C. procera plant. The method described by Naz and Bano [17] was used to prepare C. procera leaves extracts.
Three-month-old uniform C. roseus plants were collected in spring 2017 from the botanical garden of the Faculty of Agriculture, Assiut University. The plants were transferred into (30 cm in diameter and 35 cm in depth) containing 3000 gm air-dry soil (sand/clay 1:2 v/v) in rate of 5 plant/pot. The plants were kept in the greenhouse of the botanical garden of the Botany and Microbiology Department, Faculty of Science, Assiut University, Egypt (42" and 28 • 59 23" E and latitude 25 • 45 06" and 25 • 53 34" N) to secure mild climatic conditions during experimentation.

Treatment
After one week of pre-culture, the transplants were randomly divided into irrigation and foliar spraying treatment groups. Each group consists of 12 pots (3 different extracts (methanolic extract (ME), cold water extract (CWE) and autoclaved water extracts (AWE)) × 4 replicates for each). The plants not delivered any extracts (either by irrigation or spraying) were considered as control. For irrigation treatment, each replicate was treated using extract by field capacity at the rate of 250 mL kg −1 , soil with polyethylene bags to avoid soil treatment leaching. Additionally, for foliar spraying treatment, each replicate was sprayed by 250 mL of extract per pot and the soil surface was covered by polyethylene bag to avoid foliar treatment reaching soil. During the treatment, average temperature was between 21 • C and 27 • C and relative humidity varied between 65.4% and 70.5%, and plants were allowed to grow for one week. After one week of treatments, all the replicates were washed firstly thoroughly with tap water and then with distilled water to remove any extract surface deposit, rinsed in ultrapure water. Then the replicates were used for growth criteria measurements and physio-biochemical indices evaluation.

Isolation of Phytochemical Compounds from C. procera Leaves
The correct method to reduce technical variability throughout a sampling procedure is essential to stop cell metabolism and to avoid leaking of phytochemical metabolites during the various preparation steps before the actual phytochemical extraction [18,19]. Therefore, three biological replicates from the fresh leaves were immediately frozen on dry ice. In the laboratory, the frozen three biological replicates from fresh leaves samples were homogenized in cryogenic grinding for three cycles at one minute for each, after which the plant material (ca. 500 g, 280 g and 280 g) were directly soaked in 1000 mL from each of 90% methanol as a solvent, cold water and autoclaved water, respectively in Amber storage bottles, 2000 mL screw-top vials with silicone/PTFE septum lids (https://www.brecklandscientific.co.uk/aboutus.asp accessed on 1 January 2020) were used to limit dispersal of volatiles to the headspace then incubated with shaking at 37 • C and 200 rpm for 24 h [16,19]. Afterward, each one from ME, CWE and AWE were transferred and aliquoted into several 250 mL utofil ® PP centrifuge bottles with screw cap and centrifuged at 5000 rpm for 10 min at 4 • C to eliminate plant debris. The upper layers from the extract were transferred into new 1-L storage bottles. After that 3 mL from each extract was pipetted into three new glass tubes of 1.5 mL with lids to reduce a loss of volatiles, and each 1.5-mL glass tube contained 1 mL from the extract. Then all the glass tube 1.5 mL with lids were placed on the GC-MS auto-sampler for GC-MS run, or each tube was covered with paraflm after closed with screw-top vials with silicone/PTFE septum lids and stored at −20 • C until GC-MS analysis [18,19].

GC-MS Analysis of Phytochemical Compounds from C. procera Leaves
Trace GC-ISQ Quantum mass spectrometer system (Thermo Scientific, Austin, TX, USA) was used for phytochemical analysis. About 1 µL from each sample was injected into a GC-MS equipped with a TG-5MS column (30 m × 0.25 mm ID, 0.25 µm film thickness). Helium gas was used as a carrier at a constant flow of 1.0 mL min −1 . The mass spectra were observed between 50-500 m/z. Temperature was initially started at 50 • C for 10 min, then increased at a rate of 5 • C min −1 to 250 • C, and isothermal was held at 300 • C for 2 min, and finally, the held isothermal for 10 min at 350 • C. NIST, Adams, Terpenoids and Volatile Organic Compounds libraries were used to identify the phytochemical constituents by comparing the recorded mass spectra for each compound with the data stored in the previous libraries. The relative percentage amount for each component was calculated using by Retention time index and comparing its average peak area with total peak areas. And each sample was represented by three replicates [18,19].

Determination of C. roseus Plant Growth Criteria Affected by C. procera Leaves Extracts under Irrigation and Foliar Spraying Treatments
After measuring the shoot and root length, the plants were separated into shoot and root parts for measuring fresh and dry weight. Shoots and roots length and fresh weight of harvested plants were determined immediately then oven-dried at 60 • C for 2 days to a constant weight to evaluate dry weight.
2.6. Determination of C. roseus Physio-Biochemical Indices Affected by C. procera Leaves Extracts under Irrigation and Foliar Spraying Treatments 2.6.1. Photosynthetic Pigment Content The component of leaf photosynthetic pigments; chlorophyll a, chlorophyll b, and carotenoids were quantified as mg g −1 FW adopting the method used by Abeed and Dawood [20].

Primary Metabolites
Primary metabolites in terms of carbohydrate content, free amino acids content and soluble proteins content all expressed as mg g −1 DW were estimated according to the published methods of Abeed et al. [21] and Iqpal [22], respectively.

Statistical Analysis
A completely randomized design (CRD) was utilized for the pot experiments. Obtained data were expressed as means ± SE. SPSS 10.0 software program was used for performing the statistical analysis. Comparisons between control and treatments were assessed by one-way ANOVA using the least significant differences (LSD) test. Difference from control was counted significant at the probability levels of 0.05 or very significant at the probability levels of 0.01.

Identification of Phytochemical Components of C. procera Used Extracts
According to GC-MS analysis, 126 bioactive phytochemical compounds were identified in ME, CWE and AWE of C. procera leaves. The numbers of obtained bioactive phytochemical compounds in ME, CWE and AWE were 68, 38 and 20, respectively. The results of the qualitative and quantitative analyses of all phytochemicals in the three extracts are recorded in (Table 1 and Figure 1. The identified phytochemical compounds are listed based on the retention time, compound formula, compound molecular mass, CAS registry number and percentage of peak area (Table 1). In ME, guanosine was recorded as the major compound (15.57%), followed by 1-heptatriacotanol compound, neophytadiene compound and trans-phytol compound that registered as (4.57%), (4.53%) and (4.53%), respectively.

Effect of C. procera Leaf Extracts on the Growth Criteria of C. roseus under Irrigation and Foliar Spraying Treatments
The shoot and root length of C. roseus increased significantly (p < 0.05 or p < 0.01) upon irrigation and spraying treatments. These increments ranged between 18-46% and 18-105% for shoot and root length, respectively, when compared with the control (Table 2 and Figure 2). The difference between the different types of extracts is significant in most cases (p ≤ 0.05) whereas their promoting effect was comparable regardless of the used application mode. On the other hand, highly significant (p < 0.01) growth promotion in terms of fresh and dry weight compared to control was registered by C. procera treatments which ranged between 25-160% and 55-85% for shoot and root fresh weight, respectively, when compared with the control. While dry weight increments ranged between 90-199% and 73-160% for shoot and root, respectively, as compared with the control. This effect upon fresh and dry weights was extract type and application mode independent as the values of fresh and dry weight statistically showed no significant differences between treatments (p > 0.05, bearing the same difference letters).

Effect of C. procera Leaf Extracts on the Growth Criteria of C. roseus under Irrigation and Foliar Spraying Treatments
The shoot and root length of C. roseus increased significantly (p < 0.05 or p < 0.01) upon irrigation and spraying treatments. These increments ranged between 18-46% and 18-105% for shoot and root length, respectively, when compared with the control ( Table  2 and Figure 2). The difference between the different types of extracts is significant in most cases (p ≤ 0.05) whereas their promoting effect was comparable regardless of the used application mode. On the other hand, highly significant (p < 0.01) growth promotion in terms of fresh and dry weight compared to control was registered by C. procera treatments which ranged between 25-160% and 55-85% for shoot and root fresh weight, respectively, when compared with the control. While dry weight increments ranged between 90-199% and 73-160% for shoot and root, respectively, as compared with the control. This effect upon fresh and dry weights was extract type and application mode independent as the values of fresh and dry weight statistically showed no significant differences between treatments (p > 0.05, bearing the same difference letters).

Figure 2.
Illustration of growth pattern of C. roseus shoots and roots treated with three leaves extracts of C. procera (ME, CWE and AWE) applied by two different method (irrigation and foliar spraying) and control plants. Table 2. Effect of three types of C. procera leaf extracts (ME, CWE and AWE) applied by two different application modes (irrigation and foliar spraying) on growth criteria of C. roseus. Each value represents a mean value of four replicates ±SE. * and ** donate the difference significantly from the control at the probability levels of 0.05 and 0.01, respectively. Different letters within row (a, b, c) indicate significant differences (p ≤ 0.05) between the types of extracts of the two application modes irrigation and spraying.

Parameters
Irrigation Foliar Spraying Figure 2. Illustration of growth pattern of C. roseus shoots and roots treated with three leaves extracts of C. procera (ME, CWE and AWE) applied by two different method (irrigation and foliar spraying) and control plants. Table 2. Effect of three types of C. procera leaf extracts (ME, CWE and AWE) applied by two different application modes (irrigation and foliar spraying) on growth criteria of C. roseus. Each value represents a mean value of four replicates ±SE. * and ** donate the difference significantly from the control at the probability levels of 0.05 and 0.01, respectively. Different letters within row (a, b, c) indicate significant differences (p ≤ 0.05) between the types of extracts of the two application modes irrigation and spraying. The obtained promotion effect on growth criteria is in agreement with the data recorded from studying the impacts of aqueous extracts of four medicinal plants (Eclipta prostrate, Wood fordia fructicosa, Ageratum conyzoides and Cannabis sativa) on the seed germination, seedling growth and biomass production of Triticum aestivum (wheat) and Pisum sativum (pea) [40]. Additionally, the obtained results are in line with Draz et al. [41] who demonstrated that the extracts from lantana, Lantana camara; henna, Lawsonia inermis; pomegranate, Punica granatum; acalypha, Acalypha wilkesiana and chinaberry, Melia azedarach significantly increased wheat yield components in terms of 1000-kernel and spike weight compared to the non-treated control. Moreover, Shabana et al. [42] and Nagwa and Iman [43] found that the foliar spraying of plant extracts (Brazilian pepper, pomegranate, neem, garlic, cactus, and eucalyptus) significantly increased wheat yield components, including spike weight and 1000-kernel weight. Naz and Bano [44] reported that C. procera leaves extract has growth-promoting effects on maize crop and this was attributed to the macro and micronutrients present in C. procera extracts easily absorbed by target plants that exhibit an influential role in the vital metabolism within the plant [45].

Photosynthetic Pigments Content
The results derived from growth-performance screening of C. roseus leaves and roots were depicted in Tables 3 and 4. All C. procera extracts induce significant (p < 0.05) stimulant effect on photosynthetic pigments accumulation which recorded a considerable increment ranged between 60-179%, 71-156% and 32-158% for chlorophyll a, chlorophyll b and carotenoids, respectively, compared to control (Table 3). Statistically, in most cases, there is no significant difference between treatments. Stimulation effect was comparable between the different extracts of the two modes of application.
It is worthy to note that chlorophylls and carotenoids are ubiquitous and essential photosynthetic pigments, which are intricately linked with plant growth criteria in terms of shoot and root length, fresh and dry weight indicating enhancing carbon allocating process [46]. Draz et al. [41] found that plant extracts of Acalypha wilkesiana, Melia azedarach, Lawsonia inermis, Lantana camara and Punica granatum enhanced the total chlorophyll (a + b) content in wheat leaves. Table 3. Effect of three types of C. procera leaves extracts (ME, CWE and AWE) applied by two different application modes (irrigation and foliar spraying) on physio-biochemical indices of C. roseus. Each value represents a mean value of four replicates ± SE. * and ** donate the difference significantly from the control at the probability levels of 0.05 and 0.01, respectively. Different letters (a, b, c) within row indicate significant differences (p ≤ 0.05) between the types of extracts of the two application modes irrigation and foliar spraying.  Table 4. Effect of three types of C. procera leaves extracts (ME, CWE and AWE) applied by two different application modes (irrigation and foliar spraying) on physio-biochemical indices of C. roseus. Each value represents a mean value of four replicates ±SE. * and ** donate the difference significantly from the control at the probability levels of 0.05 and 0.01, respectively. Different letters (a, b, c) within row indicate significant differences (p ≤ 0.05) between the types of extracts of the two application modes irrigation and foliar spraying.  Table 3 revealed that three different types of extracts significantly (p < 0.05 or p < 0.01) intensified the primary metabolites production in terms of carbohydrates, amino acids and proteins in both leaves and root with percent increase ranged between 3-60%, 43-61% and 25-81% in leaves and 7-75%, 10-63% and 14-70% in root for carbohydrates, amino acids and proteins, respectively, in comparison to control. The overproduction of some primary metabolites (i.e., carbohydrates, amino acids and proteins) and consequently investment in C. roseus shoot and root biomass may go in line with the enhancing of chlorophyll content in the plant, presumably as a consequence of improved levels of photosynthetic pigments, leading to an increased photosynthetic rate have stimulated the source-to-sink transport of sugars thereby increasing carbohydrates content. The increasing in amino acids content goes parallel with the enhancing of proteins manufacturing indicating anabolism pathway under C. procera treatments.

Physio-Biochemical Indices
These results were concomitant with Gamal et al. [47] who showed that the treatment with aqueous extracts of Malva parviflora L. and Artemisia ludia L. significantly increased the protein content, yield and growth of cowpea (Vigna unguiculata (L.) Walp.). Cheema et al. [6] and Gamalero and Glick [48] found that the phytochemicals can promote growth through optimum CO 2 fixation upon normal conditions and they operate a positive role in physiological processes such as chlorophyll accumulate, photosynthesis, leaf expansion, translocation and genetic encodings, greater uptake and use efficiency of nitrogen resulting in a greater synthesis of protein.

Superoxide Anion (O 2 •− ), Hydroxyl Free Radical ( • OH) and Hydrogen Peroxide (H 2 O 2 ) Accumulation and Membrane Damage Trait (MDA)
Effect of Calotropis treatments on oxidative cell status of C. roseus was evaluated by determination of the stress markers (ROS), such as superoxide radical (O 2 •− ), hydroxy radical ( • OH), hydrogen peroxide (H 2 O 2 ), besides ROS, oxidative burst to cellular membranes (membrane damage trait) assessed as lipid peroxidation level (malondialdehyde concentration, MDA) all of these were significantly (p < 0.05 or p < 0.01) and interestingly lowered than control by all applied treatments. Statistically (p ≤ 0.05) and in the most investigated cases of Calotropis treatments a comparable effect on MDA reduction was pronounced among all types of extracts submitting a comparable protective effect on C. roseus via reducing the accumulation of each O 2 •− , • OH and H 2 O 2 [49,50] consequently low MDA content in leaves and roots of C. roseus. In the present study, CLEs-devoid control plants exhibited a substantial accumulation of ROS, including O 2 •− , • OH and H 2 O 2 which was accompanied by a concomitant increase in MDA content indicating that the control plants might have suffered from oxidative damage under field conditions. The reduction in ROS level was concomitant with enhancing of membrane integrity and stability evidenced by low MDA concentration that may be another main reason for growth improvement under C. procera treatments compared to control reflecting high optimal condition for efficient cellular metabolism and performance that can be ascribed to stabilization of cell redox status [49,51]. The improving mechanism displayed by Calotropis treatments thus was appraised by oxidative cell status maintenance ensuring normal cellular function and cell metabolism by Calotropis that was associated with enhancing in growth criteria, advocating bio-stimulant effect of Calotropis extracts upon C. roseus.

Anthocyanin, Phenolics, Flavonoid, Alkaloids, Ascorbic Acid, Reduced Glutathione and α-Tocopherol Contents
Catharanthus roseus is one of the very extensively investigated medicinal plants due to its powerful antioxidants. It produces a wide spectrum of secondary metabolites viz., anthocyanins, phenolics and alkaloids. We further elucidated the beneficiary role of CLEs by quantifying the amount of non-enzymatic antioxidants. The protective impact of C. procera was explained by exacerbation of low molecular weight non-enzymatic antioxidants viz. ascorbic acid, reduced glutathione and α-tocopherol (which registered percent increase fluctuated between 19-40, 2-7 and 211-411, respectively) besides boosting of secondary metabolites pathway as manifested by elevated leaves anthocyanins, phenolics, flavonoids and alkaloids contents (Table 4) with percent increase ranging between 47-87, 6-31, 10-370 and 37-197, respectively, when compared to control. C. procera treatments significantly (p < 0.05 or p < 0.01) increased the antioxidant potential of C. roseus plant whatever extract type and application mode. These compounds play multiple functions concerning antioxidant properties and the ability to diminish free oxygen radicals conferring membrane stability, in turn, restricts the dispersal of free radicals and minimizes membrane lipids peroxidation. This may be partly accounted for upholding the low level of lipid peroxidation in plants subsequently higher membrane integrity compared to control plants [49,51]. That was a promising result to approve C. procera as a prospective protective agent. Moreover, the exacerbation of both primary and secondary metabolites production by C. procera extracts indicates the highly prompted and efficiently up-regulated carbon metabolism that may encourage the translocation of carbon to its sinks with an increment in the carbon pool ultimately allocated for secondary metabolism and thus greater production of secondary metabolites as well as growth and development [52,53], the matter that may catapult C. procera as an inducible agent. In the present work, the increase in the production of secondary metabolites: anthocyanin, phenolics and flavonoids could be witnessed by increasing in PAL activities submitting an up-regulation of plant secondary metabolite production jointed with enhanced PAL activity that is efficiently prompted by CLEs. This is mainly due to the fact that PAL is an enzyme, which synthesizes a precursor for various secondary metabolites production and a crucial regulation factor between primary and secondary metabolism [54]. On the other hand, the elevated amino acids content might be legalized the availability of the phenylalanine (Phe) as an elite substrate for PAL thus more Phe is available for secondary metabolites production [55].

Enzymatic Antioxidants
The alternations in the capacities of enzymatic antioxidant of C. roseus leaves and roots under irrigation and spraying treatments with CLEs (ME, CWE and AWE) were showed in Table 4. All C. procera treatments significantly (p < 0.05 or p < 0.01) triggered the enzymatic antioxidants activities in C. roseus leaves and roots compared to control thus the stabilizing of cell oxidative status may be due to boosting of quenching hydrogen peroxide enzymes viz. CAT, APX, PPO, POD and GST. The highest increment in leaves enzymes activities was registered for both enzymes PPO and GST and amounted to 389% and 384%, respectively, when compared to control. In root CAT activity, the highest increment value that amounted as 217% with respect to control was recorded.
The increment of various ascorbate-glutathione pathway enzymes, POD and GST, could interpret the trapping of oxidative stress burst and healthiness of C. roseus plants [49]. The stimulation of PAL activity by various extracts was the main way for secondary metabolites enhancement as phenolics, flavonoids and anthocyanins as was reported by Ghasemzadeh et al. [53,54]. The activation of PPO could increase the plant defense responses against the pathogen attack. C. procera extracts and whatever the method of application efficiently induced the activity of PPO under natural conditions. Thus, further studies should be done to evaluate the role of C. procera in induction biochemical changes under biotic and a biotic stress.
Overall, The enhanced antioxidative capacity via increasing activities of CAT, APX, PPO, POD, GST and PAL as well as healthy promoting non-enzymatic antioxidants such as ascorbic acid, (AsA), glutathione (GSH), α-tocopherols, function together to maintain membrane integrity which advocated by lower lipid peroxidation indicating the potential role of phytochemicals in CLEs at the cellular level as modulators of gene expression, plant growth regulators and in signal transduction [54,56,57] indicating that CLEs mediated the promotion of antioxidant enzyme activities. These results were concomitant with several studies that confirmed that many plants extracts have defense enzymes stimulation properties. Goel and Paul [58] found that the PPO in tomato was stimulated by Azadirachta indica aqueous fruit extracts. Additionally, PAL, POD and PPO in cotton were activated by zimmu (Allium cepa L. and Allium sativum L.) [59], and PAL in barely prompted by aqueous extract of leaves of Azadirachta indica [60].

The Interplay between Phytochemical Compounds from C. procera Extracts and Metabolism of C. roseus Plant
Based on the statistical components accompanied with the data in Tables 2-4 where comparable enhancing effects were achieved by either two modes of application regarding growth criteria or antioxidants potential, it can be suggested that the behavior of phytochemicals of the tested extracts in the soil (rooting medium) was not the dominant factor determining the phytochemical performances but spraying (leave contact surface) treatments can produce comparable results as well. Besides, the nature of phytochemicals mostly differs according to the type of extract however the same result was also pronounced. Accordingly, the reaction between C. procera phytochemicals and of C. roseus metabolism herein may be received plant species dependence regardless of the chemical nature of phytochemical or contact surface (foliar spraying or rooting medium) explaining the superiority of Catharanthus in cellular up-regulate, utilize and successful management of Calotropis phytochemicals. Thereby, the current investigation could serve as a matrix for further studies to seek elucidation of several plants' responses to C. procera extracts as a bio-stimulant agent.
In an attempt to reveal the mechanism of phytochemical components action and mapping out how receiver plant could respond to them, we try to know the various putatively biological activities of each component that has been identified in the used extracts by comparing against the Chemical Entities of Biological Interest (ChEBI) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway databases [61].
Moreover, the guanosine triphosphate (GTP; C00044) that produced from a small guanosine triphosphatases enzyme (small GTPases) are involved in nearly every aspect of cell biology such as, source of energy, an activator of substrates in metabolic reactions and it is one of the building blocks needed for the synthesis of RNA during the transcription process that may be a prerequisite for enzyme synthesis and eliciting in C. roseus [62] 3.4.2. Biocytin (C05552) Biocytinisan amide compound, a result from the vitamin biotin and the amino acid L-lysine, plays an important role in various pathways such as (biotin metabolism; KEGG: map00780, metabolic pathways; KEGG: map01100, vitamin digestion and absorption; KEGG: map04977) through different biochemical reactions such as (KEGG: R01077 and R04869) (KEGG: https://www.kegg.jp/dbget-bin/www_bget?C05552 (accessed on 1 January 2020)).

Phthalates (C01606)
Phthalatesor phthalate esters, are esters of phthalic anhydride and used in numerous applications especially as plasticizers to increase the durability, transparency, flexibility and longevity of plastics. Furthermore, in some edible plants, the exposure of phthalates has unequivocally affected the physiology, growth and development of edible plants [65,66] (KEGG: https://www.kegg.jp/dbget-bin/www_bget?C01606 (accessed on 1 January 2020)). Arpna and Rajinder [67], found that the barley seedlings treated with phthalate significantly increase the contents of proline, pigment, carbohydrate, soluble protein in barley seedlings shoots and roots. Additionally, CAT, SOD, POD, GR and APX activities were significantly increased in shoots and roots of barley seedlings after exposure by phthalate.

Digitoxin (D00297)
Digitoxin is a phytosteroid and similar in structure to acetyldigitoxin (D01972), acetyldigoxin (D07556) and beta-Acetyldigoxin (D06881) (KEGG: https://www.kegg.jp/dbgetbin/www_bget?PATH:map07233 (accessed on 1 January 2020)). Phytosteroids in plants shoulder an important task as components of cell membranes and growth hormones serving as safeguarding factor for membrane function and stability that evidenced by low level of MDA in C. roseus by CLES supplementation [68]. Moreover, the digitoxin or other similar structure play roles in principal biological functions as signaling molecules, and can also modulate the activity of membrane-bound enzymes [69].