Figure 1 shows the ameliorating effect of CuNP and CuONP based nanofertilizers compared to bulk fertilizers (CuSO4) on germinating barley seeds. Therefore, we were interested in exploring potential side-effects and risks of the use of Cu-based agricultural nanofertilizers on food quality of concern to Human health.
We found increased production of H2O2 in Cu NP and CuO NP treated H. vulgare L. plants (Fig. 2A). This increase in the level of reactive oxygen species (ROS), especially with CuO NP, may be harmful to biological molecules like proteins, amino acids and lipids. ROS can also alter antioxidant concentration and activity. Similar findings were reported in other studies that found a negative effect of Cu-based NPs on plant cellular components. These included alteration of important structural and functional proteins and enzymatic systems which may lead to metabolic dysfunction 14. In addition, exposure to Cu NPs increased the total amounts of phenolic acids and flavonoids (Fig. 2B). CuO NP also triggered a significant increase of the level of total flavonoids (Fig. 2B). When compared with the bulk form of Cu sulfate, we may attribute these changes to the effects both of Cu ions released and the nanosized particles. Polyphenolic compounds are secondary metabolites that are involved in plant defense against biotic and abiotic environmental stress and they offer promising health benefits. Polyphenolic compounds are derivatives of hydroxybenzoic acids or hydroxycinnamic acids. Hydroxybenzoic acid derivatives include compounds such as gallic, vanillic, p-coumaric, hydroxybenzoic, and syringic acids. Hydroxycinnamic acids include derivatives such as ferulic acid, p-coumaric acids and dehydrodimers.
Liquid chromatography-mass spectrometry (HPLC-MS) analysis (Figs. 3–6) revealed qualitative and quantitative changes in phenolic acids (Table 1) and flavonoids (Table 2) in H. vulgare L. plants after exposure to NPs. Overall data showed that treatment with Cu NP and CuO NP decreased some polyphenolic compounds, such as quercetin, syringic acid, caffeic acid, p-coumaric and o-coumaric acids. Conversely, Cu NPs induced appearance of gallic acid, naringenin, apegenin, luteolin and cirsilineol. CuO NPs increased levels of catechin(+), naringenin, silymarin, 1,3-di-o-caffeoyquinic acid, epicatechin, hyperoside (quercetin-3-o-galactoside), luteolin-7-o-glucoside and acacetin. Both types of NP increased synthesis and quantity of salviolinic acid, quinic acid and transfrulic acid. On the other hand, Cu sulfate also triggered an increase in content of several polyphenolics and flavonoids, such as quinic acid, p-coumaric acid, silymarin, acacetin and transcinnamic, versus a decrease of other compounds such as transfrulic acid and cirsiliol, when compared to controls. A comparison of effects between the nanosized forms of Cu and Cu sulfate suggests that the changes observed may be attributable to either NPs effects or to both NP and released Cu ions. An increase of quinic acid may be due to Cu ions, and also to the significant involvement of NPs themselves. On the contrary, an increase in transfrulic acid and cirsiliol due to Cu NP and CuO NP is due to NPs, because they decreased markedly on exposure to CuSO4. The increase of salviolonic acid is also attributable to NPs since no change was observed with CuSO4 as compared with control. The increase of silymarin in the presence of Cu NP may be associated with Cu ions because a similar increase was evident with CuSO4 treatment. The absence of caffeic acid, o-coumaric acid, quercetin and syringic acid after NP exposure is similar to CuSO4, which suggests an involvement of Cu ions. Induction of gallic acid, naringenin, cirsilineol, luteolin and apegenin with only Cu NP versus their absence on CuO NP and CuSO4 treatment suggests an effect of NP. An increase in catechin(+), naringenin, 1,3-di-o-caffeoquinic acid, epicatechin and luteolin-7-o-glucoside under CuO NP exposure when compared to respective controls suggests an effect attributable to CuO NP.
Table 1
Quantitative and qualitative analysis by HPLC-MS of phenolic acids in barley (Hordeum vulgare L. var. Ardhaoui) seedlings germinated for 6 days in the presence of distilled water (H2O) or 500 mg L− 1 of solutions of Cu NP, CuO NP and CuSO4.
Treatment
|
|
Quinic acid
|
Caffeic acid
|
p-coumaric acid
|
transfrulic acid
|
o-coumaric acid
|
Salviolinic acid
|
1,3-di-O-caffeoyquinic acid
|
Syringic acid
|
Gallic acid
|
H2O
|
Mean
|
19.08 b
|
13.72 a
|
43.58 a
|
1.92 b
|
4.74 a
|
0 b
|
0 b
|
3.96 a
|
0 b
|
±SE
|
10.65
|
4.97
|
3.53
|
0.014
|
2.45
|
0
|
0
|
2.30
|
0
|
CuNP
|
Mean
|
519.57 a
|
0 b
|
27.07 a
|
6.97 a
|
0 b
|
1.05 a
|
0 b
|
0 a
|
2.25 a
|
±SE
|
260.14
|
0
|
4.00
|
1.73
|
0
|
0.61
|
0
|
0
|
1.28
|
CuONP
|
Mean
|
64.52 c
|
0 b
|
31.53 a
|
6.18 a
|
0 b
|
1.18 a
|
0.724 a
|
0 a
|
0 b
|
±SE
|
2.59
|
0
|
0.65
|
0.69
|
0
|
0.60
|
0.43
|
0
|
0
|
CuSO4
|
Mean
|
52.35 c
|
0 b
|
46.97 a
|
1.54 b
|
0 b
|
0 b
|
0 b
|
0 a
|
0 b
|
±SE
|
6.26
|
0
|
11.37
|
0.87
|
0
|
0
|
0
|
0
|
0
|
ANOVA α = 0.05
|
***
|
*
|
NS
|
*
|
NS
|
***
|
***
|
NS
|
**
|
Values are means of 3 replicates ± SE. Letters (a-d) denote the statistical classes based on the differences between treatments according to Duncan test (α = 0.05). ANOVA (p < 0.05): Significance of difference in comparison with control (H2O): Not significant: NS; Significant *: p < 0.05; Very significant **: p < 0.01; Highly significant ***: p < 0.001.
Table 2
Quantitative and qualitative analysis by HPLC-MS of flavonoids in barley (Hordeum vulgare L. var. Ardhaoui) seedlings germinated for 6 days in the presence of distilled water (H2O) or 500 mg L− 1 of solutions of Cu NP, CuO NP and CuSO4
Treatment
|
|
Apegenin
|
Cirsiliol
|
Quercetin
|
Luteolin
|
Cirsilineol
|
Catechin
(+)
|
Naringenin
|
Naringin
|
Epicatechin
|
Silymarin
|
Hyperoside (quercetin-3-o-galactoside
|
Luteolin-7-o-glucoside
|
Acacetin
|
trans cinnamic
|
H2O
|
Mean
|
0 b
|
514.18 a
|
1.59 a
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
±SE
|
0
|
13.01
|
0.36
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
Cu NP
|
Mean
|
5.836 a
|
517.66 a
|
0 b
|
3.44 a
|
15.85 a
|
0 b
|
0 b
|
3.59 a
|
0 b
|
4.927 a
|
0 b
|
0 b
|
0 b
|
0 b
|
±SE
|
3.21
|
42.03
|
0
|
1.98
|
7.99
|
0
|
0
|
1.93
|
0
|
2.84
|
0
|
0
|
0
|
0
|
CuO NP
|
Mean
|
0 b
|
472.08 a
|
0 b
|
0 b
|
0 b
|
4.83 a
|
680.15 a
|
0 b
|
3.807 a
|
0 b
|
0.47 a
|
1.09 a
|
0.47 b
|
0 b
|
±SE
|
0
|
6.07
|
0
|
0
|
0
|
2.7
|
296.47
|
0
|
2.19
|
0
|
0.25
|
0.60
|
0.23
|
0
|
CuSO4
|
Mean
|
0 b
|
194.02 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
0 b
|
4.35 a
|
0 b
|
0 b
|
10.26 a
|
76.91 a
|
±SE
|
0
|
15.85
|
0
|
0
|
0
|
0
|
0
|
0
|
0
|
2.49
|
0
|
0
|
3.98
|
44.35
|
ANOVA α = 0.05
|
***
|
***
|
***
|
***
|
***
|
***
|
***
|
*
|
**
|
**
|
***
|
***
|
***
|
**
|
Values are means of 3 replicates ± SE. Letters (a-d) denote the statistical classes based on the differences between treatments according to Duncan test (α = 0.05). ANOVA (p < 0.05): Significance of difference in comparison with control (H2O): Not significant: NS; Significant *: p < 0.05; Very significant **: p < 0.01; and Highly significant ***: p < 0.001.
Taken together, qualitative and quantitative changes in phenolic acids and flavonoids, suggest that the uptake of Cu NP and CuO NP by H. vulgare L. alters antioxidant status including phytochemical profiles. Consumption of H. vulgare L. derived products are thought to lead to positive effects on human health. For example, p-coumaric acid (4‐hydroxy‐cinnamic acid) is classified as a phytochemical and nutraceutical 17. Coumaric acid is a hydroxy derivative of cinnamic acid and naturally occurs in three isomers (ortho‐, meta‐ and para‐). In our study, depletion of p-coumaric acid may result in decrease of antioxidant and antimicrobial effects. Similarly, deprivation of other phenolic compounds like rutin, ferulic acid, and quercetin may reduce their efficiency in preventing ROS activity such as lipid peroxidation 18. Ferulic acid is another promising antioxidant of cereals such as wheat and barley which has been shown to induce apoptotic cell death in human breast cancer cells such as MCF-7, MDA-MB-231, osteosarcoma 143B, and MG63 cells lines 19,20.
Gas chromatography-mass spectrometry (GC-MS) analysis revealed changes in levels of various volatile organic compounds (fatty acids, phenolic acids, esters, alcohols) with treatments (Figs. 7, 8, Table S1); Some of these compounds appeared with Cu NP and CuO NP, such as 5-H-1-pyrindine. Other phytocompounds appeared with CuO NP and CuSO4 treatments, like 13-docsenoic acid and oleic acid with Cu NPs, CuO NP and CuSO4, as well as cholest-5-en-3-ol and 24-propylidene (3β) with Cu NP. Some phytochemicals increased, such as 5-hydroxymethyl furfural with Cu NP, CuO NP and CuSO4, as well as 2-methoxy-4-vinylphenol and 9,12-octadecadienoic acid (linoleate = linoleic acid) in the presence of Cu NP, CuO NP and CuSO4. However, Cu NP and CuO NP triggered the complete absence of indolizine, and the depletion of most volatiles, notably n-hexadecanoic acid, 9-octadecenamide, campesterol, stigmasterol and β-sitosterol. Indolizine was absent in Cu NP and CuO NP treated barley. This effect could be attributed to the nanosized particles because indolizine was also detected in CuSO4 treated seedlings.
The percentage of 2-methoxy-4-vinylphenol inversely increased with all forms of Cu treatment. However, n-hexadecanoic acid (palmitic acid) decreased from 15% (H2O) to around 6.6% with Cu NP, 4.8% with CuO NP and 6% with CuSO4. Similar decreases of the percentages of erucid acid were estimated at approximately 1% and 2.7%, respectively, with Cu NP and CuSO4. Heptadecanoic acid also decreased from 0.85% (H2O) to 0% with Cu NP, but increased with CuO NP (around 4.5%) and CuSO4 (around 3%). A decrease of 9-octadecenamide under Cu NP and CuO NP treatment may be attributed to the effect of Cu ions because CuSO4 induced complete disappearance of 9-octadecenamide. On the other hand, all Cu based treatments led to complete deficiency in many phytochemicals such as 8,11-octadecadienoic acid, trans-13-octadecenoic acid, octadecanoic acid, 9-hexadecenoic acid, cis-13-eicosenoic acid, eicosanoic acid (also known as arachic acid), 9,12-octadecadienoic acid (also known as α-glyceryl linoleate and linoleic acid). Campesterol, stigmasterol and β-sitosterol also declined with Cu NP and CuO NP, as well as with CuSO4.
The above-described findings suggest that deficiency in some phytocompounds may disrupt the homeostatic control of their concentration in plant tissues. This may lead to decreased health and nutritional benefits for this food. The effect of indolizine (pyrrolo[1,2-a]pyridine) in barley exposed to Cu-based NP, may impair multiple biological properties, such as antioxidant activity 21, anticancer activity 22, antitubercular 23, antimicrobial 24, anti-inflammatory 25, and antifungal 26 activities. Moreover, lack of indolizine may result in deprivation of indolizidine alkaloids, which are known for their wide range of beneficial pharmacological properties 27.
On the other hand, in our study, effects on palmitic acid in barley-based diets may influence its putative health effects. In general, its level is maintained steady via metabolic regulation. Decrease of palmitic acid’s uptake in food does not affect significantly its tissue concentration, since the exogenous source is counterbalanced by its endogenous biosynthesis. However, due to other factors such as excessive intake of carbohydrates and a sedentary lifestyle, reduced tissue content of palmitic acid may cause disruption of membrane phospholipid balance and an over-accumulation of palmitic acid in tissues. This may result in dyslipidemia, hyperglycemia, increased ectopic fat accumulation and increased inflammation via toll-like receptor 4. A shortage of trans-13-octadecenoic acid in barley after exposure to NP may cause the loss of important bioactivities including anti-inflammatory, anticancer, hypocholesterolemic and anemiagenic activities 28. Similarly, eicosanoids are crucial for normal metabolic functioning of cells and tissues. A decrease in cis-13-eicosenoic acid and eicosanoic acid (also known as arachic acid) in barley may cause impairment of the anti-inflammatory effects of these bioactive compounds. Anti-inflammatory properties and cardiovascular health effects of erucic acid may also be reduced or lost, respectively, by Cu NP and CuO NP treatments. Additionally, a decrease in 9,12-octadecadienoic acid, also known as linoleic acid, may lead to reduced energy and altered phospholipid structure, fluidity and function of membrane in the epidermis. Conjugated linoleic acid (CLA) is a mixture of isomers of octadecadienoic acid, which are polyunsaturated fatty acids that provide essential nutritional benefits, including immune modulatory and anti-inflammatory activities. Dietary CLA have been shown to enhance proliferation of immune cells, like CD8⁺ lymphocytes and thymocytes, and to play a role in prevention or treatment of diseases (e.g. nutraceuticals). Linoleic acid cannot be synthesized by humans, and thus must be consumed in food. Therefore, a deprivation of linoleic acid, as found in this study, may contribute to disorders such as scaly skin lesions, growth retardation, altered plasma fatty acid patterns and thrombocytopenia.
Furthermore, our findings suggest that the use of Cu based fertilizers may impact on health-promoting effects of bioactive compounds in a barley-based diet. For instance, alteration of phenolic acid and flavonoid levels may reduce their efficiency as antioxidants, immune enhancers and tumor suppressors 29–34. The roles of barley in facilitating digestion and protection against disorders may also be altered, leading to decreased efficiency against obesity, hypertension, oxidative stress, cardiovascular diseases and type II diabetes 29–34. Similarly, other biological benefits in barley may be disrupted or lost, resulting in reduced anticancer effects and a less valuable role in prevention of oxidative damage to DNA, proteins, and membrane lipids 35,36. Moreover, a scarcity in adequate antioxidants may limit protection of human cells from free radical-induced oxidative damage 37. This failure may also cause elevation of concentrations of triglycerides, low-density lipoprotein (LDL), ROS, thiobarbituric acid-reactive substances (TBARS), and reduce concentrations of high-density lipoprotein (HDL), vitamin E, and vitamin A 38. In addition, phytosterols such as sitosterol, campesterol and stigmasterol are natural compounds with established and emerging health benefits. A deficiency in sterols will impact on their bioactive properties and benefits to human health, such as reduction of intestinal cholesterol absorption, and protection against cancer. A deficiency of β-sitosterol may reduce its immune stimulating and anti-inflammatory activities. Compounds like Z-9-octadecenamide (oleamide), squalene, n-hexadecanoic acid, linoleic acid, octacosane, tetratriacontane and 𝛼-tocopherol have been reported to give beneficial effects on human health. Free phenolic acids are considered precursors for the bioactive aromes such as vinylbenzene (styrene). The 2-methoxy-4-vinylphenol and 4-vinylphenol can be formed by enzymatic decarboxylation of ferulic acid and p-coumaric acid. Decrease of availability of polyphenols from barley may reduce protection against human cancer due to reduced antioxidant potential and reduced apoptosis 39. In addition, an increase in steroid (e.g. cholest-5-en-3-ol, 24-propylidene) levels may trigger psychological effects such as aggressive behavior, mood swings and impulsivity in male teenagers and adults. Long-term consumption of treated barley may lead to other harmful health effects such as acne, male-pattern baldness, liver damage, increased risk of heart attack and stroke, high blood pressure and liver, kidney or prostate cancers. The steroids also can affect bone development and linear growth, and cause hormone imbalance leading to body image disorders and infertility 40.
Furthermore, Cu is persistent in the environment, so it can bioaccumulate within barley plants. Therefore, an additional potential risk may be posed by elevated Cu concentrations in plants treated with Cu NP (4-fold) and CuO NP (10-fold) (Table 3). Redox properties of Cu can contribute to its toxicity, as free ions trigger production of damaging radicals 41. In the literature, Cu addition to crops led to several benefits in agriculture 42–44, however it was harmful in other reports 45. In fact, excessive Cu intake can lead to toxicity and a drop in Zn and vitamin C levels. Effects of consumption of Cu contaminated barley may include acute hemolysis, hepatic necrosis, cardio-toxicity with hypotension, tachycardia and tachypnea, as well as central-nervous-system manifestations leading sometimes to coma 46. Our data revealed that Cu excess altered availability of several mineral elements: macronutrients (Ca, Mg, K), micronutrients (Fe, Mn, Zn, Cu) and Na. A decrease in Fe, Mg, Zn and Ca contents were observed in Cu NP treated seedlings. A similar decrease was more significantly evident after treatment with CuO NP (Table 3). Conversely, content of Mn and K increased in the presence of Cu NP and CuO NP.
Table 3 Content of essential mineral elements determined by SAA in barley (Hordeum vulgare L. var. Ardhaoui) seedlings germinated for 6 days in the presence of distilled water (H2O) or 500 mg L− 1 of solutions of Cu NP, CuO NP and CuSO4.
Values are means of 3 replicates ± SE. Letters (a-d) denote the statistical classes based on the differences between treatments according to Duncan test (α = 0.05). ANOVA (p < 0.05): Significance of difference in comparison with control (H2O): Not significant: NS; Significant *: p < 0.05; Very significant **: p < 0.01; Highly significant ***: p < 0.001.
The content of Na increased with CuO NP but decreased with Cu NP. This may be due to effects of Cu ions since similar effects were observed with CuSO4. A balanced diet usually provides all essential minerals. Alteration of Na and K levels may disrupt fluid balance, nerve transmission and muscle contraction. A deficiency of Ca may affect health of bones and teeth, muscle and nerve functioning, blood pressure regulation and the immune system. Ca deficiency can also lead to osteoporosis, dysfunction of bone and muscle, as well as cardiovascular irregularities. Similarly, a shortage of Mg may alter several bodily functions including immune system health. Lack of Fe also alters energy metabolism and hemoglobin function, leading to anemia, tissue inflammation and fatigue, while Zn deficiency may result in delayed sexual maturation and a depressed immune response.
It was reported in a study conducted on chitosan-coated mesoporous silica nanoparticles in Citrullus lanatus that applied nanoparticles were not entering the fruits at any appreciable rate when compared to untreated controls 47. Other authors 13 found that the efficiency of foliar applications of NP and their translocation to roots may require applications of low levels of NP to young plants, thereby minimizing human and environmental exposure to these particles. Other studies also reported the interaction between agricultural practices notably the use of fertilizers and nanomaterials on the nutritional properties of vegetables and foods 7,48−50.