DPPH scavenging and metal chelating activity of sage

In order to support the protective role of sage against uranyl acetate toxicity, DPPH scavenging and metal chelating activities, which indicate antioxidant activity, were investigated and the results are given in Fig.2. DPPH is a stable free radical in aqueous solutions, and the decrease in the absorbance of the DPPH radical indicates antioxidant activity. Sage was determined to exhibit a dose-dependent increasing DPPH scavenging effect. DPPH scavenging activities of 200mg/mL sage, BHA and BHT were determined as 72.9%, 67.9% and 89.1%, respectively. The metal chelating activities of sage and standards were determined by evaluating their ability to compete with ferrozine for the ferrous ions. A dose-dependent increasing activity was also obtained in metal chelating activity. Metal chelating activities of 200mg/mL sage, BHA and BHT were determined as 82.8%, 76.3% and 88.5%, respectively. These results show that sage has a free radical scavenging activity that is higher than the standard antioxidant BHA and lower than BHT. Its high DPPH removal and metal chelating activity indicate the antiradical and metal chelating properties of sage, as well as its strong antioxidant capacity. There are also studies in the literature that draw attention to the similar features of sage. Emre et al.30 reported that different Salvia species grown in Turkiye exhibited metal chelating activity in the range of 45.080.48%. Roman et al.31 investigated the antiradical properties of S. officinalis extract and stated that it exhibited more than 85% DPPH removal activity. With the powerful antioxidant property, Sage has a protective role against much toxicity, and the results obtained in the Allium test in this study confirm this hypothesis.

DPPH scavenging (DSCA) and metal chelating activity (MCA) of sage.

The effects of uranyl acetate and sage application on selected physiological parameters are shown in Table 1. The maximum germination percentage, root length and weight gain were measured in the control group and Group II and Group III, which were exposed to two different doses of sage. No statistically significant difference was found between the physiological parameter values measured in these groups (p>0.05). In Group IV, in which 0.1mg/mL of uranyl acetate was administered, statistically significant decreases were found in all investigated physiological parameter values compared to the control group (p<0.05). It was observed that this decrease was approximately 2.1 times for germination percentage, about 7 times for root length and about 4.8 times for weight gain. The application of sage together with uranyl acetate caused a statistically significant (p<0.05) increase in the values of all investigated physiological parameters, although not as much as the control group. It was determined that these increases were more pronounced at the 380mg/L dose of sage. Compared to Group IV, germination percentage increased approximately 1.2 times, root length approximately 1.9 times and weight gain approximately 1.8 times in Group VI.

Although there is no comprehensive study in the literature on the effects of uranium or uranyl acetate application on the physiological properties of plants, there are some studies on the effects of other heavy metals. For example, avuolu et al.32 determined that Pb and Hg heavy metal application at 10 and 50ppm doses caused dose-dependent decreases in the germination percentage, root length and weight gain of Cicer arietinum L. seeds. They also reported that these decreases were more pronounced in the group exposed to the 50ppm dose of Hg. avuolu and Yaln33 determined that 25 and 50ppm doses of Al and Co application caused a dose-dependent decrease in the germination percentage, root length and weight of Phaseolus vulgaris L. cv. kidney bean seeds. They also observed that these decreases were more pronounced at the 50ppm dose of Al. Grel et al.34 observed that 2.4, 8.0 and 12.5mg/L Cr doses caused dose-related decreases in germination percentage, root length and weight gain in A. cepa. Girasun et al.35 determined that Pb application at 50, 100 and 200mg/L doses caused a dose-dependent decrease in physiological parameters such as germination percentage, root length and weight gain in A. cepa. Macar et al.36 found statistically significant reductions in germination percentage, root length and weight gain in A. cepa bulbs exposed to 5.5mg dose of Co for 72h.

In this study, it is thought that the abnormalities in physiological parameters as a result of uranium exposure are due to the reduction of A. cepa roots' intake of water and inorganic substances. Because it has been reported in the literature that high doses of heavy metal exposure in different plant species reduce the water and mineral substance uptake of the roots, and their productivity decreases by affecting the photosynthesis reactions and nitrogen metabolisms. On the other hand, it has been reported that exposure to heavy metals causes root, shoot, plant growth and plant weight reduction, deterioration of grana structure, inhibition of chlorophyll synthesis and respiration and development of apoptosis and necrosis processes in plants. ROS produced by heavy metals is shown as the main reason for the processes that encourage all these negative effects in the plant. Sage, which exhibits strong DPPH removal and metal chelating activity, protected against oxidative stress induced by uranium and exhibited a toxicity-reducing effect with its antioxidant property. It has also been stated in the literature that plants have developed some effective defense mechanisms to combat ROS-induced oxidative stress37. Therefore, it is considered that these defense mechanisms developed by A. cepa to prevent uranium from entering the cell may be another reason for the decrease in the investigated physiological parameter values. Because the excessive increases in the number/frequency of epidermis and cortex cells observed in the microscopic examination of root tip meristematic cells support this idea.

The genotoxicity induced by uranyl acetate application and the protective role of sage against this toxicity are shown in Figs. 3, 4 and Table 2. Statistically insignificant (p>0.05) MN formations were found in the control group and Group II and Group III, which were exposed to two different doses of sage. In addition, CAs in the form of a few sticky chromosome and unequal distribution of chromatin was detected in these groups, which was not statistically significant (p>0.05). On the other hand, the highest MI value (741.30, 747.90 and 743.80, respectively) was also determined in these groups. The application of 0.1mg/mL uranyl acetate caused the highest rate (82.40) of MN formation (p<0.05) in the root tip cells of the bulbs in Group IV, and promoted CAs such as fragment, vagrant chromosome, sticky chromosome, bridge and unequal distribution of chromatin and caused significant decreases (p<0.05) in the MI value. The greatest effect of uranyl acetate application on chromosomes occurred in the form of fragment formation. The application of sage together with uranyl acetate decreased the genotoxic effects of uranyl acetate, and caused a statistically significant (p<0.05) decrease in the frequencies of MN and CAs, and a significant (p<0.05) increase in the MI value, depending on the dose. It was determined that these alterations observed in the investigated genotoxic parameters were more pronounced in Group VI, where 380mg/L dose of sage was administered. Compared to Group IV, the frequency of fragment decreased approximately 1.5 times, the MN frequency decreased approximately 1.4 times, and the MI rate increased approximately 1.3 times in Group VI.

CAs induced by uranyl acetate. MN in interphase (a), fragment in metaphase (b), vagrant chromosome in anaphase (c), sticky chromosome in prophase (d), bridge in early anaphase (e), unequal distribution of chromatin in anaphase (f).

The effects of uranyl acetate and sage on DCN and MI (%). Group I: Control, Group II: 190mg/L sage, Group III: 380mg/L sage, Group IV: 0.1mg/mL uranyl acetate, Group V: 0.1mg/mL uranyl acetate+190mg/L sage, Group VI: 0.1mg/mL uranyl acetate+380mg/L sage. MI was calculated by counting 10,000 cells in each group. *indicates statistical difference between Groups I and IV, **indicates statistical difference between Groups IV and VI (p<0.05). DCN: dividing cell number, MI: mitotic index.

Although there is no comprehensive study in the literature on genotoxicity caused by exposure to uranium or uranyl acetate in plants, there are some studies conducted with experimental animals. For example, avuolu et al.38 observed MN formation in erythrocyte and buccal mucosal epithelial cells of Swiss albino mice exposed to 5mg/kg b.w of uranyl acetate by oral gavage for 5days. In addition, they reported a decrease in MI value with CAs in the form of break, fragment, gap, acentric and ring chromosomes in bone marrow cells. In addition, there are some studies in the literature investigating the genotoxicity induced by other heavy metal ions in plants. For example, avuolu et al.32 determined that exposure to Pb and Hg at two different doses (10 and 50ppm) caused an increase in the frequency of MN in C. arietinum root tip cells and promoted CAs in the form of sticky chromosome and bridge. avuolu and Yaln33 observed that exposure to Al and Co at 25 and 50ppm doses caused MN formation in P. vulgaris cv. kidney bean root cells. Grel et al.34 reported that administration of three different doses of Cr (2.4, 8.0 ve 12.5mg/L) caused a dose-dependent decrease in MI in A. cepa root tip cells. In addition, they found an increase in the frequency of MN and the numbers of CAs such as fragments, unequal distribution of chromatin, sticky chromosomes, bridges, reverse polarization and c-mitosis. Girasun et al.35 showed that exposure to three different doses (50, 100 ve 200mg/L) of Pb decreased the MI value, increased the frequency of MN, and caused damage in the form of fragments, adhesions, bridges and c-mitosis in A. cepa root tip cells, depending on the application dose. Macar et al.36 observed a decrease in MI, an increase in MN formation and an increase in the number of CAs in root tip cells of A. cepa, where 5.5mg of Co was applied. They also determined that Co application promotes CAs in the form of fragment, sticky chromosome, bridge, unequal distribution of chromatin, multipolar anaphase, nucleus damage, and irregular mitosis.

In our study, it is thought that the main reason for the decrease in MI value and the increase in the numbers of MN and CAs in Group IV treated with uranyl acetate may be due to the direct or indirect interaction of uranium with chromosomes. Because it has been reported in the literature that heavy metals disrupts the structure of DNA directly or indirectly by producing ROS, promoting DNA damages. On the other hand, some heavy metals have been reported to cause disruptions in DNA repair processes. For example, while Cr causes damage by reacting directly with DNA, As, Ni and Cd act by preventing the repair processes of DNA double-strand breaks. Damages such as MN, fragments, breaks, sister chromatid exchanges and variation are other CAs promoted by heavy metal ions39. The genotoxic and cytotoxic effects induced by uranyl acetate may be related to the occurrence of oxidative stress in general. Sage protects the integrity of the genome by reducing the oxidative load in the cell, especially with its strong metal chelating activity and antioxidant power. The reductions in MN and CAs frequencies observed in groups V and VI treated with sage+uranyl acetate confirm this idea.

The effects of uranyl acetate and sage application on selected biochemical parameters are shown in Fig.5. No statistically significant difference was observed between the root MDA levels and SOD and CAT activities of the control group and Group II and Group III exposed to two different doses of sage (p>0.05). Uranyl acetate application at 0.1mg/mL dose caused statistically significant (p<0.05) increases in root MDA level, which is an indicator of lipid peroxidation, and in SOD and CAT activities, which are antioxidant enzymes. Compared to the control group, these increases were found to be approximately 3.8 times for MDA level, approximately 3.2 times for SOD activity and approximately 2.7 times for CAT activity in Group IV. It was determined that the application of sage together with uranyl acetate again promoted statistically significant (p<0.05) decreases in MDA levels, SOD and CAT activities, depending on the dose. These decreases were even more pronounced in Group VI exposed to 380mg/L of sage. Compared to Group IV, approximately 2.1-fold decrease in MDA level, approximately 1.3-fold decrease in SOD activity and approximately 1.3-fold decrease in CAT activity was detected in Group VI.

Effect of uranyl acetate and sage application on selected biochemical parameters. Group I: Control, Group II: 190mg/L sage, Group III: 380mg/L sage, Group IV: 0.1mg/mL uranyl acetate, Group V: 0.1mg/mL uranyl acetate+190mg/L sage, Group VI: 0.1mg/mL uranyl acetate+380mg/L sage. * indicates statistical difference between Groups I and IV, ** indicates statistical difference between Groups IV and VI (p<0.05).

Although there is no comprehensive study in the literature of biochemical toxicity induced by exposure to uranium or uranyl acetate in plants, there are some studies with Swiss albino mice. avuolu et al.38 reported a significant increase in blood MDA levels and a significant decrease in GSH levels in Swiss albino mice exposed to 5mg/kg b.w of uranyl acetate by oral gavage for 5days. In a similar study, Yapar et al.40 found significant increases in MDA levels and significant decreases in GSH levels in liver and kidney tissues of Swiss albino mice exposed to 5mg/kg b.w of uranyl acetate. In addition, there are some studies dealing with the biochemical toxicity induced by other heavy metals other than uranium in plants. avuolu et al.32 reported that MDA levels in C. arietinum root tip cells exposed to Pb and Hg heavy metals at 10 and 50ppm doses increased dose-dependently, and these increases were even more pronounced at 50ppm doses of Hg. avuolu and Yaln33 stated that the application of Al and Co at two different doses (25 and 50ppm) caused dose-related increases in the MDA levels of P. vulgaris cv. kidney bean root cells, and these increases were higher et al. doses than at Co doses. Macar et al.36 observed that Co application at 5.5mg dose caused significant increases in MDA levels and SOD and CAT enzyme activities of A. cepa root tip cells.

MDA is a 3-carbon aldehyde, which is one of the most important markers of cell membrane damage, in other words, lipid peroxidation. Lipid peroxidation is a reaction caused by free radicals that cause oxidative damage of unsaturated fats. A free radical can then abstract the H atom and form an oxidized lipid free radical, producing a peroxyl radical. The peroxyl radical can remove an electron and produce a lipid hydroperoxide and another lipid free radical. This process can continue as a chain reaction. Since lipid hydroperoxide is unstable, it decomposes to form MDA and 4-hydroxy-2-nonenal products. In cases where the increase of free radicals in the cell, enzyme systems and antioxidant molecules in the cell are not sufficient for protection, these free radicals attack cell membranes, cause lipid peroxidation and increase MDA levels41. Therefore, the increase in MDA levels in A. cepa root cells treated with uranyl acetate can be explained by the fact that uranium causes free radical production and these free radicals cause damage to the membranes of the root cells.

SOD and CAT enzymes are known as antioxidants that prevent the formation of free radicals in the cell or eliminate or neutralize their effects. While the SOD enzyme neutralizes the superoxide radical, the CAT enzyme catalyzes the conversion of H2O2, which is highly toxic for the cell, into water and oxygen42. Therefore, the increase in SOD and CAT enzyme levels in A. cepa root cells exposed to uranyl acetate can be explained by the fact that uranium causes free radical production and increases SOD and CAT enzyme levels as a defense mechanism of the cell to minimize the harmful effects of free radicals. The fact that uranyl acetate increases MDA levels and induces antioxidant enzyme activities can be explained by triggering oxidative stress. The decrease observed in MDA, SOD and CAT levels in Group V and Group VI treated with sage+uranyl acetate shows that sage provides protection against the biochemical toxicity of uranyl acetate. This healing property is closely related to the antioxidant, antiradical and metal chelating activities of sage.

The effects of uranyl acetate and sage application on the root anatomy of A. cepa are shown in Fig.6 and Table 3. No damage was observed in the root meristem cells of Group II and Group III, which were exposed to two different doses of sage with the control group. In Group IV exposed to uranyl acetate at a dose of 0.1mg/mL, epidermis and cortex cell damage, as well as meristematic cell damage in the form of flattened cell nucleus were observed. Co-administration of sage with uranyl acetate caused reductions/improvements in the severity of observed meristematic cell damage by reducing the negative effects of uranyl acetate, depending on the dose. It was determined that this decrease in the severity level was more pronounced at 380mg/L dose of sage.

Meristematic cell damages induced by uranyl acetate. Normal appearance of epidermis cells (a), normal appearance of cortex cells (b), normal appearance of cell nucleus-oval (c), epidermis cell damage (d), cortex cell damage (e), flattened cell nucleus (f).

Although there is no study in the literature that deals with the anatomical changes caused by uranium or uranyl acetate exposure in plant root tip meristematic cells, there are some studies on the anatomical effects of other heavy metals. Grel et al.34 reported that 2.4, 8.0 and 12.5mg/L Cr doses caused anatomical damage in the form of cell deformation, thickening of the cortex cell wall, flattened cell nucleus and necrosis in root tip meristematic cells of A. cepa. They also stated that the severity of these damages was dose dependent. avuolu et al.43 observed anatomical damage such as cell deformation, necrosis, flattening cell nucleus, thickening of the cortex cell wall, inclearly vascular tissue and accumulation of some substances in cortex cells in A. cepa root tip meristematic cells exposed to Hg at 25, 50 and 100mg/L doses. Girasun et al.35 detected cell damage such as thickening of the cortex cell wall, cell deformation, inclearly vascular tissue and necrosis in A. cepa root tip meristem cells of 50, 100 and 200mg/L doses of Pb exposure, the severity of which increased with the application dose. Macar et al.36 reported that 5.5mg Co dose promoted damages such as epidermis cell deformation, thickening of the cortex cell wall and flattened cell nucleus in A. cepa root tip meristematic cells.

This suggests that this epidermis and cortex cell damage induced by uranium occurs as a result of the defense mechanisms developed by plants against heavy metal ions. Because the roots have increased the number and frequency of the epidermis and cortex cells in order to prevent uranium from entering the cell, and these damages may have occurred as a result of the compression/suppression of the cells. The information in the literature that plants develop different defense mechanisms against heavy metal toxicity, such as accumulation, storage and crystallization of metals in certain regions, or changes in the cell membrane and cell wall, increase in vacuole numbers and metal-binding protein synthesis44, supports our this idea.

In recent studies, different plant extracts such as lycopene, carotene, Ginkgo biloba L., green coffee, green tea and stinging nettle are used to reduce toxicity promoted by toxic agents such as heavy metal ions. In this study, sage treatment provided significant protection against the physiological, biochemical, cytogenetic and anatomical abnormalities exhibited by the application of uranyl acetate in the A. cepa root tip cells. It provided improvement in germination-related parameters such as root length and weight gain, and decreased MN and CAs frequencies, which were detected at high rates after uranyl acetate application. These improvements increased depending on the dose and the highest protection was obtained at the dose of 380mg/L. In this study, it was determined that sage has antiradical property and scavenges the DPPH radical at a rate of 72.9%. Sage is a powerful antioxidant compound, which also exhibits an important metal chelating activity. These powerful properties of sage are related to the active ingredients it contains. The greatest role in the protective role of sage is the antioxidant activity exhibited due to phenolic compounds such as carnosic acid, carnosol, rosmarinic acid and camphor in the content. There are some studies in the literature focused on the antioxidant role of sage. For example, Lima et al.45 investigated the antioxidant potential of traditional water infusion (tea) of sage in vivo in mice and rats. In conclusion, it was determined that replacing the water in the diet of rodents with sage for 14days did not affect the body weight and food consumption of the animals. They also reported that sage did not cause liver toxicity, liver GST activity was increased in rats (24% rate) and mice (10% rate) drinking sage, on the other hand, sage caused an improvement in the antioxidant status of hepatocytes, increased GSH levels and provided a protection against lipid peroxidation. Horvthov et al.46 investigated the protective effect of sage extract against oxidative stress to which liver cells of SpragueDawley rats are exposed. As a result, no negative effects were observed on basal DNA damage levels and SOD activities in hepatocyte cells of animals that drank sage for 14days, and no changes were detected in the biochemical parameters of blood plasma. On the contrary, they determined that sage extract significantly increased GPx activity, decreased DNA damage levels caused by oxidants, and provided antioxidant protection by increasing GSH levels. Alshubaily and Jambi47 investigated the possible protective role and antioxidant activity of sage extract against metabolic disorders caused by hypercholesterolemic diet in heart and testicular tissues of rats. In conclusion, they determined that the hypercholesterolemic diet significantly increased serum lipid content, cardiac marker enzyme activities, MDA levels, and significantly decreased high-density lipoprotein-cholesterol levels in testes and heart tissues. They observed that the co-administration of hypercholesterolemic diet and sage extract reduced the damage caused by the hypercholesterolemic diet by causing a decrease in lipid peroxidation, induction of heart and testis functions, and increased activity. They reported that essential oil, phenolic contents and other antioxidant components contained in sage extract were effective in this.

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Metal chelating and anti-radical activity of Salvia officinalis in the ameliorative effects against uranium toxicity | Scientific Reports - Nature.com