Phenolic profile and protective role of Calendula officinalis against molybdenum-induced toxiciy in Allium cepa

Environmental pollution increases the level of hazardous chemical agents that adversely affect all organisms. Chemicals that cause various toxic effects exhibit cytotoxic effects through oxidative stress associated with the production of free radicals and reactive oxygen species (ROS) and cause cell death¹. In the reduction of these toxic effects, especially cytotoxicity, herbal extracts attract attention with their natural structure and strong antioxidant properties. Despite the plethora of studies conducted on these natural extracts, the vast array of plant biodiversity present in both Turkiye and the world renders existing research insufficient. In this study, the protective effects of Calendula officinalis L., which was used for wound healing, digestive problems and various skin lesions in the Middle Ages in the XIIth century, against the toxicity of molybdenum were investigated. C. officinalis belongs to the Asteraceae family and is distinguished by its herbaceous stem and whole or slightly dissected leaves². The plant is capable of attaining a height of between 25 and 80 centimetres. The leaves measure between 5 and 15 centimetres in length and between 1 and 3 centimetres in width, with the leaves clasping the stem. The corollary is yellow or orange, with a length approximately twice that of the involucre and a width of 4–5 mm at the base. The tubular flowers are triangular-lanceolate and lobed. Notably, under optimal conditions, these flowers exhibit a persistent blooming capacity throughout the year. The flowers close when there is a chance of rain and are thus used in weather forecasting^3,4. In ethnobotanical studies conducted in Turkiye, C. officinalis has been recorded as a treatment for psoriasis among the local population. It has been reported that C. officinalis flowers are used externally for eczema and psoriasis in the form of a cream, and that the above-ground parts are used externally for eczema and psoriasis in the form of an ointment⁵. C. officinalis has been reported to be used in the treatment of jaundice, as an antispasmodic and as an effective agent for blood purification. The large colourful flowers of the plant also have important biological and pharmacological properties. In India, ointments obtained from the flowers are reported to be used in the treatment of herpes, wounds, scars and blood purification^6,7.

Plants realise their protective effects especially through antioxidant activity. There are studies investigating the antioxidant activity of C. officinalis in the literature. Coyago-Cruz et al.⁸ reported that C. officinalis flowers collected from Ecuador have significant radical scavenging activity and exhibit strong antioxidant activity, calculating ABTS radical scavenging activity to be 88%. Preethi et al.⁹ found that C. officinalis flower extract scavenged superoxide and hydroxyl radicals, exhibiting IC₅₀ values of 500 µg/mL and 480 µg/mL, respectively, thereby supporting its antioxidant effect. The pharmacological and biological effects of C. officinalis have been associated with the active components it contains. A diversity of phytochemicals has been observed in C. officinalis collected from different regions. In addition, the level of the same phytochemical varies according to the region from which it is collected. Terpenoids, flavonoids and phenolics, sterols, saponins, amino acids and essential oils are the main categories of phytochemicals contained in C. officinalis¹⁰. Flavonoids represent the most prevalent components in C. officinalis extracts, and the presence of flavonoids with diverse structures, including neohesperidoside, rutinoside, quercetin-3-O-glucoside, sorhamnetin-3-O-2G-rhamnosyl rutinoside, and isorhamnetin-3-O-2G, has been documented in the literature^11,12. A study of essential oils derived from C. officinalis revealed the presence of compounds with significant antimicrobial and antioxidant properties. The essential oils detected in plant extracts collected from different regions include thujene, limonene, terpene-4-ol, 2-pentylfuran, γ-terpinene, terpinolene, α-cadinol, safranal, carvone, p-thymol, δ-cadinene, calamemene and cadalene^13,14,15.

Molybdenum is a trace element for many organisms and is required in small amounts in biochemical reactions, particularly in cofactor function. Some of the metabolic pathways in which molybdenum is involved are nitrogen metabolism, purine catabolism, hormone biosynthesis and sulphur metabolism¹⁶. Extensive studies on abnormalities that may occur in plants in molybdenum deficiency are available in the literature^17,18 but studies on abnormalities that may occur in plants in the presence of molybdenum above the required amount are insufficient. Özkan et al.¹⁹ reported that molybdenum exposure at doses above trace levels induced oxidative stress and exhibited genotoxic effects. In this study, the phytochemical content of flower parts of C. officinalis collected from Giresun (Türkiye) was investigated by LC-MS/MS and its protective effect against molybdenum-induced toxicity was investigated. The protective effect of C. officinalis against morpho-physiological, cytogenetic, biochemical and anatomical abnormalities induced by molybdenum was determined and the radical scavenging activity of the extract was tested to determine the dose of C. officinalis to be tested. Studies investigating the effects of heavy metals on agriculturally important plants, such as in this study, are very valuable, but investigating the solution while identifying a problem increases the original value and impact of the study. In this context, this study investigates the toxic effect of molybdenum and the solution that can be applied to eliminate this effect.

Calendula officinalis collection, characterization and exposure

Calendula officinalis was collected from Giresun (Türkiye) and the identification of the plant was made in Giresun University Department of Botany by Dr. Zafer TÜRKMEN. A sample was stored in the herbarium of the department with the voucher number BIO-C-off253/2024. Experimental research and field studies on plants, including the collection of plant material, comply with relevant institutional, national, and international guidelines and legislation. The colored flower parts of the plant were separated and dried at 35 °C for 2 days. 2 g of dry sample was incubated in 100 mL ethanol in a shaking oven. At the end of the incubation period, the liquid phase was cleaned of solid particles and evaporated with an evaporator^20,21. The residue obtained at the end of evaporation was collected and used to determine the protective effect of the plant. Plants provide protection against many diseases and toxicities and this protective effect is closely related to the antioxidant effect and the antioxidant effect is closely related to the phenolic compounds contained in the plant. For this purpose, the superoxide removal activity of C. officinalis was first determined. In this way, both the antioxidant effect and the dose to be used in the study were determined. The method used in superoxide scavenging activity is given in Supp. Mat. The IC₅₀ value expresses the amount of extract required for the reduction of 50% of the superoxide radical²². The IC₅₀ value of C. officinalis was determined as 100 µg/mL (Sup. Fig. 1). IC₅₀ and IC₅₀ × 2 values as 100 µg/mL and 200 µg/mL were used to determine the protective effect in the study. The dose of molybdenum used as toxic agent was chosen as 4000 mg/L, which was reported in the literature that the toxic effect was clearly observed¹⁹. The quantitative determination of phenolic compounds in C. officinalis was conducted through LC-MS/MS analysis. For this purpose, approximately 1 g of dried C. officinalis flower parts were powdered and extracted with dichloromethane-methanol solvent (1:4) and analysed by LC-MS/MS (Thermo Scientific, ODS Hypersil 4.6 × 250 mm column). The analytical conditions, mobile phase and gradient profile are given in Sup.Mat. The LC-MS/MS analysis was performed at HUBTUAM (Hitit University)^23,24.

Experimental groups and morpho-physiological parameters

Allium cepa bulbs were purchased from Akdeniz Tarım (TR -55-K-009228) and used as a bioindicator in toxicity assays. Sodium Molybdate Dihydrate was obtained from Merck. Six groups were formed for molybdenum (Mo) and C. officinalis applications. Group I: Distilled water (control); Group II: 100 µg/mL C. officinalis extract (Cos1), Group III: 200 µg/mL C. officinalis extract (Cos2). In Group II and Group III, only the extract was applied based on IC₅₀ values. The data from these two groups were used to assess whether the extract alone had any toxic effect. Group IV was treated with 4000 mg/L Sodium Molybdate Dihydrate (Mo). In this group only the level of abnormalities induced by Mo was determined. Group V and Group VI were treated with 4000 mg/L Sodium Molybdate Dihydrate + 100 µg/mL C. officinalis extract (MoCos1), Group VI: 4000 mg/L Sodium Molybdate Dihydrate + 200 µg/mL C. officinalis extract (MoCos2). The determination of the protective effect was calculated using the data of groups V and VI, based on the reductions in toxicity induced by molybdenum alone. Each bulb was germinated separately in glass beakers, Following a seven-day germination period at room temperature (20 °C) and 60% humidity, root tissues and leaves were collected for further analysis^25,26.

Morpho-physiological parameters, especially those related to germination and growth, were used to determine morpho-phiological parameters. For germination percentage and weight gain, 10 bulbs were used in each group, and for determination of root length, 5 random roots were taken from each bulb, totaling 50 measurements for each group. Root length was determined by measuring the radicle length (cm) with the use of a caliper. Weight gain was calculated by weighing the bulbs at the commencement and conclusion of the germination period using a precision balance. Germination percentage was established by calculating the ratio of the number of germinated bulbs to the total number of bulbs^27,28.

Biochemical parameters

The impact of Mo and C. officinalis extract treatments on antioxidant-oxidant dynamics was assessed through the analysis of various parameters, malondialdehyde (MDA), chlorophyll a and b, superoxide dismutase (SOD) and catalase (CAT). The activity of SOD was determined in accordance with the methodology proposed by Macar et al.²⁹with the results expressed as U/mg. The activity of CAT was determined by monitoring the decrease in absorbance at 240 nm, and defined as OD_240nm min/g³⁰. MDA levels were quantified in accordance with the methodology outlined by Demirtaş et al.³¹ and expressed as µM/g. The extraction and measurement of chlorophyll were done using the protocol recommended by Kaydan et al.³². Analyses were performed in triplicate and the detailed analytical procedures for all parameters are provided in the supplementary material.

Cytogenetic parameters

Cytogenetic effects of Mo and C. officinalis extract were determined by mitotic index (MI), micronucleus (MN), chromosomal abnormality (CAs) test³³. Analyses were performed in triplicate and the detailed analytical procedures for all parameters are provided in the supplementary material.

Anatomical changes

Five root tips were randomly selected from each group and microscopic slides were prepared. Transverse sections of the roots were taken and stained with 5% methylene blue. A total of 100 images were obtained for each group by analysing 20 images from each sample. Anatomical damage was documented using a research microscope with a camera attachment³⁴.

Statistical analysis

The results were statistically evaluated using the SPSS Statistics 22 (IBM SPSS, Türkiye) package program. The data were presented as mean ± SD (standard deviation) in the one-way ANOVA and Duncan tests. A p-value of less than 0.05 was deemed statistically significant.

Morpho-pysiolocial alterations

The morpho-physiological alterations induced by Mo and C. officinalis in A. cepa root meristem cells were analyzed using germination percentage, root length and weight gain (Table 1). It was determined that the Cos1 and Cos2 groups treated with flower extract exhibited 100% germination, comparable to the control group. Additionally, the mean root length and weight gain of the Cos1 and Cos2 groups did not demonstrate statistical differences when compared with the control group (p > 0.05). Therefore, the selected extract doses did not function as a “growth suppressor” for A. cepa. In contrast, the Mo group demonstrated a decline in germination percentage to 73%, accompanied by a significant reduction in root length and weight gain values in comparison to the control values. The root length and weight gain in the Mo group were 39.47% and 56.32% lower than those observed in the control group, respectively. In accordance with the results obtained, Özkan et al.¹⁹ also found that Mo caused growth inhibition in A. cepa and this effect was strengthened at higher doses of Mo. Yurdakul et al.³⁵ reported that elevated levels of Mo in Triticum aestivum resulted in spindly and unhealthy plant growth, as well as a decline in yield. Additionally, Gopal et al.³⁶ demonstrated that over-exposure to Mo reduced the total dry matter content of Vigna mungo. Furthermore, McGrath et al.³⁷ revealed that Mo induced retardation in shoot growth of Brassica napus L., Trifolium pretense L., Lolium perenne L., and Solanum esculentum plants. In another study, high dosage of Mo (1 mM) tested on Pisum sativum L. plants decreased the growth of the shoot by 35% and the root by 50%³⁸. In addition to its role as an essential component of nitrogenase and various other vital enzymes involved in redox reactions in plants, such as nitrate reductase, xanthine oxidase, sulfite oxidase, and aldehyde oxidase, Mo is also vital for the growth of the majority of other species. However, plants require extremely little levels of Mo and there is a thin line between toxicity and shortage^37,39. Although it has been reported that the toxicity of Mo in plants can be understood by leaf pigmentation abnormality¹⁷the results of this study demonstrate that Mo in the root environment is also effective without being transported to the upper parts. Indeed, Xu et al.⁴⁰ reported that roots exhibited a greater accumulation of Mo than leaves in soybean seedlings. The underlying causes of Mo-induced growth retardation in A. cepa may be the result of either the restriction of water or macro- and microelement uptake necessary for growth, or the disruption of the anatomical structure of the root¹⁹. In addition, Macar et al.²⁹ reported that the toxicity of trace elements was responsible for the inhibition of cell division in the root tips of plants. Therefore, the decrease in mitotic activity in these regions may also be a contributing factor to Mo-induced delay of root formation in A. cepa. Mo is a heavy metal as well as a trace element. Another factor in the inhibition of growth may be oxidative stress, which develops due to heavy metal application and has the potential to damage cellular structures related to substance uptake and mitotic division^33,41,42.

In the MoCos1 and MoCos2 groups, where C. officinalis extract was applied in mixture with Mo, germination percentages increased compared to the Mo group, and were determined as 79% and 86%, respectively (Table 1). These groups also exhibited greater root length and weight increase values compared to the Mo-only treated group. Although the physiological parameter values in the MoCos1 and MoCos2 groups were unable to approach the levels of the control group, C. officinalis extract considerably reduced Mo-induced physiological deterioration when used in combination with Mo. This study provides the first evidence of the ameliorative effect of C. officinalis extract against Mo-induced growth inhibition in plants. On the other hand, it has been reported that this extract accelerates cell division and wound healing in animals and humans⁴³. Hormozi et al.⁴⁴ demonstrated that C. officinalis extract increased the expression of growth factors and stimulated cell proliferation in mouse embryonic fibroblasts. C. officinalis extract has also been documented to alleviate oxidative stress induced by deleterious agents such as aflatoxin and hydrogen peroxide, through its bioactive constituents and radical scavenging substances⁴⁵. The findings of our study align with those of previous studies, which demonstrate that plant extracts with high antioxidant properties can mitigate heavy metal-induced growth retardation in A. cepa^46,47,48.

Biochemical alterations

The biochemical alterations resulting from the application of Mo, C. officinalis extract and Mo + C. officinalis mixture in A. cepa were evaluated by measuring the levels of MDA and chlorophyll pigment, along with SOD and CAT enzyme activities (Table 2). Statistically significant biochemical alterations were not identified in either the Cos1 or Cos2 groups treated with C. officinalis extract in comparison to the control group (p > 0.05). These results suggest that the selected doses of C. officinalis did not induce any biochemical toxicity in A. cepa roots. However, the Mo group exhibited an increase in MDA, SOD activity, and CAT activity, reaching 2.51, 1.68, and 2.64 times the levels observed in the control group, respectively. Conversely, Mo administration caused a remarkable decrease in chlorophyll levels in the fresh leaves of A. cepa. The levels of chlorophyll a and b in the Mo group decreased by 62.25% and 63.04% respectively, compared to the levels in the control group. The present findings are in alignment with those of Özkan et al.¹⁹who demonstrated that Mo increased both MDA accumulation and SOD and CAT activities in A. cepa root meristem cells. It has also been documented that the activity of antioxidant enzymes, including SOD and CAT, was considerably elevated in soybean seedlings under extreme Mo stress compared to normal Mo conditions⁴⁰. Furthermore, Boojar and Tavakkoli⁴⁹ revealed that the SOD, CAT, APX, and GR enzymes of plants grown in the Mo-contaminated zone in the mining region were higher than those grown in uncontaminated soil. Our results are also in agreement with the studies in the literature showing that Mo has an indisputable influence on chlorophyll levels of plants. Gupta et al.¹⁷ reported that when Mo toxicity does occur, it is generally marked by chlorosis, yellowing, and other forms of leaf discoloration. Indeed, a recent study demonstrated that elevated doses of Mo resulted in reduced levels of chlorophyll a and b in T. aestivum⁵⁰. Nautiyal and Chatterjee⁵¹ proposed that the chlorosis resulting from elevated levels of Mo in Cicer arietinum crops was attributable to the inhibition of iron translocation. Both low and high doses of Mo have also been shown to reduce iron uptake and accumulation in tomato plants⁵². This may finally limit the manufacture of chlorophyll by impeding the transformation of Mg-Proto-Me to protochlorophyll⁴⁵. In addition to the decrease in chlorophyll biosynthesis, the destruction due to changes in membrane permeability and reactive oxygen species (ROS) production may also represent significant mechanisms underlying the observed reduction of chlorophyll pigment levels⁵³.

As posited by Singh et al.⁵⁴the toxicity that results from elevated trace element doses, such as Mo, can be attributed to a number of factors. These include a decline in chlorophyll levels, interference of antioxidant enzymes, impairment to nutrient transport and metabolic activities, and initiation of lipid peroxidation. ROS are continuously produced by plants during standard metabolic processes, particularly as byproducts of respiration, photosynthesis, and photorespiration. Nonetheless, when optimal conditions are disrupted, an uncontrolled increase in ROS production occurs in plants, leading to oxidative stress. Once cells undergo an oxidative burst, the structural integrity and function of critical cellular components such as membranes, proteins, enzymes, photosynthetic apparatus and DNA become jeopardized and are susceptible to damage. MDA accumulation and changes in the activities of SOD and CAT enzymes are among the biological markers of oxidative stress^55,56,57. Thus, in our study, A. cepa root tip cells exposed to Mo underwent oxidative stress. MDA is a metabolite that is formed as a consequence of the peroxidation of polyunsaturated fatty acids within cell membranes. Both ROS and the lipoxygenase enzyme have the capacity to initiate membrane peroxidation⁵⁸. It can influence ion transitions across cell membranes and inhibits genes involved in plant defense against stressors by altering DNA structure^33,34. In circumstances where plants are subjected to substantial levels of heavy metals, the antioxidant enzyme system assumes a pivotal function in mitigating oxidative damage. Superoxide radicals within cells are converted to hydrogen peroxide radicals by SOD enzyme, while CAT enzyme acts on hydrogen peroxide, facilitating its conversion to water and oxygen^58,59. Even though enzymatic defense activities increased in A. cepa root meristem cells exposed to Mo, the increase in MDA and the decrease in chlorophyll in this group indicated that oxidative stress was not suppressible in the Mo group.

The MoCos1 and MoCos2 groups, in which C. officinalis extract was applied in mixture with Mo, exhibited a significant increase in chlorophyll pigment levels and a significant decrease in MDA levels and SOD and CAT activities, in comparison to the group treated with Mo alone (p < 0.05) (Table 2). The severity of these alterations became more pronounced as the dose of C. officinalis extract in the mixture increased. MDA level, SOD enzyme activity and CAT enzyme activity values in the MoCos2 group decreased by 37.76%, 23.12% and 46.97%, respectively, compared to the Mo group. On the other hand, the values of chlorophyll a and chlorophyll b in this group were 1.65 and 1.87 times those of the Mo group, respectively. To the best of our knowledge, this is the first study demonstrating the mitigating effect of C. officinalis against heavy metal stress in plants. However, it has been shown that C. officinalis extract can partially suppress iron-induced oxidative stress in brain and liver homogenates of rats. In addition, there are many studies in the literature proving the protective potential of natural plant extracts against heavy metal-related stress in various organisms^60,61,62. Preethi et al.⁹ demonstrated that C. officinalis extract has the capacity to scavenge superoxide radical as well as hydroxyl radical, which is produced from hydrogen peroxide radicals via Fenton reactions. Phenolic compounds in plants have the potential to show antioxidant effects by chelating metals thanks to their hydroxyl and carboxyl groups^63,64.

Genotoxicity parametres

The protective role of C. officinalis extract against Mo-induced genotoxicity is given in Fig. 1; Table 3. Types of chromosomal abnormalities induced by Mo were given in Fig. 2. The protective effects of C. officinalis extract was determined by calculating the regression of chromosomal abnormalities induced by Mo. Very low and statistically insignificant abnormalities were detected in the control group, Cos1 and Cos2 groups (p > 0.05). The statistically insignificant difference between these groups indicates that C. officinalis extract alone is not genotoxic. High levels of chromosomal abnormalities and micronucleus formation were detected in the Mo-only group. The highest chromosomal abnormality type in this group was fragment and a level of 24.8 ± 2.16 was detected in 1000 counted cells. Other chromosomal abnormalities induced by Mo were vagrant chromosome, sticky chromosome, multipolar anaphase, disorientation. Fragments that cannot be incorporated into the nucleus at the end of mitosis form small nucleus-like structures, micronuclei, by accumulation of nuclear envelope around them^63,64. The high frequency of fragments and MNs in this study strengthens this claim. In total, 28.5 ± 2.23 micronuclei were detected in 1000 cells counted in Mo-treated root tips. MN is recognized as a biomarker of genotoxic stress and genetic instability. MN can originate not only from fragments but also from vagrant chromosomes and damage to the spindle structure. Therefore, multiple mechanisms through multiple pathways are involved in MN formation^63,65.

Genotoxic effects observed in the cell also affect cell proliferation. In this study, it was determined that mitotic index decreased by 25.7% in the Mo-treated group compared to the control group. This decrease in cell proliferation may be associated with possible pauses in the cell cycle as a result of chromosomal abnormalities in the cell, spindle thread damage, oxidative stress or direct interactions of molybdenum with DNA¹⁹. C. officinalis extract co-administration resulted in a regression in genotoxic effects. The fragment with the highest chromosomal abnormality was reduced by 25.8% in the MoCos1 group and 45.2% in the MoCos2 group compared to the Mo-only group. Similar regressions were observed for all chromosomal abnormalities. C. officinalis extract treatment at two different doses together with Mo resulted in a 27.7% − 47% reduction in MN frequency. Along with these reductions, increases in cell proliferation were also detected. Mitotic index, which decreased with Mo application, increased with Mo + C. officinalis extract applications. MI increased by 6.7% in the MoCos1 group and by 12.2% in the MoCos2 group. This result indicates that C. officinalis extract provides protection against Mo-induced genotoxic effects and the protection is closely related to the active ingredients in the extract, especially phenolic substances. Similarly, Leffa et al.⁶⁶ show that Calendula officinalis extract repairs chemical-induced DNA damage, reduces the formation of micronuclei and contains protective substances that reduce damage to genetic material.

Anatomical alterations

The roots are the primary route of entry for toxic substances that contaminate water or soil. As a result, any damage caused by heavy metals to a plant can be most easily and quickly recognized in the roots. The damage caused by Mo on root meristem, and the mitigative effect of C. officinalis extract against these damages, are presented in Table 4; Fig. 3. As a result of the analyses, similar to the control group, no meristematic cell damage was determined in Cos1 and Cos2 groups in which 100 mg/L and 200 mg/L C. officinalis extract doses applied (Fig. 3a,c,f,h). In accordance with the other findings of this investigation, these results show that the applied dosages of C. officinalis did not result in any toxicity in A. cepa. In contrast to the Cos1 and Cos2 groups, the Mo group treated with 4000 mg/L molybdenum showed major levels of epidermis cell damage (Fig. 3b), cortex cell damage (Fig. 3d), thickened cortex cell wall (Fig. 3e), flattened cell nucleus (Fig. 3g) and thickened conduction tissue (Fig. 3i). Conversely, the severity of Mo-induced meristematic cell damage was reduced when C. officinalis extract was applied in conjunction with Mo. The mitigating effect of C. officinalis extract was found to be dose-dependent, being more pronounced in the MoCos 2 group. Although the deleterious effects of heavy metals on root meristem structure of A. cepa have been reported previously^67,68studies on Mo-induced damage to root meristem cells are extremely limited. In agreement with the results of the present study, Özkan et al.¹⁹ reported that an excessive dose of Mo increased the incidence of epidermis cell damage, cortex cell wall thickening, cell cortex cell damage, nucleus flattening, and vascular tissue thickening in A. cepa. In the same study, it was proposed that Mo-induced damage may be caused by membrane degradation due to oxidative stress, increased intracellular pressure, and changes in DNA and nuclear proteins¹⁹. However, a counter-argument has been posited that the adaptation of plants to prevent hazardous substances from entering the conduction bundles, where they can travel to the upper parts of the plant, may lead to the formation of defense barriers such as thickened cortical cell walls and conduction tissue⁶⁹. This is the first study to demonstrate the protective effect of C. officinalis extract against the detrimental effects of Mo on the meristem of A. cepa root. However, it is documented that herbal extracts characterized by notable antioxidant and chelating capacity are able to effectively counteract the deleterious effects of heavy metal toxicity on root meristem tissue^48,70. In cases of heavy metal-induced toxicity and poisoning, oxidative stress and the resulting genetic damage as well as metabolic disorders play an important role⁷¹. The abovementioned findings of the current study demonstrate that C. officinalis extract exerts a substantial mitigating effect on the oxidative stress triggered by Mo. In conclusion, it can be suggested that C. officinalis extract showed a protective effect against toxicity-induced damage in root meristem structure by reducing Mo-induced oxidative stress.

Protective effects of C. officinalis flower extract

Mo caused toxicity in different parameters of Allium cepa root tip cells. A regression of these toxic effects was observed in the groups that were treated with both Mo and C. officinalis extract. The protective property was calculated based on the regression of abnormalities observed in the molybdenum-alone-treated group (Fig. 4). The protection exhibited by C. officinalis extract increased in a dose-dependent manner, but this increase was not directly proportional. The decrease in root elongation, a critical parameter in physio-morphological development, was regressed as 6.75% in the MoCos1 group and 20.6% in the MoCos2 group. In the genotoxic parameters of fragment, bridge, and MN frequencies, 100 µg/mL C. officinalis extract application provided protection ranging from 25.8 to 34.9%, while 200 µg/mL C. officinalis extract showed protection ranging from 37.7 to 62.4%. Among all parameters, the highest protection was obtained against bridge. A decline in MDA levels, a hallmark of oxidative stress, was also observed, with enhancement ranging from 18.1 to 37.7% in the C. officinalis extract-treated groups.

The improvements observed in SOD and CAT levels as well as MDA levels indicate that the oxidative stress induced by Mo regressed. Antioxidant activity lies at the basis of the protective properties of C. officinalis extract in all parameters. Phenolic compounds have an important role in the emergence of this activity. The protective role of C. officinalis extract against the toxic effects induced by Mo is closely related to the active ingredients in its content, especially phenolic compounds with antioxidant role. To this end, the phenolic components of C. officinalis were subjected to an LC/MS-MS investigation, and the results are presented in Table 5 and chromatograms in Fig. 5. Chlorogenic acid, which was detected at 645,937 µg/g, was determined as the major component and its percentage among all phenolics was calculated as 61.8%. This was followed by salicylic acid (99,724 µg/g), 4_OH benzoic acid (95,679 µg/g), caffeic acid (87,819 µg/g) and rutin (50,151 µg/g). Minor amounts of p-coumaric acid, ferulic acid, sinapic acid, rosmarinic acid, quercetin, protecateuic aldehyde, sesamol, vannilin and apigenin were detected. All these phenolic substances contribute to the biological activity of C. officinalis extract. The major component chlorogenic acid provides protection by exhibiting antioxidant activity through many different mechanisms. Some of these mechanisms include direct scavenging of free radicals by polyhydroxyl groups in its structure, activation of antioxidant signaling pathway, up-regulation of related genes, and increasing endogenous antioxidant levels. Chlorogenic acid reduces free radicals by donating hydrogen atoms in its structure and thus prevents oxidation reactions^72,73. Salicylic acid, the second major compound detected in the extract, has an important role in protection against toxicity. Salicylic acid has a strong antioxidant role and makes important contributions to the development of cell tolerance to oxidative stress. It also increases the production of antioxidant substances in cells⁷⁴. Caffeic acid has an important role in the defense mechanism of plants against insects, fungi, bacteria, ultraviolet radiation and infections^75,76. Rutin is a potent antioxidant and exhibits various pharmacological activities including anti-inflammatory, antibacterial, antiviral and antiprotozoal properties⁷⁷. Quercetin and quercetin glycosides, the most abundant of the flavonoid molecules, are widely available in plants and are excellent free radical scavenging antioxidants^78,79. P-coumaric acid, which was detected in the LC-MS/MS analysis, is a strong antioxidant and exhibits scavenging activity against reactive oxygen species and other free radicals. Vanillin, exhibiting notable antioxidant and antimicrobial properties. Additionally, the antimutagenic potential of vanillin has been widely investigated in previous research²². As a result of a cumulative effect of all these phenolic substances, C. officinalis extract exhibited a dose-dependently increasing protection by reducing Mo toxicity.

1. Jomova, K. et al. Reactive oxygen species, toxicity, oxidative stress, and antioxidants: chronic diseases and aging. Arch. Toxicol. 97 (10), 2499–2574 (2023).

   CAS  PubMed  PubMed Central  Google Scholar 

2. Güven, U. M., Arslan, S., Çıracı, M. B. & Kayıran, S. D. Morphological characteristics of Calendula officinalis L. plant, development and in vitro evaluation of extract loaded topical drug formulation. Mersin Univ. School Med. Lokman Hekim J. History Med. Folk Med. 12, 105–115 (2022).

   Google Scholar 

3. Kemper, K. J. Calendula (Calendula officinalis). Longwood Herbal Task Force, The Center for Holistic Pediatric Education and Research. 1–13 (1999).

4. Jan, N. & John, R. Calendula officinalis-an important medicinal plant with potential biological properties. Proc. Indian Natl. Sci. Acad. 83, 769–787 (2017).

   Google Scholar 

5. Erarslan, Z. B., Ecevit-genç, G. & Kültür, Ş. Medicinal plants traditionally used to treat skin diseases in Turkey–eczema, psoriasis, vitiligo. J. Fac. Pharm. Ankara. 44, 137–166 (2020).

   Google Scholar 

6. Muley, B., Khadabadi, S. & Banarase, N. Phytochemical constituents and pharmacological activities of Calendula officinalis Linn (Asteraceae): a review. Trop. J. Pharm. Res. 8, 455–465 (2009).

   CAS  Google Scholar 

7. Leach, M. J. Calendula officinalis and wound healing: a systematic review. Wounds 20, 236–243 (2008).

   PubMed  Google Scholar 

8. Coyago-Cruz, E. et al. Functional, antioxidant, antibacterial, and antifungal activity of edible flowers. Antioxidants 13, 1297 (2024).

   CAS  PubMed  PubMed Central  Google Scholar 

9. Preethi, K. C., Kuttan, G. & Kuttan, R. Antioxidant potential of an extract of Calendula officinalis flowers in vitro and in vivo. Pharm. Biol. 44, 691–697 (2006).

   Google Scholar 

10. Sapkota, B. & Kunwar, P. A. Review on traditional uses, phytochemistry and Pharmacological activities of Calendula officinalis Linn. Nat. Prod. Commun. 19 https://doi.org/10.1177/1934578X24125 (2024).

11. Santosh Ferreira, C. D. et al. Phenolic compounds in extracts from Eucalyptus globulus leaves and Calendula officinalis flowers. J. Nat. Prod. Resour. 2, 53–57 (2016).

    Google Scholar 

12. Raal, A., Orav, A., Nesterovitsch, J. & Maidla, K. Analysis of carotenoids, flavonoids and essential oil of Calendula officinalis cultivars growing in Estonia. Nat. Prod. Commun. 11, 1157–1160 (2016).

    PubMed  Google Scholar 

13. Lohani, A., Mishra, A. K. & Verma, A. Cosmeceutical potential of geranium and calendula essential oil: determination of antioxidant activity and in vitro sun protection factor. J. Cosmet. Dermatol. 18, 550–557 (2019).

    PubMed  Google Scholar 

14. Gudzenko, A. V. et al. Study of volatile compounds of Сalendula officinalis L. flowers by the method of gas chromatography with MAS detection. Farm. Zh. 1, 75–81 (2023).

    Google Scholar 

15. Raal, A. & Kirsipuu, K. Total flavonoid content in varieties of Calendula officinalis L. originating from different countries and cultivated in Estonia. Nat. Prod. Res. 25, 658–662 (2011).

    CAS  PubMed  Google Scholar 

16. Sardesai, V. M. Molybdenum: an essential trace element. NCP. 8 (6), 277–281 (1993).

    CAS  PubMed  Google Scholar 

17. Gupta, U. C. Molybdenum in agriculture: symptoms of molybdenum deficiency and toxicity in crops. Agric. Food Sci. 2, 160–170 (1997).

    Google Scholar 

18. Kaiser, B. N., Gridley, K. L., Brady, N., Phillips, J., Tyerman, S. The role of molybdenum in agricultural plant production. Ann. Bot. 96, 745–754 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

19. Özkan, B., Çavuşoğlu, K., Yalçin, E. & Acar, A. Investigation of multidirectional toxicity induced by high-dose molybdenum exposure with allium test. Sci. Rep. 14 (1), 8651 (2024).

    ADS  PubMed  PubMed Central  Google Scholar 

20. Üst, Ö., Yalçin, E., Çavuşoğlu, K. & Özkan, B. LC–MS/MS, GC–MS and molecular docking analysis for phytochemical fingerprint and bioactivity of beta vulgaris L. Sci. Rep. 14 (1), 7491 (2024).

    ADS  PubMed  PubMed Central  Google Scholar 

21. Topatan, Z. Ş. et al. Alleviatory efficacy of Achillea millefolium L. in etoxazole-mediated toxicity in Allium cepa L. Sci. Rep. 14, 31674 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

22. Kesti Usta, S., Yalçın, E. & Çavuşoğlu, K. Synergistic and antagonistic contributions of main components to the bioactivity profile of anethum graveolens extract. Sci. Rep. 15 (1), 21465 (2025).

    PubMed  PubMed Central  Google Scholar 

23. Akman, T. C. et al. LC-ESI-MS/MS chemical characterization, antioxidant and antidiabetic properties of propolis extracted with organic solvents from Eastern Anatolia Region. Chem. Biodivers. 20(5), e202201189 (2023).

24. Kayir, Ö., Doğan, H., Alver, E. & Bilici, İ. Quantification of phenolic component by LC-HESI-MS/MS and evaluation of antioxidant activities of Crocus ancyrensis extracts obtained with different solvents. Chem. Biodivers. 20, 202201186 (2023).

    Google Scholar 

25. Macar, T. K., Macar, O., Yalçın, E. & Çavuşoğlu, K. Resveratrol ameliorates the physiological, biochemical, cytogenetic, and anatomical toxicities induced by copper (II) chloride exposure in Allium Cepa L. Environ. Sci. Pollut. Res. 27 (1), 657–667 (2020).

    CAS  Google Scholar 

26. Çavuşoğlu, D., Macar, O., Kalefetoğlu Macar, T., Çavuşoğlu, K. & Yalçın, E. Mitigative effect of green tea extract against mercury (II) chloride toxicity in Allium Cepa L. model. Environ. Sci. Pollut. Res. 29 (19), 27862–27874 (2022).

    Google Scholar 

27. Cavusoglu, K. et al. Protective role of Ginkgo Biloba on petroleum wastewater-induced toxicity in Vicia Faba L.(Fabaceae) root tip cells. J. Environ. Biol. 31 (3), 319 (2010).

    CAS  PubMed  Google Scholar 

28. Kesti, S., Macar, O., Kalefetoğlu Macar, T. & Çavuşoğlu, K. Yalçın, E. Investigation of the protective role of Ginkgo Biloba L. against phytotoxicity, genotoxicity and oxidative damage ınduced by trifloxystrobin. Sci. Rep. 14, 19937 (2024).

    CAS  PubMed  PubMed Central  Google Scholar 

29. Macar, O., Kalefetoğlu Macar, T., Çavuşoğlu, K. & Yalçın, E. Protective effects of anthocyanin-rich Bilberry (Vaccinium myrtillus) extract against copper(II) chloride toxicity. Environ. Sci. Pollut. Res. 27, 1428–1435 (2020a).

    CAS  Google Scholar 

30. Kurt, D., Yalçin, E. & Çavuşoğlu, K. GC-MS and HPLC supported phytochemical analysis of watercress and the protective role against Paraben toxicity. Environ. Sci. Pollut. Res. 30, 6033–6046 (2023).

    CAS  Google Scholar 

31. Demirtaş, G., Çavuşoğlu, K. & Yalçın, E. Aneugenic, clastogenic, and multi-toxic effects of diethyl phthalate exposure. Environ. Sci. Pollut. Res. 27, 5503–5510 (2020).

    Google Scholar 

32. Kaydan, D., Yagmur, M. & Okut, N. Effects of Salicylic acid on the growth and some physiological characters in salt stressed wheat (Triticum aestivum L). J. Agric. Sci. 13, 114–119 (2007).

    Google Scholar 

33. Altunkaynak, F., Çavuşoğlu, K. & Yalçin, E. Detection of heavy metal contamination in Batlama stream (Turkiye) and the potential toxicity profile. Sci. Rep. 13, 11727 (2023).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

34. Doğan, M., Çavuşoğlu, K., Yalçın, E. & Acar, A. Comprehensive toxicity screening of Pazarsuyu stream water containing heavy metals and protective role of lycopene. Sci. Rep. 12, 16615 (2022).

    ADS  PubMed  PubMed Central  Google Scholar 

35. Yurdakul, I., Kalinbacak, K., Altinkaynak, D. & Peker, R. M. Determination of the effects of molybdenum and arsenic on yield and toxicity of wheat plant in field conditions. J. Fac. Agric. 18, 25–34 (2023).

    Google Scholar 

36. Gopal, R., Sharma, Y. K. & Shukla, A. K. Effect of molybdenum stress on growth, yield and seed quality in black gram. J. Plant. Nutr. 39, 463–469 (2016).

    CAS  Google Scholar 

37. McGrath, S. P. et al. Predicting molybdenum toxicity to higher plants: Estimation of toxicity threshold values. Environ. Pollut. 158, 3085–3094 (2010a).

    CAS  PubMed  Google Scholar 

38. Kevresan, S., Petrovic, N., Popovic, M. & Kandrac, J. Nitrogen and protein metabolism in young pea plants as affected by different concentrations of nickel, cadmium, lead, and molybdenum. J. Plant. Nutr. 24, 1633–1644 (2001).

    CAS  Google Scholar 

39. Roychoudhury, A. & Chakraborty, S. Cobalt and molybdenum: deficiency, toxicity, and nutritional role in plant growth and development. In Plant Nutrition and Food Security in the Era of Climate Change (eds Kumar, V., Srivastava, A. K. & Suprasanna, P.) 255–270 (Academic, 2022).

40. Xu, S., Hu, C., Tan, Q., Qin, S. & Sun, X. Subcellular distribution of molybdenum, ultrastructural and antioxidative responses in soybean seedlings under excess molybdenum stress. Plant. Physiol. Biochem. 123, 75–80 (2018).

    CAS  PubMed  Google Scholar 

41. Üst, Ö. et al. A multidimensional study on the effects of Abelmoschus esculentus (L.) Moench extract in uranyl acetate-exposed Allium Cepa L. Sci. Rep. 15, 26565 (2025).

    PubMed  PubMed Central  Google Scholar 

42. Sipahi Kuloğlu, S., Çavuşoğlu, K. & Yalçın, E. LC–MS/MS phenolic profile and remedial role of Urtica dioica extract against Li₂CO₃-induced toxicity. Environ. Sci. Pollut. Res. 31, 54589–54602 (2024).

    Google Scholar 

43. Ashwlayan, V. D., Kumar, A., Verma, M., Garg, V. K. & Gupta, S. K. Therapeutic potential of Calendula officinalis. Pharm. Pharmacol. Int J. 6, 149–155 (2018).

    Google Scholar 

44. Hormozi, M., Gholami, M., Babaniazi, A. & Gharravi, A. M. Calendula officinalis stimulate proliferation of mouse embryonic fibroblasts via expression of growth factors TGFβ1 and bFGF. Inflamm. Regen. 39, 7 (2019).

    PubMed  PubMed Central  Google Scholar 

45. Hamzawy, M. A., El-Denshary, E. S., Hassan, N. S., Mannaa, F. A. & Abdel-Wahhab, M. A. Dietary supplementation of Calendula officinalis counteracts the oxidative stress and liver damage resulted from aflatoxin. Int. Sch. Res. Notices. 2013, 538427 (2013).

46. Yalçın, E., Macar, O., Kalefetoğlu Macar, T., Çavuşoğlu, D. & Çavuşoğlu, K. Multi-protective role of Echinacea purpurea L. water extract in Allium Cepa L. against mercury (II) chloride. Environ. Sci. Pollut. Res. 28, 62868–62876 (2021).

    Google Scholar 

47. Aydin, D., Yalçin, E. & Çavuşoğlu, K. Metal chelating and anti-radical activity of Salvia officinalis in the ameliorative effects against uranium toxicity. Sci. Rep. 12, 15845 (2022).

    CAS  PubMed  PubMed Central  Google Scholar 

48. Kalefetoğlu Macar, T. & Macar, O. A study on the effect of Hypericum perforatum L. extract on vanadium toxicity in Allium Cepa L. Sci. Rep. 14, 28486 (2024).

    Google Scholar 

49. Boojar, M. M. A. & Tavakkoli, Z. New molybdenum-hyperaccumulator among plant species growing on molybdenum mine-a biochemical study on tolerance mechanism against metal toxicity. J. Plant. Nutr. 34, 1532–1557 (2011).

    CAS  Google Scholar 

50. Kumar, A. & Aery, N. C. Biochemical changes, biomass production, and productivity of Triticum aestivum as a function of increasing molybdenum application. J. Plant. Nutr. 46, 2351–2362 (2023).

    CAS  Google Scholar 

51. Nautiyal, N. & Chatterjee, C. Molybdenum stress-induced changes in growth and yield of Chickpea. J. Plant. Nutr. 27, 173–181 (2004).

    CAS  Google Scholar 

52. Berry, J. A. & Reisenauer, H. M. The influence of molybdenum on iron nutrition of tomato. Plant. Soil. 27, 303–313 (1967).

    CAS  Google Scholar 

53. Yılmaz, H., Kalefetoğlu Macar, T., Macar, O., Çavuşoğlu, K. & Yalçın, E. DNA fragmentation, chromosomal aberrations, and multi-toxic effects induced by nickel and the modulation of Ni-induced damage by pomegranate seed extract in Allium cepa L. Environ. Sci. Pollut. Res. 30 (51), 110826–110840 (2023).

    Google Scholar 

54. Singh, A. L., Jat, R. S., Chaudhari, V., Bariya, H. & Sharma, S. J. Toxicities and tolerance of mineral elements boron, cobalt, molybdenum and nickel in crop plants. Plant. Stress. 4, 31–56 (2010).

    Google Scholar 

55. Elkelish, A. & Abu-Elsaoud, A. M. Oxidative stress management in plants: a balancing act between antioxidants and redox signaling. Spectr. Sci. J. 1, 35–46 (2024).

    Google Scholar 

56. Seven, B., Kültiğin, Çavuşoğlu, Yalçin, E. & Acar, A. Investigation of Cypermethrin toxicity in Swiss albino mice with physiological, genetic and biochemical approaches. Sci. Rep. 12 (1), 11439 (2022).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

57. Gündüz, A., Yalçın, E. & Çavuşoğlu, K. Combined toxic effects of aflatoxin B2 and the protective role of resveratrol in Swiss albino mice. Sci. Rep. 11(1), 18081 (2021).

58. Carmo de Carvalho e Martins, da Silva Santos Oliveira, M. D. M., da Silva, A. S. & Primo, L. A. A. M. G. S. & de Carvalho Lira, V. B. Biological indicators of oxidative stress [malondialdehyde, catalase, glutathione peroxidase, and superoxide dismutase] and their application in nutrition. iIn Biomarkers in Nutrition (ed. Packer, L.) 1–25 (Springer International Publishing, 2022).

59. Zhou, Y. Y., Wang, Y. S. & Inyang, A. I. Ecophysiological differences between five Mangrove seedlings under heavy metal stress. Mar. Pollut. Bull. 172, 112900 (2021).

    CAS  PubMed  Google Scholar 

60. Shahid, M. et al. Heavy metal stress and crop productivity. In Crop Production and Global Environmental Issues (ed. Hakeem, K.) 1–25 (Spiringer, 2015).

61. Macar, O., Kalefetoğlu Macar, T., Çavuşoğlu, K. & Yalçın, E. Determination of protective effect of Carob (Ceratonia siliqua L.) extract against Cobalt (II) nitrate-induced toxicity. Environ. Sci. Pollut. Res. 27, 40253–40261 (2020b).

    CAS  Google Scholar 

62. Kalefetoğlu Macar, T., Macar, O., Çavuşoğlu, K., Yalçın, E. & Yapar, K. Turmeric (Curcuma longa L.) tends to reduce the toxic efects of nickel (II) chloride in Allium cepa L. roots. ESPR. 29 (40), 60508–60518 (2022).

    PubMed  Google Scholar 

63. Kutluer, F., Güç, İ., Yalçın, E. & Çavuşoğlu, K. Toxicity of environmentally relevant concentration of esfenvalerate and Taraxacum officinale application to overcome toxicity: a multi-bioindicator ın-vivo study. Environ. Pollut. 373, 126111 (2025).

    CAS  PubMed  Google Scholar 

64. Ayhan, B. S. et al. A comprehensive analysis of royal jelly protection against cypermethrin-ınduced toxicity in the model organism Allium cepa L., employing spectral shift and molecular docking approaches. Pestic Biochem. Physiol. 203, 105997 (2024).

    CAS  PubMed  Google Scholar 

65. Zhang, C. Z. et al. Chromothripsis from DNA damage in micronuclei. Nature. 522, 179–184 (2015).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

66. Leffa, D. D. et al. Genotoxic and antigenotoxic properties of Calendula officinalis extracts in mice treated with Methyl methanesulfonate. Adv. Life Sci. 2, 21–28 (2012).

    Google Scholar 

67. Tümer, C., Çavuşoğlu, K. & Yalçin, E. Screening the toxicity profile and genotoxicity mechanism of excess manganese confirmed by spectral shift. Sci. Rep. 12, 20986 (2022).

    ADS  PubMed  PubMed Central  Google Scholar 

68. Üstündağ, Ü., Çavuşoğlu, K. & Yalçın, E. Comparative analysis of cyto-genotoxicity of zinc using the comet assay and chromosomal abnormality test. Environ. Sci. Pollut. Res. 31, 56140–56152 (2024).

    Google Scholar 

69. Emamverdian, A., Ding, Y., Mokhberdoran, F. & Xie, Y. Heavy metal stress and some mechanisms of plant defense response. Sci. World J. 2015, 756120 (2015).

70. Çavuşoğlu, D., Macar, O., Kalefetoğlu Macar, T., Çavuşoğlu, K. & Yalçın, E. Mitigative effect of green tea extract against mercury (II) chloride toxicity in Allium cepa L. model. Environ. Sci. Pollut. Res. 29, 27862–27874 (2022).

    Google Scholar 

71. Mehrandish, R., Rahimian, A. & Shahriary, A. Heavy metals detoxification: a review of herbal compounds for chelation therapy in heavy metals toxicity. J. Herbmed Pharmacol. 8, 69–77 (2019).

    CAS  Google Scholar 

72. Wang, L. et al. The biological activity mechanism of chlorogenic acid and its applications in food industry: A review. Front. Nutr. 9, 943911 (2022).

    PubMed  PubMed Central  Google Scholar 

73. Liang, N. & Kitts, D. D. Role of chlorogenic acids in controlling oxidative and inflammatory stress conditions. Nutrients 8, 16 (2015).

    PubMed  PubMed Central  Google Scholar 

74. Raskin, I. Role of salicylic acid in plants. Annu. Rev. Plant. Biol. 43 (1), 439–463 (1992).

    CAS  Google Scholar 

75. Gould, K. S., Markham, K. R., Smith, R. H. & Goris, J. J. Functional role of anthocyanins in the leaves of Quintinia Serrata A. Cunn. J. Exp. Bot. 51, 1107–1115 (2000).

    CAS  PubMed  Google Scholar 

76. Tošović, J. Spectroscopic features of caffeic acid: theoretical study. Kragujevac J. Sci. 39, 99–108 (2017).

    Google Scholar 

77. Calabro, M. L. et al. The rutin/β-cyclodextrin interactions in fully aqueous solution: spectroscopic studies and biological assays. J. Pharm. Biomed. Anal. 36, 1019–1027 (2005).

    CAS  PubMed  Google Scholar 

78. Bors, W., Michel, C. & Saran, M. Flavonoid antioxidants: rate constants for reactions with oxygen radicals. Methods Enzymol. 234, 420–429 (1994).

    CAS  PubMed  Google Scholar 

79. Onur, M., Yalçın, E., Çavuşoğlu, K. & Acar, A. Elucidating the toxicity mechanism of AFM2 and the protective role of Quercetin in albino mice. Sci. Rep. 13, 1237 (2023).

    ADS  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Authors and Affiliations

1. Department of Herbal and Animal Production, Kırıkkale Vocational School, Kırıkkale University, Kırıkkale, Turkey

   Fatih Kutluer

Contributions

F.K.: Investigation, methodology, visualization, writing-review and editing., methodology, software, visualization.

Corresponding author

Correspondence to Fatih Kutluer.

Competing interests

The authors declare no competing interests.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions