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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

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69607
10.1038/s41598-024-69607-w
Article
Phytochemical, biological, and computational investigations of Ephedra alata Decne. growing in salinity conditions of Arabian Peninsula
Mohammed Hamdoon A. 1
Said Rana 23
Abbas Manal M. 34
Al-Najjar Belal O. 23
Abd-Elmoniem Essam 5
Khan Riaz A. 1
Alsohim Abdullah S. 6
Almahmoud Suliman A. 1
Kedra Taha A. 7
Shehata Safia M. 8
http://orcid.org/0000-0003-0266-8760
Ismail Ahmed Ahmed_ph_fut@yahoo.com

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1 https://ror.org/01wsfe280 grid.412602.3 0000 0000 9421 8094 Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, Qassim University, Qassim, 51452 Saudi Arabia
2 https://ror.org/00xddhq60 grid.116345.4 0000 0004 0644 1915 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Al-Ahliyya Amman University, Amman, 19328 Jordan
3 https://ror.org/00xddhq60 grid.116345.4 0000 0004 0644 1915 Pharmacological and Diagnostic Research Laboratory, Al-Ahliyya Amman University, Amman, 19328 Jordan
4 https://ror.org/00xddhq60 grid.116345.4 0000 0004 0644 1915 Department of Medical Laboratory Sciences, Faculty of Allied Medical Sciences, Al-Ahliyya Amman University, Amman, 19328 Jordan
5 https://ror.org/01wsfe280 grid.412602.3 0000 0000 9421 8094 Department of enviromental and natural resources, College of Agriculture and Food, Qassim University, P.O. Box 6622, Buraydah, 51452 Qassim Saudi Arabia
6 https://ror.org/01wsfe280 grid.412602.3 0000 0000 9421 8094 Department of Plant Production, College of Agriculture and Food, Qassim University, P.O. Box 6622, Buraydah, 51452 Qassim Saudi Arabia
7 https://ror.org/05fnp1145 grid.411303.4 0000 0001 2155 6022 Department of Pharmacognosy and Medicinal Plants, Faculty of Pharmacy, Al-Azhar University, Cairo, 11371 Egypt
8 https://ror.org/00cb9w016 grid.7269.a 0000 0004 0621 1570 Clinical Pathology Department, Ain Shams University Hospitals, Cairo, Egypt
9 https://ror.org/023gzwx10 grid.411170.2 0000 0004 0412 4537 Pharmacognosy Department, Faculty of Pharmacy, Fayoum University, Fayoum, 63514 Egypt
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© The Author(s) 2024
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Ephedra alata Decne is a medicinal plant widely used in traditional medicine for the management of bronchial asthma and cancer. Phytochemical analysis and biological activities, including antioxidant and anticancer effects, were investigated in the current work as new findings for the plant E. alata, a species growing wildly in the marsh and saline environments of the central area of Saudi Arabia. The Ultra Pressure Liquid Chromatography coupled with Electron spray ionization-Quadropole-Time of flight (UPLC-ESI-Q-TOF) system was used for the phytochemical analysis of the plant constituents. In addition, Polyphenolic profiling including the total phenolic (TPC) and flavonoid (TFC) contents of the plant extracts were measured. Phenolic acids were found at the highest relative percentages among all the identified compounds and were measured at 66.07 mg GAE (Gallic acid equivalent). The UPLC analysis of the E. alata extract indicated the presence of chlorogenic acid, syringic acid, caffeic acid, vanillic acid, rosmarinic acid, umbelliferone, isorhoifolin, and apigenin at the highest relative percentages. Mineral analysis indicated that the microelement content of E. alata was relatively low, except for magnesium (Mg). In vitro antioxidant assays revealed the ability of the plant to scavenge DPPH free radicals, reduced molybdenum ions, and ferrous at levels of 14.63, 19.97, and 27.78 mg Trolox equivalents, respectively. The extract induced transition metal chelation at 31.36 mg EDTA equivalents. The extract induced cytotoxic effects against MDA-231 and A549 cell lines at IC50 levels of 25.31 and 39.81 µg/mL, respectively. The plant extract inhibited the colonization and migration of cancer cells as part of its potential anticancer effects. In addition, major E. alata constituents like isorhoifolin, chlorogenic acid, apigenin, and rosmarinic acid exhibited the lowest binding energy to the CAIX enzyme at − 8.41, − 6.64, − 6.32, and − 6.26 kcal/mol, respectively, compared to the binding energy (− 7.72 kcal/mol) of the co-crystallized ligand (Y0R). The docking results further supported the selection of the CAIX enzyme as a standard predictive therapeutic target, since it exhibited significant binding interactions with the major constituents of the plant.

Keywords

Ephedra alata Decne
Polyphenolic profiling
UPLC-ESI-Q-TOF
Antioxidants
Anticancer
Docking analysis
Subject terms

Biological techniques
Plant sciences
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Medicinal plants play a major role in the lives of people. They represent one of the pillars of traditional medicine around the world, and their use in treating diseases has been established since ancient times1–3. Owing to the effectiveness of these plants (in most cases), their use have become widespread, and people have passed on their experiences regarding their benefits, harms, and how to use them. Therefore, many traditional healers specialize in herbal and traditional medicine styles4.

Medicinal effects of these plants have been reflected in the fact that they are phytochemically varied. In other words, the active substances produced by medicinal plants are the main reason for their effectiveness in treating various diseases. The quantitative and qualitative differences of these active substances in medicinal plants are the reason that these plants treat different diseases and do not participate as a whole in treating a specific disease5,6. The active constituents of some plant families and individual plants are always used as a chemical definition map (chemomarkers) for these plants. In addition, several studies have shown that the active constituents of a particular plant may vary in quantity and quality as a result of the different environmental conditions in which the plant grows7–9.

More than 65 plant species have been taxonomically listed in the Ephedra genus (Ephedraceae family). These plants are geographically distributed in Europe, Asia, America, and Africa10,11. The plants of Ephedra have long been used in traditional medicine, such as traditional Chinese medicine, Kompo, and African and Arab traditional medicine11–14. For example, Ephedra altissima has been used to treat asthma and bronchitis in the Algerian Central Sahara area, China, and India14,15. They also reported some toxicity by inducing hypertension, dizziness, insomnia, anxiety, and urine retention owing to the presence of the known alkaloid ephedrine15. In addition to ephedra alkaloids such as ephedrine, norephedrine, pseudoephedrine, and methyl ephedrine, several other constituents have been identified in the genus, such as phenolic acids, tannins, terpenoids, and flavonoids10. However, the traditional uses, biological activities, and toxicities of Ephedra species are thought to be due to the presence of alkaloid constituents10,16.

Ephedra alata belongs to the genus Ephedra, with the Arabic common name “Alanda.” The plant is native to several Middle Eastern countries, including Saudi Arabia, Egypt, Iran, Algeria, Lebanon, Jordan, Iraq, Libya, Tunisia, and Morocco17. The plant's use in traditional medicine is prevalent for specific diseases such as bronchial asthma; however, it has also been reported to be used in managing digestive system disorders, cancer, and fungal and bacterial infections18. Several phytochemicals, such as kaempferol, quercetin, resveratrol, p-coumaric acid, and caffeic acid, have been identified in high quantities in the aerial parts of the plant19. However, quercetin, naringin, epicatechin, and caffeic acid were detected as the major constituents in the seeds of the plant20. Several biological activities of E. alata have been recorded for E. alata, including anti-inflammatory, antiproliferative, antioxidant, antimicrobial, and antidiabetic effects17,18,20–22.

This study is aimed to investigate the phytochemical and biological properties of E. alata grown in the central area of Saudi Arabia. The study provides information for plant species growing under environmental salt stress, and discusses the phytochemical and biological variations compared to the same species growing in different locations. This study focused on the anticancer activity of the plant and provided detailed information on the potential underlying anticancer mechanisms of the plant by docking analysis of the plant constituents with carbonic anhydrase IX as a promising therapeutic target for anticancer drugs. This study also provides information on the mineral contents of plant and their plausible antioxidant effects.

Materials and methods

Plant materials and extraction method

The plant material was collected from the Asyah Governorate in the Qassim region during March 2022. The plant is registered as endangered, and was collected in limited quantities under the supervision of the Department of Plant Production and Protection, College of Agriculture and Veterinary Medicine, Qassim University, and identified as Ephedra alata Decne by Dr. Abdullah S. Alsohim, Department of Plant Production and Protection, College of Agriculture and Veterinary Medicine, Qassim University. The authors adhered to the guidelines of the Convention on International Trade in Endangered Species of Wild Fauna and Flora (https://cites.org/eng) and the IUCN Policy Statement on Research Involving Species at Risk of Extinction (https://portals.iucn.org/library/efiles/documents/PP-003-En.pdf) during the plant collection process. Plant sample was stored under the number of QPP-136 at the College of Pharmacy, Qassim University. The plant powder materials (100 g) was soaked in 500 mL of aqueous ethanol (aq. ETOH) solution (7:3, ethanol: water) and shaken in a stirring mixer for one day. The extract was then filtered and dried over a rotary evaporator (BÜCHI Rotavapor R-114, BÜCHI, Switzerland). The extraction yield was calculated as 2.7% (w/w) and the dried extract was stored in a freezer at – 20 °C to save the constituents until further analysis8.

Phytochemical analysis

Polyphenols quantification

Phenolic and flavonoid contents of the plant were quantified as equivalents of gallic acid and quercetin using spectrophotometric assays, as demonstrated in the literature23.

Determination of total phenolics

The phenolic contents of the plant extracts was determined using diluted Folin Ciocalteu reagent in water (1:5) and concentrated Na2CO3 solution (10%). Both reagents (200 µL) were added to 1600 µL of plant extract at a concentration of 0.1 mg/mL. After 30 min, the absorbance of the mixture was measured at 760 nm against a blank solution. A calibration curve for gallic acid with the line equation (Absorbance + 0.4346)/0.2563 was used to determine the phenolic content. The total phenolic contents were calculated as gallic acid equivalents per gm of the dried extract from three measurements and expressed as mean ± standard deviation.

Determination of total flavonoids

The Flavonoids were measured using 100 µL of AlCl3 (10%) and 100 µL of CH3CO2K (0.1 mM) solutions, which were thoroughly mixed with 2000 µL of the extract (0.1 mg/mL). After 30 min, the absorbance of the mixture was measured at 415 nm against a blank solution. A calibration curve of quercetin with the line equation (Absorbance + 0.1646)/0.4195 was used to determine the flavonoid content. The total flavonoid contents were calculated as quercetin acid equivalents per gm of the dried extract from three measurements and expressed as mean ± standard deviation.

UPLC-ESI-Q-TOF analysis of the E. alata extract

Stock solution and sample preparation for polyphenolic profile

The substance was dissolved in analytical-grade dimethyl sulfoxide (DMSO) to obtain a stock solution, which was then diluted with acetonitrile and utilized to determine the precise MS and retention period. For the plant extract, the sample was dissolved in 2.0 mL of DMSO and added to acetonitrile (50 mL by Acetonitrile. Each sample was centrifuged at 4000 rpm for two minutes. One milliliter was transferred to the HPLC vial and 3.0 µL was the injection volume. The reagents used, such as acetonitrile, methanol, water, and formic acid, were of LC–MS grade8.

Mass and chromatographic conditions

A Bruker Daltonik (Bremen, Germany) Impact II ESI-Q-TOF (Apollo II ion funnel electrospray source system) equipped with a Bruker Daltonik Elute UPLC system (Bremen, Germany) was used to screen compounds of interest. Standards were used to identify m/z with high resolution and the exact retention time of each analyte after chromatographic separation. A summary of the chromatographic and Mass Spectrometric conditions is presented in Table 1.Table 1 Chromatographic and mass spectrometry conditions.

HPLC (high-performance liquid chromatography) conditions	Pump initial flow rate	Auto sampler injection volume	Auto sampler temp	Column oven temp	
Chromatography	0.51 mL/min	3 µL	15 °C	40 °C	
Total run time	35 Min	
Mobile phase linear gradient elution	Step	Total time (min)	Flow rate (µL/min)	A (%) water with 0.05% formic acid	B (%) (ACN)	
0	27	0.51	95 (95–20)	5 (5–80)	
27	2	0.51	5	95	
	29.1	6	0.51	95	5	
Column type	Bruker solo 2.0_C-18 UHPLC column (100 mm × 2.1 mm × 2.0 μm)	
MS conditions	TOF repetition	Nebulizer gas	Capillary voltage	TEM	Flow rate dry gas	FSR	
Positive	20 kHz	2.0 bar	2500	200	8 mL/min	50,000	

Soil and trace elements analysis

The E. alata plant was collected from the Asyah Governorate in the Qassim region. In general, the Qassim region is in the middle of the desert of the Kingdom of Saudi Arabia, where the climate is hot, especially in summer, when the temperature exceeds 45 °C. We collected eight soil samples from the Asyah government around the ephedra plant. Each sample was collected from a depth of 0–30 cm. Soil samples were collected in clean polythene bags, dried, milled, and passed through a 2 mm sieve. Each soil sample had nine chemical characteristics. The parameters (pH, electrical conductivity (EC), total dissolved solids (TDS), three soluble cations (Ca++, Mg++, Na+), and three soluble anions (CO3−−, HCO3 −, and Cl−) were determined using different techniques according to Jones24. The soil pH and EC were measured by preparing a saturated soil paste. The pH of saturated soil paste (pH) was measured using a benchtop pH meter (Hanna 210). The EC of the saturated extract was measured using a conductivity meter (Jenway 4310). In addition, in the extract of saturated soil paste, Ca++ and Mg++ were analyzed by titrating against 0.005 N EDTA solution using NaOH and Murexid indicator for Ca++ as well as NH4Cl + NH4OH buffer solution and Eriochrome Black T indicator for Mg++. In addition, CO3−−, HCO3− were analyzed by titrating by HCl 0.01N with phenolphthalein and methyl orange as indicators for CO3- and HCO3−, respectively. Titration with silver nitrate 0.01 using chromate potassium indicator, ion chloride was analyzed. The available Fe, Mn, Zn, and Cu were extracted using DTPA25 and measured using an Atomic Absorption Spectrophotometer (Shimadzu AA-6000).

Antioxidant assays

Four different assays were used to determine the in vitro antioxidant activity of E. alata.

Total antioxidant capacity (TAC)

According to the reported method, TAC of the E. alata extract was measured using freshly prepared ammonium molybdate reagent26. The reagent (4000 µL) was thoroughly agitated with 400 µL of extract (containing 200 µg of extract). TAC was measured using the Trolox calibration curve with the line equation (Absorbance + 0.1788)/0.1954.

DPPH scavenging activity

The method reported by Shimada et al.27 was used. Equal volumes (1000 µL) of E. alata extracts containing 200 µg of the dried materials and DPPH (2,2-diphenyl-1-picrylhydrazyl) solution (12 mg in 100 mL of methanol) were mixed and the reduction in DPPH color was measured at 517 nm after 30 min of keeping the mixture in the dark. The DPPH-scavenging activity of the extract was calculated to be equivalent to that of Trolox using the line equation (Absorbance + 0.1296)/0.1835.

Ferric reducing antioxidant power (FRAP)

This method was performed as reported by Benzie and Strain28. The FRAP reagent (2000 µL) and 100 µL of the extract containing 200 µg of dried material were thoroughly mixed, and the developed color was measured at 593 nm after 30 min of incubation. The FRAP activity of E. alata was measured to be equivalent to that of Trolox using a standard calibration curve with the line equation (Absorbance + 0.3754)/0.359.

Iron chelating activity assay (ICA)

The iron-chelating activity of E. alata was measured using the method of Zengin et al.29. The E. alata extract (2000 µL) containing 200 µg of dried materials, 25 µL of FeCl2 (2 mM), and 100 µL of ferrozine were mixed. Absorbance was recorded at 562 nm against a blank. The ICA of E. alata was measured to be equivalent to that of EDTA using a standard calibration curve with the line equation (Absorbance + 0.1296)/0.1835.

Antiproliferative activity of E. alata extract

Cell proliferation assay

The MDA-MB-231 (Human Caucasian breast adenocarcinoma) and A549 (Human Caucasian lung carcinoma) cell lines were acquired from The European Collection of Authenticated Cell Cultures (UK). The cell lines were cultured in a humidified 5% CO2 atmosphere incubator at 37 °C and in Dulbecco’s modified Eagle’s medium (DMEM) high glucose (Euroclone, S.p.A) containing 10% fetal bovine serum, 10 g/L penicillin/streptomycin, and 10 g/L l-glutamine. A stock solution of Ephedra extract was freshly prepared in twofold serial dilutions. The impact of ephedra extract on cell lines was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Cells were plated in 96-well plates at a concentration of 7000 cells per well in a suitable medium. The plates were incubated for 24 h in CO2 incubator and the cells were treated with the extract, 500–7.8 µg/mL for 24 h. Doxorubicin was used as a positive antiproliferative control. MTT was then performed using an MTT kit (Catalog: G4000, Promega, USA), where 15 µL of the staining reagent was added to each well, and incubated for four h at 37 °C, followed by the addition of 100 µL of the solubilization stop/mix solution and incubated for 1 h before measuring the absorbance using microplate reader (Biotech, USA) at 590 nm8.

Scratch assay

The scratching assay was performed according to the reported assay30. The cells were cultured in (DMEM) medium containing 10% fetal bovine serum (FBS) and incubated under 5% CO2 and 95% humidity. 3 × 105 cells/mL) were seeded into 24-wells tissue culture dishes for 24 h at 37 °C. When cultured cells were confluent, a monolayer was formed, and a linear wound was created using a sterile 100 μL plastic pipette tip. The plates were rinsed with phosphate-buffered saline (PBS) to eliminate any residual cellular debris. A negative control consisting of untreated cells in a complete medium was used. The Ephedra extract was tested at a concentration equal to the IC50, was applied to the experimental wells, and incubated for 24 h at 37 °C with 5% CO2. The surface area of the scratch was measured at zero time when it was created, and 24 h later. The relative migration of the cells under each condition was estimated by capturing three representative images of the scratch area from different wells. Motic software version 2 (Motic China group Co, Ltd) was used to analyze the data. Experiments were performed in triplicates. The closure rate was calculated using the formula provided by the CytoSelect™ 24-well wound healing assay (Cell Biolabs, INC, USA) as follows:%Closure=100-((areaafter24hrs/areaatday1)×100)

Colony assay

The colony assay was performed on MDA-231 and A549 cell lines using the IC50 of Ephedra extract, and untreated cells of both cell lines were used as controls. 600 cells/well) were seeded in 6 wells plate in DMEM media and incubated overnight at 37°C and 5% CO2. Both cell lines were treated with the IC50 of Ephedra extract. Cell lines were incubated for 14 days at 37 °C and 5% CO2. After 14 days, the media was removed and the plate was washed with phosphate buffer saline (PBS) (Euro-clone, Europe), afterwards the cells were fixated by adding 50 µL of 4% paraformaldehyde, and incubated for 1 h31. After this, the cells were stained with 50 µL crystal violet and incubated for 30 min, washed with tap water, and dried for 2 days.

Molecular docking analysis

Study specifically targeted the plant constituents of the utmost prevalence, outlined in Fig. 1, ensuring a concentrated exploration of the most abundant and potentially bioactive compounds within the plant extracts. To investigate the potential anticancer properties inherent to these chosen constituents, a molecular docking study using Autodock 4 software was conducted32,33.Figure 1 2D chemical structure of the selected plant constituents (Prepared by ChemDraw).

Carbonic anhydrase IX as a therapeutic target

Carbonic anhydrase IX (CAIX) has emerged as a promising therapeutic target for anticancer drugs because of its unique expression pattern and involvement in tumor biology34,35. CAIX is a transmembrane protein that catalyzes the reversible hydration of carbon dioxide to bicarbonate and protons, and plays a critical role in maintaining the intracellular pH homeostasis36. In contrast to other carbonic anhydrase isoforms, the CAIX is highly overexpressed in various types of cancers but is not significantly expressed in most of the normal tissues36. This differential expression pattern makes CAIX an attractive target for selective cancer therapy, minimizing the potential side effects on normal cells.

Molecular docking procedure

In this study, we sought to gain deeper insights into the molecular mechanisms underlying the anticancer activities of the identified compounds (Fig. 1). To elucidate the mechanism, molecular docking simulations were conducted using AutoDock 432, using the protein crystal structure (5FL6)37 with a crystallized ligand (code: Y0R) serving as a control for comparative analysis.

The preparation of both the protein and ligands for docking simulations was carried out using AutoDock Tools 1.5.7. Initially, the protein crystal structure was loaded into AutoDock Tools 1.5.7, where polar hydrogens were added and Kollman charges were assigned, further enhancing the accuracy of the protein representation. Subsequently, each ligand molecule was loaded separately and Gasteiger charges were assigned, taking into account the specific chemical properties of each ligand. Furthermore, a grid box with a volume of 15 Å3 and default grid spacing of 0.375 Å was established. The center of this grid box was defined at coordinates − 30.702 (x), − 1.353 (y), and − 1.510 (z), ensuring an appropriate spatial context for the docking calculations.

Finally, actual docking simulations were executed using default parameters, and a Lamarckian genetic algorithm was employed across 100 runs38. Subsequently, the results were analyzed by examining the AutoDock log files. Of particular interest were the lowest energy of binding (LEB) values for each ligand, with a focus on identifying the conformer with the most favorable binding energy. To further refine our selection, we considered the conformer with the highest cluster number, thus ensuring robustness in our choice of ligand conformation. These selected conformers were exported and visualized using BIOVIA Discovery Studio Visualizer 16.1, providing insights into the binding interactions of the identified compounds and the reference compound, Y0R, with the CAIX enzyme.

Statistical analysis

The results of the antioxidant and antiproliferative activities, and scratching assay were calculated as the mean ± standard deviation. The results of the colony assays were expressed as the mean ± standard error of the mean (SEM). Differences between control and tests were analyzed using one-way ANOVA and Tukey’s multi-group comparison test.

Results and discussion

Phytochemical analysis

Polyphenol quantification

The total phenolic and flavonoid contents of E. alata growing in the central area of the Kingdom of Saudi Arabia were investigated in this study. The results indicated the presence of 66.07 ± 7.65 mg and 8.96 ± 0.24 mg of the phenolic acids and flavonoids, as equivalents to the gallic acid and quercetin, respectively. These results differed from the reported polyphenolic contents of the E. alata  plant species growing in different areas. For instance, the plant species E. alata, growing in the Jenin region of Palestine, has been investigated for its phenolic and flavonoid quantities by Jaradat et. al., that found total phenolics and flavonoids in the ethanol extract of the plant at the concentration of 19.17 and 5.44 mg gallic acid and rutin equivalents39. Additionally, Al-Rimawi et al., found the concentration of the phenolic acids and flavonoids as 40.9–101.2 and 9.8–19.5 mg gallic acid and catechin equivalents, respectively in the ethanol extracts of the plant40. On the other hand, the phenolic and flavonoids of E. alata growing in Algeria have been measured at 157.54–214.92 and 21.86–30.74 mg gallic acid and catechin equivalents, respectively, in the methanol extracts of the plant18. The polyphenolic constituents of plants growing in Tunisia have also been measured by Soumaya et al., They have found much higher quantity of the phenolic acids compared to our findings for the plant species growing in Saudi Arabia (164.58 mg compared to 66.07 mg gallic acid equivalents)10. The significantly higher percentages of phenolic acids and flavonoids in the E. alata species growing in Tunisia compared to our current findings have also been reported by Elhadef et al., who measured the polyphenolic constituents of the plants from different locations in Tunisia, and found some significant differences among them41. One more example for the plant species collected form the Botanical Garden of the University of Hamburg, Germany, which has been investigated by Ibragic and Sofić, found 53.3 and 2.8 mg gallic acid and quercetin equivalents for the total phenolics and flavonoid contents, respectively. The overall results of the reported literature indicated that E. alata growing in moderate to cold temperate regions produced less phenolic acids and flavonoids as compared to the plant species growing in the Sahara and relatively higher temperature regions. Comparing the current findings with those reported from plants growing in different regions also indicated substantial differences in the number and structure-types of phenolic acids and flavonoids, which can be attributed to variations in the environmental conditions.

UPLC-ESI-Q-TOF profiling

Some of the effects of environmental conditions on plants can be easily recognized in plant secondary metabolite profiling, and several studies have supported this notion by investigating the phytochemical constituents of plants growing under different environmental conditions42–44. In the current study, the secondary metabolites of E. alata growing in the central area of Saudi Arabia were profiled using LC–MS analysis. This analysis was carried out to identify the phenolic acids and flavonoids of the plant, which are thought to be responsible for its various biological activities and traditional applications. The relative percentages of identified compounds were calculated using the peak areas corresponding to the total peak area in the chromatogram (Supplementary information file, Supplementary Fig. S1). The compounds were tentatively identified using the standard machine library, and mass fragments of each identified peaks were compared to those reported in the literature. Some of the compounds "assigned by asterisk (*)" were identified based on the injected standard compounds (Supplementary information file, Supplementary Table S1 and Supplementary Fig. S2). A total of 29 compounds were identified in the extract of E. alata with total calculated relative percentage of 36.57% (Table 2). Fifteen peaks of these compounds were identified as phenolic acids, three peaks were identified as phenolic coumarins, and ten compounds were identified as flavonoids. The results also indicated that phenolic acids were represented as major constituents, with calculated relative percentages of 22.18%. However, the relative concentrations of the flavonoids and phenolic coumarins were calculated as 6.59% and 7.20%, respectively. In addition to the polyphenolic compounds, the triterpene ursolic acid was detected at a retention time of 28.17 min at a relative concentration of 0.59% (Table 2). The identified compounds in the plant, their relative percentages in the extract, and the quantitative percentages of phenolic acids and flavonoids indicated the potential health and food-based benefits of the plant. The identity of the compounds also revealed some similarities among the active constituents of the plant, E. alata, growing in different locations with the species growing in the central area of Saudi Arabia, from the perspective that phenolic acids and flavonoids are dominant in the plant20,40,45,46. A review of the literature indicated the presence of vanillic acid, rosmarinic acid, caffeic acid, syringic acid, quercetin, kaempferol, and naringenin as common constituents of plants growing in different locations10,20,40,45–47, which were also detected in the plants growing in Saudi Arabia as observed in the current study. The overall phytochemical investigations revealed the ability of the plants from different locations to biosynthesize polyphenols; however, these polyphenols could vary in their concentrations (abundancy) and structural identity in E. alata growing under different environmental conditions.Table 2 LC–MS based constituents profiling of the E. alata extract.

	RT (min)	m/z [M–H]–	M	Name	Molecular formula	Area	R%	
1	1.95	153.01940	154.02668	2,5-Dihydroxybenzoic acid	C7H6O4	6920	0.80%	
2	2.8	137.02447	138.03175	4-Hydroxybenzoic acid	C7H6O3	6118	0.71%	
3	3.15	353.08763	354.09491	Chlorogenic acid	C16H18O9	50,268	5.83%	
4	3.49	167.03511	168.04239	Vanillic acid	C8H8O4	16,248	1.89%	
5	3.56	179.03488	180.04215	Caffeic Acid	C9H8O4	12,216	1.42%	
6	3.85	197.04569	198.05297	Syringic acid	C9H10O5	24,128	2.80%	
7	4.26	193.05064	194.05791	Ferulic acid (trans)	C10H10O4	2718	0.32%	
8	4.76	163.04000	164.04728	p-Coumaric acid	C9H8O3	10,186	1.18%	
9	5.02	301.03850	302.04577	3,7,3′,4′,5′-Pentahydroxyflavone (Robinetin)	C15H10O7	1198	0.14%	
10	5.07	195.06600	196.07328	3,5-Dimethoxy-4-hydroxyacetophenone	C10H12O4	26,900	3.12%	
11	5.13	161.02435	162.03163	Umbelliferone (phenolic coumarin)	C9H6O3	32,246	3.74%	
12	5.34	121.02936	122.03663	Benzoic acid	C7H6O2	4840	0.56%	
13	5.36	191.03503	192.04231	Scopoletin	C10H8O4	5410	0.63%	
14	5.6	623.19916	624.20644	Acteoside	C29H36O15	1116	0.13%	
15	5.81	607.16641	608.17369	Diosmin	C28H32O15	1458	0.17%	
16	5.97	433.11466	434.12193	Naringenin-7-O-glucoside	C21H22O10	1296	0.15%	
17	6.22	175.04002	176.04730	4-Methylumbelliferone (phenolic coumarin)	C10H8O3	24,400	2.83%	
18	6.41	577.15579	578.16310	Isorhoifolin	C27H30O14	22,710	2.64%	
19	7.12	447.09484	448.10211	Kaempferol-7-O-glucoside	C21H20O11	2558	0.30%	
20	7.22	359.07706	360.08433	Rosmarinic acid	C18H16O8	23,540	2.73%	
21	7.24	301.00221	302.00949	Ellagic acid	C14H6O8	1720	0.20%	
22	8.35	459.13105	460.13833	6,4′-Dimethoxyisoflavone-7-glucoside (Wistin)	C23H24O10	1222	0.14%	
23	8.37	149.06034	150.06761	Hydrocinnamic acid	C9H10O2	2502	0.29%	
24	8.96	301.03505	302.04233	Quercetin	C15H10O7	9648	1.12%	
25	10.27	269.04561	270.05289	Apigenin	C15H10O5	13,422	1.56%	
26	10.5	285.04117	286.04845	Kaempferol	C15H10O6	2146	0.25%	
27	11.83	299.09550	300.10277	Farrerol	C17H16O5	1156	0.13%	
28	19.15	331.19052	332.19780	Carnosic acid	C20H28O4	1684	0.20%	
29	28.17	455.35066	456.35793	Ursolic acid	C30H48O3	5112	0.59%	
Total	36.57%	
Phenolic acids	22.18%	
Flavonoids	6.59%	
Phenolic coumarins	7.20%	

Soil and trace element analysis

Environmental conditions, including biotic and abiotic stresses, are well-known factors affecting the phytochemicals, and, subsequently, the biological and therapeutic effects of the plants. In this context, several reports have investigated the secondary metabolites, physiological, and pharmacological properties of plants growing under drought and high salinity conditions2,48. The reports have highlighted the positive effect of environmental salinity and drought conditions on the quantity and diversity of the polyphenolic constituents, including phenolic acids and flavonoids, of the plants growing under these harsh circumstances43,49. In addition, the elevated levels of such constituents have been reported to enhance certain pharmacological effects related to the plants, including the antioxidant and anticancer effects2,43,48). From these points of view, the current study investigated the soil contents and mineral contents of the plant, E. alata, to demonstrate the growth circumstances of the plant, and how these conditions of drought and high salinity affected the plant constituents and their pharmacological effects.

The environment for E. alata growth was represented by eight samples of soil collected from the Asyah governorate. The chemical characteristics of the soil samples were determined, and are summarized in Table 3. The pH, electrical conductivity (EC), and total dissolved solids (TDS) ranged between 7.62–8.4, 6.34–25.5 dS/m and 4057–16,320 ppm, respectively. Therefore, E. alata shows good tolerance to salt50. The water-soluble cations in soil, Ca++, Mg++, and Na+ ranged between 20.7–80, 7.8–50, and 25.1–135 meq/l, respectively; whereas the water-soluble anions, HCO3−, SO4−− and Cl− ranged between 1.5–2.5, 5.1–65.1 and 50–200 meq/L, respectively. Moreover, CO3− was found zero in all the samples. In addition, the available microelement concentrations in soil were, Fe, 1.47–1.7; Mn, 0.49–0.69; Zn, 0.21–0.35 and Cu, 0.22–0.26 mg kg−1 (Table 3). The table 4 shows the mineral contents of E. alata collected from the Asyah governorate in the Qassim region. The plant’s mineral contents were Fe, 60.5; Cu, 11.0; Mn, 8.84; Co, 4.50; Zn, 12.2 and Mg, 2898 mg kg−1. The microelement contents of ephedra were relatively low, except for Mg. The main source of elements in plants is soil, and the soil analysis data showed that these soils were low in elements except, sodium, calcium, and magnesium due to the high concentration of salts in most soils in this zone. Soil pH is a soil parameter that affects the availability of elements for plants. The soil pH was found slightly basic (> 7.5). It is well known that the availability of most microelements decreases with increasing the soil pH51. The concentration of microelements in E. alata was found low. In contrast, Mg in E. alata was relatively high, whereas Mg ion concentrations in the soil extracts (Table 3). With an increased concentration of certain soluble elements in the soil, the concentration of the same elements in plants increases52. The Qassim region is also described as arid, meaning that the soil moisture content is very low, wherein the mean monthly precipitation is 11.4 mm with mean monthly air temperature of 15–34 °C53. This dry environment negatively affects the elemental contents of the plants. Therefore, E. alata shows good tolerance to drought54. The soil and mineral analyses of the region revealed that E. alata, as a native plant to the Qassim region, has the remarkable ability to adapt to the high salinity and drought conditions prevalent in the region. These findings also encouraged us to investigate the phytochemical constituents of the plant, with a particular focus on phenolic acids and flavonoids. Based on the plant's demonstrated tolerance to the challenging high salinity and drought environmental factors, we hypothesized that these secondary metabolites would be present in E. alata at elevated levels. In addition, the anticancer and antioxidant potentials of the plant extract were also expected to be affected, as these bioactivities are often associated with the presence of phenolic compounds.Table 3 Soil analysis of different locations around the habitat of E. alata in Asyah, Qassim region.

Soil samples	pH 1:2.5	EC dS/m	TDS ppm	EC dS/m	Milliequivalent/L	
Soluble cations	Soluble anions	
Ca++	Mg++	Na+	CO3−	HCO3−	SO4−	Cl−	
1	7.97	6.50	4160	6.5.0	20.7	7.80	36.9	0	1.5	13.9	50	
2	7.88	12.9	8256	12.9	38.0	26.6	66.1	0	1.9	13.8	115	
3	8.01	8.70	5568	8.70	42.0	21	26.2	0	2.5	6.7	80	
4	7.62	8.50	5440	8.5	30.0	25	29.1	0	1.8	12.3	70.0	
5	8.40	13.7	8768	13.7	60.0	15.2	58	0	2.3	10.9	120	
6	8.20	6.34	4057	6.34	25.6	16.2	25.1	0	1.8	5.10	60	
7	8.32	25.5	16,320	25.5	80.0	50.0	137	0	1.9	65.1	200	
8	12.0	7.90	7680	7.9.0	65.0	25.0	50.2	0	2.0	11.0	102	
Min	7.62	6.34	4057	6.34	20.7	7.80	25.1	0	1.5	5.10	50.0	
Max	8.40	25.5	16,320	25.5	80.0	50.0	137	0	2.5	65.1	200	

Table 4 Concentration of micronutrients (mg kg−1) in the soil of the Qassim region.

Site	Fe	Mn	Zn	Cu	
1	1.48	0.58	0.24	0.24	
2	1.47	0.69	0.34	0.23	
3	1.57	0.59	0.29	0.23	
4	1.52	0.56	0.21	0.26	
5	1.70	0.58	0.35	0.22	
6	1.64	0.49	0.24	0.23	
7	1.63	0.59	0.27	0.22	
8	1.67	0.59	0.31	0.25	
Min	1.47	0.49	0.21	0.22	
Max	1.70	0.69	0.35	0.26	

Antioxidant activity

The potential antioxidant effects of E. alata growing in Saudi Arabia was estimated through four in vitro assays that measured the free radical capture and reducing potential of the extract, which were expressed as Trolox equivalents, and the iron chelating potency, expressed as EDTA equivalents. The results demonstrated in Fig. 2 indicated the ability of the plant extract to chelate iron metal at the level of 31.36 mg EDTA equivalent. This effect of E. alata extract contributes to the antioxidant activity of the plant by reducing the Fenton reaction and subsequent free radicals formation. The metal-chelating effect of the plant might be attributed to the presence of several flavonoids in the plant extract, such as quercetin, apigenin, and kaempferol, which have been identified in the plants and are known for their transition metal-chelating effects55. These flavonoids have specific structural features that enable them to chelate transition metals, especially the presence of 5-hydroxy along with the presence of 4-keto groups in their structures56. The metal-chelating effects of E. alata growing in different locations have been reported to have better activity than that of vitamin C57. These results also indicated that E. alata has the ability to reduce molybdate and ferric ions as part of its antioxidant activity. Plant-induced reduction to molybdate (Mo (VI) to Mo (V)) and ferric (Fe3+ to Fe2+) ions by 19.97 and 27.78 mg Trolox equivalents, respectively (Fig. 2). The reducing power of plant species growing in different locations has also been reported10,40. The reducing power of the plant extract is expected to be due to the presence of condensable quantities of phenolic acids and flavonoids, and the variety of polyphenols identified in the plant extract by LC–MS analysis. LC–MS analysis indicated the presence of well-known reducing phenolic acids, such as chlorogenic acid, caffeic acid, p-coumaric acid, and rosmarinic acid58 at high relative concentrations (Table 2). The extract of the plant also induced a free radical scavenging effect of 14.63 mg Trolox equivalent (Fig. 2). This effect has also been reported in plant species growing in different areas and is mostly attributed to the presence of flavonoids and phenolic acids. Most of the identified polyphenols in Table 2 have been reported for their potential free radical scavenging activity as their major mechanisms for antioxidant activity. For instance, the flavonoids, kaempferol, naringenin, quercetin, apigenin, and farrerol have been reported for their free radical scavenging effects59–61. Moreover, the identified phenolic acids, such as hydrocinnamic acid, rosmarinic acid, carnosic acid, p-coumaric acid, vanillic acid, caffeic acid, ferulic acid, and chlorogenic acid, have also been reported for their antioxidant activity and contributed together with the flavonoids to the antioxidant potential of several medicinal plants such as rosemary and Pulicaria7,62,63. In general, the current findings further highlight the antioxidant potential of E. alata, regardless of its growing location, and the role of polyphenolics as plant constituents in that effect.Figure 2 In vitro antioxidant effect of E. alata extract. ICA iron chelating activity, FRAP ferric reducing antioxidant power, DPPH-SA 1,1-diphenyl-2-picrylhydrazyl-scavenging activity, TAC total antioxidant capacity. The results are expressed as means of three consequetive measurements ± standard deviation.

Antiproliferative activity of E. alata extract

Ephedra alata extracts have been reported to have antiproliferative effects against several cancer cell lines, such as CMF-7 and MDA-MB-231 breast cancer, HeLa cervical adenocarcinoma, HepG2 liver cancer, PC-3 prostate cancer, and PANC-1 pancreatic cancer17,64–67. The current investigation of the cytotoxic effects of E. alata growing in the salty area of the central region of Saudi Arabia is part of the novelty of the present work. The findings demonstrated in Fig. 3 indicated the dose-dependent inhibition of cancer cells, MDA-231 and A549, in response to exposure to E. alata extract. The viabilities of MDA-231 and A549 cells were substantially and proportionally reduced with increasing plant extract's concentration. The cytotoxic IC50s for both cell lines, MDA-231 and A549, were calculated as 25.31 and 39.81 µg/mL of the E. alata extract, respectively. Current cytotoxic findings indicated the potential anticancer effects of the plant. The standard cytotoxic compound, doxorubicin, was used as positive control, and induced IC50s of 1.984 and 2.302 µg/mL against MDA-231 and A549 cells, respectively. The plant cytotoxic effects could be attributed, but not limited, to specific phytochemicals identified in Table 2, such as chlorogenic acid, caffeic acid, syringic acid, isorhoifolin, rosmarinic acid, apigenin, and quercetin, which were identified in the plant extract at higher relative concentrations and have been reported in several potent cytotoxic plants, such as Suaeda vermiculata and Pulicaria undulata, which grow under the same environmental conditions7,68. In addition, the cytotoxic effects of the plant could be attributed to the presence of coumarins, umbelliferone, and 4-methylumbelliferone, which have been reported to have anticancer activities69,70 at a relative concentration of 6.57% (Table 2).Figure 3 The Log-dose response curve of Ephedra extract on MDA-231 and A549 cell lines. The results are expressed as means of three measurements ± standard deviation.

The extract of E. alata was tested in two different cancer cell lines, MDA-231 and A549, for ability to enhance and/or decrease cell migrations in the scratch assay. Therefore, this study was conducted to evaluate the effects of the plant extract on scratch closure, which is the ability of the cells to migrate and close a scratch, or a wound. In the case of MDA-231 cells (Fig. 4A–D), the scratch size increased by 55% within 24 h. This suggested that the plant extract limited, or inhibited the migration, and closure of scratch in MDA-231 cells. Essentially, the cells treated with the plant extract were less efficient in closing the scratch than the untreated cells. Similarly, in the case of A549 (Fig. 4E–H), the scratch size increased by 4% within 24 h. This indicated that the plant extract also limited the scratch closure in A549 cells, although to a lesser extent than in MDA-231 cells. Overall, the results suggested that the plant extract had an inhibitory effect on the migration and closure of scratches in both MDA-231 and A549 cells (Table 5).Figure 4 Scratched area (mm2) of MDA-123 (A–D) A549 (E–H) Cell line before and after treatment with E. alata extract. (A) Untreated MDA-123 cells in day 1, (B) Untreated MDA-123 cells in day 2, (C) E. alata extract treated MDA-123 cells in day 1, (D) E. alata extract treated MDA-123 cells in day 2, (E): Untreated A549 cells in day 1, (F) Untreated A549 cells in day 2, (G) E. alata extract treated A549 cells in day 1, and (H) E. alata extract treated A549 cells in day 2.

Table 5 Percentage of closures of MDA-123 and A549 cell lines before and after treatments with E. alata extract.

Cell lines	MDA-123	A549	
Parameters	% Closure	% Closure	
Control	16.15 ± 0.21	39.90 ± 0.15	
E. alata	− 55.00 ± 0.02	− 0.46 ± 0.07	
The closure percentages were calculated from two measurements and expressed as means ± standard deviation.

MDA-231 and A549 cells were subjected to a colony assay test after treatment with the E. alata extract. In the case of MDA-231 cells, 103 colonies were observed in untreated cells. However, in the plate treated with E. alata extract, the number of colonies was diminished (Fig. 5A,B). These results suggested that E. alata had a significant inhibitory effect on the proliferation of MDA-231 cells, as indicated by the absence of visible colonies. The results indicated a potential cytotoxic effect of the plant extract against MDA-231 cells, which is consistent with the results of the MTT assay. In A549 cells, 96 colonies were observed in the untreated plate. However, in the plate treated with the E. alata extract, the number of colonies decreased to six (Fig. 5C,D). While the reduction in colony count was not as drastic as in the case of MDA-231 cells, which is still indicated a substantial inhibitory effect of the E. alata extract on the growth of A549 cells. The results in Table 6 and Fig. 5 are consistent with the MTT results shown in Fig. 3 for the reduction in the viability of A549 cells after treatment with the E. alata extract. The colony assay results indicated that the E. alata extract has anti-proliferative effects on both MDA-231 and A549 cell lines, with varying degrees of potency, indicating the potential anticancer effects of the plant.Figure 5 Colony of MDA-231 and A549 cell lines after 14 days of seeding., (A) Untreated MDA-231 plate, (B) MDA-231 plate treated with the IC50 concentration of E. alata extract, (C) Untreated A549 plate, (D) A549 plate treated with the IC50 concentration of E. alata extract.

Table 6 Colonies number of MDA-231 and A549 cell lines after extract treatment and untreated cells.

Treatment	MDA-231	Colony count of A549	
Control untreated plate	103A	96A	
E. alata extract	0B	6B	
The results were obtained from two trials. Values are expressed as means ± SEM. Statistical significance was performed using one-way ANOVA The mean values that do not share a superscript letter (A,B) are significantly different (p < 0.0001) using Tukey’s multi-group comparisons.

Docking analysis

In support of our in vitro findings, molecular docking simulations were conducted to elucidate the molecular mechanisms underlying the anticancer activity of the identified compounds against CAIX. To validate the docking procedure, the co-crystal structure of Y0R was redocked, yielding an RMSD value of 0.81 Å, as depicted in Fig. 6. RMSD values below the 2 Å threshold confirmed the reliability of the docking protocol for subsequent analyses71. As presented in Table 7, the Ligand Binding Energies (LBEs) and binding interactions of the constituents, along with Y0R as a control molecule, were examined. Notably, isorhoifolin, chlorogenic acid, apigenin, and rosmarinic acid exhibited the lowest binding energies of − 8.41, − 6.64, − 6.32, − 6.26 kcal/mol, respectively. The co-crystallized ligand (Y0R), on the other hand, exhibited binding energy of − 7.72 kcal/mol. Y0R demonstrated hydrogen bond interactions with GLN92, HIS94, HIS96, and THR200, along with hydrophobic interactions involving VAL130 and LEU199 AA (amino acids residues (Fig. 7A). Conversely, isorhoifolin interacted with TRP9, LEU91, GLN92, GLU106, and PRO202, while participating in hydrophobic interactions with HIS94, VAL130, MET547, and LEU199 residues (Fig. 7B). Moreover, chlorogenic acid interacted with TRP9, GLN71, GLU106, and PRO202, and hydrophobic interactions with one AA residue (LEU199) (Fig. 7C). Apigenin engages in hydrogen bonding interactions with GLU106, HIS119, and THR201, whereas HIS94, VAL121, and LEU199 participated in the hydrophobic interactions (Fig. 7D).Figure 6 Solid ribbon representation of CAIX (PDB ID: 5FL6) with cocrystal (gray) and redocked (blue) Y0R. (Prepared by BIOVIA Discovery Studio Visualizer).

Table 7 Lowest binding Energy in Kcal/mol of the identified plant constituents.

#	Compound	Binding energy (Kcal/mol)	
1	Isorhoifolin	− 8.41	
2	Chlorogenic acid	− 6.64	
3	Apigenin	− 6.32	
4	Rosmarinic acid	− 6.26	
5	3,5-Dimethoxy-4-hydroxyacetophenone	− 5.85	
6	4-Methylumbelliferone	− 5.98	
7	Umbelliferone	− 5.62	
8	Syringic acid	− 5.06	
9	Vanillic acid	− 4.81	
10	Co-crystallized Ligand (Y0R)	− 7.72	

Figure 7 Stick representation of (a) co-crystallized ligand (Y0R), (b) Isorhoifolin, (c) Cholorogenic acid and (d) Apigenin docked within CAIX (PDB ID: 5FL6) binding site. (Prepared by BIOVIA Discovery Studio Visualizer).

Conclusion

In the current study, the phytochemical profile and associated bioactivities of E. alata, a plant known for its tolerance to the high salinity and drought challenging environmental conditions of the Qassim region, were investigated. These findings indicated that the plant was enriched in polyphenols, which are expected to play an important role in its remarkable ability to adapt to the high salinity and drought conditions prevalent in the plant habitat , and the surrounding environment of the region. The plant extract exerted potential antioxidant and anticancer activities, as demonstrated by the results of in vitro assays. In addition, the extract reduced the viability of MDA-231 and A549 cancer cells and inhibited their migration and colonization. The molecular docking simulations conducted in this study provided valuable insights into the potential mechanisms underlying the anticancer activity of the identified compounds, particularly isorhoifolin, chlorogenic acid, apigenin, and rosmarinic acid. These compounds demonstrated strong binding affinities for the CAIX enzyme, suggesting their potential as therapeutic agents for cancer treatment. The docking results further supported the selection of CAIX as a standard for predicting anti-cancer activity and a therapeutic agent, as it exhibited significant binding interactions with the tested compounds which have been identified from the plant extract. Further studies, including in vitro and in vivo investigations, are warranted to fully elucidate the anticancer potential and clinical efficacy of these compounds.

Supplementary Information

Supplementary Information.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-69607-w.

Acknowledgements

The authors are grateful for institutional support.

Author contributions

Conceptualization, H.A.M. and A.S.A.; methodology, H.A.M., R.S., E.S.-E., B.O.A.-L., M.M.A., S.M.S., T.A.K. and A.I.; software, R.A.K., B.O.A.-L., M.M.A., and H.A.M.; validation, R.S., E.S.-E., A.I. and H.A.M.; formal analysis, H.A.M.; investigation, H.A.M., R.S., E.S.-E., B.O.A.-L., M.M.A., A.I., S.M.S., and R.A.K.; data curation, S.A.A., A.S.A., and H.A.M.; writing—original draft preparation, S.-E, B.O.A.-L., R.S., and H.A.M.; writing—review and editing, S.-E., B.O.A.-L., R.S., R.A.K., A.I. and H.A.M. All authors have read and agreed to the published version of the manuscript.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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.
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