The many pollutants detected in water represent a global environmental issue. Emerging and persistent organic pollutants are particularly difficult to remove using traditional treatment methods. Electro-oxidation and sulfate-radical-based advanced oxidation processes are innovative removal methods for these contaminants. These approaches rely on the generation of hydroxyl and sulfate radicals during electro-oxidation and sulfate activation, respectively. In addition, hybrid activation, in which these methods are combined, is interesting because of the synergistic effect of hydroxyl and sulfate radicals. Hybrid activation effectiveness in pollutant removal can be influenced by various factors, particularly the materials used for the anode. This review focuses on various organic pollutants. However, it focuses more on pharmaceutical pollutants, particularly paracetamol, as this is the most frequently detected emerging pollutant. It then discusses electro-oxidation, photocatalysis and sulfate radicals, highlighting their unique advantages and their performance for water treatment. It focuses on perovskite oxides as an anode material, with a particular interest in calcium copper titanate (CCTO), due to its unique properties. The review describes different CCTO synthesis techniques, modifications, and applications for water remediation. It results from the release into the environment of harmful substances that can cause harm to human health, wildlife and the natural environment. Environmental pollution, particularly water pollution, is a significant issue worldwide and its effects can be severe and long-lasting ,2,3,4. PThis review is composed of three main sections. The first section describes water pollution. The second section presents AOPs for water treatment. The third section describes perovskite materials, their synthesis and modification techniques, and their potential applications in water treatment.Water pollution is caused by the introduction of harmful substances or contaminants  into water bodies. The sources of water pollution can be natural or human-made, including industrial activities, agriculture, urbanization, and sewage disposal ,14,15. WPharmaceuticals are commonly used to prevent and treat diseases in humans and animals. They are considered emerging contaminants due to their persistent nature and potential to harm aquatic ecosystems ,23. TherIn 2021, a global-scale study on the active pharmaceutical ingredient (API) pollution of the world’s rivers was published . This sThese results are in agreement with a previous study at different groundwater locations in France published in 2011 . SpecifiDue to its low cost and availability without prescription, low risk of side effects and wide use as self-medication, paracetamol is the most used drug worldwide for minor pain, headache and fever ,36,37. PThese findings stress the need of effective water treatment technologies to reduce the presence of pharmaceuticals in wastewater and mitigate their potential effects on the environment and human health.Water pollution, specifically from emerging pharmaceutical contaminants, is a worldwide problem. However, the methods commonly used in wastewater treatment plants such as filtration, coagulation and sedimentation may not completely remove such pollutants, leading to their accumulation in surface water and groundwater ,42. Ther3 and UV/H2O2) based on UV sources for activation, catalytic AOPs  that require a catalyst, and ozone-based AOPs. These AOPs have been assessed in many studies to remove paracetamol from wastewater  based on the application of electrical energy, physical AOPs that rely on sound waves (ultrasound and microwave), UV-based AOPs  pollutant diffusion from the solution to the anode surface; and (ii) pollutant oxidation .In this process, a strong oxidizing agent, produced at the anode surface via an electrochemical process, diffuses into the bulk solution to oxidize and destroy the pollutant .Electro-oxidation is influenced by the anode composition and operation conditions, such as pH, temperature and currents density .2, and Ti/SnO2) on the degradation of 1 mM paracetamol at an applied current of 500 mA [−1. Several electrode types, including graphite, SnO2, RuO2 and TiO2, perovskite and BDD, have been investigated and all showed significant oxygen transient progression and ability to produce enough HO● radicals [The choice of anodic material is critical for electrochemical reactions and significantly influences the electrocatalytic degradation efficiency. A good anode must display high electrical conductivity, catalytic selectivity and activity, high physical and chemical stability, and low cost. Waterston et al. analyzed the effect of different anodes (boron-doped diamond (BDD), Ti/IrOf 500 mA . They foradicals ,57,58.2. Paracetamol degradation is also influenced by the electrolyte concentration. Indeed, Periasamy et al. used three different initial concentrations of Na2SO4  in an electrocatalytic system with a graphite anode as a working electrode [2SO4, 90% of paracetamol was removed in 240 min (current density of 5.1 mA/cm2) due to the formation of sulfate radicals and stable oxidants  on the electro-oxidation process efficiency using BDD as an anode . At a cusolution e. After Electro-oxidation has been used in different studies due to its numerous advantages: (i) easy operation with simple equipment; (ii) environmentally friendly process with clean reagents (electrons) and without additional chemicals; (iii) versatile technology that can be used for the removal of a wide range of contaminants, including organic pollutants, heavy metals, and microorganisms; and (iv) need of lower pressure and lower temperatures compared with non-electrochemical alternatives ,62,63. T2-based materials have been extensively studied for their photocatalytic applications, there has been a growing interest in exploring ternary and other complex oxide systems as alternative photocatalysts. However, TiO2 has limitations, such as low sunlight absorption and limited recovery and fast recombination of photogenerated charges. The use of TiO2 perovskites, which can be modified by doping, is a promising method for reducing these limitations [3 perovskite materials to degrade rhodamine B under visible light [3 doping improved charge separation, and increased the photocatalytic degradation by 40% compared with MnTiO3, as shown in Photocatalysis is recognized as an environmentally friendly process referred to as “green technology”. It operates by utilizing the energy from light to activate a semiconductor material. When the photocatalyst is exposed to light, it absorbs photons and generates electron–hole pairs. The excited electrons and holes can participate in various redox reactions with surrounding molecules, including the degradation of pollutants ,66. Whilitations . Dong etle light . MnTiO3 4●−) are produced to degrade organic and inorganic contaminants in wastewater [Sulfate-radical-based AOPs have also been widely studied. In this catalytic AOP, sulfate radicals  activation are described in the following sections and summarized in Heat (>60 °C) is used to activate PMS and PDS by breaking the peroxide bond to generate sulfate or mono-sulfate radicals as follows ,75,76:S2Thermal activation of PMS/PDS has been used to degrade different emergent contaminants, such as fluoroquinolones , tetracyDifferent radiation types can be used to activate PDS and PMS, such as ultraviolet (UV), gamma rays, and ultrasound ,83.UV irradiation is a cost-effective activation system. It involves a photochemical process that results in the cleavage of the peroxide bonds, producing sulfate and hydroxyl radicals .PMS + hSimilarly, the general reaction for PDS activation by UV radiation is:Dibene et al. evaluated the importance of coupling UV and PDS for paracetamol degradation . They obUltrasonic radiation also can activate PDS/PMS through peroxide bond cleavage, as described for UV irradiation (Equations (3) and (4) but by changing hν with ultrasound).2+, Co2+, Mn2+, Cu2+, and Ni2+) for drug removal from water (Equations (5) and (6)).PMS/PDS can also be activated using various metal ions  to mineralize atrazine (The best performance (PMS activation) was obtained with Co2+ . These m2+ . Li et a removal . They shatrazine a.2O4 and MnFe2O4 for paracetamol degradation (10 mg/L). Both catalysts efficiently degraded paracetamol via PMS activation by Mn2+-Mn3+/Fe3+-Fe2+ recycle and Co2+-Co3+/Fe3+-Fe2+ [Tan et al. assessed PMS activation by CoFee3+-Fe2+ .PDS and PMS activation are influenced by the solution pH. At alkaline pH, PDS forms sulfate radicals that are then transformed into hydroxyl radicals, as described by Equations (7)–(9) .S2O82− Qi et al. first reported the pathway of peroxymonosulfate activation by alkaline conditions . The resCarbon-based materials also can activate PDS/PMS and display several advantages, particularly a large specific surface area and cost-effectiveness. PDS/PMS activation is facilitated by the donation of electrons from carbon-based materials following Equations (10) and (11).Yao et al. showed that PDS alone cannot degrade p-chloroaniline , and actTo decrease the amount of energy needed for electro-oxidation, the synergistic effects of hydroxyl and sulfate radicals can be combined in electro-oxidation to optimize PMS/PDS activation . As acti5−/S2O82−) with high activity for the degradation of pharmaceutical compounds [For anode activation, a non-radical route of PMS/PDS activation leads to the formation of a special transition state  and the sulfate-radical-based advanced oxidation process (AOP). EO utilizes a power supply, cathode, anode, and electrolyte to achieve water treatment. The performance of these methods is influenced by various parameters, including operating conditions and anode materials. In addition, the activation of sulfate radicals has been explored in this review. Among different activation methods, hybrid activation has demonstrated the highest removal efficiency for degrading trace contaminants. Notably, anode materials play a crucial role in activating the EO-PMS system. Extensive studies have emphasized the impact of transition metals present in the anode on the effectiveness of the hybrid system.3Ti4O12 is highlighted due to its unique combination of two oxide materials and the numerous advantages it offers. Overall, through the exploration of electro-oxidation and sulfate-radical-based AOPs, along with the significance of anode materials and the potential of perovskite structures like CaCu3Ti4O12, this review sheds light on advanced techniques for efficient water treatment.This section provides an overview of the perovskite structure, which serves as a promising candidate for anode materials. Specifically, the compound CaCu3 (CaTiO3): A and B are cations and X are anions . The radius of the A ions is larger than that of B metal ions [2BB’O6 or AA’B2O6 in which two different cations occupy the B site in the cubic lattice  is a complex double-perovskite crystal structure with a cubic group space Im-3. In CCTO, Ca2+ and Cu2+ ions are on the A and A’ sites, respectively, and Ti4+ ions occupy the B site [4+ and open-shell Cu2+ ions into the structure. A Cu2+ presence causes a Jahn–Teller distortion that leads to a square planar structure in the TiO6 octahedra [Perovskite materials can be classified in two categories: single and double perovskite structures. The formula of single perovskite a is ABX3tal ions ,99,100. tal ions ,102. Howtal ions . The for lattice b. A catielectric . In doubelectric . For inse B site ,107. CCTctahedra ,111,112.CCTO can be synthesized using the solid state, wet chemical, combustion and microwave-assisted methods.3, CuO, and TiO2) are mixed in the appropriate stoichiometric ratio with or without a suitable liquid (acetone or ethanol) using ball-milling to obtain a fine powder. Ball milling relies on the application of mechanical force to the precursor material with grinding balls that are agitated by rotating the milling jar at a constant rate  to prepare CCTO. After calcination at 900 °C in air for 3 h, they obtained a pure and homogeneous CCTO powder without impurities [The mechanical reaction can be influenced by different factors, thus also affecting the properties of the obtained CCTO. These factors include the grinding time and speed and the ball/powder ratio ,118,119.nd balls . After 1purities ,122.As an alternative to the solid-state reaction method, chemical techniques, such as sol–gel, hydrothermal, and co-precipitation, allow producing CCTO powders with enhanced purity, particle size distribution, and reactivity. Consequently, these powders require much lower sintering temperatures.For the sol–gel method, metal alkoxides, such as calcium nitrate, copper(II) nitrate, and titanium(IV) isopropoxide, are used as precursor materials. They are hydrolyzed and condensed in the presence of a suitable solvent  in controlled conditions . The resulting gel is dried, calcined, and sintered to produce the final CCTO product b 123,12,125.Liu et al. used the sol–gel method to prepare fine CCTO powders. First, they dissolved tetrabutyl titanate, calcium nitrate, and copper(II) nitrate (in stoichiometric amounts) in ethanol . Second,The co-precipitation method, described in 2, TiCl3, and CuCl2·2H2O) in water and then added them to the precipitating agent  [Barbier et al. prepared CCTO using this method. First, they dissolved metal chlorides (CaClethanol) . This waKumari et al. produced CCTO using the co-precipitation method, as explained by Equation (15) :CaCl2 + 2) and copper nitrate trihydrate (Cu(NO3)2·3H2O) in water, and then added a titanium tetrachloride (4TiCl4)-HCl saturated solution. After stirring, they added the obtained solution, drop by drop, to an aqueous solution of 0.8 M NaOH that was stirred at a constant speed. This resulted in a milky blue color solution. Then, they washed the obtained precipitation, followed by pulverization into a fine powder and calcination to obtain the CCTO powder.Briefly, they dissolved calcium chloride (CaClThe combustion synthesis method (or self-propagating high-temperature synthesis method) is a cheap approach to fabricate homogeneous nanopowders and single-phase materials, such as ceramics, catalysts, composites, alloys, intermetallics, and nanomaterials. The method is based on a self-sustaining solid flame-combustion reaction for a short period, and does not need the high reaction temperatures that are typically required for ceramic synthesis. Instead of placing the sample in a furnace, high temperature is achieved via an exothermic chemical reaction between a fuel and an oxidant present in the precursor solution that maintains the high temperature for the time required to form the final product . During this chemical reaction to produce heat, a large amount of gas is released, leading to the synthesis of a nanosized, porous, and foamy product ,132.3 and CuO, that can affect its properties. Additionally, the prolonged heat treatment caused the formation of CuO that led to an increase in the grain size to 600 nm [Kumari et al. used metal nitrates, titanium isopropoxide, anhydrous citric acid, and acetyl acetone as raw materials. The auto-combustion involved mixing citric acid dissolved in de-ionized water with the titanium precursor dissolved in acetyl acetone. This was followed by an addition to the titanium solution of copper nitrate trihydrate and calcium nitrate tetrahydrate dissolved in de-ionized water. The solution was stirred and after the addition of ammonia, it was heated to allow for water evaporation and the formation of a viscous blue gel. Then, a porous structure was obtained by heating the gel at 450 °C for 15 min, followed by grinding into a fine powder and heating at 950 °C for 8 h to obtain oxides. The obtained CCTO powder contained some impurities, such as CaTiOo 600 nm .Microwave energy may be used at the place of calcination to prepare pure-phase CCTO powders. The microwave heating process generates heat rapidly and uniformly throughout the reaction vessel, resulting in a shorter reaction time and lower processing temperature. Microwave-assisted synthesis relies on the heating effect generated by the microwave energy. This effect is primarily due to the interaction of microwaves with the polar molecules present in the precursor solution ,134.3, CuO and TiO2) in a ball mill for 24 h and pressed them into pellets [Yu et al. used the microwave-assisted solid-state method to fabricate CCTO pellets. Briefly, they mixed the precursors  play a crucial role in determining its properties. Sol–gel methods produce large amounts of CCTO with controlled composition, but require long reaction times, expensive starting materials and the production of secondary phases due to the used solvent ,137. ComIn addition to the synthesis parameters, researchers have also explored various methods to modify CCTO properties, particularly doping with different metal ions and changing the atmosphere treatment. Metal doping can alter CCTO electronic and structural properties and improve its performance in various applications. Changing the atmosphere treatment, such as oxygen, nitrogen, and hydrogen, also significantly affects CCTO properties. These modifications have shown promising results for improving CCTO performance in various applications, making it a promising material for future research and development.Doping is a commonly used method to modify CCTO properties . Various types of dopants, such as Fe, Co, and Mn, have been tested. Indeed, the dopant type and concentration can significantly influence CCTO properties, and the optimal doping conditions may vary in function of the intended application. CCTO can be doped with different elements (one or two elements) and at different sites  to modify its features. 2O, TiO2 and CaTiO3. Zaho et al. [3, and TiO2 peaks, confirming CCTO decomposition upon nitrogen treatment.CCTO can also be treated in different atmospheres, such as oxygen, nitrogen, and hydrogen, to modify its properties. However, the literature on this topic is relatively limited, and the effects of these treatments are not fully understood. Some articles reported promising results in terms of enhancing CCTO specific properties, but more research is needed to determine the optimal conditions for each application ,154,155.o et al.  studied 3, decreased the direct current loss, and increased the grain boundary activation energy to 0.85 eV.In energy storage applications, an oxidizing atmosphere increased the CCTO storage energy density from 7.7 to 26.5 kJ/mTo summarize, CCTO proprieties can be modified by different methods. CCTO can be doped with different metals  to modify its resistance, grain size, and dielectric constant. These modifications improve its performance for various applications. Changing the atmosphere treatment also significantly influences CCTO properties. The atmosphere treatment leads to CCTO decomposition into other heterostructure composites that can be used in many applications. The next section presents some applications of CCTO materials.−5 mol L−1 tetracycline solution was degraded in the presence of 40 mg CCTO [CCTO is a ceramic material that displays a high dielectric constant, low dielectric loss, high temperature stability, and nonlinear current–voltage characteristics ,111,112. mg CCTO  after 30CCTO materials can also be used in hybrid systems and for the activation of sulfate radicals. Zhu et al. studied ibuprofen degradation in the presence of CCTO powder . They usAfter confirming the efficiency of CCTO materials in the laboratory for degrading different pollutants, it will be important to investigate their performance using real water samples. Indeed, real samples may contain other impurities and contaminants that can affect the performance of CCTO materials. These impurities and contaminants present in real water can include organic matter, heavy metals, inorganic ions, and various organic pollutants. The presence of such substances can introduce challenges and complexities in the degradation process, potentially affecting the reaction kinetics and overall performance of the CCTO materials ,168. HenTo further enhance the practical applicability of CCTO materials in water treatment, it is important to investigate their performance in continuous processes at a larger scale. Lab-scale experiments provide valuable insights into the material’s efficiency, but translating these results to continuous operations is essential for real-world implementations ,170. ScaDoping CCTO with cobalt offers several advantages for water treatment such as improving the activation of sulfate radicals. However, this approach is limited by cobalt leakage into the solution and CCTO membrane instability. To address these limitations, the membrane can be stabilized using various methods, such as a thin coating of the membrane surface by an atomic layer deposition or by a thermal treatment of the membrane after its use.In addition, CCTO has many interesting advantages for numerous applications, specifically for water treatment and in various fields. Therefore, it is important to further study the potential of CCTO membranes in different conditions, such as electro-Fenton, or by coupling different processing techniques.2 and copper. TiO2 is the most promising phase for photocatalytic applications, particularly when using UV light. Moreover, the presence of copper in CCTO facilitates the shift in TiO2 adsorption to visible light wavelengths [Electro-oxidation processes require a significant amount of electrical energy to drive the oxidation reactions. This could be addressed by using solar energy. CCTO contains TiOelengths . Therefo2, CaTiO3, and CuO. CCTO materials can reduce emerging compounds using different systems, such as photocatalysis, PMS activation by light, and photo-electrocatalysis. The future trend in the field of water treatment applications is to develop continuous systems for water treatment by exploring synergistic approaches (electro-oxidation/PMS) to enhance the overall efficiency and degradation capabilities. In addition, extensive studies should be performed on the performance of CCTO materials and technologies using real water samples to take into account the presence of impurities and variations in water composition. Better understanding the degradation mechanisms and the formation of harmful by-products will help to design targeted treatment strategies and optimize the performance of water treatment systems.This review described the harmful effects of water pollution on human health and the environment and the detection of various pollutant types  in wastewater. Among them, chemical pollution, specifically emerging pollutants, is particularly concerning due to the persistence of such molecules. Paracetamol presents the highest concentration in surface water worldwide. Similarly, in France, paracetamol was the most commonly detected (27%) emerging pollutant. AOPs could be part of the solution to this issue because they can mineralize persistent compounds. Many studies showed that different AOPs can remove paracetamol. In this review, particular attention was focused on electro-oxidation and the parameters affecting its performance. Sulfate-based AOPs and their activation methods for the formation of sulfate radicals were also discussed. The hybrid activation process (electro-oxidation/PMS) combines the synergic effect of the two systems for the degradation of water pollutants. In these systems, the anode material strongly influences the performance. This review showed that the CCTO perovskite material has several advantages, such as a high dielectric constant, low dielectric loss, high-temperature stability, and nonlinear current–voltage characteristics. The synthesis method, doping, and atmosphere treatment significantly influence the CCTO performance. Doping decreases the dielectric loss and increases the grain boundary size. Furthermore, nitrogen treatment leads to CCTO decomposition into TiO


Saccharomyces cerevisiae strains, selected from different matrices - CHE-3 (cherry), P4 (sourdough) and TA4-10 (grape must) - on characteristics of Italian Grape Ale (IGA) beers obtained at microbrewery scale. A multidisciplinary approach, combining results from analysis of chemical, volatile and organoleptic profiles of the beers, was adopted to underline the relationships between yeast starter and the quality of final products. Detection volatile organic compounds (VOCs) by Gas-Chromatography coupled with Mass Spectrometry (GC-MS) after extraction carried out by head-space micro-extraction (HS-SPME) revealed that the beer obtained by P4 strain differed from the others for its higher concentrations of esters, alcohols, and terpenes as confirmed by PCA  and Cluster heatmap. Furthermore, sensorial analysis and consumer test showed that this sample differed from others by more pronounced notes of “fruity smell and floral” and “olfactory finesse,” and it was the most appreciated beer for smell, taste, and overall quality. Conversely, CHE-3 was the sample with the lowest concentrations of the identified volatiles and, together TA4-10, showed the highest scores for smoked, yeast, malt, and hop notes. As far as we know, these are the first results on the application of indigenous S. cerevisiae strains in the production of craft IGA beers analyzed through a complex multivariate approach.This study investigated the influence of three  Saccharomyces cerevisiae species. Esters, alcohols, acids, aldehydes, ketones, hydrocarbons, sulfur compounds and volatile phenols are the main volatiles detected in beer, they being able to influence their aroma and flavor both individually and in a synergistic or antagonistic way  recently recognized a new subcategory of special fruit beers, denoted as Italian Grape Ale IGA; . Producestic way . Differeompounds .The use of pure yeast cultures, pioneered by Christian Hansen in the 1880s, greatly improved the consistency and quality of beer. Moreover, the increasing popularity of both the craft beer market  and the S. cerevisiae selected from different matrices, CHE-3 (cherry), P4 (sourdough) and TA4-10 (grape must), were recently applied in brewing trials for IGA production at a laboratory scale; these selected yeast strains resulted potentially able for differentiation and quality improvement of IGA beer production  already tested during laboratory scale trials . Ten hectoliters of wort, prepared by using the commercial extract malt for Pale Ale beer , a yeast differential culture medium .The primary fermentation lasted 10 days, and then the samples were transferred into other steel tanks for the maturation step, performed at low temperatures (10°C) for 1 month. The secondary fermentation was carried out by disposing the samples in 500 mL bottles in the presence of 5 g/L of sucrose for 2 weeks at 10°C. At the end of secondary fermentation, the samples were maintained at 4°C for 2 months in order to allow flavor maturation and stability. Afterward, the beers were evaluated for analytical and aromatic composition.During fermentation, the starter evolution was monitored by yeast viable cell counts on WL nutrient agar medium  and by plate incubation at 26°C for 5 days. In details, the following sampling points were considered: after 24 h of inoculation, at the end of primary and secondary fermentations. For each isolation time, the plates containing a statistically representative number of colonies were counted and around 20 colonies were purified on YPD plates for identification by 5.8S ITS-RFLP analysis.The content of total soluble solids (°Plato) was determined by using a refractometer . For bitterness determination, craft beers were decarbonated and 10 mL of samples were submitted to extraction of bitter substances by using 1 mL of hydrochloric acid 3M and 20 mL of pure iso-octane . After tIBU = 50 × A275where A275 correspond to the absorbance at 275 nm.The beer color, was expressed as European Brewing Convention (EBC) units, determined by a colorimeter  and calculated as follows:EBC color: 25.5 × (A430 -A700)where A430 and A700 correspond to the absorbance at 430 and 700 nm, respectively.®, Loveland, Colorado, US) and expressed in nephelometric turbidity units (NTU).The turbidity was measured in a turbidimeter . The temperature of the column compartment was maintained at 70°C. The flow rate applied was 0.4 mL/min with a run time of 30 min. The phase was represented by 4.0 mM/L H2 ≥ 0.99) based on authentic standards.The concentrations of maltodextrin, sucrose, maltose, maltotriose, glucose, fructose, glycerol, and ethanol were quantified using the same type of column used for organic acids. The mobile phase was represented by deionized water and a constant flow rate of 0.6 mL/min. A run time of 30 min was applied. Sugar detection was carried out through a Refractive Index Detector (RID). Quantification of individual organic acids and sugars was performed directly by the Chem-Station software (Agilent) using a five-point regression curve  by using an Agilent 7890A Gas-Chromatograph, following the protocol previously reported .Splitless mode was selected as injection mode. GC-MS analyses were performed on a GC 6890  coupled to an Agilent MSD 5973 Network detector using a HP-INNOWAX capillary column  as reported by Volatile organic compounds (VOCs) were identified and quantified by Gas-Chromatography coupled with Mass Spectrometry (GC-MS) after extraction carried out by head-space micro-extraction (HS-SPME) according to A trained panel of ten judges between 40 and 65 years of age, experienced in alcoholic beverage sensory evaluation, carried out a Quantitative Descriptive Analysis (QDA) in a room free of noise, odors and with white light. Fifty milliliters of each beer were served to the panelists in crystal goblets at a temperature of 5 ± 1°C. The parameters evaluated by the judges were selected among those found in the literature  and thosThe consumer test was conducted with 42 consumers , with age ranging between 21 and 75 years. The prerequisites for participating in the study were that the individual habitually consumed beer and they were selected on the basis of other criteria, such as not reporting any conditions affecting the senses of sight, taste or smell, The test was conducted in the sensory laboratory from the School of Agricultural, Forestry, Food and Environmental Sciences of Basilicata University using individual sensory booth equipped with sensory data collection software . The test was conducted to assess liking for appearance, odor, taste, and overall liking, using a using a 9-point hedonic scale in which 1 = dislike extremely, 5 = neither like nor dislike and 9 = like extremely . The samOne-way ANalysis Of VAriance (ANOVA) applied to chemical, sensory, and volatolomic data determined significant quantitative differences among samples. Differences were considered statistically significant for p-values of the null hypothesis less than or equal to 0.05. Partial Least Squares Correlation (PLSC) was applied to volatile compound data and sensory analysis data to study their multivariate covariance. PLSC  is a mulS. cerevisiae species in order to confirm that no contamination occurred during the fermentation trials. The theoretical inoculum ratio resulted as planned; in fact, 24 h after inoculation, for all the trials the yeast population ranging between 7.1 and 7.9 Log CFU/mL. At this time, the three indigenous strains reached levels higher than those of the control strain US-05 (7.15 Log CFU/mL); the highest count (7.94 Log CFU/mL) were displayed by the strain CHE-3. At the end of primary fermentation, an increase of yeast population was observed for P4 and CHE-3 strains , a slight decrease to 6.96 Log CFU/mL was observed for the control strain US-05, whereas a 0.7 Log cycles decrease was registered for the strain TA4-10. After the secondary fermentation, the end of monitoring, all the fermentations showed a decrease of yeast levels; the highest value (7.05 Log CFU/mL) was detected for CHE-3 strain, whereas the lowest level was found for the control strain US-05, which was the strain showing the lowest count along all the process. However, the high yeast vitality found during the process for all the starters is crucial for improving fermentation and product quality as it is related to the efficiency and predictability of fermentation. This aspect, together the presence of only S. cerevisiae cells, are pivotal for beer quality, in particular for craft beers, which are more prone to spoilage than beer produced in large-scale breweries, in consequence of absence of pasteurization or sterile-filtration processes.The strains growth was monitored during the fermentation process by a microbiological assay . The viaThe IGA beers were analyzed for the main quality attributes, such as ethanol content, attributes related to turbidity, color and bitterness . The ethAll the beers presented a bitterness  ranging between 8 and 13.3. The lowest values, found in TA4-10 and US-05 samples , were slightly lower than indications given by the Beer Judges Certification Program (BJCP), whereas IBU values significantly higher were found in beers fermented with CHE-3 and P4. Hops are the raw material in beer mainly responsible for beer bitterness. Indeed, it provides α-acids, which during the technological process are transformed into the more bitter iso-α-acids. By considering that the variety and quantity of hop were kept constant among all beer samples, the difference in beer IBU might be correlated to the different capacity of yeast strain used for fermentation to preserve the α-acids from hop. These results enhance the role of starter on beer bitterness, conversely to data reported in other studies in which no differences in IBU were found among beers fermented with different starters . Beer coS. cerevisiae yeast is not able to utilize the dextrins  were found for 2-methyl-1-butanol, 3-methyl-1-butanol and isobutanol. The higher alcohols may influence the sensory profile of beer by giving an alcoholic or solvent-like aroma and a warm mouthfeel , ethyl caproate, ethyl caprylate (sour apple-like flavor and aroma), isoamyl acetate , isobutyl acetate, phenylethyl acetate, and ethyl octanoate . In our samples, ethyl esters and acetates represented the second most abundant group of volatile compounds , rangingPhenyl acetate and ethyl hexanoate were above their perception thresholds for TA4-10 and P4 beers, contributing with rose, honey, and green notes to the aroma of these samples. Among esters, ethyl hexanoate, isoamyl acetate and phenyl acetate were in concentrations above their respective perception thresholds in P4 contributing significantly to the overall aroma . HoweverAcids are the third most abundant group in beers studied, but not statistically significant differences have been detected in the amount found in the different samples. The acids are produced during fermentation process but their amount depend both on yeast activity and on the composition of raw materials .Three terpenes have been found in IGA beers, i.e., linalool, citronellol and nerolidol. In all samples, linalool and citronellol were found in concentrations above their odor thresholds, thus enhancing the aromatic profiles of IGA beers with fruity and floral notes. Moreover, IGA are beer made with a variable percentage of grape must, which is mainly characterized by primary aromatic compounds derived from grapes, such as terpenes, sulfur compounds, pyrazines, and norisoprenoids .Pearson principal component analysis (PCA) was carried out. To highlight the effects of yeast strains on volatile molecules concentration, a Overall, the samples were characterized by different standardized concentrations of the analyzed volatiles. Indeed, US-05, the commercial strain used as control, was characterized by higher production of molecules ranging from styrene to isobutanol; sample obtained with P4 strain showed high values of compounds ranging from phenylethyl acetate and ethyl acetate; however, this beer was characterized by the highest amounts of most detected volatiles, confirming the interesting findings of this strains previously obtained during brewing trials at laboratory scale , 2021. TFinally, CHE-3 beer appeared to be the sample with fewer dominant volatile molecules, including 2-octanone, styrene, isoamyl alcohols, 2-ethyl-1-hexanol, acetoin and 1-hexanol. Our results underscore the ability of the yeast strain to influence the volatolomic profile, from a quantitative point of view, by affecting the sensory perception of notes associated with the detected molecules.In order to investigate the impact of the three cross-over strains on the sensory quality of beers obtained by using malt fortified with grape must, a sensory analysis was carried out. This study offers valuable insight into the sensory response of Italian consumers on this new version of beer, which has a particular aromatic complexity strongly influenced by the combination of malt and grape must.p < 0.05) affect some of the attributes considered, including foam color, amount and persistence of foam, effervescence, olfactory finesse, floral, yeast and fruity smell, acidity, and finally overall quality. Spider plots for the aroma  and taste profiles, reporting the mean values of samples, are shown in The The comparison of the ANOVA results  and the A PCA was applied to analyze the covariance of sensory descriptors judged by panelists . The firp-value = 0.013), whereas for the second components the correlation coefficient was 0.9825 .In our research, Partial Least Square-Correlation (PLSC) analysis was applied to study the relationship between two different groups of data obtained with different techniques, as reported in literature . In partThe P4 strain contributed to the overall correlation between the datasets with higher concentration of ethyl esters , acetate esters , hexanoic acid. These molecules were correlated to the fruity smell and olfactory finesse, indicating the contribution of esters on fruity notes in IGA beers. US-05 was positively correlated with terpenes , ethyl octanoate, ethyl acetate, isobutanol and was characterized by “floral, spicy and caramel” notes and more pronounced “overall olfactory intensity.”Finally, CHE-3 and TA4-10, differed from the others for a positive correlation with few volatiles, including 2-ethyl-1-hexanol, acetic acid, methyl dodecanoate and nerolidol, while, on the sensorial-olfactory level, they displayed high correlation with “smoked, yeast and malt smell” and then a good quality of foam.p < 0.05), for beer sample, gender and age class. No statistically significant interactions were found.In the last step of the research, the beer samples were submitted to evaluation of consumer liking. This is fundamental to identify the reason for the success (or failure) of a product and its market opportunities, giving useful indication about the suitability of a testing beer for full scale production in the breweries. The panel was composed of 69% females and 31% males, with an average consumer age of 44 ± 14 years old and ranged from 21 to 75 years old. The recruited consumers rated the following parameters: appearance liking, taste liking, odor liking and then overall liking. The mean values and standard deviations for each descriptor are reported in A PCA was applied also to liking attributes judged by the consumers . AlthougBoth sensory analysis and consumers’ test indicated that beer obtained by using P4 .S. cerevisiae strains selected from different food matrices for the production of craft IGA beers was investigated for the first time through a multidisciplinary chemistry-multivariate approach. Overall, the strains tested showed the ability to degrade maltose during fermentation and contribute to the qualitative complexity of the final products. In particular, beer obtained by inoculating P4 strain showed high content of some classes of volatile molecules, including alcohols, esters, and volatile acids, as shown by the cluster heatmap and PCA. In agreement with the chemical data, the sensory analysis performed by a trained panel on P4 sample revealed more pronounced fruity and floral notes. On the other hand, TA4-10 differed from the control (US-05) and from the other samples  especially for the visual attributes “quantity and persistence of foam,” for the taste descriptor “acidity” and for its “overall quality.”In the present study, the applicability of S. cerevisiae strains.In terms of consumer acceptability, the overall liking, odor-taste-appearance liking were not significantly different among four beers, but consumer group showed a preference for P4, concerning odor-taste-overall liking, whereas CHE-3 and TA4-10 were preferred for appearance. An interesting result is the highest consumer preference toward beers obtained by using indigenous Moreover, the PLSC clearly indicated how crucial the interaction between analytical and sensory data is in obtaining a snapshot of the overall quality of the fermented beverage, which is useful in standardizing a fermentation process. In conclusion, this work represents the first phase of a wider project for the qualitative improvement of IGA beers, which will industrially employ indigenous cross over starter cultures as potential approach to improve the organoleptic profile of this innovative beverage and to tie it to the culture and history of the production area.The original contributions presented in this study are included in the article/MT: conceptualization, investigation, data curation, writing–original draft, and writing–review and editing. LP: data curation, formal analysis, data acquisition, software, validation, writing–original draft, and writing–review and editing. FG: conceptualization, writing–original draft, and writing–review and editing. CG, ABr, and FGe: data acquisition and writing–review and editing. AC: conceptualization, funding acquisition, investigation, data acquisition, writing–original draft, and writing–review and editing. GS and RP: investigation, methodology, data acquisition, data curation, and writing–review and editing. ABa: data acquisition, writing–original draft, and writing–review and editing. All authors contributed to the article and approved the submitted version.


Nitrogen-containing bisphosphonates lead to the depletion of geranylgeranyl pyrophosphate involved in the mevalonate pathway. The effect of geranylgeraniol (GGOH) on human osteoblast and osteoclast activities suppressed by zoledronate was investigated in this study. The effect of GGOH on human osteoblasts and osteoclasts subjected to treatment with zoledronate was analyzed by assessing cell viability, osteoclast differentiation, resorption ability, gene expression, and protein synthesis. Cell viability suppressed by bisphosphonates in osteoblasts and osteoprogenitor cells was restored with GGOH. Osteoclast differentiation was analyzed by vitronectin receptor immunofluorescence staining, and the addition of GGOH to zoledronate significantly increased osteoclast differentiation compared with zoledronate alone. A trend of reversal of osteoclast resorption by GGOH was observed; however, it was not significant in all groups. The expression of ALP, type 1 collagen, and RUNX2 in osteoblasts was recovered by the addition of GGOH. Only CALCR expression in osteoclasts was significantly recovered by GGOH addition in the zoledronate group. Although the activities of osteoblasts and osteoclasts were not entirely restored, the possibility that the topical application of GGOH in MRONJ patients or patients with dental problems and bisphosphonates might lessen the risk of development and recurrence of MRONJ is shown. Bisphosphonates are pharmacological agents that have been used for over 40 years for the treatment of bone diseases, such as osteoporosis, Paget’s disease, osteogenesis imperfecta, multiple myeloma, fibrous dysplasia, and bone metastasis mainly caused by prostate and breast cancer ,4,5,6,7.Nitrogen-containing bisphosphonates inhibit the actions of farnesyl pyrophosphate synthase, an enzyme involved in the mevalonate pathway, leading to the depletion of geranylgeranyl pyrophosphate (GGPP), an intermediate in the mevalonate pathway ,15. GGPPAlthough clear standards for treatment of MRONJ have not been determined thus far, early surgical interventions are gaining preference among oral and maxillofacial surgeons, as higher success rates for the management of MRONJ have been reported . HoweverThus, this study aimed to investigate the effect of GGOH on human osteoblast and osteoclast activities, including cell proliferation, osteoclastogenesis, resorption ability, gene expression, and protein synthesis, in the presence of various bisphosphonates.2, and the media were replaced with fresh media every three days. The osteoblasts were passaged when they reached 70–80% confluence in a 75 cm2 culture flask  using Accutase solution . Osteoblasts from passages 4 to 7 were used in the experiments and seeded onto appropriate plates at a density of 2 × 104 cells/cm2. The osteoclast precursors were seeded onto appropriate plates for each experiment at a density of 4 × 104 cells/cm2, and human receptor activators of nuclear factor kappa-B ligand  and human macrophage colony stimulating factor  were added to induce osteoclast differentiation.Commercially available human osteoblast cells (PromoCell) and human osteoclast precursors (Lonza) were cultured in their respective culture media (PromoCell and Lonza) at 37 °C with 5% CONitrogen-containing bisphosphonates, namely, Zometa  and Alendron , and a non-nitrogen-containing bisphosphonate, Bonefos , were used for the experiments, and geranylgeraniol (GGOH) purchased from Sigma-Aldrich was supplemented along with bisphosphonate treatment. The alendronate tablet was pulverized and dissolved in phosphate-buffered saline (PBS). Subsequently, the pH of the solution was adjusted, and the solution was sterilized by filtration.4 cells/cm2 and incubated for 24 h. Bisphosphonates, namely, zoledronate (Zol), alendronate (Aln), and clodronate (Clod), were added to the wells  with or without GGOH (10 μM). The above-mentioned range of concentrations was selected, because in vivo concentrations in plasma shortly after zoledronate infusion are approximately 5 μM, and these doses roughly correspond to the standard oncology dose regimens of 1 year and 4 years . The assay was conducted in triplicate, with three replicates for each experiment. After incubation for a duration of 72 h, MTT solution was added to the wells and the plates were incubated for 4 h. Cells were subjected to lysis for 30 min using a lysis buffer , and the optical density (OD) was measured at 570 nm using a spectrophotometric microplate reader .The viability of human osteoblasts and human osteoclast precursors was determined using the MTT colorimetric assay (Sigma-Aldrich). Osteoblasts and osteoclast precursor cells were seeded onto 24-well plates at a density of 2 × 104 cells/cm2, and human receptor activators of nuclear factor kappa-B ligand  and human macrophage colony stimulating factor  were added. The assay was conducted in triplicate. After incubation for a duration of 24 h, bisphosphonates (50 μM) with or without GGOH (10 μM) were added and the plates were incubated for 72 h. Subsequently, the cells were subjected to fixation in 3.7% paraformaldehyde for 20 min at 25 °C and blocked with goat serum  for 30 min at 25 °C. The cells were then incubated with anti-human CD51/61 antibody  for 1 h at 25 °C and with a secondary fluorescent antibody  for 1 h at 25 °C. Images were obtained using a fluorescence microscope  and subsequently merged with the DAPI images for nuclei staining. For each group, fifteen different areas, each measuring 0.01 cm2, were selected, and a cell count was performed using ImageJ software. The proportion of cells with more than two nuclei among the total cell population was calculated.To analyze osteoclast differentiation, vitronectin receptor (VNR) immunofluorescence staining was performed. VNR-positive multinucleated cells (cells with more than 2 nuclei) were considered osteoclasts. The osteoclast precursors were seeded onto 96-well plates at a density of 4 × 104 cells/cm2. The assay was conducted in quadruplicate with two replicates for each experiment. After incubation for a duration of 24 h, bisphosphonates (10 and 50 μM) with or without GGOH (10 μM) were added. Following incubation for 72 h, resorption pits were observed using an inverted phase-contrast microscope and analyzed by measuring the resorbed area using ImageJ software.After incubation of osteoclast precursor cells with M-CSF and RANKL for 72 h as per the methods described above, the cells were counted and seeded onto 24-well hydroxyapatite-coated plates (Corning) at a density of 2 × 10Osteoblasts were seeded onto 6-well plates and incubated with Aln and Zol (50 μM) with or without GGOH (10 μM) for 72 h. Osteoclast precursors were also seeded onto 6-well plates and differentiated into osteoclasts by RANKL and M-CSF stimulation. Thereafter, Aln and Zol, with or without GGOH, were added for the conduction of treatment for 72 h. Subsequently, the cells were harvested using Accutase solution, and mRNA extraction was performed. The amount of extracted mRNA was measured spectrophotometrically at 260 nm. Complementary DNA synthesis was performed using a cDNA synthesis kit  and a thermal cycler . The sequences of primers used in the experiment are listed in Osteoblasts were incubated with zoledronate (50 μM) for 72 h. Subsequently, the cells were subjected to lysis with RIPA buffer (Sigma-Aldrich) containing a phosphatase inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined using a BCA protein assay kit. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins, which were then transferred onto a PVDF membrane, followed by blocking for 1 h. The membrane was then incubated for 24 h at 4 °C with primary antibodies against the following molecules: alkaline phosphatase (ALP), macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor kappa-B ligand (RANKL), and type 1 collagen , RUNX2 , β-actin . Subsequently, they were incubated with anti-mouse and anti-rabbit secondary horseradish peroxidase-conjugated antibodies  for 1 h at room temperature. The immunoreactive proteins were determined using enhanced ECL chemiluminescence and a detection device .U test with Bonferroni correction were used to perform statistical analyses. Two-way ANOVA with Dunnett’s T3 test was used to analyze the osteoclast differentiation count, as the normality of data was proven. All statistical analyses were performed using SPSS . Statistical significance was set at p < 0.05.The Kruskal–Wallis test and the Mann–Whitney p < 0.001). The Zol 50 μM group showed the most considerable decrease among the bisphosphonate-treated groups. The addition of GGOH demonstrated a statistically significant improvement in cell viability in the Aln and Zol groups .Based on the statistical analysis results, a significant difference was observed between the groups in osteoblasts . Bisphosp < 0.001). Zol 50 μM was determined as the most potent inhibitor of cell viability. Cell viability was significantly improved by GGOH supplementation (p < 0.001), except in the Zol 10 μM group (p = 0.089).Regarding osteoclast progenitors, a significant difference was observed between the groups (p = 0.019). The number of osteoclasts decreased with bisphosphonate treatment, and Zol showed the most considerable potency among bisphosphonates, followed by Aln and Clod. The addition of GGOH to Zol significantly increased osteoclast differentiation compared to that observed with Zol alone (p = 0.027). In the Clod and Aln groups, although the differentiation increased with GGOH, the increase was not found to be statistically significant.Osteoclast differentiation was evaluated by counting the number of cells with more than two nuclei, showing positive results for VNR, and by calculating the proportion of such cells among the total cell population . A statip < 0.001). Although a trend of reversal of the resorption of osteoclasts by GGOH was observed, it was not statistically significant in all groups .To analyze the resorption ability of osteoclasts and the extent of osteoclast differentiation, a resorption pit assay was conducted . A signiqRT-PCR was performed to analyze the gene expression of the osteogenic markers and osteoclastogenesis-stimulating genes, including ALP, RUNX2, type 1 collagen, osteocalcin, M-CSF, and RANKL .p < 0.001). The addition of GGOH reversed the effects of bisphosphonates on the expression of the above-mentioned genes (p < 0.001). Although no statistical significance was observed in the gene expression of M-CSF, bisphosphonates decreased the expression and GGOH reversed the action of Zol (p = 0.836). RANKL and osteocalcin gene expression levels increased with bisphosphonate treatment, and GGOH significantly enhanced the expression (p = 0.001).The gene expression of ALP, type 1 collagen, and RUNX2 in osteoblasts significantly decreased as bisphosphonates were administered compared to the control group , TRAP, calcitonin receptor (CALCR), colony stimulating factor 1 receptor (CSF1R), c-Fos, and osteoclast-associated receptor (OSCAR) .p = 0.011). In the case of TRAP and CSF1R, GGOH treatment did not cause a significant difference in expression . The gene expression of RANK and c-Fos in the bisphosphonate-treated group was significantly decreased compared to that observed in the control. The effect of GGOH increased the gene expression of RANK; however, the difference was not statistically significant (p = 0.895). GGOH had a significant effect on c-Fos expression after Zol treatment (p = 0.030).Bisphosphonate treatment significantly increased the gene expression of TRAP, CALCR, CSF1R, and OSCAR compared to the control group. However, only CALCR expression was significantly recovered by GGOH in the Zol group , an enzyme involved in the mevalonate pathway, leading to the depletion of geranylgeranyl pyrophosphate (GGPP), an essential substrate for the post-translational prenylation of small GTP-binding proteins, such as those belonging to Rab and Rho subfamilies ,15. The Certain researchers have focused on the application of geranylgeraniol (GGOH) as a potential therapeutic approach, as it serves as a substrate for GGPP and undergoes conversion to GGPP . A studyDecreased osteoclasts and their suppressed resorption ability under bisphosphonates have a critical effect on the occurrence of MRONJ due to impaired bone turnover. However, they have not been investigated in human osteoclasts, despite the importance of doing so. The present study focused on analyzing human osteoclast differentiation and activities, and the results showed positive effects of GGOH exerted on human osteoblasts and osteoclasts subjected to treatment with Zol. Notably, osteoclast differentiation compromised by Zol treatment was restored to a certain extent, although this level was not sufficient to reach values demonstrated by the control. Given that the expression levels of M-CSF and RANKL reduced by BPS in osteoblasts were also recovered by GGOH, it is assumed that it has the potential to not only promote increased bone formation but also enhance osteoclastogenesis. The addition of GGOH also tends to improve hydroxyapatite resorption. Given that the expression levels of M-CSF and RANKL reduced by BPS in osteoblasts were also recovered by GGOH, it is assumed that it has the potential to not only promote increased bone formation but also enhance osteoclastogenesis and bone resorption in vivo. Gene expression, such as ALP, collagen 1, RANKL, and RUNX2 expression in osteoblasts and C-Fos and CALCR expression in osteoclasts, was significantly recovered with the addition of GGOH. Interestingly, not all genes and proteins were affected. Since protein prenylation is mediated not only by the action of geranylgeranyl transferase, but also by the action of farnesyltransferase for farnesylation of proteins ,34, the Aln and Clod also exhibited suppressive effects on cell survival, osteoclastogenesis, resorption, and gene expression. As Clod is not a nitrogen-containing BP that acts via the mevalonate pathway, it is not surprising that GGOH has no impact on cells treated with Clod. However, in the case of Aln, the effect of GGOH was also not profound. Although 10 μM of GGOH increased the viability of osteoclast precursors treated with Aln, similar to Zol, there was no statistically significant impact on osteoclast differentiation. Considering that the drug potency of Aln is lower than that of Zol, resulting in a lesser effect on bone turnover, higher concentrations of Aln could be comparable to Zol, and supplementation with varying amounts of GGOH might lead to improved results.As demonstrated by the present study, GGOH addition antagonized the effect of Zol and, as a result, the activities of osteoblasts and osteoclasts were restored. However, the effect of GGOH was exerted on a limited number of genes. The Ras superfamily, influenced by FPPS, comprises various subfamilies, including Ras, Rho, Rab, Ran, and Arf , and theTo manage the conditions associated with MRONJ, various adjuvant therapies, including hyperbaric oxygen (HBO) therapy, parathyroid hormone analog teriparatide treatment, and laser treatment, have been reported . However


Migraine, a common primary headache disorder, is associated with various factors such as stress, hormones in women, fasting, weather, and sleep disturbance as well as odors. We aimed to categorize odors associated with migraine and explore their relationships with clinical characteristics. A total of 101 migraineurs answered a questionnaire to determine the odors associated with migraine attacks. We performed factor analysis to explore the common factors of the odors and the relationship between these factors and the clinical characteristics. The factor analysis estimated six common factors: factor 1, fetid odor; factor 2, cooking products; factor 3, oil derivatives and others; factor 4, shampoo and conditioner; factor 5, cleaning products; factor 6, perfumes, insecticides, and rose. Factor 5 also included hair styling preparations, laundry detergent, and fabric softener, usually those with floral fragrances, and factor 5 was more likely to be associated with migraine attacks in patients with chronic migraine than in those with episodic migraine (P = 0.037). Our study showed that odors associated with migraine attacks could be categorized into six groups and suggested that some chemicals were more likely associated with migraine attacks in patients with chronic migraine than in those with episodic migraine. Migraine attacks are believed to be provoked by various triggers such as stress, hormones in women, fasting, weather, and sleep disturbance3. Determining causality in headache triggers identified three basic assumptions, including constancy of the sufferer, constancy of the trigger effect, and constancy of the trigger presentation4. In clinical practice, however, these assumptions are very difficult to identify4. The study of agreement between self-reported triggers and early premonitory symptoms showed that some patient-reported triggers, such as light, sound, food, and skipping meals, were consistent with early premonitory symptoms5. This study has suggested that some patient-reported triggers may represent early brain manifestations of the premonitory phase of the migraine attack5. Therefore, we believe that what is considered to be a trigger is more appropriately considered an associated factor.Migraine is a common primary headache disorder with a prevalence of 8.4% in Japan3. Many studies have shown that odor sensitivity is a fairly specific symptom of migraine12. Although specific, odor sensitivity does not necessarily add further ability to discriminate patients in comparison to existing diagnostic criteria13. In contrast, few studies have reported the types of odors that are associated with migraine attacks16.Odors are also common associated with migraine attacks18. The pathway for each system is the olfactory nerve projection to the olfactory bulb, the trigeminal nerve projection to the somatosensory and insular cortex, and projection from the accessory olfactory bulb to the hypothalamus18. In addition, imaging studies showed that different odors activate different regions of the brain20.There are three anatomical systems for processing odorous stimuli, namely, the olfactory, trigeminal, and pheromone systemsWe hypothesized that different odors are likely to cause headaches depending on age, sex, and type of migraine, such as episodic (EM) or chronic (CM). Therefore, this study aimed to categorize odors associated with migraine and to explore their relationships with clinical characteristics. We used factor analysis to find common factors in odors associated with migraine and cluster analysis to explore the relationship between odors and migraine onset/severity.One hundred two patients were enrolled in this study. However, one patient was excluded because of insufficiently described data. Table The most frequently reported odors associated with migraine attacks were perfume (55.4%), tobacco (47.5%), fabric softener 32.7%), body odor (32.7%), garbage (24.8%), hairdressing products (22.8%), cars (22.8%), and sweat (19.8%) , followed by homes (40.6%), restaurants (28.7%), and hospital waiting rooms (5.0%). No patient reported migraine attacks in the consultation or examination room. The most frequent places besides the questionnaire were on the train (7.9%), in town (5.9%), and in department stores (5.0%).Forty-two percent of the patients used masks, 22.8% used air cleaner, and 21.8% used deodorant spray to prevent odors. In the free-text field, 16.8% of the patients reported moving out of the place, whereas 5.0% reported holding their noses with a handkerchief.The relationships between odors and age, sex, or types of migraine are described below. Two items showed significant age differences with or without odors. The patients with migraine attacks associated with tobacco or soap were significantly younger than those without . Only women had migraine attacks associated with body odor (37%) and garbage (28%) . Fabric softener, sweat, socks, coffee, excrement, vomit, and animals were associated with migraine attacks in significantly more patients with CM than those with EM .Factor analysis was performed on 35 items of odors, excluding three items that were not associated with migraine attacks. The analysis estimated six common factors with reference to the elbow chart Fig. A.Figure 12, we defined factor 1 as fetid odor, factor 2 as cooking products, factor 3 as oil derivatives and others, factor 4 as shampoo and conditioner, factor 5 as cleaning products, and factor 6 as perfumes, insecticides, and rose.The cumulative variance was 53.2%. The factor structure matrix and loading values on the six factors are presented in Fig. The Spearman correlation coefficient test revealed that factors 3 , 1 , and 2  seemed to be particularly important as hidden factors in headache-inducing odors, in that order. CM was likely induced by factor 5 compared with EM (P = 0.037). Factors 2, 3, 4, 5, and 6 had a stronger influence on headache aggravation .21 was 0.471268, suggesting that 3 is the appropriate number of clusters. The clusters were plotted in the three-dimensional space, with the axes calculated using principal component analysis consisting of the six factors  and mental state, i.e., people are generally more relaxed while bathing, were mathematically calculated separately was considered.22. Chronification of migraine may increase sensitivity to certain chemicals.Compared with EM, CM was likely to be associated with factor 5. Factor 5 included hairdressing products, laundry detergent, and fabric softener; thus, factor 5 was defined as cleaning products. Odors in cleaning products arise due to the use of odorant substances such as floral fragrances (rose oxide)18. The hypothalamus is activated in women and men by the smell of an androgen-like and estrogen-like substance, respectively. Body odor is mainly induced by axillary odor, foot odor, and diacetyl decomposed by lactic acid, occurring in the occiput and around the neck23. Axillary odor is caused by a combination of sweat gland secretions. The exact composition of human sweat contains a mixture of different compounds, including androgen-like androstenone, androstanol, and androstadienone25. The reason why body odor was associated with migraine attacks in women only is presumed to be related to the sex-specific effects of pheromones. The main component of garbage is methyl mercaptan, which is not a pheromone23. In Japan, women usually take out the garbage and have more contact with it. This social context could be the reason that garbage was associated with migraine attacks only in women.Non-parametric analysis showed that body odor or garbage was associated with migraine attacks in women only. Savic et al. reported the sex-specific effects of pheromones23. The scent of soap is derived from synthetic fragrances such as floral and aldehyde bouquets24. Therefore, it is unlikely that the chemical substances common to both tobacco and soap were associated with more migraine attacks among younger migraineurs than older migraineurs. We hypothesize that younger people have less exposure to tobacco and soap odors and are, therefore, more sensitive to unfamiliar odors. This can be due to various reasons. First, there is lower exposure to tobacco odor in public places than that in the past, and smoking rates are declining gradually26. Second, the use of solid soap is higher among older people, whereas younger people often use liquid body soaps, which vary from floral to citrus, herbal, and fruit scents. The strength of the odor also varies (from unscented and slightly scented to scented)28.The non-parametric analysis also showed that the patients with migraine attacks associated with tobacco or soap were significantly younger than those with attacks associated with other factors. The chemical substances in tobacco are different from those in soap. Tobacco smoke contains more than 4,000 chemicals, mainly nicotine, tar, carbon monoxide, carbon dioxide, nitrogen oxides, ammonia, hydrogen sulfide, aldehydes, and ketones30. Prospective confirmation is needed to determine whether or not this is an actual association.This study has limitations. It was conducted at a single headache clinic with the role of a regional headache center. This setting limits the generalizability of the results and may introduce a selection bias toward patients with more severe outcomes. The main limitations were the retrospective design of the study and the use of a questionnaire to retrospectively identify potential associations in their cohort. This limitation could lead to misattribution or recall bias. Osmophobia is reported in a large number of patients, and early manifestation of osmophobia may be the reason for a large proportion of self-reported odorants as triggers, i.e., an odorant with an associations vs. early manifestation of osmophobiaIn conclusion, this retrospective study suggests that odors associated with migraine attacks could be categorized into six groups using factor analysis. These results suggest that the chronification of migraine is associated with increased sensitivity to certain chemicals. Further prospective studies are needed to confirm these findings.31, as determined by a headache specialist certified by the International Headache Society. The exclusion criteria were as follows: tension-type headache for > 5 days per month, history of any other primary headache, pregnancy, breastfeeding, cardiovascular or cerebrovascular disease, uncontrolled psychiatric disorder, and drug abuse. The Japanese Red Cross Shizuoka Hospital Ethics Committee approved the study protocol , and the study was conducted in accordance with the Helsinki II Declaration of 1964 with later revisions. Written informed consent was obtained from all participants. The sample size was set at 100 cases based on other studies of odor10.Patients with migraine who consulted the Department of Neurology, Japanese Red Cross Shizuoka Hospital, were enrolled from April 28 to September 8, 2020. All patients fulfilled the criteria of the International Classification of Headache Disorders, 3rd Edition for migraine without aura, migraine with aura, or CM15. The locations were homes, workplaces, restaurants, and hospitals. The questionnaire items for preventing odors were masks, air cleaners, and deodorant spray. Free-text fields were created for all items so that participants could describe odors, locations, and remedies not included in the items.A questionnaire was used to determine whether odors induced migraine attacks, which odors or location induced migraine attacks, and how the participants cope with the odors. The questionnaire included 35 items of odors selected according to previous studies to determine the types of odors that trigger migraine attacksDemographics and relationships between clinical characters and odors were analyzed using t-tests for continuous data with normal distribution, Mann‒Whitney U test or Kruskal‒Wallis test for non-parametric variables, and Fisher’s exact test for classified data using the SPSS Statistics 20.0 . The significance level was defined as P < 0.05. We classified migraine without aura and migraine with aura as EM and compared them with CM.33.We conducted a factor analysis using the maximum-likelihood method with the promax rotation using SPSS Statistics 28.0 (IBM Corp.) to explore common factors of odors that cause migraine. The number of common factors was decided with reference to the elbow chart. The relationship between the common factors and migraine occurrences was tested using Spearman’s correlation coefficient. Furthermore, to investigate the migraineurs’ sensitivities against the common factors calculated by the factor analysis, we performed non-stratified clustering using k-means ++ using Python 3.9.0, scikit-learn 0.24.1, and Matplotlib 3.4.3. The clusters were plotted in the three-dimensional space, the axes of which were calculated using principal component analysisSupplementary Legends.Supplementary Video 1.


Mosquitoes are the primary vector for West Nile virus, a flavivirus. The virus’s ability to infiltrate and establish itself in increasing numbers of nations has made it a persistent threat to public health worldwide. Despite the widespread occurrence of this potentially fatal disease, no effective treatment options are currently on the market. As a result, there is an immediate need for the research and development of novel pharmaceuticals. To begin, molecular docking was performed on two possible West Nile virus target proteins using a panel of twelve natural chemicals, including Apigenin, Resveratrol, Hesperetin, Fungisterol, Lucidone, Ganoderic acid, Curcumin, Kaempferol, Cholic acid, Chlorogenic acid, Pinocembrin, and Sanguinarine. West Nile virus methyltransferase (PDB ID: 2OY0) binding affinities varied from −7.4 to −8.3 kcal/mol, whereas West Nile virus envelope glycoprotein affinities ranged from −6.2 to −8.1 kcal/mol (PDB ID: 2I69). Second, substances with larger molecular weights are less likely to be unhappy with the Lipinski rule. Hence, additional research was carried out without regard to molecular weight. In addition, compounds 01, 02, 03, 05, 06, 07, 08, 09, 10 and 11 are more soluble in water than compound 04 is. Besides, based on maximum binding affinity, best three compounds  has been carried out molecular dynamic simulation (MDs) at 100 ns to determine their stability. The MDs data is also reported that these mentioned molecules are highly stable. Finally, advanced principal component analysis (PCA), dynamics cross-correlation matrices (DCCM) analysis, binding free energy and dynamic cross correlation matrix (DCCM) theoretical study is also included to established mentioned phytochemical as a potential drug candidate. Research has indicated that the aforementioned natural substances may be an effective tool in the battle against the dangerous West Nile virus. This study aims to locate a bioactive natural component that might be used as a pharmaceutical. BesidesThe majority of people who have been diagnosed with the West Nile virus do not experience any symptoms of illness. Fever and other symptoms like headaches, body aches, joint pains, diarrhea, vomiting, or a rash arise in around one of every five infected patients. About one in every 150 infected persons may get a severe disease, which can often be deadly. Over the age of 60, people have the greatest chance of developing this life-threatening disease. Positive cases in New York City had a median age of 62. Those who already have preexisting health problems, such as cancer, diabetes, or hypertension, may be at a greater risk of developing severe further health issues. In extreme cases of West Nile virus, recovering might require several weeks or months, but impairment to the central nervous system can be incurable .The West Nile virus infection should be included in the differential diagnosis, and particular laboratory test findings should raise a high index of clinical suspicion. The IgM-capture enzyme-linked immunosorbent test is the most often applied diagnostic technique. This test has a sensitivity range of 95 to 100% in serum and spinal fluid. The IgM is usually detected in the serum and cerebrospinal fluid by the time the sickness manifests itself. When a person has encephalitis or meningitis, a high IgM of West Nile virus antibodies is undoubtedly indicative of infection; however, the IgM may last for many months to more than a year because humoral IgM antibodies do not penetrate the blood–brain barrier, an intrathecal West Nile virus-specific IgM strongly supports a central nervous system infection .The clinical care of West Nile virus diseases is considered supportive since neither vaccination nor any drug can be used to treat or prevent West Nile virus in humans. Patients experiencing severe meningeal symptoms typically require pain relief for their headaches, antiemetic treatment, and rehydration for the nausea and vomiting associated with their condition .Although, a huge number of people are affecting in recent time, but no authorized medication or potential vaccine is available still to fight the West Nile virus . Therefoin silico research is to develop a drug design strategy to predict the inhibitory activity of a series of natural compounds against West Nile virus with the help of advanced in silico method such as molecular docking, molecular dynamics simulations, ADMET, principal component analysis (PCA), dynamics cross-correlation matrices (DCCM) analysis, binding free energy and dynamic cross correlation matrix (DCCM). The molecular docking studies was conducted to determine the binding affinity of the selected phytocompounds with the target proteins of the West Nile virus and then based on maximum affinity, three most potential compounds were carried out to the molecular dynamics simulations and determine the stability and binding energies of the protein-ligand complexes. Ultimately the results of this investigation have the potential to contribute to the development of novel drugs for the treatment of West Nile virus infection.To development of a novel drug candidate, required more than 10 years of times, huge amount of money and resources . So, the2.2.1.2.1.1.Apigenin (APG) is a flavonoid found in high concentrations in many fruits, vegetables, and Chinese medicinal herbs. It has a wide range of physiological effects, including anti-inflammatory, antioxidant, antibacterial, anti-viral, and blood pressure-lowering properties . Apigeni2.1.2.Resveratrol is a natural chemical produced by certain plants . Resvera2.1.3.in vitro against a few RNA viruses  has been shown to have anti-viral, antihypertensive, anti-cancer, and immune-modulatory effects . Anti-vi2.1.7.It has also been demonstrated that Curcumin prevents the development of several viruses and cancerous cells. Curcumin can block the production of HSV-1’s immediate early genes and the action of the HIV-1 integrase, both of which are required for the virus’s replication . Through2.1.8.Previous reports indicate flavanol Kaempferol and its glycosides have strong anti-viral properties . Kaempfe2.1.9.Since both Cholic acid and metal ions have pharmacological effects, such as antibacterial, anti-viral, antifungal, antimalarial, antitubercular, anti-cancer, spermicidal, and antiallergic, their organometallic complexes were created to have a synergistic effect . It has 2.1.10.in silico investigation has reported that Chlorogenic acid might be effective against SARS-CoV-2 . The virion has a single, plus-sense RNA genome about 11 kilobases in length and is divided into a 5′ untranslated region (UTR). The conserved dinucleotide AG follows the 5′ caps of the flavivirus genome, and the conserved dinucleotide CUOH finishes off the 3′ ends of the genome. The 5′ UTR and 3′ UTR of genomic RNA each have a length of about 100 and 500 to 700 nucleotides, respectively. Viral and cellular proteases co-translate and post-translate the encoded polyprotein into seven nonstructural proteins  and three structural proteins . The nonstructural proteins function primarily as replication complex components to ensure the successful replication of viral RNA. For RNA replication to take place, NS1 must interact with NS4a. Hydrophobic NS2a was shown to have a role in flavivirus particle formation and release. With NS3, NS2b generated a complex essential for NS3’s serine protease activity. A serine protease, 5′-RNA triphosphatase, nucleoside triphosphatase, and RNA helicase are all functions of NS3, a multifunctional protein. Yet, the roles of NS4a and NS4b, two membrane-associated proteins, remain unknown. The NS5 protein is an RNA-dependent RNA polymerase and a methyltransferase that modifies the 5’ RNA cap structure. When a flavivirus replicates, a replication complex is assembled on the 3′ UTR of the genomic RNA during the process of co-translation. When the positive-sense RNA is copied, the resulting negative-sense RNA is still base-paired with the original replicative form (RF). After that, the RF is reused as a starting point for the asymmetric production of more positive-sense RNA  Tilgner.2.3.The West Nile virus infection is a mosquito-borne disease that may infect people, birds, and animals. According to the CDC, the primary host for the virus is birds, and mosquitoes acquire the virus by biting affected birds. Health experts have announced that it cannot be transmitted from direct contact from person to person . Figure 3.3.1.By utilizing vibrational frequency from the DMol3 code of Material Studio 08, a method known as DFT functional was used to accomplish molecular optimization . The mol3.2.To assess their drug likeliness, we examine the ADMET  pharmacological characteristics of chosen compounds . With th3.3.The crystal structure of several west Nile virus strains was retrieved from the Protein Data Bank (PDB) of the RCSB Protein online portal . Proteinvia the use of a computer approach known as “in silico molecular docking”  target receptor. The simulation system was set up using the CHARMM-GUI web-based graphical interface and produced the force field for both the ligands and the proteins . The six-ligand compound can successfully pass the Lipinski rule of five without any violations  have revealed that drugs like Apigenin, and Curcumin, had the greatest binding score. On the other hand, compounds such as Ganoderic acid, and Cholic acid have been found to dock with the West Nile virus envelope glycoprotein (PDB ID: 2I69) with the highest binding score. So, the protein-ligand interaction valuation of these mentioned compounds was carried out by BIOVIA Discovery Studio 2016 v16.1.0 and Pymol v2.4.1 software . CurcumiBesides, Un-ionized medications are administration into the body more effectively than their ionized counterparts, which are also known as charged drugs. A drug that is a weak acid will be absorbed predominantly in the acidic medium if it is administered orally; on the other hand, a drug that is a weak base will be captured in the alkaline system of the small intestines if it is administered orally . In 6C, ionizabi4.4.The molecular dynamics simulation was launched to comprehend the stability of the complex in depth. This approach is essential for understanding the structure–function relationship of macromolecules. In this study, Apigenin, Curcumin, and Ganoderic Acid were determined to be the best leaders among all the natural compounds evaluated due to their consistent performance in terms of docking score and protein-ligand interactions. To validate this further, the docked complexes of selected compounds, i.e., apigenin and curcumin with West Nile virus methyltransferase (PDB ID 2OY0) and Ganoderic acid with West Nile virus envelope glycoprotein (PDB ID 2I69), were studied for the complexes’ stability and intermolecular interaction with respect to time by 100 ns molecular dynamics (MD) simulation. When simulations were completed, the root mean square deviation (RMSD), root mean square fluctuation (RMSF), the radius of gyration (Rg), H-bond analysis, and principal component analysis (PCA) were determined for each frame of the trajectory.4.4.1.RMSD refers to the occurrence of deviations that were noticed during the evolution of the simulation . FurtherDerived from a 100 ns MD simulation, the RMSD of the ligand, protein backbone, and complex RMSD values for apigenin, curcumin, and Ganoderic acid are displayed. The RMSD of the protein backbone is displayed in red. The complex RMSD (root-mean-square deviation) is depicted in black. The RMSD (root mean square deviation) of the ligand is depicted in green.4.4.2.RMSF measures the amount by which atomic locations have deviated from their initial positions and illustrates the dynamic nature of the protein-ligand interaction. In other words, RMSF exemplifies how dynamic the interaction between the protein and the ligand is. As a result, the RMSF data of the protein backbone and complexes were plotted to visualize the average fluctuation of all the amino acid residues, as depicted in 4.4.3.To evaluate structural compression changes, the gyration radius diagram of each structure was recorded over the course of the simulation. To determine the compactness of the system over time, Rg was calculated, with higher Rg values indicating less compactness (more unfolded) with conformational entropy and lower Rg values indicating high compactness with greater structural stability (more folded). 4.4.4.It is worthwhile to investigate hydrogen bonding interactions because they contribute to the binding process and, in particular, persistent hydrogen bonds. Using the VMD hydrogen bond analysis tool, all conceivable hydrogen bonding interactions between the two specified areas, in this case the protein and the ligand, have been explored throughout time. Hydrogen bonds are recognized, and the output contains the overall number of hydrogen bonds as well as their occupancy over time. The ‘Percentage occupancy of the Hbond’ output of the hydrogen bond analysis tool provides access to these values . In dockIn the case of the apigenin complex, during MD, the protein-apigenin complex stability was maintained by interactions with GLY 81, LYS 105, and ILE 147 residues. It has hydrogen bonds with the highest occupancy of 11.28%, where apigenin forms the hydrogen bond with LYS 105. Then 0.90% occupancy, where apigenin plays a role as an acceptor for GLY 81, and 0.10% with ILE 147. Although the Apigenin complex has 50 hydrogen bonds more than the other complexes, the compound (Apigenin) formed only two hydrogen bonds, which were found in docked simulation. Curcumin has 28 total hydrogen bonds, with the highest occupancy of 80%, where curcumin and VAL 132 form hydrogen bonds. Then 40% occupancy, where curcumin plays a role as an acceptor of ASP 131, and 1.20% with ASP 131. Only three of the 28 hydrogen bonds were formed in the docked simulation. We conclude that, during MD, the curcumin complex has more stability than the apigenin complex. Ganoderic acid has 32 total hydrogen bonds with the highest occupancy of 29%, where Ganoderic acid has the role of a donor and SER 341 has the role of an acceptor of hydrogen bond formation. Then 19% occupancy where Fisetin plays a role as an acceptor to ILE 340 and 19% with PRO 339. Only two of the 32 hydrogen bonds were formed in the docked simulation. In the system, hydrogen bonds were stable, and most of them appeared between 0 and 15 ns and 40 and 50 ns. Hydrogen bond occupancy with a score of more than 100% indicates that more than one atom pair interacts to form hydrogen bonds. Meanwhile, curcumin has a lower total hydrogen bond but a higher hydrogen bond occupancy than Ganoderic acid. This total hydrogen bond and hydrogen bond occupancy determine the stability of each system. The number and occupancy of hydrogen bonds are the keys to the interaction stabilization of the protein-ligand complex.4.4.5.Principal component analysis was used to identify and understand significant concerted motions in different regions of the protein. PCA extracts the most variable dynamic motions in simulations, which are required for biological function. In addition, it may be used to examine the effect of various parameters on the collective motion and to reduce the motion’s complexity, which is related to the system’s stability and protein functions. Also, it can be utilized to characterize the many conformational variations that are associated with the process of protein folding as well as the open-close mechanism of ion channels. The conformational alterations of West Nile virus methyltransferase and West Nile virus envelope glycoprotein were further analyzed using dynamic cross correlation matrix (DCCM) analysis. Principal component analysis (PCA) and a DCCM were used to identify protein backbone conformation shifts. PCA and DCCM were performed using RStudio and Bio3d; axes, each dot represents a different protein configuration. The distribution of blue and red dots represented the extent of conformational changes in the simulation, where the color ranges from blue to white to red corresponds to simulation duration. The color blue represents the initial timestep, the color white represents the intermediate, and the color red represents the final timestep . Various colors were used to denote varying degrees of association between residues, with darker colors indicating stronger correlations. Correlations closer to 1 indicated that the residues were moving in the same direction, while correlations closer to −1 indicated that the residues were moving in the opposite direction. In order to see the relationship between the I and J residue indices, we constructed pairwise correlated graphs. Colors such as dark cyan, white, and pink were used to analyze the predicted map results. Fully correlated pairs are denoted by the color cyan, while anti-correlated pairs are denoted by the color pink. Comparative results reveal that the atomic motions in the Apigenin complex resemble more closely those of the Curcumin structure. But, Comparing the DCCM diagrams of the three systems, it could be found that the correlated motions of the apigenin and curcumin systems were noticeably distinct from the Ganoderic complex. We postulate that the simultaneous appearance of positively and negatively correlated movements destabilizes the domain. For this reason, we observed high anti-correlation in the apigenin complex compared to the curcumin complex, indicating a more compact structure of the apigenin. For the Ganoderic acid complex, which showed less anticorrelated and correlated motions and more noncorrelated motions than the Apigenin and Curcumin complexes in all regions of the protein  was calculated using MM-PBSA, which considers both bonded and non-bonded  interactions. Using MMPBSA and the final 20 ns of the trajectory, the binding free energies of the protein-ligand complexes were calculated. Using the MMGBSA approach, the binding free energy (∆ Gbind) of 12 natural compounds, of which top nine were chosen based on binding affinity score, was determined. The greater the negative values, the more favorable the binding free energy between proteins and ligands. As shown in 4.5.By utilizing the pkCSM web server, ADMET  properties were performed. The 5.in-silico research has been reported that all the natural molecules have better binding affinity, high solubility in aqueous system, free from hepatic toxicity, and skin sensitization, most of them are accepted by lipinski rule. Besides, different types of active amino acid are seen during the formation of drug protein complexes such as VAL-A:132, ASP-A:131, PHE-A:133, LYS-A:105, ASP-A:146, TRP-A:87, GLY-A:58, ASP. The most active compounds were reported Apigenin against West Nile virus methyltransferase (PDB ID 2OY0), and Fungisterol, & Sanguinarine against West Nile virus envelope glycoprotein (PDB ID 2I69) with maximum binding affinity − 8.1.3 kcal/mol, and − 8.1 kcal/mol. The drug-likeness properties, and the theoretical ADMET data is accepted by our reported phytocompounds. After that, their stability is confirmed by molecular dynamic simulation at 100 ns which is also confirmed that the molecules are highly stable when form protein ligands complex. As, there is currently neither an antiviral medicine nor a vaccination that can treat WNV infection in people. In the course of drug discovery and repurposed research, various potential natural molecules have been studied in this investigation that capable to inhibit WNV in silico model; however, none of these candidates have performed it to the stage of clinical assessment. Now, further experimental studies should be conducted and determine their practical value to establish them as potential drug candidate for further use.Our The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.SA, IB, MR, NM, SM, MI, SR, AG, SA-H, MZ, VJ, SS, JB, and RS: data collection, conceptualization, methodology, software, validation, formal analysis, investigation, resources, data curation, writing—original draft preparation, writing—review and editing, visualization, supervision, project administration, and funding acquisition. All authors contributed to the article and approved the submitted version.This research was supported by the Deanship of Scientific Research, Imam Mohammad Ibn Saud Islamic University (IMSIU), Saudi Arabia.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.