Zanthoxylum piperitum) contains several types of sanshools belonging to N-alkylamides. Because of the long-chain unsaturated fatty acids present in their structure, sanshools are prone to oxidative deterioration, which poses problems in processing. In this paper, we evaluated the effects of antioxidants from the genus Zanthoxylum in preventing sanshool degradation using accelerated tests. An ethanolic extract of segment membranes of the sansho fruit pericarp was incubated at 70 °C for 7 days with different antioxidants to determine the residual amount of hydroxy-α-sanshool (HαS) in the extract. α-Tocopherol (α-Toc) showed excellent HαS-stabilizing activity at low concentrations. Among phenolic acids, we noted that the HαS-stabilizing activity increased with the number of hydroxy groups per molecule. For example, gallic acid and its derivatives exhibited excellent sanshool-stabilizing activity. Quercetin was found to be a superior HαS stabilizer compared with hesperetin and naringenin. However, the effective concentration was much higher for phenolic compounds than for α-Toc. These substances are believed to play a role in preventing the decomposition of sanshools in the pericarp of sansho. These sanshool stabilizers should be useful in the development of new beverages, foods, cosmetics, and pharmaceuticals that take advantage of the taste and flavor of sansho.Japanese pepper (sansho,  Zanthoxylum piperitum) has been used as a spice and a traditional herbal medicine (Kampo medicine) in Japan since ancient times. Wakayama in central Japan cultivates the variety Budo Sansho, which accounts for 60–70% of Japan’s sansho production [The Japanese pepper  [Sansho has a long history of being used as a spice, and in recent years, its pungent taste accompanied with numbness has led to a boom in its production. The pungent taste of Japanese peppers arises from sanshools. To date, 13 types of sanshools have been identified in the genus thoxylum . Among tngeanum) ,3,4,5,6.ngeanum) ,8,9. Forngeanum) . HuajiaoKampo medicines have been used traditionally for more than 1500 years in Japan. The sansho-containing Kampo medicines used in Japan are Daikenchuto (DKT), Chukenchuto, Tokito, and Shukubaito. Among these, DKT is one of the most frequently prescribed medicines for various digestive disorders ,12. It cIn addition to its gastrointestinal effects, strong salivating effects of HαS have been reported . It is wZanthoxylum, and measured their stabilizing activity against sanshools using a screening system which we developed ourselves.Sanshools are long-chain polyunsaturated amides. Therefore, it is conceivable that the instability of sanshools may be due to the oxidation of the polyunsaturated chain or amide hydrolysis under acidic conditions. We selected antioxidants reported to be present in the genus Zanthoxylum piperitum fruits  were harvested from Kainan, Wakayama, Japan. Ripened fruits were dried in a drier at 40 and 50 °C for 6 h each, and then at 60 °C for 2 h. Upon drying, the fruit split, and the pericarp was separated via sieving off the seeds. HαS and hydroxyl-β-sanshool (HβS) were obtained from MedChemExpress . Hydroxyl-ε-sanshool (HεS) was purchased from MuseChem . Hydroxyl-γ-sanshool (HγS) was obtained from Chengdu Biopurify Phytochemicals Ltd. . High performance liquid chromatography (HPLC) grade acetonitrile, α-tocopherol (α-Toc), 2,2,5,7,8-pentamethyl-6-chromanol, phytol -3,7,11,15-tetramethyl-2-hexadecen-1-ol), gallic acid monohydrate and ascorbic palmitate were purchased from Fujifilm-Wako Chemicals . 2-Hydroxybenzoic acid , 4-hydroxy benzoic acid (PHBA), ethyl gallate, propyl gallate, 4-hydroxy-3-methoxybenzoic acid (vanillic acid), hydroxyquinone, and 3,4-dihydroxybenzoic acid (protocatechuic acid) were purchased from Tokyo Chemical Industry . Quercetin, naringenin, hesperetin, caffeic acid, p-coumaric acid, and ferulic acid were purchased from Sigma Aldrich . All other chemicals were of analytical grade.The pericarp of sansho mainly consists of flavedo and segment membranes . The segg for 2 min, and the supernatant was collected. The amount of HαS in the alcohol extract of the segment membrane powder was determined using the HPLC method described below, and ethanol was added to bring the final HαS amount to 2.5 μmole/mL. Briefly, 2 mL of supernatant and 2 mL of the same volume of ethanol containing the candidate stabilizer were mixed in a 10 mL Spitz tube and incubated in an oil bath at 70 °C. The amount of HαS in the sample on day 7 of incubation was determined using HPLC. The ratio of the amount of HαS on day 7 of incubation to the amount on day 0 was calculated and expressed as survival rate (%). When ethanol without stabilizer was used, the amount of HαS remaining in the test solution was 61.35 ± 1.18% in the mean ± SD value. If it was above this value, it was judged to have a stabilizing effect, and if it was below, it was judged to have no stabilizing effect or to destabilize HαS. Three lots of sansho fruit were harvested from three sansho trees and dried separately. From the dried pericarps of the three lots (2 kg each), 200 g of pericarps were used for segmental membrane separation. Segment membranes (50 g each) were obtained. Segment membranes from each lot were powdered as described above. To perform the assay for one candidate compound in triplicate, segment membranes powder (0.4 g) was suspended in ethanol (8 mL) and mixed via vortexing. The suspension was centrifuged at 2000× HPLC was performed on a SHIMADZU LC-2010 instrument . All samples were injected into an InertSustainSwift C18 4.6 × 250 mm column . The analytes were eluted with mixed solvent A (30% acetonitrile): B (80% acetonitrile) at a flow rate of 1.0 mL/min. The conditions were as follows: 0 min: 0% B; 35 min: 45% B; 50 min: 100% B; 51 min: 0% B; 55 min: 0% B. All analyses were performed at 40 °C and the absorbance of eluate was monitored at 270 nm. Liquid chromatography–mass spectrometry (LC-MS) was performed via electrospray ionization (ESI)-MS in a positive mode using a Bruker micrOTOF™ ESI-TOF mass spectrometer  interfaced with an Agilent 1200 HPLC system . The MS conditions are follows: capillary voltage, 4500 V; drying gas temperature, 200 °C; drying gas flow, 8.0 L/min; nebulizer pressure, 1.6 Bar. A portion of the sample was loaded onto an Inertsil ODS-3 (2.1 × 150 mm) column. Separation was achieved via elution with a mixture of solvents A (30% acetonitrile) and B (80% acetonitrile) at a flow rate of 1.0 mL/min. The conditions were as follows: 0 min: 0% B; 35 min: 45% B; 50 min: 100% B; 51 min: 0% B; 55 min: 0% B. All analyses were performed at 40 °C, and the absorbance of eluate was monitored at 270 nm.n = 3) under the conditions described above. In this concentration range, a linear relationship was obtained between the HαS concentration and the HPLC peak areas. This calibration curve was used to determine the HαS content in the sample. The experiment was also performed three times on different days, but no daily difference was found. The limit of detection in this method was 0.00163 µmole/mL.HαS of the samples listed in Materials and Methods were diluted to 0.38, 0.76, 1.90, and 3.8 mmole/mL, respectively, using an ethanol solution containing 0.1% α-Toc, and analyzed using HPLC (v/v/v) at a flow rate of 1.5 mL/min at 40 °C. The excitation and emission wavelengths were 298 and 325 nm, respectively.Analysis of α-, β-, γ-, and δ-Tocs was performed using HPLC by Japan Food Research Laboratories . The method is shown below. NaCl , 6% pyrogallol in ethanol (10 mL), ethanol (1 mL) incorporating 2,2,5,7,8-pentamethyl-6-hydroxychroman , and 60% KOH (1.5 mL) were added to the crushed pericarp (0.5 g) or seed (1 g). The mixture was kept at 70 °C for 30 min. After cooling in an ice–water mixture, the unsaponified compound was extracted three times with 1% NaCl (15 mL) and 1:9 ethyl acetate/hexane (15 mL). The organic solvent layer was combined and evaporated to dryness under vacuum . The rest-test. p < 0.05 and are indicated by a single asterisk (*) or different letters.All data are expressed as the mean ± standard deviation (SD) values for each group. Statistical analysis was performed using one-way analysis of variance with Student’s Sansho fruits are harvested in three seasons in the central prefecture of Wakayama, Japan . The unr+) and 286.1778 (M + Na+) based on high-resolution LC-MS  data. Similarly, we found that peak E compound had the elemental composition of C16H25NO due to the signals of 248.2009 (M + H+) and 270.1828 (M + Na+), and peak F compound had the elemental composition of C18H27NO due to the signals of 274.2165 (M + H+) and 296.1985 (M + Na+). Therefore, peaks B, E, and F were believed to be for HαS, α-sanshool (αS), and γ-sanshool (γS), respectively. From HPLC of references performed under the similar conditions [A chromatogram of the ethanolic extract of pericarp is shown in nditions ,21,22, wg for 2 min, and the supernatant was recovered. The amount of HαS in each supernatant was measured using HPLC. As shown in The pericarp powder (0.4 g) was suspended in aqueous ethanol solutions of various concentrations (20 mL). After vigorous mixing for 5 min at about 20 °C, the mixture was centrifuged at 2000× HαS stabilizer, found to be present in the sansho pericarp, was apparently extracted with ethanol. Therefore, to search for stabilizing substances, we considered it necessary to build a system that minimized contamination of stabilizers derived from sansho. We collected segment membranes attached to the inside of the pericarps and prepared their ethanolic extract. The amount of HαS in the segment membrane was approximately 5 mg/g dry weight, which was equivalent to approximately 1/20 of that in the pericarp. As shown in The two ethanolic extracts were incubated at 60, 70, and 80 °C for 1 week and the residual amounts of HαS in them was compared . The amoTocopherol is a fat-soluble antioxidant vitamin. Hisatomi et al. reported that the α-Toc content in the pericarp of sansho is 35 mg/100 g, which is approximately 11.7 times that in the seeds . This viAscorbic acid is a well-known water-soluble antioxidant vitamin normally present in plants. However, because it is insoluble in ethanol, the HαS-stabilizing activity of ascorbic acid could not be investigated using our assay system, and we believe that it is not a stabilizer of HαS. Ascorbic palmitate is an ester formed from ascorbic acid and palmitic acid, which is hydrolyzed into fat-soluble ascorbic acid and is used as a food additive. We investigated the HαS-stabilizing activity of ascorbic acid using ascorbic palmitate. It showed the highest HαS-stabilizing activity at approximately 2 mM, and the activity decreased at higher concentrations . Zanthoxylum [Many phenolic acids, including hydroxycinnamic acid, flavonoids, lignans, and coumarins, have been reported to be present in the genus thoxylum ,26,27,28Gallic acid and its related compounds showed high HαS-stabilizing activity at low concentrations . This wap-coumaric and ferulic acids  , and theIn this study, several compounds with stabilizing activity for HαS, which is the main pungent substance sansho, were found. Among these, α-Toc was found to substantially stabilize HαS in sansho pericarp. This appears to be one of the reasons for the abundance of α-Toc in sansho pericarp, which is an amphiphilic compound present in cell membranes. α-Toc, which is synthesized in chloroplasts and is present in cell membranes, plays an important role in preventing lipid oxidation ,31. The Zanthoxylum exhibited high HαS-stabilizing activity. However, because these compounds are believed to be bound to sugars and organic acids, which makes them hydrophilic, they are difficult to access HαS. Therefore, we believe that they are not involved in the stabilization of HαS in the pericarp. Furthermore, the concentration of phenolic compounds, which are effective in stabilizing HαS, was much higher than that of α-Toc; it may, therefore, be difficult to practically use these phenolic compounds. Furthermore, as shown in Some phenolic compounds reported in We believe that the mechanism by which antioxidants stabilize HαS is the same as that involved in the prevention of oxidation of vegetable oils rich in unsaturated fatty acids. In this case, the antioxidants do not bind to the unsaturated fatty acids at a specific site through a special binding, such as that seen for enzyme–substrate binding. The antioxidants are instead believed to react with oxygen radicals present in the environment surrounding the unsaturated fatty acid, reducing the frequency with which oxygen radicals approach the unsaturated fatty acid. HαS has also potential use for the treatment of diseases and in healthcare. Therefore, for further pharmacological research or development of the medicinal uses of sanshools, it is important to identify the conditions or substances that stabilize sanshools. Methods for the synthesis of sanshools have also been reported ,35,36, aN-alkylamide, which is one of the active ingredients of Echinacea, poses a problem, and its content is not guaranteed for each preparation [Echinacea purpurea contains eight isobutylamides, all of which are susceptible to oxidative degradation and are very similar to sanshools. They found that phenolic acids in Echinacea prevented the degradation of these isobutylamides depending on the experimental conditions [N-alkylamides is prevented to some extent by the antioxidant action of these phenolic acids. However, because N-alkylamides are vulnerable to acids, further stability can be expected using α-Toc.Echinacea is a popular herbal medicine in Western countries that has been used to treat colds and influenza ,38. Howeparation . Echinacnditions . It is bWe constructed a system for stabilizing substances in sanshools and found that α-Toc exhibits excellent activity. Stabilization of sanshools can be employed for developing new sansho products that exploit their excellent functionality. However, since sanshools and α-Toc are not strongly bonded, they are likely to separate easily when turned into liquids or powders. In the future, it will be necessary to develop a technology to make sanshools and α-Toc always coexist in close proximity.TM is named an inventor on a patent .


The detector (PDA) was set at 239 nm. The mobile phase consisted of methanol and ammonium acetate (8.1818 mM) in a ratio of 75.7: 24.3 (v/v). The flow rate was set at 0.048 mL min−1. The overall separation time was 9.5 min. The retention times of oseltamivir phosphate, dexamethasone, daclatasivir dihydrochloride, and remdesivir were 6.323 ± 0.145, 7.166 ± 0.036, 8.078 ± 0.124, and 8.572 ± 0.166 min (eight replicates), respectively. The proposed method demonstrated linearity in the ranges of 10.0–500.0 (ng mL−1) and 0.5–30.0 (µg mL−1) for oseltamivir phosphate, 50.0–5000.0 (ng mL−1) for dexamethasone, 25.0–1000.0 (ng mL−1) and 0.5–25.0 (µg mL−1) for daclatasvir dihydrochlorde, and 10.0–500.0 (ng mL−1) and 0.5–30.0 (µg mL−1) for remdesivir. The coefficients of determination (R2) were greater than 0.9999, with percentage recoveries greater than 99.5% for each drug. The limits of quantitation were 6.4, 1.8, 7.8, and 1.6 ng mL−1, and the limits of detection were 1.9, 0.5, 2.0, and 0.5 ng mL−1 for oseltamivir phosphate, dexamethasone, daclatasivir dihydrochloride, and remdesivir, respectively. The proposed method was highly precise, as indicated by the low percentage of relative standard deviation values of less than 1.2% for each drug. The average content and uniformity of dosage units in the studied drugs' dosage forms were determined. The average contents of oseltamivir phosphate, dexamethasone, daclatasivir dihydrochloride, and remdesivir were nearly 93%, 102%, 99%, and 95%, respectively, while the uniformity of dosage unit values were nearly 92%, 102%, 101%, and 97%. Two novel methods were established in this work. The first method was used to assess the stability of standard solutions. This novel method was based on the slope of regression equations. The second was to evaluate the excipient's interference using an innovative instrumental standard addition method. The novel instrumental standard addition method was performed using the UPLC instrument program. It was more accurate, sensitive, time-saving, economical, and eco-friendly than the classic standard addition method. The results showed that the proposed method can estimate the tested drugs' concentrations without interference from their dosage form excipients. According to the Eco-score (more than 75), the Green Analytical Procedure Index (GAPI), and the AGREE criteria , the suggested method was considered eco-friendly.A green, rapid, and simple RP-UPLC method was developed and optimized by full factorial design for the simultaneous separation of oseltamivir phosphate, daclatasivir dihydrochloride, and remdesivir, with dexamethasone as a co-administered drug. The separation was established on a UPLC column BEH C COVID-19 was caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Corona viruses are enclosed viruses with a positive-polarity single-stranded RNA genome. Because of this lethal sickness, researchers worldwide are forced to produce appropriate medicines for its prevention and treatment. Oseltamivir phosphate (OSTP) Fig. A, dexame1. REM is a nucleoside analog. It prevents viral replication by inhibiting the RNA-dependent RNA polymerase enzyme. In October 2020, the FDA approved it for the treatment of COVID-19.OSTP is a neuraminidase inhibitor that inhibits the viral neuraminidase enzyme present on the virus' surface. This action prevents the budding of the virus from the host cell, replicating, and infecting. It is used to treat and prevent infections with influenza viruses A (including pandemic H1N1) and B. It was approved by the U.S. Food and Drug Administration (FDA) in August 2016. DEX is a glucocorticoid. It is used for the treatment of inflammatory diseases. It was approved by the FDA in October 1958. It is used for COVID-19 patients with severe respiratory symptoms and is given in conjunction with other antiviral medications. DAC is a direct-acting antiviral drug. It was approved by the FDA in July 2015. It is used in conjunction with other antiviral medications to treat hepatitis C virus (HCV) infections. The daclatasivir/sofosbuvir combination was observed to enhance clinical outcomes in individuals who had moderate-to-severe COVID-19 symptoms6. Since 2011, OSTP has been determined by UPLC7, RP-HPLC8, LC–MS/MS9, potentiometric10, spectrofluorimetric11, and spectrophotometric12 methods. OSTP in combination with compounds other than DEX, DAC, and REM was determined by RP-HPLC14, LC–MS/MS15, and mass spectrometric (MS)16. DEX has been determined by RP-HPLC-UV, UPLC-UV, LC–MS/MS, and spectrophotometric methods since it was approved by FDA until 202217. DAC has been determined by RP-HPLC18, UPLC18, spectrofluorimetric19, spectrophotometric20 techniques since it was approved by FDA in 2015,. DAC in combination with compounds other than OSTP, DEX, and REM was determined by RP-HPLC22, electrochemical23, spectrofluorimetric24, and CE25 techniques. REM has been determined by RP-HPLC26, LC–MS/MS27, spectrofluorimetric28, and electrochemical23 techniques. REM with DEX has been determined by the UPLC29 method. No analytical method was reported to estimate OSTP, DEX, DAC, and REM simultaneously.Several analytical methods for the determination of OSTP, DEX, DAC, and REM in dosage forms and plasma have been documented in the literature. According to literature studies, OSTP was determined by chromatographic, capillary zone electrophoresis (CE), enzymatic, spectrofluorimetric, mass spectrometric (MS), colorimetric, and spectrophotometric techniques since it was approved by FDA until 2011During the development of the proposed method, two challenges were anticipated. The first was due to the high degree of ionization of DAC and OSTP, so they would create tailed peaks. The second was peak overlapping between OSTP and DEX and between DAC and REM because of their close log P values. The aim of this work was to develop and validate a green, simple, highly-sensitive, fast, and accurate RP-UPLC-PDA technique for the simultaneous determination of OSTP, DEX, DAC, and REM without interference from their dosage form excipients.30. DOE is a statistics-based technique. It has numerous benefits in optimization and the development of a robust method31. DOE optimization requires significantly less investment, effort, and resources than univariate procedures32. Because of the DOE's research, it is now possible to understand and improve the method's performance by identifying key method parameters and designing plots that ensure the method's stability and best performance33. The robustness and system suitability parameters of the developed method were estimated using mathematical equations. These mathematical equations were derived from the estimated coefficients (in uncoded units), which were estimated by the full factorial design (23 FFD)34.The proposed method was optimized using the design of experiments (DOE) techniqueTwo novel methods were established in this work. The first method was used to assess the stability of standard solutions. This novel method was based on the slope of regression equations. The second was to evaluate the excipient's interference using an innovative instrumental standard addition method. The novel instrumental standard addition method was performed using the UPLC instrument program. The novel instrumental standard addition method was more accurate, sensitive, time-saving, economical, and eco-friendly than the classic standard addition method. The results showed that the proposed method can estimate OSTP, DEX, DAC, and REM without interference from their dosage form excipients.35, the suggested method was used to calculate the average content and uniformity of dosage unit. The results were compared to previously published RP-HPLC-UV methods36. The suggested method will be useful for quality control evaluation of the pharmaceuticals under investigation in their dosage units, as well as for future polypill formulations. The proposed method is appropriate for UV detectors (PDA at one wavelength) and LC–MS/MS method development since the mobile phases utilized (methanol and ammonium acetate) are appropriate for mass detectors. The proposed method is simple in sample preparation. In addition, Eco-Scale, the Green Analytical Procedure Index (GAPI) guidelines, and AGREE  analysis were used to assess the suggested method's greenness39. The proposed method is a more eco-friendly, time-saving, and cost-saving method than the reported methods36.According to BP18 1.7-µm (2.1 The developed method was carried out on an ACQUITY UPLC H-Class PLUS System (U S A) outfitted with a WATERS quaternary solvent manager (M20QSP 471A), a WATERS column heater compartment (K20CHA 249G) with an ACQUITY Waters UPLC column BEH CStatistical analysis of the factorial design was performed using MINITAB statistical software .Water deionizer , calibrated sensitive balance , pH meter , ultrasonic , mixer , oil-free vacuum pump , and nylon membrane filters  were used for mobile phase preparation. Pipet4u pro manual pipettes 2–20, 10–100, 20–200, and 100–1000 µL  were used for the preparation of standard and sample solutions. Syringe filters  were used to filtrate standard and sample solutions before injection at UPLC.36 and they were found to be 99.976% w/w, 99.307% w/w, 101.171% w/w, and 99.841% w/w, respectively.Qseltamivir phosphate (OSTP), dexamethasone (DEX), daclatasvir dihydrochloride (DAC), and remdesivir (REM) standard powders were obtained from NODCAR, Cairo, Egypt. Their purities were assessed against the reported methodsOSELTAMIVIR hard gelatin capsules  were purchased from a local pharmacy. Each hard capsule contains 98.50 mg of OSTP . DEXAZONE tablets were purchased from a local pharmacy . Each tablet contains 0.50 mg of DEX. JAVIDACLA film-coated tablets were kindly donated by Multicare Egypt for Pharmaceutical Industries, batch No. 160404 . Each film-coated tablet contains 65.92 mg of DAC, equivalent to 60.00 mg of DAC. Aliquots (100 µL) of ten REMDESIVIR vials  were separately obtained from Mahalla General Hospital, Egypt. Each vial of 20 mL of concentrated solution contains 100.00 mg of REM.Methanol HPLC grade  and ammonium acetate (AA)  were purchased from CORNELL LAB—Fine Chemicals & LAB Equipment, Cairo, Egypt.−1 for each drug) was prepared in a 10-mL volumetric flask using methanol as a solvent. The stock quaternary solution was further diluted with methanol to obtain a working solution of 500 µg mL−1 for each drug.A stock solution containing a quaternary mixture of OSTP, DEX, DAC, and REM  into a series of 10-mL volumetric flasks, which were then completed to the mark with 30.0% (v/v) methanol. All prepared quaternary solutions were kept in the freezer (− 15 °C to − 28 °C) and shielded from light.Standard quaternary solutions with concentrations ranging from 10 to 30 µg mLVolumes of 10 µL (in triplicate) were injected under optimal chromatographic conditions after filtering each prepared quaternary solution using a 0.22 mm membrane filter. To create the calibration curves, the peak areas were plotted against the final concentrations. The corresponding regression equations were then derived.The UPLC vials containing laboratory-prepared standard quaternary solutions were frozen at (− 15 °C to − 28 °C). Each vial was analyzed by the proposed method at time intervals over eight days. For each drug, calibration curves were obtained every day. Percentages total), then evacuated, and the evacuated powder was homogeneously mixed. The empty hard gelatin capsules were washed using diethyl ether and then dried in the air before being weighed (Wcap). Then, 25 mL of methanol were used to transfer an accurately weighed amount of evacuated powder proportional to the estimated average weight /5) into a 100-mL volumetric flask. Another five JAVIDACLA film-coated tablets were weighed and then finely powdered. A carefully weighed amount equal to the average weight of five JAVIDACLA film-coated tablets was placed into the same flask with another 25 mL of methanol and sonicated for 15 min. After sonication, the flask was allowed to cool for 5 min before being filled to the mark with methanol (solution A).Five OSELTAMIVIR hard gelatin capsules were weighed (WFive DEXAZONE tablets were finely powdered and transferred quantitatively into a 10-mL volumetric flask using 5 mL of methanol. The flask was sonicated for 15 min and then kept at room temperature for 5 min before being filled with methanol to the mark (solution B). To reach the average content of the five DEXAZONE tablets, solution B was diluted to its fifth strength. 2 mL of solution B were transferred into a 10-mL volumetric flask, which was then filled with methanol to the mark (solution C).To estimate remdesivir's average content, 25 µL of REMDESIVIR was transferred into an Eppendorf tube from each of the five vials and then vortexed for 30 s (solution D).Finally, aliquots of 250 µL, 500 µL, and 25 µL were transferred from solutions A, C, and D, respectively, into 10-mL volumetric flasks and completed with 30.0% (v/v) methanol (solution E). Later, solution E was diluted by transferring an aliquot of 100 µL into a 10-mL volumetric flask, which was then filled to the mark with 30.0% (v/v) methanol (solution F).The suggested method was used to analyze the final quaternary mixes (solutions E and F) after filtration through a 0.22-mm membrane filter. The concentrations of the four drugs were determined using the corresponding regression equations.The above procedure under the "total) and individually (Wn) weighed. The empty hard gelatin capsules were separately washed with diethyl ether, and the washings were transferred into ten test tubes. Both the empty capsules and the ten test tubes were dried by air, and then the empty capsules were weighed (Wcap) and separately weighed (Wncap). Then one residue from a dried test tube was transferred into a 100-mL volumetric flask with its corresponding evacuated powder using 50 mL of methanol. One JAVIDACLA film-coated tablet was inserted in this flask, and the flask was sonicated for 15 min. After sonication, the flask was allowed to cool for 5 min before being filled to the mark with methanol (solution G).Ten OSELTAMIVIR hard gelatin capsules were collectively .Finally, aliquots of 250 µL, 500 µL, and 25 µL were transferred from solutions G, H, and one REMDESIVIR vial, respectively, into a 10-mL volumetric flask and marked with 30.0% (v/v) methanol (solution I). Later, solution I was diluted by transferring an aliquot of 100 µL into a 10-mL volumetric flask, which was then filled to the mark with 30.0% (v/v) methanol (solution J). This procedure was repeated for the rest of the nine dosage units.The resultant quaternary mixtures (solutions I and J) were filtered through a 0.22-mm membrane filter and analyzed by the suggested method. The concentrations of the four drugs were determined using the regression equations that corresponded to them.−1) into a series of 10-mL volumetric flasks holding 100 µL of solution E and then filling them to the mark with 30.0% (v/v) methanol.Two procedures were used. The first procedure was applied to the low linearity ranges of OSTP, DAC, and REM. This was accomplished by transferring various aliquots  of the standard quaternary solution (250 ng mL−1) into a series of 10-mL volumetric flasks holding aliquots of 250 µL, 500 µL, and 25 µL from solutions A, C, and D, respectively, and then completing them with 30.0% (v/v) methanol.The second procedure was used for OSTP, DAC, and REM at high linearity ranges and also for DEX. This was accomplished by transferring various aliquots  of the standard solutions  were estimated using “Eq. ” and ploFrom the corresponding regression equations, the average content concentrations of the four drugs were calculated [Eq. ]:2\docum−1 for each drug) and the other containing solution E, were utilized. The UPLC was programmed to mix various aliquots  of the first vial with 0.1 µL of the second , then analyze them using the proposed method. The final standard amounts (ng) were estimated by “Eq. , response optimizer, optimization plot, and estimated coefficients of the independent factors (data in uncoded units) were studied in this work.DOE refers to the process of making systematic plans to extract maximum data with minimum experimentation. From these extracted data, meaningful conclusions are drawn using statistical models3 total) were conducted. In the response optimizer, the lower, target, and higher values for dependent responses as well as their importance values were determined. The importance value has a range of 0.1 to ten. Because all responses are equally significant, the default value of one was applied to each.In the full factorial design (FFD), three factors with two levels were chosen. As a result, eight designed experiments  and composite desirability (D). The optimal UPLC conditions with the highest composite desirability (D) value were determined. Individual desirability (d) measures how well the conditions optimize a single response, whereas composite desirability (D) measures how well the conditions optimize all responses in the set. Desirability is measured on a scale of zero to one.The optimization plot showed the effect of each independent factor (columns) on the dependent responses (rows). The vertical red lines on the graph represent the optimum settings of the independent factors. The red numbers displayed at the top of the columns showed the optimum values of the independent factors. The horizontal dashed blue lines and numbers (y) represented the responses to the optimum settings Table .31 and system suitability parameters.Furthermore, factorial design analysis provides estimated coefficients of the independent factors (data in uncoded units), from which mathematical equations [Eqs. (S1)–(S12)] for chromatographic responses can be created. These equations can predict the proposed method's robustnessAfter many experimental trials, well-defined symmetrical peaks were achieved. These experiments can be summarized as follows:2-CH2-) Bridged Hybrid (BEH) C18 columns as the first choice. BEH groups have three functions. The first function is due to the ability of the surface BEH groups to conceal the surface free silanol groups, which results in improving the tailing factor and, thus, the peak shape. The second function is due to the cross-linking BEH groups, which improve the column's chemical and mechanical stability. The third function is due to the internal hydrophobic BEH groups that have the best pH stability in the range of pH 1.0 to 12.0. These trifunctionally bonded alkyl columns can use the power of pH to influence the retention, selectivity, and sensitivity of ionizable molecules. They enable a robust separation technology for method development40.The most common UPLC separations use the 1.7-µm UPLC Ethylene  efficiently preserves UPLC column performance. Furthermore, during the manufacturing of guard columns, the same UPLC column stationary phase and column frits are used, resulting in an improvement in the overall separation efficiency and resolution. Finally, the guard columns are connected directly to the UPLC column's inlet, so extra-column volumes are minimized, and mobile-phase leaks caused by additional connections are almost avoided42). On the other hand, the aqueous mobile phase was intended to be water (pH 7.0), 0.1% (v/v) acetic acid (pH 4.3), or ammonium acetate solution (pH 7.043). Water is widely available, inexpensive, and eco-friendly. Ammonium acetate solution is widely available, chemically stable, non-toxic, and inexpensive. It is totally ionized, almost neutral in water, and an effective buffering medium. It is extremely soluble in methanol and acetonitrile and serves as an effective masking agent for residual silanol groups on the chromatographic media, resulting in greatly enhanced separations. It can speed up proton equilibrium rates, which is essential for the chromatography of ionic compounds. It is also relatively volatile and easy to remove after preparative separation or when used with a mass spectrometer. It is compatible with all frequently used liquid chromatographic detectors44.The retention time and peak tailing are greatly affected by the pH of the mobile phase. The eco-friendly biodegradable methanol was intended to be utilized as the organic mobile phase  acetic acid) or 7.5 ± 0.5 (in the case of water and ammonium acetate).45. Both methanol and ammonium acetate have UV cutoffs at 205 nm, while acetic acid solution has a UV cutoff at 210 nm. As a result, the PDA-detector should theoretically be set above 230 nm to reduce baseline noise during the baseline stabilization process47. It was found that the absorbance at 240 nm of the degassed methanol, 0.1% (v/v) acetic acid, and 10.0000 mM ammonium acetate is 0.05, 0.01, and less than 0.01, respectively48. As a result, during optimization, data was taken at 239 or 240 nm from the PDA scan , REM, and DAC were the drugs under investigation. Theoretically, according to log P (Table 49.The compounds' log P (log Kow) and pka values influence their separation order P Table , these dk′) of DEXP created during optimization would determine which of DEXP and DEX should be used. DAC acidic and basic groups, on the other hand, would be ionized, and REM molecules would be totally unionized. As a result, it was predicted that DAC would be eluted before REM.It was observed that the log P values of DEXP, DEX, and OSTP are close, as are the log P values of DAC and REM. As a result, the ionization of these drugs was the primary factor influencing their elution sequence. The drugs under investigation had both acidic and basic functional groups. Table Two difficulties were anticipated with UPLC analysis. The first was due to the high degree of ionization of each of DEXP, DAC, and OSTP. It was hypothesized that when using water as an aqueous mobile phase, they would produce tailed peaks. This is due to interactions between ionized DEXP, DAC, and OSTP and free silanol groups. So, it was necessary to use a low pH (0.1% v/v) acetic acid solution or an ammonium acetate solution to hide free silanol groups in the stationary phase and hence decrease tailing (T).The second difficulty was peak overlap between DEXP and OSTP, between OSTP and DEX, and between DAC and REM due to their close log P values and use of methanol as an organic mobile phase. This problem was solved by gradually decreasing the flow rate while increasing the methanol percentage.k').Over the course of 13 days, 23 different conditions were used to develop the method Table . Method −1 dissolved in 60.0% (v/v) methanol) was detected at 37.500 min (min) with a flow rate of 0.200 mL min−1 and a mobile phase of methanol and water (50:50). Under identical conditions, the peak of a freshly prepared OSTP solution (100 µg mL−1 dissolved in 50.0% (v/v) methanol) was detected at 15.197 min. So, the resulting capacity factors (k') of REM and OSTP were very high   acetic acid (pH 4.2) was used with methanol instead of water to mask free silanol groups and hence decrease T and W of the OSTP peak and hence increase its N Table , OSTP pe−1 was tested. They exhibited promising system suitability parameters for OSTP. Therefore, under the conditions of Method 10 (Table k′ was very low (0.2).During Methods 8, 9, and 10 Table , the tem10 Table , REM and−1 each) in three different solvents were separately prepared from a methanolic stock solution of DEX, and then they were analyzed by Method 18  values were found to be promising.A ternary mixture of OSTP, DEX, and REM in 10.0% (v/v) methanol was then tested. The system suitability parameters of their resulted peaks were good. Therefore, DAC was evaluated both alone and then in combination with DEX, OSTP, and REM by Method 18 Table . Their TFinally, Methods 18–23 Table  were tesDuring the UPLC analysis of factorial designs, the quaternary mixtures were diluted with 30.0% (v/v) methanol. This was because samples diluted with 30.0% (v/v) methanol had higher responses (higher AUC) than ones diluted with water Table , which l31. These wavelengths (λ) were 239 nm (λmax of DEX), 245 nm (λmax of REM), 315 nm (λmax of DAC), 274 nm , and 340 nm . It was found that 239 nm was the most suitable one with satisfactory responses for the four drugs  of the analyzed drugs Fig.  to deterugs Fig. . As a reugs Fig. .Stabilities per day were determined for each drug according to the procedure mentioned under the "The interference of excipients was assessed using the classical standard addition method and the innovative instrumental standard addition method. The instrumental standard addition method outperformed the traditional method in that it was more accurate and sensitive because it was more dependent on the instrument (mixing procedure); it was time-saving and had fewer human errors because only one sample and one standard were needed to be prepared; it was economical because it required only a small amount of solvents; and thus, it was more eco-friendly. The only disadvantage was that the UPLC had to be calibrated.−1  (Table k′ (OSTP), T1 (OSTP), and RS3 (REM).The chromatographic performance was found to be affected by three independent factors: methanol percentage (v/v) , ammonium acetate concentration mM , and flow rate mL min1) Table . Accordik′ (OSTP), T1 (OSTP), and RS3(REM) results for all eight designed experiments were within the (0.48–0.91), (1.30–2.30), and (1.39–2.83) ranges. However, the acceptable limit for k' is greater than 0.551, T is from 0.8 to 2, and RS is greater than 1.552. As a result, these dependent responses were fed into the response optimizer program. The lower and target values for k′ (OSTP), T1 (OSTP), and RS3 (REM) (Table 31.The M) Table  were ent31, was 75.7% (v/v) methanol with 24.3% (v/v) AA (8.1818 mM) at a flow rate of 0.048 mL min−1 and an injection volume of 10 mL , T1(OSTP), and RS3(REM), which were methanol percentage (A), ammonium acetate concentration in mM (B), and flow rate in mL min−1 (C).Three independent factors were shown to have an effect on k'(OSTP) since it was the longest, but it was not statistically significant at a 95% confidence level. This was supported by its sharp slope in the main effects plot  decreased because OSTP interacted more effectively with methanol than the reversed C18 stationary phase, which was consistent with the solvophobic theory54. Solvophobic theory describes the hydrophobic interaction among analytes, the stationary phase, and the solvent55.This meant that as the percentage of methanol (A) increased, k'(OSTP) values were larger when methanol percentage interacted with 20.0000 mM ammonium acetate or a 0.070 mL min−1 flow rate based on BA and CA interactions, respectively.According to the interaction plot Fig. j, a simik'(OSTP) were AC (flow rate with 73.0 or 77.0% (v/v) methanol) and AB (ammonium acetate concentration with 73.0 or 77.0% (v/v) methanol), respectively. Moreover, flow rate and ammonium acetate concentration, when interacting with methanol percentage at AC and AB interactions, both had a positive influence on k'(OSTP). Additionally, at 73.0% (v/v) methanol, they produced greater k'(OSTP) values than at 77.0% (v/v) methanol.According to the Pareto chart Fig. a and thek'(OSTP) was at 73% (v/v) methanol , 20.0000 mM ammonium acetate , and a flow rate of 0.070 mL min−1 . However, to maintain the optimum conditions, 75.7% (v/v) methanol, 8.1818 mM ammonium acetate, and a flow rate of 0.048 mL min−1 were used.Finally, the interaction plot Fig. j reveale1(OSTP) since it was the longest, and it was statistically significant at a 95% confidence level. This was supported by its sharp slope in the main effects plot  decreased. This was because the addition of ammonium acetate rendered the stationary phase's free silanols inactive50.The Pareto chart of standardized effects Fig. b demonstlot Fig. . Based olot Fig.  and the 1(OSTP) values were smaller when ammonium acetate concentration interacted with 77.0% (v/v) methanol based on the AB interaction. The interaction of ammonium acetate concentration with flow rate (CB interaction), on the other hand, was divided into two phases. T1(OSTP) values were lower when ammonium acetate concentrations ranging from 5.0000 to 13.1818 mM interacted with a 0.070 mL min−1 flow rate, as well as when ammonium acetate concentrations ranging from 13.1818 to 20.0000 mM interacted with a 0.030 mL min−1 flow rate.According to the interaction plot Fig. k, a simik'(OSTP) were BC (flow rate with 5.0000 or 20.0000 mM ammonium acetate) and AC (flow rate with 73 or 77.0% (v/v) methanol), respectively. Furthermore, ammonium acetate concentration and methanol percentage both affected T1 (OSTP) positively at their high levels and negatively at their low levels when they interacted with flow rate at BC and AC interactions.According to the Pareto chart Fig. b and int1(OSTP) was at 77.0% (v/v) methanol , 20.0000 mM ammonium acetate , and a flow rate of 0.030 mL min−1 . However, to maintain the optimum conditions, 75.7% (v/v) methanol, 8.1818 mM ammonium acetate, and a flow rate of 0.048 mL min−1 were used.Finally, the interaction plot Fig. k reveale3 (REM) since it was the longest, and it was statistically significant at a 95% confidence level. This was supported by its sharp slope in the main effects plot   Fig. c.S3 (REM) decreased.Based on the estimated effects Table  and the 44. The ionization of DAC molecules into DAC+ cations occurred at pH 7.5 ± 0.5  values were higher when ammonium acetate concentrations ranging from 5.0000 to 15.0000 mM interacted with 73.0% (v/v) methanol, as well as when ammonium acetate concentrations ranging from 15.0000 to 20.0000 mM interacted with 77.0% (v/v) methanol.According to the interaction plot Fig. l, a simi3 (REM) were AB (ammonium acetate concentration with 73.0 or 77.0% (v/v) methanol) and BC (flow rate with 5.0000 or 20.0000 mM ammonium acetate), respectively. Moreover, ammonium acetate concentration and methanol percentage, when interacting with flow rate at BC and AC interactions, both had a negative effect on Rs3 (REM). Furthermore, at their low levels, they yielded lower Rs3 (REM) values than at their high ones.According to the Pareto chart Fig. c and int3 (REM)was at 73.0% (v/v) methanol , 5.0000 mM ammonium acetate , and a flow rate of 0.030 mL min−1 . However, to maintain the optimum conditions, 75.7% (v/v) methanol, 8.1818 mM ammonium acetate, and a flow rate of 0.048 mL min−1 were used.Finally, the interaction plot Fig. l reveale−1 of each drug was prepared. The mixture's 23 full factorial designs were applied using 30.0% (v/v) methanol as a blank and saved in the computer. The dependent factors at 239 nm were determined using the chromatograms of the combination  suggested that the calibration curves were linear.A PDA-detector analysis of six concentrations was used to assess the method's linearity for each drug Table . The sug57:They were calculated using two methods, according to ICH recommendations (Q2 R1)The first was the S/N ratio method, which used peak height data. In this method, a signal-to-noise ratio (S/N) of 3:1 was applied for the limits of detection (LOD) and 10:1 for the limits of quantitation (LOQ). The second method involved the use of the following “Eqs.  and 5)””5)”:4\do−1 for OSTP, 0.5 ng mL−1 for DEX, 2.0 ng mL−1 for DAC, and 0.5 ng mL−1 for REM, so the proposed method is suitable for their assay in human plasma. To achieve these low concentrations in plasma, solid-phase extraction (SPE), liquid–liquid extraction (LLE) with the salting-out technique, or liquid-phase microextraction (LPME)59 are excellent choices. They clean up the sample and preconcentrate the target analytes in very small volumes. This increases the concentration to a limit that can be detected by UV or PDA detectors. In addition, these techniques reduce the consumption of organic solvents, saving time, effort, and money.The LOD and LOQ values for the proposed method are shown in Table 36. The standard deviation (SD) and percentage relative standard deviation (% RSD) of the obtained findings were calculated. The RSD was found to be extremely small , indicating that the proposed method had appropriate accuracy and precision (Table The suggested method's accuracy and precision (intra-day and inter-day) were determined using three separate laboratory-prepared quaternary mixtures covering low, middle, and high levels at each linearity range (three replicates) Table . The obton Table .36 indicated no significant differences in statistical analysis (Student's t-test and Variance Ratio F-test) (Tables The findings obtained by the suggested and reported methods) Tables  and S8.Blank (30.0% (v/v) methanol), OSTP, DEX, DAC, and REM were all tested separately. The acquired blank chromatograms revealed no peaks during the retention times of the drugs under consideration. Furthermore, no peaks were noticed on any of the drug chromatograms except for their own. As a consequence, they were prepared in a quaternary solution ].k′ is the analyte peak's capacity factor, commonly known as the mass distribution ratio (Dm) or retention factor . Its suggested value is [0.5–10.0]51, where tR is the analyte peak retention time (min) and tm is the solvent front peak retention time (min).S is greater than 1.5, which represents the resolution of two consecutive peaks . Peak tR represents peak retention time, n represents peak serial number, and W represents peak width.As). Its recommended range is [0.8–2]52. W5% is the width of the analyte peak at 5% height, and f is the distance from the peak maximum to the leading edge of the peak measured at 5% of the peak height from the baseline.tR stands for retention time (min); and W50% stands for analyte peak width at 50% height.The robustness and system suitability parameters were determined both experimentally and theoretically using 2ng “Eqs. –9”31. ThAccording to Table 3 FFD estimated coefficients in uncoded units. The comparison revealed no significant differences, demonstrating that factorial design may take the place of the practical method.ANOVA-F at alpha = 0.05 and F-critical = 9.552 was used to compare the robustness and system suitability data obtained from the practical applications of the proposed method with those calculated from equations derived from 2According to Table 35. The collected data was compared to those from previously reported techniques36 using the Student's t-test and F-Test. The comparison indicated that there was no statistically significant difference between the suggested and reported methods36  without interference from excipients “” section36 Table .35, the uniformity of the dosage unit test of each dose form was also assessed. One of two methods (weight variation or content uniformity) can be used to demonstrate the dosage unit test's uniformity. The weight variation method is applied to hard capsules or film-coated tablets containing 25 mg or more of a drug substance that comprised 25% or more, by weight, of the dosage unit. The content uniformity method is applied to solutions in unit-dose containers or uncoated/film-coated tablets containing less than 25 mg of a drug substance or comprising less than 25% by weight of the dosage unit.According to BP standardsThe weight variation method was applied to OSELTAMIVIR hard capsules (containing 98.5 mg of OSTP and comprising 41.9% by weight of the dosage unit), while the content uniformity method was applied to DEXAZONE tablets (containing 0.5 mg of DEX and comprising less than 25 mg of a drug substance), JAVIDACLA film-coated tablets (containing 24.9% by weight of DAC in the dosage unit), and REMDESIVIR vials. The ANOVA test (P = 0.05) indicated that there was no statistically significant difference between the two obtained data sets for OSTP, DAC, and REM Tables  and S12.With such positive analytical results, the method could well be routinely used to evaluate various formulations, including the investigated drugs.38. The proposed method for the quaternary mixture was determined to be an excellent green analysis with low laboratory needs (Eco-score 78). Furthermore, the suggested method provided excellent green analysis when applied to the assessment of each drug separately (Eco-score greater than 75)  Table .38, the suggested method for the quaternary mixture was a direct procedure (no extraction process), as shown in Fig. −162) and environmentally friendly organic solvents. Furthermore, the suggested method was for both qualifying and quantifying. Figure According to the Green Analytical Procedure Index (GAPI)39.The greenness of the proposed method was also assessed by the AGREE method  Fig. . Analytik'(OSTP), T1(OSTP), and RS3(REM) of OSTP, DEX, DAC, and REM peaks were studied. They were affected by the interaction of three independent factors . The optimized conditions were then validated using the ICH Q2 R1 guideline. Without the influence of excipients, the proposed method was effectively used to estimate the average content and uniformity of dosage units of the drugs under study in their pharmaceutical formulations. The interference of excipients was assessed using the classic standard addition method and the innovative instrumental standard addition method. The instrumental standard addition method outperformed the classic standard addition method in that it was more accurate and sensitive, had fewer human errors, and was time-saving, economical, and more eco-friendly. The proposed method should be considered for the formulation of polypills. The developed method has high sensitivity, so it is suitable for assaying in human plasma. Additionally, the analytical Eco-Scale, GAPI, and AGREE proved the greenness of the proposed method, which aids its utility in routine applications.The suggested method is eco-friendly, sensitive, accurate, and precise, with a wide range of linearity. In terms of cost, the procedure uses inexpensive analytical reagents that are readily available in any analytical laboratory. The factorial design was used to create and optimize this procedure. The suggested method's robustness and system suitability parameters were predicted using the factorial design. The factors affecting the Supplementary Information.


For lignification, Phenylalanine ammonia-lyase (PAL) might start the phenylpropanoid pathway of lignin synthesis. Cinnamoyl-CoA reductase 4 (CCR4), cinnamyl alcohol dehydrogenase 2 (CAD2), and 4-coumarate--CoA ligase like 7 (4CL7) were up-regulated to promote the lignin monomer’s synthesis. Peroxidase 1 (POD1) and Laccase 3 (LAC3) were up-regulated to promote the oxidative polymerization of lignin monomers. These results suggest that changes of the cell wall structure and cell wall metabolism, as well as lignification, are involved in the senescence and quality deterioration of the banana after chilling injury.The banana is prone to chilling injury (CI) at low temperature and showing a series of chilling symptoms, such as peel browning, etc. Lignification is a response to abiotic stress and senescence, which is an important manifestation of fruits and vegetables during chilling exposure. However, little is known about the lignification of bananas during low-temperature storage. Our study explored the characteristics and lignification mechanism of banana fruits during low-temperature storage by analyzing the changes of chilling symptoms, oxidative stress, cell wall metabolism, microstructures, and gene expression related to lignification. The results showed that CI inhibited post-ripening by effecting the degradation of the cell wall and starch and accelerated senescence by increasing O Low-temperature storage, as one of the most useful technologies, can slow down the speed of cell metabolism and delay plant senescence to extend the post-harvest life of horticultural products ,2. HowevLignification, a response to abiotic stress and natural senescence, is observed in many chilling-sensitive horticultural products, such as loquat , mangostIn this study, the objective was to investigate the characteristics of CI as well as the mechanism of lignification induced in harvested banana fruits during storage at a low temperature. Some related physiological indexes, including chilling symptoms, cell wall component, enzyme activity, observation of tissue structure, and related gene expression, were tested and analyzed. This study provides a theoretical basis for the study of alleviating the symptoms of banana chilling injury by exploring its mechanism.Musa spp., AAA group cultivar “Brazil″) fruit (approximately 110 days after anthesis) was harvested from an orchard in Shawei Village, Wanqingsha Town, Nansha District, Guangzhou city, Guangdong province, China  and transported to the laboratory within 2 h. Banana fruit fingers were cut and then dipped in 0.1% Sportak®  fungicide solution for 3 min and finally allowed to air-dry. Fruit with uniform shape, color, and size were selected and randomly divided into two groups, which were stored at 25 °C or 6 °C respectively with 85–90% relative humidity depending on the allocated group. During storage, samples were periodically taken to measure the related physiological indexes. Some indicators related to the appearance, senescence, cell wall metabolism were measured. Some enzymes were related to the phenylpropanoid pathway and peel browning, including the up-stream enzymes PAL, PPO, and POD, were analyzed. Banana peel tissues were collected, frozen in liquid nitrogen, and stored at −80 °C for RNA extraction and physicochemical indexes analysis. Each treatment consisted of three biological replicates, with each replicate containing 60 fruit fingers.Green mature banana  by recording the force needed to penetrate 5 mm into the middle part of whole fruit. The flat probe of penetrometer is 8 mm in diameter. The results were expressed as N.Peel color. Banana peel color was measured with a Konica Minolta CR-400 colorimeter  in the CIEL*a*b mode. L* indicated the lightness ranging from black to white, C* represented the color saturation that varied from dull to vivid, and hue angle (h*) referred to a color wheel: red at an angle of 0°, yellow at 90°, green at 180°, and blue at 270° in accordance with the method by McGuire .Fo, Fm) were measured on dark-adapted (30 min) fruit. Fo was measured with a measuring beam at a light intensity less than 0.05 μmol m−2 s−1, while Fm was obtained by measuring chlorophyll fluorescence during a 2.5 s pulse of saturating light . The maximal variable fluorescence (Fv = Fm-Fo) and PSII quantum yield (Fv/Fm) were calculated from the Fo and Fm values.Chlorophyll fluorescence. Chlorophyll fluorescence of the banana peel was determined using a portable chlorophyll fluorometer . Minimum and maximum fluorescence yields (g) for 10 min at 25 °C and the supernatant was removed. Then, the residue was washed twice with 10.0 mL ethanol and the supernate was removed. The precipitate was hydrolyzed in H2SO4 (0.5 M) at 90 °C for 1 h. After cooling to room temperature, the sample was centrifuged (8000× g) for 15 min at 25 °C and the supernate was collected for protopectin analyzation. The following experimental operations mainly referred to corresponding assay kits  in accordance with the manufacturer’s instructions. The absorbance was analyzed by Multiskan Spectrum  at 530 nm. Protopectin content (mg g−1 FW) = mst * VTS * ΔA2/(ΔA1 * m * VS); ‘mst’—the quality of the standard substance, mg; ‘VTS’—total volume of enzyme-extracting solution, mL; ‘VS’—volume of the reactive enzyme liquid added; ‘m’—fresh weight of banana peel, g; ΔA1530nm = Astandard − Ablank, ΔA2530nm = Atest − Acontrol.Protopectin content. Protopectin was extracted and determined according to the method of Lu et al.  with somg) for 10 min at 25 °C and the supernate was removed. The precipitate was added to 1.0 mL DMSO for 15 h to remove starch and then centrifuged (6000× g) at 25 °C for 10 min to remove the supernate. The precipitate was dried as cell wall materials (CWM). A total of 50.0 mg of dried CWM was homogenized in distilled water (5.0 mL) and then added 7.5 mL H2SO4 slowly and incubated in ice water for 30 min. Then, the homogenate was centrifuged at 4 °C for 10 min (8000× g) and the supernate was collected for cellulose content analysis. After adding anthrone reagent and incubating at 95 °C for 10 min, the absorbance of sample was measured against a distilled water blank at 620 nm. Cellulose content (mg−1 g−1 DW) = [(ΔA620nm + 0.0043) * VS/5.25]/(m * VS/Vt); ‘ΔA620nm’ = Atest − Ablank; ‘VS’—volume of the extracting solution added, mL; ‘VT’—total volume of extracting solution, mL; ‘m’—dry weight of banana peel, g; ‘20’—dilution ratio of sample; ‘0.0043’ and ‘5.25’ were from the regression equation  under standard conditions.Cellulose content. Cellulose contents were determined using the anthrone method. Frozen banana peel (3.0 g) was homogenized in 10.0 mL ethanol of 80% for 20 min at 90 °C. After cooling to room temperature, the homogenate was centrifuged (6000× v/v) ethanol and centrifuged at 20,000× g for 20 min. The pellet was dried overnight at room temperature. Fifty milligrams of dried residue was suspended with 5.0 mL of 2 M HCl and 0.5 mL of thioglycolic acid. The sample was heated at 100 °C for 8 h and cooled on ice, then centrifuged at 20,000× g for 20 min (4 °C) and the supernate was remove. The pellet was washed with distilled water and re-suspended in 5.0 mL 1 M NaOH. The solution was agitated gently at 25 °C for 18 h and then centrifuged at 20,000× g for 20 min. One milliliter of concentrated HCl was added to the supernate and the lignin thioglycolic acid was allowed to precipitate at 4 °C for 4 h. After centrifugation at 20,000× g for 20 min, the pellet was dissolved in 1.0 mL of 1 M NaOH. The absorbance was measured against a NaOH blank at 280 nm, and data was expressed on a dry weight basis. Lignin content (mg g−1 DW) = [(ΔA−0.0068) * VTA * 10−3 * N]/(0.0347 * m); ‘ΔA’ = Atest − Abalnk; ‘VTA’—total volume of the reaction system, mL; ‘m’—dry weight of banana peel, g; ‘N’: ‘0.0068′ and ‘0.0347′ were from the regression equation  under standard conditions.Lignin content. Lignin was extracted and measured by the method of Bruce and West . Three g2O2 content. Superoxide anion and H2O2 content were determined using corresponding assay kits  in accordance with the manufacturer’s instructions.Superoxide anion and Hg and 4 °C, the supernate was collected as the crude enzyme extract of the analysis of PPO and POD activity. PAL activity was extracted with 50 mM sodium phosphate buffer (pH 8.8) containing 5 mM β-mercaptoethanol.Extration and assay of PPO, POD and PAL. Peel tissues (2.0 g) from three fruit were ground in liquid nitrogen and homogenized in 20.0 mL of 0.05 M phosphate buffer (pH 7.0) and 0.5 g of polyvinylpyrrolidone (insoluble) at 4 °C. After centrifugation for 20 min at 19,000× 525nm value was measured. One unit of enzyme activity was defined as the amount that cause a change of 0.005 in the absorbance at 525 nm per minute. PPO activity (OD525nm g−1 min−1 FW) = ΔOD525nm * Vt/(Vs * m * ΔT * 0.005); ‘Vt’—total volume of enzyme-extracting solution, mL; ‘Vs’—volume of the reactive enzyme liquid added, mL; ‘m’—fresh weight of banana peel, g; ‘ΔT’—reaction time, min.According to the method of Jiang  with som470nm g−1 min−1 FW) = ΔOD470nm * Vt/(Vs * m * ΔT * 0.005).POD activity was measured with 0.1 mL guaiacol (4.0%), 0.1 mL hydrogen peroxide (0.46%), 2.75 mL phosphate buffer (pH 6.8), and 0.05 mL enzyme-extracting solution. After adding enzyme fluid, the change value of the absorbance of 0.005 at 470 nm was measured. One unit of enzyme activity was defined as the amount that caused a change of 0.01 in the absorbance at 470 nm per minute. PPO activity (OD290nm g−1 min−1 FW) = ΔOD290nm * Vt/(Vs * m * ΔT * 0.005). All absorbance values were analyzed by using Multiskan Spectrum and the results were expressed on a fresh weight basis.PAL activity was assayed with the method of Assis et al.  with somg for 30 min at 4 °C and the supernate was collected for enzyme activity analysis. The enzyme was added in sodium acetate buffer  containing carboxy methyl cellulose (1.0% w/v) in a final volume of 10.0 mL. The mixture was incubated at 37 °C with vibration for 1 h and was then incubated at 90 °C for 15 min. After cooling down, the mixture was centrifuged at 8000× g for 10 min at 25 °C. The supernate was saccharification solution added with authrone and then incubated at 90 °C for 10 min. After another cooling, the samples were analyzed at 620 nm. One unit of cellulase activity is defined as the amount of the enzyme that releases 1.0 μg glucose per milligram of fresh weight per minute. Cellulase activity (μg min−1 g−1 FW) = [1000 * (ΔA + 0.0462) * VTA * VT]/[2.509 * (m * Vs) * T]; ‘1000′—1 mg mL−1 = 1000 μg mL−1; ‘VTA’—total volume of the reaction system, mL; ‘Vs’—volume of sample added, mL; ’VT’—total volume of enzyme-extracting solution, mL; ’T’—reaction time, min; ‘0.0462’ and ‘2.509’ were from the regression equation  under standard conditions.Cellulase activity. Cellulase activity was determined by the anthrone colorimetry method. A total of 2 g frozen tissue powder was dissolved in 20.0 mL sodium phosphate buffer  containing cysteine, HCl (20 mM), EDTA (20 mM), and TritonX-100 (0.05%). Then, the homogenate was centrifuged at 15,000× α-amylase and β-amylase activity. α-amylase and β-amylase were determined with the 3, 5-dinitrosalicylic acid method according to the characteristics that α-amylase was not resistant to acid and β-amylase was not resistant to the heat (70 °C). Assay kits  were used to analysis enzyme activity in accordance with the manufacturer’s instructions.Scanning electron microscopy observation and analysis. The banana peel was cut into regular squares and then fixed with glutaraldehyde  overnight and vacuumed. The glutaraldehyde was washed with 0.1 M phosphate buffer for 40 min, three times in total, and the samples were then dehydrated with 30%, 50%, and 70% ethanol for 20 min each time, followed by further dehydration with 80%, 90%, and 100% ethanol for 20 min each time. Finally, the samples were washed three times with tert-butanol for 20 min each time. The samples were finally freeze-dried and sprayed with gold  using the JFC-1600  ion sputtering instrument. The specimens were observed with a JSM-6360 LV  scanning electron microscope (SEM) at 15 KV.MaActin-3 was selected as the reference gene [−ΔΔCT. Three independent biological replicates were performed in the analysis.Quantitative real-time PCR analysis. DNA-free RNA was reverse-transcribed for first-strand cDNA synthesis. The gene-specific oligonucleotide primers were used for qRT-PCR analysis . The qRTnce gene . qRT-PCRThe experiments were arranged in a completely randomized design with three replicates, and the data were expressed as the mean ± SD (standard deviation). Data were analyzed by SPSS version 19.0. Least significant differences (L.S.D.) were calculated to compare significant effects at the 5% level.Fv/Fm ratio, which refers to chlorophyll content, also decreased as the CI symptoms appeared  storage ,19,20,21appeared C, which appeared D. Soften2 fixation is limited by environment stresses, such as CI, an excess of photosystem I reduction and ROS production is observed [2− [2− in banana peels stored at 6 °C increased during first two days and then decrease in the later storage days  is a vital enzyme between the shikimate pathway and secondary phenylpropanoid pathway, which can convert phenylalanine to mono- and diphenols, which are substrates of PPO . PAL actIt had been reported that the texture changes of certain CI-sensitive fruits under low-temperature storage were due mainly to disorders of cell wall metabolism, including a decrease in the solubilization and depolymerization of pectin .−1 DW, while the lignin content of banana peels stored at 25 °C did not change significantly  observation. As shown in POD1, PAL, LAC3, CCR4, CAD2, and 4CL7. Studies have shown that POD and PAL are the key enzymes of lignification, and the enzyme activities increase significantly as lignification occurs [POD1 for chilled banana peels was down-regulated at the early storage stage, then up-regulated from the second day, and finally higher than that of the control group at the end of the storage period (AtmiR397b can reduce the lignin deposition by negatively regulating LAC4 [ZmmiR528 regulates lignin biosynthesis and lignification by negatively regulating LAC3 and LAC5 [LAC3 increased dramatically, especially on the fourth day (PAL, POD, and LAC play important roles in the lignification of banana peels under chilling injury.To further understand the mechanism of lignification in bananas after CI, some genes possibly involved in the lignification process were selected for gene expression analysis, as shown in n occurs ,53. POD n occurs . In our e period A. This ie period B. LAC haing LAC4 . ZmmiR52and LAC5 . In our urth day C, which CCR4, CAD2, and 4CL7 were significantly up-regulated during the storage for chilled banana fruit, which was involved in the lignification process for bananas after chilling injury (Lignin is one of the important products of the phenylpropane metabolism pathway, and its biosynthesis involves many enzymes, such as CCR, CAD, and 4CL. CCR is the first rate-limiting enzyme in the lignin synthesis pathway, which has a potential regulatory effect on lignin monomer biosynthesis . CAD is g injury D–F. It i2− and hydrogen peroxide. In addition, chilling injury affected the normal post-harvest ripening process of banana fruit by inhibiting the normal degradation of starch grains and metabolism of the cell wall. We found that lignification, as a response to abiotic stress and senescence, is an important manifestation of banana fruits after chilling injury. The study showed that PAL might start the phenylpropanoid pathway of lignin synthesis, while the synthesis of lignin monomers was promoted by up-regulating the expression of the CCR4, CAD2, and 4CL7 genes. Then, the POD1 and LAC3 genes were up-regulated to promote the oxidation of lignin monomers. POD1 and LAC3 were finally up-regulated to promote the oxidative polymerization of lignin monomers. These results suggest that changes in cell wall structure and metabolism, as well as lignification, are involved in the senescence and quality deterioration of bananas after chilling injury. Studies on the characteristics and lignification mechanisms of bananas involved in chilling injury will provide a theoretical basis for heat-sensitive fruits and vegetables to alleviate CI.Low-temperature storage can improve the storage quality of fruits and vegetables but also has adverse effects on heat sensitive fruits and vegetables. The study comprehensively elucidated the involvement of chilling symptoms and the mechanism of lignification for bananas after chilling injury. In the present study, banana peel browning induced by chilling injury was promoted by the degradation of chlorophyll and the increased activity of POD and PAL. Chilling injury accelerated the senescence of banana fruit by increasing the content of O


S. aureus and MB removal was evaluated. EDX, XRD, and FTIR confirm the presence of the elemental compositions of CAZ. Friable CuO nanorods (10–70 nm in diameter) existed on the surface of the zeolite. Pure zeolite had a higher band gap (5.433 eV) and lower MB removal efficiency than CAZ. The adsorption method by CAZ was more effective at removing MB than photodegradation. 0.10 CAZ had the highest removal effectiveness (~ 99%) and adsorption capacity (~ 70.4 mg g−1) of MB. The inhibitory zone diameter for 0.005 CAZ against S. aureus was 20 mm, while 0.01 CAZ had a diameter of 17 mm. Azithromycin, ceftriaxone, and erythromycin antibiotics demonstrated lower or no efficacy against S. aureus than CAZ. Significant antibacterial activities and wastewater treatment were achieved by CAZ. The combination of photodegradation and adsorption enhanced pollutant removal. It will be interesting to study further the optimal molar ratio for MB removal (0.10 CAZ) in future investigations.Novel CuO/Ag nanocomposites added zeolite (CAZ) were successfully fabricated, and their effectiveness as an antibacterial on  The discharge of dyes into surface water has an impact on the photosynthesis process of aquatic plants, which is one of the most significant food sources for living things there. Moreover, dyes negatively impact human and animal health because they are toxic, carcinogenic, and resistant to oxidizing and biodegrading agents3. Consequently, polluted water must be treated before being discharged into the waste stream. However, dyes appear to be challenging to remove from wastewater using traditional methods due to their relative stability and high-water solubility. Several methods have been used to remove pollutants from wastewater, with adsorption and photocatalytic degradation being the most commonly used. The adsorption method is inexpensive, simple, and effective for a wide range of dye concentrations5. Methylene blue (MB), methyl orange (MO), malachite green (MG), congo red (CGR), and rhodamine B (RHB) are examples of organic dye pollution6.Large quantities of stable industrial effluents with high organic dye content continue to be a significant barrier to achieving clean water using traditional methods. Most dyes are toxic and commonly used in coloring industrial products like leather, cosmetics, textiles, drugs, paper, and printing inks. The majority of dye binds to the surfaces of products during the dyeing process, and the rest is released into surface water (rivers and lakes) without treatment, resulting in polluted water, solar cells, and gas sensors, among other applications11. CuO is a p-type semiconductor with a small band gap (Eg = 1.2 up to 1.9 eV), low-cost, and non-toxic nature. It has been used to enhance the photocatalytic activity of some wide-band gap semiconductors12. Silver and copper are among the most important toxic elements for gram positive and negative bacteria14. Furthermore, Ag nanoparticles in Na+-Y-zeolite and Cu2+ nanoparticles in X-zeolite have excellent antibacterial activity16. The polypyrrole-Ag/graphene/O-carboxymethyl chitosan nanocomposite and polypyrrole-Ag NPs have shown excellent antibacterial action, successfully inhibiting the growth of E.coli and S. aureus bacteria18. Electrostatic interactions allowed the cationic Ag nanoparticles to bind to the bacterial surface19. Ag nanoparticles can interact with the cytoplasm of E. coli and S. aureus bacteria and destroy them20. Different silver nitrate molar ratios have been examined in Ag/zeolite nanocomposite, and Ag (0.004 M)/zeolite showed the best photocatalytic activity for dye removal and good antibacterial activity21. CuO has also been synthesized with several composite materials, including zeolite/Fe3O4, TiO2, WO3/CdS, and Ce/Zn, and demonstrated excellent photocatalytic activity for producing hydrogen and dye pollution removal26. Generally, nanoscale materials have a higher specific surface area than that in their bulk. The higher surface area makes available more atoms with a reduction in coordination and can cause rapid agglomeration. Because of agglomeration, the exceptional properties of nanoparticles such as catalysts may be lost. Therefore, zeolites are used as host matrices and supported for nanoparticle applications such as dye photodegradation and adsorption in addition to being a catalyst30. Zeolites interact with other materials on their surfaces and throughout the bulk. Among all the available natural materials adsorbents, zeolite is commonly used for the removal of toxic pollutants from water because of its low-cost, negative charge, open structure, and high ability to exchange ions31. Zeolites are microporous aluminosilicate crystalline materials formed of silicon, aluminum, and oxygen in a framework. According to the ratio of aluminum and silicon, there are several kinds of zeolites such as A-zeolite, β-zeolite, X-zeolite, and Y-zeolite. Zeolites have exceptional chemical and physical properties due to their unique structure and act as adsorbents, antibacterial agents, and molecular sieves (for separation)34. The materials that destroy bacteria without being toxic to surrounding tissues or the environment are known as antibacterial agents. They have a variety of uses, including the filtration of bacterially polluted water, the packaging of food and medicine, and the production of antibacterial textiles.Nanomaterials have been widely researched for their capability to remove dyes from wastewater and eliminate bacteria. CuO and Ag have been synthesized as nanoparticles or nanocomposites and examined to use as photocatalysts, antimicrobials, anticancer30. Also, the existence of antibiotic resistant bacteria is one of the biggest challenges to universal health because of their growing resistance to various antimicrobial substances13. Besides that, several components can be added with particular advantages to improve the dye removal and antibacterial performance of a single component. Therefore, it would be interesting to investigate the effects of CuO/Ag nanocomposite, CA added zeolite, Z (CAZ) on dye removal in addition to inhibiting or destroying the bacteria.Some adsorbents have low adsorption capabilities and can only be used under certain circumstances for the removal of dyes35. Moreover, the potential of CAZ as an adsorbent for MB removal in the dark from polluted water was studied. The antibacterial activities of CAZ were estimated by using bacteria of Staphylococcus aureus (S. aureus), which usually exist in wastewater16. The antibacterial effects of azithromycin, ceftriaxone, and erythromycin antibiotics were compared with CAZ. We investigated the impacts of several molar ratios of Cu2+, C , AgNO3, A (0.004 M), and zeolite, Z (0.5 gm) contents in CAZ. Pure Z was also examined for comparison. We characterized CAZ by SEM, TEM, EDX, and XRD techniques to investigate their morphology, elemental compositions, and structure. The optical properties of the prepared samples were characterized. The optical gap energies of samples were estimated. Spectra of FTIR and TGA of CAZ were also recorded.In this work, CAZ were successfully fabricated by a simple technique, co-precipitation as adsorbents, photocatalytic, and antibacterial. The photocatalytic activities of CAZ were estimated through the degradation of toxic dye  under various UV irradiation times. MB causes eye burns, shock, dyspnea, increased heart rate, cyanosis, vomiting, nausea, diarrhea, and tissue necrosis2:Al2O3 mole ratio 13:1, Alfa Aesar), Methylene blue (MB), , Silver nitrate , Copper (II) nitrate. (Cu(NO3)2.2.5H2O, 98+%, Chem-Lab NV), Sodium hydroxide .Zeolite mordenite, sodium (SiO36. Constant concentrations of AgNO3 (0.004 M) and zeolite (0.5 gm) with a different molar ratio of Cu (NO3)2.2.5H2O  were combined to obtain 0.005, 0.01, 0.05, and 0.10 CAZ. Zeolite powder was suspended in deionized water (75 ml) and stirred for 10 min at 60 °C. AgNO3 solution (5 ml and 0.004 M) was slowly added to the zeolite solution and stirred for a further 10 min. Following that, Cu(NO3)2.2.5H2O (20 ml) with different molar ratios was slowly added. The mixture was stirred for 2 h (500 rpm) at 60 °C, followed by the slow addition of NaOH , and left to stir for a further 1 h at that temperature. The mixture of CAZ was filtered and dried at room temperature. The obtained samples  were heated in a furnace for 2 h at 550 °C. A schematic illustration of CuO/Ag nanocomposite added zeolite is shown in Scheme CuO/Ag nanocomposite added zeolite mordenite (CAZ) was synthesized by the co-precipitation technique2+  were added to the polluted water (50 ml). The suspension was stirred at room temperature in the dark for 30 min and then under UV irradiation (λ = 365 nm) for 30, 60, 90, 120, and 150 min for adsorption and photodegradation tests. Every 30 min, ~ 7 ml liquor was collected and centrifuged  to remove the solid CAZ catalyst particles. By using a UV/VIS spectrophotometer at λ = 664 nm, the filtrates were analyzed. The MB removal efficiency (Reff.) and adsorption capacity (Ac) of samples were obtained by the following eqations5.V are the initial and final concentrations and volume of MB solution, respectively; m is the weight of CAZ.The photocatalytic and adsorption activity of CAZ were examined by reducing methylene blue  in deionized water at room temperature. 0.14 gm of CAZ with different molar ratios of CuStaphylococcus aureus were defined by specialists at the medical lab department, Aljanad University for Science and Technology, Taiz, Yemen. These were kept on nutrient agar at 4 °C. A loopful of cells from the stock cultures was transferred to a tube containing 1 ml of ordinary saline for bacteria, which was cultured for 24 h at 37 °C to create the active cultures for the tests. The McFarland 0.5 turbidity level was then attained by diluting the cultures with ordinary saline37.Stock cultures of 38.Mueller–Hinton agar aliquots of 20 ml were put into sterile Petri plates. Using a sterile cotton swab, the isolates and standardized bacterial stock suspension were streaked on Mueller Hinton agar medium plates after being adjusted to 0.5 McFarland. A disc of 3 mm diameter was put on sterile Mueller–Hinton agar plates. Each disc was filled with 500 µg of the chemical agents tested. The plates were incubated at 37 °C for 24 h. The diameters of inhibitory zones were measured using a ruler in millimetersThe crystalline structure of pure Z and CAZ was analyzed by X-ray powder diffraction (XRD) using Bruker, D8 Discover with CuKα radiation (λ = 1.5406 Å). The field-emission scanning electron microscope , transmission electron microscope  and energy-dispersive X-ray  spectroscopy were used to observe the morphology and estimate elemental compositions of the samples, respectively. Spectra of UV–visible absorbance and Fourier transform infrared (FTIR) of samples were recorded using spectrophotometer Lambda 950 and Nicolet 380, respectively. Thermal gravimetric analysis (TGA) of samples was recorded using LTG-A10, LABTRON. PL spectra of samples were investigated using PL system S/N: 1001.2+ molar ratios are shown in Fig. 2θ = 6.50°, 8.61°, 9.75°, 13.41°, 13.82°, 14.56°, 15.27°, 19.59°, 22.19°, 25.55°, 26.23°, 27.83°, and 30.82°. These peak positions correspond to the planes of (110), (020), (200), (111), (130), (021), (310), (400), (150), (202), (440), (530), and (402), respectively. Also, a small number of peaks corresponding to nano CuO (PDF# 02–1040) were observed in the added samples (CAZ) at 2θ = 35.8°, 38.7°, 48.5°, 66.2°, and 68.4° as a secondary phase. The peak of a low Ag molar ratio (0.004 M) did not appear. MDI Jada 6 software and the following Scherrer’s equation39 were used to determine the crystalline size (Cs) of CuO.The X-ray diffraction patterns for the pure Z and CAZ with different Cu2+ molar ratios are shown in Fig. 2+ amounts (0.05 and 0.10 M). Nano CuO was also described as having a nanorod-like structure with those of other studies40. The increase of CuO nanorods peak in the X-ray diffraction patterns confirmed the results shown in Fig. 10 as shown in Fig. 41. However, the peaks at about 0.25 keV and between 3 to 3.5 keV signified the binding energies of Ag nanoparticles42.FESEM images of CuO/Ag nanocomposite added Zeolite with different Cu−1. The spectra of added samples are almost similar to that of the pure Z. A new peak appeared at ~ 1385 cm−1 in the spectra of added samples (0.05 CAZ and 0.10 CAZ) belonging to CuO, respectively43. Ag peak with a low molar ratio (0.004 M) did not appear in the FTIR spectra. The vibrations bands in the 3454–3623 are attributed to the hydroxyl group, OH45, whereas the peaks between 1600 and 3700 cm−1 are attributed to the zeolitic water. The peak of O–Al–O was observed at ~ 1081, while the O–Si–O peak was presented at ~ 700 cm−1. The located peak at 1636 cm−1 belongs to Si–OH vibration.Figure −1. The initial degradation process was in the range between 80 and 700 °C because of the loss of physical water and chemically absorbed water evaporation. The major thermal degradation process (second stage) was in the range of 700– 800°C. This can be explained by separating the macromolecular framework and forming stable metal oxalate intermediates46. The third stage was in the range higher than 800 °C due to more decomposition of metal oxalates and the formation of metal oxide. The thermal degradation of pure and added zeolite started at around 80 °C and continued up to 1000 °C. The mass loss was about 48.4% for the pure Z and 64.7% for 0.05 CAZ. It was clear that the pure sample had higher thermal stability than the added samples.TGA analysis was used to investigate the thermal stability of added samples and compare them with pure zeolite. Figure 47. For example, the direct and indirect energy gaps for natural zeolite clinoptilolite were 4.46 and 4.26 eV, respectively48. The band gap of CuO nanoparticles with zeolite was 4.35 eV43. A theoretical energy gap value for an idealized clinoptilolite (44 Si and 76 O atoms) was 5.593 eV49.The optical characteristics of zeolites have been investigated in a few earlier research. Natural zeolites recorded a band gap energy lower than the standard aluminosilicate zeolites (7 eV)2+ in Z (0.005 CAZ and 0.01 CAZ) exhibited a high absorption beak in the range of ~ 221.5 nm and had similar absorbance spectra. However, the maximum absorption peak of a large amount of Cu2+ (0.05 and 0.10 M) was observed at 233 nm and 237 nm, respectively. The existence of CuO in/on zeolite may clarify the absorption peaks increased with the increasing molar ratio of Cu2+ and extended to a wider wavelength  compared to the pure Z. Large concentrations of Cu2+ (0.05 and 0.10 M) displayed an extra absorbance peak at 238 and 240 nm, respectively, which could be attributed to CuO nanorods8. The direct transition band gap energy (Eg) was determined using Tauc's equation 39.n = 1/2, is the type of transition, hν) and extrapolating the linear part near the onset on the curve to the axis of hv. Figure 2+ molar ratio increased. That could be explained as the result of structural deformation, in which Ag ion was replaced by Cu2+ in the CuO lattice. These values were consistent with the results of other research40.In this study, the optical absorptions of pure Z and CAZ were examined at room temperature in the wavelengths between 200 and 850 nm through UV–visible spectrophotometer. As shown in Fig. 2+ molar ratios were also investigated through the degradation of MB under various UV irradiation times. Figure Reff.) in the dark and UV irradiation. CAZ was a more effective removal efficiency of MB than the pure Z. A slight increase in the MB removal efficiency was observed when exposed to UV light for longer than 30 min compared with efficiency in the dark. The MB removal effectiveness by pure Z was about 93% in the 30 min dark condition, while CAZ demonstrated removal efficiencies of between 96 and 97.5%. When exposed to UV light for longer than 30 min, pure Z presented removal efficiencies of between ~ 93 and ~ 96%, whereas CAZ exhibited efficiencies of between ~ 96 and ~ 99%. The maximum MB removal efficiency was achieved by 0.10 CAZ with the highest Cu2+ concentration and lowest energy gap during 30 min of darkness and 120 min of UV exposure. The adsorption capacity (Ac) of pure Z and CAZ to the MB removal is shown in Fig. −1 in the dark and increased to 68.52 mg g−1 with increasing the UV irradiation time. However, the adsorption capacity of CAZ was higher than that of pure Z. The adsorption capacity of 0.10 CAZ was up to 70.37 mg g−1  and superoxide  pairs could be created because the CAZ photocatalysts in the MB solution absorb light energy (hv) equal to or higher than their energy gap (Eg). Electrons in CuO/Ag could be excited from the valence band to the conduction band, resulting in the immediate production of holes in the valence band  pairs was determined during the activation of photocatalysis. Figure 61. Pure zeolite displayed a peak at 425 nm and fluorescence intensities greater than 0.01 CAZ, 0.05 CAZ, and 0.10 CAZ. The PL spectra of CAZ samples showed an emission peak at 500 nm. In comparison to pure Z, nanocomposite CuO/Ag-modified zeolite (CAZ) samples were more successful in reducing e/h pair recombination rates, hence improving photocatalysis. 0.10 CAZ was the most effective at slowing e/h pair recombination and increasing photocatalysis.The process of photogenerated electron transfer in CAZ can be examined using PL spectroscopy. The rate of recombination for electron/hole  was examined using the disc diffusion technique. The antibacterial performance of CAZ was examined based on the diameter of the inhibitory zone around the tested chemicals. Figure S. aureus bacteria. The bacterial inhibition zones indicate that 0.005 CAZ and 0.01CAZ had antibacterial activity against S. aureus bacteria. The inhibitory zone diameter for 0.005 CAZ was 20 ± 1.5 mm, while it was 17 ± 0.8 mm for 0.01 CAZ. No bacterial inhibition zones were detected for 0.05 CAZ and 0.10 CAZ. The antibacterial effect of azithromycin (AZM), ceftriaxone (CTR), and erythromycin (E) antibiotics were compared to that of CAZ  than CAZ. The removal efficiency and adsorption capacity of MB were higher in CAZ than in pure zeolite. The highest MB removal efficiency of ~ 99% was achieved by 0.10 CAZ. A combination of CAZ photodegradation and adsorption enhanced the efficacy of MB removal. The adsorption process had the most significant effects on the MB removal rate compared to the photodegradation process. S. aureus bacteria was inhibited by a 25 mm inhibition zone formed by 0.005 CAZ and a 17 mm inhibition zone formed by 0.01 CAZ. Azithromycin, ceftriaxone, and erythromycin were less effective or ineffective against S. aureus than CAZ. These findings demonstrated that CAZ had significant antibacterial properties in addition to its capacity for wastewater treatment. In future work, examining the effect of CuO/Ag nanocomposites addition with different Ag and zeolite concentrations will be interesting.Novel nanocomposite CuO/Ag-modified zeolite (CAZ) with different Cu