Helicobacter pylori. The current investigation was carried out as a single center, open label, two phase, single dose, randomized way in cross over manner to evaluate the potential of pharmacokinetic interaction among amoxicillin formulation and ranitidine in adult healthy male volunteers. Post dosing blood samples were collected at multiple time points that are 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 10 hours after administering amoxicillin 250mg capsule with and without ranitidine. For estimation of amoxicillin concentration in plasma, an HPLC method was developed and validated. The solvent system consisted of 0.025M phosphate buffer: acetonitrile (94:6 v/v). C18 column was employed with a flow rate of 1.0 ml/minute and at 230nm. A linear pattern with a correlation coefficient of 0.999 in the concentration ranges of 25μg/mL to 0.097μg/mL for amoxicillin and 25μg/mL to 0.048μg/mL for ranitidine was observed. Amoxicillin retention time was about 8 minutes and ranitidine retention time was around 12 minutes. Amoxicillin levels were computed and the concentrations were applied to calculate the pharmacokinetic parameters. Pharmacokinetic parameters were estimated by Kinetica TM 4.4.1 (Thermo Electron Corp. USA). The analysis of variance (two way) and t test (two one sided) were applied on log transformed pharmacokinetic parameters of amoxicillin. The Tmax was determined between amoxicillin alone and amoxicillin with ranitidine by Friedman test. The 90% confidence interval values for Cmax (0.687–0.743) and Tmax (1.148–1.742) for amoxicillin with or without ranitidine were not found within the FDA acceptable limits of 0.8–1.25. Study demonstrated the significant reduction in peak plasma levels of amoxicillin in presence of ranitidine. It is advisable to administer both drugs with time interval to avoid such interactions and increases in the bactericidal efficacy of amoxicillin.Polypharmacy may be considered as the customary practice to provide optimum care services to patients but inter resulted in augmented probability of multiple drug interaction. Keeping in view the importance of drug interaction possibility, this study was designed to evaluate the effect of ranitidine on pharmacokinetics of amoxicillin in the local population of Karachi, Pakistan. Amoxicillin and ranitidine are the most commonly prescribed drugs to treat duodenal ulcer caused by  In order to treat a patient with a single disease or multiple diseases, different drugs are usually administered to the same patient. During hospitalization, inpatients usually receive multiple drugs due to various comorbid situations including cardiovascular, gastrointestinal or neurological diseases, dermatological disorders, hepatic or renal impairment or irrational use of medicines. Drug interaction may occur during multiple drug therapies –3 and onIn relation to drug-drug interactions, it is crucial to know whether co-administration of another drug therapy influences the effectiveness and safety of one drug therapy or not. A drug-drug interaction arises when one drug’s pharmacodynamics and/or pharmacokinetics are changed by another drug. Co-administration of two drugs may result in one of the most precarious forms of drug interactions. Reported results indicate that drug-drug interaction can affect various aspects of pharmacokinetics. Most common evidences were found to be alteration in the absorption  –6, distrAmoxicillin (semi synthetic penicillin) is a frequently prescribed drug due to its good oral absorption. Amoxicillin is a broad spectrum β-lactam antibiotic that is commonly used in clinical practice –16. AmoxRanitidine is a H2 receptor blocker, it is the most frequently prescribed drug and is used to treat gastro esophageal reflux disease and gastric or duodenal ulcer . It achiet al. also reported pharmacokinetic interaction of ranitidine with ABT-773 on 12 healthy volunteers [Pletz lunteers ,21. The lunteers .Helicobacter pylori infection [et al. evaluated the pharmacokinetic interaction of ranitidine 300mg tablets and daijokito (Kampo medicine) on seven healthy volunteers [Different studies reported the occurrence of gastroduodenal ulcer in local population of Karachi, Pakistan in association with nfection ,24. A confection . In Pakilunteers .Hence, in this study the pharmacokinetic (PK) interaction between Amoxicillin 250 mg capsules and ranitidine 300mg tablets has been studied in a randomized, two phasic, crossover, single dose study following FDA guidelines in local population.rthophosphoric acid, acetonitrile (HPLC grade) and methanol were of HPLC grade, Merck Germany. Deionizer; Elga, High Wycombe, England. Borosilicate Glassware  was used. HPLC system was coupled with LC-10 AT VP pump, detector specifications were SPD-10 A UV detector and Class GC software from Shimadzu were used, system efficiency was also optimized with guard column . Method developmenta was carried out using Purospher star RP-18  column, a highly sensitive analytical balance , centrifuge , pH meter , microliter syringe , filtration assembly , vortex mixer , swinney membrane filter , sonicator (LC20H) and Millipore filtration assembly  were used.Amoxicillin trihydrate capsules (250mg) and Ranitidine hydrochloride tablets (300mg) manufactured by GlaxoSmithKline Pakistan Ltd. were purchased from the community market. Crucial information like manufacturing and expiry dates were noted along with the batch number. Ranitidine hydrochloride and amoxicillin trihydrate reference standards used were gifts by Central Drug Laboratory, Karachi, Pakistan and Indus Pharma (Pvt) Limited respectively. Disodium hydrogen phosphate, OIn the present study Amoxicillin 250 mg capsules sold under the name of Amoxil and ranitidine 300mg tablets sold under the name of Zantac were selected as reference products for the PK interaction study as both are the innovator products of GlaxoSmithKline, Pakistan Ltd.A PK interaction study was conducted in a hospital setting to estimate various pharmacokinetic parameters of both products and then variables were compared to estimate the possibility of PK interaction between Amoxicillin 250 mg capsules and ranitidine 300 mg tablets, selected as reference products. Batch number, date of manufacturing and expiry are mentioned in In the present study, twelve healthy male volunteers having code as V1 –V12 with age group between 21–28 years (mean age was 23.5 years), weight between 55–74 kg (mean weight was 64.33 kg) and height between 5.4–5.9 ft.in (mean height was 5.50 ft.in) were selected for the present study  of Ziauddin University, Karachi, Pakistan (0440612SPPHAR). Following ICH guidelines, all the subjects were notified about the possible events of the present study. A written consent in English and in local language (Urdu) was signed by all participants involved in the study. Volunteers were allowed to leave the study at any time without difficulty, however all volunteers were able to complete the study duration.The present PK interaction study was designed as a randomized, two phasic, crossover, single dose. Before the drug administration, all volunteers were asked to fast for 10 hours. The Amoxicillin 250 mg capsules were coded as “A”, and co-administration of Amoxicillin 250 mg capsules and ranitidine 300mg tablets were coded as “B”. The present study was conducted in two phases. Each subject randomly received A and B with 240 ml of drinking water following fasting for an overnight period. One week wash out period was given in between the doses  with pH 3.1 was used as the mobile process. For separation, C18 column was used with a flow rate of 1.0 ml/minute and at a wavelength of 230nm. ICH Q2-B guidelines were taken into consideration for validation of the bioanalytical method.All the volunteers were assessed in order to determine any adverse effects related to the drug by physical examination and other clinical tests. Volunteers were encouraged to immediately notify in case of any unusual event. Complete physical examinations and vital signs determinations were carried out at screening period and on study days of each treatment phase. Subjects were asked for any significant signs of GI disturbances, pain or any noticeable conditions.max, Cmax, AUC0-α, AUC0-t, λz, T1/2λz, AUMC and MRT. The pharmacokinetic data was fitted in one compartment oral model. Different parameters like sequence effect, subject effect, period effect were calculated by Latin ANOVA (two–way) using Kinetica® version 4.4.1. ANOVA was applied. The PK variables were analyzed on log transformed pharmacokinetic data of Amoxicillin  and Amoxicillin and ranitidine (in combination). The 90 percent CIs were determined using the acceptance limits of 0.8–1.25 and 0.8–1.2 respectively, based on differences in geometric mean values of log transformed results.In the present study non-compartmental analysis using Kinetica® version 4.4.1, Thermo electron Corp., USA has been applied for the estimation of various PK parameters and variables including TThe pharmacokinetic parameters of amoxicillin were calculated and compared to those obtained in combination with ranitidine to see if there was any pharmacokinetic interaction. An analytical method validation was performed for the determination of plasma drug concentration profile under the guidelines of ICHQ2B. Intraday and interday accuracy and precision were found within acceptable limit and method showed the linearity with a correlation coefficient of 0.999 , p value of less than 0.05 was measured statistically significant to note the geometric mean value differences of log transformed results. For pharmacokinetic analysis of amoxicillin, PK/PD software, Kinetica® version 4.4.1, Thermo electron Corp., USA was used. These variables were statistically tested to see whether there was any pharmacokinetic interaction between the two products. To compare the pharmacokinetic parameters of amoxicillin given alone and in combination with ranitidine, Analysis of variance (ANOVA) latin square was used. To compare Tmax were 3.113± 0.064 μg/mL for amoxicillin given alone and 2.288± 0.043 μg/mL when it is given with ranitidine. When amoxicillin was given with or without ranitidine, there was a very large difference in measured Cmax (p = 0.0008). Non compartmental analysis exhibited that the geometric mean of observed Cmax for amoxicillin alone was 3.099±0.141μg/mL and 2.477± 0.190 μg/mL for amoxicillin administered with ranitidine. When p value is equal to 0.015, there was a substantial difference between amoxicillin administered alone and with ranitidine. For amoxicillin alone, the geometric mean for calculated Tmax was 1.912± 0.019 hrs and 2.228± 0.031 hrs for amoxicillin given with ranitidine. The 90 percent confidence interval was 114.8 to 117.4 percent. There was a significant difference in Tmax of amoxicillin in the presence and absence of ranitidine (p = 0.0001). AlGaai et al. conducted a bioequivalence study on two amoxicillin formulations and found Tmax values of 1.92 ± 0.76 and 2.02 ± 0.62 hr for test and reference formulation respectively [0-t was 12.582±0.272 mg/L.hr and for amoxicillin given with ranitidine, it was 10.611±0.359 mg/L.hr. A highly significant difference in AUC0-t of amoxicillin administered alone and in presence of ranitidine with p value of 0.0033. The geometric mean of AUCtotal for amoxicillin given alone and coadministered with ranitidine were 12.783±0.301 mg/L.hr and 11.192±0.182 mg/L.hr correspondingly. For amoxicillin, the geometric mean of clearance was 19.817±0.394 L/hr and 23.524±0.411 L/hr for amoxicillin plus ranitidine. In presence of ranitidine, amoxicillin clearance was significantly reduced (p = 0.001). Amoxicillin’s geometric mean volume of distribution (Vc) was 31.383±0.604 L, and amoxicillin’s volume of distribution in the presence of ranitidine was 46.137±0.503 L. The volume of distribution (Vc) of amoxicillin in presence or absence of ranitidine was significantly different (p = 0.0007). ANOVA was used to test the log transformed data of amoxicillin, with less than 0.05 p value deemed statistically relevant at 90% confidence interval and at 0.8–1.2 acceptance limit. No significant effect of subject, time or sequence on measured Cmax, observed Cmax or Tmax of amoxicillin was found as p values were more than 0.05. When amoxicillin was co-administered with ranitidine, the AUC0-t, AUClast and AUCtotal values were significantly modified (p < 0.05), but there was no significant impact of covariables. The 90 percent confidence intervals on log transformed Cmax values of amoxicillin alone and in presence of ranitidine were found to be beyond the acceptance limits of 0.8–1.2 and 0.8–1.25 respectively   .el) and end half-life (T1/2Kel) managed alone were discovered to be 0.636 ± 0.008 hr-1 and 1.088 ± 0.019 hr while when given with ranitidine; Kel was decreased to 0.511 ± 0.011 hr-1 and T ½ Kel was expanded to 1.357 ± 0.019 hr. The elimination half-life and rate constant of amoxicillin was markedly altered when administered with ranitidine (p = 0.003). Guoxin et al., 2002 announced that the elimination half-lives calculated by amoxicillin administration of test and reference formulation were 1.09±0.22 hr and 1.43±0.44 hr respectively [el and half life are dependent upon clearance and volume of distribution. However, it would be unreasonable to make any assumptions about the parameters of a drug solely on the basis of its half life. For amoxicillin, the geometric mean of absorption rate constant was observed to be 0.715 ± 0.015 hr-1 when given alone and 0.653 ± 0.012 hr-1 when given along with ranitidine. The values for amoxicillin absorption half-life were 0.973 ± 0.016 hr and 1.069 ± 0.016 hr when amoxicillin given alone and in combination with ranitidine respectively. There was a significant effect exhibited on amoxicillin absorption rate constant and amoxicillin absorption half-life when given with ranitidine with p value of 0.0004. . Drug interaction was considered significant if the difference in amoxicillin parameters did not fall between 0.80 and 1.25 using 90% CI after log transformation of data. The geometric mean ratios of Cmax, Tmax, AUCtot. AUC0-t. AUC0-∞, T1/2Kel and MRT of log-transformed data were calculated as 0.715, 1.161, 0.870, 0.831, 0.840, 1.233 and 1.263 respectively Click here for additional data file.


Locusta migratoria manilensis is one of the most important agricultural pests in China. The locust has high fecundity and consumes large quantities of food, causing severe damage to diverse crops such as corn, sorghum, and rice. Immunity against pathogens and reproductive success are two important components of individual fitness, and many insects have a trade-off between reproduction and immunity when resources are limited, which may be an important target for pest control. In this study, adult females L. migratoria manilensis were treated with different concentrations (5 × 106 spores/mL or 2 × 107 spores/mL) of the entomopathogenic fungus Paranosema locustae. Effects of input to immunity on reproduction were studied by measuring feeding amount, enzyme activity, vitellogenin (Vg) and vitellogenin receptor (VgR) production, ovary development, and oviposition amount. When infected by P. locustae, feeding rate and phenol oxidase and lysozyme activities increased, mRNA expression of Vg and VgR genes decreased, and yolk deposition was blocked. Weight of ovaries decreased, with significant decreases in egg, length and weight.Thus, locusts used nutritive input required for reproduction to resist invasion by microsporidia. This leads to a decrease in expression of Vg and VgR genes inhibited ovarian development, and greatly decreased total fecundity. P. locustae at 2 × 107 spores/mL had a more obvious inhibitory effect on the ovarian development in migratory locusts. This study provides a detailed trade-off between reproduction and immune input of the female, which provides a reliable basis to find pest targets for biological control from those trade-off processes. Paranosema (Nosema) locustae is an entomopathogenic fungus that only infects locusts. Since the 1980s, the fungus has shown great potential as a biological control in locust control . The feeding temperature was set at 30 ± 2°C with a photoperiod L16: D8 and 50% relative humidity. Locusts were hatched in small, clean boxes (10 cm × 15 cm × 20 cm) and then released into large, dry, well-ventilated cages of 50 cm × 50 cm × 50 cm at a density of 200 to 300 insects per cage. Locusts were provided fresh wheat seedlings and wheat bran for at least two generations before experimentation. A suspension of P. locustae provided by the China Agricultural University  was diluted to either 5 × 106 spores/mL or 2 × 107 spores/mL and maintained at 4°C.Colonies of 6 spores/mL and another with 2 × 107 spores/mL of P. locustae, and both were dried at 37°C for 10 min. The third portion was used as a control treatment. Food was provided from emergence, and on days 1–4 post emergence, the average daily weight of wheat seedlings consumed was recorded. The three groups of locusts were reared in the same conditions, and males and females were paired. Each treatment included three biological replications of five locust individuals each.Fresh wheat seedlings were divided into three equal portions of 100g each. One portion was soaked with 5 × 10P. locustae-infection and control groups, with five individuals per replicate. In the three groups of locusts, fat bodies and ovaries were dissected at 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 d. Fat bodies and ovaries were frozen with liquid nitrogen and stored at −80°C.There were three biological replicates of 595 nm value was determined. In a blank control, 60 μL of CAC buffer and 60 μL of 3 g/L L-dopa were used. Detection time of PO activity was 30 min. Total protein content was determined using a Coomassie bright blue protein kit following the manufacturer’s instructions . The OD495 value was calculated as sample OD495 value − control OD495 value. One unit of PO activity (U) was defined as U = OD495 value/min/mg protein.To detect PO activity, 2 µL of serum was diluted to 20 µL using 0.9% normal saline, followed by addition of 40 μL of CAC buffer and 60 μL of 3 g/L L-dopa. Solutions were mixed and placed in a 25°C water bath for 30 min, and then, the OD0). Lysozyme activity (U) was calculated as U = (A0 − A)/A.The detection method for lysozyme activity was adjusted according to Hultmark . To prep® RT Reagent Kit with gDNA Eraser  following the manufacturer’s protocol. All primers were designed to determine expression of the genes VgA, VgB, VgR1, and VgR2  following the manufacturer’s instructions. Each treatment included three biological replicates of five locust individuals each. Dissecting time was 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 d after emergence. The RNA concentration was determined by measuring the absorbance at 260 nm with a spectrophotometer. First-strand cDNA synthesis was performed using a PrimeScriptand VgR2 Table 1.P. locustae, ovarian tissue sections were prepared by a paraffin method. Female locusts 8 days after eclosion were immersed in 10% formaldehyde solution for 12 h. Fixed samples were then dehydrated in an ethanol series of 50%, 75%, 90%, and 100%, with at least 30 min in each step. Following dehydration, samples were cleared with xylene. Cleared samples were embedded in paraffin and sectioned. Sections were dewaxed with xylene and ethanol, hydrated, and stained with hematoxylin and eosin. After staining, samples were dehydrated with anhydrous alcohol for 30 s and 1 to 2 drops of neutral glue were added to seal the samples. Samples were observed and photographed under a microscope. Each treatment included three biological replicates of five locust individuals each.In controls and groups infected with P. locustae, the length and weight of egg pods and the length and width of each egg were measured. Each treatment included three biological replicates of eight individuals each. In an additional set of experiments, amount of oviposition was measured in the two treatments and control. Each treatment included three biological replicates of eight individuals each. After hatching, offspring were fed individually in transparent plastic feeding tubes and maintained in an artificial climate chamber. Each treatment included more than 30 individuals. Locust nymph development was recorded twice a day at 8:00 AM and 8:00 PM.In controls and groups infected with P < 0.05 was considered statistically significant.Statistical analyses were performed with the GraphPad Prism 8.3.0 software. Except for the data from sections of ovarian tissue, statistical significance analysis of the remaining data presented in this study were analyzed using one-way or two-way ANOVA followed by Tukey’s multiple comparisons test. The confidence interval was set to 95%, and L. migratoria manilensis metabolism after microbial infection, effects of infection with different concentrations of P. locustae on locust feeding behavior were determined. Compared with uninfected control females, infected locusts consumed more food . When infected with a concentration of 2 × 107 spores/mL, food intake increased significantly and was more than 1.48-fold higher than that in the control , duration of infection , and their interaction . PO activity in 5 × 106 and 2 × 107 spores/mL treatments was higher than that in the control from day 2 to 12 but was significantly lower from day 16 to 20. PO activity was 1.33-fold and 1.38-fold higher in 5 × 106 and 2 × 107 spores/mL treatments, respectively,than that in the control at 6 days , duration of infection , and their interaction . Compared with the control, microsporidian infection increased lysozyme activity from day 2 to 8 but decreased it from day 10 to 20. Lysozyme activity was strongly induced at 4 days and was 1.28-fold (5 × 106 spores/mL) and 1.52-fold (2 × 107 spores/mL) higher than that in the control. Lysozyme activity at the high concentration (2 × 107 spores/mL) began to decrease significantly by day 10. However, enzyme activity at the low concentration (5 × 106 spores/mL) was not significantly different from that in the control from day 10 to 18 , duration of infection, and their interaction  . The low concentration treatment had no effect on oviposition amount, whereas at the high concentration, total number of eggs laid decreased by 17%  and width  of eggs decreased significantly after P. locustae infection  decreased significantly, and nutritional status of eggs also decreased. Overall, the results of this study suggest the immune response to P. locustae reduced energy resources required for locust reproduction. In addition, new ideas were generated in this study for further studies on molecular mechanisms of the trade-offs between immunity and reproduction in locusts. Further studies are warranted on allocation of energy resources between immune responses and reproductive output and on identification of potential regulatory mechanisms, including energy-metabolizing and nutrient signaling, that could be targets for control of L. migratoria.In this study, decreased expression of The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.Paranosema locustae for this research. Y-WH wrote the initial manuscript. S-HW, BT, LZ, S-GW and YT involved in interpreting data and revising manuscript. All authors read and approved the final manuscript.S-GW and BT designed the work. Y-WH, G-QX, Y-JD and Q-YX carried out the experiments. Y-WH, S-HW, and YT performed the analysis. LZ provides This work was supported by National Key Research and Development Program of China (Grant No. 2017YFD0201000) and National Natural Science Foundation of China (Grant Nos. 31270459).Thanks to China Agricultural University for providing Paranosema locustae for this research.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.


Staphylococcus aureus (ATCC 25923), and Pseudomonas aeruginosa (ATCC 27853). The results indicated spherical ZnO nanoparticles on the surface of malachite and strong antibacterial activity and antioxidant properties. The ointments produced from the nanocomposites also exhibited wound healing activity. The administration of the ointments prepared from ZnO/Mlt, and ZnO/Mlt/Chsn NCs decreased the expressions of IL-1β, IL-6, and TNF-α, while it increased the expressions of IL-10, TGF-β and bFGF. In sum, the nanocomposites produced from ZnO, malachite, and chitosan had better biological activity than ZnO/Malachite nanocomposites. We suggest applying ZnO/Mlt/Chsn nanocomposites in the structure of ointments to treat infected wounds after future clinical studies.Recently, nanocomposites produced from clays and metals coated with chitosan have shown wound healing activity. This study aimed to synthesize the zinc oxide/malachite nanocomposite (ZnO/Mlt-NC) and its coating form with chitosan (ZnO/Mlt/Chsn-NC). Physicochemical characterization of the produced nanocomposites was investigated. Biomedical effects of nanocomposites, such as in vivo and in vitro antibacterial activity, antioxidant properties, cytotoxicity, and modulation in the gene expressions of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), and transforming growth factor-β (TGF-β) and histopathological parameters, were also investigated. Expression intensities of basic fibroblast growth factor (bFGF) and tumor necrosis factor alpha (TNF-α) were also investigated by immunofluorescence staining. To investigate biomedical effects under in vivo conditions, infected wounds were induced and inoculated with  Wounds are classified as acute and chronic based on the pathogenesis and consequences2. Acute wounds induce molecular processes to obtain structural integrity. Immune cells and factors play pivotal roles in acute wound healing3. The faulted regulation of the immune response results in the formation of chronic wounds4. Infectious wounds are a form of acute wounds characterized by the presence of bacteria in viable tissue and damage to tissues5. The infections start with bacteria colonization and can cause systemic infection. Staphylococcus aureus and Pseudomonas aeruginosa are the most common bacteria in infected wounds6. In infected wounds, the wound healing process is delayed7. Infected wounds also cause overproduction of reactive oxygen species and induce faults in antioxidant systems8. Antibiotics are used to treat infected wounds; however, they cause antimicrobial resistance. Therefore, it is essential to find safe and novel agents for the treatment of infected wounds9.Skin wounds cause damage to healthcare systems and loss economic10. Copper oxide is extensively used for wound healing activity owing to its biomedical properties like antibacterial activity3. Malachite is a form of copper oxide ore derivatives as Cu2CO(OH)211. It has been reported that malachite exhibits antibacterial activity against S. aureus and P. aeruginosa by changing the integrity of the cell membrane and antioxidant properties12. Recently, studies have reported synergistic interaction effects between clays and metal nanoparticles15. Metallic nanoparticles, including ZnO nanoparticles (ZnONPs), are extensively used in medicine. ZnONPs exhibit antioxidant properties via the electron donation property of the oxygen atom in ZnO nanomaterial and antibacterial properties via the electrostatic activity between the negative charge of bacterial cells and the positive charge of ZnO nanoparticles that can be used in the wound healing process16. ZnONPs also influence oxidative stress and increase the generation of reactive oxygen species16. They also promote the regeneration of damaged tissues by activating collagen synthesis17. Metal oxide NPs, including ZnO NPs, may be stabilized by being mixed with other inorganic structures like malachite19. This study aims to synthesize ZnO/Mlt-NC as an agent for the wound healing process using the external biofabrication method. This method requires plant active compounds, including phenolic compounds for capping and stabilizing15. In this study, we used pennyroyal (Mentha pulegium) extract to synthesize ZnO/Mlt-NC as a capping and stabilizing agent. M. pulegium belongs to the mint family, Lamiaceae. It contains some phenolic compounds, such as syringic acid, ferulic acid, and flavonoid compounds, including isorhamnetin-3-O-glucoside and kaempferol-3-O-rutinoside that can contribute to capping and stabilizing. Therefore, in the present study, for the first time, M. pulegium extract was used to synthesize ZnO/Mlt-NC20.Metal oxides nanomaterials are safe and cheap structures used to treat wounds21 and anti-inflammatory22 properties and exhibits skin regenerative behavior, biocompatibility, and biodegradability23. It is also a safe compound and has very low toxicity21. It shows antibacterial activity via penetration in the cellular membrane21 and clears free radicals via adsorption, ion-exchange, and chelation13.To improve biological properties, the synthesized nanocomposites were coated with chitosan. Chitosan is known to have antibacterialIn this study, ZnO/malachite nanocomposites coated with chitosan (ZnO/Mlt/Chsn-NC) were synthesized to heal infected wounds in a mouse model. In addition, not only the structural properties of ZnO/Mlt/Chsn-NC were investigated, but also the efficiency of ZnO/Mlt/Chsn-NC and ZnO/Mlt-NC was evaluated. Furthermore, antibacterial activity, antioxidant properties, and the genes expression of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-10 (IL-10), transforming growth factor-β (TGF-β), basic fibroblast growth factor (bFGF), and tumor necrosis factor alpha (TNF-α) were investigated.Figure 2, Cu(CO3)2, Cu(OH)2, Fe2O3, Al2O3, and other metallic oxides, such as Na, Ca, K and Mg oxides11. Figure 15.Malachite contains SiO11. In addition to malachite, a small amount of quartz was also observed. The results indicated strong and sharp diffraction lines originated from ZnO with a hexagonal structure. The peaks observed at 31.76°, 34.42°, 36.25°, 47.53°, 56.59°, 62.85°, 67.94° and 69.08° show (100), (002), (101), (102), (110), (103), (112) and (202) planes, respectively. The results demonstrate the presence of ZnO nanoparticles, being in accordance with the standard spectrum of the JCPDS file No. 01-079-0206 13. A band at 1558 cm−1 is observed for the N–H bending of the primary amine. The CH2 bending and CH3 symmetrical deformations were approved by bands found at around 1423 and 1393 cm−1, respectively. Figure The ZnO/Mlt-NC produced in this research showed stability due to in situ bio-capping by the phytochemical metabolites in pennyroyal derivations. The results of FTIR confirm the results Fig. F–H. The Figure max of the malachite, ZnO/Mlt-NC, and ZnO/Mlt/Chsn-NC was in the range of 102.2, 124.8, and 236.3 nm, respectively. The values obtained for the zeta potential of colloidal suspensions in the malachite, ZnO/Mlt-NC and ZnO/Mlt/Chsn-NC were found at + 17.2, + 18.9, and + 24.5 mV, respectively.Figure Figure P. aeruginosa and S. aureus. ZnO/Mlt/Chsn-NC showed better antibacterial activity than the antibiotics did in MIC and MBC tests.Table P. aeruginosa, but it formed a small inhibition zone for S. aureus (0.50 ± 0.707 mm). ZnO/Mlt nanocomposite had a higher inhibition zone than Mlt. The results revealed that ZnO/Mlt created an inhibition zone of 21.50 ± 1.41 mm for S. aureus, while it formed an inhibition zone of 18.50 ± 1.41 mm for P. aeruginosa (P = 0.021). The highest inhibition zone was observed in ZnO/Mlt/Chsn-NC than in the other nanocomposites. The results demonstrated that ZnO/Mlt/Chsn-NC created inhibition zones of 25.25 ± 2.12 mm and 23.25 ± 1.41 mm for S. aureus, and P. aeruginosa bacteria, respectively, which were significantly different(P = 0.0285). The diameters of the bacitracin antibiotic zone as a standard for S. aureus and the polymixin B antibiotic as a standard for P. aeruginosa were 20.10 ± 1.83 mm and 18.21 ± 1.12 mm for S. aureus and P. aeruginosa bacteria, respectively, which were significantly different (P = 0.0375). The results did not exhibit significant differences (P > 0.05) between ZnO/Mlt/Chsn-NC and antibiotics for the zone diameter.The results of the well test in Fig. The results of the kinetic section demonstrated that all nanocomposites showed antibacterial activity from 1 to 24 h. Mlt, ZnO/Mlt, and ZnO/Mlt/Chsn nanocomposites demonstrated antibacterial activity at all times. The results of the kinetic time confirmed other antibacterial results. Based on kinetic findings, ZnO/Mlt/Chsn-NC exhibited the most powerful antibacterial activity, so that ZnO/Mlt/Chsn-NC decreased bacteria colony from 3 h after incubation, and the colony number was almost zero in 12 h after incubation; antibiotics showed similar effects after 24 h, and ZnO/Mlt and antibiotics exhibited similar effects.P = 0.0001). Production of ROS was greater in NCs than in the control treatment (P = 0.0001). Parallel with other findings, the greatest release of LDH was observed in bacteria exposed to ZnO/Mlt/Chsn, ZnO/Mlt, Mlt and control groups, respectively. Production of ROS was also greater in ZnO/Mlt/Chsn, ZnO/Mlt, Mlt and control groups.As the results show, NCs exhibited antibacterial activity via the release of LDH and production of ROS Fig. . The res12. Malachite shows its antibacterial activity via the change in the integrity of the cell membrane and antioxidant properties12. The findings also revealed the antioxidant activity of Mlt in the DPPH test. Apparently, Mlt exhibited antibacterial activity in a longer time. The findings for the kinetic time indicated that Mlt showed antibacterial activity in a longer time. Moreover, ZnO/Mlt had better antibacterial activity that could be attributed to the presence of ZnO. The results are similar to the ones reported by Ehsani et al.24, demonstrating that loading ZnO on clays could improve antibacterial properties. ZnO shows antibacterial activity via the electrostatic activity between the negative charge of bacterial cells and the positive charge of ZnO nanoparticles. The results show a positive interaction between Mlt and ZnO for antibacterial activity. ZnO/Mlt/Chsn-NC had better antibacterial activity than ZnO/Mlt that could be attributed to the antibacterial activity of chitosan. Chitosan is known to have antibacterial properties by mechanisms, including penetration in the cellular membrane21. In the current study, the release of LDH and the production of ROS were investigated. Based on findings, NCs penetrate into bacterial membrane integrity, disrupt it, and increase the release of LDH in bacteria. In addition, the produced ROS directly attacks bacterial membrane, destroys it, and finally causes cellular death. The results show that Mlt, ZnO, and Chsn have a synergism interaction effect on antibacterial activity. The effects of ZnO/Mlt/Chsn-NC were almost similar for both bacteria, revealing its efficiency for both bacteria.The findings indicated that Mlt showed weak antibacterial properties. The results are similar to the ones reported by Anju et al.12 reported the antioxidant activity of Mlt via decreasing reactive oxygen species. Loading ZnO on Mlt improved antioxidant properties that could be attributed to the electron donation property of the oxygen atom in the ZnO nanomaterial26. Addition of chitosan to ZnO/Mlt increased antioxidant properties that might be attributed to the antioxidant properties of chitosan. Chitosan clears free radicals by mechanisms, such as adsorption, ion-exchange, and chelation21. In sum, antioxidant structures of ZnO, Mlt, and Chsn exhibit synergistic effects for antioxidant activity, since the values were higher for all concentrations in ZnO/Mlt/Chsn than for similar concentrations in Mlt and ZnO/Mlt.Figure 27 showed that malachite at higher concentrations exhibited cytotoxicity owing to the involvement in antioxidant activity. The results showed that addition of ZnO decreased cytotoxicity that could be attributed to coating effects of ZnO. Gharehpapagh et al.13 showed that nanoparticles had lower cytotoxicity that coated the surface of the malachite and decreased its toxicity. Loading chitosan improved viability that might be attributed to coating effects of chitosan and its low toxicity. In sum, viability was almost 100.00% in ZnO/Mlt/Chsn, indicating it as a safe nanocomposite. Although Mlt showed cytotoxicity, it only lowered 10% viability.Figure P = 0.925) between the groups on day 3. The highest wound contraction was observed in the ZnO/Mlt/Chsn group than in the other groups on day 7 (P = 0.0001). The mice in polysporin and ZnO/Mlt exhibited similar wound contraction on day 7 (P = 0.485). The lowest contraction was observed in Mlt and Cnl groups on day 7. The mice in the Cnl group showed the lowest wound contraction on day 12. The mice exhibited a higher wound contraction in the Mlt group than in the Cnl group on day 12 (P = 0.025). The results indicate that Mlt has poor wound healing activity. Studies have not reported the wound healing activity of Mlt. However, ZnO/Mlt/Chsn revealed good wound healing activity and could compete with the standard ointment of polysporin. Studies have shown synergism interaction effects between ZnO and Chsn for the wound contraction24. ZnO and Chsn have antibacterial properties and improve histological parameters, such as edema, fibroblast, and collagen. The results suggest that ZnO/Mlt/Chsn is an appropriate for wound healing activity.Figure 28.The results of the total bacterial count in Fig. 15. The findings show that prepared ointments decrease the inflammation, and the results are consistent with the results of the antibacterial section.Figure P < 0.05) higher in the mice treated with polysporin and ZnO/Mlt/Chn on day 12 . Epithelium did not have any thickness on day 3. The results indicated that the epithelium scores were significantly higher in the mice treated with polysporin and ZnO/Mlt, ZnO/Mlt/Chsn than in the control group on day 12.Table  12 Fig. . All the24. Anti-inflammatory properties and proliferative promoting effects are the mechanism suggested for the wound healing activity of ZnO nanoparticles24. Regarding the effects of chitosan as the wound healing structure, it must be stated that it causes to produce blood vessels, growth factors, and activating endothelial cells15. Chitosan and ZnO are antibacterial structures that accelerate wound healing, decrease the inflammation, and move the wound toward the proliferative phase. All the ointments increased collagen synthesis, thereby contracting the wound and closing it.Malachite decreased edema and immune cells. In literature review, there was no paper investigating the effects of malachite on histological parameters. Notably, malachite shows weak antibacterial properties that decrease the edema and promote the proliferative phase. The results of histological parameters for ZnO are similar to the results reported by Ehsani et al.P < 0.05) higher and lower in the control groups than in the other groups, respectively  compared to the control group. The administration of ZnO/Mlt and ZnO/Mlt/Chn ointments significantly (P < 0.05) increased the expression of IL-10 on days 3 and 7, and TGF-β on day 7 compared to the control group. The lowest expression for IL-1β and the highest expression for IL-10 were observed in the mice treated with ZnO/Mlt/Chsn ointment and polysporin.The results of immunofluorescence staining showed that expression intensities of TNF-α and bFGF were significantly  were purchased from Merk and Sigma companies, respectively. Medicinal grade of malachite powder (free from hazardous elements) was prepared from Iranshid Co. Mashhad, Iran. To investigate morphological properties, EDX  was used. Hydrodynamic diameter andsize distribution were evaluated, and zeta potential of the samples was investigated by DLS . FTIR spectra (Shimadzu model FTIR 8101N spectrometer) was also used.M. pulegium were collected from  Botanical Garden of Tabriz University, washed with distilled water, shaken for 24 h, and placed in an ultrasound bath under reduced pressure. It was then exposed to microwave radiation and filtered to obtain the supernatant and stored in a refrigerator for further use based on other studies15.Leaves of 2 was mixed with 0.60 g of malachite powder in 75 ml of the extract, and then vigorously stirred at 65 °C for 12 h. The obtained raw product was then separated after centrifuging. This precipitate was washed with distilled water and ethyl alcohol to remove the remained ions and other impurities. Afterward, it was dried at 70 °C for 1 h to prepare ZnO/Mlt/Chsn-NC. One gram of ZnO/Mlt-NC powder was gradually added into 50 mL protonated chitosan solution (acetic acid 1% w/w) at room temperature, stirred for 24 h, and ZnO/Mlt/Chsn-NC was obtained. The therapeutic ointments (2% w/w) were formulated as reported by Mahmoudabadi et al.14. In sum, a certain amount of NCs (2 g) was mixed with 98 g base ointment to prepare NC ointments.To prepare ZnO/Mlt-NC, 0.20 g of ZnCl36, for S. aureus (ATCC 25923), and P. aeruginosa (ATCC 27853). In short, the wells were filled with 100 μL of fresh culture media . Then, 100 μL of the prepared nanomaterials were added to it at the highest concentration (64 µg/mL). Serial concentrations were prepared up to the tenth well. Additionally, 100 μL of the bacterial suspension (5 × 105 CFU/mL) was added for all the wells except for the first well (negative control). The OD values were investigated in a wavelength of 630 nm after 24 h and at 37 °C. First well lack of turbidity was considered as MIC. To determine MBC, 10 µL was produced from wells lacking turbidity, negative wells, and positive wells with turbidity and then cultured in nutrient agar media. MBC was considered the minimum concentration of nanomaterial that inhibited bacteria growth after 24 h. The results were compared to the control antibiotics of bacitracin 10%  for S. aureus and to polymixin B for P. aeruginosa.MIC and MBC methods were assessed as reported by Koeth et al.S. aureus (ATCC 25923), and P. aeruginosa (ATCC 27853), as reported by Ehsani et al.24. Summary, 100 μL of 2% Mlt and ZnO/Mlt and ZnO/Mlt/Chsn were administrated into each well, and the diameter of the inhibition zone was measured at 37 °C after 24 h. The findings were compared to the control antibiotics of bacitracin for S. aureus and to polymixin B for P. aeruginosa.The antimicrobial activity of the nanocomposites was evaluated with the agar diffusion method against S. aureus and P. aeruginosa were investigated at different times as reported by Nguyen et al.37. Briefly, bacterial suspension solution was added to the Muller-Hinton media (Merk Company- Germany) culture containing nanocomposites with the same MIC concentrations and incubated in a shaker incubator at 37 °C, 200 rpm and for 24 h. Samples were collected at time intervals of 0, 1, 3, 6, 12 and 24 h and cultured on the Muller-Hinton media culture. The colony forming unit (CFU) of the organisms was investigated in triplicate, and a graph of the log CFU/mL was illustrated against time as reported by Nguyen et al.37.The growth curves of S. aureus and P. aeruginosa) were cultured on foils (Mlt and ZnO/Mlt and ZnO/Mlt/Chsn) located on inserted in 6-well plates (200 μL MH broth with 5 × 103 cells per foil) and incubated for overnight. Cells without treatment were regarded as control. The samples were then transferred into microcentrifuge tubes and centrifuged at 1200 rpm for 5 min. Additionally, 100 µL of supernatants were transferred to 96-well plates, and 100 μL of the LDH assay mixture were included into each well. The plate was coated and incubated for 30 min at room temperature. The optical density of per well was registered at 450 nm on an ELISA reader, and the LDH leakage was reported as follows:To evaluate lactate dehydrogenase (LDH), bacterial cells , the cellular reactive oxygen species detection assay Kit  was used, and all the protocols were conducted based on the producer Company. All the conditions for culturing and incubation were similar to those for LDH. Following the incubation, the samples were transferred to microcentrifuge tubes and centrifuged at 1200 rpm for 5 min. Then, 100 µL of the supernatants were transferred into 96-well plates, and 100 μL of the diluted DCFDA were added to each well and incubated for an additional 45 min at 37 °C in the dark. Production of DCF was evaluated by fluorescence spectroscopy with an excitation wavelength at 485 nm and an emission wavelength at 535 nm on an ELISA reader.13 using 0.1 mL Mlt, ZnO/Mlt, and ZnO/Mlt/Chsn diluted at concentrations of 25, 50, 75, and 100 μg/mL and 2.9 mL of 0.025 g/L DPPH in DMSO. In sum, after incubation at 25 °C for 45 min, the absorbance was then recorded at 517 nm using a PowerWave XS microplate spectrophotometer.2,2-diphenyl-1-picrylhydrazyl analysis was used to investigate antioxidant activity as reported by Gharehpapagh et al.5–1 × 106 cells/mL) were investigated as previously reported by Tominaga et al., (1999) using staining with calcein-AM and a fluorescence microscope  with 490 nm excitation to monitor viable cells for Mlt, ZnO/Mlt and ZnO/Mlt/Chsn38.To evaluate cytotoxicity, L929 cells   aged 10–12 weeks with a weight of 28 ± 4 g were prepared. The animals had unlimited access to water and food. They were kept under lab conditions prior to the start of study as the conditioning period. The mice were kept under a lighting diet of 12 h. This study lasted for 12 days in accordance with the Iranian ethical guidelines for the use of animals. All the used protocols, such as study design, sample size, randomization, outcome measures, data analysis, experimental procedures, and report of results, were in agreement with the ARRIVE guidelines,. The protocols were approved by the Committee on the Ethics of Animal Experiments of Veterinary Faculty and the Islamic Azad University Council on Animal Care, Tehran, Iran (IR.IAU.SRB.REC.1399.180), and were in compliance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health . We declare that all methods were performed in accordance with the relevant guidelines and regulations.7 bacterial cell of S. aureus. Furthermore, P. aeruginosa was immediately administrated into the wound site as reported previously39. The treatment was started 24 h after inoculation of bacteria. The mice were divided into 5 groups and daily treated with 0.5 g of ointments of polysporin® , Malachite (Mlt), ZnO/Mlt, and ZnO/Mlt/Chsn. A group was regarded as control (Cnl) and was not treated. To prepare ointments, 2 g Mlt, ZnO/Mlt, and ZnO/Mlt/Chsn were mixed with base ointment , and 2%-ointments were prepared. The administration of ointments lasted for 12 days. The wound area was investigated with the help of a transparent paper over the wound as reported previously39. In summary, the wound site was measured by placing a transparent paper over the wound and tracing it using a graph sheet and formula calculations.To induce wound, the general anesthesia was conducted by administrating the ketamine (50 mg/kg)/xylazine (10 mg/kg) cocktail intraperitoneally. After induction of anesthesia, the hair was shaved, and a circular full-thickness wound (with a diameter of 7 mm) was created by surgical cutaneous punch on the skin, and 0.5 McFarland Standard suspension in Muller-Hinton media (Merk Company- Germany) containing 1039 with the help of sterile swab and cultured on plate count agar on days 3, 7 and 12, and the results were reported as CFU/g of granulation tissue. In short, 0.1 g of the sample was crushed, minced, and homogenized in a sterile mortar containing 10 mL of sterile saline and then diluted in tubes containing 9 mL of sterile saline. They were then cultured on plate count agar, incubated and the colonies were calculated.Bacterial colony counts were investigated as reported previously2 healthy tissue, fixed in formalin, embedded in paraffin, and stained with hematoxylin and eosin and sirius red staining, and they were investigated by a light microscope as reported previously on days 3, 7, and 1239. Two blinded pathologists investigated the samples by a microscope and reported the results.To investigate histopathological parameters, the samples were prepared from granulation tissue, along with 1–2 mmLPS was investigated as recommended by the producer company and using commercial kits .40. Briefly, skin samples were collected from the wound bed with 1–2 mm outside the wound. Paraffin-embedded tissue sections were dewaxed, rehydrated and immersed in citrate buffer (pH 7.4) for 20 min to retrieve antigen. The used antibodies included bFGF , TNF-α , Goat Anti-Mouse IgG , and Goat Anti-Rabbit IgG .Immunofluorescence staining was conducted as reported by previous study on days 3 and 739 and assessed by a spectrophotometer (260 nm and 260/280 = 1.8–2.0). The used primers included IL-1β, forward (5′-AACAAACCCTGCAGTGGTTCG-3′) and reverse (5′-AGCTGCTTCAGACACTTGCAC-3′); IL-6, forward (5′-CTTCCATCCAGTTGCCTTCTTG-3′) and reverse (5′- AATTAAGCCTCCGACTTGTGAAG-3′); IL-10, forward (5′-CCATCATGCCTGGCT CAGCAC-3′) and reverse (5′-TGTACTGGCCCCTGCTGATCC-3′); and TGF-β, forward (5-CCAAACGCCGAAGACTTATCC-3′) and reverse (5′-CTTATTACCGATGGGATGGGATAGCCC-3′). Finally, mRNA expressions were measured by quantitative real-time polymerase chain reaction using the 2_ΔΔCt method and b-actin as an internal control.On days 3 and 7, the wound tissues were isolated as previously reported, and RNA was extracted by applying a standard TRIZOL procedure based on previous studyThe data were investigated for normality by the Shaipro-wilk test in the SPSS software (version 23). The data were normal for all the parameters excluding histopathological parameters. The data for histological parameters were analyzed by Kruskal–wallis, and other data were analyzed by the one-way analysis.All the used protocols were approved by the Committee on the Ethics of Animal Experiments of Veterinary Faculty and the Islamic Azad University Council on Animal Care, Tehran, Iran (IR.IAU.SRB.REC.1399.180), and also by the US National Institutes of Health .


Historical land-use practices have caused forest loss in Cambodia’s Tonle Sap Lake area (TSLA), the largest freshwater lake in Southeast Asia. However, it remains unclear if this deforestation trend had continued since 2001 when the land was designated as protected areas. Using satellite imagery, we investigated forest conversion flows and fragmentation patterns in the TSLA for 1992–2001, 2001–2010, and 2010–2019, respectively. Results show substantial forest losses and fragmentations occurring at the lower floodplain where the protected areas are located until 2010, with some forest regain during 2010–2019. The land conversions indicated that forest clearing and agricultural farming were the primary causes for observed extensive forest loss during 1992–2010. Hence, despite the creating of protected areas in 2001, our findings reveal the persistence of alarming forest loss in the TSLA until 2010. On the other hand, while net forest loss has stopped after 2010, forest regain during 2010–2019 is way too small to restore the region’s total forest area to even the level when the protected areas were established. Thus, more effective planning and implementations of forest management and restoration policies are needed for the TSLA.The online version contains supplementary material available at 10.1007/s13280-022-01704-4. Forests provide essential ecosystem services including mitigating greenhouse gas emissions, regulating water flow, supporting aquatic ecosystem and freshwater fisheries, maintaining soil fertility, sustaining wildlife and biodiversity, and providing a source of income for residents Keskinen . Globall2 in the dry season to ~ 15 000 km2 in the wet season, creating a so-called annual monomodal flood pulse . At the same time, agricultural land expanded by 34%, making it one of the leading factors of the diminishing forests in the TSLA  since the 1960s, characterized by rapid deforestation and agricultural expansion ADB . Forest The critical question is: as one of the most prominent protected areas in Cambodia and even the Mainland Southeast Asia, has the declaration of the TSLA as a key protected area in 2001 helped slow down, stop, or even reverse the deforestation in this region? Lohani et al.  analyzedHere, we assessed the LUCC with a particular focus on the forest cover change in the TSLA during 1992–2019, which was further equally divided into three sub-periods , using the European Space Agency (ESA) Climate Change Initiative (CCI) Land Cover product. ESA CCI provides the first long-term and successive annual time series of land cover change for 1992–2019. It has been validated by external parties and applied in various high-impact societal issues, for instance, analyzing carbon and water budgets with land surface models  during dry and wet seasons, respectively  offers annual global land cover classifications with 300 m spatial resolution from 1992. It is developed with machine learning and unsupervised classifications based on the global daily surface reflectance observations from AVHRR, MERIS, PROBA-V, SPOT-VGT, Sentinel-3 OLCI, and SLSTR .The ‘Fragmentation Model’ developed by Riitters et al. , 2016 hawww.nis.gov.kh) (NIS p < 0.05) and Sen's slope  NIS . Althoug.kh) NIS . Therefo.kh) NIS  with a clope Sen  for measlope Sen .2 in 1992, which decreased to 1935.8 km2 in 2001, 958.5 km2 in 2010, but slightly increased to 1018.8 km2 in 2019 . As a result, the net forest loss was about 1640.3 km2  in 1992–2019. Meanwhile, about 95.9 km2 of shrubs were lost (−1.4%/year) during this period. On the other hand, wetlands have considerably increased by 1620 km2 over the years. Urban areas have expanded from 11.1 to 63.3 km2, croplands have increased by 57.2 km2 simultaneously, while water bodies remained roughly unchanged.Figure 2  in 1992 to 196.4 km2 (1.5%) in 2001, 43.3 km2 (0.3%) in 2010, and then slightly recovered to 52.7 km2 (0.4%) in 2019. In fact, any forest blocks greater than 1.5 × 1.5 km2 had hardly remained since 2010. Such forest fragmentation trends primarily took place in the protected areas at the lower floodplain, where most of the forest interior area were located in 1992. By 2001, however, big patches of forest interior area in the northwest had gone. This fragmentation trend of forest patches had continued until 2010. Similar forest fragmentation patterns can also be revealed by applying other ‘windows’ sizes  Fig. S1). Overall, although the TSLA was declared a protected area in 2001, rapid forest fragmentations continued until 2010.Along with the overall trend of forest loss, we also found rapid forest fragmentations since 1992 in the TSLA Fig. a–d. The Figure Croplands in the TSLA, on the other hand, experienced an increase from 1992 to 2011, followed by a slight decrease. The annual change rates of croplands in the TSLA were 0.07%/year, 0.13%/year, and −0.11%/year for 1992–2001, 2001–2010, and 2010–2019, respectively. Compared to the TSLA, Cambodia as a whole experienced a more prominent cropland expansion till the 2010s. The annual change rates of croplands in Cambodia were 1.17%/year, 0.98%/year, and 0.06%/year for 1992–2001, 2001–2010, and 2010–2019, respectively.The TSLA’s upper and lower floodplains are dominated by different land cover types and subject to different social-economic and management activities. We found that the two subzones possessed diverging land cover changes during the study period. Figure S2, and Tables S1–S3), the most extensive land-use conversion was from forests to wetlands, followed by conversions from forests to cropland and shrubs to croplands. Statistically, 91.9% of the forest loss was converted to wetlands (691.1 km2), 5.4% of the forest loss was directly converted to croplands (40.5 km2), and 54.7% of the shrub loss was converted to croplands (16.1 km2). Land conversions in the whole TSLA between 2001 and 2010 presented a similar pattern: 91.8% and 2.3% of the forests were lost to wetlands (960.5 km2) and croplands (24.4 km2), respectively; and 84.1% of shrubs were converted to croplands (76.1 km2). Between 2010 and 2019, land conversions showed very different patterns and were dominated by some forests regain (60.3 km2). During this sub-period, 5.6% of forests were still lost to wetlands (59 km2), while less than 1 km2 of forests were lost to other land cover types. However, more forests were reclaimed from shrubs (5.2 km2), wetlands (79.9 km2), and croplands (34.9 km2). In addition, the growth of urban area was most prominent (42.9 km2) since 2010, mostly converted from croplands.Conversion flows among the land cover types at the upper and lower floodplains were used to disclose the land conversion processes in the TSLA Fig. . In termS4–S6). For 1992–2001, the most extensive land conversion was from shrubs to croplands (13.5 km2). And about three times more shrubs (49.4 km2) were converted into croplands in 2001–2010 than in 1992–2001. This shrub-to-cropland conversion was reduced to 3.2 km2 in 2010–2019. In addition, urban area expansion mainly occurred in the upper floodplain, primarily from croplands. Temporally speaking, the urban area only expanded by 1 km2 between 1992 and 2001; this value increased to 8.0 km2 and then surged to 42.3 km2 during the latter two sub-periods. In the lower floodplain, forest-to-wetland was the dominant land conversion type . About 682.8, 906.3, and 55.3 km2 of forests were transformed into wetlands in 1992–2001, 2001–2010, and 2010–2019, respectively. Another 33, 19.1, and 0.2 km2 of forests were converted to croplands in the three sub-periods, respectively. And 2.6 km2 shrubs were changed to croplands in 1992–2001; this value rose to 26.7 km2 in 2001–2010, and dropped to 1.4 km2 in 2010–2019.The upper and lower floodplains showed different land cover conversion patterns during 1992–2019. In the upper floodplain, croplands played a significant role among the conversion flows in the three sub-periods Fig. S4–S. For 199Comparisons of land cover conversions across the three sub-periods revealed similar but larger forest loss-related conversions in 2001–2010 than in 1992–2001. This extensive forests loss was primarily caused by farming expansion, forest clearing, and forest product collecting during 1992 and 2010.This forest loss-related conversion was significantly reduced after 2010. Overall, the land-use change was intensified after 2001 and the establishment of the protected areas did not curb the trend of forest loss in the TSLA until 2010. On the other hand, although the apparent forest loss was reduced and there had some net gain of forests after 2010, hardly any pristine forests have been left in the TSLA.S3a). There has been almost no forest loss since 2011, accompanied by some forest regain. Since the forest-to-wetland conversion was the dominant flow, the annual change of forests to wetlands showed a similar temporal pattern as that of forest loss . Before 2001, the annual rate of forest-to-cropland conversion increased from 1992 and reached a peak in 2000. Since 2001, this type of land conversion continued to decrease and was nearly zero after 2011 . Annual changes of shrubs to croplands showed a monotonous peak in 2002 . Overall, the results indicated an increase in forest loss before 2001, which stayed relatively stable during 2001–2010 and reached close to zero after 2010.Examining the annual time series of forest loss and land conversion flows in the TSLA for 1992–2019, we also found that the amount of forest loss steadily increased from 1992, peaked in 1999 and 2003, and decreased afterward . This forest loss was close to the result of Lohani et al. . Many satellite-based land cover data are available, including the MODIS MCD12Q1   forests were fragmented with barely any intact forest left from 2010, (ii) forest loss hotspots were at the lower floodplain where protected areas are located, and (iii) notable cropland expansion was mainly located at the intersection area between the lower and upper floodplains. Compared with forest cover change in 1992–2001, evidence shows that the forest reforms did not halt forest loss in the TSLA in 2001–2010. And the forest loss rate was much higher than the Cambodian national rate for 2001–2010. This forest loss in the TSLA has been paused since 2010 with even some forest regain. However, forest recovery since 2010 was almost negligible and the large net forest loss over the entire period (1992–2019) still posed a grand challenge for restoring the ecological integrity of this internationally important protected area. Generally, population growth, firewood collection, and logging are leading causes of forest loss. Meanwhile, intensified agriculture activity in the TSLA and the upstream hydropower development further increases the risk of forest loss by reducing the buffer to natural habitats. Facing the challenge of meeting human demands and maintaining the functional capacity of the ecosystem in the TSLA in the long term, this study offers scientific evidence for understanding the human interference on forests that can be useful for future sustainable forest management to achieve a healthy ecosystem in the TSLA, the rest of Cambodia, and other areas facing similar socioeconomic and forest loss issues.Supplementary file1 (DOCX 1075 kb)Below is the link to the electronic supplementary material.


Diabetes is a metabolic disease linked to multiple comorbidities, such as low-grade inflammation. β-pinene  or glibenclamide (5 mg/kg), for seven consecutive days. Diabetes was induced by alloxan (40 mg/kg) through the penile vein. On the seventh day of treatment, blood samples were collected for biochemical analysis. The anti-inflammatory effect of β-pinene was evaluated using the carrageenan-induced paw edema model, followed by the carrageenan-induced peritonitis.  Male Wistar rats (200–250 g) were treated orally with β-pinene decreased plasma glucose, triglyceride, VLDL, LDL, and HDL levels, when compared to those of the control group. In addition, the association β-pinene 10 mg/kg + glibenclamide 2 mg/kg significantly decreased blood glucose, total cholesterol, and triglyceride level. Finally, oral treatment with β-pinene reduced carrageenan-induced paw edema and leukocyte migration in the peritoneum. Taken together, our results indicate that β-pinene shows hypoglycemic and hypolipemic effects, which may involve some common mechanisms of glibenclamide. Besides, the monoterpene presented an anti-inflammatory action in diabetic rats that needs further investigation in order to clarify such effect and its correlation with the alterations observed in plasma parameters of β-pinene-treated diabetic rats. The treatment with  Accordifections .Currently, DM still remains a serious public health problem. It is associated worldwide with high rates of morbidity and mortality due to complications related to DM and dyslipidemia and to alterations that involve renal and hepatic oxidative damage and that worsen the clinical picture and increase mortality rates . One wayThe search for medicinal plants capable of treating diseases has awakened researchers and the pharmaceutical industry to invest more in new drugs endowed with relevant therapeutic action . Indeed,β-pinene is a cyclic monoterpene commonly found in essential oils from various aromatic medicinal plants [β-pinene may act as a hypoglycemic agent given its regenerating capacity in pancreatic beta cells [β-pinene-mediated anti-inflammatory effects and the involvement of inflammation in DM, we sought to investigate the possible hypoglycemic, hypolipidemic, and anti-inflammatory effects of β-pinene in diabetic rats.Terpenes are substances derived from a plant's secondary metabolism and are classified based on the number of a five-carbon molecule, the isoprene unit, as follows: hemiterpenes (5C), monoterpenes 10C), sesquiterpenes (15C), diterpenes (20C), sesterterpenes (25C), triterpenes (30C), tetraterpenes (35C), and polyisoprenoids (>35 carbons). C, sesquil plants . In addita cells . Thus, i50 . Glibenclamide (5 mg/kg) was purchased from Biosynthetic Laboratory  and used as control. Alloxan (40 mg/kg) and Tween-80 (2% v/v) were purchased from Sigma Chemical Co. . All other substances were donated by the Faculty of Medicine of Juazeiro do Norte.Beta-pinene , batch: MKBP3888V, was sponsored by the Regional University of Cariri-URCA . In the This study was carried out in the Laboratory of Biophysiology of the Faculty of Medicine of Juazeiro do Norte, and it was approved by the Faculty's Ethics Committee on the Use of Animals, under the process no. 2018.01.018. A total of 150 adult male Wistar rats (200 to 250 g) were donated from the vivarium of The Faculty of Medicine of Juazeiro do Norte. The animals were housed in groups of five, for at least 24 h prior to the experiments, in plastic boxes, receiving water and food ad libitum in a controlled environment with a 12 h light/dark cycle and regulated temperature (22 ± 2°C) and humidity (70%).β-pinene (BP) and glibenclamide (Glib 5 mg/kg) orally for 7 days. Untreated diabetic controls (DC) received vehicle. After this period, the animals underwent fasting and blood collection for biochemical analysis. At the end of the experiment, the animals were euthanized with sodium thiopental. A lethal dose of thiopental (150 mg/kg) and lidocaine (10 mg/mL) was used intraperitoneally [Alloxan  was injected in male Wistar rats (250 g) and after 48 h, animals were subjected to blood collection for measurements of blood glucose (mg/dL). Only those showing glycemia levels higher than 250 mg/dL were submitted to the study. After this period, the animals were administered daily doses of 25, 50 and 100 mg/kg of toneally .Blood samples were centrifuged at 3000 rpm for 10 min to obtain the serum, which was used to determine glucose, total cholesterol (TC), HDL, LDL, and triglycerides (TG) levels and AST and ALT activities by standard enzymatic colorimetric methods, according to the manufacturer's instructions .β-pinene, rats were treated for seven consecutive days with vehicle or β-pinene (10 mg/kg) or glibenclamide (2 mg/kg) or the association of β-pinene (10 mg/kg) plus glibenclamide (2 mg/kg). On the last day (7th day), blood samples were collected to determine the blood glucose, cholesterol, and triglyceride levels.To investigate the possible mechanism of action of µL/paw of a 1% w/v carrageenan solution was given to the right hind paw. Paw volumes were then measured at intervals of 1, 2, 3, 4, and 24 h (standard times), for atypical progressive edema evaluation, which occurs in diabetic rats. The edema volume (EV) resulting from an acute inflammatory reaction was determined by the difference between the final (FV) and initial (IV) paw volumes [On the last day of treatment, 1 h after the drug's administration, an intraplantar injection of 40  volumes .β-pinene (50 mg/kg). After 60 min of treatment, each rodent received a 1% intraperitoneal injection of carrageenan. The fifth group consisted of healthy animals that did not receive any substance. After 4 h, a 3 mL volume of heparinized PBS was administered into the peritoneal cavity of the animals. Then, a gentle massage was performed in the abdominal region of the mouse and the peritoneal fluid was collected for a subsequent leukocyte count, which was performed by using the ABX Micros 60 device [Diabetic rats were orally treated with vehicle, glibenclamide (5 mg/kg), dexamethasone (5 mg/kg), and 0 device .p < 0.05.The results that presented a normal distribution were represented as mean ± standard error of the mean (SEM) and analyzed by one-way or two-way analysis of variance (ANOVA) followed by the Bonferroni test. The results were considered significant at β-pinene significantly decreased blood glucose, VLDL, HDL, and LDL levels , when administered for 7 days, significantly decreased the blood glucose level by 41.6% , when compared to the vehicle (365.1 ± 27.51). On the other hand, such effect was not observed in groups that were treated with β-pinene alone, at 10 mg/kg (323.3 ± 41.11) and glibenclamide alone, at 2 mg/kg (347.6 ± 48.5), when compared to the vehicle (The association of glibenclamide (2 mg/kg) plus  vehicle .p < 0.001) and 29.1% , respectively, when compared to the respective control groups (105.9 ± 2.7), and (338.1 ± 47.86)  and glibenclamide (5 mg/kg) significantly reduced the edema at the 3rd h of evaluation, by 29.6% (p < 0.001) and 38.4% (p < 0.001), respectively, when compared to the diabetic group (91.66 ± 4.3). In addition, β-pinene at 50 significantly reduced the edema at the 4th h of evaluation by 36% (p < 0.001) and 26.8% (p < 0.001) when compared to the diabetic (80.8 ± 6.1) and normal control (25.3 ± 4.5) groups. Glibenclamide at 5 mg/kg also reduced paw edema by 68.6% (p < 0.001). After 24 h of edema induction, β-pinene at 50 reduced paw edema by 32.7% (p < 0.001) and 43.9% (p < 0.001), respectively, when compared to the diabetic (49.2 ± 5.3) and normal control (9.2 ± 1.3) groups. It should be noted that this time point (T24), represents the 7th and last day of experiments. During this period, the normal control group presented a regression in the animal's paw edema, returning to a volume similar to that observed during the 1st h of edema induction . Similarly, it significantly reduced monocyte migration by 44.4% and lymphocyte migration by 37.9% compared to the control group (6.12 ± 0.3) and (60.8 ± 3.1), respectively , also showed a significant reduction of leukocyte migration in 18.5% in granulocytes, 71.8% in monocytes, and 61.3% in lymphocytes in the control group.β-pinene is a monoterpene that is found in essential oils as the enantiomers (+)-β-pinene and (−)-β-pinene [β cell performance and to decrease plasma hyperglycemia, in addition to other biological effects [β cells despite causing DM2, it rather triggers a reduction in these cells. Thus, in the present study, we decided to use this method of inducing DM to test the hypoglycemic and hypolipidemic activities of β-pinene in rats.β-pinene . It is k effects . Such ef effects , 18. The effects . Alloxan effects , 20 as w effects , alloxanβ-pinene reduced glycemic levels in alloxan-induced diabetes in rats. Kahn et al. [β-pinene and α-pinene have hypoglycemic effects in streptozotocin-treated mice. In addition, D'Adamo and Caprio [Origanum vulgare L. has hypoglycemic effects in streptozotocin-induced diabetic rats, possibly by the presence of α-pinene, a β-pinene isomer in the composition of the plant. Although the authors used a distinct diabetogenic agent, these findings contribute to the statement that chemical components present in plants are responsible for their pharmacological action. The data obtained from these authors also reinforces the fact that the isolated terpenic substances have similar effects on the glycemic level.Our results showed that n et al.  report td Caprio  showed td Caprio  also shoα- and β-pinene, which caused a significant reduction in serum TG, TC, and high-density lipoprotein (HDL) levels in hyperglycemic rats treated with aqueous, ethanolic, and hexane leaf extracts from species belonging to this family. According to Menezes et al. [β-pinene reduced such biochemical parameters  and β-pinene (10 mg/kg) were tested alone and then in combination. The association of the two substances led to a reduction in glucose, cholesterol, and triglyceride levels in the blood of diabetic rats, while the use of both substances alone did not modify such biochemical parameters. Briefly, glibenclamide stimulates insulin secretion by blocking ATP-dependent K+ channels in pancreatic β cell membranes, causing depolarization and Ca2+ influx [β-pinene may present a mechanism like that of glibenclamide.Changes in the metabolism of plasma lipoproteins in patients with diabetes mellitus are quite common . Valdes + influx . The resβ-pinene has anti-inflammatory effects, we investigated the anti-inflammatory effect of β-pinene in diabetic animals. Carrageenan is a sulfated polysaccharide obtained from some red algae species that promotes different inflammatory stimuli and recruits several inflammatory mediators to the injured site [α) and prostaglandins PGE2 [β-pinene in diabetic rats. Our results showed that non-diabetic animal animals presented an edema elevation peak in the 3rd h of inflammation, which later regressed to baseline levels within 24 h. This can be explained through studies by Wold [Based on the involvement of inflammation with diabetes and with strong evidence that red site . Brieflyins PGE2 , followeins PGE2 . This edins PGE2 . Carrage by Wold  that shoth h of the experiment. On the other hand, β-pinene reduced edema volume by up to 29.6% in the 3rd h, when used at 50 mg/kg. Moreover, after 24 h, β-pinene also reduced edema by 32.7% at 50 mg/kg. Pioneering studies by Di Rosa [nd phase, evidenced only after 3 h of induction, cytokines, prostaglandins, and NO participate in the inflammatory process, further increasing the inflammatory reaction. Thus, β-pinene may possibly act by inhibiting inflammatory mediators that participate in the 2nd phase of the process; however, more studies are necessary to test such a hypothesis.As DM is a chronic disease, the entire inflammatory process has a longer time period. This is observed in the results presented in this study, in which untreated diabetic rats showed that edema lasted longer, until the 24 Di Rosa  have preβ, and IL-6), nitric oxide, peptide fragments of the complement system , histamine, leukotrienes, prostaglandins, thromboxane, and PAF52 [Carrageenan-induced peritonitis is a model that involves cell migration and plasma exudation into the peritoneal cavity during an inflammatory process . Neutropnd PAF52 .Bumelia sartorum Mart. The aqueous extract, which is rich in terpenes, used the carrageenan-induced paw edema method in diabetic rats and reported a significant reduction in edema. This finding is similar to those obtained in our study, as the authors used a substance belonging to the terpene class and analyzed its anti-inflammatory activity using the same method. Nonetheless, although the considerations reported in this experiment show that there is an increase in the inflammatory process in diabetic rats when compared to normal ones, these findings cannot elucidate the exact mechanism by which β-pinene reduces edema in this model and further studies in this field are needed.De Carvalho et al.  indicateβ-pinene, for seven consecutive days, presented hypoglycemic and hypolipidemic effects, which seems to involve, at least in part, the blocking of ATP-dependent K+ channels and the activation of LPL, respectively. The treatment with β-pinene also revealed an important anti-inflammatory effect, possibly by inhibiting inflammatory mediators that participate in the 2nd phase of the process, possibly by decreasing leukocyte migration, indicating that its action may be linked to the inhibition of cytokine production. It is important to stress that other studies are necessary to provide more information about the mechanisms underlying the hypoglycemic, hypolipidemic and anti-inflammatory effects of beta-pinene in diabetic rats.The oral administration of


Rhodotorula glutinis can make full use of TWW to ferment and reproduce yeast cells, produce carotenoids and other nutrients, improve the utilization value of TWW, and reduce environmental pollution and resource waste.Tofu whey wastewater (TWW) is the wastewater of tofu processing, which is rich in a variety of nutrients. Rhodotorula glutinis were analyzed to reformulate TWW medium, and the optimal composition and proportion of TWW medium that can improve the biomass and carotenoids production of Rhodotorula glutinis were explored. Meanwhile, the Rhodotorula glutinis liquid obtained under these conditions was used to prepare biological feed for laying hens, and the effect of Rhodotorula glutinis growing on TWW as substrate on laying performance and egg quality of laying hens were verified.In this study, the nutrient composition changes of TWW treated by Rhodotorula glutinis fermentation increased by 62.30%, the phosphorus content decreased by 42.31%, and the contents of vitamin B1, B2 and B6 increased to varying degrees. The optimal fermentation conditions of Rhodotorula glutinis in the TWW medium were as follow: the initial pH was 6.40, the amount of soybean oil, glucose and zinc ions was 0.80 ml/L, 16.32 g/L, and 20.52 mg/L, respectively. Under this condition, the biomass of Rhodotorula glutinis reached 2.23 g/L, the carotenoids production was 832.86 μg/g, and the number of effective viable yeast count was 7.08 × 107 cfu/ml. In addition, the laying performance and egg quality of laying hens fed Rhodotorula glutinis biological feed were improved.The results showed that the zinc content of TWW after Rhodotorula glutinis in TWW medium, explored the influence of Rhodotorula glutinis utilizing TWW on laying layers, and provided a new idea for the efficient utilization of TWW.In this study, we analyzed the composition changes of TWW, optimized the fermentation conditions of  The pH of the medium was adjusted to 6.2 and then sterilized at 121°C for 21 min.The LB solid culture medium was formulated as follow: peptone (10.0 g/L), yeast extract (3.0 g/L), glucose (3.0 g/L), NaCl (5.0 g/L), and agar powder (15.0 g/L). The pH of medium was adjusted to 6.2 and then sterilized at 121°C for 21 min.Rhodotorula glutinis was melted at room temperature and transferred to LB medium  to obtain the activated Rhodotorula glutinis culture. The activated Rhodotorula glutinis culture was then added to LB medium at 10% (v/v), and incubated at 30°C, 180 rpm for 48 h. The viable yeast count of SRY (Rhodotorula glutinis SRY) was 1.3 × 107 cfu/ml.The 2.3.Rhodotorula glutinis SRY seed medium was added to YWW medium at 5% (v/v) and incubated at 180 rpm, 30°C for 48 h. The supernatant was collected by centrifugation  and the yeast cells were filtered out through a sterile filter (0.22 μm).The Rhodotorula glutinis fermented TWW samples were tested by Qingdao Zhongyi Monitoring Co., Ltd.The protein content, fat content, reducing sugar content, mineral elements, vitamin B1, vitamin B2, vitamin B6 were determined according to the Chinese National Standard Method GB5009.5-2016, GB5009.6-2016, GB5009.7-2016, GB5009.268-2016, GB5009.84-2016, GB5009.85-2016, GB5009.154-2016, respectively –30. The 2.4.2.4.1.R. glutinis SRY grown. The effects of initial pH value, glucose content, manganese ion content, copper ion content, magnesium ion content, zinc ion content and ferrous iron ion content on SRY biomass, carotenoids content and viable yeast count were investigated.The medium was prepared according to factors and levels as shown in 2.4.2.SRY, eight factors were selected as the initial pH value (A), soybean oil content (B), glucose content (C), hydrogen peroxide content (D), vitamin B1 content (E), manganese ion content (F), magnesium ion content (G) and zinc ion content (H) for Plackett-Burman test. The range of optimal culture conditions for the 8 factors was taken at high (+1) and low (−1) levels, respectively, and the PB test design factors and levels are shown in Using the biomass, carotenoids content and viable yeast count of 2.4.3.SRY were used as the response values to find reasonable optimization conditions , carotenoids content (Y2), and effective viable number (Y3) of nditions . The des2.4.4.Rhodotorula glutinis SRY broth (1 L), dried to constant weight at 50°C, weighed and biomass was calculated. The viable yeast count of Rhodotorula glutinis SRY in the culture medium was determined by solid plate counting methods. Briefly, Rhodotorula glutinis SRY seed culture was diluted at appropriate multiples, evenly spread on LB solid medium. The culture was incubated at 30°C for 72 h and the viable yeast counts of Rhodotorula glutinis SRY were counted.The cell pellet were obtained by centrifugation  of 2.4.5.Rhodotorula glutinis was determined by the hydrochloride acid thermal breakage method, as follows: the liquid culture (30 ml) was centrifuged . 3 M HCl (30 ml) was added to the yeast cell pellet, shaken for 45 min, and then heated at 100°C for 5 min. The mixture was cooled and centrifuged . The sterilized water (30 ml) was added to the yeast cell pellet. And the above process was repeated three times. Acetone (10 ml) was added to the yeast cell pellet, shaken for 1 h and centrifuged  to obtain carotenoids extracts. The absorbance of the carotenoids extracts was measured at 475 nm. Formula for calculating the carotenoids content:Carotenoids content in c is carotenoids content of fermentation broth, μg/g; Amax is absorbance at 475 nm; D is dilution of sample; V is organic solvent volume for pigment extraction, ml; m is dry yeast mass, g; 0.16 is molar extinction coefficient of carotenoids.where W2.5.2.5.1.The Luhua chicken used in the experiment is a local chicken breed bred by Jilin Academy of Agricultural Sciences. Ninety-six 21-week-old Luhua chickens of similar weight were selected and randomly divided into 6 groups of 16 in each group, and grouped as shown in The laying hens in each treatment group were raised in a three-layer chicken cage, with each two birds kept in one cage, and a nipple drinking fountain was used to provide drinking water. The daily feed mass of each layer was 150 g and the feed formula was shown in 2.5.2.The laying number and egg weight of laying hens were recorded daily, and the laying rate and average egg weight were calculated. Ten eggs were selected from each group every week. Fifteen grade color fans were used to contrast the color of the egg yolk. Protein height was determined using a protein height meter, and the average value was taken based on the protein height and egg weight, and the Haugh unit value was query on the unit table.where Haugh unit is a quality standard for the internal quality of eggs, and the parameter is expressed as a score between 0 and 100; H is the protein height, mm; W is egg weight, g.2.5.3.The well-stirred egg yolk (0.5 g) was added to acetone (10 ml), vortexed for 15 min and then centrifuged at 4000 rpm for 15 min. The absorbance value of the supernatant at 475 nm was determined by UV–visible spectrophotometer. Carotenoids content was calculated according to the following formula:where A is absorbance value at 475 nm; V is the volume of acetone being added, ml; 0.16 is molar extinction coefficient of carotene; W is weight of the egg yolk used in the measurement process, g.2.6.p < 0.05 represents significance.Test data were analyzed and processed using Design-Expert software  and SPSS software. Group mean values were compared using a one-way analysis of variance and tested for multiple comparisons by the LSD test. Data are presented as mean ± standard deviation (sd), and 3.3.1.Rhodotorula glutinis fermented TWW. The test results showed that the Rhodotorula glutinis consumed 41.58% of the protein in the TWW, and produced more vitamin B1, vitamin B2, and vitamin B6. The content of most amino acids in the TWW after Rhodotorula glutinis fermentation was greatly reduced, but the content of serine and glycine was somewhat increased. In addition, we found that the content of potassium and magnesium in TWW was relatively high, reaching 369.00 and 63.80 mg/kg, respectively. After Rhodotorula glutinis fermentation, the content of these two elements did not change significantly, while the content of zinc and phosphorus was significantly changed, among which the zinc content increased by 62.30% and the phosphorus content decreased by 42.31%.TWW is rich in nutrients, and we detected the changes in protein, fat, reducing sugar, vitamin B1, vitamin B2, vitamin B6, amino acids, and mineral elements  in the u3.2.3.2.1.Rhodotorula glutinis is largely influenced by the components of the culture matrix. On this basis, we added glucose to the TWW medium to explore the effect of glucose on the growth of Rhodotorula glutinis. As can be seen from Rhodotorula glutinis biomass, viable yeast count and carotenoids content. When the glucose concentration was 15 g/L, the viable yeast count and carotenoids content of Rhodotorula glutinis reached the highest value, which was 2.80 × 107 cfu/ml and 725.36 μg/g, respectively, the carotenoids content was also at a relatively high level.The growth metabolism of 3.2.2.Rhodotorula glutinis. As shown in SRY were not significant when the initial pH was 4.0 compared to that at an initial pH of 3.0. Then, as the initial pH value increases, these indexes showed a tendency to increase first and then decrease. When the initial pH was 6.0, the Rhodotorula glutinis SRY biomass, carotenoids content, and viable yeast count were all high, reaching 1.94 g/L, 476.36 μg/g, and 1.18 × 108 cfu/ml, respectively.We set five initial pH as: 3.0, 4.0, 5.0, 6.0, and 7.0 to further investigate the effect of the initial pH on the growth of 3.2.3.Rhodotorula glutinis. We measured the effects of metal ions of different species and concentrations on Rhodotorula glutinis biomass, viable yeast count, and carotenoids content. As shown in SRY biomass, carotenoids content and viable yeast count of Rhodotorula glutinis all increased first and then decreased with the increase of zinc ion concentration. When the zinc ion content was 20 mg/L, the maximum values of SRY biomass, carotenoids content and viable yeast count were 1.50 g/L, 650.68 μg/g and 3.50 × 107 cfu/ml, respectively. When the manganese ion content was 20 mg/L, the copper ion content was 30 mg/L, and the ferrous iron ion content was 40 mg/L, the Rhodotorula glutinis SRY biomass, carotenoids content and viable yeast count were all at higher levels . The value of “Lack of Fit” was 0.6353, inferring that “Lack of Fit” was not significant relative to the pure error (p > 0.05); therefore, the model was reliable. The R2 was 0.9846, indicating that the total variable of response values over 98.46% could be represented by this model. The Adj.R2 was 0.9692, confirming that the model reflected the relationship between each single factor, with a high fitting degree and small experimental error. The fit of the quadratic multivariate equation of SRY fermentation is as follows:As shown in Rhodotorula glutinis. The optimized Rhodotorula glutinis fermentation condition obtained biomass was 2.23 g/L, the carotenoids content was 832.86 μg/g, and the viable yeast count was 7.08 × 107 cfu/ml.The secondary multiple regression model was optimized, and the optimal fermentation conditions were as follow: the initial pH was 6.40, soybean oil content was 0.80 ml/L, glucose content was 16.32 g/L, and zinc ion content was 20.52 mg/L, predicting the overall desirability of 0.492 under the above conditions. Three parallel tests were carried out according to the best fermentation conditions obtained in the experiment, and the average value of the fermentation overall desirability was 0.471 ± 0.03, which was close to the theoretical predicted value, indicating that the model can better reflect the actual situation of the fermentation condition optimization of 3.4.Rhodotorula glutinis fermentation condition, and the effects of the initial pH versus soybean oil content on the overall desirability of the Rhodotorula glutinis fermentation condition are shown in Rhodotorula glutinis fermentation condition, which was consistent with the significance test results of the overall desirability partial regression coefficient.The effects of the initial pH and zinc ion content on the overall desirability of the 3.5.3.5.1.Rhodotorula glutinis diet, the laying rate and egg weight of this group increased significantly in the fourth week. The results showed that, the laying rate of laying hens decreased gradually as the weather turned cooler, and feeding the Rhodotorula glutinis feed could change this trend to some extent.As shown in the 3.5.2.Rhodotorula glutinis in laying hens had significantly higher than those in the NC group (p < 0.05). The Haugh unit was determined by the relationship between the height and the weight of the albumin. The Haugh unit is a parameter indicative of the egg quality. Our results indicated that the yolk color and carotenoids content in all Rhodotorula glutinis treated groups were significantly higher than those in untreated groups (p < 0.05).According to the 4.Rhodotorula glutinis biomass and carotenoids. Study by Aleksandr et al. showed that the addition of K+ and Na+ to Rhodotorula toruloides cultures induced glucose utilization and increased β-carotene production by 60%   . Zinc caas 59.3% . Our stun in TWW , 37. TheRhodotorula glutinis, we further explored the effect of metal ion addition in TWW on Rhodotorula glutinis. When the zinc ion content increased from 0 to 50 mg/kg, the Rhodotorula glutinis biomass, viable yeast count and carotenoids content showed a trend of increasing in the first step and then decreasing. Zinc ions could promote carotenoids production in Rhodotorula glutinis, and this promotion might result from the activation of specific desaturases involved in carotenoids synthesis by zinc ions , expressing the influence of different variables (factors) on a specific index , Changchun Science and Technology Bureau Key Research and Development Project (21ZGN36), and Jilin Province Science and Technology Development Plan (202202051NC).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.