Citrus aurantium L., Cymbopogon citratus (Stapf.), and Cinnamomum verum (J. Presl.) were selected for topical assay of their ovicidal activity against Aedes aegypti (Linnaeus) and Aedes albopictus (Skuse). Their efficacy was compared to that of 1% (w/w) temephos. In addition, their non-toxicity against aquatic mosquito predators, Poecilia latipinna and Poecilia reticulata, was tested. Found by GC–MS analysis, the major constituent of C. verum EO was trans-cinnamaldehyde, of C. aurantium EO was d-limonene, and of C. citratus EO was geranial. Both C. verum EO and trans-cinnamaldehyde at a high concentration  exhibited high ovicidal activity against Ae. aegypti and Ae. albopictus eggs after 48 h of incubation with an inhibition rate of 91.0–93.0% for C. verum EO and 96.7–95.2% for trans-cinnamaldehyde. The combination of C. verum EO + geranial exhibited the strongest synergistic inhibition activity (100%) against the two mosquito vectors and was five times more effective than temephos. Moreover, they were not toxic to the non-target fishes. As a safe ovicidal agent for mosquito egg control, the combination of C. verum EO + geranial has excellent potential.Plant essential oil (EO) is a natural alternative to synthetic chemical insecticides for mosquito control. EOs from  Aedes aegypti L. and Aedes albopictus (Skuse) mosquitoes that have bitten viral-infected humans transmit the disease to other humans. These mosquitoes are major vectors of arboviruses such as Zika, yellow fever, dengue, and chikungunya3. Dengue is one of the most dangerous arboviruses, causing high morbidity and mortality rates in several countries around the world including many parts of Thailand4. According to a report by the Thai Ministry of Public Health, there were 9084 and 19,380 total dengue cases in Thailand, in 2021 and 2022, respectively, with 9 and 17 fatalities, calculated into a morbidity rate of 0.06% and 0.09%, respectively5. Since vaccines and other drugs have limited effectiveness in controlling dengue, controlling the population of mosquitoes with pesticides is the most effective measure to avoid this contagious disease6.8. Generally, temephos, a common synthetic organophosphate insecticide, is used extensively around the world, especially in Thailand, for killing mosquito larvae. Unfortunately, its efficacy has been reduced drastically because populations of mosquitoes have developed resistance to it1. Moreover, temephos incurs serious negative side effects. It causes irreversible damage to non-target aquatic predators and humans, thus limiting its use10. In this glum context, many researchers have urgently developed alternative strategies that are safer for humans and the environment7.Controlling the mosquitoes at their embryonic and larval stages is the key strategy in controlling mosquito populations11. The EOs of Cinnamomum verum (J. Presl.), Citrus aurantium L., and Cymbopogon citratus (Stapf.) were investigated in this study. They have been reported safe for humans and mammals as well as having low toxicity on non-target predators because they have been long used as feed in food industry as well as antibiotic and antioxidant in folk medicine for thousands of years14.Because plant essential oils (EOs) are natural substances, they and their phytochemical constituents are good, green alternatives to temephos. They are not harmful or only slightly harmful to mammals and non-target aquatic predators of mosquitoes at a practical pesticidal level, plus they degrade rapidly in the environmentAe. aegypti (Order Diptera)15, Musca domestica (Order Diptera)16, Haemaphysalis longicornis (Order Ixodidae)17, Pediculus humanus capitis (Order Phthiraptera)18, Spodoptera littoralis (Order Lepedroptera)19, and Periplaneta americana (Order Blattodea)20. Specifically, EOs from C. verum, Coccinia indica, C. citratus, Illicium verum (Hook.f.), and Moringa oleifera (Lam.) and their major constituents  exhibited strong ovicidal activity against many mosquitoes species like Anopheles indica7, Ae. aegypti23, and Culex quinquefasciatus24 and housefly (M. domestica)16 with an LC50 ranging from 3.31 to 303,200 ppm. Furthermore, combined formulations of EOs and EO constituents showed even higher efficacy than their individual EOs26. For example, Soonwera et al.27 reported that a combined trans-anethol + I. verum EO formulation provided complete mortality against Ae. aegypti and Ae. albopictus larvae. Another group of researchers, Andrade-Ochoa et al.28, showed that a combined trans-cinnamaldehyde + trans-anethol formulation and a combined trans-cinnamaldehyde + (–)-limonene formulation were highly insecticidal against Cx. quinquefasciatus larvae and pupae, synergistically high. Youssefi et al.29 stated that a combination of thymol + carvacrol provided strong ovicidal and larvicidal activities against Cx. pipiens.Regarding plant EOs insecticidal efficacy, they are strongly insecticidal against many insect pests, such as d-limonene, geranial, and trans-cinnamaldehyde show several dominant activities for mosquito and other insect pest control. Single and combined formulations of d-limonene showed a strong larvicidal activity against Ae. aegypti25 and also shown a strong pupicidal activity against Ae. aegypti and Ae. albopictus27. Geranial showed a strong adulticidal activity against Ae. aegypti, Ae. albopictus, and M. domestica31. Single and combined formulations of trans-cinnamaldehyde also showed a strong adulticidal activity against Ae. aegypti and Ae. albopictus15.To conclude, Ae. aegypti and Ae. albopictus incurred by EOs from C. aurantium, C. citratus, and C. verum, their major constituents, and several of their combinations. In addition, the biosafety of the EO treatment was evaluated against two common, non-target predators of mosquitoes, Poecilia latipinna and Poecilia reticulata fishes.From these pieces of studies, our group was inspired to investigate the egg mortality against —C. aurantium, C. citratus, and C. verum EOs—gave different EO chemical profiles, and hence can make an accurate efficacy comparison between studies meaningless.GC–MS analysis of EOs was necessary because different parts of the three plant speciesv/w, was recovered from C. aurantium EO, followed by from C. citratus EO (1.14% v/w), and from C. verum EO (1.01% v/w). C. aurantium EO, 21 chemical constituents were found to compose 96.67% of its chemical composition. d-limonene (78.15%) was the major constituent. Some other main constituents were linalool (4.80%), δ-3-carene (2.40%), and β-myrcene (2.00%). For C. citratus EO, 9 chemical constituents were found to compose 96.54% of its chemical profile. The major constituent was geranial (45.41%). A few other main constituents were neral (24.80%), 1,8-cineole (10.59%), and geraniol (4.70%). For C. verum EO contained 14 constituents as 97.26% of its chemical profile. Trans-cinnamaldehyde (73.21%) was the major constituent. Some other main constituents were benzyl alcohol (12.87%), cinnamyl acetate (2.50%), and eugenol (2.35%).All EOs were pale yellow. Table 50), 50% Lethal concentration (LC50), effective inhibition rate index (EII) versus temephos are tabulated in Tables C. aurantium EO at 30,000 ppm alone provided an egg inhibition rate of 78.1%, an LT50 of 55.3 h, and an LC50 of 15,071.7 h, with EII = 2.56 against Ae. aegypti. From Table C. aurantium EO at 30,000 ppm alone provided an egg inhibition rate of 76.3%, an LT50 of 51.7 h, and an LC50 of 16,592.2 h, with EII = 2.59 against Ae. albopictus. Regarding the synergistic index (SI) column, since this formulation is of single C. aurantium EO, the synergistic index of combined formulation is not applicable. Several individual EOs and EO constituents at 30,000 ppm showed a significantly higher efficacy than at a lower concentration. The highest egg inhibition rate of individual EOs was at 91.0% against Ae. aegypti and 93.0% against Ae. albopictus, achieved by C. verum EO. At 30,000 ppm. It provided an LT50 of 29.7 h against Ae. aegypti and 31.1 h against Ae. albopictus. In contrast, at 10,000 ppm, C. aurantium EO provided the lowest egg inhibition rate, at 59.7%, against Ae. aegypti and 53.2% against Ae. albopictus, with an LT50 of 83.6 h and 85.8 h, respectively. The highest egg inhibition rate of EO constituents was 96.7% against Ae. aegypti and 95.2% against Ae. albopictus, achieved by trans-cinnamaldehyde, the major constituent of C. verum EO. At 30,000 ppm, trans-cinnamaldehyde provided an LT50 of 24.3 h against Ae. aegypti and 24.7 h against Ae. albopictus. In contrast, at 10,000 ppm, d-limonene, the major constituent of C. aurantium EO, provided the lowest egg inhibition rate against Ae. aegypti at 76.4% and against Ae. albopictus at 72.1%, with an LT50 of 39.5 h and 36.3 h, respectively. To conclude, C. verum EO exhibited a stronger ovicidal activity (lower LC50) against Ae. aegypti, but trans-cinnamaldehyde was stronger against Ae. albopictus.The following ovicidal activity indexes for each formulation against the two mosquito species: inhibition rate, 50% Lethal time  10,000 ppm. It provided an LT50 of 17.7 h against Ae. aegypti and an LT50 of 16.9 h against Ae. albopictus. In contrast, the combination of C. aurantium EO + geranial (2:1) 10,000 ppm provided the lowest egg inhibition rate, at 81.0% against Ae. aegypti and 80.0% against Ae. albopictus, with an LT50 of 35.8 h and 34.3 h, respectively. To conclude, C. verum EO + geranial (2:1) 10,000 ppm exhibited a stronger ovicidal activity against both Ae. aegypti and Ae. albopictus, in terms of low LC50.Moreover, the ovicidal efficacy of all combined formulations against the eggs of the two mosquito vectors was greater than the efficacy of individual EOs and EO constituents, with a synergistic index (SI) in the range of 0.26–0.49. The highest egg inhibition rate was at 100% against both Ae. aegypti and Ae. albopictus than 1 ppm temephos was.Regarding the effective inhibition rate index (EII), every combination of separate EOs and EO components as well as all combined formulations showed higher than 1.0 EII—they were more toxic to the eggs of 50 values against P. latipinna and P. reticulata, two fish species, of all formulations are summarized in Table trans-cinnamaldehyde, d-limonene, and geranial EO constituents than every combined formulation, in terms of LC50. The range of LC50 values against the two fish species of those EOs and EO constituents was from 8165.5 to 57,232.5 ppm, while for the combined formulations, the range was 4091.6–5921.3 ppm. On the other hand, the range for 1 ppm temephos was high toxic to both species with LC50 from 298.7 to 526.7 ppm.The estimated LCOn the biosafety index (BI) are shown in Table 32. The extraction yield of C. verum EO was 1.05% v/w compared to 1.1% v/w found by Aungtikun and Soonweera4 and Soonweera et al.20; the extraction yield of C. aurantium EO was 1.30% v/w compared to 1.4% v/w found by Bnina et al.33; and the extraction yield of C. citratus EO was 1.14% v/w, compared to 1.2% v/w found by Soonwera et al.20. The slight differences can be attributed to many factors, e.g., the harvesting season, the integrity of the plant species, the adequate plant management, and the degree of fertility of the soil 32.Regarding extraction yield, the extraction yields of all tested plants were the same or only slightly different from the corresponding yields reported by previous studies20. The major chemical constituent and key component (the active component20) of C. verum EO was trans-cinnamaldehyde (73.21% of the profile), very close to 72.2% reported by Soonwera et al.20; the major chemical constituent and key component13 of C. aurantium EO was d-limonene (78.15%), agreeing well with 73.6% supported by Bnina et al.33; and the major chemical constituent and key component14 of C. citratus EO was geranial (45.41%), agreeing well with 49.4% found by Chanthai et al.34. Nevertheless, some papers report larger differences. For C. verum EO, Chansang et al.15 reported a higher percentage of trans-cinnamaldehyde (90.2% compared to 73.21%); for C. aurantium EO, Zarrad et al.32 reported a higher percentage of d-limonene (87.5% compared to 78.15%); and for C. citratus EO, Brügger et al.35 reported a lower percentage of geranial (31.5% compared to 45.41%). This variation can be attributed to seasonal fluctuations, differences in temperature at the farms where these plants were cultivated, geographic location, ontogenetic variables, the growth stage of the plant at the time of harvest (pest management), and extraction method32, and all key components were robustly identified and quantified.On GC–MS analysis results, the chemical profiles of all tested EOs agreed well with those found in previous studies50, the ranking of inhibition against Ae. aegypti and Ae. albopictus eggs was as follows: (1) C. verum EO + geranial (2:1), (2) geranial + trans-cinnamaldehyde (1.5:1.5), (3) d-limonene + trans-cinnamaldehyde (1.5:1.5), (4) C. citratus EO + d-limonene (2:1), (5) d-limonene + geranial (1.5:1.5), and (6) C. aurantium EO + geranial (2:1). All tested formulations were more effective than temephos, and one formulation was outstanding. The outstanding combination of C. verum EO + geranial (2:1), at the final concentration of 10,000 ppm each, showed the shortest lethal time (LT50 ranging from 16.9 to 17.7 h) and the smallest lethal concentration . In contrast, temephos showed a lethal time in the range of 60.2–61.3 h, which is longer than that (ranging from 34.3 to 35.8 h) of the least effective EO formulation, C. aurantium EO + geranial (2:1) 10,000 ppm. Previous works27 have already established the potent toxicity of several combined EOs and EO constituents against mosquitoes at most stages in their life cycle, except at the egg stage. The effectiveness of the formulations that combined EO and EO constituents and targeted mosquitoes at the egg stage of their life cycles was first reported in this study. Regarding the most effective combined formulation, it was not surprising that it was so effective since its individual components, C. verum EO and geranial, have already been shown to be effective against several pest insects, as presented in the following papers. Nakasen et al.24 supported that C. verum EO at 12.5 ppm showed high ovicidal activity against Cx. quinquefasciatus with a 100% inhibition rate and an LC50 of 3.31 ppm. Soonwera et al.20 reported that C. verum EO had strong ovicidal activity against Periplaneta americana. Dias et al.36 indicated that trans-cinnamaldehyde exhibited a strong insecticidal effect against Mahanarva spectabilis eggs. Trans-cinnamaldehyde showed a toxic effect against the eggs of P. humanus capitis37, and Spodoptera littoralis19. Finally, Castillo-Morales et al.38 reported that geranial provided strong ovicidal activity against Ae. aegypti. Regarding the low efficacy of temephos, It can be inferred that it was low because it was not designed specifically to kill mosquitoes at the egg stage but at the larval stage. The mosquito subjects were a laboratory-selected strain, not field-collected, and hence the larvae have not developed resistance to temephos. Their morbidity was confirmed. The low egg-inhibition activity of temephos is supported by previous works of Puwanard and Soonwera23 and Cotchakaew and Soonwera39, indicating that 1% (w/w) temephos showed an inhibition rate ranging from 9.3 to 34.6% against the eggs of Ae. aegypti and Ae. albopictus, while the EOs showed a 47.0–100% inhibition rate.Regarding ovicidal efficacy results, based on LT41. Moreover, the cell borders and papillae as well as aeropyles were covered with a layer that was assumed to be an oil layer, which would explain the ovicidal mechanism of the EO, discussed in the paragraph below.On egg morphology, its SEM images in Figs. 19C. verum EO induced mortality of embryo and egg by forming a thin film of oil over the outer egg surface and blocking the egg respiration by sealing the aeropyles. Nakasen et al.24 concluded that C. verum EO destroyed chitin wall by the oil penetrating the eggshell pore leading to embryo death. Another possible mechanism of action is the mechanism that trans-cinnamaldehyde, the major constituent of C. verum EO, acts on the egg. Trans-cinnamaldehyde reduces the ATPase activity in the cell membrane of the respiratory system and inhibits the enzymes involved in cytokinesis as well as retards juvenile hormone production and cell growth in the immune system of mosquito42. In short, C. verum and trans-cinnamaldehyde act mainly on the respiratory system of mosquito eggs. Contrarily, geranial affects the egg's neurological system. Geranial inhibits the acetylcholinesterase (AChE) enzymes of neural cells and neuroreceptors30. Castillo-Morales et al.38 concluded that geranial penetrates through the serosal cuticle of an embryo and disturbs the embryogenesis process. Hence, the synergistic effect of the combination may stem from the fact that both substances acted along two different pathways, reinforcing one another.Regarding the mechanisms of ovicidal action, as stated in the paragraph above, the aeropyles seemed to be blocked by an oil layer, making respiration difficult or impossible. This respiration inhibition mechanism has been reported by Khedr et al.P. latipinna and P. reticulata, two species of predator fish, because its BI was more than 1 and its high lethal concentration (LC50). EOs are also generally considered safe for other arthropods and fishes43. Other authors have supported the conclusion that EOs are safe for non-target organisms. Alsalhi et al.43 supported that trans-cinnamaldehyde showed very low toxicity on Gambusia affinis (LC50 = 3960.6 ppm). Nwanade et al.17 reported that trans-cinnamaldehyde provided a less toxic effect on Tenebrio molitor (LC50 = 28.4 μL/mL). In addition, Hýbl et al.44 indicated that C. zeylanicum EO did not show toxicity against honey bee, Apis mellifera (LC50 = 4.542 μL). Sabahi et al.45 reported that C. citratus EO was not toxic to A. mellifera . It has also been shown that geranial, the major constituent of C. citratus EO, had a low negative effect on a predatory bug, Podisus nigrispinus (LD50 = 25.56 μg/insect−1)35. In contrast, temephos is highly toxic to several non-target organisms e.g., Acilius sulcatus, Anisops bouvieri, and G. affinis with LC50 ranging from 0.957 to 4.817 ppm43. Chellappandian et al.11 reported that temephos showed a highly toxic effect against aquatic mosquito larvae predator, Toxorhynchites splendens. Along the same line, USA EPA46 concluded that temephos showed highly acute toxicity to risk quotients for freshwater fish: the LC50 against rainbow trout was 3490 ppb. Similarly, in this study, 1% (w/w) temephos showed a high level of toxicity to two fish species, P. latipinna and P. reticulata with LC50 ranging from 298.7 to 526.7 ppm. Furthermore, temephos resists degradation and accumulates in the environment at a high level, thus harming non-target organisms46. On the contrary, EOs and their constituents are natural substances that degrade quickly in the environment and do not accumulate in the environment, hence much safer for the environment. More than one BI for all formulations verified that those formulations were absolutely safe for these non-target aquatic predators. The mortality rate after the treatment of the eggs was much higher than the mortality rate against the fishes , it is still necessary to investigate other factors that affect to mortality of mosquito vectors and their eggs, e.g., a field study and a study of the post-application temperature effect.On the biosafety of non-target aquatic predators of mosquito eggs, the combined EO formulation was deemed safe for All plants were obtained under national and international guidelines. The plants were collected under the supervision and permission of the School of Agricultural Technology, KMITL. All of the authors complied with all local and national guidelines.C. verum were purchased from a local Chinese pharmacy in Thailand . Peels of C. aurantium fruit were obtained from a farm in Nakhon Ratchasima province, Thailand (14° 58′ 47.6400″ N/102° 5′ 51.9756″ E). Fresh stems of C. citratus were obtained from a farm in Chanthaburi province, Thailand (12° 36′ 34″ N/102° 06′ 16″ E) in July–October 2021. All plant species were identified by Mr. Tanapoom Moungthipmalai, a herbal specialist at the KMITL herbal museum, and some of the specimens were kept at the KMITL herbal museum, School of Agricultural Technology, KMITL.Dried barks of Plant part (1000 g) was washed and extracted by hydro-distillation in 2000 mL of distilled water at 100 °C for 5 h. The rate of distillation was two drops of EO per second. The EO was then filtered and stored in a tea color bottle at 4 °C.C. aurantium, C. citratus, and C. verum EOs were analyzed by an Agilent 6890 N gas chromatograph GC–MS at the central Laboratory, KMITL, following our previous protocol27. Serving as the mobile phase is 1 mL per min flow of helium (99.99%). To start, 0.2 μL of extract in ethyl alcohol solution (split ratio = 1:100) of each EO was injected into the column. A 5973-N mass spectrometer (using an HP-5 MS fused silica capillary column (30 m × 0.25 mm ID with 0.25 m film thickness of 5% phenyl-methylpolysiloxane coating), an electron ionization system with 70 eV electron energy (30–500 m z−1), and an Agilent 6890-N gas chromatograph (USA) made up the GC–MS system. The column temperature was programmed to increase gradually from room temperature to 50 °C and stay there for 2 min. The column temperature was then increased to 200 °C and maintained there for 3 min at a rate of 10 °C min−1. In the final stage, the column temperature was raised to 260 °C at a rate of 15 °C min−1 and held there for 20 min. The injector and detector temperature were held at 270 °C. The total running time was 40 min. A mass spectra search program with Wiley 7 N library was used for identifying all components of EOs. The mass spectra of peaks were compared with those stored in Adams53 and NIST 1754 libraries. Temperature-programmed retention indices (RI) were determined using n-alkanes (C7–C30). The experiment was performed in three replicates.Samples of C. verum EO, d-limonene (96% pure), a major constituent of C. aurantium EO, and geranial (96% pure), a major constituent of C. citratus EO together with standard n-alkanes (C7–C30) were supplied by Sigma-Aldrich company (USA). Temephos (1 ppm), the positive control, was obtained from Thailand’s Government Pharmaceutical Organization (GPO) . Ethyl alcohol (95% v/v) was supplied by Thailand’s Liquor Distillery Organization . All chemicals used in this study were reagent-grade.Cinnamaldehyde (98% pure), a major constituent of Ae. aegypti and Ae. albopictus, reared in the Entomology laboratory at the School of Agricultural Technology, KMITL. They were reared under the conditions of 26.5 ± 2 °C temperature, 75.0 ± 2% RH, and an 11 ± 13 h photoperiod. Female adult mosquitoes were fed with 2.5% glucose solution + 2.5% multivitamin syrup solution and periodically blood-fed via membrane by an artificial membrane method1. The first generation of eggs was used in various experiments.The mosquito eggs used in this experiment were freshly laid eggs of mosquitoes of two species, 23. The eggs used for this bioassay were stored at 26.5 ± 2 °C for 7 days after female mosquitoes had laid their eggs on a Whatman No.1® filter paper. Eggs were selected under a stereomicroscope (Nikon® Type 102): abnormal eggs were discarded, and normal eggs were collected for the bioassay. For each mosquito species, 25 eggs were suspended in 99 mL of distilled water in a 150 mL plastic cup. A treatment was added to the cup: 1 mL of each EO formulation. This assay was done in ten replicates for each treatment, with positive, negative, and neutral controls: 1 ppm temephos  for destroying mosquito larvae), 70% (v/v) ethyl alcohol, and pure water, respectively. The numbers of hatched larvae at 30 min, 1, 6, 24, and 48 h post-treatment were observed and recorded because it was easier and more practical to count live larvae than to count dead eggs under a stereomicroscope. The percentage inhibition rate after 48 h was determined by the formulas23 below,Ovicidal activity bioassay was performed on the eggs following the method of Puwanard and Soonwera4 below,The effective inhibition rate index (EII) as a comparative efficacy index between an EO and temephos, was determined by the formulaEII < 1 indicates that the EO formulation was not as effective as temephos; EII = 1 indicates that the EO formulation was as effective as temephos; and IRI > 1 indicates that the EO formulation was more effective than temephos.31,Synergistic index (SI) is an efficacy comparison index between a combined formulation and the corresponding individual EO or individual EO constituent. It was calculated by the following formulaSI < 1 indicates synergistic; SI > 1 indicates antagonistic; and SI = 0 indicates not either one.The experimental methods and procedures were performed in accordance with the guidelines and regulations of the National Research Council of Thailand guide for the care and use of laboratory animals and approved by the King Mongkut’s Institute of Technology Ladkrabang of animal care and use committee. This study was carried out in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.P. latipinna and P. reticulata, was analyzed with a modified technique reported by Rajeswary et al.55. The test used four concentrations  of treatment that corresponded to the estimated LC50 against the two mosquito species. Both fish species were purchased from a farm in Nakhon Pathom province, Thailand. They were separately kept in a glass container containing 10.5 L of water at 35 ± 2 °C and 77 ± 5% RH. With the registration number KDS2021/002 , the King Mongkut's Institute of Technology Ladkrabang's Ethics committee had approved each bioassay used in this study. One adult P. latipinna or P. reticulata was put in a glass jar containing 99 mL of water and contaminated with a treatment at a specified concentration. Four replicates were done for each treatment with positive control. Data on mortality and swimming sluggishness were recorded for 5 days post-treatment.The effect of individual EOs, EO constituents, and combined formulations against non-target aquatic predators, 55 below,The biosafety index (BI) was determined by the formulaBI > 1 indicates that the EO formulation was safe for the non-target organisms, and BI < 1 indicates that the EO formulation was not safe for non-target organisms.2 critical point drier. Each dehydrated sample was mounted on a stub coated with gold–palladium and examined with a JSM-5800 LV  SEM. Photographs of the egg surface morphology were taken.After 48 h of treatment, the morphology of the external surface of the eggs of each mosquito species that underwent a treatment or control was observed under scanning electron microscopy (SEM) at the Scientific and Technological Research Equipment Centre, Chulalongkorn University, Thailand. Samples were placed in a fixative, 2.5% glutaraldehyde for 30 min in 0.1 M phosphate buffer. Thoroughly washed with the same buffer, the eggs were dehydrated by soaking in a series of alcohol solutions in water . Each 1-h soaking process with an alcohol solution was replicated three times with an automatic tissue processor. Then, the eggs were dried with a COP < 0.05 were applied to the mortality data of mosquito eggs. The treatment time to produce 50% egg mortality (LT50) was determined by probit analysis. The eggs were observed at 30 min, 1, 6, 24, and 48 h after the treatment. The concentration of a treatment that provided 50% mortality (LC50) against mosquito eggs was determined. The tested concentrations were 10,000 and 30,000 ppm for individual EOs and 5000, 10,000, and 30,000 ppm for individual EO constituents. The LC50 values against the two species of fish were determined at 500, 1000, 2500, and 5000 ppm. SPSS Statistical Software Package version 22 was the statistical software package used.The design of the experiments was completely randomized. Analysis of variance (ANOVA) and Duncan’s multiple range test at Supplementary Information.


N-acetyl-β-D-glucosaminidase), amino acids (leucine aminopeptidase), and of organic phosphorus  and sulfur compounds (sulfatase). Remarkably, all fungal strains were capable of hydrolyzing all the offered substrates. However, the hydrolysis rate (Vmax) and half-saturation constant (Km) varied among the fungal strains depending on the enzyme type. Temperature had a strong impact on the EEAs, resulting in Q10 values of up to 6.1 and was species and substrate dependent. The observed impact of temperature on fungal EEA suggests that warming of the global ocean might alter the contribution of pelagic fungi in marine biogeochemical cycles.Although terrestrial and aquatic fungi are well-known decomposers of organic matter, the role of marine fungi remains largely unknown. Recent studies based on omics suggest that marine fungi potentially play a major role in elemental cycles. However, there is very limited information on the diversity of extracellular enzymatic activities performed by pelagic fungi in the ocean and how these might be affected by community composition and/or critical environmental parameters such as temperature. In order to obtain information on the potential metabolic activity of marine fungi, extracellular enzymatic activities (EEA) were investigated. Five marine fungal species belonging to the most abundant pelagic phyla (Ascomycota and Basidiomycota) were grown at 5 °C and 20 °C, and fluorogenic enzymatic assays were performed using six substrate analogues for the hydrolysis of carbohydrates (β-glucosidase, β-xylosidase, and  Fungi are eukaryotic and osmoheterotrophic organisms depending on organic matter to grow and obtain energy . OsmotroApproximately 70% of the Earth’s biosphere is composed of persistently cold environments, from the deep sea to polar regions ,12. Depemax and Km) of the EEA of five oceanic fungal isolates using six different fluorogenic substrate analogues. β-glucosidase and β-xylosidase were used as a proxy for the degradation of plant-derived matter; N-acetyl-β-D-glucosaminidase were used for the utilization of animal and fungal chitinous compounds; alkaline phosphatase and leucine aminopeptidase were used for the cleavage of phosphate moieties and peptides, respectively; and sulfatase was used for the degradation of sulfate esters in macromolecules. We used five fungal strains isolated from the oceanic water column, with two strains belonging to the phylum Ascomycota and three to Basidiomycota. We selected these strains because Ascomycota and Basidiomycota are the most abundant pelagic fungal phyla  using the equation obtained from the standard calibration lines of MCA and MUF. The resulting hydrolysis rates were fitted directly with the Michaelis–Menten equation using nonlinear least-squares regression analysis with R software [max) and half-saturation constant (Km) were calculated. To obtain the biomass-specific activity, the Vmax was normalized to the fungal biomass obtained from the dry weight. Additionally, the Q10 value was calculated to identify the dependence of enzyme activity on temperature [The increase in fluorescence over time (3 h) was transformed into hydrolysis rate [μmol Lsoftware . The enzperature .max and Km, a one-way analysis of variance (ANOVA) was performed to determine differences among species and temperature. Tukey’s Honestly Significant Difference (Tukey’s HSD) was used for a multiple and simultaneous comparison between species and to identify significance at the species level. A Student’s T-test was used to test the normal distribution of the data. A principal component analysis (PCA) was performed to analyze the fungal enzymatic activity by species and substrate. For this purpose, the prcomp command of the R software was used. To maximize the sum of the variance of the squared loadings, Varimax rotation was performed.For the kinetic parameters Vmax and Km of the EEA varied, however, among the different fungal strains and substrates  at 5 °C (6.4 ± 3.2 μmol/g biomass/h) and 20 °C (7.4 ± 3.0 μmol/g biomass/h)  A. The otlaginosa .m was significantly higher in S. dacryoidea than in the other fungal strains  and ranged between 139.6 ± 19.1 μM and 78.6 ± 11.9 μM . Nonetheless, the other two species of the phylum Basidiomycota, R. sphaerocarpa and R. mucilaginosa, showed low Km values, especially the latter. Finally, for the Ascomycota species, the Km was highest at 5 °C  with 12.9 ± 3.4 μM for B. parvus and 38.7 ± 14.6 for M. australis.The K 11.9 μM A. MoreovB. parvus exhibited a significantly higher Vmax for BXY than the other strains . In B. parvus, the Vmax was 0.8 ± 0.3 μmol/g biomass/h at 5 °C and 1.0 ± 0.5 μmol/g biomass/h at 20 °C. In the other strains, the hydrolysis rates were higher at 20 °C than at 5 °C, amounting to 0.5 and 0.1 μmol/g biomass/h, respectively. When all the fungal species were compared, the temperature had a greater effect in M. australis and S. dacryoidea with Q10 values of 2.1 and 1.8, respectively, than in the other fungal strains  (S. dacryoidea showed a high Km (49.7 ± 27.6 μM) followed by B. parvus with a Km value of 37.2 ± 8.5 μM. In contrast, at 20 °C, the Km decreased in both B. parvus and S. dacryoidea but increased in M. australis. The other two species, R. sphaerocarpa and R. mucilaginosa, showed low Km values corresponding to their low Vmax.The K = 0.90) B. At 5 °B. parvus exhibited similar NAG hydrolysis rates at 5 °C and 20 °C   C. At 5 °ectively .B. parvus coincided with a high Km , except in M. australis   at 5 °C resulting in a Q10 of 6.1  A. The LA0 of 6.1 . All the strains .B. parvus and S. dacryoidea exhibited significantly higher Km values than the other fungal strains   A. The KmM. australis exhibited a significantly higher Vmax (8.8 ± 3.7 μmol/g biomass/h) than the other fungal isolates  (max was about 10-fold lower than at 5 °C (0.6 ± 0.2 μmol/g biomass/h). In contrast, in all the other fungal strains, Vmax slightly increased with temperature  B. At 20 perature B. The Q1ustralis .m was significantly higher in the Ascomycota than in the Basidiomycota strains   than at 20 °C, whereas the Km in B. parvus was higher at 20 °C (85.7 ± 10.5 μM) than at 5 °C. The other fungal species belonging to the Basidiomycota phylum exhibited Km values ranging from 55.1 μM to 4.1 μM (The K = 0.01) B. M. auso 4.1 μM B.max values were determined for B. parvus and S. dacryoidea at 5 °C   than at 20 °C . In contrast, for M. australis, R. mucilaginosa, and R. sphaerocarpa, the SUL activity increased with temperature. The Q10 value in M. australis was 1.9 while all the other fungal strains were <1.0  (Sulfatase (SUL) activity was generally low compared to the other extracellular enzymatic activities C. Higherere <1.0 . With th≤ 0.002) C.R. mucilaginosa and R. sphaerocarpa belonging to the same genera (Rhodotorula) indicated very similar activity levels and extracellular enzyme characteristics. In contrast, the other fungal strains substantially differed in their extracellular enzymatic activity and enzyme characteristics, independent of their taxonomic affiliation.PCA analysis allowed for the comparison of the five studied marine fungal species and the different extracellular enzymatic activities determined. The explained variance of the dataset was 63.9% . The minB. parvus, M. australis, R. mucilaginosa, R. sphaerocarpa, and S. dacryoidea, produced extracellular enzymes to degrade substrate analogues of carbohydrates such as cellulose, chitin, and xylan. Additionally, they all produced enzymes to cleave off amino acids, phosphate, and sulfate esters from organic compounds.All the studied marine pelagic fungi species, Rhodotorula (R. mucilaginosa and R. sphaerocarpa) exhibited similar kinetic parameters (Vmax and Km) for the majority of extracellular enzymes  seem to be more suitable to overcome this P-limitation, but a higher amount of substrate, and hence of organic matter, might be needed for this purpose.Inorganic phosphate (Pi) is the preferred phosphorus source for microbial uptake. In surface waters, however, Pi frequently limits phytoplankton productivity . To over high Km B. In conFusarium proliferatum was reported to produce sulfatase for the assimilation of sulfated fucoidans of brown algae [Rhodotorula maintained a low sulfatase activity, whereas it varied among the other species. The Vmax and Km were high in S. dacryoidea, indicating fast hydrolysis and low substrate affinity. As all the fungal species showed different sulfatase activity, we can deduce that these marine fungi might be capable to use sulfated amino acids as well as carbohydrates.In living organisms, sulfur is the sixth most abundant element as it can be found in amino acids, such as cysteine and methionine, but also in polysaccharides and proteoglycans . In contwn algae . In the In this study, even though we analyzed just a few hydrolysis possibilities, the functional diversity seems to be broad in marine fungi. As suggested by Berlemont  and BaltThe ocean is a complex and diverse ecosystem composed of microorganisms such as archaea, bacteria, fungi, protists, and viruses. As osmoheterotrophic organisms, fungi might provide intermediate decomposition products needed for other microorganisms. These also might lead to the proliferation or inhibition of other microbes as well as enzymatic activities. As marine fungi can utilize a wide range of organic substrates , nutrien10 values lower than 1.0 would indicate a reaction rate completely independent of temperature, whereas values above 1 indicate thermodependency [Temperature is considered one of the most important abiotic factors because it influences essentially all biochemical reactions . Enzymespendency .R. mucilaginosa and R. sphaerocarpa maintained Vmax values as low as 0.1 μmol/g biomass/h, whereas the other species exhibited only half of the activity at 5 °C, particularly for BGL and ALP  is due to a conformational change in proteins and indicates the need for high activation energy. We found that only for LAP, all the examined fungal species expressed Q10 values higher than 1. Generally, the temperature where an enzyme can achieve its highest activity does not match the optimal growth temperature of the microorganism that is producing it [B. parvus, M. australis, R. sphaerocarpa, and S. dacryoidea, can respond to a temperature rise by increasing enzymatic activity. According to the Arrhenius equation, temperature can influence the activation energy needed to initiate a chemical reaction, and hence, its rate. At a higher temperature, the molecules gain energy to move faster, which also increases the collisions between enzymes and substrates. As a result, elevated extracellular enzymatic activity at increasing temperatures in the surface waters might lead to changes in cleavage and uptake rate of organic matter in oceanic fungi.Microorganisms isolated from cold environments can also display kinetic parameters similar to those of their mesophilic counterparts ,90. Ito ucing it . Apparenmax and Km values varying over a range of one order of magnitude. Although the fungal species were isolated from coastal Antarctic waters , and hence are potentially adapted to low temperatures, they exhibited higher extracellular enzymatic activity at 20 °C than at 5 °C, with some exceptions. While in some fungal strains the Km values for specific extracellular enzymes were higher at low temperatures, for other enzymes they were lower. Additionally, there was considerable species-specific variability in the extracellular enzymatic activity. Taken together, our study indicates that temperature might be one of most important physical factors controlling marine fungal extracellular enzymatic activity. Thus, species composition and temperature determine the role of marine fungi in organic matter cleavage in the global ocean.In the present study, we have shown that different marine fungal strains exhibit varying extracellular enzyme characteristics with V


Q-Exactive Orbitrap high-resolution mass spectrometry, 16 chemical components such as gallic acid, protocatechuic acid, and neochlorogenic acid in QZC were characterized and unambiguously identified based on comparison with the corresponding reference standards. In addition, the correlation between the focused components and their corresponding raw herbs from QZC prescription was investigated. For the first time, the relationship between the components mentioned above and their anti-inflammatory activity was explored via network pharmacology analysis, and a visualized network of “medicinal materials-QZC-compounds-targets-pathways” was established. Based on the brief prediction results of network pharmacological analysis, ultra-performance liquid chromatography coupled with photodiode array detector method was validated in terms of linearity, limit of detection, limit of quantification, precision, repeatability, stability, and recovery test and was successfully employed to determine 16 compounds in 28 batches of QZCs, which confirmed the feasibility and reliability of the established method for the quantitative analysis of 16 compounds in QZC. Considering the content and bioactivity of the tested components, four compounds were recommended as candidate indicators for quality evaluation ultimately. The potential value of this study could not only support a quality evaluation of QZC but also provide a theoretical basis for further in-depth research of QZC in clinical research.Qufeng Zhitong capsule (QZC) is a well-known Chinese patent medicine that has been widely applied for the clinical treatment of rheumatoid arthritis and other inflammatory diseases. To date, its material basis is still unclear, which has greatly limited its clinical application. In this study, by taking advantage of ultra-high-performance liquid chromatography tandem  As a clinically effective Chinese patent medicine, Qufeng Zhitong capsule (QZC), which consists of seven herbs, including Radix Angelicae Pubescentis , Herba Geranii , Radix Dipsaci , Radix et Rhizoma Clematidis , Flos Carthami , Herba Visci , and Radix Aconiti Kusnezoffii Cocta  , is commNetwork pharmacology is a comprehensive analysis method based on the interaction network of disease, gene, target protein, and drug, which has been proved to be a practical way to explore the potential targets and pathways of traditional Chinese medicine (TCM) in the treatment of diseases . By applAt present, the research on QZC mostly focuses on its clinical application, while much less attention has been paid to the identification and quantitative analysis of its chemical components. Moreover, studies on the underlying mechanism related to the effects of QZC are negligible because of its complex chemical components and the multi-targets networks. As is known to us, an effective quality control system is the key to ensuring the quality, safety, and efficacy of TCM in clinical use. Various chemical analysis methods such as thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and other detection techniques have been employed for quality assessment and standardization of QZC. As the standard to evaluate the quality of QZC, ChP stipulates that the content of akebia saponin D (AsD) should not be less than 1.5 mg per capsule and gallic acid (Gaa) should not be less than 0.30 mg per capsule, which indicate that there are certain limitations such as using the limited quality indicators and the laborious operation for preparing sample solution , 12. TheQ-Exactive Orbitrap high-resolution mass spectrometry (UHPLC/Q-Orbitrap-MS) within 35 min. Besides, the relationship between the 16 compounds and their anti-inflammatory activity was investigated by network-based pharmacology analytical approaches, which provided the basis for the quantitative analysis of QZC. We also used the “spider-web” mode to optimize the sample preparation process so as to improve the efficiency of the extraction method comprehensively. For the quantitative analysis of the multi-components in QZC, a novel method was established by ultra-performance liquid chromatography with photodiode array detector (UPLC-PDA), which was validated in terms of linearity, limit of detection (LoD), limit of quantification (LoQ), precision (intra- and inter-day), repeatability, stability, and recovery test and successfully applied to the quantitative determination of 16 compounds mentioned above in 28 batches of QZCs. The results confirmed its feasibility and reliability in practice with the advantages of simple sample preparation, good chromatographic peak shape, and high repeatability.In this study, 16 compounds, including iridoids, coumarins, phenolic acids, and triterpenoid saponin, were identified and traced to their herbal sources by ultra-high-performance liquid chromatography tandem Q water purification system .Methanol was purchased from Thermo Fisher Scientific . Formic acid was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . Water used in the experiment was purified by a Milli-Reference standards including Gaa, protocatechuic acid (Pra), neochlorogenic acid (Nea), chlorogenic acid (Cha), cryptochlorogenic acid (Cra), loganic acid (Loa), corilagin (Cor), loganin (Log), isochlorogenic acids A−C (IaA−C), angelol A (AnA), columbianetin acetate (Coa), osthole (Ost), columbianadin (Col), and AsD were obtained from Shanghai Yuanye Bio-Technology Co., Ltd. . The purities of these reference standards were determined to be above 98% by UPLC analysis. All the 28 batches of QZCs were provided by Shaanxi Buchang Pharmaceutical Co., Ltd.  and numbered as S1-S28. Duhuo, Laoguancao, Xuduan, Weilingxian, Honghua, Hujisheng, and Zhicaowu were also provided by Shaanxi Buchang Pharmaceutical Co., Ltd., and identified by Prof. Yuefei Wang. All samples were deposited in the State Key Laboratory of Component-based Chinese Medicine, Tianjin University of Traditional Chinese Medicine .https://pubchem.ncbi.nlm.nih.gov/) was used to search the structural formulas and IUPAC International Chemical Identifiers (InChI) of the tested compounds. The targets of the compounds were collected from the Swiss Target Prediction database (https://www.swisstargetprediction.ch/) and Bioinformatics Analysis Tool for Molecular Mechanism of TCM  and analyzed by Ingenuity Pathway Analysis (IPA). The protein-protein interaction (PPI) data were obtained from the STRING database (https://string-db.org/). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis on target proteins was performed based on the KEGG database (https://www.kegg.jp/). Ranked by the KEGG analysis, the top 30 pathways related to inflammation were screened out and irrelevant pathways were eliminated. Origin 9.6 software was used to build a visualized network of “medicinal materials-QZC-compounds-targets-pathways.”We followed the network pharmacology analysis methods of Xue et al. . The PubQ-Exactive Orbitrap MS equipped with heated electrospray ionization (HESI) source. Chromatographic separation was performed using an ACQUITY UPLC® BEH C18 column  at 40°C. The mobile phase system consisted of 0.1% formic acid aqueous solution (v/v) (A) and methanol (B) at a flow rate of 0.3 mL min−1 with the following gradient program: 0–5 min, 3–9% B; 5–9 min, 9–15% B; 9–11 min, 15–17% B; 11–15 min, 17–27% B; 15–18 min, 27–28% B; 18–26 min, 28–50% B; 26–31 min, 50–66% B; 31–35 min, 66–74% B; 35–36 min, 74–3% B. The injection volume for all samples was 2 μL. The MS method used in this study was adopted from Xue et al. [2 (TopN) modes simultaneously. Ions were scanned at high resolution , and the MS scan range was 100–1500 m/z at both MS1 and MS2 levels. The maximum injection time of 50 and 100 ms was used for MS1 and MS2, respectively. The automatic gain control (AGC) target was set to 3e6 and 1e5 in MS1 and MS2, respectively. The optimal MS parameters were as follows: spray voltage, −2.5 kV (in negative ion mode), 3.5 kV (in positive ion mode); sheath gas flow rate, 35 Arb; aux gas flow rate, 10 Arb; capillary temperature, 350°C; aux gas heater temperature, 350°C. All data were acquired and processed using the Thermo Scientific™ Xcalibur™ system.The qualitative analysis was performed on a Thermo Scientific UltiMate 3000 instrument  coupled with a e et al. . The masEquipped with a binary solvent manager, an autosampler, and a PDA detector, an ACQUITY UPLC H-class PLUS system (Waters) was used to perform the quantitative analysis. A multiwavelength switching method was adopted on the PDA detector, in which the detection wavelength was set as follows: 0.00–2.20 min at 300 nm, 2.20–3.50 min at 259 nm, 3.50–5.20 min at 300 nm, 5.20–18.00 min at 239 nm, and 18.00–35.00 min at 325 nm. The wavelength was set at 211 nm separately for the detection of AsD in consideration of its maximum response at 211 nm. Other liquid chromatography parameters were the same as the qualitative analysis described above.v/v) to prepare stock solutions, respectively. A certain amount of each stock solution was placed in a 10 mL volumetric flask and diluted to volume with 10% methanol aqueous solution at the concentration of 24.89 μg mL−1 Gaa, 1.332 μg mL−1 Pra, 3.034 μg mL−1 Nea, 7.442 μg mL−1 Cha, 3.036 μg mL−1 Cra, 33.92 μg mL−1 Loa, 10.68 μg mL−1 Cor, 7.686 μg mL−1 Log, 2.490 μg mL−1 IaB, 1.747 μg mL−1 IaA, 3.192 μg mL−1 IaC, 9.231 μg mL−1 AnA, 6.269 μg mL−1 Coa, 10.86 μg mL−1 Ost, 1.602 μg mL−1 Col, and 138.2 μg mL−1 AsD. The mixed standard solution was diluted stepwise with 50% methanol solution to obtain seven different concentrations for plotting the calibration curves. All the standard solutions were stored at 4°C when not in use.A total of 16 reference standards were accurately weighed and dissolved in 10–50% methanol aqueous solutions (QZC powder (0.5 g) precisely weighed was transferred into a 25 mL volumetric flask and then ultrasonically extracted with 75% methanol aqueous solution at 60°C for 30 min. After cooling down to room temperature, the extracted solution was diluted to scale by 75% methanol aqueous solution and centrifuged at 12700 rpm for 10 min.Weilingxian and Duhuo were pulverized into homogeneous powder and about 0.5 g of the above powder was weighted accurately and then extracted with ultrasonic assistance in 25 mL of 75% methanol aqueous solution at 60°C for 30 min separately. The solutions were centrifuged at 12700 rpm for 10 min. Honghua, Laoguancao, Hujisheng, Zhicaowu, and Xuduan were refluxed twice with water for 3 h each time, respectively, and the filtrates were subsequently centrifuged at 12700 rpm for 10 min.QZC, Weilingxian, and Duhuo solutions were diluted 25 times with water for UHPLC-MS/MS analysis, respectively, while Honghua, Laoguancao, Hujisheng, Zhicaowu, and Xuduan solutions were diluted 25 times with 10% methanol aqueous solution for UHPLC-MS/MS analysis, respectively. QZC solution was diluted twice with water for UPLC-PDA analysis.x-axis and the peak area as the y-axis. The LoD and LoQ were measured as concentrations corresponding to a signal-to-noise ratio of 3 : 1 and 10 : 1, respectively. The intra- and inter-day precisions were carried out by six repetitive injections on the same day and for three consecutive days. The stability test was evaluated by injecting the same sample solution at 0, 2, 4, 6, 8, 10, and 12 h after preparation, respectively. The repeatability was determined by analyzing six prepared samples from the same source. The recovery was investigated by adding an accurate amount of standard solutions to 0.25 g sample powder. Six samples were prepared in parallel according to the preparation method of sample solution.The calibration curves were plotted with the concentration of tested reference as the The heat map, box plot, and parallel coordinate plot were performed using Origin 2019 software .Q-Orbitrap-MS method was established to characterize the chemical constituents in QZC. As a result, sixteen compounds, including two iridoids, four coumarins, nine phenolic acids, and one triterpenoid saponin, were identified by comparing the retention times and mass spectrometry data with reference standards. Then, the established method was applied to detect components in raw herbs from the QZC prescription to trace the herbal sources of the identified components. The information of all the compounds is summarized in A UHPLC/Based on computational biology, network analysis, and other disciplines, network pharmacology can reveal the complex network relationship among drugs, targets, and diseases so as to display the network of drugs-compounds-targets-pathways visually , 15. Givγ, TNF-α, and IL-17A to ease alveolitis and pulmonary fibrosis in the bleomycin-induced mouse pulmonary fibrosis model [P value <1.0e −16. These results indicated that all the 16 compounds exhibit potential anti-inflammatory pharmacological activity.Zhang et al. found that QZC could upregulate the level of inflammatory cytokine IL-10, inhibit synovial hyperplasia, and reduce cartilage damage in rats with arthritis . Relevanis model . In addiis model , 21. The−1), extraction time , and extraction temperature  were investigated by single-factor experiment to obtain higher extraction efficiency of the tested compounds. To show the optimization process of the extraction method intuitively, the “spider-web” mode proposed by our research group in 2016 [Cm-k; the maximum content value of each compound in the tested extracting conditions was assigned as Ck(max); the normalized value of content was assigned as Em-k. Among them, m was denoted as different extract condition, namely extraction solvent , ratio of material/solvent , extraction time , and extraction temperature . K stood for the different compounds, namely Gaa, Pra, Nea, Cha, Cra, Loa, Cor, Log, IaB, IaA, IaC, AnA, Coa, AsD, Ost, and Col. The calculation formula is shown inExtraction variables, including extraction solvent , ratio of material/solvent  of the “spider-web” mode. α, the angle between two adjacent dimensions, was 22.5°. By calculating the regression area of the “spider-web,” the efficiency of extracting index compounds was comprehensively evaluated. The regression area of the “spider-web” was marked as S and calculated to be 2.78. The calculation formula is shown in  is the optimal extraction condition with a “spider-web” area at 2.84, which is shown in The chromatographic conditions were optimized to accurately determine the 16 chemical components in QZC with satisfactory peak shape, sensitivity, and resolution. Adding 0.1% formic acid to the mobile phase helped achieve satisfactory peak symmetry, and methanol was chosen to enhance sensitivity and resolution. Therefore, a gradient elution system composed of 0.1% formic acid-methanol was chosen. The column temperature was set at 40°C, improving the separation and peak shape. To attain lower noise and satisfactory sensitivity, we established a UV wavelength switching method using a variable-wavelength detector and four wavelengths, including 239, 259, 300, and 325 nm, were selected as the detective wavelengths for determining the different compounds in QZC.R2) values greater than 0.999, which was qualified to be used for quantitative analysis of the focused compounds in QZC. The LoD and LoQ values were 0.02118–1.728 μg mL−1 and 0.08707–3.455 μg mL−1 for the 16 compounds quantified in this study, respectively. The RSD values of the measured intra- and inter-day precisions were lower than 2.6% and 2.9%, respectively, indicating that the instrument has good precision. The experimental method had good repeatability with RSD values below 2.9% and the RSD values of the sample stability test were lower than 2.4%, showing that the sample solution could remain stable within 12 h. In addition, the recovery rates for spiked samples ranged from 92.1% to 108.2%. In summary, all the results of the experiments were within an acceptable range of quantitative research, which demonstrated that the established method is rapid and simple with good accuracy and reproducibility for the content determination of 16 compounds in different batches of QZCs. Compared to the previously reported analytical methods [Up to now, there have been limited studies on quantitative analysis of chemical compounds in QZC, especially the multi-components quantitative analysis. Therefore, a method for the simultaneous determination of 16 compounds in QZC by UPLC-PDA was established for the first time. In addition, the UV wavelength switching method was employed, which made the 16 compounds have satisfactory peak shape, sensitivity, and resolution. The typical chromatograms of sample and mixed standard solutions are shown in −1, the detailed content of which is listed as follows: Gaa between 2.644 and 4.049 mg·g−1, Pra between 0.1175 and 0.1846 mg·g−1, Nea between 0.3662 and 0.5921 mg·g−1, Cha between 1.354 and 1.609 mg·g−1, Cra between 0.4363 and 0.6689 mg·g−1, Loa between 3.640 and 6.008 mg·g−1, Cor between 0.9905 and 2.316 mg·g−1, Log between 0.6843 and 1.003 mg·g−1, IaB between 0.7053 and 0.8372 mg·g−1, IaA between 0.3942 and 0.5321 mg·g−1, IaC between 0.6495 and 0.8388 mg·g−1, AnA between 0.8528 and 1.245 mg·g−1, Coa between 0.7487 and 0.8916 mg·g−1, AsD between 9.652 and 18.24 mg·g−1, Ost between 1.567 and 1.777 mg·g−1, and Col between 0.2017 and 0.2359 mg·g−1, respectively. The RSD values of the different detected components ranged from 4.2% to 19.1%, showing that the content of 16 compounds in different batches of QZCs varied to a certain extent. And fluctuation in the content of the tested compounds may due to the fact that the raw materials come from different sources. In order to intuitively display the trend of content distribution, we adopted a heat map and box plot, which are shown in The validated UPLC-PDA method was subsequently used to determine the content of 16 compounds in 28 batches of QZCs. As shown in Required by ChP, the quality of QZC is evaluated primarily by detecting the content of AsD and Gaa, proved reasonable by this study. Besides, in the current study, we also found that Cha, Loa, Cor, and Ost have potential as an evaluation index of QZC quality control. Cha, a common component contained in Xuduan, Honghua, and Hujisheng, can exert a neuroprotective effect . MoreoveIn our study, the anti-inflammatory components in QZC were mined through network pharmacology and quantified by using the UPLC-PDA analysis method for the first time. Moreover, 16 compounds extracted from QZC were identified and traced to their herbal sources by UHPLC/Q-Orbitrap-MS. The established method for simultaneous determination of 16 compounds in QZC by UPLC-PDA integrated with the UV wavelength switching method has been demonstrated to be rapid and simple with good accuracy and reproducibility, which was used to investigate the focused compounds in 28 batches of QZCs. Eventually, four compounds, Ost, Cha, Loa, and Cor, were preferentially recommended as candidate components in QZC. The results of the study could help to uncover the chemical basis of QZC and pave the way for evaluation purposes for the quality of QZC.


During past decades, there was a positive trend in growth and nutrition status of adolescents in China, but there was significant regional disparity. The purpose of this study is to investigate the trends in growth and nutritional status of high school graduates in Hangzhou between 2011 and 2020.2, for thinness, overweight and obesity respectively. High school graduates (Grade 12) who finished the physical examination of the national college entrance examination between 2011 and 2020 were included in this study. Data were obtained from the database of physical examination of the national college entrance exam. Height and weight were measured; body mass index (BMI) was calculated from height and weight. Thinness, overweight and obesity were defined according to the International Obesity Task Force criteria. For the vast majority of the high school graduates were 18 years old or nearly 18 years old, the cutoffs of 18 years were adopted. Those are 18.5, 25 and 30 kg/mP < 0.001). Height increased by 1.80 cm in boys and 1.45 cm in girls. Weight increased by 4.62 kg in boys and 2.51 kg in girls. BMI increased by 1.09 kg/m2 in boys and 0.60 kg/m2 in girls. An increase trend was found in the prevalence of overweight and obesity in both sexes (P < 0.001). Overweight increased by 7.43% (from 9.05 to 16.48%) among boys and 4.05% (from 4.57 to 8.62%) among girls. Obesity increased by 3.85% (from 2.29 to 6.14%) among boys and 1.76% (from 0.64 to 2.40%) among girls. The prevalence of thinness fluctuated in both boys and girls, 12.42–15.59% among boys and 18.97–23.68% among girls. Boys had higher odds of overweight and obesity and lower odds of thinness than girls (P < 0.001).There was a significant growth trend in height, weight and BMI in both sexes (There is a positive trend in growth and nutritional status of high school graduates in Hangzhou. However, there is still a considerable prevalence of thinness, it indicates a double burden of undernutrition and overnutrition. Growth and nutritional status in adolescence play an important role in the whole human life. Both undernutrition and overnutrition in adolescence are associated with adverse health consequences even in adulthood –6. UnderThe trends of growth and nutritional status of adolescents are associated with socioeconomic development –12. In dHangzhou is a developed city located in the southeast of China. During the last decade, the social economy of Hangzhou developed rapidly , 15. We In China, every high school graduate who apply to college is required to take a physical examination before the national college entrance examination(Gaokao). The current study is based on the data of physical examination of high school graduates in Hangzhou.A total of 481,353 students finished the physical examination of the national college entrance examination between 2011 and 2020. Data were exported from the database of physical examination of the national college entrance examination. 2 students were excluded because of obvious mistakes of height and weight, one with a height of 1 cm and the other with a height of 0 cm and a weight of 0 kg. And thus, a total of 481,351 students were finally included, 229,555 boys and 251,796 girls.Height and weight were measured by trained doctors or nurses. Height was measured without shoes to the nearest 1 cm and weight was measured with light clothes to the nearest of 1 kg. Every student had a chance of remeasurement after the physical examination, if he or she questioned the results.2, for thinness, overweight and obesity respectively.Body mass index(BMI) was calculated as weight in kilograms divided by the square of height in meters. Thinness, overweight and obesity were defined according to the International Obesity Task Force(IOTF) criteria , 17. ForContinuous variables including height, weight and BMI were expressed as mean and SD, differences between sexes were examined by the t-test. Linear regression was used to examine the trends in height, weight and BMI during the study period. Categorical variables including the prevalence of thinness, overweight and obesity were expressed as proportions and 95% confidence interval (CI), differences between sexes were examined by Pearson’s chi-squared test, odds ratio(OR) was calculated. Linear-by-linear association test was used to examine the trends in prevalence of thinness, overweight and obesity during the study period.P < 0.05 was considered statistically significant.All statistical analyses were performed with the statistical software SPSS version 24. A two-sided P < 0.001). Height increased by 1.80 cm (from 171.95 ± 5.85 to 173.74 ± 6.04 cm) in boys and increased by 1.45 cm(from 159.84 ± 5.38 to 161.30 ± 5.59) in girls, except a slight decrease in 2017. Weight increased by 4.62 kg (from 63.01 ± 10.53 to 67.63 ± 13.65 kg) in boys and increased by 2.51 kg (from52.00 ± 7.78 to 54.51 ± 9.86 kg) in girls, except a slight decrease in 2017.Table P < 0.001). BMI increased by 1.09 kg/m2(from 21.28 ± 3.24 to 22.37 ± 4.20 kg/m2) in boys, except a slight decrease in 2018. It increased by 0.60 kg/m2(from 20.34 ± 2.76 to 20.93 ± 3.53 kg/m2) in girls, except a slight decrease in 2020. Boys had significantly higher BMI than girls.A significant trend of increase in BMI was found in both sexes between 2011 and 2020(P < 0.001). The prevalence of overweight increased by 7.43%(from 9.05 to 16.48%) in boys and increased by 4.05%(from 4.57 to 8.62%) in girls. There was a positive trend in the prevalence of obesity in both sexes between 2011 and 2020 (P < 0.001). The prevalence of obesity in boys increased by 3.85%(from 2.29 to 6.14%), except a slight decrease in 2018. The prevalence of obesity in girls increased by 1.76%(from 0.64 to 2.40%), except slight decreases in 2013 and 2017.Table p < 0.001), there was no trend of increase or decrease between 2011 and 2020. It fluctuated between 12.42 and 15.59% in boys and between 18.97 and 23.68% in girls.The prevalence of thinness was 14.33% in boys and 21.95% in girls in the whole sample. Boys had lower odds of thinness than girls. Although the prevalence of thinness significantly different among years(During the last century, a large progression in body height and weight was observed all over the world in both sexes, regardless of the country, climate, race or socioeconomic situation , 18–20. Overweight and obesity of children and adolescents is a public health challenge in both developing and developed countries , 27, 28.Although overweight and obesity increase globally, underweight is still an important public health problem, especially in developing countries . In ChinIn China, boys have higher prevalence of overweight and obesity and lower prevalence of thinness than girls , except Because this study is a retrospective study based on the physical examination of the national college entrance examination. There are some limitations to this study. Firstly, data of exact age was not available. Therefore, thinness, overweight and obesity were defined according to the cutoffs of 18 years old, but not the exact age of every subject. Secondly, the measurement was carried out at several medical stations and the equipments were not unified. And the accuracy of height and weight was up to 1 cm and 1 kg. But the large sample size may compensate for the measurement error to some extent. Thirdly, this study didn’t exclude students who applied to college and took physical examination several times in different years. It may affect the independence of sample from years, although they were only a very small part of study population. Fourthly, urban and rural areas were not distinguished in this study, although the growth and nutrition status of adolescents are different between urban and rural areas , 24.This study demonstrates that there is a positive trend of growth and nutritional status of high school graduates in Hangzhou. However, there is still a considerable prevalence of thinness without significant decrease, especially in girls. It indicates a double burden of undernutrition and overnutrition in high school graduates in Hangzhou.The results of Hangzhou add more understanding of growth and nutritional status of adolescents in China. Double duty actions and sex specific actions were needed for public health policies.


Staphylea holocarpa wood as feedstock to evaluate its potential as energy source. We characterized Staphylea holocarpa wood by utilizing FT–IR, GC–MS, TGA, Py/GC–MS and NMR. The results showed that Staphylea holocarpa wood contained a large amount of oxygenated volatiles, indicating that it has the ability to act as biomass energy sources which can achieve green chemistry and sustainable development.Energy is indispensable in human life and social development, but this has led to an overconsumption of non-renewable energy. Sustainable energy is needed to maintain the global energy balance. Lignocellulose from agriculture or forestry is often discarded or directly incinerated. It is abundantly available to be discovered and studied as a biomass energy source. Therefore, this research uses  Human civilization has reached a new historical height with current developments in science and technology, but it has also increased its utilization of energy simultaneously. Energy is extremely important for the development of the material basis and society, and is particularly important economically . As an iBiomass is an important energy source in renewable energy because it can achieve carbon neutrality. Biomass has many advantages, such as diversity, practicality and sustainability . BiomassEucalyptus pellita and Hevea brasiliensis clones are potential woods for bioenergy production, with a net calorific value of 16,502 kJ kg−1 and 19,757 kJ kg−1, respectively [Tachigali vulgaris planted at a land greater than 6 m2 has a net calorific value more than 7.95 MJ/kg, and a medium basic density which is suitable for bioenergy production [Pinus spp. and Quercus spp. were pressed into solid biofuel blocks that can be used to meet the need to generate low-power heat for the residential sector. The preparation of solid biofuel blocks from biomass residues is efficient, economical and easy to manufacture and use [Bioenergy is obtained mostly from wood biomass, and wood-based bioenergy development can play a vital role in achieving energy independence, reducing carbon emissions and promoting rural development . With thectively . Tachigaoduction . Buss etoduction . Pinus s and use .Lignocellulosic biomass is the most widespread biological resource on earth and can be transformed into value-added by-products, including biomass materials, bio-oils and biofuels ,16. LignStaphylea holocarpa Hemsl. (S. holocarpa) is an endemic plant in China; it is a small deciduous tree belonging to staphyleaceae. S. holocarpa has compound leaves (three leaflets), and smooth and glabrous branchlets. Its flowers are pear-shaped bulging capsules, and usually pink in color. It blooms from April to May and ripens in September, which is of ornamental value [S. holocarpa can be found throughout China, including in Henan, Anhui and Shanxi, generally on limestone slopes of deciduous forests at altitudes of about 900–1000 m [S. holocarpa is rich in cellulose. The seeds are pressed and used in soap and paint production [S. holocarpa root has the functions of moisturizing the lungs and relieving coughs, dispelling wind and dampness; this is of great developing value in the utilization of wild economic plants [S. holocarpa plants, including 13 distribution types and 10 variations. According to the distribution area types of the genus, S. holocarpa has obvious temperate characteristics. With the increase in altitude, the population structure changed from growing to stable [al value . S. holo0–1000 m . The baroduction . S. holoc plants . Tian sto stable .S. holocarpa wood, especially in the field of bioenergy. Therefore, this article begins with S. holocarpa wood as a subject of research on its latent as a lignocellulosic bioenergy source. The active component of S. holocarpa wood was extracted and was characterized with FT–IR, GC–MS and TGA. The Py/GC–MS and NMR were used to detect the pyrolysis product and characterize the composition, functional groups and thermal stability from pyrolysis of S. holocarpa wood.To the best of author’s knowledge, limited research has been reported about S. holocarpa wood extracts were analyzed in the range of 4000–550 cm–1  and 1051 cm–1 (the other three extracts) indicate –O stretching in the tetrahedral sheet [–1 was due to C–O–C and –OH vibrations. Thus, –OH, together with the peak assigned to the aromatic ring (1655 cm–1), indicates the presence of phenols [In this experiment, the FT–IR of four 550 cm–1 . Differear H–O–H ,34. The –O–H [–1 . Due to  [–1 [–1 . Bands cretching . There ionds [–1 . In part–1 -2-acrylic acid, which has the potential to inhibit thrombosis, reduce inflammation, inhibit tumors and enhance sperm vitality. Clinically, 3-(4-hydroxy-3-methoxy-methoxy)-2-acrylic acid is mainly used for the vaso-assisted treatment of atherosclerosis, coronary heart disease, cerebrovascular disease, glomerular disease, pulmonary hypertension, etc., for phenyl. Furthermore, 3-(4-hydroxy-3-methoxy-methoxyacid)-2-acrylic acid can be used to treat migraines and vascular headaches, enhance hematopoietic function and treat leukocytopenia and thrombocytopenia [It can also be seen from ytopenia ,46,47. dytopenia ,49.S. Holocarpa wood extracts shows its potential in biomedicine and bioenergy. Thus, S. Holocarpa wood has the potential to be used as a lignocellulosic biomass source for bioenergy production.In addition, many chemical components that are conducive to the development of biomass energy were detected in the four extracts. For example, furfural is an important biomass-derived platform molecule that can be used to synthesize a variety of value-added chemicals. Furfural and its derivatives are promising alternatives to traditional petrochemicals . The furS. holocarpa wood was studied by TGA method in the range of 30–300 °C (rate of 20 °C/min). The changes in sample mass (TGA) and thermal degradation rate (DTG) are shown in S. holocarpa wood. The first stage occurs at temperatures around 83 °C and the weight of the wood was slightly reduced by 3.33%. As shown in the DTG curve, the maximum mass loss rate for this stage occurs at the first peak of 54 °C. There were reports that this stage was a drying process, where mass loss represents the removal of moisture and volatiles [S. holocarpa wood (3.33%) indicates that the wood has a certain moisture. The second phase of the mass reduction occurred in the range of 190–300 °C, and the wood mass was reduced by a total of 20.63% [The decomposition process of olatiles . The weif 20.63% .J. nudiflorum wood biomass [S. holocarpa wood has good thermal stability. In addition, the temperature set by this project was far from the carbonization temperature > 300 °C. Therefore, more volatile components can be obtained using this process [The decomposition temperature of the wood biomass was about 190 °C; however, this decomposition temperature was delayed compared to some reported plants, such as  biomass . The DTG biomass . The mas biomass . The mas process .S. holocarpa wood pyrolysis at 500 °C were: ethyne, fluoro- (5.96%), dihydroxyacetone (4.22%), acetaldehyde (3.31%), ethyl ether (2.81%), hexadecanenitrile (2.33%), 2-propanone, 1-hydroxy- (2.11%), methyl glyoxal (2.07%), furfuryl alcohol, tetrahydro-5-methyl-, cis- (2.05%), etc.  were mainly distributed between 0–80 ppm and 10–160 ppm. The signals at 20.3 ppm and 172.9 ppm shifts were mainly assigned to hemicellulose acetyl [Organic elements are the skeleton of carbon, currently spectrum . For care acetyl ,83,84.1H and 13C–NMR spectra, and analysis of two different aromatic groups [In order to further understand the sample structure in more detail, 2D–HSQC NMR analysis was also carried out in this study. 2D–HSQC NMR is currently one of the most widely used technologies. Its ability to categorize functional groups clearly from 2D–HSQC maps provides an important structural shift in the sample and provides a resolution of overlapping signals in both c groups . In Figuc groups . δC/δH92c groups . As can c groups ,89.S. holocarpa wood was collected from Xiaoqinling National Forest Park, Henan Province, and the wood sample was obtained from the trunk. The sample was processed into two different particle sizes, which were 40–60 μm and 200 μm powder. The powder with particle size of 40–60 μm was prepared for extraction, while the 200 μm powder was prepared for pyrolysis. About 15 g of the 40–60 µm powder was weighted, and extraction was performed in different organic solvents. Four types of organic solvent were prepared with optimum extraction temperature, including ethanol (75 °C), methanol (65 °C), benzene/ethanol (1:1) (90 °C) and ethanol/methanol (1:1) (70 °C). The extraction was performed for 5 h. After extraction, the four extracts were separated, and the excess solvent was removed with a rotary evaporator. The concentrated extracts were now ready for FT–IR and GC–MS analysis.About 1 mL of extractives and 1 g KBr were mixed and pressed into KBr tablets which were, respectively, detected using a Thermo Fisher Nicolet iS10 FT–IR spectrophotometer  by KBr method .Each of the four extracts was tested on the Agilent–7890B–5977A  instrument. Elastic quartz capillary columns (HP–5MS: 30 m × 250 μm × 0.25 μm) were used. The carrier gas flow rate was 1 mL/min; the carrier gas used was high purity helium, and the split ratio was set at 2:1. First, the temperature rises gradually from 50 °C to 250 °C at a rate of 8 °C/min, and then from 250 °C to 300 °C at a rate of 5 °C/min. The program was set to scan quality ranges, ionization voltages, ionization currents, ion sources and four-pole data for 30–600 amu, 70 eV, 150 ua, 230 °C and 150 °C .Analysis of the 1 mg 200 μm powder was performed on a Perkin Elmer–STA8000  instrument. The temperature was set to rise from 30 °C–300 °C at the rate of 5 °C/min. The carrier gas rate was set at 60 mL/min and the gas used was nitrogen .About 0.1 mg of the powder sample (200 mu) was placed in an Agilent–7890B–5977A CDS5000  instrument for analysis and detection. The carrier gas, pyrolysis temperature, heating rate and pyrolysis time used were helium (high purity), 500 °C, 20 °C/ms and 15 s, respectively. The capillary column (HP-5MS: 60 m × 250 μm × 0.25 μm), pyrolysis product delivery line, injection valve temperature, shunt ratio and shunt velocity were 300 °C, 300 °C, 1:60 and 50 mL/min, respectively. The GC was programmed to last for 2 min at 40 °C, increasing from 40 °C to 120 °C at a rate of 5 °C/min. At 10 °C/min, it went from 120 °C to 200 °C for 15 min. The MS ion source temperature and scanning range was 230 °C and 28–500 amu. .1H–NMR, 13C–NMR, and 2D–NMR. The duration, sample-and-hold time, pulse, pulse width and frequency of 1H–NMR were 1.000 s, 2.556 s, 45 degrees, 6410.3 Hz and 399.79 MHz, respectively. The duration, sample-and-hold time, pulse, pulse width, frequency, hydrogen decoupling frequency and power of 13C–NMR are 1.000 s, 1.311 s, 45 degrees, 25,000 Hz, 100.53 MHz, 399.79 MHz and 38 dB, respectively. The duration, sample-and-hold time, pulse, pulse width, frequency, frequency of carbon decoupling and power of 2D–HSQC are 1.000 s, 0.150 s, 4807.7 Hz, 20,105.6 Hz, 399.79 MHz, 100.54 MHz and 38 dB, respectively [The NMR polarizer model is Agilent–400 MR  and methanol-d4 was the solvent used in the testing process. The entire assay was performed with the same NMR probe: ectively .S. holocarpa wood as the research object to evaluate its potential as a lignocellulosic biomass source for bioenergy production The results proved that S. holocarpa wood contains a large amount of organic chemicals that can be used in the bioenergy and chemical industries. This project also provides the research basis of S. holocarpa wood pyrolysis, in which a large number of volatile compounds  were detected, which are proven to be important organic components of bio-oil. The NMR detection method used to identify the distribution of functional groups also proved that S. holocarpa wood has the potential to become a biomass energy source. This study explores the relationship between potential of the extraction and the pyrolysis of S. holocarpa wood, which is helpful for the exploitation and utilization of S. holocarpa wood as for bioenergy production. For the first time, NMR technology has been applied to S. holocarpa wood, and pyrolysis products show the potential to become high-value products. In the future, pyrolysis with various parameters can be optimized to maximize bioenergy production.This project utilized


In silico molecular docking studies of embelin and α-synuclein (α-syn) fibrils were also performed. Embelin alone or in combination with LD ameliorated oxidative stress and gut damage. TH and Nurr1 protein levels were also significantly restored. Docking studies confirmed the affinity of embelin toward α-syn. Taken together, embelin could be a promising drug for the treatment of PD, especially when combined with LD.Parkinson’s disease (PD), a progressive neurodegenerative disorder, affects dopaminergic neurons. Oxidative stress and gut damage play critical roles in PD pathogenesis. Inhibition of oxidative stress and gut damage can prevent neuronal death and delay PD progression. The objective of this study was to evaluate the therapeutic effect of embelin or the combination with levodopa (LD) in a rotenone-induced PD mouse model. At the end of experimentation, the mice were sacrificed and the midbrain was used to evaluate various biochemical parameters, such as nitric oxide, peroxynitrite, urea, and lipid peroxidation. In the substantia nigra (midbrain), tyrosine hydroxylase (TH) expression was examined by immunohistochemistry, and Nurr1 expression was evaluated by western blotting. Gut histopathology was evaluated on tissue sections stained with hematoxylin and eosin.  It has a complex etiology and mostly affects elderly people . PathophDysfunctional mitochondria show a reduction in energy production and utilization. The effect of environmental toxins such as herbicides, pesticides, fungicides, and insecticides on health is a major concern ,10. ManyThe enteric nervous system (ENS), also referred to as the “second brain,” is a bidirectional integrated system that communicates extensively with the central nervous system (CNS) ,33,34. REmbelia ribes Burm. f. (Family: Myrsinaceae). E. ribes berry extract has been extensively investigated for its various pharmacological properties, including antioxidant, anxiolytic, antidepressant, anticonvulsant, antidiabetic, and antimicrobial effects ); groups II–VII, rotenone ; group III, embelin ; group IV, embelin (40 mg kg−1 p.o.); group V, embelin (20 mg kg−1 p.o.) and LD (7.5 mg kg−1 p.o.); group VI, embelin (40 mg kg−1 p.o.) and LD (7.5 mg kg−1 p.o.); and group VII, LD (7.5 mg kg−1 p.o.). The treatment protocol in mice as indicated above was for a period of 21 days.The mice were randomly divided into seven groups (10 per group). The treatments were as follows: group I, vehicle control, namely olive oil .2.5Previous work has shown that chronic daily intraperitoneal injection of rotenone that is prepared using a natural oil causes behavioral (locomotor) deficits and neurochemical abnormalities characteristic of PD ,55. Ther2.6At the end of the experiment, the animals were anesthetized using isoflurane and subjected to cardiac perfusion using normal saline (0.9% NaCl) to eliminate the blood. The skull of the mice was opened and the midbrain carefully isolated and washed with ice-cold phosphate-buffered saline (PBS), pH 7.4. The whole midbrain tissue isolated from each mouse was kept on ice and homogenized with 0.1 M PBS (pH 7.0) using a Potter–Elvehjem PTFE-coated pestle and glass tube .2.7N,N,N′,N′-tetraacetic acid (EGTA), 5 mm HEPES-KOH (pH 7.2), and 1 mg mL−1 of essential fatty acid-free bovine serum albumin (BSA) in a 5 mL plastic tube. The tissue was homogenized at 25,300g using a tissue homogenizer  and transferred into a 1.5 mL microcentrifuge tube. The brain homogenate was centrifuged at 1,100g for 2 min in a refrigerated centrifuge . The supernatant was placed aside and the pellet was resuspended in 0.2 mL of the buffer and centrifuged again at 1,100g for 2 min. The pellet was discarded and the supernatant was combined with that from the first centrifugation. Next, 0.5 mL of combined supernatants was mixed with 0.07 mL of 80 vol% Percoll solution, carefully layered on top of 0.7 mL of 10% Percoll solution, and centrifuged at 18,500g for 10 min. Then, 80% Percoll was diluted in buffer to prepare the 10% solution. The mitochondria-enriched fraction was collected at the bottom of the tube and resuspended in 0.7 mL of washing buffer composed of 250 mM sucrose, 5 mM HEPES-KOH (pH 7.2), 0.1 mM EGTA, and 1 mg mL−1 BSA. The suspension was centrifuged at 10,000g for 5 min. The final mitochondrial pellet was resuspended in 0.07 mL of washing buffer and stored on ice. To isolate mitochondria, the same procedure was employed as described above, except that IB was supplemented with 0.01% digitonin at the tissue homogenization step, to release mitochondria. The mitochondrial protein content was measured with a Lowry assay kit  according to the manufacturer’s instructions. After removing the pellet, the postmitochondrial supernatant fraction was used to estimate the levels of nitric oxide (NO), urea, peroxynitrite, and malondialdehyde (MDA), a measure of lipid peroxidation (LPO).The midbrains were immediately immersed in 0.7 mL of ice-cold isolation buffer (IB) composed of 225 mm mannitol, 75 mm sucrose, 1 mm ethylene glycol-bis(β-aminoethyl ether)-2.8g for 10 min, the organic layer was removed and its absorbance was read at 532 nm. The standards and blanks were treated in a similar manner. The MDA concentration, an indicator of LPO, is expressed as nmol mL−1.The MDA content was determined by the thiobarbituric acid (TBA) reaction as described previously . To 0.5 2.9Nitrite .Peroxynitrite levels were measured according to the method described by Beckman et al.  Peroxyni2.11Elevated brain urea levels in neurodegenerative Huntington’s disease patients were reported as a major metabolic defect ; hence, 2.12g for 15 min at 4°C. Supernatants were collected and the protein concentration was determined using a Bradford protein assay kit. Equal amounts of protein (40 μg) were separated on 10% acrylamide gels using SDS–polyacrylamide gel electrophoresis and electrotransferred onto a polyvinylidene difluoride membrane. Membranes were incubated with 5% nonfat milk for 1 h at room temperature (to block nonspecific protein binding) prior to incubation overnight at 4°C in a solution of rabbit polyclonal anti-β-actin  and rabbit polyclonal anti-Nurr1 . The next day, membranes were washed three times with Tris-buffered saline with 0.5% Tween 20 (TBST), incubated with goat anti-rabbit IgG conjugated to horseradish peroxidase  for 1 h at room temperature, and then washed three times with TBST. The target proteins were visualized using ECL reagent and exposed to a piece of X-ray film. The densities of specific protein bands were determined using Adobe Photoshop CS6 software (version 12.0). The Nurr1 (66 kDa) protein level was normalized to the β-actin (42 kDa) protein level (loading control).The SN tissue homogenate was prepared in lysis buffer containing 1% protease inhibitor (HiMedia Laboratories) for 1 h at 4°C. Samples were then centrifuged at 10,0002.13Gut tissue was fixed in 10% neutral buffered formalin, dehydrated, embedded in paraffin, and then sectioned at a thickness of 5 μm and stained with hematoxylin and eosin (H&E) for histopathological investigation. For histomorphometry analysis, the gut tissue samples were well-oriented with longitudinally cut crypts to precisely assess alterations in the overall tissue architecture. Regarding gut tissue samples, at least 4–5 consecutive villi well distended from the base to tip were rated. The number of goblet cells per villus was counted in 10 well-oriented and adjacent crypt villi on each section stained with H&E. All measurements were made at 100× magnification. On each slide, one section that represented the best view of the villus and crypts was selected for analysis. The slides were examined under an Olympus light microscope and photomicrographs were taken with a Sony camera.F was calculated as: F = N/A, where N is the number of protein expression patches in the image and A is the total area (in pixels) of the protein signal.For brain tissue, mice in each group were anesthetized with isoflurane and then transcardially perfused with 0.9% normal saline to clear the blood from the brain. The brain from each mouse was immersed in 10% neutral buffered formalin (the fixative solution) for 4 h. The tissues were cryoprotected in 30% sucrose, embedded in tissue-freezing medium with liquid nitrogen, and sectioned at 3–5 µm using a cryostat. Sections were stored under anti-freeze buffer. Parallel free-floating sections were subjected to endogenous peroxidase quenching with 1% hydrogen peroxide in PBS, followed by treatment with blocking buffer  to block nonspecific protein binding. Sections were incubated overnight at 4°C with mouse monoclonal anti-tyrosine hydroxylase . After washing with PBS, tissues were incubated for 2 h at room temperature with affinity-purified rabbit anti-mouse IgG antibody . Then, the sections were exposed to an avidin–biotin peroxidase complex for 2 h. The peroxidase activity was visualized using a stable diaminobenzidine solution. All immunoreactions were observed using a compound light microscope and these results were quantified using the ImageJ 1.46 . For quantification, fragmentation (F) reflects that TH expression (brown patches in the image) that appeared as either single, homogeneous, and continuous or complex, heterogeneous, and discontinuous patches. Based on this protein expression classification, 2.14in silico bonding mode of ligand with the receptor was carried out using the empirical free energy function and the Lamarckian genetic algorithm, applying the following standard protocol with an initial population of 150 randomly placed individuals, a maximum of 2.5 × 107 energy evaluations, GA crossover mode = two points. A genetic algorithm of 100 independent docking cycles was carried out for the ligand. A genetic algorithm of 100 independent docking conformers was carried out for the compound, and the best conformer was taken based on their lowest binding energy for further analysis. PyMOL (https://pymol.org/2/) and LigPlot 2.2 tools were used to visualize the docking results [Molecular docking studies were executed with Autodock4.2 software  using th results . The ent2.15F and P values for the one-way ANOVA are shown in each panel of each figure. Significant differences between groups are denoted by asterisks, with details indicated in each figure legend. SigmaPlot 14.5  was used for statistical analysis.The data are presented as the mean ± standard error of the mean. Differences among the groups were compared with a one-way analysis of variance (ANOVA), followed by the Bonferroni test for multiple comparisons. The 33.1−1, respectively; P < 0.001). Notably, the combination of 40 mg kg−1 embelin and 7.5 mg kg−1 LD rendered even better protection against LPO. The mean peroxynitrite level in the rotenone-treated mice (group ІI) was markedly higher than the control mice . Rotenone-treated mice that received 40 mg kg−1 embelin or 40 mg kg−1 embelin plus 7.5 mg kg−1 LD showed almost similar peroxynitrite levels to the control mice . In particular, the NO levels in the rotenone- and drug-treated mice (40 mg kg−1 embelin plus 7.5 mg kg−1 LD) were not different from the control group.The mean NO level in rotenone-treated mice was significantly higher than in the control mice . Indeed, treatment returned the urea level to near what was found in control mice. Based on the above findings, treatment with embelin alone or embelin plus LD reversed the biochemical alterations caused by rotenone administration.The mean brain urea in the rotenone-treated mice was nearly 75% higher than the control mice (57.60 ± 2.59 and 33.30 ± 7.51 nmol mg3.2P < 0.05) compared with the control group. Nurr1 expression in the rotenone-treated mice administered LD standard therapy (7.5 mg kg−1) was restored to the control level. Embelin (40 mg kg−1) plus LD (7.5 mg kg−1) combination therapy in rotenone-treated mice also reverted the Nurr1 protein expression to almost the control level. However, the effect of embelin alone (40 mg kg−1) in restoring the Nurr1 protein level was comparatively less than the embelin plus LD combination therapy. Therefore, embelin plus LD combination therapy has a better protective effect on the brain dopaminergic system.3.3−1 embelin, there were epithelial destruction, mucosal edema , intense inflammatory cell infiltration, and crypt distortion  is most favorable for interaction with site 3/13. For this interaction, two hydrogen bonds are formed. The first hydrogen bond is between the second oxygen atom of embelin with the ND1 group of the polar residue His50(D), with a bond distance of 3.0 Å  was administered to the rotenone-treated mice [Chronic intraperitoneal administration of rotenone induces a gradual movement disorder that recapitulates PD symptoms . The rotted mice , a previted mice .After entering neurons, rotenone blocks mitochondrial complex I activity, increases ROS production, and inhibits proteasome activity, generating proteolytic stress. Rotenone decreases DA and reduced glutathione (GSH) levels and increases LPO in dopaminergic neurons, resulting in oxidative damage ,73. The 2) and dinitrogen trioxide (N2O3). The reaction of NO with the superoxide anion mania . Moreovein silico molecular docking. The findings emphasize the ability of embelin to bind to and dissolve α-syn fibrils. These fibrils are 1,000-fold more toxic than the oligomers or other precursors with regard to motor impairment, dopaminergic neuronal loss, and synaptic damage [The survival of dopaminergic neurons relies on two key proteins, namely HIF-1α and Nurr1. Therefore, researchers have proposed that PD pathogenesis could be due to the deficiencies in these genes . Therapec damage ,95. TherSeveral works have substantiated gut pathology and damage in patients with PD as well as PD animal models. Some authors have speculated that PD originates outside of the CNS through some involvement with the ENS ,97. IndeEscherichia coli [Several studies indicate that patients with inflammatory bowel disease, which is known to increase intestinal permeability, have an increased risk of developing PD. This eventuality suggests a role for GI inflammation in the development of PD . PD is ahia coli . Evidenchia coli . Both inhia coli . The reshia coli , antioxihia coli ,110,111,hia coli ,112,113 hia coli ,115 and hia coli . Overallhia coli  and the in vitro and in vivo models of PD [We used the well-established rotenone model of PD, which was first reported in 2000 . Since tls of PD ,122, the5in silico binding studies revealed that embelin can bind to α-syn fibrils and break up these fibrils. This could ameliorate the deficits caused by α-syn aggregation. Overall, embelin is a promising molecule for the treatment of PD.The findings from this study demonstrate that embelin could be a potential drug for PD treatment, either alone or in combination with LD. Embelin attenuates rotenone toxicity to protect the brain through antioxidant mechanisms as well as by the upregulation of Nurr1 and TH proteins. Notably,