Astragalus membranaceus and Codonopsis lanceolata are two important medical herbs used in traditional Oriental medicine for preventing cancer, obesity, and inflammation. Night temperature is an important factor that influences the plug seedling quality. However, little research has focused on how the night temperature affects the growth and development of plug seedlings of these two medicinal species. In this study, uniform plug seedlings were cultivated in three environmentally controlled chambers for four weeks under three sets of day/night temperatures , the same relative humidity (75%), photoperiod (12 h), and light intensity (150 μmol·m−2·s−1 PPFD) provided by white LEDs. The results showed that night temperature had a marked influence on the growth and development of both species. The night temperature of 15 °C notably enhanced the quality of plug seedlings evidenced by the increased shoot, root, and leaf dry weights, stem diameter, and Dickson’s quality index. Moreover, a night temperature of 15 °C also stimulated and increased contents of primary and secondary metabolites, including soluble sugar, starch, total phenols and flavonoids. Furthermore, the 15 °C night temperature increased the chlorophyll content and stomatal conductance and decreased the hydrogen peroxide content. Analysis of the gene expression showed that granule-bound starch synthase (GBSS), ribulose bisphosphate carboxylase large chain (RBCL), and ferredoxin (FDX) were up-regulated when the night temperature was 15 °C. Taken together, the results suggested that 15 °C is the optimal night temperature for the growth and development of plug seedlings of A. membranaceus and C. lanceolata. Astragalus membranaceus and Codonopsis lanceolata are mainly distributed in East Asia, especially in Korea, China, and Japan [C. lanceolata has been consumed as food for several centuries, especially in ancient Korea [As two important medicinal herbs in Oriental medicine, nd Japan ,2. It hand Japan ,4,5. In nt Korea ,7. Nowadnt Korea ,8,9. Focnt Korea . HoweverOryza sativa L.) was the greatest in plants grown at the highest night temperature (27 °C) rather than in those grown with lower night temperatures (17 °C and 22 °C). Moreover, there is little research regarding the effects of the night temperature on the growth and development of plug seedlings. Therefore, further research is still needed to clarify how the night temperature influences the growth, biomass, and quality, especially in plug seedlings of medicinal plants.Temperature is an important abiotic factor that has a great influence on the growth, yield, and quality of crop plants and shapes the morphology of seedlings ,12,13. HFDX gene, is an important iron–sulfur protein that accepts electrons produced from sunlight-excited chlorophyll and mediates them to NADP+ in the electron transport chain [RBC gene) is a crucial enzyme in the initial step of the Calvin cycle in plants, since it catalyzes the primary CO2 fixation step and controls the photosynthesis rate [GBSS gene, is indispensable for the synthesis of linear glucan (amylose) in starch and also takes part in building the final structure of amylopectin [FDX, GBSS, and RBC genes are suppressed by high or low day temperatures, resulting in a reduction of crop yield [Photosynthesis is an important physiological process that is strictly regulated by a series of enzymes . In lighrt chain . In darksis rate . Additiolopectin ,31,32. Pop yield ,34,35. HA. membranaceus and C. lanceolata plug seedlings. In order to test the above hypothesis, uniform plug seedlings were cultivated at night temperatures of 10 °C, 15 °C, and 20 °C for four weeks. Characteristics of the growth, development and morphology of A. membranaceus and C. lanceolata plug seedlings grown with different night temperatures were evaluated. The contents of the primary and secondary metabolites such as soluble sugar, starch, total phenols, and flavonoids, were further determined. Moreover, photosynthesis-related parameters including the chlorophyll content and stomatal conductance were measured. In addition, the expression of FDX, RBC, and GBSS genes were assayed. These data could provide a theoretical and practical basis not only for enhancing the quality of plug seedlings by manipulating the night temperature, but also for guarding the management of other medicinal plants.In the current study, it was hypothesized that a suitable night temperature (15 °C) could enhance the plug seedling quality and too high or low temperatures would have a negative effect on the growth and development of A. membranaceus and C. lanceolata were shaped by different night temperatures. Compared with 15 °C and 20 °C, a night temperature of 10 °C markedly inhibited the growth and development of both species with dwarfed shoots, curved stems, and sparse roots. Plug seedlings grew better with a night temperature of 15 °C than with 20 °C, evidenced by the slightly higher biomass, stem diameter, and Dickson’s quality index (DQI), although differences in the growth parameters of seedlings between these two treatments were not significant  significantly increased the H2O2 content.As shown in A. membranaceus and C. lanceolata  increased the chlorophyll content in plug seedlings of both nceolata A,B. Slig−2s−1 in plug seedling of A. membranaceus and C. lanceolata, respectively.In the current study, the stomatal conductance significantly increased with the increase of the night temperature from 10 °C to 15 °C, and then decreased slowly C,D. The A. membranaceus, a night temperature of 15 °C significantly increased the expression of granule-bound starch synthase (GBSS), and ribulose bisphosphate carboxylase large chain (RBCL), more than those at a night temperature of 10 °C. Although there were no significant differences between those at 15 °C and 20 °C, the expression of these two genes was higher at 15 °C than at 20 °C. Similarly, the highest expression of ferredoxin (FDX) was also at 15 °C in C. lanceolata. The expression of RBCL in C. lanceolata was remarkably greater at 15 °C and 20 °C than that at 10 °C. However, the RBCL expression at night temperatures of 15 °C and 20 °C was not significantly different.The data showed that expression of the photosynthetic genes was influenced by the night temperature . In A. mA. membranaceus and C. lanceolata plug seedlings, evidenced by the higher biomass, stem diameter, and DQI. These results are supported by other studies. For instance, Jing et al. [O. sativa L.) was lower with a low night temperature than with a high night temperature. In addition, Cheng et al. [2 levels on the rice production. However, an opposite conclusion in rice had been reported from Kanno et al. [Temperature is one of crucial abiotic factors that greatly decides the quality and yield of crops ,36. Receg et al.  found thg et al.  reportedg et al.  found tho et al. , who fouGossypium hirsutum L.). The reason is that a high night temperature contributed to a boosted respiration, leading to an increased consumption of carbohydrates [A. membranaceus and C. lanceolata. Similarly, Ryu et al. [Vitis vinifera. As antioxidants in nature, these two phytochemicals are beneficial to humans in preventing diseases, such as cancer, inflammation, and stroke [The night temperature had an influence on the primary and secondary metabolism ,42. In thydrates ,44,45. Au et al.  also fouu et al.  reportedu et al.  found thd stroke . A high GBSS, RBCL, and FDX. In the photosynthetic chain, FDX codes the ferredoxin protein, which accepts and transfers electrons to the next receptor, NADP+ [FDX with a night temperature of 15 °C implies an enhanced photosynthesis, resulting in higher biomass. In CO2 assimilation, RBCL plays a key role in CO2 fixation and regulates the photosynthesis rate. Up-regulated expression of RBCL suggested that a 15 °C night temperature promoted CO2 assimilation and synthesized more carbohydrates, which is consistent with the higher contents of soluble sugar and starch observed. In addition, expression of GBSS was positively modulated by the 15 °C night temperature, indicated by increased biomass and accumulation of starch. The lower expression of GBSS, RBCL, and FDX at night temperatures of 10 °C and 20 °C was possibly related to metabolic master regulators such as SnRKs which have specialized roles in controlling growth, stress responses, and photosynthesis program in plants [2O2  leaves. Jin et al. [Euonymus japonicas seedlings. Phan et al. [Granule-bound starch synthase in grains during the early grain filling in rice (O. sativa L.).Photosynthesis is an important physiological process for biomass production in plants , and is r, NADP+ . A highen plants ,50. Moren plants . Anothernts [2O2 , since Hproteins . Those rproteins  reportedn et al.  found thn et al.  reportedA. membranaceus and C. lanceolata plug seedlings  increased the chlorophyll content in both eedlings A,B. The  bicolor . Lesjak  bicolor  also fou bicolor ,58,59. A bicolor . This alA. membranaceus and C. lanceolata plug seedlings. Similarly, Machado et al. [2 uptake rate in Phalaenopsis aphrodite were significantly greater than those in the control (28 °C) [Stomatal conductance was affected by the night temperature. In this study, the maximum stomatal conductance was found at 15 °C in both o et al.  reported (28 °C) . In the  (28 °C) ,64 . After germination, uniform plug seedlings were selected and cultivated in three environmentally controlled chambers under the same day temperature (25 °C) and different night temperatures: 10 °C, 15 °C, or 20 °C. Each treatment was set up with the same relative humidity (75%), photoperiod (12 h), and light intensity (150 μmol·m−2·s−1 PPFD) provided by white LEDs. Before the experiment, the light intensity in all treatments was adjusted and calibrated using a photoradiometer . The plug seedlings were subirrigated once every two days with a multipurpose greenhouse nutrient solution (in mg·L−1 Ca(NO3)2·4H2O 737.0, KNO3 343.4, KH2PO4 163.2, K2SO4 43.5, MgSO4·H2O 246.0, NH4NO3 80.0, Fe-EDTA 15.0, H3BO3 1.40, NaMoO4·2H2O 0.12, MnSO4·4H2O 2.10, and ZnSO4·7H2O 0.44 ) throughout the experiment. After four weeks of cultivation, some plug seedlings were harvested at 10:00 during the daytime for the measurement of the growth parameters, and others were frozen immediately in liquid nitrogen for further analyses. The Dickson’s quality index was calculated according to a previous formula [ formula . The forFor extracting the soluble sugar and starch, frozen leaves (0.2 g) were ground and then mixed with distilled water (14 mL) for 30 min at 100 °C. After centrifugation at 3000 rpm for 15 min, the supernatant was transferred into new tubes for the assay of soluble sugar. The residue was re-extracted in distilled water mixed with perchloric acid  for the measurement of starch.Contents of soluble sugar and starch were determined by the anthrone colorimetric method . In summFor extracting total phenols and flavonoids from frozen leaves, 80% methanol was prepared and used. The contents of total phenols and flavonoids were measured by the methods described by Manivannan et al. . In brie2O2) content in frozen leaves was assayed as previously done [The hydrogen peroxide . The stomatal conductance was measured on leaves at 10 am before harvesting using a Decagon Leaf Porometer SC-1 , according to the introduction of this instrument.A. membranaceus and C. lanceolata were ground and homogenized in lysis buffer under an RNase-free condition. For RNA extraction and cDNA synthesis, a total RNA extraction kit  and the PrimeScript RT Reagent Kit  were used, according to manufacturer’s protocols. Total RNA was reverse transcribed to cDNA using the GoScript Reverse Transcription System . The gene expression was assayed using the Rotor-Gene Q detection system , and was calculated using the 2−∆∆Ct method [Leaf tissues of t method . All prip < 0.05) using SPSS . All figures in this study were prepared using the OriginPro software (version 9.0).The data were collected with three individual biological repeats and presented as the mean ± standard error. One-way analysis of variance (One-way ANOVA) was performed to estimate the differences among treatments, followed by Duncan’s multiple range test , stem diameter, and DQI. Moreover, high and low night temperatures had a negative effect on primary and secondary metabolites, and a night temperature of 15 °C significantly increased the contents of soluble sugar, starch, total phenols, and flavonoids in A. membranaceus and C. lanceolata. Furthermore, the stomatal conductance and chlorophyll content were improved by a night temperature of 15 °C and decreased with night temperatures of 10 °C and 20 °C. An analysis of the gene expression showed that a 15 °C night temperature enhanced photosynthesis by up-regulating the expression of the GBSS, RBCL, and FDX genes. In conclusion, the results suggested that a night temperature of 15 °C is recommended for producing high quality plug seedlings of A. membranaceus and C. lanceolata. Many further studies should be carried out in the future, such as how night temperature affects carbohydrates allocation and partition from source to sink, how night temperature influences the expression of genes related to secondary metabolite biosynthesis and degradation, and how night temperature impacts the absorption and transport of mineral elements.In this study, the effects of the night temperature on the growth and development of plug seedling of


Penicillium italicum, which reduces its storage life and commercial value. Normally, to reduce the losses caused by fungal deterioration of harvested fruit, polysaccharide-based edible coating, containing natural antimicrobial agents , have been applied. In current study, we evaluated the effects of Ficus hirta Vahl. fruits extract (FFE)–incorporated chitosan (CS) edible coating on Xinyu tangerines during cold storage at 5 °C. The results showed FFE has efficacy as an antifungal against P. italicum in a dose-dependent manner in vivo, with an EC50 value of 12.543 mg·mL−1. It was found that the edible coating of FFE–CS exhibited a higher reduction of total soluble solid (TSS), titrable acid (TA), and ascorbic acid (AsA) content by reducing the fruit decay rate, weight loss, respiration rate, and malondialdehyde (MDA) content during cold storage at 5 °C. Moreover, the activities of protective enzyme such as superoxide dismutase (SOD), peroxidase (POD), and phenylalanine ammonia-lyase (PAL), which have been linked with reactive oxygen species (ROS) and the phenylpropanoid pathway, were higher in the FFE–CS-coated fruits. On the basis of these study results, the FFE–CS edible coating could reduce postharvest loss and enhance the storability of Xinyu tangerines due to the in vivo antifungal activity of FFE.Xinyu tangerine is a citrus fruit that has enjoyed great popularity in China for its fewer dregs and abundant nutrients. However, it is considered an easily perishable fruit that is vulnerable to various pathogenic fungal infections, especially by  Citrus reticulata Blanco) cv. Pengjia no. 39, a local mandarin bred from ‘běn dì zǎo’ (Citrus reticulata Blanco) cv. Succosa in Huangyan City , enjoy great popularity in China for their delicious taste, abundant nutrients, uniform color, fewer dregs, and delicate pulp. However, the fruits are highly susceptible to fungal pathogen infection and mechanical injury during storage due to their rich nutritional content and tender peel. Moreover, the harvested Xinyu tangerines have a high respiration rate and water loss, and are easily attacked by pathogens at room temperature [Xinyu tangerines  is widely distributed in Southern China and is used for the treatment of constipation, inflammation, postpartum hypogalactia, and tumors and cancers [F. hirta against Escherichia coli, Staphylococcus aureus, Alternaria citri, Botrytis cinereal, and many others in vitro [F. hirta Vahl. is a famous herbal medicine in Southern China, named ‘Wú Zhǐ Máo Táo Guǒ’ in Chinese pharmacopoeia, and a traditional plant resource used both as medicine and food by Hakka people [F. hirta fruits, has potent antifungal effects against Penicillium italicum in citrus fruit. However, hardly any published reports exist on the effects of Ficus hirta Vahl. fruits extract (FFE)–incorporated chitosan (CS) FFE–CS coating for Xinyu tangerine preservation. Thus, the aims of the current study were to investigate the in vivo antifungal efficacy of FFE for controlling blue mold caused by P. italicum in citrus fruit, and to evaluate the preservation effect of FFE incorporated into CS-based edible coatings on harvested Xinyu tangerines during cold storage. cancers ,12,13,14in vitro ,16. Morea people ,17,18. Wa people  have repCitrus reticulata Blanco) cv. Pengjia No. 39 used throughout this study were harvested from Mahong Garden-Spot, located in the Yushui district of Xinyu City , during late October 2016. The fruits were selected on the basis of health, consistent size (72–85 g), uniform color (2.5–3.2), and features of commercial maturity . Xinyu tangerines  and identified by Miaolian Xiang . Potato dextrose agar  medium was used for the maintenance of P. italicum, and the pure culture was sustained at 25 °C for 7 days.F. hirta Vahl.  were purchased from Huafeng Pharmacy in Zhangshu city  and authenticated by Shouran Zhou . The dried fruits were powdered in a FW-100 grinder  after drying below 40 °C for 15 h. The FFE was obtained using an ultrasonic-assisted method described by Chen et al [w/v) for further use. The fruits of en et al . The dryP. italicum spore (106 CFU/mL). There were three replicate trials of fifteen fruits per treatment with completely random allocation, and all experiments were performed twice. The lesion diameters of the fungal colonies were recorded in millimeters after 7 days of incubation at 25 °C. The control efficacy was expressed in terms of percentage of mycelia growth inhibition (MGI) and calculated using Equation (1): The selected Xinyu tangerines were washed with 0.5% sodium hypochlorite for 2 min, rinsed with distilled water, and air-dried on a benchtop before wounding [w/v) CS solution was prepared by dissolving 10.0 g of chitosan  in 400 mL of 0.5% (v/v) acetic acid solution. Dry FFE (6.25 g) was dissolved in moderate distilled water and put into the above CS coating. After agitation for 60 min, the pH of the solution was adjusted to pH 5.4 using 1.0 M NaOH, and the total volume of FFE–CS coating was made up to 500 mL. The 2.0% CS coating was prepared in the same way without addition of FFE. 2.0%  for 1 min, while the control group was dipped in 0.5% acetic acid solution (pH 5.4). After drying, the coated and control fruits were individually film -packaged. After being pre-cooled at 10–12 °C for 12 h, all fruits were stored at 5 ± 0.5 °C, and 85–90% relative humidity (RH), for 90 days. The TSS and TA contents of juice extracted from 10 fruits from the coated and control groups was measured using a RA-250WE Brix-meter  and PAL-ACID1 digital acidimeter (Atago), and the results were expressed as a percentage. AsA content in the extracted juice was determined by titration with 2,6-dichlorophenol indophenol and expressed in mg per 100 g juice (mg/100 g) Decay rate was visually evaluated using the same 120 fruits per treatment per replicate, and expressed as the percentage of rotted fruits. For this test, 360 fruits from each treatment were used and evaluated on days 0, 15, 30, 45, 60, 75, and 90.0W and iW are the initial and final weight (g), respectively, of the same fruits.Weight loss was determined by single fruit weighting, using Equation (2). For this test, 15 fruits were used and evaluated on days 0, 15, 30, 45, 60, 75, and 90.2 production, using Equation (3), and expressed as mg·kg−1·h−1.2∆CO is the increment of CO2 concentration, headspaceV is the empty volume of the container (mL), m is the mass of Xinyu tangerines (g), and t is recording time (min), respectively.The respiration rate was determined based on a method described by our previous study . Respiraw/v) polyvinylpyrrolidone (PVP), and centrifuged at 12,000 × g for 20 min at 4 °C. 2 mL of the collected supernatant was mixed with 2 mL of 0.5% (w/v) thiobarbituricacid (TBA), and further incubated in boiling water for 30 min. After being cooled and centrifuged at 6000 × g  for 10 min, the absorbance of supernatant was measured at three different wavelengths  using an M5 microplate reader . The MDA content was calculated according to Equation (4) and expressed as mmol·g−1 FW.MDA content was measured according to the method described by Hodges and coworkers . Pericarw/v) PVP for superoxide dismutase ; 8 mL of 100 mM ice-cold acetate buffer (pH 5.5) containing 1 mM polyethylene glycol (PEG), 4% (w/v) PVP, and 1% (w/v) Triton X-100 for peroxidase  and polyphenol oxidase ; 5 mL of 50 mM ice-cold Tris-HCl buffer (pH 8.8) containing 15 mM β-mercaptoethanol, 5 mM AsA, 5 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 0.15% (w/v) PVP for phenylalanine ammonia-lyase . All homogenates were centrifuged at 12,000 × g  for 30 min at 4 °C. The supernatants were then collected and used for the enzyme activity assays.Aliquots of fruit peel powder (2.0 g) were homogenized with various ice-cold extraction buffers to prepare extracts for assay of the following protective enzymes: 10 mL of 50 mM ice-cold phosphate buffer (pH 7.8) containing 1 mM EDTA, 5 mM DTT, and 2% (2 (0.1 mM), 0.3 mL riboflavin (20 µM), 0.1 mL enzyme extract, and 0.5 mL distilled water in a total volume of 3.3 mL. The mixtures were illuminated by light (4000 Lx) for 20 min at 28 °C, and the absorbance was then determined at 560 nm . One unit of SOD activity was defined as the amount of enzyme that would inhibit 50% of NBT photoreduction, and expressed as U min−1·g−1.SOD activity was assayed by measuring its ability to inhibit the photoreduction of nitroblue tetrazolium (NBT) according to the method of Sala and Lafuente, with slight modifications . The rea2O2. The collected supernatant (100 μL) was mixed with 3.0 mL of 25 mM guaiacol and 200 μL of 50 mM H2O2. Oxidation of guaiacol was determined at 470 nm for 3 min. One unit of POD activity was defined as an increment of 0.01 in absorbance per minute at 470 nm (Shimadzu UV-2600), and expressed as U min−1 g−1.POD activity was based on the measurement of guaiacol oxidation at 470 nm in the presence of H−1·g−1.PAL activity was determined by using a PAL assay kit  monitoring the absorbance of the tested sample at 290 nm using an M5 microplate reader  and expressed as U·h−1·g−1.PPO activity was based on the measurement of catechol oxidation at 420 nm. The collected supernatant (200 μL) was mixed with 4.0 mL of 50 mM acetate buffer (pH 5.5) and 1.0 mL of 50 mM catechol. Oxidation of catechol was determined at 420 nm for 5 min. One unit of PPO activity was defined as an increment of 0.01 in absorbance per hour at 420 nm (Shimadzu UV-2600), and expressed as U·hP < 0.01 and P < 0.05, respectively.All data calculated from three physical and chemical experiments was expressed as the mean with standard error (SE). The SPSS software  was applied to determine the mean differences using Duncan’s multiple range test at in vivo antifungal efficacy of postharvest blue mold in Xinyu tangerines inoculated with P. italicum were significantly reduced by FFE treatment at various concentrations. As shown in P < 0.01). Concurrently, the MGI of blue mold in FFE-treated  Xinyu tangerines were 63.68%, 42.34%, 24.96%, and 17.38%, which indicates that FFE possessed strong antifungal efficacy and efficiently inhibited in vivo mycelial growth of P. italicum in a dose-dependent manner . In addition, the FFE–CS coating effectively reduced natural infection and prolonged storage life of Xinyu tangerines during storage at 5 °C for 90 days. These results were in agreement with previous reports that application of plant extracts could reduce Penicillium decay of citrus fruit [Previously, our studies have demonstrated that FFEggplants . In partitalicum ,19. In tus fruit ,23,24,25P < 0.05). Therefore, the TSS content in FFE–CS-coated fruits were generally higher than 2.0% CS-coated and control in the later period of storage (45 days to 90 days). Similar results were reported by Chien and Chou [−1) incorporated into 1.0% CMC-based edible coating had a striking efficiency in prevention of the loss of TSS. Therefore, the 2.0% CS edible coating with FFE formed a semi-permeable coating around Xinyu tangerines which inhibited their respiration and nutrient consumption, and maintained the high level of TSS.TSS in citrus juice is made up of sugars, acids, soluble pectins, vitamins, and some soluble proteins . TSS conand Chou  and Tao and Chou , for TanP < 0.05). At the end of storage (90 days), TA content with FFE–CS coating was 0.539%, while it was 0.468% and 0.357% in 2.0% CS coating and control groups, respectively. The high reduction in TA content of FFE–CS coating could have contributed to the slowing of the respiration rate, thereby delaying the degradation of TA [The main components of TA are organic acids which participate in the respiration of the plant, and the TA content is regarded as an important indicator for evaluation of the respiration rate of horticultural crops . TA conton of TA ,31,32,33P < 0.05). The AsA content of FFE–CS-coated fruits was significantly higher than those with 2.0% CS coating and controls. These results are highly consistent with those of Chen et al. [AsA is regarded as one of key determinants in evaluation of citrus fruit quality. As illustrated in n et al.  and Gao n et al. , where ain vivo ability of FFE to inhibit fungal growth and the fresh preservation effect of FFE–incorporated chitosan coating to reduce decay rate have not yet been investigated for Xinyu tangerines. According to Chen et al. [Ficus hirta Vahl.) fruits exhibited a variety of biological activities and inhibited the fungal growth of pathogens . In line with our findings, FFE may be considered as a safe natural preservative for reducing fungal decay in horticultural crops.The percentages of decay rate and weight loss are two important indicators for evaluating storability of harvested citrus fruit ,32. As sn et al. , FFE froP < 0.05). It is apparent that FFE–CS coating showed the lowest values during the whole cold storage. The weight loss in FFE–CS-coated fruits declined by 31.41% at 90 days compared with that of the control. Several previous reports have confirmed that coatings of almond carboxymethyl cellulose (CMC) [Weight losses in coated and control groups increased with storage time as presented in se (CMC) ,37,38,39se (CMC) ,32, hydrse (CMC) ,41, algise (CMC) ,42, and se (CMC)  could re−1·h−1 at the beginning of the storage. It was observed in P < 0.05) between two coated groups of FFE–CS and 2.0% CS after 15 days of storage. Coated Xinyu tangerines generally had lower respiration rates than control fruits  higher than that in the two coated groups. Xinyu tangerines coated with FFE–CS showed a lower MDA content compared to 2.0% CS coating and control. These results are highly consistent with those of Chen et al. [MDA is the final product of lipid peroxidation, related to senescence, and is used as one of the direct indices of cell oxidative damage. As shown in n et al.  and Shahn et al. , where aP < 0.05). Therefore, the SOD activity in FFE–CS-coated fruits was generally higher than 2.0% CS-coated and control in the later stage of storage period (45 days to 90 days). The activities of SOD, POD, PAL, and PPO are closely related to the resistance of oxidation and disease in plant tissue . As disp−1·g−1, significantly (P < 0.05) higher than that in 2.0% CS-coated and control fruits. It was confusing that the overall POD activity in FFE–CS-coated fruits was significantly (P < 0.05) higher than that of the control fruits, while the MDA accumulation was inhibited with significant differences compared to 2.0% CS-coated and control groups  higher than that of the 2.0% CS-coated and control fruits. As shown in −1·g−1 for control, 18.40 ± 0.21 U·h−1·g−1 for 2% CS, and 17.64 ± 0.76 U·h−1·g−1 for FFE–CS at 30 days of cold storage. It is apparent that FFE–CS coating showed the minimum values of PPO activity during the whole cold storage. PPO activity in the coated and control fruits followed a clear tendency of increasing dramatically to reach their peaks at 30 days of storage and then decreasing as storage time increased D. The peFruit senescence leads to the overproduction and accumulation of reactive oxygen species (ROS); this is considered the most important metabolic process in plants that causes damage to cell membranes and reduces the storability of fruits . High acin vivo antifungal efficacy for controlling blue mold in citrus fruit caused by P. italicum. Our work also showed the benefit of FFE–CS coating to delay the fruit senescence process, improve postharvest quality, and prolong storage life of Xinyu tangerines. This coating helped to maintain the lower reduction of TSS, TA, and AsA content by depressing the respiration rate. In addition, the FFE–CS coating significantly increased SOD, POD and PAL activities and decreased PPO activity and MDA content, thus reducing oxidative stress and delaying lipid peroxidation to membranes. Xinyu tangerines subjected to the FFE–CS coating showed slower rates of fruit decay and weight loss, which corresponded to improved storability and prolonged storage life. According to the results from this study, FFE–CS coating treatment showed the best preservation effect for harvested Xinyu tangerines. In this study, treatments with FFE at different concentrations demonstrated its


Camellia sinensis plays a well-established neuroprotective role in several neurodegenerative diseases, including intracerebral hemorrhage (ICH). However, the other teas of the same plant do not have their properties well understood; but they can be as effective as green tea as an alternative therapy. In this study, we investigated the effects of supplementation with green tea and red tea from Camellia sinensis on motor deficits and striatum oxidative damage in rats submitted to hemorrhagic stroke (ICH). Male Wistar rats were supplemented with green tea, red tea, or vehicle for 10 days prior to ICH induction. After injury, the rats were submitted to motor tests ) 1, 3, and 7 days after ICH induction, while the tea supplementation was maintained. Subsequently, the rats were euthanized to striatal tissue dissection for biochemical analyzes . ICH caused locomotor and balance deficits, as well as increased the neurological deficit (NDS). Only red tea prevented locomotor deficits after injury. Green tea and red tea prevented balance deficits on the seventh day after ICH. On NDS evaluation, green tea presented a better neuroprotection than red tea (until day 3 after ICH injury). In addition, ICH increased reactive oxygen species and lipid peroxidation levels, without altering antioxidant markers. Green and red teas were effective in decreasing the lipid peroxidation levels. Therefore, green and red teas partially prevented the motor deficits and striatal oxidative damage induced by ICH. Based on our results, we can consider that the two teas seem to be equally effective to prevent motor deficits and striatal oxidative damage induced by hemorrhagic stroke in rats.Green tea from  Intracerebral hemorrhage (ICH) is a common type of stroke associated with a considerable socioeconomic impact, disability, and mortality  and reprSome regions of the brain are more susceptible to stroke damage, including striatum, which is one of the most important regions for voluntary motor control . After ICamellia sinensis as an antioxidant strategy [Despite the breakthrough in research, ICH treatments deserve great attention and further investigation. So, to seek adjuvant therapies that may prevent the progression of ICH secondary damage is important . In thisstrategy , 12.Camellia sinensis contains a high content of flavonoids: epigallocatechin-3-gallate (EGCG), which accounts for approximately 59% of its total catechin content, epigallocatechin, epicatechin, and others [Camellia sinensis may be responsible for different neuroprotective effects of each one of them.Green tea derived from d others . Severald others . Howeverd others . So, theCamellia sinensis to protect from memory deficits in ischemia reperfusion (IR) [Camellia sinensis in the prevention of possible motor deficits and striatal oxidative damage in a model of intracerebral hemorrhage in adult rats.Recent studies comparing the therapeutic potential of the different teas of ion (IR)  and in aion (IR)  have shoion (IR) ; howeverad libitum. All the experiments were performed in accordance with the standards of the “Principles of laboratory animal care”  and were previously approved by the Institutional Animal Care and Use Committee (protocol #002/2017).Forty male Wistar rats (250–350 g) were obtained from the Central Vivarium of the Federal University of Santa Maria . The rats were housed 4 per cage and kept in a temperature-controlled room (24°C ± 1), with light/dark cycle of 12 h and food and water The rats were randomly divided into four groups: sham; intracerebral hemorrhage (ICH); intracerebral hemorrhage + green tea (ICH + GT); and intracerebral hemorrhage + red tea (ICH + RT). Rats received specific tea or vehicle for ten days prior to ICH surgery . After, μl of sterile saline containing 0.2 U of bacterial collagenase (type IV). The needle was left in place for 5 min and then slowly withdrawn. The hole was sealed with a metal screw, suture threads were used to close the wound, and lidocaine was used at the suture site [Surgical procedures were performed aseptically. The rats were anesthetized with ketamine and xylazine . Body temperature was maintained at 37°C during surgery using a surgical warming table. After placing the animals on the stereotactic table, a 3.5 mm hole was made to the right and 0.5 mm anterior to bregma. A 26-gauge needle  was inserted unilaterally into 6.5 mm in the right striatum to infuse 1 ure site . The samCamellia sinensis teas (green and red) were purchased directly from the same company , and prepared daily in the same way.The two types of The teas were prepared with distilled water 95°C ± 5°C) and then the infusion was maintained rested and muffled for approximately 3 minutes, and after was filtered. Teas were administered via gavage (400 mg/ml/day), at room temperature [5°C ± 5°CTea samples used in this study were analyzed by high-performance liquid chromatography (HPLC) to analyze the presence of epicatechin (EC), epigallocatechin (EGC), epigalocatechin gallate (EGCG), and epicatechin gallate (ECG)  Table 1Table 1.To verify possible changes and/or deficits in motor function, the rats were trained in OF, RR, and NDS tasks 24 h prior to ICH or sham surgery. The tests were performed 24 h, 3 days and 7 days after the surgeries.To evaluate the locomotor activity and the exploratory behavior of the rats, we used the open field apparatus. The open field consists in a box (60 cm in diameter × 45 cm in height) divided into 12 quadrants of the same surface area. The animals were gently placed in the open field arena , so that they could freely explore it for 5 min. The number of crossed lines (crossings) was counted . After eIn order to evaluate the influence of ICH in rats' motor coordination and balance, the RR test was used. The apparatus consists of a rotating cylinder (5 cm diameter × 8 cm wide × 20 cm high), with an automatic fall register. On the training day, the animals were placed in the static cylinder for 2 minutes for habituation. After the habituation time, the cylinder rotation was set at 16 rpm for 6 minutes, and the number of falls and latency for the first fall were recorded. In the test sessions, the animals were suspended for 6 minutes in the cylinder at a speed set in 20 rpm. The number of falls and latency for first fall were again recorded [This test set is sensitive to striatal/motor damage . The ratw/v). The samples were centrifuged at 2400g for 10 min, and the supernatants (S1) were used for assay.Rats were sacrificed 24 hours after the last test day. Their brains were removed, and the striatum ipsilateral to the lesion was quickly dissected and homogenized in 50 mM Tris HCl, pH 7.4  test . S1 aliqμl of DCFH-DA (1 mM). Oxidation monitoring was made of DCFH-DA to dichlorofluorescein (DCF) fluorescence by reactive oxygen species. The fluorescence emission intensity was performed at 520 nm (with excitation at 480 nm) for 60 minutes after the addition of DCFH-DA in spectrofluorimeter (Shimadzu RF-5301PC Model).ROS levels were determined indirectly by fluorimetric method spectrum using 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). The samples were incubated in the dark with 5 4) and centrifuged at 3000g for 10 min. This mixture was centrifuged, the protein pellet was discarded, and free thiols (SH) groups were determined in the clear supernatant. An aliquot of supernatant was incubated with orthophthaladehyde and fluorescence was measured at excitation of 350 nm and emission of 420 nm. Results were normalized by mg of protein and expressed as percent of control.GSH levels were fluorometrically determined . An aliquot of homogenized sample was mixed (1 : 1) with perchloric acid . A standard curve with 10 μl trolox  and 300 μl working FRAP reagent was used. The microplate was incubated at 37°C for 15 min before reading in SpectraMax M5 Microplate Reader at 593 nm. Each sample was analyzed in triplicate.Total antioxidant capacity was measure by ferric reducing/antioxidant power assay (FRAP). Working FRAP reagent was prepared by mixing 25 ml acetate buffer, 2.5 ml TPTZ solution, and 2.5 ml FeCl3.6H2O solution, and 10 −1) as standard.Protein content was measured colorimetrically by the method of Bradford  using boData were checked for normality of distribution using Shapiro-Wilk test. Results are presented as mean ± standard error of the mean (SEM). Results were analyzed by one-way ANOVA followed by Tukey's post hoc test, when appropriate. Significance level was set at 0.05 for all analyses.F = 4.621, P = 0.0078, F = 4.771, P = 0.0067, F = 1.563, P = 0.215, P < 0.05, P < 0.05, P > 0.05). In addition, there are no differences on crossings between ICH and ICH + green tea groups on days 1 and 3 , while green tea did not present the same effect.The locomotor activity was assessed using the OF test. The locomotor activity was different between the groups on days 1  = 5.264, P = 0.004, F = 2.925, P = 0.046, F = 6.352, P = 0.001, P < 0.05, P < 0.05 for GT; P < 0.01 for RT; P > 0.05, ICH versus ICH + teas; P = 0.04, In rotarod test, there was a difference between the groups on days 1  = 5.698, P = 0.0027, F = 4.832, P = 0.0063, F = 5.921, P = 0.002, P < 0.05, P < 0.05, P < 0.01, P < 0.05; D7: P < 0.01; P > 0.05, Considering the number of falls (rotarod test), we observed difference between the groups on days 1 (F(3.36) = 20.47, P < 0.0001, F(3.36) = 7.704, P = 0.0004, F(3.36) = 4.21, P = 0.013, P < 0.0001, P < 0.001, P < 0.05, P < 0.05, RT: P < 0.05, P < 0.05, On neurological deficit (NDS) evaluation, we found differences between the groups on days 1 , and the total antioxidant capacity in the striatum of rats.F = 6.850, P = 0.0137, P < 0.01). Neither green tea (P > 0.05) nor red tea (P > 0.05) was able to reverse striatal oxidative stress.A significant difference on the striatal level of oxidative species (DCFH)  = 7.144; P = 0.0019, P < 0.05). Both green tea and red tea avoided this increase   = 0.4608, P = 0.7127, F = 0.2767, P = 0.8415, Considering the antioxidant measures, we did not find differences between the groups in the GSH levels  and balance deficits (RR), general neuromotor function deficits (NDS), and contributing to avoid the increase of lipid peroxidation (TBARS).ICH is associated with motor damage , 26 and Strategies that propose the administration of antioxidant agents have been widely tested to prevent or treat hemorrhagic stroke , 31. PreCamellia sinensis and EGCG (the main component of green tea) has already been described as neuroprotective by its antioxidant effects in different models of brain injury [Camellia sinensis  in an ischemic stroke model showed that the greatest protective effect on memory and oxidative stress is from green tea and red tea, since the best effects associated with green tea [Camellia sinensis, Schimidt and colleagues suggest that supplementation with green tea and red tea may avoid deficits in social and object recognition memories related to Alzheimer's disease, but only green tea avoids the hippocampal oxidative stress and damage induced in Alzheimer model [Camellia sinensis teas, although these mechanisms are not completely clear.The administration of tea from n injury , 28. A rreen tea . In anoter model . These eer model , 34, siner model . Howeverer model  of the CThe great amount of compounds in teas emphasizes the differences of mixture use or its isolated components. Here, considering that the neuroprotection effects could be related to the combined activity of the varios teas' compounds , we deciGreen and red teas have been shown to be effective strategies, with similar effectiveness, to prevent some motor deficits and the striatal oxidative damage induced by ICH. The mechanisms involved in their protective role include the decrease of oxidative damage, that is, lipid peroxidation.


Thaumastocoris peregrinus Carpintero et Dellape (Heteroptera: Thaumastocoridae) is a eucalyptus pest. The objective of this study was to determine biological and life table parameters of T. peregrinus on Eucalyptus benthamii at five temperatures  with a relative humidity (RH) of 70 ± 10% and photoperiod of 12 hours. The duration of each instar and the longevity of this insect were inversely proportional to the temperature, regardless of sex. The nymph stage of T. peregrinus was 36.4 days at 18 °C and 16.1 days at 30 °C. The pre-oviposition period was 5.1 days at 30 °C and 13.1 days at 18 °C and that of oviposition was 7.6 days at 30 °C and 51.2 days at 22 °C. The generation time (T) of T. peregrinus was 27.11 days at 22 °C and 8.22 days at 30 °C. Lower temperatures reduced the development and increased the life stage duration of T. peregrinus. Optimum temperatures for T. peregrinus development and reproduction were 18 and 25 °C, respectively.Temperature affects the development, population dynamics, reproduction and population size of insects.  Thaumastocoris peregrinus Carpintero & Dellapé (Hemiptera: Thaumastocoridae), an Australian eucalyptus pest was first recorded in Brazil in 20091. At high infestations, this insect decreases the photosynthetic rate, leading to partial or total plant defoliation, and in some cases, plant death3.The frequent introduction and establishment of exotic insect pests on eucalyptus plantations in Brazil are impacting and reducing productivity. The bronze bug 6, chemical control7, chemical ecology9, morfology10, remote sensing for monitoring12 and biological control16 of this pest, aiming to minimize losses. However, the effect of temperature on the biological parameters of this species is not yet well known.Studies have focused on the biology20. In fact, each insect species has an optimum temperature at which they thrive, with lower and upper limits for development22. High temperatures can decrease fecundity, hatching and survival of these organisms23, while low temperatures can affect the sex ratio , behavior, and population distribution of insects24.Ambient temperature is one of the most important abiotic factors affecting the survival, development rate, abundance, behavior and fitness of insects25. The temperature decrease Parapoynx crisonalis  life tables26 and Brachmia macroscopa (Lepidoptera: Gelechiidae) development and fecundity27. Thus, the objective of this study was to evaluate the effect of different temperatures on biological parameters of T. peregrinus.The study of temperature in life-history variables, such as nymph development period, adult longevity and fecundity is crucial to the development of pest-management strategiesT. peregrinus differed across temperatures   to 5 (30 °C) days (Table The pre-oviposition period of ys Table . The ferys Table .Table 2DT. peregrinus was longest at 22 °C (53 days) and that of males at 18 to 22 °C      Fig. , females01) Fig. , and mal01) Fig. .Figure 1T. peregrinus as temperature increased  for nymphs and adults  of ed Table  and  per instar was 79.34 degree-day (DD) (first), 58.58 DD (second), 55.57 DD (third), 62.24 DD (fourth) and 88.91 DD (fifth). The accumulated temperature for the nymph-to-adult period of this insect was 338.50 DD, with a temperature limit of 9.93 °C  of T. peregrinus was higher at 25 °C (6.39) and 18 °C (4.45), the latter being similar to that at 22 °C (4.00). The net reproduction rate was lower at 30 °C (0.13). The generation time (T) of T. peregrinus varied between 27.11 days at 22 °C to 8.22 at 30 °C, and the intrinsic growth rate (rm) and finite increase (λ) of T. peregrinus were higher at 25 °C  and lower at 30 °C   (Hemiptera: Tingidae) and Loxostege sticticalis (L.) (Lepidoptera: Crambidae)30.Temperature strongly influences insect development in both single generation progeny and in organisms that are established and successfully continued for multiple generationsT. peregrinus at higher temperatures is due to increased metabolism, food intake and energy, allowing the insect to reach the next stage32. Other factors, such as poor food quality34, decreased the survival and/or insect growth rate35. The ladybird Harmonia axyridis  (Coleoptera: Coccinellidae)37, the dragonfly Ischnura verticalis (Odonata: Coenagrionidae)38, and the locust Romalea microptera 39 had shorter juvenile stages at increased temperatures.The shorter duration of each instar and of the adult period of Thaumastocoris peregrinus had a shorter pre-oviposition period with increased temperature, indicating the effect of this parameter on this organism. This is also reflected in the mating and egg laying of T. peregrinus as reported for Phenacoccus madeirensis Green (Hemiptera: Pseudococcidae)40 and Leptocoris achinensis  (Hemiptera: Alydidae)41 and food/temperature and bioecology interactions42 as reported for Cimex lectularius 43. The number of eggs per T. peregrinus female at 26 °C on Eucalyptus urophylla x Eucalyptus camaldulensis5 and Eucalyptus scoparia at different temperatures4 and with E. tereticornis at 25 °C44 varied within certain limits45. The longer pre-oviposition period, at least for some T. peregrinus females at lower temperatures could be due to the longer time required for this predator to develop its ovary46.Thaumastocoris peregrinus female and male longevity was increased at temperatures between 18 to 22 °C, which could be due to reduced metabolic processes at lower temperatures, affecting development and life history47. At low metabolic rates, certain physiological processes are suppressed, for example reproduction48, in order to maintain more crucial processes for survival. The effect of low temperatures on longevity have been reported for Monosteira unicostata Mulsant & Rey 1852 (Hemiptera: Tingidae) and Cleruchoides noackae Lin & Huber, 2007 (Hymenoptera: Mymaridae)50.T. peregrinus development and reproduction between 25 and 30 °C was similar to those reported for egg, nymph and egg-adult periods, respectively, for this bug51, as well as for Nezara viridula (L.) (Hemiptera: Pentatomidae) collected in soybean fields at climatically different locations28. The linear increase in the ratio between instars and of the adult stage duration of T. peregrinus (1/D) confirms the energy gain for its physiological processes52.The optimal temperature range for T. peregrinus in different environments53.The low survival at high temperatures indicates a phenotypic plasticity for T. peregrinus nymph development, 338.50 DD with a minimum of 9 °C shows the impact of low temperatures on this insect54. This result was also observed for Axinoscymnus cardilobus (Ren and Pang) (Coleoptera: Coccinellidae), with 204 DD; it took 67 days at a minimum of 9.07 °C to complete one generation, while this was 120 days55 at 17 °C. However, the accumulated temperature for the nymph-to-adult period of T. peregrinus, with 395.43 DD with a temperature limit of 9.93 °C shows its high adaptive potential. This species needed 905.65 DD in Canberra, Australia, to complete a generation and survived at temperatures below 1.5 °C, with adults recovering at higher temperatures51.The higher thermal constant of 0, rm, T and λ of T. peregrinus showed shorter development periods and higher growth rates with increased temperature, similar to that reported for Megacopta cribraria (F.) (Hemiptera: Plastaspidae)56 and Jakowleffia setulosa  (Hemiptera: Lygaeidae)57. These characteristics are important to understand the impact of temperature on insect growth, survival, reproduction and population increase59. This is necessary because the energy generated by the anabolism and catabolism metabolic processes for insect growth and reproduction depends on the environmental temperature60.The e RT. peregrinus. Thus, the definition of thermal requirements for T. peregrinus can assist traditional techniques in managing this pest. As well, this important data can be used in simulating population dynamics, monitoring, population peaks, occurrence, ecological zoning and modeling in order to manage this pest.The environmental temperature affected the development, fertility, longevity and mortality of Thaumastocoris peregrinus was reared in the laboratory at 24 ± 2 °C, 60 ± 10% RH, and a photoperiod of 12:12 h L:D on bouquets of Eucalyptus benthamii Maiden & Cambage  branches. The branches were fixed in a piece of foam to prevent drowning the insects in a 500-mL glass flask filled with water62. The effect of temperature on various biological parameters of T. peregrinus was evaluated at five constant temperatures  with a photoperiod of 12:12 L: D and RH 70 ± 10% in climatic chambers .The experiments were conducted at the Forest Entomology Laboratory of Embrapa Florestas in Colombo, Paraná state, Brazil. T. peregrinus nymphs were individually placed in acrylic plates (2.8 cm diameter × 1.5 cm) with a Eucalyptus benthamii fresh leaf disk (2.1 cm diameter) with its petiole introduced in a hydrogel layer  to maintain the leaf turgor. The eucalyptus leaf discs were replaced every two days. The duration and viability of T. peregrinus instars were assessed daily. Instar changes were evaluated based on the exuvia presence. Survival was evaluated in relation to the number of live individuals beginning each instar.Newly hatched Thaumastocoris peregrinus adults (<24 h old) were sexed based on its morphological characteristics6. A couple of this insect was placed per Petri dish (5.0 cm in diameter) with a fresh E. benthamii leaf disc (4.9 cm diameter). The pre-oviposition  and oviposition period, fecundity , longevity and mortality of T. peregrinus were evaluated. The males were not replaced. Mortality data were used to calculate longevity. Females were maintained until death, and egg numbers were use in the analysis.T. peregrinus were estimated using the hyperbole method63, based on the duration of the different instars, the nymph stage and the egg-adult period at 18, 22, 25, 27 and 30 ± 2 °C. The T. peregrinus instar development rate and nymph-to-adult period was regressed against temperature using a linear equation given by the formula: 1/D = a + bT, where, 1/D is the insect development time (D) in days, and T is the temperature (°C). The intercept ratio over the slope of the regression line corresponds to the threshold temperature (Tt) and the thermal constant (K) was estimated by taking the inverse of the slope (1/b)64.The temperature development threshold (Tt) and thermal constant (K) of T. peregrinus fertility life table at each temperature was built with specific survival at age x (lx), specific fertility (mx) and number of offspring reaching the age x in the next generation (lxmx). These data were used to calculate the net reproductive rate (R0), time between generations (T), innate ability to increase (rm) and finite rate of increase (λ) of this insect65.The 66. Sex ratio was analyzed using a generalized linear model (GLM) assuming binomial distribution68. The analyses were performed with the software R, version 3.3.2. The fertility life table was analyzed by Jackknife and the averages compared by Student’s t-test using the software SAS version 9.169.All data were first analyzed using the Shapiro-Wilk and Bartlett tests to determine data normality and homogeneity. The data related to each instar duration and of the nymph-to-adult period did not conform to normality, even after log transformation. Therefore, the comparisons were made using the non-parametric Kruskal-Wallis test. Pre-oviposition, oviposition, fecundity, oviposition rate and female and male longevity data were normally distributed, and thus they were analyzed using a linear model followed by a post hoc pairwise comparisons performed using Tukey HSD test survival”, “survminer” packages)71 followed by a pairwise comparisons Mantel-Haenszel Test (Log-Rank test) and Cox Proportional-Hazard Model (PH Model). The data evaluated at 30 °C was used as the reference for the other treatments (temperatures) on Multivariate Cox regression. Individuals who did not die by the end of the nymph period were censored . The adult individuals were not censored, because the experiment finished with the death of all insects .Survival curves were fitted and analysed using Kaplan-Meier survival probabilities (R version 3.3.2, “


Inthis study, to improve its solubility and bioavailability, the nanomicelles were preparedwith TPGS and soluplus as carriers for the first time. The particle size, Zeta potential,encapsulation efficiency, drug loading, stability, cytotoxicity, cellular uptake, andmetabolites in rats were studied. Cytotoxicity, cellular uptake, and metabolites in ratsof AMF-loaded TPGS/soluplus mixed micelles were compared with those of AMF. As a result,AMF-loaded TPGS/soluplus mixed micelles with a particle size of 67.33 ± 2.01 nm and Zetapotential of −0.84133 ± 0.041405 mV were successfully prepared. The encapsulationefficiency and drug loading of the mixed nanomicelles were 99.18 ± 0.76% and 2.47 ± 0.01%,respectively. The physical and chemical properties of the mixed micelles were stablewithin 60 d, and the cytotoxicity of the mixed micelles was much greater than that of AMFmonomers. Thirty-four kinds of metabolites of AMF were identified in rats. The metaboliteswere mainly distributed in rat feces. No metabolites were detected in bile and plasma. 14kinds of metabolites of the mixed micelles in rats were detected, including 11 in feces, 6in urine, and 3 in plasma, which indicated that the bioavailability of AMF has beenimproved. And the toxicity to cancer cells was enhanced, which laid a foundation for thedevelopment of new drugs.Amentoflavone (AMF) is a kind of biflavonoids existing in Ginkgo biloba leaves. It hasmany biological activities, such as antioxidant, anti-inflammatory, anti-bacterial,antiviral, hypoglycemic, anti-tumor and inducing apoptosis. However, its solubility andbioavailability are poor and there are a few studies on it  It is an ancient gymnospermwith a growth history of several hundred million years  transforming into inactive substances; (2)transforming the drug with no pharmacological activity into active metabolites; (3) changingthe types of pharmacological actions of drugs; (4) producing toxic substances  were used as carriers to prepare mixed micelles ofAMF. Soluplus has good solubilization for the drugs with poor water solubility, and canreduce the critical micellar concentration (CMC) value (0.76 × 10–3% w/v) and increase thedrug loading ;Ultra-high performance liquid chromatography system ; D3024R bench refrigerated centrifuge ; EYELLA N1100 rotary evaporator ;Ultrasonic crushing instrument ; Nano-ZS particle size tester ;Ultimate3000 high performance liquid chromatography ; pectraMax Plus384 Molecular Devices ; HF240cell culture box ;SW-CJ-2FD clean bench ; T9S dual-beamultraviolet-visible spectrophotometer ; FV1200MPE laser confocal microscope .2.2.J.T.-Baker Chemical Company .HPLC-grade formic acid was provided by Diamond Technology . CCK-8 was purchased from Beijing Zoman Biotechnology Co., Ltd., Beijing,China. Dimethyl sulphoxide (DMSO), iodine (I2), potassium iodide (KI) andsodium carboxymethyl cellulose (CMC-Na) were purchased from Tianjin Yongda Reagent Co.,Ltd., Tianjin, China. Pure water was purchased from Hangzhou Wahaha Group Co., Ltd.,Zhejiang, China.AMF  was purchased from Shanghai Shifeng Biological TechnologyCo., Ltd., Shanghai, China. Soluplus was purchased from BASF .Vitamin E polyethylene glycol succinate (TPGS) was purchased from Shanghai YuanyeBio-Technology Co., Ltd., Shanghai, China. HPLC-grade methanol and acetonitrile werepurchased from American 2.3.2.3.1.The preparation of AMF-loaded TPGS/soluplus mixed micelles was carried out by membranehydration method  is an important indicator for evaluating thestability of micelles. The lower the CMC, the more stable the nanomicelles. In thisexperiment, the CMC of AMF-loaded TPGS/soluplus mixed nanomicelles was measured usingthe iodine hydrophobic probe method and determined by an ultraviolet spectrophotometer.To prepare I2.3.3.The particle size and Zeta potential of AMF-loaded TPGS/soluplus mixed micelles weremeasured by dynamic light scattering technique, and each sample was measured intriplicate. The results were expressed as mean size ± standard deviation (SD) for threeseparate experiments.2.3.4.The encapsulation efficiency (EE) and drug loading (DL) were determined by HPLC. Achromatographic column was used. The mobile phases were water  and acetonitrile (B),and the flow rate was 1 mL/min. Gradient elution was adopted and the elution procedurewas as follows: 35–45% B (0–10 min), 45–35% B (10–10.1 min), and 35% B (10.1–15 min).The sample was injected at a volume of 20 μL and the detection wavelength was338 nm.For the EE determination: the prepared mixed micelles solution(200 μL) was dissolved in 2 mL methanol, and then treated with ultrasonic for 10 min torelease the encapsulated drug. The solution was then centrifuged twice at21,380×g for 10 min. Take the supernatant sample for analysis.For the DL determination: The prepared micelle solution wasfreeze-dried. And 45 mg of the powder was dissolved in 1 mL water. Subsequently, 200 μLthe solution was added to 2 mL methanol, and then treated with ultrasonic for 10 min.The solution was then centrifuged twice at 21,380×g for 10 min. Takethe supernatant sample for analysis: AMF is the weight of drug in micelles; WAMF’ is the weight offeeding drug; Wmicelle is the total weight of feeding soluplus, TPGS, anddrug in micelles.W2.3.5.Because the ratio of drugs and excipients has a great influence on the particle size,encapsulation efficiency, and drug loading of the micelle, the ratio of AMF andexcipients was optimized in this experiment. Particle size, PDI, Zeta potential, andencapsulation efficiency were used as the indexes. The results are shown in 2.3.6.To test the optimal formulation’s storage stability, the AMF-loaded TPGS/soluplus mixedmicelles was stored at 4 °C for 60 d. Particle size, PDI, Zeta potential, andencapsulation efficiency of the drug-loaded micelles were measured at 0 d, 15 d, 30 dand 60 d, respectively, and the clarification of the micelles was observed.2.3.7.2.A549 cells were used in the cytotoxicity and cellular uptake experiments of theAMF-loaded TPGS/soluplus mixed micelles. A549 cells were cultured in F-12 mediumcontaining 1% penicillin–streptomycin and 10% fetal bovine serum. And the cells weremaintained in an incubator at 37 °C in a humidified atmosphere of 5% CO2.3.8.In vitro cytotoxicity of AMF-loaded TPGS/soluplus mixed micelles wasdetermined using the CCK-8 assay. The cells were seeded in 96-well plates at a densityof 5000 cells/well, and incubated 24 h to allow cell attachment. After 24 h, theoriginal medium was sucked out. Then, the cells were incubated with AMF and AMF-loadedTPGS/soluplus mixed micelles of different concentration. The concentrations of AMF wasranged from 2000 μg/mL to 15.6 μg/mL, and AMF-loaded TPGS/soluplus mixed micelles rangedfrom100 μg/mL to 0.78 μg/mL. The cells were incubated for another 24 h. And then themedium was removed, fresh medium and CCK-8 (10 μL) were added and the cells wereincubated for 2 h. Finally, the absorbance at 450 nm was measured using a microplatereader, and each treatment was measured for three times /intracellular proteinconcentration (mg/mL)5 cells/well,cultured overnight at 37 °C for 24 h. Then the old medium was removed and washed withPBS for three times. Add the prepared culture medium with the drug concentration at30 μg/mL, and continue to culture at 37 °C for 1 h, 2 h, 4 h and 6 h. The fluorescenceintensity of cells was observed under the laser confocal microscope at different timepoints. The intensity of fluorescence represented the amount of drug intake.In the study, to analyze the results of cellular uptake more comprehensively, a visualqualitative analysis was carried out. The detail method was as follows: A549 cells withgood growth status were seeded in 4× culture dishes at 1.5 × 102.3.10.2.3.10.1.+ MS/MS system. The chromatographic separation was carried on Poroshell120 EC-C18  with a Security Guard® UHPLC C18pre-column (Poroshell). The mobile phases were water  andacetonitrile (B), and the elution procedures were as follows: 2 0–36% B (0–5 min), 36–59% B (5–14 min), 59–73% B (1 4–18 min), 73–95%B (18–21 min), and 95% B (21–25 min).The flow rate was 0.3 mL/min and the injection volume was 2 μL. The column temperaturewas 40 °C, and the automatic injector temperature was 4 °C.UHPLC was performed on Shimazu’s ultra-high performance liquid chromatography system, which was equipped with a triple TOF™ 5600A Triple TOF™ 5600 system with Duo-Spray™ ion sources operating in the negativeelectrospray ionization mode was used for the detection. The optimized conditions ofmass spectrometry as follows: ion source voltage −4.5 kV; ion source temperature,550 °C; declustering potential (DP), 60 V; collision energy (CE), −40 V + 15 eV.Atomization gas , heat gas , and curtain gas pressure was55 psi, 55 psi, and 35 psi, respectively. The parent ion and the daughter ion scanningrange were 50–800 Da and 50–1000 Da, respectively. Data were collected in IDA mode,and an automated calibration delivery system (CDS) was used to calibrate the MS andMS/MS automatically. The data were collected using the Analyst TF 1.6.1 software for25 min.2.3.10.2.Sprague–Dawley (SD) male rats  were provided bythe experimental animal center of Hebei Medical University. All animal experimentsfollowed the guidelines of the experimental animal management committee of HebeiMedical University. The rats were housed under standard conditions  for 5 dprior to use, and fasted 12 h before experiment, but free to water.The experimental rats were randomly divided into 12 groups with 3 rats per group. The volumeof gavage was 16 mL/kg. The prepared AMF suspension was orally administered to 9 ratsof groups 7, 9 and 11 at a dose of 100 mg/kg, and the prepared AMF-loadedTPGS/soluplus mixed micelles was orally administered to 9 rats of groups 8, 10, and 12at a dose of 100 mg/kg . Groups 1, 3, and 5 were given aqueoussolution of 0.5% CMC-Na, and groups 2, 4, and 6 were given blank micelle of the sameconcentration as the IGG-loaded micelle.2.3.10.3.Plasma samples: Approximately 300–500 μL blood was collected fromthe canthus of each rat at 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 3 h, 6 h, 9 h,12 h, and 24 h after gavage. The blood samples were centrifuged at1920×g for 10 min to obtain the plasma, and then the samples weremerged.Bile samples: After intragastric administration, each rat wassubcutaneously injected with urethane solution at a dose of 1.0 g/kg. Afteranesthesia, the bile duct was intubated and the bile was collected for 24 h.Feces and urine samples: The rats were put into a metabolic cageafter administration, fasted, but can drink freely. The urine and feces samples werecollected at regular intervals for 72 h, and the urine and feces were merged,respectively.2.3.10.4.g. The supernatant was taken and dried under nitrogen gas atroom temperature.For plasma, bile and urine pretreatment, an aliquot of 2 mL bio-sample was vortexedwith 6 mL of methanol for 5 min and centrifuged for 10 min at 4 °C and21,380×g for 10 min, and thesupernatant was taken. Repeat the above process once, and the supernatant was merged.The supernatants were evaporated to dryness under nitrogen gas.For feces, 6 mL methanol was added to 1 g feces, ultrasonically extracted for 30 minat 25 °C after vortexed, centrifuged at 21,380×g for 10 min. The supernatant wasinjected for analysis.Before analysis, the residue was dissolved with 100 mL methanol, vortexed andcentrifuged twice at 21,380×3.3.1.- toI2 in the solution. Therefore, the ultraviolet absorption of the solutionchanged, and it can be measured with an ultraviolet spectrophotometer. The measured massratios of TPGS and soluplus were 0: 4, 1: 3, 2: 2, 3: 1 and 4: 0. Their CMC values areshown in in vivo . 3.4.The AMF-loaded mixed micelles was stored at 4 °C for 60 d. Particle size, PDI, Zetapotential, and encapsulation efficiency of micelle were determined and the clarity of themicelles was observed at 0 d, 15 d, 30 d, and 60 d. The results are shown in 3.5.50 value of AMF and AMF-loaded mixed micellescalculated by GraphPad Prism 5  were 83.09 ± 0.65 μg/mLand 6.11 ± 0.74 μg/mL, respectively. As can be seen from the figure and the calculationresults, the IC50 value of AMF-loaded micelles was almost one-twelfth of thatof AMF, indicating that AMF-loaded micelles have much greater toxicity to A549 cells thanAMF. Therefore, it was speculated that AMF-loaded micelles had much higher lethality tocancer cells than AMF, and had the potential to treat cancer. At the same time, it can beseen that when AMF was at a lower concentration, the fatality rate of A549 cells was lessthan 0, which indicating that the low concentration of AMF had a small inhibitory effecton A549 cells, and the cells continued growing under conditions of sufficient nutrition,so the cell concentration showed an increasing trend, and the cell inhibition rate wasless than 0.A549 cells were commonly used in the study of cytotoxicity. In this study, the cellfatality rates were measured after incubated in different concentrations of AMF andAMF-loaded mixed micelles. The results are shown in 3.6.p < .05), which wasdue to the negative charge on the micelle and the negative charge on the outer surface ofthe cell membrane, resulting in a lower uptake of the micelle than that of themonomer.The results of cellular uptake of AMF monomers and AMF-loaded TPGS/soluplus mixedmicelles are shown in As shown in 3.7.3.7.1.In this study, a total of 34 metabolites of AMF were found. Metabolites were mainlydistributed in feces, including 34 metabolites (M1–M34), and 3 metabolites were in urine. No metabolite of AMF was found in bile or plasma. The result wasshown in In the metabolism, metabolites were mainly distributed in feces, and none of them werefound in bile or plasma, which indicated that the bioavailability of AMF was low. Themain metabolic pathways were oxidation, methylation, oxidation and methylation,acetylation, hydrogenation. Oxidation is its main metabolic reaction, which is relatedto its antioxidant properties. The results are shown in 3.7.2.Fourteen metabolites of AMF-loaded micelles were found in rats, including three inplasma , six in urine , and 11 in feces. No metabolites were found in bile, as shown in Among the metabolites, five were phase I metabolites and nine were phase IImetabolites. The main metabolic pathways were oxidation, methylation, oxidation andmethylation, loss of O, hydrogenation, loss of O and glycine conjugation andphosphorylation. Methylation and oxidation were the main metabolic pathways. The resultsare shown in 4.In the determination of encapsulation efficiency of AMF-loaded micelles, the micelles werefirst diluted with methanol, then sonicated in water-bath, filtered by 0.22 μm nylon filtermembrane, and injected for analysis. During the process, it was found that the encapsulationefficiency of micelles prepared by the same ratio and method was unstable, and theencapsulation efficiency of micelles prepared by different ratios was low. Because themethod of micelles preparation was the same, it was speculated that nylon filter membraneshad the sorption at different degrees to AMF. Therefore, after demulsification, the samplewas centrifuged twice but not filtered.The CMC of different carrier ratios measured in the experiment were all low, and when theTPGS/soluplus was 1: 3, the CMC was the lowest, indicating that the mixed nanomicellesprepared at this ratio had good stability. AMF-loaded TPGS/soluplus mixed micelles exhibitedgood stability, which was determined by the structure of micelles. AMF could be wrapped bythe two carriers in the core of the nanomicelles, which avoided the direct contact with thematrix and prevented the degradation of drugs by gastric acid and P450 enzyme. As a result,the drug could get into the blood to cycle in the form of micelles, and also delayed therelease of drugs, to ensure that it can play a more effective pharmacological role to the antineoplastic drug over the first 6 h ofexposure (Cagel et al., In the experiments of cytotoxicity and cellular uptake, we found that the ICin vitro through feces, which proved once again that AMF monomerswere water-insoluble and ester-insoluble compounds with very low bioavailability.Metabolites in micelles are also mainly distributed in feces, with 11 metabolites and nometabolites found in bile. However, three metabolites were detected in plasma and six inurine. This indicated that AMF could be absorbed into blood and urine or be absorbed moreinto the blood when it was prepared into micelles, and the metabolites could be detected.The wider distribution of metabolites indicated that more drugs can enter the bloodcirculation. Especially in plasma, there was a significant increase in metabolites, whichreflected indirectly that the bioavailability of AMF was improved. However, the metabolitesof micelles in feces were much less than those of monomers in feces, which illuminated thatafter encapsulated in micelles, it was difficult for the AMF monomers to be metabolized byenzymes and intestinal flora in the gastrointestinal tract, and they were directly excretedin the form of micelles, which also reflected that the stability of AMF-loaded micelles wasgood in vivo. In the metabolite analysis, not only a variety of AMFmetabolites were identified but also the indirect response to the changes inbioavailability, which was killing two birds with one stone.By comparing the metabolites of AMF monomers with those of AMF-loaded micelles in rats, itwas found that the metabolites of AMF monomers were mainly distributed in feces, 34metabolites were found, only 3 metabolites were found in urine, and no metabolites werefound in plasma and bile, which indicated that AMF monomers were difficult to enter blood,urine and bile. And most drugs passed through stomach, after intestinal metabolism, it wasexcreted In this experiment, AMF-loaded TPGS/soluplus mixed nanomicelle with good solubility andstability, high encapsulation efficiency and drug loading, and a particle size of less than100 nm, which was good for oral absorption, highly toxic to cancer cells and released slowlyin the body. It can provide a good choice for the development of anti-cancer drugs.5.in vivo. So in this experiment, theAMF-loaded TPGS/soluplus mixed micelles were prepared, and the properties of the micellesin vivo and in vitro were evaluated. The size of micellewas small and its physicochemical properties remained relatively stable within 60 d. In thecytotoxicity test, the IC50 value of micelles to A549 cells was much smaller thanthat of monomers, which was about one-twelfth of that of monomers. And the cellular uptakewas lower than that of AMF monomers due to the negative electricity on the surface ofmicelles and cell membranes. The metabolites of AMF monomer and AMF-loaded mixed micelleswere also studied. A total of 34 metabolites of AMF monomer were detected. The mainmetabolic pathways were oxidation, oxidation and methylation, methylation and acetylation.In rats, AMF was metabolized mainly through the gastrointestinal tract and then dischargedfrom the body. It was difficult to be absorbed into the blood and exert its pharmacologicaleffect and its bioavailability was low. Nevertheless, AMF-loaded mixed micelles had 14metabolites in rats, 11 in feces, 6 in urine, and 3 in plasma. The increase of metabolitesin plasma and urine indicated that the bioavailability of AMF was improved. But it wasdifficult for AMF to be metabolized by intestinal flora and various enzymes after beingencapsulated in micelles.The solubility and the stability of AMF were poor. And literatures have reported that thebioavailability of AMF was very low


The experiments were performed at air temperatures of 50, 60, and 70°C and air velocities of 0.6, 1.2, and 1.8 m/s. The thermal parameters were determined using thermodynamic relations. Increasing air temperature and air velocity increased the effective moisture diffusivity (effD), uE, EUR, exergy efficiency, and exergy loss. The value of the effD was varied from 4.19 × 10–10 to 1.18 × 10–9 m2/s. The highest value uE, EUR, and exergy loss and exergy efficiency were calculated 0.0694 kJ/s, 0.882, 0.044 kJ/s, and 0.879, respectively. Midilli et al. model, ANNs, and ANFIS model, with a determination coefficient (R2) of .9992, .9993, and .9997, provided the best performance for predicting the moisture ratio of quince fruit. Also, the ANFIS model, in comparison with the artificial neural networks model, was better able to predict uE, EUR, exergy efficiency, and exergy loss, with R2 of .9989, .9988, .9986, and .9978, respectively.This study aimed to predict the drying kinetics, energy utilization ( R2) of .9992, .9993, and 0.9997 provided the best performance for predicting the moisture ratio of quince fruit. Also, the ANFIS model in comparison with the artificial neural network model was better able to predict Eu, EUR, exergy efficiency, and exergy loss, with the R2 of .9989, .9988, .9986, and .9978, respectively.In this study, drying kinetics, effective moisture diffusivity (Deff), activation energy (Ea), specific energy consumption (SEC), energy utilization (Eu), energy utilization ratio (EUR), exergy loss, and exergy efficiency in a hot air (HA) dryer are presented for quince fruit slices. Midilli et al. model, ANNs, and ANFIS model, with a determination coefficient ( Drying is defined as the reduction in moisture from the products and is the most important process for preserving agricultural products since it has a significant effect on the quality of the dried products. The principle objective in drying agricultural products is the reduction in the moisture content to a level that allows safe storage over an extended period  and drying rate) and thermodynamic parameters (energy and exergy efficiency) of drying banana in HA flow dryer with the help combined structure ANNs‐RSM showed that this structure is able to predict drying index and thermodynamic parameters with R2 > .96 and RMSE < 0.060  and recorded in a computer.The relative temperature and moisture in the ambient around the dryer are two determining variables in drying foodstuff. Thus, in each experiment, the relative temperature and moisture around the dryer were measured and recorded using a digital Testo 925 thermometer with the accuracy of ±0.1°C and a Testo 400 moisture meter with the accuracy of ±0.1%.During the drying experiments, the average ranges for changes in ambient temperature and relative air moisture were 25 ± 4°C and 17 ± 5%, respectively. The experiments were conducted at temperatures  and three input air velocities . The thickness of 3 mm was selected for the samples in this study.MR) of the quince fruit samples can be obtained through Equation (The relative moisture ratio (Equation  (JahanbaEquation .(1)MR=Mt2.3MR of the quinces in different treatments was calculated using Equation , and the appropriate fitting can be determined using an index called root mean square error (RMSE). The model, which has the highest R2 and the lowest RMSE, will be the best model for treatment. These values can be calculated using Equations One of the most important criteria used for specifying the best model is the coefficient of determination R, and the2.4Fick's second law is used extensively to describe diffusivity in the process of drying agricultural products  is obtained through Equation (K) of the Ln (MR) graph over time:Effective moisture diffusivity coefficient  in front of (a1/T), a line with the gradient 1K is obtained:By drawing the graph 2.6SEC. Value of the SEC used in an HA dryer is taken from two sources. These energies are (a) heat energy  and (b) blower energy . The heat generator energy is obtained from Equation EUteaρ can be obtained by Equation ρa=1The mechanical energy obtained from the blower is calculated through Equation  Rad, Ka:(12)EUmeSEC of the quince fruit in the HA drying is obtained from Equation SEC=2.7uE), mass flow of air (daṁ), dry air enthalpy (dah), specific heat of input and output air (pdaC), air humidity ratio (kg water/kg dry air) (w), ratio of air humidity to the inlet and outlet (wdao), mass transfer rate (kg water/s) (vṁ), and energy utilization ratio (EUR) calculated through the first law of thermodynamics can be expressed as follows (Table The energy utilization (ws Table :2.8yĖ x), and exergy efficiency (Exη) obtained using (Equations 21–24) in  for the quince fruit drying process. It is difficult to determine the optimal number of neurons in the hidden layer, and it usually depends on the type and complexity of the work. Thus, it is determined by trial and error.The network architecture consists of an input layer with three neurons, an output layer and one or two hidden layers Figure . The inpFor artificial neural networks, single‐layer and double‐layer cascade Forward Back Propagation (CFBP) and Feed Forward Back Propagation (FFBP) networks were used, with different numbers of neurons varying between 3 and 15. Moreover, the Levenberg‐Marquardt (LM) and Bayesian Regularization (BR) algorithms were used. Three threshold functions namely sigmoid activation function (Logsig), linear activation function (Purelin), and hyperbolic tangent activation function (Tansig) were used to predict the proposed parameters  Summation . Training data and data simulation were conducted in Toolbox of Matlab R2014a software. For network modeling, the data were randomly assigned to two groups, of which 75% were used for training and 25% were applied for testing models.The present study was modeled using ANNs and ANFIS to predict experimental data on quince fruit drying  and the data from the models (predicted data) for each of the temperatures and input air velocities are reported in Table R2 value and the lowest RMSE value was selected as the appropriate model. According to Table R2 of .9992 and RMSE of 9.1 × 10−3.The values of 3.3effD of the quince fruit at different temperatures and input air velocities. The results show that the effD increases with increase in drying temperature and air velocity. The effD varies from 4.19 × 10−10 to 1.18 × 10−9 for temperature range of 50 to 70°C and air velocity of 0.6 to 1.8 m/s. By increasing the temperature of the dryer chamber, the moisture transfer rate from inside the fruit to its surface increases and the effD increases around three times  for agricultural products  and output exergy is determined by the temperature of the output air (Equation 23). The results for exergy loss are shown in Figure Aghbashlo, Kianmehr, and Arabhosseini  conducte3.8.2To determine the exergy loss, the experiments were performed in the velocity range of 0.6 to 1.8 m/s. The results shown in Figure 3.9Exergy efficiency was obtained for quince fruit in the drying experiments at different temperatures and air velocities. The highest amount of exergy efficiency was about 0.879 at the temperature of 70°C and an air velocity of 1.8 m/s. The lowest exergy efficiency was approximately 0.344 at the temperature of 50°C and an air velocity of 0.6 m/s.3.9.1Exergy efficiency in an HA dryer within the temperature range of 50 to 70°C is shown in Figure 3.9.2Exergy efficiency increases by raising the air velocity Figure . This inThe dryer's thermodynamic efficiency can be improved by the insulation of the drying chamber, designing and choosing the right components, and selecting optimum drying conditions, using other drying techniques such as microwave, infrared, etc. Exergy efficiency is a valuable tool for detecting key system losses and the optimal performance of industrial dryers.3.10MR, uE, and EUR as well as exergy loss and exergy efficiency. Air velocity, temperature, and drying time were considered as inputs of the network. Moreover, kinetic parameters of drying, uE, and EUR as well as exergy loss and exergy efficiency were selected as network output. The training of the network was based on the two algorithms of BR and LM. Before data entering to the network, they were normalized between 0 and 1.In this study, a multilayer perceptron (MLP) was used to predict 3.10.1MR, (b) uE, (c) EUR, (d) exergy loss, and (e) exergy efficiency are reported in Table The results of modeling artificial neural networks for predicting (a) MR of the FFBP network, with the topology 3‐12‐12‐1, the tansig–tansig–tansig transfer functions and the LM training algorithm with R2 = .9993 and RMSE = 0.0016, had the best performance. The results indicate that the ANNs modeling technique can be used effectively to predict MR. Similar results have been reported by other researchers for other products such as almond  To predict the uE during the process of drying quince fruits are given in Table RMSE = 0.0037 and R2 = .9985, the best FFBP network has a topology of 3‐10‐10‐1, with the tansig–logsig–tansig transfer functions and LM training algorithm. Similar results have been reported to predict uE by other authors including Nikbakht et al. (2) ANNs modeling results for predicting t et al.  for pomet et al.  for potaEUR. In addition, in this network, RMSE = 0.0029 and R2 = .9977. Nikbakht et al.  According to Table t et al.  used ANNRMSE = 0.0032 and R2 = .9980, had the best prediction for exergy loss.4) The CFBP network with BR algorithm, the tansig–tansig–tansig transfer function, with eight neurons in the first and second hidden layers, and RMSE = 0.0047 and R2 = .9970, was selected as the best network. Taheri‐Garavand et al.  The results of ANNs prediction for exergy efficiency in drying quince fruit in a HA dryer showed that the FFBP network, with a topology 3‐10‐10‐1, the transfer function of tansig–logsig–logsig and the LM training algorithm with d et al.  used ANNMadadlou  also pre3.11MR, uE, EUR, exergy loss, and exergy efficiency. These changes include the type of membership function, the number of membership functions, and the number of cycles.Some changes are required in order to find the best ANFIS network for predicting The best results for predicting the parameters in question are shown in Table R2 for MR, uE, EUR, exergy loss, and exergy efficiency was .9997, .9989, .9988, .9986, .9978, respectively. Also, the values RSME for MR, uE, EUR, exergy loss, and exergy efficiency were 0.0011, 0.0028, 0.0022, 0.0030, and 0.0046, respectively.According to Table 3.12MR, uE, EUR, exergy loss, and exergy efficiency). According to Tables R2 for each of the five parameters predicted in the ANFIS model was higher than the artificial neural networks model. Moreover, the value for the ANFIS in all the predicted parameters was lower than the artificial neural networks model.Comparison of statistical parameters between ANFIS and ANNs models confirms that the ANFIS model has a more accurate performance for predicting each of the five parameters  in drying potato, garlic, and cantaloupe using HA dryers. The ANFIS model showed a higher ability to predict these parameters than artificial neural networks.Khoshnevisan, Rafiee, Omid, and Mousazadeh  used art, et al.  predicte4effD, SEC, uE, EUR, exergy loss, and exergy efficiency was investigated. The results of the research showed that among the experimental models, the model of Midilli et al. to describe the kinetics of drying the thin layer of quince fruit slices can be introduced as the most suitable model. The range of effD in quince fruit samples varied from 4.19 × 10−10 to 1.18 × 10−9 m2/s, regardless of shrinkage, in the range of temperatures and velocities studied. The highest value of effD was obtained at the highest levels of temperature and inlet air velocity. With increasing air temperature and air velocity, specific energy consumption, energy utilization, energy utilization ratio, exergy loss, and exergy efficiency increased. The highest uE and EUR were 0.0694 and 0.882 kJ/s at 70°C, respectively. The lowest uE and EUR were 0.009 and 0.061 kJ/s at 50°C, respectively. The highest exergy losses and exergy efficiencies were calculated as 0.044 and 0.879 kJ/s, respectively. Also, the lowest exergy losses and exergy efficiencies were calculated as 0.0088 and 0.344 kJ/s, respectively. ANFIS was one of the fastest methods compared to artificial neural networks with lower RMSE and higher R2 for estimating MR, uE, EUR, exergy loss, and exergy efficiency for quince fruit. By insulating the drying chamber, designing and selecting the right components, as well as selecting the optimum drying conditions, the thermodynamic efficiency of the hot air dryer can be increased. Exergy efficiency is a valuable tool for identifying key system losses and optimizing the performance of hot air dryers.In this research, the effect of temperature and air velocity on drying time, dcA2)Surface area drying chamber (mpaCSpecific heat (kJ/kg C)pdaCSpecific heat of inlet and outlet airEuEnergy utilization (kJ/s)EUREnergy utilization ratio (dimensionless)EUmecMechanical energy consumption (Kwh/kg)terEUThermal energy consumption (Kwh/kg)EaActivation energy (kJ/mol)E˙xdciExergy inlet air (kJ/s)E˙xdcoExergy outlet air (kJ/s)E˙xyExergy loss (kJ/s)0DWidth from the source, which is a constant valueeffD2/s)Effective moisture diffusivity coefficient fghLatent heat of vaporization of water (kJ/kg)dahSpecific enthalpy drying air (kJ/kg)daihSpecific enthalpy of inlet air (kJ/kg dryair)daohSpecific enthalpy of outlet airdaehSpecific enthalpy of air environment (kJ/kg dry air)pdaCSpecific heat drying air (kJ/kg K)m˙vMass transfer rate (kg water/s)m˙daMass flow rate of drying air (kg/s)MRMoisture ratioeMEquilibrium moisture content (% d.b.)tMMoisture content (% d.b.)bMInitial moisture content (% d.b.)nindex of a summation and the number of terms taken into considerationNNumber of observationsPAtmospheric pressure (kPa)vsPSaturated pressure (kPa)R2Correlation coefficientRMSERoot Mean Square ErrorgRUniversal gas constant equal to 8.3143 kJ/molSECSpecific energy consumption (kWh/kg)tDrying timeaTAir temperature inside the drying chamber (K)refTRefers to characteristic valueTTemperature (K)dciTInlet air temperature of drying chamber (K)dcoTOutlet air temperature of drying chamber (K)T∞Temperature of outlet air (°C)VaInput air velocity (m/s)wt+ΔtWeight of product at time t + t (kg)wHumidity ratio of air (kg water/kg drying air)twt andWeight of product at time daowOutlet humidity ratio of air (kg water/kg drying airdaiwInlet humidity ratio of air (kg water/kg drying air)yStands for the experimental valuesy′Predicted values by calculating from the model for this measurementsy~The average predicted valuesΔtTime between two sample weighing (s)ϕRelative humidity of airρa3)Air density (kg/mΔPPressure difference (mbar)ΔtTemperature difference (°C)ηExexergy efficiencyThe authors have declared no conflict of interest.This study does not involve any human or animal testing.


Brassica rapa subsp. chinensis) is cultivated for its nutritional value, particularly with regard to vitamins, minerals and dietary fibers. However, limited metabolic information is available on the phyto-nutritional traits of pakchoi. Our GC-TOF MS analysis showed that green pakchoi has higher contents of carbon metabolism-associated metabolites such as sugars, sugar derivatives and inositol, while purple pakchoi has higher levels of nitrogen metabolism-associated metabolites such as amino acids and amino acid derivatives. To compare the content and composition of secondary metabolites in green and purple pakchoi, we analyzed phenylpropanoid-derived compounds and anthocyanins in mature leaves using an HPLC-UV system. This analysis identified 9 phenylpropanoid-derived compounds and 12 anthocyanins in the mature leaves of green and purple pakchoi. The level of rutin was significantly higher in purple pakchoi compared with green pakchoi, consistent with the expression of phenylpropanoid biosynthetic genes in the two pakchoi cultivars. The data obtained from this comprehensive metabolic profiling would be helpful to improve our understanding of the nutritional values of pakchoi cultivars as food sources.Pakchoi ( Brassica genus is the largest and most diverse within the Brassicaceae family and includes many economically important crop plants that are cultivated worldwide. This genus includes vegetables such as Chinese cabbage, turnip, cabbage, kale, broccoli, cauliflower, kohlrabi and Brussels sprouts that are known to be rich in phytochemicals and soluble fibers [Brassica species are used in the production of the condiment mustard.The e fibers ,2,3. In Brassica rapa subsp. chinensis) is an important crop that is widely cultivated and consumed in Asia and the level of consumption is also rising in countries in Northern Europe and elsewhere. The young leaves of pakchoi are consumed as part of salads or a garnish, the mature plants can be eaten steamed or briefly cooked. The plant is known by a variety of common names, such as “bok choy, pak choy and buk choy”. Pakchoi contains numerous health-benefiting compounds, such as glucosinolates, flavonoids, vitamins and minerals [Pakchoi  and predictive ability (Q2). The R2 and Q2 values were 0.802 and 0.999, respectively. The model “if Q2 > 0.8” is usually considered to have an excellent predictive ability. The corresponding loading plot shows the metabolites responsible for separation on the score plot  cycle, such as citric acid, fumaric acid and malic acid, showed few significant changes between green pakchoi and purple pakchoi. Citric acid was increased 1.35-fold, whereas fumaric acid and maleic acid were 1.57- and 1.30-fold lower in purple pakchoi compared with green pakchoi, respectively. The level of malic acid was considerably higher than any other organic acids. The content of malic acid in the green pakchoi was 45.0, 29.5 and 5.9 times higher than that in the fumaric acid, succinic acid and citric acid, respectively. In the red pakchoi, the content of malic acid was 54.2, 22.7 and 3.4 times higher than that in the fumaric acid, succinic acid and citric acid, respectively. The high content of malic acid may play an important role in energy source and carbon skeletons for the formation of amino acids in cell. Succinic acid did not show any significant differences between green pakchoi and purple pakchoi.As shown in the loading plot data, most of the amino acids and amino acid derivatives were higher in purple pakchoi than in green pakchoi, excepting leucine and phenylalanine B. Among BrPAL1, BrPAL2, BrC4H, Br4CL1, BrCHS, BrCHI, BrF3H, BrF3′H, BrFLS, BrDFR and BrANS in green and purple pakchoi diglucoside-5-glucoside was the most abundant (0.35 mg/g dry weight), followed by cyanidin 3-(sinapoyl)(sinapoyl)diglucoside-5-glucoside (0.34 mg/g dry weight). The composition of anthocyanins found in purple pakchoi was similar to that reported by Zhang et al. (2014). In their study, Zhang and coworkers showed that 15 anthocyanins were present in ‘Zi He’, a purple cultivar and that no anthocyanins were detected in the mature leaves of ‘Su Zhouqing’, a green cultivar. The principal anthocyanins identified in ‘Zi He’ were cyanidin and cyanidin derivatives but not petunidin 3,5-diglucoside.To determine whether the pigmentation of purple pakchoi is caused by anthocyanins, we analyzed soluble anthocyanins in mature leaves of green and purple pakchoi using an HPLC-Q-Trap MS system. Anthocyanins were not detected in green pakchoi, whereas 12 anthocyanins were separated and characterized from purple pakchoi . The majThe green pakchoi cultivar ‘8210’ and purple pakchoi cultivar ‘8389’ were grown into seedbeds containing soil to 3 weeks after germination. The seedlings were grown under field conditions at Asia Seeds Ltd., Korea with mean minimum temperature 15 °C and mean maximum temperature 27 °C. Mature plants at approximately two months after sowing were separated from root tissue. Harvested samples were frozen in liquid nitrogen and stored at −70 °C until it could be used for QRT-PCR and HPLC-UV analysis.v/v/v) methanol:water:chloroform and performed mixing at 1200 rpm for 30 min at 37 °C. The polar phase was transferred into a new tube after centrifugation and 0.4 mL of water was added. The mixed samples were centrifuged at 16,000× g for 5 min and then the methanol/water phase was freeze-dried for 16 h. The samples were redissolved in 80 μL of methoxyamine hydrochloride (20 mg/mL) in pyridine and incubated for 90 min at 30 °C. This samples were derivatized with 80 μL of MSTFA at 37 °C for 30 min. Sixty microliter ribitol (0.2 mg/mL) was used as an internal standard (IS). After derivatization, 1 µL samples were analyzed into an Agilent 7890A gas chromatograph  connected to a Pegasus HT TOF mass spectrometer  with a split ratio of 1:25. The column was 30 m × 0.25 mm ID fused-silica capillary column coated with 0.25-µm CP-SIL 8 CB low bleed . The injection temperature was set to 230 °C. The helium gas (1.0 mL/min) was used as carrier gas. The temperature program was as follows: 2 min isothermal heating at 80 °C, followed by an increase to 320 °C at 15 °C/min and a 10 min hold at 320 °C. The transfer line and ion-source temperatures were set at 250 °C and 200 °C, respectively. Mass scanning range was 85–600 m/z and the detector voltage was set at 1700 V.Polar metabolites were extracted and analyzed as described previously ,29. The Component detection and automated deconvolution were conducted with a ChromaTOF software. The compounds identified in libraries derived from the National Institute of Standards and Technology (NIST) and the in-house libraries for standard chemicals.BrEF1α gene, used as a housekeeping gene. All QRT-PCR assays were conducted as the mean of three different biological experiments and subjected to statistical analysis. QRT-PCR product sizes and primer sequences are provided in The pakchoi cultivar were immediately frozen in liquid nitrogen following treatment. Total RNA was isolated from frozen pakchoi cultivars using total RNA mini kit . QRT-PCR analysis was conducted using a QuantiTect SYBR Green RT-PCR kit  in a CFX96TM real-time PCR detection system . Data analysis and determination of reaction specificities were performed as described previously . Gene ex18 column  and monitored at 280 nm. A 20 µL injection was separated using a mixture of 0.15% acetic acid (solvent A) and 100% methanol (solvent B) at a flow rate of 1.0 mL/min. The elution condition was set as: 5% solvent B, followed by a linear gradient from 0 to 80% solvent B over 93 min and then a hold at 5% solvent B for an additional 5 min. Chlorogenic acid, caffeic acid, epicatechin, p-coumaric acid, ferulic acid, rutin, trans-cinnamic acid, quercetin and kaempferol were purchased from Sigma-Aldrich . Different compounds were quantified on the basis of peak areas and the concentrations were calculated as equivalents of representative standard compounds. The linear equations and regression coefficients for chlorogenic acid, caffeic acid, epicatechin, p-coumaric acid, ferulic acid, rutin, trans-cinnamic acid, quercetin and kaempferol were y = 52135.7x − 57351.96 with r2 = 0.999, y = 101427.93x − 19999.04 with r2 = 0.999, y = 16656.06x − 8837.11 with r2 = 0.998, y = 172376.91x − 2288.50 with r2 = 0.998, y = 115029.24x − 16713.48 with r2 = 0.998, y = 25662.41x – 3352.89 with r2 = 0.999, y = 323515.13x – 3738.31 with r2 = 0.998, y =40520.94x + 12547.81 with r2 = 0.997 and y = 68166.24x – 4265.15 with r2 = 0.998, respectively. Means ± SD were determined from three replicates.Phenylpropanoids were extracted from 100 mg dried samples with 3 mL 80% methanol at 25 °C for 1 h. The extracts were filtered through a 0.45 μm PTFE syringe filter  after centrifugation. Phenylpropanoids were measured on a NS-4000 HPLC apparatus  with a Cv/v) in a sonicator for 20 min and filtered in a brown vial. Anthocyanins were measured by an Agilent 1200 series HPLC  connected to a 4000 Q-Trap LC-ESI-MS/MS system  with a 4 μm POLAR-RP 80A column , as described previously [O-glucoside  as an external standard. Means ± SD were determined from three replicates.For anthocyanin analysis, 100 mg of dried powder was extracted with 2 mL water:formic acid  to inspect which components separated between green and purple pakchoi samples . The OPLIn conclusion, we obtained a comprehensive metabolic profile, including secondary metabolites, of green and purple pakchoi cultivars. Overall, the content of phenylpropanoids and anthocyanins was higher in purple pakchoi than in green pakchoi. In particular, the amount of rutin was considerably higher in purple pakchoi than in green pakchoi. Quercetin, kaempferol and anthocyanins were only detected in purple pakchoi. These phenylpropanoid compounds and their precursors act as antioxidants to inhibit the synthesis of reactive oxygen species (ROS) in plants ,22. The