Salmonella infection that can lead to health issues. Artificial antibacterial compounds used to preserve fresh meat can have negative health effects. We investigated the potential of natural essential oils (EOs), namely Mentha arvensis (mint) and Cinnamomum cassia (cinnamon) EOs, to prevent contamination of the food pathogen, Salmonella enterica subsp. enterica serotype Typhimurium, in vitro and on chicken skin. The gas chromatography–mass spectrometry (GC-MS) technique was used to determine the compositions of mint EO (MEO) and cinnamon EO (CEO); the most abundant compound in MEO was menthol (68.61%), and the most abundant compound was cinnamaldehyde (83.32%) in CEO. The antibacterial activity of MEO and CEO were examined in vapor and direct contact with S. typhimurium at temperatures of 4 °C, 25 °C, and 37 °C. The minimal inhibitory concentration at 37 °C for MEO and CEO reached 20.83 µL/mL, and the minimal bactericidal concentration of CEO was the same, while for MEO, it was two-fold higher. We report that in most tested conditions in experiments performed in vitro and on chicken skin, CEO exhibits a stronger antibacterial effect than MEO. In the vapor phase, MEO was more effective against S. typhimurium than CEO at 4 °C. In direct contact, the growth of S. typhimurium was inhibited more efficiently by MEO than CEO at small concentrations and a longer exposure time at 37 °C. The exploration of CEO and MEO employment for the inhibition of Salmonella bacteria at different temperatures and conditions expands the possibilities of developing more environment- and consumer-friendly antibacterial protection for raw meat.The bacterial contamination of meat is a global concern, especially for the risk of  Salmonella, which is one of the most common foodborne pathogens [EOs are hydrophobic extracts, which are rich in plant secondary metabolites, and they are intensively studied for their antimicrobial and other valuable properties. EOs extracted from mint, cinnamon, clove, basil, lavender, thyme, sage, etc., have GRAS  statuses provided by the Food and Drug Administration (FDA) and are considered to be safe for human consumption. Researchers have successfully demonstrated the potential of several plant EOs for the microbiological protection of various food products ,2,3. Theathogens ,6. Synthathogens . Thus, tathogens ,9,10,11.Salmonella and Staphylococcus bacteria by analyzing transcriptional responses, cell membrane properties, oxidative damage, and metabolism [The properties of EOs, mainly their high hydrophobicity and volatility, make the investigation challenging. The efficacy of EOs and their individual constituents on the survival of microorganisms has been studied extensively by measuring the effects of direct contact with the oils and the impact of EO vapor. However, there is still a lack of standard testing procedures, especially for the investigation of the vapor phase, but attempts to standardize the methods are emerging ,13. Temptabolism ,19,20,21tabolism ,23.Staphylococcus aureus [Mentha genus plants, and their individual components have shown inhibitory activity towards various bacteria [MEO has a wide range of positive health effects and is used for the treatment of various skin infections, headaches, and respiratory and gastrointestinal tract problems ,25. The s aureus . EOs, exbacteria ,26,27, ybacteria , fungi [bacteria , and insbacteria .S. aureus and S. typhimurium [S. typhimurium to adhere to cells, thus reducing the possibility of infection [α-pinene, and linalool [CEO has strong broad-spectrum antibacterial, antifungal, antioxidant, and insecticidal activities ,31. Cinnhimurium ,33, and nfection . Other clinalool . The meclinalool .S. typhimurium bacteria in vitro and on chicken skin at different temperatures, namely 4 °C, 25 °C, and 37 °C. The main compositions of MEO and CEO were determined via GC-MS. The minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of MEO and CEO in direct contact with S. typhimurium were established. The EO vapor’s antibacterial activity against S. typhimurium and Staphylococcus aureus bacteria was tested at different temperatures in vitro, and CEO showed greater inhibition than MEO. In the vapor phase, the efficacy of CEO against S. typhimurium on chicken skin was greater than the activity of MEO.The aim of this work is to investigate the antibacterial activity of CEO and MEO via vapor and direct contact with Mentha arvensis) and Chinese cinnamon bark (Cinnamomum cassia) used in this research were bought from one retailer in Lithuania, JSC Kvapu namai. Both EOs were labeled with traditional and botanical names of the plant they were produced from via hydrodistillation, the series number, country of origin, and expiry date (EOs of wild mint (iry date .m/z. The oven temperature of the gas chromatograph was set at 50 °C (for 1 min) and then increased by 5 °C per minute to reach 160 °C; then, it was held for 2 min and programmed to reach 250 °C at the increasing rate of 10 °C/min and was held at the final temperature for 4 min. He with a flow rate of 1.0 mL/min was used as carrier gas. Detector and injector temperatures were 250 °C, and the ion source temperature was 220 °C. The qualitative analysis and identification of compounds were based on the comparison of time and retention indexes of the column, with corresponding data in the literature [7–C30) (≥99% (GC), Supelco, Bellefonte, PA, USA) with linear interpolation. The relative percentage of EO constituents was computed from the chromatogram peak areas with none of the correction factors.GC-MS was applied to determine the chemical compositions of MEO and CEO. A total of 10 µL of each EO was dissolved in 1 mL of pentane (≥99% (GC), Sigma-Aldrich, Darmstadt, Germany) and diethyl ether , Sigma-Aldrich, Darmstadt, Germany) (1:1) mixture. An amount of 1 µL of the prepared solution was injected into the GC/MS system. Analyses were performed using Shimadzu GC/MS-Q2010 PLUS interfaced to a GC-MS-QP2010 ULTRA mass spectrometer  and fitted with a non-polar Rxi-5MS (30 m × 0.25 mm × 0.25 µm)  capillary column. Mass spectra in electron impact mode were generated at 70 eV, with 0.97 scans per second and a mass range of 33–400 terature , as wellSalmonella enterica subsp. enterica serotype Typhimurium LT2  and Staphylococcus aureus ATCC 29213  were used in this study. Bacteria were grown on LB agar , 0.5% yeast extract , 0.5% NaCl , 2% agar  on solid medium plates at 37 °C. Freshly grown cells were inoculated in LB broth and grown overnight (16–18 h) at 37 °C with continuous shaking. The optical density (OD) of cells was determined spectrophotometrically (600 nm), and the cell number was adjusted to an OD of 1 in LB broth medium. Reagents from Liofilchem, Italy were microbiological grade.Bacterial strains S. typhimurium cells (about 1 × 107 cells/plate) was evenly spread onto the plates with LB agar medium. For the measurement of direct contact between the cells and EO, 10 µL of pure EO was placed on the surface of the LB agar medium covered with bacteria. The antibacterial effect of EO vapor was tested using the inverted Petri dish method. A total of 25 µL of pure EO was placed on the inside surface of the plate cover, and LB agar plates with bacteria were covered and sealed with parafilm. Plates were incubated at 4 °C, 25 °C, and 37 °C overnight (24 h). Three plate replicates of each condition were tested. After incubation, the size of the growth inhibition zone was evaluated.To determine the antibacterial activity of EO in agar plates, 20 µL of S. typhimurium cells (about 1 × 107 cells/mL) was mixed with an equal amount of the suspension containing EO to achieve the final concentration of 5 × 105 cells/well. Suspension containing EO consisted of EO premixed with 75% ethanol  at a 1:1 ratio (v/v), diluted with LB broth. Control samples containing concentrations of ethanol corresponding to EO samples were used to confirm that the amount of alcohol added does not have a significant impact on the survival of cells. Tested final concentrations of EO varied from 0.008% to 12.5% and for ethanol—from 0.006 to 9.37% per well. Samples were incubated at 37 °C overnight (24 h). The following day, the wells with bacterial suspensions were visually inspected, and the smallest concentration of EO at which no signs of bacterial growth were observed (liquid turbidity or cell sedimentation) was considered the MIC. To determine MBC, 25 µL of bacterial suspension in the wells was distributed on plates with LB agar medium and incubated at 37 °C overnight (24 h). The following day, the growth of bacteria in each plate was evaluated. The smallest concentration of EO that reduced the number of viable S. typhimurium cells by more than 99%  was considered the MBC.To determine MIC, 120 µL of v/v). The MIC of each EO was diluted 10-fold, 100-fold, and 1000-fold with LB broth. In control samples, EO was replaced with an equal amount of sterile distilled water. Each EO dilution at equal volume was mixed with 100 µL of S. typhimurium cells  and incubated for 10, 20, and 40 min at temperatures of 4 °C, 25 °C, and 37 °C. Serial dilutions were made, and 50 µL of each solution was spread onto the LB agar plates and incubated at a 37 °C temperature for 24 h. Colonies on plates were counted as colony-forming units (CFUs), and the mean values of CFU/mL were calculated.To investigate the rate of the inhibition of bacterial survival via direct contact with the EO, several EO concentrations at different temperatures were tested. Each EO was premixed with 75% ethanol at a 1:1 ratio , and 25 µL of pure EO was placed into the inner side of the tube cap. In control samples, LB broth was used instead of EO. Tubes were positioned horizontally to avoid contact between the cells and the EO. Tubes were incubated at temperatures of 4 °C, 25 °C, and 37 °C for 1 h, 4 h, 24 h, 48 h, and 120 h. Serial dilutions were made, and 50 µL of each solution was spread onto the LB agar plates and incubated at a 37 °C temperature for 24 h. All experiments were performed in triplicate. The number of survived cells in each sample was calculated, and the results were expressed as mean value lg (CFU/mL) ± standard deviation.EO vapor antibacterial activity was tested against Gram-negative 2. On each skin sample, 25 µL of S. typhimurium (1 × 107 cells/mL) was placed. Plates were incubated at 4 °C for 30 min to enhance the adhesion of bacteria on the skin’s surface. In the center of the inner Petri dish cover, 25 µL of the pure EO was placed. In the control plates, a sterile 0.9% NaCl solution was used instead of EO. Plates were covered with lids and insulated with parafilm. Plates were incubated at temperatures of 4 °C, 25 °C, and 37 °C for 1 h, 4 h, 24 h, 48 h, and 120 h. After incubation, skin samples were transferred to sterile 50 mL conical centrifuge tubes. To each tube, 15 mL of sterile 0.9% NaCl solution was added, and tubes were gently inverted continuously for 5 min to detach the bacteria from the skin’s surface. Serial dilutions were made, and 50 µL of each solution was spread onto the LB agar plates, followed by overnight incubation at 37 °C and counting of colony-forming units (CFUs). Experiments were performed in triplicate, and the mean value of CFU/mL was calculated.Fresh chicken pieces were bought from the local supermarket in Vilnius, Lithuania. The chicken skin was removed from the flesh and cut into 2 cm × 2 cm pieces. The skin was rinsed with cold running tap water and with sterile distilled water. The excess water from the washed skin was removed via absorption with sterile paper. Dry pieces of chicken skin were placed onto sterile Petri dishes and sterilized with a UV dose of 12 mJ/cmp < 0.05) was used to define statistically significant results. All experiments were performed in triplicate, and the treatment efficiency is expressed as mean ± standard deviation. Statistical analysis was carried out using Microsoft Office Excel.A one-way analysis of variance -cinnamaldehyde in CEO), accounting for more than 50% of the total compounds identified (E)-methoxy-cinnamaldehyde (7.62%) and (E)-cinnamyl acetate (4.69%), respectively. In the latter, all other identified compounds were present in very small quantities, not exceeding 1%. In MEO, α-pinene, β-pinene, limonene, geraniol, and menthyl acetate were present in amounts exceeding 1% of the total composition, with other compounds being present in minor or trace levels. When comparing the compositions of the two EOs, it should be noted that there are only a few compounds in common between them, and these compounds are present in very modest amounts. Both predominant compounds, in addition to being plant-specific odorants, have strong antifungal, insecticidal, and antibacterial properties [Both EOs used in the study contained one predominant compound . In the agar diffusion experiments of MEO and CEO, and in the vapor diffusion experiment for CEO, the antibacterial effect was stronger at 37 °C than at 25 °C b. The MEectively . CEO andat 37 °C , respectSalmonella and other bacteria [β-pinene [β-pinene was shown to have little to no impact on Salmonella sp. propagation [M. piperita EO addition to model food systems incubated at 4 °C and 10 °C showed diminished S. enteritidis numbers during a period of one week [Salmonella spp. in several foods, such as tahini [Salmonella spp. at 37 °C and 10 °C, where it was also tested, and it was lower at 25 °C, which is consistent with our findings. CEO has a prominent antibacterial effect, which is determined by CEO components, mainly cinnamaldehyde, since the aldehyde functional group provides strong antibacterial properties, while cinnamyl acetate shows lower activity than other major CEO components [Methoxy-cinnamaldehyde likely has similar antibacterial properties to cinnamaldehyde because of the aldehyde group, although to our knowledge, this has not been demonstrated yet; however, o-methoxy-cinnamaldehyde has been proven to have antifungal activity against pathogenic Candida species [Salmonella sp. and Candida albicans were the same for several MEO components, supporting the efficacy of the tested oils not only against bacteria but also against yeasts [The agar diffusion experiments previously demonstrated the antibacterial effect of MEO against bacteria ,44. A prpagation ,44. Thusone week , confirms tahini . CEO is s tahini . Accordis tahini , in a dimponents . Methoxy species . The MICS. typhimurium is observed at 35–37 °C, while typically, it can still grow at lower rates in the range between 5 and 47 °C on chicken meat [S. typhimurium growth on agar plates at 25 °C and 37 °C, likely as a result of both the action of EO and the speed of growth of the cells at particular temperatures.The optimal growth of ken meat . Temperaken meat . Temperaken meat . We demoS. typhimurium were determined at 37 °C. The MICs of MEO and CEO were 20.83 µL/mL, while the MBC for CEO was the same as the MIC, and for MEO, it was 41.67 µL/mL, two-fold higher. Because of differences in the compositions of EOs, the comparison between their MIC values is complicated; thus, the indicative range of the MICs of EOs and their components is relevant. The reported MIC at 28 °C of the main component in MEO—menthol—for S. typhimurium was 1.0 µg/mL, and the MBC was 1.5 µg/mL, while for limonene, α-pinene, and β-pinene, the MIC and MBC values were between 8.0 and 10.0 µg/mL [Salmonella sp. at 37 °C of menthol was 6 µg/mL, while for menthone, menthyl acetate, and limonene, the value was 10-fold greater—60 µg/mL [S. enteritidis and S. gallinarum at 37 °C [Salmonella spp., as published by most authors, is about 0.32–1 mg/mL [The MIC and MBC for .0 µg/mL . The est60 µg/mL . CEO wasat 37 °C . The MIC–1 mg/mL .S. typhimurium growth inhibition by EOs at different temperatures, the MICs at 37 °C were selected as reference points for the concentrations to be tested. Serial dilutions of EOs at a 20.83 µL/mL concentration were tested at 4 °C, 25 °C, and 37 °C at different timepoints, namely 10, 20, and 40 min. As fast as after 10 min, at all tested temperatures, both EOs at 20.83 µL/mL (1-fold dilution) killed all the cells—7.6 logs  as soon as after 20 min of exposure, regardless of the temperature. After 10 min, MEO inhibited the growth of about 3.2-fold more cells than CEO at 25 °C and 37 °C. CEO at a 100-fold dilution (0.21 µL/mL) at 25 °C exhibited 100 and 1000 times higher antibacterial activity after 20 and 40 min, respectfully, than MEO. At 37 °C, CEO’s antibacterial activity after 20 and 40 min incubation was only up to 7-fold higher than at the corresponding timepoints at 25 °C, whereas MEO showed a robust increase in killing, and there were no viable cells after 40 min, which was even 3 logs less than the amount of cells seen in the CEO samples at the same EO concentration. MEO also exhibited significant killing at the lowest concentration tested—1000-fold dilution (0.021 µL/mL)—where only 3 logs out of 7.6 logs of live cells remained at 37 °C. Previous research has demonstrated that EOs from distinct plants, despite having the same MIC values, can have different inhibition mechanisms, as it was in the case of hyme EOs . It may S. typhimurium. At 25 °C, CEO shows more efficient killing than MEO at a lower concentration (100-fold dilution). However, at 37 °C after 40 min of exposure, MEO shows significantly stronger killing than CEO at lower concentrations (100-fold and 1000-fold dilution), where the antibacterial activity with a 10 times smaller concentration than that of MEO equals that of more concentrated CEO (S. typhimurium growth kinetics during the exposure to thyme and cinnamon EOs [At 4 °C and at the highest concentrations (MIC and 10-fold dilution) at all tested temperatures, both EOs have similar antibacterial activity against ated CEO . A similamon EOs , which samon EOs ,50. CEO S. typhimurium cells in vitro. The viable cell count was measured at 4 °C, 25 °C, and 37 °C at different timepoints for 120 h. The growth of S. typhimurium cells exposed to MEO vapor at 25 °C and 37 °C was similar to the control cell growth at 4 °C with no EO  to about 4.6 logs in the samples with EOs: the viability decreased at 4 °C with CEO (p = 0.025) and at 37 °C for MEO (p = 0.021) and CEO (p = 0.003) (We aimed to investigate the effects of MEO and CEO vapors against = 0.003) . At 25 °S. typhimurium, CEO was more effective than MEO. The number of surviving cells significantly decreased under 24 h of treatment with CEO compared with the control and MEO samples at 25 °C (p = 0.0002 for both) and 37 °C . An exposure to CEO reduced the count of S. typhimurium cells by 1.8 logs and 2.4 logs at 25 °C and 37 °C, respectively , but MEO remained a weaker alternative because of a two-fold higher MBC value (41.67 µL/mL) than that of CEO. However, MEO surpassed CEO in S. typhimurium killing via direct contact with diluted EOs at a longer exposure time and higher temperature . Also, MEO vapor showed a higher suppression of S. typhimurium at 4 °C than CEO in vitro, but no such effect was detected for Gram-positive S. aureus bacteria or in the experiments on the contaminated chicken skin samples. CEO was more effective than MEO at slowing down the proliferation of S. typhimurium on chicken skin at 25 °C and 37 °C, but for 4 °C, neither of the storage vapors of the tested oils had a role in controlling bacterial growth in a 24 h period.MEO consisted mostly of menthol (68.61%), while in CEO, (E)-cinnamaldehyde (83.32%) was dominant. Our findings show that in most conditions, CEO possesses stronger antibacterial properties against


Asarum essential oil (AEO) has been shown to have good pharmacological activities for the anti-inflammatory and analgesic effects, but increasing the dose may cause toxicity. Therefore, we studied the toxic and pharmacodynamic components of AEO by molecular distillation (MD). Anti-inflammatory activity was assessed using RAW264.7 cells. Neurotoxicity was assessed in PC12 cells and the overall toxicity of AEO was evaluated in the mouse acute toxicity assay. The results showed that AEO is primarily composed of safrole, methyl eugenol, and 3,5-dimethoxytoluene. After MD, three fractions were obtained and contained different proportions of volatile compounds relative to the original oil. The heavy fraction had high concentrations of safrole and methyl eugenol, while the light fraction contained high concentrations of α-pinene and β- pinene. The original oil and all three fractions exhibited anti-inflammatory effects, but the light fraction demonstrated more excellent anti-inflammatory activity than the other fractions. Asarum virgin oil and MD products are all neurotoxic. The exposure of PC12 cells to high concentrations of AEO resulted in abnormal nuclei, an increased number of apoptotic cells, increased ROS formation, and decreased SOD levels. Moreover, the results of acute toxicity tests in mice revealed that the light fractions were less toxic than virgin oils and other fractions. In summary, the data suggest that the MD technology enables the enrichment and separation of essential oil components and contributes to the selection of safe concentrations of AEO. Asarum can dispel wind and cold, relieve pain through the orifice, and warm the lungs  is isolated from the dry rhizome of the Chinese herb Asarum [Asarum heterotropoides Fr. Schmidt var. mandshuricum (Maxim.)Kitag.] (The “Chinese Pharmacopoeia” records that he lungs . It is u)Kitag.] , the mai)Kitag.] . AEO is )Kitag.] . It has )Kitag.]  (Wu et a)Kitag.]  (Akhlaq )Kitag.] .in vivo and in vitro . Overdosaging may lead to Qi stuffiness and congestion, ultimately causing death  is a unique liquid-liquid separation technology that can separate different substances based on their molecular characteristics, such as the mean free path of different molecules under high vacuum conditions  Reza Sh. MD can n-Hexane (F2124114GC > 99%) was obtained from Aladdin Biotechnology . Butyl acetate (RH269812GC ≥ 97.7%) was obtained from Shanghai Yien Chemical Technology Co., Ltd. . Anhydrous sodium sulfate (1804081) was procured from Xilong Scientific .Lipopolysaccharide (LPS), dimethyl sulfoxide (DMSO), and dexamethasone (Dex) were purchased from Sigma-Aldrich . Cell culture medium RPMI 1640, DMEM, phosphate buffer saline solution (PBS), and trypsin were obtained from Solarbio Life Sciences . Fetal bovine serum (FBS) was obtained from Gibco BRL Life Technology . A cell counting kit 8 (CCK-8) was obtained from Meilunbio . Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) kits were purchased from Meimian Biotechnology . Superoxide dismutase (SOD) assay kits were procured from Nanjing Jiancheng Bioengineering Institute . 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was purchased from Solarbio Life Sciences . Annexin V-FITC apoptosis detection kit, mitochondrial membrane potential assay kit, and reactive oxygen species (ROS) assay kit were obtained from Beyotime Biotechnology .Asarum (jk20220118008) was purchased from Chengdu and authenticated as genuine Chinese herbal medicine by Professor Zhang Puzhao of the School of Pharmacy, Jiangxi University of Chinese Medicine. AEO was obtained by steam distillation , with an oil yield of approximately 1%. The volatile oil was stored in a refrigerated cabinet at 4°C.AEO was spread on a thin film on a heated surface to ensure even heating and improve evaporation efficiency and shorten the distillation time. The feed flow rate and scraping speed had a relatively minor impact on the separation results, unlike the evaporation temperature and vacuum degree, which significantly affected the degree of evaporation . The optTo a 10-mL volumetric flask, 1,000 μL of butyl acetate was added and the flask was accurately weighed on an analytical balance. The solution was then diluted to 10 mL with n-Hexane and allowed to mix well.Asarum crude oil was dried with anhydrous Na2SO4. Next, 50 μL of crude oil and 50 μL of each distillate were pipetted into four 10-mL volumetric flasks. Then, 50 μL of the internal standard solution was added to each flask and diluted to 10 mL with n-hexane. The mixture was evenly mixed and thereafter filtered with a 0.22-μm microporous membrane to obtain the GC sample.Gas phase conditions: A gas chromatograph-mass spectrometer  and HP-5MS columns were used. The carrier gas was He, the flow rate was 1.0 mL/min, and the split ratio was 10:1. The inlet temperature was 250°C. The heating conditions are shown in MS conditions: Electrospray ionization (ESI) was performed. The electron energy was 70 eV, the ion source temperature was 230°C, and the quadrupole temperature was 150°C. Then, the scan range was defined as m/z 30–650 amu, and the injection volume was 0.3 μL. The results were searched with the NIST14. L database in combination with related literature.2 incubator at 37°C. The cells were harvested using 0.25% trypsin/EDTA when they reached 80%–90% confluency. The cells were treated with different concentrations of the previously obtained four distillates for the following experiments.PC12  cells were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. . The cells were cultured in RPMI 1,640 medium containing 1% penicillin/streptomycin and 10% FBS in a 5% CO2 incubator at 37°C. After the cells reached 80%–90% confluency, they were collected using 0.25% trypsin/EDTA for the subsequent experiments. Then, the cells were treated with 0.5, 0.25, 0.125, and 0.0625 μL/mL concentrations of each of the four distillates.RAW264.7 murine macrophage cells were kindly given by the Research and Experiment Center, College of Traditional Chinese Medicine, Jiangxi University of Chinese Medicine. The cell medium was prepared with DMEM (containing 1% penicillin/streptomycin) and 10% FBS and was cultured in a humidified 5% COLPS was diluted to 1 mg/mL in sterile PBS stored at −20°C and then diluted to 100 μg/mL in DMEM medium. Dex was diluted to 4 μg/mL in PBS. LPS and Dex were used in the anti-inflammatory experiment with RAW264.7 cells.4 per well. Then, the cells were treated with eight different concentrations  of four distillates  in an incubator for 24 h. According to the kit instructions, RPMI 1,640 containing 10% CCK-8 reagent was added to each well. After incubation at 37°C for 2 h, the optical density (OD) value of each well was measured at 450 nm using a microplate reader .PC12 and RAW264.7 cells were harvested by centrifugation and seeded in 96-well plates at 1 × 104 RAW264.7 cells per well were cultured in 24-well plates. Four concentrations  of distillates C, H, M, and L were added for intervention on the second day. After 2 h of incubation, the cells were induced by LPS (100 μg/mL). The untreated RAW264.7 cells served as the control group, the cells induced by LPS without dosing were used as the model group, and dexamethasone was added for the Dex group. After 24 h of incubation, cell supernatants were collected for further experiments with commercial IL-6 and TNF-α kits. Then, after rinsing the plate five times, the kit reagents were added to the reaction plate and incubated at 37°C for 30 min. After washing the samples five times, reagents were added following the manufacturer’s instructions and reacted for 10 min. The OD values of the wells were detected at 450 nm via a microplate reader, and the anti-inflammatory activity of the cells was calculated.A total of 4 × 105 cells per well. The cells were cultured overnight and exposed to 1, 0.5, 0.25, and 0.125 μL/mL of the four distillates . Then, the cells were incubated in an incubator for 24 h. The cells and cell supernatants were centrifuged and collected in 2 mL EP tubes. Thereafter, the cells were broken using a cell disruptor and stored at −80°C for subsequent assays for the determination of oxidative enzymes. The SOD levels in PC12 cells were measured using the 96-well plates in the commercial assay kits with five replicate wells per sample. The OD values were measured via a microplate reader.PC12 cells were collected by trypsin and seeded in 6-well plates with 4 × 105 PC12 cells were cultured in each well of the 6-well plates. Four different concentrations  of distillates C, H, M, and L were separately added to the cells and incubated for 24 h. After incubation, the cell supernatants were discarded. The cells were washed twice in PBS, and fixed with 4% paraformaldehyde (PFA)  for 15 min. Thereafter, the DAPI reagent was added for 5–10 min at room temperature (RT), followed by washing with PBS twice. The stained cells were observed under a fluorescence microscope .DAPI and Annexin V-FITC were used to detect the apoptosis of PC12 cells. For DAPI staining, 4 × 10Annexin V-FITC assay was performed according to the manufacturer’s instructions. PC12 cells were collected after trypsin digestion and centrifuged . After resuspending with PBS, the cells were centrifuged again, and the supernatants were discarded. Next, 195 μL Annexin V-FITC binding reagent was added. The cells were resuspended and 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added. The samples were incubated for 10–20 min at RT under a dark condition. A flow cytometer  was used to analyze the stained cells.5 cells were seeded in each well of a 6-well plate, and the ROS level was measured after treatment with the distillates  for 24 h. To each well, 2 mL of DCFH-DA was added, followed by incubation at 37°C for 20 min. Thereafter, the cells were rinsed thrice with serum-free RPMI 1,640 medium. The cells were then studied using a flow cytometer with an excitation wavelength of 488 nm and an emission wavelength of 525 nm.The ROS levels in PC12 cells were determined using the fluorescent probe DCFH-DA. Approximately 4 × 105 per well) were seeded in 6-well plates and incubated with four concentrations  of the four distillates  for dosing intervention. After 24 h of intervention, the experiment was performed according to the manufacturer’s instructions. JC-1 staining solution was added to the cells and cultured at 37°C for 20 min. A fluorescence microscope was used to observe the fluorescence of the cells.JC-1 was used as a fluorescent probe in the Mitochondrial Membrane Potential Assay Kit (JC-1) for early apoptosis detection. PC12 cells , control (20% Tween-80), and 20 experimental groups . According to the pre-experimental results, the doses of both crude oil and H distillate were 1.0792, 1.3489, 1.6862, 2.1077, and 2.6347 g/Kg; the concentrations of M were 2.1077, 2.6347, 3.2865, 4.1080, 5.1349 g/Kg, and the administration concentrations of L distillate were 2.6347, 3.2865, 4.1080, 5.1349, 6.4185 g/Kg. The mice fasted without water for 12 h before the experiment. After 12 h, the four distillates were administered by gavage at 0.2 mL/10 g per mouse.A total of 90 SPF KM mice , half male and half female, were obtained from the Experimental Animal Center of the Jiangxi University of Chinese Medicine. This experiment was approved by the Animal Ethics Committee of the university  and strictly followed the Guidelines for the Management and Use of Laboratory Animals. The acute toxicity test was performed to assess the toxicity of AEO, and the p < 0.05 was considered statistically significant.Data were processed using SPSS 21.0  for plotting and statistical analysis. Graphs were constructed using GraphPad Prism 9.0  and SIMCA 14.1 . Data were expressed as mean ± standard deviation (‾X ± SD) and compared using one-way analysis of variance (ANOVA). Asarum volatile crude oil (C) was divided into three distillates, heavy oil (H), medium oil (M), and light oil (L) via MD. A total of 34 components were identified and isolated from the Asarum crude oil, among which methyl eugenol had the highest content (24.02%), and the others were mainly safrole (17.08%), 3,5-dimethoxytoluene (10.62%), myristyl ether (8.20%), 3,4,5-trimethoxytoluene (7.04%), 3-carene (4.77%), β-pinene (4.51%), eugenolone (3.00%), lobenol (1.21%), and levulinic-limonene (1.05%) ( (1.05%) , S4. The (1.05%) .From these results, the contents of monoterpenes in crude oil, H, M, and L were 13.58, 1.31, 53.72, and 77.25%, respectively, whereas the contents of ethers in C, H, M, and L were 69.44, 86.21, 27.86, and 5.83%, respectively. The monoterpenes, with boiling points generally in the range of 140°C–180°C, exhibit analgesic, antibacterial, detoxification, and diuretic effects. For example, the boiling point of β-pinenelaevo is 155°C–156°C, and (+) -3-caren has a boiling point of 168°C–169°C. However, the ethers in AEO had higher boiling points, such as safrole 232°C–234°C and methyl eugenol 254°C–255°C. These ether components exhibited neuroleptic effects and were also the primary toxic components of AEO. From these results, it can be concluded that the light distillate is enriched in low-boiling-point components, while the heavy distillate is enriched in high-boiling-point components.To investigate the safety concentrations of the four distillates C, H, M, and L on RAW264.7 cells and PC12 cells, eight different concentrations  of these distillates were added to the RAW264.7 cells and PC12 cells to detect cell viability for 24 h.The cell viability results of RAW264.7 cells showed that the survival rate was close to 0 at a concentration of 2 μL/mL. However, the survival rates increased with the decrease in the distillate concentration for all the distillates. Especially at 1 μL/mL concentrations, the cell viability with heavy, medium, and light distillates was significantly higher than that with crude oil. Meanwhile, when the concentrations of crude oil, heavy distillate, medium distillate, and light distillate were all less than or equal to 0.5 μL/mL, the cell viability of RAW264.7 cells was greater than 85%. Therefore, four concentrations  of C, H, M, and L were selected for the following experiments on RAW264.7 cells .Furthermore, the cell viability results from PC12 cells showed an increase in survival rate with the decrease of concentrations of C, H, M, and L. At 2 μL/mL concentration of the four distillates, the survival rates of PC12 cells were almost 0, while at a concentration of 1 μL/mL of C, H, M, and L, the cell viability of the C group was lower than that of the H, M, and L groups. Notably, the survival rates of PC12 cells were greater than 85% at a concentration of 0.5 μL/mL for all four distillates. This indicates that 0.5 μL/mL was a safe distillate concentration for PC12 cells .p < 0.05, vs. model group). These results indicated that distillates H, M, and L, obtained from AEP by MD, could reduce the contents of pro-inflammatory cytokine IL-6 compared with that of the model group  of crude oil, heavy distillate, medium distillate, and light distillate were compared. The results showed that compared with the LPS-induced model group, there were decreasing trends in IL-6 levels in the Dex groups and four concentration groups of H, M, and L. Furthermore, the IL-6 levels in 0.5 and 0.25 μL/mL groups of C were decreased (el group .p < 0.05). When the concentrations were 0.25, 0.12,5, and 0.0625 μL/mL, the IL-6 levels IL-6 in H, M, and L groups were clearly decreased . This indicated that distillates H, M, and L showed a relative decrease in the content of pro-inflammatory cytokine IL-6 at the same concentrations compared to crude oil in a dose-dependent manner of distillates. Compared with the C group, the IL-6 levels were significantly lower in M and L groups for 0.5 L/mL distillate concentration (t manner .p < 0.05)  when the concentration was 0.25 μL/mL  of the H, M, and L groups showed a decreasing trend ( < 0.05) . Further25 μL/mL . These rPrincipal component analysis was used to analyze the relationship between AEO and the IL-6 and TNF-α levels via SIMCA14.1software. The results are shown in The correlation analysis between anti-inflammatory activity and components of AEO revealed that the anti-inflammatory activity was primarily associated with methyl eugenol, α-pinene, β-pinene, α-phellandrene, 1,8 eucalyptol, camphorene, and β-laurelene. Therefore, the strong anti-inflammatory activity of L may be related to the high relative percentage content of α-pinene and β-pinene.p < 0.01). Fluorescence intensities corresponding to all four concentrations of H were statistically significant, while only C4 (0.125 μL/mL) had a significant change in M distillate (p < 0.01). Finally, all four concentrations  of H distillate caused significant changes in the cellular oxidative stress levels compared to the normal group (p < 0.01) . Moreover, the fluorescence intensity tended to be normal as the drug concentration decreased. In addition, the statistical results showed that under C intervention, the fluorescence intensities of A2 (0.5 μL/mL), A3 (0.25 μL/mL), and A4 (0.125 μL/mL) were significantly changed compared with that of the normal group ( < 0.01) .p < 0.01, compared with that with the normal group). In M groups, the apoptosis rates of 1, 0.5, and 0.25 μL/mL were significantly different from those of the normal group. In distillate L groups, the apoptosis rates were significantly changed for the concentrations of 1, 0.25, and 0.125 μL/mL  (The Annexin V-FITC assay kits were used to investigate the cell apoptosis rates of crude oil and distillates with PC12 cells. The results of this study showed that the high concentration (1 μL/mL) of these distillates  obtained after MD exhibited a higher apoptosis rates compared to the normal group (blank group). Furthermore, the apoptosis rate varied in a concentration-dependent manner. Lower concentration resulted in lower cell apoptosis rate. In addition, the statistical findings revealed that A2 (0.5 μL/mL) in distillate C and B1 (1 μL/mL) in distillate H exhibited a significant increase in apoptosis rate (l group) .Asarum volatile crude oil and the different distillates on PC12 cell morphology by DAPI staining were observed. Cells in 1 μL/mL of each of C, H, M, and L  groups demonstrated significant morphological changes with abnormal nuclei and increased apoptotic cells. However, as the concentration of the added distillate decreased, the morphology of cells in C, H, M, and L  groups at 0.12 5 μL/mL of C, H, M, and L cells demonstrated similar morphology to that of the normal group (blank group), with intact nuclei and significantly more viable cells. These results suggested thatthat the survival rates of PC12 cells is concentration-responsively changed following treatment with AEO and its different distillates. (The effects of illates. .).Oxidative damage can induce apoptosis. The expression levels of superoxide dismutase (SOD) in the different distillates of AEO at different concentrations are shown in. The SOD levels were significantly decreased at high concentrations of AEO, suggesting that AEO affects the oxidative stress system at high concentrations. Notably, the SOD expression of H was significantly lower than that of the other three groups  when the concentration was 0.25 μL/mL, which may be related to the higher safrole content in H .LD50 of C, H, and M was 1.8852, 1.9566, and 3.6741 g/Kg, respectively, and the toxicity of the M group was lower than that of the other two groups. The mortality rate of mice was only 16.67% when the administration concentration of L was 6.4185 g/Kg, indicating that the toxicity of L was significantly lower than the other distillates  to 31.3644% (H). However, the olefin components in the middle and light distillates achieved enrichment. The ratios of α-pinene and β-pinene contents in crude oil, heavy distillate, middle distillate, and light distillate are 1:0.1:2.5:4.7 and 1:0.1:2.8:4.4, respectively, and the total amount of α-pinene and β-pinene accounts for 39.5% of the light oil distillate, which is five times more than that of crude oil. Furthermore, significant differences in the relative contents of methyl eugenol, safrole, and 3,5-dimethyl methylbenzene were observed in each distillate, with 3,5-dimethyl methylbenzene in C, H, M, and L in the ratio 1:1.1:0.8:0.2 and safrole in C, H, M, and L in the ratio 1:1.1:0.7:0.2. The ratio of methyl eugenol in C, H, M, and L was 1:1.3:0.2:0.02. The methyl eugenol content was significantly reduced in the light fraction, and the safrole content was only 18.44% of the crude oil. The difference in the composition of each distillate was influenced by the different boiling points of the components, providing a basis for further toxicological investigation.in vivo and in vitro (Numerous experiments have shown that the AEO exhibits a significant anti-inflammatory effect both in vitro  (Antsyshin vitro . During in vitro  (Choudhuin vitro . TherefoAsarum, including hepatotoxicity, nephrotoxicity, and neurologic reactions (LD50 assessment confirmed that the toxicity of the crude oil was similar to that of the heavy distillate. In contrast, the toxicities of the middle and light distillates were remarkably reduced. A comparison of the components revealed that the reduction in toxicity was associated with a reduction in the contents of safrole, methyl eugenol, and 3,5-dimethyl methylbenzene.A previous study by our group demonstrated that AEO at a dose of 1.8852 g/kg inhibited the normal functioning of the central nervous system of KM mice and impaired their balance and coordination, with clinical manifestations including an intoxicated state, abnormal gait, limping, slowed movement, and the loss of appetite. These results are consistent with the reported adverse events of eactions . Accordieactions . Becausein vitro model to explore AEO neurotoxicity in this study. According to the flow cytometric detection, biochemical methods, and the DAPI staining, it was demonstrated that AEO administration induced oxidative damage and dysfunction in the mitochondria of PC 12 cells, with significant changes in cell morphology, abnormal nuclei, and an increase in apoptotic cells. Moreover, we also noted that the apoptosis rate of PC12 cells was increased after AEO treatment, indicating that AEO could induce apoptosis in PC12 cells. In summary, the enrichment of the AEO by MD confirmed that the primary anti-inflammatory components in the AEO were methyl eugenol, α-pinene, β-pinene, α-phellandrene, 1,8 eucalyptol, camphorene, and β-myrcene, and the main toxic components were safrole, methyl eugenol and, 3,5-Dimethoxytoluene. This finding is consistent with previous literature reports. Methyl eugenol and myristicin in AEO have been reported to have hepatorenal toxicity and genotoxicity (Only a few studies are available on the mechanism of neurotoxic effects of AEO, which mainly focus on cell apoptosis, oxidative stress, and the inhibition of neuraxial growth . Prior etoxicity  (Akhlaq toxicity  (Carvalhtoxicity . A studytoxicity . In the toxicity .in vitro findings were also validated by in vivo experiments. The experimental results in mice demonstrated that the light distillates of AEO exhibited low toxicity. However, the characteristic components of AEO were safrole, methyl-eugenol, and 3,5-Dimethoxytoluene, and the light distillates contained relatively low levels of the characteristic components and were not representative of AEO. Therefore, further investigations are needed to apply the MD technique to enrich the distillates containing the characteristic constituents of AEO and identify a safety window for clinical applications by studying the varying proportions of the constituents.The Asarum are based on increasing the decoction time and reducing the essential oil content. However, studies have indicated that the essential oil of Asarum exhibits a significant anti-inflammatory effect, and reducing the essential oil content also reduces its effectiveness. Therefore, determining the safety window is essential for the clinical applications of AEO, both to exploit its medicinal properties and avoid the toxicity of the essential oil. In the current study, the MD technique was used to enrich and separate the Asarum essential oil and provide methods and ideas for future experiments.In summary, the MD technique can effectively enrich and separate essential oils. AEO exhibits good anti-inflammatory activity and considerable toxicity. The potential mechanism of neurotoxicity is related to oxidative stress and apoptosis. Most of the current methods of reducing the toxicity of Based on our previous studies, AEO has anti-inflammatory, antibacterial and analgesic effects. Through molecular distillation technology to reduce its toxicity, it is expected to be developed as a spray gel for treating oral ulcers.


Multiple Sclerosis (MS) is an autoimmune disease believed to be secondary to T cells autoreactive against myelin, leading to demyelination, neurodegeneration and, eventually, disability . Four su9/L, platelets of 10 × 109/L, hemoglobin of 6 g/dl) with 5% cellularity on the bone marrow biopsy. He underwent allo-SCT from his matched brother using fludarabine/cyclophosphamide (flu/cy) conditioning and bone marrow as a stem cell source. His course was complicated by mixed lymphoid chimerism. His GvHD prophylaxis consisted of short methotrexate (MTX) course and cyclosporine (CSA). Nine months post-transplant CSA tapering was started. However, 6 weeks after tapering he developed unsteady gait and, after an extensive workup, he was diagnosed with MS, based on the detection of typical MRI T2 lesions in periventricular, cortical and infratentorial areas of the brain, . However, a month after the first dose, he developed pancytopenia, and an extensive workup showed hypocellular bone marrow (5%) with full donor chimerism . Simultaneously, his MS symptoms got worse, the expanded disability status scale (EDSS) was 6.5 and an MRI showed new spinal cord demyelinating lesions. He then underwent a second allo-SCT from the same donor using FCC conditioning and mycofenolate mofetil (MMF) for GvHD prophylaxis. He engrafted by day 14 and his sensory symptoms improved as well.8/kg nucleated cells infused, including 11.25 × 106/kg CD34 stem cells and 39.98 × 107/kg CD3 + /kg). Currently, 56 months after his stem cell boost, he remains in remission of the severe AA, and his MS is stable, with an EDSS of 6, and no further clinical or radiological progression.His second post-transplant course was complicated by poor graft function and mixed chimerism Fig.  which ev9/L, HGB 81 g/L ANC 0.33 × 109/L, platelet 3 × 109/L). IFN-β 1a was suspended, and she was started on horse ATG at 40 mg/m2 and CSA, with no improvement. She then underwent matched sibling donor allo-SCT in 2020, with Flu/Cy/ATG/TBI conditioning. She engrafted quickly with full donor chimerism and, currently, 27 months post allo-SCT, she is off immunosuppression, with no further progression of her MS, despite being off MS medications since the AA diagnosis.A 42-year-old woman, diagnosed with MS in 2014, started on IFN-β 1a with good control, and no further relapses. In 2019, she was diagnosed with AA, after presenting with vaginal bleeding, and was found to have pancytopenia (WBC 3.49 × 10The association of MS and SAA has been reported in the literature . The HLAOur cases highlight the association of MS and AA. These cases also highlight the issue of CNS GvHD and how to differentiate this from MS.


Listeria monocytogenes and Salmonella enterica on enoki and wood ear mushrooms. Fresh mushrooms were inoculated with four strain cocktails of either L. monocytogenes or S. enterica and dried at ambient conditions for 10 min. Following drying of the inoculum, mushrooms were placed into food dehydrators preheated to 70, 80, or 90°C and treated for up to 24 h. At treatment intervals, mushrooms were removed from the dehydrators for pathogen enumeration. Inactivation kinetics for both pathogens were modeled using the Weibull, log-linear with tail, and log-linear with shoulder models. Pathogen reductions of >4 log CFU/g were achieved on both enoki and wood ear mushrooms during dehydration at 90°C after only 2–4 h. At 70 and 80°C, log reductions of >4 log CFU/g were observed on wood ear mushrooms after 4–8 h. On enoki mushrooms, a tailing effect was observed with residual populations (>2 log CFU/g) of L. monocytogenes and S. enterica remaining even after 24 h of treatment at both 70 and 80°C. This study emphasizes the need for an individualized dehydration strategy for each mushroom type to ensure the effectiveness of dehydration as a process to reduce pathogen populations. Results of this study will aid in informing proper time and temperature combinations for dehydration of specialty mushrooms to ensure product safety.Foodborne illness outbreaks in the U.S. associated with consumption of both fresh and dried specialty mushrooms have recently occurred. Dried wood ear mushrooms were implicated in a salmonellosis outbreak in 2020, while fresh enoki mushrooms were associated with two listeriosis outbreaks in 2020 and 2023. These specialty mushrooms are commercially available in both their fresh and dried states. Due to the short shelf life of mushrooms, dehydration is a common method used in both industry and by consumers to extend the shelf life and preserve quality. Therefore, the aim of this study was to evaluate the use of dehydration on the inactivation kinetics of both  Drying mushrooms is also convenient for transportation and storage as the product requires a smaller footprint and its weight is greatly reduced. Nonetheless, drying methods relying on heated air are often conducted at low temperatures (<80°C) for short periods of time in order to retain the organoleptic properties of the product  , various product . In an i product . Therefow < 0.70, where no pathogen growth would occur, minimal reductions in pathogen populations due to drying would be expected.Even though the enoki mushrooms involved in both listeriosis outbreaks were sold to consumers in their fresh state, enoki mushrooms, as with other types of specialty mushrooms, are also commercially available in their dried state. Consumers may also choose to dry fresh mushrooms in their homes using household food dehydrators. Consumers looking for instructions on how to dehydrate mushrooms may find information online at educational extension websites  suggestiL. monocytogenes and Salmonella enterica serovar Typhimurium on sliced portobello mushrooms during hot-air drying in a household food dehydrator  and carrots (1.6–1.7 log) during dehydration at 60°C for 6 h in household food dehydrators  were weighed into 6-cm foil pans.2.2.L. monocytogenes or S. enterica was used. The L. monocytogenes strains included LS806 (isolated from hummus), LS3132 (isolated from avocado), LS0352 (isolated from cream cheese), and ScottA . The S. isolate ], Alachu isolate ], and Po2.3.L. monocytogenes or S. enterica cocktail (10 spots of 10 μL each for each sample) at approximately 6.5 log CFU/g. Inoculated mushrooms were dried at ambient for 10 min. Five household food dehydrators  were pre-heated for 10 min at 70, 80, or 90°C. Triplicate mushroom samples were placed onto the middle shelf in the pre-heated dehydrators. The circulating air temperature at the center of the middle shelf as well as the internal temperature of the mushrooms during dehydration was continuously monitored using probe thermometers coupled to a data logger . To avoid temperature fluctuations due to opening the dehydrator doors, only one sampling timepoint was conducted per dehydrator at one time and a different dehydrator was used for each trial. Sampling timepoints included 0, 0.5, 1, 1.5, 2, 4, 6, 8, and 24 h. Triplicate samples were assessed at each timepoint and three independent trials were conducted (n = 9).Mushrooms (10 g per sample) were placed into aluminum foil trays (60 mm) and spot inoculated with the diluted 2.4.L. monocytogenes and S. enterica were enumerated after each dehydration timepoint. Mushrooms  were homogenized 1:10 with Buffered Listeria Enrichment Broth  or BPB, for L. monocytogenes and S. enterica respectively, for 1 min in at 185 rpm a stomacher . Homogenates were serially diluted and plated onto BHIA supplemented with 100 μg/mL of rifampicin (BHIArif) for enumeration. All agar plates were incubated at 37°C for 24–48 h. Data were expressed as log CFU/g per initial 10-g sample.2.5.n = 9).The moisture content of the freshly chopped enoki and wood ear mushrooms (10 g) and the mushrooms after each dehydration sampling timepoint were assessed. To determine the moisture content of the freshly chopped mushrooms, the samples were dried in an oven at 100°C for 24 h and the remaining solid weight was measured. After each dehydration timepoint, the samples were also measured to determine the remining solid weight. Moisture content results were reported on a wet basis. Triplicate samples were assessed at each timepoint and three independent trials were conducted  during dehydration at 70, 80, and 90°C were fitted to the Weibull model (The populations (log CFU/g) of ll model . Populatll model . All modll model .The Weibull model is displayed in N (t) is the population at time t, N0 is the initial population, ρ is the shape parameter , and δ is the time for the first decimal reduction in microbial population. This model is commonly used when examining microbial inactivation and has been shown to provide the best fit in low aw (<0.60) food matrices at a wide range of temperatures (21 to 80°C) (where to 80°C) , 2017.The log-linear with tail and the log-linear with shoulder models are displayed in N is the population at time t, N0 is the initial population, Nres is the residual or remaining population, kmax is the maximum inactivation rate, and Sl is the shoulder length (time unit).where 2.7.α = 0.05). The populations of L. monocytogenes and S. enterica on both mushroom types after inoculation and during dehydration were also statistically compared using ANOVA. For primary modeling, the goodness of fit parameters, mean square error (MSE) and coefficient of determination (r2), were reported by GInaFiT.The moisture contents of both mushroom types after chopping and during dehydration were statistically compared using ANOVA with Tukey’s post-hoc test  were not significantly different. After 1 h of dehydration, the moisture content at 90°C (20.96 ± 5.84%) was significantly lower than that at the other two temperatures. After 2 h of dehydration, the moisture content of the wood ear mushrooms equilibrated across temperatures; there was no significant difference between the moisture contents at 70, 80, or 90°C . Dehydration for longer time periods also did not result in a significant decrease in the moisture content of the wood ear mushrooms.3.2.L. monocytogenes and S. enterica on the fresh enoki mushrooms prior to dehydration were 6.40 ± 0.60 and 6.51 ± 0.50 log CFU/g, respectively , whereas at 70 and 80°C, significant population reductions were observed after 4 and 2 h, respectively . After 2 and 4 h of dehydration at 90°C, the population of L. monocytogenes was reduced by 3.38 and ≥ 4.70 log CFU/g, respectively. A tailing effect was observed during dehydration of enoki mushrooms at both 70 and 80°C, with remaining populations of L. monocytogenes of 3.50 ± 0.43 and 2.19 ± 0.30 log CFU/g, respectively .The populations of ectively . For L. S. enterica, significant population reductions were observed on enoki mushrooms dehydrated at 80 and 90°C after only 2 and 1.5 h , whereas at 70°C, a significant population reduction was observed after 6 h (reduction of 2.52 log CFU/g). After 2 and 4 h of dehydration at 90°C, the population of S. enterica was reduced by 4.37 and ≥ 4.70 log CFU/g, respectively. As observed with L. monocytogenes, a similar tailing effect occurred with S. enterica during dehydration of enoki mushrooms at both 70 and 80°C, with remaining populations of 3.92 ± 0.59 and 2.08 ± 0.17 log CFU/g, respectively .For L. monocytogenes and S. enterica on enoki mushrooms during dehydration at all temperatures examined (ρ (0.39–0.71) indicated that the survival of L. monocytogenes and S. enterica on enoki mushrooms followed an upward concavity at all temperatures, with the exception of S. enterica at 90°C (ρ = 1.95), indicating a convex survival curve. According to the Weibull model, a 4 log CFU/g reduction of L. monocytogenes would occur after 24.00, 14.16, and 3.16 h on enoki mushrooms during dehydration at 70, 80, and 90°C, respectively. While a 4 log CFU/g reduction of S. enterica was not determined to occur at 70°C, reductions would occur at 80 and 90°C after 9.12 and 1.96 h, respectively.The Weibull model was first utilized to describe the inactivation of examined . The parL. monocytogenes and S. enterica on enoki mushrooms during dehydration (L. monocytogenes and 2.25–3.25 log CFU/g for S. enterica). While a 4 log CFU/g decrease in population was not predicted to occur for either pathogen at 70°C, 4 log CFU/g decreases were determined to occur at 80°C after 5.04 and 5.28 h for L. monocytogenes and S. enterica, respectively. No tailing was observed for S. enterica on enoki mushrooms during dehydration at 90°C, and hence the log-linear with shoulder model was used; the maximum inactivation rate was 6.35 ± 0.71 log CFU/g/h. For L. monocytogenes, neither of the log-linear models adequately described the inactivation.Secondly, the log-linear model, with either tail or shoulder, was also used to describe the inactivation of ydration . Due to 3.3.L. monocytogenes and S. enterica on the fresh wood ear mushrooms prior to dehydration were 6.26 ± 0.33 and 6.38 ± 0.35 log CFU/g, respectively  and after only 1 h at both 80 and 90°C . After 2 and 4 h of dehydration at 90°C, the population of L. monocytogenes was reduced by 4.25 and ≥ 4.56 log CFU/g, respectively. After 4 and 6 h of dehydration at 80°C, the population of L. monocytogenes was reduced by 4.35 and ≥ 4.56 log CFU/g, respectively. At 70°C, a 3.26 log CFU/g reduction in population was observed after 6 h, followed by a reduction of ≥4.56 log CFU/g after 8 h.The populations of ectively . For L. S. enterica, significant population reductions were observed on wood ear mushrooms after 1 h of dehydration at all temperatures; population reductions after 1 h were 0.82, 1.03, and 1.20 log CFU/g at 70, 80, and 90°C, respectively. After 2 and 4 h of dehydration at 90°C, the population of S. enterica was reduced by 4.22 and ≥ 4.68 log CFU/g, respectively. After 4 h of dehydration at 70 and 80°C, the population of S. enterica was reduced by 4.43 and 4.19 log CFU/g, respectively. A reduction of ≥4.68 log CFU/g was observed on wood ear mushrooms after dehydration at both 70 and 80°C after 6 h.For L. monocytogenes and S. enterica on wood ear mushrooms during dehydration . The survival curves for both pathogens on wood ear mushrooms during dehydration at 90°C were convex. According to the Weibull model, a 4 log CFU/g reduction of L. monocytogenes would occur on wood ear mushrooms after 6.96, 3.16, and 1.86 h during dehydration at 70, 80, and 90°C, respectively. Similarly, for S. enterica, 4 log CFU/g reductions would occur after 3.52, 3.44, and 1.88 h during dehydration at 70, 80, and 90°C, respectively.As with enoki mushrooms, the Weibull model was first utilized to describe the inactivation of ydration . The timL. monocytogenes and S. enterica on wood ear mushrooms during dehydration . While 80°C resulted in a greater reduction of both pathogens on enoki mushrooms, residual populations were still observed after 24 h . The tailing effect observed at both 70 and 80°C was most likely attributed to the increased thermal resistance of the pathogens during dehydration at those temperatures. This is not surprising as the lower moisture content of the food matrix would result in increased thermal resistance of both pathogens.Pathogen inactivation during dehydration was dependent on the mushroom type as well as the dehydration time and temperature combination. For enoki mushrooms, this study observed similar pathogen reductions as when portobello mushrooms were dehydrated at 55°C for 8 h . Log redL. monocytogenes and S. enterica on dehydrated wood ear mushrooms resulted in reductions of 2.49 and 2.26 log CFU/g, respectively, whereas no significant decrease was observed on enoki mushrooms under the same conditions  had the highest microbial load (9.4 log CFU/g) out of 22 tested mushroom varieties from retail markets in Spain; wood ear also had the highest Enterobacteriaceae population (6.5 log CFU/g) (Enterobacteriaceae populations on enoki mushrooms (Flammulina velutipes) were 7.1 and 5.9 log CFU/g, respectively. Taken together with the published literature, the results of this study and the specific conditions examined suggest that the use of dehydration alone at temperatures below 90°C would not be an effective preventive control to ensure the safety of all mushrooms.This study determined that for enoki mushrooms, dehydration temperatures below 90°C did not achieve >4 log CFU/g reduction of either og CFU/g . L. monoin Spain  and Chinin Spain  and in min Spain . In addi markets . Wood eag CFU/g) . In the It is noted that the use of higher drying temperatures have been shown to negatively affect the organoleptic properties or the rehydration ratio for some mushrooms; for example, button mushrooms dehydrated at 50°C, compared to 70°C, had better rehydration characteristics and were lighter in color . HoweverL. monocytogenes and S. enterica on enoki and wood ear mushrooms during dehydration. Of the three temperatures evaluated, 90°C was most effective at pathogen reduction on both mushrooms, while temperatures of 70 and 80°C were not as effective for enoki mushrooms. Due to the striking difference in the inactivation of both pathogens between the two types of specialty mushrooms, this study highlights the need for individual mushroom type assessments. Results from this study suggest that dehydration at the examined temperatures would not serve as a preventive control for specialty mushrooms.In conclusion, this study aimed to understand the inactivation kinetics of both The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.JS: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. MF: Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review & editing. BK: Data curation, Investigation, Methodology, Writing – review & editing. NC: Data curation, Investigation, Methodology, Writing – review & editing. GP: Data curation, Investigation, Methodology, Writing – review & editing. DI: Data curation, Investigation, Writing – review & editing.


Cinnamomum osmophloeum Kanehira (C. osmophloeum), a broad-leaved tree species of Taiwan, contains phenolic acids, flavonoids, and phenylpropanoids such as cinnamaldehyde and cinnamic acid in leaves. Many reports have shown that the cinnamon leaf extract possesses anti-inflammatory, hypoglycemic, hypolipidemic and neuroprotective functions. This study aims to analyze bioactive compounds in C. osmophloeum (cinnamon leaves) by UPLC-MS/MS and prepare hydrosol, cinnamon leaf extract and cinnamon leaf nanoemulsion for comparison in improving Parkinson’s disease (PD) in rats.After extraction and determination of total phenolic and total flavonoid contents, cinnamaldehyde and the other bioactive compounds were analyzed in cinnamon leaves and hydrosol by UPLC-MS/MS. Cinnamon leaf nanoemulsion was prepared by mixing a suitable proportion of cinnamon leaf extract, soybean oil, lecithin, Tween 80 and deionized water, followed by characterization of particle size and polydispersity index by dynamic light scattering analyzer, particle size and shape by transmission electron microscope, encapsulation efficiency, as well as storage and heating stability. Fifty-six male Sprague-Dawley rats aged 8 weeks were divided into seven groups with group 1 as control (sunflower oil) and group 2 as induction , while the other groups including rotenone injection (2 mg/kg bw) followed by high-dose of 60 mg/kg bw (group 3) or low-dose of 20 mg/kg bw (group 4) for tube feeding of cinnamon leaf extract or cinnamon leaf nanoemulsion at the same doses (groups 5 and 6) every day for 5 weeks as well as group 7 with rotenone plus hydrosol containing 0.5 g cinnamon leaf powder at a dose of 10 mL/kg bw. Biochemical analysis of brain tissue (striatum and midbrain) was done to determine dopamine, α-synuclein, tyrosine hydroxylase, superoxide dismutase, catalase, glutathione peroxidase and malondialdehyde contents by using commercial kits, while catalepsy performed by bar test.An extraction solvent of 80% ethanol was found to be the most optimal with a high yield of 15 bioactive compounds being obtained following UPLC analysis. A triple quadrupole tandem mass spectrometer with electrospray ionization mode was used for identification and quantitation, with cinnamaldehyde present at the highest amount (17985.2 µg/g). The cinnamon leaf nanoemulsion was successfully prepared with the mean particle size, zeta potential, polydispersity index and encapsulation efficiency being 30.1 nm, -43.1 mV, 0.149 and 91.6%, respectively. A high stability of cinnamon leaf nanoemulsion was shown over a 90-day storage period at 4 and heating at 100 for 2 h. Animal experiments revealed that the treatments of cinnamon leaf extract, nanoemulsion and hydrosol increased the dopamine contents from 17.08% to 49.39% and tyrosine hydroxylase levels from 17.07% to 25.59%, while reduced the α-synuclein levels from 17.56% to 15.95% in the striatum of rats. Additionally, in the midbrain of rats, an elevation of activities of superoxide dismutase (6.69-16.82%), catalase (8.56-16.94%), and glutathione peroxidase (2.09-16.94%) was shown, while the malondialdehyde content declined by 15.47-22.47%. Comparatively, the high-dose nanoemulsion exerted the most pronounced effect in improving PD in rats and may be a promising candidate for the development of health food or botanic drug. Studies have shown that the cinnamon leaves contained a variety of bioactive compounds such as cis-cinnamaldehyde, trans-cinnamaldehyde, isobornyl acetate, eugenol, cinnamyl acetate, phenolic acids and flavonoids , a neurodegenerative disease, is becoming more common worldwide due to a rise in aging population. The World Health Organization statistics show that about 18 million people are expected to suffer from PD in 2030 and the number continues to rise by multiple growths every year, thereby creating a huge burden for health care in many countries . Accordiin vivo reacting with lipids in the brain cell membrane to generate lipid hydroperoxides and degradation products, leading to cell membrane damage and neuron death  were prepared to compare their efficiency in improving PD in rats.2.2.1.2.1.1.Fu-Tai Co.  provided the cinnamon leaves for this study which was harvested in November from Pinlin district and Taitung County, Taiwan. A total of 1 kg cinnamon leaves was transported to our lab, cleaned, oven-dried at 60°C for 2 h and ground into powder (550 g) for subsequent experiments. The moisture contents in fresh and oven-dried leaves were 45.1 and 5.8%, respectively.2.1.2.The TPC in cinnamon leaves was determined by Folin–Ciocalteu’s phenol method as reported by Abeysekera et al.  and Kao 2.1.3.The TFC in cinnamon leaves was determined based on two methods reported by Abeysekera et al.  and Kao 2.1.4.in vivo. Furthermore, the cinnamaldehyde content remains the same in both nanoemulsion and extract for the animal study for comparison of the efficiency in improving PD.Cinnamon leaf nanoemulsion is mainly composed of cinnamon leaf extract, Tween 80, soybean oil, lecithin and deionized water. Of the various bioactive compounds, cinnamaldehyde is the dominant one in both cinnamon leaf extract and cinnamon leaf nanoemulsion. However, the prepared nanoemulsion was used to encapsulate cinnamaldehyde and the other bioactive compounds in the extract for elevation of their stability and bioavailability A method reported by Waty and Suryanto  was modi2.1.5.Using a Luna Omega C18 100 Å LC column  with 0.025% acetic acid in water (A) and 0.025% acetic acid in methanol (B) as mobile phase, flow rate at 0.3 mL/min, column temperature at 30°C, the elution was carried out in a gradient mode with 83% A and 17% B initially, raised to 20% B in one min, 40% B in 5 min, 55% B in 10 min, 99% B in 14 min, and finally returned to the initial ratio. A total of 15 components were separated within 14 min, including 5-O-caffeoylquinic acid, caffeic acid, p-coumaric acid, coumarin, benzoic acid, rutin, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin, cinnamyl alcohol, cinnamaldehyde, kaempferol, kaempferol-3-β-D-glucopyranoside, trans-cinnamic acid and eugenol. But the peaks of quercetin-3-O-galactoside and quercetin-3-O-glucoside overlapped.The MS/MS detection by multiple reaction monitoring (MRM) mode was done in both ESI positive and negative ions as reported by Huang and Chen . Identif2.1.6.y = 95.239x–1811.4 for 5-O-caffeoylquinic acid, y = 575.78x + 663.16 for caffeic acid, y = 172.24x + 500.63 for p-coumaric acid, y = 868.53x–12,024 for coumarin, y = 96x + 1360.7 for benzoic acid, y = 349.3x + 593.46 for rutin, y = 447.31x–936.38 for quercetin 3-O-galactoside, y = 447.31x–936.38 for quercetin 3-O-glucoside, y = 21.489x–242.09 for quercetin, y = 135.37x + 3793.5 for cinnamyl alcohol, y = 120.98x–576.95 for cinnamaldehyde, y = 1413.6x + 2789.1 for kaempferol, y = 447.87x–2,624 for kaempferol-3-β-D-glucopyranoside, y = 200.98x–137.52 for trans-cinnamic acid and y = 9.5249x + 61.521 for eugenol.All the standards including 5-O-caffeoylquinic acid, caffeic acid, p-coumaric acid, coumarin, benzoic acid, rutin, quercetin 3-O-galactoside, quercetin 3-O-glucoside, quercetin, cinnamyl alcohol, cinnamaldehyde, kaempferol, kaempferol-3-β-D-glucopyranoside, trans-cinnamic acid and eugenol were each dissolved in 80% ethanol and 8 concentrations including 500, 400, 300, 200, 100, 50, 20, and 10 ng/mL for each standard were prepared. After UPLC-MS/MS analysis, the calibration curves were obtained by plotting concentration against peak area and the linear regression equations used for calculation of the content of each bioactive compound were 2.2.The cinnamon leaf extract containing 5,000 ppm cinnamaldehyde extract  was prepared by mixing 100 g of dried cinnamon leaves with 500 mL of 80% ethanol and stirring for 1 h, after which a portion (20 mL) was collected for preparation of cinnamon leaf nanoemulsion. To prepare 10 mL of cinnamon leaf nanoemulsion containing 10,000 ppm of cinnamaldehyde, 0.1 g (1%) of soybean oil, 0.2 g (2%) of lecithin and 0.6 g (6%) of Tween 80 were mixed and dissolved in 99% ethanol, followed by mixing thoroughly in a round-bottomed flask, adding 20 mL of the cinnamon leaf extract, mixing thoroughly, evaporating to dryness under vacuum to form a thin film, adding 9.1 g of deionized water (91%), and ultrasonicating for 30 min. Hydrosol (30 L) was the aqueous portion obtained through separation from essential oil by mixing 8 kg of fresh cinnamon leaves with 50 L of pure water in a distiller for steam distillation at 100°C for 2 h. Due to the presence of a large proportion of water (97%) and lower amount of cinnamaldehyde and the other bioactive compounds in hydrosol, 0.5 g of cinnamon leaf powder was dissolved in 10 mL of hydrosol to elevate the content of cinnamaldehyde and the other compounds. Specifically, the cinnamaldehyde content in hydrosol was increased to 17 mg/kg to attain a dose of 10 mL/kg bw for tube feeding in rats.2.3.2.3.1.2PO4 buffer (pH 5.5), filtering through a 0.22 μm membrane filter and pouring into a polystyrene colorimetric tube for analysis. A portion (100 μL) of this nanoemulsion was diluted 200 times with deionized water for determination of zeta potential at 25°C. In addition, by using a transmission electron microscope (TEM), both the particle size and shape of the nanoemulsion was determined by diluting 50 times with deionized water and 20 μL was collected and dropped on a carbon-coated copper grid. After 90 s, the excessive sample was removed with a filter paper for negative staining with 20 μL of phosphotungstic acid (2%) for 30 s, followed by removing excessive stain with a filter paper and drying overnight in an oven.The prepared nanoemulsion was characterized for both particle size and polydispersity index (PDI) in a dynamic light scattering (DLS) analyzer by diluting 50 times with 25 mM of KH2.3.2.Initially, free cinnamaldehyde was extracted from the nanoemulsion by mixing 400 μL of n-hexane and 100 μL of nanoemulsion for dissolving cinnamaldehyde into the n-hexane phase (upper layer). In addition, 100 μL of nanoemulsion was mixed with 400 μL of ethanol and the mixture was ultrasonicated for 2 h to release total cinnamaldehyde for analysis by HPLC with UV detection at 280 nm. The encapsulation efficiency of cinnamaldehyde was then calculated using the formula shown below:Encapsulation efficiency of cinnamaldehyde.2.3.3.The storage stability was determined by pouring a 1 mL nanoemulsion into an eppendorf tube in triplicate and storing for a total of 18 tubes at 4°C for 3 mo for subsequent analysis of mean particle size, polydispersity index and zeta potential every week, while the heating stability was conducted by collecting a 0.2 mL nanoemulsion in an eppendorf tube and heating in a water bath separately at 40, 70, and 100°C for 0.5, 1, 1.5, and 2 h in triplicate for a total of 36 tubes, and analyzing the above 3 parameters for each heating time length.2.4.Fifty-six male Sprague–Dawley rats, aged 6 weeks, was obtained from Taiwan BioLASCO Co , and these rats were individually housed in ventilated cages at Fu Jen University Animal Center maintained at a temperature of 21 ± 2°C and relative humidity of 55 ± 10% in a 12 h dark/light cycle. The ethical approval to conduct this animal study was previously obtained from the Fu Jen University Animal Care and Use Committee (permission no. A11053) and all the procedures adhered to approved guidelines. Throughout the study, the rats were provided with a standard laboratory rodent diet  and uninterrupted access to water. Weekly measurements of body weight and water intake was recorded over a period of 5 weeks. After a 2 week acclimatization period, the rats reached 8 weeks of age and were ready for conducting experiments.2.4.1.The rats were then randomly divided into seven different groups with 8 rats each: Group 1 (control group): subcutaneous (SC) injection with sunflower oil at a dose of 1 mL/kg body weight (bw) every day followed by tube feeding 30 min later with normal saline at a dose of 10 mL/kg bw as a vehicle for a period of 5 weeks; Group 2 (rotenone-induced group): SC injection with rotenone at a dose of 2 mg/kg bw (dissolved in sunflower oil at 2 mg/mL) every day followed by tube feeding 30 min later with normal saline (10 mL/kg bw) for 5 weeks successively; Group 3: SC injection with rotenone (2 mg/kg bw) every day followed by tube feeding 30 min later with HDCE (high-dose cinnamon leaf extract) at 60 mg/kg bw for 5 weeks successively; Group 4: SC injection with rotenone (2 mg/kg bw) every day followed by tube feeding 30 min later with LDCE (low-dose cinnamon leaf extract) at 20 mg/kg bw for 5 weeks; Group 5: SC injection with rotenone (2 mg/kg bw) every day followed by tube feeding 30 min later with HDCN (high-dose cinnamon leaf nanoemulsion) at 60 mg/kg bw for 5 weeks; Group 6: SC injection with rotenone (2 mg/kg bw) every day followed by tube feeding 30 min later with LDCN (low-dose cinnamon leaf nanoemulsion) at 20 mg/kg bw for 5 weeks; Group 7: SC injection with rotenone (2 mg/kg bw) every day followed by tube feeding 30 min later with cinnamon leaf powder dissolved in hydrosol (0.5 g/10 mL) at a dose of 10 mL/kg bw for 5 weeks.2.4.2.Catalepsy test was assessed using the bar test, in which the rats were positioned in a half-rearing stance with their front paws resting on a horizontal bar located 9 cm above and parallel to the bottom surface. The rats were then carefully observed to measure the duration until one paw was lifted from the bar. After the maximum cut-off time of 180 s for observation, the rats were subjected to carbon dioxide asphyxiation before sacrifice and then both striatum and midbrain were collected separately and weighed, followed by adding homogenate for extraction with a homogenizer. The extracts were then stored at −80°C for subsequent analysis.2.4.3.Dopamine content was analyzed with a commercial kit . In brief, the antibody-coated 96-well plate was washed with wash buffer twice, followed by adding 50 μL of samples or standards of each concentration , 50 μL of antibody (biotin-labeled antibody working solution), mixing and reacting at 37°C for 45 min, washing with wash buffer solution 3 times, adding 100 μL of horseradish peroxidase polymer (HRP-streptavidin conjugate), mixing at 37°C for 30 min, washing again with wash buffer solution 5 times, adding 90 μL of TMB  substrate, reacting at 37°C for 15 min in the dark, adding 50 μL of stop solution, and the absorbance was measured at 450 nm immediately after mixing. Then the dopamine content of each sample was calculated based on the standard curve.The 𝛼-synuclein content was analyzed with a commercially available kit . Briefly, the antibody-coated 96-well plate was washed with wash buffer solution twice, and then 100 μL of samples or standards of each concentration  were added and reacted at 37°C for 90 min. Each solution was washed twice with wash buffer solution and mixed with 100 μL of antibody (biotin-labeled antibody working solution) for reaction at 37°C for 60 min, followed by washing 3 times with wash buffer solution, adding 100 μL of horseradish peroxidase polymer (HRP-streptavidin conjugate), reacting at 37°C for 30 min, washing 5 times with wash buffer solution, adding 90 μL of TMB as substrate, reacting at 37°C for 15 min in the dark, adding 50 μL of the stop reagent, and measuring the absorbance at 450 nm immediately after mixing. The content of 𝛼-synuclein in each sample was then calculated based on the standard curve.Tyrosine hydroxylase (TH) content was also analyzed with a commercial kit . Briefly, the antibody-coated 96-well plate was washed twice with wash buffer solution, and 100 μL of samples or standards of each concentration  were added for reaction at 37°C for 90 min, followed by washing twice with wash buffer solution twice, adding 100 μL of antibody (biotin-labeled antibody working solution), reacting at 37°C for 60 min, washing 3 times with wash buffer solution, adding 100 μL of horseradish peroxidase polymer (HRP-streptavidin conjugate), reacting at 37°C for 30 min, washing 5 times with wash buffer solution, adding 90 μL of TMB as substrate, reacting at 37°C for 15 min in the dark, adding 50 μL of the stop reagent, and measuring the absorbance at 450 nm immediately after mixing. Then, the TH content in each sample was calculated based on the standard curve.Superoxide dismutase (SOD) activity was assayed with a commercial kit . One unit of SOD is defined as the amount of enzyme required for 50% superoxide radical dismutation. In brief, the bovine erythrocyte SOD (Cu/Zn) standard including 0.005, 0.010, 0.020, 0.030, 0.040, and 0.050 U/mL were prepared separately. Then a 10 μL of sample or each concentration from standard was collected and added to a 96-well plate, and then 200 μL of the radical detector was added. After mixing thoroughly, 20 μL of xanthine oxidase was added for the initial reaction to proceed, followed by shaking at room temperature for 30 min, and the absorbance at 450 nm was measured. Then the SOD activity of each sample was measured based on the standard curve.Catalase (CAT) activity was assayed with a commercial kit . Initially, a total of 6 concentrations of the formaldehyde standard including 5, 15, 30, 45, 60, and 75 μM were prepared separately. Then a 20 μL sample or standard was added to a 96-well plate, followed by adding 100 μL of buffer and 30 μL of methanol, mixing well and then adding 20 μL of hydrogen peroxide to start the reaction, shaking at room temperature for 20 min, adding 30 μL of potassium hydroxide to stop the reaction, adding 30 μL of catalase purpald, shaking for 10 min at room temperature, and finally adding 10 μL of potassium periodate. After 5 min, the absorbance at 540 nm was measured. Then the CAT activity of each sample was calculated based on the standard curve.Glutathione peroxidase (GSH-Px) activity was assayed with a commercially available kit . In brief, a 20 μL sample or GSH-Px standard was added to a 96-well plate, followed by adding 50 μL of buffer, 50 μL of co-substrate mixture and 50 μL of NADPH, mixing well and then adding 20 μL of cumene hydroperoxide to start the reaction. After shaking at room temperature for a few seconds, the absorbance at 340 nm was measured once every minute for 5 times for each concentration. Then the values at two time points were used to calculate the GSH-Px activity of each sample.The malondialdehyde (MDA) content was analyzed with a commercial kit . Briefly, a total of 7 MDA standard concentrations including 0.625, 1.25, 2.5, 5, 10, 25, and 50 μM were prepared separately. Then a 100 μL sample or standard was collected and mixed with 100 μL of the trichloroacetic acid reagent and 800 μL of the coloring reagent, after which the mixture was boiled in a water bath (100°C) for 1 h, followed by cooling on ice for 10 min and centrifuging at 1600 g at 4°C for 10 min. The supernatant (200 μL) was collected and added to a 96-well plate, and the absorbance at 535 nm was measured. Then the MDA content in each sample was calculated based on the standard curve.2.5.p < 0.05).Statistical Analysis System (SAS) with a statistical software package  was used3.3.1.Cinnamomum cassia bark was reported to be higher with 80% ethanol than that with 50% or 100% ethanol  was shown for cinnamaldehyde.As indicated above, cinnamaldehyde is the most abundant bioactive compound in cinnamon leaves. But due to poor aqueous solubility of cinnamaldehyde, its bioactivity In several previous studies Mukerjee et al.  prepared3.4.p < 0.05) by 56.33 ng/g (58.64%), while for the HDCE, LDCE, HDCN, LDCN, and HP groups, the dopamine contents were raised by 26.78 ng/g (40.27%), 8.18 ng/g (17.08%), 38.77 ng/g (49.39%), 15.74 ng/g (28.38%), and 27.85 ng/g (41.22%). Thus, the HDCN was the most effective in raising dopamine contents in the striatum in the rotenone-induced PD rats.Dopamine was analyzed as PD is a movement disorder disease caused by degeneration of brain cells of the basal ganglia and substantia nigra for inadequate production of dopamine as mentioned above. This symptom occurs when the dopamine level secreted is lower than 80% of normal. Additionally, the striatum in the human midbrain possesses the function of regulating muscle tension and coordinating complex movements, both of which need to rely on dopamine to work normally . Therefop < 0.05) of 56.5, 54.5, and 44.4% was observed for the treatments of two doses (1.0 and 0.1 mmol/kg) before induction and one dose (1.0 mmol/kg) after induction, respectively. This outcome also implied that in addition to cinnamaldehyde, eugenol also plays a critical role in improving PD in rats. In addition to cinnamaldehyde and eugenol, the effect of flavonoids such as quercetin and phenolic acids such as caffeic acid on improving PD syndrome in rats cannot be ignored. For instance, compared to the 6-OHDA induced group, the dopamine content in the striatum of PD rats was raised by 34.56% following intake of quercetin at 30 mg/kg for 14 days  by 12.79%. Similarly, for the HDCE, LDCE, HDCN, LDCN and HP groups, the SOD activities rose by 13.00, 9.36, 16.82, 8.62, and 6.69%, respectively, implying that the high-dose nanoemulsion showed the most prominent effect in enhancing SOD activity.As reactive oxygen species (ROS) have been demonstrated to be a vital factor in causing neurodegenerative disease such as PD, the effect of various treatments on activities of antioxidant enzyme in the midbrain of PD rats needs to be explored. The SOD activity in the midbrain of PD rats as affected by various treatments is shown in Tribulus terrestris extract at 1000 mg/kg for 21 days  at 200 mg/kg for 20 days  extract at 100, 300, and 1,000 mg/kg for 21 days, the catalepsy time was lowered by 54.6, 54.6, and 81.9%, respectively . The nanoemulsion composed of soybean oil, lecithin, Tween 80 and deionized water was successfully prepared with a mean particle size of 30.1 nm, PDI of 0.149, zeta potential of −43.1 mV and encapsulation efficiency of 91.6%. Additionally, a high storage ability (4°C for 3 mo) and heating stability (100°C for 2 h) was observed. Of the various treatments, the high-dose nanoemulsion showed the most pronounced effect in elevating both dopamine and tyrosine hydroxylase contents, as well as, the antioxidant enzyme activities including SOD, CAT and GSH-Px, while decreasing 𝛼-synuclein aggregation and MDA contents. Taken together, the high-dose nanoemulsion showed the most pronounced effect in improving Parkinson’s disease in rats and should possess a great potential to be developed into a health food or botanic drug. Nevertheless, a future study is warranted to evaluate the cinnamon leaf nanoemulsion by a 3D The original contributions presented in the study are included in the article/The animal study was reviewed and approved by Fu Jen University Animal Care and Use Committee.YW: methodology, investigation, formal analysis, software, data curation, validation, and writing – original draft. VW: investigation, formal analysis, data curation, validation, visualization, and resources. BC: conceptualization, methodology, resources, validation, visualization, writing – original draft, writing – review and editing, project administration, supervision, and funding acquisition. All authors contributed to the article and approved the submitted version.The authors declare that this study received funding from Tou-Fu Investment Co, Taipei, Taiwan (Grant no. 7100339). The funder was not involved in the study design, collection, analysis, interpretation of data, the writing of this article, or the decision to submit it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fnut.2023.1229192/full#supplementary-materialThe Supplementary material for this article can be found online at: Click here for additional data file.


Inappropriate variety use in different agroecology is one of the primary issues which significantly affect garlic phenotype, growth, yield, and nutritional quality. For this reason, a trial was done during the 2022 main season at the demonstration site of Bule Hora University, to see the impact of genotypes on phenotype, growth, yield-related attributes, and nutritional quality of garlic. The experiment was composed of four varieties namely Kuriftu, Holleta, Chafe, Tsedey 92, and one local cultivar. The trial was set up in a randomized complete block design replicated four times. The output showed that garlic varieties statistically (p < 0.05) affected all phenotype, growth, yield-related attributes, and nutritional qualities. Using a variety of Holleta and kuriftu minimize days of emergence by 9.82 and 10.33 days after planting and 75% physiological maturity by 23.6 and 25.90 days after planting, respectively, beyond variety Tsedey 92. The highest marketable bulb yield  was observed from Kuriftu and Holleta, respectively. While the lowest (4.39 t/ha) was from Tsedey 92. The highest Ash, energy, and carbohydrate were recorded from Kuriftu and Holleta, while the least from the Local cultivar. However, the highest moisture content, protein, and fat were observed from local cultivars, while the least from Kuriftu, and Holleta. In general, garlic varieties significantly affected all the phenotype, growth, bulb yield-related, and Nutritional quality. Therefore, it can be concluded that using the variety Kuriftu, and Holleta at Bule Hora agroecology is suggested to boost early germination, better performance, marketable bulb yield, and nutritional quality of garlic. Even thAccording to Ref. , there w(Allium sativum L.) genotypes on phenotype, growth, yield, yield related, and nutritional quality at Bule Hora agroecology.Garlic grows and has been cultivated in a broad range of agroecology conditions . While i22.1https://en.db-city.com/Ethiopia--Oromia--West -Zone--Bule-Hora). They are characterized by bimodal rainfall with 1250 mm and 600 mm of highest and lowest average rainfall, respectively [The study was conducted at the demonstration site, east Africa during the growing period of 2022. The site is geographically between a latitude of 5.58333, a Longitude of 38.25, 5° 34′ 60″ north, 38° 15′ 0″ East, and an altitude of 1836 m (6024 ft) with an Oceanic climate (Köppen climate classification: Cfb)  were selected based on their availability and adaptability. The varieties were obtained from Debrziet/Bishoftu Agricultural Research Center, Institute of Ethiopia Agriculture Research (DZARC/EIAR). The description of the varieties is stated in Debrziet/Bishoftu Agricultural Research Center, Institute of Ethiopia Agriculture Research (DZARC/EIAR).2.3After plowing, and well harrowing, the study area was leveled thereby 20 cm beds high were prepared. 1 m between blocks and 0.5 m between plots were used, respectively. 2 square meters of plot area were used. Five rows per plot with the distance between rows and plant was 20 cm × 10 cm were employed. Before planting, the bulb was separated into cloves and upright planting was employed . Uniform2.4After the experimental site was selected, the place to be sampled was taken based on the experimental total area. The zigzag method was used to sample the soil of the experimental site. The sampling was taken from an average depth of garlic root as described by Ref. . One kil2.5−1); cloves number per bulb (count/number); length of clove (cm); marketable  (tha−1), and non-marketable cloves  (tha−1); and total garlic bulb (tha−1) has been measured from three representatively random sampling plants per row, three mid rows of each experimental unit (plot), nine plant per plot and total of 180 plants indicated by the [Phonological data such as 50% emergence (days after planting) and 75% maturity time (days after planting); growth data like the height of the plant (cm), leave number per plant (count), length, and diameter of the leaf (cm); and yield traits such as length (cm), diameter (cm), and weight of dry bulb  grade from Fisher Scientific  [According to 99.9% of Acetonitrile, 95% of ortugal) . A purifBefore nutritional analysis, a bulb of garlic was peeled to get individual cloves, peeled, and cut into small sizes as slices. Nine bulbs were sampled from each plot and were exposed to −80 °C freezing conditions and dried by freezing before analysis. The dried samples from each plot were fined by pestle and mortar. The fined samples were stored and exposed to −20 °C freezing conditions until analysis. American Organization of Analytical Chemists International  procedures were employed to determine Ash, proteins, carbohydrates, and fat, which are standard analysis methods . The mac2.7The data collected from Phonological data such as 50% emergence and 75% maturity time; growth data like the height of plant leave number per plant, length, and diameter of the leaf; and yield traits such as length, diameter, and dry weight of bulb; cloves number per bulb; length of clove; marketable and non-marketable cloves; and total garlic bulb was computed using ANOVA by SAS programming language version 9.2 and LSD was employed to indicate the variation among varieties in mean values with a probability level of 5% .33.1The current study indicated that varieties were statistically (p < 0.01) varied for the investigated phonological traits such as today to 50% emergency and 75% physiological maturity, indicating the existence of genetic variations among the considered varieties .Table 4MThe present study revealed that Kurftu  and Holleta (8.51 DAP) were the earliest varieties, followed by variety Chafe 15 DAP), while Tsedey 92 (18.33 DAP) and local cultivar (18.01 DAP) was the latest variety. Furthermore, the study indicated that there was no significant difference between Kurftu and Holleta varieties. Similar to the present study , reveale DAP, whiGarlic varieties' days of 75% physiological maturity were significantly varied . The pre3.23.2.110.13039/100014337Furthermore, the non-significance (p < 0.05) difference observed in plant height among varieties Holleta, and Kuriftu has been supported by previous experiments [There was a statistical (p > 0.05) difference between varieties in the pseudo height of the garlic plant. The longest plant height was measured from variety Holleta (50.43 cm), and Kuriftu (50.3 cm), while the shortest pseudo stem height was recorded from the local cultivar (40.35 cm), and chafe (42.21 cm). However, there was no significant difference between the variety Holleta, and Kuriftu . Previoueriments . Contraderiments . The con3.2.2The present finding indicated that different garlic varieties significantly (p > 0.05) varied in leaf number. The highest leaf number was counted from the variety Holleta (16.1 leaves per plant) and kuriftu (15.6 leaves per plant) followed by the variety Chafe (8.2 leaves per plant). However, the minimum number of leaves was recorded from the Local cultivar (5.5 leaves per plant), and Tsedey 92 (5.6 leaves per plant) . The varLength and diameter of garlic leaf were statistically (p > 0.05) affected due to varieties. The longest leaf length of 32.31 cm, and 31.64 cm; and the wider leaf diameter of 2.12 cm, and 2.03 cm were recorded from varieties Holleta and Kuriftu, respectively . Whereas3.33.3.1The bulb length and diameter of the garlic plant were statistically (p > 0.05) influenced due to varieties . The lon3.3.2−1), and Holleta (42.74 q ha−1), respectively  affected due to varieties . It has ectively . While tdry bulb . Furtherdry bulb ,18,28. C3.3.3According to 3.3.4−1, and 8.18 t ha−1, respectively followed by variety chafe with the marketable bulb of 5.97 t/ha  varied in the marketable and unmarketable bulbs . The max.97 t/ha . Howevercultivar . The curectively . The stuvely  different variations among varieties in the total bulb . The max8 t ha−1 . Howeverfference . In linent areas . Besides3.4In terms of nutritional quality, the present study reported that different varieties statistically (p < 0.05) varied in the water content, protein, ash, energy, fat, and carbohydrates at Bule Hora agroecology . The valp-hydroxybenzoic acid. On the other hand, similarity in other phenolic compounds has been reported in their experimental set-up for garlic cultivar analysis [The highest protein (7.97 g/100 g f.w) was detected from commonly cultivated, local cultivar, and, while the lowest was harvested from Kuriftu (5.65 g/100 g f.w) followed by Holleta (6.31 g/100 g f.w) as presented under analysis . The varThe highest mean of garlic ash was recorded at Kuriftu (1.99 g/100 g f.w) followed by Holleta (1.75 g/100 g f.w), respectively . While tThe energy content of the garlic bulb was statistically (p > 0.05) influenced due to varieties at Bule Hora agroecology . The higThere was also a statistical (p > 0.05) difference between varieties in the fat content of garlic bulbs . The Loc4The main objective of the current study was to evaluate effect of phenotype, growth, yield-related attributes and nutritional quality of garlic genotypes, and local cultivar at Bule Hora agroecology. The experiment was conducted using completely randomized block design (RCBD) with four replication. Generally, the garlic genotypes and local cultivar tested revealed a significant (p > 0.05) variation in phenotype, growth, yield, and yield-related traits and nutritional quality at Bule Hora agroecology. This implies that different genotypes and local cultivars at Bule Hora condition shall have a paramount impact on phenotype, growth, yield, yield-related traits, and nutritional quality resulting in good quality of the pharmaceutical, and other medical sector input for their end garlic processed product. The presence of variability in the quality of nutrition is paramount for the production and productivity of garlic genotypes as a medicinal plant. Therefore, considerations might be devotedly attributed to select variety during production thereby boosting yield and productivity. Finally, Variety Kuriftu and Holleta can be recommended for production at Bule Hora agro ecology due to their advantageous phenotype, growth, high-yielding, and nutritional quality. Further study is also suggested for updating yield and revealing other nutrients like minerals, vitamins, specific proteins, carbohydrates, and other molecular characteristic of different garlic accession, germplasms, genotypes, and cultivar.Not applicable.Included in the main article.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.