The study aimed to assess the prevalence and associated factors of suicidal behaviour (SB) among adults in Malawi.Data were analysed from 4187 persons aged 18–69 years (median age: 32 years) that took part in the nationally representative cross-sectional “2017 Malawi STEPS survey.”The prevalence of ever suicide attempt was 0.8%, and the prevalence of SB  in the past year was 7.9% (6.0% among men and 9.6% among women). In adjusted logistic regression analysis, among men, being 30 years and older and having an alcohol family problem were positively and obesity was negatively associated with SB in the past year. Moreover, in univariate analysis, early smoking initiation was positively and not using active transportation was negatively associated with SB in the past year. Among women, having an alcohol family problem, death from suicide of a close family member, having a cardiovascular incident  and not using active transportation increased the odds of SB in the past year. In addition, in univariate analysis, low systolic blood pressure (< 100 mmHg), not taking antihypertensive medication, and current alcohol use were associated with past year SB.Almost one in ten participants engaged in SB in the past year and several associated factors were identified that can inform intervention programmes.The online version contains supplementary material available at 10.1186/s13033-021-00483-x. Sociodemographic factors associated with SB may include family history of suicide, younger age, female sex , 8, 10, It has been hypothesised that psychosocial distress, physical factors, health risk behaviours, and sociodemographic factors are associated with SB in Malawi. The study aimed to assess for the first time the national prevalence and associated factors of SB among persons aged 18–69 years in Malawi.Data were analysed from adults that participated in the cross-sectional nationally representative “2017 Malawi STEPS survey” . Based oOutcome variables included lifetime suicide attempt, SB in the past year , methods used for suicide attempts, and care seeking for SB , measured diabetes ; raised total cholesterol (TC) (“fasting TC ≥ 5.0 mmol/L or currently on medication for raised cholesterol”); history of “heart attack or chest pain from heart disease (angina) or a stroke (cerebrovascular accident or incident)”, age of smoking initiation, current smokeless tobacco consumption, current alcohol use, alcohol dependence, past month passive smoking, and based on the “Global Physical Activity Questionnaire” no active transportation  and sedentary behaviour (≥ 7 h/day) [Alcohol dependence was sourced from three items the “Alcohol Use Disorder Identification Test = AUDIT” (items 4–6), e.g., “How often during the last year have you found that you were not able to stop drinking once you had started?” Responses ranged from “0 = never to 4 = daily or almost daily”. Scores were summed, with ≥ 4 scores indicating alcohol dependence [7 h/day) . Alcoholpendence .Statistical analyses were done with “STATA software version 15.0 ,” taking into account the complex study design. Unadjusted and adjusted logistic regression was utilised to estimate predictors of ever suicide attempt and SB  in the past year. Variables found significant in univariate analyses were subsequently added in the multivariable regression model. Covariates were included based on literature review –31. The The sample consisted of 4,187 individuals , 51.7% were female, 58.7% had Standard 5 or more education, 73.0% were married or cohabiting, 43.8% were employed or students, 89.1% lived in rural areas and 89.1% were Chewa speakers. Almost one in ten participants (8.3%) reported alcohol family problems, 5.4% and 2.5% had a close family member who attempted suicide and died from suicide, respectively, and 20.7% were exposed to passive smoking. Almost one in five participants (18.5%) were overweight/obese, 6.4% had a systolic blood pressure of < 100 mmHg, 5.6% were not taking antihypertensive medications, 1.7% had prediabetes and 1.3% had diabetes, 8.2% had raised total cholesterol, 6.5% had a history of angina, heart attack, or stroke, 0.5% initiated smoking early (≤ 14 years), 1.4% were current smokeless tobacco users, 5.0% had alcohol dependence, 90.4% had inadequate intake of fruit and vegetables, 8.0% did not engage in active transportation and 5.6% engaged in sedentary behaviour (see Table P < 0.001)  was 7.9% , and not using active transportation were associated with SB in the past year. Moreover, in univariate analysis, low systolic blood pressure (< 100 mmHg), not taking antihypertensive medication, and current alcohol use were associated with SB in the past year  was similar to national studies among adults in Nigeria 0.7%) , but low% , but lThe study found that psychosocial distress  and female sex were associated with SB in the past year. Major suicide attempt methods were to “hang on a rope”, “overdose of medication” and “poisoning with pesticides”, which are similar to global suicides in low- and middle-income countries . In a reIn line with previous findings , 8, 10, Having a family history of suicide has been a known risk factor for SB , 8, 10, In agreement with previous studies , 44, havStudy limitations include the cross-sectional design of the study, which hinders us to draw causative conclusions. In addition, most of the data were assessed by self-report, which may lead to biased responses. Furthermore, certain variables, such as parental psychopathology and mental disorders of the study participants, were not assessed in Malawi STEPS survey and should be included in future research. The variable on household income was not included, because too many values were missing.Findings of this nationally representative community survey of persons aged 18 to 69 years in Malawi showed that 0.8% had ever attempted suidide and that almost one in ten had engaged in SB in the past year. Several risk factors of SB for men and/or women were identified, including female sex, older age among men, alcohol family problems (both men and women), death from suicide of family members among women, history of cardiovascular disease and no active transportation among women, which may assist in designing programmes to prevent SB in the Malawian population. Furthermore, the low health care utilization for SB in this study in a low-income country versus high receipt for treatment of SB in high-income countries may call for the integration of mental health into existing primary and community health services to increase access to services for people with mental health problems.Additional file 1. Description of study variables.


Streptococcus bovis, but knowledge of its role in response to different pH is still limited. In this study, a ccpA-knockout strain of S. bovis S1 was constructed and then used to examine the effects of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at pH 5.5 or 6.5.Catabolite control protein A (CcpA) regulates the transcription of lactate dehydrogenase and pyruvate formate-lyase in max) and growth lag period (λ), which caused a lowest μmax and a longest λ in ccpA-knockout strain at pH 5.5. Deletion of ccpA decreased the concentration and molar percentage of lactic acid, while increased those of formic acid. Strains at pH 5.5 had decreased concentrations of lactic acid and formic acid compared to pH 6.5. The significant interaction between strain and pH caused the highest production of total organic acids and acetic acid in ccpA-knockout strain at pH 6.5. The activities of α-amylase and lactate dehydrogenase decreased in ccpA-knockout strain compared to the wild-type strain, and increased at pH 5.5 compared to pH 6.5. There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-knockout strain at pH 6.5. The expression of pyruvate formate-lyase and acetate kinase was higher in the ccpA-knockout strain compared to wild-type strain. The lower pH improved the relative expression of pyruvate formate-lyase, while had no effect on the relative expression of acetate kinase. The strain × pH interaction was significant for the relative expression of lactate dehydrogenase and α-amylase, both of which were highest in the wild-type strain at pH 5.5 and lowest in the ccpA-knockout strain at pH 6.5.There was a significant interaction between strain and pH for the maximum specific growth rate (μS. bovis S1, but did not affect the fermentation pattern. CcpA regulated S. bovis S1 growth and organic acid fermentation pattern. Moreover, there seemed to be an interaction effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1.Overall, low pH inhibited the growth of The online version contains supplementary material available at 10.1186/s12866-021-02404-x. Streptococcus bovis is the major cause of increasing lactic acid and declining ruminal pH, which will result in the inability of most ruminal bacteria, and the acid-tolerant Lactobacilli becoming predominant [S. bovis is considered the major etiologic agent of rumen lactic acidosis, and suppressing its overgrowth and overproduction of lactic acid is vital to prevent rumen lactic acidosis when animals are supplied with a high grain diet.Generally, high-concentrate diets based on grains are applied in modern intensive ruminant production to increase production. However, ruminants fed on high-concentrate diets display increased yields of volatile fatty acids (VFA) and lactic acid in the rumen and decreased rumen pH, which may cause subacute ruminal acidosis , 2. In tdominant , 5. TherS. bovis is a gram-positive bacterium that utilizes starch and soluble sugars as substrate in the rumen, thereby producing lactic acid, formic acid, acetic acid and ethanol. Several previous studies have shown that the fermentation products of S. bovis are regulated by the pH, carbon source and substrate concentration [S. bovis S1 demonstrated that the carbohydrate source (soluble starch vs. glucose) had a major effect on lactic acid production due to the transcriptional regulation of metabolic genes [S. bovis S1 and found that the fermentation of the strain was more sensitive to the pH changes [S. bovis is also controlled by the catabolite control protein A (CcpA) [cre) [S. bovis S1, which might be is affected by pH. To test this hypothesis, we constructed the ccpA gene knockout mutant of S. bovis S1 using homologous recombination technology, and used it to investigate the effect of ccpA gene deletion on the growth and fermentation characteristics of S. bovis S1 at different pH.ntration –10. Our  changes . In addiA (CcpA) , which iA) [cre) –15. The A) [cre) –18 and sA) [cre) , but alsA) [cre) –22. BaseS. bovis S1 (CCTCC AB 2016240) used in this study was previously isolated from the rumen fluid of Saanen dairy goats (late lactation) in our laboratory [ccpA-disrupted mutant was constructed by homologous recombination as follows. First, DNA fragments corresponding to the upstream . The recombinant vector pCE was electroporated into S. bovis S1 cells using an electroporation system at 2.5 kV, 200 Ω, and 25 μF. Finally, knockout mutants were selected on MRS plates containing 1 μg/mL erythromycin at 37 °C for 3–4 days.Strain boratory . Its ccpS. bovis S1 wild-type strain and its mutant strain were extracted with the TIANamp Bacteria DNA Kit  and used as the templates. Primers used for verification are shown in Table ccpA gene (ccpA1-F/ccpA1-R) or across the upstream and downstream sequences of the ccpA gene (ccpA2-F/ccpA2-R). PCR analysis was performed using the 2 × Taq PCR MasterMix II Kit  according to the manufacturer’s instructions. The amplified PCR products were electrophoresed on a 1% (wt/vol) agarose gel. Sequence analysis for wt2 and ko2, which were the products of primer ccpA2 amplified from S. bovis S1 wild-type strain and the ccpA-knockout strain respectively, was performed by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. for further verification.The result of knockout was validated by PCR and DNA sequencing. Briefly, genomic DNA of S. bovis S1 wild-type strain and its ccpA-disrupted mutant respectively, were cultured in the media at pH 5.5 or pH 6.5. Specific operations are as follows. First, the seed cultures of S. bovis S1 wild-type strain and its mutant strain were cultured in a modified MRS medium [2PO4, 0.45 g/L K2HPO4, 0.9 g/L NaCl, 0.9 g/L (NH4)2SO4, 0.12 g/L CaCl2·2H2O, 0.19 g/L MgSO4·7H2O, 1.0 g/L tryptone, 1.0 g/L yeast extract, 0.6 g/L cysteine hydrochloride, and 3.0 g/L soluble starch . The original pH of the medium was 4.2, which was adjusted to 5.5 and 6.5 using 10% NaOH (w/v) before sterilization. The cultivation was performed in an anaerobic thermostat shaker at 37 °C and 160 rpm, and the pH of the medium was monitored using a pH meter  and adjusted to 5.5 or 6.5 by continuous titration with 10% NaOH (w/v) using an injection pump . Three replicates were set for each treatment.The experiment treatments were arranged as a 2 × 2 factorial design: two strains, which were S medium  in an anS medium , and it Cell growth were monitored by measuring OD values at 600 nm every hour using SpectraMax M5 plate reader . The cultures of each sample were harvested in duplicate when they reached the exponential growth phase. A portion of the samples was quickly placed in liquid nitrogen and stored at − 80 °C for enzyme activity assay, while the remaining samples were centrifuged at 13,400×g for 2 min at 4 °C. The obtained cell pellets were quickly frozen in liquid nitrogen for 15 min, then stored at − 80 °C for further RNA isolation; the supernatant was filtered using a 0.22 μm filter membrane and stored at − 80 °C for the determination of organic acids.2SO4 with the flow rate set at 0.5 mL/min. Organic acids were then measured with a UV detector set at 210 nm. Commercial assay kits  were used to measure the activities of α-amylase (Cat. No. DFMA-1-Y), lactate dehydrogenase (Cat. No. LDH-1-Y), and acetate kinase (Cat. No. ACK-1-Y). Before the measurement, 2 mL of bacterial fluid was centrifuged at 8000×g for 10 min at 4 °C and the cell pellets were resuspended in the extracting solution from the kit. To lyse the bacterial cells, the suspension was then sonicated on an ice bath using a VCX-130 Sonicator  for 10 min with 30 s pulse on and 30 s pulse off, at 100 W. Unbroken cells were removed by centrifugation at 8000×g for 10 min at 4 °C, and the supernatant was collected for the determination of enzyme activities according to the corresponding kit instructions.A high performance liquid chromatographer  equipped with an acclaim OA column (Sepax Carbomix H-NP) and a UV detector was used to determine concentrations of organic acids  in the supernatant. The column temperature was kept at 55 °C, and the mobile phase was 2.5 mM HS. bovis S1 wild-type and mutant strains using the RNAprep Pure Bacteria Kit  according to the manufacturer’s instructions. The quantity and quality of total RNA were evaluated using Agilent 2100 Bioanalyzer  and agarose gel electrophoresis, respectively. Next, total RNA was reverse-transcribed to cDNA in a 20 μL reaction mixture using the Primer-Script™ Reagent Kit  according to the manufacturer’s instructions. The reverse transcriptase quantitative PCR (RT-qPCR) was performed with the ABI Step-One-Plus RT-PCR system  using the TB Green Premix Ex Taq™ II Kit . The reaction was first performed in a 20 μL reaction solution containing 10 μL 2 × TB Green Premix Ex Taq II, 1.6 μL primer, 1 μL cDNA, 0.4 μL 50 × ROX, and 7.0 μL RNase-free water. The RT-qPCR conditions were as follows: 95 °C for 30 s, followed by 40 cycles of the amplification at 95 °C for 5 s and 60 °C for 34 s. Primers of α-amy (gene encoding α-amylase), ldh (gene encoding lactate dehydrogenase), pfl (gene encoding pyruvate formate lyase), and ack (gene encoding acetate kinase) were designed using Primer-Blast and the 16S rRNA was used as an internal reference gene  at room temperature for 10 min to break the cell walls. Total RNA was extracted from ccpA-knockout strain) and pH (6.5 or 5.5), as well as their interaction. Graphs were drawn using GraphPad Prism 8. The logistic function model [t/y0) = D/(1 + exp.((4 × μmax/D)(λ-t) + 2))”, where yt is OD value at time t, y0 is initial OD value, t is hour of the incubation, μmax is the maximum specific growth rate, D is the limit of ln (yt/y0), and λ is the growth lag period (h).Results were expressed as means and SEMs. All data were analyzed as a 2 × 2 factorial design by ANOVA using GLM procedures of SPSS  software. The statistical model included the effects of strain  and maximum specific growth rate (μmax) (P < 0.05). Compared with the wild-type strain at pH 6.5, the μmax of other groups were reduced (P < 0.05), and the ccpA-deficient strain at pH 5.5 had the lowest μmax (P < 0.05). The λ values of ccpA-deficient strain at both pH were increased compared with those of the wild-type strain, and the ccpA-deficient strain at pH 5.5 had the highest λ (P < 0.01).Nonlinear fitting of the growth curves of S. bovis S1 wild-type strain and ccpA deletion strain at different pH. Results indicated that both strains produced lactic acid, formic acid, and acetic acid at different pH. Among them, lactic acid made up the largest proportion of organic acids followed by formic acid, with acetic acid being the least abundant. The interaction between strain and pH affected the concentration of total organic acids (P < 0.05) and acetic acid (P < 0.01), and the acetic acid molar percentage to total organic acids (P < 0.05). Compared with the wild-type strain at pH 6.5, the total organic acids concentrations of ccpA-deficient strain at pH 6.5 were increased, but those of both strains at pH 5.5 were decreased. The acetic acid concentration and its molar percentage to total organic acids of ccpA-deficient strain were higher than those of wild-type strain at both pH 6.5 and pH 5.5, with the highest at pH 6.5. The significant strain × pH interaction was not observed for lactic acid and formic acid concentration, and their molar percentages to total organic acids. Deletion of ccpA gene decreased the lactic acid concentration (37.38 vs 40.15 mM) and its molar percentage to total organic acids (73.83% vs 81.66%), while increased the formic acid concentration (8.07 vs 5.46 mM) and molar percentage (15.93% vs 11.10%) compared to the wild-type strain (P < 0.01). Strains at pH 5.5 had decreased concentrations of lactic acid  and formic acid , while an increased lactic acid molar percentage to total organic acids  compared to those at pH 6.5.Table S. bovis S1 wild-type strain and ccpA-deficient strain at different pH. The interaction between strain and pH did not affect the activities of α-amylase and lactate dehydrogenase. Lower activities of α-amylase (32.74 vs 37.07 U/L) and lactate dehydrogenase (18.75 vs 22.06 U/L) were observed for ccpA-deficient strain compared to the wild-type strain (P < 0.01), and higher activities of these two enzymes (38.67 vs 31.14 U/L and 21.65 vs 19.16 U/L) were observed at pH 5.5 than at pH 6.5 (P < 0.01). There was a significant interaction between strain and pH for the activity of acetate kinase, which was the highest in the ccpA-deficient strain at pH 6.5 (P < 0.05).Table α-amy, ldh, pfl, and ack of S. bovis S1 wild-type strain and ccpA-deficient strain at different pH. The strain × pH interaction was significant for the relative expression of ldh (P < 0.05) and α-amy (P < 0.01), both of which were the highest in the wild-type strain at pH 5.5 and the lowest in the ccpA-deficient strain at pH 6.5. The interaction between strain and pH did not affect the relative expression of pfl and ack. The relative expressions of pfl and ack in the ccpA-deficient strain were increased compared to the wild-type strain (P < 0.01). The lower pH improved the relative expression of pfl (P < 0.01), while did not affect the relative expression of ack.Figure S. bovis and Lactobacillus [Ruminal pH generally fluctuates within a physiological range of about 5.5–7.0 in a 24-h period, which is driven by the amount of fermentable carbohydrate in each meal, innate buffering capacity of animals, and the absorption rate of organic acids , 26. HowS. bovis can grow in the pH range of 4.5–6.7 with the highest growth rate at pH 6.4 [S. bovis S1 wild-type strain was greater at pH 6.5 than pH 5.5, indicating that the low pH inhibited the growth of S. bovis S1. This finding is consistent with the characteristic of this strain found by Chen et al. [ccpA deletion does not alter this characteristic. The absence of ccpA, at both pH 6.5 and 5.5, caused an extended lag phase and decreased maximum specific growth rate of S. bovis S1, which was consistent with the results obtained in other lactic acid-producing bacteria, such as Lactobacillus bulgaricus [Lactobacillus casei [ccpA-disrupted mutant of S. bovis 12 U1 have not been previously observed [max in this study, which caused a lowest maximum specific growth rate and a longest lag phase in the ccpA-deficient strain at pH 5.5. The finding suggests a possible synergic effect between low pH and ccpA deletion on suppressing the growth of S. bovis S1, which is probably because that both deletion of ccpA and low pH could inhibit its growth, and the gene expression of ccpA in S. bovis S1 wild-type strain is influenced by the pH [ccpA gene would have a better effect on controlling the overgrowth of S. bovis S1 in rumen when ruminal pH is low, which need to be verified in the further study. We did not observe significant differences in maximum specific growth rate between wild-type strain at pH 5.5 and ccpA-disrupted mutant at pH 6.5, indicating that ccpA deletion inhibited the growth of S. bovis S1 only if the strain grown at the same pH condition.n et al. , and ccplgaricus  and Lactus casei . Howeverobserved . Besidesy the pH . The resS. bovis is regulated by extracellular pH and growth rate [S. bovis JB1 was grown with a relatively fast growth rate, while it changed to formic acid, acetic acid and ethanol fermentation when the strain was grown slower in continuous culture [S. bovis JB1 mainly produced lactic acid even at slow growth rate, which is because the intracellular pH at this condition is similar to the optimal pH of S. bovis LDH [S. bovis S1 in the present study did not change when the extracellular pH dropped from 6.5 to 5.5, and the molar percentage of lactic acid is more than 80% of total organic acids at both pH, which is similar to the outcome previously observed in our lab [S. bovis S1 as extracellular pH dropped. Although the fermentation pattern was not affected by the extracellular pH in the present study, low pH reduced the final concentrations of lactic acid, formic acid and total organic acids of S. bovis S1 wild-type strain. Similarly, this pattern of changes was also found after ccpA knockout. The production of organic acids is associated with the specific activity and amount of enzymes. In Streptococcal, the LDH has a requirement for FDP [S. bovis S1 at pH 5.5 is lower than pH 6.5 [ldh gene expression in both strains were significantly higher at pH 5.5 than pH 6.5.The organic acid fermentation pattern of wth rate , 33. An  culture . As the ovis LDH , 34, 35. our lab . The use for FDP , and then pH 6.5 , which mccpA gene significantly decreased lactic acid production, and increased the production of formic acid and acetic acid of S. bovis S1 growing at both pH 6.5 and pH 5.5. The findings are consistent with the results of Asanuma et al. [ccpA-disrupted mutants. Similar results were found in other lactic acid bacteria. For example, Asanuma et al. [S. bovis 12 U1 increased significantly after ccpA gene deletion, and this change was directly associated with changes in related enzyme activities and gene expression. The transcription of the las operon encoding phosphofructokinase (pfk), pyruvate kinase (pk), and lactate dehydrogenase (ldh) in L. lactis was reduced by 75% in the ccpA deletion mutant strain compared to the wild-type strain, which increased ethanol and acetic acid and decreased lactic acid production [L. bulgaricus were significantly reduced after ccpA gene deletion, which resulted in reduced lactic acid yield [S. mutans, CcpA has been demonstrated to regulate the transcription of ackA (acetate kinase A) and pta (phosphotransacetylase) in response to pH [ccpA-disrupted mutant at pH 6.5. Moreover, a significant interaction between strain and pH was observed for the activity of acetate kinase and gene expressions of ldh and α-amy. The interaction effect between pH and ccpA gene deletion on these indicators is possibly caused by the fact that the expression of ccpA gene is regulated by extracellular pH in S. bovis S1 [Inactivation of the a et al. . Subsequa et al.  found thoduction . Moreoveid yield . In S. mse to pH . In the bovis S1 . HoweverS. bovis S1, and lowers the production of organic acids but does not alter the production pattern despite the increased activities of α-amylase and LDH. The deletion of ccpA gene inhibits the growth of S. bovis S1 and regulates the organic acid fermentation pattern towards lower lactic acid and higher formic acid and acetic acid, suggesting that CcpA is probably involved in the carbon metabolism of S. bovis S1. Moreover, there seems to be an additive effect between pH and ccpA deletion on regulating the growth and organic acids production of S. bovis S1. The mechanism underlying this interaction remains to be clarified.Low pH inhibits the growth of Additional file 1.


Ligusticum sinense Oliv.cv. Chaxiong has been used to treat cardiovascular and cerebrovascular diseases, atherosclerosis, anemia and stroke. A high purity extract from chaxiong  was extracted and separated. Its main components were senkyunolide A , N-butylphthalide , Neocnidilide , Z-ligustilide , and butenyl phthalide , respectively. Little is known about the pharmacokinetics of these phthalides in Chaxiong, and different preparations to improve the physicochemistry and pharmacokinetics of VOC have not been investigated.The dried rhizome of At different predetermined time points after oral administration or intravenous administration, the concentrations of SA, NBP, NOL, ZL and BP in the rat plasma were determined using LC-MS/MS, and the main PK parameters were investigated. VOC-P188 solid dispersion and VOC-β-CD inclusion compound were prepared by melting solvent method and grinding method, respectively. Moreover, the physicochemical properties, dissolution and pharmacokinetics of VOC-P188 solid dispersion and VOC-β-CD inclusion compound in rats were assessed in comparison to VOC.The absorptions of SA, NBP, NOL, ZL and BP in VOC were rapid after oral administration, and the absolute bioavailability was less than 25%. After the two preparations were prepared, dissolution rate was improved at pH 5.8 phosphate buffer solution. Comparing VOC and physical mixture with the solid dispersion and inclusion compound, it was observed differences occurred in the chemical composition, thermal stability, and morphology. Both VOC-P188 solid dispersion and VOC-β-CD inclusion compound had a significantly higher AUC and longer MRT in comparison with VOC.SA, NBP, NOL, ZL and BP in VOC from chaxiong possessed poor absolute oral bioavailability. Both VOC-P188 solid dispersion and VOC-β-CD inclusion compound could be prospective means for improving oral bioavailability of SA, NBP, NOL, ZL and BP in VOC. Ligusticum sinense Oliv.cv. Chaxiong has been used to treat cardiovascular and cerebrovascular diseases, atherosclerosis, anemia and stroke [Stroke, a common neurological disease with a high risk of suddenness, disability or death, is mainly caused by atherosclerosis, vasospasm, cerebral ischemia and hypoxia. It is the second largest cause of death in the world , 27. It d stroke , 41. Chad stroke . Some mod stroke . Some ned stroke , 36.At present, a high purity extract from chaxiong  was extracted and separated in our labs. Its main components were phthalides, and the contents were senkyunolide A SA, 33.81%), N-butylphthalide , Neocnidilide , Z-ligustilide , and butenyl phthalide , respectively . The che%, N-butyZL provided the potent neuroprotective effects against hemorrhagic stroke by inhibiting Prx1/TLR4/NF-kB signaling, the subsequent immune and neuroinflammation lesions . In addiP < 0.05) [Pharmacokinetic studies play an increasingly important role in drug discovery and development processes, a poor pharmacokinetic profile due to lower bioavailability and rapid clearance limits drug efficacy . NBP und < 0.05) , 33. The < 0.05) . Until nPreliminary studies and pharmacokinetic experiments showed that VOC had poor water solubility and instability, and the bioavailability of each active ingredient was no more than 25% in rats, and therefore, VOC was prepared into solid dispersions and inclusion compounds to improve that , 46. SolIn this work, VOC-P188 solid dispersion and VOC-β-CD inclusion compound were prepared by melting solvent method and grinding method, respectively. The physicochemical properties of the formulations were investigated using SEM (scanning electron microscopy), DSC , PXRD (powder X-ray diffraction) and FT-IR (Fourier Transform Infrared Spectrometer). Furthermore, studies of the dissolution in vitro and pharmacokinetics in rats were carried out in comparison with VOC. In view of the irreplaceable role of animals in pharmacokinetic studies, this study used Sprague Dawley rats.SA (purity> 98%), NBP (purity> 98%), ZL (purity> 98%) and dehydrocostus lactone  were purchased from Desite Biotech Co., Ltd., Chengdu, China. NOL (purity> 98%) and BP (purity> 98%) were purchased from Chroma Biotechnology Co., Ltd., Chengdu, China.β-cyclodextrin (β-CD) was purchased from Damao Chemical Reagent Co., Ltd., Tianjin, China. Poloxamer 188 (P188) was purchased from BASF Co., Ltd. Potassium bromide  were purchased from Silaikejingda Laboratory animals Co., Ltd., Hunan, China, the number of animal Quality License: SCXK 2016–0002. The animals were maintained in a room with a constant temperature of 23 ± 1 °C; A relative humidity of 30–40%; light for 12 h from 06:00 to 18:00; and ad libitum food and purified water. The studies were approved by the animal ethics committee of Jiangxi University of Traditional Chinese Medicine (jzllsc0137). All animals were maintained in accordance with the guidelines outlined by the Chinese legislation on the ethical use and care of laboratory animals. All efforts were made to minimize both animal suffering and the number of animals used to produce reliable data.Ligusticum sinense Oliv.cv. Chaxiong were extracted two times with 95% ethanol, each for 1.5 h. After filtration and removal of the solvent under reduced pressure, the residue was partitioned into H2O and extracted with EtOAc. The EtOAc fraction was separated by Pope Molecular Distillation after solvent was recovered, for the light component and heavy component. The light component was VOC  [The crushed air-dried aerial parts of ld, w/w) .Chinese Traditional Patent Medicine [The constituents of VOC were identified by high performance liquid chromatography (HPLC), which had been published in Medicine . The higSolid dispersion of VOC was prepared by using the solvent melting method. Briefly, VOC was dissolved in minimum volume of absolute alcohol, and P188 at drug to carrier ratios  was melted at 60 °C under water bath, then the VOC solution was added to P188 to obtain a homogeneous mixture. The solvent was removed under vacuum in a rotary evaporator at 40 °C and 45 rpm for 2 h, and the resulting dispersion was kept in refrigerator at − 80 °C. After 4 h, the dispersion was crushed and sieved through 40-mesh, and stored in a desiccator at room temperature for use , 33.The preparation of the β-CD-VOC was carried out by co-dissolution. A solution of β-CD in three times purified water (v/w), was added a solution of VOC in minimum volume of absolute alcohol at drug to carrier ratios 1:8, w/w), and the resulting solution was allowed to stir for 1.5 h. The mixture was then filtered, washed by a little water and an appropriate amount of ether for three times to remove the unencapsulated VOC in the outer layer, and dried at 40 °C for 6 h. Finally, the inclusion compound was crushed and sieved through 65-mesh, and stored in a desiccator at room temperature for use [:8, w/w, − 6 cm/s by the everted intestinal sac model. The cumulative absorption of the five components in VOC was duodenal > colon > ileum > jejunum, which indicated that the small intestine was the main absorption site of the drug, so pH 5.8 phosphate buffer solutions was selected as its dissolution medium. Dissolution studies were performed according to the USP XXI method 2 (paddle method) at 37 ± 0.5 °C at 50 rpm. 900 ml of dissolution medium were used as standard volume. Dissolution samples (1 mL) were withdrawn at the specified times of 15, 30, 45, 60, 90, 120 min from Dissolution cups, and 1 ml of dissolution medium was replenished in the same time. Samples were filtered through a 0.45 μm nylon disc filter and were measured absorbance at 230 nm by HPLC [The results of our previous study showed that the apparent permeability coefficient of SA, NBP, NOL, ZL and BP in duodenum, jejunum, ileum and colon of rats were greater than 2.0 × 10 by HPLC .− 3 mbar) for 4 min at a current of 15 mA and 100% turbo speed.Their morphological aspects were assessed using the Quanta250 scanning electron microscope . The samples were put on a brass disc by double-sided adhesive carbon tape. Employing an EMI Teck Ion Sputter (K575K), they were sputter-coated with platinum under vacuum . Sample was placed in alumina crucible and scanned at a 10 °C/min linear rate from − 40 °C to 300 °C in an atmosphere of nitrogen.Their crystallinity was determined utilizing a powder X-ray diffractometer (PXRD) . The analysis was carried out at 25 °C using a Cu Kα1 monochromatic radiation source at a current of 40 mA and a voltage of 40 kV. PXRD patterns were obtained in the range of 4–50° with a 2θ scanning mode, a rate meter with time constant of 0.02 pulse/sec and a scan speed of 5°/min [− 1, and the resolution is 4 cm− 1.The FT-IR spectra of the samples were recorded on a Sprucm two spectrometer  using the (KBr) disc technique. For FT-IR measurements, all the samples were mixed with KBr separately in a clean glass mortar and compressed to obtain a thin tablet. Background spectra were obtained with a KBr tablet for each sample. The wave number range is 4000 ~ 400 cmThe intravenous injection solution was prepared by dissolving 0.15 g of VOC in 25 ml water containing 3 ml 10% Tween-80 as solubilizer. The oral solution was prepared by dissolving 0.35 g of VOC in 25 ml normal saline containing 3 ml 10% Tween-80 as solubilizer. The VOC-P188 solution was prepared by mixing VOC-P188 5 g in 25 ml water with magnetic stirring apparatus. The VOC-β-CD solution was prepared by mixing VOC-β-CD 3.5 g in 25 ml water with magnetic stirring apparatus.Twenty-four male SD rats (220 ~ 250 g) were randomly divided into four groups, 6 in each group. After fasting for 12 h before administration, the rats in group 1 were given VOC by tail vein injection at a dose of 30 mg/Kg, the rats at second group were oral administration of VOC solution, the dosage was 120 mg/Kg based on the mass of VOC. The rats at groups 3 and 4 were given VOC-P188 and VOC-β-CD, respectively. The dosage of each active component was shown in Table Blood samples (0.3 ml) were collected from the eyes at 0.08, 0.17, 0.33, 0.5, 1, 1.5, 2, 4, 6, 8, 12 and 24 h after administration, and they were immediately transferred into a heparinized Eppendorf (EP) tube. The blood samples were then centrifuged at 13,000 r/min for 10 min. The plasma samples were stored at − 80 °C for later analysis , 21.The rats were anaesthetised with an intraperitoneal injection of 10% pentobarbital sodium (4 ml/kg) and subsequently sacrificed by rapid decapitation after experimentation.The thawed plasma sample (0.1 ml) was transferred to another EP tube and 20 μl of DL, 50 μl of 2% formic acid and 500 μl of methanol were added. The mixture was vortexed for 2 min, and centrifuged at 13000 r/min for 10 min. The supernatant was transferred into a clean EP tube, and evaporated to dryness under a stream of nitrogen at 40 °C. The residue was dissolved in 100 μl of methanol. The supernatant was injected into the HPLC system .An HPLC system consisting of a solvent-delivery system LC-30 AD, an autosampler SIL-30 AC, a column oven CTO-30 AC, a solvent degasser DGU-20A3, and a controller CBM-20A from AB Sciex  was used in the study. Separation was conducted using a Shim-pack GIST C18-AQ HP column . The column oven was maintained at 35 °C. The mobile phase was 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B. The linear gradient elution program was set according to preliminary tests: 45–60% B, 0–2 min; 60–65% B, 2–6 min; 65–95% B, 6–7 min; 95–45% B, 7–9 min and 45% B, 9–10 min, with the flow rate kept at 0.3 ml/min. The injection volume was set at 2 μl.The MS analysis was performed on a 4500 QTRAP™ system from Applied Biosystems (AB Sciex) equipped with Turbo V sources and Turbo Ion Spray™ (TIS) interface. Electrospray ionization was performed in positive mode. Ion source: electrospray ion source (ESI source); detection method: multiple reaction monitoring (MRM); ion source temperature: 500 °C; desolvent gas temperature: 550 °C; desolvent gas flow rate: 800 l/h. The detection ion pair, retention time, declustering voltage and collision energy of each index component and internal standard were shown in Table The standard stock solution was prepared by dissolving 34.13 mg of SA, 10.41 mg of NBP, 36.56 mg of NOL, 5.11 mg of ZL and 11.39 mg of BP in 25 ml methanol. The mixed stock solutions were diluted in methanol to prepare a series of standard working solutions. All the stock solutions were kept at 4 °C before use. An IS solution of DL was diluted with methanol to yield a final concentration of 3.38 μg/ml. Calibration samples were prepared by spiking 100 μl standard working solutions into 100 μl blank rat plasma at the following concentrations: 84.33, 168.67, 337.34, 647.68 and 5397.40 ng/ml for SA, 5.12, 10.25, 20.50, 41.00 and 327.92 ng/mL for NBP, 25.16, 50.32, 100.65, 201.30 and 1610.40 ng/ml for NOL, 90.42, 180.84, 361.69, 723.38 and 5787.01 ng/ml for ZL and 5.70, 11.39, 22.78, 45.55 and 364.48 ng/ml for BP. Quality control (QC) samples including 168.67, 337.34 and 647.68 ng/ml for SA, 10.25, 20.50 and 41.00 ng/ml for NBP, 50.32, 100.65 and 201.30 ng/ml for NOL, 180.84, 361.69 and 723.38 ng/ml for ZL and 11.39, 22.78 and 45.55 ng/ml for BP were prepared in a similar way to the calibration standards.An aliquot of 20.0 μl IS  was pipetted into 100 μl of the plasma sample. After that, 50 μl of 2% formic acid and 500 μl of methanol were added. The mixture was vortexed for 2 min, and centrifuged at 13000 r/min for 10 min. The supernatant was transferred into a clean EP tube, and evaporated to dryness under a stream of nitrogen at 40 °C. The residue was dissolved in 100 μl of methanol. The supernatant was injected into the HPLC system. The method was verified according to the following criteria: specificity, linearity, accuracy, precision, extraction recovery, matrix effect and stability. Precision and accuracy were carried out by quantifying QC samples at low, medium and high concentrations in six replicates on a single day (intra-day) and on five successive validation days (inter-day). The extraction recoveries were evaluated by comparing the peak areas obtained from extracted spiked samples  with those from post-extraction blank matrix spiked with the appropriate analytes. The matrix effect was investigated by comparing the peak areas obtained from the extracted matrix spiked with standard solutions with the corresponding standard solutions. The stability of the analytes in rat plasma was investigated by analyzing QC samples under three different storage conditions: 12 h at room temperature, three freeze (− 80 °C) and thaw (room temperature) cycles, and keeping at 4 °C for 24 h. All stability studies utilizing QC samples were determined using a calibration curve of freshly prepared solutions.0 → t value was calculated using the trapezoidal rule. The absolute bioavailability of VOC and the relative bioavailability of VOC-P188 and VOC-β-CD were calculated, the formula for calculating absolute bioavailability (F1) and relative bioavailability (F2):The plasma SA, NBP, NOL, ZL and BP concentrations were evaluated using the equation from the standard curves that were run with each batch of samples. The plasma concentration-time data were analyzed with the Drug and Statistics 2.0 software package to determine the pharmacokinetic parameters. The observed Cmax value was obtained from the observed data and the observed AUCWhere AUC is the area under the temporal curve of blood drug concentration (μg/l*h), D is administration dosage (mg/kg); the subscripts iv and po are injection and oral administration, respectively; the subscript R is reference drug (VOC); the subscript T is solid dispersion or inclusion compound.p < 0.05 were considered statistically significant.The results were expressed as the mean ± SD. The differences in the pharmacokinetic parameters among the four groups were tested using one-way analysis of variance (ANOVA), followed by the Fisher’s least significant difference (LSD) test to analyze differences among multiple groups . Values of Typical chromatograms obtained for VOC samples were shown in Fig. Dissolution curve of SA, NBP, ZOL, ZL and BP in VOC, VOC-P188 and VOC-β-CD in pH 5.8 PBS in vitro were shown in Fig. The SEM images of VOC-P188, VOC-β-CD, carrier and their physical mixture were shown in Fig. β-CD Fig. d mainly The DSC spectra of VOC-P188, VOC-β-CD, carrier and its physical mixture were shown in Fig. − 1 and a strong C = O stretching vibration at 1756 cm− 1. Combining with the VOC molecular structure formula, the absorption peak at 1650 ~ 1800 cm− 1 was the characteristic absorption peak of its functional group. P188 had C-H plane of stretching vibration at 2850 cm− 1, the characteristic absorption peak of the physical mixture of VOC and P188 had weakened at 1750 cm− 1, and the characteristic absorption of solid dispersion of VOC-P188 at 1750 cm− 1 peak almost disappeared completely, and the absorption peak at 900 ~ 1400 cm− 1 was also greatly reduced, indicating that VOC and P188 had formed a solid dispersion, and the drug molecules mainly existed in amorphous form [− 1, but the characteristic absorption peak of VOC-β-CD almost disappeared, indicating that the drug molecule was included in the cavity, inclusion compounds had been formed.The infrared spectrum of VOC-P188, VOC-β-CD, VOC, carrier and their physical mixture were shown in Fig. ous form . Figure The PXRD spectrum of VOC-F188, VOC-β-CD, carrier and their physical mixture were shown in Fig. There were no interference peak close to that of SA, NBP, NOL, ZL and BP in the blank plasma. The standard curves of the analytes all exhibited good linearity within the described ranges for each analyte. Linear ranges, regression equations, lower limit of quantification (LLOQs) and correlation coefficients obtained from typical calibration curves were listed in Table max of SA, NOL and ZL were about 12 min, which meant they absorbed more quickly than NBP and BP [Pharmacokinetic parameters of VOC by oral and intravenous administration were listed in Table P and BP , 33. ComP and BP , 40. SA P and BP . NBP wasP and BP , 6. The P and BP .Table 5p < 0.05). The reason may that VOC was highly dispersed in P188, but VOC was encapsulated in the β-CD molecular cavity and it took some time for VOC to be released from β-CD molecular cavity.Pharmacokinetic parameters of VOC-P188 and VOC-β-CD by oral administration were listed in Table Tmax value of SA, NBP, NOL and ZL in VOC-P188 and VOC-β-CD was prolonged. The formulation of VOC-P188 and VOC-β-CD could slow down the absorption process of the analytes. The Cmax value of the five components in VOC-β-CD was lower, when compared with VOC, which indicated that this formulation made the components release more smoothly in vivo. Both VOC-P188 and VOC-β-CD had a significantly higher AUC in comparison with VOC, and gave between 1.3-fold to 3.6-fold improved oral bioavailability of five compounds in VOC (Table p < 0.01), while the solid dispersion showed higher AUC of SA and NOL than did the inclusion compound .Except for BP, the In addition, the stability test results showed that the content of SA and ZL in VOC-P188 was decreased by 15.8 and 38.9% respectively at 60 °C for 10 days, and the content of SA and ZL in VOC-β-CD was decreased by 9.0 and 20.3% respectively at the same temperature. Compared with VOC, the two preparations reduced the active ingredient content to a lesser extent. Humidity had a great influence on the stability of VOC in the two preparations. The contents of SA, NBP, NOL, ZL and BP decreased in different degrees at 92.5% relative humidity for 10 days. Furthermore, the contents of the five phthalides in the two preparations were decreased by less than 10% under the illumination condition (4500 lx ± 500 lx). In general, the five phthalides in the inclusion compound were more stable than in the solid dispersion. VOC-β-CD had a white appearance and was similar in color to β-CD, which indicated that VOC have been encapsulated in the cavity of β-CD. More importantly, VOC was brownish yellow oil and had extremely pungent smell, while VOC-β-CD had almost no pungent smell for people in our lab. Literatures showed that the inclusion complex not only effectively enhanced the dissolution of drugs in aqueous solutions, but also showed the potential to mask its bitter taste in the oral cavity , 42, 48.In fact, both the bioavailability of SA, NBP, NOL, ZL and BP in the two preparations was still less than 50%, although the relative bioavailability and the stability had been significantly improved. Other new dosage forms such as microcapsule , micelleSA, NBP, NOL, ZL and BP in VOC from chaxiong possessed poor absolute oral bioavailability, which was less than 25%. In this paper, VOC were prepared as VOC-P188 solid dispersions and VOC-β-CD inclusion compound, and the physicochemical property which was done with FT-IR, TEM, DSC, PXRD, indicated that these two formulations were successfully formed. Furthermore, both VOC-P188 solid dispersions and VOC-β-CD inclusion compound significantly increased the dissolution in vitro and improved oral bioavailability of these five phthalides in vivo when compared with VOC. However, the bioavailability of SA, NBP, NOL, ZL and BP in the two preparations was still less than 50%, other new dosage forms could be used to further improve the bioavailability.


Previous studies have indicated that the deposition of abdominal adipose tissue was associated with the abnormalities of cardiometabolic components. The aim of this study was to examine the relationship of visceral adipose tissue (VAT), subcutaneous adipose tissue (SAT) and metabolic status and the different effects between males and females.The 1388 eligible subjects were recruited in a baseline survey of metabolic syndrome in China, from two communities in Hangzhou and Chengdu. Areas of abdominal VAT and SAT were measured by magnetic resonance imaging (MRI). Serum total triglycerides (TG), high-density lipoprotein cholesterol (HDL-C) were measured by an automated biochemical analyzer. Metabolic abnormality (MA) was defined more than one abnormal metabolic components, which was based on the definition of metabolic syndrome (IDF 2005). Multiple logistic regression was used to calculate the odds ratios (ORs) and 95% confidence intervals (95%CI). Predictive value was assessed by area under the curve (AUC), net reclassification improvement (NRI), and integrated discrimination improvement (IDI), respectively.2, and 44.8% of the subjects were male. Both male and female with MA had higher VAT levels compared to subjects with normal metabolism (MN), and male had higher SAT levels than female (P < 0.05). Higher VAT was significantly associated with MA with ORs in the fourth quartile (Q4) of 6.537 (95% CI = 3.394–12.591) for male and 3.364 (95% CI = 1.898–5.962) for female (P for trend < 0.05). In female, VAT could increase the risk of metabolic abnormalities, but SAT could increase the risk of MA in the second and fourth quartiles (Q2 and Q4) only at BMI > 24 kg/m2. In male, VAT improved the predictive value of MA compared to BMI and waist circumference (WC), the AUC was 0.727 (95% CI = 0.687–0.767), the NRI was 0.139 (95% CI = 0.070–0.208) and 0.106 (95% CI = 0.038–0.173), and the IDI was 0.074 (95% CI = 0.053–0.095) and 0.046 (95% CI = 0.026–0.066). Similar results were found in female.Their mean age was 53.8 years (SD: 7.1 years), the mean body mass index (BMI) was 23.7 kg/m2 and at BMI ≥ 24 kg/m2. In female, VAT could increase the risk of metabolic abnormalities but SAT could increase the risk of MA in the second and fourth quartiles (Q2 and Q4) only at BMI > 24 kg/m2. Deposition of abdominal adipose tissue was associated with metabolic abnormalities. VAT improved the predictive power of MA.In male, VAT and SAT could increase the risk of metabolic abnormalities both at BMI < 24 kg/mThe online version contains supplementary material available at 10.1186/s12986-022-00651-x. Obesity, especially central obesity, is a well-established risk factor for a several diseases, such as dyslipidemia, type 2 diabetes (T2DM), cardiovascular diseases (CVD), and all-cause mortality , 2. BodyOne possible reason for these sophisticated associations is the different fat distribution in different ethnic groups , 13. In These subjects were recruited in a baseline investigation of metabolic syndrome investigation in China in 2010. The participants were recruited if they were ≥ 18 years old, detailed information has been described in our previous study . In thisThis study was approved by the institutional Review Board of Zhejiang University, China. All participants provided their written informed consents.Abdominal adipose tissue was measured by magnetic resonance imaging (MRI) using a whole-body imaging system  with TR-500 and TE-200 of SE. MRI scans were performed at the interface of the umbilicus (approximately the lower edge of L4) with the subject in the supine position. The area of subcutaneous and visceral adipose tissue was calculated using sliceOmatic software (version 4.2).With a standard questionnaire, the information including age, sex, smoking , alcohol drinking behaviors , menstrual history  and disease history were collected. Current smoking was defined as smoking at least one cigarette per day and lasting for one year. Previous smoking was considered to have quit smoking for at least one year. They were classified as heavy drinkers, moderate drinkers and never drinkers according to the frequency of alcohol consumption: more than 3 times a week was classified as heavy drinking. A local nurse or investigator asked the subjects if they have any diseases such as hypertension and if they use medication. Anthropometric variables were collected by trained investigators following a standard protocol  and inclThe overnight fasting blood samples were collected for each subject. Total triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C) were measured by biochemical auto-analyzers . Fasting plasma glucose (FPG) was analyzed using the glucose oxidase method with a Beckman Glucose Analyzer . A 2 h oral glucose tolerance test (OGTT-2h) was performed as a routine procedure for the subjects, except for patients with previously diagnosed diabetes.2 [The metabolic abnormality component was defined according to the 2005 International Diabetes Federation (IDF) criteria for metabolic syndrome , includi2  was usedP < 0.05 was considered statistically significant. Two packages of “Predict ABEL” and “pROC” were used to calculate the net reclassification improvement (NRI), integrated discrimination improvement (IDI), area under curve (AUC) and so on. The software IBM SPSS Statistics version 25.0 and R 3.6.3 were used to analyze the data.Continuous variables are presented as mean ± standard deviation (SD) or median and inter-quartile range (IQR). Categorical variables were shown as numbers (%). Student’s t-test or the Wilcoxon rank-sum test was used to compare continuous variables. Chi-square tests were used to compare categorical variables. The subjects were divided into four groups by quartiles of SAT and VAT, with the first quartile (Q1) as the reference group. The ORs and 95%CIs for each quartile using multiple logistic regression, adjusted for age, BMI , smoke, drink, menstrual history . A two-tailed 2 (55.1–127.4 cm2) higher than 60.4 cm2 (43.3–79.6 cm2) for female (P < 0.05), and the median SAT for male was 123.2 cm2 (98.1–149.8 cm2) lower than 178.2 cm2 (139.1–221.6 cm2) for female (P < 0.05). Compared to female, male had higher BMI, WC, WHR, VAT, SBP, DBP, FPG, OGTT-2 h, TG, and higher prevalence of metabolic abnormality; however, female had higher TC and HDL-C .The baseline characteristics by sex are summarized in Table 2 (106.2–159.5 cm2) higher than 110.8 cm2 (80.3–141.3 cm2) for MN group. The median VAT for MA was 110.5 cm2 (75.2–136.8 cm2) higher than 64.5 cm2 (32.1–101.2 cm2) for MN group . Similar results were found in female .When stratified by levels of BMI at 24 kg/mP for trend < 0.05). Compared with the reference group for the first quartile (Q1), the ORs in fourth quartile (Q4) were 6.537 (95% CI = 3.394–12.591) for male and 3.364 (95% CI = 1.898–5.962) for female, respectively. However, there was no association between SAT and MA when BMI was not grouped. Since there are relatively few Q3 and Q4 males with metabolic abnormality when BMI < 24 kg/m2, so we put Q3 and Q4 males together for analysis.Table 2, VAT was found to be significantly associated with MA in both male and female. However, for SAT, different effects were found between males and females. In male, SAT were consistently associated with the risk of MA for both BMI < 24 kg/m2 and BMI ≥ 24 kg/m2 (P for trend < 0.05). In female, SAT could increase the risk of MA only when BMI ≥ 24 kg/m2. Additional File P for trend < 0.05).When stratified by BMI level of 24 kg/mP values < 0.05). VAT could improve the predictive value of MA compared with BMI or WC, with NRIs (95%CI) of 0.139  and 0.106 , respectively; and the IDIs (95%CI) were 0.074  and 0.046 , respectively. But SAT was less predictive of metabolic abnormalities than WC and BMI.Table P values < 0.05). Compared with BMI and WC, VAT improved the predictive value.Similar results were found in female Table , with an2. Compared with BMI and WC, VAT improved the predictive power of MA. Deposition of abdominal adipose tissue was associated with the risk of MA.In this cross-sectional study, we found that higher VAT, but not SAT, was associated with the risk of MA when BMI was used as a covariate. However, after BMI stratification, SAT and VAT in men could increase the risk of MA at all levels of BMI. For women, SAT could increase the risk of MA in the second and fourth quartiles (Q2 and Q4) only at BMI > 24 kg/mIn fact, there are some differences between SAT and VAT in anatomy, cytology, molecular, physiology, clinical and so on . The VATSAT is known to have adverse effects on a variety of metabolic risk factors and may have unique pathogenic properties independent of BMI , 25, 29,Previous studies have shown that baseline and changes in VAT were independent predictors of future dyslipidemia, but BMI and SAT were not associated with future development of atherosclerotic dyslipidemia . This reThere are some advantages in our study. Areas of SAT and VAT were measured using MRI, which is the gold standard method of determining abdominal adipose tissue . The dat2 and at BMI ≥ 24 kg/m2. In female, VAT could increase the risk of metabolic abnormalities but SAT could increase the risk of MA in the second and fourth quartiles (Q2 and Q4) only at BMI > 24 kg/m2. Deposition of abdominal adipose tissue was associated with metabolic abnormalities. VAT improved the predictive power of MA.In male, VAT and SAT could increase the risk of metabolic abnormalities both at BMI < 24 kg/mAdditional file 1. Supplementary Tables.


Helicobacter pylori eradication. Thus, appropriate management including chemoprevention is required. The aim of this study was to evaluate the association between nonsteroidal anti‐inflammatory drugs (NSAIDs) and the incidence of post‐eradication gastric cancer in PPI users.Proton pump inhibitors (PPIs) are a potential cause of gastric carcinogenesis after H. pylori eradication between 2014 and 2019 were analyzed in nine hospital databases. Gastric cancer incidence was a primary outcome, and their association with NSAIDs use and clinical factors was evaluated. Hazard ratios were adjusted by age, sex, smoking, and Charlson Comorbidity Index.A multicenter retrospective cohort study was conducted. Patients who used a PPI (≥30 days) after P = 0.005). No gastric cancer was observed in the cyclooxygenase‐2 inhibitor users (n = 256). NSAID use with high dose and long duration was significantly associated with a lower incidence of gastric cancer.During the mean follow‐up period of 2.38 years, 1.13% (31/2431) of all patients developed gastric cancer. The cumulative incidence of gastric cancer in PPI users was 0.25% at 1 year, 0.51% at 3 years, and 1.09% at 5 years in the NSAID users and 0.89% at 1 year, 2.32% at 3 years, and 3.61% at 5 years in nonusers. NSAIDs were associated with a lower gastric cancer risk  users, with dose and duration response effects. NSAIDs may be effective for chemoprevention against PPI‐related gastric cancer.Nonsteroidal anti‐inflammatory drugs (NSAIDs) were associated with a 60% decrease in the gastric cancer incidence in  Helicobacter pylori is the most important carcinogen for gastric cancer,H. pylori eradication reduces gastric cancer risk by 47%. However, the annual incidence rate of post‐eradication gastric cancer is approximately 1.4%,Gastric cancer is the third leading cause of cancer‐related mortality worldwide.H. pylori post‐eradication gastric cancer (post‐eradication gastric cancer).H. pylori‐eradicated PPI users.Previous reports revealed that proton pump inhibitor (PPI) use was associated with a 2.4‐fold increase in the risk of H. pylori infection,H. pylori‐eradicated PPI users. However, NSAIDs increase gastrointestinal (GI) bleeding risk and potentially cardiovascular risk. The balance between the benefits and risks of NSAID use must be considered.Nonsteroidal anti‐inflammatory drugs (NSAIDs) are potential chemopreventive drugs for gastric cancer. NSAIDs were associated with a 58% decrease in gastric cancer incidence in a country with a high rate of To address these issues, we performed a multicenter retrospective cohort study of patients who used PPIs after eradication and evaluated the associations between NSAID use and both gastric cancer development and adverse events, including upper GI bleeding and cardiovascular diseases. Furthermore, we evaluated the association between gastric cancer and patients' genotype of CYP2C19, which is involved in PPI metabolism. The findings may be useful for identification of optimal PPI users who require additional chemopreventive intervention.We performed a retrospective cohort study using the diagnostic procedure combination databases from nine hospitals between April 2014 and March 2019. The combined database provided records from all inpatients and outpatients of the University of Tokyo Hospital, Shuto General Hospital, Fukui Prefectural Hospital, Nerima Hikarigaoka Hospital, St. Leuk's International Hospital, Toyonaka Municipal Hospital, Ishikawa Prefectural Central Hospital, and Nagasaki Minato Medical Center and from inpatients of Tonan Hospital. The database included diagnosis, comorbidities, and adverse events using the International Classification of Diseases, tenth revision (ICD‐10), and drugs and procedures coded according to the original Japanese code.H. pylori infection between April 2014 and March 2019 using the drug codes .From the database, we extracted the data of patients who used a 7‐day course of clarithromycin‐based or metronidazole‐based triple therapy for es Table . We exclThe primary outcome was the development of gastric cancer, as defined by the ICD‐10 codes  or procedure codes for endoscopic and surgical resection .The secondary outcomes were upper GI bleeding and cardiovascular diseases, including cerebrovascular diseases and ischemic heart diseases. Upper GI bleeding was defined as performing endoscopic hemostasis for GI bleeding, with a procedure code of K654. Cerebrovascular disease was defined by ICD‐10 codes G450–469, H340, I600–639, I64, and I650–699, and ischemic heart disease was defined by ICD‐10 codes I210–I229 and I252.We evaluated the following clinical factors: age, sex, smoking, comorbidities, and mediation use. Age was categorized into two groups: >70 and ≤70 years. The following comorbidities were included based on ICD codes: atrial fibrillation, acquired immunodeficiency syndrome, arterial thrombosis, carotid disease, cerebrovascular disease, chronic heart failure, chronic kidney disease (stage 5 or lower), dementia, diabetes mellitus with or without complications, deep vein thrombosis, hemiplegia, dyslipidemia, ischemic heart diseases, liver disorder (mild/severe), malignancy with or without metastasis, pulmonary embolism, peripheral vascular disease, pulmonary disease, rheumatic disease, transient ischemic attack, peptic ulcer disease, unstable angina disease, and valvular disease. The Charlson Comorbidity Index was calculated using these data.Usage of NSAIDs, including COX2 (cyclooxygenase‐2) inhibitors, aspirin, metformin, and statins with other lipid‐lowering agents (fibrates and others), was assessed. NSAIDs were defined as loxoprofen, sulindac, diclofenac, flurbiprofen, ibuprofen, indomethacin, ketoprofen, oxaprozin, naproxen, mefenamic acid, flufenamate aluminum, acemetacin, proglumetacin maleate, mofezolac, pranoprofen, tiaprofenic acid, zaltoprofen, tiamide hydrochloride, etodolac, meloxicam, nabumetone, zaltoprofen, lornoxicam, and piroxicam, including COX2 inhibitors (celecoxib). Statins included pitavastatin, simvastatin, pravastatin, fluvastatin, atorvastatin, and rosuvastatin. Rosuvastatin, pitavastatin, and atorvastatin were defined as strong statins. Fibrates included fenofibrate, bezafibrate, clinofibrate, and clofibrate. The duration of NSAID use was categorized as short‐term (<30 days) or long‐term (≥30 days). The NSAID dose was categorized as low (one tablet) or high (two tablets or more). The details of the medication codes of the medications are shown in Table Genotyping of the CYP2C19 wild‐type gene and two mutated alleles (mutations CYP2C19m1 in exon 5 and CYP2C19m2 in exon 4) was performed using polymerase chain reaction (PCR) restriction fragment length polymorphism (TaqMan Sample‐to‐SNP kit) for patients who consented to the examination. The patients were categorized into three groups: extensive metabolizers (*1/*1), intermediate metabolizers (*1/*2 and *1/*3), and poor metabolizers .n = 1355) and age (n = 1355) were imputed using the fitted values from general linear models for comorbidities.The primary endpoint, gastric cancer development, was censored at the date of the final visit. The Kaplan–Meier method was used to calculate the cumulative probability of gastric cancer development at 5 years. Univariate and multivariate Cox models were used to estimate hazard ratios (HRs) and 95% confidence intervals (CIs); the multivariate Cox proportional hazards models were adjusted for age and sex. Missing data for sex . We calculated the posterior probabilities of gastric cancer using the Bayesian model in patients who underwent CYP2C19 genotyping.P value <0.05 was considered statistically significant, and HRs with 95% CIs were determined. All statistical analyses were performed using SAS software v. 9.4 .A Patients and the public were neither involved in the study design nor the recruitment.This study received ethics approval from the institutional review boards of the University of Tokyo (ID: 2058‐(2)).H. pylori eradication therapy. After excluding 7802 patients, 2431 H. pylori‐eradiated PPI users were analyzed  were male (missing data n = 1355), and 41.01% used NSAIDs. High‐ and low‐dose and short‐term and long‐term NSAID users made up 2.71, 38.30, 23.36, and 17.65% of the study population, respectively. The percentages of aspirin, statin, strong statin, fibrate, other lipid lowering agent, and metformin users were 17.03, 26.00, 22.75, 2.38, 3.50, and 6.38%, respectively. The baseline characteristics of the cohort are shown in Table A total of 10 233 patients received PPIs were principally used for gastroesophageal reflux related symptoms. In addition, PPI users had histories of 14.64% of gastroesophageal reflux disease, 8.68% of cardiovascular diseases, and 10.94% of previous GI bleeding. NSAIDs were principally used for orthopedic disease related pain. In addition, NSAID users had histories of 10.83% of cardiovascular disease, 2.01% of rheumatic diseases, and 2.51% of orthopedic disease including osteoarthritis, spinal disc herniation, and spinal stenosis.P = 0.006, log‐rank test).During the mean follow‐up period of 2.38 years, 31 patients (1.13%) developed gastric cancer . The cumulative incidence of gastric cancer was 0.24% at 1 year, 0.52% at 3 years, and 1.09% at 5 years in the patients using NSAIDs and PPI, and 0.89% at 1 year, 2.32% at 3 years, and 3.61% at 5 years in the patients using PPI alone Fig. . NSAID uP = 0.044, log‐rank test).The cumulative incidence of gastric cancer was 0% at 1, 3, and 5 years in the patients who used COX2 inhibitors with PPI, and 0.69% at 1 year, 1.72% at 3 years, and 2.81% at 5 years in the patients who used PPI but not COX2 inhibitors Fig. . The useH. pylori‐eradicated PPI users, NSAID use was associated with a lower incidence of gastric cancer compared with non‐use . The use of other drugs including aspirin, statin, fibrate, and metformin was not significantly associated with gastric cancer incidence. Analysis of other factors showed that a higher incidence of gastric cancer was associated with age >70 years , chronic kidney disease stage 5 , and malignancy without metastasis .The results of a Cox model evaluating factors associated with gastric cancer are shown in Table Of the 931 patients using low‐dose NSAIDs, six patients developed gastric cancer (0.64%). None of the 66 high‐dose NSAID users developed gastric cancer. The associations between dose or duration of NSAID use and gastric cancer incidence were assessed by using a Cox model .Figure In order to investigate the effects by PPI metabolism in cancer incidence, CYP2C19 genotype was measured in a total of 199 patients. Of these patients, 78.9% were aged >70 years, 72.4% were men. Genotyping results indicated that 77, 90, and 32 patients were CYP2C19 extensive, intermediate, and poor metabolizers, respectively. A total of five patients had gastric cancer. The risk of gastric cancer (aHR 2.47) in the CYP2C19 non‐extensive metabolizers, that is, the intermediate and poor metabolizers, was elevated compared with that in extensive metabolizers; however, this difference was not statistically significant . NeverthIn this multicenter cohort study, we found that NSAID use was associated with a 60% decrease in the post‐eradication gastric cancer incidence in PPI users, and dose– and duration–response relationships were confirmed. NSAID use was not associated with increased GI bleeding or cerebrovascular events. In addition, COX2 inhibitors showed relatively higher chemopreventive effects, and we identified CYP2C19 poor metabolizers as a potential high‐risk population for post‐eradication gastric cancer.H. pylori eradication therapy has been widely encouraged for prevention of peptic ulcers, as well as subsequent gastric cancers. However, a significant population in H. pylori‐eradicated patients often need the prolonged use of PPIs, due to refractory acid‐reflux symptoms. Given the potential cancer‐promoting effects by PPIs in such patients,H. pylori‐eradicated PPI users. Our current finding that NSAIDs efficiently prevent gastric cancer development in PPI users would be helpful for solving this problem, that is, additional chemopreventive use of NSAIDs may be considered for a subgroup of H. pylori‐eradicated PPI users.H. pylori eradication. In addition, given that past history of gastric cancers and advanced histopathological changes such as gastric atrophy and intestinal metaplasia are known to be a risk factor for post‐eradication gastric cancer, patients with these factors may also be a potential target of chemopreventive approach using NSAIDs.We found that CYP2C19 non‐extensive metabolizers who use PPIs are at high risk of gastric cancer. NSAID use as chemoprevention may be beneficial in these patients following Since NSAIDs are anti‐inflammatory drug, their anti‐inflammatory effects may contribute to a low incidence of gastric cancer. In addition, several in vitro and in vivo studies suggested that COX2 may play a critical role in proliferation and tumor growth in gastric cancers.H. pylori eradication therapy and used PPIs, which theoretically lower the incidence of upper GI bleeding. In our study, the rate of comorbidities associated with a cardiovascular risk, such as carotid, cerebrovascular, peripheral vascular, and ischemic heart diseases, was also fewer (5%) than in previous studies (more than 10%).H. pylori‐eradicated PPI users.Contrary to previous reports,H. pylori eradication in PPI users. Second, this was a large multicenter cohort study. Nevertheless, our study has several limitations. First, this study was retrospective. Second, our diagnostic procedure combination database lacked data on successful eradication of H. pylori. However, the success rate of H. pylori eradication was expected to be greater than 90%, based on the primary use of vonoprazan‐based drugs in Japan.Our study has several strengths. First, to our best knowledge, this was the first study to evaluate the association between NSAID use and the incidence of gastric cancer after H. pylori eradication, without adverse events. NSAIDs may be a candidate for prevention of PPI‐related gastric cancer.In conclusion, NSAIDs were associated with a decreased risk of gastric cancer with dose– and duration–response relationships among PPI users after Figure S1. Cumulative incidence of gastric cancer according to (A) CYP2C19 extensive vs non‐extensive metabolizers and (B) CYP2C19 extensive vs intermediate vs poor metabolizers.Figure S2. Posterior probability of gastric cancer in CYP2C19 intermediate and poor metabolizers using a Bayesian modelFigure S3. Cumulative incidence of gastric cancer in (A) PPI+NSAID high‐dose and low‐dose users and PPI‐users, (B) PPI+NSAID long‐term and short‐term users and PPI‐users.Table S2. ICD10 codes of comorbidities.Table S3. Association between CYP2C19 extensive and non‐extensive metabolizers and gastric cancer incidence (n = 199).Table S4. Associations of gastric cancer incidence with CYP2C19 extensive, intermediate, and poor metabolizers (n = 199).Click here for additional data file.Table S1. Drug codes.Click here for additional data file.


Elsholtzia beddomei C. B. Clarke ex Hook. f. was investigated for its chemical composition and tested for antioxidant and antimicrobial activities. The E. beddomei essential oil was extracted using hydrodistillation for 4 h (yield of 1.38% w/w). Forty-three volatile compounds were identified in the E. beddomei essential oil, including linalool (83.67%), perillaldehyde (4.68%), neral (3.68%), perillene (1.65%), E-caryophyllene (1.55%), and α-zingiberene (1.06%) as the major compounds. The antioxidant activity of the E. beddomei essential oil was determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical cation scavenging activity. The IC50 values calculated using the DPPH and ABTS methods were 148.31 and 172.22 µg/mL, respectively. In addition, using disc diffusion and broth microdilution methods, the antimicrobial activities of the E. beddomei essential oil against Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus subtilis, and Candida albicans were evaluated. The E. beddomei essential oil possessed an inhibitory effect with the minimum inhibitory concentration in the range of 31.25–250.00 µg/mL among these pathogens. The results indicated that E. beddomei essential oil is an alternative raw material of food, and medicinal products for use in pharmaceutical applications.The essential oil of  According to an increase of microbial resistance, several antimicrobial agents are losing their treating potential1. The therapeutic alternatives for treatments of the microbial infection may be limited or unavailable. In addition, World Health Organization (WHO) reported that infectious diseases have been evaluated as the second death cause around the world3. It was found that more than two million illnesses are caused from antimicrobial resistance with more than 20,000 deaths per year in the United States and these cases have been increased every year1. Therefore, searching of new alternative antimicrobial agents is necessary to decrease the microbial resistance problem.The serious problem of antimicrobial resistance has been increased for public health threats due to rapid evolution and spread of microbial resistance especially clinically important bacterial species1. These compounds are mainly employed by plant as a defense mechanism against plant pathogens, herbivores, and competitors1. The major secondary metabolites produced by plants are essential oils, phenolic compounds, alkaloids, lectins/polypeptides, and polyacetylenes1. Essential oils are complex volatile liquids containing a complex variety of terpenes and their derivatives. They are conventionally extracted using hydrodistillation, steam distillation, or mechanical processes4. The yield and composition of essential oil varies depending on ecological, onto genetic, climatic, post-harvest effects, and intra-species genetic factors6. Essential oils possess biological activities, including antibacterial, antiviral, antifungal, antitoxigenic, antiparasitic, and insecticidal activities5. They have also been reported to decrease the risks of diabetes, cancer, and cardiovascular diseases7. Therefore, researches on their use in several pharmaceutical, food, agricultural, and cosmetic industry applications have recently increased.Plants are evaluated as an enormous source of active secondary metabolites with medicinal propertiesElsholtzia, a genus of the family Lamiaceae, consists of at least 33 species. They are widely distributed in Europe, Africa, North America, and Asia, especially in China, Japan, Korea, and India in various cultivation conditions and altitudes8. Most Elsholtzia plants are aromatic and used as food, spices, herbal tea, beverages, folk medicine, cosmetics, andperfume8. Moreover, they have been used to treat colds, headaches, fever, pharyngitis, diarrhea, nephritises, rheumatic arthritis, and nyctalopia in China8.They have been evaluated to strongly inhibit the central nervous system and show an analgesic effect9. The essential oils of Elsholtzia species, including E. cristata, E. blanda, E. bodinieri, E. ciliata, E. eriostachya, E. densa, E. rugulosa, E. ianthina, E. stauntonil, and E. splendens, have been shown to possess antimicrobial activities8. Elsholtzia species also displayed other activities including antiviral activity (from E. rugulosa and E. blanda), antioxidant activity , and anti-Alzheimer property (from E. rugulosa)8. Thus, the pharmacological use of the genus Elsholtzia have extensively increased.E. beddomei C. B. Clarke ex Hook. f. and E. stachyodes (Link) Raiz. and Saxena have been discovered in Doi Hua Mod, Umpang, Tak province and in Doi Angkang, Fang, Chiang Mai province, respectively. Both plant species are evaluated as indigenous and aromatic plant species that are widely used as a local herb by hill tribes10.The essential oil of E. stachyodes has been described as an inhibitor of some bacterial strains, such as Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae. Moreover, E. stachyodes oil also has antioxidant abilities as revealed by radical scavenging assay measurements and the β-carotene bleaching method10. In addition, carvacrol together with γ-terpinene have been found in the essential oil from aerial parts of E. beddomei11. It also possessed antibacterial activity against S. aureus and Staphylococcus epidermidis. However, less information is available on the chemical composition, antimicrobial activity, as well as antioxidant properties of E. beddomei essential oil. The purposes of this study are to identify the volatile compounds of the E. beddomei essential oil, and to determine its antioxidant activity based on the DPPH radical and ABTS radical cation scavenging activity.Recently, E. beddomei essential oil provided a yield of 1.38% v/w. The volatile compounds of the E. beddomei essential oil are shown in Fig. E. beddomei essential oil. These results showed a high content of monoterpene hydrocarbons (over 95%) in E. beddomei essential oil.The hydrodistillation of the E. beddomei essential oil using the DPPH and ABTS scavenging assays are shown as IC50 values in Table 50 of E. beddomei essential oil was measured to be 148.31 ± 0.23 µg/mL and 172.22 ± 0.32 µg/mL using the DPPH and ABTS assays, respectively. Trolox showed significantly higher antioxidant activity with IC50 of 7.34 ± 0.12 µg/mL and 11.31 ± 0.15 µg/mL from DPPH and ABTS assays, respectively. Linalool and perillaldehyde, the major components of E. beddomei essential oil, also showed significant lower antioxidant activity than those of E. beddomei essential oil with IC50 of 201.23 ± 0.26 µg/mL and 216.28 ± 0.18 µg/mL, and 186.43 ± 0.27 µg/mL and 197.61 ± 0.63 µg/mL using the DPPH and ABTS assays, respectively.The antioxidant activities of E. beddomei essential oil against seven pathogenic microorganisms was evaluated using disc diffusion and broth microdilution methods. The zone of inhibition diameter, minimum inhibitory concentration (MIC), and minimum microbicidal (MMC) of the E. beddomei essential oil and chloramphenicol are shown in Table C. albicans were suppressed by the E. beddomei essential oil. The results from the disc diffusion method indicated that Gram-positive bacteria were the most significantly sensitive to 10 µg/mL of E. beddomei essential oil with a zone diameter ranging between 25.17 and 29.91 mm, compared to those obtained from other tested pathogens ranging between 19.34 and 22.23 mm. The MIC of the bacterial pathogens ranged from 3.91 to 7.81 µg/mL, whereas the MIC for C. albicans was 15.62 µg/mL. The MIC values of chloramphenicol were 31.25–125 µg/mL and 125 µg/mL for bacterial pathogens and C. albicans, respectively. The results of the MMC determination indicated that the E. beddomei essential oil had the lowest MMC value (3.91 µg/mL) for all Gram-positive bacteria.The antimicrobial activity of the Elsholtzia plants species differed from previous studies. Previously, α-pinene and β-pinene were evaluated as the major volatile compounds in at least 15 species of Elsholtzia plants12. The essential oils of E. rugusola and E. splendens revealed thymol and carvacrol as major compounds14, whereas E. ciliata and E. patrini essential oils showed high content of β-dehydroelsholtzia ketone and elsholtzia ketone15. It was indicated that species have specific volatile profiles. Moreover, carvacrol and γ-terpinenewere found as the main compound in E. beddomei essential oil reported by Phetsang et al.11 which was different from this study. Plant age, geography, day length, temperature, cultivation, climatic conditions, selection of plant organ, and harvest period, as well as extraction method, may influence the essential oil composition. These factors may result in the biosynthesis pathways of the plant and, consequently, the chemical composition and content, leading to the production of diverse chemotypes17. Similarly, the variety of essential oil compositions of Elsholtzia species was reported in previous studies. Fang et al.18 and Ren et al.14 described that the content of α-pinene in the essential oil of E. blanda growing in Yunnan province in China was4.84% while those cultivated in Sichuan province in China was 1.43%. In addition, the volatile components of the essential oil from E. stauntonii obtained from different extraction methods significantly varied in terms of content and type20.The obtained results on the chemical composition of essential oils extracted from 22. Several studies on the antioxidant activity of extracts obtained from Elsholtzia species have been previously reported. High antioxidant activity was found in the extracts from E. ciliata23, E. rugulosa24, E. bodineri25, E. ciliataobtained from CO2 supercritical fluid extraction26, E. splendens27, and E. blanda28. Although the antioxidant activity of Elsholtzia plants has been studied extensively, the antioxidant activity of the E. beddomei essential oil have not been reported yet. The antioxidant activity of E. beddomei essential oil may be influenced by non-phenolic constituents such as monoterpenic compounds and its derivatives. The combination of major and minor compounds in the essential oil may enhance antioxidant activity via the synergistic effects among these compounds producing an effective defense system against free radical attack. Several previous studies also confirmed linalool as a valuable antioxidant compound with promising nutraceutical applications due to its donation of hydrogen atoms and removing the electron from DPPH30. Perillaldehyde was also evaluated mainly as an antioxidant compound of essential oil of Perilla frutescens (L.) Britton inhibiting inflammatory skin diseases or disorders related to oxidative stress33. The antioxidant mechanism of perillaldehyde has been found by inhibiting BaP-induced AHR activation and ROS production and BaP/AHR-mediated release of the CCL2 chemokine while activating the NRF2/ HO1 antioxidant pathway40. Other compounds, such as neral, E-caryophyllene, and α-zingiberene, were reported to exhibit antioxidant activity due to the activated methylene group in their chemical structures34.The potential of essential oil for free radical inhibition may involve the specific compounds eliminating and preventing free radical formationElsholtzia plants against pathogenic bacteria has been previously investigated. Essential oils of E. splendens showed antibacterial inhibitory effects against S. aureus, S. epidermidis, and Propionibacterium acnes35. Similarly, E. ciliata and E. rugulosa essential oils also inhibited S. aureus, P. aeruginosa, Bacillus enteritidis, B. subtilis, Proteus vularis, Shigella dysenteriae, and E. coli37.Essential oils from several Elsholtzia species revealed extensive antibacterial activity against some bacteria involved inhuman respiratory infections such as Aeruginosus bacillus and Diplococcus intracellularis32. Moreover, the E. blanda and E. rugulosa essential oils showed significant inhibitory activity against methicillin resistant S. aureus strain33. A report on the antibacterial activity of essential oil extracted from E. beddomei was found in the study of Phetsang et al.11E. beddomei displayed an inhibitory effect against S. aureus and S. epidermidis.The antibacterial activity of essential oils of E. beddomei essential oil may be correlated mainly with the presence of active components, including monoterpenes, sesquiterpenes, and their derivatives, as reported by Burt5. The major active compound enhancing the antimicrobial property of E. beddomei essential oil is linalool, which is abundant in thyme essential oils and has been reported to show antimicrobial properties5. In addition, the significant antimicrobial activity of E. beddomei essential oil could result from the synergetic effects of the essential oil composition37. Various studies proposed that the mechanism of action of monoterpenes and its derivatives was membrane permeability, based on their ability to disrupt the cell wall and cytoplasmic membrane resulting in lysis and leakage of intracellular components37. The interaction of the antimicrobial compounds of the essential oil with the membrane can disturb the transportation of nutrients and ions, the membrane potential, and the overall cell permeability47. However, there is limited information about the in vivo antimicrobial activity mechanism of E. beddomei essential oil. Thus, in vivo experiments should be improved in order to apply E. beddomei essential oil to the best of its functional potential. In addition, toxicological and regulatory investigations are also necessary before using E. beddomei essential oil as a biological agent.The antimicrobial activity of E. beddomei essential oil were linalool, perillaldehyde, neral, perillene, E-caryophyllene, and α-zingiberene. The essential oil of E. beddomei showed antioxidant activity as determined by DPPH and ABTS assays. It also showed inhibitory effects against Gram-negative and Gram-positive bacteria, as well as fungus C. albicans, based on the presence of linalool. These findings demonstrate that E. beddomei essential oil may be an alternative natural product and a potential source of pharmaceutical agent. Toxicological and clinical tests are further needed before applying it in humans.The major volatile components of the E. beddomei were collected from Doi Hua Mod, Umpang, Tak Province, Thailand in July 2020. This plant species is not endangered. It was identified by taxonomist Dr. Jantrararuk Tovaranonte and the voucher specimen MFU 10,232 was deposited at the Mae Fah Luang Botanical Garden, Mae Fah Luang University, Chiang Rai, Thailand. The specimen was collected in the field with permission from the Department of National Parks, Wildlife and Plant Conservation, Umpang, Tak Province, Thailand. The study on this plant species has comply with relevant institutional, national, and international guidelines and legislation.The aerial parts of E. beddomei were subjected to hydrodistillation using a Clevenger-type apparatus for 4 h. The obtained essential oil was dried with anhydrous sodium sulfate to eliminate the water. The essential oil was kept in a sealed vial and stored at 4 °C for further use.The fresh aerial parts of 9–C16 n-alkanes and were compared with the relevant literature reported by Adams38 and the mass spectra from the Wiley 7 N library. Semi-quantitative analysis (given as % peak area of the particular component) was determined by area normalization method. Calculation of area percent is assumed to be equal to weight percent. The %area calculation procedure reports the area of each peak in the run as a percentage of the total area of all peaks in the run. Area percentage does not require prior calibration and does not depend upon the amount of sample injected within the limits of the detector so response factors were not used.A total of 0.5% v/v essential oil was prepared by diluting with dichloromethane. A total of 1.0 μL of solution was injected into the gas chromatography-mass spectrometry (GC–MS) using an Agilent 6890 N gas chromatograph  equipped with electron impact ionization and mass-selective detector . A fused-silica capillary DB5-MS   was used with helium, a carrier gas, with a rate of 1.0 mL/min. The injector temperature was set at 250 °C. The ionization energy was 70 eV in electron impact ionization mode with an ion source temperature of 250 °C and interface temperature of 250 °C. The oven temperature program was initiated at 60 °C and then increased to 240 °C with a rate of 3 °C/min. The acquisition was performed in scan mode (m/z 30–300). The volatile components were identified using the computer matching method and by comparing the mass spectra with the Wiley 7 N and W8N08 libraries. The Kovats retention indices were calculated using linear interpolation of the retention times of CE. beddomei essential oil, similar to the method reported by Insawang et al.39 A 60 µM DPPH solution was prepared in methanol. The essential oil was diluted in 10% dimethyl sulfoxide (DMSO) in concentrations of 1000, 500, 250, 125, 62.50, 31.25, 15.62, 7.81 and 3.91 µg/mL. A mixture consisting of 50 µL of essential oil or standard solutions and 1950 µL of DPPH solution was then made and the reaction was monitored in the dark at room temperature. The solution was then incubated for 30 min. The absorbance of the mixture was determined at 517 nm using a PerkinElmer spectrophotometer. Methanol was used as a blank solution. The scavenging capacity were calculated using following equation.E. beddomei essential oil, trolox, linalool and perillaldehyde were reported as IC50. Each sample was tested for antioxidant activity in triplicate.The DPPH radical was used in the antioxidant investigation of E. beddomei essential oil against ABTS was investigated in a manner similar to Insawang et al.39 The ABTS radical cation reagent was prepared by combining 7 mM ABTS solution with 2.45 mM potassium persulfate. The reagent was kept in the dark at room temperature. The E. beddomei solution was prepared in methanol . For each sample, a reaction was prepared by mixing 50 µL of the essential oil or standard solutions and 1950 µL of the ABTS solution before shaking vigorously and then kept in the dark at room temperature for 30 min. The absorbance of the solution was determined as 734 nm using a PerkinElmer spectrophotometer. Methanol was used as a blank solution. The scavenging capacity was calculated using the equation described above. The antioxidant activity of E. beddomei essential oil, trolox, linalool and perillaldehyde were reported as IC50. Each sample was tested for antioxidant activity in triplicate.The scavenging activity of E. beddomei essential oil against seven human pathogens  was measured. These pathogens were obtained from the culture collection of the Thailand Institute of Scientific and Technological Research, Bangkok, Thailand.The antimicrobial activity of E. beddomei essential oil was screened by the disc diffusion method according to Lu et al.40 with some modifications. All bacterial pathogens were cultured in Mueller Hinton agar while C. albicans was cultured in potato dextrose agar (PDA). All bacterial and fungal pathogens were then sub-cultured in nutrient broth and potato dextrose broth (PDB), respectively. The bacterial pathogens were incubated at 37 °C for 24 h while the fungus pathogen was incubated at 28 °C for 48 h. The active bacterial pathogens were prepared in a nutrient broth until it matched the 0.5 McFarlane standard (1 × 108 colony-forming unit/mL). The bacterial pathogens were spread out on the dried surface of the nutrient agar plates. The serialized 6 mm diameter paper discs  were impregnated with 30 µL of E. beddomei essential oil solution (10 µg/mL) preparing in 10% DMSO. The paper discs were then placed on the nutrient agar plate. These plates were incubated at 37 °C for 24 h. The fungus plates were incubated at 28 °C for 48 h. The diameter of the zone inhibition was determined. Chloramphenicol (10 µg/mL) and 10% DMSO were used as the positive and negative controls, respectively. Each experiment was performed in replicates.The antimicrobial activity of 41 The E. beddomei essential oil was prepared in 10% DMSO. The dilution series of E. beddomei essential oil were performed by two-fold dilution in concentrations of 1000, 500, 250, 125, 62.50, 31.25, 15.62, 7.81, and 3.91 µg/mL. The reaction was achieved by mixing 50 µL of essential oil, 10 µL of microbial suspension, and 10 µL of 0.675% of resazurin  on a 96-well microtiter plate. Chloramphenicol and 10% DMSO as the positive and negative controls, respectively. The bacterial and fungal plates were then incubated at 37 °C for 4.5 h and 28 °C for 48 h, respectively. The MIC was determined by considering the pink color of resazurin. Furthermore, MMC was also determined by mixing the solution from the well with a violet color of resazurin and spreading it on nutrient agar or PDA plate prior to incubation at 37 °C for 24 h and 28 °C for 48 h for bacteria and fungus, respectively. The MMC was determined by evaluating the plates without microbial colony. Each experiment was performed in triplicate.The MIC and MMC were determined according to the modifications of Teh et al.p < 0.05. All statistical tests were performed using the SPSS statistics software .Results are expressed as mean ± standard deviation. All experiments were performed in triplicate. Analysis of variance (ANOVA) was performed to measure the antimicrobial and antioxidant activities. The mean comparison was based on the Student's t-test at