To summarize the clinical distribution of Modic changes in patients with low back pain and explore the related factors.A total of 153 patients were enrolled. Gender, age, disk degeneration, herniation, involved segments, lumbar lordosis angle, and endplate concave angle were recorded, respectively. Patients were divided into two or more groups according to a different classification. The relevant factors were studied with a multivariate logistic regression analysis to analyze their correlation.Y = 2.410 − 1.361S − 0.633A − 0.654P + 1.106L − 0.990D . The OR values were S = 0.256, A = 0.531, P = 0.520, L = 3.022, D = 0.372, respectively.A total of 35 patients with type I changes, 110 patients with type II changes, and 8 patients with type III changes. In total, 204 disks were found with Modic changes, L1/2 (10 disks), L2/3 (18 disks), L3/4 (17 disks), L4/5 (76 disks), and L5/S1 (81 disks). Type I changes were distributed mainly under the age of 50. Multivariate regression showed that gender, age, disk degeneration, lumbar lordosis, L4/5 segment lordosis angle, and L5 lower endplate concave angle were related with different types of Modic changes. The regression equation Type II changes are the most common, followed by type I. Modic changes mostly occur in L4/5 and L5/S1; young, male, lower-grade disk degeneration, normal physiological curvature of the lumbar spine, and normal endplate concave angle were associated with type I changes; gender and lumbar curvature were the most relevant factors for different types. Low back pain (LBP) is a common disease, and up to 80% of adults have suffered from it during their lifetime . The lumPatients who took lumbar MRI for LBP in our hospital from January 2015 to December 2017 and diagnosed with endplate Modic changes were enrolled. According to the following inclusion/exclusion criteria, 153 patients were included in the study.Patient inclusion criteria are as follows: (1) a typical endplate Modic changes on T1-weighted image and T2-weighted image and (2) clear and complete imaging data.Exclusion criteria are: (1) fresh compression fracture of the lumbar vertebral; (2) lumbar spondylolisthesis, scoliosis; (3) tuberculosis, tumor, etc.; (4) Schmorl’s node; and (5) a history of back surgery.According to the classification criteria proposed by Modic et al. , Modic cAccording to the classification raised by WHO in 2012, the patients were divided into three groups: young group—under 44 years old; middle-aged group—between 45 and 59 years; elderly group—over 60 years.According to the classification raised by Pfirrmann et al. , all casAccording to the classification raised by the American Association of Orthopedic Surgeons, all cases of disk herniation were divided into three types: protruded, extruded, and sequestrated Fig. .Fig. 3TyLumbar lordosis (LL), sacral slope (SS), the lordosis angle of the L4/5 vertebral segment, and the lordosis angle of the L5/S1 vertebral segment were measured  of the L4 lower endplate, the L5 upper endplate, and the L5 lower endplate were measured , L2/3 (18 disks), L3/4 (17 disks), L4/5 (76 disks), and L5/S1 (81 disks). Patients under 50 years were more likely to have type I changes, and those over 50 years were more likely to have type II changes.There were 15 patients in the young group, 14 patients in the middle-aged group, and 6 patients in the elderly group with type I changed; there were 23 patients in the young group, 50 patients in the middle-aged group, and 37 patients in the elderly group with type II changes. There was a statistical difference among the three groups  was considered as the affected segment. There were 27 L1/4 segments, 55 L4/5 segments, and 71 L5/S1 segments. For patients with type I changes, there were 6 L1/4 segments, 14 L4/5 segments, and 15 L5/S1 segments; for patients with type II changes, there were 13 L1/4 segments, 41 L4/5 segments, and 56 L5/S1 segments. There was no significant difference between the three groups  was considered as the affected segment. For patients with type I changes, there were 7 grade II, 10 grade III, 12 grade IV, and 6 grade V; for patients with type II changes, there were 6 grade II, 19 grade III, 31 grade IV, and 54 grade V. There was a statistical difference among all groups , sacral slope (SS), and the lordosis angles of the L4/5 and L5/S1 vertebral segments were divided into two groups according to the mean degree. The mean degree of LL was 34.7°, SS was 29.7°, and L4/5 and L5/S1 vertebral segment lordosis angles were 9.4° and 17.2°, respectively. Patients with type I changes had larger LL and L4/5 vertebral segment lordosis angle, with statistical differences. There was no significant difference for SS and L5/S1 vertebral segment lordosis angle (Table CAVE was divided into two groups according to the mean degree. The mean degree of L4 lower was 164.8°, that of L5 upper was 168.6°, and that of L5 lower was 165.2°. Patients with type I changes had a smaller degree than type II changes for each CAVE. However, there was a statistical difference only for L5 lower CAVE (Table Y = 2.410 −1.361S −0.633A −0.654P + 1.106L −0.990D (Y means type I changes, S means gender, A means age, P means the disk degeneration, L means L4/5 segment lumbar lordosis, and D means L5 upper endplate concave angle. The OR values are S = 0.256, A = 0.531, P = 0.520, L = 3.022, D = 0.372, respectively (Table Univariate Chi-square test showed that age, gender, disk degeneration, lumbar lordosis, L4/5 segment lumbar lordosis, and L5 lower CAVE were associated with different types. After correlation analysis of the above factors by binary logistic regression analysis, no significant correlation was found between lumbar lordosis and types. Binary logistic regression analysis shows that the regression equation is Our results showed that type II changes are most common and the proportion is 71.9% for type II, 22.9% for type I, and 5.2% for type III. Modic changes mainly occur in L4/5 and L5/S1 segments, accounting for 77% of all cases. These findings are consistent with previous reports , 14. ManOur results show that age is one of the factors associated with different types. Modic changes are degenerative disease, with a higher incidence rate over 45 . In thisOur results show that gender is one of the factors leading to different types of Modic changes. Men patients are associated with type I changes, and women patients are associated with type II changes. Endplate microfractures may be a reason for it. Adams et al.  found thOur results show that disk degeneration is one of the factors leading to the different types of Modic changes. We believe this may be related to the biomechanical effects of disk degeneration on the entire lumbar spine. Intervertebral disk is the largest bloodless tissue in the human body, it largely depends on the nutrients supplied by the cartilaginous endplate; the vascular channels in the cartilaginous endplate are particularly important for the nucleus pulposus . Holm etOur results show that lumbar curvature is an important factor leading to the different types of Modic changes. Lumbar spine curvature is associated with type II changes, which we believe may be related to biomechanical factors. Lumbar lordosis plays a key role in maintaining the sagittal balance in the standing position of the body, and its curvature will have an effect on the biomechanics of the entire lumbar spine . The preOur results show that CAVE is one of the relevant factors leading to the different types of Modic changes. Small CAVE is related to type I changes, and large CAVE is related to the type II changes. We think this may be related to disk degeneration and difference in biomechanics. Endplate is a thin layer structure located between the vertebral body and disk, which can be divided into bony endplate and a cartilage endplate. The cartilage endplate is a part of the intervertebral disk, which is directly connected with the nucleus pulposus and annulus fibrosus, and serves to isolate the nucleus pulposus from contact with the vertebral cancellous bone. Wang et al.  used lasIn conclusion, type II changes are most common in patients with LBP, followed by type I, and type III is the least common. Modic changes occur mostly in L4/5 and L5/S1 segments. Age, gender, disk degeneration, lumbar curvature, VACE was associated with different types, among these factors, gender, and lumbar curvature have the strongest correlations. Therefore, for patients with type I changes, we believe that simple conservative treatment can achieve satisfactory results, and an extended period of conservative treatments is recommended. For patients with type II changes, when conservative treatments cannot achieve satisfactory effects, other aggressive treatment options can be taken to prevent further disease progression. The study is a preliminary analysis, and the specific clinical efficacy needs further investigation.


PubMed was searched from 1935 to December 2017 with a variety of search phrases among article titles. The references of the identified manuscripts were then manually searched. The inclusion criteria were as follows: (1) the paper presented data on measured normal body temperature of healthy human subjects ages 18 and older, (2) a prospective design was used, and (3) the paper was written in or translated into the English language. Thirty-six articles met the inclusion criteria. This comprised 9227 measurement sites from 7636 subjects. The calculated ranges (mean ± 2 standard deviations) were 36.32–37.76 , 35.76–37.52 (tympanic), 35.61–37.61 (urine), 35.73–37.41 , and 35.01–36.93 (axillary). Older adults (age ≥60) had lower temperature than younger adults (age <60) by 0.23°C, on average. There was only insignificant gender difference. Compared with the currently established reference point for normothermia of 36.8°C, our means are slightly lower but the difference likely has no physiological importance. We conclude that the most important patient factors remain site of measurement and patient’s age. Human body temperature is well established as one of the key vital signs. It is measured at regular intervals in the medical setting and often at home to try estimate the degree of “sickness” of an individual . It had The peer-reviewed literature was searched using PubMed . The timPooled standard deviations were calculated using the pooled standard deviation formula:For equal sample sizes, the formula was simplified as follows:For the data in which standard deviation for the measured temperatures was not reported in the original articles, the standard deviation was estimated via extrapolation from a plot of the known standard deviations and the corresponding sample sizes. The search hits are summarized in The average temperatures per measurement site, in decreasing order, were rectal at 37.04 ± 0.36, tympanic at 36.64 ± 0.44, urine at 36.61 ± 0.5, oral at 36.57 ± 0.42, and axillary at 35.97 ± 0.48 , Table 3The quest for understanding human body temperature and defining normothermia is ongoing, as is evidenced by the steady number of published prospective studies depicted in Knowing that body temperature is influenced by the measurement site, we calculated average temperatures, in decreasing order, rectal at 37.04°C, tympanic at 36.64°C, urine at 36.61°C, oral at 36.57°C, and axillary at 35.97°C. The trend is similar to the one reported by Sund-Levander et al ; howeverWith regards to age, our analysis confirmed that, on average, healthy elderly people have lower body temperature  comparedFinally, our analysis demonstrated only a trivial difference in body temperature between the genders , with woHuman body temperature is a highly variable vital sign and known to be influenced by several variables, most prominently the person’s age and the site of measurement. Our systematic review is the largest of its kind and provides clinicians with evidence-based normal temperature ranges to guide their evaluation of patients with possible fever or hypothermia.


Coriolus versicolor in mice infected by intracellular bacteria Neisseria gonorrhoeae.For many years, people use natural products from the plant and fungal to improve immune response against microorganism. This study aimed to investigate the immunomodulatory properties of polysaccharides (PS) from N. gonorrhoeae was infected twice with 2 weeks gap from the first to second exposure with a dose of 106 cells. 1 week after the end of treatment, level of oxidants, innate immune responses, and adaptive immune responses were measured.Thirty-six female BALB/C mice were divided into six groups: Normal control, negative control, positive control, P1 (PS before infection), P2 (PS after infection), and P3 (PS before and after infection). PS were administrated for 10 days. This study showed that PS administration could restore the number of leukocytes as normal but could not enhance the number of phagocytes and its activity. PS administration also showed immunosuppression activity by lowering nitric oxide levels in P2 and P3 groups (p<0.05). This result showed that PS prevent over-expression of pro-inflammatory cytokines by decreasing phagocytic activity. Contrast with innate immune response result; PS administration could significantly increase interferon-gamma level in P1, P2, and P3 groups (p<0.05). Level of antibodies was significantly increased in the P3 group (p<0.05). PS administration also showed an increased level of tumor necrosis factor-α, but the difference was not significant (p>0.05).N. gonorrhoeae that able to survive and replicate in phagocytes. Thus, PS from C. versicolor could be potentially be used as a natural immunomodulator against intracellular bacteria.PS enhance adaptive immunity due to the capability of  Neisseria gonorrhoeae infections are approximately 60 million cases each year worldwide ). K+ group also showed a significant increase in the number of leukocytes compared to normal control (p<0.05). P1 group (3560±860 [cell/mm3]) showed a higher number of leukocytes compared to normal control, but the difference was not significant (The highest number of leukocytes was shown by K+ group (4430±735 [cell/mmnificant .The highest phagocytic activity was shown in K+ group (25.4±8.8%). P1, P2, and P3 groups showed a significant decrease in phagocytic activity compared to the normal control group (p<0.05). Phagocytic activity of P1, P2, and P3 groups was 13.8±1.6%, 8.8±2.3%, and 15.4±3.2%, respectively .P3 group showed a significant level of antibody compared to the normal control group (p=0.05). P3 group also had the highest level of antibody (0.498±0.048). P1 group showed an increase in the level of antibody, but the difference was not significant .Serum levels of IFN-γ were significantly increased in P1 group (99.6±62 pg/mL) and P2 group (206.7±146 pg/mL) compared to normal control and positive group (p=0.05). P3 group showed the highest level of IFN-γ (735±130.7 pg/mL) and was significantly different compared to all groups. Meanwhile, there was no difference in the result of the level of TNF-α. P1, P2, and P3 groups showed an increase in the level of TNF-α, but the difference was not significant .P3 and P4 groups showed a significant decrease in the level of NO compared to the normal control group. NO level of P3 and P4 was 0.481±0.01 M and 0.470±0.005 M, respectively. NO level of P1 group was the same with normal control and did not show a significant difference .Defense against microbes is mediated by innate immunity as the first response and the later response of adaptive immunity. The immune system offers a layered defense against microbes . An unbaN. gonorrhoeae is intracellular bacteria. Like most of the intracellular bacteria, N. gonorrhoeae enter the host through the mucosa [N. gonorrhoeae could not enhance number of phagocytes and number of leukocytes significantly. In line with that result, this study also showed a decrease in phagocytic activity in all groups with the administration of PS.Different microbes require a different mechanism for elimination. Adaptive immunity is able to differ bacteria compound from other microbes specifically. Immune system gives different response against intracellular and extracellular bacteria. e mucosa . The chaet al. [C. versicolor possessed to stimulate macrophage and phagocytes activity. N. gonorrhoeae as an antigen may able to resist phagocytic killing, escape the phagolysosome, and survive inside macrophages [C. coriolus might increase adaptive immune cells responses.Contrast with our result, Meng et al.  stated trophages . Our resN. gonorrhoeae activates sensor cells of the innate immune system to trigger an adaptive immune response. Phagocytes as antigen presenting cell stimulate differentiation of Th-cell. Th-cell that specifically gives response to the infection of intracellular bacteria is Th-1 cells [I. obliquus and C. versicolor significantly increased secretion of IFN-γ [C. versicolor could enhance cytokines production.Murphy and Weaver  stated t-1 cells . Related-1 cells . In thisof IFN-γ ,18. Meanet al. [N. gonorrhoeae as intracellular bacteria is resistant to phagocytosis. Adaptive immune compounds are needed to eliminate it. The statement is in line with this study. This study showed a decrease in phagocytic activity in all treatment groups. Meanwhile, there was increase in production of TNF-α and IFN-γ. Our result showed that C. versicolor modulates immune system through the production of cytokines, which lead to activation of adaptive immune cells.According to Abbas et al. , N. gonoet al. [TNF-α and IFN-γ are cytokines produced by macrophage and Th cell due to all kinds of antigen infections. In this study, TNF-α and IFN-γ were not dominantly produced by macrophages because there is no relationship between phagocytic activity with the production of TNF-α and IFN-γ. Based on D’Elios , Th-1 ceet al.  also repWe found that the insignificant result of the increase in TNF-α level was beneficial. TNF-α is one of the important pro-inflammatory cytokines. The overproduction of TNF-α will induce the development of various disease and inflammation . InsigniN. gonorrhoeae requires mechanism of cell-mediated immunity and adaptive immunity [C. versicolor increases level of antibody significantly.Elimination of immunity . ActivatC. versicolor are due to the β-glucan compound. Chan et al. [et al. [Immunomodulatory activities of PS from n et al.  stated t [et al. , β-glucaC. versicolor could become the new candidate of immunomodulator. PS from C. versicolor enhance mainly adaptive immunity by increasing the production of cytokines and antibody. PS from C. versicolor also possessed immunosuppression activity by decreasing the level of phagocytic activity and NO to prevent tissue damage. Immunomodulation consist enhancement of the activity of immune cells, suppress component of immune that leads to tissue damage and restores the response of immune cells. In this study, PS from C. versicolor could do all immunomodulation mechanisms.Based on all of the result in this study, PS from C. versicolor could restore a number of leukocytes, increase antibody level, TNF-α level, and IFN-γ level but decrease phagocytic activity and NO level to prevent over-expression of pro-inflammatory cytokine. This study suggests that PS from C. versicolor could act as an effective compound to modulate the immune response.We concluded that PS from in vivo experiment and collected the samples. SPAW and MP analyzed the data. All authors drafted, read, and approved the final manuscript.SPAW designed the study. MP performed the


The method utilized atmospheric pressure chemical ionization (APCI) in multiple reaction monitoring (MRM) mode for the quantitation of impurities. The method employed an ODS column as the stationary phase and water-acetonitrile as the solvents for gradient elution without derivatization steps. The method was specific, linear, accurate, precise, and robust. Recoveries of the sulfonic esters from three drug matrices were observed in the range of 91.6∼109.0% with an RSD of not greater than 17.9% at the concentration of the LOQ and in the range of 90.4%∼105.2% with an RSD of not greater than 7.1% at the concentration of 50 ng/mL for the methanesulfonates and 10 ng/mL for the benzenesulfonates and p-toluenesulfonates. The LOD was not greater than 15 ng/mL, 2 ng/mL, and 1 ng/mL for the methanesulfonate, benzenesulfonate, and p-toluenesulfonate esters, respectively. This method was sufficiently sensitive to detect the 15 PGIs in the phentolamine mesylate tablet, amlodipine besylate tablet, and tosufloxacin tosylate tablet. This analytical method is a direct, specific, rapid, and accurate quality control tool for the determination of the 15 sulfonate esters that are most likely to exist in drug products.Sulfonate esters have been recognized as potential genotoxic impurities (PGIs) in pharmaceuticals. An LC-MS/MS method was developed and validated for the simultaneous determination of 15 sulfonate esters, including methyl, ethyl, propyl, isopropyl, and  Genotoxicity of different sulfonate esters, including methanesulfonate, benzenesulfonate, and eus test . Because0 years) . In addi0 years) , which r0 years) .p-toluenesulfonates [p-toluenesulfonate, and other sulfates in APIs [The low-acceptable intake level of PGIs in drug substances/products sets higher demands and challenges for establishing analytical methods with respect to sensitivity and specificity. Considering the differentiated volatility of sulfonate esters, gas chromatography- (GC-) based and liquid chromatography- (LC-) based methods have been developed. Methyl, ethyl, and isopropyl mesylate esters in APIs or final products have mostly been quantified by gas chromatography-mass spectrometry (GC-MS) –6. A higlfonates . Howeverlfonates  is pract in APIs . In addi in APIs . Moreove in APIs  demonstrp-toluenesulfonates in pharmaceuticals. The method was rapid, specific, accurate, and sensitive for the detection of mesylate, besylate, and tosylate drug products.The aim of the current study was to develop one simple and rapid separation method for analysis of multiple sulfonate esters, which does not require derivatization. An HPLC-APCI-MS/MS method was developed for the simultaneous determination of 15 PGIs, including the methyl, ethyl, propyl, isopropyl, and butyl esters of methanesulfonates, benzenesulfonates, and p-toluenesulfonate were purchased from Sigma-Aldrich Co. ; the propyl (98.0%) and isopropyl (98.0%) esters of methanesulfonate, ethyl (98.0%) and n-butyl (98.0%) esters of benzenesulfonate, and methyl (98.0%), propyl (98.0%), and n-butyl (97.0%) esters of p-toluenesulfonate were obtained from Tokyo Chemical Industry Co., Ltd. ; the propyl ester of benzenesulfonate (97%) was provided by Bide Pharmatech Ltd. ; the isopropyl ester of benzenesulfonate (98%) was purchased from Adamas Reagent Co., Ltd. ; and the n-butyl ester of methanesulfonate (98.5%) was provided from J&K Scientific Ltd. .Reference standards were obtained as follows: the methyl (99%) and ethyl (98%) esters of methanesulfonate, methyl ester of benzenesulfonate (98%), and ethyl (98.0%) and isopropyl (97%) esters of 4Ac) were purchased from Fisher Scientific Products . HPLC grade acetic acid (HAc) was obtained from Tedia company, Inc. . Ultrapure water was obtained from a Milli-Q water purification system .HPLC grade methanol, acetonitrile, phosphoric acid (85%), and ammonium acetate , and the oven temperature was set to 30°C. The gradient elution was employed with solution A (water) and solution B (acetonitrile). The gradient solvent program was set as follows: time/% solution B, 0/40, 5.0/90, 7.5/90, 8.0/40, and 11.0/40. The flow rate was set to 1.0 mL/min, and the injection volume was 10 μL.Chromatographic separations were performed on a Shimadzu UFLC-20AD XR system with an autoinjector and binary solvent manager . The column was a Kromasil C18 column , 20 psi; nebulizer pressure, 40 psi; collision exit potential (CXP), −8 V; entrance potential (EP), 10 V; declustering potential (DP), −10 V; and collision energy (CE) of 25 eV for methanesulfonate esters and 30 eV for benzenesulfonate and p-toluenesulfonate esters. The MRM ion pairs were m/z 95 ⟶ 80, m/z 157 ⟶ 93, and m/z 171 ⟶ 107 for methanesulfonate, benzenesulfonate, and p-toluenesulfonate esters, respectively. The HPLC fraction corresponding to the API that eluted before 3.3 min was diverted to waste. Analyst 1.6 software was used for the control of equipment, data acquisition, and analysis.The Shimadzu UFLC-20AD XR system was coupled online to an Applied Biosystems Sciex Qtrap 5500 mass spectrometer  with an atmospheric pressure chemical ionization (APCI) interface. The typical operating source conditions for the MS scan in negative APCI mode were optimized as follows: corona current, −5 p-toluenesulfonate esters. The limit of detection (LOD) and limit of quantitation (LOQ) were estimated at a signal-to-noise ratio of 3 : 1 and 10 : 1, respectively. Precision was determined by analyzing six replicates at the concentrations of the LOQ and 50 ng/mL. In the recovery test, the reference samples of the standards were added at concentrations of LOQ and 50 ng/mL for the methanesulfonate esters and 10 ng/mL for the benzenesulfonate and p-toluenesulfonate esters in powdered tablets with six replicates. The matrix effect was quantitated by comparing the peak area of six replicates of the mixed standards (100 ng/mL) added into an extracted drug matrix with that of the standards at the same concentration in the absence of matrix. Both the reference samples from the recovery test and the extracted matrices from the matrix effect evaluation followed the sample preparation procedure described in A standard stock solution (10 mg/mL) of each of the 15 sulfonate esters was prepared by dissolving the accurately weighed reference substance in methanol and storing at 2°C–6°C until further use. A series of mixed standard working solutions were freshly prepared from the standard stock solution and diluted with methanol containing 0.25% HAc before use, giving concentrations in the range of LOQ-200 ng/mL for propyl methanesulfonate, LOQ-150 ng/mL for other methanesulfonate esters, LOQ-400 ng/mL for benzenesulfonate esters, and LOQ-100 ng/mL for μm membrane syringe filter , and the subsequent filtrate was used as the test solution.A quantity of powdered drug tablets equivalent to 10 mg of the drug substance was weighed accurately and extracted with 5 mL of methanol containing 0.25% HAc in an ultrasound bath for 10 min. The extracted solution was filtered through a 0.45 p-toluenesulfonate esters in the presence of vinpocetine in acetonitrile/water  were found to be stable for an hour [Stability of the sample solution is an important consideration in a solvent selection. We selected acetonitrile, acetonitrile with acetic acid, methanol, and methanol with acetic acid as sample solutions, and the 15 analytes were stable for less than 4 h in acetonitrile with or without acetic acid, for 16 h in methanol, and for 24 h in methanol with 0.25% acetic acid. As shown in  an hour .μA to −5 μA and −5 μA produced the strongest signal response. The vaporizer temperature had a significant effect on the ion response. Temperatures of 200∼500°C were carefully evaluated and 300°C was optimal. In addition, a declustering potential set at 10 V was suitable for all the 15 analytes. The optimal collision energy was found to be 25 eV for methanesulfonate esters and 30 eV for benzenesulfonate and p-toluenesulfonate esters. The esters in each group shared the same multiple reaction monitoring (MRM) ion pairs , 100 mm × 3.0 mm (2.7 μm), and 250 mm × 4.6 mm (5 μm), were employed and compared, and the column with a 250 mm length showed a better separation capability to resolve the 15 sulfonate esters within a short period of time. The investigation of mobile phase included water, 0.1% HAc, and NH4Ac-HAc buffer in varying combinations with methanol or acetonitrile. The results showed that the MS responses of the standards were higher in acetonitrile than in methanol, and there was no difference between acetonitrile with water, 0.1% HAc, or acetate buffer, so the combination of acetonitrile and water was selected as the mobile phase. Furthermore, different gradient programs as well as the column temperature were optimized to finally obtain a satisfactory separation of the 15 analytes (Octadecyl silica (ODS) columns in different sizes, including 75 mm × 3.0 mm , vaporizer temperature, and collision energy. The results found that the 15 sulfonate esters were well separated when the parameters were in the following ranges: flow rate within 0.9∼1.0 mL/min, column temperature within 30∼40°C, initial proportion of mobile phase B in the gradient elution within 38%∼42%, vaporizer temperature within 290∼310°C, and collision energy within 23∼27 eV for methanesulfonate esters and within 28∼32 eV for benzenesulfonate and μg/g, 1 μg/g, and 0.5 μg/g for the methanesulfonate, benzenesulfonate, and p-toluenesulfonate esters, respectively, which corresponded to the drug products. As the ICH M7 [μg/day for a long duration of treatment. The upper limit daily dose was 60 mg, 10 mg, and 0.6 g for phentolamine mesylate, amlodipine, and tosufloxacin tosylate, respectively (The results showed that the 15 sulfonate esters were not detected in the phentolamine mesylate tablet, amlodipine besylate tablet, and tosufloxacin tosylate tablet. The LOD values in this method were not greater than 7.5 e ICH M7  suggesteectively . This mep-toluenesulfonates within a single HPLC run. This method was sufficiently sensitive to detect the 15 PGIs in the phentolamine mesylate tablet, amlodipine besylate tablet, and tosufloxacin tosylate tablet and can be further studied for its application to other drug substances or products.A simple, rapid, and reliable HPLC-APCI-MS/MS method has been developed for the determination of three types of sulfonate esters in pharmaceuticals. The advantage of the proposed method was that it required no derivatization and was capable of simultaneously separating the 15 sulfonic acid esters most likely to exist, covering methanesulfonates, benzenesulfonates, and


We aim to discover whether HbA1c affects incident cardiovascular disease (CVD) through regulating endogenous metabolites.IE from 0.997 to 6.098, false discovery rate q < 0.05, mediated proportion from 0.4% to 85.4%). Pathway enrichment analysis indicated that different metabolic pathway showed significant IE  cycle metabolism ORIE = 1.048, mediated proportion = 13.8%; phenylalanine metabolism ORIE = 1.067, mediated proportion = 18.4%; glycerophospholipid metabolism ORIE = 3.007, mediated proportion = 82.2%; all the P < .01).Totally, 2019 plasma samples were analyzed by liquid chromatography‐quadrupole time‐of‐flight mass spectrometry. Logistic regression and linear regression were used to screen metabolites which were associated with both CVD and HbA1c. The VanderWeele's mediation approach was performed to assess the direct effect and indirect effect (IE) in the counterfactual model. Forty‐eight metabolites showed an association with both HbA1c and CVD risk. Forty‐four of the 48 metabolites worked as mediators mediated in HbA1c's effect on CVD (odds ratio [OR]Our findings suggest that metabolites mediate the effect of HbA1c on incident CVD and provide a new study sight into pathogenesis of CVD. Here, we carried a hospital‐based case control study with 2019 participants and aimed to explore a potential mechanism whether endogenous metabolites are involved in effect of HbA1c on incident CVD.22.1A total of 2019 participants were recruited from the Affiliated Wujin Hospital of Jiangsu University during August 2009 and December 2015. Each participant underwent coronary angiography for confirmation the presence of CVD.Plasma samples were collected before the coronary angiography surgery and quickly stored at −80°C for further metabolomic analyses. Blood biochemical indices were measured, and the history of diseases and smoking history were recorded using questionnaire. Patients with other cardiac‐related diseases, blood‐related disorders, infectious diseases, and malignant tumors were excluded. All subjects signed the informed consent forms. This study was approved by the ethics committee of Affiliated Wujin Hospital of Jiangsu University and complied with the Helsinki Declaration.2.2To eliminate the protein in the plasma, 150 μL of acetonitrile was added to a 50 μL aliquot of plasma and vortexed for 10 seconds. Precipitated protein was subsequently removed by centrifugation  at 4°C. Then, 150 μL of the supernatant was transferred to a tube and dried under a gentle stream of nitrogen gas at room temperature. Finally, the supernatant was reconstituted in 100 μL of aqueous acetonitrile  for LC/MS (Liquid Chromatography‐Mass Spectrometry) detection.2.3To ensure data quality for metabolic profiling, quality control (QC) sample was proceeded. The detail process of QC referred to Fan et al.2.4Liquid chromatographic separation was conducted using a 1290 Infinity System , with 100 × 2.1‐mm Zorbax Eclipse Plus 1.8‐mm C18 column maintained at 45°C. The mobile phase consisted of water with 5 mM ammonium acetate (A) and 10% aqueous acetonitrile with 5 mM ammonium acetate (B). Gradient program of elution was: 5 to 80% B at 0 to 7 minutes, 80 to 100% B at 7 to 12 minutes, 100% B at 12 to 13 minutes, and then back to initial conditions, and 2 minutes for equilibration. The sample volume injected was 1 μL and the flow rate was 0.4 mL/min.The mass spectrometric detection was performed on an Agilent 6530 Q/TOF‐MS system  in positive mode. The parameters were set as: the fragmental voltage at 100 V, nebulizer gas at 35 psig, capillary voltage at 3500 V, drying gas flow rate at 10 L/minute, and temperature at 300°C. Reference masses at m/z 121.0509 and 922.0098 were introduced for accurate mass calibration.MassHunter Workstation Software  was used to convert mass spectrometry data (.d) into data format (.mzdata) files. XCMS package  was used to conduct the data pre‐treatment, including nonlinear retention time alignment, peak discrimination, filtering, alignment, matching, and identification. The detailed information of the experiment has been described in previous study.2.52.5.1P ≤ .05 were considered statistically significant unless stated otherwise.All metabolites concentrations were first log‐transformed prior to analyses to obtain approximately normal distributions. The false discovery rate (FDR) using Benjamini and Hochberg method was calculated to correct the multiple test adjustment. All tests were two‐sided, and 2.5.2β), odds ratio (OR) and 95% confidence interval (95% CI) were calculated.Initially, we performed non‐conditional logistic regression to explore the association between HbA1c and CVD risk with adjusting or no‐adjusting potential covariates . Then, the associations between (a) HbA1c and metabolites, (b) metabolites and CVD risk were assessed by multiple linear regression and non‐conditional logistic respectively, and the beta value  of HbA1c on CVD risk that was mediated through metabolites (explained by the change of metabolites per 1 SD increase of log‐transformed level), as well as the proportion of the effect mediated.2.5.4MetaboAnalyst” , so that, an elaboration of comprehensive metabolites will be accomplished. Principal component analysis was used to extract the first principal component (PC1) from metabolites by pathways. Afterwards, mediation analysis was conducted to seek for the potential relationship among HbA1c, metabolic pathways, and CVD.We classified the metabolites into different pathways using “2.5.5The whole participants were divided into young (age < 65) and old (≥65) groups by age, we conducted the mediation analyses in subsets. Potential confounders incorporating age, gender, smoking and drinking history were used as covariates and considerably adjusted in all models. All analyses and visualizations were conducted using R version 3.4.1 (The R Foundation).33.1A total of 2019 subjects (1784 CVD patients and 235 at‐risk controls) were included in this study. Each sample underwent coronary angiography and plasma samples were analyzed by liquid chromatography‐quadrupole time‐of‐flight mass spectrometry. The mean age of 2019 participants was 62.9 ± 9.6 years, of which males and females were 1276 (63.1%) and 743 (36.9%), 75% was non‐smoker, and 93% was non‐drinker. No significant differences were observed in smoke and drink history distributions among CVD and non‐CVD groups. Age and gender were found as unbalanced distributions between CVD and non‐CVD subjects Figure .3.2P = 3.79 × 10−9), which was consistent with a previous follow‐up study.P = 2.91 × 10−9). A total of 2059 positive ions were confirmed in our study. We performed a non‐condition logistic regression to uncover the relationship between CVD risk and metabolism features, and identified 176 metabolites associated with the incident CVD (P ≤ 0.05). Seventy‐five metabolites reached statistical significance after correction for multiple testing by FDR‐q ≤ 0.05. Acting as CVD risk factors, those metabolites might be influenced by HbA1c. We further explored the relationship between each significant metabolite and HbA1c. Out of 75 metabolites, 48 were then detected to be affected by HbA1c  . In detail, HbA1c decreased the level of methylglutarylcarnitine which further reduced CVD risk. In another word, methylglutarylcarnitine alleviated the effect of HbA1c on CVD risk (masking effect), and the mediated proportion was not estimated due to the opposite signs . 2α‐Methyl‐5α‐androstane‐3,17‐dione and glycocholic acid revealed a wholly mediate effect . More generally, adjusting with 2α‐Methyl‐5α‐androstane‐3,17‐dione and glycocholic acid, the effect of HbA1c on CVD was non‐significantly. Besides, the remaining 41 metabolites showed a positive IE explaining partial phenotypic variance .Given these association between HbA1c, metabolites, and CVD risk, it may be possible that metabolites act as mediators of the effect of HbA1c on CVD risk Figure . To veri3.4P values, and we extracted PC1s from the corresponding five metabolic pathways . The corresponding PC1s' proportion of variance was 0.950, 0.898, 0.994, 0.897, and 0.947, respectively, which indicated an appropriate representativeness of pathway metabolites. We assessed the proportion of HbA1c effect on risk of CVD that was mediated through metabolic PC1s. As shown in Table IE = 1.058, FDR‐q = 0.001, mediated proportion = 16%; alanine, aspartate and glutamate metabolism ORIE = 1.082, FDR‐q = 0.002, mediated proportion = 21.8%; TCA cycle metabolism ORIE = 1.048, FDR‐q = 0.003, mediated proportion = 13.8%; phenylalanine metabolism ORIE = 1.067, FDR‐q = 0.004, mediated proportion = 18.4%; glycerophospholipid metabolism ORIE = 3.007, FDR‐q = 5.72 × 10−9, mediated proportion = 82.2%. The IE among those pathways ranged from 1.048 to 3.001, which averagely explained 30.4% total effect (IE + DE). The linear combination of metabolites in certain pathway uncovered a potential mediator in HbA1c caused CVD.Considering an overall interpretation for metabolic IDE, pathway enrichment was investigated. Figure 3.5IE = 1.050, P = .01, mediated proportion = 14.7%; alanine, aspartate and glutamate metabolism ORIE = 1.062, P = 0.025, mediated proportion = 17.9%; TCA cycle IE = 1.049, P = 0.011, mediated proportion = 14.5%; phenylalanine metabolism ORIE = 1.075，P = .003, mediated proportion = 20.6%; glycerophospholipid metabolism ORIE = 3.561，P = 8.25 × 10−8, mediated proportion = 86.5%). However, butanoate metabolism, citrate cycle, and phenylalanine metabolism in old group failed .Based on the age division standard from world health organization (WHO), we divided participants into young (<65) and old (≥65) groups. Re‐extracting PC1s from both young and old groups, we obtained analogous results in 5 metabolic pathways Tables  and S3. 4As previous reported, metabolic biomarkers always play a critical role in CVD.In this study, significant IEs were identified among 42 metabolites independent from established CVD risk factor, and nine metabolites belonged to lysophosphatidylcholine (LPC) category. Ganna et alPathway analysis classified metabolic mediators into five categories, and principal analysis assembles those metabolites by pathway. The blood glucose can be metabolized into small molecule metabolites and then enter the TCA‐cycle,butanoate metabolism, TCA cycle, phenylalanine metabolism) in old group failed to revel a significant IE.This research is absence in external validation. In order to enhance the reliability, we performed a bootstrap when estimating the SE of DE and IE, and a stratified analysis was performed to validate the results. During stratified analysis, we confirmed the robustness performance of our results in young group, but three metabolic pathways (In this study, we captured 48 metabolites associated with both HbA1c and CVD risk and tested their mediation effect. It was revealed that metabolite may act as a mediator between the connection of HbA1c and CVD incidence and has a potential regulatory mechanism. we also recognized some limitations here. First, because of the uncommercial reference compounds obtained, identification of metabolites is still a challengeable work. Second, future prospective confirmation in independent cohorts is warranted. Third, the targeted metabolomics was not done, and only the untargeted metabolomics for screening of significant metabolites was studied. Fourth, the distributions of age and gender between case and control subjects were identified, indeed we adjusted those potential confounders among all models.5Metabolites were the downstream end products in endogenous matrix. High level HbA1c contributed to incident CVD, which may be mediated by metabolite functions. Explanations of the relationship among HbA1c, metabolites and CVD were beneficial to the etiological study of CVD.The authors declare no potential conflict of interest.F.C., Y.L., and G.M. conceived and designed the research study. X.D. and W.Z. analyzed the data and finished the manuscript writing. F.C. oversaw all parts of the analysis. X.Y. and Y.F. contributed to project development and manuscript revision. H.L. revised the manuscript All authors approved the final submission.Figure S1. Clinical characteristics.Figure S2. Schematic diagram of mediation analysis.Table S1. Metabolites associated with incident cardiovascular disease and HbA1c.Table S2. HbA1c associated with incident cardiovascular disease mediated by first principal component in various metabolic pathway in young group.Table S3. HbA1c associated with incident cardiovascular disease mediated by first principal component in various metabolic pathway in old group.Click here for additional data file.


Our results suggest that chemical modification of therapeutics with warheads of glucose and Vc represents a promising and efficient strategy for the development of brain-targeting prodrugs by utilizing the endogenous transportation mechanism of the warheads.Ibuprofen is one of the most potent non-steroid anti-inflammatory drugs (NSAIDs) and plays an important role in the treatment of neurodegenerative diseases. However, its poor brain penetration and serious side effects at therapeutic doses, has hindered its further application. Thus, it is of great interest to develop a carrier-mediated transporter (CMT) system that is capable of more efficiently delivering ibuprofen into the brain at smaller doses to treat neurodegenerative diseases. In this study, a dual-mediated ibuprofen prodrug modified by glucose (Glu) and vitamin C (Vc) for central nervous system (CNS) drug delivery was designed and synthesized in order to effectively deliver ibuprofen to brain. Ibuprofen could be released from the prepared prodrugs when incubated with various buffers, mice plasma and brain homogenate. Also, the prodrug showed superior neuroprotective effect  Recently, non-steroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, naproxen and indomethacin have been utilized as neuroprotective agents in treating central nervous system (CNS) diseases , monocarboxylic acid transporter (MCT1), sodium-dependent vitamin C transporter-2 (SVCT2) and glucose transporter (GLUT1). GLUT1 and SVCT2 expressed on the surface of brain capillary endothelial cells and choroid plexus epithelium cells, respectively, are considered as the most efficient transportation systems  system seems to be one of the most promising methods to facilitate the delivery of drugs into brain. There are plenty of physiological transport systems for nutrients and endogenous compounds, for example, the large neutral amino acid transporter . The other way is through the transporter SVCT2 which transports Vc into cerebrospinal fluid (CSF) at the choroid plexus, from which Vc can be further diffused to brain extracellular fluid (ECF), and then taken up into the brain cells from ECF. Given the superior ability of Vc in crossing the BBB, it is promising to be used as the carrier for brain-targeting drugs too. Some recent studies also explored the possibility of using Vc as a carrier to improve the BBB permeation and hence to promote the delivery of brain drugs  using CDCl3, dimethylsulfoxide-d6 (DMSO-d6) and D2O as solvent. High-resolution mass spectroscopy data of the product were collected on a Waters Micromass GCT or a Bruker Apex IV FTMS instrument. Reversed-phase chromatography performed on C18 chromatographic analysis was carried out using the high-performance liquid chromatography (HPLC) system  consisted of a RF-530 fluorescence detector (Shimadzu) and Allchorom plus data operator, respectively. A Diamonsil column  was used. A LC-10A liquid chromatographic system (Shimadzu) and a reverse-phase HPLC column  were used.The melting point was measured on a YRT-3 melting point apparatus . Infrared (IR) spectra were obtained on a Perkin Elmer983 . 2–9 was reported in our previous work  in CH2Cl2 (20 ml) was added DCC  and DMAP , and the reaction was stirred at −5 °C for 30 min. Then compound 9  in CH2Cl2 (10 ml) was added promptly. After stirring for another 6 h at 30 °C, the mixture was filtered and the filtrate was concentrated. Then, the residue was purified by chromatography to give 10  as a white solid. 1H-NMR  δ: 2.62 , 3.48–3.58 , 3.65 , 4.04–4.05 , 4.19–4.38 , 4.49–4.96 , 5.12 , 5.14–5.21 , 7.19–7.37 . HRMS: (ESI+) calculated for C58H58O14Na [M + Na]+ 1001.3724, found 1001.3720. Elemental analysis: C, 71.15; H, 5.97, found C, 71.10; H, 6.03.To a solution of compound 2Cl2 (15 ml) was added DCC  and DMAP , and the reaction was stirred at −5 °C for 30 min. Then, compound 10  in CH2Cl2 (10 ml) was added dropwise. After stirring for another 10 h at room temperature, the mixture was filtered and the filtrate was concentrated. Then the residue was purified by chromatography to give 11  as a white solid. 1H-NMR  δ: 0.89 , 1.45 , 1.79–1.87 , 2.16–2.58 , 3.44–3.86 , 4.03–5.09 , 5.34–5.35 , 6.97–7.38 . HRMS: (ESI+) calculated for C71H74O15Na [M + Na]+ 1189.4925, found 1189.4927. Elemental analysis: C, 73.05; H, 6.39, found C, 73.17; H, 6.30.To a solution of ibuprofen  in CH11  in methanol (15 ml), Pd/C  was added. Then, the mixture was stirred in hydrogen atmosphere at room temperature for 6 h. Pd/C was filtered, and the filtrate was concentrated to give prodrug Glu-Vc-Ibu  as a colorless oil. 1H-NMR  δ: 0.88 , 1.42 , 1.79–1.86 , 2.41–2.55 , 3.11–3.48 , 3.58–3.72 , 3.93–3.97 , 4.14–4.88 , 5.44–5.46 , 7.07–7.19 . HRMS: (ESI+) calculated for C29H38O15Na [M + Na]+ 649.2108, found 649.2105. Elemental analysis: C, 55.59; H, 6.11, found C, 55.52; H, 6.17.To a solution of compound Supplementary material.The detailed synthetic procedures of prodrug Glu-Ibu and Vc-Ibu were given in the in vitro chemical stability of prodrugs was investigated by incubating the prodrugs in pH 2.5, 5.0, 7.4 and 8.0 phosphate buffers, respectively. Briefly, 1 ml of the prodrugs methanol solution (250 μg/ml) was added into 4 ml different buffer separately. The entire system was kept at 37 ± 0.5 °C with continuous shaking at 100 rpm/min. 200 μl medium samples were taken at the following time points: 0, 0.5, 1, 2, 3, 6, 12, and 24 h, and then replaced by fresh medium. The samples were analyzed by HPLC (Shimadzu). The analysis was accomplished on a SinoChrom ODS-C18 column  , thermostated at 35 °C. The mobile phase was composed of methanol/water (73:27), and the pH was adjusted to 2.86 with 10% phosphoric acid. The UV wavenumber was 225 nm and the flow rate was set at 1.0 ml/min with a 20 μl injection volume.The The preliminary metabolic stability of prodrugs was studied by incubating in plasma and brain homogenate, respectively. 1 ml of the prodrugs methanol solution (250 μM) was added into 4 ml plasma or brain homogenate. Then, the entire system was kept at 37 ± 0.5 °C with continuous shaking at 100 rpm/min. 200 μl samples were taken at the following time points: 0, 0.5, 1, 2, 3, 6, 12, and 24 h, and then replaced by plasma or brain homogenate. The internal standard  was added to each sample followed by 200 μl acetonitrile. After centrifuging for 15 min, the supernatants of the samples were analyzed by HPLC method described before.2 humidified environment incubator .The PC12 cells were purchased from the American Type Culture Collection . Cells were cultured in DMEM  supplemented with 10% fetal bovine serum (FBS) , 100 U/ml penicillin and streptomycin. Cells were cultured at 37 °C in a 5% CO4 cells/well and cultured for 24 h at 37 °C. The cells were then treated with prodrugs and ibuprofen , respectively and incubated for 24 h. After incubation, 20 μl MTT solution (5.0 mg/ml) was added to the medium and incubated for another 4 h at 37 °C. After removal of the culture medium, the reduced MTT dye was solubi1ized by DMSO (100 μl) and the absorbance was read at 490 nm wavelength on an automatic microplate spectrophotometer. The cell viability = A490 nm for the treated cells/A490 nm for the control cells ×100%.The cytotoxicity of prodrugs and ibuprofen was assayed in PC12 cells using the -2,5-diphenyl-tetrazolium (MTT) assay. Briefly, PC12 cells were seeded in 96-well plates at a density of 6 × 10in vitro neurotoxicity model was established using H2O2 to induce oxidative stress and injury in PC12 cells. The cell viability, reactive oxygen species (ROS) level and superoxide dismutase (SOD) activity were then determined to measure the protection of ibuprofen and the prodrugs.The 2O2-induced cytotoxicity, the cell viability of PC12 cells was evaluated using the MTT assay. Briefly, PC12 cells were seeded in 96-well plates at the density of 6 × 104 cell/well and incubated at 37 °C for 24 h. Then, the cells were treated with ibuprofen or the prodrugs (10 μM) in FBS-free medium for 4 h and then exposed to H2O2 (1 μM) for 2 h. After the treatment, the cells were observed under inverted phase contrast microscopy and cell images were recorded. Subsequently, the cells viability was assessed by MTT assay using a microplate reader.To investigate the protective effects of ibuprofen and prodrugs against H5 cells/well and incubated at 37 °C for 24 h. After cultured with or without drugs  in FBS-free medium for 4 h, cells were treated with H2O2 (1 μM) for 2 h. Then trypsin–EDTA was used to harvest the cells, which were washed three times with PBS afterward, and finally stained with 2′,7′-dichlorofluorescein diacetate  and incubated for 20 min at 37 °C in the dark. After centrifugation , the cell pellet was washed three times with PBS, resuspended in PBS before flow cytometer analysis (Ex 488 nm and Em 525 nm). SOD is an index of superoxide level which neutralizes free oxygen radicals, usually being tested along with ROS level showing oxidative stress. In brief, after harvesting, the cells were resuspended in PBS and lysed by Ultrasonic cell grinder  to obtain the cell lysis solution. Then, the SOD activity was evaluated using colorimetry kits . Untreated cells were used as the control.The ROS assay kit  in DCFH-DA method was used to determine the intracellular ROS generation. PC12 cells were seeded in six-well plates at a density of 5 × 10Adult male Wistar rats weighing 250 ± 20 g were purchased from DOSSY Experimental Animal Center of Chengdu . All animal procedures for this study were approved by the Experiment Animal Administrative Committee of Sichuan University (China).in vivo study, cerebral ischemia model was induced by bilateral common carotid artery occlusion according to the previously published methods  and received an injection of saline or drugs  through the tail vein. After 30 min, the animals were anesthetized with 10% chloral hydrate , tied to the operating table, and exposed the bilateral common carotid artery through a middle incision of the neck and careful separation from the vagal nerves. Then, the arteries were enduringly ligated with bulldog clamps for 1.5 h. The brains were isolated, washed with PBS, frozen at −20 °C for 30 min and cut into 2 mm slices. The brain slices were stained in 1% 2,3,5-triphenyltetrazolium chloride (TTC) at 37 °C for 30 min in dark. Stained sections were photographed with digital camera and the staining rate was calculated by ImageJ .For the Furthermore, the remained brain was adequately homogenized with ice-cold saline to determine the levels of SOD, malondialdehyde (MDA) and glutathione (GSH). The activity of SOD, the content of MDA and the level of GSH were measured by using commercial assay kits . The tests were performed according to the manufacturer’s instructions.Kunming mice weighing 20–22 g were purchased from DOSSY Experimental Animal Center of Chengdu . All animal procedures for this study were approved by the Experiment Animal Administrative Committee of Sichuan University (P.R. China). The aqueous solutions of ibuprofen and prodrugs  were prepared with 0.1% Tween-80 to give a final concentration of 1 mg/ml. The mice were injected the ibuprofen or prodrugs solutions  through the tail vein.At appropriate time interval , blood was collected from the eye socket of mouse into a tube containing heparin, and centrifuged at 5000 rpm for 5 min. The supernatant was collected as plasma sample. The animals were killed by cervical dislocation, and the brain tissue was removed, and then washed with saline for three times to remove the remained blood and rolled over on the filter paper to remove the main vessel. All the brain tissues were homogenized with twice amount of saline. An aliquot of 10 μl of internal standard  was added into 200 μl plasma or 200 μl brain homogenate, the mixture was vortexed with 200 μl methanol for 5 min. Then, an aliquot of 50 μl of 6 M NaOH aqueous solution was added to the spiking mixture. After 10 min of hydrolysis at room temperature, ibuprofen was released from the prodrugs, and 50 μl of 6 M HCl was added to neutralize NaOH. Followed by vortexing for 5 min, 0.6 ml acetonitrile was added and the mixture was centrifuged at 15,000 rpm for 10 min. The separated supernatant was evaporated to dryness at 40 °C under air flow. The residues were redissolved in 50 μl methanol and centrifuged at 15,000 rpm for 10 min, and then 20 μl of the supernatant was injected into the HPLC system for analysis. What’s more, the concentrations of released ibuprofen and prodrugs in blood and brain were also analyzed before hydrolysis.0–t) and the maximal concentration (Cmax) were calculated by Data and Statistics . The statistical analysis of the samples was performed by using a Student’s t-test with p values < .05 as the minimal level of significance. The relative uptake efficiency (RE) and concentration efficiency (CE) were calculated to evaluate the brain-targeting property of prodrugs. The values of REbrain and CEbrain are defined as follows:The area under the curve (AUCt-test. The threshold for significance was p < .05.All the data were presented as mean ± standard deviation (SD). Statistical comparisons were performed by analysis of variance (ANOVA) for multiple groups followed by Student’s 5 and the synthetic route was demonstrated in 1 with benzyl was furnished by treating with benzyl bromide. Then, selectively acetolysis of the benzyl protecting group at C-6 in compound 2 using ZnCl2-Ac2O-AcOH gave 3. Subsequently, deacetylation of 3 under Zemplén condition gave compound 4 in 98.9% yield, which then coupled with succinic anhydride to give acid 5 by using DMAP as catalyst. The synthesis of intermediate 9 started from the available material Vc. Treatment of 6 with acetyl chloride in acetone following our earlier method afforded 5,6-O,O-isopropylidene protected Vc ketol 7  and half-lives (t1/2) of these prodrugs in aqueous solutions were calculated by linear regression of the peak area against time, and were illustrated in In order to investigate the chemical stability of the prodrugs, Glu-Vc-Ibu, Glu-Ibu and Vc-Ibu were incubated in pH 2.5, 5.0, 7.4 and 8.0 phosphate buffers, respectively. These solutions were maintained at 37 °C, and the aliquots were withdrawn in predetermined time intervals, then the concentrations of the prodrugs were determined by HPLC method. The pseudo first order rate constants . What’s more, the three prodrugs significantly relieved this damage and improved the viability of H2O2-treated cells , which indicated that the dual-targeting prodrug Glu-Vc-Ibu showed a significant efficacy compared with others (p < .05). Generation of intracellular ROS is another mechanism of oxidative damage to PC12 cells. It could be seen from 2O2. Pretreatment with the prodrugs significantly counteracted the ROS generated by H2O2, showing the better antioxidant effect than that of ibuprofen. What’s more, SOD is antioxidant defense system in cells to prevent ROS damage. Therefore, the activity of SOD was measured to evaluate the neuroprotective effect of ibuprofen and the prodrugs .The neuroprotective effect of ibuprofen and prodrugs was measured on a Hprodrugs . The res2O2 disrupted the dendritic networks and markedly decreased the cell viability , which was ascribed to the stability of these prodrugs in plasma. Hence, the prodrugs had enough time to reach the brain before they were metabolized.The curves in in vivo pharmacokinetic evaluation of prodrugs gives new insight into the possibility of utilizing glucose and Vc as means for brain targeting. It is obvious that the prodrugs could be delivered to the brain after intravenously administration . The AUC0–t and Cmax of Glu-Vc-Ibu in brain were obviously higher than that of the parent drug (Supplementary Table S2), although the opposite trend was seen in plasma. What’s more, the brain concentration of ibuprofen released from Glu-Vc-Ibu was approximately three times of that found in brain after ibuprofen was administrated for 45 min, which suggested that the prodrugs might have a slow release, long-term effect. These data further indicated our design was feasible, that the prodrug Glu-Vc-Ibu could maintain sufficient levels and deliver ibuprofen into the brain.The stration . The conbuprofen . The relin vivo with the improved neuroprotective effect compared with naked ibuprofen or other singly-modified prodrugs, suggesting that both glucose and Vc transporters (GLUT1 and SVCT2) might be involved to help the prodrug to penetrate the BBB. Therefore, the Glu-Vc modification represents a promising strategy for the development of future brain-specific drug delivery systems.In order to develop an efficient brain-targeting drug delivery system to greatly improve the brain permeability of the drug, we used ibuprofen as a model parent drug, designed, synthesized and evaluated a dual-targeting prodrug co-modified by glucose and Vc. The Glu-Vc moiety significantly increased the brain uptake of ibuprofen


Bacteroides galacturonicus, Bifidobacterium catenulatum, Lactobacillus rhamnosus GG, and Enterococcus caccae, was examined by measuring their anaerobic growth profiles. The effect of apigenin on a gut microbiota community was studied by culturing a fecal inoculum under in vitro conditions simulating the human ascending colon. 16S rRNA gene sequencing and GC-MS analysis quantified changes in the community structure. Single molecule RNA sequencing was used to reveal the response of Enterococcus caccae to apigenin. Enterococcus caccae was effectively inhibited by apigenin when cultured alone, however, the genus Enterococcus was enhanced when tested in a community setting. Single molecule RNA sequencing found that Enterococcus caccae responded to apigenin by up-regulating genes involved in DNA repair, stress response, cell wall synthesis, and protein folding. Taken together, these results demonstrate that apigenin affects both the growth and gene expression of Enterococcus caccae.Apigenin is a major dietary flavonoid with many bioactivities, widely distributed in plants. Apigenin reaches the colon region intact and interacts there with the human gut microbiota, however there is little research on how apigenin affects the gut bacteria. This study investigated the effect of pure apigenin on human gut bacteria, at both the single strain and community levels. The effect of apigenin on the single gut bacteria strains  The polyphenol apigenin  is one oScutellaria barbata D. Don (Lamiaceae) [Castanea sativa Mill. (Fagaceae) [Portulaca oleracea L. [Marrubium globosum ssp. Libanoticum [Combretum erythrophyllum (Combretaceae) [Aquilegia oxysepala [2O2 or Fe2+ [Research has found that a diet rich in the flavonoid apigenin has multiple beneficial properties. Apigenin has been identified as an active ingredient in the traditional, antibacterial, herbal medicines miaceae) , Castaneagaceae) , Portularacea L. , Marrubianoticum , Combretetaceae) , and Aquxysepala . These hxysepala , digestixysepala , abdominxysepala , gynopatxysepala  in diffexysepala , includixysepala . In eukaxysepala  and is c or Fe2+ .O-glucoside, and acylated derivatives [Apigenin is a flavonoid, which is a type of plant polyphenol. It is present in food and ingested as its glycoside conjugates, primarily as apigenin-7-ivatives . These fivatives . About 5ivatives . Since oivatives . One stuivatives . In anotivatives . These rivatives ,18.Eubacterium ramulus and Bacteroides distasonis were found to be capable of converting apigenin-7-glucoside to apigenin, however, this was not the case with Escherichia coli (Migula) [Clostridium orbiscindens, was found to degrade apigenin to 3-(4-hydroxyphenyl)propionic acid with phloretin and naringenin as intermediates [The human colon gut microbiota has been found to harbor enzymes that could degrade apigenin ,20. In gmediates . TherefoKlebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Escherichia coli (Migula), Salmonella typhimurium, Vibrio harveyi, Proteus mirabilis, and Enterobacter aerogens, but not, or only mildly inhibiting, Staphylococcus aureus, Escherichia coli (MG 1655), Bacillus subtilis or Proteus vulgaris [Previous reports have found that apigenin can also affect the growth of some bacteria, inhibiting the growth of species such as vulgaris ,8,21,22.Bacteroides galacturonicus, Bifidobacterium catenulatum, Lactobacillus rhamnosus GG, and Enterococcus caccae by analyzing their anaerobic growth profiles for 24 h. Results of this initial study indicated that apigenin had a varying effect on the growth of the different types of bacteria. Based on these results, whether or not apigenin would have an effect on a gut microbiota community structure, or short chain fatty acid production, was examined by culturing a fecal inoculum in the presence of apigenin under in vitro anaerobic growth conditions simulating the human ascending colon region. These results indicated that apigenin may have a unique interaction with Enterococcus. In order to better understand this interaction at the molecular level, single molecule RNA sequencing was performed on Enterococcus caccae, with and without apigenin, to produce a full transcriptome profile for gene expression. Comparison of these profiles revealed the effect of apigenin on Enterococcus caccae at genotypic level. Taken together, these results provide a detailed analysis on the effect of apigenin on the gut microbiota community, and in particular Enterococcus caccae.The objective of this paper was to study how apigenin, a dietary flavone with various pharmacological activities, impacts the growth and metabolism of the gut microbiota. Initially, a reductionist model was adopted to determine if apigenin would have an effect on the growth of representative gut microbial strains Lactobacillus rhamnosus GG (L. rhamnosus GG), Bacteroides galacturonicus , Enterococcus caccae (E. caccae), and Bifidobacterium catenulatum (B. catenulatum) was examined by comparing the growth profile of bacteria treated with increasing concentrations of apigenin to the control group containing no apigenin (The effect of apigenin on the growth of apigenin . Such coL. rhamnosus GG. The lowest dose of 25 µg/mL had no effect on growth at any time point (p > 0.05).Apigenin had a minimal effect on growth of me point A. The onme point A. The McB. galacturonicus was inhibited in a dose dependent manner by the presence of apigenin at both 12 and 24 h post inoculation  and the other as apigenin treated (100 µg/mL). Samples were harvested at 4, 8, 12, 24 and 48 h post inoculation to measure culture density, microbial composition, and SCFAs production.600 for the control group at 8, 12, 24, and 48 h were 0.573 ± 0.003, 0.557 ± 0.002, 0.365 ± 0.002, 0.358 ± 0.031 respectively; and for the apigenin-treated group were 0.593 ± 0.002, 0.603 ± 0.004, 0.412 ± 0.002, and 0.395 ± 0.001. Analysis of the alpha diversity for both communities using the Shannon index found that microbial diversity increased with fermentation time, with the apigenin treated group having a significantly higher Shannon index at 8, 24, and 48 h post inoculation  that were more than 0.1% of the community for at least one time point were generated . In bothThe addition of apigenin differentially affected the four major phyla in the human gut microbiota, which are Firmicutes, Bacteroidetes, Proteobacteria, and Actinobacteria . For botFor Bacteroidetes, the percentages in both groups increased with time B. At 4 hThe percent of phylum Proteobacteria increased over time for both the control and apigenin treated groups C. The adFor both the control and apigenin groups, the percentage of phylum Actinobacteria increased over time D. At 8 hE. caccae under axenic conditions demonstrated that the addition of apigenin influenced strain growth  detected in the culture were acetate, propionate, and butyrate. Other SCFAs, including 2-methylpropionate, 2-methylbutyrate, pentanoic acid, 2-methylpentanoic acid and 4-methylpentanoic acid were also detected, but only in low or trace concentrations. The amounts of acetate, propionate, and butyrate produced all increased with time for both the control and apigenin-treated groups . AcetateE. caccae grown with and without the presence of 25 µg/mL apigenin were assembled and compared. A total of 764 individual genes were identified; 141 of these had expression levels significantly different from the control (p < 0.05). Among the 141 genes, 97 were up-regulated, and 44 were down-regulated. For brevity, only genes with a greater than 1.5-fold change are considered in the Discussion. Setting the threshold at a 1.5-fold change, 60 genes were up-regulated and 30 were down-regulated. Their fold change, number, and brief description of function can be found in The gene expression profiles of L. rhamnosus GG and B.catenulatum were not affected by apigenin. This is not surprising, considering that L. rhamnosus GG is specifically used as a probiotic because it is unaffected by harsh conditions such as acid and bile [B. catenulatum is unaffected by other polyphenols, such as quercetin [L. rhamnosus GG and B. catenulatum may respond to apigenin at a higher concentration, however, the low solubility of apigenin in media limits the concentration to no more than 100 µg/mL.Apigenin is a flavonoid widely consumed in the diet, yet little is known regarding its ability to alter the gut microbiota. Using a reductionist model, the effect of apigenin on the growth of single gut commensal bacteria was first analyzed . These rand bile  and B. cuercetin . It is pE. caccae and B. galacturonicus. Among the four strains, B. galacturonicus is the only Gram-negative strain. However, the observed inhibitory effect is not considered to be determined by Gram stain status. This was concluded based on the observation that both Gram-positive and Gram-negative strains were inhibited to some extent , however, not all G+ strains were inhibited.Apigenin effectively inhibited growth of both E. caccae specifically revealed a divergent pattern between the treatment groups. For the 5 and 12.5 µg/mL apigenin groups, inhibition was observed during the early exponential phase. Yet, the rate of cell density increase was similar between the control and apigenin treated groups after 8 h, with a potential enhancement of growth due to the addition of apigenin  tracts, as well as in traditional fermented food and dairy products, and in various extraenteric environments [Enterococcus is considered a commensal opportunist and they emerged in the 1970s as some of the leading causes of multidrug-resistant, hospital-acquired infections [Enterococci have been known to cause multidrug-resistant infections, and how close E. caccae is related to the most abundant Enterococcus species in the human gut, the inhibitory effect of apigenin provides an opportunity for developing new antibiotics.Species of the ronments ,26. Entefections . ConsideThe results of the single strain testing revealed that the ability of apigenin to influence growth of the gut commensal bacteria was individualistic. Some species were affected and others were not, and among those affected the growth profiles were modified in different ways. Therefore, whether or not apigenin would have an effect on a complete gut community was questioned. Since a community has hundreds of species that are able to interact, a reductionist model cannot be used to determine a community effect. To examine the effect of apigenin on a complete community, bioreactors were set to simulate the ascending colon region only using a batch culture model. The ascending colon region was chosen because it is where the apigenin in foods first interacts with the gut bacteria; the other colon regions would be interacting with a lower concentration of apigenin or its degradation products as it has been passed from previous regions. It should be noted that the effects of apigenin could very possibly vary across different regions or microenvironments of the colon.In batch culture of the microbiota, it was observed that in both the control and apigenin groups, microbial diversity increased with time, represented by an increasing number of OTUs of higher abundance over the course of 48 h. This is not surprising since after inoculation the bacteria were able to enter into an exponential growth phase, allowing the community to mature and species that were below detection level in the fecal sample to develop. The amount of growth for both the control and apigenin was similar, with enhancement by apigenin, as determined by optical density and alpha diversity using the Shannon index. These observations are important because they indicate that apigenin did not inhibit growth and maturation of the gut microbiota, but may function to enhance overall diversity of the community.E caccae was tested under axenic conditions, a reduction of 47.1% compared to control with presence of 25 µg/mL of apigenin. This is not entirely surprising since phyla contain a large number of OTUs, which make them more robust than a single strain, and the effect of apigenin may occur at an intra-phylum level. However, the small changes noted did result in a change to the Firmicutes/Bacteroidetes (F/B) ratio, which was lower in the apigenin-treated microbiota. Since a higher F/B ratio has been associated with obesity [There were some statistically different changes between the control and apigenin treated groups at the phylum level. However, other than for phylum Proteobacteria at 48 h, those differences were not as drastic as the inhibitory effect observed when  obesity , lowerinEnterococci in the community. On the contrary, growth of genus Enterococci was enhanced with apigenin at 4 and 8 h post inoculation. This is opposite to what happened when apigenin was tested with E. caccae alone. One possible explanation is that apigenin was degraded or taken up by other species in the community, and as a result Enterococci were exposed to less apigenin. Other bacteria more susceptible to apigenin were inhibited and thus allowed for an enhancement of Enterococci. It is also possible that the Enterococci in this fecal sample were not sensitive to apigenin. Determination if this is the case would require identification of the Enterococcus species and examination of their growth in the presence of apigenin.Interestingly, apigenin did not inhibit growth of The gut microbiota community not only resides in the colon, but is a functional part of the environment. Fermentation of substrates by the gut microbiota produces short chain fatty acids, which are absorbed by the colonic cells. Therefore, the effect of apigenin on the gut microbiota may not be limited to community structure, but could also result in changes to the production of SCFAs. In this case, it is not just about an effect on who is there, but what they are doing, and what they are producing.Akkermansia muciniphila. However, this effect is yet to be tested in vivo.The results of this study are in accordance with previous findings in that the most abundant SCFAs produced were acetate, followed by propionate, and butyrate . In thisButyrate, which was significantly higher in apigenin-treated samples at all three time points, is almost completely consumed by the colonic epithelium, and it is a major source of energy for colonocytes . It has E. caccae, inhibiting growth in a single strain setting and enhancing growth in a community setting. In order to elucidate how apigenin and E. caccae may interact at the genetic level, resulting in a change in growth status, RNA expression was evaluated. Among all the 764 genes identified, 290 were described as hypothetical proteins, including the most upregulated and the most downregulated ones, thus their identities and functions are unknown. Setting the threshold at a fold change of 1.5 times among those are not hypothetical proteins, 43 genes were up-regulated and 12 were down regulated.The results of single strain and community studies demonstrated that apigenin had a divergent effect on growth of d-alanine:d-alanine ligase in bacteria. Related genes could be upregulated by apigenin as the bacteria compensates for the inhibition exerted on the end products, e.g., enzymes. The results of the gene expression for this experiment demonstrated that Tyrosine recombinase XerC, which is responsible for chromosome dimer resolution during DNA replication [d-Alanine-poly(phosphoribitol) ligase subunit 2, which participates in cell wall and cell membrane metabolism [d-alanine ligase.It has been previously indicated that apigenin affects nucleic acids, type II fatty acids and lication , was uprlication ,35,36,37lication ,39,40,41tabolism  was alsoEnterococcus caccae. Both the d-alanine:d-alanine ligase and Type II fatty acid synthetic pathway are involved in cell membrane/wall synthesis. The Type II fatty acid synthetic pathway is the principal route for membrane phospholipid acyl chains production [N-acetylmuramoyl-l-alanine amidase, upregulated 1.7-fold, participates in cell separation [The results of gene expression analysis in this study indicate that cell membrane and cell wall synthesis is likely a major target of apigenin in oduction . Severaloduction ,45; phosoduction ,47; acyloduction ,49; penioduction ,51; N-acparation ; undecapparation .Several general stress response genes were also found to be upregulated due to apigenin treatment. The MarR family transcriptional regulator was upregulated 2.5-fold compared to the control , spx/MgsProtein chaperones facilitate correct folding of proteins and help with the refolding of misfolded proteins . SeveralAmong the genes that were down-regulated, two participate in iron-sulfur (FeS) cluster formation and one is an element in FeSr biosynthesis: FeS assembly protein SufB by 1.6-fold; iron-sulfur cluster assembly scaffold protein by 1.6-fold; and SufS family cysteine desulfurase by 1.6-fold. Fe-S cluster is regulated under homeostatic control, meaning that it is regulated according to cellular requirements . A decreE. caccae and B. galacturonicus, but not L. rhamnosus GG or B.catenulatum. In apigenin-treated microbiota, phylum percentages were different from the control, with a lowered Firmicutes/Bacteroidetes (F/B) ratio. Contrary to the inhibition on E. caccae when cultured alone, apigenin enhanced growth of Enterococci in the community. Production of short chain fatty acids was promoted by apigenin. Gene expression profiles changed with apigenin-treatment showing that apigenin had a negative effect on cell wall/cell membrane synthesis, protein folding, and triggered overall stress response.The results of this study demonstrated that apigenin was able to influence growth of some gut microbes in a species dependent manner: effectively inhibiting growth of both TM Lactobacilli MRS broth  was made by resuspending 55.00 g of MRS powder in a total volume of 1 L deionized, distilled water. The solution was heated with agitation until the powder was completely dissolved.DifcoBacteroides galacturonicus medium was made using the following ingredients: Sodium polygalacturonate 4.00 g, trypticase peptone 5.00 g (Becton Dickinson), yeast extract 2.50 g, MgSO 4 × 7 H2O 2.50 g, CaCl 2 × 2 H2O 0.15 g, FeSO4 × 7 H2O 20.00 mg, (NH4)2SO4 1.40 g, l-cysteine 1.00 g, resazurine stock solution 1.5 mL, NaHCO3 40 mL 5% (w/v) solution, in a final volume of 1 L deionized, distilled water. Final pH was adjusted to 7.1 using 10 M NaOH or 37% HCl.Enterococcus caccae was made with the following ingredients: 30.00 g trypticase soy broth (Becton Dickinson), and 3.00 g yeast extract, in a final volume of 1 L deionized, distilled water. Final pH was adjusted to 7.0–7.2 using 10 M NaOH or 37% HCl.Tryticase Soy Yeast Extract Medium for Bifidobacterium catenulatum medium was made with the following ingredients: Casein peptone tryptic digest 10.00 g (Becton Dickinson), yeast extract 5.00 g, meat extract 5.00 g, Bacto Soytone 5.00 g (Becton Dickinson), glucose 10.00 g, K2HPO4 2.00 g, MgSO4 × 7 H20 0.20 g, Tween 80 1.00 mL, NaCl 5.00 g, cysteine-HCl × H2O 0.50 g, resazurin stock solution 1 mL, salt solution 40 mL in a final volume of 1 L deionized, distilled water. Cysteine-HCl × H2O was added after the medium had been boiled and cooled. Final pH was adjusted to 6.8 using 10 M NaOH or 37% HCl. Resazurin stock solution was made by dissolving 0.1 g resazurin in 100 mL deionized, distilled H2O. Salt solution was made by dissolving the following in 1 L deionized, distilled water: CaCl2 × 2 H2O 0.25 g, MgSO4 × 7 H2O 0.50 g, K2HPO4 1.00 g, KH2PO4 1.00 g, NaHCO3 10.00 g, NaCl 2.00 g, then filter sterilized.l-cysteine 0.5 g, NaCl 0.1 g, K2HPO4 40 mg, KH2PO4 40 mg, MgSO4 × 7H2O 10 mg, CaCl2 × 2H2O 6.7 mg, Tween80 2 mL, resazurine stock solution 1.5 mL , with a final volume of 1 L deionized, distilled water. The final pH was adjusted to 5.8 using 10M NaOH or 37% HCl.Basal medium  was madeAll broths were autoclaved at 121 °C for 30 min. Before being transferred into a Bactron anaerobic chamber to cool overnight, the broth was boiled under negative pressure using nitrogen gas for 10 min to remove any oxygen from the solution. All above anaerobic broths were made fresh every two to three weeks and stored at room temperature under oxygen free conditions after cooling in the anaerobic chamber. All ingredients were purchased from Sigma unless otherwise labeled.Apigenin  was purchased from Cayman Chemical Company  and was stored at −20 °C prior to use. For the single strain bacteria growth tests, stock solutions were made at 500 times the final concentration in an Eppendorf tube with DMSO  before being transferred into the anaerobic chamber. Each tube was vortexed to ensure homogenization. Stock solutions were diluted with media at a 1:9 volume ratio and a final volume of 100 µL of each diluted solution was injected into a sealed Hungate tube  containing 5 mL of media. Final concentrations of apigenin tested were 5, 12.5, 25, 50, and 100 µg/mL.For use in the batch culture experiment, apigenin was made into stock solutions 500 times the final concentration in DMSO to increase solubility before being transferred into the anaerobic chamber, as described above. The stock was then diluted with basal medium at 1:1 ratio in the anaerobic chamber to avoid corrosion of pure DMSO on plastics. Final concentration for apigenin in each bioreactor was 100 µg/mL.Lactobacillus rhamnosus GG ) was purchased from ATCC . The other four strains were ordered from the company Deutsche SammLung von Mikroorganismen und Zellkulturen GmbH : freeze dried ampoule of 3978, type strain; Bacteroides galacturonicus N6, freeze dried ampoule of 19829, type strain; Enterococcus caccae SS-1777 (E. caccae), freeze dried ampoule of 16992, type strain, Bifidobacterium catenulatum B669 (B. catenulatum). Each bacterial strain was recovered from frozen aliquots in strain specific broths (described above) and grown overnight in the anaerobic chamber at 37 °C sequentially at least twice prior to use in order to ensure recovery from freezing.All steps of microbial culture were performed using a Bactron anaerobic chamber to ensure oxygen free conditions. For the single strain experiments, anaerobic broth was aliquoted into Hungate tubes prior to starting an experiment, 5 mL per tube. The tubes were sealed with a rubber septum (Chemglass) and screw cap lid (Chemglass), and stored at room temperature in the anaerobic chamber until needed. Each Hungate tube containing 5 mL pre-aliquoted broth was injected with 100 µL of the corresponding diluted stock solution of apigenin with a 1 mL needle and a 25-gauge syringe.The cultures of bacteria grown overnight were diluted in their specific broth to 0.5 McFarland units above background. Each 5 mL Hungate tube containing the appropriate anaerobic broth and the desired concentration of apigenin was injected with 100 µL of this culture using a 1 mL syringe and a 25-gauge needle. Each Hungate tube was briefly vortexed after injection to ensure proper distribution and the McFarland units for each culture were determined using a DEN-1 densitometer . After adding apigenin and the bacteria, the McFarland units were determined as the time 0 read. The cultures were then placed into the anaerobic incubator set to 37 °C. Each culture was removed temporarily from the incubator at 4, 8, 12, and 24 h post inoculation, briefly vortexed, and the McFarland units measured using a densitometer. For each strain at each concentration of apigenin, six Hungate tubes of broth were used. Three tubes were designated as a broth control, consisting of broth containing the desired concentration of apigenin only. The other three tubes were designated as the experimental group, consisting of broth containing apigenin which were also inoculated with bacteria. The sets with DMSO only, with 0 µg/mL of apigenin, were designated as the control group.t-test was run to determine if the difference between the control and the experimental groups was statistically significant, p < 0.05.For the growth of a single strain bacteria, the McFarland readings from each group were adjusted by subtracting the broth control read from the experimental read. The adjusted numbers were plotted in a growth curve as McFarland units above broth-only over time. For each time point, a 2-tailed, unpaired homoscedastic Student’s In order to prepare a fecal inoculum to use in the batch culture experiment, 630 mL of basal medium was sterilely transferred into a bioreactor after the empty vessel had been flushed sterilely with nitrogen for 10 min. The transferred medium was then sparged with nitrogen for 20 min at 37 °C to achieve anaerobic conditions. Resazurin color indicator was used to confirm anaerobic conditions. The temperature was maintained at 37 °C with constant stirring, and the pH was computer controlled, and set to 5.8 ± 0.1 using 0.5 M HCl and 0.5 M NaOH.After the removal of oxygen from the basal media, 70 mL of defrosted human gut microbiota preparation  was added into the bioreactor, mixed three times with a syringe and allowed to grow for 16 h overnight. The donor of the gut microbiota preparation was a healthy female between 25–45 years of age, with a normal BMI, who had been following a non-vegetarian American diet, and had been antibiotic free for at least six months. The preparation was stored at −80 °C and thawed at 37 °C on the day of use. After complete thawing and thorough mixing, half of the preparation was used in the first replicate of batch fermentation experiment. The other half was frozen at −80 °C immediately after aliquoting and was used in the second replicate of the batch fermentation experiment.®, Simulator of the human intestinal microbial ecosystem . SHIME® was originally designed to simulate the complete GI tract, but for this experiment it was set up to simulate only the ascending colon. The glass bioreactors have double jackets allowing heating water to circulate across all the vessels, maintaining 37 °C. The lids were assembled with two head space flush ports, one media sparging port, one sample port for removing medium during the experiment, acid and base ports, and a pH probe. The entire system was sterilized before use. pH probes were sterilized with 75% ethanol and the rest were autoclaved at 121 °C for 15 min.Batch culture was performed in a series of bioreactors using SHIME600 in a G10S UV-Vis spectrophotometer .Prior to the start of the experiment, 540 mL of basal medium was sterilely transferred into each bioreactor using the same method as described above. Apigenin stock solution was made as described above and added to the vessel using a syringe. One vessel received DMSO only was deemed as the control group. After adding the basal medium and the apigenin, 3 mL of sample was harvested and tested for OD600 values were measured. Another set of samples were centrifuged at 5000× g for 10 min and the supernatants were then filtered with 0.20 Micron PES filters . Both the pellets and filtered supernatant were immediately stored at −80 °C until further analysis.At time 0 h, 60 mL of inoculum prepared overnight was added into each bioreactor. Nitrogen was sparged through each vessel for 10 min to ensure anaerobic condition. Growth condition was maintained for 48 h at 37 °C with constant stirring and pH controlled at 5.8 ± 0.1. Samples were harvested at 4, 8, 12, 24, and 48 h post inoculation and ODTM microtube homogenizer (Benchmark Scientific) for two, 20-s bead-beating rounds, with a 20 s interval in between. Tubes were centrifuged at 3000× g for 5 min. 300 µL of the supernatant were transferred to a new 1.5 mL Eppendorf tube. Five hundred µL of CTAB buffer were added to the original bead-beating tube. Tubes were homogenized again with two rounds of 20-s bead-beatings and a 20 s interval in between. Tubes were centrifuged at 3000× g for 5 min. 300 µL of the supernatant were transferred and combined with the previous 300 µL. Six hundred µL of Chloroform-isoamyl alcohol(CI) were added into the 1.5 mL Genemate tube, inverted and spun for 10 s with a minifuge . Five hundred µL of the upper aqueous phase were transferred to a new 2 mL Eppendorf tube. One thousand µL PEG-6000 precipitation solution were added and incubated at room temperature for 2 h. Tubes were then centrifuged for 10 min at 18,200× g. Supernatant was removed and the pellet was washed with 1 mL ice cold 70% ethanol. Tubes were centrifuged again for 10 min at 18,200× g. Supernatant was removed and the tubes were air-dried for 30 min under laminar flow in a Biosafety Cabinet . Samples were then eluded with 75 µL of PCR grade water .A DNA extraction was performed on harvested samples using a bead beater-CTAB extraction method. For each sample, 500 µL CTAB buffer and 500 µL of phenol-chloroform-isoamyl alcohol (PCI) were added to a pellet spun down from the collected samples. After vortexing, all liquid was transferred to a bead-beating tube, prefilled tube kit, with Triple-PureTM High Impact Zirconium Beads of diameter 0.1 mm . Bead beating tubes were placed in a BeadBug2HPO4 and 4.091 g NaCl in 200 mL deionized, distilled water. The solution was autoclaved at 120 °C for 30 min. The day before use, 10 g of Hexadecyltrimethylammonium bromide (CTAB) was added to the autoclaved solution and heated to 60 °C with agitation until the particles were completely dissolved. PEG-6000 precipitation solution was made by completely dissolving 300 g of polyethylene glycol 6000  and 93.5 g NaCl in 1000 mL deionized, distilled water. The solution was autoclaved at 120 °C for 30 min and later stored at room temperature.CTAB buffer was made by dissolving 4.20 g KBarcoded PCR primers annealing to the V1-V2 region of the 16S rRNA gene was used for library generation using the primer sequences 27F (AGAGTTTGATCCTGGCTCAG) and 338R (TGCTGCCTCCCGTAGGAGT) ,62. PCR t-test was run to determine if the percentage difference of a given OTU, genus, order, or phylum between the control and the experimental groups was statistically significant, p < 0.05.Sequence data was processed using QIIME version 1.9 . Read paEnterococcus caccae cultured with DMSO only and 25 µg/mL of apigenin were harvested at 8 h post inoculation. Two samples were collected from each group, centrifuged at 5000× g for 5 min at 4 °C to pellet the cells, and the supernatant discarded. RNA from these samples was purified using an Ambion PureLink® RNA Mini Kit .Ribosomal RNA depletion of the bacterial total RNA was performed using the Illumina Bacterial Ribo-Zero kit (#MRZMB126). Magnetic beads were prepared by vortexing and aliquoting 225 µL per reaction into RNase-free tubes, placing on magnetic stand, and removing supernatant. Beads were washed twice with RNase free water, followed by addition of 65 µL of Magnetic Bead Resuspension solution and 1 µL of RiboGuard RNase Inhibitor.RNA was treated with rRNA Removal solution by combining ~1 µg of total RNA with RNase-free water, 4 µL of Ribo-zero rRNA Reaction Buffer, and 8 µL Ribo-zero Removal Solution to a total of 40 µL, and incubating at room temperature for 5 min.The probe-hybridized RNA solution was then transferred to the magnetic beads and the solution was mixed, incubated at room temperature for 5 min, and subsequently at 50 °C for 5 min. Tubes were removed from the heat and placed on the magnetic stand. Supernatant containing 90 µL of rRNA depleted sample was removed and placed on ice until purified.g for 30 min and supernatant was carefully removed. The pellet was washed twice with a 70% ethanol solution, air dried, and dissolved in RNase free water. Concentration was measured using the nanodrop.Ethanol precipitation was performed by adjusting the volume of sample to 180 µL with water, followed by addition of 18 µL of 3M sodium acetate, 2 µL of glycogen (10 mg/mL), and 600 µL of 100% ethanol. This solution was gently vortexed and placed at 80 °C for overnight. After approximately 16 h, the solution was centrifuged at 10,000× First strand cDNA synthesis was carried out by initially incubating 7 µL of sample plus water (~100 ng RNA) at 95 °C for 5 min and chilling on ice for 2 min. While on ice, 5 µL of 50 ng/µL random hexamers and 1 µL of dNTP mix were added to the RNA and the mixture was incubated at 65 °C for 5 min. While on ice, 4 µL of 5 × buffer and 1 µL of 0.1 M DTT were mixed in to the solution and incubated at 15 °C for 20 min. Finally, 1 µL of RNase Inhibitor and 1.5 µL of Superscript III Reverse Transcriptase (Invitrogen #18080-044) were added. The following program was run on the thermal cycler: 25 °C for 10 min, 40 °C for 40 min, 55 °C for 50 min, and 85 °C for 5 min. Upon completion, 1 µL each of RNase H and RNase If was used to digest the RNA strand at 37 °C for 30 min. Performa DTR Gel filtration cartridges  were used to purify the cDNA. 3’ Poly A tailing was initiated by addition of 4 µL of 10 × TdT buffer and 4 µL of 2.5 mM CoCl2 and water to 9 µL of cDNA in a final volume of 33 µL, denatured at 95 °C for 5 min, and snap cooled. Then 2 µL of 1 mM dATP and 0.5 µL of 20 U/µL Terminal Transferase  were added and the solution was incubated for 10 min at 37 °C. Finally, 2 µL of 1 mM ddATP was spiked into the reaction and it was incubated at 37 °C for ½ h, and 70 °C for 10 min to inactivate the enzyme.Enterococcous caceae gene sequences were downloaded from the National Center for Biotechnology Information  under accession number NZ_KB946335. Then UCLUST [E. caceae. Gene expression was measured by reads per kilobase of mapped reads (RPKM [The n UCLUST  with defds (RPKM ), where ds  at 5000 rpm for 10 min. The supernatant was drawn into a syringe and sterile filtered using a 0.22 PES (Corning) syringe filter and transferred new 5 mL vials. The fluid was then stored in −80 °C freezer until analysis.To begin SCFA quantification, the samples were thawed at 40 °C for 30 min. The total SCFAs were extracted from the media with diethyl ether for GCMS  analysis. The GC-MS method involved injecting 1 µL of sample into the 260 °C injection port. Using a split ratio of 1:20 and a flow rate of 1.00 mL/min of helium, the sample was deposited on the Stabilwax-DA 30 m × 0.25 mm column  which was held at 125 °C for 1 min and then ramped to 170 °C at 30 °C/min, then to 220 °C at 10 °C/min and then to 250 °C at 50 °C/min where the temperature was held for 3 min. The interface temperature between the GC and MS was held at 250 °C and the ion source temperature was 220 °C.t-test was run to determine if the concentration difference between the control and the experimental groups was statistically significant, p < 0.05.For each time point, a 2-tailed, unpaired homoscedastic Student’s E. caccae. Analysis of RNA expression indicated that apigenin affects E. caccae cell wall/membrane synthesis and increased the incidence of protein misfolding. B. galacturonicus growth was also inhibited by apigenin, while B. catenulatum and L. rhamnosus GG were not affected. In the bacterial community cultured in vitro, apigenin promoted overall growth and diversity, lowered Firmicutes to Bacteroidetes ratio, and promoted production of short chain fatty acids, including butyrate, which is associated with health. Taken together, these observations may explain the health benefits of an apigenin-rich diet.For the first time, the effect of apigenin on the human gut microbiota was documented. The results of testing using a reductionist model demonstrated that this effect varied with species. Among the species tested, the most effective inhibition by apigenin was observed on