Screening of active fractions of Jiegu ointment for promoting fracture healing in New Zealand rabbits / 中国药房

OBJECTIVE To screen the active site of Jiegu ointment in promoting fracture healing in New Zealand rabbits. METHODS The ethanol extract of Jiegu ointment， as well as the ethyl acetate and n-butanol parts， were prepared and mixed with honey to form a plaster with appropriate viscosity. The radial fracture model of left forelimb in New Zealand rabbit was established and divided into model control group， ethanol extract group， ethyl acetate fraction group and n-butanol fraction group， with 6 rabbits in each group. Except for model control group， rabbits of all other groups were treated with corresponding polar part of Jiegu ointment for external application， for 4 weeks. The radial fracture healing of rabbits was studied by X-ray examination. Enzyme-linked immunosorbent assay was used to detect the serum levels of interleukin-6 （IL-6）， tumor necrosis factor α （TNF-α）， osteocalcin （OC）， vascular endothelial growth factor A （VEGFA）， basic fibroblast growth factor 2 （bFGF2） and alkaline phosphatase （ALP）. HE staining was adopted to observe the changes of pathological morphology of rabbit fracture site， and immunohistochemical method was used to detect the protein expression of bFGF2 in fracture site of rabbits. RESULTS The healing speed of the fracture site in the n-butanol fraction group was the fastest， followed by ethanol extract group， and the ethyl acetate fraction group was the slowest； the serum levels of TNF-α and IL-6 in n-butanol fraction group decreased the fastest， while the levels of ALP， bFGF2， OC and VEGFA increased the fastest [significant increase compared with ethanol extract group （P＜0.01）]； the chondrocytes at the fracture fraction completely disappeared， forming a large number of bone marrow cavities， and the bone trabeculae in the bone marrow cavity were officially formed. The expression level of bFGF2 was also higher than ethanol extract group. CONCLUSIONS The effect of n-butanol fraction on promoting fracture healing is more significant than ethyl acetate fraction and ethanol extract， and n-butanol fraction is the active fraction of Jiegu ointment to promote fracture healing.

Mechanism of n-butanol alcohol extract of Baitouweng Decoction in treatment of vulvovaginal candidiasis based on negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis / 中国中药杂志

This study aimed to explore the mechanism of n-butanol alcohol extract of Baitouweng Decoction(BAEB) in the treatment of vulvovaginal candidiasis(VVC) in mice based on the negative regulation of NLRP3 inflammasome via PKCδ/NLRC4/IL-1Ra axis. In the experiment, female C57BL/6 mice were divided randomly into the following six groups: a blank control group, a VVC model group, high-, medium-, and low-dose BAEB groups(80, 40, and 20 mg·kg~(-1)), and a fluconazole group(20 mg·kg~(-1)). The VVC model was induced in mice except for those in the blank control group by the estrogen dependence method. After modeling, no treatment was carried out in the blank control group. The mice in the high-, medium-, and low-dose BAEB groups were treated with BAEB at 80, 40, and 20 mg·kg~(-1), respectively, and those in the fluconazole group were treated with fluconazole at 20 mg·kg~(-1). The mice in the VVC model group received the same volume of normal saline. The general state and body weight of mice in each group were observed every day, and the morphological changes of Candida albicans in the vaginal lavage of mice were examined by Gram staining. The fungal load in the vaginal lavage of mice was detected by microdilution assay. After the mice were killed, the degree of neutrophil infiltration in the vaginal lavage was detected by Papanicolaou staining. The content of inflammatory cytokines interleukin(IL)-1β, IL-18, and lactate dehydrogenase(LDH) in the vaginal lavage was tested by enzyme-linked immunosorbent assay(ELISA), and vaginal histopathology was analyzed by hematoxylin-eosin(HE) staining. The expression and distribution of NLRP3, PKCδ, pNLRC4, and IL-1Ra in vaginal tissues were measured by immunohistochemistry(IHC), and the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues were detected by immunofluorescence(IF). The protein expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by Western blot(WB), and the mRNA expression of NLRP3, PKCδ, pNLRC4, and IL-1Ra was detected by qRT-PCR. The results showed that compared with the blank control group, the VVC model group showed redness, edema, and white secretions in the vagina. Compared with the VVC model group, the BAEB groups showed improved general state of VVC mice. As revealed by Gram staining, Papanicolaou staining, microdilution assay, and HE staining, compared with the blank control group, the VVC model group showed a large number of hyphae, neutrophils infiltration, and increased fungal load in the vaginal lavage, destroyed vaginal mucosa, and infiltration of a large number of inflammatory cells. BAEB could reduce the transformation of C. albicans from yeast to hyphae. High-dose BAEB could significantly reduce neutrophil infiltration and fungal load. Low-and medium-dose BAEB could reduce the da-mage to the vaginal tissue, while high-dose BAEB could restore the damaged vaginal tissues to normal levels. ELISA results showed that the content of inflammatory cytokines IL-1β, IL-18, and LDH in the VVC model group significantly increased compared with that in the blank control group, and the content of IL-1β, IL-18 and LDH in the medium-and high-dose BAEB groups was significantly reduced compared with that in the VVC model group. WB and qRT-PCR results showed that compared with the blank control group, the VVC model group showed reduced protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues of mice and increased protein and mRNA expression of NLRP3. Compared with the VVC model group, the medium-and high-dose BAEB groups showed up-regulated protein and mRNA expression of PKCδ, pNLRC4, and IL-1Ra in vaginal tissues and inhibited protein and mRNA expression of NLRP3 in vaginal tissues. This study indicated that the therapeutic effect of BAEB on VVC mice was presumably related to the negative regulation of NLRP3 inflammasome by promoting PKCδ/NLRC4/IL-1Ra axis.

Effects of N-butanol extract of Amygdalus mongolica on rats with bleomycin‐induced pulmonary fibrosis based on metabolomics

Pulmonary fibrosis (PF) is a major public health issue with limited treatment options. As the active ingredient of the n-butanol extract of Amygdalus mongolica (BUT), amygdalin inhibits PF. However, its mechanisms of action are unclear and need further verification. Therefore, the purpose of the present studies was to investigate the anti-fibrotic effects of BUT on PF by serum metabolomics and the transforming growth factor β (TGF-β) pathway. Sixty male Sprague-Dawley rats were randomly divided into control, untreated PF, prednisone-treated (5 mg/kg), and BUT-treated (1.75, 1.25, 0.75 g/kg) groups, and the respective drugs were administered intragastrically for 21 days. The serum metabolomics profiles were determined by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) and metabolism network analysis. The expression of TGF-β1, Smad-3, Smad-7, and α-smooth muscle actin (α-SMA) was measured using a real-time polymerase chain reaction in the lung tissue. BUT significantly alleviated fibrosis by reducing the mRNA expressions of TGF-β1 (from 1.73 to 1.13), Smad-3 (from 2.01 to 1.19), and α-SMA (from 2.14 to 1.19) and increasing that of Smad7 (from 0.17 to 0.62). Twenty-eight potential biomarkers associated with PF were identified. In addition, four key biomarkers were restored to baseline levels following BUT treatment, with the lowest dose showing optimal effect. Furthermore, A. mongolica BUT was found to improve PF by the pentose phosphate pathway and by taurine, hypotaurine, and arachidonic acid metabolism. These findings revealed the mechanism of A. mongolica BUT antifibrotic effects and metabolic activity in PF rats and provided the experimental basis for its clinical application.

Effect of Polysaccharides and n-Butanol Fractions of Atractylodis Rhizoma Stir-fried with Bran on Metabonomics in Rats with Spleen Deficiency / 中国实验方剂学杂志

ObjectiveTo explore the regulatory effect of polysaccharides and n-butanol fractions of Atractylodis Rhizoma stir-fried with bran on the plasma metabolites of spleen-deficient rats， and then to elucidate their mechanisms of spleen-enhancing effects. MethodForty male SD rats were randomly divided into the blank group， model group， polysaccharide group （FD group， 0.075 6 g·mL-1·d-1）， n-butanol fractions group （FZ group， 0.012 1 g·mL-1·d-1）， with 10 rats in each group. Except the blank group， the other three groups used the compound factors of overwork， dietary disorders and intragastric administration of Sennae Folium decoction to replicate the rat model of spleen deficiency. After the end of modeling， the FD group and FZ group were given the corresponding medicinal solution by gavage for 7 d， meanwhile， the blank group and model group were given an equal volume of saline. The plasma samples from rats in the blank， model， FZ and FD groups were analyzed by ultra-high performance liquid chromatography-quadrupole/electrostatic field orbitrap high-resolution mass spectrometry （UHPLC-Q-Orbitrap-MS）， multivariate statistical methods were used to process the data and screen differential metabolites， and metabolic pathway enrichment analysis of the screened differential metabolites was performed using the Kyoto Encyclopedia of Genes and Genomes （KEGG)）database and MetaboAnalyst 5.0. ResultThe results of multivariate statistical analysis showed that there were significant differences in plasma metabolites between the model group and blank group， FZ group and model group， FD group and model group. There were 380 differential metabolites between the blank group and the model group， of which 78 and 57 were called back by polysaccharides and n-butanol fractions of Atractylodis Rhizoma stir-fried with bran， respectively. Metabolic pathway enrichment results showed that the n-butanol fractions mainly affected glycine， serine and threonine metabolism， alanine， aspartate and glutamate metabolism， D-arginine and D-ornithine metabolism， which were summarized as amino acid metabolism， while the polysaccharides mainly affected glycine， serine and threonine metabolism， alanine， aspartate and glutamate metabolism， tricarboxylic acid cycle， biotin metabolism and thiamine metabolism. ConclusionBoth of polysaccharides and n-butanol fractions of Atractylodis Rhizoma stir-fried with bran have significant regulating effects on the metabolic abnormalities in spleen-deficient rats， in which the n-butanol fractions is mainly involved in amino acid metabolism， and the polysaccharides are involved in energy metabolism and cofactor and vitamin metabolism in addition to regulating amino acid metabolism.

n-butanol part from Qubai tablet and study on its pharmacodynamics to de melanocyte model / 中国药房

OBJECTIVE To stud y the chemical cons tituents of n-butanol part of Qubai tablet and its pharmacodynamic effect on the model of de melanocyte. METHODS The n-butanol part of Qubai tablet was prepared. The chemical constituents were analyzed by ultra-high performance liquid chromatography-mass spectrometry （UPLC-MS）. Taking mice B 16 melanoma cells as the research object ，the de melanocyte model was established and divided into model group ，positive control different concentration groups（8-methoxypsoralen 10，50，100，150，200 μmol/L），solvent group （diluted with DMSO ）and Qubai tablet n-butanol part different concentration groups （10，50，100，150，200 μmol/L）. The number of cells were observed by inverted microscope ，and the cell proliferation rate ，the rate of melanin production and promotion rate of tyrosinase activity were also detected. RESULTS In the positive and negative ion mode ，53 compounds in the n-butanol part of Qubai tablet were preliminarily determined （29 in the positive ion mode ，33 in the negative ion mode ，overlapping 9），of which coumarins accounted for the largest proportion ， followed by flavonoids. The n-butanol part of Qubai tablet could significantly increase the number of cells ，which was positively correlated with the action time and administration concentration. It could significantly increase the proliferation rate of cells ，the rate of melanin production and promotion rate of tyrosinase activity （P＜0.01）. CONCLUSIONS Coumarins and flavonoids may be the material basis for the anti-vitiligo effect of n-butanol part from Qubai tablet ；anti-vitiligo effect of n-butanol part of Qubai tablet may be realized by promoting tyrosinase activity.

Analysis and Identification of Chemical Constituents in Hypoglycemic Effective Fractions of Longan Folium Based on UPLC-Q-Orbitrap HRMS / 中国实验方剂学杂志

Objective:Chemical constituents in hypoglycemic effective fractions of Longan Folium were isolated and identified by ultra performance liquid chromatography-quadrupole/electrostatic field orbitrap high resolution mass spectrometry （UPLC-Q-Orbitrap HRMS） to clarify the hypoglycemic substance basis of Longan Folium. Method:Chemical constituents in hypoglycemic effective fractions of Longan Folium were isolated on a Thermo Hypersil GOLD C₁₈ column （2.1 mm×100 mm， 1.9 μm）， the mobile phase was 0.1% formic acid acetonitrile solution and 0.1% formic acid solution （containing and 10 mmol ammonium acetate） for gradient elution. HRMS was operated in the positive and negative ion modes with the scanning range of <italic>m</italic>/<italic>z</italic> 100-1 500. Result:The secondary fragment ion information of target compounds was selected and compared with the compounds reported in the databases and related literature to further confirm these compounds. Nine compounds were identified in the ethanol fraction of Longan Folium， including cynaroside， kaempferol， quercitrin， luteolin， shikimic acid， citric acid， <italic>L</italic>-tyrosine， adenosine and nicotinamide. A total of 11 compounds were determined in the ethyl acetate fraction （cynaroside， quercitrin， kaempferol， luteolin， shikimic acid， gallic acid， protocatechuic acid， adenosine， nicotinamide， <italic>L</italic>-phenylalanine and scopoletin）， and 10 compounds were identified in the <italic>n</italic>-butanol fraction （cynaroside， kaempferol-3-<italic>O</italic>-rutinoside， kaempferol， astragalin， luteolin， citric acid， gallic acid， adenosine， nicotinamide and 5-hydroxymethylfurfural）. And five common compounds were identified in these three hypoglycemic effective fractions. Conclusion:The established UPLC-Q-Orbitrap HRMS can quickly identify chemical constituents in three hypoglycemic effective fractions of Longan Folium， their main chemical constituents are flavonoids and their glycosides， organic acids and nitrogen-containing compounds， which provides technical support and scientific evidence for the study on pharmacodynamic material basis and quality control of Longan Folium.

Antidepressant Effect of n-Butyl Alcohol Extracts from Xiaoyaosan via IGF-1Rβ/PI3K/Akt Pathway / 中国实验方剂学杂志

Objective:To investigate the intervention effect of <italic>n</italic>-butyl alcohol extracts from Xiaoyaosan against depression-like behavior induced by chronic unpredictable mild stress （CUMS） in model mice and the role of insulin-like growth factor-1 receptor <italic>β</italic> （IGF-1R<italic>β</italic>）/phosphatidylinositol-3 kinase （PI3K）/protein kinase B （Akt） signaling pathway in such intervention. Method:The effective dose of n-butyl alcohol extracts from Xiaoyaosan was preliminarily determined in model mice with behavioral despair. Then the male C57BL/6 mice were randomly divided into the blank group， model group， fluoxetine group， Xiaoyaosan group， and the low- （20 g·kg^-1） and high-dose （40 g·kg^-1） <italic>n</italic>-butyl alcohol extract groups. The mice in all groups except for the blank group were exposed to CUMS for inducing the depression-like behavior， which was judged by the sucrose preference test （SPT）. The successfully modeled mice in the medication groups were intragastrically administered with the corresponding drugs， whereas those in the blank and model groups were treated with an equal volume of solvent for five successive weeks. Following the SPT， tail suspension test （TST）， and novelty suppressed feeding test （NSFT） at the end of the fifth week， the insulin-like growth factor-1 （IGF-1） levels in mouse serum and hippocampus were measured by enzyme-linked immunosorbent assay （ELISA）. The average optical density （<italic>IA</italic>） of Nissl bodies in mouse hippocampal CA3 region was detected by toluidine blue staining. The 5-bromo-2-deoxyuridine （Brdu） and doublecortin （DCX） expression in the dentate gyrus （DG） was assayed using immunofluorescence method. The protein expression levels of IGF-1R<italic>β</italic>， PI3K， phosphorylated-PI3K （p-PI3K）， Akt， p-Akt， cysteine aspartic acid-specific protease 3 （Caspase-3）， and cleaved Caspase-3 in the hippocampus were determined by Western blot. Result:The results of forced swimming test and TST showed that n-butyl alcohol extracts from Xiaoyaosan at 9.1 and 40 g·kg^-1 both significantly shortened the immobility time of mice （<italic>P</italic><0.05， <italic>P</italic><0.01）， indicating that the effective dose ranged from 9.1-40 g·kg^-1. Compared with the model control， the n-butyl alcohol extracts from Xiaoyaosan at 20 and 40 g·kg^-1 significantly increased the sucrose preference percentage （<italic>P</italic><0.05， <italic>P</italic><0.01）， shortened the immobility time in TST （<italic>P</italic><0.01） and the feeding latency in NSFT （<italic>P</italic><0.01）， reversed the down-regulated IGF-1 content in mouse serum and hippocampus （<italic>P</italic><0.01）， increased the AOD of Nissl bodies in the hippocampal CA3 region （<italic>P</italic><0.01）， promoted the expression of Brdu and DCX in DG （<italic>P</italic><0.05， <italic>P</italic><0.01）， and down-regulated the protein expression levels of IGF-1R<italic>β</italic> （<italic>P</italic><0.05， <italic>P</italic><0.01）， p-PI3K/PI3K （<italic>P</italic><0.05， <italic>P</italic><0.01）， p-Akt/Akt （<italic>P</italic><0.05）， and cleaved Caspase-3/Caspase-3 in the hippocampus of CUMS mice. Conclusion:The n-butyl alcohol extracts from Xiaoyaosan are equivalent to Xiaoyaosan in inhibiting expression. They alleviate the depression-like behavior in CUMS mice， induce the production of Nissl bodies in hippocampal CA3 region， enhance neuronal proliferation and differentiation in DG， and facilitate neurogenesis. All these may be related to the inhibition of over-activated IGF-1R<italic>β</italic>/PI3K/Akt pathway and the reduction of neuronal apoptosis.

Anti-acetylcholinesterase Active Ingredients from Aconitum tanguticum by UPLC-Q-TOF-MS/MS / 中国实验方剂学杂志

Objective:To identify the anti-acetylcholinesterase active ingredients in <italic>Aconitum tanguticum</italic>， so as to lay the foundation for finding new anti-Alzheimer's disease （AD） drugs. Method:The anti-acetylcholinesterase active fractions of <italic>A. tanguticum</italic> were screened by the modified Ellman's method， and the chemical composition of the active fraction was analyzed by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-Q-TOF-MS/MS）. The chromatographic separation was performed on an ACQUITY UPLC BEH C₁₈ column （2.1 mm×50 mm， 1.7 μm） with acetonitrile （A）-0.4% ammonia aqueous solution （B） as mobile phase for gradient elution， and the column temperature was set at 30 ℃ with the flow rate of 0.4 mL·min^-1. Phase A of the dichloromethane fraction changed with time as follows：0-3 min， 5%A； 3-7 min， 5%-20%A； 7-11.5 min， 20%-33%A； 11.5-15.5 min， 33%-50%A； 15.5-20.5 min， 50%-80%A； 20.5-23 min， 80%-85%A； 23-25 min， 85%-95%A. Phase A of the <italic>n</italic>-butanol fraction changed with time as follows：0-2 min， 5%A； 2-8 min， 5%-20%A； 8-11 min， 20%-33%A； 11-15 min， 33%-95%A. Mass spectrometry was performed on electrospray ionization， data were collected in positive ion mode， and the detection range was <italic>m</italic>/<italic>z</italic> 100-1 500. Result:Both the dichloromethane and <italic>n</italic>-butanol fractions had a certain inhibitory effect on acetylcholinesterase， their half inhibitory concentration （IC₅₀） values were （64±4.4） mg·L^-1 and （85.7±3.8） mg·L^-1， respectively. By UPLC-Q-TOF-MS/MS analysis， a total of 21 alkaloids were identified from the dichloromethane fraction， and 11 alkaloids were identified from <italic>n</italic>-butanol fraction. Guan-fu base Ⅰ， found in both fractions， was first discovered in <italic>A. tanguticum</italic>. Conclusion:Diterpene alkaloids are the main anti-acetylcholinesterase substances of <italic>A. tanguticum</italic>， which is worth further exploration.

Chemical constituents from the stem of Callerya nitida Benth.var.hirsutissima Z.Wei / 中国药科大学学报

@#The chemical constituents from 70% ethanol petroleum ether and n-butanol extractions of Callerya nitita Benth.var.hirsutissima.Z.Wei. were separated by preparative high-performance liquid chromatographic techniques, including repeated column chromatography over macroporous adsorption resin, silica gel, ODS, Sephadex LH-20. The structures of the compounds were identified by their physicochemical properties, spectral data, and mass spectrometry data, in comparison with literature. In our research, one triterpenoids, taraxerone (1), and twenty flavonoids, including genistein-4′-O-β-glucoside (2), 5-hydroxy-4′-methoxyisoflavone-7-O-β-D-glucopyranosyl-(1→6)-β-D-glucopyranoside (3), biochanin A 7-O-β-D-apiofuranosyl-(1→5)-β-D-apiofuranosyl- (1→6)-β-D-glucopyranoside (4), formononetin-7-O-β-D-galactopyranoside (5), 5,7-dihydroxy-3′,4′-dimethoxyisoflavone (6), biochanin A-7-O-β-D-apiofuranosyl-(1→2)-β-D-glucopyranoside (7), 5, 7-dihydroxyisoflavone-4′-O-α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranoside (8), formononetin-7-O-D-apio-β-D-furanosyl(l→2)-β-D-glucopyranoside (9), 4′-hydroxy-3′-methoxyisoflavone-7-O-β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside (10), prunetin (11), prunetin 4′-O-β-D-glucopyranoside (12), pratensein7-O-β-D-glucoside (13), 8-methoxyisoformononetin (14), genistein (15), 3′-hydroxybiochanin A (16), biochanin A (17), 5,7-dihydroxy-3′,5′-dimethoxyisoflavone (18), ononin (19), isoformononetin (20), 5,7,3′,4′-tetrahydroxyflavone (21) were isolated from the two extract parts.Compounds 1-10, 12-14, 16-18, 20 were obtained from this plant, and it is the first time to investigate the plant for the first time.

Chemical Constituents of n-Butanol Extract Part of Akebia trifoliata Caulis / 中国实验方剂学杂志

<b>Objective::To study the chemical constituents from <italic>n</italic>-butanol extract of <italic>Akebia trifoliata</italic> caulis. <b>Method::The 100 kg caulis of <italic>A</italic>. <italic>trifoliata</italic> was extracted with 75% ethanol (EtOH) for three times by heating reflux. These 3 extracts were decompressed and concentrated, and then dissolved in water. The solvent was successively extracted with dichloromethane, ethyl acetate and <italic>n</italic>-butanol. The chemical constituents from the <italic>n</italic>-butanol fraction were isolated by macroporous, silica gel, sephadex LH-20 and ODS columns, and semi-preparative high performance liquid chromatography, and their chemical structures were determined through MS, NMR analysis (¹H and ¹³C-NMR) and spectroscopic data from literatures. <b>Result::Totally 14 compounds were isolated and identified as mutongsaponin B(<bold>1</bold>), mutongsaponin C(<bold>2</bold>), saponin PH(<bold>3</bold>), begoniifolide A(<bold>4</bold>), 2<italic>α</italic>, 3<italic>β</italic>, 23-trihydroxy-30-noroleana-12, 19-dien-28-oicacid-<italic>O</italic>-<italic>β</italic>-<italic>D</italic>-xylopyranosyl-(1→3)-<italic>α</italic>-<italic>L</italic>-rhamnopyranosyl-(1→4)-<italic>β</italic>-<italic>D</italic>-glucopyranosyl-(1→6)-<italic>β</italic>-<italic>D</italic>-glucopyranosyl ester(<bold>5</bold>), akemisaponins D(<bold>6</bold>), akemisaponins E(<bold>7</bold>), asiaticoside(<bold>8</bold>), saponin PJ1(<bold>9</bold>), scheffoleoside A(<bold>10</bold>), symplocosneolignan A(<bold>11</bold>), kalopanax-saponins D(<bold>12</bold>), leonticin E(<bold>13</bold>), ciwujianoside A₁(<bold>14</bold>). <b>Conclusion::Compounds <bold>1-4</bold>, <bold>11</bold>, <bold>13, 14</bold> were isolated from this plant for the first time. The discovery of these compounds further enriched the chemical constituents of <italic>A</italic>. <italic>trifoliata</italic>, and provided experimental and scientific basis for the comprehensive development and utilization of <italic>A</italic>. <italic>trifoliata</italic>.

Effects of Jingfang n-butanol extraction isolated fraction A on LPS-induced inflammation in RAW264.7 cells / 中国中药杂志

The LPS-induced RAW264. 7 cells inflammation model was used as a carrier to investigate the in vitro anti-inflammation effects of Jingfang n-butanol extraction(JFNE) isolated fraction A and explore its preliminary anti-inflammation mechanism by observing the regulatory effect on PI3 K/AKT signaling pathway and NF-κB pathway. The RAW264. 7 cells inflammation model was established by stimulating with LPS for 12 h. After 3 h pre-treatment with fraction A,the contents of interleukin-6(IL-6),interleukin-1β(IL-1β) and tumor necrosis factor(TNF-α) in the supernatant of RAW264. 7 cells inflammation model were determined by ELISA and the contents of NO in supernatant were assayed by Griess. Reverse transcription-polymerase chain reaction(RT-PCR) method was used to determine the expression of IL-6,IL-1β,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammatory cells,and the expression levels of phosphorylated and total PI3 K/AKT protein,NF-κB p50,p65,p-p65,p105 protein in cells were determined via Western blot. In addition,LC-MS and database were used to identify the possible chemical constituents in fraction A. The results showed that fraction A could significantly reduce the release levels of NO,IL-6,IL-1β and TNF-α in the supernatant and the expression of IL-6,IL-1β,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammation model cells(P<0. 05 or P<0. 01) and significantly inhibit the phosphorylation expression levels of PI3 K and AKT protein and mRNA expressions(P<0. 05 or P<0. 01). Moreover,fraction A could significantly reduce the levels of NF-κB p50,p-p65 and i NOS protein,as well as NF-κB1,Rela mRNA expressions in RAW264. 7 cells,and increase the expression of CHUK gene.A total of 196 compounds were identified from fraction A in the composition analysis,and isoobtusilactone,5-O-methyl-vismitol,emebel(embelin) and prim-O-glucosylcimifugin showed high contents. The results all above showed that fraction A had a certain antiinflammatory effect in LPS-induced RAW264. 7 inflammation model cells,and its anti-inflammatory effects may be related to its regulatory effect on the activation of PI3 K/AKT signaling pathway and NF-kappa B signaling pathway. In addition,emblin may be its effective anti-inflammation chemical composition.

Spectrum-effect Relationship of Anti-inflammatory Active Components from Nonvolatile Fraction of Blumea balsamifera Based on Gray Correlation Analysis / 中国实验方剂学杂志

Objective: To study the correlation between UPLC fingerprint of anti-inflammatory effect of active components from nonvolatile fraction of Blumea balsamifera, and to provide the basis for clarifying the anti-inflammatory material basis of B. balsamifera. Method: UPLC was used to establish fingerprint of nonvolatile fraction of 12 batches of B. balsamifera and their common fingerprint peaks were identified by UPLC-Q-TOF-MS.The corresponding pharmacodynamic data were obtained by auricle swelling and inflammation model mice induced by xylene, and spectrum-effect relationship was established by gray correlation analysis. Result: A total of 14 common peaks in nonvolatile fraction of B. balsamifera were established by UPLC fingerprint and 9 common peaks of them were identified.The correlation between UPLC fingerprint and the anti-inflammatory activity was from 0.717 1 to 0.550 5.The contribution of chemical compositions represented by each characteristic peak to the anti-inflammatory efficacy was in the order of peak 3 > peak 9 > peak 4 > peak 11 > peak 2 > peak 1 > peak 14 > peak 7 > peak 6 > peak 5 > peak 12 > peak 8 > peak 10 > peak 13, and the top two peaks with strong contribution to anti-inflammatory effect were peak 3 and peak 9, they were 3-O-caffeoylquinic acid and 3, 5-di-O-caffeoylquinic acid identified by contrast reference substances, respectively. Conclusion: The active substances in nonvolatile fraction of B. balsamifera are obtained through the study on the relationship between spectrum and efficiency, and the anti-inflammatory efficacy of the nonvolatile fraction is the result of combination of various components.It is clear that the caffeoylquinic acid derivates act as predominant anti-inflammatory active substance of nonvolatile fraction of B. balsamifera.

Study on Chemical Constituents of n-Butanol Part from the Roots of Camellia oleifera / 中国药房

OBJECTIVE： To study the chemical constituents of n-Butanol part from the roots of Camellia oleifera， and to provide reference for elucidating the pharmacodynamic substance basis of it’s anti-tumor effect. METHODS： The ethanol extracts were obtained by using 95％ ethanol as extraction solvent to extract the roots of C. oleifera， and the n-Butanol part was obatined after the extracts were extratced with ethyl acetate and water-saturated n-butanol solution in turn. The chemical constituents of n-butanol part were isolated and purified by D101 macroporous resin column， silica gel， atmospheric pressure reversed phase column chromatography， sephadex gel SephadexLH-20 column chromatogram and preparative HPLC. The structure of the compounds was identified by spectroscopic analysis of physicochemical constants， electrospray ionization mass spectrometry， 1H-NMR and 13C-NMR. RESULTS： Eight compounds were isolated from the roots of C. oleifera and elucidated as quercetin 3′ -O-β-D-glucoside （compound 1）， apigenin-7-O-β-D-glucoside （compound 2）， （+）-lyoniresinol-3α-O-β-D-glucopyranoside （compound 3）， rubusoside （compound 4）， dulcoside B （compound 5）， 4-hydroxy-3-methoxyphenol 1-O-β-D-[6-O-（4-hydroxy-3，5-dimethoxylbenzoate）-glucopyranoside （compound 6）， 3，4，5-trimethoxyphenyl-6-O- syringoyl-β-D-glucopyranoside （compound 7）， gordonoside P （compound 8）.  CONCLUSIONS： Compounds 1-8 were isolated from this plant for the first time. This not only enriches the chemical constituents of this genus， but also provides a reference for elucidating the anti-tumor bioactive substances in this part.

Separation and Purification of Alhagi sparsifolia n -butanol Extract Monomeric Compounds and Study on Their Effects on Human Cervical Cancer HeLa Cells / 中国药房

OBJECTIVE： To separate and purify Alhagi sparsifolia n-butanol extract monomeric compounds， and to investigate its effects on the proliferation and metastasis of human cervical cancer HeLa cells. METHODS： The n-butanol extract was separated and purified by silica gel column， Sephadex LH-20 gel column and prep-HPLC. The structures of compounds were analyzed and identified according to physicochemical properties and spectrum （mass spectrum， hydrogen spectrum， carbon spectrum） data. Using human cervical cancer HeLa cells as objects， 5-FU as positive control， MTT assay was used to detect the inhibitory rate of HeLa cells pretreated with different doses of compounds （6.25， 12.5， 25， 50， 100， 200 μg/mL）； IC50 was calculated to screen active monomers. Scratch test was used to investigate the effects of above active monomers （all 50 μg/mL） on the migration ability of HeLa cells. Kim’s formula was used to evaluate the effects of 5-FU separately combined with above active monomers [（3.125+6.25），（6.25+12.5），（12.5+25），（25+50）μg/mL]. RESULTS： Six compounds were isolated from the n-butanol extract part of A. sparsifolia and identified as butin （Ⅰ）， 3′，4′，7-trihydroxyisoflavone （Ⅱ）， p-methoxyphenylacetic acid （Ⅲ）， 4-hydroxyacetophenone （Ⅳ）， aurantiamide acetate （Ⅴ）， protocatechualdehydea （Ⅵ）. Compared with blank control group， 5-FU and each compound （5-FU：6.25-200 μg/mL， compound Ⅰ： 12.5-200 μg/mL； compound Ⅱ： 25， 50， 200  μg/mL； compound Ⅲ： 6.25， 100， 200 μg/mL； compound Ⅳ： 50， 100， 200 μg/mL； compound Ⅴ： 12.5， 25， 200 μg/mL； compound Ⅵ： 6.25-200 μg/mL） could significantly increase the cell inhibition rate. IC50 of compound Ⅰ， Ⅴ， Ⅵ were decreased significantly （P＜0.05 or P＜0.01）， and those of compound Ⅰ and Ⅵ were lower relatively. The migration distance of cells in 5-FU and compound Ⅰ and Ⅵgroups were decreased significantly， compared with blank control group （P＜0.05 or P＜0.01）. 5-FU separately combined with compound Ⅰ and Ⅵ showed additive and enhanced inhibitory effects on the proliferation of HeLa cells （synergistic index＞0.9）. CONCLUSIONS： Compounds Ⅰ-Ⅵ are isolated from Alhagi for the first time. Butin and protocatechualdehydea are active monomers of its n-butanol extract part. Above two monomers can inhibit the proliferation and migration of human cervical cancer Hela cells， with strong inhibitory effect in vitro， and stronger inhibitory effect combined with 5-FU than any compound alone.

Therapeutic potential of n-butanol extract of Pulsatilla decoction in a murine model of ulcerative colitis induced by DSS combined with Candida albicans colonization / 中国中药杂志

To investigate the mechanism of n-butanol extract of Pulsatilla decoction (BAEB) against murine ulcerative colitis (UC) model induced by DSS combined with Candida albicans (CA) colonization, mice were randomly divided into normal control group, DSS group, DSS+CA group, BAEB high, medium and low dose group, and positive drug Mesalazine group. The general condition of mice was observed, fungal loads of murine intestinal contents were detected by plate method, colonic pathological change of mice was examined by HE staining. ASCA in serum and IL-6, IL-8, IL-1β, HBD-2, HBD-3 in colonic mucosa were detected by ELISA. The results showed that, compared with DSS group, the general condition and ASCA in serum had no obvious change for DSS+CA group, but the fungal loads in intestinal contents, the colonic pathological damage, and the levels of IL-6, IL-8, IL-1β, HBD-2, HBD-3 in colonic mucosa were greater than that of DSS group. High dose of BAEB group and Mesalazine group could improve the colonic pathology, decrease IL-6, IL-8, IL-1β, HBD-2, HBD-3 expression level. In conclusion, BAEB could effectively improve the UC symptoms in mice induced by DSS combined with CA colonization, and inhibit the inflammatory factors such as IL-6, imply that BAEB is of important value for the treatment of intestinal fungal-related colitis.

Chemical Constituents from n-Butanol Part in Toddalia asiatica Root Bark / 中国药学杂志

OBJECTIVE: To study the chemical constituents from n-butanol part in Toddalia asiatica root bark. METHODS: A variety of chromatographic techniques were applied to extract, isolate and purify natural products from extract of Toddalia asiatica root bark, their structures were identified on the basis of spectral data. RESULTS: Thirteen compounds were isolated from n-butanol part in Toddalia asiatica root bark, there were seven coumarin compounds, including six furocoumarin compounds, identified as pimpinellin (1), bergapten (2), alloimperatorin (3), isopimpinellin (4), moellendorffiline (5), phellopterin (6), toddalolactone (7); four organic acid ester compounds identified as mono (2-ethylhexyl) terephthalate (8), ferulic acid methyl ester (9), 4-methoxycinnamic acid (10), methyl trans-4-hydroxycinnamate (11); one alkaloid identified as nitidine (12) and one flavonoid compound which was hesperidin (13). CONCLUSION: The compound 8 is isolated from the Rutaceae family for the first time, compound 3, 5, 9, 10, 11 are isolated from this plant for the first time.

Study on exosite inhibitor of cathepsin K from Herba Ecliptae fractions / 药学学报

Cathepsin K (CTSK) is considered a critical pharmaceutical target in the treatment of osteoporosis. CTSK exerts proteolytic activities against regulatory proteins besides its collagenase function, which may account for some of the adverse reactions when blocked by active site-directed inhibitors. Exosite inhibitors that can discriminate between the therapeutic collagenase and other biological activities of CTSK specifically inhibit the collagenase activity of CTSK without interfering with the other proteolytic activities of the protease. Active recombinant CTSK was expressed in Pichia pastoris, and purified by n-butyl sepharose and SP sepharose column chromatography. Herba Ecliptae is a common traditional Chinese medicine in the treatment of bone diseases. Collagenase assay and benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin (Z-FR-MCA) substrate assay based on CTSK are applied to verify the exosite inhibitors. n-Butanol extract of Herba Ecliptae are the most active fraction and eclalbasaponin IX isolated from n-butanol fraction is the potential exosite inhibitor of CTSK.

Study on the Mechanism of Celosia cristata N-butanol Extracts in the Improvement of Dysfunctional Uter-ine Bleeding of Rats / 中国药房

OBJECTIVE:To study the improvement effect of Celosia cristata n-butanol extracts on dysfunctional uterine bleed-ing of rats,and explore its mechanism. METHODS:60 pregnant SD rats were randomly divided into blank group,model group, Gongxuening capsule group (positive control,0.07 g/kg) and C. cristata n-butanol extracts high-dose,medium-dose,low-dose groups(4.32,2.16,1.08 g/kg),10 in each group. Except for the blank group,rats in other groups were intragastrically given mife-pristone and misoprostol on 7th of pregnancy for resulting incomplete abortion to induce models of dysfunctional uterine bleeding. Then rats in administration groups were intragastrically given relevant medicines,rats in blank group and model group were intra-gastrically given normal saline once every morning and evening,for 7 d. On 8th d of pregnancy,uterine bleeding amount,and thromboxane (TXA2),prostacyclin (PGI2) and tumor necrosis factor α(TNF-α) contents in serum were determined. RESULTS:Compared with blank group,uterine bleeding amount in model group was significantly increased(P<0.01),TXA2 content in se-rum was significantly reduced,PGI2 and TNF-α contents were significantly increased(P<0.01). Compared with model group,uter-ine bleeding amounts in administration groups were significantly reduced,TXA2 content in serum was significantly increased(P<0.01);PGI2 and TNF-α contents in serum in Gongxuening capsule group and C. cristata n-butanol extracts high-dose group and TNF-α content in serum in C. cristata n-butanol extracts medium-dose group were significantly reduced (P<0.01). CONCLU-SIONS:C. cristata n-butanol extracts show obvious improvement effect on incomplete drug abortion-induced dysfunctional uterine bleeding of rats,and the mechanism may be related to the regulation of TXA2/PGI2 dynamic balance and inhibition of TNF-α tran-sient secretion.

Simultaneous Determination of 6 Residual Organic Solvents in Xingnaojing Injection by Headspace GC / 中国药房

OBJECTIVE:To establish the method for simultaneous determination of 6 residual organic solvents in Xingnaojing injection,such as methanol,ethanol,isopropanol,n-butanol,ethyl acetate and acetonitrile.METHODS:Headspace GC method was adopted.The determination was performed on DB-624 capillary column by temperature programming with the injector temperature of 200 ℃;flame ionization detector was adopted with the temperature of 250 ℃;carrier gas was nitrogen with flow rate of 25 mL/min and split ratio of 35 ∶ 1;headspace sampling size was 1 mL,and heating temperature of headspace sampling was 80 ℃;equilibrium time was 15 min.RESULTS:The linear ranges of methanol,ethanol,isopropanol,n-butanol,ethyl acetate and acetonitrile were 15.00-240.00 μg/mL (r =0.999 9),25.00-400.00 μg/mL (r =0.999 9),25.00-400.00 μg/mL (r =0.999 9),25.00-399.99 μg/mL(r=0.999 9),25.00-399.99 μg/mL(r=0.999 8) and 5.00-80.00 μg/mL(r=0.999 9).The LOQ were 5.98,3.94,2.05,2.13,1.39,1.24 μg/mL,and the LOD were 2.01,2.11,1.18,1.56,1.15,0.01 μg/mL,respectively.RSDs of precision tests were all less than 2.0％,stability and repetitive tests only ethyl acetate was detected,RSD＜2.0％;the recoveries were 93.59％-99.02％ (RSD=2.62％,n=6),92.42％-98.40％ (RSD=2.43％,n=6),94.81％-104.64％ (RSD=3.47 ％,n=6),94.56％-106.73％ (RSD=4.21％,n=6),97.04％-106.33％(RSD=3.50％,n=6)and 98.40％-107.97％ (RSD=3.37％,n=6).CONCLUSIONS:The method is specific,rapid,simple and accurate,and can be used for simultaneous determination of 6 residual organic solvents in Xingnaojing injection.

Simultaneous Determination of 6 Residual Organic Solvents in Xingnaojing Injection by Headspace GC / 中国药房

OBJECTIVE:To establish the method for simultaneous determination of 6 residual organic solvents in Xingnaojing injection,such as methanol,ethanol,isopropanol,n-butanol,ethyl acetate and acetonitrile.METHODS:Headspace GC method was adopted.The determination was performed on DB-624 capillary column by temperature programming with the injector temperature of 200 ℃;flame ionization detector was adopted with the temperature of 250 ℃;carrier gas was nitrogen with flow rate of 25 mL/min and split ratio of 35 ∶ 1;headspace sampling size was 1 mL,and heating temperature of headspace sampling was 80 ℃;equilibrium time was 15 min.RESULTS:The linear ranges of methanol,ethanol,isopropanol,n-butanol,ethyl acetate and acetonitrile were 15.00-240.00 μg/mL (r =0.999 9),25.00-400.00 μg/mL (r =0.999 9),25.00-400.00 μg/mL (r =0.999 9),25.00-399.99 μg/mL(r=0.999 9),25.00-399.99 μg/mL(r=0.999 8) and 5.00-80.00 μg/mL(r=0.999 9).The LOQ were 5.98,3.94,2.05,2.13,1.39,1.24 μg/mL,and the LOD were 2.01,2.11,1.18,1.56,1.15,0.01 μg/mL,respectively.RSDs of precision tests were all less than 2.0％,stability and repetitive tests only ethyl acetate was detected,RSD＜2.0％;the recoveries were 93.59％-99.02％ (RSD=2.62％,n=6),92.42％-98.40％ (RSD=2.43％,n=6),94.81％-104.64％ (RSD=3.47 ％,n=6),94.56％-106.73％ (RSD=4.21％,n=6),97.04％-106.33％(RSD=3.50％,n=6)and 98.40％-107.97％ (RSD=3.37％,n=6).CONCLUSIONS:The method is specific,rapid,simple and accurate,and can be used for simultaneous determination of 6 residual organic solvents in Xingnaojing injection.