复方甘草片中甘草酸、异甘草苷、异甘草素的测定

目的：建立复方甘草片中甘草酸、异甘草苷和异苷草素的含量定量分析的高效液相色谱(HPLC)方法。方法：采用色谱柱ODS-C_(18)(4．6mm×250mm,5μm),甘草酸流动相为甲醇：冰醋酸：水(70：1：30)；检测波长254nm。异甘草苷与异甘草素在同一条件下检测,其流动相为甲醇：冰醋酸：水(50：2：50)；检测波长346nm；三者流速均为1ml /min；进样量为10μl；柱温为25℃。结果：复方甘草片中甘草酸的含量为7．51mg/g,异甘草苷和异甘草素的含量分别为3．01mg/g,0．448mg/g,上述3种组分的平均回收率分别为98．32%,99．57%和98．59%。结论：本方法可作为复方甘草片中的甘草酸、异甘草苷和异甘草素的含量质量控制的一种有效方法。     

HPLC测定逍遥丸中甘草苷、异甘草素和甘草酸的含量

目的 建立测定逍遥丸中甘草苷、异甘草素和甘草酸含量的方法。 方法 色谱柱：Shim-packVP-ODS(150 mm× 4.6 mm,5.0 μm)。流动相：甲醇-0.5%冰醋酸梯度洗脱;流速1 mL·min^-1;检测波长0～7 min为276 nm,7～18 min为370 nm,18 min以后为254 nm。 结果 甘草苷的线性范围是0.018 40～0.368 0 μg(r=0.999 8,平均回收率为97.96%,RSD为0.60%);异甘草素的线性范围是0.015 2～0.304 0 μg(r=0.999 9,平均回收率为97.17%,RSD为1.02%);甘草酸的线性范围是0.441 8～8.835 0 μg (r=0.999 9,平均回收率为97.52%,RSD为1.06%)。 结论 所建立的方法简便可行、重复性好,可用于逍遥丸的质量控制。     

HPLC法同时测定炙甘草汤中甘草苷、甘草素、异甘草素的含量

目的:建立同时测定炙甘草汤中甘草苷、甘草素、异甘草素含量的方法。方法:采用高效液相色谱法。色谱柱为Waters Sunfire C18(250mm×4.6mm,5μm),流动相为乙腈-0.5%冰醋酸(50:50,V/V),检测波长为278nm,流速为1.0mL·min-1。结果:甘草苷、甘草素、异甘草素的检测浓度分别在1.2～24.0、64.0～1280.0、50.0～1000.0μg·mL-1范围内与各自峰面积积分值呈良好的线性关系(r分别为0.9998、0.9999、0.9999);三者平均加样回收率分别为100.38%、99.45%、97.46%,RSD分别为0.45%、0.53%、0.96%(n=6)。结论:该方法准确、可靠,可为炙甘草汤的质量控制提供检测依据。     

单壁碳纳米管-异甘草素复合物在大鼠体内缓释性能的研究

目的：研究单壁碳纳米管（SWNTs）-异甘草素复合物在大鼠体内的缓释性能。方法：以大鼠为动物模型,将碳纳米管-异甘草素复合物以聚乙二醇400稀释液分散灌胃,不同时间点取血,以高效液相色谱法测定血浆中异甘草素浓度,探讨复合物的体内缓释性能。结果：单壁碳纳米管-异甘草素复合物在大鼠体内具有明显缓释作用,t_max和C_max分别为11.2 h和201.3 ng·mL^-1,相对于异甘草素组的相对生物利用度为141.8%。结论：单壁碳纳米管具有良好的缓释性能,具有作为缓释载体材料的潜力。     

甘草中异甘草素和甘草素含量测定方法研究

目的 建立同时测定甘草中异甘草素和甘草素含量的方法。方法 采用高效液相色谱（HPLC）等度洗脱和检测波长时间序列采样的方法,流动相为甲醇-水=（45∶55）,检测波长0~25min时为276nm,25~70min时为360mn,流速为0.8mL/min,同时测定甘草中异甘草素和甘草素的含量。结果 异甘草素和甘草素进样量线性范围分别为0.028~0.28μg和0.064~0.64μg,平均加样回收率分别为97.92%和98.27%,RSD分别为1.29%和1.67%。结论 该方法准确、灵敏度高、重现性好、操作简单,可为甘草药材的质量评价提供参考。     

碳纳米管-异甘草素复合物体内外释药相关性研究

目的：研究碳纳米管-异甘草素复合物体内外释药相关性,考察该复合物缓释性能。方法：采用转篮法对复合物进行体外释放度测定;以大鼠为实验对象对复合物进行体内吸收实验,按照Wagner-Nelson公式计算吸收分数。结果：碳纳米管-异甘草素复合物的体外释放度（F）与体内吸收分数（f）的回归方程为：F=2.980 2 f-35.948,相关系数为0.960 8。结论：碳纳米管-异甘草素复合物体内外释药相关性良好,表现出良好的缓释性能。     

黄芩苷在多壁碳纳米管修饰玻碳电极上的电化学行为及其测定

目的建立了检测黄芩苷含量的电化学分析新方法。方法采用循环伏安法研究黄芩苷在多壁碳纳米管修饰玻碳电极上的电化学行为及其电极反应机理,以差示脉冲伏安法建立了检测黄芩苷含量的电化学分析新方法。结果在优化的条件下,电化学信号强度电流值I与黄芩苷浓度在1×10-9~5×10-7mol.L-1和6×10-7~5×10-6mol.L-1范围呈线性关系,相关系数为0.9956和0.9934。该方法的黄芩苷浓度检出限为1×10-10mol.L-1,平行测定6次得RSD为1.04%,回收率为98.0~102.0%。结论利用多壁碳纳米管修饰的玻碳电极分析法,建立了测定黄酮类药物黄芩苷的含量新方法,并应用于清开灵注射液中黄芩提取物（黄芩苷计）的含量测定,结果令人满意。     

HPLC-CAD法测定小建中片中氧化芍药苷、芍药内酯苷、芍药苷、甘草苷、异甘草苷、桂皮酸、桂皮醛和甘草酸

目的 采用高效液相色谱–电喷雾检测器(HPLC-CAD)法同时测定小建中片中氧化芍药苷、芍药内酯苷、芍药苷、甘草苷、异甘草苷、桂皮酸、桂皮醛、甘草酸.方法 采用Agilent Zorbax Eclipse XDB C18色谱柱(250 mm×4.6 mm,5μm);流动相:90％甲醇–10 mmol/L甲酸铵(甲酸调p...     

甘草素与异甘草素的合成

甘草素与异甘草素的合成杨立，沈凤嘉（兰州大学化学系，兰州７３００００）甘草（Ｇｌｙｃｙｒｒｈｉｚａｕｒａｌｅｎｓｉｓｆｉｓｃｈ．）为我国一项宝贵的中药资源，历来受到人们重视。甘草素（ｌｉｑｕｉｒｉｔｉｇｅｎｉｎ）和异甘草素（ｉｓｏｌｉｑｕｉｒｉｔｉｇ...     

HPLC法测定甘草附子汤中甘草苷和香豆素的含量

目的建立HPLC法测定甘草附子汤中甘草苷和香豆素含量的方法。方法采用HypersilC18色谱柱 (2 0 0mm× 4 6mm ,5 μm) ,流动相为甲醇 乙腈 水 冰醋酸 (体积比 5∶1 5∶80∶1 ) ,流速0 8mL·min-1,检测波长 2 5 4nm。结果甘草苷在 2 0～ 3 0 0mg·L-1内成良好线性关系 ,平均回收率 95 9% ,RSD为 3 1 %。香豆素在 1 7～ 1 6 7mg·L-1内成良好线性关系 ,平均回收率 95 9% ,RSD为 2 0 %。结论本法可作为甘草附子汤质量控制的手段之一     

Drug targeting to arthritic region via folic acid appended surface-engineered multi-walled carbon nanotubes

This study was aimed at developing and investigating folate anchored carbon nanotubes for targeting an anti-arthritic drug, Methotrexate (MTX) to inflammatory arthritic region. The folic acid (FA) was conjugated to amidated multi-walled carbon nanotubes (MWCNTs) and confirmed by Fourier transform infrared (FTIR), ¹H NMR spectroscopy and X-ray diffraction analysis. The MTX was loaded into the pristine and functionalized-MWCNTs and extensively characterized in vitro and in vivo studies. The drug entrapment efficiency was found high in folate conjugated MWCNTs. In vitro drug release in PBS (pH 7.4) from pristine MWCNTs and folate conjugated MWCNTs formulation was found to be 66.35?±?2.3 and 56.88?±?1.9% in 24?h, respectively. Folate conjugated MWCNTs significantly increased (p?<?0.005) the percentage inhibition of arthritis, biological half-life and volume of distribution of MTX as compared to MTX-loaded naked MWCNTs as well as free MTX. In in vivo biodistribution studies, MTX was found to be significantly higher (p?<?0.005) in arthritic joints from folate functionalized MWCNTs as compared to free drug as well as drug-loaded naked MWCNTs. The present outcomes highlights the propensity of drug-loaded functionalized MWCNTs to alter the pharmacokinetics as well as sustained and targeted drug delivery system as well.     

Proteomics-based safety evaluation of multi-walled carbon nanotubes

This study evaluated the biological responses to multi-walled carbon nanotubes (MWCNTs). Human monoblastic leukemia cells (U937) were exposed to As-grown MWCNTs and MWCNTs that were thermally treated at 1800 °C (HTT1800) and 2800 °C (HTT2800). Cell proliferation was highly inhibited by As-grown but not HTT2800. However, both As-grown and HTT1800, which include some impurities, were cytotoxic. Proteomics analysis of MWCNT-exposed cells revealed 37 protein spots on 2-dimensional electrophoresis gels that significantly changed (p < 0.05) after exposure to HTT1800 with a little iron and 20 spots that changed after exposure to HTT2800. Peptide mass fingerprinting identified 45 proteins that included heat shock protein β-1, neutral α-glucosidase AB, and DNA mismatch repair protein Msh2. These altered proteins play roles in metabolism, biosynthesis, response to stress, and cell differentiation. Although HTT2800 did not inhibit cell proliferation or cause cytotoxicity in vitro, some proteins related to the response to stress were changed. Moreover, DJ-1 protein, which is a biomarker of Parkinson's disease and is related to cancer, was identified after exposure to both MWCNTs. These results show that the cytotoxicity of MWCNTs depends on their impurities, such as iron, while MWCNTs themselves cause some biological responses directly and/or indirectly in vitro. Our proteomics-based approach for detecting biological responses to nanomaterials is a promising new method for detailed safety evaluations.     

The increases in relative mRNA expressions of inflammatory cytokines and chemokines in splenic macrophages from rats exposed to multi-walled carbon nanotubes by whole-body inhalation for 13 weeks

Abstract

Background: The toxicity of multi-walled carbon nanotubes (MWCNT) may be related to the immune system. The objective of this study was to obtain information for immunotoxic mechanisms of MWCNT in situ.

Methods: Using whole-body inhalation, male and female rats were exposed to 0, 0.2, 1 or 5?mg MWCNT/m³ for 13 weeks. Thereafter, spleens were recovered from the rats. Real-time PCR was done to assess expression of TNFα, IL-1β, IL-6, IL-10, MCP-1 and MIP-1α mRNA in the splenic macrophages; splenic T-lymphocytes were examined for IL-2 and TGF-β₁ mRNA expression.

Results: The relative expression of IL-1β mRNA in the cells from female rats exposed to 5?mg MWCNT/m³ was significantly higher than that in control cells. For IL-6 and IL-10, cells from rats in the 0.2 and 5?mg MWCNT/m³ had significantly higher mRNA expressions than did cells from controls. Expression of IL-1β, IL-6 and TNFα genes in cells from males in all exposure groups were higher than in control cells. Expression of MIP-1α in the cells from female 5-mg group was significantly higher than that in cells in the control. Only IL-2 was expression reduced, i.e. cells from male and female rats in all MWCNT groups had significantly lower mRNA expressions than control cells.

Conclusions: Systemic inflammation would likely occur in rats (or other hosts) exposed to MWCNT via inhalation due to increases in the expression of inflammatory cytokines in splenic macrophages. Moreover, decreases in IL-2 expression in T-lymphocytes may be critical to the potential reductions in anti-tumor responses in MWCNT-exposed hosts.     

TIMP1 promotes multi-walled carbon nanotube-induced lung fibrosis by stimulating fibroblast activation and proliferation

Pulmonary exposure to multi-walled carbon nanotubes (MWCNTs) may cause fibrosing lesions in animal lungs, raising health concerns about such exposure in humans. The mechanisms underlying fibrosis development remain unclear, but they are believed to involve the dysfunction of fibroblasts and myofibroblasts. Using a mouse model of MWCNT exposure, we found that the tissue inhibitor of metalloproteinase 1 (Timp1) gene was rapidly and highly induced in the lungs by MWCNTs in a time- and dose-dependent manner. Concomitantly, a pronounced elevation of secreted TIMP1 was observed in the bronchoalveolar lavage (BAL) fluid and serum. Knockout (KO) of Timp1 in mice caused a significant reduction in fibrotic focus formation, collagen fiber deposition, recruitment of fibroblasts and differentiation of fibroblasts into myofibroblasts in the lungs, indicating that TIMP1 plays a critical role in the pulmonary fibrotic response to MWCNTs. At the molecular level, MWCNT exposure significantly increased the expression of the cell proliferation markers Ki-67 and PCNA and a panel of cell cycle-controlling genes in the lungs in a TIMP1-dependent manner. MWCNT-stimulated cell proliferation was most prominent in fibroblasts but not myofibroblasts. Furthermore, MWCNTs elicited a significant induction of CD63 and integrin β1 in lung fibroblasts, leading to the formation of a TIMP1/CD63/integrin β1 complex on the surface of fibroblasts in vivo and in vitro, which triggered the phosphorylation and activation of Erk1/2. Our study uncovers a new pathway through which induced TIMP1 critically modulates the pulmonary fibrotic response to MWCNTs by promoting fibroblast activation and proliferation via the TIMP1/CD63/integrin β1 axis and ERK signaling.     

In vitro and in vivo genotoxic effects of straight versus tangled multi-walled carbon nanotubes

Some multi-walled carbon nanotubes (MWCNTs) induce mesothelioma in rodents, straight MWCNTs showing a more pronounced effect than tangled MWCNTs. As primary and secondary genotoxicity may play a role in MWCNT carcinogenesis, we used a battery of assays for DNA damage and micronuclei to compare the genotoxicity of straight (MWCNT-S) and tangled MWCNTs (MWCNT-T) in vitro (primary genotoxicity) and in vivo (primary or secondary genotoxicity). C57Bl/6 mice showed a dose-dependent increase in DNA strand breaks, as measured by the comet assay, in lung cells 24?h after a single pharyngeal aspiration of MWCNT-S (1–200?μg/mouse). An increase was also observed for DNA strand breaks in lung and bronchoalveolar lavage (BAL) cells and for micronucleated alveolar type II cells in mice exposed to aerosolized MWCNT-S (8.2–10.8?mg/m³) for 4 d, 4?h/d. No systemic genotoxic effects, assessed by the γ-H2AX assay in blood mononuclear leukocytes or by micronucleated polychromatic erythrocytes (MNPCEs) in bone marrow or blood, were observed for MWCNT-S by either exposure technique. MWCNT-T showed a dose-related decrease in DNA damage in BAL and lung cells of mice after a single pharyngeal aspiration (1–200?μg/mouse) and in MNPCEs after inhalation exposure (17.5?mg/m³). In vitro in human bronchial epithelial BEAS-2B cells, MWCNT-S induced DNA strand breaks at low doses (5 and 10?μg/cm²), while MWCNT-T increased strand breakage only at 200?μg/cm². Neither of the MWCNTs was able to induce micronuclei in vitro. Our findings suggest that both primary and secondary mechanisms may be involved in the genotoxicity of straight MWCNTs.     

Genotoxicity and carcinogenicity risk of carbon nanotubes

Novel materials are often commercialized without a complete assessment of the risks they pose to human health because such assessments are costly and time-consuming; additionally, sometimes the methodology needed for such an assessment does not exist. Carbon nanotubes have the potential for widespread application in engineering, materials science and medicine. However, due to the needle-like shape and high durability of multiwalled carbon nanotubes (MWCNTs), concerns have been raised that they may induce asbestos-like pathogenicity when inhaled. Indeed, experiments in rodents supported this hypothesis. Notably, the genetic alterations in MWCNT-induced rat malignant mesothelioma were similar to those induced by asbestos. Single-walled CNTs (SWCNTs) cause mitotic disturbances in cultured cells, but thus far, there has been no report that SWCNTs are carcinogenic. This review summarizes the recent noteworthy publications on the genotoxicity and carcinogenicity of CNTs and explains the possible molecular mechanisms responsible for this carcinogenicity. The nanoscale size and needle-like rigid structure of CNTs appear to be associated with their pathogenicity in mammalian cells, where carbon atoms are major components in the backbone of many biomolecules. Publishing adverse events associated with novel materials is critically important for alerting people exposed to such materials. CNTs still have a bright future with superb economic and medical merits. However, appropriate regulation of the production, distribution and secondary manufacturing processes is required, at least to protect the workers.     

Macrophage polarization and activation at the interface of multi-walled carbon nanotube-induced pulmonary inflammation and fibrosis

Pulmonary exposure to carbon nanotubes (CNTs) induces fibrosing lesions in the lungs that manifest rapid-onset inflammatory and fibrotic responses, leading to chronic fibrosis in animals and health concerns in exposed humans. The mechanisms underlying CNT-induced fibrogenic effects remain undefined. Macrophages are known to play important roles in immune regulation and fibrosis development through their distinct subsets. Here we investigated macrophage polarization and activation in mouse lungs exposed to multi-walled CNTs (MWCNTs). Male C57BL/6J mice were treated with MWCNTs (XNRI MWNT-7) at 40?μg per mouse (～1.86?mg/kg body weight) by oropharyngeal aspiration. The treatment stimulated prominent acute inflammatory and fibrotic responses. Moreover, it induced pronounced enrichment and polarization of macrophages with significantly increased M1 and M2 populations in a time-dependent manner. Induction of M1 polarization was apparent on day 1 with a peak on day 3, but declined rapidly thereafter. On the other hand, the M2 polarization was induced on day 1 modestly, but was remarkably elevated on day 3 and maintained at a high level through day 7. M1 and M2 macrophages were functionally activated by MWCNTs as indicated by the expression of their distinctive functional markers, such as iNOS and ARG1, with time courses parallel to M1 and M2 polarization, respectively. Molecular analysis revealed MWCNTs boosted specific STAT and IRF signaling pathways to regulate M1 and M2 polarization in the lungs. These findings suggest a new mechanistic connection between inflammation and fibrosis induced by MWCNTs through the polarization and activation of macrophages during MWCNT-induced lung pathologic response.     

Uptake and cytotoxic effects of multi-walled carbon nanotubes in human bronchial epithelial cells

Carbon nanotubes (CNT) are cytotoxic to several cell types. However, the mechanism of CNT toxicity has not been fully studied, and dosimetric analyses of CNT in the cell culture system are lacking. Here, we describe a novel, high throughput method to measure cellular uptake of CNT using turbimetry. BEAS-2B, a human bronchial epithelial cell line, was used to investigate cellular uptake, cytotoxicity, and inflammatory effects of multi-walled CNT (MWCNT). The cytotoxicity of MWCNT was higher than that of crocidolite asbestos in BEAS-2B cells. The IC₅₀ of MWCNT was 12 μg/ml, whereas that of asbestos (crocidolite) was 678 μg/ml. Over the course of 5 to 8 h, BEAS-2B cells took up 17-18% of the MWCNT when they were added to the culture medium at a concentration of 10 μg/ml. BEAS-2B cells were exposed to 2, 5, or 10 μg/ml of MWCNT, and total RNA was extracted for cytokine cDNA primer array assays. The culture supernatant was collected for cytokine antibody array assays. Cytokines IL-6 and IL-8 increased in a dose dependent manner at both the mRNA and protein levels. Migration inhibitory factor (MIF) also increased in the culture supernatant in response to MWCNT. A phosphokinase array study using lysates from BEAS-2B cells exposed to MWCNT indicated that phosphorylation of p38, ERK1, and HSP27 increased significantly in response to MWCNT. Results from a reporter gene assays using the NF-κB or AP-1 promoter linked to the luciferase gene in transiently transfected CHO-KI cells revealed that NF-κB was activated following MWCNT exposure, while AP-1 was not changed. Collectively, MWCNT activated NF-κB, enhanced phosphorylation of MAP kinase pathway components, and increased production of proinflammatory cytokines in human bronchial epithelial cells.