Total Alkaloids from Sophora Alopecuroides L.Increase Susceptibility of Extended-Spectrum β-Lactamases Producing Escherichia coli Isolates to Cefotaxime and Ceftazidime

Objective： To evaluate the antimicrobial activity of total alkaloids extracted from Sophorea alopecuroides L. （TASA） against clinical isolated extended-spectrum beta-lactamases （ESBLs） producing Escherichia coil （E.. coil） strains. Methods： The antibacterial activity of TASA either itself or in combination with cefotaxime （CTX） or ceftazidime （CAZ） was investigated by using the microbroth dilution method and phenotypic confirmatory disk diffusion test against three clinical isolated ESBLs-producing E. coil strains; the interactions of TASA and C＇I＇X or CAZ were ascertained by evaluating the fractional inhibitory concentration index （FICI）. Results： The antibacterial activity of either TASA itself or in combination with C＇IX or CAZ was found. The minimum inhibitory concentration （MICs） of TASA against the ESBLs producing isolates was 12.5 mg/mL. In the combinations with a sub-inhibitory concentration of TASA, a synergistic effect on CTX and CAZ against the ESBLs producing isolates was observed. Similarly, the isolates exposed to lower dose of TASA yielded an increased susceptibility to CTX and CAZ by 8-16 folds determined by microdilution assay. Moreover, enzymatic detection of ESBLs demonstrated that TASA induced reversal resistance to CTX and CAZ partially by a mechanism of inhibition of ESBLs activity in these isolates. Additionally, in the tested isolates following the exposure of TASA, molecular analysis verified the SHV-type beta-lactamase encoding ESBL gene in these isolates, and no mutation was introduced into the ESBL gene. Conclusions： These results suggest that TASA could be used as a source of natural compound with pharmacological activity of reversal resistance to antimicrobial agent. These findings also indicated that the application of the TASA in combination with antibiotics might prove useful in the control and treatment of infectious diseases caused by the ESBLs producing enterobacteriaceae.

Protective Effect of Total Alkaloids of Sophora Alopecuroides on Dextran Sulfate Sodium-Induced Chronic Colitis

Objective：To investigate the effect of total alkaloids of Sophora alopecuroides（TASA） on dextran sulfate sodium（DSS）-induced colitis in mice.Methods：Chronic experimental colitis was induced by administration of 4 cycles of 4%DSS.Fifty mice were randomly distributed into 4 groups（normal,DSS,DSS/high-dose TASA, and DSS/low-dose TASA groups） by a random number table with body weight stratification.Mice in the normal group（n=11） and DSS-induced colitis control group（n=15） received control treatment of 20 mL/kg distilled water; DSS plus TASA high- and low-dose groups（n=12 each） were treated with TASA solution（20 mL/kg） at the doses of 60 mg/kg and 30 mg/kg,respectively.The severity of colitis was assessed on the basis of clinical signs, colon length,and histology scores.Moreover,secretory immunoglobulin A（slgA） and haptoglobin（HP） were analyzed by enzyme linked immunosorbent assay;intercellular adhesion molecule 1（ICAM-1） and macrophage-migration inhibitory factor（MIF） gene expressions were analyzed by quantitative reverse transcriptase realtime polymerase chain reaction（qRT-PCR） using SYBA greenⅠ;and nuclear factorκB（NF-κB） expression and activation and p65 interaction with the promoter of ICAM-1 gene were assessed by Western blotting and chromatin immunoprecipitation assay.Results：TASA administration significantly attenuated the damage and substantially reduced HP elevation and maintained the level of cecum slgA.TASA inhibited the ICAM-1 gene expression and had no effect on MIF gene expression.Also,TASA was able to reduce phospho-lκBα（p-lκBα） protein expression;however,it had no effect on the activation of IκB kinaseα（IKKα） and inhibitor of NF-κBα（IκBα）.Moreover,TASA inhibited the p65 recruitment to the ICAM-1 gene promoter.Conclusions：TASA had a protective effect on DSS-induced colitis.Such effect may be associated with its inhibition of NF-κB activation and blockade of NF-κB-regulated transcription activation of proinflammatory mediator gene.

Anti-helicobacter pylori effect of total alkaloids of sophora alopecuroides in vivo

Background Helicobacterpylori （H.pylori） infection could lead to most gastroduodenal diseases and is even identified as a carcinogen of gastric cancer.Total alkaloids of sophora alopecuroides （TASA） is widely used in herbal remedies to treat various infectious diseases,including stomach-associated diseases.This study is aimed at evaluating the antimicrobial activity of TASA on H.pylori-infected BALB/c mice mouse gastritis.Methods Totally 120 BALB/c mice were orally inoculated with H.pylori Bacterial liquid to construct BALB/c mice H.pylori infection gastritis animal model,after the model was successfully created.We randomly assigned 100 infected mice into 10 treatment groups,the first group （normal saline）; the second group （bismuth pectin）; the third group （omeprazole）; the fourth group （TASA 2 mg/d）; the fifth group （TASA 4 mg/d）; the sixth group （TASA 5 mg/d）; the seventh group （TASA ＋ bismuth pectin）; the eighth group （TASA ＋ omeprazole）; the ninth group （bismuth pectin ＋ clarithromycin ＋ metronidazole）;the tenth group （omeprazole ＋ clarithromycin ＋ metronidazole）,5 other non-infected mice as negative control.Mice were orally inoculated twice a day and 7 days continuously.Then the mice were killed 4 weeks after treatment,we used realtime PCR to detect 16sDNA of H.pylori to test both the colonization and the clearance mice of bacteria of each treatment.We applied hematoxylin and eosin （HE） staining and immunostaining of mice gastric mucosa to observe the general inflammation and related factors interleukin 8 （IL-8）,cyclooxygenase 2 （COX-2）,and nuclear factor-kappa B （NF-KB） expression change after treatments.Results Firstly,we ensured that after 6-week intragastric administration,the bacteria colonization reached an exceed peak which is far higher than positive threshold （P ＜0.001）; secondly,after treatments,it is revealed that TASA combined with omeprazole or bismuth pectin showed promising antimicrobial activity against H.pylori as well as conventional triple therapy （P ＜0.001）; thirdly,HE staining showed that the inflammation on mice gastric mucosal membrane were also relieved obviously in TASA combined treatments and conventional triple therapy compared with normal saline treated mice,moreover,from immunohistochemistry results,H.pylori-induced IL-8,COX-2,and NF-KB were consistently suppressed in seventh,eighth,ninth,and tenth group to a certain extent.Conclusion These results open the possibility of taking TASA as an anti-inflammatory agent for H.pylori gastritis.

Total alkaloids of Sophora alopecuroides-and matrine-induced reactive oxygen species impair biofilm formation of Staphylococcus epidermidis and increase bacterial susceptibility to ciprofloxacin

Objective:To investigate the mechanism by which total alkaloids of Sophora alopecuroides(TASA)and matrine(MT)impair biofilm to increase the susceptibility of Staphylococcus epidermidis(S.epidermidis)to ciprofloxacin.Methods:The minimum biofilm inhibitory concentration(mBIC)was determined using a 2-fold dilution method.Structure of biofilm of S.epidermidis was examined by Confocal Laser Scanning Microscope(CLSM).The cellular reactive oxygen species(ROS)was determined using a DCFH-DA assay.The key factors related to the regulation of ROS were accessed using respective kits.Results:TASA and MT were more beneficial to impair biofilm of S.epidermidis than ciprofloxacin(CIP)(P<0.05).TASA and MT were not easily developed resistance to biofilm-producing S.epidermidis.The mBIC of CIP decreased by 2-6-fold following the treatment of sub-biofilm inhibitory concentration(sub-BIC)TASA and MT,whereas the mBIC of CIP increased by 2-fold following a treatment of sub-BIC CIP from the first to sixth generations.TASA and MT can improve the production of ROS in biofilmproducing S.epidermidis.The ROS content was decreased 23%-33%following the treatment of submBIC CIP,whereas ROS content increased 7%-24%following treatment with TASA+CIP and MT+CIP combination from the first to sixth generations.Nitric oxide(NO)as a ROS,which was consistent with the previously confirmed relationship between ROS and drug resistance.Related regulatory factorssuperoxide dismutase(SOD)and glutathione peroxidase(GSH)could synergistically maintain the redox balance in vivo.Conclusion:TASA and MT enhanced reactive oxygen species to restore the susceptibility of S.epidermidis to ciprofloxacin.

Optimizing pH-sensitive and time-dependent polymer formula of colonic pH-responsive pellets to achieve precise drug release

Time-sensitive and pH-dependent polymers are generally employed to prepare colon-site delivery system, and their coating thickness and order are very important in controlling the drug release. The traditional colon-site delivery systems consist of time-dependent polymers as inner layer and pH-sensitive polymers as outer layer. However, they suffer from low drug-loading rate and immature drug release. In this study, total alkaloids of sophora alopecuroides(TASA)-loaded pellets were prepared by extrusion-spheronization method and coated with Eudragit RS30D and Eudragit S100. Pellets using Eudragit RS30D as inner layer and Eudragit S100 as outer layer were named as ERS-ES100 TCO, while pellets with Eudragit S100 as inner layer and Eudragit RS30D as outer layer were ES100-ERS NCO. Both types of formulations with varying coating ratios and orders of Eudragit S100 and Eudragit RS30D were designed and prepared. The following in vitro drug release and SEM studies indicated that ERS-ES100 TCO(F2) with 12.8% Eudragit RS30D as inner layer and 21% Eudragit S100 as outer layer released up to 42% drug in 5 h. Interestingly, ES100-ERS NCO(F4) coated with 12.8% Eudragit S100 and 14.8% Eudragit RS30D showed optimal drug release in colon. In conclusion, ES100-ERS NCO colonic delivery system achieved reduced coating thickness and improved colonic targeting compared with traditional delivery system(ERS-ES100 TCO). In addition, the similarity factors( f 2) value of sophoridine and matrine for investigated formulation were within 50–100 and > 80, demonstrating that sophoridine and matrine in all formulations achieved a synchronous release.

苦豆子种子中生物碱的分离及lehmannine的结构确定

目的 研究苦豆子种子中生物碱的分离及槐果碱转移氢化制备苦参碱的研究。方法 利用离子交换、萃取和柱层 ,并结合转移氢化增大不同生物碱理化性质差异进行分离。结果 从苦豆子种子中分得 5种生物碱 ,其结构经元素分析、IR、MS、1 H NMR和 1 3CNMR光谱鉴定为氧化苦参碱、氧化槐定碱、苦参碱、槐定碱和 lehannine。结论 首次从苦豆子种子中分得 lehm

TLC和HPLC法同时测定苦豆子总碱中槐定碱和苦参碱的体系优化

采用薄层层析法（TLC）,展开剂为氯仿-甲醇-氨水=8∶2∶0.1;同时采用高效液相色谱法（HPLC）,色谱柱为Ultra II^TM C18（5μm×250mm×4.6mm）、流动相为0.01mol/L磷酸盐缓冲液-甲醇（45∶55）、检测波长为216nm、流速为1.0mL/min、柱温为30℃、进样量为10μL;建立TLC和HPLC同时分离测定苦豆子生物碱体系的优化方法。结果表明：槐定碱和苦参碱在200-900μg/mL浓度范围内呈良好的线性关系,R2分别为0.9994和0.9996。平均回收率（n=9）分别为99.47%和99.82%;试验表明可通过TLC和HPLC法同时测定苦豆子总碱中槐定碱和苦参碱的含量,并可用该方法进行苦豆子药材及含苦豆子药用成分的质量控制。

HPLC法测定苦豆子总碱结肠定位释放片中生物碱

目的建立苦豆子总碱结肠定位释放片中生物碱的测定方法。方法采用HPLC法,乙腈-0.05 mol/L磷酸二氢钾(三乙胺调pH值大于6.45),梯度洗脱,柱温35℃,检测波长205 nm,体积流量1.0 mL/min。结果野靛碱、苦豆碱、槐定碱、氧化苦参碱、氧化槐果碱、槐果碱、苦参碱、莱曼碱8种生物碱分离良好,线性范围分别为0.044 8～0.896 0μg、0.113 0～2.260 0μg、1.536 6～30.732 0μg、0.322 2～6.444 0μg、0.108 4～2.168 0μg、0.404 8～8.096 0μg、0.033 2～0.664 0μg、0.036 2～0.724 0μg,加样回收率大于96%,供试品溶液在24 h内稳定性良好,8种生物碱总量的转移率大于90%。结论该方法简便,检出率高,可以作为苦豆子总碱结肠定位释放片的测定方法。

宁夏产苦豆子不同器官HPLC指纹图谱的分析研究

目的用HPLC指纹图谱的方式研究苦豆子不同器官中各生物碱的变化。方法采用HPLC检测方法,色谱柱Waters X-Brige C18(4.6 mm×250 mm,5μm);流动相为乙腈-0.05 mol/L的磷酸二氢钾溶液(2.0ml/L三乙胺),进行梯度洗脱;检测波长为205 nm,进样量10μl,流速1.0 ml/min。结果 10批不同器官的苦豆子样品得到7个共有峰,通过与对照品比较,2、3、5、6、7号峰确定为野靛碱、槐定碱、氧化苦参碱、氧化槐果碱、苦参碱。结论该指纹图谱的分析体现了不同器官的苦豆子样品所含各成分及各成分的含量变化较大。

苦豆子抗内毒素效应的实验研究

目的:研究苦豆子中提取的苦豆子总碱及氧化苦参碱、槐定碱、槐果碱对内毒素肺损伤小鼠模型的干预作用及其作用机制。方法:以清开灵注射液作为本研究的阳性对照药物。分别连续3天腹腔注射给予ICR小鼠各个生物碱及清开灵,均末次给药后1 h腹腔注射LPS(E.coil,055∶B5)9 mg/kg造成小鼠内毒素性肺损伤,观察小鼠一般状况、白细胞数量、肺W/D比值、肺组织病理形态学改变,以及免疫组织化学法检测肺组织CD14和清道夫受体(SR-A)的表达,放免法检测血清肿瘤坏死因子(TNF-α)和白细胞介素-6(IL-6)含量。结果:预防性给予内毒素肺损伤小鼠苦豆子总碱和三种苦豆子单体碱,均不同程度改善了模型鼠的一般状况,并可不同程度地升高外周血白细胞,降低肺脏W/D比值,减轻肺组织病理改变,降低模型鼠肺组织CD14表达,增加SR-A表达,降低血清中TNF-α和IL-6的水平。结论:苦豆子总碱和三种苦豆子单体碱可不同程度地减轻内毒素肺损伤小鼠的病理损害,它们的抗内毒素机制可能与调节LPS的识别受体,并进而影响下游炎症因子表达有关。

微波法提取苦豆子生物碱的研究

苦豆子资源在国内比较丰富,其有效成分生物碱含量较高,目前国内外对苦豆子的研究和利用重点放在了生物碱上,本文对苦豆子生物碱传统提取法和微波辅助提取法进行了试验比较,在单因素实验的基础上,采用正交设计方法,考察微波时间、微波功率、料液比3个因素对苦豆子生物碱提取的影响,结果表明:微波辅助提取生物碱效果较好,最佳条件是微波提取时间为30S、微波功率为360W、料液比为(g/mL)1∶5。利用这种方法生物碱的提取效率较高,生产成本较低,工序较为简单,适宜于工业化生产。

几种苦豆子生物碱对小菜蛾部分生理指标的影响

四龄小菜蛾幼虫经苦豆子7种生物碱处理后，表现呼吸强度增加，完成完整呼吸次数减少，其中，苦参碱、苦豆碱和槐胺碱均显著促进试虫二氧化碳释放量和释放过程。槐果碱处理试虫体内总糖含量显符降低。所有处理试虫的生长发育均显著受到抑制。

苦豆子生物碱单体的分离纯化工艺研究

在对苦豆子的酸水浸提液进行了超滤和脱色预分离的基础上，采用不同的有机溶剂萃取和硅胶柱层析对其中主要的生物碱进行分离纯化及条件优化。结果表明合适的生物碱萃取富集条件是用氯仿为溶剂，pH=10．5。该氯仿萃取液用硅胶柱层析分离，洗脱剂为氯仿．甲醇．氨水（5：0．4：0．01，V/V/V），洗脱速度1柱床体积·h^-1，得到峰1～峰3。峰1和峰2中的化合物经丙酮结晶和重结晶分别得到纯度99％以上的氧化槐果碱和氧化苦参碱；峰3对应的组分再经硅胶柱层析，洗脱剂为丙酮．甲醇（10：1，V／V），洗脱速度1柱床体积·h^-1，得到峰4～峰6，峰4中的化合物经石油醚结晶和重结晶，可以得到纯度95．8％的槐定碱。采用了HPLC-MS，IR，熔点测定和薄层色谱等方法进行上述生物碱单体的定性定量及分子结构确定。本研究为深度开发苦豆子生物碱资源和为制备医药工业所需的高纯度生物碱原料提供了依据。

苦参碱和苦豆子总碱光分解及光分解抑制的研究

研究了苦参碱(从苦豆子中提取分离获得)和苦豆子总碱在太阳光及紫外光照射下的光分解现象,发现两者均属于光易分解物质,其光分解半衰期均不超过1h。运用11种不同的光稳定剂处理均能有效地抑制苦参碱和苦豆子总碱光分解,其中以TBHQ(Tertiary-butylhydroquinone,特丁基对苯二酚)的抑制效果最为明显。在此基础上探索了不同浓度TBHQ对苦参碱和苦豆子总碱光分解的抑制效应,结果显示其抑制效果与稳定剂的浓度相关,处理浓度越大,其抑制苦豆子总碱光分解的作用越强。

苦豆子总碱对SW480细胞株及裸鼠移植瘤的影响

目的:探讨苦豆子总碱(TASA)对SW480细胞株及裸鼠移植瘤的抑制作用及机制。方法:利用MTT法检测不同浓度TASA对SW480细胞的增殖抑制影响;Annexin V-FITC标记法检测SW480细胞凋亡;建立裸鼠移植性实体瘤SW480模型,观察TASA作用下移植性实体瘤的体积和质量,用荧光定量PCR法检测移植性实体瘤组织中Caspase-3、Caspase-9及BCL-2 mRNA的表达。结果:实验结果显示TASA对体外培养的SW480细胞增殖具有明显抑制作用,并可诱导其发生凋亡,其作用呈剂量和时间依赖性,48 h IC50=0.92 mg/mL;TASA高、低剂量组裸鼠移植性实体瘤SW480的体积和质量、BCL-2表达低于模型组,Caspase-3、Caspase-9表达则显著高于模型组,其抑瘤率分别达到57.3%、37.4%。结论:TASA对SW480细胞株及裸鼠移植瘤均有抑制作用,其机制可能与诱导凋亡有关。

苦豆子种子中生物碱的冷浸提取实验研究

目的 考察苦豆子种子中生物碱的冷浸工艺条件。方法 在室温下用稀盐酸水提取苦豆籽中的生物碱 ,考查 p H值、浸提时间、豆籽粉粒度及保持负压、超声波振荡下总碱浸出率 ,并对比热浸状况。结果 总生物碱浸出率可达 3 .7%以上。结论 在优选的工艺条件下 ,可能提高收率和浸提速度。

苦豆子内生真菌对宿主培养物生长及喹诺里西啶类生物碱合成的影响

利用苦豆子健康植株中分离鉴定的8株内生真菌菌株为真菌诱导子,分别制备灭活菌丝和菌液浓缩物,研究内生真菌诱导子不同种类、浓度和诱导时间对苦豆子无菌苗和愈伤组织的生长以及喹诺里西啶生物碱含量的影响。结果表明:8株苦豆子内生真菌诱导子中,菌液浓缩物的诱导效果要强于灭活菌丝。菌株HMGKDF1菌液浓缩物和灭活菌丝都能明显促进愈伤组织的生长,净生长率是对照的1.82和1.42倍;菌株NDZKDF13菌液浓缩物对愈伤组织生物碱的合成效果明显,生物碱含量为0.5483 mg·g-1,是对照的23.8倍;在一定浓度范围内(0.01~1.0 mg·L-1),苦豆子内生真菌诱导子能够促进宿主植物喹诺里西啶生物碱的合成。内生真菌诱导子处理苦豆子无菌苗12 d时,喹诺里西啶生物碱含量最高,是对照的2.65倍。在苦豆子无菌苗或愈伤组织中添加一定量的苦豆子内生真菌诱导子,对宿主的生长以及提高喹诺里西啶生物碱含量是一种有效的方法。

新疆苦豆子种子中生物碱类化学成分的研究

采用硅胶柱层析法、氧化铝柱层析法、Sephadex LH-20柱色谱法,以及重结晶等方法对新疆苦豆子种子中生物碱类化学成分进行研究,根据化合物的理化性质和光谱数据鉴定其结构。结果从新疆苦豆子种子中分离得到4个化合物,分别鉴定为9α-羟基苦参碱(Ⅰ)、苦参碱(Ⅱ)、槐定碱(Ⅲ)、氧化苦参碱(Ⅳ)。其中化合物Ⅰ为首次从本植物中分离得到。

不同酯化度果胶对苦豆子总碱水凝胶骨架片体外释放的影响

目的:制备苦豆子总碱水凝胶骨架结肠定位释放片,研究不同酯化度果胶对其释放行为的影响。方法:采用湿法制粒压片法制备不同酯化度果胶骨架片,分别考察其在模拟胃液、肠液中6 h释放情况,在此基础上采用Kollicoat MAE 30DP包衣,分析其在模拟胃液、肠液、盲肠液介质中的释放行为。结果:不同酯化度果胶能够明显影响其在模拟胃液、肠液中的释放。采用Kollicoat MAE 30DP包衣的低酯果胶配方E能够使苦豆子总碱中槐定碱结肠释放,近似零级释放模型,属骨架溶蚀释药机制。结论:肠溶包衣的低酯果胶骨架片靶向性强,能够使苦豆子总碱定位释放,从而提高疗效。

苦豆草中抑制杉炭疽菌分生孢子萌发的物质

预试验发现苦豆草地上部分乙醇提取物对杉炭疽菌有较强的抑制活性 ,进一步用不同溶剂 ,在不同 pH条件下萃取将苦豆草乙醇提取物分离为 4个组分。生测表明其生物碱部分对该菌的抑菌活性最强。将生物碱部分用氧化铝柱层析分离得到 7种单体结晶。用熔点、旋光度、红外、和GC MS得到的MS数据与文献相应数据对照鉴定了这 7种生物碱 ,它们分别是 :槐果碱、苦参碱、槐胺碱、莱曼碱、槐定碱、苦豆碱、野靛碱。用纸环法生测表明这 7种生物碱对杉炭疽菌分生孢子萌发均具有较强的抑制活性 ;它们的EC50 为 :苦豆碱 72 0 μg/mL ,野槐胺碱 1 98 0 μg/mL ,苦参碱 86 0 μg/mL ,槐果碱 2 4 0 μg/mL ,槐定碱 2 6 0 μg/mL ,莱曼碱 1 2 6 0 μg/mL ,而相同条件下的常用杀菌剂 75 %百菌清和 70 %甲基托布津的EC50 值分别为 82 0和 1 1 8 0μg/mL。