Study of Total Alkaloids from Rhizoma Coptis Chinensis on Experimental Gastric Ulcers

Objective： To study the effects of total alkaloids （TA） extracted from Rhizoma Coptis Chinensis on experimental gastric ulcer models. Methods： Four kinds of experimental ulcer models were established respectively by water-immersion stress, intragastric ethanol, acetic acid erosion, and pylorus ligation. The anti-ulcer effects of TA were evaluated, and compared with that of berberine （Bet） and cimetidine （Oim）. Results： TA showed significant inhibitory effects on ulcerative formation induced by water-immersion stress, intragastric ethanol, and pylorus l igation in dose-dependent manner, and showed therapeutic effect on acetic acid erosion-inducing ulcer, in comparison with the control group. The anti-ulcer activity of Bet was less than TA containing equal content of Bet. TA significantly reduced the free acidity, total acidity and total acid output, but didn＇t affect the gastric juice volume, gastric pepsin activity, adherent mucus quantity of stomach wall and free mucus dissolving in gastric juice. The suppressive activities of TA on gastric acid secretion didn＇t occur when it was administered into dodecadactylon at a dose of 360 mg/kg wt. Moreover, when compared with Oim, the inhibitory effect of TA on gastric acid secretion isn＇t proportional to the inhibitory effects on the formation of the 4 kinds of experimental ulcers. Conclusion： TA is a potent candidate in therapeutic drugs for treating gastric ulcer. Its anti-ulcer effective components and mechanism is not only related to Bet and inhibition of gastric acid, but also to other ingredients of TA and mechanism so far unknown.

Analysis of Hupehenine in the Total Alkaloids from Fritillaria hupehensis by HPLC-ELSD

A reverse-phase high pressure liquid chromatography （HPLC） method with evaporative light scattering detection （ELSD） has been developed for the quantitative analysis of hupehenine in the total alkaloids from Fritillaria hupehensis. Samples were analyzed on a reverse-phase column （Hypersil C-18） with a mobile phase of methanol：water：chloroform： triethylamine （85：15：1：0.6）. The ELSD was set at the drift tube temperature of 68.3℃ and gas flow rate of 1.8 L/min. Hupehenine＇s retention time was 13.7 min with an asymmetry factor of 1.2. The validity of the method has been verified with linearity, limit of detection, accuracy and precision. The logarithmic linear curve was obtained from 8.936 to 134.04 μg/mL （r=0.9993）. The detection limit （S/N〉3） of hupehenine was 1.79 μg/mL on the column. Intra-day RSD was 1.42% and inter-day RSD was 2.26% （3 days within a week）. The average recovery of hupehenine was 101.50%, and RSD was 1.62%.

Antihyperglycemic and antioxidant activities of total alkaloids from Catharanthus roseus in streptozotocin-induced diabetic rats

We evaluated the hypoglycemic and antioxi- dant effects of the total alkaloids of leaves and twigs of Catharanthus roseus Linn. （CTA） in streptozotocin （STZ）- induced diabetic rats. The hypoglycemic effect was mea- sured by blood glucose and plasma insulin level. Oxidative stress was measured in heart, liver and kidney by levels of antioxidant markers, free radical scavengers and lipid peroxides i.e. superoxide dismutase （SOD）, catalase （CAT）, glutathione （GSH） and thiobarbituric acid reactive substances （TBARS）. Biochemical parameters, i.e. aspar- tate aminotransferase （AST）, alanine aminotransferase （ALT）, alkaline phosphate （ALP） were observed in diabetic control and treated rats. Oral administration of CTA for 30 days was followed by a significant （P 〈 0.05） decrease in fasting blood glucose and increase in insulin level as compared with untreated diabetic rats. Also it significantly （P 〈 0.05） reduced ALT, AST and ALP. The treatment also resulted in significant （P 〈 0.05） reductions in GSH, SOD, CAT, and decrease in TBARS in the heart, liver and kidney of diabetic rats. The results suggest that CTA can effectively normalize the impaired antioxidant status in STZ-induced diabetes in a dose-dependent manner.CTA exerted rapid protective effects against lipid peroxi- dation by scavenging of free radicals and reducing the risk of diabetic complications.

Effect of Different Processing Techniques on the Content of Total Alkaloids in Toddalia asiatica Lam.

[Objectives]Effects of different processing techniques on the total alkaloid content in Toddalia asiatica Lam.were investigated by ultraviolet spectrophotometry.[Methods]T.asiatica was processed by the water washing method,water boiling method,water steaming method,wine steaming method and wine processing method,and the total alkaloid content was determined by ultraviolet spectrophotometry.[Results]Different processing methods can affect the total alkaloid content in the medicinal materials.Among them,the water steaming method and the wine processing method can increase the total alkaloid content of the medicinal materials,and the degree of improvement was higher in the water steaming method;and the water boiling method,water washing method and wine steaming method can reduce the total alkaloid content of the materials,and showed a reduction order of water boiling method>water washing method>wine steaming method.[Conclusions]The water steaming method and the wine processing method can increase the total alkaloid content in T.asiatica,and the water boiling method,water washing method and wine steaming method can reduce the total alkaloid content of the medicinal material.

Comparison of Extraction Methods for Total Alkaloids from Abrus Cantoniensis Hance

[Objectives] To compare different extraction methods for total alkaloids from Abrus Cantoniensis Hance taking chloroform as solvent. [Methods] The ethanol extraction method,ultrasonic extraction method,acid water extraction method,and Soxhlet extraction method were used to extract total alkaloids,and the total alkaloids of Abrus Cantoniensis Hance were measured at 238 nm wavelength. [Results] The extraction rate of ethanol extraction method was 14. 76 mg/100 g; the extraction rate of ultrasonic extraction method was 56. 85 mg/100 g; the extraction rate of acid water extraction method was 56. 63 mg/100 g; the extraction rate of Soxhlet extraction method was 58. 19 mg/100 g.[Conclusions]Results indicate that Soxhlet extraction method can obtain the highest extraction rate.

Effect of Liposome-Encapsulated Total Alkaloid of Harmaline on Rabbit Lens Epithelial Cells:Experimental Study on the Prevention of Posterior Capsule Opacification

Purpose: To investigate whether liposome encapsulated total alkaloid of Harmaline (TAH) as a therapeutic agent is beneficial to prevention of posterior capsular opacifi-cation (PCO).Methods: Liposome-encapsulated TAH was prepared by modified freeze-thawing method. 0. 1ml of liposome-encapsulated TAH (0. 2mg/ml) was injected into the capsular bag during extracapsular lens extraction (ECLE) of each eye in total 10 rabbit eyes. Blank liposome or balance salt solution (BSS) was used as control. Slit-lamp examination and histopathological examination was used to evaluated capsule opacifica-tion. Intraocular pressure (IOP) , density and morphology of corneal endothelia cells, the amplitude and latency of b wave of ERG were measured.Results: The inflammatory response was mild both in TAH treated and the control group. PCO formation occurred in the control group 2 weeks postoperatively, but the posterior capsule was clear in TAH treated eyes. 4 weeks and 8 weeks after operation, PCO occurred both in TAH

Pinellia Total Alkaloids Modulate the GABAergic System in Hippocampal Formation on Pilocarpine-Induced Epileptic Rats

Objective:To investigate the neuromodulatory effect of pinellia total alkaloids(PTA)on the gamma-aminobutyric acidergic(GABAergic)system in epileptic rats,and preliminarily evaluate the anti-epileptic effect of PTA.Methods:Ninety-one male Sprague-Dawley rats were randomized to a control group(n=17)or an epileptic group(n=74)using computer-generated random numbers.Status epilepticus(SE)was induced with pilocarpine in the epileptic group.Epileptic rats that survived SE were randomly divided into 4 groups,namely an epilepsy group(n=13),a topiramate(TPM,60 mg/kg)group(n=12),a high-dose PTA(800 mg/kg)group(n=12),and a low-dose PTA(400 mg/kg)group(n=10).Treatments were given intragastrically once daily for 14 days.The control group and epilepsy group received normal saline.Spontaneous recurrent seizures(SRSs)were monitored 8-h daily for 7 days after treatment.Then,the hippocampal formation tissues were collected.GABA level was measured using enzyme-linked immunosorbent assay.Protein and mRNA expression levels of glutamate decarboxylase 65(GAD65),GABA transporter-1(GAT-1),GABA transaminase(GABA-T),and GABAA receptor(GABAAR)α4,α5,γ2 andδsubunits were measured using Western-blotting analysis and quantitative polymerase chain reaction.Results:PTA lowered the incidence and frequency of SRS(both doses vs.the TPM group,P>0.05).Compared with the epilepsy group,PTA increased the levels of GABA(both doses P<0.01)and GAD65(mRNA,800 mg/kg,P<0.01),and suppressed the levels of GAT-1(mRNA,800 mg/kg,P<0.01;400 mg/kg,P<0.05),GABA-T(mRNA,both doses P<0.01),and GABAARδsubunit(protein,800 mg/kg,P<0.05)andγ2 subunit(protein,both doses P<0.01).PTA upregulated the low-expressed mRNA levels of GABAARα5 subunit(400 mg/kg,P<0.01),δsubunit(800 mg/kg,P<0.05),andγ2 subunit(400 mg/kg,P<0.05).Conclusions:PTA regulated the GABAergic system through modulating GABA levels and the expression levels of GAD65,GAT-1,GABA-T,and GABAARα4,α5,γ2 andδsubunits.PTA may exert antiepileptic effects on the pilocarpine-induced epilepsy model.

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.

Effects of Total Alkaloids in Buxus microphylla Leaves on Aorta Smooth Muscle of Rats and Their Mechanisms

Objective To investigate the effects of total alkaloids in Buxus microphylla leaves(ABML)on isolated rats thoracic aorta rings,and then to explore the possible mechanisms underlying the effects.Methods Thoracic aortas of Wistar rats were isolated,removed,and mounted onto an organ bath.The effects of ABML at different concentration on the contraction of isolated thoracic aorta rings(with and without endothelium)precontracted with KCl or PE were observed with organ bath technique.Dose-effect curves of CaCl2 were recorded by organ bath technique.The concentration of intracellular Ca 2+ ([Ca 2+ ]i)increased by PE,KCI,and caffeine in the presence of ABML was determined using Ca 2+ sensitive fluorescence indicator Fura-2/AM loaded thoracic aorta vascular smooth muscle (VSM)cells of rats.Results In aorta rings precontracted with PE and KCl,ABML produced concentration- dependent relaxation in both intact and denuded endothelium ring groups.There was no difference in the inhibition of contraction between the intact and denuded endothelium ring groups at the same concentration.Exposure of isolated thoracic aorta rings to ABML led to a significant reduction in the contracting response induced by CaCl2,and shifted the cumulative concentration-response curves to right.ABML could significantly inhibit the extracellular Ca 2+ influx induced by PE and KCl under[Ca 2+ ]0 of 1.5 mmol/L,with inhibitory ratios of 40.2%and 49.9%,respectively.In the case of Ca 2+ -free,ABML could significantly inhibit the intracellular Ca 2+ release induced by PE,with inhibitory ratio of 72.4%.Conclusion ABML relaxes thoracic aorta VSM cells by suppressing influx of extracellular Ca 2+ via voltage-dependent Ca 2+ channel and receptor-operated Ca 2+ channel.

Research on Hypnotic and Anticonvulsant Activities of Total Alkaloids in Leaves of Eucommia ulmoides

Objective To study the hypnotic and anticonvulsant activities of the leaves of Eucommia ulmoides（LEU）,the total alkaloids were extracted from LEU using water-acid method.Methods Mice were divided into five groups,including groups contained total alkaloids in three different doses,negative and positive control groups.Direct hypnotic experiment in mice,pentobarbital sodium synergistic experiment,and anticonvulsant experiment were used.The numbers of spontaneous activities,sleep rate,sleep latency,sleep time,convulsion rate,and convulsion latency of the mice were recorded and analyzed.Results The total alkaloids from LEU could increase the sleep rate,significantly lengthen the sleep time,and shorten the sleep latency of mice.Even the low dose of total alkaloids（0.33 g/kg）showed significantly different activities with negative control group（physiological saline）with the synergistic effect of the superthreshold dose of pentobarbital sodium.Furthermore,the total alkaloids efficiently inhibited the convulsion caused by nikethamide.Conclusion The total alkaloids from LEU have the excellent sedative,hypnotic,and anticonvulsant activities in mice,with high safety and little drug side effects.Therefore,they have the protential development prospects in sedative-hypnotic drugs.

Absorption characteristics of the total alkaloids from Mahonia bealei in an in situ single-pass intestinal perfusion assay

AIM: To investigate the absorption characteristics of the total alkaloids from Mahoniae Caulis(TAMC) through the administration of monterpene absorption enhancers or protein inhibitors. METHOD: The absorption behavior was investigated in an in situ single-pass intestinal perfusion(SPIP) assay in rats. RESULTS: The intestinal absorption of TAMC was much more than that of a single compound or a mixture of compounds(jatrorrhizine, palmatine, and berberine). Promotion of absorption by the bicyclic monoterpenoids(borneol or camphor) was higher than by the monocyclic monoterpenes(menthol or menthone), and promotion by compounds with a hydroxyl group(borneol or menthol) was higher than those with a carbonyl group(camphor or menthone). The apparent permeability coefficient(Papp) of TAMC was increased to 1.8-fold by verapamil, while it was reduced to one half by thiamine. The absorption rate constant(Ka) and Papp of TAMC were unchanged by probenecid and pantoprazole. CONCLUSION: The intestinal absorption characteristics of TAMC might be passive transport, and the intestinum tenue was the best absorptive site. In addition, TAMC might be likely a substrate of P-glycoprotein(P-gp) and organic cation transporters(OCT), rather than multidrug resistance protein(MRP) and breast cancer resistance protein(BCRP). Compared with a single compound and a mixture of compounds, TAMC was able to be absorbed in the blood circulation effectively.

STUDIES ON GLYCOSIDES ⅤⅢ.TOTAL SYNTHESIS OF NEW ALKALOID GLYCOSIDES FROM SELAGINELLA DOEDERIENII

Two new alkaloids,hordenine-O- -L-rhamnopyranoside(Ⅰ)and hordenine -0-(6′-trans-cinnamoyl)-4′-O-β-D-glucopyranosyl- -L-rhamnopyranoside(Ⅲ) isolated from Selaginella doederleinii have been synthesized the first time. A new method,in which trifluoroacetoxyl group was used both as a good leaving group and a protecting group for synthesis of O-glycosides with high stereo- selectivity was reported.

3种类型烟草种质资源烟叶生物碱等化学成分差异性分析

为筛选能为科研、生产和育种利用的优异烟草种质资源。对来源于国内外的141份烤烟、18份白肋烟和6份雪茄烟的烟叶常规化学成分和主要生物碱进行检测、比较和聚类分析。结果表明:总糖和还原糖含量以烤烟最高,总氮、烟碱和氧化钾以白肋烟种质最高。总植物碱和烟碱含量以烤烟种质自来黄2243最高,雪茄烟种质Dutch(Ohio)最低;降烟碱和烟碱转化率以烤烟种质平板柳叶最高,以烤烟种质二性子和白肋烟种质Burley11B最低。聚类分析将165份种质分成4类,烤烟种质主要集中在第1和第4类,其主要特征是总糖和还原糖含量较高,烟碱转化率较低;第2类的总糖和还原糖含量低,总氮、总植物碱和氧化钾含量高;第3类的烟碱和总植物碱含量低,降烟碱含量高,烟碱转化率高。不同类型种质资源的化学成分和生物碱差异明显,筛选出的不同梯度烟碱的种质可为烟碱的进一步研究提供材料支撑。

金属离子对Trichoderma longibrachiatum UN32石斛碱型生物碱产量的影响

石斛碱是传统植物金钗石斛(Dendrobium nobile)的特征药用活性成分,结构上属于石斛型生物碱(dendrobine-type total alkaloids,DTTAs)。石斛碱的积累受周期和环境的影响,仅从植物中获取难以满足需求。该研究的对象Trichoderma longibrachiatum UN32是实验室前期通过复合诱变获得的产DTTAs的正突变株。为提高UN32的生物碱产量,使产物表达水平趋于稳定,该研究从UN32发酵培养基中无机盐的添加量出发,利用响应面法确定金属离子的最优配比。根据Plackett-Burman法设计筛选出对响应结果影响程度最显著的3个因素分别为Zn^(2+)、Cu^(2+)、Fe^(2+),其最佳添加时间和添加浓度分别是Cu^(2+)0.5 mmol/L(第6天)、Fe^(2+)0.4 mmol/L(第6天)和Zn^(2+)0.7 mmol/L(第3天)。响应面统计的结果显示,当模型响应值生物碱量达最大值时,Cu^(2+)、Zn^(2+)和Fe^(2+)的浓度分别为0.54、0.69、0.39 mmol/L,此时生物碱产量达到(317.36±6.48)μg,比初始培养基提高了72.68%。综上,金属离子及其不同组合对菌株UN32生物碱的积累具有促进作用,该方法具有较好的开发价值和应用前景。

夏桑菊中药渣发酵菌种筛选及其发酵条件优化

该研究以总生物碱含量为评价指标,从长期堆积的夏桑菊中药渣中分离筛选提高夏桑菊中药渣生料发酵中总生物碱含量的菌株,通过形态学特征、生理生化试验和16S rDNA序列分析对菌株进行鉴定,并通过单因素试验及响应面法对菌株发酵条件进行优化。结果表明,共分离纯化出6株可提高夏桑菊中药渣中总生物碱的菌株(L-1~L-6),其中菌株L-1发酵物料中总生物碱含量最高(24.89 mg/g),其被鉴定为枯草芽孢杆菌(Bacillus subtilis)。最优发酵条件为:物料(夏桑菊中药渣80 g+麦麸10 g+豆饼粉10 g)与水的固水比1∶1(g∶mL),装料量50 g/250 mL,接种量6.7%,发酵时间6 d,初始pH值6.5。在此优化条件下,总生物碱含量达31.15 mg/g。

钩吻总碱诱导慢性粒细胞白血病K562细胞凋亡的机制分析

目的 探讨钩吻总碱对慢性粒细胞白血病K562细胞的抑杀作用及其机制,为其抗白血病研究提供科学依据。方法 以不同浓度钩吻总碱(25、50、100、200、400μg·mL-1)干预K562细胞,MTT法测定其对K562细胞活力的影响;倒置相差显微镜观察其对K562细胞形态的影响;DAPI染色法观察K562细胞核形态变化;Annexin V FITC/PI流式细胞术检测K562细胞凋亡情况;比色法测定caspase-3活性变化;RT-PCR检测Bax和Bcl-2基因的表达水平。结果 钩吻总碱对K562细胞抑制效果呈时间、剂量依赖性,24 h时IC50为122μg·mL^(-1);TAG干预K562细胞后细胞形态逐渐不规则、胞质不清、胞核皱缩,最终胞膜的完整性破坏;DAPI染色发现细胞体积变小,整体皱缩,胞核固缩、生成典型的凋亡小体;Annexin V FITC/PI流式细胞测定显示K562细胞凋亡以早凋为主,存在量效关系;不同浓度钩吻总碱作用于K562细胞24 h后caspase-3的活性提高;RT-PCR检测发现TAG干预后会下调Bcl-2基因表达、上调Bax基因表达,二者均表现出量效关系。结论 钩吻总碱可能通过上调Bax基因表达、下调Bcl-2基因表达,活化caspase-3,从而诱导慢性粒细胞白血病K562细胞发生早期凋亡,抑制其细胞活力。