新疆紫草毛状根的诱导及培养

将处于对数生长期(A600为0.5)的发根农杆菌MSU440、A4、R1000、15834、1025和R1601与新疆紫草子叶外植体共培养.结果表明:(1)发根农杆菌不同菌种对转化率有显著影响,供试6个菌种中只有MSU440菌株获得转化株.PCR及序列分析表明发根农杆菌Ri质粒的rolC基因已在新疆紫草毛状根基因组中整合并得到表达,转化率达4.5%.(2)子叶较真叶的不定根发生率高,且生根持续时间长.(3)在B5无铵无激素固体培养基上,毛状根分支多且根较长,达2~3 cm,毛状根鲜重月平均增殖达7~9倍,是固体培养毛状根的适宜培养基.(4)毛状根在MS无铵无激素液体培养基中培养12 d时,毛状根鲜重平均增殖达12倍,MS无铵液体培养基有利于毛状根的扩大生产.首次获得了激素自主、快速伸长生长、多分支、多根毛的新疆紫草毛状根株系,初步建立了新疆紫草毛状根诱导体系,为大规模培养、生产紫草素奠定了基础.     

遮荫对新疆紫草育苗的影响

目的 探讨新疆紫草育苗的最佳遮荫条件.方法 采用不同的遮荫条件,研究了遮荫处理对新疆紫草原生苗生长量和成活率的影响.结果 结果表明,不同强度的遮荫处理对原生苗的叶面积、成活率、地下生长量影响显著.适度遮荫可以增加叶面积和地下生长量,提高幼苗成活率,结论 采用30%遮荫率的遮荫处理能够同时满足增加生长量和提高成活率的要求,同时避免了叶片先端的日灼伤害.     

新疆紫草毛状根总糖及多糖含量分析

采用超声提取法,对新疆紫草固体培养毛状根、液体培养毛状根以及2～3年野生根中的总糖和多糖进行了提取测定.结果表明:毛状根中水溶性总糖含量为25.573%,是野生根总糖含量的3.4倍;固体培养的毛状根多糖含量最高,达4.289%,是液体培养毛状根的3.37倍,是野生根的4倍.稳定性试验和重复性试验表明,该研究结果可靠性强,重复性好且稳定.研究发现,新疆紫草毛状根中水溶性总糖和多糖的含量高于野生根.     

真菌诱导子与吸附树脂对新疆紫草毛状根中萘醌积累的影响

通过考察真菌诱导子与吸附树脂对新疆紫草毛状根中萘醌积累的影响,获得真菌诱导子与吸附树脂对萘醌类物质积累的最佳处理,为规模化生产提供依据.以新疆紫草毛状根为试验材料,将黑曲霉、米曲霉诱导子及其混合诱导子、大孔吸附树脂添加到M-9培养基中,采用分光光度法测定毛状根总萘醌含量.试验结果表明:在毛状根培养10d时以2.5∶50的比例添加混合诱导子,总萘醌含量是对照的2.28倍;在此结果基础上,在培养第0天添加大孔吸附树脂NKA-9,总萘醌含量最高是对照的3.71倍;黑曲霉诱导子与米曲霉诱导子有协同效应;在生物反应器中添加混合诱导子及大孔吸附树脂NKA-9,其总萘醌含量是对照的4.17倍.米曲霉诱导子、混合诱导子对毛状根增殖有促进作用;同时添加大孔吸附树脂NKA-9及混合诱导子能提高毛状根总萘醌含量.生物反应器培养毛状根为今后利用新疆紫草毛状根规模化生产总萘醌提供了理论参考.     

Effect of l-phenylalanine on PAL activity and production of naphthoquinone pigments in suspension cultures of Arnebia euchroma (Royle) Johnst

The effects of l-phenylalanine (PHE) on cell growth and production of shikonin and its derivatives, acetylshikonin (ACS) and isobutyrylshikonin (IBS), in suspension cultures of Arnebia euchroma were examined. Supplementing media using PHE have been successfully utilized to enhance shikonin production in cell cultures of other species of Boraginaceae. l-Phenylalanine, the key compound in the phenylpropanoid pathway, is converted by phenylalanine ammonia lyase (PAL) to trans-cinnamic acid, which is the precursor of p-hydroxybenzoic acid (PHB). Coupling of PHB and geranyl pyrophosphate (derived from mevalonate pathway) by p-hydroxybenzoate-m-geranyltransferase leads later to biosynthesis of shikonins. The addition of 0.01 or 0.1?mM PHE to the culture medium stimulated cell proliferation, where the highest observed increase in fresh cell biomass (measured as a ratio of final weight to initial weight) was 12-fold, in contrast to an eightfold increase in control cultures. Whereas, growth media supplemented with 1?mM PHE markedly reduced the rate of cell growth (to only twofold). Precursor feeding had detrimental effects on both ACS and IBS production in all PHE-supplemented media. The highest total content (intracellular + extracellular) of the investigated red pigments (9.5?mg per flask) was detected in the control culture without PHE. ACS was the major component of the naphthoquinone fraction determined in cells and post-culture media. Shikonin itself was found only in the post-culture media from cultures supplemented with 0.01 or 0.1?mM PHE. Increases in PAL activity corresponded well with the accumulation of investigated naphthoquinones in control culture. However, peak PAL activity did not directly correlate with maximum production of shikonin derivatives. Cytotoxicity of extracts, prepared from the cells cultivated in the presence of PHE or in control cultures, was tested on three cancer cell lines: HL-60, HeLa, and MCF-7. The extracts prepared from the untreated control cultures proved to be the most potent against the examined cancer cell lines. The mean inhibitory concentration values were 0.3, 13, and 8???g?ml^?1 for the HL-60, HeLa, and MCF-7 cells, respectively.     

Direct shoot regeneration from intact leaves of Arnebia euchroma (Royle) Johnston using thidiazuron

The present study highlights the importance of preculture time and concentration of TDZ (thidiazuron) for direct regeneration from in vitro leaves (attached to shoots) in Arnebia euchroma. Shoot buds proliferated to form multiple shoots on MS medium (Murashige and Skoog medium) with 5.0 μM Kn. Different additives viz. ascorbic acid, PVP (polyvinylpyrrolidone), PVPP (polyvinylpolypyrrolidone) or activated charcoal (50, 100 and 250 mg/l each) were used to check the phenolic exudations. Direct shoot regeneration was obtained when shoots were initially precultured for 40 days on medium with a higher concentration of TDZ (20.0 μM) and then transferred to a lower concentration (5.0 μM TDZ). The identity of shoot buds was confirmed by histological studies. Regenerated shoots were cultured for 30 days on medium containing Kn (5.0 μM) for proliferation and then transferred to IBA (0.25 μM)‐containing medium for rooting. Rooted plantlets were transferred to greenhouse with 45–50% survival.     

外循环气升式反应器培养新疆紫草细胞

采用两步培养法进行新疆紫草细胞悬浮培养及5L外循环气升式反应器扩大培养,探讨了培养过程中细胞生长、紫草色素合成与培养液的电导率、可溶性糖含量变化之间的关系。第一步培养时细胞生长迅速,但也有一部分色素合成,电导率及可溶性糖含量迅速下降;第二步培养初期电导率也开始下降,但当色素合成达到高峰并有一部分外泌到培养基后,电导率又开始回升。可溶性糖捎耗很快,到后期巳测不出其存在。因此通过监测培养液中电导率及可溶性糖的变化情况,可以为新疆紫草细胞大规模培养与色素合成提供有用的参数指标。     

Determination of metronidazole in vaginal tissue by high-performance liquid chromatography using solid-phase extraction

A sensitive high-performance liquid chromatographic (HPLC) method for the determination of metronidazole in vaginal tissue is reported. The method uses a Zorbax SB phenyl column with a 0.01 M aqueous monobasic potassium phosphate buffer (pH 4.0)-absolute methanol (85:15, v/v) as mobile phase at a flow-rate of 1.0 ml/min and detection at 313 nm. Tinidazole was used as the internal standard. The method employed homogenization of tissue followed by solid-phase extraction. The quantitation was achieved within 30 min with sensitivity in the ng/g range. Metronidazole was linear in the 100–2000 ng/g range. The accuracy and precision were in the 1–4% range for the drug and the limit of detection was approximately 100 ng/g based on a signal-to-noise ratio of 3 and a 100-μl injection.     

Determination of glibenclamide in human plasma by solid-phase extraction and high-performance liquid chromatography

A sensitive high-performance liquid chromatographic method for determination of intact glibenclamide in human plasma has been developed. Sample clean-up prior to chromatographic analysis was accomplished by extraction of the drug using a solid-phase RP-8 or RP-18 cartridge instead of the conventional liquid-liquid extraction methods described. For the separation of the drug from the endogenous components a reversed-phase column (LiChrosorb RP-8) of 5 μm particle size and 250×4 mm I.D., together with a mobile phase consisting of acetonitrile-12 μM perchloric acid (47:53) was selected. The method employs progesterone as an internal standard, and a reversed-phase column combined with UV detection of the drug at 230 nm. The detector response was linear up to the concentration of 400 ng/ml and the average recovery was 100.36%. The sensitivity of the method was 5 ng/ml.     

Determination of cimetidine in human plasma by high-performance liquid chromatography following liquid-liquid extraction

A new method is described for the determination of cimetidine in human plasma. The drug and internal standard (ranitidine) were separated on a Nucleosil C₁₈ 5 μm (25 × 4.6 mm I.D.) column using a mobile phase of acetonitrile-phosphate buffer, pH 6.2 (25:75, v/v) containing 2.5 g/l heptane sulphonic acid. The mobile phase was delivered at a flow-rate of 0.9 ml/min, detection was by ultraviolet absorption at 228 nm and concentrations were calculated on the basis of peak areas. The drugs were extracted from alkaline plasma into ethyl acetate using a salting out procedure which involved the addition of 100 ml of a saturated solution of K₂CO₃ to each 250-μl plasma aliquot. The method was validated over the concentration ranges 50–3000 ng/ml and 100–7000 ng/ml for two separate studies. Mean coefficients of variation were less than 6% for both intra- and inter-assay in both studies and recoveries varied between 71 and 81%. The method was successfully applied to the determination of cimetidine in plasma for a pharmacokinetic study.     

Determination of linezolid in growth media by high-performance liquid chromatography with on-line extraction

An isocratic high-performance liquid chromatography (HPLC) method with on-line extraction has been developed to determine linezolid in Mueller-Hinton broth. The loading mobile phase consisting of water-acetonitrile 99:1 (v/v) allowed retention of the analyte on a LiChrocart 4-4 pre-column filled with a LiChrospher 100 RP-8, 5 microm. The transfer of the analyte by a backflush mode to a 150 mm x 4.6 mm I.D. Kromasil C8 5 microm column was performed using a mobile phase of water-acetonitrile 80:20 (v/v). UV detection at 254 nm allowed a quantification limit of 0.39 microg/mL with a 50-microL sample size. The method was successfully applied to in vitro pharmacokinetic-pharmacodynamic studies.     

Determination of penicillin-V in human plasma by high-performance liquid chromatography and solid-phase extraction

A high-performance liquid chromatographic method has been developed for the determination of penicillin-V concentrations between 0.1 and 19 μg/ml in human plasma. Penicillin-V was isolated from plasma by solid-phase extraction on a C₁₈/OH cartridge. The extracts were injected onto a reversed-phase HPLC system. A 125×4 mm C₁₈ column was used to separate penicillin-V from its main metabolites, 5R- and 5S-penicilloic acid and endogenous compounds. The eluent consisted of 66% 0.02 M phosphoric acid buffer, to which tetrabutylammonium dihydrogenphosphate and 34% acetonitrile were added. The column effluent was monitored by ultraviolet spectrophotometry at 269 nm. Using this method, penicillin-V concentrations in plasma could be determined with an accuracy between −5.4 and 5.2% and a precision between 0.8 and 1.6%. The method has proved to be reliable and was used in biovailability studies for the development of a new oral penicillin-V formulation.     

Determination of dopaminergic prodrugs by high-performance liquid chromatography followed by post-column ion-pair extraction

One possibility to optimize the therapeutic application of dopaminergic compounds with a catechol function is the reversible protection of this moiety using a prodrug approach. Important features in this respect are a proper chemical stability in the gastrointestinal tract, an adequate release rate after arrival in the blood stream or the possibility to cross the blood–brain barrier. A HPLC method was developed to measure the hydrolysis of prodrugs of dopamine and epinine directly. The method is based on reversed-phase separation followed by post-column ion-pair extraction with a fluorescent counter-ion. The separation of di-isobutyryl esters of dopamine and epinine is obtained within 10 min while the more hydrophobic dopaminergic esters, di-benzoyl and di-pivaloyl dopamine, are retained for 30 min. The precision of the assay measuring 160 ng dibudop and 100 ng ibopamine was 1.2 and 1.0%, respectively. The detection limit of all prodrugs tested was approximately 10 ng.     

Determination of omeprazole in rat plasma by high-performance liquid chromatography without solvent extraction

A HPLC method without solvent extraction and using ultraviolet detection at 302 nm for the determination of omeprazole in rat plasma has been validated. Plasma samples after pretreatment with acetonitrile to effect deproteinization were dried under N(2) at 40 degrees C and reconstituted with mobile phase. The standard calibration curve for omeprazole was linear (r(2)=0.9999) over the concentration range of 0.02-3 microgml(-1). The intra- and inter-day assay variability range was 4.8-9.2% and 5.2-10.3% individually. This method has been successfully applied to a pharmacokinetic study of omeprazole in rats.     

Determination of cystamine by high-performance liquid chromatography

A highly sensitive and specific assay method for cystamine using high-performance liquid chromatography has been developed. The method is based on postcolumn derivatization of cystamine with o-phthaladehyde in the presence of 2-mercaptoethanol and sodium hypochlorite. The separation of cystamine was achieved using a cation exchange column (ISC-05/S0504). The assay was linear over the concentration range of 2 to 200 pmol. For the application of this assay method to biological materials, the pretreatment with a cation exchange column (Dowex 50W X 8) was necessary to remove interfering o-phthaladehyde-reactive substances. Since cysteamine in biological materials was quantitatively converted to cystamine during these sampling procedures, this method was found to be suitable for assaying the cysteamine plus cystamine content in various organs and tissues. The cysteamine-cystamine content in various tissues of rat determined by the present assay method has been presented.     

Determination of finasteride in human plasma by liquid–liquid extraction and high-performance liquid chromatography

A high-performance liquid chromatographic method for the quantitation of finasteride in human plasma is presented. The method is based on liquid–liquid extraction with hexane–isoamylalcohol (98:2, v/v) and reversed-phase chromatography with spectrophotometric detection at 210 nm. The mobile phase consists of acetonitrile–15 mM potassium dihydrogenphosphate (40:60, v/v). Clobazam is used as the internal standard. The limit of quantitation is 4 ng/ml and the calibration curve is linear up to 300 ng/ml. Within-day and between-day precision expressed by relative standard deviation is less than 5% and inaccuracy does not exceed 8%. The assay was used for pharmacokinetic studies.     

Determination of phenothiazine derivatives in human urine by using ionic liquid-based dynamic liquid-phase microextraction coupled with liquid chromatography

A simple and rapid method for the determination of seven phenothiazines derivatives (chlorpromazine, promethazine, levomepromazine, prochlorperazine, trifluoperazine, fluphenazine and thioridazine) in human urine samples is presented. The analytes are extracted from the sample in 50 μL of the ionic liquid 1-butyl-3-methyl-imidazolium hexafluorophosphate working in an automatic flow system under dynamic conditions. The chemical affinity between the extractant and the analytes allows a good isolation of the drugs from the sample matrix achieving at the same time their preconcentration. The separation and detection of the extracted compounds is accomplished by liquid chromatography and UV detection. The proposed method is a valuable alternative for the analysis of these drugs in urine within the concentration range 0.07–10 μg mL^?1. Limits of detection were in the range from 21 ng mL^?1 (thioridazine) to 60 ng mL^?1 (levomepromazine). The repeatability of the proposed method expressed as RSD (n = 5) varied between 2.2% (levomepromazine) and 3.9% (chlorpromazine).