Studies on rhizosphere microbial diversity of some commercially important medicinal plants

A preliminary study was made on four medicinal plants viz., Ocimum sanctum L., Coleus forskholii Briq, Catharanthus roseus (L.) G. Don. and Aloe vera in order to identify and enumerate the rhizosphere, non-rhizosphere and diazotrophic microorganisms in soil. The diazotrophic bacterial population studied includes Azospirillum, Azotobacter and Pseudomonas. The rhizosphere bacterial populations were 23.33 x 10(6)g(-1) in O. sanctum followed by C. roseus (20.46 x 10(6)g(-1)), A. vera (18.44 x 10(6)g(-1)) and C. forskholii (16.64 x 10(6)g(-1)). The fungi populations were 19.44 x 10(4)g(-1) in C. roseus, 18.66 x 10(4)g(-1) in O. sanctum, 16.44 x 10(4)g(-1) in A. vera and 14.22 x 10(4)g(-1) in C. forskholii. The actinomycetes population was 12.22 x 10(5)g(-1) in O. sanctum, 10.44 x 10(5)g(-1) in C. roseus, 8.44 x 10(5)g(-1) in A. vera and 6.22 x 10(5)g(-1) in C. forskholii. The diazotrophic bacterial population of Azospirillum, Azotobacter and Pseudomonas is 8.2 x 10(4)g(-1), 12 x 10(4)g(-1), 6 x 10(4)g(-1) in the rhizosphere soil. In all the four medicinal plants the microbial population is more in the rhizosphere soil, when compared to non-rhizosphere soil. These results are helpful in developing a biofertilizer consortium for these commercially grown medicinal plants.     

In vitro accelerated mass propagation and ex vitro evaluation of Aloe vera L. with aloin content and superoxide dismutase activity

An innovative protocol on accelerated in vitro propagation and acclimatisation was developed in Aloe vera L. Culture was initiated with rhizomatous stem where Murashige and Skoog (MS) medium fortified with 0.5?mg?L(-1) α-naphthalene acetic acid and 1.5?mg?L(-1) N(6)-benzylaminopurine (BAP) promoted earliest shoot induction. Maximum shoot multiplication was achieved in MS medium supplemented with 2.5?mg?L(-1)BAP. The best in vitro rooting was observed in the MS medium with 0.5?mg?L(-1) indole-3-acetic acid plus 2?g?L(-1) activated charcoal. The simple acclimatisation process, primarily with a combination of sand and soil (1?:?1 v/v) and finally with a blend of sand, soil and farm yard manure (2?:?1?:?1 v/v), ensured a 98% survival rate. Overall, 192 true-to-type plantlets were achieved from a single explant within 85 days. Morphologically, in vitro generated plants performed better than conventionally propagated plants; nevertheless the similarity in aloin content, gel content and superoxide dismutase activity was corroborated.     

毛细管区带电泳测定芦荟中的有效成分

 建立了毛细管区带电泳测定中药芦荟中的有效成分芦荟甙和芦荟大黄素的分析方法。采用24 mmol/L的磷酸盐(pH 10.85)作为缓冲溶液,在电压为15 kV和检测波长为254 nm的条件下进行分离实验,芦荟提取液中有效成分获得基线分离。定量分析表明,芦荟甙的质量浓度为0.029 g/L～1.00 g/L时,其峰面积与质量浓度呈线性关系(r=0.997);芦荟大黄素的质量浓度为5.4 mg/L～84 mg/L时,其峰面积与质量浓度呈线性关系(r=0.999)。此外,还讨论了缓冲溶液的pH值、有机改性剂对两种有效成分迁移行为的影响。     

Identification and determination of active anthraquinones in Chinese teas by micellar electrokinetic capillary chromatography

The simultaneous determination of emodin, aloe-emodin and rhein, in Semen Cassiae, Qinghai Wild Dahuang tea, Ningxia Juemingzi tea, Lanzhou Juemingzi tea and Surong Juemingzi tea, has been investigated by micellar electrokinetic capillary chromatography for the fi rst time. With an electrolyte containing 15 mm borax, 30 mm SDS, 10% (v/v) ethanol, at pH 9.60 and 20 kV applied voltage, the three analytes were completely separated within 12 min. The effects of the concentration of borax, electrolyte pH, the concentrations of SDS and organic modifier and the applied voltage on electropheoretic behavior and separation were studied. The linear calibration range was 4-120 micro g/mL (r = 0.9921) for emodin, 10-200 micro g/mL (r = 0.9970) for aloe-emodin and 2-100 micro g/mL (r = 0.9971) for rhein, respectively. Under the optimum conditions, the relative standard deviation (RSD) values (n = 6) of the migration time and the peak area of each peak was 0.59-0.80% and 1.30-3.22%, respectively. The contents of the analytes in Rheum, Qinghai Wild Dahuang tea, Semen Cassiae and three other kinds of teas were easily determined with recoveries ranging from 95.3 to 104.4%.     

芦荟大黄素在水和非水溶剂中的电化学性质

在HAc-NaAc水溶液或二甲亚砜(DMSO)非水溶剂中,芦荟大黄素于铂电极上均显示一个准可逆的双电子转移过程,与HAc-NaAc溶液相比,芦荟大黄素在DMSO溶剂中的峰电位明显负移,且波形改变很大;HAc-NaAc溶液中,峰电流Ipa与芦荟大黄素的浓度在1.0×10-5~6.0×10-4mol/L范围内成线性关系,检测限为1.0×10-6mol/L.溶剂对芦荟大黄素的电化学性能有甚大的影响.     

Determination of six bioactive components of Saussurea katochaete by capillary electrophoresis

The simultaneous determination of 3,4-dimethoxy-3'-hydroxy propiophenone, 5-hydroxy-7-methoxy coumarin, 7-hydroxy coumarin, 3',5'-dimethoxy apigenin, apigenin and 4-hydroxy cinnamic acid in the extract of S. katochaete has been investigated by capillary electrophoresis for the first time. The six active components were completely separated within 10 min in 20 mM Na(2)HPO(4) buffer at pH 11.00 with 10% (v/v) methanol and detected at 214 nm. The applied voltage was 15 kV and the temperature was kept at 25 degrees C. The effects of buffer pH, the concentration of Na(2)HPO(4) and the concentration of methanol on the separation efficiency were studied systematically. The regression equations revealed good linear relationships (correlation coefficients were 0.9987-0.9998) between the peak area of each analyte and its concentration. The relative standard deviations (RSD) of migration time and peak areas were <1.63 and <4.03%, respectively. The application of this method for the separation and determination of the six bioactive components in S. katochaete was reported. The contents of the six analytes ranged from 0.023 to 0.131 mg/g and recoveries ranged from 94.7 to 104.8%.     

Analysis of tetramethylpyrazine in Ephedrae herba by gas chromatography-mass spectrometry and high-performance liquid chromatography

A simple and reliable HPLC method was developed for the determination of 2,3,5,6-tetramethylpyrazine (TMP) in Ephedrae herba. Further identification of TMP was achieved using GC-MS. The mobile phase used was methanol-water-35% acetic acid (35:65:0.5, v/v/v) at a flow-rate of 0.8 ml/min. The detection wavelength was set at 290 nm. The linear range of the peak area calibration curve of TMP was 2.64-264 mg/l (r=0.9987) and the recovery for TMP in Ephedrae herba extracts was 101.1-106.9%. The relative standard deviations of retention time and peak area were 0.18 and 1.5% (n=6), respectively. The detection limit of TMP was 0.03 mg/l. The contents of TMP in Ephedrae herba could easily be determined within 10 min.     

气相色谱-三重四极杆串联质谱法同时测定调味品中的氨基甲酸乙酯和氯丙醇

建立了气相色谱-三重四极杆串联质谱(GC-MS/MS)同时测定调味品中氨基甲酸乙酯(EC)和氯丙醇(3-氯-1,2-丙二醇(3-MCPD)和2-氯-1,3-丙二醇(2-MCPD))的方法。样品添加同位素内标后,采用Extrelut^TM NT有机硅藻土填料进行基质固相分散萃取,先用正己烷淋洗除杂,再用乙酸乙酯-乙醚(20:80, v/v)混合溶剂洗脱被测物,洗脱液经浓缩后采用GC-MS/MS多反应监测(MRM)模式测定。EC、3-MCPD和2-MCPD的方法检出限依次为2、5和5 μg/kg,线性范围依次为5~1000 μg/kg(r=0.9997)、10~1000 μg/kg(r=0.9991)和10~1000 μg/kg(r=0.9995)。酱油、料酒(黄酒)、沙拉酱和方便面调味料4种基体中在20、100和400 μg/kg 3个水平加标测定的平均回收率和相对标准偏差(RSD,n=7)范围分别为EC: 87.7%~104%(RSD为4.3%~10.7%)、3-MCPD: 90.1%~109%(RSD为2.6%~10.2%)、2-MCPD: 90.9%~103%(RSD为3.0%~9.5%)。在部分酱油、料酒和方便面调味料中同时检测到了EC、3-MCPD和2-MCPD,在部分沙拉酱中检测到了EC或3-MCPD。该法准确、快速,适用于调味品中EC、3-MCPD和2-MCPD的同时检测。     

In vitro and in silico study of Aloe vera leaf extract against human breast cancer

Abstract

Aloe vera leaf contains some bioactive compounds that have a strong binding affinity toward estrogen receptor as compared to standard drug tamoxifen. In this study, we have found that the IC₅₀ of Aloe vera leaf extract against breast cancer cell line (MCF-7) is 23?µg/mL which is much lower than the IC₅₀ (332?µg/mL) of Aloe vera leaf extract against non-cancerous cell line (NIH-3T3). We have also calculated the total concentration of phenolic acid (385.662?µg/mL), flavonoids (160.402?µg/mL) and alkaloids (276.754?µg/mL) in Aloe vera leaf extract. The free radical scavenging activity of Aloe vera leaf extract is 67% to 89% (at 50 to 300?µg/ml). Our virtual molecular docking study suggests that bioactive compounds like Aloe-emodin (?8.8?Kcal/mol), 7-hydroxy-2,5 dimethylchromone (?7.5?Kcal/mol), Beta-sitosterol (?7.3?Kcal/mol) etc. have a greater binding affinity toward estrogen alpha receptor as compared to standard drug Tamoxifen (?6.4?Kcal/mol).

     

Densitometric thin-layer chromatographic determination of artemisinin and its lipophilic derivatives,artemether and arteether

A thin-layer chromatograpy (TLC) method is developed to analyze artemisinin (AT) and its derivatives, artemether (AM) and arteether (AE), using a silica-gel plate with a mobile phase containing pure chloroform. After development, all products are visualized after dipping in a 4-methoxybenzaldehyde dipping reagent of 1% (v/v) in an acidic solution of sulphuric acid (98%, v/v) and acetic acid (96-98%, v/v) (respectively, 2% and 10%, v/v in alcohol-water, 60:30, v/v), presenting a purple color against a slightly colored background. This TLC system is quantitatively evaluated in terms of stability of the color, precision, accuracy, and calibration. Activation is performed at 110 degrees C. Stability of the color of both analytes is reached after 12 min. Precision, less than 5%, is obtained at two levels. Good linearity is obtained in the range of 0.5-8 micro g for all analytes. Some applications show its utility in the quality control of capsules. The prederivatization technique, applying the described dipping reagent before development, reveals the presence of various reaction products, possibly isomers. These results prove that TLC can be a cheap and easy alternative for the analysis of AT and its lipophilic derivatives, AM and AE, as pure powder and in pharmaceutical-dosage forms.     

A simple high-performance liquid chromatography assay for the major cisapride metabolite, norcisapride, in human urine

A simple high-performance liquid chromatography assay using fluorescence detection for the major metabolite of the gastric prokinetic drug cisapride, norcisapride, is presented. Analysis is performed using an Alltech Platinum EPS C8 column with a mobile phase made up of methanol and 0.02M sodium dihygrogen phosphate (45:55, v/v) containing triethylamine (1 g/L). Complete resolution is achieved among norcisapride, the internal standard (metoclopramide), and endogenous urinary components. The assay is linear over the range 50-2000 ng/mL with a mean recovery of 71.2% across the analytical range following solvent extraction with toluene-isoamyl alcohol (95:5, v/v). Intraday coefficients of variation (precision) determined at 200 and 1000 ng/mL are 6.0 and 9.8%, respectively, and interday coefficients of variation are 8.8 and 6.6%, respectively. Intra- and interassay accuracy (as mean relative error) determined at the same concentrations is within 10% in all cases. An analysis of urine samples from a healthy volunteer following the administration of a single 10-mg oral dose of cisapride is shown.     

直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相拆分布地奈德对映体及其制剂含量的测定

22R-布地奈德的药物活性比22S-布地奈德的强2~3倍,开发布地奈德对映体拆分和定量分析方法,可为其药物研发及质量控制提供重要依据。目前,主要以反相C₁₈固定相对布地奈德对映体进行拆分,而采用手性固定相对其进行拆分少有报道。通过考察固定相、流动相和柱温对布地奈德对映体拆分的影响,建立了基于直链淀粉-三[(S)-1-苯乙基氨基甲酸酯]手性固定相快速拆分和检测布地奈德对映体的高效液相色谱方法,其色谱条件如下:色谱柱为Chiralpak AS-RH色谱柱(150 mm×4.6 mm, 5.0 μm),流动相为乙腈-水(45∶55, v/v),柱温40 ℃,流速1.0 mL/min,二极管阵列检测器(DAD),检测波长246 nm,进样量10 μL。在该色谱条件下,布地奈德的两个对映体得到较好拆分,22R-布地奈德和22S-布地奈德的保留时间分别6.40 min和7.77 min,分离度为4.64; 22R-布地奈德和22S-布地奈德分别在各自范围内线性关系良好,相关系数(R²)均为0.9999,检出限分别为0.05 μg/mL和0.07 μg/mL,定量限分别为0.16 μg/mL和0.20 μg/mL; 4个添加水平的样品加标回收率为102.63%~104.17%,相对标准偏差(RSD)为0.08%~0.57%(n=6)。将该方法应用于1批次4个吸入用布地奈德混悬液实际样品进行检测,22R-布地奈德和22S-布地奈德的含量分别为283.15~284.63 μg/mL和259.86~261.51 μg/mL。该方法操作简便,分析时间短,重复性好,准确度高,可用于布地奈德对映体的拆分及其制剂的质量控制。     

Spectroscopic behavior of 1,1′-diethyl-2,2′-dicarbocyanine iodide in ethanol/water solutions with high ionic strength

Absorption spectra of 1,1′-diethyl-2,2′-dicarbocyanine iodide (DDI) have been obtained in ethanol/water mixtures. Increase of ionic strength obtained by addition of salts (e.g. NaCl) to the solvent mixture containing 10% of H₂O and 90% of EtOH leads to appearance of a new absorption band at 849 nm. Intensity of this band increases with salt concentration which suggests that this spectral feature is related to a DDI aggregate. However, no emission has been observed with excitation at 850 nm indicating that this feature does not correspond to a J-type aggregate. Addition of NaCl to DDI solutions possessing the EtOH/H₂O (v:v) ratio different from 1:9 does not induce appearance of the new absorption band.     

Determination of uric acid in human urine by capillary zone electrophoresis with indirect laser-induced fluorescence detection

A CZE with indirect LIF detection method was used for the determination of uric acid (UA) in human urine. UA and its coexisting analytes (i.e. hypoxanthine, xanthine and ascorbic acid) could be well separated within 4.5?min at a voltage of 25?kV with 25°C cartridge temperature in a running buffer composed of 5?mM sodium borate, 10% methanol (v/v) and 0.3?μM fluorescein sodium (apparent pH of the final mixed hydro-organic solution of sodium borate, methanol and fluorescein is 9.5). Under the optimum condition, the method has good linearity relationships (correlation coefficients: 0.9973-0.9987) with ranges of 25-500, 25-350, 25-250 and 25-300?μg/mL for hypoxanthine, ascorbic acid, xanthine and UA, respectively. The detection limits for the analytes were in the range of 0.29-0.90?μg/mL. The intra-day RSD values for migration times and peak areas were less than 0.43 and 3.27%, respectively. This method was applied to the quantitation of UA in human urine with recoveries in the range of 93.1-107.3%.     

毛细管电泳-间接紫外检测法测定蜂蜜中的氨基酸

采用毛细管电泳-间接紫外检测法同时分离测定蜂蜜中的赖氨酸、色氨酸、谷氨酸等9种氨基酸。考察了磷酸浓度、进样方式和缓冲液pH对分离效率和重现性的影响。在分离电压为-15 kV、检测波长为220 nm条件下,以含有0.5 mmol/L十六烷基三甲基溴化铵、20 mmol/L烟酸、10%甲醇的10 mmol/L磷酸二氢钠缓冲溶液(pH 10.2)为运行缓冲液,9种组分在11 min内达到基线分离;检出限最低可达到0.3 mg/L;线性范围为1.0~1000 mg/L;日间及日内精密度为0.64%~5.83%。实际样品中除甲硫氨酸外的8种氨基酸的加标回收率为60.00%~118.37%。将该方法应用于不同蜜源植物和产地的蜂蜜样品的测定,在市售的5种蜂蜜中均检测到脯氨酸、丝氨酸和天冬氨酸,而只在荔枝蜜中检测到苏氨酸。该方法可以为蜂蜜的蜜源鉴别及质量评估提供借鉴方法。     

Simple method for the determination of rosiglitazone in human plasma using a commercially available internal standard

To the best of our knowledge, bioanalytical methods to determine rosiglitazone in human plasma reported in literature use internal standards that are not commercially available. Our purpose was to develop a simple method for the determination of rosiglitazone in plasma employing a commercially available internal standard (IS). After the addition of celecoxib (IS), plasma (0.25 mL) samples were extracted into ethyl acetate. The residue after evaporation of the organic layer was dissolved in 750 microL of mobile phase and 50 microL was injected on to HPLC. The separation was achieved using a Hichrom KR 100, 250 x 4.6 mm C(18) with a mobile phase composition potassium dihydrogen phosphate buffer (0.01 m, pH 6.5):acetonitrile:methanol (40:50:10, v/v/v). The flow-rate of the mobile phase was set at 1 mL/min. The column eluate was monitored by fluorescence detector set at an excitation wavelength of 247 nm and emission wavelength of 367 nm. Linear relationships (r(2) > 0.99) were observed between the peak area ratio rosiglitazone to IS vs rosiglitazone concentrations across the concentration range 5-1000 ng/mL. The intra-run precision (%RSD) and accuracy (%Dev) in the measurement of rosiglitazone were <+/-10.69 and <-12.35%, respectively across the QC levels (50-1000 ng/mL). The extraction efficiency was >80% for both rosiglitazone and IS from human plasma. The lower limit of quantitation of the assay was 5 ng/mL. In summary, the methodology for rosiglitazone measurement in plasma was simple, sensitive and employed a commercially available IS.     

Determination of cimetidine,famotidine, and ranitidine hydrochloride in the presence of their sulfoxide derivatives in pure and dosage forms by high-performance thin-layer chromatography and scanning densitometry

A selective, precise, and accurate method was developed for the determination of cimetidine (C), famotidine (F), and ranitidine hydrochloride (R x HCl) in the presence of their sulfoxide derivatives. The method involves quantitative densitometric evaluation of mixtures of the drugs and their derivatives after separation by high-performance thin-layer chromatography on silica gel plates (10 x 20 cm) with ethyl acetate-isopropanol-20% ammonia (9 + 5 + 4, v/v) as the mobile phase for both C and F and ethyl acetate-methanol-20% ammonia (10 + 2 + 2, v/v) as the mobile phase for R x HCl; Rf values for C, F, and R x HCl and their corresponding derivatives were 0.85 and 0.59, 0.73 and 0.41, and 0.56 and 0.33, respectively. Developing time was approximately 20 min. For densitometric evaluation, peak areas were recorded at 218, 265, and 313 nm for C, F, and R x HCl, respectively. The relationship between concentration and the corresponding peak area was plotted for the ranges of 5-50 microg/spot for C and 2-20 microg/spot for F and R x HCl. Mean recoveries were 100.39 +/- 1.33, 99.77 +/- 1.30, and 100.09 +/- 0.69% for C, F, and R x HCl, respectively. The proposed method was used successfully for stability testing of the pure drugs in the presence of up to 90% of their degradates, in bulk powder and dosage forms. The results obtained were analyzed statistically and compared with those obtained by the official methods.     

芦荟色酮的高速逆流色谱分离制备方法研究

芦荟色酮是在芦荟叶中特有的一类具有抗炎和抑制酪氨酸酶等作用的活性物质。以芦荟全叶为原料,经过一系列的预处理手段,从脱色剂活性炭中获得芦荟色酮粗提物,再经过溶剂分配和富集后采用高速逆流色谱（HSCCC）对其中的色酮成分进行分离纯化。研究结果表明,采用氯仿-甲醇-水(体积比为4∶3∶2)混合溶液和二氯甲烷-甲醇-水(体积比为5∶4∶2)混合溶液作为溶剂分离系统,经过两步HSCCC可以分离纯化出色谱纯度在95％以上的芦荟色酮单体。经过紫外检测、快原子轰击质谱及核磁共振等方法的结构分析鉴定,证实分离所得物质为肉桂酰基-C-葡萄糖甙芦荟色酮。     

Simultaneous determination of lappaconitine hydrobromide and isopropiram fumarate in rabbit plasma by capillary electrophoresis with electrochemiluminescence detection

A CE electrochemiluminescence (CE-ECL) method for simultaneous determination of lappaconitine hydrobromide (LH) and isopropiram fumarate (IF) has been first established, with a chemically modified platinum electrode by europium (III)-doped Prussian blue analogue film as a working electrode. The conditions for CE separation and ECL detection are discussed and optimized in detail. It has been proved that 20 mmol/L phosphate buffer (pH 8.5) containing 5% (v/v) ACN and 0.17 mol/L SDS could achieve the most favorable resolution, and the high sensitivity of detection was obtained by maintaining the detection potential at 1.23 V. Under optimized conditions, a baseline separation for the two analytes was achieved within 6 min, and the standard curves were linear in the range of 1.0×10(-7) ～ 5.0 × 10(-5) g/mL for LH and 4.0 × 10(-8) ～ 1.0 × 10(-5) g/mL for IF with the detection limits (3σ) of 6.6 × 10(-8) g/mL for LH and 3.7 × 10(-8) g/mL for IF, respectively. The precisions of intra- and interday measurements for LH and IF were less than 4.21 and 2.61%, respectively. The applicability of the proposed method was illustrated in the determination of LH and IF in rabbit plasma with recoveries between 95.6 and 103.0%.     

Studies of the interaction between Aloe-emodin and DNA and preparation of DNA biosensor for detection of PML-RARalpha fusion gene in acute promyelocytic leukemia

The interaction of Aloe-emodin (AE) with salmon sperm DNA in 0.1M Tris-HCl buffer (pH 4.4) and at the DNA-modified glassy carbon electrode (GCE) was systemically studied with voltammetry and ultraviolet-visible (UV-vis) spectroscopy. AE had excellent electrochemical activity on the GCE with a couple of redox peaks. We propose that AE can intercalate into DNA strands forming a nonelectroactive complex, which results in the decrease of the reduction peak current of AE. The Langmuir adsorption constants of AE at ss- and dsDNA/GCE were (2.1+/-0.4)x10(5) and (2.7+/-0.2)x10(5)M(-1), respectively. The difference between AE at ss- and dsDNA has been used for the preparation of a sequence-specific DNA electrochemical biosensor for detection of PML-RARalpha fusion gene in acute promyelocytic leukemia (APL) with a detection limit of 6.7x10(-8)M and a linear range from 1.5x10(-8) to 1.5x10(-7)M. The selectivity of ssDNA-modified electrode was also described.