Determination Of Active Components Research Articles

Phytochemical analysis, UPLC-ESI-Orbitrap-MS analysis, biological activity, and toxicity of extracts from Tripleurospermum limosum (Maxim.) Pobed

Tripleurospermum limosum (TL) has been used in folk medicine to treat gastritis. Toward the further development and use of TL, we report the phytochemical profiling, determination of active components, and antioxidant and enzyme inhibitory activities of TL. Nineteen compounds were identified by ultra-performance liquid chromatography-electrospray ionization-orbitrap-mass spectrometry for the first time in this plant. Phytochemical studies indicated that TL contained 11 types of phytochemicals. The active components [total carbohydrate content (TCC), total protein content (TProC), total triterpenoid content (TTC), total phenolic content (TPheC), total flavonoid content (TFC), total phenolic acid content (TPAC), condensed tannin content (CTC), and gallotannin content (GC)] of eight different solvent extracts were determined by ultraviolet–visible spectrophotometry. Aqueous extract had highest TProC, TPheC, and GC values. Methanol extract exhibited highest TCC and TFC values. Ethanol extract showed highest TPAC and CTC values and dichloromethane extract exhibited highest TTC value. Methanol extract showed strongest ability to scavenge 2,2-diphenyl-1-picrylhydrazyl radicals, 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt cation radicals, and hydroxyl radicals, and also exhibited highest antioxidant activity in ferric-reducing antioxidant power and cupric ion-reducing antioxidant capacity assays. Best iron and copper chelating activity and H2O2 scavenging ability were shown by aqueous extract. Ethanol extract showed strongest ability to scavenge superoxide radicals and effectively prevent β-carotene bleaching. Acetone extract had highest inhibitory activity toward α-glucosidase, α-amylase, and xanthine oxidase. Ethyl ether extract had highest inhibitory activity towards urease and angiotensin converting enzyme. Aqueous and ethanol extracts had strongest inhibitory activity toward acetylcholinesterase. Methanol extract showed highest inhibitory activity toward tyrosinase. Methanol extract showed good stability and antioxidant capacity during heating, at different pH values, and after in vitro digestion and had low toxicity. The efficacy of methanol extract in stabilizing olive and sunflower oils was studied, the results suggested that methanol extract had a protective effect on the primary oxidation of the two oils. TL may be useful as a source of active components for application in human nutrition and/or phytomedicine and methanol extract of TL could be used as a natural oil stabiliser.

High performance liquid chromatographic method for determination of active components in lithospermum oil and its application to process optimization of lithospermum oil prepared by supercritical fluid extraction

紫草提取制备成的紫草油能够预防及治疗婴儿尿布疹、皮肤溃烂、湿疹等多种皮肤疾患,临床应用非常广泛,超临界流体萃取是紫草有效成分提取的优选方法。该文建立了紫草油有效成分的高效液相色谱(HPLC)测定方法,并以紫草油所含的有效成分含量为评价指标,采用三因素三水平正交试验法对紫草超临界流体萃取制备过程中的几个重要因素(萃取压力、萃取温度和CO2流量)进行考察,确定了紫草超临界流体萃取的最佳工艺流程。所建立的HPLC方法如下:Diamonsil C18色谱柱(250 mm×4.6 mm, 5 μm),流动相为含0. 1%甲酸的乙腈-含5 mmol/L甲酸铵的0.1%甲酸水溶液(75:25, v/v),等度洗脱,流速为1 mL/min;进样量为15 μL;柱温为室温;二极管阵列检测器(PAD)检测波长为275 nm。该法可在30 min内同时测定紫草油中的有效成分紫草素、乙酰紫草素、β-乙酰氧基异戊酰阿卡宁、异丁酰紫草素、β,β-二甲基丙烯酰紫草素和2-甲基丁酰基紫草素的含量,方法精密度、准确度、重复性好。在萃取压力23 MPa、萃取温度40 ℃、CO2流量27 L/h这一优化工艺流程下得到的紫草油有效成分含量最高。所建立的HPLC-PAD法简便可行、准确可靠,可用于超临界流体萃取制备紫草油的工艺过程优化和质量控制,也可为紫草及其制剂的质量评价提供参考。优化后的工艺条件能够满足紫草油制备需求,也符合生产实际需求。

Разработка и валидация методики количественного определения действующего вещества в субстанции фенилсалицилата методом газожидкостной хроматографии

Целью исследования являлась разработка и валидация новой методики количественного определения действующего вещества в субстанции фенилсалицилата методом газожидкостной хроматографии с пламенно-ионизационным детектированием (ГХ/ПИД). Исследования выполнены на капиллярной кварцевой колонке HP-5 с химически привитой фазой: 5 % дифенил, 95 % диметилполисилоксан («Agilent Technologies» США), разработаны условия хроматографического разделения. Определены параметры пригодности хроматографической системы. Содержание фенилсалицилата в субстанции определяли по методу внутреннего стандарта. Валидационную оценку методики проводили по показателям: специфичность, линейность, прецизионность, устойчивость (робастность). Установлено ее соответствие основным критериям приемлемости.

UPLC-MS/MS determination of active components from Wuzhuyu Decoction in plasma and brain tissues of nitroglycerin-induced migraine rats

A sensitive and specific ultra-performance liquid chromatography-mass spectrometry(UPLC-MS/MS) method was deve-loped for analysis of rutaecarpine(Ru), evodiamine(Ev), rutaevine(Rv), limonin(Li), ginsendside Rb_1(Rb_1), ginsendside Re(Re) in rat plasma and brain tissues of nitroglycerin-induced migraine rats. Male healthy Sprague-Dawley(SD) rats were orally given multiple dose of optimized(OS) and un-optimized Wuzhuyu Decoction(UNOS), and their blood samples and brainstem were collected at different time points after injection of nitroglycerin(10 mg·kg~(-1)) into the frontal region. The drug concentrations of the 6 analytes in plasma and brainstem were determined by UPLC-MS/MS method. Subsequently, the main pharmacokinetics parameters of plasma were calculated by using Phoenix WinNolin 5.2.1 software. The methodological test showed that all of analytes in both plasma and brainstem homogenate exhibited a good linearity within the concentration range(r>0.994 7). The intra-day and inter-day accuracy, precision, matrix effect, stability of the investigated components meet the requirements for biopharmaceutical analysis. The developed method was successfully applied in pharmacokinetic studies on abovementioned ingredients in rat plasma and brain stem. The plasma pharmacokinetic parameters of active ingredients in two different Wuzhuyu Decoction group were compared, it was found that Rb_1 had higher t_(1/2), T_(max), C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. Meanwhile, Ev had higher t_(1/2) and T_(max) but lower C_(max), AUC_(0-24 h) and AUC_(0-∞), Ru has higher t_(1/2 )but lower C_(max), AUC_(0-24 h) and AUC_(0-∞ )in OS group. The brain tissue distribution of each component were compared between the two groups, the component with higher content in OS, such as Ru at 30 min and 2 h after administration, Ev at 30 min, Rb_1 at 30 min and Rb_1 at 2 h after administration have lower brain tissue distribution than those in UNOS group, while the component with higher content in UNOS, such as Rv at 30 min, 2 h and 12 h after administration had higher brain tissue distribution than those in OS group.

Homogenate-assisted negative-pressure cavitation extraction for determination of organic acids and flavonoids in honeysuckle (Lonicera japonica Thunb.) by LC–MS/MS

In the present study, a homogenate-assisted negative-pressure cavitation extraction (H-NPCE) is developed for the extraction and determination of the main organic acids and flavonoids in honeysuckle by LC–MS/MS. The influential parameters of the H-NPCE procedure were optimized, and the optimal conditions were as follows: homogenate time 45s, negative pressure −0.07MPa, ethanol concentration 70%, liquid/solid ratio 20:1mL/g, NPCE time 20min and 3 extraction cycles. Furthermore, a rapid LC–MS/MS analysis method was established and validated, and it exhibited satisfactory linearity (R2>0.996), precision (RSD⩽4.87%) and recovery (96.94–104.32%). H-NPCE followed by LC–MS/MS was successfully applied for the extraction and quantification of ten organic acids and flavonoids in honeysuckle. This work provided a fast and effective method for the sample preparation and determination of active components from plants.

Electroanalysis for Quality Control of Acerola (Malpighia emarginata) Fruits and their Commercial Products

The use of natural antioxidants is widespread around the world since they may delay oxidative stress and, consequently, degenerative diseases. Among the prescribed healthy habits for humans is the consumption of fruits containing high levels of phenolic compounds and other radical scavengers. Acerola (Malpighia emarginata) is very appreciated for its high content of ascorbic acid (AA) and rutin, two remarkable natural antioxidants (NA). Industrialized as frozen pulp and processed as juice, it is also a good source of carotenoids and anthocyanins. Nevertheless, its commercialization is hindered by its low stability. Considering the great potential of acerola frozen and dried products as rich sources of NA, the development of analytical methods focusing on the determination of active components is imperative for their quality control. Therefore, the aim of this work was to apply differential pulse voltammetry (DPV) for quality control of different products made of acerola fruits. Quality control samples consisted of acerola products spiked with AA, which was the biomarker, compared with its contents in samples analyzed by high-performance liquid chromatography coupled with photodiode array detection (HPLC-PDA). DPV was employed for quantification of AA in all samples. The achieved limits of detection and quantification were 0.31 and 0.96 mM, respectively, whereas the recovery range was from 97.4 to 102.2 %; the results presented good accuracy and correlation with HPLC-PDA method. In addition to its use in identification, DPV proved to be helpful to check the integrity of natural products and avoid adulteration. Moreover, the electrochemical index proposed is in accordance with traditional methods for estimating antioxidant capacity.

Content Determination of Active Component in Huangqi Yinyanghuo Group and Its Effects on hTERT and Bcl-2 Protein in Osteosarcoma.

To screen the optimal extraction process and content determination of active component of Huangqi Yinyanghuo group (HYG) and to study the effects of HYG on human telomerase reverse transcriptase (hTERT) and Bcl-2 protein in osteosarcoma (HOS) cells, providing the theoretical basis for clinical application of HYG in treatment of osteosarcoma, orthogonal design table L9(43) was used to design the extraction process of HYG, and icariin was taken as the investigation index to optimize the extraction process of HYG. 0.125, 0.25, 0.5, 1, 2, 4, and 8 μmol/L HYG were taken to act separately on logarithmic growth phase osteosarcoma HOS cells, CCK-8 assay was used to determine cell viability, and immunohistochemical SP assay was used to determine the expression of hTERT and Bcl-2 protein. Apoptosis rate was positively correlated with the dose of HYG, and the expressions of hTERT and Bcl-2 protein were significantly decreased with the prolonged duration of action. Under the effect of HYG, dose was negatively correlated with osteosarcoma cell survival fraction; osteosarcoma cell survival fraction was positively correlated with hTERT and Bcl-2 protein; duration of action was negatively correlated with hTERT and Bcl-2 protein; and hTERT and Bcl-2 protein were in a synchronous relationship.

HPTLC determination of chemical composition variability in raw materials used in botanicals

Besides the chemotaxonomic value, nowadays determination of biodiversity and chemical variability has a commercial impact. The exact identity of raw material and constituents of botanical products, such as food supplements or herbal remedies, is a very important argument, being the real prerequisite for quality control and traceability, followed by the determination of active components. However, the analytical approach must consider the natural great variability in secondary metabolites and product form, such as in extracts. Against the reductive approach, on the basis of single chemical standards, so far dominant in Pharmacopoeias monographs, we report applications and utility of the high-performance thin-layer chromatography fingerprint in determination of species of the same genus, of populations of the same species and of different drugs of the same plant.

Separation and Determination of Active Components in <i>Evodiae fructus</i> <i></i> and its Pharmaceutical Preparations by Non-Aqueous Micellar Electrokinetic Chromatography

In this paper, an easy, economical and effective nonaqueous micellar electrokinetic capillary chromatography (NAMECC) method for the simultaneous determination of two bioactive components (evodiamine and rutaecarpine) in Evodiae Fructus and its pharmaceutical preparations was developed for the first time. After optimization of the separation conditions and concentration conditions, the two alkaloids can be separated by using a fused-silica capillary column (57 × 75 μm I.D.) with a running buffer of 140 mM sodium cholate (SC) in methanol and detected with a diode-array detector (228nm). The second-order derivative electropherograms were applied for resolving overlapping peaks in complicated preparations. Regression equations revealed good linear relationships (correlation coefficients 0.9969-0.9999) between peak heights in second-order derivative electropherograms and concentrations of the two analytes. The relative standard deviations (RSD) of the migration times and the peak height of the two constituents were less than 0.49% and 1.6%. The recoveries of two constituents ranged from 93.5 to 105.6%. The results indicated that baseline separation of the analytes was hard to be achieved in real samples sometimes and second-order derivative electropherograms was applicable for the resolving and analysis of overlapping peaks.

Determination of active components in silymarin by UPLC-ESIMS

A UPLC-ESIMS method was developed for simultaneous analysis of seven major bioactive flavonolignans in silymarin including silychristins A(1) and B(2), silydianin(3), silybins A(4) and B(5), and isosilybins A(6) and B(7). In this study, the seven major active flavonolignans including the diastereomers 1/2, 4/5, and 6/7 were completely separated using UPLC with an ACQUITY UPLC C18 column and MeOH-water (formic acid) mobile phase system. The collision-induced dissociation (CID) MS/MS spectra of these flavonolignans were studied systematically using ESI-MS. The results with the present methodology showed that UPLC-MS/MS can be used for general screening of active natural products from plant extracts and for the specific quality control of silymarin.

Fast determination of active components in <I>Angelica dahurica</I> extract using capillary electrochromatography with methacrylate ester-based monolithic columns

The separation and determination of four important active components (imperatorin, isoimperatorin, phelloptorin and falcarindiol) from Angelica dahurica extract has been performed using capillary electrochromatography (CEC) with a methacrylate ester-based monolithic column. The effect of the porogen ratio on the column preparation was studied. The mobile phase composition, such as the concentration of organic solvent, the ionic strength and the pH of the buffer were also optimized. Under the optimized conditions (50% acetonitrile and 50% of a 20 mmol/L sodium dihydrogen phosphate electrolyte at pH 4.95, - 25 kV), a fast and baseline separation of the four analytes was achieved. The calibration curves showed a good linearity (r2 > 0.997) and the limits of detection were lower than 0.34 mg/L. The mean recoveries of the studied components ranged between 95.18% and 98.44%. The method developed is sensitive, reliable and suitable for the quality control. With this CEC system, the quality of Angelica dahurica extracts from 18 various regions was evaluated.

Simultaneous Determination of Active Components and Anti-diabetic Drugs in Propolis Health Foods by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

Simultaneous Determination of Active Components and Anti-diabetic Drugs in Propolis Health Foods by Ultra Performance Liquid Chromatography Tandem Mass Spectrometry

Quantitative hplc determination of antigrippin drug components

An HPLC technique for simultaneous quantitative determination of antigrippin components ascorbic acid, paracetamol, and chlorpheniramine maleate has been developed. Optimum conditions are determined for chromatographic separation on a Symmetry C18 column of the poorly retained impurity 4-aminophenol and ascorbic acid. The proposed method provides high sensitivity and rapid analysis and ensures accurate and reproducible results in the quantitative determination of active components (ascorbic acid, paracetamol, and chlorpheniramine maleate), a controlled toxic impurity (4-aminophenol), and an excipient (sodium saccharinate) in antigrippin effervescent tablets for children and adults.

Application of experimental design and radial basis function neural network to the separation and determination of active components in traditional Chinese medicines by capillary electrophoresis

Orthogonal design has been used to the optimization of separation and determination of two active components in traditional Chinese medicines by capillary electrophoresis. The concentration of phosphate, applied voltage, organic modifier content and buffer pH were selected as variable parameters. Their different effects on peak resolution were studied by the experimental design method. Optimized separation conditions were obtained and successfully applied to the separation and determination of aconitine and hypaconitine in Aconitum medicinal herbs. Good separation was achieved within 7 min using a buffer system composed of 20 mmol L −1 phosphate and 35% acetonitrile at pH 9.5. The applied voltage was 14 kV and the detection was set at 235 nm. In addition, a radial basis function neural network with a “4-18-1” structure was developed based on the experimental results of orthogonal design and uniform design, and was applied to the prediction of peak resolution of the two active components under the optimum separation conditions given by orthogonal design. The predicted results were in good agreement with the experimental values, indicating that radial basis function neural network is a potential way for the selection of separation conditions in capillary electrophoresis.

Determination of active components in Chinese medicinal preparations by capillary electrophoresis

AbstractThe determination of paeoniforin, paeonol, and censenoside Rg1 in traditional Chinese medicinal preparations, Tze Po San Pien pills and Liuwen Dihuang pills has been investigated by micellar electrokinetic capillary electrophoresis with borate buffer (20 mM), sodium dodecylsulfate (30 mM) and acetonitrile (20%) as background electrolytes (pH 9.30), 20 kV applied voltage and 203 nm UV detection. The effects of SDS concentration, borate, buffer pH, and organic modifier on electrophoretic behavior and separation are discussed. Regression equations revealed linear relationships between the peak-area of each component and the content with the correlation coefficients from 0.9982 to 0.9999. In addition, the levels of the active compounds in two kinds of traditional Chinese medicinal preparations were easily determined with the recoveries from 93.1% to 108.2%.

A new capillary zone electrophoresis method for the analysis of Ginkgo biloba extracts and Ginkgo biloba pharmaceutical products

Ginkgo biloba, traditional Chinese medicine is now generally accepted. Separation and determination of active components in G. biloba is important for the product quality control. Therefore, the development of an effective and reliable separation method is important. In this work, a new capillary electrophoretic (CZE) method for separation of the G. biloba leaf extracts components was developed and optimized by the use of experimental design and artificial neural network (ANN). Under best separation conditions, in gamma-CD-modified buffer, the separation was reached within 10 min (36 mM borate BGE, pH 9.2, 1 mM gamma-CD), while the hydrodynamic mode for sample injection (2 s) and UV detection at 270 nm were applied. The method developed was validated and applied for analysis of various extracts and G. biloba products.

Determination of active components in rosemary by capillary electrophoresis with electrochemical detection

Rosemary ( Rosmarinus officinalis L.) is a spice and medicinal herb widely used around the world. Among natural antioxidants, rosemary has been widely accepted as one of the spices with the highest antioxidant activity. A capillary electrophoresis method for the determination of its active components using electrochemical detection was developed. Effects of several important factors were investigated to acquire the optimum conditions. The detection electrode was a 300 μm carbon disc electrode at a working potential of +0.90 V (versus SCE). The analytes can be well separated with 25 min in a 75 cm length fused-silica capillary at a separation voltage of 16 kV in an 80 mmol/l borate buffer (pH 9.0). The current response was linear over about three orders of magnitude with detection limits (S/N = 3) ranging from 2 × 10 −7 to 1 × 10 −6 g/ml for all the analytes. The method was successfully used in the analysis of rosemary with relatively simple extraction procedures, and the assay results were satisfactory.

Phosphorimetric Determination of Active Components in Medicinal Preparations

The use of room-temperature phosphorescence was proposed for the determination of active components containing naphthalene rings in their structures in medicinal preparations. It was demonstrated that this method can be used for the determination of active components in combined preparations.

Determination of Active Components in St. John's Wort (Hypericum Perforatum) by Capillary Electrophoresis with Electrochemical Detection

St. John's Wort (Hypericum perforatum) is a medicinal plant which has been known in traditional medicine as an anti-inflammatory and healing agent. Nowadays, the alcoholic extract of its aerial parts finds wide application for its antidepressant activity. A capillary electrophoresis method for the identification of its constituents using electrochemical detection was developed. Effects of several important factors were investigated to acquire the optimum conditions. The working electrode was a 300 μm carbon disc electrode at a working potential of +0.90 V (vs. SCE). The analytes can be well separated within 20 min in a 75 cm length fused-sillica capillary at a separation of 16 kV in 50 mmol L−1 borate buffer (pH=9.0). The current response was linear over about three orders of magnitude with detection limits (S/N=3) ranging from 0.2 to 0.5 mg L−1 for all the analytes. The method was successfully used in the analysis of Hypericum perforatum with relatively simple extraction procedures, and the assay results were satisfactory.

Separation and determination of active components inSchisandra chinensis Baill. and its medicinal preparations by non-aqueous capillary electrophoresis

A simple, economical and effective non-aqueous capillary electrophoresis separation and detection method was developed for the quantification of deoxyschizandrin and gamma-schizandrin in Schisandra chinensis Baill. and its medicinal preparations for the first time. After optimization of separation conditions, a buffer of 140 mmol/L sodium cholate in methanol was selected for separating the two analytes, but baseline separation of SA and SB in real samples was not obtained. Therefore second-order derivative electropherograms were applied for resolving overlapping peaks. Regression equations revealed good linear relationships (correlation coefficients 0.9975--0.9988) between peak heights in second-order derivative electropherograms and concentrations of the two analytes. The relative standard deviations (RSD) of the migration times and the peak height of the two constituents were in the ranges 0.62--0.79% and 0.25--2.17% (intra-day) and 1.43--2.06 and 4.08--5.72% (inter-day), respectively. The recoveries of the two constituents ranged from 93.2 to 103.0%. The results indicated that baseline separation of the analytes was sometimes hard to obtain in real samples and second-order derivative electropherograms were applicable for the resolution and analysis of overlapping peaks.