反相高效液相法测定穿心莲药材及其制剂中的穿心莲内酯和脱水穿心莲内酯

 发展了穿心莲药材及其中成药中两种主要成分穿心莲内酯和脱水穿心莲内酯的反相高效液相测定方法。采用甲醇振荡提取法进行样品前处理 ,在以乙腈 水为流动相作梯度洗脱、ODS柱、检测波长为 2 2 5nm的条件下 ,穿心莲内酯和脱水穿心莲内酯在 1 5min内可达到基线分离。两种内酯在 1 0mg/L～ 1 0 0mg/L时其浓度与峰面积成良好的线性关系 ,加标回收率为 96 %～ 1 0 4 %。     

Chionanthus virginicus L.: phytochemical analysis and quality control of herbal drug and herbal preparations

Root barks of Chionanthus virginicus L. are used in homeopathic medicines in the treatment of icterus and hepatitis. The objective of this study is to identify novel secoiridoids and lignans and to develop a simple and reliable HPLC method for the determination of oleuropein, phillyrin, total secoiridoids and total lignans for quality control and stability studies of C. virginicus herbal drug and preparations. Secoiridoids and lignans were purified by preparative HPLC. Compounds previously described were identified by HPLC according to their retention times and UV spectra. Structures of new compounds were determined by NMR. Two compounds namely excelside B and acetoxypinoresinol-4"-O-beta-D-glucoside are described for the first time in the drug. HPLC separation was performed on Symmetry C18 (Waters) by gradient elution using acetonitrile and 0.2% aqueous phosphoric acid. The method was validated for specificity, linearity, precision, accuracy, limits of detection and quantification for simultaneous determination of secoiridoids and lignans in herbal drug and herbal preparations as mother tinctures. The proposed HPLC method is linear in the range studied (r2 > or = 0.9989) for all the analytes. The method is precise with intra- and inter-day variations of less than 4%. The mean recoveries of the analytes range from 99.65 to 102.81%. The method is successfully applied to the quantification of nine compounds belonging to secoiridoids and lignans and for the stability studies of these compounds. The study allowed completing the phytochemical knowledge of C. virginicus. This simple developed assay could be used as tools for routine quality control of C. virginicus herbal drug and herbal medicinal products.     

Identification and determination of geniposide contained in Gardenia jasminoides and in two preparations of mixed traditional Chinese medicines

A high-performance liquid chromatographic method was applied to the determination of the geniposide concentration in Gardenia fruit and preparations of traditional Chinese medicine using a mobile phase of acetonitrile-methanol-5 mM monosodium phosphate (pH 4.6) (5:15:80, v/v/v). Intra-assay and inter-assay accuracy and precision of the analyses were < or = 10% in the range of 0.1 through 50 microg/ml. The presence of geniposide in the medicinal herb and its preparations was ascertained by retention time, spiking with an authentic standard, change of detection wavelength and change of the composition of the mobile phase. The concentration of geniposide in the fruit of Gardenia jasminoides Ellis var. grandiflora Nakai is higher than that in Gardenia jasminoides Ellis. The concentration of geniposide in the traditional Chinese herbal medicine preparations, Huang-Lian-Jiee-Dwu-Tang (66.27 +/- 1.98 mg/g) and In-Chern-Hau-Tang (68.54 +/- 2.62 mg/g) was less than in the herb Gardenia jasminoides Ellis (73.44 +/- 2.62 mg/g) itself.     

On-line coupling of dynamic microwave-assisted extraction with high-performance liquid chromatography for determination of andrographolide and dehydroandrographolide in Andrographis paniculata Nees

A novel technique based on dynamic microwave-assisted extraction (DMAE) coupled on-line with high-performance liquid chromatography (HPLC) through a flow injection interface has been developed for determination of andrographolide and dehydroandrographolide in Andrographis paniculata Nees. A TM(010) microwave resonance cavity built in the laboratory was applied to concentrating the microwave energy. An extraction vessel was placed in microwave irradiation zone. The extraction was performed in a recirculating system. When a number of extraction cycles were completed, the fractional extract (20muL) was driven to the analytical column by 65% aqueous methanol and was measured by diode array detector (DAD) at 225nm. The optimized extraction conditions are follows: extraction solvent 60% aqueous methanol; microwave forward power 80W; extraction time 6min; extraction solvent flow-rate 1.0mLmin(-1). The detection and quantification limits obtained are 0.5 and 1.7microgmL(-1) for andrographolide and 0.6 and 1.9microgmL(-1) for dehydroandrographolide, respectively. The within-day and between-day precision (RSD) are 2.1% and 3.7% for andrographolide and 1.7% and 4.1% for dehydroandrographolide, respectively. Mean recoveries for andrographolide and dehydroandrographolide are 97.7% and 98.7%, respectively. Compared with ultrasonic extraction used in the Chinese pharmacopoeia, the proposed method was demonstrated to obtain higher extraction yield in a shorter time. In addition, only small quantities of solvent (5mL) and sample (10mg) were required.     

Effects of PAR and UV‐B Radiation on Herbal Yield,Bioactive Compounds and Their Antioxidant Capacity of Some Medicinal Plants Under Controlled Environmental Conditions

Photosynthetically active radiation (PAR) and Ultraviolet B (UV‐B) radiation are among the main environmental factors acting on herbal yield and biosynthesis of bioactive compounds in medicinal plants. The objective of this study was to evaluate the influence of biologically effective UV‐B light (280–315 nm) and PAR (400–700 nm) on herbal yield, content and composition, as well as antioxidant capacity of essential oils and polyphenols of lemon catmint (Nepeta cataria L. f. citriodora), lemon balm (Melissa officinalis L.) and sage (Salvia officinalis L.) under controlled greenhouse cultivation. Intensive UV‐B radiation (2.5 kJ m^?2 d^?1) influenced positively the herbal yield. The essential oil content and composition of studied herbs were mainly affected by PAR and UV‐B radiation. In general, additional low‐dose UV‐B radiation (1 kJ m^?2d^?1) was most effective for biosynthesis of polyphenols in herbs. Analysis of major polyphenolic compounds provided differences in sensitivity of main polyphenols to PAR and UV‐B radiation. Essential oils and polyphenol‐rich extracts of radiated herbs showed essential differences in antioxidant capacity by the ABTS system. Information from this study can be useful for herbal biomass and secondary metabolite production with superior quality under controlled environment conditions.     

Determination of andrographolide and dehydroandrographolide in rabbit plasma by on-line solid phase extraction of high-performance liquid chromatography

The high-performance liquid chromatography (HPLC) coupled with on-line solid phase extraction (SPE) and ultraviolet (UV) detection was developed for determining andrographolide and dehydroandrographolide in rabbit plasma. Plasma samples (100 μL) were injected directly into a C18 SPE column and the biological matrix was washed out for 6 min using 15% aqueous methanol. By rotation of the switching valve, andrographolide and dehydroandrographolide were eluted in the back-flush mode and transferred to the analytical column by the chromatographic mobile phase consisted of methanol:acetonitrile (ACN):water (50:10:40; v/v). The UV detection was performed at 225 nm. The calibration curves showed excellent linear relationship (R ≥ 0.9993) over the concentration range of 0.05-5.0 μg mL⁻¹. The within- and between-day precisions (R.S.D.) of two analytes were in the range of 1.2-6.5% and the accuracies were between 92.0% and 102.1%. Their recoveries were all greater than 94%. The limits of detection were 0.019 μg mL⁻¹ for andrographolide and 0.022 μg mL⁻¹ for dehydroandrographolide. This method was successfully applied to the plasma concentration-time curve study after oral administration of Andrographis paniculata Nees extract in rabbit.     

A strategy for the identification of plants in illegal pharmaceutical preparations and food supplements using chromatographic fingerprints

The detection of regulated and forbidden herbs in pharmaceutical preparations and nutritional supplements is a growing problem for laboratories charged with the analysis of illegal pharmaceutical preparations and counterfeit medicines. This article presents a feasibility study of the use of chromatographic fingerprints for the detection of plants in pharmaceutical preparations. Fingerprints were developed for three non-regulated common herbal products—Rhamnus purshiana, Passiflora incarnata L. and Crataegus monogyna—and this was done by combining three different types of detection: diode-array detection, evaporative light scattering detection and mass spectrometry. It is shown that these plants could be detected in respective triturations of the dry extracts with lactose and three different herbal matrices as well as in commercial preparations purchased on the open market.

The Potential Use of Herbal Fingerprints by Means of HPLC and TLC for Characterization and Identification of Herbal Extracts and the Distinction of Latvian Native Medicinal Plants

The growing market of herbal medicines, the increase in international trade in Latvia, and the lack of adequate analytical methods have raised the question of the potential use of herbal fingerprinting methods. In this study, high-performance liquid chromatography (HPLC) and thin layer chromatography (TLC) methods were developed for obtaining chromatographic fingerprints of four taxonomically and evolutionary different medicinal plants (Hibiscus sabdariffa L., Calendula officinalis L., Matricaria recutita L., Achillea millefolium L.). Retention time shifting, principal component analysis (PCA), hierarchical cluster analysis (HCA), and orthogonal projections to latent structures (OPLS) analysis were used to improve and analyze the obtained fingerprints. HPLC data detection at 270 nm was determined superior to 360 nm for the distinction of medicinal plants and used data alignment method significantly increased similarity between samples. Analyzed medicinal plant extracts formed separate, compact clusters in PCA, and the results of HCA correlated with the evolutionary relationships of the analyzed medicinal plants. Herbal fingerprinting using chromatographic analysis coupled with multivariate analysis has a great potential for the identification of medicinal plants as well as for the distinction of Latvian native medicinal plants.     

Separation and purification of verticine and verticinone from Bulbus Fritillariae Thunbergii by high-speed counter-current chromatography coupled with evaporative light scattering detection

High-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) was successfully applied to preparative separation and purification of verticine and verticinone from crude extracts of Bulbus Fritillariae Thunbergii by a one-step separation, using chloroform–ethanol–0.2 mol L⁻¹ hydrochloric acid (3:2:2, v/v/v) as a solvent system. HPLC analysis of the fractions collected on the preparative HSCCC of 200 mg of crude extracts showed that the purity of verticine (25.6 mg) was 96.8% and that of verticinone (10.3 mg) was 95.4%. The chemical identities of these components were confirmed by ¹H NMR and EI–MS.     

Second‐order calibration serves as a remedial measure for the simultaneous determination of andrographolide and dehydroandrographolide in Andrographis paniculata and its preparations by HPLC without complete baseline separation

In this work, high‐performance liquid chromatography with diode array detection was applied for the simultaneous determination of andrographolide and dehydroandrographolide in Andrographis paniculata and its preparations. As a result of the incomplete baseline separation caused by complex backgrounds, the classical univariate calibration method failed to determine accurate contents of the analytes. On this occasion, chemometric second‐order calibration based on the well‐known alternating trilinear decomposition algorithm was then explored to serve as a post‐experimental remedial tool to solve this problem. By using the intelligent “mathematical separation” of alternating trilinear decomposition, the peak areas of the analytes do not need to be directly measured and the predictive results become accurate. The contents of andrographolide and dehydroandrographolide were determined to be (7.95 ± 0.15) and (1.85 ± 0.02) μg/mL for Andrographis paniculata, (1.34 ± 0.01) and (5.53 ± 0.04) μg/mL for its preparations, which was in agreement with those obtained by a reference liquid chromatography with mass spectrometry method. This study showed the superiority of second‐order calibration method over classical univariate calibration method for simultaneous determination of multi‐analytes in complex samples. It also proved that second‐order calibration may be a good choice for remedying incomplete baseline separation problem, with the accompanied reduction of experimental burden and toxic organic solvents as well as analysis time and cost.     

Optimization and validation of post-column assay for screening of radical scavengers in herbal raw materials and herbal preparations

On-line method, which combines HPLC distribution and post-column reaction, was designed for the search of individual antioxidants. Optimization of the assay was performed evaluating optimal ABTS(+) radical cation concentration in the reactor, reaction time, impact of flow rate, reaction coil length. HPLC-ABTS assay validation in this work was performed by assessing reference antioxidant negative peak areas in radical scavenging chromatogram. Sample free radical scavenging activity is expressed as trolox equivalent antioxidant capacity (TEAC). Optimized and validated method was applied in detection of compounds possessing free radical scavenging ability in complex mixtures. Antioxidant compounds were studied in perilla (Perilla frutescens (L.) Britton var. crispa f. viridis) herbal raw material and its preparations. The HPLC-separated antioxidant compounds were identified using HPLC-photodiode array coupled to mass spectrometer, using a reference mass for determining accurate masses. Radical scavenging characteristics of rosmarinic acid, which is the dominant phenolic compound in medicinal herbal raw material of perilla and its preparations, were confirmed by the calculated TEAC values. Compounds responsible for antioxidant effect in herbal raw materials and herbal preparations were identified, evaluated and compared.     

Simultaneous determination of total arsenic and total selenium in Chinese medicinal herbs by hydride generation atomic fluorescence spectrometry in tartaric acid medium

By HG-AFS, a new method was proposed for simultaneous determination of total arsenic and total selenium existed in the Chinese medicinal herbs in tartaric acid medium. The effects of analytical conditions and coexisting ions on the fluorescence signal intensity of analytes were investigated. The proposed method was provided with linear response ranges above 22 μg l⁻¹ for As and 44 μg l⁻¹ for Se, and the detection limits of 0.13 and 0.12 μg l⁻¹ were obtained for As and Se respectively. The recoveries of 93.8–96.1% for As and 95.3–99.1% for Se, and the precision of 1.2–3.8% and 2.4–5.3% (R.S.D., n = 8) respectively, were obtained via simultaneous determined four samples of Chinese medicinal herbs and three certified botanic reference materials successfully. The proposed method has the advantages of simple operation, high sensitivity and high efficiency.     

Similarity analyses of chromatographic herbal fingerprints: A review

Herbal medicines are becoming again more popular in the developed countries because being “natural” and people thus often assume that they are inherently safe. Herbs have also been used worldwide for many centuries in the traditional medicines. The concern of their safety and efficacy has grown since increasing western interest. Herbal materials and their extracts are very complex, often including hundreds of compounds. A thorough understanding of their chemical composition is essential for conducting a safety risk assessment. However, herbal material can show considerable variability. The chemical constituents and their amounts in a herb can be different, due to growing conditions, such as climate and soil, the drying process, the harvest season, etc. Among the analytical methods, chromatographic fingerprinting has been recommended as a potential and reliable methodology for the identification and quality control of herbal medicines. Identification is needed to avoid fraud and adulteration. Currently, analyzing chromatographic herbal fingerprint data sets has become one of the most applied tools in quality assessment of herbal materials. Mostly, the entire chromatographic profiles are used to identify or to evaluate the quality of the herbs investigated. Occasionally only a limited number of compounds are considered. One approach to the safety risk assessment is to determine whether the herbal material is substantially equivalent to that which is either readily consumed in the diet, has a history of application or has earlier been commercialized i.e. to what is considered as reference material. In order to help determining substantial equivalence using fingerprint approaches, a quantitative measurement of similarity is required. In this paper, different (dis)similarity approaches, such as (dis)similarity metrics or exploratory analysis approaches applied on herbal medicinal fingerprints, are discussed and illustrated with several case studies.     

Identification of chemical ingredients of peanut stems and leaves extracts using UPLC‐QTOF‐MS coupled with novel informatics UNIFI platform

Peanut stems and leaves have been used traditionally as both herbal medicines and special food in Asia. In this study, the main functional compounds of peanut stems and leaves extracts were identified using UPLC separation coupled to high resolution mass spectrometry (QTOF‐MS), and a traditional medicine library. Three different extraction solvents (ethyl acetate, petroleum ether and n‐butanol) were evaluated to prepare the extracts of peanut stems and leaves. A total of 283 chemical compounds were identified in peanut stems and leaves extracts, of which 207 compounds are tentatively new identifications in Genus Arachis. The integration of data acquisition and processing with the traditional medicine library provides a simple, efficient process to effectively facilitate the identification of chemical ingredients in complex natural product extracts. The integrated workflow for separation, detection and identification of functional compounds in natural products using UPLC/QTOF‐MS greatly improves productivity for development of traditional herbal medicines. Copyright © 2016 John Wiley & Sons, Ltd.     

Diagnosis of public programs focused on herbal medicines in Brazil

The present study is aimed to diagnose the current public programs focused on herbal medicines in Brazil by means of in loco visits to 10 programs selected by means of questionnaires sent to 124 municipalities that count on herbal medicine services. The main purpose of the implementation of program programs is related to the development of medicinal herbs. 70% of them are intended for the production of herbal medicines and 50% are aimed to ensure the access of the population to medicinal plants and or herbal medicines. The initiative of the implementation of these programs was related to the managers (60%). The difficulties in this implementation were due to the lack of funding (100%) of the programs. In 60% of the programs, the physicians did not adhere to herbal medicine services due to the lack of knowledge of the subject. Training courses were proposed (80%) to increase the adhesion of prescribers to the system. Some municipalities use information obtained from patients to assess the therapeutic efficiency of medicinal plants and herbal medicines. Of the programs underway, cultivation of medicinal plants was observed in 90% and 78% of them adopt quality control. In most programs, this control is not performed in accordance with the legal requirements. The programs focused on medicinal plants and herbal medicines implemented in Brazil face some chronic problems of infrastructure, management, operational capacity and self-sustainability, which can be directly related to the absence of a national policy on medicinal plants and herbal medicines.     

Optimisation of sample treatment for arsenic speciation in alga samples by focussed sonication and ultrafiltration

A procedure for arsenic species fractionation in alga samples (Sargassum fulvellum, Chlorella vulgaris, Hizikia fusiformis and Laminaria digitata) by extraction is described. Several parameters were tested in order to evaluate the extraction efficiency of the process: extraction medium, nature and concentration (tris(hydroxymethyl)aminomethane, phosphoric acid, deionised water and water/methanol mixtures), extraction time and physical treatment (magnetic stirring, ultrasonic bath and ultrasonic focussed probe). The extraction yield of arsenic under the different conditions was evaluated by determining the total arsenic content in the extracts by ICP-AES. Arsenic compounds were extracted in 5 mL of water by focussed sonication for 30 s and subsequent centrifugation at 14,000 × g for 10 min. The process was repeated three times. Extraction studies show that soluble arsenic compounds account for about 65% of total arsenic.

An ultrafiltration process was used as a clean-up method for chromatographic analysis, and also allowed us to determine the extracted arsenic fraction with a molecular weight lower than 10 kDa, which accounts for about 100% for all samples analysed.

Speciation studies were carried out by HPLC–ICP-AES. Arsenic species were separated on a Hamilton PRP-X100 column with 17 mM phosphate buffer at pH 5.5 and 1.0 mL min⁻¹ flow rate. The chromatographic method allowed us to separate the species As(III), As(V), MMA and DMA in less than 13 min, with detection limits of about 20 ng of arsenic per species, for a sample injection volume of 100 μL. The chromatographic analysis allowed us to identify As(V) in Hizikia (46 ± 2 μg g⁻¹), Sargassum (38 ± 2 μg g⁻¹) and Chlorella (9 ± 1 μg g⁻¹) samples. The species DMA was also found in Chlorella alga (13 ± 1 μg g⁻¹). However, in Laminaria alga only an unknown arsenic species was detected, which eluted in the dead volume.     

Determination of phenolic compounds in medicinal plants from the Lamiaceae family

A procedure for the determination of Phenolic compounds in extracts from the medicinal plants of the Lamiaceae family—garden sage (Salvia officinalis L.), creeping thyme (Thymus serpyllum L.), wild marjoram (Origanum vulgare L.), and common balm (Melissa officinalis L.)—obtained under different extraction conditions was developed. The identification of the extracted compounds was performed and their qualitative and quantitative composition was established by HPLC with diode array and mass-spectrometric detection with consideration for the obtained characteristics of the standard samples of individual components. The test samples of medicinal herbs contained caffeic acid (0.19–0.62 mg/g) and rosmaric acid (4–23 mg/g); the highest rosmaric acid content (23 mg/g) was found in wild marjoram, and the lowest content (4 mg/g), in creeping thyme. The extracts of wild marjoram contained the greatest amounts of Phenolic compounds; rosmaric acid and luteolin-7-O-β-D-glucuronide were major components, whereas protocatechuic, 3-O-caffeoylquinic, and caffeic acids were minor components.     

腰果酚键合硅胶固定相的制备及其色谱性能北大核心CSCD

天然产物作为一种绿色低毒、来源广泛、功能位点丰富的单体,已被广泛应用于色谱固定相的研制与开发。该文以天然可再生资源腰果酚为配体,通过一步法开环反应将其接枝到由γ-缩水甘油醚氧丙基三甲氧基硅烷(KH-560)修饰的硅胶上,制备得到腰果酚键合硅胶固定相。利用傅里叶红外光谱、元素分析、热失重分析和N_(2)吸附脱附实验对固定相进行表征,结果表明成功制备了腰果酚键合硅胶色谱固定相。采用Tanaka实验试剂、烷基苯、多环芳香烃、苯酚类化合物和芳香族位置异构体为探针评价其分离性能和保留机制,并与C_(18)柱进行对比。研究发现,腰果酚键合固定相除疏水作用外,还具有π-π和氢键作用。基于上述保留作用,腰果酚键合硅胶固定相对测试探针表现出良好的分离性能。重复进样10次,各探针保留时间的RSD为0.052%~0.079%,峰面积的RSD为0.104%~0.847%,峰高的RSD为0.081%~0.272%,表明该色谱柱具有良好的重复性和稳定性。此外,腰果酚键合硅胶色谱柱对中药喜树果和吴茱萸果的粗提物具有良好的分离性能,验证了其在实际样品分析中的巨大潜力。将天然产物腰果酚用于色谱固定相的制备,为分离纯化喜树碱和吴茱萸提供了新的方法,同时拓展了腰果酚在色谱分离材料方面的应用。     

Simultaneous determination of six Aconitum alkaloids in proprietary Chinese medicines by high-performance liquid chromatography

By optimizing the extraction, separation and analytical conditions, a reliable and accurate high-performance liquid chromatography (HPLC) method coupled with photodiode array detector (DAD) was developed for simultaneous quantitative determination of six Aconitum alkaloids, i.e., aconitine, mesaconitine, hypaconitine, benzoylaconine, benzoylmesaconine and benzoylhypaconine, in Chinese medicinal herbs, aconite roots, and 12 proprietary Chinese medicines containing processed aconite roots. The separation of these Aconitum alkaloids was achieved on an ODS column with gradient elution using solvents of acetonitrile and ammonium bicarbonate buffer (pH 10.0+/-0.2). Intra-assay and inter-assay precision of the analytes were less than 2.97%, and the average recovery rates obtained were in the range of 90-103% for all with RSDs below 3.28%. Good linear relationships were showed with correlation coefficients for the analytes exceeded 0.999. Quantitative analysis of the six Aconitum alkaloids in the unprocessed and processed aconite roots and in twelve proprietary Chinese medicines containing processed aconite roots showed that the contents of the alkaloids varied significantly. This method and quantitation results can provide a scientific and technical platform to the products manufacturers for setting up a quality control standard as well as to the public for quality and safety assurance of the proprietary Chinese medicines and other herbal preparations containing aconite roots.     

Rapid Determination of Diterpenoids in Andrographis paniculata by Microemulsion Electrokinetic Capillary Chromatography with Short-End Injection

A rapid, simple, and reliable method has been developed for routine capillary electrophoretic analysis of two diterpenoids, andrographolide and dehydroandrographolide, in Andrographis paniculata. For this system, using ethyl acetate oil-based microemulsion electrokinetic chromatography (MEEKC) and the short-end injection technique, analysis was complete in less than 2.5 min. In method validation the relative standard deviations of migration time and peak area of the two constituents were, respectively, 0.54% and 1.70% for andrographolide and 0.45% and 2.11% for dehydroandrographolide. Regression equations revealed linear relationships (correlation coefficients 0.9994 for andrographolide and 0.9993 for dehydroandrographolide) between peak area and concentration. The effects of buffer pH, borate concentration, SDS concentration, co-surfactant type and concentration, injection time, and running potential were systematically investigated. The method can be successfully implemented in routine quality-control testing.