Preparative isolation of pseudolaric acids A and B,and their glucosides from the root bark of Pseudolarix kaempferi using high‐speed counter‐current chromatography

In order to provide the chemical markers for the quality control of herbal medicines, four diterpenoids, pseudolaric acids A and B (PAA and PAB), and their glucosides were isolated from the methanol extract of the Chinese herb Pseudolarix kaempferi using high‐speed counter‐current chromatography (HSCCC). The diphase solvent system was n‐hexane/EtOAc/MeOH/H₂O which was used at two ratios (5:5:5:5 and 1:9:4:6 by volume) in the separation of pseudolaric acids and their glycosides, respectively. As a result, PAA (14 mg), PAB (129 mg), PAA‐O‐β‐D ‐glucopyranoside (8 mg, PAAG), and PAB‐O‐β‐D ‐glucopyranoside (42 mg, PABG) were obtained from 0.5 g of the crude extract. Their purities were determined to be above 97% by HPLC analysis. Their chemical structures were confirmed by ¹H and ¹³C NMR analysis or HPLC comparison with the reference compounds.     

Rapid analysis of pseudolaric acids in Cortex Pseudolaricis and related medicinal products by high performance liquid chromatography

A simple, rapid, reverse-phase high performance liquid chromatographic method was developed for the quantitative analysis of pseudolaric acids in Cortex Pseudolaricis and its related medicinal products. With a C₁₈ analytical column (4.6 mm × 150 mm i.d.), five pseudolaric acids, namely pseudolaric acids A-C, pseudolaric acid A-O-β-d-glucopyranoside and pseudolaric acid B-O-β-d-glucopyranoside, were well separated within 7 min. Acetonitrile and 0.10% acetic acid were used as the mobile phase in a gradient program. The UV detection wavelength was set at 260 nm. The detection limits and quantification limits ranged in 8.26-16.66 ng/ml and 27.54-55.53 ng/ml, respectively. The intra- and inter-day variations were less than 1% for all five compounds. The recovery of all spiked pseudolaric acids ranged from 99.1% to 101.9%. Compared to existing analytical methods, this new method not only used two more important chemical markers but also provided a fivefold reduction in analysis time. In addition, the extraction method of herb sample was also modified by an orthogonal array experiment on three variable parameters: extraction time, solvent volume, and extraction cycles. The optimized extraction method was much simpler and could be efficiently used to analyse large set of herbal materials and related medicinal products. Nineteen herb samples collected from different regions of China and five related products were examined with this new analytical method. The results showed that this method is effective in distinguishing adulterants and unqualified products.     

Total synthesis of (-)-pseudolaric acid B

We report the enantioselective synthesis of pseudolaric acid B (1a), a diterpene acid isolated from the bark of Pseudolarix kaempferi Gordon, which displays interesting antifungal, antifertility, and cytotoxic activity against multidrug resistant cell lines. Our synthesis utilizes a highly efficient metal-catalyzed [5 + 2] vinylcyclopropane-alkyne intramolecular cycloaddition to construct the polyhydroazulene core of the natural product. Elaboration to the tricyclic scaffold of the pseudolaric acids was completed with an intramolecular alkoxycarbonyl radical cyclization to form the quaternary center and a highly diastereoselective cerium acetylide addition to a methyl ketone for introduction of the acid side chain.     

Chromatographic fingerprinting and metabolomics for quality control of TCM

Chromatographic fingerprinting technique of traditional Chinese medicine (TCM) has proved to be a comprehensive strategy for assessing the intact quality of herbal medicine. In general, one could use the chromatographic techniques to obtain a relatively complete picture of herbal medicines, which are in common called chromatographic fingerprints of herbal medicines to represent the so-called phytoequivalence. Based on this, the features of chromatographic fingerprints of herbal medicines have been discussed in some detail. The technique based on chromatographic fingerprinting is essentially a kind of high-throughput and integral tools to explore the complexity of herbal medicines. In order to further control the comprehensive quality of TCMs, some new strategies are proposed to trace the chemical changes of chromatographic fingerprints both in product processing and/or after their administration by modern chromatographic techniques and chemometrics. Combined with metabolomics, it seems possible for one to reveal the working mechanism of TCMs and to further control their intrinsic quality. Finally, the intensive study of chromatographic fingerprinting coupled with multivariate analysis tools developed in bioinformatics and chemometrics are emphasized in order to achieve the aim to reveal the working mechanisms of TCMs and to further control and strengthen TCMs' intrinsic quality in a comprehensive manner.     

Chromatographic separation techniques and data handling methods for herbal fingerprints: a review

As herbal medicines have an important position in health care systems worldwide, their current assessment and quality control are a major bottleneck. Over the past decade, major steps were taken not only to improve the quality of the herbal products but also to develop analytical methods ensuring their quality. Nowadays, chromatographic fingerprinting is the generally accepted technique for the assessment and quality control of herbal products. This paper briefly considers the evolution of the regulations and guidelines on the quality control of herbal medicines, and reviews the established analytical techniques for herbal fingerprinting with an emphasis on the most recent developments, such as miniaturized techniques, new stationary phases, analysis at high temperatures and multi-dimensional chromatography. Accessory to the new analytical techniques, the chemometric data handling techniques applied are discussed. Chemometrics provide scientists with useful tools in understanding the huge amounts of data generated by the analytical advances and prove to be valuable for quality control, classification and modelling of, and discrimination between herbal fingerprints.     

A concise approach for the total synthesis of pseudolaric acid A

A new strategy for the stereoselective total synthesis of natural product pseudolaric acid A (1) was accomplished in 16 steps from commercially available starting material, featuring a samarium diiodide (SmI(2))-mediated intramolecular alkene-ketyl radical cyclization and a ring-closing metathesis (RCM) reaction to stereoselectively cast the unusual trans-fused [5-7]-bicyclic core of pseudolaric acid A (1).     

Chemical fingerprint analysis for quality assessment and control of Bansha herbal tea using paper spray mass spectrometry

Chemical fingerprinting methodology is an approach for quality assessment and control of herbal medicines and related products based on the holistic chemical profile obtained by various analytical techniques. This study demonstrates the first application of paper spray mass spectrometry (PS-MS) as a chemical fingerprinting methodology for tracing the origins, establishing the authenticity, and assessing the overall quality of a famous herbal product, Bansha herbal tea (BHT). A negative ion PS-MS spectrum yielded the best chemical profiling information and was most appropriate for fingerprint analysis of BHT. In addition to the identification of active ingredients, various compounds present in BHT were simultaneously detected without any sample pretreatment and chromatographic separation, providing valuable information for the quality assessment and control of this herbal product. According to the principal component analysis of the PS-MS fingerprints, two sources of commercially available BHT products made by different manufacturers were easily differentiated. Qualified and expired products from the two manufacturers were also successfully distinguished, and the consistency of the quality between the manufacturers was assessed. Our experimental data demonstrated that the PS-MS chemical fingerprinting is a simple, rapid, and robust methodology for pharmaceutical analysis, with promising prospects for quality assessment and control of herbal medicines and related products with high-throughput.     

On-line two-step stacking in capillary zone electrophoresis for the preconcentration of strychnine and brucine

An on-line sample preconcentration method by two-step stacking i.e., sweeping and micelle to solvent stacking, in capillary zone electrophoresis (CZE) has been developed for the determination of strychnine and brucine in traditional Chinese herbal medicines. After experimental optimizations, the best separation was achieved by using 75 mM phosphate buffer (pH 2.5) with 30% methanol (v/v). Compared with normal CZE injection, 51- and 38-fold improvement in concentration sensitivity was achieved for strychnine and brucine, respectively. The calibration curve was linear in the range of 0.1–5.0 μg mL⁻¹ for both strychnine and brucine, with the correlation coefficients of 0.9998 and 0.9997, respectively. The limits of detection (S/N = 3) for both alkaloids were 0.01 μg mL⁻¹. The inter-day (n = 8) and intra-day (n = 5) reproducibilities expressed as the relative standard deviations for corrected peak area were less than 9.5%. The method was applied to determine strychnine and brucine in two Chinese herbal medicines, with recoveries ranging from 94.2% to 105.4%. The results indicated that the method is simple, rapid, reliable, and can be applied to determine strychnos alkaloids in traditional Chinese herbal medicines.     

Multiple chromatographic fingerprinting and its application to the quality control of herbal medicines

Recently, chromatographic fingerprinting has become one of the most powerful approaches to quality control of herbal medicines. However, the performance of reported chromatographic fingerprinting constructed by single chromatogram sometimes turns out to be inadequate for complex herbal medicines, such as multi-herb botanical drug products. In this study, multiple chromatographic fingerprinting, which consists of more than one chromatographic fingerprint and represents the whole characteristics of chemical constitutions of the complex medicine, is proposed as a potential strategy in this complicated case. As a typical example, a binary chromatographic fingerprinting of “Danshen Dropping Pill” (DSDP), the best-sold traditional Chinese medicine in China, was developed. First, two HPLC fingerprints that, respectively, represent chemical characteristics of depsides and saponins of DSDP were developed, which were used to construct binary chromatographic fingerprints of DSDP. Moreover, the authentication and validation of the binary fingerprints were performed. Then, a data-level information fusion method was employed to capture the chemical information encoded in two chromatographic fingerprints. Based on the fusion results, the lot-to-lot consistency and frauds can be determined either using similarity measure or by chemometrics approach. The application of binary chromatographic fingerprinting to consistency assessment and frauds detection of DSDP clearly demonstrated that the proposed method was a powerful approach to quality control of complex herbal medicines.     

Involvement of JNK-initiated p53 accumulation and phosphorylation of p53 in pseudolaric acid B induced cell death

A terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay was used to determine that apoptosis causes HeLa cell death induced by pseudolaric acid B. The c-Jun N-terminal kinase (JNK) inhibitor SP600125 decreased p53 protein expression during exposure to pseudolaric acid B. SP600125 decreased the phosphorylation of p53 during pseudolaric acid B exposure, indicating that JNK mediates phosphorylation of p53 during the response to pseudolaric acid B. SP600125 reversed pseudolaric acid B-induced down-regulation of phosphorylated extracellular signal-regulated protein kinase (ERK), and protein kinase C (PKC) was activated by pseudolaric acid B, whereas staurosporine, calphostin C, and H7 partly blocked this effect. These results indicate that p53 is partially regulated by JNK in pseudolaric acid B-induced HeLa cell death and that PKC participates in pseudolaric acid B-induced HeLa cell death.     

Development of a polymer system for the delivery of daunorubicin to tumor cells to overcome drug resistance

Method for the synthesis of polymeric nanoparticles (NP) with encapsulated daunorubicin (DNR) was developed on the basis of double emulsion solvent evaporation technique using biodegradable poly(lactide-co-glycolide) (PLGA), which is aimed at customization of pharmacokinetic properties of the preparation, enhanced accumulation of DNR in tumor cells and prolongation of its action. The obtained polymer nanoparticles (DNR-PLGA) had average size ranging around 138±36 nm, with zeta-potential of –25.3 mV and the polydispersity index (PDI) of 0.072. The release kinetics of DNR from polymer nanoparticles at pH 7.4 and 5.0 has been studied. In vitro studies showed similar specific activity of DNR- PLGA in K562 and MCF-7 cancer cell lines together with an increase in activity in K562 Adr and MCF-7 Adr cell lines, which are anthracycline resistant, by 1.6 and 3.4 times. The study demonstrated the efficacy of the developed PLGA-based DNR delivery system in the improvement of antitumor effect of DNR, overcoming multidrug resistance in cancer cells, and also in the decrease in nonspecific toxicity of the preparation.     

Antioxidant, antitubercular and cytotoxic activities of Piper imperiale

Phenolic compounds are widely distributed in Nature and act as pharmacologically active constituents in many herbal medicines. They have multiple biological properties, most notably antioxidant, antibacterial and cytotoxic activities. In the present study an attempt to correlate the phenolic composition of leaf, flower and wood extracts of Piper imperiale, with antioxidant, antitubercular and cytotoxic activities was undertaken. The total phenol content ranged from 1.98 to 6.94 mg GAE/gDW among ethanolic extracts, and gallic acid, catechin, epicatechin, ferulic acid, resveratrol and quercetin were identified and quantified by HPLC. DPPH and ABTS assays showed high antioxidant activity of the leaf extract (EC(50ABTS) = 15.6 μg/mL, EC(50DPPH) = 27.3 μg/mL) with EC?? in the same order of magnitude as the hydroxyquinone (EC(50ABTS) = 10.2 μg/mL, EC(50DPPH) = 15.7 μg/mL). The flower extract showed strong antimicrobial activity against Mycobacterium tuberculosis H??Rv. All the extracts exhibited dose-dependent cytotoxic effects against MCF-7 cancer cells. This is the first time that a Piper extract has been found to be highly active against M. tuberculosis. This study shows the biological potential of Piper imperiale extracts and gives way to bio-guided studies with well-defined biological activities.     

Chemometrics and modernization of traditional Chinese medicine

Development of chromatographic fingerprinting and its related chemometric methods in the research of quality control of traditional Chinese medicines(TCMs) are discussed. The quality control methods for guarantying the authentication and stability of products and semi-products of TCMs are firstly assessed. The technique based on chromatographic fingerprinting is essentially a kind of high-through put and integral tools to explore the complexity of herbal medicines. In order to further control the comprehensive quality of TCMs,confirmation and identification of their important chemical components are necessary. Some new strategies are proposed to trace the chemical changes of chromatographic fingerprints both in product processing and/or after their administration by modern chromatographic techniques and chemometrics. Combined with systems biology and bioinformatics,it seems possible for one to reveal the working mechanism of TCMs and to further control their intrinsic quality comprehensively.     

采用隔离池的毛细管电泳-间接紫外吸收法测定茶叶中的氨基酸

改进的毛细管电泳-间接紫外吸收法采用了自制隔离池,以对氨基苯甲酸(PAB)为背景电解质,对茶叶中的氨基酸进行了测定。PAB能够提高分离效率,降低检出限。隔离池的使用避免了PAB的电极反应,降低了基线噪声,维持了两缓冲液池间的电流导通。研究了背景电解质的浓度、pH值以及电渗流改性剂的种类和浓度对氨基酸分离的影响。在优化的实验条件下,16种氨基酸在14 min内达到了基线分离,峰面积的相对标准偏差小于5%(n=5),检出限为1.7~4.5 μmol/L,回收率为83.0%~106%。该法快速、便捷和灵敏,已成功应用于茶叶中11种游离氨基酸的检测。     

Metabolite Fingerprinting Based on 1H-NMR Spectroscopy and Liquid Chromatography for the Authentication of Herbal Products

Herbal medicines (HMs) are regarded as one of the traditional medicines in health care to prevent and treat some diseases. Some herbal components such as turmeric and ginger are used as HMs, therefore the identification and confirmation of herbal use are very necessary. In addition, the adulteration practice, mainly motivated to gain economical profits, may occur by substituting the high price of HMs with lower-priced ones or by addition of certain chemical constituents known as Bahan Kimia Obat (chemical drug ingredients) in Indonesia. Some analytical methods based on spectroscopic and chromatographic methods are developed for the authenticity and confirmation of the HMs used. Some approaches are explored during HMs authentication including single-component analysis, fingerprinting profiles, and metabolomics studies. The absence of reference standards for certain chemical markers has led to exploring the fingerprinting approach as a tool for the authentication of HMs. During fingerprinting-based spectroscopic and chromatographic methods, the data obtained were big, therefore the use of chemometrics is a must. This review highlights the application of fingerprinting profiles using variables of spectral and chromatogram data for authentication in HMs. Indeed, some chemometrics techniques, mainly pattern recognition either unsupervised or supervised, were applied for this purpose.     

A quantitative study of the enzymatic activity of horseradish peroxidase at a planar poly(acrylic acid) brush

The enzymatic activity of horseradish peroxidase (HRP) has been quantified at a planar poly(acrylic acid) (PAA) brush. A PAA brush is known to exhibit an unusual protein binding affinity, since proteins adsorb at low ionic strength only. At elevated ionic strengths of a few 100 mM proteins desorb, and the PAA brush becomes largely protein resistant. In this study, HRP was used to catalyze the oxidation of Amplex Red to resorufin by hydrogen peroxide. The fluorescence of resorufin was recorded using a microplate reader. As compared to a bare silica surface, the enzymatic activity of HRP is higher by more than one order of magnitude at a PAA brush. This increase results from a higher degree of adsorption and a reasonable preservation of the HRP activity. Upon adsorption at a PAA brush from a 20 mU/mL (0.1 μg/mL) solution, the molecular enzymatic activity of HRP is reduced to about 11%. However, when a HRP molecule is desorbed from a PAA brush by increasing the ionic strength, a gain of the molecular enzymatic activity by only 52% can be observed. Overall, this study illustrates the potential applicability of a PAA brush as a biocompatible material coating.     

土槿皮新二萜成分的研究 IV: 土槿丁酸和土槿戊酸的分离和结构测定

从土槿皮(Pseudolarix kaempferi, Gord.)中分离到两个新二萜酸, 命名为土槿丁酸(1)和土槿戊酸(2), 经理化性质和对它们的IR、^1H NMR、^1^3C NMR、MS数据的分析, 鉴定1为 18-羟-对映-贝壳杉-15-烯-17-羧酸; 2为对映-贝壳杉-15-烯-17, 18-二羧酸. 1的结构经X衍射得到了证实.     

Biological analysis of herbal medicines used for the treatment of liver diseases

Herbal medicines have been used to treat liver disorders for thousands of years in the East and have now become a promising therapy internationally for pathological liver conditions. Biological analysis of hepatoprotective herbs is an important issue from the pharmacokinetic perspective in developing new therapeutic managements for liver disease. The biological analysis focuses on the pretreatment methods, separation and quantification of herbal medicines in biological samples. We have compiled and discuss the biological analytical method of six herbal medicines for liver protection containing Silybum marianum(silymarin), Glycyrrhiza glabra, Scutellaria baicalensis, Schisandra chinensis, Salvia miltiorrhiza and Astragalus membranaceus. This review provides a convenient reference for researchers to reduce time-consuming method optimization.     

Non-alkaloids extract from Stemona sessilifolia enhances the activity of chemotherapeutic agents through P-glycoprotein-mediated multidrug-resistant cancer cells

One of the major impediments to the successful treatment of cancer is the development of resistant cancer cells, which could cause multidrug resistance (MDR), and overexpression of ABCB1/P-glycoprotein (P-gp) is one of the most common causes of MDR in cancer cells. Recently, natural products or plant-derived chemicals have been investigated more and more widely as potential multidrug-resistant (MDR) reversing agents. The current study demonstrated for the first time that non-alkaloids extract from Stemona sessilifolia significantly reversed the resistance of chemotherapeutic agents, adriamycin, paclitaxel and vincristine to MCF-7/ADR cells compared with MCF-7/S cells in a dose-dependent manner. The results obtained from these studies indicated that the non-alkaloids extract from S. sessilifolia plays an important role in reversing MDR of cancer as a P-gp modulator in vitro and may be effective in the treatment of multidrug-resistant cancers.     

Colorimetric Analysis of the Acrosin Inhibitor Para-Aminobenzamidine

Abstract

A method is presented for the colorimetric assay of para-aminobenzamidine (PAB) in blood. Essentially, the PAB in trichloroacetic acid extracts is coupled to N-(l-napthyl)ethylenediamine in a typical diazotization reaction. The colored complex formed exhibits a peak absorption at 540 nm. This complex in distilled water is very stable and loses less than 5% of its absorptivity over a 24 hour period. Trichloroacetic acid extracts of whole blood containing PAB are likewise stable, exhibiting less than a 7% mean reduction in absorbance after standing for 24 hours. A plot of optical density versus concentration of the PAB diazo complex over a range of 1–20 μg/ml shows excellent linerarity. The method can accurately quantitate as little as 1 μg/ml at a dilution of 3.5.