Preparative isolation of bufalin and cinobufagin from Chinese traditional medicine ChanSu

In this investigation, a method is developed for the isolation and purification of bufadienolides (resibufogenin, cinobufagin, and bufalin) from the Chinese traditional medicine ChanSu. The crude ChanSu extract is prepared with solvent refluxing under optimal conditions. A new chromatographic approach for separating bufadienolides utilizes silica gel column chromatography with isocratic elution using cyclohexane-acetone (5:1) as the mobile phase to separate resibufogenin, followed by reversed-phase C18 preparative HPLC column using isocratic elution of methanol-water (72:28) to separate bufalin and cinobufagin. Combining these two methods, bufalin and cinobufagin could be completely separated in high purity and recovery, and the amounts obtained were 1.9 and 3.1 g, respectively, from 500 g of ChanSu. These two compounds have been identified by mass spectrometry and 1H NMR, and their purities were quantitated by HPLC at 99% and 98%, respectively.     

Simultaneous determination of four bufadienolides in human liver by high-performance liquid chromatography

A high-performance liquid chromatographic method for the simultaneous quantitation of four bufadienolides-cinobufotalin, bufalin, cinobufagin and resibufogenin-in human liver was investigated. The procedure involved solid phase extraction of human liver with an Oasis HLB cartridge coupled with reversed-phase HPLC and photodiode array detection. Recoveries obtained from spiked liver for the bufadienolides were better than 70%. The linearity was studied up to 1.2 mg/kg and the detection limits of the method were 0.4 ng for cinobufotalin and bufalin and 0.5 ng for cinobufagin and resibufogenin. The developed method was successfully applied to a lethal poisoning case.     

Simultaneous determination of three bufadienolides in rat plasma after intravenous administration of bufadienolides extract by ultra performance liquid chromatography electrospray ionization tandem mass spectrometry

A novel, rapid and specific ultra performance liquid chromatography electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS) method has been developed for simultaneous determination and pharmacokinetic studies of three bufadienolides (bufalin, cinobufagin and resibufogenin) in rat plasma. The analytes, bufalin, cinobufagin, resibufogenin and the internal standard, diphenhydramine were extracted from rat plasma samples by a one-step liquid–liquid extraction and separated on an ACQUITY UPLC™ BEH C₁₈ column with gradient elution using a mobile phase composed of acetonitrile and water (containing 0.1% formic acid) at a flow rate of 0.20 mL min⁻¹. Detection was carried out on a triple-quadrupole tandem mass spectrometer in the multiple reaction monitoring (MRM) mode via an electrospray ionization (ESI) interface. The three bufadienolides could be simultaneously determined within 3.0 min. Linear calibration curves were obtained over the concentration ranges of 1.0–200 ng mL⁻¹ for all the analytes. The intra- and inter-day precisions (relative standard deviation (R.S.D.)) were less than 11.35 and 10.87%, respectively. The developed method was applied for the first time to the pharmacokinetic studies of bufadienolides in rats following a single intravenous administration of 2.10 mg kg⁻¹ bufadienolides.     

Separation and determination of resibufogenin and cinobufagin in Chansu using reversed-phase liquid chromatography with γ-cyclodextrin as mobile-phase modifier

A simple, accurate, sensitive, and robust reversed-phase high-performance liquid chromatography (HPLC) method employing cyclodextrins as mobile phase additives has been developed in order to separate and determine resibufogenin and cinobufagin. Various factors affecting the separation for them, such as the nature of cyclodextrins, organic solvent, the concentration of γ-cyclodextrin, and temperature, were systematically studied. γ-cyclodextrin, as an effective mobile phase additive, can markedly improve the separation for resibufogenin and cinobufagin. The role of γ-cyclodextrin in the developed HPLC method is attributed to the formation of the inclusion complex between resibufogenin (or cinobufagin) and γ-cyclodextrin. So, the apparent formation constant (K(f) ) of the resibufogenin (or cinobufagin)/γ-cyclodextrin inclusion complex and the thermodynamic parameters of the inclusion process also were investigated. Resibufogenin (or cinobufagin) forms the 1:1 inclusion complexes with γ-cyclodextrin, and the resibufogenin/γ-cyclodextrin complex is more stable than the cinobufagin/γ-cyclodextrin complex. The K(f) values of resibufogenin and cinobufagin decrease with the increase of the temperature. The thermodynamic parameters of the inclusion reveal that the inclusion process between resibufogenin (or cinobufagin) and γ-cyclodextrin is spontaneous, exothermic, and enthalpically driven. Finally, the optimized method was successfully applied to separate and determine of resibufogenin and cinobufagin in the different Chansu (Bufonis venenum) samples.     

Evaluation of phenyldimethylethoxysilane treated high-performance thin-layer chromatographic plates. Application to analysis of flavonoids inScutellariae radix

Summary The retention and selectivity of flavonoids (baicalin, baicalein, wogonin, oroxylin A) inScutellariae radix have been studies by high-performance thin-layer chromatography on phenyldimethylethoxysilane-treated silica plates. The silica plates treated with phenyl groups were used for physical and chemical analysis. From elemental carbon analysis, the maximum number of bonded phenyl surface groups per gram was calculated to be 0.467×10²¹ (Oginal silica plate: Merck Art. 15109, Silica gel 100 F₂₅₄). With methanol-1/15 M phosphate buffer (pH 6.2) mixtures as mobile phase, baicalin, baicalein, wogonin, and oroxylin A inScutellariae radix were separated. It has been shown that phenyl-treated plates are more suitable for selective separation of baicalin, baicalein, wogonin, and oroxylin A than octadecyl-treated plates.     

反相高效液相色谱法测定蟾酥中的3种蟾毒内酯

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索,获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息;再以此为定位依据,在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。。     

Determination of resibufogenin and cinobufagin in heart-protecting musk pill by HPLC

A high-performance liquid chromatographic (HPLC) method for the simultaneous determination of resibufogenin and cinobufagin in traditional Chinese medicine (heart-protecting musk pill, shexiang baoxin wan in Chinese) was developed. A reversed-phase system with a Hypersil (ODS2) C(18) column and tetrahydrofuran: methanol: water (8:31:61) mobile phase was employed for the separation of resibufogenin and cinobufagin. The detection was set at 299 nm and ethinyl estradiol was chosen as the internal standard. The limit of detection was 1.5 ng for resibufogenin and 2.0 ng for cinobufagin at a signal-to-noise ratio of 4:1. It is a rapid, simple and accurate method for quantitative analysis of resibufogenin and cinobufagin in heart-protecting musk pill.     

Identification of Cinobufagin and Resibufogenin as Inhibitors of Enterovirus 71 Infection

In this paper, cinobufagin and resibufogenin were found to inhibit enterovirus 71（EV71） infection in vitro in cell viability and plaque reduction assays. The 50% inhibitory concentrations（lCs0） of einobufagin and resibufoge- nin were （10.94±2.36） and （218±31） nmol/L, respectively, the 50% cytotoxic concentrations（CCs0） of them were （1277±223） and （1385±254） nmol/L, respectively, and the anti-EV71 selectivity index（SI50） of cinobufagin was 116.7, which are promisingly developed into drug. Using a VP1 detection assay and a constructed reporter luciferase, we found that cinobufagin and resibufogenin disrupted the synthesis of EV71 protein. However, neither of them inhibited EV71 RNA replication. Our study suggests that cinobufagin and resibufogenin are the promising candidates that should he fllrther investigated for the treatment of EV71 caused disease.     

Microemulsion electrokinetic chromatographic determination of bufadienolides in toad venom and in traditional Chinese medicine

A microemulsion electrokinetic chromatographic (MEEKC) method has been developed and validated for determination of resibufogenin and cinobufagin in toad venom and in traditional Chinese medicine prepared from the venom. The MEEKC method involved use of sodium dodecyl sulfate (SDS) as surfactant, heptane as organic solvent, and butan-1-ol as co-solvent. To improve the separation, the effect of temperature and running buffer pH were evaluated. The optimized conditions (heptane 0.81% (w/w), SDS 3.31% (w/w), butan-1-ol 6.61% (w/w), and 10 mmol L⁻¹ sodium tetraborate buffer, pH 9.2, and 298 nm as the detection wavelength) enabled useful and repeatable separation of the analytes.     

Quantitative analysis of multicomponents by a single marker and quality evaluation of Venenum Bufonis from different geographical origins

Bufadienolides are the main bioactive components of Venenum Bufonis (VB) and have been widely used to treat different types of human cancers for decades. The bufadienolide content in VB varies significantly in materials from different geographical origins. In this work, a new strategy for the quality assessment of VB was developed through quantitative analysis of multi‐components by single marker (QAMS). Cinobufagin was selected as the internal reference substance; seven bufadienolides were separated and simultaneously determined based on relative correction factors. The correlation coefficient value (r ≥ 0.9936) between QAMS and the normal external standard method proved the consistency of the two methods. According to the outcomes of 30 batches of VB samples, the contents of the seven bufadienolides were used for further chemometric analysis. All of the samples of VB from various geographical origins were divided into three categories based on hierarchical cluster analysis and radar plot, which indicated the crucial influence of geographical origins on VB. This study showed that QAMS combined with chemometristry could be used to comprehensively evaluate and effectively control the quality of VB from different geographical origins.     

Chiral separation of dansyl amino acids by capillary electrophoresis: Comparison of formamide andN-Methylformamide as background electrolytes

Summary Chiral separation of 12 dansyl amino acids has been achieved by capillary electrophoresis using -cyclodextrin dissolved inN-methylformamide or formamide. The viscosity and the dielectric constants of these two solvents are very different, giving them disimilar electrophoretic properties. Intense electroosmotic flow, high electrophoretic mobilities and high efficiency were achieved inN-methylformamide, but high chiral selector concentration was needed for sufficient resolution. In contrast, the separation of dansyl amino acids in formamide was characterized by longer analysis time and lower efficiency, but the resolution and selectivity of the separation were better and baseline separation could be achieved at lower -cyclodextrin concentrations.     

Isomerization ofn-pentane andn-hexane on modified zeolites

The activity and selectivity of catalysts based on TsVM (an analog of ZSM-5), Beta, and La-H-Beta zoelites modified by Pt, Pt−Fe, and Pt−Ga were studied in the isomerization of C₅ and C₆ linear alkanes. The Pt/HTsVM, Pt/H-Beta, and Pt/La-H-Beta catalysts are efficient inn-pentane isomerization, whereas the Pt/H-Beta and Pt/La-H-Beta are most active inn-hexane isomerization. Nearly equilibirum isoparaffin yield at a selectivity of at least 95–96% is reached on these catalysts unlike other zeolite systems. The overall yield of 2,2-and 2,3-dimethylbutanes is 22 wt.%. The hexane isomers are not formed over the Pt/HTsVM catalyst due to the molecular-sieve properties of this type of zcolites. Published inIzvestiya Akademii Nauk. Seriya Khimicheskaya No. 11, pp. 1866–1869, November, 2000.     

Preparative separation and purification of bufadienolides from Chinese traditional medicine of ChanSu using high‐speed counter‐current chromatography

A preparative high‐speed counter‐current chromatography method for isolation and purification of bufadienolides from ChanSu was developed by using a stepwise elution with two‐phase solvent system composed of n‐hexane/ethyl acetate/methanol/water at the ratios of 4:6:2:4 v/v, 4:6:2.5:4 v/v and 4:6:3.2:4 v/v. A total of 3.8 mg of gamabufotalin (1), 7.2 mg of arenobufagin (2), 3.4 mg of telocinobufagin (3), 5.3 mg of bufotalin (4), 8.5 mg of cinobufotalin (5) and 8 mg of bufalin (6) were obtained in one‐step separation from 80 mg of the crude extract with purity of 92.7, 96.7, 87.2, 97.3, 94.9 and 99.4%, respectively. Their chemical structures were identified on the basis of ¹H‐NMR and ¹³C‐NMR technology.     

Anti-proliferative and pro-apoptotic effects of cinobufagin on human breast cancer MCF-7 cells and its molecular mechanism

Cinobufagin (CBF) is an active ingredient isolated from Venenum Bufonis extracted and dried from the secretory glands of Bufo gargarizans Cantor. The purpose of the study was to investigate the effects and underlying mechanisms of CBF on human breast cancer MCF-7 cells in vitro. Our results showed that CBF exhibited obvious cytotoxicity on MCF-7 cells in a dose- and time-dependent manner, as indicated by CCK-8 assays. Also, Hoechst 33258 staining and flow cytometry assays showed that CBF strongly induced MCF-7 cell apoptosis and G1 phase arrest. In addition, further molecular mechanistic investigation demonstrated that cinobufagin significantly increased Bax expression, decreased Bcl-2 expression level and up-regulated the ratio of the pro-apoptosis/anti-apoptosis protein Bax/Bcl-2, which were demonstrated by RT-qPCR and western blot assays. Taken together, our data confirm that CBF inhibits growth and triggers apoptosis of MCF-7 cells by affecting the expression of Bax and Bcl-2 in vitro.     

Impact of column and stationary phase properties on the productivity in chiral preparative LC

By generating 1500 random chiral separation systems, assuming two‐site Langmuir interactions, we investigated numerically how the maximal productivity (P_R,max) was affected by changes in stationary phase adsorption properties. The relative change in P_R,max, when one adsorption property changed 10%, was determined for each system and for each studied parameter the corresponding productivity change distribution of the systems was analyzed. We could conclude that there is no reason to have columns with more than 500 theoretical plates and larger selectivity than 3. More specifically, we found that changes in selectivity have a major impact on P_R,max if it is below ∼2 and, interestingly, increasing selectivity when it is above ∼3 decreases P_R,max. Increase in relative saturation capacity will have a major impact on P_R,max if it is below ∼40%, but only modest above this percent. Increasing total monolayer saturation capacity, or decreasing the first eluting enantiomer's retention factor, will have a modest effect on P_R,max and increased efficiency will have almost no effect at all on P_R,max unless it is below ∼500 theoretical plates. Finally, we showed that chiral columns with superior analytic performance might have inferior preparative performance, or vice versa. It is, therefore, not possible to assess columns based on their analytical performance alone.     

Marinobufagenin extraction from Rhinella marina toad glands: Alternative approaches for a systematized strategy

Marinobufagenin is a bufadienolide compound detected mainly in skin and parotoid gland secretions of Rhinella marina (L.) toad. Bufadienolides regulate the Na⁺/K⁺‐ATPase pump by inhibiting the cardiotonic steroid dependent‐site and act as cardiac inotropes with vasoconstrictive properties. Marinobufagenin and other bufadienolides, such as telocinobufagin and bufalin, are thought to be found endogenously in mammals in salt‐sensitive hypertensive states such as essential hypertension, congestive heart‐failure, and preeclampsia. The role of marinobufagenin as antimicrobial agent and its cytotoxic potential have also been recognized. The particular interest around marinobufagenin prompts us to consider the Rhinella marina toad venom as a possible source for molecules with pharmacological and/or diagnostic potential. In this article, two different approaches of extraction and purification of marinobufagenin from Rhinella marina (L.) venom are studied: (i) Preparative thin‐layer chromatography combined to mass spectrometry and/or ultraviolet detection and (ii) solid‐phase extraction coupled with fractionation on high‐performance liquid chromatography. Different chromatographic conditions are tested for each approach. The solid‐phase extraction combined with high‐performance liquid chromatography fractionation approach was preferred as it offered a greater yield, was less time‐consuming and allowed us to selectively isolate marinobufagenin. Both protocols aim to provide efficient and convenient methods for toad venom extraction, based on an easily automatable and systematized strategy.     

Efficient isolation and purification of five products from microbial biotransformation of cinobufagin by high‐speed counter‐current chromatography

An efficient separation method of using high‐speed counter‐current chromatography was successfully established to directly purify cytotoxic transformed products of cinobufagin by Cordyceps militaris. The two‐phase solvent system composed of n‐hexane–ethyl acetate–methanol–water (4:6:3:4, v/v) was used in high‐speed counter‐current chromatography. A total of 9 mg of 4β,12α‐dihydroxyl‐cinobufagin ( 1 ), 15 mg of 12β‐hydroxyl‐cinobufagin ( 2 ), 8 mg of 5β‐hydroxyl‐cinobufagin ( 3 ), 12 mg of deacetylcinobufagin ( 4 ) and 6 mg of 3‐keto‐cinobufagin ( 5 ) were obtained in a one‐step separation from 400 mg of the crude extract with purity of 98.7, 97.2, 90.6, 99.1 and 99.4%, respectively, as determined by HPLC. Their chemical structures were identified on the basis of ¹H‐NMR and ¹³C‐NMR technology. All products ( 1 – 5 ) showed the potent activities against human carcinoma cervicis (Hela) and malignant melanoma (A375) cells in vitro.     

Phosphorus macrocycles and cryptands

The reviews covers authors" studies dealing with the synthesis of P^III- and P^V-containing macrocycles and cryptands. The separation, structural characterization, and the chemical properties of a number of homeomorphic compounds with in,out bridgehead phosphorus atoms are described. Modification of the in-positions in macrobicyclic compounds with bulky groups is described for the first time.     

A study of the behaviour of some new column materials in the chromatographic analysis ofCatharanthus alkaloids

Summary New column-packing materials specially designed for the HPLC analysis of basic compounds have been tested for the analysis ofCatharanthus alkaloids. Mobile phase optimization was performed for each column tested. The influence of mobile phase pH, nature and content of the organic modifier and salt concentration on retention, selectivity and resolution was studied. An important factor in the separation proved to be the pH of the eluent, because of the widely different pK _a values of the analytes. Complete separation was easily achieved on ODS columns, but polymeric materials also gave acceptable results. Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th September, 1996     

n-Alkane isomerization on heteropolyacids

Acid-base properties of bulk 12-tungstophosphoric heteropolyacid (HPWA) and HPWA supported on alumina and fluorinated alumina were studied by DRIFT spectroscopy using adsorption of probe molecules. Catalytic properties of Pt-containing catalysts carried by these supports were studied inn-pentane isomerization. It was shown that the Pt/HPWA/Al₂O₃ system is almost inactive in isomerization as a result of the interaction of HPWA with basic sites of the alumina support. On the contrary, the presence of acidic sites in fluorinated alumina prevents HPWA destruction and favors the uniform distribution of HPWA on the support surface. As a result, enhancement of the activity and selectivity of the Pt/HPWA/Al₂O₃-F catalyst inn-pentane isomerization is observed. Published inIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 10, pp. 1744–1750, October, 2000.