Preliminary Screening and Analysis of Biomembrane Permeable Compounds in Herbal Medicines: Hollow Fiber Liposome Microscreening Combined with HPLC

An advantageous method based on hollow fiber liposome microscreening (HFLMS) combined with high-performance liquid chromatography (HPLC) has been developed and used in preliminary screening and analysis of biomembrane permeable compounds from herbal medicines (HMs). In the method, a liposome hollow fiber was prepared as a screening tool; permeable compounds that could specifically bind to liposomes were screened from herbal extract to liposome in the pores and lumen of hollow fiber, then permeable compounds were dissociated from liposomes and analyzed by HPLC. The chromatographic separation was applied with a Zorbax Eclipse XDB-C18 column by a gradient elution using acetonitrile, methanol, and phosphoric acid aqueous solution as the mobile phases. In the study, influencing factors of the screened target compounds were investigated and optimized. Under optimum conditions, the surface properties of liposome hollow fibers were characterized, the nonspecific binding between the active center of hollow fiber and the bioactive components were investigated, the repeatability of this method was tested. And eight permeable compounds of flavonoids and anthraquinones from HMs were preliminarily screened and analyzed by HFLMS-HPLC. The results demonstrated that new method is a simple, fast, effective, and reliable method for preliminary screening and analyzing biomembrane permeable ingredients in HMs, and it can be used in screening other permeable compounds in HMs.

     

Fast Screening of Biomembrane-Permeable Compounds in Herbal Medicines Using Bubble-Generating Magnetic Liposomes Coupled with LC–MS

A novel strategy based on the use of bionic membrane camouflaged magnetic particles and LC–MS was developed to quickly screen the biomembrane-permeable compounds in herbal medicines. The bionic membrane was constructed by bubble-generating magnetic liposomes loaded with NH₄HCO₃ (BMLs). The lipid bilayer structure of the liposomes enabled BMLs to capture biomembrane-permeable compounds from a herbal extract. The BMLs carrying the compounds were then separated from the extract by a magnetic field. Upon heat treatment, NH₄HCO₃ rapidly decomposed to form CO₂ bubbles within the liposomal bilayer, and the captured compounds were released from BMLs and analyzed by LC–MS. Jinlingzi San (JLZS), which contains various natural ingredients, was chosen to assess the feasibility of the proposed method. As a result, nine potential permeable compounds captured by BMLs were identified for the first time. Moreover, an in vivo animal study found that most of the compounds screened out by the proposed method were absorbed into the blood. The study provides a powerful tool for rapid and simultaneous prediction of multiple biomembrane-permeable components.     

Screening and quantification of anticancer compounds in traditional Chinese medicine by hollow fiber cell fishing and hollow fiber liquid/solid‐phase microextraction

Hollow fiber cell fishing, based on HepG‐2, SKOV‐3, and ACHN cancer cells, and hollow fiber liquid/solid microextraction with HPLC were developed and introduced for researching the anticancer activity of Rhizoma Curcumae Longae, Radix Curcumae, and Rhizoma Curcumae. The structures of curcumin, demethoxycurcumin, and bisdemethoxycurcumin screened were identified and their contents were determined. The compound target fishing factors and cell apoptosis rates under the effect of the three medicines were determined. The binding sites (cell membrane and cell organelle) and binding target (phospholipase C) on the cell were researched. Hollow fiber liquid/solid‐phase microextraction mechanism was analyzed and expounded. Before the application, cell seeding time, growth state and survival rate, compound nonspecific binding, positive and negative controls, repeatability in hollow fiber cell fishing with high‐performance liquid chromatography; extraction solvent, sample pH, salt concentration, agitation speed, extraction time, temperature and sample volume in hollow fiber liquid/solid‐phase microextraction with high‐performance liquid chromatography were investigated. The results demonstrated that the proposed strategy is a simple and quick method to identify bioactive compounds at the cellular level as well as determine their contents (particularly trace levels of the bioactive compounds), analyze multicompound and multitarget entirety effects, and elucidate the efficacious material base in traditional medicine.     

Adsorption and desorption behaviors of cationic liposome-DNA complexes upon lipofection in inside and outside biomembrane models using a dynamic quasi-elastic laser scattering method.

The dynamic behaviors of cationic liposome-DNA complexes in inside and outside biomembrane models upon lipofection were investigated using the time-resolved quasi-elastic laser scattering (QELS) method. Inside and outside biomembrane models with similar phospholipid compositions to those in living cells were formed at a tetradecane/phosphate buffered saline (TD/PBS) interface. Cationic liposome-DNA complexes were injected into the buffer subphase, and their adsorption/desorption behaviors at the biomembrane models were monitored through changes in the interfacial tension. We found that the adsorption rate of the complexes increased 2.6 times more in the outside model than in the inside one. The adsorption rate of DNA alone did not show a remarkable difference from one side to the other; however, the adsorption rate of the cationic liposome alone showed a similar tendency to that of the liposome-DNA complex. These results indicated that the difference in lipid composition induced a different dynamic behavior of exogenous biomolecules and that the cationic liposomes played an important role in the faster incorporation of DNA into cells upon lipofection.     

Effect of Different Pretreatment Methods on the Measurement of the Entrapment Efficiency of Liposomes and Countermeasures in Free Drug Analysis

Further investigation into the methods for the analysis of the entrapment efficiency of liposomes has been prompted by the urgent need to improve traditional methods which offer more risk to liposome leakage. The aim of this study was to investigate the suitability of hollow fiber centrifugal ultrafiltration (HF-CF-UF) coupled with HPLC as an alternative method for the routine determination of the entrapment efficiency of liposomes and to compare it with previously developed nonequilibrium procedures based on size-exclusion chromatography (SEC). By comparison, evaluation of the entrapped fraction with SEC resulted in 3 or 4 % lower entrapment values. More importantly, the free drug concentrations were three- to sixfold higher than with HF-CF-UF. In addition, we investigated the reasons for the different values obtained with SEC and a dynamic equilibrium theory was put forward based on this. In addition, we have developed and characterized an equilibrium method of separating free and liposomal drugs for liposomal formulations based on HF-CF-UF. The method was validated over the concentration range of 7.9–235 μg mL^?1 for indomethacin and 0.258–8.24 μg mL^?1 for vitamin A. Inter- and intra-day precision (RSD %) were ≤1.2 % for indomethacin and ≤1.8 % for vitamin A, respectively. The recoveries of both free drug and total drug were higher than 96.0 % with RSD ≤1.3 % (n = 5). Taken together, our improved method differs from those previously reported with regard to higher recovery, small sample volume, less laborious, and most importantly without damaging or disturbing liposomes. Validation results suggested that our method was sufficiently accurate and sensitive to be used to evaluate the entrapment efficiency of a liposome formulation without complicated pretreatment.     

脂质体毛细管电泳方法评价有机化合物在体内的吸收

有机化合物在脂质体毛细管电泳中的保留值大小可以体现化合物的亲脂性大小。比较了9种有机化合物在脂质体毛细管电泳中脂质体/水的疏水参数(log Plw)(由保留因子k的对数值(log k)转化而来)及其在正辛醇/水体系中的疏水参数(log Pow)与其渗透系数的对数值(log Pm)的相关性,log Plw与log Pm的相关系数为0.94,log Pow与log Pm的相关系数仅为0.78,说明脂质体/水模型较正辛醇/水模型更加接近于生物膜模型。脂质体毛细管电泳可以快速、准确地测定化合物的疏水参数,通过化合物在脂质体毛细管中的保留可以快速、初步地预测化合物在体内的吸收情况。     

Hollow‐fiber double‐solvent synergistic microextraction with high‐performance liquid chromatography for the determination of antitumor alkaloids in Coptis chinensis

A new hollow‐fiber double‐solvent synergistic microextraction method was proposed for the extraction and concentration of trace active compounds in traditional Chinese medicine. The main variables affecting the method were investigated and optimized. Under the optimized conditions, linearities were 0.01–10 μg/mL, detection limits were lower than 0.8 ng/mL, and interday, and intraday relative standard deviations were <9.20%. Furthermore, average recoveries ranged from 102.8 to 104.1%, and enrichment factors were 6–70 for the four alkaloids tested. The antitumor alkaloid group in Coptis chinensis was screened and identified by hollow‐fiber cell fishing with high‐performance liquid chromatography. The four alkaloids were then enriched and quantified by hollow‐fiber double‐solvent synergistic microextraction with high‐performance liquid chromatography. The mechanism of the proposed microextraction method was described, and results demonstrated that the approach was a simple and reliable sample‐preparation procedure. This method, as well as hollow‐fiber cell fishing combined with high‐performance liquid chromatography can be adopted to study the different characteristic effects of the multiple components and multiple targets of traditional Chinese medicine. The approach can also be used to conduct tailored quality control of the active compounds associated with therapeutic efficacy.     

Adsorbed hollow fiber‐based biological fingerprinting for the discovery of platelet aggregation inhibitors from Danshen–Honghua decoction

For lead compound discovery from natural products, hollow fiber cell fishing with chromatographic analysis is a newly developed method. In this study, an adsorbed hollow fiber‐based biological fingerprinting method was firstly developed to discover potential platelet aggregation inhibitors from Danshen–Honghua decoction. Platelets were seeded on the fiber and their survival rate was tested. Results indicated that more than 92% platelets survived during the whole operation process. Ranitidine and tirofiban were used as positive and negative control respectively to verify the reliability of the presented approach. The main variables such as amount of extract and stirring time that affect the adsorbed hollow fiber‐based biological fingerprinting process were optimized, and the repeatability of this method was also investigated. Finally, 12 potential active compounds in Danshen–Honghua decoction were successfully detected using the established approach and structures for nine of them were tentatively identified by liquid chromatography with mass spectrometry analysis. In addition, the in vitro platelet aggregation inhibition test was carried out for five of the nine hit compounds, and three active components, namely, lithospermic acid, salvianolic acid A, and salvianolic acid B were confirmed. These results proved that the proposed method could be an effective approach for screening platelet inhibitors from plant extracts.     

Hollow fiber membrane supported thin liquid film extraction for determination of trace phenoxy acid herbicides and phenols in environmental water samples

Traces of phenoxy acid herbicides and phenols were determined in environmental water samples by high performance liquid chromatography (HPLC) coupled to thin liquid film extraction (TLFE). A TLFE sampling device was prepared by dipping pieces of a polypropylene microporous hollow fiber membrane into dihexyl ether (containing 10% tri-n-octylphosphine oxide as carrier) for a few minutes to impregnate the pores of the hollow fiber wall. Extraction of analytes takes place from the outer aqueous phase into the immobilized solvent. After extraction the removal of the organic solvent was accomplished with a few µl of methanol which was used for HPLC analysis. Enrichment factors as high as 446 were obtained for the target compounds. The method provided detection limits as low as 0.4–1.2 µg L^?1, good repeatability (the RSD ranging from 2.1 to 6.3%, n?=?5) and a linear range from 2 to 200 µg L^?1 for the target compounds. Real sample analysis showed recoveries between 84.6% and 112% for all compounds investigated.     

Immobilized liposome chromatography to study drug-membrane interactions. Correlation with drug absorption in humans

For rapid screening of drug-membrane interactions and predicting drug absorption in vivo, unilamellar liposomes were stably immobilized in the pores of gel beads by avidin-biotin binding. Interactions of a diverse set of well-described drugs with the immobilized liposomal membranes were reflected by their elution profiles. The membrane partitioning coefficients (KLM) of the drugs were determined from the retention volumes. The drug retentions on egg phosphatidylcholine (EPC)-phosphatidylserine (PS)-cholesterol (chol) and EPC-PS-phosphatidylethanolamine (PE)-chol columns intended to mimic small intestine membranes were similar, although the positively-charged drugs were more strongly retarded on the negatively-charged liposomes than the negatively-charged drugs. The relationship between log KLM with the drug fraction absorbed in humans showed that the log KLM values obtained with unilamellar liposomes can be used to predict drug passive transcellular absorption, similarly to that previously shown for entrapped multilamellar liposomes. The immobilized liposome chromatography method should be useful for screening compounds at an early stage of the drug discovery process. The avidin-biotin immobilization of the liposomes prolongs the lifetime of the columns.     

Herbal medicine analysis by liquid chromatography/time-of-flight mass spectrometry

The fact that the effects of herbal medicines (HMs) are brought about by their chemical constituents has created a critical demand for powerful analytical tools performing the chemical analysis to assure their efficacy, safety and quality. Liquid chromatography coupled to mass spectrometry (LC–MS) is an excellent technique to analyze multi-components in complex herbal matrices. Due to its inherent characteristics of accurate mass measurements and high resolution, time-of-flight (TOF) MS is well-suited to this field, especially for qualitative applications. The purpose of this article is to provide an overview on the potential of TOF, including the hybrid quadrupole- and ion trap-TOF (QTOF and IT-TOF), hyphenated to LC for chemical analysis in HMs or HM-treated biological samples. The peculiarities of LC–(Q/IT)TOF-MS for the analysis of HMs are discussed first, including applied stationary phase, mobile-phase selection, accurate mass measurements, fragmentation and selectivity. The final section is devoted to describing the applicability of LC–(Q/IT)TOF-MS to routine analysis of multi-components, including target and non-target (unknown) compounds, in herbal samples, emphasizing both the advantages and limitations of this approach for qualitative and quantitative purposes. The potential and future trends of fast high-performance liquid chromatography (HPLC) (e.g. rapid resolution LC and ultra-performance LC) coupled to (Q)TOF-MS for chemical analysis of HMs are highlighted.     

Development of a novel 96‐well format for liquid–liquid microextraction and its application in the HPLC analysis of biological samples

A novel 96‐well liquid–liquid microextraction system combined with modern HPLC was developed and used for the simultaneous analysis of 96 biological samples. The system made use of hollow fibers, a 96‐well plate, and a plastic base with a center hole and a side hole. One end of the hollow fiber was sealed, while the other end was attached to one of the holes positioned at the center for the plastic base. The needle was inserted into the liquid from inside or outside of the hollow fiber through the center or the side holes, respectively. The system was tested with plasma samples containing three compounds, acidic indomethacin, neutral dexamethasone, and basic propafenone. Some parameters, such as the kind and dimension of hollow fiber, pH and salt concentration of the donor phase, the selection of organic solvent for the acceptor phase, and the extraction time were investigated. Under the optimization conditions, the Log D and drug concentration of indomethacin, dexamethasone, and propafenone in plasma and urine samples were analyzed. Then, the methodology was validated. The results demonstrated that ng/mL levels could be exactly and rapidly analyzed by our system, which was equipped with an auto‐injection sampler, making sample analysis more convenient.     

Screening of bioactive compounds in Rhizoma Polygoni Cuspidati with hepatocyte membranes by HPLC and LC–MS

A new method employing HPLC, LC–MS, and hepatocyte membranes for the screening of bioactive compounds in traditional Chinese medicines (TCMs) has been proposed. We hypothesized that exposure of the TCM extracts to hepatocyte membranes should decrease the concentration of membrane‐permeable compounds in the solution. Using this approach, the permeability of the compounds in Rhizoma Polygoni Cuspidati was investigated. By comparing chromatograms of samples prepared both before and after interaction with hepatocyte membranes, seven permeable compounds of Rhizoma Polygoni Cuspidati were identified. Additionally, it was found that piceid, resveratrol, emodin‐8‐β‐d‐glucoside, physcion‐8‐β‐d‐glucoside, aloe‐emodin, emodin, and physcion combined specifically with hepatocyte membranes, which might indicate a useful approach for revealing the antiatherosclerotic effects of Rhizoma Polygoni Cuspidati. Therefore, the proposed method could be a good approach to predict the potential bioactivities of multiple compounds in TCMs simultaneously. Based on the significance of these results, this method could be a novel approach for identifying potentially bioactive components in other TCMs.     

中空纤维膜液相微萃取-高效液相色谱法测定醋酸氯己定痔疮栓中的醋酸氯己定

建立了中空纤维膜液相微萃取-高效液相色谱（HPLC）测定醋酸氯己定痔疮栓中醋酸氯己定含量的方法。将样品用醋酸水溶液萃取5次,配成样品溶液,然后用装有正辛醇的中空纤维膜进行液相微萃取,萃取液用高效液相色谱测定。醋酸氯己定的质量浓度为0.5 ～ 16 mg/L时与其峰面积呈良好的线性关系,加样回收率大于98.0%,相对标准偏差低于4.0%。该方法样品处理简单、快速,灵敏度与不经过液相微萃取的HPLC方法相比提高了约24倍,醋酸氯己定痔疮栓中的其他物质对测定无干扰。     

Adamantyl ferrocene derivatives: Antioxidant abilities and effects on model lipid membranes

A series of homoannularly and heteroannularly substituted adamantyl ferrocene derivatives has been synthesized and their effects on membrane fluidity were investigated using liposomes as the membrane models. The liposome formulations of adamantyl ferrocene derivatives were characterized by using dynamic light scattering, differential scanning calorimetry and fluorescence anisotropy measurements. It was demonstrated that adamantyl ferrocene derivatives incorporated into the liposome significantly affect the structure of the lipid bilayer. The results of the study have revealed that adamantyl ferrocene derivatives, compounds 9 – 12, partition into the hydrophobic/hydrophilic interface of the membrane, causing a significant decrease in membrane fluidity. The antioxidant potential of synthesized compounds was assessed with DPPH method and it was shown that the examined compounds possess certain antioxidant activity.     

透析-高效液相色谱法在当归补血汤药效物质基础研究中的应用

将脂质体作为模拟生物膜,采用平衡透析与液相色谱联用技术,建立了一种研究中药成分与模拟生物具有相互作用的新方法。应用该方法对当归补血汤进行了分析,同时考察了模拟生物膜的浓度、pH值、缓冲系统和胆固醇的加入等因素对当归补血汤与模拟生物膜相互作用的影响。结果表明:当归补血汤中有7个组分与模拟生物膜相互作用明显;模拟生物膜的浓度影响最大,pH值对酸性组分阿魏酸的作用影响较大,其它因素的影响较小。该方法可用于预测药物在体内的吸收情况,进而研究中草药及复方的药效物质基础。     

Physicochemical aspects of the liposome-wool interaction in wool dyeing

Despite the promising application of liposomes in wool dyeing, little is known about the mechanism of liposome interactions with the wool fiber and dyestuffs. The kinetics of wool dyeing by two dyes, Acid Green 27 (hydrophobic) and Acid Green 25 (hydrophilic), were compared in three experimental protocols: (1) without liposomes, (2) in the presence of phosphatidylcholine (PC) liposomes, and (3) with wool previously treated with PC liposomes. Physicochemical interactions of liposomes with wool fibers were studied under experimental dyeing conditions with particular interest in the liposome affinity to the fiber surface and changes in the lipid composition of the wool fibers. The results obtained indicate that the presence of liposomes favors the retention of these two dyes in the dyeing bath, this effect being more pronounced in case of the hydrophobic dye. Furthermore, the liposome treatment is accompanied by substantial absorption of PC by wool fibers with simultaneous partial solubilization of their polar lipids (more evident at higher temperatures). This may result in structural modification of the cell membrane complex of wool fibers, which could account for a high level of the dye exhaustion observed at the end of the liposome dyeing process.