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Cent. Eur. J. Chem. • 10(3) • 2012 • 417-432 DOI: 10.2478/s11532-011-0141-4

Central European Journal of Chemistry

Developments of instruments and methods related with high-speed countercurrent chromatography and their applications in research of natural medicines

Review Article

Shun Yao, Yu Cao, Chun-mei Jia, Yan Wang, Hang Song*

Department of Pharmaceutical and Biological Engineering, Sichuan University, Chengdu 610065, R. P. China

Received 7 July 2011; Accepted 25 September 2011

Abstract: High-speed countercurrent chromatography (HSCCC) is a liquid-liquid separation chromatographic technique which uses no solid supporting medium. During its rapid development in the last 30 years, great progress has been made in the instrumentation, the basic study and application of HSCCC. It has significant advantages over other instrumental separation techniques, in its high efficiency and continuous processing capability. In recent years, HSCCC has been widely used in research and development of natural medicines and functional foods, including preparative separation, fingerprint analysis and screening of bioactive constituents. A review of the technique is presented in this paper. Keywords: HSCCC • Instrument • Method • Application • Natural medicines © Versita Sp. z o.o.

1. Introduction such as rotation locular countercurrent chromatography (RLCCC), droplet countercurrent chromatography High-speed counte+r-current chromatography (DCCC), and centrifugal partition chromatography (CPC). (HSCCC) is one form of support-free all-liquid partition Recent years have seen great advances and blossoming chromatography, which was originally invented by Ito [1] of this kind of CCC technology, with increasing numbers in order to improve the existing techniques of counter- of studies reported about the basic theory, instrumental current chromatography (CCC). and relies on the improvement, method development, skills summary and repeated partition of a sample between the two phases various applications of HSCCC (see Fig. 1). HSCCC has of an immiscible solvent system. The basis of all of these undoubtedly become one of the mainstream techniques techniques lies in the well known process of liquid-liquid in separation science. extraction. There are many potential variations (especially In the last decade, HSCCC has been used widely for column geometries and rotation axes) in the design of for preparative separation, fingerprint analysis, and HSCCC instruments, which allows much greater scope screening of a wide variety of bioactive components for its development and application than is available with from natural medicines and functional foods using many early CCC equipment. As no solid stationary phase (SP) different aqueous-organic two-phase solvent systems exists, CCC eliminates irreversible adsorption of samples [3-5]. It is clear that this all-liquid technique is an attractive onto the support matrix and the tailing of solute peaks proposition for the study of these natural products, due seen with traditional chromatographic columns [2]. With to its ability to handle complex polar or non-polar crude its new hydrodynamic equilibrium system, HSCCC can material extracts with sample quantities from milligrams provide more efficient mixing and excellent separation to tens of grams. This review will offer a brief overview of efficiency compared with the hydrostatic CCC systems, HSCCC and survey the recently published applications

* E-mail: [email protected] 417 Developments of instruments and methods related with high-speed countercurrent chromatography and their applications in research of natural medicines

(6) general improvement of performance. Generally, there are major relationships between instrumental parameters and performance which are summarized in Fig. 2. Different types of HSCCC instruments are continually being developed and and are continuously applied in daily separation tasks. These instruments differ most notably in the geometrical design of column, the relative angle of the two axes of rotation (e.g. J-type, X-type, L-type, I-type and their hybrids), the ratio of central to

Figure 1. The numbers of published articles about HSCCC per planetary radius (β-value) and capacity of coiled tube year (2000-2010) on account of Scifinder Scholar data (analytical type, semi-preparative type or preparative type). Among them, the most commercially successful in various subject areas. For each area an overview of has been the J-type apparatus, in which the two axes the progress of development will be provided, and some are parallel to each other and the rotation and revolution important applications will be given. The references proceed in the same direction and same angular velocity. provided here represent only a fraction of the current But it has a lower capability for retention of the stationary literature available. Additional references can be found phase in hydrophilic solvent systems. The X-axis CCC in the articles cited. system (L-X type, a cross-axis system) was developed to raise the level of SP retention in these conditions. Ito et al. also invented the multiple spiral disk assembly 2. Development and innovation [6-11] for J-type HSCCC to resolve the problem. All of instruments and techniques kinds of channels in spiral disks were designed and investigated, even including the use of glass beads to As mentioned above, HSCCC instruments are a modern interrupt the laminar flow and enhance mixing of the and popular type of counter-current chromatography two phases. Moreover, Guan and his colleagues [12,13] apparatus. Many prototype HSCCC instruments have have constructed toroidal columns (length 26–140 m, been developed by Ito and other researchers, and column volume 51–280 mL, bore size 1.6 mm) suitable continuing innovation and improvement have been for mounting onto the commercially available preparative proceeding in the labs of National Institutes of Health CCC apparatus to provide pseudo-hydrostatic CCC, (USA), Brunel University (UK), Lausanne and Geneva in which much improved retention of SP and bi-phase universities (CH), Lyon University (FR), Kinjogakuen mixing were obtained. University (JPN), Technical University of Braunschweig In order to perform high-throughput fractionation (DE) and Beijing Institute of New Technology Application of bioactive natural products, Wu and his coworkers (CN), etc. The key and characteristic component of designed and fabricated a multi-channel counter-current these machines mainly is helically coiled inert tubing chromatograph [14]. It has multiple independent CCC (equivalent to a separation column) around a cylindrical channels and each channel connects independent column holder with its own axis. A planetary gear and separation column(s) with parallel flow tubes in order stationary sun gear coupling is employed to produce to achieve two or more independent chromatographic the double axis gyratory motion (planetary motion) of separations simultaneously. All separation columns the coils of the spiral tube together with the contained undergo identical synchronous J-type planetary motions biphasic fluid system in the whole separation process. (shown in Fig. 3a). This group also developed various The design seeks to achieve efficient retention of high-throughput CCC fractionation methods have stationary phase under a variable gravity field (G-force been developed using a combination of the new three- field). The sample solution and solvent system(s) are channel CCC apparatus and conventional parallel introduced from the inlet of the spiral tube and separated chromatographic devices for separation of three natural fractions elute from the outlet. In recent years, most products in the crude extract at the gram level. Their effort has been devoted to (1) improving the retention research proved that in spite of the solvent balancing of stationary phase, (2) increasing the sample requirement and the lower resolution caused by the loading capacity, (3) speeding up the separation shorter CCC columns, the multi-channel CCC was process, (4) providing more choices to deal with still very useful for high-throughput fractionation of different fluid mechanics behaviors, (5) interfacing natural products for drug discovery. Then Wu’s team to other separation and analytical instruments and invented a unique cone-shaped holder shaft (Fig. 3b)

418 S. Yao et al.

Figure 2. Major relationships between instrumental parameters and performance of HSCCC. for their three-channel CCC with the coiled column throughput and preparative quantities up to gram mounted coaxially around [15]. This special design was level be met at the same time? In order to overcome expected to strengthen the centrifugal force field for the this well-known bottleneck, high performance counter- efficient retention of stationary liquid phase, resulting in current chromatography (HPCCC, easily confused improved resolution. It could easily be generalized for with HSCCC) was developed in the early 2000 s. incorporation in different assemblies. With the same The chemists can solve their separation problems, with aim of improved fractionation efficiency, dual counter- cycle times of minutes or 10 s of minutes, similar to those current chromatography (DuCCC) was developed of preparative high performance liquid chromatography to change the one-way flow of mobile phase (MP) in (prep-HPLC). The maximum preparative amount HSCCC into a two-way flow of MP and SP. For the of 240 g also greatly exceeds those of conventional same planetary motion, the components of hardware HSCCC (80–100 g) and HPLC (milligrams). Moreover, an for DuCCC are very similar to those for HSCCC; with extra advantage of HPCCC is found when polar biphasic only minor modifications needed in the sample injection solvent systems are adopted, because it can produce arrangements, and in the inlet and outlet parts. A centre relatively high SP retention across the complete range tap is fitted in the middle of the coil for sample introduction of liquid-liquid systems. Low-speed/slow rotary counter- so that the two phases can be simultaneously eluted current chromatography (LSRCC or SRCCC) [17,18] through the column in opposite directions while the is another technique for large preparative quantities sample solution is continuously fed from the centre tap. (hundreds of grams) and it can easily be scaled up for There is no obvious loss of the stationary phase after industrial production through volume enlargement of the multiple sample injections and high separation efficiency convoluted multilayer coil It is commonly operated at a can be achieved for the complex constituents with large very low rotation speed (less than 100 rpm, generally differences in polarity, which is nearly impossible in 10–50 rpm) and long separation time of a few days ordinary HSCCC [16]. restricts its application to some extent. Generally, HSCCC users have to employ low MP Besides the enlargement of the column size for large- flow rate to maintain high SP retention, which means scale preparation, some interest and effort has also that hundreds of minutes are needed in a cycle of been directed at decreasing the column size (including separation. How can the requirements of high speed volume and i.d.) for analytical purposes and for basic

419 Developments of instruments and methods related with high-speed countercurrent chromatography and their applications in research of natural medicines

Figure 3. Three-channel CCC apparatus (a) and its modification (b).

study. A small-volume column allows for reliable and fast with minimum overlapping that elute according to their determination of solute partition coefficients, with high pKa values and hydrophobicities [21]. The pH value of peak resolution and efficiency. Thus it is very suitable each eluted fraction can be measured with a portable pH for such tasks as separation condition exploration and meter, so that effluents without a chromophore can also quantitative analysis. If narrow-bore tubing is used, be monitored. Compared with common HSCCC, it can solvent interactions with tubing wall will increase, and provide more than a 10-fold increase in sample loading so higher centrifugal force and higher rotating speed capacity, with high concentration of fractions and minor (1500-2000 rpm) are required for adequate stationary impurities. Ion-pair HSCCC is another technique used phase retention. Berthod et al. [19] compared the to separate those compounds with ionization tendency performance of a new commercial hydrodynamic 18 mL and a series of related applications have been reported column made with narrow-bore 0.8 mm Teflon tubing [22-25]. Chemical affinity to organic stationary CCC with that of older hydrodynamic CCC columns and a solvent phases leading to the retention of highly polar similar 19 mL 1.6 mm column. The results proved the constituents could be significantly improved by using fact that the two older CCC columns could not compete ion-pair forming reagents. This method has proved to with the new small i.d. column for peak resolution, but be extremely effective for fractionating the complex the 19 mL column with 1.6 mm bore tubing can be useful crude extract of natural products into narrower ‘polarity- when fast results are desired without a need for optimum windows’. resolution. A series of studies was conducted to evaluate Many analytical instruments can be interfaced performance of coiled columns with i.d.s ranging from to HSCCC separation systems online [26], such as 0.10 to 0.55 mm in analytical HSCCC [20]. Comparison mass spectrum (MS), Ultraviolet-Visible (UV), Fourier of the results indicated that multilayer coils of 0.55 mm Transform Infrared (FT-IR), Optical Rotation Dispersion and 0.30 mm i.d. could provide satisfactory retention (ORD) spectrometers and Evaporative Light-Scattering of the SP while the 0.10 mm i.d. column failed to yield Detector (ELSD), pH meter, etc. Their satisfactory reproducible retention. The best partition efficiency compatibility with HSCCC makes it very easy to develop was obtained from the 0.30 mm i.d. multilayer coil many new analytical methods. Coupling CCC to MS with a 6 mL capacity which produced theoretical plate with various types of ion source and mass analyzer numbers ranging from 5500 to 10500. integrates the advantages of HSCCC with the low New techniques are widening the range of detection limit and identification capability of MS. CCC- applications for HSCCC; one of them is pH-zone- MS thus becomes a helpful complementary method refining counter-current chromatography, which can to LC-MS [27,28]. The many on-line technical and achieve the separation of ionic or ionizable compounds hardware problems have gradually been resolved and such as organic acids and bases into a succession of several applications have been reported in the field of highly-concentrated rectangular peaks fused together natural medicines [29-36]. In the interface arrangement,

420 S. Yao et al.

Tee-split is employed to reduce the effluent velocity and and monographs [26,49-54] dealing with the selection, the amount of sample introduced into the MS. To detect development and optimization of solvent systems, the separation in analytical samples of components including Ito’s famous eighteen golden rules [50], that without UV absorption, ELSD is interfaced to HSCCC in provide guidance for HSCCC users. HSCCC has many more and more studies [37]. more options for solvent selection and elution mode than It has also proved useful and possible to combine HPLC, which is both an obvious advantage and a great separation apparatus with HSCCC on line. With a simple challenge for researchers (especially for beginners). adaptation of the fittings of a Linomat C device, Diallo There are three questions that must be addressed: (1) et al. [38] realized the first direct interfacing of HSCCC what factors in the solvent system will affect separation with thin-layer chromatography (TLC) in an efficient selectivity and retention of SP? (2) Whether the target mode for on-line monitoring of the column effluents. Two compounds are known or not, and how should the dimensional (2D) chromatography between HSCCC composition of solvent system be determined in each and HPLC [39] (detected with UV/MS/MSn), GC [40] case? (3) which phase should be chosen as MP or SP: or HSCCC [41-44] can also be easily achieved using the upper (lighter) or lower (heavier) phase? a switch valve (see Fig. 4) to determine the purities or Separation selectivity is mainly correlated with the structures of target constituents online or offline. Many type of solvent system. Researchers have adopted some modern extraction techniques have also been combined physical parameters to describe, compare and classify with HSCCC, such as supercritical fluid extraction [45] different solvent systems, such as the Hilderbrand and microwave-assisted extraction [46]. Supercritical solubility parameter (δ), Snyder solvent strength and fluid and microwave can effectively improve the adsorption parameters (ε0), Rohrschneider-Snyder extraction yield of desired materials for further HSCCC polarity parameters (P’), Reichardt polarity parameter preparation, with better performance in the mass transfer (ET), etc. Among them ET is known as a simpler and process than with conventional extraction conditions. more useful parameter, based on direct UV analysis. A series of studies and inventions have addressed UNIversal Functional Activity Coefficient (UNIFAC) actual and potential problems with hardware and and UNIversal QUAasi Chemical (UNIQUAC) and software for HSCCC. It is widely recognized among Non-Random Two-Liquid (NRTL) models can be used users that higher rotation speeds lead to greater noise to predict the biphasic composition of a multi-solvent levels. With high-speed rotation, powerful circular airflow system. Density, viscosity and interfacial tension of can arise naturally in the chamber of the apparatus, solvents are the three major factors that determine the increasing the noise. There is then an increased settling time of the two-phase solvent system, which probability of faults due to imbalance of the different is recognised as the barometer for the retention of components, and of material impairment due to rising SP. It has been suggested that the ideal settling time temperature during a longer separation run. Wu [47] should be less than 20 seconds, and usually over 50% made a fairing on the major rotating structure to block air SP retention can be obtained with the proper flow rate motion and protect the internal components. To address [26,50]. the problem of insufficient control functions, Shi [48] In their definitive and instructive studies, Ito [50] and developed an automated control system to integrate Oka [55] designed various solvent screening systems the control and detection of rotation speed, temperature and systematic search methods for unknown samples and input/output (I/O) channel, using a programmable on the basis of the Hexane–Ethyl acetate–1-Butanol– logic controller (PLC) and a single-chip microcomputer Methanol–Water (HEBMW) system. If the sample is a (SCM). This system can prevent burning caused by methanol extract (equivalent to polar constituents), it uncontrolled temperature rise with low boiling point is suggested the search may start at 1-butanol–water solvents, and automate collection of different fractions (1:1). Whereas if the sample is an ethyl acetate extract by switching I/O channels. (relatively hydrophobic constituents), the search may start at hexane–ethyl acetate–methanol–water (1:1:1:1). Organic modifiers or aqueous modifiers can 3. Selection and optimization of be added into the solvent system to modify the K value solvent systems of acid or basic compounds, which commonly include triethylamine, hydrochloric acid, trifluoroacetic acid, An optimal solvent system must not only generate buffer salts, formic acid and even ionic liquids [56]. enough difference in partition coefficients for target Other screening protocols have also been explored compounds in two phases, but also ensure adequate to minimize the experimental effort required to find an retention of stationary phase. There are many reviews optimal solvent system. These include the Berthod

421 Developments of instruments and methods related with high-speed countercurrent chromatography and their applications in research of natural medicines

Figure 4. Schematic diagram of the 2D HSCCC system with a six port injection valve and a six port switching valve, the first separation in HSCCC 1 (position A) and the further separation in HSCCC 2 (position B) [41].

method, the Abbott-Kleiman method, the Glyme system, solvent systems, respectively. ‘Haloalkane’ refers to

the HBAW method, the ARIZONA method, the GUESS CH2Cl2, CHCl3 and CCl4; ‘alcohol’ includes MeOH, EtOH, method, inter alia [57-61]. A quantum chemical method BuOH, etc; and ‘alkane’ means C5–C8 alkanes and combined with statistical thermodynamics (conductor- petroleum ether. Fig. 5c shows the percent of different like screening model for real solvents, COSMO-RS) was numbers of diverse solvent systems used in separation recently devised by Minceva and co-workers to predict of a certain natural extract in these reports. Besides the the partition coefficients [62], This has potential as a step conventional organic solvents in Fig. 5, supercritical fluid

towards the development of a systematic method for the CO2 [63] and surfactant-containing refrigerant R134a selection of solvent systems based on thermodynamics. (1,1,1,2-tetrafluoroethane) [64] have also been used Ternary diagrams are extensively used to map every in solvent systems as mobile phase for their unusual possible combination of 3–4 member solvent system properties. The former has the advantages of high families. compressibility, liquid-like density, high diffusivity, When working with known target compounds, it is low viscosity, low surface tension and environmental necessary firstly to comprehensively search existing friendliness, and the latter can achieve better SP references and reported solvent systems for all of the retention in polar systems than conventional solvents. CCC and CCD (counter-current distribution) techniques Both of them are gases at atmospheric pressure and involving separation of similar compounds. From the room temperature, so the separated products can be experimental results with solvent systems reported in easily recovered as soon as they are eluted from the references, users usually need to make appropriate column. adjustments to take account of the practical situation. In HSCCC, either phase (upper or lower) can be Fig. 5 provides data compiled from existing articles used as the mobile phase to provide the K values of the about using HSCCC in separation of natural medicines. targets in a proper range. Once the MP of the binary Parts (a) and (b) of the figure display the percent of system is chosen, it may be necessary to interchange systems with different numbers of solvents and the the inlet and outlet tubing. If K ≤ 1.0 and the lower percent of systems with various compositions in total phase is used as MP, the related compound might

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Figure 5. The percent data about the using condition of HSCCC solvent systems in study of natural medicines. be eluted in a shorter time without perfect resolution natural medicines and functional foods. It enables from other coexisting constituents. The decision about more “dirty” samples to be handled directly and the which phase will be the mobile phase is less important strict pretreatment procedure need not be repeated, if K = 1, because in this case the retention time and so long as there are not obvious visible particles in volume of the target compounds will be very similar in the sample solution. When strong adsorption on a either mode. The lower phase may often be employed solid support is not a possibility, the loss of separated as the mobile phase, because the system can provide products can be reduced to the lowest level, which is more stable retention of the stationary phase. For similar very benefit for the discovery of trace novel compounds considerations, with SP a lower (heavier) mobile phase with special activities. No more expensive commercial is suggested for introduction through the head toward columns are needed; the requirements of solvents for the tail, with the upper (lighter) mobile phase moving in HSCCC are also very low; and there are fewer precisely the opposite direction engineered instrumental components needed, ensuring that the experimental and maintenance costs are very low. Without the effects of differences in commercial .4. Applications of HSCCC for natural LC columns, the HSCCC methods that have been medicines developed are more easily copied, more reproducible, and easier to produce in different workshops than those Though HSCCC is not currently as widely used as LC and for HPLC. Furthermore, the separation mechanism of Capillary Electrophoresis (CE), its advantages cannot HSCCC is completely different from HPLC and CE, so be neglected for preparative separation, fingerprint it could become a useful supplement to them in daily analysis and screening of bioactive constituents from separation tasks.

423 Developments of instruments and methods related with high-speed countercurrent chromatography and their applications in research of natural medicines

4.1. Application to preparative separations not achieve complete separation, additional instruments So far, several hundred natural medicines have been and techniques (HSCCC, prep-LC, prep-TLC, etc.) may studied and more than 1000 compounds have been be used to perform further online or offline resolution. In successfully prepared by HSCCC for both commercial order to separate bioactive compounds in a well-targeted and research purposes. The major classes of natural manner, a bioassay-guided fractionation method has bioactive constituents include alkaloids, , been employed to link the purification and antimicrobial flavonoids, phenolics, , phenylpropanoids, activity of constituents in green tea extract quinones, stilbenes, , peptides and organic acids. [129]. Among them the more noteworthy representatives are HSCCC has had perfect performance in the [65], taxol [66], vincamine [67], silycristin separation of isomers of natural active compounds [68], caffeine [69], strychnine [70], resveratrol [71], with its special liquid-liquid mechanism. The resolution [72], daidzin [73], tanshinone IIA [74,75], of barbinervic acid and its epimer (rotungenic acid) in salvianolic acid B [76], huperzine A [77], [78], the extract from leafs of could not easily and ginkgolides [79]. Table 1 summarizes the popular be achieved by common column chromatography, so a solvent systems in separation of various bioactive single step HSCCC method was developed to separate compounds. Pharmaceutical chemists are usually them together with the other two pentacyclic concerned with how to prepare several important lead acids, using a solvent system composed of n-hexane– compounds with HSCCC for potential new drugs, and ethyl acetate–methanol–water (3:6:4:2, v/v/v/v) [130]. phytochemists tend to use HSCCC in comprehensive As a result, 49.6 mg of barbinervic acid (3a,19a,24- investigations of the chemical composition of plant trihydroxy-urs-12-en-28-oic acid) and its epimer, extracts to supplement the most favourable liquid-solid 32.2 mg of rotungenic acid (3b,19a,24-trihydroxy- chromatography. Kong and his team introduced the urs-12-en-28-oic acid) were obtained from 750 mg concept of systematic separation into the application of the extract. Successful resolution among isomers of HSCCC for traditional Chinese medicines (TCMs) of dihydroxybenzoic acid [131], catechins research. They separated the crude extracts with simple [132], (quadrangularin A and liquid-liquid partition into several fractions with different parthenocissin A) [133], gambogic acid [134] and polarity, and then adopted well-directed HSCCC methods tetrahydropalmatine [135] have also been reported. to further separate the constituents involved in each All of the above studies provide a sufficient fraction according to their polarity features [125]. For demonstration that HSCCC is an effective method for systematic separation applications, general procedures the preparative separation of natural products and their and recommended solvent systems are summarized in isomers. Fig. 6. Natural medicines contain hundreds, or even 4.2. Analytical and quality control applications thousands, of constituents. Some compounds have Analytical HSCCC can be used either for the preliminary similar retention behaviors on HSCCC, arising from selection of a suitable solvent system or for rapid analysis their similar structures. Conventional elution conditions of chemical composition in extracts and fractions. It has could not always achieve perfect resolution with a single proved to be efficient in the analysis of natural active solvent system and isocratic elution mode. Special elution pigments such as anthocyanins, , modes are helpful in solving such problems and saving and chlorophyll-related pigments [136]. Especially separation time. Gradient elution is the most popular when it is interfaced to MS [82], analytical HSCCC choice, with 2 or 3 systems containing the same solvents has the capability of providing perfect resolution and in different percent composition, and gradient formation identification for tracking active ingredients of natural can be either pump-based or valve-based. In pump- medicines in a quick, efficient, and high-recovery based style, two or more pumps are needed to provide manner. In Chen’s researches [33,137], two analytical a steady linear change in MP solvent composition. In HSCCC instruments were successfully employed to valve-based style, a switch valve is adopted to achieve separate and identify flavonoids from an ethyl acetate step-wise gradient elution [126] from one MP system to extract of the seeds of Oroxylum indicum. These another. Obviously, the latter is less costly, with only a instruments were interfaced directly with electrospray single pump, but it results in poorer SP retention than the ionization (ESI) and atmospheric pressure chemical former. Apart from gradient elution, special elution modes ionization (APCI) mass spectrometry. Hexane–ethyl involving gradient flow rate [127], column temperature acetate–methanol–water solvent systems with different [78] and pH value of MP [128] have been developed to ratios were chosen and optimized for the best resolution. address similar problems. If a HSCCC instrument can The online data of APCI-MS and APCI-MS-MS provided

424 S. Yao et al.

useful information for the identification of the respective Many analytical tasks have also been completed compounds. Besides ESI and APCI-MS, fast atom with the involvement of semi-preparative HSCCC in bombardment (FAB)-MS and thermospray (TSP)-MS sample preparation. There have been some related has been successfully coupled with HSCCC and applied studies about the comparison of analytical performance to the analyses of alkaloids, triterponic acids, and ligans between HSCCC and HPLC [139], HSCCC and CE [30,82,138]. [140], or HSCCC and TLC [141], especially about

Table 1. Summary of most popular solvent systems used in separation of typical bioactive compounds.

No. Structural Solvent systems Typical compounds Major functions types

n-Hexane–CHCl3–MeOH–H2O andrographolide, neoandrographolide [80]

n-Hexane–EtOAc–MeOH–H2O evodiamine, rutaecarpine [81] anti-cancer, anti-bacterial, n-Hexane–EtOH–H2O vincamine and vincine [82] anti- inflammatory, smooth muscle 1 Alkaloids palmatine, berberine, epiberberine relaxation, cardiovascular CHCl3–MeOH–HCl and coptisine [83] and central nervous system functions gentianine, gentianadine,[84] caffeine, CHCl –MeOH–Na H PO solution 3 2 2 3 theophylline [69]

n-BuOH–Me2CO–H2O venecurine, panarine [85,86]

n-Hexane–n-BuOH–MeOH–H2O icariin [87]

n-Hexane–EtOAc–n-BuOH–MeOH–HAc–H2O ginkgo flavonol [88]

n-Hexane–EtOAc–MeOH–H2O casticin [89] anti-oxidization, enzyme

CHCl3–MeOH–H2O quercetin, rutin [90] inhibitor, immunity, 2 Flavonoids hormone-like function, CHCl –MeOH–n-BuOH–H O soybean flavonoids [91] antivirus, anti-cancer and 3 2 neuron protection

EtOAc–n-BuOH–H2O puerarin, daidzin [73] apigenin-7-O-neohesperidoside, luteolin-7-O-glucoside, EtOAc–EtOH–HAc–H O 2 apigenin-7-O-glucoside and kaempferol-7-O-glucoside [92]

n-Hexane–EtOAc–MeOH–H2O emodin, chrysophanol, rhein [93] anti-bacterial,

n-Hexane–EtOH–H2O tanshinones [74,75] hemostatic, 3 Quinones anti- inflammatory, purgative and Light petroleum–EtOAc–MeOH–H2O emodin, physcion [94] cardiovascular activities

CHCl3–MeOH–H2O aloin and aloe-emodin [95]

n-Hexane–EtOH–H2O 9’-cis-neoxanthin [96]

anti-cancer, anti- n-Hexane–EtOAc–MeOH–H O ursolic acid [97] pacilitaxol [66] 2 inflammatory, anti- 4 Terpenoids bacterial, anti-viral and CHCl3–MeOH–H2O bilobalide [98] immunity activities geniposide, cis-crocin EtOAc–n-BuOH–H O 2 and trans-crocin [99]

n-Hexane–EtOAc–MeOH–H2O psoralen and isopsoralen [100]

mperatorin, isoimperatorin anti-coagulation, anti- n-Hexane–MeOH/EtOH–H O 2 and oxypeucedanine [101] bacterial, anti-viral, 5 Phenylpropanoids muscle relaxation and Light petroleum–EtOAc–MeOH–H2O notopterol and isoimperatorin [102] liver protection 7-hydroxy coumarin, 7-methoxycoumarin, CHCl –MeOH–H O 3 2 7 –hydroxyl-6-methoxyl coumarin [103]

425 Developments of instruments and methods related with high-speed countercurrent chromatography and their applications in research of natural medicines

ContinuedTable 1. Summary of most popular solvent systems used in separation of typical bioactive compounds.

Structural No. Solvent systems Typical compounds Major functions types

n-Hexane–EtOAc–MeOH–H2O theaflavins [104]

n-Hexane–EtOAc–H2O catechins [105]

anti-oxidization, anti- MtBE-n–BuOH–MeOH/ACN–H2O procyandins [106] bacterial, anti-viral and 6 Polyphenols cerebro-cardiac vascular EtOAc–MeOH–H O gallic acid [107] 2 activities

EtOAc–n-BuOH–H2O salidroside [108]

CHCl3–MeOH–H2O resveratrol [71]

n-Hexane–EtOAc–MeOH–H2O phillyrin [109]

n-Hexane–n-BuOH–H2O ginsenosides [72]

MtBE–n-BuOH–MeOH/ACN–H2O goyaglycosides [110] anti-cancer, liver CHCl –MeOH–H O aloin [111] protection, anti- Polysaccharides 3 2 coagulation, anti- 7 and glycosides viral, improving CHCl –MeOH– BuOH–H O -saponins [112] 3 n- 2 microcirculation and immunity activities trans-3,5,4’-trihydroxystilbene 4’-O-β-D- EtOAc–MeOH/EtOH–H O 2 glucopyranoside [113]

EtOAc–n-BuOH–H2O geniposide [114]

n-BuOH–MeOH–HAc clemastanin B, indigoticoside A [115]

n-Hexane–EtOAc–MeOH–H2O salvianolic acid B [76]

anti-bacterial, digestive, 8 Carboxylic acids n-Heptane–ACN–HAc–MeOH free fatty acids [116] immunity and central nervous system functions

n-BuOH–HAc–H2O chlorogenic acid [117]

n-Hexane–EtOAc–ACN α-spinasterol [118]

n-Heptane–ACN–EtOAc β-sitosterol [119] anti-allergic, anti-shock, 9 Sterols and lipids anti- inflammatory, anti- bacterial activities n-Hexane–EtOH–H2O glycolipids [120] neutral phospholipids and n-Hexane–EtOAc–MeOH/EtOH–H O 2 glycoglycerolipids [121] cuminaldehyde and p-menta-1,4-dien-7-al n-Hexane–MeOH/EtOH–H O 2 [122] anti-spasmodic, analgesic, anti-bacterial, coriandrin, dihydrocoriandrin, coriandrone 10 Essential oils n-Hexane–EtOAc–MeOH–H O anti-oxidant, anti- 2 A and B [123] depressant, disinfectant germacrone and curdione functions Light petroleum–EtOH–Et O–H O 2 2 [124]

MeOH = methanol, EtOAc = acetidin, CHCl3 = chloroform, BuOH = butanol, HAc = acetic acid, ACN = acetonitrile, MtBE = methyl-tert-butyl ether, Et2O = diethyl ether. The same solvent systems are in the same colorful unit grids.

their application in fingerprinting of TCMs [142-144]. the studies cited above [142], 12 quinone components The components of natural medicines may be influenced were well separated within 13 hours by HSCCC, with by many environmental factors such as soil, climate, good correspondence and precision. The relative and growth stage. Fingerprinting can be employed to amounts of each component varied greatly in various provide a relatively complete picture for their quality samples from different regions. The relative standard control as an important and effective means. In one of deviation of the HSCCC retention time was less than

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Figure 6. General procedure and recommendable solvent systems in HSCCC systematic separation. 3%, which satisfied the requirement of the national of natural products in order to accelerate the drug standard reference index. The results of these related discovery process for high-throughput screening studies showed that HSCCC had the best performance (HTS). The integration of a large number of automated for fingerprinting of tanshinones in roots of Salvia chromatographic and biological screening techniques miltiorrhiza Bunge (Dansheng, shown in Fig. 7). It had with multiple channels provides an efficient platform for certain advantages in scales of crude samples and non- drug discovery and succeeds in quick identification of common peak area compared with HPLC; meanwhile it the active constituents. Han et al. [145] developed a could provide more chemical information (peak number) high throughput screening method based on analytical than CE. These comparisons provided preliminarily HSCCC and novel fluorescence-based biosensor indications that HSCCC could be a feasible and cost detection, which was proved to be very sensitive in effective method in the development of analysis and detecting apoptosis induced by various known anticancer quality control for TCMs but further studies are required drugs. Guided by apoptosis detection, the active fraction for other aspects of methodology: detection limit (LOD), was tracked and purified continuously using HSCCC. range of linearity, repeatability, application range, etc. As a result, the most potent apoptosis inducer was discovered by analytical HSCCC equipped with a 16 mL 4.3. High throughput screening mini-coil column, using less than 50 mL diphase solvent, High-throughput methods were applied to the from about 50 mg active fraction from Isodon eriocalyx. analysis, production, and characterization of libraries It finally was identified as eriocalyxin B.

427 Developments of instruments and methods related with high-speed countercurrent chromatography and their applications in research of natural medicines

Figure 7. Fingerprinting chromatograms of HSCCC (a, peaks (7) cryptotanshinone; (8) tanshinone I; (11) tanshinone IIA), HPLC (b, peaks (1) cryptotanshinone; (3) tanshinone IIA; (5) tanshinone I), CE (c, peaks (1) cryptotanshinone; (3) tanshinone IIA; (5) tanshinone I), and TLC scanning (d) for crude samples of Dansheng from Hebei province, China [142-144].

Wu and his coworkers [146] established a high- and 100 mg extract was separated into six fractions in throughput CCC fractionation method for natural products only 100 min with a 140 mL-capacity CCC instrument. by combining their new three-channel CCC apparatus Each fraction was subjected to antioxidant activity assay with conventional parallel chromatographic devices. Its and fifty compounds were successfully characterized by performance has been displayed on the fractionation LC/MS/MS analysis. of ethyl acetate extracts of three herbs (Solidago canadensis, Suillus placidus, and Trichosanthes kirilowii) 5. Conclusions in the course of screening the antitumor candidates. The results obtained indicated that multi-channel CCC was In the last 30 years, the technique of HSCCC has made very useful for high-throughput fractionation of natural a great contribution to the development of effective products for drug discovery in spite of the solvent chromatographic methods. It can simplify the processing balancing requirement and the lower resolution of the procedure and speed up the throughput screening for shorter CCC columns. An effective method has been researchers in the field of natural medicines and functional developed for rapid screening of antioxidant phenolic foods. In view of its important position in the field of compounds in Chinese Rhubarb [147]. An integrated separation science, many reviews and monographs three-coil CCC column (40 mL each coil) was used to have been published about HSCCC instruments, accomplish optimization of the biphasic liquid system, method development and related applications. Of these,

428 S. Yao et al.

references [2-5,26,49-55,57,61,136] provide particularly cannot prevent HSCCC from moving forward. At useful guidance, and attract the most attention. Some present, more and more HSCCC instruments are being recent articles [148-153] are also recommended to deployed by scientists in related fields as a major item readers. The geographical location of all the HSCCC of laboratory equipment for handling complex natural authors is relatively well balanced between USA, Asia samples. Compared with HPLC and CE, HSCCC has (mainly China and Japan) and Europe. Chromatography more diverse possibilities, with attractive prospects for journals are the chosen and appropriate venue for half of its development. New basic studies and modifications the published articles [3]. The separation and purification to apparatus are stimulating new applications over a of natural compounds has become the dominant theme wider range and are pushing these applications forward in HSCCC applications at present. relentlessly. It is worth mentioning that HSCCC has Admittedly, the shortcomings of HSCCC are great potential and may soon achieve prevalent usage as obvious as its advantages, such as the difficult in quantitative analysis and quality control for natural exploration required to find ideal solvent systems, a medicines. With time and experience this technique can less than ideal number of theoretical plates, and the be expected to provide new technologies and advances, slow equilibration process between the two phases and deliver their promise of potential benefits in possible before sample can be injected. Moreover, there is a applications. risk of losing valuable compounds. But these problems

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