REVIEWS Chinese Journal of Chemical Engineering, 21(9) 937 952 (2013) DOI: 10.1016/S1004-9541(13)60560-1

Recent Advances in Separation of Bioactive Natural Products*

REN Qilong ( )**, XING Huabin ( ), BAO Zongbi ( ), SU Baogen ( ), YANG Qiwei ( ), YANG Yiwen ( ) and ZHANG Zhiguo ( ) Key Laboratory of Biomass Chemical Engineering of Ministry of Education, Department of Chemical and Biologi-cal Engineering, Zhejiang University, Hangzhou 310027, China

Abstract Bioactive natural products are a main source of new drugs, functional foods and food additives. The separation of bioactive natural products plays an important role in transformation and use of biomass. The isolation and purification of bioactive principle from a complex matrix is often inherent bottleneck for the utilization of natural products, so a series of extraction and separation techniques have been developed. This review covers recent advances in the separation of bioactive natural products with an emphasis on their solubility and diffusion coeffi-cients, recent extraction techniques and isolation techniques. This overview of recent technological advances, dis-cussion of pertinent problems and prospect of current methodologies in the separation of bioactive natural products may provide a driving force for development of novel separation techniques. Keywords biomass, chromatography, extraction, isolation, natural products

1 INTRODUCTION

Bioactive natural products are the main source of new drugs, functional foods and food additives. They are secondary metabolites of plants and animals gen-erated through various biological pathways in secon-dary metabolism processes. Typical features of bioac-tive natural products include: (1) diverse structures, i.e. flavonoids, alkaloids, sterols, terpenes, quinones and phenylpropanoid, etc.; (2) molecular mass between 200 and 1000, usually with complex structures con-taining a skeleton of aromatic rings or multi-rings and a number of functional groups; (3) various physiologi-cal activities; (4) boiling points mostly above 200 °C, some of which are heat-sensitive. Nowadays, more than 80% food active compounds and more than 30% drugs are produced from bioactive natural products, and the annual growth rate of global natural prod-ucts-derived drugs is up to 20% [1, 2]. As a result, the research on the separation and purification of bioactive natural products from plants, animals and microor-ganisms has attracted much attention in academia and industry. In view of tremendous components and low contents of target compounds (0.01% 10%) in a plant, separation is often a complex process, which may last from days to months, depending on the problem being tackled.

Generally, the separation of target products in-cludes two steps, extraction and purification. In the extraction, studies are mainly focused on the application of environmentally friendly solvents and intensification of extraction process by means of physical fields. In the isolation and purification, novel separation tech-nologies such as chromatography techniques, membrane separation techniques and ionic liquids-mediated ex-traction have been developed and brought a major

impact on this respect. Since the separation efficiency is relevant with the solubility and diffusion coefficient, research efforts in measuring and modeling the solu-bility and diffusion coefficient of bioactive natural products are also included in this review. In the past decades, there has been considerable evolution of separation knowledge, theory, and technology. It is the goal of this review to give an insight into the techniques and some future challenges.

2 SOLUBILITY AND DIFFUSION COEFFI-CIENT OF BIOACTIVE NATURAL PRODUCTS

2.1 Solubility

Solubility is of particular importance for the de-sign and optimization of separation processes. Bioac-tive natural products usually have complex scaffolds bearing a number of functional groups [3]. Hydropho-bic and hydrophilic segments are relatively common in bioactive natural products, which render their solu-bility different from conventional small organic mole-cules. So far, a large number of solubility data of bio-active natural products in organic solvents have been reported, such as flavonoids [4 7], terpenes [8] and steroids [9 11]. However, due to the highly cohesive energy of some bioactive natural products, it is diffi-cult to select suitable solvents for separation process [12, 13]. Some bioactive natural products have the maximum solubility in binary solvents [14, 15]. Chen et al. [16] have found that the solubility of cholesterol in n-hexane-ethanol binary solvent reaches its peak at the mole fraction of ethanol close to 0.45 (shown in Fig. 1). The rationale is presumably that the synergis-tic effect of mixed solvents meets the polarity re-quirements of cholesterol.

Received 2013-03-17, accepted 2013-05-20.

* Supported by the National Natural Science Foundation of China (20936005, 21076175 and 21076178), the National High Technology Research and Development Program of China (2012AA040211), and the Program for Zhejiang Leading Team of S&T Innovation (2011R50002).

** To whom correspondence should be addressed. E-mail: renql@zju.edu.cn

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Ionic liquid (IL) is a new class of solvent con-sisting of entirely ionic species. As they consist of at least two components that can be varied (anion and cation), the solvent can be designed to dissolve bioac-tive natural products that scarcely dissolve in conven-tional solvents [17 20]. Thus ILs have the potential to replace traditional organic solvents for extraction of bioactive natural compounds from plants. Different from ILs, supercritical carbon dioxide (scCO2) dissolves lipophilic or weak polar bioactive natural products easily, e.g. artemisinin [21], natural vitamin E and carotene [22].

It is highly desirable to establish models for cor-relation and prediction of solubility, but it is challeng-ing for bioactive natural products owing to the struc-ture complexity. For simple organic molecules, mod-els such as UNIFAC group contribution method, NRTL-SAC and COSMO are in good agreement with experimental data, while those for bioactive natural products are still under investigation.

UNIFAC model has been used to predict the solubility of some bioactive natural products [23]. However, this model is based on the assumption that there is no interaction in the molecule and the interaction between groups is equivalent in different chemical

environment, which is obviously not the case in bio-active natural products. As a consequence, the solubil-ity prediction with such model is often in error, espe-cially with several non-alkyl groups in bioactive natural products [23, 24]. Besides, some groups widely present in bioactive natural products such as steroid nucleus are not defined in UNIFAC model, which makes pre-diction difficult. Thus it is essential to define new groups. With C5H2N+, ArCOOH and steroid nucleus defined [25, 26], the prediction for solubility of ber-berine chloride, p-toluic acid and cholesterol coincides well with experimental data.

Based on the “virtual site” concept, the NRTL-SAC model describes the surface property of a molecule in a new way and successfully solves the problems re-sulted from the complexity of structure, especially in bioactive natural products. This model has been used to predict the solubility of drugs with a medium size [27] and other bioactive natural products [28], giving better accuracy than UNIFAC model (Fig. 2). Fur-thermore, it is possible to use the NRTL-SAC model to describe the solubility variation in binary solvents [27]. COSMO-RS and COSMO-SAC have been used to compute molecular interactions through calculation of electronic density in the molecular surface,

Figure 1 Mole fraction solubility of cholesterol in n-hexane (1) + ethanol (2) mixed solvents vs. mole fraction of ethanol on solute-free basis [16]

293.2 K; 298.2 K; 303.2 K; 313.2 K; 323.2 K

(a) (b)

Figure 2 NRTL-SAC (a) and UNIFAC (b) results for aspirin solubility at 298.15 K, with solubility data for 14 solvents fittedwith NRTL-SAC [23]

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improving the prediction accuracy [28, 29]. These models are far more convenient than other models as they can be used without any experimental data. Such models have been successfully used to predict the solubility of artemisinin [30], nitro compounds [31] and other substances [32, 33].

The study on solubility models of bioactive natural products has some progress, but the accuracy is not satisfactory especially to substances with complex structures. Development of new models with better accuracy is the direction of future study. In view of the advantages of COSMO-RS and COSMO-SAC models, they also need further study because of great conven-ience in solubility prediction and solvent screening.

2.2 Diffusion coefficient

Diffusion and mass transfer coefficient are essen-tial for process design, scale-up and optimization. Ear-lier research efforts were focused on determination of diffusion coefficients of bioactive natural products in organic solvents or in water. In recent years, studies were concentrated on the diffusion behavior of bioac-tive natural products in scCO2.

Hills et al. [34] summarized the diffusion coeffi-cients of 139 organic compounds with molecular mass ranging from 2 to 235 g·mol�1 in water at 298 K, which varies from 0.23×10�5 to 4.4×10�5 cm2·s�1. For bioactive natural products with higher molecular weights, such as amino acids [35] and sugars [36], their diffusion coefficients in water at 298 K range from 0.602×10�5 to 1.040×10�5 cm2·s�1 and from 0.546×10�5 to 0.773×10�5 cm2·s�1, respectively. Funazukuri et al. [37, 38] measured the diffusion coefficients of some small organic molecules including acetone, benzene and bioactive natural products, such as vitamin E, vitamin K1 and linoleic acid methyl ester, in organic solvents. The diffusion coefficients of vitamin E are 70% lower than that of acetone. In general, due to their relatively large molecular volumes, bioactive natural substances have smaller diffusion coefficients than small organic molecules.

Because of the low viscosity of scCO2, which is close to that of gas, the self diffusion coefficient and molecular diffusion coefficient of solutes in scCO2 are distinctly higher than that in organic solvents. Diffu-sion coefficients of various bioactive natural products in scCO2 have been reported, including C18 C22 satu-rated and unsaturated fatty acids and esters [39 48], terpenes (citral [49], pinene [50], L-carvone [51] and linalool [52]), natural pigments (�-carotene [53] and malvidin 3,5-diglucoside [54]), and other active com-pounds such as tocopherol [53], CoQ10 [55], indole [56], and vitamin K3 [57]. The diffusion coefficients of bioactive natural substances in pure scCO2 are ten times that in organic solvents or water.

The diffusion of bioactive natural compounds in the mixture of scCO2 and cosolvent was investigated by Dong et al [51, 58]. The diffusion coefficients of L-menthone and L-earvone in the mixture of scCO2

and ethanol increase with temperature, and decrease with the increase of pressure, density or viscosity at constant temperature, and ethanol mole fraction in the mixture due to the chemical association between the two solutes and ethanol [51]. The experimental results show that for cosolvents without hydrogen-bond abil-ity, the dispersion force between solute and cosolvent is the primary factor affecting the diffusion of solute. For cosolvents with only HBA basicity and amphi-protic cosolvents, the hydrogen-bond interaction be-tween solute and cosolvent influences the diffusion of solute significantly [58].

At present, the study on the diffusion coefficient is focused on its determination. The investigation on solute-solvent interaction effect on diffusion coeffi-cients from a microscopic aspect is quite challenging but deserves study.

3 EXTRACTION TECHNIQUES

The first step to isolate bioactive natural com-pounds is generally the extraction of target compound from plant or animal tissues. Recent study on extraction of bioactive natural compounds focused on intensifi-cation of extraction process and application of novel solvents, such as scCO2, subcritical water, and ILs.

3.1 Multi-stage countercurrent extraction and extraction intensification

Extraction of natural substances using conven-tional solvents such as water has been a popular method for more than 2000 years. Maceration (batch single pot extraction) [59 62], percolation [63 65] and countercurrent extraction [66, 67] are commonly used solvent extraction methods in industry [68 72]. Multi-stage countercurrent extraction (MCE) tech-nique attracts much attention due to its high efficiency. The comparison in Table 1 [73] shows that the extrac-tion efficiency of MCE for extraction of glycyrrhizic acid from licorice (Glycyrrhizauralensis Fisch) pre-sents better results than other extraction techniques including microwave-assisted extraction, ultrasonic extraction, Soxhlet extraction and room temperature extraction, offering the highest yield, short extraction time, and the least solvent consumption.

For improvement of extraction efficiency, various types of physical field such as microwave and ultra-sound have been used in the extraction of bioactive natural products to strengthen the mass transfer [74 77]. Microwave-assisted extraction is a technology devel-oped recently for extracting soluble products into a fluid aided by microwave energy, which can signifi-cantly increase the mass transfer rate, reduce the ex-traction time and extract bioactive natural products more rapidly and selectively in comparison to tradi-tional extraction processes [78]. This technique has been successfully applied to the extraction of natural compounds such as glycosides, alkaloids, carotenoids,

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terpenes, and essential oils [79 84]. For extracting ac-tive components in Chinese quince, with 44.15% ethanol/water as solvent and microwave power of 102.1 W, the product was obtained with the yield more than 77.47% in 5 min [85]. Although the improvement of extraction efficiency via microwave energy is ob-vious in analytical scale, its wide applications just appear around one decade since the invention of sev-eral extraction instruments. The results obtained so far show that microwave radiation does not damage the extracted compounds, unless the temperature in the extraction instrument increases dramatically.

Ultrasonic cavitation is another recently devel-oped technique to improve traditional extraction proc-esses [86 88]. The extraction of saponins, steroids and triterpenoids from chresta spp was three times faster than that with traditional extraction methods [89]. Scanning electron micrographs of sonicated samples showed the structural disruption of soy �akes (Fig. 3). The particle size decreased nearly 10-fold with ultra-sonic treatment at high amplitudes. Sonication at high amplitude for 120 s gave the highest increase in total sugar release (50%) and protein yield (46%) compared with non-sonicated samples (control). The use of ul-trasound can significantly improve protein yield and reduce the overall cost in producing soy protein from

�akes [90]. However, microwave and ultrasound-assisted ex-

traction may cause local high temperature, which may not be suitable for heat-sensitive natural substances. In addition, it may not be easy to realize industrial pro-duction for lacking of corresponding industrial facili-ties [76].

3.2 Supercritical/subcritical fluid extraction

Supercritical/subcritical fluids are regarded as al-ternatives to conventional solvents. The properties of supercritical fluids (SCFs) can be simply changed by changing pressure and temperature, rendering them potential applications in a range of industrial and laboratory processes. Carbon dioxide (CO2) and water are the most commonly used supercritical/subcritical fluids. Supercritical CO2 is an excellent solvent for the extraction of bioactive compounds, especially heat-sensitive substances because of its mild critical condition, and has been widely used for extraction of essential oils, fatty acids and antioxidants [91 96]. Table 2 is a summary of scCO2 extraction of natural substances recently. Lipid-soluble bioactive substances such as essential oil and germ oil can be efficiently

Table 1 Comparison of extraction processes for extraction of glycyrrhizic acid from licorice [73]

Method Solvent usage/mg·ml�1 Extraction time/min Extraction yield/% (by mass)

multi-stage countercurrent extraction (MSE) 6 60 97.2

microwave-assisted extraction 16 54 86.6

ultrasonic extraction 16 40 93.8

Soxhlet extraction 16 240 87.3

room temperature extraction 16 2660 87.3

Figure 3 SEM images of defatted soy �akes at high amplitude (84 �mpp) [90] (a) control (0 s); (b) 15 s; (c) 60 s; (d) 120 s; bar: 10 �m

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extracted by pure CO2 [97 102]. For polar bioactive substances (e.g. flavonoid), modifiers are added to increase the polarity of CO2 [103 108]. Another advan-tage of scCO2 extraction is the easy operation and ef-ficient separation of products by simple decompres-sion. With a refrigerator installed to the separator, highly volatile substances are able to be collected with-out organic solvent residue [109]. However, collection of products by decompression would consume power since CO2 needs to be re-pressurized to complete a cycle. Researchers are making their efforts on coupling extraction-absorption and extraction-adsorption. By using selective solvent or adsorbent, separation of products without decompression has been successfully carried out [110]. After thirty years of development, supercritical fluid extraction (SFE) technology has become a mature technique and has been used in a number of large-scale extractions for natural substances, such as the removal of caffeine from coffee [111 115] and industrial extraction of hops [116 119].

In recent years, SFE model has become a useful tool for design of extraction processes. With appropri-ate models, one can obtain more useful information about extraction mechanisms and optimize extraction conditions. Many models [119 124], such as diffusion model and hot-ball model, have been developed, among which the simple and practically useful one is the hot-ball model developed by Bartle et al [125]. Fischer and Jefferies used this model to predict ex-traction of nicotine from tobacco using methanol modified scCO2 [120] and the prediction was in accord with experimental data.

Extraction of bioactive compounds from natural substances using subcritical water was in the rapid development over the past decade [126] because it is a cheap and pollution-free extraction solvent [127]. Sereewatthanawut et al. [128] compared the recovery of protein and amino acids by subcritical water extrac-tion at various temperatures and time with those by alkaline method at 30 °C for 45 min. The protein con-tent in the extraction by subcritical water at 160 °C for 20 min or longer and at 220 °C for more than 5 min was higher than that by alkaline extraction. However, subcritical extraction technology usually operates at a high temperature (more than 150 °C), so degradation of bioactive compounds may occur in the extraction process [129].

3.3 Ionic liquid extraction

Ionic liquids attract great interest as alternative solvents in recent years, due to their unique features such as ultra-low vapor-pressure, wide liquid range and good thermal stability [130]. Moreover, ILs are “designable” solvents whose physicochemical proper-ties can be well tuned by introducing various func-tional groups into the structures of cations and anions [131, 132], so they often dissolve many organic materials. Using ILs as solvents in extracting bioactive natural compounds from biomass not only leads to a green process, but also improves the extraction efficiency from a thermodynamic point of view.

Usuki et al. utilized [bmim]Cl as solvent to

Table 2 Summary on the extraction of bioactive natural compounds from plant by SFE in recent years

Natural material Compounds of interest Related functional

activity Extraction conditions Ref.

Rhodiola rosea roots rosavin antioxidant, anti-stress,among others

CO2 + water (10%), 20 MPa, 80 �C, 3 h [103]

ginger (Zingiber corallinum Hance) essential oil antipyretic CO2 + methanol, 10 MPa, 30 �C, 40 min [104]

hyssop (Hyssopus of�cinalis L.) essential oil antipyretic CO2, 9 MPa, 40 �C (dynamic) [97]

lotus (Nelumbo nucifera) germ oil antioxidant CO2, 32 MPa, 50 �C, 2 h (dynamic) [98]

Garcinia mangostana xanthones antioxidant CO2 + ethanol (4%), 20 MPa, 40 �C [105]

Pinus sp. flavonoids antioxidant activity CO2 + ethanol (3%, by volume), 20 MPa, 40 �C

[106]

Hibiscus cannabinus oil antioxidant CO2, 20 MPa, 80 �C, 150 min [100]

Hippophae rhamnoides coagulation related compounds

antithrombotic and antiaterogenic

CO2, 45 MPa, 60 �C [99]

Artemisia annua L. scopoletin and artemisinin anti-malarial CO2 + ethanol (16.25%, by volume), 24.2 MPa, 40 �C

[108]

northern shrimp (Pandalus borealis Kreyer)

polyunsaturated acids antioxidant CO2, 35 MPa, 40 �C [101]

coffee husks caffeine stimulant CO2, 30 MPa, 100 �C [102]

green tea leaves caffeine stimulant CO2 + ethanol (95%, by volume), 23 MPa, 63 �C

[107]

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extract shikimic acid from Ginkgo biloba leaves at 423.15 K and obtained an extraction yield of 2.3% (by mass), which was 2.5 times that using methanol (0.93%, by mass) and 2 times that using DMF (1.1%, by mass) [133]. The enhanced yield is ascribed to the great solubility of [bmim]Cl for cellulose, which is the primary component of the wall of plant cells, with the evidence from SEM micrographs showing the change of microscopic morphology of leaves after extraction (Fig. 4). The dissolved shikimic acid was isolated from [bmim]Cl by using anion exchange resin. Chowdhury et al. employed a distillable IL, N,N-dimethylammonium N�N�-dimethylcarbamate (DIMCARB), to extract hy-drolysable tannin materials from plant sources [134]. For the samples of catechu (Acacia Catechu) and my-robolan nut (Terminalia Chebula), the content of tan-nins in DIMCARB extract was 56% and 23%, respec-tively, much higher than those using water as solvent (27% and 21%). Moreover, the DIMCARB extract showed a better selectivity of ellagic acid than the water extract. Bioniqs Ltd. investigated the extraction of artemisinin from dry solid materials with ILs [135]. In comparison with hexane, ILs were superior because of elevated equilibrium concentration of artemisinin or reduced extraction time. Further purification of ar-temisinin after the IL extraction was performed by an IL/water distribution-recrystallization method.

The relatively large viscosity of ILs is one of the main problems in IL extraction, which may impede the mixing of ILs with biomass and diffusion of solutes. Lu et al. studied the IL-based microwave-assisted ex-traction of phenolic alkaloids from medicinal plant Nelumbo nucifera Gaertn [136]. Compared with heat-reflux extraction, this method improved the ex-traction efficiency by 20% 50% within 90 s. Du et al. utilized the aqueous solution of [bmim]Br in the mi-crowave-assisted extraction of trans-resveratrol from Rhizma Polygoni Cuspidati [137]. With the liquid/solid ratio of 20 1, an extraction yield of 92.85% was achieved after 10 min. Row et al. employed the aque-ous solution of chloride-anion ILs in the ultrasonica-tion-assisted extraction of medicinal products from herb, such as cryptotanshinone, tanshinone I and tan-shinone II [138], and found that the extraction effi-ciency in aqueous ILs was much higher than that in pure water or n-hexane. Among these ILs used, the length of alkyl chain of cation is proportional to the

extraction efficiency, probably owing to the increase in hydrophobicity of ILs.

In spite of a series of advantages provided by ILs, efficient recovery methods of bioactive natural com-pounds from ILs are not yet resolved due to their high-boiling points or heat-sensitive properties, greatly hindering its industrial applications. Therefore, devel-opment of highly effective isolation/recovery methods is needed in future researches.

4 ISOLATION AND PURIFICATION TECH-NIQUES

4.1 Chromatography separation techniques

Bioactive natural products are often a mixture of a number of compounds with similar structures and polarities. Chromatography related technologies are the most often used methods for separation of pure natural products. The separation efficiency of targeted compounds is highly dependent on their adsorption affinity to stationary phase. In the past decades, chro-matography techniques have seen an explosion of in-terests and have been successfully applied to the sepa-ration of natural products. This part of review mainly focuses on recent progress of high speed counter cur-rent chromatography, supercritical fluid chromatogra-phy, simulated moving bed and gel permeation chro-matography, and their applications in the separation of bioactive natural products are briefly presented.

4.1.1 High speed countercurrent chromatography High speed counter current chromatography

(HSCCC) is a support free liquid-liquid partition chro-matographic technique, which eliminates irreversible adsorption of samples on solid support in the conven-tional chromatographic column. Compared with tradi-tional chromatography, HSCCC offers various advan-tages such as rapid separation, low solvent consump-tion and high recovery. It has been successfully applied to isolate and purify a number of natural products, especially for polar substances. The crucial factor for the HSCCC is the selection of the two-phase solvent system. With appropriate solvent systems, HSCCC has been widely used for the isolation of flavonoid compounds from natural products. Preparative isolation and purification of flavonoid glycosides from Chinese

Figure 4 SEM micrographs of leaves before extraction (left), after extraction by methanol (middle), and after extraction by [bmim]Cl (right) [133]

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medicinal herbs, including Taraxacum mongolicum [139 141], Nelumbo nucifera [142, 143], Radix Astragali [144], Ziziphus jujube [145], sarcandra glabra [146], hedyotis diffusa [147], oroxylum indicum [148], etc., have been broadly reported. It has been proven that n-hexane-ethyl acetate-methanol-water solvent system is effective to separate and purify phenolic acids [149], anthraquinones [150], tetrahydropalmatine [151], gin-gerols [152], ferulic acid [153] and liensinine homo-logues [154] from corresponding natural products. The solvent systems such as ethyl acetate/2-propanol/water and ethyl acetate/1-butanol/water were successfully used for the fractionation of dimeric to tetrameric procyanidins [155]. In order to achieve fast and effi-cient separation, gradient mode in HSCCC was often employed. Peng et al. [156] employed flow rate gradient HSCCC to separate five diterpenoids from Tripery-gium wilfordii. Niu et al. [157] first demonstrated that preparative isolation of alkaloids from Corydalis bungeana Turcz. was possible with gradient elution. For both development and practical experience with existing HSCCC technology, an expected major area of expansion is the development of new HSCCC in-strumentation. In order to bring the numerous benefits of HSCCC to an increasing number of research and application laboratories, factors such as dependability and simplicity, paired with a healthy degree of auto-mation, can be considered key criteria of successful progression.

4.1.2 Supercritical fluid chromatography Supercritical fluid chromatography (SFC) is a

chromatographic technique generally using super-critical carbon dioxide (scCO2) instead of organic solvents as mobile phase. Compared to traditional or-ganic solvents, scCO2 has higher diffusivity and lower viscosity, which give rise to better separation efficien-cies and lower pressure drops. SFC is thus allowed to be operated at higher flow-rate, leading to shorter run time. In addition, SFC significantly reduces solvent consumption and subsequent energy consumption for solvent recovery. Ren et al. [158 160] developed a process for separating DHA-EE and EPA-EE from fish oil using SFC without co-solvent. The separation of arachidonic acid ethyl ester and linoleic acid ethyl ester by SFC on RP C18 stationary phase was reported [161]. Because of poor solubility of polar solutes in scCO2, addition of co-solvents such as methanol, ethanol and 2-propanol is a general solution to elute the solutes that interact strongly with stationary phase. Jiang et al. [162] found that baseline resolution of to-copherol homologues could be achieved on C18 col-umn using unmodified scCO2 in temperature range from 303 K to 343 K and pressure range of 16 22 MPa. In the absence of co-solvents, it was difficult to elute these solutes from silica gel in a reasonable time. Nevertheless, resolutions and peak profiles were sig-nificantly improved by adding 5% (by mass) ethanol or 2-propanol to the mobile phase. Recently, SFC was also coupled with SFE to isolate desired ingredient from natural products. Ramírez et al. [163] demonstrated the

potential use of preparative-supercritical fluid chro-matography to fractionate complex supercritical rose-mary extracts. The fractions containing carnosic acid, carnosol and methyl carnosate were obtained by SFC in the presence of 10% of ethanol as the modifier in LC-Diol packed column at 80 °C and 13 MPa. SFC was also employed to fractionate thyme (Thymus vul-garis L.) extracts, which were obtained by supercriti-cal carbon dioxide extraction of thyme leaves [164]. Solanesol [165] and artemisinin [166] were fractionated from the supercritical extracts by preparative SFC. Desmortreux et al. [167] utilize SFC to improve sepa-ration of furocoumarins of essential oils. SFC with inert scCO2 could effectively prevent some oxygen-sensitive bioactive products from oxidation, which likely occurs in liquid chromatography. SFC has attracted much attention in purification of bioactive natural products as standard of residual solvent is becoming more rig-orous in recent years.

4.1.3 Simulated moving bed Simulated moving bed (SMB) is a continuous

chromatography, in which the feed entrance and the analyte recovery are simultaneous and continuous by properly simulating the countercurrent movement be-tween stationary phase and mobile phase. The separa-tion efficiency of SMB is much higher than that of batch liquid chromatography, and high purity of product may be obtained even if the selectivity of two solutes is too low to be baseline resolved. The high productivity of SMB has made it practical for indus-trial applications.

SMB has been widely used to separate racemates since 1990s [168 171]. It also serves to separate bioac-tive natural products. Lu et al. [172] separated toco-pherol homologues using SMB for the first time, in which �- and �-tocopherols with purity greater than 98% were obtained. The productivity was as high as 6 g·L�1·h�1, which was three times higher than that of batch chromatography at the same feeding concentra-tion of 28 g·L�1. The SMB was used for separations of capsaicin and dihydrocapsaicin, L-arabinose and D-xylose in xylose mother liquor, mono- and di-d-�-tocopherol polyethylene glycol 1000 succinate [173 175]. Sun et al. [176] separated xylose and xylitol in xylose mother liquor, and obtained xylose with pu-rity of 99.3% and xylitol with purity of 99.8% though the resolution of the two components was only 0.97. Wei and Zhao [177] purified capsaicinoids by combin-ing SMB with adsorption technique with macroporous resin. Cong et al. [178] reported the purification of liquiritin with SMB after suitable post-treatment and the purity of product was around 85%, achieving the purity of 99% by further recrystallization. Long et al. [179] separated D-psicose from D-fructose and ob-tained product with purity higher than 99%. By opti-mizing the SMB process using multiple objective op-timization method, a maximum productivity reached as high as 103 g·L�1·h�1, and the purities of succinic acid and lactic acid were up to 98% [180]. Although SMB has been proven to be a promising technique for

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pilot preparation of racemates, isomers and bioactive homologues, the cost of bulk packing materials and equipment limits its industrial applications.

4.1.4 Gel permeation chromatography Gel permeation chromatography (GPC) is a form

of liquid chromatography also known as size exclu-sion chromatography or gel filtration chromatography. Generally, Sephadex is the most widely used stationary phase in GPC because it could filter molecules ac-cording to their sizes. While some smaller molecules enter the pores of gel and travel longer distance, larger molecules shows much shorter retention time. In view of its separation mechanism, GPC is often applied to the purification of water soluble macromolecules. Lu et al. [181] tried to remove macromolecular impurities from penicillin G sodium, which may cause allergic reactions. The impurities could be completely sepa-rated using HW-40F as stationary phase and 0.005 mol·L�1 citrate buffer with pH close to 7.0 as mobile phase. Aryusuk et al. [182] separated rice bran wax and its major impurity triglyceride using 10 nm Phenogel column as stationary phase and isooctane-toluene (volume ratio 65/35) as mobile phase. Du et al. [183] separated theaflavins from the extract of black tea leaves using conventional Sephadex LH-20 column chromatography. Cara et al. [184] reported a separation of olive tree pruning oligomers from olive tree biomass hydrolyzates using preparative gel filtration chroma-tography. Zhang [185] separated immunoglobulin IgG and IgM in colostrum by combining GPC and ion ex-change chromatography and obtained sIgA with purity of 96.88%. The major challenges of GPC technique in practical applications are the scale-up of chroma-tographic process and the life period of bulk packing because of its relatively weak pressure resistance.

4.2 Membrane separation technology

Advances in material science and membrane manufacturing technology have made membrane technique grow to be an important technology for separation of natural products. The general principle of membrane separation is based on the selective per-meability of the membrane to allow the target sub-stances to penetrate through the membrane, whilst unwanted substances are normally rejected by the membrane. Membrane-based processes are generally operated at room temperature, and there are no phase changes and chemical reactions in the process, so it is especially suitable for separation and purification of thermal-sensitive bioactive substances.

Nowadays, membrane technologies are still re-stricted to desalinate, recover or remove protein from extracts of natural products because of poor selectivity of membrane for similar molecules with close size and structures. Cho et al. reported that the extracted pectin solution was concentrated with the cross flow micro-filtration using a 0.2 �m regenerated cellulose mem-brane. The filtration process was effective to remove

flavonoids, polyphenols and carotenoids, with the ga-lacturonic acid content of pectin increased from 68.0% to 72.2% [186]. With ultrafiltration, Denis et al. con-centrated and pre-purified R-phycoerythrin solution extracted from macro-algae [187], Xu et al. achieve high purity of flavoniods from Ginkgo Biloba leaf extract [188], Goulas et al. obtained the yields of 19% (by mass) for monosaccharides and 88% (by mass) for di- and oligosaccharides [189], and Nabarlatz et al. investigated purification of xylo-oligosaccharides from almond shells [190]. With nanofiltration technol-ogy, the refinement of rice bran oil increased the con-tent of �-oryzanol in oil from 0.95% (by mass) to 4.1% (by mass) [191], and the purity was over 90% in fructooligosaccharides with the yield around 80% [192]. Ion-exchange membrane was used to remove pectinesterase from Marsh grapefruit pulp extract ef-fectively, preventing pectin from esterification [193].

Membrane separations were coupled recently when a single membrane filtration step is not satisfac-tory. A coupling membrane process was investigated for the separation and concentration of polyphenols in bergamot juice, in order to develop a natural product enriched in polyphenols [194]. Kamada et al. examined the effectiveness of combined membrane process with ultrafiltration and nanofiltration for purification and concentration of oligosaccharides from chicory root-stock [195]. Bazinet et al. evaluated the feasibility of the production of a cranberry juice enriched with natural phenolic antioxidant compounds using an ul-trafiltration membrane stacked in an electrodialysis cell [196]. A process for isolating tea polyphenol and caffeine from green tea leaves was developed using extraction followed by ultrafiltration with CA-Ti composite membrane and adsorption with PA resin and a purified product containing more than 90% of tea polyphenol was obtained [197].

In summary, membrane separation technology has demonstrated potential application in the separa-tion of bioactive products with the development of novel membrane materials. The main problem en-countered in membrane-based separation is the decay of permeate flux caused by concentration polarization and fouling, increasing operating cost and shortening membrane life. As a result, much effort has been di-rected to modify the structure and chemistry of mem-branes and develop new membrane systems with re-duced fouling characteristics and enhanced permeate flux. Another important trend in membrane separation is to improve the selectivity for target substances by incorporation of appropriate functional groups onto membrane surface. With these contributions, mem-brane separation techniques will remain a promising tool in the separation of bioactive natural products.

4.3 Ionic liquids-mediated separation of bioactive natural homologues

The most challenging problem in the separation of bioactive natural products from other compounds is

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the separation of bioactive homologues [1, 198], which has become a critical procedure in producing highly bioactive products. Current methods for separating bioactive natural homologues mainly include adsorp-tion [199], high-speed countercurrent chromatography and crystallization [200, 201], but they usually suffer from problems such as large consumption of solvent and energy, environmental pollution, and high produc-tion cost. The application of liquid-liquid extraction is limited by lacking of appropriate extractants and bi-phasic systems as well as poor selectivity for different homologues.

In virtue of the unique features of ionic liquids, such as ultralow vapor pressure, non-flammability, designable structure and property, and the facility of forming biphasic systems with other solvents, Ren and co-workers developed a novel method for separating bioactive natural homologues based on IL-mediated liquid-liquid extraction [202 205], showing a very promising method for separating typical bioactive homologues including polyphenols, flavonoids and

terpenes. A series of IL/hexane and IL/polar cosol-vent/hexane biphasic systems that have low mutability and different microscopic solvent properties were prepared and utilized to separate the mixture of toco-pherol homologues, which are the main components of natural vitamin E (Fig. 5) [202, 203, 205]. The selec-tivity of �-tocopherol to �-tocopherol (the difference of their structures is in two methyl groups) was re-markably increased to more than 20.0, which is much more superior to common biphasic systems (e.g., se-lectivities in DMF/hexane and methanol/hexane sys-tems are 1.8 and 1.3, respectively). The use of cosol-vent is very important, because it could not only reduce the viscosity of extraction system, but also enhance the tunability of the physicochemical properties of extractant such as dipolarity/polarizability, hydrogen- bond basicity and hydrogen-bond acidity. A synergistic effect between IL and cosolvent has been revealed by Ren and his coworkers through the discovery of a maximum point in the distribution coefficient-IL con-centration plot (Fig. 6) [203], showing that cosolvent

Compound R1 R2

�-tocopherol CH3 CH3

�-tocopherol CH3 H

�-tocopherol H CH3

�-tocopherol H H

Figure 5 Structure of tocopherol homologues [202]

Figure 6 Distribution coefficient of �-tocopherol (D�) using different [bmim]Cl-cosolvent mixtures as extraction solvent at 303.15 K [203] initial concentration of tocopherol in hexane/mg·cm�3: � 1.00; � and � 0.98; � 0.20

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may improve the extraction efficiency of ILs in some extent. With cosolvent, a large separation selectivity, adequate distribution coefficient, low viscosity, and low consumption of IL can be simultaneously achieved in the extractive separation of tocopherol homologues. Based on this strategy, [bmim]Cl-acetonitrile mixture with a molar ratio of 2 98 as the extractant (with viscosity of about 0.44 m·Pa·s only) at 303.15 K re-sulted in the selectivity of �-tocopherol to �-tocopherol of 11.3, four times larger than that using pure acetoni-trile, and the distribution coefficients of �-, �- and �-, and �-tocopherol were 4.07, 2.02, and 0.36, respec-tively, at least eighteen times larger than those using pure [bmim]Cl. A correlation between the selectivity of different tocopherol homologues and the Kamlet- Taft hydrogen-bond basicity � of IL-cosolvent mix-tures was established, and the essential role of hydrogen- bonding recognition in separating homologues was also illustrated by quantum chemical study.

In order to apply liquid-liquid extract techniques to scarcely soluble natural homologues, Ren’s group developed novel IL + water + ethyl acetate biphasic systems to separate those “sparingly aq-/lipo-soluble” bioactive homologues with high efficiency [204 206]. For ginkgolide homologues and soybean isoflavone homologues, the distribution coefficients of gink-golides obtained with [emim]Br-water-ethyl acetate system at 303.2 K were about 10 1000 times larger than those obtained with conventional biphasic sys-tems and a selectivity of ginkgolide C to ginkgolide B was up to 12 [206]. With the same biphasic system, adequate distribution coefficients and selectivities over 7.0 were also achieved for the separation of dif-ferent soybean isoflavone homologues [204]. Through a laboratory-scale simulation of fractional extraction process containing four extraction stages and four scrubbing stages, genistein was separated from other soybean isoflavone homologues with a purity of 95.3% and a recovery >90%.

In general, recent studies have demonstrated that IL-mediated liquid-liquid extraction is a promising method for separation of bioactive natural homologues, which results in low solvent/energy consumption, high selectivity, large throughput capacity and low envi-ronment pollution. With the application of this tech-nique, it is expected that the production cost of highly-bioactive natural products will reduce remarka-bly. IL-mediated liquid-liquid extraction can be ex-tended to the separation of other natural analogues, e.g., Ni et al. prepared several amino acid-functionalized ILs to separate �-tocopherol from its structural ana-logue methyl linoleate by liquid-liquid extraction [207]. With the 15 85 (molar ratio) IL-DMF mixture as extractant, the selectivity was as high as 29.0 with an adequate extraction capacity. Future research efforts should be made to promote IL-mediated liquid-liquid extraction as an industrial technology, which include the effective recovery of ILs, design of more efficient IL extractants, study on mass transfer, and intensification

of large-scale extraction.

5 CONCLUSIONS AND OUTLOOK

Numerous approaches have been developed for efficient separation of bioactive natural products. Cru-cial breakthroughs in separation technologies have greatly lowered the hurdles in the isolation of structur-ally complex molecules or natural homologues. How-ever, lacking of systematical thermodynamic theory and effective models, solvent selection and process optimization rely mainly on random screening. Since the majority of separation methods are related with solubility, liquid/liquid, solid/liquid partition equilib-rium and other thermodynamic properties, the study on those properties remains to be an important subject in separation of natural products. On the one hand, the collection of a large amount of thermodynamic data is important to establish quantitative structure-property relationships for bioactive natural products. On the other hand, molecular simulation, spectral and other advanced methods should be utilized to study the inter-actions between bioactive natural products and separa-tion medium (solvent, extractant, adsorbent, etc.) at micro-scale and meso-scale, which may provide an insight into the separation mechanism for bioactive products. Accordingly, a rational optimization of separation process may be achieved.

The advance of separation technologies is highly dependent on the evolution of separation media, e.g. adsorbents and solvents. As a consequence, develop-ment of novel adsorbents is the focus of research efforts. Ideal adsorptive materials, such as adsorptive macro-porous resins, mesoporous molecular sieves and mono-lithic continuous beds, are expected to have low cost, specific pore diameters, adjustable pore structures and enriched porous surfaces with functional groups. For alternative solvents, SCF and ILs are increasingly considered to replace conventional solvents, because they are generally regarded as environmental benign solvents and are superior in the reduction of solvent consumption, energy consumption and wastes emission in the separation process. Moreover, in the realm of liquid-liquid extraction, ILs have showed great advan-tages in the separation of natural homologues, which is generally considered as the most challenging in iso-lation of natural products.

Some physical fields or methods, such as ultrasonic extraction, microwave-assisted extraction, near-critical water extraction, and supercritical chromatography, have been utilized to improve the separation efficiency of bioactive natural products on laboratory-scale or analytical-scale. However, the application of such techniques in industry still remains to be fully explored. For some bioactive compounds, mutual interplay or combination of different separation techniques are essential for their separation. It is thus of great impor-tance to study the combination ways of different separation techniques in the future.

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87 Li, M.F., Sun, S.N., Xu, F., Sun, R.C., “Ultrasound-enhanced ex-traction of lignin from bamboo (Neosinocalamus affinis): Charac-terization of the ethanol-soluble fractions”, Ultrason. Sonochem., 19 (2), 243 249 (2012).

88 Pan, Z.L., Qu, W.J., Ma, H.L., Atungulu, G.G., Mchugh, T.H., “Continuous and pulsed ultrasound-assisted extractions of antioxi-dants from pomegranate peel”, Ultrason. Sonochem., 19 (2), 365 372 (2012).

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90 Karki, B., Lamsal, B.P., Jung, S., van Leeuwen, J., Pometto, A.L., Grewell, D., Khanal, S.K., “Enhancing protein and sugar release from defatted soy flakes using ultrasound technology”, J. Food Eng., 96 (2), 270 278 (2010).

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