Natural medicines were the only option for the prevention and treatment of human diseases for thousands of years. Natural products are important sources for drug development. The amounts of bioactive natural products in natural medicines are always fairly low. Today, it is very crucial to develop effective and selective methods for the extraction and isolation of those bioactive natural products. This paper intends to provide a comprehensive view of a variety of methods used in the extraction and isolation of natural products. This paper also presents the advantage, disadvantage and practical examples of conventional and modern techniques involved in natural products research.
The amounts of active ingredients in natural medicines are always fairly low. The lab-intensive and time-consuming extraction and isolation process has been the bottle neck of the application of natural products in drug development. There is an urgent need to develop effective and selective methods for the extraction and isolation of bioactive natural products. This review intends to provide a comprehensive view of a variety of methods used in the extraction and isolation of natural products.
Extraction is the first step to separate the desired natural products from the raw materials. Extraction methods include solvent extraction, distillation method, pressing and sublimation according to the extraction principle. Solvent extraction is the most widely used method. The extraction of natural products progresses through the following stages: (1) the solvent penetrates into the solid matrix; (2) the solute dissolves in the solvents; (3) the solute is diffused out of the solid matrix; (4) the extracted solutes are collected. Any factor enhancing the diffusivity and solubility in the above steps will facilitate the extraction. The properties of the extraction solvent, the particle size of the raw materials, the solvent-to-solid ration, the extraction temperature and the extraction duration will affect the extraction efficiency [6,7,8,9,10].
The conventional extraction methods, including maceration, percolation and reflux extraction, usually use organic solvents and require a large volume of solvents and long extraction time. Some modern or greener extraction methods such as super critical fluid extraction (SFC), pressurized liquid extraction (PLE) and microwave assisted extraction (MAE), have also been applied in natural products extraction, and they offer some advantages such as lower organic solvent consumption, shorter extraction time and higher selectivity. Some extraction methods, however, such as sublimation, expeller pressing and enfleurage are rarely used in current phytochemical investigation and will not discussed in this review. A brief summary of the various extraction methods used for natural products is shown in Table 1.
Pressurized liquid extraction has been successfully applied by the researchers at the University of Macau and other institutes in extracting many types of natural products including saponins, flavonoids and essential oil from TCM [8, 25,26,27]. Some researchers believed PLE could not be used to extract thermolabile compounds due to the high extraction temperature, while others believed it could be used for the extraction of thermolabile compounds because of the shorter extraction time used in PLE. Maillard reactions occurred when PLE was used at 200 °C to extract antioxidants from grape pomace . Anthocyanins are thermolabile. Gizir et al. successfully applied PLE to obtain an anthocyanin-rich extract from black carrots because the degradation rate of anthocyanins is time-dependent, and the high-temperature-short-duration PLE extraction conditions could overcome the disadvantage of high temperature employed in the extraction .
Supercritical fluid extraction (SFE) uses supercritical fluid (SF) as the extraction solvent. SF has similar solubility to liquid and similar diffusivity to gas, and can dissolve a wide variety of natural products. Their solvating properties dramatically changed near their critical points due to small pressure and temperature changes. Supercritical carbon dioxide (S-CO2) was widely used in SFE because of its attractive merits such as low critical temperature (31 °C), selectivity, inertness, low cost, non-toxicity, and capability to extract thermally labile compounds. The low polarity of S-CO2 makes it ideal for the extraction of non-polar natural products such as lipid and volatile oil. A modifier may be added to S-CO2 to enhance its solvating properties significantly.
Ultrasonic-assisted extraction (UAE), also called ultrasonic extraction or sonication, uses ultrasonic wave energy in the extraction. Ultrasound in the solvent producing cavitation accelerates the dissolution and diffusion of the solute as well as the heat transfer, which improves the extraction efficiency. The other advantage of UAE includes low solvent and energy consumption, and the reduction of extraction temperature and time. UAE is applicable for the extraction of thermolabile and unstable compounds. UAE is commonly employed in the extraction of many types of natural products [32, 33].
The structure of the cell membrane and cell wall, micelles formed by macromolecules such polysaccharides and protein, and the coagulation and denaturation of proteins at high temperatures during extraction are the main barriers to the extraction of natural products. The extraction efficiency will be enhanced by EAE due to the hydrolytic action of the enzymes on the components of the cell wall and membrane and the macromolecules inside the cell which facilitate the release of the natural product. Cellulose, α-amylase and pectinase are generally employed in EAE.
The components in the extract from above methods are complex and contain a variety of natural products that require further separation and purification to obtain the active fraction or pure natural products. The separation depends on the physical or chemical difference of the individual natural product. Chromatography, especially column chromatography, is the main method used to obtain pure natural products from a complex mixture.
Adsorption column chromatography is widely used for the separation of natural products, especially in the initial separation stage, due to its simplicity, high capacity and low cost of adsorbents such as silica gel and macroporous resins. The separation is based on the differences between the adsorption affinities of the natural products for the surface of the adsorbents. The selection of adsorbents (stationary phase) as well as the mobile phase is crucial to achieve good separation of natural products, maximize the recovery of target compounds and avoid the irreversible adsorption of target compounds onto the adsorbents.
Alumina (aluminum oxide) is a strong polar adsorbent used in the separation of natural products especially in the separation of alkaloids. The strong positive field of Al3+ and the basic sites in alumina affecting easily polarized compounds lead to the adsorption on alumina that is different from that on silica gel. The application of alumina in the separation of natural products has decreased significantly in recent years because it can catalyze dehydration, decomposition or isomerization during separation. Zhang and Su reported a chromatographic protocol using basic alumina to separate taxol (74, Fig. 11) from the extract of Taxus cuspidate callus cultures and found the recovery of taxol was more than 160%. They found that the increase of taxol came from the isomerization of 7-epi-taxol (75) catalyzed by alumina. It was also found that a small amount of taxol could be decomposed to baccatin III (76) and 10-deacetylbaccatin III (77) in the alumina column . Further investigation into the separation of taxol on acidic, neutral and basic alumina indicated that the Lewis souci and the basic activity cores on the surface of alumina induced the isomerization of 7-epi-taxol to taxol .
The structures of polyamides used in chromatography contain both acryl and amide groups. Hydrophobic and/or hydrogen bond interaction will occur in polyamide column chromatography depending on the composition of the mobile phase. When polar solvents such as aqueous solvents are used as the mobile phase, the polyamides act as the non-polar stationary phase and the chromatography behavior is similar to reversed-phase chromatography. In the contrast, the polyamides act as the polar stationary phase and the chromatography behavior is similar to normal phase chromatography. Polyamide column chromatography is a conventional tool for the separation of natural polyphenols including anthraquinones, phenolic acids and flavonoids, whose mechanisms are ascribed to hydrogen bond formation between polyamide absorbents, mobile phase and target compounds. Gao et al. studied the chromatography behavior of polyphenols including phenolic acids and flavonoids on polyamide column. It was found that the polyamide functioned as a hydrogen bond acceptor, and the numbers of phenolic hydroxyls and their positions in the molecule affected the strength of adsorption . In addition to polyphenols, the separation of other types of natural products by polyamide column chromatography were also reported. The total saponins of Kuqingcha can be enriched by polyamide column chromatography, which significantly reduced the systolic pressure of SHR rat . Using a mixture of dichloromethane and methanol in a gradient as the eluent, the seven major isoquinoline alkaloids in Coptidis Rhizoma including berberine (39), coptisine (40), palmatine (41), jatrorrhizine (42), columbamine (78), groenlandicine (79) (Fig. 4), and magnoflorine (80, Fig. 11) were separated in one-step polyamide column chromatography .
Adsorptive macroporous resins are polymer adsorbents with macroporous structures but without ion exchange groups that can selectively adsorb almost any type of natural products. They have been widely used either as a standalone system, or as part of a pretreatment process for removing impurities or enriching target compounds due to their advantages, which include high adsorptive capacity, relatively low cost, easy regeneration and easy scale-up. The adsorptive mechanisms of adsorptive macroporous resins include electrostatic forces, hydrogen bonding, complex formation and size-sieving actions between the resins and the natural products in solution. Surface area, pore diameter and polarity are the key factors affecting the capacity of the resins . 20(S)-protopanaxatriol saponins (PTS) (81) and 20(S)-protopanaxadiol saponins (PDS) (82, Fig. 11) are known as two major bioactive components in the root of Panax notoginseng. PTS and PDS were successfully separated with 30 and 80% (v/v) aqueous ethanol solutions from the D101 macroporous resin column, respectively. The chromatography behaviors of PDS and PTS were close to reversed-phase chromatography when comparing the chromatographic profiles of macroporous resin column chromatography to the HPLC chromatogram on a Zorbax SB-C18 column . Recently, Meng et al. obtained the total saponins of Panacis Japonici Rhizoma (PJRS) using D101 macroporous resin. The contents of the four major saponins, chikusetsusaponins V (55), IV (56) and IVa (57), and pseudoginsenoside RT1 (58) (Fig. 8), in the obtained PJRS was more than 73%. The PJRS served as the standard reference for quality control of Panacis Japonici Rhizoma . Some researchers assumed that the principal adsorptive mechanism between macroporous resins and polyphenols was associated with the hydrogen bonding formation between the oxygen atom of the ether bond of the resin and the hydrogen atom of phenolic hydroxyl group of the phenol. The hydrogen bonding interaction force was significantly affected by the pH value of the solution [57, 58]. 2b1af7f3a8