INTRODUCTION Liquid–liquid extraction, also known as solvent extraction and partitioning, is a method to separate compounds based on their relative
solubilities in two different immiscible liquids, usually water and an organic solvent. It is an extraction of a substance from one liquid phase into another liquid phase. Liquid–liquid extraction is a basic technique in chemical laboratories, where it is performed using a separating funnel (Hu, 1999).
Figure 1: Feed phase or feed solution contains the target compound and the feed phase is called the raffinate after the extraction (Hu, 1999). Kupchan’s extraction method is probably the most popular liquid-liquid fractionation scheme for plant extracts. Medicinal plants possess various medicinal properties; have been serving as the major sources of therapeutic agents for maintenance of human health. These medicinal plants were used by the early human beings, as are done now, in a variety of forms, such as in the entire
form, and as powders, pastes, juices, infusions and decoctions for the treatment of their various diseases and ailments. This way, the medicinal plants formed an integral part of the health management practices and constituted important items of medicines used in the treatment of diseases from the very early days of human civilization (Sarker et al., 2006). HISTORY In the early 1960's, Jonathan Hartwell of the United States National Cancer Institute organized collection of plants from the U.S. for evaluation as potential sources of anticancer drugs. These collections were conducted by botanists from the U.S. Department of Agriculture. Plants were shipped from the field to chemistry laboratories where they were extracted with solvents to dissolve agents present in the plant for testing as possible cancer drug candidates. Monroe Wall, Morris Kupchan, Jack Cole, Norman Farnsworth, and G. Robert Pettit were some of the chemists involved (American Society of Pharmacognosy; 2011). S. Morris Kupchan was a chemist and cancer researcher and is best known for his work with plant-derived drugs. While his continuing search for tumor inhibitors from plant sources has yielded over two hundred extracts with reproducible growth-inhibitory activity. He discovered a systematic fractionation method, now known as Kupchan extraction method, guided by assay in cell culture and animal tumor systems, has led to the isolation of the active principles of more than sixty plants. His systemic extraction procedure lead to the extraction of even minute amount of compounds like the isolation of the tumor-inhibitory principle, withaferin A, as shown in the flow chart below (Kupchan, 1969).
Figure 2: Fractionation of tumor-inhibitory extract from Acuistus arborescens (Kupchan, 1969). KUPCHAN EXTRACTION PROCEDURE The fresh leaves were collected, cut into small pieces and air dried for several days. The plant materials were then ground into coarse powder. The dried and ground plant powder (500g) was extracted with methanol (2.5 liters) in an air tight, clean flat bottomed container for seven days at room temperature with occasional stirring and shaking. The extract was then filtered first through a fresh cotton plug and finally with a Whitman filters paper. The filtrate was concentrated using a rotary evaporator at low temperature (30ºC) and pressure. The weight of the crude extract was measured (Muhit et al., 2010).
Figure 3: Schematic representation of the modified Kupchan partitioning of methanolic crude extracts (Muhit et al., 2010). Solvent-solvent partitioning was done by using the protocol designed by Kupchan and Tsou (1973) and modified version of Wagenen et al. (1993). 5gm of the crude extract was triturated with 90% methanol. The prepared solution was then fractionated successfully using solvents of increasing polarity, such as n-hexane (HX: 820mg), carbon tetrachloride (CT: 550mg) and chloroform (CF: 665mg). All these three fractions were evaporated to dryness by using rotary evaporators at low temperature of 390ºC and kept in air tight containers for further analysis (Muhit et al., 2010).
Later the fractions are analyzed to evaluate the in vitro antimicrobial, antioxidant, cytotoxic and/or thrombolytic activities of n-hexane, ethyl acetate and methanol extracts of leaves. The leaves extracts were screened for major phytochemicals using established procedures. Phytochemical screenings of the extracts of leaves are also done to investigate if various bioactive components such as alkaloids, anthraquinones flavonoids, saponins and steroids are present (Apu et al., 2012). ADVANTAGES AND DISADVANTAGES OF KUPCHAN PARTITIONING METHOD This method is the most well-known liquid-liquid extraction process since even the most minute quantity of the substance can be easily extracted and this help in the discovery of many new compounds which were impossible to obtain while following the classical extraction approach. The polar and the non-polar compounds can be easily seperated (Kupchan, 1969). However problems related to this process includes wastage of a large quantity of solvents, very time consuming and tedious, and many precautions need to be followed, like the densities between the feed phase and the organic solvent need to be high in order to prevent formation of stable emulsions; targeted compound must have higher affinity towards the organic solvent or otherwise extraction of target compound will not take place; the temperature should be maintained or otherwise a change in temperature may cause a decrase or increase of solubility of target compound in the organic solvent. CONCLUSION Over the years Kupchan’s method of extraction has been modified as shown in the figure below. Here most fats will go with the n-hexane fraction, while inorganic salts will go with the aqueous one. The advantage of the method is total recovery of the target compounds. Drawbacks are the
problems of emulsion formation, time ineffectiveness, and use of large volume of solvents (Sarkar et al., 2006).
Figure 4: Modified Kupchan’s partion scheme (Sarkar et al., 2006). Kupchan’s fractionation is probably the most popular liquid-liquid fractionation scheme for plant extracts, but it is exceedingly replaced by solid phase extraction protocols based on absorption on a solid matrix and de-absorption with solvents of growing affinity for the solid phase, e.g. petroleum ether/ethyl acetate/acetone for silica gel, mixture of water/methanol (ethanol) with increasing amounts of alcohol for RP-18 silica gel or its cheaper polystyrene resin alternative (Buss and Bulter, 2010).
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Buss, A., D. and Butler, M., S. (Ed.) (2010). Natural Product Chemistry for Drug Discovery. UK: The Royal Society of Chemistry
Hu, X. (1999). Liquid-Liquid Extraction (LLX). Retrieved February 6, 2013. Web site: http://ihome.ust.hk/~kexhu/ceng3210/221-08.pdf Kupchan, S., M. (1969). Recent Advances in the Chemistry of Terpenoid Tumor Inhibitors. University of Wisconsi. Retrieved February 6, 2013. Web site: www.iupac.org/publications/pac/21/2/0227/pdf/ Muhit, M., A., Tareq, S.,M., Apu A.,S., Basak D. and Islam, M.,S. (2010). Isolation and Identification of Compounds from the Leaf Extract of Dillenia indica Linn. Bangladesh Pharmaceutical Journal, 13(1). Sarker, S., D., Latif, Z. AND Gray, A., I. (2006). Natural Products Isolation. New Jersey; Humana Press Inc. The American Society of Pharmacognosy (2011). The Story of Taxol. Retrieved February 6, 2013. Web site: http://www.pharmacognosy.us/wordpress/wp-content/uploads/The-Storyof-Taxol.pdf