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Extraction, Isolation and Characterization of Bioactive Compounds from Plants' Extracts

Views:38     Author:Natalia     Publish Time: 2015-12-22      Origin:African Journal of Traditional, Complementary, and Alternative Medicines

Abstract

Natural products from medicinal plants, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug leads because of the unmatched availability of chemical diversity. Due to an increasing demand for chemical diversity in screening programs, seeking therapeutic drugs from natural products, interest particularly in edible plants has grown throughout the world. Botanicals and herbal preparations for medicinal usage contain various types of bioactive compounds. The focus of this paper is on the analytical methodologies, which include the extraction, isolation and characterization of active ingredients in botanicals and herbal preparations. 


The common problems and key challenges in the extraction, isolation and characterization of active ingredients in botanicals and herbal preparations are discussed. As extraction is the most important step in the analysis of constituents present in botanicals and herbal preparations, the strengths and weaknesses of different extraction techniques are discussed. The analysis of bioactive compounds present in the plant extracts involving the applications of common phytochemical screening assays, chromatographic techniques such as HPLC and, TLC as well as non-chromatographic techniques such as immunoassay and Fourier Transform Infra Red (FTIR) are discussed.


Keywords: Bioactive compound, Plant Extraction, Isolation, Herbal preparations, Natural products Introduction


Natural products, such as plants extract, either as pure compounds or as standardized extracts, provide unlimited opportunities for new drug discoveries because of the unmatched availability of chemical diversity (Cos et al., 2006). According to the World Health Organization (WHO), more than 80% of the world's population relies on traditional medicine for their primary healthcare needs. The use of herbal medicines in Asia represents a long history of human interactions with the environment. Plants used for traditional medicine contain a wide range of substances that can be used to treat chronic as well as infectious diseases (Duraipandiyan et al., 2006). Due to the development of adverse effects and microbial resistance to the chemically synthesized drugs, men turned to ethnopharmacognosy. They found literally thousands of phytochemicals from plants as safe and broadly effective alternatives with less adverse effect. 


Many beneficial biological activity such as anticancer, antimicrobial, antioxidant, antidiarrheal, analgesic and wound healing activity were reported. In many cases the people claim the good benefit of certain natural or herbal products. However, clinical trials are necessary to demonstrate the effectiveness of a bioactive compound to verify this traditional claim. Clinical trials directed towards understanding the pharmacokinetics, bioavailability, efficacy, safety and drug interactions of newly developed bioactive compounds and their formulations (extracts) require a careful evaluation. Clinical trials are carefully planned to safeguard the health of the participants as well as answer specific research questions by evaluating for both immediate and long-term side effects and their outcomes are measured before the drug is widely applied to patients.


According to the World Health Organization (WHO), nearly 20,000 medicinal plants exist in 91 countries including 12 mega biodiversity countries. The premier steps to utilize the biologically active compound from plant resources are extraction, pharmacological screening, isolation and characterization of bioactive compound, toxicological evaluation and clinical evaluation. A brief summary of the general approaches in extraction, isolation and characterization of bioactive compound from plants extract can be found in Figure 1. This paper provides details in extraction, isolation and characterization of bioactive compound from plants extract with common phytochemical screening assay, chromatographic techniques, such as HPLC, and HPLC/MS and Fourier Transform Mass Spectrometry (FTMS).


                                                  Figure 1

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                               An external file that holds a picture, illustration, etc. Object name is AJT0801-0001Fig1.jpg



Figure 1

A brief summary of the general approaches in extraction, isolation and characterization of bioactive compound from plants extract

 

Extraction

Extraction is the crucial first step in the analysis of medicinal plants, because it is necessary to extract the desired chemical components from the plant materials for further separation and characterization. The basic operation included steps, such as pre-washing, drying of plant materials or freeze drying, grinding to obtain a homogenous sample and often improving the kinetics of analytic extraction and also increasing the contact of sample surface with the solvent system. Proper actions must be taken to assure that potential active constituents are not lost, distorted or destroyed during the preparation of the extract from plant samples. If the plant was selected on the basis of traditional uses (Fabricant and Farnsworth, 2001), then it is needed to prepare the extract as described by the traditional healer in order to mimic as closely as possible the traditional ‘herbal’ drug. The selection of solvent system largely depends on the specific nature of the bioactive compound being targeted. Different solvent systems are available to extract the bioactive compound from natural products. The extraction of hydrophilic compounds uses polar solvents such as methanol, ethanol or ethyl-acetate. For extraction of more lipophilic compounds, dichloromethane or a mixture of dichloromethane/methanol in ratio of 1:1 are used. In some instances, extraction with hexane is used to remove chlorophyll (Cos et al., 2006).

 

As the target compounds may be non-polar to polar and thermally labile, the suitability of the methods of extraction must be considered. Various methods, such as sonification, heating under reflux, soxhlet extraction and others are commonly used (United States Pharmacopeia and National Formulary, 2002; Pharmacopoeia of the People's Republic of China, 2000; The Japanese Pharmacopeia, 2001) for the plant samples extraction. In addition, plant extracts are also prepared by maceration or percolation of fresh green plants or dried powdered plant material in water and/or organic solvent systems. A brief summary of the experimental conditions for the various methods of extraction is shown in Table 1. 


    Table 1

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Table 1

A brief summary of the experimental conditions for various methods of extraction for plants material

The other modern extraction techniques include solid-phase micro-extraction, supercritical-fluid extraction, pressurized-liquid extraction, microwave-assisted extraction, solid-phase extraction, and surfactant-mediated techniques, which possess certain advantages. These are the reduction in organic solvent consumption and in sample degradation, elimination of additional sample clean-up and concentration steps before chromatographic analysis, improvement in extraction efficiency, selectivity, and/ kinetics of extraction. The ease of automation for these techniques also favors their usage for the extraction of plants materials (Huie, 2002).


Identification and characterization

Due to the fact that plant extracts usually occur as a combination of various type of bioactive compounds or phytochemicals with different polarities, their separation still remains a big challenge for the process of identification and characterization of bioactive compounds. It is a common practice in isolation of these bioactive compounds that a number of different separation techniques such as TLC, column chromatography, flash chromatography, Sephadex chromatography and HPLC, should be used to obtain pure compounds. The pure compounds are then used for the determination of structure and biological activity. Beside that, non-chromatographic techniques such as immunoassay, which use monoclonal antibodies (MAbs), phytochemical screening assay, Fourier-transform infrared spectroscopy (FTIR), can also be used to obtain and facilitate the identification of the bioactive compounds.

Chromatographic techniques

 

Thin-layer chromatography (TLC) and Bio-autographic methods

TLC is a simple, quick, and inexpensive procedure that gives the researcher a quick answer as to how many components are in a mixture. TLC is also used to support the identity of a compound in a mixture when the Rf of a compound is compared with the Rf of a known compound. Additional tests involve the spraying of phytochemical screening reagents, which cause color changes according to the phytochemicals existing in a plants extract; or by viewing the plate under the UV light. This has also been used for confirmation of purity and identity of isolated compounds.


Bio-autography is a useful technique to determine bioactive compound with antimicrobial activity from plant extract. TLC bioautographic methods combine chromatographic separation and in situ activity determination facilitating the localization and target-directed isolation of active constituents in a mixture. Traditionally, bioautographic technique has used the growth inhibition of microorganisms to detect anti-microbial components of extracts chromatographed on a TLC layer. This methodology has been considered as the most efficacious assay for the detection of anti-microbial compounds (Shahverdi, 2007). Bio-autography localizes antimicrobial activity on a chromatogram using three approaches: (i) direct bio-autography, where the micro-organism grows directly on the thin-layer chromatographic (TLC) plate, (ii) contact bio-autography, where the antimicrobial compounds are transferred from the TLC plate to an inoculated agar plate through direct contact and (iii) agar overlay bio-autography, where a seeded agar medium is applied directly onto the TLC plate (Hamburger and Cordell, 1987; Rahalison et al., 1991). The inhibition zones produced on TLC plates by one of the above bioautographic technique will be use to visualize the position of the bioactive compound with antimicrobial activity in the TLC fingerprint with reference to Rf values (Homans and Fuchs, 1970). Preparative TLC plates with a thickness of 1mm were prepared using the same stationary and mobile phases as above, with the objective of isolating the bioactive components that exhibited the antimicrobial activity against the test strain. These areas were scraped from the plates, and the substance eluted from the silica with ethanol or methanol. Eluted samples were further purified using the above preparative chromatography method. Finally, the components were identified by HPLC, LCMS and GCMS. Although it has high sensitivity, its applicability is limited to micro-organisms that easily grow on TLC plates. Other problems are the need for complete removal of residual low volatile solvents, such as n-BuOH, trifluoroacetic acid and ammonia and the transfer of the active compounds from the stationary phase into the agar layer by diffusion (Cos et al., 2006). Because bio-autography allows localizing antimicrobial activities of an extract on the chromatogram, it supports a quick search for new antimicrobial agents through bioassay-guided isolation (Cos et al., 2006). The bioautography agar overlay method is advantageous in that, firstly it uses very little amount of sample when compared to the normal disc diffusion method and hence, it can be used for bioassay-guided isolation of compounds. Secondly, since the crude extract is resolved into its different components, this technique simplifies the process of identification and isolation of the bioactive compounds (Rahalison et al., 1991).


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