Review Article

Novel Techniques for Isolation and Extraction of Phyto-Constituents from Herbal Plants
Shah Megha.V*, Rohit Minal. C.
Department of Quality Assurance, Pioneer Pharmacy Degree College, Ajwa-Cross Road, N.H.-8, Sayajipura, Vadodara-390019, India.

ABSTRACT The techniques used for extraction since decades have the limitations of requiring longer extraction times, large solvent volumes and cause degradation of thermo labile components. Modern methods of extraction are effective in advancing the development of traditional herbal remedies. Various novel techniques including ultrasonicationassisted extraction, microwave-assisted extraction,counter-current extraction and accelerated solvent extraction have been developed for the extraction of nutraceuticals from plants in order to shorten the extraction time, decrease the solvent consumption, increase the extraction yield, and enhance the quality of extracts. A new advancement in the field of extraction was introduction of Phytonics Process, a new solvent based on hydrofluorocarbon-134a technology used for extraction of fragrant components of essential oils and biological or phyto-pharmacological extracts which can be used directly without further physical or chemical treatment. The present article portrays a comprehensive discussion on principles, methodology, benefits and applications of these innovative extraction technologies for procuring active metabolites from different phytoconstituents. Keywords: Ultrasonication-assisted extraction, Microwave-assisted extraction, Counter-current extraction, Accelerated solvent extraction, Phytonics. INTRODUCTION Extraction, as the term is used pharmaceutically, involves the separation of medicinally active portions of plant or animal tissues from the inactive or inert components by using selective solvents in standard extraction procedures. Plants contain a broad range of bioactive compounds such as lipids; phytochemicals, pharmaceutics, flavours, fragrances and pigments.1ExtractionTechniques have been

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American Journal of Phytomedicine and Clinical Therapeutics

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Shah et al__________________________________________________ ISSN 2321 2748 widely investigated to obtain such valuable natural compounds from plants for commercialization. Thereis an increasing demand for new extraction techniques with shortened extraction time, reduced organic solvent consumption, and increased pollution prevention. Novel extraction methods including ultrasonication-assisted extraction, microwave-assisted extraction,accelerated solvent extraction and Phytonics processare fast and efficient for extracting chemicals from solid plant matrixes.2, 3These techniques have the possibility of working at elevated temperatures and/or pressures, greatly decreasing the time of extraction.There has been a need for better and newer extraction techniques, in the herbal drug industry so that the extraction time and the cost of solvent consumption are decreased. EXTRACTION TECHNIQUES FOR PREPARATION OF HERBAL PLANT EXTRACTS Ultrasonication-Assisted Extraction Principle The procedure involves the use of ultrasound waves, which have frequencies higher than 20 kHz, have great effects on extraction yield and kinetics. Unlike electromagnetic waves, sound waves must travel in a matter and they involve expansion and compression cycles during travel in the medium. Expansion pulls molecules apart and compression pushes them together.4The expansion can create bubbles in a liquid and produce negative pressure. The bubbles form, grow and finally collapse. UAE involves ultrasonic effects of acoustic cavitations. Under ultrasonic action solid and liquid particles are vibrated and accelerated and, because of that solute quickly diffuses out from solid phase to solvent.5 Mechanism Several probable mechanisms for ultrasonic enhancement of extraction, such as cell disruption, improved penetration, and enhanced swelling, capillary effect, and hydration process have been proposed. If the intensity of ultrasound is increased in a liquid, then it reaches at a point at which the intramolecular forces are not able to hold the molecular structure intact, so it breaks down and bubbles are created, this process is called cavitation.6 Collapse of bubbles can produce physical, chemical and mechanical effects which result in the disruption of biological membranes to facilitate the release of extractable compounds and enhance penetration of solvent into cellular materials. Thebeneficial effects of sound waves on extraction are attributed to the formation and asymmetrical collapse of micro cavities in the vicinity of cell walls leading to the generation of micro jets rupturing the cells.7 Extraction of the tea solids from dried leaves with water using ultrasound gave 20% improvement in extraction yield. UAE also proved better for extraction of carnosic acid by using different solvents viz. ethanol, ethyl acetate, and butanone and also reduced the extraction time. UAE provides better extraction of the vanillin in shorter time period for different solvents as compared to the Soxhlet method.8 Types of Ultrasound Extractors Two general designs of ultrasoundassisted extractors are ultrasonic baths or closed extractors fitted with an ultrasonic horn transducer. The mechanical effects of ultrasound induce a greater penetration of solvent into cellular materials and improve mass transfer. Ultrasound in extraction can also disrupt biological cell walls, facilitating the release of contents. Therefore, efficient cell disruption and effective mass transfer are cited as two major factors leading to the enhancement of extraction with ultrasonic power. The use of ultrasound allows changes

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Shah et al__________________________________________________ ISSN 2321 2748 in the processing condition such as a decrease of temperature and pressure from those used in extractions without ultrasound. Therefore, use of ultrasound-assisted extraction is advisable for thermolabile compounds, since ultrasound generates heat, it is important to accurately control the extraction temperature. The sonication time should also be considered carefully as excess of sonication can damage the quality of extracts.9 Advantages of Ultra sonicated extraction Ultrasound-assisted extraction is an inexpensive, simple and efficient alternative to conventional extraction technique. The main benefits of use of ultrasound in solidliquid extraction include the increase of extraction yield andfaster kinetics. Ultrasound can also reduce the operating temperature allowing the extraction of thermolabilecompounds. 10 Compared with other novel extraction techniques such as microwave-assisted extraction, the ultrasound apparatus is cheaper and its operation is easier. The ultrasound-assisted extraction, like Soxhlet extraction, can be used with any solvent for extracting a wide variety of natural compounds.10 Disadvantages of Ultra sonicated extraction A known deleterious effect of ultrasound energy (more than 20 kHz) on the active constituents of medicinal plants through formation of free radicals and consequently undesirable changes in the drug molecules.40 Applications of Ultrasound-assisted Extraction 1. Ultrasound-assisted extraction has been used to extract nutraceuticals from plants such as essential oils and lipids dietary supplements. It found that ultrasonication was a criticalpre-treatment to obtain high yields of oils from almond, apricot and rice bran.11, 12 For ultrasound-assisted extraction of saponinfrom ginseng, the observed total yield and saponin yield increased by 15 and 30%, respectively.13 Ultrasound can increase extraction kinetics and even improve the quality of extracts.It wasfound that rice bran oil extraction can be efficientlyperformed in 30 min under high-intensity ultrasound eitherusing hexane or a basic aqueous solution. 14 Extraction ratesof carvone and limonene by ultrasound-assisted extractionwith hexane were 1.32 times more rapid than those by the conventional extraction depending on temperature.15 The ultrasound was also applied to the cartridge of a Soxhlet extraction for the extraction of total fat from oleaginous seeds such as sunflower, rape and soybean seeds. The use of ultrasound reduced the extraction at least to half of the time needed by conventional extraction methods without any change in the composition of extracted oils.11 Ultrasound-assisted extraction was considered as an efficient method for extracting bioactive compounds from Solvia officinalis16 and Hibiscus tiliaceus L. flowers,17 antioxidants from Rosmarinus officinalis,18 and steroids and triterpenoids from Chresta spp.19

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MICROWAVE-ASSISTED EXTRACTION Microwaves are electromagnetic fields in the frequency range of 300 MHz to 300 GHz or between wavelengths of 1 cm and 1m. Microwaves heat the whole sample simultaneously. In the case of extraction, the advantage of microwave heating is the disruption of weak hydrogen bounds promoted by the dipole rotation of the

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Shah et al__________________________________________________ ISSN 2321 2748 molecules.20 The effect of microwave energy is strongly dependent on the nature of both the solvent and the solid matrix. Solvents generally used cover a wide range of polarities, from heptane to water.Sometimes the matrix itself interacts with microwaves while the surrounding solvent possesses a low dielectric constant which is advantageous for thermo-sensitive compounds and hence, useful for extraction of essential oils. Solvent free MAE (SFMAE) has been designed, where the moisture content within the plant matrix itself serves extraction and no solvent are used.21 Principle Microwave-assisted extraction (MAE) offers a rapid delivery of energy to a total volume of solvent and solid plant matrix with subsequent heating of the solvent and solid matrix, efficiently and homogeneously.22 Components of the sample absorb microwave energy in accordance to their dielectric constants. When plant material is immersed inside a microwave transparent solvent, the heat of microwave radiation directly reaches to the solid without being absorbed by the solvent, resulting in instantaneous heating of the residual moisture in the solid. Heating causes the moisture to evaporate and creates a high vapour pressure that breaks the cell wall of substrate and releases the content into solvent. The extracting selectivity and the ability of the solvent tointeract with microwaves can be modulated by usingmixtures of solvents.23 One of themost commonly used mixtures is hexane-acetone. During extraction, the solvent volume must be sufficient to ensure that the solid matrix is entirely immersed.Generally, a higher ratio of solvent volume to solid matrix mass in conventional extraction techniques can increase the recovery.24 Instrumentation MAE can be practiced in two different modes- one is closed vessel operation that is under controlled (elevated) pressure and temperature, another is open vessel operation performed at atmospheric pressure. These technologies are named as pressurized microwave assisted extraction (PMAE) and focused microwave assisted extraction (FMAE), respectively.25 Closed vessel systems- Such systems are generally advised for digestions or acid mineralisation or for extractions under drastic conditions, since the solvents may be heated to ca. 100C above their atmospheric boiling point. Both extraction speed and efficiency are enhanced in this procedure. The closed vessel system is most suitable for volatile compounds.26 Open cells- These cells are quartz vessels topped by a vapour condenser. The system works at atmospheric pressure, and the maximum temperature is determined by the boiling point of the solvent used. The solvent is heated and refluxed through the sample, and in this case the microwaves are focused on the sample placed into the vessel allowing homogeneous and very efficient heating. The sample to be extracted can be placed into a Soxhlet-type cellulose cartridge in order to avoid filtration steps, or may be directly dipped into the solvent. Open cells offer increased safety in sample handling and they allow larger samples to be extracted. 27 Advantages of Microwave Assisted Extraction The main advantages of microwave assisted extraction over the conventional extraction techniques is that it reduces solvent consumption, it has a shorter operational time, it possess moderately high recoveries, has a good reproducibility and minimal sample manipulation for extraction process. MAE is also comparable to other modern extraction techniques such as supercritical fluid

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Shah et al__________________________________________________ ISSN 2321 2748 extraction due to its process simplicity and low cost.10 Disadvantages of Microwave Assisted Extraction Compared to SFE, an additional filtration or centrifugation is necessary to remove the solid residue during MAE. Furthermore, the efficiency of microwaves can be very poor when either the target compounds or the solvents are non-polar, or when they are volatile.10 Applications of Microwave-Assisted Extraction 1. MAE can extract nutraceuticals products from plant sources in a faster manner than conventional solidliquid extractions. MAE of the puerarin from the herb Radix puerariae could be completed within 1 min.28 MAE (80% methanol) could dramatically reduce the extraction time of ginseng saponin from 12 h using conventional extraction methods to a few seconds.29 2. Numerous biologically active compounds have been extracted by application of microwave-assisted extraction, such as extraction of taxanes from Taxus brevifolia needles, extraction of azadiractine related limonoids from Azadirachta indica seed kernels, extraction of glycyrrhizic acid from Glycyrrhizia glabra roots, extraction of tanshinones from Salvia miltorrhiza bung, 30 extraction of artemisinin from Artemisia annua.31 3. A higher microwave temperature and a short extraction time are more effective in extracting anti-oxidative phenolic compounds from tomato using MAE.32 4. MAE was proven as a potential alternative to traditional methods for extraction of phenols such as chlorogenic acids from green coffee beans and prepared extracts were also showing a good radical scavenging activity.33 ACCELERATED SOLVENT EXTRACTION A form of Pressurized Solvent Extraction is a solidliquid extraction process performed at elevated temperatures, usually between 50 and 200 deg C and at pressures between 10 and 15 MPa. Increased temperature accelerates the extraction kinetics and elevated pressure keeps the solvent in the liquid state, thus achieving safe and rapid extraction. Also, pressure allows the extraction cell to be filled faster and helps to force liquid into the solid matrix. Elevated temperatures enhance diffusivity of the solvent resulting in increased extraction kinetics.34 Instrumentation and Extraction Procedure The solvent used in ASE is usually organic solvents, even pressurized hot-water, or subcritical water can also be used in an ASE apparatus.34 A solid or semi-solid sample is placed into a stainless steel extraction cell which is filled with solvent and heated (50200 deg C) in an oven.33 The heating process generates solvent expansion and thus pressure in the extraction cell, typically in the region of 5003000 psi. All the solvent present in the system is then purged with a compressed gas, generally nitrogen. The Dionex apparatus for carrying out extraction offers the possibility of automation and up to 24 samples can be sequentially extracted.35 Advantages Use of non-toxic extracting solvents such as carbon dioxide and water has economic and environmental benefits. Accelerated solvent extraction is considered as a potential alternative technique to SFE for the extraction of polar compounds. 36

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Shah et al__________________________________________________ ISSN 2321 2748 Compared with traditional Soxhlet extraction, there is a dramatic decrease in the amount of solvent and the extraction time for ASE.33 Disadvantages This technique performed with high extraction temperature may cause degradation of thermolabile compounds. Applications to extraction of natural products 1. Accelerated solvent extraction (ASE) is usually used for the extraction of hightemperature stable organic pollutants from environmental matrices. 2. A single ASE treatment with 20 mL hexane (for defatting) followed by 5 min in methanol lead to the quantitative extraction of the flavonolignans, e.g. Silybin contained in milk thistle fruit.38 3. The performance of ASE with traditional Soxhlet extraction for the recovery of steroids from the leaves of Iochroma gesnerioides was compared. They found ASE produced similar results to Soxhlet in terms of recovery, repeatability and selectivity. However, both extraction time and solvent consumption were dramatically reduced with ASE.39 4. ASE with methanol was used for the extraction of taxanes from the bark of Taxus cuspidateand permitted the extraction of paclitaxel, baccatin III and 10-deacetylbaccatin III (10-DAB) in higher yields than could be obtained using classical SLE.40 5. ASE was applied to the recovery of naphthodianthrones (e.g. hypericin) from extracts and pharmaceuticals containing St Johns wort (Hypericum perforatum).41 6. PSE was applied to the rapid extraction of cocaine and benzoylecgonine from coca leaves. It was demonstrated that 10 min were sufficient to extract cocaine quantitatively at 80C and 20 MPa.37 COUNTER-CURRENT EXTRACTION A liquid-liquid extraction process in which the solvent and the process stream in contact with each other flow in opposite directions. Unlike maceration and percolation, which are batch processes, this method is continuous. Screw extractors and carousel extractors are the two type of equipments used for Counter-Current Extraction.1 In counter-current extraction (CCE), wet raw material is pulverized using toothed disc disintegrators to produce fine slurry. In this process, the material to be extracted is moved in one direction (generally in the form of fine slurry) within a cylindrical extractor where it comes in contact with extraction solvent. The further the starting material moves, the more concentrated the extract becomes. Complete extraction is thus possible when the quantities of solvent and material and their flow rates are optimized. The process is highly efficient, requiring little time and posing no risk from high temperature. Finally, sufficiently concentrated extract comes out at one end of the extractor while the marc (practically free of visible solvent) falls out from the other end.42 This extraction process has significant advantages: 1. A unit quantity of the plant material can be extracted with much smaller volume of solvent as compared to other methods like maceration, decoction, and percolation. 2. CCE is commonly done at room temperature, which spares the thermolabile constituents from exposure to heat which is employed in most other techniques.42 3. As the pulverization of the drug is done under wet conditions, the heat generated during comminution is neutralized by water. This again spares the thermolabile constituents from exposure to heat.

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Shah et al__________________________________________________ ISSN 2321 2748 4. The extraction procedure has been rated to be more efficient and effective than continuoushot extraction.1 Applications 1. DNA purification: The ability to purify DNA from a sample is important for many modern biotechnology processes. However, samples often contain nucleases that degrade the target DNA before it can be purified. It has been shown that DNA fragments will partition into the light phase of a polymersalt separation system. If ligands known to bind and deactivate nucleases are incorporated into the polymer phase, the nucleases will then partition into the heavy phase and be deactivated. Thus, this polymersalt system is a useful tool for purifying DNA from a sample while simultaneously protecting it from nucleases.43 2. Food Industry: The Polyethylene glycol NaCl system has been shown to be effective at partitioning small molecules, such as peptides and nucleic acids. These compounds are often flavorants or odorants. The system could then be used by the food industry to isolate or eliminate particular flavours.43 3. The counter current extraction process has also been applied to natural material processing such as citrus oils, unsaturated fatty acids, and squalenetocopherol. An important application is citrus oil processing, an important subject in perfumes and food industries.43 safety benefits over traditional processes for the production of high quality natural fragrant oils, flavours and biological extracts. Advanced Phytonics Limited (Manchester, UK) has developed this patented technology termed phytonics process. The products mostly extracted by this process are fragrant components of essential oils and biological or phytopharmacological extracts which can be used directly without further physical or chemical treatment. The properties of the new generation of fluorocarbon solvents have been applied to the extraction of plant materials.43 The core of the solvent is 1, 1, 2,2tetrafluoroethane, better known as hydrofluorocarbon-134a (HFC-134a). This product was developed as a replacement for chlorofluorocarbons. The boiling point of this solvent is -25 C. It is not flammable or toxic. Unlike chlorofluorocarbons, it does not deplete the ozone layer. It has a vapour pressure of 5.6 barat ambient temperature. By most standards this is a poor solvent. For example, it does not mix with mineral oils or triglycerides and it does not dissolve plant wastes. The process is advantageous in that the solvents can be customized: by using modified solvents with HFC-134a, the process can be made highly selective in extracting a specific class of phytoconstituents. Similarly, other modified solvents can be used to extract a broader spectrum of components. The biological products made by this process have extremely low residual solvent. The residuals are invariably less than 20 parts per billion and are frequently below levels of detection.44 These solvents are neither acidic nor alkaline and, therefore, have only minimal potential reaction effects on the botanical materials. The processing plant is totally sealed so that the solvents are continually recycled and fully recovered at the end of eachproduction cycle. The only utility needed to operate these systems is electricity and, even then,they do no consume much energy. There is no scope

PHYTONICS PROCESS: AN EXTRACTION METHODOLOGY A new solvent based on hydrofluorocarbon-134a and a new technology to optimize its remarkable properties in the extraction of plant materials offer significant environmental advantages and health and
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Shah et al__________________________________________________ ISSN 2321 2748 for the escape of the solvents. Even if some solvents do escape, they contain no chlorine and therefore pose no threat to the ozone layer. The waste biomass from these plants is dry and eco-friendly to handle.43 Advantages of the Process Unlike other processes that employ high temperatures, the phytonics process is cool and gentle and its products are never damaged by exposure to temperatures in excess of ambient. 1. No vacuum stripping is needed which, in other processes, leads to the loss of precious volatiles. 2. The process is carried out entirely at neutral pH and, in the absence of oxygen; the products never suffer acid hydrolysis damage or oxidation. 3. The technique is highly selective, offering a choice of operating conditions and hence a choice of end products. 4. It is less threatening to the environment. 5. It requires a minimum amount of electrical energy.45 6. It releases no harmful emissions into the atmosphere and the resultant waste products (spent biomass) are innocuous and pose no effluent disposal problems. 7. The solvents used in the technique are not flammable, toxic or ozone depleting. 8. The solvents are completely recycled within the system.46 Applications 1. The phytonics process can be used for extraction in biotechnology (e.g. for the production ofantibiotics), in the herbal drug industry, in the food, essential oil and flavour industries, and in the production of other pharmacologically active products. 2. In particular, it is used in the production of top quality pharmaceutical-grade extracts, pharmacologically active intermediates, antibiotic extracts and phytopharmaceuticals. 42 3. The technique is being used in the extraction of high-quality essential oils, oleoresins, natural food colours, flavours and aromatic oils from all manner of plant materials. 4. The technique is also used in refining crude products obtained from other extraction processes. It provides extraction without waxes or other contaminants. It helps remove manybiocides from contaminated biomass.42 REFERENCES
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