Extraction methods for preparation of bioactive plant extracts
Authors: Waghmare Deepak Harishchandra¹, Deshmukh Arti Madhukar²
Savitribai Phule Pune University, Pune¹
Vasantrao Naik Marathwada Agriculture University, Parbhani²

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. The purpose of all extraction is to separate the soluble plant metabolites, leaving behind the insoluble cellular marc (residue). The initial crude extracts using these methods contain complex mixture of many plant metabolites, viz, alkaloids, glycosides, saponins, phenolics, terpenoids and flavonoids. Some of the initially obtained extracts may be ready for use as medicinal agents in the form of tinctures and fluid extracts but some need further processing. Several extraction methods are used which are discussed below:

1. Maceration:

Maceration is a technique use in wine making and has been adopted and widely used in medicinal plants research. Maceration involved soaking plant materials (coarse or powdered) in a stoppered container with a solvent and allowed to stand at room temperature for a period of minimum 3 days with frequent agitation. The processed intended to soften and break the plant’s cell wall to release the soluble phytochemicals. After 3 days, the mixture is pressed or strained by filtration. In this conventional method, heat is transferred through convection and conduction and the choice of solvents will determine the type of compound extracted from the samples.

1. Infusion:

The infusion is carried out by immersing the plants parts to use in an amount of boiling water, allowed to stand 15 minutes and then filtered through a filter or filter paper. Infusion uses the same principle as maceration; both are soaked in cold or boiled water. However, the maceration period for infusion is shorter and the sample is boiled in specified volume of water (eg. 1:4 or 1:16) for a defined time for decoction.

1. Percolation:

The percolator is a conical vessel with a top opening in which is placed a circular drilled lid allowing the pass of liquid and subjecting the materials placed on it to a slight pressure. The bottom has an adjustable closure to allow passage of the fluid at a convenient rate. The plant material is moistened prior to their placement in the percolator with a proper amount of menstruum, it´s placed in a sealed container and leave stand for approximately four hours. After that time the plant material must be conveniently placed in the percolator so as to allow the even passage of fluid and the complete contact with the plant material. The percolator must be filled with liquid and covered up. The bottom outlet is opened until get a regular dripping and then closes. More menstruum is added to cover all the material and must stand to soak in the percolator closed for 24 hours. After this time leave it to drip slowly and added enough menstruum to a proportional volume of 3/4 of the total volume required for the final product. The wet mass is pressed to extract the maximum residual fluid retained and supplemented with sufficient menstruum to get the proper proportion, it´s filtered or clarified by decantation. The percolation process is usually done at moderate rate (e.g. 6 drops /min) until the extraction is completed before evaporation to get a concentrated extracts.

1. Decoction:

In this process the drug is boiled in water for 15 to 60 minutes (depending on the plant or the active ingredient to extract), it´s cooled, strained and added enough cold water through the drug to obtain the desired volume. Depending on the consistency of the parts to extract, decoction times will be more or less long; generally roots, leaves, flowers and leafy stems are boiled in water for about 15 minutes, while the branches and other hard parts can require up to an hour, during this time the evaporated water must be replacing. Once the decoction is done it´s necessary to filter the liquid through a cloth, squeezing very well the obtained liquid. Doses are similar to the infusion ones, i.e. a plant part per ten of water, except for the plants with high mucilage content in this case will be 1/20 to prevent the solution takes much viscosity. Decoction is mostly suitable for extracting heat-stable compounds, hard plants materials (e.g. roots and barks) and usually resulted in more oil-soluble compounds compared to maceration and infusion. The decoctions are prepared for using in the moment and shouldn’t be stored for more than 24 hours.

1. Digestion:

Digestion is a form of maceration with slight warming during the extraction process, provided that the temperature does not alter the active ingredients of plant material and so there is greater efficiency in the use of menstruum. The most used temperatures are between 35°C and 40°C., although can rise to no higher than 50°C. This process is used with the tougher plant parts or those that contain poorly soluble substances. We introduce the parts to extract in a container with the liquid pre-heated to the indicated temperatures, is maintained for a period that may vary between half an hour and 24 hours, shaking the container regularly.

1. Soxhlet extraction:

In this method, finely ground sample is placed in a porous bag or “thimble” made from a strong filter paper or cellulose, which is place, is in thimble chamber of the Soxhlet apparatus. Extraction solvents is heated in the bottom flask, vaporizes into the sample thimble, condenses in the condenser and drip back. When the liquid content reaches the siphon arm, the liquid contents emptied into the bottom flask again and the process is continued. This method requires a smaller quantity of solvent compared to maceration. However, the Soxhlet extraction comes with disadvantage such as exposure to hazardous and flammable liquid organic solvents, with potential toxic emissions during extraction. Solvents used in the extraction system need to be of high-purity that might add to cost. This procedure is considered not environmental friendly and may contribute to pollution problem compared to advance extraction method such as supercritical fluid extraction (SFE). The ideal sample for Soxhlet extraction is also limited to a dry and finely divided solid and many factors such as temperature, solvent-sample ratio and agitation speed need to be considered for this method.

1. Microwave assisted extraction (MAE):

MAE utilizes microwave energy to facilitate partition of analytes from the sample matrix into the solvent. Microwave radiation interacts with dipoles of polar and polarizable materials (e.g. solvents and sample) causes heating near the surface of the materials and heat is transferred by conduction. Dipole rotation of the molecules induced by microwave electromagnetic disrupts hydrogen bonding; enhancing the migration of dissolved ions and promotes solvent penetration into the matrix. In non-polar solvents, poor heating occurs as the energy is transferred by dielectric absorption only. This technique reduced extraction time and solvent volume as compared to conventional method (maceration & Soxhlet extraction). Improved recoveries of analytes and reproducibility were observed in MAE method but with caution of using proper conditions to avoid thermal degradation. However, this method is limited to small-molecule phenolic compounds such as phenolic acids (gallic acid and ellagic acid), quacertin, isoflavin and trans-resveratrol because these molecules were stable under microwave heating conditions up to 100°C for 20 minutes. Additional cycles of MAE (e.g. from 2×10s to 3×10s) resulted in drastic decrease in the yield of phenolics and flavanones, mainly caused by the oxidation of compounds. Tannins and anthocyanins may not be suitable for MAE as they were potentially subjected to degradation at high temperature.

1. Ultrasound-assisted extraction (UAE) or Sonication:

UAE involves the use of ultrasound ranging from 20 kHz to 2000 kHz. The mechanic effect of acoustic cavitation from the ultrasound increases the surface contact between solvents and samples and permeability of cell walls. Physical and chemical properties of the materials subjected to ultrasound are altered and disrupt the plant cell wall; facilitating release of compounds and enhancing mass transport of the solvents into the plant cells. The procedure is simple and relatively low cost technology that can be used in both small and larger scale of phytochemical extraction. The benefits of UAE is mainly due reduction in extraction time and solvent consumption. However, use of ultrasound energy more than 20 kHz may have an effect on the active phytochemicals through the formation of free radicals.

1. Accelerated solvent extraction (ASE):

ASE is an efficient form of liquid solvent extraction compared to maceration and Soxhlet extraction as the method use minimal amount of solvent. Sample is packed with inert material such as sand in the stainless steel extraction cell to prevent sample from aggregating and block the system tubing. Packed ASE cell includes layers of sand-sample mixture in between cellulose filter paper and sand layers. This automated extraction technology is able to control temperature and pressure for each individual samples and requires less than an hour for extraction. Similar to other solvent technique, ASE also critically depend on the solvent types.

1. Supercritical fluid extraction (SFE):

Supercritical fluid is a substance that shares the physical properties of both gas and liquid at its critical point. Factors such as temperature and pressure are the determinants that push a substance into its critical region. Supercritical fluid behaves more like a gas but have the solvating characteristic of a liquid. An example of Supercritical fluid is CO₂ that become Supercritical fluid at above 31.1°C and 7380kPa. Interest in Supercritical- CO₂ (SC-CO₂ ) extraction due to excellent solvent for nonpolar analytes and CO₂ is readily available at low cost and has low toxicity. Even though SC-CO₂ has poor solubility for polar compounds, modification such as adding small amount of ethanol and methanol enable it to extracts polar compounds. SC-CO₂ also produces analytes at concentrate form as CO₂ vaporizes at ambient temperature. SC-solvents strength can be easily altered by changing the temperature, pressure or by adding modifiers that lead to reduce extraction time. A major drawback of this method is the initial cost of the equipment is very high.

References:

Azwanida NN (2015) A Review on the Extraction Methods Use in Medicinal Plants, Principle, Strength and Limitation. Med Aromat Plants 4:196.

About Author / Additional Info:
I am working as Project Fellow on DST-SERB funded research project entitled "Phytochemical potential of medicinal plants as a source of Antisickling agents in management of sickle cell disease "