For millennia, natural compounds from plants have served as indispensable resources for human health and wellbeing. [1] Forming a large bulk of the natural products, these compounds have received renewed attention for their pharmaceutical, nutraceutical, agricultural, cosmetic and recreational value. [2] Increasing concerns over synthetic compounds due to their declining efficacy, adverse health effects and harmful environmental impacts, has further fuelled the demand for natural alternatives. [3]
However, the lab-scale studies used to inform and refine commercial separation processes are typically hampered by the use of inefficient and outdated techniques. [4] Hence, the commercial exploitation of natural compounds is often constrained by low yields, high operational costs and low profitability. [5], [6]
Nonetheless, the increasing global demand for natural products has led to increased investment in research and development. This has rejuvenated innovation, leading to the development of novel technologies that improve extraction, purification and production processes. These advancements have progressed sustainable commercialization of plant-based natural products, discovery of novel compounds, and effective harnessing of nature’s fragile secrets. [7]
Classification
Natural products are compounds derived from living organisms (terrestrial or marine), where those of plant origin are called phytochemicals. A large proportion of phytochemicals are secondary metabolites.
They are synthesized in relatively small quantities and are not essential to the maintenance of a plant’s life. However, phytochemicals produced through secondary metabolism enhance the plant’s adaptability and resilience to environmental stresses due to their wide-ranging physiological and biological functions [8].
According to chemical structure, they are categorized as polyphenols (phenolic acids, flavonoids and tannins), phytosterols, carotenoids, terpenes, alkaloids or sulfur-containing compounds (sulfides, glucosinolates). They exhibit multiple bioactivities such as antioxidant, antimicrobial, anti-inflammatory, and or anticancer. From a physical perspective, they can be hydrophilic or hydrophobic, polar or non-polar, stable or thermolabile and or volatile. [5], [8], [9]
To harness these useful phytochemicals, they usually have to be extracted from their natural source. How the extraction procedure is performed affects the accuracy of extraction data, extract quality and associated costs.
Analytical, Semi-Preparative and Industrial Approaches to Extraction
An analytical approach to phytochemical extraction is normally adopted for lab-scale phytochemical screening, mechanistic studies or other preliminary investigations that guide further studies.
For qualitative analysis, the focus is to confirm the presence or absence of certain phytochemicals. Here, partial extraction may suffice where qualitative studies are usually used in screening and preliminary investigations of plant compounds. Damage to the target compounds caused by degradation or chemical alterations, and the coextraction of interfering compounds, are not so critical so long as the target phytochemicals can be detected and identified.
With quantitative analysis, the extraction process must be exhaustive, meaning the target phytochemicals are extracted from the plant material as much as possible, for accurate and reproducible results. Quantified analytical data is used to precisely assess bioactivity, improve extraction processes, ensure extract consistency or to support the development of industrial processes. Quantitation often involves the optimization of extraction parameters for your target compounds. [10], [11], [12]
Semi-preparative
With semi-preparative phytochemical extractions, the goal is to produce small quantities of highly purified extract. This approach typically involves additional preparative separation techniques (crystallization, prep-HPLC) and compound characterisation techniques (MS, NMR). The semi-preparative approach is fundamental in the discovery of novel compounds, and other small-scale studies such as process optimization used to develop industrial processes. [12], [13]
Industrial
The industrial approach to phytochemical extraction involves the large-scale production or manufacture of a commercial natural product with specified characteristics at a reasonable cost. The purity of the extract can range from undefined concentrations to 98 % and above. This approach usually involves validated and optimized data from analytical and semi-preparative lab-scale studies that are then scaled-up and standardized to suit industrial application. [4], [12]
Challenges
Common challenges experienced with the three approaches include:
- Factors that limit the extraction of sufficient quantities of target compounds
- Interference from coextracted compounds
- Poor reproducibility
- Technical difficulties in achieving desired level of purity
- Time and cost constraints
- Scale-up issues
- Environmental and regulatory compliance issues
- Product stability issues
Principles of Extraction
Mechanism
Extraction is a key process used to transfer target compounds from one phase into another immiscible phase and the universal goal is to achieve this effectively, efficiently and economically. The extraction mechanism is largely dependent on the extraction method used. Most methods are solvent-based while others are solventless. Solid-liquid extraction of phytochemicals using solvents will be used as a mechanistic example as this is the most widely adopted and studied category of extraction methods.
In general, the solvent-based extraction mechanism involves initial contact of the solvent with the plant matrix, dissolution and mass transfer of surface phytochemicals into solution by convection, solvent penetration into the inert matrix, desorption of internal phytochemicals, and diffusion of phytochemicals out of the matrix. The process continues until an equilibrium is reached whereby the concentration of target phytochemicals in solution is equal to that present in and around the structural matrix. At times when equilibrium is reached there may still be significant amounts of target phytochemicals remaining in the plant’s structural matrix, and the process must be repeated for an exhaustive extraction. [12]
Main Factors that Affect Extraction
Solvents are used to dissolve the solute from the structural matrix of the plant particles. A common term is ‘like dissolves like’, hence polar solvents such as water, ethanol and methanol are used to dissolve polar target phytochemicals such as alkaloids, phenolic acids and flavonoids from the raw plant material. In contrast, non-polar solvents such as chloroform and hexane are used to dissolve non-polar phytochemicals such as terpenoids and other components of essential oils. Intermediate polarity solvents such as ethyl acetate and isopropanol are used for phytochemicals with intermediate polarities such as many flavonoids, alkaloids, terpenoids, phenolic acids etc. Sometimes mixtures of solvents are used to optimize the extraction of particular components. When selecting solvents for extraction it is not only important to consider the physiochemical properties of the target phytochemicals to enhance solubility but also solvent reactivity with target phytochemicals, safety, regulatory issues alongside economic aspects.
Solid to Solvent Ratio
The ratio between the raw material mass and solvent volume affects the solubility limit, and the concentration gradient that governs diffusion. Generally, the more solvent that is used the faster the diffusion rate and the higher the yield. However, using large amounts of solvent can be costly also due to the excessive processing requirements respective of the yield obtained.
Particle Properties
Particle size affects the surface contact area between solvent and solutes where smaller particles have a collectively larger contact area and the faster the three phases of extraction occur. Reducing particle sizes can also help expose target phytochemicals by disrupting plant cell walls. The small particle sizes also enhance diffusivity as the intraparticle diffusion length and resistance are reduced. However, particles that are too fine can lead to clogging in the extraction or filtration equipment. Porosity of the particles can also affect diffusivity, as a highly porous complex matrix of the plant particles will reduce any mass transfer resistances of solvent into the particles and solutes out, increasing extraction rate. If it is difficult for the particles to soak in solvent, it will make sense to process them into smaller particles. If the particles are easily soaked then processing them into the smallest reasonable particle size will not be necessary.
Moisture Content
The moisture content or residual water in the plant particles (structural matrix) can facilitate or more restrict extraction of target phytochemicals depending on the circumstances. The extraction rate can be enhanced with a moderate level of moisture due to increased solvent permeability into the partially soaked particles. However, high levels of moisture can limit solvent permeability, reduce the concentration gradient due to the dilution effect, hinder the extraction of non-polar phytochemicals and increase enzymatic activity and hydrolysis which alters or breaks down the bioactive phytochemicals.
Agitation
Agitation improves extraction by increasing the rate of contact between solvent and plant particles, prevents stagnation layers from developing, maintains homogenous concentration gradients, reduces variability and distributes heat evenly. However, excessive agitation can damage phytochemicals that are sensitive to shear stress and lead to foaming of the extract solution.
Temperature and Pressure
Increased temperature generally increases extraction rate by increasing the solubility of phytochemicals in the solvent, supplies more kinetic energy which boosts diffusion and mass transfer rates, and reduces solvent viscosity making the phytochemicals and solvent molecules more mobile. However, excessive temperatures can lead to the degradation of thermolabile phytochemicals and is costly. Pressure can be used to increase the density of the solvent to increase solubility solvent permeability, by forcing solvent deeper into the plant particles. In combination at very specific ranges, temperature and pressure can also be used to achieve unique solvent properties such as supercritical fluids with both gas and liquid physicochemical properties (as with CO2) and can also be used to lower the solvent’s boiling point, which can optimize the selective extraction of particular phytochemicals.
Extraction Time
In general, the longer the extraction time the more phytochemicals are extracted at varying rates. Prolonged extraction can lead to the degradation of sensitive compounds depending on the extraction conditions. Optimal extraction times achieve the highest yields with the least degradation and co-extraction of interfering compounds (determined in quantitative analytical and semi-preparative studies). Extending extraction time beyond an optimal point results in diminishing returns.
Extraction Steps
The selection of raw material for phytochemical extraction depends on the approach and purpose. Generally, it involves careful consideration of the plant species and plant part that is likely to contain the highest concentrations of the target phytochemicals. The consistency of the raw material’s physicochemical characteristics and supply is also important, especially in industrial applications alongside considerations for sustainable practices. With analytical and semi-preparative approaches, thoroughly validating and cataloging the selected raw material is essential for reproducibility (species, origin, growth conditions, time of harvest, freshness, etc).
Raw Material Pretreatment
Pretreatment of the selected raw material is a preparative step, to remove contaminants, and to prepare and characterize the raw material for the extraction process. The raw material is usually fresh and commonly washed with water where damaged parts are excised. The latter is typical for samples grown outdoors where samples grown in controlled environments usually do not need a washing step. When the sample is clean, it is then dried via various methods (oven-drying, air drying, freeze drying, etc) to reduce and characterize the moisture content. The moisture content can also be used to calculate the dry weight of the sample, where the yield or phytochemical concentrations are expressed as a proportion of dry weight.
After drying, the sample is then pulverized via various methods (grinding, milling, blending, etc) to reduce particle size and then sieved through mesh to obtain the desired particle size range for extraction. The particle size range is noted as this is also a parameter of extraction.
Note that the drying and pulverization methods consider the sensitivity of the target phytochemicals to the pretreatment processes. For example, for thermolabile and volatile phytochemicals, non-thermal drying methods such as freeze-drying are better suited. Machine milling to reduce particle size is performed at the milder settings so as not to generate too much heat.
Selection of Extraction Method
Once the raw material is cataloged, prepared and characterized for moisture content and particle size, extraction can begin. The extraction method considers the sensitivity of the target phytochemicals especially to thermal degradation, oxidation and reactivity with solvent. Non-thermal extraction methods are chosen for thermolabile compounds and the complexity of the method is determined by resource availability and cost constraints.
Post-processing
After extraction, the extract solution is usually filtered to remove any particulates and then dried by rotary evaporation. The dried extract can then be
Common Extraction Methods
- Maceration
- Percolation
- Decoction
- Reflux Extraction
- Soxhlet Extraction
Advanced Extraction Methods
- Supercritical fluid extraction
- Pressurized fluid extraction
- Microwave assisted extraction
- Ultra-sound assisted extraction
- Pulsed electric field extraction
- Enzyme assisted extraction
Concluding Remarks
The extraction of natural plant-based products is an intricate, interdisciplinary process that synchronizes knowledge from scientific disciplines such as botany, chemistry, pharmacology, and engineering. This allows for the precise manipulation of natural resources in ways that preserve bioactivity, maximize yield, and ensure safety and efficacy of the final product. With ongoing advances in extraction technologies, biotechnology, and green chemistry, alongside the increased awareness of the dangers of many synthetic compounds, the potential for plant-based products in enhancing human health and well-being continues to expand.
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