INTRODUCTION:

The utilisation of plant-derived materials can aid in the identification of new chemical compounds as well as the creation of new bioactive molecules. According to research by Newman and Cragg, between 1981 and 2019, almost half of the medications that the FDA approved were either natural or derived from natural sources. The trend of the pharmaceutical, cosmeceutical, nutraceutical, or food industries to develop and formulate natural source-based products is largely explained by the proactive ambience that botanical preparations and their bioactive constituents generally enjoy that seems to favourably influence consumers more than synthetic drugs¹.

In the past few decades, and still today, natural ingredients have aided in the development of new drugs. However, the use of natural materials in medicine research has been hampered by the labor-intensive and time-consuming extraction and isolation methods. A growing number of novel automatic and quick processes have been developed to extract and isolate natural compounds, possibly meeting the need for high-throughput screening².

The pre-extraction and extraction processes are the first important processes in the creation of bioactive phytochemical components from biomass. Due to technical choices made in these early stages, the biomolecules found within the plants must be preserved. Pre-extraction procedures often involve leaving the sample to dry in a ventilated area at room temperature (or on a low heat setting in an oven). A particle size of less than 0.5mm is generally considered to be ideal for effective extraction, hence the grinding stage is particularly crucial to enhance overall surface contact among samples and solvents. After that, the extracted substance's quality is largely dependent on the method used for extraction, which is measured by the mass percentage of chemicals that were recovered¹.

Solid-liquid extraction (SLE), liquid-liquid extraction (LLE), and Soxhlet extraction are examples of traditional extraction techniques that utilise large amounts of solvent and take a long time to finish. Using these techniques, biologically active chemicals are frequently extracted inefficiently and selectively³.

Although the introduction of current spectrometric techniques has made bioactive component analysis simpler than ever before, the efficacy mainly relies on the extraction processes, input variables, and precise nature of the plant parts. The matrix qualities of the plant component, solvent, temperature, pressure, and time are the most frequent variables influencing the extraction operations⁴.

Classification and synthesis of bioactive compounds:

Bioactive substances are still inconsistently categorised into distinct groups; instead, it depends on the classification's purpose. The scope of pharmacological categorization, for instance, will not meet the simplicity of biosynthetic classifications that serve the description of biosynthetic pathways. There are three primary kinds of bioactive compounds found in plants: terpenes and terpenoids (about 25,000 varieties), alkaloids (about 12,000 types), and phenolic compounds (approximately 8000 types)⁴.

Most bioactive substances fall into one of several families, each of which has unique structural traits resulting from the manner that they are constructed in nature (biosynthesis). For the creation of secondary metabolites or bioactive substances, there are four main pathways: (1) Shikimic acid pathway, (2) malonic acid pathway, (3) Mevalonic acid pathway and (4) non-mevalonate (MEP) pathway. Both aliphatic and aromatic amino acids can be used to make alkaloids; they both originate from the shikimic acid pathway (come from tricarboxylic acid cycle). Malonic acid and shikimic acid pathways are used to create phenolic chemicals. Terpenes are produced via the MEP pathway and the mevalonic acid pathway⁴.

The pharmacological effects of several well-known secondary metabolites are recently highlighted in a review by Kashani et al., and numerous recent publications report the activity of new and/or lesser-known alkaloids, terpenoids, and phenolic compounds, providing clear evidence of the critical function of natural products as promising sources of numerous modern pharmaceuticals. The extraction, purification, and characterization of secondary metabolites, which are frequently present in small amounts in plant material, remain a significant problem in the drug development process⁴.

Extraction of bioactive compounds:

It is vital to develop a standardised and integrated screening method to weed out bioactive chemicals that are beneficial to human health given the wide variances among them and the abundance of plant species. If you want to understand the extraction choice from various natural sources, you need utilise extraction strategies in different situations. The extraction of bioactive substances is also possible using a variety of methods, many of which have remained essentially unchanged for hundreds of years. Each of these methods share certain common goals, (a) to extract specific bioactive substances from a complicated plant material, (b) to boost the analytical techniques' selectivity, (c) to make the bioassay more sensitive by raising the concentration of the desired chemicals, (d) Changing the bioactive compounds into a more manageable form for separation and detection, and (e) to offer a reliable, repeatable approach that is not affected by changes in the sample matrix⁴.

EXTRACTION METHODS:

Extraction comes first in the process of removing the desired natural ingredient from the starting material. Examples of extraction techniques include pressurization, solvent extraction, sublimation, and distillation methods based on extraction principles. The most common method is solvent extraction².

Following are the steps involved in extracting natural goods: (a) the solvent enters the solid matrix; (b) the solvent dissolves the solute; (c) diffusion occurs as the extracted solute leaves the dense matrix; and (d) then extracted component is gathered. Extraction will be made simpler by any components that encourage solubility and diffusibility in the earlier stages. Efficiency of extraction is influenced by the kind of extraction solvent, raw material particle size, solvent to solid ratio, extraction temperature, and extraction time².

There are two types of theextraction techniques: alternative/unconventional methods and traditional/conventional methods. Infusion, percolation, maceration, pressing, decoction, water distillation, and soxhlet extractionare examples of traditional techniques. Technologies like UAE, EAE, PLE, MAE, and SFE are examples of unconventional extraction techniques³.

Several novel techniques have been developed in addition to more conventional models, but as of yet, there is not a single technique that is accepted as the gold standard for removing bioactive chemicals from plants. The effectiveness of both traditional and innovative extraction techniques largely relies on the crucial input variables, knowledge of the composition of the plant matrix, bioactive molecule chemistry, and scientific knowledge. In this review, various extraction methods for obtaining bioactive chemicals from medicinal plants will be discussed along with their fundamental workings⁴.

Traditional methods of extraction:

1. Maceration:

An old, basic method employed in the creation of medications is called maceration⁵. Using this method, solid plant materials are put into a sealed container with the entire solvent and let to stand for at least three days (or between three and seven days) while being frequently stirred, until soluble stuff is dissolved. After standing, the mixture is filtered (via nets or sieves), the marc is compressed, and the mixed liquids are cleansed (by filtration or by decantation). To stop microbial development while using water as the solvent and macerating for a long moment, a tiny amount of alcohol may be added^6,7.

Periodic shaking throughout the maceration procedure can aid in extraction by enhancing diffusion, removing saturated solution from the material surface, introducing additional solvent into the menstruum, and increasing the extraction yield. It may be used to extract compounds that are thermolabile^4,8.

2. Infusion:

This extraction procedure involves briefly immersing the solid particle in tepid or hot water⁹.

3. Percolation:

Due to the continual nature of the process, where saturated solvent was regularly replaced by fresh solvent, percolation is more effective than maceration². The plant materials are placed in a percolation tubing that has a cotton stopper or is equipped with a strainer and a stopcock. The plant material is mixed with solvent and left to stand for about 4hours in a tightly covered container before the mixture is loaded and the percolator's top is secured. The entire system is left at room temperature for 24hours, and the solvent and extracted material are gathered by releasing the stopper below. The mixed liquid is then purified by screening or by standing, followed by decanting⁶.

4. Decoction:

This method works well for extracting heat-insensitive, water-soluble components. The dried extract is combined with distilled water during the decoction and heated for a prolonged amount of time at 100°C. Before filtering, the filtrate was left to come to room temperature. Concentrating the filtrate allowed to create an extract⁹.

5. Digestion:

In this type of maceration, extraction takes place with the use of low heat (40–60^OC). When a somewhat high temperature is acceptable, it is employed. The procedure can be changed by utilising a magnetic stirrer, mechanical stirrer, or periodically shaking by hand to mix the material and solvent. The extract is strained after 8 to 12hours, a new solvent is added, and the procedure is repeated until all the necessary compounds are removed⁶.

6. Tincture:

An alcohol-based plant extract is the ingredient in concern. The typical dosage is 1:5 of fresh plant material and ethyl alcohol. Due of the alcohol concentration, tinctures can be kept at room temperature without becoming bad⁶.

7. Reflux extraction:

Maceration or percolation are less efficient than reflux extraction, which also takes less time and consumes fewer solvents². This hot extraction method involves treating the sample with a boiling solvent. An attached condenser, preferably a round-bottomed flask, is used to recycle the solvent vapour from the container. The extraction of naturally thermolabile products is not possible with it⁶.

8. Soxhlet extraction:

Franz Ritter von Soxhlet, a German scientist, developed the first technique for extracting lipids, known as the Soxhlet extraction process^4,10. It is used to separate bioactive (solid-liquid) substances from a range of natural resources. Utilizing Soxhlet extraction is simple. Fresh solvent can be used to continue the extraction process indefinitely till most of the solute in the source material has been consumed^11,12.

The Soxhlet extraction combines the advantages of both percolation and reflux by continuously extracting with a new solvent using the concepts of reflux and syphon. Soxhlet extraction is an automated, high-efficiency continuous extraction technique. It uses less solvent and takes less time than maceration or percolation. Because of the elevated temp and lengthy extraction time, thermal degradation is more likely during Soxhlet extraction^2,10.

The solid substance is ground into a powder and fed inside the Soxhlet equipment in a filter paper thimble. The device is attached to a reflex condenser and a round-bottomed (RB) flask holding the solvent. Gently boiling the solvent inside the RB flask causes the vapour to rise down the side tube, condensate in the condenser, drop into the material-containing thimble, and then slowly fill the Soxhlet. The solvent eliminates the substance it has extracted when it reaches the top of the connected tube by syphoning over into the flask⁶.

9. Steam Distillation:

It is the procedure that is typically used to separate volatile oil from raw plant material. Simple vaporisation is accomplished through steam distillation, which involves running steam through the substance.Here, oil separates from water during condensation, recovering the steam volatile essential oil⁶.

Vapour and heating have the biggest effects on the release of biologically active compounds in flower tissues. When water is indirectly cooled, the vapour mix of oil and water condenses. The separator receives the condensing solution from the condenser and automatically separates the water from the oils and biologically active substances¹³.

10. Hydro Distillation:

The most common method for isolating essential oils is this one. A heating mantle is used to boil the plant material after it has been soaked in water. The volatile oil is liberated from the plant tissues oil glands under the effect of hot water, and it then travels with the steam. The steam oil combination is condensed, the oil and water are separated using a common glass device called a Clevenger apparatus, as well as the condensed water is recycled⁶.

11. Expression:

Citrus essential oils are extracted by a process known as expression, often known as cold pressing. Sponge pressing was a common form of expression in earlier times, and it was done entirely by hand. In this procedure, the sponge absorbs the oil leak, and when the sponge is squeezed, the oil is recovered. According to reports, this approach produces oil that has the strongest fruit odour of any method used to make oil⁶.

12. Enfleurage:

In order to extract delicate scents from flowers, this procedure is used. The refined fat layer is covered with flower petals, which absorb the floral scent. The saturate fat is then treated with a solvent usually alcoholin which the aromatic elements are soluble. When the fat separates out, the remaining fat that has been dissolved in the alcohol may be eliminated by lowering the alcohol extract temperature to 20^oC. Pure oils are produced once the alcohol is evaporated under lower pressure⁶.

All of these procedures make use of solvents, high temperatures, and stirring. Traditional methods are frequently manual operations, which makes replication challenging. Low extraction rates are the result of the heating method destruction of sensitive physiologically active compounds biological activity. The active molecules can change depending on the pressure, pH, and temperature settings used. Furthermore, the necessary organic solvents are bad for the environment. Due to these limitations and the huge increase in the demands for biologically active compounds, it is essential to create efficient, selective, affordable, and environment benign extraction techniques that are quick, yield more, and abide by all current rules and regulations³.

Modern Methods of Extraction:

The creation and use of novel methods that facilitate the chemistry of natural products are investigated. Additionally, there are "green" extraction technologies, which focus on the creation and research of energy-efficient extraction techniques. Utilizing renewable natural resources in conjunction with alternative solvents can result in safe and high-quality extraction. Existing new extraction methods are likely to satisfy several of the aforementioned criteria. Among them are the technologies EAE, MAE, UAE, SFE, and PLE. These technologies principles, mechanisms of action, advantages, and disadvantages in comparison to more traditional methods, as well as the potential for obtaining biologically active compounds from natural materials, will all be examined³.

1. Ultrasound-Assisted Extraction (UAE):

Ultrasound energy is used in the extraction process via ultrasound assisted extraction (UAE), sometimes referred to as ultrasonic extraction or ultrasonic processing. The extraction efficiency is increased by ultrasound because it hastens heat transfer, solute dissolution, and diffusion in the solvent. Low energy and solvent use, as well as shortened extraction times and temperatures, are other benefits of the UAE. The UAE is frequently used to extract various natural compounds^14,15.

In the UAE, the plant material is placed with an ultrasonic bath after being covered in the extraction solvent, often in a glass container. It has grown in popularity because mechanical stress, which causes cavitations and cellular collapse, reduces extraction time and increases extraction yields.

Anthocyanidins, flavonols, and phenolic acids from Delonix regia, capsaicinoids from Capsicum frutescens at lab and pilot-plant scales, cyanidin-3-rutinosid from Litchi chinensis, or essential oils from Laurel, rosemary, thyme, oregano, and tuberose are a few examples of NPs extracted using UAE¹⁶.

2. Pressurized liquid extraction (PLE):

Extraction is the most effective method for getting biologically active compounds from various source¹⁷. In 1996, the first report on the use of PLE for biologically active material extraction was published^18,19.

Different research teams have referred to pressurised liquid extraction (PLE) as high-pressure solvent extraction, accelerated solvent extraction, enhanced solvent extraction, pressurised fluid extraction, and accelerated fluid extraction. The extraction process at PLE uses a lot of pressure. High pressure maintains solvents in a liquid condition above their boiling point, leading to high lipid solubility, high lipid solute diffusion rates, and high solvent penetration into the matrix.Compared to other procedures, PLE significantly reduced the amount of extraction time and solvent used and had superior repeatability².

High temperatures cause the solvent to become less viscous and have less surface tension, which speeds up the extraction rate by allowing the solvents to enter the matrix region. But because it uses so few organic solvents and complies with all fundamental pollution laws, PLE is normally understood as a green extraction process²⁰.

PLE can extract a greater variety of physiologically active compounds than SFE because it can adapt to a wider range of extraction solvents. Depending on how polar the target molecule is, the PLE solvent will be selected. However, compared to SFE, this approach performs much worse³.

3. Microwave assisted extraction (MAE):

With frequencies ranging from 300 MHz to 300 GHz, microwaves are a type of non-ionizing electromagnetic radiation. The use of microwaves to extract different chemicals was first documented in 1986^21,22. Mass and heat gradients in traditional heating operate in opposing directions, whereas in MAE they operate in the same direction²³. As a result, in traditional heating, only the substrate's surface is heated directly; the remainder is heated by conduction from the substrate particle's surface to the core. By producing friction between polar molecules, MAE, on the other hand, produces direct heat in the matrix²⁴.

The following two oscillating vertical fields contain microwaves: (1) magnetic field and (2) electric field, based on fundamental physics and microwave theory²⁵.

The use of MAE has various benefits, including improving extract yield, reducing thermal deterioration, and selectively heating vegetal material. Because MAE uses less organic solvent, it is also rated as a green technology. There are two different kinds of MAE techniques: solvent extraction (often for volatile chemicals) and solvent-free extraction (usually for non-volatile compounds)².

MAE has undergone several modifications, including ultrasonic microwave assisted extraction (UMAE), vacuum microwave assisted extraction (VMAE), dynamic microwave aided extraction (DMAE), and nitrogen-protected microwave assisted extraction (NPMAE), and¹⁶.

4. Enzyme assisted extraction (EAE):

The main difficulties in extracting natural products are the structure of cell membranes and cell walls, micelles generated by macromolecules such proteins and polysaccharides, and protein coagulation and denaturation at high temperatures. By hydrolysing intracellular macromolecules, cell membranes, and components of cell walls, EAE can increase the effectiveness of extraction while allowing the release of natural substances ². Under the right temperature and pH conditions, it has been discovered that cell wall degrading enzymes (like carbohydrases and proteases) can break down cell walls and release the necessary biologically active substances. These enzymes can therefore be used to impregnate tissues and break down the cell walls of natural substrates.

In this regard, a number of factors must be kept into account to ensure the success of the extraction procedure: pH of the system, particle size of the substrate temperature of the reaction, and enzyme concentration²⁶.

5. Pulsed electric field (PEF) extraction:

Pulsed electric field extraction greatly improves extraction yield while cutting down on extraction time since it can speed up mass transfer by breaking membrane structures. The field intensity, specific energy input, number of pulses, and treatment temperature are only a few of the factors that affect PEF therapy success. PEF extraction uses a non-thermal process to reduce the amount of time that thermolabile chemicals deteriorate².

6. Supercritical fluid extraction (SFE):

Supercritical fluid extraction is being used more and more, especially when supercritical CO2 is used, as a replacement for extraction with organic solvents that is less harmful to the environment and complies with increasing regulatory requirements^16,27.

Supercritical fluids are used in SFE because they perform better in transportation because they have properties that are both similar to those of liquids and gases above their critical point, such as solvent strength and low surface tension ^12,28.

A supercritical fluid is one that is thick and not condensable (SF). A fluid is referred to as supercritical when its temperature and pressure are higher than the relevant critical temperature and pressure. At the critical point, there is just one phase, which combines features of gases and liquids (such as density) (mass diffusion coefficient, viscosity, and compressibility)^29,30. Additionally, SFE employs the minimum amount of solvent compared to other extraction techniques and can be utilised to extract a variety of biologically active compounds^31,32.

The most popular supercritical fluid for extracting heat - sensitive compounds is CO2, which has suitable Tc and Pc (31.1°C and 73.9 bar). Additionally, the properties of supercritical CO2 include low density, high diffusivity, low viscosity, and low surface tension. It is also inexpensive, easy to obtain, non-toxic, non-flammable, and chemically inert in a range of circumstances. It does not require solvent evaporation after extraction because it maintains in gaseous form at ordinary pressure and temperature^33,34.

Additionally, carbon dioxide creates a non-oxidizing environment inside the extract that stops the extract from deteriorating³⁵. The primary drawback of supercritical CO2 is because it can be used to extract polar molecules³³.

A crucial component of SFE is the ability to modify the fluid's temperature and pressure in order to modify the fluid's density. This indicates that the fluid's solvency, that is based on its density, can be impacted by changes in temperature and pressure^36,37.

Recently published studies on SFE for NP extraction and modelling have used gallic acid, phloroglucinol and benzophenone derivatives from Hypericum carinatum, essential oils, quercetin, and essential oil from the flowers of Achyrocline satureioides, or phenolics including anthocyanidins from grape peels (Vitis labrusca)¹⁶.

CONCLUSION:

In the subject of natural products, there have been a number of significant advancements recognised recently. The separation of natural ingredients from plant extracts is a significant barrier for the identification and characterization processes since they typically contain multiple component mixes with variable polarity. Separation and characterisation of various natural compounds depend heavily on extraction.

Modern extraction techniques do not need organic solvents like older ones did. The extraction of biologically active chemicals using these procedures has thus far proven to be more efficient and environmentally friendly. The extraction efficiency is greatly increased by the high yield attained utilising current techniques.

Recent years have seen the designation of contemporary extraction techniques as "green extraction techniques" (such as SFE, UAE, PLE, and MAE). Due to the advantages of higher extraction rates, process safety, selectivity, and target extract stability they have attracted a lot of attention recently.

Along with being quick and requiring less solvent, PLE and SFE procedures are also very sensitive. In PLE and SFE, they can be changed by adjusting the solvent's temperature and pressure by including co-solvent, or by switching the solvent entirely (for PLE). Since CO2 is non-flammable, non-toxic, and inexpensive, as well as having a low and reachable critical temperature (31.2⁰C) and critical pressure (7.4 MPa) which may produce a non-toxic, solvent-free extract, CO2 is the most frequently used supercritical fluid in CO2-SFE. Higher extraction rates can be attained while lowering the extraction time by using mechanical (MAE and UAE), electrical (PEP), and enzymatic (EAE) approaches. Briefly said, biologically active substances will no longer be the exception but the rule relatively soon, regardless of the source.

To ensure that the industry can fully profit from the enormous advantages brought about by the extraction of bioactive chemicals, further research in this field should concentrate on finding solutions to the challenges associated with applying these new technologies on a broad scale.

CONFLICTS OF INTEREST:

There are no conflicts of interest, according to the author(s).

ACKNOWLEDGEMENT:

The authors acknowledge technical assistance from the Dr. Babasaheb Ambedkar Marathwada University's University Department of Chemical Technology in Aurangabad, Maharashtra, India.

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Received on 01.01.2023 Modified on 30.03.2023

Research J. Pharm. and Tech 2024; 17(4):1874-1880.

DOI: 10.52711/0974-360X.2024.00297