Green Extraction Techniques for Phytoconstituents from Natural Products

Abstract

The use of green extraction techniques for extracting phytoconstituents from natural sources minimizes the amount of solvents needed and the amount of waste generated during the extraction process. Traditional extraction processes generate a lot of solvent waste, which causes a lot of environmental and health issues. Furthermore, by employing automated modern processes, exposure to solvents and vapor is reduced. Green extraction is based on the analytical procedures that employ less energy, allow the use of different solvents and sustainable natural products, and provide a safe and superior extract/product. According to a life cycle analysis of waste created in Active Pharmaceutical Ingredient (API) manufacturing plants, solvent-related waste accounts for 80% of the waste. In case other pharmaceutical companies generate equal amounts of solvent waste, addressing solvent selection, use, recovery, and disposal will go a long way toward tackling the issue. Solvent considerations will feature regularly in the case histories of the drug development process. Natural extracts comprise phytoconstituents such as proteins, lipids and oils, dietary fibres, carbohydrates, antioxidants, essential oils and fragrances, and colours, and can be found in wide variety of plant materials. In this chapter, we will discuss principles, techniques, and solvents used for green extraction techniques of phytoconstituents.

1. Introduction

Medicinal plants are getting more demand because of their distinctive features as an abundant source of curative phytochemicals that may be used to develop new medications. Approximately 20% of all known plants have been employed in pharmacological investigations, positively improving the healthcare system by treating cancer and other ailments [1]. Many of these medicinal plants are good sources of phytochemicals like polyphenols, carotenoids, flavonoids, anthocyanins, and vitamins that possess antioxidant activities. Today, medicinal plants are finding diverse use in society from medicine to cosmetics, nutraceuticals, herbal drinks, herbal foods, and other articles in their daily uses. Plant phytoconstituents are created as secondary metabolites, which are produced through a variety of biological routes in secondary metabolism. The choice of solvents for extracting phytoconstituents from plants is critical. A suitable solvent has an appropriate extraction capacity as well as the ability to maintain the chemical structure of the desired molecules stable [2].

Green technologies are increasingly being employed in practically every scientific sector to promote ecologically acceptable activities that do little or no harm to humans. Ionic liquids, aqueous biphasic systems, and pressurized hot water have all become attractive research topics in recent years [3]. Traditional techniques of extracting phytoconstituents require the use of more powerful and toxic solvents (nonenvironmentally friendly), as well as more energy. Each method’s extraction time varies, ranging from minutes to 7 days in the case of maceration [4]. Another problem is that none of the current plant processing methods meets all the economical, safety, and scalability requirements. Other concerns include security hazards, solvent toxicity, and the existence of solvent remnants in the extracts. The high cost of feedstock, the high cost of extracting desirable bioactive compounds, their comparatively low yield, and the resulting substantial concentration of residual waste biomass are the major roadblocks to commercially viable phytochemical production [5]. In many process sectors, microwave-assisted extractions like ultrasound-assisted extraction, pulsed electric field extraction, and molecular distillation have been reported. Green chemistry, as ecological and economic chemistry, could be one of the solutions to humanity’s future [6]. The entire process of green extraction of phytoconstituents from natural sources is concluded in the Figure 1.

Figure 1.

Extraction of crude drugs using green solvent, green extraction process, and purification techniques.

2. Green extraction

A vast diversity of plants, animals, and microorganisms can produce a wide spectrum of chemical compounds with amazing health-care properties in nature. Science is steadily changing our world by finding the possibilities of natural products [7]. Natural product extraction has been practised since civilization. Extraction methods are used in the perfume, cosmetics, pharmaceutical, food, and chemicals sectors. Recent extraction technique advancements have mostly focused on creating solutions that employ lower solvents [8].

Soxhlet extraction, maceration, and hydro distillation (HD) are examples of traditional/conventional extraction processes. The choice of specific solvents has a considerable impact on any extraction rate. The polarity of the target biochemical is the most significant factor to consider, and when choosing a solvent, the solvent’s molecular affinity for the solute, as well as its environmental friendliness, toxicity, and economic efficiency, must all be considered. Simple, safe, repeatable, low-cost, and adaptable to a variety of applications are all desirable characteristics in an extraction procedure. High-temperature extraction (e.g., Soxhlet technique, HD) has been shown to cause changes in the composition due to phytoconstituent degradation [9].

To circumvent the constraints of classic extraction procedures, green extraction techniques can be employed to extract phytoconstituents from plants. The majority of these include less harmful chemical synthesis, nontoxic chemicals, safe solvent aides, energy efficiency patterns, use of sustainable feedstock, fewer derivatives, catalysis, design to avoid deterioration, and time scheduling for pollution avoidance, hazardous air pollutants, and naturally safer chemistry for safety programs. The development of effective and selective technologies for extracting and isolating bioactive phytoconstituent is crucial. This article aims to provide a detailed overview of green solvents employed, as well as the methods for extracting and isolating natural compounds form natural sources. Green solvents can help to improve old procedures significantly, especially when incorporated with new and novel methodologies. Hydrolysis of cellulose from biomass with supercritical water and the extraction of hydrophobic compounds using supercritical CO₂ are few examples of green extraction process.

3. Solvent selection for green extraction

Solvents, their vapors, and mists have a variety of health impacts. Many contain narcotic properties, causing lethargy, dizziness, carcinogens, etc. Solvents irritate the eyes and respiratory system, as well as causing skin problems. High doses can cause unconsciousness and death in certain people. Petroleum-based solvents, which are mostly sourced from fossil fuels, are commonly utilized in various stages of the analytical process [10]. Solution preparation, extraction, and enrichment of phytoconstituents, washing of extracts, solvent exchange, sample preservation, dilution, cleaning of glassware, liquid desorption, derivatization, analytical separation, and detection are all activities that involve solvents in phytochemistry. A suitable solvent has an appropriate extraction capacity as well as the ability to maintain the chemical structure of the desired molecules stable.

3.1 Water

Water is one such “green” solvent that can have its properties changed by changing the temperature. Water’s polarity allows it to be employed as an extraction solvent for both natural and inorganic substances that are aqueous soluble, like proteins, carbohydrates, and organic acids. Water is an important green solvent for the extraction of phytoconstituents. It has no harmful health or environmental consequences [11]. Furthermore, it is the safest and cheapest solvent. The technology used has an impact on the extractability of biologically active chemicals. Water is used as the only extractant in several ways, including decoction, infusion, and hydro distillation. Water as a solvent can be used in a variety of traditional and modern procedures. Extraction with pressurized hot water is one of the most promising new green extraction techniques and procedures, especially in a dynamic mode [11]. Water, on the other hand, has several drawbacks in terms of the less solubility of nonpolar molecules and energy required to enrich products. This difficulty can be overcome in part by employing supercritical water or a mixture of alcohol and water.

When using hydro distillation, high temperatures and long distillation times might cause volatiles to change and be lost. Supercritical water extraction (SWE) was shown to have a quicker extraction time, cheaper costs, and higher purity than hydro distillation. In terms of oxygenated components, SWE’s products yielded higher valuable essential oil. To boost extraction yields, microwave-assisted extraction with water as a solvent has been proposed.

3.2 Alcohol

Alcohols like methanol, ethanol, and isopropyl alcohol have similar solvent properties such as solvent strengths, dielectrics, critical points, and hydrogen donating abilities. However, due to its nontoxic nature, ethanol has ascended to the top [12]. Alkanes (heptane, hexane) and simple alcohols (methanol, ethanol) are healthier for the environment than dioxane, acetonitrile, acids, formaldehyde, and tetrahydrofuran [13]. The main disadvantage of alcohol is that they are flammable and some of them are toxic (i.e., methanol). In addition, extended exposure to their vapors can also lead to health problems.

3.3 Supercritical carbon dioxide (CO₂)

CO₂ as a liquid or supercritical solvent possesses multiple features of an admirable green solvent. They are incombustible, nonpoisonous, nonenvironmentally harmful, plentiful, inexpensive, easy to produce, simple to eliminate from a product, do not add to smog, and do not contribute to global warming [14]. Purified CO₂ is produced, pressurized, and cooled to a liquid state at 20 psi and −20°C before being stored or transported in insulated bulk containers for use in a variety of liquid and supercritical CO₂ processes. The viscosity of CO₂ is extremely low, and supercritical CO₂ has negligible surface tension [15]. The strong diffusivity, along with the low viscosity, causes significant improvements in the condensed phases. Supercritical fluid extraction of a crude drug is achieved by passing supercritical CO₂ over a column packed drug material. Until the substrate is depleted, supercritical CO₂ travels over the column of packed material and dissolves soluble components. The loaded solvent is then transported through a separator, where the soluble components precipitate as pressure and temperature are reduced. The CO₂ is recirculated once it has been condensed. It is employed in the removal of caffeine from coffee and tea, the removing fatty material from cacao, the production of hops extracts, sesame seed oil, and pesticide extraction from rice. Under high pressure, SC CO₂ is used to extract triglycerides and volatile compounds. Volatile, triglyceride and phenolic chemicals etc. are extricated at high pressure (300–400 bars) with EtOH. Add water or alcohols like ethanol or iso-propyl-alcohol to the SC-CO₂ extraction has already been used to modify the polarity [16].

3.4 Deep eutectic solvents (DES)

DES is formed when the melting point of a mixture of substances is much lower than the melting points of the two constituents. A hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA) are required to build a DES system, and when mixed in the right proportions, they generate a novel “mesh” of hydrogen-bond-interconnected molecules with remarkable physicochemical features [17]. Their extraordinary physicochemical features (like ionic liquids) combined with remarkable green properties, low cost, and ease of handling are piquing researchers’ attention in a variety of sectors. The eutectic composition of DESs is formed by heating and stirring two or more solid starting components to generate a transparent, viscous homogenous liquid. Other techniques involve grinding (combining and powdering solid components till clear liquid forms), evaporation (dissolving all starting elements in water and then removing the water via evaporation at reduced pressure), and freeze-drying (dissolving all starting components in water and then draining the water via evaporation at reduced pressure).

Among them, heating and stirring below the melting points of the individual constituents is possibly the most acceptable method [18]. Because DESs are nonflammable and nonvolatile, they are easier to store. They are also biodegradable, unlike standard organic solvents. Furthermore, DES manufacture is cost-effective, simple to run, and requires no modification, making their use on a broad scale possible. DESs can be made by mixing molecules derived from natural sources (e.g., glycerol and glucose), which makes them environmentally friendly. Within the HBD section, polymerized deep eutectic solvents (PDEs) are a novel category of DESs that can be polymerized [17].

The high viscosity of DES is a key disadvantage that can limit their usage as extraction solvents since it prevents the solvent from penetrating the extraction matrix. Although increasing the temperature of the extraction process helps reduce viscosity, this is not always the best solution because it consumes energy, and some heat-sensitive phytochemicals may not withstand the higher temperature. The addition of a co-solvent to the extraction medium is a straightforward technique to remedy this problem. Most of the time, this co-solvent is water, which keeps the process green; nevertheless, organic solvents like methanol have also been utilized. Alkaloids, phenolic acids, flavonoids, and saponins are all extracted using DES [19].

The DES is called natural deep eutectic solvents (NADES) when amino acids, organic acids, sugars etc. are used to make DES [20]. Due to the natural nature of its ingredients, NADESs are deemed environmentally beneficial and “readily biodegradable,” and the resulting extracts can use in food, pharmaceutical, and cosmetics preparations. Because of their great stability and solubilization properties, NADES is ideal candidates to replace traditional solvents. NADESs combinations have efficiently extracted bioactive compounds including flavonoids, phenolic acids, alkaloids, natural pigments, sugars, peptides, and volatile components from natural matrices [21].

3.5 Ionic liquids (IL)

ILs were a type of organic salt that consisted of an organic cation (e.g., imidazolium, pyrrolidinium, pyrrolidinium tetra alkyl ammonium, pyrrolidinium tetra alkyl phosphonium) and an inorganic or organic anion (e.g., tetrafluoroborate, hexafluorophosphate, and bromide) that form of liquid below 100°C [22]. Because of their distinctive and construction dependent features, like low nucleophilicity, mixability with water or organic solvents, and good extractability, ILs have been frequently used [23]. A variety of organic and inorganic substances are perhaps enriched and separated using IL-based methods. As a result, they have been frequently used in food safety, drug testing, environmental monitoring, biological analysis, and other areas. The ability of ILs could be tailor-made for the extraction of alkaloids, flavonoids, terpenoids, phenylpropanoids, quinones, and other phytoconstituents from plants. A vast number of research organizations have also created IL-based silica and polymers that can improve the extraction/separation of target chemicals.

3.6 Aqueous enzymatic extraction (AEE)

Extraction is an important step in the isolation of bioactive chemicals from plant matter. However, because of the existence of complex cell wall polysaccharides including cellulose, hemicellulose, lignin, pectin alginate, and carrageenan, the extraction yield of bioactive chemicals is poor. Researchers are now considering modern methods of extracting these compounds because of the low specific gravity of bioactive compounds, the low productivity of the solvents used to extract these compounds, high energy, high durability, solvent residue in the extracts, and the decline in the quality of the final product, as well as environmental concerns [23]. The use of enzymes to extract bioactive chemicals from plants could be a viable substitute for traditional solvent extraction methods. Enzymes are excellent catalyzers for extracting, modifying, or synthesizing complex bioactive substances from nature. The natural ability of enzymes to accelerate reactions with perfect particularity, regiospecificity, and the ability to employ under gentle processing conditions in an aqueous medium facilitate enzyme-based extraction [24]. The use of enzymes for sugar extraction is a new topic that needs further research. To improve extraction processes, custom enzymes must be developed, either by biodiversity screening, genetic engineering perspective, or a mix of the two. From plant sources, enzyme-aided extraction can be utilized to obtain lipophilic, polyphenolic, and hydrophilic chemicals [25]. Factors including high enzyme production and downstream processing costs, extended incubation times, and an extra stage (de-emulsification) in the process are still preventing aqueous enzyme extraction from becoming commercially viable. Commercial enzyme production has been accelerated, and enzyme synthesis has now become more affordable. The downstream processing expenses could be reduced by using appropriate technology rather than the traditional technique [26].

3.7 Limonene

The predominant element of essential oils derived from citrus fruit peels is d-limonene, which belongs to the terpene family. Since its cleaner and degreaser properties were discovered and considered, d-limonene has sparked a surge of interest. In this sense, this chemical has been classified as a viable alternative to halogenated carbon hydrates or traditional degreasing chemicals commonly used in industry and households. Several authors have attempted to create a commercial application for d-limonene. Sustainable chemistry has generated a lot of study into the processing of renewable fuels due to the demand for environment-friendly techniques and products [27].

Because d-limonene has a higher boiling point (175°C) than n-hexane (69°C), it uses more energy to recover the solvent by evaporation. To minimize the difficulty of solvent recovery caused by high d-limonene’s boiling point, a technique based on steam or hydro-distillation employing Clevenger can be used. Distilled water was added to the extracted oil and d-limonene mixture after Soxhlet extraction with d-limonene. D-limonene and extracted oil were separated using a Clevenger device and azeotropic water distillation at less than 100°C [28]. It is a valuable and practical method for determining the lipids and oils in olive seeds. Waste minimization, rapid operation, and energy saving are all possible with Soxhlet microwave-integrated with limonene and microwave Clevenger distillation [29]. Limonene has a dielectric constant that is very similar to that of hexane and has been used to extract rice bran oil, oil from olive leftovers, carotenoids from tomatoes or algae and, more recently, algal lipids from wet algae [30].

3.8 Solvent-free extraction

Solvent-free extraction of a variety of important natural products (essential oils, fragrances, edible oils, antioxidants, and other organic compounds) eliminates the price and threats correlated with large amounts of solvent. It minimizes the amount of wastewater after extraction and uses a fraction of the energy that a traditional solvent-solid extraction process does.

In 2008, Chemat et al. developed the MHG method, which uses in situ dielectric heating on plant cell water to stretch the structure and cause membrane and wall ruptures. As a result, plant matter is used to gather primary and secondary metabolites, as well as the water in the cells. The behavior described is known as hydro diffusion. Gravity then drops the diffused components into a container. A continuous condensation system is maintained using a perforated Pyrex disc. MHG has been used to extract pigments, aroma components, and antioxidants from a variety of natural sources on a lab and commercial scale [21].

4. Pre-treatment techniques

Crude drugs can be extracted in fresh or dried form. Grinding and drying of plant materials are examples of pre-preparation. This has an impact on the preservation of phytochemicals in final extracts. Air drying takes anywhere from 3 to 7 days. To optimize extraction operations and save energy, mechanical disruption pre-treatments can be employed alone or in combination. Bead milling, high-pressure homogenization, and hydrodynamic cavitation are all methods for mechanical disruption. The extraction of lipids has been demonstrated to be aided using a bead mill. Powdered samples, on the other hand, have a more homogenized and smaller particle size, developing in substantial surface contact with extraction solvents [31].

Nanotechnologies, including microwave, ultrasound, and pulse electric field, were found to improve operation efficacy as a pre-treatment before drying. After size reduction and before extraction, microwave pre-treatments upgraded the extraction of polyphenols, sugars, and other compounds. Pre-treatments with a pulsed electric field (PEF) improved extraction efficiencies in terms of yield and extract standard. PEF pre-treatment of rapeseed, apple, and sugar beet fruit extracts before mechanical expression resulted in higher yields [26]. Oven-drying is one more pre-extraction method that uses heat energy to eliminate moisture from substances. This procedure for preparing a sample is regarded as particular easiest and most rapid thermal processing method available for phytochemicals.

Costly drugs can be dried by freeze-drying. In freeze-drying before use, the sample is frozen at −80°C to −20°C to lyophilize any liquid (e.g., solvent, moisture) in the body samples. The mouth of the test tube or other container holding the sample is wrapped in needle-poked-parafilm to avoid sample loss during the operation. Freeze-drying resulted in a greater phenolic content compared to air-drying because most phytochemicals are preserved. This strategy is used to keep phytoconstituents safe. Freeze-drying, on the other hand, is a difficult process. Microwave drying is more expensive than traditional air drying. As a result, only fragile, heat-sensitive goods and high-quality materials are permitted [32].

5. Green technologies for extraction

The main goal of green extraction procedures is to obtain a rapid extraction, increased efficient energy usage, higher mass and heat transfer, smaller apparatus, and fewer processing stages [3]. Several novel alternatives to traditional techniques for obtaining target compounds from a variety of crude drugs have been proposed, such as ultrasound-assisted extraction (UAE), subcritical and supercritical fluid extraction (SFE), microwave-assisted extraction (MAE), and accelerated solvent extraction (ASE) [33]. These extraction methods, which are alternatives to traditional procedures, have piqued the curiosity of academics, who see future applications for recovering bioactive molecules from plants in less time using green solvents. Most of these new methods have already shown promise in extracting high-value chemicals, particularly natural antioxidants, from various sources such as plants or food processing by-products [34].

5.1 Expression

It is a physical technique in which pressure is employed to extract the oil or juice from a material. A tincture press was used to do this. When essential oils are temperature sensitive, this approach is used. It’s used to extract essential oils from citrus peels like lemons and oranges. Squeezing any plant material at high pressures to extract oils or other liquids is known as expression. In remote rural locations, hand-operated presses or crushes are used, while in industrial hubs, massive mechanical presses are used. However, the products obtained are impure and frequently contain impurities such as water, mucoid particles, and cell tissues, making them murky, and pressing the volatile oil in plants completely is difficult. As a result, the crushed residue is frequently steam distilled to remove all volatile oils. Black soybean oil, for example, is frequently extracted using the low-temperature pressing process [31].

5.2 Effleurage (extraction with cold fat)

Enfleurage is the method of extracting aroma from flowers by absorbing it through contact with cold lipids. This method is used for fragrant flowers like jasmine and tuberose, which retain their unique aroma even after being plucked. To prevent fat odors from entering, fats should be saturated and odorless. It’s best to use refined lard or beef suet. On both sides of a glass plate mounted on a rectangular hardwood frame or chassis, fat is thinly deposited. On a fat-coated chassis, fresh aromatic flowers are delicately stacked. Enfleurage produces far more floral oil than other processes.

5.3 Ultrasound extraction

Ultrasound extractions can now be finished in minutes with high reliability, reducing solvent consumption, clarifying control and work-up, improving final product purity, removing wastewater after treatment, and consuming only a fragment of the fossil energy required for a traditional extraction method [35]. USAE (ultrasound-assisted extraction) has been used to extract polyphenols from vegetable tissues, protein, sugar, and starch from cereals and legumes, oil, and flavor components. Extraction efficiency and rate are improved by sonication. It lowers the required temperature, saves solvents, and promotes the solubilization of the desired chemicals. Solubility is enhanced by a significant increase in the very temperature [36].

To extract phytochemicals from plants, both the cell wall and the cell membrane must be broken. Because of this, ultrasounds are used in ultrasound-assisted extraction for cell disintegration. Ultrasounds are sound waves that are above the human hearing range, with frequencies ranging from 20 kHz to a few gigahertz. Plant materials and liquids absorb the energy emitted by ultrasonic waves and convert it to heat. The frequency, intensity, and duration of ultrasonic therapy affect the amount of heat created in plant materials [37]. This heat energy debases proteins, destroys plant cells, and causes therapeutic substances to be released from plant cells. In most cases, the highest extraction rate is attained in the initial few minutes, which is also the most profitable time [38].

5.4 Super critical fluid extraction (SCFE)

The extraction of thermally labile chemicals is possible because of carbon dioxide’s low critical temperature (304.1 K). It can replicate a variety of organic solvents by adjusting the density of SCF carbon dioxide. Because of its variable solvating strength, this feature allows for selective extraction, purification, and fractionation techniques. SCF carbon dioxide media provide the prime possibility for fractionation of reaction products and solvent separation, which can be performed by simply depressurizing the media. This is because SCF quickly penetrates and leaves solid matrices, compared to the use of organic solvents with a higher viscosity [39]. It has a broad variety of applications, including the extraction of common spices such as black pepper, celery seed, cumin, cinnamon, clove bud, and nutmeg. Extraction of Natural Colors: Paprika Pigments, etc. Dry Ginger, Saw Palmetto, Rosemary, and other botanicals are used to extract active ingredients. Forskolin, Turmerones—from Turmeric, Oscimum sanctum, Neem Leaf, and other plants; Cholesterol and other lipids are extracted from dried egg yolks. Hops are extracted to use in the beverage sector. Precipitation of Human Immunoglobulin G (IgG), viral deactivation, and other biochemical components. The main disadvantage of supercritical carbon dioxide extraction is the high cost of the device. Because supercritical carbon dioxide is nonpolar, polar co-solvents of 5% and 10% ethanol were added to change the polarity and improve solubility.

5.5 Microwave-assisted extraction

Nontraditional ways are more prominent when it comes to improving the quality and quantity of desired items. By directly linking microwave energy with the bulk reaction mixture, microwave irradiation creates efficient internal heating. The magnitude of energy transfer is determined by the molecules’ dielectric characteristics. Radiation absorption and heating can be quite selective in this approach (Hoz et al.). The reduction in operating time and solvent use are two major benefits of microwave treatments. However, during microwave processing, acceleration in chemical reactions of target substances such as epimerization, oxidation, and polarization should be considered with dielectric heating.

Microwave-assisted extraction without solvents is a long-term technology for extracting and separating chemicals from natural plant resources. Microwave heating is directed at the moisture content of new material. Under microwave irradiation, plant cell water and charged molecules are stimulated; this internal alteration causes a significant amount of pressure to be imposed on plant cell walls, resulting in cell swelling. Due to the rupturing of plant cells, this swelling causes an increase in the mass transfer of solutes. As a result, phytochemical leaching from the plant cellular matrix into the extractant is facilitated during MAE [40]. The best extraction conditions were a microwave power of 150 W for 90 min. Concerning the efficiency and yield of essential oils, solvent-free microwave extraction was superior. As a result, increased rates of adsorption, diffusion, and separation of phytochemicals from the plant matrix into the extracting solvent are more likely [41].

An MAE can be performed using two different types of equipment. The apparatus runs at atmospheric pressure in the open mode, which is often coupled with a refluxing mechanism. Domestic microwaves are frequently modified to accommodate this model. The closed mode, on the other hand, allows for high-pressure operation. Pumping inert gas into the extraction chamber increases the pressure. During the heating of the extraction mixture, however, vapor pressure may generate a degree of pressure. Since these molecules were stable at microwave heating settings of up to 100°C for 20 min, this approach was confined to small-molecule phenolic compounds like phenolic acids (gallic acid and ellagic acid), quercetin, isoflavones, and trans-resveratrol. Due to compound oxidation, more MAE cycles (e.g., from 2 10 s to 3 10 s) resulted in a considerable reduction in phenolic and flavanone yields. Because tannins and anthocyanins are prone to temperature degradation, they may not be suitable for MAE [32].

Microwave-assisted hydro distillation (MAHD) is like standard hydro distillation, with the exception that the solvent is heated using microwaves. The solvent (typically water) and plant parts are placed inside a microwave oven (normally running at 2.45 GHz), and different output powers and reaction periods can be used to improve the extraction process. Again, using microwaves for the heating process speeds up the extraction of chemicals, requiring shorter timeframes to generate comparable amounts of extracts. Furthermore, the chemical makeup of extracts obtained by standard hydro distillation and MAHD is not comparable.

5.6 Pulsed electric field (PEF) extraction

In batch mode, the electric field strength (EFS) ranges from 100 to 300 V/cm, while in continuous mode, the EFS ranges from 20 to 80 kV/cm. An external electrical force is used in electro-permeabilization or electroporation to increase the permeability of cell membranes. The cell membrane is perforated by the formation of hydrophilic holes, which result in the opening of protein channels. When high-voltage electrical pulses are applied across the electrodes, the sample experiences a force per unit charge termed the electric field. The plant material is removed once the membrane loses its structural functioning [41]. Anthocyanin, carotenoids, lycopene, lutein, polyphenols, alkaloids, lactase, protein, polysaccharides, fat, oil, and other bioactive compounds are extracted using PEF. PEF-assisted extraction provides more bioactive component extracts, uses less energy, and takes less time to process, according to the study, resulting in the optimal process parameters [42].

6. Purification of phytoconstituents

The extracts, which contain numerous phytoconstituents, must be separated and purified further to obtain the fraction or pure phytoconstituents. The techniques utilized for isolation and purification from the extract are determined by the physical and chemical properties of the component to be separated. The physical approaches employed for this goal are as follows.

6.1 Fractional crystallization

The point of supersaturation in the solvent in which phytopharmaceuticals are soluble causes them to crystallize. The processes involved in the crystallization of phytoconstituents are slow concentration, slow evaporation, and chilling. Crystallization is an ideal purification procedure. It is operationally easy, very inexpensive, and may be done in quantities ranging from a few micrograms to hundreds of kilograms. The results are normally highly pure (unlike the mixes that can sometimes be obtained with distillation). Using chromatography to purify that much material is a nightmare. Another key point to remember about crystallization is that X-ray crystallography can be used to discover the structure of unknown molecules. With very few exceptions, X-ray crystallography is the gold standard for structure determination: if you can get a substance to crystallize, you can determine its structure. The only issue is that not all compounds crystallize, and finding circumstances that can preferentially recrystallize one chemical can take a long time [43].

6.2 Fractional distillation

This is a process of purifying phytoconstituents from a mixture. It’s commonly used to separate hydrocarbons like crude oil, citral, and eucalyptol. Purification is accomplished by comparing the boiling points of the different substances. When heat is applied, the fractional distillation equipment is built in such a way that each chemical evaporates and separates at its boiling point. As a result, each fractionated chemical will condense and be collected separately via numerous syphons coupled to fractional distillation apparatus [44].

The fractional distillation method is based on differences in compound volatility and is affected by physicochemical properties of the components, as well as the pressure and temperature of the distillation process. The mass and energy transition between the fluid and vapor stages of the mixture has an impact on separation efficiency. Most terpenes are thermally unstable, dissolving, or oxidizing when exposed to high temperatures, light, or oxygen. As a result, the distillation technique is typically used at vacuum pressures to lower the vaporization temperature of the volatile mixture. Due to the boiling temperature reduction, the vacuum also slows processes such as thermal deterioration in temperature-sensitive chemicals. In the chemical industry, vacuum fractional distillation is used to separate compounds with extremely high boiling points that would need a lot of energy to separate under atmospheric pressure [45].

6.3 Fractional liberation

Fractional liberation separates some components from a mixture. The weakest base in the free salt is liberated first when an aqueous solution of alkaloid salts is treated with aliquots of alkali, followed by base liberation in ascending order of basicity. After each addition, shake the mixture with an organic solvent to get a fractionated sequence of bases. Organic acids that are soluble in water-immiscible solvents take a similar route. It is feasible to fractionally liberate acids in this case by adding mineral acids to a mixture of acid salts.

6.4 Chromatography

6.4.1 Column chromatography

Chromatography on a column separates and purifies phytochemicals on a laboratory and industrial scale without the use of complicated technology. The “eluent” is the liquid employed as the mobile phase, and the stationary phase is usually a solid or a liquid. The sample solution is supplied to a porous stationary phase, and the mobile phase is delivered at a greater pressure via the column, causing separation depending on the solute’s affinity for the stationary phase. The development of HPLC (High-Performance Liquid Chromatography) was aided by the need for a higher degree of separation and faster analysis, which was met by refining the stationary phase packing material to a size of 3–10 m and eluent delivery via a high-pressure pump. Despite its extensive and time-consuming nature, commercial use of column chromatography is comparable to that of other techniques. The advantages of column chromatography include efficient sample handling regardless of the number or nature of the samples, the availability of a wide range of adsorbents, the selection and recyclization of a large solvent system, improved purity of the product, and minimal space requirements. Column chromatography has a few disadvantages, including the use of a large amount of mobile phase, compared to other techniques it is a complicated technique, time consumption, the requirement for an expert, and a greater cost of identifying the separated product.

6.4.2 Vacuum liquid chromatography (VLC)

The fundamental disadvantage of column chromatography is that it is a time-consuming technique; however, vacuum liquid chromatography can solve this problem. In vacuum chromatography, rather than using pressure, vacuum is employed to improve the flow rate and hence speed up the fractionation process. The stationary phase is usually 40–60 mesh particle size silica or reversed-phase silica, and the crude extracts are separated by gradient elution. TLC is a typical method for examining eluted fractions [43].

6.4.3 Simulated moving bed chromatography (SMB)

In the pharmaceutical sector, simulated moving bed (SMB) technology is an economical and eco-friendly process for purifying crude extracts and fractions [46]. It has a higher purity and yield than other techniques. A traditional Simulated Moving Bed system has 4–24 columns divided into four zones. In general, a four-column SMB should be sufficient for testing and optimizing purification conditions. Purification of sugars, proteins, monoclonal antibodies, separation of organic solvents, optical isomers, charged molecules, and desalting are all common applications. For the separation of crude medicines, the SMB technique utilizes extremely less solvent. The SMB technique is simple to adapt to a continuous process and can be integrated with other equipment such as evaporation. SMB, on the other hand, necessitates meticulous process control and is less adaptable than traditional elution chromatography.

6.5 Capillary electrophoresis (CE)

CE provides several advantages, including a smaller sample, high efficiency leads to shorter analysis time, cheap, environmental friendliness, reduced solvent usage, and a powerful tool appropriate for drug discovery [47]. CE is a new method for analyzing different phytochemical groups. Variations in mass to charge ratios are used to separate phytochemicals in capillary electrophoresis. Because borate can form compounds with the flavonoid nucleus’ ortho dihydroxyl groups and the sugar’s vicinal cis-dihydroxyl groups, borate buffers with a pH of 8–11 and a concentration of 25–200 mM are generally used [48].

Capillary zone electrophoresis (CZE) is the most basic characteristic, and it’s been utilized to isolate a variety of target molecules, especially polyphenolic compounds like epicatechin, catechin, quercetin, gentistic acid, caffeic acid, gallic acid, trans-resveratrol, myricetin, and rutin from wine and grape samples. A CZE technique was also used to isolate antioxidants in Ginkgo leaf extracts. For the separation of anthocyanins in wine, a new CZE approach was developed recently [49]. Food analysis, environmental monitoring, clinical diagnostics, and pharmaceutical analysis have mostly used capillary electrophoresis. Since it allows the use of chirality selectors with limited aqueous solubility, nonaqueous capillary electrophoresis can be utilized to separate enantiomeric drugs. Furthermore, the low dielectric constant of organic solvents can let chiral counter-ions that have less selectivity in aqueous environments form ion pairs and therefore increase their selectivity. CE-MS is one of many multidimensional techniques used in the pharmaceutical and biotechnology industries, particularly for drug development. Because high resolution and structural and/or molecular weight information of an analyte may be collected along with using a mass spectrometer as a detector for CE splitting, could be useful. CE has various advantages (for example, high speed, efficiency, and low price); yet, combining CE with MS produces several problems. CE solvents, for example, are not accepted by MS.

6.6 Molecular imprinted technology

Molecular imprinting knowledge has been a prominent isolation method in the last years because of its distinctive qualities, such as high selectiveness, economical, and ease of preparation. Many correlative cavities with the memory of the template molecules’ size, shape, and functional groups are produced when the template molecules are removed from the molecular imprinted polymer (MIP). As a result, the template molecule and its analogues will be able to recognize the MIP and adsorb it selectively. MIPs have been extensively used in the isolation of phytoconstituents and as sorbents for solid-phase extraction of herbal materials to enrich phytoconstituent components. MIP was made with methyl methacrylate as the monomer, solanesol as the template molecule, and ethylene glycol as the crosslinker by a suspension polymerization method. This technique is used for the purification of enriching in water extract of Panax notoginseng, solanesol from tobacco leaves, thermo-responsive magnetic MIP is used to isolate curcuminoids, curcumin, dimethoxy curcumin, and bisdemethoxycurcumin, from the TCM Curcumae Longa Rhizoma [50].

7. Conclusion

Plant materials go through several processes to acquire the necessary secondary metabolites and/or extract, including drying, extraction, separation, and purification. To produce better eco-friendly processes, the current investigation of the use of green solvents in the field of extraction needs more awareness for a greater perception of different factors such as innate solvent properties (polarity, viscosity, solubility, and pH), external factors (temperature, time, and solid-liquid ratio), and cytotoxicity. However, more study is needed on green or smart solvents that have high specificity for phytochemical compounds, as well as improved stability, recovery, and reduced operational costs. Until now, the framework has only been used to evaluate organic solvents. To expand the currently established techniques to new solvents, more study is required. This entails looking into novel waste-solvent treatment technologies as well as alternative solvent production techniques. Will the eventual transfer of DES/NADES-based extraction technologies to industrial sectors need further investments? Would their use result in a shorter lifespan for the extractors and the analytical tools required for their identification and quantification in the long run? All the questions are still open, and there are a lot of options for answers in the future.