Primary metabolites, such as carbohydrates, amino acids, fatty acids, and organic acids, are essential for plant growth, development, and maintenance, playing critical roles in photosynthesis, respiration, protein synthesis, and other physiological processes. They serve as the building blocks for plant growth and development, providing energy and organic compounds necessary for plant survival 11. In contrast, secondary metabolites, including flavonoids, alkaloids, phenolic acids, and others, are not essential for plant growth but play crucial roles in plant defence, stress response, and adaptation to environmental changes.

These metabolites exhibit a range of biological activities, including antimicrobial, insecticidal, antioxidant, and signalling activities, which help plants to defend against pathogens and pests, protect against oxidative stress, and communicate with other plants and organisms. Under stress conditions, plants produce secondary metabolites to enhance their adaptability, and these metabolites play critical roles in defence against pathogens and pests, antioxidant activity, and signal molecule activities, ultimately contributing to plant survival and fitness 12. Additionally, secondary metabolites can also play roles in plant-to-plant communication, attracting beneficial insects, and influencing soil microorganisms, highlighting their importance in plant ecology and interactions with the environment.

Alkaloids: Alkaloids are a diverse group of naturally occurring, organic compounds that contain nitrogen and are primarily found in plants, as well as some animals. One notable example is Catharanthus roseus, which produces an impressive array of around 120 alkaloids, with approximately 70 exhibiting pharmacological activity. The biological effects of alkaloids are wide-ranging, and their alkaline properties stem from the presence of a nitrogen atom. To date, approximately 10,000 alkaloids have been identified, with the majority being utilized for recreational and therapeutic purposes. Alkaloids are classified based on the type of nitrogen-containing ring they possess, which enables their categorization into distinct groups. Many alkaloids have been harnessed for their medicinal properties and are currently employed as drugs in modern medicine. Examples of these pharmacologically active alkaloids include morphine, nicotine, atropine, caffeine, quinine, and codeine. These compounds have been instrumental in the development of various treatments and therapies, underscoring the significant contribution of alkaloids to the field of medicine 13.

Glycosides: Glycosides are a diverse class of secondary metabolic products primarily derived from plants, characterized by their ability to break down into one or more sugars. The non-sugar portion of the glycoside is known as a glycone or genin, and glycosides play a vital role in the plant's life, regulating physiological processes, protecting against pathogens and pests, and promoting overall health. Various types of glycosides exist, including C-Glycosides, anthraquinone glycosides, saponin glycosides, cardiac glycosides (CGs), and isothiocyanate glycosides, each exhibiting unique biological activities such as anti-inflammatory, antioxidant, and antimicrobial properties 14, 15, 16, 17, 18. Many medicinal plants contain significant levels of naturally occurring glycosides, including those with anticancer properties, and the use of glycosides derived from medicinal plants as alternative medications to treat various cancers has been recognized, highlighting their potential as valuable therapeutic agents. Furthermore, glycosides have been found to exhibit a range of biological activities, including anti-diabetic, anti-hypertensive, and anti-arthritic properties, making them a promising area of research for the development of new drugs. Additionally, glycosides have been used in traditional medicine for centuries, and their potential as lead compounds for the development of new pharmaceuticals is being increasingly recognized 19, 8. Overall, glycosides are an important class of compounds with a wide range of biological activities and potential therapeutic applications.

Terpenoids: Terpenes are naturally occurring molecules composed of several isoprene units, which are the building blocks of these compounds. Terpenoids, on the other hand, refer to the various types of terpenes that differ in the number of isoprene units they contain. Monoterpenoids and sesquiterpenoids are two examples of terpenoids, each with distinct properties and functions. Terpenoids are volatile substances responsible for the fragrances of plants and flowers and are widely distributed in the leaves and fruits of higher plants, conifers, citrus, and eucalyptus 20. Herbal terpenoids, such as cadinene, caryophyllene, zingiberene, guaiazulene, and guaianolide, are widely used and characterized by their therapeutic and other effects. These compounds have been found to exhibit a range of biological activities, including anti-inflammatory, antioxidant, and antimicrobial properties, making them valuable for the development of new drugs and therapies 21. One of the most well-known and widely used terpenoids is artemisinin, which is mainly isolated from Artemisia annua. Artemisinin is a potent antimalarial agent, and its derivatives are used for the treatment of malaria. In addition to its antimalarial properties, artemisinin has also been found to exhibit anticancer, anti-inflammatory, and antioxidant activities, highlighting its potential as a versatile therapeutic agent. Terpenoids have also been found to play important roles in plant defence, signalling, and communication 22. They can act as repellents or attractants for insects and other animals and can also influence plant-plant interactions and soil microorganisms. Overall, terpenoids are an important class of compounds with a wide range of biological activities and potential therapeutic applications.

Essential oil: Volatile oil, also known as ethereal oil or essential oil, is a fragrant and unstable substance found in plants and animals. These oils are characterized by their ability to evaporate quickly when exposed to air at room temperature, releasing their distinctive aromas. Essential oils are typically extracted from various parts of plants, including flowers, leaves, stems, bark, wood, roots, seeds, fruits, rhizomes, and gums or oleoresin exudations. The production and consumption of essential oils and perfumes are increasing worldwide, driven by their growing demand in aromatherapy, cosmetics, and pharmaceutical applications 23. However, the quality and yield of essential oils can vary greatly depending on factors such as the plant material used, extraction methods, and production technologies. Therefore, advances in production technology are crucial for enhancing the overall quality and yield of essential oil. Most herbal volatile oils are used for therapeutic purposes, and their potential health benefits are being increasingly recognized 24. These oils have been found to exhibit a range of biological activities, including anti-inflammatory, antimicrobial, and antioxidant properties, making them valuable for the development of new treatments and therapies. Some essential oils, such as tea tree oil and eucalyptus oil, are also used in traditional medicine for their antimicrobial and anti-inflammatory properties 25. Overall, essential oils are a valuable resource with a wide range of applications from aromatherapy and perfumery to pharmaceuticals and traditional medicine.

Flavonoids: Flavonoids are a diverse group of phenolic substances extracted from various vascular plants, with approximately 8,000 distinct chemicals identified to date. These compounds serve multiple purposes in plants, including acting as antioxidants, antimicrobials, photoreceptors, visual attractants, feeding deterrents, and light filters 26. The health benefits of flavonoids are numerous, and include vasodilating, anti-inflammatory, antiviral, and antiallergenic properties, making them a valuable component of a healthy diet. Flavonoids have received growing attention in recent years due to their potent antioxidant properties, which help prevent the production of free radicals and scavenge existing ones, thereby reducing oxidative stress and inflammation in the body 27. These compounds are widely distributed in various plant-based foods, including vegetables, fruits, grains, roots, flowers, bark, stems, tea, and wine. Flavonoids can be classified into different types based on their chemical structure, which includes flavones, flavanones, flavonols, catechins, anthocyanins, and chalcones 28. Each of these subclasses has unique properties and biological activities, and they have been found to have therapeutic significance in the prevention and treatment of various diseases, including cardiovascular disease, cancer, and neurodegenerative disorders.

The medical industry has widely adopted flavonoids due to their potential health benefits and therapeutic applications. Research has shown that flavonoids can interact with various cellular signalling pathways, influencing gene expression, enzyme activity, and cell signalling, which can lead to improved health outcomes. Overall, flavonoids are a valuable group of compounds with a wide range of health benefits and therapeutic applications.

Coumarins: Coumarins are naturally occurring biomolecules often containing an oxygen substituent at the C-7 position. These compounds are highly valued for their bacteriostatic, physiological, and anti-tumour properties, which make them an attractive target for further derivatization and screening as potential therapeutic agents, as shown in Figure 4. Coumarin can also be found in certain fruits (such as bilberry and cloudberry), green tea, and other foods, such as chicory. Most coumarins are found in higher plants, with the richest sources being Rutaceae and Umbelliferone. They are also in significant concentrations of essential oils, including cinnamon bark oil. These compounds exhibit various effective pharmacological actions, such as anti-inflammatory, anti-cancer, anti-bacterial, etc. Synthetic coumarins are primarily used as aroma chemicals due to their odour strength, alkali stability, and relatively low cost 29, 30, 31.

Numerous plants such as Moringa oleifera, Duboisia species, Cannabis sativa, Ficus racemosa, and members of the Amaryllidaceae family have been studied to gain a better understanding of the diverse biomolecules produced by both primary and secondary metabolism.

The variations in primary and secondary metabolites may arise from differences among species, within a species, in growing regions or environments, during processing and storage, as well as from other factors such as plant age, plant part, and analytical methodologies. Since plants grow in different locations and environments, they receive varying amounts of minerals and nutrients, which can be identified using spectroscopic and chromatographic techniques illustrated in Figure 5 32.

Maceration is a process for extracting both organized and unorganized plant drugs. Multiple macerations, such as double or triple, are often used to improve extraction efficiency. During maceration, plant material is soaked at slightly elevated temperatures called digestion. There are two types of maceration: circulatory extraction and multistage extraction techniques. Additionally, there is potential for improvement with this extraction technology, mostly due to efficiency, environmental concerns, and time constraints. Significantly, polarity and consequently solubility, groups involved in the study of natural products typically perform a series of extractions using solvents with varying polarities, ranging from apolar alkane-based solvents (such as n-heptane, n-hexane, and cyclohexane) to intermediate solvents (such as dichloromethane and ethyl acetate) and ultimately to polar solvents (alcohols and water) 34. However, the maceration technique has significant disadvantages, such as excess solvent usage, time consumption, and inefficient extraction 35.

The percolation method is a more efficient extraction technique compared to maceration, offering several advantages. During this process, an imbibition process is employed, where the solvent gradually penetrates the plant material, preventing the percolator - the vessel used for extraction - from getting clogged. This results in a more consistent and controlled extraction process. However, one of the primary disadvantages of the percolation method is the potential for dilution of the extract. As fresh solvents are added during the process, the extract can become diluted, leading to a longer evaporation time. This can be a significant drawback, particularly when working with thermolabile compounds or when a concentrated extract is required. Despite this limitation, the percolation method remains a popular choice for extracting bioactive compounds from plant materials, due to its efficiency and ability to produce high-quality extracts. By optimizing the extraction conditions and solvent composition, it is possible to minimize the disadvantages of the percolation method and obtain the desired extract 36.

Soxhlet extraction is a widely used and efficient method that combines the principles of percolation and maceration techniques. This hybrid approach allows for the optimal extraction of bioactive compounds from plant materials. The Soxhlet extraction process involves the use of high temperatures and a solvent recycling system, which increases the mass transfer rate and enhances the extraction efficiency. The Soxhlet apparatus consists of a distillation flask, a condenser, and a solvent reservoir. The plant material is placed in a thimble, and the solvent is heated, causing it to evaporate and rise into the condenser. The condensed solvent then drips into the thimble, extracting the bioactive compounds from the plant material. The extracted compounds are then collected in the solvent reservoir, and the process is repeated until the desired level of extraction is achieved. Soxhlet extraction offers several advantages over conventional methods, including increased efficiency, reduced solvent consumption, and improved extract quality. The high temperatures used in Soxhlet extraction can also help to break down complex plant matrices, releasing more bioactive compounds into the solvent 37. The Soxhlet extraction is a reliable and efficient method for extracting bioactive compounds from plant materials, and is widely used in various industries, including pharmaceuticals, cosmetics, and food processing.

The Multiple Component Extraction Technique (MCET) is a versatile method that combines reflux extraction, infusion, and decoction to extract bioactive compounds from plants. Reflux extraction, a key component of MCET, involves the repeated circulation of a solvent through the plant material, allowing for more efficient extraction of compounds compared to maceration and percolation. Although reflux extraction is less efficient than the Soxhlet method, its simplicity and ease of use make it a popular choice for plant extraction 38. Decoctions, another component of MCET, are commonly used to prepare ayurvedic extracts known as "Kwaths." This traditional method involves boiling the plant material in water to extract the bioactive compounds. Decoctions are particularly useful for extracting water-soluble constituents, such as polysaccharides, glycosides, and alkaloids. Infusion, the third component of MCET, involves steeping the plant material in hot water to extract the bioactive compounds. This method is also used to extract water-soluble constituents and is commonly used to prepare herbal teas and tinctures 39. Overall, the MCET method offers a flexible and efficient approach to plant extraction, allowing for the extraction of a wide range of bioactive compounds using different solvents and techniques. Its applications are diverse, ranging from the preparation of traditional herbal remedies to the extraction of high-value compounds for pharmaceutical and cosmetic applications.

Various modern techniques are available for the extraction of HBs (Table 1), such as pressurized or accelerated solvent extraction (PSE or ASE), Microwave-assisted extraction (MAE), Ultrasonic-assisted extraction (UAE), Supercritical fluid extraction (SFE), Enzyme-assisted extraction (EAE), and Pulse electric field extraction (PEFE).

The Multiple Component Extraction Technique (MCET) is a highly efficient and versatile method that has gained widespread acceptance in the field of natural products extraction. Its flexibility in handling a diverse range of vegetative extraction samples, including wood, leaves, flowers, fruits, gums, and agricultural residues, makes it an ideal choice for extracting bioactive compounds from various plant materials. MCET is widely employed in the preparation of extracts rich in biologically active compounds, such as flavonoids, alkaloids, phenolic compounds, lipids, essential oils, and other valuable phytochemicals. The technique's ability to extract a broad spectrum of compounds has made it a valuable tool in the discovery and development of new natural products, including pharmaceuticals, nutraceuticals, and cosmeceuticals. The applications of MCET are diverse and continue to expand, with potential uses in the food, beverage, and pharmaceutical industries, as well as in the development of new sustainable products and technologies 40. Overall, MCET is a powerful technique that offers a flexible and efficient approach to extracting bioactive compounds from plant materials, and its continued development and application are expected to have a significant impact on various industries and fields of research.

MAE involves microwaves that induce ionic conduction and dipole rotation in the solvent and the sample, producing heat that helps extract biomolecules from the plant matrix. MAE systems are broadly divided into multimode or closed systems and focused or mono-mode or open systems. In both systems, extraction is carried out at elevated atmospheric pressure. As a part of improving extraction efficiency, the MAE is further modified with nitrogen-protected MAE (NPMAE), vacuum-assisted MAE (VAMAE), ultrasonic MAE (UMAE), dynamic MAE, etc. Extraction efficiency mainly depends on factors like solvent selection, solvent ratio, extraction time, microwave power and temperature, nature of plant materials, etc. 41, 42.

The UAE technique, widely used in various healthcare applications, is a testament to the industry's commitment to safety and environmental friendliness. UAE uses both high-frequency and low-frequency ultrasound waves and has emerged as a useful, safer, and greener method of extraction. It depends on factors such as the frequency and intensity of the ultrasound waves, type of solvent, and temperature 43, 44, 45.

SFE involves extracting supercritical fluids, defined as any pure homogeneous substance above their critical points. To increase the efficiency of SFE, various strategies, such as the use of ionic liquid solvents combined with enzyme-assisted, sudden decompression, etc., are used. The efficiency of the SFE depends upon factors like particle size, moisture content, pressure and temperature, solvent-to-feed ratio, solvent flow rate, etc. It is being used to harvest a wide range of extracts, bioactive molecules, and single compounds 46.

The EAE method is mainly used to overcome some demerits, like less efficiency and low quality of the extract, which is prepared through conventional methods. The basic principle of EAE is that enzymes are ideal for catalysis, which work with specificity and regioselectivity to extract bioactive compounds from the plant matrix. The efficiency of the EAE depends on various operational systems, extraction time, type, and concentration of enzyme, as well as substrate particle size. The EAE method mainly extracts different biomolecules and compounds responsible for flavour and colour, oils, juices, etc., from the plant matrix. The cost and availability of enzymes are the demerits of this method 47.

PEFE is a modern extraction technique largely used in the food industry to increase the shelf life of food products. It is efficient, non-thermal, and rapid. The basic principle of this technique is to increase the membrane permeability with the help of an electric current. The extraction yield of PEFE depends on various factors, such as the intensity of the electric field, duration of extraction, the shape of the pulse wave, type of solvent, and pH 48.

The authors declare no conflict of interest, financial or otherwise.

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