Extraction and Analysis of Chemical Compositions of Natural Products and Plants

Abstract

There are many types of natural plants in nature that contain a variety of effective and complex chemical components. These constituents can be categorized as organic acids, volatile oils, coumarins, steroids, glycosides, alkaloids, carbohydrates, phytochromes, etc., all of which play important roles in the fields of pharmaceuticals, food, nutraceuticals, and cosmetics. The study of extraction and chemical composition analysis of natural products is important for the discovery of these active ingredients and their precursors. Therefore, the aim of this article is to review the status of research on the extraction, separation and purification, and structural identification of natural products, to provide a reference for the study of natural products.

1. Introduction

Natural products originate from constituents or metabolic products found in plants, animals, minerals, marine organisms, and microorganisms, with a primary emphasis on plant sources [1]. In recent years, with the development of modernization, molecular biology, pharmacology, and other disciplines, research on natural products has yielded fruitful results, finding extensive applications in pharmaceuticals, food, and health supplements. Many drugs used in clinical settings are directly or indirectly derived from natural products, which have a long history. Especially noteworthy are natural products isolated from plants, such as morphine, artemisinin, paclitaxel quinine, atropine, etc., which play an important role in modern medicine [2,3,4,5]. Therefore, the study of extraction, separation and purification, and structural identification of natural products is of great significance for the discovery of lead compounds, the development of new drugs, and the advancement of applied chemistry. This article provides a comprehensive review from three aspects: extraction, separation and purification, and structural identification of natural products, offering valuable insights for researchers in the field of natural product studies.

2. Extraction

The chemical composition of natural products is characterized by complex composition and a low content of active ingredients. The development and utilization of natural product resources necessitate research into the complex chemical composition of these products. Extraction and separation are the initial stages of research, aiming to obtain crude extracts containing active components from natural sources, enabling subsequent work in separation, purification, and analysis. Based on the principle of extraction, the extraction of natural products from plants is mainly solvent extraction, steam distillation, sublimed method, and expelling. Based on the extraction methods, these can be categorized into traditional extraction methods (such as maceration, percolation, decoction, reflux extraction, Soxhlet extraction) and modern extraction methods (such as ultrasound, microwave-assisted extraction), as well as more environmentally friendly and efficient methods like supercritical fluid extraction and pressurized liquid extraction (Figure 1). Compared to traditional extraction methods, the aforementioned approaches offer advantages, such as solvent conservation and reduced extraction time [6].

2.1. Traditional Extraction Methods

2.1.1. Maceration

Maceration involves soaking coarse powder of natural materials in an appropriate solvent at room temperature or with gentle heating. This allows the active ingredients to diffuse into the solvent, achieving the purpose of extraction. This method is suitable for extracting natural products that are prone to heat damage and contain large amounts of starch, mucilage, gum, and pectin [7,8,9,10]. The efficiency of maceration is related to the time of impregnation, leaching solvent, the material–liquid ratio, and the particle size of raw materials [11,12]. Di et al. [13] extracted phenolic compounds from hazelnut shells by various methods, including maceration, ultrasonic bath, and high-power ultrasound, then evaluated them for their antioxidant effects. The results indicate that phenolic compounds obtained through maceration extraction exhibit stronger antioxidant effects. Although maceration is a simple and effective method for extracting active components, it is time-consuming and has a relatively low yield. Therefore, maceration is often combined with ultrasound technology to significantly enhance its effectiveness. In a study aiming to improve the nutritional value of olive oil by using ultrasound-assisted maceration to extract phenolic compounds from olive leaves, it was found that the total phenol content in olive oil obtained through ultrasound-assisted maceration was higher than that obtained through conventional maceration, and the process required less time [14]. In another study of citrus peel extraction of volatile compounds, microwave and ultrasonic assistance can significantly improve the extraction efficiency and obtain a large number of volatile compounds [15].

2.1.2. Percolation

Percolation involves putting crushed herbs in a percolation cylinder, continuously adding solvent from the top to allow it to percolate through the natural material. The solvent permeates through the medicinal herbs, leaching out the components of the raw material as it flows downward. Percolation is a dynamic leaching method with high solvent utilization, complete leaching of active ingredients, and direct collection of leachates. Percolation extraction is conducted at room temperature, providing a gentle extraction process suitable for heat-sensitive substances. However, it is not suitable for extracting natural materials that are prone to expansion and lack organized structures. The disadvantages of the percolation method are the large amount of solvent used and the time-consuming extraction [16,17]. Factors affecting the percolation process are powder size, solvent composition, extraction time, percolation flow rate, and solvent dosage [18,19,20]. The method is commonly used in different concentrations of ethanol or white wine as a solvent, so the solvent should be prevented from volatile loss. Wilson et al. [21] extracted total cannabidiol from cannabis by both maceration and percolation, respectively, and the results showed that percolation was more efficient than maceration extraction. In another study on the extraction of phenolics from Allium sativum, extraction and recovery rates were higher with percolation than with maceration [22]. Similarly, in a comparison of the extraction of volatile components from grapeseed oil, Soxhlet extraction yielded 67 volatile components, while percolation yielded 60 volatile components [23].

2.1.3. Decoction

Decoction involves heating and boiling the crude powder of natural materials in water and keeping it for a certain duration, so that the natural ingredients can be leached out. Water must be used as the solvent when extracting natural material using the decoction method. While the decoction method is quite effective in extracting, it is not recommended for materials that are high in starch, mucilaginous compounds, volatile components, or chemicals that degrade quickly in the presence of heat. The decoction method is mostly used in Chinese medicine clinics. For thousands of years in Chinese medicine clinics, a variety of Chinese medicines have been paired to form a formula, which is boiled with water and concentrated for the treatment of diseases. Yang et al. [24] found that Xuefu Zhuyu decoction had neuroprotective effects on rats in the controlled cortical influence (CCI) model and enhanced their memory and learning ability, suggesting that Xuefu Zhuyu decoction can treat traumatic brain injury. In addition, several studies have shown that the extract obtained by decoction has good antioxidant and antifungal activities, due to the high content of phenolic compounds [25,26]. Martins et al. [27,28] found that the extracts obtained by decoction had the highest content of flavonoids and showed strong antioxidant activity and antimicrobial activity after comparing the extracts obtained by decoction, infusion, and hydroalcoholic methods. Decoction does have one clear drawback, though: it can only extract natural compounds with higher water solubility. Reynoso-Camacho et al. [29] analyzed the residual chemical constituents in decocted citrus broths and citrus dregs, and found higher levels of flavanones and phytosterols in citrus dregs, which were not detected in citrus decoction. At the same time, some of the carotenoids are degraded due to high-temperature decoctions. Zan et al. [30] extracted and identified 85 volatile compounds of Yinchenzhufu decoction and clarified its pharmacokinetic characteristics. This study provided a deeper understanding of the role of volatile compound decoction in the treatment of disease.

2.1.4. Reflux Extraction

Reflux extraction is a method that uses volatile organic solvents, such as ethanol, to extract components from raw materials. The extracted liquid is heated and distilled, with the volatile solvent condensing and returning to the extraction vessel for repeated soaking of the raw material. This cycle is repeated until the complete reflux extraction of the active components. Reflux extraction improves the extraction rate and reduces the solvent usage, but it is not suitable for the leaching of raw materials that are easily damaged by heat due to the long heating time. The main factors affecting the reflux extraction method are the material–liquid ratio, the extraction time, and the concentration of organic reagents [31,32]. Ma et al. [33] used ethanol as a solvent for reflux extraction of phenolic compounds from Pleioblastus amarus shells, and optimized the extraction parameters by combining the response surface to obtain optimal extraction process parameters, including an ethanol concentration of 75%, a liquid–solid ratio of 20:1, and an extraction time of 2.1 h. In addition, it has been shown that autoclaving the raw materials before reflux extraction can significantly increase the extraction rate. The content of polysaccharide and triterpenoid from Chaenomeles fruits and the time of autoclaving treatment has a significant effect on extraction efficiency [34]. Many studies have shown that the efficiency of reflux extraction is lower than that of modern extraction methods, such as ultrasound-assisted extraction and microwave-assisted extraction [35]. Yang et al. [36] used different methods to extract apigenin from Scutellaria barbata D. Don, and the results showed that compared with heat-reflux extraction, ultrasound-assisted supercritical CO₂ extraction of apigenin took a shorter time and had a higher yield. However, in the extraction of pectin, microwave frequency may affect the structure and quality of pectin [37]. Jiang et al. [38] adopted steam distillation extraction, reflux extraction, and ultrasound-assisted extraction to extract essential oils and evaluate their quality. The yield and chemical composition of essential oils obtained by different extraction methods are different, which suggests that we should choose the appropriate extraction method when extracting essential oils.

2.1.5. Soxhlet Extraction

Soxhlet extraction, also known as the continuous reflux extraction method, uses the siphon principle and solvent reflux to extract the solid material with pure solvent each time. Soxhlet extraction offers greater extraction efficiency and uses less solvent than reflux extraction, solving the drawbacks of reflux extraction, which include high solvent consumption and many reflux extractions [39]. Similarly, heat-damaged components cannot use this method. Usually, Soxhlet extraction is used for extracting phenolic compounds and oils [40,41,42]. Alara et al. [43] extracted phenolic compounds from Vernonia cinerea leaves using the Soxhlet extraction method, and investigated the effects of extraction time, feed-to-liquid ratio, ethanol concentration on product yield, total polyphenols, and total flavonoids content. The results showed that an extraction time of 2 h, a feed solvent ratio of 1:20 g/mL, and an ethanol concentration of 60% v/v showed a higher yield.

2.1.6. Steam Distillation

Steam distillation is primarily used to extract volatile components, primarily essential oils, from chemical substances that are insoluble in water, volatile, and cannot be destroyed by steam distillation [44,45,46]. The extraction principle involves bringing out the volatile components in the raw materials, after crushing and soaking, with steam distillation, and collecting them in layers after condensation. Steam distillation has simple equipment and operational steps, but during the extraction process, the high temperature may cause the thermally unstable components in essential oils to be destroyed. Poor water and oil separation and easy emulsification of the volatile oil contribute to the low rate of oil recovery. Factors affecting the extraction of volatile oil are mainly herb-related, including moisture content, particle size, origin, etc. In addition, extraction conditions such as material–liquid ratio and distillation time also have an effect on the yield and quality of essential oils [47,48]. It was also shown that hydro distillation can obtain higher yields than steam distillation [49,50].

2.2. Modern Extraction Methods

2.2.1. Ultrasound-Assisted Extraction

The principle of ultrasonic-assisted extraction is based on the mechanical effects and cavitation effects of ultrasound, aiming to extract the chemical components of natural products by increasing the movement speed and penetration force of medium molecules. The mechanical effect refers to the propagation of ultrasound in the medium, causing the particles in the medium to vibrate within its spatial range, thereby enhancing diffusion and mass transfer in the medium. The cavitation effect of ultrasound refers to the vibration of microbubbles in the medium induced by ultrasound. When the sound pressure reaches a certain value, the bubbles enlarge due to directional diffusion, forming resonant cavities, and then collapse [51,52,53]. In comparison to traditional extraction methods, ultrasonic extraction is a green and economically viable approach for extracting natural products. It significantly reduces extraction time, enhances extraction efficiency, and concurrently reduces solvent usage. It serves as an energy-saving and emission-reducing alternative to traditional extraction methods [54].

Lin et al. [55] extracted Shatian pomelo peel polysaccharide with different extraction methods, and the results showed that ultrasonic extraction had a higher yield and stronger antioxidant activity. The main factors affecting the efficiency of ultrasonic extraction are solid–liquid ratio, ultrasonic power, ultrasonic time, and ultrasonic temperature. Most current studies have combined response surface optimization with ultrasonic extraction conditions. Achat et al. [14] investigated the possibility of extracting phenolic compounds from olive leaves by ultrasonic maceration in order to improve the nutritional value of olive oil, and determined the optimal extraction conditions as an ultrasonic power of 60 W, ultrasonic temperature of 16 °C, and ultrasonic time of 45 min. Nurkhasanah et al. [56] optimized the extraction of anthocyanins from pigmented corn using response surface, and obtained the optimum process conditions as an extraction solvent of 36% methanol, optimum pH of 7 for extraction, ultrasonic power of 73%, extraction time of 10 min, and extraction temperature of 70 °C. When extracting volatile components, compared with conventional extraction technology, ultrasound-assisted extraction of clove oil and α-humulene obtained a higher yield and required a shorter time, indicating that ultrasound-assisted extraction is an efficient and energy-saving extraction method [35].

2.2.2. Microwave-Assisted Extraction

The microwave extraction method uses microwaves to destroy the cell wall and cell membrane of plant cells, so as to achieve the purpose of extracting active ingredients within the cell. At the same time, microwaves can rapidly heat the extraction system as a whole, cutting down on both the extraction time and the volume of organic solvents required [57,58,59]. Factors affecting microwave extraction efficiency during microwave extraction are extraction time, extraction temperature, material–liquid ratio, and microwave power. Hu et al. [60] extracted polysaccharides from Camptotheca acuminata fruits using microwaves, and optimized the microwave-assisted extraction conditions with response surface methodology. The optimal extraction conditions were a material–liquid ratio of 1:40, microwave power of 600 W, extraction time of 14 min, and extraction temperature of 70 °C. Compared with traditional extraction methods, microwave extraction has the advantage of high efficiency. Ding et al. [61] compared ultrasound-assisted, microwave-assisted, and reflux extraction using ionic liquids as extraction solvents, and showed that microwave-assisted extraction had the highest extraction efficiency. In most cases, the yield of microwave-assisted extraction is better than that of traditional extraction methods. Fernandez-Pastor et al. [62] studied microwave-assisted extraction and Soxhlet extraction of triterpene acids from olive skins, and the results showed that microwave-assisted extraction saved time and increased the yield of triterpene acids. Similarly, in the study of microwave-assisted extraction of lavender, the essential oil obtained by microwave-assisted extraction showed higher antibacterial activity than that obtained by hydrodistillation [63]. This suggests that microwave-assisted extraction is an efficient and effective extraction method.

2.2.3. Supercritical Fluid Extraction

Supercritical fluid refers to the dual characteristics of liquid and gas when a substance is at its critical point (critical temperature, critical pressure, and critical density). At this point, the fluid exhibits density and solubility similar to that of a liquid, viscosity close to that of a gas, and a diffusion coefficient 100 times that of a liquid [64]. Supercritical fluid extraction is a new type of green extraction technology that uses supercritical fluid as the extraction solvent, with commonly used extraction solvents being carbon dioxide [65]. Carbon dioxide, as a supercritical fluid for extraction, has several advantages: (1) its critical temperature is close to room temperature, making it suitable for extracting heat-sensitive components; (2) the critical pressure is not excessively high, facilitating easy operation; (3) its density is highly sensitive to changes in temperature and pressure, and its solubility is proportional within a certain range, allowing for the modulation of substance solubility by controlling temperature and pressure; (4) it is non-toxic, non-flammable, chemically inert, inexpensive, high-purity, readily available, and environmentally friendly [66,67,68,69]. Compared with traditional extraction methods, supercritical fluid extraction can shorten the extraction time and increase the extraction rate [70]. Zhang et al. [71] extracted camphor tree essential oil with steam distillation and supercritical CO₂, respectively, and the results showed that supercritical fluid extraction had a higher yield of essential oil. Additionally, GC/MS analysis revealed a more varied composition of the essential oils extracted using supercritical fluid. The main factors affecting supercritical fluid extraction are extraction temperature and extraction pressure, so the extraction conditions can be optimized by combining the response surface. Zermane et al. [72] used response surface methodology to optimize the supercritical extraction conditions for essential oil from Algerian Myrtus communis L. leaves, and the results showed that the highest yield was obtained at 313 K, 30 MPa.

2.2.4. Pressurized Liquid Extraction

Pressurized liquid extraction (PLE) is an automated method of extraction with organic solvents at elevated temperatures (50~200 °C) and pressures (1000~3000 PSI) [73]. The elevated temperature significantly attenuates the interaction forces caused by van der Waals forces, hydrogen bonding, and dipole attraction between the target molecule and the active site of the sample matrix [74]. The solvency of liquids is much greater than that of gases, so increasing the pressure in the extraction cell raises the solvent temperature above its boiling point at atmospheric pressure. The advantages of this method are a small amount of organic solvent, rapidity, high recovery, and good reproducibility [75,76]. Cam et al. [77] compared conventional organic solvent extraction and PLE of polyphenols from pomegranate peels, showing that PlE is an effective polyphenol extraction technique, which can be an alternative to conventional organic solvent extraction, indicating that pressurized liquid extraction is a green extraction method. Factors affecting pressurized liquid extraction relate to solvent polarity, liquid–solid ratio, and extraction temperature [78,79,80]. Supasatyankul et al. [81] studied phenolic and flavonoid compounds from Mung Bean (Vigna radiata L.) seed coat. The effects of temperature, pressure, and ethanol concentration on the content of total flavonoids and total polyphenols were investigated by response surface, and the results showed that 160 °C, 1300 PSI, and 50% ethanol were the optimal extraction conditions. In a study on extracting volatile compounds from coffee beans, the main factor affecting extraction was temperature [82].

2.2.5. Enzyme-Assisted Extraction

Enzyme extraction is based on the ability of enzymes to degrade plant cell walls, thereby facilitating the release of intracellular compounds. Choosing specific enzymes can promote the conversion of certain low-polarity lipophilic components into glycoside-like components that are easily soluble in water, facilitating extraction. Enzymes can also break down and remove impurities such as starch, proteins, pectin, etc., which affect the clarity of the extraction liquid [83]. Giahi et al. [84] used cellulase, hemicellulase, and pectinase to pre-treat licorice roots before extraction, and the results showed that the enzyme treatments were able to increase the yield of glycyrrhizic acid. In addition, the residue after extraction using conventional extraction methods can be re-treated with enzymes to obtain non-extractable active ingredients [85]. Factors affecting enzymatic extraction are the nature of the enzyme itself (optimum pH and optimum temperature), the concentration of the enzyme, and the concentration of the substrate [86]. Since enzymes are often extracted at low temperatures, they are suitable for extracting natural active substances that decompose easily when exposed to heat [87]. In addition, enzyme extraction is often combined with other extraction methods, such as ultrasound-assisted enzyme extraction [55], enzyme-assisted supercritical fluid extraction [88], and enzyme-assisted microwave extraction [89]. The combined use of extraction methods can greatly improve the efficiency of extraction.

2.2.6. Ionic Liquid Extraction

Ionic liquids are salts composed of organic cations and organic or inorganic anions that appear liquid at room temperature, characterized by high viscosity and density [90]. Ionic liquid, as a new type of extraction agent, has the advantages of low vapor pressure, non-volatility at room temperature, good chemical and thermal stability, non-flammability, good electrical and thermal conductivity, a wide range of soluble substances, high solubility for inorganic, organic, and other substances, and designability compared to traditional organic solvents [91,92,93,94]. The mechanism by which ionic liquids can extract natural products is that they can complex with cellulose in the cell wall, break the hydrogen bonds between cellulose molecules, and make cellulose soluble in ionic liquids, thus enabling the target substances to dissolve out of the cell wall better, improving the extraction rate. Through the design of the structure of the ionic liquid, certain groups of electrostatic interaction, dispersion, hydrogen bonding intermolecular forces can be achieved, increasing the solubility of the target extract in the ionic liquid to achieve high efficiency and high selectivity extraction. Nowadays, microwave and ultrasonic methods are usually combined with ionic liquids to improve the extraction and separation of natural products [95,96,97,98]. Ding et al. [61] designed and synthesized 11 ionic liquids for the extraction of Praeruptorin A from Radix peucedani, showing that the soluble guanidinium ILs aqueous solution achieved a much higher extraction amount than the pure insoluble guanidinium ILs. It was also found that among the 11 ionic liquids, [TMG]CH 2CH(OH)COOH had the highest extraction rate of Praeruptorin A, the same as that of methanol. Wang et al. [99] extracted alkaloids from Phellodendron amurense Rupr by ultrasonic-assisted extraction, using four ionic liquids and water as solvents. The results showed that the shorter the carbon chain of the ionic solvents, the higher the extraction rate of alkaloids, related to the solubility of alkaloids. The ultrasonic conditions of ionic solvents were also optimized, and the optimal extraction conditions for alkaloids were obtained as ultrasonic power of 100 W, extraction time of 75 min, and material–liquid ratio of 1:14. Ionic liquids can also be used to extract volatile compounds. In a study of microwave-assisted extraction of the essential oil of Schisandra chinensis using 1-lauryl-3-methylimidazolium bromide ionic liquid as a solvent, ionic liquid extraction not only improved the extraction efficiency but also shortened the extraction time [100].

The above discussion has covered the characteristics and extraction examples of commonly used extraction methods. A summary of the characteristics, extraction solvents, and extraction temperatures of a commonly used extraction method are summarized in

Table 1. Summary of extraction methods.

Method	Characteristic	Solvent	Temperature
Maceration	easy to operate, time-consuming, low yield [14]	Water, organic solvents	room temperature, warm conditions
Percolation	high solvent utilization, complete leaching of active ingredients, direct collection of leachates [16,17]	ethanol, white wine	room temperature
Decoction	easy to operate, short extraction time, high production efficiency [27,28]	water	heat
Reflux extraction	high extraction rate, high solvent consumption [31,32]	volatile organic solvents	heat
Soxhlet extraction	reduces the solvent usage, high extraction efficiency [39]	volatile organic solvents	heat
steam distillation	simple equipment, simple operation steps [44,45,46]	water	heat
Ultrasound-assisted extraction	saves extraction time, improves extraction efficiency, reduces the use of solvents [54]	water, organic solvents, ionic liquid	room temperature, warm conditions
Microwave-assisted extraction	saves extraction time, improves extraction efficiency, reduces the use of solvents [57,58,59]	water, organic solvents, ionic liquid	heat
Supercritical fluid extraction	short extraction time, environmentally friendly, high extraction efficiency [66,67,68,69,70]	supercritical CO2	room temperature
Pressurized liquid extraction	reduces the solvent usage, rapidity, high recovery good reproducibility [75,76]	water, organic solvents,	heat
Enzyme-assisted extraction	simple equipment, poor selectivity [87]	water	room temperature
Ionic liquid extraction	good solubility properties, wide operable temperature [91,92,93,94]	Ionic liquid	−40~300 °C

, aiding in the selection of the appropriate extraction method for natural product extraction.

3. Separation

Natural products are extracted to obtain a complex and diverse mixture of components, which needs to be further separated to obtain pure natural compounds. The selection of the separation method is mainly based on the differences in physical or chemical properties between the compounds. At present, chromatography is an effective separation method that utilizes the differences in adsorption capacity, partition coefficient, or other affinity effects of each component of the mixture in the immiscible two phases (stationary and mobile phases) to achieve separation [101,102]. Chromatography has the advantages of high sensitivity, high selectivity, high performance, fast analysis, and a wide range of applications.

Based on the state of the mobile phase, chromatography can be categorized into gas chromatography, liquid chromatography, and supercritical fluid chromatography. Based on the fixed state of the stationary phase, it can be classified as column chromatography and thin-layer chromatography, countercurrent chromatography. According to the separation principle, it can be divided into adsorption chromatography, partition chromatography, ion exchange chromatography, and molecular exclusion chromatography [103] (Figure 2).

3.1. Adsorption Chromatography

Adsorption column chromatography uses a solid adsorbent as the stationary phase and realizes separation by taking advantage of the differences in adsorption capacity of the separated components to the active adsorption center on the surface of the stationary phase. Most stationary phases for adsorption chromatography are porous particulate materials with a large specific surface area, and the active groups on the surface are called adsorption centers. The adsorption capacity of different adsorbents depends on the number of adsorption centers and the adsorption center’s ability to form hydrogen bonds with the adsorbent. The more adsorption active centers there are and the stronger the ability to form hydrogen bonds, the stronger the adsorption capacity of the adsorbent. Adsorbents commonly used in adsorption chromatography can be divided into two categories: organic and inorganic. Organic adsorbents include activated carbon, starch, sucrose, polyamide, and macroporous adsorption resin. Inorganic adsorbents include silica gel, alumina, magnesium oxide, calcium carbonate, and diatomaceous earth. Among them, polyamide, macroporous adsorption resin, silica gel, and alumina are more commonly used.

3.1.1. Polyamide Column Chromatography

Polyamide is a class of polymer compounds made of amide polymerization, with polycaprolactam being commonly used. The amide group in the carbonyl group can form hydrogen bonds with phenols, flavonoids, and acids in the hydroxyl group, and the amino group can form hydrogen bonds with quinones and fat carboxylic acid on the carbonyl group, thus producing an adsorption effect [104,105,106]. Different compounds achieve separation due to variations in the type, number, and location of active functional groups, as well as the differing forms and capabilities of hydrogen bonding with polyamide. Currently, polyamides are widely used in the separation of flavonoids [107,108,109]. Li et al. [110] compared the separation of total flavonoids from Ginkgo biloba flowers by five kinds of macroporous resins and polyamide resins, and the separation effect of the polyamide resin was better than that of macroporous resin. Due to the good adsorption effect of polyamide on flavonoids, in order to obtain non-flavonoid target compounds, polyamide can be used to remove flavonoids first, and then further separated and purified to obtain pure compounds, using macroporous resins [111].

3.1.2. Macroporous Resins

Macroporous adsorbent resin is a kind of polymer adsorbent without dissociable group and with a large pore mesh structure, divided into two categories: non-polar and medium polar. Macroporous resins are separation materials that combine the principles of adsorption and molecular sieving. Their adsorption is due to the generation of van der Waals forces and hydrogen bonding, while their molecular sieving properties are determined by their own porous structure. The adsorption performance of macroporous resins depends mainly on the surface properties of the adsorbent, such as the specific surface area, surface electrical properties, and the ability to form hydrogen bonds with compounds. The main factors affecting the separation efficiency of large-pore resin include the characteristics of the resin, the properties of the substances being separated, and the nature of the solvent. Macroporous resins are widely used in the separation of natural compounds (flavonoids, saponins, sugars, alkaloids) because of their stable nature and insolubility in acids, alkalis, and organic solvents [112,113,114,115,116,117].

Compared to natural adsorbents, macroporous resins have a higher adsorption capacity and yield purer compounds. Ye et al. [118] compared the adsorption capacity of different natural sources of lignocelluloses and synthetic macroporous resin HPD 600 for decaffeinated catechins in tea, and the results showed that HPD 600 had the highest adsorption capacity for catechins. Dong et al. [119] used ADS-7 macroporous resin to isolate flavonoids root bark glycosides from crude extract of Lithocarpus Polystachyus Rehd with a purity of 99.87%, and the recovery could reach 40%. It has also been shown that macroporous resin chromatography coupled with other chromatography can further isolate and purify natural products to obtain pure compounds [112,120]. It is worth noting that when using macroporous resins for the separation of natural products, it is necessary to select suitable macroporous resins according to the nature of the target compounds, in order to separate the target compounds more efficiently and optimize the separation conditions at the same time. Sun et al. [121] compared the separation and purification effect of eight kinds of macroporous resins on total polyphenols from apples. The result showed that the surface X-5 resin had the best adsorption effect on total polyphenols. Further optimization of separation conditions, such as maintaining a sample solution pH of 5, using a 70% ethanol solution as the eluent, and setting the elution rate to 2.0 BV/h, increased the content of total polyphenols by 2.12 times.

3.1.3. Silica Gel

Silica gel has a silicone-oxygen crosslinked structure, and the surface has many porous particles of silanol group. The silanol group is the adsorption activity center of silica gel, that can form hydrogen bonds with polar compounds or unsaturated compounds during adsorption. Silica gel is slightly acidic and suitable for the separation of acidic and neutral substances, such as organic acids, amino acids, steroids, and glycosides [122,123,124]. Zhou et al. [125] obtained two new tetralone derivatives from the crude extract of Cyclocarya paliurus leaves by stepwise separation using silica gel chromatography, and they further structurally resolved the compounds using infrared spectroscopy and nuclear magnetic resonance spectroscopy. Zhang et al. [126] synthesized bionic multi-tentacled ionic liquid-modified silica gel as an adsorbent for the separation of polyphenols from green tea (Camellia sinensis) leaves. After optimizing the operating conditions, the adsorption capacity reached 236.84 mg/g, which was superior to that of traditional adsorbents. In addition, it has been shown that the combined use of silica gel and other adsorption chromatography methods can separate pure compounds more efficiently [127,128,129].

3.1.4. Aluminum Oxide

Alumina is a kind of adsorbent with a strong adsorption capacity, with the advantages of strong separation capacity and controllable activity. Alumina used for chromatography is divided into three types: alkaline, neutral, and acidic, according to difference in pH during preparation. Alkaline alumina is suitable for the separation of alkaloids and neutral compounds, acidic alumina is applicable to the separation of acidic compounds such as acidic pigments, amino acids, and neutral substances that are stable in acid, and neutral aluminum oxide is suitable for the separation of alkaloids, volatile oils, terpenes, steroids, and compounds such as glycosides, esters, and lactones, that are unstable in acids and bases acids and bases [130,131,132]. Feng et al. [133] used alumina column chromatography to separate glucose from cellulose hydrolysis products. Compared with other separation methods, alumina column chromatography allows for easy and quick separation of the target product without polluting the environment.

3.2. Distribution Chromatography

Distribution chromatography uses solubility differences between separated components in the stationary and mobile phases to achieve separation. Techniques, such as liquid–liquid extraction, paper chromatography, droplet countercurrent chromatography, high-speed countercurrent chromatography, gas–liquid partitioning chromatography, and liquid–liquid chromatography mostly belong to the distribution of chromatography.

However, the most commonly used method is high-speed countercurrent chromatography (HSCCC), which is widely used for the separation of natural products such as saponins, alkaloids, acidic compounds, proteins, and carbohydrates. The principle of countercurrent chromatography is based on the partitioning of a sample between two immiscible solvents, whereby the components of the sample are separated due to differences in partition coefficients during their passage through the two solvent phases. High-speed countercurrent chromatography is a new liquid chromatographic technique that utilizes countercurrent partitioning of liquid–liquid phases to separate mixtures in the absence of a solid filler [134]. HSCCC utilizes a constant force field generated by centrifugal force to retain the stationary phase in a series of chambers connected by piping. Since both the stationary and mobile phases of HSCCC are liquids without irreversible adsorption, it has the advantages of a wide range of applications, flexible operation, no loss of samples, no contamination, high efficiency, rapidity, and the ability to separate large preparative volumes [135,136,137]. HSCCC has been recognized as an effective new separation technology in the natural product industry, and is widely used in the separation of traditional Chinese medicine ingredients and natural product chemistry. The choice of the solvent system is very important in the separation of natural products by HSCCC, and two aqueous systems can be used to separate polar components [138]. Song et al. [139] utilized an aqueous two-phase system of PEG1000-K₂HPO₄-KH₂PO₄-H₂O (0.5:1.25:1.25:7.0, w/w) to isolate three polysaccharide fractions from Auricularia polytricha. In a tumor inhibition assay, APS-2 showed 40.4% tumor inhibition. Zhi et al. [140] established a hydrophilic organic/salt-containing aqueous two-phase system for the isolation of salvianolic acid B from the crude extract of Salvia miltiorrhiza, and the results showed success in the 36% (w/w) n- propanol/8% (w/w) phosphate system. pH-zone-refining countercurrent chromatography is a preparative technique developed based on HSCCC, which is often used for the separation of alkaloidal compounds [140,141,142,143,144]. In addition to changing the two-phase solvent system, HSCCC can be combined with other chromatographic techniques to rapidly separate and purify target compounds from natural products in one step. Li et al. [145] proposed a new type of expanded bed adsorption chromatography (EBAC) and high-speed countercurrent chromatography that can rapidly separate salvianolic acid B and rosmarinic acid from Salvia miltiorrhiza, as well as coptisine and berberine from Rhizoma coptidis.

3.3. Ion Exchange Chromatography

Ion exchange refers to the mutual exchange of an ion in a solution with an ion on a resin, where ions in the solution are exchanged to the resin, and ions on the resin are exchanged to the solution. Ion exchange chromatography, a liquid chromatographic method for separating ionic compounds by ion exchange, employs a commonly used stationary phase called ion exchange resin, a class of polymers with a reticulated three-dimensional structure. There are many types of ion exchange resins, with the most used being polystyrene ion exchange resin. It is formed through the polymerization of styrene as a monomer and divinylbenzene as a cross-linking agent, resulting in a spherical mesh structure. The introduction of different active groups onto the mesh skeleton allows it to become an ion exchange resin. According to the introduction of the active groups, different ion exchange resins can be divided into two major categories: cation exchange resins and anion exchange resins. Acidic groups, such as sulfonic acid groups, carboxyl groups, and phenolic hydroxyl groups, are introduced into the structure of the resin skeleton, and the hydrogen ions of these acidic groups can be exchanged with cations in the solution, so they are called cation exchange resins. If alkaline groups, such as quaternary amino groups, primary amino groups, and secondary amino groups, are introduced into the skeleton of the resin, the hydroxyl radicals of these alkaline groups can exchange with anions in the solution, leading to them being called anion exchange resins. Ion exchange resins have the advantages of fast separation speed, no use of organic solvents in the separation process, and no pollution to the environment. Ion exchange chromatography is mostly used for the separation and refinement of plant macromolecular proteins, nucleic acids, and polysaccharides [146,147,148,149]. Guo et al. [150] used a variety of separation methods to separate the mushroom extract step by step, finally separating proteins with antifungal properties through Affi-gel blue gel affinity chromatography column and CM-cellulose ion exchange chromatography column. Other studies have shown that different polysaccharides can be separated by changing the salt concentration during ion exchange chromatography, a process that can remove impurities and obtain the target compounds [151]. In addition, ion exchange chromatography can be used in conjunction with other chromatographic methods to separate pure compounds [152,153,154].

3.4. Molecular Exclusion Chromatography

Molecular exclusion chromatography, also known as volume exclusion chromatography, spatial exclusion chromatography, or gel chromatography, involves the separation of solute molecules based on their sizes. The stationary phase used is a chemically inert porous material with no interaction with the solute. Molecular exclusion chromatography uses a porous gel as the stationary phase, exploiting the presence of numerous pores with different sizes on the gel surface. When a sample solution containing molecules of varying sizes passes through the gel, smaller molecules can occupy more pores on the gel surface. As a result, they spend more time within the column, leading to a longer elution time. In contrast, larger molecules cannot enter the pores, requiring less time to pass through the chromatographic column. This allows for the separation of sample components in order of molecular size. Commercial gels are dry granular substances, and they can only be made into gels after absorbing a large amount of solvent for dissolution. Commonly used gels are the cross-linked dextran gel, polyacrylamide gel, agarose gel, and polystyrene gel [155,156,157]. Molecular exclusion chromatography is mainly used for the separation and analysis of large molecules (proteins, polysaccharides) [158,159]. Another study showed that molecular exclusion chromatography has a better separation effect on anthocyanins and other polymers [160,161]. In order to obtain better separation results, separation conditions can be optimized by changing the nature of the eluent [160]. Zhang et al. [162] separated polysaccharides from Laminaria japonica using high-performance size-exclusion chromatography and optimized the separation conditions, and the results showed that the highest separation efficiency was achieved with water as the mobile phase at a flow rate of 0.6 mL/min. Zhao et al. [163] purified polysaccharides from ginseng by first removing saponins with a macroporous resin, followed by gradual separation through molecular exclusion chromatography after alcohol precipitation to finally obtain purified polysaccharides.

3.5. Other Chromatograms

3.5.1. Membrane Filtration

Membrane separation is based on the molecular weight size of the separation, utilizing a selectively permeable membrane as the separation medium. When there is a pressure difference, concentration difference, or point difference between the two sides of the membrane, the components on the side of the crude extracts selectively permeate through the membrane, so as to achieve separation and purification [164]. Compared with traditional separation methods, membrane separation does not involve the use of organic solvents, making it suitable for heat-sensitive substance separation, and it has high separation selectivity. Tannins, starches, resins, and proteins can be removed from crude extracts by selecting suitable membrane materials [165,166]. According to the separation function, the separation membrane can be divided into microfiltration (≥0.1 µm), ultrafiltration (10~100 nm), nanofiltration (1~10 nm), and reverse osmosis (≤1 nm) [167]. Microfiltration and ultrafiltration can be used to clarify extracts, while nanofiltration allows for concentration [168]. Membrane separation technology is now widely used in the separation of natural products, especially for heat-sensitive compounds such as anthocyanins and other polyphenolic compounds [169,170,171,172]. When performing ultrafiltration, it is necessary to choose the appropriate membrane pore size and ultrafiltration conditions. Zhu et al. [173] carried out ultrafiltration of Jerusalem artichoke chrysanthemum extract by optimizing the operating conditions, and the results showed that when the trans-membrane pressure was 0.3 MPa and the rotation speed was 800 rpm, filtrate with inulin purity of 98% was obtained using a 50-kDa membrane. Balyan et al. [174], in the separation of phenolic compounds from Syzygium cumini (L.) Skeels leaves, achieved high purity in the separation of polyphenolic compounds with a 0.45 μm membrane.

3.5.2. Molecular Distillation

Molecular distillation differs from general distillation techniques in that it utilizes the difference in the mean free path of gas molecules of different substances to achieve the separation of substances [175]. Heating a liquid substance causes a sufficient number of molecules to overflow the liquid surface. Light molecules have a larger average free path, while heavy molecules have a smaller average free path. If a collector is placed where the distance from the liquid surface is less than the average free path of light molecules but greater than the average free path of heavy molecules, light molecules can be continuously captured. This disrupts the dynamic equilibrium of light molecules, causing them to continuously overflow from the mixture. On the other hand, heavy molecules, unable to reach the collector, achieve dynamic equilibrium and no longer overflow from the mixture, thereby achieving separation. Compared to conventional distillation techniques, molecular distillation operates at lower temperatures, lower pressure, shorter heating times, and achieves a higher degree of separation. At present, this technology is widely used in the separation of high-purity substances. In the separation of natural products, it can be used to extract volatile components and heat-sensitive substances [176,177,178,179]. Deng et al. [180] obtained grapefruit essential oil prepared by molecular distillation, which had good in vitro antioxidant, antibacterial, and antitumor activities. Ruben et al. [181] found that compounds obtained from oregano essential oils by distillation showed better in vitro antioxidant activity than whole oregano essential oil. In the molecular distillation process, distillation temperature and pressure are two important factors. Chen et al. [182] separated cuminaldehyde and p-mentha-1,4-dien-7-al from cumin (Cuminum cyminum L.) oil and found that the compounds had the best yield and purity at a distillation temperature of 30 °C and a distillation pressure of 19 Pa. Liang et al. [183] studied the molecular distillation conditions of allicin, and the results showed that at a distillation temperature of 50 °C, absolute pressure of 200 Pa, feed flow rate of 15 mL/min, and distillation frequency of 3 times, allicin had higher purity and content.

3.5.3. Supercritical Fluid Chromatography

The principle of supercritical fluid chromatography (SFC) is similar to that of liquid chromatography, which involves normal phase chromatography using a supercritical fluid as the mobile phase and a solid adsorbent (e.g., silica gel) or organic polymers bonded to the carrier (or capillary wall) as the stationary phase, and it is suitable for separating and analyzing stable substances that are difficult to volatilize and have poor thermal stability [184]. In recent years, SFC is no longer limited to the analysis of non-polar or moderately polar components. It exhibits good separation efficiency for polar natural products such as alkaloids, anthraquinones, saponins, and phenolic acids [185,186,187,188]. Supercritical fluid chromatography is a highly efficient separation and preparation method, that has the advantages of being green, having a wide choice of mobile phases that are readily available and inexpensive, not destroying the activity of natural products, and enabling quick separations [189]. Usually, CO₂ is used as a supercritical fluid mobile phase in SFC. However, non-polar CO₂ can effectively extract only non-polar lipophilic substances with low molecular weight and has limited solubilization ability for polar compounds. It is usually necessary to add appropriate non-polar or polar solvents as co-reagents, also known as modifiers, to the mobile phase to change the polarity, improve the solvation effect, and enhance the elution ability [190,191]. Barbini et al. [192] used supercritical carbon dioxide for the multistage fractionation of pine bark, in which varying the temperature, pressure, and polarity of the mobile phase allowed for the separation of different substances.

3.5.4. Preparative Gas Chromatography

Gas chromatography is a method of chromatographic analysis in which a gas is used as the mobile phase, called the carrier gas. According to the aggregation state of the stationary phase, gas chromatography is divided into gas-solid chromatography (GSC) and gas-liquid chromatography (GLC). GSC belongs to adsorption chromatography and GLC belongs to partition chromatography. At present, the flame ionization detector (FID) is one of the most used detectors for gas chromatography, which has the characteristics of high sensitivity, wide linear range, and wide range of applications. Gas chromatography has the advantages of high column efficiency, high selectivity, high sensitivity, small number of samples used, and fast analysis speed. The disadvantage is that it is limited by the vapor pressure of the samples; therefore, in the analysis of natural products, gas chromatography is suitable for the separation of volatile substances, such as essential oils [193]. Li et al. [194] characterized Ruta graveolens L. essential oil, isolating 17 compounds by gas chromatography. Sciarrone et al. [195] extracted essential oil from Clausena lansium Skeels leaves and isolated terpenoids using multidimensional preparative gas chromatography. Aimila et al. [196] isolated essential oil from Mentha asiatica Boriss., screened isolated essential oil fractions for bacteriostatic activity, separated the fractions using preparative gas chromatography, and identified the fractions with strong bacteriostatic ability. The results showed that 4-hydroxypiperone and thymol had strong bacteriostatic activity. At present, gas chromatography is mostly used in conjunction with other methods to realize the separation and analysis of natural products more quickly and accurately. Gas chromatography-mass spectrometry (GC-MS) plays an important role in the identification of unknown components of natural products [197]. Additionally, the combination of gas chromatography with olfactometry is an important method for assessing wine and fruit flavors [198,199].

In addition, the combination of gas chromatography-mass spectrometry and the solid-phase microextraction (SPME) technique enables the qualitative and quantitative analysis of volatile components in plant extracts. The solid-phase microextraction technique is based on the use of fused silica fibers coated with a stationary phase to adsorb and enrich the substances to be measured in the sample, and has the advantages of convenient operation, short processing time, and efficient determination [200]. Yang et al. [201] analyzed the volatile metabolites in different parts of Medicago sativa L. by using SPME-GC-MS, and they detected 87 volatile metabolites in glandular trichomes, 59 volatile metabolites in stems, and 99 volatile metabolites in leaves, indicating that SPME-GC-MS is a highly efficient and rapid method for extracting, detecting, and analyzing volatile compounds in plants.

3.5.5. Preparative Liquid Chromatography

Preparative liquid chromatography is a method for separating crude extracts of natural products based on classical liquid chromatography, using a high-pressure infusion system, a high-efficiency stationary phase, and a high-sensitivity detector, capable of purifying or separating crude extracts in large quantities [202]. Currently, the commonly used model is reversed-phase prep-HPLC [203]. Preparative liquid chromatography can be used in combination with other chromatographic methods to further separate mixtures and obtain pure compounds. The basic procedure involves the separation of crude extracts in the first dimension, followed by preparative chromatography to obtain the pure compounds [204,205]. Wen et al. [206] extracted flavonoids from leaves of Crataegus pinnatifida, separated them by HSCCC, and further purified the resulting mixture through preparative liquid chromatography to obtain three pure compounds. Qiu et al. [207] developed a two-dimensional preparative liquid chromatography system, first using MPLC to separate R. hotaoense extract, followed by preparative chromatographic separation, capable of efficiently separating the compounds in natural products compared to one-dimensional separations. Zou et al. [208] isolated gallic acid from Terminalia bellirica (Gaertn.) Roxb using macroporous adsorbent resina and preparative high-performance liquid chromatography. First, gallic acid was prepared by chromatography using AB-8 macroporous adsorbent resina, and then the prepared gallic acid was purified by preparative high-performance liquid chromatography. Chromatographic coupling allows for obtaining gallic acid with a purity of 99.1% in a short time. In addition, the combination of high-performance liquid chromatography—diode-array detector (HPLC-DAD) was effective in separating and characterizing the composition of natural products [209].

The above discussed the separation and application of chromatography in natural products. And the separation mechanisms of chromatography and the components suitable for separation are summarized in

Table 2. Summary of separation methods.

Method	Mechanism	Application
polyamide column chromatography	adsorption	flavonoids [108,109,110]
macroporous resins	adsorption	flavonoids, saponins, and alkaloids [112,113,114,115,116,117]
silica gel	adsorption	acidic and neutral substances [122,123,124]
aluminum oxide	adsorption	depend on pH [130,131,132]
distribution chromatography	distribution	saponins, alkaloids, acidic compounds, and polysaccharides [139,140,141,142,143,144,145]
ion exchange chromatography	ion exchange	plant macromolecular proteins, and polysaccharides [146,147,148,149]
molecular exclusion chromatography	sizes of molecules	plant polysaccharides [162]
membrane filtration	sizes of molecules	tannins, starches, resins, and proteins [165,166]
molecular distillation	mean free path	volatile compounds [176,177,178,179,180,181]
supercritical fluid chromatography	adsorption	alkaloids, saponins and phenolic acids [185,186,187,188]
preparative gas chromatography	adsorption	volatile compounds [193,194,195]
preparative liquid chromatography	adsorption	flavonoids, polysaccharides and phenolic acids [206,207,208]

, facilitating the selection of different chromatographic methods for separating various compounds such as polyphenols, flavonoids, saponins, polysaccharides, and so on.

4. Structural Identification

Compared with chemically synthesized compounds, the structures of natural compounds obtained after extraction, isolation, and purification are unclear. To further advance the research, it is necessary to conduct structural identification of natural compounds. In the first half of the 20th century, structural studies of natural products relied on classical chemical methods, such as chemical degradation and derivative synthesis. These methods required large quantities of samples, and the elucidation of structures often took several decades. But after 1960, UV-visible absorption spectroscopy (UV-vis), infrared spectroscopy (IR), nuclear magnetic resonance (NMR) spectroscopy, and mass spectrometry (MS) were developed. The combination of these four spectroscopic methods to analyze the structure of natural products has become more and more mature, and the four spectroscopies have become the conventional means of structural characterization nowadays (Figure 3). Among them, UV and IR belong to spectra, NMR belongs to wave spectra, and MS is the mass spectra of substances. The combined use of these four spectra can rapidly and accurately identify the structure of unknown natural products [210,211]. The combination of these four spectra can rapidly and accurately identify the structure of unknown natural products. Here, we briefly introduce the basic principles of the four spectra and their combined use to characterize natural products.

4.1. Ultraviolet-Visible (UV-Vis) Spectroscopy

The UV-visible region refers to the absorption of electromagnetic radiation in the wavelength range of 200~800 nm, with the wavelength in the 200~380 nm range of electromagnetic radiation called ultraviolet, and the wavelength in the range of 400~750 nm called visible light [212]. The UV-visible absorption spectrum is due to molecular absorption of ultraviolet or visible light after the valence electrons undergo a leap, produced by the molecular absorption spectrum, usually only 2~3 absorption peaks and mainly used to detect the unsaturation of molecules to determine the conjugate system [213,214]. UV spectroscopy can be used for qualitative analysis in the structural identification of compounds, which requires a comprehensive consideration of the number, position, intensity, and shape of absorption bands. Among these, the position and intensity of absorption bands are the main basis for characterization. According to the principle of UV spectroscopy and the empirical calculation method of the wavelength of absorption bands, the general laws of the relationship between UV absorption and the structure of organic compounds are summarized as follows, as listed in

Table 3. The relationship between UV absorption and structure.

Position of Absorption Bands	Intensity of Absorption	Structure
220~800 nm	0	Aliphatic hydrocarbon Alicyclic hydrocarbon
210~250 nm	≥104	Conjugated diolefine
260~300 nm	≥104	3 to 5 conjugated double bonds
250~300 nm	10~100	Hydroxyl group
250~300 nm	103	Benzene ring

.

In the ultraviolet spectrum, the unsaturated group that can produce ultraviolet or visible absorption is called a chromophore, and the group that has no ultraviolet absorption itself but can enhance the absorption peak of the chromophore is called an auxochrome [215]. The UV spectrum reflects the characteristics of the chromophore and auxochrome in the molecule, so compounds with the same chromophore and auxochrome have roughly the same UV spectrum. Based on this property, the structures of natural products and their derivatives can be determined from the spectra [216]. The typical UV spectrum of anthocyanin has two peaks, the first at 260~280 nm and the other at 490~550 nm. When the glycosyl of anthocyanin is acylated, a peak appears at 310~340 nm [217]. Flavonoids show two strong absorption peaks in the UV spectrum, at 300~380 nm and 240~280 nm, respectively. The UV spectra of flavonoids change depending on the type and number of substituent groups and their substitution positions in the molecular structures [218,219].

4.2. Infrared Spectroscopy (IR)

Organic molecules, chemical bonds, or functional groups of atoms are a constant state of vibration, with vibration frequencies comparable to the vibration frequency of infrared light. When organic molecules are irradiated with infrared light, the chemical bonds or functional groups within the molecules can undergo vibrational absorption. Different chemical bonds or functional groups have different absorption frequencies, so they appear at different positions on the infrared spectrum. This allows for obtaining information about the chemical bonds or functional groups present in the compound [220]. The near-infrared region, 780~2500 nm, is the region of X-H (X is C, N, O, S) and other telescopic vibrations, and information on hydrogen-containing groups can be obtained by scanning the near-infrared spectra [221]. Infrared spectroscopy can be used to infer functional groups in molecules, and Fourier Transform Infrared Spectroscopy (FTIR) is able to characterize the functional groups of compounds online [222]. FTIR is sensitive to the identification of aromatic rings, hydroxyl groups, amino groups, alkyne bonds, alkene bonds, and various types of carbonyl groups [223,224,225]. In the structural identification of natural products, infrared spectroscopy is usually combined with mass spectrometry and ultraviolet spectroscopy for structural analysis. Firstly, the molecular formula is calculated from the mass spectrometry results, and then the chemical bonds and functional groups are analyzed according to the ultraviolet and infrared absorption spectra [226,227].

4.3. Nuclear Magnetic Resonance Spectroscopy (NMR)

Nuclear magnetic resonance (NMR) is a physical phenomenon in which certain nuclei of a molecule absorb radio waves with frequencies between 4 and 1000 MHz in an applied magnetic field, causing a change in the direction of the spin of the nucleus [228]. NMR is an analytical method for the qualitative, quantitative, and structural identification of compounds based on the NMR phenomenon of atomic nuclei in a variety of chemical environments, and is capable of providing a complete characterization of compounds [229,230,231]. The skeletons of organic compounds are made up of carbon and hydrogen, making one-dimensional ¹H and ¹³C NMR spectroscopy commonly used to characterize the structure of natural products [232,233,234]. Ning et al. [235] established a method for the identification of flavonoid chemical constituents of homoisoflavonoids using ¹H-NMR, which can rapidly and accurately identify common flavonoids. For the structural identification of crude extracts, the NMR spectra of the crude extracts were compared with the NMR spectra of compounds already identified in the database to determine the structures of the compounds [236]. For unknown compounds, the structure of natural products can be analyzed by one- and two-dimensional NMR spectroscopy combined with mass spectrometry [237,238,239].

4.4. Mass Spectrometry (MS)

Mass spectrometry is one of the important techniques in the structural analysis of natural products. The basic principle involves ionizing the components in the crude extract within an ion source, producing charged ions with different mass-to-charge ratios. These ions are then accelerated through an electric field to form an ion beam, which enters a mass analyzer. Inside the mass analyzer, an electric field and a magnetic field are utilized to induce opposite velocity dispersion, allowing the ions to be focused separately to obtain a mass spectrum. This process helps us determine the mass of the ions [240,241]. The role of mass spectrometry is to determine the molecular weight, molecular formula, and molecular fragmentation structure, with the advantages of high sensitivity, small amounts of samples required, fast analysis, separation, and identification, to be carried out at the same time [242]. According to the physical and chemical properties of the analyte and the different internal energy transfer modes in the ionization process, different ionization methods can be used to ionize sample meteorological molecules into charged ions. Common ion sources include electron bombardment (EI) [243], chemical ionization (CI) [244], fast atom bombardment (FAB) [245], Matrix Assisted Laser Desorption Ion Sources (MALDI) [246], and Electrospray Ionization (ESI) [247].

The mass analyzer is the core of the mass spectrometer, where the ion beam formed after ionization of the sample is accelerated into the mass analyzer, which separates different types of ions according to the m/z of the ions. Commonly used mass analyzers are the quadruple-rod mass analyzer (Q), the time-of-flight mass analyzer (TOF), and the ion trap mass analyzer [248]. The orbitrap is a mass analyzer with ultra-high resolution for fast and reliable identification, quantification, and characterization of more compounds [249,250]. Tandem mass spectrometry, with multiple mass analyzers, is widely used in the structural identification of natural products, such as Q-TOF, TOF-TOF, which play an important role in the identification of mixtures [251,252]. Ion mobility spectrometry (IMS), on the other hand, enables the identification of isomers of natural products [253]. Currently, the combined use of gas chromatography (GC) and liquid chromatography (LC) with mass spectrometry (MS) allows for the rapid and accurate separation and identification of natural products. Man et al. [254] comprehensively studied the phenolic compounds in pomegranate peels using UPLC-QQQ-MS, which is a rapid and accurate method, capable of qualitatively and quantitatively analyzing phenolic compounds in pomegranate peels of different varieties from different origins. Abdallah et al. [255] analyzed the ethyl acetate fraction extracts of S. birrea stem extract, using a hybrid tandem mass spectrometry LC-MS/LC-HRMS technique to identify a new compound, vidarabine, demonstrating its bacteriostatic effects. For volatile natural compounds, GC-MS is an efficient method of separation and analysis. Vallarino et al. [256] utilized GC-TOF-MS to measure plant hormones, demonstrating that this method is a sensitive and reliable analytical approach. It provides a material foundation for further understanding the role of plant hormones in the growth and development of plants. A natural product mass spectrometry database is now available for more efficient structural characterization of natural products [257].

5. Summary

Natural products are rich in variety and are valuable natural resources with incalculable medicinal potential. Therefore, research on the extraction, separation, and identification techniques of natural products plays a pioneering role in drug development.

There are various kinds of extraction methods, ranging from traditional solvent extraction to modern methods utilizing ultrasound, microwave, and other technologies. The overarching goal is to extract natural products from raw materials as much as possible. On this basis, extraction considerations need to include the cost of extraction, the efficiency of extraction, and whether the use of extraction solvents is environmentally friendly. Taken together, modern extraction techniques, such as ultrasound-assisted extraction and microwave-assisted extraction, are more frequently used in natural product extraction due to their advantages of simple equipment, high extraction efficiency, and short time-consumption. The crude extract obtained after extraction is a complex mixture of components. To further purify single components, chromatography is used. Column chromatography is a widely used method capable of separating most compounds. It should be noted that the nature of the target compounds should be considered when selecting the appropriate packing material for separation. The structural identification of natural products is a very important part of the four major spectra jointly used to identify components of extracts or unknown compounds. This is a very mature technology, assisted by a relatively perfect database, further advancing the development and utilization of natural products.