Recent trends in extraction, purification, and antioxidant activity evaluation of plant leaf‐extract polysaccharides

This review elaborates on current advances in the extraction, purification, and antioxidant activity of plant leaf‐extract polysaccharides. Polysaccharides are widely used as important ingredients in the food, pharmaceutical, and cosmetic industries. Researchers have been investigating useful sources of natural polysaccharides and developing green and feasible extraction procedures for polysaccharides. This review examines different methods for extracting polysaccharides from leaves, and discusses their advantages and limitations. Purification techniques for plant leaf‐based polysaccharides were also highlighted, together with their antioxidant effects. Among different extraction methods, pressurized‐liquid extraction and enzyme‐assisted extraction are considered to be better for large‐scale extraction of polysaccharides from plant leaves. This review could contribute to the design of leaf waste processes at a commercial level for the sustainable recovery of polysaccharides. © 2022 The Authors. Biofuels, Bioproducts and Biorefining published by Society of Industrial Chemistry and John Wiley & Sons Ltd.

Introduction P olysaccharides have many biological functions and many applications. 1 Natural polysaccharides originate from plants, animals, and microorganisms. 2 Polysaccharides extracted from plant leaves exhibit significant differences in structure and processbility. 3 Generally, in polysaccharides, monomers (usually >10) are covalently linked either in the form of linear or branched glucosidic linkage. 4,5 The extraction of polysaccharides from plant leaves with a high yield is important for their economic application. 6 Several extraction techniques have been established for the preparation of polysaccharides but the selection of the one with best commercial feasibility is important to manage leaf waste. 7 The chemical composition and structure of polysaccharides act as key aspects to characterize, and the understanding of these is important for tailor-made applications. 8 Analytical techniques and chemical methods have been developed for the extraction and purification of polysaccharides, and an understanding of their advantages and limitations supports their use for commercial purposes.
The polysaccharides have now been broadly exploited in the medicinal, food, textile, cosmetics, leather-tanning, electronics, and mechanical industries due to their wide availability, low cost, biodegradability, non-toxic nature, renewability, environment-friendly behavior, and multiple biological functions with negligible side effects. 9,10 Previous studies have demonstrated that polysaccharides can be used as active agents in medicines due to their biological activity -for example their antioxidant, anti-tumor, anticoagulant, anti-virus, anti-radiation, anti-cancer, and immunoregulatory activity. [11][12][13][14][15][16][17] The broad range of medicinal functions of polysaccharides and their antioxidant activity have attracted wide attention due to their feasible transfer of value-added properties to the entire system with negligible disturbance. 18 In other words, the vigorous role of polysaccharides as modifiers of biological systems and to inhibit the potential of oxidative stress by scavenging the free radicals cannot be ignored. 18 The potential application and antioxidant activity of polysaccharides also depend on the structure of the molecules and functional groups. 19 Plant biomass is loaded with useful phytochemicals and biomolecules. Plant biomass commonly consists of plant leaves, branches, bark, grass, flowers, fruits, and other woody materials. The leaves of plants account for the largest portion of plant biomass. In autumn, a large amount of plant leaves drop onto the ground surface, which increases the volume of plant biomass. Plant biomass has been widely used for the removal of heavy metals, phenols, dyes, and other organic pollutants from wastewater through adsorption processes for water purification. [20][21][22] Consumption of plant biomass for adsorption purposes is not a sustainable solution for biomass management because the adsorption process generates pollutant-loaded biomass, which is more difficult to manage than the initial one. Previous studies suggest that the waste generated by plants can be managed by utilizing aerobic and anaerobic digestion techniques, but these technologies require significant investment for equipment, create several environmental issues, and might not be economically feasible on a large scale. 23 The production of biofuel, bioenergy, bioplastic, and biogas can be other possible routes for the conversion of plant biomass into value-added products but a large financial investment is required to commercialize these possibilities. There is thus a need to provide alternative cost-effective, easy, and feasible solutions to manage plant biomass on a commercial scale. Given these challenges, this review was compiled to discuss common methods for the extraction and purification of polysaccharides and resultant antioxidant activity, to manage plant leaves on an industrial scale. Several reviews have been published on the extraction of polysaccharides from plants, seaweeds, microbes, and other natural sources, with the biological potential for polysaccharides highlighted. However, these reviews did not discuss the processing of polysaccharides on a large scale. The present review is therefore designed to compare different extraction methods for polysaccharides with largescale schematic layouts and to suggest the most promising ones. The methods of purifying these polysaccharides, their antioxidant activity, and their large-scale applications are also highlighted in this review.

Extraction methods for polysaccharides
Researchers are focusing on developing a bundle of extraction procedures to prepare polysaccharides without disturbing their structural composition. 24 Polysaccharides from natural sources (e.g. plants, animals, and microbes) have been extracted successfully using several extraction techniques. Although all these extraction techniques, which are described below, can be used for the extraction of polysaccharides from different sources, the data presented in this review are to provide insights into the utilization of leaf-extract polysaccharides. In this section, different methods of polysaccharide extraction are discussed based on mechanisms and operating conditions. These methods of extracting polysaccharides are depicted in Figure 1.
a specified duration and polysaccharide-containing extracts are obtained by a jacketed vessel where the extract is cooled and separated from plant material ( Figure 2). Extraction time, temperature, and material-to-water ratio are key factors that affect the extraction efficiency of polysaccharides. 26 For the optimization of these factors, mathematical designs such as continuous single-factor and orthogonal designs and statistical designs like response surface with Box-Behnken designs (BBD) or central composite designs (CCD) can be used.
In HWE, a high temperature is required to extract plant polysaccharides. Generally, at high temperature, Maillard reaction and caramelization may occur, which degrades polysaccharides and leads to poor extraction efficiency. 6 At high temperatures, some of the reducing sugars may react with amino acids present in plant materials to produce complex compounds, which may interact with polysaccharides and cause them to degrade and thus reduce the extraction yield. Generally, in HWE, a material-to-liquid ratio of about 1:20 is used to concentrate the extract, and  thermal degradation of polysaccharides can occur. 27 Other disadvantages of this method are a long extraction time and a large volume of ethanol (4-10 times) required to precipitate crude polysaccharides. Previous studies suggested that HWE can only extract extracellular polysaccharides and cannot destroy the cell wall and plasma membrane of plant materials. 25 Polysaccharides from Hizophora mucronate leaves showed a 7.67% extraction yield when being processed using an HWE system at a solid-to-liquid ratio of 1/50 (w/v) and a temperature of 90 °C for 3 h. 28 Hot-water extraction was applied to extract polysaccharides from Aconitum carmichaelii leaves, which showed a 4.2% extraction yield when the extraction was carried out at a solid-to-liquid ratio of 1:20 (w/v) and a temperature of above 90 °C (under reflux) with a 1 h extraction time twice. 29 Polysaccharides extracted from Tymus quinquecostatus leaves showed an 86% extraction yield when being processed at a solid-to-liquid ratio of 1:20 (w/v) and a temperature of 100 °C for 3 h using HWE. 30 Many other studies have involved polysaccharide extraction using HWE from plant leaves as listed in Table 1.

Microwave-assisted extraction
The microwave-assisted extraction (MAE) method can be used for polysaccharide extraction due to its strong penetration, high selectivity, and high efficiency. 74 In an electromagnetic field, microwaves with non-ionizing radiation within the energy range 300 MHz to 300 GHz penetrate plant material and generate volumetrically distributed heat through molecular friction. 96 Microwaves break the cell wall and inactivate enzymes in the cell membrane to extract polysaccharides. In MAE, microwave and electromagnetic radiation passed through the extraction chamber, which contains water and plant material, leading to an increased temperature, the destruction of the cell wall and cell membrane, and the increased polarization of polysaccharide molecules (Fig. 3). After extraction, the resultant slurry passed through a jacketed vessel where the extract was separated from plant material and cooled to room temperature. Remarkably, MAE possesses some advantages over other conventional methods, such as being financially feasible, time saving, and eco-friendly, and possessing high extraction efficiency with the minimal use of solvents, and low energy consumption. 97 The glycosidic linkages in polysaccharides can be disturbed by intense microwave treatment for a long time. High microwave power and intense electromagnetic radiation also depolymerize polysaccharides chains. 98 Hence, the microwave power and extraction time should be controlled stringently to prevent polysaccharide degradation. A high temperature in MAE also reduces the extraction yield because, at a high temperature, a browning reaction (caramelization) takes place. Polysaccharides from Eucommia ulmoides leaves were extracted using MAE and showed a 12.31% extraction yield when being processed at a solid-toliquid ratio of 1:29 (w/v) and a temperature of 74 °C with a reaction time of 15 min. 99 Pectin from Premna microphylla leaves extracted using MAE showed an 18.25% extraction yield when the extraction was carried out at a solid-to-liquid ratio of 1:50 (w/v), a temperature of 90 °C, and a pH of 2 for 2 h. 69 Different polysaccharides extracted from leaves using MAE are listed in Table 1.

Ultrasound-assisted extraction
In UAE, ultrasonic wavse ruptures plant cells, thus leading to polysaccharide extraction. 100 Ultrasound waves generated from the probe travel to the medium (water) and produce cavitation bubbles, which strike plant cells in the extraction chamber and destroy plant cells at a certain temperature, leading to polysaccharide extraction (Fig. 4). After extraction, the whole extract, including plant material, pass through a jacketed vessel where the extract is separated from plant material and cool to room temperature. Ultrasound waves with a power level of 10-100 kHz are normally used to generate cavitation bubbles, and the cavitation process makes polysaccharides diffuse from the cell wall, which can increase extraction efficiency significantly. 101 In UAE, a higher temperature increases the kinetic energy of gas-phase cavitation bubbles, which weakens the cell-wall polysaccharides, thus leading to high extraction efficiency. A great advantage of ultrasonic extraction is that it is fast. The extraction efficiency of polysaccharides by using ultrasonic extraction is affected by extraction time, ultrasonic power, and temperature. When high ultrasound power is applied for long periods, a decrease in extraction efficiency was observed due to the destruction of glycosidic linkages and depolymerization. 102 To overcome these drawbacks, it is necessary to optimize parameters such as extraction time, ultrasound power, and temperature. Ultrasound-assisted extraction was applied to polysaccharides from Sorghum bicolor leaves and showed a 9.23% extraction yield at a solidto-liquid ratio of 1:20 (w/v), an ultrasound frequency of 60 kHz, an ultrasound power of 240 W, and a temperature of 70 °C, with an extraction time of 70 min. 103 Some other leafextract polysaccharides using UAE are listed in Table 1.  104 For polysaccharide extraction, water is applied to the extraction chamber with nitrogen purged by a purge valve, and the extraction is carried out at high pressure and high temperature (Fig. 5). After extraction, the extract was separated and cooled with a jacketed vessel attached to the extraction unit. High pressure and increased critical temperature improve the solubility and mass transfer rate, which results in a high extraction yield. Extraction time and solvent volume can be reduced with the help of PLE. Polysaccharides from Sagittaria sagittifolia L. leaves were extracted using PLE and showed a significant yield (Table 1) in the optimal conditions (a pH of 7, a temperature of 170 °C, a duration of 45 min, and a materialto-liquid ratio of 1:30). 94 Pressurized-liquid extraction was applied to extract β-glucans from barley bran and showed a 16.39% extraction yield in the optimal conditions (pressure of 10 MPa, a temperature of 70 °C, and an extraction time of 9 min). 105

Supercritical-fluid extraction
Supercritical-fluid extraction (SFE) is an efficient extraction technique that gives an extraordinary yield and high purity and is commonly employed for the fractionation of low-molecular-weight polysaccharides. In SFE, gasphase extraction, solvent (argon or carbon dioxide) is used, which is first cooled and then heated in a preheated column to maintain its high pressure and temperature, and then allowed to pass from the extraction chamber to extract polysaccharides from plant material (Fig. 6). Then, the extract is passed through the jacketed vessel where the extract is separated from plant material and cooled to room temperature. Supercritical fluid (SCF) is more applicable when carbon dioxide is used instead of argon, and polysaccharides are fractionated based on their solubility in carbon dioxide, and sometimes the solubility, and thus the yield, can be enhanced by introducing some organic solvents combined with water as a modifier. 106 On a commercial scale, SFE is widely used for the selective fractionation of lactulose from different aldoses. 107 Polysaccharides from Phyllostachys heterocycle leaves were extracted using SCF (supercritical CO 2 ) with ethanol as a modifier and there was a yield of 2.47% when the extraction was operated at a temperature of 50 °C and a pressure of 40 MPa for 2 h with 20 mL of ethanol. 87 Supercritical-fluid extraction was applied for polysaccharide extraction from Artemisia sphaerocephala Krasch seeds and there was an 18.59% extraction yield when the extraction was performed under selected conditions such as a temperature of 45 °C, a pressure of 10 MPa, a CO 2 flow rate of 20 L/h, and an extraction time of 2 h. 108 Polysaccharides from Ginkgo leaves were extracted using SFE-CO 2 and showed a 10.13% extraction yield when being processed with the optimized conditions including a duration of 99 min, a temperature of 63 °C, and a pressure of 42 MPa. 109 For polysaccharide extraction, SFE is more advantageous than other extraction methods because it can provide maximum extraction efficiency with negligible degradation. Nonetheless, this extraction method is not very selective and the capital investment is high.

Enzyme-assisted extraction
Enzyme-assisted extraction (EAE) is another extraction method that is conventional, selective, specific, and efficient. Enzymes such as cellulase, amylase, hemicellulose, pectinase, and papain have the potential to break down the cell wall of plant material and release polysaccharides without affecting its structure by hydrolysis. 110 In EAE, selected enzymes are applied to the extraction chamber under a controlled temperature to destroy the cell wall of plant material and hydrolyze the polysaccharides, which further passed through the centrifugation and filtration process in a jacketed vessel to make crude polysaccharides (Fig. 7). Enzyme-assisted extraction has several advantages over other traditional methods, such as high extraction yield, high reaction compatibility, short extraction time, low cost, greenness, and mild operation conditions. 111 This extraction method has also attracted the attention of researchers because it is time and energy efficient and can be operated with a lower volume of extraction solvent. 112 Sometimes, other constituents of plants, like proteins, lipids, phenolics, flavonoids, pigments, nucleic acids, and other small organic and inorganic compounds can interact with enzymes, which reduces the extraction   89 Some other leaf-extracted polysaccharides using EAE are given in Table 1.

Combined extraction methods
Combined extraction techniques can also be used for the extraction of polysaccharides to overcome the limitations

Subcritical-water extraction
Subcritical-water extraction (SWE) is an extraction technique that is becoming popular due to its green nature. Another name for SWE is superheated-water extraction. Briefly, in SWE, polysaccharides can be extracted using a small quantity of solvent, especially water, under a high pressure ranging from 0.35 to 2.1 MPa and a high temperature ranging from 100 to 374 °C for a short time. 114 Temperature, pressure, solid-to-liquid ratio, solvent flow rate, pH, and extraction time are important parameters that affect the extraction yield using SWE. 115 Using SWE, polysaccharides from Sagittaria sagittifolia leaves were extracted, leading to a high yield of 24.57% at a solid-to-liquid ratio of 1:30 (w/v), a temperature of 170 °C, a pH of 7, and a pressure of 1 MPa, with an extraction time of 16 min. 94 Subcritical-water extraction was successfully utilized for polysaccharide extraction from the stems of Dendrobium Lindl and showed a maximum extraction yield of 21.88% at a temperature of 129.83 °C, a pressure of 1.12 MPa, and a solid-to-liquid ratio of 1:25 (w/v), with an extraction time of 16.71 min. 116 Thlaspi arvense leaves showed a 6.26% yield of selenium-containing polysaccharides when SWE was used at a temperature of 140 °C and a pressure of 8 MPa for 15 min. 117

Ultra-high-pressure extraction
Ultra-high-pressure extraction (UPE) under high pressure at a low temperature can be used for the extraction of polysaccharides as a novel and green technique. It is one of the eco-friendly extraction methods approved by the US Food and Drug Administration (FDA) and has been widely adopted in the food industry. 118 The UPE process can be operated at high pressure ranging from 100 to 1000 MPa with a low temperature of around 50 °C. 115 This technique requires less time and solvent volume since high pressure enhances the mass transfer by disrupting the cell wall, and thus the polysaccharide extraction yield is increased. Ultra-highpressure extraction showed a 3.07% polysaccharide yield from the root of Morinda officinalis at a pressure of 420 MPa and a solid-to-liquid ratio of 1:12 (w/v) for 6.5 min. 119 In the optimal conditions using a UPE system, polysaccharides from Litdshi chinesis Sonn. showed a 12.01% extraction yield when being processed at a solid-to-liquid ratio of 1:15 (w/v) and a pressure of 460 MPa for 17 min. 120 The polysaccharide content in yellow tea leaf extracts increased by 1.31, 128.28, and 19.86% when UPE was operated at 200, 400, and 600 MPa respectively at 25 °C for 5 min. 121

Ionic-liquid extraction
Paul Walden reported ionic liquids for the first time in 1914, and he is known as the father of ionic liquids (ILs). 122,123 It was a time when scientists were not very familiar with the importance of ILs, but over time, the worth of these liquids expanded exponentially. 124 In the present century, ILs are well known due to their versatile properties and wide range of applications in the field of chemical sciences. 122 Ionic liquids are liquids formed by cations and anions linked with different types of chemical bonds and possesses a bundle of chemical and physical properties including low vapor pressure, good thermal stability, and high tunability, and can be used as solvents, catalysts, extraction liquids, etc., in various fields. 125 They are composed of large organic cations and small organic or inorganic anions and have a melting temperature below 100 °C. The low melting temperature of ILs is suggested by their low ionic symmetry composition and low charge density. 126 Singh and Savoy 125 classified ILs into several categories including task-specified ILs, chiral ILs, switchable polarity solvent ILs, bio-ILs, poly-ILs, energetic ILs, neutral ILs, protic ILs, metallic ILs, basic ILs, and supported ILs. Among other ILs, task-specified ILs including alkyl phosphate-type ILs and imidazolium-based hydrophilic ILs have been most used in the IL extraction (ILE) of polysaccharides. 127 Ionic-liquid extraction has been used frequently for cellulose extraction from biomass using those hydrophilic ILs that have good hydrogen-bond-accepter capability and moderate hydrogen-bond-donor behavior because the solubility of cellulose is increased in those ILs at room temperature. 128 The extraction yields of cellulose from corn stover using three ILs, tetra-butylphosphonium 2-  123 reported that some of the ILs were considered to be green, non-flammable, non-volatile, and stable in air and water, but recently, many of them have been found to be flammable, volatile, unstable, and even toxic. On a large scale, ILE cannot be used because some ILs are hazardous and non-biodegradable, and the large-scale synthesis of ILs is expensive and difficult. 123

Deep eutectic solvents
Deep eutectic solvents (DESs) are alternatives to ILs and are known to be less hazardous, less expensive, more stable, and biodegradable in comparison with ILs. 134 Deep eutectic solvents possess some advantages like the ease of synthesis, non-flammability, non-volatility, low cost for large-scale production, high biocompatibility, and the wide availability of their primary ingredients. 135 They represent a new class of IL formed from a eutectic mixture of Lewis or Bronsted acids and bases, with several anionic and/or cationic species, and they differ from ILs, which are composed of one type of discrete anion and cation. 136 According to the first described concept, DESs are liquids formed by mixing a variety of quaternary ammonium salts and carboxylic acid. 137 They are synthesized by combining hydrogen-bond donors (HBDs) and hydrogen-bond acceptors (HBAs) to form eutectic mixtures. 138 Based on the complexing agent, DESs are classified into four categories including Type I (composed of a quaternary ammonium salt (QAS) and a metal chloride), Type II (composed of a QAS and a metal chloride hydrate), Type III (composed of QAS and a hydrogen bond donor), and Type IV (composed of a metal chloride and a hydrogen bond donor). 138 The most widely used hydrogen-bond acceptor in DESs is choline chloride (ChCl), and choline-derived DESs have been reported extensively for the dissolution of cellulose. 139 Hemicellulose and amorphous cellulose were extracted from rice straw and showed 16.71 and 9.60% extraction yields when being treated with ChCl/urea at a temperature of 130 °C for 4 h. 140 Polysaccharides from Fucus vesiculosus were extracted with a DES of ChCl and 1,4-butanediol at a molar ratio of 1:5, and showed an 11.63% extraction yield when being processed at a temperature of 168 °C for 35 min. 141 Lotus leaf polysaccharides showed a 5.38% extraction yield when being extracted with a DES of ChCl and ethylene glycol at a molar ratio of 1:3, a solid-to-liquid ratio of 1:31 (w/v), and a temperature of 92 °C for 126 min. 142 In DES extraction, two or more organic or inorganic liquids or combinations of both types of liquid can also be used to obtain specific properties for polysaccharide extraction. 143 Pulsed electric field-assisted extraction In pulsed electric field-assisted extraction (PEF), an electric field ranging from 0.1 to 80 kV is applied to plant cells. This permeabilizes the cell membrane to release constituent compounds such as carbohydrates, polyphenolic compounds, and flavonoids in the solvent system at ambient temperature. 115 Pulsed electric field-assisted extraction is a novel, nonthermal and efficient extraction method that is capable to extract high-purity polysaccharides within seconds with less energy. 144 Corn bran polysaccharides were extracted using water-assisted and enzyme-assisted PEF, which showed 6.4 and 15.36% extraction rates in the optimal conditions (a solid-to-liquid ratio of 1:42 (w/v), an electric field intensity of 25 kV/cm, and an electric field frequency of 1080 Hz). 145 Corn silk polysaccharides were extracted using PEF and a 7.31% extraction yield was achieved when the extraction was carried out at an electric intensity of 30 kV/cm and a solid-toliquid ratio of 1:50 (w/v) for 6 μs. 146 Negative pressure cavitation

Comparison of different extraction methods
Carbohydrates are produced in plants by photosynthetic CO 2 fixation and are a central source of energy in the global bioeconomy. There are three reasons for the importance of investigating polysaccharides. First, the emergence of the bioeconomy highlights the contribution of natural products, especially biobased products. Second, while polysaccharides have been utilized widely in material science, health care, food, and nutrition, it is important to evaluate the exceptional properties of polysaccharides to open possible routes to novel applications. One more reason is associated with environmental concerns, and regarding this, the adoption of polysaccharides can contribute to sustainability because of their ubiquitous presence and renewability. 149 The several potential benefits include an increase in biodiversity, food safety, sustainability, and fuel production, and a decrease in CO 2 emission and pollution, which could be achieved by proper utilization of polysaccharides. Polysaccharides are widely distributed due to their different structures and are classified based on origin, shape, structure, charge, monosaccharide unit, and chemical and functional properties. 6 Generally, polysaccharides obtained from plant leaves with long chains and complex structures with branches have attracted much attention because they have the potential to regulate a variety of biochemical functions like cell proliferation, immune response, and cell differentiation, inflammation, and adhesion. 150 Biochemical functions including cell proliferation, immune response, cell differentiation, inflammation, and adhesion are related to the biological activities of polysaccharides. Polysaccharides with long chains, complex structures, and clusters of branches have been reported to perform excellent biological activities in comparison with short-chain, linear, and simple structured polysaccharides. 18,151 The recovery of such polysaccharides without disturbing their composition and structure is much more important. In this sense, a bundle of extraction methods discussed above can be used for polysaccharide extraction from plant leaves, but this section differentiates the extraction methods according to their performance. Hotwater extraction is an easy and commonly used extraction method but it requires a high temperature and time, whereas these issues were not seen with UAE and MAE. Mulberry leaf polysaccharides were extracted using HWE and MAE, and HWE has an 18.64% extraction yield, whereas MAE provides a yield of 9.41% under the optimal conditions (Table 1).
Hot-water extraction is superior to MAE considering extraction yield. High-molecular-weight polysaccharides could be extracted using HWE and EAE, whereas UAE and MAE mostly tend to extract low-molecular-weight polysaccharides (Table 1). Hot-water extraction and EAE were utilized respectively to extract polysaccharides from Camellia sinensis. A 1.28% extraction yield was obtained using HWE while an extraction yield of 4.08% using EAE under the optimal conditions. Enzyme-assisted extraction performed better over HWE regarding extraction yield and molecular weight distribution (Table 1). Corn silk polysaccharides were extracted using HWE and PEF respectively and showed a 5.46% yield at 100 °C temperature with an extraction duration of 60 min using HWE, while a 7.31% yield was obtained using PEF under the optimal conditions such as an electric field intensity of 30 kV/ cm and a reaction time of 6 μs. 146 Pulsed electric fieldassisted extraction is a fast, nonthermal and efficient extraction technique but it has not been widely utilized for polysaccharide extraction. It is currently not feasible on a large scale due to the complexity of setting it up.
Polysaccharides from Eriobotrya japonica leaves have been extracted using HWE, MAE, PLE, UAE, U-EAE, and U-MAE, respectively (Table 1). Extraction yields of 2.95, 3.11, 5.05, 4.53, 4.73, and 4.93% were obtained using HWE, MAE, PLE, UAE, U-EAE, and U-MAE, under the following operating conditions: solid-to-liquid ratio 1:20 (w/v), temperature 95 °C, and extraction time 2 h for HWE; solidto-liquid ratio 1:30, temperature 80 °C, time 6.5 min, and microwave power 500 W for MAE; solid-to-liquid ratio 1:10, temperature 55 °C, time 40 min, and pressure 1.8 MPa for PLE; solid-to-liquid ratio 1:40, time 20 min, and ultrasound power 450 W for UAE; cellulase dosage 50 mg, time 20, and ultrasound power 450 W for U-EAE; and time 6.5 min, ultrasound power 450 W, and microwave power 500 W for U-MAE. The results showed that PLE provided the highest extraction yield in the optimal conditions. Ginkgo biloba leaf polysaccharides were extracted using three extraction methods including UAE, EAE, and U-EAE and showed different yields of 8.36, 7.92, and 12.85% respectively at the optimal conditions of a solid-to-liquid ratio of 1/50 (w/v), a temperature of 50 °C, ultrasound treatment for 30 min, and enzyme hydrolysis with 0.8% cellulase for 40 min. 113 The extraction yield of polysaccharides has increased significantly with the combination of two methods, which suggests that combining extraction methods provides other possible options for the extraction of polysaccharides on a large scale. In comparison, HWE, MAE, and PLE extracted highmolecular-weight polysaccharides while UAE, U-EAE, and U-MAE extracted low-molecular-weight polysaccharides.

Purification of polysaccharides
After extraction followed by the ethanol precipitation process, purification and fractionation are important procedures to remove residues of other constituents such as proteins, lipids, phenolics, flavonoids, pigments, nucleic acids, and other small organic and inorganic compounds conjugated with plant polysaccharides. 150 Conventional methods used for the purification of crude polysaccharides include ethanol treatment, hydrogen peroxide treatment, activated carbon treatment, amylase hydrolysis, ultrafiltration through a membrane, dialysis against water (ultrapure, distilled), and Sevag reagent treatment (n-butanol and chloroform, 1:5). 152 Polysaccharides can be fractionated based on their size, charge, and chemical interaction, with the help of column chromatography, gel permeation chromatography, ionexchange chromatography, and affinity chromatography.

Column chromatography
Column chromatography is one of the simplest, conventional methods used for the fractionation of polysaccharides, and nowadays it has attracted wide attention due to its excellent performance in the fractionation of plant polysaccharides. Cellulose is a commonly used stationary phase in column chromatography. It is equilibrated with ethanol to avoid the nonspecific adsorptive forces of cellulose molecules to polysaccharides. 150 Fractions of polysaccharides can be eluted by using state-of-the-art eluents such as water, buffers, and some organic solvents. Polysaccharides, with short chains and low molecular weight, have weak interactions with ethanolequilibrated cellulose and are eluted first. Polysaccharides have moderate branches and possess medium molecular weight. They interact moderately with ethanol-equilibrated cellulose and are eluted in the second place. Polysaccharides have long branches and possess high molecular weight. They have strong interactions with ethanol-equilibrated cellulose and are eluted last. However, the lower the cellulose particle size, the greater is the surface area, and the higher is the number of theoretical plates. Hence there is higher fractionation efficiency for polysaccharides. 153 The low flow rate and time-consuming behavior of column chromatography make it less usable.

Gel permeation chromatography
Polysaccharides can be fractionated based on their size and shape by using gel permeation chromatography (GPC). In GPC, gels with pores of different sizes are used to separate polysaccharide molecules. 154 Polysaccharides with large molecules and high molecular weight do not enter the pores of gel and are eluted first. Polysaccharides with small molecules and low molecular weight obstruct the pores of the gel and are eluted last. Some commonly used gel-packing materials include Sepharose, Sephacryl, Superdex, Bio Gel, and Sephadex. The selection of gel is strongly dependent on the nature and source of polysaccharides that are to be fractionated. 155 For polysaccharide fractionation, GPC with a refractive index detector (RID) is most widely used. The peaks of different polysaccharide fractions are generated based on RID elution by using deionized/distilled water and sodium chloride solution with different concentrations along with buffer solutions. 156 In GPC-RID, some ionic solvents can be run before the introduction of samples to reduce the nonspecific adsorptive forces of the gel to polysaccharides, and hence increase the purity. 157 There is no doubt that GPC is a powerful analytical technique for polysaccharide fractionation but it possesses some limitations such as expensive instrumentation, low efficiency, a lack of automation, and difficulty in scaling up.

Ion-exchange chromatography
Polysaccharides can be fractionated with the help of ionexchange chromatography (IEC). In IEC, polysaccharides are separated based on charge and polarity index. 158 Ion-exchange chromatography is operated with either cation exchange resins or anion exchange resins but anion exchange resins are commonly preferred for polysaccharides fractionation. 159 Anion resins separate neutral and acidic fractions of polysaccharides. Acidic polysaccharides having a high uronic acid content can bind strongly to anion resin, and neutral polysaccharides do not have interaction with anion resin and hence are eluted first. Furthermore, neutral and acidic polysaccharides are fractionated by using gradient elution practices with a combination of different ionic eluents. 160 Diethylaminoethyl (DEAE) containing anion exchange resins such as DEAE-cellulose, DEAE-Sepharose, DEAE-Sepharose fast flow, DEAE-Sephadex, DEAE-Sephadex fast flow, and Q-Sepharose are most widely used for polysaccharide fractionation. 161,162 The selection of an anion exchanger depends on small-scale experimental trials and the concentration of uronic acid in polysaccharides.

Affinity chromatography
Selective liquid adsorption chromatography, commonly known as affinity chromatography (AC) is another analytical tool for polysaccharide fractionation. In AC, the polysaccharides are separated based on adsorptive forces to the stationary phase. 163 Immobilized ligands act as stationary phases in affinity chromatography. Many commercially available immobilized lectins can be used as ligands in AC. Practically, Concanavalin A, wheat germ agglutinin, and Sepharose have been used for glycoprotein fractionation. 164 Many standard polysaccharides such as mannan, chitin, alginate, and other polysaccharides from Azospirillum brasilense and P. aeruginosa interact well with these ligands for fractionation. 165,166 Affinity chromatography can be used as a potential separating tool on small and large scales but the selection of ligands for typical polysaccharides might be timeconsuming. This fractionation tool demands more research work in the future to explore further fundamental trends of polysaccharides fractionation.

Antioxidant values of polysaccharides
A large number of free radicals produced in different products during chemical degradation, which occurs under different conditions, are detrimental to the entire product and the human body. 90 These free radicals are harmful to the entire body and generate oxidative stress in biological systems. 166,167 Free radicals can oxidize biological macromolecules like lipids, proteins, and carbohydrates, causing serious biological disorders such as inflammation, aging, cancer, hepatoxicity, and diabetes. 168 Polysaccharides play a meaningful role as modifiers towards biological response and reduce or inhibit oxidative stress by scavenging free radicals. 18 Polysaccharide antioxidants can be used to prevent a wide range of oxidative disorders as well as preservatives in cosmetic and food products. 169 Previous studies indicate that polysaccharides extracted from plant leaves have good antioxidant activities by scavenging free radicals like 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, 2,2-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (ABTS), and superoxide, and have also shown excellent reducing power (Table 2).

1,1-Diphenyl-2-picrylhydrazyl radical antioxidant activity
The antioxidant assay based on DPPH is a direct, simple, and reliable method. DPPH is a very stable free radical because free electrons are delocalized over the whole molecule without dimerizing, and in the presence of hydrogen species, the DPPH radical is reduced into 1,1-diphenyl-2-picrylhydrazine by changing the color from violet to yellow. 170,171 Plant leaf-extract polysaccharides have good DPPH antioxidant activity at the optimal dose (see Table 2). At a 2.8 mg/mL concentration, the maximum DPPH antioxidant activity of 94% was obtained for Zagros oak leaf-extracted polysaccharides. 86 Besides, polysaccharides extracted from leaves of Leonurus cardiaca showed 92.8% DPPH antioxidant activity when it was applied with a concentration of 12.14 mg/ mL. 46 In the same way, polysaccharides separated from leaves of Mentha haplocalyx, Acanthopanax senticosus, and mulberry showed DPPH antioxidant activities of 91.88, 91.75, and 91% when they were used at concentrations of 2, 2, and 0.24 mg/ mL, respectively. 39,47,82 Hydroxyl radical antioxidant activity The hydroxyl radical (OH • ) is one of the most reactive radicals that can attack the biological system. 172 Hydroxyl radical scavenging assay is based on the inhibition of OH • radicals that can be produced during the Fenton reaction. In this reaction, hydrogen peroxide is introduced to ferrous ions, which results in ferric ions through the oxidation process. This oxidation is based on the availability of hydroxyl radicals to oxidize Fe 2+ to Fe 3+ . In other words, the production of Fe 3+ ions increased by increasing the number of hydroxyl radicals. 173 Hydroxyl radicals can be captured with the help of antioxidant species that donate hydrogen atoms and this capturing can be determined by the salicylic acid method, which forms a purple-color complex (absorb at 510 nm) with Fe 3+ ions. If OH • radicals are scavenged by species under study, the absorbance at 510 nm is reduced compared to the control solution.
The hydroxyl radical antioxidant activity of Lilium lancifolium leaf-extract polysaccharides was found to be about 96.16% when it was loaded with a concentration of 3 mg/mL. 41 Leaf-extract polysaccharides of Leonurus cardiaca at a 13.5 mg/mL concentration showed the best OH • scavenging activity (about 94.8%), and Althaea officinalis leaf-extract polysaccharides at a concentration of 20 mg/mL showed 94.8% OH • scavenging activity. 46  close to this radical, this radical accepts an electron from polysaccharides and transforms into a non-radical form. The extent of ABTS radical scavenging is assayed by the reduction of the blue-green solution of ABTS at 734 nm using a spectrophotometer. 173 The ABTS radical antioxidant activity of Phyllostachys pubescens leaf-extract polysaccharides at a concentration of 3 mg/mL was found to be 99.98%. 75 Morus alba leaf-extract polysaccharide showed high ABTS-radical-scavenging activity of 99.33% when being applied at a concentration of 4 mg/mL. 43 Polysaccharides separated from leaves of Silphium perfoliatum displayed 93.69% ABTS radical antioxidant activity at a low concentration of 1.2 mg/mL. 90  ) belong to one of the most toxic radicals commonly generated during biological and photochemical reactions. 84 The superoxide radical scavenging potential of polysaccharides is commonly assayed by the NADH-NBT-PMS system. In this system, the superoxide radical is generated by the reaction of β-nicotinamide adenine dinucleotide (NADH) and phenazine methosulfate (PMS). The PMS is reduced by NADH and generates O − 2 • , which is further reduced by nitroblue tetrazolium (NBT). 174,175 This radical is scavenged by antioxidant species like polysaccharides and decreases the reducing extent of NBT, which is monitored at 569 nm using a spectrophotometer. 82 The superoxide radical antioxidant activity of Lilium lancifolium leaf-extract polysaccharides was found to be 96.83% when it was applied at a concentration of 1 mg/   6 ]) spectrophotometric method at 700 nm. The greater the reducing potential of polysaccharides, the greater is the absorbance of the ferric-ferrocyanide complex at 700 nm. Ferric reducing antioxidant power of plant leaf-extract polysaccharides can be described in terms of absorbance at 700 nm. The greater the absorbance, the greater is the reducing power of plant leaf-extract polysaccharides ( Table 2). Polysaccharides obtained from olive leaves showed a high reducing potential (3 absorbances at 700 nm) at a low concentration of 0.7 mg/mL. 36 In the same way, the reducing potential of plant leaf-extract polysaccharides extracted from different sources follows the order of Gynura procumbens > Silphium perfoliatum > Mentha haplocalyx > Malva sylvestris > Acanthopanax senticosus > Phyllostachys pubescens at the optimal concentration.

Large-scale applications of plant polysaccharides
Starch, hemicellulose, cellulose, pectin, and gums are the most important plant polysaccharides for industrial applications.
Starch is a fundamental polysaccharide for human life and is found in leaves, root tubers, fruits, and seeds of plants as a storage polysaccharide. Starch is composed of amylose and amylopectin, which are glucose chains with different lengths and degrees of branching depending on origin. 178 In the food industry, starch is widely used as an additive to improve the thickening and adhesion of liquid and paste products.
Cationic starches as wet-end additives are used extensively in the paper industry. In the textile industry, starch is used for wrap sizing and fabric printing. Starches are used widely as excipients, diluents, disintegrants, binders, lubricants, glidants, and drug deliverers in pharmaceutical products. 179 Hemicellulose polysaccharides (xyloglucans) have a linear structure and are present in the cell walls of higher plants. 180 Tamarind seed xyloglucan is one of the most studied hemicellulose polysaccharides regarding rheological behavior and different applications. 181 Xyloglucans are widely used in the food industry (as, e.g., stabilizers, thickeners, gelling agents, and modifiers), the pharmaceutical industry (for drug delivery systems due to hydrophilic and mucoadhesive properties), and the cosmetic industry (as ultraviolet protective agents). 181 One of the richest polysaccharides in nature is cellulose, which is typically found in plant and fungi cell walls and is also synthesized by some bacteria. 182,183 In cellulose, glucose molecules are joined together by β (1)(2)(3)(4) linkage. It is hydrophilic, biodegradable, and insoluble in water and most organic solvents. 184 Cellulose acetates, cellulose nitrates, cellulose propionates, cellulose sulfates, and cellulose ethers are the most commonly used derivatives of cellulose. 185 The most significant types of cellulose that are widely used for commercial purposes are cellulose esters and cellulose ethers. 185 Cellulose is used on a large scale in the paper industry (to make paper), the textile industry (e.g. for making fabric), the pharmaceutical industry (e.g. as an additive, thickening agent, and drug delivery and gelling agent), and the food industry (e.g. as an additive, thickening agent, and viscosifier).
The potential application of pectins -structural acidic heteropolysaccharides contained in the primary and middle lamella and cell walls of terrestrial plants -are being recognized increasingly and have been widely studied due to their complex structures. 185 Commercially, pectin can be extracted from citrus peels and some fruits, like apple under acidic conditions. 100 Pectin is widely used in the food industry as a gelling agent, stabilizer, thickening agent, and viscosifier. 186 At low concentrations, pectin binds water to form gel. 187 Pectin is extensively used in child food items like toffees, jellies, and jams.
Gums are high-molecular-weight macromolecules obtained from plant exudates, which are soluble in water and have stabilizing and thickening effects. 188 In gums, monomers like glucose, mannose, galactose, xylose, amylose, and arabinose are joined together by glycosidic linkage with a perspective anomeric conformation. Gums differ in their properties (e.g. pH, solubility, gelling power, and viscosity) and source. Some gums are found in associated forms with terpenoids or proteins. 189 Gum Arabic, gum tragacanth, gum karaya, and gum ghatti are obtained from plant exudates whereas locust bean gum, guar gum, and tamarind are obtained from the seeds of plants. Gums are widely used in the food industry, pharmaceutical industry, cosmetic industry, and the chemical industry. 190

Conclusions and future projections
This review highlighted the importance of plant leaf-extract polysaccharides and explored major aspects of the utilization of polysaccharides, including extraction, purification, and their antioxidant potential. Several commonly used extraction methods such as HWE, MAE, UAE, PLE, SFE, and EAE provide meaningful extraction efficiency for plant leaf-extract polysaccharides. All these extraction techniques have appropriate uses but consideration of several factors differentiates them. Hot-water extraction is timeconsuming and requires high temperatures to obtain the best polysaccharide extraction efficiency, whereas UAE and MAE are time-saving. Normally, UAE and MAE favor extracting low-molecular-weight polysaccharides, while HWE is better at extracting high-molecular-weight polysaccharides. In our opinion, EAE and PLE are the only extraction methods that can be used for polysaccharide extraction on a large scale. Pressurized-liquid extraction and SWE are very similar to each other in terms of basic principles. Subcritical-water extraction is operated at high temperature and so it is also called superheated extraction. At high temperatures, extraction efficiency is reduced due to the degradation of polysaccharides. Although ILE provides good extraction efficiency for selective polysaccharides like cellulose, chitin, and pectin, technical development is still needed to make it fully cost effective. Supercritical-fluid extraction, UPE, and NPC are complicated processes and are not being widely applied to extract plant leaf polysaccharides due to high costs and some operational limitations. Pulsed electric fieldassisted extraction is a novel, nonthermal, efficient and fast extraction method that is capable of extracting high-purity polysaccharides within seconds by consuming less energy, but further research is needed to use it on a commercial scale. Numerous conventional techniques can be used to remove residues of other constituents like proteins, lipids, phenolics, flavonoids, pigments, nucleic acids, and other small organic and inorganic compounds that are conjugated with leaf polysaccharides, but there are still some gaps in these methods related to their optimization. The major impurities in leaf polysaccharides are proteins. Hence, several quick, feasible, and novel protein-removal protocols should be established. The high antioxidant activity of plant leaf-extract polysaccharides suggests that garden waste can be regarded as a healthy source of antioxidant polysaccharides. Finally, this review advises developing a commercial-scale setup to convert garden waste into polysaccharides that can play a vital role in functional applications as value-added products and stabilizing agents.