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<journal-id journal-id-type="publisher-id">Front. Nutr.</journal-id>
<journal-title>Frontiers in Nutrition</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Nutr.</abbrev-journal-title>
<issn pub-type="epub">2296-861X</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
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<article-meta>
<article-id pub-id-type="doi">10.3389/fnut.2024.1359813</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Nutrition</subject>
<subj-group>
<subject>Review</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Recent trends in extraction, purification, structural characterization, and biological activities evaluation of <italic>Perilla frutescens</italic> (L.) Britton polysaccharide</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author"><name><surname>Zhu</surname> <given-names>Ling</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author" corresp="yes"><name><surname>Guan</surname> <given-names>Lijun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="corresp" rid="c001"><sup>&#x002A;</sup></xref>
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<contrib contrib-type="author"><name><surname>Wang</surname> <given-names>Kunlun</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Ren</surname> <given-names>Chuanying</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Gao</surname> <given-names>Yang</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Li</surname> <given-names>Jialei</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Yan</surname> <given-names>Song</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Zhang</surname> <given-names>Xindi</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Yao</surname> <given-names>Xinmiao</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Zhou</surname> <given-names>Ye</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author"><name><surname>Li</surname> <given-names>Bo</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="aff" rid="aff2"><sup>2</sup></xref>
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<contrib contrib-type="author" corresp="yes"><name><surname>Lu</surname> <given-names>Shuwen</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="corresp" rid="c002"><sup>&#x002A;</sup></xref>
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<aff id="aff1"><sup>1</sup><institution>Institute of Food Processing, Heilongjiang Province Academy of Agricultural Sciences</institution>, <addr-line>Harbin</addr-line>, <country>China</country></aff>
<aff id="aff2"><sup>2</sup><institution>Heilongjiang Province Key Laboratory of Food Processing</institution>, <addr-line>Harbin</addr-line>, <country>China</country></aff>
<author-notes>
<fn fn-type="edited-by" id="fn0001">
<p>Edited by: Sa&#x0161;a &#x0110;urovi&#x0107;, Institute of General and Physical Chemistry, Serbia</p>
</fn>
<fn fn-type="edited-by" id="fn0002">
<p>Reviewed by: Gu Haofeng, Ankang University, China</p>
<p>Sne&#x017E;ana Filip, University of Novi Sad, Serbia</p>
<p>Guangping Lyu, Nanjing Normal University, China</p>
</fn>
<corresp id="c001">&#x002A;Correspondence: Lijun Guan, <email>qqaipphh@sina.com</email></corresp>
<corresp id="c002">Shuwen Lu, <email>shuwenlu@sina.com</email></corresp>
</author-notes>
<pub-date pub-type="epub">
<day>22</day>
<month>02</month>
<year>2024</year>
</pub-date>
<pub-date pub-type="collection">
<year>2024</year>
</pub-date>
<volume>11</volume>
<elocation-id>1359813</elocation-id>
<history>
<date date-type="received">
<day>22</day>
<month>12</month>
<year>2023</year>
</date>
<date date-type="accepted">
<day>13</day>
<month>02</month>
<year>2024</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#x00A9; 2024 Zhu, Guan, Wang, Ren, Gao, Li, Yan, Zhang, Yao, Zhou, Li and Lu.</copyright-statement>
<copyright-year>2024</copyright-year>
<copyright-holder>Zhu, Guan, Wang, Ren, Gao, Li, Yan, Zhang, Yao, Zhou, Li and Lu</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p><italic>Perilla frutescens</italic> (L.) Britton is an annual herb plant of the <italic>Perilla</italic> genus in the Labiatae family, which is commonly utilized as an edible and medicinal resource. Polysaccharides are among the major components and essential bioactive compounds of <italic>P. frutescens</italic>, which exhibit a multitude of biological activities, including antioxidant, antitumor, anti-fatigue, immunoregulation, hepatoprotective, anti-inflammatory, and lipid-lowering effects. As a natural carbohydrate, <italic>P. frutescens</italic> polysaccharide has the potential to be utilized in the development of drugs and functional materials. In this paper, we provide an overview of progress made on the extraction, purification, structural characterization, and bioactivity of polysaccharides from different parts of <italic>P. frutescens</italic>. The challenges and opportunities for research are discussed, along with the potential development prospects and future areas of focus in the study of <italic>P. frutescens</italic> polysaccharides.</p>
</abstract>
<kwd-group>
<kwd>
<italic>Perilla frutescens</italic>
</kwd>
<kwd>polysaccharides</kwd>
<kwd>extraction</kwd>
<kwd>structural characterization</kwd>
<kwd>bioactivities</kwd>
</kwd-group>
<counts>
<fig-count count="5"/>
<table-count count="3"/>
<equation-count count="0"/>
<ref-count count="79"/>
<page-count count="19"/>
<word-count count="10272"/>
</counts>
<custom-meta-wrap>
<custom-meta>
<meta-name>section-at-acceptance</meta-name>
<meta-value>Food Chemistry</meta-value>
</custom-meta>
</custom-meta-wrap>
</article-meta>
</front>
<body>
<sec sec-type="intro" id="sec1">
<label>1</label>
<title>Introduction</title>
<p><italic>Perilla frutescens</italic> (L.) Britton, also known as zisu in China, is an annual herb plant of the Labiatae family (<xref ref-type="bibr" rid="ref1">1</xref>, <xref ref-type="bibr" rid="ref2">2</xref>). Although <italic>P. frutescens</italic> is widespread in Asian countries (<xref ref-type="fig" rid="fig1">Figure 1A</xref>) (<xref ref-type="bibr" rid="ref3">3</xref>, <xref ref-type="bibr" rid="ref4">4</xref>), China is proposed to be the main genetic center of this species, where it has been cultivated for more than 2,000 years (<xref ref-type="bibr" rid="ref5">5</xref>, <xref ref-type="bibr" rid="ref6">6</xref>). According to the variation of plant leaf color, <italic>P. frutescens</italic> can be divided into two main varieties circulating in China: <italic>P. frutescens</italic> var. <italic>arguta</italic> (the lower part of the leaf is red or purple) and <italic>P. frutescens</italic> var. <italic>frutescens</italic> (the upper and lower leaf surfaces are green) (<xref ref-type="bibr" rid="ref7">7</xref>, <xref ref-type="bibr" rid="ref8">8</xref>). <italic>P. frutescens</italic> is considered a medicine and food homologous plant, as the dried stems, leaves (<xref ref-type="fig" rid="fig1">Figure 1B</xref>), and seeds (<xref ref-type="fig" rid="fig1">Figure 1C</xref>) can be used as a natural herbal medicine for pain relief, hemostasis, relief of cough, purgative, detoxification, relief of stomach upset, dissipating colds, and anti-inflammation (<xref ref-type="bibr" rid="ref8 ref9 ref10">8&#x2013;10</xref>). As part of the daily diet, <italic>P. frutescens</italic> leaves can be used in barbecue, sashimi, sushi, and as a culinary condiment given their aromatic flavor (<xref ref-type="bibr" rid="ref9">9</xref>, <xref ref-type="bibr" rid="ref11">11</xref>). Additionally, the oil of <italic>P. frutescens</italic> seeds can be used in baking pastries as an alternative to hydrogenated oils or cream (<xref ref-type="bibr" rid="ref9">9</xref>). <italic>P. frutescens</italic> is becoming a more popular item in home cooking as it is increasingly recognized as a health-promoting food source.</p>
<fig position="float" id="fig1">
<label>Figure 1</label>
<caption>
<p>Distribution diagram of <italic>P. frutescens</italic> in the world <bold>(A)</bold>; stems and leaves of <italic>P. frutescens</italic> <bold>(B)</bold>; seeds of <italic>P. frutescens</italic> <bold>(C)</bold>.</p>
</caption>
<graphic xlink:href="fnut-11-1359813-g001.tif"/>
</fig>
<p>In recent years, the chemical composition of <italic>P. frutescens</italic> has been extensively investigated (<xref ref-type="bibr" rid="ref12">12</xref>). <italic>P. frutescens</italic> contains multiple active ingredients, including polysaccharides, anthocyanins, flavonoids, terpenoids, phenols, volatile oils, fatty acids, and proteins (<xref ref-type="bibr" rid="ref13">13</xref>, <xref ref-type="bibr" rid="ref14">14</xref>). Among these compounds, polysaccharide is an essential component, which has been increasingly researched for various applications owing to its advantages of natural origin, safety, and low toxicity (<xref ref-type="bibr" rid="ref15">15</xref>). The polysaccharides obtained from <italic>P. frutescens</italic> have proven to exhibit antioxidant, hepatoprotective, antitumor, and immunomodulatory activities (<xref ref-type="bibr" rid="ref16 ref17 ref18 ref19">16&#x2013;19</xref>), highlighting their broad application prospects in the food and biomedicine industries. To date, studies in this field have mainly focused on the small-molecule compounds of <italic>P. frutescens</italic> and their pharmacological effects; however, a comprehensive review of the macromolecular polysaccharides of <italic>P. frutescens</italic> is lacking. Hence, in the present paper, the extraction, purification, structural characteristics, and biological activities of <italic>P. frutescens</italic> polysaccharides are systematically summarized with a view to further expand their application areas.</p>
</sec>
<sec id="sec2">
<label>2</label>
<title>Extraction and purification of polysaccharides from <italic>Perilla frutescens</italic></title>
<sec id="sec3">
<label>2.1</label>
<title>Extraction of polysaccharides from <italic>Perilla frutescens</italic></title>
<p>The extraction of polysaccharides from raw materials is the first and key step in their effective utilization. Therefore, identifying optimal extraction methods has been a primary research focus. <italic>P. frutescens</italic> polysaccharides are mainly extracted from the leaf and seed meal (a by-product of the seed after oil extraction) of the plant. Typically, a suitable solvent (such as petroleum ether, butane, or <italic>n</italic>-hexane) is selected to degrease the <italic>P. frutescens</italic> raw material in a Soxhlet extractor prior to polysaccharide extraction (<xref ref-type="bibr" rid="ref15">15</xref>, <xref ref-type="bibr" rid="ref18">18</xref>, <xref ref-type="bibr" rid="ref20">20</xref>). Polysaccharides are a class of highly polar, highly water-soluble, and ethanol-insoluble substances that can be obtained by hot water extraction (HWE) in combination with ethanol precipitation (<xref ref-type="bibr" rid="ref21">21</xref>). For instance, Zhou and Sheng (<xref ref-type="bibr" rid="ref22">22</xref>) prepared polysaccharides from <italic>P. frutescens</italic> seeds using HWE at 80 &#x00B0;C followed by 70% ethanol alcohol precipitation. To increase the polysaccharide yield, response surface methodology (RSM) can be used to optimize the extraction conditions. For example, Ding (<xref ref-type="bibr" rid="ref23">23</xref>) determined the following optimized HWE conditions for the extraction of <italic>P. frutescens</italic> leaf polysaccharide using RSM: material-to-liquid ratio of 1:30, extraction time of 4 h, extraction three times, and ethanol concentration for precipitation of 60% (<italic>v/v</italic>). Under the above conditions, the extraction yield of crude <italic>P. frutescens</italic> leaf polysaccharide reached up to 5.22&#x2009;&#x00B1;&#x2009;0.17%. HWE has advantages of being a relatively simple, low-cost, and non-polluting process (<xref ref-type="bibr" rid="ref24">24</xref>); however, this method suffers from the drawbacks of a long operating time, low yield, and the need for repetitive operations (<xref ref-type="bibr" rid="ref25">25</xref>). Thus, HWE should be combined with other innovative technologies such as ultrasonic-assisted extraction (UAE), microwave-assisted extraction (MAE), and ultrasonic-assisted enzyme extraction (UAEE).</p>
<p>UAE can promote the release and dissolution of intracellular and cell wall polysaccharides through the high shear pressure generated via cavitation, which has the benefits of a high extraction rate, simple operation, and low solvent dosage (<xref ref-type="bibr" rid="ref26">26</xref>). Zhang et al. (<xref ref-type="bibr" rid="ref18">18</xref>) optimized the experimental scheme for the extraction of polysaccharides from <italic>P. frutescens</italic> seed meal (PSMP) by UAE through RSM, obtaining an average extraction yield of 6.137&#x2009;&#x00B1;&#x2009;0.062% using a liquid-to-solid ratio of 26.00&#x2009;mL/g, extraction temperature of 43.00&#x00B0;C, ultrasonic time of 52.00&#x2009;min, and ultrasonic power of 229.00&#x2009;W. Compared with the time required using HWE, the extraction time for obtaining <italic>P. frutescens</italic> polysaccharides could be shortened by two-thirds and the extraction temperature was reduced by 33.70&#x2013;57.40% with UAE (<xref ref-type="bibr" rid="ref20">20</xref>).</p>
<p>MAE has the advantages of high permeability, selectivity, and extraction efficiency (<xref ref-type="bibr" rid="ref27">27</xref>), demonstrating its suitability for the extraction of <italic>P. frutescens</italic> polysaccharide. Microwave is an electromagnetic wave with a frequency in the range of 300&#x2009;MHz to 300&#x2009;GHz (<xref ref-type="bibr" rid="ref24">24</xref>). Microwave heating is produced by the ionic conduction of dissolved ions and molecular friction due to the dipolar rotation of polar solvents (<xref ref-type="bibr" rid="ref28">28</xref>). The optimized conditions for the MAE of <italic>P. frutescens</italic> seed polysaccharides were determined to be a liquid-to-feed ratio of 25 mL/g, microwave power of 480 W, and microwave processing time of 3 min, which resulted in a yield of 9.06% with subsequent HWE (90&#x00B0;C, 3&#x2009;h) (<xref ref-type="bibr" rid="ref29">29</xref>). In another study, using the MAE method to extract polysaccharides from <italic>P. frutescens</italic> leaf powder with a short microwave treatment (800 W) of only 30&#x2009;s after hot water immersion (80&#x00B0;C) for 2&#x2009;h resulted in improvement of the yield to 3.99% compared to a yield of only 2.21% obtained with the HWE method, which required a total extraction time of 6&#x2009;h at 80&#x00B0;C (<xref ref-type="bibr" rid="ref30">30</xref>). Therefore, the extraction of <italic>P. frutescens</italic> polysaccharides by MAE can save substantial time while improving the efficiency compared with the HWE method.</p>
<p>Enzyme-assisted extraction of polysaccharides involves the use of enzymes capable of breaking down the cell wall, which offers benefits of environmental friendliness, low-energy consumption, and high extraction efficiency (<xref ref-type="bibr" rid="ref31">31</xref>, <xref ref-type="bibr" rid="ref32">32</xref>). Under the optimal conditions of 1771.85&#x2009;U/g cellulase, enzyme activation temperature of 53.7&#x00B0;C, and enzyme digestion time of 36.2&#x2009;min, the extraction rate of <italic>P. frutescens</italic> leaf polysaccharides reached 17.91&#x2009;mg/g (<xref ref-type="bibr" rid="ref33">33</xref>). Ultrasonic extraction can further enhance the affinity of the enzyme for the substrate and thereby increase the speed of the enzymatic reaction (<xref ref-type="bibr" rid="ref24">24</xref>). Therefore, enzyme-assisted extraction can be combined with UAE (i.e., UAEE) to effectively increase the extraction rate. Recently, Li et al. (<xref ref-type="bibr" rid="ref34">34</xref>) optimized the UAEE process to extract polysaccharides from <italic>P. frutescens</italic> leaves according to a single-factor test and Box-Behnken design (BBD), obtaining a yield of 3.84% when the liquid-to-solid ratio was 41:1, enzymatic time was 40 min, enzymatic temperature was 49 &#x00B0;C, and ultrasonic power was 204 W. Subsequently, Zhang et al. (<xref ref-type="bibr" rid="ref35">35</xref>) applied RSM analysis with a BBD to identify the optimal conditions for the UAEE of <italic>P. frutescens</italic> seed meal polysaccharides (PSMP). With the optimized conditions of a compound enzyme dose of 6.6%, liquid-to-solid ratio of 25&#x2009;mL/g, extraction time of 61 min, and extraction temperature of 62&#x00B0;C, the PSMP yield was 7.711&#x2009;&#x00B1;&#x2009;0.201%.</p>
<p>In addition to the methods described above, various novel approaches for extracting polysaccharides have also been proposed, including pressurized-liquid extraction, supercritical-fluid extraction, ionic-liquid extraction, and pulsed electric field-assisted extraction (<xref ref-type="bibr" rid="ref27">27</xref>, <xref ref-type="bibr" rid="ref32">32</xref>). However, these novel approaches have not yet been employed for extracting polysaccharides from <italic>P. frutescens</italic>, requiring further investigation in the future.</p>
</sec>
<sec id="sec4">
<label>2.2</label>
<title>Purification of polysaccharides from <italic>Perilla frutescens</italic></title>
<p>The crude polysaccharide obtained after ethanol precipitation normally contains impurities such as proteins, pigments, and small molecules, necessitating subsequent separation and purification processes to obtain the pure polysaccharide (<xref ref-type="bibr" rid="ref36">36</xref>). The Sevag method is commonly used to remove proteins from crude polysaccharide solutions, which is based on the principle of protein denaturation in chloroform and other organic solvents (<xref ref-type="bibr" rid="ref17">17</xref>). In brief, the crude polysaccharide solution is blended with Sevag&#x2019;s reagent (1-butanol/chloroform, 1/4 <italic>v/v</italic>) at a certain proportion, the mixture is shaken well, and the supernatant is collected after centrifugation to remove some of the proteins (<xref ref-type="bibr" rid="ref37">37</xref>, <xref ref-type="bibr" rid="ref38">38</xref>). The Sevag method offers advantages of mild conditions, without causing the denaturation of polysaccharides (<xref ref-type="bibr" rid="ref39">39</xref>), and can achieve a good effect of removing proteins from the crude <italic>P. frutescens</italic> polysaccharide solution after multiple repeated operations (<xref ref-type="bibr" rid="ref20">20</xref>, <xref ref-type="bibr" rid="ref23">23</xref>, <xref ref-type="bibr" rid="ref34">34</xref>). Zhang (<xref ref-type="bibr" rid="ref30">30</xref>) discovered that under the conditions of a 4:1 ratio of chloroform to n-butanol, 1:1 ratio of polysaccharide solution to Sevag reagent, 35 min of deproteinization time, and two deproteinization steps, the deproteinization rate of <italic>P. frutescens</italic> leaf polysaccharide reached 73.6%. However, the deproteinized <italic>P. frutescens</italic> polysaccharide solution needs to be further dialyzed to remove residual Sevag reagent and other small-molecule impurities (<xref ref-type="bibr" rid="ref15">15</xref>, <xref ref-type="bibr" rid="ref35">35</xref>).</p>
<p>To obtain homogeneous polysaccharides, further isolation and purification of the crude polysaccharides is required. Column chromatography is the most extensively applied approach for the purification of polysaccharides owing to its good purification effect and simple operation procedures (<xref ref-type="bibr" rid="ref40 ref41 ref42">40&#x2013;42</xref>). Column chromatography mainly includes ion-exchange column chromatography and gel-column chromatography (<xref ref-type="bibr" rid="ref43">43</xref>). Ion-exchange chromatography involves the use of either cation- or anion-exchange resins (<xref ref-type="bibr" rid="ref44">44</xref>), with anionic columns most frequently used in the purification of <italic>P. frutescens</italic> polysaccharides. Anion-exchange chromatography enables strong binding to acidic polysaccharides without interacting with neutral polysaccharides, thus, the neutral polysaccharides are eluted first (<xref ref-type="bibr" rid="ref45">45</xref>) and those obtained from different fractions can be separated by a stepwise elution process using solutions with different ionic strengths (<xref ref-type="bibr" rid="ref46">46</xref>). For example, Li et al. (<xref ref-type="bibr" rid="ref34">34</xref>) eluted four types of <italic>P. frutescens</italic> leaf polysaccharides on a DAE-Cellulose 52 column (2.6&#x2009;&#x00D7;&#x2009;30&#x2009;cm) at a flow rate of 0.6 mL/min using different concentrations of sodium chloride solution (0, 0.1, 0.2, 0.3, and 0.5&#x2009;mol/L).</p>
<p>Gel-column chromatography (Sephadex-G series and Sephacryl-S series) is an effective method to separate polysaccharides with different molecular weights (<xref ref-type="bibr" rid="ref47">47</xref>). High-molecular-weight polysaccharides will be eluted first with the mobile phase, while the low-molecular-weight polysaccharides will diffuse into the pores of the gel and elute later (<xref ref-type="bibr" rid="ref48">48</xref>). Ding et al. (<xref ref-type="bibr" rid="ref15">15</xref>) used a Sephadex G-200 gel column (1.6&#x2009;cm&#x2009;&#x00D7;&#x2009;30&#x2009;cm) to purify and obtain a <italic>P. frutescens</italic> leaf polysaccharide (PFP) with purity reaching up to 89.73%. Different columns can be used synergistically in the separation and purification of polysaccharides to achieve a better effect. Kim et al. (<xref ref-type="bibr" rid="ref16">16</xref>) first used a DEAE-Toyopearl 650 M column (4.0&#x2009;&#x00D7;&#x2009;30&#x2009;cm) to isolate one polysaccharide, designated PFB-1-0. This polysaccharide was then eluted using a Sephadex G-100 gel column (2.5&#x2009;&#x00D7;&#x2009;94&#x2009;cm) with 0.2&#x2009;mol/L NaCl at a flow rate of 0.2&#x2009;mL/min to obtain fractions PFB-1-0i and PFB-1-0ii with 43.2&#x2009;&#x00B1;&#x2009;2.5% and 82.8&#x2009;&#x00B1;&#x2009;4.8% purity, respectively. In the majority of studies, DEAE-52 cellulose, DEAE-Toyopearl 650 M, Sephadex G-100 gel, and Sephadex G-200 gel chromatography columns are employed for the separation of <italic>P. frutescens</italic> polysaccharides, the eluate is collected, concentrated, and freeze-dried to finally obtain purified <italic>P. frutescens</italic> polysaccharides. The flow chart of the extraction and purification procedure of <italic>P. frutescens</italic> polysaccharides is displayed in <xref ref-type="table" rid="tab1">Table 1</xref> and <xref ref-type="fig" rid="fig2">Figure 2</xref>.</p>
<table-wrap position="float" id="tab1">
<label>Table 1</label>
<caption>
<p>Extraction and purification of polysaccharides from the different parts of <italic>P. frutescens</italic>.</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th align="left" valign="top">Polysaccharide name</th>
<th align="left" valign="top">Source</th>
<th align="left" valign="top">Extraction method</th>
<th align="center" valign="top">Crude polysaccharide yield</th>
<th align="left" valign="top">Separation and purification progress</th>
<th align="center" valign="top">References</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="top">PFP</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">Petroleum ether, degreasing, HWE, solid to liquid ratio of 1:30, 100&#x00B0;C, 3 replicate extractions, EtOH precipitation</td>
<td align="center" valign="top">5.22&#x2009;&#x00B1;&#x2009;0.17%</td>
<td align="left" valign="top">Water dialysis for 2&#x2009;days, Sephadex G-200 gel column chromatography</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref15">15</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFB-1-0-ii</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">HWE, 20&#x2009;mL/g liquid-solid ratio, 100&#x00B0;C, methanol reflux, 80% EtOH precipitation</td>
<td align="center" valign="top">9.8%</td>
<td align="left" valign="top">DEAE-Toyopearl 650 M, Sephadex G-100 column chromatography</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref16">16</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PEPF</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">HWE, 10&#x2009;mL/g liquid-solid ratio, 90&#x00B0;C, 2&#x2009;h, 70% EtOH precipitation</td>
<td align="center" valign="top">ND</td>
<td align="left" valign="top">Dialysis</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref14">14</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PLP1, PLP2, PLP3, PLP4</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">UAE, liquid-to-solid ratio of 41:1, 49&#x00B0;C, 40&#x2009;min, ultrasonic power of 204&#x2009;W, dehydrated ethanol precipitation</td>
<td align="center" valign="top">3.84%</td>
<td align="left" valign="top">Sevag method deproteinization, DEAE-52, Sephadex G-100 column chromatography</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref34">34</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFB-1-0</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">HWE, 20&#x2009;mL/g liquid-solid ratio, 100&#x00B0;C, methanol reflux, 80% EtOH precipitation</td>
<td align="center" valign="top">98&#x2009;g/kg</td>
<td align="left" valign="top">DEAE-Toyopearl 650 M chromatography, tap water dialysis</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref49">49</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P.</italic> leaf polysaccharide</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">UAEE, enzymolysis time 40&#x2009;min, enzymolysis temperature 20.4&#x00B0;C, cellulases quantity 2000&#x2009;U/g, ultrasonic time 60&#x2009;min, 80% EtOH precipitation</td>
<td align="center" valign="top">4.54%</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref50">50</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFPS</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">HWE, 20&#x2009;mL/g liquid-solid ratio, 80&#x00B0;C, 3&#x2009;h, 2 replicate extractions, 95% EtOH precipitation, Sevag method deproteinization, dialysis</td>
<td align="center" valign="top">3.57%</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref51">51</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">EAE, 40&#x2009;mL/g liquid-solid ratio, 1771.85&#x2009;U/g cellulase, enzyme action temperature 53.7&#x00B0;C, enzyme treatment time 36.2&#x2009;min, EtOH precipitation</td>
<td align="center" valign="top">17.91&#x2009;mg/g</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref33">33</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFP-1, PFP-2, PFP-3, PFP-4</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">Ethanol degreasing, HWE, 20&#x2009;mL/g liquid-solid ratio, 95% EtOH precipitation</td>
<td align="center" valign="top">ND</td>
<td align="left" valign="top">DEAE-52 cellulose column chromatography, dialysis</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref52">52</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PLP-0.1-I, PLP-0.2-I, PLP-0.3-I</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">UAE, 35.3&#x2009;mL/g liquid-solid ratio, 210&#x2009;W, 66.7&#x00B0;C, 51.5&#x2009;min</td>
<td align="center" valign="top">6.853&#x2009;&#x00B1;&#x2009;0.321%</td>
<td align="left" valign="top">Macroporous adsorbent resin D101, DEAE 52, Sephadex G-200, column chromatography, Sevag method deproteinization</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PLP</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">UEAE, compound enzyme dosage of 5.54%, 15&#x2009;mL/g liquid-solid ratio, 48&#x2009;min, 40&#x00B0;C</td>
<td align="center" valign="top">7.326&#x2009;&#x00B1;&#x2009;0.291%</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">HWE, 30&#x2009;mL/g liquid-solid ratio, 80&#x00B0;C, 6&#x2009;h; Firstly, soak in hot water at 70&#x00B0;C for 3&#x2009;h, then, using MAE to extract, loading volume of 10&#x2009;mL, microwave time of 30&#x2009;s, microwave power of 800&#x2009;W</td>
<td align="center" valign="top">2.21%; 3.99%</td>
<td align="left" valign="top">Activated carbon decolorization, Sevag method deproteinization, DEAE-cellulose column chromatography</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref30">30</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFSP-2-1</td>
<td align="left" valign="top">Seed</td>
<td align="left" valign="top">Petroleum ether degreasing, HWE, 20&#x2009;mL/g liquid-solid ratio, 85&#x00B0;C, 2.5&#x2009;h, 3 replicate extractions, 75% EtOH precipitation</td>
<td align="center" valign="top">3.42&#x2009;&#x00B1;&#x2009;1.97%</td>
<td align="left" valign="top">Sevag method deproteinization, polyamide chromatography column method decolorization, DEAE 52, Sephacryl S-500 HR column chromatography</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref53">53</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> seed polysaccharide</td>
<td align="left" valign="top">Seed</td>
<td align="left" valign="top">WAE, 25 (mL/g) liquid-to-feed ratio, 480&#x2009;W microwave power, 3&#x2009;min microwave processing time, hot water extraction at 90&#x00B0;C for 3&#x2009;h, Sevag method deproteinization, EtOH precipitation</td>
<td align="center" valign="top">9.06%</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref29">29</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> polysaccharide</td>
<td align="left" valign="top">Seed</td>
<td align="left" valign="top">Petroleum ether de-oiling, HWE, 80&#x00B0;C, 4 h, 70% EtOH precipitation</td>
<td align="center" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref22">22</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP-0.1-I; PSMP-0.2-I; PSMP-0.3-I</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top">UAE, 26.1&#x2009;mL/g liquid-solid ratio, 229&#x2009;W, 42&#x00B0;C, 51.8&#x2009;min</td>
<td align="center" valign="top">6.137&#x2009;&#x00B1;&#x2009;0.062%</td>
<td align="left" valign="top">Macroporous adsorbent resin D101, DEAE 52, Sephadex G-200, column chromatography, Sevag method deproteinization</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top">UEAE, compound enzyme dosage of 6.60%, 24.7&#x2009;mL/g liquid-solid ratio, 60.5&#x2009;min, 62&#x00B0;C</td>
<td align="center" valign="top">7.611&#x2009;&#x00B1;&#x2009;0.20%</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP-1</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top">UAEE, 6.6% compound enzyme, 25&#x2009;mL/g liquid-solid ratio, 62&#x00B0;C, 61&#x2009;min, 80% ethanol (EtOH) precipitation</td>
<td align="center" valign="top">7.711&#x2009;&#x00B1;&#x2009;0.201%</td>
<td align="left" valign="top">D101 resin adsorption, Sevag method deproteinization, DEAE-52 column chromatography</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref35">35</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top"><italic>N</italic>-hexane degreasing, UAE, liquid&#x2013;solid ratio of 26.00&#x2009;mL/g, 43&#x00B0;C, 52.00&#x2009;min, 229.00&#x2009;W ultrasonic power</td>
<td align="center" valign="top">6.137&#x2009;&#x00B1;&#x2009;0.062%</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref18">18</xref>)</td>
</tr>
<tr>
<td align="left" valign="top" char="&#x00D7;">PFSP-1; PFSP-2; PFSP-3</td>
<td align="char" valign="top" char="&#x00D7;">Seed meal</td>
<td align="char" valign="top" char="&#x00D7;">Petroleum ether, degreasing 6&#x2009;h, HWE, 20&#x2009;mL/g liquid-solid ratio, 85&#x00B0;C, 2&#x2009;h, 3 times, EtOH precipitation, Sevag method deproteinization</td>
<td align="char" valign="top" char="&#x00D7;">8.38%</td>
<td align="char" valign="top" char="&#x00D7;">DEAE-52 Cellulose, Sephadex G-100 column chromatography</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref17">17</xref>)</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<p>ND stands for not detected.</p>
</table-wrap-foot>
</table-wrap>
<fig position="float" id="fig2">
<label>Figure 2</label>
<caption>
<p>Schematic diagram of the extraction and purification process of <italic>P. frutescens</italic> polysaccharide.</p>
</caption>
<graphic xlink:href="fnut-11-1359813-g002.tif"/>
</fig>
</sec>
</sec>
<sec id="sec5">
<label>3</label>
<title>Structure of <italic>Perilla frutescens</italic> polysaccharides</title>
<p>Structural characterization of polysaccharides is important since the structure is closely related to biological activity (<xref ref-type="bibr" rid="ref54">54</xref>). The key structural features of polysaccharides include their monosaccharide composition, molecular weight (M<sub>w</sub>), chemical components, and bonding information (<xref ref-type="bibr" rid="ref55">55</xref>). Different plant parts (i.e., the roots, leaves, stems, or seeds), extraction methods, and isolation and purification steps can lead to variations in polysaccharide structure (<xref ref-type="bibr" rid="ref56">56</xref>). The structural characteristics of polysaccharides derived from different parts of <italic>P. frutescens</italic> are summarized in <xref ref-type="table" rid="tab2">Table 2</xref>.</p>
<table-wrap position="float" id="tab2">
<label>Table 2</label>
<caption>
<p>Structural characterization of polysaccharides from the different parts of <italic>P. frutescens</italic>.</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th align="left" valign="top">Polysaccharide name</th>
<th align="left" valign="top">Source</th>
<th align="left" valign="top">Molecular weight</th>
<th align="left" valign="top">Monosaccharide composition</th>
<th align="left" valign="top">Structural characterization</th>
<th align="left" valign="top">Analysis technique</th>
<th align="center" valign="top">References</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="top">PFP</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">1.18&#x2009;&#x00D7;&#x2009;106&#x2009;Da</td>
<td align="left" valign="top">Rha, Ara, Gal, Glc, Xyl, and GalA in a molar ratio of 0.13: 0.55: 1.40: 1.00: 0.13: 0.22</td>
<td align="left" valign="top">&#x03B1;-L-Ara<italic>f</italic>-(1&#x2192;, &#x2192;6)-&#x03B2;-D-Gal<italic>p</italic>-(1&#x2192;, &#x2192;4)-&#x03B1;-D-Glc<italic>p</italic>-(1&#x2192;, &#x2192;1,4)-&#x03B2;-D-Xyl<italic>p</italic>(1&#x2192;, &#x2192;4)-&#x03B1;-Gal<italic>p</italic>A-(1&#x2192;, &#x2192;2,4)-&#x03B1;-L-Rha<italic>p</italic>-(1&#x2192;</td>
<td align="left" valign="top">HPGPC, FT-IR, IC, NMR</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref15">15</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFB-1-0-ii</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">10&#x2009;kDa</td>
<td align="left" valign="top">Ara (9.6%), Xyl (0.2%), Man (39.8%), Gal (14.5%) and Glu (45.6%)</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">HPLC, GLC</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref16">16</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PEPF</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref14">14</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PLP1, PLP2, PLP3, PLP4</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref34">34</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFB-1-0</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref49">49</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P.</italic> leaf polysaccharide</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref50">50</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFPS</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">N-H(-CONH-), C-O-C glycosidic bonds</td>
<td align="left" valign="top">FT-IR</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref51">51</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref33">33</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFP-1, PFP-2, PFP-3, PFP-4</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND; 2.2 kDa; 5.2 kDa; 1.6 kDa</td>
<td align="left" valign="top">Man, Glu, Gal, Ara in a ratio of 51.3:32.1; Man, Rha, GluA, GalA, Glu, Gal, Ara, fructose (Fuc) in a ratio of 1.7:7.2:1.2:65.9:0.8:11.3:10.7:1.1; Man, Rha, GluA, GalA, Glu, Gal, Ara, Fuc in a ratio of 5.8:15.7:2.9:33.1:2.3:24.7:14.5:0.9; Man, Rha, GluA, GalA, Glu, Gal, Ara, Fuc in a ratio of 4.8:5.7:5.6:8.3:14.4:34.9:23.8:2.5</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">HPGPC, HPLC</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref52">52</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PLP-0.1-I, PLP-0.2-I, PLP-0.3-I</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">Rha, Fuc, Ara, Xyl, Man, Glu, Gal in a molar ratio of 22.88, 2.20:14.41:1.00:3.88:4.47: 24.55; Rha, ribose (Rib), Fuc, Ara, Man, Glu, Gal in a molar ratio of 20.44:1.00:1.98:6.55:4.58:15.45:21.52; Rha, Rib, Fuc, Ara, Xyl, GluA, Man, Glu, Gal in a molar ratio of 27.55:2.91:5.07:16.04:4.03:1.00:18.66:39.62:64.26</td>
<td align="left" valign="top">&#x03B2;-D-Glucopyranose</td>
<td align="left" valign="top">GC-MS, FT-IR</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PLP</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top">Leaf</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref30">30</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PFSP-2-1</td>
<td align="left" valign="top">Seed</td>
<td align="left" valign="top">8.81&#x2009;&#x00D7;&#x2009;106&#x2009;Da</td>
<td align="left" valign="top">Ara, Gal, Glu, Xyl, GluA in a molar ratio of 20.207:11.223:1.228:18.232:0.331</td>
<td align="left" valign="top">The main chain is &#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Gal<italic>p</italic>-(6&#x2192;1)-Gal<italic>p</italic>-(6&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Gal<italic>p</italic>-(6&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Araf-(3&#x2192;1)-Xyl<italic>p</italic>-(4&#x2192; with side chains of &#x2192;1,6)-Gal<italic>p</italic>-(3&#x2192;1)-Ara<italic>p</italic>, &#x2192;1,6)-Gal<italic>p</italic>-(3&#x2192;1,3)-Gal<italic>p</italic>-(6&#x2192;1)-Ara<italic>f</italic>, &#x2192;1,6)-Gal<italic>p</italic>-(3&#x2192;1)-Ara<italic>p</italic>, and &#x2192;1,3)-Ara<italic>f</italic>-(5&#x2192;1)-Glc<italic>p</italic>-(4&#x2192;1)-Gal<italic>p</italic>-(3&#x2192;1)-Glc<italic>p</italic></td>
<td align="left" valign="top">HPGPC, IC, FT-IR, methylation analysis, NMR</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref53">53</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> seed polysaccharide</td>
<td align="left" valign="top">Seed</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref29">29</xref>)</td>
</tr>
<tr>
<td align="left" valign="top"><italic>P. frutescens</italic> polysaccharide</td>
<td align="left" valign="top">Seed</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref22">22</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP-0.1-I; PSMP-0.2-I; PSMP-0.3-I</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">Rha, Ara, Xyl, GalA, Man, Glu, Gal in a molar ratio of 1.31:22.98:5.60:2.72:3.21:1.00:12.44; Rha, Fuc, Ara, Xyl, GalA, Man, Glu, Gal in a molar ratio of 8.58:2.08:20.36: 6.39:3.63:1.00:6.10:18.49; Rha, Rib, Fuc, Ara, Xyl, GalA, Man, Glu, Gal in a molar ratio of 12.91:1.00:2.21:9.55:8.03:6.71:1.42:29.58:22.24</td>
<td align="left" valign="top">&#x03B2;-D-Glucopyranose</td>
<td align="left" valign="top">GC-MS, FT-IR</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP-1</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">Rha, Ara, Xyl, Man, Glu, and Gal in a molar ratio of 0.69: 11.13:5.55:0.90:1.00:4.71</td>
<td align="left" valign="top">&#x03B2;-D-glucopyranose</td>
<td align="left" valign="top">FT-IR, GC-MS</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref35">35</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">PSMP</td>
<td align="left" valign="top">Seed meal</td>
<td align="left" valign="top">ND</td>
<td align="left" valign="top">Rha, Ara, Xyl, Man, Glu, and Gal in the mass ratio of 3.196%: 43.901%:21.956%:4.244%:4.706%:21.997%</td>
<td align="left" valign="top">&#x03B2;-D-Xyl, &#x03B1;-L-Ara, &#x03B2;-D-Gal, &#x03B2;-L-Ara, (1&#x2192;6) glycosidic, and (1&#x2192;3/4) glycosidic</td>
<td align="left" valign="top">GC-MS, FT-IR, NMR</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref18">18</xref>)</td>
</tr>
<tr>
<td align="left" valign="top" char="&#x00D7;">PFSP-1; PFSP-2; PFSP-3</td>
<td align="char" valign="top" char="&#x00D7;">Seed meal</td>
<td align="left" valign="top">1.06&#x2009;&#x00D7;&#x2009;105; 5.96&#x2009;&#x00D7;&#x2009;104; 3.72&#x2009;&#x00D7;&#x2009;104</td>
<td align="char" valign="top" char="&#x00D7;">Man, Gal, Xyl, Ara in a molar ratio of 0.01:0.06:0.11:0.81; Man, Xyl, Ara in a molar ratio of 0.28:0.28:0.41; Rha, glucuronic acid (GluA), Glu, Gal, Xly, Ara with a molar ratio of 0.013:0.024:0.040:0.080:0.120:0.700</td>
<td align="char" valign="top" char="&#x00D7;">Pyranose ring, C-O glycosidic bond</td>
<td align="char" valign="top" char="&#x00D7;">HPLC, FT-IR</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref17">17</xref>)</td>
</tr>
</tbody>
</table>
</table-wrap>
<sec id="sec6">
<label>3.1</label>
<title>Monosaccharide composition</title>
<p>Monosaccharide composition is an essential foundation for the structural analysis of polysaccharides. The acid hydrolysis method is used to completely break the glycosidic bonds of polysaccharides; after hydrolysis, the polysaccharide sample is neutralized, filtered, and derivatized for analysis of monosaccharide composition (<xref ref-type="bibr" rid="ref57">57</xref>). High-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), gas chromatography (GC), gas-liquid chromatography, and ion chromatography (IC) are common methods employed to measure the monosaccharide composition and molar ratio of polysaccharides (<xref ref-type="bibr" rid="ref15">15</xref>, <xref ref-type="bibr" rid="ref16">16</xref>, <xref ref-type="bibr" rid="ref47">47</xref>, <xref ref-type="bibr" rid="ref48">48</xref>). Using GC-MS analysis, the monosaccharide composition of a PSMP was determined to comprise rhamnose (Rha), arabinose (Ara), xylose (Xyl), mannose (Man), glucose (Glu), and galactose (Gal) at a mass ratio of 3.196%: 43.901%: 21.956%: 4.244%: 4.706%: 21.997% (<xref ref-type="bibr" rid="ref18">18</xref>). Using IC analysis, the PFP extracted from the leaves was reported to comprise six major monosaccharides, including Rha, Ara, Gal, Glu, Xyl, and galacturonic acid (GalA), with the Glu and Gal monosaccharides being the most abundant (<xref ref-type="bibr" rid="ref15">15</xref>). Furthermore, analysis of <italic>P. Frutescens</italic> polysaccharides extracted from the leaves and purified with DEAE-Toyopearl 650 M and Sephadex G-100 column chromatography showed that the proportions of Ara, Xyl, and Gal were gradually reduced during the progression of purification, whereas the proportion of Man and Glu significantly increased, which ultimately accounted for the major portion of the obtained <italic>P. frutescens</italic> leaf polysaccharides (<xref ref-type="bibr" rid="ref16">16</xref>). Subsequent GC analysis showed that the PFB-1-0 fraction consisted of Ara (8.9%), Xyl (0.9%), Man (25.9%), Glu (16.8%), and Gal (24.9%) after DEAE-Toyopearl 650 M column chromatography purification, and the PFB-1-0-ii fraction consisted of Ara (9.6%), Xyl (0.2%), Man (39.8%), Glu (45.6%), and Gal (14.5%) after Sephadex G-100 column chromatography purification.</p>
</sec>
<sec id="sec7">
<label>3.2</label>
<title>Molecular weight</title>
<p>The <italic>M</italic><sub>w</sub> not only affects the physical properties of polysaccharides, such as viscosity and solubility, but also affects their biological activities, forming the basis for characterizing the properties of polysaccharides (<xref ref-type="bibr" rid="ref58">58</xref>). To date, the <italic>M</italic><sub>w</sub> of <italic>P. Frutescens</italic> polysaccharide has mainly been analyzed using HPLC and high-performance gel-permeation chromatography (HPGPC) detection techniques (<xref ref-type="bibr" rid="ref16">16</xref>, <xref ref-type="bibr" rid="ref23">23</xref>, <xref ref-type="bibr" rid="ref51">51</xref>). Based on the available literature, the <italic>M</italic><sub>w</sub> of <italic>P. frutescens</italic> polysaccharide ranges from approximately 1.6&#x2009;&#x00D7;&#x2009;103&#x2009;Da to 1.18&#x2009;&#x00D7;&#x2009;106&#x2009;Da. For example, PFB-1-0-ii obtained from extraction of <italic>P. frutescens</italic> leaves using HWE and ethanol precipitation, followed by further separation and purification on a DEAE-Toyopearl 650 M column and a Sephadex G-100 chromatographic column, had an <italic>M</italic><sub>w</sub> of 10&#x2009;kDa determined by HPGPC (<xref ref-type="bibr" rid="ref16">16</xref>). By contrast, Niu et al. (<xref ref-type="bibr" rid="ref52">52</xref>) obtained three main polysaccharide fractions from <italic>P. frutescens</italic> leaves by HWE, ethanol precipitation, and further purification on a DEAE-52 column with an <italic>M</italic><sub>w</sub> of 2.2&#x2009;kDa and 1589.8&#x2009;Da for PFP-2 and PFP-4, respectively, whereas PFP-3 was mainly composed of two segments with an <italic>M</italic><sub>w</sub> of 5.2 and 40.0&#x2009;kDa, respectively, as determined by HPGPC. The difference in <italic>M</italic><sub>w</sub> among these <italic>P. frutescens</italic> polysaccharides is mainly attributed to variations in factors such as source, treatment method, and separation equipment. Similar variations in <italic>M</italic><sub>w</sub> have also been observed in studies on polysaccharides from Bupleuri Radix, and <italic>Nelumbo nucifera</italic> Gaertn. (lotus), and Radix Hedysari (<xref ref-type="bibr" rid="ref26">26</xref>, <xref ref-type="bibr" rid="ref59">59</xref>, <xref ref-type="bibr" rid="ref60">60</xref>).</p>
</sec>
<sec id="sec8">
<label>3.3</label>
<title>Chemical structures</title>
<p>The biological significance of polysaccharides is intimately associated with their complex structural properties and unique backbone features (<xref ref-type="bibr" rid="ref47">47</xref>). Therefore, determining the chemical structure of polysaccharides is an important element in the investigation of their pharmacological effects and improvement of their applications (<xref ref-type="bibr" rid="ref61">61</xref>). Chromatographic techniques, spectroscopic analysis, and other chemical analyses are effective methods for studying the structural characteristics of <italic>P. frutescens</italic> polysaccharides, including the types and linkages of sugar residues. According to the results of Fourier-transfer infrared spectroscopy (FT-IR), <italic>P. frutescens</italic> seed meal polysaccharide had five absorption peaks at 3,356&#x2009;cm<sup>&#x2212;1</sup> (O&#x2212;H), 2931 cm<sup>&#x2212;1</sup> (C&#x2212;H), 1658 cm<sup>&#x2212;1</sup> (&#x2212;C=O and &#x2212;CHO), 1415 cm<sup>&#x2212;1</sup> (C&#x2212;O), and 1,317 and 1,245 cm<sup>&#x2212;1</sup> (&#x2212;COOH). The strong peaks noted at 1,072 and 1,047&#x2009;cm<sup>&#x2212;1</sup> suggested the presence of galactopyranose and arabinofuranose in the backbone and branches of the sugar chain. GC-MS and one-dimensional nuclear magnetic resonance (NMR) spectrum (1H, 13C) analyses showed that <italic>P. frutescens</italic> seed meal polysaccharide was composed of &#x03B2;-<sc>d</sc>-Xyl, &#x03B1;-<sc>l</sc>-Ara, &#x03B2;-<sc>d</sc>-Gal, and &#x03B2;-<sc>l</sc>-Ara sugar residues, and was free of glucuronic acid (<xref ref-type="bibr" rid="ref18">18</xref>). Moreover, Li et al. (<xref ref-type="bibr" rid="ref53">53</xref>) obtained a homogenous polysaccharide faction of PFSP-2-1 from <italic>P. frutescens</italic> seed. Based on FT-IR, IC, methylation, and one-and two-dimensional NMR analysis, the structure of PFSP-2-1 showed a backbone of &#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Gal<italic>p</italic>-(6&#x2192;1)-Gal<italic>p</italic>-(6&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Gal<italic>p</italic>-(6&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Ara<italic>f</italic>-(5&#x2192;1)-Araf-(3&#x2192;1)-Xyl<italic>p</italic>-(4&#x2192;; and three branches consisting of &#x2192;1,6)-Gal<italic>p</italic>-(3&#x2192;1)-Ara<italic>p</italic>, &#x2192;1,6)-Gal<italic>p</italic>-(3&#x2192;1,3)-Gal<italic>p</italic>-(6&#x2192;1)-Ara<italic>f</italic>, &#x2192;1,6)-Gal<italic>p</italic>-(3&#x2192;1)-Ara<italic>p</italic>, and &#x2192;1,3)-Ara<italic>f</italic>-(5&#x2192;1)-Glc<italic>p</italic>-(4&#x2192;1)-Gal<italic>p</italic>-(3&#x2192;1)-Glc<italic>p.</italic> The link sites were at position C-3 of &#x2192;3,6)-Gal<italic>p</italic>-(1&#x2192;, C-3 of &#x2192;3,6)-Gal<italic>p</italic>-(1&#x2192;, and C-5 of &#x2192;3,5)-Ara<italic>f</italic>-(1&#x2192;, respectively (<xref ref-type="fig" rid="fig3">Figure 3</xref>). Using FT-IR, IC, and NMR spectroscopy, PFP was confirmed to be an acidic polysaccharide with a backbone comprising six sugar residues: &#x03B1;-<sc>l</sc>-Ara<italic>f</italic>-(1&#x2192;, &#x2192;6)-&#x03B2;-<sc>d</sc>-Gal<italic>p</italic>-(1&#x2192;, &#x2192;4)-&#x03B1;-<sc>d</sc>-Glc<italic>p</italic>-(1&#x2192;, &#x2192;1,4)-&#x03B2;-<sc>d</sc>-Xyl<italic>p</italic>-(1&#x2192;, &#x2192;4)-&#x03B1;-Gal<italic>p</italic>A-(1&#x2192;, and &#x2192;2,4)-&#x03B1;-<sc>l</sc>-Rha<italic>p</italic>-(1&#x2192; (<xref ref-type="bibr" rid="ref15">15</xref>). However, the detailed chemical structures of polysaccharides from <italic>P. frutescens</italic> leaf, such as the linkage of sugar residues, have been not reported to date. Therefore, the detailed structures of <italic>P. frutescens</italic> polysaccharides from different sources and parts of the plant need to be analyzed more deeply and comprehensively to further understand the physicochemical and biological properties of <italic>P. frutescens</italic> polysaccharides.</p>
<fig position="float" id="fig3">
<label>Figure 3</label>
<caption>
<p>Diagram of the planar structure of <italic>P. frutescens</italic> polysaccharide (PFSP-2-1) (<xref ref-type="bibr" rid="ref53">53</xref>).</p>
</caption>
<graphic xlink:href="fnut-11-1359813-g003.tif"/>
</fig>
</sec>
<sec id="sec9">
<label>3.4</label>
<title>Conformational features</title>
<p>Conformation refers to the three-dimensional structure created by the penetrating bonds formed by the molecular structure of the polymer and the physical force penetrating the space (<xref ref-type="bibr" rid="ref62">62</xref>). Polysaccharides possess complicated substituents and diversified chemical structures, and their spatial conformation largely determines the various biological functions (<xref ref-type="bibr" rid="ref63">63</xref>). Scanning electron microscopy (SEM) observations showed that the apparent morphology of polysaccharides obtained from <italic>P. frutescens</italic> leaves represents a combination of reticulated, lamellar, and chained forms, while the surface of the polysaccharides extracted from the <italic>P. frutescens</italic> seed meal is dominated by meshes and sheets (<xref ref-type="bibr" rid="ref20">20</xref>) (<xref ref-type="fig" rid="fig4">Figure 4</xref>). Ding (<xref ref-type="bibr" rid="ref23">23</xref>) carried out a comprehensive characterization of the morphological features of <italic>P. frutescens</italic> leaf polysaccharides via Congo red staining, circular dichroism, and SEM observations. The results revealed an overall orderly spatial structure of the <italic>P. frutescens</italic> polysaccharide, without a triple-helix structure, and the surface was smooth and flaky. Zhao et al. (<xref ref-type="bibr" rid="ref51">51</xref>) similarly found a smooth lamellar structure on the surface of a <italic>P. Frutescens</italic> polysaccharide extracted from the leaves based on SEM observations.</p>
<fig position="float" id="fig4">
<label>Figure 4</label>
<caption>
<p>SEM micrographs of <italic>P. frutescens</italic> leaf polysaccharides (<bold>A</bold>: PLP-0.1-I; <bold>C</bold>; PLP-0.2-I; <bold>E</bold>: PLP-0.3-I) and <italic>P. frutescens</italic> seed meal polysaccharides (<bold>B</bold>: PSMP-0.1-I; <bold>D</bold>: PSMP-0.2-I; <bold>F</bold>: PSMP-0.3-I) (<xref ref-type="bibr" rid="ref20">20</xref>).</p>
</caption>
<graphic xlink:href="fnut-11-1359813-g004.tif"/>
</fig>
</sec>
</sec>
<sec id="sec10">
<label>4</label>
<title>Biological activities</title>
<p><italic>P. frutescens</italic> exhibits numerous edible and medicinal benefits, and its polysaccharide is an essential component conferring these characteristics. Indeed, <italic>P. frutescens</italic> polysaccharide possess a variety of biological activities, including antioxidant, antitumor, anti-fatigue, immunoregulation, hepatoprotective, anti-inflammatory, and lipid-lowering effects, which are summarized in <xref ref-type="fig" rid="fig5">Figure 5</xref>.</p>
<fig position="float" id="fig5">
<label>Figure 5</label>
<caption>
<p>The biological activity of <italic>P. frutescens</italic> polysaccharide.</p>
</caption>
<graphic xlink:href="fnut-11-1359813-g005.tif"/>
</fig>
<sec id="sec11">
<label>4.1</label>
<title>Antioxidant activity</title>
<p>Reactive oxygen species (ROS) are oxygen radicals in biological bodies, consisting of oxygen and oxygenated highly active molecules (e.g., superoxide anion, tissue peroxidation products, and free radicals). Typically, appropriate levels of ROS can facilitate immunity, repair, survival, and growth (<xref ref-type="bibr" rid="ref64">64</xref>, <xref ref-type="bibr" rid="ref65">65</xref>). However, the excessive production of ROS will disrupt the dynamic redox balance, causing the human body to suffer damage, leading to multiple diseases (<xref ref-type="bibr" rid="ref66">66</xref>). Hence, supplementation of antioxidants exogenously is essential for the body to fight or mitigate oxidative stress damage. <italic>P. frutescens</italic> polysaccharides demonstrate potential as effective antioxidants. The antioxidant activities of <italic>P. frutescens</italic> polysaccharides have been evaluated <italic>in vitro</italic> by examining the potential for scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2&#x2032;-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), hydroxyl (&#x2022;OH), superoxide anion (O2-&#x2022;), and nitrite ion (NO<sub>2</sub>-) radicals, along with iron-reduction capacity assays (<xref ref-type="bibr" rid="ref14">14</xref>, <xref ref-type="bibr" rid="ref20">20</xref>, <xref ref-type="bibr" rid="ref37">37</xref>). For example, 1,000&#x2009;&#x03BC;g/mL of a <italic>P. frutescens</italic> seed meal polysaccharide (PMP) showed scavenging rates on &#x2022;OH, O2-&#x2022;, NO<sub>2</sub>-, and DPPH radicals of 49.70&#x2009;&#x00B1;&#x2009;2.10%, 21.76&#x2009;&#x00B1;&#x2009;1.09%, 60.75&#x2009;&#x00B1;&#x2009;2.19%, and 72.95&#x2009;&#x00B1;&#x2009;4.19%, respectively (<xref ref-type="bibr" rid="ref67">67</xref>). The scavenging ability of PMP for O2-&#x2022; showed a certain dose-dependent effect at a polysaccharide mass concentration range of 62.5&#x2013;1,000&#x2009;&#x03BC;g/mL. However, Zhu et al. (<xref ref-type="bibr" rid="ref68">68</xref>) showed that the scavenging rate of <italic>P. frutescens</italic> seed meal polysaccharide on O2-&#x2022; radicals exhibited a trend of initial increase followed by a decrease; when the concentration of the polysaccharide reached 1,000&#x2009;&#x03BC;g/mL, the formation of free radicals was promoted rather than suppressed. The reason for these discrepant effects could be attributed to the different structures and compositions of polysaccharides. Moreover, a PSMP exhibited a 73.83% scavenging rate of &#x2022;OH radicals at 5,000&#x2009;&#x03BC;g/mL and a 91.10% scavenging rate of ABTS radicals at 600&#x2009;&#x03BC;g/mL (<xref ref-type="bibr" rid="ref18">18</xref>). In general, the high antioxidant activity of polysaccharides is related to the enrichment of uronic acid groups (<xref ref-type="bibr" rid="ref39">39</xref>). However, no uronic acid was detected in the PSMP, and the antioxidant properties were instead attributed to the presence of Man and Glu (<xref ref-type="bibr" rid="ref18">18</xref>). Overproduction of ROS induced by oxidative stress also plays a key role in neuronal damage. In H<sub>2</sub>O<sub>2</sub>-induced neurotoxic cellular assays, <italic>P. frutescens</italic> polysaccharide reduced ROS production, lowered malondialdehyde (MDA) levels, increased intracellular superoxide dismutase (SOD) activity, and counteracted H<sub>2</sub>O<sub>2</sub>-induced neuronal cell death by activating the PI3K/AKT pathway and negatively regulating the mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-&#x03BA;B) pathways in HT22 hippocampus cells (<xref ref-type="bibr" rid="ref14">14</xref>).</p>
</sec>
<sec id="sec12">
<label>4.2</label>
<title>Antitumor activity</title>
<p>Cancer remains a major global public health challenge. In the last few years, polysaccharides have attracted considerable attention in cancer research owing to their reported antitumor activity and low side effects (<xref ref-type="bibr" rid="ref69">69</xref>). The antitumor effects of <italic>P. Frutescens</italic> polysaccharides could be attributed to their strong immunomodulatory abilities (<xref ref-type="bibr" rid="ref15">15</xref>, <xref ref-type="bibr" rid="ref17">17</xref>). Treatment of H22 tumor-bearing mice with a <italic>P. frutescens</italic> leaf polysaccharide (PFP) at doses of 100 mg/kg and 300 mg/kg for three weeks significantly decreased tumor volume and weight and reduced thymic atrophy and splenomegaly in mice in a dose-dependent manner (<xref ref-type="bibr" rid="ref15">15</xref>). <italic>In vitro</italic> investigations further showed that the PFP also inhibited tumor proliferation by blocking the S-phase of the cell cycle and decreasing mitochondrial membrane potential. The antitumor effect of PFP may be related to the Gal component. Treatment of tumor-bearing mice with <italic>P. frutescens</italic> seed polysaccharide (0.1, 0.3, and 0.5&#x2009;mg for 10&#x2009;days) significantly reduced the levels of lactate dehydrogenase, aldolase, and interleukin (IL)-10; increased the levels of IL-2 and tumor necrosis factor-alpha (TNF-&#x03B1;) in the serum of mice; and down-regulated the expression of the anti-apoptotic protein Bcl-2 and up-regulated the expression of the pro-apoptotic protein Bax (<xref ref-type="bibr" rid="ref17">17</xref>). Collectively, these results indicated that <italic>P. frutescens</italic> seed polysaccharide can inhibit the growth of tumor cells <italic>in vivo</italic> by enhancing the autoimmunity of mice. Li et al. (<xref ref-type="bibr" rid="ref53">53</xref>) found that a <italic>P. frutescens</italic> seed polysaccharide (PFSP-2-1) could significantly inhibit the growth of three types of hepatocellular carcinoma cells (HepG2, Hep3b, and SK-Hep-1), and the inhibitory effect gradually increased with the increase of PFSP-2-1 concentration. It has been hypothesized that Ara and Xyl in PFSP-2-1 are some of the factors inhibiting the growth of liver cancer cells. Moreover, the triple-helix high-level structure of PFSP-2-1 is also an important factor in its anti-tumor activity. In summary, <italic>P. frutescens</italic> polysaccharides exhibit excellent antitumor properties; however, the therapeutic effects will need to be further investigated on more cancer models along with determination of the conformational relationships.</p>
</sec>
<sec id="sec13">
<label>4.3</label>
<title>Hepatoprotective effect</title>
<p>The liver is a crucial organ with essential functions in regulating metabolism, biotransformation, and detoxification (<xref ref-type="bibr" rid="ref70">70</xref>). Factors such as autoimmune aggression of liver cells, medication misuse, alcohol consumption, infections with viruses, and cardiovascular disease predispose the liver to injury (<xref ref-type="bibr" rid="ref71">71</xref>, <xref ref-type="bibr" rid="ref72">72</xref>). Several studies have demonstrated that <italic>P. frutescens</italic> polysaccharides have a hepatoprotective effect, which may be associated with their antioxidant and anti-inflammatory activities (<xref ref-type="bibr" rid="ref19">19</xref>, <xref ref-type="bibr" rid="ref73">73</xref>). Li et al. (<xref ref-type="bibr" rid="ref29">29</xref>) reported that <italic>P. frutescens</italic> seed meal polysaccharide administered at doses of 100, 300, and 500&#x2009;mg/kg for 7&#x2009;days alleviated CCl<sub>4</sub>-induced acute liver injury in an animal model. Specifically, the <italic>P. frutescens</italic> seed meal polysaccharide decreased serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities, as well as liver and spleen coefficients. In a type 2 diabetic rat liver injury model induced by high-fat and high-sugar feeding combined with intraperitoneal injection of streptozotocin, gavage of <italic>P. frutescens</italic> leaf polysaccharide at 0.15, 0.30, 0.60&#x2009;g/kg for 28&#x2009;days significantly reduced the levels of MDA, C-reactive protein, IL-6, TNF-&#x03B1;, and acetylated forkhead transcription factor protein in liver tissues, while the activities of glutathione peroxidase (GSH-Px), catalase (CAT), and SOD as well as the expression of sirtuin 1 (SIRT1) and FoxO1 proteins in liver tissues were significantly elevated (<xref ref-type="bibr" rid="ref73">73</xref>). Thus, the <italic>P. frutescens</italic> leaf polysaccharide could improve type 2 diabetes-induced rat liver injury by inhibiting oxidative stress and inflammation, while regulating activation of the SIRT1/FoxO1 signaling pathway. Additionally, Tao et al. (<xref ref-type="bibr" rid="ref19">19</xref>) found that <italic>P. frutescens</italic> leaf polysaccharide significantly improved hepatocyte inflammatory cell infiltration, bleeding, and liver steatosis in a mouse model of alcoholic liver injury disease (ALD) induced by chronic ethanol gavage. After gavage of the <italic>P. frutescens</italic> polysaccharides, the contents of IL-1&#x03B2;, IL-6, TNF-&#x03B1;, and MDA in the liver tissues of ALD mice were significantly decreased; the SOD and GSH-Px activities were significantly increased; and the relative expression levels of p-AMPK&#x03B1;/AMPK&#x03B1; and SIRT1 were significantly increased. These findings suggested that <italic>P. frutescens</italic> polysaccharide from the leaves might ameliorate liver injury in ALD model mice by modulating the activity of the SIRT1-AMPK signaling pathway in the liver tissues.</p>
</sec>
<sec id="sec14">
<label>4.4</label>
<title>Immunomodulatory effect</title>
<p>Immunomodulation involves recognition of the body&#x2019;s own and external substances, along with maintenance of the body&#x2019;s physiological balance via the immune response to external antigens, which plays an essential role in the resistance to infections, tumors, and other diseases (<xref ref-type="bibr" rid="ref31">31</xref>, <xref ref-type="bibr" rid="ref74">74</xref>). Natural polysaccharides play a vital role in immune regulation by stimulating immune cells such as T lymphocytes, B cells, macrophages, and cytotoxic T cells (<xref ref-type="bibr" rid="ref75">75</xref>). To date, the immunomodulatory activity of <italic>P. frutescens</italic> polysaccharides has been demonstrated in a variety of <italic>in vitro</italic> and <italic>in vivo</italic> models. Kwon et al. (<xref ref-type="bibr" rid="ref49">49</xref>) obtained a fraction from the crude polysaccharides extracted from <italic>P. frutescens</italic> leaves (PFB-1) by DEAE-Toyopearl 650 M chromatographic column elution named PFB-1-0, which significantly increased lysosomal enzyme activity as well as nitric oxide and TNF-&#x03B1; levels in mouse peritoneal macrophages. Moreover, PFB-1 could stimulate the production of IL-6 and granulocyte-macrophage colony-stimulating factors in mice. In a corroborative study, Kim et al. (<xref ref-type="bibr" rid="ref16">16</xref>) showed that the active polysaccharide fractions (PFB-1, PFB-1-0, PFB-1-0-ii) from <italic>P. frutescens</italic> leaves at a concentration range of 1&#x2013;100&#x2009;&#x03BC;g/mL promoted macrophage lysosomal enzyme activity in a concentration-dependent manner. The immunomodulatory activity of these four <italic>P. frutescens</italic> leaf polysaccharides was attributed to the Man, Gal, and Glu components. Moreover, the high-molecular-weight <italic>P. Frutescens</italic> polysaccharide exhibited more effective macrophage-stimulating activity than the low-molecular-weight <italic>P. Frutescens</italic> polysaccharide. PFB-1-0-ii showed the best effect among all polysaccharide fractions, with 100&#x2009;&#x03BC;g/mL PFB-1-0-ii increasing macrophage lysosomal enzyme activity by 245% in comparison to that of the untreated control. Together, these studies indicate that <italic>P. Frutescens</italic> polysaccharides have the potential to be developed into drugs and functional foods with novel immunostimulatory activities; however, the interaction mechanism between polysaccharides and the immune system remains unclear, warranting further research.</p>
</sec>
<sec id="sec15">
<label>4.5</label>
<title>Other activities</title>
<p>In addition to the above biological activities, <italic>P. Frutescens</italic> polysaccharides also contribute to other health dimensions, including anti-fatigue, anti-inflammatory, and lipid-lowering effects. In a mouse model of fatigue induced by exhaustive swimming, gavage of <italic>P. Frutescens</italic> polysaccharides extracted from the leaves (5, 10, and 20&#x2009;mg/kg for 4&#x2009;weeks) significantly increased the forceful swimming time of mice, as well as the blood glucose level, liver glycogen content, and muscle glycogen content after exercise (<xref ref-type="bibr" rid="ref76">76</xref>). These findings demonstrated a protective effect of the <italic>P. Frutescens</italic> polysaccharide in accelerating the removal of certain fatigue-causing metabolic substances, maintaining the normal physiological function of cells, and delaying fatigue. Wang et al. (<xref ref-type="bibr" rid="ref77">77</xref>) tested the effectiveness of <italic>P. Frutescens</italic> leaf polysaccharide as an anti-inflammatory agent in a rat model of smoking combined with lipopolysaccharide-induced chronic obstructive pulmonary disease (COPD); treatment with 20&#x2009;mg/kg <italic>P. Frutescens</italic> polysaccharide for 4&#x2009;weeks <italic>in vivo</italic> resulted in improvement of lung function in COPD model rats. <italic>P. Frutescens</italic> polysaccharide treatment significantly decreased the expression levels of IL-8, TNF-&#x03B1;, Ras homologous protein A, Wnt5a, and p-JNK; reduced the thickness of bronchial wall and smooth muscle; and improved lung histopathologic changes. The inhibitory effect of <italic>P. Frutescens</italic> leaf polysaccharide on inflammation in COPD rats was mainly achieved by inhibiting the Wnt/PCP signaling pathway. In addition, Liu et al. (<xref ref-type="bibr" rid="ref78">78</xref>) demonstrated the ameliorative effects of a <italic>P. frutescens</italic> seed polysaccharides (PFSP) on abnormal lipid metabolism and oxidative stress induced by a high-fat diet in mice. Supplementation of the PFSP (50, 100, and 200&#x2009;mg/kg) in the feed for 8 weeks significantly decreased the levels of AST, ALT, triglyceride, total cholesterol, low-density lipoprotein, blood glucose, MDA, and the mRNA and protein expression levels of fatty acid synthesis-related genes. By contrast, PFSP supplementation increased the levels of high-density lipoprotein, fatty acid catabolism-related genes (<italic>CPT-1, ATGL</italic>) mRNA and protein expression, and the activities of SOD, GSH-Px, and CAT. Collectively, PFSP ameliorated high-fat diet-induced oxidative stress and fatty liver in mice. Based on the above research, the detailed biological effects or mechanisms of <italic>P. Frutescens</italic> polysaccharides are summarized in <xref ref-type="table" rid="tab2">Table 2</xref>.</p>
</sec>
</sec>
<sec id="sec16">
<label>5</label>
<title>Conclusion and future prospects</title>
<p><italic>Perilla frutescens</italic> is a highly promising edible and medicinal homologous resource; as the main components of <italic>P. frutescens</italic>, the polysaccharides of this herb have received increasing research attention in recent years. In this review, the extraction, purification, structural characterization, and associated biological activities of <italic>P. Frutescens</italic> polysaccharides have been summarized. Despite the remarkable advances achieved in <italic>P. Frutescens</italic> polysaccharide research, there are still several opportunities and challenges that remain to be resolved (<xref ref-type="table" rid="tab3">Table 3</xref>).</p>
<table-wrap position="float" id="tab3">
<label>Table 3</label>
<caption>
<p>Summary of biological activities of polysaccharides from <italic>P. frutescens</italic> (&#x201C;&#x2193;,&#x201D; decrease; &#x201C;&#x2191;,&#x201D; increase).</p>
</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th align="left" valign="top">Biological activities</th>
<th align="left" valign="top">Polysaccharide name</th>
<th align="left" valign="top">Types</th>
<th align="left" valign="top">Testing subjects</th>
<th align="left" valign="top">Doses/duration</th>
<th align="left" valign="top">Effects</th>
<th align="left" valign="top">Mechanisms</th>
<th align="center" valign="top">References</th>
</tr>
</thead>
<tbody>
<tr>
<td align="left" valign="top">Antioxidant</td>
<td align="left" valign="top">PSMP</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">DPPH, hydroxyl, ABTS radicals</td>
<td align="left" valign="top">2,000&#x2009;&#x03BC;g/mL for DPPH radical, 5,000&#x2009;&#x03BC;g/mL for hydroxyl radical, 600 &#x03BC;g/mL for ABTS radical</td>
<td align="left" valign="top">DPPH, hydroxyl, and ABTS scavenging rate was 75.00%, 73.83%, and 91.10%, respectively</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref18">18</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PEPF</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">DPPH, ABTS radicals, reducing power, ferric antioxidant power, H<sub>2</sub>O<sub>2</sub>-induced HT22 hippocampus cells</td>
<td align="left" valign="top">0.25&#x2013;2 mg/mL in DPPH, and ABTS scavenging test, 0.25&#x2013;2 mg/mL in reducing power and ferric antioxidant power assay, 500&#x2009;&#x03BC;g/mL for oxidatively damaged HT22 hippocampus cells</td>
<td align="left" valign="top">Radical-scavenging activities, reducing power, and ferric antioxidant power&#x2191;; MDA, Bax, sub-G1 cells phase population&#x2193;; SOD, PARP, Bcl-2&#x2191;</td>
<td align="left" valign="top">Activating PI3K/AKT, negatively regulating the MAPK and NF-&#x03BA;B pathways</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref14">14</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PSMP-1</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">DPPH, ABTS radicals</td>
<td align="left" valign="top">0.10&#x2013;5.00&#x2009;mg/mL in DPPH scavenging assay, 0.0625&#x2013;2.00&#x2009;mg/mL in ABTS scavenging assay</td>
<td align="left" valign="top">Scavenging DPPH and ABTS radicals, the IC<sub>50</sub> was 2.078&#x2009;&#x00B1;&#x2009;0.092 and 0.266&#x2009;&#x00B1;&#x2009;0.009&#x2009;mg/mL, respectively</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref35">35</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PLP1, PLP2, PLP3, PLP4</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">Reducing power, DPPH, hydroxyl, ABTS radicals</td>
<td align="left" valign="top">0.1&#x2013;5.0&#x2009;mg/mL for reducing power test, 0.1&#x2013;5.0&#x2009;mg/mL for DPPH, ABTS, hydroxyl radicals</td>
<td align="left" valign="top">DPPH, ABTS, hydroxyl radical-scavenging capacity and reducing power&#x2191;; antioxidant power of the four fractions was in the order of PLP3 &#x003E; PLP2 &#x003E; PLP1 &#x003E; PLP4</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref34">34</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PFP-1, PFP-2, PPFP-3, PFP-4</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">DPPH, ABTS radicals, iron ion reduction ability</td>
<td align="left" valign="top">0.03&#x2013;5.0&#x2009;mg/mL for DPPH radical, 0.25&#x2013;7.0&#x2009;mg/mL for ABTS radical, 0.5&#x2013;7.0&#x2009;mg/mL in iron ion reduction ability assay</td>
<td align="left" valign="top">DPPH and ABTS scavenging capacity and ion reduction ability&#x2191;</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref52">52</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">DPPH, hydroxyl radicals</td>
<td align="left" valign="top">0.1&#x2009;mg/mL in DPPH scavenging test; 1.0 mg/mL in hydroxyl scavenging test</td>
<td align="left" valign="top">DPPH scavenging rate (94.17%) and hydroxyl scavenging rate (90.06%)</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref50">50</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PMP</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">Hydroxyl, O<sub>2</sub>-&#x2022;, NO<sub>2</sub>-, DPPH radicals, H<sub>2</sub>O<sub>2</sub>-induced oxidative damaged LO2 hepatocytes</td>
<td align="left" valign="top">62.5&#x2013;1,000&#x2009;&#x03BC;g/mL for hydroxyl, O<sub>2</sub>-&#x2022;, NO<sub>2</sub>-, and DPPH radicals; 200, 400, 800, 1,000&#x2009;&#x03BC;g/mL for LO2 cell</td>
<td align="left" valign="top">Scavenging hydroxyl, O<sub>2</sub>-&#x2022;, NO<sub>2</sub>-, and DPPH radicals, the EC<sub>50</sub> was 964.59, 6376.84, 275.24, and 333.55&#x2009;&#x03BC;g/mL, respectively; LO2 cell survival rate, lactic dehydrogenase, mitochondrial membrane potential, GSH, SOD, CAT, Bcl-2 expression&#x2191;; ROS, MDA, Bax expression, PARP expression&#x2193;</td>
<td align="left" valign="top">Regulation of the mitochondria-mediated apoptosis signaling pathway</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref67">67</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PLP-0.1-I, PLP-0.2-I, PLP-0.3-I, PSMP-0.1-I, PSMP-0.2-I, PSMP-0.3-I</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">DPPH, hydroxyl, ABTS radicals</td>
<td align="left" valign="top">1&#x2013;5,000&#x2009;&#x03BC;g/mL for DPPH, hydroxyl, ABTS radicals, 1&#x2013;500&#x2009;&#x03BC;g/mL for reducing power assay</td>
<td align="left" valign="top">DPPH, hydroxyl, and ABTS radical-scavenging activities, and reducing power&#x2191;; ABTS scavenging ability was in the order of PLP-0.1-I &#x003E; PLP-0.2-I &#x003E; PLP-0.3-I &#x003E; PSMP-0.1-I &#x003E; PSMP-0.2-I &#x003E; PSMP-0.3-I</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref20">20</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">Antitumor</td>
<td align="left" valign="top">PFSP-2-1, PFSP-2-2</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">HepG2, Hep3b, SK-Hep-1 cells</td>
<td align="left" valign="top">100, 200, 400, 800, and 1,600&#x2009;&#x03BC;g/mL</td>
<td align="left" valign="top">Ability to inhibit the activity of HepG2, Hep3b, and SK-Hep-1 cells&#x2191;; HepG2, Hep3b, and SK-Hep-1 cells survival rates were 53.34%, 70.33%, 71.06% and 61.07%, 75.58%, 64.02% treating with 1600&#x03BC;g/mL of PFSP-2-1 and PFSP-2-2, respectively</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref53">53</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PFP</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">H22 hormonal mice</td>
<td align="left" valign="top">100 and 300&#x2009;mg/kg for 3&#x2009;weeks</td>
<td align="left" valign="top">Thymus index, tumor weight and volume, mitochondrial membrane potential&#x2193;; spleen index and tumor cell apoptosis&#x2191;</td>
<td align="left" valign="top">Interference with the transformation of cells in S phase</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref15">15</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PFSP-2</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">H22 hormonal mice</td>
<td align="left" valign="top">0.1, 0.3, 0.5&#x2009;mL (1&#x2009;mg/mL), 10&#x2009;days</td>
<td align="left" valign="top">LDH, aldolase, IL-10, Bcl-2 protein expression&#x2193;; IL-2, TNF-&#x03B1;, and proapoptotic protein Bax expression&#x2191;</td>
<td align="left" valign="top">Activates the ability of immune cells to produce cytokines</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref79">79</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">Hepatoprotective</td>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">ALD mice</td>
<td align="left" valign="top">0.3, 0.6&#x2009;g/kg for 60&#x2009;days</td>
<td align="left" valign="top">Hepatic fat cell degeneration score, ALT, AST, TG, TC, LDL, IL-1<italic>&#x03B2;</italic>, IL-6, TNF-&#x03B1;, MDA, and SREBP1c relative expression&#x2193;; HDL, SOD, GSH-Px, p-AMPK&#x03B1;/AMPK&#x03B1;, and SIRT1 relative expression&#x2191;</td>
<td align="left" valign="top">Activation of the SIRT1-AMPK signaling pathway, reduction in oxidative stress and inflammation in the body</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref19">19</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">Type 2 diabetic rat liver injury model</td>
<td align="left" valign="top">0.15, 0.30, 0.60&#x2009;g/kg for 28&#x2009;days</td>
<td align="left" valign="top">Fasting blood-glucose, liver index, TG, TC, AST, ALT, MDA, CRP, IL-6, TNF-&#x03B1;, Ac-FoxO1 expression&#x2193;; body mass, GSH-Px, CAT, SOD, SIRT1, FoxO1 expression&#x2191;</td>
<td align="left" valign="top">Inhibition of oxidative stress, anti-inflammation, modulation of SIRT1/ FoxO1 signaling pathway activation</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref73">73</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top"><italic>P. frutescens</italic> seed polysaccharide</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">CCl4-induced acute liver injury in mice</td>
<td align="left" valign="top">100, 300, 500&#x2009;mg/kg for 7&#x2009;days</td>
<td align="left" valign="top">ALT, AST, liver coefficient, spleen coefficient, and spotted necrotic lesions on the surface of the liver&#x2193;</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref29">29</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">Immunomodulatory</td>
<td align="left" valign="top">PFB-0, PFB-1, PFB-1-0, PFB-1-0-ii</td>
<td align="left" valign="top"><italic>In vitro</italic></td>
<td align="left" valign="top">Male ICR mice macrophages</td>
<td align="left" valign="top">1, 10, 100&#x2009;&#x03BC;g/mL</td>
<td align="left" valign="top">Macrophage lysosomal relative activities&#x2191;; stimulatory effect of four components on macrophage lysosomal enzyme activity in the order PFB-1-0-ii &#x003E; PFB-1-0 &#x003E; PFB-1 &#x003E; PFB-0</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref16">16</xref>)</td>
</tr>
<tr>
<td/>
<td align="left" valign="top">PFB-1-0, PFB-1</td>
<td align="left" valign="top"><italic>In vitro, in vivo</italic></td>
<td align="left" valign="top">Murine peritoneal macrophages</td>
<td align="left" valign="top">100&#x2009;&#x03BC;g/mL for in vitro test; 0.5, 0.75, 1.0 and 1.25&#x2009;g/kg for specific pathogen free ICR mice for 8&#x2009;days</td>
<td align="left" valign="top">Murine peritoneal macrophages lysosomal enzyme activity, mouse peritoneal macrophages phagocytic activity, nitric oxide, tumor necrosis factor, IL-6, and GMCSF&#x2191;</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref49">49</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">Anti-fatigue</td>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">5, 10, 20&#x2009;mg/kg for 4&#x2009;weeks</td>
<td align="left" valign="top">Exhaustive swimming induced exercise fatigue in mice</td>
<td align="left" valign="top">Liver glycogen, muscle glycogen, and exhaustion swimming time, blood glucose level&#x2191;</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref76">76</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">Anti-inflammatory</td>
<td align="left" valign="top"><italic>P. frutescens</italic> leaf polysaccharide</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">20&#x2009;mg/kg for 4&#x2009;weeks</td>
<td align="left" valign="top">Chronic obstructive pulmonary disease rat</td>
<td align="left" valign="top">Forced vital capacity, forced expiratory volume in one second, peak expiratory flow, and average alveolar number&#x2191;; IL-8, TNF-&#x03B1;, alveolar expansion, inflammatory cell infiltration, mean interalveolar lining, Wnt5a, RhoA, and p-JNK protein relative expression&#x2193;</td>
<td align="left" valign="top">Inhibition of the Wnt/PCP pathway</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref77">77</xref>)</td>
</tr>
<tr>
<td align="left" valign="top">Lipid-lowering</td>
<td align="left" valign="top">PFSP</td>
<td align="left" valign="top"><italic>In vivo</italic></td>
<td align="left" valign="top">50, 100, 200&#x2009;mg/kg for 8&#x2009;weeks</td>
<td align="left" valign="top">High-fat diet-induced mice</td>
<td align="left" valign="top">Liver index, AST, ALT, triglyceride, total cholesterol, low-density lipoprotein, blood glucose level, MDA, and <italic>CPT-1</italic> and <italic>ATGL</italic> mRNA expression&#x2193;; high-density lipoprotein, SOD, peroxidase, glutathione peroxide enzyme, and <italic>FAS</italic> mRNA expression&#x2191;</td>
<td align="left" valign="top">ND</td>
<td align="center" valign="top">(<xref ref-type="bibr" rid="ref78">78</xref>)</td>
</tr>
</tbody>
</table>
<table-wrap-foot>
<p>ND stands for not detected.</p>
</table-wrap-foot>
</table-wrap>
<p>First, studies have been carried out to extract <italic>P. Frutescens</italic> polysaccharides by HWE, MAE, UAE, enzyme-assisted extraction, and UAEE. There is still a great deal of scope for the development of other extraction methods. In particular, efforts should be focused on developing new environmentally friendly and high-yield extraction technologies. Second, the isolation and purification methods of <italic>P. Frutescens</italic> polysaccharides are currently limited to the laboratory level; thus, these methods should be further optimized to obtain reproducible and stable products that are suitable for industrial-scale production. Third, investigations on the structure of <italic>P. Frutescens</italic> polysaccharides have primarily focused on the monosaccharide composition and molecular weight. However, research is lacking on the more detailed structures of the sequence of monosaccharides, types, and positions of glycosidic bonds, and structural fragments. Various classical approaches, including FT-IR, NMR, X-ray diffraction, atomic force microscopy, and methylation analysis, can be applied to characterize the chemical structure of <italic>P. Frutescens</italic> polysaccharides. Fourth, to date, researchers have mostly focused on the antioxidant activity of <italic>P. Frutescens</italic> polysaccharides, with fewer studies on other bioactivities and their relationship with <italic>P. Frutescens</italic> polysaccharides structure. Therefore, it is imperative to establish more appropriate pharmacological models to investigate the additional biological activities of <italic>P. Frutescens</italic> polysaccharides and further study the structure-activity relationships of the polysaccharide, as well as the exact molecular mechanisms underlying the observed activities. Finally, research on <italic>P. Frutescens</italic> polysaccharides is still limited to theoretical studies in animal and <italic>in vitro</italic> experiments, with a notable lack of relevant clinical, toxicity, and pharmacokinetic studies. Developing and applying <italic>P. Frutescens</italic> polysaccharides as nutraceuticals or natural medicines is a huge challenge. Hence, a large number of clinical trials should be carried out to ensure the efficacy and safety of <italic>P. Frutescens</italic> polysaccharides toward creating novel products with <italic>P. Frutescens</italic> polysaccharides as the active ingredient.</p>
<p>In summary, <italic>P. Frutescens</italic> polysaccharides possess diverse biological functions, which offer extensive prospects in the fields of food and medicine; however, there is still a long way to go to achieve the transformation of <italic>P. Frutescens</italic> polysaccharides into practical functional foods, nutraceuticals, or natural medicines. We hope that more researchers will pay attention to PFP in the future and further investigate its precise structure, active mechanism, and clinical utilization, to offer a scientific foundation for its further development and utilization as functional foods and therapeutic drugs.</p>
</sec>
<sec sec-type="author-contributions" id="sec17">
<title>Author contributions</title>
<p>LZ: Validation, Writing &#x2013; original draft, Writing &#x2013; review &#x0026; editing. LG: Writing &#x2013; review &#x0026; editing. KW: Conceptualization, Writing &#x2013; review &#x0026; editing. CR: Data curation, Writing &#x2013; review &#x0026; editing. YG: Methodology, Writing &#x2013; review &#x0026; editing. JL: Investigation, Software, Writing &#x2013; review &#x0026; editing. SY: Project administration, Writing &#x2013; review &#x0026; editing. XZ: Formal analysis, Writing &#x2013; review &#x0026; editing. XY: Resources, Writing &#x2013; review &#x0026; editing. YZ: Methodology, Writing &#x2013; review &#x0026; editing. BL: Writing &#x2013; review &#x0026; editing. SL: Writing &#x2013; review &#x0026; editing.</p>
</sec>
</body>
<back>
<sec sec-type="funding-information" id="sec18">
<title>Funding</title>
<p>The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the National Key Research and Development Program of China (2021YFD2100902) and the Agricultural Science and Technology Innovation Project of Heilongjiang Academy of Agricultural Sciences (CX23GG16-04).</p>
</sec>
<sec sec-type="COI-statement" id="sec19">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="sec100" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
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