Pollen polysaccharides, methods for their isolation, and use

Abstract

The present invention belongs to the technical field of plant extract separation, and specifically relates to pollen polysaccharide 1-4, its separation method and use. The present invention extracts, separates and purifies the polysaccharide in rapeseed pollen to obtain pollen polysaccharide 1-4, and tests it. It has been shown that pollen polysaccharide 1-4 has a certain effect in the direction of promoting plant growth and stress tolerance. In addition, the present invention has found that the pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, and pollen polysaccharide active site Fr-1-5 of the rapeseed pollen crude separation components also have the effects of promoting plant growth and stress tolerance.

Description

[Technical Field] 【0001】 The present invention belongs to the technical field of plant extract separation, and more specifically relates to pollen polysaccharides 1, 2, 3, or 4, methods for separating them, and their uses. [Background technology] 【0002】 In the inventors' previous research (Patents CN113133454B and CN113133455B), rapeseed pollen extract was used to improve stress tolerance and promote growth in plants, including vegetables and fruit trees such as baby's lettuce, stem lettuce, lettuce, bok choy, wheat, chili peppers, tomatoes, citrus fruits, kiwis, cherries, pears, and apples. Analysis of its components revealed that it contains various components such as carbohydrates, proteins, amino acids, and lipids. However, the specific active ingredients have not yet been investigated. 【0003】 JPEG0007876054000001.jpg17170 【0004】 While rapeseed pollen extract may contain numerous substances that promote plant growth and stress tolerance, those skilled in the art currently cannot determine which substances play a dominant role in promoting plant growth and stress tolerance. Therefore, further research on rapeseed pollen extract is necessary. [Overview of the Initiative] 【0005】 Polysaccharides are complex sugar substances formed by the dehydration condensation of multiple monosaccharide molecules, with their constituent units linked by glycosidic bonds. Common types of glycosidic bonds include α-1,3 glycosidic bonds, β-1,6 glycosidic bonds, β-1,4 glycosidic bonds, α-1,4 glycosidic bonds, and β-1,3 glycosidic bonds. Polysaccharides are widely present in animals, plants, and microorganisms, and polysaccharides from different sources possess different biological activities. Current research suggests that plant polysaccharides have biological activities such as immunomodulation, antitumor, antioxidant, anti-aging, blood glucose lowering, and lipid lowering. They also have a wide range of sources, are biodegradable, highly safe, easily modifiable, and highly environmentally friendly, making them widely used in industries such as food, pharmaceuticals, livestock farming, and aquaculture. 【0006】 On the other hand, plant polysaccharides are a type of natural polymer with a complex structure, and currently there is little research on the specific molecular structure of pollen polysaccharides and their structural effectiveness regarding their physiological activity. Therefore, it is necessary to isolate and identify pollen polysaccharide compounds and provide theoretical and experimental evidence for future development research of pollen polysaccharide resources. 【0007】 In this invention, a novel pollen polysaccharide was obtained by extracting, separating, and purifying polysaccharides from rapeseed pollen. When this polysaccharide was tested, it was found to have a certain effect in both promoting plant growth and improving stress tolerance. Specifically, the present invention provides pollen polysaccharide 1, the main repeating structural unit shown below. JPEG0007876054000002.jpg48170 However, R1 is T-α-L-Araf-(1→5)-α-L-Araf-(1→ R2 is T-β-D-Galp-(1→6)-β-D-Galp-(1→ R3 is T-α-D-Glcp-(1→4)-α-D-Glcp-(1→). In the polysaccharide skeleton of the present invention, R1, R2, and R3 are attached to the 5th position of arabinose. 【0008】 In the present invention, Ara represents arabinose; Gal represents galactose; Glc represents glucose; f represents a furanose configuration; p represents a pyranose configuration; T represents the terminal (end group) of a polysaccharide molecule. Here, the molar content ratio of arabinose:galactose:glucose is 0.671:0.132:0.120. In the pollen polysaccharide of the present invention, in addition to the above main repeating structural units, it contains trace amounts of monosaccharide units such as fucose, xylose, mannose, etc. In the present invention, the average molecular weight of pollen polysaccharide 1 is 20 - 30 KDa, Furthermore, the average molecular weight of pollen polysaccharide 1 is 23 - 26 KDa, In the present invention, the average molecular weight of pollen polysaccharide 1 is 24 KDa - 25 KDa, for example 24774.22 Da. 【0009】 The infrared spectrum of pollen polysaccharide 1 of the present invention has at least 3342 cm -1 , 1641 cm -1 , 1536 cm -1 , 1440 cm -1 , 1147 cm -1 , 1103 cm -1 , 1076 cm -1 , 1311 cm -1 , 1241 cm -1 , 1027 cm -1 , 894 cm -1 , 873 cm -1 , 873 cm -1 including one or more absorption peaks at 873 cm. 【0010】 Here, 3342 cm -1 is the stretching vibration absorption peak of O - H and is a characteristic peak of saccharides. The absorption peak at 1641 cm -1 is attributed to crystal water; the absorption peak at 1536 cm -1 is attributed to the N - H bending vibration; the absorption peaks at 1440 cm -1 , 1147 cm -1 , 1103 cm -1 , 1076 cm -1The absorption peak at 1311cm is attributed to CO stretching vibrations; -1 , 1241cm -1 , 1027cm -1 The absorption peak at 894 cm is attributed to OH bending vibrations; -1 The absorption peak at 873 cm² is attributed to the CH bending vibration of the epimer of the β-terminal group of the pyran ring; -1 The absorption peak at this point is attributed to the CH bending vibrations of equatorial bonds other than CH of the epimer of the terminal group of the pyran ring. The present invention provides pollen polysaccharide 2, the main repeating structural unit shown below. JPEG0007876054000003.jpg25170 However, R1' is T-α-L-Araf-(1→5)-α-L-Araf-(1→, R2' is T-β-D-Galp-(1→6)-β-D-Galp-(1→ In the polysaccharide main chain of the present invention, R1' is attached to the 5th position of arabinose, and R2' is attached to the 6th position of galactose. 【0011】 In this invention, Ara represents arabinose; Gal represents galactose; f represents furanose; p represents pyranose; and T represents the end (terminal group) of a polysaccharide molecule. Here, the molar ratio of arabinose to galactose is 0.597:0.283. The pollen polysaccharide of the present invention contains, in addition to the main repeating structural units described above, trace amounts of monosaccharide units of fucose, glucose, xylose, and mannose. In this invention, the average molecular weight of pollen polysaccharide 2 is 5 to 15 kDa; Furthermore, the average molecular weight of pollen polysaccharide 2 is 9-11 kDa; In this invention, the average molecular weight of pollen polysaccharide 2 is 10 to 11 kDa, for example, 10718.15 Da. 【0012】 The infrared spectrum of the pollen polysaccharide 2 of the present invention is at least 3288m -1 , 2931 cm -1 , 2871cm -1 , 1641m-1 , 1548cm -1 , 1440cm -1 , 1402cm -1 , 1309cm -1 , 1243cm -1 , 1081cm -1 , 896cm -1 It includes one or more absorption peaks. 【0013】 Here, 3288cm -1 This is the stretching vibration absorption peak of OH, and is a characteristic peak of sugars. 2931cm -1 , 2871cm -1 There is one absorption peak, which can be attributed to CH stretching vibrations. 1641m -1 There is one absorption peak, which can be attributed to water of crystallization. 1548 cm -1 There is an absorption peak, which can be attributed to C=O stretching vibration. 1440cm -1 , 1402cm -1 There is an absorption peak at 1309cm, which can be attributed to CO stretching vibration. -1 , 1243cm -1 , 1081cm -1 There is an absorption peak, which can be attributed to OH bending vibration. 896cm -1 There is an absorption peak, which can be attributed to the CH bending vibration of the epimer of the β-terminal group of the pyran ring. The present invention provides pollen polysaccharide 3, the main repeating structural unit shown below. JPEG0007876054000004.jpg64170 【0014】 In the formula, Ara represents arabinose; Gal represents galactose; f represents the furanose configuration; p represents the pyranose configuration; and T represents the end (terminal group) of the polysaccharide molecule. Here, the molar ratio of arabinose to galactose is 0.328:0.399. 【0015】 In addition to the main repeating structural units mentioned above, the pollen polysaccharide of the present invention contains trace amounts of monosaccharides such as fucose, rhamnose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid. In this invention, the average molecular weight of pollen polysaccharide 3 is 60-70 kDa; Furthermore, the average molecular weight of pollen polysaccharide 3 is 65-70 kDa; In this invention, the average molecular weight of pollen polysaccharide 3 is 66-68 kDa, for example, 66911.38 kDa. 【0016】 The infrared spectrum of the pollen polysaccharide 3 of the present invention is at least 3426 cm⁻¹. -1 , 2939 cm -1 , 1734 cm -1 , 1619cm -1 , 1423cm -1 , 1145 cm -1 , 1091 cm -1 , 895cm -1 It includes one or more absorption peaks. 【0017】 The absorption zone for pollen polysaccharide 3 is 3600-3200 cm. -1 At this point, there is an absorption peak for the stretching vibration of the -OH group, and absorption peaks in this region are characteristic peaks of sugars. Specifically, at 3426 cm⁻¹, there is an absorption peak for the stretching vibration of the -OH group. -1 There is an OH stretching vibration absorption peak, which is a characteristic peak for sugars. 2939cm² -1 There is one absorption peak, which can be attributed to CH stretching vibration. 1734cm -1 The weak absorption peaks observed in the vicinity are attributed to the stretching vibration of the C=O carboxyl group, indicating that pollen polysaccharide 3 contains some uronic acid. 1619cm -1 There is one absorption peak, which can be attributed to water of crystallization. 1423cm -1 , 1145cm -1 , 1091cm -1 There is an absorption peak, which can be attributed to CO stretching vibration. 895cm -1 There is an absorption peak, which can be attributed to the CH bending vibration of the epimer of the β-terminal group of the pyran ring. The present invention provides a pollen polysaccharide 4 whose main repeating structural units are shown below. JPEG0007876054000005.jpg37170 Here, R1'' is T-α-L-Araf-(1→5)-α-L-Araf-(1→). 【0018】 In this invention, Ara represents arabinose; Gal represents galactose; f represents a furanose configuration; p represents a pyranose configuration; and T represents the end (terminal group) of a polysaccharide molecule. Here, the molar ratio of arabinose to galactose is 0.686:0.243. 【0019】 In addition to the main repeating structural units described above, the pollen polysaccharide 4 of the present invention contains trace amounts of monosaccharide units of fucose, glucose, xylose, mannose, and galacturonic acid. In this invention, the average molecular weight of pollen polysaccharide 4 is 5 to 15 kDa; Furthermore, the average molecular weight of pollen polysaccharide 4 is 9-11 kDa; In this invention, the average molecular weight of pollen polysaccharide 4 is 10-11 kDa, for example, 10328.089 kDa. 【0020】 The infrared spectrum of the pollen polysaccharide 4 of the present invention is at least 3322m -1 , 2931 cm -1 , 2875cm -1 , 1641m -1 , 1544cm -1 , 1407cm -1 , 1309cm -1 , 1241cm -1 , 1076cm -1 , 1047cm -1 , 894cm -1 It includes one or more absorption peaks. 【0021】 The infrared absorption band for pollen polysaccharide 4 is 3600-3200 cm². -1 At this point, there is an absorption peak for stretching vibrations of the -OH group, and absorption peaks in this region are characteristic peaks of sugars. Specifically, at 3322 cm⁻¹, there is an absorption peak for stretching vibrations of the -OH group. -1 This is the stretching vibration absorption peak of OH, and is a characteristic peak of sugars. 2931cm -1, 2875cm -1 There is one absorption peak, which can be attributed to CH stretching vibration. 1641cm -1 There is an absorption peak at 1544 cm², which can be attributed to water of crystallization. -1 There is an absorption peak, which can be attributed to C=O stretching vibration. 1407cm -1 There is one absorption peak, which can be attributed to CO stretching vibration. 1309cm -1 , 1241cm -1 , 1076cm -1 , 1047cm -1 There is an absorption peak, which can be attributed to OH bending vibration. 894cm -1 There is an absorption peak, which can be attributed to the CH bending vibration of the epimer of the β-terminal group of the pyran ring. 【0022】 In this invention, the values ​​of n1 to n5 depend on the molecular weight of the polysaccharide. The present invention further aims to provide a method for separating pollen polysaccharides, comprising the following steps. The above-mentioned method for separating pollen polysaccharides, wherein the method for separating pollen polysaccharide 1 is: (1) The rapeseed pollen polysaccharide extract is purified using a macroporous adsorption resin column and eluted with water to obtain the pollen polysaccharide active site Fr-1. (2) Pass Fr-1 through an ion exchange chromatography column and elute the pollen polysaccharide active site Fr-1 with water to obtain the pollen polysaccharide active site Fr-1-1. (3) Further purification of the pollen polysaccharide active site Fr-1-1 using a Sephadex chromatography column, and obtaining pollen polysaccharide 1 using an aqueous solution of ammonium bicarbonate as the eluent. Includes, The method for separating pollen polysaccharide 2 is: (1) The rapeseed pollen polysaccharide extract is purified using a macroporous adsorption resin column and eluted with water to obtain the primary purified pollen polysaccharide active site Fr-1. (2) The pollen polysaccharide active site Fr-1 is passed through an anion exchange chromatography column, and gradient elution is performed with a 0-0.025 mol / L NaCl solution. The 0.025 mol / L NaCl eluate is collected to obtain the pollen polysaccharide active site Fr-1-2. (3) Further purification of the pollen polysaccharide active site Fr-1-2 using a Sephadex chromatography column, and obtaining pollen polysaccharide 2 using an aqueous solution of ammonium bicarbonate as the eluent. Includes, The method for separating pollen polysaccharide 3 is: (1) The rapeseed pollen polysaccharide extract is purified using a macroporous adsorption resin column and eluted with water to obtain the primary purified pollen polysaccharide active site Fr-1. (2) The pollen polysaccharide active site Fr-1 is passed through an anion exchange chromatography column and gradient elution is performed with a 0-0.05 mol / L NaCl solution to collect the pollen polysaccharide in a 0.05 mol / L NaCl solution to obtain the pollen polysaccharide active site Fr-1-3. (3) Further purification of the pollen polysaccharide active site Fr-1-3 using a Sephadex chromatography column, and obtaining pollen polysaccharide 3 using an aqueous solution of ammonium bicarbonate as the eluent. Includes, The method for separating pollen polysaccharide 4 is: (1) The rapeseed pollen polysaccharide extract is purified using a macroporous adsorption resin column and eluted with water to obtain the primary purified pollen polysaccharide active site Fr-1. (2) The pollen polysaccharide active site Fr-1 is passed through an anion exchange chromatography column and gradient elution is performed with a 0-0.25 mol / L NaCl solution to collect the pollen polysaccharide in a 0.25 mol / L NaCl solution to obtain the pollen polysaccharide active site Fr-1-4. (3) Further purification of the pollen polysaccharide active site Fr-1-4 using a Sephadex chromatography column, and obtaining pollen polysaccharide 4 using an aqueous solution of ammonium bicarbonate as the eluent. Includes. 【0023】 The pollen polysaccharide active site Fr-1 applied to the ion exchange column may be in the form of the raw eluate of the macroporous resin, a concentrated raw eluate, or a re-solution obtained after concentrating and drying the raw eluate. 【0024】 The pollen polysaccharide active sites Fr-1-1, Fr-1-2, Fr-1-3, and Fr-1-4 subjected to gel chromatography may be the raw eluate from the ion exchange column, a concentrated raw eluate, or a re-solution obtained after concentrating and drying the raw eluate. 【0025】 The concentration and drying method in the present invention includes, but is not limited to, conventional operating methods such as reduced-pressure evaporation, atmospheric-pressure evaporation, drying, vacuum drying, thin-film drying, and freeze-drying, and can be used individually or in combination. 【0026】 The rapeseed pollen polysaccharide extract in step (1) is crude rapeseed pollen polysaccharide obtained by a conventional method. The conventional method described in the present invention includes, but is not limited to, water extraction and primary purification after water extraction. The aforementioned water extraction includes, but is not limited to, conventional plant extract extraction methods such as heat extraction, ultrasonic extraction, and microwave extraction. 【0027】 In addition, depending on the circumstances, degreasing and decolorization may be performed before extraction to remove impurities. For example, extraction, degreasing, and decolorization may be performed using lipid-soluble solvents such as ethanol or petroleum ether. The primary purification means after water extraction includes, but is not limited to, various means such as alcohol precipitation and chitosan removal. 【0028】 In the purification method, auxiliary means such as protein removal, decolorization, and small molecule removal may be selectively added in some cases, which is convenient for the subsequent polysaccharide concentration process. For example, protein removal agents such as phenol, trichloroacetic acid, and tannins can be used; decolorization can be performed with adsorbents such as cellulose, diatomaceous earth, and activated carbon; and small molecules can be removed by methods such as dialysis. In some specific embodiments of the present invention, the rapeseed pollen polysaccharide extract of step (1) is a crude pollen polysaccharide obtained after aqueous extraction and alcohol precipitation of rapeseed pollen. 【0029】 The aforementioned water-extraction alcohol precipitation refers to the process where ethanol is added to an aqueous extract to achieve a certain alcohol content, and a precipitate forms due to a decrease in the solubility of some component in the mixed solution. 【0030】 In the present invention, when employing the water extraction alcohol precipitation method, in the alcohol precipitation step, ethanol is added to the water extract (or concentrated water extract) until the ethanol concentration reaches 70-90% v / v, for example, selectively 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90% v / v, etc. In some embodiments of the present invention, the rapeseed pollen polysaccharide extract in step (1) refers to a heated rapeseed pollen water extract and a chitosan-free crude pollen polysaccharide. In some embodiments of the present invention, rapeseed pollen may be ultra-finely ground to facilitate the extraction of polysaccharides. In some embodiments of the present invention, a pollen polysaccharide extract of rapeseed pollen may be vacuum freeze-dried to produce a pollen crude polysaccharide solid. In a technical embodiment of the present invention, the macroporous resin column used in step (1) is a nonpolar column. 【0031】 In the present invention, the macroporous resin column includes, but is not limited to, the DB-101 macroporous resin column, the S-8 macroporous resin column, the AB-8 macroporous resin column, and the HP-20 macroporous resin column. Furthermore, the macroporous resin column may be selected from the HP-20 macroporous resin column. 【0032】 In some embodiments of the present invention, the primary purified pollen polysaccharide active site Fr-1 in step (1) can be made usable as a solid by dialysis and freeze-drying; and in step (2), the pollen polysaccharide active sites Fr-1-1, Fr-1-2, Fr-1-3, and Fr-1-4 can be made usable as a solid by dialysis and freeze-drying. In this invention, the molecular weight of the dialysis fraction is at least 3500 Da. 【0033】 In technical embodiments of the present invention, the ion exchange column used in step (2) is an anion exchange column and includes, but is not limited to, various ion exchange columns such as DEAE-cellulose, DEAE-agarose gel, and DEAE-glucose gel, and is further selected from DEAE cellulose-52 chromatography columns. 【0034】 In the technical embodiments of the present invention, the gel chromatography column used in step (3) includes, but is not limited to, different gel chromatography columns such as Sephadex G columns and polyacrylic gel Toyopearl HW columns, and can be selected from, for example, acrylic Sephadex S-400 HR chromatography columns and Sephadex LH-20 chromatography columns. In the present invention, in step (3), the concentration of the ammonium bicarbonate aqueous solution is 0.1 to 0.3 mol / L, preferably 0.2 mol / L. 【0035】 In some embodiments of the present invention, when the sugar content of step (2) and step (3) was detected using phenol-sulfuric acid, an absorbance value was detected at 490 nm. The present invention further provides pollen polysaccharide active site Fr-1, which is obtained by passing a rapeseed pollen polysaccharide extract through a macroporous resin and eluting it with water. 【0036】 In the present invention, the macroporous resin column is a nonpolar column; further, it is one selected from DB-101 macroporous resin column, S-8 macroporous resin column, AB-8 macroporous resin column, and HP-20 macroporous resin column; further, the macroporous resin column is selected from HP-20 macroporous resin column. In some embodiments of the present invention, the pollen polysaccharide active site Fr-1, purified using a macroporous resin column, can be solidified by dialysis and freeze-drying; Furthermore, the molecular weight of the dialysis fraction is at least 3500 Da. 【0037】 The present invention further provides rapeseed pollen polysaccharide active site Fr-1-5, which is obtained by purifying a rapeseed pollen polysaccharide extract using a macroporous resin, passing it through an ion exchange chromatography column, and eluting it with a 0-0.25 mol / L sodium chloride solution. 【0038】 In the present invention, the ion exchange chromatography column is an anion exchange chromatography column; further selected from DEAE cellulose chromatography columns. In some embodiments of the present invention, the pollen polysaccharide active site Fr-1-5, purified using a macroporous resin column, can be solidified by dialysis and freeze-drying; Furthermore, the molecular weight of the dialysis fraction is at least 3500 Da. 【0039】 In some embodiments of the present invention, the rapeseed pollen polysaccharide extract is crude pollen polysaccharide obtained by removing impurities from rapeseed pollen by ethanol degreasing, heated water extraction, and alcohol precipitation. 【0040】 The present invention further provides pollen polysaccharide active sites Fr obtained by extracting rapeseed pollen with water, taking the supernatant, concentrating it, and precipitating it with anhydrous ethanol. If a normal washing operation is performed after alcohol precipitation using an organic solvent such as ether or acetone, the product after washing also falls into the category of Fr. 【0041】 In the present invention, in the alcohol precipitation step, ethanol is added to the water extract (or concentrated water extract) until the ethanol concentration reaches 70-90% v / v, allowing for selection of concentrations such as 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90% v / v, etc. In the present invention, the degree of concentration during the concentration step can be determined by those skilled in the art depending on the amount of eluate added during elution and the final requirements of the product. 【0042】 The various extracts in this invention can exist in various forms, such as solids, liquids, and suspensions. The product form can be adjusted in the same way as before, according to the needs of actual production, sales, and use. 【0043】 The present invention also relates to the use of pollen polysaccharides 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active sites Fr-1-5 in the preparation of stress-tolerant plant products. The stress tolerance of plants according to the present invention includes cold tolerance, drought tolerance, high temperature tolerance, salt and alkali tolerance, and the like. In this invention, when used, the product containing pollen polysaccharides is applied to the leaves or roots of plants. In the present invention, when a pollen polysaccharide product is prepared in solution and used, the concentration of the main active ingredient can be selected according to the actual needs. 【0044】 For example, when using pollen polysaccharide 1-4, the concentration can be selected from 0.01 ppm to 500 ppm; 0.01 ppm to 100 ppm; 0.01 ppm to 50 ppm; 0.03 ppm, ... 0.05 ppm, ... 0.1 ppm, ... 0.2 ppm, ... 0.5 ppm, ... 1.0 ppm, ... 3.0 ppm, ... 5.0 ppm, ... 10 ppm, ... 20 ppm, ... 30 ppm, etc. 【0045】 For example, when using the pollen polysaccharide active site Fr, the concentration can be selected from 0.01 ppm to 500 ppm; 0.01 ppm to 100 ppm; 0.01 ppm to 50 ppm; 0.03 ppm, ... 0.05 ppm, ... 0.1 ppm, ... 0.2 ppm, ... 0.5 ppm, ... 1.0 ppm, ... 3.0 ppm, ... 5.0 ppm, ... 10 ppm, ... 20 ppm, ... 30 ppm, etc. 【0046】 For example, when using the pollen polysaccharide active site Fr-1, the concentration can be selected from 0.01 ppm to 500 ppm; 0.01 ppm to 100 ppm; 0.01 ppm to 50 ppm; 0.03 ppm, ... 0.05 ppm, ... 0.1 ppm, ... 0.2 ppm, ... 0.5 ppm, ... 1.0 ppm, ... 3.0 ppm, ... 5.0 ppm, ... 10 ppm, ... 20 ppm, ... 30 ppm, etc. 【0047】 For example, when using the pollen polysaccharide active site Fr-1-5, the concentration can be selected from 0.01 ppm to 500 ppm; 0.01 ppm to 100 ppm; 0.01 ppm to 50 ppm; 0.03 ppm, ... 0.05 ppm, ... 0.1 ppm, ... 0.2 ppm, ... 0.5 ppm, ... 1.0 ppm, ... 3.0 ppm, ... 5.0 ppm, ... 10 ppm, ... 20 ppm, ... 30 ppm, etc. 【0048】 The present invention also aims to enable the use of pollen polysaccharides 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5 in the production of plant growth promoting products. 【0049】 The plants in this invention include, but are not limited to, commercial crops and food crops such as Chinese cabbage, baby cabbage, stem lettuce, lettuce, bok choy, wheat, chili peppers, tomatoes, citrus fruits, kiwi, cherries, pears, apples, tobacco, etc. 【0050】 The aforementioned "economic crops" encompass a wide variety of crops, including, but are not limited to, fiber crops (e.g., cotton, hemp), oil crops (e.g., sesame, peanuts), sugar crops (e.g., sugarcane, beets), beverage crops (tobacco), medicinal crops, pigment crops, ornamental crops, fruits, and other economic crops. 【0051】 The aforementioned "food crops" include, but are not limited to, cereal crops (wheat, rice, corn), tuberous crops (including sweet potatoes, potatoes, etc.), and legume crops (including soybeans, broad beans, peas, mung beans, etc.). Furthermore, the present invention provides an agricultural product in which the active ingredient comprises pollen polysaccharide 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5. 【0052】 The aforementioned plant growth promotion includes promoting the germination of plants and promoting the growth of one or more of the following: roots, stems, leaves, flowers, and fruits. For example, it includes promoting an increase in root length, stem thickness, plant height, leaf width, leaf length, number of leaves, leaf area, biomass, chlorophyll content, and yield. 【0053】 In the present invention, pollen polysaccharides 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5 may be used as single components. However, for the convenience of product stabilization, transportation, and storage, conventionally known auxiliary agents such as dispersants, wetting agents, binders, emulsifiers, stabilizers, solvents, and embedding agents may be added to formulate the product into the corresponding dosage form. 【0054】 On the other hand, the pollen polysaccharides 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5 of the present invention can be used as synergistic agents in combination with pollen fertilizers, water-soluble fertilizers, compound fertilizers, pesticides, etc. The dosage forms of the formulations in this invention include, but are not limited to, common agricultural formulations such as emulsions, suspensions, wettable powders, powders, granules, wettable powders, mother liquor, and mother powder. [Effects of the Invention] 【0055】 The advantageous effects of the present invention are shown below. This invention identifies novel pollen polysaccharides 1-4, which are useful for improving plant stress tolerance and promoting plant growth, and isolates them from rapeseed powder for the first time. Furthermore, it was found that the pollen polysaccharide active sites Fr, Fr-1, or Fr-1-5 of the crudely isolated rapeseed pollen also possess plant growth-promoting and stress-tolerant effects. 【0056】 In this invention, "rapeseed" refers to a herbaceous crop of the Brassicaceae family, Brassica genus, and generally includes, but is not limited to, Chinese cabbage-type rapeseed, cabbage-type rapeseed, black mustard-type rapeseed, mustard-type rapeseed, and Abyssinian mustard. In this invention, "pollen" is obtained from the above-mentioned "rapeseed". [Brief explanation of the drawing] 【0057】 [Figure 1] This is the gradient elution curve of pollen crude polysaccharides using a DEAE cellulose-52 anion exchange column. [Figure 2] This is the elution curve for pollen polysaccharides 1-4 using an S-400 HR acrylic Sephadex column. [Figure 3] This is the standard molecular weight distribution curve for dextran. [Figure 4] These are HPGPC chromatograms of pollen polysaccharides 1-4. [Figure 5] Monosaccharide mixture and pollen polysaccharide ion chromatogram detection chromatograms (A: monosaccharide mixture, B: pollen polysaccharide 1, C: pollen polysaccharide 2, D: pollen polysaccharide 3, E: pollen polysaccharide 4), where the monosaccharide mixture standard is: 1. rhamnose (Rha) 2. fucose (Fuc) 3. arabinose (Ara) 4. xylose (Xyl) 5. mannose (Man) 6. glucose (Glc) 7. galactose (Gal) 8. glucuronic acid (GlcA) 9. galacturonic acid (GalA). Solvent peaks: the peak at 2.0 min is sodium hydroxide, and the peak at 40 min is sodium acetate. [Figure 6] This is a pollen polysaccharide 1-methylation analysis total ion chromatogram (TIC). [Figure 7] These are the mass spectra of each methylated PMAA belonging to pollen polysaccharide 1. [Figure 8] This is the 1H-NMR spectrum of pollen polysaccharide 1. [Figure 9] This is the 13C-NMR spectrum of pollen polysaccharide 1. [Figure 10]This is the DEPT-135 NMR spectrum of pollen polysaccharide 1. [Figure 11] This is the HSQC spectrum of pollen polysaccharide 1. [Figure 12] This is the COSY spectrum of pollen polysaccharide 1. [Figure 13] This is the TOCSY spectrum of pollen polysaccharide 1. [Figure 14] This is the HMBC spectrum of pollen polysaccharide 1. [Figure 15] This is the ultraviolet absorption spectrum of pollen polysaccharide 1. [Figure 16] This is the infrared absorption spectrum of pollen polysaccharide 1. [Figure 17] This is a separation scheme for pollen polysaccharides 1-4 and the pollen polysaccharide active sites Fr, Fr-1, and Fr-1-5. [Figure 18] This is a total ion chromatogram (TIC) of pollen polysaccharide 2-methylation analysis. [Figure 19] These are the mass spectra of each methylated PMAA belonging to pollen polysaccharide 2. [Figure 20] This is the 1H-NMR spectrum of pollen polysaccharide 2. [Figure 21] This is the 13C-NMR spectrum of pollen polysaccharide 2. [Figure 22] This is the DEPT-135 NMR spectrum of pollen polysaccharide 2. [Figure 23] This is the HSQC spectrum of pollen polysaccharide 2. [Figure 24] This is the COSY spectrum of pollen polysaccharide 2. [Figure 25] This is the TOCSY spectrum of pollen polysaccharide 2. [Figure 26] This is the HMBC spectrum of pollen polysaccharide 2. [Figure 27] This is the ultraviolet absorption spectrum of pollen polysaccharide 2. [Figure 28] This is the infrared absorption spectrum of pollen polysaccharide 2. [Figure 29] This is a total ion chromatogram (TIC) of pollen polysaccharide 3-methylation analysis. [Figure 30]These are the mass spectra of each methylated PMAA belonging to pollen polysaccharide 3. [Figure 31] This is the 1H-NMR spectrum of pollen polysaccharide 3. [Figure 32] This is the 13C-NMR spectrum of pollen polysaccharide 3. [Figure 33] This is the DEPT-135 NMR spectrum of pollen polysaccharide 3. [Figure 34] This is the HSQC spectrum of pollen polysaccharide 3. [Figure 35] This is the COSY spectrum of pollen polysaccharide 3. [Figure 36] This is the NOSEY spectrum of pollen polysaccharide 3. [Figure 37] This is the HMBC spectrum of pollen polysaccharide 3. [Figure 38] This is the ultraviolet absorption spectrum of pollen polysaccharide 3. [Figure 39] This is the infrared absorption spectrum of pollen polysaccharide 3. [Figure 40] This is a pollen polysaccharide 4-methylation analysis total ion chromatogram (TIC). [Figure 41] These are the mass spectra of each methylated PMAA belonging to pollen polysaccharide 4. [Figure 42] This is the 1H-NMR spectrum of pollen polysaccharide 4. [Figure 43] This is the 13C-NMR spectrum of pollen polysaccharide 4. [Figure 44] This is the DEPT-135 NMR spectrum of pollen polysaccharide 4. [Figure 45] This is the HSQC spectrum of pollen polysaccharide 4. [Figure 46] This is the COSY spectrum of pollen polysaccharide 4. [Figure 47] This is the TOCSY profile of pollen polysaccharide 4. [Figure 48] This is the HMBC spectrum of pollen polysaccharide 4. [Figure 49] This is the ultraviolet absorption spectrum of pollen polysaccharide 4. [Figure 50] This is the infrared absorption spectrum of pollen polysaccharide 4. [Modes for carrying out the invention] 【0058】 The following describes the technical embodiments of the present invention clearly and completely; however, naturally, the examples described are only some, not all, embodiments of the present invention. All other embodiments that can be obtained by those skilled in the art without creative effort based on the embodiments of the present invention are included within the scope of protection of the present invention. Where there are processes that are not described in particular detail below, those skilled in the art can implement or understand them by referring to the prior art. Unless the manufacturer is specified, the reagents or equipment used are considered to be common products available on the market. 【0059】 <Example 1> Preparation of pollen polysaccharide extract Rapeseed pollen was ultra-finely ground into a powder to obtain pre-treated pollen powder. 5.0 g of the pre-treated pollen powder was weighed, free monosaccharides were removed with ethanol, degreased and decolorized with petroleum ether, extracted with heated water, precipitated with alcohol, the precipitate was collected, and washed in the order of anhydrous ethanol, ethyl ether, and acetone. Finally, the washed precipitate was re-precipitated with water, pre-frozen at -20°C, and freeze-dried under vacuum to obtain hot water-extracted crude pollen polysaccharide. 【0060】 <Example 2> Preparation of pollen polysaccharide extract Rapeseed pollen was mixed with water, heated and extracted, filtered, chitosan was added to the filtrate, and the mixture was kept warm and allowed to stand. The resulting liquid after solid-liquid separation was the crude pollen polysaccharide extract. The extract was concentrated under reduced pressure, pre-frozen at -20°C, and then vacuum freeze-dried to obtain crude pollen polysaccharide. 【0061】 <Example 3> Preparation of pollen polysaccharide extract Rapeseed pollen was ultra-finely ground into a powder to obtain pre-treated pollen powder. 5.0 g of the pre-treated pollen powder was weighed, free monosaccharides were removed with ethanol, degreased and decolorized with petroleum ether, ultrasonically extracted, and precipitated with alcohol. The precipitate was collected and washed in the order of anhydrous ethanol, ethyl ether, and acetone. Finally, the washed precipitate was re-precipitated with water, pre-frozen at -20°C, and freeze-dried under vacuum to obtain hot water extracted crude pollen polysaccharide. 【0062】 <Example 4> Preparation of pollen polysaccharide active site Fr Rapeseed pollen was ultra-finely ground into a powder to obtain pre-treated pollen powder. 5.0 g of the pre-treated pollen powder was weighed out, free monosaccharides were removed with ethanol, degreased and decolorized with petroleum ether, extracted with heated water, and precipitated with alcohol. When precipitation with alcohol, the ethanol concentration in the solution was approximately 75% to 85%. The precipitate was collected to obtain the active site Fr of the crude pollen polysaccharide. 【0063】 <Example 5> Isolation and Purification of Pollen Polysaccharides The separation scheme for pollen polysaccharides 1-4 and the pollen polysaccharide active sites Fr, Fr-1, and Fr-1-5 is as follows. 【0064】 Step (1) Purification using HP-20 macroporous adsorption resin chromatography column: Pre-purification of crude pollen polysaccharide is performed using an HP-20 macroporous adsorption resin chromatography column (Φ4.0cm × 40cm). Specifically, a certain amount of crude pollen polysaccharide (in this example, crude pollen polysaccharide prepared by the method of Example 4 is used as an example to explain further separation and purification, but crude pollen polysaccharide obtained by other methods can be similarly applied to the separation and purification method of the present invention) is weighed, a certain amount of water is added to prepare a 10-20 mg / mL crude pollen polysaccharide solution, and 10 mL is loaded at a time. After adsorption on the HP-20 macroporous adsorption resin chromatography column for 3 hours, the column is washed with water up to 5 times the volume of the column, a sample is taken to obtain the pollen polysaccharide active site Fr-1, concentrated under reduced pressure at 48°C, dialyzed (3500 Da), and freeze-dried. 【0065】 Step (2) Separation and purification using DEAE cellulose-52 chromatography column: Pollen polysaccharide active site Fr-1 was separated and purified using a DEAE cellulose-52 chromatography column (Φ3.5cm × 30cm). The specific steps were: 1) Load 100 mg of pollen polysaccharide active site Fr-1 solution; 2) Elute the solution, and the eluates were, in order, water, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5 mol / L NaCl solutions, with a flow rate of 1.2 mL / min, and 6 mL was collected per vial; 3) The sugar content was measured by the phenol-sulfuric acid method, and the absorbance value at 490 nm was measured using a microplate reader. An elution curve was drawn with the number of collected vials on the x-axis and the absorbance value of the collected solution on the y-axis (Figure 1). Each NaCl elution peak was collected, and the same components were combined to obtain each fractional component of pollen polysaccharide (pollen polysaccharide active site Fr-1-1, pollen polysaccharide active site Fr-1-2, pollen polysaccharide active site Fr-1-3, pollen polysaccharide active site Fr-1-4). Pollen polysaccharide active site Fr-1-5 was obtained by collecting the elution sites in 0-0.25 mol / L NaCl solution. The above active sites were concentrated under reduced pressure at 48°C, dialyzed (3500 Da) to remove salts, and freeze-dried. 【0066】 As is clear from Figure 1, the crude pollen polysaccharide was passed through a DEAE cellulose-52 anion exchange column and gradient eluted with NaCl solutions of different concentrations, resulting in four elution peaks: the water-eluted fraction (pollen polysaccharide active site Fr-1-1), the 0.025 mol / L NaCl solution-eluted fraction (pollen polysaccharide active site Fr-1-2), the 0.05 mol / L NaCl solution-eluted fraction (pollen polysaccharide active site Fr-1-3), and the 0.25 mol / L NaCl solution-eluted fraction (pollen polysaccharide active site Fr-1-4). The eluates corresponding to each elution peak were collected to obtain the primary fractions of crude pollen polysaccharide: pollen polysaccharide active site Fr-1-1, pollen polysaccharide active site Fr-1-2, pollen polysaccharide active site Fr-1-3, and pollen polysaccharide active site Fr-1-4. These were then concentrated under reduced pressure, dialyzed, and freeze-dried. 【0067】 Step (3) Purification using S-400HR Acrylic Sephadex Chromatography Column: Finally, the pollen polysaccharide fractions were further purified using an Acrylic Sephadex S-400HR chromatography column (Φ1.0cm × 100cm). The specific steps were: 1) Each fraction solution was loaded with a sample volume of 20 mg; 2) Elution was performed, and 3 mL was collected per vial at a flow rate of 0.2 mL / min using 0.2 mol / L ammonium bicarbonate as the elution phase. Elution was continued until no sugar was detected, and 40 vials were collected from each fraction eluate; 3) The samples were collected, and the sugar content was detected every other vial using the phenol-sulfuric acid method. The absorbance value at 490 nm was detected using a microplate reader to create an elution curve, collect the elution peaks, combine the same fractions, concentrate under reduced pressure at 48°C, remove ammonia by dialysis (3500 Da), and freeze-dry to obtain the purified pollen polysaccharide fraction. 【0068】 Four fractions of pollen polysaccharide (active sites Fr-1-1, Fr-1-2, Fr-1-3, and Fr-1-4) separated using a DEAE cellulose-52 anion exchange column were further purified using an S-400HR Acrylic Sephadex chromatography column, and each of the four fractions was eluted with 0.2 mol / L ammonium bicarbonate. As is clear from Figure 2, after elution of the four fractions with 0.2 mol / L ammonium bicarbonate, a single elution peak was obtained for each, and the corresponding fractions were named fraction 1, fraction 2, fraction 3, and fraction 4. The eluates corresponding to the elution peaks were collected (fraction 1: 21-31 vials, fraction 2: 20-26 vials, fraction 3: 18-26 vials, fraction 4: 20-23 vials), concentrated under reduced pressure, dialysis, and freeze-dried to obtain four purified pollen polysaccharides. 【0069】 Identification of the purity of pollen polysaccharides and measurement of their molecular weight. Measurement method: High-performance liquid gel permeation chromatography (HPGPC) was used to identify the purity and measure the molecular weight of each purified fraction of pollen polysaccharides. Measurement conditions were as follows: Agilent 1260 series high-performance liquid chromatography, differential refractive index detector (RID), Shodex OHpak SB-804 HQ (7.8 mm × 300 mm) gel chromatography column, mobile phase 0.1 mol / L Na2SO4, flow rate 0.5 mL / min, column temperature 35°C, sample injection volume 20 μL, instrument measurement time 24 min. 【0070】 Preparation of standard curves: Dextran standards of different series with molecular weights (5900, 9600, 21100, 47100, 107000, 200000, 341400 Da) were dissolved in 0.1 mol / L Na2SO4 solution to prepare standard solutions with a concentration of 5 mg / mL. These solutions were filtered through a 0.22 μm aqueous filtration membrane and measured under HPGPC measurement conditions, with the retention time recorded. A standard curve was created with retention time (min) on the x-axis and the logarithm of the dextran standard molecular weight (LogMW) on the y-axis. When this curve was applied to a regression equation to calculate the molecular weights of different pollen polysaccharides, the standard curve y = -0.322x + 9.4365 was obtained, as specifically shown in Figure 3. 【0071】 Measurement of the purity and molecular weight of pollen polysaccharides: 10 mg of purified pollen polysaccharide sample was dissolved in 2 mL of 0.1 mol / L Na2SO4 solution and thoroughly dissolved to prepare a polysaccharide solution with a concentration of 5 mg / mL. After filtration through a 0.22 μm aqueous filtration membrane, the solution was analyzed under HPGPC measurement conditions, and the chromatogram of each pollen polysaccharide was recorded. The purity of each polysaccharide sample was evaluated based on the number of peaks and peak symmetry on the chromatogram, and the molecular weight was calculated by comparing the retention time on the chromatogram of each polysaccharide sample with the dextran standard molecular weight curve. 【0072】 Figure 4 shows the results of measuring the purity and molecular weight of pollen polysaccharides 1, 2, 3, and 4 by the HPGPC method. As is clear from Figure 4, a single symmetrical peak appears in the HPGPC chromatograms of all four pollen polysaccharides, suggesting that the four types of pollen polysaccharides are highly pure. This result is consistent with the separation and purification results obtained using an S-400HR acrylic Sephadex column, and further explains that an established method for separating and purifying pollen polysaccharides is possible, and that highly pure purified pollen polysaccharides can be produced. Based on a previously established molecular weight standard curve, the average molecular weights of each pollen polysaccharide were calculated by comparing the peak times of pollen polysaccharides 1-4, which were 24774.22 Da, 10718.15 Da, 66911.38 Da, and 10328.089 Da, respectively. 【0073】 <Example 6> Structural identification of pollen polysaccharides 1. Measurement results of pollen polysaccharide monosaccharide composition Measurement Method: The monosaccharide composition of pollen polysaccharide samples was measured by ion chromatography (IC). 10 mg of the polysaccharide sample was precisely weighed into an ampoule for acid hydrolysis. The acid hydrolysis solution was accurately aspirated and transferred to a test tube, dried by blowing nitrogen, 5 mL of distilled water was added and mixed by vortexing, 50 μL was aspirated and added to 950 μL of distilled water, centrifuged at 12000 r / min for 5 minutes, and the supernatant was taken for IC analysis. Sixteen monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, aminogalactose hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, glucuronic acid, mannuronic acid) were simultaneously prepared in standard mother liquor solutions. The precise concentration standards of each monosaccharide standard solution were taken as mixed standards. The monosaccharide composition is determined based on the peak times in chromatography of hydrolyzed pollen polysaccharide samples. The masses of different monosaccharides are determined according to an absolute quantification method, and the molar ratio is calculated from the molar mass of the monosaccharides, with C(standard) / A(standard) = C(sample) / A(sample). C is the concentration, and A is the peak area. Chromatography conditions: DionexCarbopacTMPA20 (3*150); mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH and 100 mM sodium acetate; flow rate: 0.3 mL / min; sample injection volume: 5 μL; column temperature: 30°C; detector: electrochemical detector. As shown in Figure 5(A), ion chromatography can separate and detect the chromatographic peaks of 16 standard monosaccharides. The peaks are regular, the separation is high, and it is suitable for the simultaneous detection of common monosaccharide species in monosaccharide composition analysis. 【0074】 Monosaccharide composition of pollen polysaccharide 1 Figure 5(B) shows the ion chromatogram for detecting the monosaccharide composition of pollen polysaccharide 1. In Figure 5(B), no peak corresponding to uronic acid is detected, suggesting that pollen polysaccharide 1 does not contain uronic acid and is a neutral sugar. From Figure 5(B), it can be seen that pollen polysaccharide 1 is mainly composed of arabinose, galactose, and glucose, with trace amounts of xylose, fucose, and mannose. Calculating the molar ratio of the monosaccharide composition, we find that fucose:arabinose:galactose:glucose:xylose:mannose is 0.008:0.671:0.132:0.120:0.011:0.046 (Table 1). 【0075】 Monosaccharide composition of pollen polysaccharide 2 As shown in Figure 5(C), no peak corresponding to uronic acid was detected in pollen polysaccharide 2, suggesting that pollen polysaccharide 2 does not contain uronic acid and is a neutral sugar. Pollen polysaccharide 2 mainly consists of arabinose and galactose. Calculated from the correlated chromatogram peak areas, the ratios of fucose:arabinose:galactose:glucose:xylose:mannose in pollen polysaccharide 2 are 0.010:0.597:0.283:0.048:0.007:0.038 (Table 2). 【0076】 Monosaccharide composition of pollen polysaccharide 3 As shown in Figure 5(D) of JPEG0007876054000008.jpg58170, galacturonic acid and glucuronic acid were detected, suggesting that pollen polysaccharide 3 is an acidic sugar. Pollen polysaccharide 3 mainly contains arabinose and galactose. Calculated from the correlated chromatogram peak areas, the ratio of fucose:rhamnose:arabinose:galactose:glucose:xylose:mannose:galacturonic acid:glucuronic acid in pollen polysaccharide 3 is 0.006:0.095:0.328:0.399:0.024:0.027:0.010:0.096:0.014 (Table 3). 【0077】 Monosaccharide composition of pollen polysaccharide 4 As shown in Figure 5(E), a small amount of galacturonic acid was detected, suggesting that pollen polysaccharide 4 contains uronic acid and is an acidic sugar. There are two distinct spectral peaks, arabinose and galactose, from left to right. Calculated from the correlated chromatogram peak areas, the ratio of fucose:arabinose:galactose:glucose:xylose:mannose:galacturonic acid in pollen polysaccharide 4 is 0.003:0.686:0.243:0.014:0.021:0.013:0.009 (Table 4). 【0078】 2. Analysis of Pollen Polysaccharide Methylation and Nuclear Magnetic Resonance Results 2.1 Structure of pollen polysaccharide 1 2.1.1 Methylation Measurement Results To investigate the primary structure of pollen polysaccharide 1, it was methylated using the modified Needs method. However, since pollen polysaccharide 1 is a neutral sugar, uronic acid reduction was not required, and an RXI-5 SIL MS capillary column was used for gas analysis. The structure and content of related monosaccharides were detected by gas analysis, and the glycosidic bond sites related to monosaccharides were determined. 【0079】 Fully methylated pollen polysaccharide 1 was converted to partially methylated glycol acetate derivatives (PMAAs) by hydrolysis, reduction, derivatization, etc., and the total ion chromatogram of the methylation analysis of pollen polysaccharide 1 was obtained by GCMS analysis as shown in Figure 6. The mass spectra corresponding to the methylated glycosyl peaks in the figure (Figure 7) were correlated with a search of the standard spectral library (Standard spectral library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / ms / PMAA / pframe.html) to determine the type of partially methylated glycosyl. At the same time, the relative molar ratio was calculated from the peak area of ​​the chromatogram peaks, and together with the measurement results of the monosaccharide composition, structural information necessary for estimating the repeating unit structure of pollen polysaccharide 1 can be provided. 【0080】 Results of Methylation Analysis of Pollen Polysaccharide 1 JPEG0007876054000010.jpg541702,3-Me2-Araf is 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-arabinitol (1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-arabitol), and by analogy. 【0081】 Pollen polysaccharide 1 mainly contains arabinose, galactose, and glucose, and the contents of xylose, fucose, and mannose are too low. Therefore, in methylation analysis, only the PMAAs of arabinose, galactose, and glucose were detected, and the PMAAs of xylose, fucose, and mannose were not detected. However, since the contents of xylose, fucose, and mannose are low, they do not affect the analysis and determination of the main glycosidic bond linkage forms of pollen polysaccharide 1. As shown in Table 5, in pollen polysaccharide 1, arabinose exists in three linkage forms: terminal sugar, 1,5-Araf, and 1,3,5-Araf; galactose exists in two linkage forms: terminal sugar and →6)-Galp-(1→; glucose exists in two linkage forms: terminal sugar and →4)-Glcp-(1→. From the linkage forms of the sugar residue glycosidic bonds of pollen polysaccharide 1 and the characteristics of the pollen polysaccharide structure, there is a possibility that the main chain of pollen polysaccharide 1 is linked to other glycosidic bonds through 1,3,5-Araf; the branched chain is relatively complex and may mainly consist of 1,5-Araf, →6)-Galp-(1→, and →4)-Glcp-(1→. 【0082】 2.1.2 NMR Measurement Results of Pollen Polysaccharide 1 Monosaccharide Residues in Pollen Polysaccharide 1 1 H and 13 C NMR Chemical Shift Assignments JPEG0007876054000011.jpg40170 【0083】 For pollen polysaccharide 1 1 H-NMR spectrum (Figure 8), 13As shown in the figures, the 1C-NMR spectrum (Figure 9) and the DEPT-135 spectrum (Figure 10) allow us to estimate basic information such as the configuration type of glycosidic bonds and the type of monosaccharide composition from the terminal hydrogens and terminal carbons in one-dimensional NMR spectra. 1 In the 1H-NMR spectrum, five more distinct hydrogen signal peaks with chemical shifts of 5.18 ppm, 5.02 ppm, 4.95 ppm, 4.46 ppm, and 4.42 ppm are present in the terminal hydrogen-related region, suggesting that pollen polysaccharide 1 contains not only α-glycosidic bonds but also β-glycosidic bonds. Based on the monosaccharide composition and methylation detection results, as well as relevant references, it can be confirmed that 5.18 ppm and 5.02 ppm belong to the terminal proton hydrogen signals of arabinose residues, and 4.95 ppm is the terminal hydrogen signal of galactose residues. The HSQC spectrum reflects the ortho-position correlation of carbon and hydrogen in the polysaccharide glycosidic bonds. 13 The position of terminal carbons can be determined in conjunction with the 1C-NMR spectrum. The TOCSY spectrum is a two-dimensional nuclear magnetic spectrum related to the hydrogen of a monosaccharide residue, which can be used to identify the position of hydrogens on the monosaccharide residue, and the COSY spectrum is a spectrum related to the ortho-hydrogen on the monosaccharide residue. Together with the chemical shift of the terminal group proton and the associated two-dimensional spectrum, the hydrocarbons of individual monosaccharides can be assigned. 【0084】 During the monosaccharide composition analysis, the presence of xylose and mannose was detected. While they may be present during methylation, their content is relatively low, and the related carbon and hydrogen absorption peaks in the one-dimensional nuclear magnetic spectrum overlap considerably with those of the same monosaccharide in other linked forms, making differentiation difficult. Therefore, the possible types of glycosidic bonds in this region were not assigned during the methylation and nuclear magnetic analysis. However, due to their low content, this does not affect the estimation of the main constituent units of pollen polysaccharide 1. The assignment of carbon and hydrogen to the main monosaccharide residues in pollen polysaccharide 1 is as follows: 1 H-NMR spectrum and 13The two-dimensional spectra of HSQC (Figure 11), COSY (Figure 12), and TOCSY (Figure 13) obtained by 13C-NMR spectroscopy, combined with relevant literature reports and methylation detection results, are shown in Table 6. 【0085】 Since the HMBC two-dimensional nuclear magnetic spectrum reflects the spatial correlation between carbon and hydrogen atoms, the linkage order of monosaccharide residues can be estimated from the correlated two-dimensional spectrum. As is clear from Figure 14, the HMBC spectrum contains multiple cross-peaks at the carbon and hydrogen atoms associated with each monosaccharide residue, and by analyzing these, the linkage status between related monosaccharide residues can be identified. 【0086】 Nuclear magnetic analysis reveals the carbon and hydrogen assignments of the relevant monosaccharide residues in pollen polysaccharide 1. The results of the analysis of the linkage configuration of these monosaccharide residues closely match those of the methylation structure analysis, allowing for the determination of the relevant glycosidic bond configuration. Analysis of this two-dimensional spectrum determines the linkage order of different monosaccharide residues, effectively elucidating the molecular structural characteristics of pollen polysaccharide 1. Based on the results of methylation and nuclear magnetic analysis, the main chain structure of pollen polysaccharide 1 is mainly composed of linked α-L-1,3,5-Araf, and the branched chains are mainly composed of α-L-1,5-Araf, β-D-1,6)-Galp, and β-D-1,4)-Glcp. The main repeating structural units of pollen polysaccharide 1 are as follows. JPEG0007876054000012.jpg44170 However, R1 is T-α-L-Araf-(1→5)-α-L-Araf-(1→, R2 is T-β-D-Galp-(1→6)-β-D-Galp-(1→, R3 is T-α-D-Glcp-(1→4)-α-D-Glcp-(1→ 【0087】 2.1.3 Ultraviolet Spectrum Measurement Results As shown in Fig. 15, pollen polysaccharide 1 has no characteristic absorption peak in the visible light region. The main reason is that there is a lack of corresponding chromogenic groups in the molecular structure of pollen polysaccharide, which is also a factor that makes it difficult to quickly qualitatively and quantitatively detect polysaccharides. In the ultraviolet region, no absorption peak is observed at either 280 nm or 260 nm, indicating that pollen polysaccharide 1 is a simple polysaccharide with high purity. 【0088】 2.1.4 Infrared Spectroscopy Measurement Results Fig. 16 shows the typical infrared spectrum of plant polysaccharide substances. Since plant polysaccharides are high molecular compounds in which monosaccharides rich in hydroxyl groups are linked by glycosidic bonds, pollen polysaccharides all have broad and strong absorption peaks at 3400 - 3500 cm -1 As shown in Fig. 16, the absorption band of pollen polysaccharide 1 at 3600 - 3200 cm -1 is the absorption peak of the stretching vibration of -OH. The absorption peak in this region is a characteristic peak of saccharides. Specifically, there is an absorption peak of the stretching vibration of O-H at 3342 cm -1 , which is a characteristic peak of saccharides. There is an absorption peak at 1641 cm -1 , which can be attributed to crystal water. There is an absorption peak at 1536 cm -1 , which can be attributed to the bending vibration of N-H. Absorption peaks are observed at 1440 cm -1 , 1147 cm -1 , 1103 cm -1 , 1076 cm -1 , which can be attributed to the stretching vibration of C-O. Absorption peaks are observed at 1311 cm -1 , 1241 cm -1 , 1027 cm -1 , which can be attributed to the bending vibration of O-H. There is an absorption peak at 894 cm -1 , which can be attributed to the bending vibration of C-H of the epimer of the β-terminal group of the pyran ring. There is an absorption peak at 873 cm -1 , which can be attributed to the bending vibration of C-H of the equatorial bond other than C-H of the epimer of the terminal group of the pyran ring. The separation scheme of each component is shown in Fig. 17. 【0089】 2.2 Structure of Pollen Polysaccharide 2 2.2.1 Methylation Measurement Results Pollen polysaccharide 2 mainly contains arabinose and galactose, and the content of glucose, xylose, fucose, and mannose is too low. Therefore, methylation analysis detected only PMAAs of arabinose and galactose, and PMAAs of glucose, xylose, fucose, and mannose were not detected. However, the low content of glucose, xylose, fucose, and mannose does not affect the analytical determination of the main glycosidic bond configuration of pollen polysaccharide 2. Figure 18 shows the total ion chromatogram obtained by GC-MS after methylation, hydrolysis, and derivatization of pollen polysaccharide 2. From the figure, mainly six methylation-induced glycosyl group ion fragments were selected, and the mass spectra corresponding to the methylated glycosyl peaks in the figure (Figure 19) were correlated and matched with a search of the standard spectral library (Standard spectral library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / MS / pmaa / pframe.html). Combined with the monosaccharide composition measurement results, the correlation linkage morphology of individual monosaccharide residues was estimated, providing information necessary for estimating the repeating unit structure of pollen polysaccharide 2. 【0090】 Results of pollen polysaccharide dimethylation analysis JPEG0007876054000013.jpg30170JPEG0007876054000014.jpg25170 Note: 2,3-Me2-Araf is 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-arabinitol, and can be inferred sequentially. 【0091】 As shown in Table 7, the pollen polysaccharide 2 methylation analysis sample mainly contained six glycosylion fragments, each derived from galactose and arabinose. Galactose mainly existed in the form of →3,6)-Galp-(1→ linkage, and arabinose mainly existed in the forms of →3,5)-Araf-(1→ and →5)-Araf-(1→). Based on the linkage configuration of the sugar residue glycosidic bonds of pollen polysaccharide 2 and the characteristics of the pollen polysaccharide structure, it can be tentatively inferred that the pollen polysaccharide 2 main chain is linked to other glycosidic bonds via 1,3,5-Araf and 1,3,6-Galp linkages; and the branched chain may mainly consist of 1,5-Araf and 1,6-Glcp. 【0092】 2.2.2 Results of Nuclear Magnetic Resonance NMR Measurement of Pollen Polysaccharide 2 Monosaccharide residues of pollen polysaccharide 2 1 H and 13 Chemical shift assignment of 1C NMR JPEG0007876054000015.jpg32170 【0093】 Figures 20-22 show pollen polysaccharide 2. 1 H-NMR spectrum, 13 The C-NMR spectrum and DEPT-135 spectrum are shown. 1 In the 1H-NMR spectrum, the presence of five more prominent signal peaks in the terminal hydrogen-related region was observed, indicating that pollen polysaccharide 2 has five monosaccharide residues with corresponding chemical shifts of 5.18 ppm, 5.08 ppm, 4.83 ppm, 4.41 ppm, and 4.46 ppm, respectively (Table 8). The chemical shifts in the range of 4.30–3.50 ppm represent the hydrogen shifts corresponding to the 2nd to 6th carbon atoms of the five corresponding sugar residues. The chemical shifts of the terminal carbons corresponding to the glycosidic bond terminal hydrogens were observed in the HSQC spectrum and 13This can be determined from the 1C-NMR spectrum. HSQC (Figure 23), COSY (Figure 24), and TOCSY (Figure 25) are two-dimensional spectral diagrams showing the hydrocarbon assignment and ortho-hydrogen correlation and total hydrogen correlation at the same monosaccharide residue. Analysis allows for the assignment of other carbons and hydrogens at the correlated monosaccharide residue. HMBC spectra (Figure 26) allow for the determination of the spatial correlation between carbons and hydrogens, and further, the linkage order of monosaccharide glycosidic bonds can be determined. 【0094】 From methylation analysis and correlated nuclear magnetochemistry, it can be determined that pollen polysaccharide 2 forms the main chain by linking arabinose to galactose via α-L-1,3,5-Araf and β-D-1,3,6-Galp, with branching groups linked to the 5th position of arabinose and the 6th position of galactose. The branching of the linked branching groups mainly consists of α-L-1,5-Araf and β-D-1,6-Gal, and the main repeating structural units of pollen polysaccharide 2 are as follows. JPEG0007876054000016.jpg26170However, R1' is T-α-L-Araf-(1→5)-α-L-Araf-(1→ R2' is T-β-D-Galp-(1→6)-β-D-Galp-(1→). 【0095】 2.2.3 Ultraviolet Spectrum Measurement Results As shown in Figure 27, pollen polysaccharide 2 does not have a characteristic absorption peak in the visible light region. In the ultraviolet region, no absorption peaks are observed at either 280 nm or 260 nm, indicating that pollen polysaccharide 2 is a simple polysaccharide with high purity. 【0096】 2.2.4 Infrared Spectroscopic Measurement Results As shown in Figure 28, the absorption band of pollen polysaccharide 2 is 3600-3200 cm². -1 At this point, the absorption peak of the stretching vibration of -OH is present, and absorption peaks in this region are characteristic peaks of sugars. Specifically, 3288m -1 This is the stretching vibration absorption peak of OH, and is a characteristic peak of sugars. 2931cm -1 , 2871cm -1There is one absorption peak, which can be attributed to CH stretching vibrations. 1641m -1 There is an absorption peak at 1548 cm², which can be attributed to water of crystallization. -1 There is an absorption peak, which can be attributed to C=O stretching vibration. 1440cm -1 , 1402cm -1 There is an absorption peak at 1309cm, which can be attributed to CO stretching vibration. -1 , 1243cm -1 , 1081cm -1 There is an absorption peak at 896 cm, which can be attributed to OH bending vibration. -1 There is an absorption peak, which can be attributed to the CH bending vibration of the epimer of the β-terminal group of the pyran ring. 【0097】 2.3 Structure of pollen polysaccharide 3 2.3.1 Methylation Measurement Results To investigate the primary structure of pollen polysaccharide 3, pollen polysaccharide 3 was methylated using a modified Needs method. Fully methylated pollen polysaccharide 3 was converted to partially methylated glycol acetate derivatives (PMAAs) by hydrolysis, reduction, derivatization, etc., and the total ion chromatogram of the methylation analysis of pollen polysaccharide 3 was obtained by GC-MS analysis as shown in Figure 29. The mass spectra corresponding to the methylated glycosyl peaks in the figure (Figure 30) were correlated with a search of the standard spectral library (Standard spectral library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / ms / pmaa / pframe.html) to determine the type of partially methylated glycosyl group. At the same time, the relative molar ratio was calculated from the peak area of ​​the chromatogram peaks, and together with the measurement results of the monosaccharide composition, structural information necessary for estimating the repeating unit structure of pollen polysaccharide 3 can be provided. 【0098】 Results of pollen polysaccharide trimethylation analysis JPEG0007876054000017.jpg371702,3-Me2-Araf is 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-arabinitol, and can be inferred sequentially. 【0099】 Pollen polysaccharide 3 mainly contains arabinose and galactose, and the content of rhamnose, glucose, xylose, fucose, and mannose is too low. Therefore, methylation analysis detected only PMAAs of arabinose and galactose, while PMAAs of rhamnose, glucose, xylose, fucose, and mannose were not detected. However, the low content of rhamnose, glucose, xylose, fucose, and mannose does not affect the analytical determination of the main glycosidic bond configuration of pollen polysaccharide 3. Total ion chromatograms were detected by GC-MS, and mainly five types of methylation-induced glycosylated ion fragments were selected from Figure 30. 【0100】 As shown in Table 9, the pollen polysaccharide 3 methylation analysis sample mainly contained five glycosylion fragments, derived from galactose and arabinose, respectively. Galactose mainly existed in the form of →3,6)-Galp-(1→ and →3)-Galp-(1→, and arabinose mainly existed in the form of →3,5)-Araf-(1→ and →5)-Araf-(1→. Based on the linkage of the sugar residue glycosidic bonds of pollen polysaccharide 3 and the characteristics of the pollen polysaccharide structure, it can be tentatively inferred that the pollen polysaccharide 3 main chain is linked to other glycosidic bonds via →3,6)-Galp-(1→ and →3)-Galp-(1→ linkages; and the branched chain may mainly consist of →3,5)-Araf-(1→ and →5)-Araf-(1→. 【0101】 2.3.2 Results of Nuclear Magnetic Resonance NMR Measurement of Pollen Polysaccharide 3 Monosaccharide residues of pollen polysaccharide 3 1 H and 13 Chemical shift assignment of 1C NMR JPEG0007876054000018.jpg46170 【0102】 Figures 31, 32, and 33 show pollen polysaccharide 3. 1 H-NMR spectrum, 13 The 1C-NMR spectrum and DEPT-135 spectrum are shown, revealing six prominent signal peaks in the terminal hydrogen-related region, indicating that pollen polysaccharide 3 has six monosaccharide residues, with corresponding chemical shifts of 5.17 ppm, 5.15 ppm, 5.09 ppm, 4.83 ppm, 4.58 ppm, and 4.40 ppm, respectively. The chemical shifts in the range of 4.30–3.50 ppm represent the hydrogens corresponding to the 2nd to 6th carbon atoms of the six corresponding sugar residues. The chemical shifts of the terminal carbons corresponding to the glycosidic bond terminal hydrogens are shown in the HSQC spectrum and 13 This can be determined from the 1C-NMR spectrum. HSQC (Figure 34), COSY (Figure 35), and NOSEY (Figure 36) are two-dimensional spectral diagrams showing the assignment of hydrocarbons and ortho-hydrogen correlations and total hydrogen correlations at the same monosaccharide residue. Analysis allows for the assignment of other carbons and hydrogens at the correlated monosaccharide residue. HMBC spectra (Figure 37) allow for the determination of the spatial correlation between carbons and hydrogens, and further, the linkage order of monosaccharide glycosidic bonds can be determined. 【0103】 From methylation analysis and correlated nuclear magnetochemistry results, it can be determined that pollen polysaccharide 3 forms the main chain by linking from galactose via β-D-1,3,6-Galp and β-D-1,3-Galp, and that the branching groups that link mainly consist of α-L-1,3,5-Araf and α-L-1,5-Araf, and that the main repeating structural units of pollen polysaccharide 3 are as follows. JPEG0007876054000019.jpg57170 【0104】 2.3.3 Ultraviolet Spectrum Measurement Results As shown in Figure 38, pollen polysaccharide 3 does not have a characteristic absorption peak in the visible light region. In the ultraviolet region, no absorption peaks are observed at either 280 nm or 260 nm, indicating that pollen polysaccharide 3 is a simple polysaccharide with high purity. 【0105】 2.3.4 Infrared Spectroscopic Measurement Results Figure 39 shows a typical infrared spectrum of plant polysaccharides, with the absorption band for pollen polysaccharide 3 being 3600–3200 cm⁻¹. -1 There is an absorption peak for the stretching vibration of the -OH group, and absorption peaks in this region are characteristic peaks of sugars. Specifically, at 3426 cm⁻¹, there is an absorption peak for the stretching vibration of the -OH group. -1 There is an OH stretching vibration absorption peak, which is a characteristic peak for sugars. 2939cm² -1 There is an absorption peak at 1734cm, which can be attributed to CH stretching vibration. -1 The weak absorption peaks observed in the vicinity can be attributed to the stretching vibration of the C=O carboxyl group, indicating that pollen polysaccharide 3 contains some uronic acid. -1 There is an absorption peak at 1423 cm², which can be attributed to water of crystallization. -1 , 1145cm -1 , 1091cm -1 There is an absorption peak, which can be attributed to CO stretching vibration. 895cm -1 There is an absorption peak, which can be attributed to the CH bending vibration of the epimer of the β-terminal group of the pyran ring. 【0106】 2.4 Structure of pollen polysaccharide 4 2.4.1 Methylation Measurement Results To investigate the primary structure of pollen polysaccharide 4, pollen polysaccharide 4 was methylated using a modified Needs method. Fully methylated pollen polysaccharide 4 was converted to partially methylated glycol acetate derivatives (PMAAs) by hydrolysis, reduction, derivatization, etc., and the total ion chromatogram of the methylation analysis of pollen polysaccharide 4 was obtained by GC-MS analysis as shown in Figure 40. The mass spectra corresponding to the methylated glycosyl peaks in the figure (Figure 41) were correlated with a search of the standard spectral library (Standard spectral library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / ms / pmaa / pframe.html) to determine the type of partially methylated glycosyl group. At the same time, the relative molar ratio was calculated from the peak area of ​​the chromatogram peaks, and together with the measurement results of the monosaccharide composition, structural information necessary for estimating the repeating unit structure of pollen polysaccharide 4 can be provided. 【0107】 Results of Pollen Polysaccharide 4-Methylation Analysis JPEG0007876054000020.jpg691702,3-Me2-Araf is 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-arabinitol, and can be inferred sequentially. 【0108】 Pollen polysaccharide 4 mainly contains arabinose and galactose, and the content of glucose, xylose, fucose, and mannose is too low. Therefore, methylation analysis detected only PMAAs of arabinose and galactose, while PMAAs of xylose, fucose, and mannose were not detected. However, because the content of glucose, xylose, fucose, and mannose is low, it does not affect the analytical determination of the main glycosidic bond configuration of pollen polysaccharide 4. Figure 40 shows the total ion chromatogram detected by GC-MS after methylation, hydrolysis, and derivatization treatment of pollen polysaccharide 4, and Figure 41 shows that mainly nine methylation-induced glycosyl ion fragments were selected. 【0109】 As shown in Table 11, the pollen polysaccharide 4 methylation analysis sample mainly contained nine glycosylion fragments, derived from galactose, arabinose, and glucose, respectively. Galactose mainly existed in the form of →6)-Galp-(1→, and arabinose mainly existed in the forms of →3,5)-Araf-(1→ and →5)-Araf-(1→. Based on the linkage of the sugar residue glycosidic bonds of pollen polysaccharide 4 and the characteristics of the pollen polysaccharide structure, it can be tentatively inferred that the pollen polysaccharide 4 main chain is linked to other glycosidic bonds by 1,3,5-Araf and 1,6-Galp linkages; and the branched chain may mainly consist of 1,5-Araf, →3,6)-Galp-(1→, and 1,3-Galp. 【0110】 2.4.2 Results of Nuclear Magnetic Resonance NMR Measurement of Pollen Polysaccharide 4 Monosaccharide residues of pollen polysaccharide 4 1 H and 13 Chemical shift assignment of 1C NMR JPEG0007876054000021.jpg38170 【0111】 Figures 42, 43, and 44 show pollen polysaccharide 4. 1 H-NMR spectrum, 13 The 1C-NMR spectrum and the DEPT-135 spectrum are shown. 1In the 1H-NMR spectrum, seven prominent signal peaks were observed in the terminal hydrogen-related region, indicating that pollen polysaccharide 4 has seven monosaccharide residues (Table 12), with corresponding chemical shifts of 5.31 ppm, 5.14 ppm, 5.09 ppm, 4.79 ppm, 4.63 ppm, 4.57 ppm, and 4.52 ppm, respectively. The chemical shifts in the range of 4.30–3.50 ppm represent the hydrogen shifts corresponding to the 2nd to 6th carbon atoms of the seven corresponding sugar residues. The chemical shifts of the terminal carbons corresponding to the glycosidic bond terminal hydrogens can be determined from the HSQC spectrum (Figure 45) and 13C-NMR spectrum. HSQC, COSY (Figure 46), and TOCSY (Figure 47) are two-dimensional spectral diagrams of hydrocarbon assignment and ortho-hydrogen correlation and total hydrogen correlation at the same monosaccharide residue, and analysis allows for the assignment of other carbons and hydrogens at the correlated monosaccharide residue. The HMBC spectrum (Figure 48) allows us to determine the spatial correlation between carbon and hydrogen atoms, and further, to determine the linkage order of monosaccharide glycosidic bonds. 【0112】 Methylation analysis and correlated nuclear magnetochemistry analysis revealed that pollen polysaccharide 4 has a main chain formed by arabinose being linked to galactose via α-L-1,3,5-Araf and β-D-1,3)-Galp, and that branched chains consisting mainly of T-α-L-Araf-(1→5)-α-L-Araf-(1) are attached to the 5th and 3rd positions of arabinose. Furthermore, there is another branched chain structure →3)-β-D-Galp-(1→6)-β-D-Galp-(1→. The main repeating structural units of pollen polysaccharide 4 are as follows. JPEG0007876054000022.jpg35170R1'' is T-α-L-Araf-(1→5)-α-L-Araf-(1→). 【0113】 2.4.3 Ultraviolet Spectrum Measurement Results As shown in Figure 49, pollen polysaccharide 4 does not have a characteristic absorption peak in the visible light region. In the ultraviolet region, no absorption peaks are observed at either 280 nm or 260 nm, indicating that pollen polysaccharide 4 is a simple polysaccharide with high purity. 【0114】 2.4.4 Infrared Spectroscopic Measurement Results Figure 50 shows a typical infrared spectrum of plant polysaccharides, specifically the infrared result for pollen polysaccharide 4. The absorption band is 3600–3200 cm⁻¹. -1 At this point, there is an absorption peak for the stretching vibration of -OH, and absorption peaks in this region are characteristic peaks of sugars. Specifically, 3322m -1 This peak represents the absorption peak of the stretching vibration of OH, and is a characteristic peak of sugars. 2931cm² -1 , 2875cm -1 There is one absorption peak, which is attributed to CH stretching vibrations. 1641m -1 There is an absorption peak at 1544 cm², which is attributed to water of crystallization. -1 There is an absorption peak, which is attributed to the C=O stretching vibration. 1407cm -1 There is one absorption peak, which is attributed to CO stretching vibration. 1309cm -1 , 1241cm -1 , 1076cm -1 , 1047cm -1 There is an absorption peak at 894cm², which is attributed to OH bending vibrations. -1 There is an absorption peak, which is attributed to the CH bending vibration of the epimer of the β-terminal group of the pyran ring. 【0115】 <Example 7> Tobacco growth promoting effect Experimental sample: Pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, pollen polysaccharide active site Fr-1-5, and pollen polysaccharide 1-4 were all prepared in pure water to a mother liquor with a polysaccharide concentration of 10 mg / ml, and then diluted with water according to the experimental plan before use. 1. Experimental design: Drug concentration design JPEG0007876054000023.jpg1171702, Experimental method: The treatment agent was prepared according to the table above, then uniformly sprayed onto the leaves of tobacco seedlings. The treatment was repeated six times for each plant, and care was taken to prevent the agent from seeping into the soil and affecting the experimental results by avoiding dripping. The cultivation period was set under the following conditions: temperature 28°C, light 2000 lux, 14h / 10h (day / night), and humidity 65%. Tobacco RGB AREA_MM parameter values ​​(leaf area / mm²) 2 The growth rates of each drug were recorded using a plant phenotype analysis system before and 7 days after drug administration, and the growth-promoting effect of each drug was evaluated by calculating the leaf area growth rate using the following formula. Leaf area increase rate (%) = (Final leaf area - Initial leaf area) × 100% / Initial leaf area 3. Experimental results: The table below shows the leaf area growth rates of different treatment groups 7 days after drug administration. From the experimental results, each pollen polysaccharide treatment group showed a growth-promoting effect in the concentration range of 0.03 to 30 ppm. Among these, the optimal growth-promoting concentration for pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, and active site Fr-1-5 is 3 ppm, but in terms of growth-promoting effect, active site Fr-1-5 > active site Fr-1 > active site Fr. 【0116】 The optimal growth-promoting concentration for pollen polysaccharide 1 was 0.3 ppm; for pollen polysaccharide 2 it was 3 ppm; for pollen polysaccharide 3 it was 0.3 ppm; and for pollen polysaccharide 4 it was 0.03 ppm. The growth-promoting effects of all of the above polysaccharides were superior to those of the same concentrations of pollen polysaccharide active sites Fr, Fr-1, and Fr-1-5, with pollen polysaccharide 4 showing the highest effect and achieving optimal growth promotion at extremely low concentrations. 【0117】 Measurement results of tobacco growth promotion for each treatment group JPEG0007876054000024.jpg137170 【0118】 <Example 8> Growth-promoting effect on Chinese cabbage 1. Experimental sample: Pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, pollen polysaccharide active site Fr-1-5, and pollen polysaccharide 1-4 were all prepared in pure water to a mother liquor with a polysaccharide concentration of 10 mg / ml, and then diluted with water according to the experimental plan. 2. Experimental design: See Table 13. 3. Experimental method: Uniformly growing young cabbage seedlings (3-4 leaves) were selected, and the treatment chemical was prepared according to the table above. The treatment was then injected into the roots of the seedlings, and the treatment was repeated 6 times for each plant. The amount of chemical injected per plant was 80 ml, and the plants were cultured in a plant culture room at 25°C. The light irradiation intensity was 3000 lux, 14h / 10h (day / night), and the humidity was 65%, and the water and fertilizer management levels were kept uniform throughout the culture period. Leaf length, leaf width, above-ground fresh weight, and SPAD value were measured 7 days after treatment. 4. Experimental results: The table below shows the measurement results for each treatment group 7 days after administration. From the experimental results, each pollen polysaccharide treatment group showed a growth-promoting effect on small cabbage plants in the concentration range of 0.03 to 30 ppm. Among these, the optimal growth-promoting concentration for the active sites Fr, Fr-1, and Fr-1-5 of the pollen polysaccharide was 3 ppm, but it can be seen that the active site Fr-1-5 had a superior growth-promoting effect compared to the active site Fr-1. 【0119】 Pollen polysaccharide 1 has an optimal growth-promoting concentration of 0.3 ppm, pollen polysaccharide 2 has an optimal growth-promoting concentration of 3 ppm, pollen polysaccharide 3 has an optimal growth-promoting concentration of 0.3 ppm, and pollen polysaccharide 4 has an optimal growth-promoting concentration of 0.03 ppm. Its growth-promoting effect is superior to that of pollen polysaccharide active sites Fr, Fr-1, and Fr-1-5. In particular, pollen polysaccharide 4 can exert its optimal growth-promoting effect at extremely low concentrations. 【0120】 Measurement results of growth promotion of small Chinese cabbage in each treatment group 7 days after medication JPEG0007876054000025.jpg158170 【0121】 <Example 9> Low-temperature resistance 1. Experimental sample: Pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, pollen polysaccharide active site Fr-1-5, and pollen polysaccharide 1-4 were all prepared in pure water to a mother liquor with a polysaccharide concentration of 10 mg / ml, and then diluted with water according to the experimental plan. 2. Experimental design: See Table 13. 3. Experimental method: After preparing the treatment agent according to the table above, it was uniformly sprayed onto the leaves of tobacco seedlings, and the treatment was repeated six times for each plant. It was important to prevent the agent from seeping into the soil and affecting the experimental results by ensuring that the solution did not drip. One day after seedling growth, the plants were subjected to cold damage (4°C) treatment for 24 hours, followed by recovery under 28°C conditions. The chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of tobacco were recorded using a plant phenotypic meter before treatment, after 24 hours of cold treatment, and after 24 hours of recovery. 4. Experimental results: QY-max represents the theoretical maximum photosynthetic capacity of a plant, and a smaller rate of decrease indicates higher cold tolerance. Fv / Fm-lss represents the maximum photosynthetic yield of PSII, and similar to QY-max, a smaller rate of decrease indicates higher cold tolerance. As is clear from the table below, after 24 hours of cold treatment, the decrease in fluorescence values ​​of QY-max and Fv / Fm-lss in each treatment group was significant. However, the decrease in fluorescence values ​​of QY-max and Fv / Fm-lss in each treatment group using pollen polysaccharides in the concentration range of 0.03 to 30 ppm was small, indicating that the growth state of the plants was better than that of the clean water control, and that they had strong cold tolerance. 【0122】 After 24 hours of low-temperature treatment, the optimal low-temperature tolerance concentration for pollen polysaccharide active sites Fr, Fr-1, and Fr-1-5 was 3 ppm. However, the low-temperature tolerance effect of active site Fr-1-5 was superior to that of active sites Fr and Fr-1. The optimal low-temperature tolerance concentration for pollen polysaccharides 1 and 2 was 0.3 ppm, and for pollen polysaccharides 3 and 4 it was 0.03 ppm. 【0123】 Measurement results after 24 hours of low-temperature treatment JPEG0007876054000026.jpg150170 【0124】 After 24 hours of recovery at room temperature, as shown in the table below, the QY-max and Fv / Fm-lss fluorescence values ​​for each treatment group began to recover, clearly indicating that the growth state of each plant damaged by low temperature was recovering. Among these, pollen polysaccharide active sites Fr, Fr-1, and Fr-1-5 at a concentration of 3 ppm, and pollen polysaccharide 1 at a concentration of 0.3 ppm clearly promoted the recovery of the plant's growth state, pollen polysaccharide 2 at a concentration of 0.3 ppm clearly promoted the recovery of the plant's growth state, and pollen polysaccharide 3 and pollen polysaccharide 4 at a concentration of 0.03 ppm clearly promoted the recovery of the plant's growth state. 【0125】 Measurement results after 24 hours of recovery at room temperature JPEG0007876054000027.jpg161170 【0126】 <Example 10> High Temperature Resistance 1. Experimental sample: Pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, pollen polysaccharide active site Fr-1-5, and pollen polysaccharide 1-4 were all prepared in pure water to a mother liquor with a polysaccharide concentration of 10 mg / ml, and then diluted with water according to the experimental plan. 2. Experimental design: See Table 13. 3. Experimental method: Tobacco seedlings with uniform growth (3-leaf stage) were selected, and the treatment chemical was prepared according to Table 11. The chemical was then uniformly sprayed onto the leaves of the tobacco seedlings, and the treatment was repeated six times for each seedling. It was important to prevent the chemical solution from dripping into the soil to prevent it from affecting the experimental results. One day after seedling growth, the seedlings were treated at a high temperature of 40°C for 48 hours. The chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of the tobacco were recorded using a plant phenotypic meter before treatment and 48 hours after high-temperature treatment. After 48 hours of high-temperature treatment, samples were taken and the malonaldehyde content was measured. 4. Experimental results: QY-max represents the theoretical maximum photosynthetic capacity of a plant, and a smaller rate of decrease indicates a higher heat tolerance of the plant. Fv / Fm-lss represents the maximum photosynthetic quantum yield of PSII, and similar to QY-max, a smaller rate of decrease indicates a higher heat tolerance of the plant. The non-photochemical extinction coefficient (NPQ-lss) reflects the plant's ability to dissipate excess light energy as heat, and reflects its photoprotective capacity. When a plant is stressed, it avoids damage by directly converting the light energy it absorbs into heat energy and dissipating it instead of using it for photosynthesis. In this case, the plant's NPQ-lss increases, so a lower NPQ-lss value during high-temperature stress indicates less damage to the plant and a higher heat tolerance. 【0127】 As is clear from the table below, after 48 hours of high-temperature treatment, there was a significant decrease in the fluorescence values ​​of the treated groups QY-max and Fv / Fm-lss, and a significant increase in the fluorescence value of NPQ-lss. However, in the concentration range of 0.03 to 30 ppm, the decrease in fluorescence values ​​of each treated group QY-max and Fv / Fm-lss, and the increase in fluorescence value of NPQ-lss, were smaller. This indicates that the plant growth was better than that of the clean water control, and that the plants had stronger high-temperature tolerance. After 48 hours of high-temperature treatment, the optimal high-temperature tolerance concentration for the pollen polysaccharide active sites Fr, Fr-1, and Fr-1-5 was 3 ppm, but the low-temperature tolerance effect of active site Fr-1-5 was superior to that of active site Fr and active site Fr-1. The optimal high-temperature tolerance concentration for pollen polysaccharides 1 and 2 was 0.3 ppm, and the optimal high-temperature tolerance concentration for pollen polysaccharides 3 and 4 was 0.03 ppm. 【0128】 Measurement results of fluorescence parameters for each treatment group JPEG0007876054000028.jpg145170

Claims

[Claim 1] The main repeating unit structure is the isolated and purified pollen polysaccharide shown below, Pollen polysaccharide 4: However, R1'' is T-α-L-Araf-(1→5)-α-L-Araf-(1→), and the isolated and purified pollen polysaccharide 4 further contains monosaccharide units of fucose, glucose, xylose, mannose, and galacturonic acid, and has an average molecular weight of 5 to 15 kDa. 【Claim 2】 The infrared spectrum of the pollen polysaccharide 4 has at least 3322 m －１ , 2931 cm －１ , 2875 cm －１ , 1641 m －１ , 1544 cm －１ , 1407 cm －１ , 1309 cm －１ , 1241 cm －１ , 1076 cm －１ , 1047 cm －１ , and contains one or more absorption peaks at 894 cm-1. The separated and purified pollen polysaccharide according to Claim 1, characterized in that. [Claim 3] A method for separating pollen polysaccharides according to claim 1, The method for separating pollen polysaccharide 4 is: (1) Purify the rapeseed pollen polysaccharide extract using a macroporous adsorption resin column, and elute it with water to obtain the primary purified pollen polysaccharide active site Fr-1. (2) The pollen polysaccharide active site Fr-1 is passed through an anion exchange chromatography column and gradient elution is performed with a 0-0.25 mol / L NaCl solution to collect the pollen polysaccharide in a 0.25 mol / L NaCl solution to obtain the pollen polysaccharide active site Fr-1-4. (3) A method for separating pollen polysaccharides according to claim 1, characterized by further purifying the pollen polysaccharide active site Fr-1-4 using a Sephadex chromatography column and obtaining pollen polysaccharide 4 using an aqueous solution of ammonium bicarbonate as the eluent. [Claim 4] The separation method according to claim 3, characterized in that the rapeseed pollen polysaccharide extract in step (1) is crude pollen polysaccharide obtained by water extraction and alcohol precipitation of rapeseed pollen. [Claim 5] The separation method according to claim 3, characterized in that the macroporous resin column used in step (1) is selected from DB-101 macroporous resin column, S-8 macroporous resin column, AB-8 macroporous resin column, and HP-20 macroporous resin column; the ion exchange chromatography column used in step (2) is selected from DEAE cellulose chromatography columns; and the Sephadex chromatography column used in step (3) is selected from Acrylic Sephadex columns, and further selected from Acrylic Sephadex S-400HR chromatography columns. [Claim 6] The separation method according to claim 3, characterized in that in step (3), the concentration of the ammonium bicarbonate aqueous solution is 0.1 to 0.3 mol / L. [Claim 7] Use of the pollen polysaccharide according to any one of claims 1 to 2 in the preparation of a plant stress tolerance product or a plant growth promoting product, Furthermore, the plant stress tolerance includes cold tolerance, drought tolerance, salt / alkali tolerance, and high temperature tolerance. In addition, when used, a product containing pollen polysaccharide or a pollen polysaccharide active site is applied to the leaves or roots of a plant. Furthermore, the plants include tobacco, Chinese cabbage, baby cabbage, stem lettuce, lettuce, bok choy, wheat, chili pepper, tomato, citrus, kiwi, cherry, pear, and apple. The use of pollen polysaccharide active sites in the preparation of plant stress tolerance products or plant growth promoting products. [Claim 8] An agricultural product characterized in that the active ingredient contains the pollen polysaccharide described in any one of claims 1 to 2. [Claim 9] The agricultural product according to claim 8, further comprising one or more auxiliary agents selected from dispersants, wetting agents, binders, emulsifiers, stabilizers, solvents, and embedding agents. [Claim 10] The agricultural product according to claim 8, wherein the dosage form is an emulsion, suspension, wettable powder, powder, granules, aqueous solution, mother material, or mother powder.

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