Pollen Polysaccharide, Its Isolation Method 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 specifically relates to pollen polysaccharides 1, 2, 3 or 4, their separation methods and uses.

Background Art

[0002] In the previous research of the present inventor (Patent CN113133454B and Patent CN113133455B), the aqueous extract of rapeseed pollen was used for stress tolerance and growth promotion of plants including, for example, plants such as bok choy, stem lettuce, lettuce, Chinese cabbage, wheat, pepper, tomato, citrus, kiwi, cherry, pear, apple, etc. Also, when its components were detected, it was shown to contain various components such as carbohydrates, proteins, amino acids and lipids. However, specific active ingredients have not yet been studied.

[0003] JPEG2025523229000002.jpg17170

[0004] There may be many substances in the aqueous extract of rapeseed pollen that have the effects of promoting plant growth and stress tolerance. Currently, however, those skilled in the art do not know which substances play a dominant role in promoting plant growth and stress tolerance. Therefore, further research on rapeseed pollen extract is necessary.

Summary of the Invention

[0005] Polysaccharides are carbohydrate substances with a complex structure formed by the dehydration condensation of multiple monosaccharide molecules. The constituent units are linked by glycosidic bonds. Common glycosidic bonds include α-1,3 glycosidic bond, β-1,6 glycosidic bond, β-1,4 glycosidic bond, α-1,4 glycosidic bond, β-1,3 glycosidic bond, etc. Polysaccharides are widely present in animals, plants, and microorganisms. Polysaccharides from different sources have different biological activities. According to current research, plant polysaccharides have biological activities such as immunomodulation, antitumor, antioxidant, anti-aging, blood sugar lowering, and lipid lowering. They also have characteristics such as a wide range of sources, decomposability, high safety, ease of modification, and high environmental affinity, and are widely used in industries such as food, medicine, livestock farming, and aquaculture.

[0006] On the other hand, plant polysaccharides are a type of natural polymer with a complex structure. Currently, due to the lack of research on the specific molecular structure of pollen polysaccharides and the structural effectiveness of their physiological activities, it is necessary to isolate and identify pollen polysaccharide compounds to provide theoretical and experimental basis for future development research of pollen polysaccharide resources.

[0007] In the present invention, polysaccharides in rapeseed pollen are extracted, separated, and purified to obtain a novel pollen polysaccharide. When tested, this pollen polysaccharide has a certain effect in both promoting plant growth and stress tolerance. Specifically, the present invention provides pollen polysaccharide 1 whose main repeating structural unit is shown below. JPEG2025523229000003.jpg48170However, 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 backbone of the present invention, R1, R2, and R3 are bonded 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 of a polysaccharide molecule (terminal group). 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, and mannose. 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 the 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 [1311 cm] is attributed to C-O stretching vibration; -1 at [1241 cm] -1 and at [1027 cm] -1 the absorption peak is attributed to O-H bending vibration; at [894 cm] -1 the absorption peak at [873 cm] is attributed to C-H bending vibration of the epimer of the β-terminal group of the pyran ring; -1 the absorption peak at [873 cm] is attributed to C-H bending vibration of the equatorial bond other than C-H of the epimer of the terminal group of the pyran ring. The present invention provides a pollen polysaccharide 2 whose main repeating structural unit is shown below. JPEG2025523229000004.jpg25170 However, R1’ is T-α-L-Araf-(1→5)-α-L-Araf-(1→, and R2’ is T-β-D-Galp-(1→6)-β-D-Galp-(1→. In the polysaccharide main chain of the present invention, R1’ is bonded to the 5-position of arabinose, and R2’ is bonded to the 6-position of galactose.

[0011] In the present invention, Ara represents arabinose; Gal represents galactose; f represents furanose; p represents pyranose; T represents the terminal (terminal group) of the polysaccharide molecule. Here, the molar content ratio of arabinose:galactose is 0.597:0.283. In addition to the above main repeating structural unit, the pollen polysaccharide of the present invention contains trace amounts of monosaccharide units of fucose, glucose, xylose, and mannose. In the present invention, the average molecular weight of the pollen polysaccharide 2 is 5 - 15 KDa; furthermore, the average molecular weight of the pollen polysaccharide 2 is 9 - 11 KDa; In the present invention, the average molecular weight of the pollen polysaccharide 2 is 10 - 11 KDa, for example, 10718.15 Da.

[0012] The infrared spectrum of the pollen polysaccharide 2 of the present invention is at least at 3288 m -1 , 2931 cm -1 , 2871 cm -1 , 1641 m-1 、1548 cm -1 、1440 cm -1 、1402 cm -1 、1309 cm -1 、1243 cm -1 、1081 cm -1 、896 cm -1 contains one or more absorption peaks of

[0013] Here, 3288 cm -1 is the stretching vibration absorption peak of O-H and is a characteristic peak of saccharides. 2931 cm -1 、2871 cm -1 has one absorption peak and can be attributed to C-H stretching vibration. 1641 m -1 has one absorption peak and can be attributed to crystal water. 1548 cm -1 has an absorption peak and can be attributed to C=O stretching vibration. 1440 cm -1 、1402 cm -1 has an absorption peak and can be attributed to C-O stretching vibration. 1309 cm -1 、1243 cm -1 、1081 cm -1 has an absorption peak and can be attributed to O-H bending vibration. 896 cm -1 has an absorption peak and can be attributed to the C-H bending vibration of the epimer of the β-terminal group of the pyran ring. The present invention provides pollen polysaccharide 3 whose main repeating structural unit is shown below. JPEG2025523229000005.jpg64170

[0014] In the formula, Ara represents arabinose; Gal represents galactose; f represents furanose configuration; p represents pyranose configuration; T represents the terminal (end group) of the polysaccharide molecule. Here, the molar content ratio of arabinose:galactose is 0.328:0.399.

[0015] In addition to the above main repeating structural units, 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 the present invention, the average molecular weight of pollen polysaccharide 3 is 60 to 70 kDa; Furthermore, the average molecular weight of pollen polysaccharide 3 is 65 to 70 kDa; In the present invention, the average molecular weight of pollen polysaccharide 3 is 66 to 68 kDa, for example 66911.38 Da.

[0016] The infrared spectrum of pollen polysaccharide 3 of the present invention has at least one absorption peak at 3426 cm -1 , 2939 cm -1 , 1734 cm -1 , 1619 cm -1 , 1423 cm -1 , 1145 cm -1 , 1091 cm -1 , 895 cm -1 or more.

[0017] The absorption band of pollen polysaccharide 3 has an absorption peak of stretching vibration of -OH at 3600 - 3200 cm -1 . The absorption peak in this region is a characteristic peak of saccharides. Specifically, there is an absorption peak of stretching vibration of O-H at 3426 cm -1 , which is a characteristic peak of saccharides. There is one absorption peak at 2939 cm -1 , which can be attributed to C-H stretching vibration. The weak absorption peak found around 1734 cm -1 is attributed to the stretching vibration of C=O of the carboxyl group, indicating that pollen polysaccharide 3 contains a certain amount of uronic acid. There is one absorption peak at 1619 cm -1 , which can be attributed to crystal water. There are absorption peaks at 1423 cm -1 , 1145 cm -1 , 1091 cm -1 , which can be attributed to C-O stretching vibration. There is an absorption peak at 895 cm -1 , which can be attributed to the C-H bending vibration of the epimer of the β-terminal group of the pyran ring. The present invention provides pollen polysaccharide 4 whose main repeating structural unit is shown below. JPEG2025523229000006.jpg37170 Here, R1’’ is T-α-L-Araf-(1→5)-α-L-Araf-(1→.

[0018] In the present invention, Ara represents arabinose; Gal represents galactose; f represents the furanose configuration; p represents the pyranose configuration; T represents the terminal of the polysaccharide molecule (terminal group). Here, the molar content ratio of arabinose:galactose is 0.686:0.243.

[0019] In addition to the above main repeating structural units, the pollen polysaccharide 4 of the present invention contains trace monosaccharide units of fucose, glucose, xylose, mannose, and galacturonic acid. In the present invention, the average molecular weight of the pollen polysaccharide 4 is 5 - 15 KDa; Furthermore, the average molecular weight of the pollen polysaccharide 4 is 9 - 11 KDa; In the present invention, the average molecular weight of the pollen polysaccharide 4 is 10 - 11 KDa, for example 10328.089 Da.

[0020] The infrared spectrum of the pollen polysaccharide 4 of the present invention 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 , 894 cm -1 including one or more absorption peaks at.

[0021] The infrared absorption band of the pollen polysaccharide 4 has a stretching vibration absorption peak of -OH at 3600 - 3200 cm -1 , and the absorption peak in this region is a characteristic peak of saccharides. Specifically, 3322 cm -1 is the stretching vibration absorption peak of O-H and is a characteristic peak of saccharides. 2931 cm -1, 2875 cm -1 has one absorption peak, which can be attributed to C-H stretching vibration. 1641 cm -1 has an absorption peak, which can be attributed to water of crystallization. 1544 cm -1 has an absorption peak, which can be attributed to C=O stretching vibration. 1407 cm -1 has one absorption peak, which can be attributed to C-O stretching vibration. 1309 cm -1 , 1241 cm -1 , 1076 cm -1 , 1047 cm -1 has an absorption peak, which can be attributed to O-H bending vibration. 894 cm -1 has an absorption peak, which can be attributed to C-H bending vibration of the epimer of the β-terminal group of the pyran ring.

[0022] In the present invention, the values of n1 to n5 depend on the molecular weight of the polysaccharide. It is an object of the present invention to further provide a method for separating the pollen polysaccharide including the following steps. A method for separating the pollen polysaccharide described above, wherein the method for separating pollen polysaccharide 1 is (1) Purifying the rapeseed pollen polysaccharide extract with a macroporous adsorption resin column, eluting with water, and obtaining the pollen polysaccharide active site Fr-1; (2) Passing Fr-1 through an ion exchange chromatography column, eluting the pollen polysaccharide active site Fr-1 with water, and obtaining the pollen polysaccharide active site Fr-1-1; (3) Further purifying the pollen polysaccharide active site Fr-1-1 using a Sephadex chromatography column, using an ammonium bicarbonate aqueous solution as the eluent, and obtaining pollen polysaccharide 1; including The method for separating pollen polysaccharide 2 is (1) Purifying the rapeseed pollen polysaccharide extract with a macroporous adsorption resin column, eluting with water, and obtaining the primary purified pollen polysaccharide active site Fr-1; (2) Passing the pollen polysaccharide active site Fr-1 through an anion exchange chromatography column, performing gradient elution with a 0 - 0.025 mol / L NaCl solution, collecting the 0.025 mol / L NaCl eluate portion, and obtaining the pollen polysaccharide active site Fr-1-2; (3) Further purify the active site Fr-1-2 of the pollen polysaccharide using a Sephadex chromatography column, and use an aqueous ammonium bicarbonate solution as the eluent to obtain pollen polysaccharide 2. including The separation method of pollen polysaccharide 3 is (1) Purify the rapeseed pollen polysaccharide extract using a macroporous adsorption resin column, elute with water, and obtain the primary purified pollen polysaccharide active site Fr-1. (2) Pass the pollen polysaccharide active site Fr-1 through an anion exchange chromatography column, perform gradient elution with a 0-0.05 mol / L NaCl solution, collect the 0.05 mol / L NaCl solution pollen polysaccharide, and obtain the pollen polysaccharide active site Fr-1-3. (3) Further purify the pollen polysaccharide active site Fr-1-3 using a Sephadex chromatography column, and use an aqueous ammonium bicarbonate solution as the eluent to obtain pollen polysaccharide 3. including The separation method of pollen polysaccharide 4 is (1) Purify the rapeseed pollen polysaccharide extract using a macroporous adsorption resin column, elute with water, and obtain the primary purified pollen polysaccharide active site Fr-1. (2) Pass the pollen polysaccharide active site Fr-1 through an anion exchange chromatography column, perform gradient elution with a 0-0.25 mol / L NaCl solution, collect the 0.25 mol / L NaCl solution pollen polysaccharide, and obtain the pollen polysaccharide active site Fr-1-4. (3) Further purify the pollen polysaccharide active site Fr-1-4 using a Sephadex chromatography column, and use an aqueous ammonium bicarbonate solution as the eluent to obtain pollen polysaccharide 4. including.

[0023] The pollen polysaccharide active site Fr-1 applied to the ion exchange column may be in the form of the original eluate of the macroporous resin, a concentrated solution of the original eluate, or a re-solution after concentrating and drying the original eluate.

[0024] The pollen polysaccharide active sites Fr-1-1, Fr-1-2, Fr-1-3, and Fr-1-4 applied to the gel chromatography column may be the original eluate of the ion exchange column, a concentrated solution of the original eluate, or a re-solution after concentrating and drying the original eluate.

[0025] The concentration and drying method in the present invention includes ordinary operating means such as vacuum evaporation, atmospheric evaporation, drying, vacuum drying, thin-film drying, freeze-drying, etc., but is not limited thereto, and can be used alone or in combination.

[0026] The rapeseed pollen polysaccharide extract in step (1) is a crude rapeseed pollen polysaccharide obtained by a conventional method. The conventional method described in the present invention includes water extraction and primary purification means after water extraction, but is not limited thereto. The water extraction includes, but is not limited to, conventional plant extract extraction methods such as heat extraction, ultrasonic extraction, and microwave extraction.

[0027] Also, in some cases, impurity removal treatment may be performed by methods such as degreasing and decolorization before extraction. For example, extraction, degreasing, and decolorization may be performed with a fat-soluble solvent such as ethanol or petroleum ether. The primary purification means after the water extraction includes various means such as alcohol precipitation and removal of chitosan, but is not limited thereto.

[0028] In the purification method, in some cases, auxiliary means such as protein removal, decolorization, and removal of small molecules may be selectively added, which is convenient for the subsequent polysaccharide concentration process. For example, protein removal agents such as phenol, trichloroacetic acid, and tannin can be used; for example, decolorization can be performed with an adsorbent such as cellulose, diatomaceous earth, or activated carbon; small molecules can be removed by methods such as dialysis. In some specific embodiments of the present invention, the rapeseed pollen polysaccharide extract in step (1) is a crude pollen polysaccharide obtained after water extraction and alcohol precipitation of rapeseed pollen.

[0029] The water extraction - alcohol precipitation refers to adding ethanol to the water extract to reach a certain alcohol content, causing the solubility of some components in the mixed solution to decrease and precipitates to form.

[0030] In the present invention, when adopting the water extraction - alcohol precipitation method, in the alcohol precipitation step, ethanol is added to the water extract (or the concentrated solution of the 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 the rapeseed pollen hot - water extract and the crude pollen polysaccharide after chitosan removal. In some embodiments of the present invention, the rapeseed pollen may be subjected to ultrafine pulverization treatment to facilitate the extraction of polysaccharides. In some embodiments of the present invention, the pollen polysaccharide extract of rapeseed pollen may be vacuum freeze - dried to produce a crude pollen polysaccharide solid. In the technical form of the present invention, the macroporous resin column used in step (1) is a non - polar column.

[0031] In the present invention, the macroporous resin column includes, but is not limited to, DB - 101 macroporous resin column, S - 8 macroporous resin column, AB - 8 macroporous resin column, HP - 20 macroporous resin column. Furthermore, the macroporous resin column is 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 into a solid form by dialysis and freeze - drying treatment; in step (2), the pollen polysaccharide active sites Fr - 1 - 1, Fr - 1 - 2, Fr - 1 - 3, Fr - 1 - 4 can be subjected to dialysis and freeze - drying treatment to be made into a solid form for use. In the present invention, the fractionation molecular weight of dialysis is at least 3500 Da.

[0033] In the technical form of the present invention, the ion exchange column used in step (2) is an anion exchange column, including various ion exchange columns such as DEAE-cellulose, DEAE-agarose gel, and DEAE-glucose gel, but not limited thereto, and is further selected from DEAE cellulose-52 chromatography columns.

[0034] In the technical aspect of the present invention, the gel chromatography column used in step (3) includes different gel chromatography columns such as Sephadex (SephadexG) column and polyacrylamide gel ToyopearlHW column, but is not limited thereto. For example, it can be selected from acrylic Sephadex S-400 HR chromatography column, SephadexLH-20 chromatography column, etc. In the present invention, in step (3), the concentration of the ammonium bicarbonate aqueous solution is 0.1 - 0.3 mol / L, preferably 0.2 mol / L.

[0035] In some embodiments of the present invention, when the sugar content in steps (2) and (3) was detected using phenol-sulfuric acid, an absorbance value was detected at 490 nm. The present invention further provides a pollen polysaccharide active site Fr-1 obtained by passing rapeseed pollen polysaccharide extract through a macroporous resin and eluting with water.

[0036] In the present invention, the macroporous resin column is a non-polar column; and is further one selected from DB-101 macroporous resin column, S-8 macroporous resin column, AB-8 macroporous resin column, and HP-20 macroporous resin column; furthermore, 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 by the macroporous resin column can be made into a solid by dialysis and freeze-drying; Furthermore, the fractionation molecular weight of dialysis is at least 3500 Da.

[0037] The present invention further provides a rape pollen polysaccharide active site Fr-1-5 obtained by purifying a rape pollen polysaccharide extract with a macroporous resin and further passing it through an ion exchange chromatography column and eluting with a 0 to 0.25 mol / L sodium chloride solution.

[0038] In the present invention, the ion exchange chromatography column is an anion exchange chromatography column; and is further selected from a DEAE cellulose chromatography column. In some embodiments of the present invention, the pollen polysaccharide active site Fr-1-5 purified by a macroporous resin column can be made into a solid by dialysis and freeze-drying; Furthermore, the molecular weight cut-off of the dialysis is at least 3500 Da.

[0039] In some embodiments of the present invention, the rape pollen polysaccharide extract is a crude pollen polysaccharide obtained by degreasing rape pollen with ethanol, extracting with hot water, and removing impurities by alcohol precipitation.

[0040] The present invention further provides a pollen polysaccharide active site Fr obtained by extracting rape pollen with water, taking the supernatant, concentrating it, and performing alcohol precipitation with absolute ethanol. After alcohol precipitation, when normal washing operations are performed using organic solvents such as ether and acetone, the product after washing still 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 solution of the water extract) until the ethanol concentration reaches 70 to 90% v / v, and for example, 70%, 71%, 72%, 73%, 74%, 75%... 80%... 85%... 90% v / v, etc. can be selected. In the present invention, the degree of concentration during the concentration step can be determined by those skilled in the art according to the amount of eluent added during elution and the final requirements of the product.

[0042] The various extracts in the present invention can exist in various forms such as solids, liquids, suspensions, etc. According to the needs of actual production, sales, use, etc., the conventional product form can be adjusted.

[0043] The present invention also relates to the use of pollen polysaccharide 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5 in the preparation of plant stress tolerance products. The plant stress tolerance according to the present invention includes cold tolerance, drought tolerance, high temperature tolerance, salt-alkali tolerance, etc. In the present invention, when used, a product containing pollen polysaccharides is applied to the leaves or roots of plants. In the present invention, when the product of pollen polysaccharide is prepared into a solution for use, the concentration of the main active ingredient can be selected according to 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 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] An object of the present invention is also the use of pollen polysaccharide 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5 in the manufacture of plant growth promoting products.

[0049] The plants in the present invention include, but are not limited to, cash crops, food crops, such as pakchoi, baby bok choy, stem lettuce, lettuce, Chinese cabbage, wheat, pepper, tomato, citrus, kiwi, cherry, pear, apple, tobacco, etc.

[0050] The "cash crops" have many types and include, but are not limited to, fiber crops (such as cotton, hemp, etc.), oil crops (such as sesame, peanuts, etc.), sugar crops (such as sugarcane, beet, etc.), tobacco (a crop for personal consumption), medicinal crops, pigment crops, ornamental crops, fruits, and other cash crops.

[0051] The "edible crops" include, but are not limited to, cereal crops (such as wheat, rice, and corn), tuber crops (including sweet potatoes, potatoes, etc.), and legume crops (including soybeans, broad beans, peas, mung beans, etc.). In addition, the present invention provides an agricultural product whose active ingredient contains 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 plant growth promotion includes promoting the germination of plants and promoting the growth of one or more of roots, stems, leaves, flowers, and fruits. For example, it includes promoting the increase in root length, stem thickness, plant height, leaf width, leaf length, number of leaves, leaf area, biomass, chlorophyll content, yield, etc.

[0053] In the present invention, pollen polysaccharide 1-4, pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5 may be used directly as a single agent. However, for the convenience of product stabilization, transportation, and storage, conventionally known adjuvants such as dispersants, wetting agents, binders, emulsifiers, stabilizers, solvents, embedding agents, etc. may be added and formulated into corresponding dosage forms.

[0054] On the other hand, pollen polysaccharide 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 in combination with pollen fertilizers, water-soluble fertilizers, compound fertilizers, pesticides, etc. as synergists. The dosage forms of the formulations in the present invention include, but are not limited to, ordinary agricultural formulations such as emulsions, suspensions, wettable powders, powders, granules, wettable powders, mother liquors, mother powders, etc.

Advantages of the Invention

[0055] The advantageous effects of the present invention are shown below. The present invention has found a novel pollen polysaccharide 1-4, which is useful for plant stress tolerance and plant growth promotion, and was first separated from rapeseed powder. In addition, it has been found that the pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, or pollen polysaccharide active site Fr-1-5 of the rapeseed pollen crude separation component also has plant growth promotion effects and stress tolerance effects.

[0056] In the present invention, "rapeseed" refers to a cruciferous herbaceous crop of the genus Brassica. Common "rapeseed" includes, but is not limited to, Brassica rapa, Brassica napus, Brassica juncea, Brassica carinata, and Brassica abyssinica. "Pollen" in the present invention is obtained from the above-mentioned "rapeseed".

Brief Description of the Drawings

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BEST MODE FOR CARRYING OUT THE INVENTION

[0058] Hereinafter, the technical aspects of the present invention will be described clearly and completely. However, it goes without saying that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts are included in the protection scope of the present invention. In addition, if there are processes not specifically described in detail below, those skilled in the art can realize or understand them with reference to the prior art. The reagents or equipment used are considered general products available on the market if the manufacturer is not specified.

[0059] <Example 1> Preparation of pollen polysaccharide extract Rapeseed pollen was ultrafinely pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated powder of the pollen powder was weighed, free monosaccharides were removed with ethanol, degreased and decolorized with petroleum ether, extracted with hot water, precipitated with alcohol, the precipitate was collected, and washed in the order of absolute ethanol, ethyl ether, and acetone. Finally, the washed precipitate was reprecipitated with water, pre-frozen at -20 °C, and vacuum freeze-dried to obtain a crude polysaccharide of pollen extracted with hot water.

[0060] <Example 2> Preparation of pollen polysaccharide extract Rapeseed pollen was mixed with water, extracted by heating, filtered, chitosan was added to the filtrate, incubated and allowed to stand, and the liquid separated by solid-liquid separation was the crude polysaccharide extract of pollen. After the extract was concentrated under reduced pressure, it was pre-frozen at -20 °C and vacuum freeze-dried to obtain crude polysaccharide of pollen.

[0061] <Example 3> Preparation of pollen polysaccharide extract Rapeseed pollen was ultrafinely pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated powder of the pollen powder was weighed, free monosaccharides were removed with ethanol, degreased and decolorized with petroleum ether, extracted by ultrasonic wave, precipitated with alcohol, the precipitate was recovered, and washed in the order of absolute ethanol, ethyl ether, and acetone. Finally, the washed precipitate was reprecipitated with water, pre-frozen at -20 °C, and vacuum freeze-dried to obtain a crude polysaccharide of pollen extracted with hot water.

[0062] <Example 4> Preparation of the Active Fraction Fr of Pollen Polysaccharide Rapeseed pollen was ultrafinely pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated pollen powder was weighed, and free monosaccharides were removed with ethanol, degreased and decolorized with petroleum ether, extracted with hot water, and precipitated with alcohol. When precipitating with alcohol, in the solution added with ethanol, the ethanol concentration was about 75% - 85%. The precipitate was collected to obtain the crude pollen polysaccharide active fraction Fr.

[0063] <Example 5> Separation and Purification of Pollen Polysaccharide The separation schemes of pollen polysaccharide 1 - 4, and pollen polysaccharide active fractions Fr, Fr - 1, and Fr - 1 - 5 are as follows.

[0064] Step (1) Purification by HP - 20 Macroporous Adsorption Resin Chromatography Column: The crude pollen polysaccharide was preliminarily purified using an HP - 20 macroporous adsorption resin chromatography column (Φ4.0 cm × 40 cm). The specific steps were as follows: A certain amount of crude pollen polysaccharide (in this example, taking the crude pollen polysaccharide prepared by the method of Example 4 as an example for further explanation of separation and purification, but the crude pollen polysaccharide obtained by other methods is similarly applicable to the separation and purification method of the present invention) was weighed, a certain amount of water was added to prepare a crude pollen polysaccharide solution of 10 - 20 mg / mL, and 10 mL was loaded each time. After adsorbing on the HP - 20 macroporous adsorption resin chromatography column for 3 hours, the column was washed with 5 times the column volume of water, the sample was collected to obtain the pollen polysaccharide active fraction Fr - 1, concentrated under reduced pressure at 48°C, dialyzed (3500 Da), and freeze - dried.

[0065] Step (2) Separation and purification by DEAE cellulose-52 chromatography column: The active site Fr-1 of pollen polysaccharide was separated and purified using a DEAE cellulose-52 chromatography column (Φ3.5 cm × 30 cm). The specific steps were as follows: 1) 100 mg of the active site Fr-1 solution of pollen polysaccharide was loaded; 2) Elution was performed, and the eluents were water, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4, and 0.5 mol / L NaCl solutions in sequence. The flow rate was 1.2 mL / min, and 6 mL was collected per vial; 3) Samples were collected, the sugar content was measured by the phenol-sulfuric acid method, the absorbance value at 490 nm was measured with a microplate reader, and an elution curve was drawn with the number of collected vials on the horizontal axis and the absorbance value of the collected solution on the vertical axis (Figure 1). Each NaCl elution peak was collected, the same components were combined, and each fraction component of pollen polysaccharide (active site Fr-1-1 of pollen polysaccharide, active site Fr-1-2 of pollen polysaccharide, active site Fr-1-3 of pollen polysaccharide, active site Fr-1-4 of pollen polysaccharide) was obtained. The elution site with 0 - 0.25 mol / L NaCl solution was collected to obtain the active site Fr-1-5 of pollen polysaccharide. The above active sites were concentrated under reduced pressure at 48 °C, dialyzed (3500 Da) to remove salts, and freeze-dried.

[0066] As is apparent from Figure 1, by passing through a DEAE cellulose-52 anion exchange column and gradient eluting the crude pollen polysaccharide with NaCl solutions of different concentrations, four elution peaks were obtained, which were the water elution fraction (active site Fr-1-1 of pollen polysaccharide), the elution fraction with 0.025 mol / L NaCl solution (active site Fr-1-2 of pollen polysaccharide), the elution fraction with 0.05 mol / L NaCl solution (active site Fr-1-3 of pollen polysaccharide), and the elution fraction with 0.25 mol / L NaCl solution (active site Fr-1-4 of pollen polysaccharide), respectively. The eluents corresponding to each elution peak were collected respectively to obtain the primary fractions of the crude pollen polysaccharide, namely the active site Fr-1-1 of pollen polysaccharide, the active site Fr-1-2 of pollen polysaccharide, the active site Fr-1-3 of pollen polysaccharide, and the active site Fr-1-4 of pollen polysaccharide, which were concentrated under reduced pressure, dialyzed, and freeze-dried.

[0067] Step (3) Purification by Sephadex S-400HR Acrylamide Chromatography Column: Finally, the fractionated fractions of pollen polysaccharides were further purified using a Sephadex S-400HR acrylamide chromatography column (Φ1.0 cm × 100 cm). The specific steps were as follows: 1) Each fractionated fraction solution was loaded at a single sample amount of 20 mg; 2) Elution was carried out, using 0.2 mol / L ammonium bicarbonate as the elution phase, at a flow rate of 0.2 mL / min, collecting 3 mL per vial until no sugar was detected, and continuing to elute. A total of 40 vials were collected for each fraction eluate; 3) The collected samples were taken, 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, an elution curve was created, the elution peaks were collected, the same fractions were combined, concentrated under reduced pressure at 48°C, dialyzed (3500 Da) to remove ammonia, and freeze-dried to obtain the purified fraction of pollen polysaccharides.

[0068] Four fractions of pollen polysaccharide fractions (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) separated by a DEAE cellulose-52 anion exchange column were further purified using a Sephadex S-400HR acrylamide chromatography column, and the four fractions were eluted with 0.2 mol / L ammonium bicarbonate respectively. As is obvious from Figure 2, after the four fractions were eluted with 0.2 mol / L ammonium bicarbonate, single elution peaks were obtained for all of them, and the corresponding fractions were named fraction 1, fraction 2, fraction 3, and fraction 4 respectively. The eluates corresponding to the elution peaks were collected (fraction 1: vials 21 - 31, fraction 2: vials 20 - 26, fraction 3: vials 18 - 26, fraction 4: vials 20 - 23), concentrated under reduced pressure, dialyzed, freeze-dried, and four purified pollen polysaccharides were obtained.

[0069] Identification of the Purity of Pollen Polysaccharides and Measurement of Their Molecular Weights 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 polysaccharide. The measurement conditions were as follows: Agilent 1260 series high performance liquid chromatography, refractive index detector (RID), Shodex OHpak SB-804 HQ (7.8 mm × 300 mm) gel chromatography column, mobile phase of 0.1 mol / L Na2SO4, flow rate of 0.5 mL / min, column temperature of 35°C, sample injection volume of 20 μL, and instrument measurement time of 24 min.

[0070] Preparation of standard curve: Dextran standards with different series of molecular weights (5900, 9600, 21100, 47100, 107000, 200000, 341400 Da) were dissolved in 0.1 mol / L Na2SO4 solution to prepare a standard solution with a concentration of 5 mg / mL. After filtration through a 0.22 μm aqueous phase filter membrane, it was measured under the measurement conditions of HPGPC, and its retention time was recorded. A standard curve was prepared with the retention time (min) on the horizontal axis and the logarithm of the molecular weight of the dextran standard (LogMW) on the vertical axis. To calculate the molecular weights of different pollen polysaccharides, when applied to the regression equation, as specifically shown in Figure 3, a standard curve of y = -0.322x + 9.4365 was obtained.

[0071] Measurement of the purity and molecular weight of pollen polysaccharide: 10 mg of the purified pollen polysaccharide sample was dissolved in 2 mL of 0.1 mol / L Na2SO4 solution, and after sufficient dissolution, a polysaccharide solution with a concentration of 5 mg / mL was prepared. After filtration through a 0.22 μm aqueous phase filter membrane, it was analyzed under the measurement conditions of HPGPC, and the chromatogram of each pollen polysaccharide was recorded. The purity was evaluated based on the number of peaks and peak symmetry on the chromatogram of each polysaccharide sample, and the molecular weight was calculated by comparing with the molecular weight standard curve of the dextran standard based on the retention time on the chromatogram of each polysaccharide sample.

[0072] The results of measuring the purity and molecular weight of pollen polysaccharides 1, 2, 3, and 4 by the HPGPC method are shown in Figure 4. As is clear from Figure 4, a single symmetric peak appears in the HPGPC chromatograms of all four pollen polysaccharides, suggesting that the four types of pollen polysaccharides have high purity. This result is consistent with the separation and purification results using an S-400HR acryldex column, further demonstrating that the established pollen polysaccharide separation and purification method is feasible and that it was possible to produce highly pure purified pollen polysaccharides. By comparing the peak times of pollen polysaccharides 1-4 based on the previously established molecular weight standard curve, the average molecular weights of each pollen polysaccharide were calculated to be 24774.22 Da, 10718.15 Da, 66911.38 Da, and 10328.089 Da.

[0073] <Example 6> Structural Identification of Pollen Polysaccharides 1 Measurement Results of the Monosaccharide Composition of Pollen Polysaccharides Measurement method: The monosaccharide composition of the pollen polysaccharide sample 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, vortexed and mixed, 50 μL was aspirated and added to 950 μL of distilled water, centrifuged at 12,000 r / min for 5 minutes, and the supernatant was taken for IC analysis. Sixteen kinds of monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, glucuronic acid, mannuronic acid) were simultaneously prepared in a standard stock solution. The precisely arranged concentration standards of each monosaccharide standard solution were taken as a mixed standard. The monosaccharide composition was determined based on the peak time in the chromatography of the hydrolyzed sample of pollen polysaccharide. The masses of different monosaccharides were determined according to the absolute quantification method, the molar ratio was calculated from the molar masses of the monosaccharides, and C (standard) / A (standard) = C (sample) / A (sample). C is the concentration and A is the peak area. Chromatography conditions: Dionex Carbopac TMPA20 (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), the chromatogram peaks of 16 kinds of standard monosaccharides can be separated and detected using an ion chromatograph. The peaks are regular, the resolution is high, and it can preferably correspond to the simultaneous detection of common monosaccharide species in monosaccharide composition analysis.

[0074] Monosaccharide composition of pollen polysaccharide 1 JPEG2025523229000007.jpg42170 shows the monosaccharide composition detection ion chromatogram of pollen polysaccharide 1 in Fig. 5(B). In Fig. 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. It can be seen from Fig. 5(B) that pollen polysaccharide 1 is mainly composed of arabinose, galactose, and glucose, and contains trace amounts of xylose, fucose, and mannose. Calculating the molar ratio of the monosaccharide composition, 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 Fig. 5(C) of JPEG2025523229000008.jpg42170, no peak corresponding to uronic acid is detected in pollen polysaccharide 2, suggesting that pollen polysaccharide 2 does not contain uronic acid and is a neutral sugar. Pollen polysaccharide 2 is mainly composed of arabinose and galactose. Calculated from the peak area of the correlation chromatogram, the ratio of fucose: arabinose: galactose: glucose: xylose: mannose in pollen polysaccharide 2 is 0.010: 0.597: 0.283: 0.048: 0.007: 0.038 (Table 2).

[0076] Monosaccharide composition of pollen polysaccharide 3 As shown in Fig. 5(D) of JPEG2025523229000009.jpg58170, galacturonic acid and glucuronic acid are detected, suggesting that pollen polysaccharide 3 is an acidic sugar. Pollen polysaccharide 3 mainly contains arabinose and galactose. Calculated from the peak area of the correlation chromatogram, 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 Fig. 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, which are arabinose and galactose in order from left to right. Calculated from the peak area of the correlation chromatogram, the 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 Methylation and NMR Results of Pollen Polysaccharides 2.1 Structure of Pollen Polysaccharide 1 2.1.1 Methylation Measurement Results To investigate the primary structure of pollen polysaccharide 1, pollen polysaccharide 1 was methylated using the improved Needs method. Since pollen polysaccharide 1 is a neutral sugar, it does not require uronic acid reduction. Also, RXI-5 SIL MS was used for the capillary column in gas analysis. By detecting the structure and content of related monosaccharides through gas analysis, the glycosidic bond sites related to monosaccharides were determined.

[0079] Fully methylated pollen polysaccharide 1 was converted into partially methylated glycol acetate derivatives (PMAAs) through processes such as hydrolysis, reduction, and derivatization. The total ion chromatogram of the methylation analysis of pollen polysaccharide 1 was obtained as shown in Fig. 6 by GCMS analysis. The mass spectrum (Fig. 7) corresponding to the peak of methylated glycosyl in the figure was correlated with and collated by searching the standard spectrum library (Standard Spectrum Library: The CCRC Spectral Database for PMAA’s https: / / www.ccrc.uga.edu / specdb / ms / PMAA / pframe.html). While determining the type of partially methylated glycosyl, the relative molar ratio was calculated from the peak area of the chromatogram peak. Combined with the measurement results of the monosaccharide composition, it can provide the structural information necessary for estimating the repeating unit structure of pollen polysaccharide 1.

[0080] Methylation analysis results of pollen polysaccharide 1 JPEG2025523229000011.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, only the PMAAs of arabinose, galactose, and glucose were detected in the methylation analysis, 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 chains are 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 Chemical shift assignment of 13C NMR JPEG2025523229000012.jpg40170

[0083] Of pollen polysaccharide 1 1 1H-NMR spectrum (Figure 8), 13As shown in the \(^{13}\)C-NMR spectrum (Figure 9) and DEPT-135 spectrum (Figure 10), in the one-dimensional NMR spectrum, basic information such as the configuration type of the glycosidic bond and the monosaccharide composition type can be estimated based on the terminal hydrogen and terminal carbon. 1 In the \(^1\)H-NMR spectrum, in the region related to the terminal hydrogen, there are peaks of five clearer hydrogen signals with chemical shifts of 5.18 ppm, 5.02 ppm, 4.95 ppm, 4.46 ppm, and 4.42 ppm, suggesting that in pollen polysaccharide 1, there are not only α-type glycosidic bonds but also β-type glycosidic bonds. According to the detection results of the monosaccharide composition and methylation, and related references, it can be determined that 5.18 ppm and 5.02 ppm are attributed 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 correlation between carbon and hydrogen in the ortho position of the polysaccharide glycosidic bond, 13 and together with the \(^{13}\)C-NMR spectrum, the position of the terminal carbon can be determined. The TOCSY spectrum is a two-dimensional nuclear magnetic spectrum related to the hydrogen of the monosaccharide residue that can be used to identify the position of the hydrogen on the monosaccharide residue. The COSY spectrum is a spectrum related to the ortho hydrogen on the monosaccharide residue. Combining the chemical shift of the terminal group proton and the related two-dimensional spectra, the hydrocarbon of each individual monosaccharide can be attributed.

[0084] The presence of xylose and mannose was detected during the monosaccharide composition analysis. They may exist during the methylation process, but their contents are relatively low, and the related carbon and hydrogen absorption peaks in the one-dimensional nuclear magnetic spectrum overlap relatively greatly with those of the same type of monosaccharide in other linkage forms, making it not easy to distinguish. Therefore, during the analysis of the methylation results and nuclear magnetic results, the possible types of glycosidic bonds in this part were not attributed. However, due to their low contents, they do not affect the estimation of the main structural units of pollen polysaccharide 1. The attribution of carbon and hydrogen of the main monosaccharide residues in pollen polysaccharide 1 is 1 the \(^1\)H-NMR spectrum and 13The 13C-NMR spectrum, combined with the two-dimensional spectra of HSQC (Figure 11), COSY (Figure 12), and TOCSY (Figure 13), and the detection results of methylation and related literature reports, are shown in Table 6.

[0085] Since the HMBC two-dimensional nuclear magnetic spectrum reflects the spatial correlation between carbon and hydrogen, the linkage order of monosaccharide residues can be estimated by the correlation two-dimensional spectrum. As is clear from Figure 14, there are multiple cross-peaks in the HMBC spectrum for the related carbon and hydrogen of each monosaccharide residue. By analyzing this, the linkage situation between related monosaccharide residues can be identified.

[0086] Through the analysis of nuclear magnetic results, the carbon and hydrogen assignments of the related monosaccharide residues of pollen polysaccharide 1 are clarified. The analysis results of the linkage form of the monosaccharide residues are almost consistent with the methylation structure analysis results, and the related glycosidic bond configuration is determined. By analyzing this two-dimensional spectrum, the binding order of different monosaccharide residues is determined, and the molecular structure characteristics of pollen polysaccharide 1 are substantially elucidated. According to the results of methylation and nuclear magnetic analysis, the main chain structure of pollen polysaccharide 1 is mainly composed of α-L-1,3,5-Araf linked together, 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. JPEG2025523229000013.jpg44170However, 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 UV 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 the corresponding chromophore is lacking in the molecular structure of pollen polysaccharide, which is also a factor that makes it difficult to rapidly 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 macromolecular 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 found 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 contents of glucose, xylose, fucose, and mannose are too low. Therefore, in the methylation analysis, only the PMAAs of arabinose and galactose were detected, and the PMAAs of glucose, xylose, fucose, and mannose were not detected. However, since the contents of glucose, xylose, fucose, and mannose are low, it does not affect the analysis and determination of the main glycosidic bond forms of pollen polysaccharide 2. Figure 18 shows the total ion chromatogram detected by GCMS after the methylation treatment, hydrolysis, and derivatization treatment of pollen polysaccharide 2. From the figure, mainly six kinds of methylated glycosyl group ion fragments were selected, and the mass spectra (Figure 19) corresponding to the peaks of methylated glycosyls in the figure were correlated with the standard spectrum library search (Standard spectrum library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / MS / pmaa / pframe.html), and combined with the monosaccharide composition measurement results, the correlated linkage forms of individual monosaccharide residues were estimated, providing the information necessary for the estimation of the repeating unit structure of pollen polysaccharide 2.

[0090] Methylation Analysis Results of Pollen Polysaccharide 2 JPEG2025523229000014.jpg30170JPEG2025523229000015.jpg25170Note: 2,3-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 so on by analogy.

[0091] As shown in Table 7, the 2-methylation analysis sample of pollen polysaccharide 2 mainly contains six types of glycosyl ion fragments, which are derived from galactose and arabinose respectively. Galactose mainly exists in the form of →3,6)-Galp-(1→ linkage, and arabinose mainly exists in the forms of →3,5)-Araf-(1→ and →5)-Araf-(1→. Based on the linkage form of the glycoside bond of the sugar residue of pollen polysaccharide 2 and the characteristics of the pollen polysaccharide structure, it can be preliminarily inferred that the main chain of pollen polysaccharide 2 is linked to other glycoside bonds by 1,3,5-Araf and 1,3,6-Galp linkages; the branched chain may mainly consist of 1,5-Araf and 1,6-Glcp.

[0092] 2.2.2 NMR measurement results of pollen polysaccharide 2 Monosaccharide residues of pollen polysaccharide 2 1 H and 13 Chemical shift assignment of C NMR JPEG2025523229000016.jpg32170

[0093] Figures 20 to 22 show the 1 H-NMR spectrum, 13 C-NMR spectrum, and DEPT-135 spectrum of pollen polysaccharide 2, 1 In the H-NMR spectrum, the presence of five more prominent signal peaks in the terminal hydrogen-related region was shown, indicating that pollen polysaccharide 2 has five monosaccharide residues, each having corresponding chemical shifts of 5.18 ppm, 5.08 ppm, 4.83 ppm, 4.41 ppm, and 4.46 ppm (Table 8). The chemical shifts of the hydrogens corresponding to the second to sixth carbons of the five corresponding sugar residues are in the range of 4.30 to 3.50 ppm of chemical shift. The chemical shift of the terminal carbon corresponding to the terminal hydrogen of the glycoside bond is in the HSQC spectrum and 13It can be determined from the 13C-NMR spectrum. HSQC (Figure 23), COSY (Figure 24) and TOCSY (Figure 25) are two-dimensional spectral diagrams of hydrocarbon attribution and ortho-hydrogen correlation and total hydrogen correlation in the same monosaccharide residue. Through analysis, the attribution of other carbons and hydrogens in the correlated monosaccharide residues can be carried out. The HMBC spectrum (Figure 26) can determine the spatial correlation of carbon and hydrogen, and further judge the linkage order of monosaccharide glycosidic bonds.

[0094] From methylation analysis and correlated nuclear magnetic results analysis, it can be determined that pollen polysaccharide 2 is composed of arabinose linked through α-L-1,3,5-Araf to galactose and through β-D-1,3,6-Galp to form the main chain. A branched group is linked to the 5-position of arabinose and the 6-position of galactose, and the branches of the linked branched groups mainly consist of α-L-1,5-Araf and β-D-1,6-Gal. The main repeating structural unit of pollen polysaccharide 2 is as follows. JPEG2025523229000017.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 characteristic absorption peaks in the visible light region. In the ultraviolet region, no absorption peaks are seen at either 280 nm or 260 nm, indicating that pollen polysaccharide 2 is a simple polysaccharide with high purity.

[0096] 2.2.4 Infrared spectroscopy measurement results As shown in Figure 28, the absorption band of pollen polysaccharide 2 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, 3288 m -1 is the stretching vibration absorption peak of O-H, which is a characteristic peak of saccharides. 2931 cm -1 and 2871 cm -1There is one absorption peak, which can be attributed to C-H stretching vibration. 1641m -1 There is an absorption peak, which can be attributed to crystal water. 1548cm -1 There is an absorption peak, which can be attributed to C=O stretching vibration. 1440cm -1 、1402cm -1 There is an absorption peak, which can be attributed to C-O stretching vibration. 1309cm -1 、1243cm -1 、1081cm -1 There is an absorption peak, which can be attributed to O-H bending vibration. 896cm -1 There is an absorption peak, which can be attributed to the C-H 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 by the modified Needs method. The completely methylated pollen polysaccharide 3 was converted into partially methylated glycol acetate derivatives (PMAAs) by treatments such as hydrolysis, reduction, and derivatization. The total ion chromatogram of the methylation analysis of pollen polysaccharide 3 was obtained as shown in Figure 29 by GC-MS analysis. The mass spectra (Figure 30) corresponding to the peaks of methylated glycosyls in the figure were correlated with the standard spectral library search and collated (Standard spectral library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / ms / pmaa / pframe.html). While determining the type of partially methylated glycosyl groups, the relative molar ratio was calculated from the peak area of the chromatogram peaks, and the structural information necessary for estimating the repeating unit structure of pollen polysaccharide 3 could be provided in combination with the measurement results of the monosaccharide composition.

[0098] Results of methylation analysis of pollen polysaccharide 3 JPEG2025523229000018.jpg371702, 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 so on by analogy.

[0099] Pollen polysaccharide 3 mainly contains arabinose and galactose. Since the contents of rhamnose, glucose, xylose, fucose, and mannose are too low, only the PMAAs of arabinose and galactose were detected in the methylation analysis, and the PMAAs of rhamnose, glucose, xylose, fucose, and mannose were not detected. However, because the contents of rhamnose, glucose, xylose, fucose, and mannose are low, it does not affect the analysis and judgment of the main glycosidic bond linkage forms of pollen polysaccharide 3. The total ion chromatogram was detected by GC-MS, and mainly 5 kinds of methylated glycosylated ion fragments were selected from Figure 30.

[0100] As shown in Table 9, the pollen polysaccharide 3 methylation analysis sample mainly contains 5 kinds of glycosyl ion fragments, which are derived from galactose and arabinose respectively. Galactose mainly exists in the linkage forms of →3,6)-Galp-(1→ and →3)-Galp-(1→, and arabinose mainly exists in the forms of →3,5)-Araf-(1→ and →5)-Araf-(1→. Based on the linkage forms of the sugar residue glycosidic bonds of pollen polysaccharide 3 and the characteristics of the pollen polysaccharide structure, it can be preliminarily estimated that the main chain of pollen polysaccharide 3 is linked to other glycosidic bonds by →3,6)-Galp-(1→ and →3)-Galp-(1→ linkages; the branched chains may mainly consist of →3,5)-Araf-(1→ and →5)-Araf-(1→.

[0101] 2.3.2 NMR measurement results of pollen polysaccharide 3 Monosaccharide residues of pollen polysaccharide 3 1 H and 13 13C NMR chemical shift assignments JPEG2025523229000019.jpg46170

[0102] Figures 31, 32, and 33 show the 1 H-NMR spectrum, 13 C-NMR spectrum, and DEPT-135 spectrum of pollen polysaccharide 3. There are six prominent signal peaks in the terminal hydrogen-related region, indicating that pollen polysaccharide 3 has six monosaccharide residues. The corresponding chemical shifts are 5.17 ppm, 5.15 ppm, 5.09 ppm, 4.83 ppm, 4.58 ppm, and 4.40 ppm, respectively. The chemical shifts of the hydrogens corresponding to carbons 2 to 6 of the six corresponding sugar residues are in the range of 4.30 - 3.50 ppm. The chemical shift of the terminal carbon corresponding to the glycosidic bond terminal hydrogen can be determined from the HSQC spectrum and 13 C-NMR spectrum. HSQC (Figure 34), COSY (Figure 35), and NOSEY (Figure 36) are two-dimensional spectral diagrams of the hydrocarbon assignment and ortho-hydrogen correlation and total hydrogen correlation in the same monosaccharide residue. Through analysis, the assignment of other carbons and hydrogens in the correlated monosaccharide residues can be carried out. The HMBC spectrum (Figure 37) can determine the spatial correlation of carbon and hydrogen and further judge the linkage order of the monosaccharide glycosidic bond.

[0103] From methylation analysis and correlation nuclear magnetic results analysis, it can be determined that pollen polysaccharide 3 forms a main chain by linking through β-D-1,3,6-Galp and β-D-1,3-Galp from galactose, and the branching groups for linking mainly consist of α-L-1,3,5-Araf and α-L-1,5-Araf. The main repeating structural unit of pollen polysaccharide 3 is as follows. JPEG2025523229000020.jpg57170

[0104] 2.3.3 Ultraviolet Spectrum Measurement Results As shown in Figure 38, pollen polysaccharide 3 does not have characteristic absorption peaks 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 Spectroscopy Results Figure 39 is a typical infrared spectrum of plant polysaccharides. The absorption band of pollen polysaccharide 3 has an absorption peak of stretching vibration of -OH at 3600 - 3200 cm -1 There is an absorption peak of stretching vibration of O-H at 3426 cm, which is a characteristic peak of saccharides. Specifically, there is an absorption peak of stretching vibration of O-H at 3426 cm, which is a characteristic peak of saccharides. There is an absorption peak at 2939 cm -1 There is an absorption peak at 2939 cm, which can be attributed to C-H stretching vibration. There is a weak absorption peak found near 1734 cm -1 which can be attributed to the stretching vibration of C=O of the carboxyl group, indicating that pollen polysaccharide 3 contains a certain amount of uronic acid. There is an absorption peak at 1619 c -1 which can be attributed to crystal water. There are absorption peaks at 1423 cm -1 1145 cm -1 1145 cm, 1091 cm -1 1091 cm -1 which can be attributed to C-O stretching vibration. There is an absorption peak at 895 cm -1 which can be attributed to the C-H 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 by the modified Needs method. Fully methylated pollen polysaccharide 4 was converted into partially methylated glycol acetate derivatives (PMAAs) by treatments such as hydrolysis, reduction, and derivatization. The total ion chromatogram of the methylation analysis of pollen polysaccharide 4 was obtained as shown in Fig. 40 by GC-MS analysis. The mass spectra (Fig. 41) corresponding to the peaks of the methylated glycosyls in the figure were correlated with and matched by searching the standard spectrum library (Standard Spectrum Library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / ms / pmaa / pframe.html). While determining the type of the partially methylated glycosyl group, the relative molar ratio was calculated from the peak area of the chromatogram peak, and structural information necessary for estimating the repeating unit structure of pollen polysaccharide 4 could be provided in combination with the measurement results of the monosaccharide composition.

[0107] Results of Methylation Analysis of Pollen Polysaccharide 4 JPEG2025523229000021.jpg691702, 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 so on by analogy.

[0108] Pollen polysaccharide 4 mainly contains arabinose and galactose, and the contents of glucose, xylose, fucose, and mannose are too low. Therefore, only the PMAAs of arabinose and galactose were detected in the methylation analysis, and the PMAAs of xylose, fucose, and mannose were not detected. However, because the contents of glucose, xylose, fucose, and mannose are low, it does not affect the analysis and determination of the main glycosidic bond linkage forms of pollen polysaccharide 4. Fig. 40 shows that after the methylation treatment, hydrolysis, and derivatization treatment of pollen polysaccharide 4, the total ion chromatogram was detected by GC-MS, and mainly 9 kinds of methylated and derived glycosyl ion fragments were selected from Fig. 41.

[0109] As shown in Table 11, the 4-methylated analysis sample of pollen polysaccharide mainly contains nine kinds of glycosyl ion fragments, which are derived from galactose, arabinose, and glucose, respectively. Galactose mainly exists in the linked form of →6)-Galp-(1→, and arabinose mainly exists in the forms of →3,5)-Araf-(1→ and →5)-Araf-(1→. Based on the linkage form of the glycoside bond of the sugar residue of pollen polysaccharide 4 and the characteristics of the pollen polysaccharide structure, it can be preliminarily estimated that the main chain of pollen polysaccharide 4 is linked to other glycoside bonds by 1,3,5-Araf and 1,6-Galp linkages; the branched chain may mainly consist of 1,5-Araf, →3,6)-Galp-(1→, and 1,3-Galp.

[0110] 2.4.2 NMR measurement results of pollen polysaccharide 4 Monosaccharide residues of pollen polysaccharide 4 1 H and 13 C NMR chemical shift assignments JPEG2025523229000022.jpg38170

[0111] Figures 42, 43, and 44 are the 1 H-NMR spectrum, 13 C-NMR spectrum, and DEPT-135 spectrum of pollen polysaccharide 4, respectively, and 1In the H-NMR spectrum, seven prominent signal peaks exist in the terminal hydrogen-related region, indicating that pollen polysaccharide 4 has seven monosaccharide residues (Table 12). The corresponding chemical shifts are 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 of the hydrogens corresponding to the second to sixth carbons of the seven corresponding sugar residues are in the range of 4.30 - 3.50 ppm. The chemical shift of the terminal carbon corresponding to the glycosidic bond terminal hydrogen can be determined from the HSQC spectrum (Figure 45) and the 13C-NMR spectrum. HSQC, COSY (Figure 46), and TOCSY (Figure 47) are two-dimensional spectral diagrams of the hydrocarbon assignment and ortho-hydrogen correlation and total hydrogen correlation in the same monosaccharide residue. Through analysis, the assignment of other carbons and hydrogens in the correlated monosaccharide residues can be carried out. The HMBC spectrum (Figure 48) can determine the spatial correlation of carbon and hydrogen and further judge the linkage order of the monosaccharide glycosidic bond.

[0112] Through methylation analysis and correlation nuclear magnetic resonance result analysis, it was found that pollen polysaccharide 4 forms the main chain by linking arabinose through α-L-1,3,5-Araf to galactose and β-D-1,3)-Galp. It was found that branched chains mainly composed of T-α-L-Araf-(1→5)-α-L-Araf-(1 are bonded to the 5-position and 3-position of arabinose. Furthermore, there is another branched chain structure of →3)-β-D-Galp-(1→6)-β-D-Galp-(1→. The main repeating structural units of pollen polysaccharide 4 are as follows. JPEG2025523229000023.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 characteristic absorption peaks 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 Spectroscopy Results Figure 50 shows the typical infrared spectrum of plant polysaccharides and is the infrared result of pollen polysaccharide 4. Absorption bands have absorption peaks for the stretching vibration of -OH at 3600 - 3200 cm -1 There is an absorption peak for the stretching vibration of O-H at 3322 m, which is a characteristic peak of saccharides. At 2931 cm -1 There is an absorption peak for the stretching vibration of O-H, which is a characteristic peak of saccharides. At 2875 cm -1 There is one absorption peak at 2875 cm -1 which is attributed to the stretching vibration of C-H. At 1641 m -1 There is an absorption peak, which is attributed to crystal water. At 1544 cm -1 There is an absorption peak, which is attributed to the stretching vibration of C=O. At 1407 cm -1 There is one absorption peak at 1407 cm -1 which is attributed to the stretching vibration of C-O. At 1309 cm -1 At 1241 cm -1 At 1076 cm -1 At 1047 cm -1 There are absorption peaks, which are attributed to the bending vibration of O-H. At 894 cm

[0115] <Example 7> Growth Promotion Effect on Tobacco Experimental Samples: The active fraction Fr of pollen polysaccharides, the active fraction Fr-1 of pollen polysaccharides, the active fraction Fr-1-5 of pollen polysaccharides, and pollen polysaccharide 1-4 were all prepared into stock solutions with a polysaccharide concentration of 10 mg / ml in pure water and then diluted with water according to the experimental plan for use. 1. Experimental Plan: Drug Concentration Design JPEG2025523229000024.jpg1171702. Experimental Method: After the treatment agent was prepared according to the above table, it was evenly sprayed on the young leaves of tobacco seedlings. The treatment was repeated 6 times, with each repetition being for 1 plant. To prevent the chemical from seeping into the soil and affecting the experimental results, it is appropriate to ensure that the chemical solution does not drip. The culture period was set under the conditions of a temperature of 28 °C, light of 2000 lux, 14 h / 10 h (day / night), and humidity of 65%. The RGB AREA_MM parameter value of tobacco (leaf area / mm 2 ) was recorded using a plant phenotype analysis system before and 7 days after the treatment, respectively. The growth rate of the leaf area was calculated using the following formula to evaluate the growth promotion effect of each chemical agent. Leaf area increase rate (%) = (final leaf area - initial leaf area) × 100% / initial leaf area 3. Experimental results: The leaf area growth rates of different treatment groups 7 days after the treatment are shown in the following table. From the experimental results, a growth promotion effect was observed in each pollen polysaccharide treatment group within the concentration range of 0.03 - 30 ppm. Among them, the optimal growth promotion concentration of pollen polysaccharide active site Fr, pollen polysaccharide active site Fr-1, and active site Fr-1-5 is 3 ppm, and in terms of the growth promotion effect, active site Fr-1-5 > active site Fr-1 > active site Fr.

[0116] The optimal growth promotion concentration of pollen polysaccharide 1 is 0.3 ppm; the optimal growth promotion concentration of pollen polysaccharide 2 is 3 ppm; the optimal growth promotion concentration of pollen polysaccharide 3 is 0.3 ppm; the optimal growth promotion concentration of pollen polysaccharide 4 is 0.03 ppm. The growth promotion effects of the above polysaccharides are all superior to those of pollen polysaccharide active site Fr, active site Fr-1, and active site Fr-1-5 at the same concentration. The effect of pollen polysaccharide 4 is the highest, and it can exhibit the optimal growth promotion effect at an extremely low concentration.

[0117] Measurement results of tobacco growth promotion in each treatment group JPEG2025523229000025.jpg137170

[0118] <Example 8> Growth promotion effect on pakchoi 1. Experimental samples: The active fraction Fr of pollen polysaccharide, the active fraction Fr-1 of pollen polysaccharide, the active fraction Fr-1-5 of pollen polysaccharide, and pollen polysaccharide 1-4 were all prepared into a stock solution with a polysaccharide concentration of 10 mg / ml in pure water, and then diluted with water according to the experimental plan before use. 2. Experimental plan: Refer to Table 13. 3. Experimental method: Select the seedlings of pakchoi with uniform growth (3-4 leaves). After preparing the treatment agents according to the above table, perform root irrigation treatment on the seedlings, repeat the treatment 6 times each, repeat with 1 plant each, with the drug injection amount of 80 ml per plant, and place them in a plant culture room at 25°C for cultivation. Set the light irradiation intensity at 3000 lux, 14 h / 10 h (day / night), and humidity at 65%, and keep the water and fertilizer management level uniform during the cultivation period. Measure the leaf length, leaf width, above-ground fresh weight, and SPAD value 7 days after the drug treatment. 4. Experimental results: The measurement results of each treatment group 7 days after the drug treatment are shown in the following table. From the experimental results, each pollen polysaccharide treatment group showed a growth promotion effect on pakchoi in the concentration range of 0.03-30 ppm. Among them, the optimal growth promotion concentration of the active fraction Fr, the active fraction Fr-1, and the active fraction Fr-1-5 of pollen polysaccharide is 3 ppm. It can be seen that the growth promotion effect of the active fraction Fr-1-5 is better than that of the active fraction Fr-1.

[0119] The optimal growth promotion concentration of pollen polysaccharide 1 is 0.3 ppm, the optimal growth promotion concentration of pollen polysaccharide 2 is 3 ppm, the optimal growth promotion concentration of pollen polysaccharide 3 is 0.3 ppm, and the optimal growth promotion concentration of pollen polysaccharide 4 is 0.03 ppm. The growth promotion effect is better than that of the active fraction Fr, the active fraction Fr-1, and the active fraction Fr-1-5 of pollen polysaccharide. Among them, pollen polysaccharide 4 can achieve the optimal growth promotion effect at an extremely low concentration.

[0120] Measurement results of growth promotion of pakchoi in each treatment group 7 days after the drug treatment JPEG2025523229000026.jpg158170

[0121] <Example 9> Cold tolerance 1. Experimental samples: The active fraction Fr of pollen polysaccharide, the active fraction Fr-1 of pollen polysaccharide, the active fraction Fr-1-5 of pollen polysaccharide, and pollen polysaccharide 1-4 were all prepared into a stock solution with a polysaccharide concentration of 10 mg / ml in pure water, and then diluted with water according to the experimental plan for use. 2. Experimental plan: Refer to Table 13. 3. Experimental method: After the treatment agents were prepared according to the above table, they were evenly sprayed on the seedling leaves of tobacco. The treatment was repeated 6 times, with each repetition being 1 plant. To prevent the chemicals from penetrating into the soil and affecting the experimental results, it is appropriate to ensure that the chemical solution does not drip. One day after the seedling extension, after cold stress (4°C) treatment for 24 h, recovery was carried out under the condition of 28°C. Before the treatment, 24 h after the low-temperature treatment, and 24 h after the recovery, the chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of tobacco were recorded with a plant phenotype meter. 4. Experimental results: QY-max indicates the theoretical maximum photosynthetic ability of plants, and the smaller the reduction rate, the higher the low-temperature tolerance effect of plants. Fv / Fm-lss indicates the maximum photochemical quantum yield of PSII, and similar to QY-max, the smaller the reduction rate, the higher the low-temperature tolerance effect of plants. As is obvious from the following table, 24 h after the low-temperature treatment, the fluorescence values of QY-max and Fv / Fm-lss in each treatment group decreased significantly. However, in each treatment group using pollen polysaccharide in the concentration range of 0.03 - 30 ppm, the degree of decrease in the fluorescence values of QY-max and Fv / Fm-lss was small, the growth state of the plants was better than that of the water control, and it can be seen that the low-temperature tolerance was strong.

[0122] 24 h after the low-temperature treatment, the optimal low-temperature tolerance concentration of the active fraction Fr, the active fraction Fr-1, and the active fraction Fr-1-5 of pollen polysaccharide was 3 ppm. However, the low-temperature tolerance effect of the active fraction Fr-1-5 was better than that of the active fraction Fr and the active fraction Fr-1. The optimal low-temperature tolerance concentration of pollen polysaccharide 1 and 2 was 0.3 ppm, and the optimal low-temperature tolerance concentration of pollen polysaccharide 3 and 4 was 0.03 ppm.

[0123] Measurement results of 24 h of low-temperature treatment JPEG2025523229000027.jpg150170

[0124] After 24-hour recovery at room temperature, as shown in the following table, the fluorescence values of QY-max and Fv / Fm-lss in each treatment group began to recover, indicating that the growth states of the plants damaged at low temperature were recovering. Among them, the pollen polysaccharide active site Fr, active site Fr-1, and active site Fr-1-5 with a concentration of 3 ppm, and pollen polysaccharide 1 with a concentration of 0.3 ppm significantly promoted the recovery of the plant growth state. Pollen polysaccharide 2 with a concentration of 0.3 ppm significantly promoted the recovery of the plant growth state. Pollen polysaccharide 3 and pollen polysaccharide 4 with a concentration of 0.03 ppm significantly promoted the recovery of the growth state.

[0125] Measurement results of 24-hour room temperature recovery JPEG2025523229000028.jpg161170

[0126] <Example 10> High temperature tolerance 1. Experimental samples: The 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 into a stock solution with a polysaccharide concentration of 10 mg / ml in pure water and then diluted with water according to the experimental plan for use. 2. Experimental plan: Refer to Table 13. 3. Experimental method: Select tobacco seedlings (at the three-leaf stage) with uniform growth. After preparing the treatment agents according to Table 11, spray them evenly on the leaves of the tobacco seedlings. Repeat the treatment 6 times, with 1 plant for each repetition. To prevent the agent from entering the soil and affecting the experimental results, it is appropriate to ensure that the chemical solution does not drip. One day after extending the seedlings, treat them at a high temperature of 40 °C for 48 hours. Record the chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of the tobacco with a plant phenotype meter before the drug and 48 hours after the high-temperature treatment. Sample and measure the malondialdehyde content 48 hours after the high-temperature treatment. 4. Experimental results: QY-max indicates the theoretical maximum photosynthetic capacity of plants, and the smaller the reduction rate, the higher the high-temperature tolerance effect of plants. Fv / Fm-lss indicates the maximum photochemical quantum yield of PSII, and similar to QY-max, the smaller the reduction rate, the higher the high-temperature tolerance effect of plants. The non-photochemical quenching coefficient NPQ-lss reflects the ability of plants to dissipate excessive light energy as heat, reflecting the light protection ability. When plants are stressed, the light energy absorbed by plants is directly converted into thermal energy and dissipated without being used for photosynthesis to avoid damage to plants. At this time, the plant NPQ-lss increases. Therefore, the lower the NPQ-lss value during high-temperature stress, the smaller the damage to plants and the higher the high-temperature tolerance effect.

[0127] As is obvious from the following table, after 48 h of high-temperature treatment, the fluorescence values of QY-max and Fv / Fm-lss in the treatment group decreased significantly, and the fluorescence value of NPQ-lss increased significantly. However, in the concentration range of 0.03 - 30 ppm, the decrease in the fluorescence values of QY-max and Fv / Fm-lss and the increase in the fluorescence value of NPQ-lss in each treatment group using pollen polysaccharide were smaller. The growth state of plants was better than that of the water control, indicating stronger high-temperature tolerance. After 48 h of high-temperature treatment, the optimal high-temperature tolerance concentrations of the active site Fr, active site Fr-1, and active site Fr-1-5 of pollen polysaccharide were 3 ppm. However, the low-temperature tolerance effect of active site Fr-1-5 was better than that of active site Fr and active site Fr-1. The optimal high-temperature tolerance concentrations of pollen polysaccharides 1 and 2 were 0.3 ppm, and the optimal high-temperature tolerance concentrations of pollen polysaccharides 3 and 4 were 0.03 ppm.

[0128] Measurement results of fluorescence parameters of each treatment group JPEG2025523229000029.jpg145170

Claims

1. A pollen polysaccharide in which the main repeating unit structure is one of the following: Pollen polysaccharide 1: 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→, and the average molecular weight of pollen polysaccharide 1 is 20 to 30 kDa, Pollen polysaccharide 2: 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→, and the average molecular weight of pollen polysaccharide 2 is 5 to 15 kDa, Pollen polysaccharide 3: the average molecular weight of pollen polysaccharide 3 is 60 to 70 kDa, Pollen polysaccharide 4: However, R1'' is T-α-L-Araf-(1→5)-α-L-Araf-(1→, and the pollen polysaccharide 4 has an average molecular weight of 5 to 15 kDa.

2. The pollen polysaccharide according to claim 1, wherein pollen polysaccharide 1 further contains monosaccharide units of fucose, xylose and mannose, pollen polysaccharide 2 further contains monosaccharide units of fucose, glucose, xylose and mannose, pollen polysaccharide 3 further contains monosaccharide units of fucose, rhamnose, glucose, xylose, mannose, galacturonic acid and glucuronic acid, and pollen polysaccharide 4 further contains monosaccharide units of fucose, glucose, xylose, mannose and galacturonic acid.

3. The infrared spectrum of pollen polysaccharide 1 contains one or more absorption peaks at least at 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 , and the infrared spectrum of pollen polysaccharide 2 contains one or more absorption peaks at least at 3288 m －１ , 2931 cm －１ , 2871 cm －１ , 1641 m －１ , 1548 cm －１ , 1440 cm －１ , 1402 cm －１ , 1309 cm －１ , 1243 cm －１ , 1081 cm －１ , 896 cm －１ , and the infrared spectrum of pollen polysaccharide 3 contains one or more absorption peaks at least at 3426 cm －１ , 2939 cm －１ , 1734 cm －１ , 1619 cm －１ , 1423 cm －１ , 1145 cm －１ , 1091 cm －１ , 895 cm －１ , and the infrared spectrum of pollen polysaccharide 4 contains one or more absorption peaks at least at 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 , 894 cm －１ The pollen polysaccharide according to claim 1, characterized by comprising one or more absorption peaks.

4. A method for separating the pollen polysaccharide according to claim 1, wherein the method for separating pollen polysaccharide 1 is (1) Purifying the rapeseed pollen polysaccharide extract by a macroporous adsorption resin column, eluting with water to obtain the pollen polysaccharide active site Fr-1; (2) Passing Fr-1 through an anion exchange chromatography column and eluting the pollen polysaccharide active site Fr-1 with water to obtain the pollen polysaccharide active site Fr-1-1; (3) Further purifying the pollen polysaccharide active site Fr-1-1 using a Sephadex chromatography column, and using an ammonium bicarbonate aqueous solution as the eluent to obtain pollen polysaccharide 1, including the method for separating pollen polysaccharide 2 is (1) Purifying the rapeseed pollen polysaccharide extract by a macroporous adsorption resin column, eluting with water to obtain the primary purified pollen polysaccharide active site Fr-1; (2) Pass the active fraction Fr-1 of pollen polysaccharide through an anion exchange chromatography column and perform gradient elution with a 0 - 0.025 mol / L NaCl solution, and collect the 0.025 mol / L NaCl eluate fraction to obtain the active fraction Fr-1-2 of pollen polysaccharide. (3) Further purify the active fraction Fr-1-2 of pollen polysaccharide using a Sephadex chromatography column, with an aqueous ammonium bicarbonate solution as the eluent to obtain pollen polysaccharide 2. including The method for separating pollen polysaccharide 3 is (1) Purify the rapeseed pollen polysaccharide extract using a macroporous adsorption resin column, elute with water, and obtain the primary purified active fraction Fr-1 of pollen polysaccharide. (2) Pass the active fraction Fr-1 of pollen polysaccharide through an anion exchange chromatography column and perform gradient elution with a 0 - 0.05 mol / L NaCl solution, and collect the 0.05 mol / L NaCl solution of pollen polysaccharide to obtain the active fraction Fr-1-3 of pollen polysaccharide. (3) Further purify the active fraction Fr-1-3 of pollen polysaccharide using a Sephadex chromatography column, with an aqueous ammonium bicarbonate solution as the eluent to obtain pollen polysaccharide 3. including The method for separating pollen polysaccharide 4 is (1) Purify the rapeseed pollen polysaccharide extract using a macroporous adsorption resin column, elute with water, and obtain the primary purified active fraction Fr-1 of pollen polysaccharide. (2) Pass the active fraction Fr-1 of pollen polysaccharide through an anion exchange chromatography column and perform gradient elution with a 0 - 0.25 mol / L NaCl solution, and collect the 0.25 mol / L NaCl solution of pollen polysaccharide to obtain the active fraction Fr-1-4 of pollen polysaccharide. (3) Further purify the active fraction Fr-1-4 of pollen polysaccharide using a Sephadex chromatography column, with an aqueous ammonium bicarbonate solution as the eluent to obtain pollen polysaccharide 4. The method for separating pollen polysaccharide according to claim 1, characterized by including the above.

5. The method for separation according to claim 4, characterized in that the rapeseed pollen polysaccharide extract in step (1) is a crude pollen polysaccharide obtained by water extraction and alcohol precipitation of rapeseed pollen.

6. The macroporous resin column used in step (1) is one selected from a DB-101 macroporous resin column, an S-8 macroporous resin column, an AB-8 macroporous resin column, and an HP-20 macroporous resin column. The ion exchange chromatography column used in step (2) is selected from DEAE cellulose chromatography columns. The Sephadex chromatography column used in step (3) is selected from acrylic Sephadex columns and further selected from an acrylic Sephadex S-400 HR chromatography column. The separation method according to claim 4, characterized in that.

7. In step (3), the separation method according to claim 4, characterized in that the concentration of the ammonium hydrogen carbonate aqueous solution is 0.1 to 0.3 mol / L.

8. The rape pollen polysaccharide active site Fr-1, characterized in that the rape pollen polysaccharide extract is passed through a macroporous resin and eluted with water to obtain the pollen polysaccharide active site Fr-1 containing pollen polysaccharide 1, pollen polysaccharide 2, pollen polysaccharide 3, and pollen polysaccharide 4 according to claim 1.

9. The rape pollen polysaccharide extract is purified with a macroporous resin, and the water elution site is further passed through an ion exchange chromatography column, and all the elution sites of a 0 to 0.25 mol / L sodium chloride solution are collected to obtain the active site Fr-1-5 containing pollen polysaccharide 1, pollen polysaccharide 2, pollen polysaccharide 3, and pollen polysaccharide 4 according to claim 1. The rape pollen polysaccharide active site Fr-1-5, characterized in that.

10. The pollen polysaccharide active site Fr, characterized in that rape pollen is subjected to water extraction and alcohol precipitation to obtain the active site Fr containing pollen polysaccharide 1, pollen polysaccharide 2, pollen polysaccharide 3, and pollen polysaccharide 4 according to claim 1.

11. Use of the pollen polysaccharide according to any one of claims 1 to 3 or the pollen polysaccharide active site according to any one of claims 8 to 10 in the preparation of a plant stress tolerance product or a plant growth promotion product, Furthermore, the plant stress tolerance includes cold tolerance, drought tolerance, salt-alkali tolerance, and high temperature tolerance. Furthermore, when used, a product containing the pollen polysaccharide or the pollen polysaccharide active site is applied to the leaves or roots of the plant. Furthermore, the use of the plant in the preparation of a plant stress tolerance product or a plant growth promotion product of the active site of pollen polysaccharide, the plant including tobacco, pakchoi, baby bok choy, stem lettuce, lettuce, bok choy, wheat, pepper, tomato, citrus, kiwi, cherry, pear, apple.

12. An agricultural product, characterized in that the active ingredient contains the pollen polysaccharide according to any one of Claims 1 to 3, or the active site of the pollen polysaccharide according to any one of Claims 8 to 10.

13. The agricultural product according to Claim 12, further comprising one or more auxiliary agents among a dispersant, a wetting agent, a binder, an emulsifier, a stabilizer, a solvent, and an embedding agent.

14. The agricultural product according to Claim 12, wherein the dosage form is an emulsion, a suspension, a wettable powder, a powder, a granule, an aqueous solution, a mother liquor or a mother powder.

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