Pollen polysaccharide component 3, its separation method and application

Abstract

The application belongs to the technical field of plant extract separation, and particularly relates to pollen polysaccharide, a separation method thereof and application. The application extracts, separates and purifies polysaccharide in rape pollen, and obtains pollen polysaccharide component 3. The pollen polysaccharide component 3 has certain effects in the aspects of plant growth promotion and stress resistance.

Description

Technical Field

[0001] This invention belongs to the field of plant extract separation technology, specifically relating to a pollen polysaccharide component 3 and its separation method and application. Background Technology

[0002] Previous research by the inventors (patents CN 113133454B and CN113133455B) has shown that rapeseed pollen water extract can be used to enhance plant stress resistance and promote plant growth, including vegetables and fruit trees such as bok choy, lettuce, romaine lettuce, Shanghai bok choy, wheat, chili peppers, tomatoes, citrus fruits, kiwifruit, cherries, pears, and apples. The components were also analyzed, revealing the presence of various substances including sugars, proteins, amino acids, and lipids. However, specific active ingredients have not yet been investigated.

[0003] In addition, the literature “Research progress on the relationship between amino acids and plant stress resistance” (Xing Fangfang, Gao Mingfu, Zhou Chuanzhi, Xu Chunying, Fan Lingchao. [J]. Heilongjiang Agricultural Sciences, 2018(03):150-155) reported that amino acid substances can also be used for plant stress resistance.

[0004] Given that rapeseed pollen water extracts may contain various substances that promote plant growth or resist stress, it is currently impossible for those skilled in the art to determine which substance plays a dominant role in promoting plant growth or resisting stress. Therefore, it is necessary to conduct further research on rapeseed pollen extraction. Summary of the Invention

[0005] Polysaccharides are a class of complex carbohydrates formed by the dehydration condensation of multiple monosaccharide molecules. These structural units are linked by glycosidic bonds, with common glycosidic bonds including α-1,3, β-1,6, β-1,4, α-1,4, and β-1,3. Polysaccharides are widely found in animals, plants, and microorganisms, and those from different sources exhibit varying biological activities. Current research indicates that plant polysaccharides possess immunomodulatory, antitumor, antioxidant, anti-aging, hypoglycemic, and hypolipidemic activities. They also possess advantages such as wide availability, biodegradability, high safety, ease of modification, and high environmental compatibility, leading to their widespread application in the food, pharmaceutical, livestock, and aquaculture industries.

[0006] Meanwhile, plant polysaccharides are a class of natural macromolecular polymers with relatively complex structures. Currently, there is limited research on the structure-activity relationship between the specific molecular structure of pollen polysaccharides and their physiological activities. Therefore, it is necessary to isolate and identify pollen polysaccharide compounds to provide reliable theoretical and experimental basis for the future development and research of pollen polysaccharide resources.

[0007] Specifically, this invention extracts, separates, and purifies polysaccharides from rapeseed pollen to obtain a new pollen polysaccharide. Tests show that this pollen polysaccharide has certain effects on promoting plant growth and resisting stress.

[0008] Specifically, the present invention provides one pollen polysaccharide component 3, whose main repeating structural units are as follows:

[0009] Main chain

[0010] branch

[0011] In this invention, Ara represents arabinose; Gal represents galactose; f represents furanose configuration; p represents pyranose configuration; and T represents the terminal group (terminal group) of the polysaccharide molecule.

[0012] The molar ratio of arabinose to galactose is 0.328:0.399.

[0013] In addition to the main repeating structural units mentioned above, the pollen polysaccharides of the present invention also contain trace amounts of the following monosaccharides: fucose, rhamnose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

[0014] The molar ratio of fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid: glucuronic acid is 0.006:0.095:0.328:0.399:0.024:0.027:0.010:0.096:0.014.

[0015] In this invention, the average molecular weight of pollen polysaccharides is 60–70 kDa;

[0016] Furthermore, the average molecular weight of pollen polysaccharides is 65–70 kDa;

[0017] In this invention, the average molecular weight of pollen polysaccharides is 66-68 kDa, for example 66911.38 kDa.

[0018] The pollen polysaccharide of the present invention has an infrared spectrum that includes at least one or more of the following absorption peaks: 3426 cm⁻¹ -1 2939cm -1 1734cm -1 1619cm -1 1423cm -1 1145cm -1 1091cm -1 895cm -1 .

[0019] Pollen polysaccharide component 3 has an absorption band at 3600-3200 cm⁻¹.-1 This is the absorption peak of the stretching vibration of -OH, and the absorption peak in this region is a characteristic peak of carbohydrates. Specifically: 3426 cm⁻¹ -1 The peak at 2939 cm⁻¹ is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. -1 There is an absorption peak at 1734 cm⁻¹, which can be attributed to the CH stretching vibration. -1 The weaker absorption peak appearing nearby is attributed to the stretching vibration of the C=O group in the carboxyl group, indicating that pollen polysaccharide component 3 contains a certain amount of uronic acid. At 1619 cm⁻¹ -1 There is an absorption peak at 1423 cm⁻¹, which can be attributed to the water of crystallization. -1 Location, 1145cm -1 Location, 1091cm -1 An absorption peak is observed at 895 cm⁻¹, which can be attributed to the CO stretching vibration. -1 The absorption peak at this point can be attributed to the CH-angle vibration of the β-terminal epimer of the pyran ring.

[0020] Another objective of this invention is to provide a method for isolating the above-mentioned pollen polysaccharides, comprising the following:

[0021] (1) The rapeseed pollen polysaccharide extract was purified by macroporous adsorption resin column and eluted with water to obtain the preliminarily purified pollen polysaccharide active fraction Fr-1;

[0022] (2) The active part of pollen polysaccharide Fr-1 was eluted by gradient elution with 0-0.05 mol / L NaCl solution through an ion exchange chromatography column, and the 0.05 mol / L NaCl solution fraction was collected to obtain the active part of pollen polysaccharide Fr-1-3.

[0023] (3) The active part of pollen polysaccharide Fr-1-3 was further purified by gel chromatography column, wherein ammonium bicarbonate aqueous solution was used as the eluent to obtain component 3, namely the pollen polysaccharide of the present invention.

[0024] The active site of pollen polysaccharides, Fr-1, on the ion exchange column can be in the form of the original eluent of the macroporous resin, the concentrated original eluent, or a reconstituted solution after the original eluent has been concentrated and dried, etc.

[0025] The active pollen polysaccharide Fr-1-3 on the gel chromatography column can be in the form of the original eluent of the ion exchange column, the concentrated original eluent, or a reconstituted solution of the concentrated and dried original eluent, etc.

[0026] The concentration and drying methods used include, but are not limited to, conventional operating methods such as reduced pressure evaporation, atmospheric pressure evaporation, drying, vacuum drying, thin film drying, and freeze drying. These methods can be used individually or in combination.

[0027] The rapeseed pollen polysaccharide extract mentioned in step (1) refers to crude rapeseed pollen polysaccharide obtained by conventional methods. The conventional methods described in this invention include, but are not limited to, water extraction and preliminary purification after water extraction.

[0028] The water extraction includes, but is not limited to, conventional plant extract extraction methods such as heating extraction, ultrasonic extraction, and microwave extraction.

[0029] In addition, depending on the circumstances, degreasing and decolorization can be performed before extraction to remove impurities. For example, fat-soluble solvents such as ethanol and petroleum ether can be used for extraction, degreasing, and decolorization. The preliminary purification methods after water extraction include, but are not limited to, different methods such as alcohol precipitation and chitosan impurity removal.

[0030] In this invention, if the water extraction and alcohol precipitation method is selected, ethanol is added to the water extract (or the concentrate of the water extract) in the alcohol precipitation step until the ethanol concentration reaches 70-90% v / v. For example, 70%, 71%, 72%, 73%, 74%, 75%, ... 80%, ... 85%, ... 90% v / v can be selected.

[0031] In purification methods, auxiliary methods such as protein removal, decolorization, and small molecule removal can be selectively added depending on the specific circumstances to facilitate subsequent polysaccharide enrichment processes. For example, protein removal reagents such as phenol, trichloroacetic acid, and tannic acid can be used; adsorbents such as cellulose, diatomaceous earth, and activated carbon can be used for decolorization; and dialysis and other methods can be used to remove small molecules.

[0032] In some specific embodiments of the present invention, the rapeseed pollen polysaccharide extract mentioned in step (1) refers to the crude pollen polysaccharide obtained after water extraction and alcohol precipitation of rapeseed pollen.

[0033] In some specific embodiments of the present invention, the rapeseed pollen polysaccharide extract mentioned in step (1) refers to crude pollen polysaccharide obtained by heating rapeseed pollen with water and removing impurities with chitosan.

[0034] In some specific embodiments of the present invention, rapeseed pollen can be subjected to ultrafine grinding to facilitate the extraction of polysaccharides.

[0035] In some specific embodiments of the present invention, the pollen polysaccharide extract of rapeseed pollen can be freeze-dried under vacuum to produce crude pollen polysaccharide solid.

[0036] In the technical solution of the present invention, the macroporous resin column used in step (1) is a non-polar column.

[0037] In this 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, and HP-20 macroporous resin column; further, the macroporous resin column is selected from HP-20 macroporous resin column.

[0038] In the technical solution of the present invention, the ion exchange column used in step (2) is an anion exchange column, and the anion includes, but is not limited to, different ion exchange columns such as DEAE-cellulose, DEAE-agarose gel, and DEAE-glucose gel, and is further selected from DEAE cellulose-52 chromatography column.

[0039] In the technical solution of the present invention, the gel chromatography column used in step (3) includes, but is not limited to, different gel chromatography columns such as dextran gel (Sephadex G) chromatography column and polyacrylic acid gel Toyopearl HW chromatography column; and is further selected from acrylic dextran gel S-400HR chromatography column, Sephadex LH-20 chromatography column, etc.

[0040] In the technical solution of 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.

[0041] In some specific embodiments of the present invention, the pollen polysaccharide active part Fr-1 initially purified in step (1) and the pollen polysaccharide active part Fr-1-3 in step (2) can be obtained as solids by dialysis and freeze-drying for later use.

[0042] Furthermore, the dialysis refers to the removal of substances with a molecular weight cutoff of at least 3500 Da.

[0043] In some specific embodiments of the present invention, the eluent containing component 3 in step (3) is concentrated under reduced pressure, desalted by dialysis (3500 Da), and freeze-dried to obtain a solid of pollen crude polysaccharide component 3.

[0044] In this invention, the degree of concentration during the concentration step can be determined by those skilled in the art based on the amount of eluent added during elution and the final requirements of the product.

[0045] In some specific embodiments of the present invention, the sugar content in steps (2) and (3) is detected by the phenol-sulfuric acid method, and the absorbance at 490 nm is detected.

[0046] Another objective of this invention is the application of pollen polysaccharide component 3 in the preparation of plant stress-resistant products.

[0047] The plant stress resistance described in this invention includes resistance to high temperature, cold, drought, and salinity.

[0048] In this invention, the product containing pollen polysaccharides is applied to the leaves or roots of plants.

[0049] When the pollen polysaccharide product of this invention is prepared into a solution for use, the concentration of the main active ingredient can be selected according to actual needs.

[0050] For example, the concentration of pollen polysaccharide component 3 can be selected from 0.01ppm to 500ppm; it can be selected from 0.01ppm to 100ppm; it can be selected from 0.01ppm to 50ppm; it can also be selected from 0.03ppm, ... 0.05ppm, ... 0.1ppm, ... 0.2ppm, ... 0.5ppm, ... 1.0ppm, ... 3.0ppm, ... 5.0ppm, ... 10ppm, ... 20ppm, ... 30ppm, etc.

[0051] Another objective of this invention is the application of pollen polysaccharide component 3 in the preparation of products that promote plant growth.

[0052] The plants mentioned in this invention include, but are not limited to, cash crops and food crops, such as bok choy, baby bok choy, lettuce, romaine lettuce, Shanghai bok choy, wheat, chili peppers, tomatoes, citrus fruits, kiwifruit, cherries, pears, apples, and tobacco.

[0053] The term "cash crops" encompasses a wide variety of crops, including but not limited to fiber crops (such as cotton and hemp), oil crops (such as sesame and peanuts), sugar crops (such as sugarcane and sugar beets), phytosanitary crops (tobacco), medicinal crops, dye crops, ornamental crops, fruits, and other cash crops.

[0054] The term "grain crops" includes, but is not limited to, cereal crops (wheat, rice, corn), tuber crops (including sweet potatoes, potatoes, etc.) and legume crops (including soybeans, broad beans, peas, mung beans, etc.).

[0055] The present invention also provides an agricultural product, wherein the active ingredient of the product includes pollen polysaccharide component 3.

[0056] In this invention, direct pollen polysaccharide component 3 can be used as a single agent. In order to make the product stable and easy to transport and store, excipients can also be added to make corresponding dosage forms. The excipients can be conventional excipients in the art, such as dispersants, wetting agents, binders, emulsifiers, stabilizers, solvents, encapsulating agents, etc.

[0057] On the other hand, the pollen polysaccharide component 3 of the present invention can be used as a synergist in combination with foliar fertilizers, water-soluble fertilizers, compound fertilizers, pesticides and other products.

[0058] The dosage forms of the products described in this invention include, but are not limited to, conventional agricultural product preparations such as emulsifiable concentrates, suspensions, wettable powders, powders, granules, aqueous solutions, mother liquors, and mother powders.

[0059] The beneficial effects of this invention are as follows:

[0060] This invention discovers a new polysaccharide and isolates it from rapeseed pollen for the first time. This polysaccharide can be used to enhance plant stress resistance and promote plant growth.

[0061] The "rapeseed" mentioned in this invention refers to a herbaceous crop belonging to the Brassicaceae family and the Brassica genus. Common types of "rapeseed" include, but are not limited to, Chinese cabbage type rapeseed, Brassica napus type rapeseed, black mustard type rapeseed, mustard type rapeseed, and Ethiopian mustard. The "pollen" mentioned in this invention is obtained from the above-mentioned "rapeseed". Attached Figure Description

[0062] Figure 1 Gradient elution curve of crude pollen polysaccharides on a DEAE cellulose-52 anion exchange column;

[0063] Figure 2 Elution curves of the four components of pollen polysaccharides on an S-400HR propylene dextran gel column;

[0064] Figure 3 Standard curve of molecular weight distribution of dextran standards;

[0065] Figure 4 Chromatogram of HPGPC determination of pollen polysaccharide component 3;

[0066] Figure 5 Chromatograms of monosaccharide mixed standards and pollen polysaccharide components detected by ion chromatography (A: monosaccharide mixed standards; B: pollen polysaccharide components 3;). The monosaccharide mixed standards are: 1. Rhamnose (Rha) 2. Fucose (Fuc) 3. Arabinose (Ara) 4. Xylose (Xyl) 5. Mannose (Man) 6. Glucose (Glc) 7. Galactose (Gal) 8. Glucuronic acid (GlcA) 9. Galuronic acid (GalA); Solvent peaks: sodium hydroxide peak at 2.0 min, sodium acetate peak at 40 min.

[0067] Figure 6 Total ion chromatogram (TIC) for 3-methylation analysis of pollen polysaccharide components;

[0068] Figure 7 Mass spectra of methylated PMAAs belonging to pollen polysaccharide fraction 3;

[0069] Figure 8 Pollen polysaccharide component 3 1 H-NMR spectrum;

[0070] Figure 9 Pollen polysaccharide component 3 13 C-NMR spectrum;

[0071] Figure 10 DEPT-135 NMR spectrum of pollen polysaccharide fraction 3;

[0072] Figure 11 HSQC spectrum of pollen polysaccharide fraction 3;

[0073] Figure 12 COSY spectrum of pollen polysaccharide fraction 3;

[0074] Figure 13 TOCSY spectra of pollen polysaccharide fraction 3;

[0075] Figure 14 HMBC spectrum of pollen polysaccharide fraction 3;

[0076] Figure 15 : Ultraviolet absorption spectrum of pollen polysaccharide component 3;

[0077] Figure 16 Infrared absorption spectrum of pollen polysaccharide component 3. Detailed Implementation

[0078] The technical solution of the present invention will be clearly and completely described below. Of course, 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 effort are within the scope of protection of the present invention. It should be noted that any processes not specifically described in detail below are those that those skilled in the art can implement or understand by referring to the prior art. Reagents or instruments used without specifying the manufacturer are considered to be conventional products that can be purchased commercially.

[0079] Example 1: Extraction of pollen polysaccharides

[0080] Rapeseed pollen was ultra-finely pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated pollen powder was weighed, and then subjected to ethanol to remove free monosaccharides, petroleum ether for defatting and decolorization, followed by ethanol defatting, hot water extraction, and alcohol precipitation. The precipitate was collected and washed successively with anhydrous ethanol, diethyl ether, and acetone. Finally, the washed precipitate was reconstituted with water, pre-frozen at -20°C, and freeze-dried under vacuum to obtain hot water-extracted crude pollen polysaccharides.

[0081] Example 2: Extraction of pollen polysaccharides

[0082] Rapeseed pollen was mixed with water, heated for extraction, filtered, and chitosan was added to the filtrate. The mixture was kept at a constant temperature and allowed to stand, resulting in solid-liquid separation. The obtained liquid was the crude pollen polysaccharide extract. After concentration under reduced pressure, the extract was pre-frozen at -20°C and then freeze-dried under vacuum to obtain the crude pollen polysaccharide.

[0083] Example 3: Extraction of pollen polysaccharides

[0084] Rapeseed pollen was ultra-finely pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated pollen powder was weighed, and then subjected to ethanol to remove free monosaccharides, petroleum ether for defatting and decolorization, ultrasonic extraction, and alcohol precipitation. The precipitate was collected and washed successively with anhydrous ethanol, diethyl ether, and acetone. Finally, the washed precipitate was reconstituted with water, pre-frozen at -20°C, and freeze-dried under vacuum to obtain hot water-extracted crude pollen polysaccharides.

[0085] Example 4: Isolation and purification of pollen polysaccharides

[0086] Step (1) HP-20 macroporous adsorption resin chromatography column purification: The crude pollen polysaccharide was initially purified using an HP-20 macroporous adsorption resin chromatography column (Φ4.0cm×40cm). The specific steps are as follows: Weigh a certain amount of crude pollen polysaccharide, add a certain amount of water, and prepare a 10-20 mg / mL crude pollen polysaccharide solution. The single sample loading volume is 10 mL. After adsorption for 3 hours using the HP-20 macroporous adsorption resin chromatography column, wash the column with 5 column volumes of water, collect the sample, and obtain the active fraction of pollen polysaccharide, Fr-1. Concentrate under reduced pressure at 48℃, dialyze (3500 Da), and freeze-dry for later use.

[0087] Step (2) DEAE cellulose-52 chromatography column separation and purification: The active part of pollen polysaccharide Fr-1 was separated and purified using a DEAE cellulose-52 chromatography column (Φ3.5cm×30cm). The specific steps are as follows: ① Load 100mg of pollen polysaccharide active part Fr-1 solution; ② Elute, the eluent is water, 0.025, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.4 and 0.5mol / L NaCl solution in sequence, the flow rate is 1.2mL / min, and 6mL is collected from each tube; ③ Collect, the sugar content is detected by the phenol-sulfuric acid method in separate tubes, and the absorbance value at 490nm is detected by an enzyme-linked immunosorbent assay (ELISA) reader. The elution curve is plotted with the number of collection tubes as the x-axis and the absorbance value of the collected solution as the y-axis. Collect the NaCl elution peaks, combine the same components to obtain the fractional components of pollen polysaccharides (pollen polysaccharide active fraction Fr-1-1, pollen polysaccharide active fraction Fr-1-2, pollen polysaccharide active fraction Fr-1-3, pollen polysaccharide active fraction Fr-1-4). The above active fractions are concentrated under reduced pressure at 48℃, desalted by dialyzing (3500Da), and freeze-dried for later use.

[0088] Depend on Figure 1It was found that by using a DEAE cellulose-52 anion exchange column and eluting crude pollen polysaccharides with gradient NaCl solutions of different concentrations, four elution peaks were obtained: the water-eluted fraction (active part of pollen polysaccharide Fr-1-1), the 0.025 mol / L NaCl solution-eluted fraction (active part of pollen polysaccharide Fr-1-2), the 0.05 mol / L NaCl solution-eluted fraction (active part of pollen polysaccharide Fr-1-3), and the 0.25 mol / L NaCl solution-eluted fraction (active part of pollen polysaccharide Fr-1-4). The eluents corresponding to each elution peak were collected, concentrated under reduced pressure, dialyzed, and freeze-dried to obtain the preliminary fractionation of crude pollen polysaccharides into solids containing the active parts of pollen polysaccharides Fr-1-1, Fr-1-2, Fr-1-3, and Fr-1-4.

[0089] Step (3) Purification using S-400HR propylene dextran gel chromatography column: Finally, the fractions of pollen polysaccharides were further purified using an S-400HR propylene dextran gel chromatography column (Φ1.0cm×100cm). The specific steps are as follows: ① Load the fractional solutions, with a single loading volume of 20mg; ② Elute, using 0.2mol / L ammonium bicarbonate as the elution phase, with a flow rate of 0.2mL / min, collecting 3mL per tube, and continuing elution until no sugar is detected. Collect 40 tubes of elution solution for each fraction; ③ Collect, using the phenol-sulfuric acid method to detect the sugar content in separate tubes, and using an enzyme-linked immunosorbent assay (ELISA) reader to detect the absorbance at 490nm, plotting the elution curve, collecting the elution peaks, merging the same fractions, concentrating under reduced pressure at 48℃, dialysis (3500Da) to remove ammonia, and freeze-drying to obtain the purified fractions of each pollen polysaccharide.

[0090] Four pollen polysaccharide fractions (active fractions Fr-1-1, Fr-1-2, Fr-1-3, and Fr-1-4) separated by a DEAE cellulose-52 anion exchange column were further purified using an S-400HR propylene dextran gel chromatography column, eluting each fraction with 0.2 mol / L ammonium bicarbonate. Figure 2 It was found that after elution with 0.2 mol / L ammonium bicarbonate, each of the four components yielded a single elution peak, which was named component 1, component 2, component 3, and component 4, respectively. The eluents corresponding to the elution peaks were collected (component 1: tubes 21–31; component 2: tubes 20–26; component 3: tubes 18–26; component 4: tubes 20–23), and the purified pollen polysaccharide components were obtained by vacuum concentration, dialysis, and freeze-drying.

[0091] Purity identification and molecular weight determination of pollen polysaccharide purified components

[0092] Determination methods: The purity and molecular weight of each purified pollen polysaccharide fraction were determined by high-performance liquid chromatography-gel permeation chromatography (HPGPC). The test conditions were as follows: Agilent 1260 series HPLC system; refractive index detector (RID); Shodex OHpak SB-804HQ (7.8 mm × 300 mm) gel permeation column; mobile phase: 0.1 mol / L Na₂SO₄; flow rate: 0.5 mL / min; column oven temperature: 35℃; injection volume: 20 μL; instrument test time: 24 min.

[0093] Plotting the standard curve: A series of dextran standards with different 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 filter membrane, the solution was tested according to the HPGPC test conditions, and the retention time was recorded. A standard curve was plotted with retention time (min) on the x-axis and the logarithm of the molecular weight of the dextran standard (Log MW) on the y-axis. A regression equation was fitted to calculate the molecular weight of different components of pollen polysaccharides. The resulting standard curve was y = -0.322x + 9.4365. (See details below.) Figure 3 As shown.

[0094] Determination of purity and molecular weight of pollen polysaccharide purified fractions: 10 mg of pollen polysaccharide purified sample was dissolved in 2 mL of 0.1 mol / L Na2SO4 solution. After complete dissolution, a polysaccharide solution with a concentration of 5 mg / mL was prepared. The solution was filtered through a 0.22 μm aqueous filter membrane and analyzed according to HPGPC test conditions. Chromatograms of each pollen polysaccharide purified fraction were recorded. The purity of each polysaccharide sample was evaluated based on the number of peaks and the symmetry of peak shape on the chromatogram. The molecular weight of each polysaccharide sample was calculated based on the retention time on the chromatogram and compared with the molecular weight standard curve of dextran standard.

[0095] The purity and molecular weight of pollen polysaccharide fraction 3 were determined using the HPGPC method, and the results are as follows: Figure 4 As shown. By Figure 4 As can be seen, a single symmetrical peak appeared in the HPGPC chromatogram of pollen polysaccharide purified fraction 3, indicating its high purity. This result is consistent with the separation and purification results of S-400HR propylene dextran gel column, further demonstrating the feasibility of the established pollen polysaccharide separation and purification method, which can prepare pollen polysaccharides with high purity. Based on the previously established molecular weight standard curve, the average molecular weight of fraction 3 was calculated to be 66911.38 Da.

[0096] Example 5: Structural Identification of Pollen Polysaccharides

[0097] 1. Results of the determination of monosaccharide composition of pollen polysaccharides

[0098] Determination Method: The monosaccharide composition of pollen polysaccharide samples was determined by ion chromatography (IC). 10 mg of polysaccharide sample was accurately weighed and placed in an ampoule for acid hydrolysis. The hydrolysate was accurately transferred to a test tube, dried under nitrogen, and 5 mL of distilled water was added and vortexed. 50 μL of the solution was added to 950 μL of distilled water, and the mixture was centrifuged at 12000 rpm for 5 min. The supernatant was then used for IC analysis. Simultaneously, 16 monosaccharide standards (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosyl hydrochloride, glucosamine hydrochloride, N-acetyl-D-glucosamine, guluronic acid, and mannuronic acid) were prepared as standard stock solutions. Precise concentrations of each monosaccharide standard solution were prepared as mixed standards. The monosaccharide composition was determined based on the peak times of the pollen polysaccharide hydrolysate sample in the chromatogram. The masses of different monosaccharides were determined using an absolute quantitative method. The molar ratio was calculated based on the molar mass of the monosaccharides: C(standard) / A(standard) = C(sample) / A(sample). C represents the concentration, and A represents the peak area. Chromatographic conditions: Column: Dionex Carbopac™ PA20 (3*150); Mobile phase: A: H2O; B: 15mM NaOH; C: 15mM NaOH and 100mM sodium acetate; Flow rate: 0.3 mL / min; Injection volume: 5 μL; Column temperature: 30℃; Detector: Electrochemical detector. Figure 5 As shown in (A), chromatographic peaks of 16 standard monosaccharides were successfully separated and detected using ion chromatography. The peaks were well-defined and highly separated, which can well meet the requirements for simultaneous detection of common monosaccharide types in monosaccharide composition analysis.

[0099] Table 1 Monosaccharide composition of pollen polysaccharide component 3

[0100]

[0101] like Figure 5 (B) shows the ion chromatogram for the analysis of the monosaccharide composition of pollen polysaccharide component 3. Figure 5 (B) The detection of galacturonic acid and glucuronic acid indicates that pollen polysaccharide component 3 is an acidic sugar. The figure shows that pollen polysaccharide component 3 mainly contains arabinose and galactose. Calculations based on the relevant chromatographic peak areas yielded the following ratio for pollen polysaccharide component 3: fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid: glucuronic acid = 0.006:0.095:0.328:0.399:0.024:0.027:0.010:0.096:0.014 (Table 1).

[0102] 2. Analysis of pollen polysaccharide methylation and nuclear magnetic resonance results

[0103] To investigate the spatial structure of each component of pollen polysaccharide, nuclear magnetic resonance (NMR) analysis was performed on pollen polysaccharide component 3, and the spatial structure of each component was determined. 1 H-NMR spectrum, 13 C-NMR, DEPT-135, HSQC, COSY, TOCSY, HMBC, and NOESY spectra were analyzed, and the linkage sequence between different monosaccharide residues was determined through correlation analysis, elucidating the molecular structural characteristics of the PZMP1 polysaccharide components. 1 H-NMR spectrum and 13 C-NMR spectra, combined with COSY, HSQC, and TOCSY two-dimensional spectra, as well as relevant literature reports and methylation detection results, were used to assign the carbon and hydrogen atoms of the major monosaccharide residues in each component of pollen polysaccharides. HMBC two-dimensional NMR spectra reflect the spatial correlation between carbon and hydrogen atoms, while NOESY two-dimensional NMR spectra reflect the spatial correlation between hydrogen atoms. Therefore, the linkage sequence of monosaccharide residues can be inferred from the relevant two-dimensional spectra.

[0104] 2.1 Results of methylation determination of pollen polysaccharide component 3

[0105] To investigate the primary structure of pollen polysaccharide component 3, a modified Needs method was used to methylate pollen polysaccharide component 3.

[0106] The fully methylated pollen polysaccharide fraction 3, after treatment involving hydrolysis, reduction, and derivatization, was converted into partially methylated sugar alcohol acetate derivatives (PMAAs). GC-MS analysis yielded a total ion chromatogram for the methylation of pollen polysaccharide fraction 3, as shown below. Figure 6 As shown in the figure. The mass spectrum corresponding to the methylated glycosyl peaks in the figure ( Figure 7 By comparing the results with those from the standard spectral library (the CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / ms / pmaa / pframe.html), the types of partially methylated glycosyl groups can be determined. At the same time, the relative molar ratio can be calculated based on the peak area of ​​the chromatographic peaks. Combined with the results of monosaccharide composition determination, the necessary structural information can be provided for the inference of the structure of the three repeating units of pollen polysaccharide components.

[0107] Table 2 Results of 3-methylation analysis of pollen polysaccharide components

[0108]

[0109] 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-arabinitol), and so on.

[0110] Because pollen polysaccharide fraction 3 mainly contains arabinose and galactose, while the contents of rhamnose, glucose, xylose, fucose, and mannose are too low, methylation analysis only detected PMAAs of arabinose and galactose, while PMAAs of rhamnose, glucose, xylose, fucose, and mannose were not detected. However, since the contents of rhamnose, glucose, xylose, fucose, and mannose are very low, it does not affect the analysis and judgment of the main glycosidic bond linkage forms of pollen polysaccharide fraction 3. The total ion chromatogram detected by GC-MS is shown below. Figure 7 Five methylation-derived glycosyl ion fragments were mainly screened and collected.

[0111] As shown in Table 2, the methylation analysis of pollen polysaccharide component 3 mainly contained five glycosidic ion fragments, derived from galactose and arabinose, respectively. Galactose was primarily linked by →3,6)-Galp-(1→ and →3)-Galp-(1→) linkages, while arabinose was primarily linked by →3,5)-Araf-(1→ and →5)-Araf-(1→) linkages. Based on the glycosidic bond linkages of the sugar residues in pollen polysaccharide component 3 and the characteristics of the pollen polysaccharide structure, it can be preliminarily inferred that the main chain of pollen polysaccharide component 3 is likely connected to other glycosidic bonds via →3,6)-Galp-(1→ and →3)-Galp-(1→) linkages; the side chains are likely mainly composed of →3,5)-Araf-(1→ and →5)-Araf-(1→) linkages.

[0112] 2.2 Nuclear magnetic resonance (NMR) determination results of pollen polysaccharide component 3

[0113] Table 3 Monosaccharide residues in pollen polysaccharide component 3 1 H and 13 C10 NMR chemical shift assignment

[0114]

[0115] like Figure 8 , 9 Pollen polysaccharide component 3 shown in Figure 10 1 H-NMR spectrum ( Figure 8 ), 13 C-NMR spectrum ( Figure 9 ) and DEPT-135 spectrum ( Figure 10The presence of six distinct signal peaks in the terminal hydrogen-related region indicates that pollen polysaccharide component 3 contains six monosaccharide residues, with corresponding chemical shifts of 5.17 ppm, 5.15 ppm, 5.09 ppm, 4.83 ppm, 4.58 ppm, and 4.40 ppm. The chemical shifts within the range of 4.30–3.50 ppm correspond to the chemical shifts of the hydrogen atoms at carbons 2 through 6 of these six sugar residues. This information is based on HSQC spectra and... 13 C-NMR spectroscopy can determine the chemical shift of the terminal carbon corresponding to the terminal hydrogen of the glycosidic bond. HSQC( Figure 11 COSY Figure 12 ) and NOSEY Figure 13 This is a two-dimensional spectrum showing the carbon and hydrogen assignments, adjacent hydrogen correlations, and total hydrogen correlations of the same monosaccharide residue. Analysis allows for the assignment of other carbons and hydrogens within the related monosaccharide residue. This is achieved through HMBC spectroscopy (…). Figure 14 This can determine the spatial correlation of carbon and hydrogen, and thus determine the connection sequence of monosaccharide glycosidic bonds.

[0116] Based on methylation analysis and related NMR results, it can be determined that pollen polysaccharide component 3 is composed of galactose linked by β-D-1,3,6-Galp and β-D-1,3-Galp to form the main chain. The connecting branch groups are mainly composed of α-L-1,3,5-Araf and α-L-1,5-Araf. The main repeating structural units of pollen polysaccharide component 3 are as follows:

[0117] Main chain

[0118] branch

[0119] 3 Other structural measurement results

[0120] 3.1 Results of Ultraviolet Spectroscopy Measurement

[0121] like Figure 15 As shown, pollen polysaccharide component 3 showed no characteristic absorption peaks in the visible light region. This is mainly because the pollen polysaccharide molecule lacks a corresponding chromophore, which is also the primary reason why it is difficult to quickly qualitatively and quantitatively detect polysaccharide components. In the ultraviolet region, no absorption peaks were found at 280 nm and 260 nm, indicating that pollen polysaccharide component 3 is a simple polysaccharide with high purity.

[0122] 3.2 Infrared Spectroscopy Results

[0123] Figure 16 This is a typical infrared spectrum of plant polysaccharides; the absorption band of pollen polysaccharide component 3 is in the 3600-3200 cm⁻¹ range. -1This is the absorption peak of the stretching vibration of -OH, and the absorption peak in this region is a characteristic peak of carbohydrates. Specifically: 3426 cm⁻¹ 1 The peak at 2939 cm⁻¹ is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. -1 There is an absorption peak at 1734 cm⁻¹, which may be attributed to the CH stretching vibration. -1 The weaker absorption peak appearing nearby is attributed to the stretching vibration of the C=O group in the carboxyl group, indicating that pollen polysaccharide component 3 contains a certain amount of uronic acid. At 1619 cm⁻¹ -1 There is an absorption peak at 1423 cm⁻¹, which may be attributed to the water of crystallization. -1 Location, 1145cm -1 Location, 1091cm -1 An absorption peak is observed at 895 cm⁻¹, which may be attributed to the CO stretching vibration. -1 The presence of an absorption peak at this point may be attributed to the CH-angle vibration of the β-terminal epimer of the pyran ring.

[0124] Example 6: The effect of pollen polysaccharide component 3 on promoting tobacco growth

[0125] Experimental sample:

[0126] Pollen polysaccharide component 3 was prepared into a stock solution with a polysaccharide concentration of 10 mg / ml using pure water, and then diluted with water according to the experimental design before use.

[0127] 1. Experimental Design:

[0128] Table 4. Drug Concentration Design

[0129]

[0130] 2. Experimental methods:

[0131] After preparing the treatment agents according to Table 4, they were evenly sprayed onto the leaves of tobacco seedlings. Each treatment was repeated 6 times, with one plant per replicate. The spray solution should not drip to prevent it from entering the soil and affecting the experimental results. The cultivation conditions were set as follows: temperature 28℃, light intensity 2000 lux, 14h / 10h (day / night), humidity 65%. The RGB AREA_MM parameter values ​​(leaf area / mm²) of the tobacco seedlings were recorded using a plant phenotypic analysis system before and 7 days after the application. 2 To evaluate the growth-promoting effect of each agent, the leaf area growth rate is calculated. The formula for calculating the leaf area growth rate is as follows:

[0132] Leaf area growth rate (%) = (final leaf area - initial leaf area) × 100% / initial leaf area

[0133] 3. Experimental Results:

[0134] Table 5 shows the leaf area growth rate of different treatment groups 7 days after the drug treatment. The experimental results show that pollen polysaccharide component 3 has a growth-promoting effect on tobacco in the concentration range of 0.03 to 30 ppm. The optimal growth-promoting concentration is 0.3 ppm, which has a significant effect on promoting tobacco growth.

[0135] Table 5. Results of tobacco growth promotion assays in different treatment groups

[0136]

[0137]

[0138] Example 7: The growth-promoting effect of pollen polysaccharide component 3 on Chinese cabbage

[0139] 1. Experimental Samples:

[0140] Pollen polysaccharide component 3 was prepared into a stock solution with a polysaccharide concentration of 10 mg / ml using pure water, and then diluted with water according to the experimental design.

[0141] 2. Experimental Design:

[0142] Table 6. Drug Concentration Design

[0143]

[0144] 3. Experimental methods:

[0145] Select uniformly growing bok choy seedlings (3-4 leaves). Prepare the treatment agent according to Table 6 and apply it to the roots of the seedlings via root drenching. Each treatment was repeated 6 times, with one seedling per replicate. Each seedling received 80 ml of the treatment agent. The seedlings were placed in a plant culture room at 25℃ with a light intensity of 3000 lux, 14h / 10h (day / night), and a humidity of 65%. Water and fertilizer management levels were maintained consistently throughout the culture period. Leaf length, leaf width, aboveground fresh weight, and SPAD value were measured 7 days after treatment.

[0146] 4. Experimental Results:

[0147] The results of different treatment groups 7 days after the drug were measured are shown in Table 7. The experimental results show that pollen polysaccharide component 3 has a growth-promoting effect on Chinese cabbage in the concentration range of 0.03 to 30 ppm. The optimal growth-promoting concentration is 0.3 ppm, which is better than the control group.

[0148] Table 7. Results of growth promotion assays for different treatment groups of Chinese cabbage 7 days after drug administration.

[0149]

[0150]

[0151] Example 8: Low-temperature resistance test of pollen polysaccharide component 3

[0152] 1. Experimental Samples:

[0153] Pollen polysaccharide component 3 was prepared into a stock solution with a polysaccharide concentration of 10 mg / ml using pure water, and then diluted with water according to the experimental design.

[0154] 2. Experimental Design:

[0155] Table 8. Drug Concentration Design

[0156]

[0157] 3. Experimental methods:

[0158] After the treatment agents were prepared according to Table 8, they were sprayed evenly on the leaves of tobacco seedlings. Each treatment was repeated 6 times, with 1 plant per replicate. The solution should not drip to prevent the agent from entering the soil and affecting the experimental results. After 1 day of seedling establishment, the seedlings were treated with chilling injury (4℃) for 24 hours and then recovered at 28℃. The chlorophyll fluorescence QY-max parameter and Fv / Fm-lss parameter values ​​of tobacco were recorded using a plant phenotyping instrument before treatment, 24 hours after low temperature treatment, and 24 hours after recovery.

[0159] 4. Experimental Results:

[0160] QY-max represents the theoretical maximum photosynthetic capacity of a plant; a smaller reduction rate indicates better low-temperature resistance. Fv / Fm-lss represents the maximum photon yield of PSII; similar to QY-max, a smaller reduction rate indicates better low-temperature resistance. Table 9 shows that after 24 hours of low-temperature treatment, the fluorescence values ​​of QY-max and Fv / Fm-lss in all treatment groups decreased significantly. However, the reduction in fluorescence values ​​of QY-max and Fv / Fm-lss in the treatment group using pollen polysaccharide component 3 was even less, indicating that the plant's growth status was better than the water control, and its low-temperature resistance was stronger. After 24 hours of low-temperature treatment, pollen polysaccharide component 3 improved the cold resistance of tobacco within a concentration range of 0.03–30 ppm, with the optimal low-temperature resistance concentration being 0.03 ppm. After 24 hours of recovery at room temperature, as shown in Table 10, the fluorescence values ​​of QY-max and Fv / Fm-lss in each treatment group began to rise, indicating that the growth status of the plants damaged at low temperature was recovering. Pollen polysaccharide component 3 could promote the recovery of plants with chilling injury in the concentration range of 0.03 to 30 ppm, among which component 3 at a concentration of 0.03 ppm could significantly promote the recovery of plant growth status.

[0161] Table 9 Results of measurements after 24 hours of low-temperature treatment.

[0162]

[0163] Table 10 Results of measurements after 24 hours of room temperature recovery

[0164]

[0165] Example 9: High-temperature resistance test of pollen polysaccharide component 3

[0166] 1. Experimental Samples:

[0167] Pollen polysaccharide component 3 was prepared into a stock solution with a polysaccharide concentration of 10 mg / ml using pure water, and then diluted with water according to the experimental design.

[0168] 2. Experimental Design:

[0169] Table 11 Drug Concentration Design

[0170]

[0171] 3. Experimental methods:

[0172] Tobacco seedlings with uniform growth (3-leaf stage) were selected. The treatment agent was prepared according to Table 11 and sprayed evenly on the leaves of the tobacco seedlings. Each treatment was repeated 6 times, with 1 plant per replicate. The solution should not drip to prevent the agent from entering the soil and affecting the experimental results. After the seedlings had recovered for 1 day, they were treated at 40℃ for 48 hours. The chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of tobacco were recorded using a plant phenotyping instrument before the treatment and after 48 hours of high temperature treatment. The malondialdehyde content was measured after 48 hours of high temperature treatment.

[0173] 4. Experimental Results:

[0174] QY-max represents the theoretical maximum photosynthetic capacity of a plant; the smaller the rate of decrease, the better the plant's resistance to high temperatures. Fv / Fm-lss represents the maximum photon yield of PSII; similar to QY-max, a smaller rate of decrease indicates better heat resistance. The non-photochemical quenching coefficient NPQ-lss reflects the plant's ability to dissipate excess light energy as heat, reflecting its photosensitivity. When a plant is under stress, the light energy it absorbs is no longer used for photosynthesis but is directly converted into heat energy for dissipation, thus preventing damage. In this case, the plant's NPQ-lss increases. Therefore, under high-temperature stress, a lower NPQ-lss value indicates less damage and better heat resistance. Malondialdehyde (MDA) content is closely related to plant stress damage. Under stress conditions, membrane lipid peroxidation often occurs, producing cytotoxic MDA. Therefore, under high-temperature conditions, a lower MDA content indicates less damage and better heat resistance.

[0175] As shown in Table 12, after 48 hours of high-temperature treatment, the fluorescence values ​​of QY-max and Fv / Fm-lss in all treatment groups decreased significantly, while the fluorescence value of NPQ-lss increased significantly. However, the decrease in QY-max and Fv / Fm-lss fluorescence values ​​and the increase in NPQ-lss fluorescence values ​​were less pronounced in the pollen polysaccharide treatment groups, indicating that the plants were in better growth condition and had stronger high-temperature resistance than the water control. After 48 hours of high-temperature treatment, pollen polysaccharide component 3 could enhance the high-temperature resistance of tobacco in the concentration range of 0.03–30 ppm, with the optimal concentration being 0.03 ppm. The MDA content determination results for each treatment group are shown in Table 13. Compared with the control group, the pollen polysaccharide treatment groups reduced the production of MDA in the plants, thereby reducing the degree of damage to the plants. Consistent with the above fluorescence parameters, the MDA content in the plants treated with pollen polysaccharide component 3 at a concentration of 0.03 ppm was the lowest.

[0176] Table 12 Results of fluorescence parameter measurements in different treatment groups

[0177]

[0178] Table 13 Results of MDA content determination in different treatment groups

[0179]

Claims

1. A pollen polysaccharide component 3, the main repeating unit structure of which is as follows: .

2. The pollen polysaccharide component 3 according to claim 1, characterized in that, Pollen polysaccharides also contain the following monosaccharide units: fucose, rhamnose, glucose, xylose, mannose, galacturonic acid, and glucuronic acid.

3. The pollen polysaccharide component 3 according to claim 1, characterized in that, The average molecular weight of pollen polysaccharides is 60-70 kDa.

4. The pollen polysaccharide component 3 according to claim 1, characterized in that, The average molecular weight of pollen polysaccharides is 65-70 kDa.

5. The pollen polysaccharide component 3 according to claim 1, characterized in that, The average molecular weight of pollen polysaccharides is 66911.38 Da.

6. The pollen polysaccharide component 3 according to any one of claims 1-5, characterized in that, Its infrared spectrum includes at least one or more of the following absorption peaks: 3426 cm⁻¹ -1 2939 cm -1 1734 cm -1 1619cm -1 1423cm -1 1145 cm -1 1091 cm -1 895cm -1 .

7. A method for separating pollen polysaccharide component 3 as described in any one of claims 1-6, characterized in that, Includes the following: (1) The rapeseed pollen polysaccharide extract was purified by macroporous adsorption resin column and eluted with water to obtain the preliminarily purified pollen polysaccharide active fraction Fr-1; (2) The active part of pollen polysaccharide Fr-1 was eluted by gradient elution with 0~0.05 mol / L NaCl solution through an ion exchange chromatography column, and the 0.05 mol / L NaCl solution fraction was collected to obtain the active part of pollen polysaccharide Fr-1-3; (3) The active part of pollen polysaccharide Fr-1-3 was further purified by dextran gel chromatography column, with ammonium bicarbonate aqueous solution as the eluent, to obtain component 3, namely pollen polysaccharide component 3.

8. The separation method according to claim 7, characterized in that, The rapeseed pollen polysaccharide extract mentioned in step (1) refers to the crude pollen polysaccharide Fr obtained by water extraction and alcohol precipitation of rapeseed pollen.

9. The separation method according to claim 7, characterized in that, The macroporous adsorption resin column used in step (1) is a non-polar column, selected from one of the following: DB-101 macroporous resin column, S-8 macroporous resin column, AB-8 macroporous resin column, and HP-20 macroporous resin column.

10. The separation method according to claim 9, characterized in that, The macroporous adsorption resin column was selected from the HP-20 macroporous resin column.

11. The separation method according to claim 7, characterized in that, The pollen polysaccharide active fraction Fr-1 in step (1) and the pollen polysaccharide active fraction Fr-1-3 in step (2) were obtained as solids by dialysis and freeze-drying, respectively. Furthermore, the dialysis refers to the removal of substances with a molecular weight cutoff of at least 3500 Da.

12. The separation method according to claim 7, characterized in that... The ion exchange chromatography column used in step (2) is an anion exchange chromatography column; Furthermore, the dextran gel chromatography column used in step (3) is selected from propylene dextran gel columns.

13. The separation method according to claim 12, characterized in that, The ion exchange chromatography column used in step (2) is selected from DEAE cellulose chromatography column; the dextran gel chromatography column used in step (3) is selected from propylene dextran gel S-400 HR chromatography column.

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

15. The use of pollen polysaccharide component 3 according to any one of claims 1-6 in the preparation of plant stress-resistant products; Furthermore, the plant's stress resistance includes resistance to high temperatures and cold. Furthermore, when using the product, apply it to the leaves or roots of the plant; Furthermore, the plant in question is tobacco.

16. The use of pollen polysaccharide component 3 according to any one of claims 1-6 in the preparation of plant growth promoting products; Furthermore, the plants mentioned are tobacco and bok choy.

17. An agricultural product, characterized in that, The active ingredient of the product includes pollen polysaccharide component 3 as described in any one of claims 1-6.

18. The agricultural product according to claim 17, characterized in that, The product also includes excipients, which are one or more of the following: dispersants, wetting agents, binders, emulsifiers, stabilizers, solvents, and encapsulating agents.

19. The agricultural product according to claim 17, wherein the product is in the form of emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, or aqueous solution.

20. The agricultural product according to claim 17, wherein the product is used as a mother liquor or mother powder.

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