Pollen polysaccharide component 2 and 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 a new pollen polysaccharide component 2. The pollen polysaccharide component 2 has certain effects in the aspects of plant growth promotion and stress resistance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of plant extract separation, and particularly relates to a pollen polysaccharide component 2 and a separation method and application thereof. BACKGROUND

[0002] The previous researches (patents CN 113133454B and CN 113133455B) of the inventors show that the water extract of rape pollen can be used for plant stress resistance and plant growth promotion, which includes vegetables or fruit trees such as baby bok choy, asparagus lettuce, lettuce, Shanghai green, wheat, pepper, tomato, citrus, kiwi, cherry, pear, apple, etc. Meanwhile, the components in the water extract are detected, which include saccharides, proteins, amino acids and lipids, etc. However, the specific active components have not been studied.

[0003] In addition, the literature "Research Progress of 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) reports that amino acid substances can also be used for plant stress resistance.

[0004] On the basis that there may be various substances with plant growth promotion or stress resistance in the water extract of rape pollen, the skilled in the art cannot know which substance plays a leading role in plant growth promotion or stress resistance at present, and therefore it is necessary to further study the extraction of rape pollen. SUMMARY

[0005] Polysaccharides are a class of complex sugar substances formed by the dehydration condensation of multiple monosaccharide molecules, and the structural units are connected 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 and β-1, 3 glycosidic bond, etc. Polysaccharides widely exist in animals, plants and microorganisms, and polysaccharides from different sources have different biological activities. Existing studies show that plant polysaccharides have immunomodulatory, antitumor, antioxidant, anti-aging, hypoglycemic and hypolipidemic biological activities, and are widely used in food, medicine, livestock breeding and aquaculture industries, etc. due to their wide sources, degradability, high safety, easy modification and environmental affinity, etc.

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

[0007] Specifically, the polysaccharide in the rape pollen is extracted, separated and purified to obtain a new pollen polysaccharide, which has certain effects in plant growth promotion and stress resistance.

[0008] Specifically, the present application provides one pollen polysaccharide component 2, and the main repeating structural unit is as follows:

[0009] Among them

[0010] R1 is T-alpha-L-Araf-(1→5)-alpha-L-Araf-(1→

[0011] R2 is T-beta-D-Galp-(1→6)-beta-D-Galp-(1→.

[0012] In the polysaccharide main chain of the present application, the groups R1 and R3 are connected to the 5th position of arabinose, and R2 is connected to the 6th position of galactose.

[0013] In the present application, Ara represents arabinose; Gal represents galactose; f represents furanose; p represents pyranose; and T represents the end of the polysaccharide molecule (end group).

[0014] Among them, the molar content ratio of arabinose: galactose is equal to 0.597:0.283.

[0015] In addition to the above main repeating structural unit, the pollen polysaccharide of the present application also contains the following trace monosaccharide units: fucose, glucose, xylose and mannose.

[0016] Among them, the molar content ratio of fucose: arabinose: galactose: glucose: xylose: mannose is equal to 0.010:0.597:0.283:0.048:0.007:0.038.

[0017] In the present application, the average molecular weight of the pollen polysaccharide is 5-15KDa;

[0018] Further, the average molecular weight of the pollen polysaccharide is 9-11KDa;

[0019] In the present application, the average molecular weight of the pollen polysaccharide is 10-11KDa, for example, 10718.15Da.

[0020] The pollen polysaccharide of the present application has at least one or more of the following absorption peaks in its infrared spectrum: 3288m -1 , 2931cm -1 , 2871cm -1 , 1641m -1 , 1548cm -1 , 1440cm -1, 1402 cm -1 , 1309 cm -1 , 1243 cm -1 , 1081 cm -1 , 896 cm -1 .

[0021] wherein, 3288 m -1 is the O-H stretching vibration absorption peak, and is the characteristic peak of the saccharide. There is an absorption peak at 2931 cm -1 , 2871 cm -1 , which can be attributed to the C-H stretching vibration. There is an absorption peak at 1641 m -1 , which can be attributed to the crystal water. There is an absorption peak at 1548 cm -1 , which can be attributed to the C=O stretching vibration. There is an absorption peak at 1440 cm -1 , 1402 cm -1 , which can be attributed to the C-O stretching vibration. There is an absorption peak at 1309 cm -1 , 1243 cm -1 , 1081 cm -1 , which can be attributed to the O-H variable angle vibration. There is an absorption peak at 896 cm -1 , which can be attributed to the C-H variable angle vibration of the β-omeric difference isomerization of the pyran ring.

[0022] The purpose of the present application is also to provide a separation method of the pollen polysaccharide component 2, comprising the following contents:

[0023] (1) The rapeseed pollen polysaccharide extract is purified by a macroporous adsorption resin column, dialyzed and freeze-dried to obtain a preliminarily purified pollen polysaccharide active part Fr-1;

[0024] (2) The preliminarily purified pollen polysaccharide active part Fr-1 is gradient eluted by an ion exchange chromatography column with 0-0.025 mol / L NaCl solution, and the 0.025 mol / L NaCl eluate part is collected to obtain the pollen polysaccharide active part Fr-1-2;

[0025] (3) The pollen polysaccharide active part Fr-1-2 is further purified by a gel chromatography column, wherein an ammonium bicarbonate aqueous solution is used as an eluent to obtain component 2, i.e., the pollen polysaccharide of the present application.

[0026] The pollen polysaccharide active part Fr-1 eluted from the ion exchange column can be in the form of the original eluent of the macroporous resin, or a concentrated solution of the original eluent, or a reconstituted solution after the original eluent is concentrated and dried, and the like.

[0027] The pollen polysaccharide active site Fr-1-2 of the upper gel chromatography column can be in the form of the original eluent of the ion exchange column, the concentrated solution of the original eluent, the redissolved solution of the concentrated and dried original eluent, or the like.

[0028] The concentration and drying in the present application include, but are not limited to, the following methods: reduced pressure evaporation, atmospheric evaporation, drying, vacuum drying, thin film drying, freeze drying, and the like.

[0029] The oilseed rape pollen polysaccharide extract in step (1) refers to the crude oilseed rape pollen polysaccharide obtained by a conventional method.

[0030] The water extraction includes, but is not limited to, the following methods: heating extraction, ultrasonic extraction, microwave extraction, and the like.

[0031] Meanwhile, according to different conditions, the impurities can be removed by defatting and decolorization before extraction, for example, using a fat-soluble solvent for extraction, such as ethanol, petroleum ether, and the like.

[0032] The preliminary purification method after water extraction includes, but is not limited to, the following methods: alcohol precipitation, chitosan impurity removal, and the like.

[0033] In the present application, if the water extraction and alcohol precipitation method is used, 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, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 85%, 90% v / v, or the like.

[0034] In the purification method, according to different conditions, auxiliary means such as protein removal, decolorization, and small molecule removal can be selectively added to facilitate the subsequent polysaccharide enrichment process. For example, protein removal reagents such as phenol, trichloroacetic acid, tannic acid, and the like can be used; adsorbents such as cellulose, diatomite, activated carbon, and the like can be used for decolorization; and dialysis and the like can be used for removing small molecules.

[0035] In some specific embodiments of the present application, the oilseed rape pollen polysaccharide extract in step (1) refers to the crude pollen polysaccharide obtained after water extraction and alcohol precipitation of the oilseed rape pollen.

[0036] In some specific embodiments of the present application, the oilseed rape pollen polysaccharide extract in step (1) refers to the crude pollen polysaccharide obtained after water extraction and alcohol precipitation of the oilseed rape pollen.

[0037] In some specific embodiments of the present application, the oilseed rape pollen can be subjected to ultrafine grinding treatment to facilitate the extraction of polysaccharides.

[0038] In some embodiments of the present application, the pollen polysaccharide extract of rape pollen can be vacuum freeze-dried to prepare a pollen crude polysaccharide solid.

[0039] In the technical solution of the present application, the macroporous resin column used in step (1) is a non-polar column.

[0040] In the present application, 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.

[0041] In the technical solution of the present application, the ion exchange column used in step (2) is a cation exchange column, and the anion includes but is not limited to DEAE-cellulose, DEAE-agarose gel, DEAE-dextran gel, and other different ion exchange columns, and is further selected from DEAE cellulose-52 chromatography column.

[0042] In the technical solution of the present application, the gel chromatography column used in step (3); the gel chromatography column includes but is not limited to Sephadex G chromatography column, Toyopearl HW chromatography column, and other different gel chromatography columns; for example, it can be selected from propylene sephadex gel S-400HR chromatography column, Sephadex LH-20 chromatography column, and the like.

[0043] In the technical solution of the present application, in step (3), the concentration of the ammonium bicarbonate aqueous solution is 0.1-0.3 mol / L, and preferably 0.2 mol / L.

[0044] In some embodiments of the present application, the pollen polysaccharide active site Fr-1 in step (1) and the pollen polysaccharide active site Fr-1-2 in step (2) can be treated by dialysis and freeze-drying to obtain a solid for standby use.

[0045] Further, the dialysis refers to substances with a molecular weight of at least 3500 Da.

[0046] In some embodiments of the present application, the eluent containing component 2 in step (3) is concentrated under reduced pressure, dialyzed (3500 Da) to remove salt, and freeze-dried to obtain a solid of pollen polysaccharide component 2.

[0047] In the present application, when the concentration step is experienced, the degree of concentration can be determined by those skilled in the art according to the amount of eluent added during elution and the final demand of the product.

[0048] In some embodiments of the present application, the phenol-sulfuric acid method is used to detect the sugar content in steps (2) and (3), and the absorbance value at 490 nm is detected.

[0049] The application also provides the use of the pollen polysaccharide component 2 in the preparation of a plant stress resistance product.

[0050] The plant stress resistance includes high temperature resistance, cold resistance, drought resistance, salt and alkali tolerance, etc.

[0051] In the application, the product containing the pollen polysaccharide is applied to the plant leaves or the plant roots.

[0052] When the product containing the pollen polysaccharide is used in the form of a solution, the concentration of the main active ingredient can be selected according to actual needs.

[0053] For example, the concentration of the pollen polysaccharide component 2 can be selected from 0.01 ppm to 500 ppm; can be selected from 0.01 ppm to 100 ppm; can be selected from 0.01 ppm to 50 ppm; can also be selected from 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.

[0054] The application also provides the use of the pollen polysaccharide component 2 in the preparation of a plant growth promoting product.

[0055] The plants include but are not limited to economic crops and food crops, such as Chinese flowering cabbage, baby bok choy, asparagus lettuce, lettuce, Shanghai green, wheat, pepper, tomato, citrus, kiwi, cherry, pear, apple, tobacco, etc.

[0056] The economic crops are various, including but not limited to fiber crops (such as cotton, hemp, etc.), oil crops (such as sesame, peanut, etc.), sugar crops (such as sugarcane, sugar beet, etc.), tobacco crops, medicinal crops, dye crops, ornamental crops, fruits and other economic crops, etc.

[0057] The food crops include but are not limited to cereal crops (wheat, rice, corn), potato crops (including sweet potato, potato, etc.) and legume crops (including soybean, broad bean, pea, mung bean, etc.).

[0058] The application also provides an agricultural product, and the active ingredient of the product includes the pollen polysaccharide component 2.

[0059] In the present application, the direct pollen polysaccharide component 2 can be used as a single agent, and in order to stabilize the product and facilitate transportation and storage, an excipient can be added to form a corresponding dosage form. The excipient can be a conventional excipient in the art, for example, a dispersing agent, a wetting agent, a binder, an emulsifying agent, a stabilizer, a solvent, an embedding agent, and the like.

[0060] In another aspect, the pollen polysaccharide component 2 described in the present application can be used as a synergist in combination with foliar fertilizer, water-soluble fertilizer, compound fertilizer, pesticide, and the like.

[0061] The dosage form of the product described in the present application includes, but is not limited to, emulsifiable concentrate, suspension concentrate, wettable powder, powder, granule, water agent, mother liquor, mother powder, and the like conventional agricultural product formulations.

[0062] The present application has the following beneficial effects:

[0063] The present application discovers a new polysaccharide and first separates it from rape pollen, which can be used for plant stress resistance and plant growth promotion.

[0064] The "rape" described in the present application is a cruciferous, Brassica herbaceous crop. Common "rape" includes, but is not limited to, white cabbage rape, cabbage rape, black mustard rape, mustard rape, and Ethiopian mustard. The "pollen" described in the present application is obtained from the above-mentioned "rape". BRIEF DESCRIPTION OF DRAWINGS

[0065] Figure 1 : Gradient elution curve of pollen crude polysaccharide on DEAE cellulose-52 anion exchange column;

[0066] Figure 2 : Elution curve of pollen polysaccharide 4 components on S-400HR propylene dextran gel column;

[0067] Figure 3 : Standard curve of dextran standard molecular weight distribution;

[0068] Figure 4 : HPGPC determination chromatogram of pollen polysaccharide component 2;

[0069] Figure 5 : HPAEC-PAD detection chromatogram of monosaccharide mixed standard and pollen polysaccharide component (A: monosaccharide mixed standard; B: pollen polysaccharide component 2; ) wherein, monosaccharide mixture standard: 1. Rhamnose (Rha) 2. Fucose (Fuc) 3. Arabinose (Ara) 4. Xylose (Xyl) 5. Mannose (Man) 6. Glucose (Glc) 7. Galactose (Gal) 8. Glucuronic acid (GlcA) 9. Galacturonic acid (GalA); solvent peak: 2.0 min for sodium hydroxide peak, 40 min for sodium acetate peak.

[0070] Figure 6 Methylation analysis total ion chromatogram (TIC) of pollen polysaccharide component 2;

[0071] Figure 7 Mass spectra of each methylated PMAA of pollen polysaccharide component 2;

[0072] Figure 8 HSQC spectrum of pollen polysaccharide component 2; 1 H-NMR spectrum;

[0073] Figure 9 C-NMR spectrum of pollen polysaccharide component 2; 13 C-NMR spectrum;

[0074] Figure 10 DEPT-135 NMR spectrum of pollen polysaccharide component 2;

[0075] Figure 11 HSQC spectrum of pollen polysaccharide component 2;

[0076] Figure 12 COSY spectrum of pollen polysaccharide component 2;

[0077] Figure 13 TOCSY spectrum of pollen polysaccharide component 2;

[0078] Figure 14 HMBC spectrum of pollen polysaccharide component 2;

[0079] Figure 15 UV absorption spectrum of pollen polysaccharide component 2;

[0080] Figure 16 IR absorption spectrum of pollen polysaccharide component 2; DETAILED DESCRIPTION

[0081] The technical solutions of the present application will be described clearly and completely below. Of course, the described embodiments are part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application. It should be noted that if there are processes not specifically described below, they can be implemented or understood by those skilled in the art according to the prior art. If the reagents or instruments used are not marked with the manufacturer, they are considered to be conventional products that can be purchased on the market.

[0082] Example 1 Extraction of pollen polysaccharide

[0083] Rape pollen was super-micro pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated pollen powder was weighed, and then free monosaccharides were removed by ethanol, and the pollen was defatted and decolorized by petroleum ether, and then hot water extraction was performed, and the precipitate was collected and washed with anhydrous ethanol, diethyl ether and acetone in sequence. Finally, the washed precipitate was redissolved with water, pre-frozen at -20°C, and vacuum freeze-dried to obtain the hot water extracted pollen crude polysaccharide.

[0084] Example 2 Extraction of pollen polysaccharide

[0085] Rape pollen was mixed with water, heated for extraction, filtered, and then chitosan was added to the filtrate, which was incubated and separated. The obtained liquid was the pollen crude polysaccharide extract. After the extract was concentrated under reduced pressure, it was pre-frozen at -20°C and vacuum freeze-dried to obtain the pollen crude polysaccharide.

[0086] Example 3 Extraction of pollen polysaccharide

[0087] Rape pollen was super-micro pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated pollen powder was weighed, and then free monosaccharides were removed by ethanol, and the pollen was defatted and decolorized by petroleum ether, and then hot water extraction was performed, and the precipitate was collected and washed with anhydrous ethanol, diethyl ether and acetone in sequence. Finally, the washed precipitate was redissolved with water, pre-frozen at -20°C, and vacuum freeze-dried to obtain the hot water extracted pollen crude polysaccharide.

[0088] Example 4 Isolation and purification of pollen polysaccharide

[0089] Step (1) HP-20 macroporous adsorption resin column purification: HP-20 macroporous adsorption resin column (Φ4.0 cm x 40 cm) was used to preliminarily purify the pollen crude polysaccharide, and the specific steps were as follows: a certain amount of pollen crude polysaccharide was weighed, a certain amount of water was added to prepare a 10-20 mg / mL pollen crude polysaccharide solution, and the single sample loading amount was 10 mL. After being adsorbed by the HP-20 macroporous adsorption resin column for 3 hours, the column was washed with 5 times the column volume of water, and the sample was connected to obtain the pollen polysaccharide active fraction Fr-1, which was concentrated under reduced pressure at 48°C, dialyzed (3500 Da), and freeze-dried for use.

[0090] 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 eluents are 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, and 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.

[0091] Depend on Figure 1 It 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.

[0092] Step (3) S-400HR Sepharose CL-4B column purification: Finally, the S-400HR Sepharose CL-4B column (Φ1.0 cm x 100 cm) was used to further purify the pollen polysaccharide fractions. The specific steps are as follows: ① Load the fraction solution, 20 mg per time; ② Elute with 0.2 mol / L ammonium bicarbonate, flow rate 0.2 mL / min, collect 3 mL per tube, continue to elute until no sugar is detected, collect 40 tubes of each component eluate; ③ Collect, detect the sugar content by phenol-sulfuric acid method, detect the absorbance at 490 nm by enzyme label instrument, draw the elution curve, collect the elution peak, combine the same components, concentrate under reduced pressure at 48°C, dialyze (3500 Da) to remove ammonia, and freeze-dry to obtain the purified components of each pollen polysaccharide.

[0093] The four pollen polysaccharide components (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) obtained by DEAE cellulose-52 anion exchange column separation were further purified by S-400HR Sepharose CL-4B column, and 0.2 mol / L ammonium bicarbonate was used to elute the four components. Figure 2 It can be seen that after elution with 0.2 mol / L ammonium bicarbonate, four components all obtained a single elution peak, and the corresponding components were named as component 1, component 2, component 3 and component 4, respectively. The eluate corresponding to the elution peak was collected (component 1: 21-31 tubes; component 2: 20-26 tubes; component 3: 18-26 tubes; component 4: 20-23 tubes), and then concentrated under reduced pressure, dialyzed and freeze-dried to obtain four component pollen polysaccharide purified components.

[0094] Purity identification and molecular weight determination of pollen polysaccharide purified components

[0095] Determination method: The purity identification and molecular weight determination of each purified component of pollen polysaccharide were performed by high performance gel permeation chromatography (HPGPC). The test conditions were as follows: Agilent 1260 series high performance liquid chromatograph; differential refractive index detector (RID); Shodex OHpak SB-804HQ (7.8 mm x 300 mm) gel chromatographic column; mobile phase was 0.1 mol / L Na2SO4; flow rate was 0.5 mL / min; column oven temperature was 35°C; sample size was 20 μL; instrument test time was 24 min.

[0096] Preparation of standard curve: The dextran standard samples with different molecular weight (5900, 9600, 21100, 47100, 107000, 200000, 341400 Da) were dissolved in 0.1 mol / L Na2SO4 solution to prepare a standard sample solution with a concentration of 5 mg / mL. After filtration through a 0.22 μm water phase filter, the sample was tested according to the HPGPC test conditions, and the retention time was recorded. The standard curve was plotted with the retention time (min) as the abscissa and the logarithmic value of the molecular weight of the dextran standard sample (Log MW) as the ordinate. The regression equation was fitted to calculate the molecular weight of the different components of pollen polysaccharide. The standard curve obtained was y = -0.322x + 9.4365, as shown in Figure 3 .

[0097] Determination of the purity and molecular weight of the purified components of pollen polysaccharide: 10 mg of the purified pollen polysaccharide sample was dissolved in 2 mL of 0.1 mol / L Na2SO4 solution to prepare a polysaccharide solution with a concentration of 5 mg / mL. After filtration through a 0.22 μm water phase filter, the sample was analyzed according to the HPGPC test conditions. The chromatogram of each purified component of pollen polysaccharide was recorded. The purity of each polysaccharide sample was evaluated based on the number of peaks and the symmetry of the peaks in the chromatogram. The molecular weight of each polysaccharide sample was calculated based on the retention time in the chromatogram and the dextran standard molecular weight standard curve.

[0098] The purity and molecular weight of the purified component 2 of pollen polysaccharide were determined by HPGPC, and the results are shown in Figure 4 . As can be seen from Figure 4 , a single symmetrical peak appeared in the HPGPC chromatogram of the purified component 2 of pollen polysaccharide, indicating that it had high purity. This result is consistent with the results of separation and purification by S-400 HR propylene dextran gel column, further indicating that the established method for separation and purification of pollen polysaccharide is feasible and can prepare pollen polysaccharide with high purity. According to the previously established molecular weight standard curve, the average molecular weight of component 2 was calculated to be 10718.15 Da.

[0099] Example 5 Structure identification of pollen polysaccharide

[0100] 1 Determination results of monosaccharide composition of pollen polysaccharide

[0101] 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.

[0102] Table 1 Monosaccharide composition of pollen polysaccharide component 2

[0103]

[0104] Figure 5 (B) shows the ion chromatogram for the monosaccharide composition analysis of pollen polysaccharide component 2. Figure 5 (B) No chromatographic peak corresponding to uronic acid was detected, indicating that pollen polysaccharide component 2 does not contain uronic acid and is a neutral sugar. The figure shows that pollen polysaccharide component 2 is mainly composed of arabinose and galactose. Calculations based on the relevant chromatographic peak areas yielded the following ratios for pollen polysaccharide component 2: fucose: arabinose: galactose: glucose: xylose: mannose: 0.010:0.597:0.283:0.048:0.007:0.038 (Table 1).

[0105] 2. Analysis of pollen polysaccharide methylation and nuclear magnetic resonance results

[0106] To investigate the spatial structure of each component of pollen polysaccharide, nuclear magnetic resonance (NMR) analysis was performed on pollen polysaccharide component 2, and the spatial structure of each component was determined. 1H-NMR spectra, 13 C-NMR spectra, DEPT-135 spectra, HSQC spectra, COSY spectra, TOCSY spectra, HMBC spectra and NOESY spectra were used to determine the connection order between different monosaccharide residues by correlation analysis, and to clarify the molecular structure characteristics of the polysaccharide components. By 1 H-NMR spectra and 13 C-NMR spectra, combined with COSY, HSQC, and TOCSY two-dimensional spectra, as well as relevant literature reports and methylation detection results, the carbon and hydrogen of the main monosaccharide residues in the pollen polysaccharide components were assigned. HMBC two-dimensional nuclear magnetic spectra reflect the spatial correlation of carbon and hydrogen, and NOESY two-dimensional nuclear magnetic spectra reflect the spatial correlation of hydrogen and hydrogen, so the connection order of monosaccharide residues can be inferred by correlation two-dimensional spectra.

[0107] 2.1 Methylation determination results of pollen polysaccharide component 2

[0108] Table 2 Methylation analysis results of pollen polysaccharide component 2

[0109]

[0110] Note: 2,3-Me2-Araf is 1,4,5-tri-O-acetyl-2,3-di-O-methyl-L-arabinitol, and the same applies to the following.

[0111] Since pollen polysaccharide component 2 mainly contains arabinose and galactose, and the contents of glucose, xylose, fucose and mannose are too low, methylation analysis only detects the PMAAs of arabinose and galactose, and the PMAAs of glucose, xylose, fucose and mannose are not detected. However, since the contents of glucose, xylose, fucose and mannose are very low, it does not affect the analysis and judgment of the main glycosidic bond connection form of pollen polysaccharide component 2. Figure 6 The total ion chromatogram shown is the total ion chromatogram of pollen polysaccharide component 2 after methylation treatment, hydrolysis, and derivatization treatment, and detected by GC-MS. From the figure, 6 kinds of methylated sugar group ion fragments were mainly screened and collected. The mass spectrum corresponding to the methylated sugar group peak in the figure ( Figure 7 ) was compared with the standard spectrum library (standard spectrum library: The CCRC Spectral Database for PMAAs https: / / www.ccrc.uga.edu / specdb / ms / pmaa / pframe.html) by correlation, and combined with the determination results of monosaccharide composition, the relevant connection mode of each monosaccharide residue can be inferred, which provides necessary information for the speculation of the repeating unit structure of pollen polysaccharide components.

[0112] As shown in Table 2, the methylation analysis sample of pollen polysaccharide component 2 mainly contains 6 glycosyl ion fragments, which are derived from galactose and arabinose. Among them, galactose mainly exists in the form of →3,6)-Galp-(1→, and arabinose mainly exists in the form of →3,5)-Araf-(1→ and →5)-Araf-(1→. According to the glycosidic bond connection mode of the sugar residues of pollen polysaccharide component 2 and the characteristics of the structure of pollen polysaccharide, it can be preliminarily inferred that the main chain of pollen polysaccharide component 2 is possibly connected by 1,3,5-Araf and 1,3,6-Galp to connect other glycosidic bonds; the branched chain is mainly composed of 1,5-Araf and 1,6-Glcp.

[0113] 2.2 Determination results of pollen polysaccharide component 2 by nuclear magnetic resonance NMR

[0114] Table 3 Monosaccharide residues in pollen polysaccharide component 2 1 H and 13 C NMR chemical shift attribution

[0115]

[0116] As Figures 8-10 shown in the 1 H-NMR spectrum, 13 C-NMR spectrum and DEPT-135 spectrum of pollen polysaccharide component 2, in the 1 H-NMR spectrum, there are 5 relatively obvious signal peaks in the end group hydrogen related region, indicating that pollen polysaccharide component 2 has 5 monosaccharide residues, and the corresponding chemical shifts are 5.18 ppm, 5.08 ppm, 4.83 ppm, 4.41 ppm and 4.46 ppm (Table 3). In the chemical shift range of 4.30-3.50 ppm, it is the chemical shifts of the corresponding hydrogens of the 2nd to 6th carbons of the 5 corresponding sugar residues. According to the HSQC spectrum and 13 C-NMR spectrum, the chemical shifts of the end group carbons corresponding to the end group hydrogens of the glycosidic bond can be determined. HSQC( Figure 11 ), COSY( Figure 12 ) and TOCSY( Figure 13 ) are two-dimensional spectra of carbon and hydrogen attribution and adjacent hydrogen correlation and total hydrogen correlation for the same monosaccharide residue. Through analysis, other carbons and hydrogens in the related monosaccharide residues can be attributed. Through HMBC spectrum( Figure 14 ), the spatial correlation of carbon and hydrogen can be determined, and then the connection order of the glycosidic bond of the monosaccharide is judged.

[0117] Based on methylation analysis and related NMR results, it can be determined that pollen polysaccharide component 2 is composed of a main chain consisting of arabinose linked by α-L-1,3,5-Araf and galactose linked by β-D-1,3,6-Galp, with branching groups attached at the 5-position of arabinose and the 6-position of galactose. The branching groups are mainly composed of α-L-1,5-Araf and β-D-1,6-Gal. The main repeating structural units of pollen polysaccharide component 2 are as follows:

[0118]

[0119] ,in

[0120] R1 is T-α-L-Araf-(1→5)-α-L-Araf-(1→

[0121] R2 is T-β-D-Galp-(1→6)-β-D-Galp-(1→

[0122] 3 Other structural measurement results

[0123] 3.1 Results of Ultraviolet Spectroscopy Measurement

[0124] like Figure 15 As shown, pollen polysaccharide component 2 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 component 2 is a simple polysaccharide with high purity.

[0125] 3.2 Infrared Spectroscopy Results

[0126] like Figure 16 As shown, the absorption band of pollen polysaccharide component 2 is in the range of 3600-3200 cm⁻¹. -1 This is the absorption peak of the stretching vibration of -OH, and absorption peaks in this region are characteristic peaks of carbohydrates. Specifically: 3288m. -1 This is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. At 2931 cm⁻¹ -1 Location, 2871cm -1 There is an absorption peak at 1641 m, which may be attributed to the CH stretching vibration. -1 There is an absorption peak at 1548 cm⁻¹, which may be attributed to the water of crystallization. -1 An absorption peak is observed at 1440 cm⁻¹, which may be attributed to the C=O stretching vibration. -1 Location, 1402cm -1 There is an absorption peak at 1309 cm⁻¹, which may be attributed to the CO stretching vibration. -1 Location, 1243cm-1 an absorption peak at 1081 cm -1 an absorption peak at 896 cm -1 an absorption peak at 896 cm

[0127] Example 6 Growth promoting effect of pollen polysaccharide component 2 on tobacco

[0128] Experimental sample:

[0129] The pollen polysaccharide component 2 was diluted with water to a polysaccharide concentration of 10 mg / ml and used according to the experimental design.

[0130] 1. Experimental design:

[0131] Table 4 Concentration design of the agents

[0132]

[0133]

[0134] 2. Experimental method:

[0135] The agents were prepared according to Table 4 and sprayed uniformly on the leaves of the tobacco seedlings, with 6 repeats for each treatment and 1 plant for each repeat. The spraying was done without dripping of the liquid to prevent the agents from entering the soil and affecting the experimental results. During the cultivation, the temperature was 28°C, the illumination was 2000 lux, the light / dark cycle was 14h / 10h, and the humidity was 65%. The RGB AREA_MM parameter values (leaf area / mm 2 ) of the tobacco were recorded by the plant phenotype analysis system before and 7 days after the treatment, the leaf area growth rate was calculated to evaluate the growth promoting effect of the agents, and the calculation formula was as follows:

[0136] Leaf area growth rate (%) = (final leaf area - initial leaf area) x 100% / initial leaf area

[0137] The leaf area growth rates of the different treatment groups 7 days after the treatment are shown in Table 5. It can be seen from the experimental results that the pollen polysaccharide component 2 has a growth promoting effect on tobacco within the concentration range of 0.03-30 ppm, and the optimal growth promoting concentration is 3 ppm, which has a significant growth promoting effect on tobacco.

[0138] Table 5 Results of the determination of the growth promotion of tobacco in different treatment groups

[0139]

[0140] Example 7 Growth promoting effect of pollen polysaccharide component 2 on small cabbage

[0141] 1. Experimental sample:

[0142] Pollen polysaccharide component 2 was diluted with water to a polysaccharide concentration of 10 mg / ml, and then used according to the experimental design, using water.

[0143] 2. Experimental design:

[0144] Table 6 Concentration design of the agent

[0145]

[0146] 3. Experimental method:

[0147] Select small white cabbage seedlings with consistent growth (3-4 leaves), and prepare the agent according to Table 6. Irrigate the roots of the small white cabbage seedlings, with 6 replicates per treatment, 1 plant per replicate, and an irrigation volume of 80 ml. Place the plants in a plant culture room at 25°C, with a light intensity of 3000 lux, 14h / 10h (day / night), and a humidity of 65%. Maintain consistent water and fertilizer management levels during the culture period. Measure the leaf length, leaf width, aboveground fresh weight, and SPAD value 7 days after treatment.

[0148] 4. Experimental results:

[0149] The results of the different treatment groups 7 days after treatment are shown in Table 7. According to the experimental results, pollen polysaccharide treatment groups had a growth-promoting effect on small white cabbage at a concentration range of 0.03-30 ppm, with the best growth-promoting concentration of pollen polysaccharide component 2 being 3 ppm, which was superior to the control group.

[0150] Table 7 Results of small white cabbage growth promotion determination 7 days after treatment

[0151]

[0152] Example 8 Anti-low temperature experiment of pollen polysaccharide component 2

[0153] 1. Experimental sample:

[0154] Pollen polysaccharide component 2 was diluted with water to a polysaccharide concentration of 10 mg / ml, and then used according to the experimental design, using water.

[0155] 2. Experimental design:

[0156] Table 8 Concentration design of the agent

[0157]

[0158] 3. Experimental method:

[0159] The processing agent is prepared according to Table 8, and then uniformly sprayed on the tobacco seedling leaves. Each treatment has 6 repeats, and each repeat has 1 plant. The liquid is not allowed to drip, so as to prevent the agent from entering the soil and affecting the experimental results. After the seedlings are recovered for 1 day, the plants are subjected to cold damage (4°C) for 24 hours, and then recovered at 28°C. The chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of tobacco are recorded by a plant phenotype instrument before the treatment, after 24 hours of low-temperature treatment, and after 24 hours of recovery.

[0160] 4. Experimental results:

[0161] QY-max represents the theoretical maximum photosynthetic capacity of plants. The smaller the reduction rate, the better the plant's resistance to low temperature. Fv / Fm-lss represents the maximum quantum yield of PSII, which is similar to QY-max. The smaller the reduction rate, the better the plant's resistance to low temperature. As can be seen from Table 9, after 24 hours of low-temperature treatment, the fluorescence values of QY-max and Fv / Fm-lss of each treatment group decreased significantly, but the reduction degree of the treatment group using pollen polysaccharide component 2 was lower, indicating that the growth state of the plants was better than that of the water control, and the resistance to low temperature was stronger. After 24 hours of low-temperature treatment, pollen polysaccharide component 2 with a concentration of 0.03-30 ppm has the effect of improving the cold resistance of tobacco, and the best low-temperature resistance concentration is 0.3 ppm. After 24 hours of recovery at room temperature, as shown in Table 10, the fluorescence values of QY-max and Fv / Fm-lss of each treatment group began to rise, indicating that the growth state of each plant damaged by low temperature was recovered, and component 2 at a concentration of 0.3 ppm could significantly promote the recovery of the growth state of the plants.

[0162] Table 9. Measurement results after 24 hours of low-temperature treatment

[0163]

[0164]

[0165] Table 10. Measurement results after 24 hours of recovery at room temperature

[0166]

[0167] Example 9. Anti-high-temperature experiment of pollen polysaccharide component 2

[0168] 1. Experimental samples:

[0169] The pollen polysaccharide component 2 is prepared into a mother liquor with a polysaccharide concentration of 10 mg / ml, and then diluted with water according to the experimental design. The water is used as the control.

[0170] 2. Experimental design:

[0171] Table 11. Concentration design of the agent

[0172]

[0173] 3. Experimental method:

[0174] Select tobacco seedlings with consistent growth state (3-leaf stage), and prepare the treatment agent according to Table 11. Then, uniformly spray the treatment agent on the leaves of the tobacco seedlings. There are 6 repeats for each treatment, and 1 plant for each repeat. The spraying should be appropriate so that the agent does not drip and enter the soil to affect the experimental results. After the seedlings are recovered for 1 day, treat them at high temperature of 40°C for 48 h. Record the chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of the tobacco using a plant phenotype instrument before treatment and after 48 h of high-temperature treatment, respectively. Then, take samples to measure the malondialdehyde content after 48 h of high-temperature treatment.

[0175] 4. Experimental results:

[0176] QY-max represents the theoretical maximum photosynthetic capacity of the plant. The smaller the reduction rate, the better the high-temperature resistance of the plant. Fv / Fm-lss represents the maximum quantum yield of PSII, which is similar to QY-max. The smaller the reduction rate, the better the high-temperature resistance of the plant. The non-photochemical quenching coefficient NPQ-lss reflects the ability of the plant to dissipate excess light energy as heat, reflecting the light protection ability of the plant. When the plant is under stress, the light energy absorbed by the plant is no longer used for photosynthesis, but is directly converted into heat energy and dissipated, thereby avoiding damage to the plant. At this time, the NPQ-lss of the plant will increase. Therefore, the lower the NPQ-lss value under high-temperature stress, the less damage the plant suffers, and the better the high-temperature resistance. The malondialdehyde content is closely related to the stress damage of the plant. Under stress conditions, membrane lipid peroxidation often occurs, producing malondialdehyde (MDA) with cytotoxicity. Therefore, the lower the MDA content under high-temperature conditions, the less damage the plant suffers, and the better the high-temperature resistance.

[0177] As can be seen from Table 12, after high temperature treatment for 48h, the QY-max and Fv / Fm-lss fluorescence values of each treatment group obviously decreased, and the NPQ-lss fluorescence value obviously increased, but the decrease degree of QY-max and Fv / Fm-lss fluorescence values and the increase degree of NPQ-lss fluorescence value of each treatment group using pollen polysaccharide were smaller, indicating that the growth state of the plant was better than that of the water control, and the high temperature resistance was stronger. After high temperature treatment for 48h, the pollen polysaccharide component 2 in the concentration range of 0.03-30ppm had the effect of improving the high temperature resistance of tobacco, and the best high temperature resistance concentration was 0.3ppm. The MDA content determination results of each treatment group are shown in Table 13, and compared with the control group, each treatment group using pollen polysaccharide can reduce the production of MDA in the plant body, thereby reducing the damage degree of the plant. Consistent with the above-mentioned fluorescence parameters, the MDA content in the plant body of the treatment group of pollen polysaccharide component 2 at a concentration of 0.3ppm was the least.

[0178] Table 12 fluorescence parameter determination results of different treatment groups

[0179]

[0180]

[0181] Table 13 MDA content determination results of different treatment groups

[0182]

Claims

1. A pollen polysaccharide component 2, the main repeating unit structure of which is as follows: wherein, , R1 is T-α -L-Araf- (1→5) -α -L-Araf- (1→ R2 is T-β -D-Galp- (1→6) -β -D-Galp- (1→ The average molecular weight of the pollen polysaccharide component 2 is 5-15 KDa. The pollen polysaccharide component 2 further contains the following monosaccharide units: fucose, glucose, xylose and mannose.

2. Pollen polysaccharide fraction 2 according to claim 1, characterized in that, The average molecular weight of the pollen polysaccharide component 2 is 9-11 KDa.

3. Pollen p oly saccharide fraction 2 according to claim 1, characterized in that, The average molecular weight of the pollen polysaccharide component 2 is 10718.15 Da.

4. Pollen p oly saccharide fraction 2 according to claim 3, characterized in that, The following are included:

5. Pollen p oly saccharide fraction 2 according to any one of claims 1 to 4, characterized in that, The infrared spectrum thereof includes at least one or more of the following absorption peaks: 3288m -1 , 2931 cm -1 , 2871 cm -1 , 1641m -1 , 1548 cm -1 , 1440 cm -1 , 1402 cm -1 , 1309 cm -1 , 1243 cm -1 , 1081 cm -1 , 896 cm -1 .

6. A method for the isolation of a pollen polysaccharide fraction 2 according to any one of claims 1 to 5, characterized in that, (1) The rape pollen polysaccharide extract is purified by a macroporous adsorption resin column, and a preliminary purified pollen polysaccharide active part Fr-1 is obtained by elution with water; (2) The pollen polysaccharide active part Fr-1 is further purified by ion exchange chromatography column, and a pollen polysaccharide active part Fr-1-2 is obtained by gradient elution with 0-0.025 mol / L NaCl solution; (3) The pollen polysaccharide active part Fr-1-2 is further purified by a dextran gel chromatography column, and a component 2, i.e. the pollen polysaccharide component 2, is obtained by using an ammonium bicarbonate aqueous solution as an eluent. The rape pollen polysaccharide extract in step (1) refers to a crude pollen polysaccharide obtained by water extraction and alcohol precipitation of rape pollen.

7. The separation method of claim 6, wherein, The macroporous adsorption resin column used in step (1) is a non-polar column. The macroporous adsorption resin column is selected from one of DB-101 macroporous resin column, S-8 macroporous resin column, AB-8 macroporous resin column and HP-20 macroporous resin column.

8. The separation method of claim 7, wherein, The macroporous adsorption resin column is selected from HP-20 macroporous resin column.

9. The separation method of claim 8, wherein, The preliminary purified pollen polysaccharide active part Fr-1 in step (1) and the pollen polysaccharide active part Fr-1-2 in step (2) are treated by dialysis and freeze-drying to obtain their solids, respectively; the dialysis refers to the dialysis of substances with a molecular weight of at least 3500 Da.

10. The separation method of claim 6, wherein, The ion exchange chromatography column used in step (2) is an anion exchange chromatography column.

11. The separation method of claim 6, wherein, The dextran gel chromatography column used in step (3) is selected from a propylene dextran gel column. The anion exchange chromatography column is selected from a DEAE cellulose chromatography column.

12. The separation method of claim 11, wherein, The propylene dextran gel column is selected from a propylene dextran gel S-400HR chromatography column. In step (3), the concentration of the ammonium bicarbonate aqueous solution is 0.1-0.3 mol / L.

13. The separation method of claim 6, wherein, 14. Use of the pollen polysaccharide component 2 according to any one of claims 1-5 in the preparation of a plant stress resistance product; the plant stress resistance includes high temperature resistance and cold resistance. The product containing the pollen polysaccharide component 2 is applied to the leaves or roots of plants during use. The plants include tobacco.

15. Use of the pollen polysaccharide component 2 according to any one of claims 1-5 in the preparation of a plant growth promoting product; the plants include tobacco and small bok choy. The active ingredients of the product include the pollen polysaccharide component 2 according to any one of claims 1-5.

16. An agricultural product, characterized in that, The product further includes an auxiliary material, which is one or more of a dispersing agent, a wetting agent, a binder, an emulsifying agent, a stabilizer, a solvent and an embedding agent.

17. The agricultural product of claim 16, wherein, ​ 18. The agricultural product according to claim 17, the product is in the form of an emulsifiable concentrate, a suspension concentrate, a wettable powder, a dustable powder, a granule, a water soluble concentrate.

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