Pollen polysaccharide component 4, 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 polysaccharide in rape pollen is extracted, separated and purified, and the pollen polysaccharide is obtained. The pollen polysaccharide component 4 has certain effects in the aspects of plant growth promotion and stress resistance.

Description

[0001] The application belongs to the technical field of plant extract separation, and particularly relates to pollen polysaccharide component 4 and a separation method and application thereof. BACKGROUND

[0002] Previous researches (patent CN 113133454B and patent 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., and the components thereof are also detected, including sugars, proteins, amino acids and lipids. 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 promoting plant growth or stress resistance, 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 bonds, β-1, 6 glycosidic bonds, β-1, 4 glycosidic bonds, α-1, 4 glycosidic bonds and β-1, 3 glycosidic bonds. Polysaccharides widely exist in animals, plants and microorganisms, and polysaccharides from different sources have different biological activities. Existing researches 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.

[0006] At the same time, 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 polysaccharides, so as 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, and the pollen polysaccharide has certain effects in plant growth promotion and stress resistance.

[0008] Specifically, the present application provides one pollen polysaccharide component 4, and the main repeating structural unit is as follows:

[0009]

[0010] In the formula, R1 is T- α -L-Ara f -(1→5)- α -L-Ara f -(1→ 。

[0011] In the present application, Ara represents arabinose; Gal represents galactose; f represents furanose configuration; p represents pyranose configuration; T represents the end of the polysaccharide molecule (end group).

[0012] In the formula, the molar content ratio of arabinose: galactose is equal to 0.686: 0.243.

[0013] 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, mannose and galacturonic acid.

[0014] In the formula, the molar content ratio of fucose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid is equal to 0.003: 0.686: 0.243: 0.014: 0.021: 0.013: 0.009.

[0015] In the present application, the average molecular weight of the pollen polysaccharide is 5-15KDa;

[0016] Further, the average molecular weight of the pollen polysaccharide is 9-11KDa;

[0017] In the present application, the average molecular weight of the pollen polysaccharide is 10-11KDa, for example, 10328.089 Da.

[0018] The infrared spectrum of the pollen polysaccharide of the present application at least includes one or more of the following absorption peaks: 3322m -1 , 2931 cm -1 , 2875cm -1 , 1641m -1 , 1544cm -1 , 1407cm -1 , 1309cm -1 , 1241cm-1 , 1076 cm -1 , 1047 cm -1 , 894 cm -1 .

[0019] The infrared absorption band of the pollen polysaccharide component 4 is at 3600-3200 cm -1 , which is the stretching vibration absorption peak of -OH, and the absorption peak in this region is the characteristic peak of saccharides. Specifically, 3322m -1 is the stretching vibration absorption peak of O-H, which is the characteristic peak of saccharides. At 2931 cm -1 , 2875 cm -1 , there is an absorption peak that can be attributed to C-H stretching vibration. At 1641m -1 , there is an absorption peak that can be attributed to crystal water. At 1544 cm -1 , there is an absorption peak that can be attributed to C=O stretching vibration. At 1407 cm -1 , there is an absorption peak that can be attributed to C-O stretching vibration. At 1309 cm -1 , 1241 cm -1 , 1076 cm -1 , 1047 cm -1 , there are absorption peaks that can be attributed to O-H variable angle vibration. At 894 cm -1 , there is an absorption peak that can be attributed to the β-anomeric C-H variable angle vibration of the pyran ring.

[0020] The purpose of the present application is also to provide a pollen polysaccharide separation method as described above, which comprises the following contents:

[0021] (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;

[0022] (2) The pollen polysaccharide active part Fr-1 is subjected to gradient elution by an ion exchange chromatography column with 0-0.25 mol / L NaCl solution, and the 0.25 mol / L NaCl solution component is collected to obtain the pollen polysaccharide active part Fr-1-4;

[0023] (3) The pollen polysaccharide active part Fr-1-4 is further purified by a gel chromatography column, wherein ammonium bicarbonate aqueous solution is used as an eluent to obtain component 4, i.e., the pollen polysaccharide of the present application.

[0024] The pollen polysaccharide active part Fr-1 eluted by the ion exchange column can be in the form of the original eluent of the macroporous resin, or the concentrated solution of the original eluent, or the reconstituted solution after the original eluent is concentrated and dried, etc.

[0025] The pollen polysaccharide active site Fr-1-4 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.

[0026] The concentration and drying method includes but is not limited to the following methods: reduced pressure evaporation, normal pressure evaporation, drying, vacuum drying, thin film drying, freeze drying, and the like.

[0027] The oilseed rape pollen polysaccharide extract in step (1) refers to the crude pollen polysaccharide obtained by a conventional method.

[0028] The water extraction includes but is not limited to the following methods: heating extraction, ultrasonic extraction, microwave extraction, and the like.

[0029] Meanwhile, the impurities can be removed by defatting and decolorization before extraction according to different conditions, for example, the defatting and decolorization are performed by using a fat-soluble solvent such as ethanol or petroleum ether.

[0030] The preliminary purification method after water extraction includes but is not limited to the following methods: alcohol precipitation and chitosan impurity removal.

[0031] 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.

[0032] In the purification method, auxiliary means such as protein removal, decolorization, and small molecule removal can be selectively added according to different conditions, so as to facilitate the subsequent polysaccharide enrichment process.

[0033] 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.

[0034] In some specific embodiments of the present application, the oilseed rape pollen polysaccharide extract in step (1) refers to the crude pollen polysaccharide obtained by heating water extraction and chitosan impurity removal of the oilseed rape pollen.

[0035] In some specific embodiments of the present application, the oilseed rape pollen can be subjected to ultramicro-pulverization treatment to facilitate the extraction of polysaccharides.

[0036] 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.

[0037] In the technical solution of the present application, the macroporous resin column used in step (1) is a non-polar column.

[0038] 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.

[0039] 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.

[0040] 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, polyacrylic acid gel Toyopearl HW chromatography column, and other different gel chromatography columns; and is further selected from propylene dextran gel S-400 HR chromatography column and Sephadex LH-20 chromatography column.

[0041] 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.

[0042] In some embodiments of the present application, the pollen polysaccharide active site Fr-1 preliminarily purified in step (1) and the pollen polysaccharide active site Fr-1-4 in step (2) can be treated by dialysis and freeze-drying to obtain a solid for standby use.

[0043] Further, the dialysis refers to substances with a molecular weight of at least 3500 Da.

[0044] In some embodiments of the present application, the eluent containing component 4 in step (3) is concentrated under reduced pressure, dialyzed (3500 Da) to remove salt, and freeze-dried to obtain a solid of pollen crude polysaccharide component 4.

[0045] 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.

[0046] 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.

[0047] The application also provides application of the pollen polysaccharide component 4 in preparation of a plant stress resistance product.

[0048] The plant stress resistance of the application includes high temperature resistance, cold resistance, drought resistance, salt and alkali tolerance, etc.

[0049] In the application, the product containing the pollen polysaccharide is applied to the plant leaves or the plant roots.

[0050] 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.

[0051] For example, the concentration of the pollen polysaccharide component 4 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 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.

[0052]

[0053] The application also provides application of the pollen polysaccharide component 4 in preparation of a plant growth promoting product.

[0054] The plants in the application include but are not limited to economic crops and food crops, such as Chinese cabbage, baby bok choy, asparagus lettuce, lettuce, Shanghai green, wheat, pepper, tomato, citrus, kiwi, cherry, pear, apple, tobacco, etc.

[0055] 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, medicinal crops, dye crops, ornamental crops, fruits and other economic crops, etc.

[0056] 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.).

[0057] The application also provides an agricultural product, and the active ingredient of the product includes the pollen polysaccharide component 4.

[0058] ​In the present application, the direct pollen polysaccharide component 4 can be used as a single agent, and in order to stabilize the product, 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.

[0059] In another aspect, the pollen polysaccharide component 4 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.

[0060] 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.

[0061] The present application has the beneficial effect that a new polysaccharide is discovered and first isolated from rape pollen, which can be used for plant stress resistance and plant growth promotion.

[0062] 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

[0063] Figure 1 : Gradient elution curve of pollen crude polysaccharide on DEAE cellulose-52 anion exchange column;

[0064] Figure 2 : Elution curve of pollen polysaccharide component 4 on S-400 HR propylene dextran gel column;

[0065] Figure 3 : Standard curve of dextran standard molecular weight distribution;

[0066] Figure 4 : HPGPC determination chromatogram of pollen polysaccharide component 4;

[0067] Figure 5 : Monosaccharide mixed standard and pollen polysaccharide component ion chromatograph detection chromatogram (A: monosaccharide mixed standard; B: pollen polysaccharide component 3;). Among them, the 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.

[0068] Figure 6Total ion chromatogram (TIC) for 4-methylation analysis of pollen polysaccharide components;

[0069] Figure 7 Mass spectra of methylated PMAAs belonging to pollen polysaccharide fraction 4;

[0070] Figure 8 Pollen polysaccharide component 4 1 H-NMR spectrum;

[0071] Figure 9 Pollen polysaccharide component 4 13 C-NMR spectrum;

[0072] Figure 10 DEPT-135 NMR spectrum of pollen polysaccharide fraction 4;

[0073] Figure 11 HSQC spectrum of pollen polysaccharide fraction 4;

[0074] Figure 12 COSY spectrum of pollen polysaccharide fraction 4;

[0075] Figure 13 TOCSY spectra of pollen polysaccharide fraction 4;

[0076] Figure 14 HMBC spectrum of pollen polysaccharide fraction 4;

[0077] Figure 15 : Ultraviolet absorption spectrum of pollen polysaccharide component 4;

[0078] Figure 16 Infrared absorption spectrum of pollen polysaccharide component 4. Detailed Implementation

[0079] 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.

[0080] Example 1: Extraction of pollen polysaccharides

[0081] Rape pollen is super-micro pulverized into powder to obtain pretreated pollen powder. 5.0 g of the pretreated pollen powder is weighed, and then free monosaccharides are removed by ethanol, and the pollen is defatted and decolorized by petroleum ether, and then hot water extraction is performed, and the precipitate is collected and washed with anhydrous ethanol, diethyl ether and acetone in sequence. Finally, the washed precipitate is redissolved with water, pre-frozen at -20 ℃, and vacuum freeze-dried to obtain hot water extracted pollen crude polysaccharide.

[0082] Rape pollen is mixed with water, heated for extraction, filtered, and chitosan is added to the filtrate, which is incubated and separated. The obtained liquid is the pollen crude polysaccharide extract. The extract is concentrated under reduced pressure, pre-frozen at -20 ℃, and vacuum freeze-dried to obtain pollen crude polysaccharide.

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

[0084] Example 4: Isolation and purification of pollen polysaccharide

[0085] Step (1) HP-20 macroporous adsorption resin column purification: HP-20 macroporous adsorption resin column (Φ 4.0 cm × 40 cm) is used to preliminarily purify the pollen crude polysaccharide, and the specific steps are as follows: a certain amount of pollen crude polysaccharide is weighed, a certain amount of water is added to prepare a 10-20 mg / mL pollen crude polysaccharide solution, and the single sample loading amount is 10 mL. After being adsorbed by the HP-20 macroporous adsorption resin column for 3 hours, the column is washed with 5 times the column volume of water, and the sample is connected to obtain the pollen polysaccharide active fraction Fr-1, which is concentrated under reduced pressure at 48 ℃, dialyzed (3500 Da), and freeze-dried for use.

[0086] 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 (Φ 3.5 cm × 30 cm). The specific steps are as follows: ① Load 100 mg 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 0.5 mol / L NaCl solution in sequence, the flow rate is 1.2 mL / min, and 6 mL 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 490 nm 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 °C, dialyzed (3500 Da) to remove salt, and freeze-dried for later use.

[0087] 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.

[0088] Step (3) S-400 HR Sepharose CL-4B column purification: Finally, the S-400 HR Sepharose CL-4B column (Φ 1.0 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 eluent; 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, dialysis (3500 Da) to remove ammonia, freeze-drying to obtain the purified components of each pollen polysaccharide.

[0089] 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-400 HR Sepharose CL-4B column, and 0.2 mol / L ammonium bicarbonate was used to elute the four components. From Figure 2 It can be seen that after elution of the four components with 0.2 mol / L ammonium bicarbonate, single elution peaks were obtained, and the corresponding components were named as component 1, component 2, component 3 and component 4, respectively. The eluent 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.

[0090] Purity identification and molecular weight determination of pollen polysaccharide purified components

[0091] Determination method: The purity identification and molecular weight determination of each purified component of pollen polysaccharide were carried out 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-804 HQ (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; injection volume was 20 μL; instrument test time was 24 min.

[0092] Preparation of standard curve: The dextran standard with different molecular weight (5900, 9600, 21100, 47100, 107000, 200000, 341400 Da) was 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 water phase filter, the standard solution 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 (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 = -0.322 x + 9.4365, as shown in Figure 3 .

[0093] 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 pollen polysaccharide component 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.

[0094] The purity and molecular weight of the purified component 4 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 4 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 pollen polysaccharide separation and purification method 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 4 was calculated to be 10328.089 Da.

[0095] Example 5 Structure identification of pollen polysaccharide

[0096] Determination method: Ion chromatography (IC) was used to determine the monosaccharide composition of pollen polysaccharide samples. 10 mg of polysaccharide sample was precisely weighed into an ampoule and subjected to acid hydrolysis. The acid hydrolysis solution was accurately pipetted into a test tube and blown dry with nitrogen. 5 mL of distilled water was added and vortexed to mix. 50 uL was pipetted into 950 uL of distilled water, centrifuged at 12000 r / min for 5 min, and the supernatant was subjected to IC analysis. At the same time, 16 kinds of monosaccharide standard samples (fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, galactosamine hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid, mannuronic acid) were prepared into a standard mother liquor solution. The monosaccharide standard solution was precisely configured as a mixed standard. The monosaccharide composition of the pollen polysaccharide hydrolysis sample was determined according to the peak time in the chromatogram. According to the absolute quantitative method, the mass of different monosaccharides was determined, and the molar ratio was calculated according to the molar mass of the monosaccharide. C (standard) / A (standard) =C (sample) / A (sample). C is the concentration, and A is the peak area. Chromatographic conditions: Chromatographic column: Dionex Carbopac T PA20 (3*150); mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH and 100 mM sodium acetate; flow rate: 0.3 mL / min; injection volume: 5 uL; column temperature: 30ºC; detector: electrochemical detector. As shown in Figure 5 (A), 16 standard monosaccharide chromatographic peaks were successfully separated and detected using an ion chromatograph, with regular peak shape and high separation degree, which can well meet the simultaneous detection of common monosaccharide types in monosaccharide composition analysis.

[0097]

[0098] As shown in Figure 5 (B), the ion chromatogram of the monosaccharide composition analysis of pollen polysaccharide component 4 showed that a small amount of galacturonic acid was detected in Figure 5 (B), indicating that pollen polysaccharide component 4 contains uronic acid, which belongs to acidic sugar. As can be seen from the figure, there are two obvious chromatographic peaks, which are arabinose and galactose from left to right. Through the calculation of related chromatographic peak area, it can be obtained that the ratio of fucose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid in pollen polysaccharide component 4 is 0.003: 0.686: 0.243: 0.014: 0.021: 0.013: 0.009 (Table 1).

[0099] 2 Methylation and nuclear magnetic resonance analysis of pollen polysaccharide

[0100] In order to study the spatial structure of each component of pollen polysaccharide, nuclear magnetic resonance NMR analysis was performed on pollen polysaccharide component 3 to determine the 1 H-NMR spectrum,13 C-NMR, DEPT-135, HSQC, COSY, TOCSY, HMBC, and NOESY spectra were analyzed, and correlation analysis was used to determine the linkage sequence between different monosaccharide residues, 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.

[0101] 2.1 Results of methylation determination of pollen polysaccharide component 4

[0102] To investigate the primary structure of pollen polysaccharide component 4, a modified Needs method was used to methylate pollen polysaccharide component 4.

[0103] The fully methylated pollen polysaccharide fraction 4, 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 4, as shown below. Figure 6 As shown in the figure. The mass spectrum corresponding to the methylated glycosylation 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 4 repeating units of pollen polysaccharide components.

[0104] Table 2 Results of 4-methylation analysis of pollen polysaccharide components

[0105]

[0106] 2,3-Me2-Ara f For 1,4,5-tri- O -acetyl-2,3-di- O -methyl-L-arabinitol (1,4,5-tri- O -Acetyl-2,3-bis- O- methyl-L-arabinose), and so on.

[0107] Since pollen polysaccharide component 4 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 xylose, fucose and mannose are not detected. However, since the contents of glucose, xylose, fucose and mannose are very low, they do not affect the analysis and judgment of the main glycosidic bond connection form of pollen polysaccharide component 4. Figure 6 The total ion chromatogram of pollen polysaccharide component 4 after methylation treatment, hydrolysis, derivatization treatment, and detection by GC-MS is shown. Figure 7 Nine kinds of methylated derivatized sugar group ion fragments are mainly screened and collected.

[0108] As shown in Table 2, the methylation analysis sample of pollen polysaccharide component 4 mainly contains nine kinds of sugar group ion fragments, which are derived from galactose, arabinose and glucose. Among them, galactose mainly exists in the form of →6)-Gal p -(1→, arabinose mainly exists in the form of →3,5)-Ara f -(1→ and →5)-Ara

[0109] f According to the glycosidic bond connection mode of the sugar residues of pollen polysaccharide component 4 and combined with the characteristics of pollen polysaccharide structure, it can be preliminarily inferred that the main chain of pollen polysaccharide component 4 is possibly connected by 1,3,5-Ara f and 1, 6-Gal p , and other glycosidic bonds; the branch chain is mainly composed of 1,5-Ara f , →3,6)-Gal p -(1→ and 1,3-Gal p

[0110] .

[0111] 2.2 Determination results of pollen polysaccharide component 4 by nuclear magnetic resonance NMR

[0112] Table 3 Monosaccharide residues in pollen polysaccharide component 4 1 H and 13 C NMR chemical shift attribution

[0113]

[0114] As shown in Figures 8, 9 and 10, in the 1 H-NMR spectrum, 13 C-NMR spectrum and DEPT-135 spectrum of pollen polysaccharide component 4, 1In the end group hydrogen related region of H-NMR spectrum, there are 7 relatively obvious signal peaks, indicating that pollen polysaccharide component 4 has 7 monosaccharide residues (Table 3), and the corresponding chemical shifts are 5.31 ppm, 5.14 ppm, 5.09 ppm, 4.79 ppm, 4.63 ppm, 4.57 ppm and 4.52 ppm. In the range of 4.30-3.50 ppm, it is the chemical shifts of the 2nd to 6th carbon corresponding hydrogen of the 7 corresponding sugar residues. According to HSQC spectrum (Fig. 11) and 13C-NMR spectrum, the chemical shifts of end group carbon corresponding to end group hydrogen of glycosidic bond can be determined. HSQC, COSY (Fig. 12) and TOCSY (Fig. 13) are two-dimensional spectra of carbon and hydrogen attribution and adjacent hydrogen correlation and total hydrogen correlation of the same monosaccharide residue. Through analysis, other carbons and hydrogens in the related monosaccharide residue can be attributed. Through HMBC spectrum (Fig. 14), the spatial correlation of carbon and hydrogen can be determined, and then the connection order of monosaccharide glycosidic bond can be judged. Figure 11 )and 13C-NMR spectrum. HSQC, COSY (Fig. 12) and TOCSY (Fig. 13) are two-dimensional spectra of carbon and hydrogen attribution and adjacent hydrogen correlation and total hydrogen correlation of the same monosaccharide residue. Through analysis, other carbons and hydrogens in the related monosaccharide residue can be attributed. Through HMBC spectrum (Fig. 14), the spatial correlation of carbon and hydrogen can be determined, and then the connection order of monosaccharide glycosidic bond can be judged. Figure 14 )and 13C-NMR spectrum. HSQC, COSY (Fig. 12) and TOCSY (Fig. 13) are two-dimensional spectra of carbon and hydrogen attribution and adjacent hydrogen correlation and total hydrogen correlation of the same monosaccharide residue. Through analysis, other carbons and hydrogens in the related monosaccharide residue can be attributed. Through HMBC spectrum (Fig. 14), the spatial correlation of carbon and hydrogen can be determined, and then the connection order of monosaccharide glycosidic bond can be judged.

[0115] According to the methylation analysis combined with the analysis of related nuclear magnetic results, it can be determined that pollen polysaccharide component 4 is connected by arabinose through α- L - 1,3,5-Ara f and galactose through β -D-1, 3)-Gal p to form the main chain, and branched chain groups are connected at the 5th position of arabinose and the 3rd position of arabinose. The branched chain group is mainly composed of T-α-L-Ara f -(1→5)-α-L-Ara f -(1. In addition, there is another branched chain structure of →3)-β-D-Galp-(1→6)-β-D-Galp-(1→. The main repeating structural unit of pollen polysaccharide component 4 is as follows:

[0116]

[0117] Among them, R1 is T- α -L-Ara f -(1→5)- α -L-Ara f -(1→. 3 Other structure determination results

[0118] 3.1 Ultraviolet spectrum determination results

[0119] As Figure 15As shown, pollen polysaccharide component 4 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 4 is a simple polysaccharide with high purity.

[0120] 3.2 Infrared Spectroscopy Results

[0121] Figure 16 This is a typical infrared spectrum of plant polysaccharides, showing the infrared results for pollen polysaccharide component 4. The absorption band is in the 3600-3200 cm⁻¹ range. -1

[0122] This is the absorption peak of the stretching vibration of -OH, and absorption peaks in this region are characteristic peaks of carbohydrates. Specifically: 3322m -1 This is the absorption peak of the stretching vibration of OH, a characteristic peak of carbohydrates. At 2931 cm⁻¹ -1 Location, 2875cm -1 There is an absorption peak at 1641 m, attributed to the CH stretching vibration. -1 There is an absorption peak at 1544 cm⁻¹, which is attributed to the water of crystallization. -1 An absorption peak is observed at 1407 cm⁻¹, which is attributed to the C=O stretching vibration. -1 There is an absorption peak at 1309 cm⁻¹, which is attributed to the CO stretching vibration. -1

[0123] Location, 1241cm -1 Location, 1076cm -1 Location, 1047cm -1 An absorption peak is observed at 894 cm⁻¹, which is attributed to the OH- angular vibration. -1 The absorption peak is attributed to the CH-angle vibration of the β-terminal epimer of the pyran ring.

[0124] Example 6: The effect of pollen polysaccharide component 4 on promoting tobacco growth

[0125] Experimental sample:

[0126] Pollen polysaccharide component 4 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]

[0129] 2. Experimental methods:

[0130] The processing agent is prepared according to Table 4, and the tobacco seedling leaves are uniformly sprayed, 6 repeats for each treatment, 1 plant for each repeat, and the liquid is not allowed to drip to prevent the agent from entering the soil and affecting the experimental results. The conditions during cultivation are as follows: temperature 28°C, illumination 2000 lux, 14 h / 10 h (day / night), and humidity 65%. The RGB AREA_MM parameter value (leaf area / mm) of tobacco is recorded by the plant phenotype analysis system before and 7 days after the drug, and the leaf area growth rate is calculated to evaluate the growth-promoting effect of each agent. The formula for calculating the leaf area growth rate is as follows: 2

[0131] Leaf area growth rate (%) = (final leaf area - initial leaf area) x 100% / initial leaf area

[0132] 3. Experimental results:

[0133] The leaf area growth rate of different treatment groups 7 days after the drug is shown in Table 5. From the experimental results, it can be seen that pollen polysaccharide component 4 has a growth-promoting effect on tobacco in the concentration range of 0.03-30 ppm, and the best growth-promoting concentration is 0.03 ppm, which has a significant growth-promoting effect on tobacco.

[0134] Table 5 Tobacco growth-promoting determination results of different treatment groups

[0135]

[0136] Example 7 Growth-promoting effect of pollen polysaccharide component 4 on Chinese cabbage

[0137] 1. Experimental sample:

[0138] Pollen polysaccharide component 4 is prepared into a polysaccharide concentration of 10 mg / ml stock solution with pure water, and then diluted with water according to the experimental design, i.e. water.

[0139] 2. Experimental design:

[0140] Table 6 Agent concentration design

[0141]

[0142] 3. Experimental method:

[0143] Select Chinese cabbage seedlings with consistent growth (3-4 leaves), prepare the processing agent according to Table 6, and perform root irrigation treatment on the Chinese cabbage seedlings, 6 repeats for each treatment, 1 plant for each repeat, and the irrigation amount for each plant is 80 ml. Place in a plant cultivation room at 25°C, set the light intensity to 3000 lux, 14 h / 10 h (day / night), and the humidity to 65%, and maintain consistent water and fertilizer management levels during cultivation. Measure the leaf length, leaf width, aboveground fresh weight, and SPAD value 7 days after the drug.

[0144] ​4、Experimental results:

[0145] The results of different treatment groups 7 days after administration are shown in Table 7. It can be seen from the experimental results that the pollen polysaccharide treatment group 4 has a growth promoting effect on Chinese cabbage in the concentration range of 0.03-30 ppm, and the optimal growth promoting concentration is 0.03 ppm, which is better than the control group.

[0146] Table 7: Growth promotion test results of Chinese cabbage in different treatment groups 7 days after administration

[0147]

[0148] Example 8: Anti-low temperature experiment of pollen polysaccharide component 4

[0149] 1、Experimental sample:

[0150] Pollen polysaccharide component 4 was prepared into a mother liquor with a polysaccharide concentration of 10 mg / ml using pure water, and then diluted with water according to the experimental design, and the water was used.

[0151] 2、Experimental design:

[0152] Table 8: Design of agent concentration

[0153]

[0154] 3、Experimental method:

[0155] The treatment agent was prepared according to Table 8, and then uniformly sprayed on the leaves of tobacco seedlings, with 6 repeats for each treatment and 1 plant for each repeat. It is appropriate that the drug solution does not drip, to prevent the agent from entering the soil and affecting the experimental results. After 1 day of seedling, the cold damage (4℃) treatment was carried out for 24h, and then the recovery was carried out at 28℃. The chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of tobacco were recorded by plant phenotype instrument before administration, 24h after low temperature treatment and 24h after recovery.

[0156] 4、Experimental results:

[0157] QY-max represents the theoretical maximum photosynthetic capacity of the plant, and the smaller the reduction rate indicates the better low temperature resistance of the plant. Fv / Fm-lss represents the maximum quantum yield of PSII, and similar to QY-max, the smaller the reduction rate indicates the better low temperature resistance of the plant. 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 fluorescence values of QY-max and Fv / Fm-lss of the treatment group using pollen polysaccharide component 4 was smaller, indicating that the growth state of the plant was better than that of the water control, and the low temperature resistance was stronger. After 24 hours of low temperature treatment, pollen polysaccharide component 4 had the effect of improving the cold resistance of tobacco in the concentration range of 0.03-30 ppm, and the best low temperature resistance concentration was 0.03 ppm. After 24 hours of normal temperature recovery, 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 recovering, and pollen polysaccharide component 4 in the concentration range of 0.03-30 ppm could promote the recovery of tobacco growth, and component 4 at a concentration of 0.03 ppm could obviously promote the recovery of the growth state of the plant.

[0158] Table 9 Determination results after 24 hours of low temperature treatment

[0159]

[0160] Table 10 Determination results after 24 hours of normal temperature recovery

[0161]

[0162] Example 9 Anti-high temperature experiment of pollen polysaccharide component 4

[0163] 1. Experimental sample:

[0164] The pollen polysaccharide component 4 was diluted with water to prepare a mother liquor with a polysaccharide concentration of 10 mg / ml, and then used according to the experimental design, i.e., water.

[0165] 2. Experimental design:

[0166] Table 11 Design of agent concentration

[0167]

[0168] 3. Experimental method:

[0169] Select the tobacco seedlings with uniform 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. Each treatment has 6 repeats, and each repeat has 1 plant. The spraying should be appropriate without liquid dripping, so as to prevent the agent from entering the soil and affecting the experimental results. After the seedlings are recovered for 1 day, treat them at 40°C for 48 h. Record the chlorophyll fluorescence QY-max parameter value and Fv / Fm-lss parameter value of the tobacco by using a plant phenotype instrument before the treatment and after the high-temperature treatment for 48 h, respectively. Then, take samples to measure the malondialdehyde content after the high-temperature treatment for 48 h.

[0170] 4. Experimental results:

[0171] QY-max represents the theoretical maximum photosynthetic capacity of the plant. The smaller the reduction rate is, the better the high-temperature resistance of the plant is. Fv / Fm-lss represents the maximum light quantum yield of PSII, which is similar to QY-max. The smaller the reduction rate is, the better the high-temperature resistance of the plant is. NPQ-lss represents the ability of the plant to dissipate excess light energy as heat, which reflects the light protection ability of the plant. When the plant is stressed, the light energy absorbed by the plant is no longer used for photosynthesis, but is directly converted into heat energy and dissipated, so as to avoid the damage to the plant. At this time, the NPQ-lss of the plant increases. Therefore, the lower the NPQ-lss value is under high-temperature stress, the smaller the damage to the plant is, and the better the high-temperature resistance of the plant is. The malondialdehyde content is closely related to the stress damage of the plant. Under stress conditions, membrane lipid peroxidation often occurs, and malondialdehyde (MDA) with cytotoxicity is produced. Therefore, the lower the MDA content is under high-temperature conditions, the smaller the damage to the plant is, and the better the high-temperature resistance of the plant is.

[0172] As shown in Table 12, after the high-temperature treatment for 48 h, the fluorescence values of QY-max and Fv / Fm-lss of each treatment group obviously decrease, and the fluorescence value of NPQ-lss obviously increases. However, the decrease degree of the fluorescence values of QY-max and Fv / Fm-lss and the increase degree of the fluorescence value of NPQ-lss of each treatment group using the pollen polysaccharide are smaller, which indicates that the growth state of the plant is better than that of the control, and the high-temperature resistance of the plant is stronger. After the high-temperature treatment for 48 h, the pollen polysaccharide component 4 in the concentration range of 0.03-30 ppm can improve the high-temperature resistance of the tobacco, and the optimal concentration for the high-temperature resistance is 0.03 ppm. The MDA content measurement results of each treatment group are shown in Table 13. Compared with the control group, each treatment group using the pollen polysaccharide can reduce the production of MDA in the plant, thereby reducing the damage degree of the plant. Consistent with the above-mentioned fluorescence parameters, the MDA content in the plant of the treatment group using the pollen polysaccharide component 4 at the concentration of 0.03 ppm is the least.

[0173] Table 12: Fluorescence parameter measurement results of different treatment groups

[0174]

[0175] Table 13 Determination results of MDA content of different treatment groups

[0176] .

Claims

1. A pollen polysaccharide component 4, the main repeating unit structure of which is as follows: in, R1 is T- α -L-Ara f -(1→5)- α -L-Ara f -(1→ ).

2. The pollen polysaccharide component 4 according to claim 1, characterized in that, Pollen polysaccharides also contain the following monosaccharide units: fucose, glucose, xylose, mannose, and galacturonic acid.

3. The pollen polysaccharide component 4 according to claim 1, characterized in that, The average molecular weight of pollen polysaccharides is 5-15 kDa.

4. The pollen polysaccharide component 4 according to claim 3, characterized in that, The average molecular weight of pollen polysaccharides is 9-11 kDa.

5. The pollen polysaccharide component 4 according to claim 4, characterized in that, The average molecular weight of pollen polysaccharides is 10328.089 Da.

6. The pollen polysaccharide component 4 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: 3322m -1 2931 cm -1 2875cm -1 1641m -1 1544cm -1 1407cm -1 1309cm -1 1241cm -1 1076cm -1 1047cm -1 894cm -1 .

7. A method for separating pollen polysaccharide component 4 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 0~0.25 mol / L NaCl solution through an ion exchange chromatography column, and the 0.25 mol / L NaCl solution fraction was collected to obtain the active part of pollen polysaccharide Fr-1-4; (3) The active part of pollen polysaccharide Fr-1-4 was further purified by dextran gel chromatography column, with ammonium bicarbonate aqueous solution as the eluent, to obtain component 4, namely pollen polysaccharide component 4.

8. The separation method according to claim 7, characterized in that, The rapeseed pollen polysaccharide extract mentioned in step (1) refers to crude pollen polysaccharide obtained by water extraction and alcohol precipitation of rapeseed pollen; The macroporous adsorption resin column used in step (1) is a non-polar column.

9. The separation method according to claim 7, characterized in that, The macroporous adsorption resin column is 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-1-4 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 anion exchange chromatography column was selected from DEAE cellulose chromatography column, and the propylene dextran gel column was 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 4 according to any one of claims 1-5 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 the product containing pollen polysaccharide component 4 to the leaves or roots of the plant; Furthermore, the plant includes tobacco.

16. The use of pollen polysaccharide component 4 according to any one of claims 1-5 in the preparation of plant growth promoting products; Furthermore, the plants include tobacco and bok choy.

17. An agricultural product, characterized in that, The active ingredient of the product includes pollen polysaccharide component 4 as described in any one of claims 1-5.

18. The agricultural product according to claim 17, characterized in that, The product also includes excipients, which are one or more of 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.

Images