AG type tobacco leaf pectin and application thereof

Abstract

The invention relates to AG type tobacco leaf pectin and application thereof, in particular to high-purity AG type tobacco leaf pectin with uniform molecular weight, the tobacco leaf pectin is composed of galactose, arabinose and glucose, the tobacco leaf pectin has remarkable in-vitro oxidation resistance and hypoglycemic activity, and the research blank of the side chain structure and activity of the tobacco leaf pectin is filled.

Description

Technical Field

[0001] The invention belongs to the field of tobacco macromolecules, and specifically relates to AG type tobacco pectin and application thereof. Background Art

[0002] The cell wall of tobacco leaves is mainly composed of macromolecular substances such as cellulose, hemicellulose, pectin, and lignin, among which the pectin content is 5-12%. Pectin plays an important role in the growth, development, storage, transportation, and sensory quality of tobacco leaves. During the growth and development stage, the difference in the composition of pectin structural units is closely related to the permeability, water retention, mechanical properties, and ion binding of tobacco leaf cell walls. Pectin is highly sensitive to ambient air humidity. Tobacco leaves with high pectin content are prone to moisture absorption and mildew; if the pectin content is too low, the tobacco leaves have poor hardness and are difficult to transport. Studies have shown that the use of pectinase to degrade pectin, increase the content of total sugar and RG-I structural units, and improve the sensory quality of tobacco leaves.

[0003] Jiang Caixia et al. characterized the structure of flax fiber pectin extracted by ammonium oxalate (Analysis of the primary structure of flax fiber pectin [J]. Cellulose Science and Technology, 2021, 29(01): 19-24.). The main structural domain unit of flax fiber pectin molecules is galacturonic acid polysaccharide, which has 1→ or 1→6 and 1→2, 1→2,6 or 1→4, 1→4,6 bond types, and has a less branched structure. Gu Xiaojun et al. found that kiwi fruit pectin AP-A1 is mainly composed of D-galactose and D-xylose (Purification of kiwi fruit pectin and analysis of molecular structure information [J]. Food Science, 2016, 37(11): 47-51.). However, there are few studies on the structure of tobacco leaf pectin.

[0004] The RG-I structural unit skeleton of pectin is composed of [→4)-α-D-GalpA-(1→2)-α-L-Rhap-(1→]n connected in sequence to form the main chain, where Rha pThe C-4 position of pectin can be replaced by a neutral sugar side chain. The pectin RG-I side chain generally includes three types: galactan, arabinan, and arabinogalactan (AG). Wang Peng et al. (Solid-state NMR spectroscopy characterization of tobacco pectin uronic acid structure [J]. Journal of Anhui Agricultural University, 2024, 51(02): 207-211.) only characterized the uronic acid structure of tobacco pectin, and did not analyze its neutral sugar side chain structure. Its preparation method is acid extraction, which is easy to damage the structure of pectin. Chang et al. used water extraction and alcohol precipitation and ion exchange chromatography to prepare tobacco polysaccharides. The monosaccharide composition of the purified polysaccharide components included galactose, arabinose and glucose, as well as monosaccharides such as galacturonic acid, rhamnose, and mannose, and contained a variety of pectin structural units. The study did not analyze the fine structure of tobacco polysaccharides (Chang et al. In vitro study on antioxidant and lipid-lowering activities of tobacco polysaccharides[J]. Bioresources and Bioprocessing, 2024, 11:15).

[0005] my country produces a large amount of low-grade tobacco leaves every year, accounting for 20% of tobacco production, and the waste of resources is quite serious. At present, the structural research of AG-type tobacco pectin is still blank, and its biological activity research has yet to be developed. Therefore, it is of great significance to develop a preparation process for high-purity AG-type tobacco pectin and clarify the fine structure of AG-type tobacco pectin. Summary of the invention

[0006] In view of the problems existing in the above prior art, the present invention provides an AG-type tobacco pectin with uniform molecular weight, clarifies the structural unit and composition of the AG-type tobacco pectin, and finds that the tobacco pectin has significant in vitro antioxidant and hypoglycemic activities, filling the research gap in the structure and activity of AG-type tobacco pectin.

[0007] Specifically, in the first aspect of the present invention, the present invention provides an AG type tobacco pectin, wherein the AG type tobacco pectin is composed of galactose, arabinose and glucose, and the molar ratio of the galactose, arabinose and glucose is 0.55:0.33:0.12;

[0008] The main chain of the AG type tobacco pectin is composed of sugar residues →6)-β-D-Galp-(1) and →3,6)-β-D-Galp-(1) connected, and the side chains β-D-Galp-(1→4)-β-D-Galp-(1), α-L-Araf-(1→4)-β-D-Glcp-(1) and α-L-Araf-(1→5)-α-L-Araf-(1) are independently connected to the main chain through the O-3 bond of glycosidic bond →3,6)-β-D-Galp-(1). The structure of the AG type tobacco pectin is as follows: Fig.11 shown.

[0009] In some embodiments, the AG tobacco pectin has a weight average molecular weight of 62.0 kDa and a number average molecular weight of 61.1 kDa.

[0010] In a second aspect of the present invention, the present invention provides a method for preparing the AG type tobacco pectin described in any technical solution of the first aspect, comprising:

[0011] Step (1) extracting crude tobacco pectin from shredded tobacco using a water extraction and alcohol precipitation method;

[0012] Step (2) removing impurities from the crude tobacco pectin;

[0013] Step (3) purifies the crude tobacco pectin after impurities removal by ion exchange chromatography and column chromatography to obtain the AG type tobacco pectin.

[0014] In some embodiments, step (1) comprises:

[0015] Step (1-1) heating a mixture of shredded tobacco and water to obtain a hot mixture;

[0016] Step (1-2) subjecting the hot mixture obtained in step (1-1) to a first centrifugation treatment, and collecting the supernatant;

[0017] Step (1-3) mixing anhydrous ethanol with the supernatant, allowing the mixture to stand, performing a second centrifugation process, and collecting the precipitate;

[0018] Step (1-4) is to dry the precipitate to obtain crude tobacco pectin.

[0019] In some embodiments, in step (1-1), the heating treatment is performed at a temperature of 80-100° C. (eg, 90° C.).

[0020] In some embodiments, in step (1-1), the heating treatment time is 2-5 h (such as 2 h, 3 h, 4 h or 5 h), preferably 3 h.

[0021] In some embodiments, in step (1-1), the ratio of the mass of the tobacco shreds to the volume of the water (solid-liquid ratio) is 1:5-1:30 g / mL (such as 1:5 g / mL, 1:10 g / mL, 1:15 g / mL, 1:20 g / mL, 1:25 g / mL or 1:30 g / mL), preferably 1:20 g / mL.

[0022] In some embodiments, in step (1-2), the first centrifugation treatment is carried out at a rotation speed of 4000-10000 rpm (such as 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm or 10000 rpm, preferably 8000 rpm).

[0023] In some embodiments, in step (1-2), the first centrifugation treatment time is 5-15 min (such as 5 min, 8 min, 10 min, 12 min or 15 min), preferably 10 min.

[0024] In some embodiments, in step (1-3), anhydrous ethanol is added to the supernatant until the ethanol volume fraction reaches 50%-80% (such as 50%, 60%, 70% or 80%), preferably 60%.

[0025] In some embodiments, in step (1-3), the standing is performed at a temperature of 0-5°C (preferably 4°C).

[0026] In some embodiments, in step (1-3), the standing time is 10-16 h (such as 10 h, 12 h, 14 h or 16 h), preferably 12 h.

[0027] In some embodiments, in step (1-3), the second centrifugation treatment is carried out at a rotation speed of 4000-10000 rpm (such as 4000 rpm, 5000 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm or 10000 rpm, preferably 4000-5000 rpm, more preferably 4500 rpm).

[0028] In some embodiments, in step (1-3), the second centrifugation treatment time is 5-15 min (such as 5 min, 8 min, 10 min, 12 min or 15 min), preferably 10 min.

[0029] In some embodiments, after step (1-3) and before step (1-4), the method further includes step (1-i): washing the precipitate and collecting the washed precipitate; preferably, the washing is performed using 95% ethanol and anhydrous ethanol; preferably, step (1-i) is to wash the precipitate twice using 95% ethanol and then once using anhydrous ethanol, and collect the washed precipitate.

[0030] In some embodiments, in step (1-4), the drying is freeze-drying.

[0031] In some embodiments, step (2) comprises:

[0032] Step (2-1) adding water to dissolve the crude tobacco pectin, adding a macroporous adsorption resin, shaking, and then filtering to remove the macroporous adsorption resin to obtain a filtrate;

[0033] Step (2-2) subjecting the filtrate to proteolysis, inactivating the enzyme and subjecting the filtrate to a third centrifugation treatment, and collecting the supernatant;

[0034] Step (2-3) dialyzing the supernatant and collecting the permeate;

[0035] Step (2-4) dries the permeate to obtain the crude tobacco pectin after impurities are removed.

[0036] In some embodiments, in step (2-1), the volume fraction of the macroporous adsorption resin is 1-5% (such as 1%, 2%, 3%, 4% or 5%), preferably 1%.

[0037] In some embodiments, the shaking time is 2-6 h (such as 2 h, 3 h, 4 h, 5 h or 6 h), preferably 3 h.

[0038] In some embodiments, the proteolysis is performed using papain.

[0039] In some embodiments, the amount of papain added is 1%-3%, preferably 2%, of the mass of the crude tobacco pectin in step (2-1).

[0040] In some embodiments, the proteolysis is performed at a temperature of 30-40°C (such as 30°C, 32°C, 35°C, 37°C or 40°C, preferably 37°C).

[0041] In some embodiments, the proteolysis time is 1-5 h (such as 1 h, 2 h, 3 h, 4 h or 5 h), preferably 2 h.

[0042] In some embodiments, the enzyme inactivation condition is heating at 100° C. for 10 min.

[0043] In some embodiments, the third centrifugation treatment is performed at a rotation speed of 4000-6000 rpm (eg, 5000 rpm).

[0044] In some embodiments, the third centrifugation treatment lasts for 5-15 min (such as 5 min, 8 min, 10 min, 12 min or 15 min), preferably 10 min.

[0045] In some embodiments, the drying is freeze-drying.

[0046] In some embodiments, step (3) comprises:

[0047] Step (3-1) loading the impurity-removed crude tobacco pectin onto a weak anion exchange column, eluting with water and a NaCl aqueous solution, and collecting the eluate from the NaCl aqueous solution elution;

[0048] Step (3-2) dialyzing the eluate obtained in step (3-1) and collecting the permeate;

[0049] Step (3-3) concentrating and drying the permeate to obtain pre-purified tobacco pectin;

[0050] Step (3-4) loading the pre-purified tobacco pectin onto a gel column, eluting with a NaCl aqueous solution, and collecting the eluate;

[0051] Step (3-5) desalts and dries the eluate obtained in step (3-4) to obtain the AG type tobacco pectin.

[0052] In some embodiments, the weak anion exchange column is a DEAE-FF weak anion exchange column.

[0053] In some embodiments, in step (3-1), the elution is performed as follows: gradient elution is performed with water and 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L NaCl aqueous solutions, and the eluate is collected during elution with each concentration of NaCl aqueous solution, preferably the eluate is collected during elution with 0.2 mol / L NaCl aqueous solution; preferably, each gradient elution is 3 column volumes.

[0054] In some embodiments, the drying is freeze-drying.

[0055] In some embodiments, the crude pectin from tobacco leaves after impurities removal is pre-prepared into an aqueous solution and filtered (eg, filtered through a 0.45 μm filter membrane) before being loaded.

[0056] In some embodiments, the crude tobacco pectin after impurities removal is pre-prepared into an aqueous solution of 1-10 mg / mL (such as 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL, preferably 5 mg / mL) and filtered (such as 0.45 μm filter membrane filtration) before loading.

[0057] In some embodiments, the gel column is a Sepharose CL-6B gel column.

[0058] In some embodiments, step (3-4) is performed as follows: the pre-purified tobacco pectin is loaded onto a Sepharose CL-6B gel column, eluted with a 0.05M NaCl aqueous solution, and the eluate is collected; preferably, during the elution, the elution curve is determined by a phenol-sulfuric acid method and the eluate is collected; preferably, the flow rate is 1 mL / min.

[0059] In some embodiments, the pre-purified tobacco pectin is pre-configured into a solution and filtered (such as 0.45 μm filter membrane filtration) before loading. Preferably, the solvent is a NaCl aqueous solution, preferably a 0.05M NaCl aqueous solution; preferably, in the solution, the concentration of the pre-purified tobacco pectin is 40-60 mg / mL (such as 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL or 60 mg / mL, preferably 50 mg / mL).

[0060] In some embodiments, the desalting is accomplished using a 3500 Da dialysis bag.

[0061] In the third aspect of the present invention, the present invention provides an AG type tobacco pectin, which is prepared by the method described in any technical solution of the second aspect.

[0062] In the fourth aspect of the present invention, the present invention provides a composition comprising the AG tobacco pectin described in any technical scheme of the first aspect, or the AG tobacco pectin prepared by the method described in any technical scheme of the second aspect, or the AG tobacco pectin described in any technical scheme of the third aspect.

[0063] In some embodiments, the composition further comprises an excipient.

[0064] In some embodiments, the composition is a medicine, a functional product, or a health product.

[0065] In a fifth aspect of the present invention, the present invention provides use of the AG tobacco pectin described in any technical solution of the first aspect, or the AG tobacco pectin prepared by the method described in any technical solution of the second aspect, or the AG tobacco pectin described in any technical solution of the third aspect, or the composition described in any technical solution of the fourth aspect in preparing a product, wherein the product is used for one or more selected from the following (i)-(ii),

[0066] (i) used for anti-oxidation, or as a contribution to anti-oxidation;

[0067] (ii) used to lower blood sugar or help maintain healthy blood sugar levels.

[0068] In some embodiments, the product is a medicine, a functional product, or a nutraceutical. Beneficial Effects

[0069] 1. The present invention provides an AG type tobacco pectin with uniform molecular weight, and clarifies the fine structure and composition of the AG type tobacco pectin for the first time.

[0070] 2. The tobacco pectin provided by the present invention does not contain uronic acid and rhamnose, does not contain the monosaccharide components of the pectin RG-I main chain, and is a high-purity arabinogalactan (AG) type tobacco pectin polysaccharide.

[0071] 3. The present invention adopts a water extraction and alcohol precipitation method to crudely extract tobacco pectin, which has high extraction efficiency, mild reaction conditions, environmentally friendly reagents, and can retain the side chain structure of tobacco pectin.

[0072] 4. The AG tobacco pectin prepared by the present invention has significant in vitro antioxidant and hypoglycemic activities and can be used in the fields of medicines, functional products and health products.

[0073] 5. The present invention is simple to operate and can be easily prepared on a large scale. At the same time, the present invention provides technical support for further exploring the structure-activity relationship of tobacco pectin on its quality and the resource utilization of tobacco pectin. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 :(a)Ion chromatogram of 14 monosaccharide standards;(b)Ion chromatogram of TP02A tobacco leaf AG type pectin; Among them, 1. fucose; 2. galactosamine hydrochloride; 3. rhamnose; 4. arabinose; 5. glucosamine hydrochloride; 6. galactose; 7. glucose; 8. xylose; 9. mannose; 10. ribose; 11. galacturonic acid; 12. guluronic acid; 13. glucuronic acid; 14. mannuronic acid.

[0075] Figure 2 : Gel filtration chromatogram of tobacco pectin TP02A.

[0076] Figure 3 : Infrared spectrum of tobacco pectin TP02A.

[0077] Figure 4 : One-dimensional H NMR spectrum of tobacco pectin TP02A.

[0078] Figure 5 : One-dimensional C-NMR spectrum of tobacco pectin TP02A.

[0079] Figure 6 :Two-dimensional NMR Dept135 spectrum of tobacco pectin TP02A.

[0080] Figure 7 : Two-dimensional NMR HH-COSY spectrum of tobacco pectin TP02A.

[0081] Figure 8 :Two-dimensional NMR HSQC spectrum of tobacco pectin TP02A.

[0082] Fig. 9 : Two-dimensional NMR HMBC spectrum of tobacco pectin TP02A.

[0083] Fig.10 :Two-dimensional NMR NOESY spectrum of tobacco pectin TP02A.

[0084] Fig.11 : The structural formula of tobacco pectin TP02A.

[0085] Fig.12 :(a) DPPH free radical scavenging ability of tobacco pectin TP02A;(b) hydroxyl free radical scavenging ability of tobacco pectin TP02A.

[0086] Fig.13 :(a)α-glucosidase inhibition rate of tobacco pectin TP02A;(b)α-amylase inhibition rate of tobacco pectin TP02A. DETAILED DESCRIPTION

[0087] Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings and examples, but it will be appreciated by those skilled in the art that the following drawings and examples are only used to illustrate the present invention, rather than to limit the scope of the present invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments.

[0088] The inventors of the present invention have achieved the development and utilization of tobacco pectin through experimental research: using flue-cured tobacco shreds as raw materials, extracting crude tobacco pectin by water extraction and alcohol precipitation, removing impurities from the crude tobacco pectin, purifying the tobacco pectin by ion exchange chromatography, preparing AG type tobacco pectin for the first time, systematically analyzing its physical and chemical indicators, monosaccharide composition, molecular weight, functional group, glycosidic bond type, etc., clarifying the structure of AG type tobacco pectin, and the obtained tobacco pectin has in vitro antioxidant and hypoglycemic activities.

[0089] The present invention is further explained below with reference to specific embodiments.

[0090] Example 1: Preparation of tobacco pectin

[0091] The preparation of the tobacco pectin comprises the following steps:

[0092] Extraction: The crude pectin from tobacco leaves was extracted by water extraction and alcohol precipitation. Specifically, 200 g of B3F grade flue-cured tobacco from Nanping, Fujian Cuibi No. 1 was weighed and heated in pure water at 90°C for 3 h in a condensation reflux device at a solid-liquid ratio of 1:20 g / mL. The solution was concentrated to 500 mL in a rotary evaporator under reduced pressure. The concentrated solution was centrifuged at 8000 rpm for 10 min, the precipitate was removed, and the supernatant was taken. Anhydrous ethanol (v / v) was slowly added to the supernatant by a slow-speed stirring method until the ethanol volume fraction reached 60% (v / v), and the solution was allowed to stand at 4°C for 12 h, centrifuged at 4500 rpm for 10 min, and the precipitate was collected. The precipitate was washed twice with 95% ethanol and once with anhydrous ethanol. The precipitate was collected and freeze-dried to obtain the crude pectin from tobacco leaves.

[0093] Impurity removal: Dissolve the crude tobacco pectin with distilled water, add 1% by volume AB-8 macroporous adsorption resin, shake for 3 hours, remove the macroporous adsorption resin by filtration, add papain (the amount added is 2% of the mass of the crude tobacco pectin), react at 37°C for 2 hours, heat at 100°C for 10 minutes to inactivate the enzyme, centrifuge at 4000 rpm for 10 minutes, collect the supernatant, dialyze the permeate, and freeze-dry to obtain the impurity-removed tobacco pectin.

[0094] Purification: The impurity-removed tobacco pectin was prepared into a 5 mg / mL aqueous solution, filtered through a 0.45 μm filter membrane and loaded onto a DEAE-FF weak anion exchange column, and gradient eluted with water, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl aqueous solution, with 3 column volumes for each gradient elution. The eluate of each concentration of NaCl solution was collected, the permeate was dialyzed, concentrated, and freeze-dried to obtain pre-purified tobacco pectin of various concentrations.

[0095] The above-mentioned pre-purified tobacco pectin samples of various concentrations were prepared into 50 mg / ml solutions (solvent 0.05M NaCl aqueous solution), and then loaded onto a Sepharose CL-6B gel column after being filtered through a 0.45μm filter membrane, eluted with a 0.05M NaCl aqueous solution, with a flow rate of 1ml / min, and 5ml was collected in each tube. The elution curve was determined by the phenol-sulfuric acid method and different pectin fractions were collected, desalted by a 3500 Da dialysis bag, and freeze-dried to obtain purified tobacco pectin samples of various concentrations. Among them, the purified tobacco pectin sample obtained by eluting with a concentration of 0.2mol / L NaCl aqueous solution is referred to as TP02A.

[0096] Example 2: Characterization of tobacco pectin

[0097] This example provides a structural characterization of AG-type tobacco pectin, and TP02A obtained in Example 1 is used as the structural characterization sample of this example.

[0098] 2.1 Analysis of monosaccharide composition of tobacco pectin TP02A

[0099] Ion chromatography was used to detect monosaccharide composition. The detection method was based on the method in the literature (Guoliang Wang, et al. Structural characterization and immunomodulatory activity of anacidicpolysaccharide from walnut green husk[J]. Journal of FunctionalFoods2023, 110, 105877). The results are as follows Figure 1 As shown in (ab), by comparing the chromatogram with the mixed monosaccharide standard, it was found that the prepared AG type tobacco pectin TP02A was mainly composed of typical pectin monosaccharides such as galactose and arabinose and a small amount of glucose, with a corresponding molar ratio of 0.55:0.33:0.12. According to the monosaccharide composition results and literature (Yu Chengxiao, Chen Jianle, Ye Xingqian, et al. Research progress on the structural characteristics and separation and detection of rhamnogalacturonan I [J]. Journal of Shaanxi Normal University (Natural Science Edition), 2022, 50(05):51-63.), it can be concluded that the prepared tobacco pectin is a pectin with an arabinogalactan (AG) structure as the main structure, which does not contain uronic acid and rhamnose, and does not contain the monosaccharide components of the pectin RG-I main chain. It is a high-purity arabinogalactan (AG) type tobacco pectin.

[0100] 2.2 Molecular weight analysis of tobacco pectin TP02A

[0101] High performance gel permeation chromatography (HPGPC) was used to determine the molecular weight distribution of tobacco pectin samples. A 5 mg / mL tobacco pectin aqueous solution was prepared and filtered through a 0.22 μm filter membrane for testing. The molecular weight distribution of the sample was determined using a Waters high performance liquid chromatograph (detector was a differential detector) equipped with a TSK-gel G3000PWXL column. The retention time (x) of the dextran standard and the corresponding molecular weight logarithm (y) were used as a linear equation to calculate the molecular weight of TP02A based on its retention time.

[0102] Table 1 Retention time and molecular weight of TP02A

[0103] The results are as follows Figure 2 As shown in Table 1, the retention time of the TP02A sample is 35.266 min. According to the linear equation of the weight average molecular weight of the standard sample y = -0.1957x + 11.694 and the linear equation of the number average molecular weight y = -0.1948x + 11.656, the weight average molecular weight of the prepared TP02A tobacco pectin sample is calculated to be 62.0 kDa, the number average molecular weight is 61.1 kDa, and the polydispersity index (PDI = Mw / Mn) is 1.01. It can be seen from the chromatogram and PDI value that its molecular weight uniformity is good.

[0104] 2.3 Infrared spectroscopy analysis of tobacco pectin TP02A

[0105] Take 1-2 mg of dried TP02A tobacco pectin sample, add dry KBr powder and grind it. After tableting, use infrared spectrometer to collect spectrum with a scanning range of 400-4000 cm -1 , scan times 16 times.

[0106] The infrared spectrum of TP02A is shown in Figure 3 As shown in the figure, TP02A exhibits typical infrared spectral characteristics of polysaccharides. -1 2940cm is the stretching vibration absorption peak of OH, which is the characteristic peak of sugars. -1 2886cm -1 The stretching vibration absorption peak of CH is at 1633cm -1 There is an absorption peak at 1365cm, which may be attributed to C=O stretching vibration. -1 There is an absorption peak at 1419cm -1 1081cm -1 There is an absorption peak at 1303cm -1 1222cm-1 1027cm -1 There is an absorption peak at , which may be attributed to the OH angle vibration.

[0107] 2.5 Methylation analysis of tobacco pectin TP02A

[0108] Weigh TP02A sample (2-3 mg) and place it in a glass reaction bottle, add 1 mL of anhydrous DMSO, quickly add methylation reagent A solution, seal, dissolve under ultrasound, and then add methylation reagent B solution. React in a magnetic stirring water bath at 30°C for 60 minutes. Finally, add 2 mL of ultrapure water to the above mixture to terminate the methylation reaction, dialyze in a 1000Da dialysis bag for 24 hours and then freeze-dry. Accurately weigh 2 mg of the dialyzed sample and 200 mg of potassium bromide, press into tablets, and the blank control is made of potassium bromide powder tablets. Place them in a Fourier transform infrared spectrometer for scanning and recording, and make sure that the methylation is complete before conducting subsequent experiments.

[0109] Take the methylated TP02A sample, add 1ml of 2mol / L trifluoroacetic acid to hydrolyze for 90min, evaporate to dryness on a rotary evaporator, add sodium borohydride to reduce for 8 hours, add glacial acetic acid to neutralize, rotary evaporate, dry in an oven at 100℃, then add 1ml of acetic anhydride to acetylate, react at 100℃ for 1h, cool, and then add 10ml of pure water to terminate the reaction.

[0110] The acetylated product was dissolved in 3 mL of dichloromethane and transferred to a separatory funnel. A small amount of distilled water was added and shaken thoroughly, and the upper aqueous solution was removed. This was repeated 4 times. The lower extract was tested. The acetylated product samples were analyzed using a Thermo scientific, 1300-7000 gas chromatography-mass spectrometer;

[0111] GC-MS conditions: HP-INNOWAX column 30m*0.32mm*0.25um; programmed temperature conditions: starting temperature 140°C, heating to 230°C at 1°C / min; injection port temperature 250°C, detector temperature 250°C, carrier gas helium, flow rate 1mL / min.

[0112] As shown in Table 2, after methylation, hydrolysis and acetylation, the samples were determined by GC-MS and compared with the standard mass spectrum library. The main glycosidic bond connection modes of TP02A samples are 1→, 1→5, 1→4, 1→6 and 1→3,6. According to the peak area, the proportions of each glycosidic bond connection mode are 19.7%, 17.1% and 15.3% for →3,6)-Galp-(1→, →6)-Galp-(1→, →4)-Galp-(1→), followed by →4)-Glcp-(1→, →5)-Araf-(1→, Galp-(1→, Araf-(1→), with proportions of 15.1%, 12.7%, 12.3% and 7.7% respectively, indicating that TP02A is an arabinogalactan with more branches.

[0113] Table 2: Analysis of results of methylated sugar alcohol acetyl ester (PMAA) of tobacco leaf AG type pectin TP02A

[0114] 2.4 NMR spectroscopy analysis of tobacco pectin TP02A

[0115] Weigh 50 mg of dried pectin homogenous fraction and dissolve it in 1 mL of heavy water. Use Bruker Avance 600 MHz spectrometer to analyze TP02A by NMR carbon spectrum. 1 HNMR spectrum, 13 C NMR spectrum, DEPT135 one-dimensional spectrum and two-dimensional spectrum.

[0116] like Figure 4 As shown, the hydrogen spectrum signal is mainly concentrated between 3.0 and 5.5 ppm. δ3.2-4.0 ppm is the sugar ring proton signal, and the main terminal proton peaks δ4.39, 4.43, 4.46, 4.59, 4.87, 4.97, 5.12, and 5.16 are concentrated in the 4.3-5.5 ppm region. 13 C NMR (126 MHz, D2O): The nuclear magnetic carbon spectrum signal is mainly concentrated between 60-120ppm. Figure 5From the one-dimensional NMR carbon spectrum, we can see that the main anomeric carbon signal peaks are δ103.92, 104.02, 104.68, 105.13, 108.76, 108.79, 108.87, and 110.61, and the anomeric carbon region is mainly between δ93 and 105. The main signal peaks of δ75.12, 76.32, 83.52, 76.27, 61.36, 77.49, 72.90, 82.90, 76.45, 63.76, 71.30, 81.49, 69.81, 74.80, 70.75, 71.68, 69.76, 74.79, 74.80, 62.43, 82.69, 77.79, 85.09, 62.32, 72.01, 73.99, 69.81, 74.8, 70.19, 82.17, 78.11, 83.67, 68.26, 82.61, 77.96, 85.21, and 62.63 are distributed in the 60-85ppm region. According to the monosaccharide composition results, the polysaccharide is composed of galactose, arabinose, and a small amount of glucose. This indicates that the polysaccharide is mainly arabinogalactan, i.e., AG type tobacco pectin. Figure 6 According to the Dept135 spectrum analysis, the peaks at 61.36, 63.76, 70.75, 62.43, 70.19, 62.63, 62.32, and 68.26 ppm are inverted peaks, indicating chemical shifts of C6. Figure 7 From the HSQC spectrum, we can see that the anomeric carbon signal is δ108.79, and the corresponding anomeric hydrogen signal in the HSQC spectrum is δ4.43. Figure 8 The HH-COSY spectrum shows that the signals of H1-2 are 4.43 / 3.45; the signals of H2-3 are 3.45 / 3.61; the signals of H3-4 are 3.61 / 4.04; the signals of H4-5a are 4.04 / 3.86; we can infer that H1, H2, H3, H4, H5a, H5b, H6b are δ4.43, 3.45, 3.61, 4.04, 3.86, 3.84, 3.94 respectively. The corresponding C1-C6 are 108.79, 72.01, 73.99, 69.81, 74.8, 70.19; therefore, the signal should be attributed to the glycosidic bond →6)-β-D-Galp-(1→. According to similar rules and combined with HMBC ( Fig. 9 ) and NOESY spectra ( Fig.10 ), all glycosidic bond signals were assigned, as shown in Table 3:

[0117] Table 3: Attribution of hydrocarbon signals of tobacco leaf AG type pectin TP02A

[0118] In summary, the structure of tobacco pectin was characterized by combining multiple technologies. The main chain connection mode of this AG type pectin polysaccharide is →6)-β-D-Galp-(1) and →3,6)-β-D-Galp-(1→, while the side chains β-D-Galp-(1→4)-β-D-Galp-(1→, α-L-Araf-(1→4)-β-D-Glcp-(1→ and side chains α-L-Araf-(1→5)-α-L-Araf-(1→) are connected to the main chain through the glycosidic bond →3,6)-β-D-Galp-(1→ O-3 bond. The structural formula is as follows Fig.11 shown.

[0119] Example 3: Activity test of tobacco pectin

[0120] This example provides a study on the in vitro antioxidant and hypoglycemic activities of AG-type tobacco pectin, and TP02A obtained in Example 1 is used as the research object of this example.

[0121] 3.1. In vitro antioxidant activity studies

[0122] Prepare a series of tobacco pectin TP02A solutions with concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, 6.0 mg / mL, and 8.0 mg / mL, respectively. Take 1 mL of the sample solution and place it in a brown centrifuge tube. Add 4 mL of DPPH anhydrous methanol solution (DPPH mass fraction is 0.0036%), vortex mix, and react at 37°C for 35 minutes. Use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 517 nm, and use ascorbic acid (Vc) and distilled water as positive controls and blank controls, respectively. Calculate the DPPH free radical scavenging rate of the sample to be tested according to the following formula:

[0123] DPPH clearance rate = [1-(A2-A1) / A0]×100%.

[0124] A1 represents the absorbance value of the blank control measurement;

[0125] A2 represents the absorbance value measured by the sample to be tested;

[0126] A0 represents the absorbance value measured when an equal volume of methanol is used instead of DPPH solution.

[0127] Prepare a series of tobacco pectin TP02A solutions with concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, 6.0 mg / mL, and 8.0 mg / mL, take 0.5 mL of the sample solution and place it in a brown centrifuge tube, add 1 mL of 9 mM FeSO4 solution and 1 mL of 9 mM salicylic acid in anhydrous ethanol, vortex mix, add 1 mL of 8.8 mM H2O2 solution and react for 30 min, use an ultraviolet spectrophotometer to measure the absorbance at a wavelength of 510 nm, and use ascorbic acid (Vc) and distilled water as positive controls and blank controls, respectively. Calculate the hydroxyl radical scavenging rate of the sample to be tested according to the following formula:

[0128] Hydroxyl removal rate = [1-(A2-A1) / A0]×100%

[0129] A1 represents the absorbance value of the blank control measurement;

[0130] A2 represents the absorbance value measured by the sample to be tested;

[0131] A0 represents the absorbance value measured when an equal volume of distilled water is used instead of H2O2 solution.

[0132] Spectrophotometry can quickly quantify the scavenging ability of DPPH and hydroxyl radicals in the presence of free radical scavengers, such as Fig.12 As shown in the results, compared with the positive control group of vitamin C, tobacco pectin TP02A has a certain scavenging ability for both DPPH and hydroxyl free radicals, and with the increase of sample concentration, its free radical scavenging ability shows an increasing trend. At a concentration of 8 mg / mL, the scavenging rate of tobacco pectin TP02A for DPPH free radicals is 72%, and the scavenging rate for hydroxyl free radicals is 86%. The above results show that the AG type tobacco pectin prepared by the present invention has good antioxidant ability, and the IC value for scavenging DPPH free radicals is 2.34. 50 is 4.65 mg / mL, for example, superior to the polysaccharide of centipede algae (IC 50 is 12.61 mg / mL) (Chen Shengjun, Liu Huan, Yang Shaoling, et al. Extraction process and antioxidant activity analysis of polysaccharides from Ligularia scolopendra [J]. Journal of Shanghai Ocean University, 2020, 29(01): 153-160.), IC 50 is 4.03 mg / mL, for example, better than the antioxidant components of Gastrodia elata reported in the literature (IC 50 is 13.018 mg / mL) (Li Dezhu, Zhang Xingguo. Optimization of extraction process of antioxidant components from Gastrodia elata and study on their antioxidant activity [J]. Modern Chinese Medicine Research and Practice, 2019, 33(06): 46-49.).

[0133] 3.2. In vitro hypoglycemic activity studies

[0134] A series of tobacco pectin TP02A solutions with concentrations of 0.5 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 4.0 mg / mL, 6.0 mg / mL, 8.0 mg / mL, and 10.0 mg / mL were prepared, and phosphate buffer (0.1M, pH=6.8) was used as the solvent to prepare 0.5 U / mL α-glucosidase solution and 2.5 mmol / L 4-nitrophenyl-D-pyranoglucoside solution. 0.5 mL of α-glucosidase solution was taken into a centrifuge tube, and 0.5 mL of tobacco pectin TP02A solution of different concentrations was added, mixed, reacted at 37°C for 15 min, 0.25 mL of 4-nitrophenyl-D-pyranoglucoside solution was added, reacted at 37°C for 15 min, and 2 mL of 1 mol / L Na2CO3 was added to terminate the reaction, and acarbose was used as a positive control. The light absorption value at 405 nm was measured by ultraviolet spectrophotometer, and the α-glucosidase inhibition rate of the sample to be tested was calculated according to the following formula:

[0135] α-glucosidase inhibition rate = [1-(A2-A1) / A0]×100%

[0136] Wherein, A2 is the absorbance of the tobacco pectin reaction system, A1 is the absorbance of the reaction system when the α-glucosidase solution is replaced by the phosphate buffer solution, and A0 is the absorbance of the reaction system when the tobacco pectin sample solution is replaced by the phosphate buffer solution.

[0137] The α-amylase inhibition rate of the sample was determined by the DNS method. 0.5 mL of sample solution with different concentration gradients and 0.5 mL of 0.5U / mL α-amylase solution were mixed, reacted at 37°C for 20 min, 0.5 mL of 2.0% soluble starch was added, mixed, reacted at 37°C for 10 min, 1 mL of DNS reagent was added, reacted in a boiling water bath for 5 min, 10 mL of distilled water was added for dilution, cooled, and the absorbance was measured at 520 nm. Deionized water was used instead of tobacco pectin sample as the blank control group, and deionized water was used instead of α-amylase solution as the sample control. The α-amylase inhibition rate of the sample to be tested was calculated according to the following formula:

[0138] α-Amylase inhibition rate = [1-(A2-A1) / A0]×100%.

[0139] Wherein, A2 is the absorbance of the tobacco pectin reaction system, A1 is the absorbance of the reaction system when the α-amylase solution is replaced by the phosphate buffer solution, and A0 is the absorbance of the reaction system when the tobacco pectin sample solution is replaced by the phosphate buffer solution.

[0140] α-glucosidase is the main glycoside hydrolase in the human small intestine, and α-amylase is the main hydrolase in the human pancreas. Both can hydrolyze starch and cause blood sugar to rise. Therefore, the inhibition rate of α-glucosidase and α-amylase can reflect the degree of hypoglycemia. Differences in the monosaccharide composition and molecular weight of pectin will affect its hypoglycemic activity. Fig.13 As shown in (ab), compared with the positive control group of acarbose, tobacco pectin TP02A has a certain inhibitory activity on α-glucosidase and α-amylase inhibition rates, and with the increase of sample concentration, tobacco pectin has a dose-dependent effect on α-glucosidase and α-amylase inhibition rates. At a concentration of 10 mg / mL, tobacco pectin TP02A has an inhibition rate of 72% on α-glucosidase, and tobacco pectin TP02A has an inhibition rate of 59% on α-amylase (IC 50 =7.78 mg / mL), inhibitory activity of TP02A against α-glucosidase (IC 50 =6.12 mg / mL) is more significant. The above results show that the tobacco pectin prepared by the present invention has in vitro hypoglycemic activity, for example, it is superior to the α-amylase inhibitory activity (IC 50 =13.80 mg / mL) and α-glucosidase inhibitory activity (IC 50 =14.22mg / mL) (He Junye, Liu Cheng, Yu Chongyang, et al. Structural characteristics and in vitro hypoglycemic activity of polysaccharides from different parts of yellow peel [J]. Food and Machinery, 2024, 40(03): 156-164.), which is expected to be applied in the fields of medicine, functional products or health products.

[0141] Comparative Example

[0142] Using the same Fujian Nanping Cui Bi No. 1 B3F grade flue-cured tobacco as in Example 1 as raw material, citric acid was used to extract tobacco pectin. The molar percentage of its monosaccharide composition is shown in Table 4. It was found that it contained monosaccharides such as galacturonic acid, rhamnose, galactose, arabinose and glucose, indicating that the sample contained multiple pectin structural units such as HG and RG-I, and was not a single AG type tobacco pectin.

[0143] Table 4 Composition of tobacco pectin extracted by citric acid

[0144] It should be understood that the invention described herein is not limited to specific methodology, experimental protocols or reagents, as these may vary. The discussion and examples provided herein are presented only to describe specific embodiments and are not intended to limit the scope of the invention, which is limited only by the claims.

Claims

1. An AG type tobacco pectin, wherein: The AG tobacco pectin is composed of galactose, arabinose and glucose, and the molar ratio of galactose, arabinose and glucose is 0.55:0.33:0.12; The main chain of the AG type tobacco pectin is composed of sugar residues →6)-β-D-Galp-(1) and →3,6)-β-D-Galp-(1→, and the side chains β-D-Galp-(1→4)-β-D-Galp-(1→, α-L-Araf-(1→4)-β-D-Glcp-(1→, and α-L-Araf-(1→5)-α-L-Araf-(1→) are each independently connected to the main chain through the O-3 bond of glycosidic bond →3,6)-β-D-Galp-(1→.

2. The AG tobacco pectin according to claim 1, wherein The weight average molecular weight of the AG tobacco pectin is 62.0 kDa, and the number average molecular weight is 61.1 kDa.

3. A method for preparing the AG type tobacco pectin according to any one of claims 1 to 2, comprising: Step (1) extracting crude tobacco pectin from shredded tobacco using a water extraction and alcohol precipitation method; Step (2) removing impurities from the crude tobacco pectin; Step (3) purifies the crude tobacco pectin after impurities removal by ion exchange chromatography and column chromatography to obtain the AG type tobacco pectin.

4. The method according to claim 3, wherein: Step (1) includes: Step (1-1) heating a mixture of shredded tobacco and water to obtain a hot mixture; Step (1-2) subjecting the hot mixture obtained in step (1-1) to a first centrifugation treatment, and collecting the supernatant; Step (1-3) mixing anhydrous ethanol with the supernatant, allowing the mixture to stand, performing a second centrifugation process, and collecting the precipitate; Step (1-4) is to dry the precipitate to obtain crude tobacco pectin.

5. The method according to claim 4, wherein: The method further has one or more technical features selected from the following (i)-(xii): (i) In step (1-1), the heating treatment is carried out at a temperature of 80-100° C. (e.g., 90° C.); (ii) In step (1-1), the heating treatment time is 2-5 h, preferably 3 h; (iii) in step (1-1), the ratio of the mass of the shredded tobacco to the volume of the water (solid-liquid ratio) is 1:5-1:30 g / mL, preferably 1:20 g / mL; (iv) in step (1-2), the first centrifugation treatment is carried out at a rotation speed of 4000-10000 rpm (preferably 8000 rpm); (v) In step (1-2), the first centrifugation treatment time is 5-15 min, preferably 10 min; (vi) in step (1-3), anhydrous ethanol is added to the supernatant until the ethanol volume fraction reaches 50%-80%, preferably 60%; (vii) In step (1-3), the standing is carried out at a temperature of 0-5°C (preferably 4°C); (viii) In step (1-3), the standing time is 10-16 hours, preferably 12 hours; (ix) In step (1-3), the second centrifugation treatment is carried out at a rotation speed of 4000-10000 rpm (preferably 4000-5000 rpm, more preferably 4500 rpm); (x) In step (1-3), the second centrifugation treatment time is 5-15 min, preferably 10 min; (xi) after step (1-3) and before step (1-4), further comprising step (1-i): washing the precipitate and collecting the washed precipitate; Preferably, the washing treatment is performed using 95% ethanol and anhydrous ethanol; Preferably, step (1-i) is to wash the precipitate twice with 95% ethanol, then wash it once with anhydrous ethanol, and collect the washed precipitate; (xii) In step (1-4), the drying is freeze-drying.

6. The method according to any one of claims 3 to 5, wherein: Step (2) includes: Step (2-1) adding water to dissolve the crude tobacco pectin, adding a macroporous adsorption resin, shaking, and then filtering to remove the macroporous adsorption resin to obtain a filtrate; Step (2-2) subjecting the filtrate to proteolysis, inactivating the enzyme and subjecting the filtrate to a third centrifugation treatment, and collecting the supernatant; Step (2-3) dialyzing the supernatant and collecting the permeate; Step (2-4) drying the permeate to obtain the impurity-free tobacco leaf crude pectin; Preferably, in step (2-1), the volume fraction of the macroporous adsorption resin is 1-5%, preferably 1%; Preferably, the oscillation time is 2-6 hours, preferably 3 hours; Preferably, the proteolysis is performed using papain; Preferably, the amount of papain added is 1%-3% of the mass of the crude tobacco pectin in step (2-1), preferably 2%; Preferably, the proteolysis is carried out at a temperature of 30-40°C (preferably 37°C); Preferably, the proteolysis time is 1-5h, preferably 2h; Preferably, the enzyme inactivation condition is heating at 100°C for 10 minutes; Preferably, the third centrifugal treatment is carried out at a rotation speed of 4000-6000 rpm; Preferably, the third centrifugation treatment lasts for 5-15 min, preferably 10 min; Preferably, the drying is freeze-drying.

7. The method according to any one of claims 3 to 6, wherein: Step (3) includes: Step (3-1) loading the impurity-removed crude tobacco pectin onto a weak anion exchange column, eluting with water and a NaCl aqueous solution, and collecting the eluate from the NaCl aqueous solution elution; Step (3-2) dialyzing the eluate obtained in step (3-1) and collecting the permeate; Step (3-3) concentrating and drying the permeate to obtain pre-purified tobacco pectin; Step (3-4) loading the pre-purified tobacco pectin onto a gel column, eluting with a NaCl aqueous solution, and collecting the eluate; Step (3-5) desalting and drying the eluate obtained in step (3-4) to obtain the AG type tobacco pectin; Preferably, the weak anion exchange column is a DEAE-FF weak anion exchange column; Preferably, in step (3-1), the elution is performed by gradient elution with water and 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, 0.4 mol / L, and 0.5 mol / L NaCl aqueous solutions, and the eluate during elution with each concentration of NaCl aqueous solution is collected, preferably the eluate during elution with 0.2 mol / L NaCl aqueous solution is collected; preferably, each gradient elution is 3 column volumes; Preferably, the drying is freeze drying; Preferably, the crude tobacco pectin after impurities removal is pre-prepared into an aqueous solution and filtered (such as 0.45 μm filter membrane filtration) before being sampled; Preferably, the crude tobacco pectin after impurities removal is pre-prepared into a 1-10 mg / mL (such as 5 mg / mL) aqueous solution and filtered (such as 0.45 μm filter membrane filtration) before loading; Preferably, the gel column is a Sepharose CL-6B gel column; Preferably, step (3-4) is performed in the following manner: loading the pre-purified tobacco pectin onto a Sepharose CL-6B gel column, eluting with a 0.05M NaCl aqueous solution, and collecting the eluate; preferably, during the elution, determining the elution curve by a phenol-sulfuric acid method and collecting the eluate; preferably, the flow rate is 1 mL / min; Preferably, the pre-purified tobacco pectin is pre-prepared into a solution and filtered (such as 0.45 μm filter membrane filtration) before loading, preferably, the solvent is a NaCl aqueous solution, preferably a 0.05M NaCl aqueous solution; preferably, in the solution, the concentration of the pre-purified tobacco pectin is 40-60 mg / mL (such as 50 mg / mL); Preferably, the desalination is accomplished using a 3500 Da dialysis bag.

8. A composition comprising the AG tobacco pectin according to any one of claims 1 to 2 or the AG tobacco pectin prepared by the method according to any one of claims 3 to 7; optionally, further comprising an auxiliary material; Preferably, the composition is a medicine, a functional product or a health product.

9. Use of the AG tobacco pectin according to any one of claims 1 to 2, or the AG tobacco pectin prepared by the method according to any one of claims 3 to 7, or the composition according to claim 8 in preparing a product, wherein the product is used for one or more selected from the following (i) to (ii), (i) used for anti-oxidation, or as a contribution to anti-oxidation; (ii) used to lower blood sugar or help maintain healthy blood sugar levels.

10. The use according to claim 9, wherein The product is a medicine, a functional product or a health product.

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