RG-I-HG type tobacco leaf pectin and application

Abstract

The invention relates to RG-I-HG type tobacco leaf pectin and application thereof, and particularly provides RG-I-HG type tobacco leaf pectin which is clear in fine structure and uniform in molecular weight, and an innovative pectin extraction and purification process. According to the extraction and purification process, the RG-I-HG type pectin CSTP5b with the weight-average molecular weight of 65.8 kDa is separated from tobacco leaves by adopting ammonium oxalate sequential extraction combined with anion exchange and gel column chromatography purification technologies. The RG-I-HG type pectin is composed of 57.88% of galacturonic acid, 13.11% of rhamnose, 20.71% of galactose and 8.3% of arabinose, and does not contain non-pectin components such as glucose at all.

Description

Technical Field

[0001] The invention belongs to the field of tobacco macromolecules, and particularly relates to RG-I-HG type tobacco pectin and applications thereof. Background Art

[0002] Solid waste generated during tobacco processing (such as tobacco stems and low-quality tobacco leaves) accounts for about 20%-30% of the total processing volume. Conventional incineration or landfilling methods not only lead to inefficient utilization of resources, but also release harmful substances, increasing the burden on the ecological environment. Studies have shown that discarded tobacco leaves are rich in pectin polysaccharides (5-12% of dry weight). Pectin is a key component of plant cell walls. The structure of pectin, such as monosaccharide composition, molecular weight, and branching structure, is closely related to biological activity. Studies have shown that the pectin polysaccharide CMDP-4b isolated from pumpkin has a molecular weight of 31.97 kDa. Its immunoactivity is related to its α-1,4 glycosidic bond backbone and specific side chain branches. These structural features contribute to receptor recognition and signal transduction (Huang Linlin et al. Structural characterization and mechanisms of macrophage immunomodulatory activity of a pectic polysaccharide from Cucurbita moschata Duch[J]. Carbohydrate Polymers, 2021, 269, 118288.). Pectin polysaccharides extracted from Veronica peregrina L contain a high proportion of HG domains. The highly linear GalA chain enhances the free radical scavenging ability by providing a large number of carboxylic acid groups, making its scavenging activity for DPPH, hydroxyl and ABTS free radicals significantly higher than that of other components of polysaccharides. Its antioxidant activity is positively correlated with the content of HG region (Su Yan et al. Structural characterization and antioxidant activity of pectic polysaccharides from Veronica peregrina L[J]. Frontiers in Nutrition, 2023, 10, 1217862.). Tobacco pectin has a complex structure and undergoes structural changes during growth and development and a special alcoholization process. It is tightly cross-linked with cellulose and lignin. Its pectin structure is significantly different from that of conventional sources such as citrus and apple. Literature reports on tobacco pectin (Wang Peng, et al. Solid-state nuclear magnetic resonance spectroscopy characterization of tobacco pectin uronic acid structure [J]. Journal of Anhui Agricultural University, 2024, 51(2): 207-211) only characterized the uronic acid structure, and did not clarify the structural composition and branching information of tobacco HG and RG-I. There are two major gaps in existing research: 1) The RG-I / HG ratio of tobacco pectin is lacking, and the fine structure of tobacco pectin is unknown; 2) The lack of structure-activity relationship research restricts its application in high value-added fields such as daily chemicals and medicine.

[0003] Currently, the industrial production of pectin mainly relies on strong acid or high-temperature extraction methods, which can easily lead to problems such as pectin degradation and poor controllability, limiting its high-value application. Conventional alcohol precipitation methods have low selectivity for pectin (acidic heteropolysaccharide), and the product is mixed with various polysaccharide components such as starch-like polysaccharides (such as glucan), affecting subsequent activity. Low-ester HG pectin in tobacco leaves has a high proportion of unmethylated GalA residues, which can be cross-linked by calcium ions to form a stable "egg-crate" structure. Conventional hot water extraction cannot fully extract pectin, resulting in low extraction efficiency. Summary of the Invention

[0004] To address these technical bottlenecks, the present invention provides a tobacco pectin with a well-defined fine structure and uniform molecular weight, as well as an innovative pectin extraction and purification process. This extraction and purification process utilizes sequential ammonium oxalate extraction combined with anion exchange and gel column chromatography to isolate RG-I-HG pectin CSTP5b with a weight-average molecular weight of 65.8 kDa from tobacco leaves. This RG-I-HG pectin is composed of 57.88% galacturonic acid, 13.11% rhamnose, 20.71% galactose, and 8.3% arabinose, completely devoid of non-pectin components such as glucose. Its molecular weight is significantly lower than that of commercial pectins, such as citrus pectin. At the same time, methylation analysis, nuclear magnetic resonance (NMR) and other technologies confirmed that the main chain of CSTP5b is →[4)-α-D-GalpA-(1]6→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→2)-α-D-Rhap-(1→4)-α-D-GalpA-(1→2)-α-D-Rhap-(1→, the RG-I domain side chain is composed of arabinan and galactan, and the HG structure is slightly methylated. This unique composition gives it excellent moisturizing and hygroscopicity and good anti-inflammatory activity. Compared with existing pectin, the present invention not only expands the high-value utilization of tobacco resources, but also provides theoretical and technical support for the development of new moisturizing and anti-inflammatory functional materials.

[0005] To this end, in a first aspect of the present invention, the present invention provides an RG-I-HG tobacco pectin, wherein the RG-I-HG tobacco pectin is composed of galacturonic acid, rhamnose, galactose, and arabinose, and the molar ratio of the galacturonic acid, rhamnose, galactose, and arabinose is 57.88:13.11:20.71:8.3;

[0006] The main chain of the RG-I-HG tobacco pectin is connected in the following manner: →[4)-α-D-GalpA-(1]6→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→2)-α-D-Rhap-(1→4)-α-D-GalpA-(1→2)-α-D-Rhap-(1→, the branched arabinan and galactan are connected to the main chain via the O-4 bond of →2,4)-α-D-Rhap-(1→), and the main chain has a methyl ester group at the O-3 bond of →3,4)-α-D-GalpA-(1→. The structural formula of the RG-I-HG tobacco pectin is as follows: Figure 6 shown.

[0007] In some embodiments, the RG-I-HG tobacco pectin has a weight average molecular weight of 65.8 kDa and a number average molecular weight of 64.0 kDa.

[0008] In a second aspect of the present invention, the present invention provides a method for preparing the aforementioned RG-I-HG type tobacco pectin, comprising:

[0009] (1) Removing water-soluble crude polysaccharides from tobacco leaves;

[0010] (2) extracting crude pectin from the tobacco leaves obtained in step (1) using an ammonium oxalate aqueous solution and ethanol precipitation;

[0011] (3) Purifying the crude pectin by ion exchange chromatography and gel column chromatography in sequence to obtain the RG-I-HG tobacco pectin.

[0012] In some embodiments, step (1) comprises:

[0013] (1-1) mixing chopped tobacco leaves with water and subjecting the mixture to a heat reflux treatment to obtain a hot mixture;

[0014] (1-2) Filter the hot mixture and collect the filter residue.

[0015] In some embodiments, in step (1-1), the temperature of the heat reflow treatment is 100°C.

[0016] In some embodiments, in step (1-1), the heat reflux treatment time is 2.5-3.5 hours, preferably 3 hours.

[0017] In some embodiments, in step (1-1), the ratio of the mass of the chopped tobacco leaves to the volume of the water is 10-20 g / mL (such as 10 g / mL, 12 g / mL, 14 g / mL, 16 g / mL, 18 g / mL or 20 g / mL), preferably 16 g / mL.

[0018] In some embodiments, in step (1-2), the filtration is filter cloth filtration.

[0019] In some embodiments, the shredded tobacco leaves have been defatted in advance.

[0020] In some embodiments, the degreasing treatment includes: pre-immersing the shredded tobacco leaves in ethanol for degreasing or pre-heating and refluxing the shredded tobacco leaves in ethanol;.

[0021] In some embodiments, the ethanol is 95% ethanol.

[0022] In some embodiments, the soaking and degreasing treatment lasts for 48 hours, and the ethanol is replaced every 12 hours.

[0023] In some embodiments, the heating reflux treatment is performed at 80° C. for 2 hours.

[0024] In some embodiments, the soaking and degreasing treatment or the heating and reflowing treatment further includes a drying step.

[0025] In some embodiments, the drying is air-drying.

[0026] In some embodiments, step (2) comprises:

[0027] (2-1) stirring the ammonium oxalate aqueous solution and the filter residue obtained in step (1-2) to obtain a stirred product;

[0028] (2-2) filtering the stirred product, collecting the filtrate, concentrating and centrifuging the filtrate, and collecting the supernatant;

[0029] (2-3) mixing the supernatant with ethanol for alcohol precipitation, and collecting the precipitate;

[0030] (2-4) The precipitate is sequentially subjected to deproteinization, monosaccharide and salt removal, and freeze-drying to obtain crude pectin.

[0031] In some embodiments, in step (2-1), the ratio of the mass of the filter residue to the volume of the ammonium oxalate aqueous solution is 10-20 g / mL (such as 10 g / mL, 12 g / mL, 14 g / mL, 16 g / mL, 18 g / mL or 20 g / mL), preferably 16 g / mL.

[0032] In some embodiments, the concentration of the aqueous ammonium oxalate solution (w / v concentration) is 0.25%-1.0% (such as 0.25%, 0.5%, 0.75% or 1%), preferably 0.5%.

[0033] In some embodiments, in step (2-1), the stirring treatment has a rotation speed of 200-300 r / min (such as 200 r / min, 220 r / min, 240 r / min, 250 r / min, 270 r / min, 290 r / min or 300 r / min), preferably 250 r / min.

[0034] In some embodiments, the stirring is mechanical stirring.

[0035] In some embodiments, in step (2-1), the stirring treatment is carried out at a temperature of 20-30°C (such as 20°C, 22°C, 24°C, 25°C, 26°C, 28°C or 30°C, preferably 25°C) for 3-5 hours (preferably 4 hours).

[0036] In some embodiments, in step (2-2), the filtration is filtration through filter cloth; and / or the concentration is concentration under reduced pressure.

[0037] In some embodiments, in step (2-2), the centrifugation is performed at a rotation speed of 3000-5000 r / min (preferably 4000 r / min) for 15-25 min (preferably 20 min).

[0038] In some embodiments, in step (2-3), the ratio of the volume of the ethanol to the volume of the supernatant is 3:1 to 5:1, preferably 4:1.

[0039] In some embodiments, in step (2-3), the ethanol is 95% ethanol.

[0040] In some embodiments, in step (2-3), the final concentration of ethanol in the mixture of the supernatant and the ethanol is 80%.

[0041] In some embodiments, in step (2-3), the alcohol precipitation treatment is achieved by standing and centrifuging.

[0042] In some embodiments, the standing is performed at a temperature of 4°C.

[0043] In some embodiments, the standing time is 10-15 hours (such as 10, 11, 12, 13, 14 or 15 hours), preferably 12 hours.

[0044] In some embodiments, in step (2-4), the deproteinization treatment includes: repeatedly removing proteins using the Sevag method until no impurity peaks are detected by ultraviolet detection.

[0045] In some embodiments, in step (2-4), the monosaccharide and salt removal treatment comprises: dialyzing using a 3500Da dialysis bag for 45-50 hours (preferably 48 hours) to remove monosaccharides and salts.

[0046] In some embodiments, in step (2-4), the precipitate is pre-washed before the deproteinization treatment.

[0047] In some embodiments, the washing treatment is performed using ethanol.

[0048] In some embodiments, step (3) comprises:

[0049] (3-1) The crude pectin was loaded onto a DEAE column and gradient eluted with ultrapure water, 0.2 mol / L NaCl aqueous solution, 0.5 mol / L NaCl aqueous solution, and 1 mol / L NaCl aqueous solution in sequence. The eluate from the 0.5 mol / L NaCl aqueous solution was collected, and the eluate was concentrated, desalted, and lyophilized to obtain a lyophilized product.

[0050] (3-2) The lyophilized product was loaded onto a Sephadex-200 column and eluted with a 0.1 mol / L NaCl aqueous solution. The main peak (i.e., the largest and most significant peak) eluate was collected, and the eluate was dialyzed and lyophilized to obtain the RG-I-HG tobacco pectin.

[0051] In some embodiments, in step (3-1), the crude pectin is pre-configured into a crude pectin aqueous solution before being loaded, and the crude pectin aqueous solution is filtered through a filter membrane; preferably, the filter membrane is preferably a 0.45 μm filter membrane; preferably, the concentration of the crude pectin aqueous solution is 3-7 mg / mL (such as 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL or 7 mg / mL), preferably 5 mg / mL.

[0052] In some embodiments, in step (3-1), the flow rate of the gradient elution is 2.65 mL / min.

[0053] In some embodiments, in step (3-1), the desalting is achieved by using a 3,500 Da dialysis bag.

[0054] In some embodiments, in step (3-2), the lyophilized material is pre-configured into a lyophilized material aqueous solution before loading, and the lyophilized material aqueous solution is filtered; preferably, the concentration of the lyophilized material aqueous solution is 40-60 mg / mL, preferably 50 mg / mL.

[0055] In some embodiments, in step (3-2), the elution flow rate is 0.5 mL / min.

[0056] In some embodiments, in step (3-2), the dialysis is dialysis using a 3,500 Da dialysis bag.

[0057] In a third aspect of the present invention, the present invention provides a product comprising the aforementioned RG-I-HG tobacco pectin or the RG-I-HG tobacco pectin prepared by the aforementioned method.

[0058] In some embodiments, the product is a cosmetic, a medicine, a health product, a functional food, or a medical device.

[0059] In some embodiments, the drug is a skin repair drug, a wound repair drug, or an ophthalmic drug.

[0060] In some embodiments, the health care product is an oral beauty health care product or an intestinal health health care product.

[0061] In some embodiments, the medical device is a medical dressing or a prosthetic implant material.

[0062] In some embodiments, the functional food is an enhanced food or a food for special medical purposes.

[0063] In a fourth aspect of the present invention, the present invention provides the use of the aforementioned RG-I-HG tobacco pectin or the RG-I-HG tobacco pectin prepared by the aforementioned method in preparing a product, wherein the product is used for one or more of the following (i) to (iv):

[0064] (i) anti-inflammatory;

[0065] (ii) Moisturizing;

[0066] (iii) Hygroscopic;

[0067] (iv) Helps improve skin moisture.

[0068] In some embodiments, the product is a cosmetic, a medicine, a health product, a functional food, or a medical device.

[0069] In some embodiments, the drug is a skin repair drug, a wound repair drug, or an ophthalmic drug.

[0070] In some embodiments, the health care product is an oral beauty health care product or an intestinal health health care product.

[0071] In some embodiments, the medical device is a medical dressing or a prosthetic implant material.

[0072] In some embodiments, the functional food is an enhanced food or a food for special medical purposes.

[0073] Beneficial effects

[0074] 1. The present invention provides a RG-I-HG type tobacco pectin with uniform molecular weight, which has a molecular weight much lower than that of commercial pectin such as citrus pectin.

[0075] 2. The tobacco pectin provided by the present invention is composed of 57.88% galacturonic acid, 13.11% rhamnose, 20.71% galactose, and 8.3% arabinose. This tobacco pectin is completely free of non-pectin components such as glucose. Furthermore, the tobacco pectin has a galacturonic acid content of 57.88% and is composed of an RG-I domain (55.23%) and an HG domain (44.77%), representing a mixed RG-I and HG tobacco pectin.

[0076] 3. The present invention adopts ammonium oxalate sequential extraction method to crudely extract tobacco pectin, which has high extraction efficiency, avoids side chain degradation caused by β-elimination reaction under alkaline extraction conditions, and retains galactan and arabinan side chains (accounting for 29.01%).

[0077] 4. This invention clearly reveals for the first time the mixed nature of RG-I and HG structures in tobacco pectin. Its molecular weight uniformity (PDI = 1.03) and high purity (free of non-pectin components such as glucose) provide a reliable structural foundation for research. A previous invention patent (CN119708283A, "AG-type tobacco pectin and its applications") focused on the side chain structure of pectin. This structure, which lacks uronic acid and rhamnose, is a high-purity arabinogalactan (AG)-type tobacco pectin polysaccharide. Therefore, the RG-I-HG type has broader application prospects in terms of structural diversity and functional activity.

[0078] 5. The high-purity tobacco pectin prepared by the present invention has significant moisturizing and anti-inflammatory activities and can be used in the fields of medicine, cosmetics, health products, food and medical equipment.

[0079] 6. The method of the present invention is universal and applicable to tobacco processing by-products such as tobacco stems and cigar waste, and is beneficial to the resource utilization of tobacco. BRIEF DESCRIPTION OF THE DRAWINGS

[0080] Figure 1 :Elution curve of tobacco pectin CSTP on DEAE Sepharose Fast Flow column (a) and elution curve of CSTP5 component on Sephadex-200 column (b)

[0081] Figure 2 : Flow chart of tobacco pectin preparation and purification.

[0082] Figure 3HPLC chromatogram of the monosaccharide composition of a mixed monosaccharide standard solution and a CSTP5b sample (a), and HPGPC chromatogram of the CSTP5b sample (b). In Figure (a), the peaks are: 1. PMP; 2. Man; 3. Rib; 4. Rha; 5. GlcUA; 6. GalUA; 7. Glc; 8. Gal; 9. Xyl; 10. Ara.

[0083] Figure 4 : Infrared spectrum of CSTP5b sample.

[0084] Figure 5 :NMR spectrum of CSTP5b sample. 1 H NMR spectrum (a); 13 C NMR spectrum (b); HSQC NMR spectrum (c); COSY NMR spectrum (d); NOESY NMR spectrum (e).

[0085] Figure 6 : Structural formula of CSTP5b.

[0086] Figure 7 : Moisture absorption (a) and moisture retention (b) capabilities of CSTP5b in an environment with a relative humidity of 43%.

[0087] Figure 8 Effects of CSTP5b on the proliferation activity (a) and the release of TNF-α (b), IL-1β (c), and NO (d) of RAW264.7 cells. Compared with the control group: #P<0.05, ##P<0.01; compared with the LPS group: *P<0.05, **P<0.01.

[0088] Figure 9 : HPLC chromatogram of monosaccharide composition of HSTP5a sample.

[0089] Figure 10 : Infrared spectrum of HSTP5a.

[0090] Figure 11 : HPLC chromatogram of monosaccharide composition of WSTP5c.

[0091] Figure 12 : HPGPC chromatogram of WSTP5c. DETAILED DESCRIPTION

[0092] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the 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.

[0093] The present invention discloses a structure and application of RG-I-HG type tobacco pectin. The preparation method of tobacco pectin provided by the present invention comprises the following steps: extraction by ammonium oxalate sequential extraction, ion exchange column chromatography and gel column chromatography, dialysis and freeze drying to obtain RG-I-HG type pectin with low esterification degree of tobacco leaves. The fine structure of the obtained tobacco pectin is determined by multiple technical means. The main chain connection mode of the structure is →[4)-α-D-GalpA-(1]6→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→2)-α-D-Rhap- The invention discloses a novel RG-I-HG tobacco pectin with a molecular weight of 65.8 kDa. The invention discloses a RG-I-HG tobacco pectin with a molecular weight of 65.8 kDa. The RG-I-HG tobacco pectin has a glycosidic bond of (1→4)-α-D-GalpA-(1→2)-α-D-Rhap-(1→), and a branched arabinan and a galactan connected to the main chain via an O-4 bond of (→2,4)-α-D-Rhap-(1→). A methyl ester group exists in the O-3 bond of (→3,4)-α-D-GalpA-(1→). The RG-I-HG tobacco pectin has a molecular weight of 65.8 kDa. The RG-I-HG tobacco pectin has a high purity and a uniform molecular weight, and has a certain moisturizing, hygroscopic and anti-inflammatory activity, and can be used in the fields of functional moisturizing materials and daily chemical products.

[0094] The present invention will be further explained below with reference to specific embodiments.

[0095] Example 1: Preparation of RG-I-HG tobacco pectin

[0096] The preparation of the tobacco pectin comprises the following steps:

[0097] Extraction: Tobacco leaves (Fujian Nanping B03 Cuibi No. 1) were dried at 40°C for 2 hours and then crushed. 1 kg of tobacco dust was heated and refluxed at 80°C with 95% ethanol for 2 hours to remove lipids, pigments, and other components. After air-drying, sequential extractions were performed. The defatted tobacco dust was added to pure water at a liquid-to-solid ratio of 16 g / mL and refluxed at 100°C for 3 hours. The hot water extraction residue was collected after filtration through a filter cloth. The hot water extraction residue was added to a 0.5% ammonium oxalate aqueous solution at a liquid-to-solid ratio of 16 g / mL and extracted at 25°C with mechanical stirring (250 rpm) for 4 hours. The extract was filtered through filter cloth, concentrated under reduced pressure to 2 L, and then centrifuged (4,000 r / min, 20 min). Four volumes of 95% ethanol (final concentration 80%) were added to the supernatant for precipitation. The precipitate was collected by centrifugation after standing at 4°C for 12 h. After washing with ethanol, it was deproteinized. The protein was repeatedly removed by the Sevag method until no impurity peaks were detected by UV detection. The extract was dialyzed with a 3,500 Da dialysis bag for 48 h to remove small molecular monosaccharides and salts, and then freeze-dried to obtain CSTP crude pectin.

[0098] Purification: Crude pectin CSTP was prepared into a 5 mg / mL solution, filtered through a 0.45 μm filter membrane, and loaded onto a DEAE-52 column (2.6 cm × 40 cm). The column was then eluted with ultrapure water, then 0.2 / 0.5 / 1 mol / L NaCl aqueous solution (flow rate 2.65 mL / min, 8 mL / tube collection). The uronic acid content was monitored by the m-hydroxybiphenyl method (Liu Ying. Comparative analysis of the structure and antioxidant activity of lily and polygonatum pectin [D]. Northeast Normal University, 2022.). Figure 1 As shown in a, the 0.5 mol / L NaCl elution peaks were combined, concentrated, desalted, and lyophilized using a 3500 Da dialysis bag to obtain pectin CSTP5. CSTP5 was prepared into a 50 mg / mL aqueous solution, filtered, and loaded onto a Sephadex-200 column (1.6 cm × 100 cm) and eluted with 0.1 mol / L NaCl (flow rate 0.5 mL / min, 8 mL / tube). Based on the elution curve (detection by m-hydroxybiphenyl method), as shown in FIG. Figure 1 As shown in (b), the eluted fractions were collected and concentrated, dialyzed using a 3500Da dialysis bag, and freeze-dried to obtain pectin fractions, which were named CSTP5a, CSTP5b, and CSTP5c, respectively.

[0099] Sample preparation process diagram Figure 2 shown.

[0100] Example 2: Fine structure characterization of RG-I-HG tobacco pectin

[0101] This example provides a method for fine structural characterization of RG-I-HG tobacco pectin, and the refined pectin prepared in Example 1 (abbreviated as CSTP5b) is used as the structural characterization sample in this example.

[0102] 1.1 Analysis of monosaccharide composition and molecular weight of CSTP5b

[0103] Monosaccharide composition determination procedure: Dissolve 100 mg each of monosaccharide standards [galacturonic acid (GalUA), glucuronic acid (GlcUA), D-mannose (Man), D-ribose (Rib), L-rhamnose (Rha), D-anhydrous glucose (Glc), D-galactose (Gal), D-xylose (Xyl), and L-arabinose (Ara)] (McLean, ≥99%, HPLC grade) in water and dilute to 100 mL to prepare a 1 mg / mL stock solution. Dilute as needed before use. Mix 1 mL of sample with 1 mL of 0.3 mol / L NaOH and 1 mL of 0.5 mol / L PMP methanol solution. Incubate in a 70°C water bath for 30 min, cool in an ice-water bath, adjust to pH 7 with HCl, and extract three times with 3 mL of chloroform. Filter the upper aqueous phase and perform analysis.

[0104] HPLC analysis was performed using an Agilent 1260 system with an Eclipse XDB-C18 column (250 mm × 4.6 mm, 5 μm). The mobile phase consisted of 0.05 mol / L phosphate buffer (pH 7.0) and acetonitrile (84:16). The injection volume was 10 μL, the detection wavelength was 250 nm, the flow rate was 1 mL / min, and the column temperature was 30°C.

[0105] Molecular weight determination procedure: Accurately weigh 5 mg each of dextran standard and CSTP5b, dissolve in 0.5 mol / L NaCl solution to prepare a 5 mg / mL solution, and filter through a 0.22 μm filter for determination. The molecular weights of the dextran standard and polysaccharide samples were determined using a high-performance gel permeation chromatography (HPGPC) system equipped with a differential refractive index detector (DRD). Detection conditions: Column: BRT105-103-101 (7.8 mm × 300 mm); Injection volume: 100 μL; Mobile phase: 0.5 mol / L NaCl solution; Flow rate: 0.7 mL / min; Column temperature: 40°C.

[0106] The results are as follows Figure 3 As shown in (a), by comparison with the chromatogram of the mixed monosaccharide standard, it was found that the prepared tobacco pectin CSTP5b was mainly composed of typical pectin monosaccharides such as galacturonic acid (57.88%), rhamnose (13.11%), galactose (20.71%), and arabinose (8.3%). Referring to the calculation method of HG and RG-I type pectin in the literature (Shokouh Ahmadi, Chengxiao Yu, Davood Zaeim, et al. Increasing RG-I content and lipase inhibitory activity of pectic polysaccharides extracted from gojiberry and raspberry by high-pressure processing [J]. Food Hydrocolloids, 2022, 126: 107477.), HG% = GalA mol% - Rha mol%; RG-I% = GalA mol% - HG mol% + Rha mol% + Galmol% + Ara mol%. According to the monosaccharide composition results, it can be concluded that the prepared tobacco pectin contains 44.77% HG structural units and 55.23% RG-I structural units, and does not contain non-pectin components such as glucose. This confirms that CSTP5b is a high-purity HG and RG-I mixed tobacco pectin.

[0107] The results of HPGPC analysis showed that ( Figure 3b) The CSTP5b sample exhibited a single, symmetrical peak at 35.538 min, indicating high purity. A linear equation was established between the logarithm of the molecular weight (y) of the dextran standard and the retention time (x), yielding the following linear equations for the weight-average molecular weight (Mw) and number-average molecular weight (Mn): y = -0.1221x + 9.1573 and y = -0.1227x + 9.1669, respectively. Based on this model, the Mw of CSTP5b was calculated to be 65.8 kDa and the Mn was 64.0 kDa. Its polydispersity index (PDI = Mw / Mn) was only 1.03, confirming the narrow molecular weight distribution and excellent molecular weight uniformity of CSTP5b.

[0108] 1.2 Infrared spectroscopy analysis of CSTP5b

[0109] 2 mg of dried CSTP5b tobacco pectin sample was added with dry KBr powder and ground. After tableting, the spectrum was collected using an infrared spectrometer with a scanning range of 400-4000 cm -1 , scan times 64 times.

[0110] like Figure 4 As shown in the figure, the infrared spectrum of CSTP5b shows typical characteristic peaks of pectin polysaccharide. -1 The broad absorption band at 2933 cm is attributed to the stretching vibration of the hydroxyl groups in between / intramolecular pectin polysaccharides. -1 and 1421cm -1 Symmetrical / asymmetric stretching vibration and bending vibration signals of CH bonds of methyl / methylene groups were detected respectively. -1 and 1610cm -1 The stretching vibrations of the methylated carboxylic acid group (COOCH3) and the free carboxylic acid group (COO-) were respectively corresponded. The degree of methylation (DM) was calculated to be 22.34% by integrating the characteristic peak area ratio (Formula 1), proving that it is a low-esterified pectin. In addition, 1242 cm -1 The stretching vibration signals of CO bond were detected at 1101, 1016 and 958 cm -1 The absorption peak in the region confirms the pyranose ring configuration of the sample.

[0111] Formula 1: Degree of esterification (DM) = A1739 cm －1 / (A1739 cm －1 +A1610 cm －1 )

[0112] Among them, A1739 cm －1 is 1739cm －1 The characteristic peak area, A1610 cm －1is 1610cm －1 The characteristic peak area.

[0113] 1.3 Methylation analysis of CSTP5b

[0114] The methylation analysis of the samples was carried out according to the method of Huang et al. (Huang LL, Zhao J, Wei YL, et al. Structural characterization and mechanisms of macrophage immunomodulatory activity of apectic polysaccharide from Cucurbita moschata Duch[J]. Carbohydrate Polymers, 2021, 269: 118288.).

[0115] After pretreatment with uronic acid reduction, methylation, and acetylation, GC-MS was used to identify the glycosidic bond types of CSTP5b (Table 1). Combining the monosaccharide composition analysis with the methylation results, CSTP5 predominantly exhibited a (→4)-GalpA-(1→) linkage, which is closely related to the linear backbone of homogalacturonan (HG) in pectin polysaccharides. (→2,4)-Rhap-(1→) was also detected (4%), a representative glycosidic bond of RG-I pectin. The O-4 position of this residue is substituted by a small amount of arabinan and galactan, indicating that the CSTP5b backbone is composed of an RG-I domain and an HG domain, with a branched structure.

[0116] Table 1 Analysis results of glycosidic bond types of CSTP5b

[0117]

[0118] Note: Some →4)-Galp-(1→ and →3,4)-Galp-(1→ are glycosidic bonds after GalA reduction.

[0119] 1.4 Nuclear Magnetic Resonance Spectroscopy Analysis of CSTP5b

[0120] 50 mg of dried pectin homogenous fraction was weighed and dissolved in 1 mL of heavy water. CSTP5b was analyzed by carbon nuclear magnetic resonance spectroscopy using a Bruker Avance 600 MHz spectrometer at 25°C and a detection frequency of 150 MHz.

[0121] Comprehensive application of one-dimensional ( 1 H / 13C) and two-dimensional (HSQC, COSY, NOESY) NMR techniques, combined with methylation analysis and monosaccharide composition data, were used to systematically characterize its fine structure. 1 H NMR spectrum ( Figure 5 The δ3.0-5.5 ppm region of a) exhibits complex coupling signals, and chemical shifts of anomeric hydrogen at δ5.38, 5.20, and 5.19 ppm are observed, corresponding to different glycosidic bond configurations. 13 CNMR spectrum ( Figure 5 b) Multiple characteristic signals were detected in the anomeric carbon region of δ93-110 ppm, and further HSQC correlation spectrum ( Figure 5 c) Seven groups of anomeric signals (labeled as residues AG) were identified, including: δ 4.97 / 100.38, 4.87 / 101.54, 5.19 / 100.37, 5.02 / 101.3, 5.2 / 108.87, 5.38 / 99.91, and 5.1 / 99.48 ppm. The HSQC spectrum shows an anomeric carbon signal of 100.38, and the corresponding anomeric hydrogen signal in the HSQC spectrum is 4.97. Using HH-COSY, the signals for H1-2 are δ4.97 / 3.67; H2-3 are δ3.67 / 3.93; and H3-4 are δ3.93 / 4.32. We can infer that H1, H2, H3, and H4 are δ4.97, 3.67, 3.93, and 4.32, respectively, with corresponding C1-4 values ​​of δ100.38, 69.4, 70.05, and 79.15. NOESY also observed the H1 / H4 signal at δ4.97 / 4.32, confirming this assignment. Combined with HSQC, the δ4.68 peak is assigned to H6a,b, with the corresponding C6 value at δ176.7. Therefore, this signal should be attributed to the glycosidic bond →4)-α-D-GalAp-. Based on similar rules, the specific chemical shift data of other glycosidic bonds were deduced and are shown in Table 2.

[0122] Table 2 CSTP5b 1 H and 13 C signal chemical shift (ppm) assignment

[0123]

[0124] In the GalpA unit, the correlation between H1 of residue A and its own H4 (δ5.08 / 3.88) confirmed the existence of the (1→4)-α-D-GalpA repeating unit; the cross peak between H1 of sugar residue A and H4 of residue B (δ5.08 / 3.78) suggested the existence of the →4)-GalpA-(1→3,4)-GalpA-(1→). The correlation between H1 of residue B and H4 of A (δ5.12 / 3.88) verified the existence of →3,4)-α-D-GalpA→4)-GalpA-(1→.

[0125] Regarding the Rhap connection mode, the H1 of residue A correlated with the H2 of residue C (δ5.08 / 4.18), corresponding to (→4)-α-D-GalpA-(1→2,4)-α-D-Rhap, while the cross peak of the H1 of residue G and the H4 of C (δ5.22 / 3.92) corresponded to the connection mode of (1→4,6)-α-D-Galp-(1→2,4)-α-D-Rhap.

[0126] In addition, the H1 of residue D correlated with the H2 of E (δ5.28 / 4.25), corresponding to α-Arap-(1→2)-α-L-Araf, while the H1 of residue E correlated with the H4 of residue B (δ5.32 / 3.78), indicating the existence of α-D-Galp-(1→4,6)-α-D-Galp-(1→2)-α-L-Araf-(1→2,4)-α-D-Rhap-(1→2). In addition, the H1 of sugar residue F and the H4 of sugar residue G had correlated signal peaks, indicating the existence of a linkage mode of α-D-Galp-(1→4,6)-α-D-Galp-(1→2)-α-L-Araf.

[0127] According to the methylation analysis results and NMR data, the pectin is composed of galacturonic acid (GalA), rhamnose (Rha), galactose (Gal) and arabinose (Ara), and its structural characteristics are consistent with the typical characteristics of RG-I type pectin and HG type pectin. The structure of CSTP5b is shown in Figure 2. Figure 6 shown.

[0128] Example 3: Testing of Moisture Absorption and Moisture Retention Capacity of RG-I-HG Tobacco Pectin

[0129] This example provides the moisture absorption and moisture retention capabilities of an RG-I-HG tobacco pectin. The refined pectin prepared in Example 1 (abbreviated as CSTP5b) was used as the research object of this example.

[0130] CSTP5b was ground into powder and dried in an oven at 100°C for 4 hours. To determine the moisture absorption rate, 0.1 g of sample was placed in a desiccator with a relative humidity of 43% and a temperature of (20.0 ± 2.0)°C containing a saturated potassium carbonate solution. The sample weight was measured at 1, 2, 4, 8, 12, 24, and 48 hours. Three replicates were set for each group, and glycerol was used as a control. The moisture absorption rate (RH ) is calculated as R H =(M t -M0) / M0. Where: M t is the mass of samples at different time periods (g); M0 is the initial sample mass (g).

[0131] The sample was ground into powder and dried in an oven at 100°C for 4 hours. 0.1 g of the sample was placed in a silica gel desiccator containing 3 times the amount of ultrapure water and tested continuously at different time points. Three parallels were set for each group, and glycerol was used as a control. The moisturizing rate (R M )Calculation formula R M =M t / M0×100%. Where: M t is the mass of samples at different time periods (g); M0 is the initial sample mass (g).

[0132] like Figure 7 As shown in a, in an environment of 20°C and 43% relative humidity, the moisture absorption rate of CSTP5b at different time periods is better than that of glycerol. CSTP5b is composed of galacturonic acid, rhamnose, galactose, and arabinose monosaccharides. The molecule contains a large number of carboxyl and hydroxyl groups, and has a strong ability to form intermolecular hydrogen bonds with water molecules in the air, indicating that tobacco pectin polysaccharides can effectively lock in moisture and have excellent hygroscopicity.

[0133] like Figure 7 As shown in Figure b, in an environment of 20°C and 43% relative humidity, the moisture retention rate of tobacco pectin remained above 90% for 96 hours, demonstrating strong moisture retention, surpassing that of glycerol. This is primarily due to the fact that polar groups such as hydroxyl and carboxyl groups in pectin polysaccharides can form hydrogen bonds, contributing positively to water retention. Furthermore, pectin polysaccharide chains can intertwine, forming a network structure that facilitates the adsorption of water molecules.

[0134] Example 4: Evaluation of the anti-inflammatory activity of RG-I-HG tobacco pectin

[0135] This example provides an anti-inflammatory ability of RG-I-HG tobacco pectin, and the refined pectin prepared in Example 1 (abbreviated as CSTP5b) is used as the research object of this example.

[0136] In this study, mouse mononuclear macrophage RAW 264.7 cells (1×10 5Cells were seeded in 24-well plates (100 μg / well) and cultured in a 37°C, 5% CO2 incubator until the cells adhered. Subsequently, the cells were treated with lipopolysaccharide (LPS) 1 μg / mL for 1 hour to induce an inflammatory response. After treatment, CSTP5b at different concentrations (10, 50, 100, 200 μg / mL) was added and cultured for 24 hours. After 24 hours, cell debris was removed by centrifugation, and the supernatant was collected as the test sample. Three parallel experiments were set up for each group, and the culture medium of untreated cells was used as a negative control group to ensure the repeatability and accuracy of the experimental results. According to the operating instructions of the ELISA kit, the test sample and the standard were added to a 96-well plate in proportion, and three replicates were set up in each well. Subsequently, biotinylated antibody, enzyme-labeled antibody and chromogenic substrate were added in sequence according to the kit instructions. After incubation, the absorbance value was measured at a wavelength of 540 nm using a microplate reader. By drawing a standard curve, the concentration levels of anti-inflammatory factors TNF-α, IL-1β and NO in the cell culture supernatant were calculated.

[0137] Depend on Figure 8 (a) It can be seen that after 24 hours of co-culture with RAW264.7 cells, CSTP5b at concentrations of 10, 50, 100 and 200 μg / mL significantly promoted cell proliferation compared with the control group (P < 0.01).

[0138] Figure 8 (bd) show that compared with the control group, CSTP5b significantly inhibited NO production at concentrations of 10, 50, 100, and 200 μg / mL. Furthermore, the inhibitory effect of CSTP5b on TNF-α and IL-1β increased with increasing concentration, reaching its maximum inhibitory effect at 200 μg / mL. This suggests that CSTP5b exerts its anti-inflammatory effects by inhibiting the release of pro-inflammatory factors.

[0139] Comparative Example 1

[0140] Except that the 0.5% ammonium oxalate aqueous solution in Example 1 was replaced by a hydrochloric acid aqueous solution with a pH of 2, the remaining steps were exactly the same as in Example 1. The extracted pectin sample was named HSTP5a.

[0141] The characterization method is exactly the same as that in Example 2.

[0142] like Figure 9 As shown in the figure, the monosaccharide composition of purified pectin HSTP5a extracted with hydrochloric acid as solvent is mainly composed of GalA, Glc and a small amount of Man, indicating that hydrochloric acid solvent will cause the dissociation of pectin arabinan and galactan side chains, and some non-pectin components such as Glc and Man will also be extracted, indicating that the hydrochloric acid system is not suitable for the extraction of RG-I-HG structure pectin. Figure 10As shown, the infrared spectrum of HSTP5a shows the absence of 1739 cm -1 The ester absorption peak of the extract indicated that hydrochloric acid extraction would lead to the removal of the polygalacturonic acid methyl ester group in tobacco pectin, thereby affecting its functional activity.

[0143] Comparative Example 2

[0144] Except that the extraction under reflux at 100° C. for 3 h in Example 1 was replaced by continuous stirring in cold water at 25° C. for 3 h, the remaining steps were exactly the same as in Example 1. The extracted pectin sample was named WSTP5c.

[0145] The characterization method is exactly the same as that in Example 2.

[0146] The results showed that the monosaccharide composition of the pectin polysaccharide WSTP5c extracted by this method included Glc, GalA, Gal, Ara, Rha and Man ( Figure 11 ), where the molar ratio of Glc is as high as 65.6%, indicating that the cold water system cannot completely remove the neutral polysaccharides mainly composed of glucan, resulting in the simultaneous dissolution of glucan-based polysaccharides and pectin polysaccharides in the subsequent 0.5% ammonium oxalate system. Figure 12 As shown, the retention times of WSTP5c are 30.123 min (21.635%) and 34.153 min (78.365%), indicating that the molecular weight uniformity of the sample is poor.

[0147] 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 RG-I-HG type tobacco pectin, wherein: The RG-I-HG tobacco pectin is composed of galacturonic acid, rhamnose, galactose, and arabinose, and the molar ratio of galacturonic acid, rhamnose, galactose, and arabinose is 57.88:13.11:20.71:8.3; The main chain of the RG-I-HG type tobacco pectin is connected in the following manner: →[4)-α-D-GalpA-(1]6→4)-α-D-GalpA-(1→4)-α-D-GalpA-(1→2)-α-D-Rhap-(1→4)-α-D-GalpA-(1→2)-α-D-Rhap-(1→, the branched arabinan and galactan are respectively connected to the main chain through the O-4 bond of →2,4)-α-D-Rhap-(1→), and the main chain has a methyl ester group at the O-3 bond of →3,4)-α-D-GalpA-(1→.

2. The RG-I-HG tobacco pectin according to claim 1, wherein The weight average molecular weight of the RG-I-HG tobacco pectin is 65.8 kDa, and the number average molecular weight is 64.0 kDa.

3. A method for preparing the RG-I-HG tobacco pectin according to claim 1 or 2, comprising: (1) Removing water-soluble crude polysaccharides from tobacco leaves; (2) extracting crude pectin from the tobacco leaves obtained in step (1) using an ammonium oxalate aqueous solution and ethanol precipitation; (3) Purifying the crude pectin by ion exchange chromatography and gel column chromatography in sequence to obtain the RG-I-HG tobacco pectin.

4. The method according to claim 3, wherein: Step (1) includes: (1-1) mixing chopped tobacco leaves with water and subjecting the mixture to a heat reflux treatment to obtain a hot mixture; (1-2) filtering the hot mixture and collecting the filter residue; Preferably, in step (1-1), the temperature of the heat reflux treatment is 100°C; Preferably, in step (1-1), the heat reflux treatment time is 2.5-3.5 hours, preferably 3 hours; Preferably, in step (1-1), the ratio of the mass of the shredded tobacco leaves to the volume of the water is 10-20 g / mL, preferably 16 g / mL; Preferably, in step (1-2), the filtration is performed by filtering with a filter cloth; Preferably, the shredded tobacco leaves are pre-degreased; Preferably, the degreasing treatment comprises: pre-immersing the shredded tobacco leaves in ethanol for degreasing or pre-heating and refluxing the shredded tobacco leaves in ethanol; Preferably, the ethanol is 95% ethanol; Preferably, the soaking and degreasing treatment lasts for 48 hours, and the ethanol is replaced every 12 hours; Preferably, the heating reflux treatment is carried out at a temperature of 80° C. for 2 hours; Preferably, the soaking and degreasing treatment or the heating and reflowing treatment further includes a drying step; Preferably, the drying is air-drying.

5. The method according to claim 4, wherein Step (2) includes: (2-1) stirring the ammonium oxalate aqueous solution and the filter residue obtained in step (1-2) to obtain a stirred product; (2-2) filtering the stirred product, collecting the filtrate, concentrating and centrifuging the filtrate, and collecting the supernatant; (2-3) mixing the supernatant with ethanol for alcohol precipitation, and collecting the precipitate; (2-4) The precipitate is sequentially subjected to deproteinization, monosaccharide and salt removal, and freeze-drying to obtain crude pectin.

6. The method according to claim 5, wherein: The method further has one or more technical features selected from the following (i)-(xii): ( i) in step (2-1), the ratio of the mass of the filter residue to the volume of the ammonium oxalate aqueous solution is 10-20 g / mL, preferably 16 g / mL; Preferably, the concentration (w / v concentration) of the ammonium oxalate aqueous solution is 0.25%-1.0%, preferably 0.5%; (ii) In step (2-1), the stirring speed is 200-300 r / min, preferably 250 r / min; Preferably, the stirring is mechanical stirring; (iii) in step (2-1), the stirring treatment is carried out at a temperature of 20-30° C. (preferably 25° C.) for 3-5 hours (preferably 4 hours); (iv) in step (2-2), the filtration is performed by filtering with a filter cloth; and / or the concentration is performed by concentrating under reduced pressure; (v) In step (2-2), the centrifugal treatment is carried out at a rotation speed of 3000-5000 r / min (preferably 4000 r / min) for 15-25 min (preferably 20 min); (vi) in step (2-3), the ratio of the volume of the ethanol to the volume of the supernatant is 3:1 to 5:1, preferably 4:1; (vii) In step (2-3), the ethanol is 95% ethanol; (viii) in step (2-3), the final concentration of ethanol in the mixture of the supernatant and the ethanol is 80%; (ix) In step (2-3), the alcohol precipitation treatment is achieved by standing and centrifuging; Preferably, the standing is carried out at a temperature of 4°C; Preferably, the standing time is 10-15 hours, preferably 12 hours; (x) In step (2-4), the deproteinization treatment includes: repeatedly removing proteins using the Sevag method until no impurity peaks are detected by ultraviolet detection; (xi) In step (2-4), the monosaccharide and salt removal treatment comprises: dialyzing using a 3500Da dialysis bag for 45-50 hours (preferably 48 hours) to remove monosaccharides and salts; (xii) In step (2-4), the precipitate is washed before the deproteinization treatment; Preferably, the washing treatment is performed using ethanol.

7. The method according to claim 3, wherein: Step (3) includes: (3-1) The crude pectin was loaded onto a DEAE column and gradient eluted with ultrapure water, 0.2 mol / L NaCl aqueous solution, 0.5 mol / L NaCl aqueous solution, and 1 mol / L NaCl aqueous solution in sequence. The eluate from the 0.5 mol / L NaCl aqueous solution was collected, and the eluate was concentrated, desalted, and lyophilized to obtain a lyophilized product. (3-2) loading the lyophilized product onto a Sephadex-200 column, eluting with a 0.1 mol / L NaCl aqueous solution, collecting the main peak eluate, dialyzing the eluate, and lyophilizing it to obtain the RG-I-HG tobacco pectin; Preferably, in step (3-1), the crude pectin is pre-prepared into a crude pectin aqueous solution before being loaded, and the crude pectin aqueous solution is filtered through a filter membrane; preferably, the filter membrane is preferably a 0.45 μm filter membrane; preferably, the concentration of the crude pectin aqueous solution is 3-7 mg / mL, preferably 5 mg / mL; Preferably, in step (3-1), the flow rate of the gradient elution is 2.65 mL / min; Preferably, in step (3-1), the desalination is achieved by a 3500Da dialysis bag; Preferably, in step (3-2), the lyophilized material is pre-configured into a lyophilized material aqueous solution before being loaded, and the lyophilized material aqueous solution is filtered; preferably, the concentration of the lyophilized material aqueous solution is 40-60 mg / mL, preferably 50 mg / mL; Preferably, in step (3-2), the elution flow rate is 0.5 mL / min; Preferably, in step (3-2), the dialysis is performed using a 3500Da dialysis bag.

8. A product comprising the RG-I-HG tobacco pectin according to any one of claims 1 to 2 or the RG-I-HG tobacco pectin prepared by the method according to any one of claims 3 to 7; Preferably, the product is a cosmetic, medicine, health product, functional food or medical device; Preferably, the drug is a skin repair drug, a wound repair drug or an ophthalmic drug; Preferably, the health care product is an oral beauty health care product or an intestinal health health care product; Preferably, the medical device is a medical dressing or a restorative implant material; Preferably, the functional food is an enhanced food or a food for special medical purposes.

9. Use of the RG-I-HG tobacco pectin according to any one of claims 1 to 2 or the RG-I-HG tobacco pectin prepared by the method according to any one of claims 3 to 7 in preparing a product, wherein the product is used for one or more of the following (i) to (iv): (i) anti-inflammatory; (ii) Moisturizing; (iii) Hygroscopic; (iv) Helps improve skin moisture.

10. The use according to claim 9, wherein The product is a cosmetic, medicine, health product, functional food or medical device; Preferably, the drug is a skin repair drug, a wound repair drug or an ophthalmic drug; Preferably, the health care product is an oral beauty health care product or an intestinal health health care product; Preferably, the medical device is a medical dressing or a restorative implant material; Preferably, the functional food is an enhanced food or a food for special medical purposes.

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