RG-I type sweet potato pectin with anti-inflammatory activity as well as preparation method and application of RG-I type sweet potato pectin

Abstract

The invention discloses RG-I type sweet potato pectin with anti-inflammatory activity and a preparation method and application thereof, and belongs to the technical field of pectin extraction. In order to solve the technical problems that strong acid, strong base and other reagents which pollute the environment need to be used in an existing method for preparing RG-I type pectin, a purification process is complex and the like, pectin in sweet potatoes is extracted by a water extraction method, crude pectin is obtained through enzymolysis and alcohol precipitation, and then the crude pectin is purified by a dialysis bag dialysis method, so that the RG-I type pectin is obtained. The RG-I type pectin with high purity and relatively good anti-inflammatory activity is obtained. The preparation method provided by the invention has the advantages of simple process, no participation of toxic and harmful reagents, environmental friendliness, low cost and suitability for large-scale production, and the prepared pectin has relatively high purity and relatively good anti-inflammatory activity and can be used for preparing medicines and functional foods with anti-inflammatory activity.

Description

Technical Field

[0001] The invention belongs to the technical field of pectin extraction, and particularly relates to RG-I type sweet potato pectin with anti-inflammatory activity and a preparation method and application thereof. Background Art

[0002] RG-I pectin (Rhamnogalacturonan I) is a polysaccharide present in plant cell walls and is a major component of pectin. Pectin is a complex class of high molecular weight compounds that is widely present in the cell walls and middle lamina of plants, especially in the skin and flesh of citrus fruits, apples, grapes, etc. RG-I pectin is composed of α-1,4-linked D-galacturonic acid chains, which contain side chains including α-L-rhamnose, β-D-galactose, α-L-arabinose, and other sugars and non-glycosylated parts. These side chains are connected to the galacturonic acid residues on the main chain through different glycosidic bonds to form a complex three-dimensional structure. RG-I pectin plays multiple roles in plant cell walls, including maintaining the mechanical strength of the cell wall, participating in cell-to-cell interactions, and acting as a signal molecule to participate in the regulation of plant growth and development and response to environmental stress. In addition, RG-I pectin has been implicated in plant defense responses against pathogens, as it can bind to enzymes produced by certain pathogens, thereby protecting the plant from infection. In the food industry, pectin is used as a food additive for its gel-forming ability and thickening properties to make jams, jellies, and other food products that require stability and structure. The specific applications of RG-I pectin may depend on its structural properties and interactions with other ingredients. Due to the complexity and diversity of RG-I pectin, scientists are still studying its detailed structure and function to better understand its role in plant physiology and food science.

[0003] The existing extraction process of RG-I natural pectin generally requires the use of strong acid and strong alkali reagents, and / or requires high pressure and other environments. The use of strong acid and strong alkali reagents causes certain pollution to the environment, and high pressure treatment also consumes more energy. In addition, the purification steps of the existing RG-I natural pectin are relatively complicated. The crude pectin after alcohol precipitation needs to be purified by DEAE-cellulose chromatography column or other complicated purification methods before it can be converted into pectin with higher purity. For example, in the invention patent with the invention name of a method for extracting RG-I pectin rich in the patent number CN201710879850.7, an alkaline solution that pollutes the environment is used to extract pectin; in the patent application with the invention name of a tofu chayote leaf RG-I pectin with antioxidant and anti-inflammatory activity and its preparation method and application, the application number CN202110122216.5, an alkaline solution is used as the extraction solvent, and its purification step uses a complex DEAE-cellulose chromatography column purification and membrane separation technology to obtain a uniform pectin component; in the invention patent with the invention name of a method for preparing RG-I pectin polysaccharide rich in arabinose side chains, the patent number CN202110308340.0, the pectin extraction process is not only in a harsh environment of acidity and nitrogen protection, but also needs to be treated with high pressure. Therefore, it is urgent to develop a method for preparing RG-I pectin with high purity and good biological activity without using strong acid, strong alkali and other environmentally polluting reagents, and without a complex purification process. Summary of the invention

[0004] In order to solve the technical problems that the existing method for preparing RG-I type pectin requires the use of reagents such as strong acids and strong bases that pollute the environment, and the purification process is complicated, the present invention adopts a water extraction method to extract pectin from sweet potatoes, obtains crude pectin through enzymatic hydrolysis and alcohol precipitation, and then purifies the crude pectin through a dialysis bag dialysis method to obtain high-purity RG-I type pectin with good anti-inflammatory activity.

[0005] In order to solve the above technical problems and achieve corresponding technical effects, the present invention provides the following technical solutions:

[0006] The first object of the present invention is to provide an RG-I type sweet potato pectin with anti-inflammatory activity, wherein the RG-I type sweet potato pectin has a molecular weight of 58.41-62.98 KDa; mainly contains the following monosaccharides: galacturonic acid, galactose, rhamnose, arabinose, glucose, glucuronic acid, xylose and mannose, and the molar ratio of the galacturonic acid, galactose, rhamnose, arabinose, glucose, glucuronic acid, xylose and mannose is: (41.50-43.56): (33.62-34.78) (10.49-11.98): (8.58-9.56): (6.12-6.56): (1.43-1.56): (0.75-0.89): (0.45-0.53); the RG-I type sweet potato pectin has an RG-I domain, and its main chain structure is as follows:

[0007]

[0008] , wherein m+n=8-9, m and n are both integers and not 0.

[0009] The second object of the present invention is to provide a method for preparing the above-mentioned RG-I type sweet potato pectin, the preparation method comprising the following steps:

[0010] S1, impurity removal: mixing the sweet potato residue powder with anhydrous ethanol, shaking, filtering to remove soluble substances in the sweet potato residue powder, and drying the obtained filter residue to obtain the impurity-removed sweet potato residue powder;

[0011] S2, extraction: the sweet potato residue powder after impurity removal is mixed with water according to the solid-liquid ratio of 1g:25mL-1g:45mL, extracted at 80-100°C for 60-90min, centrifuged after cooling, the supernatant is collected, the precipitate is extracted again by the same method, the supernatants obtained by the two extractions are combined, and concentrated to 1 / 5-1 / 4 of the original volume at 55°C-60°C to obtain a concentrated solution;

[0012] S3, enzymolysis: α-amylase and saccharifying enzyme are added to the concentrated solution obtained in S2, incubated at 60°C for 1-2h, and the enzymolysis solution is obtained after the enzyme is inactivated;

[0013] S4, alcohol precipitation: centrifuge the enzymatic hydrolysate obtained in S3, collect the supernatant, add 2 times the volume of anhydrous ethanol of the supernatant, let stand at 4°C for 12-24h, and collect the precipitate by centrifugation to obtain crude pectin;

[0014] S5. The crude pectin obtained in S4 was dissolved in water again, dialyzed in a dialysis bag for 72 hours, and freeze-dried to obtain RG-I sweet potato pectin.

[0015] In one embodiment of the present invention, the sweet potato residue powder described in S1 is obtained by crushing dried sweet potato residue and then passing it through an 80-mesh sieve.

[0016] In one embodiment of the present invention, the solid-liquid ratio of the sweet potato residue powder to anhydrous ethanol in S1 is 1g:20mL-1g:30mL, and the shaking time is 1-2h.

[0017] In one embodiment of the present invention, the added amount of the α-amylase and saccharifying enzyme in S3 is 0.5% of the weight of the concentrate.

[0018] In one embodiment of the present invention, the enzyme inactivation in S3 is boiling.

[0019] In one embodiment of the present invention, the centrifugation in S4 is performed at a rotation speed of 5000 rpm for 15-25 min.

[0020] In one embodiment of the present invention, the molecular weight cutoff of the dialysis bag in S5 is 5000Da.

[0021] The third object of the present invention is to provide the use of the above RG-I sweet potato pectin in the preparation of a drug with anti-inflammatory activity.

[0022] The fourth object of the present invention is to provide the use of the above RG-I sweet potato pectin in preparing food.

[0023] Beneficial effects of the present invention:

[0024] The preparation method of RG-I sweet potato pectin provided by the present invention does not require the use of acidic or alkaline reagents, nor does it require high-energy-consuming equipment such as pressurization, and the extraction of pectin can be achieved only in hot water. In addition, the purification step of the method of the present invention is only dialysis in a dialysis bag (5000Da MWCO) for 72 hours to obtain pectin with a purity higher than 95%. The preparation method provided by the present invention has the advantages of simple process, no participation of toxic and harmful reagents, environmental friendliness, low cost, and suitability for large-scale production, and the prepared pectin is RG-I type pectin with high purity, a pectin molecular weight in the most appropriate range, and good anti-inflammatory activity.

[0025] In addition, the comparison of extracting sweet potato residue pectin with alkaline solvent (sodium hydroxide), acidic solvent (hydrochloric acid), and chelating agent (EDTA) showed that the pectin monosaccharide compositions extracted by these three methods and the method (water extraction method) provided by the present invention were similar, but the pectin extracted by the water extraction method (method of the present invention) was rich in galactose (content of 33.62mol%), which was significantly higher than that of the other three methods (the contents of galactose in the pectin obtained by alkali extraction pectin, acid extraction pectin, and chelating agent extraction pectin were 26.66mol%, 22.62mol%, and 25.34mol%, respectively) (as shown in Table 1). According to existing literature records, the content of galactose in pectin polysaccharides is positively correlated with its anti-inflammatory activity, so it can be explained that the sweet potato pectin of the present invention has a stronger anti-inflammatory activity relative to the sweet potato pectin extracted by other methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the high performance gel permeation chromatogram of the RG-I sweet potato pectin prepared in Example 1;

[0027] Figure 2 This is the monosaccharide composition diagram of the RG-I sweet potato pectin prepared in Example 1;

[0028] Figure 3 This is the infrared spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0029] Figure 4 This is a one-dimensional hydrogen nuclear magnetic resonance spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0030] Figure 5 This is a one-dimensional carbon nuclear magnetic resonance spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0031] Figure 6 This is the DEPT 135 spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0032] Figure 7 This is the COSY spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0033] Figure 8 This is the HMBC spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0034] Fig. 9 This is the HSQC spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0035] Fig.10 This is the NOESY spectrum of the RG-I sweet potato pectin prepared in Example 1;

[0036] Fig.11The structural formula of the RG-I sweet potato pectin prepared in Example 1 is deduced;

[0037] Fig.12 The in vitro anti-inflammatory activity and mechanism of action of RG-I sweet potato pectin HWSP-1 prepared in Example 1 are shown in FIG. Fig.12 A in the figure is the test result of the effect of different concentrations of HWSP-1 on the survival rate of iBMDM cells. Fig.12 B is the test result of the effect of different concentrations of HWSP-1 on the relative expression level of IL-1β mRNA in LPS-induced inflammatory cells. Fig.12 C in the figure is the test result of the effect of different concentrations of HWSP-1 on the relative expression level of IL-6 mRNA in LPS-induced inflammatory cells. Fig.12 D in the figure is the test result of the effect of different concentrations of HWSP-1 on the relative expression level of TNF-α mRNA in LPS-induced inflammatory cells. Fig.12 Figure E is the Western blot analysis of the protein levels of TLR4, p-p65, p65, NLRP3, pro-caspas-1, pro-IL-1β, Cleaved-caspas-1, Cleaved-IL-1β and Cleaved-GSDMD. Fig.12 F in the figure is the immunofluorescence image. Fig.12 G in the figure is the Western blot quantification data; compared with the control group, ###p<0.001; compared with the LPS group, ****p<0.0001, ***p<0.001, **p<0.01 and *p<0.05;

[0038] Fig.13 The figure shows the results of the in vivo anti-inflammatory activity test of RG-I sweet potato pectin; Fig.13 A in the figure is the experimental flow chart for in vivo anti-inflammatory activity detection. Fig.13 B is the H&E staining analysis of mouse lung tissue. Fig.13 C in the figure is the result of ELISA detection of the expression levels of IL-1β, TNF-α and IL-6 in mouse serum; compared with the control group, ###p<0.001, ##p<0.01; compared with the LPS group, ***p<0.001, **p<0.01 and *p<0.05. DETAILED DESCRIPTION

[0039] The present invention will be further described in detail below in conjunction with specific examples and accompanying drawings, and the embodiments are intended to illustrate the present invention only, but are not intended to limit the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements are within the scope of protection of the present invention. The process, conditions, experimental methods and reagents for implementing the present invention, except for the contents specifically mentioned below, are all common knowledge and conventional products in the market in the art, and the present invention does not particularly limit the contents.

[0040] Embodiment 1:

[0041] This embodiment provides a method for preparing RG-I sweet potato pectin by water extraction, which is as follows:

[0042] (1) Impurity removal: The dried sweet potato residue is crushed and passed through an 80-mesh sieve to obtain sweet potato residue powder, the sweet potato residue powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1 g:25 mL, and the mixture is shaken for 1 h and then filtered to remove soluble substances in the sweet potato residue powder, such as small molecular sugars and phenolic acids, and then the obtained filter residue is dried to obtain the impurity-removed sweet potato residue powder.

[0043] (2) Extraction: The sweet potato residue powder obtained in step (1) after impurity removal was mixed with water at a solid-liquid ratio of 1 g: 25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant was collected; water was added to the precipitate at a solid-liquid ratio of 1 g: 25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant was collected; the supernatants obtained from the two extractions were combined and concentrated at 60° C. to 1 / 5 of the original volume to obtain a concentrated solution.

[0044] (3) Enzymatic hydrolysis: adding α-amylase and saccharifying enzyme to the concentrated solution obtained in step (2), incubating at 60° C. for 2 h, and boiling to inactivate the enzymes to obtain an enzymatic hydrolyzate; the amount of α-amylase and saccharifying enzyme added is 0.5% of the weight of the concentrated solution.

[0045] (4) Alcohol precipitation: The enzymatic hydrolyzate obtained in step (3) is cooled to room temperature, and then centrifuged at 5000 rpm for 20 min. The supernatant is collected, and anhydrous ethanol twice the volume of the supernatant is added. The mixture is allowed to stand at 4°C for 12 h, and the precipitate is collected by centrifugation to obtain crude pectin.

[0046] (5) The crude pectin obtained in step (4) was dissolved in water again, dialyzed in a dialysis bag with a molecular weight cutoff of 5000 Da for 72 hours, and freeze-dried to obtain RG-I sweet potato pectin (denoted as HWSP-1).

[0047] Embodiment 2:

[0048] This embodiment provides a method for preparing RG-I sweet potato pectin by water extraction, which is as follows:

[0049] (1) Impurity removal: The dried sweet potato residue is crushed and passed through an 80-mesh sieve to obtain sweet potato residue powder, the sweet potato residue powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1 g:25 mL, and the mixture is shaken for 1 h and then filtered to remove soluble substances in the sweet potato residue powder, such as small molecular sugars and phenolic acids, and then the obtained filter residue is dried to obtain the impurity-removed sweet potato residue powder.

[0050] (2) Extraction: The sweet potato residue powder obtained in step (1) after impurity removal is mixed with water at a solid-liquid ratio of 1 g:30 mL, extracted at 90° C. for 60 min, centrifuged after cooling, and the supernatant is collected; water is added to the precipitate at a solid-liquid ratio of 1 g:30 mL, extracted at 90° C. for 60 min, centrifuged after cooling, and the supernatant is collected; the supernatants obtained from the two extractions are combined and concentrated at 60° C. to 1 / 5 of the original volume to obtain a concentrated solution.

[0051] (3) Enzymatic hydrolysis: adding α-amylase and saccharifying enzyme to the concentrated solution obtained in step (2), incubating at 60° C. for 1 h, and boiling to inactivate the enzymes to obtain an enzymatic hydrolyzate; the amount of α-amylase and saccharifying enzyme added is 0.5% of the weight of the concentrated solution.

[0052] (4) Alcohol precipitation: The enzymatic hydrolyzate obtained in step (3) is cooled to room temperature, and then centrifuged at 5000 rpm for 15 min. The supernatant is collected, and anhydrous ethanol twice the volume of the supernatant is added. The mixture is allowed to stand at 4°C for 18 h, and the precipitate is collected by centrifugation to obtain crude pectin.

[0053] (5) The crude pectin obtained in step (4) was dissolved in water again, dialyzed in a dialysis bag with a molecular weight cutoff of 5000 Da for 72 hours, and freeze-dried to obtain RG-I type sweet potato pectin (denoted as HWSP-2).

[0054] Embodiment 3:

[0055] This embodiment provides a method for preparing RG-I sweet potato pectin by water extraction, which is as follows:

[0056] (1) Impurity removal: The dried sweet potato residue is crushed and passed through an 80-mesh sieve to obtain sweet potato residue powder, the sweet potato residue powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1 g:25 mL, and the mixture is shaken for 1 h and then filtered to remove soluble substances in the sweet potato residue powder, such as small molecular sugars and phenolic acids, and then the obtained filter residue is dried to obtain the impurity-removed sweet potato residue powder.

[0057] (2) Extraction: The sweet potato residue powder obtained in step (1) after impurity removal is mixed with water at a solid-liquid ratio of 1 g:45 mL, extracted at 100° C. for 75 min, centrifuged after cooling, and the supernatant is collected; water is added to the precipitate at a solid-liquid ratio of 1 g:45 mL, extracted at 100° C. for 75 min, centrifuged after cooling, and the supernatant is collected; the supernatants obtained from the two extractions are combined and concentrated at 60° C. to 1 / 5 of the original volume to obtain a concentrated solution.

[0058] (3) Enzymatic hydrolysis: adding α-amylase and saccharifying enzyme to the concentrated solution obtained in step (2), incubating at 60° C. for 1.5 h, and boiling to inactivate the enzymes to obtain an enzymatic hydrolyzate; the amount of α-amylase and saccharifying enzyme added is 0.5% of the weight of the concentrated solution.

[0059] (4) Alcohol precipitation: The enzymatic hydrolysate obtained in step (3) is cooled to room temperature, and then centrifuged at 5000 rpm for 25 min. The supernatant is collected, and anhydrous ethanol twice the volume of the supernatant is added. The mixture is allowed to stand at 4°C for 24 h, and the precipitate is collected by centrifugation to obtain crude pectin.

[0060] (5) The crude pectin obtained in step (4) was dissolved in water again, dialyzed in a dialysis bag with a molecular weight cutoff of 5000 Da for 72 hours, and freeze-dried to obtain RG-I type sweet potato pectin (denoted as HWSP-3).

[0061] Comparative Example 1:

[0062] This embodiment provides a method for preparing sweet potato pectin using a strong alkaline solution, which is as follows:

[0063] (1) Impurity removal: The dried sweet potato residue is crushed and passed through an 80-mesh sieve to obtain sweet potato residue powder, the sweet potato residue powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1 g:25 mL, and the mixture is shaken for 1 h and then filtered to remove soluble substances in the sweet potato residue powder, such as small molecular sugars and phenolic acids, and then the obtained filter residue is dried to obtain the impurity-removed sweet potato residue powder.

[0064] (2) Extraction: The sweet potato residue powder obtained in step (1) after impurity removal was mixed with a sodium hydroxide solution having a pH value of 11.0 at a solid-liquid ratio of 1 g:25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant was collected; sodium hydroxide solution having a pH value of 11.0 was added to the precipitate at a solid-liquid ratio of 1 g:25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant was collected; the supernatants obtained from the two extractions were combined, the pH of the supernatant was adjusted to 7.0 using a 1.0 M HCl solution, and then concentrated at 60° C. to 1 / 5 of the original volume to obtain a concentrated solution.

[0065] (3) Enzymatic hydrolysis: adding α-amylase and saccharifying enzyme to the concentrated solution obtained in step (2), incubating at 60° C. for 2 h, and boiling to inactivate the enzymes to obtain an enzymatic hydrolyzate; the amount of α-amylase and saccharifying enzyme added is 0.5% of the weight of the concentrated solution.

[0066] (4) Alcohol precipitation: The enzymatic hydrolyzate obtained in step (3) is cooled to room temperature, and then centrifuged at 5000 rpm for 20 min. The supernatant is collected, and anhydrous ethanol twice the volume of the supernatant is added. The mixture is allowed to stand at 4°C for 12 h, and the precipitate is collected by centrifugation to obtain crude pectin.

[0067] (5) The crude pectin obtained in step (4) was dissolved in water again, dialyzed in a dialysis bag with a molecular weight cutoff of 5000 Da for 72 hours, and freeze-dried to obtain preliminarily purified sweet potato pectin.

[0068] Comparative Example 2:

[0069] This embodiment provides a method for preparing sweet potato pectin using a strong acid solution, which is as follows:

[0070] (1) Impurity removal: The dried sweet potato residue is crushed and passed through an 80-mesh sieve to obtain sweet potato residue powder, the sweet potato residue powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1 g:25 mL, and the mixture is shaken for 1 h and then filtered to remove soluble substances in the sweet potato residue powder, such as small molecular sugars and phenolic acids, and then the obtained filter residue is dried to obtain the impurity-removed sweet potato residue powder.

[0071] (2) Extraction: The sweet potato residue powder obtained in step (1) after impurity removal was mixed with a hydrochloric acid solution having a pH value of 3.0 at a solid-liquid ratio of 1 g:25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant was collected; hydrochloric acid solution having a pH value of 3.0 was added to the precipitate at a solid-liquid ratio of 1 g:25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant was collected; the supernatants obtained from the two extractions were combined, the pH value of the supernatant was adjusted to 7.0 using a 1.0 M NaOH solution, and then concentrated at 60° C. to 1 / 5 of the original volume to obtain a concentrated solution.

[0072] (3) Enzymatic hydrolysis: adding α-amylase and saccharifying enzyme to the concentrated solution obtained in step (2), incubating at 60° C. for 2 h, and boiling to inactivate the enzymes to obtain an enzymatic hydrolyzate; the amount of α-amylase and saccharifying enzyme added is 0.5% of the weight of the concentrated solution.

[0073] (4) Alcohol precipitation: The enzymatic hydrolyzate obtained in step (3) is cooled to room temperature, and then centrifuged at 5000 rpm for 20 min. The supernatant is collected, and anhydrous ethanol twice the volume of the supernatant is added. The mixture is allowed to stand at 4°C for 12 h, and the precipitate is collected by centrifugation to obtain crude pectin.

[0074] (5) The crude pectin obtained in step (4) was dissolved in water again, dialyzed in a dialysis bag with a molecular weight cutoff of 5000 Da for 72 hours, and freeze-dried to obtain preliminarily purified sweet potato pectin.

[0075] Comparative Example 3:

[0076] This embodiment provides a method for preparing sweet potato pectin using a chelating agent, which is as follows:

[0077] (1) Impurity removal: The dried sweet potato residue is crushed and passed through an 80-mesh sieve to obtain sweet potato residue powder, the sweet potato residue powder is mixed with anhydrous ethanol at a solid-liquid ratio of 1 g:25 mL, and the mixture is shaken for 1 h and then filtered to remove soluble substances in the sweet potato residue powder, such as small molecular sugars and phenolic acids, and then the obtained filter residue is dried to obtain the impurity-removed sweet potato residue powder.

[0078] (2) Extraction: The sweet potato residue powder after impurity removal obtained in step (1) is mixed with EDTA solution with a mass fraction of 1% at a solid-liquid ratio of 1 g: 25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant is collected; EDTA solution with a mass fraction of 1% is added to the precipitate at a solid-liquid ratio of 1 g: 25 mL, extracted at 80° C. for 90 min, centrifuged after cooling, and the supernatant is collected; the supernatants obtained from the two extractions are combined, the pH of the supernatant is adjusted to 7.0 using 1.0 M HCl solution and 1.0 M NaOH solution, and then concentrated at 60° C. to 1 / 5 of the original volume to obtain a concentrated solution.

[0079] (3) Enzymatic hydrolysis: adding α-amylase and saccharifying enzyme to the concentrated solution obtained in step (2), incubating at 60° C. for 2 h, and boiling to inactivate the enzymes to obtain an enzymatic hydrolyzate; the amount of α-amylase and saccharifying enzyme added is 0.5% of the weight of the concentrated solution.

[0080] (4) Alcohol precipitation: The enzymatic hydrolyzate obtained in step (3) is cooled to room temperature, and then centrifuged at 5000 rpm for 20 min. The supernatant is collected, and anhydrous ethanol twice the volume of the supernatant is added. The mixture is allowed to stand at 4°C for 12 h, and the precipitate is collected by centrifugation to obtain crude pectin.

[0081] (5) The crude pectin obtained in step (4) was dissolved in water again, dialyzed in a dialysis bag with a molecular weight cutoff of 5000 Da for 72 hours, and freeze-dried to obtain preliminarily purified sweet potato pectin.

[0082] The present invention adopts high performance gel permeation chromatography to detect the molecular weight of HWSP-1, HWSP-2 and HWSP-3 prepared in Examples 1-3, respectively. The specific method is as follows: the determination is carried out using a high performance gel permeation chromatography (HPGPC) system equipped with an RI-10A differential refractive index detector, using a series column BRT105-104-102, an isocratic flow rate of 0.05M sodium chloride of 0.7mL / min, the column temperature is set to 40°C, and the injection volume is 25ml. The molecular weight detection result of HWSP-1 is as follows: Figure 1 As shown, the molecular weight detection results of HWSP-1, HWSP-2 and HWSP-3 show that the molecular weight of the pectin prepared by the present invention is 58.41 KDa to 62.98 KDa.

[0083] The present invention also tested the monosaccharide composition of HWSP-1, HWSP-2 and HWSP-3 prepared in Examples 1-3. The monosaccharide composition test result of HWSP-1 is shown in FIG. Figure 2 The monosaccharide composition test results of HWSP-1, HWSP-2 and HWSP-3 show that the main monosaccharides in the pectin prepared by the present invention are galacturonic acid (41.50% to 43.56%), galactose (33.62% to 34.78%), rhamnose (10.49% to 11.98%) and arabinose (8.58% to 9.56%), in addition, it also contains a small amount of glucose (6.12 to 6.56), glucuronic acid (1.43 to 1.56), xylose (0.75 to 0.89) and mannose (0.45 to 0.53).

[0084] In addition, the present invention infers the structural characteristics of the pectin prepared by the present invention by detecting the infrared spectra, one-dimensional nuclear magnetic resonance hydrogen spectra, one-dimensional nuclear magnetic resonance carbon spectra, DEPT 135 spectra, COSY spectra, HMBC spectra, HSQC spectra and NOESY spectra of HWSP-1, HWSP-2 and HWSP-3 prepared in Examples 1 to 3. Among them, the infrared spectrum, one-dimensional nuclear magnetic resonance hydrogen spectrum, one-dimensional nuclear magnetic resonance carbon spectrum, DEPT 135 spectrum, COSY spectrum, HMBC spectrum, HSQC spectrum and NOESY spectrum of HWSP-1 are as follows: Figure 3 , Figure 4 , Figure 5 , Figure 6 , Figure 7 , Figure 8 , Fig. 9 and Fig.10According to the above spectra of HWSP-1, HWSP-2 and HWSP-3, the structural characteristics of the pectin prepared by the present invention are analyzed as follows: it mainly contains 13 glycosidic bonds, namely Araf-(1→,→2)-Rhap-(1→,Glcp-(1→,→5)-Araf-(1→,Galp-(1→,→4)-Galp-(1→,→4)-Glcp-(1→,→6-Glcp-(1→,→6)-Galp-(1→,→3,4)-Galp-(1→,→2,4)-Galp-(1→,→4,6)-Galp-(1→,→3,6)-Gal p-(1→, the molar ratio of 13 glycosidic bonds is (0.069-0.071): (0.012-0.014): (0.017-0.018): (0.051-0.055): (0.104-0.109): (0.524-0.528): (0.065-0.068): (0031-0.037): (0.011-0.015): (0.040-0.046): (0.022-0.025): (0.013-0.015): (0.011-0.014). The main glycosidic bonds are attributed to →4)-Galp-(1→ (52.4%), which indicates that the main chain of the pectin prepared by the present invention may be composed of 1,4-Galp repeating units. The presence of trisubstituted →3,4)-Galp-(1→, →2,4)-Garp-(1)→, →4,6)-Galp-1→, and →3,6)-Garp-1→ indicates that the pectin prepared by the present invention is a heteropolysaccharide with a branched structure. The analyzed primary structure is: pectin has an RG-I domain, and its main chain is →4)-α-D-GalpA(6-OCH3)-(1→ and →2)-α-L-Rap-(1→, →4)-β-D-Galp-(1→ and →5)-α-L-Araf-(1→ respectively bonded to the C-3 position of the main chain →4)-α-D-GalpA(6-OCH3)-(1→ (see Fig.11 ).

[0085] The present invention uses high performance gel permeation chromatography to detect the purity of HWSP-1, HWSP-2 and HWSP-3 prepared in Examples 1-3, and the results show that HWSP-1, HWSP-2 and HWSP-3 are uniform components with a purity of more than 95%.

[0086] The present invention also tests the monosaccharide composition of the pectin prepared by Example 1 and Comparative Examples 1-3, and the test results are shown in Table 1. As can be seen from Table 1, the pectin extracted by the water extraction method (Example 1) is rich in galactose (content is 33.62mol%), which is significantly higher than the other three methods (the galactose content in the pectin obtained by alkali extraction pectin, acid extraction pectin and chelating agent extraction pectin is 26.66mol%, 22.62mol% and 25.34mol% respectively) (as shown in Table 1). According to existing literature records, the content of galactose in pectin polysaccharides is positively correlated with its anti-inflammatory activity, so it can be explained that the sweet potato pectin of the present invention has a stronger anti-inflammatory activity than the sweet potato pectin extracted by other methods.

[0087] Table 1 Monosaccharide composition of pectin prepared in Example 1 and Comparative Examples 1-3 (unit: mol%)

[0088]

[0089]

[0090] Note: The results are expressed as the mean (± standard error) of three replicates (n=3); RG-I% = 2× rhamnose + galactose + arabinose

[0091] In addition, the present invention also verifies the anti-inflammatory activity of HWSP-1 prepared in Example 1 in vitro and in vivo.

[0092] (I) The in vitro anti-inflammatory activity test of HWSP-1 is as follows:

[0093] 1. Detection of the effect of HWSP-1 on the survival rate of iBMDM cells

[0094] (1) iBMDM cell culture

[0095] iBMDM cells were purchased from the cell bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in DMEM medium supplemented with 10% (v / v) FBS, 100 U / mL penicillin, and 100 U / mL streptomycin. iBMDM cells were cultured in a humidified incubator at 37°C with 5% CO2.

[0096] (2) Cell survival rate detection

[0097] The CCK8 assay was used to study the effect of HWSP-1 on the survival rate of iBMDM macrophages. The specific method was as follows: iBMDM cells were plated in 96-well plates at 1×10 5Cells were cultured overnight at a concentration of 10 cells / mL. Different concentrations of HWSP-1 were added to a 96-well plate and cultured for 48 hours. 10 μL of CCK8 was added to dissolve the stained formazan for 2.5 hours, and the absorbance of the reaction solution at 450 nm was measured using a microplate reader. The results are shown in Fig.12 As shown in A, after the cells were treated with different concentrations of HWSP-1 at 0.35, 0.70, 1.75, 3.50 and 7.00 mmol / L, the survival rates of iBMDM cells were all higher than 91%, indicating that HWSP-1 at a concentration below 7 mmol / L was not toxic to iBMDM cells.

[0098] 2. qPCR detection of the effect of HWSP-1 on the mRNA expression level of pro-inflammatory factors in LPS-induced inflammatory cells

[0099] The present invention uses lipopolysaccharide (LPS) to induce inflammatory cells, and then uses qPCR to detect the effect of adding different concentrations of HWSP-1 on the expression level of intracellular pro-inflammatory factor mRNA. The specific method is as follows: iBMDM macrophages are cultured at 1.0×10 6 Cells were seeded at a density of 10 cells / well in a 6-well plate, and 1 μg / mL LPS was added to stimulate the cells. The treatment group (LPS+1, 1 represents HWSP-1) was treated with HWSP-1 at concentrations of 2, 4, and 8 mM at the same time as LPS was added; the positive drug group (LPS+DEX) was treated with dexamethasone (DEX) at a concentration of 10 μM at the same time as LPS was added; the control group (ctrl) did not add any reagents; the treatment with only LPS was the model group (LPS). After 24 hours of treatment, total cell RNA was extracted using Trizol reagent and PrimeScript was used to analyze the RNA. TM RT kit to reverse transcribe RNA, using TB Premix Ex Taq TM II Real-time qPCR was performed on the CFX96 Touch real-time qPCR detection system to detect the mRNA expression levels of three pro-inflammatory factors, IL-1β, IL-6, and TNF-α. Fig.12 B in Fig.12 C and Fig.12 As shown in D, LPS significantly increased the expression of IL-1β, IL-6, and TNF-α related genes in iBMDM cells compared with the control group (P<0.001). Compared with the LPS group, HWSP-1 reduced the mRNA level of IL-1β in a dose-dependent manner ( Fig.12 B). HWSP-1 also has a significant inhibitory effect on IL-6 mRNA expression ( Fig.12C). When the concentrations of HWSP-1 were 2, 4, and 8 mmol / L, the relative mRNA expression of IL-6 was 135.33, 107.66, and 45.43, respectively, which was significantly lower than that of the LPS group. Similarly, HWSP-1 showed a significant inhibitory effect on the TNF-α gene (P<0.01) ( Fig.12 Therefore, the above results further indicate that HWSP-1 has a certain anti-inflammatory effect on LPS-induced macrophages by inhibiting the mRNA expression of pro-inflammatory cytokines.

[0100] 3. Western blot (WB) analysis of the anti-inflammatory mechanism of HWSP-1

[0101] The present invention used lipopolysaccharide (LPS) to induce inflammatory cells, and then studied the possible mechanism of HWSP-1 anti-inflammatory effect by WB analysis. The specific method is as follows: iBMDM macrophages were cultured at 1.0×10 6 The cells were seeded at a density of 10 cells / well in a 6-well plate, and 1 μg / mL LPS was added to stimulate the cells. The treatment group (LPS+1, 1 represents HWSP-1) was treated with HWSP-1 at concentrations of 2, 4 and 8 mM at the same time as LPS was added; the positive drug group (LPS+DEX) was treated with dexamethasone (DEX) at a concentration of 10 μM at the same time as LPS was added; the control group (ctrl) did not add any reagents; the treatment with only LPS added was the model group (LPS). After 24 hours of treatment, macrophages from each group were collected, washed twice with cold PBS, and lysed in RIPA buffer. Then, the cell lysate was centrifuged to extract cell proteins. After cell lysis, the protein content in the supernatant was determined by the BCA method, and SDS protein electrophoresis buffer was added to the sample, which was then refrigerated at -20°C. The obtained data were quantified using Image J software. The results are shown in Fig.12 As shown in Figure E, western blot analysis results showed that compared with the control group, the expression of TLR4 and p-NF-κB in LPS-induced cells was upregulated, and the expression of NF-κB was significantly downregulated. Compared with the cells in the LPS group, the expression of TLR4 and p-NF-κB in the LPS+DEX group and the HWSP-1 treatment group decreased. The above results indicate that HWSP-1 can inhibit the TLR4 / NF-κB signaling pathway. In addition, compared with the control group, the expression of NLRP3, cleaved caspase-1, cleaved IL-1β and cleaved GSDMD in the LPS group cells was significantly increased. All HWSP-1 treatments can downregulate the expression of NLRP3, cleaved caspase-1, cleaved IL-1β and cleaved GSDMD (see Fig.12 E and Fig.12G). The results demonstrated that HWSP-1 inhibited the TLR4 / NF-κB signaling pathway and NLRP3 inflammasome.

[0102] 4. Immunofluorescence

[0103] iBMDM macrophages were cultured at 1.0×10 6 Cells were seeded at a density of 10 cells / well in a 6-well plate, and 1 μg / mL LPS was added to stimulate the cells. The treatment group (LPS+1, 1 represents HWSP-1) was treated with 8 mM HWSP-1 at the same time as LPS was added; the positive drug group (LPS+DEX) was treated with 10 μM dexamethasone (DEX) at the same time as LPS was added; the control group (ctrl) did not add any reagents; the treatment with only LPS was the model group (LPS). After 24 hours of treatment, immunofluorescence analysis was performed: the cells were fixed, the membranes were ruptured, and blocked with 3% bovine serum albumin for 30 minutes. Subsequently, the cells were immersed in NF-κB p65 (1:100) antibody for 12 hours in a refrigerator at 4°C. The cells were washed three times with PBS to remove residual antibodies and incubated with secondary antibodies for 1 hour at room temperature. After washing the cells with PBS, the nuclei were stained with DAPI for 10 minutes and observed under a fluorescence microscope. The results showed that NF-κB p65 translocated to the nucleus in LPS-treated macrophages, which was also assessed by immunofluorescence. Increased translocation was observed in the LPS group relative to the control group, but this was significantly reduced by HWSP-1 treatment ( Fig.12 F).

[0104] (II) The in vivo anti-inflammatory activity test of HWSP-1 is as follows:

[0105] 1. Animals and experimental design

[0106] SPF female C57BL / 6J mice, 6 weeks old (18–20 g), were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. (Xuzhou, China). All mice were housed under controlled conditions with a temperature of 22 °C, a relative humidity of 55%, and a 12-h light-dark cycle, and had ad libitum access to standard chow and water.

[0107] The mice were randomly divided into six groups, with eight mice in each group. The following groups were established: control group, model group (LPS, 10 mg / kg), positive drug group (LPS+DEX, DEX concentration was 10 mg / kg) and HWSP-1 treatment group (LPS+1, 1 represents HWSP-1) (the concentrations of HWSP-1 were 100, 200 and 400 mg / kg, respectively). Except for the control group, the rest were established by intratracheal instillation of LPS. 24 hours after LPS treatment, the normal control group, positive drug group and HWSP-1 group were nebulized three times with sterile saline, DEX and corresponding doses of HWSP-1, respectively. The mice were killed 72 hours after LPS treatment, and the lung tissues were collected and stored at -80°C for further study. The animal experiment flow chart is shown below. Fig.13 As shown in A.

[0108] 2. Lung Histological Evaluation

[0109] Samples obtained from the right middle lobe of the lung were fixed in 4% paraformaldehyde for >48 h, dehydrated with graded ethanol, and embedded in paraffin. Hematoxylin and eosin-stained paraffin sections were used to assess gross morphology, lung injury, and inflammation. Images were captured using a Nikon Eclipse TS100 microscope and analyzed using a Nikon Ds-U3 image analysis system. Fig.13 As shown in B, the results demonstrated that HWSP-1 and dexamethasone significantly inhibited the pathological responses of acute lung injury (ALI) mice, including pulmonary edema, inflammatory infiltration, and necrosis.

[0110] 3. Determination of plasma cytokines

[0111] Cytokines IL-1β, TNF-α, and IL-6 in serum were detected using ELISA kits (Abcam, Shanghai, China). The colorimetric reaction was read at 540 nm using a microplate reader. Fig.13 C, the results showed that the expression of IL-1β, IL-6 and TNF-α in the LPS group mice was 40.32±3.40ng / mL, 40.40±2.25ng / mL and 26.70±1.71ng / mL, respectively, which was significantly increased compared with the control group (5.10±0.87ng / mL, 5.19±1.02ng / mL, 6.06±0.43ng / mL). The expression of IL-1β, IL-6 and TNF-α in the HWSP-1 treatment group was lower than that in the LPS group. It can be confirmed that HWSP-1 inhibited LPS-induced acute lung injury in mice by reducing the levels of inflammatory cytokines.

[0112] In summary, it can be seen from the in vitro and in vivo anti-inflammatory activity experiments that HWSP-1 effectively attenuated LPS-induced acute lung injury and reduced the expression levels of IL-6, IL-1β and TNF-α in macrophages. HWSP-1 inhibits the TLR4 / NF-κB signaling pathway and NLRP3 inflammasome. Therefore, the RG-I type sweet potato residue pectin extracted by the invention can be used as a functional food raw material in health foods and functional foods.

[0113] Although the present invention has been disclosed as above in the form of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. An RG-I sweet potato pectin with anti-inflammatory activity, It is characterized in that The molecular weight of the RG-I sweet potato pectin is 58.41-62.98 KDa; it mainly contains the following monosaccharides: galacturonic acid, galactose, rhamnose, arabinose, glucose, glucuronic acid, xylose and mannose, and the molar ratio of galacturonic acid, galactose, rhamnose, arabinose, glucose, glucuronic acid, xylose and mannose is: (41.50-43.56): (33.62-34.78) (10.49-11.98): (8.58-9.56): (6.12-6.56): (1.43-1.56): (0.75-0.89): (0.45-0.53); the RG-I sweet potato pectin has an RG-I domain, and its main chain structure is as follows: Wherein, m+n=8-9, m and n are both integers and not 0.

2. The method for preparing the RG-I sweet potato pectin according to claim 1, It is characterized in that The steps include: S1, impurity removal: mixing the sweet potato residue powder with anhydrous ethanol, shaking, filtering to remove soluble substances in the sweet potato residue powder, and drying the obtained filter residue to obtain the impurity-removed sweet potato residue powder; S2, extraction: the sweet potato residue powder after impurity removal is mixed with water according to the solid-liquid ratio of 1g:25mL-1g:45mL, extracted at 80-100°C for 60-90min, centrifuged after cooling, the supernatant is collected, the precipitate is extracted again by the same method, the supernatants obtained by the two extractions are combined, and concentrated to 1 / 5-1 / 4 of the original volume at 55°C-60°C to obtain a concentrated solution; S3, enzymolysis: α-amylase and saccharifying enzyme are added to the concentrated solution obtained in S2, incubated at 60°C for 1-2h, and the enzymolysis solution is obtained after the enzyme is inactivated; S4, alcohol precipitation: centrifuge the enzymatic hydrolysate obtained in S3, collect the supernatant, add 2 times the volume of anhydrous ethanol of the supernatant, let stand at 4°C for 12-24h, and collect the precipitate by centrifugation to obtain crude pectin; S5. The crude pectin obtained in S4 was dissolved in water again, dialyzed in a dialysis bag for 72 hours, and freeze-dried to obtain RG-I sweet potato pectin.

3. The preparation method according to claim 2, It is characterized in that S1 The sweet potato residue powder is obtained by crushing the dried sweet potato residue and then passing it through an 80-mesh sieve.

4. The preparation method according to claim 2, It is characterized in that S1: The solid-liquid ratio of the sweet potato residue powder to anhydrous ethanol is 1g:20mL-1g:30mL, and the shaking time is 1-2h.

5. The preparation method according to claim 2, It is characterized in that The added amount of α-amylase and saccharifying enzyme in S3 is 0.5% of the weight of the concentrate.

6. The preparation method according to claim 2, It is characterized in that The enzyme inactivation in S3 is boiling.

7. The preparation method according to claim 2, It is characterized in that The centrifugation in S4 is performed at a rotation speed of 5000 rpm for 15-25 min.

8. The preparation method according to claim 2, It is characterized in that The molecular weight cut-off of the dialysis bag described in S5 is 5000Da.

9. Use of the RG-I sweet potato pectin according to claim 1 in the preparation of a drug with anti-inflammatory activity.

10. Use of the RG-I sweet potato pectin according to claim 1 in preparing food.

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