Pomegranate seed pectic polysaccharide as well as preparation method and application thereof in immunoregulation

Abstract

The invention relates to the technical field of biology, in particular to pomegranate seed pectic polysaccharide as well as a preparation method and application thereof in immunoregulation, and provides novel pomegranate seed pectic polysaccharide (PGSP2) and application thereof in immunoregulation. The method comprises the following steps: extracting the waste after pomegranate seed oil extraction by using pectinase-pectin methylesterase, precipitating polysaccharide by using ethanol, removing protein and nucleic acid impurities by using a trichloroacetic acid solution, primarily separating by using DEAE-650C anion exchange resin, and separating and purifying again by using a Sephacryl S-200 gel column to obtain a uniform polysaccharide component named as PGSP2, which can regulate the immune system and improve the immunity of a human body. The capability of generating immune factors in organisms is obviously improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to a pomegranate seed pectin polysaccharide, a preparation method thereof, and an application thereof in immunomodulation. Background Art

[0002] After pomegranate seeds are pressed for oil, they are often discarded as waste, but they still contain a large number of bioactive components, such as pectin, polyphenols, and peptide substances. Pectin has a wide range of applications in the medical field, including various functions such as antibacterial, hemostatic, detumescent, detoxifying, antidiarrheal, lipid-lowering, and anti-radiation. Pectin can be used alone or in combination with other components to prepare pharmaceutical forms such as ointments, films, suppositories, and capsules. Pectin has a good effect on maintaining normal blood cholesterol levels, and at the same time can prevent cation poisoning and effectively remove harmful substances such as lead and mercury in the stomach, intestines, and respiratory tract.

[0003] Currently, industrial production of pectin usually uses orange peels and apple pomace as raw materials. However, factors such as climate and plant diseases will affect the yield and quality of pectin, resulting in instability of the yield. The molecular structure of pectin is diverse and complex, and it is one of the polysaccharides with a very complex structure in nature. The research on the structural characterization and function of pomegranate seed pectin polysaccharide is relatively lacking. Therefore, how to extract pectin with functional properties from pomegranate seeds has become a technical problem that needs to be broken through. Summary of the Invention

[0004] Aiming at the above defects, the purpose of the present invention is to provide a novel pectin polysaccharide extracted from pomegranate seeds and a preparation method thereof, which can extract a non-toxic and harmless pectin polysaccharide that can regulate the immune system and significantly improve the ability of organisms to produce immune factors from the pomegranate waste after pomegranate seeds are pressed for oil.

[0005] In order to achieve the above purpose, the first technical solution of the present invention provides a pomegranate seed pectin polysaccharide, and the polysaccharide includes the following structural fragments:

[0006] Branched structure:

[0007]

[0008] Among them, the monosaccharide composition ratio is galacturonic acid: rhamnose: arabinose: galactose: glucose: glucuronic acid: fucose = 81.23%: 3.71%: 5.47%: 4.83%: 1.50%: 0.87%: 0.24%, and its weight average molecular weight is 61.35KDa.

[0009] The second technical solution of the present invention provides a preparation method of the above pomegranate seed pectin polysaccharide, including the following steps:

[0010] The waste pomegranate seeds after oil extraction are used to obtain a crude extract by using pectinase-pectin methylesterase;

[0011] The crude extract is purified to obtain a refined pectin polysaccharide mixture;

[0012] The pectin polysaccharide mixture is separated and purified by DEAE-650C cellulose anion exchange resin, detected by the sulfuric acid-phenol method, an elution curve is plotted, each component is collected according to the peak, and then each component is separated and purified again by a Sephacryl S-200 gel column, and the single-peak component is collected to obtain pomegranate seed pectin polysaccharide.

[0013] Furthermore, the mass ratio of the pectinase-pectin methylesterase is 1:1.

[0014] Furthermore, the purification includes the following steps:

[0015] The supernatant of the crude extract separated by centrifugation is precipitated with absolute ethanol to obtain a flocculent precipitate;

[0016] The flocculent precipitate is dissolved in pure water, centrifuged to separate and the supernatant is taken, and then impurities are removed by the trichloroacetic acid method, and a refined pectin polysaccharide mixture is obtained through dialysis.

[0017] Furthermore, the concentration of the trichloroacetic acid is 2%-5%.

[0018] In addition, the pomegranate seed pectin polysaccharide prepared according to the above preparation method has the structural fragment and monosaccharide composition ratio as shown in Claim 1.

[0019] The third technical solution of the present invention discloses the application of the pomegranate seed pectin polysaccharide in immune regulation.

[0020] The beneficial effects of the present invention are as follows:

[0021] The present invention provides a novel pomegranate seed pectin polysaccharide (PGSP2) and its application in immune regulation. By extracting the waste after pomegranate seeds are pressed for oil with pectinase-pectin methylesterase, precipitating polysaccharides with ethanol, removing protein and nucleic acid impurities with trichloroacetic acid solution, initially separating with DEAE-650C anion exchange resin, and separating and purifying again with a Sephacryl S-200 gel column, a homogeneous polysaccharide component is obtained, named PGSP2, which can regulate the immune system and significantly improve the ability of organisms to produce immune factors. Description of the Drawings

[0022] Figure 1 It is the ultraviolet spectrum diagram of the pomegranate seed pectin polysaccharide prepared in Preparation Example 1;

[0023] Figure 2Gel filtration chromatogram of the obtained pomegranate seed pectin polysaccharide (PGSP2);

[0024] Figure 3 Purity diagram of the obtained pomegranate seed pectin polysaccharide (PGSP2) by high performance liquid chromatography;

[0025] Figure 4 Monosaccharide composition chromatogram of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0026] Figure 5 Infrared spectrum diagram of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0027] Figure 6 1H NMR of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0028] Figure 7 13C NMR spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0029] Figure 8 H-H COSY spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0030] Figure 9 NOESY spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0031] Figure 10 HMBC spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0032] Figure 11 HSQC spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1;

[0033] Figure 12 Immune enhancing factor (NO) spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1.

[0034] Figure 13 Immune enhancing factor (IL-1β) spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1.

[0035] Figure 14 Immune enhancing factor (TNF-α) spectrum of the pomegranate seed pectin polysaccharide (PGSP2) obtained in Preparation Example 1. Detailed implementation manners

[0036] The specific embodiments of the present invention will be described below to facilitate the understanding of those skilled in the art of the present technology. It should be clear that the present invention is not limited to the scope of the specific embodiments. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention defined and determined by the appended claims, these changes are obvious, and all inventions made using the inventive concept of the present invention are within the scope of protection.

[0037] The first embodiment of the present application provides a pomegranate seed pectin polysaccharide (named PGSP2), and the polysaccharide includes the following structural fragments:

[0038]

[0039] Branched structure:

[0040]

[0041] Among them, the monosaccharide composition ratio is galacturonic acid: rhamnose: arabinose: galactose: glucose: glucuronic acid: fucose = 81.23%: 3.71%: 5.47%: 4.83%: 1.50%: 0.87%: 0.24%, and its weight-average molecular weight is 61.35KDa.

[0042] The second embodiment of the present application provides a preparation method of the above-mentioned pomegranate seed pectin polysaccharide PGSP2, including the following steps:

[0043] Using the pomegranate seed waste after oil extraction to obtain a crude extract by using pectinase-pectin methylesterase;

[0044] Purifying the crude extract to obtain a refined pectin polysaccharide mixture;

[0045] Separating and purifying the pectin polysaccharide mixture with DEAE-650C cellulose anion exchange resin, detecting with the sulfuric acid-phenol method, plotting an elution curve, collecting each component according to the peak, and then separating and purifying each component again with a Sephacryl S-200 gel column, and collecting the single-peak component to obtain the pomegranate seed pectin polysaccharide.

[0046] In this embodiment, the purpose of using the pectinase-pectin methylesterase complex enzyme is to hydrolyze and separate the pectin in the pomegranate seed cell wall as much as possible, because galacturonan is the main component in pectin, accounting for about 60-65%, and its carboxyl group is usually esterified.

[0047] In this embodiment, the refined pectin polysaccharide mixture obtained by purifying the crude extract contains various polysaccharides, such as acidic polysaccharides, neutral polysaccharide components, etc. Therefore, through the sulfuric acid-phenol method for detection, an elution curve is plotted, and each component is collected according to the peak to separate the polysaccharide mixture and obtain a single polysaccharide component; each component is then further separated using a Sephacryl S-200 gel column, the elution peaks are detected by the sulfuric acid-phenol method, the components with a single symmetric peak are collected, and the purity is identified. The homogeneous fraction with a relatively high purity is selected and named pomegranate seed pectin polysaccharide (PGSP2).

[0048] In some specific embodiments, the mass ratio of pectinase to pectin methylesterase is 1:1.

[0049] Further, the purification includes the following steps:

[0050] The supernatant of the crude extract separated by centrifugation is precipitated with absolute ethanol to obtain a flocculent precipitate;

[0051] The flocculent precipitate is dissolved in pure water, centrifuged to separate the supernatant, and then the impurities are removed by the trichloroacetic acid method. After dialysis, a refined pectin polysaccharide mixture is obtained. The concentration of the trichloroacetic acid is 2%-5%. The above purification steps are conventional techniques in the art, and the settings of various technical parameters do not exceed the scope of knowledge of those skilled in the art. This application only provides a preferred Example 1.

[0052] Example 1 Preparation of Pomegranate Seed Pectin Polysaccharide

[0053] S1. The waste after pomegranate seed oil extraction is dried in an oven, crushed, and passed through a 60-mesh sieve. After distillation and reflux with a mixed solvent of petroleum ether-ether (3:1) three times, the organic solvent is evaporated. 500 g of pomegranate seed powder is weighed, and PBS solution with a pH value of 7.5 is added according to a solid-liquid ratio of 1:5. After preheating in a 30°C water bath for 30 minutes, 5000 U of pectinase-pectin methylesterase complex enzyme is added, stirred evenly, and after enzymatic hydrolysis for 1 hour, ultrasonic (400 w) is used for auxiliary extraction for 20 minutes;

[0054] S2. The extract is centrifuged, and then 4 volumes of absolute ethanol are added to the supernatant obtained by centrifugation to precipitate the polysaccharide, and the polysaccharide precipitate is collected by centrifugation;

[0055] S3. The polysaccharide precipitate is redissolved in pure water, the precipitate is removed by centrifugation, and the protein impurities are removed with a trichloroacetic acid solution (2-5%). Then, vacuum freeze-drying is carried out to obtain 25.6 g of a polysaccharide sample;

[0056] S4. Weigh 1 g of the polysaccharide sample and dissolve it in 100 ml of distilled water to make a 1 g / 100 ml solution. Then, directly load the solution onto a DEAE-650C anion exchange resin for enrichment and separation. First, wash the unadsorbed polysaccharide sample with 200 mL of distilled water, and then perform gradient elution with a 0 - 0.5 mol / L NaCl solution. Collect the elution fractions at 10 ml per tube to obtain the elution fractions of pomegranate seed pectin polysaccharide.

[0057] S5. Re-separate the above elution fractions using a Sephacryl S-200 gel column. Detect the elution peaks by the phenol-sulfuric acid method, collect the components of a single symmetric peak, and perform purity identification to obtain a homogeneous component, named pomegranate seed pectin polysaccharide (PGSP2).

[0058] S6. Dialyze the pomegranate seed pectin polysaccharide (named PGSP2), and store it in a freeze-dried state.

[0059] Experimental Example 1 Structure Identification of Pomegranate Seed Pectin Polysaccharide (PGSP2)

[0060] The pomegranate seed pectin polysaccharide PGSP2 obtained in Example 1 was dissolved in water to obtain a pomegranate seed pectin polysaccharide solution, which was analyzed by an ultraviolet spectrophotometer from 200 - 600 nm. The results are as Figure 1 shown. There is no ultraviolet absorption at 254 nm and 280 nm, indicating that there are no protein and nucleic acid impurities in the polysaccharide. First, it was separated by DEAE-650C anion exchange resin, and PGSP2 was collected. The pomegranate seed pectin polysaccharide (PGSP2) was separated by a Sephacryl S-200 gel column, and the elution curve is as Figure 2 shown. Collect the peak top components for subsequent structure characterization.

[0061] The pomegranate seed pectin polysaccharide (PGSP2) was analyzed by gel permeation chromatography using a series of Ohpak SB-805HQ (300×8 mm), Ohpak SB-804HQ (300×8 mm) and Ohpak SB-803HQ (300×8 mm) columns, as Figure 3 shown. The chromatographic conditions were as follows: column temperature 45°C, injection volume 100 μL, mobile phase A (0.02% NaN3, 0.1 M NaNO3), flow rate 0.4 mL / min, elution gradient: isocratic for 100 min. Chromatograph: The liquid phase system was U3000 (Thermo, USA), and the differential refractive index detector was Optilab T-rEX (Wyatt technology, CA, USA). The relative molecular mass of the pomegranate seed pectin polysaccharide (PGSP2) was determined by high performance gel filtration chromatography (HPGFC) to be 61.35 KDa.

[0062] Take about 5 mg of pomegranate seed pectin polysaccharide (PGSP2) sample, dissolve it in 3 mol / L trifluoroacetic acid solution, seal the tube with nitrogen, hydrolyze it at 100 °C for 2 hours. After the hydrolysis solution is evaporated to dryness, it is analyzed by ion chromatography. The results of monosaccharide composition analysis are as Figure 4 shown. The monosaccharide composition ratio is galacturonic acid: rhamnose: arabinose: galactose: glucose: glucuronic acid: fucose = 81.23%: 3.71%: 5.47%: 4.83%: 1.50%: 0.87%: 0.24%. Pectin PGSP2 contains a large amount of galacturonic acid, followed by arabinose, rhamnose, and galactose, and contains trace amounts of fucose, xylose, and glucuronic acid.

[0063] Take about 2 mg of pomegranate seed pectin polysaccharide (PGSP2), and use the potassium bromide tablet pressing method to measure the infrared spectrum of the novel pomegranate seed pectin polysaccharide (PGSP2). The results are as Figure 5 , 3408 cm -1 shows a strong and broad peak under the -OH stretching vibration, indicating the presence of intermolecular hydrogen bonds. 2925 cm-1 is at the -CH stretching vibration, and the absorption bands at 1325 cm-1 and 1233 cm-1 are the bending vibration absorption peaks of C-H. The absorption peaks at 764 cm-1 and 919 cm-1 represent the symmetric stretching and asymmetric stretching vibrations of D-pyranose respectively. An absorption peak is found near 1742 cm-1, indicating the presence of uronic acid in this component. There is no absorption peak at 1597 cm-1, and there is no N-H bending vibration. There are no characteristic absorption peaks of amide at 2850 cm-1, indicating that there are no nitrogen-containing impurities such as proteins. An obvious absorption peak is observed at 889 cm-1, indicating that this polysaccharide contains a β configuration, and an absorption peak appears at 834 cm-1, indicating the presence of α-pyranose in the polysaccharide. These results indicate that this polysaccharide component may be a polysaccharide containing uronic acid and has α and β configuration glycosidic bonds.

[0064] Weigh 20 mg of pomegranate seed pectin polysaccharide (PGSP2) sample, carry out reduction treatment and then methylation modification. After the modified product is completely hydrolyzed by acid, it is analyzed and detected by GC-MS. The results are shown in Table 1. Based on the matching of the mass spectrometry database, the main glycosidic bond linkages of pomegranate seed pectin polysaccharide (PGSP2) are:

[0065] →4)-GalAp-(1→,→3,4)-GalpA-(1→,→4,6)-GalpA-(1→,→2,4)-GalpA-(1→,→3,6)-GalpA-(1→, and the side chains have

[0066] →2,4)-Rhap-(1→, Araf-(1→, →5)-Araf-(1→, Galp-(1→, →4)-Glcp-(1→, →3)-Galp-(1→, →3,4)-Galp-(1→, →2,4)-Galp-(1→, →4,6)-Galp-(1→, →3,6)-Galp-(1→, Glcp-(1→ is linked to the residue of galacturonic acid.

[0067] Table 1 GC-MS analysis of methylation products

[0068]

[0069] Take 20 mg of the freeze-dried product of pomegranate seed pectin polysaccharide (PGSP2), dissolve it in 0.5 mL of D2O, centrifuge at 10000 r / min, transfer the supernatant to a nuclear magnetic resonance tube, and perform one-dimensional and two-dimensional nuclear magnetic resonance analyses on a JEOL600M nuclear magnetic resonance instrument at 25 °C. The results are as Figures 6 - 10 shown. Figure 6 Figure shows the 1H NMR spectrum of pomegranate seed pectin polysaccharide (PGSP2). The chemical shift at 5.03 ppm is attributed to the anomeric hydrogen signal of (1→4)-α-D-GalpA; 5.09 ppm is attributed to the anomeric hydrogen signal of (1→3,4)-α-D-GalpA; 4.99 ppm is attributed to the anomeric hydrogen signal of (1→4,6)-α-D-GalpA; 4.84 ppm is attributed to the anomeric hydrogen signal of T-α-D-Galp; 4.83 is attributed to (1→3,6)-α-D-GalpA;

[0070] The chemical shift at 5.18 ppm is attributed to the anomeric hydrogen signal of (1→2,4)-α-D-Rhap; 5.18 ppm is attributed to the anomeric hydrogen signal of (1→5)-α-D-Araf; 5.24 ppm is attributed to the anomeric hydrogen signal of T-α-D-Araf; 4.9 ppm is attributed to the anomeric hydrogen signal of (1→2,4)-α-D-GalpA; 4.66 ppm is attributed to the anomeric hydrogen signal of (1→3)-β-D-Galp; 4.69 ppm is attributed to the anomeric hydrogen signal of (1→6)-β-D-Galp; 4.71 ppm is attributed to the anomeric hydrogen signal of (1→4)-β-D-Glcp; the relatively strong peak at 3.74 ppm is attributed to the hydrogen signal of -OMe; 2.01 ppm is attributed to the hydrogen signal of -OAc; 1.18 ppm is the hydrogen signal of -Rhap.

[0071] Figure 7In the 13C-NMR nuclear magnetic resonance spectrum of the pectin polysaccharide, characteristic peaks of →4)-α-Galp A-(1→ can be clearly observed. Among them, the chemical shifts of 99.45 ppm, 68.68 ppm, 67.93 ppm, 77.84 ppm, and 71.32 ppm are respectively attributed to the characteristic peaks of C-1, C-2, C-3, C-4, and C-5 of →4)-α-Galp A-(1→, while 175.39 ppm is the characteristic signal peak of the carboxyl carbon (-COOH) of C-6 of α-Galp A. The C-6 carboxyl of pomegranate seed pectin polysaccharide (PGSP2) can be methyl esterified to a certain extent. Therefore, the chemical shifts of C-1 to C-6 of α-GalpA after methylation will shift. For example, the chemical shifts at 99.04 ppm, 68.61 ppm, and 70.56 ppm are respectively attributed to the characteristic peaks of C-1, C-2, and C-5 of →4)-α-Galp A-OMe-(1→, and the peak at the chemical shift of 170.8 ppm is the characteristic signal peak of the methyl ester carbon (-COOCH3) of C-6 of methyl esterified α-Galp A. The chemical shifts at 71.49 ppm and 78.3 ppm are attributed to the characteristic signal peaks of C-2 and C-4 of →3)-β-Galp-(1→; the chemical shifts at 68.77 ppm and 68.68 ppm are attributed to the characteristic signal peaks of C-2 and C-4 of →6)-β-Galp-(1→; and characteristic signal peaks of C-2 and C-4 of the terminal galactose T-Galp with chemical shifts of 71.49 ppm and 78.68 ppm are also found. There are also some characteristic signal peaks of arabinose. For example, the chemical shift at 106.02 ppm is attributed to

[0072] the anomeric carbon signal of →5)-α-Araf-(1→. The chemical shift at 52.85 ppm is the characteristic signal peak of the oxygen methyl (-OMe). The chemical shift at 20.12 ppm is the characteristic peak signal of the acetyl group (-OAc), indicating that a small amount of acetyl substitution is contained in the pomegranate seed pectin polysaccharide (PGSP2). Figure 5 the relatively weak signal at a chemical shift of 1.18 ppm in Figure 6 and the signal at a chemical shift of 16.49 ppm in

[0073] Figure 8 Indicating the COSY correlation spectrum of the pectin polysaccharide, 5.02 / 3.71 ppm is the H1 / H2 peak of →4)-α-Galp A-OMe-(1→, 5.36 / 3.57 ppm is the H1 / H2 peak of →3,4)-a-D-Rhap-(1→, 3.97 / 1.19 is

[0074] The signal cross peak of H5 / H6 of →3,4)-α-D-Rhap-(1→ or →2,4)-α-D-Rhap-(1→.

[0075] Figure 9 It indicates that the HSQC correlation spectrum of this pectic polysaccharide at 5.35 / 99.67 ppm is the H1 / C1 of →3,4)-α-D-Rhap-(1→ or →2,4)-α-D-Rhap-(1→. 5.10 / 70.54 is the H1 / C4 of GA1,4. 4.46 / 102.8 is the

[0076] H1 / C4 of →4)-β-D-Galp-(1→.

[0077] Figure 10 It indicates that in the HMBC correlation spectrum of this pectic polysaccharide, 3.75 / 170.853 is the signal of H5 / C6 of the methyl-esterified CH3 of →4)-α-Galp A-OMe-(1→ and →3,4)-α-Galp A-OMe-(1→, and 2.03 / 173.48 is the H1 / C6 of the acetylated group.

[0078] Figure 11 It indicates the NOESY correlation spectrum of this pectic polysaccharide. 5.34 / 3.58 is the

[0079] signal cross peak of H4 of →3,4)-α-D-Rhap-(1→ and H6 of →4)-α-Galp A-OMe-(1→. The rest are points on the diagonal, which do not represent the glycosidic bond type.

[0080] Combining related two-dimensional spectra such as HMBC and HSQC confirmed the assignment of related signals. Thus, the structure of this pomegranate seed pectic polysaccharide is obtained as follows:

[0081]

[0082] The branched structure is as follows:

[0083]

[0084] Experimental Example 2 Study on the Enhancement of the Immune Activity of RAW264.7 Cells by Pomegranate Seed Pectic Polysaccharide (PGSP2)

[0085] Experimental method: S1. Prepare PGSP2 solutions with concentrations of 0.05 mg / mL, 0.1 mg / mL, 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL) using a culture medium. The blank control group adds an equal amount of fresh culture medium, and the positive control group adds 0.2 mL of an LPS solution with a concentration of 0.025 mg / mL. Set 3 replicates for each condition;

[0086] S2. RAW264.7 macrophages in the logarithmic growth phase were counted using a hemocytometer to a cell concentration of 5×105 cells / mL. 0.2 mL of the cell suspension was inoculated into each well of a 96-well plate, and the plate was placed in an incubator overnight to promote cell attachment. The next day, the old medium in the wells was removed, and a mixed solution of 0.2 mL of medium and the sample was added to the wells of the experimental group.

[0087] S3. Test the results of the enhanced RAW264.7 cell analysis of immune factors of PGSP2 prepared in Example 1.

[0088] The experimental results, as Figure 12 shown, treatment of RAW264.7 cells with PGSP2 at a concentration of 0.05 mg to 1 mg / mL for 24 hours significantly enhanced NO secretion. Within this concentration range, the secretion showed an increasing trend and was significantly enhanced compared to the blank group.

[0089] At a concentration of 0.05 mg / mL, the NO level was higher than that of the blank group; after 0.5 mg / mL, the change was not obvious; the highest level was reached at 1 mg / mL, which was weaker than the LPS positive control group. (NO is a regulatory molecule of various immune cells, which can promote the activity of NK cells, activate peripheral blood monocytes, and regulate T lymphocytes. Therefore, PGSP2 can promote immune function by promoting the production of NO.)

[0090] Figure 13 As shown, treatment of RAW264.7 cells with 0.5 mg to 1 mg / mL of PGSP2 for 24 h significantly enhanced the production of IL-1β as the concentration of the drug increased. (IL-1β, as a key pro-inflammatory cytokine, is involved in various autoimmune inflammatory reactions and various cell activities, including cell proliferation, differentiation, and apoptosis. Together with IL1-α and IL18, IL-1β coordinates immune responses through multiple downstream mechanisms. PGSP2 promotes immune function by promoting the production of IL-1β.)

[0091] Figure 14 As shown, stimulation of RAW264.7 cells with PGSP2 for 24 hours significantly increased the content of TNF-α as the concentration of the drug increased. Compared with the blank control group, when the concentration was 1 mg / mL, the secretion of TNF-α reached more than 2 times. (TNF-α is mainly produced by macrophages, which can enhance the killing ability of macrophages or NK cells and stimulate the immune system to enhance the anti-infection effect. Therefore, PGSP2 can promote immunity by promoting the production of TNF-α.)

[0092] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pomegranate seed pectin polysaccharide, characterized in that, It includes the following structural fragments: Among them, the branched-chain structure is: Among them, the monosaccharide composition ratio is galacturonic acid: rhamnose: arabinose: galactose: glucose: glucuronic acid: fucose = 81.23%: 3.71%: 5.47%: 4.83%: 1.50%: 0.87%: 0.24%, and its weight-average molecular weight is 61.35 KDa.

2. A preparation method of pomegranate seed pectin polysaccharide as described in claim 1, characterized in that, It includes the following steps: Using pectinase-pectin methylesterase to extract the crude extract from the waste pomegranate seeds after oil extraction; Purifying the crude extract to obtain a refined pectin polysaccharide mixture; Separating and purifying the pectin polysaccharide mixture with DEAE-650C cellulose anion exchange resin, detecting with the sulfuric acid-phenol method, drawing an elution curve, collecting each component according to the peak, and then separating and purifying each component again with a Sephacryl S-200 gel column, collecting the single-peak component to obtain pomegranate seed pectin polysaccharide.

3. The preparation method according to claim 2, wherein The mass ratio of the pectinase-pectin methylesterase is 1:

1.

4. The preparation method according to claim 2, wherein, The purification includes the following steps: Using anhydrous ethanol to precipitate the supernatant of the centrifuged crude extract to obtain a flocculent precipitate; Dissolving the flocculent precipitate in pure water, centrifuging to separate and taking the supernatant, and then removing impurities by the trichloroacetic acid method, and obtaining a refined pectin polysaccharide mixture through dialysis.

5. The preparation method according to claim 4, characterized in that, The concentration of the trichloroacetic acid is 2%-5%.

6. The pomegranate seed pectin polysaccharide prepared by any one of the preparation methods according to claims 2-5, characterized in that, It has the structural fragment and monosaccharide composition ratio as shown in claim 1.

7. The application of the pomegranate seed pectin polysaccharide according to claim 1 or 6 in immunomodulation.

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