Lycium barbarum pectin polysaccharide as well as preparation method and application thereof

Abstract

The invention provides lycium barbarum pectin polysaccharide as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The lycium barbarum pectin polysaccharide takes Gal and Ara as main composition monosaccharides, and the core structure of the lycium barbarum pectin polysaccharide is a repeating unit of-> 6)-beta-D-Galp-(1-> 6 / 3, 6)-beta-D-Galp-(1-> and-> 4)-alpha-D-GalpA-(1-> 2 / 2, 4)-alpha-L-Rhap-(1->. According to the method disclosed by the invention, lycium barbarum L. is taken as a research material, and various analysis technical methods are comprehensively applied, so that the whole structural diversity information of the lycium barbarum pectin is obtained, and the structural characteristics of fine subfractions of the lycium barbarum L. pectin are deeply clarified. The lycium barbarum pectin polysaccharide prepared by the method has a remarkable intervention effect on intestinal inflammation, scientific data is provided for deep research and utilization of the pectin polysaccharide, and the anti-inflammatory effect and the application range of lycium barbarum pectin are known more comprehensively.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological drugs, and particularly relates to a Lycium barbarum pectic polysaccharide as well as a preparation method and application thereof. BACKGROUND

[0002] Lycium barbarum is a famous traditional Chinese medicinal material and a homology of food and medicine, and has important medicinal and nutritional values. Polysaccharide is one of the main bioactive components in Lycium barbarum, and has been confirmed to have various pharmacological activities. Existing structural studies have shown that there are abundant pectic polysaccharide components in Lycium barbarum. However, most of the existing studies on the pharmacological activities of Lycium barbarum polysaccharide take Lycium barbarum total polysaccharide as the research object. As the most widely distributed and most complex polysaccharide in the plant kingdom, pectin is an important active component of medicinal plants. The complex molecular structure endows pectin with various biological functions, and at the same time makes the structural diversity and structure-activity relationship research of pectin a difficult and frontier scientific problem in the current field of plant polysaccharide research, and Lycium barbarum polysaccharide is one of the typical examples. Lycium barbarum is a famous traditional Chinese medicinal material and a homology of food and medicine, and contains abundant pectic polysaccharide components. However, the research on the pharmacological activities of Lycium barbarum polysaccharide is still focused on the level of Lycium barbarum total polysaccharide, and the structure-activity relationship research of Lycium barbarum pectin components is not perfect. Compared with the extensive development and increasing depth of the research on the pharmacological activities of Lycium barbarum polysaccharide, the structure-activity relationship research based on the structural diversity of Lycium barbarum pectin is obviously insufficient.

[0003] The existing technology discloses that Lycium barbarum polysaccharide has an intervention effect on intestinal inflammation, but intestinal inflammations caused by different causes (such as infectious enteritis, autoimmune enteritis, etc.) have different pathogenesis and pathological characteristics, and the intervention effect of Lycium barbarum pectin may be different. In view of this, the present field urgently needs to take Lycium barbarum pectic polysaccharide as the research object, aiming to explore the structure-activity relationship between the structural diversity of Lycium barbarum pectin and its biological activity, so as to provide scientific data for the in-depth research and utilization of pectic polysaccharide, and more comprehensively understand the anti-inflammatory effect and application range of Lycium barbarum pectin. SUMMARY

[0004] The purpose of the present application is to provide a Lycium barbarum pectic polysaccharide as well as a preparation method and application thereof, which can significantly improve the intestinal inflammation of mice induced by CRS.

[0005] In order to achieve the above-mentioned purpose, the present application provides the following technical scheme:

[0006] The present application provides a Lycium barbarum pectic polysaccharide, which takes galactose and arabinose as the main monosaccharide, and the core structure is:→6)-β-D-Galp-(1→6 / 3,6)-β-D-Galp-(1→ and→4)-α-D-GalpA-(1→2 / 2,4)-α-L-Rhap-(1→ repeating unit.

[0007] Preferably, the wolfberry pectin polysaccharide includes mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose, and the molar ratio of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose is (0.3-5.5):(3.0-6.3):(0.3-9.6):(1.9-7.1):(0.9-20.6):(23.2-49.7):(33.9-45.6).

[0008] Preferably, the molecular weight (Mw) of the wolfberry pectin polysaccharide is between 3.36 kDa and 49.0 kDa.

[0009] The present invention also provides a method for preparing the aforementioned wolfberry pectin polysaccharide, the method comprising the following steps:

[0010] Total polysaccharides from Lycium barbarum were dissolved in distilled water, centrifuged, and the supernatant was collected. The supernatant was eluted using a DE-52 ion exchange chromatography column, and neutral polysaccharides were separated by elution with 400 mL of water. Elution was then carried out with 0.08 M NaCl, 0.15 M NaCl, and 0.2 M NaCl solutions. For elution with 0.08 M NaCl solution, target eluent 1 (from 11.5 to 19 min) was collected; for elution with 0.15 M NaCl solution, target eluent 2 (from 11 to 18.2 min) was collected; and for elution with 0.2 M NaCl solution, target eluent 3 (from 11.2 to 18.1 min) was collected. The elution flow rate was 10 mL / min.

[0011] The collected target eluents 1-3 were concentrated and dialyzed, then concentrated again and freeze-dried to obtain Lycium barbarum pectin polysaccharides LBPA-I, LBPA-II and LBPA-III, respectively.

[0012] Preferably, the mass-to-volume ratio of the total polysaccharides of wolfberry to distilled water is 1g:25mL.

[0013] Preferably, target eluents 1 to 3 are concentrated to 1 / 10 of their original volume; the molecular weight cutoff for dialysis is 3500 Da, and the dialysis time is 24 h.

[0014] Preferably, the total polysaccharide of wolfberry is the total polysaccharide of wolfberry extracted from wolfberry by water extraction and alcohol precipitation.

[0015] Preferably, the preparation of the total polysaccharides from wolfberry includes the following steps:

[0016] Goji berries were added to purified water and heated for 2 hours. The filtrate was then collected by filtration. The filtrate was concentrated and centrifuged to obtain the supernatant. Ethanol was added to the supernatant until the final ethanol concentration reached 80%. After standing, the mixture was centrifuged to collect the precipitate. The precipitate was washed to remove water and dried to obtain total polysaccharides from goji berries.

[0017] The present invention also provides applications of the aforementioned wolfberry pectin polysaccharide or the wolfberry pectin polysaccharide prepared by the aforementioned preparation method, wherein the applications include at least one of the following a to d;

[0018] a. The application of the aforementioned wolfberry pectin polysaccharide in the preparation of products that improve intestinal inflammation;

[0019] b. The application of the aforementioned wolfberry pectin polysaccharide in the preparation of a product that improves intestinal inflammation in mice induced by chronic restraint stress;

[0020] c. The application of the wolfberry pectin polysaccharide in the preparation of products that improve the expression of endocannabinoid system-related proteins;

[0021] d. Application of the aforementioned wolfberry pectin polysaccharide in the preparation of products that regulate intestinal flora diversity.

[0022] Preferably, the product includes pharmaceuticals.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The present invention relates to a novel homogeneous polysaccharide derived from Lycium barbarum pectin, with Gal as the main monosaccharide component. The common main chain structure is →6)-β-D-Galp-(1→6 / 3,6)-β-D-Galp-(1→), and it contains disaccharide units linked by →4)-α-D-GalpA-(1→2 / 2,4)-α-L-Rhap-(1→). Fine structural feature analysis reveals that the Lycium barbarum pectin polysaccharide contains RG-I type pectin and AG-II type pectin. This invention significantly improves CRS-induced intestinal inflammation in mice, demonstrating a significant interventional effect on intestinal inflammation. It provides scientific data for in-depth research and utilization of pectin polysaccharides, and offers a more comprehensive understanding of the anti-inflammatory effects and applicable scope of Lycium barbarum pectin. Attached Figure Description

[0025] Figure 1 The standard curves for sugar content detection in Example 1 are shown below, where (A) is the total sugar standard curve, (B) is the protein standard curve, and (C) is the uronic acid standard curve.

[0026] Figure 2 The results of ion exchange fractionation of total polysaccharides from wolfberry in Example 1 are shown.

[0027] Figure 3 This is a flowchart illustrating the preparation process of wolfberry pectin in Example 1.

[0028] Figure 4 The results of Sepharose CL-6B gel column fractionation purification in Example 1 are shown. (A) is the elution curve of LBPA-Ⅰ; (B) is the elution curve of LBPA-Ⅱ; and (C) is the elution curve of LBPA-Ⅲ.

[0029] Figure 5 The image shows the HPGPC chromatogram of the fine components of wolfberry pectin in Example 1.

[0030] Figure 6 For LBPA-Ⅰa in Example 2 1 H and 13 C-NMR spectra, where (A) represents LBPA-Ia. 1 H-NMR spectrum, (B) is LBPA-Ⅰa 13 C-NMR spectrum.

[0031] Figure 7 For LBPA-Ⅱa in Example 2 1 H and 13 C-NMR spectra, where (A) is LBPA-IIa. 1 H-NMR spectrum, (B) is LBPA-IIa 13 C-NMR spectrum.

[0032] Figure 8 For LBPA-Ⅲa in Example 2 1 H and 13 C-NMR spectra, where (A) represents LBPA-Ⅲa. 1 H-NMR spectrum, (B) is LBPA-Ⅲa 13 C-NMR spectrum.

[0033] Figure 9 The HSQC spectrum of LBPA-Ⅰa in Example 2 is shown.

[0034] Figure 10 The HMBC spectrum of LBPA-Ⅰa in Example 2 is shown.

[0035] Figure 11 The COSY spectrum of LBPA-Ⅰa in Example 2 is shown.

[0036] Figure 12 The NOESY spectrum of LBPA-Ⅰa in Example 2 is shown.

[0037] Figure 13 The HSQC spectrum of LBPA-Ⅱa in Example 2 is shown.

[0038] Figure 14The HMBC spectrum of LBPA-Ⅱa in Example 2 is shown.

[0039] Figure 15 The COSY spectrum of LBPA-Ⅱa in Example 2 is shown.

[0040] Figure 16 The NOESY spectrum of LBPA-Ⅱa in Example 2 is shown.

[0041] Figure 17 The HSQC spectrum of LBPA-Ⅲa in Example 2 is shown.

[0042] Figure 18 The HMBC spectrum of LBPA-Ⅲa in Example 2 is shown.

[0043] Figure 19 The COSY spectrum of LBPA-Ⅲa in Example 2 is shown.

[0044] Figure 20 The NOESY spectrum of LBPA-Ⅲa in Example 2 is shown.

[0045] Figure 21 For LBPA-Ⅰb in Example 2 1 H and 13 C-NMR spectra, where (A) is LBPA-Ib. 1 H-NMR spectrum, (B) is LBPA-Ib 13 C-NMR spectrum.

[0046] Figure 22 For LBPA-Ⅱb in Example 2 1 H and 13 C-NMR spectra, where (A) is LBPA-IIb. 1 H-NMR spectrum, (B) is LBPA-IIb 13 C-NMR spectrum.

[0047] Figure 23 For LBPA-Ⅲb in Example 2 1 H and 13 C-NMR spectra, where (A) represents LBPA-Ⅲb. 1 H-NMR spectrum, (B) is LBPA-Ⅲb 13 C-NMR spectrum.

[0048] Figure 24 The HSQC spectrum of LBPA-Ⅰb in Example 2 is shown.

[0049] Figure 25 The HMBC spectrum of LBPA-Ⅰb in Example 2 is shown.

[0050] Figure 26 The COSY spectrum of LBPA-Ⅰb in Example 2 is shown.

[0051] Figure 27 The NOESY spectrum of LBPA-Ⅰb in Example 2 is shown.

[0052] Figure 28 The HSQC spectrum of LBPA-Ⅱb in Example 2 is shown.

[0053] Figure 29 The HMBC spectrum of LBPA-Ⅱb in Example 2 is shown.

[0054] Figure 30 The COSY spectrum of LBPA-Ⅱb in Example 2 is shown.

[0055] Figure 31 The NOESY spectrum of LBPA-Ⅱb in Example 2 is shown.

[0056] Figure 32 The HSQC spectrum of LBPA-Ⅲb in Example 2 is shown.

[0057] Figure 33 The HMBC spectrum of LBPA-Ⅲb in Example 2 is shown.

[0058] Figure 34 The COSY spectrum of LBPA-Ⅲb in Example 2 is shown.

[0059] Figure 35 The NOESY spectrum of LBPA-Ⅲb in Example 2 is shown.

[0060] Figure 36 The images show the appearance morphology features of the six fine sub-fractions observed by SEM and AFM in Example 2.

[0061] Figure 37 The image shows the chromatograms of HPGPC obtained from the hydrolysis of LBPA-I at different times in Example 2.

[0062] Figure 38 The image shows the chromatogram of the main chain portion HPGPC in Example 3, where (A) is LBPA-I-H-3; (B) is LBPA-II-H-3; (C) is LBPA-III-H-3; and (D) is the molecular weight distribution of LBPA-I, LBPA-II, and LBPA-III.

[0063] Figure 39 LBPA-Ⅰ-H-3 in Example 3 1 H and 13 C-NMR spectrum.

[0064] Figure 40 LBPA-Ⅱ-H-3 in Example 3 1 H and 13 C-NMR spectrum.

[0065] Figure 41 LBPA-Ⅲ-H-3 in Example 3 1 H and 13 C-NMR spectrum.

[0066] Figure 42 The HSQC spectrum of LBPA-Ⅰ-H-3 in Example 3 is shown.

[0067] Figure 43 The HMBC spectrum of LBPA-Ⅰ-H-3 in Example 3 is shown.

[0068] Figure 44 The COSY spectrum of LBPA-Ⅰ-H-3 in Example 3 is shown.

[0069] Figure 45 The NOESY spectrum of LBPA-Ⅰ-H-3 in Example 3 is shown.

[0070] Figure 46 The HSQC spectrum of LBPA-Ⅱ-H-3 in Example 3 is shown.

[0071] Figure 47 The HMBC spectrum of LBPA-Ⅱ-H-3 in Example 3 is shown.

[0072] Figure 48 The COSY spectrum of LBPA-Ⅱ-H-3 in Example 3 is shown.

[0073] Figure 49 The NOESY spectrum of LBPA-Ⅱ-H-3 in Example 3 is shown.

[0074] Figure 50 The HSQC spectrum of LBPA-Ⅲ-H-3 in Example 3 is shown.

[0075] Figure 51 The HMBC spectrum of LBPA-Ⅲ-H-3 in Example 3 is shown.

[0076] Figure 52 The COSY spectrum of LBPA-Ⅲ-H-3 in Example 3 is shown.

[0077] Figure 53 The NOESY spectrum of LBPA-Ⅲ-H-3 in Example 3 is shown.

[0078] Figure 54This is a comparison diagram of the molecular weight distribution of LBPA-Ⅰ-H-3, LBPA-Ⅰa-H-3, and LBPA-Ⅰb-H-3 in Example 3.

[0079] Figure 55 This is the HPLC chromatogram of the main chain portion in Example 3.

[0080] Figure 56 This is the total ion chromatogram of methylation in Example 3.

[0081] Figure 57 This is a schematic diagram of the proposed structures of components a and b in Example 3.

[0082] Figure 58 This is a schematic diagram of the experimental animal grouping and drug administration in Example 4.

[0083] Figure 59 The results of HE staining of the colonic structure of CRS mice in the NC, CRS, ASA, LPBA-I-L, LPBA-I-M and LPBA-I-H treatment groups in Example 4 are shown. The transverse scale bar is 100 μm and the longitudinal scale bar is 100 μm.

[0084] Figure 60 The results of HE staining of the colonic structure of CRS mice in the LPBA-II-L, LPBA-II-M, LPBA-II-H, LPBA-III-L, LPBA-III-M and LPBA-III-H treatment groups in Example 4 are shown. The transverse scale bar is 100 μm and the longitudinal scale bar is 100 μm.

[0085] Figure 61 The results show the effects of wolfberry pectin on cytokines and MPO in CRS mice in Example 4.

[0086] Figure 62 The results of the effect of wolfberry pectin on the α diversity of gut microbiota in CRS mice in Example 4 are shown. (A) is the observed ASV index; (B) is the PD whole tree index; (C) is the microbiota diversity index (Simpson), N=6; * represents P<0.05, ** represents P<0.01, *** represents P<0.001 vs. NC group; # represents P<0.05, ## represents P<0.01, ### represents P<0.001 vs. CRS group.

[0087] Figure 63 Example 4 illustrates the effect of wolfberry pectin on the β-diversity of gut microbiota in CRS mice.

[0088] Figure 64The results of the effect of wolfberry pectin on the relative abundance of gut microbiota in CRS mice in Example 4 are shown. In this example, (A) represents the relative abundance of f-Helicobacteraceae, (B) represents the relative abundance of f-Muribaculaceae, and (C) represents the relative abundance of f-Ruminococcaceae. N = 6. *** represents P < 0.001 vs. NC group; # represents P < 0.05, ## represents P < 0.01, and ### represents P < 0.001 vs. CRS group.

[0089] Figure 65 The following is a cluster analysis diagram of the differential metabolites in CRS vs. NC and CRS vs. ASA in Example 4, where (A) is the CRS group vs. NC group; and (B) is the ASA group vs. CRS group.

[0090] Figure 66 The following is a cluster analysis diagram of the differential metabolites of CRS vs. LPBA-I in Example 4, where (A) is the CRS group vs. LPBA-IL group; (B) is the ASA group vs. LPBA-IM group; and (C) is the CRS group vs. LPBA-IH group.

[0091] Figure 67 The following is a cluster analysis diagram of the differential metabolites of CRS vs. LBPA-II in Example 4, where (A) is the CRS group vs. LPBA-IIL group; (B) is the ASA group vs. LPBA-IIM group; and (C) is the CRS group vs. LPBA-IIH group.

[0092] Figure 68 The following is a cluster analysis diagram of the differential metabolites of CRS vs. LBPA-III in Example 4, where (A) is the CRS group vs. LPBA-IIIL group; (B) is the ASA group vs. LPBA-IIIM group; and (C) is the CRS group vs. LPBA-IIIH group.

[0093] Figure 69 The results of the effect of wolfberry pectin on differential metabolites in CRS mice in Example 4 are shown.

[0094] Figure 70 The results of Spearman correlation analysis are shown for the intervention of wolfberry pectin in intestinal inflammation in mice in Example 4.

[0095] Figure 71 The results of metabolic pathway correlation analysis for CRS vs. NC and CRS vs. ASA in Example 4 are shown. (A) represents the CRS group vs. the NC group; (B) represents the ASA group vs. the CRS group.

[0096] Figure 72The results of metabolic pathway correlation analysis between CRS and LPBA-I in Example 4 are shown. (A) CRS group vs. LPBA-IL group; (B) ASA group vs. LPBA-IM group; (C) CRS group vs. LPBA-IH group.

[0097] Figure 73 The results of the metabolic pathway correlation analysis of CRS vs. LBPA-II in Example 4 are as follows: (A) CRS group vs. LPBA-IIL group; (B) ASA group vs. LPBA-IIM group; (C) CRS group vs. LPBA-IIH group.

[0098] Figure 74 The results of the metabolic pathway correlation analysis of CRS vs. LBPA-III in Example 4 are as follows: (A) CRS group vs. LPBA-IIIL group; (B) ASA group vs. LPBA-IIIM group; (C) CRS group vs. LPBA-IIIH group.

[0099] Figure 75 The results show the effects of the intervention in Example 4 on endocannabinoid pathway proteins in mice. (A) represents the expression levels of CB2, Bcl-2, Bax, and FAAH proteins; (B), (C), (D), and (E) represent the expression intensities of FAAH, CB2, Bax, and Bcl-2 proteins.

[0100] Figure 76 The results of the intervention in Example 4 on the effects of tight junction proteins in the mouse colon are shown. (A) represents the expression levels of Claudin-1, Occludin, and ZO-1 proteins; (B), (C), and (D) represent the expression intensities of Claudin-1, Occludin, and ZO-1 proteins, respectively. Detailed Implementation

[0101] This invention provides a Lycium barbarum pectin polysaccharide, wherein the Lycium barbarum pectin polysaccharide is mainly composed of galactose and arabinose, and its core structure is: →6)-β-D-Galp-(1→6 / 3,6)-β-D-Galp-(1→and →4)-α-D-GalpA-(1→2 / 2,4)-α-L-Rhap-(1→repeating unit).

[0102] In this invention, the wolfberry pectin polysaccharide comprises mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, and arabinose, wherein the molar ratio of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose, and arabinose is (0.3–5.5):(3.0–6.3):(0.3–9.6):(1.9–7.1):(0.9–20.6):(23.2–49.7):(33.9–45.6). The molecular weight (Mw) of the wolfberry pectin polysaccharide is 3.36 kDa to 49.0 kDa.

[0103] This invention also provides a method for preparing the above-mentioned wolfberry pectin polysaccharides, the method comprising the following steps:

[0104] Total polysaccharides from Lycium barbarum were dissolved in distilled water, centrifuged, and the supernatant was collected. The supernatant was eluted using a DE-52 ion exchange chromatography column, and neutral polysaccharides were separated by elution with 400 mL of water. Elution was then carried out with 0.08 M NaCl, 0.15 M NaCl, and 0.2 M NaCl solutions. For elution with 0.08 M NaCl solution, target eluent 1 (from 11.5 to 19 min) was collected; for elution with 0.15 M NaCl solution, target eluent 2 (from 11 to 18.2 min) was collected; and for elution with 0.2 M NaCl solution, target eluent 3 (from 11.2 to 18.1 min) was collected. The elution flow rate was 10 mL / min.

[0105] The collected target eluents 1-3 were concentrated and dialyzed, then concentrated again and freeze-dried to obtain Lycium barbarum pectin polysaccharides LBPA-I, LBPA-II and LBPA-III, respectively.

[0106] In the preparation method of the above-mentioned wolfberry pectin polysaccharide of the present invention, the mass-to-volume ratio of the total wolfberry polysaccharide to distilled water is 1g:25mL; the target eluents 1 to 3 are concentrated to 1 / 10 of their original volume; the molecular weight cutoff for dialysis is 3500Da; and the dialysis time is 24h.

[0107] As an optional implementation method, in preparing the Lycium barbarum pectin polysaccharide LBPA-I, elution is performed using 0.08M NaCl solution at a flow rate of 10 mL / min. The target eluent is collected from the 11.5 to 19 min time interval, and 115 to 190 mL of eluent is collected. The collected eluent is concentrated, dialyzed, concentrated again, and then freeze-dried to obtain the Lycium barbarum pectin polysaccharide, denoted as LBPA-I Lycium barbarum pectin (abbreviated as LBPA-I). The LBPA-I Lycium barbarum pectin is composed of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose, with a molar ratio of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose of 1.4:2.8:0.4:2.1:15.7:30.0:47.7.

[0108] As an optional implementation method, in preparing the Lycium barbarum pectin polysaccharide LBPA-II, elution is performed using 0.15M NaCl solution at a flow rate of 10 mL / min. The target eluent is collected during the period from 11 to 18.2 min. The collected eluent is concentrated, dialyzed, concentrated again, and then freeze-dried to obtain the Lycium barbarum pectin polysaccharide, denoted as LBPA-II Lycium barbarum pectin (abbreviated as LBPA-II). The LBPA-II Lycium barbarum pectin is composed of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose, with a molar ratio of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose of 2.5:8.0:1.6:5.5:7.6:35.1:39.8.

[0109] As an optional implementation method, in preparing the Lycium barbarum pectin polysaccharide LBPA-III, elution was performed using 0.2M NaCl solution at a flow rate of 10 mL / min. The target eluent was collected during the period from 11.2 to 18.1 min. The collected eluent was concentrated, dialyzed, concentrated again, and then freeze-dried to obtain the Lycium barbarum pectin polysaccharide, denoted as LBPA-III Lycium barbarum pectin (abbreviated as LBPA-III). The LBPA-III Lycium barbarum pectin is composed of mannose: glucuronic acid: rhamnose: galacturonic acid: glucose: galactose: xylose: arabinose, with a molar ratio of mannose: glucuronic acid: rhamnose: galacturonic acid: glucose: galactose: xylose: arabinose of 2.5: 4.5: 13.5: 7.6: 7.0: 24.6: 0.3: 40.0.

[0110] The wolfberry pectin polysaccharides LBPA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ prepared above were further purified by fractionation using a Sepharose CL-6B gel chromatography column to obtain fine subfractions of wolfberry pectin: LBPA-Ⅰa, LBPA-Ⅰb, LBPA-Ⅱa, LBPA-Ⅱb, LBPA-Ⅲa, and LBPA-Ⅲb.

[0111] As an optional implementation method, in preparing the fine subfraction LBPA-Ia of wolfberry pectin, the LBPA-I wolfberry pectin polysaccharide component is dissolved in a 0.15M NaCl solution, with a mass-to-volume ratio of total wolfberry pectin to 0.15M NaCl solution of 40g:1mL. After dissolution, the supernatant is collected by centrifugation, and the supernatant is eluted using a Sepharose CL-6B gel chromatography column (2.6×100cm) with a 0.15M elution solution. NaCl solution was used for elution at a flow rate of 0.45 mL / min. The eluent was collected using an automatic collector at 18 mL / tube. The neutral sugar and uronic acid content in each tube of eluent was determined using the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves were plotted based on the results. The target eluent was collected based on the characteristic peak of the elution curve. Preferably, arabinose was used as the standard, with a sugar content of 87.0%. The molecular weight was collected based on the characteristic peak of the elution curve, with a molecular weight of 49.0 kDa. The eluent was lyophilized after dialyzing to obtain the fine subfraction of wolfberry pectin, LBPA-Ia. The molecular weight cutoff during dialysis was 3500 Da. The LBPA-Ⅰa is composed of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose, and the molar ratio of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose is 0.3:3.0:0.3:1.9:0.9:49.6.

[0112] : 44.0; protein content is 0%; molecular weight (Mw) is 49.0 kDa.

[0113] As an optional implementation method, in preparing the fine subfraction LBPA-Ib of wolfberry pectin, the LBPA-I wolfberry pectin polysaccharide component is dissolved in a 0.15M NaCl solution, with a mass-to-volume ratio of total wolfberry pectin to 0.15M NaCl solution of 40g:1mL. After dissolution, the supernatant is collected by centrifugation, and the supernatant is eluted using a Sepharose CL-6B gel chromatography column (2.6×100cm) with a 0.15M elution solution. NaCl solution was used for elution at a flow rate of 0.45 mL / min. The eluent was collected using an automatic collector at 18 mL / tube. The neutral sugar and uronic acid content in each tube of eluent was determined using the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves were plotted based on the results. The target eluent was collected based on the characteristic peak of the elution curve. Preferably, arabinose was used as the standard, with a sugar content of 87.8%. The molecular weight was collected as 3.36 kDa based on the characteristic peak of the elution curve. The eluent was lyophilized after dialyzing to obtain the fine subfraction of wolfberry pectin, LBPA-Ib. The molecular weight cutoff during dialysis was 3500 Da. The LBPA-Ⅰb is composed of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose, with a molar ratio of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose of 4.0:4.3:2.5:3.5:16.5:33.6:35.6; a protein content of 6.6%; and a molecular weight (Mw) of 3.36 kDa.

[0114] As an optional implementation method, in preparing the fine subfraction LBPA-IIa of wolfberry pectin, the LBPA-II wolfberry pectin polysaccharide component is dissolved in a 0.15M NaCl solution, with a mass-to-volume ratio of total wolfberry pectin to 0.15M NaCl solution of 40g:1mL. After dissolution, the supernatant is collected by centrifugation, and the supernatant is eluted using a Sepharose CL-6B gel chromatography column (2.6×100cm) with a 0.15M elution solution. NaCl solution was used for elution at a flow rate of 0.45 mL / min. The eluent was collected using an automatic collector at 18 mL / tube. The neutral sugar and uronic acid content in each tube of eluent was determined using the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves were plotted based on the results. The target eluent was collected based on the characteristic peak of the elution curve. Preferably, arabinose was used as the standard, with a sugar content of 74.6%, and the molecular weight was collected as 47.2 kDa based on the characteristic peak of the elution curve. The eluent was lyophilized after dialyzing to obtain the fine subfraction of wolfberry pectin, LBPA-IIa. The molecular weight cutoff during dialysis was 3500 Da. The LBPA-IIa is composed of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose, with a molar ratio of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:arabinose of 0.4:6.2:0.4:3.4:1.1:45.6:42.9; a protein content of 1.7%; and a molecular weight (Mw) of 47.2 kDa.

[0115] As an optional implementation method, in preparing the fine subfraction LBPA-IIb of wolfberry pectin, the LBPA-II wolfberry pectin polysaccharide component is dissolved in a 0.15M NaCl solution, with a mass-to-volume ratio of total wolfberry pectin to 0.15M NaCl solution of 40g:1mL. After dissolution, the supernatant is collected by centrifugation, and the supernatant is eluted using a Sepharose CL-6B gel chromatography column (2.6×100cm) with a 0.15M elution solution. NaCl solution was used for elution at a flow rate of 0.45 mL / min. The eluent was collected using an automatic collector at 18 mL / tube. The neutral sugar and uronic acid content in each tube of eluent was determined using the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves were plotted based on the results. The target eluent was collected based on the characteristic peak of the elution curve. Preferably, arabinose was used as the standard, with a sugar content of 79.1%. The molecular weight was collected as 4.66 kDa based on the characteristic peak of the elution curve. The eluent was lyophilized after dialyzing to obtain the fine subfraction of wolfberry pectin, LBPA-IIb. The molecular weight cutoff during dialysis was 3500 Da. The LBPA-IIb is composed of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:xylose:arabinose, with a molar ratio of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:xylose:arabinose of 5.5:3.1:6.6:5.2:20.6:24.2:0.2:34.6; a protein content of 18.3%; and a molecular weight (Mw) of 4.66 kDa.

[0116] As an optional implementation method, when preparing the fine subfraction LBPA-IIIa of wolfberry pectin, the LBPA-III wolfberry pectin polysaccharide component is dissolved in a 0.15M NaCl solution, with a mass-to-volume ratio of total wolfberry pectin to 0.15M NaCl solution of 40 g: 1 mL. After dissolution, the supernatant is collected by centrifugation, and the supernatant is eluted using a Sepharose CL-6B gel chromatography column. The CL-6B gel chromatography column has a specification of 2.6 × 100 cm, and the elution solution is 0.15 M NaCl solution. The elution flow rate is 0.45 mL / min, and the eluent is collected using an automatic collector at a rate of 18 mL / tube. The neutral sugar content and uronic acid content in each tube of eluent are determined using the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves are plotted based on the results, and the target eluent is collected according to the characteristic peak of the elution curve. Preferably, arabinose is used as the standard, with a sugar content of 82.2%, and the molecular weight is collected based on the characteristic peak of the elution curve at 45.9 kDa. The eluent is lyophilized after dialyzing to obtain the fine subfraction of wolfberry pectin LBPA-Ⅲa. The molecular weight cutoff during dialysis is 3500 Da. The LBPA-Ⅲa is composed of mannose: glucuronic acid: rhamnose: galacturonic acid: glucose: galactose: arabinose, with a molar ratio of mannose: glucuronic acid: rhamnose: galacturonic acid: glucose: galactose: arabinose of 0.8:6.2:2.7:5.2:2.4:37.2:45.6; a protein content of 8.9%; and a molecular weight (Mw) of 45.9 kDa.

[0117] As an optional implementation method, in preparing the fine subfraction LBPA-Ⅲb of wolfberry pectin, the LBPA-III wolfberry pectin polysaccharide component is dissolved in a 0.15M NaCl solution, with a mass-to-volume ratio of total wolfberry pectin to 0.15M NaCl solution of 40 g: 1 mL. After dissolution, the supernatant is collected by centrifugation, and the supernatant is eluted using a Sepharose CL-6B gel chromatography column. The CL-6B gel chromatography column has a specification of 2.6 × 100 cm, and the elution solution is 0.15 M NaCl solution. The elution flow rate is 0.45 mL / min, and the eluent is collected using an automatic collector at a rate of 18 mL / tube. The neutral sugar content and uronic acid content in each tube of eluent are determined using the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves are plotted based on the results, and the target eluent is collected according to the characteristic peak of the elution curve. Preferably, arabinose is used as the standard, with a sugar content of 85.3%, and the molecular weight is collected as 5.62 kDa based on the characteristic peak of the elution curve. The eluent is lyophilized after dialyzing to obtain the fine subfraction of wolfberry pectin LBPA-Ⅲb. The molecular weight cutoff during dialysis is 3500 Da. The LBPA-Ⅲb is composed of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:xylose:arabinose, with a molar ratio of mannose:glucuronic acid:rhamnose:galacturonic acid:glucose:galactose:xylose:arabinose of 2.9:6.3:9.6:7.1:16.6:23.2:0.4:33.9; a protein content of 15.7%; and a molecular weight (Mw) of 5.62 kDa.

[0118] In this invention, the total polysaccharide of Lycium barbarum is obtained by water extraction and alcohol precipitation from Lycium barbarum. The specific preparation method of the total polysaccharide of Lycium barbarum includes the following steps: Lycium barbarum is added to purified water, heated and extracted for 2 hours, then filtered and the filtrate is collected. The filtrate is concentrated and centrifuged to obtain the supernatant. Ethanol is added to the supernatant until the final ethanol concentration reaches 80%. After standing, centrifugation is performed, the precipitate is collected, the water in the precipitate is washed to remove water, and the precipitate is dried to obtain the total polysaccharide of Lycium barbarum. As an optional embodiment, 1.0 kg of dried Lycium barbarum is weighed and added to 10 L of purified water. Extraction is performed at 100℃ under a slight boiling state for 2 hours. The filtrate is filtered through a 400-mesh nylon mesh and collected. The extraction is performed 4 times, and all extracts are combined and concentrated to 4-5 L in a water bath at 75-80℃. The mixture is centrifuged at 4000 rpm for 10 min and the supernatant is collected. 95% ethanol is slowly added to the supernatant while stirring continuously until the final ethanol concentration reaches 80%. The mixture is then allowed to stand overnight. Centrifuge at 4000 rpm, collect the precipitate, and wash it alternately with 95% ethanol and anhydrous ethanol to remove water. Evaporate the ethanol and dry to obtain total polysaccharides from wolfberry.

[0119] In this invention, LBPA-Ⅰa, LBPA-Ⅱa, and LBPA-Ⅲa have similar structural features and are divided into component a, which is mainly composed of β-D-Galp and α-L-Araf residues, and is presumed to be AG-Ⅱ type pectin. LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb have similar structural features and are divided into component b, which contains β-D-Galp, α-L-Araf residues, and →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→disaccharide unit, and is presumed to be RG-Ⅰ type pectin.

[0120] The fine structure of component a includes a main chain and side chains. The main chain is composed of β-D-Galp residues, and the side chains contain α-L-Araf and β-D-Galp residues. The connection mode is →1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→1), where Gal is replaced by 5 side chains at the O-3 positions.

[0121] ①→1)-α-L-Araf-(3→1)-α-L-Araf-(3→1)-α-L-Araf;

[0122] ②→1)-α-L-Araf-(5→1)-α-L-Araf-(5→1)-α-L-Araf;

[0123] ③→1)-α-L-Araf-(5→1)-α-L-Araf-(5→1)-β-L-Araf;

[0124] ④→1)-β-D-Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap;

[0125] ⑤→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→;The proposed structural diagram is as follows Figure 57 Component a is shown in the figure.

[0126] The fine structure of component b includes a main chain and side chains. The main chain is composed of β-D-Galp, and also contains disaccharide units of →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→) and a similar AG-II type side chain structure to component a. The connection mode is mainly →1)-β-D-Galp-(3→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→); and mainly →2,4)-α-L-Rhap-(1→4)-α-D-G alpA-(1→) forms a short chain structure, and also contains →1)-β-D-Galp-(6→1)-β-D-Galp-(3,6→1;→1)-α-L-Araf-(3→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-β-L-Araf and →1)-β-D-Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap; the proposed structural diagram is shown below. Figure 57 Component b is shown in the figure.

[0127] In this invention, component a is mainly composed of 1,6-Galp and does not contain glycosidic bonds of 1,4-Galp and 1,2,4-Rhap, which conforms to the structural characteristics of AG-II type; component b is mainly composed of 1,6-Galp and contains glycosidic bonds of 1,4-Galp and 1,2,4-Rhap, which conforms to the pectin characteristics of RG-I type.

[0128] The present invention also provides applications of the aforementioned wolfberry pectin polysaccharide or the wolfberry pectin polysaccharide prepared by the aforementioned preparation method, wherein the applications include at least one of the following a to d;

[0129] a. The application of the aforementioned wolfberry pectin polysaccharide in the preparation of products that improve intestinal inflammation;

[0130] b. The application of the aforementioned wolfberry pectin polysaccharide in the preparation of a product that improves intestinal inflammation in mice induced by chronic restraint stress;

[0131] c. The application of the wolfberry pectin polysaccharide in the preparation of products that improve the expression of endocannabinoid system-related proteins;

[0132] d. Application of the aforementioned wolfberry pectin polysaccharide in the preparation of products that regulate intestinal flora diversity.

[0133] The product described in this invention is a pharmaceutical product.

[0134] In this invention, intervention with three pectin polysaccharide components, LPBA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ, all inhibited the increase of pro-inflammatory cytokines TNF-α, LPS, IL-1β, and IL-6, the decrease of anti-inflammatory cytokine IL-10, and the increase of the inflammatory marker MPO activity. Overall comparison revealed that low doses of LPBA-Ⅰ, medium doses of LBPA-Ⅱ, and high doses of LBPA-Ⅲ showed better effects on improving key inflammatory factors in the colonic tissue of the CRS model.

[0135] In this invention, the three Lycium barbarum pectin components LPBA-Ⅰ, LBPA-Ⅱ and LBPA-Ⅲ can restore the relative abundance of f-Helicobacteraceae, f-Muribaculaceae and f-Ruminococcaceae; among them, the LBPA-Ⅲ component has the best effect, and the medium dose (100mg / kg) is better than the other doses and close to the positive control (ASA).

[0136] In this invention, medium-dose intervention with Lycium barbarum pectin LPBA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ improved the expression of CB2, Bcl-2, Bax, and FA AH. Among them, LBPA-ⅢM (medium dose 100 mg / kg) showed the best effect, approaching that of the positive control drug (ASA).

[0137] In this invention, medium-dose intervention with Lycium barbarum pectin LPBA-Ⅰ, LBPA-Ⅱ and LBPA-Ⅲ can effectively improve the effect of CRS on the intestinal barrier function of mice. Among them, LBPA-ⅢM has the best effect, which is close to that of the positive control drug (ASA).

[0138] In this invention, wolfberry pectin LPBA-I, LBPA-II, and LBPA-III exert their intervention effect on intestinal inflammation by regulating the endocannabinoid system (ECS) and influencing apoptosis and inflammatory responses of intestinal cells. Specifically, wolfberry pectin LPBA-I, LBPA-II, and LBPA-III regulate the synthesis and metabolism of endocannabinoids, increase the activation of CB2 receptors, thereby inhibiting the release of inflammatory cytokines and promoting the repair and regeneration of intestinal cells.

[0139] This invention also provides the aforementioned wolfberry pectin polysaccharide, which can promote the synthesis and assembly of tight junction proteins, enhance intercellular tight junctions, and thus maintain the integrity of the intestinal barrier. Furthermore, wolfberry pectin may also promote the repair of damaged intestinal barriers by regulating the proliferation and differentiation of intestinal epithelial cells.

[0140] In this invention, the medium dose is 100 mg / kg.

[0141] In the following examples, the reagents included PBS buffer (1×) purchased from Wuhan Pronosei Biotechnology Co., Ltd.; 4% PFA purchased from Beijing Regen Biotechnology Co., Ltd.; mesalazine enteric-coated tablets (ASA) purchased from Losan Pharma GmbH; the kit included: mouse tumor necrosis factor α (TNF-α) purchased from Wuhan Genemei Biotechnology Co., Ltd.; mouse lipopolysaccharide (LPS) purchased from Wuhan Genemei Biotechnology Co., Ltd.; mouse interleukin 1β (IL-1β) purchased from Wuhan Genemei Biotechnology Co., Ltd.; mouse interleukin 6 (IL-6) purchased from Wuhan Genemei Biotechnology Co., Ltd.; mouse interleukin 10 (IL-10) purchased from Wuhan Genemei Biotechnology Co., Ltd.; and mouse myeloperoxidase (MPO) purchased from Wuhan Genemei Biotechnology Co., Ltd.

[0142] In the following embodiments, the restraint tube is self-made. The preparation method is as follows: the restraint tube is a modified 50mL centrifuge tube with appropriate openings at the front end and rear cap, allowing the mouse to move back and forth within a small range. This invention does not further limit the 50mL centrifuge tube, as long as it can establish a mouse intestinal inflammation model induced by chronic restraint stress (CRS).

[0143] Unless otherwise specified, the test methods used in the following examples are conventional test methods; the materials and reagents used are commercially available unless otherwise specified.

[0144] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments thereof.

[0145] Example 1

[0146] 1. Isolation, preparation and composition analysis of wolfberry pectin

[0147] 1.1 Experimental Materials

[0148] The dried goji berries used in the experiment were naturally air-dried red goji berries, produced in the Nuomuhong Goji Berry Industrial Park (Qingqi No. 1) of Dulan County, Chaidamu (National) Circular Economy Experimental Zone, Qinghai Province. The harvesting period was from July to August, and the berries were naturally air-dried.

[0149] 1.2 Experimental Methods

[0150] 1.2.1 Extraction of total polysaccharides from Lycium barbarum

[0151] Total polysaccharides from Lycium barbarum were extracted using a water extraction-ethanol precipitation method: 1.0 kg of dried Lycium barbarum was weighed and added to 10 L of purified water. The mixture was extracted at 100℃ under gentle boiling for 2 hours. The extract was filtered through a 400-mesh nylon mesh and the filtrate was collected. The extraction was repeated four times, and all extracts were combined and concentrated to 4 L in a water bath at 80℃. The solution was centrifuged at 4000 rpm for 10 min, and the supernatant was collected. 95% ethanol was slowly added to the supernatant while continuously stirring until the final ethanol concentration reached 80%. The solution was allowed to stand overnight. The precipitate was collected by centrifugation at 4000 rpm and washed alternately with 95% ethanol and anhydrous ethanol to remove water. The ethanol was evaporated to remove the precipitate, and the precipitate was dried to obtain total polysaccharides from Lycium barbarum.

[0152] 1.2.2 Preparation and grading of wolfberry pectin

[0153] (1) Elution concentration of wolfberry pectin

[0154] Weigh 400.0 mg of the total polysaccharides from Lycium barbarum prepared above and dissolve them in 10 mL of purified water to prepare a polysaccharide solution. Heat the solution in a 40℃ water bath until fully dissolved, centrifuge at 10000 rpm for 10 min, and collect the supernatant. Load the supernatant onto a pre-equilibrated DE-52 ion exchange chromatography column (Cl- type, 2.6 × 30 cm; empty glass columns were custom-made by Shanghai Huxi Analytical Instrument Factory Co., Ltd.), and elute at a flow rate of 0.45 mL / min. Collect the eluent using an automatic fraction collector (8 mL / tube). First, perform isocratic elution with 300 mL of purified water, followed by linear gradient elution with 0–0.25 M NaCl solution (1000 mL) using a gradient mixing system (Shanghai Huxi TH-1000). Using distilled water (500 mL) as the initial concentration, elution was performed under linear concentration elution conditions of 0 M–0.25 M NaCl within a 1000 mL elution volume range. The neutral sugar and uronic acid contents in each eluent tube were determined by the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves were plotted based on the results to determine the optimal elution concentration of wolfberry pectin.

[0155] The results showed that the optimal elution concentrations were 0.08 M NaCl, 0.15 M NaCl, and 0.2 M NaCl.

[0156] (2) Preparation of wolfberry pectin

[0157] 20.0g of total polysaccharides from Lycium barbarum were dissolved in 500mL of distilled water and heated in a water bath at 40℃ to ensure complete dissolution. The solution was centrifuged at 4000rpm for 10min. The supernatant was loaded onto a pre-equilibrated DE-52 ion exchange chromatography column (Cl-type, 7.5×60cm, DEAE cellulose DE-52, CAS No.: 9013-34-7, Shanghai Yuanye Biotechnology Co., Ltd.). Neutral polysaccharides were first separated by elution with 400mL of purified water. The phenol-sulfuric acid method was used for continuous monitoring. After the neutral sugar components were completely eluted, stepwise elution was performed according to the NaCl solution concentrations (0.08M NaCl, 0.15M NaCl, and 0.2M NaCl) determined in 1.2.2(1) at a flow rate of 10mL / min. The eluents containing sugars as determined by the phenol-sulfuric acid method were collected separately. That is, 0.08M NaCl, 0.15M NaCl, and 0.2M NaCl were used respectively. Elution with NaCl solution: during elution with 0.08M NaCl solution, target eluent 1 is collected during the time period from 11.5 to 19 min; during elution with 0.15M NaCl solution, target eluent 2 is collected during the time period from 11 to 18.2 min; and during elution with 0.2M NaCl solution, target eluent 3 is collected during the time period from 11.2 to 18.1 min. Target eluents 1 to 3 are characteristic fractions of wolfberry pectin (i.e., wolfberry pectin polysaccharides) obtained by elution with specific concentrations (0.08M NaCl, 0.15M NaCl, and 0.2M NaCl) NaCl solutions. The characteristic pectin fraction (i.e., wolfberry pectin polysaccharides) was concentrated to 1 / 10 of its original volume by water bath at 75-80℃. Then, it was dialyzed with running water and pure water for 24 hours to remove salt using hollow fiber (molecular weight cutoff 3500 Da). The fraction was concentrated to a small volume and freeze-dried to obtain wolfberry total pectin of specific concentrations with relatively uniform charge (0.08M NaCl concentration, 0.15M NaCl concentration, and 0.2M NaCl concentration).

[0158] 24-hour running water dialysis refers to using running tap water for 24 hours; 24-hour pure water dialysis refers to complete immersion in pure water, with the pure water being changed every 6-8 hours.

[0159] The specific concentrations of total pectin in wolfberry refer to LBPA-I (0.08M NaCl concentration), LBPA-II (0.15M NaCl concentration), and LBPA-III (0.2M NaCl concentration).

[0160] (3) Fractionation and purification of wolfberry pectin

[0161] Weigh 800.0 mg of total pectin from wolfberry at various concentrations (0.08 M NaCl, 0.15 M NaCl, and 0.2 M NaCl) with relatively uniform charge, dissolve in 20 mL of 0.15 M NaCl solution, centrifuge (10000 rpm, 10 min), and load the supernatant onto a pre-equilibrated Sepharose CL-6B gel chromatography column [2.6 × 100 cm, Sepharose CL-6B packing material, catalog number 17016001, manufacturer: Cytiva; empty glass column was custom-made by Shanghai Huxi Analytical Instrument Factory Co., Ltd.]. Use 0.15 M NaCl solution as the eluent, elute at a flow rate of 0.45 mL / min, and collect the eluent (18 mL / tube) using an automatic collector. The neutral sugar and uronic acid contents in each eluent tube were determined by the phenol-sulfuric acid method and the m-hydroxybiphenyl method, respectively. Elution curves were plotted based on the results. Target eluent fractions were collected according to the characteristic peaks of the elution curves. After desalting by dialyzing (molecular weight cutoff 3500 Da), the target eluent fractions were freeze-dried (freeze-drying conditions: cold trap temperature ≤ -60℃, vacuum degree <10Pa, time: 24-48h) to obtain fine subfractions of wolfberry pectin with relatively uniform charge and molecular weight distribution.

[0162] 1.2.3 Composition and content determination

[0163] (1) Determination of sugar content of wolfberry pectin by phenol-sulfuric acid method.

[0164] Preparation of 100 μg / mL sugar standard solution: Accurately weigh 10 mg of each monosaccharide, including Man (mannose), GlcA (glucuronic acid), Rha (rhamnose), GalA (galacturonic acid), Glc (glucose), Gal (galactose), Xyl (xylose), Ara (arabinose), Fuc (fucose), and dextran standard into a small beaker, dissolve in a small amount of ultrapure water, and dilute to 100 mL in a 100 mL volumetric flask.

[0165] Standard curve plotting: Add reagents to glass test tubes (1.8×18cm) according to Table 1, and repeat three samples for each concentration.

[0166] Table 1 Preparation of glucose standard solution

[0167] Sugar standard solution content (pg) 0 20 40 60 80 100 Sugar standard solution (mL) 0 0.2 0.4 0.6 0.8 1.0 Distilled water (mL) 1.0 0.8 0.6 0.4 0.2 0

[0168] Add 0.5 mL of 6% phenol reagent and 2.5 mL of concentrated sulfuric acid to each test tube, shake rapidly to mix, and after reacting for 10 min, measure the absorbance (A490) at 490 nm. Plot a standard curve with absorbance (A490) on the ordinate and sugar content (μg) of the standard on the abscissa.

[0169] Determination of sugar content in samples: Take 1 mL of sample solution with a concentration of 0.1 mg / mL, operate according to the standard curve plotting method, measure the absorbance at 490 nm, and calculate the total sugar content based on the calibration curve and the sample absorbance.

[0170] (2) Determination of uronic acid content in wolfberry pectin by m-hydroxybiphenyl method.

[0171] Using galacturonic acid as a standard, a 100 μg / mL D-galacturonic acid standard solution was prepared. Each reagent was added to a glass test tube (1.8 × 18 cm) (Table 2), and three samples were repeated for each concentration.

[0172] Table 2 Preparation of D-galacturonic acid standard solution

[0173]

[0174]

[0175] Add 40 μL of sulfamic acid (pH 2.5) to each tube, shake well, then add 2.5 mL of concentrated sulfuric acid and mix thoroughly. Heat in a boiling water bath for 20 min, then immediately cool to room temperature with cold water. Add 40 μL of 0.3% m-hydroxybiphenyl (the 0.3% m-hydroxybiphenyl solution was prepared with 0.5% sodium hydroxide solution) to each tube and shake well. Let stand at room temperature for 15 min. Measure the absorbance A525 at 525 nm. Plot a standard curve with the D-galacturonic acid standard solution content (μg) on ​​the x-axis and the absorbance A525 on the y-axis.

[0176] (3) Determination of protein content in wolfberry pectin using the Coomassie brilliant blue method.

[0177] Coomassie Brilliant Blue Reagent: Dissolve 10 mg of Coomassie Brilliant Blue Reagent in 95% ethanol (5 mL), add 10 mL of 85% phosphoric acid, dilute to volume with 100 mL volumetric flask (ultrapure water), mix well, and filter with filter paper for later use.

[0178] Prepare a 50 μg / mL protein standard solution using bovine serum albumin as the standard. Take the protein standard solution according to Table 3 into glass test tubes, add distilled water to a final volume of 1.0 mL, and perform three replicates for each concentration.

[0179] Table 3 Preparation of protein standard solutions

[0180] Protein standard solution concentration (pg) 0 8 16 24 32 40 Protein standard solution (mL) 0 0.2 0.4 0.6 0.8 1.0 Distilled water (mL) 1.0 0.8 0.6 0.4 0.2 0

[0181] Add 4 mL of freshly prepared Coomassie Brilliant Blue reagent to each test tube, shake rapidly until homogeneous, and let stand in the dark for 5 min. Then, take the upper layer and measure the absorbance at a wavelength of 595 nm. Plot a standard curve with absorbance on the ordinate and bovine serum albumin content (μg) on ​​the abscissa.

[0182] Protein content determination in samples: Prepare a sample solution with a concentration of 0.1 mg / mL, take 1 mL and operate according to the standard curve plotting method, and calculate the protein content based on the standard curve and the sample absorbance value.

[0183] 1.2.4 Molecular weight distribution and uniformity analysis

[0184] Weigh 8.0 mg of wolfberry pectin and each fine subfraction into a 1.5 mL ep tube, add 0.5 mL of 0.15 M NaCl solution to dissolve, filter using a 0.22 μm aqueous filter membrane, load onto a Shimadzu high performance liquid chromatography system (LC-10ADvp) equipped with a TSK-gel G3000PWXL (7.8 × 300 mm) or TSK-gel G4000PWXL (7.8 × 300 mm) column, use 0.15 M NaCl as the mobile phase, flow rate of 0.6 mL / min, and injection volume of 20 μL. Among them, fine sub-grade a uses TSK-gel G4000PWXL (7.8×300mm), manufacturer: Tosoh Corporation, Japan, part number: 0008022; fine sub-grade b uses TSK-gel G3000PWXL (7.8×300mm), manufacturer: Tosoh Corporation, Japan, part number: 0008021.

[0185] Under identical conditions, T-dextran series dextran standards with known molecular weights were tested, and a standard curve was generated. The effective partition coefficient Kav was calculated using the formula Kav = (Ve - Vo) / (Vt - Vo), where Vo is the volume of water outside the column, Vt is the total volume, and Ve is the actual elution volume of the sample. The standard curve was plotted with Kav on the ordinate and lg Mw on the abscissa. The molecular weight (Mw) of the sample was estimated based on the standard curve.

[0186] 1.2.5 Monosaccharide Composition Analysis

[0187] Weigh 1.0 mg of wolfberry pectin and each fine subfraction into a hydrolysis flask, add 1 mL of 2M trifluoroacetic acid (TFA) solution, and place in a preheated 120℃ oven for hydrolysis for 2 h. After cooling, transfer the solution to an evaporating dish, and add anhydrous ethanol in small amounts several times to thoroughly remove residual trifluoroacetic acid. Then add 1 mL of LPMP and NaOH solution (0.5 M PMP: 0.3 M NaOH = 1:1, V / V) to the evaporating dish, mix well, and take 0.3 mL of the sample solution into a 1.5 mL ep tube. React in a water bath at 70℃ for 30 min. Add 150 μL of distilled water and 150 μL of 0.3 M HCl solution to the tube, mix thoroughly, add 1 mL of CHCl3 for extraction, discard the CHCl3 layer, and repeat the extraction three times.

[0188] The extracted sample solution was filtered through a 0.22 μm organic microporous membrane and loaded onto a Thermo Fisher Ultimate 3000 two-dimensional liquid chromatography system. The chromatographic column was: 2.6μm C18 (4.6×100mm); Detector: DAD; Mobile phase: 20.0% acetonitrile (v / v), 80.0% phosphate buffer (50mM, pH 7.0); Elution flow rate: 0.5mL / min; Column temperature: 35℃; Injection volume: 5μL.

[0189] 1.3 Experimental Results

[0190] 1.3.1 Preparation and Composition Analysis of Total Polysaccharides from Lycium barbarum

[0191] Standard curve plotting: Plot a standard curve for sugar content detection with glucose (or arabinose) content (mg) on ​​the x-axis and absorbance (λ = 490nm) on the y-axis. Plot a standard curve for protein detection with bovine serum albumin content (μg) on ​​the x-axis and absorbance (λ = 562nm) on the y-axis. Plot a standard curve for uronic acid detection with D-galacturonic acid content (μg) on ​​the x-axis and absorbance (λ = 525nm) on the y-axis. Figure 1 As shown, the regression equation for the total sugar standard curve is A490 nm=0.0149C-0.0173, R 2 =0.9945; the regression equation for the protein standard curve is A562 nm = 0.0055C - 0.0013, R 2 =0.9979; the regression equation for the uronic acid standard curve is A525nm = 0.0012C - 0.0059, R 2 =0.9907.

[0192] The total polysaccharide yield of wolfberry was 21.0%, and the composition analysis results are shown in Table 4. Among them, the total sugar content, measured using glucose as a standard, was 90.5%, the uronic acid content was 10.7%, and the protein content was 0.4%. The monosaccharide composition results showed that wolfberry mainly contained Glc, and also contained Rha, GalA, Gal, and Ara, indicating that the total polysaccharide of wolfberry contains pectin components.

[0193] Table 4. Yield, content and monosaccharide composition of total polysaccharides from Lycium barbarum

[0194]

[0195] Note: 'a' represents the yield of total polysaccharides from wolfberry relative to the dry weight of the raw material.

[0196] 1.3.2 Ion fractionation purification of total polysaccharides from Lycium barbarum

[0197] Total polysaccharides from Lycium barbarum were separated and purified using a DE-52 anion exchange column. First, neutral polysaccharides were collected by isocratic elution with pure water. Then, a linear gradient elution was performed using 0–0.25 M NaCl solution. The sugar and uronic acid contents were measured, and elution distribution curves were plotted. The elution distribution curves are shown below. Figure 2 As shown in the figure. The analysis results show that three characteristic elution components, LBPA-Ⅰ, LBPA-Ⅱ and LBPA-Ⅲ, can be separated during the linear gradient elution process of 0M to 0.25M NaCl solution.

[0198] 1.3.3 Preparation of Lycium barbarum pectin

[0199] Further, a preparative DE-Cellulose ion-exchange chromatography column (Cl-type, 7.5×60cm; empty column: Jiangsu Hanhuang Technology Co., Ltd., catalog number: LCC75 / 60; chromatographic packing material: DE-Cellulose, Shanghai Yuanye, catalog number S28556, CAS number: 9013-34-7) was used with 0.08M, 0.15M, and 0.2M NaCl solutions as eluents to separate and prepare three characteristic total pectin fractions, LBPA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ, with relatively uniform charge. The specific flow chart is shown below. Figure 3 As shown.

[0200] The sugar contents of LBPA-I, LBPA-II, and LBPA-III, measured using glucose as a standard, were 24.9%, 29.0%, and 42.7%, respectively; the sugar contents of arabinose as a standard were 86.9%, 71.5%, and 84.4%, respectively; and the uronic acid contents were 2.5%, 4.0%, and 13.1%, respectively. The monosaccharide composition consisted of varying degrees of Man, Rha, GalA, Glc, Gal, and Ara, with Rha, GalA, Gal, and Ara accounting for over 80.0% of the total, indicating that the composition was consistent with the monosaccharide characteristics of pectin. The results are shown in Table 5. Based on the monosaccharide composition, it is inferred that it contains a type I rhamnose-galacturonic acid (RG-I) / galactan / arabinogalactan / arabinogalactan (AG) series structure.

[0201] Table 5. Yield and monosaccharide composition of wolfberry pectin

[0202]

[0203] Note: b indicates the yield of wolfberry pectin relative to total wolfberry polysaccharides.

[0204] 1.3.4 Gel fractionation and purification of wolfberry pectin

[0205] Three types of total pectin from wolfberry (LBPA-I, LBPA-II, and LBPA-III) were fractionated and purified using a Sepharose CL-6B gel chromatography column (2.6 × 100 cm; empty column: Jiangsu Hanhuang Technology Co., Ltd., catalog number: LCC26 / 100; chromatographic packing material: Cytiva, catalog number 17016001). Sugar and uronic acid contents were determined, and elution curves were plotted. Figure 4 As shown in (A), (B), and (C) in the figure. After separation and purification, LBPA-I, LBPA-II, and LBPA-III yielded six fine subfractions: LBPA-Ia, LBPA-Ib, LBPA-IIa, LBPA-IIb, LBPA-IIIa, and LBPA-IIIb, respectively. Their yields relative to the total pectin content of the three Lycium barbarum fractions (LBPA-I, LBPA-II, and LBPA-III) were 22.4%, 26.6%, 23.2%, 19.0%, 20.0%, and 22.2%, respectively, and their sugar contents were 87.0%, 87.8%, 74.6%, 79.1%, 82.2%, and 85.3%, respectively.

[0206] 1.3.5 Molecular weight distribution of fine subfractions of wolfberry pectin

[0207] High-performance gel permeation chromatography (HPGPC) was used to determine the molecular weight distribution and uniformity of six fine subfractions of wolfberry pectin, such as... Figure 5 As shown. The six fine subfractions exhibit relatively narrow single symmetrical peaks on the HPGPC spectrum: LBPA-Ⅰa, LBPA-Ⅰb, LBPA-Ⅱa, LBPA-Ⅱb, LBPA-Ⅲa, and LBPA-Ⅲb. Figure 5 The polydispersity index (PDI) of (A) to (F) were 1.71, 2.17, 1.85, 2.75, 2.64 and 3.69, respectively, indicating good homogeneity. The molecular weight (Mw) was 49.0 kDa, 3.36 kDa, 47.2 kDa, 4.66 kDa, 45.9 kDa and 5.62 kDa, respectively. It was found that the Mw of LBPA-Ⅰa, LBPA-Ⅱa and LBPA-Ⅲa were all greater than those of LBPA-Ⅰb, LBPA-Ⅱb and LBPA-Ⅲb.

[0208] 1.3.6 Monosaccharide composition analysis of fine subfractions of wolfberry pectin

[0209] The yield, protein content, and monosaccharide composition analysis results of the six wolfberry pectin subfractions are shown in Table 6. Monosaccharide composition analysis showed that the main monosaccharides in LBPA-Ia, LBPA-IIa, and LBPA-IIIa fractions were Gal and Ara; the main monosaccharides in LBPA-Ib, LBPA-IIb, and LBPA-IIIb fractions were also Gal and Ara, but the contents of monosaccharides such as Rha, GalA, Glc, and GlcA were higher than those in LBPA-Ia, LBPA-IIa, and LBPA-IIIa. The Gal+Ara content in all six fine subfractions was greater than 50%, with even higher contents (greater than 80%) in LBPA-Ⅰa, LBPA-Ⅱa, and LBPA-Ⅲa fractions. Their Rha / GalA values ​​were 0.2, 0.1, 0.5, 0.7, 1.3, and 1.4, respectively, indicating that the Rha / GalA values ​​in LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb were closer to 1. Based on the structural characteristics of pectin, it is speculated that the LBPA-Ⅰa, LBPA-Ⅱa, and LBPA-Ⅲa fractions may contain arabinogalactan (AG-Ⅱ) structures; while the LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb fractions may contain type I rhamnose-galacturonic acid (RG-Ⅰ) structures, and based on their chemical composition and monosaccharide composition analysis, they may contain side chains with AG structures.

[0210] Table 6. Yield, molecular weight, and monosaccharide composition of refined components of wolfberry pectin

[0211]

[0212] Note: c represents the yield of fine components of wolfberry pectin relative to wolfberry pectin.

[0213] In summary, the content and molecular weight of monosaccharides Gal+Ara in the six fine subfractions of wolfberry pectin (LBPA-Ia, LBPA-IIa, and LBPA-IIIa) are higher than those in fractions (LBPA-Ib, LBPA-IIb, and LBPA-IIIb). Conversely, fractions (LBPA-Ib, LBPA-IIb, and LBPA-IIIb) all contain high levels of GalA, Rha, Glc, and protein. Based on these characteristics, it is indicated that the compositions and molecular weights of LBPA-Ia, LBPA-IIa, and LBPA-IIIa are similar among the six fine subfractions of wolfberry pectin; similarly, the compositions and molecular weights of LBPA-Ib, LBPA-IIb, and LBPA-IIIb are also similar.

[0214] 1.4 Summary

[0215] This embodiment mainly studies the separation, preparation, and composition of wolfberry pectin. The main research results are as follows:

[0216] (1) Crude polysaccharide (LBP) was extracted from wolfberry by hot water extraction, with a yield of 21.0%.

[0217] (2) Total polysaccharides from Lycium barbarum were purified by fractionation using a DE-52 anion exchange chromatography column. Three Lycium barbarum total pectin fractions (LBPA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ) with relatively uniform charge were prepared by using 0.08M, 0.15M, and 0.2M NaCl solutions as eluents, respectively. The sugar contents were 86.9%, 71.5%, and 84.4%, respectively, and the uronic acid contents were 2.5%, 4.0%, and 13.1%, respectively.

[0218] (3) The total pectin of three wolfberries was further separated and purified by Sepharose CL-6B gel chromatography column, and six fine subfractions (LBPA-Ⅰa, LBPA-Ⅰb, LBPA-Ⅱa, LBPA-Ⅱb, LBPA-Ⅲa and LBPA-Ⅲb) were prepared respectively.

[0219] (4) The homogeneity test results showed that the polydispersity index (PDI) of the six fine subfractions were 1.71, 2.17, 1.85, 2.75, 2.64 and 3.69, respectively, indicating good homogeneity; among them, the molecular weight (Mw) of LBPA-Ⅰa, LBPA-Ⅱa and LBPA-Ⅲa were much higher than those of LBPA-Ⅰb, LBPA-Ⅱb and LBPA-Ⅲb, which were 49.0kDa, 47.2kDa, 45.9kDa, 3.36kDa, 4.66kDa and 5.62kDa, respectively. Monosaccharide composition analysis showed that the main monosaccharides in LBPA-Ia, LBPA-IIa, and LBPA-IIIa were Gal and Ara, with a combined proportion of more than 80%, and the Rha / GalA ratios were 0.2, 0.1, and 0.5, respectively. In contrast, the combined proportion of Gal and Ara in LBPA-Ib, LBPA-IIb, and LBPA-IIIb was more than 50%, and the Rha / GalA ratios were 0.7, 1.3, and 1.4, respectively.

[0220] (5) In summary, the six Lycium barbarum pectin fine subfractions, LBPA-Ia, LBPA-IIa, and LBPA-IIIa, have similar compositions and close molecular weights; LBPA-Ib, LBPA-IIb, and LBPA-IIIb also have similar compositions and close molecular weights. It is preliminarily speculated that the structural characteristics of LBPA-Ia, LBPA-IIa, and LBPA-IIIa may be similar; the structural characteristics of LBPA-Ib, LBPA-IIb, and LBPA-IIIb may also be similar.

[0221] Example 2

[0222] 2. Analysis of the overall structural characteristics of wolfberry pectin

[0223] 2.1 Experimental Methods

[0224] 2.1.1 Nuclear Magnetic Resonance Spectroscopy Analysis

[0225] Six finely fractionated pectin samples, 0.5 mg each, were dissolved in 0.5 mL L2O. The 1H-NMR spectra of the samples were determined using a Bruker AV600 nuclear magnetic resonance spectrometer. 13 C-NMR, HSQC, HMBC, NOESY, and COSY spectra.

[0226] 2.1.2 SEM Analysis

[0227] The microstructure of the samples was observed using a SU-8010 field emission scanning electron microscope at an observation voltage of 5.0 kV and a magnification of 400–5000.

[0228] 2.1.3 AFM Analysis

[0229] Six fine subfractions were dissolved in ultrapure water to prepare a 5 μg / mL solution. Then, 5 μL of this solution was dropped onto a clean mica sheet, allowed to air dry at room temperature, and observed under a Dimension Icon atomic force microscope in tapping mode, with a scan frequency of 1 Hz and a scan range of 3000 nm. Finally, the atomic force microscope images were analyzed and processed using NanoscopeAnalysis 1.7 software.

[0230] 2.2 Experimental Results

[0231] 2.2.1 Nuclear Magnetic Resonance Spectroscopy Analysis

[0232] Based on the monosaccharide composition and molecular weight analysis results, LBPA-Ⅰa, LBPA-Ⅱa, and LBPA-Ⅲa may have similar structural characteristics, and their 1D-NMR spectra are as follows: Figure 6 , Figure 7 and Figure 8 As shown. The signal appearing in the range of 102–105 ppm is the C-1 signal peak of β-Galp, while the signal in the range of 107–110 ppm is the C-1 signal peak of α-Araf. This result is consistent with the monosaccharide composition analysis results, indicating that β-Galp and α-Araf are the main monosaccharide residues in the structure. By resolving the 1D / 2D-NMR spectra, it was found that LBPA-Ⅰa, LBPA-Ⅱa, and LBPA-Ⅲa have similar structural features, such as... Figure 6 to Figure 20 As shown. Therefore, the signal attribution of LBPA-Ⅰa will be explained as an example.

[0233] Analyze the HSQC, HMBC, COSY, and NOESY spectra of LBPA-Ia. Based on the chemical shift of H, analyze the HSQC (HMBC, COSY, and NOESY spectra). Figure 9The chemical shifts of major sugar residues in LBPA-Ia were assigned and analyzed using COSY ( Figure 11 The chemical shift signals of other H atoms were derived (Table 7). The signal appearing at δ 5.26 / 109.31 ppm was attributed to C-1 of 1,5-α-L-Araf; the signal at δ 5.25 / 109.39 ppm was attributed to C-1 of 1,3-α-L-Araf. The signals appearing at δ 5.10 / 107.48 ppm and δ 5.10 / 101.48 ppm were the C-1 signals of T-α-L-Araf and T-β-L-Araf residues, respectively. The signals near δ 4.71 / 103.54 ppm, δ 4.54 / 103.54 ppm, and δ 4.49 / 103.22 ppm were attributed to 1,3-, 1,3,6-, and 1,6-β-D-Galp, respectively. In addition, the signal appearing at δ4.52 / 102.69ppm belongs to C-1 of 1,4-α-D-GlcpA; the signal near δ4.75 / 100.81ppm belongs to C-1 of T-α-L-Rhap; and the signal at δ1.26, 1.27 / 16.57ppm is the characteristic signal peak of -CH3.

[0234] HMBC ( Figure 10 In the spectrum, the correlation peak (δ66.73 / 5.10ppm) indicates that the C-5 of residue 1,5-α-L-Araf is correlated with the H-1 of T-α-L-Araf and T-β-L-Araf; the correlation peak (δ79.19 / 4.75ppm) indicates that the C-4 of residue 1,4-α-D-GlcpA is correlated with the H-1 of T-α-L-Rhap; and the correlation peak (δ107.48 / 3.80ppm) indicates that the T-α-L-Araf residue is correlated with the H-1 of T-α-L-Rhap. C-1 of 1,3-β-D-Galp is correlated with H-5 of 1,3-α-L-Araf and 1,5-α-L-Araf; the correlation peak (δ 103.54 / 3.74 ppm) indicates a correlation between C-1 of 1,3-β-D-Galp and H-3 of 1,3,6-β-D-Galp; the correlation peak (δ 103.54 / 3.67 ppm) indicates a correlation between C-1 of 1,3-β-D-Galp and H-3 of 1,3-β-D-Galp. NOESY Figure 12 In the spectrum, the correlation peaks (δ 5.26 / 3.80, 3.89 ppm) indicate spatial coupling between H-1 and H-5 of 1,5-α-L-Araf; the correlation peaks (δ 5.25 / 3.77, 3.80 ppm) indicate spatial coupling between H-1 and H-3 of 1,3-α-L-Araf; the correlation peaks (δ

[0235] The concentrations of 5.10 / 3.80 ppm indicate spatial coupling between H-1 of T-α-L-Araf and T-β-L-Araf and H-5 of 1,5-α-L-Araf; the correlation peak (δ)

[0236] The correlation peak (δ 4.71 / 3.74ppm) indicates spatial coupling between H-1 of 1,3-β-D-Galp and H-3 of 1,3,6-β-D-Galp; the correlation peak (δ 4.49 / 3.74ppm) indicates spatial coupling between H-1 of 1,6-β-D-Galp and H-3 of 1,3,6-β-D-Galp; the correlation peak (δ 4.49 / 4.05ppm) indicates spatial coupling between H-1 of 1,6-β-D-Galp and H-6 of 1,6-β-D-Galp or H-6 of 1,3,6-β-D-Galp; the correlation peak (δ

[0237] The correlation peaks (δ 4.52 / 4.05ppm) indicate spatial coupling between H-1 of 1,4-α-D-GlcpA and H-6 of 1,6-β-D-Galp; the correlation peaks (δ 4.75 / 3.59ppm) indicate spatial coupling between H-1 of T-α-L-Rhap and H-4 of 1,4-α-D-GlcpA; and the correlation peaks (δ 5.25 / 3.74ppm) and (δ 5.26 / 3.74ppm) indicate spatial coupling between H-1 of 1,3-α-L-Araf and 1,5-α-L-Araf and H-3 of 1,3,6-β-D-Galp.

[0238] 1D / 2D-NMR spectral analysis of LBPA-Ia, LBPA-IIa, and LBPA-IIIa, combined with monosaccharide composition and molecular weight results, demonstrates that the overall structural characteristics and linkage modes of each sugar residue in LBPA-Ia, LBPA-IIa, and LBPA-IIIa are similar.

[0239] Take →1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→;

[0240] →1)-α-L-Araf-(3→1)-α-L-Araf-(3→1)-α-L-Araf;

[0241] →1)-α-L-Araf-(5→1)-α-L-Araf-(5→1)-α-L-Araf;

[0242] →1)-α-L-Araf-(5→1)-α-L-Araf-(5→1)-β-L-Araf;

[0243] →1)-β-D-Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap and 1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→connected AG-Ⅱ type pectin structures.

[0244] Table 7 Component a 13 C-NMR spectral chemical shift signal assignment

[0245]

[0246]

[0247]

[0248] Similarly, based on the monosaccharide composition and molecular weight analysis results, LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb may have similar structural characteristics, as shown in the 1D-NMR spectra. Figure 21 , Figure 22 and Figure 23 As shown, the signal peaks overlap significantly. A C-1 signal peak for β-Galp exists in the 102–105 ppm range, and a C-1 signal peak for α-Araf exists in the 107–110 ppm range. This result is consistent with the monosaccharide composition analysis, indicating that β-Galp and α-Araf are the main monosaccharide residues in the main chain structure. The signal peaks in the 97–105 ppm range may be chemical shifts of α-D-GalpA and α-L-Rhap; however, due to their weak signals, further analysis is needed. Analysis of their 1D / 2D-NMR spectra revealed that LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb have similar structural characteristics. Figure 21 to Figure 35 (As shown). Therefore, the signal attribution of LBPA-Ⅰb will be explained as an example.

[0249] Analyze the HSQC, HMBC, COSY, and NOESY spectra of LBPA-Ib. Similarly, based on the chemical shift of H, analyze the HSQC (HMBC, COSY, and NOESY spectra). Figure 24 The chemical shifts of major sugar residues in LBPA-Ib were assigned and analyzed using COSY ( Figure 26The chemical shift signals of other H atoms were derived (Table 8). The signal appearing at δ 5.25 / 109.34 ppm was attributed to C-1 of 1,5-α-L-Araf; the signal at δ 5.24 / 109.34 ppm was attributed to C-1 of 1,3-α-L-Araf. The signals appearing at δ 5.09 / 107.56 ppm and δ 5.09 / 101.47 ppm were the C-1 signals of T-α-L-Araf and T-β-L-Araf residues, respectively. Signals near δ 4.69 / 103.22 ppm, δ 4.51 / 103.22 ppm, and δ 4.47 / 103.14 ppm are attributed to the C-1 of 1,3-, 1,3,6-, and 1,6-β-D-Galp, respectively. Comparison revealed that the chemical shifts and characteristics of these anomeric carbon signals are very similar to those of the C-1 of 1,3-, 1,3,6-, and 1,6-β-D-Galp in LBPA-Ⅰa, suggesting the possible presence of an AG structure. The signal at δ 5.09 / 99.81 ppm is the C-1 signal of 1,4-α-D-GalpA, with its C-6 signal appearing at δ - / 175.30 ppm. The signal at δ 5.05 / 97.92 ppm is the C-1 signal of 1,2,4-α-L-Rhap, the C-6 signal appears at δ 1.25,126 / 16.78 ppm, and the signal at δ 1.25,126 / 16.45 ppm is the C-6 signal of 1,2-α-L-Rhap. Furthermore, the signal at δ 4.51 / 102.66 ppm belongs to the C-1 of 1,4-α-D-GlcpA; the signal near δ - / 100.75 ppm belongs to the C-1 of T-α-L-Rhap; and the signal at δ 1.26,1.27 / 16.45 ppm is the characteristic peak of -CH3.

[0250] Comprehensive utilization of HMBC ( Figure 25 ) and NOESY ( Figure 27In the HMBC spectrum, the correlation peak (δ66.93 / 5.09ppm) indicates that the C-5 of residue 1,5-α-L-Araf is correlated with the H-1 of T-α-L-Araf and T-β-L-Araf. Comparison with the HMBC of LBPA-Ⅰa suggests that the C-4 of 1,4-α-D-GlcpA is correlated with the H-1 of T-α-L-Rhap, but no C-4 signal of 1,4-α-D-GlcpA and H-1 signal of T-α-L-Rhap were found in LBPA-Ⅰb. The correlation peak (δ 107.56 / 3.80 ppm) indicates a correlation between the C-1 residue of T-α-L-Araf and the H-5 residue of 1,5-α-L-Araf; the correlation peak (δ 103.22 / 3.66 ppm) indicates a correlation between the C-1 residue of 1,3,6-β-D-Galp and the H-3 residue of 1,3-β-D-Galp, and also indicates a correlation between the C-1 residue of 1,3-β-D-Galp and the H-3 residue of 1,3-β-D-Galp. In the NOESY spectrum, the correlation peaks (δ 5.26 / 3.80, 3.89 ppm) indicate spatial coupling between H-1 and H-5 of 1,5-α-L-Araf; the correlation peaks (δ 5.25 / 3.77, 3.80 ppm) indicate spatial coupling between H-1 and H-3 of 1,3-α-L-Araf; and the correlation peak (δ 5.10 / 3.80 ppm) indicates spatial coupling between T-α-L- Spatial coupling exists between H-1 of Araf and T-β-L-Araf and H-5 of 1,5-α-L-Araf; the correlation peak (δ 4.47 / 3.73 ppm) indicates spatial coupling between H-1 of 1,6-β-D-Galp and H-3 of 1,3,6-β-D-Galp; the correlation peak (δ 4.52 / 4.05 ppm) indicates spatial coupling between H-1 of 1,4-α-D-GlcpA and H-6 of 1,6-β-D-Galp; and the correlation peaks (δ 5.24 / 3.73 ppm) and (δ 5.25 / 3.73 ppm) indicate spatial coupling between H-1 of 1,3-α-L-Araf and 1,5-α-L-Araf and H-3 of 1,3,6-β-D-Galp.

[0251] 1D / 2D-NMR spectral analysis of LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb, combined with their monosaccharide composition and molecular weight results, indicates that the structures and linkage modes of the sugar residues of LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb are similar, with →1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→ and →2,4)-α-L-Rhap-(1→4)- The RG-Ⅰ structure composed of α-D-GalpA-(1→), and the side chain structure connected by →1)-β-D-Galp-(6→1)-β-D-Galp-(3,6→;→1)-α-L-Araf-(5→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-β-L-Araf and →1)-β-D-Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap.

[0252] Table 8 Component b 13 C-NMR spectral chemical shift signal assignment

[0253]

[0254]

[0255]

[0256] In summary, by analyzing the full spectrum information of the six fine subfractions, 239 C signals (including 63 anodic carbon signals) were obtained. Analysis revealed that the anodic carbon signal characteristics of LBPA-Ia, LBPA-IIa, and LBPA-IIIa are similar, with consistent major monosaccharide residues; similarly, the anodic carbon signal characteristics of LBPA-Ib, LBPA-IIb, and LBPA-IIIb are also similar, with consistent major monosaccharide residues. This result demonstrates that LBPA-Ia, LBPA-IIa, and LBPA-IIIa have similar structural characteristics; and LBPA-Ib, LBPA-IIb, and LBPA-IIIb have similar structural characteristics. Therefore, based on the above data analysis combined with the monosaccharide composition and molecular weight results, the six fine subfractions can be divided into two categories: component a (LBPA-Ia, LBPA-IIa, and LBPA-IIIa) and component b (LBPA-Ib, LBPA-IIb, and LBPA-IIIb). Among them, component a may be AG-II type pectin composed of a main chain of β-D-Galp residues and a side chain structure composed of α-L-Araf; component b may be RG-I type pectin composed of a main chain of β-D-Galp residues, including a disaccharide unit structure of →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→) and an AG-II structure similar to component a.

[0257] 2.2.4 SEM and AFM analysis of wolfberry pectin

[0258] like Figure 36 As shown, the surface morphology of the six fine subfractions was observed using SEM and AFM. The results showed that components a and b both exhibited a predominantly lamellar, loosely structured structure, indicating the presence of branching structures. LBPA-Ⅰa had a lamellar surface covered with regularly arranged banded structures, with irregular protrusions or depressions observed on the edges or surface of the bands. LBPA-Ⅱa showed reduced banded structures, exhibiting a lamellar, porous structure, with irregular protrusions or depressions also appearing at the fracture surface edges. LBPA-Ⅲa showed granular and porous structures, while the irregular protrusions or depressions at the fracture surface edges decreased. In comparison, LBPA-Ⅰb showed reduced porous structures and relatively thicker lamellar layers; LBPA-Ⅱb had a relatively smooth surface with granular structures; and LBPA-Ⅲb had an even smoother surface.

[0259] LBPA-Ia forms long fibrous structures with most nodes not cross-linked, indicating a potentially ordered structure on the sample surface. This orderliness may be due to the directional arrangement of polysaccharide molecules on the surface. 3D images show regularly ordered protrusions, which may be related to the aggregation mode or intermolecular interactions of polysaccharide molecules. LBPA-IIa forms a relatively tight macromolecular network; this ring-like arrangement may be due to interactions between polysaccharide molecules. 3D images show dense protrusions on the sample surface, suggesting the possible presence of interwoven polysaccharide structures. LBPA-IIIa exhibits spherical aggregates on the surface, and 3D images show some relatively high protrusions, possibly due to the formation of larger aggregates or polymers of polysaccharide molecules in localized areas. LBPA-Ib forms alternating light and dark spots, and 3D images show densely packed, unevenly distributed protrusions. This structure may be due to the non-uniformity of polysaccharide molecule deposition or diffusion on the surface. Similar to LBPA-Ib, LBPA-IIb forms dense, loose protrusions, with most protrusions becoming smaller. A few protrusions remain relatively high, which may be due to the aggregation of some polysaccharide molecules into larger aggregates, or due to the presence of regions with different properties on the sample surface. The smoother surface of LBPA-IIIb may suggest that the polysaccharides have formed a more compact arrangement or structure in this region, possibly due to the interaction forces between polysaccharide molecules. These findings demonstrate that changes in the structure of wolfberry pectin can cause changes in its morphology.

[0260] 2.3 Discussion

[0261] 2.3.1 Structural characteristics of component a

[0262] Analysis of the NMR spectra of component a revealed similarities in the 1D / 2D-NMR patterns of LBPA-Ⅰa, LBPA-Ⅱa, and LBPA-Ⅲa, indicating identical monosaccharide residues. This suggests that component a shares similar structural features, consistent with its monosaccharide composition analysis and molecular weight distribution. Therefore, 2D-NMR analysis was performed using LBPA-Ⅰa, which exhibited the clearest and most complete signal. The analysis revealed that its main sugar residues are 1,5-α-L-Araf (C-1 signal δ5.26 / 109.31ppm), 1,3-α-L-Araf (C-1 signal δ5.25 / 109.39ppm), T-α-L-Araf (C-1 signal δ5.10 / 107.48ppm), and T-β-L-Araf (C-1 signal δ5.10 / 107.48ppm). 10 / 101.48ppm) and 1,3-, 1,3,6- and 1,6-β-D-Galp (C-1 signal δ4.71 / 103.54ppm, δ4.54 / 103.54ppm and δ4.49 / 103.22ppm), as well as 1,4-α-D-GlcpA (C-1 signal δ4.52 / 102.69ppm) and T-α-L-Rhap (C-1 signal δ4.75 / 100.81ppm). Analysis of the HMBC and NOESY spectra revealed the following correlations: C-5 of residue 1,5-α-L-Araf is correlated with H-1 of T-α-L-Araf and T-β-L-Araf; C-4 of 1,4-α-D-GlcpA is correlated with H-1 of T-α-L-Rhap; C-1 of 1,3-β-D-Galp is correlated with H-3 of 1,3,6-β-D-Galp; C-1 of 1,3-β-D-Galp is correlated with H-3 of 1,3-β-D-Galp; H-1 of 1,5-α-L-Araf and H-5 of 1,5-α-L-Araf are spatially coupled; and H-1 of 1,3-α-L-Araf and 1,3-α-L-Araf... Spatial coupling exists between H-3 of 1,3,6-β-D-Galp and H-3 of 1,3-β-D-Galp; spatial coupling exists between H-1 of 1,6-β-D-Galp and H-6 of 1,6-β-D-Galp or H-6 of 1,3,6-β-D-Galp; spatial coupling exists between H-1 of 1,4-α-D-GlcpA and H-6 of 1,6-β-D-Galp; spatial coupling exists between H-1 of T-α-L-Rhap and H-4 of 1,4-α-D-GlcpA; and spatial coupling exists between H-1 of 1,3-α-L-Araf and 1,5-α-L-Araf and H-3 of 1,3,6-β-D-Galp.

[0263] Based on the above analysis, the overall structural characteristics of component a are inferred to be: mainly composed of 1,6-β-D-Galp as the backbone, with possible branch substitution at O-3, and the branches are likely mainly Araf, similar to the structure of AG-II type pectin. Based on the monosaccharide composition, molecular weight distribution, and 1D-NMR spectral characteristics, the main differences between LBPA-Ia, LBPA-IIa, and LBPA-IIIa are in the length of the branched chains, etc.

[0264] 2.3.2 Structural characteristics of component b

[0265] Nuclear magnetic resonance (NMR) spectral analysis of component b revealed similar 1D / 2D-NMR characteristics in LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb, indicating identical monosaccharide residues. This suggests that components b also share similar structural features, consistent with their monosaccharide composition analysis and molecular weight distribution. 2D-NMR analysis of LBPA-Ⅰb, which exhibited the clearest signal, revealed its main sugar residues to be 1,5-α-L-Araf (C-1 signal δ 5.25 / 109.34 ppm); 1,3-α-L-Araf (C-1 signal δ 5.24 / 109.34 ppm); T-α-L-Araf and T-β-L-Araf residues (C-1 signals δ 5.09 / 107.56 ppm and δ 5.09 / 101.47 ppm, respectively); 1, The C-1 signals of 3-, 1,3,6-, and 1,6-β-D-Galp (C-1 signals δ 4.69 / 103.22ppm, δ 4.51 / 103.22ppm, and δ 4.47 / 103.14ppm, respectively) were found to be very similar to the C-1 chemical shifts and characteristics of 1,3-, 1,3,6-, and 1,6-β-D-Galp in component LBPA-Ⅰa, suggesting that LBPA-Ⅰb may have a structure similar to LBPA-Ⅰa. Simultaneously, anomeric carbon signals were observed for 1,4-α-D-GalpA (C-1 signal δ 5.09 / 99.81 ppm), 1,2,4-α-L-Rhap (C-1 signal δ 5.05 / 97.92 ppm), and 1,2-α-L-Rhap (C-6 signal δ 1.25, 126 / 16.45 ppm), suggesting the presence of an RG-I structure in the LBPA-Ⅰb structure. The linkage sequence of each monosaccharide residue was determined using a combination of HMBC and NOESY spectroscopy. The results showed that C-5 of residue 1,5-α-L-Araf was correlated with H-1 of T-α-L-Araf and T-β-L-Araf; C-1 of 1,3,6-β-D-Galp was correlated with H-3 of 1,3-β-D-Galp; C-1 of 1,3-β-D-Galp was correlated with H-3 of 1,3-β-D-Galp; H-1 of 1,6-β-D-Galp was spatially coupled with H-6 of 1,3,6-β-D-Galp; H-1 of 1,4-α-D-GlcpA was spatially coupled with H-6 of 1,6-β-D-Galp; and H-1 of 1,3-α-L-Araf and 1,5-α-L-Araf was spatially coupled with H-3 of 1,3,6-β-D-Galp.

[0266] Based on the above analysis, the overall structural characteristics of component b are inferred as follows: it is mainly composed of 1,6-β-D-Galp, with the 1-position of Galp linked to the RG-I backbone. There are branch substitutions at the O-3 positions, with the branch mainly composed of 1,3-β-D-Galp. It also contains branching similar to that of component a (AG-II). Furthermore, a high Glc content was found in all components b (16.5% in LBPA-Ib), with Glc present in the side chain structure of pectin. Due to severe overlap in the 1D / 2D-NMR spectra of component b, no Glc signal was found. Based on the similarity characteristics combined with monosaccharide composition analysis, the main differences between LBPA-Ib, LBPA-IIb, and LBPA-IIIb are likely in the alternating linkages of the RG-I backbone with 1,4-α-D-GalpA and 1,2- / 1,2,4-α-L-Rhap residues, and the length of the branching chains.

[0267] 2.3.3 Apparent morphological characteristics of components a and b

[0268] The morphological characteristics of pectin (e.g., gel, solution, or solid state) are closely related to its molecular structure, molecular weight, and environmental conditions (e.g., pH, temperature). These factors collectively determine the solubility of pectin and its interactions with other molecules, thus affecting its bioactivity. For example, gel-state pectin is more effective in drug sustained-release applications. In this study, both components a and b exhibited a relatively loose structure dominated by lamellar structures, indicating the presence of branched structures in both. Scanning electron microscopy (SEM) revealed that component a exhibited various morphologies, including fibrous and network structures. This may be due to its larger molecular weight, resulting in a higher electron density and a greater tendency to aggregate, thus presenting a network structure. Component b, on the other hand, exhibited a dispersed network or granular structure, possibly due to its smaller molecular weight, lower electron density, and less tendency to aggregate. Atomic force microscopy (AFM) further clarified the differences in surface morphology between components a and b.

[0269] Monosaccharide composition significantly influences the charge distribution and intermolecular forces of pectin, thereby determining its gel-forming ability and bioactivity. For example, pectin with higher galacturonic acid (GalA) content typically exhibits stronger immunomodulatory effects. Components a and b are predominantly composed of galactose (Gal) and arabinose (Ara), but component b also contains higher levels of rhamnose (Rha) and galacturonic acid (GalA). Due to the higher GalA content, component b may exhibit more pronounced aggregation and a smoother morphology in SEM; in AFM, it displays a smooth network structure. Therefore, it is hypothesized that disaccharide units containing alternating 1,4-α-D-GalpA and 1,2- / 1,2,4-α-L-Rhap units are associated with the immunomodulatory activity of pectin.

[0270] 2.4 Summary

[0271] This embodiment uses nuclear magnetic resonance spectroscopy to study the overall structural characteristics of six fine subfractions of wolfberry pectin. The results are as follows:

[0272] (1) 1D / 2D-NMR spectral analysis results show that LBPA-Ⅰa, LBPA-Ⅱa, and LBPA-Ⅲa have structural similarities. Their overall structural characteristics and the linkage of each sugar residue are as follows: →1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→1); →1)-α-L-Araf-(3→1)-α-L-Araf-(3→1)-α-L-Araf; →1)-α-L-Araf-(5→1)-α-L-Araf f-(5→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-α-L-Araf-(5→1)-β-L-Araf;→1)-β-D-Galp-(6→1 )-α-D-GlcpA-(4→1)-α-L-Rhap; 1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→.

[0273] (2) 1D / 2D-NMR spectral analysis results showed that LBPA-Ⅰb, LBPA-Ⅱb, and LBPA-Ⅲb have similar structures. Their overall structure and the connection mode of each sugar residue are as follows: →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1;→1)-β-D-Galp-(3→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→;→1)-β-D-Galp-(6→1)-β-D-G alp-(3,6→1;→1)-α-L-Araf-(3→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-α-L-Araf;→1 )-α-L-Araf-(5→1)-β-L-Araf;→1)-β-D-Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap.

[0274] (3) Based on the 1D / 2D-NMR spectral analysis, monosaccharide composition and molecular weight distribution of the six fine subfractions, they were divided into two categories: component a (LBPA-Ⅰa, LBPA-Ⅱa and LBPA-Ⅲa) and b (LBPA-Ⅰb, LBPA-Ⅱb and LBPA-Ⅲb).

[0275] (4) SEM and AFM analyses showed that both components a and b exhibited a relatively loose structure dominated by lamellar structures, indicating that both had branched structures. Component b showed a smoother morphology, which may be related to the increased content of galacturonic acid (GalA).

[0276] (5) It is speculated that component a may be AG-II type pectin composed of a main chain of β-D-Galp residues and a side chain structure composed of α-L-Araf; component b may be RG-I type pectin composed of a main chain of β-D-Galp residues, including a disaccharide unit structure of →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→) and a structure similar to component a (AG-II), wherein the disaccharide unit structure of alternating 1,4-α-D-GalpA and 1,2- / 1,2,4-α-L-Rhap is related to the immunomodulatory activity of pectin.

[0277] Example 3

[0278] 3. Analysis of the fine structural features of wolfberry pectin

[0279] 3.1.1 Partial acid hydrolysis

[0280] Weigh 40.0 mg each of the pectin samples from Example 2 (LBPA-I, LBPA-II, and LBPA-III) and Example 2 (LBPA-Ia and LBPA-Ib) into hydrolysis flasks, add 8 mL of 0.05 M trifluoroacetic acid, and hydrolyze at 100°C for 1 h, 2 h, 3 h, 4 h, and 5 h. Dialyze the hydrolysate (molecular weight cutoff 3500 Da), collecting the dialysate from the inside and outside of the dialysis bag. Dry to remove excess trifluoroacetic acid, dissolve in an appropriate amount of distilled water, and freeze-dry to obtain partial acid hydrolysis products at different hydrolysis times. HPLC and HPGPC were used to analyze the optimal hydrolysis time.

[0281] 3.1.2 Nuclear Magnetic Resonance Spectroscopy Analysis

[0282] Weigh 0.5 mg each of the pectin samples from Example 2 after partial acid hydrolysis in 3.1.1 above: LBPA-Ⅰ, LBPA-Ⅱ and LBPA-Ⅲ, and the pectin samples from Example 2: LBPA-Ⅰa and LBPA-Ⅰb, and dissolve them in 0.5 mL L2O. Use a Bruker AV600 nuclear magnetic resonance spectrometer to determine the 1H-NMR, 13C-NMR, HSQC, HMBC, NOESY and COSY spectra of the samples.

[0283] 3.1.3 Methylation Analysis

[0284] Methylation analysis is an effective method for analyzing the substitution sites and linkage modes of polysaccharides. Weigh 5.0 mg each of thoroughly dried sample from Example 2 (LBPA-I) and samples from Example 2 (LBPA-Ia and LBPA-Ib), add 1 mL of anhydrous dimethyl sulfoxide (DMSO, dehydrated using 4A molecular sieve), seal under N2, and stir at 40°C (overnight) until the pectin sample is fully dissolved. Add 20 mg of dried NaOH powder to the fully dissolved sample, seal under N2, and stir magnetically until fully dissolved. Then, slowly add 1 mL of iodomethane under ice bath conditions and react for 2 h. After terminating the reaction with 1 mL of distilled water, extract the methylated sample with an equal volume of dichloromethane, collect the dichloromethane layer, repeat the extraction three times, combine the dichloromethane layers, and back-extract with distilled water three times, then dry the dichloromethane. Detect the methylated pectin sample at 3400 cm⁻¹ using Fourier transform infrared spectroscopy. -1 Check if a hydroxyl (-OH) absorption peak appears nearby. If no absorption peak appears, it indicates that the sample is completely methylated; otherwise, a second methylation is required (until methylation is complete).

[0285] 1 mL of 2M trifluoroacetic acid was added to the fully methylated pectin sample, and hydrolysis was carried out at 110 °C for 6 h. Methanol was repeatedly added to the hydrolyzed pectin sample, and the mixture was dried until neutral. 1 mL of freshly prepared 30 mg / mL NaBD4 (sodium borodeuteride) solution was added to the hydrolyzed sample, and the mixture was stirred overnight at room temperature. Then, 0.5 mL of acetic acid-methanol solution was added, and the mixture was dried until no boric acid was present. 0.5 mL each of acetic anhydride and anhydrous pyridine were added to the dried sample, and the mixture was sealed under N2 and acetylated at 100 °C for 2 h. 1 mL of distilled water was added, and excess acetic anhydride was removed by vortexing. The sample was extracted three times with 2 mL of dichloromethane, and the chloroform layer was collected. Then, the sample was back-extracted three times with distilled water, and the dichloromethane was dried to obtain the methylated sample to be tested. The dried methylated sample was redissolved in 0.5 mL of dichloromethane and analyzed by GC-MS.

[0286] 3.2 Experimental Results

[0287] 3.2.1 Results of partial acid hydrolysis of wolfberry pectin

[0288] Two types of pectin polysaccharides (components a and b) are fine subfractions of total pectin, and their structures are relatively conserved. Therefore, partial acid hydrolysis was first performed on three total pectin compounds (LBPA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ) to lay the foundation for further analysis of the main and side chain structural composition of components a and b. After hydrolysis with 0.05M TFA at 100℃ for 1h, 2h, 3h, 4h, and 5h, dialyzing (3500Da) was performed, and the dialysate inside and outside the dialysis bag was collected. The high molecular weight component inside the bag is represented by H; the low molecular weight component outside the bag is represented by L. Taking LBPA-I as an example, the solutions inside the bag after hydrolysis were named LBPA-I-H-1, LBPA-I-H-2, LBPA-I-H-3, LBPA-I-H-4, and LBPA-I-H-5, respectively; the solutions outside the bag were named LBPA-I-L-1, LBPA-I-L-2, LBPA-I-L-3, LBPA-I-L-4, and LBPA-I-L-5, respectively. HPGPC analysis showed that the molecular weight distribution of the solutions inside the bag gradually became a single, relatively symmetrical peak with increasing hydrolysis time. When the hydrolysis time was 3 h, LBPA-I-H-3 (Mw = 4.04 kDa) showed a single relatively symmetrical peak in the Vo and Vt ranges. However, as the hydrolysis time continued to increase to 4 h and 5 h, the molecular weight distributions of LBPA-I-H-4 (Mw = 3.98 kDa) and LBPA-I-H-5 (Mw = 3.07 kDa) no longer changed significantly, indicating that 3 h may be the optimal hydrolysis time, during which most of the side chain structures were hydrolyzed, and LBPA-I-H-3 became the main chain portion of LBPA-I. Figure 37 Similarly, the hydrolysis trends of LBPA-II and LBPA-III are also similar, with LBPA-II-H-3 and LBPA-III-H-3 being their main chain components, respectively.

[0289] like Figure 38 As shown in (A), (B), (C), and (D), in contrast, the Mw values ​​of the three main chain fractions of total pectin after acid hydrolysis—LBPA-Ⅰ-H-3, LBPA-Ⅱ-H-3, and LBPA-Ⅲ-H-3—are 4.04 kDa, 5.97 kDa, and 5.65 kDa, respectively. These Mw values ​​exhibit relatively narrow, single, symmetrical peaks on the HPGPC spectrum, indicating that they are all homogeneous components. Monosaccharide composition results show that after hydrolysis, the content of the monosaccharide Gal in the main chain fractions (LBPA-Ⅰ-H-3, LBPA-Ⅱ-H-3, and LBPA-Ⅲ-H-3) significantly increases, while the content of Ara significantly decreases; the contents of other monosaccharides do not change much (Table 9). This suggests that the main chain fractions of the three total pectin fractions may primarily consist of Gal as the main monosaccharide, with Ara possibly appearing at the outer ends of the side chains. By analyzing the monosaccharide composition and molecular weight distribution, it is speculated that the structural characteristics of the main chain portions of the three total pectins (LBPA-Ⅰ-H-3, LBPA-Ⅱ-H-3 and LBPA-Ⅲ-H-3) may be similar.

[0290] Table 9 shows the monosaccharide composition of the inner and outer components of the acid hydrolysis bag.

[0291]

[0292]

[0293] 3.2.2 Results of Nuclear Magnetic Resonance Spectroscopy Analysis of the Main Chain

[0294] The above analysis indicates that the main chain structures of the three total pectin groups may be similar. To further verify this, the 1D / 2D-NMR spectra of the three wolfberry total pectin main chain components (LBPA-Ⅰ-H-3, LBPA-Ⅱ-H-3, and LBPA-Ⅲ-H-3) were analyzed. Figure 39 , Figure 40 and Figure 41 As shown, the H-1 signals all appeared in the range of 4.4–4.7 ppm, and the C-1 signals all appeared in the range of 95–105 ppm. The signals in the range of 102–105 ppm were the C-1 signal peaks of β-Galp, which is consistent with the monosaccharide composition analysis and partial acid hydrolysis results, indicating that the main chain structure is dominated by β-Galp monosaccharide residues. The 1D / 2D-NMR spectra of LBPA-Ⅰ-H-3, LBPA-Ⅱ-H-3, and LBPA-Ⅲ-H-3 were analyzed. Figure 39 to Figure 53 The results showed that the overall signal characteristics of the three wolfberry total pectin backbones were similar, with the main monosaccharide residues being the same, indicating that they have similar structural features. The LBPA-Ⅲ-H-3 spectrum showed a more comprehensive anodic carbon signal; therefore, LBPA-Ⅲ-H-3 was used as an example to illustrate the signal attribution of each sugar residue.

[0295] like Figure 50 , Figure 51 , Figure 52 and Figure 53 The figures shown are the HSQC, HMBC, COSY, and NOESY spectra of LBPA-Ⅲ-H-3, respectively. Based on the chemical shift of H, the HSQC (… Figure 50The chemical shifts of the major sugar residues in LBPA-Ⅲ-H-3 were assigned, and the chemical shift signals of other H residues were deduced using COSY (Table 10). Signals at δ 5.09 / 99.55 ppm and 173.89 ppm were assigned to C-1 and C-6 of 1,4-α-D-GalpA. Signals at δ 5.22 / 99.01 ppm and δ 5.03 / 97.73 ppm were 1,2- and 1,2,4-α-L-Rhap residues, respectively. Comparison revealed similarities to the signal peaks of LBPA-Ⅰb in the 97–105 ppm range. The chemical shifts at C-6 of LBPA-Ⅲ-H-3 at 1.25 ppm and 1.26 ppm are characteristic signal peaks of methyl groups. This indicates the presence of disaccharide units with alternating α-D-GalpA and α-L-Rhap residues in LBPA-Ⅲ-H and LBPA-Ⅰb, suggesting a RG-Ⅰ pectin backbone structure. The signals appearing near δ4.70 / 103.88ppm, δ4.63 / 103.38ppm, and δ4.44 / 103.47ppm are close to and similar in characteristics to the corresponding chemical shifts in LBPA-Ⅰa, and are attributed to 1,3-, 1,3,6-, and 1,6-β-D-Galp, respectively, indicating that β-D-Galp monosaccharide residues are also present in the main chain structure.

[0296] To further determine the linkage sequence between individual sugar residues, HMBC ( Figure 51 ) and NOESY ( Figure 53 In the HMBC spectrum, the correlation peak (δ78.15 / 5.22ppm) indicates a correlation between C-4 of residue 1,4-α-D-GalpA and H-1 of residue 1,2-α-L-Rhap; the correlation peak (δ76.30 / 5.09ppm) indicates a correlation between C-2 of residues 1,2-α-L-Rhap and 1,2,4-α-L-Rhap and H-1 of residue 1,4-α-D-GalpA, indicating the presence of the RG-I backbone structure. The correlation peak (δ68.63 / 4.54ppm) indicates a correlation between C-6 of 1,6-β-D-Galp and H-1 of 1,4-α-D-GlcpA; the correlation peak (δ81.66 / 4.44ppm) indicates a correlation between C-4 of 1,2,4-α-L-Rhap and H-1 of 1,6-β-D-Galp. In the NOESY spectrum, the correlation peak (δ4.7 / 3.86ppm) indicates spatial coupling between H-1 of 1,3-β-D-Galp and H-3 of 1,3-β-D-Galp and 1,3,6-β-D-Galp; the correlation peak (δ4.44 / 3.94ppm) indicates spatial coupling between H-1 of 1,6-β-D-Galp and H-6 of 1,3,6-β-D-Galp.

[0297] Analysis of the monosaccharide composition and 1D / 2D-NMR spectral resolution revealed the main chain structure of LBPA-Ⅲ-H-3: disaccharide units linked by →4)-α-D-GalpA-(1→2 / 2,4)-α-L-Rhap-(1→), and the presence of →6)-β-D-Galp-(1→6 / 3,6)-β-D-Galp-(1→ linkages, consistent with the RG-Ⅰ type structure. It is speculated that this structural feature is also a common main chain structure characteristic of the three types of total pectin from wolfberry.

[0298] Table 10 Main Chain Section 13 C-NMR spectral chemical shift signal assignment

[0299]

[0300]

[0301]

[0302] 3.2.3 Results of partial acid hydrolysis of components a and b

[0303] To further investigate the fine structural characteristics of components a and b, LBPA-Ia and LBPA-Ib were used as representative components of the two types of pectin polysaccharides, respectively, and partial acid hydrolysis analysis was performed. Under the optimal hydrolysis time of 3 h, their main chain components were LBPA-Ia-H-3 and LBPA-Ib-H-3, respectively. HPGPC results showed that they were homogeneous components, with LBPA-Ia-H-3 (5.28 kDa) having a higher molecular weight than LBPA-Ib-H-3 (3.81 kDa). Monosaccharide composition results showed that LBPA-Ia-H-3 was composed of Gal, Ara, and GlcA, with Gal as the main monosaccharide; while LBPA-Ib-H-3 was composed of Man, GlcA, Rha, GalA, Glc, Gal, Xyl, and Ara, with Glc and Gal as the main monosaccharides (Table 11), indicating that the main chain components of components a and b have different monosaccharide compositions. To further analyze the relationship between the main chain portions of components a and b and the three main chain portions of total pectin, the monosaccharide composition, molecular weight, and peak times on the HPGPC spectrum of LBPA-Ia-H-3, LBPA-Ib-H-3, and LBPA-I-H-3 (the main chain portion of total pectin LBPA-I) were compared. Figure 54As shown, HPGPC results indicate that the molecular weight of LBPA-I-H-3 (4.04 kDa) is close to that of LBPA-I-Ib-H-3 (3.81 kDa), and their peak times are similar (peaks almost overlap); while the molecular weight of LBPA-I-Ia-H-3 (5.28 kDa) is larger. Monosaccharide composition comparison analysis shows that the monosaccharide composition of LBPA-Ib-H-3 is highly similar to that of LBPA-I-H-3; however, it differs from that of LBPA-I-Ia-H-3 in several ways, such as... Figure 55 As shown.

[0304] The above data indicate that LBPA-Ia-H-3 and LBPA-Ib-H-3 have different molecular weights and different monosaccharide compositions; LBPA-Ib-H-3 and LBPA-I-H-3 have similar molecular weights and highly similar monosaccharide compositions; this suggests that LBPA-Ib-H-3 and LBPA-I-H-3 have structural homology, but are different from the structure of LBPA-Ia-H-3. It is speculated that the main chain portion of the three total pectins is structurally similar to that of component b, but different from that of component a.

[0305] Table 11 Monosaccharide composition of LBPA-Ia-H-3 and LBPA-Ib-H-3

[0306]

[0307] 3.2.4 Results of partial methylation of the pectin backbone of wolfberry

[0308] The above analysis indicates that the main chain portion of the three total pectin compounds from *Lycium barbarum* may be similar, showing homology with component b; the main chain structure of component a differs from it. To further verify this hypothesis, LBPA-I-H-3, LBPA-Ia-H-3, and LBPA-Ib-H-3 were used as representative components of the three total pectin compounds and two types of pectin polysaccharides (components a and b), and methylation analysis was performed to determine the glycosidic bond linkage type of their hydrolyzed main chain portion. Figure 56As shown, the total ion chromatograms of LBPA-I-H-3 and LBPA-Ib-H-3 are similar; while the total ion chromatogram of LBPA-Ia-H-3 is significantly different from that of LBPA-Ib-H-3 and LBPA-I-H-3. This result confirms the above analysis, indicating that the main chain portion of the three total pectins is highly similar in structure to the main chain portion of component b, but different from the main chain portion of component a. Specific results of methylation analysis show that the glycosidic bond linkage types of LBPA-I-H-3 and LBPA-Ib-H-3 are highly consistent, mainly including T-Glcp, T-Galp, 1,4-Galp, 1,3-Galp, 1,6-Galp, and 1,3,6-Galp. All show that 1,6-Galp has the largest proportion at 33.3% and 27.2%, respectively, followed by 1,3-Galp and 1,3,6-Galp, and also contains 1,4-Galp and 1,2,4-Rhap (Table 12). In contrast, the glycosidic bond linkage types of LBPA-Ia-H-3 mainly include T-Araf, T-Arap, 1,3-Araf, 1,5-Araf, T-Galp, 1,3-Galp, 1,6-Galp, and 1,3,6-Galp. Among them, 1,6-Galp accounts for the largest proportion at 29.7%, followed by 1,3,6-Galp at 19.5%. This indicates that the backbone of LBPA-Ia-H-3 is mainly composed of 1,6-Galp, but there are no 1,4-Galp and 1,2,4-Rhap glycosidic bond types, which is significantly different from LBPA-I-H-3 and LBPA-Ib-H-3.

[0309] The methylation analysis results above indicate that the three total pectins share similar main chain structural characteristics (represented by LBPA-Ⅰ-H-3), with 1,6-Galp as the predominant component and a small number of 1,4-Galp and 1,2,4-Rhap glycosidic bonds, indicating the presence of a relatively short RG-Ⅰ type backbone structure. Simultaneously, it demonstrates that the main chain structures of the three total pectins are highly similar to those of component b, but different from those of component a. Component b is predominantly 1,6-Galp, with the presence of 1,4-Galp and 1,2,4-Rhap glycosidic bonds, consistent with the characteristics of RG-Ⅰ type pectin; component a is predominantly 1,6-Galp, lacking 1,4-Galp and 1,2,4-Rhap glycosidic bonds, consistent with the structural characteristics of AG-Ⅱ type pectin.

[0310] Table 12 Glycosidic bond types in the main chain structure

[0311]

[0312] In summary, analysis of the monosaccharide composition, molecular weight distribution, methylation, and 1D / 2D-NMR spectra of the main chain of three total pectins (represented by LBPA-Ⅰ-H-3) revealed similar structural characteristics: predominantly 1,6-Galp, with glycosidic bonds of 1,4-Galp and 1,2,4-Rhap, classifying them as RG-Ⅰ type pectins. Partial acid hydrolysis analysis of the fine structural composition and characteristics of components a and b showed that their main chain composition was predominantly Gal, and their side chain composition was predominantly Ara. Methylation analysis revealed that the glycosidic bond linkages of the main chains of components a and b were different. Component b had 1,6-Galp as the main glycosidic bond and also contained 1,4-Galp and 1,2,4-Rhap, indicating that its main chain contained a relatively short type I rhamnose-galacturonic acid (RG-I) backbone structure. In contrast, the main chain of component a had 1,6-Galp as the main glycosidic bond, exhibiting the structural characteristics of type II arabinogalactan (AG-II).

[0313] By comparing and analyzing the main chain monosaccharide composition, molecular weight distribution, and methylation analysis results of three total pectins, components a and b (LBPA-Ⅰ-H-3, LBPA-Ⅰa-H-3, and LBPA-Ⅰb-H-3 as representative components), it was found that the three total pectins from wolfberry share a common main chain structure with component b, while their main chain structure differs from that of component a. Combining the compositional analysis, molecular weight analysis, main chain structure, side chain structure, and 1D / 2D-NMR spectral analysis results of the three total pectins, components a and b, the fine structural characteristics of the two types of pectin polysaccharides were finally obtained:

[0314] Fine structure of component a (AG-II): Main chain → 1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→, where Gal is replaced by 5 side chains at the O-3 position: ①→1)-α-L-Araf-(3→1)-α-L-Araf-(3→1)-α-L-Araf, ②→1)-α-L-Araf-(5→1)- α-L-Araf-(5→1)-α-L-Araf, ③→1)-α-L-Araf-(5→1)-α-L-Araf-(5→1)-β-L-Araf, ④→1)-β-D-Galp- (6→1)-α-D-GlcpA-(4→1)-α-L-Rhap, ⑤→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→.

[0315] The fine structure of component b is characterized by a predominantly →1)-β-D-Galp-(3→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→); and a short-chain structure consisting of →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→), along with →1)-β-D-Galp-(6→1)-β-D-Galp The AG-II structure of similar component a is linked by -(3,6→1;→1)-α-L-Araf-(3→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-β-L-Araf and →1)-β-D-Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap.

[0316] like Figure 57 The diagram shows the proposed structures of components a and b.

[0317] 3.3 Discussion

[0318] 3.3.1 Main chain structural characteristics of wolfberry pectin

[0319] Three main chain fractions of total pectin from wolfberry were obtained through partial acid hydrolysis. The monosaccharide changes inside and outside the dialysis bag during hydrolysis were similar, indicating that the composition and connection mode of the main and side chains are similar: Gal is the main monosaccharide in the main chain fraction, and Ara is the main monosaccharide in the side chains. Nuclear magnetic resonance (NMR) spectroscopy analysis revealed very close H-1 and C-1 chemical shifts and consistent signal peak characteristics in the main chain fractions of the three total pectin fractions (LBPA-Ⅰ-H-3, LBPA-Ⅱ-H-3, and LBPA-Ⅲ-H-3), indicating very similar main chain structures. 2D-NMR analysis of LBPA-Ⅲ-H-3, which showed the most comprehensive signal, revealed that the main sugar residues in LBPA-Ⅲ-H-3 include 1,4-α-D-GalpA (C-1 signal δ 5.09 / 99.55 ppm), 1,2- and 1,2,4-α-L-Rhap (C-1 signal δ 5.22 / 99.01 ppm and δ ...

[0320] (5.03 / 97.73ppm). Also present were 1,3-, 1,3,6- and 1,6-β-D-Galp (C-1 signal δ4.70 / 103.88ppm, δ4.63 / 103.38ppm and δ4.44 / 103.47ppm) sugar residues. Next, by combining HMBC and NOESY spectra, the connection sequence between various sugar residues was analyzed. It was found that in the HMBC spectrum, the C-4 of sugar residue 1,4-α-D-GalpA was correlated with the H-1 of 1,2-α-L-Rhap; the C-2 of 1,2-α-L-Rhap and 1,2,4-α-L-Rhap were correlated with the H-1 of 1,4-α-D-GalpA; and the C-4 of 1,2,4-α-L-Rhap was correlated with the H-1 of 1,6-β-D-Galp. This indicates that there are disaccharide units with alternating α-D-GalpA and α-L-Rhap residues in LBPA-Ⅲ-H, which is a characteristic of the main chain structure of RG-Ⅰ type pectin. At the same time, 1,6-β-D-Galp sugar residues are connected to the O-4 of 1,2,4-α-L-Rhap. In the NOESY spectrum, spatial coupling exists between H-1 of the sugar residues 1,3-β-D-Galp and H-3 of 1,3-β-D-Galp and 1,3,6-β-D-Galp; spatial coupling also exists between H-1 of 1,6-β-D-Galp and H-6 of 1,3,6-β-D-Galp, indicating the presence of 1,3- and 1,6-linked Galp in LBPA-Ⅲ-H-3. Combined with the changes in monosaccharide composition of the main chain LBPA-Ⅲ-H-3 after hydrolysis (a significant increase in Gal content, while Ara is hydrolyzed), this suggests that the 1,6- and 1,3,6-β-D-Galp sugar residues may constitute the main chain of AG-Ⅱ type pectin. The analysis revealed that the main chain structure of the three types of total pectin from wolfberry was very similar. Their common main chain structure was dominated by 1,6-Galp, and contained glycosidic bonds of 1,4-Galp and 1,2,4-Rhap, which conforms to the characteristics of RG-I type pectin.

[0321] 3.3.2 Fine structural features of components a and b

[0322] By analyzing the relationship between the main chains of total pectin in wolfberry and its fine subfractions (represented by LBPA-I and its subfractions), it was found that the main chains of LBPA-I-H-3 and LBPA-Ib-H-3 may be similar, but different from those of LBPA-Ia-H-3. To verify this finding, methylation analysis was performed on the glycosidic bond composition of LBPA-I-H-3, LBPA-Ia-H-3, and LBPA-Ib-H-3. The results showed that LBPA-I-H-3 and LBPA-Ib-H-3 had the same major glycosidic bond types, mainly T-Glcp, T-Galp, 1,4-Galp, 1,3-Galp, 1,6-Galp, and 1,3,6-Galp, with 1,6-Galp being the predominant glycosidic bond (33.3% and 27.2%), followed by 1,3-Galp (20.3% and 23.7%). Both also contained 1,4-Galp (4.0% and 7.6%) and 1,2,4-Rh... The total pectin LBPA-Ia-H-3 contains 0.7% and 2.0% glycosidic bonds; while the main glycosidic bond types of LBPA-Ia-H-3 include T-Araf, T-Arap, 1,3-Araf, 1,5-Araf, T-Galp, 1,3-Galp, 1,6-Galp, and 1,3,6-Galp. Although it also has 1,6-Galp glycosidic bonds as the main component (29.7%), followed by 1,3,6-Galp (19.5%), this is different from LBPA-I-H-3 and LBPA-Ib-H-3. Therefore, it indicates that the main chain of total pectin LBPA-I and subfraction LBPA-Ib is similar, but different from subfraction LBPA-Ia. This further illustrates that the main chain structure of the three total pectin components of wolfberry is similar to that of component b, but different from that of component a. The results showed that the three total pectins of wolfberry and component b had short chain structures with alternating 1,4-α-D-GalpA and 1,2- / 1,2,4-α-L-Rhap residues; while component a had long chains with 1,6- and 1,3,6-β-D-Galp (19.5%) linked together, which is consistent with the main chain structure characteristics of AG-II type pectin.

[0323] 3.3.3 Structural characteristics of total pectin in wolfberry

[0324] The above analysis indicates that two types of pectin polysaccharides (components a and b) constitute the complex structure of total pectin from *Lycium barbarum* (LBPA-I, LBPA-II, and LBPA-III) as fine subfractions. The structure of these three total pectin groups is likely that of RG-I type pectin, with component b as the main chain and component a as the side chain. Although the main chain structures of the three total pectin groups are similar, the compositional and NMR analysis results of LBPA-I, LBPA-II, and LBPA-III show that the number of disaccharide units (1,4-α-D-GalpA and 1,2- / 1,2,4-α-L-Rhap alternating links) in RG-I is greater in LBPA-III. This difference may reflect the different biological activities of LBPA-I, LBPA-II, and LBPA-III.

[0325] 3.4 Summary

[0326] This chapter employs partial acid hydrolysis, nuclear magnetic resonance spectroscopy combined with methylation analysis to study the main chain structure of wolfberry pectin and the fine structural characteristics of components a and b (LBPA-Ia and LBPA-Ib). The results are as follows:

[0327] (1) First, partial acid hydrolysis was performed on the three total pectin fractions (LBPA-I, LBPA-II, and LBPA-III). The results showed that after hydrolysis, the content of monosaccharide Gal in the main chain fractions (LBPA-I-H-3, LBPA-II-H-3, and LBPA-III-H-3) increased significantly, while the content of Ara decreased significantly. This indicates that the main chain fractions of the three total pectin fractions may be mainly composed of Gal as the main monosaccharide, and Ara may appear at the outer end of the side chain. By analyzing the monosaccharide composition and molecular weight distribution, it is speculated that the structural characteristics of the main chain fractions of the three total pectin fractions (LBPA-I-H-3, LBPA-II-H-3, and LBPA-III-H-3) may be similar.

[0328] (2) The 1D / 2D-NMR spectrum analysis results show that the three total pectin backbones have similar structures. Their common backbone structure is a disaccharide unit linked by →4)-α-D-GalpA-(1→2 / 2,4)-α-L-Rhap-(1→, and there is a →6)-β-D-Galp-(1→6 / 3,6)-β-D-Galp-(1→, which is consistent with the RG-I type structure characteristics.

[0329] (3) Using LBPA-Ia and LBPA-Ib as representative components, partial acid hydrolysis analysis was further performed on components a and b. The results showed that the main chain of LBPA-Ia-H-3 was composed of Gal, Ara, and GlcA, with Gal as the main monosaccharide; while the main chain of LBPA-Ib-H-3 was composed of Man, GlcA, Rha, GalA, Glc, Gal, Xyl, and Ara, with Glc and Gal as the main monosaccharides, indicating that the main chain monosaccharide composition of components a and b was different. By analyzing the monosaccharide composition, molecular weight, and peak times in the HPGPC spectra of LBPA-Ia-H-3, LBPA-Ib-H-3, and LBPA-I-H-3 (the main chain portion of total pectin LBPA-I), the following connections were found between the main chain portions of components a and b and the three total pectin main chain portions: LBPA-Ia-H-3 and LBPA-Ib-H-3 have different molecular weights and monosaccharide compositions; LBPA-Ib-H-3 and LBPA-I-H-3 have similar molecular weights and highly similar monosaccharide compositions. This indicates that LBPA-Ib-H-3 and LBPA-I-H-3 share structural homology but are different from the structure of LBPA-Ia-H-3. It is speculated that the main chain portions of the three total pectin components are structurally similar to the main chain portion of component b, but different from the main chain portion of component a.

[0330] (4) Methylation analysis results showed that the three total pectins had similar main chain structure characteristics (represented by LBPA-Ⅰ-H-3), with 1,6-Ga lp as the main component and a small number of 1,4-Galp and 1,2,4-Rhap glycosidic bonds, indicating that they contained a relatively short RG-Ⅰ type backbone structure. Simultaneously, it was demonstrated that the main chain structure of the three total pectins was highly similar to that of component b, but different from that of component a. Component b was dominated by 1,6-Galp and contained 1,4-Galp and 1,2,4-Rhap glycosidic bonds, consistent with the characteristics of RG-Ⅰ type pectin; component a was dominated by 1,6-Galp and lacked 1,4-Galp and 1,2,4-Rhap glycosidic bonds, consistent with the characteristics of AG-Ⅱ type structure.

[0331] (5) Combining the 1D / 2D-NMR spectrum analysis results of the three total pectin and components a and b, the fine structural features of the two types of pectin polysaccharides were obtained:

[0332] The fine structure of component a (AG-II): Main chain → 1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→1)-β-D-Galp-(3,6→1)-β-D-Galp-(6→, where Gal is replaced by 5 side chains at the O-3 position: ①→1)-α-L-Araf-(3→1)-α-L-Araf-(3→1)-α-L-Araf, ②→1)-α-L-Araf-(5 →1)-α-L-Araf-(5→1)-α-L-Araf, ③→1)-α-L-Araf-(5→1)-α-L-Araf-(5→1)-β-L-Araf, ④→1)-β-D -Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap, ⑤→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→1)-β-D-Gal p-(3,6→.

[0333] The fine structure of component b is characterized by a predominantly →1)-β-D-Galp-(3→1)-β-D-Galp-(3→1)-β-D-Galp-(3,6→1)-β-D-Galp-(3→); and a short-chain structure consisting of →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→), along with →1)-β-D-Galp-(6→1)-β-D-Galp The AG-II structure of similar component a is linked by -(3,6→1;→1)-α-L-Araf-(3→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-α-L-Araf;→1)-α-L-Araf-(5→1)-β-L-Araf and →1)-β-D-Galp-(6→1)-α-D-GlcpA-(4→1)-α-L-Rhap.

[0334] Example 4

[0335] 4. Study on the intervention effect of Lycium barbarum pectin on intestinal inflammatory damage induced by chronic restraint stress in mice.

[0336] 4.1 Experimental Materials

[0337] (1) Laboratory animals

[0338] The experimental mice were purchased from SPF-grade male KM mice (130 mice, 5 weeks old, weighing 16–20 g) from SPF (Beijing) Biotechnology Co., Ltd. Before the experiment, all animals were housed in cages and acclimatized for one week in a 22±2℃, 55±10%RH, and 12 / 12h light / dark cycle environment, with free access to food and water. All animal experiments were conducted in accordance with the ARRIVE (Animal Research: In Vivo Reporting) guidelines, following the "Guidelines for the Care and Use of Laboratory Animals," and were approved by the Northwest Plateau Institute of Biology, Chinese Academy of Sciences (License No.: NWIPB20171106-01).

[0339] 4.2 Experimental Methods

[0340] 4.2.1 Establishment of an intestinal inflammation model

[0341] A mouse model of intestinal inflammation induced by chronic restraint stress (CRS) was established by using a 14-day modeling method of restraint in plastic centrifuge tubes for 6 hours daily (References: QIAO Y, CHEN H, GUO J, et al. A study on the effects of metacinnabar (β-HgS) on weight and appetite recovery in stressed mice[J]. Journal of ethnopharmacology, 2024:118663. and ZHANG X, QIAO Y, LI G, et al. Exploratory studies of the antidepressant effect of Cordyceps sinensis polysaccharide and its potential mechanism[J]. International journal of biological macromolecules, 2024:134281.). The restraint tubes were modified 50mL centrifuge tubes with appropriate openings at the front and back ends, allowing the mice to move back and forth within a small area. During CRS modeling, mice were placed in restraint tubes at regular intervals (10:00 to 16:00) each day and then returned to their cages.

[0342] 4.2.2 Grouping and Dosing

[0343] After the adaptation period, mice were randomly divided into 12 groups (n=10 for all groups except the NC and CRS groups, n=15): normal control group (NC), model group (CRS), model group + low-dose group (CRS+LBPA-IL, CRS+LBPA-IIL, and CRS+LBPA-IIIL); model group + medium-dose group (CRS+LBPA-IM, CRS+LBPA-IIM, and CRS+LBPA-IIIM); model group + high-dose group (CRS+LBPA-IH, CRS+LBPA-IIH, and CRS+LBPA-IIIH); model group + positive control group (CRS+ASA), etc. Figure 58 As shown in the figure. The normal and model groups were administered pure water by gavage, the positive control group was administered mesalazine enteric-coated tablets (ASA) 200 mg / kg by gavage, and the other groups were administered the corresponding polysaccharides (L: 50 mg / kg, M: 100 mg / kg, H: 200 mg / kg) by gavage daily for 14 consecutive days. On day 15, after collecting blood from the orbital cavity, the mice were euthanized by cervical dislocation, and the abdomen was disinfected and dissected to collect cecal contents and colon tissue.

[0344] 4.2.3 H&E staining

[0345] A small segment of tissue from the middle of the mouse colon was fixed in 4% paraformaldehyde for 24 hours. The fixed tissue was dehydrated using an automated dehydrator and embedded in paraffin. Sections were dewaxed to 95% ethanol; stained with blue solution overnight at room temperature; excess staining solution was washed away with 95% ethanol, and the tissue was rinsed with distilled water; the tissue was differentiated with blue differentiation solution for 10–15 seconds; it was then differentiated with 70% ethanol for 30 seconds until the gray-white matter was clearly visible, and rinsed with distilled water; counterstained with eosin solution for 30 seconds–2 minutes; dehydrated with graded alcohols; cleared with a clearing agent; and mounted with neutral resin. A panoramic scan of the sections was performed using 3DHISTECH (Hungary).

[0346] 4.2.4 Detection of cytokines in colon tissue

[0347] The level of CRS-induced intestinal inflammation was evaluated by detecting key inflammatory cytokine markers in mouse colon tissue. Small segments of proximal and distal mouse colon tissue were weighed and placed in 1.5 mL centrifuge tubes, which were immediately refrigerated for later use. Nine times the volume of PBS solution was added to the centrifuge tubes, along with 2-3 small steel balls, and the tubes were homogenized for 2 minutes. The homogenate was then centrifuged (4°C, 800 rpm). The levels of TNF-α, LPS, IL-1β, IL-6, IL-10, and MPO cytokines were detected using an enzyme-linked immunosorbent assay (ELISA) kit, following the manufacturer's instructions.

[0348] 4.2.516S rDNA sequencing analysis

[0349] 16S rDNA amplicon sequencing typically involves selecting one or more variable regions, designing universal primers for PCR amplification using conserved regions, and then performing sequencing analysis and species identification on hypervariable regions.

[0350] After filtering and splicing, the raw data (Clean Data) underwent noise reduction analysis to generate amplicon sequence variants (ASVs). Based on the ASV analysis results, corresponding species information and species-based abundance distribution were obtained, and abundance and alpha diversity were calculated. PCA dimensionality reduction analysis was used to explore differences in community structure among different samples or groups. To further explore the differences in community structure among grouped samples, the T-test statistical analysis method was used to test for significant differences in species composition and community structure among grouped samples.

[0351] 4.2.6 Metabolomics Analysis

[0352] The intestinal metabolite profiles of mice in each experimental group were obtained by non-targeted UPLC-MS / MS metabolomics technology, and differentially metabolized molecules (mainly endogenous small molecule metabolites with a relative molecular mass of less than 1000) were screened. Subsequently, the differences and similarities of differentially metabolites in each group were compared by cluster analysis, heat map drawing, IPA pathway and network analysis, so as to elucidate the pectin metabolism-related processes. Sample extraction procedure: (1) The sample was thawed on ice; (2) 400 μL of 70% methanol-water internal standard extraction solution was added to 20 mg of sample and vortexed for 3 min; (4) The sample was sonicated for 10 min (ice-water bath), the sample was removed and vortexed for another 1 min, and placed in a -20℃ refrigerator for 30 min; (5) The sample was centrifuged for 10 min (4℃, 12000 r / min), 300 μL of supernatant was taken, centrifuged for 3 min (4℃, 12000 r / min), and 200 μL of supernatant was taken into the inner liner of the corresponding sample bottle for instrument detection. Chromatographic and mass spectrometry conditions: Column: Waters ACQUITY Premier HSS T3 Column 1.8 μm, 2.1 mm × 100 mm; Mobile phase A: 0.1% formic acid / water; Mobile phase B: 0.1% formic acid / acetonitrile; Column temperature: 40℃; Flow rate: 0.4 mL / min; Injection volume: 4 μL.

[0353] The raw data was converted to mzXML format using Proteo Wizard, and peak extraction, alignment, and retention time correction were performed using XCMS. Peaks with a missing rate >50% in each sample group were filtered, and blank values ​​were filled using KNN. The SVR method was used to correct peak area, and metabolites were identified by searching a self-built database, integrating public libraries, prediction libraries, and using metDNA methods. Finally, substances with a comprehensive score of 0.5 or higher and a QC sample CV value of less than 0.3 were extracted and identified. Positive and negative patterns were then merged (retaining the substance with the highest qualitative grade and the smallest CV value), resulting in the data file (Clean Data.xlsx) for data analysis.

[0354] 4.2.7 Western blotting to detect protein expression

[0355] Small segments of tissue from the proximal and distal ends of the mouse colon were taken, placed in 1.5 mL centrifuge tubes, and rapidly frozen in liquid nitrogen. They were then stored at -80°C for later use.

[0356] (1) Extraction of total protein: Weigh about 0.1g of tissue sample, add 1mL of RIPA lysis buffer, homogenize using a tissue homogenizer; lyse at 4℃ for 30min, then centrifuge at 4℃ for 10min (12000rpm), take the supernatant for BCA protein quantification, and store at -80℃ for a long time.

[0357] (2) SDS-PAGE electrophoresis: Prepare fresh electrophoresis buffer and electrophoresis at 140V until the loading just runs out of the separating gel.

[0358] (3) Transfer: Take out the gel after electrophoresis and put it into the prepared transfer solution (1×) for equilibration for 20 min; PVDF membrane and thin filter paper (cut to appropriate size), after activation with methanol, put the PVDF membrane and thin filter paper into the transfer solution for equilibration (20 min); place them in the order of negative electrode-thin filter paper-gel-PVDF membrane-thin filter paper-positive electrode, and transfer the membrane at a constant current of 200mA for 1 h.

[0359] (4) Blocking and antibody incubation: Block with 5% skim milk powder solution prepared with 1×TBST; add 6μL of primary antibody according to the antibody instructions (1:1000), incubate overnight on a shaker at 4℃, and then wash 3 times with 1×TBST for 10min each time; put the washed membrane into secondary antibody diluted 1:3000, incubate on a shaker at room temperature for 1h, and then wash 3 times with 1×TBST for 10min each time.

[0360] (5) ECL color development: Follow the instructions for the ECL color development kit. Use the grayscale software Gelpro32 to analyze the grayscale values ​​of the bands.

[0361] 4.2.8 Data Analysis

[0362] One-way ANOVA was performed using SPSS 22.0 statistical software. Experimental results are expressed as mean ± standard deviation. A p-value < 0.05 was considered statistically significant (* or #), and a p-value < 0.01 was considered highly significant (** or ##). Data visualization was performed using GraphPad Prism 9 and Origin 2024 software.

[0363] 4.3 Results and Analysis

[0364] 4.3.1 H&E staining results

[0365] like Figure 59 to 60 As shown in the H&E stained sections, the characteristic finger-like processes on the surface of the small intestinal mucosa of the colon tissue in the NC group mice were intact, and the small intestinal villi formed by the epithelium and lamina propria were dense. Compared with the Control group, the colon tissue of the CRS group mice showed epithelial cell shedding and scattered inflammatory cell infiltration. After intervention with wolfberry pectin, all three wolfberry pectin components could restore the morphology of colonic villi and crypt damage in CRS model mice compared with the CRS group. Among them, the medium and high doses of LBPA-III significantly alleviated the colonic pathological state in CRS model mice.

[0366] 4.3.2 Cytokine content and MPO level in colon tissue

[0367] The levels of cytokines TNF-α, LPS, IL-1β, IL-6, IL-10, and the inflammatory marker MPO were detected in the colon of mice. Figure 61 As shown, compared with the NC group, the levels of TNF-α (P < 0.001), LPS (P < 0.01), IL-1β (P < 0.001), IL-6 (P < 0.001), and MPO (P < 0.001) in the colon tissue of mice in the CRS group were significantly increased, while the level of IL-10 (P < 0.05) was significantly decreased. Compared with CRS, the TNF-α content in all groups of mice was decreased after intervention with different Lycium barbarum pectin. Among them, the high dose of LPBA-I component, the low, medium, and high doses of LBPA-III were close to the positive control group, and the high dose of LBPA-III component was close to the blank group. However, the low, medium, and high doses of LBPA-II component were not statistically significant compared with the model group. Figure 61 A); After intervention, the LPS content of mice in all groups decreased. Among them, the low, medium and high doses of LPBA-I, the low, medium and high doses of LBPA-II, and the low and medium doses of LBPA-III were close to the positive drug and tended to be similar to the blank group. However, the high dose of LBPA-III was not statistically significant compared with the model group. Figure 61B); After intervention, the IL-1β levels in mice in all groups decreased. Among them, the low, medium, and high doses of LPBA-I, the low, medium, and high doses of LBPA-II, and the low, medium, and high doses of LBPA-III were close to those of the positive control drug, and all were statistically significant. Figure 61 C); After intervention, the IL-10 levels in all groups of mice increased, with the medium dose of LPBA-I and the low, medium, and high doses of LBPA-III showing statistical significance. Figure 61 D); After intervention, the IL-6 levels in all groups of mice decreased. The low and high doses of LPBA-I and the low, medium, and high doses of LBPA-III were close to those of the positive control group, and the high dose of LBPA-III was close to that of the blank group. However, the medium dose of LPBA-I was not statistically significant compared with the model group. Figure 61 E); After intervention, the MPO content of mice in all groups decreased, with statistically significant differences observed in low, medium, and high doses of LPBA-I, medium and high doses of LBPA-II, and low, medium, and high doses of LBPA-III. Figure 61 F).

[0368] Comprehensive analysis showed that intervention with the three pectin polysaccharide components LPBA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ could inhibit the increase of pro-inflammatory cytokines TNF-α, LPS, IL-1β, and IL-6, the decrease of anti-inflammatory cytokine IL-10, and the increase of the inflammatory marker MPO activity. Overall comparison revealed that low doses of LPBA-Ⅰ, medium doses of LBPA-Ⅱ, and high doses of LBPA-Ⅲ showed the best improvement in key inflammatory factors in the colonic tissue of the CRS model. Among these, the medium dose of pectin LBPA-Ⅲ was statistically significant compared with the CRS model group.

[0369] 4.3.3 Analysis of Microbiome Results

[0370] 4.3.3.1 Diversity Analysis

[0371] (1) α-diversity analysis

[0372] like Figure 62As shown, α-diversity analysis revealed that compared with the NC group, the CRS group had significantly higher observed ASV index, PD wholetree index, and Simpson's index, indicating that CRS affects the gut microbiota diversity of mice. After intervention with Lycium barbarum pectin, medium and high doses of LPBA-I, LBPA-II, and LBPA-III significantly reduced the observed ASV index; medium doses of LPBA-I, LBPA-II, and LBPA-III significantly reduced the PD wholetree index; and low, medium, and high doses of LPBA-I, LBPA-II, and LBPA-III significantly reduced the Simpson's index, indicating that medium and high doses of LBPA-II and LBPA-III can significantly reduce the observed ASV index, PD wholetree index, and Simpson's index.

[0373] (2) β-diversity analysis

[0374] β-diversity analysis between groups showed that the CRS group was completely separated from the NC group, indicating that chronic restraint caused significant changes in the gut microbiota of mice. Figure 63 A). After three interventions with wolfberry pectin, all groups showed recovery.

[0375] 4.3.3.2 Analysis of Differences in Subject Levels

[0376] Differential microbial community analysis at the family level revealed that, compared with the NC group, the relative abundance of f-Helicobacteraceae (P < 0.01) and f-Ruminococcaceae (P < 0.01) was significantly increased in the CRS group. Figure 64 A and Figure 64 C); f-Muribaculaceae (P < 0.01) was significantly reduced ( Figure 64 B). After intervention with Lycium barbarum pectin, compared with the model group, all three Lycium barbarum pectin components, LPBA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ, were able to restore the relative abundance of f-Helicobacteraceae, f-Muribaculaceae, and f-Ruminococcaceae. Among them, component LBPA-Ⅲ showed better results, with the medium dose being superior to the other doses and approaching the level of the positive control.

[0377] 4.3.4 Results of Metabolomics Analysis

[0378] 4.3.4.1 Screening for Differential Metabolites

[0379] like Figure 65 ABFigure 66 AC, Figure 67 AC and Figure 68 As shown in AC, compared with the NC group, the relative mass of 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine and PE (16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)) increased in the CRS group; compared with the CRS group, the relative mass of 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine and PE (16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)) was increased in the CRS group; compared with the CRS group, the relative mass of the three wolfberry pectin components LPBA-Ⅰ, LBPA-Ⅱ and LBPA-Ⅲ could be restored to 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine and PE (16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)) was restored. Among them, the LBPA-III component showed better efficacy, with the medium dose being superior to other doses. Its regulatory effect on 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine was close to blank, while its regulatory effect on PE (16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)) was close to that of the positive control. Figure 69 ).

[0380] 4.3.5 Correlation Analysis

[0381] Spearman correlation analysis was performed on differentially expressed microbiota, differentially expressed metabolites, inflammatory factors, and MPO activity. The results showed that 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine was significantly positively correlated with TNF-α, LPS, IL-1β, IL-6, IL-10, f-Helicobacteraceae, and f-Muribaculaceae; PE16:0 / 20:5 (5Z, 8Z, 11Z, 14Z, 17Z) was significantly positively correlated with TNF-α, LPS, IL-1β, IL-6, MPO, f-Helicobacteraceae, f-Muribaculaceae, and f-Ruminococcaceae. Figure 70 ).

[0382] 4.3.6 Metabolic pathway analysis

[0383] By comparing the CRS group and the NC group ( Figure 71 A) CRS group and ASA group ( Figure 71 B), CRS group and LPBA-IL group ( Figure 72 A), CRS group and LPBA-IM group ( Figure 72B), CRS group and LPBA-IH group ( Figure 72 C), CRS group and LBPA-IIL group ( Figure 73 A), CRS group and LBPA-IIM group ( Figure 73 B), CRS group and LBPA-IIH group ( Figure 73 C), CRS group and LBPA-IIIL group ( Figure 74 A), CRS group and LBPA-IIIM group ( Figure 74 B), CRS group and LBPA-ⅢH group ( Figure 74 C) The first 30 common pathways were found to be Retrograde endocannabinoid signaling.

[0384] 4.3.7 Effects of Lycium barbarum pectin on the endocannabinoid system in mice

[0385] Studies have found that at least one-third of the anti-inflammatory effects produced by gut microbiota are related to elevated levels of endocannabinoids, with CB2 being closely associated with gut inflammation. The endocannabinoid system (ECS) mainly consists of three parts: endocannabinoids (such as arachidonic acid ethanolamine (AEA) and 2-arachidonic acid glyceride (2-AG)); cannabinoid receptors (such as CB1 and CB2); and enzymes involved in synthesis and metabolism (such as fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL)). Figure 75 As shown, compared with the NC group, the expression level of CB2 in the CRS group was significantly decreased (P < 0.001), leading to a significant decrease in the expression of the anti-apoptotic protein Bcl-2 (P < 0.001) and a significant increase in the expression of the pro-apoptotic protein Bax (P < 0.001), accompanied by a significant increase in the expression of the metabolic enzyme FAAH (P < 0.001), indicating inflammation in the mouse colon. Compared with CRS, the expression of CB2, Bcl-2, Bax, and FAAH were all improved after medium-dose intervention with Lycium barbarum pectin LPBA-Ⅰ, LBPA-Ⅱ, and LBPA-Ⅲ. Among them, LBPA-ⅢM showed the best effect, approaching the positive control drug level.

[0386] 4.3.8 Effects of Lycium barbarum pectin on intestinal barrier function in mice

[0387] The above analysis revealed that the three medium-dose groups (-M) of Lycium barbarum pectin all showed good intervention effects on CRS model mice, with LBPA-IIIM showing the best effect. To further verify the intervention effect of LBPA-IIIM on intestinal inflammation in CRS model mice, Western blot was used to measure the expression of intestinal epithelial tight junction proteins. Figure 76As shown, compared with the NC group, the expression of Claudin-1, Occludin, and ZO-1 in the colonic epithelium of CRS model mice was significantly reduced (P < 0.001), while the medium-dose groups (-M) of the three Lycium barbarum pectin groups significantly inhibited the reduction of Claudin-1, Occludin, and ZO-1 protein expression (P < 0.001). The results indicate that the medium-dose intervention of the three Lycium barbarum pectin groups can effectively improve the effect of CRS on the intestinal barrier function of mice, with LBPA-ⅢM showing the best effect, approaching that of the positive control drug (ASA).

[0388] 4.3.9 Comparative Results of the Efficacy of Total Polysaccharides and Pectin from Lycium barbarum in Improving CRS-Induced Intestinal Inflammation

[0389] A comparative experiment was conducted to investigate the effects of total polysaccharides and pectin from Lycium barbarum on improving CRS-induced intestinal inflammation. The results are shown in Table 13.

[0390] Table 13 Effects of total polysaccharides and pectin from Lycium barbarum on the content of inflammatory factors in the colon tissue of mice with CRS-induced intestinal inflammation

[0391]

[0392] Note:( (n=8) Compared with the blank control group: #P<0.05, ##P<0.01, ###P<0.001; Compared with the model group: *P<0.05, **P<0.01, ***P<0.001.

[0393] By comparing the regulatory effects of the total polysaccharide group (LBWP) and the three pectin groups (LPBA-Ⅰ, LBPA-Ⅱ, LBPA-Ⅲ) on inflammatory factors, it is evident that pectin has a unique advantage in improving CRS-induced intestinal inflammation:

[0394] 1. It has a stronger inhibitory effect on pro-inflammatory factors.

[0395] TNF-α (a pro-inflammatory core factor): In the total polysaccharide group of wolfberry, the level could only be reduced from 1742.7 pg / mL to 1523.6 pg / mL (*, P<0.05), while in the pectin group, LBPA-II and LBPA-III could be further reduced to 1255.5 pg / mL and 1204.3 pg / mL (**, P<0.01), with a significantly greater reduction than that of total polysaccharide, and the significance was even higher.

[0396] IL-1β (a key pro-inflammatory factor): The total polysaccharide group of Lycium barbarum did not show a statistically significant reduction in IL-1β (1759.4 pg / mL → 1545.6 pg / mL, not marked with *), while all three pectin groups significantly reduced this factor (e.g., LBPA-II decreased to 1294.3 pg / mL, *, P<0.05), demonstrating the specific inhibitory effect of pectin on this factor.

[0397] IL-6 (pro-inflammatory mediator): The LBPA-III group showed the most significant reduction in IL-6, decreasing from 1104.5 pg / mL to 681.6 pg / mL (**, P<0.001), which was far superior to the 1022.6 pg / mL of the total polysaccharide group of Lycium barbarum (*, P<0.05), with a reduction of 38% and extremely high statistical significance.

[0398] MPO (an inflammatory marker reflecting neutrophil infiltration): In the total polysaccharide group of wolfberry, MPO was reduced from 2889.6 IU / mL to 2396.2 IU / mL (**, P<0.01), while in the pectin group, LBPA-I and LBPA-II were reduced to 1983.3 IU / mL and 1825.7 IU / mL respectively (**, P<0.01), close to the level of the blank control group (1985.3 IU / mL), indicating that pectin can more effectively reduce intestinal inflammatory cell infiltration.

[0399] 2. It has a more significant activation effect on anti-inflammatory factors.

[0400] The increase in the anti-inflammatory factor IL-10 is a key indicator for evaluating the improvement of inflammation.

[0401] The total polysaccharide group of wolfberry could only increase IL-10 from 402.8 pg / mL to 535.2 pg / mL (*, P<0.05), while in the pectin group, the LBPA-III group could significantly increase it to 887.9 pg / mL (**, P<0.001), which is 1.66 times that of the total polysaccharide group and much higher than the blank control group (522.7 pg / mL). This indicates that pectin can more strongly activate anti-inflammatory pathways and regulate inflammation bidirectionally by "inhibiting pro-inflammatory + enhancing anti-inflammatory".

[0402] 3. Structure-specific gradient activity was observed within the pectin group.

[0403] The efficacy of the three pectin groups (LPBA-Ⅰ, LBPA-Ⅱ, LBPA-Ⅲ) was not simply a repetition, but rather showed a trend of gradient optimization:

[0404] Based on the decrease in TNF-α and IL-6 and the increase in IL-10, the activity order was LBPA-III > LBPA-II > LPBA-I > total polysaccharides of Lycium barbarum. This indicates that the structural differences of pectin (such as molecular weight, monosaccharide composition, substituents, etc.) are directly related to its activity. It is not a "pan-active component" in total polysaccharides, but a highly efficient active component with a specific structure.

[0405] This demonstrates that wolfberry pectin possesses a unique "structure-activity" relationship. While the anti-inflammatory activity of total wolfberry polysaccharides, a known natural product, has been studied, the data in this study show that wolfberry pectin (especially LBPA-II and LBPA-III) significantly outperforms total polysaccharides in regulating multiple key inflammatory factors, both in terms of the intensity of its effects (e.g., the increase in IL-10 and the decrease in IL-6) and statistical significance (more marked with ** and ***), exhibiting a structure-dependent gradient activity.

[0406] This "superiority" and "specificity" indicate that wolfberry pectin is not a simple component of total polysaccharides, but an active substance with a unique chemical structure and mechanism of action. Its effect on improving CRS-induced intestinal inflammation cannot be derived from the known activity of total polysaccharides. Therefore, it possesses the inventiveness required for an invention patent—that is, compared with the existing technology (wolfberry total polysaccharides), wolfberry pectin has superior efficacy, and this advantage is unpredictable.

[0407] Studies have found that stress induces intestinal barrier dysfunction in mice, while chronic stress induces reversible intestinal mucosal inflammation. In this chapter, we first used a chronic restraint stress model lasting 14 days (6 hours per day) to study the effects of stress on intestinal health in mice. Pathological results showed scattered inflammatory cell infiltration in the colonic tissue of CRS model mice; significantly elevated levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and LPS), significantly decreased levels of anti-inflammatory cytokines (IL-10), and significantly increased activity of inflammatory markers (MPO) in the colonic tissue. These changes indicate that CRS causes intestinal inflammation in mice. However, this experiment found that the CRS-induced intestinal inflammation in mice did not produce typical colitis symptoms such as diarrhea, bloody stools, and erosive lesions of the colonic mucosa. Colitis is a type of inflammatory bowel disease (IBD), a chronic, idiopathic, multifactorial inflammatory disease of the gastrointestinal tract. Its pathogenesis includes multiple factors such as genetics, environmental factors, persistent epithelial damage, and intestinal microbial dysbiosis. It is mainly manifested as mixed symptoms such as erosive lesions of the colonic mucosa, diarrhea, and hematochezia. Chronic restraint stress is an important factor in the triggering and reactivation of colitis.

[0408] The gut microbiota, as an important component of the gut microecology, plays an indispensable role in maintaining gut health. CRS (Corrective Reproductive Stress) significantly alters the composition and diversity of the gut microbiota. In this study, we observed a significant increase in the α-diversity indices (such as the observed ASV index, PD whole tree index, and Simpson index) of the gut microbiota in CRS model mice. This may indicate a disruption in the stability of the gut microbiota, a weakening of the dominance of beneficial bacteria, and the proliferation of harmful bacteria. For example, the relative abundance of f-Helicobacteraceae and f-Ruminococcaceae was significantly increased in the CRS model group, while f-Muribaculaceae was significantly decreased. Some bacteria in the Helicobacteraceae family are closely related to the occurrence of gastrointestinal diseases, and their increase may increase the risk of intestinal infection and inflammation; although Ruminococcaceae has certain physiological functions in the normal gut, excessive proliferation under stress may disrupt the balance of the gut microbiota; Muribaculaceae is considered to be positively correlated with gut health, and its reduction is detrimental to maintaining normal gut function. These changes in the gut microbiota can further affect gut immune regulation, nutrient metabolism, and barrier function, promoting the development and worsening of gut inflammation.

[0409] In this experiment, intervention with Lycium barbarum pectin significantly regulated the diversity of gut microbiota in CRS model mice. For example, medium and high doses of LBPA-II and medium doses of LBPA-III significantly reduced the observed ASV index, PD whole tree index, and Simpson's diversity index, restoring the disordered gut microbiota structure to normal. On one hand, Lycium barbarum pectin effectively restored the relative abundance of key bacterial groups, such as f-Helicobacteraceae, f-Muribaculaceae, and f-Ruminococcaceae. Studies have reported that arabinogalactan (AG), with Gal and Ara as its main monosaccharides, has been shown to have Bifidobacterium proliferative capacity and can significantly increase the relative abundance of Lactobacillus. On the other hand, the regulation of the retrograde endocannabinoid signaling pathway by Lycium barbarum pectin may also be one of the important mechanisms by which it exerts its anti-inflammatory effects. The endocannabinoid system (ECS) plays a crucial role in maintaining intestinal homeostasis, and its dysfunction is closely related to the occurrence and development of intestinal inflammation. The ECS is mainly composed of endocannabinoids (such as arachidonic acid ethanolamine (AEA) and 2-arachidonic acid glyceride 2-AG), cannabinoid receptors (such as CB1 and CB2), and enzymes involved in synthesis and metabolism (such as fatty acid amide hydrolase FAAH and monoacylglycerol lipase MAGL). In a CRS-induced intestinal inflammation model in mice, we observed a significant decrease in CB2 expression, a significant decrease in the expression of the anti-apoptotic protein Bcl-2, and a significant increase in the expression of the pro-apoptotic protein Bax, accompanied by a significant increase in the expression of the metabolic enzyme FAAH. This series of changes indicates inflammation in the mouse colon. After intervention with Lycium barbarum pectin, the expression of CB2, Bcl-2, Bax, and FAAH were all improved, with LBPA-III showing the best effect, approaching that of the positive control drug (ASA). This suggests that Lycium barbarum pectin may exert its intervention effect on intestinal inflammation by regulating the ECS and affecting intestinal cell apoptosis and inflammatory responses. Specifically, goji berry pectin may increase the activation of CB2 receptors by regulating the synthesis and metabolism of endocannabinoids, thereby inhibiting the release of inflammatory cytokines and promoting the repair and regeneration of intestinal cells.

[0410] In terms of intestinal barrier function, wolfberry pectin exhibited a good protective effect. The intestinal barrier is the body's first line of defense against external pathogens, and its integrity is crucial for maintaining intestinal health. In this study, CRS induced impaired intestinal barrier function in mice, manifested as decreased expression of colonic epithelial tight junction proteins Claudin-1, Occludin, and ZO-1. Intervention with medium-dose groups of three wolfberry pectin groups significantly inhibited the decrease in Claudin-1, Occludin, and ZO-1 protein expression, effectively reversing the effects of CRS on mouse intestinal barrier function. LBPA-ⅢM showed the best effect, approaching that of a positive control (ASA). Wolfberry pectin may maintain intestinal barrier integrity by regulating intracellular signaling pathways, promoting the synthesis and assembly of tight junction proteins, and enhancing intercellular tight junctions. Furthermore, wolfberry pectin may also promote the repair of damaged intestinal barriers by regulating the proliferation and differentiation of intestinal epithelial cells.

[0411] Finally, from an immunomodulatory perspective, wolfberry pectin can effectively regulate the secretion of inflammatory cytokines. Inflammatory cytokines play a crucial role in the occurrence, development, and resolution of intestinal inflammation. Overexpression of pro-inflammatory cytokines (such as TNF-α, IL-1β, IL-6, and LPS) exacerbates the inflammatory response, while insufficient secretion of anti-inflammatory cytokines (such as IL-10) leads to ineffective inflammation control. In this study, after wolfberry pectin intervention, the levels of pro-inflammatory cytokines TNF-α, LPS, IL-1β, and IL-6 in all groups of mice decreased, while the level of the anti-inflammatory cytokine IL-10 increased. This indicates that wolfberry pectin can regulate the balance of the immune system and reduce damage to intestinal tissues. The specific mechanism may be that wolfberry pectin affects the secretion and release of inflammatory cytokines by regulating the activity of immune cells (such as macrophages, T cells, and B cells). For example, wolfberry pectin may inhibit macrophage activation, reducing the production of pro-inflammatory cytokines; at the same time, it promotes the differentiation of T cells into anti-inflammatory cell subsets, increasing the secretion of anti-inflammatory cytokines.

[0412] 4.4 Summary

[0413] This chapter systematically investigates the intervention effect of wolfberry pectin on intestinal inflammatory damage by establishing a chronic restraint stress (CRS) mouse model of intestinal inflammation. The main findings are as follows:

[0414] (1) Pathological studies showed that CRS caused intestinal damage and intestinal inflammation in mice. Epithelial cells in the colonic tissue of CRS model mice were sloughed off, and scattered inflammatory cells infiltrated. The levels of pro-inflammatory cytokines TNF-α, IL-1β, IL-6 and LPS in the colonic tissue were significantly increased, the level of anti-inflammatory cytokine IL-10 was significantly decreased, and the activity of inflammatory markers (MPO) was significantly increased. The stability of the intestinal flora was disrupted, the relative abundance of f-Helicobacteraceae and f-Ruminococcaceae was significantly increased, while f-Muribaculaceae was significantly decreased. The expression of colonic epithelial tight junction proteins Claudin-1, Occludin and ZO-1 was reduced.

[0415] (2) After intervention with Lycium barbarum pectin, the symptoms of intestinal damage in mice were significantly improved. Among them, the medium dose of LBPA-III component had the most significant effect, which could effectively regulate the diversity of intestinal flora; restore the relative abundance of key flora f-Helicobacteraceae, f-Ruminococcaceae and f-Muribaculaceae; restore the relative quality of differential metabolites 1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine and PE (16:0 / 20:5(5Z,8Z,11Z,14Z,17Z)); regulate metabolic pathways such as retrograde endocannabinoid signaling; effectively improve the effect of CRS on the intestinal barrier function of mice; improve the expression of endocannabinoid system-related proteins and alleviate colonic inflammation in CRS mice.

[0416] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A type of wolfberry pectin polysaccharide, characterized in that, The pectin polysaccharide of wolfberry is mainly composed of galactose and arabinose, and its core structure is: →6)-β-D-Galp-(1→6 / 3,6)-β-D-Galp-(1→and →4)-α-D-GalpA-(1→2 / 2,4)-α-L-Rhap-(1→repeating unit).

2. The wolfberry pectin polysaccharide according to claim 1, characterized in that, The wolfberry pectin polysaccharide includes mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose, and the molar ratio of mannose, glucuronic acid, rhamnose, galacturonic acid, glucose, galactose and arabinose is (0.3~5.5):(3.0~6.3):(0.3~9.6):(1.9~7.1):(0.9~20.6):(23.2~49.7):(33.9~45.6).

3. The wolfberry pectin polysaccharide according to claim 1, characterized in that, The molecular weight (Mw) of the wolfberry pectin polysaccharide ranges from 3.36 kDa to 49.0 kDa.

4. The method for preparing Lycium barbarum pectin polysaccharides according to any one of claims 1 to 3, characterized in that, The method includes the following steps: Total polysaccharides from Lycium barbarum were dissolved in distilled water, centrifuged, and the supernatant was collected. The supernatant was eluted using a DE-52 ion exchange chromatography column, and neutral polysaccharides were separated by elution with 400 mL of water. Elution was then carried out with 0.08 M NaCl, 0.15 M NaCl, and 0.2 M NaCl solutions. For elution with 0.08 M NaCl solution, target eluent 1 (from 11.5 to 19 min) was collected; for elution with 0.15 M NaCl solution, target eluent 2 (from 11 to 18.2 min) was collected; and for elution with 0.2 M NaCl solution, target eluent 3 (from 11.2 to 18.1 min) was collected. The elution flow rate was 10 mL / min. The collected target eluents 1-3 were concentrated and dialyzed, then concentrated again and freeze-dried to obtain Lycium barbarum pectin polysaccharides LBPA-I, LBPA-II and LBPA-III, respectively.

5. The preparation method according to claim 4, characterized in that, The mass-to-volume ratio of the total polysaccharides from wolfberry to distilled water was 1g:25mL.

6. The preparation method according to claim 4, characterized in that, The target eluents 1-3 are concentrated to 1 / 10 of their original volume; the molecular weight cutoff for dialysis is 3500 Da, and the dialysis time is 24 h.

7. The preparation method according to claim 4, characterized in that, The total polysaccharides of wolfberry are obtained by water extraction and alcohol precipitation.

8. The preparation method according to claim 7, characterized in that, The preparation of the total polysaccharides from wolfberry includes the following steps: Goji berries were added to purified water and heated for 2 hours. The filtrate was then collected by filtration. The filtrate was concentrated and centrifuged to obtain the supernatant. Ethanol was added to the supernatant until the final ethanol concentration reached 80%. After standing, the mixture was centrifuged to collect the precipitate. The precipitate was washed to remove water and dried to obtain total polysaccharides from goji berries.

9. The application of the Lycium barbarum pectin polysaccharide according to any one of claims 1 to 3 or the Lycium barbarum pectin polysaccharide prepared by any one of claims 4 to 8, characterized in that, The application includes at least one of the following a to d; a. The application of the aforementioned wolfberry pectin polysaccharide in the preparation of products that improve intestinal inflammation; b. The application of the aforementioned wolfberry pectin polysaccharide in the preparation of a product that improves intestinal inflammation in mice induced by chronic restraint stress; c. The application of the wolfberry pectin polysaccharide in the preparation of products that improve the expression of endocannabinoid system-related proteins; d. Application of the aforementioned wolfberry pectin polysaccharide in the preparation of products that regulate intestinal flora diversity.

10. The application according to claim 9, characterized in that, The products include pharmaceuticals.

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