Pogostemonis polysaccharide pc-b-1, preparation method and application thereof

Abstract

The application provides a uniform polyose PC-B-1 of pachouli, a preparation method and application thereof, and belongs to the technical field of biological medicine. The application provides the uniform polyose PC-B-1 of pachouli and application thereof in preparation of a medicine for treating ulcerative colitis. Through a DSS-induced mouse ulcerative colitis model, it is confirmed that PC-B-1 can significantly improve disease symptoms, including inhibiting body weight loss, relieving colon shortening and regulating immune organ index. Histopathological analysis (hematoxylin-eosin staining and alcian blue-periodic acid Schiff staining) further shows that PC-B-1 can effectively reduce colon mucosa inflammatory infiltration and promote repair of goblet cells and mucus. The application separates and identifies the novel polyose PC-B-1 from pachouli, and confirms that the polyose has significant anti-inflammatory and intestinal mucosa protection activity, and is expected to be developed into a medicine for treating ulcerative colitis.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, specifically to the homogeneous polysaccharide PC-B-1 of patchouli, its preparation method, and its applications. Background Technology

[0002] Patchouli is a plant of the Lamiaceae family, specifically patchouli (Paeonia lactiflora). Pogostemon cablin (Blanco) Benth The dried aerial parts of patchouli are washed, cut into sections, and sun-dried or shade-dried. This product is light, brittle, aromatic, and slightly bitter. It consists of irregular, often curled fragments. The stem is slightly square-shaped, grayish-brown, grayish-yellow, or reddish-brown on the surface, covered with soft hairs. The cut surface contains white pith. The leaves are broken or shriveled into clumps; intact leaves, when flattened, are ovate or elliptical, covered with grayish-white hairs on both sides. Modern pharmacological studies have shown that patchouli possesses aromatic dampness-resolving, heat-relieving, diaphoretic, anti-inflammatory, antibacterial, and immunomodulatory pharmacological activities. Its main chemical components are volatile oils (containing patchouli alcohol, patchouli ketone, etc.), polysaccharides, flavonoids, and terpenoids.

[0003] Polysaccharides are natural polymers composed of repeating units of carbohydrates or sugars, consisting of more than 10 monosaccharides linked together by glycosidic bonds. These units form the molecular chains of polysaccharides, which can be linearly arranged, branched, or cross-linked. Polysaccharides have a wide molecular weight range, from thousands of Daltons to over one million Daltons, and their complex linkages result in extremely intricate overall structures, which has significantly limited research progress. However, in recent years, with the continuous innovation and rapid development of analytical instruments, research on polysaccharides has become increasingly in-depth, and various biological activities of polysaccharides have been revealed. Modern scientific research shows that polysaccharides are found in most higher plants, animals, microorganisms, lichens, and algae in nature. Polysaccharides exhibit numerous biological activities, including antioxidant, immunomodulatory, antitumor, blood sugar-lowering, and antibacterial effects.

[0004] Currently, research on patchouli has been quite comprehensive regarding its volatile oil components, but research on its polysaccharides is relatively scarce. However, patchouli actually contains a considerable amount of polysaccharides. Therefore, it is essential to utilize existing polysaccharide extraction, separation, and identification techniques to discover novel polysaccharides from patchouli and to further explore their bioactivity. Summary of the Invention

[0005] The purpose of this invention is to provide a structurally well-defined homogeneous polysaccharide PC-B-1 extracted and isolated from patchouli, and to demonstrate its application in the prevention and treatment of ulcerative colitis, so as to realize the high-value utilization of patchouli resources.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] A homogeneous patchouli polysaccharide PC-B-1 with a relative molecular weight of 6073 Da is composed of α-L-Araf-(1→,β-D-Xylp-(1→,→5)-α-L-Araf-(1→, →2)-α-D-Glcp-(1→, →2)-α-D-GlcpA-(1→, →2,3,4)-α-L-Rhap-(1→, →4)-β-D-Galp-(1→, →3)-β-D-Man-(1→, →6)-α-D-Manp-(1→, →4,6)-β-D-Galp-(1→, →3,6)-β-D-Galp-(1→).

[0008] It was characterized by the following nuclear magnetic resonance spectroscopy data: 12 related peaks were observed in the anodic region of the HSQC spectrum, with ¹H / ¹³C chemical shifts of 5.08 / 107.4, 4.35 / 103.3, 4.96 / 107.3, 5.18 / 100.8, 4.93 / 101.1, 5.13 / 100.9, 4.46 / 97.0, 4.81 / 99.1, 5.19 / 97.8, 4.47 / 102.2, 4.39 / 97.1 and 5.12 / 92.5 ppm.

[0009] To achieve the objectives of this invention, this invention also provides a method for preparing patchouli homogeneous polysaccharide PC-B-1, comprising the following steps:

[0010] (1) Alkali extraction: Patchouli was used as raw material and cold-soaked in 0.3M-0.5M sodium hydroxide solution. The extracts were combined and neutralized to neutral. After concentration, the extracts were precipitated with alcohol to obtain crude patchouli polysaccharide.

[0011] (2) Protein removal: Mix the crude polysaccharide solution with Sevag reagent and shake. After standing and separating the layers, take the supernatant and repeat the operation to remove the protein.

[0012] (3) Primary purification: The polysaccharide solution after protein removal is loaded onto a DEAE ion exchange chromatography column and eluted with a gradient of 0-0.15M NaCl solution. The target eluted fraction is collected.

[0013] (4) Fine purification: The primary purified product was loaded onto a Sephadex G-75 gel chromatography column, eluted with deionized water, and the homogeneous fraction was collected, concentrated and lyophilized to obtain the homogeneous polysaccharide PC-B-1 of patchouli.

[0014] Preferably, in step (1), the sodium hydroxide solution used for alkaline extraction has a concentration of 0.3M, the cold soaking extraction time is 3 hours, and the final ethanol concentration used for alcohol precipitation is 75%.

[0015] The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned patchouli homogeneous polysaccharide PC-B-1 and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier includes sodium carboxymethyl cellulose.

[0016] To achieve the objectives of this invention, a technical solution is also provided: the application of patchouli homogeneous polysaccharide PC-B-1 in the preparation of a medicament for the prevention and / or treatment of ulcerative colitis.

[0017] The present invention also includes the application of patchouli homogeneous polysaccharide PC-B-1 in the preparation of intestinal mucosal protectants.

[0018] Compared with the prior art, the present invention achieves the following technical effects:

[0019] This invention not only yields a novel, active polysaccharide molecule, but also provides a safe and highly effective natural candidate for the treatment of ulcerative colitis, and realizes the high-value utilization of patchouli resources. Specifically, this invention successfully extracts PC-B-1, which has a clear chemical structure and strong biological activity, from stem and leaf residues traditionally considered waste through optimized processes.

[0020] PC-B-1 significantly improved macroscopic symptoms and microscopic pathological damage in ulcerative colitis at extremely low doses (5 mg / kg), and its mechanism of action is closely related to the regulation of key inflammatory factor levels. These effects suggest that PC-B-1 has the potential to be developed into a novel colonic mucosal protectant, thus providing a completely new solution for the treatment of inflammatory bowel disease. Simultaneously, it transforms waste into a valuable resource, greatly reducing the waste of patchouli resources, and providing a safe and efficient natural drug source for the treatment of ulcerative colitis and other inflammatory bowel diseases. Attached Figure Description

[0021] For ease of explanation, the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0022] Figure 1 The column elution curve of DEAE Sepharose Fast Flow for PCB-1.

[0023] Figure 2 Elution curve of PC-B-1 on Sephadex G-75 column.

[0024] Figure 3 This is a molecular weight diagram of PC-B-1.

[0025] Figure 4 HPLC chromatograms of PMP derivatization; (A) mixture of monosaccharide standards; (B) PC-B-1 hydrolysate.

[0026] Figure 5 The image shows the 1H-NMR spectrum of PC-B-1.

[0027] Figure 6 The 13C-NMR spectrum of PC-B-1.

[0028] Figure 7 The HSQC spectrum of PC-B-1.

[0029] Figure 8 The HMBC map of PC-B-1.

[0030] Figure 9 The 1H-1H COSY spectrum of PC-B-1.

[0031] Figure 10 This is a structural diagram of 10 PC-B-1.

[0032] Figure 11 PC-B-1 was used to improve symptoms in UC mice (n=6). (A) UC model; (B) Body weight change; (C) DAI score; (D) Colon morphology; (E) Colon length; (F) Thymus index; (G) Spleen index; Compared with the control group:** p <0.01, **** p <0.0001;

[0033] Compared with the DSS group # p <0.05, ## p <0.01, ### p <0.001, #### p <0.001.

[0034] Figure 12 The levels of cytokines in mouse serum and colon tissue (n=6) were measured. (A) Serum interleukin-6 level; (B) Serum interleukin-1β level; (C) Serum interleukin-10 level; (D) Colon tissue interleukin-6 level; (E) Colon tissue interleukin-1β level; (F) Colon tissue interleukin-10 level; Compared with the control group:** p <0.01, *** p <0.001, **** p <0.0001;

[0035] Compared to the DSS group: # p <0.05, ## p <0.01, ### p <0.001, #### p <0.01.

[0036] Figure 13 Improve the colonic tissue damage of UC mice by PC-B-1 (n = 6). (A) Hematoxylin-eosin staining, Alcian blue-periodic acid Schiff staining; (B) Histopathological score of hematoxylin-eosin staining, the proportion of the goblet cell area to the goblet cell area of the normal group; compared with the control group: **** p <0.0001; compared with the DSS group # p <0.05, ## p <0.01, # p <0.01. Specific implementation manners

[0037] The following are specific embodiments of the present invention and in combination with the accompanying drawings, the technical solutions of the present invention are further described, but the present invention is not limited to these embodiments; in the following description, providing specific details such as specific configurations is only to help a comprehensive understanding of the embodiments of the present invention. Therefore, those skilled in the art should clearly understand that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of the present invention.

[0038] It should be noted that, without conflict, the implementation manners and features in the implementation manners of the present invention can be combined with each other.

[0039] The materials, practices and experimental equipment involved in the embodiments of the present invention, unless otherwise specified, all conform to the commercially available products in the relevant chemical engineering and biotechnology fields.

[0040] Experimental material description:

[0041] 1. Experimental animals

[0042] The male 6-8-week-old SPF-grade C57BL / 6 mice used in this experiment, weighing 18-20 g, were purchased from the Guangdong Provincial Medical Animal Experiment Center (Experiment Unit License Number: SYXK (Guangdong) 2022-0125). The mice were housed in the SPF-grade laboratory of the Experimental Animal Center of Guangdong Pharmaceutical University, with a light-dark cycle of 12 hours each, the environmental temperature was 24 ± 2 °C, the relative humidity was 55 ± 5%, and sufficient feed and drinking water were provided every day. In addition, all animal experiments were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University and were carried out in accordance with the "Animal Experiment Guide" of Guangdong Pharmaceutical University.

[0043] 2. Experimental materials and reagents

[0044] Table 1 Experimental materials and reagents

[0045]

[0046] 3. Experimental instruments

[0047] Table 2 Experimental Instruments

[0048]

[0049] Example 1: Extraction and Separation of PC-B-1 Polysaccharides

[0050] 1. Extraction:

[0051] 10 kg of dried patchouli was completely submerged in 10 times its volume of 0.3 M sodium hydroxide solution. The patchouli residue was then extracted at room temperature for 3 hours. This process was repeated three times. After combining the extracts, the alkaline extract was slowly neutralized to neutral using 0.5 M hydrochloric acid solution. After concentration and centrifugation, the supernatant was precipitated with alcohol until the final ethanol concentration was 75%, yielding crude polysaccharides from the alkaline extract of patchouli.

[0052] Take appropriate amounts of crude polysaccharides from different parts of patchouli, add sufficient deionized water to completely dissolve them, add Sevag reagent at a volume ratio of 5:1, mix, shake for 30 minutes to allow the Sevag reagent to fully contact the patchouli crude polysaccharide solution, let stand and wait for the solution to separate into layers, discard the lower layer, take the supernatant and repeat the operation more than 3 times. After removing the protein, concentrate the supernatant under reduced pressure to remove the residual Sevag reagent, aliquot the supernatant into dialysis bags (cutoff: 1000 Da), dialyze with running water for 48 hours, combine the dialysates, concentrate and freeze dry.

[0053] The crude polysaccharide from patchouli after protein removal was prepared into a 20 mg / mL solution with deionized water. After centrifugation, 10 mL of the solution was loaded onto a DEAE-52 anion exchange chromatography column (Ø 2.5 × 40 cm). The column was eluted sequentially with sodium chloride solutions of different concentrations: 0 M, 0.05 M, 0.1 M, and 0.15 M. The elution tubes were then stained using the sulfuric acid-phenol method. The absorbance was measured using an ELISA reader (wavelength 490 nm), and elution curves were plotted. The stained tubes were then combined, concentrated, dialyzed, and lyophilized.

[0054] The lyophilized sample was separated using a DEAE-52 column, and a solution with a concentration of 20 mg / mL was prepared using deionized water. After centrifugation, 1 mL of the solution was loaded onto a G-75 M column (Ø 1.5 × 100 cm). Elution was performed with deionized water, and each eluent tube was colored using the sulfuric acid-phenol method. The absorbance was measured using a microplate reader (wavelength 490 nm), and elution curves were plotted. The purity of the colored eluent tubes was determined by gel permeation chromatography (GPC). The eluent fractions that met the purity criteria were collected, concentrated, and lyophilized to obtain homogeneous refined polysaccharides.

[0055] 2. Structural characterization of homogeneous patchouli polysaccharides

[0056] Determination of the relative molecular weight of PC-B-1:

[0057] The homogeneity and molecular weight of the polysaccharide were determined using a high-performance gel permeation chromatography (HPGPC) system. 3 mg of polysaccharide was dissolved in 1 mL of deionized water and analyzed using a Waters 1525 HPLC system equipped with a Waters 2414 differential refractive index detector. The system used two columns in tandem: a Waters Ultrahydrogel 1000 (7.8 × 300 mm) and a Waters Ultrahydrogel 500 (7.8 × 300 mm) (Milford, USA). The mobile phase was 0.02 M KH₂PO₄ solution, and the flow rate was 0.5 mL / min. A standard curve was constructed using the T-series dextran standards (T5, T10, T40, T70, T500, T1000), and the molecular weight was determined accordingly. The sample concentration was 2.0 mg / mL, and the injection volume was 20 μL.

[0058] Monosaccharide composition analysis of PC-B-1:

[0059] Weigh 5 mg of sample and dissolve it in 2 mL of 2 M trifluoroacetic acid (TFA) solution. Hydrolyze the solution in a 120°C oil bath for 5 hours. The hydrolysis product and monosaccharide standard were separately derivatized using 1-phenyl-3-methyl-5-pyrazolone (PMP) before the column. An HPLC system equipped with an Agilent ZORBAX Eclipse XDB-C18 column (5 μm, 4.6 mm × 250 mm) and a UV detector (Shimadzu LC20AT, Kyoto, Japan) was used. p The derivatives were analyzed using a mobile phase consisting of a mixture of 0.05 M phosphate buffer (pH 6.7) and acetonitrile (83:17, v / v), with isocratic elution and a flow rate of 1.0 mL / min.

[0060] Methylation and GC-MS analysis of PC-B-1:

[0061] Dissolve 10 mg of polysaccharide in 5 mL of deionized water. Weigh 120 mg of carboxymethyl cellulose (CMC) and dissolve it in 5 mL of deionized water. After thoroughly mixing the CMC solution and the polysaccharide solution, add 0.01 M HCl solution dropwise to adjust the pH to 4.75 and react at room temperature for 2 h. Then add 15 mL of 2 mol / L sodium borohydride (NaBH4) while maintaining the pH at 7.0 using 4 mol / L hydrochloric acid (HCl) solution. After reacting for 60 min, dialyze the mixture (molecular weight cutoff: 1000 Da) and freeze-dry. Repeat the above steps three times to obtain the reduced polysaccharide.

[0062] Six mg of polysaccharide, both before and after uronic acid reduction, were used for subsequent reactions. The polysaccharide was dissolved in 5 mL DMSO and sonicated for 1 h. 400 mg of NaOH was weighed and added to 5 mL DMSO, then ground to form a suspension. The polysaccharide-DMSO solution was mixed with the NaOH-DMSO solution and sonicated for 1 h. Under light-protected conditions, 500 μL of iodomethane (CH3I) was added to the mixture, and sonication was performed for 15 min. Subsequently, 500 μL of iodomethane was added twice, with sonication times of 15 min and 1 h, respectively. After the reaction was complete, 8 mL of deionized water and 2 mL of chloroform were added, and the mixture was vigorously shaken. The chloroform layer was collected and extracted three times with 2 mL of deionized water to obtain the methylated product. Finally, the methylated product was hydrolyzed using the same method as the polysaccharide hydrolysis, followed by reduction with NaBH4 and acetylation, and then analyzed by gas chromatography-mass spectrometry. The GC-MS conditions were as follows: an Agilent 7890B-7000D GC-MS system (Santa Clara, USA) equipped with a TG-SQC column (0.25µm, 15 m × 0.25 mm) was used; the carrier gas was helium (99.999% purity); the temperature program was: initial 80 °C for 1 min, increased to 280 °C at a rate of 5 °C / min, and finally held at 280 °C for 1 min; the flow rate was 1.2 mL / min; and the injection volume was 2 μL.

[0063] Nuclear magnetic resonance analysis of PC-B-1:

[0064] Each 65 mg sample was weighed and dissolved in 700 μL of deuterated water (D2O). After centrifugation and filtration, the samples were analyzed using a nuclear magnetic resonance spectrometer (AVANCE NEO 500 M, Bremen, Germany) at 25 °C.

[0065] 3. Pharmacological evaluation of patchouli polysaccharides in mice with ulcerative colitis

[0066] Drug preparation:

[0067] Preparation of 3% sodium dextran sulfate solution (3% DSS solution): Prepare fresh daily. Accurately weigh 18g of sodium dextran sulfate powder using a balance, place it in a 50ml centrifuge tube, add 40ml of purified water, vortex to mix, and then add 560ml of purified water and mix well to obtain the 3% DSS solution.

[0068] Preparation of 0.5% sodium carboxymethyl cellulose (CMC-Na) solution: Accurately weigh 0.4g of sodium carboxymethyl cellulose and add it in small amounts several times to a beaker containing 80mL of boiling distilled water. Heat and stir until completely dissolved. After cooling, add distilled water to make up the weight. Store at 4℃ for later use.

[0069] Preparation of mesalazine solution (5-ASA): Prepare fresh before use. Weigh an appropriate amount of mesalazine powder and dissolve it in 0.5% CMC-Na solution. Vortex mix well to prepare a 30 mg / ml 5-ASA solution. Store at 4°C protected from light. The gavage volume for mice is 0.01 mL / g, and the dose is 300 mg / kg.

[0070] Preparation of patchouli polysaccharide solution: Weigh an appropriate amount of patchouli polysaccharide powder and dissolve it in 0.5% CMC-Na solution to prepare a 0.5 mg / ml polysaccharide solution. The gavage volume for mice was 0.01 mL / g, and the dose was 5 mg / kg.

[0071] Experimental plan:

[0072] After a week of acclimatization, mice were randomly divided into four groups (n=6 per group): control group (Con), DSS group (DSS), mesalazine treatment group (5-ASA, 300 mg / kg), and patchouli polysaccharide treatment group (PC-B-1, 5 mg / kg). The Con group had free access to regular water, while the other groups had free access to 3% DSS solution to induce ulcerative colitis. After 7 days, they switched to regular water. Simultaneously, the Con and DSS groups were administered a blank 0.5% CMC-Na solution by gavage, while the treatment groups received the drug daily at a gavage volume of 0.01 ml / g. This regimen was implemented for 10 consecutive days, with administration once daily. Disease activity index was recorded daily. On day 11, mice were euthanized by cervical dislocation. The cecum, colon, thymus, and spleen were collected. The thymus and spleen were weighed, and the colon length was recorded. The cecal contents were collected and rapidly frozen in liquid nitrogen, then stored at -80°C. The distal colon was placed in a tissue cell fixative (4% paraformaldehyde) and left at room temperature for 24 hours for subsequent HE staining. The remaining colon tissue was cut open, the contents were removed, rinsed with physiological saline, and then rapidly frozen in liquid nitrogen and stored in a freezer at -80°C for later use.

[0073] Colitis assessment:

[0074] Throughout the experiment, daily observations and records were kept of mouse weight changes, fecal characteristics, and bloody stools, and scores were calculated according to Table 3. The Disease Activity Index (DAI) was the sum of the three scores; a higher score indicated a more severe disease. After euthanizing the mice, the colon, thymus, spleen, and other organs were quickly removed, rinsed thoroughly with PBS, and the length of the colon was measured using a ruler. The thymus and spleen were weighed using an analytical balance, and the immune organ index was calculated.

[0075] The formula for calculating the immune organ index is: Immune organ index (mg / g) = Spleen weight (mg) / Body weight (g)

[0076] Table 3. Disease Activity Index Scoring Criteria

[0077]

[0078] Serum cytokine level detection:

[0079] After collecting blood samples, they were first allowed to stand at room temperature for 1 hour, and then centrifuged at 4000 rpm for 15 minutes at 4°C. The supernatant was collected to prepare serum. A certain weight of colon tissue was added to phosphate buffer solution containing 1% protease inhibitor at a weight-to-volume ratio of 1:9 for tissue homogenization. The homogenate was centrifuged at 5000Xg for 8 minutes, and the supernatant was collected as the sample. Following standard operating procedures, a mouse ELISA kit was used to quantitatively detect three key inflammatory cytokines in the serum—interleukin-6 (IL-6), interleukin-1β (IL-1β), and interleukin-10 (IL-10)—to systematically assess the levels of relevant indicators of inflammatory response.

[0080] Histopathological evaluation:

[0081] Hematoxylin and eosin (H&E) staining of mouse colon tissue:

[0082] The collected distal colon tissue was fixed in 4% paraformaldehyde for 24 hours, followed by routine dehydration, clearing, and paraffin embedding to prepare paraffin-embedded blocks. The tissue blocks were serially sectioned into 4 μm thick sections using a microtome. After dewaxing and graded ethanol hydration, the sections were stained with hematoxylin and eosin sequentially. The stained sections were then dehydrated, cleared, and mounted with neutral resin before being scanned for image acquisition. Histological scoring of the acquired images was performed according to the criteria in Table 4 to assess the overall morphology, inflammatory cell infiltration, and epithelial damage of the colon tissue.

[0083] Table 4. Histopathological Scoring Criteria

[0084]

[0085] Alcian blue-periodic acid Schiff (AB-PAS) staining of mouse colon tissue:

[0086] The collected distal colon tissue was fixed in 4% paraformaldehyde for 24 hours, followed by routine dehydration, clearing, and paraffin embedding to prepare paraffin-embedded blocks. The tissue blocks were serially sliced ​​into 4 μm thick sections using a microtome. After dewaxing and graded ethanol hydration, the sections were stained sequentially with Alcian blue, periodic acid, and Chevron. After dehydration, clearing, and mounting with neutral resin, the stained sections were then imaged using a scanner. The percentage of AB-PAS-positive stained areas was quantitatively analyzed using ImageJ software to serve as a quantitative indicator of the number and functional status of mucus-secreting cells.

[0087] Example 2: Isolation and purification results, molecular weight and monosaccharide composition analysis of crude polysaccharides from patchouli.

[0088] 1. Results of isolation and purification of patchouli crude polysaccharide PCB:

[0089] After loading the crude patchouli polysaccharide PCB onto a DEAE Sepharose Fast Flow column, a gradient elution method was performed using NaCl solutions of different concentrations. Four main peaks were observed at NaCl solution concentrations of 0 M, 0.05 M, 0.1 M, and 0.15 M, indicating that four polysaccharides were obtained from PCB after separation by the DEAE Sepharose Fast Flow column. The polysaccharide obtained with the 0.05 M gradient was named PCB-1. The results are as follows... Figure 1 As shown in the figure. The four components (collection times are shown in red circles in the figure) were collected and accumulated separately. The eluents of each concentration of patchouli polysaccharide obtained were concentrated, dialyzed, lyophilized, and then further purified by Sephadex G-75 column. The purification results are shown in the figure. Figure 2 .

[0090] 2. Molecular weight and monosaccharide composition analysis of PC-B-1

[0091] The molecular weight of patchouli polysaccharide PC-B-1, determined by HPGPC, is 6073 Da. Figure 3 The mixture of monosaccharide standards and the hydrolysate of PC-B-1 were derivatized by PMP, and the results were obtained by HPLC analysis as follows: Figure 4 A and Figure 3 From the B in the formula, we know that the monosaccharide composition of PC-B-1 contains mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, and arabinose.

[0092] 3. Methylation and GC-MS analysis of PC-B-1

[0093] The fully methylated PC-B-1-M was hydrolyzed, reduced, and acetylated, and then analyzed by GC-MS to obtain fragment ion peaks. Comparison with standard spectra revealed the polysaccharide linkage modes and proportions. The results showed that PC-B-1 has 10 linkage modes: L-Araf-(1→, D-Xylp-(1→, →5)-L-Araf-(1→, →2)-D-Glcp-(1→,→2)-D-GlcpA-(1→, →2,3,4)-L-Rhap-(1→, →4)-D-Gal p-(1→, →3)-D-Man-(1→,→6)-D-Manp-(1→, →4,6)-D-Galp-(1→, →3,6)-D-Galp-(1→, whose relative proportions are 3.91, 2.31, 13.31, 2.89, 1.23, 1.00, 9.76, 1.13, 2.26, 1.33, 1.07.

[0094] 4. Nuclear magnetic resonance spectroscopy analysis of PC-B-1

[0095] The HSQC spectrum of PC-B-1 showed 12 distinct correlation peaks in the anodic region, with the following ¹H / ¹³C chemical shifts: 5.08 / 107.4, 4.35 / 103.3, 4.96 / 107.3, 5.18 / 100.8, 4.93 / 101.1, 5.13 / 100.9, 4.46 / 97.0, 4.81 / 99.1, 5.19 / 97.8, 4.47 / 102.2, 4.39 / 97.1, and 5.12 / 92.5 ppm. These observations clearly indicate that PC-B-1 consists of twelve glycosyl residues, named A, B, D, E, F, G, I, J, K, L, M, and N. This assignment is consistent with the GC-MS results. Based on the references, the C-H chemical shifts of the glycosyl residues in PC-B-1 were assigned, and the results are shown in Table 5.

[0096] The HMBC spectrum revealed correlation peaks between ¹H and ¹³C, demonstrating the connection sequences and sites between different residues. In the HMBC spectrum, the correlation peak at 4.93 / 69.6 ppm (F H-1 / D C-5) confirmed the connection between O-1 of Residue F and C-5 of Residue D. Similarly, the correlation signal of 4.96 / 69.6 ppm (DH-1 / DC-5) indicates that the O-1 of Residue D is connected to the C-5 of Residue D; the correlation signal of 5.18 / 68.5 ppm (EH-1 / KC-6) indicates that the O-1 of Residue E is connected to the C-6 of Residue K; the correlation signal of 4.35 / 69.6 ppm (BH-1 / DC-5) indicates that the O-1 of Residue B is connected to the C-5 of Residue D; the correlation signal of 5.19 / 68.5 ppm (KH-1 / KC-6) indicates that the O-1 of Residue K is connected to the C-6 of Residue K; and the correlation signal of 5.19 / 68.7 ppm (KH-1 / LC-6) indicates that the O-1 of Residue K is connected to the C-6 of Residue L. The correlation signals of 4.35 / 69.0 ppm (B H-1 / M C-6) indicate that O-1 of Residue B is connected to C-6 of Residue M; 5.08 / 81.0 ppm (A H-1 / G C-2) indicate that O-1 of Residue A is connected to C-2 of Residue G; 5.13 / 81.5 ppm (G H-1 / I C-4) indicate that O-1 of Residue G is connected to C-4 of Residue I; 5.18 / 82.1 ppm (E H-1 / E C-2) indicate that O-1 of Residue E is connected to C-2 of Residue E; 5.08 / 82.1 (A H-1 / E C-2) indicate that O-1 of Residue A is connected to C-2 of Residue E. The correlation signal of 4.96 / 76.9 ppm (DH-1 / F C-2) indicates that O-1 of Residue D is connected to C-2 of Residue F; the correlation signal of 4.96 / 81.5 ppm (DH-1 / L C-4) indicates that O-1 of Residue D is connected to C-4 of Residue L; 4.96 / 81.5 ppm (DH-1 / L C-4) indicates that O-1 of Residue D is connected to C-4 of Residue L;The correlation signal of 5 ppm (DH-1 / IC-4) indicates that the O-1 of Residue D is connected to the C-4 of Residue I; the correlation signal of 4.46 / 69.6 ppm (ICH-1 / DC-5) indicates that the O-1 of Residue I is connected to the C-5 of Residue D; the correlation signal of 4.47 / 79.6 ppm (LH-1 / GIC-3) indicates that the O-1 of Residue L is connected to the C-3 of Residue G; the correlation signal of 4.96 / 81.2 ppm (DH-1 / JIC-3) indicates that the O-1 of Residue D is connected to the C-3 of Residue J; the correlation signal of 3.82 / 97.1 ppm (GH-4 / MC-1) indicates that the O-4 of Residue G is connected to the C-1 of Residue M; the correlation signal of 4.81 / 69.6 ppm (JIC-1 / MC-4) indicates that the O-1 of Residue G is connected to the C-1 of Residue M; The correlation signals for H-1 / D C-5 indicate that O-1 of Residue J is connected to C-5 of Residue D; the correlation signals for 4.96 / 82.1 ppm (D H-1 / N C-2) indicate that O-1 of Residue D is connected to C-2 of Residue N.

[0097] Table 5 Chemical shift values ​​of sugar residues in PC-B-1

[0098]

[0099] 5. Structural information of PC-B-1

[0100] Based on the monosaccharide composition, methylation, and GC-MS and NMR analyses of PC-B-1, it can be seen that PC-B-1 is composed of α-L-Araf-(1→, β-D-Xylp-(1→,→5)-α-L-Araf-(1→, →2)-α-D-Glcp-(1→, →2)-α-D-GlcpA-(1→, →2,3,4)-α-L-Rhap-(1→, →4)-β-D-Galp-(1→, →3)-β-D-Man-(1→, →6)-α-D-Manp-(1→, →4,6)-β-D-Galp-(1→, →3,6)-β-D-Galp-(1→), and its primary structure is as follows. Figure 10 As shown.

[0101] 6. Patchouli polysaccharides alleviate DSS-induced ulcerative colitis in mice.

[0102] This study successfully established an ulcerative colitis model in mice by allowing them free access to sodium dextran sulfate (DSS) solution. Figure 11 (A) To macroscopically assess the overall disease status of the mice, we systematically monitored their weight changes, fecal characteristics, and rectal bleeding, and calculated the Disease Activity Index (DAI) score accordingly. Compared with the normal group, the model group mice experienced more significant weight loss throughout the experiment and consistently exhibited symptoms such as loose stools and gross rectal bleeding, resulting in a significantly higher DAI score on day 10. p <0.001)( Figure 11 (B and C in the text). However, after treatment with the positive control drug and PC-B-1, the macroscopic symptoms of the mice were significantly improved, manifested as a slowdown in weight loss, and the DAI scores were significantly lower than those of the DSS model group (B and C in the text). p <0.01 and p <0.001).

[0103] To visually evaluate the therapeutic effect of PC-B-1, we measured the colon length of mice in each group. Figure 11 As shown in D, compared with the normal group (colon length 7.75±0.23 cm), the colon length in the model group was significantly shortened to 5.56±0.66 cm. p <0.0001), indicating successful modeling and inducing typical intestinal structural damage. After intervention with the positive control drug 5-ASA, colon length was partially improved, recovering to 7.05±0.25 cm (compared to the model group). p <0.0001. The PC-B-1 treatment group showed efficacy comparable to the positive control group, with colon length recovering to 6.93±0.29 cm, a significant difference compared to the model group. p <0.001).

[0104] Further analysis of immune organ indices ( Figure 11 The F and G in the model group (DSS) significantly affected the immune organ status of mice with DSS-induced colitis. Compared with the normal group (thymus index: 1.76±0.24 mg / g; spleen index: 2.91±0.42 mg / g), the thymus index of the model group mice was significantly reduced (0.56±0.30 mg / g). p <0.0001), while the spleen index was significantly elevated (4.66±0.97 mg / g, p <0.01) indicates atrophy and compensatory enlargement of immune organs in the disease state. After treatment with 5-ASA-positive drugs, the thymus index recovered to 1.22±0.22 mg / g (compared to the DSS group). p <0.05), the thymus index in the PC-B-1 treatment group was 1.20±0.49 mg / g (compared to the DSS group).p <0.05 indicates that both treatments could partially reverse thymic atrophy. Regarding the spleen index, although the positive control group (4.38±1.12 mg / g) and the PC-B-1 group (4.17±0.53 mg / g) showed a decreasing trend, there was no statistically significant difference compared to the DSS model group. p >0.05).

[0105] 7. Patchouli polysaccharides reduce the levels of pro-inflammatory cytokines in mouse serum.

[0106] Figure 12 As shown, compared with the normal group, the levels of pro-inflammatory factors interleukin-6 and interleukin-1β in the serum and colon tissue of mice in the model group were significantly increased. p <0.01 or 0.0001), while the level of the anti-inflammatory factor interleukin-10 was significantly reduced ( p <0.001 indicates that the inflammatory response was successfully induced and the anti-inflammatory mechanism was suppressed. Compared with the model group, the levels of pro-inflammatory factors interleukin-6 and interleukin-1β in the serum and colon tissue of mice in the positive drug group and PC-B-1 group were significantly reduced ( p <0.05 or 0.001), indicating that both have certain anti-inflammatory effects. Among them, compared with the model group, the level of the anti-inflammatory factor interleukin-10 in the serum of mice in the positive drug group was significantly increased ((…). p <0.05), interleukin-10 in the tissues of mice in the positive drug group showed an increasing trend, but the increase was not significant. Figure 12 (C and F in the model group). Compared with the model group, the serum interleukin-10 level in the PC-B-1 group mice showed a trend of increasing but not statistically significant. Notably, PC-B-1 significantly increased the level of interleukin-10 in mouse tissues (C and F in the model group). p <0.05).

[0107] 8. Patchouli polysaccharides reduce colonic tissue damage induced by colitis.

[0108] Figure 13 The results showed that the colon tissue of the normal group mice had normal morphology, intact epithelial structure, and no obvious pathological damage. In contrast, the colon tissue of the model group was severely damaged, with mucosal ulceration, epithelial cell shedding, a large number of necrotic fragments and inflammatory cells on the surface, obvious inflammatory cell infiltration, irregular morphology of surrounding intestinal glands, significantly reduced number of goblet cells, unclear boundary between local mucosa and submucosa, large lymph nodes, submucosal edema, loose tissue structure, and a large number of inflammatory cell infiltrations. Figure 13 (A) After intervention with positive control drugs and PC-B-1, the pathological condition of colonic tissue was significantly improved, mucosal damage was reduced, and inflammatory infiltration was also significantly decreased. Figure 13A in the text). Hematoxylin-eosin staining histological score showed ( Figure 13 In the B group, the score was 0.67±0.47 in the normal group and 7.67±0.47 in the DSS group, indicating the most severe colonic damage. The scores in the positive drug group and the PC-B-1 group were 2.67±0.47 and 2.32±0.47, respectively, all showing significant protective effects on colonic tissue. p <0.0001,) among which group PC-B-1 showed better results.

[0109] The goblet cells and intestinal glands of the colonic mucosa mainly secrete acidic mucus. After Asin blue-periodic acid-Schiff staining (AB-PAS), the mucus glycoprotein in the goblet cells appears blue. Figure 13 In the normal group, A shows that the colonic mucosa of the mice is intact, with abundant and evenly distributed goblet cells, indicating normal mucus secretion function and no significant loss of mucus glycoproteins. In the model group, the colonic mucosa shows severe damage, a significant reduction in goblet cells, and marked epithelial cell shedding. The area of ​​AB-PAS-stained positive cells is only 0.22±0.04 of that in the normal group, indicating a significant decrease in positive staining. Figure 13 (C) The ratio of AB-PAS-positive cell area in the positive drug group and the PC-B-1 group was 0.42±0.08 and 0.57±0.08, respectively, compared with the normal group. Figure 13 In the C group, compared with the model group, the area ratio of positive cells was significantly increased (C). p <0.05). This indicates that after intervention with positive control drugs and PC-B-1, the colonic mucosa of mice was effectively protected, goblet cell function was restored, and the loss of mucus glycoproteins was reduced.

[0110] Those skilled in the art to which this application pertains may modify or supplement the specific embodiments described or use similar methods to replace them, but without departing from the inventive concept of this application or exceeding the scope defined by the appended claims.

Claims

1. Patchouli homogeneous polysaccharide PC-B-1, characterized in that, Its relative molecular weight is 6073 Da, and it is composed of the following monosaccharide residues linked together in the manner and relative proportions shown: PC-B-1 consists of α-L-Araf-(1→, β-D-Xylp-(1→,→5)-α-L-Araf-(1→, →2)-α-D-Glcp-(1→, →2)-α-D-GlcpA-(1→, →2,3,4)-α-L-Rhap-(1→, →4)-β-D-Galp-(1→, →3)-β-D-Man-(1→, →6)-α-D-Manp-(1→, →4,6)-β-D-Galp-(1→, →3,6)-β-D-Galp-(1→Composition; Their relative proportions are 3.91, 2.31, 13.31, 2.89, 1.23, 1.00, 9.76, 1.13, 2.26, 1.33, and 1.

07. The preparation method of the patchouli homogeneous polysaccharide PC-B-1 includes the following steps: (1) Alkali extraction: Patchouli was used as raw material and cold-soaked in 0.3M sodium hydroxide solution. The extracts were combined and neutralized to neutral. After concentration, the extracts were precipitated with alcohol to obtain crude patchouli polysaccharide. (2) Protein removal: Mix the crude polysaccharide solution with Sevag reagent and shake. After standing and separating the layers, take the supernatant and repeat the operation to remove the protein. (3) Primary purification: The polysaccharide solution after protein removal is loaded onto a DEAE ion exchange chromatography column and eluted with a gradient of 0-0.15M NaCl solution. The target eluted fraction is collected. (4) Fine purification: The primary purified product was loaded onto a Sephadex G-75 gel chromatography column, eluted with deionized water, and the homogeneous fraction was collected, concentrated and lyophilized to obtain the homogeneous polysaccharide PC-B-1 of patchouli. In step (1), the cold soaking extraction time is 3 hours, and the final ethanol concentration used for alcohol precipitation is 75%.

2. The patchouli homogeneous polysaccharide PC-B-1 according to claim 1, characterized in that, It was characterized by the following nuclear magnetic resonance spectroscopy data: 12 related peaks were observed in the anodic region of the HSQC spectrum, with ¹H / ¹³C chemical shifts of 5.08 / 107.4, 4.35 / 103.3, 4.96 / 107.3, 5.18 / 100.8, 4.93 / 101.1, 5.13 / 100.9, 4.46 / 97.0, 4.81 / 99.1, 5.19 / 97.8, 4.47 / 102.2, 4.39 / 97.1 and 5.12 / 92.5 ppm.

3. A pharmaceutical composition, characterized in that, The product comprises a therapeutically effective amount of the patchouli homogeneous polysaccharide PC-B-1 as described in claim 1 or 2, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, characterized in that, Pharmaceutically acceptable carriers include sodium carboxymethyl cellulose.

5. The use of the patchouli homogeneous polysaccharide PC-B-1 according to claim 1 or 2 in the preparation of a medicament for the prevention and / or treatment of ulcerative colitis.

6. The use of the patchouli homogeneous polysaccharide PC-B-1 according to claim 1 or 2 in the preparation of an intestinal mucosal protectant.

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