Preparation method and application of blue cloth polysaccharide extract

Abstract

The application discloses a preparation method and application of a blue cloth polysaccharide extract, and belongs to the field of traditional Chinese medicine. The preparation method comprises the following steps: taking the whole plant of blue cloth, drying, crushing and sieving, adopting an acid-assisted extraction method, using an acid aqueous solution with pH 2-4 as a solvent, a material-liquid ratio of 1:10-1:30, extracting at 60-90 DEG C for 1-3 hours, and obtaining the extract through concentration, ethanol precipitation, deproteinization, ultrafiltration and freeze-drying. In a mouse model of anemia induced by chemotherapy with cyclophosphamide, the extract can significantly increase the levels of peripheral red blood cells, hemoglobin and platelets, and improve the atrophy of immune organs. The mechanism of action is as follows: regulating the intestinal microbiota (enriching beneficial bacteria such as cryptosporidium and akkermansia, and inhibiting pro-inflammatory bacteria such as prevotella), and then regulating glycerophospholipid metabolism and amino acid metabolism. The application provides a safe and effective natural product source for anemia caused by chemotherapy, and is expected to be used for preparing related drugs.

Description

Technical Field

[0001] This invention relates to the field of traditional Chinese medicine, specifically to a method for preparing a polysaccharide extract of *Lysimachia christinae* and its application in the preparation of drugs for the prevention and / or treatment of chemotherapy-induced anemia. Background Technology

[0002] Chemotherapy-related anemia (CRA) is one of the most common complications during chemotherapy in patients with malignant tumors. It manifests as a decrease in peripheral blood red blood cells, white blood cells, platelets, and hemoglobin levels, often accompanied by immunosuppression and bone marrow suppression. Currently, commonly used clinical interventions include iron supplementation, erythropoietin, and blood transfusions; however, these methods have limitations such as iron overload, thrombosis, increased risk of tumor progression, and blood shortages. Therefore, developing safe, naturally sourced products that can be used long-term for dietary support during chemotherapy is of significant clinical importance.

[0003] *Geum japonicum* Thunb. var. *chinense* Bolle, a plant belonging to the genus *Geum japonicum* in the Rosaceae family, is a characteristic plant with a long history of edible and medicinal use in Miao ethnic regions, commonly used in folk medicine for replenishing blood and treating dizziness and fatigue. Previous research by our group has shown that the aqueous extract of *Geum japonicum* can improve hematopoietic dysfunction induced by cyclophosphamide, and its total tannin components can alleviate hematopoietic inhibition by activating the JAK2 / STAT3 signaling pathway. Phytochemical analysis shows that the total sugar content in *Geum japonicum* extract is as high as 41% or more, suggesting that polysaccharides may be the main material basis for its blood-replenishing activity. The applicant further compared the effects of seven extraction methods on the physicochemical properties and antioxidant activity of *Geum japonicum* polysaccharides (see *Molecules* 2025, 30, 148), finding that the polysaccharides obtained by acid-assisted extraction had higher contents of neutral sugars and uronic acids. However, the in vivo anti-anemia activity of *Geum japonicum* polysaccharides obtained by different extraction methods has not been reported, and its mechanism of action in alleviating chemotherapy-related anemia, especially its regulatory role based on the gut microbiota-metabolite axis, remains unclear. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for preparing blue cloth polysaccharide extract and its application in the preparation of drugs for the prevention and / or treatment of chemotherapy-induced anemia.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] A method for preparing a polysaccharide extract of *Lysimachia foetida* includes the following steps: taking the whole herb of *Lysimachia foetida*, drying, pulverizing and sieving, extracting using an acid-assisted extraction method, concentrating the extract, precipitating with ethanol, removing proteins, ultrafiltration and freeze-drying to obtain the polysaccharide extract of *Lysimachia foetida*; the conditions for the acid-assisted extraction method are: using an acidic aqueous solution with pH 2-4 as the solvent, a material-to-liquid ratio of 1:10-1:30 (g / mL), extracting at 60-90 ℃ for 1-3 hours, and extracting 1-3 times.

[0007] Furthermore, the deproteinization is performed using the Sevag method, with a volume ratio of chloroform to n-butanol of 4:1, and the deproteinization is performed 1-5 times; the ultrafiltration uses an ultrafiltration membrane with a molecular weight cutoff of 3500 Da.

[0008] Furthermore, the preferred conditions for the acid-assisted extraction method are: pH 2.5, material-to-liquid ratio 1:20 (g / mL), extraction temperature 80 ℃, extraction time 2 hours, and extraction twice.

[0009] This application also provides the use of the blue cloth polysaccharide extract obtained by the above preparation method in the preparation of drugs for the prevention and / or treatment of chemotherapy-induced anemia.

[0010] Furthermore, the chemotherapy-induced anemia was induced by cyclophosphamide.

[0011] Compared with the prior art, the present invention has the following beneficial effects:

[0012] 1. This invention is the first to systematically compare the in vivo anti-anemia activity of blue cloth polysaccharides obtained by five different extraction methods. It was found that the blue cloth polysaccharides prepared by acid-assisted extraction method showed the best hematopoietic recovery effect in a cyclophosphamide-induced anemia mouse model, which could significantly increase the levels of peripheral blood erythrocytes, hemoglobin, and platelets, and improve the atrophy of immune organs.

[0013] 2. This invention reveals for the first time that blue cloth polysaccharide extract alleviates chemotherapy-induced anemia by reshaping the gut microbiota, specifically by increasing the relative abundance of beneficial bacteria such as Cryptobacterium and Akkermansia, and decreasing the relative abundance of pro-inflammatory bacteria such as Prevotella, thus establishing a new mechanism of action of the gut-bone marrow axis.

[0014] 3. This invention elucidates for the first time that the extract of blue cloth polysaccharide can regulate glycerophospholipid metabolism (decreasing lysophosphatidic acid and increasing lysophosphatidylcholine) and amino acid metabolism (decreasing L-phenylalanine and increasing 2-oxoarginine), explaining its role in supporting hematopoiesis from a metabolic perspective.

[0015] 4. The blue cloth polysaccharide extract provided by this invention is derived from a traditional edible plant, has high safety, and a stable preparation process. It provides a new natural drug source for chemotherapy-induced anemia and has broad application prospects. Attached Figure Description

[0016] Figure 1 This invention relates to the effects of the blue cloth polysaccharide extract on peripheral blood counts and immune organ indices in chemotherapy-induced anemia mice; where A: schematic diagram of the experimental protocol; B: white blood cell count (WBC); C: red blood cell count (RBC); D: hemoglobin concentration (HGB); E: hematocrit (HCT); F: platelet count (PLT); G: spleen index; H: thymus index. Data are expressed as mean ± SEM, compared with the control group, ## This indicates that P < 0.01; compared with the model group, ns This indicates no significant difference. * This indicates that P < 0.05. ** This indicates that P < 0.01.

[0017] Figure 2 This invention relates to the regulatory effect of the blue cloth polysaccharide extract on the intestinal microbiota of chemotherapy-induced anemia mice; wherein AC: Alpha diversity index (Chao1, Shannon, Simpson); DF: Beta diversity analysis (PCA, PLS-DA score plot); GI: relative abundance bar chart at the phylum, family, and genus levels; JL: differential abundance analysis of key bacterial genera (Prevotella, Cryptosporidium, etc.); MO: LEfSe analysis branch diagram and LDA score histogram.

[0018] Figure 3 This invention provides a multivariate statistical analysis of serum metabolomics in mice with chemotherapy-induced anemia after intervention with the blue cloth polysaccharide extract. AC represents the PCA score plot, OPLS-DA score plot for Control vs. CTX, and OPLS-DA score plot for CTX vs. ACE-H under positive ion mode; DF represents the PCA score plot, OPLS-DA score plot for Control vs. CTX, and OPLS-DA score plot for CTX vs. ACE-H under negative ion mode; G represents the volcano plot for Control vs. CTX; and H represents the volcano plot for CTX vs. ACE-H.

[0019] Figure 4This invention provides an analysis of differential metabolites and metabolic pathways regulated by the blue cloth polysaccharide extract; where AB: bubble charts of KEGG pathway enrichment for Control vs CTX and CTX vs ACE-H; C: cluster heatmap of differential metabolites; D: bar chart of relative abundance of key metabolites (lysophosphatidic acid, lysophosphatidylcholine, L-phenylalanine, 2-oxoarginine, etc.); EF: ROC curves (AUC>0.8) of potential biomarkers.

[0020] Figure 5 The correlation analysis of the gut microbiota regulated by the blue cloth polysaccharide extract of the present invention with serum metabolites is shown in Figure A: Spearman correlation heatmap between gut microbiota (genus level) and differential metabolites; and Figure B: correlation network diagram between key gut microbiota and differential metabolites (P<0.05, P<0.01). Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments and accompanying drawings. The following embodiments are for illustrative purposes only and are not intended to limit the invention.

[0022] Example 1: Preparation of blue cloth polysaccharide extract (acid-assisted extraction method)

[0023] Referring to the acid-assisted extraction (ACE) method in the applicant's previous research (Chen et al., Molecules 2025, 30, 148), the following specific procedures were performed:

[0024] (1) Dry the fresh whole plant of *Lysimachia nummularia* at 50 °C, pulverize it and pass it through a 60-mesh sieve to obtain *Lysimachia nummularia* powder.

[0025] (2) Take the blue cloth powder, add hydrochloric acid aqueous solution with pH 2.5 at a material-to-liquid ratio of 1:20 (g / mL), extract at 80 °C for 2 hours, filter, repeat the extraction twice, and combine the filtrates.

[0026] (3) Concentrate the combined filtrate under reduced pressure to 1 / 5 of the original volume, add anhydrous ethanol to a final concentration of 80% (v / v), let stand overnight, and centrifuge to collect the precipitate.

[0027] (4) The precipitate was deproteinized three times using the Sevag method (chloroform: n-butanol = 4:1), and then ultrafiltered through an ultrafiltration membrane with a molecular weight cutoff of 3500 Da until the conductivity was constant. After freeze-drying, a white flocculent blue cloth polysaccharide extract was obtained.

[0028] The total sugar content of the extract was determined to be consistent with that reported in the literature, and it can be used for subsequent pharmacodynamic experiments.

[0029] Example 2: Pharmacodynamic experiment of blue cloth polysaccharide extract on chemotherapy-induced anemia mice

[0030] 2.1 Animal Model and Grouping

[0031] SPF-grade female ICR mice, weighing 20±2 g, were purchased from Henan Scibes Biotechnology Co., Ltd. After 7 days of acclimatization, they were randomly divided into 8 groups (n=8): control group, model group (CTX), low-dose ACE group (ACE-L, 100 mg / kg), high-dose ACE group (ACE-H, 200 mg / kg), high-dose AAE group (AAE, 200 mg / kg), high-dose PLE group (PLE, 200 mg / kg), high-dose EAE group (EAE, 200 mg / kg), and high-dose DESE group (DESE, 200 mg / kg). All groups were administered the drugs by gavage for 13 consecutive days. The control and CTX groups were administered an equal volume of physiological saline by gavage. Except for the control group, the other groups were intraperitoneally injected with cyclophosphamide (100 mg / kg / day) on days 8, 9, and 10 to establish a chemotherapy-induced anemia model. Figure 1 A). All animal experiments were approved by the Animal Ethics Committee of Zunyi Medical University (Ethics No.: ZMU21-2510-003).

[0032] 2.2 Peripheral blood count

[0033] After a 12-hour fast following the last dose, blood was drawn from the orbital rim. White blood cells (WBC), red blood cells (RBC), hemoglobin (HGB), hematocrit (HCT), and platelets (PLT) were measured using a fully automated blood analyzer (XS-1000i, Sysmex, Japan). Results are as follows: Figure 1 As shown in Figure 1, compared with the Control group, the CTX group showed significantly lower levels of WBC, RBC, HGB, HCT, and PLT (P<0.01), indicating successful establishment of the anemia model. Compared with the CTX group, the ACE-H group showed significantly higher levels of the above indicators (P<0.05 or P<0.01); the ACE-L group showed a significant increase in PLT (P<0.05). In the AAE, PLE, EAE, and DESE groups, only some indicators (such as HGB and HCT) showed an increasing trend, but there were no statistically significant differences (P>0.05). These results indicate that the blue cloth polysaccharide extract prepared by acid-assisted extraction has the best hematopoietic recovery activity.

[0034] 2.3 Measurement of Immune Organ Index

[0035] After euthanizing the mice, the thymus and spleen were harvested, rinsed with pre-cooled physiological saline, blotted dry with filter paper, and weighed. The organ index (organ weight / body weight × 100%) was calculated. Results are as follows: Figure 1As shown in the GH results, the thymus index and spleen index in the CTX group were significantly lower than those in the Control group (P<0.01). Compared with the CTX group, the ACE-H group showed a significant increase in the thymus index and spleen index (P<0.05), while other extract groups showed an increasing trend but no statistically significant difference (P>0.05). This indicates that ACE-GJPs can effectively alleviate CTX-induced atrophy of immune organs.

[0036] Example 3: The regulatory effect of blue cloth polysaccharide extract on the intestinal microbiota of anemic mice

[0037] 3.1 Fecal sample collection and 16S rDNA sequencing

[0038] Fresh fecal samples were collected from mice in the Control, CTX, and ACE-H groups (n=8), and total genomic DNA was extracted using the QIAamp PowerFecal Pro DNA Kit. Full-length 16S rDNA sequencing was performed using the PacBio Sequel II platform. High-quality amplicon sequence variants (ASVs) were obtained through circular consensus (CCS) analysis, and bioinformatics analysis was performed using the QIIME2 platform (version 2023.5).

[0039] 3.2 Alpha and Beta Diversity Analysis

[0040] like Figure 2 As shown in AC, there were no significant differences in Chao1, Shannon, and Simpson indices among the three groups (P>0.05), indicating that CTX and ACE-GJPs treatments did not significantly alter the richness and evenness of the gut microbiota. Principal coordinate analysis (PCoA) and partial least squares discriminant analysis (PLS-DA) based on the Bray-Curtis distance matrix results showed that... Figure 2 The microbial community structures of the DF, Control, CTX and ACE-H groups were significantly separated, indicating that ACE-GJPs can reshape the overall structure of the gut microbiota.

[0041] 3.3 Changes in microbial community composition

[0042] At the gate level ( Figure 2 On the G and J scales, compared with the Control group, the CTX group showed increased relative abundance of Firmicutes and Proteobacteria, and decreased relative abundance of Bacteroidetes; the ACE-H group reversed these changes, decreasing the abundance of Firmicutes and Proteobacteria, and increasing the abundance of Bacteroidetes. At the family level ( Figure 2At the genus level (H), the relative abundance of Akkermansiaceae, UBA932, and Bacteroidaceae in the CTX group was significantly lower than that in the Control group (P<0.05); after intervention in the ACE-H group, the abundance of the above beneficial bacterial families all significantly increased (P<0.05). Figure 2 In the I and KL groups, compared with the Control group, the relative abundance of Prevotella was significantly increased in the CTX group (P<0.05), while the relative abundance of Cryptobacterium was significantly decreased (P<0.05); the ACE-H group could significantly reverse these two changes, decreasing the abundance of Prevotella and increasing the abundance of Cryptobacterium (P<0.05).

[0043] 3.4 LEfSe Analysis

[0044] The linear discriminant analysis (LEfSe) method was used, with an LDA score > 3.0 as the threshold, to identify biomarkers with statistically significant differences among the groups. Figure 2 From the phylum to the genus level, 11, 8, and 6 significantly enriched bacterial groups were identified in the Control, CTX, and ACE-H groups, respectively. The CTX group showed abnormal enrichment of Eubacterium, indicating severe dysbiosis; the ACE-H group featured Campylobacteria and other characteristic bacterial groups. These results further confirm that ACE-GJPs can reshape the CTX-induced pro-inflammatory gut microbiota structure, shifting it towards a more anti-inflammatory and homeostatic ecological pattern.

[0045] In summary, ACE-GJPs, as a prebiotic preparation, can alleviate the intestinal microecological imbalance caused by CTX by enriching beneficial bacteria such as Cryptobacterium, Akkermansia, and Bacteroides and inhibiting pro-inflammatory bacteria such as Prevotella and Eubacterium.

[0046] Example 4: Effects of Blue Cloth Polysaccharide Extract on Serum Metabolomics in Anemic Mice

[0047] 4.1 Serum Sample Pretreatment

[0048] Take 100 μL of serum from each of the Control, CTX, and ACE-H groups of mice, add 400 μL of methanol, vortex for 30 s, and centrifuge at 13,000 rpm for 10 min at 4 ℃. Collect 400 μL of the supernatant, dry it under nitrogen, and reconstitute the residue with 100 μL of anhydrous methanol. Filter the solution through a 0.22 μm filter membrane and perform UPLC-Q-TOF-MS / MS analysis. Prepare quality control samples (QC) by mixing equal volumes of serum from each group.

[0049] 4.2 Chromatographic-Mass Spectrometry Conditions

[0050] An ACQUITY UPLC BEH Amide column (1.7 μm, 2.1 × 100 mm) was used at a column temperature of 40 °C and a flow rate of 0.5 mL / min. Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution program was: 0–12 min, 2%–62% B; 12–12.1 min, 62%–2% B; 12.1–15 min, 2% B. The injection volume was 2 μL. Mass spectrometry was performed using an electrospray ionization (ESI) source in both positive and negative ion modes. The capillary voltage was 2.0 kV, the ion source temperature was 110 °C, the nebulizer gas temperature was 350 °C, the nebulizer gas flow rate was 800 L / h, and the mass-to-charge ratio acquisition range was 50–1500 m / z.

[0051] 4.3 Multivariate Statistical Analysis

[0052] The raw data were preprocessed using Progenesis QI software and then imported into MetaboAnalyst 6.0 for multivariate statistical analysis. (PCA score plot) Figure 3 (A) and (D) show that the QC samples are tightly aggregated, indicating good instrument stability. OPLS-DA analysis ( Figure 3 BC and EF showed that the metabolic profiles of the Control group, CTX group and ACE-H group were significantly separated, and the ACE-H group regressed to the Control group, indicating that ACE-GJPs can partially reverse the metabolic disorders caused by CTX.

[0053] 4.4 Screening and Identification of Differential Metabolites

[0054] Differential metabolites were screened based on the criteria of variable importance projection (VIP) > 1.0, P < 0.05, and fold change (FC) > 1.5 or < 0.67. Figure 3 GH). Compared with the Control group, the CTX group screened out 17 differential metabolites (Table 1); compared with the CTX group, the ACE-H group screened out 21 differential metabolites (Table 2). The structures of the above metabolites were identified by comparing the HMDB database and secondary mass spectrometry fragment information.

[0055] 4.5 Metabolic pathway enrichment analysis

[0056] Differential metabolites were imported into the KEGG database for pathway enrichment. Figure 4 AB) and heat map ( Figure 4C) Analysis. The results showed that the main metabolic pathways disrupted in the CTX group included glycerophospholipid metabolism, purine metabolism, thiamine metabolism, and niacin and nicotinamide metabolism. After ACE-GJPs intervention, glycerophospholipid metabolism and amino acid metabolism (phenylalanine metabolism and arginine metabolism) were mainly regulated.

[0057] 4.6 Analysis of changes in key metabolites

[0058] like Figure 4 As shown in D, compared with the CTX group, the ACE-H group significantly reversed the abnormal levels of the following key metabolites: in glycerophospholipid metabolism, the level of lysophosphatidic acid (16:0 / 0:0) was decreased (P<0.01), and the level of lysophosphatidylcholine (O-18:0) was increased (P<0.05); in amino acid metabolism, the level of L-phenylalanine was decreased (P<0.05), and the level of 2-oxoarginine was increased (P<0.05).

[0059] 4.7 Identification of Potential Biomarkers

[0060] ROC curve analysis Figure 4 The EF results showed that the area under the curve (AUC) of the above key metabolites was greater than 0.8, indicating high diagnostic value and suggesting that they could serve as potential biomarkers for the treatment of chemotherapy-induced anemia by blue cloth polysaccharide extract.

[0061] Example 5: Correlation analysis between gut microbiota and serum metabolites

[0062] 5.1 Analytical Methods

[0063] To explore the potential association between ACE-GJPs-regulated gut microbiota and serum metabolites, Spearman correlation analysis was performed on the top 10 gut microbiota with relative abundance at the genus level and the differentially expressed metabolites screened in Example 4. The correlation coefficient (r) and significance level were calculated using SPSS 29.0 software, with P < 0.05 considered statistically significant. The analysis results are presented as a correlation heatmap (…). Figure 5 A) and network diagram ( Figure 5 B) Display only associations that are statistically significant.

[0064] 5.2 Correlation between Cryptosporidium and glycerophospholipid Metabolites

[0065] Cryptobacterium showed significant correlations with multiple glycerophospholipid metabolites. Specifically, it was significantly positively correlated with lysophosphatidylcholine (O-18:0) (r=0.72, P<0.01); significantly negatively correlated with lysophosphatidic acid (16:0 / 0:0) (r=-0.65, P<0.05); and also significantly negatively correlated with glycerophosphatidylcholine (r=-0.58, P<0.05). These results suggest that ACE-GJPs may regulate glycerophospholipid metabolism by enriching Cryptobacterium, reducing pro-inflammatory lysophosphatidic acid levels, and increasing membrane-protective lysophosphatidylcholine levels, thereby alleviating bone marrow microenvironment inflammation and protecting cell membrane integrity.

[0066] 5.3 Correlation between Akkermansia spp. and metabolites

[0067] Akkermansia showed significant correlations with multiple metabolites: a significant positive correlation with nicotinamide adenine dinucleotide phosphate (NADP) (r=0.68, P<0.05); a significant positive correlation with phosphatidylethanolamine (PE (P-18:1 / 18:3)) (r=0.61, P<0.05); and a significant negative correlation with lysophosphatidylcholine (16:0 / 0:0) (r=-0.54, P<0.05). These results suggest that ACE-GJP enrichment in Akkermansia may help maintain energy homeostasis and mitigate lipid-mediated inflammatory responses.

[0068] 5.4 Correlation between Prevotella spp. and amino acid metabolites

[0069] Prevotella showed a significant negative correlation with amino acid metabolites: a significant negative correlation with 2-oxoarginine (r = -0.62, P < 0.01) and a significant negative correlation with N-acetylsorinoline (r = -0.55, P < 0.05). These results suggest that ACE-GJPs may increase 2-oxoarginine levels and improve the nutritional supply required for hematopoiesis by inhibiting the excessive growth of Prevotella and relieving the negative regulation of arginine metabolism.

[0070] 5.5 Correlation between other bacterial genera and metabolites

[0071] Bacteroides showed a positive correlation with lysophosphatidylcholine (O-18:0) (r=0.59, P<0.05) and a negative correlation with L-phenylalanine (r=-0.51, P<0.05). Eubacterium showed a positive correlation with multiple pro-inflammatory metabolites and a negative correlation with beneficial metabolites, further validating the CTX-induced dysbiosis and the corrective effect of ACE-GJPs.

[0072] 5.6 Summary

[0073] Based on the correlation analysis results above, ACE-GJPs may exert their anti-anemia effects through the following two key "microbe-metabolite" axes: first, the Cryptosporidium-glycerophospholipid metabolic axis, which involves enriching Cryptobacterium, regulating glycerophospholipid metabolism, reducing lysophosphatidic acid, and increasing lysophosphatidylcholine, thereby alleviating inflammation and protecting cell membrane integrity; second, the Prevotella-amino acid metabolic axis, which involves inhibiting Prevotella, relieving negative regulation of arginine metabolism, and increasing 2-oxoarginine, thereby improving nutrient supply and promoting hematopoiesis. These correlations provide strong evidence for the mechanism by which ACE-GJPs regulate host metabolism by remodeling the gut microbiota.

[0074] Example 6: Summary of the mechanism of action of blue cloth polysaccharide extract in alleviating chemotherapy-induced anemia

[0075] Based on the research results of Examples 2-5 above, the mechanism of action of the blue cloth polysaccharide extract (ACE-GJPs) proposed in this invention in alleviating chemotherapy-induced anemia can be summarized as follows.

[0076] After oral administration, ACE-GJPs enter the intestine and initially exert a prebiotic-like effect, reshaping the gut microbiota structure. Specifically, this manifests as an increase in the relative abundance of beneficial bacteria such as Cryptosporidium, Akkermansia, and Bacteroides, while decreasing the relative abundance of pro-inflammatory bacteria such as Prevotella and Eubacterium. Subsequently, changes in the gut microbiota regulate the host's serum metabolic profile, primarily involving two key metabolic pathways. One is glycerophospholipid metabolism: ACE-GJPs significantly reduce the level of pro-inflammatory lysophosphatidic acid (16:0 / 0:0) and increase the level of membrane-protective lysophosphatidylcholine (O-18:0), thereby alleviating inflammatory damage to the bone marrow microenvironment and maintaining the integrity of hematopoietic cell membranes. Secondly, amino acid metabolism: ACE-GJPs significantly reduce L-phenylalanine levels (associated with inflammation) and increase 2-oxoarginine levels (arginine metabolites that support hematopoietic stem cell function), thereby improving the nutritional supply required for hematopoiesis.

[0077] Correlation analysis further confirmed the existence of two key microbial-metabolite regulatory pathways: the Cryptosporidium-glycerophospholipid metabolic axis and the Prevotella-amino acid metabolic axis. Ultimately, the above synergistic effects alleviated cyclophosphamide-induced bone marrow suppression, promoted the recovery of peripheral blood erythrocytes, hemoglobin, and platelets, and improved immune organ atrophy. This invention is the first to systematically compare the anti-anemia activity of blue broom polysaccharides obtained by different extraction methods, confirming that the polysaccharide prepared by acid-assisted extraction (ACE) is the most effective; it also reveals for the first time a novel mechanism by which ACE-GJPs alleviate chemotherapy-induced anemia through the gut microbiota-glycerophospholipid / amino acid metabolism axis, providing a scientific basis for its development as a natural anti-anemia drug.

[0078] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0079] Table 1. Differential metabolic markers between the model group and the control group

[0080]

[0081] Table 2. Differential metabolic biomarkers between the high-dose acid extraction group and the model group

[0082]

Claims

1. A method for preparing a polysaccharide extract of *Cymbidium goeringii*, characterized in that, The process includes the following steps: taking the whole herb of *Lysimachia foenum-graecum*, drying, pulverizing and sieving, and extracting using an acid-assisted extraction method. The extract is then concentrated, precipitated with ethanol, deproteinized, ultrafiltered and freeze-dried to obtain *Lysimachia foenum-graecum* polysaccharide extract. The conditions for the acid-assisted extraction method are: using an acidic aqueous solution with pH 2-4 as the solvent, a material-to-liquid ratio of 1:10-1:30 (g / mL), and extracting at 60-90 ℃ for 1-3 hours.

2. The preparation method according to claim 1, characterized in that: In the acid-assisted extraction method, the extraction is performed 1-3 times.

3. The preparation method according to claim 1, characterized in that: The blue cloth polysaccharide extract contained 39.82±0.37% neutral polysaccharides and 55.62±2.9% uronic acid.

4. The preparation method according to claim 1, characterized in that: The deproteinization process employs the Sevag method, with a chloroform to n-butanol volume ratio of 4:1, and the deproteinization is performed 1-5 times. The ultrafiltration process uses an ultrafiltration membrane with a molecular weight cutoff of 3500 Da.

5. The use of the blue cloth polysaccharide extract obtained by the preparation method according to any one of claims 1 to 4 in the preparation of drugs for the prevention and / or treatment of chemotherapy-induced anemia.

6. The application according to claim 5, characterized in that: The chemotherapy-induced anemia was induced by cyclophosphamide.

7. The application according to claim 5, characterized in that: The blue cloth polysaccharide extract exerts its anti-anemia effect by increasing the relative abundance of Cryptobacterium and / or Akkermansia in the intestine, and / or decreasing the relative abundance of Prevotella.

8. The application according to claim 5, characterized in that: The blue cloth polysaccharide extract exerts its anti-anemia effect by reducing serum lysophosphatidic acid (16:0 / 0:0) levels and / or increasing lysophosphatidylcholine (O-18:0) levels.

9. The application according to claim 5, characterized in that: The blue cloth polysaccharide extract exerts its anti-anemia effect by reducing serum L-phenylalanine levels and / or increasing 2-oxoarginine levels.

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