Chlorella pyrenoidosa polysaccharide XQZ3, and preparation method and use thereof

Abstract

The application discloses a Chlorella pyrenoidosa polysaccharide XQZ3, a preparation method and application thereof. The weight average molecular weight of the polysaccharide XQZ3 is 10-500 kDa, the relative molecular mass is 29.13 kDa, and the polysaccharide XQZ3 contains 53.1 wt% galactose, 12.8 wt% mannose, 12.2 wt% rhamnose, 11.7 wt% glucuronic acid, 5.4 wt% glucosamine and 4.6 wt% arabinose. The Chlorella pyrenoidosa is used as raw material, and the Chlorella pyrenoidosa polysaccharide XQZ3 is obtained through the following steps: water extraction, concentration, dialysis, alcohol precipitation and protein removal. The Chlorella pyrenoidosa polysaccharide XQZ3 can inhibit the proliferation of tumor cells and the clonal ability of tumor cells in vitro and inhibit the growth of transplanted tumors of nude mice in vivo, and has the tumor inhibition effect. The Chlorella pyrenoidosa polysaccharide XQZ3 has obvious proliferation inhibition effect on various tumors, especially PDACs pancreatic tumors, and is expected to become a potential polysaccharide drug for treating tumors.

Description

Technical Field

[0001] This invention belongs to the field of polysaccharide extraction and application technology, specifically relating to a Chlorella polysaccharide XQZ3, its preparation method, and its uses. Specifically, it relates to a method for extracting Chlorella polysaccharide XQZ3 from Chlorella, the Chlorella polysaccharide XQZ3 extracted by this method, and the use of the Chlorella polysaccharide XQZ3 in the preparation of antitumor drugs. Background Technology

[0002] Given the anticipated increase in cancer diagnosis, finding effective anticancer drugs as quickly as possible is crucial. However, most anticancer chemotherapeutic drugs kill tumor cells while severely damaging normal cells. Therefore, the search for highly effective and low-toxicity antitumor drugs is of great significance. Evidence accumulated over recent decades has shown that polysaccharides possess significant anticancer activity with relatively few toxic side effects on the human body. Currently, researchers have discovered various polysaccharides from different organisms and elucidated information regarding their structure and functional activities. Among these, research on the antitumor effects of natural polysaccharides has become one of the hot topics in drug development and functional product development.

[0003] Chlorella proteoglycans, a type of green microalgae, has been designated as a "green and healthy food" by the Food and Agriculture Organization of the United Nations (FAO). Polysaccharides are one of the main active components of Chlorella, possessing various biological activities such as immunomodulation, antioxidation, lipid-lowering, asthma relief, antitumor activity, and neuroprotection. In recent years, the extraction, purification, structural characterization, and bioactivity studies of Chlorella polysaccharides have been extensively investigated, indicating that Chlorella polysaccharides are an important functional food ingredient or potential drug. However, research on the antitumor effects of Chlorella polysaccharides remains limited, and detailed studies on their in vivo antitumor activity are lacking; the underlying mechanisms remain unknown. Summary of the Invention

[0004] Based on this, the inventors isolated and purified a homogeneous polysaccharide XQZ3 from Chlorella proteoglycans and conducted research on its anticancer activity and mechanism of action. Polysaccharide XQZ3 exhibited broad-spectrum antitumor properties, particularly showing significant effects against pancreatic cancer cells. Through in vitro and in vivo evaluations, the antitumor activity of polysaccharide XQZ3 was assessed using patient-derived three-dimensional organoid models and xenograft models. This provides a theoretical and practical basis for polysaccharide XQZ3 as a natural saccharide-based anticancer agent.

[0005] One objective of this invention is to provide a novel acidic polysaccharide, XQZ3, extracted from Chlorella protozoa, which exhibits excellent antitumor activity, particularly against pancreatic cancer. Pharmacological experiments show that polysaccharide XQZ3 can significantly inhibit the proliferation of tumor cells (e.g., pancreatic cancer cells) and cancer cell cloning at the cellular level. In vitro and in vivo experiments also demonstrate that this polysaccharide exerts anticancer activity, indicating that Chlorella protozoa polysaccharide XQZ3 possesses good antitumor effects and holds promise as a potential polysaccharide drug for cancer treatment.

[0006] Specifically, the weight-average molecular weight of the Chlorella polysaccharide XQZ3 is 10-500 kDa, and the relative molecular mass is 29.13 kDa. It is mainly composed of galactose, containing 53.1 wt% galactose, 12.8 wt% mannose, 12.2 wt% rhamnose, 11.7 wt% glucuronic acid, 5.4 wt% glucosamine, and 4.6 wt% arabinose.

[0007] The infrared spectrum of the protein-nucleated Chlorella polysaccharide XQZ3 shows the following absorption peak: 3358 cm⁻¹ -1 The absorption peak near the OH stretching vibration is at 2935 cm⁻¹. -1 The nearby peak is the CH stretching vibration absorption peak, at 1731 cm⁻¹. -1 The absorption peak is located near the C=O stretching vibration at 1644 cm⁻¹. -1 The nearby peak is the NH variable-angle vibration absorption peak, at 1373 cm⁻¹. -1 The absorption peak is located near the C=O symmetric stretching vibration at 1238 cm⁻¹. -1 The absorption peak near the S=O stretching vibration (sulfate ion) is 1061 cm⁻¹. -1 The nearby peak is the OH angle vibration absorption peak, at 879 cm⁻¹. -1 The peak value corresponds to the glucose ring linked by a β-glycosidic bond.

[0008] The second objective of this invention is to provide a method for preparing the above-mentioned Chlorella polysaccharide XQZ3, specifically including the following steps:

[0009] Step a, Polysaccharide extraction: The dried Chlorella pulveratum powder was successively defatted with ethanol, extracted with water, and filtered. The filtrate was concentrated, dialyzed, concentrated again, precipitated with alcohol, centrifuged, vacuum dried, and then freeze-dried after protein removal to obtain water-extracted crude polysaccharide from Chlorella pulveratum.

[0010] Step b, Polysaccharide Purification: The water-extracted Chlorella vulgaris crude polysaccharide was fractionated and purified using a DEAE cellulose anion exchange column. The elution was performed sequentially with distilled water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions. Sulfuric acid-phenol detection was performed. The eluted fraction from the 0.3 mol / L NaCl solution was collected, concentrated, dialyzed, and freeze-dried to obtain Chlorella vulgaris polysaccharide XQZ3.

[0011] Preferably, the dialysis bag used in the dialysis has a molecular weight cutoff of 3500 Da.

[0012] Preferably, in step a, the method for polysaccharide extraction includes: soaking dried Chlorella pulveratum powder in 95% ethanol for one week, air-drying, adding 20 times its weight of deionized water, extracting at 100°C, filtering, and extracting the residue again with deionized water. This extraction is repeated three times, each time for 3 hours. The combined supernatant is concentrated and treated with 15% trichloroacetic acid at 4°C for 3 hours to remove proteins. The supernatant is then centrifuged, concentrated, dialyzed, concentrated again, and 3 times the volume of 95% ethanol is added. The mixture is centrifuged to obtain a precipitate, which is then freeze-dried to obtain water-extracted crude Chlorella pulveratum polysaccharide.

[0013] Preferably, in step b, the method for purifying the polysaccharide includes: taking crude polysaccharide from Chlorella proteoglycans, dissolving it in 10 times its weight of water, centrifuging, separating the supernatant by passing it through a DEAE cellulose anion exchange column, eluting it sequentially with distilled water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions, detecting it with sulfuric acid-phenol, collecting and combining the 0.3 mol / L NaCl solution eluent, concentrating it, dialyzing it, and freeze-drying it to obtain Chlorella proteoglycans polysaccharide XQZ3.

[0014] A third objective of this invention is to provide a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned Chlorella polysaccharide XQZ3 as an active ingredient. The composition may further include pharmaceutically acceptable excipients, such as carriers, excipients, adjuvants, and / or diluents, and may be prepared as an injection, emulsion, tablet, powder, granule, ointment, liposome, or oral liquid.

[0015] The fourth objective of this invention is to provide the use of the aforementioned Chlorella polysaccharide XQZ3 or pharmaceutical compositions containing it in the preparation of antitumor drugs. The tumor refers to a new growth formed by the proliferation of local tissue cells under the influence of various tumorigenic factors. Because such new growths often present as space-occupying, blocky protrusions, they are also called growths. This invention screened 11 common tumors, including esophageal squamous cell carcinoma (ESCC), bladder cancer (UBC), colorectal cancer (CRC), ovarian cancer (OC), prostate cancer (PCa), hepatocellular carcinoma (HCC), melanoma (MM), non-small cell lung cancer (NSCLC), breast cancer (BC), pancreatic ductal adenocarcinoma (PDAC), and pancreatic neuroendocrine tumor (PEEN), and found that Chlorella polysaccharide XQZ3 has a significant inhibitory effect on the proliferation of these tumor cells, especially on pancreatic ductal adenocarcinoma and pancreatic tumors.

[0016] In application, the protein-nucleated Chlorella polysaccharide XQZ3 can be administered alone or in combination with other pharmaceutically acceptable therapeutic agents, particularly with other drugs used for the prevention or treatment of tumors or cancer. These therapeutic agents include, but are not limited to: nitrogen mustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan, thiotepa, carmustine, semustine, busulfan, cisplatin, carboplatin, oxaloplatin, and mitomycin.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention proposes a simple and effective method for polysaccharide extraction and purification, using *Chlorella proteoglycans* as raw material to obtain a novel functional *Chlorella proteoglycans* polysaccharide XQZ3. Experimental verification shows that *Chlorella proteoglycans* polysaccharide XQZ3 can inhibit tumor cell proliferation and cloning ability in vitro, and inhibit the growth of xenograft tumors in nude mice in vivo, thereby exerting an anti-tumor effect. The *Chlorella proteoglycans* polysaccharide XQZ3 of this invention has a significant inhibitory effect on the proliferation of various tumors, especially pancreatic tumors with pDAS (proliferative dysplasia of the pancreas), and has potential application value in the development of novel anti-tumor glycoside drugs. Attached Figure Description

[0019] Figure 1 The IR, monosaccharide, and molecular weight spectra of the Chlorella polysaccharide XQZ3 prepared in Example 1 are shown.

[0020] Figure 2 This is a schematic diagram illustrating the effect of Chlorella polysaccharide XQZ3 on the growth inhibition of various cancer cells and normal human cells as a function of concentration, as shown in Example 2.

[0021] Figure 3This is a schematic diagram illustrating the inhibitory effect of Chlorella polysaccharide XQZ3 on the growth of organoid pancreatic cancer tumors in Example 2. A represents the cell diameter of PDAC-1 and PDAC-2 PDO after culturing for 0, 3, and 9 days after treatment with different concentrations of XQZ3, respectively; B represents the cell viability of PDAC-1 PDO after treatment with different concentrations of XQZ3; C represents the cell viability of PDAC-2 PDO after treatment with different concentrations of XQZ3; and D represents the dependence of cell viability of PDAC-1 and PDAC-2 PDO on different concentrations of XQZ3.

[0022] Figure 4 This is a schematic diagram illustrating the inhibitory effect of Chlorella polysaccharide XQZ3 on the formation of plate colonies in PDAC cell lines BxPC-3, Miapaca-2, Capan-1, SW1990, Hup-T4, and AsPC-1 in Example 2.

[0023] Figure 5 This is a schematic diagram illustrating the inhibitory effect of Chlorella polysaccharide XQZ3 on xenograft tumors in nude mice and the histopathological analysis of tumor tissue in Example 3. Detailed Implementation

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0025] Example 1: Preparation of Chlorella polysaccharide XQZ3

[0026] a. Polysaccharide extraction:

[0027] Dried Chlorella proteoglycans were defatted with 95% ethanol for one week (the ethanol was replaced every 3 days), and then allowed to dry naturally at room temperature. 1000g of the dried Chlorella proteoglycans were extracted three times with 20L of boiling water (deionized water), 3 hours each time. After a sulfuric acid-phenol test showed no significant reaction, the extracts were filtered. The combined extracts were heated and concentrated, dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da, and concentrated to 2L. Then, 3 times the volume (6L) of 95% ethanol was added with stirring, and the mixture was allowed to stand overnight. The supernatant was discarded, and the mixture was centrifuged. The resulting precipitate was washed with 3 times the volume of anhydrous ethanol, centrifuged again, and vacuum dried. The precipitate was then treated with 15% trichloroacetic acid at 4℃ for 3 hours to remove protein. Finally, it was freeze-dried at -40℃ under vacuum to obtain 118g of crude Chlorella proteoglycans.

[0028] b. Polysaccharide purification:

[0029] 10 g of the crude polysaccharide from *Chlorella proteoglycans* prepared above was dissolved in 100 mL of water. The insoluble matter was removed by centrifugation, and the supernatant was preliminarily purified by fractionation using a Cl-type DEAE-cellulose anion exchange column. The solution was eluted sequentially with distilled water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions, with sulfuric acid-phenol detection. The combined 0.3 mol / L NaCl eluent was collected, concentrated, dialyzed, and freeze-dried to obtain 31.4 g of *Chlorella proteoglycans* polysaccharide XQZ.

[0030] c. Identification and analysis of polysaccharide structure:

[0031] Molecular weight was determined using high-performance gel permeation chromatography (HPGPC). HPGPC employed tandem columns of Ultrahydrogel™ 2000 (25 cm × 0.75 cm, Waters Corporation, USA) and Ultrahydrogel™ 500 (25 cm × 0.75 cm, Waters Corporation, USA), and standard curves were constructed using T-series dextran standards of different molecular weights. HPGPC analysis showed that the weight-average molecular weight of polysaccharide XQZ3 ranged from 10 to 500 kDa, with a relative molecular weight of 29.13 kDa.

[0032] Monosaccharide composition analysis was performed using PMP-pre-column derivatization high-performance liquid chromatography (HPLC). The process involved complete hydrolysis of the polysaccharide, PMP derivatization, chloroform extraction, and the upper aqueous phase was then sent for HPLC analysis. An Agilent 1260Seri HPLC system (Agilent Technologies, USA), a TSKgel GMPWXL column (7.5 × 300 mm), a TSKgel guard column (6.0 × 40 mm, 12 μm, purchased from TOSOH, Japan), and a BDS HYPESIL C18 column (250 × 4.60 mm, 6 μm, purchased from Thermo) were used. Monosaccharide composition analysis showed that the polysaccharide XQZ3 from Chlorella contained 53.1 wt% galactose, 12.8 wt% mannose, 12.2 wt% rhamnose, 11.7 wt% glucuronic acid, 5.4 wt% glucosamine, and 4.6 wt% arabinose.

[0033] Infrared analysis was performed on the polysaccharide XQZ3 from Chlorella proteoglycans using a Perkin-Elmer 599B infrared spectrophotometer (Perkin-Elmer, USA). The infrared spectrum of Chlorella proteoglycan XQZ3 is shown below. Figure 1 As shown, 3358cm -1 The absorption peak near the OH stretching vibration is at 2935 cm⁻¹. -1 The nearby peak is the CH stretching vibration absorption peak, at 1731 cm⁻¹. -1The absorption peak is located near the C=O stretching vibration at 1644 cm⁻¹. -1 The nearby peak is the NH variable-angle vibration absorption peak, at 1373 cm⁻¹. -1 The absorption peak is located near the C=O symmetric stretching vibration at 1238 cm⁻¹. -1 The absorption peak near the S=O stretching vibration (sulfate ion) is 1061 cm⁻¹. -1 The nearby peak is the absorption peak of the OH angle vibration, and the peak at 879 cm⁻¹ corresponds to the glucose ring linked by the β-glycosidic bond.

[0034] Example 2: Antitumor activity of Chlorella polysaccharide XQZ3

[0035] ①CCK8 experiment

[0036] The following cell lines were used: human in situ pancreatic cancer cells BxPC-3, human pancreatic cancer cells Miapaca-2, HPAC, Capan-1, Capan-2, PSN1, SW1990, PANC-1, human pancreatic cancer resuscitation cells Hup-T4, human metastatic pancreatic adenocarcinoma cells AsPC-1, human prostate cancer cells LNCaP, human gastric cancer cells SNU-5, human liver cancer cells HepG2, human malignant melanoma cells A375, human non-small cell lung cancer cells A549, human esophageal squamous cell carcinoma cells KYSE-150, human colon cancer cells HT-29, human ovarian cancer cell line Caov-3, human breast cancer cells MCF-7, and PANC-1 (American Type Culture). Collection (Rockville, USA) and QGP-1, as well as human normal immortalized epidermal cells Hacat, human normal umbilical vein endothelial cells HUVEC (Cobioer, Nanjing, China), human normal hepatocyte line LO2, human normal brain microvascular endothelial cells HUVEC, and human normal intestinal epithelial cells HIEC (Minnesota Biotechnology, Ningbo, China), these cells were added to DMEM or 1640 medium (Solaibao, Beijing, China) containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 U / mL streptomycin (Beyotime Biotechnology, Shanghai, China) and cultured in an incubator containing 5% CO2 at 37°C.

[0037] Logarithmically growing cells were used for cell seeding. After trypsin digestion and preparation of a single-cell suspension with culture medium, cells were counted and seeded at 2000-5000 cells / well in a 96-well plate. A blank control well was also included, containing only complete culture medium. Cells were cultured overnight at 37°C with 5% CO2 and saturated humidity. After cell attachment, different concentrations of Chlorella proteoglycan XQZ3 were used for treatment, with a final volume of 100 μL per well and three replicates per group. After 72 hours of treatment, 20 μL of CCK8 solution (5 mg / mL, purchased from Sigma-Aldrich) was added to each well, and the plates were cultured for another 4 hours. The culture medium was carefully aspirated from the plate, and 150 μL of LDMSO solution was added to each well. The plate was then shaken on a microplate shaker for 10 minutes to fully dissolve any crystals. The optical density (OD) value of each well was measured at 570 nm using a microplate reader. The OD value of the blank control well was subtracted from the OD value of each well, and the results were recorded.

[0038] The results are as follows Figure 2 As shown, to verify the anti-cancer potential of XQZ3, CCK8 assays were performed on various tumor cells treated with XQZ3. The following types of cancer were observed: esophageal squamous cell carcinoma (ESCC), bladder cancer (UBC), colorectal cancer (CRC), ovarian cancer (OC), prostate cancer (PCa), hepatocellular carcinoma (HCC), melanoma (MM), non-small cell lung cancer (NSCLC), breast cancer (BC), pancreatic neuroendocrine tumor (pNEN), and pancreatic ductal adenocarcinoma (PDAC). Figure 2 (AB, DE). 2F is the half-maximal inhibitory concentration (IC50) value obtained after XQZ3 administration, indicating that XQZ3 inhibits the growth of different tumor types in a dose-dependent manner. These results show that XQZ3 exhibits the highest sensitivity against PDAC cells (BxPC-3), with an IC50 value of 0.05026 mg / mL.

[0039] Then, another nine PDAC cell lines were selected for efficacy testing. The results showed that XQZ3 had significant inhibitory effects on all ten PDAC cell lines, with IC50 values ​​ranging from 0.05026 to 0.6678 mg / ml. Figure 2 DF). The potential cytotoxic effects of XQZ3 on non-cancerous cells were also investigated. Results showed that XQZ3 did not exhibit any noteworthy cytotoxicity to various normal cell types, such as human hepatocytes, umbilical vein endothelial cells, brain microvascular endothelial cells, intestinal epithelial cells, and keratinocytes. Figure 2 C).

[0040] ②PDO evaluation of XQZ3 anti-proliferation experiment

[0041] It is well known that solid tumors respond less to chemotherapy drugs compared to cells cultured in monolayers. To more accurately replicate the microenvironment and predict treatment outcomes, researchers often turn to diseased human organs (PDOs). In this example, PDOs from pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNEN) were used to evaluate the antiproliferative effect of XQZ3. The diameter and cell viability of the PDOs were measured, such as… Figure 3 As shown, compared with the group treated with PBS, the diameter of the two PDAC PDOs treated with XQZ3 and cultured for three days was significantly reduced, indicating that the cell proliferation rate of PDAC was significantly decreased. Figure 3 AC). After 9 days of XQZ3 treatment, a sustained reduction in PDO size was observed in the PDAC compared to the PBS group. Specifically, the PDOs in the PBS-treated group showed solid spheres with well-defined borders, while those in the XQZ3-treated group showed diffuse spheres with blurred edges. Figure 3 A). In the XQZ3 treatment group, after 9 days of treatment, the size of PDOs was slightly increased compared to the 3-day treatment group, which was attributed to the scattering of PDOs within compact spheres. Figure 3 A). Following a 9-day treatment regimen, PDO models were obtained from each group, and the viability of 3D-cultured PDO was assessed using the CellTiter-Glo 3D cell viability assay. Figure 3 C). These findings indicate that XQZ3 exhibits a concentration-dependent inhibitory effect on the cell viability of both PDAC and PDO cells.

[0042] ③ Plate colony formation experiment

[0043] Cells in the logarithmic growth phase were trypsinized and pipetted into single cells. Single-cell suspensions were prepared with culture medium and counted. BxPC-3 cells (100 cells / well), SW1990 cells (100 cells / well), Miapaca cells (100 cells / well), Hup-T4 cells (100 cells / well), AcPc-1 cells (100 cells / well), and Capan-1 cells (100 cells / well) were seeded in 6-well plates. The cells were gently rotated to ensure even dispersion. Chlorella polysaccharide XQZ3 was added to achieve final concentrations of 0 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL. Cells were incubated at 37°C, 5% CO2, and saturated humidity for 1–2 weeks. Frequent observation was performed; culture was terminated when visible clones appeared in the culture dish. The supernatant was discarded, and the cells were carefully washed twice with phosphate-buffered saline (PBS). Fixed cells with 4% paraformaldehyde for 15 minutes, removed the fixative, stained with 0.1% crystal violet dye (Beyotime Biotechnology, Shanghai, China) for 10–30 minutes, slowly washed away the dye with running water, and air-dried. The number of clones was counted directly with the naked eye and statistically analyzed.

[0044] Results of plate colony formation experiment as follows Figure 5 As shown, administration of Chlorella proteoglycan XQZ3 significantly reduced the proliferation of BxPC-3, Miapaca2, HuP-T4, Capan-1, AsPC-1, and SW1990 cells (5H). Plate clone experiments showed that Chlorella proteoglycan XQZ3 exhibited significant efficacy in inhibiting the growth of various types of tumor cells, while showing negligible cytotoxicity to healthy cells, with the most significant inhibitory effect on PDACs.

[0045] Example 3: Chlorella polysaccharide XQZ3 inhibits tumor xenograft activity in nude mice

[0046] Experiments with nude mouse xenografts derived from pancreatic cancer patients

[0047] Female BALB / c nu / nu mice (6-8 weeks old, weighing 18-20g, purchased from Gempharmatech Biotechnology, Nanjing, China) were used in the experiment at the Laboratory Animal Center of Shanghai Changzheng Hospital. They were housed under constant temperature (24-26℃) and Specific Pathogen Free (SPF) conditions. Cages, bedding, water, and feed were all sterilized by autoclaving and ultraviolet light. Aseptic procedures were performed, and all mouse programs were conducted in accordance with the guidelines for laboratory animal care and use issued by the National Institutes of Health (NIH). This experiment was approved by the Animal Ethics Committee of Shanghai Changzheng Hospital.

[0048] Pancreatic cancer tissue, including pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNEN), was obtained from patients who underwent surgical resection at Shanghai Changzheng Hospital. This study was approved by the Research Ethics Committee of Shanghai Changzheng Hospital. Written informed consent was obtained before obtaining tissue from all patients. The samples were confirmed as pancreatic tumors by pathologists.

[0049] Tumor tissue was chopped into pieces approximately 4 mm in a high-concentration Matrigel™ basement membrane matrix (BD Biosciences, Franklin Lakes, USA). 3 The fragments were directly implanted into the subcutaneous space of female BALB / c nude mice aged 6 to 8 weeks.

[0050] Subsequently, the effect of XQZ3 on tumor progression was evaluated using the PDAC PDX model. PDAC PDX models were randomly assigned to three groups: PBS, 10 mg / kg XQZ3 treatment, and 20 mg / kg XQZ3 treatment, with 10 models in each group. PBS or XQZ3 was administered intraperitoneally every 4 days, and body weight and tumor growth were measured at equal time intervals until the endpoint was reached. After 36 days of treatment, 4 mice from each group were sacrificed, and their subcutaneous tumors were harvested and weighed. The internal organs of each mouse were harvested for staining, and blood was collected for biochemical tests. Tumor growth and survival were observed in the remaining 6 mice in each group. Animal body weight and tumor size were measured every 4 days during the experiment until the tumor exceeded 2000 mm. 3 Mice may die naturally. Clinical symptoms should be observed and recorded daily. At the end of administration, the tumor size should be photographed and recorded. Tumor tissue should be taken, weighed, fixed in 4% paraformaldehyde, or protein should be taken or frozen in liquid nitrogen. The formula for calculating tumor volume (TV) is: TV = (L × W2) / 2.

[0051] The results showed that administration of XQZ3 significantly inhibited the in vivo growth of pancreatic cancer. Figure 5 (A, D). Tumor growth curve results showed that, compared to the initial tumor volume, the tumor volume of mice receiving a dose of 10 mg / kg XQZ3 increased by an average of 5.24 times, that of mice receiving a dose of 20 mg / kg XQZ3 increased by an average of 3.21 times, while that of mice receiving PBS increased by an average of 11.35 times. Figure 5 A). After 42 days of treatment, a 52.92% reduction in tumor growth was observed in the group treated with 10 mg / kg XQZ3, while a 70.27% reduction in tumor growth was observed in the group treated with 20 mg / kg XQZ3. Figure 5 A, D). The results showed that XQZ3 exhibited a concentration-dependent inhibitory effect on tumor weight, with a maximum inhibition rate of 79.96% observed in the group treated with 20 mg / kg XQZ3. Figure 5 CD).

[0052] Furthermore, XQZ3 treatment significantly prolonged the lifespan of xenograft models derived from patients with pancreatic ductal adenocarcinoma. Figure 5 E). After 70 days of monitoring, the survival rate was observed to be 0% in the PBS group, 33.33% in the 10 mg / kg XQZ3 group, and 66.67% in the 20 mg / kg XQZ3 group. Figure 5 E).

[0053] In addition to evaluating the efficacy of XQZ3, its in vivo biosafety was also assessed. Results showed that XQZ3 administration did not cause a statistically significant change in mouse body weight compared to mice treated with PBS. Figure 5 B). Furthermore, biochemical analysis of mouse blood showed that administration of XQZ3 did not cause any significant changes in the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in the blood. Figure 5 F). Histological morphology of mouse organs treated with XQZ3 or PBS was evaluated, and XQZ3 did not cause any significant changes. Figure 5 G). The results showed that XQZ3 had a strong inhibitory effect on HSP90, exhibiting noteworthy anti-pancreatic cancer properties both in vitro and in vivo. The results also indicated that XQZ3 demonstrated good safety, making it a promising candidate drug for the treatment of pancreatic cancer.

[0054] The above description represents a preferred embodiment of the present invention, but the present invention should not be limited to the content disclosed in this embodiment. Therefore, any equivalent or modified versions made without departing from the spirit of the present invention fall within the scope of protection of the present invention.

Claims

1. The application of Chlorella polysaccharide XQZ3 in the preparation of antitumor drugs, characterized in that, The tumors mentioned are esophageal squamous cell carcinoma, bladder cancer, colorectal cancer, ovarian cancer, prostate cancer, melanoma, breast cancer, and pancreatic cancer; The protein nucleus Chlorella polysaccharide XQZ3 has a weight-average molecular weight of 10-500 kDa and a relative molecular mass of 29.

13. It contains 53.1 wt% galactose, 12.8 wt% mannose, 12.2 wt% rhamnose, 11.7 wt% glucuronic acid, 5.4 wt% glucosamine, and 4.6 wt% arabinose.

2. The application according to claim 1, characterized in that, The tumor is pancreatic cancer, including pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumor.

3. The application according to claim 1, characterized in that, The preparation method of the protein nucleus Chlorella polysaccharide XQZ3 includes the following steps: Step a, Polysaccharide extraction: The dried Chlorella pulveratum powder was successively defatted with ethanol, extracted with water, and filtered. The filtrate was concentrated, dialyzed, concentrated again, precipitated with alcohol, centrifuged, vacuum dried, and then freeze-dried after protein removal to obtain water-extracted crude polysaccharide from Chlorella pulveratum. Step b, Polysaccharide Purification: The water-extracted Chlorella vulgaris crude polysaccharide was fractionated and purified using a DEAE cellulose anion exchange column. The elution was performed sequentially with distilled water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions. Sulfuric acid-phenol detection was performed. The eluted fraction from the 0.3 mol / L NaCl solution was collected, concentrated, dialyzed, and freeze-dried to obtain Chlorella vulgaris polysaccharide XQZ3.

4. The application according to claim 3, characterized in that, The dialysis bag used in the dialysis has a molecular weight cutoff of 3500 Da.

5. The application according to claim 3, characterized in that, In step a, the method for polysaccharide extraction includes: soaking dried Chlorella pulveratum powder in 95% ethanol for one week, air-drying, adding 20 times its weight of deionized water, extracting at 100°C, filtering, and extracting the residue again with deionized water. This extraction is repeated three times, each time for 3 hours. The combined supernatant is concentrated and treated with 15% trichloroacetic acid at 4°C for 3 hours to remove proteins. The supernatant is then centrifuged, concentrated, dialyzed, concentrated again, and 3 times the volume of 95% ethanol is added. The mixture is centrifuged to obtain a precipitate, which is then freeze-dried to obtain water-extracted crude Chlorella pulveratum polysaccharide.

6. The application according to claim 3, characterized in that, In step b, the method for purifying the polysaccharide includes: taking crude polysaccharide from Chlorella proteoglycans, dissolving it in 10 times its weight of water, centrifuging, separating the supernatant by passing it through a DEAE cellulose anion exchange column, eluting it sequentially with distilled water, 0.1 mol / L, 0.2 mol / L, and 0.3 mol / L NaCl solutions, detecting it with sulfuric acid-phenol, collecting and combining the 0.3 mol / L NaCl solution eluent, concentrating it, dialyzing it, and freeze-drying it to obtain Chlorella proteoglycans polysaccharide XQZ3.

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