CN117820508A - Chlorella pyrenoidosa polysaccharide XQZ3 and preparation method and application thereof - Google Patents
Chlorella pyrenoidosa polysaccharide XQZ3 and preparation method and application thereof Download PDFInfo
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- CN117820508A CN117820508A CN202311495951.6A CN202311495951A CN117820508A CN 117820508 A CN117820508 A CN 117820508A CN 202311495951 A CN202311495951 A CN 202311495951A CN 117820508 A CN117820508 A CN 117820508A
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- China
- Prior art keywords
- polysaccharide
- chlorella pyrenoidosa
- xqz3
- carcinoma
- tumor
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- Medicines Containing Plant Substances (AREA)
Abstract
The invention discloses a chlorella pyrenoidosa polysaccharide XQZ3, a preparation method and application thereof, wherein the weight average molecular weight of the polysaccharide XQZ3 is 10-500 kDa, the relative molecular weight is 29.13kDa, the polysaccharide contains 53.1wt% galactose, 12.8wt% mannose, 12.2wt% rhamnose, 11.7wt% glucuronic acid, 5.4wt% glucosamine and 4.6wt% arabinose, the chlorella pyrenoidosa is taken as raw materials, crude polysaccharide is obtained through water extraction, concentration, dialysis, alcohol precipitation and protein removal, and the obtained chlorella pyrenoidosa polysaccharide XQZ3 can inhibit tumor cell proliferation and tumor cell cloning capacity in vitro and inhibit nude mouse transplanted tumor growth in vivo, 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
The invention belongs to the technical field of polysaccharide extraction and application, and particularly relates to chlorella pyrenoidosa polysaccharide XQZ3, and a preparation method and application thereof, wherein the method comprises the following steps: a method for extracting chlorella pyrenoidosa polysaccharide XQZ3 from chlorella pyrenoidosa, chlorella pyrenoidosa polysaccharide XQZ3 extracted by the method and application of the chlorella pyrenoidosa polysaccharide XQZ3 in preparing antitumor drugs.
Background
In view of the expected growth in cancer diagnosis, it is important to find effective anticancer drugs as soon as possible. However, most anticancer chemicals kill tumor cells, severely damaging normal cells. Therefore, the search for high-efficiency low-toxicity antitumor drugs has important significance. Evidence accumulated in recent decades shows that polysaccharides have remarkable anticancer activity and have relatively small toxic and side effects on human bodies. Currently, researchers have found a variety of polysaccharides from different species of organisms, and have elucidated information about their structural and functional activities, wherein the anti-tumor effect research of natural polysaccharides has become one of the hot spots for drug development and functional product development.
Chlorella pyrenoidosa, a green microalgae, has been designated as a "green health food" by the United nations Food and Agricultural Organization (FAO). The polysaccharide is one of main active ingredients of chlorella, and has various biological activities such as immunoregulation, antioxidation, blood lipid reduction, asthma relieving, anti-tumor, neuroprotection and the like. In recent years, extraction, purification, structural characterization and biological activity research of chlorella polysaccharide are widely studied, which shows that the chlorella polysaccharide is an important functional food raw material or potential drug. However, so far, there are few related studies on the antitumor effect of Chlorella polysaccharides, and there is a lack of detailed studies on antitumor activity in vivo, and the potential mechanism thereof is still unknown.
Disclosure of Invention
Based on the above, the inventor separates and purifies a uniform polysaccharide XQZ3 from chlorella pyrenoidosa, and researches on anticancer activity and action mechanism, wherein the polysaccharide XQZ3 shows wider anti-tumor property and particularly has remarkable effect on pancreatic cancer tumor cells. By in vitro and in vivo evaluation, the antitumor activity of polysaccharide XQZ3 was evaluated using a three-dimensional organoid model derived from a patient and a xenograft model, and polysaccharide XQZ provides a theoretical and practical basis as a natural saccharide anticancer agent.
The invention aims to provide chlorella pyrenoidosa polysaccharide XQZ3 which is an acidic polysaccharide extracted from chlorella pyrenoidosa and has a novel structure and good anti-tumor activity, in particular pancreatic cancer activity. Pharmacological experiments show that the polysaccharide XQZ3 can obviously inhibit proliferation and cancer cell cloning of tumor cells (pancreatic cancer tumor cells are examples), and in-vitro and in-vivo experimental results also show that the polysaccharide can play an anticancer role, and the chlorella pyrenoidosa polysaccharide XQZ3 has a good anti-tumor effect and is expected to become a potential polysaccharide medicament for treating tumors.
Specifically, the weight average molecular weight of the chlorella pyrenoidosa polysaccharide XQZ is 10-500 kDa, the relative molecular weight is 29.13kDa, and the chlorella pyrenoidosa polysaccharide XQZ mainly consists of galactose, and contains 53.1wt% galactose, 12.8wt% mannose, 12.2wt% rhamnose, 11.7wt% glucuronic acid, 5.4wt% glucosamine and 4.6wt% arabinose.
The infrared spectrum of the chlorella pyrenoidosa polysaccharide XQZ3 has the following absorption peaks: 3358cm -1 Near the absorption peak of O-H stretching vibration, 2935cm -1 C-H telescopic vibration absorption peak is 1731cm nearby -1 Near C=O stretching vibration absorption peak, 1644cm -1 Near the N-H angle-changing vibration absorption peak, 1373cm -1 The vicinity is C=O symmetrical telescopic vibration absorption peak, 1238cm -1 Near the S=O stretching vibration absorption peak (sulfate ion), 1061cm -1 The vicinity is O-H angle-changing vibration absorption peak, 879cm -1 Corresponds to the peak of the beta-glycosidic linkage to the glucose ring.
The second purpose of the invention is to provide a preparation method of the chlorella pyrenoidosa polysaccharide XQZ3, which comprises the following steps:
step a, polysaccharide extraction: sequentially degreasing the dried chlorella pyrenoidosa powder with ethanol, extracting with water, filtering, concentrating the obtained filtrate, dialyzing, concentrating again, precipitating with ethanol, centrifuging, vacuum drying, deproteinizing, and freeze-drying to obtain crude polysaccharide of the chlorella pyrenoidosa;
step b, polysaccharide purification: the crude polysaccharide of the water extracted chlorella pyrenoidosa is subjected to fractional purification by using a DEAE cellulose anion column, sequentially eluted by distilled water, 0.1mol/L, 0.2mol/L and 0.3mol/L NaCl solution, detected by sulfuric acid-phenol, and eluted components of the 0.3mol/L NaCl solution are collected, concentrated, dialyzed and freeze-dried to obtain the chlorella pyrenoidosa polysaccharide XQZ.
Preferably, the dialysis employs a dialysis bag having a molecular weight cut-off of 3500Da.
Preferably, in the step a, the method for extracting polysaccharide comprises: soaking dried Chlorella pyrenoidosa powder in 95% ethanol for 1 week, air drying, adding 20 times of deionized water, extracting at 100deg.C, filtering, extracting the residue with deionized water again, repeatedly extracting for 3 times each for 3 hr, concentrating the combined supernatant, treating with 15% trichloroacetic acid at 4deg.C for 3 hr to remove protein, centrifuging, concentrating, dialyzing, concentrating again, adding 3 times of 95% ethanol, centrifuging to obtain precipitate, and lyophilizing to obtain crude Chlorella pyrenoidosa polysaccharide.
Preferably, in step b, the method for purifying polysaccharide comprises: dissolving crude chlorella pyrenoidosa polysaccharide in 10 times of water, centrifuging, separating supernatant with DEAE cellulose anion column, eluting with distilled water, 0.1mol/L, 0.2mol/L and 0.3mol/L NaCl solution sequentially, detecting with sulfuric acid-phenol, collecting eluate of the combined 0.3mol/L NaCl solution, concentrating, dialyzing, and lyophilizing to obtain chlorella pyrenoidosa polysaccharide XQZ.
It is still another object of the present invention to provide a pharmaceutical composition comprising a therapeutically effective amount of the above-mentioned Chlorella pyrenoidosa polysaccharide XQZ3 as an active ingredient, which may further comprise pharmaceutically acceptable pharmaceutical excipients, such as carriers, excipients, adjuvants and/or diluents, and may be formulated as injections, emulsions, tablets, powders, granules, ointments, liposomes or oral liquids.
The fourth purpose of the invention is to provide the application of the chlorella pyrenoidosa polysaccharide XQZ3 or a pharmaceutical composition containing the same in preparing antitumor drugs. The tumor refers to a new organism formed by local tissue cell proliferation under the action of various tumorigenic factors, because the new organism is in the form of occupying massive protrusions, which are also called neoplasms. The invention screens 11 common tumors including Esophageal Squamous Cell Carcinoma (ESCC), bladder carcinoma (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 discovers that the chlorella pyrenoidosa polysaccharide XQZ has obvious proliferation inhibition effect on the tumor cells, especially for pancreatic ductal adenocarcinoma pancreatic tumor.
For use, the Chlorella pyrenoidosa polysaccharide XQZ3 may be administered alone or in combination with other pharmaceutically acceptable therapeutic agents, particularly with other drugs for the prevention or treatment of tumors or cancers. Such therapeutic agents include, but are not limited to: nitrogen mustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan, thiotepa, carmustine, semustine, busulfan, cisplatin, carboplatin, platinum oxalate, mitomycin.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides a simple and effective polysaccharide extraction and purification method, which takes chlorella pyrenoidosa as a raw material to obtain novel functional chlorella pyrenoidosa polysaccharide XQZ. Experiments prove that the chlorella pyrenoidosa polysaccharide XQZ3 can inhibit tumor cell proliferation in vitro, inhibit tumor cell cloning capacity and inhibit nude mice transplanted tumor growth in vivo, thereby playing a role in inhibiting tumor. The chlorella pyrenoidosa polysaccharide XQZ3 has obvious proliferation inhibition effect on various tumors, especially PDACs pancreatic tumors, and has potential application value in developing novel antitumor saccharide medicaments.
Drawings
FIG. 1 is an IR, monosaccharide and molecular weight spectrum of Chlorella pyrenoidosa polysaccharide XQZ3 prepared in example 1.
FIG. 2 is a graph showing the concentration-dependent growth inhibition of Chlorella pyrenoidosa polysaccharide XQZ3 of example 2 on various cancer cells and normal human cells.
FIG. 3 is a schematic representation of the growth inhibition effect of Chlorella pyrenoidosa polysaccharide XQZ3 of example 2 on organoid pancreatic cancer tumors, wherein A is the cell diameter of PDAC-1 and PDAC-2 after culturing for 0, 3 and 9 days after treatment with XQZ3 at different concentrations, respectively; cell viability of PDAC-1 PDO cultured after treatment at different concentrations of XQZ 3; cell viability of PDO of PDAC-2 cultured after treatment with XQZ3 at different concentrations; d is the cell viability of PDO of PDAC-1 and PDAC-2 as a function of XQZ3 at different concentrations.
FIG. 4 is a schematic representation of the inhibition of the formation of P.pyrenoidosa polysaccharide XQZ3 on the cloning of PDAC cell lines human pancreatic cancer cells BxPC-3, miapaca-2, capan-1, SW1990, hup-T4 and AsPC-1 plates in example 2.
FIG. 5 is a schematic diagram showing the inhibition effect of Chlorella pyrenoidosa polysaccharide XQZ3 on nude mice transplantation tumor and histopathological analysis of tumor tissue in example 3.
Detailed Description
The invention is further described below with reference to the drawings and specific examples.
Example 1: preparation of Chlorella pyrenoidosa polysaccharide XQZ3
a. Polysaccharide extraction:
the dried Chlorella pyrenoidosa was defatted with 95% ethanol for one week (ethanol was replaced every 3 days), and then naturally dried at room temperature. 1000g of dried Chlorella pyrenoidosa was extracted 3 times with 20L of boiling water (deionized water) for 3 hours each. The solution is filtered after being combined, heated and concentrated, dialyzed by a dialysis bag with molecular weight cut-off of 3500Da, concentrated to 2L, added with 3 times volume of 6L of 95% ethanol under stirring, and stood overnight. Pouring out the supernatant, centrifuging, washing the obtained precipitate with 3 times of absolute ethyl alcohol, centrifuging, vacuum drying the precipitate, treating with 15% trichloroacetic acid at 4deg.C for 3 hr to remove protein, and vacuum freeze drying at-40deg.C to obtain crude Chlorella pyrenoidosa polysaccharide 118g.
b. Polysaccharide purification:
taking 10g of the prepared chlorella pyrenoidosa crude polysaccharide, dissolving with 100mL of water, centrifuging to remove insoluble substances, and carrying out preliminary fractionation and purification on the supernatant by using a Cl-DEAE-cellulose anion column. Sequentially eluting with distilled water, 0.1mol/L, 0.2mol/L and 0.3mol/L NaCl solution, detecting with sulfuric acid-phenol, collecting and mixing 0.3mol/L NaCl eluates, concentrating, dialyzing, and freeze-drying to obtain Chlorella pyrenoidosa polysaccharide XQZ31.4g.
c. Polysaccharide structure identification and analysis:
molecular weight measurements were performed using High Performance Gel Permeation Chromatography (HPGPC) using columns in series of UltraHydrogel lTM 2000 (25 cm. Times.0.75 cm, waters, USA) and UltraHydrogel lTM 500 (25 cm. Times.0.75 cm, waters, USA) to make standard curves with T-series standard glucans (Dextran) of different molecular weights. Analysis by high performance gel permeation chromatography shows that the weight average molecular weight of polysaccharide XQZ is 10-500 kDa and the relative molecular weight is 29.13kDa.
Adopting PMP-pre-column derivatization high performance liquid chromatography to analyze monosaccharide composition, namely: the polysaccharide was completely hydrolyzed, PMP derivatized, chloroform extracted, and the upper aqueous phase was fed to HPLC analysis, which was performed using an Agilent 1260Seri high performance liquid system (Agilent Co., USA), a TSKgel GMPWXL column (7.5X100 mm), a TSKgel guard column (6.0X10 mm,12 μm, available from TOSOH Co., japan), a BDS HYPESIL C18 column (250X 4.60mm,6 μm, available from Thermo). The monosaccharide composition analysis result shows that the Chlorella pyrenoidosa polysaccharide XQZ3 contains 53.1wt% galactose, 12.8wt% mannose, 12.2wt% rhamnose, 11.7wt% glucuronic acid, 5.4wt% glucosamine and 4.6wt% arabinose.
The Chlorella pyrenoidosa polysaccharide XQZ3 was subjected to infrared analysis using a Perkin-Elmer 599B type infrared spectrophotometer (Perkin-Elmer Co., U.S.A.). The infrared spectrum of Chlorella pyrenoidosa polysaccharide XQZ3 is shown in figure 1, 3358cm -1 Near the absorption peak of O-H stretching vibration, 2935cm -1 C-H telescopic vibration absorption peak is 1731cm nearby -1 Near C=O stretching vibration absorption peak, 1644cm -1 Near the N-H angle-changing vibration absorption peak, 1373cm -1 The vicinity is C=O symmetrical telescopic vibration absorption peak, 1238cm -1 Near s=o telescopic vibrationKinetic absorption peak (sulfate ion), 1061cm -1 Near the O-H angular vibration absorption peak, the peak of 879cm-1 corresponds to the β -glycosidic linkage glucose ring.
Example 2: chlorella pyrenoidosa polysaccharide XQZ3 inhibits tumor activity
(1) CCK8 experiment
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 resuscitating 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 A549, human esophageal squamous carcinoma cells KYSE-150, human colon cancer cells HT-29, human ovarian cancer cell strain Caov-3, human breast cancer cells MCF-7, PANC-1 (American Type Culture Collection, rockville, USA) and QGP-1 and human normal immortalized epidermal cells Hacat, human normal umbilical vein endothelial cells HUVEC (Cobioer Corp., nanjing, china), human normal hepatic cell line LO2, human normal brain microvascular endothelial cells HUVEC and human intestinal epithelial cells HIEC (Mingzhou biosome, ningbo, china) added to DMEM or 1640 medium (Solaibao, beijing, china) containing 10% fetal bovine serum, 100U/mL penicillin and 100U/mL streptomycin (Beyotime Biotechnology, shanghai, china) containing 5% CO at 37% 2 Culturing in an incubator.
The inoculated cells are taken in logarithmic phase, digested by pancreatin, and are counted after being made into single cell suspension by using culture medium, and are inoculated into 96-well culture plate by using 2000-5000 cells/well, and in addition, blank control well is arranged, and only complete culture medium is added. At 37deg.C, with 5% CO 2 The culture was carried out in a saturated humidity incubator for a period of time. After the cells are attached, the cells are treated by using the chlorella pyrenoidosa polysaccharide XQZ with different concentrations, the final volume of each hole is 100 mu L, and 3 compound holes are arranged in each group. After 72h of drug treatment, 20. Mu.L per well of CCK8 (5 mg/mL purchased from Sigma-Aldrich) was added and incubation continued for 4h. The culture medium in the plate was carefully aspirated, 50. Mu. LDMSO solution was added to each well, and the plate was placed on a microplate shaker for 10min to allow the crystals to dissolve well. The OD value of the optical density of each well was measured at 570nm by using an ELISA reader,OD values of the blank wells were subtracted from each well and the results were recorded.
Results as shown in fig. 2, CCK8 assays were performed on various tumor cells treated with XQZ to verify the anticancer potential of XQZ3. Cancers of the above type were observed, namely: esophageal Squamous Cell Carcinoma (ESCC), bladder carcinoma (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) (FIGS. 2A-B, D-E). 2F is the half maximal inhibitory concentration (IC 50) value obtained after XQZ3 administration, indicating XQZ inhibits the growth of different types of tumors in a dose dependent manner. These results indicate that XQZ3 showed the highest sensitivity to PDAC cells (BxPC-3) with an IC50 value of 0.05026mg/mL.
Then, another 9 PDAC cell lines were selected for efficacy testing. The results showed that XQZ3 had a remarkable inhibitory effect on all 10 PDAC cell lines, with IC50 values ranging from 0.05026 to 0.6678mg/ml (FIGS. 2D-F). The potential toxic effects of XQZ3 on non-cancerous cells were also investigated. The results showed that XQZ3 did not exhibit any significant cytotoxicity to various normal cell types, such as human hepatocytes, umbilical vein endothelial cells, brain microvascular endothelial cells, intestinal epithelial cells, and keratinocytes (fig. 2C).
(2) PDO evaluation XQZ3 anti-proliferation assay
It is well known that solid tumors respond to chemotherapeutic agents to a lesser extent than monolayer cultured cells. Researchers often turn to diseased human organs (PDOs) in order to more accurately replicate the microenvironment and predict treatment outcome. The antiproliferative effect of XQZ3 was evaluated in this example using PDO from Pancreatic Ductal Adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNEN). The diameter and cell viability of PDOs were measured, as shown in fig. 3, and it can be seen that there was a significant decrease in the diameter of two PDAC PDOs treated with XQZ3 and cultured for three days, indicating a significant decrease in the cell proliferation rate of PDACs, as compared to the PBS-treated group (fig. 3A-C). After 9 days of XQZ treatment, a sustained decrease in PDO size was observed for PDAC compared to PBS group. Specifically, PDOs of PBS treated group showed solid spheres with distinct boundaries, while XQZ treated group showed diffuse spheres with blurred edges (fig. 3A). After 9 days of treatment, the group receiving XQZ3 treatment had a slight increase in PDO size compared to 3 days of treatment due to scattering of PDO in compact spheres (fig. 3A). After a 9 day treatment regimen, PDO models were obtained from each group and viability of 3D cultured PDO was assessed using CellTiter-Glo 3D cell viability assay (fig. 3C). These findings indicate that XQZ3 exhibits a concentration-dependent inhibition of cell viability of both PDAC PDOs.
(3) Plate cloning experiments
Cells in the logarithmic growth phase are taken, digested by pancreatin and blown into single cells, and the single cells are counted after being prepared into single cell suspension by culture medium. 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 inoculated in 6-well plates, gently rotated to disperse the cells uniformly, and then treated with Chlorella pyrenoidosa polysaccharide XQZ3 to give final concentrations of 0. Mu.g/mL, 50. Mu.g/mL, 100. Mu.g/mL, 200. Mu.g/mL. Placing at 37deg.C and 5% CO 2 Culturing in a saturated humidity cell culture box for 1-2 weeks. It is often observed that the culture is terminated when macroscopic clones appear in the culture dish. The supernatant was discarded and carefully rinsed 2 times with Phosphate Buffered Saline (PBS). Cells were fixed with 4% paraformaldehyde for 15 min, the fixation solution was removed, stained with 0.1% crystal violet dye (Beyotime Biotechnology, shanghai, china) for 10-30 min, the stain was slowly washed off with running water, and air dried. The number of clones was counted directly with naked eyes and counted.
As shown in FIG. 5, it can be seen that administration of Chlorella pyrenoidosa polysaccharide XQZ3 resulted in a significant decrease in proliferation of BxPC-3, miapaca2, huP-T4, capan-1, asPC-1 and SW1990 cells (5H), and that Chlorella pyrenoidosa polysaccharide XQZ3 showed significant efficacy in inhibiting growth of various types of tumor cells while showing negligible cytotoxicity to healthy cells, and most significant inhibition of PDACs.
Example 3: chlorella pyrenoidosa polysaccharide XQZ3 inhibits nude mice engraftment tumor activity
Nude mouse transplantation tumor experiment of pancreatic cancer patient source
Female BALB/c nu/nu mice (6-8 weeks old, 18-20g weight, purchased from Gempharmatech Biotechnology, nanjin, china) were bred under conditions of constant temperature (24-26 ℃) Specific Pathogen Free (SPF) at Shanghai Hospital laboratory animal center, and subjected to high pressure and ultraviolet sterilization treatment with cages, pads, drinking water and feeds, and were subjected to aseptic procedures, all of which were approved by the Shanghai Hospital animal ethics committee according to the national institutes of health's laboratory animal care and use guidelines.
Pancreatic cancer tissues, including Pancreatic Ductal Adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNEN), are from patients undergoing surgical resection in Shanghai long sign hospitals. The study was approved by the ethics committee of Shanghai long sign hospital research. Written informed consent was obtained prior to obtaining the tissues of all patients. Samples were confirmed as pancreatic tumors by pathologist evaluation.
Tumor tissue was chopped into a high concentration matrigel (TM) basement membrane matrix (BD Biosciences, franklin Lakes, USA) to about 4mm 3 Directly into the subcutaneous space of 6 to 8 week old female BALB/c nude mice.
Subsequently, the effect of XQZ on tumor progression was assessed using the PDAC PDX model. The PDAC PDX model is randomly assigned to three groups: PBS, 10mg/kg XQZ3 treatment and 20mg/kg XQZ3 treatment, each group included 10 models. PBS or XQZ3 was injected intraperitoneally every 4 days and body weight and tumor growth were measured at the same time interval until endpoint was reached. After 36 days of treatment, 4 mice per group were sacrificed and their subcutaneous tumors were harvested and weighed. The viscera of each mouse were picked for staining and blood was collected for biochemical testing while tumor growth and survival of the remaining 6 mice in each group were observed. Animal body weight and tumor size were measured every 4 days during the experiment until tumors exceeded 2000mm 3 Or the mice die naturally. Daily observations record clinical symptoms. At the end of dosing, photographs were taken to record tumor size. Tumor tissue is taken, weighed, fixed in 4% paraformaldehyde or protein is taken orThe calculation formula of Tumor Volume (TV) frozen in liquid nitrogen is: tv= (l×w2)/2.
The results showed that XQZ3 administration had a significant inhibitory effect on the in vivo growth of pancreatic cancer (fig. 5a, d). Tumor growth curve results showed an average 5.24-fold increase in tumor volume for mice receiving the 10mg/kg XQZ3 dose compared to the original tumor volume, 3.21-fold increase in mice receiving the 20mg/kg XQZ3 dose, and 11.35-fold increase in mice receiving PBS (FIG. 5A). A52.92% reduction in tumor growth was observed with the 10mg/kg XQZ3 group, while the 20mg/kg XQZ3 group showed a 70.27% reduction in tumor growth (FIGS. 5A, D) over the 42 day treatment. The results showed that XQZ3 showed a concentration-dependent inhibition of tumor weight, with a maximum inhibition of 79.96% observed in the group treated with 20mg/kg XQZ3 (fig. 5C-D).
Furthermore, XQZ treatment significantly prolonged the life of the pancreatic ductal adenocarcinoma patient-derived xenograft model (fig. 5E). After 70 days of monitoring, the survival rate was observed to be 0% for the PBS group, 33.33% for the 10mg/kg XQZ3 group and 66.67% for the 20mg/kg XQZ3 group (FIG. 5E).
Furthermore, while evaluating the effectiveness of XQZ3, the in vivo biosafety thereof was also evaluated. The results showed that administration of XQZ3 did not result in statistically significant changes in mouse body weight compared to PBS-treated mice (fig. 5B). 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 (fig. 5F). The histological morphology of organs of mice treated with XQZ or PBS were evaluated and XQZ was found to not cause any significant changes (fig. 5G). The results indicate that XQZ3 has a strong inhibition effect on HSP90 and exhibits remarkable anti-pancreatic cancer properties in vitro and in vivo. The results show that XQZ3 shows good safety, and makes it a promising candidate drug for pancreatic cancer treatment.
The foregoing is a preferred embodiment of the present invention, but the present invention should not be limited to the disclosure of this embodiment. So that equivalents and modifications will fall within the scope of the invention, all within the spirit and scope of the invention as disclosed.
Claims (10)
1. The chlorella pyrenoidosa polysaccharide XQZ3 has weight average molecular weight of 10-500 kDa and relative molecular weight of 29.13kDa, and contains galactose 53.1wt%, mannose 12.8wt%, rhamnose 12.2wt%, glucuronic acid 11.7wt%, glucosamine 5.4wt% and arabinose 4.6 wt%.
2. The method for preparing the chlorella pyrenoidosa polysaccharide XQZ3 as claimed in claim 1, comprising the following steps:
step a, polysaccharide extraction: sequentially degreasing the dried chlorella pyrenoidosa powder with ethanol, extracting with water, filtering, concentrating the obtained filtrate, dialyzing, concentrating again, precipitating with ethanol, centrifuging, vacuum drying, deproteinizing, and freeze-drying to obtain crude polysaccharide of the chlorella pyrenoidosa;
step b, polysaccharide purification: the crude polysaccharide of the water extracted chlorella pyrenoidosa is subjected to fractional purification by using a DEAE cellulose anion column, sequentially eluted by distilled water, 0.1mol/L, 0.2mol/L and 0.3mol/L NaCl solution, detected by sulfuric acid-phenol, and eluted components of the 0.3mol/L NaCl solution are collected, concentrated, dialyzed and freeze-dried to obtain the chlorella pyrenoidosa polysaccharide XQZ.
3. The method for preparing chlorella pyrenoidosa polysaccharide XQZ3 as claimed in claim 2, wherein the dialysis bag has a molecular weight cut-off of 3500Da.
4. The method for preparing chlorella pyrenoidosa polysaccharide XQZ3 as claimed in claim 2, wherein the method for extracting polysaccharide in the step a comprises: soaking dried Chlorella pyrenoidosa powder in 95% ethanol for 1 week, air drying, adding 20 times of deionized water, extracting at 100deg.C, filtering, extracting the residue with deionized water again, repeatedly extracting for 3 times each for 3 hr, concentrating the combined supernatant, treating with 15% trichloroacetic acid at 4deg.C for 3 hr to remove protein, centrifuging, concentrating, dialyzing, concentrating again, adding 3 times of 95% ethanol, centrifuging to obtain precipitate, and lyophilizing to obtain crude Chlorella pyrenoidosa polysaccharide.
5. The method for preparing chlorella pyrenoidosa polysaccharide XQZ3 as claimed in claim 2, wherein the method for purifying the polysaccharide in the step b comprises: dissolving crude chlorella pyrenoidosa polysaccharide in 10 times of water, centrifuging, separating supernatant with DEAE cellulose anion column, eluting with distilled water, 0.1mol/L, 0.2mol/L and 0.3mol/L NaCl solution sequentially, detecting with sulfuric acid-phenol, collecting eluate of the combined 0.3mol/L NaCl solution, concentrating, dialyzing, and lyophilizing to obtain chlorella pyrenoidosa polysaccharide XQZ.
6. A pharmaceutical composition comprising a therapeutically effective amount of the chlorella pyrenoidosa polysaccharide XQZ3 of claim 1 as an active ingredient.
7. The pharmaceutical composition of claim 6, further comprising a pharmaceutically acceptable pharmaceutical adjuvant selected from one of a carrier, excipient, adjuvant, and/or diluent;
and/or a pharmaceutically acceptable therapeutic agent selected from the group consisting of nitrogen mustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan, thiotepa, carmustine, semustine, busulfan, cisplatin, carboplatin, platinum oxalate, mitomycin.
8. Use of the chlorella pyrenoidosa polysaccharide XQZ of claim 1 or the pharmaceutical composition of claim 6 or 7 in the manufacture of an anti-tumor medicament.
9. The use according to claim 8, wherein the tumor comprises esophageal squamous cell carcinoma, bladder carcinoma, colorectal carcinoma, ovarian carcinoma, prostate carcinoma, hepatocellular carcinoma, melanoma, non-small cell lung carcinoma, breast carcinoma, pancreatic carcinoma.
10. The use of claim 8, wherein the tumor is a pancreatic cancer, including pancreatic ductal adenocarcinoma and pancreatic neuroendocrine tumors.
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