Glucomannan and application thereof in preparation of antitumor drugs

Abstract

The invention discloses a structure of glucomannan and application of the glucomannan in antitumor drugs, the glucomannan is mainly composed of mannose (Man) and glucose (Glc), the molar ratio of the mannose to the glucose is 1.0-4.0: 1.0, the mannose and the glucose are mainly connected through a 1-4 beta bond (as shown in the specification), and the molecular weight range of the glucomannan is 3.0 * 10 < 4 >-2.0 * 10 < 5 > Da. The glucomannan is a homogeneous polysaccharide obtained by carrying out ethanol degreasing, water extraction and alcohol precipitation and Sevag method deproteinization on dried pseudobulb of orchid rhododendron, and then carrying out ion exchange resin column chromatography, and the structure of the glucomannan is determined through UV, IR, HPLC, GC-MS, NMR and other technologies. Animal in-vivo tests show that the glucomannan can effectively inhibit growth of tumors such as breast cancer and liver cancer of mice, has small toxic and side effects, has an effect of treating solid tumors, and can be used as an anti-tumor polysaccharide drug. Wherein the range of m is from 1.0 to 4.0, and the range of n is from 30 to 920; r1 and R2 are hydroxyl (-OH) or acetyl (CH3COO-).

Description

Technical Field

[0001] The invention relates to a polysaccharide compound extracted from natural products: the structure of a type of glucomannan, a pharmaceutical composition containing the glucomannan, and the use of the glucomannan and the pharmaceutical composition thereof in anti-tumor drugs, belonging to the field of medical technology. Background Art

[0002] Cancer is one of the major threats to human health worldwide today. Treating cancer and protecting life safety has become a top priority for the global medical community. Currently, chemotherapy and radiotherapy commonly used to treat tumors work by directly killing tumor cells, but these two methods will also damage other healthy cells adjacent to cancer cells to varying degrees, and can also cause serious side effects, such as anemia, immune deficiency, fatigue, fertility problems, and neurological diseases. [2] .

[0003] Active polysaccharides obtained from natural products have gradually become one of the important sources of anti-tumor drugs due to their significant anti-tumor activity, good biocompatibility, and low toxicity and side effects, such as Poria polysaccharide injection, ginseng polysaccharide, and Lingsuo polysaccharide injection. [3] Glucomannan is an important natural polysaccharide, mainly composed of mannose and glucose. [4] . The glucomannan currently on the market is mainly extracted from the tubers of konjac, and its main uses are as a thickener and food satiety substitute. There are only two types of glucomannan recorded as medicines by the State Food and Drug Administration, which are the raw materials produced by the same company - glucomannan coarse powder and its capsule preparation - glucomannan capsules, which are used to treat constipation and prevent and treat diabetes. Existing functional research on glucomannan is mainly focused on the above two aspects. The number of glucomannans with anti-tumor activity discovered so far is relatively small, and they are mainly concentrated in the Dendrobium plants of the Orchidaceae family. The research is in its infancy. There are reports that Dendrobium officinale glucomannan may have good anti-melanoma cell activity, and Huoshan Dendrobium polysaccharide can induce apoptosis of gastric cancer cells in MFC mice. [5-6] For the glucomannan with antitumor activity of Dendrobium, some structures are relatively clear, while others are not. The structures of the glucomannan with reported structures are quite different from those of the glucomannan contained in the present invention. Except for the polysaccharide of Dendrobium huoshanense, the mannose content is generally high, and the ratio of mannose to glucose is in the range of 5.3 to 11.0:1.0. The molecular weight of the polysaccharide of Dendrobium huoshanense is greater than the maximum molecular weight of the present invention, and its molecular weight is 2.59×10 5 Da. Therefore, the present invention determines the molecular structure of this type of glucomannan by separating, purifying and analyzing its structure, and finds that it has obvious inhibitory activity against various tumors at the animal level, which has certain uniqueness and depth.

[0004] References

[0005] 【1】Zheng RS,Zhang SW,Zeng HM,Cancer incidence and mortality in China,2016[J]Journal of the National Cancer Center,2022,2(1):1-9.

[0006] 【2】Sung H,FerlayJ,Siegel RL,et al.Global Cancer Statistics 2020:GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185Countries[J]CA Cancer J Clin,2021,71(3):209-249.

[0007] 【3】Khan T,Date A,Chawda H,Patel K,et al..Polysaccharides as potentialanticancer agents—A review of their progress[J]Carbohydrate Polymers,2019,210:412-428.

[0008] 【4】Shi XD,Yin JU,Steve W,et al.Plant-derived glucomannans:Sources,preparation methods,structural features,and biological properties[J]Trends inFood Science&Technology,2020,99:101-116.

[0009] 【5】Yu,W.X.,Z.Y.Ren,X.F.Zhang,et al.Structural characterization ofpolysaccharides from Dendrobium officinale and their effects on apoptosis ofHeLa cell line[J]Molecules,2018,23:2484.

[0010] 【6】Liu, B., ZZShang, QMLi, et al. Structural features and anti-gastriccancer activity of polysaccharides from stem, root, leaf and flower of cultivated Dendrobium huoshanense [J] International Journal of Biological Macromolecules, 2020, 143: 651–664. Summary of the invention

[0011] The purpose of the present invention is to provide a type of glucomannan structure with anti-tumor effect in view of the shortcomings of the prior art.

[0012] Another object of the present invention is to provide a type of use of natural glucomannan in drugs for treating tumors.

[0013] In order to achieve the above object, the present invention adopts the following technical scheme:

[0014] The first aspect of the technical solution of the present invention provides a type of glucomannan, characterized in that it is composed of mannose and glucose, the molar ratio of mannose to glucose is 1.0-4.0:1.0, mainly connected by 1→4β bonds, and the molecular weight range is 3.0×10 4 ~2.0×10 5 Da.

[0015] The glucomannan is characterized in that it has the following structure:

[0016]

[0017] Wherein: m ranges from 1.0 to 4.0, n ranges from 30 to 920; R1 and R2 are hydroxyl or acetyl.

[0018] The first aspect of the technical solution of the present invention provides a pharmaceutical composition, characterized in that the pharmaceutical composition contains the therapeutically effective amount of glucomannan described in the first aspect, and a pharmaceutically acceptable carrier and excipient.

[0019] The pharmaceutical composition includes injection, tablet, capsule, pill, oral solution or dripping pill dosage form.

[0020] The preparation method of natural glucomannan is as follows:

[0021] The bulb of the orchid plant azalea orchid is dried, crushed, refluxed and defatted with 95% ethanol twice, decocted with water, 95% ethanol is added to the extract until the final concentration of ethanol is 75%, and the extract is left to stand at 4°C for 48 hours, filtered, and the filter cake is freeze-dried. The freeze-dried sample is dissolved in water, the protein is removed by the Sevag method, the water layer is collected, centrifuged, and the supernatant is collected for dialysis to obtain crude polysaccharides. The crude polysaccharides are dissolved in water, separated by an ion exchange column, and the sugar content is detected by the phenol-sulfuric acid method to obtain an elution curve, and the sugar-containing solution is collected to obtain natural glucomannan.

[0022] The invention performs purity analysis and molecular weight determination, total sugar content and protein content determination, and monosaccharide composition analysis on the obtained glucomannan, and then uses infrared spectroscopy, high performance liquid chromatography, gas chromatography-mass spectrometry, nuclear magnetic resonance and other methods to determine its structure.

[0023] The first aspect of the technical solution of the present invention provides the use of the glucomannan described in the first aspect and the pharmaceutical composition described in the second aspect in the preparation of anti-tumor drugs.

[0024] The present invention also relates to the use of such glucomannan in tumor treatment drugs. Animal experiments have shown that such glucomannan has a high inhibition rate on the tumor volume of H22 liver cancer-bearing mice and 4T1 breast cancer-bearing mice, and does not cause low immunity. Such glucomannan has potential use as an anti-tumor drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The ultraviolet spectrum (UV) of the glucomannan of Example 1

[0026] Figure 2 This is a diagram of the size exclusion method combined with multi-angle laser scattering (SEC-MALLS) of glucomannan in Example 1.

[0027] Figure 3 HPLC chromatogram of the pre-column derivative of 1-phenyl-3-methyl-5-pyrazolone (PMP) of the standard monosaccharide and hydrolysis product of Example 2

[0028] Figure 4 Gas chromatogram (GC) of the sugar alcohol acetate derivative of the glucomannan hydrolyzate of Example 2

[0029] Figure 5 The infrared spectrum (IR) of glucomannan of Example 2 is shown in FIG.

[0030] Figure 6 is the hydrogen spectrum of glucomannan in Example 2 ( 1 H NMR)

[0031] Figure 7 The carbon spectrum of glucomannan in Example 2 (13 C NMR)

[0032] Figure 8 The graph of the change of tumor volume over time in 4T1 tumor-bearing mice in Example 3 DETAILED DESCRIPTION

[0033] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments. The described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. The embodiments of the present invention and all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Example 1 Preparation of Glucomannan

[0035] 1. Extraction and purification of glucomannan

[0036] 200g of dried pseudobulbs of azalea were selected, crushed, soaked in 95% ethanol (solid-liquid ratio of 1:3) for 12h, refluxed and degreased with 95% ethanol twice, each time for 2h, and the medicinal materials after evaporation of ethanol were boiled and extracted with 12 times water for 3 times, each time for 2h, and the extracts were combined to obtain 5L, and then 95% ethanol was added to the alcohol content of 75%, refrigerated at 4℃ for 48h, filtered, and the precipitate was collected and freeze-dried; 10g of the freeze-dried product was weighed, water was added, and heated and stirred to dissolve (60℃, 400r / min), Obtain about 800 mL of solution, transfer to a separatory funnel, add 5 times the volume of Sevage reagent to remove protein, after standing, collect the upper solution and centrifuge (8000r / min, 10min); refer to the Bradford method to detect the protein content, repeat the above deproteinization process until the Coomassie brilliant blue solution does not change color, and dialyze the crude polysaccharide after protein removal with a dialysis bag with a molecular weight of 3500Da; freeze-dry the polysaccharide solution in the dialysis bag to obtain crude glucomannan polysaccharide.

[0037] Take 200 mg of crude glucomannan polysaccharide, dissolve it in water, centrifuge (8000 r / min, 10 min), take the supernatant and load it on DEAE Sepharose TM The Fast Flow anion exchange column was eluted with 150 mL of water, 100 mL of 0.1 M NaCl, 50 mL of 1 M NaCl, and 50 mL of 2 M NaCl in sequence, and the fractions were collected. The fractions were detected by the phenol-sulfuric acid method, and the main peak of the elution curve was collected, concentrated, and dried to obtain glucomannan homogeneous polysaccharide.

[0038] 2. Purity detection and molecular weight determination of glucomannan

[0039] 1. Ultraviolet spectroscopy (UV) method

[0040] Glucomannan was prepared into a 1 mg / mL aqueous solution and scanned in the range of 190-600 nm using a JASCO V-650 UV spectrophotometer. The UV spectrum was as follows: Figure 1 As shown, there are no obvious absorption peaks at 260nm and 280nm, indicating that glucomannan does not contain protein and nucleic acid impurities.

[0041] 2. Determination of total sugar content of glucomannan

[0042] The total sugar content of glucomannan was determined by the phenol-sulfuric acid method, and the absorbance was used as the ordinate and the concentration was used as the abscissa (μg / mL) to draw a graph. A standard curve for sugar content determination was drawn: y=5.1482x+0.0003, with a correlation coefficient of 0.9997. The total sugar content of glucomannan was finally measured to be 98.23%.

[0043] 3. Determination of protein content of glucomannan

[0044] The protein content of glucomannan was determined by Bradford method, with absorbance as the abscissa and concentration as the ordinate (μg / mL) and a standard curve for protein content determination was drawn: y=2124.1x-11.407, with a correlation coefficient of 0.9996. The protein content of glucomannan was finally measured to be 0.06%.

[0045] 4. Determination of molecular weight by size exclusion chromatography coupled with multi-angle laser scattering (SEC-MALLS)

[0046] The molecular weight was determined by size exclusion chromatography coupled with multi-angle laser scattering (SEC-MALLS). The experimental detectors were Wyatt DAWN HELEOS-II light scattering detector and Wyatt Optilab rex differential refractometer. The analytical column was ShodexOH-pakSB-806M-HQ gel column. The mobile phase was 0.2M NaCl solution. The flow rate was 0.5mL / min, the column temperature was 35°C, and the sample concentration was 2mg / mL. The results are shown in Figure 2 The glucomannan chromatogram shows a single symmetrical peak, indicating that the glucomannan prepared by the present invention is a uniform polysaccharide. The molecular weight of glucomannan is shown in Table 1, and the molecular weight distribution range is shown in Table 2.

[0047] Table 1 Molecular weight test results of glucomannan

[0048]

[0049] Mw: weight average molecular weight; Mn: number average molecular weight; Mz: higher average molecular weight; Mp: molecular weight of the highest peak; Mw / Mn: polydispersity index

[0050] Table 2 Molecular weight distribution range of glucomannan

[0051]

[0052] Example 2 Structural Characterization of Glucomannan

[0053] 1. Analysis of monosaccharide composition of glucomannan

[0054] 1.1-Phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivative high performance liquid chromatography (HPLC) method

[0055] 5 mg of glucomannan was placed in a stoppered reaction bottle, 2M trifluoroacetic acid (TFA) solution was added, dissolved and reacted in a sealed container at 120°C for 2h, and TFA was removed by evaporation under reduced pressure to obtain glucomannan hydrolyzate. 100 μL of 4 mg / mL glucomannan complete hydrolyzate and 4 mg / mL standard monosaccharide solution were accurately measured and placed in a centrifuge tube, 300 μL of 0.3M NaOH solution was added first, and then 300 μL of 0.5M PMP methanol solution was added after mixing, and the reaction was carried out in a 70°C water bath for 90 min. After the reaction stopped, it was cooled to room temperature, and then the reaction solution was neutralized with 0.5M HCl to a weak acid. 2 mL of chloroform was added for extraction, centrifugation was performed, and the aqueous layer was collected. The above operation was repeated 3 times, and the aqueous layer was concentrated and filtered with a 0.45 μm microporous filter head, and analyzed by Agilent 1260 high performance liquid chromatograph. The results are as follows: Figure 3 It can be seen that glucomannan is composed of mannose and glucose only. According to the ratio of its peak areas, the molar ratio of mannose to glucose is determined to be 3.10:1.00.

[0056] 2. Sugar alcohol acetate method

[0057] The derivatization reaction process of glucomannan is as follows:

[0058]

[0059] The standard monosaccharides mannose and glucose were derivatized in the same way. Figure 4 By comparing the retention time of the products of sugar alcohol acetate derivatization with those of standard monosaccharides mannose and glucose, it can be determined that the monosaccharide composition of glucomannan is mannose and glucose. According to the ratio of their peak areas, the molar ratio of mannose to glucose in glucomannan is determined to be 2.90:1.00.

[0060] 2. Infrared Spectroscopy (IR) Analysis of Glucomannan

[0061] The infrared spectra of glucomannan were determined by Nicolet 5700 Fourier transform microscope transmission spectrometer (FT-IR) with a scanning range of 4000-500 cm -1, the results are shown in Figure 5 , in the FT-IR spectrum of glucomannan, 3264.4 cm -1 The broad peak at 2883.6cm is the stretching vibration peak of OH, which is caused by hydrogen bonds within and between molecules. -1 The absorption peak at 1376.5cm is caused by the stretching vibration of CH. -1 The absorption peak near is the bending vibration of OH, 1248.6 cm -1 The absorption peak at 1151.7 cm is the bending vibration of CH. -1 、1061.5cm -1 and 1028.6cm -1 The absorption peak at 1726.4cm is caused by the vibration of COC and COH. -1 The absorption peak at is caused by the stretching vibration of C=O.

[0062] 3. GC-MS analysis of methylation of glucomannan

[0063] The glucomannan was completely methylated by the Needs method, and then subjected to hydrolysis, reduction and acetylation. The acetylation product was analyzed by GC-MS (the results are shown in Table 3). The main reaction steps are as follows:

[0064]

[0065] Table 3 GC-MS analysis results of glucomannan

[0066]

[0067] a 2,3,4,6-Me4-Manp stands for 1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-mannose

[0068] By comparing the main fragment ions in the mass spectra of peaks 1 and 2 with the standard spectra containing such fragment ions in the database, it is inferred that the methylated sugar alcohol acetate corresponding to peak 1 is 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-mannose, and the methylated sugar alcohol acetate corresponding to peak 2 is inferred to be 1,4,5-tri-O-acetyl-2,3,6-tri-O-methyl-glucose. At the same time, according to the regularity of fragment ions, the rationality of the inference can also be confirmed. Taking peak 1 as an example, C-2 and C-3 are carbons with methoxy groups, and the carbon-carbon bond here is most likely to break, generating fragment ions m / z 117.0 and 233.1. C-3 and C-4 are carbons with one methoxy group and one acetoxy group, and the carbon-carbon bond breakage here is easy to generate m / z 161.0. For some fragment examples, m / z 161.0 loses the neutral fragment acetic acid (m / z 60) to obtain a fragment ion m / z 101.0. Based on the above analysis, it is speculated that the structure of glucomannan is mainly a linear polysaccharide composed of glucose and mannose connected at the 1 and 4 positions.

[0069] 4. Nuclear Magnetic Resonance (NMR) Analysis of Glucomannan

[0070] 11 mg of glucomannan was dissolved in 0.5 mL of D2O and freeze-dried, and then the freeze-dried powder was dissolved in 0.5 mL of D2O again, and repeated three times to fully exchange active hydrogen. The sample was dissolved in 0.5 mL of D2O and measured at 25 °C using a Bruker Avance Ⅲ HD 600 NMR spectrometer (Bruker Biospin, Germany). 1 H NMR ( Figure 6 )and 13 C NMR ( Figure 7 ).from 1 The characteristic hydrogen signal of sugar can be seen in the H NMR spectrum: δ H 4.50~4.80 is the terminal hydrogen signal of sugar, δ H 3.37~4.14 are the hydrogen signals on the oxymethylene and methine of sugar; 13 The characteristic carbon signal of sugar can be seen in the C NMR spectrum: δ C 105.69-102.07 is the terminal carbon signal of sugar, δ C63.08~81.45 are the carbon signals of the oxymethylene and methine of the sugar. The above information indicates that the compound is a polysaccharide. Comparing the carbon signals of the sugar with the data of the standard methyl mannopyranoside, it is found that the carbon signal at C-4 is shifted to the low field, indicating that the 4-position may be a glycoside. Further combined with the results of GC-MS analysis, it can be seen that the monosaccharide composition of glucomannan and the connection mode of the glycosidic bond are mainly 1,4-linked. When performing FT-IR spectral analysis on glucomannan, it was found that it is possible to have acetyl groups. With the help of NMR spectral analysis, the existence of acetyl groups was further confirmed. 1 In the H NMR spectrum, δ H The hydrogen signal at 2.20 is the hydrogen signal of the methyl group connected to the carbonyl group. 13 In C NMR spectra, δ C The carbon signals at 176.40 and 175.97 are the carbonyl carbon signals of the ester on the acetyl group. C The carbon signals at 23.54 and 23.28 are the methyl carbon signals on the acetyl group.

[0071] Example 3 Study on the anti-tumor activity of glucomannan

[0072] 1. Experiment on the inhibition of H22 liver cancer in mice by glucomannan

[0073] 1. Glucomannan inhibits the growth of liver cancer cells in mice

[0074] Forty male BALB / C mice aged 4-6 weeks were randomly divided into four groups: oral glucomannan group, oral glucomannan control group, intraperitoneal glucomannan group and intraperitoneal glucomannan control group, with 10 mice in each group. Five days after subcutaneous injection of mouse liver cancer cell H22, the tumor volume reached 100mm 3 , treated with glucomannan. The oral administration group had a dose of 200 mg / kg / day, a dosing volume of 100 μL, and a solvent of H2O; the control group was only given 100 μL H2O by oral gavage every day. The intraperitoneal injection group had a dose of 20 mg / kg / day, a dosing volume of 100 μL, and a solvent of H2O; the control group was only given 100 μL H2O by intraperitoneal injection every day. Tumor size was recorded with a vernier caliper. Tumor volume = (length × width × width) / 2; experimental results are expressed as mean ± SEM, and t test was used to test the difference between the glucomannan group and the control group.

[0075] After 3 weeks of administration, the subcutaneous tumor volume and tumor weight of mice in each group are shown in Table 4 (oral administration) and Table 5 (intraperitoneal injection administration).

[0076] Table 4 Oral administration of glucomannan inhibits the growth of H22 liver cancer cells in mice

[0077]

[0078]

[0079] Table 5 Intraperitoneal injection of glucomannan inhibits the growth of H22 liver cancer cells in mice

[0080]

[0081] The larger the tumor volume, the faster the tumor grows. According to the data in Tables 4 and 5, after continuous oral or intraperitoneal administration to mice for 3 weeks, glucomannan can significantly inhibit tumor growth compared with the control group, P<0.01 (tumor volume), P<0.05 (tumor weight).

[0082] 2. Glucomannan enhances CD8 in mouse tumors + T cell activity assay

[0083] The mice were killed by cervical dislocation, and the tumors were removed. The removed tumor nodes were placed in MACS buffer at room temperature. The tumor was placed in a 100mm culture dish and chopped. The chopped tissue pieces were placed in Digest Buffer (Collagenase IV (200U / mL) and DNase I (100μg / mL) in HBSS buffer.) and incubated on a shaker at 37°C for 30-60 minutes. The digested tissue pieces and liquid were passed through a 70μm Strainer, and the red blood cells were ruptured with red blood cell lysis buffer. Surface antibody staining, including Anti-CD45, Anti-CD3, and Anti-CD8. Stain at room temperature in a dark place for 15-20 minutes. After washing the cells with MACS buffer, resuspend the cells in 250μL MACS buffer (0.5% heat-inactivated FCS, 2mM EDTA in PBS), and detect CD8 in the tumor on a flow cytometer. + The results are shown in Table 6.

[0084] Table 6 Effect of oral (po) / intraperitoneal (ip) injection of glucomannan on CD8 infiltrating tumor cells + Effect of T cell ratio

[0085]

[0086] From the data in Table 6, it can be seen that oral administration or intraperitoneal injection of glucomannan has an effect on CD8 + There was no significant effect on the proportion of T cells.

[0087] To detect CD8 + To activate T cells, proceed as follows after step 4:

[0088] Take 15mL of lymphocyte separation solution (Lymphoprep TM ) was placed in a 50mL centrifuge tube. 30mL of cell suspension resuspended in the above MACS buffer was slowly superimposed on the stratified liquid surface along the tube wall with a dropper. Pay attention to maintaining a clear interface. Horizontal centrifugation at 2,000rpm for 20 minutes (both centrifugal acceleration and deceleration were adjusted to 2) to remove dead cells, tumor cells, and red blood cells. Lymphocytes in the stratified phase were collected. The obtained stratified phase lymphocyte suspension was diluted with 30mL MACS buffer and centrifuged at 2,000rpm for 5min at room temperature. The supernatant was discarded. Lymphocytes were resuspended with 2mL R10 medium. The resuspended cells were placed in a flow tube, and 5ng / mL PMA, 1μL / mL Ionomycin (500ng / μL), 1μL / mL Brefeldin A, and 1μL / mL GolgiStop were added to the cell suspension and incubated at 37℃ for 4 hours. Add surface staining related antibodies, including Anti-CD45, Anti-CD3, and Anti-CD8, mix with Vortex and place in a dark place for staining at room temperature for 15-20 minutes. After centrifugation, resuspend the cell clumps with 1mL of freshly prepared Fixation / Perm buffer for fixation and perforation. Incubate the fixed perforation at 4℃ for 1 hour. Add 4mL Perm / Wash buffer to wash the cells, centrifuge and discard the supernatant. Add the corresponding antibody Anti-Granzyme B to about 50μL Perm / Wash buffer for intracellular molecule staining. Incubate at 4℃ for 30 minutes. For example, intracellular molecules: Wash the cells with about 4mL Perm / Wash buffer, centrifuge at 2,000rpm for 5min, and discard the supernatant. Resuspend the cells with about 250μL Perm / Wash buffer, pass through a 300-mesh sieve and put them on the machine. The results are shown in Table 7:

[0089] Table 7. Effects of oral / intraperitoneal injection of glucomannan on CD8 infiltrating tumor cells + Effects of T cell activity

[0090]

[0091] From the data in Table 7, it can be seen that oral or intraperitoneal injection of glucomannan can increase the number of CD8 + There was a statistically significant difference in the activity of T cells.

[0092] Based on the above data analysis, it can be seen that glucomannan can significantly inhibit the growth of tumors in H22 liver cancer-bearing mice. Its mechanism of action may be through enhancing the CD8 + T cell activation and exert anti-tumor effects.

[0093] 2. Glucomannan inhibits 4T1 breast cancer tumor in mice

[0094] Thirty-two 4T1 tumor-bearing mice (female BALB / C) with similar tumor volumes and weights were selected and randomly divided into four groups: model group, positive drug group, high-dose glucomannan group, and low-dose glucomannan group, with 8 mice in each group. The high-dose glucomannan group was intraperitoneally injected with glucomannan at a dose of 50 mg / kg / day, and the administration volume was 100 μL; the low-dose glucomannan group was intraperitoneally injected with glucomannan at a dose of 20 mg / kg / day, and the administration volume was 100 μL; the model group was only intraperitoneally injected with 100 μL of normal saline; the positive drug group was intraperitoneally injected with paclitaxel (PTX) at 10 mg / kg / day, and the administration volume was 100 μL. After 14 days of continuous administration, the tumors, livers, spleens, and thymus were dissected and weighed, and the liver index, spleen index, thymus index, and in vivo tumor inhibition rate were calculated. The data are shown in Table 8. The changes in mouse tumor volume over time are shown in Table 8. Figure 8 shown.

[0095] Table 8. Tumor inhibition rate and liver, spleen and thymus index data of each group of mice

[0096]

[0097] Compared with the saline group: * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001

[0098] According to the above data, glucomannan can significantly inhibit the growth of tumors in 4T1 breast cancer-bearing mice. Compared with the model group, the spleen and thymus indexes of mice in the high-dose and low-dose glucomannan groups did not change significantly, which shows that glucomannan does not damage the immune system of tumor-bearing mice while exerting its anti-tumor effect. This type of glucomannan may become a polysaccharide drug with less toxic side effects in the treatment of tumors such as liver cancer and breast cancer.

[0099] So far, the above embodiments are the best implementation methods of the present invention. Various changes, modifications, substitutions and variations of these embodiments without departing from the principle and spirit of the present invention are all included in the protection scope of the present invention.

Claims

1. A type of glucomannan, characterized in that: It is composed of mannose and glucose, with a molar ratio of mannose to glucose of 1.0-4.0:1.0, mainly connected by 1→4β bonds, and a molecular weight range of 3.0×10 4 ~2.0×10 5 Da.

2. The glucomannan according to claim 1, characterized in that It has the following structure: Wherein: m ranges from 1.0 to 4.0, n ranges from 30 to 920; R1 and R2 are hydroxyl or acetyl.

3. A pharmaceutical composition, characterized in that The pharmaceutical composition contains the therapeutically effective amount of glucomannan according to any one of claims 1 or 2, and a pharmaceutically acceptable carrier and excipient.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition includes injection, tablet, capsule, pill, oral solution or dripping pill dosage form.

5. Use of the glucomannan according to any one of claims 1 and 2 and the pharmaceutical composition according to any one of claims 3 to 4 in the preparation of anti-tumor drugs.

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