Metaplexis japonica herb polysaccharide LMP-1 with antitumor activity as well as extraction method and application of metaplexis japonica herb polysaccharide LMP-1

Abstract

The invention discloses metaplexis japonica herb polysaccharide LMP-1 with anti-tumor activity as well as an extraction method and application thereof, the metaplexis japonica herb polysaccharide LMP-1 has a structural formula as follows: # imgabs0 #, has definite targeting property, can specifically act on tumor cells, inhibit proliferation of the tumor cells, induce apoptosis of the tumor cells and reduce damage to normal cells, and can be used for preparing anti-tumor drugs. The treatment effect is improved, the side effect is reduced, and meanwhile, the compound has a remarkable proliferation promoting effect on RAW264.7 immune cells.

Description

Technical Field

[0001] The present invention relates to the preparation and application of polysaccharides from the whole herb of Metaplexis japonica, and specifically belongs to a polysaccharide LMP-1 from the whole herb of Metaplexis japonica with anti-tumor activity, its extraction method and application. The polysaccharide LMP-1 from the whole herb of Metaplexis japonica can be applied in the preparation of anti-tumor drugs and natural health foods. Background Art

[0002] The statements herein only provide background art related to the present invention and do not necessarily constitute prior art.

[0003] Metaplexis japonica Makino., also known as Zhuo He Zi, Yang Po Nai, Po Po Zhen Xian Bao, Yang Jiao, Tian Jiang Ke, etc. It is a plant under the family Asclepiadaceae of the order Contortae in the class Dicotyledoneae, widely distributed in the northeastern, northern, eastern China, and provinces such as Gansu, Shaanxi, Guizhou, Henan, and Hubei. It grows in forest margins, wasteland, foot of mountains, riversides, and roadside bushes. It is also distributed in Japan, Korea, and Russia.

[0004] "Jiuhuang Bencao" records Metaplexis japonica: "For famine relief, pick young leaves, scald them, change water and soak to remove bitterness and pathogenic qi, wash them clean, and season with oil and salt for eating". "Bencao Shiyi" records: "Emperor Gaozu of the Han Dynasty used its seeds to dress the soldiers' golden sores, so it was named Zhuo He Zi". "Bencao Huiyan" records: "Metaplexis japonica is a medicine for tonifying deficiency and benefiting essence". Currently, there are a large number of literature reports: it contains a variety of medicinal components, has high edible and medicinal values, and has effects such as tonifying deficiency and benefiting essence, and is a good health food for both medicine and food.

[0005] Polysaccharides refer to a class of natural macromolecular compounds composed of 10 or more monosaccharide molecules and are one of the macromolecular substances maintaining normal life activities. Existing studies have shown that the polysaccharides from the pericarp of Metaplexis japonica have anti-tumor and immune function promoting activities. However, it is difficult to obtain specific active substances, which are generally mixtures, difficult to ensure the uniformity of drugs, and have extremely large molecular weights, and are difficult to prepare except by natural extraction, resulting in high costs. Summary of the Invention

[0006] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a brand-new polysaccharide LMP-1 from the whole herb of Metaplexis japonica with anti-tumor activity, its extraction method and application, aiming to solve the problems of large drug side effects, poor patient tolerance, and drug resistance of some tumors to traditional chemotherapy drugs in current anti-tumor treatments. The small molecule polysaccharide LMP-1 from the whole herb of Metaplexis japonica in the present invention has a lower molecular weight, and its structure has been analyzed. Based on this, chemical or biological synthesis of LMP-1 can be achieved, thus completely replacing the traditional extraction method and ensuring the uniformity of drugs.

[0007] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0008] In a first aspect, the present invention provides a Metaplexis japonica whole herb polysaccharide LMP-1 with anti-tumor activity, and its structural formula is:

[0009]

[0010] In a second aspect, the present invention provides a method for extracting the Metaplexis japonica whole herb polysaccharide LMP-1 with anti-tumor activity, including the following steps:

[0011] After drying and pulverizing the Metaplexis japonica whole herb, it is soaked in ethanol for a set time. After soaking, the ethanol is volatilized and dried to obtain the pretreated Metaplexis japonica whole herb powder.

[0012] The pretreated Metaplexis japonica whole herb powder is extracted with hot water for a set time, and the solid-liquid separation is carried out. The extract is evaporated and concentrated, and then absolute ethanol is added thereto for ethanol precipitation. The obtained precipitate is the crude Metaplexis japonica polysaccharide.

[0013] The crude Metaplexis japonica polysaccharide is dissolved in water, and a Sevag solution is added thereto. After shaking and centrifuging, the protein impurities are removed to obtain the crude polysaccharide.

[0014] The crude polysaccharide is dissolved in water, activated carbon is added thereto, and after stirring, the solid-liquid separation is carried out.

[0015] The obtained polysaccharide solution is passed through a DEAE-52 cellulose chromatography column and then eluted with water to obtain the Metaplexis japonica whole herb polysaccharide LMP-1.

[0016] The inventors found through experiments that after drying and pulverizing the Metaplexis japonica whole herb, soaking it in ethanol first can remove the fat-soluble components in the Metaplexis japonica whole herb in advance, which is convenient for subsequent impurity removal, and is also convenient for the extraction and purification of the Metaplexis japonica whole herb polysaccharide LMP-1, and plays a crucial role in the successful separation of the Metaplexis japonica whole herb polysaccharide LMP-1.

[0017] In some embodiments, in the step of soaking the Metaplexis japonica whole herb in ethanol after drying and pulverizing, the concentration of ethanol is 90%-97%, and % is the mass percentage.

[0018] Preferably, the soaking time in ethanol is 8-20 h.

[0019] In some embodiments, after soaking, the temperature for volatilizing ethanol and drying is 50-60 °C.

[0020] In some embodiments, when extracting with hot water, the temperature of the hot water is 70-90 °C, and the extraction time is 2-5 h.

[0021] Preferably, the temperature of the hot water is 75-85 °C, and the extraction time is 2-4 h.

[0022] Further preferably, the temperature of the hot water is 80 °C and the extraction time is 3 h.

[0023] Preferably, during hot water extraction, the solid-liquid ratio is 1:10 - 20, preferably 1:15.

[0024] In some embodiments, during alcohol precipitation, in the alcohol precipitation system, the ethanol concentration is 60% - 80%, where % is mass percentage.

[0025] Preferably, during alcohol precipitation, in the alcohol precipitation system, the ethanol concentration is 65% - 75%, where % is mass percentage.

[0026] Further preferably, during alcohol precipitation, in the alcohol precipitation system, the ethanol concentration is 70%, where % is mass percentage.

[0027] In some embodiments, in the Sevag solution, the volume ratio of chloroform to n-butanol is 4:1.

[0028] In a third aspect, the present invention provides the application of the whole grass polysaccharide of Metaplexis japonica LMP-1 with anti-tumor activity in the preparation of anti-tumor drugs or tumor vaccine adjuvants.

[0029] The beneficial effects obtained from one or more of the above embodiments of the present invention are as follows:

[0030] The in vitro anti-tumor activity of the whole grass polysaccharide of Metaplexis japonica LMP-1 obtained in the present invention was detected, and the results showed that when cells were treated with different concentrations of the whole grass polysaccharide of Metaplexis japonica LMP-1 for 72 hours, the inhibition rates of the six cells used in the experiment were all about 20% or more, showing extremely significant differences compared with the control group (P < 0.001); for the RAW264.7 immune cells treated for 24 h, it showed a significant proliferation-promoting effect.

[0031] The whole grass polysaccharide of Metaplexis japonica LMP-1 has clear targeting, can specifically act on tumor cells, inhibit their proliferation, induce their apoptosis, while reducing damage to normal cells, improving the treatment effect and reducing side effects.

[0032] Through response surface analysis of the traditional extraction method, the present invention obtained the theoretically optimal extraction method, providing highly efficient and mass-produced whole grass polysaccharide of Metaplexis japonica for subsequent pharmacological activity evaluation and structural analysis of the whole grass polysaccharide of Metaplexis japonica. The results showed that the optimal extraction and separation process conditions for the whole grass polysaccharide of Metaplexis japonica are a solid-liquid ratio of 1:15, an extraction temperature of 80 °C, an extraction time of 3 h, and an extraction frequency of 2 times, and the maximum yield of the whole grass polysaccharide of Metaplexis japonica can reach 4.986%.

[0033] The Cynanchum bungei stem and leaf polysaccharide LMP-1 of the present invention can not only be applied to the research and development of natural new foods and healthy beverages, providing a theoretical basis for the in-depth development and effective utilization of the Chinese medicinal herb Cynanchum bungei resources; but also is expected to be developed and applied as a natural small-molecule polysaccharide anti-tumor drug and immune-promoting drug in the fields of cancer treatment adjuvant drugs, tumor vaccine adjuvants, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The attached drawings of the specification, which form a part of the present invention, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0035] Figure 1 is the extraction and purification flow chart of Cynanchum bungei polysaccharides LMP-1, LMPA-1 and LMPA-2 in the embodiments of the present invention;

[0036] Figure 2 is the influence of the solid-liquid ratio (A), extraction temperature (B), extraction time (C) and extraction times (D) on the extraction rate of Cynanchum bungei polysaccharides in the embodiments of the present invention;

[0037] Figure 3 is the response surface diagram of the variable parameters on the yield of Cynanchum bungei polysaccharides in the embodiments of the present invention, wherein, A is the solid-liquid ratio - times; B is the solid-liquid ratio - time; C is the solid-liquid ratio - temperature; D is the time - times; E is the temperature - times; F is the temperature - time;

[0038] Figure 4 is the column chromatography result diagram of Cynanchum bungei polysaccharides in the embodiments of the present invention;

[0039] Figure 5 is the high performance gel permeation chromatography diagram of Cynanchum bungei polysaccharides LMP-1 (A), LMPA-1 (B) and LMPA-2 (C) in the embodiments of the present invention;

[0040] Figure 6 is the monosaccharide composition result diagram of Cynanchum bungei polysaccharide LMP-1 in the embodiments of the present invention;

[0041] Figure 7 is the infrared spectrum diagram of Cynanchum bungei polysaccharide LMP-1 in the embodiments of the present invention;

[0042] Figure 8 is of Cynanchum bungei polysaccharide LMP-1 in the embodiments of the present invention 1 1H-NMR spectrum diagram;

[0043] Figure 9 is the structure diagram of Cynanchum bungei polysaccharide LMP-1 in the embodiments of the present invention;

[0044] Figure 10 The influence result of the proliferation toxicity of Cynanchum bungei polysaccharide LMP-1 on tumor cells;

[0045] Figure 11 The results of the proliferation toxicity of Metaplexis polysaccharides LMPA-1 (A) and LMPA-2 (B) on tumor cells;

[0046] Figure 12 The change curves of body weight and administration days of tumor-bearing mice in each group;

[0047] Figure 13 The organ indices of tumor-bearing mice in each group;

[0048] Figure 14 The comparison chart of tumor volumes of tumor-bearing mice in each group. Among them, A is the CTX group, B is the LMP-1 group, C is the PBS group, and D is the combined comparison chart of the CTX group, LMP-1 group, and PBS group;

[0049] Figure 15 The comparison chart of tumor weights of tumor-bearing mice in each group;

[0050] Figure 16 The comparison chart of cytokine contents of tumor-bearing mice in each group. Detailed implementation manners

[0051] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0052] The present invention will be further described below in conjunction with embodiments.

[0053] Example 1 The best preparation method of Metaplexis japonica whole herb polysaccharide sample

[0054] 1) Removal of fat-soluble impurities: Take a certain amount of dried Metaplexis japonica whole herb and crush it. The powder is soaked in 95% ethanol overnight, and the ethanol is volatilized and dried (55 °C).

[0055] 2) Single-factor investigation: Fix the reaction conditions as extraction temperature 80°C, extraction time 2 h, and extraction times 2 times, and investigate the effects of different solid-liquid ratios (1:10, 1:15, 1:20, 1:25, 1:30) on the polysaccharide yield; fix the reaction conditions as solid-liquid ratio 1:15, extraction time 2 h, and extraction times 2 times, and investigate the effects of different temperatures (50°C, 60°C, 70°C, 80°C, 90°C) on the polysaccharide yield; fix the reaction conditions as solid-liquid ratio 1:15, extraction temperature 80°C, and extraction times 2 times, and investigate the effects of different extraction times (1 h, 2 h, 3 h, 4 h, 5 h) on the polysaccharide yield; fix the reaction conditions for each time as solid-liquid ratio 1:15, extraction temperature 80°C, and extraction time 2 h, and investigate the effects of different extraction times (1 - 5 times) on the polysaccharide yield. Conduct single-factor experiments to investigate the effects of various factor variables on the yield of Metaplexis japonica polysaccharide. The experimental results are as Figure 2 shown.

[0056] 3) Response surface method to investigate the optimal preparation method: On the basis of single-factor experiments, select three levels with greater influence on the polysaccharide yield for each factor, establish a four-factor and three-level Box - Benhnken central composite experiment, use the polysaccharide yield as the response value, and code the three levels of each factor as -1, 0, 1, as shown in Table 1. According to the single-factor experimental results, design the experimental scheme using Design-Expert 8.0.6 statistical analysis software and perform extraction according to the method in 2.2.1. Calculate the polysaccharide extraction rate in turn. Among them, there are 24 factorial experimental points and 5 central experimental points to calculate the experimental error. Conduct response surface analysis on the experimental results, and conduct multiple parallel experiments on the obtained optimal extraction method to verify the accuracy of the response surface method. The experimental results are as Figure 3 shown. After regression analysis, the functional relationship between the extraction rate Y and each independent variable is:

[0057] Y = 5.03 + 0.3367A + 0.7008B + 0.2558C + 0.3033D + 0.3050AB +

[0058] 0.0600AC + 0.0700AD - 0.200BC + 0.0925BD + 0.1125CD - 0.5665A 2 - 0.6367B 2 - 0.2868C 2 - 0.3230D 2 ;

[0059] Y has an actual maximum value of 4.986% when the solid-liquid ratio is 1:15, the extraction temperature is 80°C, the extraction time is 3 h, and the extraction times are 2 times.

[0060] 4) Alcohol precipitation: Combine the extracted liquids and filter, then concentrate under reduced pressure until slightly viscous. While stirring, add anhydrous ethanol to the extract for alcohol precipitation until the final ethanol concentration reaches 70%. Leave it overnight at 4°C, centrifuge at 7000 rpm for 30 min, and vacuum-dry the resulting precipitate to obtain crude Metaplexis japonica polysaccharide.

[0061] 5) Removal of protein impurities: Dissolve the obtained powder in 400 ml of distilled water, prepare 100 ml of Sevag solution [chloroform:n-butanol = 4:1 (v:v)], add it to the crude polysaccharide solution, shake well for 40 min, centrifuge for 10 min (7500 r / min) to separate the layers, and use a dropper to suck out the upper aqueous phase. Repeat this process 5 times. Freeze-dry the solution obtained by sucking after the fifth centrifugation to obtain crude polysaccharide with protein impurities removed.

[0062] 6) Removal of pigments: Dissolve the crude polysaccharide powder after removing protein impurities above in 500 ml of distilled water, weigh about 15 g of activated carbon powder with a mass percentage of 3% and add it to the solution, put in a magnetic stir bar, stir well in a water bath at 80°C for 1 h, centrifuge for 10 min (8000 r / min), pour out the upper clear liquid, and discard the solid. Repeat this process 5 times. Freeze-dry the finally obtained solution to obtain a basically pure crude polysaccharide powder, named LMPC, and store it at 4°C.

[0063] 7) Detection of polysaccharide content: Use the sulfuric acid-phenol method to determine the content of crude polysaccharide from the whole herb of Metaplexis japonica with glucose as the standard.

[0064] 8) Fractionation of polysaccharides by chromatography column: Separate using a DEAE-52 cellulose chromatography column (Φ2 cm × 40 cm), elute with distilled water at a flow rate of 1.5 ml / min, collect 10 ml per tube fractionally, detect the sugar content in each tube by the sulfuric acid-phenol method, draw an elution curve, collect the elution peaks, and freeze-dry to obtain 10.034 g of Metaplexis japonica whole herb polysaccharide LMP-1. The specific process is shown in Figure 3 and the elution results are shown in Figure 4 。

[0065] Example 2 Chemical Structure Identification of Metaplexis japonica Whole Herb Polysaccharide

[0066] Molecular weight

[0067] Precisely weigh the polysaccharides LMP-1, LMPA-1, LMPA-2 in the present invention and dextran standard products with different molecular weights (molecular weight 1000, 5000, 12000, 25000, 50000, 80000, 150000, 270000, 410000, 670000 series analysis standard products), make a standard curve through high-performance gel permeation chromatography (HPGPC) tandem column analysis, and determine the purity and relative molecular weight of the polysaccharides. The results are from Figure 5It can be seen that LMP-1 peaks at 44 min with good homogeneity. Combining with the standard curve, the molecular weight of LMP-1 is calculated to be 1679 Da, the molecular weight of LMPA-1 is 1752 Da, and the molecular weight of LMPA-2 is 1779 Da.

[0068] Monosaccharide composition

[0069] After the LMP-1 in the present invention is hydrolyzed into monosaccharides and derivatized with PMP to obtain derivatives, through HPLC analysis, the hydrolysis product of LMP-1 contains a main peak, and its retention time is as Figure 6 shown, indicating that LMP-1 is composed of one kind of monosaccharide. The monosaccharide standard was operated in parallel, and the peak time of the standard was consistent with the peak time of the main peak in the LMP-1 chromatogram, indicating that LMP-1 is composed of mannose.

[0070] Infrared spectrum

[0071] Take 2 mg of the dried and constant-weight Cynanchum bungei polysaccharide LMP-1, add 200 mg of dried KBr crystals, grind and mix them, then press into tablets for infrared absorption spectrum analysis, as shown in Figure 7 . The infrared absorption peak at 1102 cm -1 in the infrared spectrum of LMP-1 reflects the pyranose ring structure of LMP-1. The absorption peak at 2930 cm -1 is the absorption peak of C-H, and the absorption peaks in this region are characteristic peaks of carbohydrates. The absorption peak at 3236 cm -1 is the stretching vibration absorption peak of hydroxyl groups.

[0072] Nuclear magnetic resonance hydrogen spectrum

[0073] Take about 30 mg of LMP-1 in the present invention after freeze-drying, dissolve it in 0.5 ml of D 2 O after vacuum drying at room temperature, and detect the 1 H-NMR spectrum at 25 °C in a nuclear magnetic resonance instrument. As shown in Figure 8 , the hydrogen spectrum signals are concentrated between 3 and 4 ppm. The H signal attribution of LMP-1 is shown in Table 1.

[0074] Based on the above analysis, the following results can be obtained:

[0075] The results of molecular weight determination show that LMP-1 in the present invention is a homogeneous polysaccharide with a molecular weight of about 1679 Da;

[0076] The results of monosaccharide composition analysis show that LMP-1 is a mannan;

[0077] The infrared results show that the absorption peaks of LMP-1 in this region are 3236 cm -1 , 2931 cm -1 , 1576 cm-1 、1398 cm -1 、1102 cm -1 , indicating that it is a pyranose ring;

[0078] NMR analysis showed that the polysaccharide structure of LMP-1 is β-D-Manp-(1→[→4)-β-D-Manp-(1→] 8 →6)-β-D-Manp;

[0079] Therefore, the structural derivation result of the whole grass polysaccharide LMP-1 of Metaplexis japonica in the present invention is as Figure 9 , which is a novel and simple mannan isolated from the whole grass of Metaplexis japonica for the first time.

[0080] Example 3

[0081] Materials and Reagents: Whole grass polysaccharide LMP-1 of Metaplexis japonica (self-made, detected as a homogeneous polysaccharide by HPLC), PBS (Servicebio), CCK8 (Service bio), RPMI medium (Service bio), DMEM medium (Service bio).

[0082] Cytotoxic effects of three polysaccharides on various tumor cells and immune cells

[0083] RAW264.7 cells, B16F10 cells, 4T1 cells, MC38 cells, MCF-7 cells, HepG2 cells, and MDA-MB-231 cells in the logarithmic growth phase were trypsinized for about 1 min, then centrifuged to remove the supernatant, added with medium and diluted, and then seeded in 96-well plates at the corresponding densities (except for the RAW264.7 cell line seeded at a density of 5000 cells / well, other cell lines were seeded at a density of 3000 cells / well). After that, they were incubated in an incubator at 37 °C and 5% CO2 for 12 h. After 12 h, the medium was discarded, and 200 μl of medium prepared with different concentrations of the whole grass polysaccharide LMP-1 of Metaplexis japonica (final concentrations of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mg / ml) was added to each well. After incubation for a period of time, 20 μl of CCK8 solution was added, and then incubated in the incubator for another 1 h. The absorbance (Abs) of each well was measured at a wavelength of 450 nm.

[0084] The cell survival rate was calculated according to the following formula.

[0085]

[0086] The whole grass polysaccharide LMP-1 of Metaplexis japonica obtained in the present invention was subjected to the above in vitro anti-tumor activity detection. As Figure 10The results showed that: The inhibition rates of the whole grass polysaccharide of Metaplexis japonica (Thunb.) Makino LMP-1 (0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, 2.0 mg / ml) on the six kinds of tumor cells used in the experiment were all about 20% and above after treating the cells for 72 hours, showing extremely significant differences compared with the control group (P<0.001); for the RAW264.7 immune cells treated for 24 h, it showed a significant proliferation-promoting effect. As Figure 11 The results showed that LMPA-1 and LMPA-2 also had anti-proliferative activities against a variety of tumor cells, but the activities were not as good as those of the whole grass polysaccharide of Metaplexis japonica (Thunb.) Makino LMP-1.

[0087] Table 1 Assignment of each signal in the 1H NMR spectrum of the whole grass polysaccharide of Metaplexis japonica (Thunb.) Makino LMP-1

[0088]

[0089] Animal experiment of the polysaccharide of Metaplexis japonica (Thunb.) Makino LMP-1 and its results

[0090] 1) Establishment of tumor-bearing mouse model

[0091] The mouse melanoma B16F10 cell line was cultured routinely. After the cells entered the logarithmic growth phase, the cell density was resuspended to 5×10 5 cells / mL, and then 100 μL was taken and inoculated subcutaneously into the axilla of the right forelimb of the mouse to establish a melanoma mouse model.

[0092] 2) Grouping and administration

[0093] On the 7th day after inoculating the tumor cells, the tumor-bearing mice were randomly divided into 3 groups, with 4 mice in each group. Experimental grouping: negative control group (PBS), positive drug cyclophosphamide group (40 mg / kg), polysaccharide of Metaplexis japonica (Thunb.) Makino LMP-1 group (40 mg / kg). Intraperitoneal injection was given once a day for 10 consecutive days. The mice were weighed every day, and the tumor volume was measured.

[0094]

[0095] 3) Determination of physiological and biochemical indexes of mice

[0096] After the last administration for 24 h, the mice were weighed. After taking whole blood from the orbital cavity, the serum was separated, and the levels of cytokines IFN-α, TNF-γ, IL-4, and IL-6 were detected using an ELISA kit. All the mice were sacrificed by cervical dislocation, and the main organs of the mice were separated and weighed, including the heart, liver, spleen, lung, and kidney. Then the tumor mass was separated and weighed; the tumor mass was peeled off and weighed, and the tumor inhibition rate and immune organ index were calculated.

[0097]

[0098] 4) Experimental results

[0099] The change curve of the body weight of mice with the number of days of drug administration is as Figure 12 shown. It can be seen that the body weight of mice in the positive drug group decreased. This may be due to the side effects such as reduced appetite of tumor-bearing mice caused by the chemotherapy drug cyclophosphamide during the process of inhibiting tumors. Compared with the positive group, the change in the body weight of tumor-bearing mice in the LMP-1 group was smaller before and after, indicating that LMP-1 has very few side effects on mice and can prevent the further development of tumors without damaging the normal tissues of the body as much as possible.

[0100] The organ indices of tumor-bearing mice in each group are as Figure 13 shown. It can be seen that the hearts, livers, and kidneys of mice in the positive drug group were significantly enlarged, and obvious lesions could also be seen during dissection. The spleen was significantly shrunk, indicating that the side effects of the positive drug cyclophosphamide were obvious, while there were no significant differences in the various indices of mice in the LMP-1 group, indicating that LMP-1 has very few side effects on the organs of mice.

[0101] The comparison chart of the tumor volumes of tumor-bearing mice in each group is as Figure 14 shown. It can be seen that the tumor volumes of tumor-bearing mice in each group from high to low are: PBS group > LMP-1 group > positive drug group. As shown in the figure, compared with the PBS model group, the tumor volume of mice in the LMP-1 administration group also decreased significantly, indicating that this polysaccharide has a significant anti-tumor effect.

[0102] The comparison chart of the tumor weights of tumor-bearing mice in each group is as Figure 15 shown. It can be seen that the tumor weights of tumor-bearing mice in each group from high to low are: positive drug group > LMP-1 group > PBS group. As shown in the figure, compared with the PBS model group, the tumor weight of mice in the LMP-1 administration group decreased significantly (P < 0.01), indicating that this polysaccharide has a significant anti-tumor effect.

[0103] The comparison chart of the cytokine contents of tumor-bearing mice in each group is as Figure 16 shown. It can be seen that compared with the positive drug group, the content levels of TNF-α, IFN-γ, IL-4, and IL-6 in the serum of mice in the LMP-1 group increased significantly (P < 0.01), and the increase in the content level of IL-4 was the most obvious, indicating that LMP-1 may inhibit tumor growth by stimulating the body's immune response and increasing the expression level of IL-4.

[0104] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A polysaccharide from the whole herb of Asclepias rapa LMP-1 having anti-tumor activity, characterized in that: Its structural formula is:

2. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 1, characterized in that: The steps include: After the whole herb of Radix Corydalis is dried and crushed, it is soaked in ethanol for a set time. After the soaking is completed, the ethanol is volatilized, and the whole herb is dried to obtain the pre-treated Radix Corydalis powder; The pretreated whole herb powder of Radix Astragali is extracted with hot water for a set time, solid-liquid separation is performed, the extract is evaporated and concentrated, and then anhydrous ethanol is added thereto for alcohol precipitation, and the obtained precipitate is the crude polysaccharide of Radix Astragali; Dissolving the crude polysaccharide of Radix Astragali in water, adding Sevag solution thereto, shaking, centrifuging, removing protein impurities, and obtaining the crude polysaccharide; The crude polysaccharide is dissolved in water, activated carbon is added thereto, and after stirring, the solid and liquid are separated; The obtained polysaccharide solution was passed through a DEAE-52 cellulose chromatography column and then eluted with water to obtain the whole herb polysaccharide LMP-1 of Radix Astragali.

3. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 2, characterized in that: After the whole herb of Radix Astragali is dried and crushed, the ethanol concentration is 90%-97% in the ethanol soaking step, where % is the mass percentage; Preferably, the ethanol soaking time is 8-20 hours.

4. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 3, characterized in that: After soaking, the ethanol is evaporated and the drying temperature is 50-60°C.

5. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 2, characterized in that: When hot water extraction is performed, the temperature of the hot water is 70-90°C and the extraction time is 2-5h; Preferably, the temperature of the hot water is 75-85°C and the extraction time is 2-4h; Preferably, the temperature of the hot water is 80°C and the extraction time is 3h; Preferably, during hot water extraction, the solid-liquid ratio is 1:10-20, preferably 1:

15.

6. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 2, characterized in that: When alcohol precipitation is carried out, the ethanol concentration in the alcohol precipitation system is 60%-80%, where % is the mass percentage.

7. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 6, characterized in that: When alcohol precipitation is carried out, the ethanol concentration in the alcohol precipitation system is 65%-75%, where % is the mass percentage.

8. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 7, characterized in that: When alcohol precipitation is carried out, the ethanol concentration in the alcohol precipitation system is 70%, where % is the mass percentage.

9. The method for extracting LMP-1 polysaccharide from the whole herb of Asclepias rapa with anti-tumor activity according to claim 2, characterized in that: In the Sevag solution, the volume ratio of chloroform to n-butanol is 4:

1.

10. Use of the whole herb polysaccharide LMP-1 with anti-tumor activity as claimed in claim 1 in the preparation of anti-tumor drugs or tumor vaccine adjuvants.

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