CN118085120A - Qihong polysaccharide KBPW1 with anti-tumor activity and application thereof in preparation of anti-tumor drugs - Google Patents
Qihong polysaccharide KBPW1 with anti-tumor activity and application thereof in preparation of anti-tumor drugs Download PDFInfo
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- CN118085120A CN118085120A CN202410402982.0A CN202410402982A CN118085120A CN 118085120 A CN118085120 A CN 118085120A CN 202410402982 A CN202410402982 A CN 202410402982A CN 118085120 A CN118085120 A CN 118085120A
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Abstract
The invention discloses a Qihong polysaccharide KBPW1 with anti-tumor activity and application thereof in preparing anti-tumor drugs, and relates to the technical field of plant polysaccharide active substances and application thereof. The repeatable primary structural unit of the keemun polysaccharide KBPW contains →4)-β-D-Glcp-(1→、α-L-Araf-(1→、→3)-β-D-Galp-(1→、→5)-α-L-Araf-(1→、→3,5)-β-L-Araf-(1→、β-D-Glcp-(1→、→4,6)-β-D-Glcp-(1→、→6)-β-D-Glcp-(1→ eight glycosidic bonds, which consists of arabinose, galactose and glucose, wherein the molar ratio of the arabinose to the galactose to the glucose is 2.62:1.02:6.36; the relative molecular mass is 3.8X10 3 Da. The beneficial effects are that: as an anticancer active ingredient, the composition can obviously inhibit the growth, proliferation and migration invasion of colorectal cancer CT26WT cells, reduce the tumor volume and weight of CT26WT tumor-bearing mice, has the characteristics of small toxic and side effects on organisms, good biocompatibility, strong specificity, low toxic and side effects and the like, can make up the defects of the traditional cancer treatment means, and can be used for developing new anticancer drugs.
Description
Technical Field
The invention relates to the technical field of plant polysaccharide active substances and application thereof, in particular to a keemun polysaccharide KBPW1 with anti-tumor activity and application thereof in preparing anti-tumor drugs.
Background
Tumors have been one of the major public health problems worldwide, severely compromising human health. In recent years, the morbidity and mortality of tumors are gradually increased, and according to the global cancer statistics report in 2020, 457 cases of new cancer cases and 300 cases of death cases are newly increased in China every year, accounting for 23.7% of the world, and the new cases and the death population of the world are far beyond those of other countries. The traditional treatment method of the tumor at present mainly adopts the combination of surgery, chemotherapy and radiotherapy, but the curative effect is not ideal, the toxic and side effects of the traditional anti-tumor medicine are large, normal cells and immune cells of a body are destroyed while tumor cells in a patient are killed, various tissues and organs are damaged, the prognosis is poor, and the malignant progress is rapid. Therefore, there is a need to develop new and highly targeted cancer treatment methods and drugs to improve the quality of life and survival time of cancer patients, and to find safe, efficient and specific natural antitumor new drugs gradually becomes the goal of the middle and outer scientific research workers.
The Qimen black tea (hereinafter called Qimen for short) is one of three-major high-aroma black tea in the world and ten-major famous tea in China, and is mainly produced in the Qimen county of Huang Shanshi Anhui province in China, and is popular among consumers all the time because the special fragrance like flowers, fruits and honey, namely the fragrance like Qimen, is known as the fragrance of group. The literature records that the medical history of the keemun has more than one hundred years, and the long-term drinking of the tea has various health effects of nourishing stomach, protecting stomach, promoting the production of body fluid, clearing heat, promoting urination, diminishing inflammation, sterilizing, refreshing, relieving fatigue, resisting aging, preventing cardiovascular and cerebrovascular diseases and the like. Modern pharmacological researches show that tea polysaccharide is a key component of tea to exert various biological activities, and has remarkable curative effects on aspects of resisting tumor, improving immunity, resisting oxidation and the like. The chemical structure of tea polysaccharide is the material basis for exerting biological activity, while keemun has different structure and physiological activity from other tea leaves due to the special fermentation process and production place, and deserves intensive research.
At present, the main active components of the keemun polysaccharide are not clear, the active substance basis and the efficacy of the keemun polysaccharide are still to be clear, meanwhile, the fine chemical structure of the keemun polysaccharide is not reported, and the keemun polysaccharide with the anticancer activity, which has the clear chemical structure and uniform composition, is prepared by a suitable extraction and purification process, so that the research and development of novel anticancer drugs of the keemun polysaccharide are limited.
The Chinese patent application document with publication number of CN105859903A discloses a radix glehniae polysaccharide, a preparation method and application thereof, and relates to the technical field of polysaccharides. The molecular weight of the glehnia littoralis polysaccharide is 26.3kDa; the monosaccharide composition and the molar ratio of the glehnia littoralis polysaccharide are as follows: rhamnose: galactose: glucose = 2.05:1.00:7.06; and provides a primary structure repeating unit of the glehnia littoralis polysaccharide. The radix Glehniae polysaccharide has the advantages of simple extraction process, high component purity, good anti-tumor and antioxidant activity, and small toxic and side effects. However, the patent does not disclose the keemun polysaccharide KBPW1 and its use according to the present invention.
Disclosure of Invention
The technical problem to be solved by the invention is how to provide the keemun polysaccharide with anti-tumor activity.
The invention solves the technical problems by the following technical means:
in a first aspect of the present invention, a keemun polysaccharide KBPW with anti-tumor activity is provided, wherein a repeatable primary structural unit of the keemun polysaccharide KBPW1 contains →4)-β-D-Glcp-(1→、α-L-Araf-(1→、→3)-β-D-Galp-(1→、→5)-α-L-Araf-(1→、→3,5)-β-L-Araf-(1→、β-D-Glcp-(1→、→4,6)-β-D-Glcp-(1→、→6)-β-D-Glcp-(1→ eight glycosidic bonds; the connection mode of the glycosidic bond is shown as formula 1:
The beneficial effects are that: the keemun polysaccharide KBPW-1 provided by the invention has novel structure, definite chemical structure and uniform composition, and the in-vitro and in-vivo experiments of cells prove that the keemun polysaccharide KBTP-1 has good anti-tumor effect.
Preferably, the relative molecular mass of the keemun polysaccharide KBPW is 3.8X10 3 Da.
Preferably, the keemun polysaccharide KBPW is composed of arabinose, galactose and glucose, and the molar ratio of the arabinose, galactose and glucose is 2.62:1.02:6.36.
The second aspect of the invention provides an application of the Qihong polysaccharide KBPW1 in preparing an anti-tumor medicament.
Preferably, the tumor comprises colorectal cancer (CRC), cholangiocarcinoma, gastric cancer, glioblastoma (GBM), leiomyosarcoma, melanoma, lung cancer, neuroblastoma (NB), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, breast cancer, or any combination thereof.
The third aspect of the present invention provides an antitumor pharmaceutical composition, which comprises the above-mentioned keemun polysaccharide KBPW.
Preferably, the composition further comprises one or more pharmaceutically acceptable carriers.
Preferably, the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant.
Preferably, the pharmaceutically acceptable carrier is a diluent.
Preferably, the oral dosage form of the pharmaceutical composition comprises a tablet, a capsule, a lozenge, an oral liquid, a granule, a pill or a powder.
Compared with the prior art, the invention has the beneficial technical effects that:
1. The Qihong polysaccharide KBPW1 provided by the invention has novel structure, definite chemical structure and uniform composition, and the in-vitro and in-vivo experiments of cells prove that the Qihong polysaccharide KBPW1 has good anti-tumor effect, can relieve weight loss caused by 5-Fu, improve the immune organ index and liver function of mice, and overcomes the defect of large side effect of the existing chemical anticancer drugs on organisms.
2. The Qihong polysaccharide KBPW1 provided by the invention can obviously inhibit the growth, proliferation and migration invasion of colorectal cancer CT26 WT cells, the tumor growth of tumor-bearing mice given with the polysaccharide in animal experiments can be effectively inhibited, the tumor inhibition rate can reach 71.26%, and the vital signs of the tumor-bearing mice are still obviously better than those of control groups without the polysaccharide and the positive drug 5-FU when the administration concentration reaches high concentration of 200 mg/kg.
3. The keemun polysaccharide has good anti-tumor activity in vitro and in vivo, can obviously inhibit the growth and proliferation of colorectal cancer, has no toxic or side effect, and has not been reported at present.
4. The invention provides a brand-new keemun polysaccharide compound, which has a definite chemical structure, and through high performance liquid chromatography (HPGPC) analysis, the keemun polysaccharide prepared by the method has only one absorption peak, has the molecular weight of 3.8X10 3 Da, has stronger anti-tumor activity, and can be used as a novel active ingredient for preparing anti-colorectal cancer drugs.
5. The method for preparing the active polysaccharide has high efficiency and stable activity.
Drawings
FIG. 1 is a graph showing the effect of the feed to liquid ratio (A), temperature (B) and time (C) on the yield of Keemun polysaccharide in example 1 of the present invention;
FIG. 2 is a response surface analysis chart of the factor design of the preparation process of the Keemun polysaccharide in example 1 of the invention; a-B: feed liquid ratio and temperature; C-D: feed liquid ratio and time; E-F: time and temperature;
FIG. 3 is a graph showing the elution of the Qihong polysaccharide KBPW1 in example 2 of the present invention; a is DEAE-Sepharose Fast Flow ion exchange chromatographic column elution curve of Qihong polysaccharide KBPW; b is the elution curve of a Sephadex G-100 gel column of the Qihong polysaccharide KBPW;
FIG. 4 is an Ultraviolet (UV) full-wavelength scan (A) and HPGPC molecular weight chromatogram (B) of Qihong polysaccharide KBPW1 of example 3 of the present invention;
FIG. 5 is a Fourier infrared spectrum (FT-IR) of the Qihong polysaccharide KBPW of example 3 of the invention;
FIG. 6 is a Scanning Electron Microscope (SEM) image of the Qihong polysaccharide KBPW1 of example 3 of the present invention;
FIG. 7 is a diagram showing the monosaccharide composition Ion Chromatography (IC) of the Qihong polysaccharide KBPW1 of example 3 of the present invention;
FIG. 8 is a chart of a glycosidic bond type mass spectrum (GC-MS) of the Qihong polysaccharide KBPW1 of example 4 of the present invention;
FIG. 9 is a nuclear magnetic resonance spectrum of the Qihong polysaccharide KBPW1 of example 4 of the present invention; a is 13 C. Carbon spectrum; b is 1 H hydrogen spectrum; c is HSQC spectrum; d is 1H-1 H COSY spectrum; e is HMBC spectrum;
FIG. 10 is a graph showing the inhibition of CT26 WT colorectal cancer cells by Qihong polysaccharide KBPW1 of example 5 of the present invention; wherein A is the inhibition of the proliferation of CT26-WT cells by Qihong polysaccharide KBTP-1; b is a migration condition diagram of cell scratches after carrying out treatment on the Qihong polysaccharide for 0, 24 and 48 hours by KBTP-1 with different concentrations;
FIG. 11 is a graph showing the tumor-inhibiting effect of the Qihong polysaccharide KBPW1 of example 6 on CT26 WT colorectal cancer tumor-bearing mice.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The test materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Those of skill in the art, without any particular mention of the techniques or conditions, may follow the techniques or conditions described in the literature in this field or follow the product specifications.
Example 1: response surface optimized keemun polysaccharide extraction process
(1) Removing impurities, taking proper amount of tea leaves, crushing by a crusher, sieving by a 80-mesh sieve, and drying and preserving in dark place for standby; accurately weighing tea powder, adding 95% ethanol according to a feed-liquid ratio of 1:20g/mL, stirring at 25deg.C and a rotation speed of 120rpm/min for 24h, suction filtering, collecting precipitate, and drying in a 55 deg.C oven to obtain decolorized and defatted dry tea powder;
(2) And (3) a single factor test, namely respectively setting the factors such as the feed liquid ratio (1:10, 1:20, 1:30, 1:40, 1:50 g/mL), the time (1, 1.5, 2, 2.5, 3 h), the temperature (60, 70, 80, 90, 100 ℃) and the like of the extracted keemun polysaccharide, taking the yield of the keemun polysaccharide as an index, examining the influence of various factor variables on the extraction rate of the keemun polysaccharide, and determining the optimal extraction process parameters.
(3) And adopting Box-Benhnken L 17(33) Design test scheme in Design-Expert 13 software to extract the feed liquid ratio, temperature and time of the Keemun polysaccharide as dependent variables and the polysaccharide yield as variables for response surface analysis. Each independent variable level was coded with-1, 0, 1, respectively, and experimental factors and level designs are shown in Table 1.
Table 1 coding level of response surface test independent variables
(4) Single factor experimental results:
As shown in fig. 1A, as the feed-to-liquid ratio increases, the extraction rate of the keemun polysaccharide tends to increase, and the extraction rate reaches the highest value at a feed-to-liquid ratio of 1:30g/mL, but as the feed-to-liquid ratio continues to increase, the polysaccharide yield starts to decrease. It is presumed that when the feed liquid is relatively low, the polysaccharide is not completely swelled, the extract is difficult to separate, the polysaccharide is affected to be precipitated, when the feed liquid is relatively high, the solution becomes thin, the intermolecular adsorption is enhanced, thereby causing the reduction of the polysaccharide extraction rate and the increase of the subsequent treatment cost of the solvent. Therefore, the optimal feed-to-liquid ratio is 1:30g/mL.
FIG. 1B shows that as the extraction temperature increases, the yield of the Keemun polysaccharide tends to increase and then decrease, and the polysaccharide yield reaches a maximum when the temperature is 90 ℃. The diffusion coefficient of polysaccharide increases with increasing extraction temperature, so that the content of polysaccharide in the aqueous solution increases. However, excessive temperatures can cause degradation of the polysaccharide, resulting in reduced polysaccharide yields, while increasing energy consumption. Therefore, the optimum extraction temperature is 90 ℃.
FIG. 1C shows that polysaccharide extraction rate showed a slow increasing trend with increasing extraction time, with a maximum polysaccharide yield of 5.35.+ -. 0.35% when time was 2.5h, but polysaccharide yield began to decrease after time exceeded 2.5h. This is because the extraction time is prolonged to make the dissolution of polysaccharide into the solvent simpler and faster, but too long an extraction time also results in degradation of polysaccharide, which reduces its yield. Therefore, the optimal extraction time is 2.5h.
(5) Response surface test results, on the basis of single factor test, the response surface test of L 17(33) is carried out by taking the liquid-material ratio, the extraction temperature and the extraction time as test factors, the extraction process of the Qihong polysaccharide is optimized, and the test design and the result are shown in Table 2. Performing multiple regression fitting on the data of table 2 by using Design-Expert 13.0 software to obtain a quadratic multiple regression equation of the yield (Y) of the keemun polysaccharide to the feed-liquid ratio (A), the temperature (B) and the time (C):
Y=7.48+0.06400A+0.3563B+0.2013C-0.1750AB-0.0450AC+0.1475BC-0.6878A2-0.91
53B2-1.01C2。
TABLE 2 response surface test factor level and results
TABLE 3 analysis of variance
Note that: * p <0.05, representing a significant difference; * P <0.01, representing a very significant difference
Analysis of variance was performed on the regression mathematical model to verify the validity of the equation and the partial regression coefficient of each factor, as shown in table 3, the model selected for the test was extremely significant (P < 0.01), the mismatching term of variance was not significant (p=0.5787 > 0.05), and it was demonstrated that the influence of the non-test factors on the test results was not great. In addition, the value of the decision coefficient R 2 of the selected model is 0.9760, which indicates that the model is better fitted with an actual test, the value of the corrected decision coefficient R 2 Adj is 0.9451 and is close to the value of R 2, and the model has sufficient accuracy and universality. The influence of each factor on polysaccharide yield can be seen from the F value as: feed to liquid ratio > temperature > time. It can also be seen from the table that A, B, A 2、B2、C2 has a very significant effect on polysaccharide yield (P < 0.01), and that C has a significant effect on polysaccharide yield (P < 0.05); the effect of other factors is insignificant.
The response curve and contour diagram of interaction among the factors of the regression model are shown in fig. 2, and the optimal extraction process of the keemun crude polysaccharide obtained by the regression model comprises the following steps: the feed liquid ratio is 1:34.418g/mL, the temperature is 91.6057 ℃ and the time is 2.55097 hours, under the condition, the extraction rate of polysaccharide reaches 7.65811 percent, the feasibility is considered from the practical operation point of view, and the optimal extraction parameters are set as follows according to the model result and the practical situation: the feed liquid ratio is 1:30g/mL, the temperature is 90 ℃ and the time is 2.5 hours, the average yield of the obtained crude polysaccharide is 7.48+/-0.22%, and the average yield is basically consistent with the predicted result. The results show that optimal polysaccharide extraction process parameters can be obtained by using the method.
Example 2: preparation and purification of keemun polysaccharide
(1) Uniformly mixing the decolorized and defatted tea powder obtained in the example 1 with distilled water according to a feed liquid ratio of 1:30g/mL, stirring and extracting at 80 ℃ and a rotation speed of 120rpm/min for 2.5 hours, filtering, collecting filtrate, repeating for 2 times, and combining 3 times of filtrate to obtain a tea water extract;
(2) Concentrating the tea water extract in a rotary evaporator (35 rpm/min,55 ℃ and 0.09-0.10 MPa) under reduced pressure to 1/4 of the original volume, centrifuging at 4 ℃ and 10000rpm/min for 10min, discarding the precipitate, and collecting the supernatant concentrate; adding 4 times volume of absolute ethanol (to make final concentration of ethanol 80%) into the concentrated solution, precipitating (at room temperature) for 12 hr, filtering with gauze, collecting precipitate, and drying in oven at 55deg.C to obtain tea alcohol precipitate;
(3) Taking a tea alcohol sediment, adding a proper amount of deionized water (100 mL) until the tea alcohol sediment is completely dissolved, adding 1/4 volume (25 mL) of Sevag reagent (chloroform/n-butanol=4/1), magnetically stirring for 30min at room temperature, repeating for 4 times until no white protein layer exists, and collecting a supernatant polysaccharide solution; placing the polysaccharide solution with protein removed in a plate, sealing with a preservative film, freezing in a refrigerator at-20deg.C for 12 hr, and freeze-drying in a vacuum freeze dryer (-50deg.C, 10 Pa) for 48 hr to obtain tea polysaccharide sample;
(4) Using tea polysaccharide sample, preparing 20mg/mL tea polysaccharide solution, eluting with DEAE-Sepharose Fast Flow ion exchange column, sequentially gradient eluting with deionized water and 0.1, 0.2, 0.3, 0.4 and 0.5mol/L NaCl solution at 5mL/min, detecting polysaccharide content in the eluent by phenol-sulfuric acid colorimetric method, collecting sugar-containing eluent to obtain Qihong polysaccharide extract, and drawing elution curve as shown in figure 3A.
(5) Collecting the eluent of the main component deionized water, further purifying by using a Sephadex G-100 gel column, eluting with deionized water at a flow rate of 0.5mL/min, detecting the polysaccharide content in the eluent by a phenol-sulfuric acid colorimetric method, and drawing an elution curve as shown in figure 3B. Collecting polysaccharide-containing eluate, concentrating under reduced pressure, dialyzing, desalting, and vacuum lyophilizing to obtain Qihong polysaccharide component KBPW.
Example 3: physicochemical properties and structural characterization of Qihong polysaccharide KBPW1
(1) Carbohydrate content determination of Qihong polysaccharide KBPW1
The carbohydrate content of KBPW1 obtained in example 1 was determined by the phenol-sulfuric acid method, and the carbohydrate content in KBPW1 was 83.60.+ -. 0.79% by spectrophotometry at 490 nm.
(2) Ultraviolet spectral analysis of Qihong polysaccharide KBPW1
The Qihong polysaccharide KBPW1 is prepared into 0.1mg/mL solution, and is scanned in the wavelength range of 190-400 nm by using a full-wavelength scanning microplate reader, as shown in figure 4A, KBPB 1 has no absorption peak at 260nm and 280nm, which shows that KBPW1 almost contains no pigment, protein and nucleic acid.
(3) Homogeneity and weight average molecular weight determination of keemun polysaccharide KBPW1
The retention time of dextran standards of different molecular weights (5 KD, 25KD, 80KD, 150KD, 420KD, 670 KD) was determined by high performance gel permeation chromatography HPGPC, a molecular weight standard curve was drawn, and then the relative molecular weight was calculated from the standard curve and the peak time of the Qihong polysaccharide KBPW1 sample.
HPGPC detection conditions: chromatographic column: BRT105-103-101 series gel column (8X 300 mm); mobile phase: 0.05M NaCl solution; flow rate: 0.8mL/min; column temperature: 40 ℃; sample injection amount: 25 μl; a detector: differential detector RID-20A.
As a result, as shown in FIG. 4B, the Qihong polysaccharide KBPW is a homogeneous polysaccharide having a weight average molecular weight of: 3.8X10 3 Da.
(4) Apparent morphology analysis of Qihong polysaccharide KBPW1
Analyzing the apparent morphology of the Qihong polysaccharide KBPW by adopting a scanning electron microscope, weighing 2mg of KBPB 1, adhering the obtained product to a metal iron block by using conductive adhesive, spraying a layer of gold on the surface of a polysaccharide sample by using a spraying instrument, and then observing the apparent morphology by using the scanning electron microscope. As a result, as shown in FIG. 6, KBPW a surface was uniformly smooth and exhibited an irregular lamellar structure.
(5) Characteristic group analysis of Qihong polysaccharide KBPW1
Analyzing characteristic groups of the Qihong polysaccharide KBPW mg, the Qihong polysaccharide KBPW and the potassium bromide 100mg by adopting a Fourier infrared spectrometer, uniformly mixing, grinding, drying, fixing and tabletting, and then placing on a Nicolet 67 type Fourier infrared spectrometer for scanning analysis, wherein the scanning range is 4000cm -1-500cm-1. As a result, as shown in FIG. 5, the Qihong polysaccharide KBPW has typical characteristic peaks of polysaccharide, a strong absorption peak is a stretching vibration of-OH at 3405.19cm -1, an absorption peak at 2923.07cm -1 is a stretching vibration of C-H in CH 2 group, an absorption peak at 1617.33cm -1 is a stretching vibration of C=O, and an absorption peak at 1401.24cm -1 is related to a C-H bending vibration; the strong absorption peak at 1100.29cm -1, which is due to the stretching vibration of the pyran ring of the glycosyl residue, is related to the β -and α -glycosidic bonds contained in the polysaccharide at 963.75cm -1 and 640.73cm -1.
(6) Monosaccharide composition analysis of Qihong polysaccharide KBPW1
The monosaccharide composition of the Qihong polysaccharide KBPW is measured by using a Thermo ICS5000 ion chromatograph, 5mg KBPB 1 is precisely weighed and placed in an ampoule bottle, 3mol/L trifluoroacetic acid (TFA) 2mL is added, hydrolysis is carried out for 3h at 120 ℃, the acid hydrolysis solution is accurately sucked and transferred into a tube, after nitrogen is blown dry, 5mL ultra-pure water is added, vortex mixing is carried out, and centrifugation is carried out for 5min at 12000 rpm. Taking supernatant and transferring the supernatant into a chromatographic bottle for testing. And accurately weighing the monosaccharide standard substances of 5mg each, including fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, glucuronic acid, amino galactose hydrochloride, glucosamine hydrochloride, N-acetyl-D glucosamine, guluronic acid and mannuronic acid, and preparing monosaccharide mixed standard solutions with different concentration gradients, determining the mass of different monosaccharides according to an absolute quantitative method and calculating the molar ratio according to the molar mass of the monosaccharides.
IC detection conditions: chromatographic column: dionex Carbopac TM PA20 (3 x 150 mm); mobile phase: h 2 O; b15 mM NaOH; 15mM NaOH&100mM NaOAc; flow rate: 0.3mL/min; sample injection amount: 25 μL; column temperature: 30 ℃; elution gradient: 0min A/B/C (98.8:1.2:0, V/V), 18min A/B/C (98.8:1.2:0, V/V), 20min A/B/C (50:50:0, V/V), 30min A/B/C (50:50:0, V/V), 30.1min A/B/C (0:0:100, V/V), 46min A/B/C (0:0:100, V/V), 46.1min A/B/C (0:100:0, V/V), 50min A/B/C (0:100:0, V/V), 50.1min A/B/C (98.8:1.2:0, V/V), 80min A/B/C (98.8:1.2:0, V/V). A detector: an electrochemical detector.
As a result, as shown in FIG. 7, KBPW1 was composed of arabinose Ara, galactose Gal, and glucose Glc in a molar ratio of 2.62:1.02:6.36.
Example 4: structure identification of Qihong polysaccharide KBPW1
(1) Analysis of the glycosidic bond composition of Qihong polysaccharide KBPW1
And adopting gas chromatography-mass spectrometry (GC-MS) to analyze the type of the glycosidic bond of KBPW1.20 mg KBPB 1, placing the mixture in a glass reaction bottle, adding 1mL anhydrous DMSO, adding a methylating reagent A liquid for sealing according to a methylation kit (BRT-JJH) instruction book, adding a methylating reagent B liquid after ultrasonic dissolution, adding 2mL ultrapure water after stirring in a water bath at 30 ℃ for 60min for reaction, terminating the methylation reaction, and finally concentrating, dialyzing and freeze-drying the reaction liquid to obtain a completely methylated polysaccharide sample.
Taking a methylated polysaccharide sample, placing the sample in an ampoule bottle, adding 1mL of TFA (2 mol/L), hydrolyzing for 90min at 110 ℃, and steaming until the TFA is completely removed, thus obtaining a complete hydrolysate. 2mL of double distilled water and 60mg of sodium borohydride (NaBH 4) are added to the hydrolysate for reduction reaction, glacial acetic acid is added to the hydrolysate for neutralization after 8 hours, after the redundant NaBH4 is removed by rotary evaporation, 1mL of acetic anhydride is added for reaction at 100 ℃ for 1 hour, 3mL of toluene is added after cooling, after the redundant acetic anhydride is evaporated to dryness by decompression concentration, dichloromethane (CH 2Cl2) is added for extracting an acetylation product, and the extract is dried by anhydrous sodium sulfate and then subjected to GC-MS measurement.
GC-MS detection conditions: agilent 7890A-5977B system, column: RXI-5SIL MS (30 m.times.0.25 mm.times.0.25 um); temperature programming conditions: heating to a starting temperature of 120 ℃ at 3 ℃/min to 250 ℃/min; maintaining for 5min; sample inlet temperature: 250 ℃, detector temperature: the carrier gas is helium at 250 ℃ with a flow rate of 1mL/min; ion flame detector, mass scan range (m/z): 50-350.
As a result, as shown in FIG. 8, the KBPW methylation products showed eight methylation derivative ion peaks in the gas chromatograph, and the type of glycosidic bond and the molar ratio of the Qihong polysaccharide KBPW1 were obtained by analyzing each mass spectrum by on-line searching according to the retention time of each peak as shown in Table 4.
(2) Glycoside linkage analysis of Qihong polysaccharide KBPW1
The glycosidic linkage mode of nuclear magnetic resonance analysis KBPW is adopted: 50mg KBPB 1 was dissolved in 1mL of heavy water, the solution was transferred into a nuclear magnetic resonance tube, and the sample was scanned using AGILENT VNMRS600 superconducting nuclear magnetic resonance spectrometer. One-dimensional 1H-NMR、13 C-NMR spectra and two-dimensional 1H-1 H COSY, HMBC, HSQC spectra were obtained (FIG. 9).
From FIG. 9, it is understood that the chemical shift assignment of 13C、1 H of each sugar residue KBPW is shown in Table 5, and substantially corresponds to the monosaccharide composition and methylation structure. Through the above structural characterization of KBPW, the chemical structural formula of KBPW1 according to the present invention can be obtained as follows:
Table 4KBPW type of glycosidic bond
Sequence number | Retention time (min) | Glycosyl derivatives | Molar ratio of | Glycosidic bond connection mode |
1 | 10.412 | 2,3,5-Me3-Araf | 16.2 | Araf-(1→ |
2 | 15.098 | 2,3-Me2-Araf | 8.5 | →5)-Araf-(1→ |
3 | 16.862 | 2,3,4,6-Me4-Glcp | 4.8 | Glcp-(1→ |
4 | 18.412 | 2-Me1-Araf | 7.2 | →3,5)-Araf-(1→ |
5 | 20.768 | 2,3,6-Me3-Glcp | 45.5 | →4)-Glcp-(1→ |
6 | 21.322 | 2,4,6-Me3-Galp | 11.4 | →3)-Galp-(1→ |
7 | 22.06 | 2,3,4-Me3-Glcp | 2.6 | →6-Glcp-(1→ |
8 | 26.55 | 2,3-Me2-Glcp | 3.8 | →4,6)-Glcp-(1→ |
TABLE 5 chemical shifts of the glycosidic linkages 1 H and 13 C
Example 5: migration inhibition effect of Qihong polysaccharide KBPW1 on CT26 WT colorectal cancer cells in vitro
(1) Cell proliferation assay
The effect of Qihong polysaccharide KBPW1 prepared in example 1 on the activity of CT26 WT of colorectal cancer cells of mice was detected by CCK8, CT26.WT cells in the logarithmic phase of growth were taken, their cell concentration was adjusted to 5X 10 4/mL, inoculated into 96-well plates, 100. Mu.L of cell suspension per well was placed in an incubator at 37℃and 5% CO 2 for wall-attached culture for 24 hours, KBPW1 was added at final concentrations of 50, 100, 200, 400, 600, 800. Mu.g/mL, and a blank medium control group was set at the same time, and after incubation was continued for 24 hours, CCK-8 (10. Mu.L) was added to each well for incubation for 2 hours. Finally, the OD value of each hole is measured by an enzyme-labeled instrument at the wavelength of 450nm, and the effect of the Qihong polysaccharide KBPW on the tumor cell activity is calculated. Cell viability (%) = (OD dosing group-OD blank)/(OD control group-OD blank) ×100% the cell viability of the control group was set to 100%.
As shown in FIG. 10A, after incubation of CT26.WT cells with Qihong polysaccharide KBPW, the inhibitory activity of KBTP-1 on CT26-WT cells increased with increasing KBPW1 concentration, and the cell viability was less than 50% when KBPW concentration was 800. Mu.g/mL, demonstrating that Qihong polysaccharide KBPW1 inhibited proliferation of CT26 WT cells to some extent.
(2) Cell scratch assay: CT26-WT cells were inoculated into 6-well plates at a cell density of 1X 10 6 cells per well, and after 24 hours of adherent culture, vertically and uniformly streaked at the center of each well with a 200. Mu.L gun head, and then cultured in a serum-free medium containing KBPW1 at 100, 200, 400. Mu.g/mL. At time nodes of 0h,24h and 48h of culture, the scratch change condition is observed under a fluorescence inversion microscope, and the change of the CT26-WT cell migration capacity is recorded.
As shown in FIG. 10B, it can be seen that CT26-WT cells have a greatly reduced migration capacity after KBPW's 1 incubation. And the cell migration capacity is reduced along with the increase of KBPW1 concentration, which shows that KBPW can obviously inhibit the migration of colon cancer cells CT26-WT, and the inhibition effect is dose-dependent.
Example 6: research on antitumor activity of Qihong polysaccharide KBPW in vivo
Colorectal cancer CT26 WT cell xenograft is adopted to construct a tumor-bearing mouse model, different doses of Qihong polysaccharide KBPW1 are given to oral gastric lavage, and the anti-tumor activity of KBPW is analyzed.
(1) Colorectal cancer tumor-bearing mouse model construction and drug administration intervention
After 5-week-old male BALB/C mice were adaptively bred in SPF-class animal houses at 23.+ -. 2 ℃ under conditions of relative humidity of 50% -60%, 12h light and 12h darkness for one week, 72 mice were randomly picked up, and CT26 WT colorectal cancer was inoculated to the remaining mice except for the Normal group (Normal, 12 mice), CT26 WT cell suspensions (2X 10 6/mL) in the log phase of growth were extracted by a 1mL microinjector, inoculated to the right underarm of 60 BALB/C mice after sterilization, each inoculated with 0.2mL (2X 10 6/mL), and the injection points were pressed to prevent extracellular leakage, and tumor-bearing mice after CT26.WT colorectal cancer cell inoculation were randomly divided into 5 groups of 12 mice each: model, lavage sterile saline; positive group (Positive, lavage 5-fluorouracil, 20 mg/kg. DBW); KBPW1 low dose group (KBPW-L, 50 mg/kg.dBW), KBPW1 medium dose group (KBPW-M, 100 mg/kg.dBW), KBPW1 high dose group (KBPW-H, 200 mg/kg.dBW), wherein the solvents configuring the Positive and KBPW1 groups were sterile physiological saline. Each group was given by oral gavage 1 time a day, and the mice were sacrificed by CO 2 asphyxiation and tested for relevant indicators at a gavage volume of 0.1mL/10g for 15 days continuously.
(2) Detection index of in-vivo antitumor activity of mice
General observations: the mice were observed daily for their mental state, activity, hair gloss, etc.
Body weight and diet drinking water detection: during the experiment, from day 0 of starting the gavage to day 15 of ending the gavage, the weight, diet and drinking water changes of each group of mice were monitored and recorded every 3 days at regular intervals, and weight and diet change curves were plotted.
Determination of tumor volume: during the period of gastric lavage administration, the length and the diameter of the tumor of the mice are measured by a vernier caliper every 3 days, and the growth condition of the tumor of the mice is monitored and recorded regularly; the tumor volume was calculated according to the formula v=0.5ab 2 (long diameter of a-tumor, short diameter of b-tumor), the tumor volume change curve was drawn, and the relative volume of tumor and relative tumor proliferation rate were calculated.
Tumor inhibition rate assay: after the gastric lavage administration treatment is finished, the axillary tumor of each group of mice is dissected and taken out, and the tumor inhibition rate is calculated after weighing, measuring and photographing. Tumor inhibition% = (average tumor weight of 1-administration group/average tumor weight of model control group) ×100%
Measurement of organ index: after the experiment, the thymus of the mice was taken out, the physiological saline was rinsed, the filter paper was dried with the physiological saline, and then the samples were sequentially weighed, and the thymus index= (thymus weight/animal weight) ×100%.
(3) Experimental results
In vivo experiments of anti-CT 26 WT colorectal cancer tumor-bearing mice of the Qihong polysaccharide KBPW1 show that the volume and weight of the right armpit tumor mass of the mice in the model control group are increased and enlarged continuously along with the time, and the KBPW1 administration group can obviously inhibit the tumor growth of the tumor-bearing mice, and the tumor inhibition effect is a concentration dependent effect; although the mice of the 5-FU positive administration group showed higher tumor inhibiting activity, the mice had obvious side effects, which resulted in weight loss, decreased immune organ index, obvious decline of vitality and poor physiological condition (Table 6 and FIG. 11). Further, the results of statistical analysis of the tumor inhibition rates of different groups show that the tumor inhibition rate of the high-dose group of the Qihong polysaccharide KBPW on CT26 WT colorectal cancer tumor-bearing mice reaches 71.26 +/-4.34%, and the immune organ thymus index of the tumor-bearing mice can be obviously improved. The results show that the Qihong polysaccharide KBPW has good in vivo effect of resisting CT26 WT colorectal cancer, and the toxic and side effects are far smaller than those of the traditional antitumor drug 5-FU, so that the influence on the physiological state of mice is small, and adverse reactions are avoided after long-term use.
Table 6 inhibition of tumor growth in CT26 WT colorectal cancer tumor-bearing mice by Qihong polysaccharide KBPW1
Note that: the data in the table are **P<0.01,* P <0.05, representing a comparison with the model set; ##P<0.01,# P <0.05, indicating comparison with the normal group.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (10)
1. A keemun polysaccharide KBPW with anti-tumor activity, wherein the repeatable primary structural unit of the keemun polysaccharide KBPW contains →4)-β-D-Glcp-(1→、α-L-Araf-(1→、→3)-β-D-Galp-(1→、→5)-α-L-Araf-(1→、→3,5)-β-L-Araf-(1→、β-D-Glcp-(1→、→4,6)-β-D-Glcp-(1→、→6)-β-D-Glcp-(1→ eight glycosidic linkages; the connection mode of the glycosidic bond is shown as formula 1:
2. The antitumor active muntin polysaccharide KBPW a 1 according to claim 1, wherein the muntin polysaccharide KBPW a has a relative molecular mass of 3.8x10 3 Da.
3. The antitumor active muntin polysaccharide KBPW a 1 according to claim 1, wherein said muntin polysaccharide KBPW a is composed of arabinose, galactose and glucose in a molar ratio of 2.62:1.02:6.36.
4. Use of a keemun polysaccharide KBPW1 with anti-tumor activity according to any one of claims 1-3 for the preparation of an anti-tumor medicament.
5. The use of claim 4, wherein the tumor comprises colorectal cancer (CRC), cholangiocarcinoma, gastric cancer, glioblastoma (GBM), leiomyosarcoma, melanoma, lung cancer, neuroblastoma (NB), ovarian cancer, pancreatic cancer, prostate cancer, thyroid cancer, breast cancer, or any combination thereof.
6. An antitumor pharmaceutical composition comprising the keemun polysaccharide KBPW1 having antitumor activity according to any one of claims 1 to 3.
7. The anti-tumor pharmaceutical composition according to claim 6, wherein the composition further comprises one or more pharmaceutically acceptable carriers.
8. The anti-tumor pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier is a diluent, excipient, filler, binder, wetting agent, disintegrant, absorption enhancer, adsorption carrier, surfactant or lubricant.
9. The anti-tumor pharmaceutical composition according to claim 6, wherein the pharmaceutically acceptable carrier is a diluent.
10. The anti-tumor pharmaceutical composition according to claim 6, wherein the oral dosage form of the pharmaceutical composition comprises a tablet, a capsule, a lozenge, an oral liquid, a granule, a pill or a powder.
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