Crocus sativus polysaccharide, preparation method and application of crocus sativus polysaccharide in anti-hepatic fibrosis
The method of extracting polysaccharides from the whole flower of saffron has solved the problem of lack of effective drugs for treating liver fibrosis in the prior art, and achieved a significant inhibition of liver stellate cell activation and liver fibrosis.
Patent Information
- Application Number
- CN202410159126.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-02-04
- Publication Date
- 2025-03-21
AI Technical Summary
The prior art has not used saffron whole flower to extract polysaccharides for the treatment of liver fibrosis, and there is a lack of effective drug solutions.
By designing a method for extracting saffron polysaccharides, including ethanol degreasing, water extraction, filtration, concentration, dialysis, alcohol precipitation, centrifugation and vacuum drying of dried saffron, water extraction, saffron crude polysaccharides were obtained, and purified by DEAE cellulose anion column, finally obtaining saffron polysaccharides XHH0, XHH1 and XHH2.
Saffron polysaccharide significantly inhibits the activation of hepatic stellate cells and the generation of extracellular matrix. In vivo experiments have shown that it can significantly inhibit liver fibrosis induced by carbon tetrachloride and has a good inhibitory effect.
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Figure CN119661731A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant polysaccharides, and in particular to saffron polysaccharide, a preparation method and use thereof in preventing liver fibrosis. Background Art
[0002] Liver fibrosis refers to a pathological process in which various pathogenic factors cause abnormal proliferation of connective tissue in the liver, leading to excessive precipitation of diffuse extracellular matrix in the liver. Its pathogenesis is that when the liver is invaded by pathogens, it can cause liver damage and inflammatory response, and the liver tissue immune system is activated to repair tissue. When this tissue repair process is excessive and out of control, excessive proliferation and abnormal precipitation of extracellular matrix in the liver tissue will lead to abnormal changes in liver structure and liver function. It is not an independent disease. Many chronic liver diseases can cause liver fibrosis, such as acute and chronic hepatitis, fatty liver, liver cancer, etc., which is the inevitable path to the development of cirrhosis. Therefore, finding a drug that can effectively treat liver fibrosis is an important treatment strategy for liver disease.
[0003] Liver fibrosis has many causes and a particularly complex pathogenesis. Therefore, polysaccharides have outstanding advantages in the clinical treatment of liver fibrosis and have broad application prospects because of their multi-link, multi-pathway, multi-level, multi-target action characteristics and the ability to regulate the pathological process of organ tissues as a whole.
[0004] Saffron, a Chinese medicine name, is the dried stigma of Crocus sativus L, a plant of the Iridaceae family. Saffron is a traditional Chinese medicine in my country, which has the effects of promoting blood circulation, relieving menstruation, removing blood stasis and relieving pain. At present, the research on the chemical composition of saffron mainly focuses on small molecular compounds such as crocin. However, there are few reports on the extraction, separation, structure and pharmacological activity of saffron whole flower polysaccharides. At present, there is no research on the extraction of polysaccharides using the whole flower of saffron as raw material and using it as a drug for the treatment of liver fibrosis. Summary of the invention
[0005] In view of the deficiencies in the prior art, the present invention designs a method for extracting saffron polysaccharide, a drug for treating liver fibrosis.
[0006] One of the purposes of the present invention is to provide a saffron polysaccharide, including saffron polysaccharide XHH0, saffron polysaccharide XHH1 and saffron polysaccharide XHH2, wherein the saffron polysaccharide XHH0 is composed of mannose, glucose, galactose, xylose and arabinose, and the molar ratio thereof is 1.0-3.0:5.0-15.0:10.0-50.0:1.0-5.0:1.0-3.0; the saffron polysaccharide XHH1 is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, The molar ratio is 0.3-1.6: 1.0-3.0: 1.0-4.0: 1.0-4.0: 0.5-4.0: 10.0-50.0: 1.0-8.0: 3.0-20.0; saffron polysaccharide XHH2 is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molar ratio is 0.3-1.5: 1.0-15.0: 0.3-18.0: 1.0-18.0: 0.3-6.0: 8-50.0: 5.0-30.0.
[0007] Furthermore, the weight average molecular weight of the saffron polysaccharide XHH0 is 10 to 500 kDa; the weight average molecular weight of the saffron polysaccharide XHH1 is 5 to 500 kDa; and the weight average molecular weight of the saffron polysaccharide XHH2 is 10 to 500 kDa.
[0008] Furthermore, the saffron polysaccharide XHH0 contains 60-100% neutral sugar, 0.5-20% uronic acid and 0.5-10% protein respectively; XHH1 contains 60-100% neutral sugar, 5-30% uronic acid and 0.5-20% protein respectively; XHH2 contains 10-70% neutral sugar, 5-30% uronic acid and 0-10% protein respectively.
[0009] The second object of the present invention is to provide a method for preparing saffron polysaccharide, comprising the steps of defatting dried saffron by ethanol, extracting with water, filtering, concentrating the obtained filtrate, dialyzing, concentrating, precipitating with alcohol, centrifuging and vacuum drying to obtain water-extracted saffron crude polysaccharide; subjecting the water-extracted saffron crude polysaccharide to graded purification using a DEAE cellulose anion column, eluting with water, 0.1M NaCl and 0.2M NaCl in sequence, and collecting the corresponding water-washed component ZHH0, 0.1M NaCl eluted component ZHH1, and 0.2M NaCl eluted component ZHH2, and further subjecting the purified product to gel permeation to obtain saffron polysaccharide XHH0, saffron polysaccharide XHH1 and saffron polysaccharide XHH2, respectively.
[0010] Furthermore, the molecular weight cut-off of the dialysis bag during dialysis is 3500Da.
[0011] Furthermore, when the boiling water extraction is performed, the number of extractions is 1 to 5 times, the amount of boiling water used in each extraction is 1 to 10 times the total mass of saffron, and the extraction time for each extraction is 1.0 to 5 hours.
[0012] Furthermore, when the water-extracted saffron crude polysaccharide is subjected to graded purification using a DEAE cellulose anion column, the crude saffron polysaccharide is first dissolved in water and centrifuged, and the supernatant is then separated by passing through a DEAE cellulose anion column.
[0013] The third object of the present invention is to provide the use of saffron polysaccharide in the preparation of drugs for inhibiting liver fibrosis.
[0014] Among them, inhibiting liver fibrosis is to inhibit the activation of hepatic stellate cells, thereby achieving the purpose of inhibiting liver fibrosis.
[0015] Experiments show that the three saffron polysaccharides prepared in this application are pectin polysaccharides and can significantly inhibit the activation of hepatic stellate cells. In vivo experiments show that the polysaccharide can significantly inhibit carbon tetrachloride-induced liver fibrosis, which indicates that the polysaccharide has a good inhibitory effect on fibrosis and is expected to become a potential polysaccharide drug for the treatment of fibrosis. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 This is a standard curve diagram for the determination of the physicochemical properties of saffron polysaccharides XHH0, XHH1 and XHH2 prepared in Example 1;
[0017] Figure 2 This is a graph showing the molecular weight test results of saffron polysaccharides XHH0, XHH1 and XHH2 prepared in Example 1;
[0018] Figure 3 This is a diagram showing the results of monosaccharide composition analysis of saffron polysaccharides XHH0, XHH1 and XHH2 prepared in Example 1;
[0019] Figure 4 This is the 13C NMR spectrum of saffron poly-XHH2 prepared in Example 1;
[0020] Figure 5 This is a graph showing the concentration-dependent inhibitory effects of saffron polysaccharides XHH0, XHH1, and XHH2 on hepatic stellate cell activation;
[0021] Figure 6 This is a diagram showing the improvement effect of saffron polysaccharide XHH2 on the carbon tetrachloride-induced liver fibrosis model. DETAILED DESCRIPTION
[0022] The following is further described in detail through specific implementation methods:
[0023] Example 1: Preparation of Crocus polysaccharide XHH0, Crocus polysaccharide XHH1 and Crocus polysaccharide XHH2
[0024] a. Western polysaccharide extraction:
[0025] The dried whole saffron flower was soaked in 95% industrial ethanol for 1 week, then dried naturally at room temperature. 1 kg of dried saffron was extracted 3 times with 10 L of boiling water (deionized water), each time for 3 hours. The extracts were combined and filtered, and the extracts were concentrated to about 5 L under normal pressure and dialyzed against running water for 3 days. The liquid in the dialysis bag was concentrated to about 2.5 L, the concentrate was centrifuged, and the supernatant was added with 4 times the volume of 95% industrial ethanol under stirring, and allowed to stand for 24 hours. The precipitate was collected by centrifugation, and the precipitate was washed with anhydrous ethanol and acetone twice in sequence, and then dried in a vacuum dryer at 50°C to obtain saffron crude polysaccharide (yield 10.54%).
[0026] b. Polysaccharide purification:
[0027] Take 6g of the above-prepared saffron crude polysaccharide, dissolve it in 100mL water, centrifuge to remove insoluble matter, and preliminarily separate the supernatant through a Cl-type DEAE-cellulose anion column. Eluted with distilled water, 0.1M NaCl and 0.2M NaCl in turn, detected by sulfuric acid-phenol, eluted with distilled water, 0.1M NaCl and 0.2M NaCl in turn, detected by sulfuric acid-phenol, collect and combine the eluates of distilled water, 0.1M NaCl and 0.2M NaCl, concentrate, dialyze, and freeze-dry to obtain secondary components ZHH0, ZHH1 and ZHH2 respectively. The three components were further purified by gel column to obtain 45.27mg of saffron polysaccharide XHH0, 79.12mg of saffron polysaccharide XHH1 and 65.30mg of saffron polysaccharide XHH2, which correspond to the homogeneous saffron polysaccharides.
[0028] c. Polysaccharide structure identification:
[0029] 1. Determination of molecular weight by high performance gel permeation chromatography (HPGPC)
[0030] The instrument was an Agilent 1260Seri high performance liquid phase system (Agilent, USA). The chromatographic columns used were Sugar KS-802 and KS-804 (Showa Denko, Japan) in series, with a column temperature of 40°C; 0.1 M sodium nitrate was used as the mobile phase, the sample injection volume was 10 μL, and the flow rate was 0.5 mL / min; it was equipped with a G1362A differential detector.
[0031] The molecular weight (Mp), number average molecular weight (Mn), weight average molecular weight (Mw), higher average molecular weight (Mz) and dispersion coefficient (P) of XHH0 were calculated by Agilent workstation and matching chromatography GPC software, which were 2207Da, 1240Da, 1978Da, 2769Da and 1.60 respectively. Figure 2 As shown, the molecular weight (Mp), number average molecular weight (Mn), weight average molecular weight (Mw), higher average molecular weight (Mz) and dispersion coefficient (P) of the highest peak of XHH1 are 61008Da, 44037Da, 61824Da, 80294Da and 1.40, respectively. The molecular weight (Mp), number average molecular weight (Mn), weight average molecular weight (Mw), higher average molecular weight (Mz) and dispersion coefficient (P) of the highest peak of XHH2 are 39635Da, 30589Da, 35671Da, 40273Da and 1.17, respectively.
[0032] 2. Determination of monosaccharide composition by PMP-HPLC pre-column derivatization method
[0033] 1) Complete acid hydrolysis: Take 2-4 mg of polysaccharide sample, add 4 mL of 2M trifluoroacetic acid, seal, and hydrolyze in an oven at 110°C for 4 h. After the reaction is completed, cool, add methanol several times to remove trifluoroacetic acid, and redissolve the residue in 200 μL of deionized water.
[0034] 2) PMP derivatization: Take 100 μL of the above hydrolyzed sample solution and 100 μL of the mixed standard solution, add them to 2 mL EP tubes respectively, then add 100 μL of 0.6 M sodium hydroxide solution, mix well, add 200 μL of 0.5 M PMP methanol solution, vortex and mix well, and react in a 70 ° C water bath for 100 min. After the reaction is completed, add 200 μL of 0.3 M hydrochloric acid solution to neutralize, then add 400 μL of deionized water and 1 mL of chloroform for extraction, vortex and shake, and let stand for 2 hours or overnight. Take 850 μL of the upper aqueous phase, add 850 μL of chloroform for extraction, and let stand for 1 hour. The upper aqueous phase (750 μL) is extracted with an equal volume of chloroform and allowed to stand for stratification. After filtering with a microporous filter membrane (0.22 μM), it is placed in a liquid phase bottle and detected by HPLC.
[0035] The results of monosaccharide composition analysis are as follows Figure 3 As shown, saffron polysaccharide XHH0 contains mannose, glucose, galactose, xylose and arabinose, and the molar ratio is 1.79:11.61:38.23:3.16:1.46. XHH1 contains mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose, and the molar ratio is 0.67:1.84:0.93:0.87:1.50:37.94:4.53:9.61. XHH2 contains mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molar ratio is 0.71:7.53:0.97:5.84:2.13:26.00:14.97, which is a RG~Ⅰ pectin polysaccharide.
[0036] 3. Nuclear magnetic resonance measurement:
[0037] Take 20-30 mg of polysaccharide sample and dissolve it in 400-500 μL D2O. After freeze drying, dissolve the sample in 400-500 μL D2O. Centrifuge, draw the supernatant along the wall and add it to the NMR tube, then quickly add 2-2.5 μL acetone as an internal reference (δH=2.29ppm, δ31.5), and use Bruker AM-500 (500Hz) NMR instrument to perform 13C-NMR measurement at 25°C.
[0038] 13C NMR spectrum ( Figure 4 ), the anomeric carbon signals at δ110~δ108 are the C~1 signals of terminal arabinose and 1,5-arabinose respectively; the terminal carbon signals at δ106~δ104 are the C~1 signals of terminal galactose, 1,3-galactose, 1,4-galactose and 1,4,6-galactose respectively; the terminal carbon signals at δ100~δ99 are the C~1 signals of 1,2,4-rhamnose, 1,2-rhamnose and 1,4-galacturonic acid carboxyl. In addition, at δ17~δ18 are the signal peaks of rhamnose methyl carbon, at δ171 is the signal peak of galacturonic acid carboxyl carbon, from which it can be seen that galacturonic acid has undergone methyl esterification, and at δ54 is the signal peak of methoxy carbon. From the above results, it can be inferred that XHH2 is RG~Ⅰ pectin polysaccharide.
[0039] Example 2: Crocus polysaccharides XHH0, XHH1 and XHH2 inhibit the activation activity of hepatic stellate cells
[0040] Hepatic stellate cells (HSCs) account for 15% of the total number of liver intrinsic cells and about 30% of non-parenchymal cells. HSCs exist in the Disse cavity and are spindle-shaped or polygonal. There are multiple lipid droplets rich in vitamin A in the cytoplasm. Their slender processes extend outward and surround the outside of the sinusoidal endothelial cells. They are the primary site for storing retinal derivatives in the body.
[0041] In a normal liver, hepatic stellate cells are in a quiescent state, do not express α-smooth muscle actin (α-SMA), have low proliferation activity, and low ability to synthesize collagen. Their main function is to store retinaldehydes.
[0042] Under normal circumstances, hepatic stellate cells are in a quiescent state. When the liver is damaged by inflammation or mechanical stimulation, hepatic stellate cells are activated and their phenotype changes from quiescent to activated. On the one hand, activated hepatic stellate cells participate in the formation of liver fibrosis and the reconstruction of intrahepatic structure through proliferation and secretion of extracellular matrix, and on the other hand, they increase the intrahepatic sinusoidal pressure through cell contraction.
[0043] In vitro stimulation with TGF-β induces the activation of hepatic stellate cells, thereby transforming hepatic stellate cells into myofibroblasts, expressing α-SMA protein, and producing a large amount of extracellular matrix (ECM) deposition including (Fibronectin, Collagen-α1).
[0044] Spread Matrigel on a 24-well plate, 235μl per well (pre-cooled serum-free medium to prepare Matrigel, 200μl contains 1μl Matrigel) and shake well to cover the bottom of the well. 20min. Discard the Matrigel in the well and wash it once with PBS. Treat LX2 cells, trypsinize, centrifuge (300g, 3min), resuspend, and count. Inoculate 24-well plates (15w / ml, 500μl per well) according to experimental requirements. After the cells adhere to the wall, starve the cells with HG medium containing 0.5% FBS for 12h. Add TGF-β (10ng / ml) to 0.5% FBS HG, and dilute the compound to the corresponding concentration with this medium. Aspirate the medium used for starved cells, and then put the diluted compound into the well. After 24h, wash once with PBS and add 100μl 1x loading protein sample. Western blot was used to detect fibnenctin, α-SMA, and GAPDH as loading control. Figure 5 As shown, from the experimental results it can be seen that XHH0, XHH1 and XHH2 can inhibit the activation of hepatic stellate cells and the production of extracellular matrix in a concentration-dependent manner at 0.125 and 0.25 mg / ml.
[0045] Example 3: Crocus polysaccharide XHH2 inhibits carbon tetrachloride-induced liver fibrosis in mice
[0046] The experiment used male C57BL / 6J mice, and CCl4 was injected intraperitoneally to establish the model (10% CCL4 in olive oil, 2mL / kg IP; three times a week, 50μl was injected for mice weighing more than 22.5g, and 40μl was injected for mice weighing less than 22.5g). Ten mice of the same litter were injected intraperitoneally with olive oil only as the normal control group. The experiment was conducted in the Experimental Animal Center of Shanghai Institute of Materia Medica, and the mice were kept at a constant temperature (24-26℃) and under Specific Pathogen Free (SPF) conditions. The cages, bedding, drinking water, and feed were sterilized by high pressure and ultraviolet light, and the operation was aseptic. The experiment was carried out in accordance with the regulations of the Experimental Animal Use and Management Committee of Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Two weeks after the modeling, serum samples were collected from the orbital blood to detect ALT, AST, etc. According to the above indicators and body weight, the mice with abnormal values were eliminated and randomly divided into groups for drug administration (while continuing to inject CCl4 intraperitoneally to establish the model), with 10 mice in each group. The drug was administered for 3 weeks, during which the food intake and body weight were monitored. After each 3-week administration, blood was collected from the eye sockets at 48 hours after CCl4 injection to detect ALT and AST. Blood was collected after the administration, and the mice were sacrificed. The liver was taken and weighed, and part of the liver was fixed with 4% paraformaldehyde and part of the liver was frozen at ~80°C.
[0047] After the experiment, serum ALT and AST levels, liver hydroxyproline content, α~SMA and Col1~α1 mRNA expression levels, and liver tissue pathological sections were detected.
[0048] like Figure 6 As shown in the figure, compared with WT (normal control group), the serum ALT AST level in the vehicle group was significantly increased; compared with the vehicle group, after 3 weeks of administration, the serum ALT AST level in the saffron polysaccharide XHH2 (30 mg / kg) group was significantly reduced. Compared with the WT group, the expression of α~SMA and Col1α1 mRNA in the liver of the vehicle group was significantly upregulated, and the XHH2 (30 mg / kg) group could significantly reduce the expression of α~SMA and Col1α1 mRNA. The area of collagen stained with Sirius red and the content of hydroxyproline in the vehicle group were significantly increased compared with the normal group. Compared with the vehicle group, XHH2 (30 mg / kg) reduced the area of collagen stained with Sirius red and the content of hydroxyproline.
[0049] The above is only an embodiment of the present invention, and the common knowledge such as the known specific structure and characteristics in the scheme is not described in detail here. It should be pointed out that for those skilled in the art, several deformations and improvements can be made without departing from the structure of the present invention, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the specification can be used to interpret the content of the claims.
Claims
1. A saffron polysaccharide, characterized in that: The invention comprises saffron polysaccharide XHH0, saffron polysaccharide XHH1 and saffron polysaccharide XHH2. The saffron polysaccharide XHH0 is composed of mannose, glucose, galactose, xylose and arabinose in a molar ratio of 1.0-3.0:5.0-15.0:10.0-50.0:1.0-5.0:1.0-3.0; the saffron polysaccharide XHH1 is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 0.3-1.0:1.0-3.
0. 1.6:1.0~3.0:1.0~4.0:1.0~4.0:0.5~4.0:10.0~50.0:1.0~8.0:3.0~20.0; Crocus polysaccharide XHH2 is composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose and arabinose, and the molar ratio is 0.3~1.5:1.0~15.0:0.3~18.0:1.0~18.0:0.3~6.0:8~50.0:5.0~30.
0.
2. The saffron polysaccharide according to claim 1, characterized in that: The weight average molecular weight of the saffron polysaccharide XHH0 is 10 to 500 kDa; the weight average molecular weight of the saffron polysaccharide XHH1 is 5 to 500 kDa; and the weight average molecular weight of the saffron polysaccharide XHH2 is 10 to 500 kDa.
3. The saffron polysaccharide according to claim 2, characterized in that: The saffron polysaccharide XHH0 contains 60-100% of neutral sugar, 0.5-20% of uronic acid and 0.5-10% of protein respectively; XHH1 contains 60-100% of neutral sugar, 5-30% of uronic acid and 0.5-20% of protein respectively; XHH2 contains 10-70% of neutral sugar, 5-30% of uronic acid and 0-10% of protein respectively.
4. The method for preparing saffron polysaccharide according to any one of claims 1 to 3, characterized in that: The dried saffron is defatted by ethanol, extracted by water, and filtered. The obtained filtrate is concentrated, dialyzed, concentrated, precipitated with alcohol, centrifuged, and vacuum dried to obtain water-extracted saffron crude polysaccharide; the water-extracted saffron crude polysaccharide is graded and purified by DEAE cellulose anion column, eluted with water, 0.1M NaCl and 0.2M NaCl in sequence, and the corresponding water-washed component ZHH0, 0.1M NaCl elution component ZHH1, and 0.2M NaCl elution component ZHH2 are collected, and further purified by gel permeation to obtain saffron polysaccharide XHH0, saffron polysaccharide XHH1 and saffron polysaccharide XHH2, respectively.
5. The preparation method according to claim 4, characterized in that: The molecular weight cut-off of the dialysis bag during dialysis is 3500Da.
6. The preparation method according to claim 5, characterized in that: When the boiling water extraction is performed, the number of extractions is 1 to 5 times, the amount of boiling water used in each extraction is 1 to 10 times the total mass of saffron, and the extraction time for each extraction is 1.0 to 5 hours.
7. The preparation method according to claim 6, characterized in that: When the water-extracted saffron crude polysaccharide is subjected to graded purification using a DEAE cellulose anion column, the crude saffron polysaccharide is first dissolved in water and centrifuged, and the supernatant is then separated by passing through a DEAE cellulose anion column.
8. Use of the saffron polysaccharide according to any one of claims 1 to 3 in the preparation of drugs for inhibiting liver fibrosis.
9. The use according to claim 8, characterized in that: The inhibition of liver fibrosis is to inhibit the activation of hepatic stellate cells, thereby achieving the purpose of inhibiting liver fibrosis.