WO2020147350A1 - 阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用 - Google Patents

阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用 Download PDF

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WO2020147350A1
WO2020147350A1 PCT/CN2019/111301 CN2019111301W WO2020147350A1 WO 2020147350 A1 WO2020147350 A1 WO 2020147350A1 CN 2019111301 W CN2019111301 W CN 2019111301W WO 2020147350 A1 WO2020147350 A1 WO 2020147350A1
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arabinoxylan
sulfonate
group
application according
cartilage
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PCT/CN2019/111301
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English (en)
French (fr)
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王磊
张厚瑞
陈海珊
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广西壮族自治区中国科学院广西植物研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

Definitions

  • the invention relates to an application of arabinoxylan sulfonate in the preparation of a medicine for treating osteoarthritis, and belongs to the technical field of medicines.
  • Osteoarthritis is a chronic degenerative disease of articular cartilage.
  • the main clinical manifestations are slow-developing joint pain, stiffness, swelling and restricted mobility.
  • the prevalence of OA in people over 60 years old in my country has reached 50%, and 80% over 75 years old.
  • the disease has become one of the most common diseases that seriously affect the quality of life of the elderly.
  • the treatment of OA mainly includes three options: oral medication, joint cavity injection and surgery.
  • the joint cavity injection is a puncture operation, which requires high operation requirements and is not easy to use repeatedly. It is mainly aimed at relieving symptoms and improving the condition of moderate and severe patients.
  • Surgical treatment is mainly for severe patients in the middle and late stages, and joint replacement can be used to completely eliminate pain.
  • oral drugs including two categories of analgesics and chondroprotective drugs. Usually the two types of drugs are used in combination to achieve the purpose of analgesia and promote cartilage repair.
  • Analgesics mainly include non-steroidal anti-inflammatory drugs, such as diclofenac sodium, meloxicam, celecoxib, etc.; antipyretic analgesics, acetaminophen, aspirin, etc.; opioid analgesics such as codeine, tramaine hydrochloride Wait a lot.
  • non-steroidal anti-inflammatory drugs such as diclofenac sodium, meloxicam, celecoxib, etc.
  • antipyretic analgesics such as diclofenac sodium, meloxicam, celecoxib, etc.
  • opioid analgesics such as codeine, tramaine hydrochloride Wait a lot.
  • the most commonly used chondroprotective drugs are glucosamine, chondroitin sulfate, and diacerein.
  • some external preparations of the above analgesics and external Chinese medicines also have clinical applications.
  • drug treatment can only improve the joint inflammation of patients within a period of time, and there is no drug that
  • chondroitin sulfate and hyaluronic acid play important physiological functions in the body, such as lubricating joints, regulating the permeability of blood vessel walls, regulating proteins, and preventing adhesions. Therefore, chondroitin sulfate and hyaluronic acid themselves are also used in the repair and rehabilitation of articular cartilage damage, so the preparation of related polysaccharide derivatives through sulfation or sulfonation has become an important direction for finding arthritis treatment drugs.
  • Arabinoxylan is a new type of dietary fiber approved by the FDA in 2018. The current research on it is mainly focused on its own physiological effects, and there are few studies on its derivatives. At present, there is no relevant report on the application of arabinoxylan sulfonate in the preparation of drugs for treating osteoarthritis.
  • the purpose of the present invention is to overcome the shortcomings of the prior art and provide an application of arabinoxylan sulfonate in the preparation of a medicine for treating osteoarthritis.
  • the inventor of the present application has discovered through a large number of studies that arabinoxylan sulfonate has a very obvious therapeutic effect on arthritis, which can not only eliminate inflammation, but also promote cartilage repair, and can reverse the disease to a certain extent. Therefore, arabinoxylan Sugar sulfonates can be used to prepare medicines for treating osteoarthritis.
  • the technical scheme of the present invention to solve the above technical problems is as follows: the application of arabinoxylan sulfonate in the preparation of medicines for treating osteoarthritis.
  • arabinoxylan sulfonate exerts the therapeutic effect of osteoarthritis by eliminating inflammation and promoting cartilage nutrition. It has a significant effect on osteoarthritis model rabbits, and its effect is significantly better than that of the commercially available oral drug combination (celecoxib and Glucosamine sulfate).
  • the second point is that the efficacy of arabinoxylan sulfonate by injection is better than that of oral administration.
  • oral administration may be due to the macromolecular structure that is not conducive to digestive tract absorption or may be decomposed by various enzymes in the digestive tract and intestinal microbes, and cannot reach the affected area in the form of effective ingredients, so injection administration is better s Choice.
  • arabinoxylan sulfonate itself has a significant effect on the curative effect.
  • arabinoxylan sulfonate prepared from raw materials with a branched structure and high arabinose content has better efficacy.
  • the present invention can also be improved as follows.
  • the sum of x, y, and z is 1-19;
  • the side chain sulfonated arabinose is connected to the 2- or/and 3-position of the main chain sulfonated xylose through the 1-position oxygen glycosidic bond, and the two The molar ratio is 1:2-1:20;
  • the side chain heterosaccharide is connected to the 2- or/and 3-position of the main chain sulfonated xylose through the 1-position oxo-glycoside bond;
  • the heterosaccharide in the sulfonated heterosaccharide It is one or more of glucose, glucuronic acid, 4-methylglucuronic acid, galactose, rhamnose, galacturonic acid, and xylose.
  • the above further beneficial effect is that the above arabinoxylan sulfonate is obtained by sulfonating arabinoxylan.
  • the increase in the number of side chain sulfonated arabinose on the polymer backbone of arabinoxylan sulfonate is beneficial to improve the curative effect of arabinoxylan sulfonate in treating osteoarthritis.
  • the preparation method of the arabinoxylan sulfonate includes the following steps:
  • Step 1 Put arabinoxylan and sulfonating agent in a molar ratio of 1:(1-5) together in a reactor to obtain a first mixture;
  • Step 2 According to the ratio of adding 100mL polar organic solvent to the arabinoxylan described in step 1 per (1-8) g, add polar organic solvent to the first mixture obtained in step 1, and heat to 55°C while stirring -95°C, after reacting for 1-3 hours, lower to room temperature to obtain the second mixture;
  • Step 3 Add lye to the second mixture obtained in step 2, and the molar ratio of the alkali in the lye to the sulfonating agent described in step 1 is 1:1. After fully stirring, add the resulting reaction mixture to In the polar organic solvent of 2-5 times the volume of ethanol in step 2, filter after sufficient precipitation to obtain a filter residue;
  • Step 4 Dissolve in water according to the ratio of 10g filter residue to (50-100) mL of water, adjust the pH to 7 with the same lye as in step 3, add 2-5 times the volume of the above water in ethanol for alcohol precipitation, Filter the precipitate, repeat the operations of water dissolution, alcohol precipitation, and filtration twice, collect the final precipitate, dissolve it in deionized water, pass through an ultrafiltration membrane, and freeze-dry the resulting retentate to obtain the arabinoxylan Sulfonate.
  • arabinoxylan and sulfonating agent are used to prepare arabinoxylan sulfonate in a polar organic solvent, which has high sulfonation efficiency and avoids the need for pyridine as a solvent in the prior art. The resulting serious environmental pollution burden.
  • the sulfonating agent is one of sulfur trioxide trimethylamine complex, sulfur trioxide triethylamine complex, sulfur trioxide pyridine complex or sulfur trioxide N,N-dimethylformamide complex Species;
  • the polar organic solvent is one of dimethylsulfoxide, N,N-dimethylformamide or N,N-dimethylacetamide;
  • the room temperature is 20-25°C;
  • the The lye is one of NaOH solution, KOH solution, and Ca(OH) 2 solution; the molecular retention of the ultrafiltration membrane is 600 Da.
  • arabinoxylan is extracted from monocotyledonous plants.
  • arabinoxylan is the xylan mainly present in monocotyledonous plant hemicellulose, and the corresponding xylan is mainly present in dicotyledonous plant hemicellulose.
  • dicotyledonous xylan is mainly single-stranded, completely free of branched chains or occasionally very small amounts.
  • the glucuronic acid branch is attached to the 2-position of xylose.
  • the monocotyledon arabinoxylan has a branched structure with a large number of branched chains, and the branched chains exist at the 2- and 3-position hydroxyl groups of the main chain xylose.
  • the main form of the branched chains is arabinose and a small amount Other miscellaneous sugars.
  • the inventor of the present application found through a large number of experiments that although the two types of plant-derived xylans have a certain ability to treat model rabbit osteoarthritis after sulfonation, the arabinoxylan sulfonate derived from monocotyledonous plants The curative effect of acid salt is more significant, and the content of arabinose branches in its structure has a positive correlation with the overall effect.
  • the monocot plant is one or more of sugarcane, bamboo, corn, oats, reed, rice, and wheat.
  • the further beneficial effect of adopting the above is: extracting arabinoxylan from the above monocot plant is more conducive to industrial production.
  • the drug is a pharmaceutical preparation prepared with an effective amount of arabinoxylan sulfonate as an active ingredient, plus pharmaceutically acceptable carriers and/or auxiliary materials.
  • the dosage form of the drug is injection.
  • the injection can allow the medicine to directly enter the blood circulation to reach the affected area without causing the degradation of arabinoxylan sulfonate medicine due to the digestive tract.
  • the injection includes an aqueous solution or a water composite solution.
  • the drug administration mode is one or more of subcutaneous injection, intramuscular injection and intravenous injection.
  • the above-mentioned further beneficial effect is that the drug can directly enter the blood circulation to reach the affected area by injection administration, and will not cause the degradation of arabinoxylan sulfonate drugs due to the digestive tract.
  • arabinoxylan sulfonate has a very obvious therapeutic effect on arthritis. It can not only eliminate inflammation, but also promote cartilage repair, which can reverse the disease to a certain extent. Therefore, Arab Xylan sulfonate can be used to prepare medicine for treating osteoarthritis.
  • the inventor of the present application found that the drug effect of arabinoxylan sulfonate by injection is better than that of oral administration.
  • arabinoxylan sulfonates prepared from raw materials with a branched structure and high arabinose content have better medicinal effects.
  • Figure 1 is an anatomical photograph of the knee joint of the blank group A in Example 1 of the present invention.
  • Figure 2 is an anatomical photograph of the knee joint of model group B in Example 1 of the present invention.
  • Figure 3 is an anatomical photograph of the knee joint of administration group C (arabinoxylan sulfonate sodium salt oral group) in Example 1 of the present invention.
  • Figure 4 is an anatomical photograph of the knee joint of administration group D (arabinoxylan sulfonate sodium salt subcutaneous injection group) in Example 1 of the present invention.
  • Fig. 5 is an anatomical photograph of the knee joint of administration group E (celecoxib and glucosamine sulfate oral group) in Example 1 of the present invention.
  • Fig. 6 is a comparison of the uronic acid content of the cartilage uronic acid in each group of experimental rabbits in Example 1 of the present invention.
  • Figure 7 is one of the HE stained cartilage sections of the blank group A in Example 2 of the present invention.
  • Fig. 8 is the second section of HE-stained cartilage section of blank group A in Example 2 of the present invention.
  • Figure 9 is the third section of the HE stained cartilage section of the blank group A in Example 2 of the present invention.
  • Fig. 10 is the fourth section of the HE-stained cartilage section of the blank group A in Example 2 of the present invention.
  • Figure 11 is one of HE stained cartilage sections of model group B in Example 2 of the present invention.
  • Example 12 is the second section of HE stained cartilage section of model group B in Example 2 of the present invention.
  • Figure 13 is the third section of HE stained cartilage section of model group B in Example 2 of the present invention.
  • Example 14 is the fourth section of HE stained cartilage section of model group B in Example 2 of the present invention.
  • Figure 15 is one of HE stained cartilage sections of administration group C (bagasse source) in Example 2 of the present invention.
  • Figure 16 is the second section of HE stained cartilage section of administration group C (bagasse source) in Example 2 of the present invention.
  • Fig. 17 is the third section of HE-stained cartilage section of administration group C (bagasse source) in Example 2 of the present invention.
  • Example 18 is the fourth section of HE stained cartilage section of administration group C (bagasse source) in Example 2 of the present invention.
  • Figure 19 is one of HE stained cartilage sections of administration group D (maize seed coat source) in Example 2 of the present invention.
  • Figure 20 is the second section of HE stained cartilage section of administration group D (maize seed coat source) in Example 2 of the present invention.
  • Fig. 21 is the third section of HE stained cartilage section of administration group D (maize seed coat source) in Example 2 of the present invention.
  • Fig. 22 is the fourth section of HE stained cartilage section of administration group D (maize seed coat source) in Example 2 of the present invention.
  • Figure 23 is one of the HE-stained cartilage sections of administration group E (Birchwood source) in Example 2 of the present invention.
  • Figure 24 is the second section of HE stained cartilage section of administration group E (Birchwood source) in Example 2 of the present invention.
  • Figure 25 is the third section of HE stained cartilage section of administration group E (Birchwood source) in Example 2 of the present invention.
  • Fig. 26 is the fourth section of HE stained cartilage section of administration group E (Birchwood source) in Example 2 of the present invention.
  • Figure 27 is a comparison of uronic acid content in cartilage of rabbit knee joints in each group in Example 2 of the present invention.
  • Example 1 The effect of different administration methods on the treatment of rabbit knee arthritis and comparison with commercially available drugs
  • Osteoarthritis is a chronic, progressive, and degenerative joint disease. It is clinically characterized by joint pain, deformation and restricted mobility. The pathological manifestations are softening, fibrosis, ulcers, cartilage reduction, and sclerosis of subchondral bone. , Subchondral bone cyst, etc.
  • the mode of administration has an important influence on the effectiveness of the drug. This experiment investigated the effects of oral and subcutaneous injection of arabinoxylan sulfonate on the treatment of arthritis through different modes of administration, and combined the commercially available oral drug combination (celecoxib) And glucosamine sulfate) and sodium arabinoxylan sulfonate.
  • PBS powder (Beijing Zhongshan), absolute ethanol (Sinopharm Group), 0.9% sodium chloride injection (Hebei Tiancheng Pharmaceutical Co., Ltd.), paraformaldehyde (Shanghai Shenggong), hematoxylin stain (Nanjing Jiancheng), eosin Dye (Biyuntian), neutral resin (Sinopharm Group), bamboo-derived arabinoxylan (Guilin fiber), celecoxib capsule (Pfizer), glucosamine sulfate capsule (Aippu Bio), chloral hydrate, Carbazole, sulfur trioxide pyridine, etc. (Shanghai Aladdin).
  • Disposable syringe Choengdu Xinjinshifeng Medical Equipment Co., Ltd.), electronic balance (Precisa12A, Switzerland), centrifuge tube (Shanghai Wohong Biotechnology Co., Ltd.), pyrogen-removing ultrapure water system (Millipo 3150, USA), autoclaving Pot (Shanghai Saiyang), Microsurgery Equipment Package (Shanghai Surgical Instrument Factory), Pipette (Eppendorf, Germany), Electric Pressure Steam Sterilizer (Shaoxing Medical Equipment General Factory), Constant Temperature Drying Box (Shanghai Yuejin Medical Equipment Factory), inverted microscope (Olympus, Japan), microtome (Leica, Germany), spreading machine (Leica, Germany), baking sheet machine (Leica, Germany), UV-Vis spectrophotometer (Shanghai Jingke).
  • Preparation method Weigh 45g of raw bamboo powder arabinoxylan (its monosaccharide composition: the molar ratio of xylose to arabinose is 17:1), and place it in a reactor together with 210g of sulfur trioxide pyridine complex, and add 750mL DMA, heated to 55°C with stirring, cooled down after reacting for 1 hour. Dissolve 41 g of NaOH in water and add it to the reaction system. After fully stirring, add the reaction mixture to 2L of ethanol, and filter after sufficient precipitation. Dissolve the filter residue in water, adjust the pH to neutral with NaOH, add ethanol for precipitation, filter, and repeat the water-soluble alcohol precipitation operation twice.
  • the obtained product was dissolved in deionized water, ultrafiltration was performed in a membrane separator, and the molecular weight cutoff was 600Da.
  • the obtained retentate was freeze-dried to obtain sodium arabinoxylan sulfonate derived from bamboo.
  • the specific structural formula is as follows:
  • the animal room alternates day and night for 12h-12h.
  • the only protection animals are drinking water and eating at a temperature of 23-25°C, and entering the experiment a week later.
  • Groups B, C, D, and E construct arthritis models.
  • test rabbits are weighed, and after the anesthesia is satisfied, they are placed on the experimental table and fixed.
  • the right hind leg is pulled to make the right knee joint in an extended position.
  • the medical gauze wraps 3-4 layers to relieve the necrosis and ulcers caused by direct contact with the plaster
  • the plaster cloth Wrap and brake wrap the plaster with a bandage, keep the rabbit free to move after the wrap is completed, and brake for 6 weeks.
  • Administration group C was administered with arabinoxylan sulfonate sodium salt 10 mg/kg/day (dissolved in drinking water); administration group D was a modeled leg with local subcutaneous injection of arabinoxylan sulfonate sodium salt 6 mg/kg Dosage administration (normal saline is formulated into an aqueous solution, twice a week); administration group E is administered with celecoxib 7mg/kg/day (the capsule is removed and mixed in the feed) and glucosamine sulfate 15mg/kg/day (peeling Remove the capsules and dissolve in drinking water) dose administration; A and B groups are not administered; administration is continued for four weeks.
  • the animals were sacrificed, the knee joints were taken, and a small amount of cartilage on the articular surface of the femoral condyle was left for the test of uronic acid content.
  • the rest of the joints were fixed with 4% (v/v) paraformaldehyde for decalcification and used for tissue after decalcification was completed. slice.
  • Penetrating wax soak the transparent treated tissue in melted paraffin for 3 hours.
  • Sectioning Use a microtome to slice the tissue in the paraffin block into 5 ⁇ m thick slices, and spread them flat on a glass slide.
  • Dewaxing soak the paraffin sections in xylene for 10 minutes, twice, and a mixed solvent of 50% (v/v) ethanol and 50% (v/v) xylene for 10 minutes.
  • Eosin dye solution is dyed for 30 seconds.
  • Group A The test rabbits have strong hind legs and can move freely.
  • Group B The experimental rabbits do not like activities, the modeled legs do not participate in body support, and even move in a sitting position only with the support of the front legs.
  • Group C The experimental rabbits do not like activities, and there is no significant difference from the model group.
  • Group D In some experimental rabbits, the support of the modeled legs was slightly weak occasionally, and the modeled legs of the remaining test rabbits were vigorously moving normally.
  • Group E The experimental rabbits are not restricted by the modeling legs, but it can be seen that the support of the modeling legs is still slightly weak, which is obviously inferior to the blank group.
  • knee joint specimens were grossly observed and scored according to the Pelletier scoring principle. See Table 1 for details, and Table 2 for results. Some photos of knee joint specimens are shown in Figure 1-5.
  • A is significantly different from group A (P ⁇ 0.01)
  • b is significantly different from group B (P ⁇ 0.01)
  • c is significantly different from group C (P ⁇ 0.01)
  • d is significantly different from group D Sexual difference (P ⁇ 0.01).
  • the surface of the articular cartilage of the blank group A was smooth, without cracks or defects, with clear four-layer structure and complete tide line.
  • the articular cartilage layer of group B became thin, the four-layer structure was difficult to distinguish, the surface cartilage defect was serious, the chondrocyte arrangement was disordered, the cells were clustered, proliferated and fibrosis, and the tide line was severely damaged.
  • group C the articular cartilage layer was thinned, the four-layer structure was difficult to distinguish, the surface cartilage defect was severe, the chondrocyte arrangement was disordered, the cells were clustered, proliferated and fibrosis, and the tide line was severely damaged.
  • the articular cartilage cells in group D of the drug treatment group are arranged regularly, and the hierarchy is basically complete. The cells are occasionally missing in the surface part, and the tide line is still intact.
  • group E the articular cartilage layer of the drug group became thin, the four-layer structure was difficult to distinguish, the surface cartilage defects were serious, the chondrocytes were arranged disorderly, the cells were clustered proliferation and fibrosis, and the tide line was damaged more seriously.
  • the loss of articular cartilage proteoglycan is an important sign of osteoarthritis.
  • the content of proteoglycan and uronic acid in cartilage is significantly reduced than normal.
  • the change of uronic acid content in cartilage is inversely proportional to the severity of osteoarthritis.
  • Figure 6 shows the comparison of the uronic acid content in the cartilage of the rabbit knee joints in each group.
  • the oral administration group C is significantly different from the other groups (P ⁇ 0.05), and the administration group D is compared with the model group B. There were significant differences between drug group C and E (P ⁇ 0.01), and drug group E was significantly different from other groups (P ⁇ 0.01).
  • subcutaneous injection is significantly better than oral administration, indicating that the macromolecular structure of sodium arabinoxylan sulfonate is not conducive to the absorption of the digestive tract or may be decomposed by various enzymes of the digestive tract and intestinal microbes, and cannot reach the affected area in the form of effective ingredients. .
  • Example 2 The effect of xylan sulfonates of different plant sources and different structures on rabbit knee arthritis
  • Example 1 bagasse source, arabinoxylan from corn seed coat (Guilin Fibre), and birch wood source xylan (Sigma-Aldrich).
  • the method is the same as in Example 1.
  • the raw materials are arabinoxylan derived from bagasse (branched structure, the molar ratio of xylose to arabinose is 11.2:1), and arabinoxylan derived from corn husk (branched structure, xylose)
  • the molar ratio to arabinose is 3.5:1
  • birch wood-derived xylan direct structure, no arabinose
  • the alkali used is KOH.
  • the potassium sulfonate prepared from the above raw materials are serially numbered as sample one (bagasse source), sample two (corn seed coat source), and sample three (birch source).
  • the structural formulas of the three samples are as follows:
  • Group A The test rabbits have strong hind legs and can move freely.
  • Group B The experimental rabbits do not like activities, the modeled legs do not participate in body support, and even move in a sitting position only with the support of the front legs.
  • Group C The modeled legs of the experimental rabbits are strong and their activities are normal, which is not significantly different from the blank group.
  • Group D The experimental rabbits have strong modeling legs and normal activities, which are not significantly different from the blank group.
  • Group E Individual test rabbits occasionally saw a slight weakness in the modelling leg support, while the rest of the test rabbits were able to move normally.
  • test rabbit joint tissues were removed, the knee joint specimens were grossly observed and scored according to the Pelletier scoring principle (same as Example 1). The results are shown in Table 3. .
  • a is significantly different from group A (P ⁇ 0.01)
  • b is significantly different from group B (P ⁇ 0.01)
  • c is significantly different from group D (P ⁇ 0.05).
  • FIG. 7- Figure 26 The results of tissue sections are shown in Figure 7- Figure 26.
  • the surface of the articular cartilage of the blank group A is smooth, without cracks or defects, the four-layer structure is clear, and the tide line is complete, as shown in Figure 7-10.
  • the articular cartilage layer of the model group B is thinner, the four-layer structure is difficult to distinguish, the surface cartilage defect is serious, the chondrocyte arrangement is disordered, the cells are clustered proliferation and fibrosis, and the tide line is severely damaged. See Figure 11-14 for details.
  • the articular chondrocytes in group C of the drug treatment group are arranged regularly, the hierarchy is basically complete, some cells in the surface layer are complete, and the tide line is still complete, see Figure 15-18 for details.
  • the articular chondrocytes in group D of the drug treatment group are arranged regularly, the hierarchy is basically complete, some cells in the surface layer are complete, and the tide line is still complete, as shown in Figure 19-22.
  • the articular chondrocytes in group E of the drug treatment group are arranged regularly, the hierarchy is basically complete, the cells are occasionally missing in the surface part, and the double tide line can be seen in the samples where the tide line is still intact, as shown in Figure 23- Figure 26.
  • the arabinoxylan from corn seed coat with the most branched and branched chain has the best effect, and the birch wood xylan with linear and without arabinose has the weakest effect. Therefore, wood with high arabinose content is used. Potassium arabinoxylan sulfonate prepared from glycan is beneficial to improve the efficacy of the drug.

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Abstract

一种阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用，属于药品技术领域。经过大量的研究发现，阿拉伯木聚糖磺酸盐具有非常明显的关节炎治疗作用，不仅可以消除炎症，还可以促进软骨修复，一定程度上可以逆转病情，因此，阿拉伯木聚糖磺酸盐可以用于制备治疗骨关节炎的药物。

Description

阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用 技术领域
本发明涉及一种阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用，属于药品技术领域。
背景技术
骨关节炎(Osteoarthritis，OA)是关节软骨的慢性退化性疾病，临床主要表现为缓慢发展的关节疼痛、僵硬、肿胀和活动受限。我国60岁以上人群OA的患病率已经高达50％，而75岁以上则达到80％，该疾病已经成为严重影响老年人生活质量的最常见疾病之一。
目前，对OA的治疗主要有口服药物、关节腔注射药物和手术治疗三种方案。其中，关节腔注射属于穿刺术，对操作要求高且不易反复多次使用，主要针对中、重度患者的症状缓解和改善病情。手术治疗则主要针对严重的中晚期患者，可以采用关节置换术彻底消除疼痛。临床中应用最多的是口服药物，包括镇痛药和软骨保护药两大类，通常两类药物联合使用，同时达到镇痛和促进软骨修复的目的。镇痛药主要包括非甾体消炎药，如双氯芬酸钠、美洛昔康、塞来昔布等；解热镇痛药乙酰氨基酚、阿司匹林等；阿片类止痛药如可待因、盐酸曲马多等。软骨保护药最常用的是氨基葡萄糖、硫酸软骨素、双醋瑞因等。另外，以上镇痛药的某些外用制剂和中药外用药在临床上也有应用。但是药物治疗只能在一段时间内对患者的关节炎症改善，目前尚无药物可逆转病情、彻底治愈。
动物关节细胞中大量存在酸性粘多糖，包括硫酸软骨素、透明质酸等， 它们在机体内发挥着重要的生理功能，例如润滑关节、调节血管壁通透性、调节蛋白质、预防粘连等。因此，硫酸软骨素、透明质酸本身也被用于关节软骨损伤的修复及康复治疗，所以通过硫酸化或磺化的手段制备相关多糖衍生物便成为寻找关节炎治疗药物的重要方向。
阿拉伯木聚糖是2018年被FDA批准的新型膳食纤维，目前对它的研究主要集中在其本身的生理作用上，而对它衍生物的研究甚少。目前，尚未有关于阿拉伯木聚糖磺酸盐在制备治疗骨关节炎药物中的应用的相关报道。
发明内容
本发明的目的是克服现有技术的不足，提供一种阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用。本申请的发明人经过大量的研究发现，阿拉伯木聚糖磺酸盐具有非常明显的关节炎治疗作用，不仅可以消除炎症，还可以促进软骨修复，一定程度上可以逆转病情，因此，阿拉伯木聚糖磺酸盐可以用于制备治疗骨关节炎的药物。
本发明解决上述技术问题的技术方案如下：阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用。
本申请的发明人经过研究，得到如下结论：
第一点：阿拉伯木聚糖磺酸盐通过消除炎症和促进软骨营养发挥骨关节炎治疗作用，对骨关节炎模型兔疗效显著，其效果明显好于市售口服药物组合(塞来昔布和硫酸氨基葡萄糖)。
第二点，阿拉伯木聚糖磺酸盐注射给药的药效优于口服给药的药效。发明人推测，口服给药有可能由于大分子结构不利于消化道吸收或者有可能受到消化道各种酶及肠道微生物的分解，不能以有效成分的形式到达患处，因此注射给药是更好的选择。
第三点，阿拉伯木聚糖磺酸盐本身的结构对疗效有明显的影响，其中支链结构且阿拉伯糖含量高的原料制备的阿拉伯木聚糖磺酸盐的药效更好。
在上述技术方案的基础上，本发明还可以做如下改进。
进一步，所述阿拉伯木聚糖磺酸盐的结构通式为：
Figure PCTCN2019111301-appb-000001
式中，x、y和z的总和为1-19；侧链磺化阿拉伯糖通过1-位氧苷键与主链磺化木糖的2-或/和3-位连接，且两者的摩尔比为1:2-1:20；侧链杂糖通过1-位氧苷键与主链磺化木糖的2-或/和3-位连接；所述磺化杂糖中的杂糖为葡萄糖、葡萄糖醛酸、4-甲基葡萄糖醛酸、半乳糖、鼠李糖、半乳糖醛酸、木糖中的一种或多种。
采用上述进一步的有益效果是：上述阿拉伯木聚糖磺酸盐是由阿拉伯木聚糖经过磺化反应后获得的。阿拉伯木聚糖磺酸盐高分子主链上的侧链磺化阿拉伯糖的数量增加，有利于提升阿拉伯木聚糖磺酸盐治疗骨关节炎的疗效。
进一步，所述阿拉伯木聚糖磺酸盐的制备方法，包括如下步骤：
步骤1：将阿拉伯木聚糖与磺化剂按摩尔比1:(1-5)，一同置于反应釜中，得到第一混合物；
步骤2：按照每(1-8)g步骤1所述阿拉伯木聚糖加入100mL极性有机溶剂的比例，向步骤1得到的第一混合物中加入极性有机溶剂，边搅拌边升温至55℃-95℃，反应1-3小时后降至室温，得到第二混合物；
步骤3：向步骤2得到的第二混合物中加入碱液，所述碱液中的碱与步骤1所述磺化剂的摩尔比为1:1，充分搅拌后，将得到的反应混合物加入到 步骤2所述极性有机溶剂2-5倍体积的乙醇中，待沉淀充分后过滤，得到滤渣；
步骤4：按照每10g滤渣溶于(50-100)mL水的比例进行水溶，用与步骤3中相同的碱液调节pH值为7，加入上述水2-5倍体积的乙醇进行醇沉，将沉淀物过滤，重复上述水溶、醇沉、过滤的操作两次，收集最后的沉淀物，溶于去离子水中，过超滤膜，所得截留液经冷冻干燥，即得到所述阿拉伯木聚糖磺酸盐。
采用上述进一步的有益效果是：在极性有机溶剂中，以阿拉伯木聚糖和磺化剂来制备阿拉伯木聚糖磺酸盐，磺化效率高，也避免了现有技术中吡啶作为溶剂所导致的严重环境污染负担。
更进一步，所述磺化剂为三氧化硫三甲胺复合物、三氧化硫三乙胺复合物、三氧化硫吡啶复合物或三氧化硫N,N-二甲基甲酰胺复合物中的一种；所述极性有机溶剂为二甲基亚砜、N,N-二甲基甲酰胺或N,N-二甲基乙酰胺中的一种；所述室温为20-25℃；所述碱液为NaOH溶液、KOH溶液、Ca(OH) 2溶液中的一种；所述超滤膜的分子截留量为600Da。
更进一步，所述阿拉伯木聚糖提取于单子叶植物。
采用上述进一步的有益效果是：阿拉伯木聚糖是主要存在于单子叶植物半纤维素中的木聚糖，与之相对应的是主要存在于双子叶植物半纤维素中的木聚糖。两种木聚糖除了来源的植物明显不同外，本身的化学结构也存在巨大的差别，最大的区别是双子叶植物木聚糖以单链为主，完全不含支链或者偶有非常少量的葡萄糖醛酸支链连接在木糖的2-位上。而单子叶植物阿拉伯木聚糖是支链结构，含有大量支链，并且支链同时存在于主链木糖的2-和3-位羟基上，支链的主要形式是阿拉伯糖同时伴有少量其它杂糖。本申请的发明人通过大量的试验发现，虽然两类植物来源的木聚糖经过磺化反应后都具备一定的治疗模型兔骨关节炎的能力，但是来源于单子叶植物的阿拉伯木 聚糖磺酸盐疗效更为显著，其结构中的阿拉伯糖支链含量对整体的效果有正相关作用。经过比较不同植物来源，结构以及阿拉伯糖含量不同的木聚糖制备的磺酸盐，可以发现支链越丰富、阿拉伯糖含量越高的单子叶植物阿拉伯木聚糖磺酸盐对兔骨关节炎的治疗效果越好。因此，该来源于单子叶植物的阿拉伯木聚糖磺酸非常有潜力成为骨关节炎的治疗药物。
更进一步，所述单子叶植物为甘蔗、竹子、玉米、燕麦、芦苇、水稻、小麦中的一种或多种。
采用上述进一步的有益效果是：从上述单子叶植物中提取阿拉伯木聚糖，更有利于工业化生产。
进一步，所述药物是以有效量的阿拉伯木聚糖磺酸盐为活性成分，加上药学上可接受的载体和/或辅料制备而成的药物制剂。
采用上述进一步的有益效果是：提高阿拉伯木聚糖磺酸盐的成型性、有效性和稳定性。
进一步，所述药物的剂型为注射剂。
采用上述进一步的有益效果是：相较于其它的剂型，注射剂可以让药物直接进入血液循环到达患处，不会因为消化道造成阿拉伯木聚糖磺酸盐药物的降解。
更进一步，所述注射剂包括水溶液或水复合溶液。
进一步，所述药物的给药方式为皮下注射、肌内注射、静脉注射中的一种或多种。
采用上述进一步的有益效果是：注射给药可以让药物直接进入血液循环到达患处，不会因为消化道造成阿拉伯木聚糖磺酸盐药物的降解。
本发明的有益效果是：
1.本申请的发明人经过大量的研究发现，阿拉伯木聚糖磺酸盐具有非常明显的关节炎治疗作用，不仅可以消除炎症，还可以促进软骨修复，一定程度上可以逆转病情，因此，阿拉伯木聚糖磺酸盐可以用于制备治疗骨关节炎的药物。
2.本申请的发明人发现，阿拉伯木聚糖磺酸盐注射给药的药效优于口服给药的药效。
3.本申请的发明人发现，支链结构且阿拉伯糖含量高的原料制备的阿拉伯木聚糖磺酸盐的药效更好。
附图说明
图1为本发明的实施例1中空白组A的膝关节解剖照片。
图2为本发明的实施例1中模型组B的膝关节解剖照片。
图3为本发明的实施例1中给药组C(阿拉伯木聚糖磺酸钠盐口服组)的膝关节解剖照片。
图4为本发明的实施例1中给药组D(阿拉伯木聚糖磺酸钠盐皮下注射组)的膝关节解剖照片。
图5为本发明的实施例1中给药组E(塞来昔布和硫酸氨基葡萄糖口服组)膝关节解剖照片。
图6为本发明的实施例1中各组试验兔膝关节软骨糖醛酸含量比较。
图7为本发明的实施例2中空白组A的HE染色软骨切片之一。
图8为本发明的实施例2中空白组A的HE染色软骨切片之二。
图9为本发明的实施例2中空白组A的HE染色软骨切片之三。
图10为本发明的实施例2中空白组A的HE染色软骨切片之四。
图11为本发明的实施例2中模型组B的HE染色软骨切片之一。
图12为本发明的实施例2中模型组B的HE染色软骨切片之二。
图13为本发明的实施例2中模型组B的HE染色软骨切片之三。
图14为本发明的实施例2中模型组B的HE染色软骨切片之四。
图15为本发明的实施例2中给药组C(甘蔗渣源)的HE染色软骨切片之一。
图16为本发明的实施例2中给药组C(甘蔗渣源)的HE染色软骨切片之二。
图17为本发明的实施例2中给药组C(甘蔗渣源)的HE染色软骨切片之三。
图18为本发明的实施例2中给药组C(甘蔗渣源)的HE染色软骨切片之四。
图19为本发明的实施例2中给药组D(玉米种皮源)的HE染色软骨切片之一。
图20为本发明的实施例2中给药组D(玉米种皮源)的HE染色软骨切片之二。
图21为本发明的实施例2中给药组D(玉米种皮源)的HE染色软骨切片之三。
图22为本发明的实施例2中给药组D(玉米种皮源)的HE染色软骨切片之四。
图23为本发明的实施例2中给药组E(桦木源)的HE染色软骨切片之一。
图24为本发明的实施例2中给药组E(桦木源)的HE染色软骨切片之二。
图25为本发明的实施例2中给药组E(桦木源)的HE染色软骨切片之三。
图26为本发明的实施例2中给药组E(桦木源)的HE染色软骨切片之四。
图27为本发明的实施例2中各组试验兔膝关节软骨糖醛酸含量比较。
具体实施方式
以下结合附图对本发明的原理和特征进行描述，所举实例只用于解释本发明，并非用于限定本发明的范围。
实施例1：不同给药方式对治疗兔膝关节炎的影响以及与市售药物的对比
骨关节炎是一种慢性、进行性、退行性关节疾病，临床上以关节疼痛、变形和活动受限为特点，病理表现为关节软骨软化、纤维化、溃疡、软骨减少、软骨下骨的硬化、软骨下骨囊肿等。给药方式对药效的发挥有着重要影响，本实验通过不同给药方式考察阿拉伯木聚糖磺酸盐口服和皮下注射对治疗关节炎的影响，并将市售口服药物组合(塞来昔布和硫酸氨基葡萄糖)与阿拉伯木聚糖磺酸钠进行比较研究。
1.材料
1.1.试验动物
成年新西兰大白兔40只，体重2.8±0.2kg，雌雄各半，分笼饲养。
1.2.主要试剂
PBS粉末(北京中杉)，无水乙醇(国药集团)，0.9％氯化钠注射液(河北天成药业有限公司)，多聚甲醛(上海生工)，苏木素染液(南京建成)，伊红染液(碧云天)，中性树脂(国药集团)，竹子源阿拉伯木聚糖(桂林纤元)，塞来昔布胶囊(辉瑞制药)，硫酸氨基葡萄糖胶囊(爱普生物)，水合氯醛、咔唑、三氧化硫吡啶等(上海阿拉丁)。
1.3.主要仪器
一次性注射器(成都新津事丰医疗器械有限公司)，电子天平(Precisa12A，瑞士)，离心管(上海卧宏生物科技有限公司)，除热原超纯水系统(Millipo3150，美国)，高压灭菌锅(上海赛洋)，显微外科手术器械包(上海手术器械厂)，移液器(Eppendorf，德国)，电热压力蒸汽消毒器(绍兴医疗器械总厂)，恒温干燥箱(上海跃进医疗器械厂)，倒置显微镜(日本奥林巴斯)，切片机(德国徕卡)，展片机(德国徕卡)，烤片机(德国徕卡)，紫外-可见分光光度计(上海精科)。
1.4.制备阿拉伯木聚糖磺酸钠盐
制备方法：称取原料竹粉阿拉伯木聚糖(其单糖组成为：木糖与阿拉伯糖的摩尔比为17:1)45g，与210g三氧化硫吡啶复合物一同置于反应釜中，加入750mL DMA，搅拌的条件下升温至55℃，反应1小时后降温冷却。将41g NaOH溶于水加入反应体系，充分搅拌后将反应混合物加入2L的乙醇中，待沉淀充分后过滤。将滤渣溶于水，NaOH调节pH中性，加乙醇析出，过滤，重复水溶醇沉操作两次。将所得产物溶于去离子水，于膜分离机内进行超滤，截留分子量为600Da，所得截留液经冷冻干燥，得到竹子来源的阿拉伯木聚糖磺酸钠，具体结构式如下：
Figure PCTCN2019111301-appb-000002
2.实验部分
2.1.实验分组
成年新西兰大白兔40只，体重2.8±0.2kg，雌雄各半，平均分为5组，分笼饲养。分组情况如下：
A：正常空白组
B：模型组
C：阿拉伯木聚糖磺酸钠盐口服组
D：阿拉伯木聚糖磺酸钠盐皮下注射组
E：塞来昔布和硫酸氨基葡萄糖口服组
2.1.适应性饲养
动物房12h-12h昼夜交替，保护动物只有饮水、进食，温度23-25℃，一周后进入实验。
2.2.构建关节炎模型
B、C、D、E组构建关节炎模型。
试验兔称重，麻醉满意后放于实验台上固定，牵拉右后腿使右膝关节处于伸直位，医用纱布包裹3-4层(缓解石膏直接接触皮肤导致坏死、溃疡)，石膏布包裹制动，石膏外层缠绷带，包裹完成后保持兔子自由活动，制动时间6周。
2.3.分组给药
给药组C按阿拉伯木聚糖磺酸钠盐10mg/kg/天(溶于饮水中)剂量给药；给药组D为造模腿局部皮下注射阿拉伯木聚糖磺酸钠盐6mg/kg剂量给药(生理盐水配制成水溶液，每周二次)；给药组E按塞来昔布7mg/kg/天(剥除胶囊后拌于饲料中)和硫酸氨基葡萄糖15mg/kg/天(剥除胶囊后溶于饮水中)剂量给药；A、B组不给药；连续给药四周。
2.4.取材
动物处死，取膝关节，取股骨外髁关节面软骨少量留作糖醛酸含量测试，其余整个关节于4％(v/v)多聚甲醛固定后进行脱钙，脱钙完成后用于组织切片。
2.5.制作石蜡样本，方法如下：
(1)取材后，用PBS清洗，4％多聚甲醛固定。
(2)脱水：将组织以此浸泡在75％(v/v)乙醇、85％(v/v)乙醇、95％(v/v)乙醇二次、100％(v/v)乙醇中，二次，各10分钟。
(3)透明：将脱水后的组织依次浸泡在50％(v/v)乙醇和50％(v/v)二甲苯的混合溶剂中10分钟，二甲苯10分钟，二次。
(4)透蜡：将透明处理过的组织浸泡于融化的石蜡中3小时。
(5)包埋。
(6)切片：用切片机将石蜡块中的组织切片成5μm厚的薄片，并平铺于防脱玻片上。
(7)烤片：将切片放置于55℃烤片机上，使组织片紧贴于防脱玻片上。
2.6.石蜡切片脱蜡复水：
(1)脱蜡：将石蜡切片分别浸泡于二甲苯10分钟，二次，50％(v/v)乙醇和50％(v/v)二甲苯的混合溶剂10分钟。
(2)复水：将脱蜡后的石蜡切片浸泡于100％(v/v)乙醇二次、95％(v/v)乙醇二次、85％(v/v)乙醇、75％(v/v)乙醇中，各5分钟。
(3)双蒸水清洗3次，每次2分钟。
2.7.苏木精-伊红染色：
(1)苏木素染液染色10分钟。
(2)自来水冲洗多余的染色液，约5分钟。
(3)蒸馏水再洗涤一遍。
(4)镜检，若苏木素着色过深可用1％盐酸乙醇进行分脱色，脱去细胞质中多余的苏木素染色液。
(5)伊红染液染色30秒。
(6)自来水冲洗多余染液，约3分钟。
(7)蒸馏水再洗涤一遍。
2.8.脱水封片：
(1)95％(v/v)乙醇脱水2分钟二次。
(2)二甲苯透明5分钟二次。
(3)中性树脂封片。
(4)镜检，细胞核成蓝色，细胞质成红色或粉红色。
2.9.软骨中糖醛酸含量测定
(1)留取的关节软骨样品，精确称取5mg，置于试管中，稀释至10％的盐酸1mL逐滴加入，封口，于90℃水浴中加热2小时，冷却至室温，加入0.2mL的0.1％咔唑试液显色，静置2小时后与紫外可见分光光度计中测试530nm的吸光度。
(2)标准曲线的绘制，分别取不同浓度的标准糖醛酸样品1mL，如上方法测量吸光度，并绘制拟合浓度-吸光度曲线，即为标准曲线。
(3)利用标准曲线的拟合方程计算样品中糖醛酸的含量。
3.实验结果
3.1.动物行为观察
动物处死前活动情况如下：
A组：试验兔后腿支撑有力，行动自如。
B组：试验兔不爱活动，造模腿不参与身体支撑，甚至仅靠前腿支撑以坐姿移动。
C组：试验兔不爱活动，与模型组没有明显差别。
D组：个别试验兔偶见造模腿支撑略显无力，其余试验兔造模腿有力活动正常。
E组：试验兔活动不受造模腿限制，但可见造模腿支撑仍略显无力，明显不如空白组。
3.2.Pelletier评分
试验兔关节组织取下后，进行膝关节标本大体观察，按Pelletier评分原则进行评分，详见表1，结果详见表2。部分膝关节标本照片见图1-5。
表1 Pelletier评分原则
Figure PCTCN2019111301-appb-000003
表2 Pelletier评分结果
Figure PCTCN2019111301-appb-000004
注：a与A组有显著性差异(P<0.01)，b与B组有显著性差异(P<0.01)，c与C组有显著性差异(P<0.01)，d与D组有显著性差异(P<0.01)。
由表2可见，给药组D和给药组E与其它各组间均有显著性的差异，而阿拉伯木聚糖磺酸钠口服给药组C与模型组B之间并无显著性差异。而皮下注射阿拉伯木聚糖磺酸钠盐的D组效果明显优于口服市售药物组合的E组。
3.3.组织病理评价
空白组A组关节软骨表面光滑，无裂隙或缺损，四层结构清晰，潮线完整。模型组B组关节软骨层变薄，四层结构难以分辨，表层软骨缺损严重，软骨细胞排列紊乱，细胞大量簇聚增生及纤维化，潮线破坏严重。给药组C 组关节软骨层变薄，四层结构难以分辨，表层软骨缺损严重，软骨细胞排列紊乱，细胞大量簇聚增生及纤维化，潮线破坏严重。给药组D组关节软骨细胞排列规则，层次基本完整，表层部分偶见细胞缺失，潮线尚完整。给药组E组关节软骨层变薄，四层结构难以分辨，表层软骨缺损严重，软骨细胞排列紊乱，细胞有簇聚增生及纤维化现象，潮线破坏较严重。
3.4.软骨中糖醛酸的含量
关节软骨蛋白多糖丧失是骨关节炎发生的重要标志，软骨中蛋白多糖和糖醛酸含量较正常会明显减少，软骨中糖醛酸的含量变化与骨关节炎的严重程度呈反比。如图6是各组中试验兔膝关节软骨糖醛酸含量的比较,其中口服给药组C与其它各组均有显著性差异(P<0.05)，给药组D与模型组B、给药组C和E均有显著性差异(P<0.01)，给药组E与其它各组均有显著性差异(P<0.01)。
4.结论
通过动物行为观察、解剖和组织病理观察以及软骨中糖醛酸含量测定，可以发现，各给药组的药效结果为D组强于E组，E组又强于C组。阿拉伯木聚糖磺酸钠皮下注射给药组在各个指标上都明显优于其它各组，不仅优于阿拉伯木聚糖磺酸钠的口服给药组，而且也明显优于市售药物组合(塞来昔布和硫酸氨基葡萄糖)。从软骨中糖醛酸含量大幅回升以及组织病理观察可以推测阿拉伯木聚糖磺酸钠对受损软骨的营养和再生有显著的促进作用；解剖大体观察可以发现阿拉伯木聚糖磺酸钠注射给药的试验兔关节标本炎症已经基本消失；两方面的实验证据可知阿拉伯木聚糖磺酸钠是通过消除炎症和促进软骨营养发挥骨关节炎治疗作用的。而且皮下注射明显优于口服给药说明阿拉伯木聚糖磺酸钠的大分子结构不利于消化道吸收或者有可能受到消化道各种酶及肠道微生物的分解，不能以有效成分的形式到达患处。
实施例2不同植物来源、不同结构的木聚糖磺酸盐对兔膝关节炎的影响
虽然几乎所有的陆生植物细胞壁内都含有各种类型的木聚糖，但木聚糖本身的结构差别却极为显著，甚至截然不同，因此本实施例的目的是研究不同植物来源、结构差异的木聚糖磺酸盐对治疗兔骨关节炎的影响，说明原料中不同的高分子结构与其治疗效果的关系。
1.材料
1.1.试验动物
成年新西兰大白兔40只，体重2.8±0.2kg，雌雄各半，分笼饲养，平均分为5组，分别是：
A：正常空白组
B：模型组
C：样品一给药组
D：样品二给药组
E：样品三给药组
1.2.主要试剂
除原料来源不同外，其余同实施例1，具体为：甘蔗渣源、玉米种皮源阿拉伯木聚糖(桂林纤元)，桦木源木聚糖(Sigma-Aldrich)。
1.3.主要仪器
同实施例1。
1.4.制备阿拉伯木聚糖磺酸钠盐
方法同实施例1，原料分别为甘蔗渣来源的阿拉伯木聚糖(支链结构，木糖与阿拉伯糖的摩尔比为11.2:1)，玉米皮来源阿拉伯木聚糖(支链结构，木糖与阿拉伯糖的摩尔比为3.5:1)和桦木来源木聚糖(直连结构，无阿拉伯糖)，所用的碱均为KOH。以上原料所制备的磺酸钾盐依次编号为样品一 (甘蔗渣源)、样品二(玉米种皮源)和样品三(桦木源)，三个样品的结构式分别如下：
样品一：
Figure PCTCN2019111301-appb-000005
样品二：
Figure PCTCN2019111301-appb-000006
样品三：
Figure PCTCN2019111301-appb-000007
2.实验部分
除给药方式全部为皮下注射外，包括注射剂量在内其它试验方法等均与实施例1相同。
3.实验结果
3.1.动物行为观察
动物处死前活动情况如下：
A组：试验兔后腿支撑有力，活动自如。
B组：试验兔不爱活动，造模腿不参与身体支撑，甚至仅靠前腿支撑以坐姿移动。
C组：试验兔造模腿有力，活动正常，与空白组没有明显区别。
D组：试验兔造模腿有力，活动正常，与空白组没有明显区别。
E组：个别试验兔偶见造模腿支撑略显无力，其余试验兔造模腿有力活动正常。
3.2.Pelletier评分
试验兔关节组织取下后，进行膝关节标本大体观察，按Pelletier评分原则进行评分(同实施例1)，结果详见表3。。
表3 Pelletier评分结果
Figure PCTCN2019111301-appb-000008
注：a与A组有显著性差异(P<0.01)，b与B组有显著性差异(P<0.01)，c与D组有显著性差异(P<0.05)。
由表3可见，相对于模型组，三个给药组的样本关节软骨的损伤都更小，说明三个样品都能够达到促进关节软骨恢复的作用，尤其D组表现最好。三个给药组之间只有D、E二组存在显著性差异(P<0.05)。
3.3.组织病理评价
组织切片结果见图7-图26。其中，空白组A组关节软骨表面光滑，无裂隙或缺损，四层结构清晰，潮线完整，详见图7-图10。模型组B组关节软骨层变薄，四层结构难以分辨，表层软骨缺损严重，软骨细胞排列紊乱，细 胞大量簇聚增生及纤维化，潮线破坏严重，详见图11-图14。给药组C组关节软骨细胞排列规则，层次基本完整，表层部分细胞完整，潮线尚完整，详见图15-图18。给药组D组关节软骨细胞排列规则，层次基本完整，表层部分细胞完整，潮线尚完整，详见图19-图22。给药组E组关节软骨细胞排列规则，层次基本完整，表层部分偶见细胞缺失，潮线尚完整部分样本可见双潮线，详见图23-图26。
3.4.软骨中糖醛酸的含量
各组中试验兔膝关节软骨糖醛酸含量的比较,详见图27。其中模型组与其它各组均有显著性差异(P<0.01)，给药组C与空白组A和给药组E有显著性差异(P<0.05)，给药组D与给药组E有显著性差异(P<0.01)。
4.结论
从动物活动情况可见，三个给药组均表现出了明显的关节炎治疗效果；解剖大体观察和病理切片都可以发现D组实验动物关节软骨恢复状况最好，C组次之，E组相对略差；软骨中糖醛酸含量的结果与解剖和病例切片结果基本一致，同样是给药组D的关节软骨恢复情况最好，E组恢复稍慢。由此可见，原料木聚糖的来源和结构对骨关节炎疗效影响明显，三个木聚糖的来源中玉米种皮和甘蔗渣均为单子叶植物，桦木为双子叶植物。其中，支链型且支链最丰富的玉米种皮源的阿拉伯木聚糖效果最好，直链型且不含阿拉伯糖的桦木源木聚糖效果最弱，所以采用阿拉伯糖含量高的木聚糖制备的阿拉伯木聚糖磺酸钾有利于提升药效。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述阿拉伯木聚糖磺酸盐的结构通式为：
    Figure PCTCN2019111301-appb-100001
    式中，x、y和z的总和为1-19；侧链磺化阿拉伯糖通过1-位氧苷键与主链磺化木糖的2-或/和3-位连接，且两者的摩尔比为1:2-1:20；侧链杂糖通过1-位氧苷键与主链磺化木糖的2-或/和3-位连接；所述磺化杂糖中的杂糖为葡萄糖、葡萄糖醛酸、4-甲基葡萄糖醛酸、半乳糖、鼠李糖、半乳糖醛酸、木糖中的一种或多种。
  3. 根据权利要求1所述的应用，其特征在于，所述阿拉伯木聚糖磺酸盐的制备方法，包括如下步骤：
    步骤1：将阿拉伯木聚糖与磺化剂按摩尔比1:(1-5)，一同置于反应釜中，得到第一混合物；
    步骤2：按照每(1-8)g步骤1所述阿拉伯木聚糖加入100mL极性有机溶剂的比例，向步骤1得到的第一混合物中加入极性有机溶剂，边搅拌边升温至55℃-95℃，反应1-3小时后降至室温，得到第二混合物；
    步骤3：向步骤2得到的第二混合物中加入碱液，所述碱液中的碱与步骤1所述磺化剂的摩尔比为1:1，充分搅拌后，将得到的反应混合物加入到步骤2所述极性有机溶剂2-5倍体积的乙醇中，待沉淀充分后过滤，得到滤渣；
    步骤4：按照每10g滤渣溶于(50-100)mL水的比例进行水溶，用与步骤3中相同的碱液调节pH值为7，加入上述水2-5倍体积的乙醇进行醇沉，将沉淀物 过滤，重复上述水溶、醇沉、过滤的操作两次，收集最后的沉淀物，溶于去离子水中，过超滤膜，所得截留液经冷冻干燥，即得到所述阿拉伯木聚糖磺酸盐。
  4. 根据权利要求3所述的应用，其特征在于，所述磺化剂为三氧化硫三甲胺复合物、三氧化硫三乙胺复合物、三氧化硫吡啶复合物或三氧化硫N,N-二甲基甲酰胺复合物中的一种；所述极性有机溶剂为二甲基亚砜、N,N-二甲基甲酰胺或N,N-二甲基乙酰胺中的一种；所述室温为20-25℃；所述碱液为NaOH溶液、KOH溶液、Ca(OH) 2溶液中的一种；所述超滤膜的分子截留量为600Da。
  5. 根据权利要求3所述的应用，其特征在于，所述阿拉伯木聚糖提取于单子叶植物。
  6. 根据权利要求5所述的应用，其特征在于，所述单子叶植物为甘蔗、竹子、玉米、燕麦、芦苇、水稻、小麦中的一种或多种。
  7. 根据权利要求1所述的应用，其特征在于，所述药物是以有效量的阿拉伯木聚糖磺酸盐为活性成分，加上药学上可接受的载体和/或辅料制备而成的药物制剂。
  8. 根据权利要求1所述的应用，其特征在于，所述药物的剂型为注射剂。
  9. 根据权利要求8所述的应用，其特征在于，所述注射剂包括水溶液或水复合溶液。
  10. 根据权利要求8所述的应用，其特征在于，所述药物的给药方式为皮下注射、肌内注射、静脉注射中的一种或多种。
PCT/CN2019/111301 2019-01-15 2019-10-15 阿拉伯木聚糖磺酸盐在制备治疗骨关节炎的药物中的应用 WO2020147350A1 (zh)

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