WO2016123795A1 - 绿原酸在制备治疗骨硬化病的药物中的用途 - Google Patents

绿原酸在制备治疗骨硬化病的药物中的用途 Download PDF

Info

Publication number
WO2016123795A1
WO2016123795A1 PCT/CN2015/072415 CN2015072415W WO2016123795A1 WO 2016123795 A1 WO2016123795 A1 WO 2016123795A1 CN 2015072415 W CN2015072415 W CN 2015072415W WO 2016123795 A1 WO2016123795 A1 WO 2016123795A1
Authority
WO
WIPO (PCT)
Prior art keywords
chlorogenic acid
group
mice
preparation
bone
Prior art date
Application number
PCT/CN2015/072415
Other languages
English (en)
French (fr)
Inventor
张洁
张梦甜
黄望
Original Assignee
四川九章生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 四川九章生物科技有限公司 filed Critical 四川九章生物科技有限公司
Priority to PCT/CN2015/072415 priority Critical patent/WO2016123795A1/zh
Publication of WO2016123795A1 publication Critical patent/WO2016123795A1/zh

Links

Images

Classifications

    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/216—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acids having aromatic rings, e.g. benactizyne, clofibrate

Definitions

  • the present invention relates to the use of chlorogenic acid for the preparation of a medicament for the treatment of osteopetrosis.
  • Osteosclerosis also known as marble osteopetrosis, or primary fragile sclerosis
  • the skull is one of the most common sites.
  • the bones are extremely dense and lose their original structure, just like marble, but the increase in bone fragility is prone to fracture.
  • the cause of the osteopetrosis is not clear, and may be related to abnormal bone resorption, resulting in excessive deposition of calcium salts in the bone, the appearance of marble or ivory, increased fragility.
  • the disease has a family history and is more common among children who are married to close relatives.
  • osteosclerosis is only hematopoietic stem cell transplantation, but it is very difficult to find transplant donors of hematopoietic stem cells.
  • a combination of symptomatic treatments such as the treatment of anemia with prednisone, Shujinghuoxue tablets for bone damage, but the treatment effect is not good, and the side effects Large, for example, prednisone can cause femoral head necrosis, stress ulcers, steroid-induced hyperglycemia, easy infection and mental disorders.
  • the technical solution of the present invention is to provide a new use of chlorogenic acid.
  • the present invention provides the use of chlorogenic acid for the preparation of a medicament for treating osteopetrosis.
  • the medicament is prepared by using an effective amount of chlorogenic acid as an active ingredient, and adding a pharmaceutically acceptable auxiliary or auxiliary component.
  • the pharmaceutical preparation contains 1 to 1000 mg of chlorogenic acid per preparation unit.
  • the medicinal preparation uses chlorogenic acid in a dose of 1 to 100 mg/kg.
  • the agent is an oral preparation or an injection.
  • the chlorogenic acid of the present invention can effectively treat osteopetrosis, and the therapeutic effect is better than the combination of the positive drug prednisone and Shujinghuoxue tablets, and it has been proved to be a safe drug with little side effects and clinical Good application prospects.
  • Figure 2 shows the results of detection of type II carbonic anhydrase in osteoclasts of each group of mice. *p ⁇ 0.05, **p ⁇ 0.01 compared with the normal group; #p ⁇ 0.05, ##p ⁇ 0.01 compared with the model group; ⁇ p ⁇ 0.05, ⁇ p ⁇ 0.01 compared with the combined administration group.
  • Example 1 In vivo pharmacodynamic study of chlorogenic acid in the treatment of osteosclerosis
  • the animal model used in this example is a directly purchased BALB/C mouse suffering from osteopetrosis (this mouse is also a common model for basic research on osteosclerosis at home and abroad).
  • this mouse model due to abnormal bone growth, the bones are hard, the bone marrow cavity disappears, and all bones are hardened with edentulism.
  • This mouse lesion is similar to human sclerosis.
  • Chlorogenic acid prednisone (Hebei Xinyinhe Chemical Co., Ltd.), Shujin Huoxue Tablet (Henan Zhongjie Pharmaceutical Co., Ltd.), double distilled water, 12-well sterile plate, 96-well sterile plate, high power microscope, Electronic balance (one hundred thousandth).
  • Osteosclerosis is a rare disease, and its clinical treatment and research are relatively lacking. There is no specific drug for treating osteopetrosis at home and abroad. Most of the drugs stay at the level of symptomatic treatment.
  • a combination of prednisone and Shujin Huoxue tablets was used as a positive control group. According to the dose: prednisone (6mg / kg), Shujin Huoxue tablets (1 tablet / only) for drug treatment. Ordinary BALB/C mice were used as a normal control group.
  • Blood was taken from each group of mice by blood sampling from the tail vein, and then the red blood cells and platelet indexes in the blood were detected.
  • mice The appearance characteristics, body weight changes, and the like of each group of mice were examined. After the end of the experiment, the mice were sacrificed, and the liver and spleen weights of each group of mice were weighed, and the percentage of liver and spleen (liver or spleen weight/mouse body weight) was calculated.
  • the long bones of the limbs of the mouse dissected in the previous step were separated, and the soft tissues and osteophytes attached to the surface of the bone were carefully removed.
  • the bone portion was washed with PBS and placed in a culture medium containing 15% 199 (containing 10% calf serum, 5%).
  • the inner surface of the bone was gently scraped into the culture solution with a scalpel, and the bone fragments were repeatedly blown for 2 min with a round head.
  • the upper cell suspension density of 1 ⁇ 10 6 /mL
  • the number of absorption lacuna on each bone fragment was counted 100 times under the bone plate absorption tract by toluidine blue staining light microscopy, and recorded and analyzed.
  • mice The results of the detection of red blood cells and platelets in mice showed that the number of red blood cells and platelets in the model group of patients with osteopetrosis was significantly lower, which was significantly different from the normal control group (p ⁇ 0.01).
  • the number of red blood cells and platelets in the co-administered group was significantly higher than that in the model group, and there was a significant difference between the two groups (p ⁇ 0.01), but it was significant compared with the normal control group. Difference (p ⁇ 0.05).
  • the number of blood cells and platelets also increased significantly, compared with the model group, there was a very significant difference (p ⁇ 0.01), and medium and high doses
  • the chlorogenic acid administration group had already reached the normal value of red blood cells and platelets, and there was no statistical difference between the normal control group and the normal control group (p>0.05).
  • the number of red blood cells and platelets in the mice treated with different doses of chlorogenic acid was higher than that in the co-administered group.
  • the data of each group showed significant difference compared with the co-administered group (p ⁇ 0.05).
  • mice in the model group died severely, and 7 out of 10 died, and the surviving mice were stiff and the muscles were stiff. With concomitant convulsions.
  • the relative mortality was low, 4 mice died, and the mice's activities were basically normal, the bone stiffness was weak, the facial muscle stiffness was severe, and small was observed.
  • the rat has convulsions.
  • the mortality was significantly reduced, and the mice in the low-dose chlorogenic acid treatment group died, and the mice were basically normal, and the bones were not obviously stiff, only two small The mouse's face has muscle stiffness.
  • mice in the middle-dose chlorogenic acid treatment group did not die, and the activities were normal, the bones were not stiff, and the muscles were not stiff.
  • One of the mice in the high-dose chlorogenic acid treatment group died, and its activity was basically normal.
  • the bones of the mice showed no obvious rigidity and the face was normal.
  • Liver and spleen enlargement is one of the clinical manifestations of osteopetrosis.
  • the liver and spleen after dissection of the mice in each group are weighed to obtain the percentage index of liver and spleen weight.
  • the results show that compared with the normal group, the model The percentage of liver and spleen weight of the mice in the group was significantly increased, suggesting that the liver and spleen were enlarged.
  • the percentage of liver and spleen weight decreased significantly in mice treated with combination therapy, and there was a significant difference between the model group and the model group (p ⁇ 0.05).
  • the percentage of liver and spleen in the mice treated with chlorogenic acid also decreased significantly, and there was a significant difference between the model group and the model group (p ⁇ 0.05).
  • the results of all measurements are shown in Table 3.
  • mice in the osteosclerosis model group have bone fragment absorption pockets compared with normal control mice. The number of traps and the area of the abscess are very small, suggesting that there is a serious obstacle to osteoclastic bone resorption.
  • mice in the high, medium and low dose chlorogenic acid administration group had significantly increased absorption pocket area, and the increase range of each group was different. Compared with the model group, they were very different. Significant difference (p ⁇ 0.01).
  • mice with osteosclerosis were used as experimental models.
  • the combination of prednisone and Shujinhuoxue tablets is used.
  • the method was used as a positive control for the treatment of osteosclerosis mice.
  • the appearance of the mice, the liver and spleen weight index, the survival rate, the number and area of red blood cells, platelets, bone resorption nests and the area of the mice showed that the survival status of the osteosclerosis mice after chlorogenic acid treatment Good, appearance and other signs are normal, liver and spleen weight tend to normalize, and red blood cells and platelets have been significantly improved.
  • Example 2 Effect of chlorogenic acid on the expression of CA2 gene and its corresponding type II carbonic anhydrase in osteoclasts in a mouse model of osteopetrosis.
  • the animal model used in this example was a directly purchased BALB/C mouse suffering from osteopetrosis.
  • the bones due to abnormal bone growth, the bones are hard, the bone marrow cavity disappears, and all bones are hardened with edentulism.
  • This mouse lesion is similar to human sclerosis.
  • Chlorogenic acid prednisone (Hebei Xinyinhe Chemical Co., Ltd.), Shujin Huoxue Tablet (Henan Zhongjie Pharmaceutical Co., Ltd.), type II carbonic anhydrase ELISA kit, PCR instrument, RNA extraction kit, cDNA Chain synthesis kit, double distilled water, 12-well and 96-well plates, microplate reader, electronic balance.
  • Osteosclerosis is a rare disease, and its clinical treatment and research are relatively lacking. There is no specific drug for treating osteopetrosis at home and abroad. Most of the drugs stay at the level of symptomatic treatment.
  • prednisone (6mg / kg)
  • Shujin Huoxue tablets (1 tablet / only) for drug treatment.
  • Ordinary BALB/C mice were used as a normal control group.
  • prednisone prednisone
  • the specific grouping and administration methods are shown in Table 5:
  • the corresponding first strand cDNA is synthesized using the cDNA first strand synthesis kit and the above extracted RNA solution.
  • the specific reverse transcription process follows the instructions and is briefly described as follows:
  • RNA template 5 mL Oligo (dT) 2Ml, Super Pure dNTP 2Ml, RNase-Free ddH2O 5.5 mL.
  • EvaGreen fluorescent dyes bind to double-stranded DNA to produce strong fluorescence. By detecting the final fluorescence intensity, we can get the total amount of DNA generated by the reaction. Fluorescent dye was added to the test tube, the cDNA product synthesized in (1) (2), and the CA2 primer were uniformly mixed and subjected to RT-PCR. The system used RNase-free water instead of cDNA products as the NTC control. Each sample was set to a relative value of the ⁇ -actin primer set for relative quantification.
  • the group B osteoclast suspension described in 2.2.1 (1) was taken and evenly whipped, carefully incubated in a 12-well plate, and tested after 24 hours. After protein quantification by the BCA method, a subsequent test was carried out using a type II carbonic anhydrase ELISA enzyme-linked immunoassay kit to determine type II carbonic anhydrase activity.
  • CA2 gene The detection of CA2 gene showed that the expression of CA2 was relatively high in the osteoclasts of the normal group, while the expression of CA2 gene in the osteoclasts was significantly down-regulated in the mice of the osteosclerosis model group.
  • After treatment with co-administration and chlorogenic acid there was no significant increase in CA2 gene in mouse osteoclasts in the co-administered group, which was not statistically different from the model group (p>0.05).
  • the chlorogenic acid-treated mice significantly increased the expression of CA2 gene in mouse osteoclasts, which was significantly different from the model group and the co-administered group (**p ⁇ 0.01, ##p ⁇ 0.01).
  • the experimental results of the gene assay are shown in Figure 1.
  • type II carbonic anhydrase showed that the content of type II carbonic anhydrase in osteoclasts of osteopetrosis model mice was significantly lower than that of the normal control group, while type II carbonic anhydrase was osteoclast bone resorption. The key to promoting enzymes, this decline is shown to be a severely insufficient bone resorption capacity of osteoclasts in a mouse model of osteopetrosis.
  • the type II carbonic anhydrase content was significantly different from the control group (**p ⁇ 0.01). There was no significant change in type II carbonic anhydrase content in mouse osteoclasts after combination therapy, and there was no statistical difference between the model group and the model group (p>0.05).
  • BALB/C mice suffering from osteopetrosis were used as model animals, and prednisone and Shujinhuoxue tablets were combined as a positive control group.
  • the expression of CA2 gene and corresponding type II carbonic anhydrase in osteoclasts of each group were determined.
  • the results showed that chlorogenic acid can effectively up-regulate the expression of CA2 gene in mouse osteoclasts and promote the content of corresponding type II carbonic anhydrase in comparison with the co-administered group.
  • the main function of type II carbonic anhydrase is to promote bone resorption of osteoclasts, and its activation can significantly improve osteosclerosis caused by poor absorption of osteoclasts.
  • chlorogenic acid can significantly activate type II carbonic anhydrase activity and has a certain therapeutic effect on osteopetrosis.
  • the chlorogenic acid raw material used in the present embodiment is obtained by extracting and purifying from the leaves of Eucommia ulmoides Oliv. The degree is 99.38%.
  • the above prescription was completely dissolved in water for injection, filtered, and then finely filtered with a 0.22 ⁇ m sterile microfiltration membrane to adjust the pH. After the conventional operation of the sterile powder injection, 1000 ml of 2 ml powder injections were prepared. Contains 40mg of chlorogenic acid.
  • the chlorogenic acid used in the present example was obtained by extraction and purification from Eucommia ulmoides leaves, and the purity was 99.21%.
  • Chlorogenic acid with a purity of 99.21% (main drug) 1g Starch (diluent) 50g Dextrin powder (adhesive) Moderate amount Anhydrous ethanol (wetting agent) Moderate amount
  • the chlorogenic acid used in the present example was extracted and purified from the burdock leaves, and the purity was 99.29%.
  • each oral solution is 100mL, containing green
  • the original acid is 200mg.
  • the chlorogenic acid used in the present embodiment is extracted and purified from honeysuckle, and the purity is obtained.
  • a chlorogenic acid tablet is prepared by a wet granule tableting method.
  • (1) Prepare an aqueous solution of hypromellose according to the prescription; (2) mix the prescribed amount of chlorogenic acid, powdered sugar and lactose, add the hypromellose aqueous solution, stir well and make it soft. (3) the prepared soft material according to the conventional wet granulation operation procedure, sieving, drying and granulating to obtain granules of uniform size; (4) mixing the prepared granules with magnesium stearate uniformly After compression, a total of 1000 tablets were prepared, each containing 100 mg of chlorogenic acid.
  • the chlorogenic acid used in the present example was obtained by extraction and purification from Eucommia ulmoides leaves, and the purity was 99.25%.
  • each capsule contains 50mg of chlorogenic acid.
  • the chlorogenic acid used in the present example was extracted and purified from Eucommia ulmoides leaves, and the purity was 98.92%.
  • Chlorogenic acid with a purity of 98.92% 200g Mannitol (diluent) 100g Sucrose (diluent) 400g Povidone K30 (adhesive) Moderate amount
  • Povidone K30 was taken and water for injection was added to prepare a solution. After the prescription amount of chlorogenic acid, mannitol and lactose were uniformly mixed, the povidone K30 solution was added to prepare a soft material. According to the conventional preparation process of the granules, the granules are obtained after sieving, drying and granulating the soft materials. The granules were dispensed under aseptic conditions to prepare 400 bags of granules, each bag containing 500 mg of chlorogenic acid.
  • the chlorogenic acid raw material used in the present embodiment is obtained by extracting and purifying the burdock leaves, and the purity is 98.82%.
  • the chlorogenic acid of the invention can effectively treat osteopetrosis, and the therapeutic effect is obviously superior to the currently used ossification treatment drug: the combination of prednisone and Shujinghuoxue tablets, with small side effects and good clinical application prospects. .

Landscapes

  • Health & Medical Sciences (AREA)
  • Emergency Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

Abstract

本发明提供绿原酸在制备治疗骨硬化病的药物中的用途。绿原酸可以有效治疗骨硬化病，治疗效果优于阳性药物强的松与舒经活血片的联合用药，并且副作用较小。

Description

绿原酸在制备治疗骨硬化病的药物中的用途 技术领域
本发明涉及绿原酸在制备治疗骨硬化病的药物中的用途。
背景技术
骨硬化病，又称大理石骨症，或称原发性脆骨硬化症，是一种少见的全身性骨结构发育异常的先天性疾病，颅骨为好发部位之一。骨质极为密，并失去原来的结构，犹如大理石，但骨脆性增加易发生骨折。尚可以伴贫血、眼萎缩及耳聋等情况。一般认为，绝大多数病人在出生前即已开始有病变。根据临床表现分为恶性(幼儿型)及良性(成人型)两种，前者常为死产或出生后死于贫血，预后差。石骨症的发病原因尚不明确，可能与骨吸收异常有关，致使钙盐过量沉积于骨内，外观呈大理石或象牙样，脆性增加。本病有家族史，多见于近亲结婚的子女中。
目前，骨硬化病的有效治疗途径只有造血干细胞移植，但是找到造血干细胞的移植供者却非常困难。基于治疗的难度和发病机理的不明确，通常情况下均采用联合给药对症治疗的治疗方案，如用强的松治疗贫血，舒经活血片治疗骨损伤，但是治疗效果不佳，且毒副作用大，如，强的松会引起股骨头坏死，应激性溃疡，类固醇性高血糖，容易合并感染及精神异常等等。
未见绿原酸治疗骨硬化病的报道。
发明内容
本发明的技术方案是提供了绿原酸的新用途。
本发明提供了绿原酸在制备治疗骨硬化病的药物中的用途。
其中，所述的药物是以有效量的绿原酸为活性成分，加上药学上可接受的辅料或者辅助性成分制备而成的制剂。
其中，所述的药物制剂中每制剂单位含有绿原酸1～1000mg。
其中，所述的药物制剂中绿原酸的使用剂量为1～100mg/kg。
其中，所述的药剂是口服制剂或者注射剂。
综上，本发明绿原酸可以有效治疗骨硬化病，治疗效果比阳性药物强的松与舒经活血片的联合用药还要好，而且其已经被证明是一种安全的药物，副作用小，临床应用前景良好。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、 替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1各组小鼠破骨细胞中CA2基因的表达情况
图2各组小鼠破骨细胞中Ⅱ型碳酸酐酶的检测结果。与正常组相比*p<0.05，**p<0.01；与模型组相比#p<0.05，##p<0.01；与联合给药组相比△p<0.05，△△p<0.01。
具体实施方式
实施例1：绿原酸治疗骨硬化病体内药效学试验研究
1.实验材料
1.1动物
骨硬化病BALB/C小鼠50只。
本实施例中所使用的动物模型，是直接购买的患有骨硬化症BALB/C小鼠(该小鼠也是国内外骨硬化病基础研究的常用模型)。该小鼠模型由于病鼠骨质生长异常，骨质硬，骨髓腔消失，所有骨骼都硬化，并伴有无齿症。这种鼠的病变的与人类硬骨病相似。
1.2实验药物及仪器
绿原酸，强的松(河北鑫银河化工有限公司)，舒筋活血片(河南中杰药业有限责任公司)，双蒸水，12孔无菌板，96孔无菌板，高倍显微镜，电子天平(十万分之一)。
2.实验方法
2.1实验动物分组及给药
骨硬化病属于罕见病，临床对其的治疗和研究都比较欠缺，国内外尚没有治疗骨硬化病的特效药，用药大多停留在对症治疗的层面上。本实验采用强的松和舒筋活血片联合用药的给药方案作为阳性对照组。按给药剂量：强的松(6mg/kg)，舒筋活血片(1片/只)进行给药治疗。以普通BALB/C小鼠作为正常对照组。
将骨硬化病小鼠模型随机平均分为5组，分别命名为模型对照组(模型组，n＝10)、强的松和舒筋活血片联合治疗组(联合治疗组，n＝10)、绿原酸低剂量治疗组(L-LYS组，n＝10)、绿原酸中剂量治疗组(M-LYS组，n＝10)和绿原酸高剂量治疗组(H-LYS组，n＝10)，连同正常对照组(NC组，n＝10)，共6个实验组，共60只。具体分组及给药方式如表1所示：
表1.实验分组、给药方式及给药量(注：所有给药组的给药体积相同)
Figure PCTCN2015072415-appb-000001
2.2.检测指标和实验方法
2.2.1小鼠红细胞和血小板指标检测
采用尾静脉取血的方式对各组小鼠进行取血，之后对血中的红细胞和血小板指标等进行检测。
2.2.2小鼠外貌特征、生存率和肝脾重量的检测
对各组小鼠的外貌特征、体重变化等进行检测。实验结束后，处死小鼠，称取各组小鼠的肝脏和脾脏重量，计算肝、脾百分比(肝脏或脾脏重量/小鼠体重)。
2.2.3小鼠骨片吸收陷窝计数及吸收陷窝面积测定
①破骨细胞的分离
将上步解剖后的小鼠的四肢长骨进行分离，仔细清除附着于骨头表面的软组织和骨骺，骨干部分用PBS清洗后放入盛有15％199培养液(含10％小牛血清、5％胎牛血清、100μg/mL硫酸链霉素、100U/mL青霉素钠，pH7.2)的玻璃平皿中。用解剖刀将骨质内表面轻刮入培养液，再用圆头习惯反复吹打骨质碎片2min，静置30s后将上层细胞悬液(密度为1×106/mL)均匀接种于预置薄骨片的24孔培养板内。每孔分别加入破骨细胞悬液1mL及含血清的细胞培养液1mL，进行培养。
②骨片吸收陷窝技术及吸收陷窝面积测定
采用骨片吸收陷窝甲苯胺蓝染色光镜法100倍下计数各组骨片上的吸收陷窝数，进行记录和分析。
3.实验结果
3.1小鼠红细胞和血小板指标检测
小鼠的红细胞和血小板指标检测的结果显示，患有骨硬化病的模型组小鼠，其红细胞和血小板数显著降低，其与正常对照组相比，具有非常显著性差异(p<0.01)。联合给药组的小鼠的红细胞和血小板数较模型组相比有了明显升高，二者之间有非常显著性差异(p<0.01)，但与正常对照组相比，尚有显著性差异(p<0.05)。绿原酸治疗组的小鼠，其血细胞和血小板数也有显著升高，与模型组相比，有非常显著性差异(p<0.01),且中、高剂量的 绿原酸给药组，已经能使红细胞和血小板数达到正常值，与正常对照组之间没有统计学差异(p>0.05)。不同剂量绿原酸给药组小鼠的红细胞和血小板数，均高于联合给药组，各组数据分别与联合给药组相比后显示有显著性差异(p<0.05)。
表2 各组小鼠的红细胞和血小板测定结果
Figure PCTCN2015072415-appb-000002
3.2小鼠外貌特征、体重、生存率和肝、脾重量百分比的检测
治疗60天后，观察小鼠的各种外貌和体态特征，结果发现：(1)模型组的小鼠死亡严重，10只中有7只死亡，且存活的小鼠骨头僵直，肌肉也大面积僵硬，且伴随间隔性抽搐。(2)联合给药治疗组的小鼠，相对死亡率较低，有4只小鼠死亡，且小鼠的活动基本正常，骨头僵直程度较弱，面部肌肉僵硬较严重，且也观察到小鼠有抽搐现象。(3)绿原酸治疗的小鼠中，其死亡率明显降低，低剂量绿原酸治疗组的小鼠死亡1只，且小鼠活动基本正常，骨头也未见明显僵直，只有两只小鼠的面部有肌肉僵硬的现象。而中剂量绿原酸治疗组的小鼠未见死亡，且活动正常，骨头未僵直，肌肉也未僵硬。高剂量的绿原酸治疗组的小鼠死亡一只，其活动基本正常，小鼠骨头未见明显僵直，面部正常。
肝、脾脏肿大，是骨硬化病的临床表现之一，对各组的小鼠解剖后的肝脏和脾脏进行称重后得到肝、脾重量百分比指数，结果显示，与正常组相比，模型组的小鼠肝、脾重量百分比明显升高，提示其肝、脾肿大程度较大。而联合给药治疗的小鼠，其肝、脾重量百分比均有明显下降，与模型组之间有显著性差异(p<0.05)。而绿原酸治疗的各组小鼠，其肝、脾脏的重量百分比，也有较为明显的下降，与模型组之间存在显著性差异(p<0.05)。所有测定结果如表3所示。
表3 外貌特征和肝、脾重量百分比的检测
Figure PCTCN2015072415-appb-000003
Figure PCTCN2015072415-appb-000004
与正常对照组相比a:P<0.01 b:p<0.05与模型组相比c:p<0.01 d:p<0.05
与联合给药组相比e:p<0.01 f:p<0.05
3.3小鼠骨片吸收陷窝计数及吸收陷窝面积测定
骨硬化病的根本是由于破骨细胞性骨吸收严重障碍，本部分实验的结果显示：(1)骨硬化病模型组的小鼠，与正常对照组的小鼠相比，其骨片吸收窝陷数目和吸收窝陷面积非常少，提示其破骨细胞性骨吸收发生严重障碍。(2)联合给药治疗组的小鼠，其骨片吸收陷窝数无明显升高，与模型组之间无统计学差异(p>0.05)。(3)高、中、低剂量绿原酸给药组的小鼠，其吸收窝陷面积均有明显的升高，各组的增加幅度有所不同，分别与模型组相比，均有非常显著性差异(p<0.01)。高、中剂量绿原酸治疗组的小鼠，其骨片吸收陷窝计数和吸收陷窝面积的测定结果，与正常对照组之间没有统计学差异(p>0.05)。测定结果显示，绿原酸能够显著增加骨片吸收陷窝计数及吸收陷窝面积，促进破骨细胞性骨吸收。而联合给药组没有表现出相应的实验结果。所有测定结果如表4所示。
表4 骨片吸收陷窝计数及吸收陷窝面积测定结果
Figure PCTCN2015072415-appb-000005
Figure PCTCN2015072415-appb-000006
4统计学处理
连续型变量形式的实验数据以
Figure PCTCN2015072415-appb-000007
表示，2组数据间的比较用两样本t检验。采用SPSS13.0统计软件，P<0.05为有统计学意义。
5.结论
本实施例以骨硬化的BALB/C小鼠作为实验模型，鉴于国内外尚没有治疗骨硬化病的特效药，故而结合临床的实际治疗方案，采用强的松和舒筋活血片联合给药的方法来作为阳性对照治疗骨硬化病小鼠。通过各组小鼠的外貌体征、肝脏和脾脏重量指数、生存率、红细胞、血小板、骨吸收窝陷数目和面积的测定，结果显示，绿原酸治疗后的骨硬化病小鼠，其生存状况良好，外貌体征等较为正常，肝、脾脏重量趋于正常化，且红细胞和血小板有了显著提高，另外，破骨细胞骨吸收窝陷数目和面积有了显著的提高，高、中、低剂量绿原酸治疗组的测定结果均高于强的松和舒筋活血片联合给药治疗组，提示绿原酸能够有效促进破骨细胞的骨性吸收，显著改善小鼠的红细胞和血小板数目，对骨硬化病的生存状况等都有非常明显的提高，表现出良好的药效。
实施例2：绿原酸对骨硬化病小鼠模型破骨细胞中的CA2基因及其对应的Ⅱ型碳酸酐酶表达情况的影响。
1.实验材料
1.1动物
骨硬化病BALB/C小鼠30只。
本实施例中所使用的动物模型，是直接购买的患有骨硬化症BALB/C小鼠。该小鼠模型由于病鼠骨质生长异常，骨质硬，骨髓腔消失，所有骨骼都硬化，并伴有无齿症。这种鼠的病变的与人类硬骨病相似。
1.2实验药物及仪器
绿原酸，强的松(河北鑫银河化工有限公司)，舒筋活血片(河南中杰药业有限责任公司)，Ⅱ型碳酸酐酶ELISA检测试剂盒，PCR仪，RNA提取试剂盒，cDNA链合成试剂盒，双蒸水，12孔和96孔板，酶标仪，电子天平。
2.实验方法
2.1实验动物分组及给药
骨硬化病属于罕见病，临床对其的治疗和研究都比较欠缺，国内外尚没有治疗骨硬化病的特效药，用药大多停留在对症治疗的层面上。本实验采用强的松和舒筋活血片联合用药的方式作为阳性对照组。按给药剂量：强的松(6mg/kg)，舒筋活血片(1片/只)进行给药治疗。以普通BALB/C小鼠作为正常对照组。
将骨硬化病小鼠模型随机平均分为3组，分别命名为模型对照组(模型组，n＝10)、强的松和舒筋活血片联合治疗组(联合治疗组，n＝10)和绿原酸治疗组(LYS组，n＝10)，连同正常对照组(n＝10)，共4个实验组，共40只。具体分组及给药方式如表5所示：
表5.实验分组、给药方式及给药量(注：所有给药组的给药体积相同)
Figure PCTCN2015072415-appb-000008
2.2检测实验
2.2.1RT-PCR法检测各组小鼠骨细胞中的CA2基因的表达情况。
(1)破骨细胞总mRNA的提取
①破骨细胞的分离：实验终止后，将小鼠经75％酒精浸泡消毒5min，拉颈处死后分离四肢长骨，仔细清楚附着于骨头表面的软组织和骨骺，骨干部分用PBS清洗后放入盛有15％199培养液(含10％小牛血清、5％胎牛血清、100μg/mL硫酸链霉素、100U/mL青霉素钠，pH7.2)的玻璃平皿中。用解剖刀将骨质内表面轻刮入培养液，再用圆头习惯反复捶打骨质碎片2min，静置30s后收集上层的细胞悬液，所有实验组均分成两份A和B，备用。
②将上述细胞悬液A进行离心，收集破骨细胞，分别编号命名，向每份破骨中加入350μl的预先加有β-巯基乙醇的裂解液RL，静置5min。
③将上述液体在12,000rpm/min的条件下离心3min，小心吸取上清液使用；
④向上清液中缓慢加入0.5倍体积的无水乙醇，反复吹打，直至混合均匀后转移至吸附柱CR3中，12,000rpm离心2min，弃去废液，留吸附柱；
⑤加入去蛋白液RW1至吸附柱中，去除蛋白，之后离心1min，弃去废液；
⑥向吸附柱CR3中加入DNase I工作液，降解其中的DNA；
⑦.用去蛋白液和漂洗液分别清洗吸附柱后，将吸附柱放于收集管中，开启通风，充分挥干柱子上的残留液体；
⑧向吸附柱中加入40μlRNase free ddH2O，放置2min后，12,000rpm离心2min，即得mRNA样品；
(2)mRNA的逆转录
利用cDNA第一链合成试剂盒和上述提取得到的RNA溶液合成对应的第一链cDNA。具体逆转录过程按照说明书操作，简述如下：
1.取200μl的无酶离心管置于冰浴上，并于其中加入下列溶液：RNA模板5mL，Oligo(dT)2Ml,Super Pure dNTP2Ml,RNase-Free ddH2O5.5mL。
2.离心后，将装有上述液体的离心管放置于PCR仪中，70℃孵育5min后，简短收集液体后，迅速将其转移到冰上冷却2min后补加如下试剂：5×first strand buffer(含DTT)4mL，RNasin0.5mL，TIANScriptM-MLV(200U)1mL。
3.将离心管轻轻混匀并简短离心后，设置PCR仪42℃孵育50min，之后95℃加热5min。最后向得到的cDNA溶液中加入20ul RNase-Free ddH2O稀释至40ul，即得第一链cDNA。
4.将得到的产物仔细编号后置于－20℃条件下保存。
(3)RT-PCR定量
EvaGreen荧光染料与双链的DNA结合会产生很强的荧光，通过检测最终的荧光强度，我们可以得到反应生成DNA的总量。在试管中加入荧光染料、(1)(2)步中所合成的cDNA产物、CA2引物混合均匀后进行RT-PCR检测本体系使用RNase-free water代替cDNA products组为NTC对照。每个样品均设β-actin引物组为相对值，进行相对定量。
2.2.2各组小鼠破骨细胞中Ⅱ型碳酸酐酶的测定
取2.2.1(1)中所述的B组破骨细胞悬液，吹打均匀后，小心的于12孔板中进行孵育，24后进行试验。用BCA法进行蛋白定量后，利用Ⅱ型碳酸酐酶ELISA酶联免疫试剂盒进行后续试验，测定Ⅱ型碳酸酐酶活性。
3.实验结果
3.1各组小鼠骨细胞中的CA2基因的表达情况
CA2基因的检测结果显示，在正常组小鼠的破骨细胞中CA2的表达相对较高，而骨硬化病模型组的小鼠中，其破骨细胞中的CA2基因表达显著下调。在分别用联合给药及绿原酸治疗后，在联合给药组中，小鼠破骨细胞中的CA2基因没有明显的上升，其与模型组相比没有统计学差异(p>0.05)，而绿原酸治疗的小鼠，能够显著的提高小鼠破骨细胞中CA2基因的表达，其与模型组及联合给药组相比，均有非常显著性的差异(**p<0.01,##p<0.01)。基因检测的实验结果如图1所示。
3.2各组小鼠破骨细胞中Ⅱ型碳酸酐酶的测定结果
Ⅱ型碳酸酐酶的检测结果显示，骨硬化病模型小鼠的破骨细胞中Ⅱ型碳酸酐酶的含量相比于正常对照组，显著下降，而Ⅱ型碳酸酐酶是破骨细胞骨吸收的关键促进酶，此种下降显示在骨硬化病的小鼠模型中破骨细胞的骨吸收能力严重不足。其Ⅱ型碳酸酐酶含量与对照组相比，具有非常显著性的差异(**p<0.01)。在经过联合给药治疗后，小鼠破骨细胞中的Ⅱ型碳酸酐酶含量没有明显变化，其与模型组之间没有统计学的差异(p>0.05)。而绿原酸治疗的小鼠中，其破骨细胞中的Ⅱ型碳酸酐酶含量有了非常显著的上调，与联合给药组之间具有非常显著性的差异(△△p<0.01),但与正常对照组相比，其值尚有显著性差异(*p<0.05)。
结果显示，绿原酸能高效的促进Ⅱ型碳酸酐酶的表达，从而促进破骨细胞性的骨吸收。具体的实验结果如图2所示。
4.统计学处理
连续型变量形式的实验数据以
Figure PCTCN2015072415-appb-000009
表示，2组数据间的比较用两样本t检验。采用SPSS13.0统计软件，P<0.05为有统计学意义。
5.结论
本实施例中，采用患有骨硬化病的BALB/C小鼠作为模型动物，以联合给予强的松和舒筋活血片作为阳性治疗对照组。分别测定了各组小鼠破骨细胞中的CA2基因及对应的Ⅱ型碳酸酐酶的表达情况。结果显示，与联合给药组相比，绿原酸能够有效的上调小鼠破骨细胞中CA2基因的表达，同时促进其对应Ⅱ型碳酸酐酶的含量提升。Ⅱ型碳酸酐酶主要功能是促进破骨细胞的骨性吸收，对其的激活能够显著的改善因破骨细胞吸收不良而导致的骨质硬化症。综上，绿原酸能够显著的激活Ⅱ型碳酸酐酶活性，对骨硬化病具有一定的治疗作用。
实施例3：用绿原酸制备冻干粉针剂
1.绿原酸的提取：
本实施例中所用的绿原酸原料药，系由杜仲叶中提取、纯化得到，纯 度为99.38％。
2.绿原酸冻干粉针剂的制备
2.1处方：
纯度为99.38％的绿原酸(主药) 40g
甘露醇(支撑剂) 55g
亚硫酸氢钠(抗氧化剂) 4.5g
将以上处方完全溶解于注射用水中，过滤后，再用0.22μm的除菌微孔滤膜精滤，调节pH后，按照无菌粉针剂的常规操作共制成2ml粉针剂1000支，每支含绿原酸40mg。
实施例4：用绿原酸制备丸剂
1.绿原酸的提取
本实施例中所使用的绿原酸，系由杜仲叶中提取、纯化得到的，纯度为99.21％。
2.绿原酸丸剂的制备
2.1处方
纯度为99.21％的绿原酸(主药) 1g
淀粉(稀释剂) 50g
糊精粉(粘合剂) 适量
无水乙醇(润湿剂) 适量
2.2.制法：
取适量的聚维酮K30，用无水乙醇配制成溶液，再取处方量的绿原酸和淀粉，采用等量稀释法混合均匀之后，加入糊精粉的乙醇溶液中，充分搅拌后制得软材，采用搓丸法制得绿原酸丸剂1000粒，每粒丸剂含绿原酸1mg。
实施例5：用绿原酸制备口服溶液剂
1.绿原酸的提取
本实施例中所使用的绿原酸，系由牛蒡叶中提取、纯化得到，纯度为99.29％。
2.绿原酸口服溶液剂的制备
2.1处方
纯度为99.29％的绿原酸(主药) 200g
亚硫酸氢钠(抗氧化剂) 10g
注射用水(溶剂) 100L
2.2制法
取处方量的绿原酸和亚硫酸氢钠，溶解于5L注射用水中，按照口服液的常规制备工艺，过滤后，无菌灌装成1000支口服液，每支口服液为100mL，含绿原酸200mg。
实施例6：用绿原酸制备片剂
1.绿原酸的提取：
本实施例中所使用的绿原酸，系由金银花中提取、纯化得到，纯度
为98.57％。
2.绿原酸片剂的制备
2.1处方：
Figure PCTCN2015072415-appb-000010
2.2制法：
本实施例采用制湿颗粒压片法制备绿原酸片剂。(1)按处方量取羟丙甲纤维素制成水溶液；(2)取处方量的绿原酸、糖粉和乳糖混合均匀后，加入羟丙甲纤维素水溶液，充分搅拌均匀后制成软材；(3)将制备好的软材按常规的湿法制粒的操作规程，过筛、干燥和整粒后得到大小均一的颗粒；(4)将制得的颗粒与硬脂酸镁混合均匀后压片，共制成1000片剂，每片含绿原酸100mg。
实施例7：用绿原酸制备胶囊剂
1.绿原酸的提取：
本实施例中所使用的绿原酸，系由杜仲叶中提取、纯化得到的，纯度为99.25％。
2.绿原酸胶囊剂的制备：
2.1处方：
纯度为99.25％的绿原酸 100g
糖粉 200g
2.2制法：
取处方量的绿原酸和淀粉，混合均匀，加入80％乙醇溶液制成软材，干燥，整粒后按照胶囊剂的常规制备工艺制备2000粒胶囊，每粒胶囊含绿原酸50mg。
实施例8：用绿原酸制备颗粒剂
1.绿原酸的提取
本实施例中所使用的绿原酸，系由杜仲叶中提取、纯化得到的，纯度为98.92％。
2.绿原酸颗粒剂的制备
2.1处方：
纯度为98.92％的绿原酸(主药) 200g
甘露醇(稀释剂) 100g
蔗糖(稀释剂) 400g
聚维酮K30(粘合剂) 适量
2.2制法：
取聚维酮K30，加入注射用水，制成溶液。取处方量的绿原酸、甘露醇和乳糖混合均匀之后，加入聚维酮K30溶液，制成软材。按照颗粒剂的常规制备工艺，对软材进行过筛、干燥和整粒之后，得到颗粒剂。在无菌条件下分装颗粒剂，制备400袋颗粒剂，每袋颗粒剂含绿原酸500mg。
实施例9：用绿原酸制备散剂
1.绿原酸的提取：
本实施例所用的绿原酸原料药，系由牛蒡叶中提取、纯化得到的，纯度为98.82％。
2.绿原酸散剂的制备：
2.1处方
纯度为98.82％的绿原酸1000g
2.2制法
取处方量绿原酸过筛后，按照散剂的常规制备工艺，无菌分装成含1000瓶/袋散剂，每瓶/袋散剂含绿原酸1000mg。
综上，本发明绿原酸可以有效治疗骨硬化病，且治疗效果明显优于目前通常使用的骨化病治疗药物：强的松与舒经活血片的联合用药，副作用小，临床应用前景良好。

Claims (5)

  1. 绿原酸在制备治疗骨硬化病的药物中的用途。
  2. 根据权利要求1所述的用途，其特征在于：所述的药物是以有效量的绿原酸为活性成分，加上药学上可接受的辅料或者辅助性成分制备而成的制剂。
  3. 根据权利要求2所述的用途，其特征在于：所述的药物制剂中每制剂单位含有绿原酸1～1000mg。
  4. 根据权利要求3所述的用途，其特征在于：所述的药物制剂中绿原酸的使用剂量为1～100mg/kg。
  5. 根据权利要求3或4所述的用途，其特征在于：所述的药剂是口服制剂或者注射剂。
PCT/CN2015/072415 2015-02-06 2015-02-06 绿原酸在制备治疗骨硬化病的药物中的用途 WO2016123795A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/072415 WO2016123795A1 (zh) 2015-02-06 2015-02-06 绿原酸在制备治疗骨硬化病的药物中的用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/072415 WO2016123795A1 (zh) 2015-02-06 2015-02-06 绿原酸在制备治疗骨硬化病的药物中的用途

Publications (1)

Publication Number Publication Date
WO2016123795A1 true WO2016123795A1 (zh) 2016-08-11

Family

ID=56563339

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/072415 WO2016123795A1 (zh) 2015-02-06 2015-02-06 绿原酸在制备治疗骨硬化病的药物中的用途

Country Status (1)

Country Link
WO (1) WO2016123795A1 (zh)

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101120936A (zh) * 2006-08-11 2008-02-13 四川九章生物化工科技发展有限公司 绿原酸在制备具有雌激素样或促雌激素样作用药物中的应用

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101120936A (zh) * 2006-08-11 2008-02-13 四川九章生物化工科技发展有限公司 绿原酸在制备具有雌激素样或促雌激素样作用药物中的应用

Similar Documents

Publication Publication Date Title
JP2022548997A (ja) 多能性幹細胞、医薬組成物、並びにそれらの調製方法及びそれらの適用
Nada et al. Heme oxygenase 1-mediated neurogenesis is enhanced by Ginkgo biloba (EGb 761®) after permanent ischemic stroke in mice
WO2018120683A1 (zh) 一种多组分注射液
TW200904979A (en) Reoviruses having modified sequences
WO2016127787A1 (zh) 绿原酸在制备治疗心肌病的药物中的用途
TW201805010A (zh) 用於促進血管生成活性之含衍生自成體幹細胞之擬胞外體奈米囊胞之組成物及其製造方法
JP2012523414A5 (zh)
JP6995125B2 (ja) Ｍ２表現型に極性化されたマクロファージを作る方法及びその用具
WO2024001265A1 (zh) 麦冬皂苷d在制备抗轮状病毒药物中的应用
CN113456645A (zh) Dmxaa在制备防治骨质疏松症药物中的应用
CN106110325A (zh) 一种新型 glp‑1 受体激动剂的制备方法及其在神经退行性疾病治疗领域的应用
CN113679737A (zh) 间充质干细胞在对成年抑郁症防治中的应用
CN105920044B (zh) 一种水螅干细胞制剂及其制备方法与应用
WO2016123795A1 (zh) 绿原酸在制备治疗骨硬化病的药物中的用途
CN104606179B (zh) 绿原酸在制备治疗骨硬化病的药物中的用途
WO2021009660A1 (en) Composition and methods for improving thickness and receptivity of endometrial lining
CN112316100B (zh) 人丹中药组合物用于防醉解酒或治疗酒精性肝损伤的新用途
CN107281177B (zh) 促进造血干细胞归巢和植入的方法
CN115364129B (zh) 九节茶或其提取物、清热消炎宁制剂在制备抗冠状病毒药物中的应用及其制备方法
CN116077609B (zh) 治疗ibd、ibs的黄厚止泻速释微丸及其制备方法
CN112972466B (zh) 5-羟基吲哚-3-羧酸酯类化合物在抑制外周炎症及中枢神经系统炎症方面的用途
CN118184736A (zh) 活性肽、含有活性肽的组合物及其应用
WO2021219112A1 (zh) 异戊酰螺旋霉素类化合物或其组合物在制备治疗脓毒症疾病药物中的应用
WO2022126857A1 (zh) 二维纳米材料在抑制冠状病毒的应用
JP7054956B2 (ja) リゾビウム属細菌由来のナノ小胞およびその用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15880751

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15880751

Country of ref document: EP

Kind code of ref document: A1