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WO2021128232A1 - 一种麦冬降解提取物及其在制备降糖药物中的应用 - Google Patents

一种麦冬降解提取物及其在制备降糖药物中的应用 Download PDF

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Publication number
WO2021128232A1
WO2021128232A1 PCT/CN2019/128981 CN2019128981W WO2021128232A1 WO 2021128232 A1 WO2021128232 A1 WO 2021128232A1 CN 2019128981 W CN2019128981 W CN 2019128981W WO 2021128232 A1 WO2021128232 A1 WO 2021128232A1
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extract
ophiopogon japonicus
polysaccharide
degradation
hours
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PCT/CN2019/128981
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English (en)
French (fr)
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林爱华
刘奕明
王永刚
陈宝婷
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广东省中医院
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Publication of WO2021128232A1 publication Critical patent/WO2021128232A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/896—Liliaceae (Lily family), e.g. daylily, plantain lily, Hyacinth or narcissus
    • A61K36/8968—Ophiopogon (Lilyturf)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/50—Methods involving additional extraction steps
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization

Definitions

  • the invention relates to a degraded extract of Ophiopogon japonicus and its application in the preparation of hypoglycemic drugs.
  • Ophiopogon is the dried root tuber of Ophiopogon japonicus (L.f) Ker-Gawl in the lily family.
  • Ophiopogon is one of the traditional Chinese medicines for nourishing yin. Its taste is sweet, slightly bitter, and slightly cold in nature. It has the effects of nourishing yin, nourishing body fluid, nourishing the lungs and clearing the heart, and is used for dryness of the lungs, coughs, coughs, and body fluids. Wound thirst, upset, insomnia, internal heat to reduce thirst, dry intestines and constipation.
  • the active ingredients of Ophiopogon japonicus mainly include steroidal saponins, polysaccharides, and high isoflavones.
  • the activity of polysaccharides is mainly determined by the molecular mass. Polysaccharides with smaller molecular mass have higher activity and are easier to be absorbed and utilized by the human body.
  • the conventional extraction method of Ophiopogon japonicus polysaccharide is the water extraction and alcohol precipitation method, and the obtained Ophiopogon japonicus polysaccharide has a larger molecular mass.
  • the small-molecule Ophiopogon japonicus polysaccharide is prepared by the ultrafiltration method, and the polysaccharide with a molecular weight greater than 10,000 is filtered and removed. Through the degradation of the polysaccharide, the macro-molecular polysaccharide becomes the small-molecular polysaccharide. The polysaccharide can be fully utilized and the activity is increased.
  • the purpose of the present invention is to provide a degraded extract of Ophiopogon japonicus with higher activity, and the preparation method is simple and easy to implement.
  • the degraded extract of Ophiopogon japonicus of the present invention is prepared by the following method, including the following steps:
  • Extract the crude polysaccharides of Ophiopogon japonicus add water to extract 1 ⁇ 3 times of Ophiopogon japonicus according to the liquid-to-material ratio of 5-20, each extraction for 0.5-2 hours, filter, and concentrate the filtrate to a raw material content of 0.5-2g/ml Extract the extract, add ethanol to make the alcohol content 75%-90%, stand still, take the precipitate and evaporate to dry to obtain the crude polysaccharide of Ophiopogon japonicus;
  • Degradation The polysaccharide of Ophiopogon japonicus in step (2) is fully dissolved with water to a transparent state, and based on the mass of Ophiopogon japonicus medicinal material, 2mol/L acid is added according to the material-to-liquid ratio of 10:1, degraded for 4-6 hours, and NaOH is added. The solution is neutralized to neutrality, ultrafiltration intercepts the degradation products with a molecular weight between 500 and 2500, and is concentrated and freeze-dried to obtain the degradation extract of Ophiopogon japonicus.
  • the step 1 process is preferably: extract Ophiopogon japonicus medicinal materials with water twice, with a liquid-to-material ratio of 10, and extract for 2 hours each time, filter, combine the filtrate, concentrate the filtrate to an extract with a raw material content of 1g/ml, add ethanol to make The alcohol content reaches 80%, and it is allowed to stand overnight; the supernatant is filtered off, precipitated and evaporated to dryness to obtain the crude polysaccharide of Ophiopogon japonicus.
  • the acid described in step 3 is preferably hydrochloric acid or sulfuric acid, and the degradation temperature is preferably 50°C to 100°C, and most preferably 50°C.
  • step 2 takes the crude polysaccharide of Ophiopogon japonicus, add distilled water to dissolve it, add chloroform: n-butanol (4:1), stir to dissolve fully, let the separatory funnel stand still, take the lower layer liquid in a high-speed centrifuge at 5000r/min After centrifugation for 10 minutes, the precipitate was removed, and the clear liquid was ethanol-precipitated and dried to obtain purified Ophiopogon japonicus polysaccharide.
  • the present invention has the following beneficial effects: the degraded extract of the present invention has higher activity in lowering blood sugar, and is significantly better than Ophiopogon japonicus crude polysaccharide and other small-molecule polysaccharides of Ophiopogon japonicus obtained by other existing methods.
  • the yield can be as high as 98%, and the method is simple and easy to implement, and can be produced on a large scale.
  • the crude polysaccharide solution of Ophiopogon japonicus was deproteinized by the Sevage method, ethanol precipitation, and dried to obtain the purified Ophiopogon japonicus polysaccharide; the purified Ophiopogon japonicus polysaccharide was fully dissolved in water to a transparent state, added with 500ml of 2mol/L sulfuric acid, and degraded at 50°C for 4 hours , NaOH solution is added to neutralize to neutrality, the degradation liquid is ultrafiltration to retain the degradation products with a molecular weight below 10,000, the ultrafiltrate is then concentrated by nanofiltration technology, and the concentrate is freeze-dried to obtain the degradation extract of Ophiopogon japonicus.
  • the crude polysaccharide solution of Ophiopogon japonicus was deproteinized by the Sevage method, ethanol precipitation, and dried to obtain the purified Ophiopogon japonicus polysaccharide; the purified Ophiopogon japonicus polysaccharide was fully dissolved in water to a transparent state, and 500ml of 2mol/L hydrochloric acid was added and degraded at 70°C for 5 hours. , NaOH solution is added to neutralize to neutrality, the degradation liquid is ultrafiltration to retain the degradation products with a molecular weight below 10,000, the ultrafiltrate is then concentrated by nanofiltration technology, and the concentrate is freeze-dried to obtain the degradation extract of Ophiopogon japonicus.
  • the Ophiopogon japonicus crude polysaccharide solution was deproteinized by Sevage method, ethanol precipitation, and dried to obtain purified Ophiopogon japonicus polysaccharide; the purified Ophiopogon japonicus polysaccharide was fully dissolved in water to a transparent state, and 2mol/L sulfuric acid 500ml was added, and degraded at 80°C for 3 hours.
  • NaOH solution is added to neutralize to neutrality
  • the degradation liquid is ultrafiltration to retain the degradation products with a molecular weight below 10,000
  • the ultrafiltrate is then concentrated by nanofiltration technology, and the concentrate is freeze-dried to obtain the degradation extract of Ophiopogon japonicus.
  • the crude polysaccharide solution of Ophiopogon japonicus was deproteinized by the Sevage method, ethanol precipitation, and dried to obtain the purified Ophiopogon japonicus polysaccharide; the purified Ophiopogon japonicus polysaccharide was fully dissolved in water to a transparent state, and 500ml of 2mol/L hydrochloric acid was added and degraded for 1 hour at 100°C. , NaOH solution is added to neutralize to neutrality, the degradation liquid is ultrafiltration to retain the degradation products with a molecular weight below 10,000, the ultrafiltrate is then concentrated by nanofiltration technology, and the concentrate is freeze-dried to obtain the degradation extract of Ophiopogon japonicus.
  • Determination of polysaccharide content in degraded extract of Ophiopogon japonicus Determination of polysaccharide content by anthrone sulfate method
  • the sugar content in the degraded extract of Ophiopogon japonicus was measured.
  • the sugar content of Example 1 was greater than 98%
  • the sugar content of Example 2 was 97%
  • the sugar content of Examples 3 and 4 was 95%.
  • each degraded extract of Ophiopogon japonicus was made into a 2 ⁇ g/ml solution, and 0.5ul sample solution and 5mg/ml matrix 2,5-dihydroxybenzoic acid (DHB) were mutually dissolved and evaporated to dryness.
  • model model Establish the model according to the following method. After the model is successfully established, the animals in the model group are randomly divided into a model control group, a positive drug group, and corresponding doses of Ophiopogon japonicus crude polysaccharide group and Ophiopogon japonicus degraded extract group. The administration method is the same as before. . Continuous administration for 1 week.
  • Rat model preparation rats in the normal group were given ordinary drinking water every day, and rats in the high-fat group were given homemade fat emulsion (1ml/100gBW) every morning and evening. After continuous gavage of fat emulsion for 2 weeks, the animals were fasted without water for 24 hours, 10 rats in the blank control group were injected with normal saline through the tail vein, and the other rats were injected with 30 mg/kg BW streptozotocin solution (prepared before use) through the tail vein. After 48 hours of administration, fasting without water for 12 hours, taking blood from the orbital venous plexus every 3 hours, and measuring the fasting blood glucose level according to the blood glucose measurement kit operation. The fasting blood glucose value is measured for 3 times. The model is successful if the fasting blood glucose value is ⁇ 16.7mmol/L Rat.
  • Ophiopogon japonicus crude polysaccharide high dose group has the effect of lowering blood sugar; the Ophiopogon degraded extract high, medium, and low dose groups all have the blood sugar lowering effect, and the high and medium dose groups have extremely significant significance (P ⁇ 0.01)
  • the hypoglycemic effect of Ophiopogon japonicus degraded extract group was better than that of Ophiopogon japonicus crude polysaccharide group.
  • Example 8 Effect of Ophiopogon japonicus degraded extract (Example 4) on alloxan-induced diabetic mice
  • Test indicators Observe the mice's food intake, drinking water, body weight, mental state, coat color and other conditions during the experimental period; take blood after 2h, 3d, and 14d after administration, respectively, to determine the mice after 12h fasting At the end of the experiment, the serum insulin of mice after fasting for 12 hours was measured. Use SPSS11.0 statistical software for data analysis.
  • mice modeled with alloxan showed polydipsia, polyphagia, polyuria, weight loss, slow response, messy coat, and other symptoms. After medication, the above symptoms of each group of mice have varying degrees The improvement, the response is more flexible, the hair is flat and shiny.
  • the middle and high-dose groups can significantly reduce the blood glucose value of the mice (P ⁇ 0.01), Ophiopogon japonicus
  • the degraded extract group is better than the Ophiopogon japonicus crude polysaccharide group. The results are shown in Table 3.
  • Ophiopogon japonicus degraded extract After 14 days of continuous oral administration of Ophiopogon japonicus degraded extract, the serum insulin level of mice increased significantly compared with the model group, among which high and medium doses were extremely significant (**P ⁇ 0.01), and there was a dose-effect relationship.
  • the degradation effect of Ophiopogon japonicus extract is better than that of Ophiopogon japonicus crude polysaccharide group, as shown in Table 4.

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Abstract

一种具有更高降糖活性的麦冬降解提取物。制备方法为：药材按液料比5～20，水提取1～3次，每次提取0.5～2小时，滤过，滤液浓缩至原料含量为0.5～2g/ml的浸膏，加乙醇使含醇量达75％～90％，沉淀蒸干得麦冬粗多糖；采用Sevage法脱蛋白纯化；纯化后加水充分溶解至透明状态，按料液比10:1加入2mol/L的酸，降解4～6h，加碱溶液至中性，超滤截留得分子量范围在500－2500之间的降解物，浓缩冷冻干燥，即得。与现有技术相比，上述提取物在降血糖方面具有更高的活性，明显优于麦冬粗多糖及其他现有方法获得的麦冬小分子多糖。产率可以高达95％以上，且方法简单易行，可规模化生产。

Description

一种麦冬降解提取物及其在制备降糖药物中的应用 技术领域
本发明涉及一种麦冬降解提取物及其在制备降糖药物中的应用。
背景技术
麦冬为百合科植物麦冬Ophiopogon japonicus(L.f)Ker-Gawl的干燥块根。麦冬为传统滋阴中药之一，其味甘、微苦，性微寒，归心、肺、胃经，有养阴生津，润肺清心的功效，用于肺燥干咳、虚痨咳嗽、津伤口渴、心烦失眠、内热消渴、肠燥便秘。麦冬的活性成分主要有甾体皂苷、多糖、高异黄酮类等。
药理及临床试验研究表明，从麦冬中提取得到的粗多糖能有效的降低实验性糖尿病小鼠的血糖水平，降低II型糖尿病患者空腹血糖和餐后2h血糖水平，改善胰岛素抵抗。
多糖的活性主要由分子质量决定，分子质量较小的多糖活性更高，更容易被人体吸收利用。麦冬多糖常规提取方法为水提醇沉法，得到的麦冬多糖分子质量较大。现有技术制备小分子麦冬多糖采用超滤法，大于10000分子量的多糖被过滤除去，通过多糖的降解，使大分子多糖变成小分子多糖，多糖可以得到充分的利用，并且活性增加。
为了用较简便易行的方法得到分子质量较小的低聚麦冬多糖，我们采用化学降解法，进行多糖分子的降解，得到活性更强的小分子多糖。
发明内容
本发明的目的为提供一种具有更高活性的麦冬降解提取物，且制备方法简单易行。
本发明的麦冬降解提取物由以下方法制备，包括以下步骤：
1.提取麦冬粗多糖：将麦冬药材按液料比5～20，加水提取1～3次，每次提取0.5～2小时，滤过，滤液浓缩至原料含量为0.5～2g/ml的浸膏，加乙醇使含醇量达75％～90％，静置，取沉淀蒸干得到麦冬粗多糖；
2.脱蛋白：麦冬粗多糖采用Sevage法脱蛋白纯化；
3.降解：将步骤(2)所述麦冬多糖加水充分溶解至透明状态，以麦冬药材的质量计，按料液比10:1加入2mol/L的酸，降解4～6h，加入NaOH溶液中和至中性,超滤截留得分子量范围在500－2500之间的降解物，浓缩冷冻干燥，即得麦冬降解提取物。
其中，步骤1工艺优选为：将麦冬药材加水提取2次，液料比10，每次提取2小时，滤过，合并滤液，滤液浓缩至原料含量为1g/ml的浸膏，加乙醇使含醇量达80％，静置过夜；滤去上清液，沉淀蒸干得到麦冬粗多糖。
步骤3所述的酸优选盐酸或硫酸，降解温度优选50℃～100℃，最优为50℃。
步骤2优选工艺为：取麦冬粗多糖，加蒸馏水溶解，加入三氯甲烷：正丁醇(4：1)， 搅拌充分溶解，分液漏斗静置，取下层液于高速离心机5000r/min离心10分钟，除沉淀，清液经乙醇沉淀，干燥，得纯化的麦冬多糖。
与现有技术相比，本发明具有如下有益效果：本发明的降解提取物在降血糖方面具有更高的活性，明显优于麦冬粗多糖及其他现有方法获得的麦冬小分子多糖。产率可以高达98％以上，且方法简单易行，可规模化生产。
具体实施方式
以下通过具体实施例对本发明作进一步说明。
实施例1：
取麦冬药材5000克，加水煎煮提取1次，加水20倍量，煎煮2小时，滤过，合并滤液，滤液浓缩至原料含量为1g/ml的浸膏，加入乙醇至含醇量为80％，放置过夜，沉淀经过滤干燥后得到麦冬粗多糖。麦冬粗多糖溶液采用Sevage法脱蛋白，乙醇沉淀，干燥，得纯化的麦冬多糖；将纯化后的麦冬多糖加水充分溶解至透明状态，加入2mol/L硫酸500ml，50℃降解4个时，加入NaOH溶液中和至中性,降解液经超滤截留分子量10000以下的降解物，超滤液再采用纳滤技术进行浓缩，浓缩液经冷冻干燥，即得麦冬降解提取物。
实施例2：
取麦冬药材5000克，加水煎煮提取2次，加水10倍量，煎煮2小时，滤过，合并滤液，滤液浓缩至原料含量为1.5g/ml的浸膏，加入乙醇至含醇量为85％，放置过夜，沉淀经过滤干燥后得到麦冬粗多糖。麦冬粗多糖溶液采用Sevage法脱蛋白，乙醇沉淀，干燥，得纯化的麦冬多糖；将纯化后的麦冬多糖加水充分溶解至透明状态，加入2mol/L盐酸500ml，70℃降解5个时，加入NaOH溶液中和至中性,降解液经超滤截留分子量10000以下的降解物，超滤液再采用纳滤技术进行浓缩，浓缩液经冷冻干燥，即得麦冬降解提取物。
实施例3：
取麦冬药材5000克，加水煎煮提取1次，加水20倍量，煎煮2小时，滤过，合并滤液，滤液浓缩至原料含量为1g/ml的浸膏，加入乙醇至含醇量为80％，放置过夜，沉淀经过滤干燥后得到麦冬粗多糖。麦冬粗多糖溶液采用Sevage法脱蛋白，乙醇沉淀，干燥，得纯化的麦冬多糖；将纯化后的麦冬多糖加水充分溶解至透明状态，加入2mol/L硫酸500ml，80℃降解3个时，加入NaOH溶液中和至中性,降解液经超滤截留分子量10000以下的降解物，超滤液再采用纳滤技术进行浓缩，浓缩液经冷冻干燥，即得麦冬降解提取物。
实施例4：
取麦冬药材5000克，加水煎煮提取2次，加水5倍量，煎煮1小时，滤过，合并滤液，滤液浓缩至原料含量为0.5g/ml的浸膏，加入乙醇至含醇量为75％，放置过夜， 沉淀经过滤干燥后得到麦冬粗多糖。麦冬粗多糖溶液采用Sevage法脱蛋白，乙醇沉淀，干燥，得纯化的麦冬多糖；将纯化后的麦冬多糖加水充分溶解至透明状态，加入2mol/L盐酸500ml，100℃降解1个时，加入NaOH溶液中和至中性,降解液经超滤截留分子量10000以下的降解物，超滤液再采用纳滤技术进行浓缩，浓缩液经冷冻干燥，即得麦冬降解提取物。
实施例5：
麦冬降解提取物多糖含量的测定:采用硫酸蒽酮法测定多糖含量
(1)标准溶液的配制：
取经105℃干燥至恒重的无水葡萄糖对照品33mg，精密称定，置100ml量瓶中，加水溶解并稀释至刻度，摇匀，即得。
(2)标准曲线的制备
标准曲线的制备精密量取对浑品溶液0.1ml、0.2ml、0.3ml、0.4ml、0.5ml、0.6ml，分别置10ml具塞刻度试管中，各加水至2.0ml，摇匀，在冰水浴中缓缓滴加0.2％蒽酮-硫酸溶液至刻度，混匀，放冷后置水浴中保温10分钟，取出，立即置冰水浴中冷却10分钟，取出，以相应试剂为空白。照紫外-可见分光光度法，在582nm波长处测定吸光度。以吸光度为纵坐标，浓度为横坐标，绘制标准曲线。
(3)样品溶液的制备
精密称取降解提取物约10mg，加水溶解后转移至50ml容量瓶中，加水至刻度，摇匀。
(4)样品测定
精密吸取样品溶液0.1ml，按“标准曲线制备”项下操作，测得样品的吸光度值，计算含量。
测得麦冬降解提取物中糖的含量，其中实施例1的糖含量大于98％，实施例2的糖含量为97％，实施例3和4的糖含量为95％。
实施例6:
麦冬降解提取物分子量范围的测定：将各麦冬降解提取物配成2μg/ml溶液，分别取0.5ul样品溶液与5mg/ml的基质2，5-二羟基苯甲酸(DHB)互溶挥干溶液，MALDI-TOF-MASS进样检测，结果测得麦冬降解提取物的分子量范围圴在500－2500之间。
实施例7：
麦冬降解提取物(实施例4)对实验性II型糖尿病大鼠的作用
(1)动物分组:健康Wistar大鼠(SPF级)，雄性，体重180-220g(由南方医科大学 实验动物中心提供)，自由饮水进食，随机分为正常对照组和造模组，造模组按如下方法建立模型，造模成功后再将模型组动物随机分为模型对照组、阳性药物组、并设相对应剂量的麦冬粗多糖组、麦冬降解提取物组，给药方式同前。连续给药1周。
(2)大鼠模型制备:正常组大鼠每天灌胃普通饮用水，高脂组大鼠每天早晚灌胃自制脂肪乳(1ml/100gBW)。连续灌胃脂肪乳2周后，动物禁食不禁水24h，空白对照组10只尾静脉注射生理盐水，其余大鼠均尾静脉注射30mg/kgBW链脲佐菌素溶液(临用前配制)。给药48h后，禁食不禁水12h，每隔3小时眼眶静脉丛取血，按照血糖测定试剂盒操作测定空腹血糖值，连续测定3次，空腹血糖值≧16.7mmol/L的为造模成功大鼠。
(3)血糖测定:末次给药后，动物禁食不禁水12h，眼眶静脉丛取血，按照试剂盒的方法分别测定血糖值。采用SPSS11.0统计软件，分析并比较各组血糖值及给药前后血糖值的变化情况，结果见表1。
表1给药后血糖值比较
Figure PCTCN2019128981-appb-000001
注：*P<0.05vs模型组**P<0.01vs模型组ΔP<0.05vs同组给药前ΔΔP<0.01vs同组给药前(4)口服葡萄糖耐量实验
连续给药7天后，动物禁食不禁水12h，眼眶静脉丛取血测定血糖值。各组均ig.葡萄糖2.0g/kgBW，分别在给予葡萄糖后0.5h、1h、2h，从眼眶静脉丛采血，按血糖试剂盒的方法测定血糖值，结果见表2。
表2口服糖耐量实验
Figure PCTCN2019128981-appb-000002
Figure PCTCN2019128981-appb-000003
注：*P<0.05 vs模型组 **P<0.01 vs模型组
以上结果说明麦冬粗多糖高剂组具有降血糖作用；麦冬降解提取物高、中、低剂量组均具有降血糖的作用，其中高、中剂量组具有极显著意义(P<0.01)，麦冬降解提取物组的降血糖效果优于麦冬粗多糖组。
实施例8：麦冬降解提取物(实施例4)对四氧嘧啶诱导的糖尿病小鼠的作用
(1)健康昆明种小鼠(SPF级)，雌雄各半，体重18～20g(由南方医科大学实验动物中心提供)。
(2)糖尿病小鼠造模:采用四氧嘧啶(ALX)致小鼠高血糖模型的方法，取小鼠(雌雄各半)禁食(不禁水)12h后，分别腹腔注射2％的新鲜ALX溶液(220mg/kg)2次(第1次注射量为总量的70％，第2次为总量的30％，两次间隔12h)。3天后，禁食12h后断尾取血，用血糖试纸及测试仪测定小鼠血糖，并测定体重。FPG＞11.1mmol/L，定为糖尿病模型小鼠。
(3)检测指标:在实验期观察小鼠的采食、饮水、体质量、精神状态、毛色等状况；分别在给药后的2h、3d、14d断尾取血测定小鼠禁食12h后的血糖值，实验结束时测定小鼠禁食12h后的血清胰岛素。采用SPSS11.0统计软件进行数据分析。
(4)麦冬降解提取物对四氧嘧啶糖尿病小鼠空腹血糖的影响
用四氧嘧啶造模后的小鼠出现了多饮、多食、多尿、体重减轻，并有反应迟钝，被毛杂乱无光等症状，在用药后各组小鼠以上症状均有不同程度的改善，反应较灵活，毛平伏且有光泽。对四氧嘧啶糖尿病小鼠在灌胃麦冬降解提取物后2h、3d和14d后与模型组比较，中、高剂量组能极显著地降低小鼠的血糖值(P＜0.01)，麦冬降解提取物组优于麦冬粗多糖组。结果见表3.
表3麦冬降解提取物对四氧嘧啶糖尿病小鼠空腹血糖的影响
Figure PCTCN2019128981-appb-000004
Figure PCTCN2019128981-appb-000005
注:与模型组比较，*P＜0.05，**P＜0.01
(5)麦冬降解提取物对糖尿病小鼠血清胰岛素水平的影响
连续灌服麦冬降解提取物14d后，与模型组比较小鼠血清胰岛素水平有显著升高，其中高、中剂量给具有极显著意义(**P＜0.01)，并有剂量－效应关系，麦冬降解提取物效果优于麦冬粗多糖组，见表4。
表4.麦冬降解提取物对糖尿病小鼠血清胰岛素水平的影响
Figure PCTCN2019128981-appb-000006
注:与模型组比较，*P＜0.05，**P＜0.01

Claims (8)

  1. 一种麦冬降解提取物，其特征在于由以下方法制备：
    (1)提取麦冬粗多糖：将麦冬药材按液料比5～20，加水提取1～3次，每次提取0.5～2小时，滤过，滤液浓缩至原料含量为0.5～2g/ml的浸膏，加乙醇使含醇量达75％～90％，静置，取沉淀蒸干得到麦冬粗多糖；
    (2)脱蛋白：麦冬粗多糖采用Sevage法脱蛋白纯化；
    (3)降解：将步骤(2)所述麦冬多糖加水充分溶解至透明状态，以麦冬药材的质量计，按料液比10:1加入2mol/L的酸，降解4～6h，加入NaOH溶液中和至中性,超滤截留得分子量范围在500－2500之间的降解物，浓缩冷冻干燥，即得麦冬降解提取物。
  2. 根据权利要求1所述的提取物，其特征在于步骤(1)具体为：将麦冬药材加水提取2次，液料比10，每次提取2小时，滤过，合并滤液，滤液浓缩至原料含量为1g/ml的浸膏，加乙醇使含醇量达80％，静置过夜；滤去上清液，沉淀蒸干得到麦冬粗多糖。
  3. 根据权利要求1所述的提取物，其特征在于步骤(3)具体为：所述的酸是盐酸或硫酸，降解温度为50℃～100℃。
  4. 根据权利要求3所述的提取物，其特征在于步骤(3)具体为：所述的酸降解温度为50℃，降解时间为4小时。
  5. 根据权利要求1所述的提取物，其特征在于步骤(2)具体为：取麦冬粗多糖，加蒸馏水溶解，加入三氯甲烷：正丁醇(4：1)，搅拌充分溶解，分液漏斗静置，取下层液于高速离心机5000r/min离心10分钟，除沉淀，清液经乙醇沉淀，干燥，得纯化的麦冬多糖。
  6. 权利要求1到5所述的任一提取物在制备降血脂药物中的应用。
  7. 权利要求1到5所述的任一提取物在制备清除体内自由基，防止细胞脂质过氧化药物中的应用。
  8. 权利要求1到5所述的任一提取物在制备抗动脉粥样硬化药物中的应用。
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