[go: up one dir, main page]

WO2009140814A1 - 从肾蕨属植物中获得含有西曲依醇的提取物的方法和用途 - Google Patents

从肾蕨属植物中获得含有西曲依醇的提取物的方法和用途 Download PDF

Info

Publication number
WO2009140814A1
WO2009140814A1 PCT/CN2008/071006 CN2008071006W WO2009140814A1 WO 2009140814 A1 WO2009140814 A1 WO 2009140814A1 CN 2008071006 W CN2008071006 W CN 2008071006W WO 2009140814 A1 WO2009140814 A1 WO 2009140814A1
Authority
WO
WIPO (PCT)
Prior art keywords
fern
kidney
obtaining
extract
column chromatography
Prior art date
Application number
PCT/CN2008/071006
Other languages
English (en)
French (fr)
Inventor
孙天宇
王艳丽
王音音
陆游
王霆
Original Assignee
广州联创思远利生物科技有限公司
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 广州联创思远利生物科技有限公司 filed Critical 广州联创思远利生物科技有限公司
Priority to JP2011509837A priority Critical patent/JP2011520926A/ja
Priority to EP08748612A priority patent/EP2277525A4/en
Priority to US12/993,149 priority patent/US20110071225A1/en
Priority to PCT/CN2008/071006 priority patent/WO2009140814A1/zh
Publication of WO2009140814A1 publication Critical patent/WO2009140814A1/zh

Links

Classifications

    • 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
    • A61K36/11—Pteridophyta or Filicophyta (ferns)
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • 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
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • 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

Definitions

  • the present invention relates to a method and a use for obtaining an extract containing citrifloxacin, and more particularly to a method for obtaining an extract containing cetroxol from a plant of the genus Renilla, and the method of using the tretric acid obtained by the method An alcohol extract and its use. Background technique
  • Diabetes is a metabolic disease characterized by high blood sugar, which is a serious hazard to human health. It can easily cause vascular lesions in the heart, brain, kidney, lower limbs, etc., and eventually lead to hypertension, heart disease, diabetic nephropathy and lower limbs. Numerous consequences such as numbness and gangrene. At present, the pathogenesis of diabetes is still not very clear. The main means of treating diabetes is through lowering blood sugar and preventing complications.
  • the therapeutic drugs for diabetes have been classified into 8 categories according to their mechanism of action and structure: insulin, sulfonylureas, biguanides, ⁇ -glucosidase inhibitors, insulin release enhancers, insulin sensitizers, aldehydes A sugar reductase inhibitor, a gluconeogenesis inhibitor.
  • the above-mentioned drugs have large side effects, often at the expense of other organs of the human body, and have inevitable shortcomings such as strong drug resistance. Therefore, finding a safe and effective drug for treating diabetes from natural plants is an urgent problem to be solved by human beings.
  • Inositol molecules represented by myo-inositol and D-chiral-inositol, play an important role in the signaling pathway of insulin action.
  • inositol can promote insulin function, lower blood glucose concentration, blood insulin, blood triglyceride levels, etc., and have a certain protective effect on diabetic retinal capillary pericytes, and its inositol methyl derivatives It also has the effect of lowering blood sugar.
  • the invention extracts citritriol from natural plants and belongs to the class of myo-inositol derivatives.
  • the compound has been shown to have the effect of preventing and treating diabetes by pharmacological studies.
  • the application number of the invention is disclosed in Chinese Patent Application No. 02144302.5.
  • the synthetic silica produced from the yew can significantly reduce hyperglycemia, inhibit glycogen decomposition and glucose absorption in the diabetic model, and its toxicity is extremely low. It is a valuable anti-diabetic drug.
  • the yew is a national protected plant with limited resources and cannot be studied and developed on a large scale.
  • the extraction process has a very low content of cetroxol. The process cannot meet the requirements of industrial production from the aspects of extraction cost and process route. Summary of the invention
  • Another object of the present invention is to provide an extract containing cetroxol prepared by the above method.
  • a further object of the present invention is to provide an application of the above-mentioned extract containing cetroxol or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating diabetes and its complications.
  • a method for obtaining an extract containing cetroxol from a kidney fern comprising the steps of: extracting with a solvent, recovering a solvent to obtain an extract, extracting the extract with two-phase extraction and column chromatography, and collecting the mixture containing cisplatin The alcohol fraction is concentrated, filtered, and dried.
  • the kidney fern is selected from the group consisting of: kidney fern, long leaf kidney fern, round leaf kidney fern, eucalyptus kidney fern, thin leaf kidney fern, double-toothed kidney fern, ear leaf Kidney fern, tall kidney fern, hairy kidney fern, mini kidney fern and Boston kidney fern
  • the solvent in the solvent extraction is selected from the group consisting of: ethanol, methanol, acetone, and water.
  • the extraction temperature is from 0 ° C to the reflux temperature of the solvent, preferably from room temperature to the reflux temperature of the solvent, more preferably to the reflux temperature of the solvent.
  • the weight to volume ratio of the solvent to the kidney fern is from 1 : 1 to 20, preferably from 1: 2 to 10, more preferably from 1: 3 to 5.
  • the number of extractions with the solvent is 2 to 5 times.
  • the solvent used for the two-phase extraction is an organic solvent incompatible with water, water.
  • the water-incompatible organic solvent is selected from the group consisting of ethyl acetate, chloroform, dichloromethane, diethyl ether, petroleum ether, and n-butanol.
  • the volume ratio of the water-incompatible organic solvent to water is: 1 : ⁇ .
  • the column chromatography is macroporous resin column chromatography, dextran column chromatography, modified dextran column chromatography, cellulose column chromatography, activated carbon column chromatography, and ion exchange resin column chromatography.
  • the present invention also provides an extract prepared according to the above-described method for obtaining an extract containing cetroxol.
  • the present invention also provides the use of the above-prepared extract or a pharmaceutically acceptable salt thereof for the preparation of a medicament for treating diabetes and its complications.
  • the pharmaceutically acceptable salt thereof comprises the following salts of cetroxol or a group thereof: citrate, tartrate, acetate, lactate, sodium salt, potassium salt, calcium salt , ammonium salts, magnesium salts, iron salts, zinc salts and aluminum salts.
  • the complications include ocular lesions, renal lesions, neuropathy, coronary heart disease, cerebrovascular disease, peripheral vascular disease.
  • the method for obtaining an extract containing citrifloxacin from the kidney fern plant according to the present invention is based on the characteristics of the kidney fern, and an optimized extraction process is proposed, which can be used for forming Industrial production route.
  • the extract of cetroxol obtained by this method has anti-diabetic activity and low toxicity, and the main active ingredient of the extract is citritriol, and the content may be 10.00% -99.99%, and the animal experiment proves that the extract can Significantly lower blood sugar levels.
  • Fig. 1 is a graph showing the peak time of cetroxol in Example 9. detailed description
  • the present invention provides a method for extracting and isolating an extract containing citritriol having anti-diabetic activity and low toxicity, which is extracted from nephrons such as Nephrolepidaceae, including kidney Ferns (Nephrolepis auriculata (L.) Trimen), N. biserrata, N. duffii Moore, N. falcate (Cav.) C. Chr. N. strigula (Decne.) Pichi-Serm., N. biseroata (Sw.) Schott., Ear leaf kidney N. biserrata var. auriculata Ching, N. exaltata (Sw.) Schott., N. hirsutula (Forst.) Presl, N. cordifolia, Boston kidney fern (N. exaltata cv. Bostoniensis) and so on.
  • nephrons such as Nephrolepidaceae, including kidney Ferns (Ne
  • a method for obtaining an extract containing cetroxol from a kidney fern comprising the steps of: extracting with a solvent, recovering a solvent to obtain an extract, extracting the extract with two-phase extraction and column chromatography, and collecting the mixture containing cisplatin The alcohol fraction is concentrated, filtered, and dried.
  • the extract containing cetroxol obtained by this method is usually a crystalline powder, and the content of cetroxol is usually more than 60%.
  • the citritriol-containing extract obtained by the above method can be used for preparing a medicament or a pharmaceutical composition for treating diabetes and its complications, and can be prepared by combining one or more adjuvants and/or excipients, Pharmaceutical dosage forms such as capsules, tablets, granules, dragees, solutions, and the like, and sustained release preparations and targeted preparations thereof.
  • the thiotriol may be used in the preparation, or a pharmaceutically acceptable salt of cetromethine may be obtained by a reaction of ciscidol with a corresponding acid or base by a known method.
  • the extract containing cetroxol obtained by the above method can be used for preparing a medicament for treating diabetes, can significantly reduce diabetes, hyperglycemia, inhibit liver glycogen decomposition and glucose absorption, lower blood lipids, and improve free radical metabolism and protect islet ⁇ . Cells, and the toxicity is extremely low.
  • the extract containing cetroxol obtained by the above method can also be used for the prevention and treatment of diabetes and its complications, other diseases associated with disorders of glucose metabolism, improving free radical metabolism, preventing and treating type II diabetes and complications.
  • the method for obtaining an extract containing citritriol from the kidney fern plant of the present invention often includes the following steps: drying and pulverizing roots, stems, leaves and the like of the kidney fern;
  • the solvent such as ethanol, methanol, acetone, their hydrates or a mixture thereof is preferably extracted at a temperature from 0 ° C to reflux, preferably from room temperature to reflux, more preferably at reflux temperature, and the amount of solvent used is preferably 1 : 1 to 20 (weight/volume), preferably 1: 2 to 10 (weight/volume), more preferably 1: 3 to 5 (weight/volume), preferably 2 to 5 times; concentrated under reduced pressure to obtain an extract
  • the extract is extracted with a water-incompatible organic solvent (for example, ethyl acetate, chloroform, dichloromethane, diethyl ether, petroleum ether, etc.) / water to remove the organic layer, and the extractant is preferably 1:1.
  • the fractions containing cetroxol are combined, concentrated under reduced pressure, allowed to stand, and filtered to obtain a solid, which is recrystallized from, for example, ethanol, methanol, acetone, methyl ethyl ketone, their hydrates or a mixture thereof to obtain a dried plant extract powder.
  • the final product is a crystalline powder, the main active ingredient is citriol, and the content is from 10.00% to 99.99%.
  • Thin layer chromatography Using F254 thin layer plate, separately absorb the test solution and the control solution (aqueous solution of cetroxol) 5 ⁇ 1 (microliter), point to the thin layer plate, with water: ethyl acetate: isopropanol (6 : 11 : 83 (volume ratio)) is a developing agent. It is developed with potassium permanganate-sodium hydroxide. The main spot of the solution is the same as the Rf value of the reference spot.
  • the content of the main active ingredient of the extract of the present invention, cetroxol can be detected by the following high performance liquid chromatography (HPLC) method:
  • Determination of content 1 Take about 5mg (mg) of this product, accurately weighed, placed in a 10ml (ml) volumetric flask, add dilute acid anhydride solution (1: 50 volume ratio) 0.25ml, and heat in a water bath for 30min (minutes) ), cool to room temperature, add methanol 5ml, shake well, add water to the mark, shake well, get the test solution, HPLC chromatographic conditions: C18 column, 5 ⁇ 1, 4.6x250mm (mm); detection wavelength 220nm (nano); injection 20 ⁇ l; mobile phase, methanol-water (50: 50 by volume), filtered through a 0.45 ⁇ m (micron) organic filter and degassed at a flow rate of 1.0 ml/min.
  • Determination of content 2 Take 5mg of this product, accurately weigh it, put it in a 10ml volumetric flask, dissolve in volume with water, and shake well.
  • HPLC chromatographic conditions Amino column, 5 ⁇ , 4.6x250mm, Detector: Evaporative light scattering detector (ELSD), drift tube temperature: 40 ° C, pressure: 3.5 bar (bar), injection volume: 20 ⁇ 1.
  • the mobile phase was acetonitrile-water (75:25 by volume), and degassed by suction filtration through a 0.45 ⁇ m organic filter before use at a flow rate of 0.8 ml/min.
  • kidney fern extract various forms of medicines or health care products and the like can be prepared using the obtained kidney fern extract.
  • dosage forms such as capsules, tablets, granules, and dragees may contain one or more commonly used excipients, specifically:
  • Fillers and solubilizers starch, microcrystalline fibers, etc.; Binder: carboxymethyl cellulose, polyvinylpyrrolidone, etc.;
  • Moisturizer : glycerin, etc.
  • Disintegrator calcium carbonate, etc.
  • Lubricant : Talc powder, etc.
  • the pharmaceutically acceptable excipients may be in various forms such as solid, semi-solid, liquid, etc., and the formulation adjuvant may be of various types.
  • the solution may contain a general solvent, a solubilizer, an emulsifier, a preservative, etc., such as water, ethanol, glycerin, polyethylene glycol, benzyl benzoate and the like.
  • a general solvent such as water, ethanol, glycerin, polyethylene glycol, benzyl benzoate and the like.
  • Dosage forms such as capsules, tablets, granules, dragees, solutions, and the like can be prepared by a known conventional method, and a plant extract can be mixed with one or more excipients to prepare a dosage form.
  • the invention can prepare a traditional Chinese medicine extract preparation for treating diabetes, and injects new vitality into the pharmaceutical market for treating diabetes.
  • the kidney fern is a common Chinese herbal medicine in southern China. It is also widely cultivated in the region as a food. It is easy to obtain as a raw medicine, and has a simple preparation process and low cost. It provides a natural treatment for diabetes. drug.
  • Example 3 Preparation of lower purity plant extracts 2 60 kg of kidney fern, extracted three times with 3 volumes of 90% methanol at 50 ° C, combined extracts, concentrated to dryness under reduced pressure, extracted with 1:1 (vol/vol) of ethyl acetate / water, combined Water layer, filtered, macroporous resin column (MN-200 type, domestic) column chromatography, eluted with distilled water, aqueous ethanol (ethanol: 5% ⁇ 20%) gradient, each eluent was identified by identification test solution The eluate of the positive reaction was combined, concentrated under reduced pressure, and dried at 70 ° C to obtain a plant extract (yellow-yellow powder) in which the content of citriol was 29.5%.
  • the low-purity plant extract is dissolved in water, and subjected to cellulose column chromatography, and eluted with a gradient of distilled water and aqueous ethanol (ethanol: 5% to 20%). Each eluate is separately identified by a differential test solution, and the positive reaction is combined. The eluent was concentrated under reduced pressure, and recrystallized from ethanol. The crystals were filtered, dried, dried at 70 ° C to obtain a plant extract (yellow powder), which was detected by HPLC method. Up to 78%.
  • the low-purity plant extract is dissolved in water, ion-exchange column chromatography, and eluted with a gradient of distilled water and aqueous ethanol (ethanol: 5% ⁇ 20%). Each eluate is separately identified by the identification test solution, and the positive reaction is combined. The eluate was concentrated to dryness under reduced pressure, and then recrystallized from methanol. The crystals were crystallized, filtered, dried, and dried at 70 ° C to obtain a plant extract (yellow powder), which was detected by HPLC. The alcohol content is up to 75%.
  • the low-purity plant extract is dissolved in water, and subjected to gel column chromatography, and eluted with a gradient of distilled water and aqueous ethanol (ethanol: 5% to 20%). Each eluate is separately identified by a differential test solution, and the positive reaction is combined. The eluate was concentrated to dryness under reduced pressure. The crystals were recrystallized from acetone, filtered to give a crystalline powder, dried, and dried at 70 ° C to obtain a plant extract (yellow powder) which was detected by HPLC method. The alcohol content is up to 68%.
  • Example 8 Preparation of high purity plant extracts A medium-purity plant extract was repeatedly recrystallized with methanol or the like, filtered to obtain a crystalline powder, dried, and dried at 70 ° C to obtain a plant extract of high-purity citriol. After repeated recrystallization, the purity of cetriol can reach 99% or more.
  • High-performance liquid chromatography was used to take 5 mg of high-purity extract sample, accurately weighed, placed in a 10 ml volumetric flask, dissolved in water to volume, and shaken.
  • HPLC chromatographic conditions amino column, 5 ⁇ , 4.6x250mm; detector: evaporative light scattering detector (ELSD); drift tube temperature: 40 °C; pressure: 3.5 bar; injection volume: 20 ⁇ 1; mobile phase: acetonitrile-water ( 75:25), degassed by suction filtration through a 0.45 ⁇ m organic filter before use, at a flow rate of 1.0 ml/min.
  • the excipients are placed in the grinder, then the plant extract is added, and the mixture is ground and mixed for 10 to 30 minutes, until evenly, the mixed powder is canned in the No. 1 capsule, and the random sample is controlled at 300 mg per dose.
  • the plant extract content is 30 mg.
  • a three-month stability test was conducted on three batches of capsules, including items such as appearance, identification, and content.
  • the method of identifying (2) is to take the appropriate amount of the product (about 0.2g of plastic capsule), shake it with water 10ml, and filter it. 2 ml of the filtrate was taken, 1 ml of a basic lead acetate solution was added, shaken, and heated in a water bath for 5 min to become a translucent gel.
  • Table 1 Three batches of capsules sample stability test results
  • the purified cisplatin alcohol extract (purity of 98.00% to 99.99%) was prepared into a raw material for injection.
  • the oxreol alcohol has good water solubility, and the cisplatin is dissolved in water, filtered and sterilized, and the injection preparation process is carried out to prepare an injection solution.
  • the powder injection is prepared according to the freeze-drying powder process procedure.
  • the lyophilized powder injection is prepared by using the refined cetroxol raw material for injection and operating according to the lyophilized powder needle process procedure. Take the cilostatin raw material for injection, dissolve it with water for injection, add 20% low molecular weight dextran solution pre-depyrogenation, mix it thoroughly, and finally quantify the volume so that the final concentration is oxime alcohol O.lg/ml. Dextran 0.05g/ml, activated carbon to remove pyrogen, 0.22 ⁇ microporous membrane filtration sterilization, in a 100-class clean zone, 1ml per bottle was filled into pre-treated vials.
  • freeze-drying box pre-freeze at -40 ° C for 2 hours, then sublimate at minus 25 ° C, 1.33Pa vacuum, free water removal 90% After heating, the temperature is controlled to not exceed 35 ° C until the end of lyophilization, and the lyophilization box is sealed with a stopper.
  • Test Determination of uniformity: Two tablets of cisplatin extract dispersible tablets were shaken in 100 ml of water, and completely disintegrated at 20 ° C ⁇ 1 ° C for 180 seconds and passed through a No. 2 sieve. 2) Measurement of dissolution: The dissolution rate was measured using artificial gastric juice 0.1 mol/L hydrochloric acid. According to the second method of dissolution measurement in the appendix of the second part of the Pharmacopoeia (pulp method), the pulp speed is 100 rpm, 37 ° C, and 900 ml of the eluate.
  • the oxime alcohol extract raw material was prepared according to the following formula: 60 g of ticloxol extract, 4.2 g of tartaric acid, 17.5 g of sodium bicarbonate, 1.2 g of Tween-80, 3.2 g of polyvinylpyrrolidone (PVP), 1.6 g of low-substituted hydroxypropylcellulose (LHPC) and 0.64 g of magnesium stearate were prepared, and the above reagents were thoroughly mixed and tableted to prepare a total of 1,000 tablets. Each tablet contains 60 mg of cefixol extract.
  • mice 80 male mice, weighing 22-25g, were randomly selected from the normal group, and the other mice were injected with tetraoxypyrimidine 65mg/kg in the tail vein.
  • the model was established according to the literature method and divided into 3 groups according to the blood glucose level. Two groups were orally administered with 60 mg/kg kidney fern extract and Jiangtangling (positive control drug) 75 mg/kg according to 0.1 ml/10 g body weight. The normal group and the model group were given an equal volume of distilled water for 7 consecutive days. Blood glucose was measured by the glucose oxidase method.
  • mice 120 male mice, weighing 22-25g, were randomly divided into 6 groups.
  • kidney fern extract 65% cisplatin
  • the normal group and the control group were given an equal volume of distilled water for 7 days. Except the normal group, the other groups were intraperitoneally injected with 2 g/kg of glucose, and the normal group was injected with an equal volume of normal saline. After 30, 60, 90, and 120 minutes after injection, blood was taken from the posterior venous plexus of the mice, and serum was separated and oxidized by glucose. Enzymatic determination of blood sugar.
  • mice 40 normal mice, male and female, were given the kidney fern extract at 800 mg/kg once, and the reaction of the animals was observed. After 7 days, the animals were sacrificed and the organs were observed with the naked eye. Results: None of the animals died, the health was good, the fur was shiny, the eyes were bright, and the activity was good. The mice were sacrificed 7 days later, and no abnormalities were observed by the naked eye.
  • the above is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can also make several improvements and retouchings without departing from the principles of the present invention. It should be considered as the scope of protection of the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Diabetes (AREA)
  • Urology & Nephrology (AREA)
  • Medical Informatics (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Mycology (AREA)
  • Microbiology (AREA)
  • Cardiology (AREA)
  • Botany (AREA)
  • Biotechnology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Vascular Medicine (AREA)
  • Ophthalmology & Optometry (AREA)
  • Neurosurgery (AREA)
  • Neurology (AREA)
  • Biomedical Technology (AREA)
  • Obesity (AREA)
  • Hematology (AREA)
  • Endocrinology (AREA)
  • Emergency Medicine (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Description

从腎蕨属植物中获得含有西曲依醇的提取物的方法和用途 技术领域
本发明涉及一种获得含有西曲依醇的提取物的方法和用途，尤其涉及一种 从肾蕨属植物中获得含有西曲依醇的提取物的方法、利用该方法得到的含有西 曲依醇的提取物及其用途。 背景技术
近年来， 随着我国国民经济的快速发展， 人民生活水平的不断提高， 一些 危害人民健康的疾病的发病率也随之发生了相应的改变。其中糖尿病发病率的 上升及患者的低龄化尤显突出。
糖尿病是一种以高血糖为特征的代谢性疾病，严重危害人体健康，极易引 起心、 脑、 肾、 下肢等脏器的血管病变， 并最终导致高血压、 心脏病、 糖尿病 性肾病及下肢麻木、 坏疽等严重后果。 目前， 糖尿病的发病机理仍然不是十分 清楚， 治疗糖尿病的主要手段是通过降低高血糖和防治并发症。 从 50年代至 今， 糖尿病的治疗药物按照作用机制和结构类型主要分为 8类： 胰岛素、磺酰 脲类、 双胍类、 α-葡萄糖苷酶抑制剂、 胰岛素释放促进剂、 胰岛素增敏剂、 醛 糖还原酶抑制剂、糖元异生抑制剂。 上述药品副作用大， 往往以损害人体其它 器官为代价，且抗药性强等不可避免的缺点， 因此从天然植物中寻找一种安全 有效的治疗糖尿病的药物是人类亟待解决的课题。
肌醇（ inositol )类分子以 myo-肌醇和 D-手性-肌醇为代表， 在胰岛素作用 的信号传递过程中扮演了重要的角色。有研究发现正常人血液及尿液中都含有 一定浓度的 D-手性-肌醇， 而在 2型糖尿病人的血液中几乎检测不到， 在尿液 中的含量则高于正常人数倍， 因此， 提示这些病人可能由于代谢紊乱， 导致 D-手性-肌醇流失太快， 最终造成胰岛素作用的信号传递受阻。 通过动物实验 结果表明肌醇能够促进胰岛素功能， 降低血糖浓度、血中胰岛素、血甘油三酯 水平等，且对糖尿病视网膜毛细血管周细胞有一定的保护作用，其肌醇类的甲 基衍生物同样具有降低血糖的作用。
本发明就是从天然植物中提取得到西曲依醇，属于 myo -肌醇衍生物类化 合物， 通过药理研究表明其具有预防和治疗糖尿病的作用。
梁敬钰等发明的申请号为 02144302.5 中国专利申请中公开了： 从红豆杉 中分离得到的西曲依醇能显著降低糖尿病模型高血糖、抑制肝糖原分解和葡萄 糖的吸收， 而且其毒性极低， 是一个很有价值的抗糖尿病药物。 但是红豆杉是 国家保护植物， 资源有限， 不能进行大规模的研究和开发， 为了寻找红豆杉的 替代资源，我们开发了肾蕨属的植物作为西曲依醇的来源，有人报道肾蕨属植 物中含有西曲依醇，但是所采用的工艺只是以研究确认为目的，提取工艺所获 得的西曲依醇含量极低，该工艺从提取成本、工艺路线等方面无法达到工业化 生产的要求。 发明内容
本发明的一个目的是提供一种从肾蕨属植物中获得含有西曲依醇的提取 物的方法。
本发明的另一个目的是提供一种利用上述方法制备的含有西曲依醇的提 取物。
本发明的又一目的是提供一种上述含有西曲依醇的提取物或其可药用盐 在制备治疗糖尿病及其并发症药物中的应用。
一种从肾蕨属植物中获得含有西曲依醇的提取物的方法， 包括以下步骤： 用溶剂提取， 回收溶剂得浸膏， 浸膏用两相萃取和柱层析， 收集含有西曲依醇 的馏分， 浓缩、 过滤、 干燥。
优选地， 所述肾蕨属植物选自下列物质或者它们组成的群组： 肾蕨、 长叶 肾蕨、 圆叶肾蕨、 镰叶肾蕨、 薄叶肾蕨、 双齿肾蕨、 耳叶肾蕨、 高大肾蕨、 毛 叶肾蕨、 迷你肾蕨和波士顿肾蕨
优选地， 所述用溶剂提取中的溶剂选自下列物质或者它们组成的群组： 乙 醇、 甲醇、 丙酮和水。 优选地， 提取的温度为 0°C至所述溶剂的回流温度， 优 选室温至所述溶剂的回流温度， 更优选为所述溶剂的回流温度。 优选地， 所述 溶剂与肾蕨属植物的重量体积比为 1 : 1〜20,优选为 1 : 2-10, 更优选为 1 : 3〜5。 优选地， 所述用溶剂提取次数为 2〜5次。
优选地，所述两相萃取所用的溶剂为与水不相容的有机溶剂、水。优选地， 所述与水不相容的有机溶剂选自下列物质或者它们组成的群组： 乙酸乙酯、氯 仿、二氯甲烷、 乙醚、石油醚和正丁醇。优选地， 所述与水不相容的有机溶剂、 水的体积比为： 1 : ΐ〜ΐο。
优选地， 所述柱层析为大孔树脂柱层析、 葡聚糖柱层析、 改性葡聚糖柱层 析、 纤维素柱层析、 活性炭柱层析和离子交换树脂柱层析。
优选地，在过滤之后干燥之前有一个纯化步骤，此纯化步骤优选为柱层析。 本发明还提供根据上述的获得含有西曲依醇的提取物的方法制备的提取 物。
本发明还提供上述的制备的提取物或其可药用盐在制备治疗糖尿病及其 并发症药物中的应用。优选地， 所述其可药用盐包括下列西曲依醇的盐类或者 它们组成的群组： 枸橼酸盐、 酒石酸盐、 醋酸盐、 乳酸盐， 钠盐、钾盐、钙盐、 铵盐、 镁盐、 铁盐、 锌盐和铝盐。 优选地， 所述并发症包括眼部病变、 肾部病 变、 神经病变、 冠心病、 脑血管病变、 外周血管病变。
本发明所述的从肾蕨属植物中获得含有西曲依醇的提取物的方法与现有 技术相比， 是根据肾蕨属植物的本身特点， 提出了优化的提取工艺， 形成了可 用于工业化的生产工艺路线。采用这种方法获得的西曲依醇的提取物具有抗糖 尿病活性且毒性低， 所述提取物的主要有效成分为西曲依醇， 含量可在 10.00 % -99.99 % , 动物实验证明提取物能够明显的降低血糖浓度。 附图说明
图 1是实施例 9中西曲依醇的出峰时间测定图。 具体实施方式
下面对本发明的技术方案做伴细的说明。
本发明提供了一种从植物肾蕨（ Nephrolepidaceae )等肾蕨属植物中提取、 分离的具有抗糖尿病活性且毒性低的含有西曲依醇的提取物的方法，所述肾蕨 属植物包括腎蕨 ( Nephrolepis auriculata (L.) Trimen )、 长叶腎蕨 (N. biserrata)、 圆叶肾蕨（ N. duffii Moore )、镰叶肾蕨（ N. falcate (Cav.) C. Chr. )、薄叶肾蕨（ N. delicatula (Decne.) Pichi-Serm. ), 双齿肾蕨 ( N. biseroata (Sw.) Schott. ), 耳叶肾 蔽 ( N. biserrata var.auriculata Ching )、 高大腎蔽 ( N. exaltata (Sw.) Schott. )、 毛 叶肾蕨（ N. hirsutula (Forst.) Presl )、 迷你肾蕨（ N. cordifolia )、 波士顿肾蕨（ N. exaltata cv. Bostoniensis )等。
一种从肾蕨属植物中获得含有西曲依醇的提取物的方法， 包括以下步骤： 用溶剂提取， 回收溶剂得浸膏， 浸膏用两相萃取和柱层析， 收集含有西曲依醇 的馏分， 浓缩、 过滤、 干燥。 经过此方法得到的含有西曲依醇的提取物通常为 晶状粉末， 通常西曲依醇的含量可达 60%以上。
利用上述方法获得的含有西曲依醇的提取物，可以用于制备治疗糖尿病及 其并发症的药物或者药物组合物 , 可以结合一种或多种辅助剂和 /或赋型剂制 备成针剂、 胶嚢剂、 片剂、 颗粒剂、 糖衣丸剂、 溶液剂等药物剂型及其上述剂 型的緩释制剂及靶向制剂等。
在制剂中使用的可以是西曲依醇，也可以是用已知的方法通过对西曲依醇 与相应的酸、 碱进行反应得到西曲依醇的可药用盐。
利用上述方法获得的含有西曲依醇的提取物可以用于制备治疗糖尿病的 药物， 能显著降低糖尿病高血糖、抑制肝糖元分解和葡萄糖的吸收、 降低血脂 以及改善自由基代谢和保护胰岛 β细胞， 而且毒性极低。 利用上述方法获得的 含有西曲依醇的提取物也可用于预防和治疗糖尿病及其并发症，其它与糖代谢 紊乱有关的疾病， 改善自由基代谢， 预防和治疗 II型糖尿病以及并发症。
更具体的说，本发明所述从肾蕨属植物中获得含有西曲依醇的提取物的方 法经常包括如下操作步骤： 将肾蕨属植物的根、 茎、 叶等部位晒干粉碎； 用例 如乙醇、 甲醇、 丙酮、 它们的含水物或它们的混合物等溶剂在 o°c至回流的温 度下， 优选室温至回流的温度下， 更优选在回流的温度下提取， 所用溶剂的量 优选为 1 : 1至 20 (重量 /体积）， 优选 1 : 2至 10 (重量 /体积）， 更优选 1 : 3至 5 (重量 /体积）， 优选提取 2〜5次； 经减压浓缩得浸膏， 将浸膏用与水不相容的 有机溶剂 （例如乙酸乙酯、 氯仿、 二氯甲烷、 乙醚、 石油醚等） /水两相萃取， 除去有机层， 所述萃取剂优选为 1 : 1至 10 (体积 /体积） 的有机溶剂 /水； 合并 水层， 滤过， 用柱层析分离， 所述柱层析可以为大孔树脂柱层析（例如 D101 型、 MN-200型 （PUROLITE ) 等）、 葡聚糖 G或改性葡聚糖柱层析 （例如 sephadex-LH-20等）、 纤维素柱层析、 活性炭柱层析以及离子交换柱层析， 用 相应的洗脱液洗脱， 同时检测洗脱液； 收集含有西曲依醇的馏分， 减压浓缩， 用柱层析纯化， 所述柱层析可以为大孔树脂柱层析（例如 D101型、 MN-200型 ( PUROLITE )等）、 葡聚糖 G或改性葡聚糖柱层析（例如 sephadex-LH-20等）、 纤维素柱层析、活性炭柱层析以及离子交换树脂柱层析。合并含有西曲依醇的 馏分， 减压浓缩、 静置、 过滤得固形物， 用例如乙醇、 甲醇、 丙酮、 甲乙酮、 它们的含水物或它们的混合物重结晶，得到干燥的植物提取物粉末。 最终产物 为晶状粉末， 主要有效成分为西曲依醇， 含量在 10.00 % - 99.99 %。
本发明所述方法中对柱层析洗脱液的检测可以使用以下方法：
薄层层析： 采用 F254薄层板， 分别吸取待测液与对照溶液（西曲依醇水溶 液） 5μ1 (微升）， 点于薄层板， 以水： 乙酸乙酯： 异丙醇（6: 11 : 83 (体积 比））为展开剂， 用高锰酸钾-氢氧化钠显色， 溶液主斑点与对照品斑点 Rf值相 同为正反应。
本发明所述提取物的主要有效成分西曲依醇的含量可以用下述高效液相 ( HPLC )方法检测：
含量测定 1 取本品约 5mg (毫克）， 精密称定， 置 10ml (毫升）量瓶中 , 加稀^ ^酸-醋酐液（1 : 50体积比） 0.25ml， 置水浴加热 30min (分钟）， 冷却至 室温， 加甲醇 5ml， 摇匀，加水至刻度，摇匀， 得供试品溶液， HPLC色谱条件： C18柱， 5μ1, 4.6x250mm (毫米）； 检测波长 220nm (纳米）； 进样 20μ1; 流动 相， 甲醇-水（50: 50体积比）， 使用前经 0.45μπι (微米）有机滤膜抽滤并脱气， 流速 1.0ml/min。
含量测定 2 取本品 5mg, 精密称定， 置 10ml量瓶中， 以水溶解定容， 摇 匀。 HPLC色谱条件： 氨基柱， 5μπι, 4.6x250mm, 检测器： 蒸发光散射检测 器（ELSD ), 漂移管温度： 40°C , 压力： 3.5bar (巴）， 进样量： 20μ1。 流动相 为乙腈 -水 （ 75:25体积比）， 使用前经 0.45μπι有机滤膜抽滤脱气， 流速 0.8ml/min。
临床上可以用所获得的肾蕨提取物制备成各种形式的药品或保健品等。 胶嚢剂、片剂、颗粒剂、糖衣丸剂等剂型可以含有一种或多种常用赋型剂 , 具体可以为：
填料和增溶剂： 淀粉， 微晶纤维等； 粘合剂：： 羧甲基纤维素， 聚乙烯吡咯烷酮等;
致湿剂： ： 甘油等；
崩解剂: ： 碳酸钙等；
吸收剂： ： 高岭土等；
润滑剂: ： 滑石粉等。
药用赋形剂可以是固体、 半固体、 液体等各种形态的， 配方辅助剂可以是 各种类型的。
溶液剂可以含有一般的溶剂、 增溶剂、 乳化剂、 防腐剂等， 如水、 乙醇、 甘油、 聚乙二醇、 苯甲酸苄酯等。
胶嚢剂、 片剂、 颗粒剂、 糖衣丸剂、 溶液剂等剂型可以用已知的常规方法 制备， 可以将植物提取物与一种或多种赋型剂混合， 制备剂型。
本发明可以制备治疗糖尿病的中药提取物制剂，为治疗糖尿病的药物市场 注入了新的活力。 另外， 肾蕨属植物是华南地区常见易得的中草药， 也有地区 大量种植作为食用， 作为原药材， 较易获得， 且制备工艺简单， 成本低廉， 为 社会提供了一种具有明显治疗糖尿病的天然药物。
下面结合实施例对本发明的技术方案作进一步的阐述。
实施例 1 : 低纯度植物提取物的制备
肾蕨粗粉 100kg (千克）， 以 3倍体积 80%乙醇回流提取三次， 合并提取液， 减压浓缩， 用 1 : 1 (体积 /体积） 的乙酸乙酯 /水两相萃取， 合并水层， 滤过， 大孔树脂柱（D101型， 国产）层析， 以蒸馏水、 含水乙醇梯度洗脱， 收集含 有西曲依醇部分， 减压浓缩干燥， 得植物提取物（微黄色粉末）， 其中西曲依 醇含量在 10.2%。
实施例 2: 较低纯度植物提取物的制备 1
肾蕨粗粉 50kg, 以 3倍体积 90%乙醇 60 °C回流提取三次， 合并提取液， 减 压浓缩至干， 用 1 : 1 (体积 /体积）的二氯甲烷 /水两相萃取， 合并水层， 滤过， 大孔树脂柱（D101型， 国产）柱层析， 以蒸馏水、含水乙醇（乙醇： 5%〜20% ) 梯度洗脱，各洗脱液分别用鉴别试液鉴别，合并显正反应的洗脱液，减压浓缩， 于 70°C下干燥， 得植物提取物 （微黄色粉末）， 其中西曲依醇含量达 28.9%。
实施例 3: 较低纯度植物提取物的制备 2 肾蕨粗粉 60kg, 以 3倍体积 90%甲醇 50 °C回流提取三次， 合并提取液， 减 压浓缩至干， 用 1 : 1 (体积 /体积）的乙酸乙酯 /水两相萃取， 合并水层， 滤过， 大孔树脂柱（MN-200型，国产）柱层析，以蒸馏水、含水乙醇（乙醇： 5%〜20% ) 梯度洗脱，各洗脱液分别用鉴别试液鉴别，合并显正反应的洗脱液，减压浓缩， 于 70°C下干燥， 得植物提取物 （微黄色粉末）， 其中西曲依醇含量达 29.5%。
实施例 4: 较低纯度植物提取物的制备 3
肾蕨粗粉 70kg, 以 3倍体积 70%丙酮 45 °C回流提取三次， 合并提取液， 减 压浓缩至干， 用 1 : 1 (体积 /体积） 的氯仿 /水两相萃取， 合并水层， 滤过， 活 性炭柱层析， 以蒸馏水、 含水乙醇（乙醇： 5%〜25% )梯度洗脱， 各洗脱液分 别用鉴别试液鉴别， 合并显正反应的洗脱液， 减压浓缩， 于 70°C下干燥， 得植 物提取物（微黄色粉末）， 其中西曲依醇含量达 30.2%。
实施例 5: 中等纯度植物提取物的制备 1
取低纯度的植物提取物用水溶解，纤维素柱层析，以蒸馏水、含水乙醇（乙 醇： 5%〜20% )梯度洗脱， 各洗脱液分别用鉴别试液鉴别， 合并显正反应的洗 脱液， 减压浓缩， 乙醇重结晶， 过滤得晶状粉末， 抽干， 于 70°C下干燥， 得植 物提取物（微黄色粉末）， 用 HPLC方法进行检测， 其中西曲依醇含量达 78%。
实施例 6: 中等纯度植物提取物的制备 2
取低纯度的植物提取物用水溶解， 离子交换柱层析， 以蒸馏水、含水乙醇 (乙醇： 5%〜20% )梯度洗脱， 各洗脱液分别用鉴别试液鉴别， 合并显正反应 的洗脱液， 减压浓缩至干， 甲醇重结晶， 过滤得晶状粉末， 抽干， 于 70°C下干 燥， 得植物提取物（微黄色粉末）， 用 HPLC方法进行检测， 其中西曲依醇含量 达 75%。
实施例 7: 中等纯度植物提取物的制备 3
取低纯度的植物提取物用水溶解， 凝胶柱层析， 以蒸馏水、 含水乙醇（乙 醇： 5%〜20% )梯度洗脱， 各洗脱液分别用鉴别试液鉴别， 合并显正反应的洗 脱液， 减压浓缩至干， 丙酮重结晶， 过滤得晶状粉末， 抽干， 于 70°C下干燥， 得植物提取物 （微黄色粉末）， 用 HPLC方法进行检测， 其中西曲依醇含量达 68%。
实例 8: 高纯度植物提取物的制备 采用中等纯度的植物提取物， 用甲醇等反复重结晶， 过滤得晶状粉末， 抽 干， 于 70°C下干燥， 得到高纯度西曲依醇的植物提取物。 反复重结晶后， 西曲 依醇的纯度可以达到 99 %以上。
实施例 9: 高纯度西曲依醇的含量测定
采用高效液相色谱法， 取高纯度提取物样品 5mg, 精密称定， 置 10ml量瓶 中， 以水溶解定容， 摇匀。 HPLC色谱条件： 氨基柱， 5μπι, 4.6x250mm; 检 测器： 蒸发光散射检测器（ELSD ); 漂移管温度： 40 °C ; 压力： 3.5bar; 进样 量： 20μ1; 流动相： 乙腈-水（75:25 )， 使用前经 0.45μπι有机滤膜抽滤脱气， 流速 1.0ml/min。
图语如图 1，从图 1可以看出西曲依醇出峰时间 12.312min。
实施例 10: 胶嚢剂的制备
处方： 每粒 300mg, 含植物提取物 30mg。
微晶纤维素 270mg
植物提取物 30mg
1000粒
首先将辅料置于研磨器中， 之后加入植物提取物， 研磨混合 10〜30min， 至均匀止，将混匀的药粉，罐装于 1号胶嚢中，随机抽样每粒装量控制在 300mg, 其中植物提取物含量为 30mg。
实施例 11 : 胶嚢剂稳定性研究
对三批胶嚢剂进行了 9个月的稳定性试验， 考察内容包括外观、 鉴别、 含 量等项目。
鉴别 （1 ) 的方法为， 取本品 20粒， 倾出内容物， 称取内容物粉末适量加 水溶解， 过滤。 取滤液 5ml于试管， 加入 0.01mol/L (摩尔 /升）的高碘酸 ]溶液 2ml和浓硝酸 2滴后， 滴加硝酸银试液， 有白色沉淀产生。 3批胶嚢剂的检测结 果见表 1。
鉴别 （2 )的方法为， 取本品内容物适量（约相当于胶嚢 0.2g )， 加水 10ml 振摇， 滤过。取续滤液 2ml，加碱式醋酸铅溶液 lml，摇匀，置水浴中加热 5min， 即成为半透明胶状物。 3批胶嚢剂的检测结果见表 1。 表 1 三批胶嚢剂样品稳定性试验结果
时 间 批 号 夕卜 观 崩 解 时 鉴 别 鉴 别 含 量
(月） 色 泽 限（ min ) ( 1 ) ( 2 ) ( % )
051116 类白色 <10 符合 符合 72.8
0 051117 类白色 <10 付合 付合 69.5
051118 类白色 <10 付合 付合 70.6
051116 类白色 <10 付合 付合 71.0
3 051117 类白色 <10 付合 付合 70.5
051118 类白色 <10 付合 付合 68.9
051116 类白色 <10 付合 付合 72.0
6 051117 类白色 <10 付合 付合 68.7
051118 类白色 <10 付合 付合 69.1
051116 类白色 <10 付合 付合 71.5
9 051117 类白色 <10 付合 付合 70.5
051118 类白色 <10 付合 付合 69.3 实施例 12: 片剂的制备
植物提取物 25g (克）， 微晶纤维素 49g， 硬脂酸镁 lg, 充分混合； 混合物 用单冲压片机压成直径 6mm, 重 300mg的片剂， 每片含植物提取物 100mg。
实施例 13: 颗粒剂的制备
植物提取物 20g， 加玉米淀粉 180g， 充分混合， 加适量乙醇（60% )制成 软材，过 12目筛造颗粒， 经干燥后得颗粒剂，每 lg颗粒剂含植物提取物 100mg。
实施例 14: 注射剂的制备
采用精制后的西曲依醇提取物 （纯度在 98.00%〜99.99% )， 制备成注射用 原料。
西曲依醇水溶性好， 将西曲依醇水溶后， 过滤灭菌， 按注射液制备工艺程 序操作， 制备注射液。 按冻干粉针工艺程序操作， 制备粉针剂。
采用精制后的注射用西曲依醇原料，按冻干粉针工艺程序操作， 制备冻干 粉针剂。取注射用西曲依醇原料， 加注射用水溶解， 加入预先除热原的 20 %低 分子右旋糖酐溶液， 充分混勾， 定体积最后定量， 使得最后浓度为西曲依醇 O.lg/ml,右旋糖酐 0.05g/ml,采用活性炭去除热原， 0.22μπι微孔滤膜过滤除菌， 在 100级洁净区内按每瓶 lml灌装入预先处理好的西林瓶中。 在冻干箱内， - 40°C预冻 2小时， 然后在零下 25°C , 1.33Pa真空度下升华， 游离水分去除 90 % 后， 加温干燥， 温度控制不超过 35°C至冻干结束， 冻干箱内加塞密封。
实施例 15: ^片的制备
称取西曲依醇干燥提取物 60g、微晶纤维素 20g， 交联聚乙烯吡咯烷酮 10g， 低取代羟丙纤维素 10g， 混合均匀； 滴加 5 %聚乙烯吡咯烷酮 K30的乙醇液制软 材， 过 30目筛制粒， 湿粒于 60°C条件下进行干燥， 用 40目筛整粒， 加入交联聚 乙烯吡咯烷酮 10g，低取代羟丙纤维素 10g，硬脂酸镁 5g，混匀，压片，制成 1000 片。
测试： 均匀性的测定： 取 2片西曲依醇提取物分散片置于 100ml水 中振摇， 在 20°C±1 °C的温度下， 180秒钟全部崩解并通过 2号筛。 2 )溶出情况 的测定： 采用人工胃液 0.1mol/L盐酸进行溶出度的测定。 按照药典二部附录中 溶出度测定法第二法（浆法）， 浆速采用 100转 /分， 37°C , 900ml溶出液。 取西 曲依醇提取物分散片在上述条件下， 分别于 2、 4、 6、 8、 10、 15、 20、 30、 45、 60分钟定时定点取样 5毫升 （同时补充同样的介质）、 45μπι微孔滤膜过滤， 测 定溶出量， 计算相对累积溶出百分率。 试验结果见表 2。
表 2: 西曲依醇提取物分散片溶出度
时间
2 4 6 8 10 15 20 30 45 60
( min )
溶出度
73.24 81.34 85.54 91.36 92.42 94.19 95.29 96.26 96.42 97.28 ( % ) 实施例 16: 泡腾片的制备
西曲依醇提取物原料按如下配方制备泡腾片： 西曲依醇提取物 60克， 酒石 酸 4.2克， 碳酸氢钠 17.5克， 吐温 -80 1.2克， 聚乙烯吡咯烷酮 (PVP)3.2克， 低取 代羟丙基纤维素 (LHPC)1.6克， 硬脂酸镁 0.64克， 将上述试剂充分混合， 压片， 共制成 1000片。 每片含西曲依醇提取物 60mg。
检查：取内径 1.5cm 、 25ml具塞刻度试管 10支，精密加水 2ml，置 37°C±1°C 水浴中 5分钟后， 管中分别投入供试品 1片， 密塞， 20分钟内观察最大发泡量的 体积， 平均发泡体积为 4.6ml, 少于 3ml的 1片。
实施例 17: 糖浆剂的制备
取蔗糖 600g, 置 1000毫升烧杯中， 加 800毫升蒸榴水， 加热使溶解， 然后 加入西曲依醇提取物 20g， 搅拌使溶解， 冷却置室温， 滤过， 收集滤液， 自滤 器上添加适量蒸馏水使成 1000毫升，充分混匀后，分装于 10毫升安瓿瓶， 100°C 灭菌 30分钟， 即得。
以下用按照实施例 1所制得的植物提取物进行药效学实验和毒理学研究。 主要药效学实验
植物提取物对四氧嘧啶诱发小鼠血糖升高的影响
实验方法： 采用雄性小鼠 80只， 体重 22-25g, 随机取出 20只为正常组， 其 余小鼠尾静脉注射四氧嘧啶 65mg/kg, 按文献方法造模， 根据血糖值分为 3组， 其中 2组分别按 0.1ml/10g体重经口给予 60mg/kg肾蕨提取物、 降糖灵（阳性对 照药） 75mg/kg, 正常组及模型组给予等体积的蒸馏水， 连续 7天。 以葡萄糖氧 化酶法测定血糖。
实验结果：与正常组相比，对照组小鼠血糖极显著升高（升高 312.7mg/dl )。 与对照组相比，降糖灵 7511^/1¾组（降低 152.8 mg/dl )、肾蕨提取物组（降低 110.6 mg/dl ) 能明显降低四氧嘧啶诱发的高血糖。
植物提取物对葡萄糖诱发的小鼠血糖升高的影响
实验方法： 采用雄性小鼠 120只， 体重 22-25g， 随机分为 6组，
其中 4组分别按 0.1ml/10g体重经口给予 60mg/kg肾蕨提取物 （含西曲依醇 65 % )， 正常组及对照组给予等体积的蒸榴水， 连续 7天。 除正常组外， 其余各 组腹腔注射葡萄糖 2g/kg, 正常组注射等体积的生理盐水， 注射后 30、 60、 90、 120分钟， 小鼠眼眶后静脉丛取血， 分离血清， 以葡萄糖氧化酶法测定血糖。
实验结果： 与正常组相比，对照组小鼠腹腔注射葡萄糖后 30、 60、 90、 120 分钟，血糖极显著升高。与对照组相比，肾蕨提取物组在腹腔注射葡萄糖后 30、 60、 90、 120分钟， 显著降低葡萄糖诱发的高血糖， 其中 90分钟时， 肾蕨提取 物组降低 15.5mg/dl。
急性毒性实验
实验方法： 取正常小鼠 40只， 雌雄各半， 给予肾蕨提取物按 800mg/kg—次 灌服， 观察动物的反应， 7天后处死， 肉眼观察各脏器。 结果： 动物无一死亡， 健康情况良好、 皮毛光泽、 眼红明亮、 活动度好。 7天后处死小鼠， 肉眼观察 主要脏器均无异常。 以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通 技术人员来说， 在不脱离本发明原理的前提下， 还可以做出若干改进和润饰， 这些改进和润饰也应视为本发明的保护范围。

Claims

权 利 要 求
I、 一种从肾蕨属植物中获得含有西曲依醇的提取物的方法， 包括以下步 骤： 用溶剂提取， 回收溶剂得浸膏， 浸膏用两相萃取和柱层析， 收集含有西曲 依醇的馏分， 浓缩、 过滤、 干燥。
2、根据权利要求 1所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述肾蕨属植物选自下列物质或者它们组成的群组： 肾蕨、 长叶肾蕨、 圆叶肾 蕨、 镰叶肾蕨、 薄叶肾蕨、 双齿肾蕨、 耳叶肾蕨、 高大肾蕨、 毛叶肾蕨、 迷你 肾蕨和波士顿肾蕨。
3、根据权利要求 1所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述溶剂选自下列物质或者它们组成的群组： 乙醇、 甲醇、 丙酮和水。
4、根据权利要求 3所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述提取的温度为 0°C至所述溶剂的回流温度， 优选室温至所述溶剂的回流温 度， 更优选为所述溶剂的回流温度。
5、根据权利要求 4所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述溶剂与肾蕨属植物的重量体积比为 1 : 1-20, 优选为 1 : 2-10, 更优选为 1 : 3〜5。
6、根据权利要求 1所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述用溶剂提取次数为 2〜5次。
7、根据权利要求 1所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述两相萃取所用的溶剂为与水不相容的有机溶剂、 水。
8、根据权利要求 7所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述与水不相容的有机溶剂选自下列物质或者它们组成的群组： 乙酸乙酯、氯 仿、 二氯甲烷、 乙醚、 石油醚和正丁醇。
9、根据权利要求 7所述的获得含有西曲依醇的提取物的方法，其特征在于， 所述与水不相容的有机溶剂、 水的体积比为： 1 : 1〜10。
10、 根据权利要求 1所述的获得含有西曲依醇的提取物的方法， 其特征在 于， 所述柱层析为大孔树脂柱层析、 葡聚糖柱层析、 改性葡聚糖柱层析、 纤维 素柱层析、 活性炭柱层析和离子交换树脂柱层析。
II、 根据权利要求 1所述的获得含有西曲依醇的提取物的方法， 其特征在 于， 在过滤之后干燥之前有一个纯化步骤。
12、根据权利要求 11所述的获得含有西曲依醇的提取物的方法， 其特征在 于， 所述纯化为柱层析。
13、根据权利要求 1〜12中任意一项所述的获得含有西曲依醇的提取物的方 法制备的提取物。
14、根据权利要求 13所述的制备的提取物或其可药用盐在制备治疗糖尿病 及其并发症药物中的应用。
15、根据权利要求 14所述的应用， 其特征在于， 所述其可药用盐包括下列 西曲依醇的盐类或者它们组成的群组：枸橼酸盐、酒石酸盐、醋酸盐、乳酸盐， 钠盐、 钾盐、 钙盐、 铵盐、 镁盐、 铁盐、 锌盐和铝盐。
16、根据权利要求 14所述的应用，其特征在于，所述并发症包括眼部病变、 肾部病变、 神经病变、 冠心病、 脑血管病变、 外周血管病变。
PCT/CN2008/071006 2008-05-19 2008-05-19 从肾蕨属植物中获得含有西曲依醇的提取物的方法和用途 WO2009140814A1 (zh)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2011509837A JP2011520926A (ja) 2008-05-19 2008-05-19 ネフロレピス属からセクオイトール含有抽出物を得る方法及びその使用
EP08748612A EP2277525A4 (en) 2008-05-19 2008-05-19 PROCESS FOR THE PREPARATION OF AN EXTRACTION PRODUCT CONTAINING SEQUOYITOL FROM THE FAMILY OF NEPHROLEPIS AND APPLICATION
US12/993,149 US20110071225A1 (en) 2008-05-19 2008-05-19 Method for obtaining a sequoyitol-containing extract from genus nephrolepis and uses thereof
PCT/CN2008/071006 WO2009140814A1 (zh) 2008-05-19 2008-05-19 从肾蕨属植物中获得含有西曲依醇的提取物的方法和用途

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2008/071006 WO2009140814A1 (zh) 2008-05-19 2008-05-19 从肾蕨属植物中获得含有西曲依醇的提取物的方法和用途

Publications (1)

Publication Number Publication Date
WO2009140814A1 true WO2009140814A1 (zh) 2009-11-26

Family

ID=41339725

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2008/071006 WO2009140814A1 (zh) 2008-05-19 2008-05-19 从肾蕨属植物中获得含有西曲依醇的提取物的方法和用途

Country Status (4)

Country Link
US (1) US20110071225A1 (zh)
EP (1) EP2277525A4 (zh)
JP (1) JP2011520926A (zh)
WO (1) WO2009140814A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104435014B (zh) * 2014-12-29 2018-10-30 广西梧州制药（集团）股份有限公司 肾蕨在制备治疗和/或预防阿尔兹海默病药物方面的用途

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1957992A (zh) * 2005-11-01 2007-05-09 广州威尔曼新药开发中心有限公司 一种含有西曲依醇的植物提取物的制备方法及药用用途
CN101185662A (zh) * 2006-11-15 2008-05-28 广州联创思远利生物科技有限公司 从肾蕨中制备的用于治疗糖尿病新药的方法和用途

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE60230455D1 (de) * 2002-10-17 2009-01-29 Jingyu Liang Natürliche verbindung für die behandlung von diabetes, deren herstellung und verwendung
CN1706369A (zh) * 2004-06-11 2005-12-14 广州威尔曼新药开发中心有限公司 用于糖尿病预防和治疗的红杉醇和红杉醇植物提取物

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1957992A (zh) * 2005-11-01 2007-05-09 广州威尔曼新药开发中心有限公司 一种含有西曲依醇的植物提取物的制备方法及药用用途
CN101185662A (zh) * 2006-11-15 2008-05-28 广州联创思远利生物科技有限公司 从肾蕨中制备的用于治疗糖尿病新药的方法和用途

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP2277525A4 *
WANG HENGSHAN ET AL.: "Studies on the chemical constituents from the Chinese traditional edicine Nephrolepis cordifolia", GUIHAIA, vol. 24, no. 2, March 2004 (2004-03-01), pages 155 - 157, XP008143891 *

Also Published As

Publication number Publication date
US20110071225A1 (en) 2011-03-24
JP2011520926A (ja) 2011-07-21
EP2277525A1 (en) 2011-01-26
EP2277525A4 (en) 2011-11-09

Similar Documents

Publication Publication Date Title
CN101185692B (zh) 一种胆木提取物及其制剂与用途
JP6091651B2 (ja) 頭痛治療用医薬組成物、及びその調製法
AU2003260985B2 (en) Extraction and purification method of active constituents from stem of Lonicera japonica Thunb., its usage for anti-inflammatory and analgesic drug
WO2008145064A1 (fr) Procédé d&#39;obtention d&#39;un extrait contenant du séquoyitol à partir d&#39;une espèce du genre trifolium, de soja et de ginkgo biloba, et utilisation de celui-ci
WO2015081703A1 (zh) 一种含有广金钱草总黄酮的固体分散体及其制备方法和用途
CN101703588A (zh) 双黄连原位凝胶制剂及其制备方法
CN102233001B (zh) 一种治疗心脑血管疾病的中药组合物及其制法和检测方法
CN104435034A (zh) 一种三七总皂苷及其制备方法
JP2021512997A (ja) 分離された防風多糖及びその用途
CN101756955B (zh) 知母宁复合物及其制备方法和应用
CN106421075B (zh) 一种竹柳抗氧化活性组分的制备方法和应用
CN101559066B (zh) 荭草素-2&#34;-O-β-L-半乳糖苷及组合物在制备抗心肌缺血药物中的应用
CN103083370B (zh) 沙棘总黄酮的用途
CN103816147B (zh) 藤黄酸、新藤黄酸及其组合物的医药用途
CN101904894B (zh) 独一味总苷在制备药物中的用途
WO2009140814A1 (zh) 从肾蕨属植物中获得含有西曲依醇的提取物的方法和用途
CN101185662A (zh) 从肾蕨中制备的用于治疗糖尿病新药的方法和用途
CN109453149B (zh) 吉马酮在制备防治银屑病的药物中的应用
CN102302545A (zh) 一种具有降血糖活性的傣药提取物、制法、组合物与用途
CN102119984B (zh) 含羟基红花黄色素a的红花有效组分制备方法和用途
CN112870208A (zh) Pubescenoside A在制备防治心肌缺血再灌注损伤药物中的应用
CN1857385B (zh) 一种治疗颈椎病的药物组合物及其制备方法
CN103505498A (zh) 胡芦巴总黄酮有效部位制备及应用
CN103495156B (zh) 一种重组人胰岛素缓释片及其制备方法
CN1679698A (zh) 一种由三七、川芎制成的治疗心脑血管疾病的药物制剂及其制备方法

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: 08748612

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 2008748612

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2011509837

Country of ref document: JP

WWE Wipo information: entry into national phase

Ref document number: 12993149

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE