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WO2003105829A1 - Formulation de remede traditionnel chinois pour la prevention et le traitement de l'osteoporose - Google Patents

Formulation de remede traditionnel chinois pour la prevention et le traitement de l'osteoporose Download PDF

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Publication number
WO2003105829A1
WO2003105829A1 PCT/CN2003/000466 CN0300466W WO03105829A1 WO 2003105829 A1 WO2003105829 A1 WO 2003105829A1 CN 0300466 W CN0300466 W CN 0300466W WO 03105829 A1 WO03105829 A1 WO 03105829A1
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WIPO (PCT)
Prior art keywords
methanol
icariin
preparation
solution
rats
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PCT/CN2003/000466
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English (en)
Chinese (zh)
Inventor
Quan Hai Liu
Ming Min Wang
Si Qi Luo
Jing Zhi Dai
Yi Jiang
Original Assignee
Shanghai Institute Of Pharmaceutical Industry
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Application filed by Shanghai Institute Of Pharmaceutical Industry filed Critical Shanghai Institute Of Pharmaceutical Industry
Priority to AU2003244067A priority Critical patent/AU2003244067A1/en
Publication of WO2003105829A1 publication Critical patent/WO2003105829A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/29—Berberidaceae (Barberry family), e.g. barberry, cohosh or mayapple
    • A61K36/296—Epimedium
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis

Definitions

  • the present invention belongs to the technical field of traditional Chinese medicine preparations. Specifically, it relates to an effective component of a traditional Chinese medicine for anti-osteoporosis and a preparation thereof.
  • BACKGROUND ART Epimedium total flavonoid powder is an effective part extracted from Chinese medicine Epimedium.
  • Chinese medicine Epimedium has the following varieties: Epimedium brevicornum Maxim., Epimedium sagittatum (Sieb. Et Zucc) Maxim., Epimedium pubescens Maxim., Wushan Sheep Epimedium wushanense TS Ying, or Kusuki Fresh Epimedium Epimedium koreanumNakai.
  • the medicinal part of the traditional Chinese medicine Epimedium is a dry aerial part, and rough stems and impurities are removed.
  • Korean Epimedium (Epimedium koreanuni Nakai), also known as Northeast Epimedium ("National Compendium of Chinese Herbal Medicine” :). Most of the chemical components in its medicinal parts are flavonoids. Existing chemical composition studies have shown that it contains icariin and icariin A, icaridin A, B, C, icaridin A, B1, Quercetin, dehydrated icariin-3-rhamnoside, epimedokoreanoside I, II.
  • Icariin a flavonoid glycoside, combines the hydroxyl groups at the C3 and C7 positions with sugars to form a glycoside. Its chemical structure is as follows:
  • icariin should have the characteristic UV-ray yield of flavonoids. UV scanning confirmed that icariin has strong absorption around 270nm. Therefore, when the content is measured by high-performance liquid phase, 270nm is selected as the measurement wavelength. There is no anti-osteoporosis agent prepared with the Chinese medicine Epimedium. SUMMARY OF THE INVENTION The technical problem to be solved by the present invention is to develop a Chinese medicine preparation for anti-osteoporosis.
  • the invention provides a traditional Chinese medicine preparation for preventing osteoporosis, Xianling Gukang Tablets, which contains Chinese medicine Epimedium.
  • the preparation is composed of the active ingredients extracted by Epimedium and medicinal excipients.
  • the pharmacodynamics of the traditional Chinese medicine preparations for anti-osteoporosis according to the present invention are as follows: Pharmacodynamics studies of the effective components extracted from the Chinese medicine Epimedium (hereinafter referred to as 9412): 1. 9412 caused a large amount of retinoic acid Effect of rat osteoporosis-preventive effect
  • the oral dose is 1ml / 100g rats, and the suspension is formulated with 0.5% CMC, 70mg / kg dose, poX 14.
  • DPX-L dual-energy X-ray bone densitometer produced by LunAR, USA, provided by Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.
  • Rats, SD provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences.
  • the rats were randomly divided into 6 groups, the normal control group, the model control group, the 9412 low, medium, and high dose groups, and the positive control group. Except the normal control group, rats in each group were given orally retinoic acid 70mg / kg, and the amount was 1ml.
  • the rats in each group were orally administered the following samples in sequence
  • the normal control group and the retinoic acid model group were the corresponding solvents 10ml / kg, 9412 low dose group 4mg / kg, medium dose group 12mg / kg, tritium dose group 36mg / kg, positive drug sodium hydroxyethyl phosphonate sodium 50mg / kg, the dosage was 1ml / 100g rats, once a day for 28 consecutive days, during which the body weight was weighed once a week. Dosing weight adjustment.
  • the animals were killed by decapitation, blood was collected to separate the serum, and the contents of S-Ca and SP were measured according to the kit method.
  • the bilateral femurs of the rats were removed, and the meat and other tissues were stripped.
  • One of the femurs was in a dual-energy X-ray bone.
  • the other side of the femoral head was decalcified with 3% HN0 3 for paraffin sectioning, HE staining, and bone trabecular width was measured under a microscope.
  • Model control group 10 117.2 + 4.4 125.5 + 5.7 133.8 ⁇ 14.5 ⁇ 178.7 + 19.5 ⁇
  • the bone ash weight and bone Ca and bone P contents in the ash were reduced, but the bone ash weight, bone Ca, and bone P contents in the medium and high-dose groups and the hydroxyethylphosphonate sodium group in the 9412 group after treatment were relatively low. increase.
  • Bone density tester DPX-L dual-energy X-ray bone density meter, produced by LunA, USA, provided by Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.
  • Rats, SD provided by Shanghai Experimental Animal Center, Chinese Academy of Sciences.
  • the rats were randomly divided into 6 groups, the normal control group, the model control group, the 9412 low, medium, high dose group, and the positive drug control group. Except the normal control group, rats in each group were given orally retinoic acid 70mg / kg. lml / 100g rats, once a day for 14 consecutive days. After the end, each drug group will be administered orally once a day for 28 consecutive days. The normal control group and the model group will be given the corresponding solvent. 100 g body weight, during which the dose was adjusted according to body weight.
  • the animals were decapitated and the blood was collected to separate the serum.
  • the S-Ca and SP contents were measured according to the kit method.
  • the bilateral femurs of the rats were taken and the meat and other tissues were stripped.
  • One of the femurs was on the dual energy X-ray bone.
  • the other side of the femoral head was decalcified with 3% HN0 3 for paraffin sectioning, HE staining, and the width of the trabecular bone was measured under a microscope.
  • Table 5 shows that after the administration of retinoic acid, the weight of the animals in each group decreased significantly compared with the normal control group, but after 9412, the weight of the administration group began to recover, and the retinoic acid model group also began to recover.
  • the W, L, d, W / L, and W / Ld of osteoporosis in the model group were significantly reduced, compared with the high dose group of the model group 9412 and the sodium hydroxyethylphosphonate group.
  • the ash weight and the content of bone Ca and bone P in the model group were reduced, but the content of bone ash, bone Ca, and bone P in the medium and high-dose groups and the hydroxyethylphosphonate sodium group were all reduced Relative increase.
  • DPX-L dual-energy X-ray bone densitometer produced by LunAR, USA, provided by Shuguang Hospital, Shanghai University of Traditional Chinese Medicine.
  • Source species, strain, certificate-rat, SD line, provided by Shanghai Laboratory Animal Center, Chinese Academy of Sciences.
  • Weight 110 ⁇ 130g for females, 130 ⁇ 150g for males
  • the sham operation group was only sterilized by abdominal surgery and then sutured.
  • the model group was given the corresponding solvent at a dose of 1ml / 100g. Body weight, during which the dose is adjusted according to body weight.
  • the animals were decapitated and the blood was collected to separate the serum.
  • the S-Ca and SP contents were measured according to the kit method.
  • the bilateral femurs of the rats were taken and the meat and other tissues were stripped.
  • One of the femurs was on the dual energy X-ray bone.
  • the other side of the femoral head was decalcified with 3% HN0 3 for paraffin sectioning, HE staining, and the width of the trabecular bone was measured under a microscope.
  • ⁇ , L, d, W / L, and W / Ld in the model group were significantly decreased, and the doses of each group in the model group could increase the femoral weight of rats (P ⁇ 0.05, P ⁇ 0.01), which is most obvious in the middle-dose group, and the W / L and W / Ld of the middle-dose group and sodium hydroxyethylphosphonate group are significantly different from the model group.
  • Dosage form extract, test for sterile filtration after dissolving in PBS
  • L 3 H-TdR is continued after 48 hours of incubation After 8 hours of incubation, the cells were collected with a multi-head cytometer after digestion with 0.5% trypsin, and the CPM value was measured on a liquid scintillator. The results were compared with the blank control tube.
  • the active ingredient extracted from the ⁇ 9412 Chinese herbal medicine Epimedium is mainly flavonoids.
  • a series of tests showed that 9412 with a flavonoid content of 60% has an anti-osteoporosis effect.
  • 9412 in the trial of retinoic acid-induced osteoporosis in rats, 9412, whether in low, medium or high dose groups, can increase femoral bone mineral density (P ⁇ 0.05, P ⁇ 0.01), increase the weight of femur and ashes, and femur. Content of calcium and phosphorus in ashes.
  • the low, medium, and high dose groups of 9412 can increase bone density of femur in rats (P ⁇ 0.05, P ⁇ 0.01), and it is more obvious in the middle dose group (P ⁇ 0.01), the middle and high dose groups can increase the femur ash weight and bone calcium and bone phosphorus content, and the effect of low dose is not obvious.
  • the medium and high dose groups could increase the bone density of femur in the rats (P ⁇ 0.05), but the low dose group was not obvious. Influence of serum calcium and phosphorus content, However, it can increase the femur weight and the calcium and phosphorus content in the ashes of rats in different degrees, and the medium dose group is better.
  • 5 9412 has a certain growth-promoting effect on osteoblasts of newborn rats.
  • Liquid preparation 0.5% CMC + Tween 80
  • Words are based on standard full-price nutritional pellets, free to drink and drink.
  • Test method In the preliminary test, 9412 20% solution was orally administered to rats in an amount of 0.5 ml, and no death was observed in the animals.
  • mice were orally administered with a 20% 9412 suspension, 0.5 ml per mouse, twice a day, and observed the immediate response after administration.
  • the weight changes of the mice were recorded within two weeks.
  • the animals were sacrificed at the end of the observation period, and the histopathological changes of the animals were observed with the naked eye.
  • the animals did not have any adverse symptoms after administration, and the active diet was normal. At the end of the observation period, the animals were necropsied, and no pathological abnormalities were visible to the naked eye.
  • mice The maximum tolerated amount of oral 9412 in mice was above 10 g / kg.
  • WBC was found to be slightly lower in the three dose groups of male rats compared to the control group when administered for three months, and WBC and RBC were slightly lower in the high dose group of male rats compared to the control group when administered for six months.
  • the RBC in the low and high dose groups was slightly lower compared to the control group. Two weeks after discontinuation, the RBC in the medium-dose group was slightly lower than that in the control group.
  • BUN was found to be higher in the middle-dose group of male rats than in the control group, and ALP in the low- and middle-dose group of female rats was slightly higher compared to the control group.
  • BUN was found to be slightly higher in the medium-dose group than in the control group.
  • SD rats were orally administered with Xianling Gukang Tablets continuously for six months, and its non-toxic dose was 480 mg / kg / d (equivalent to 40 times the effective dose).
  • the content and other indicators were used as the basis for the determination.
  • the results showed that all the indicators in the medication group were significantly improved, especially in the middle and high dose groups, and the efficacy was not less than 50 mg / kg sodium carboxyethyl phosphate.
  • Effect on osteoporosis caused by retinoic acid in rats ——Research on therapeutic effect Retinoic acid 70 mg / kg.d for 14 consecutive days, resulting in osteoporosis in rats.
  • Epimedium total flavones 4 mg / kg, 12 mg / kg, and 36 mg / kg, and sodium carboxyethyl phosphate 50 mg / kg were used as a positive control, which was administered continuously for 28 days.
  • Beagle mg / kg dog long-term toxicity test three dose groups of 36, 120, and 480 mg / kg, administered continuously for 9 months, discontinued for two weeks, and the results showed no significant toxicity.
  • Another object of the present invention is to provide a process for preparing the Chinese medicine preparation (Xianling Gukang Tablet) for anti-osteoporosis.
  • the process includes the following steps:
  • the amount of water used each time is 20 times the amount of the medicinal material.
  • the first decoction is 2 hours and the second decoction is 1.5 hours. Filter and combine the filtrates and cool to room temperature.
  • the upper macroporous adsorption resin (1 liter of resin in the treated water state per kilogram of medicinal materials). After the sample is loaded, it is first eluted with deionized water, replaced with 15% ethanol, and then replaced with 30%. Elute with ethanol, then with 40% ethanol, and finally with 50% ethanol. At this time, monitor the ethanol concentration of the eluent. When the alcohol concentration reaches 45%, start to collect the eluate to the end. This part of the eluate was concentrated to dryness under reduced pressure to obtain it.
  • This product is tan powder.
  • Preparation of reference solution Precisely weigh the appropriate amount of icariin reference substance, and make 60% methanol to make a solution containing 10 g of icariin per ml.
  • Assay method Take the reference solution and the test solution separately, take 50% methanol as the blank, and measure it at 270nm by spectrophotometry.
  • This product uses icariin as a reference to calculate the total flavonoid content of not less than 60%. Determination of icariin content.
  • Octadecylsilane-bonded silica gel was used as the filler; the mobile phase was acetonitrile: water (30:70); the flow rate was 1.0 ml / min; the column temperature was 30 ° C and the detection wavelength was 270nm.
  • the number of theoretical plates should not be less than 1500 calculated from the peak of icariin.
  • the reference solution and the test solution were each precisely sucked into 20 ⁇ 1 of each solution, and then injected into liquid chromatography for determination.
  • This product uses icariin as a reference to calculate the content of icariin not less than 20%.
  • Functions and Indications Bushen Yang strong bones and bones are used to fight osteoporosis.
  • Extract powder, starch, lactose, and microcrystalline cellulose were weighed according to the prescription ratio, sieved through an 80-mesh sieve, mixed with a hook, and sieved three times with a 40-mesh sieve.
  • the above mixed powder was made of 7% starch slurry into a soft material, passed through a 24 mesh sieve, and dried at 50 ° C for 2 hours. The dried granules are sieved through a 30-mesh sieve, added with the prescribed amount of magnesium stearate, mixed, pressed, coated, and air-dried to obtain.
  • This product is a tablet made from Epimedium extract.
  • the coated tablets are brown-red tablets, and the plain tablets are tan tablets.
  • the same dark red spots were displayed at the corresponding positions of the chromatogram of the reference substance; after spraying with the aluminum trichloride test solution, the light was inspected under an ultraviolet lamp (365nm), showing the same orange-red fluorescent spots. .
  • test solution Take 10 tablets of this product, mix and mix thoroughly, accurately weigh about 32mg of the sample, put it in a 10ml volumetric flask, add about 9ml of 50% methanol for ultrasonic extraction for 20 minutes, add 50% methanol to the mark , Shake well, accurately suck 0.5ml, set the volume to a 50ml volumetric flask, shake well, and get.
  • This product each containing icariin to calculate the total amount of flavones reference, not less than 60mg o
  • test solution Take 10 tablets of this product, and after measuring the fine mixing hook, accurately weigh about 15mg of the sample, put it in a 50ml volumetric flask, add about 45ml of 50% methanol for ultrasonic extraction for 20 minutes, add 50% methanol to Scale, shake well, filter through a microporous membrane (0.45 ⁇ ), and take the filtrate to obtain.
  • Each tablet contains icariin as a reference, and the content of icariin should not be less than 20mg.
  • DETAILED DESCRIPTION Example 1 Take 100 kg of Epimedium net medicinal material, decoction it with water and extract it twice, each time using about 20 times the amount of medicinal material. Cook for 2 hours for the first time, 1.5 hours for the second time, and strain. The filtrates were combined and cooled to room temperature. Load the macroporous resin. After the sample is loaded, elute with deionized water, and then elute with 15% ethanol, then elute with 30% ethanol, then elute with 40% ethanol, and finally elute with 50% ethanol. At this time, the ethanol concentration of the eluate is monitored. When the alcohol concentration reaches 45%, the eluate is collected to the end, and the part of the eluate is concentrated to dryness under reduced pressure.
  • Example 2 Take 100 kg of Epimedium net medicinal material, and after the above process, obtain an extract amount of 1.28 kg, a total flavonoid amount of 73.6%, and an icariin content of 27.5%.
  • the active ingredient extracted from the 9412 series of Chinese medicine Epimedium is mainly flavonoids.
  • a series of tests showed that 9412 with 60% flavonoid content has an anti-osteoporosis effect.
  • the low, medium, and high dose groups of 9412 can increase femoral bone mineral density in rats (P ⁇ 0.05, P ⁇ 0.01), and it is more obvious in the medium dose group (P ⁇ 0.01), the middle and high dose groups can increase the femur ash weight and bone calcium and bone phosphorus content, and the effect of low dose is not obvious.
  • the medium and high dose groups could increase bone mineral density in the femur of the rats (P ⁇ 0.05), but it was not obvious in the low dose group.
  • Serum calcium and phosphorus content affect, but can increase the rat femur weight and calcium and phosphorus content in the ashes to varying degrees, and the middle dose group is better.
  • 5 9412 has a certain growth-promoting effect on osteoblasts of newborn rats, and the most obvious promotion effect is 0.01 ⁇ ⁇ / ⁇ .
  • the preparation of the present invention has been confirmed by experimental animals to prevent and treat osteoporosis, and can increase femoral bone density, the content of bone calcium and bone phosphorus, and improve the femoral index.
  • the invention provides a preparation method.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Physical Education & Sports Medicine (AREA)
  • General Health & Medical Sciences (AREA)
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  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
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  • Rheumatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
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  • Organic Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Alternative & Traditional Medicine (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention concerne la formulation pharmaceutique de remède traditionnel chinois. L'invention concerne notamment une formulation constituée de principes actifs extraits d'Epidium et de véhicules pharmaceutique. Des études expérimentales sur l'animal ont confirmé que les formulations de l'invention peuvent prévenir et traiter l'ostéoporose, augmenter la densité du fémur et la teneur en calcium et en phosphore de l'os, et améliorer l'index du fémur. L'invention porte également sur le procédé de préparation de ladite formulation.
PCT/CN2003/000466 2002-06-18 2003-06-16 Formulation de remede traditionnel chinois pour la prevention et le traitement de l'osteoporose WO2003105829A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2003244067A AU2003244067A1 (en) 2002-06-18 2003-06-16 A formulation of traditional chinese medicine for preventing and treating osteoporosis

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN02112108.7 2002-06-18
CNB021121087A CN100409857C (zh) 2002-06-18 2002-06-18 一种用于抗骨质疏松的中药制剂

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Publication number Priority date Publication date Assignee Title
CN1332676C (zh) * 2004-10-11 2007-08-22 江西本草天工科技有限责任公司 补钙制剂及制备方法
CN101953881B (zh) * 2010-10-18 2012-03-07 山东省中医药研究院 一种抗骨质疏松的中药制剂及其制备方法
CN105687317B (zh) * 2016-03-08 2021-01-01 宁夏医科大学 阴香叶总黄酮作为制备治疗骨质疏松症药物的应用及药物组合物
CN107655994B (zh) * 2017-09-30 2020-07-03 天津中医药大学 淫羊藿提取物中16种化学成分含量的测定方法

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