CN1387888A - Chinese medicine prepn for resisting osteoporosis - Google Patents

Chinese medicine prepn for resisting osteoporosis Download PDF

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Publication number
CN1387888A
CN1387888A CN02112108A CN02112108A CN1387888A CN 1387888 A CN1387888 A CN 1387888A CN 02112108 A CN02112108 A CN 02112108A CN 02112108 A CN02112108 A CN 02112108A CN 1387888 A CN1387888 A CN 1387888A
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solution
preparation
reference substance
methanol
icariin
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CN02112108A
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CN100409857C (en
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刘全海
王明民
罗思齐
戴静芝
蒋毅
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Shanghai Institute of Pharmaceutical Industry
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Shanghai Institute of Pharmaceutical Industry
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Priority to CNB021121087A priority Critical patent/CN100409857C/en
Publication of CN1387888A publication Critical patent/CN1387888A/en
Priority to PCT/CN2003/000466 priority patent/WO2003105829A1/en
Priority to AU2003244067A priority patent/AU2003244067A1/en
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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

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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)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Epidemiology (AREA)
  • Rheumatology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Medical Informatics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • General Chemical & Material Sciences (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Biotechnology (AREA)
  • Botany (AREA)
  • Organic Chemistry (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • 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

The present invention belongs to the field of Chinese medicine preparation and is specially the preparation and active components of one Chinese medicine for treating osteoporosis. The Chinese medicine preparation consists of extracted effective component of epimedium and medicinal supplementary material. Animal experiment shows that the medicine can be used to prevent and treat osteoporosis, raise thighbone density, increase calcium and phosphorus content in bone and improve thighbone index.

Description

A kind of Chinese medicine preparation that is used for osteoporosis
Technical field
The invention belongs to the Chinese medicine preparation technical field.Be specifically related to a kind of active component and preparation thereof that is used for the Chinese medicine of osteoporosis.
Background technology
The Herba Epimedii total flavones powder is to extract moral effective site from the Chinese medicine Herba Epimedii.The Chinese medicine Herba Epimedii has following kind: Berberidaceae plant Herba Epimedii Epimedium brevicomumMaxim., arrow leaf Herba Epimedii Epimedium sagittatum (Sieb.et Zucc) Maxim., pubescence Herba Epimedii Epimedium pubescens MaXim., Epimedium wushanense Epimediumwushanense T.S.Ying or Herba Epimedii Epimedium koreanum Nakai.The medicinal part of Chinese medicine Herba Epimedii is dry aerial parts, and removes thick stalk and impurity.
Herba Epimedii (Epimedium koreanum Nakai) has another name called northeast Herba Epimedii (" national Chinese herbal medicine compilation ").Its medicinal part chemical constituent is in the majority with flavone, existing chemical constitution study shows: it contains icariin and icariine A, epimidin A, B, C, epimidin A,, B1, Quercetin, dehydration 3,5,7-trihydroxy-2-(4-methoxy-phenyl)-8-(3-methyl-but-2-enyl)-chromen-4-one-3-rhamnoside, Herba Epimedii glycosides (epimedokoreanoside) I, II.
Icariin is the flavonoid glycosides composition, and the hydroxyl in the molecule on C3, the C7 position combines with sugar and forms glycoside, and its chemical constitution is as follows:
From above structure as can be known icariin should have the uv absorption of flavone compound feature,
From above structure as can be known icariin should have the uv absorption of flavone compound feature, UV scanning confirms that icariin has stronger absorption about 270nm, so when carrying out assay with high performance liquid chromatogram, select 270nm to be the mensuration wavelength.
Do not see at present osteoporosis medicament with the preparation of Chinese medicine Herba Epimedii.
Summary of the invention
Technical problem to be solved by this invention is a kind of Chinese medicine preparation that is used for osteoporosis of development.
The invention provides a kind of celestial clever bone-recovering tablets of the Chinese medicine preparation that is used for osteoporosis that contains the Chinese medicine Herba Epimedii, active ingredient that said preparation is extracted by Herba Epimedii and pharmaceutic adjuvant are formed.
(1) the Chinese medicine preparation pharmacodynamic study that is used for osteoporosis of the present invention is as follows:
The pharmacodynamic study of the active component (hereinafter referred is 9412) that the Chinese medicine Herba Epimedii extracts: 1.9412 pairs of retinoic acid cause the influence-preventive effect of rats with osteoporosis
Experiment purpose: observe 9412 pairs of retinoic acid and cause the rats with osteoporosis preventive effect.1.1. material 1.1.1. test sample
Title: 9412
The unit of providing: Shanghai Institute of Pharmaceutical Industry
Lot number: 20001017
Dosage form: extractum
Consumption: 1ml/100g rat
Route of administration: po
Dosage: 4,12,36mg/kg, po * 281.1.2. positive control drug
The ethoxy Alendronate:
Chemistry division department of Shanghai Institute of Pharmaceutical Industry provides lot number 991010, oral 50mg/kg, po * 28.1.1.3. other
Retinoic acid:
Shanghai No.6 Pharmaceutical Factory produces, the yellow crystal powder, and oral, as causing rats with osteoporosis model usefulness, oral dose 1ml/100g rat is made into suspension with 0.5%CMC, 70mg/kg dosage, po * 14.
The bone densitometry instrument:
DPX-L type dual intensity X line borne densitometers, U.S. LunAR company produces, and Shuguang Hospital provides.
Test kit: Calcium, Shanghai Biological Products Inst., Ministry of Public Health.
The phosphorus test kit, the prompt pupil's thing in Shanghai technology company.
Muffle furnace: the PVG glad scientific instrument factory that jumps.
722S spectrophotometer: Shanghai science precision instrument company limited.1.2. animal:
Source, kind, strain, the quality certification:
Rat, SD system, Chinese Academy of Sciences's Shanghai Experimental Animal Center provides.
The quality certification: No. 005, the moving Guan Huidi of middle section.
Body weight: 110～130g
Sex: male and female all have
Every treated animal number: 10
Laboratory temperature: 24～26 ℃, relative humidity: 60～70%.1.3. test method: 1.3.1. sets up Induced by Retinoic Acid rats with osteoporosis model
Rat is divided into 6 groups at random, the normal control group, model control group, 9412 is low, in, high dose group, positive controls, except that the normal control group, each organizes all oral retinoic acid 70mg/kg of rat, dose 1ml/100g rat, every day 1 time, continuous 14 days, respectively organize the oral successively following sample of rat, normal control group simultaneously every day, retinoic acid group is coordinative solvent 10ml/kg, 9412 low dose group 4mg/kg, middle dosage group 12mg/kg, high dose group 36mg/kg, positive drug ethoxy Alendronate 50mg/kg, dose is the 1ml/100g rat, every day 1 time, continuous 28 days, weigh weekly therebetween 1 time, adjust the administration consumption with body weight.1.3.2. observation index
Administration finishes, and the sacrificed by decapitation animal is got blood system from serum, presses kit method and surveys S-Ca, S-P content, gets rat bilateral femur, peels meat and other tissue off, and wherein a side femur is done rat femur scanning on dual intensity X line borne densitometers, measures bone density (g/cm 2), claim bone heavy (W), survey bone long (L), bone diameter (D) calculates bone lines of expression density (W/L), apparent surface density (W/LD), dried one hour for 110 ℃ then, place interior 200,400,600,800 ℃ of each ashing of muffle furnace 2 hours again, after ashing finishes cooling, claim ash to weigh (Washg), extract the back with 6NHCl and survey bone ash Ca, bone ash P content, the gram numerical table that contains Ca or P with the 100g bone ash shows.
Opposite side femoral head 3%HNO 3Make paraffin section after the decalcification, HE dyeing, microscopically is surveyed the bone trabecula width.
Above result all compares with administration group and model group, and model group and normal control group are relatively.1.4. result: 1.4.1. body weight:
Each treated animal body weight no significant difference (P＞0.05) in the experiment same day and the 1st week, 2,4 all retinoic acid group weight ratio normal control groups lose weight (P＜0.01, P＜0.05) after the administration, but each treated animal body weight all obviously increases after the medication, wherein low dose group (4mg/kg) has weight increase but no statistical significance, and the 9412 middle and high dosage groups (12mg/kg, 36mg/kg) and the body weight in ethoxy Alendronate group the 2nd, 4 weeks of rat all increase (P＜0.05) than model group, and each dosage group compares no significant difference mutually.
9412 pairs of retinoic acid of table 1 cause the body weight change of rats with osteoporosis preventive effect
x±SD
Group Dosage mg/kg Number of animals (only) Week time (g)
????0 ???1 ????2 ??4
The normal control group Coordinative solvent ????8 ???117.5±10.3 ???131.2±8.6 ????166.7±20.4 ??220.7±17.7
Model control group The 70mg/kg coordinative solvent ????10 ???117.2±4.4 ???125.5±5.7 ????133.8±14.5 ΔΔ ??178.7±19.5 Δ
????9412 ????4 ????10 ???119.8±6.4 ???129.8±6.6 ????139.4±7.2 ??200.4±32.7
????12 ????10 ???118.6±3.6 ???128.1±4.0 ????155.5±6.2 * ??216.0±24.0 *
????36 ????11 ???121.5±5.1 ???131.4±5.2 ????153.2±16.1 * ??216.1±19.5 *
The ethoxy Alendronate ????50 ????11 ???118.2±7.6 ???127.5±8.6 ????146.7±13.7 * ??210.0±11.6 *
ΔP＜0.05, The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *P＜0.01 and model control group compare the variation of 1.4.2. rat femur bone density and SCa, SP
Table 2 as a result display model group rat femur bone density than normal control group low (P＜0.01), serum Ca content is also than model group low (P＜0.05), the serum paraoxonase changes of contents is little, but medication group rat femur density all increases (P＜0.05, P＜0.01) than model group, the content of serum calcium and phosphorus all raises than model group, but 9412 middle and high dosage group no significant differences.The rat femur bone density of positive drug ethoxy Alendronate and serum calcium serum paraoxonase content also have evident difference (P＜0.05, P＜0.01) than model group.
Femoral bmd when 9412 pairs of retinoic acid of table 2 cause the rats with osteoporosis preventive effect,
SCa, S pVariation x ± SD
Group Dosage mg/kg Number of animals (only) Femoral bmd g/cm 2 ????SCa ??mmol/L ???Spi ??mmol/L
The normal control group Coordinative solvent ????8 ?0.219±0.006 2.09±0.15 2.48±0.22
Model control group Coordinative solvent ????10 ?0.194±0.013 ΔΔ 1.78±0.11 Δ 2.42±0.15
9412 ????4 ????10 ?0.213±0.011 * 1.90±0.07 * 2.40±0.13
????12 ????10 ?0.217±0.009 ** 2.01±0.10 ** 2.65±0.21 *
????36 ????10 ?0.217±0.015 * 2.01±0.14 ** 2.61±0.18 *
The ethoxy Alendronate ????50 ????11 ?0.213±0.012 * 2.00±0.06 ** 3.91±0.43 **
ΔP＜0.05, The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *P＜0.01 and the relatively exponential influence of 1.4.3. 9412 pairs of Induced by Retinoic Acid rats with osteoporosis preventive effect bone of model control group
Compare with matched group, the W of model group, L, d, W/L, W/Ld all obviously descend, with respect to all obviously raisings of W, W/Ld of model group 9412 middle and high dosage groups and ethoxy Alendronate group.
9412 pairs of retinoic acid of table 3. cause the exponential influence of rats with osteoporosis preventive effect bone
x±SD
Group Dosage mg/kg Number of animals (only) The heavy W (g) of femur bone Femur length L (cm) Femur diameter d (cm) Femur apparent density W/L (g/cm) The apparent surface density W/L.d (g/cm of femur 2)
The normal control group Coordinative solvent ????8 ??0.431±0.04 ??3.225±0.193 ??0.338±0.023 ??0.129±0.006 ??0.381±0.018
Model control group Coordinative solvent ????10 ??0.302±0.016 ΔΔ ??2.716±0.134 ΔΔ ??0.318±0.03 ??0.111±0.007 ΔΔ ??0.349±0.024 Δ
9412 ????4 ????10 ??0.338±0.035 * ??3.056±0.068 ??0.308±0.019 ??0.111±0.011 ??0.359±0.043
????12 ????10 ??0.359±0.03 * ??3.107±0.093 ??0.311±0.016 ??0.115±0.007 ??0.380±0.026 *
????36 ????10 ??0.370±0.047 ** ??3.092±0.121 ??0.306±0.009 ??0.119±0.011 ??0.384±0.017*
The ethoxy Alendronate ????50 ????11 ??0.367±0.05 ** ??3.090±0.18 ??0.295±0.017 ??0.118±0.011 ??0.400±0.032 **
ΔP＜0.05, The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *The influence of 1.4.4. to femur ash, bone Ca and bone P compared with model control group in P＜0.01
With matched group relatively, the bone ash of model group reaches heavily that bone Ca and bone P content all reduce in the ash, but 9412 middle and high dosage groups and ethoxy Alendronate group bone ash weight, bone Ca, all increases relatively of bone P content after the medication.
Femur ash, bone when 9412 pairs of retinoic acid of table 4 cause the rats with osteoporosis preventive effect
Ca and bone Pi content influence x ± SD
Group Dosage mg/kg Number of animals (only) The heavy Wash (g) of femur bone ash Femur bone ash Ca Ca g/100g bone ash Femur bone ash P Pig/100g bone ash
The normal control group Coordinative solvent ????8 ?0.253±0.01 ?32.62±0.80 18.84±0.43
Model control group Coordinative solvent ????10 ?0.228±0.01 ΔΔ ?30.43±0.24 ΔΔ 17.42±0.17 ΔΔ
9412 ????4 ????10 ?0.236±0.006 ?30.92±0.24 ** 17.77±0.24 *
????12 ????10 ?0.239±0.007 * ?31.08±0.24 ** 17.94±0.29 **
????36 ????10 ?0.239±0.005 * ?31.46±0.24 ** 17.98±0.29 **
The ethoxy Alendronate ????50 ????11 ?0.251±0.005 ** ?30.81±0.18 ** 18.01±0.19
ΔP＜0.05, The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *P＜0.01 and 2. 9412 pairs of retinoic acid of model control group comparison cause the influence-therapeutical effect of rats with osteoporosis
Experiment purpose: observe the therapeutical effect that 9412 pairs of retinoic acid cause rats with osteoporosis.2.1. material 2.1.1. test sample:
Title: 9412
The unit of providing: Shanghai Institute of Pharmaceutical Industry
Lot number: 20001017
Dosage form: extractum
Consumption: 1ml/100g rat
Route of administration: po
4,12,36mg/kg dosage:, po * 142.1.2. positive control drug: ethoxy Alendronate: chemistry division department of Shanghai Institute of Pharmaceutical Industry provides lot number 991010, oral 50mg/kg, po * 14.2.1.3 other: retinoic acid: Shanghai No.6 Pharmaceutical Factory produces, the yellow crystal powder, oral, as causing rats with osteoporosis model usefulness, dose 1ml/100g rat is made into suspension with 0.5%CMC, 70mg/g dosage, po * 14.The bone densitometry instrument: DPX-L type dual intensity X line borne densitometers, U.S. LunAR company produces, and Shuguang Hospital provides.Test kit: Calcium, Shanghai Biological Products Inst., Ministry of Public Health.
The phosphorus test kit, the prompt pupil's thing in Shanghai technology company.
Muffle furnace: the PVG glad scientific instrument factory that jumps.
722S spectrophotometer: Shanghai science precision instrument company limited.2.2. animal:
Source, kind, strain, the quality certification:
Rat, SD system, Chinese Academy of Sciences's Shanghai Experimental Animal Center provides.
The quality certification: No. 005, the moving Guan Huidi of middle section.
Body weight: 110～130g
Sex: male and female all have
Every treated animal number: 10
Laboratory temperature: 24～26 ℃, relative humidity: 60～70%.2.3. test method: 2.3.1. sets up Induced by Retinoic Acid rats with osteoporosis model
Rat is divided into 6 groups at random, normal control group, model control group, 9412 basic, normal, high dosage groups, positive drug control group, except that the normal control group, each organizes all oral retinoic acid 70mg/kg of rat, dose 1ml/100g rat, every day 1 time, continuous 14 days, finish each medication group of back by each dosage group drug administration oral administration every day 1 time, continuous 28 days, normal control group and model group give coordinative solvent, and dose is the 1ml/100g body weight, adjust dosage according to body weight therebetween.2.3.2. observation index:
Administration finishes, and the sacrificed by decapitation animal is got blood system from serum, presses kit method and surveys S-Ca, S-P content, gets rat bilateral femur, peels meat and other tissue off, and wherein a side femur is done rat femur scanning on dual intensity X line borne densitometers, measures bone density (g/cm 2), claim femur heavy (W), survey bone long (L), bone diameter (D) calculates bone lines of expression density (W/L), apparent surface density (W/L.d), dried one hour for 110 ℃ then, place interior 200,400,600,800 ℃ of each ashing of muffle furnace 2 hours again, after ashing finishes cooling, claim ash to weigh (Wash..g), extract the back with 6N HCl and survey bone ash Ca, bone ash P content, the gram numerical table that contains Ca or P with the 100g bone ash shows.
Opposite side femoral head 3%HNO 3Make paraffin section after the decalcification, HE dyeing, microscopically is surveyed the bone trabecula width.
Above result all compares with administration group and model group, and model group and normal control group are relatively.2.4. result: 2.4.1. body weight:
After table 5 demonstration gave retinoic acid, each treated animal weight ratio normal control group body weight all obviously descended, but after giving 9412, administration group body weight begins to recover, and retinoic acid group also begins to recover.2.4.2. the variation of rat femur bone density and SCa, SP
9412 pairs of retinoic acid of table 5 cause the body weight change of rats with osteoporosis therapeutical effect
x±SD
Group Dosage mg/kg Number of animals (only) Week time (g)
????0 ???1 ???2 ???3 ???6
The normal control group Coordinative solvent ????8 ????117.9±9.3 ???136.2±9.4 ???155.4±7.8 ???165.0±17.7 ???256.2±11.9
Model control group The 70mg/kg coordinative solvent ????10 ????117.2±4.2 ???126.7±5.7 ???138.2±4.2 ???149.9±6.8 ???202.1±5.9
??9412 ????4 ????10 ????117.3±7.4 ???129.8±7.9 ???137.9±9.9 ???155.9±9.9 ???214.9±10.7
????12 ????10 ????118.5±3.6 ???128.9±4.4 ???139.4±3.2 ???167.3±3.9 ???247.4±11.7
????36 ????11 ????118.0±4.6 ???131.6±6.2 ???143.3±7.1 ???167.4±4.6 ???250.81±7.2
The ethoxy Alendronate ????50 ????11 ????116.6±6.6 ???125.4±5.7 ???140.9±8.2 ???163.6±8.3 ???246.5±94
Table 6 as a result display model group rat femur bone density than normal control group low (P＜0.01), SCa content is also than model group (P＜0.01), and SP does not have significant change, the rat femur density of 9412 each dosage group and model group are relatively after the medication, the effect of femur density and SCa is significantly improved, the most obvious with 9412 12mg/kg group effects, the ethoxy Alendronate also has the effect that improves rat femur density and SCa, but SP content model group of each group, medication group all with normal control group no significant difference.
Femoral bmd reached when 9412 pairs of retinoic acid of table 6 caused the rats with osteoporosis therapeutical effect
Variation x ± SD of SCa, SP
Group Dosage mg/kg Number of animals (only) Femoral bmd g/cm 2 ????SCa ???mmol/L ????Spi ??mmol/L
The normal control group Coordinative solvent ????8 ?0.236±0.01 2.47±0.14 ?2.05±0.14
Model control group Coordinative solvent ????14 ?0.215±0.005 ΔΔ 2.14±0.09 ΔΔ ?2.04±0.08
9412 ????4 ????16 ?0.225±0.009 * 2.22±0.14 ?2.01±0.18
????12 ????16 ?0.228±0.007 ** 2.33±0.24 * ?2.05±0.21
????36 ????15 ?0.225±0.011 * 2.33±0.12 ** ?2.19±0.20
The ethoxy Alendronate ????50 ????15 ?0.233±0.008 ** 2.33±0.11 ** ?2.13±0.20
The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *P＜0.01 and the relatively exponential influence of 2.4.3. 9412 pairs of Induced by Retinoic Acid rats with osteoporosis therapeutical effect bone of model control group
With matched group relatively, the W of the osteoporosis of model group, L, d, W/L, W/Ld all have obvious decline, obviously improve (P＜0.01) with respect to the high dose group and the ethoxy Alendronate group W/Ld of model group 9412.
9412 pairs of retinoic acid of table 7. cause the exponential influence of rats with osteoporosis therapeutical effect bone
x±SD
Group Dosage mg/kg Number of animals (only) The heavy W (g) of femur bone Femur length L (cm) Femur diameter d (cm) Femur apparent density W/L (g/cm) The apparent surface density W/L.d (g/cm of femur 2)
The normal control group Coordinative solvent ????9 ??0.445±0.055 ??3.359±0.22 ??0.364±0.05 ??0.131±0.008 ??0.378±0.018
Model control group Coordinative solvent ????10 ??0.392±0.028 ΔΔ ??3.280±0.12 ??0.353±0.022 ??0.119±0.006 ΔΔ ??0.337±0.006
9412 ????4 ????10 ??0.415±0.03 ??3.213±0.11 ??0.366±0.019 ??0.129±0.015 ??0.351±0.015
????12 ????10 ??0.424±0.036 ??3.193±0.11 ??0.347±0.022 ??0.131±0.007 ??0.377±0.01 **
????36 ????10 ??0.414±0.022 ??3.136±0.085 ??0.348±0.014 ??0.131±0.011 ??0.378±0.003 **
The ethoxy Alendronate ????50 ????11 ??0.422±0.034 ??3.110±0.10 ??0.356±0.025 ??0.135±0.007 ??0.378±0.015 **
The Δ ΔCompare with the normal control group P＜0.01
*The influence of 2.4.4. to femur ash, bone Ca and bone P compared with model control group in P＜0.01
Compare with matched group, the bone ash of model group heavily reaches bone Ca in the ash, bone P content all reduces, but 9412 middle and high dosage groups and ethoxy Alendronate group bone ash weight, bone Ca, bone P content all increase relatively after the medication.
Femur ash, bone when 9412 pairs of retinoic acid of table 8 cause the rats with osteoporosis preventive effect
Ca and bone Pi content influence x ± SD
Group Dosage mg/kg Number of animals (only) The heavy Wash (g) of femur bone ash Femur bone ash Ca Ca g/100g bone ash Femur bone ash P Pig/100g bone ash
The normal control group Coordinative solvent ????8 ?0.261±0.006 ?32.79±0.42 ?18.99±0.22
Model control group Coordinative solvent ????10 ?0.218±0.006 ?30.54±0.24 ΔΔ ?17.29±0.23 ΔΔ
9412 ????4 ????10 ?0.232±0.003 ?30.88±0.33 ?17.43±0.21
????12 ????10 ?0.239±0.003 ** ?30.98±0.19 * ?17.79±0.14 **
????36 ????10 ?0.240±0.003 ** ?30.94±0.40 ?17.77±0.10 **
The ethoxy Alendronate ????50 ????10 ?0.243±0.004 ** ?31.45±0.38 ** ?17.98±0.17 **
The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *The therapeutical effect of 3. 9412 pairs of castrated rats osteoporosis diseases is compared with model control group in P＜0.01
Experiment purpose: the curative effect of observing 9412 pairs of castrated rats osteoporosis.3.1. material 3.1.1. test sample:
Title: 9412
The unit of providing: Shanghai Institute of Pharmaceutical Industry
Lot number: 20001017
Dosage form: extractum
Consumption: 1ml/100g rat
Route of administration: po
Dosage: 4,12,36mg/kg, po * 903.1.2. positive control drug:
The ethoxy Alendronate:
Chemistry division department of Shanghai Institute of Pharmaceutical Industry provides lot number 991010, oral 50mg/kg, po * 90.3.1.3. other:
The bone densitometry instrument:
DPX-L type dual intensity X line borne densitometers, U.S. LunAR company produces, and Shuguang Hospital provides.
Test kit: Calcium, Shanghai Biological Products Inst., Ministry of Public Health.
The phosphorus test kit, the prompt pupil's thing in Shanghai technology company.
Muffle furnace: the PVG glad scientific instrument factory that jumps.
722S spectrophotometer: Shanghai science precision instrument company limited.3.2. animal:
Source, kind, strain, the quality certification:
Rat, SD system, Chinese Academy of Sciences's Shanghai Experimental Animal Center provides.
The quality certification: No. 005, the moving Guan Huidi of middle section.
Body weight: female 110～130g, male 130～150g
Every treated animal number: 10
Laboratory temperature: 24～26 ℃, relative humidity: 60～70%.3.3. test method: 3.3.1. sets up castrated rats osteoporosis model
The male and female rat is anaesthetized with 3% pentobarbital sodium, and routine disinfection is extractd bilateral testes or ovary under aseptic condition, sew up the incision immediately, sham operated rats is only done to sew up (not excising testis and ovary) again after the abdominal operation sterilization is cut, with the rat random packet, every group 10～12, the back grouping of one week of operation, by each medication group administration, every day 1 time, continuous 90 days, model group gives coordinative solvent, and dose is the 1ml/100g body weight, adjusts dosage according to body weight therebetween.3.3.2. observation index:
Administration finishes, and the sacrificed by decapitation animal is got blood system from serum, presses kit method and surveys S-Ca, S-P content, gets rat bilateral femur, peels meat and other tissue off, and wherein a side femur is done femur scanning on dual intensity X line borne densitometers, measures bone density (g/cm 2), claim femur heavy (W), survey bone long (L), bone diameter (d) calculates bone lines of expression density (W/L), apparent surface density (W/Ld), dries one hour for 110 ℃ then, place interior 200,400,600,800 ℃ of each ashing of muffle furnace 2 hours again, ashing finishes cooling and claims ash to weigh (Wash), uses 6N HNO 3After the extraction, survey bone ash Ca, bone ash P content, contain Ca with the 100g bone ash 2+Or the gram numerical table of Pi shows.
Opposite side femoral head 3%HNO 3Make paraffin section after the decalcification, HE dyeing, microscopically is surveyed the bone trabecula width.
Above result all compares with administration group and model group, and model group and normal control group are relatively.3.4. result: 3.4.1. body weight:
No significant difference between each treated animal.
9412 pairs of castrated rats body weight change of table 9 (my god)
x±SD?????(g)
Group ????????????????????????????????♂ ??????????????????????????????????♀
????????0 ??????30 ??????60 ??????90 ???????0 ??????30 ??????60 ??????90
The normal control group ??163.1±9.9 ?286.6±8.1 ?350.0±20.6 ?382.3±28.8 ?169.1±4.2 ?265.0±22.2 ?304.5±14.6 ?315.8±7.2
Model control group ??161.0±8.7 ?292.6±13.8 ?355.3±17.2 ?351.7±22.5 ?167.1±10.1 ?262.0±22.0 ?306.5±33.8 ?304.4±24.6
9412 ????4 ??164.0±9.2 ?292.3±15.6 ?323.7±31.1 ?328.7±23.8 ?168.2±8.6 ?248.3±27.5 ?273.8±34.3 ?302.4±21.2
????12 ??165.4±6.4 ?301.2±15.7 ?314.2±29.8 ?345.1±12.8 ?169.3±6.3 ?264.5±19.3 ?274.7±33.4 ?304.9±41.2
????36 ??162.7±6.1 ?289.2±18.9 ?319.4±28.5 ?344.0±21.8 ?170.2±5.7 ?269.3±16.3 ?281.0±21.5 ?294.0±34.5
The ethoxy Alendronate ??161.2±11.9 ?294.1±18.7 ?323.6±23.4 ?318.6±23.4 ?167.8±6.2 ?249.6±28.7 ?268.6±30.9 ?282.3±37.5
3.4.2. the variation of rat femur bone density and SCa, SP
Table 9 as a result display model group rat femur bone density than normal control group low (P＜0.01), 9412 middle and high dosage groups and ethoxy Alendronate all have significant rising (P＜0.05) than model group, the serum Ca of model group, blood-serum P are all low than normal control group, serum Ca improves than model group after the medication, but no statistical significance, blood-serum P changes little, and ethoxy Alendronate serum Ca is than model group P＜0.05 that raises), blood-serum P i changes little.
9412 pairs of castrated rats femoral bmds of table 10, bone trabecular influence
x±SD
Group Dosage mg/kg Number of animals (only) Femoral bmd g/cm Bone trabecula width (μ)
Matched group Coordinative solvent ????8 ????0.251±0.011 ????109.5±7.9
Model control group Coordinative solvent ????10 ????0.230±0.007 ΔΔ ????80.1±6.5 ΔΔ
9412 ????4 ????11 ????0.236±0.007 ????84.0±6.6
????12 ????10 ????0.241±0.008 * ????91.6±5.9 **
????36 ????12 ????0.237±0.004 * ????91.8±4.5 *
The ethoxy Alendronate ????50 ????10 ????0.242±0.012 * ????93.2±6.3 **
The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *Compare with model control group P＜0.01
3.4.3. 9412 pairs of exponential influences of castrated rats bone
Compare with matched group, the W of model group, L, d, W/L, W/Ld all significantly decrease, respectively organizing dosage and all can increase rat femur weight (P＜0.05, P＜0.01) with respect to model group 9412, the most obvious with middle dosage group, middle dosage group and ethoxy Alendronate group W/L, W/Ld all have notable difference than model group.
The variation of 9412 pairs of castrated rats serum calciums of table 11., serum paraoxonase
x±SD
Group Dosage mg/kg Number of animals (only) ???????SCa ??????mmol/L ???????Sp ??????mmol/L
Matched group Coordinative solvent ????8 ????2.39±0.09 ????2.05±0.14
Model control group Coordinative solvent ????10 ????2.03±0.16 ΔΔ ????1.64±0.27 ΔΔ
9412 ????4 ????11 ????2.09±0.14 ????1.74±0.008
????12 ????10 ????2.14±0.19 ????1.89±0.26
????36 ????12 ????2.16±0.14 ????1.84±0.26
The ethoxy Alendronate ????50 ????10 ????2.20±0.09 * ????1.86±0.17
The Δ ΔCompare with the normal control group P＜0.01
9412 pairs of exponential influences of castrated rats bone of table 12.
x±SD
Group Dosage mg/kg Number of animals (only) The heavy W (g) of femur bone Femur length L (cm) Femur diameter d (cm) Femur apparent density W/L (g/cm) The apparent surface density W/L.d (g/cm of femur 2)
The normal control group Coordinative solvent ????8 ??0.8931±0.08 ??3.551±0.21 ??0.396±0.04 ??0.252±0.02 ??0.608±0.03
Model control group Coordinative solvent ????10 ??0.8057±0.06 ΔΔ ??3.663±0.11 ??0.418±0.02 ??0.219±0.01 ΔΔ ??0.552±0.03 ΔΔ
9412 ????4 ????10 ??0.8364±0.06 ??3.702±0.08 ??0.405±0.01 ??0.225±0.01 ??0.558±0.04
????12 ????10 ??0.9118±0.06 * ??3.684±0.13 ??0.437±0.04 ??0.247±0.01 * ??0.569±0.04 *
????36 ????10 ??0.8717±0.07 ** ??3.735±0.11 ??0.424±0.02 ??0.233±0.01 ??0.550±0.01
The ethoxy Alendronate ????50 ????11 ??0.8609±0.08 ** ??3.613±0.01 ??0.420±0.03 ??0.237±0.02 * ??0.567±0.03 *
The Δ ΔCompare with the normal control group P＜0.01
*P＜0.05, *Compare with model control group P＜0.01
3.4.4. influence to femur bone ash branch, bone Ca and bone P
Compare with matched group, the bone ash of model group heavily reach bone ash divide in Ca, bone Pi content all reduce, but after the medication, femur bone ash weight, bone Ca, P content have in various degree raising (P＜0.05) than model group.
The influence of 9412 pairs of castrated rats femurs of table 13 bone ash, bone ore deposit Ca, P content
x±SD
Group Dosage mg/kg Number of animals (only) The heavy Wash (g) of femur bone ash Femur bone ash Ca Ca g/100g bone ash Femur bone ash Pi Pi g/100g bone ash
The normal control group Coordinative solvent ????8 ??0.373±0.04 ????35.91±3.0 ????19.85±1.7
Model control group Coordinative solvent ????10 ??0.360±0.02 ????32.97±1.1 Δ ????18.39±1.0
9412 ????4 ????10 ??0.360±0.04 ????34.34±2.6 ????19.42±1.5
????12 ????10 ??0.356±0.03 ????35.36±2.6 * ????20.37±1.5 **
????36 ????10 ??0.361±0.02 ????34.97±2.1 * ????19.23±1.0
The ethoxy Alendronate ????50 ????10 ??0.363±0.03 ????35.58±2.4 * ????19.48±1.4
ΔCompare with the normal control group P＜0.05
*P＜0.05, *Compare with model control group P＜0.01
4. 9412 pairs of former foster influences of being commissioned to train of rat osteoblast
Experiment purpose: observe the influence of 9412 pairs of rat osteoblast primary cell growths.
4.1. material:
4.1.1. test sample:
Title: 9412
The unit of providing: Shanghai Institute of Pharmaceutical Industry
Lot number: 20001017
Dosage form: extractum, test is to dissolving back aseptic filtration with PBS
4.1.2. other reagent:
Culture medium: DMEM, the Difco product
Calf serum: Hangzhou Sijiqing Biological Engineering Material Co., Ltd.
Collagenase: biochemistry division department of Shanghai Institute of Pharmaceutical Industry
Pancreatin: Difco product
Culture medium: DMEM+10% calf serum
Digestive system: PBS+EDTA (4mM)+0.1% collagenase+0.05% pancreatin is formed
3H-TdR: the 1mci/ml of Shanghai nuclear research institute
PBS：PH?7.2，0.5M
4.2. animal:
Source, kind, strain, the quality certification:
Rat, SD system (newborn 24 hours rats) Chinese Academy of Sciences's Shanghai Experimental Animal Center provides
The quality certification: No. 005, the moving Guan Huidi of middle section
4.3. method:
Aseptic condition takes out down the skull of 10 of the SD newborn rats of giving birth in 24 hours, remove periosteum and surrounding soft tissue, be cut into small pieces with shears, with 37 ℃ of digestion of Digestive system 20 minutes, constantly jolting in the digestion process, digestion finishes centrifugal, supernatant inclines, collect postdigestive remnant tissue and cell and be put in the Tissue Culture Flask, add 37 ℃ of DMEM (containing 10% calf serum), 5%CO 2The middle cultivation after 10 days, abandoning supernatant, attached cell becomes individual cells with trypsinization, and is centrifugal, collects, and counting and adjustment cell concentration are 5 * 10 5Individual/ml, on 96 porocyte culture plates, every hole adds 100 μ l cells, 9412 of different diluted concentrations are treated test agent 100 μ l (sample concentration is 1,0.1,0.01,0.001 μ g/ml), control tube replaces with culture medium, establishes three multiple pipes again, cultivates to add 0.5 μ ci/ hole after 48 hours 3H-TdR continues to cultivate 8 hours, with bull cell instrument collecting cell, measures the CPM value on the liquid scintillation instrument after reuse 0.5% trypsinization, and the result compares with sample and blank pipe.4.4. result:
The results are shown in Table, the sample sets of 1 μ g/ml concentration is low than matched group to the incorporation efficiency of newborn rat skull cell, and all the other each groups all have the osteoblastic growth of promotion in various degree, and it is the highest wherein to mix number with 0.01 μ g/ml, 9412 samples.
The influence of 9412 pairs of neonate rat osteoblast of table 14 primary cell growth
x±SD
Form Drug level μ g/ml The CPM value
????9412 ????0.001 ????1179±196
????0.01 ????2562±234 **
????0.1 ????1114±62
????1 ????96.3±3.3
Matched group ????1116±249
*5. conclusions are compared with matched group in P＜0.01:
9412 is the active component of extracting in the Chinese medicine Herba Epimedii, is mainly flavone, and a series of tests show to have the effect that 9412 of 60% flavones content has osteoporosis disease.
1. retinoic acid is caused in the test of rats with osteoporosis preventive effect,, increase femur and bone ash weight, the content of femur bone ash calcium and phosphorus no matter 9412 basic, normal, high dosage groups all can improve rat femur bone density (P＜0.05, P＜0.01).
2. retinoic acid is caused in the test of rats with osteoporosis therapeutical effect, 9412 basic, normal, high dosage groups can improve rat femur bone density (P＜0.05, P＜0.01), and with middle dosage group comparatively obviously (P＜0.01), middle and high dosage group can increase the content that the femur bone ash heavily reaches bone calcium bone phosphorus, and the low dosage effect is not obvious.
3. after 9412 pairs of castrated rats cause osteoporosis, have middle and high dosage group can improve rat femur bone density (P＜0.05), low dose group is not obvious.The influence of serum calcium, phosphorus content, but calcium, phosphorus content and better in raising rat femur weight that can be in various degree and the bone ash with middle dosage group.
4. can increase the width and the length of rat femur skull girder to castrated rats, illustrate that also the osteoporosis of 9412 pairs of castrated rats has certain therapeutical effect.
5. 9412 pairs of effects that the newborn rat osteoblast has certain promotion to grow are the most obvious with the facilitation of 0.01 μ g/ml.
(2) acute toxicity test that is used for the Chinese medicine preparation (celestial clever bone-recovering tablets) of osteoporosis of the present invention: 1. test objective:
After observing 9412 pairs of Kunming mouse oral administrations, the toxic reaction that animal produces.2. given the test agent:
Title: 9412 extractum
The unit of providing: Shanghai Institute of Pharmaceutical Industry
Lot number: 20001017
Content: every 1g extractum is equivalent to the g crude drug
The medicinal liquid preparation: 0.5%CMC+ Tween 80 hydrotropy is made into suspension 3. animal origins, kind, strain, the quality certification:
Mice, Kunming kind, Shanghai Institute of Pharmaceutical Industry's Animal House supply.
The quality certification: Shanghai is moving closes the card word No. 107.
Body weight: 20 ± 1g
Sex: male and female half and half
Number of animals: 20 every group, male and female half and half
Laboratory temperature: 25 ± 1 ℃, relative humidity: 65 ± 10%.
Raise with standard full nutrition pellet the drinking-water of freely ingesting.4. test method
Dosage:
9,412 20% solution in the prerun, the oral 0.5ml of every Mus, animal does not see death, now increases to a twice-daily oral administration.
Liquor strength:
Be made into 20% concentration, every 1ml contains 0.2g extractum, the oral 0.5ml/20g body weight of every Mus, a twice-daily.
Route of administration: oral
Method:
Oral 20%9412 suspensoids of mice, every Mus 0.5ml, a twice-daily, immediate reaction after the observation administration, record mice body weight change in two weeks, the observation period finishes the execution animal, and the histopathology of perusal animal changes.5. result
Two weeks of animal, interior body weight change saw Table.
No any ill symptoms behind the animals administer, movable diet is as usual.Observation period finishes, and the postmortem animal does not see that macroscopic pathological tissue is arranged is unusual yet.6. conclusion
Oral 9412 maximum tolerated doses of mice are more than the 10g/kg.
The body weight change of oral 9412 mtd tests of table 15 mice
x±SD
Sample Sex Dosage g/kg Number of animals (only) Body weight change (g)
0 day 2 days 4 days 7 days 9 days 11 days 14 days
9412 ??♂ ??10 ????10 ?19.6±0.45 ?21.1±0.75 ?22.5±0.75 ?23.4±0.55 ?25.3±0.40 ?27.2±0.58 ?28.9±0.67
??♀ ??10 ????10 ?18.5±0.33 ?20.1±0.65 ?21.7±0.65 ?22.8±0.69 ?25.0±0.64 ?26.5±0.61 ?27.3±0.85
Matched group ??♂ ????10 ?19.3±0.63 ?20.7±1.22 ?21.7±1.91 ?22.7±2.40 ?25.2±2.60 ?26.8±2.70 ?28.4±2.80
??♀ ????10 ?18.1±0.10 ?19.8±0.93 ?21.0±1.20 ?22.5±1.40 ?24.1±1.70 ?25.9±2.10 ?26.3±2.10
(3) celestial clever bone-recovering tablets is as follows to the long term toxicity test of rat:
The SD rat is adopted in this experiment, and 30 every group, male and female half and half are divided into 36,120, three administration groups of 480mg/kg/d and matched group, and continuous oral was irritated weisen spirit bone-recovering tablets six months, and the drug withdrawal observation period was 2 weeks.
The clever bone-recovering tablets of the oral celestial being of SD rat is six months long term toxicity test results show, to general symptom, compares obviously influence of nothing with matched group as body weight gain, food ration.Other vigor as animal, the mental status, feces etc. are not seen the overt toxicity reaction relevant with medicine.
It is low slightly that administration finds in the time of three months that the WBC of three dosage groups of male rat compares with matched group, and it is low slightly that the male rat high dose group is found in administration in the time of six months WBC and RBC compare with matched group, and female rats is low, the RBC of high dose group compares low slightly with matched group.Drug withdrawal finds during two weeks that the RBC of dosage group in the male rat compares low slightly with matched group.
Administration is found the BUN of dosage group in the male rat than the matched group height in the time of three months, the ALP of low, the middle dosage group of female rats compares high slightly with matched group.Administration finds in the time of six months that the BUN of dosage group in the female rats is high slightly than matched group.Drug withdrawal finds during two weeks that the BUN of female rats high dose group is than the matched group height.More than the variation of these indexs all in normal range.
Inspections such as histopathology there is no the overt toxicity reaction relevant with administration, and the variation of part index number is all in normal range.
The continuous per os of SD rat is irritated stomach and is given celestial clever bone-recovering tablets six months, and its nontoxic dose is 480mg/kg/d (is equivalent to drug effect dosage 40 times).
(4) celestial clever bone-recovering tablets is as follows to the long term toxicity test of Beagle Canis familiaris L.:
The celestial clever bone-recovering tablets of Beagle Canis familiaris L. is nine months long term toxicity test results show, inspections such as general symptom (activity of animal, body weight, the mental status, feces etc.), hematology, serum biochemistry, routine urinalysis, system's postmortem, histological examination and organ coefficient be there is no the overt toxicity reaction relevant with medicine.
The overt toxicity reaction relevant with medicine do not seen in administration in 2 weeks of drug withdrawal yet after nine months.
The celestial clever bone-recovering tablets of Beagle Canis familiaris L. continuous oral 9 months, its nontoxic dose are 480mg/kg/d (be equivalent to drug effect dosage 40 times).
As follows through above-mentioned result of study:
1. to retinoic acid 70mg/kg.d, caused rats with osteoporosis in continuous 14 days, and simultaneously three grades give Herba Epimedii total flavones 4mg/kg, 12mg/kg and 36mg/kg, carboxyethyl sodium phosphate 50mg/kg makes positive controls, and continues administration 14 days (administration is 28 days altogether) after the retinoic acid drug withdrawal again.With the weight of animals, bone density, bone weight, long, the bone diameter of bone, calculate bone lines of expression density, apparent surface density, bone ash weight, bone calcium, bone phosphorus, and bone slice survey bone trabecula is wide, the indexs such as content of serum calcium, phosphorus are as the basis of judging, found that each index of medication group all has clear improvement, especially obvious with middle and high dosage group, its curative effect is not less than 50mg/kg carboxyethyl sodium phosphate group at least.
2. to influence---the research of therapeutical effect of rats with osteoporosis due to the retinoic acid
Retinoic acid 70mg/kg.d, continuous 14 days, cause osteoporosis rat, after drug withdrawal, divide three grades and give Herba Epimedii total flavones 4mg/kg, 12mg/kg and 36mg/kg, carboxyethyl sodium phosphate 50mg/kg makes positive control, successive administration 28 days.
3. to the castrated rats function of resisting osteoporosis
The one all administrations of rat castration postoperative, the grouping situation is the same, successive administration 90 days, testing index is the same.
The result shows that middle and high dosage group all has tangible osteoporosis disease curative effect.
General pharmacology research:
General pharmacology studies show that 10,30,100mg/kg, and oral blood pressure to Canis familiaris L., breathing and electrocardiogram all produce obviously influence.50,150,500mg/kg is oral does not see significant change to mice nerve and behavior.
Studies on acute toxicity:
Mice LD 50＞10g/kg.
Long term toxicity research:
The rat long term toxicity test: three dosage groups 36,120 and 480mg/kg, successive administration 6 months, in two weeks of drug withdrawal, the result does not see overt toxicity.
Beagle mg/kg Canis familiaris L. long term toxicity test: three dosage groups 36,120 and 480mg/kg, successive administration 9 months, in two weeks of drug withdrawal, the result does not see overt toxicity.
So the long term toxicity test of rat and Canis familiaris L. shows that 480mg/kg is its nontoxic dose.
Another object of the present invention has provided the above-mentioned preparation technology who is used for the Chinese medicine preparation (celestial clever bone-recovering tablets) of osteoporosis.
This technology comprises the following steps:
(1) preparation Herba Epimedii dry extract
Get epimedium herb, with the water boiling and extraction secondary, each water consumption is 20 times of amounts of medical material, decocts 2 hours for the first time, decocts 1.5 hours for the second time, filter, merging filtrate also is cooled to room temperature, and last macroporous adsorbent resin (1 liter of the resin under the per kilogram medical material usefulness water state of having handled well) is behind the end of the sample, earlier with the deionized water eluting, change with 15% ethanol elution, change again, then with 40% ethanol elution with 30% ethanol elution, carry out eluting with 50% ethanol at last, the concentration of alcohol that play the monitoring eluent this moment rises when determining alcohol reaches 45%, begins to collect eluent to finishing, this part eluent is evaporated to dried, promptly.
The raw materials quality draft standard
Prescription: Herba Epimedii
Method for making:
With the Herba Epimedii leaf, add 20 times of water gagings at every turn and extract secondary, decocted 2 hours for the first time, decocted 1.5 hours for the second time, filter, merging filtrate also is cooled to room temperature, last macroporous adsorbent resin, with the Different concentrations of alcohol eluting, collect the eluent of respective streams part, be evaporated to dried extract powder.
Character: this product is a chocolate brown powder.
Differentiate: get extract powder 0.01g, add methanol to 10ml, supersound extraction 20 minutes filters, and filtrate evaporate to dryness, residue add methanol 2ml dissolving, as need testing solution.Other gets the icariin reference substance, adds dissolve with methanol, is mixed with the solution that every ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (2000 editions appendix VIB of Chinese Pharmacopoeia), draw need testing solution and each 5ul of reference substance solution, put respectively in same be on the silica GF254 lamellae of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-n-butyl alcohol-formic acid-water (10: 1: 1: 1) be developing solvent, launch, take out, dry, put under the uviol lamp (254nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical kermesinus speckle; Spray with the aluminum chloride test solution after, put again under the uviol lamp (365nm) and inspect, show identical fluorescent red-orange speckle.
Check: every regulation that should meet quality standard.
Determination of total flavonoids:
Measure according to spectrophotography (2000 editions appendix VA of Chinese Pharmacopoeia).
It is an amount of that the preparation precision of reference substance solution takes by weighing the icariin reference substance, makes the solution that every 1ml contains icariin 10 μ g with 60% methanol, promptly.
The preparation of need testing solution:
Precision takes by weighing the about 16mg of extract powder, places the 10ml volumetric flask, adds about 9ml50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, the accurate 0.5ml that draws, and standardize solution shakes up in the volumetric flask of 50ml, promptly.
Algoscopy:
Getting reference substance solution and need testing solution respectively, is blank with 50% methanol, shines spectrophotometry at 270nm wavelength place, promptly.
This product is that reference substance calculating content of total flavone must not be less than 60% with the icariin.
The icariin assay:
Measure according to high performance liquid chromatography (2000 editions appendix VID of Chinese Pharmacopoeia).
Chromatographic condition:
With octadecylsilane chemically bonded silica is filler; Mobile phase is acetonitrile: water (30: 70); Flow velocity is 1.0ml/min; Column temperature is that 30 ℃ of detection wavelength are 270nm.Number of theoretical plate calculates by the icariin peak should be not less than 1500.
The preparation of reference substance solution:
It is an amount of that precision takes by weighing the icariin reference substance, makes the solution that every 1ml contains icariin 0.1mg with 50% methanol, promptly.
The preparation of need testing solution:
Precision takes by weighing the about 7.5mg of extract powder, places the 50ml volumetric flask, adds about 45ml 50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate, promptly.
Algoscopy:
Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly.
This product is that reference substance calculating content Determination of Icariin must not be less than 20% with the icariin.
Function with cure mainly: the kidney-replenishing bone and muscle strengthening is used for osteoporosis.
Storage: airtight, put shady and cool dry place and preserve.
(2) preparation preparation
Prescription:
Preparation prescription:
Extract powder 100g
Starch 40g
Lactose 30g
Microcrystalline Cellulose 30g
7% starch slurry is an amount of
Magnesium stearate 1%
The film coating prescription
Stomach dissolved film coating pre-mix dose 12g
50% ethanol is an amount of
Above dosage is made 1000 by technology.
Preparation method:
Take by weighing extract powder, starch, lactose, microcrystalline Cellulose in the prescription ratio, cross 80 mesh sieves respectively, mixing is after 40 mesh sieves three times.Above-mentioned mixed powder with 7% starch slurry system soft material, is crossed 24 mesh sieve grains, 50 ℃ of dryings 2 hours.Dried particles is crossed 30 mesh sieve granulate, adds the recipe quantity magnesium stearate, mixing, and tabletting, coating, the sheet that dries in the air, promptly.
The tablet that this strain is made by Herba Epimedii extract.
Character: this product coated tablet is the brownish red tablet, and plain sheet is the sepia tablet.
Differentiate:
Get 5 of this product, porphyrize takes by weighing 0.02g, adds methanol to 10ml, and supersound extraction 20 minutes filters, and filtrate evaporate to dryness, residue add methanol 2ml dissolving, as need testing solution.Other gets the icariin reference substance, adds dissolve with methanol, is mixed with the solution that every ml contains 1mg, in contrast product solution.Test according to thin layer chromatography (2000 editions appendix VIB of Chinese Pharmacopoeia), draw need testing solution and each 5ul of reference substance solution, put respectively in same be on the silica GF254 lamellae of adhesive with the sodium carboxymethyl cellulose, with ethyl acetate-n-butyl alcohol-formic acid-water (10: 1: 1: 1) be developing solvent, launch, take out, dry, put under the uviol lamp (254nm) and inspect.In the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical kermesinus speckle; Spray with the aluminum chloride test solution after, put again under the uviol lamp (365nm) and inspect, show identical fluorescent red-orange speckle.
Check:
Should meet the every regulation under 2000 editions tablets of Chinese Pharmacopoeia (appendix ID) item.
Determination of total flavonoids:
Measure according to spectrophotography (2000 editions appendix VA of Chinese Pharmacopoeia).
The preparation of reference substance solution: see raw material.
The preparation of need testing solution:
Get 10 of this product, behind the porphyrize mixing, precision takes by weighing the about 32mg of sample, places the 10ml volumetric flask, adds about 9ml50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, the accurate 0.5ml that draws, and standardize solution shakes up in the volumetric flask of 50ml, promptly.
Algoscopy: see raw material.
It is the amount that reference substance calculates total flavones that every of this product contains with the icariin, must not be less than 60mg.
The icariin assay:
Measure according to high performance liquid chromatography (2000 editions appendix VID of Chinese Pharmacopoeia).
Chromatographic condition: see raw material.
The preparation of reference substance solution: see raw material.
The preparation of need testing solution:
Get 10 of this product, behind the porphyrize mixing, precision takes by weighing the about 15mg of sample, places the 50ml volumetric flask, adds about 45ml 50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, and filters with microporous filter membrane (0.45 μ m), gets subsequent filtrate, promptly.
Algoscopy: see raw material.
It is that reference substance calculating content Determination of Icariin must not be less than 20mg that every of this product contains with the icariin.
Function with cure mainly: the kidney-replenishing bone and muscle strengthening is used for osteoporosis.
Usage and consumption: oral, every day 3 times, each 1
Specification: 100mg/ sheet
Storage: airtight, put shady and cool dry place and preserve.
The specific embodiment
Embodiment 1
Get the clean medical material 100kg of Herba Epimedii, with water boiling and extraction 2 times, each water consumption is about 20 times of amounts of medical material.Decocted 2 hours for the first time, decocted 1.5 hours for the second time, filter.Merging filtrate also is cooled to room temperature.Last macroporous resin, elder generation is with the deionized water eluting behind the end of the sample, change with 15% ethanol elution, change with 30% ethanol elution, the back is with 40% ethanol elution, at last with 50% ethanol elution more again, play the concentration of alcohol of monitoring eluent this moment, when determining alcohol reaches 45%, rise, begin to collect eluent, be evaporated to this part eluent dried to finishing.
Detect extractum amount 1.21kg, general flavone content 71.8%, icariin content 25.8%.Embodiment 2
Get the clean medical material 100kg of Herba Epimedii, after above-mentioned PROCESS FOR TREATMENT, obtain extractum amount 1.28kg, total flavones amount 73.6%, icariin content 27.5%.
Embodiment 3 gets the clean medical material 100kg of Herba Epimedii, obtains extractum amount 1.19kg after above-mentioned PROCESS FOR TREATMENT, general flavone content 69.2%, icariin content 24.4%.

Claims (2)

1. Chinese medicine preparation that is used for osteoporosis is characterized in that what active ingredient that said preparation is extracted by Herba Epimedii and pharmaceutic adjuvant were formed.
2. a preparation technology who is used for the Chinese medicine preparation of osteoporosis as claimed in claim 1 is characterized in that this technology comprises the following steps:
(1) preparation Herba Epimedii dry extract
Get epimedium herb, with the water boiling and extraction secondary, each water consumption is 20 times of amounts of medical material, decocts 2 hours for the first time, decocted 1.5 hours for the second time, filter, merging filtrate also is cooled to room temperature, last macroporous adsorbent resin, 1 liter of resin under the per kilogram medical material usefulness water state of having handled well, behind the end of the sample,, change with 15% ethanol elution earlier with the deionized water eluting, change again with 30% ethanol elution, then, carry out eluting with 50% ethanol at last, play the concentration of alcohol of monitoring eluent this moment with 40% ethanol elution, when reaching 45%, rises determining alcohol, begin to collect eluent to finishing, this part eluent is evaporated to dried, promptly;
The raw materials quality draft standard
Prescription: Herba Epimedii
Method for making:
With the Herba Epimedii leaf, add 20 times of water gagings at every turn and extract secondary, decocted 2 hours for the first time, decocted 1.5 hours for the second time, filter, merging filtrate also is cooled to room temperature, last macroporous adsorbent resin, with the Different concentrations of alcohol eluting, collect the eluent of respective streams part, be evaporated to dried extract powder;
Character: this product is a chocolate brown powder;
Differentiate: get extract powder 0.01g, add methanol, supersound extraction 20 minutes to 10ml, filter, filtrate evaporate to dryness, residue add methanol 2ml dissolving, as need testing solution, other gets the icariin reference substance, add dissolve with methanol, be mixed with the solution that every ml contains lmg, in contrast product solution, according to thin layer chromatography test, 2000 editions appendix VIB of Chinese Pharmacopoeia.Draw need testing solution and each 5ul of reference substance solution, put respectively in same be on the silica GF254 lamellae of adhesive with the sodium carboxymethyl cellulose, with 10: 1: 1: ethyl acetate-n-butyl alcohol of 1-formic acid-water was developing solvent, launch, take out, dry, put under the 254nm uviol lamp and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical kermesinus speckle; Spray with the aluminum chloride test solution after, put again under the 365nm uviol lamp and inspect, show identical fluorescent red-orange speckle;
Check: every regulation that should meet quality standard;
Determination of total flavonoids:
According to spectrophotometry, 2000 editions appendix VA of Chinese Pharmacopoeia;
The preparation of reference substance solution:
It is an amount of that precision takes by weighing the icariin reference substance, makes the solution that every 1ml contains icariin 10 μ g with 50% methanol, promptly;
The preparation of need testing solution:
Precision takes by weighing the about 16mg of extract powder, places the 10ml volumetric flask, adds about 9ml 50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, the accurate 0.5ml that draws, and standardize solution shakes up in the volumetric flask of 50ml, promptly;
Algoscopy:
Getting reference substance solution and need testing solution respectively, is blank with 50% methanol, shines spectrophotometry at 270nm wavelength place, promptly;
This product is that reference substance calculating content of total flavone must not be less than 60% with the icariin;
The icariin assay:
According to high effective liquid chromatography for measuring, 2000 editions appendix VID of Chinese Pharmacopoeia;
Chromatographic condition:
With octadecylsilane chemically bonded silica is filler; Mobile phase is acetonitrile: water, 30: 70; Flow velocity is 1.0ml/min; Column temperature is that 30 ℃ of detection wavelength are 270nm, and number of theoretical plate calculates by the icariin peak should be not less than 1500;
The preparation of reference substance solution:
It is an amount of that precision takes by weighing the icariin reference substance, makes the solution that every 1ml contains icariin 0.1mg with 50% methanol, promptly;
The preparation of need testing solution:
Precision takes by weighing the about 7.5mg of extract powder, places the 50ml volumetric flask, adds about 45ml 50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, and filters with 0.45 μ m microporous filter membrane, gets subsequent filtrate, promptly;
Algoscopy:
Accurate respectively reference substance solution and each 20 μ l of need testing solution of drawing inject chromatograph of liquid, measure, promptly;
This product is that reference substance calculating content Determination of Icariin must not be less than 20% with the icariin;
Function with cure mainly: be used for osteoporosis;
Storage: airtight, put shady and cool dry place and preserve;
(2) preparation preparation
Prescription:
Preparation prescription:
Extract powder 100g
Starch 40g
Lactose 30g
Microcrystalline Cellulose 30g
7% starch slurry is an amount of
Magnesium stearate 1%
The film coating prescription:
Stomach dissolved film coating pre-mix dose 12g
50% ethanol is an amount of
Above dosage is made 1000 by technology;
Preparation method:
Take by weighing extract powder, starch, lactose, microcrystalline Cellulose in the prescription ratio, cross 80 mesh sieves, mixing respectively, after 40 mesh sieves three times, above-mentioned mixed powder with 7% starch slurry system soft material, is crossed 24 mesh sieve grains, 50 ℃ of dryings 2 hours, dried particles is crossed 30 mesh sieve granulate, adds the recipe quantity magnesium stearate, mixing, tabletting, coating, the sheet that dries in the air, promptly;
The tablet that this strain is made by Herba Epimedii extract;
Character: this product coated tablet is the brownish red tablet, and plain sheet is the sepia tablet;
Differentiate:
Get 5 of this product, porphyrize takes by weighing 0.02g, add methanol to 10ml, supersound extraction 20 minutes filters the filtrate evaporate to dryness, residue adds methanol 2ml dissolving, as need testing solution, other gets the icariin reference substance, adds dissolve with methanol, be mixed with every ml and contain the solution of 1mg, product solution is tested 2000 editions appendix VIB of Chinese Pharmacopoeia according to thin layer chromatography in contrast, draw need testing solution and each 5ul of reference substance solution, put respectively in same be that with 10: 1: 1: ethyl acetate-n-butyl alcohol of 1-formic acid-water was developing solvent on the silica GF254 lamellae of adhesive with the sodium carboxymethyl cellulose, launch, take out, dry, put under the 254nm uviol lamp and inspect, in the test sample chromatograph, with the corresponding position of reference substance chromatograph on, show identical kermesinus speckle; Spray with the aluminum chloride test solution after, put again under the 365nm uviol lamp and inspect, show identical fluorescent red-orange speckle;
Check:
Should meet 2000 editions tablets of Chinese Pharmacopoeia, appendix ID, the every regulation under;
Determination of total flavonoids:
According to spectrophotometry, 2000 editions appendix VIB of Chinese Pharmacopoeia;
The preparation of reference substance solution: see raw material;
The preparation of need testing solution:
Get 10 of this product, behind the porphyrize mixing, precision takes by weighing the about 32mg of sample, places the 10ml volumetric flask, adds about 9ml50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, the accurate 0.5ml that draws, and standardize solution shakes up in the volumetric flask of 50ml, promptly;
Algoscopy: see raw material;
It is the amount that reference substance calculates total flavones that every of this product contains with the icariin, must not be less than 60mg;
The icariin assay:
According to high effective liquid chromatography for measuring, 2000 editions appendix VID of Chinese Pharmacopoeia;
Chromatographic condition: see raw material;
The preparation of reference substance solution: see raw material;
The preparation of need testing solution:
Get 10 of this product, behind the porphyrize mixing, precision takes by weighing the about 15mg of sample, places the 50ml volumetric flask, adds about 45ml50% methanol supersound extraction after 20 minutes, adds 50% methanol to scale, shakes up, and filters with 0.45 μ m microporous filter membrane, gets subsequent filtrate, promptly;
Algoscopy: see raw material;
It is that reference substance calculating content Determination of Icariin must not be less than 20mg that every of this product contains with the icariin;
Function with cure mainly: be used for osteoporosis;
Usage and consumption: oral, every day 3 times, each 1;
Specification: 100mg/ sheet;
Storage: airtight, put shady and cool dry place and preserve.
CNB021121087A 2002-06-18 2002-06-18 Chinese medicine prepn for resisting osteoporosis Expired - Fee Related CN100409857C (en)

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CNB021121087A CN100409857C (en) 2002-06-18 2002-06-18 Chinese medicine prepn for resisting osteoporosis
PCT/CN2003/000466 WO2003105829A1 (en) 2002-06-18 2003-06-16 A formulation of traditional chinese medicine for preventing and treating osteoporosis
AU2003244067A AU2003244067A1 (en) 2002-06-18 2003-06-16 A formulation of traditional chinese medicine for preventing and treating osteoporosis

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CN1332676C (en) * 2004-10-11 2007-08-22 江西本草天工科技有限责任公司 Preparation for enriching calcium, and preparation method
CN101953881A (en) * 2010-10-18 2011-01-26 山东省中医药研究院 Anti-osteoporosis traditional Chinese medicinal preparation and preparation method thereof
CN105687317A (en) * 2016-03-08 2016-06-22 宁夏医科大学 Application of total flavonoids in leaves of cinnamomum burmanni in preparation of medicine for treating osteroporosis and medicine composition
CN107655994A (en) * 2017-09-30 2018-02-02 天津中医药大学 The assay method of 16 kinds of chemical composition contents in Shorthorned Epimedium P.E

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CN1047082C (en) * 1992-05-15 1999-12-08 北京蓉生医药科技发展中心 Process of preparation of medicine tonifying kidney and bone
CN1038813C (en) * 1993-07-02 1998-06-24 周勇 Traditional Chinese medicine for curing osteoporosis
CN1112010A (en) * 1995-03-24 1995-11-22 广东医学院医药科技开发中心 Compound traditional Chinese medicine preparation for treatment of osteoporosis and preparing method thereof
CN1200279A (en) * 1997-05-27 1998-12-02 贵州绿色实业有限公司 Medicine for preventing and treating osteoporosis
CN1087619C (en) * 1998-11-06 2002-07-17 卿多舜 Medicines for treating osteoporosis and their preparing process
AUPQ127399A0 (en) * 1999-06-29 1999-07-22 Guangzhou University Of Traditional Chinese Medicine Compositions and methods for treating or preventing osteoporosis
CN1133450C (en) * 2001-06-18 2004-01-07 佛山市中医院 Medicine for preventing and curing asteoporosis and promoting union and its production method
CN1133454C (en) * 2001-10-19 2004-01-07 江苏省中医药研究院 Bone invigorating composition containing yak bone and its prepn

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1332676C (en) * 2004-10-11 2007-08-22 江西本草天工科技有限责任公司 Preparation for enriching calcium, and preparation method
CN101953881A (en) * 2010-10-18 2011-01-26 山东省中医药研究院 Anti-osteoporosis traditional Chinese medicinal preparation and preparation method thereof
CN105687317A (en) * 2016-03-08 2016-06-22 宁夏医科大学 Application of total flavonoids in leaves of cinnamomum burmanni in preparation of medicine for treating osteroporosis and medicine composition
CN105687317B (en) * 2016-03-08 2021-01-01 宁夏医科大学 Application of total flavonoids of cinnamomum burmannii leaves in preparation of medicine for treating osteoporosis and medicine composition
CN107655994A (en) * 2017-09-30 2018-02-02 天津中医药大学 The assay method of 16 kinds of chemical composition contents in Shorthorned Epimedium P.E
CN107655994B (en) * 2017-09-30 2020-07-03 天津中医药大学 Method for measuring content of 16 chemical components in epimedium extract

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