CN1626074A - Preparation of Chinese traditional medicine - Google Patents
Preparation of Chinese traditional medicine Download PDFInfo
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Abstract
The present invention relates to application of Osthole preparation for preventing and curing osteoporosis, which is characterized in that said Chinese traditional medicine preparation is Osthole having purity not less than 90%, which is used for preparing drug for preventing and curing osteoporosis. In said Chinese traditional medicine preparation, the weight ratio of high purity Osthol is from 10% to 90%, and that of icariine is from 90% to 10%. In said Chinese traditional medicine preparation, the weight ratio of high purity Osthol is from 10% to 90%, and that of Daidzein is from 90% to 10%. The Chinese traditional medicine preparation applies high purity Osthol (osthole not less than 90%) for preparing preparation for preventing and curing osteoporosis, the preparation can prevent further loss of osseous, increases bone density, accelerates form of new bone. And the preparation has low toxin, exact curative effect, little bad reaction, easy control of curing dosage, and satisfies the standard of international officinal preparation.
Description
Technical field: the present invention is the preparation of protect against osteoporosis, particularly about a kind of application of using the osthole preparation at the sick medicine of protect against osteoporosis.
Background technology: osteoporosis (Osteoporosis is called for short OP) is that the phalanges amount reduces, the osseous tissue fibre structure is impaired, the sclerotin attenuation, fluff, bone fragility increases, anti-brute force weakens and easily send out fracture, losing mobility is a kind of systematicness, the general skeletal diseases of feature.Be a kind of common complaint among the elderly, frequently-occurring disease, account for the 7th in the world in the commonly encountered diseases.Along with global aged tendency of population and human living standard's raising, osteoporosis and treatment thereof have become heat subject in the recent medical research field.International medical community has been listed as osteoporosis and diabetes, cardiovascular diseases etc. serious harm senior health and fitness's major disease.China is the maximum country of aging population in the world, accounts for 1/5 of world's aging population, accounts for 1/2 of Asia aging population.Patient's number of Chinese osteoporosis in 1997 is about 8,390 ten thousand people, and incidence rate accounts for 6.6% of country's total population.As time goes on the aging of human age composition, such disease patient's number also will constantly increase.Modern medicine is still not fully aware of to the osteoporotic cause of disease, but research thinks relevant with estrogen deficiency, calcium Deficiency of Intake, physical exertion minimizing, solar radiation deficiency, vitamin D deficiency and bad habit at present.Be Different types of etiopathogenises, the coefficient result of a plurality of link, and old and feeble closely related with human body.This class disease common method of treatment is controversies in hormone replacement in the elderly and additional calcium preparation at present, though the former effect is better, owing to have more side effect in clinical treatment, to be difficult to all the time promote, so the latter produces little effect owing to not solving substantive issue.Prevention of osteoporosis disease has great social significance.Chinese scholars all striving to find the osteoporotic specific drug of treatment, confirms after deliberation and clinical practice that invigorating the kidney and strengthening the bones treatment by Chinese herbs osteoporosis has obtained certain effect, and toxic and side effects is little at present, and moderate cost can be taken for a long time.Yet at present also obviously to exist dosage big for the Chinese medicine of osteoporosis, and effective ingredient is indeterminate, the problem that is difficult to carry out necessary quality control and can't makes an explanation with the modern medicine theory.Therefore, the modern Chinese medicine of exploitation osteoporosis disease, and the Chinese medicine with the effect of osteoporosis disease carried out the research of the modernization of Chinese medicine, the active component of seeking out wherein is very urgent and necessary.Domestic scholars finds that the Chinese medicine Fructus Cnidii has certain curative effect to osteoporosis in recent years, the preparation made from it can stop further losing of sclerotin, to forming the sclerotin of losing the new osteoplastic effect of promotion is arranged also, and propose that it is applied to patent in a kind of compound Chinese medicinal preparation for the treatment of osteoporosis as raw material of Chinese medicine and require that (number of patent application is respectively: 93106922.X, 93121565.X and 95100697.5), also there is the scholar Chinese medicine Fructus Cnidii to be extracted, and the Fructus cnidii extract that obtains is applied to the preparation of protect against osteoporosis preparation by research.The patent that proposes " a kind of goods of protect against osteoporosis is characterized in that these goods contain the extract of the fruit Fructus Cnidii of umbrella shape Cnidium Cusson plant cnidium monnieri ... " requires (number of patent application: 92105338.X).In above-mentioned patent, the patent No. is: 93106922.X, 93121565.X and three patents of 95100697.5, all are the compound Chinese medicinal preparation that make an addition to protect against osteoporosis with Fructus Cnidii as a kind of raw material of Chinese medicine, contain many compositions in the Fructus Cnidii, comprising the composition of an effective ingredient and a toxic and side effects.And the patent No. is: the patent of 92105338.X, the inventor also points out with " Fructus cnidii extract comprises Fructus Cnidii total coumarins; osthole; bergapton; cupreol; isopimpinellin; xanthotoxol, xanthotoxin, ammidin and α-Oleum Pini bicyclic hydrocarbons, lauro lene, bornival " etc. multiple components; but that its Fructus cnidii extract constitutes is complicated; the pharmacological action of various compositions is indeterminate; simultaneously because the difference of Fructus Cnidii raw material; cause in the Fructus cnidii extract of different batches various components in proportions such as total coumarins also inequality; cause in the clinical application effective ingredient dosage wayward, has increased unnecessary toxic and side effects (the oral LD50 of Fructus Cnidii total coumarins mice is that 2.449/kg " poisonous Chinese herbal medicine voluminous dictionary " Guo Xiao suzerain compiles the p493-494. of Tianjin Scientific English Translation publishing company); And owing to complicated component in the Fructus cnidii extract, be difficult to reach the basic standard of international pharmaceutical formulation, thereby make this series products can't move towards international drug market, influence the internationalization of this product, do not meet the requirement of the modernization of Chinese medicine yet.
Summary of the invention: the purpose of this invention is to provide a kind of Chinese medicine preparation, it is with high purity cnidicin (osthole, Osthol 〉=90%) is used for preparing the preparation of protect against osteoporosis, this preparation can stop further losing of sclerotin, bone density improving, promote the formation of new bone, have low toxicity, determined curative effect, untoward reaction is few, therapeutic dose is easy to control, meet international pharmaceutical formulation standard-required.
Technical scheme of the present invention is: design a kind of Chinese medicine preparation, it is characterized in that: described Chinese medicine preparation is that purity is the osthole Osthol more than 〉=90%, and it is as the application of the sick medicine of preparation protect against osteoporosis.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and the icariine weight ratio is chosen in 90%-10%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and big legumin weight ratio is chosen in 90%-10%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and the genistein weight ratio is chosen in 90%-10%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and the genistein weight ratio is chosen in 90%-10%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of daidzin is chosen in 90%-10%%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of Genistin is chosen in 90%-10%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of calcium preparation is chosen in 90%-10%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of puerarin is chosen in 90%-10%.
Described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of soybean isoflavone is chosen in 90%-10%.
Characteristics of the present invention are: the present invention is with various compositions in the Fructus cnidii extract, and (this high purity cnidicin is meant the osthole of purity 〉=90%, Osthol) to have obtained high purity cnidicin through any special measures separation.The different name of osthole is: osthole; Osthole.English name: Osthol; Osthole; Osthola. chemical name is: 7-methoxyl group-8-isopentenyl coumadin (2H-1-Benzopyran-2-one, 7-methoxy-8-(3-methyl-2-butenyl)-); Molecular formula: C
15O
16H
3, molecular weight: 224.28 its structural formulas are seen Fig. 1, and nuclear magnetic resonance map is seen Fig. 2 and Fig. 3.Its preparation technology be with the Fructus Cnidii raw material with alcohol reflux extract, concentrate, water cleaning, aqueous alkali cleaning, pure dissolving, activated carbon decolorizing, pure water crystallization, drying, products obtained therefrom purity can reach more than 90% (sees number of patent application 03134435.6), this preparation technology can realize large-scale industrial production fully, the osthole (Osthol 〉=90%) that obtains by this technology passes through high performance liquid chromatograph (Waters Corporation, the U.S.) detect, the result shows that the obvious existence that has reduced other compositions sees Fig. 4; The acute toxicity test in mice that uses this osthole to carry out, the result shows: the acute toxicity of this osthole is low, toxic and side effects little (seeing test 1); The mice pharmacological testing that uses this osthole to carry out, the result shows: osthole does not have tangible excitement or inhibitory action to the central nervous system, coordination exercise is not had tangible influence (seeing test 2, test 3, test 4) yet this osthole is applied to the osteoporosis experimental animal model tests, data show that osthole has good preventive and therapeutic effect to the reduction of removal ovary rat femur bone density.(see test 5) makes dosage be easy to control, thereby makes such preparation meet the requirement of the modernization of Chinese medicine, also can reach the standard-required of international pharmaceutical formulation.
Main test:
Test 1:
Mouse gavaging osthole acute toxicity test report
One, summary
The LD50 of mouse gavaging osthole is 6.241g/kg, the 95% credible 5.373g/kg～7.249g/kg that is limited to.The movable minimizing of mice after the administration, it is for sleeping in to lie prone, and indivedual mices have sialorrhea, and 4 hours mices begin death after the administration, and dead preceding mice is little convulsions of persistence and tic, the last death owing to dyspnea.In the survival mice, higher dosage group mice weight loss, food ration reduces, and small dose group mice body weight slightly increases.The dissection visible lungs of mice perusal of being poisoned to death have in various degree edema, and internal organs such as spleen, kidney have slight hyperemia, intestinal tympanites.The execution survival mice that becomes celestial except that high dose group mice intestinal tympanites, does not find that other main organs has macroscopic pathologic to change.
Two, test objective
Observe the acute toxic reaction and the death condition that are produced behind the mouse gavaging osthole.
Three, be subjected to the reagent thing
1 title: osthole
2 units of providing: Xi provides
3 preparations, lot number: osthole, be off-white powder, purity is 95.00%, lot number: 030516, facing the time spent is mixed with the suspension of 25.00% cigarette, 21.25%, 18.06%, 15.35% and 13.05% concentration with 0.5% sodium carboxymethyl cellulose and 0.1% tween 80, uses for experiment.
Four, laboratory animal
Kunming mouse, male and female half and half, body weight 20.5 ± 1.6g, the supply of University Of Suzhou Experimental Animal Center, cleaning level, laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.18 ± 2 ℃ of experiment receptacle temperature.
Five, experimental technique
By prerun, the dosage range of osthole acute toxicity is 5.0～10.0g/kg according to a preliminary estimate, carries out formal test on this basis.Get 50 of Kunming mouses, male and female half and half are divided into 5 groups at random, fasting is after 10 hours before the experiment, every treated animal respectively by 10.000,8.500,7.224,6.140 and 5.220g/kg gavage osthole, the administration volume is the 0.4ml/10g body weight, dose ratio r=0.85.
Six, experimental result
Behind the mouse gavaging osthole, the movable minimizing, it is for sleeping in to lie prone, and indivedual mices have sialorrhea, and 4 hours mices begin death after the administration, and the most of mice in the death produces dead (seeing Table 1) in 24 hours after administration.Dead mice is little convulsions of persistence and tic before death, the last death owing to dyspnea.In the survival mice when experiment finishes, higher dosage group mice weight loss, food ration reduces, and small dose group mice body weight slightly increases (seeing Table 2).The dissection visible lungs of mice perusal of being poisoned to death have in various degree edema, and internal organs such as spleen, kidney have slight hyperemia, intestinal tympanites.Become celestial and put to death survival mice except that high dose group mice intestinal tympanites, do not find that other main organs has macroscopic pathologic to change.The LD50 measurement result of mouse gavaging osthole sees Table 3.
Dead mouse situation during the acute toxicity test of table 1 mouse gavaging osthole
Be subjected to amount of reagent number of animals death toll (only)
(g/kg) (only) the 1st day the 2nd day the 3rd day the 4th day the 5th day the 6th day the 7th day
10.000???????10????8????????1????????0????????0????????0????????0???????0
8.500????????10????6????????2????????0????????0????????0????????0???????0
7.224????????10????5????????3????????0????????0????????0????????0???????0
6.140????????10????2????????1????????1????????0????????0????????0???????0
5.220????????10????2????????0????????0????????0????????0????????0???????0
Mice body weight (g) changes during the acute toxicity test of table 2 mouse gavaging osthole
(n=10) administration is after 7 days before being subjected to amount of reagent (g/kg) administration
10.000???????????????????20.8±1.6????????????????16(1)
8.500????????????????????20.7±1.5????????????????20.5±2.1(2)
7.224????????????????????20.5±1.5????????????????22.2±3.9(2)
6.140????????????????????20.2±2.1????????????????16.5±3.3(6)
5.220????????????????????20.4±1.4????????????????22.8±2.6(8)
Annotate: in the bracket is the surviving animals number.
Table 3 mouse gavaging osthole The acute toxicity tests
Be subjected to the amount of reagent log10 dose number of animals death toll mortality rate LD of probit
50And 95% fiducial limit
(g/kg) (X) (only) (only) (%) (Y) (g/kg)
10.000????1.000????????10????????9????????90??????6.282
8.500?????0.929????????10????????8????????80??????5.842???????6.241
7.224?????0.859????????10????????8????????80??????5.842???????5.373～7.249
6.140?????0.788????????10????????4????????40??????4.747
5.220?????0.718????????10????????2????????20??????4.158
According to above-mentioned experimental result, press the LD that the Bliss method is calculated the mouse gavaging osthole
50Be 6.241g/kg, the 95% credible 5.373g/kg～7.249g/kg that is limited to.
Seven, conclusion
The LD of mouse gavaging osthole
50Be 6.241g/kg, the 95% credible 5.373g/kg～7.249g/kg that is limited to.Little (the oral LD50 of Fructus Cnidii total coumarins mice is 2.44g/kg " a poisonous Chinese herbal medicine voluminous dictionary " than total coumarins acute toxicity
This experimental technique meets bureau of drug administration of Ministry of Health of the People's Republic of China: study of tcm new drug guide (pharmacy, pharmacology, toxicology) 1994,203-204.With chief editors such as Xu Shuyun: pharmacological experimental methodology, second edition, Beijing: People's Health Publisher, 1991:201-203.
Test 2:
Oral osthole is to the influence of spontaneous activity in mice
One, summary
With the active situation of trembling after the cage method writes down the oral osthole of mice, the result shows, after giving mouse gavaging osthole 10,20,40mg/kg, the spontaneous activity of mice does not see and is significantly increased or reduces that the prompting osthole does not have tangible excitement or inhibitory action to the central nervous system.
Two, test objective
The influence of osthole to central nervous system of mice understood in spontaneous activity behind the oral osthole of observation mice.
Three, be subjected to the reagent thing
1. osthole, purity is 95%, and lot number is provided by Xi: 030516, be mixed with 0.1%, 0.2%, 0.4% suspension with 0.5% tween 80, standby.
2. SHULE ANDING PIAN, the 1mg/ sheet, produced authentication code by Pharmaceutical Co Ltd, Changzhou Pharmaceutical Factory No.4: the accurate word H32020699 of traditional Chinese medicines, product batch number: KC9327 is mixed with 0.01% solution with 0.5% tween 80.
Four, experimental apparatus
Tonotransducer, range 0～30g is produced by Anhui sensing system engineering (group) company Technology Development Department.
Medlab bio signal acquisition processing system is provided by Meiyi Science ﹠ Technology Co., Ltd., Nanjing.
Five, laboratory animal
Kunming mouse, male and female half and half, body weight 20.3 ± 1.4g, the supply of University Of Suzhou Experimental Animal Center, cleaning level, laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.18 ± 2 ℃ of experimental session receptacle temperature.
Six, experimental technique
Get 60 of Kunming mouses, be divided into 6 groups at random, 10 every group, male and female half and half with trembling the cage method, place cage with mice, and silk thread links to each other with the pcs signal acquisition processing system by tonotransducer under the cage.After the mice fasting 10 hours, irritate stomach respectively and give osthole 10,20,40mg/kg, estazolam 1mg/kg and solvent group (0.5% tween 80), the administration volume is the 0.1ml/10g body weight, the normal control group gives the distilled water of respective volume, before writing down administration respectively, after the administration 15,30,45,60,75,90,105 minutes mice event trace write down 2 minutes at every turn, with the event trace area of graph on the correct oscillogram chart of SigmaScan Pro4.0 software, carry out statistical procedures then.
Seven, experimental result
By Fig. 5 table 4 and Fig. 6, Fig. 7, Fig. 8, Fig. 9, Figure 10, Figure 11 as seen, solvent group mice is after gavaging 0.5% tween 80, spontaneous activity is compared with the normal control group and is not seen have a significant effect (P＞0.05), mouse gavaging osthole 10,20,40mg/kg group each time period after administration, spontaneous activity is compared with the solvent group and is not also seen have a significant effect (P＞0.05), and the estazolam group is after administration 15,30,45, event trace area in the time of 60 minutes obviously reduces, compare with the solvent group, significant difference (P＜0.01) is then arranged.Prompting thus, the oral osthole 10,20 of mice, 40mg/kg do not have obvious excitement or inhibitory action to the central nervous system.
Eight, conclusion
Under this experiment condition, behind mouse gavaging osthole 10,20, the 40mg/kg spontaneous activity in mice is not seen that tangible increase or minimizing are arranged, prompting does not have obvious excitement or inhibitory action to the central nervous system.
This test method meets: Xu Shuyun, Bian Rulian, Chen Xiu chief editor: pharmacological experimental methodology (second edition), People's Health Publisher,, 652-653 and Qi Chen chief editor: herbal pharmacology research methodology, People's Health Publisher,, 667-669 in 1993 in 1991.
Test 3
The oral osthole of mice is to the influence of pentobarbital sodium syngignoscism
One, summary
The oral osthole 10,20 of mice, 40mg/kg add three dosage groups of lumbar injection pentobarbital sodium (30mg/kg) and add lumbar injection pentobarbital sodium group with solvent (0.5% tween 80) and compare, mice time for falling asleep and sleep time there are no significant difference.Prompting: the oral osthole 10,20 of mice, 40mg/kg do not have obviously collaborative or antagonistic effect to lumbar injection pentobarbital sodium (30mg/kg) syngignoscism.
Two, test objective
Whether the oral osthole of observation mice has collaborative or antagonism to the syngignoscism of lumbar injection pentobarbital sodium.
Three, be subjected to the reagent thing
1. osthole, purity is 95%, and lot number is provided by Xi: 030516, be mixed with 0.1%, 0.2%, 0.4% suspension with 0.5% tween 80, standby.
2. SHULE ANDING PIAN, the 1mg/ sheet, produced authentication code by Pharmaceutical Co Ltd, Changzhou Pharmaceutical Factory No.4: the accurate word H32020699 of traditional Chinese medicines, product batch number: KC9327 is mixed with 0.01% solution with 0.5% tween 80.
3. pentobarbital sodium: China Medicine (Group) Shanghai Chemical Reagent Co., provides, lot number: 20030816.Be made into 0.30% solution with normal saline.
Four, laboratory animal
Kunming mouse, male and female half and half, body weight 19.6 ± 1.0g, the supply of University Of Suzhou Experimental Animal Center, cleaning level, laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.18 ± 2 ℃ of experimental session receptacle temperature.
Five, experimental technique
Get 80 of Kunming mouses, male and female half and half are divided into 8 groups at random.Wherein irritate stomach respectively and give osthole 10,20,40mg/kg for 6 groups, estazolam 1mg/kg, solvent (0.5% tween 80) and equal-volume distilled water, the administration volume is the 0.1ml/10g body weight, 1 hour lumbar injection pentobarbital sodium (30mg/kg) behind the filling stomach, in addition two groups lumbar injection pentobarbital sodiums (30mg/kg) and filling stomach give estazolam (1mg/kg) respectively, observe and write down time and righting reflex loss and recovery time after mouse peritoneal is injected pentobarbital sodium, calculate time for falling asleep and the sleep time of mice by following formula.Time for falling asleep=righting reflex loss time-lumbar injection finishes the time, sleep time=righting reflex recovery time-righting reflex loss time.
Six, experimental result
By table 5 as seen, solvent+pentobarbital sodium group is compared with distilled water+pentobarbital sodium group, the equal no significant difference of mice time for falling asleep and sleep time (P＞0.05), osthole 10,20, three dosage groups of 40mg/kg+ pentobarbital sodium (30mg/kg) are compared with solvent+pentobarbital sodium group, the also equal no significant difference (P＞0.05) of mice time for falling asleep and sleep time, the time for falling asleep of oral estazolam+pentobarbital sodium group mice is compared remarkable quickening with solvent+pentobarbital sodium group, sleep time significant prolongation (P＜0.01).
The oral osthole of table 5 mice to the influence of pentobarbital sodium syngignoscism (x ± SD, n=10)
Group number of animals mice time for falling asleep (minute) the mice sleep persistent period (minute)
Pentobarbital sodium (30mg/kg) group 10 5.2 ± 1.5 61.7 ± 33.6
Estazolam (1mg/kg) group 10 is not sleeping not sleeping
Distilled water+pentobarbital sodium
10????????????5.9±1.5???????????????60.3±32.5
(30mg/kg) group
Solvent+pentobarbital sodium
10????????????6.3±1.9???????????????58.9±33.9
(30mg/kg) group
Osthole 10mg/kg+ penta crust
10?????????????6.1±1.3??????????66.3±50.5
Organize than appropriate sodium (30mg/kg)
Osthole 20mg/kg+ penta crust
10?????????????5.7±1.2??????????72.6±42.3
Organize than appropriate sodium (30mg/kg)
Osthole 40mg/kg+ penta crust
10?????????????6.0±0.9??????????52.2±40.7
Organize than appropriate sodium (30mg/kg)
Estazolam 1mg/kg+ penta crust
10?????????????2.7±1.3
**???????169.0±46.4
**
Organize than appropriate sodium (30mg/kg)
Compare with solvent+pentobarbital sodium group,
*P＜0.01
Seven, conclusion
Under this experiment condition, the oral osthole 10,20 of mice, 40mg/kg do not have obviously collaborative or antagonistic effect to the syngignoscism of pentobarbital sodium, and the oral osthole 10～40mg/kg of prompting mice is to nervus centralis unrestraint or excitation.
Test 4:
Oral osthole is to the influence of mice pole-climbing coordination exercise
One, summary
Situation with coordination exercise behind the oral osthole of pole-climbing method observation mice, the result shows, after giving mouse gavaging osthole 10,20,40mg/kg, each organizes pole-climbing time of mice and the number of animals that each grading system of coordination exercise occurs, compare with the solvent matched group and not see that notable difference is arranged, the prompting osthole does not have tangible harmful effect to coordination exercise.
Two, test objective
After observing oral osthole whether the mice coordination exercise had bad influence.
Three, be subjected to the reagent thing
1. osthole, purity is 95%, and lot number is provided by Xi: 030516, be mixed with 0.1%, 0.2%, 0.4% suspension with 0.5% tween 80, standby.
2. chlorpromazine injection, 2ml/ props up, and 250mg/mL is provided by Shanghai Hefeng Pharmaceutical Co., Ltd., authentication code: the accurate word (1995) of medicine is defended No. 010048 in Shanghai, product batch number: 020801, be made into 0.25% solution with distilled water.
Four, laboratory animal
Kunming mouse, male and female half and half, body weight 19.0 ± 1.0g, the supply of University Of Suzhou Experimental Animal Center, cleaning level, laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.18 ± 2 ℃ of experimental session receptacle temperature.
Five, experimental technique
Get 60 of Kunming mouses, be divided into 6 groups at random, 10 every group, male and female half and half.With a slick metal bar, diameter is 1.5cm, and the long 70cm of rod is placed on the top that stands vertically rod to mice, and head allows it creep naturally downwards.After the mice fasting 10 hours, irritate stomach respectively and give osthole 10,20,40mg/kg, chlorpromazine 25mg/kg and solvent (0.5% tween 80), the administration volume is the 0.1ml/10g body weight, and the normal control group gives the distilled water of respective volume, before writing down administration respectively, 30,90 minutes mices creep on bar 3 times the used time after the administration, and the kinestate when observing mice simultaneously and creeping is judged coordination exercise obstacle situation by following standard, and mark 0 grade: creep step by step downwards; 1 grade: slide downwards; 2 grades: can not catch rod; 3 grades: righting reflex loss.Establish 0.5 grade, 1.5 grades and 2.5 grades again at 0～3 inter-stage.The pole-climbing time, the result represented with x ± SD, and grading system result represents with the number of animals that respective level occurs, carries out statistical procedures then respectively.
Six, experimental result
By table 6 and table 7 as can be known, behind oral osthole 10,20 of mice and the 40mg/kg, the number of animals that its pole-climbing time and each grading system occur is compared with the solvent matched group and is not seen that notable difference is arranged, and points out osthole that coordination exercise is not had tangible influence.
Behind the oral osthole of table 6 mice to the influence of pole-climbing time (x ± SD, n=10)
| Group | The pole-climbing time (second) | ||
| Before the administration | 30min after the administration | 90min after the administration | |
| The normal control group | ????33.2±11.1 | ????29.7±12.6 | ????35.3±22.7 |
| The solvent matched group | ????30.7±7.9 | ????35.3±9.8 | ????45.0±25.78 |
| Osthole 10mg/kg group | ????30.0±14.9 | ????26.2±12.4 | ????28.4±12.5 |
| Osthole 20mg/kg group | ????30.1±7.2 | ????30.1±10.7 | ????43.0±20.4 |
| Osthole 40mg/kg group | ????25.7±4.4 | ????33.4±14.5 | ????40.5±31.1 |
| Chlorpromazine 25mg/kg group | ????30.6±7.4 | Can not catch rod | Can not catch rod |
Pole-climbing coordination exercise test appraisal result (n=10) behind the oral osthole of table 7 mice
| Group | Grading system (routine number occurring) | |||||||
| 0 grade | 0.5 level | 1 grade | 1.5 level | 2 grades | 2.5 level | 3 grades | ||
| The normal control group | Behind the medicine prodrug behind 30 minutes medicines 90 minutes | ???4 ???8 ???5 | ????6 ????2 ????5 | ???0 ???0 ???0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ???0 ???0 ???0 |
| The solvent matched group | Behind the medicine prodrug behind 30 minutes medicines 90 minutes | ???3 ???5 ???7 | ????7 ????5 ????3 | ???0 ???0 ???0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ???0 ???0 ???0 |
| Osthole 10mg/kg group | Behind the medicine prodrug behind 30 minutes medicines 90 minutes | ????6 ????8 ????7 | ????4 ????2 ????3 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 |
| Osthole 20mg/kg group | Behind the medicine prodrug behind 30 minutes medicines 90 minutes | ????4 ????6 ????5 | ????6 ????4 ????5 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 |
| Osthole 40mg/kg group | Behind the medicine prodrug behind 30 minutes medicines 90 minutes | ????4 ????5 ????5 | ????6 ????5 ????5 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 | ????0 ????0 ????0 |
| Chlorpromazine 25mg/kg group | Behind the medicine prodrug behind 30 minutes medicines 90 minutes | ????3 ????0 ????0 | ????7 ????0 ????0 | ????0 ????0 ????1 | ????0 ????0 ????0 | ????0 ????10 ????9 | ????0 ????0 ????0 | ????0 ????0 ????0 |
Annotate: the standard of coordination exercise obstacle can be divided into 0 grade: creep downwards step by step; 1 grade: slide downwards; 2 grades: can not catch rod; 3 grades: righting reflex loss.Establish 0.5 grade, 1.5 grades and 2.5 grades again at 0～3 inter-stage.The average progression of intact animal is 0～0.3 grade.
Seven, conclusion
Under this experiment condition, behind the oral osthole 10～40mg/kg of mice, coordination exercise there is not tangible harmful effect.
This test method meets. Xu Shuyun, Bian Rulian, Chen Xiu chief editor: pharmacological experimental methodology (front page), People's Health Publisher,, the 470th page in 1985.
Test 5:
The experimentation of osthole prevention osteoporosis rat
(1), summary
The influence that this experimentation osthole forms osteoporosis rat.After the ovariectomized rats, continuous oral osthole 5,10,12 weeks of 20mg/kg, detect rat body weight, uterus weight, serum calcium, phosphorus, alkali phosphatase, hydroxyproline/creatinine, E2, CT, TGF-β, BGP, indexs such as femoral bmd, osseous tissue form, and with model group and positive control medicine nilestriol group relatively.The result shows: osthole group rat body weight is compared no significant difference with model group, but uterus weight and uterus/body weight ratio has certain reduction.After rat gives osthole, serum calcium levels obviously increases, serum paraoxonase content descends, serum alkaline phosphatase has increase trend, hydroxyproline/creatinine has reduction trend, and change of serum C T and TGF-β raise, and descends when E2 is low dose of, in, raise when heavy dose of, serum BGP progressively raises with the increase of dosage.Osthole group rat femur bone density and bone trabecula area percentage all obviously increase than model group, and femur osseous tissue form obviously improves than model group, moves closer to sham operated rats.These experimental results show that osthole can prevent the osteoporotic formation of castrated rats.
(2), experiment purpose
Observe the influence that the oral osthole of castrated rats forms osteoporosis.
(3), be subjected to the reagent thing
1, osthole, purity is 95%, and lot number is provided by Xi: 030516, be mixed with 0.05%, 0.1%, 0.2% suspension with 0.2% tween 80, standby.
2, nilestriol, Hualian Pharmaceutical Co., Ltd., Shanghai produces, and authentication code: the accurate word (1995) of medicine is defended No. 012090 in Shanghai, product batch number: 030801.Get a tablet 5mg, abundant porphyrize, the dissolving of 50ml 0.2% tween 80, standby.
(4), reagent
1, rat transforming growth factor (TGF-β) EIA test kit, the bio tech ltd import packing of west, Shanghai Tang, lot number: 040418.
2, clear Bone Gla protein (BGP), calcitonin (CT) radioimmunoassay kit, PLA General Hospital Science and Technology Development Center puts and exempts from institute lot number is provided: 20040423.
3, estradiol (E2) radioimmunoassay kit, Depew, Tianjin biotechnology and medical product company limited provide lot number: 20040420.
4, hydroxyproline determination test kit (extraction process), Nanjing is built up Graduate School of Engineering and is provided, lot number: 040310.
(5), instrument and equipment
1, U.S.'s moral spirit Dimension RXL automatic clinical chemistry analyzer.
2, U.S. GE LUNAR DPXIQ dual intensity X line borne densitometers, the toy analysis software.
3, radioimmunoassay system, Shanghai nuclear research manufacturing.
(6), animal
60 of SD rats, female, body weight 234 ± 20g, cleaning level, University Of Suzhou's Experimental Animal Center provides, laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.
(7), experimental technique
Get 50 of female rats, after the laboratory adaptability is raised 7 days, 10% chloral hydrate 0.3ml/100g body weight intraperitoneal injection anesthesia, the ventricumbent position is fixed, 1/3 place cropping in the back, iodine tincture, ethanol partly sterilised skin, do one downwards along back lumbar vertebra median line and be about 2～3 centimetres otch, cut skin, skin is led earlier to a side, cut fascia, the passivity separating muscle, after entering peritoneum, on cornua uteri, prick fallopian tube, cut off fallopian tube with toe-in, mention ovary (being the rediance graininess), silk thread is pricked its tissue that links to each other, spay, peritoneal suture otch on every side.With operating offside, skin suture otch with quadrat method.Rat is divided into 5 groups after making the castration model at random, i.e. model group, nilestriol 1mg/kg group, osthole 5mg/kg group, osthole 10mg/kg group, osthole 20mg/kg group.Other gets 10 female rats, and operation method is the same, but does not cut off fallopian tube, and spay is not only extractd and is equivalent to the mesentery of ovary size as sham operated rats.Every rat postoperative is intramuscular injection penicillin 100,000 u for three days on end, the prevention traumatic infection.
After the modeling 7 days, osthole group rat every morning 8:00～9:00 gastric infusion, the administration volume is the 1ml/100g body weight, and the nilestriol group is administered once weekly, model group and sham operated rats then give isopyknic 0.2% tween 80 every day.Successive administration is after 12 weeks, and 10% chloral hydrate 0.3ml/100g body weight is anaesthetized, abdominal aortic blood, and weigh in the complete uterus of taking off, and the uterus is fixed with 10% formalin, the conventional organization section, HE dyeing, pathomorphism is observed.Separate the bilateral femur, remove muscle.The right side femur is measured bone density, and the left side femur is fixed with 10% formalin, conventional decalcification, embedding, makes the thick tissue slice of 5mm, and HE dyes, and PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM is also taken the photograph sheet.
Rat blood serum is got upper serum and is measured TGF-β, BGP, CT, E2, hydroxyproline by kit method after centrifugalize, and automatic clinical chemistry analyzer is measured serum calcium, phosphorus, alkali phosphatase and creatinine.
(8), experimental result
1, osthole is to the influence of castrated rats body weight and uterus weight
The model group rat body weight is compared obvious increase (P＜0.01) with sham operated rats, and uterus weight, uterus/body weight are all than sham operated rats low (P＜0.01), each dosage group rat body weight of osthole is compared no significant difference (P＞0.05) with model group, but uterus weight and uterus/body weight ratio has certain reduction, especially the effect of osthole 10mg/kg group is comparatively obvious, compares P＜0.01 with model group.The uterus pathological section shows, osthole each dosage group endometrial gland number and model group zero difference illustrate that osthole does not have tangible estrogen-like influence to the castrated rats endometrium, the results are shown in Figure 12, Figure 13, Figure 14, Figure 15, Figure 16, Figure 17.Nilestriol group body weight, uterus weigh, uterus/body weight is all approaching with sham operated rats, and there were significant differences (P＜0.01) with model group.The results are shown in Table 8.
Table 8 osthole is to the influence of castrated rats body weight and uterus weight (n=10, x ± s)
| 3 groups of Nilestriol groups of 2 groups of Ostholes of 1 group of Osthole of group sham-operation group model group Osthole | Dosage (mg/kg) 5 10 20 1 | Body weight (g) 252 ± 25 328 ± 34 ▲▲????????330±35 ????308±35 ????326±32 ????260±23 ** | Heavy (g) 0.439 in uterus ± 0.109 0.136 ± 0.026 ▲▲????????0.103?±0.036 ????0.092±0.012 **????0.120±0.027 ????0.368?±0.076 ** | Uterus/body weight 0.00175 ± 0.00046 0.00042 ± 0.00009 ▲▲0.00031±0.00010 0.00030±0.00005 **0.00037±0.00008 0.00142±0.00032 ** |
Annotate: compare with sham operated rats,
▲ ▲P＜0.01; Compare with model group,
*P＜0.05,
*P＜0.01
1, osthole is to the influence of castrated rats serum calcium, phosphorus, alkali phosphatase, hydroxyproline/creatinine
Model group rat blood serum calcium is than the obvious reduction of sham operated rats (P＜0.01), and serum paraoxonase slightly raises, give the osthole of doses after serum calcium levels obviously increase, serum paraoxonase content descends, its result is basic similar to positive control nilestriol group.Though rat blood serum alkali phosphatase, hydroxyproline/creatinine all do not have significant difference (P＞0.05) between each treated animal, give osthole after, alkali phosphatase has increase trend, hydroxyproline/creatinine has reduction trend.These results all point out osthole to have certain inhibition bone resorption, promote the effect of skeletonization.The results are shown in Table 9.
Table 9 osthole is to the influence of castrated rats serum calcium, phosphorus, alkali phosphatase, hydroxyproline/creatinine (n=10, x ± s)
| Group sham operated rats model group | Dosage (mg/kg) | Calcium (mmol/L) 2.47 ± 0.06 2.31 ± 0.08 ▲▲ | Phosphorus (mmol/L) 1.87 ± 0.23 2.16 ± 0.30 | Alkali phosphatase (U/L) 58.3 ± 9.9 64.8 ± 16.5 | Hydroxyproline/creatinine 0.058 ± 0.013 0.070 ± 0.014 |
| 3 groups of nilestriol groups of 2 groups of ostholes of 1 group of osthole of osthole | ????5 ????10 ????20 ????1 | ??2.42±0.06 *??2.30±0.14 ??2.45±0.07 **??2.49±0.07 ** | ??2.09±0.17 ??1.83±0.21 *??1.91±0.15 ??2.05±0.19 | ??69.7±11.5 ??68.4±16.3 ??62.3±10.0 ??56.6±11.2 | ??0.064±0.011 ??0.061±0.007 ??0.069±0.013 ??0.071±0.012 |
Annotate: compare with sham operated rats,
▲ ▲P＜0.01; Compare with model group,
*P＜0.05,
*P＜0.01
3, osthole is to the influence of castrated rats serum E2, CT, TGF-β, BGP
Model group rat blood serum E2, CT, TGF-β all significantly are lower than sham operated rats (P＜0.05 or P＜0.01), and CT and TGF-β go up behind the oral osthole, and especially TGF-β is tangible dose dependent.Osthole is when low dose of (5mg/kg), serum E2 level is lower, then serum E2 level is higher when middle dosage (10mg/kg) is above, in conjunction with uterus weight and pathological variation, the prompting osthole estrogen-like effects a little less than, when higher dosage, may have certain estrogen antagonist sample effect on the contrary.The model group serum BGP is than sham operated rats height (P＜0.01), after giving osthole, serum BGP further raises with the increase of dosage, osthole 20mg/kg group serum BGP is apparently higher than model group (P＜0.05), and nilestriol group serum BGP is starkly lower than model group (P＜0.01), after illustrating that castrated rats gives osthole, osteoblast is active obviously to be strengthened.The results are shown in Table l0.
Table 10 osthole is to the influence of castrated rats serum E2, CT, TGF-β, BGP (n=l0, x ± s)
| 3 groups of Nilestriol groups of group sham-operation group model group Osthole l group 2 groups of Ostholes of Osthole | Dosage (mg/kg) 5 10 20 1 | E2(pg/ml) 21.8±10.5 9.7±3.2 ▲▲4.1±2.2 **16.5±9.2 *12.1±6.7 11.4±5.2 | CT(ng/L) 552±237 276±115 ▲479±178 **414±93 **438±104 *436±125 * | TGF-β(pg/ml) 26309±4883 12746±3979 ▲▲13856±6987 17081±7428 19906±5594 **21745±4618 ** | BGP(ng/ml) 0.119±0.086 0.341±0.149 ▲▲0.411±0.204 0.427±0.132 0.584±0.211 *0.128±0.094 ** |
Annotate: compare with sham operated rats,
▲P＜0.05,
▲ ▲P＜0.01; Compare with model group,
*P＜0.05,
*P＜0.01
4, osthole is to the influence of castrated rats bone density
Model group rat femur near-end metaphysis bone density obviously reduces (P＜0.01) than sham operated rats, bone density progressively raises after giving the various dose osthole, it is similar substantially that osthole 20mg/kg group and positive control nilestriol group are compared effect, illustrate that osthole can prevent the decline of removal ovary rat bone density.The results are shown in Table 11.
Table 11 osthole is to the influence of castrated rats near end of thighbone metaphysis bone density (n=10, x ± s)
| 1 group of group sham operated rats model group osthole | Dosage (mg/kg) 5 | Bone density (g/cm 2) ????0.084±0.022 ????0.057±0.016 ▲▲????????0.084±0.023 * |
| 3 groups of nilestriol groups of 2 groups of ostholes of osthole | ????10 ????20 ????1 | ????0.088±0.017 **????0.107±0.028 **????0.112±0.019 ** |
Annotate: compare with sham operated rats,
▲ ▲P＜0.01; Compare with model group,
*P＜0.05,
*P＜0.01
1. osthole is to the influence of castrated rats osseous tissue form
The visible sham operated rats bone trabecula of rat femur near-end metaphysis pathological section light microscopy checking is many, thicker, is interconnected to net, and pulp cavity is less.Model group rat femur bone trabecula obviously reduces, and attenuates, and major part can not connect into net, and pulp cavity is bigger.After giving osthole, the bone trabecula showed increased increases slightly, is netted, and pulp cavity diminishes.Total coumarins group rat femur bone trabecula is more, thicker, and connects into net.The nilestriol group is similar substantially to the morphology performance of sham operated rats.The results are shown in Figure 18, Figure 19, Figure 20, Figure 21, Figure 22, Figure 23.Osseous tissue form machine image analysis software Sigmascan Pro 5.0 is as calculated analyzed, model group rat femur near-end metaphysis bone trabecula area percentage rate (bone trabecula area/visual field area) is starkly lower than sham operated rats (P＜0.01), giving can obviously increase bone trabecula area percentage rate behind the osthole, compare obvious increase (P＜0.05) with model group, the result is similar to positive control nilestriol group.Illustrating that osthole has significantly castrated rats femur bone trabecula increases, increases thick effect.The results are shown in Table 12.
Table 12 osthole is to the influence of castrated rats near end of thighbone metaphysis bone trabecula area percentage (n=10, x ± s)
| 3 groups of Nilestriol groups of 2 groups of Ostholes of 1 group of Osthole of group sham-operation group model group Osthole | Dosage (mg/kg) 5 10 20 1 | Bone trabecula area percentage (%) 52.9 ± 2.8 41.0 ± 5.8 ▲▲????????48.9±7.6 *????50.6±7.5 *????49.2±5.6 *????50.9±4.3 ** |
Annotate: compare with sham operated rats,
▲ ▲P＜0.01; Compare with model group,
*P＜0.05,
*P＜0.01
(9), conclusion
Castrated rats is taken osthole 5 ~ 20mg/kg after 12 weeks continuously, can obviously prevent its osteoporotic generation.
This test method meets: Chen Dong, Wang Liantang, Chen Guodong.The bone histomorphometry of rat different parts and Study of bone mineral density behind the removal ovary.China's osteoporosis magazine, 2002,8 (3): 208
Description of drawings: Fig. 1 is the osthole molecular structural formula.
Fig. 2 is a nuclear magnetic resonance map.
Fig. 3 is a nuclear magnetic resonance map.
Fig. 4 is high purity cnidicin (Osthol 〉=a 90%) high performance liquid chromatograph diagram.
Fig. 5 is that table 4 is promptly trembled the cage method and measured behind the oral osthole of mice influence to spontaneous activity track area.
Fig. 6 trembles the cage method to measure behind the oral osthole of mice normal control figure group to the tension force signal of telecommunication figure of spontaneous activity;
Fig. 7 trembles the cage method to measure behind the oral osthole of mice solvent contrast figure group to the tension force signal of telecommunication figure of spontaneous activity;
Fig. 8 trembles the cage method to measure behind the oral osthole of mice osthole 10mg/kg figure group to the tension force signal of telecommunication figure of spontaneous activity;
Fig. 9 trembles the cage method to measure behind the oral osthole of mice osthole 20mg/kg figure group to the tension force signal of telecommunication figure of spontaneous activity;
Figure 10 trembles the cage method to measure behind the oral osthole of mice osthole 40mg/kg figure group to the tension force signal of telecommunication figure of spontaneous activity;
Figure 11 trembles the cage method to measure behind the oral osthole of mice stable 1mg/kg figure group to the tension force signal of telecommunication figure of spontaneous activity;
Figure 12 sham operated rats uterus slice map, HE dyeing * 200
Figure 13 model group uterus slice map, HE dyeing * 200
The uterus section of Figure 14 osthole 5mg/kg group, HE dyeing * 200
The uterus section of Figure 15 osthole 10mg/kg group, HE dyeing * 200
The uterus section of Figure 16 osthole 20mg/kg group, HE dyeing * 200
The section of Figure 17 nilestriol group uterus, HE dyeing * 200
Figure 18 sham operated rats rat femur near-end metaphysis tissue slice, visible normal bone trabecula and pulp cavity.HE dyeing * 100
Figure 19 model group rat femur near-end metaphysis tissue slice, visible bone trabecula obviously reduces, attenuates, and major part can not connect into netted, and pulp cavity obviously increases.HE dyeing * 100
Figure 20 osthole 5mg/kg group rat femur near-end metaphysis tissue slice, visible bone trabecula is coarser than model group, but can connect into net, and pulp cavity is bigger.HE dyeing * 100
Figure 21 osthole 10mg/kg group rat femur near-end metaphysis tissue slice, visible bone trabecula obviously increases slightly, connects into net, and pulp cavity is little than model group.HE dyeing * 100
Figure 22 osthole 20mg/kg group rat femur near-end metaphysis tissue slice, visible bone trabecula obviously increases slightly, connects into net, and pulp cavity is less.HE dyeing * 100
The section of Figure 23 nilestriol group rat femur, visible bone trabecula increases slightly, and it is similar substantially to sham operated rats to connect into net.HE dyeing * 100
The specific embodiment:
Osthole purification production preparation process among the embodiment is seen number of patent application 03134435.6, it be with the Fructus Cnidii raw material with alcohol reflux extract, concentrate, water cleaning, aqueous alkali cleaning, pure dissolving, activated carbon decolorizing, pure water crystallization, drying, products obtained therefrom purity can reach more than 90%.Also can extract high purity cnidicin with other method.
Embodiment 1, and get purity and can reach high purity cnidicin (osthole, Osthol 〉=90%) more than 90%, be a dosage unit by 25mg-200mg, through pulverizing, make the preparation of protect against osteoporosis.This preparation can be conventional tablet, capsule, electuary, drop, granule.
Embodiment 2, get high purity cnidicin (osthole, Osthol 〉=90%), and by weight 80%, add 20% icariine again, through pulverizing, mix, make the preparation of protect against osteoporosis.This preparation can be conventional tablet, capsule, electuary, drop, granule.
Case study on implementation 3 is got high purity cnidicin (osthole, Osthol 〉=90%), and 60% adds 40% genistein more by weight, through pulverizing, mixes, and makes the preparation of protect against osteoporosis.This preparation can be conventional tablet, capsule, electuary, drop, granule.
Embodiment 4, get high purity cnidicin (osthole, Osthol 〉=90%), and 50% adds 35% genistein and 15% icariine more by weight, through pulverizing, mix, and make the preparation of protect against osteoporosis.This preparation can be conventional tablet, capsule, electuary, drop, granule.
Embodiment 5, get high purity cnidicin (osthole, Osthol 〉=90%), icariine 15% by weight 30%,, big legumin 12%, daidzin 10%, Genistin 10%, genistein 10%, calcium preparation 13%, through pulverizing, mixing, make the preparation of protect against osteoporosis.
Test the experimentation of 6 ostholes and Herba Epimedii, genistein, soybean isoflavone use in conjunction prevention osteoporosis rat.
The influence that this experimentation osthole and Herba Epimedii, genistein, soybean isoflavone use in conjunction form osteoporosis rat.After the ovariectomized rats, continuous oral osthole and Herba Epimedii, genistein, 12 weeks of soybean isoflavone complex, detect indexs such as rat femur bone density, osseous tissue form, and compare with model group and positive control medicine nilestriol group.The result shows: behind osthole and Herba Epimedii, genistein, the soybean isoflavone drug combination, each organizes the rat femur bone density all obviously increases than model group, and femur osseous tissue form obviously improves than model group, near sham operated rats.These experimental results show that osthole and Herba Epimedii, genistein, soybean isoflavone drug combination can prevent the osteoporotic formation of castrated rats.
One, experiment purpose
The influence that observation oral osthole of castrated rats and Herba Epimedii, genistein, soybean isoflavone drug combination form osteoporosis.
Two, reagent thing
1. osthole, purity is 95%, is provided lot number by Xi: 030516
2. Herba Epimedii, purity is 30%, is provided lot number by Xi: 030722
3. genistein, purity is 95%, provides lot number by Xi: 030706
4. soybean isoflavone, purity is 40%, provides lot number by Xi: 030412
5. nilestriol, Hualian Pharmaceutical Co., Ltd., Shanghai produces, and authentication code: the accurate word (1995) of medicine is defended No. 012090 in Shanghai, product batch number: 030801.Get a tablet 5mg, abundant porphyrize, the dissolving of 50ml 0.2% tween 80, standby.
Three, instrument and equipment
U.S. GE LUNAR DPXIQ dual intensity X line borne densitometers, the toy analysis software.
Four, laboratory animal
60 of SD rats, female, body weight 234 ± 20g, cleaning level, University Of Suzhou's Experimental Animal Center provides, laboratory animal production licence number: XCYK (Soviet Union) 2002-0008, laboratory animal occupancy permit number: SYXK (Soviet Union) 2002-0037.
Five, experimental technique
Get 50 of female rats, after the laboratory adaptability is raised 7 days, 10% chloral hydrate 0.3ml/100g body weight intraperitoneal injection anesthesia, the ventricumbent position is fixed, 1/3 place cropping in the back, iodine tincture, ethanol partly sterilised skin, do one downwards along back lumbar vertebra median line and be about 2～3 centimetres otch, cut skin, skin is led earlier to a side, cut fascia, the passivity separating muscle, after entering peritoneum, on cornua uteri, prick fallopian tube, cut off fallopian tube with toe-in, mention ovary (being the rediance graininess), silk thread is pricked its tissue that links to each other, spay, peritoneal suture otch on every side.With operating offside, skin suture otch with quadrat method.Rat is divided into 5 groups after making the castration model at random, and promptly model group, nilestriol 1mg/kg organize, osthole 5mg/kg adds Herba Epimedii 4mg/kg group, osthole 5mg/kg adds genistein 3.5mg/kg group, osthole 5mg/kg adds Semen sojae atricolor isoflavone 5mg/kg group.Other gets 10 female rats, and operation method is the same, but does not cut off fallopian tube, and spay is not only extractd and is equivalent to the mesentery of ovary size as sham operated rats.Every rat postoperative is intramuscular injection penicillin 100,000 u for three days on end, the prevention traumatic infection.
After the modeling 7 days, osthole group rat every morning 8:00～9:00 gastric infusion, the administration volume is the 1ml/100g body weight, and the nilestriol group is administered once weekly, model group and sham operated rats then give isopyknic 0.2% tween 80 every day.Successive administration is after 12 weeks, and the anesthesia of 10% chloral hydrate 0.3ml/100g body weight separates the bilateral femur, removes muscle.The right side femur is measured bone density, and the left side femur is fixed with 10% formalin, conventional decalcification, embedding, makes the thick tissue slice of 5mm, and HE dyes, and PATHOMORPHOLOGICAL OBSERVATION OF PULLORUM is also taken the photograph sheet.
Six, experimental result
Osthole is to the influence of castrated rats bone density
Model group rat femur near-end metaphysis bone density obviously reduces (P＜0.01) than sham operated rats, and bone density all had rising after each organized the osthole drug combination, illustrates that the osthole drug combination can prevent that the removal ovary rat bone density from descending.The results are shown in Table 13.
Table 13 osthole is to the influence of castrated rats near end of thighbone metaphysis bone density (n=10, x ± s)
Osthole agent drug combination dosage
Group bone density (g/cm
2)
Amount (mg/kg) (mg/kg)
Sham operated rats 0.084 ± 0.022
Model group 0.057 ± 0.016
▲ ▲
Osthole adds Herba Epimedii group 54 0.094 ± 0.021
*
Osthole adds genistein group 5 3.5 0.108 ± 0.024
*
Osthole adds Semen sojae atricolor isoflavone group 55 0.097 ± 0.019
*
Nilestriol group 1 0.112 ± 0.019
*
Annotate: compare with sham operated rats,
▲ ▲P＜0.01; Compare with model group,
*P＜0.01
Seven, conclusion
Castrated rats is taken osthole and Herba Epimedii, genistein, soybean isoflavone drug combination after 12 weeks continuously, can obviously prevent the osteoporotic formation of castrated rats.
Claims (10)
1. Chinese medicine preparation, it is characterized in that: described Chinese medicine preparation is that purity is the osthole Osthol more than 〉=90%, it is as the application of the sick medicine of preparation protect against osteoporosis.
2. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and the icariine weight ratio is chosen in 90%-10%.
3. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and big legumin weight ratio is chosen in 90%-10%.
4. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and the genistein weight ratio is chosen in 90%-10%.
5. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and the genistein weight ratio is chosen in 90%-10%.
6. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of daidzin is chosen in 90%-10%.
7. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of Genistin is chosen in 90%-10%.
8. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of calcium preparation is chosen in 90%-10%.
9. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of puerarin is chosen in 90%-10%.
10. a kind of Chinese medicine preparation according to claim 1 is characterized in that: described Chinese medicine preparation includes that high purity cnidicin Osthol weight ratio is chosen in 10%-90% and its weight ratio of soybean isoflavone is chosen in 90%-10%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103141488A (en) * | 2013-01-25 | 2013-06-12 | 浙江农林大学 | Pesticide compounded by azadirachtin and cnidium lactone and preparation method thereof |
| CN103141488B (en) * | 2013-01-25 | 2014-07-02 | 浙江农林大学 | Pesticide compounded by azadirachtin and cnidium lactone and preparation method thereof |
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