CN102008533B - Medicinal application and preparation method of shorthorned epimedium P.E - Google Patents
Medicinal application and preparation method of shorthorned epimedium P.E Download PDFInfo
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- 241000893536 Epimedium Species 0.000 title claims abstract description 35
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- 238000002360 preparation method Methods 0.000 title claims abstract description 25
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Abstract
The invention discloses medicinal application and an extracting method of shorthorned epimedium P.E, belonging to the field of medicines. The shorthorned epimedium P.E containing icariin, icariin A, icarisid II and epimedin B is applied to preparing medicines for treating chronic bacterial prostatitis, chronic nonbacterial prostatitis and hyperplasia of the prostate. The preparation method comprises the following steps: adding water or ethanol to epimedium for extraction; concentrating the extracting solution until the relative density is 1.01-1.10 at the temperature of 70 DEG C; and refining the filter liquor by a polyamide adsorption method, thus obtaining the shorthorned epimedium P.E. The invention has the following beneficial effects: by comparing data obtained in pharmacological tests with the data in related documents and patents, the medicines for treating chronic prostatitis and chronic hyperplasia of the prostate, which are prepared from the ehorthorned epimedium P.E, have definite curative effect, have no side effects and also have treatment effect superior to that of the prior art.
Description
Technical field
The invention belongs to medical reason, be specifically related to the pharmaceutical applications of Herba Epimedii extract.
Background technology
Herba Epimedii is the dry aerial parts of Berberidaceae plant Herba Epimedii Epimedium brevicornum Maxim., arrow leaf Herba Epimedii Epimedium sagiyyatum (Sieb.et Zucc.) Maxim., pubescence Herba Epimedii Epimedium pubescens Maxim., Epimedium wushanense Epimedium wushanense T.S.Ying or Herba Epimedii Epimedium koreanum Nakai.When summer, autumn, stem and leaf was in great numbers, tap, remove thick stalk and impurity, dry or dry in the shade.Herba Epimedii is the traditional kidney-nourishing tcm drug of China, on < < Compendium of Materia Medica > >, record effects such as " beneficial vital essence, hard muscles and bones, benefit waist knee joint, heart tonifying power ", on 2010 editions < < Chinese Pharmacopoeia > >, record effects such as " kidney-replenishing, bone and muscle strengthening, wind-damp dispelling ".Herba Epimedii mainly contains the chemical compositions such as flavone, polysaccharide, lignanoid, alkaloid.Modern pharmacology experimentation proof Herba Epimedii total flavones has the effects such as the immunocompetence of adjusting, adjusting sexual function, Cardiovarscular, osteoporosis, antiinflammatory.
At present, Herba Epimedii is mainly divided into following several respects at the report aspect treatment prostatosis:
The Chinese traditional compound medicine or the compositions that comprise Herba Epimedii, as the Chinese medicine composition in CN101007143 " a kind of treat prostatic Chinese medicine composition ", evident in efficacy, without any side effects.Chinese medicine compound in CN1689594 " a kind of prostatitis Chinese medicine for the treatment of ", can treat monomer prostatitis, prostatitis merging urethritis and prostatic hyperplasia.
The compositions of Herba Epimedii total flavones and composition of Salvia polysaccharide, as Herba Epimedii total flavones and the polysaccharide composition in ZL02148571.2 " Herba Epimedii extract for the treatment of prostatic hyperplasia and the application in preparing medicine thereof ", both are 2-8 part by weight: 8-2 part, can treat prostatitis and prostatic hyperplasia.
One of icariin, icariin I, epimidin A, arrow leaf icariine A or their mixture, as one of the icariin in ZL200710104013.3 " containing the medical composition and its use of epimedium active constituent ", icariin I, epimidin A, arrow leaf icariine A or their mixture, can treat prostatitis and prostatic hyperplasia.
Herba Epimedii related in above-mentioned report does not indicate concrete kind, Herba Epimedii is the dry aerial parts of Berberidaceae plant Herba Epimedii Epimedium brevicornum Maxim., arrow leaf Herba Epimedii Epimedium sagiyyatum (Sieb.et Zucc.) Maxim., pubescence Herba Epimedii Epimedium pubescens Maxim., Epimedium wushanense Epimedium wushanense T.S.Ying or Herba Epimedii Epimedium koreanum Nakai, Herba Epimedii difference in quality between different genera is very large, and effective substance is also different.
Summary of the invention
The object of the invention is for the application of a kind of Herba Epimedii extract that comprises icariin, icariine A, icariside I I, Epimedin B in preparation treatment chronic bacterial prostatitis, chronic nonbacterial prostatitis and prostatic hyperplasia medicine is provided.
Herba Epimedii extract of the present invention is for take the extract that Herba Epimedii is raw material, and defines the picking season, guaranteed the quality of crude drug, therefore guaranteed the quality homogeneity of prepared extractive of general flavone.
Based on optimum curative effect requirement, in extract of the present invention, contain 50%--80% Herba Epimedii total flavones, the content of icariin is greater than 5%.
Good effect of the present invention is: the Data Comparison in process pharmacological evaluation and pertinent literature, patent, find that the therapeutic effect of Herba Epimedii extract prepared in this patent in chronic prostatitis and chronic prostate hyperplasia disease is better.Therefore, the present invention has any different in prior art, and its determined curative effect, has no side effect substantially.
The extracting method of extract of the present invention is:
Method for making 1
(1) get the alcohol reflux 2～4 times that epimedium herb adds concentration 30～90%, add 10～20 times of weight ethanol of medical material used at every turn, each time is 0.5～3h;
(2) extracting solution of combining step (1) gained, being concentrated into relative density in the time of 70 ℃ is 1.01～1.10;
(3) will by polycaprolactam method or with macroreticular resin absorbing method, refine through the concentrated filtrate of step (2), with polycaprolactam method purification condition, be: applied sample amount is that epimedium herb is 1～5: 1 than polyamide, first use 1～5 times of column volume water elution, discard water elution liquid, use again 2～10 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii total flavones: the condition refining with macroreticular resin absorbing method is, applied sample amount is that epimedium herb is 1～5: 1 than macroporous resin, first use 1～5 times of column volume water elution, discard eluent, use again 10～30% ethanol elutions of 1～5 times of column volume, discard eluent, finally use 5～20 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.
Method for making 2
(1) get epimedium herb and decoct with water 2～4 times, add 10～20 times of weight water of medical material used at every turn, each time is 0.5～3h,
(2) combining step (1) gained extracting solution, being concentrated into relative density in the time of 70 ℃ is 1.01～1.10;
(3) will add ethanol through the concentrated extracting solution of step (2), making containing alcohol amount is 60～80%, and standing 12～48h gets supernatant and reclaims ethanol extremely without alcohol taste, and adding water to medicinal liquid is 1～3: 1 than medical material, and standing 12～48h filters;
(4) step (3) gained filtrate is refining by polycaprolactam method or macroreticular resin absorbing method, the condition refining by polycaprolactam method is: applied sample amount is that epimedium herb is 1～5: 1 than polyamide, first use 1～5 times of column volume water elution, discard water elution liquid, use again 2～10 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain excessive sheep extract; The condition refining with macroreticular resin absorbing method is: applied sample amount is that epimedium herb is 1～5: 1 than macroporous resin, first use 1～5 times of column volume water elution, discard eluent, use again 10～30% ethanol elutions of 1～5 times of column volume, discard eluent, finally use 5～20 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.
The content assaying method of total flavones in Herba Epimedii extract
The preparation of 1.1 reference substance solution
Precision takes icariin reference substance 5.0mg, with dissolve with methanol standardize solution, in 50mL measuring bottle, shakes up, and makes the reference substance storing solution of 0.1mg/mL.
The preparation of 1.2 need testing solutions
Get the about 5.0mg of Herba Epimedii extract, accurately weighed, be placed in 25mL measuring bottle, add methanol-water (60: 40, v/v) dissolve, and be settled to scale, shake up, then get 1.0mL in 10mL measuring bottle, with methanol-water (60: 40, v/v) be settled to scale, shake up, obtain need testing solution.
The preparation of 1.3 standard curves
Get respectively reference substance storing solution 0.5mL, 1.0mL, 1.5mL, 2.0mL, 2.5mL and be placed in 10mL measuring bottle, respectively by methanol constant volume to scale, shake up, according to ultraviolet spectrophotometry, take methanol as blank, under 270nm wavelength, measure absorbance, with absorbance (A), concentration (C) is made to standard curve.Result shows, the concentration of icariin is within the scope of 5.0～25.0 μ g/mL, linear with trap.A＝0.03746C+0.01230，r＝0.9996。
1.4 precision test
Measure respectively 15.0 μ g/mL reference substance solution 1.0mL, according to operating under standard curve preparation, five absorbances of METHOD FOR CONTINUOUS DETERMINATION, the RSD of result absorbance is 1.2%.
1.5 replica test
Get with 5 parts of a collection of test samples, make need testing solution, according to operating under standard curve preparation, measure respectively absorbance, and calculate general flavone content and RSD.Result RSD is 1.6%.
1.6 stability test
Get the need testing solution of handling well, respectively after preparing sample 0,1,2,3,4,5h measures absorbance, calculates RSD, the RSD of result absorbance is respectively 1.0%, result shows that need testing solution is stable in 5 hours.
1.7 recovery test
Get five parts of known content test samples, each about 2.5mg, accurately weighed, precision adds 1.0mg/mL icariin reference substance solution 1.0mL respectively, according to operating under standard curve preparation, measures absorbance, calculate recovery rate, the result response rate is that 100.4%, RSD is 3.6%.
The content assaying method of icariin, icariine A, icariside I I, Epimedin B in Herba Epimedii extract
The compositions such as icariin, icariine A, icariside I I, Epimedin B from Herba Epimedii total flavones extract, have been isolated, and identified their structure, by RP-HPLC linear gradient elution method, measured the content of these four kinds of compositions simultaneously, can take into account the quality control of main component, can rarely have the icariside I I containing quantity research that reference is provided to this again.Icariin
The molecular structural formula of A, icariin, Epimedin B, icariside I I is as follows:
Icariine A icariin
Epimedin B icariside I I
2.1 chromatographic conditions and system suitability
Chromatographic column: DIAMONSILC18 (250mm * 4.6mm, 5 μ m);
Mobile phase: methanol: water (v: v), gradient elution, elution program is in Table 1;
Wavelength: 270nm; Flow velocity: 0.8mL/min; Column temperature: 25 ℃; Sample size: 5 μ L.
Table 1 eluent gradient elution program
2.2 system suitability
Under above-mentioned chromatographic condition, the chromatogram of icariine A, Epimedin B, icariin, icariside I I reference substance and test sample is shown in accompanying drawing 1 and accompanying drawing 2, the separating degree of each chromatographic peak and adjacent chromatographic peak is greater than 1.5, theoretical cam curve is calculated and is not less than 3000 by icariin, and tailing factor is between 0.95～1.05.
The preparation of 2.3 solution
The preparation of reference substance solution: precision takes reference substance icariine A, Epimedin B, icariin, icariside I I each is appropriate respectively, with methanol, be made into the reference substance stock solution that mass concentration is respectively 1.65g/L, 3.06g/L, 9.60g/L, 0.15g/L, get respectively each reference substance solution of equivalent, mix, shake up, must mix reference substance storing solution.Every 1mL is containing icariine A 0.413mg, Epimedin B 0.765mg, icariin 2.40mg, icariside I I 0.0375mg.
The preparation of need testing solution: precision takes the about 25.0mg of Herba Epimedii extract, is placed in 25mL volumetric flask, methanol-water (60: 40, v/v) dissolve, and be settled to scale, shake up, with microporous filter membrane (0.45 μ m), filter, get subsequent filtrate as need testing solution.
2.4 linear relationships are investigated
Accurate absorption mixed reference substance storing solution 1mL, 2mL, 3mL, 4mL, 5mL, be placed in 25mL volumetric flask, with methanol, be diluted to scale, shake up, with microporous filter membrane (0.45 μ m), filter, by chromatographic condition, measure peak area, the reference substance peak area A of take is vertical coordinate, the mass concentration C of reference substance solution of take is abscissa, and drawing standard curve, tries to achieve regression equation (n=5).Data result is in Table 2.
The standard curve (n=5) of table 2 icariine A, Epimedin B, icariin, icariside I I
Result icariine A mass concentration is in 16.5～82.5mg/L, icariin mass concentration is in 96.0～480.0mg/L, Epimedin B mass concentration is in 30.6～153.0mg/L, and icariside I I mass concentration, in 1.5～7.5mg/L, has good linear relationship with peak area.
2.5 precision test
Get mixing reference substance solution, by above-mentioned chromatographic condition, repeat sample introduction 5 times, each 5 μ L, measure peak area, and the RSD of result icariine A, icariin, Epimedin B, icariside I I peak area is respectively 0.97%, 1.2%, 0.56%, 1.1%.
2.6 replica test
Get with 5 parts of a collection of test samples, make need testing solution, by above-mentioned chromatographic condition, measure, the RSD of result icariine A, icariin, Epimedin B icariside I I is respectively 1.1%, 1.8%, 0.99%, 1.4%.
2.7 stability test
Need testing solution is pressed to above-mentioned chromatographic condition, respectively after preparing sample 0,4,6,8,10,12h sample introduction, measure peak area, the RSD of result icariine A, icariin, Epimedin B, icariside I I is respectively 0.88%, 0.90%, 1.3%, 1.2%, and result shows that need testing solution is stable in 12 hours.
2.8 recovery test
Get five parts of the same batch samples of known content, each about 12.5g, accurately weighed, precision adds icariine A, icariin, Epimedin B, icariside I I reference substance solution a certain amount of respectively, press need testing solution preparation method and process, measure peak area, calculate recovery rate, average recovery rate 102.1%, RSD is 1.2%.
Herba Epimedii extract extract preparation technology of the present invention is simple, has significant treatment prostatic hyperplasia and prostatitic effect.
Accompanying drawing explanation
Fig. 1 is icariine A, Epimedin B, icariin, icariside I I standard solution chromatogram.
In Fig. 1, sequence number 1 is icariine A, and sequence number 2 is Epimedin B, and sequence number 3 is icariin, and sequence number 4 is icariside I I.
Fig. 2 is Herba Epimedii extract sample solution chromatogram of the present invention.
In Fig. 2, sequence number 1 is icariine A, and sequence number 2 is Epimedin B, and sequence number 3 is icariin, and sequence number 4 is icariside I I.
The specific embodiment
Get epimedium herb and add 70% alcohol reflux 2 times, add 15 times of ethanol, each time is 1.5h, merge extractive liquid, at every turn, being concentrated into relative density in the time of 70 ℃ is 1.02, filtrate is refining by polycaprolactam method, and applied sample amount is epimedium herb: polyamide=2: 1, first use 1 times of column volume water elution, discard water elution liquid, use again 5 times of column volumes, 50% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.Wherein general flavone content is 57.6%, and Icariin content is 10.2%.
Getting epimedium herb decocts with water 3 times, add 20 times of water at every turn, each time is 1h, merge extractive liquid,, being concentrated into relative density in the time of 70 ℃ is 1.05, add ethanol make containing alcohol amount be 70%, standing 24h, get supernatant and reclaim ethanol to without alcohol taste, add water to medicinal liquid: medical material is 2: 1, standing 48h, filter, filtrate is refining by polycaprolactam method, and applied sample amount is epimedium herb: polyamide=2: 1, first use 1 times of column volume water elution, discard water elution liquid, use again 3 column volume 70% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.Wherein general flavone content is 66.7%, and Icariin content is 11.4%.
Get epimedium herb and add 70% alcohol reflux 2 times, add 15 times of ethanol at every turn, each time is 1.5h, merge extractive liquid,, being concentrated into relative density in the time of 70 ℃ is 1.02, filtrate is refining with macroreticular resin absorbing method, applied sample amount is epimedium herb: macroporous resin=2: 1, first use 1 times of column volume water elution, discard eluent, then use 20% ethanol elution of 2 times of column volumes, discard eluent, finally use 10 column volume 80% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.Wherein general flavone content is 68.4%, and Icariin content is 9.7%.
Getting epimedium herb decocts with water 3 times, add 15 times of water at every turn, each time is 1h, merge extractive liquid, being concentrated into relative density in the time of 70 ℃ is 1.04, add ethanol make containing alcohol amount be 70%, standing 24h, get supernatant and reclaim ethanol extremely without alcohol taste, add water to medicinal liquid: medical material is 2: 1, standing 24h, filter, filtrate is refining with macroreticular resin absorbing method, applied sample amount is epimedium herb: macroporous resin=2: 1, first use 1 times of column volume water elution, discard eluent, use again 20% ethanol elution of 2 times of column volumes, discard eluent, finally use 10 column volume 80% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.Wherein general flavone content is 57.0%, and Icariin content is 12.1%.
The preparation of embodiment 5 Herba Epimedii extract conventional tablets
Get embodiment 1 Herba Epimedii extract 200g, microcrystalline Cellulose 200g, cross-linking sodium carboxymethyl cellulose 100g cross 80 mesh sieves and mix, add 3% hydroxypropyl methylcellulose solution soft material processed in right amount, crossing 18 mesh sieves granulates, put into 50 ℃ of oven dryings 12 hours, use afterwards 16 mesh sieve granulate, add 1% magnesium stearate, mix homogeneously, tabletting.Make 1000 and get final product, every containing Herba Epimedii extract 200mg.
The preparation of embodiment 6 Herba Epimedii extract conventional capsule agent
Get embodiment 1 Herba Epimedii extract 200g, microcrystalline Cellulose 200g, cross-linking sodium carboxymethyl cellulose 100g cross 80 mesh sieves and mix, add 3% hydroxypropyl methylcellulose solution soft material processed in right amount, crossing 18 mesh sieves granulates, put into 50 ℃ of oven dryings 12 hours, use afterwards 16 mesh sieve granulate, add 1% magnesium stearate, mix homogeneously, fill capsule.Make 1000 and get final product, every containing Herba Epimedii extract 200mg.
The test of pesticide effectiveness
The effect of I Herba Epimedii extract to rat chronic nonbacterial prostatitis.
1, laboratory animal, medicine and reagent
1.1 laboratory animal
Healthy male wistar rat, 200 ± 20g, male, purchased from preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SCXK (Ji) 2007-0003.
1.2 tested medicines
The prepared Herba Epimedii extract (B) of Herba Epimedii extract (A), embodiment 3 that embodiment 1 is prepared.
1.3 reagent
The kidney warming prostatitis capsule: Xiuzheng Pharmaceutical Group Co., Ltd, specification: 0.5g/ grain, lot number: 081005.
Chloral hydrate: Solution on Chemical Reagents in Shanghai company of Chinese Medicine group, lot number: 20040206.
XIAOZHILING ZHUSHEYE: Jian-Yisheng Pharmaceutical Co., Ltd., Jilin, specification: 10ml/ props up, lot number: 0907242.
2, experimental technique
The foundation of 2.1 experimental nonbacterial prostatitis rat models
70 of male rats, 10% chloral hydrate (0.3mL/100g) intraperitoneal injection of anesthesia, the about 1cm of sterile working's hypogastric region median incision, through abdominal cavity, bladder and bilateral seminal vesicle are proposed, exposure is attached to the prostate notopodium of seminal vesicle inner side, and 25% XIAOZHILING ZHUSHEYE is injected to each 0.1mL of prostate bilateral notopodium, suture muscles skin.10 of sham operated rats are in same position same method saline injection 0.2mL, and wound place coating penicillin is in case infect.After modeling, raise 14 days.
2.2 grouping and administrations
Rat is divided into 7 groups at random, i.e. model group, sham operated rats, positive drug (the kidney warming prostatitis capsule) group, A high dose group, A low dose group, B high dose group, B low dose group, 10 every group.Rat oral gavage administration, dosage and approach: model group: normal saline gavage, volume 0.5mL/100g; Sham operated rats: normal saline gavage, volume 0.5mL/100g; A low dose group: give sample A 20mg/kg, gavage, volume 0.5mL/100g; A high dose group: give sample A40mg/kg, gavage, volume 0.5mL/100g; B low dose group: give sample B 35mg/kg, gavage, volume 0.5mL/100g; B high dose group: give sample B 70mg/kg, gavage, volume 0.5mL/100g; Positive drug group: give and the kidney warming prostatitis capsule 1g/kg, gavage, volume 0.5mL/100g.
Rat administration every day 1 time, successive administration 14 days.After last administration 24 hours, rat was weighed, puts to death.Carefully cut open and get prostate, with micro-electronic balance, take prostate weight in wet base, and calculate prostate index (prostate index=prostate weight in wet base mg/ rat body weight g); With toes capacity measurer, measure prostate volume.Afterwards with the fixing ，Song histopathologic examination of 10% formaldehyde.
3, experimental result:
The impact of 3.1 Herba Epimedii extracts on nonbacterial prostatitis rat prostate index and volume
Two kinds of Herba Epimedii are always got thing and all can obviously be reduced the nonbacterial prostatitis rat prostate exponential sum volume that xiaozhiling injection causes, compare significant difference (p < 0.05 or p < 0.01) with model group, the results are shown in Table 3.
The impact of table 3 Herba Epimedii extract on rat prostate index and volume
Compare △ p < 0.05 with model group, △ △ p < 0.01, compares ※ p < 0.05 with sham operated rats.
3.2 histopathology histological observations
Rats in sham-operated group prostata tissue body of gland out-of-shape, visible more crowfoot cracks, glandular epithelium is monolayer cube or simple columnar epithelium, accidental pseudostratified columnar epithelium, alveolar lumen is arranged closely, and intracavity dyes secretions containing homogenizing powder, and body of gland wraps up a small amount of blood vessel and connective tissue around.Model group rat acinous cell is destroyed, even distortion, fibrosis disappear, the epithelial cell and the cell debris that in alveolar lumen, come off in a large number as seen.In interstitial, visible more cell infiltration, take lymphocyte as main, and fibrous connective tissue hypertrophy is obvious.Two kinds of high low dose group of technique Herba Epimedii extract all have some improvement to prostatitis rat lesion degree, and extent of disease, cell infiltration, interstitial proliferation all have alleviating in various degree, and indivedual rat prostate tissues are with normal approaching.Lesion degree is roughly divided into 4 grades by following standard: I level: normal or approaching normal; II level: acinous cell form approaches normal, in lumen of gland without or have a small amount of secretions, interstitial is without extremely; III level: part acinous cell is destroyed obviously, has more foreign body in lumen of gland, and interstitial is without abnormal or slight hypertrophy; IV level: acinous cell is destroyed obviously, has a large amount of foreign bodies in lumen of gland, and cell infiltration is obviously accompanied in interstitial proliferation, and each group the results are shown in Table 4.
Table 4 Herba Epimedii extract is on the histopathologic impact of prostatitis rat model
4, conclusion
Two kinds of Herba Epimedii extracts all can significantly reduce experimental prostatitis rat prostate index, volume; Histopathological examination shows that two kinds of Herba Epimedii extracts all have some improvement to prostatitis lesion degree.
The effect of II Herba Epimedii extract to rat chronic bacterial prostatitis
1, laboratory animal, reagent and instrument
1.1 laboratory animal
Healthy male wistar rat, 200 ± 20g, male, purchased from preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SCXK (Ji) 2007-0003.
1.2 tested medicines
The prepared Herba Epimedii extract (B) of Herba Epimedii extract (A), embodiment 3 that embodiment 1 is prepared.
1.3 reagent
The kidney warming prostatitis capsule: Xiuzheng Pharmaceutical Group Co., Ltd, specification: 0.5g/ grain, lot number: 081005.
Ofloxacin: Shanxi Qian Hui pharmaceutcal corporation, Ltd, specification 0.1g/ sheet, lot number: 20081202.
Chloral hydrate: Solution on Chemical Reagents in Shanghai company of Chinese Medicine group, lot number: 20040206.
Escherichia coli: Jilin Renmin Hospital provides.
2, experimental technique
The foundation of 2.1 experimental bacterial prostatitis rat models
60 of male rats, 10% chloral hydrate (0.3mL/100g) intraperitoneal injection of anesthesia, the about 1cm of sterile working's hypogastric region median incision, through abdominal cavity, proposes bladder and bilateral seminal vesicle, exposes the prostate notopodium that is attached to seminal vesicle inner side, by 1.5 * 10
8the escherichia coli liquid of/mL mixes each 0.1mL of injection prostate bilateral notopodium, suture muscles skin with 0.2% agar that is chilled to 45 ℃.10 of sham operated rats are in same position same method saline injection 0.2mL, and wound place coating penicillin is in case infect.After modeling, raise 28 days.
2.2 grouping and administrations
Rat is divided into 6 groups at random, i.e. model group, sham operated rats, positive drug (the kidney warming prostatitis capsule) group, positive drug (ofloxacin) group, A group, B group, 10 every group.Rat oral gavage administration, dosage and approach: model group: normal saline gavage, volume 0.5mL/100g; Sham operated rats: normal saline gavage, volume 0.5mL/100g; A group: give sample A40mg/kg, gavage, volume 0.5mL/100g; B group: give sample B 70mg/kg, gavage, volume 0.5mL/100g; Positive drug (the kidney warming prostatitis capsule) group: give and the kidney warming prostatitis capsule 1g/kg, gavage, volume 0.5mL/100g; Positive drug (ofloxacin) group: 10mg/kg, gavage, volume 0.5mL/100g.Rat administration every day 1 time, successive administration 28 days.After last administration 24 hours, rat was weighed, puts to death.Under aseptic condition, cut open and get prostate and smash to pieces at once, get 50mg and add 200ul leukocyte diluent and 200ul normal saline, fully mix, record leukocyte count under microscope, centrifugal elutriation liquid, taking precipitate carries out Bacteria Identification.
3, experimental result:
The impact of 3.1 Herba Epimedii extracts on rat chronic bacterial prostatitis model leukocyte and bacterium colony number
Two kinds of Herba Epimedii extracts all can obviously reduce bacterial prostatitis rat leukocyte that escherichia coli causes and the number of bacterium colony, compare significant difference (p < 0.05 or p < 0.01) with model group, the results are shown in Table 5.
The impact of table 5 Herba Epimedii extract on rat chronic bacterial prostatitis model leukocyte and bacterium colony number
Compare △ p < 0.05 with model group, △ △ p < 0.01, compares ※ p < 0.05 with sham operated rats.
The effect of III Herba Epimedii extract to mice prostatic hyperplasia
1, laboratory animal, reagent and instrument
1.1 laboratory animal
Healthy male mice in kunming, 20 ± 2g, male, purchased from preclinical medicine institute of Jilin University Experimental Animal Center, the quality certification number: SCXK (Ji) 2007-0003.
1.2 tested medicines
The prepared Herba Epimedii extract (B) of Herba Epimedii extract (A), embodiment 3 that embodiment 1 is prepared.
1.3 reagent
The kidney warming prostatitis capsule: Xiuzheng Pharmaceutical Group Co., Ltd, specification: 0.5g/ grain, lot number: 081005.
Chloral hydrate: Solution on Chemical Reagents in Shanghai company of Chinese Medicine group, lot number: 20040206.
Testosterone Propionate injection: Shanghai General Pharmaceutical Co., ltd., lot number: 080303.
2, experimental technique
The foundation of 2.1 Experimental Mice prostatic hyperplasia model
70 of male mices, wherein 6 groups with Testosterone Propionate every day according to the modeling of 5mg/kg subcutaneous injection, continuous 21 days.
2.2 grouping and administrations
Mice is divided into 7 groups at random, i.e. blank group, model group, positive drug (the kidney warming prostatitis capsule) group, A high dose group, A low dose group, B high dose group, B low dose group, 10 every group.Mouse stomach administration, dosage and approach: model group: normal saline gavage, volume 0.5mL/100g; Model group: normal saline gavage, volume 0.5mL/100g; A low dose group: give sample A 20mg/kg, gavage, volume 0.5mL/100g; A high dose group: give sample A40mg/kg, gavage, volume 0.5mL/100g; B low dose group: give sample B 35mg/kg, gavage, volume 0.5mL/100g; B high dose group: give sample B 70mg/kg, gavage, volume 0.5mL/100g; Positive drug group: give and the kidney warming prostatitis capsule 1g/kg, gavage, volume 0.5mL/100g.Every day 1 time, continuous 3 weeks, another 1 group was blank group.After last administration, 1h weighs, and then de-cervical vertebra is put to death mice, gets rapidly prostata tissue, weighs.And calculate prostate index (prostate index=prostate weight in wet base mg/ Mouse Weight g).
3, experimental result:
The impact of 3.1 Herba Epimedii extracts on mice prostatic hyperplasia model prostate weight and prostate index
Compare with blank group, the prostate of model group mice weighs and prostate index all significantly increases.Compare two kinds of Herba Epimedii extracts with model group and all can obviously reduce prostatic hyperplasia mice prostate weight and the prostate index that Testosterone Propionate causes, there is significant difference (p < 0.05 or p < 0.01), the results are shown in Table 6.
The impact of table 6 Herba Epimedii extract on mice prostatic hyperplasia model prostate weight and prostate index
Compare △ p < 0.05 with model group, △ △ p < 0.01, compares ※ p < 0.05 with blank group.
Claims (3)
1. a Herba Epimedii extract that comprises icariin, icariine A, icariside II and Epimedin B is treated the application in chronic bacterial prostatitis, chronic nonbacterial prostatitis and prostatic hyperplasia medicine in preparation;
Aforementioned Herba Epimedii extract need meet following speciality;
A, contain 50%--80% Herba Epimedii total flavones, wherein the content of icariin is greater than 5%;
B, with Herba Epimedii, be that raw material extracts;
C, with following process, make;
Method one
(1) get the alcohol reflux 2～4 times that epimedium herb adds concentration 30～90%, add 10～20 times of weight ethanol of medical material used at every turn, each time is 0.5～3h,
(2) extracting solution of combining step (1) gained, being concentrated into relative density in the time of 70 ℃ is 1.01～1.10,
(3) will by polycaprolactam method or with macroreticular resin absorbing method, refine through the concentrated filtrate of step (2), with polycaprolactam method purification condition, be: applied sample amount is that epimedium herb is 1～5:1 than polyamide, first use 1～5 times of column volume water elution, discard water elution liquid, use again 2～10 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract; The condition refining with macroreticular resin absorbing method is, applied sample amount is that epimedium herb is 1～5:1 than macroporous resin, first use 1～5 times of column volume water elution, discard eluent, 10～30% ethanol elutions of using again 1～5 times of column volume, discard eluent, finally use 5～20 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract;
Method two
(1) get epimedium herb and decoct with water 2～4 times, add 10～20 times of weight water of medical material used at every turn, each time is 0.5～3h,
(2) combining step (1) gained extracting solution, being concentrated into relative density in the time of 70 ℃ is 1.01～1.10,
(3) will add ethanol through the concentrated extracting solution of step (2), making containing alcohol amount is 60～80%, and standing 12～48h gets supernatant and reclaims ethanol to without alcohol taste, adds water to medicinal liquid: medical material is 1～3:1, and standing 12～48h, filters,
(4) step (3) gained filtrate is refining by polycaprolactam method or macroreticular resin absorbing method, the condition refining by polycaprolactam method is: applied sample amount is that epimedium herb is 1～5 to 1 than polyamide, first use 1～5 times of column volume water elution, discard water elution liquid, use again 2～10 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract; The condition refining with macroreticular resin absorbing method is: applied sample amount is that epimedium herb is 1～5:1 than macroporous resin, first use 1～5 times of column volume water elution, discard eluent, use again 10～30% ethanol elutions of 1～5 times of column volume, discard eluent, finally use 5～20 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.
2. a preparation method for Herba Epimedii extract claimed in claim 1, is characterized in that comprising the following steps:
(1) get the alcohol reflux 2～4 times that epimedium herb adds concentration 30～90%, add 10～20 times of weight ethanol of medical material used at every turn, each time is 0.5～3h,
(2) extracting solution of combining step (1) gained, being concentrated into relative density in the time of 70 ℃ is 1.01～1.10;
(3) will by polycaprolactam method or with macroreticular resin absorbing method, refine through the concentrated filtrate of step (2), with polycaprolactam method purification condition, be: applied sample amount is that epimedium herb is 1～5:1 than polyamide, first use 1～5 times of column volume water elution, discard water elution liquid, use again 2～10 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii total extract; The condition refining with macroreticular resin absorbing method is, applied sample amount is that epimedium herb is 1～5:1 than macroporous resin, first use 1～5 times of column volume water elution, discard eluent, 10～30% ethanol elutions of using again 1～5 times of column volume, discard eluent, finally use 5～20 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.
3. a preparation method for Herba Epimedii extract claimed in claim 1, is characterized in that comprising the following steps:
(1) get epimedium herb and decoct with water 2～4 times, add 10～20 times of weight water of medical material used at every turn, each time is 0.5～3h,
(2) combining step (1) gained extracting solution, being concentrated into relative density in the time of 70 ℃ is 1.01～1.10;
(3) will add ethanol through the concentrated extracting solution of step (2), making containing alcohol amount is 60～80%, and standing 12～48h gets supernatant and reclaims ethanol to without alcohol taste, adds water to medicinal liquid: medical material is 1～3:1, and standing 12～48h, filters;
(4) step (3) gained filtrate is refining by polycaprolactam method or macroreticular resin absorbing method, the condition refining by polycaprolactam method is: applied sample amount is that epimedium herb is 1～5 to 1 than polyamide, first use 1～5 times of column volume water elution, discard water elution liquid, use again 2～10 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract; The condition refining with macroreticular resin absorbing method is: applied sample amount is that epimedium herb is 1～5:1 than macroporous resin, first use 1～5 times of column volume water elution, discard eluent, use again 10～30% ethanol elutions of 1～5 times of column volume, discard eluent, finally use 5～20 column volume 50～90% alcoholic solution eluting, collect eluent, reclaim ethanol and obtain Herba Epimedii extract.
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| CN104711301B (en) * | 2015-03-24 | 2018-07-24 | 北京盛诺基医药科技有限公司 | A kind of preparation method of icariine |
| CN104688795A (en) * | 2015-03-24 | 2015-06-10 | 北京盛诺基医药科技有限公司 | Method for preparing general flavone through epimedium |
| CN107028999A (en) * | 2017-06-02 | 2017-08-11 | 广州和匠科技有限公司 | Eye-drops preparations containing barrenwort and preparation method thereof |
| CN107669695B (en) * | 2017-11-16 | 2019-07-16 | 遵义医科大学 | Icariside II is preparing the application in anti-hepatitis B virus product |
| CN109459515B (en) * | 2018-07-05 | 2021-09-17 | 广州科曼生物科技有限公司 | Herba epimedii control extract (arrow leaf) and application thereof |
| CN110251549B (en) * | 2019-06-24 | 2021-12-03 | 浙江省肿瘤医院 | Application of epimedium total flavone extract in preparing medicine for preventing and treating hashimoto thyroiditis |
| CN111759880A (en) * | 2020-06-09 | 2020-10-13 | 广东金骏康生物技术有限公司 | Epimedium herb extract and application thereof in preventing or treating coronavirus |
| CN111643538A (en) * | 2020-07-20 | 2020-09-11 | 成都农业科技职业学院 | Epimedium root extract and its prepn and use |
| CN112089786A (en) * | 2020-10-19 | 2020-12-18 | 琛蓝(美国)营养制品股份有限公司 | Multifunctional traditional Chinese medicine compound composition and application thereof in pharmaceutical preparation |
| CN116942767A (en) * | 2023-06-05 | 2023-10-27 | 广西万通制药有限公司 | Medicine application of compound desmodium granules for treating prostatitis |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101057855A (en) * | 2007-05-11 | 2007-10-24 | 澳美制药厂有限公司 | Medicinal composition containing epimedium active constituent and its application |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101057855A (en) * | 2007-05-11 | 2007-10-24 | 澳美制药厂有限公司 | Medicinal composition containing epimedium active constituent and its application |
Non-Patent Citations (1)
| Title |
|---|
| 董自艳等.RP-HPLC法测定朝鲜淫羊藿提取物中4种成分的含量.《沈阳药科大学学报》.2010,第27卷(第8期),第652－655页. * |
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