CN101700368B - Method for detecting components of pharmaceutical composition for treating diseases of urinary system - Google Patents

Method for detecting components of pharmaceutical composition for treating diseases of urinary system Download PDF

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CN101700368B
CN101700368B CN2009102240244A CN200910224024A CN101700368B CN 101700368 B CN101700368 B CN 101700368B CN 2009102240244 A CN2009102240244 A CN 2009102240244A CN 200910224024 A CN200910224024 A CN 200910224024A CN 101700368 B CN101700368 B CN 101700368B
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poria cocos
solution
medicinal material
water
acid
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CN101700368A (en
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何彦杰
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Jiangxi Hongxing Pharmaceutical Co Ltd
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Jiangxi Hongxing Pharmaceutical Co Ltd
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Abstract

The invention relates to a method for detecting components of a pharmaceutical composition for treating diseases of the urinary system, which comprises the following steps: taking the pharmaceutical composition as the sample solution; making a herba plantaginis medicine into a control medicine solution; and absorbing the two solutions, respectively dropping on the same silica gel G thin-layer plate using sodium carboxymethyl cellulose as the adhesive, developing by using ethyl acetate-methanoic acid-water as the developing agent, taking out, drying in the air, spraying a vanillina-sulphuric acid solution, heating until the spots become clear, and putting in a sample chromatogram, wherein in the positions corresponding to the control medicine chromatogram, the spots with the same colors are displayed. The invention has the advantages of low cost of preparation technique and detecting technique, and easy processing, reduces the heating time of the medicine and is suitable for wide application in clinical treatment.

Description

The detection method of the components of pharmaceutical composition of treatment disease in the urological system
Technical field
The present invention relates to a kind of detection method for the treatment of the components of pharmaceutical composition of disease in the urological system.
Background technology
Disease in the urological system is human common disease, its cardinal symptom have frequent micturition short red, urinate puckery cusalgia, serious occurred heating, thirsty, abdominal pain, waist is ached, blood urine, suppurate, dry and hard excrement or completely mashed.Calculi in urinary system belongs to the urolithiasis in the traditional Chinese medicine stranguria.Traditional Chinese medicine is thought by damp invasion of lower energizer, and turning into fire and then burning YIN decocts urine, becomes sandstone, and the alluvial water channel forms.Dialectical real example based on part of the body cavity below the umbilicus, housing the bladder, kidneys and bowels dampness and heat stasis.Control suitable clearing heat and promoting diuresis, Tonglin Paishi.So get rid of the key that the water channel sandstone will become the treatment stone in urinary system.At present the western medicine therapy to kidney, urinary tract, wing skin calculus is that surgical calculus removing and external instrument shake broken discharging calculus method, operates on modus operandi expense height, patient's misery and postoperative recurrence rate height; Though it is fast that row's stone method speed is impacted in instrument concussion, short treating period, the equipment manufacturing cost height is not suitable for the extensive patients in samll cities and towns or rural area.The traditional Chinese medical science then is to adopt the agent of various row's cubic meter of stone, and its cost is lower, but most prescription among the people does not pass through strict pharmacodynamic experiment and toxicity test, and also each is variant for result of treatment; To acute pyelonephritis, wing skin inflammation, the methods of treatment of the present doctor trained in Western medicine of disease that urethritis and venereal disease cause adopts antibiotic therapy mostly, the result of treatment of antibiotic is obvious, speed is fast, but it is not remarkable to the chronic disease result of treatment, the medication that has also can produce the drug-resistant effect to bacterium too for a long time, so the traditional Chinese medical science has been studied some preparations at present, in order to treatment calculi in urinary system and urinary tract infection, as three gold plaques (capsule), the treating stranguria oral medicine of money, bazheng mixture, bazheng heji, Jisheng Shenqi Wan, composite shi-wei tablet etc., these Chinese medicine preparations have certain therapeutic action to illnesss such as treatment calculi in urinary system and urinary tract infections, but do not obtain satisfactory effect yet.
Summary of the invention
The present invention provides a kind of detection method for the treatment of the components of pharmaceutical composition of disease in the urological system just from prior art, thereby its cost is reduced, and handling ease is suitable in the widespread use clinical treatment.
The detection method of the components of pharmaceutical composition of treatment disease in the urological system of the present invention, the composition of the pharmaceutical composition of wherein said treatment disease in the urological system is Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Poria cocos, Asiatic plantain, Herba lygodii, cogongrass rhizome, and each composition percentage by weight is as follows:
Desmodium styracifolium 21～24%
Corn stigma 12～18%
Pyrrosia lingua 9～13%
Canton love-pea vine 8～13%
Poria cocos 8～12%;
Asiatic plantain 7～10%
Herba lygodii 7～10%
Cogongrass rhizome 8～13%
And chlorogenic acid (C in the described pyrrosia lingua 16H 18O 9) content, must not be less than 0.3% of general assembly (TW), above-mentioned medicinal material is obtained described pharmaceutical composition by following method for making:
Step 1: get described Poria cocos and be divided into two parts, it is 80～90% that a copy of it accounts for the Poria cocos total weight percent, and it is 10～20% that another part accounts for the Poria cocos total weight percent;
Step 2: will account for the Poria cocos total weight percent and be 80～90% portion and be ground into fine powder;
Step 3: will remain Poria cocos and all the other seven flavor medicine material boilings 1～3 time, each 2～4 hours, collecting decoction filtered, and filtrate is condensed into the clear cream that relative density is 1.19～1.20 (90 ℃);
Step 4: add the Poria cocos fine powder, mixing, drying is ground into fine powder, sieves, and adds appropriate amount of auxiliary materials, and mixing is granulated, drying, whole grain;
Described detection method comprises:
Compositions 5～15g gets it filled, add water 30～70ml and make dissolving, in 60～90 ℃ of extractions 1～3 time, each 30～50ml discards sherwood oil liquid with sherwood oil, water liquid extracts 1～3 time with ethyl acetate, each 20～40ml merges ethyl acetate liquid, evaporate to dryness, residue adds ethanol 1～3ml makes dissolving, as need testing solution;
Other gets Asiatic plantain control medicinal material 1～3g, adds 70% ethanol, 100～300ml, refluxes 2～3 hours, puts coldly, filters, and filtrate evaporate to dryness, residue add water 30～50ml makes dissolving, extracts 2～4 times for 60～90 ℃ with sherwood oil, gets control medicinal material solution with legal system;
Draw above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with the sodium carboxymethyl cellulose, with ethyl acetate-formic acid-water is developping agent, launches, and takes out, dry, spray is heated to clear spot with the vanillic aldehyde sulfuric acid solution, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Preferably, the compositions 10g that gets it filled adds water 50ml and makes dissolving, extracts 2 times for 60～90 ℃ with sherwood oil, each 40ml discards sherwood oil liquid, and water liquid extracts 2 times with ethyl acetate, each 30ml, merge ethyl acetate liquid, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution;
Other gets Asiatic plantain control medicinal material 1g, adds 70% ethanol 100ml, refluxes 2 hours, puts coldly, filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, extracts 2 times with 60～90 ℃ of sherwood oils and rises, and gets control medicinal material solution with legal system;
Draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with the sodium carboxymethyl cellulose, with ethyl acetate-formic acid of 6: 1: 1-water is developping agent, launches, and takes out, dry, spray is heated to clear spot at 105 ℃, in the test sample chromatogram with 5% vanillic aldehyde sulfuric acid solution, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
On the other hand, detection method also comprises chlorogenic acid (C 16H 18O 9) the step of assay, described assay adopts high effective liquid chromatography for measuring, condition determination is as follows:
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; Acetonitrile-0.5% phosphoric acid solution (11: 89) is a moving phase; The detection wavelength is 326nm, and number of theoretical plate calculates by the chlorogenic acid peak should be not less than 3000;
It is an amount of that the preparation precision of reference substance solution takes by weighing the chlorogenic acid reference substance, adds methyl alcohol and make the solution that every 1ml contains 60 μ g, promptly;
The about 1.0g of this product content under the content uniformity item is got in the preparation of need testing solution, the accurate title, decide, and puts in the 100ml round-bottomed flask, added the 50ml methanol eddy 2 hours, filter, with the about 20ml gradation washing dregs of a decoction of methyl alcohol and filter, merging filtrate, evaporate to dryness, residue add methyl alcohol make in right amount the dissolving and be transferred in the 10ml measuring bottle, add methyl alcohol to scale, shake up, promptly;
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
Preferably, wherein said Desmodium styracifolium principal ingredient is soyasaponin I, puriri glycosides-1, puriri glycosides-3, Xia Futa basket glycosides, described corn stigma principal ingredient is a fat oil, volatile oil, alkaloid, ascorbic acid, sitosterol, described pyrrosia lingua principal ingredient is the bracken triterpene, cupreol, Kaempferol, Quercetin, isoquercitrin, trifolin, described Canton love-pea vine principal ingredient is a triterpenes: sophoradiol, abrisapogenol A-G. glycyrrhizin, described Poria cocos principal ingredient is β-pachyman, the acetyl pachymic acid, pachymic acid, chitin, protein, choline, described Asiatic plantain principal ingredient is an aucubin, Homoplantaginin, ursolic acid, cupreol, described Herba lygodii principal ingredient is an amino acid, carbohydrate, flavonoid glycoside and phenols, described cogongrass rhizome principal ingredient is an arundoin, the Yin Baimao element, Isoarborinol alcohol.
Preferably, amount of water is 10～12 times of amounts of medicinal material weight in the step 3.
Preferred, amount of water is 10 times of amounts of medicinal material weight in the step 3.
Preferred, boiling secondary in the step 3, each 3 hours, collecting decoction filtered, and filtrate is concentrated into the clear cream that relative density is 1.20 (90 ℃).
Preferred, wherein the optimum weight number percent of each composition is:
Desmodium styracifolium 22.8%
Corn stigma 15.1%
Pyrrosia lingua 11.4%
Canton love-pea vine 11.4%
Poria cocos 11.4%
Asiatic plantain 8.25%
Herba lygodii 8.25%
Cogongrass rhizome 11.4%
Preparation technology of the present invention and characterization processes cost are low, and handling ease reduces the medicine heated time, is suitable in the widespread use clinical treatment.Adopt the pharmaceutical composition of the inventive method preparation, have broad-spectrum antiseptic, bacteriostasis, significant diuresis is arranged and regulate the effect of ureter smooth muscle.And have the effect of clearing heat and expelling damp, anti-inflammatory analgetic, diuresis and stone expeling, and can be used for treating illnesss such as calculi in urinary system and urinary tract infections, have diuresis simultaneously, make the urine dilution, can reduce crystal and form., urinary tract infections damp and hot for the wing skin, lithangiuria, renal colic etc. have good supplemental treatment effect.
Description of drawings
The canonical plotting that Fig. 1 chlorogenic acid is measured
Embodiment
Below, provide some specific embodiments that the present invention treats the preparation of pharmaceutical compositions of disease in the urological system.
Embodiment 1
Desmodium styracifolium 285g corn stigma 188.8g pyrrosia lingua 142.5g
Canton love-pea vine 142.5g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 15g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 2
Desmodium styracifolium 285g corn stigma 188.8g pyrrosia lingua 187.5g
Canton love-pea vine 97.5g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 20g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 3
Desmodium styracifolium 285g corn stigma 188.8g pyrrosia lingua 100g
Canton love-pea vine 260g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 10g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 4
Desmodium styracifolium 285g corn stigma 188.8g pyrrosia lingua 222.5g
Canton love-pea vine 62.5g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 23g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 5
Desmodium styracifolium 285g corn stigma 188.8g pyrrosia lingua 135g
Canton love-pea vine 142.5g Poria cocos 150g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 14g
[method for making] above eight flavor medicines are got Poria cocos 130g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 6
Desmodium styracifolium 312.5g corn stigma 188.8g pyrrosia lingua 115g
Canton love-pea vine 142.5g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 12g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 7
Desmodium styracifolium 312.5g corn stigma 62.5g pyrrosia lingua 268.8g
Canton love-pea vine 142.5g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 28g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 8
Desmodium styracifolium 162.5g corn stigma 188.8g pyrrosia lingua 158.8g
Canton love-pea vine 142.5g Poria cocos 142.5g Asiatic plantain 125g
Herba lygodii 87.5g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 16g
[method for making] above eight flavor medicines are got Poria cocos 133g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 9
Desmodium styracifolium 300g corn stigma 150g pyrrosia lingua 142.5g
Canton love-pea vine 142.5g Poria cocos 142.5g Asiatic plantain 105g
Herba lygodii 125g cogongrass rhizome 142.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 15g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 10
Desmodium styracifolium 291.3g corn stigma 225g pyrrosia lingua 142.5g
Canton love-pea vine 100g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 142.5g
Green former (the C of pyrrosia lingua 16H 18O 9) content 15g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 11
Desmodium styracifolium 300g corn stigma 218.8g pyrrosia lingua 162.5g
Canton love-pea vine 162.5g Poria cocos 100g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 100g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 17g
[method for making] above eight flavor medicines are got Poria cocos 86g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 12
Desmodium styracifolium 285g corn stigma 223.8g pyrrosia lingua 142.5g
Canton love-pea vine 142.5g Poria cocos 150g Asiatic plantain 103.1g
Herba lygodii 103.1g cogongrass rhizome 100g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 15g
[method for making] above eight flavor medicines are got Poria cocos 120g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
Embodiment 13
Desmodium styracifolium 285g corn stigma 188.8g pyrrosia lingua 112.5g
Canton love-pea vine 142.5g Poria cocos 142.5g Asiatic plantain 103.1g
Herba lygodii 113.1g cogongrass rhizome 162.5g
Pyrrosia lingua chlorogenic acid (C 16H 18O 9) content 11g
[method for making] above eight flavor medicines are got Poria cocos 95g and are ground into fine powder, and are standby; Residue Poria cocos and Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Asiatic plantain, Herba lygodii and cogongrass rhizome seven flavor medicine merge boiling twice, each 3 hours, merging filtrate, filter, filtrate is concentrated into the medicinal extract of 1.19-1.20 (90 ℃), adds the Poria cocos fine powder, mix, be dried to dried cream, be ground into fine powder, sieve, add appropriate amount of auxiliary materials, mixing, granulate drying, whole grain, incapsulate, make 1000, promptly.
The present invention treat the percentage by weight of each composition of pharmaceutical composition preferred implementation of disease in the urological system can following subordinate list 1 shown in:
Subordinate list 1:
Desmodium styracifolium 22.80％ 21.00％ 24.00％ 23.30％ 24.00％ 22.80％ 22.80％
Corn stigma 15.10％ 15.10％ 12.00％ 18.00％ 17.50％ 17.90％ 15.10％
Pyrrosia lingua 11.40％ 12.70％ 11.40％ 11.40％ 13.00％ 11.40％ 11.40％
Canton love-pea vine 11.40％ 11.40％ 11.40％ 8.00％ 13.00％ 11.40％ 11.40％
Poria cocos 11.40％ 11.40％ 11.40％ 11.40％ 8.00％ 12.00％ 11.40％
Asiatic plantain 8.25％ 10.00％ 8.40％ 8.25％ 8.25％ 8.25％ 7.00％
Herba lygodii 8.25％ 7.00％ 10.00％ 8.25％ 8.25％ 8.25％ 7.90％
Cogongrass rhizome 11.40％ 11.40％ 11.40％ 11.40％ 8.00％ 8.00％ 13.00％
In addition,, further provide the detection method of its component, provide some specific embodiments below in conjunction with the pharmaceutical composition of treatment disease in the urological system of the present invention, as follows:
Embodiment 14
Get this product 5g, add water 30ml and make dissolving, extract 1 time, each 30ml with sherwood oil (60～90 ℃), discard sherwood oil liquid, water liquid extracts 1 time with ethyl acetate, and each 30ml merges ethyl acetate liquid, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution.Other gets Asiatic plantain control medicinal material 1g, adds 70% ethanol 100ml, refluxes 2 hours, puts coldly, filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, and personal sherwood oil (60～90 ℃) extracts 2 times and rises, and gets control medicinal material solution with legal system.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with the sodium carboxymethyl cellulose, be developping agent with ethyl acetate-formic acid-water (6: 1: 1), launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, be heated to clear spot at 105 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Embodiment 15
Get this product 10g, add water 50ml and make dissolving, extract 2 times, each 40ml with sherwood oil (60～90 ℃), discard sherwood oil liquid, water liquid extracts 2 times with ethyl acetate, and each 30ml merges ethyl acetate liquid, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution.Other gets Asiatic plantain control medicinal material 1g, adds 70% ethanol 100ml, refluxes 2 hours, puts coldly, filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, and personal sherwood oil (60～90 ℃) extracts 2 times and rises, and gets control medicinal material solution with legal system.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with the sodium carboxymethyl cellulose, be developping agent with ethyl acetate-formic acid-water (6: 1: 1), launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, be heated to clear spot at 105 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Embodiment 16
Get this product 15g, add water 70ml and make dissolving, extract 3 times, each 50ml with sherwood oil (60～90 ℃), discard sherwood oil liquid, water liquid extracts 3 times with ethyl acetate, and each 40ml merges ethyl acetate liquid, evaporate to dryness, residue add ethanol 3ml makes dissolving, as need testing solution.Other gets Asiatic plantain control medicinal material 3g, adds 70% ethanol 300ml, refluxes 3 hours, puts coldly, filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, and personal sherwood oil (60～90 ℃) extracts 4 times, gets control medicinal material solution with legal system.Test according to thin-layered chromatography (an appendix VI of Chinese Pharmacopoeia version in 2000 B), draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with the sodium carboxymethyl cellulose, be developping agent with ethyl acetate-formic acid-water (6: 1: 1), launch, take out, dry, spray is with 5% vanillic aldehyde sulfuric acid solution, be heated to clear spot at 105 ℃, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
Simultaneously, the present invention also provides a kind of method of pyrrosia lingua medicinal material determination of chlorogenic acid and related assays result to show that embodiment is as follows:
1, instrument and reagent
American SS I PC2000 type high performance liquid chromatograph, 500 type UV, visible light detecting devices; The ShimpackC18 post (6.0 * 150mm), the Anastar chromatographic work station;
Germany's BP211D Sartorius electronic balance (100,000/);
The clear device of JL-120 type ultrasound wave;
Chlorogenic acid reference substance: provide lot number by Nat'l Pharmaceutical ﹠ Biological Products Control Institute: 110753-200212
Agents useful for same is analyzes pure or chromatographically pure.
2, the selection of extraction separation method
Physicochemical property according to chlorogenic acid, adopt methyl alcohol ultrasonic method and methanol eddy legal system to be equipped with need testing solution respectively, through injecting hplc determination, methyl alcohol content that ultrasonic method is surveyed is 0.148% as a result, circumfluence method is 0.265%, so select the methanol eddy legal system to be equipped with test liquid, carried out the tracking of return time simultaneously, the results are shown in following table.
The tracking of table 1 reflux extracting time
Figure G2009102240244D00121
By above-mentioned experimental result as can be known, refluxing extraction can be extracted chlorogenic acid fully in 4 hours substantially.
3, the preparation of need testing solution
Get the about 0.5g of this product meal, accurate claim surely, put in the 100ml round-bottomed flask, added the 50ml methanol eddy 4 hours, filter, the methanol solution water-bath volatilizes, and residue adds dissolve with methanol and constant volume in the 25ml measuring bottle, shakes up, promptly.
4, the preparation precision of reference substance solution takes by weighing chlorogenic acid reference substance 2.95mg, puts in the 50ml measuring bottle, adds methyl alcohol ultrasonic dissolution and constant volume in scale, shakes up, promptly.
5, the selection of chromatographic condition
Adopting acetonitrile-0.5% phosphoric acid solution (11: 89) is moving phase; The detection wavelength is 326nm, and flow velocity is 1.0ml/min, and column temperature is 30 ℃, under this chromatographic condition, chlorogenic acid peak energy and other peaks reach baseline separation in the test sample chromatogram, and peak shape is better, consistent with chlorogenic acid reference substance peak retention time, relevant HPLC chromatogram is seen Fig. 5.
6, chromatographic condition and system suitability test
The chromatographic condition octadecylsilane chemically bonded silica is a filling agent, and acetonitrile-0.5% phosphoric acid solution (11: 89) is a moving phase; The detection wavelength is 326nm, flow velocity: 1.0ml/min, column temperature: 30 ℃.
The accurate test liquid 10 μ l that draw of system suitability test inject liquid chromatograph, measure, and press the chlorogenic acid peak and calculate number of theoretical plate n=3014, tailing factor T=1.037, degree of separation R=5.329.
The accurate chlorogenic acid reference substance solution 10 μ l that draw of replica test inject liquid chromatograph, and continuous sample introduction 5 times calculates RSD, the results are shown in following table.
Table 2 replica test
Figure G2009102240244D00131
7, the investigation of the preparation of typical curve and linear relationship
Accurate reference substance solution (59 μ g/ml) 2.5,5.0,7.5,10.0, the 12.5 and 15 μ l that draw inject liquid chromatograph, measure, with the chlorogenic acid amount is horizontal ordinate, the chlorogenic acid peak area is an ordinate, drawing standard curve (see figure 1), experimental data returns through linearity, the typical curve equation is: Y=2048948.184X-28108.333r=0.99997 (n=6), chlorogenic acid reference substance amount is good linear relationship with its peak area in 0.1475 μ g～0.8850 μ g scope, the results are shown in following table.
The preparation of table 3 typical curve
Figure G2009102240244D00132
8, precision test
The accurate need testing solution 10 μ l that draw inject liquid chromatograph, repeat sample introduction 5 times, the results are shown in following table.
The test of table 4 precision
Figure G2009102240244D00133
9, stability test
Get need testing solution, the 1 hour sample introduction at room temperature every interval is once measured peak area, calculates RSD, and experiment shows that chlorogenic acid is basicly stable in 4 hours of being surveyed, and the results are shown in following table.
Table 5 stability test
Figure G2009102240244D00141
10, reappearance test
Get with 5 parts of batch medicinal materials, measure, the results are shown in following table by the method under the text assay item.
The test of table 6 reappearance
Figure G2009102240244D00142
11, average recovery test
Get 5 parts of this product meal (content is 0.274%) of known content, accurately claim surely, the accurate respectively chlorogenic acid reference substance that adds is an amount of, presses the method mensuration text assay item under, the results are shown in following table.
Table 7 average recovery test findings
Figure G2009102240244D00143
The clinical observation curative effect:
[0022] the calculus open capsule is a kind of Chinese patent drug that Desmodium styracifolium, pyrrosia lingua, Poria cocos, Asiatic plantain, Lygodium japonicum etc. are made, the clinical observation curative effect of its treatment calculi in urinary system;
[0023] therapeutic scheme: 180 examples are divided into treatment at random and organize 130 examples, oral calculus open capsule (producing) by Jiangxi Red Star pharmaceutcal corporation, Ltd, and lot number: (20060701), each 5, every day 3 times, oral.Control group 50 examples, each 5 of oral stranguria-treating and calculus-removing tablet (manufacturing medicine company limited in Zhanjiang produces), every day 3 times, oral.
[0024] course of treatment and other.20 is 1 course of treatment, and state of an illness no change continues to observe second course of treatment.Advise the patient to drink water more than 1500ml every day during the treatment, do jumping more.
[0025] observational technique: all objects of observation are all at treatment before and after look routine blood test, and routine urinalysis, liver, renal function, B ultrasonic, plain abdominal radiograph are observed drug side-effect such as indigestion and loss of appetite, abdominal distention and pain, n and V, dizzy, headache etc., and liver, kidney function damage.
[0026] criterion of therapeutical effect, cure: calculus is discharged, transference cure.B ultrasonic or KUB check calculus disappear; Take a turn for the better: doing well,improving, B ultrasonic or KUB check calculus is dwindled or the position moves down; Invalid: symptom and check B ultrasonic or KUB calculus do not have change.
[0027] result:
[0028] 12 group of curative effect relatively
Group ?n Cure Invalid Total effective rate %
The treatment group ?130 81 35 89.2
Control group ?50 26 12 76.0
Group is imitated relatively, P＜0.05
[0029] 22 group of symptom relatively
Figure G2009102240244D00151
Compare P＜0.05 with control group
[0030] 2 group of each position calculus curative effect relatively
Figure G2009102240244D00162
Compare P＜0.05 with control group
[0031] relatively by 2 groups of clinical test results, calculus open capsule treatment group cure rate 62.3%, total effective rate 89.2%, control group is respectively 52.0%, 76.0%, 2 group more all significant difference (P＜0.05), calculus open capsule, removing damp-heat is arranged, Tonglin Paishi, the significant curative effect of pain easing and hemostasis is simultaneously anti-infective and improve and significant curative effect is also arranged aspect the renal function.
[0032] calculus open capsule toxicological experiment is as follows:
[0033] sample is capsule from Jiangxi Red Star pharmaceutcal corporation, Ltd, and content is tan powder, every 0.35g, and the oral recommended amounts of human body is 12 of every days, becomes body weight for humans to press 60kg and calculates, and amounts to dosage 0.07g/kg.
[0034] acute toxicity test in mice adopts mtd test.Selecting body weight for use is 20 of the Kunming mouses of 18g～22g, male and female half and half.If dosage is 40000mg/kg.Get calculus open capsule 100g adding distil water to 100ml, get in this liquid 24 hours at interval and to irritate stomach 2 times to its mouse oral in 4 hours, irritate at every turn that body of stomach is long-pending to be 0.2ml/10g.Irritate the preceding fasting of stomach 16 hours first.Observed for two weeks record poisoning manifestations and death condition after irritating stomach continuously.
[0035] acute toxicity testing result
The calculus open capsule is to chmice acute toxicity
Figure G2009102240244D00171
According to above result, after irritating stomach for the Kunming mouse of two kinds of sexes, the calculus open capsule of producing with the Jiangxi Red Star medicine company of 40000mg/kg dosage do not see obvious toxicity symptom, observe and do not have death in 14 days.Animal subject put to death in the 15th day and dissect main organs such as inspection, liver,spleen,kidney, stomach, intestines, the heart, lung, show no obvious abnormalities change.To female, male Kunming mouse maximal tolerance dose (MTD) greater than 40000mg/kg.According to acute toxicity grading criteria, belong to nontoxic.
[0036] mouse sperm deformity test: get 25 of the male mice in kunming of body weight 25g～35g, be divided into 5 groups at random, 5 every group, with the positive contrast of the endoxan of 40mg/kg dosage, the negative contrast of distilled water.3 dosage of test group are respectively 10000mg/kg, 5000mg/kg, 2500mg/kg, get calculus open capsule 50.0g, 25g, 12.5g adding distil water to 100ml respectively, the test solution that is subjected to of preparation corresponding dosage is given mouse stomach (0.20ml/10g), irritate stomach every day once, for three days on end, Yu Weici irritates the 30th day execution animal behind the stomach, gets the epididymis smear, Yihong dyeing, the sperm of 1000 structural integrities of every animal counting calculates distortion spermatogenesis rate, presses x 2Inspection statistics is handled.
[0037] mouse sperm deformity test findings
The calculus open capsule is to the influence of mouse sperm deformity incidence
Group Number of animals (only) Examined sperm count (individual) Teratospermia number (individual) Rate of teratosperm (%)
High dose 5 5000 101 2.02
Middle dosage 5 5000 98 1.96
Low dosage 5 5000 108 2.16
Negative control 5 5000 94 1.88
Positive control 5 5000 504 10.08
By table as seen, sample does not produce obvious change to the mouse sperm deformity incidence, and each dosage group of sample and negative control group comparing difference do not have conspicuousness (P＞0.05) and endoxan positive controls and negative control group comparing difference have conspicuousness (P＜0.05) calculus open capsule that mouse sperm is not produced the distortion effect.
[0038] 30 day feeding trial: the dosage group selection with tried thing and given mode: get 80 of SD rats, male and female half and half, male mouse body weight is 85.31 ± 6.95g, female mouse 83.04 ± 6.83g.Animal used as test is divided into four groups at random, and promptly control group and three are tried the thing group, and 20 every group, male and female half and half.If the basic, normal, high dosage of calculus open capsule is respectively 3.00g/kg, 4.50g/kg, 6.00g/kg, basic, normal, high dosage is tried gets calculus open capsule 60g, 90g respectively when thing is prepared, the 120g adding distil water is settled to 200ml, control group gives equal-volume distilled water and irritates stomach once every day, irritate the long-pending 1.0ml/100g of being of body of stomach, continuous 30 days.
[0039] experimental technique, all animals of experiment periods give normal diet, single cage is raised, the drinking-water of freely ingesting, observe animal activity and growing state every day, appetite and surplus appetite given in record, claim body weight weekly one time, calculate food-intake weekly, food utilization and total food utilization, test not, fasting 14 hours, haemoglobin is measured in blood sampling, packed cell volume, carry out red blood cell, leucocyte, platelet count and leukocyte differential count are measured total protein behind the separation of serum, albumin, glutamic-pyruvic transaminase, glutamic-oxalacetic transaminease, urea nitrogen, the liver acid anhydride, cholesterol, triglyceride, blood sugar, zootomy is put to death in the blood sampling back, gets liver, kidney, spleen, stomach, duodenum, the section of ovary malingering.
[0040] experimental result, during the nursing in 30 days, each treated animal grows well, and no abnormal behavior and toxicity symptom do not have death the every index of rat are not produced obvious toxic-side effects.
[0041] pharmacy test of calculus open capsule is as follows
Modern pharmacological research is found, Desmodium styracifolium can make the litholysis in the urine, its polysaccharide component can suppress water-calcium oxalate crystal and increase, reduce the degree of crystal accumulation, chronic experiment clear-headed rabbit shows, Poria cocos tool diuresis, with ash content in contrast, prove that its diuresis is the effect of other compositions beyond the sylvite, and influence renal tubule Na +Heavily absorption relevant, the ethanol extract of Asiatic plantain has dose dependent ground and suppresses kidney Na +-K +-atpase activity and produce diuresis, Lygodium japonicum can be by increasing ureteral peristalsis and ureter epimere cavity pressure being increased, help that calculus moves down and the row's of generation masonry is used, zoopery shows that Lygodium japonicum also has diuresis, points out thus by last medicine to have row's stone diuresis as the calculus open capsule of Main Ingredients and Appearance.
[0042] observes the diuresis of calculus open capsule and arrange masonry usefulness by effect experiment
[0043] diuresis experiment
[0044] medicine: calculus open capsule (Jiangxi Red Star pharmaceutcal corporation, Ltd), hydrochlorothiazide tablets (Danyang City, Jiangxi pharmaceutcal corporation, Ltd), ethylene glycol, ammonium chloride all is Guangzhou Chemical Reagent Factory production
[0045] method: 28 of qualified rats are tested in the screening diuresis, body weight 150～200g, the fasting feedwater is after one day, be divided into 4 groups at random, each organizes mouse all by 2ml/100g body weight intraperitoneal injection of saline, and gently press belly to make the bladder emptying, positive controls gives 0.05% Hydrochioro aqueous solution, negative control group waits the physiological saline of capacity, the high dose group low dose group gives 9% and 3% calculus water flowing solution respectively, each group is all irritated stomach by 2ml/100, immediately rat is placed in the metabolic cage after the administration.Compare between each treated animal urine amount of collection and total volume of urine are organized after 1 hour, and take statistics is handled.
The calculus open capsule to the diuresis of mouse (X ± S, n=7)
Figure G2009102240244D00191
Compare 1 with the physiological saline group) P＜0.05 2) P＜0.01
[0046] suppresses kidney stone and form test
[0047] animal model preparation and medication: 54 of rats, be divided into 3 groups at random, freely drink the water liquid (containing 1.6g ethylene glycol and 0.8g ammonium chloride in every 1000ml aqueous solution) that contains ethylene glycol and ammonification ammonium for every day rat and fed continuously 30 days, to form calculus.Give rat oral gavage 1 time every day, irritating the stomach amount is the 2ml/100g body weight, and experiment is grouped as follows: (1) control group: physiological saline group; (2) low concentration medicine group: 2% calculus water flowing solution; (3) high concentration medicine group: 6% calculus water flowing solution
[0048] detect index and method: rat is put to death in the cervical vertebra dislocation, cuts open the belly and takes out two kidneys, weighs, and vertically cuts kidney open, observes to have or not obvious crystalline deposit thing and free calculus or calcified plaque as judging the calculus standard under dissecting microscope.Divide 5 grades " (-) " according to lithogenous what and kidney shape degree of injury, all visible calculus of cortex renis, medullary substance and renal papilla and crystalline deposit thing, as seen the no abnormal variation of kidney profile " (+) " cortex renis, medullary substance and renal plevis are dispersed in the crystalline deposit thing on a small quantity; The visible crystalline deposit thing of cortex renis, medullary substance and renal plevis or little free crystallization " (+++) " is arranged; Kidney portion and visible more crystallization also have free calculus to form, the visible calcified plaque of renal papilla, renal papilla expansion ponding; " ++ ++ "; Cortex renis, the visible bulk crystallization of medullary substance have free calculus to form, the visible calcified plaque of renal papilla, and renal plevis is obviously expanded ponding, and the gained enumeration data adopts Chi-square Test to carry out statistical study.
Calculus is logical to the lithogenous influence of kidney of rats
Figure G2009102240244D00201
The formation rate that found that administration group kidney of rats calculus all significantly reduces, and along with the rising effect of drug concentration is more obvious.
[0049] experiment shows: 3% calculus water flowing solution (being equivalent to clinical consumption) has tangible diuresis, can make the rat total volume of urine increase about 50%, and acting duration is longer, increase the logical consumption (3 times of amounts) of calculus and only occurring the diuresis peak after the medication between 1～2 hour, total volume of urine and 3% concentration group be there was no significant difference relatively also, may be that body fluid volume is limited, only can strengthen diuretic effect again when acquiring a certain degree by increasing the logical consumption of calculus when urinating.With the Hydrochioro control group relatively, the logical diuresis of calculus presents gentleness, lasting characteristics, the formation rate of administration group kidney of rats calculus all significantly reduces in the experiment of row's stone, and along with the rising effect of drug concentrations is more obvious.
[0050] to the cystolithic influence test of the rat opposite sex
[0051] gets 60 of male rats, body weight is 200～250g, be divided into 3 groups at random, every mouse all does foreign matter vesical calculus moulding: IP injection and defends barbital anesthesia, the sterilization wooden stick after will weighing of cutting open the belly is inserted in the bladder and is sewed up the bladder otch, lumbar injection Benzylpenicillin sodium salt 80,000 U are anti-infective when closing abdomen, and postoperative is normally fed.4 weeks beginning ig administration after the type selecting: experiment is grouped as follows, three groups are given and are subjected to reagent thing (production of Jiangxi Red Star medicine company), negative control group is given JIESHITONG PIAN (production of Guangxi Wuzhou Pharmaceutical incorporated company), and model control group is given distilled water, once a day, it is 0.60g/kg that dosage is tried three groups, 1.2g/kg, 2.4g/kg, negative control group is 1.2g/kg, the administration volume is 10ml/kg, continuous 30 days.Do not put to death rat next day after time administration, takes out the wooden stick of inserting, and weighs after 60 ℃ of oven dry in 24 hours, deducts original rod and heavily be foreign body of bladder calculus weight and ordinary water group relatively, and seven check between the work group, see Table.
To the cystolithic influence of the rat opposite sex
Group Dosage (g/kg) N Calculus weight (mg.x ± s)
Ordinary water 10ml 12 2.52±0.67
Calculus is logical 1.2 12 1.66±0.41 *
Be subjected to reagent 0.6 12 2.44±0.71
Be subjected to reagent 1.2 12 1.69±0.81
Be subjected to reagent 2.4 12 1.57±0.32 *
[0051] experimental result is found: the weight that heavy dose is subjected to reagent thing group to form calculus is significantly less than the ordinary water group, the difference significance, and prompting this product is brought out the rat bladder calculus to foreign matter certain inhibiting effect.
[0051] human body lithangiuria (external molten stone) test
[0052] will weigh in 1 branch harness of human body lithangiuria plug test tube of big or small basically identical, totally 40 manage, with being subjected to the reagent thing to be mixed with the soup of two kinds of concentration, do not prepare JIESHITONG PIAN contrast liquid, and establish the deionized water control group, the soup of every kind of concentration is established 10 pipes, being subjected to reagent liquid concentration is 88.0%, 20.0%, the logical concentration fan 100% of calculus, every pipe by calculus weight add a certain amount of soup (g: ml=1: 30) put 37 ℃ of constant temperature water-soluble in, each jolting 1 time (putting upside down several times).Soak and take out calculus after 7 days, weigh after 60 ℃ of 5h oven dry, calculate molten stone rate, soak solution is got supernatant in semiautomatic biochemistry analyzer mensuration calcium content after centrifugal, and relatively seven checks between the work group of deionized water group:
Group Concentration (%) Weight (mg) before soaking Soak back calcium content (mg) Molten stone rate
Be subjected to the reagent thing 88 79 1.172 2.5
Be subjected to the reagent thing 20 81 0.645 2.1
Calculus is logical 100 84 1.204 1.9
Water - 82 0.533 3.7
[0053] experimental result shows that low concentration is subjected to the molten stone rate of reagent thing close with deionized water, the difference nonsignificance, but the calcium content of high concentration soak solution is greater than deionized water, the difference significance, and this product has certain litholytic effect to the human body lithangiuria when high concentration.
[0054] influence of calculus open capsule Dichlorodiphenyl Acetate induced mice pain
Get 50 of mouse, be divided into 5 groups at random.The administration group is pressed 2.4g/kg, 1.2g/kg, 0.6g/kg dosage ig administration respectively, positive controls is given rotundin, the blank group is given ordinary water, every day, the ig administration was 1 time, the administration volume is 20ml/kg, successive administration 3 days, and the equal ip of each mouse of 1n injects 0.7% acetic acid 10ml/kg after the last administration, and write down behind each ip in mice injection acetic acid and to turn round body number and the relatively t check between the work group of ordinary water group in the 15min.
[0055] influences the result to what mouse acetic acid caused pain
Group n Dosage g/kg 15 minutes object condition numbers (x ± s) Inhibiting rate (%)
Control group 10 - 42.9±8.9
The rotundin group 10 80mg 21.6±6.9 xxx 49.7
Be subjected to the medicine group 10 2.4 27.0±5.9 xxx 37.0
Be subjected to the medicine group 10 1.2 30.7±7.7 xx 28.4
Be subjected to the medicine group 10 0.6 35.9±5.3 x 15.5
The result as seen, the logical Dichlorodiphenyl Acetate induced mice of calculus is ached significant reaction is arranged.
[0056] mouse hot plate piece is caused influence bitterly.
[0057] 50 of mouse are male, be divided into 5 groups at random, first group gives ig injection ordinary water, give rotundin for second group, its excess-three group is pressed 2.4g/kg, 1.2g/kg, 0.6g/kgig injection respectively, and the administration volume is 20ml/kg, measured the pain threshold values of each mouse respectively after the administration in 1,2,3 hour with method, the difference that deducts basic value with value behind the medicine as medicine after the increment of pain valve, with the ordinary water group relatively, do t check between group
[0058] to the result that influences of pain due to the mouse heat
Figure G2009102240244D00231
[0058] experimental result shows: be subjected to the increment of 3 hours pains of reagent thing heavy dose group administration valve greater than the ordinary water group, and the difference significance, prompting this product has certain analgesia valve action bitterly due to mouse heat.
[0059] dimethyl is caused the effect of the ear swelling of mouse.
[0060] gets 40 of mouse, body weight 20g～25g, male and female half and half are divided 4 groups at random, 10 every group, first group for being subjected to reagent 2.4g/kg, second group of reagent 1.2g/kg, the 3rd group of positive contrast aspirin, every day, gastric infusion was 1 time, continuous 3d, after last 1 administration 1 hour, evenly be coated with dimethyl 0.04ml at every mouse left side ear and cause inflammation, put to death animal behind the 30min, cut the mouse ears along auricle, respectively in the punching of left and right sides auricle same area, the both sides auricle of laying is weighed respectively, calculates swelling value and inhibitory rate of intumesce with the 6mm card punch, the swelling value is checked with t value and t ' value method, and compared between organizing.
Inhibitory rate of intumesce: 1 (%)=(EC-EC)/EC * 100%Et: the average swelling value of administration group, the average swelling rate of Ec control group
[0061] dimethyl is caused the result of mice ear effect
Group Dosage g/kg Swollen film value (mg) Inhibitory rate of intumesce (%)
Control group 20.4±4.7
Be subjected to the medicine group 2.4 14.8±5.3 * 27.5
Be subjected to the medicine group 1.2 17.6±3.9 13.7
Aspirin 0.2 14.5±5.5 * 28.9
Through comparing, be subjected to heavy dose of 2.4g/kg of reagent thing and aspirin 0.2g/kg all can obviously suppress the mice ear that causes by dimethyl with control group.
[0062] to the effect of mouse agar granuloma
[0063] get 40 of 200g ± 20g rat, male and female half and half are divided 4 groups at random, every group 10, under sterile working, carry out the dorsal line hypodermic injection, every mouse 2% agar solution 2ml, the gastric infusion that divides into groups then, first group is physiological saline, 2nd, 3 groups for being subjected to reagent 2.4g/kg, 1.2g/kg, and the 4th group is aspirin 0.2g/kg, each organizes equal every day of gastric infusion 1 time, and successive administration was put to death animal after 8 days, peel off granuloma, take by weighing the granuloma weight in wet base with electronic scales.The result is with comparing between t value method and the check of t ' value method and Construction Bank's group.
[0064] to mouse agar granulation effect test findings
Group Dosage (g/kg) Agar granuloma weight in wet base (g)
Control group ?1.99±0.32
Be subjected to the reagent group 2.4 ?1.45±0.56 x
Be subjected to the reagent group 1.2 ?1.59±0.43 x
Be subjected to the reagent group 0.2 ?14.2±0.50 xx
Compare p＜0.05, p＜0.01 with control group
[0065] extracorporeal bacteria inhibitor test, tube dilution method is adopted in this test, and experimental result is subjected to the reagent thing to beta hemolytic streptococcus, and the minimum inhibitory concentration of Diplococcus pneumopniae (MIC) is 25mg/ml, to staphylococcus aureus, the minimum inhibitory concentration of Pseudomonas aeruginosa (MIC) is 50mg/ml.

Claims (8)

1. detection method for the treatment of the components of pharmaceutical composition of disease in the urological system, the raw medicinal material of the pharmaceutical composition of wherein said treatment disease in the urological system is Desmodium styracifolium, corn stigma, pyrrosia lingua, Canton love-pea vine, Poria cocos, Asiatic plantain, Herba lygodii, cogongrass rhizome, and each medicinal material percentage by weight is as follows:
Desmodium styracifolium 21～24%
Corn stigma 12～18%
Pyrrosia lingua 9～13%
Canton love-pea vine 8～13%
Poria cocos 8～12%;
Asiatic plantain 7～10%
Herba lygodii 7～10%
Cogongrass rhizome 8～13%
And chlorogenic acid contents in the described pyrrosia lingua must not be less than 0.3% of general assembly (TW), and above-mentioned medicinal material is obtained described pharmaceutical composition by following method for making:
Step 1: get described Poria cocos and be divided into two parts, it is 80～90% that a copy of it accounts for the Poria cocos total weight percent, and it is 10～20% that another part accounts for the Poria cocos total weight percent;
Step 2: will account for the Poria cocos total weight percent and be 80～90% portion and be ground into fine powder;
Step 3: will remain Poria cocos and all the other seven flavor medicine material boilings 1～3 time, each 2～4 hours, collecting decoction filtered, and it is 1.19～1.20 clear cream that filtrate is condensed into 90 ℃ relative density;
Step 4: add the Poria cocos fine powder, mixing, drying is ground into fine powder, sieves, and adds appropriate amount of auxiliary materials, and mixing is granulated, drying, whole grain;
Described detection method comprises:
Compositions 5～15g gets it filled, add water 30～70ml and make dissolving, in 60～90 ℃ of extractions 1～3 time, each 30～50ml discards sherwood oil liquid with sherwood oil, water liquid extracts 1～3 time with ethyl acetate, each 20～40ml merges ethyl acetate liquid, evaporate to dryness, residue adds ethanol 1～3ml makes dissolving, as need testing solution;
Other gets Asiatic plantain control medicinal material 1～3g, adds 70% ethanol, 100～300ml, refluxes 2～3 hours, puts coldly, filters, and filtrate evaporate to dryness, residue add water 30～50ml makes dissolving, extracts 2～4 times for 60～90 ℃ with sherwood oil, gets control medicinal material solution with legal system;
Draw above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with the sodium carboxymethyl cellulose, with ethyl acetate-formic acid-water is developping agent, launches, and takes out, dry, spray is heated to clear spot with the vanillic aldehyde sulfuric acid solution, in the test sample chromatogram, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
2. detection method as claimed in claim 1,
The compositions 10g that gets it filled adds water 50ml and makes dissolving, extracts 2 times each 40ml for 60～90 ℃ with sherwood oil, discard sherwood oil liquid, water liquid extracts 2 times with ethyl acetate, and each 30ml merges ethyl acetate liquid, evaporate to dryness, residue add ethanol 1ml makes dissolving, as need testing solution;
Other gets Asiatic plantain control medicinal material 1g, adds 70% ethanol 100ml, refluxes 2 hours, puts coldly, filters, and filtrate evaporate to dryness, residue add water 30ml makes dissolving, extracts 2 times with 60～90 ℃ of sherwood oils and rises, and gets control medicinal material solution with legal system;
Draw each 5 μ l of above-mentioned two kinds of solution, put respectively in same be on the silica gel g thin-layer plate of bonding agent with the sodium carboxymethyl cellulose, with ethyl acetate-formic acid of 6: 1: 1-water is developping agent, launches, and takes out, dry, spray is heated to clear spot at 105 ℃, in the test sample chromatogram with 5% vanillic aldehyde sulfuric acid solution, with the corresponding position of control medicinal material chromatogram on, show the spot of same color.
3. detection method as claimed in claim 1 also comprises the step that chlorogenic acid contents is measured, and described assay adopts high effective liquid chromatography for measuring, and condition determination is as follows:
Chromatographic condition and system suitability test are filling agent with octadecylsilane chemically bonded silica; 11: 89 acetonitrile-0.5% phosphoric acid solution is a moving phase; The detection wavelength is 326nm, and number of theoretical plate calculates by the chlorogenic acid peak should be not less than 3000;
It is an amount of that the preparation precision of reference substance solution takes by weighing the chlorogenic acid reference substance, adds methyl alcohol and make the solution that every 1ml contains 60 μ g, promptly;
The about 1.0g of this product content under the content uniformity item is got in the preparation of need testing solution, the accurate title, decide, and puts in the 100ml round-bottomed flask, added the 50ml methanol eddy 2 hours, filter, with the about 20ml gradation washing dregs of a decoction of methyl alcohol and filter, merging filtrate, evaporate to dryness, residue add methyl alcohol make in right amount the dissolving and be transferred in the 10ml measuring bottle, add methyl alcohol to scale, shake up, promptly;
Accurate respectively reference substance solution and each the 10 μ l of need testing solution of drawing of determination method inject liquid chromatograph, measure, promptly.
4. detection method as claimed in claim 1, wherein said Desmodium styracifolium principal ingredient is soyasaponin I, puriri glycosides-1, puriri glycosides-3, Xia Futa basket glycosides, described corn stigma principal ingredient is a fat oil, volatile oil, alkaloid, ascorbic acid, sitosterol, described pyrrosia lingua principal ingredient is the bracken triterpene, cupreol, Kaempferol, Quercetin, isoquercitrin, trifolin, described Canton love-pea vine principal ingredient is a triterpenes: sophoradiol, abrisapogenol A-G. glycyrrhizin, described Poria cocos principal ingredient is β-pachyman, the acetyl pachymic acid, pachymic acid, chitin, protein, choline, described Asiatic plantain principal ingredient is an aucubin, Homoplantaginin, ursolic acid, cupreol, described Herba lygodii principal ingredient is an amino acid, carbohydrate, flavonoid glycoside and phenols, described cogongrass rhizome principal ingredient is an arundoin, the Yin Baimao element, Isoarborinol alcohol.
5. detection method as claimed in claim 1, amount of water is 10～12 times of amounts of medicinal material weight in the step 3.
6. detection method as claimed in claim 5, amount of water is 10 times of amounts of medicinal material weight in the step 3.
7. detection method as claimed in claim 1, boiling secondary in the step 3, each 3 hours, collecting decoction filtered, and it is 1.20 clear cream that filtrate is concentrated into 90 ℃ relative density.
8. detection method as claimed in claim 1, wherein the percentage by weight of each medicinal material is:
Desmodium styracifolium 22.8%
Corn stigma 15.1%
Pyrrosia lingua 11.4%
Canton love-pea vine 11.4%
Poria cocos 11.4%
Asiatic plantain 8.25%
Herba lygodii 8.25%
Cogongrass rhizome 11.4%
And chlorogenic acid contents in the described pyrrosia lingua must not be less than 1% of general assembly (TW).
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Denomination of invention: Method for detecting drug composition components for treating urinary system diseases

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