CN101066292A - Clerodendranthus spiatus extract for treating urinary calculus and its prepn process - Google Patents
Clerodendranthus spiatus extract for treating urinary calculus and its prepn process Download PDFInfo
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Abstract
The present invention discloses clerodendranthus spiatus extract for treating urinary calculus and its preparation process, and belongs to the field of Chinese herbal medicine extract and application technology. The clerodendranthus spiatus extract preparing process includes the following steps: soaking clerodendranthus spiatus in 4-10 times methanol to extract and concentrating to obtain extractum; dissolving the extractum in solvent in the same volume; extracting the solution first with petroleum ether and then with ethyl acetate and concentrating to obtain coarse extract; and chromatographic separation, eluting, collecting and concentrating to operate clerodendranthus spiatus extract A. The clerodendranthus spiatus extract A is liposoluble flavone glycoside compound and has obvious urinary calculus treating effects.
Description
[technical field]
The present invention relates to the extract and the applied technical field of Chinese herbal medicine, relate in particular to Clerodendranthus spiatus extract for treating urinary calculus and preparation method thereof.
[background technology]
Renal calculus, vesical calculus, lithangiuria etc. are the commonly encountered diseases of urinary system, and bibliographical information Europe is 5%～9%, and Canada is 12%, and the U.S. is about 13%, China's sickness rate about 9%～15%, and the sickness rate of area, Guangdong urinary calculi is up to 20%.Morbidity increased with the age, and danger also increases.Weather heat the area, have a liking for food animal proteinum crowd, especially 30～50 years old green grass or young crops of male, middle age, sickness rate is higher, men and women's ratio is 2～3: 1.In recent years Epidemiological study, its sickness rate is in rising trend, and especially young and middle-aged increase is more obvious, has had a strong impact on people's health.
Present urinary system calculus, especially renal calculus treatment main utilization extracorporeal shock-wave lithotripsy (ESWL) and the operation of intracavity Urology Surgery, often cause the damage and the severe complications of kidney, can make the nephridial tissue degeneration, produce glomerule vitreous degeneration and fibrosis, a matter hyperemia, inflammatory cell infiltration, and ESWL and operative treatment all are difficult to effect a radical cure renal calculus, its postoperative recurrence rate high about 78%～80%.How adopting the medicine noinvasive to get rid of calculus and prevent the formation and the recurrence of calculus, is the emphasis research topic in modern medicine field.And traditional Chinese medicine resource of China is urinary system medicine calculus, prevents calculus formation and recurrence to open up wide prospect.
Kidney tea is Labiatae kidney Camellia Clerodendranthus plant kidney tea C.spicatus (Thunb.) C.Y.Wu ex H.W.Li, has another name called Herba clerodendranthi spicati.Be perennial herb; Stem is upright, high 0.5～1m, four prismatics; Leaf is to life, the avette or ovum shape lanceolar of Pedicellus et Pericarpium Trapae shape, and long 3～10cm, wide 1.5cm～5cm, the thick tooth of leaf margin tool, the two sides is by hair, tool gland point, petiole reaches 1.2cm; 4～8 of wheel umbrella inflorescences are lined up the top and are given birth to raceme, the flower lip, and corolla is pale purple or white, 4 pieces on stamen, 2 long 2 is short, and filigree exceeds corolla 2～4cm; Pyrene is avette, the tool wrinkle; 5～October of flowering fruit bearing stage.Gas is light, and it is little sweet to distinguish the flavor of, and is bitter slightly.The kidney bitter edible plant is liked warm and humid climate, 19.6～23.8 ℃ of original producton location average temperatures of the whole year, and annual rainfall 1157.6～2103mm, the growth preference temperature is 26～30 ℃.
The kidney tea whole world has five kinds approximately, is distributed widely in ground such as India east, Thailand, Indonesia and Australia.At Indonesia's kidney tea among the people as a kind of active drug for the treatment of diabetes, hypertension, rheumatism, tonsillitis, menoxenia etc.; Kidney tea aerial parts is used to treat urinary system calculus, edema, febris acuta, influenza, hepatitis and jaundice etc. in Vietnam; Kidney tea is considered to a kind of antidiabetic drug in Burma, and the water decoction of cured leaf is used for the treatment of urethra and kidney disease; In Japanese Okinawa, kidney tea is known as Java tea can promote the health detoxifcation.It is kidney tea [C.spicatus (Thunb) .C.Y.Wu ex H.W.Li] that China only has a kind of, main product in Guangxi, ground such as Guangdong, Yunnan, Fujian, Taiwan.China's use kidney among the people tea is with a long history, in Xishuangbanna, and Dai Nationality's medicine title " refined Na is wonderful ".All herbal medicine, think diuresis is arranged, effect such as antibiotic, antiinflammatory, lithodialysis, calculus, be usually used in acute and chronic nephritis, cystitis, lithangiuria, pharyngitis and rheumatic arthritis.From clinical literature and medication follow-up investigation among the people, kidney tea can prevent and reduce the renal calculus recurrence, promotes lithangiuria to discharge, and improves patient's quality of life.
At present extract 14 flavone compounds from the aerial parts of kidney tea, 12 flavone aglycone, 2 is flavonol glycosides.8 liposoluble ingredients, i.e. caffeic acid, caffeinic dimer rosmarinic acid, caffeic acid and tartaric condensation substance caffeoyl tartrate and dicaffeoyl tartrate and four caffeic acid tetramers.Also have eupatorin, succinic acid, benzoic acid, lactic acid, daucosterol, ursolic acid, glucose, pentose, oils and fats, tartaric acid, limonene, Borneolum Syntheticum, thymol, despinning Inositol and an Ingredients Such As Coumarin in addition.
Kidney tea is all the time as medication among the people, and is of many uses, and there is obvious effect its diuresis, calculus aspect, get more and more people's extensive concerning, and the world of medicine has also carried out certain research to it.The clinical research that the Premgamone of Thailand etc. have carried out reaching 18 months to kidney tea shows that the effect of kidney tea treatment renal calculus is better than potassium citrate sodium.Clinical researches such as Huang Ronggui proof kidney tea has better curative effect to urinary system infection, treatment lithangiuria.Domestic and international in recent years chemical constituent and the bioactive research thereof to kidney tea mainly concentrates on the action face of antitumor, anti-inflammation, diuresis and the kidney invigorating, is that understandings such as what active chemical components plays a leading role, what mechanism of its effect is are very few and its diuresis at urinary system, calculus, antiinflammatory etc. are specifically used actually.Urinate the Application and Development that binds cubic meter of stone face in order to deepen kidney tea at excretion, we have done deep research to effect, the mechanism of action of kidney tea 3 kinds of extracts relevant with anti-urinary system calculus.
[summary of the invention]
Purpose of the present invention, be that to have not clear its effectively leading composition at present kidney tea in anti-urinary system calculus utilization be the defective of what and the mechanism of action thereof, in verifying clerodendranthus spicatus extract with calculus, oliguria, urinary tract infection and calculus the action effect and mechanism based thereof of urinary system to the symptom related substances such as damage of internal organs on, propose 3 kinds respectively effectively to the calculus of urinary system, calculus, diuresis, antiinflammatory, anti-oxidative damage etc., can be direct, compound or can be developed further into the kidney tea activity extract of medicinal product as intermediate products; Another object of the present invention is the concrete preparation method that proposes these 3 kinds of kidney tea activity extracts.
The object of the invention is achieved through the following technical solutions:
(1) clerodendranthus spicatus extract A component can make by the following method:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks 3 times, and each 10 days, merging, concentrated lixiviating solution got the extractum mixture;
(2) earlier water is mixed into lysate with methanol by 1: 5～12 volume ratios, again with step (1) extractum mixture and lysate by gram weight: ml volumes is 1: 1 mixed, is stirred to abundant dissolving;
(3) with step (2) extractum lysate earlier with petroleum ether extraction 4 times, fully remove low polar impurity component such as resin, chlorophyll after, reuse ethyl acetate extraction 4 times merges, concentrates extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and concentrate the thick clerodendranthus spicatus extract A component of pitchy.
Clerodendranthus spicatus extract A component is fat-soluble glycosides million compounds through identification and analysis.
Clerodendranthus spicatus extract A component has the obvious calculus symptom of improving, and the content of calcium and oxalic acid alleviates the renal tissue lesion degree in the minimizing renal tissue, reduces the calculus amount; Increase renal calculus person's urine amount, especially the application point urine amount increase when 0～4h is more remarkable; Increase the smooth muscle of bladder shrinkage amplitude, accelerate the rhythm and pace of moving things, loose ureter, urethral smooth muscle help the discharge to urinary system calculus; Antiinflammatory; Reduce renal calculus nephridial tissue LPO content to a certain extent, improvement is to the effects such as damage of nephridial tissue, can be direct, compound or as the further separation and Extraction drug of intermediate effective active components, be used for the medicine of urinary system calculus, calculus, diuresis and antiinflammatory etc.
The preparation method of described clerodendranthus spicatus extract A component, carry out according to the following steps:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks 3 times, and each 10 days, merging, concentrated lixiviating solution got the extractum mixture;
(2) earlier water is mixed into lysate with methanol by 1: 5～12 volume ratios, again with step (1) extractum mixture and lysate by gram weight: ml volumes is 1: 1 mixed, is stirred to abundant dissolving;
(3) with step (2) extractum lysate earlier with petroleum ether extraction 4 times, fully remove low polar impurity component such as resin, chlorophyll after, reuse ethyl acetate extraction 4 times merges, concentrates extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and concentrate clerodendranthus spicatus extract A component product.
(2) clerodendranthus spicatus extract B component can make by the following method:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks altogether 3 times, each 10 days, merges, concentrated lixiviating solution gets the extractum mixture;
(2) earlier water is mixed into lysate with methanol by 1: 5～12 volume ratios, again with step (1) extractum mixture and lysate by gram weight: ml volumes is 1: 1 mixed, is stirred to abundant dissolving;
(3) step (2) extractum lysate is extracted 3-5 time respectively with petroleum ether and ethyl acetate earlier, fully remove low polar impurity such as resin, chlorophyll and liposoluble constituent after, reuse n-butanol extraction 4 times, merging, concentrated extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and concentrate light brown thick clerodendranthus spicatus extract B component.
Clerodendranthus spicatus extract B component is fat-soluble flavonol glycoside compound through identification and analysis.
Clerodendranthus spicatus extract B component has the obvious calculus symptom of improving, and the content of calcium and oxalic acid alleviates the renal tissue lesion degree in the minimizing renal tissue, reduces the calculus amount; Increase renal calculus person's urine amount; Increase the smooth muscle of bladder shrinkage amplitude, accelerate the rhythm and pace of moving things, loose ureter, urethral smooth muscle help the discharge to urinary system calculus; Antiinflammatory; Reduce renal calculus nephridial tissue LPO content to a certain extent, improvement is to the effects such as damage of nephridial tissue, can be direct, compound or further isolate as intermediate and to extract medicinal effective active components, be used for the medicine of urinary system calculus, calculus, diuresis, antiinflammatory etc.
The preparation method of described clerodendranthus spicatus extract B component, carry out according to the following steps:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks altogether 3 times, each 10 days, merges, concentrated lixiviating solution gets the extractum mixture;
(2) earlier water is mixed into lysate with methanol by 1: 5～12 volume ratios, again with step (1) extractum mixture and lysate by gram weight: ml volumes is 1: 1 mixed, is stirred to abundant dissolving;
(3) step (2) extractum lysate is extracted 3-5 time respectively with petroleum ether and ethyl acetate earlier, fully remove low polar impurity such as resin, chlorophyll and liposoluble constituent after, reuse n-butanol extraction 4 times, merging, concentrated extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and concentrate clerodendranthus spicatus extract B component product.
(3) clerodendranthus spicatus extract C component can prepare by the following method:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks 2-3 after week, and the elimination methanol solution is to abundant volatile dry, and is standby;
(2) step (1) is handled metanephros tea, boil 3-4 time after the water submergence, each 4 hours, merge water cooking liquid, concentrate;
(3) with step (2) concentrated solution, be that 65%, 75%, 85%, 95% ethanol carries out class settling with concentration respectively, after the collection the thick clerodendranthus spicatus extract C component of black.
Clerodendranthus spicatus extract C component is a polysaccharide compound through identification and analysis.
Clerodendranthus spicatus extract C component has the obvious calculus symptom of improving, and the content of calcium and oxalic acid alleviates the renal tissue lesion degree in the minimizing renal tissue, reduces the calculus amount; Can prolong the diuretic time, obviously increase renal calculus person's urine amount; Increase the smooth muscle of bladder shrinkage amplitude, accelerate the rhythm and pace of moving things, loose ureter, urethral smooth muscle help the discharge to urinary system calculus; Obviously reduce renal calculus nephridial tissue LPO content, improvement is to the effects such as damage of nephridial tissue, can be direct, compound or as the further separation and Extraction drug of intermediate effective active components, be used for the medicine of urinary system calculus, calculus, diuresis, anti-renal calculus damage etc.
The preparation method of described clerodendranthus spicatus extract C component, carry out according to the following steps:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks 2-3 after week, and the elimination methanol solution is to abundant volatile dry, and is standby;
(2) step (1) is handled metanephros tea, boil 3-4 time after the water submergence, each 4 hours, merge water cooking liquid, concentrate;
(3) with step (2) concentrated solution, be that 65%, 75%, 85%, 95% ethanol carries out class settling with concentration respectively, after the collection clerodendranthus spicatus extract C component product.
The invention has the beneficial effects as follows:
One, clerodendranthus spicatus extract A component, clear and definite by animal experiment: in calcium oxalate renal calculus model test, calculus mice, rat are losing weight [1], the kidney body than the symptom that increases etc. contrast be improved significantly (see Table 1, table 4, table 5); Obviously minimizing (table 2, table 6) of contrast of the content of calcium and oxalic acid in the renal tissue; Renal calculus mice, rat kidney lesion tissue degree, nephridial tissue calcium oxalate stone scoring contrast obviously alleviates (table 3, table 7); The mensuration of above-mentioned nephridial tissue mesoxalic acid and calcium content is consistent with the result of nephridial tissue pathology section examination, shows the effect that clerodendranthus spicatus extract A component has anti-renal calculus.[2] in the Mus urine amount test, kidney tea A component is more remarkable to the application point urine amount increase of normal mouse when 0～4h, has tangible statistical significance (P＜0.01); And the urine amount of renal calculus mice is had increase, but compare not statistically significant (P＞0.05) (seeing Table 9) with model group; In addition, clerodendranthus spicatus extract A component can obviously increase the urine amount of large mouse with renal calculs, relatively has statistical significance (P＜0.05) (seeing Table 10) with model group.[3] in the test to animal urinary system smooth muscle, kidney tea A component has the increase shrinkage amplitude to Cavia porcellus, new zealand rabbit smooth muscle of bladder, accelerates the effect of the rhythm and pace of moving things; And ureter, urethral smooth muscle are all had relaxation effect (seeing Fig. 1, Fig. 2, Fig. 3), this helps the discharge to urinary system calculus.[4] in the antiinflammatory test to mice caused by dimethylbenzene xylene otitis, compare with matched group, kidney tea A component otitis swelling degree reduced the same with the indometacin group has tangible statistical significance (P＜0.01) (seeing Table 11), and tool is antiinflammation preferably.[5] in test to renal calculus mouse anti lipid peroxide, after mice forms renal calculus, nephridial tissue LPO content obviously rises, with matched group statistical significance (P＜0.05) is arranged relatively, the same nephridial tissue LPO content of renal calculus mice that makes with the PAISHI KELI group of kidney tea A component descends, but compares not statistically significant (P＞0.05) (seeing Table 12) with model group.Therefore, kidney tea A component can be direct, compound or as the further separation and Extraction drug of intermediate effective active components, is used for the medicine of urinary system calculus, calculus, diuresis and antiinflammatory etc.
Two, clerodendranthus spicatus extract B component, clear and definite by animal experiment: in calcium oxalate renal calculus model test, calculus mice, rat are losing weight [1], the kidney body than the symptom that increases etc. contrast be improved significantly (see Table 1, table 4, table 5); Obviously minimizing (table 2, table 6) of contrast of the content of calcium and oxalic acid in the renal tissue; Renal calculus mice, rat kidney lesion tissue degree, nephridial tissue calcium oxalate stone scoring contrast obviously alleviate (see Table 3, table 7); The mensuration of above-mentioned nephridial tissue mesoxalic acid and calcium content is consistent with the result of nephridial tissue pathology section examination, shows the anti-renal calculus effect of clerodendranthus spicatus extract B component tool.[2] in to the test of Mus urine amount, after mice formed renal calculus, the urine amount obviously descended, and kidney tea B component can increase the urine amount of renal calculus mice, but compared not statistically significant (P＞0.05) (seeing Table 9) with model group; Kidney tea B component can obviously increase the urine amount of large mouse with renal calculs, relatively has statistical significance (P＜0.05) (seeing Table 10) with model group.[3] in the test to animal urinary system smooth muscle, kidney tea B component has the increase shrinkage amplitude to Cavia porcellus, new zealand rabbit smooth muscle of bladder, accelerates the effect of the rhythm and pace of moving things; Ureter, urethral smooth muscle are all had relaxation effect (seeing Fig. 1, Fig. 2, Fig. 3), and this helps the discharge to urinary system calculus.[4] in antiinflammatory test to mice caused by dimethylbenzene xylene otitis, the obvious swelling of auris dextra after the control group mice caused by dimethylbenzene xylene inflammation, and kidney tea B component otitis swelling degree reduced the same with the indometacin group has tangible statistical significance (P＜0.01) (seeing Table 11), and tool is antiinflammation preferably.[5] in the test to renal calculus mouse anti lipid peroxide, after mice formed renal calculus, nephridial tissue LPO content obviously rose, and with matched group statistical significance (P＜0.05) is arranged relatively; The same nephridial tissue LPO content of renal calculus mice that makes with the PAISHI KELI group of kidney tea B component descends, but compares not statistically significant (P＞0.05) (seeing Table 12) with model group.Therefore, kidney tea B component can be direct, compound or as the further separation and Extraction drug of intermediate effective active components, is used for the medicine of urinary system calculus, calculus, diuresis, antiinflammatory etc.
Three, clerodendranthus spicatus extract C component, clear and definite by animal experiment: in calcium oxalate renal calculus model test, calculus mice, rat are losing weight [1], the kidney body than the symptom that increases etc. contrast be improved significantly (see Table 1, table 4, table 5); In the renal tissue content of calcium and oxalic acid contrast obviously reduce (see Table 2, table 6); Renal calculus mice, rat kidney lesion tissue degree, nephridial tissue calcium oxalate stone scoring contrast obviously alleviate (see Table 3, table 7); The mensuration of above-mentioned nephridial tissue mesoxalic acid and calcium content is consistent with the result of nephridial tissue pathology section examination, shows the anti-renal calculus effect of clerodendranthus spicatus extract C component tool.[2] in to the test of Mus urine amount, after mice formed renal calculus, the urine amount obviously descended, and kidney tea C component can increase the urine amount of renal calculus mice, but compared not statistically significant (P＞0.05) (seeing Table 9) with model group; But kidney tea C component can obviously increase the urine amount of large mouse with renal calculs, and its action time is longer, and sustainable 24 hours effective (seeing Table 10) has diuresis preferably.[3] in the test to animal urinary system smooth muscle, kidney tea C component all has the increase shrinkage amplitude to Cavia porcellus, new zealand rabbit smooth muscle of bladder, accelerates the effect of the rhythm and pace of moving things; Ureter, urethral smooth muscle are all had relaxation effect (seeing Fig. 1, Fig. 2, Fig. 3), and this helps the discharge to urinary system calculus.[4] in antiinflammatory test to mice caused by dimethylbenzene xylene otitis, the obvious swelling of auris dextra after the control group mice caused by dimethylbenzene xylene inflammation, the auricle swelling degree of kidney tea C component mice descends to some extent, but not obvious, compares not statistically significant (P＞0.05) (seeing Table 11) with matched group.[5] in test to renal calculus mouse anti lipid peroxide, after mice forms renal calculus, nephridial tissue LPO content obviously rises, with matched group statistical significance (P＜0.05) is arranged relatively, the same nephridial tissue LPO content of renal calculus mice that makes with the PAISHI KELI group of kidney tea C component obviously descends, relatively has statistical significance (P＜0.05) (seeing Table 12) with model group, to renal calculus anti-peroxidation lipid tool better action.Therefore, kidney tea C component can be direct, compound or as the further separation and Extraction drug of intermediate effective active components, is used for the medicine of urinary system calculus, calculus, diuresis, anti-renal calculus damage etc.
Four, clerodendranthus spicatus extract A, B, C component only are single extracts, and the calculus symptom that can therefrom select to use separately or be used as medicine and treat and eliminate urinary system with different combinations according to different demands is as calculus, calculus, diuresis, antiinflammatory, anti-damage etc.
[description of drawings]
Fig. 1 clerodendranthus spicatus extract A, B, C component are to the effect curves figure of Cavia porcellus smooth muscle of bladder;
Fig. 2 clerodendranthus spicatus extract A, B, C component are to the effect curves figure of Cavia porcellus ureter smooth muscle;
Fig. 3 clerodendranthus spicatus extract A, B, C component are to the effect curves figure of Cavia porcellus urethral smooth muscle.
[specific embodiment]
By following examples and test example the present invention is described in further detail, but this is not to concrete qualification of the present invention.
The material relevant with following examples, equipment etc. explain as follows:
Kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li]
Lysate: water and methanol form by 1: 5～12 volume mixture
Petroleum ether: lot number 20030610, Zhejiang chemical reagent work of Juhua Group Co. produces
Ethyl acetate: lot number 20031009, Zhejiang chemical reagent work of Juhua Group Co. produces
N-butyl alcohol: lot number 20021217, Zhejiang chemical reagent work of Juhua Group Co. produces
Methanol: lot number 20021201, Zhejiang chemical reagent work of Juhua Group Co. produces
Ethanol (95%): lot number 2003042012, the special wine head factory in Anhui is produced
Column chromatography silica gel: Haiyang Chemical Plant, Qingdao produces
Rotary Evaporators: the R203B type, Shensheng Science ﹠ Tech. Co., Ltd., Shanghai produces
Adjuvant: Semen Plantaginis, Lignum Aquilariae Resinatum, the Radix Paeoniae Alba, Endothelium Corneum Gigeriae Galli are respectively Plantago asiatica L. (Semen Plantaginis), Aquilaria sinensis (Lour.) Gilg (Lignum Aquilariae Resinatum), Paeonialactiflora Pall. (Radix Paeoniae Alba), Gallus gallus domesticus Brisson (Endothelium Corneum Gigeriae Galli) all available from shop of Chinese medicines, Wenzhou through evaluation.
Concentrate: comprise the lixiviating solution, extract and the water cooking liquid that relate to, all adopting under 40-60 ℃ of water temperature control decompression that moisture is fully distilled and concentrating (except the special instruction).
The preparation 1 of embodiment 1:(clerodendranthus spicatus extract A component)
The preparation of described clerodendranthus spicatus extract A component, carry out according to the following steps:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 4, soak altogether 3 times, each 10 days, merge lixiviating solution after distilling under reduced pressure concentrates under 60 ℃ of water temperature controls, get the extractum mixture;
(2) with step (1) extractum mixture and water-methanol by the lysate of 1: 5 volume mixture (g) by weight: volume (ml)=1: 1 mixed, be stirred to abundant dissolving;
(3) step (2) extractum lysate is used petroleum ether extraction 4 times earlier, fully remove low polar impurity component such as resin, chlorophyll, reuse ethyl acetate extraction 4 times merges, concentrates extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and after distilling under reduced pressure concentrates under 60 ℃ of water temperatures controls the thick clerodendranthus spicatus extract A component of pitchy.
The preparation 2 of embodiment 2:(clerodendranthus spicatus extract A component)
In this preparation example, described kidney tea dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 7 is soaked; Merging lixiviating solution distilling under reduced pressure under 50 ℃ of water temperature controls concentrates; Water-methanol becomes lysate by 1: 9 volume mixture; The distilling under reduced pressure under 50 ℃ of water temperature controls of chromatography, eluting, collection liquid concentrates, and all the other preparation processes and process conditions all are same as embodiment 1.
The preparation 3 of embodiment 3:(clerodendranthus spicatus extract A component)
In this preparation example, described kidney tea dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 10 is soaked; Merging lixiviating solution distilling under reduced pressure under 40 ℃ of water temperature controls concentrates; Water-methanol becomes lysate by 1: 12 volume mixture; The distilling under reduced pressure under 40 ℃ of water temperature controls of chromatography, eluting, collection liquid concentrates, and all the other preparation processes and process conditions all are same as embodiment 1.
Embodiment 4:(kidney tea Sal Nitri soup A)
With Semen Plantaginis 30 grams, Lignum Aquilariae Resinatum 3 grams, the Radix Paeoniae Alba 10 grams, Endothelium Corneum Gigeriae Galli 15 grams, add water 1000ml and soak half an hour, decoct and poured out decoct in 15 minutes, decoct again 1 time with method, merge decoct twice, after complementing to 1200ml with sterilized water, add prepared clerodendranthus spicatus extract A components 1 gram of embodiment 1,2 or 3, be stirred to the dissolving back kidney tea Sal Nitri soup A.1 dose of every day, take medicine and carried out jumping exercise 15 minutes in back 20 minutes, and drink water as much as possible, urinate more, three months is a course of treatment, tool calculus, calculus lithodialysis, diuresis, anti-inflammatory analgetic effect.
The preparation 1 of embodiment 5:(clerodendranthus spicatus extract B component)
The preparation of described clerodendranthus spicatus extract B component, carry out according to the following steps:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 10, soak altogether 3 times, each 10 days, merge lixiviating solution after distilling under reduced pressure concentrates under 40 ℃ of water temperature controls, get the extractum mixture;
(2) with the lysate of step (1) extractum mixture and water-methanol (by 1: 5 volume mixture) (g) by weight: volume (ml)=1: 1 mixed, be stirred to abundant dissolving;
(3) step (2) extractum lysate is extracted respectively 3 times with petroleum ether and ethyl acetate, fully remove low polar impurity such as resin, chlorophyll and liposoluble constituent, the reuse n-butyl alcohol fully extracts 4 times, merges to concentrate extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and after distilling under reduced pressure concentrates under 40 ℃ of water temperatures controls light brown thick clerodendranthus spicatus extract B component.
The preparation 2 of embodiment 6:(clerodendranthus spicatus extract B component)
In this preparation example, described kidney tea dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 8 is soaked; Merging lixiviating solution distilling under reduced pressure under 60 ℃ of water temperature controls concentrates; Water-methanol becomes lysate by 1: 9 volume mixture; Petroleum ether extraction extractum lysate 4 times; Ethyl acetate extraction extractum lysate 4 times; The distilling under reduced pressure under 50 ℃ of water temperature controls of chromatography, eluting, collection liquid concentrates, and all the other preparation processes and process conditions all are same as embodiment 5.
The preparation 3 of embodiment 7:(clerodendranthus spicatus extract B component)
In this preparation example, described kidney tea dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 5 is soaked; Merging lixiviating solution distilling under reduced pressure under 50 ℃ of water temperature controls concentrates; Water-methanol becomes lysate by 1: 12 volume mixture; Petroleum ether extraction extractum lysate 5 times; Ethyl acetate extraction extractum lysate 5 times; The distilling under reduced pressure under 60 ℃ of water temperature controls of chromatography, eluting, collection liquid concentrates, and all the other preparation processes and process conditions all are same as embodiment 5.
Embodiment 8:(kidney tea Sal Nitri soup B)
With Semen Plantaginis 30 grams, Lignum Aquilariae Resinatum 3 grams, the Radix Paeoniae Alba 10 grams, Endothelium Corneum Gigeriae Galli 15 grams, add water 1000ml and soak half an hour, decoct and poured out decoct in 15 minutes, decoct again 1 time with method, merge decoct twice, after complementing to 1200ml with sterilized water, add prepared clerodendranthus spicatus extract B components 0.5 gram of embodiment 5,6 or 7, be stirred to the dissolving back kidney tea Sal Nitri soup B.1 dose of every day, take medicine and carried out jumping exercise 15 minutes in back 20 minutes, and drink water as much as possible, urinate more, three months is a course of treatment, tool calculus, calculus lithodialysis, diuresis, anti-inflammatory analgetic effect.
The preparation 1 of embodiment 9:(clerodendranthus spicatus extract C component)
The preparation of described clerodendranthus spicatus extract C component, carry out according to the following steps:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 5, soak 2-3 after week, the elimination methanol solution is to abundant volatile dry, and is standby;
(2) step (1) is handled metanephros tea, boil after the water submergence 3 times, each 4 hours, merge water cooking liquid, distilling under reduced pressure concentrates under 50 ℃ of water temperature controls;
(3) with step (2) concentrated solution, be that 65%, 75%, 85%, 95% ethanol carries out class settling with concentration respectively, after the collection the thick clerodendranthus spicatus extract C component of black.
The preparation 2 of embodiment 10:(clerodendranthus spicatus extract C component)
In this preparation example, described kidney tea dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 8 is soaked; Kidney tea behind the extremely abundant volatile dry of elimination methanol solution boils after the water submergence 4 times; Distilling under reduced pressure concentrates under 60 ℃ of water temperature controls, and all the other preparation processes and process conditions all are same as embodiment 9.
The preparation 3 of embodiment 11:(clerodendranthus spicatus extract C component)
In this preparation example, described kidney tea dry product is pulverized back and methanol (g) by weight: the ratio of volume (ml)=1: 10 is soaked; Kidney tea behind the extremely abundant volatile dry of elimination methanol solution boils after the water submergence 4 times; Distilling under reduced pressure concentrates under 40 ℃ of water temperature controls, and all the other preparation processes and process conditions all are same as embodiment 9.
Embodiment 12:(kidney tea Sal Nitri soup C)
With Semen Plantaginis 30 grams, Lignum Aquilariae Resinatum 3 grams, the Radix Paeoniae Alba 10 grams, Endothelium Corneum Gigeriae Galli 15 grams, add water 1000ml and soak half an hour, decoct and poured out decoct in 15 minutes, decoct again 1 time with method, merge decoct twice, after complementing to 1200ml with sterilized water, add prepared clerodendranthus spicatus extract C components 6 grams of embodiment 9,10 or 11, be stirred to the dissolving back kidney tea Sal Nitri soup C.1 dose of every day, take medicine and carried out jumping exercise 15 minutes in back 20 minutes, and drink water as much as possible, urinate more, three months is a course of treatment, tool inducing diuresis for treating stranguria syndrome, anti-inflammatory analgetic, anti-renal calculus damage effect.
The material relevant with following test example, equipment etc. explain as follows:
1 experiment material
1.1 animal
The ICR mice, the SD rat is provided by Zhejiang Province's Experimental Animal Center, laboratory animal certification of fitness number: SCXK (Zhejiang) 2003-0001.New zealand rabbit, DPH Cavia porcellus, laboratory animal certification of fitness number: SCXK (Zhejiang) 2003-0002.Provide by Zhejiang Province's Experimental Animal Center.The raising condition: 18 ℃～20 ℃ of room temperatures, relative humidity 40%～70% is fed with the full nutrition pellet, freely drinks water.
1.2 medicine
Clerodendranthus spicatus extract A component (embodiment 1,2 or 3 products): it is an amount of to take by weighing kidney A, and milling with 1% sodium carboxymethyl cellulose (CMC-Na) evenly is made into desired concn, standby.(mice working concentration 6.4mg/ml irritates the long-pending 0.5ml/20g of body of stomach, and dosage is 160mg/kg; Rat working concentration 16mg/ml irritates the long-pending 1ml/100g of body of stomach, and dosage is 160mg/kg.Down together.)
Clerodendranthus spicatus extract B component (embodiment 5,6 or 7 products): it is an amount of to take by weighing kidney B, and milling with distilled water evenly is made into desired concn, standby.
Clerodendranthus spicatus extract C component (embodiment 9,10 or 11 products): it is an amount of to take by weighing kidney C, and milling with distilled water evenly is made into desired concn, standby.
Indomethacin (indometacin): crude drug, Ningbo Medicine Mfg. Factory is so kind as to give, and facing mills in order to 1% sodium carboxymethyl cellulose (CMC-Na) evenly is made into desired concn, standby.
FUSAIMI PIAN (furosemide): the 20mg/ sheet, lot number 0311131, Jiangsu vast stretch of wooded country pharmaceutcal corporation, Ltd makes branch company, and (CMC-Na) is made into desired concn with 1% sodium carboxymethyl cellulose, and be standby.
PAISHI KELI: the 20g/ bag, lot number 050109, Nanjing Tongrentang Pharmaceutical Co., Ltd., milling with distilled water evenly is made into desired concn, standby.
1.3 reagent
Normal saline, lot number 20040115, Shapuaisi Pharmaceutical Co., Ltd., Pinghu City, Zhejiang Prov..
Ethylene glycol, lot number 990332, Yuhang city south of a city chemical reagent factory.
Ammonium chloride, lot number 20030603, Quzhou hugeization reagent company limited.
Calcium determination test box (methylthymol blue method): lot number 030927, kind city, Ningbo City biochemical reagents factory.
Tyrode's solution (autogamy): dissolve 8g NaCl, 2ml10%KCl, 2.6ml10%MgSO successively
47H
2O, 1g NaHCO
3In the 500ml distilled water (reagent I), 1.8ml 1mol/L CaCl
2Be dissolved in 200ml distilled water (reagent II) separately, slowly reagent II added among the reagent I, the limit edged stirs, and is settled to 1000ml (reagent III).The 1g glucose is tyrode's solution in facing time spent adding reagent III.
Malonaldehyde MDA measures test kit, lot number 20040219, and bio-engineering research institute is built up in Nanjing.
Other reagent is analytical pure.
1.4 instrument
721 type spectrophotometers, Shanghai the 3rd analytical tool factory.
Centrifuge, LXJ-II, Shanghai medical analytical instrument factory.
Electronic analytical balance, AEL-200, Tianjin, island.
Medlab-U/4cs bio signal acquisition processing system, Meiyi Science ﹠ Technology Co., Ltd., Nanjing.
SC-15 numerical control super constant temperature trough, the permanent instrument plant in sky, Ningbo.
The Motic microscopic image processing system, Maike Aodi Industry Group Co Ltd.
Test the mensuration of routine 1:(kidney mesoxalic acid and calcium content)
Carry out according to the following steps:
1) nephridial tissue is handled: get for examination animal one side nephridial tissue and add 1M HCl, make tissue homogenate with homogenizer, the centrifugal 10min of 3000r/min gets supernatant 2ml and transfers pH to 5.0 with the 1M sodium hydroxide, replenishes deionized water to 5ml, shakes up;
2) kidney mesoxalic acid Determination on content (potassium permanganate fade method): get 200 μ l step 1) nephridial tissue treatment fluids, add 500mM calcium chloride 0.1ml mixing, add the abundant mixing of dehydrated alcohol 2ml, 4 ℃ of placements are spent the night, the centrifugal 10min of 3000r/min adds the abundant mixing of 0.02M acetic acid one calcium oxalate balance liquid 5ml after abandoning supernatant.The centrifugal 10min of 3000r/min abandons supernatant.Add deionized water 5ml, abundant mixing, the centrifugal 10min of 3000r/min abandons supernatant.Test tube is inverted on the filter paper and drains away the water, and precipitation adds 1mol/L sulphuric acid 1ml and the abundant mixing of deionized water 1ml, dissolution precipitation thing.Add potassium permanganate and use liquid 3.0ml mixing, 60 ℃ of water-bath 10min, the centrifugal 10min of 3000r/min.Get supernatant 525nm colorimetric.Do simultaneously blank and standard test pipe (standard pipe oxalic acid 1mM, 1ml).Calculate by following formula:
Measure pipe concentration of oxalic acid=(blank pipe A-measures pipe A)/(blank pipe A-standard pipe A) * 1
3) mensuration of calcium content (methylthymol blue method) in the kidney: other gets kidney treatment fluid 50 μ l, adds the 3ml developer, mixing, make blank and standard pipe (standard calcium 2.5mM) simultaneously, use the 610nm wavelength, blank pipe zeroing, read mensuration pipe and standard pipe and divide absorbance (A), calculate by following formula:
Measure pipe calcium concentration=mensuration pipe A/ standard pipe A * 2.5
Test routine 2:(kidney stone pathological study)
Collect the 6h urine for water load method behind the examination animal last medicine.Put to death animal, after kidney is weighed, get a side kidney 10% formaldehyde fixed, conventional section is carried out the kidney stone pathological study after the HE dyeing.Wherein, pathological study kidney stone standards of grading are: 0 minute: no crystallization; 1 minute: local or be dispersed in and arrive the sand-like crystallization of volume less; 2 minutes: there was single tiny crystallization the part; 3 minutes: there was the tiny crystallization of volume the part or 1 large-particle monocrystal is arranged; 4 minutes: only have 3～4 large-particle monocrystals to be dispersed in or the tiny crystallization of volume; 5 minutes: the part had 3～4 place's volume maximum size productive sets in heaps; 6 minutes: full visual field volume maximum size productive set was in heaps.
Test of the influence of routine 3:(clerodendranthus spicatus extract) to mice calcium oxalate renal calculus model
Get 66 of mices, be divided into 6 groups at random, 11 every group, except that control group mice is drunk the normal water every day, other each group is all drunk into stony edema (1% ethylene glycol+1% ammonium chloride), drinks for 6 weeks continuously, causes mice renal calculus model.Following dosed administration is pressed in modeling each group simultaneously.1. matched group, ig water; 2. model group, ig water; 3. PAISHI KELI group, ig PAISHI KELI 6g/kg; 4. kidney tea A organizes, ig kidney tea A solution 160mg/kg; 5. kidney tea B organizes, ig kidney tea B solution 160mg/kg; 6. kidney tea C organizes, ig kidney tea C solution 160mg/kg; Administration every day 1 time, administration volume 0.5ml/20g, continuous 6 weeks.
Water load method is collected the 6h urine behind the last medicine, puts to death animal, and after kidney was weighed, a side kidney was measured oxalic acid and calcium content in the nephridial tissue by test example 1 method; The opposite side kidney carries out the kidney stone pathological study by test example 2 methods, and the result is as follows:
(1) ordinary circumstance: the renal calculus composition accounts for 80%～84% of whole lithangiuriaes based on calcium oxalate and calcium phosphate calculus, and wherein calcium oxalate stone accounts for 58.8%.Ethylene glycol is the precursor of oxalic acid, change into glycolic after entering in the body, the latter both can be converted into oxalic acid under the oxidasic effect of glycolic, also can be by the glyoxalic acid that is catalytically converted into of lactic acid dehydrogenase, and glyoxalic acid can directly be converted into oxalic acid again under enzyme or non-enzymatic catalysis.Therefore, finally be converted into oxalic acid after entering internal metabolism for feeding animal ethylene glycol, discharge through urine, increase the concentration of urine mesoxalic acid, add ammonium chloride, the calculus formation rate is high in the kidney.Experimental result shows, adds 1% ethylene glycol+1% ammonium chloride in drinking-water, can form ideal renal calculus model for experimentation.Mice becomes stony edema after 4 weeks, lethargy, and fear of cold is crispaturaed, and chaeta is withered, tarnish, the hair color flavescence is and alarms the hair shape, and it is more obvious to lose weight, and kidney body ratio is obvious to be increased, and 6 weeks, inner branch animal was because of the serious pathological changes death of kidney.And each group of PAISHI KELI group and the administration of kidney tea is compared with model group, and symptom makes moderate progress.The results are shown in Table 1.
Table 1 kidney tea is to the influence of renal calculus mice body weight and kidney weight (x ± s)
| Group | Number of animals | Dosage (mg/kg) | Mortality rate (%) | Body weight (g) | The kidney body is than (mg/g) |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | 11 11 11 11 11 11 | - - 6000 160 160 160 | 0 64 18 27 0 36 | 40.0±3.4 29.3±5.1 *** 32.3±2.6 34.0±4.1 33.6±4.1 32.4±2.6 | 17.2±2.8 24.2±4.5 *** 20.8±4.6 23.1±4.5 20.7±3.4 21.6±4.6 |
Annotate: compare with matched group
* *P＜0.001
(2) kidney tea is to the influence of renal calculus mouse kidney calcium and oxalic acid content: after mice was drunk 1% ethylene glycol and 1% ammonia chloride, calcium oxalate crystal was built up in kidney in a large number, and experiment shows that calcium, oxalic acid content significantly increase in the nephridial tissue.And kidney calcium, the kidney oxalic acid content of kidney A, B, C group obviously are less than model group, and wherein, the kidney oxalic acid content and the model group of kidney A group relatively have evident difference (P＜0.001), even obviously are better than the PAISHI KELI group.The results are shown in Table 2.
Table 2 kidney tea is to the influence of renal calculus mouse kidney calcium and oxalic acid content (x ± s)
| Group | Number of animals | Dosage (mg/ kg) | Kidney calcium content (mg/g) | Kidney oxalic acid content (μ g/g) |
| Control group model group PAISHI KELI group kidney A group kidney B group kidney C group | 11 11 11 11 11 11 | - - 6000 160 160 160 | 4.36±1.66 10.89±1.02### 4.32±3.50 *** 5.89±2.11 *** 6.35±1.98 *** 8.14±3.50 * | 1.43±0.80 3.66±0.82### 2.10±0.94 *** 1.66±0.60 *** 2.09±0.92 *** 2.01±0.94 *** |
Annotate: compare ###P＜0.001 with matched group; Compare with model group
* *P＜0.001,
*P＜0.05
(3) nephridial tissue morphology and nephridial tissue calcium oxalate crystal are observed: the renal tissue perusal, and model group animal kidney edema, the surface is pale, the quality hardening, as seen the kidney surface is dispersed in white point.And that PAISHI KELI group and kidney tea A, B, C respectively organize symptom is lighter, and table is and ruddy, and quality is normal.The pathological section histological observation, model group animal kidney lesion degree is more serious, has a large amount of calcium oxalate stones in renal cortex portion, marrow, renal calices, the renal pelvis, and the in heaps or full visual field of severe patient volume maximum size productive set volume maximum size productive set is in heaps.With model group relatively, PAISHI KELI group and kidney tea A, that B, C respectively organize the nephridial tissue lesion degree is lighter, the scoring of nephridial tissue calcium oxalate stone is lower, showing has anti-renal calculus effect.The results are shown in Table 3.
The influence that table 3 kidney tea forms renal calculus mouse kidney calcium oxalate stone (n=11, x ± s)
| Group | Dosage (mg/kg) | Calculus rate % | Renal cortex portion | Shen Marrow matter portion | The renal calices renal pelvis | Grand mean |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | - - 6000 160 160 160 | 0 100(11/11) 55(6/11) 64(7/11) 55(6/11) 64(7/11) | 0.6±0.6 2.2±2.3 0.9±1.8 0.7±1.4 * 0.5±0.6 ** 0.5±0.9 * | 0.3±0.5 4.7±1.3 1.7±2.0 *** 2.4±2.1 *** 1.4±1.6 * 2.7±1.9 ** | 0±0 3.0±2.3 0.3±1.0 *** 0.1±0.5 *** 0.1±0.25 *** 0.4±1.1 *** | 0.9±0.8 9.9±4.2 2.8±3.3 * 3.3±2.9 *** 2±1.9 *** 3.6±2.8 *** |
Annotate: compare with model group
* *P＜0.001,
*P＜0.01,
*P＜0.05
Test of the influence of routine 4:(clerodendranthus spicatus extract) to rat calcium oxalate renal calculus model
Get 30 of male rats, be divided into 6 groups at random, 5 every group.Except that control rats is drunk the normal water every day, other each group is all drunk into stony edema (1% ethylene glycol+1% ammonium chloride), drinks continuously 10 days, causes kidney of rats calculus model.Following dosed administration is pressed in modeling each group simultaneously.1. matched group, ig water; 2. model group, ig water; 3. PAISHI KELI group, ig PAISHI KELI 6g/kg; 4. kidney tea A organizes, ig kidney tea A solution 160mg/kg; 5. kidney tea B organizes, ig kidney tea B solution 160mg/kg; 6. kidney tea C organizes ig kidney tea C solution 160mg/kg; Administration every day 1 time, administration volume 1ml/100g, continuous 10 days.Behind the last medicine, rat metabolic cage method is collected the 24h urine, behind survey amount of urine and the urine pH, puts to death animal, gets kidney and weighs, and a side kidney is measured interior oxalic acid of nephridial tissue and calcium content by test example 1 method; The opposite side kidney carries out the kidney stone pathological study by test example 2 methods, and the result is as follows:
(1) ordinary circumstance: rat becomes stony edema after 5 days, begins to occur lethargy, and fear of cold is crispaturaed, and chaeta is withered, tarnish, and the hair color flavescence is and alarms the hair shape, and individual animal still has depilation to take place, and it is more obvious to lose weight, and the kidney body is than obviously increasing.And the PAISHI KELI group is compared with model group with kidney tea A, each group of B, C, and symptom makes moderate progress.The results are shown in Table 4,5.
Table 4 kidney tea is to the influence of large mouse with renal calculs body weight (x ± s)
| Group | Dosage (mg/ kg) | n | Body weight (g) | ||
| 0d | 5d | 10d | |||
| Control group model group PAISHI KELI group kidney A group kidney B group kidney C group | - - 6000 160 160 160 | 5 5 5 5 5 5 | 171.2±10.1 172.2±11.2 171.6±10.4 170.8±13.8 170.0±10.3 172.4±14.1 | 196.6±15.9 168.8±16.8 # 179.8±12.2 176.0±27.8 167.6±13.2 188.6±9.6 * | 222.0±10.1 158.2±26.1## # 189.2±15.0 * 185.2±37.0 167.2±16.5 204.8±10.1 ** |
Annotate: compare ###P＜0.001, #P＜0.05 with matched group; Compare with model group
* *P＜0.001,
*P＜0.01,
*P＜0.05
Table 5 kidney tea is to the influence of large mouse with renal calculs kidney body ratio (x ± s)
| Group | Number of animals | Dosage (mg/kg) | Body weight (g) | Kidney weight (g) | The kidney body is than (mg/g) |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | 5 5 5 5 5 5 | - - 6000 160 160 160 | 222.0±10.1 158.2±26.1 189.2±15.0 185.2±37.0 167.2±16.5 204.8±10.1 | 1.57±0.28 1.61±0.32 1.70±0.34 2.01±0.65 1.73±0.19 1.88±0.17 | 7.0±0.6 10.4±2.3 9.0±2.1 10.3±2.9 10.4±1.0 9.2±1.2 |
(2) kidney tea is to the influence of large mouse with renal calculs kidney calcium and oxalic acid content: after rat was drunk ethylene glycol and ammonia chloride, calcium oxalate crystal was built up in a large number in the kidney, showed that calcium, oxalic acid content significantly increase in the nephridial tissue.Kidney A, B, C group kidney calcium, kidney oxalic acid content obviously descend (rat has more obvious effect than mice), and there were significant differences to compare kidney A, C group with model group.The results are shown in Table 6.
Table 6 kidney tea is to the influence of large mouse with renal calculs kidney calcium and oxalic acid content (x ± s)
| Group | Number of animals | Dosage (mg/ kg) | Kidney calcium content (mg/g) | Kidney oxalic acid content (μ g/g) |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | 5 5 5 5 5 5 | - - 6000 160 160 160 | 3.76±0.93 18.75±6.33### 8.74±6.21 * 3.94±3.92 ** 4.65±3.28 ** 3.69±2.00 *** | 0.53±0.27 1.59±0.14### 0.81±0.27 *** 0.64±0.1 *** 0.64±0.15 *** 0.69±0.2 *** |
Annotate: compare ###P＜0.001 with matched group; Compare with model group
* *P＜0.001,
*P＜0.01,
*P＜0.05
(3) nephridial tissue morphological examination and nephridial tissue calcium oxalate crystal are observed: the renal tissue perusal, and model group animal kidney edema, the surface is pale, and as seen the kidney surface is dispersed in white point.And that PAISHI KELI group and kidney tea are respectively organized symptom is lighter, and the surface is ruddy.The renal tissue pathological observation, model group animal kidney lesion degree is more serious, has a large amount of calcium oxalate stones in renal cortex portion, marrow, renal calices, the renal pelvis, and the in heaps or full visual field of severe patient volume maximum size productive set volume maximum size productive set is in heaps.Compare with model group, it is lighter that PAISHI KELI group and kidney tea are respectively organized the nephridial tissue lesion degree, and the scoring of nephridial tissue calcium oxalate stone is lower, and showing has anti-renal calculus effect.The results are shown in Table 7.
The influence that table 7 kidney tea forms large mouse with renal calculs kidney calcium oxalate stone (n=5, x ± s)
| Group | Dosage (mg/ kg) | Calculus rate % | Renal cortex portion | Renal medulla portion | The renal calices renal pelvis | Grand mean |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | - - 6000 160 160 160 | 0(0/5) 80(4/5) 40(2/5) 100(5/5) 40(2/5) 80(4/5) | 0.0±0.0 2.6±2.4# 1.0±1.7 2.6±1.5 0.8±1.8 0.6±0.9 | 0.0±0.0 4.0±2.5## 1.8±2.5 3.0±1.4 0.0±0.0* 1.4±1.3 | 0±0 0.6±1.3 1.8±2.5 1.8±1.8 1.4±1.9 1.6±2.2 | 0.0±0.0 7.0±5.2# 4.6±6.4 7.4±3.4 2.2±3.2 3.6±3.0 |
Annotate: compare with model group
* *P＜0.001,
*P＜0.01,
*P＜0.05
Test routine 5:(clerodendranthus spicatus extract to influence little, rat urine amount)
(1) clerodendranthus spicatus extract is to the influence of normal mouse diuresis: get 55 of normal mouses and be divided into 5 groups at random, 11 every group.1. matched group, ig equal-volume water; 2. furosemide group, ig 25mg/kg; 3. kidney tea A organizes, ig 160mg/kg; 4. kidney tea B organizes, ig 160mg/kg; 5. kidney tea C organizes, ig 160mg/kg.Each organizes continuous gastric infusion 3 times, and the 1st the sky, afternoon each 1 time (8:30AM, 4:30PM), morning 1 time next day.Fasting 16h (preceding 1 day 5:00PM～next day 9:00AM) before the experiment, lumbar injection 0.9% sodium chloride solution 1ml/ only give the water load, and extruding mice hypogastric region, make the bladder emptying.The water load began administration after 0.5 hour, and administration finishes, and immediately mice is put into the wide mouthed bottle of 350ml, and the bottle end puts the cotton balls of having weighed, and collect cotton balls every 2h and weigh, be the mice voided volume with its weight difference, continuous 6h, the comparable group differences, the result is as follows:
Compare with matched group, the mice voided volume increased after mice gave furosemide 25mg/kg, and diuresis is strong, long action time, and the difference highly significant has tangible statistical significance (P＜0.001); And after mice gives kidney tea A, B, C, have only 0～4h application point urine amount increase of kidney tea A more remarkable, have tangible statistical significance (P＜0.01); Kidney tea B, C effect are not obvious.Kidney tea A, each group of B, C compare, diuresis A＞C＞B, but relatively do not have tangible statistical significance (P＞0.05) between three groups.The results are shown in Table 8.
Table 8 clerodendranthus spicatus extract is to the diuresis of normal mouse (x ± s)
| Group | Dosage (mg/kg) | 6h urine amount (ml) | 18h urine amount (ml) | 24h urine amount (ml) |
| Matched group furosemide group kidney A group kidney B group kidney C group | - 25 160 160 160 | 0.47±0.33 1.86±0.34 *** 0.59±0.28 0.39±0.28 0.64±0.27 | 1.04±0.37 2.68±0.27 *** 1.33±0.23 ** 1.11±0.45 1.27±0.30 | 1.67±0.37 3.07±0.24 *** 1.87±0.23 1.69±0.49 1.84±0.30 |
Annotate: compare with matched group
*P＜0.01;
* *P＜0.001
(2) clerodendranthus spicatus extract is to the influence of renal calculus mouse retention amount: get 66 of mices, be divided into 6 groups at random, 11 every group.Except that control group mice is drunk the normal water every day, other each group is all drunk into stony edema (1% ethylenediamine+1% ammonium chloride), drinks for 6 weeks continuously, and each is organized by following dosed administration simultaneously in modeling.1. matched group, ig equal-volume water; 2. model group, ig equal-volume water; 3. PAISHI KELI group, ig 600mg/kg; 4. kidney tea A organizes, ig 160mg/kg; 5. kidney tea B organizes, ig 160mg/kg; 6. kidney tea C organizes, ig 160mg/kg, and the diuresis experiment is carried out in the last administration later on, and method is the same, and the result is as follows:
Table 9 kidney tea is to the influence of renal calculus mouse retention amount (x ± s)
| Group | Dosage (mg/kg) | n | Urine amount (ml) |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | - - 6000 160 160 160 | 10 9 10 10 10 10 | 2.0450±0.6229 0.9542±0.5995*** 1.0721±0.4986 1.2206±0.5739 1.2013±0.5752 1.2904±0.6579 |
Annotate: compare with matched group
* *P＜0.001
After mice formed renal calculus, the urine amount obviously descended, and with matched group significant statistical significance (P＜0.001) is arranged relatively.Each component of kidney tea can increase the urine amount of renal calculus mice, but compares not statistically significant (P＞0.05) with model group.The results are shown in Table 9.
(3) clerodendranthus spicatus extract is to the influence of large mouse with renal calculs urine amount: get 30 of rats, be divided into 6 groups at random, 5 every group.Except that control rats is drunk the normal water every day, other each group is all drunk into stony edema (1% ethylenediamine+1% ammonium chloride), drinks continuously and causes the renal calculus model in 10 days.Following dosed administration, continuous 10 days are pressed in modeling each group simultaneously.1. matched group, ig equal-volume water; 2. model group, ig equal-volume water; 3. PAISHI KELI group, ig 600mg/kg; 4. kidney tea A organizes, ig 160mg/kg; 5. kidney tea B organizes, ig 160mg/kg; 6. kidney tea C organizes, ig 160mg/kg, and the diuresis experiment is carried out in the last administration later on, and method is the same, and the result is as follows:
Clerodendranthus spicatus extract A, B, C can obviously increase the urine amount of large mouse with renal calculs, and its middle kidney C is longer action time, and sustainable 24 hours effectively.The results are shown in Table 10.
Table 10 kidney tea is to the influence of large mouse with renal calculs urine amount (x ± s)
| Group | Number of animals | Dosage (mg/kg) | 6h urine amount (ml) | 18h urine amount (ml) | 24h urine amount (ml) |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | 5 5 5 5 5 5 | - - 6000 160 160 160 | 2.04±0.98 2.16±0.81 2.26±0.81 4.01±1.49 * 3.72±0.84 * 4.10±1.78 * | 9.04±5.1 3.80±0.29# 5.26±2.51 5.37±0.25 5.58±2.48 10.82±3.38 ** | 11.08±5.76 9.58±4.41 7.52±2.58 13.51±6.78 9.30±3.19 14.92±5.07 |
Annotate: compare #P＜0.05 with matched group; Compare with model group
*P＜0.01,
*P＜0.05
Modern medicine is thought the cause of disease of lithiasis, be owing to reasons such as hypourocrinia, dehydration or urine concentrate cause that the crystalline solid concentration in the urine increases on the one hand, make the dysequilibrium between crystal in the urine (as materials such as calcium oxalate, calcium phosphate) and the colloid (as materials such as mucin), the separating out of crystalline solid in causing urinating, deposition and the calculus that forms.Therefore the increase of urine amount can reduce the concentration of crystalline solid in the urine, in time discharges crystal in the urine, prevents crystalline solid precipitation in the urine, adheres to, and is condensed into renal calculus, and its effect may be one of urinary calculus removing mechanism.
Test of the influence of routine 6:(clerodendranthus spicatus extract) to animal urinary system smooth muscle
Cavia porcellus or new zealand rabbit are used in experiment, and male and female are regardless of.Taking-up bladder, ureter, urethra place 37 ℃ of tyrode's solutions, fat and other connective tissues on removal surface rapidly to put to death the back.Make bladder ring and ureter, urethra bar, with surgical thread ligation two ends, an end is fixed on the specimen hook, and the other end then links to each other with the 10g tonotransducer, and places the Magnus' bath that contains the 8ml tyrode's solution, 37 ℃ of insulations, logical oxygen.Resting tension 0.5g stablizes behind the 30min the easypro variation of contracting by Medlab-U/4cs bio signal acquisition processing system record smooth muscle.After treating that baseline steadily, in bath, add medicine (kidney A/ or kidney B/ or kidney C), observe also that the record smooth muscle reacts about 5～10min,, continuous 3 times, after stablize 15min approximately and treating that baseline recovers substantially, add the medicine observation again with the tyrode's solution flush away medicine of pre-temperature.Other processing is the same.Kidney A, kidney B, the kidney C influence to Cavia porcellus (new zealand rabbit) bladder, ureter, urethral smooth muscle observed in record.
The result is as follows:
Kidney tea A, B, three components of C all have the increase shrinkage amplitude to Cavia porcellus, new zealand rabbit smooth muscle of bladder, accelerate the effect of the rhythm and pace of moving things.And the three all has relaxation effect to ureter, urethral smooth muscle, as Figure of description Fig. 1, Fig. 2, shown in Figure 3.Kidney tea may shrink bladder-dilating urethra by the expansion Uretero-, thereby makes the unobstructed discharge calculus of urinary tract.
Test of the influence of routine 7:(clerodendranthus spicatus extract) to mouse corrosion disease effect caused by dimethylbenzene xylene otitis
Get 60 of mices and be divided into 5 groups at random, 12/group,, male and female half and half.1. matched group, ig equal-volume water; 2. indometacin group, ig 20mg/kg; 3. kidney tea A organizes, ig 160mg/kg; 4. kidney tea B organizes, ig 160mg/kg; 5. kidney tea C organizes, ig 160mg/kg.Successive administration 5 days, 30min after the last administration embrocates mouse right ear with dimethylbenzene and causes inflammation, causes scorching back 15min and puts to death mice, cuts left and right sides auricle, lays round auricle with the 9mm card punch in same area, and the difference of calculating left and right sides auricle weight is as the swelling degree.The result is as follows:
The obvious swelling of auris dextra after the control group mice caused by dimethylbenzene xylene inflammation, and the auricle swelling degree of respectively organizing mice all has decline.Compare with matched group, the swelling degree of indometacin group, kidney tea A and B group reduces tangible statistical significance (P＜0.01).But it is not obvious that kidney tea C group swelling degree reduces, not statistically significant (P＞0.05).The results are shown in Table 11.
Table 11 clerodendranthus spicatus extract is to the influence of mouse corrosion disease effect (x ± s)
| Group | Dosage (mg/kg) | Number of animals | Auricle swelling degree (mg) |
| Matched group indometacin group kidney A group kidney B group kidney C group | - 20 160 160 160 | 12 12 12 12 12 | 7.6±2.4 4.9±1.9 ** 5.7±1.9 * 5.6±2.1 * 7.3±3.7 |
Annotate: compare with matched group
*P＜0.01,
*P＜0.05
Test of the influence of routine 8:(clerodendranthus spicatus extract) to renal calculus mouse anti lipid peroxide
Get 60 of mices, be divided into 6 groups at random, 10 every group.Except that control group mice is drunk the normal water every day, other each group is all drunk into stony edema (1% ethylenediamine+1% ammonium chloride), drinks for 6 weeks continuously, and each group is by following dosed administration simultaneously.1. matched group, ig equal-volume water; 2. model group, ig equal-volume water; 3. PAISHI KELI group, ig 600mg/kg; 4. kidney tea A organizes, ig 160mg/kg; 5. kidney tea B organizes, ig160mg/kg; 6. kidney tea C organizes, ig 160mg/kg, after the last administration, put to death animal, dissect mice, kidney is weighed, get the normal saline that a side kidney adds pre-cooling, grind evenly with glass homogenizer, make 10% homogenate, draw homogenate 0.1mL and survey LPO (lipid peroxide) content (measuring kit method by malonaldehyde MDA carries out).Experimental data is represented with x ± s.Through the t check, P＜0.05 is for there being statistical significance.The result is as follows:
After mice formed renal calculus, nephridial tissue LPO content obviously rose, and with matched group statistical significance (P＜0.05) is arranged relatively.Each group of kidney tea and PAISHI KELI can make the nephridial tissue LPO content of renal calculus mice descend, and with model group comparison kidney C group statistical significance (P＜0.05) is arranged, and other organize equal not statistically significant (P＞0.05).The results are shown in Table 12.
Table 12 clerodendranthus spicatus extract is to the influence of renal calculus mice LPO (x ± s)
| Group | Dosage (mg/ kg) | n | LPO(nmol/mgprot) |
| Matched group model group PAISHI KELI group kidney A group kidney B group kidney C group | - - 600 160 160 160 | 10 9 10 10 10 10 | 2.705±0.4173 3.9433±1.3622# 3.3511±0.8111 2.9702±1.1329 3.1647±0.4341 2.7241±1.0958 * |
Annotate: compare #P＜0.05 with matched group; Compare with model group
*P＜0.05
Normal person's renal cells can resist crystalline adhesion, also contains calcium oxalate crystal growth and accumulative mortifier in the urine and promotes thing.When renal cells damaged, crystal can adhere to renal pelvis inwall, ureter inwall etc. and locate mucosa, and the calculus mechanical stimulus can cause inflammatory reactions such as the hyperemia, edema of tissue.Originally experimental results show that clerodendranthus spicatus extract has antiinflammatory action, pointed out it to reduce the adhesion probability of crystal, be beneficial to little crystalline discharge, reached the effect that suppresses renal calculus by suppressing the damage of renal tissue at renal epithelial cell.
Claims (2)
1, clerodendranthus spicatus extract B component is characterized in that and can make by the following method:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks altogether 3 times, each 10 days, merges, concentrated lixiviating solution gets the extractum mixture;
(2) earlier water is mixed into lysate with methanol by 1: 5～12 volume ratios, again with step (1) extractum mixture and lysate by gram weight: ml volumes is 1: 1 mixed, is stirred to abundant dissolving;
(3) step (2) extractum lysate is extracted 3-5 time respectively with petroleum ether and ethyl acetate earlier, fully remove resin, the low polar impurity of chlorophyll and liposoluble constituent after, reuse n-butanol extraction 4 times, merging, concentrated extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and concentrate light brown thick clerodendranthus spicatus extract B component.
2, a kind of method for preparing the described clerodendranthus spicatus extract B of claim 1 component is characterized in that carrying out according to the following steps:
(1) kidney tea [C.spicatus (Thunb.) C.Y.Wu ex H.W.Li] dry product is pulverized back and methanol by gram weight: ml volumes is 1: 4～10 ratio, soaks altogether 3 times, each 10 days, merges, concentrated lixiviating solution gets the extractum mixture;
(2) earlier water is mixed into lysate with methanol by 1: 5～12 volume ratios, again with step (1) extractum mixture and lysate by gram weight: ml volumes is 1: 1 mixed, is stirred to abundant dissolving;
(3) step (2) extractum lysate is extracted 3-5 time respectively with petroleum ether and ethyl acetate earlier, fully remove resin, the low polar impurity of chlorophyll and liposoluble constituent after, reuse n-butanol extraction 4 times, merging, concentrated extract;
(4) step (3) extract is carried out silica gel column chromatography, gradient elution, collection and concentrate clerodendranthus spicatus extract B component product.
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| CN102309542A (en) * | 2011-09-23 | 2012-01-11 | 上海交通大学 | Orthosiphon n-butanol fraction medicine for treating chronic nephritis and preparation method thereof |
| CN101669988B (en) * | 2009-07-31 | 2012-06-27 | 芜湖梁氏新材料有限公司 | Extraction process of clerodendranthus spicatus |
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| CN108685979A (en) * | 2018-08-16 | 2018-10-23 | 曲阜师范大学 | The application of cat's whisker leaf extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101669988B (en) * | 2009-07-31 | 2012-06-27 | 芜湖梁氏新材料有限公司 | Extraction process of clerodendranthus spicatus |
| CN102309542A (en) * | 2011-09-23 | 2012-01-11 | 上海交通大学 | Orthosiphon n-butanol fraction medicine for treating chronic nephritis and preparation method thereof |
| CN102309542B (en) * | 2011-09-23 | 2013-06-19 | 上海交通大学 | Orthosiphon n-butanol fraction medicine for treating chronic nephritis and preparation method thereof |
| CN103735614A (en) * | 2014-01-11 | 2014-04-23 | 西双版纳版纳药业有限责任公司 | Method for processing raw folium orthosiphoni and folium orthosiphoni product |
| CN105832849A (en) * | 2016-05-03 | 2016-08-10 | 王吉祥 | Traditional Chinese medicine composition for treating kidney calculi and preparation method thereof |
| CN108685979A (en) * | 2018-08-16 | 2018-10-23 | 曲阜师范大学 | The application of cat's whisker leaf extract |
| CN108685979B (en) * | 2018-08-16 | 2021-04-13 | 曲阜师范大学 | Application of radix ranunculi ternati leaf extract |
| CN114558065A (en) * | 2022-03-23 | 2022-05-31 | 桂林医学院 | Kidney tea fermented composition for reducing uric acid and preparation method thereof |
| CN114558065B (en) * | 2022-03-23 | 2024-03-08 | 桂林医学院 | Kidney tea fermentation composition for reducing uric acid and preparation method thereof |
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