CN103623150B - Novel processing technology for lowering alisma nephrotoxicity - Google Patents

Novel processing technology for lowering alisma nephrotoxicity Download PDF

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CN103623150B
CN103623150B CN201310667448.4A CN201310667448A CN103623150B CN 103623150 B CN103623150 B CN 103623150B CN 201310667448 A CN201310667448 A CN 201310667448A CN 103623150 B CN103623150 B CN 103623150B
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rhizoma alismatis
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alisol
monoacetate
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CN103623150A (en
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许枬
贾天柱
张宏达
李岚
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Dalian Quanjian Herbal Medicine Co Ltd
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Dalian Quanjian Herbal Medicine Co Ltd
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Abstract

The invention discloses a novel processing technology for lowering alisma nephrotoxicity and belongs to the technical field of traditional Chinese medicine processing. According to the technology, crude alisma decoction pieces, 9-degree vinegar and dried orange peel decoction are mixed according to the ratio of 10 kg : (1 to 1.5) L : 1 L, after being moistened for 2 hours, the mixture is added in a frying medicinal pot containing wheat bran, wherein the mass ratio of the crude alisma decoction pieces to the wheat bran is 5:1-3:1, the mixture is fried for 10 to 15 min at the temperature of 150 DEG C to 160 DEG C and then taken out, and the wheat bran is screen out, so that an alisma processed product is obtained; the section of the processed product is yellowish brown and has vinegar flavor, wherein the content of 24-monoacetate A is 0.025% to 0.026%, and the content of 23-monoacetate B is 0.069% to 0.071%. The technology has the advantages that the 24-monoacetate A is promoted to be converted into another component, the content of the 24-monoacetate A is reduced, and the stimulation of the 24-monoacetate A to innate cells of the kidneys is inhibited, so that harm to the kidneys can be reduced.

Description

A kind of reformed processing method reducing Rhizoma Alismatis Toxicity of Kidney
Technical field
The present invention relates to a kind of reformed processing method reducing Rhizoma Alismatis Toxicity of Kidney, it belongs to Chinese medicine processing technical field.Its object mainly promotes the conversion of triterpenes components in Rhizoma Alismatis by processing technique, thus reduces Rhizoma Alismatis to the toxic action of kidney.
Background technology
Rhizoma Alismatis is the dry tuber of Notes On Alism At Aceae Rhizoma Alismatis Alisma orientalis (Sam.) Juzep..Theory of Chinese medical science thinks that it is cold in nature, has effect of promoting diuresis to eliminate damp pathogen.Modern pharmacology research shows, Rhizoma Alismatis has all multiactions such as diuresis, blood sugar lowering, blood fat reducing, blood pressure lowering, be used widely in clinical practice, and be used to the Chinese patent medicines such as infirmity clear sheet, WULING SAN, XUEZHILING PIAN, mountain chrysanthemum hypertension pill and LIUWEI DIHUANG WAN, be used for the treatment of the diseases such as hyperlipemia, fatty liver, obesity, aural vertigo, ascites.
Rhizoma Alismatis mainly uses with the form of decoction pieces clinically.The dry tuber of Rhizoma Alismatis claims Rhizoma Alismatis raw product decoction pieces through the machining object that the process such as softening, cutting, drying is made.Chinese Pharmacopoeia has also recorded its salt product except having recorded the raw product decoction pieces of Rhizoma Alismatis, and namely Rhizoma Alismatis gives birth to the decoction pieces that product decoction pieces is prepared through salt method for making (according to Chinese Pharmacopoeia annex concocting method).Rhizoma Alismatis also has bran goods in actual applications, and namely Rhizoma Alismatis gives birth to the decoction pieces that product decoction pieces is prepared through bran method for making (according to Chinese Pharmacopoeia annex concocting method).
The chemical composition of Rhizoma Alismatis, based on triterpenoid compound such as 24-alisol acetyl A, Alisol B monoacetate, Alisol A, alisol B, damp alcohol F, separately contains sterol, alkaloid, organic acid, polysaccharide, volatile oil, fatty acid, resin, starch etc.Wherein 24-alisol acetyl A, Alisol B monoacetate are the main active of Rhizoma Alismatis, and total content reaches about 0.08%.
Modern clinic application finds, life-time service Rhizoma Alismatis is to the toxic effect of kidney, and main manifestations is the infringement to renal tubules and glomerule, and pharmacodynamic experiment also demonstrate that this result.Zhu Jianhui etc. study discovery, Rhizoma Alismatis extract powder 2g and 4g/kg (be equivalent to clinical dosage 20 and 40 times) is mixed in feedstuff the rat 3 months feeding side kidney excision, pathological section display rat kidney proximal convoluted tubule has cloudy swelling in various degree and degeneration, and heavy dose of group smaller dose group is obvious.Rhizoma Zingiberis Recens is drenched the healthy Kunming mouse of clean employing 140 and carries out testing (male and female half and half, be divided into 7 groups, often organize 20, be respectively normal group, Rhizoma Alismatis total extract high dose group, Rhizoma Alismatis total extract low dose group, component 1 high dose group, component 1 low dose group, component 2 high dose group and component 2 low dose group).Normal group gives distilled water 20mL/kg/d, Rhizoma Alismatis total extract high and low dose group gives Rhizoma Alismatis water extract 13.75g/kg/d and 8.25g/kg/d respectively, and component 1 and component 2 high and low dose group give Rhizoma Alismatis component 1 and component 2 extract 5.5g/kg/d and 3.2g/kg/d respectively.Administration time 70 days, administration terminates rear collection mice urine, and eyeball is got blood and cut kidney, weighs, prepares tissue slice.Result shows, and high dose Rhizoma Alismatis component 1 extract obviously can raise the content (P<0.01) of BUN, CR, NAG and γ-GT of mice.High dose Rhizoma Alismatis extract also can improve the above-mentioned biochemical indicator (P<0.05) of mice.CG and the NAG content of high dose Rhizoma Alismatis component 2 extract to the serum of mice has a significant effect (P<0.05).Examine under a microscope, normal group Renal Structure is complete, and renal cells and renal tubules are all normal.In male mice, total extract high dose group Mouse Renal Tubular Epithelial Cells, renal tubules are degenerated, and observe lymphocytic infiltration in focal zone; Component 1 high dose group Mouse Kidney tubular atrophy, have non-structure secretions, and there is necrosis phenomena focal zone in tube chamber.In female mice, total extract high dose group compared with component 1 high dose group, medullary substance and cortex structure clear, and component 1 high dose group murine glomerular basement membrane fracture and Zao Zhuan district have bleeding.It is dead that each administration group is showed no obvious renal interstitial fibrosis and extension, the lymphocytic infiltration of dispersion and renal cells, and prompting Rhizoma Alismatis component 1 extract high dose can cause serious harm to mouse kidney.Zhao Xiaoping etc. adopt renal cells to test, high pressure preparative chromatography is used to be separated and preparation Rhizoma Alismatis chemical constituent sample, by FDA fluorescent labeling and MTT2 kind method examination nephrotoxicity component, liquid-mass chromatography qualitative analysis is carried out to the most obvious chemical constituent of toxicity.Result shows, mtt assay and FDA fluorescent marker method testing result basically identical, in Rhizoma Alismatis, C13 component is in the most obvious toxicity, is accredited as alisol C, 16,23-epoxy alisol B and alisol O through hygroplasm combination analysis.Prompting, in Rhizoma Alismatis, triterpenes components may cause nephrotoxicity.
As fully visible, as conventional important Chinese medicine, Rhizoma Alismatis long-term, high-dose uses can cause Toxicity of Kidney.Therefore, the Toxicity of Kidney reducing Rhizoma Alismatis has major application and is worth.But up to the present, except the concocting method of inventor's research, about the effective ways reducing the effect of Rhizoma Alismatis Toxicity of Kidney have no report.
Summary of the invention
From existing toxicity Study of Medicinal Herbs, the toxicity of medicine is caused by its toxic component.If reduce the toxic component content of medicine, effectively can reduce the toxicity of medicine, improve Drug safety.Therefore, our technical scheme can promote by Chinese medicine processing the technology that the chemical composition of Chinese medicine changes, according to organic chemical reactions mechanism and cooked theory, through loose pattern draft experiment and decoction pieces experiment, use with vinegar, Pericarpium Citri Reticulatae decocts juice and through high temperature frying production method, Testa Tritici promotes that the toxic component of Rhizoma Alismatis transforms, thus reduce the toxicity of Rhizoma Alismatis kidney.
Technical scheme of the present invention is: a kind of reformed processing method reducing Rhizoma Alismatis Toxicity of Kidney, according to the raw product decoction pieces of Rhizoma Alismatis: 9 degree vinegars: it is 10kg:(1-1.5 that Pericarpium Citri Reticulatae decocts juice) amount of L:1L mixes, after mixture is carried out moistening 2h, add and be placed with in the medicine cookeding pot of Testa Tritici, the mass ratio of Testa Tritici and the raw product decoction pieces of described Rhizoma Alismatis is 5:1-3:1, at the temperature of 150-160 DEG C, parch 10-15min, take out, sieve removes Testa Tritici, obtains Rhizoma Alismatis processed product.
Described vinegar is rice vinegar, white vinegar or alimentary acetic acid.
The Pericarpium Citri Reticulatae decocting liquid concentrated juice of described Pericarpium Citri Reticulatae decocts juice to be every mL containing the crude drug amount of Pericarpium Citri Reticulatae be 1g.
The tangent plane of described Rhizoma Alismatis processed product is fallow, has vinegar fragrance; In described Rhizoma Alismatis processed product, the content of 24-alisol acetyl A is 0.025-0.026%, and the content of Alisol B monoacetate is 0.069-0.071%.
Beneficial effect of the present invention is: the reformed processing method of this reduction Rhizoma Alismatis Toxicity of Kidney, according to the raw product decoction pieces of Rhizoma Alismatis: 9 degree vinegars: it is 10kg:(1-1.5 that Pericarpium Citri Reticulatae decocts juice) amount of L:1L mixes, after mixture is carried out moistening, add and be placed with in the medicine cookeding pot of Testa Tritici, at the temperature of 150-160 DEG C, parch 10-15min, takes out, sieve removes Testa Tritici, obtains Rhizoma Alismatis processed product.The tangent plane of this Rhizoma Alismatis processed product is fallow, has vinegar fragrance; In Rhizoma Alismatis processed product, the content of 24-alisol acetyl A is 0.025-0.026%, and the content of Alisol B monoacetate is 0.069-0.071%.This technique, by controlling the processing procedure parameters such as supplementary product consumption, parch temperature, parch time, promotes that in Rhizoma Alismatis concocting process, 24-alisol acetyl A is converted into other compositions, suppresses it to the stimulation of the intrinsic cell of kidney, thus reduces it to Toxicity of Kidney.
Accompanying drawing explanation
Fig. 1 is the pyrolytic conversion thing (No. 2 peaks are 24-alisol acetyl A) of 24-alisol acetyl A.
Fig. 2 is the path for transformation of 24-alisol acetyl A.
Fig. 3 is the pyrolytic conversion thing of 24-alisol acetyl A and the chromatograms of Rhizoma Alismatis raw product.
In Fig. 3: 1, the pyrolytic conversion product of 24-alisol acetyl A, 2, Rhizoma Alismatis processed product, 3, the raw product of Rhizoma Alismatis.
Fig. 4 is the HPLC spectrogram of the raw product of Rhizoma Alismatis and different process bran goods.
In Fig. 4: S1,24-alisol acetyl A, S2, Alisol B monoacetate, 1, the Rhizoma Alismatis processed product prepared of the application's patent technique, 2, bran Rhizoma Alismatis (stir-fry of Rhizoma Alismatis plus wheat bran), 3, the raw product of Rhizoma Alismatis.
Fig. 5 is the section of different experiments group rat kidney tissue morphology.
Specific embodiment
In conjunction with above-mentioned accompanying drawing, carry out following research:
The conversion under the high temperature conditions of 1.24-alisol acetyl A and Alisol B monoacetate
The hot conditions that simulation is concocted, observes predominant amount composition 24-alisol acetyl A and the Alisol B monoacetate change under the high temperature conditions of Rhizoma Alismatis.Late Cambrian, Alisol B monoacetate is at high temperature without significant change, and 24-alisol acetyl A then significant change occurs, and concrete outcome is shown in Fig. 1.
Fig. 1 is 24-alisol acetyl A(2 peak) the HPLC chromatogram of (180 DEG C) conversion product under the high temperature conditions.As seen from Figure 1, under the high temperature conditions, 24 alisol acetyl A can be converted into number of chemical composition.Above-mentioned conversion product prepares purification through HPLC, obtains 6 compounds respectively.Its spectroscopic data, through contrast with document, is accredited as 24-alisol acetyl E(1 peak respectively), 24-acetyl-25 dewaters Alisol A (No. 3 peaks), 24-acetyl-25-dehydration-alisol E(4 peak), 23-acetyl-25-dehydration-alisol E(5 peak), alisol G(6 peak).Visible according to above-mentioned experimental result, can there is conversion in 24-alisol acetyl A under the high temperature conditions.Inferring according to Synthetic Organic Chemistry mechanism, may there is the conversion process as Fig. 2 in 24-alisol acetyl A under the high temperature conditions.
From above-mentioned experimental result, 24-alisol acetyl A change at high temperature may relate to dehydration, ester linkage breaking and esterification.
2. in processing procedure process, the 24-alisol acetyl A of Rhizoma Alismatis decoction pieces changes
In order to promote that in Rhizoma Alismatis, chemical composition transforms in concocting process, under reaction mechanism under 24-alisol acetyl A hot conditions instructs, through repeatedly testing, after final discovery Rhizoma Alismatis adopts 9 degree tablet vinegar, Pericarpium Citri Reticulataes to decoct juice moistening, the method that stirs of Testa Tritici is carried out the process of preparing Chinese medicine and obviously can be promoted chemical composition conversion in Rhizoma Alismatis decoction pieces, and this change and 24-alisol acetyl A are to simulate the change under high temperature process of preparing Chinese medicine condition basically identical.Rhizoma Alismatis processed product chemical composition prepared by contrast 24-alisol acetyl A pyrolytic conversion thing, the raw product of Rhizoma Alismatis and the application's patent technique, the results are shown in Figure 3.As seen from Figure 3, Rhizoma Alismatis processed product composition prepared by this processing procedure and 24-alisol acetyl A pyrolytic conversion thing composition quite similar, illustrate that this technique can promote that the pool of the 24-acetyl in Rhizoma Alismatis alcohol A transforms really.
The HPLC spectrogram of the raw product of contrast Rhizoma Alismatis and different process bran goods is visible, the Rhizoma Alismatis processed product adopting this technique to prepare and traditional bran technique processed product and the product of giving birth to component difference larger.The most obvious component difference is that in the Rhizoma Alismatis processed product prepared of this technique, 24-alisol acetyl A content obviously reduces, and Alisol B monoacetate content obviously raises (see figure 4).Experimental result shows, and the one-tenth adopting this concocting method can change Rhizoma Alismatis processed product is grouped into, and by promoting that 24-alisol acetyl A is converted into other compositions, can reduce the content of 24-alisol acetyl A while increasing the content of Alisol B monoacetate.
3. preparation technology
3.1 processing procedure experiment of single factor are investigated
3.1.1 the investigation of parch time
Get the raw product decoction pieces 4 parts of Rhizoma Alismatis, each 30g, add rice vinegar-Pericarpium Citri Reticulatae respectively and decoct juice blend 6mL(9 degree rice vinegar 3mL and add Pericarpium Citri Reticulatae and decoct juice 3mL) moistening 2h, mix with Testa Tritici 10g, carry out parch in 160 DEG C, the parch time is respectively 1min, 5min, 10min, 15min.Take out, cool.Adopt HPLC method to measure the content of 24-alisol acetyl A and Alisol B monoacetate in above-mentioned sample, the results are shown in Table 1(parallel sample 3 parts).
The table 1 different parch time is on the impact (n=3) of 24-alisol acetyl A and Alisol B monoacetate content in Rhizoma Alismatis goods
Sample Decoction pieces amount (g) Time (min) 24-alisol acetyl A(%) Alisol B monoacetate (%)
1 30 1 0.03643 0.06329
2 30 5 0.03831 0.06611
3 30 10 0.02515 0.07029
4 30 15 0.02487 0.06942
Result shows, under experimental conditions, along with the prolongation of concocted time, the content of 24-alisol acetyl A first increases and reduces, and the content of Alisol B monoacetate then presents the trend continued to increase.Under experimental conditions, concocted time is the key factor affecting the two content in processed product in prompting.And 24-alisol acetyl A content is lower within the scope of 10-15min, Alisol B monoacetate content is higher.
3.1.2 with the investigation of vinegar amount
Get the raw product decoction pieces 4 parts of Rhizoma Alismatis, each 30g, add respectively 9 degree of rice vinegars 1.5,3.0,4.5,6.0mL(Pericarpium Citri Reticulatae decocts juice consumption and is 3mL) moistening 2h, mix with Testa Tritici 10g, carry out parch 10min in 160 DEG C.Take out, cool.Adopt HPLC method to measure the calculating content of 24-alisol acetyl A and Alisol B monoacetate, the results are shown in Table 2(parallel sample 3 parts).
The table 2 different parch time is on the impact (n=3) of 24-alisol acetyl A and Alisol B monoacetate content in Rhizoma Alismatis goods
Sample Decoction pieces amount (g) Vinegar addition (mL) 24-alisol acetyl A(%) Alisol B monoacetate (%)
1 30 1.5 0.03013 0.06338
2 30 3.0 0.02538 0.06951
3 30 4.5 0.02515 0.07079
4 30 6.0 0.02487 0.06071
Result shows, under experimental conditions, when rice vinegar consumption is effectively to promote within the scope of 3.0-4.5mL that in Rhizoma Alismatis processed product, 24-alisol acetyl A transforms, namely the material medicine of rice vinegar and Rhizoma Alismatis is than being 10-15%(mL/g) in scope time can reduce 24-alisol acetyl A content in Rhizoma Alismatis processed product, raise the content of Alisol B monoacetate simultaneously.
3.1.3 Pericarpium Citri Reticulatae decocts the investigation of juice consumption
Get the raw product decoction pieces 4 parts of Rhizoma Alismatis, each 30g, add respectively rice vinegar 3mL and Pericarpium Citri Reticulatae decoct juice 0,1.5,3,4.5,6.0mL(9 degree rice vinegar consumption 3mL) moistening 2h, mix with Testa Tritici 10g, carry out parch 10min in 160 DEG C, taking-up, dries in the air cool.Adopt HPLC method to measure the calculating content of 24-alisol acetyl A and Alisol B monoacetate, the results are shown in Table 3(parallel sample 3 parts).
Table 3 Pericarpium Citri Reticulatae decocts the impact (n=3) of juice usage ratio on 24-alisol acetyl A and Alisol B monoacetate content in goods
Sample Decoction pieces amount (g) Pericarpium Citri Reticulatae decocts juice consumption (mL) 24-alisol acetyl A(%) Alisol B monoacetate (%)
1 30 0 0.02784 0.06247
2 30 1.5 0.02665 0.06468
3 30 3.0 0.02530 0.06940
4 30 4.5 0.02558 0.06890
5 30 6.0 0.02645 0.06487
From the above results, under experimental conditions, Pericarpium Citri Reticulatae decocts juice consumption effectively can promote that in the scope of 3.0-4.5mL 24-alisol acetyl A transforms, and namely Pericarpium Citri Reticulatae decocts the spice of juice and Rhizoma Alismatis than being 10-15%(mL/g) scope in Rhizoma Alismatis can be made to concoct in 24-alisol acetyl A content reduce while the content of Alisol B monoacetate raise.If when concocting process processing aids only has rice vinegar, the conversion ratio of 24-acetyl alcohol A decocts juice blend not as using rice vinegar and Pericarpium Citri Reticulatae.
3.1.4 the investigation of Testa Tritici consumption
Get the raw product decoction pieces 5 parts of Rhizoma Alismatis, each 30g, add rice vinegar-Pericarpium Citri Reticulatae respectively and decoct juice blend 6mL(9 degree rice vinegar 3mL and add Pericarpium Citri Reticulatae and decoct juice 3mL) moistening 2h, respectively with not commensurability Testa Tritici 4g, 6g, 8g, 10g, 12g mix, and take out after 160 DEG C of parch 10min, dry in the air cool.The content of 24-alisol acetyl A and Alisol B monoacetate in working sample, the results are shown in Table 4(parallel sample 3 parts).
The different bran consumption of table 4 is on the impact (n=3) of 24-alisol acetyl A and Alisol B monoacetate content in Rhizoma Alismatis goods
Sample Decoction pieces amount (g) Testa Tritici consumption (g) 24-alisol acetyl A(%) Alisol B monoacetate (%)
1 30 4 0.026841 0.06519
2 30 6 0.02507 0.07069
3 30 8 0.02494 0.06990
4 30 10 0.02509 0.06915
5 30 12 0.02538 0.06697
Result shows, under experimental conditions, when Testa Tritici by the ratio with Rhizoma Alismatis decoction pieces in the scope of 1:5-1:3 time, in Rhizoma Alismatis processed product, the changes of contents of 24-alisol acetyl A and Alisol B monoacetate is less, and when can ensure that 24-alisol acetyl A is lower, Alisol B monoacetate content is higher.
3.1.4 the investigation of parch temperature
Get the raw product decoction pieces 5 parts of Rhizoma Alismatis, each 30g, add rice vinegar-Pericarpium Citri Reticulatae respectively and decoct juice blend 6mL(9 degree rice vinegar 3mL and add Pericarpium Citri Reticulatae and decoct juice 3mL) moistening 2h, mix with Testa Tritici 10g, respectively at 140 DEG C, 150 DEG C, 160 DEG C, 170 DEG C, take out after parch 10min at 180 DEG C of temperature, cool.The content of 24-alisol acetyl A and Alisol B monoacetate in working sample, the results are shown in Table 5(parallel sample 3 parts).
The impact (n=3) of temperature on 24-alisol acetyl A and Alisol B monoacetate content in Rhizoma Alismatis goods concocted by table 5
Result shows, concocting the content of temperature to 24-alisol acetyl A and Alisol B monoacetate has considerable influence.But when temperatures as high more than 170 DEG C, the content of Alisol B monoacetate obviously reduces.Therefore, the content increasing Alisol B monoacetate while 24-alisol acetyl A content reduces can be guaranteed during 150-160 DEG C.
The investigation of 3.2 moistening conditions
3.2.1 the investigation of moistening time
Get the raw product decoction pieces 4 parts of Rhizoma Alismatis, each 30g, the mixture 6mL(9 degree rice vinegar 3mL adding rice vinegar and Pericarpium Citri Reticulatae decocting liquid respectively adds Pericarpium Citri Reticulatae and decocts juice 3mL) moistening 0.5,1.0,1.5,2.0,2.5h, investigate the moistening degree of Rhizoma Alismatis decoction pieces, the results are shown in Table 6(parallel sample 3 parts).
The table 6 different moistening time is on the impact (n=3) of Rhizoma Alismatis decoction pieces moistening degree
Sample Decoction pieces amount (g) Moistening time (h) Moistening degree
1 30 0.5 Do not run through
2 30 1.0 Do not run through
3 30 1.5 Do not run through
4 30 2.0 Run through
5 30 2.5 Run through
From table 6, under experimental conditions, Rhizoma Alismatis decoction pieces can run through more than 2h by the moistening time.
3.2.2 the investigation of Auxiliary Liquid Material consumption
Get the raw product decoction pieces 4 parts of Rhizoma Alismatis, each 30g, add respectively equal proportion rice vinegar and Pericarpium Citri Reticulatae decoct juice mixture 3,4.5,6,7.5,9.0mL moistening 2h, the moistening degree of investigation Rhizoma Alismatis decoction pieces, the results are shown in Table 7(parallel sample 3 parts).
Different rice vinegar-the Pericarpium Citri Reticulatae of table 7 decocts the impact (n=3) of juice blend usage ratio on Rhizoma Alismatis decoction pieces moistening degree
Sample Decoction pieces amount (g) Spice ratio (g/mL) Moistening degree
1 30 10:1 Do not run through
2 30 10:1.5 Do not run through
3 30 10:2 Run through
4 30 10:2.5 Run through
5 30 10:3 Run through
From the above results, the ratio of Rhizoma Alismatis decoction pieces and Auxiliary Liquid Material (rice vinegar-Pericarpium Citri Reticulatae decocts juice blend) is at 10:2-10:3(g/mL) scope in, Rhizoma Alismatis decoction pieces can run through by moistening 2h.Be the principal element affecting 24-alisol acetyl A and Alisol B monoacetate changes of contents in Rhizoma Alismatis concocting process owing to concocting adjuvant juice used (rice vinegar and Pericarpium Citri Reticulatae juice), therefore need before parch with adjuvant juice, decoction pieces to be run through as far as possible.Therefore the moistening time is defined as 2h.
4. toxicity test
4.1 cytotoxicity experiment
4.1.1 the toxicity assessment of monomer component and component
In order to determine toxicity and the composition relation of Rhizoma Alismatis, adopting kidney effector lymphocyte (mesangial cell) to carry out toxicity test, evaluating 24-alisol acetyl A and pyrolytic conversion thing and Alisol B monoacetate thereof to the quantity of mesangial cell and activity influence.
Tested composition: 24-alisol acetyl A and Alisol B monoacetate are two monomer components of extraction purification from Rhizoma Alismatis; 24-alisol acetyl A pyrolytic conversion thing is previous experiments products therefrom.In experiment, its weight is by the 24-alisol acetyl A Weight computation before conversion.
Experimental technique: above-mentioned tested one-tenth is distributed into variable concentrations, jointly hatches with mesangial cell, measures cytoactive and quantity, judges that composition is to the toxicity of kidney.From liquid nitrogen, take out cryopreservation tube, be placed in 37 DEG C of warm water and constantly shake, freeze-stored cell is melted within l min.By cell suspension inoculation in 25cm 2in culture bottle, add the appropriate DMEM culture fluid containing 10%FBS, in 37 DEG C, 5%CO 2and saturated humidity is cultivated.Culture fluid is discarded when cell fusion is 70%-80%, digest under adding 0.25% trypsin room temperature, after digestion 3-5min, observe under culture bottle being placed on inverted microscope, as found cell cytoplasm retraction, intercellular substance increase, the culture fluid added containing hyclone stops digestion, is blown and beaten by cell with suction pipe from bottle wall, and adjustment cell concentration is 5 × 10 6mL -1, be inoculated in respectively in new culture bottle.Put 5%CO 2, 37 DEG C of saturated humidities incubator in cultivate, every 2-3 days goes down to posterity once.
Cell is counted after trypsinization, makes 5 × 10 5the cell suspension of individual/mL, be inoculated in 96 orifice plates with every hole 200 μ L, after cultivating 24h, every hole adds drug solution 200 μ L, the total liquid measure in every hole is 200 μ L, and each drug level establishes 6 multiple holes, and (each compound DMSO adds the Contained Serum that 10%FBS makes the variable concentrations containing 0.1%DMSO after dissolving to establish blank, wherein the weight of 24-alisol acetyl A conversion product is by the 24-alisol acetyl A Weight computation before conversion, and blank group is the 10%FBS containing 0.1%DMSO).After cultivating 24h, discard original fluid, every hole adds DMEM180 μ L culture fluid, then adds 5mgmL -1mTT20 μ L, continue to cultivate 4h, abandon supernatant, every hole adds DMSO150 μ L, after vibration 5min, measures the OD value at 490nm place by microplate reader.
Toxicity screening result: compare with DMSO group, 24-alisol acetyl A is 10 -4, 10 -5, 10 -6, 10 -7gmL -1time can obviously promote messangial cell breed (P<0.05); The 24-alisol acetyl A pyrolytic conversion thing of same concentrations and Alisol B monoacetate are then without obviously promoting cel l proliferation (see table 8).
In table 8 Rhizoma Alismatis, main component affects rat mesangial cell in vitro
Note: compare with DMSO group, *: P<0.05
Because the main pathology of compromised kidneys wound shows as the mesangial region hypertrophy and sclerosis that are caused by proliferation of glomerular mesangial cells.24-alisol acetyl A obviously can urge Glomerular Mesangial Cells Proliferation and experimental result in dose-dependence is pointed out, and 24-alisol acetyl A is the Toxicity of Kidney composition of Rhizoma Alismatis, and its toxic action becomes positive correlation with its content.The conversion product Toxicity of Kidney of 24-alisol acetyl A is starkly lower than 24-alisol acetyl A, and the Toxicity of Kidney of Alisol B monoacetate is lower than 24-alisol acetyl A conversion product.Point out thus, if promote in concocting process that 24-alisol acetyl A transforms, while namely reducing 24-alisol acetyl A content, improve the Toxicity of Kidney that Alisol B monoacetate content effectively can reduce processed product.
4.1.2 the toxicity assessment of Rhizoma Alismatis raw product
Originally declaring the Toxicity of Kidney of the Rhizoma Alismatis goods of technique for evaluating, adopting Integral animal experiment, compare and investigate it with the Rhizoma Alismatis goods that Rhizoma Alismatis gives birth to product, prepared by traditional bran technique to Toxicity of Kidney size, whether have reduction toxic action to evaluate it.
Test medicine: the raw product of Rhizoma Alismatis (the raw product decoction pieces of Rhizoma Alismatis), bran Rhizoma Alismatis (adopting Rhizoma Alismatis processed product prepared by traditional bran method for making), Rhizoma Alismatis processed product (adopting processed product prepared by the processing procedure of the application's patent).Below this is all adopted to claim in research.
Experimental technique: get SPF level SD rat and be divided into 10 groups, the raw product groups of 3 dosage Rhizoma Alismatis, 3 dosage Rhizoma Alismatis processed product groups, 3 dosage bran Rhizoma Alismatis groups and blank group.The raw product group of Rhizoma Alismatis, bran Rhizoma Alismatis group and Rhizoma Alismatis processed product group give the raw product of Rhizoma Alismatis and Rhizoma Alismatis processed product decocting liquid (1gkg respectively -1, 0.1gkg -1, 0.01gkg -1), blank group gavage gives isopyknic normal saline.Successive administration is after 7 days, abdominal aortic blood, separation of serum.Take 5 × 10 5the orifice plate (96T) of the mesangial cell that the cell suspension of individual/mL is completed, raw product group gives corresponding administration group rat Contained Serum 200 μ L respectively, and blank group gives blank serum 200 μ L.Administration, after cultivating 24h, mtt assay measures OD value (see table 9).
Table 9 Rhizoma Alismatis raw product affects rat mesangial cell in vitro
Note: compare with blank group, *: P<0.01; Compare with the raw product group of same dose Rhizoma Alismatis, #: P<0.01.
From table 9, Rhizoma Alismatis processed product and the raw product of same dose Rhizoma Alismatis are to proliferation of glomerular mesangial cells difference between the effects significantly (P<0.01).Compared with blank group, the raw product of variable concentrations Rhizoma Alismatis and bran Rhizoma Alismatis all have promotion proliferation of glomerular mesangial cells effect (P<0.01), and Rhizoma Alismatis processed product is then without obviously promoting proliferation of glomerular mesangial cells effect (P>0.05).
Proliferation of glomerular mesangial cells is one of important pathological manifestations of kidney injury, and multiple nephritis all shows as glomerule propagation.Cause extracellular matrix to increase after glomerular mesangium propagation, cause glomerule rate to filter and reduce, serious caused renal fibrosis.Point out thus, this processing procedure method applied for a patent effectively can reduce the effect of Rhizoma Alismatis to Toxicity of Kidney.
4.2 whole animal toxicological experiments
Animal and grouping: SD rat (SPF level) male and female half and half 40.
Test medicine: the raw product of Rhizoma Alismatis, bran Rhizoma Alismatis and Rhizoma Alismatis processed product.
Experimental technique: each group rat excision side kidney, recovers 7 days, is divided at random 4 groups (often organizing 10), namely blank group, Rhizoma Alismatis life product group, bran Rhizoma Alismatis and Rhizoma Alismatis processed product group, each group gives corresponding test medicine.The raw product group of Rhizoma Alismatis gives Rhizoma Alismatis extract powder 4g/kg (being equivalent to clinical dosage 40 times), Rhizoma Alismatis processed product group and bran Rhizoma Alismatis group give Isodose corresponding Rhizoma Alismatis goods extract powder, blank group is to same volume normal saline, administration time 3 months, experimental session is weighed weekly once, and each treated animal presses weight regulation dosage.As a child, respectively organize rat anesthesia, ventral aorta gets whole blood in last administration 1, measures the content of BUN, Scr and circulating immune complex in blood, the results are shown in Table 10 with automatic clinical chemistry analyzer.Put to death rat afterwards, cut kidney, normal saline flushing, 10% formaldehyde is fixed, and prepares tissue slice, and HE dyes.
Table 10 Rhizoma Alismatis raw product is on the impact (x ± s) of kidney BUN, Scr
Note: compare with blank group, *: P<0.05
Experimental result: renal function biochemical indicator test result display (see table 10), heavy dose of Long-term Oral gives the effect that the side kidney excision raw product of rat Rhizoma Alismatis and bran Rhizoma Alismatis are improved kidney of rats merit index-serum urea nitrogen and creatinine content, points out and causes certain damage to kidney.Rhizoma Alismatis processed product then without rising rat blood serum blood urea nitrogen and creatinine effect, points out it to kidney without obvious damage.
Renal tissue section result: the glomerular volume of blank group rat is normal, and mesangial region is without obvious hypertrophy, and extracellular matrix has no increase, and capillary loops is open good.The glomerular volume of the raw product group of Rhizoma Alismatis and bran Rhizoma Alismatis group rat increases, mesangial cell and extracellular matrix moderate hypertrophy, part tubular epithelial cloudy swelling, stove columnar epithelium cell detachment.The glomerular volume of Rhizoma Alismatis processed product group rat slightly increases, and mesangial region is broadening not obvious, mesangial cell and extracellular matrix without hypertrophy, the rare cloudy swelling (see figure 5) of renal tubular epithelial.Prompting, the toxicity of Rhizoma Alismatis processed product is starkly lower than raw Rhizoma Alismatis group and bran Rhizoma Alismatis, points out new processing procedure to have " Attenuation ".
5 drug actions
Test medicine: the raw product decoction pieces of Rhizoma Alismatis, bran Rhizoma Alismatis (the bran Rhizoma Alismatis of preparing with traditional bran method for making), Rhizoma Alismatis processed product (the Rhizoma Alismatis processed product prepared with the application's patent technique)
5.1 effect for reducing blood fat
Administration and modeling: by laboratory animal after experimental situation is raised one week, 6 groups are divided at random by body weight, normal saline blank group, model group, the raw product group of Rhizoma Alismatis, bran Rhizoma Alismatis group, Rhizoma Alismatis processed product group and positive control zhibituo group is divided at random by rat, often organize 10, male and female half and half.Every morning gastric infusion, the isometric normal saline of blank group gavage, except blank group, each treated animal equal gavage Adeps Sus domestica in afternoon reagent 10mlkg -1, every day 1 time, the normal saline of blank group gavage respective volume, model group and test medicine group gavage Adeps Sus domestica Emulsion modeling 2 weeks, period freely absorbs normal feedstuff, freely drinks water.
After 2 weeks, get blood with glass capillary method eyeball, the centrifugal 15min of 3000r.Separation of serum, measures serum TC by test kit description, TG, LDL, HDL, calculates atherogenic index according to atherogenic index=LDL-c/HDL-c, model group TC, TG, LDL, HDL, LDL/HDL ratio, indices is respectively worth with blank group respectively and carries out t inspection, decision model whether success, the TC of each prevention administration group, TG, LDL, HDL, the ratio of LDL/HDL, carries out t inspection with model control group respectively, to evaluate Rhizoma Alismatis raw product prevention administration to the regulating action of rat fat.
Experimental result: effect for reducing blood fat no significant difference compared with traditional bran goods of Rhizoma Alismatis processed product, it regulates high density lipoprotein and serum cholesterol effect to be better than raw product.Prompting, the blood fat reducing drug action of Rhizoma Alismatis processed product is suitable with the drug action of traditional bran goods, but is better than the raw product of Rhizoma Alismatis, serves " potentiation " after the process of preparing Chinese medicine.
The effect that table 11 Rhizoma Alismatis raw product prevention high density lipoprotein reduces
Group Dosage (g/kg) Number of animals (only) High density lipoprotein (mmol ﹒ L -1)
Blank group --- 10 0.95±0.19*
Model control group --- 10 0.80±0.17 ＃
The raw product group of Rhizoma Alismatis 3.50 10 0.85±0.21*
Bran Rhizoma Alismatis group 3.50 10 0.89±0.20* △
Rhizoma Alismatis processed product group 3.50 10 0.90±0.22* △
Zhibituo 1.50 10 0.92±0.18*
Note: compare with model control group, * P<0.05; Compare with blank group, #p<0.05, goods compare with raw product suspension group, △p<0.05.
The effect that table 12 Rhizoma Alismatis raw product prevention low density lipoprotein, LDL raises
Group Dosage (g/kg) Number of animals (only) Low density lipoprotein, LDL (mmol ﹒ L -1）
Normal group --- 10 1.34±0.18**
Model control group --- 10 2.91±0.19 ＃＃
The raw product group of Rhizoma Alismatis 3.50 10 2.25±0.20**
Bran Rhizoma Alismatis group 3.50 10 2.22±0.21**
Rhizoma Alismatis processed product group 3.50 10 1.85±0.19** △△
Zhibituo 1.50 10 2.42±0.18**
Note: compare with model control group, * * P<0.01; Compare with blank group, ##p<0.01; Compare with the raw product group of Rhizoma Alismatis, △ △p<0.01.
The effect of index rising that table 13 Rhizoma Alismatis raw product prevention of arterial is atherosis
Group Dosage (g/kg) Number of animals (only) Atherogenic index
Blank group --- 10 1.43±0.18**
Model control group --- 10 3.79±0.60 ＃＃
The raw product group of Rhizoma Alismatis 3.50 10 2.23±0.34**
Bran Rhizoma Alismatis group 3.50 10 2.52±0.31**
Rhizoma Alismatis processed product group 3.50 10 2.54±0.41**
Zhibituo 1.50 10 2.71±0.37**
Note: compare with model control group, * * P<0.01; Compare with blank group, ##p<0.01.
Table 14 Rhizoma Alismatis raw product preventative adjustment serum total cholesterol metabolism
Group Dosage (g/kg) Number of animals (only) T-CHOL (mmol ﹒ L -1)
Blank group --- 10 1.19±0.27**
Model control group --- 10 2.12±0.28 ＃＃
The raw product group of Rhizoma Alismatis 3.5 10 1.56±0.30**
Bran Rhizoma Alismatis group 3.5 10 1.77±0.27* △
Rhizoma Alismatis processed product group 3.5 10 1.79±0.31* △
Zhibituo 1.5 10 1.68±0.29**
Note: compare with model control group, * P<0.05, * * P<0.01; Compare with blank group, ##p<0.01; Compare with the raw product group of Rhizoma Alismatis, △ P<0.05.
Table 15 Rhizoma Alismatis raw product preventative adjustment serum triglycerides metabolism
Group Dosage (g/kg) Number of animals (only) Triglyceride (mmol ﹒ L -1)
Blank group --- 10 0.77±0.27**
Model control group --- 10 1.24±0.26 ＃＃
The raw product group of Rhizoma Alismatis 3.50 10 0.87±0.27**
Bran Rhizoma Alismatis group 3.50 10 0.76±0.28** △
Rhizoma Alismatis processed product group 3.50 10 0.74±0.31** △
Zhibituo 1.50 10 1.00±0.32**
Note: compare with model control group, * * P<0.01; Compare with blank group, ##p<0.01.Compare with the raw product group of Rhizoma Alismatis, △p<0.05.
Above-mentioned experimental result display, Rhizoma Alismatis processed product has good hypolipidemic activity (P<0.05).5.2 function of invigorating spleen
5.2.1 on the impact of normal rabbit serum gastrin content
The preparation of sample: get SPF level SD rat 70 (male and female half and half), be divided into blank group, the raw product low dose group of Rhizoma Alismatis, the raw product high dose group of Rhizoma Alismatis, bran Rhizoma Alismatis low dose group, bran high dose group, Rhizoma Alismatis processed product low dose group and Rhizoma Alismatis processed product high dose group at random, totally 7 groups.Each group of rat oral gavage administration, blank group gives the carboxymethylcellulose sodium solution of 0.3%, and each dosage of low dose group rat is 2.0gkg -1, each dosage of high dose group rat is 6.0gkg -1.Each administration group gastric infusion every day 2 times.Administration is after 7 days, abdominal aortic blood, after 4-8 DEG C of standing 10min, and 3000rmin -1centrifugal, get serum for careful point; Get duodenum simultaneously, clean up with normal saline.Serum and duodenum sample are stored in not containing in the Ep pipe of thermal source ,-80 DEG C of preservations.
The mensuration of GAS content:
1) at enzyme mark bag by the accurate sample wells 10 of bidding on plate hole, in first, second hole, add standard substance 100 μ L respectively, in first, second hole, then add standard dilutions 50 μ L, mixing; Then from the first hole, the second hole, respectively get 100 μ L be added to the 3rd hole and the 4th hole respectively, then add standard dilutions 50 μ L respectively in the 3rd, the 4th hole, mixing; Then in the 3rd hole and the 4th hole, first respectively get 50 μ L to discard, respectively get 50 μ L and be added to respectively in the 5th, the 6th hole, then in the 5th, the 6th hole, add standard dilutions 50 μ L respectively, mixing; From the 5th, the 6th hole, respectively get after mixing that 50 μ L are added to the 7th respectively, in octal, again the 7th, add standard dilutions 50 μ L respectively in octal, after mixing from the 7th, get 50 μ L respectively octal and be added in the 9th, the tenth hole, add standard dilutions 50 μ L respectively in the 90 hole again, from the 90 hole, respectively get 50 μ L after mixing and discard.(after dilution, each hole application of sample amount is all 50 μ L, and concentration is respectively 240ngL -1, 160ngL -1, 80ngL -1, 40ngL -1, 20ngL -1).2) blank well (blank control wells does not add sample and enzyme marking reagent, and respectively step operation is identical for all the other), testing sample hole is established respectively.In enzyme mark bag is by testing sample hole on plate, first add sample diluting liquid 40 μ L, and then to add the final dilution factor of testing sample 10 μ L(sample be 5 times), rock mixing gently.
3) incubation: use the rearmounted 37 DEG C of incubations of shrouding film shrouding 30 minutes.
4) dosing: by for subsequent use after 20 times of concentrated cleaning solution distilled water 20 times dilution.
5) wash: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, leaves standstill and discards after 30 seconds, so repeats 5 times, pats dry.
6) enzyme-added: every hole adds enzyme marking reagent 50 μ L, except blank well.
7) incubation: use the rearmounted 37 DEG C of incubations of shrouding film shrouding 30 minutes.
8) wash: carefully take shrouding film off, discard liquid, dry, cleaning mixture is filled it up with in every hole, leaves standstill and discards after 30 seconds, so repeats 5 times, pats dry.
9) develop the color: every hole first adds developer A50 μ L, then adds developer B50 μ L, and shake mixing gently, 37 DEG C of lucifuges develop the color 15 minutes.
10) stop: every hole adds stop buffer 50 μ L, cessation reaction (now blue standing turns yellow).
11) measure: with blank well zeroing, the absorbance (OD value) in each hole is sequentially measured at 450nm wavelength place.
Table 16GAS content standard curve
Absorbance 4.139 2.654 1.481 0.699 0.349
GAS content/ngL -1 240 160 80 40 20
Use Excel computed in software, obtain standard curve: y=58.824x-1.6716, r=0.999.The OD value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, be the actual concentrations of sample.Measurement data with represent, adopt SPSS15.0 software kit to carry out correlation analysis, data information compares with t inspection and variance analysis, and P<0.05 is for there being statistical significance.
Experimental result: with blank group and etc. dosage raw product Rhizoma Alismatis group compare, the content of high dose Rhizoma Alismatis processed product to rat blood serum gastrin has rising effect (P<0.05); Low dosage Rhizoma Alismatis bran goods group also shows certain rising trend (see table 17) to the content of rat blood serum gastrin.Compared with blank group, though bran Rhizoma Alismatis is improved the effect of rat blood serum gastrin, compare there was no significant difference with waiting dosage raw product Rhizoma Alismatis group.
Table 17 Rhizoma Alismatis raw product is on the impact of normal rabbit serum gastrin content
Group Dosage (gkg -1） Content (ngL -1）
Blank group - 30.1±3.8
The raw product low dose group of Rhizoma Alismatis 2.0 30.7±3.5
The raw product high dose group of Rhizoma Alismatis 6.0 32.4±1.3
Bran Rhizoma Alismatis low dose group 2.0 31.2±2.5
Bran Rhizoma Alismatis high dose group 6.0 35.6±4.1*
Rhizoma Alismatis processed product low dose group 2.0 32.8±3.4
Rhizoma Alismatis processed product high dose group 6.0 37.7±4.9*#
Note: compare with blank group, *: P<0.01; Compare with the raw product group of same dose Rhizoma Alismatis, #: P<0.05
5.2.2 to normal rat preduodenal Na +-K +the impact of-ATP enzyme content
The preparation of sample: get each group rat intestinal 5-20mg in 4.2.1 and put in homogenizer, adds dehydrated alcohol and normal saline makes the homogenate of 10% in 1:9 ratio, 1500rmin -1centrifugal 10min, gets supernatant 0.2mL, adds the homogenate that 0.8mL normal saline dilution becomes 2%, to be measured.
Na +-K +the mensuration of-ATP enzyme content: at enzyme mark bag by the accurate sample wells 10 of bidding on plate hole, adds standard substance 100 μ L respectively in first, second hole, in first, second hole, then add standard dilutions 50 μ l, mixing; Then from the first hole, the second hole, respectively get 100 μ L be added to the 3rd hole and the 4th hole respectively, then add standard dilutions 50 μ L respectively in the 3rd, the 4th hole, mixing; Then in the 3rd hole and the 4th hole, first respectively get 50 μ L to discard, respectively get 50 μ L and be added to respectively in the 5th, the 6th hole, then in the 5th, the 6th hole, add standard dilutions 50 μ L respectively, mixing; From the 5th, the 6th hole, respectively get after mixing that 50 μ L are added to the 7th respectively, in octal, again the 7th, add standard dilutions 50 μ L respectively in octal, after mixing from the 7th, get 50 μ L respectively octal and be added in the 9th, the tenth hole, add standard dilutions 50 μ L respectively in the 90 hole again, from the 90 hole, respectively get 50 μ L after mixing and discard.Subsequent operation operates with GAS content.
Table 18 duodenum Na +-K +-ATP enzyme content standard curve
Absorbance 2.589833 1.857333 1.433333 0.7585 0.375167
Na +-K +-ATP enzyme content 60 40 30 15 7.5
Use Excel computed in software, obtain standard curve: y=23.623x-2.6396, r=0.998.The OD value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, be the actual concentrations of sample.Measurement data with represent, adopt SPSS15.0 software kit to carry out correlation analysis, comparison t inspection and the variance analysis of data information rate, P<0.05 is for there being statistical significance.
Experimental result: compare with blank group, Rhizoma Alismatis processed product has significantly rising rat preduodenal Na +-K +the effect (P<0.01) of-ATP enzyme content, it acts on the raw product group of comparatively Rhizoma Alismatis obviously (P<0.05), in table 19.
Table 19 Rhizoma Alismatis raw product is to rat preduodenal Na +-K +-ATP enzyme content affects
Group Dosage (gkg -1) Content (pmolg -1)
Blank group - 10.39±0.54
The raw product low dose group of Rhizoma Alismatis 2.0 10.15±2.90
The raw product high dose group of Rhizoma Alismatis 6.0 11.77±1.37
Bran Rhizoma Alismatis low dose group 2.0 10.46±1.72
Bran Rhizoma Alismatis high dose group 6.0 12.33±2.10
Rhizoma Alismatis processed product low dose group 2.0 11.37±1.63#
Rhizoma Alismatis processed product high dose group 6.0 13.31±1.99*#
Note: compare with blank group, *: P<0.01; Compare with the raw product group of same dose Rhizoma Alismatis, #: P<0.05
5.2.3 on the impact of isolated rat duodenum smooth muscle movement function
Grouping and assay method: experiment is divided into 7 groups: the raw product low dose group of Rhizoma Alismatis (2mgmL -1), the raw product high dose group of Rhizoma Alismatis (6mgmL -1), bran Rhizoma Alismatis low dose group (2mgmL -1), bran Rhizoma Alismatis high dose group (6mgmL -1), Rhizoma Alismatis processed product low dose group (2mgmL -1), Rhizoma Alismatis processed product high dose group (6mgmL -1) and blank group, each group liquor strength is final concentration, often organizes 8 sections of intestinal tubes.Rat Fast 24h before SPF level SD rat (male and female half and half) experiment, freely drink water, after de-cervical vertebra is put to death, abdominal cavity is opened immediately along ventrimeson, find rapidly stomach bottom, be about 1cm place in distance stomach bottom and get the one section of duodenum being about 2cm, be placed in the plate filling cold krebs solution, plate is placed on ice cube, passes to 95%O in plate 2and 5%CO 2mist.Careful removal mesentery and fatty tissue, clean intestinal contents.By the threading ligation of diagonal angle, intestinal tube two ends, lower end lead-in wire is connected on fixation hook, and upper end lead-in wire is connected and fixed with micro-pulling-force transducer reed, and load 3g, immerses in the nutritional solution of 37 DEG C of constant temperature completely by intestinal tube, continue to pass to 95%O in bath 2and 5%CO 2mist, the individual bubble/min of throughput (60 ~ 80).First 10-6molL is added in bath after intestinal contraction is stable -1acetylcholine (final concentration) check the energy of intestinal tube, after its effect obviously, rinse 3 times with fresh 37 DEG C of krebs solutions, after stablizing 1h, start administration.Open RM6240B biological functional system, selector channel, and select " tension force " signal, controling parameters is set, filtering F:30Hz, scanning speed: 10.00sdiv -1, load: 3g.With Contraction of The Ileum From A White amplitude, tension force and contraction frequency for statistical indicator, adopt interval measure method to calculate after the administration of each dosage group in 3min with administration before the difference of ileum contraction amplitude and contraction frequency average in 3min, comparing various dose and matched group before and after Rhizoma Alismatis bran system has there was no significant difference.Measurement data with represent, adopt SPSS15.0 software to carry out correlation analysis, comparison t inspection and the variance analysis of data information rate, P<0.05 is for there being statistical significance.
Experimental result: compare with blank group, Rhizoma Alismatis processed product has raising isolated rat duodenum smooth muscle contraction amplitude and relative tension effect (P<0.05), less on contraction frequency impact.In table 20.
The raw product goods of table 20 Rhizoma Alismatis are on the impact of isolated rat duodenum smooth muscle
Note: compare with blank group, *: P<0.01; Compare with the raw product group of same dose Rhizoma Alismatis, #: P<0.05
Above-mentioned experimental result illustrates, the Rhizoma Alismatis processed product adopting the preparation technology applied for a patent to prepare has function of invigorating spleen, and action intensity is higher than traditional bran Rhizoma Alismatis.Its mechanism of action regulates digestive enzyme activity.
5.3 diuresis
5.3.1 to normal rat kidney Na +-K +the impact of-ATP enzyme
The making of sample: get large 70 of SD and be divided into blank group, the raw product low dose group of Rhizoma Alismatis, the raw product high dose group of Rhizoma Alismatis, bran Rhizoma Alismatis low dose group, bran Rhizoma Alismatis high dose group, Rhizoma Alismatis processed product low dose group, Rhizoma Alismatis processed product high dose group at random, totally 7 groups.Each group of rat oral gavage administration, blank group gives the carboxymethylcellulose sodium solution of 0.3%, and each dosage of low dose group rat is 2.0gkg -1, each dosage of high dose group rat is 6.0gkg -1.Each administration group gastric infusion every day 2 times.Administration is after 7 days, abdominal aortic blood, and after 4-8 DEG C of standing 10min, 3000rmin-1 is centrifugal, carefully gets serum; Get kidney simultaneously, clean up (indicating left and right) with normal saline.Serum and kidney samples A are stored in not containing in the Ep pipe of thermal source ,-80 DEG C of preservations.
Assay method: operating procedure is with duodenum Na +-K +the mensuration of-ATP enzyme.
Table 21 kidney Na +-K +-ATP enzyme standard curve
Absorbance 2.589833 1.857333 1.433333 0.7585 0.375167
Standard concentration 60 40 30 15 7.5
Use Excel computed in software, standard curve: y=23.623x-2.6396, r=0.998.The OD value of sample is substituted into equation, calculates sample concentration, then be multiplied by extension rate, be the actual concentrations of sample.Measurement data with represent, adopt SPSS15.0 software kit to carry out correlation analysis, between group, data information compares with t inspection and variance analysis, and P<0.05 is for there being statistical significance.
Experimental result: compare with blank group, the raw product low dose group of Rhizoma Alismatis and Rhizoma Alismatis processed product have significantly reduction rat kidney Na +-K +the effect (P<0.01) of-atpase activity.Compared with the raw product low dose group of Rhizoma Alismatis, Rhizoma Alismatis processed product can obviously reduce rat kidney Na +-K +-atpase activity (P<0.01), in table 22.
Table 22 Rhizoma Alismatis raw product is to rat kidney Na +-K +-ATP enzyme content affects
Group Dosage (gkg -1) Content (pmolg -1)
Blank group - 173.62±5.93
The raw product low dose group of Rhizoma Alismatis 2.0 148.12±5.82*
The raw product high dose group of Rhizoma Alismatis 6.0 158.25±10.95
Bran Rhizoma Alismatis low dose group 2.0 171.83±5.39
Bran Rhizoma Alismatis high dose group 6.0 169.88±4.53
Rhizoma Alismatis processed product low dose group 2.0 167.38±2.93* #
Rhizoma Alismatis processed product high dose group 6.0 159.58±5.34*
Note: compare with blank group, *: P<0.01; Compare with the raw product group of same dose Rhizoma Alismatis, #: P<0.01
5.3.2 to Na in normal rat blood +, K +content impact
Experimental technique: grouping and administration are with kidney Na +-K +-atpase assay method.Each group of rat aorta gets blood, prepares rat blood serum, adopts Na in direct Potentiometric Determination serum +, K +ion concentration.
Experimental result: compare with blank group, Rhizoma Alismatis raw product heavy dose group all obviously can increase Na ion and K ion concentration (P<0.01) in rat blood serum, and compared with raw product group (P<0.01), Na in heavy dose of goods group rat blood serum +, K +increase significantly (P<0.01) (see table 23).
Table 23 Rhizoma Alismatis raw product is to Na in rat blood serum +, K +content affects
Note: compare with blank group, *: P<0.01; Compare with the raw product group of Rhizoma Alismatis, #: P<0.01
The prompting of above-mentioned experimental result, adopt and apply for a patent Rhizoma Alismatis processed product prepared by technique and have diuresis, its mechanism may be by regulating body Na +-K +the activity of-ATP enzyme realizes.
5.3.3 rat urine volume is affected
Experimental technique: get 70 SPF level SD rats, be divided into 5 groups at random, namely blank group, the raw product low dose group of Rhizoma Alismatis, raw product high dose group, Rhizoma Alismatis processed product low dose group and Rhizoma Alismatis processed product high dose group.All before administration, 1h presses 5mLkg -1the normal saline that body weight gavage is 38 DEG C, makes Water l oad.The hypogastric region of light pressure rat before administration, to drain the urine in bladder, then by body weight respectively gavage give each experimental group rat corresponding medicine, the blank group of normal saline giving same volume.After administration, immediately rat is put into metabolic cage respectively, collect its urine, each group rat isuria amount in 12h after record administration.
Experimental result: as can be seen from Table 24: compare with blank group, the raw product group of Rhizoma Alismatis and Rhizoma Alismatis processed product group all have obvious diuresis.Analyze through statistic software SPSS 15.0, each administration group difference is not remarkable, i.e. the diuresis there was no significant difference of the raw product of Rhizoma Alismatis and Rhizoma Alismatis processed product.Prompting, the Rhizoma Alismatis processed product adopting the processing procedure applied for a patent to prepare has diuresis, and its diuresis is no significant difference compared with raw product.
The impact of table 24 Rhizoma Alismatis raw product rat urine volume
Group Dosage (gkg -1) The urine volume (mL) of 12h
Blank group — 7.1±1.5
The raw product low dose group of Rhizoma Alismatis 2.0 21.3±4.0*
The raw product high dose group of Rhizoma Alismatis 6.0 26.6±7.4*
Bran Rhizoma Alismatis low dose group 2.0 9.21±2.5
Bran Rhizoma Alismatis high dose group 6.0 11.40±5.3
Rhizoma Alismatis processed product low dose group 2.0 23.5±6.9*
Rhizoma Alismatis processed product high dose group 6.0 25.4±7.6*
Note: compare with blank group, *: P<0.01
Embodiment one
Rhizoma Alismatis 30kg, adds the mixture that Pericarpium Citri Reticulatae decocts juice 3L and 9 degree of rice vinegar 3L, mixes thoroughly, moistening 2h.In 160 DEG C of medicine roasting machine, first drop into Testa Tritici 10kg, then drop into the Rhizoma Alismatis decoction pieces after moistening, stir-fry 10min, takes out, and sieve removes Testa Tritici, to obtain final product.
Embodiment two
Rhizoma Alismatis 30kg, evenly spray into the mixture that 9 degree of rice vinegar 45L and Pericarpium Citri Reticulatae decoct juice 3L, add Testa Tritici 6kg after 2h, be placed in 150 DEG C of medicine roasting machine, stir-fry 15min, takes out, and sieve removes Testa Tritici, to obtain final product.
Embodiment three
Rhizoma Alismatis 30kg, evenly spray into the mixture moistening 2h that 9 degree of rice vinegar 3L and Pericarpium Citri Reticulatae decoct juice 3L, drop into and first add in 160 DEG C of medicine roasting machine of Testa Tritici 10kg, stir-fry 10min, takes out, and sieve removes Testa Tritici, to obtain final product.
Embodiment four
Rhizoma Alismatis 30kg, evenly spray into the mixture moistening 2h that 9 degree of vinegar 45L and Pericarpium Citri Reticulatae decoct juice 3L, drop in 150 DEG C of medicine roasting machine of first plus wheat bran amount 6kg, stir-fry 15min, takes out, and sieve removes Testa Tritici, to obtain final product.

Claims (4)

1. one kind is reduced the reformed processing method of Rhizoma Alismatis Toxicity of Kidney, it is characterized in that: according to the raw product decoction pieces of Rhizoma Alismatis: 9 degree vinegars: it is 10kg:(1-1.5 that Pericarpium Citri Reticulatae decocts juice) amount of L:1L mixes, after mixture is carried out moistening 2h, add and be placed with in the medicine cookeding pot of Testa Tritici, the mass ratio of Testa Tritici and the raw product decoction pieces of described Rhizoma Alismatis is 5:1-3:1, at the temperature of 150-160 DEG C, parch 10-15min, take out, sieve removes Testa Tritici, obtains Rhizoma Alismatis processed product.
2. a kind of reformed processing method reducing Rhizoma Alismatis Toxicity of Kidney according to claim 1, is characterized in that: described vinegar is rice vinegar, white vinegar or alimentary acetic acid.
3. a kind of reformed processing method reducing Rhizoma Alismatis Toxicity of Kidney according to claim 1, is characterized in that: the Pericarpium Citri Reticulatae decocting liquid concentrated juice of described Pericarpium Citri Reticulatae decocts juice to be every mL containing the crude drug amount of Pericarpium Citri Reticulatae be 1g.
4. a kind of reformed processing method reducing Rhizoma Alismatis Toxicity of Kidney according to claim 1, is characterized in that: the tangent plane of described Rhizoma Alismatis processed product is fallow, has vinegar fragrance; In described Rhizoma Alismatis processed product, the content of 24-alisol acetyl A is 0.025-0.026%, and the content of Alisol B monoacetate is 0.069-0.071%.
CN201310667448.4A 2013-12-09 2013-12-09 Novel processing technology for lowering alisma nephrotoxicity Expired - Fee Related CN103623150B (en)

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CN110314122B (en) * 2018-03-30 2023-04-07 广州箴美科技有限公司 Alisma extract and preparation method and application thereof

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