CN101289494B - Cyclic peptides with vasodilator action, preparation and uses thereof - Google Patents
Cyclic peptides with vasodilator action, preparation and uses thereof Download PDFInfo
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Abstract
The invention discloses a compound of fructus lycii cyclic peptides with a relaxing function, which is extracted from the fructus lycii of natural medical plants and the preparation method and the application thereof. In the preparation method, steps such as refluxing extraction, filtering, concentration, chromatography, etc., are adopted; at the same time, the invention also provides the qualitative detective method of the peptides, the thin and in-situ chemical reaction method and great amount of experiments prove that the cyclic peptides have a strong relaxing function.
Description
Technical field
The present invention relates to a kind of cyclic peptide compound and its production and use, particularly a kind of from the natural medicinal plant wolfberry fruit, extract have wolfberry fruit cyclic peptide compound of vasodilator effect and its production and use.
Background technology
Wolfberry fruit is the traditional Chinese medicine material of China, is the dry mature fruit of plant of Solanaceae lycium barbarum Lycium barbarum.L, and its nature and flavor are sweet, flat, function tonification liver kidney, and replenishing vital essence to improve eyesight, it is thanks to smart to cure mainly consumptive disease, soreness of waist and knee joint; Pharmacological research shows, wolfberry fruit has strengthening immunity, antitumor, anti-aging, the pharmacological action that increases aspects such as hemopoietic function, anti-fatty liveranti-fatty liver; In recent years the chemical constitution study of lycium plant is found to contain multiple amino acids, flavones, lycium barbarum polysaccharide, superoxide-dismutase, alkaloid, sterol isoreactivity composition, the cyclic peptide compound is also found in the dry root skin of lycium plant to some extent, but do not see the report of cyclic peptide compound in the wolfberry fruit so far, do not see that wolfberry fruit has the report of vasodilator effect yet.
Summary of the invention
The objective of the invention is to announce a kind of cyclic peptide compound with vasodilator effect that extracts from the natural traditional Chinese medicine wolfberry fruit, another object of the present invention is to announce the preparation method and the purposes of this compounds.
The present invention seeks to be achieved through the following technical solutions:
The preparation method of cyclic peptide compound of the present invention comprises the steps:
A with 85%～100% alcohol reflux 2-4 time, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.1～1.4 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.0～1.2 suspension, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, the reclaim under reduced pressure ethyl acetate, ethyl acetate extract;
The c ethyl acetate extract separates 1-3 time with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=5-15: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 100-120 ℃ baking oven heating hydrolysis 1-3h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 6-10: 1-3: 1-3; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 100--110 ℃ baking oven heating 2-10 minute, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 1-3 time repeatedly, the cyclic peptide compound.
The preparation method of cyclic peptide compound of the present invention is preferably following steps:
A with 90% alcohol reflux 3 times, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.2～1.3 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.0～1.1 suspensions, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, and the reclaim under reduced pressure ethyl acetate gets ethyl acetate extract;
The c ethyl acetate extract separates 1-3 time with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=10: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 110 ℃ of baking oven heating hydrolysis 1.5h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 8: 2: 2; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 105 ℃ baking oven heating 5 minutes, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 3 times repeatedly, the cyclic peptide compound.
Description of drawings
Fig. 1: single-phase thin-layer chromatogram;
Fig. 2: two-phase thin-layer chromatogram;
Fig. 3: blank group vascular circle tension force record diagram;
Fig. 4: cyclic peptide group vascular circle tension force record diagram.
The present invention extracts the fruit of Chinese wolfberry with alcohol reflux, and extract is used macroporous absorbent resin, silica gel separation and purification successively, extracts and obtains a kind of cyclic peptide compound, and external isolated test shows that this cyclic peptide compound has stronger vasodilator effect; Carry out qualitative screening by the In Situ Thin Layer chemical reaction method and find that the fruit of Chinese wolfberry is positive to this reaction, and favorable reproducibility.
Following experimental example and embodiment are used for further specifying but are not limited to the present invention.
The vasodilatory isolated experiment of experimental example 1 cyclic peptide compound of the present invention: the preparation of myocardium vessel ring and tension detection
Laboratory apparatus: BIOPAC Systems MP-100;
Laboratory sample: the cyclic peptide sample adds the water-soluble solution of 3ml;
Experimental technique: behind the rat anesthesia, win rapidly aorta pectoralis, (g/L:NaCl 7.5, and KCl 0.35, CaCl to place the krebs nutrient solution that leads to oxygen2 2.4，NaHCO
3 1.0～1.25，MgCl
2 0.05，NaH
2PO
40.1, glucose 1.0, pH:7.3～7.4) in; Connective tissue around the separating blood vessel, be cut into the arterial ring of long 3mm, stainless steel wire with two diameter 0.1mm carefully penetrates respectively, make the triangle ring-type, hang in the bath that fills the 20ml nutrient solution, constant temperature is in 37 ℃, one end of sample links to each other with the ventilation hook, the other end links to each other with tonotransducer, and the tension variation of vascular circle continues to pass into oxygen by power-time reflection; Arterial ring is balance 2h under 1.2g tranquillization load, and every 20min changes liquid 1 time.
1, blank group: give 10-6G/ml epinephrine solution 0.2ml excites vascular circle to shrink, and records half an hour.
The result: after adding adrenaline, continuous vessel is shunk, and contraction is slightly on the rise, sees Fig. 3.
2, cyclic peptide group: give 10-6G/ml epinephrine solution 0.2ml excites vascular circle to shrink, and after waiting to shrink the 3min that no longer rises, adds cyclic peptide sample 0.2ml, records half an hour.
The result: after adding cyclic peptide sample 3min, antiotasis descends comparatively obvious, namely begins diastole.
Conclusion: the vasoconstriction effect that the cyclic peptide sample has pair antiadrenergic drug to cause.
Experimental example 2 cyclic peptide compounds of the present invention are tested the effect of isolated rat thoracic aorta vascular circle
1. experiment material: wolfberry fruit cyclic peptide compound, preparation voluntarily.Repone K (KCl), phyenlephrinium (PE), calcium sequestrant (EGTA) are U.S. Sigma company product.Laboratory animal is that male rat (220-270g) is provided by Provincial Medicine Research Institute, Shanxi Province Animal House.
2. experimental technique: after rat was put to death, the thoracic aorta that dissociates was cut into the long vascular circle of 2.0 ~ 3.0mm, vascular circle was placed fill 5ml K-H liquid (37 ℃ of constant temperature, and continue to feed 95%O
2And 5%CO
2Mixed gas) bath in, tension variation is transmitted and is recorded in the Medlab bio signal acquisition processing system.Vascular circle gives 2g tension force and stablizes 60min after stablizing 30min under the 0g tension force, during every 15min change K-H liquid once.KCl with 60mol/L concentration stimulates vascular circle, and vascular circle is shunk, and reaches the maximum amplitude post-flush 3 times, and vascular circle is returned to stimulates preceding state, totally 3 times.The last shrinkage amplitude that causes that stimulates is defined as the contraction standard.Stimulate vascular circle with the PE of 10-6mol/L concentration, give the vagusstoff (ACH) of 10-5mol/L concentration and measure its diastole amplitude after reaching maximum shrinkage amplitude.If the diastole amplitude＞and 80% expression endothelium is complete, shows in the operating process minimumly to the damage of vascular circle endothelium, and the endothelium integrity is good, for the endothelium group is arranged; If not diastole or diastole amplitude＜30% expression endothelial injury is big, endothelial function is imperfect, behind endothelium-denuded group detection endothelial function, flushing is to stimulating preceding state, after stablizing 30min again, during every 15min change liquid once, then further detection of drugs to the influence of vascular circle.
3. grouping: (1) is to causing the effect that vascular circle shrinks to PE in no calcium K-H liquid; (2) add in advance in the K-H liquid of KCl at no calcium calcium chloride is caused the effect that vascular circle shrinks; (3) matrimony vine cyclic peptide compound (2ml/L) incubate bathe the vascular circle 20min of endothelium is arranged after, the ACH of different concns stimulates vascular circle to shrink the influence of back diastole effect to 10-6mol/L PE in advance.
4. statistical procedures: the per-cent (%) that all data all change relative value with the vascular circle shrinkage amplitude is represented and through test for normality, detected result is with mean ± standard deviation (x ± s) expression, (data are represented with mean ± standard error in the table) data information is by computer utilization SPSS10.0 software processes, mean is relatively done the t check between two groups, and mean is relatively done variance analysis between many groups.
5. experimental result
(1) after matrimony vine cyclic peptide compound (2ml/L) is incubated the vascular circle 20min that bathes endothelium-denuded in no calcium K-H liquid (adding 0.1mol/L EGTA in advance), PE to 10-6mol/L concentration causes that the vascular circle contraction has the obvious suppression effect, the inhibition amplitude reaches 70%, (through t check P＜0.01) sees Table 1:
Table 1 matrimony vine cyclic peptide compound is to suppressing the effect data sheet that vascular circle shrinks
(2) after matrimony vine cyclic peptide compound (2ml/L) is incubated and bathed endothelium-denuded vascular circle 20min, calcium chloride is caused that with different concns the effect that vascular circle shrinks sees Table 2 in no calcium K-H liquid (KCl that adds 60mol/L concentration in advance):
Table 2 matrimony vine cyclic peptide compound causes the effect that vascular circle shrinks to calcium chloride with different concns
Table 2 shows that vascular circle obviously reduces the caused contractile response of calcium chloride that adds different concns, and two groups of caused contractile responses of same concentrations calcium chloride through t check P all＜0.01.
6. experiment conclusion
Wolfberry fruit cyclic peptide compound does not have significantly directly effect (comparing P all＞0.05 with control group) to vascular circle; The contraction that PE and KCl are caused all have obvious suppression (with control group than P＜0.05); Internally calcium discharge and outer calcium in stream institute cause contraction all have obvious suppression (with control group compare P equal＜0.01).Therefore, wolfberry fruit cyclic peptide compound has vasodilatory function.
The qualitative detection screening experiment of experimental example 3 wolfberry fruit cyclic peptide compounds
1, specimen preparation
The material 50g that gets it filled extracts three times with methanol eddy, and each 3 hours, extracting solution was condensed into medicinal extract, with the less water dissolving, uses petroleum ether degreasing, uses ethyl acetate extraction again, and is with ethyl acetate part evaporated under reduced pressure, standby with the 1ml dissolve with methanol at last.
2, single-phase thin-layer chromatography
A. get 10mm * 3mm silica-gel plate (plate A1 and plate A2) of two 5 μ L samples are put on two boards respectively, use chloroform: methyl alcohol (10: 1) launches;
B. after waving solvent, plate A1 places, and plate A2 is suspended from the bottom and is added with in the airtight glass jar of the about 2mL concentrated hydrochloric acid of 6mol/L, places the baking oven heating hydrolysis 1-2h that is warming up to 110 ℃;
C. plate A2 is taken out in the cooling back, waves hydrochloric acid in stink cupboard, will spray 0.2% triketohydrindene hydrate acetone reagent simultaneously without the plate A1 and the plate A2 after the hydrolysis of hydrolysis then, places 105 ℃ baking oven heating colour developing in 5 minutes.
The result: mauve spot appears in the plate A2 through hydrolysis, and does not have colour-change without the plate A1 of hydrolysis in the corresponding position.
3, two-phase thin-layer chromatography
A. the silica-gel plate (plate B) of getting a 10mm * 10mm according to two-way thin layer chromatography board point sample method onboard with the sample 5 μ L points that are positive on the single-phase plate, use chloroform: methyl alcohol (10: 1) does first to expansion;
B. after waving solvent, earlier wave hydrochloric acid by the cooling of preceding method concentrated hydrochloric acid pyrohydrolysis after, plate is revolved turn 90 degrees expansion in chloroform-methanol-Glacial acetic acid (8: 2: 2) then;
C. after waving solvent, plate B sprays 0.2% triketohydrindene hydrate acetone reagent, places 105 ℃ baking oven heating colour developing in about 5 minutes.
4, conclusion
Carry out qualitative screening by thin layer in-situ chemical reaction method and find that wolfberry fruit is positive to this reaction, and favorable reproducibility.
Following embodiment all can realize the effect of above-mentioned experimental example.
Embodiment
Embodiment 1: the preparation of cyclic peptide compound of the present invention
A with 90% alcohol reflux 3 times, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.2～1.3 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.0～1.1 suspensions, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, and the reclaim under reduced pressure ethyl acetate gets ethyl acetate extract;
The c ethyl acetate extract separates 3 times with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=10: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 110 ℃ of baking oven heating hydrolysis 1.5h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 8: 2: 2; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 105 ℃ baking oven heating 5 minutes, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 3 times repeatedly, the cyclic peptide compound.
Embodiment 2: the preparation of cyclic peptide compound of the present invention
A with 95% alcohol reflux 4 times, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.1～1.2 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.1～1.2 suspension, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, the reclaim under reduced pressure ethyl acetate, ethyl acetate extract;
The c ethyl acetate extract separates 2 times with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=13: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 105 ℃ baking oven heating hydrolysis 2.5h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 7: 3: 3; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 102 ℃ baking oven heating 8 minutes, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 2 times repeatedly, the cyclic peptide compound.
Claims (8)
1. cyclic peptide compound with vasodilator effect is characterized in that the preparation method of this compounds is:
A with 85%～100% alcohol reflux 2-4 time, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.1～1.4 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.0～1.2 suspension, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, the reclaim under reduced pressure ethyl acetate, ethyl acetate extract;
The c ethyl acetate extract separates 1-3 time with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=5-15: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 100-120 ℃ baking oven heating hydrolysis 1-3h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 6-10: 1-3: 1-3; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 100--110 ℃ baking oven heating 2-10 minute, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 1-3 time repeatedly, the cyclic peptide compound.
2. as cyclic peptide compound as claimed in claim 1, it is characterized in that the preparation method of this compounds is:
A with 90% alcohol reflux 3 times, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.2～1.3 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.0～1.1 suspensions, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, and the reclaim under reduced pressure ethyl acetate gets ethyl acetate extract;
The c ethyl acetate extract separates 1～3 time with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=10: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 110 ℃ of baking oven heating hydrolysis 1.5h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 8: 2: 2; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 105 ℃ baking oven heating 5 minutes, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 3 times repeatedly, the cyclic peptide compound.
3. the preparation method of cyclic peptide compound as claimed in claim 1 or 2 is characterized in that this method comprises the steps:
A with 85%～100% alcohol reflux 2-4 time, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.1～1.4 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.0～1.2 suspension, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, the reclaim under reduced pressure ethyl acetate, ethyl acetate extract;
The c ethyl acetate extract separates 1-3 time with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=5-15: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 100-120 ℃ baking oven heating hydrolysis 1-3h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 6-10: 1-3: 1-3; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 100--110 ℃ baking oven heating 2-10 minute, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 1-3 time repeatedly, the cyclic peptide compound.
4. the preparation method of cyclic peptide compound as claimed in claim 3 is characterized in that this method comprises the steps:
A with 90% alcohol reflux 3 times, merges leaching liquid with wolfberry fruit, leaves standstill, and filters, and filtrate decompression concentrates and reclaims ethanol, makes relative density and be 1.2～1.3 ethanol extraction medicinal extract;
B ethanol extraction medicinal extract adds water, and to make relative density be 1.0～1.1 suspensions, uses sherwood oil, ethyl acetate extraction successively, collects the ethyl acetate part, and the reclaim under reduced pressure ethyl acetate gets ethyl acetate extract;
The c ethyl acetate extract separates 1～3 time with styrenic DIAION HP20 macroporous adsorbent resin column chromatography, and the methanol-water with 0%, 20%, 40%, 60%, 80%, 100% is that eluent carries out gradient elution, collects elutriant by gradient; Each gradient eluent uses the two dimensional develoooment mode to carry out thin-layer chromatography respectively and detects: get the silica-gel plate of 10mm * 10mm respectively, according to two-way thin-layer chromatography point sample method point sample, use chloroform: methyl alcohol=10: 1 carries out first to expansion as developping agent; After expansion finishes and waves solvent, silica-gel plate is suspended from the bottom is added with in the airtight glass jar of 2mL 6mol/L concentrated hydrochloric acid, place 110 ℃ of baking oven heating hydrolysis 1.5h, silica-gel plate is taken out in the cooling back, waves hydrochloric acid in stink cupboard; Silica-gel plate revolved turn 90 degrees, carry out second to expansion as developping agent with chloroform-methanol-Glacial acetic acid of 8: 2: 2; After expansion finishes and waves solvent, spray 0.2% triketohydrindene hydrate--acetone reagent, place 105 ℃ baking oven heating 5 minutes, observe the colour developing spot; Collect the identical flow point of spot colors, promptly get the flow point that contains the cyclic peptide compound; With silica gel column chromatography 3 times repeatedly, the cyclic peptide compound.
5. cyclic peptide compound as claimed in claim 1 or 2 has application in the medicine of vasodilator effect in preparation.
6. application as claimed in claim 5 is characterized in that wherein said vasodilator effect is meant the vasoconstriction effect that antiadrenergic drug is caused.
7. application as claimed in claim 5, it is characterized in that wherein said vasodilator effect is meant to have suppresses phyenlephrinium and Repone K to the caused contraction of vascular circle.
8. application as claimed in claim 5, it is characterized in that wherein said vasodilator effect be meant have suppress in calcium discharge and outer calcium in flow the contraction that is caused.
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| Title |
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| 贾月霞等.枸杞多糖对肾性高血压大鼠血管活性物质的影响.《北京医科大学学报》.1997,第29卷(第5期),429-432. * |
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