CN102861123B - Traditional Chinese medicine extract with effect on promoting angiogenesis as well as preparation method and application thereof - Google Patents
Traditional Chinese medicine extract with effect on promoting angiogenesis as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a traditional Chinese medicine extract with an effect on promoting angiogenesis as well as a preparation method and application of the traditional Chinese medicine extract. The effective parts with pharmaceutical activity are obtained by carrying out water extract and alcohol precipitation, resin separation and purification, organic solvent extraction and chromatographic separation on radix astragali and radix angelicae sinensis as the raw materials. The effective parts as the pharmaceutical components are prepared into an appropriate Chinese patent drug preparation which is convenient to use and improves the pharmaceutical safety. The traditional Chinese medicine extract with the effect on promoting angiogenesis provided by the invention can be applied to a series of diseases caused by angiogenesis insufficiency or decreased angiogenesis, and can reach the purpose of preventing or treating the diseases through adjusting the angiogenesis and the functional state of the organisms or tissues.
Description
Technical field
The invention belongs to plant medicinal active ingredient technical field, relate to and a kind of there is Chinese medicine extract of angiogenesis promoting effect and its preparation method and application.
Background technology
Angiogenesis to refer on original blood capillary basis by the propagation of vascular endothelial cell and migration, generates the process of new blood capillary from the blood vessel of preexist.Angiogenesis plays an important role in physiology, the pathological states such as fetal development, wound healing, endometrical cycle propagation, tumor, proliferative retinopathy.Regulate the unbalance process of body angiogenesis, become the New Policy of some disease treatment.By promoting angiogenesis, can treat and manyly there is angiogenesis deficiency disease, as coronary heart disease, wound and fracture delayed union, thromboangiitis obliterans etc.Owing to not yet there being the effective medicine promoting angiogenesis at present, therefore from Chinese medicine and classical compound recipe, screening extraction is the important channel of new drug research.
Summary of the invention
The problem that the present invention solves is that providing a kind of has Chinese medicine extract of angiogenesis promoting effect and its preparation method and application, is that track and extract screening obtains effective site from Chinese medicine classics recipe Radix Angelicae Sinensis decoction for tonifying blood, can be applicable to short vascularization and/or new life.
The present invention is achieved through the following technical solutions:
A kind of Chinese medicine extract with angiogenesis promoting effect, the extraction crude drug of this Chinese medicine extract is the Radix Astragali and Radix Angelicae Sinensis, it extracts is in mass ratio for 5:1 mixes by the Radix Astragali and Radix Angelicae Sinensis, the concentrated solution reclaiming ethanol gained after water extract-alcohol precipitation is splined on macroporous adsorbent resin, wash with water colourless to effluent after use the alcoholic solution eluting of volumetric concentration 30 ~ 90% again, ethanol is reclaimed after collecting eluting fraction, then organic solvent extraction is used, stationary phase chromatography separation is carried out again by after organic solvent extraction liquid recycling design, dry after collecting mobile phase eluting gained fraction, obtain Radix Angelicae Sinensis decoction for tonifying blood effective site.
Described Chinese medicine extract is in the mass fraction of astragaloside, ferulic acid and Calycosin-7-O-BETA-D-glucoside, and contained astragaloside is no less than 5%, contained ferulic acid is no less than 2%, contained Calycosin-7-O-BETA-D-glucoside is no less than 2%.
A kind of preparation method with the Chinese medicine extract of angiogenesis promoting effect, comprise the following steps: be 5:1 mixing in mass ratio by the Radix Astragali and Radix Angelicae Sinensis, the concentrated solution reclaiming ethanol gained after water extract-alcohol precipitation is splined on macroporous adsorbent resin, wash with water colourless to effluent after the alcoholic solution eluting of volumetric concentration 30 ~ 90% again, ethanol is reclaimed after collecting eluting fraction, then organic solvent extraction is used, stationary phase chromatography separation is carried out again by after organic solvent extraction liquid recycling design, dry after collecting mobile phase eluting gained fraction, obtain Radix Angelicae Sinensis decoction for tonifying blood effective site.
The described preparation method with the Chinese medicine extract of angiogenesis promoting effect, comprises the following steps:
1) Radix Astragali and Radix Angelicae Sinensis are pulverized rear mixing respectively for 5:1 in mass ratio, after being soaked in water, decoct and extract or heating and refluxing extraction, extracted rear collection extracting solution, concentrating under reduced pressure obtains concentrated solution; Concentrated solution adds ethanol to reaching 10 ~ 95% containing alcohol volumetric concentration, leaves standstill after stirring under room temperature or refrigerated condition, to be precipitated completely after filter to obtain supernatant, supernatant reclaims ethanol to tasteless, obtains alcohol deposit fluid;
2) concentrated solution alcohol deposit fluid being reclaimed ethanol gained is splined on macroporous adsorbent resin, first wash with water to effluent colourless, and then with the alcoholic solution eluting of volumetric concentration 30 ~ 90%, collect eluting fraction, reclaim the ethanol in eluting fraction, obtain secondary concentration liquid;
3) secondary concentration liquid is added water after fully stirring and add organic solvent extraction, be separated organic facies extracting solution and concentration and recovery solvent, obtain three concentrated solutions;
4) three concentrated solutions are fixed phase chromatography, immobile phase is non-polar stationary phase or aluminium sesquioxide, the mixed liquor of organic solvent that mobile phase is water and dissolves each other with water, three concentrated solutions are splined on after immobile phase through mobile phase eluting, collect eluting fraction, after vacuum or normal temperature drying, obtain the Chinese medicine extract with angiogenesis promoting effect of powdery.
Described the Radix Astragali and Radix Angelicae Sinensis are pulverized and mixed after, after the water soaking 1 ~ 5h by its quality 5 ~ 10 times, decoct extraction 1.5 ~ 5h or heating and refluxing extraction 1 ~ 5h, extracted rear collection extracting solution, concentrating under reduced pressure obtains concentrated solution;
Described macroporous adsorbent resin is D-101, HPD722, AB-8, DA-201, DS401, DM-301, HPD-100, DM130 or H-103, wash with water to after colourless again with the alcoholic solution eluting that volumetric concentration is 30 ~ 90%, the flow velocity of eluting is 1.0 ~ 2.0ml/min, collects alcohol eluen;
The organic solvent of described extraction is ethyl acetate, chloroform or n-butyl alcohol;
Described non-polar stationary phase is C18, C8 or macro porous silica gel etc., and the described organic solvent dissolved each other with water is methanol, acetonitrile, isopropyl alcohol, acetone or oxolane, the retention time adding to regulate in immobile phase of the organic solvent dissolved each other with water; The flow velocity of stationary phase chromatography separated flow phase is 1.0 ~ 100ml/min.
The relative density of described concentrated solution or secondary concentration liquid is 1.06 ~ 1.10g/ml, and the relative density of concentrated solution is 0.90 ~ 0.95g/ml for the third time;
The volume ratio of the organic solvent that described water and water dissolve each other is 85:15 ~ 20:80.
The application of the described Chinese medicine extract with angiogenesis promoting effect in the short vascularization of preparation and/or rebirth medicine;
Or the application of the not enough or low medicine of angiogenesis is improved in preparation;
Or the application of body or the angiogenesis of tissue and the medicine of state is adjusted in preparation.
Described medicine comprises:
Resist myocardial ischemia, the medicine of anti-brain tissue ischemia, anti-peripheral tissues ischemia;
The medicine of antiulcer, anti-thromboangiitis obliterans, anti-diabetic complication;
Promote wound healing, improve the medicine of union of fracture delay.
With described Chinese medicine extract for medicinal ingredient, be prepared into the preparation of solid, semisolid or liquid form.
Described solid form is tablet, powder, granule, injectable powder or capsule; Semi-solid form is Emulsion, cream, ointment, paste; Liquid form is injection, solution, suspensoid, syrup or the spray made with water or organic solvent, and organic solvent includes but are not limited to ethanol, propylene glycol, vegetable oil.
Compared with prior art, the present invention has following useful technique effect:
The Chinese medicine extract with angiogenesis promoting effect provided by the invention is using the Radix Astragali and Radix Angelicae Sinensis as raw material, the effective site with medical active obtained by water extract-alcohol precipitation, resin separation purification, organic solvent extraction, chromatography.This effective site can make suitable Chinese patent medicine preparation as medicinal ingredient, easy to use, improves medicinal safety.
The Chinese medicine extract with angiogenesis promoting effect provided by the invention, be through and multiple Chinese herbal medicine and compound preparation are screened, and in conjunction with multiple separation method, the active extract part with stronger angiogenesis promoting activity followed the tracks of, finally determine progressively is carried out to the Radix Angelicae Sinensis decoction for tonifying blood of screening gained.On chick embryo allantois membrane modle, be equivalent to crude drug in whole 1gmL
-1radix Angelicae Sinensis decoction for tonifying blood effective site shows and significantly promotes angiogenesis effect, and its effect is better than contained ferulic acid and the main component such as astragaloside, Calycosin-7-O-BETA-D-glucoside.
The Chinese medicine extract with angiogenesis promoting effect provided by the invention, can be applicable to the disease that a series of angiogenesis is not enough or lowly caused, the angiogenesis and functional status thereof that also can be used for by adjusting body or tissue reaches the object of prevention or disease therapy.
Figure of description
Fig. 1-1 ~ 1-9 is different Chinese drug-treated group CAM vascular morphology comparative result figure;
Fig. 2 is that the immobile phase of astragaloside detects collection of illustrative plates;
Fig. 3 is that the immobile phase of ferulic acid detects collection of illustrative plates.
Detailed description of the invention
The invention provides a kind of Chinese medicine extract and the Synthesis and applications thereof with angiogenesis promoting effect, extract the effective site with medicinal effects from Radix Angelicae Sinensis decoction for tonifying blood.Below in conjunction with the compliance test result of specific embodiment and angiogenesis promoting, the present invention is described in further detail, and the explanation of the invention is not limited.
Embodiment 1
The screening of Chinese medicine and the effect of Chinese medicine compound angiogenesis promoting
With the pharmacological action of Chinese crude drug or Chinese medicine compound for foundation, selection may have the Chinese medicine astragalus of angiogenesis promoting effect, Radix Salviae Miltiorrhizae, Radix Angelicae Sinensis, Radix Notoginseng and Chinese medicine compound and return blood-enriching soup, TAOHONG SIWU TANG and decoction for removing blood stasis to be object, adopt chick embryo allantois membrane modle (CAM) to be means of testing, screening assessment wherein acts on the strongest Chinese medicine or Chinese medicine compound.
1, instrument and material
Abundant rich Full-automatic hatching machine (Dezhou, Shandong Ke Yu incubation equipment company limited); Superclean bench (Changping County, Beijing Great Wall air elite engineering company); BFGF bovine basic fibroblast growth factor externally used solution (bFGF, 63000IU/15mL, Yisheng Biological Pharmaceutical Co., Ltd., Shuhai, lot number 20110908); The prepared slices of Chinese crude drugs (Xi'an southern suburbs company of Chinese medicine group, through the qualification of crude drug teaching and research room of department of pharmacy of Xi'an Jiaotong University Medical College); Hatching egg is purchased from agricultural avocation production base, the military region, Shaanxi Province, and every piece heavy 50-60g, for testing in hatching egg output one week; Gelfoam (Jinling Pharmaceutical Co., Ltd., Jinling Pharmaceutical Factory, specification: 6cm × 2cm × 0.5cm).
2, method
2.1 sample preparations take Radix Angelicae Sinensis decoction for tonifying blood (Radix Astragali 90g, Radix Angelicae Sinensis 18g), TAOHONG SIWU TANG (Semen Persicae 9g, Flos Carthami 6g, Radix Rehmanniae Preparata 12g respectively by side, Rhizoma Chuanxiong 6g, Radix Paeoniae Alba 9g, Radix Angelicae Sinensis 9g), decoction for removing blood stasis (Radix Angelicae Sinensis 9g, Semen Persicae 12g, Flos Carthami 9g, Radix Rehmanniae 9g, Fructus Aurantii 6g, Radix Paeoniae Rubra 6g, Radix Bupleuri 3g, Radix Glycyrrhizae 6g, Radix Platycodonis 4.5g, Rhizoma Chuanxiong 4.5g, Radix Achyranthis Bidentatae 9g) and the Radix Astragali, Radix Salviae Miltiorrhizae, Radix Angelicae Sinensis, each 10g of Radix Notoginseng; Above-mentioned single or herbal mixture decoct with water 2 times (first time 2h, second time 1h) respectively, and merge decoction liquor and be concentrated into 1g/mL, filter, get supernatant moist heat sterilization, 4 DEG C save backup.
2.2 gelfoam carrier preparations
Making diameter with card punch under gelfoam aseptic condition is the carrier of 0.5cm, and normal saline infiltrates, and extracts for subsequent use.
2.3 hatching egg are hatched and are selected the fertilized eggs that eggshell is clean, nothing is damaged, fresh (in 1 week output time), and after warm water cleaning, in 1:1000 bromogeramine solution, soak 3-5min, wipe dry, air chamber end upward, is placed in hatch machine and hatches 7d.Temperature controls at 37.8 ± 0.5 DEG C, relative humidity 50% ~ 60%RH, and ventilation and egg-turning cycle are 2h.
2.4 medicines are to the effect of CAM
Hatching egg hatches the 8th day, checks that embryo is hatched and survival condition, discard the hatching egg of necrospermia egg and dysontogenesis under egg lamp.Adopt full random test method for designing, by hatching egg random packet, i.e. saline control group, bFGF positive controls and sample sets, often organizes about 20 eggs.75% alcohol disinfecting egg plenum surface, opens the window of about diameter about 1cm with emery wheel and ophthalmic tweezers, expose CAM, on carrier, reference substance and different testing sample 20uL is dripped with micro sample adding appliance, to be placed in the rare district of CAM central vessel containing drug carrier, adhesive tape closed window, continues to hatch 3d.After 72h, cut off adhesive tape, drip formalin solution 5-10 by observation window and drip, fixation of C AM 15-20min, removes chorion and the membrana putaminis of more than CAM plane, cuts the test zone CAM centered by carrier, launches to be laid on filter paper.
2.5 vascular counts take filter paper as background, take off CAM sample, observe number of blood vessel in carrier edge 1mm so that macro mode shooting is fresh.Send centered by carrier, being less than 45 ° of persons with the angle of carrier radius can include counting in.
2.6 statistical data analysis are with mean ± standard deviation
represent, application SPSS 13.0 statistics software processes experimental data, compares between two between different pharmaceutical and matched group group.P<0.05 is for there being statistical significance.
3, result
3.1 it is vein shape that different Chinese drug-treated group CAM vascular morphology compares Embryo Gallus domesticus CAM angiogenic growth under naturalness, venation is evenly distributed.(see Fig. 1-1 ~ Fig. 1-3) is generated without specificity thick blood vessels around normal saline, Radix Angelicae Sinensis, Radix Notoginseng group carrier; BFGF group and all the other administration groups can be seen new capillary vessel and vertically send from carrier edge around carrier, in radial dense growth in various degree, or have clear and definite tropism angiogenic growth district, the results are shown in Figure 1-1 ~ Fig. 1-9.Wherein, Fig. 1-1 is normal saline group, and Fig. 1-2 is Radix Angelicae Sinensis extract, and Fig. 1-3 returns extract for Radix Notoginseng, Fig. 1-4 returns extract for the Radix Astragali, and Fig. 1-5 is bFGF matched group, and Fig. 1-6 is Radix Angelicae Sinensis decoction for tonifying blood, Fig. 1-7 is decoction for removing blood stasis, and Fig. 1-8 is Radix Salviae Miltiorrhizae extract, and Fig. 1-9 is TAOHONG SIWU TANG.
3.2 different Chinese drug-treated group CAM vascular counts results contrast
Radix Angelicae Sinensis, Radix Notoginseng group compare CAM angiogenesis number to be increased not obvious (P > 0.05) with negative control group, and the Radix Astragali, Radix Salviae Miltiorrhizae, decoction for removing blood stasis, Radix Angelicae Sinensis decoction for tonifying blood, TAOHONG SIWU TANG compare with negative control, CAM angiogenesis number increases more (P < 0.05), especially Radix Angelicae Sinensis decoction for tonifying blood increases obviously, shows its angiogenesis promoting effect obvious.The results are shown in Table 1.
Table 1 different pharmaceutical is on the impact of CAM angiogenesis
| Group | n | Angiogenesis number |
| Normal saline group | 18 | 11.9±3.9 |
| BFGF group | 17 | 26.7±17.4 * |
| The Radix Astragali | 19 | 16.0±9.7 △ |
| Radix Salviae Miltiorrhizae | 17 | 19.0±4.9 △ |
| Radix Angelicae Sinensis | 17 | 14.2±5.3 |
| Radix Notoginseng | 16 | 15.3±4.8 |
| Radix Angelicae Sinensis decoction for tonifying blood | 18 | 23.3±7.9 * |
| Decoction for removing blood stasis | 18 | 18.2±2.2 △ |
| TAOHONG SIWU TANG | 19 | 17.0±12.5 |
Note: compare with normal saline,
△p<0.05,
*p<0.01, increases obviously so select Radix Angelicae Sinensis decoction for tonifying blood to carry out the extraction of effective site due to Radix Angelicae Sinensis decoction for tonifying blood.
Embodiment 2
Angelicae Decoction for Activating Blood is utilized to extract the preparation method of this effective site as follows:
The Radix Astragali and Radix Angelicae Sinensis (5:1) supernatant concentration ethanol after water extract-alcohol precipitation, be concentrated into and measure without after alcohol taste, concentrated solution is splined on macroporous adsorbent resin, wash with water colourless to effluent after use the alcoholic solution eluting of volumetric concentration 30 ~ 90% again, ethanol is reclaimed after collecting eluting fraction, then organic solvent extraction is used, be separated after organic solvent extraction liquid recycling design through stationary phase chromatography, dry after collecting mobile phase eluting gained fraction, obtain the Chinese medicine extract (Chinese angelica blood supplementing decoction extract effective site) with angiogenesis promoting effect.
Concrete, the preparation method of Radix Angelicae Sinensis decoction for tonifying blood effective site, comprises the following steps:
1) Radix Astragali 1000 grams chosen and Radix Angelicae Sinensis 200 grams is got, respectively through being ground into coarse powder mixing after rinsing, drying, soaking by crude drug 8 times amount, 6 times amount and 6 times amount, decocting by 2 hours, 2 hours and 1.5 hours respectively and extracting, merge concentration of liquid medicine, obtain the concentrated solution of 1500 milliliters;
Concentrated solution adds ethanol and reaches 80% to alcohol content, leaves standstill 24 hours under the rear 4 DEG C of refrigerated conditions that stir, and completely rear filtration to be precipitated, must precipitate (A) and supernatant (B), supernatant decompression recycling ethanol, to concentrated solution without alcohol taste, obtain concentrated solution 1700ml;
2) by concentrated solution to processed good D101 resin column, first wash with water to effluent colourless, and then carry out eluting with 80% ethanol, the flow velocity of eluting is 1.0ml/min, collection eluent;
Eluent recycling design, is concentrated into relative density 1.06-1.10(60 DEG C of survey); Obtain water elution liquid extract (C) and alcohol eluen extract (D) respectively; Alcohol eluen extract is effective ingredient, and water elution extracts physical opinion is impurity.For the ease of the screening of active site, therefore water elution and alcohol eluting extract are all kept, to carry out follow-up step.
3) alcohol eluting extract is dissolved with the dilution of appropriate water, add ethyl acetate half amount 300ml to extract, extract three times (each addition is 300ml), combined ethyl acetate extract, carry out concentrating under reduced pressure, thickening temperature is 50 DEG C, and vacuum is 0.09Mpa, be concentrated into solution relative density 0.90-0.95(50 DEG C of survey), obtain acetic acid ethyl ester extract (E) and the hydrotrope (F);
4) acetic acid ethyl ester extract stationary phase chromatography is separated, concrete use high-pressure liquid phase is prepared liquid phase and is prepared separation, it is 0.09Mpa that separating medium carries out vacuum drying vacuum, temperature is 60 DEG C of dry 10-20 hour, acetonitrile-water (volume ratio 17:83) and acetonitrile-water (volume ratio 32:68) is used to carry out eluting respectively, obtain acetonitrile-water (17:83) system elution fraction (G) and acetonitrile-water (32:68) system elution fraction (H), Radix Angelicae Sinensis decoction for tonifying blood effective site G is 0.5g, H is 0.7g.
Angelicae Decoction for Activating Blood extract promotes that angiogenesis effect aspect effective ingredient is containing the composition such as astragaloside and ferulic acid, Calycosin-7-O-BETA-D-glucoside and esters, by high performance liquid chromatography with astragaloside (evaporative light scattering detector) and ferulic acid (UV-detector) for contrast is carried out detecting (HPLC-UV detection is shown in Fig. 1, Fig. 2), ferulic acid determined wavelength is 316nm; In Angelicae Decoction for Activating Blood extract, effective site is in astragaloside, ferulic acid, Calycosin-7-O-BETA-D-glucoside, and astragaloside is no less than 5%, ferulic acid is no less than 2%, Calycosin-7-O-BETA-D-glucoside is no less than 2%
The screening of angiogenesis promoting action component in embodiment 3 Radix Angelicae Sinensis decoction for tonifying blood
1, instrument, material and method are all with example 1
2, test material: attempt differently carrying out extraction and isolation to the composition of Radix Angelicae Sinensis decoction for tonifying blood, using CAM model as method of testing, progressively follows the tracks of active site or composition.Final extraction flow process as described in Example 2, obtains precipitate A (being mainly polysaccharide) and the molten part B of alcohol of water extract-alcohol precipitation; After the molten partially recycled ethanol of alcohol, gained concentrated solution carries out water elution liquid extract C and the alcohol eluen extract D of macroporous resin separation; The E and hydrotrope F that obtain is extracted by alcohol eluen extract portion of ethyl acetate; Acetic acid ethyl ester extract adopts chromatography method to be separated, and obtains acetonitrile-water (17:82) system elution fraction G and acetonitrile-water (32:68) system elution fraction H.Above-mentioned test material at the trial all with normal saline dilution to 1g(crude drug)/ml concentration.
3, result
As table 2, the equal explicitly of angiogenesis number of positive control bFGF group and alcohol molten group (A) is more than saline control group, and precipitate with ethanol group (B), then without explicitly difference, shows that the angiogenesis promoting composition of Radix Angelicae Sinensis decoction for tonifying blood is mainly in the molten part of alcohol.
The angiogenesis promoting Functional observation of table 2 Radix Angelicae Sinensis decoction for tonifying blood water extract-alcohol precipitation composition on CAM model
| Group | n | First order vessel generates number |
| Normal saline group | 16 | 10.9±2.8 |
| BFGF group | 18 | 16.1±3.2 # |
| Precipitate with ethanol group (A) | 15 | 11.9±2.9 |
| Molten group of alcohol (B) | 17 | 15.1±4.7 # |
Note: compare with normal saline group,
#p<0.05
The further extraction test that Radix Angelicae Sinensis decoction for tonifying blood alcohol melt into divides is shown, the alcohol eluen extract D that macroporous resin is separated is the same with positive control bFGF all has obvious facilitation to chick chorioallantoic membrane angiogenesis, and water elution thing is then without significantly effect (the results are shown in Table 3).
The angiogenesis promoting Functional observation of table 3 Radix Angelicae Sinensis decoction for tonifying blood and separated component on CAM model
| Group | n | First order vessel generates number |
| Normal saline group | 18 | 11.5±2.9 |
| BFGF group | 16 | 17.5±4.0 # |
| Water elution thing (C) | 15 | 13.0±3.6 |
| Ethanol elution thing (D) | 16 | 16.3±4.8 # |
Note: compare with normal saline group, #P<0.05
Active extraction and tracking is proceeded to above-mentioned macroporous adsorbent resin ethanol elution thing.As shown in table 4, after this ethanol elution thing extracts by ethyl acetate, the angiogenesis promoting effect of ethyl acetate Extraction parts is obvious, water portion then with negative control group without explicitly difference.
The angiogenesis promoting Functional observation of table 4 Radix Angelicae Sinensis decoction for tonifying blood and separated component on CAM model
| Group | n | First order vessel generates number |
| Normal saline group | 15 | 13.9±3.9 |
| BFGF group | 17 | 26.7±17.4 # |
| Acetic acid ethyl ester extract group (E) | 14 | 23.0±9.7 # |
| Aqueous solution group (F) | 15 | 16.3±7.9 |
Note: compare with normal saline group, #P<0.05
Adopt stationary phase chromatography method to proceed separation and purification above-mentioned acetic acid ethyl ester extract, obtain separated component G and H.After tested, the angiogenesis promoting successful of G part is better than H.Therefore G part is Radix Angelicae Sinensis decoction for tonifying blood effective ingredient.
The angiogenesis promoting Functional observation of table 5 Radix Angelicae Sinensis decoction for tonifying blood and separated component on CAM model
| Group | n | First order vessel generates number |
| Normal saline group | 16 | 14.8±4.9 |
| BFGF group | 15 | 27.1±16.1# |
| Acetonitrile-water (17:82) eluting group (G) | 18 | 24.2±9.2# |
| Acetonitrile-water (32:68) eluting group (H) | 16 | 17.5±8.2 |
Note: compare with normal saline group, #P<0.05
After testing, containing ferulic acid 3.3%, astragaloside 8.5%, Calycosin-7-O-BETA-D-glucoside 7.8% in the Radix Angelicae Sinensis decoction for tonifying blood effective ingredient of said extracted.Therefore on CAM model, compare Radix Angelicae Sinensis decoction for tonifying blood effective ingredient and ferulic acid, astragaloside generate quantity impact on new vessels further.Radix Angelicae Sinensis decoction for tonifying blood effective ingredient is prepared into the solution (being respectively 0.4mg/mL and 4mg/mL) being equivalent to 1g/mL and 10g/mL medical material concentration, separately gets ferulic acid, astragaloside and be mixed with 0.4mg/mL concentration respectively and test.Result is as shown in table 6, and compared with negative control, the ferulic acid of 0.4mg/mL, astragaloside are to the increase not statistically significant promoting CAM new vessels quantity; With this simultaneously, the Radix Angelicae Sinensis decoction for tonifying blood effective ingredient of two kinds of concentration all has obvious facilitation to CAM angiogenesis, and has dependency in certain dosage.The angiogenesis promoting effect of display Radix Angelicae Sinensis decoction for tonifying blood effective ingredient is better than ferulic acid or astragaloside.
The angiogenesis promoting effect of table 6 Ferulic Acid in Tangkuei Blood-Supplementing Decoction-astragaloside mixture (G) component
| Group | n | First order vessel generates number |
| Normal saline group | 15 | 16.6±3.3 |
| BFGF group | 17 | 22.4±5.6 # |
| Ferulic acid group | 14 | 19.4±3.9 |
| Astragaloside group | 15 | 18.2±3.0 |
| Radix Angelicae Sinensis decoction for tonifying blood effective ingredient (1g/mL) | 16 | 22.6±3.2 # |
| Radix Angelicae Sinensis decoction for tonifying blood effective ingredient (10g/mL) | 19 | 27.6±5.0 # |
Note: compare with normal saline group,
#p<0.05
Embodiment 4
There is the preparation method of the Chinese medicine extract of angiogenesis promoting effect, comprise the following steps:
1) Radix Astragali and Radix Angelicae Sinensis are pulverized rear mixing respectively for 5:1 in mass ratio, after being soaked in water, decoct and extract or heating and refluxing extraction, extracted rear collection extracting solution, concentrating under reduced pressure obtains concentrated solution; Concentrated solution adds ethanol to reaching 50% containing alcohol volumetric concentration, leaves standstill after stirring under room temperature or refrigerated condition, to be precipitated completely after filter to obtain supernatant, supernatant reclaims ethanol to tasteless, obtains alcohol deposit fluid;
2) after alcohol deposit fluid being reclaimed ethanol, the concentrated solution of gained is splined on macroporous adsorbent resin, first washes with water to effluent colourless, and then with the alcoholic solution eluting of volumetric concentration 50%, collects alcohol eluen, reclaims the ethanol in eluting fraction, obtains secondary concentration liquid;
Described macroporous adsorbent resin is D-101, DA-201, DS401, DM-301, DM130 or H-103,
3) secondary concentration liquid is added water after fully stirring and making it dissolve, after add organic solvent extraction, be separated organic facies extracting solution and concentration and recovery solvent, obtain three concentrated solutions;
The organic solvent of described extraction is ethyl acetate, chloroform or n-butyl alcohol;
4) three concentrated solutions are fixed phase chromatography, immobile phase is aluminium sesquioxide, mobile phase is the mixed liquor of water and methanol, three concentrated solutions are splined on after immobile phase through mobile phase eluting, the volume ratio of water and methanol is 85:15, collect eluting fraction (time to peak that can refer to respective standard thing is collected), after normal temperature drying, obtain the Chinese medicine extract with angiogenesis promoting effect of powdery.
Embodiment 5
There is the preparation method of the Chinese medicine extract of angiogenesis promoting effect, comprise the following steps:
1) Radix Astragali and Radix Angelicae Sinensis are pulverized rear mixing respectively for 5:1 in mass ratio, after being soaked in water, decoct and extract or heating and refluxing extraction, extracted rear collection extracting solution, concentrating under reduced pressure obtains concentrated solution; Concentrated solution adds ethanol to reaching 60% containing alcohol volumetric concentration, leaves standstill after stirring under room temperature or refrigerated condition, to be precipitated completely after filter to obtain supernatant, supernatant reclaims ethanol to tasteless, obtains alcohol deposit fluid;
2) after alcohol deposit fluid being reclaimed ethanol, the concentrated solution of gained is splined on macroporous adsorbent resin, first washes with water to effluent colourless, and then with the alcoholic solution eluting of volumetric concentration 80%, collects alcohol eluen, reclaims the ethanol in eluting fraction, obtains secondary concentration liquid;
Described macroporous adsorbent resin is HPD722, AB-8, HPD-100 or H-103;
3) secondary concentration liquid is added water after fully stirring and making it dissolve, after add organic solvent extraction, be separated organic facies extracting solution and concentration and recovery solvent, obtain three concentrated solutions;
The organic solvent of described extraction is chloroform or n-butyl alcohol;
4) three concentrated solutions are fixed phase chromatography, immobile phase is C8 or macro porous silica gel, mobile phase be a kind of mixed liquor in water and isopropyl alcohol, acetone or oxolane (wherein, the volume ratio of water and isopropyl alcohol is 80:20, the volume ratio of water and acetone is 40:60, the volume ratio of water and acetone is 70:30), three concentrated solutions are splined on after immobile phase through mobile phase eluting, collect eluting fraction (time to peak that can refer to respective standard thing is collected), after normal temperature drying, obtain the Chinese medicine extract with angiogenesis promoting effect of powdery.
The phenomenon of body new vessels hypofunction or deficiency can be present in myocardial ischemia, brain tissue ischemia, peripheral tissues's ischemia, ulcer, thromboangiitis obliterans, diabetic complication, wound healing slowly, the numerous disease such as union of fracture delay occurs in the pathophysiological process of development, is even likely cause or impel the pathogenetic direct or indirect reason of this type of disease.
Find by following the tracks of screening study, Radix Angelicae Sinensis decoction for tonifying blood effective ingredient has significantly promotion angiogenesis effect.Therefore, by local or Formulations for systemic administration, extract obtained, having and can be applied to a series for the treatment of of diseases that there is the not enough or low state of angiogenesis, also by adjusting angiogenesis and the functional status thereof of body or tissue, using it for the object preventing disease or promote rehabilitation.
There is the application of Chinese medicine extract in the short vascularization of preparation and/or rebirth medicine of angiogenesis promoting effect;
Or the application of the not enough or low medicine of angiogenesis is improved in preparation;
Or the application of body or the angiogenesis of tissue and the medicine of state is adjusted in preparation.
Concrete, the medicine of described angiogenesis promoting effect for being applied to prevention and treatment of diseases such as including but are not limited to myocardial ischemia, brain tissue ischemia, peripheral tissues's ischemia, ulcer, thromboangiitis obliterans, diabetic complication, and promoting wound healing, improving the therapeutic processes such as union of fracture delay.
And for the preparation of Chinese medicine extract, with described Chinese medicine extract for medicinal ingredient, be prepared into the preparation of solid, semisolid or liquid form.
Concrete, described solid form is tablet, powder, granule, injectable powder or capsule; Semi-solid form is Emulsion, cream, ointment, paste; Liquid form is injection, solution, suspensoid, syrup or the spray made with water or organic solvent, and organic solvent includes but are not limited to ethanol, propylene glycol, vegetable oil.
Claims (7)
1. have a Chinese medicine extract for angiogenesis promoting effect, it is characterized in that, the extraction crude drug of this Chinese medicine extract is the Radix Astragali and Radix Angelicae Sinensis, and it extracts is be 5:1 mixing in mass ratio by the Radix Astragali and Radix Angelicae Sinensis, and this Chinese medicine extract is obtained according to following steps:
1) the 1000g Radix Astragali and 200g Radix Angelicae Sinensis is got, respectively through being ground into coarse powder mixing after rinsing, drying, soaking by crude drug 8 times amount, 6 times amount and 6 times amount, decocting by 2 hours, 2 hours and 1.5 hours respectively and extracting, merge concentration of liquid medicine, obtain 1500mL concentrated solution;
Concentrated solution adds ethanol and reaches 80% to alcohol content, leaves standstill 24 hours under the rear 4 DEG C of refrigerated conditions that stir, and completely rear filtration to be precipitated, obtains precipitate A and supernatant B, supernatant B decompression recycling ethanol, to concentrated solution without alcohol taste, obtain 1700mL concentrated solution;
2) by concentrated solution to processed good D101 resin column, first wash with water to effluent colourless, and then carry out eluting with 80% ethanol, the flow velocity of eluting is 1.0mL/min, collection eluent;
Eluent recycling design, be concentrated into 60 DEG C survey time relative density 1.06-1.10; Obtain water elution liquid extract C and alcohol eluen extract D respectively;
3) alcohol eluting extract D dilute with water is dissolved, add ethyl acetate to extract, extract three times, each addition is 300mL, and combined ethyl acetate extract carries out concentrating under reduced pressure, thickening temperature is 50 DEG C, vacuum is 0.09Mpa, is concentrated into 50 DEG C of solution relative densities 0.90 ~ 0.95 when surveying, obtains acetic acid ethyl ester extract E and hydrotrope F;
4) adopt high-pressure liquid phase to prepare liquid phase and phase chromatography is fixed to acetic acid ethyl ester extract E, described immobile phase is C8 or C18, separating medium is 0.09Mpa in vacuum, temperature is at 60 DEG C, vacuum drying 10 ~ 20 hours, carrying out eluting with the acetonitrile-water eluent of volume ratio 17:83, obtain elution fraction G, is Chinese medicine extract effective ingredient.
2. there is the Chinese medicine extract of angiogenesis promoting effect as claimed in claim 1, it is characterized in that, described Chinese medicine extract is in the mass fraction of astragaloside, ferulic acid and Calycosin-7-O-BETA-D-glucoside, and contained astragaloside is no less than 5%, contained ferulic acid is no less than 2%, contained Calycosin-7-O-BETA-D-glucoside is no less than 2%.
3. there is a preparation method for the Chinese medicine extract of angiogenesis promoting effect, it is characterized in that, comprise the following steps:
1) the 1000g Radix Astragali and 200g Radix Angelicae Sinensis is got, respectively through being ground into coarse powder mixing after rinsing, drying, soaking by crude drug 8 times amount, 6 times amount and 6 times amount, decocting by 2 hours, 2 hours and 1.5 hours respectively and extracting, merge concentration of liquid medicine, obtain 1500mL concentrated solution;
Concentrated solution adds ethanol and reaches 80% to alcohol content, leaves standstill 24 hours under the rear 4 DEG C of refrigerated conditions that stir, and completely rear filtration to be precipitated, obtains precipitate A and supernatant B, supernatant B decompression recycling ethanol, to concentrated solution without alcohol taste, obtain 1700mL concentrated solution;
2) by concentrated solution to processed good D101 resin column, first wash with water to effluent colourless, and then carry out eluting with 80% ethanol, the flow velocity of eluting is 1.0mL/min, collection eluent;
Eluent recycling design, be concentrated into 60 DEG C survey time relative density 1.06-1.10; Obtain water elution liquid extract C and alcohol eluen extract D respectively;
3) alcohol eluting extract D dilute with water is dissolved, add ethyl acetate to extract, extract three times, each addition is 300mL, and combined ethyl acetate extract carries out concentrating under reduced pressure, thickening temperature is 50 DEG C, vacuum is 0.09Mpa, is concentrated into 50 DEG C of solution relative densities 0.90 ~ 0.95 when surveying, obtains acetic acid ethyl ester extract E and hydrotrope F;
4) adopt high-pressure liquid phase to prepare liquid phase and phase chromatography is fixed to acetic acid ethyl ester extract E, described immobile phase is C8 or C18, separating medium is 0.09Mpa in vacuum, temperature is at 60 DEG C, vacuum drying 10 ~ 20 hours, carrying out eluting with the acetonitrile-water eluent of volume ratio 17:83, obtain elution fraction G, is Chinese medicine extract effective ingredient.
4. the application of Chinese medicine extract in the short vascularization of preparation and/or rebirth medicine with angiogenesis promoting effect according to claim 1;
Or improve the application in the not enough or low medicine of angiogenesis in preparation;
Or the application in preparation adjustment body or the angiogenesis of tissue and the medicine of state.
5. apply as claimed in claim 4, it is characterized in that, described medicine comprises:
Resist myocardial ischemia, the medicine of anti-brain tissue ischemia, anti-peripheral tissues ischemia;
The medicine of antiulcer, anti-thromboangiitis obliterans, anti-diabetic complication;
Promote wound healing, improve the medicine of union of fracture delay.
6. based on the preparation with the Chinese medicine extract of angiogenesis promoting effect according to claim 1, it is characterized in that, with described Chinese medicine extract for medicinal ingredient, be prepared into the preparation of solid, semisolid or liquid form.
7. have the preparation of the Chinese medicine extract of angiogenesis promoting effect as claimed in claim 6, it is characterized in that, described solid form is tablet, powder, granule, injectable powder or capsule; Semi-solid form is Emulsion, cream, ointment, paste; Liquid form is injection, suspensoid, syrup or the spray made with water or organic solvent, and organic solvent is ethanol, propylene glycol or vegetable oil.
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| CN103432191B (en) * | 2013-09-02 | 2015-11-18 | 南京中医药大学 | A kind of Danggui Buxue Formula microcapsule that can promote angiogenesis and preparation method thereof |
| CN103860744B (en) * | 2014-03-12 | 2016-04-13 | 郑鑫 | A kind of Chinese medicine being used for the treatment of central retinal vein occlusion and preparation method thereof |
| CN106176905A (en) * | 2016-08-29 | 2016-12-07 | 湖南中医药大学 | A kind of Radix Astragali and the application in treatment of vascular neointimal hyperplasia of the angelica medicament compositions |
| CN107441294A (en) * | 2017-08-15 | 2017-12-08 | 上海市中西医结合医院 | A kind of Chinese medicine preparation for treating the concurrent Hypoproteinemia of diabetic foot ulcer |
| CN111249293A (en) * | 2020-03-13 | 2020-06-09 | 湖南中医药大学第一附属医院((中医临床研究所)) | Application of astragaloside in preparation of medicine for promoting angiogenesis |
| CN113082014B (en) * | 2021-04-27 | 2022-05-17 | 正大青春宝药业有限公司 | Salvianolic acid B compound and preparation and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108072A (en) * | 2009-12-24 | 2011-06-29 | 上海张江中药现代制剂技术工程研究中心 | Method for preparing senkyunolide I from extract of Chinese angelica |
| CN102138963A (en) * | 2010-02-03 | 2011-08-03 | 高建 | Chinese angelica and astragalus extract, and preparation method and application thereof |
| CN102241685A (en) * | 2010-05-11 | 2011-11-16 | 四川科伦药业股份有限公司 | Separation method of monomeric compound from astragaloside for injection |
-
2012
- 2012-10-22 CN CN201210405699.0A patent/CN102861123B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102108072A (en) * | 2009-12-24 | 2011-06-29 | 上海张江中药现代制剂技术工程研究中心 | Method for preparing senkyunolide I from extract of Chinese angelica |
| CN102138963A (en) * | 2010-02-03 | 2011-08-03 | 高建 | Chinese angelica and astragalus extract, and preparation method and application thereof |
| CN102241685A (en) * | 2010-05-11 | 2011-11-16 | 四川科伦药业股份有限公司 | Separation method of monomeric compound from astragaloside for injection |
Non-Patent Citations (2)
| Title |
|---|
| 中医药与血管生成研究进展;闫奎坡;《光明中医》;20091231;第24卷(第12期);2410、封三 * |
| 黄芪皂苷的提取及含量测定;王培培等;《中国实验方剂学杂志》;20100430;第16卷(第4期);27-30 * |
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