CN101342259A - Method for extracting alkaloid valid target from hooked uncaria leaf, with hook stem branch or full grass and uses thereof - Google Patents
Method for extracting alkaloid valid target from hooked uncaria leaf, with hook stem branch or full grass and uses thereof Download PDFInfo
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Abstract
The present invention belongs to the technical field of medicine, and discloses a method or an application extracting the valid target of alkaloid from ramulus uncariae cum uncis leaves, stems with hooks or whole grass. The preparation method of the invention comprises: adding hydrous alcoholic solution to ramulus uncariae cum uncis leaves, stems with hooks or whole grass to perlocate or extract with hot reflux; filtering; decompressing; recycling to nonalcoholic taste; adding water or alcoholic solution to concentrated extracting solution; filtering; making filtrate run through a adsorption column filled with adsorbing material; washing the adsorption column with water to the point that effluent liquid is colorless before elution; eluting with 20 to 75 percent of alcoholic solution; collecting eluting liquid; decompressing; recycling solvent and obtaining thick paste; drying and obtaining the valid target of alkaloid. The method does not use toxic organic solvent and does not pollute environment, which has the advantages of less time consumption, high efficiency, simple extracting technology and low cost. The invention can realize industrialization and meet environment protection requirement. The extracted valid target of alkaloid can remedy or prevent senile dementia and other neurodegenerative diseases, high blood pressure and other cardiovascular and cerebrovascular diseases.
Description
Technical field
The invention belongs to medical technical field, relate to a kind of method and purposes of from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, extracting alkaloid valid target, be specifically related to from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, to extract the application in the medicine of cardiovascular and cerebrovascular diseases such as neurodegenerative disease such as preparation treatment or prevention senile dementia and hypertension of the method for alkaloid valid target and effective site thereof.
Background technology
Along with the aging of social population, senile dementia and cerebrovascular disease, malignant tumor have been listed in three big old people's diseases.Senile dementia (Alzheimer Disease is called for short AD) is a kind of agnogenic constitutional brain degenerative disease, is old people's frequently-occurring disease and commonly encountered diseases.AloisAlzheimer at first described this disease in 1907, and at present the cause of disease is still indeterminate, and clinical finally the forfeiture with the memory of carrying out property and the forfeiture of acquired knowledge and viability is feature.The AD sickness rate accounts for dull-witted patient's 40%-60%, and pathological characters is mainly the cortical neuron fibre matting, and (Neurofibrillary tangles is NFTs) with neural inflammatory speckle (Senile plaques, appearance SP) and a large amount of neuronic death.According to statistics, the world today has more than 5,000 ten thousand old peoples to suffer from various degree dementia approximately.AD is 7-8 making a definite diagnosis back mean survival time (MST).The annual need of western countries this class patient spends about 1.5 ten thousand dollars, though China does not have the statistics of this respect, estimates about 20,000,000,000 RMB of annual total cost.This shows that AD has been the heavy burden of family and society, is not only a great medical problem, and is a serious social concern.The research of nearly ten years AD has obtained remarkable progress, and external analyst's prediction AD patient in following 10 years will be increased to about 20%, and the growth rate of AD drug market will have rising by a relatively large margin.
Hypertension refers to that systemic arterial blood pressure increases, it is common clinical syndrome, also can say that it is to increase with contraction and diastolic pressure, often change into the systemic disease of feature with organ dysfunction such as heart and brain, kidney or device matter, this disease can be by due to the pathogenesis of multiple pathogenic factors and complexity, central nervous system function imbalance, the heredity of body fluid endocrine, the dysfunction of kidney cerebrovascular pressure receptor, and the cell membrane ion transport is unusual etc. all can cause hypertension.Hypertension is a commonly encountered diseases, it is again the most important risk factor that causes human dead principal disease such as coronary heart disease, cerebrovascular disease etc., the sickness rate of the heart, cerebrovascular disease and case fatality rate are all closely related with patient's blood pressure level, therefore, control and the research to hypertension is all paid much attention in countries in the world.Show that according to national hygiene department statistics the existing patients with hypertension of China has surpassed 100,000,000 people, more than annual newly-increased 3,000,000 people; Surplus the existing patients with cerebral apoplexy 500 ten thousand, annual neopathy 1,500,000 people, dead 200,000 people.And mondial hypertension sickness rate is still in rising trend, and China has become the most serious country of hypertension harm in the world.
Therefore, the medicine of cardiovascular and cerebrovascular diseases such as neurodegenerative disease such as research and development treatment or prevention senile dementia and hypertension has huge market prospect.
Ramulus Uncariae Cum Uncis, original name " Ramulus Uncariae cum Uncis ", the beginning is stated from " Mingyi Bielu ", classifies low-grades as.Cool in nature, sweet in the mouth are returned liver, pericardium channel, and the heat clearing away suppressing the hyperactive liver is arranged, the effect of dispelling wind and relieving convulsion." Chinese pharmacopoeia has stipulated that the Chinese medicine Ramulus Uncariae Cum Uncis is the dry stem branch of buckle of Maguireothamnus speciosus Ramulus Uncariae Cum Uncis Uncaria.rhynchophylla (Miq.) Jacks., Uncaria sinensis (Oliv.) Havil. U.sinensis (Oliv.) Havil., Ramulus Uncariae macrophyllae U.macrophylla Wall., Ramulus Uncariae Cum Uncis U.hirsuta Havil., stockless fruit Ramulus Uncariae Cum Uncis U.sessilifructus Roxb. to version in 2005.Bibliographical information, Ramulus Uncariae Cum Uncis alkaloid composition has effects such as calmness, blood pressure lowering, convulsion, epilepsy, neuroprotective cell, anti-cerebral ischemia/reperfusion injury.Clinically, there is the Rhomotoxine sheet of Ramulus Uncariae Cum Uncis preparation to can be used for treating hypertension; Ramulus Uncariae Cum Uncis and Rhizoma Gastrodiae compatibility can be used for treating vascular dementia, can obviously improve patient's cognitive function and behavioral competence, can also treat diseases such as liver-Yang sthenia type hypertension, hypertensive cerebral hemorrhage.
But do not see that at present the total alkaloid content that extracts with Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb is the oral formulations listing of cardiovascular and cerebrovascular diseases such as neurodegenerative disease such as primary raw material treatment or prevention senile dementia and hypertension greater than 50% alkaloid valid target.
Summary of the invention
The object of the invention has been to provide a kind of method of extracting alkaloid valid target from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, and the method is easily gone, and easy and simple to handle, with low cost, the alkaloid component extraction ratio is higher in the effective site.Total alkaloid contents is 50-90% in effective site, and wherein the content sum of Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine, isocorynoxeine is greater than 40%.
The application of the alkaloid valid target that another object of the present invention is to provide extraction in the medicine of cardiovascular and cerebrovascular diseases such as neurodegenerative disease such as preparation treatment or prevention senile dementia and hypertension.
The objective of the invention is to be achieved through the following technical solutions:
A kind of method of from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, extracting alkaloid valid target, its step is as follows:
A. adding determining alcohol in Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb is that 40～95% aqueous alcohol solutions mercerations, percolation or hot reflux are extracted, the aqueous alcohol solutions consumption is Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb weight 6～30 times, filter, merging filtrate, reclaim under reduced pressure gets Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb concentrated extracting solution to there not being the alcohol flavor.
B. add the water or the alcoholic solution of 4～10 times of Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb weight in Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb concentrated extracting solution, determining alcohol is 10～60% in the solution, filter, filtrate.
C. filtrate is through being filled with the adsorption column of adsorbing material, and the adsorbing material of selecting for use is any in macroporous adsorbent resin or the ion exchange resin.
D. before the eluting, selecting volume for use is that the water of 4～8 times of adsorbing materials or 20～40% pure drip washing adsorption column to effluent are colourless.
E. with the alcoholic solution eluting that contains 40～90%, the eluent consumption is 4～10 times of adsorbing material, collects eluent.
F. decompression and solvent recovery, get thick paste, thick paste gets Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb alkaloid valid target after drying, and total alkaloid content is 50～90%, and wherein the content sum of Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine and isocorynoxeine is greater than 40%.The dried cream of gained alkaloid valid target is adopted conventional pulverizing or solid dispersion technology or micronizing, get the different-grain diameter powder.
Used alcohol is preferably a kind of in ethanol or the methanol in the described alcoholic solution.
The model of described macroporous resin is wherein a kind of of AB-8, SP-70, D101, DM-130, S-8, D3520, D4020, HP-20.
Prepared Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb alkaloid valid target among the present invention, add an amount of adjuvant or substrate (as starch, dextrin, microcrystalline Cellulose, lactose, low-substituted hydroxypropyl cellulose, octyldodecanol, mannitol etc.), be prepared into the oral formulations that is used for the treatment of or prevents cardiovascular and cerebrovascular diseases such as neurodegenerative disease such as senile dementia or hypertension, comprise tablet (plain sheet, coated tablet, Film coated tablets, dispersible tablet), capsule (hard capsule, soft capsule), granule and drop pill.
The pharmacodynamics of the alkaloid valid target that the present invention is prepared and preliminary toxicologic study:
1, the anti-microglia activating activities of Ramulus Uncariae Cum Uncis leaf alkaloid valid target and main alkaloid monomeric compound
The chronic inflammatory reaction of microglia mediation be several main neurodegenerative diseases such as Alzheimer (Alzheimer ' s disease, AD), parkinson disease (Parkinson ' s disease, PD) etc. take place, the important step in the evolution, a variety of causes activates startup such as proinflammatory cytokine that microglia discharged and free radical or has increased the weight of neuronic damage, and neuronic damage can further activate microglia conversely, forms the pathological process of a vicious cycle.Thereby, suppress microglial activation and can effectively block or alleviate the consequence that this vicious cycle causes.This experiment activates microglia abnormal activation model by external lipopolysaccharide (LPS); to activate microglia release NO is index, estimates Ramulus Uncariae Cum Uncis leaf alkaloid valid target and main alkaloid composition by suppressing the neuroprotective systemic effect of microglia abnormal activation performance.
A, former generation microglia cultivation
Get neonate rat (giving birth in the 2d of back) cerebral cortex under the aseptic condition, place D-Hanks liquid, divest meninges and blood vessel.0.25% trypsinization, 10～15min stops digestion with the IMDM culture medium that contains 10% hyclone, pasteur pipet piping and druming, 200 eye mesh screen mechanical filters, preparation mixed cell suspension.Counting is adjusted into cell density (3～5) * 10
6Cells/mL plants in 75cm2 band screw socket cap cell culture bottle by every bottle of 5～10mL kind, and the culture medium composition: the IMDM culture fluid, 5% hyclone is added antibiotics (penicillin 100 μ g/mL, streptomycin 100 μ g/mL).The pine lid leaves standstill and is incubated at CO
2Incubator (37 ℃, 5%CO
2, 95% air) in, liquid changed once to remove the dead cell fragment with the equivalent culture medium behind the 48h.3～4d changes liquid once at interval later on, is cultured to 11～14d, collecting cell.The results proxima luce (prox. luc) changes serum-free medium, tightens bottleneck next day, puts 37 ℃ of constant temperature shaking table shakes about 2 hours, and 250rev/min collects supernatant, and this moment, the supernatant cell density generally was not less than 5 * 10
5Cells/mL.CO is planted in the 75mL Tissue Culture Flask in the cell suspension transfer that collection obtains again
2Incubator leaves standstill to be cultivated after about 4 hours, and the jog culture bottle adds fresh medium to remove the adherent not composition (being mainly oligodendrocyte and astrocyte) in jail under the room temperature after the sucking-off.
B,
Riess method detection Ramulus Uncariae Cum Uncis leaf alkaloid valid target and main alkaloid monomeric compound activate the inhibitory action of microglia to LPS
The take the logarithm rat microglia of former generation of trophophase transfers to 5 * 10 with the fresh IMDM culture medium that contains 5% hyclone with cell density
5Cells/mL is inoculated in 96 holes pull, 100 μ L/well, and in 37 ℃, 5%CO
2Incubator in cultivate.Change the fresh medium of serum-free behind the cell attachment cultivation 24h into, carry out dosing simultaneously and handle, with the LPS combined effect.Each concentration is established three parallel holes.Establish blank and positive control (resveratrol) simultaneously.The LPS final concentration is 1 μ g/mL in each administration group and the positive controls.After continuing after the cell dosing to cultivate 48h, collect supernatant, the Griess colorimetry detects NO in the supernatant
2 -Content.The NO burst size of LPS stimulating group is set at 100%, and the NO burst size of all the other each groups is expressed as relatively and the percentage composition of LPS stimulating group.The results are shown in Table 1.
The result: Ramulus Uncariae Cum Uncis leaf alkaloid valid target induces microglia NO to discharge median effective dose (IC to LPS
50) be 6.3 μ g/mL, the IC of alkaloid monomer chemical compound
50The value scope all shows significant inhibitory effect at 13.7-19.0 μ M.But Ramulus Uncariae Cum Uncis leaf or stem and branch with belt hook crude extract all fail to show the obvious suppression effect.Therefore, adopt purification by macroporous resin and the alkaloid valid target enrichment Ramulus Uncariae Cum Uncis alkaloids composition, improved pharmacologically active, and reduced dosage, have medical value.
Table 1 Ramulus Uncariae Cum Uncis leaf alkaloid valid target and main alkaloid monomeric compound
LPS is induced the inhibitory action of microglia NO release
2, the acute toxicity test of Ramulus Uncariae Cum Uncis leaf alkaloid valid target
50 of mices, male and female half and half, body weight are 18-20g.Be divided into 5 groups at random by body weight and sex, 10 every group.Fasting be can't help water after 16 hours, every treated animal is irritated the Ramulus Uncariae Cum Uncis leaf alkaloid valid target that stomach gives various dose respectively, initial dose is 2500mg/kg, the administration capacity is 0.2mL/kg, the dosage ratio is 1: 0.75 between adjacent set, observes record chmice acute toxic reaction and death toll then continuously 7 days, dissect dead mice and survival mice, observing each main organs tissue has no abnormal.Use composite accounting to calculate the LD of Ramulus Uncariae Cum Uncis leaf alkaloid valid target gastric infusion
50Value.The results are shown in Table 2.
The result shows: mouse stomach gave Ramulus Uncariae Cum Uncis leaf alkaloid valid target respectively after 5 minutes, and the part laboratory animal range of activity of dosage＞1054mg/kg reduces, and rapid breathing, trembled, and struggled and dead at last, and be 60 minutes dead rush hour.But dead mice and survival mice, each main organs is organized all no abnormal.Mouse stomach gives the LD of Ramulus Uncariae Cum Uncis leaf alkaloid valid target
50Value is 1406.4 ± 198.9mg/kg.Because the dull-witted active ED of Rhomotoxine antihypertensive active and resisting cerebrovascular
50Value is respectively 20.0mg/kg and 15.0mg/kg, and therefore, this Ramulus Uncariae Cum Uncis leaf alkaloid valid target has higher therapeutic index, and the clinical drug safety scope is bigger.
Table 2 mouse stomach gives the LD of Ramulus Uncariae Cum Uncis leaf alkaloid valid target
50Value
3, the cytotoxicity rapid screening of Ramulus Uncariae Cum Uncis alkaloid monomer chemical compound test
(neutral red uptake assay, NRU): Chinese hamster pneumonocyte (CHL) and Chinese hamster ovary cell (CHO) are with containing the MEM culture medium of 10% Ox blood serum at 5%CO to adopt the dimethyl diaminophenazine chloride absorption process
2, the conventional cultivation in 37 ℃ of saturated humidity incubators.The cell growth is added respectively for test agent in CHL and Chinese hamster ovary celI near monolayer behind the 24h, continues to cultivate 24h.Cultivate and finish preceding 2h, every hole adds dimethyl diaminophenazine chloride (NR) solution (250 μ g/mL) 50 μ L.After cultivating end, use the PBS washed cell, add extract (1% acetic acid-50% ethanol water) 100 μ L in every hole, room temperature is placed 20min, makes the whole strippings of NR in the cell.Fully the vibration back uses microplate reader to measure absorbance under wavelength 540nm.The cell survival rate of blank group is set at 100%, and the cell survival rate of all the other each groups is expressed as relatively and the percentage composition of blank group.The results are shown in Table 3.
Table 3 Ramulus Uncariae Cum Uncis alkaloids monomeric compound is to the toxic action of CHL and Chinese hamster ovary celI
The result as seen, the alkaloid monomer chemical compound of each constituent is not seen overt toxicity to Chinese hamster ovary celI, IC in the Ramulus Uncariae Cum Uncis leaf alkaloid valid target
50＞300 μ M; But Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine, isocorynoxeine is bigger to the survival rate influence of CHL cell, IC
50Value is respectively 273,10.9,180,70.9 μ M, and vincoside-lactam is not seen overt toxicity to the CHL cell, IC
50＞300 μ M.Conclusion: be equipped with constituent in the Ramulus Uncariae Cum Uncis leaf alkaloid valid target and under each dosage, Chinese hamster ovary celI do not seen overt toxicity, only under higher dosage, the CHL cell is shown certain cytotoxicity, belong to low toxicity material.
4, Morris water maze laboratory
75 of a, animal and model: 12-24 monthly age Healthy female SD rats, body weight 340 ± 37g with 70 of the good rats of Morris water maze laboratory screening learning and memory, is divided into blank group, operation group and sham operated rats at random.
B, model production method: permanent ligation bilateral common carotid arteries method.Three groups of rats are raised with under the condition, carry out the Morris water maze test in 1 month after surgery, and observation index is an average escape latency, tries to achieve blank cell mean and standard deviation, is standard with the blank group, are judged to the ability of learning and memory obstacle greater than this value.From the operation group, filter out the rat of ability of learning and memory obstacle, be divided into model group, administration group and blank group once more at random.
C, administrated method: the administration group is irritated stomach and is given Ramulus Uncariae Cum Uncis leaf alkaloid valid target 15mg/kg, and every day 1 time, dosage changes 5mg/kg into after 1 week.Behind medication 1.5 months and 2.5 months (be postoperative 2.5 and 3.5 months), carry out the Morris water maze test respectively to detect ability of learning and memory.The blank group substitutes Ramulus Uncariae Cum Uncis leaf alkaloid valid target with normal saline, and all the other are consistent with the administration group of methods.
D, Morris water maze: the pool diameter 100cm of water maze, dark 50cm, depth of water 30cm, 26 ± 2 ℃ of water temperatures, the water surface covers one deck foam plastics bits, and four equidistant points diversion basins are four quadrants on the pool wall, and an optional quadrant is placement platform in the central.The platform water white transparency, diameter 6cm, high 28cm, platform be not in underwater 2cm, and the peripheral pool object of reference remains unchanged.
E, orientation navigation test: experiment lasts 5, every day 4 times.The operator is put rat people's water towards pool wall from different quadrants, and the record rat is found the time (escape latency) of platform, if can not find platform in the 120s, then helps its upper mounting plate by the operator.After rat stands on platform 10s, rat is lifted down rest 60s from platform, test by next quadrant people water according to the order of sequence again.
F, observation index: escape latency.
G, data statistics: with the SPSS.10.0 statistical software average escape latency is carried out the variance analysis of one factor analysis of variance (one-way ANOVA) and repeated measure (repeated measures) data, Dunnett-t check and LSD check.The results are shown in Table 4.
H, result: postoperative 3.5 months (being medication 2.5 months), administration group rat average escape latency obviously shortens than model group and blank group rat.The result shows that Ramulus Uncariae Cum Uncis leaf alkaloid valid target can improve the vascular dementia rats ability of learning and memory.
Table 4 is respectively organized the comparison (s) of rat in different period average escape latency
5, Ramulus Uncariae Cum Uncis leaf alkaloid valid target is to the hypertensive influence of rat experiment
A, survey rat normal arterial pressure: get the rat about healthy weight 200g, random packet is surveyed normotensive value, continuous measurement 3 days, and record with the rat measuring cell.
B, duplicate hypertension model: the METHOD FOR CONTINUOUS DETERMINATION blood pressure is after 3 days, every day subcutaneous injection androlin 4mg, totally 14 days.When rat blood pressure obviously is increased to 1.3kPa (10mmHg), treatment can experimentize.
Table 5 Ramulus Uncariae Cum Uncis leaf alkaloid valid target is to the hypertensive influence of rat experiment
C, experimental therapy: irritate stomach every day and give and rat Ramulus Uncariae Cum Uncis leaf alkaloid valid target 20mg/kg, continuous 16 days, 1 blood pressure of survey in per then 4 days; Compare for respectively reserpine and distilled water for two groups in addition.The results are shown in Table 5.
E, result show: Ramulus Uncariae Cum Uncis leaf alkaloid valid target has obvious hypotensive effect.
6, Ramulus Uncariae Cum Uncis leaf alkaloid valid target is to the influence of platelet aggregation
Animal SD rat, male, body weight 250～350g is divided into 4 groups.Intravenous injection gives Ramulus Uncariae Cum Uncis leaf alkaloid valid target, aspirin (ASA) or the contrast liquid of various dose respectively, get blood behind the 20min, add 3.8% sodium citrate (9: 1) anticoagulant, centrifugal, separate platelet rich plasma (PRP) and platelet poor plasma (PPP), platelet count is 4 * 10 among the adjusting PRP
8/ mL by turbidimetry, measures the platelet aggregation reaction with PAM-2 type PPP autobalance platelet aggregation instrument (Shanghai Medical Univ's product).Derivant be arachidonic acid (from) 200 μ mol/L, adenosine diphosphate sodium salt (ADP) 4.0 μ mol/L and collagen 40 μ g/mL.
As a result, after rat intravenous injection gave Ramulus Uncariae Cum Uncis leaf alkaloid valid target 10,20mg/kg or aspirin 30mg/kg, its platelet aggregation rate obviously descended than control rats, sees Table 6.The result shows that Ramulus Uncariae Cum Uncis leaf alkaloid valid target might become a kind of clinical effective medicament for resisting platelet aggregation.
Table 6 Ramulus Uncariae Cum Uncis leaf alkaloid valid target is to the inhibitory action of AA, ADP or collagen-induced platelet aggregation
The present invention compared with prior art has the following advantages and effect:
Along with the arrival of world's aging society, the control of senile memory dysfunctions such as senile hypomnesis, alzheimer disease seems and becomes more and more important.A large amount of research data both domestic and external shows that per ten old peoples just have a dementia symptom that manifests in various degree, are having a strong impact on people's Health and Living quality.In addition, hypertension also is to cause human dead principal disease, as the main paathogenic factor of coronary heart disease, cerebrovascular, is the commonly encountered diseases and the frequently-occurring disease of harm humans health.Therefore, the new drug of cardiovascular and cerebrovascular diseases such as neurodegenerative disease such as research and development treatment or prevention senile dementia and resisting hypertension has huge market prospect.
The preparation of this effective site need not deleterious organic solvent, free from environmental pollution, be on the basis of conventional aqueous vehicle extracting method, adopted the adsorption separation technology of adsorbing material, consuming time few, the efficient height, extraction process is simple, with low cost, can carry out industrialization production, and reach environmental protection requirement.And ethanol can reclaim repeated use, uses after macroporous resin and ion exchange resin also can be handled again, and has reduced production cost, is convenient to promote, and therefore, powerful competitiveness is arranged on the market at home and abroad.Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb are taken from natural resources; the alkaloid valid target toxic and side effects of extracting in Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or the herb is less; safe and reliable; has multiple biological activity; its intensive neurocyte protection effect and hypotensive effect are very useful to cardiovascular and cerebrovascular diseases such as treatment or prevention neurodegenerative disease such as senile dementia and hypertension.Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb are very abundant in Natural Resources in China, and from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, extract the alkaloids effective ingredient and be used for new drug development, be an effective measure effectively utilizing natural resources.The present invention has protection and therapeutical effect and antihypertensive function by the alkaloid valid target that extracts in test of pesticide effectiveness proof Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or the herb to neurocyte, for clinical application provides theoretical foundation.
The specific embodiment
Embodiment 1
The 75% alcoholic solution percolation that adds 8 times of Ramulus Uncariae Cum Uncis stem and branch with belt hook coarse powder weight extracts, filters, and merging filtrate, reclaim under reduced pressure obtains Ramulus Uncariae Cum Uncis stem and branch with belt hook concentrated extracting solution to there not being the alcohol flavor.Concentrated extracting solution is diluted with water to the medicinal liquid of 6 times of Ramulus Uncariae Cum Uncis stem and branch with belt hook amounts, filters, filtrate.With filtrate flow through being filled with DM-130 macroporous resin absorption post, 80% ethanol elution with 6 times of amounts, decompression and solvent recovery, get thick paste, the thick paste drying must contain the dried cream that total alkaloids is 76.0% (survey of HPLC method), and wherein the content sum of Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine and isocorynoxeine is 40.1% (survey of HPLC method).Dried cream adopts micronizing technology to pulverize, and gets 1 part of dried cream powder and 0.1 part of microcrystalline Cellulose, granulate with 70% alcoholic solution, and drying, granulate adds 1% magnesium stearate, mixing, fill capsule, every 15mg.
Embodiment 2
The 75% alcoholic solution percolation that adds 10 times of Ramulus Uncariae Cum Uncis leaf coarse powder weight extracts, filters, and merging filtrate, reclaim under reduced pressure obtains Ramulus Uncariae Cum Uncis leaf concentrated extracting solution to there not being the alcohol flavor.Concentrated extracting solution with the medicinal liquid of 10% ethanol dilution to 8 times on Ramulus Uncariae Cum Uncis leaf, is filtered, get filtrate.With filtrate flow through being filled with D101 macroporous resin absorption post, extremely colourless with 30% ethanol drip washing of 6 times of amounts earlier to effluent, 75% ethanol elution of 6 times of amounts of reuse, decompression and solvent recovery, get thick paste, the thick paste drying must contain the dried cream that total alkaloids is 70.3% (survey of HPLC method), and wherein the content sum of Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine and isocorynoxeine is 42.4% (survey of HPLC method).Dried cream powder after 1 part of pulverizing is mixed the back granulate with 0.1 part of microcrystalline Cellulose with 80% alcoholic solution, drying, granulate adds 1% magnesium stearate, mixing, tabletting, the bag film-coat gets Film coated tablets, every 15mg.
Embodiment 3
The 75% alcoholic solution merceration that adds 12 times of Ramulus Uncariae Cum Uncis leaf coarse powder weight extracts 12h, filters, and merging filtrate, reclaim under reduced pressure obtains Ramulus Uncariae Cum Uncis leaf concentrated extracting solution to there not being the alcohol flavor.Concentrated extracting solution with the medicinal liquid of 20% ethanol dilution to 6 times of Ramulus Uncariae Cum Uncis leaf amounts, is filtered, get filtrate.With filtrate flow through being filled with AB-8 macroporous resin absorption post, extremely colourless with 30% ethanol drip washing of 6 times of amounts earlier to effluent, 6 times of amounts of reuse, 55% ethanol elution, collect 55% ethanol elution, decompression and solvent recovery, get thick paste, the thick paste drying must contain the dried cream that total alkaloids is 76.6% (survey of HPLC method), and wherein the content sum of Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine and isocorynoxeine is 41.5% (survey of HPLC method).Among the PEG that the broken postposition of dried cream powder has melted, treat that medicine is dispersed in the liquid carrier fully after, put in the drilling pill device, splash in the liquid paraffin, drip and make piller.
Embodiment 4
The 75% alcoholic solution hot reflux that adds 8 times of Ramulus Uncariae Cum Uncis herb coarse powder weight is extracted 2 times, and each return time 1.5 hours filters, merging filtrate, and reclaim under reduced pressure obtains Ramulus Uncariae Cum Uncis herb concentrated extracting solution to there not being the alcohol flavor.Concentrated extracting solution with the medicinal liquid of 10% ethanol dilution to 6 times of Ramulus Uncariae Cum Uncis herb amounts, is filtered, get filtrate.With filtrate flow through being filled with the macroporous resin adsorption post of HP-20, extremely colourless with 20% ethanol drip washing of 4 times of amounts earlier to effluent, 75% ethanol elution of 6 times of amounts of reuse, decompression and solvent recovery, get thick paste, the thick paste drying must contain the dried cream that total alkaloids is 69.8% (survey of HPLC method), and wherein the content sum of Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine and isocorynoxeine is 40.3% (survey of HPLC method).Be distributed in the octyldodecanol after dried cream powder is broken, and be processed into soft capsule, every 15mg.
Embodiment 5
Adopt the preparation technology's system among the embodiment 2 to get dry extract, dried cream powder is broken, get 1 part of dried cream powder and 0.1 part of low-substituted hydroxypropyl cellulose mixing, to granulate with 80% alcoholic solution, drying adds 1% magnesium stearate, mixing, tabletting, the bag film-coat promptly gets dispersible tablet.Every 15mg.
Claims (6)
1, a kind of method of from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, extracting alkaloid valid target, it is characterized in that: its extraction step is as follows:
A. adding determining alcohol in Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb is 40～95% aqueous alcohol solutions, extract through merceration, percolation or hot reflux, the aqueous alcohol solutions consumption is Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb weight 6～30 times, filter, merging filtrate, reclaim under reduced pressure obtains Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb concentrated extracting solution to there not being the alcohol flavor;
B. add the water or the alcoholic solution of 4～10 times of Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb weight in Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb concentrated extracting solution, determining alcohol is 10～60% in the solution, filter, filtrate;
C. filtrate is through being filled with the adsorption column of adsorbing material, and the adsorbing material of selecting for use is any one or two kinds of and usefulness in macroporous adsorbent resin or the ion exchange resin;
D. before the eluting, selecting volume for use is that the water of 4～8 times of adsorbing materials or 20～40% pure drip washing adsorption column to effluent are colourless;
E. with the alcoholic solution eluting that contains 40～90%, the eluent consumption is 4～10 times of adsorbing material, collects eluent;
F. decompression and solvent recovery gets thick paste, and thick paste gets Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb alkaloid valid target after drying.
2. a kind of method of extracting alkaloid valid target from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb according to claim 1 is characterized in that alcohol used in the described alcoholic solution is a kind of in ethanol or the methanol.
3. a kind of method of extracting alkaloid valid target from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb according to claim 1, the model that it is characterized in that described macroporous resin have a kind of among AB-8, SP-70, D101, DM-130, S-8, D3520, D4020, the HP-20.
4, a kind of method of from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, extracting alkaloid valid target according to claim 1, it is characterized in that: total alkaloid content is 50～90% in the described alkaloid valid target, and wherein the content sum of Ramulus Uncariae cum Uncis alkali, isorhynchophylline, corynoxeine and isocorynoxeine is greater than 40%.
5, a kind of method of extracting alkaloid valid target from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb according to claim 1, it is characterized in that: the alkaloid valid target of extraction can combine with pharmaceutically acceptable carrier or excipient and make tablet, capsule, drop pill or granule.
6, the application of the alkaloid valid target that from Ramulus Uncariae Cum Uncis leaf, stem and branch with belt hook or herb, extracts in cardiovascular and cerebrovascular diseases medicaments such as neurodegenerative disease such as preparation treatment or prevention senile dementia and hypertension.
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| CN101869556A (en) * | 2010-07-23 | 2010-10-27 | 贵州百花医药股份有限公司 | Uncaria slow release capsule preparation and preparation method thereof |
| CN101884683A (en) * | 2010-07-23 | 2010-11-17 | 贵州百花医药股份有限公司 | Uncaria slow-release pill preparation and preparation method thereof |
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- 2008-06-18 CN CN 200810011900 patent/CN101342259B/en not_active Expired - Fee Related
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| CN104262341A (en) * | 2014-10-10 | 2015-01-07 | 潍坊医学院 | Method for extracting vasodilatation active compound rhynchophylline |
| CN107184625A (en) * | 2017-05-25 | 2017-09-22 | 辽宁大学 | The method and purposes of alkaloid valid target are extracted from catharanthus roseus, stem branch or herb |
| CN108245512A (en) * | 2018-01-10 | 2018-07-06 | 南方科技大学 | A kind of application of isocorynoxeine in the drug with neuroprotection is prepared |
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