CN1143850C - Menispermum dauricum extract, its extractive process and application - Google Patents

Abstract

The present invention relates to asiatic moonseed rhizome phenol alkali extracted from asiatic moonseed rhizomes. Firstly, biological total alkali is extracted from the coarse powder of the asiatic moonseed rhizomes; coarse total alkali precipitates are obtained by alkalizing the biological total alkali; after the phenolic alkali is extracted by alcohol, brown paste objects are obtained; after the brown paste objects are dissolved by alkali and filtered, and acid is added to a phenol alkali water solution to be neutralized; finally, precipitates are filtered and dried to obtain a phenol alkali product. The asiatic moonseed rhizome phenol alkali has arrhythmia preventing action and has usage dependency if being used correctly, and the asiatic moonseed rhizome phenol alkali can overcome the defects of early afterdepolarization, triggering activity, the twisting chamber speed of tips, etc., caused by the reverse usage dependency of arrhythmia resisting medicines. In addition, poisonous benzene and chloroform are replaced by the alcohol in the extracting process. The asiatic moonseed rhizome phenol alkali has the advantages of simple technology, no pollution, high yield and low cost, and is suitable for medicine preparation.

Description

The application of yellow parilla extract and extraction process thereof and extract

Technical field

The present invention relates to alkaloid and the extraction process and the application in pharmacy of from menispermaceous plants yellow parilla rhizome, extracting.

Background technology

Yellow parilla total alkaloids Ceng Yibei beans heel piece name is referred to as heat-clearing and detoxicating drug, is used for the treatment of swelling and pain in the throat, tonsillitis, and chronic bronchitis is stated from Pharmacopoeia of People's Republic of China nineteen ninety version, nineteen ninety-five version, version in 1999.We found once in 1980 that wherein dauricine had antihypertensive function; again find effects such as it has anti-arrhythmia, resists myocardial ischemia, platelet aggregation-against thereafter; our clinical trial once in 1985 proves that this medicine has good efficacy to patients with arrhythmia, efficiently reaches 91.5%.Because of dauricine cell cube separating and extracting method more loaded down with trivial details and yield is low [CN 1047861A] thus its drug development work can not realize so far.

Cardiovascular disorder is clinical to be frequently-occurring disease, irregular pulse is the dangerous sign of painstaking effort disease especially, at present existing antiarrhythmic drug still has many shortcomings, a lot of medicines all have reverse use dependency, just therefore cause after depolarization early easily as Quinidine, sotalol, triggered activity, even torsades de pointes type chamber speed.

Summary of the invention

The objective of the invention is from new angle exploitation yellow parilla, a kind of Rhizoma Menispermi stem extract and extracting method thereof are provided, make that this extract not only has anti-arrhythmia, resists myocardial ischemia, the effect of platelet aggregation-against, also have forward and use dependency, can not cause after depolarization symptom early, but also it is simple to have a separating and extracting method, cost is low, the yield height is suitable for advantages such as industrial production.

Realize that technical scheme of the present invention is, Rhizoma Menispermi stem extract provided by the invention is the yellow parilla phenol alkaloid that can be produced by following method:

1) from yellow parilla rhizome meal, extracts biology total alkali;

2) extracting solution that contains biology total alkali is separated out thick total alkali precipitation through the NaOH alkalization;

3) utilize the fat-soluble feature of phenolic alkaloid efficient part, from thick total alkali, extract phenolic alkaloid with organic solvent and get brown paste;

4) will be molten through NaOH alkali with the brown paste that organic solvent extracts, remove by filter the non-phenol oil-soluble impurities of insoluble, the phenolic alkaloid aqueous solution adds the acid neutralization again, filter collection throw out, drying gets the high light yellow phenolic alkaloid product of purity.

The above-mentioned method of extracting biology total alkali from yellow parilla rhizome meal is the acid extraction method, with 0.2% sulfuric acid dilute solution with percolation leaching percolate; The extracting solution of saying that will contain biology total alkali is separated out the sedimentary method of thick total alkali through the NaOH alkalization and is to use 10%NaOH to alkalize to pH8-8.5, obtains the thick total alkali of throw out; Extracting the employed organic solvent of phenolic alkaloid from thick total alkali is ethanol; The organic solvent extract is added the 1%NaOH dissolving, filter, under agitation add 10%H to filtering solution ₂SO ₄Transfer PH8-8.5, filter collection throw out, washing, 50-55 ℃ of air seasoning gets the faint yellow phenolic alkaloids of menispermum dauricum of high purity.

Craft screening process of the present invention is as follows:

1. extracting method

The acid extraction method can be subdivided into decocting method, percolation and pickling process again by the working method difference, and wherein pickling process is because of drawback such as easily going mouldy, extraction efficiency is low in time-consuming length, summer, and we do not give consideration when formulating technology.At present domestic manufacturer when producing the Rhizoma Menispermi sheet general adopt for to have continued to use the sour water decocting method of two more than ten years, be with the extractor of packing into after the medicinal material chopping, add dilute sulphuric acid heat and carry three times, united extraction liquid, alkalize total alkali precipitation slightly, be directly used in preparation production then.According to the reflection of Rhizoma Menispermi sheet factory Anshan First Pharmaceutical Factory of domestic maximum, this decocting method exposes some problems already, and the content of the most outstanding is thick total alkali is not high and unstable, fluctuates in the 10%-32% scope.For this reason, be necessary existing thick total alkali extraction process is improved, we compare situations such as the extraction yield of decocting method and percolation, thick total alkali, circulation ratios.

At first, with the total alkali of neutralisation determination test medicinal material, method is as follows: precision takes by weighing the about 1g of dry medicinal powder, puts in the round-bottomed flask, drips strong aqua 1ml, and airtight, jolting 10 minutes was placed 2 hours again, added CHCl ₃50ml, heating in water bath refluxed 2 hours, filtered, and the dregs of a decoction wash with a small amount of solvent, and filtrate decompression is concentrated into dried, adds 95%EtOH 1ml dissolution residual substance, and heating boils off EtOH, removes residual ammoniacal liquor.The accurate 0.01mol/LH2SO4 liquid 20ml that adds, heat makes moltenly a little, and 2 of the red indicator of methylate become orange with 0.02mol/L NaOH drop calmly to solution from redness.Every 1ml 0.01mol/L H2SO4 liquid is equivalent to the C38H44N2O6 (dauricine) of 6.248mg, calculates the percentage composition of total alkaloids in the medicinal material.

It is known with 10 parts of a collection of medicinal material coarse powder to get total alkali, every part of 200g, and wherein 5 parts of aforementioned diacolation technologies of usefulness are extracted; Other 5 parts are extracted with decocting method, method is as follows: medicinal material coarse powder is packed in the 2000ml triangular flask, add 0.2%H2SO4 solution 900ml, heating is 2 hours in water-bath, extracts altogether 3 times, united extraction liquid, transfer pH8-8.5 with 10%NaOH liquid, filter collection precipitation is washed to nearly neutrality, drying promptly gets thick total alkali.

The thick total alkali of each part is measured content with neutralisation, and method is: precision takes by weighing the about 0.15mg of thick total alkali powder, adds CHCl ₃Refluxed 1 hour in the 10ml water-bath, filter, filter residue adds a small amount of CHCl ₃Flushing, merging filtrate, evaporate to dryness.Then by above-mentioned medicinal material content determination direct titration and calculate content.

Table 1. decocting method and percolation extract the result relatively

	Numbering	Temperature ℃	Thick total alkali yield	Total alkali	Extraction yield *
						Decocting method	1 2 3 4 5	75 80 85 90 95	6.5％ 6.8％ 6.5％ 5.8％ 8.0％	16.2％ 14.6％ 7.5％ 10.3％ 5.1％	87.7％ 82.7％ 40.6％ 49.8％ 34.0％
Percolation	6 7 8 9 10	Room temperature	2.7％ 2.6％ 2.6％ 2.5％ 2.6％	41.0％ 42.0％ 42.3％ 43.5％ 42.5％	92.2％ 91.0％ 91.6％ 90.6％ 92.1％

* testing the medicinal material total alkaloid content is 1.2%; Extraction yield=thick total alkali yield * total alkali ÷ medicinal material total alkali

Table 1 has been summed up the result that decocting method and percolation extract thick total alkali.As can be seen, decocting method can obtain more thick total alkali, and yield is more than the twice of percolation, but its total alkaloid content is below 1/2 of percolation, and the content fluctuating range is very big, and the actual extracting rate is low.The thick total alkali yield of percolation is about 2.6%, and total alkali is up to more than 40%, and extraction yield is stabilized near 91%.

Obviously, percolation is better than decocting method.Except good, the thick total alkali height of aforementioned stable, extract and to wait fully the strong point, percolation also tool is saved the energy, simple to operate, effective constituent not because of advantages such as the destructions of being heated.

The medicinal material granular size has certain influence to the diacolation extraction yield.Therefore segment and the medicinal material extract rate pulverized is lower approximately by 10% than pulverizing medicinal material is necessary to pulverize; Granularity is advisable with the 10-20 order, and is meticulous as fruit granule, as 60 orders, then can influence the percolate flow velocity, causes extraction time long.

2. refining solvent

The sour water diacolation carry thick total alkali outward appearance be Vandyke brown or black, become to be grouped into very complicated, fat-soluble total alkaloid content only accounts for about 40%, 60% the component of also having an appointment it be unclear that; Thick total alkali reaches more than 12% through ash content after the calcination, and visible inorganic components accounts for higher proportion.As if the impurity in the thick total alkali also has the soda acid both sexes, and is consistent with phenolic alkaloids of menispermum dauricum with the solubility behavior in the buck at sour water, all dissolve in sour water and strong alkali aqueous solution, and the neutralization back is another separates out with precipitating in the aqueous solution.Therefore, only handle and be difficult to remove impurity with the soda acid aqueous solution, must utilize the suitable organic solvent of fat-soluble feature selection of phenolic alkaloid efficient part that it is proposed, remove other high polarity impurity, as inorganics, polysaccharide, glucoside etc., discard the dross and select the essential, improve drug effect, reduce toxic purpose thereby reach.

We are to benzene,toluene,xylene, CHCl commonly used ₃, organic solvents such as EtOAc, EtOH investigate, wherein benzene kind solvent polarity is less, fair to the dauricine dissolving, then dissolve not good enough to the bigger alkaloid of polarity that contains two above phenol OH, extraction yield is low, add that this kind solvent also has shortcomings such as toxicity is remarkable, cost height, so be unsuitable for industrial production.CHCl ₃All big with EtOAc to the solubleness of phenolic alkaloid, the extraction yield height, but also have cost and environmental issue.We have adopted EtOH at last.To EtOH and CHCl ₃The phenolic alkaloid that extracts has carried out qualitative and quantitative comparison, and the result shows the two no significant difference.

EtOH and CHCl ₃The HPLC collection of illustrative plates of extract reflects that the two has identical chromatographic peak and similar relative content ratio.

Get with four parts of batch thick total alkalis, every part of 10g puts in the round-bottomed bottle, adds CHCl respectively ₃, 85%EtOH, 90%EtOH and each 50ml of 95%EtOH, in water-bath, refluxed 3 hours, filter, extracting solution is after evaporated under reduced pressure, residue under equal conditions neutralizes with the dissolving of NaOH solution again and obtains phenolic alkaloid.Measure the content of each phenolic alkaloid with reference to above-mentioned thick total alkali measuring method, and be HPLC and analyze, the results are summarized in table 2.

Table 2.CHCl ₃Extract the phenolic alkaloid result relatively with different concns EtOH

Solvent phenolic alkaloid yield phenolic alkaloid content (Dau+DS) %* extraction yield

CHCl ₃ 40.1％ 94.0％ 75.1％ 92.8％

85％EtOH 42.2％ 93.1％ 67.0％ 96.8％

90％EtOH 41.5％ 93.5％ 71.6％ 95.6％

95％EtOH 41.4％ 94.8％ 74.0％ 96.7％

* thick total alkali is 40.6%; (Dau+DS) % refers to dauricine (Dau) and daurisoline (DS) relative content sum in the phenolic alkaloid that the HPLC normalization method records.

Table 2 shows, EtOH and CHCl ₃The yield and the content that extract phenolic alkaloid are all close, and the extraction yield of EtOH is high slightly; Dauricine and daurisoline relative content sum are a little less than CHCl in the EtOH phenolic alkaloid ₃Phenolic alkaloid, this is because due to improving than the polyphenol OH alkaloid ratio of high polarity in the EtOH phenolic alkaloid.In addition, the concentration of EtOH influences not quite extracting the result between 85%-95%.Consider that lower concentration EtOH might introduce more much polar impurities, this technology is defined as 90%-95% with EtOH concentration; EtOH refluxing extraction 2 times, extraction yield can reach more than 98%, so regulation is carried 2 times in the technology.

To sum up, though EtOH is the high polarity solvent, the phenolic alkaloid that extracts with its aspect the quality and quantity two all and CHCl ₃Can replace lipophilic solvent as refining solvent quite, fully.

3. alkali is molten

EtOH extractives may mix non-phenol alkaloid or other impurity, the molten purpose of alkali is to utilize efficient part can take place in the NaOH aqueous solution that phenol OH dissociates and water-soluble character is removed the non-phenol oil-soluble impurities of insoluble, the phenolic alkaloid aqueous solution is again through neutralization, phenol OH is free, from water, precipitate again and separate out, realize solidifying, further remove water-soluble impurity simultaneously, last drying obtains purity height, the pale yellow powdery phenolic alkaloid finished product of color and luster, for preparation production lays the foundation.

4. precipitate

Precipitation operation is arranged in this technology twice, be that acid water extracting liquid precipitates thick total alkali with 10%NaOH for the first time, here select 10%NaOH rather than laboratory weak base ammoniacal liquor or carbonate commonly used, reason is: 1. NaOH low price, 2. 3. the pungent odour that does not have ammoniacal liquor can not resemble and produce a large amount of CO2 foams the Na2CO3.Should be noted that NaOH is a highly basic, excessive alkali can make phenol OH dissociate, and phenolic alkaloid is soluble in water again, so pH should be controlled at 8-8.5.For the second time precipitation is with NaOH solution phenolic alkaloid being separated out among the 10%H2SO4, should control pH similarly at 8-8.5, and excessive acid can make atom of tertiary amine N protonated, causes alkaloid soluble in water again.

5. dry

Because the fusing point of phenolic alkaloid principal constituent dauricine is lower, have only 63 ℃, and this Alkaloid all there is phenol OH, be heated deterioration by oxidation easily takes place, color and luster is deepened, so temperature should not be too high when dry, should be controlled at 50-55 ℃, ventilate, the centre is stirred, and prevents the moisture content alluvial.

The pharmacology of phenolic alkaloids of menispermum dauricum provided by the invention and toxicity test

One, main pharmacodynamics---antiarrhythmic effect

1. anti-ouabain causes the effect of cavy irregular pulse: phenolic alkaloids of menispermum dauricum iv3mg/kg, ig30mg/kg can significantly resist due to the ouabain the chamber early, quiver in the chamber and heartbeat stops, and the ouabain consumption is significantly increased, and be dosage and rely on the property effect.Quinidine iv6mg/kg, the same significantly antagonistic action of ig80mg/kg tool.

2. anti-napelline causes the rat ventricular effect: phenolic alkaloids of menispermum dauricum iv6mg/kg, ig60mg/kg can significantly resist the napelline chamber of causing early, quiver in the chamber and heartbeat stops, the consumption of napelline is significantly increased, the remarkable antagonistic action of the same tool of Quinidine iv6mg/kg, ig80mg/kg.

3. anti-calcium chloride causes the effect of rat ventricular: phenolic alkaloids of menispermum dauricum iv6mg/kg, ig120mg/kg, Quinidine iv6mg/kg, ig80mg/kg all have remarkable antagonism calcium chloride to cause the effect that quiver in the rat chamber and heartbeat stops.Quivered and the moving effect that stops of heartbeat in the chamber.Make the chamber quiver significantly to reduce and take place, exempt to cause heartbeat and stop.

4. improve the effect of rabbit electricity ventricular fibrillation threshold: phenolic alkaloids of menispermum dauricum iv1.5mg/kg, ig30mg/kg have the effect that improves electric ventricular fibrillation threshold.The effect that Quinidine iv3mg/kg, ig40mg/kg also have the electric chamber of raising to quiver.

5. anti-calcium chloride-vagusstoff causes the effect of mouse atrial fibrillation (pouncing on).Phenolic alkaloids of menispermum dauricum ig.120mg, Quinidine ig80mg/kg all have antagonism CaCl2-Ach to bring out the effect of atrial fibrillation (pouncing on).

6. Chinese People's Anti-Japanese Military and Political College mouse coronary ligation arrhythmia: phenolic alkaloids of menispermum dauricum iv3mg/kg, ig60mg/kg, Quinidine iv6mg/kg, ig80mg/kg all have antagonism of irritating arrhythmia again and provide protection, cause ARR animal to occur and reduce.Escalated dose is quivered by speed chamber, chamber and death does not all take place.

7. to the influence of guinea-pig ventricular's papillary muscle action potentials of cells

Phenolic alkaloids of menispermum dauricum (10mg/L100mg/L) and dauricine (10mg/L100mg/L) are all influential to each parameter of guinea pig papillary muscle electrical activity, the equal tool concentration dependent of two medicines ground prolongs APD50, APD90 and ERP reduce amplitude of action (APA), and action potential slows down.Revise the effect of maximum depolarization speed (Vmax).

8. to the influence of rabbit sinus node pacemaker cell: phenolic alkaloids of menispermum dauricum (10mg/L, 100mg/L) and dauricine (10mg/L, 100mg/L) all tool concentration dependent ground prolong APD50, APD90 and SCL, and reduction APA, the effect of slow down SP0 and SP4.

Two, general pharmacological action

(1) to the influence of central nervous system

1. anti-Ventramine causes the convulsions effect: phenolic alkaloids of menispermum dauricum 50,100mg/kg irritate stomach makes Ventramine cause that mice convulsion and death obviously prolong latent period, but the equal no significant difference of mice convulsion rate and mortality ratio.Sodium phenobarbital is then fainted from fear, all obviously prolongations in dead latent period, and convulsions, mortality ratio obviously reduce.

2. hypnosis acts synergistically: phenolic alkaloids of menispermum dauricum 50,100mg/kg irritate stomach, do not have the syngignoscism that strengthens pentobarbital sodium sub-threshold lull dosage, and chlorpromazine 10mg/kg irritates stomach then obvious enhancing syngignoscism, makes obviously to prolong (10/10) length of one's sleep.

3. to influence---the tilting plate method of coordinated movement: phenolic alkaloids of menispermum dauricum 50,100mg/kg irritate stomach and give mouse, and mouse falls number from sheet glass not to be had and change, and after vetanarcol 20mg/kg irritated stomach, mouse all fell, and (10/10) is positive.

4. to the influence of mouse general activity: phenolic alkaloids of menispermum dauricum 50,100mg/kg irritate stomach, after 30 minutes, carrying the mouse tail turn-takes 2-3 time, back its unusual attitude of landing of observation of dishing out (side position or following dorsad), be zero level by legal its progression of Itwin behavior classification, rank and physiological saline group do not have significant difference, and vetanarcol 20mg/kg, 5.8 ± 1.3 grades of the Irwin behavior rank average out to (p＜0.001) of mouse.

(2) to rat blood pressure, breathing and Electrocardiographic influence:

1. to the influence of blood pressure:

Give quiet notes phenolic alkaloids of menispermum dauricum 5 of rat or 10mg/kg, blood pressure all descends rapidly, and 1 minute begins to descend, and reaches maximum value in 20 minutes, recovers about 20 minutes to give preceding blood pressure level, and heavy dose has the change of statistical significance, and low dose of influence is not obvious.

Give phenolic alkaloids of menispermum dauricum 50,100mg/kg to rat oral gavage, blood pressure all began to descend after 30 minutes, reached maximum value in 50 minutes, returned to the preceding level of administration in about 1.5 hours, and heavy dose has statistical significance to change, and low dose of (50mg/kg) do not have obviously influence.

2. the influence to breathing:

To the quiet notes phenolic alkaloids of menispermum dauricum 5 of rat difference, 10mgkg, irritate stomach 50,100mg/kg and physiological saline group relatively, respiratory rate and amplitude can't obviously change.

3. to Electrocardiographic influence:

Give the quiet notes phenolic alkaloids of menispermum dauricum of rat 5mg/kg, to HR, P-R interval, QRS and Q-Tc all do not have influence.During quiet notes 10mg/kg, each parameter value of electrocardiogram(ECG all has change in various degree.When irritating stomach 50mg/kg, each parameter of electrocardiogram(ECG is not all had influence, and when irritating stomach 100mg/kg, P-R and QRS are produced significantly influence.

Three, acute toxicity test

The quiet notes phenolic alkaloids of menispermum dauricum of mouse LD50=37mg/kg, mouse stomach LD50=605mg/kg.

The quiet notes phenolic alkaloids of menispermum dauricum of rat LD50=45mg/kg, 8 of rats, male and female half and half, ig administration 3000mg/kg (20%15ml/kg), in 7 days dead 3.So ig administration rat LD50 is greater than 3000mg/kg.

Poisoning manifestations all has ataxia behind two administrations of two kinds of animals, expiratory dyspnea, mouth and nose cyanosis, and poor appetite, recording ecg when flat coexistence is in imminent danger chamber speed all occurs, quiver in the chamber or severe arrhythmia such as III atrioventricular block.Iv administration death occurs in the 5-8min, and is dead in 24h after the ig administration.Have activity to recover normal respectively in 4h and 24h, dead animal is dissected and is seen cardiac dilatation.

Four, long term toxicity test

(1) rat long term toxicity test

6 the week age SD be 120 of big white mouse, male and female half and half are divided into four groups at random, 3 dosage groups of phenolic alkaloids of menispermum dauricum (27,81 and 270mg/kg/ day), a control group, single cage is fed, the drinking-water of freely ingesting with drinking-water administration 24 weeks of successive administration, is adjusted dosage once by body weight weekly.Observe the animal generalized case, carry out blood test, and blood parameters detects.Animal finish is put to death in experiment, and animal viscera is carried out visual inspection and weighs, and gets various internal organs and does the pathology cut sections for microscopic examination.

Experimental result:

1. body weight and appetite: control group, low, middle dosage group, food-intake and body weight gain all belong to normally.High dose group (270mg/kg/ day) is appetite decline gradually then, and body weight gain slows down, and movable the minimizing is loose by hair.The high dose group rat successively has dead mouse in the 12nd week of administration, sees lung's extravasated blood and point-like inflammatory lesions through dissecting inspection, and wherein a mouse also has fatty degeneration of liver, inflammatory cell infiltration and hyperemia, the cause of death: pneumonia, a mouse have the drug intoxication performance.

2. laboratory examination: routine blood test and platelet count and control group be no difference of science of statistics relatively all, carry? this medicine does not have obvious damage to the rat hemopoietic system.

3. blood biochemical: after 2 usefulness, high dose group rat blood alkaline phosphatase (ALP) significantly raises after the administration, in other, low dose group rat blood biochemical indicator and control group relatively do not have statistical discrepancy.

4. organ coefficient: 24 weeks after 12 weeks and the administration after the administration, the organ coefficient and the control group no difference of science of statistics of each dosage group rat vitals.

5. pathology and histological examination: tangible pathological change is not all found in the heart of each test group and control rats, spleen, suprarenal gland, Tiroidina, stomach, thymus gland, testis and epididymis, prostate gland, ovary and uterus.Each organizes rat all has individual animal lung that point-like garnet focus is arranged, and this variation has nothing to do with being subjected to the reagent thing.After 24 weeks of administration, middle dosage group (81mg/kg/ day), most animals have fatty degeneration of liver and slight downright bad, and in 2 weeks of drug withdrawal, hepatic cell fattydegeneration can recover, but in a short time, necrosis fails to repair fully.

(2) dog long term toxicity test

16 of 4-6 monthly age domesticated dogs, male and female half and half, body weight 5-7kg is divided into 3 groups, low dose group (20mg/kg/ day) high dose group (100mg/kg/ day), control group is given cold boiling water.Every day, 8Am gave the dog feeding preceding gastric infusion, and in 24 weeks of successive administration, medication is 6 days weekly, and drug withdrawal 1 day is adjusted dosage once by body weight weekly.Observe the animal generalized case every day.Laboratory hematology and blood biochemical are learned and are checked same big white mouse, each treated animal all before experiment, in, experiment respectively carry out once after finishing back and 2 weeks of drug withdrawal more than inspection.Dog is also changed the variation of recorded heart rate (HR), P-R interval, QRS and QT with the electrocardiogram(ECG machine check animal II electrocardio that leads.

Animal dead and execution animal all carry out systems inspection, claim each organ weights, calculate organ coefficient, and formalin is fixed, will be cut into slices and use light microscopy.

Experimental result:

1. daily inleting appetite and body weight change; Each treated animal does not relatively have remarkable significant difference in the per day food-intake of different time, and depressor body weight change and control group do not have obvious significant difference more yet.

2. death condition: the high dose group dog is in giving dead one of the 3rd week of back and the 4th week, and lungs extravasated blood and point-like inflammatory lesions, cause of death pneumonia are seen in gross anatomy.Wherein one also has the sex change of liver fat-like.

3. laboratory examination: each phase hematological examination, high and low two dosage treated animals and control animals be no difference of science of statistics relatively all.

Blood biochemical is learned the index inspection: after 12 weeks of medication, T-BIL and the AKP of high dose group animal significantly raise; 24 week of administration, GPT, T-BIL, the AKP of back high dose group animal all raise, and with control group utmost point significant difference were arranged relatively.

4. Electrocardioscopy: after 24 weeks of administration, the ECG P of high dose group dog-R interval, obviously prolong, and shows that high dosage Pueraria lobota phenol alkali has the effect that prolongs the cardiac muscle conduction, and HR, QRS and Q-T are not had obvious influence.

5. pathologic finding

The form of system's each important organ of necrotomy, size, color are not all found macroscopic variation.The organ coefficient of each dosage animal vitals and control group be no difference of science of statistics relatively all.

Histological examination: each is organized most organs and is not all found obvious pathological change.Each organizes part animal lungs all has the interstitial pneumonia pathology and is tried thing irrelevant.

After 24 weeks of medication, pathologies such as bigger fat vacuole, little focal necrosis, steatosis, extravasated blood appear in the liver cell endochylema of high dose group part dog; Slight steatosis appears in the indivedual dog liver cells of low dose group.

The result of long term toxicity test: phenolic alkaloids of menispermum dauricum took medicine for 24 weeks for rat and dog for a long time, when high dosage, can make animal toxicity occur, mainly liver there is certain damaging action, showing as GPT rising and T-BIL raises, hepatic cell fattydegeneration, little focal necrosis is arranged individually, can recover after the drug withdrawal.

The invention provides Rhizoma Menispermi stem extract---phenolic alkaloids of menispermum dauricum, has antiarrhythmic effect, and have forward and use dependency, can not cause symptoms such as after depolarization morning, so this medicine has its superiority, can overcome the existing reverse use dependency of antiarrhythmic drug, cause after depolarization early easily, triggered activity, even the deficiency of torsades de pointes type chamber speed; Production technique of the present invention has been got rid of traditional use benzene or chloroform extraction separation processes, has substituted deleterious benzene and chloroform with ethanol, and production technique is simpler in addition, the non-environmental-pollution problem, and yield increases, and cost reduces, and reaches and uses in pharmacy so be suitable for industrial production.

Description of drawings

Fig. 1 is a process flow sheet of the present invention;

Fig. 2 is the refining phenolic alkaloids of menispermum dauricum HPLC color atlas of ethanol, chloroform.

Embodiment

Embodiment:

1. extract

Getting yellow parilla rhizome meal (10-20 order) 1kg packs in the percolator, compacting, soaked 2 hours with 0.2%H2S04 earlier, open outlet then, the control flow velocity is about 10ml/min, till the no obvious sediment of ammoniacal liquor alkalization was separated out, this moment, alkaloid was carried to the greatest extent substantially, collected percolate 10L altogether until effluent liquid in continuous diffusion.

2. precipitate

Stir down and slowly splash into 10%NaOH solution in the sour water percolate, transfer pH to 8-8.5, visible a large amount of tawny precipitations are separated out; Left standstill 5 hours, and inhaled and remove supernatant liquor, filter, precipitation washes with water to nearly neutrality, drains, and oven dry about 50-55 ℃ gets the thick total alkali 26g of beige.

3. alcohol is molten

Thick total alkali is ground into 60 order left and right sides fine powders, in the container of packing into, with 90-95%EtOH refluxing extraction (2 times * 100ml * 1.5 hours) in water-bath, filter, filter residue washes with a small amount of EtOH, merging filtrate, decompression and solvent recovery, concentrated brown paste.

4. alkali is molten

Alcohol extract adds 1%NaOH 200ml dissolving, stirs hydrotropy, impels the phenolic alkaloid dissolving fully, removes by filter insoluble non-phenol impurity.

5. precipitate and drying

Stir and splash into 10%H2SO4 down in phenolic alkaloid NaOH solution, transfer pH8-8.5, filter the collection precipitate, residual acid, alkali and inorganic salt are removed in washing, drain, and precipitation is put 50-55 ℃ of air seasoning in the baking oven, gets faint yellow phenolic alkaloids of menispermum dauricum 10g, yield about 1%.

Claims (4)

1. Rhizoma Menispermi stem extract, it is the phenolic alkaloid of being produced by following method:

1) from yellow parilla rhizome meal, extracts biology total alkali with percolation with 0.2% sulfuric acid dilute solution;

2) extracting solution that contains biology total alkali alkalizes to pH8-8.5 through 10%NaOH and separates out thick total alkali precipitation;

3) thick total alkali is ground into 60 order left and right sides fine powders, with 85-95% ethanol refluxing extraction in water-bath, filter, concentrating under reduced pressure gets brown paste;

4) it is molten brown paste to be added 1%NaOH alkali, removes by filter the non-phenol oil-soluble impurities of insoluble, under agitation adds 10%H to filtering solution ₂SO ₄Transfer PH to 8-8.5, filter collection throw out, washing, 50-55 ℃ of air seasoning gets the faint yellow phenolic alkaloids of menispermum dauricum of high purity.

2. from the yellow parilla rhizome, extract the preparation method of phenolic alkaloids of menispermum dauricum, may further comprise the steps:

1) from yellow parilla rhizome meal, extracts biology total alkali with percolation with 0.2% sulfuric acid dilute solution;

2) extracting solution that contains biology total alkali alkalizes to pH8-8.5 through 10%NaOH and separates out thick total alkali precipitation;

3) thick total alkali is ground into 60 order left and right sides fine powders, with 85-95% ethanol refluxing extraction in water-bath, filter, concentrating under reduced pressure gets brown paste;

4) it is molten brown paste to be added 1%NaOH alkali, removes by filter the non-phenol oil-soluble impurities of insoluble, under agitation adds 10%H to filtering solution ₂SO ₄Transfer PH to 8-8.5, filter collection throw out, washing, 50-55 ℃ of air seasoning gets the faint yellow phenolic alkaloids of menispermum dauricum of high purity.

3. the application of phenolic alkaloids of menispermum dauricum in the preparation antiarrhythmic drug of the described preparation method's preparation of claim 2.

4. the application of phenolic alkaloids of menispermum dauricum in the preparation antiarrhythmic drug.

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