CN1283650C - Process of extracting total flavone glycoside from mistletoe and medicine composition thereof - Google Patents
Process of extracting total flavone glycoside from mistletoe and medicine composition thereof Download PDFInfo
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- CN1283650C CN1283650C CN 03111554 CN03111554A CN1283650C CN 1283650 C CN1283650 C CN 1283650C CN 03111554 CN03111554 CN 03111554 CN 03111554 A CN03111554 A CN 03111554A CN 1283650 C CN1283650 C CN 1283650C
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Abstract
The present invention provides a preparation process for extracting total flavonoid glycoside from mistletoe. The raw materials of mistletoe are extracted via alcohol or water, filtered, purified via an adsorption resin column with large holes, and are eluted to be colorless via alcohol; eluent is collected after the raw materials are eluted via 50% to 95% of the alcohol; the alcohol is recovered until the scent of the alcohol disappears; the eluent is concentrated into extract or dry powder in a decompression mode of or in an ordinary pressure; the extract or the dry powder is dissolved in 60% to 95% of alcohol, and the extract or the dry powder are filtered; deposit is removed, and filtrate is recovered; the desired total flavonoid of mistletoe is obtained after the alcohol is recovered in a decompression mode. The present invention also discloses a medicine with a primary activity ingredient of the total flavonoid of mistletoe for preventing the rigidification and the thrombosis of blood vessels of brains and hearts and arrhythmia, and lowering blood pressure. The medicine has the medicinal purposes on treating and preventing pathology conditions such as the rigidification and the thrombosis of the blood vessels of brains and hearts, arrhythmia, hypertension, etc.
Description
Technical field:
The present invention discloses a kind of method and pharmaceutical composition thereof that extracts general flavone glycoside from mistletoe, specifically be to be the pharmaceutical composition of primary activity composition with the Herba Visci total flavonoid glycosides that extracts in the mistletoe, also relate to the application of the new medical use of the preparation method of this Herba Visci total flavonoid glycosides and mistletoe total flavones, belong to Chinese medicine extraction process of effective component and new medical use technical field thereof.
Background technology:
Mistletoe Viscum coloratum (komar.) Nakai. belongs to Loranthaceae plant mistletoe on plant taxonomy.The evergreen undershrub of semiparasitism, main producing region northeastward, main parasitic is on tree such as elm, birch, willow, maple, poplar.Mistletoe is used for the treatment of diseases such as rheumatic arthralgia, soreness of the waist and knees, threatened abortion by prescription in traditional Chinese medicine.With the Chinese medicine mistletoe is raw material, prepares Herba Visci total flavonoid glycosides (content reaches 50% above w/w) in a large number, and makes the prevention and the treatment that are used for cerebrovascular sclerosis or embolism, irregular pulse and high blood pressure disease and do not appear in the newspapers as yet so far.
Summary of the invention:
The invention provides a kind of method that from mistletoe, prepares Herba Visci total flavonoid glycosides.
The pharmaceutical composition that the present invention further provides Herba Visci total flavonoid glycosides be used to prepare resisting cardiovascular sclerosis and embolism and anti-arrhythmia, medicine brings high blood pressure down.
The invention discloses with the Herba Visci total flavonoid glycosides is activeconstituents, is used to prepare the medicine of pathological conditions such as effect such as treatment and prevention cardiovascular and cerebrovascular embolism and cerebrovascular sclerosis, irregular pulse, hypertension.
The invention also discloses a kind of is the primary activity composition with the Herba Visci total flavonoid glycosides, and preparation contains the medicine and the pharmaceutical composition thereof of one or more pharmaceutically acceptable carriers or vehicle.
The content of the primary activity composition Herba Visci total flavonoid glycosides in the above-mentioned pharmaceutical composition of the present invention is at least 50% (w/w).
Preparation technology of the present invention may further comprise the steps: be raw material with the mistletoe, extract 1-10 time with 0% to 95% pure liquid, preferably use 70% pure liquid to extract, also can adopt water extraction, the extracting method of 50%-95% alcohol liquid precipitate, or water extraction alkali precipitation method also can, preferably extract 3 times, extracting solution merges, filter, filtrate is through the macroporous adsorptive resins purifying, with the pure liquid of 0%-10% be eluted to colourless after, with collecting elutriant behind the 50%-95% alcohol liquid wash-out, the best reclaims pure liquid with 70% pure liquid wash-out, to there being pure liquid flavor, decompression is condensed into medicinal extract shape or dry powder down or under the normal pressure, with the dissolving of 60%-95% alcohol liquid, the most handy 80% pure liquid dissolving filters, disgorging also reclaims filtrate, obtains required Herba Visci total flavonoid glycosides behind the reclaim under reduced pressure alcohol liquid.Chemical analysis shows that gained end product Herba Visci total flavonoid glycosides content is approximately more than 50%.
Pure liquid described in the above-mentioned preparation method comprises other alcohols chemistry solvent such as ethanol, methyl alcohol, butanols liquid.
According to pharmaceutical composition of the present invention, wherein except that the Herba Visci total flavonoid glycosides that contains at least 50% (w/w), a small amount of triterpene, protein, peptide, amino acid, the polysaccharide that also can contain mistletoe plant residual in Herba Visci total flavonoid glycosides separation and purification process itself, and chemical ingredients such as trace element.It will be appreciated by those skilled in the art that, these impurity components bring disadvantageous effect can for basically the pharmacological activity of Herba Visci total flavonoid glycosides in the pharmaceutical composition of the present invention, on the contrary, in some cases in addition can be with general flavone glycoside performance collaborative or booster action, and wherein also may exist new, the related activity composition that it be not immediately clear.
In addition, what should particularly point out is, other has the activeconstituents of collaborative or booster action with Herba Visci total flavonoid glycosides can to add one or more natural or synthetic as required in pharmaceutical composition of the present invention. the natural or synthetic auxiliary activity composition that these may be added into be well known by persons skilled in the art with can expect.
Pharmaceutically acceptable carrier of Herba Visci total flavonoid glycosides and one or more or vehicle can be mixed by suitable proportion, make the pharmaceutical composition that is applicable to the different dosage form that uses clinically. for example said composition can be mixed with and can supply intravenously, the injection of drug administration by injection is perhaps made the tablet, granule, dripping pill, electuary, liquid preparation, the powder agent that are suitable for oral administration in intramuscular, intraperitoneal, subcutaneous, the myelencephalon chamber etc.Pill, capsule and suspension agent, and the sprays, aerosol, creme, ointment and the suppository that are suitable for topical.
Medicinal composition of the present invention preferably contains the Herba Visci total flavonoid glycosides of 1%-99% and the vehicle of 99%-1%.
Medicinal composition of the present invention preferably contains the Herba Visci total flavonoid glycosides of 40%-60% and the vehicle of 60%-40%.
In order to prepare the solution that is suitable for the outer approach medicine of gi tract, for example can use distilled water, water for injection, isotonic sodium chlorrde solution or glucose solution, perhaps lower concentration (for example 1-100mM) phosphate buffered saline (PBS) (PBS) is as carrier or vehicle.Can in the preparation of these gi tract external administrations, add one or more other ancillary components or additives, for example can use xitix as antioxidant, use Sodium Benzoate or Metagin junket as sanitas, use dimethyl sulfoxide (DMSO) as absorption enhancer.
In order to prepare tablet, the powder agent that is suitable for oral administration, suspension agent or capsule can use sucrose, semi-lactosi, W-Gum, gelatin, lipid, Microcrystalline Cellulose, talcum powder etc. as carrier or vehicle.Be suitable in the preparation of oral administration at these, also can contain other suitable additive, for example solubilizing agent, disintegrating agent, lubricant, absorption enhancer, sanitas, correctives, thinner, sorbent material, vehicle, dispersion agent, tensio-active agent, flavor flavor agent or tinting material etc.
Preferably pharmaceutical composition of the present invention being made route of administration is the injection liquid that intravenously or intramuscular injection are suitable for these route of administration, for example injection, lyophilized injectable powder etc.The unitary dose of these preparations generally comprises the 10-300mg Herba Visci total flavonoid glycosides for example for some ARR patient, the injection formulation of pharmaceutical composition of the present invention can be added in etc. and ooze in glucose solution or the isotonic sodium chlorrde solution, constantly dropleting medicine-feeding.The dosage of intravenous administration is generally 20-100mg every day.Another preferred route of administering of pharmaceutical composition of the present invention is the various oral formulations of cheating medicine that are suitable for, for example tablet, powder agent, capsule, or oral liquid or emulsion.The unitary dose of these oral preparations generally comprises the 20-1000mg Herba Visci total flavonoid glycosides.
Following experiment has shown the pharmacological action of medicine of the present invention and composition:
The pharmacological testing effect research of Herba Visci total flavonoid glycosides
Experiment material:
Animal: Kunming mouse, body weight 18～22g, 30～35g male and female half and half, rat, Wistar body weight 200～250g, all available from animal housing of agriculture and animal husbandry university, the conformity certification numbering is respectively 10-5112,10-5113.
Reagent: trichloromethane Shenyang reagent one factory's lot number: 900201
The bariumchloride chemical reagent factory lot number of increasing income: 881004
Chloral Hydrate Shenyang chemical reagent three factory's lot numbers: 97902
Vetanarcol Shanghai chemical reagent packing factory lot number: 84-06-12
Racemic isoproterenol Shanghai Hefeng Pharmaceutical Co., Ltd. lot number: 010701
Instrument: two road physiographs, make in Chengdu Instruement Factory Ki Ku Sui South Korea
Medicine: this laboratory of Herba Visci total flavonoid glycosides provides
Puerarin glucose injection liquid Dongya Medicine Co., Ltd., Jiangxi Prov. lot number 220,104 0641
Verapamil hydrochloride injection (isoptin) Shanghai Hefeng Pharmaceutical Co., Ltd. lot number 981101
1) chloroform is brought out the influence of quivering in the mouse chamber
Get 60 of mouse, divide seven groups at random, 10 every group, be respectively control group: intravenous injection iv 10ml/kg physiological saline; Puerarin group: iv 20mg puerarin injection; Isoptin group: iv 0.5mg/kg verapamil hydrochloride injection, the high, medium and low dosage group of Herba Visci total flavonoid glycosides, iv 12,6,3mg/kg rhythm of the heart peace respectively.In 2-3 minute, mouse is put into the inversion 500ml beaker that contains 4ml chloroform cotton balls one by one after the administration, whenever change a mouse, need to add the 0.4ml chloroform, allow mouse suck chloroform until breath stopped.Take out immediately, two lead electrocardiogram are observed whether generation chamber quiver [1] of mouse heart on oscilloscope.The counting chamber incidence of quivering, the first chi sees Table 1. as a result
Table 1 mistletoe flavonoid glycoside brings out the influence of quivering in the mouse chamber to chloroform
| Group (mg/kg) | Number of animals (only) | The chamber number of animals (only) of quivering | Rate % quivers in the chamber | P |
| Control group-Puerarin 20 isoptins 0.5 mistletoe flavonoid glycoside 12 mistletoe flavonoid glycosides 6 mistletoe flavonoid glycosides 3 | 10 10 10 10 10 10 | 9 3 1 0 2 4 | 90 30 10 0 20 40 | ＜0.05 ＜0.01 ＜0.001 ＜0.01 ＞0.05 |
Mistletoe flavonoid glycoside 12,6mg/kg, can significantly reduce mouse chamber that chloroform the brings out incidence of quivering.
2) influence of Herba Visci total flavonoid glycosides rat ventricular that bariumchloride is brought out
Get 48 of rats, divide 6 groups at random, the grouping dosage sees Table 2, rat is fixed in the operation entablature with chloral hydrate anesthesia (10% Chloral Hydrate 0.3ml/100 body weight) rear portion, separate femoral venous catheter, the fixing bariumchloride usefulness that is equipped with, iv 0.2%Bacl2,2mg/kg, irregular pulse appears and after, immediately respectively with the tail vein injection administration, observe that the rhythm of the heart recovers the normal time length in 1 hour.And to 60 minutes normal cardiac rhythm number of animals [2].The results are shown in Table 2.
Table 2 Herba Visci total flavonoid glycosides is to Bacl
2The influence of the rat ventricular that brings out
| Group (mg/kg) | Keep the time of normal cardiac rate in 60 minutes | Recover normal cardiac rhythm number of animals ratio |
| Control group-mistletoe flavonoid glycoside 10 5 2.5 Puerarins 20 isoptins 0.5 | 1.25±3.53 57.5±7.07*** 48.0±13.9*** 31.8±21.1*** 27.8±11.4*** 8.2±9.3 | 0 7/8*** 4/8*** 2/8* 0 0 |
Herba Visci total flavonoid glycosides 10,5,2.5mg/kg are to Bacl
2The rat ventricular that brings out has therapeutic action.
3) to the influence of asphyxiating anoxic mouse heart rate extinction time
Get 40 of mouse, divide 4 groups at random, grouping iv 10ml/kg physiological saline, 12,6,3mg/kg mistletoe flavones, every day 1 time, continuous 3 days, after the last administration,, separate tracheae with vetanarcol 5mg/kg anesthesia, mouse is led switch by two and two road physiographs link, observe the mouse core electrograph by oscilloscope, the ligation tracheae is observed mouse electrocardio extinction time [3].The results are shown in Table 3.
Table 3 mistletoe flavones is to the influence of asphyxiating anoxic mouse heart rate extinction time
| Group (mg/kg) | Number of animals (only) | Heart rate extinction time (second) | P |
| Control group-mistletoe flavonoid glycoside 12 mistletoe flavonoid glycosides 6 mistletoe flavonoid glycosides 3 | 10 10 10 10 | 384.7±101.2 523.2±107.2 502.9±96.9 465.3±99.5 | ＜0.05 ＜0.05 ＞0.05 |
Mistletoe flavonoid glycoside 12,6mg/kg can obviously prolong the extinction time of asphyxiating anoxic mouse heart rate.
4) to the influence of anoxic mouse survival time due to the Racemic isoproterenol
Get 30 of mouse, random packet, control group iv 10ml/kg physiological saline, mistletoe flavones high and low dose group, iv12,6mg/kg mistletoe flavones, subcutaneous injection Racemic isoproterenol (5mg/10g) immediately respectively, after 15 minutes, mouse is put into 250ml triangular pyramidal bottle, and bottle includes 2g sodica calx, 4g sawdust, cover plug, observe mouse diing time.The results are shown in Table 4.
Table 4 pair Racemic isoproterenol to the influence of anoxic mouse survival time
| Group (mg/kg) | Survival time (branch) | P |
| Control group-mistletoe flavonoid glycoside 12 6 | 7.5±0.62 12.7±2.81 9.87±2.04 | ＜0.01 ＜0.05 |
Mistletoe flavonoid glycoside 12,6mg/kg can obviously prolong the anoxic mouse survival time due to the Racemic isoproterenol.
5) to the influence of normal rat blood pressure and heart rate
Get rat and anaesthetize the 0.4ml/100g body weight with 25% urethane, fixedly on the rat autopsy table, separate right side neck point artery, link with pressure transducer, the subcutaneous insertion needle-like of three limbs electroplax, the II recording ecg that leads was stablized 20 minutes, record normal arterial pressure and electrocardiogram(ECG, the tail vein is given after physiological saline or 20,10mg/kg mistletoe flavones, the record administration 1,5,15,30 minute blood pressure and electrocardiogram(ECG, and calculating systolic pressure, diastolic pressure and heart rate the results are shown in Table 5,6.
Table 5 mistletoe flavonoid glycoside is to the influence (mmHg) of normal rat blood pressure
| Group (mg/kg) | Blood pressure before the administration | Different time after the administration (branch) blood pressure difference | |||
| 1’ | 5’ | 15’ | 30’ | ||
| The yellow diastolic pressure ketoside of the yellow diastolic pressure ketoside of control group diastolic pressure-systolic pressure mistletoe 20 systolic pressure mistletoes 10 systolic pressures | 83.9±14.7 113.6±19.2 82.8±15.7 112.6±19.4 82.7±13.9 118.7±17.6 | 0±3.6 0.8±3.4 -13.9±14.0** -16.4±13.4** -6.5±9.3 -7.7±9.8 | -0.3±3.7 -0.8±2.7 -16.3±15.6 -20.5±19.2* -3.0±7.0 -1.7±8.8 | -0.3±2.5 1.3±2.5 -4.3±12.4 -9.7±10.6 -2.0±10.7 -4.1±9.7 | 2.8±3.3 2.8±4.7 1.5±13.6 -1.2±14.9 -2.5±9.8 -2.8±10.4 |
Table 6 mistletoe flavonoid glycoside is to the influence of normal rat heart rate (inferior/minute)
| Group mg/kg | Blood pressure before the administration | Different time after the administration (branch) blood pressure difference | |||
| 1’ | 5’ | 15’ | 30’ | ||
| Control group mistletoe flavones 20 glycosides mistletoe flavones 10 glycosides | 372.5±19.8 371.2±18.5 371.5±16.6 | 369.1±20.2 369.6±13.8 367.4±15.9 | 368.8±22.4 350.8±13.9* 359.3±13.7 | 366.5±17.2 368.9±18.3 368.2±13.6 | 372.3±18.6 364.6±20.3 367.4±16.1 |
Mistletoe flavonoid glycoside 20mg/kg group can obviously reduce rat blood pressure, and 5 minutes is the peak period, and the time length is in 1-15 divides, the rat heart rate that on 5 fen hour, slowed down, and mistletoe flavones 10mg/kg group does not have obvious influence to rat blood pressure and heart rate.
6) to the influence of normal anoxia in mice survival time
Get 30 of mouse, divide 3 groups at random, control group iv 10ml/kg physiological saline, mistletoe flavones high and low dose group, 300ml Erlenmeyer flask (including 2g sodica calx, 5g sawdust) after 30 minutes put into mouse by respectively iv 12,6mg/kg mistletoe flavones, cover plug, observe mouse diing time.The results are shown in Table 7.
Table 7 mistletoe flavonoid glycoside is to the influence of normal anoxia in mice survival time
| Group (mg/kg) | Number of animals (only) | Survival time (branch) | P |
| Contrast-mistletoe flavonoid glycoside 12 mistletoe flavonoid glycosides 6 | 10 10 10 | 23.6±3.0 27.5±4.6 26.2±3.8 | ＜0.05 ＞0.05 |
Mistletoe flavonoid glycoside 12mg/kg can obviously prolong the anoxia in mice survival time.
Conclusion: Herba Visci total flavonoid glycosides 12,6mg/kg, can significantly reduce mouse chamber that chloroform the brings out incidence of quivering.Herba Visci total flavonoid glycosides 10,5,2.5mg/kg are to Bacl
2The rat ventricular that brings out has therapeutic action.Mistletoe flavonoid glycoside 12,6mg/kg can obviously prolong the extinction time of asphyxiating anoxic mouse heart rate.
Mistletoe flavonoid glycoside 12,6mg/kg can obviously prolong the anoxic mouse survival time due to the Racemic isoproterenol.Mistletoe flavonoid glycoside 20mg/kg group can obviously reduce rat blood pressure, and 5 minutes is the peak period, and the time length is in 1-15 divides, the rat heart rate that on 5 fen hour, slowed down, and mistletoe flavonoid glycoside 10mg/kg group does not have obvious influence to rat blood pressure and heart rate.Mistletoe flavonoid glycoside 12mg/kg can obviously prolong the anoxia in mice survival time.
Herba Visci total flavonoid glycosides has anti-arrhythmia, reduces oxygen-consumption, the effect of bringing high blood pressure down, for the clinical application Herba Visci total flavonoid glycosides provides the pharmacology foundation.
Embodiment:
The following example is intended to further describe for example the present invention, rather than limits the present invention by any way.Under the prerequisite that does not deviate from the spirit and principles in the present invention, any change or change that the ability city those of ordinary skill that the present invention did is realized easily all will fall within the claim scope that awaits the reply of the present invention.
Embodiment 1: the preparation of Herba Visci total flavonoid glycosides
Get the water that exsiccant mistletoe 1kg adds 8kg, heated and boiled three times, each 40 minutes.Collect and merge the aqueous extract of gained, extracting solution is with 2% medical active carbon decoloring, or through the ion exchange resin decolouring, filter, filtrate is added on macroporous adsorbent resin (the Chinese Tianjin of the routine AB-8 Nankai University chemistry) post of handling with bronsted lowry acids and bases bronsted lowry in advance, washing post with deionized water does not have color up to elutant, back with 70% ethanol liquid (about 500ml) wash-out.Collect eluate, and reclaim under reduced pressure, drying is dissolved the gained enriched material with 80% ethanol liquid, place, and filter, and the filtrate decompression concentrate drying obtains yellow powder shape Herba Visci total flavonoid glycosides.
Embodiment 2:
Get the water that mistletoe 1kg adds 6kg, heated and boiled three times, each 40 minutes.Collect and merge the water extract of gained, concentrate, adding ethanol liquid to pure liquid concentration is 70%, or extract with 85% ethanol liquid, the heavy liquid of extracting solution or pure liquid can be with 2% medical active carbon decoloring, or through the ion exchange resin decolouring, or without the decolouring direct filtration, reclaim ethanol liquid to there not being pure liquid flavor, soup is added on macroporous adsorbent resin (the Chinese Tianjin of the routine AB-8 Nankai University chemistry) post of handling with bronsted lowry acids and bases bronsted lowry in advance, washing post with deionized water does not have color up to elutant, back with 70% ethanol liquid (about 500ml) wash-out.Collect effusive eluate and concentrate it, obtain yellow powder shape Herba Visci total flavonoid glycosides in reducing pressure to reflux down.
Embodiment 3
Get Herba Visci total flavonoid glycosides 100g, make 5% total flavones solution with the dissolving of 75% ethanol liquid, use activated carbon decolorizing, filter, the concentrating under reduced pressure drying, add the injection water and make 5% the aqueous solution, filter to clear and bright sterilization with millipore filtration (φ 0.20 μ m), aseptic subpackaged in cillin bottle, every bottle of 1ml puts lyophilize in the freeze drier, promptly.
Embodiment 4
Get Herba Visci total flavonoid glycosides 100g, make 5% total flavones solution with the dissolving of 75% ethanol liquid, use activated carbon decolorizing, filter, the concentrating under reduced pressure drying, add the injection water and make 5% the aqueous solution, filter to clear and bright sterilization with millipore filtration (φ 0.20 μ m), aseptic subpackaged, every bottle of 1ml seals, promptly.
Embodiment 5
Get Herba Visci total flavonoid glycosides 1kg, medical starch 0.5kg, with 80% ethanol liquid wet granulation, whole grain, dress 1# capsule, every 0.2g.
Other project should meet 2000 editions capsule projects of Pharmacopoeia of People's Republic of China relevant requirements.
Embodiment 6
Get Herba Visci total flavonoid glycosides 1kg, medical starch 0.5kg, dextrin 1kg, 50 ethanol liquid are granulated in right amount, whole grain, drying, compressing tablet.Every 0.3g.
Other project should meet 2000 editions tablet projects of Pharmacopoeia of People's Republic of China relevant requirements.
Claims (1)
1, a kind of preparation technology who extracts general flavone glycoside from mistletoe may further comprise the steps:
A) with the mistletoe be raw material,, when water extraction,, filter with 60～95% ethanol sedimentations, united extraction liquid with 0% to 95% extraction using alcohol 1-10 time;
B) filtrate is through the macroporous adsorptive resins purifying, after colourless, with collecting elutriant behind the 50%-95% ethanol elution, reclaims pure liquid with the 0%-10% ethanol elution, do not distinguish the flavor of to there being alcohol, is condensed into the medicinal extract shape;
C) use the 60%-95% dissolve with ethanol, filter, disgorging also reclaims filtrate, gets Herba Visci total flavonoid glycosides behind the decompression recycling ethanol.
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