CN103690580A - Microemulsion extraction method and microemulsion extract of Andrographis paniculata - Google Patents
Microemulsion extraction method and microemulsion extract of Andrographis paniculata Download PDFInfo
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Abstract
The present invention discloses a microemulsion extraction method of Andrographis paniculata. The microemulsion extraction method is characterized in that an Andrographis paniculata herb completely contact a biocompatible microemulsion for a certain time such as 3 days, and suction filtration is performed to obtain the filtrate, wherein the biocompatible microemulsion comprises lecithin, cholesterol, bile salts and water, and a weight ratio of the lecithin to the cholesterol to the bile salts to the water is 1:0.013:1:18. The invention further discloses an Andrographis paniculata microemulsion extract obtained according to the microemulsion extraction method, and drugs, health care food, nutrition food or cosmetic compositions containing the microemulsion extract.
Description
Technical field
The present invention relates to a kind of microemulsion extracting method and extract thereof of Herba Andrographis, especially biocompatibility microemulsion extracting method and extract thereof.
Background technology
Microemulsion (microemulsion, ME) be by the spontaneous a kind of low-viscosity forming of proper proportion, isotropism and thermodynamically stable colloidal dispersion system by water, oil phase, surfactant and cosurfactant, outward appearance is transparent or semitransparent, particle diameter is generally between 10～100nm, it can solve the various problems such as poor stability, poorly soluble, irritant, bioavailability is low, drug release is too fast as the carrier of medicine.Especially can increase the dissolubility of insoluble drug, be the ideal carrier of insoluble drug.
Microemulsion, as a kind of new drug carrier, has great application potential.Mainly there is following advantage:
1. microemulsion formulation can improve the dissolubility of insoluble drug, and the medicine of solubilising opposed polarity simultaneously.
2. microemulsion formulation particle diameter is little, can pass through effectively and rapidly gastrointestinal tract epithelial cell, greatly improves the bioavailability of medicine.
3. microemulsion formulation can be made multiple dosage form, comprises oral formulations, ejection preparation and transdermal absorption formulation.
4. it is write out a prescription once determining, preparation method is simple.
Although microemulsion formulation is having one's own knack aspect raising bioavailability, but the problem of its existence also can not be ignored: in microemulsion, contain a large amount of surfactants and cosurfactant, they are large multipair gastrointestinal tract mucous irritant, and life-time service may cause whole body chronic toxicity.Thereby strive to find surfactant and the cosurfactant of high-efficiency low-toxicity, be the effective way that microemulsion formulation is able to extensive use.
Biocompatibility pharmaceutic adjuvant:
Because pharmaceutic adjuvant and medicine together play a role, its safety issue is to affect the major issue that it applies in pharmaceutical preparation always.Desirable pharmaceutic adjuvant should have following characteristic: good moldability, consumption is little, effect is strong, physiological security is large, inanimate object is active, stable chemical nature etc.The pharmaceutic adjuvant (biocompatibility pharmaceutic adjuvant) wherein with biological degradability and histocompatibility is ideal.The pharmaceutic adjuvant with biocompatibility, does not reply and rejection with body, is difficult for producing adverse reaction; And dissolubility is good in body, can reduce consumption, promote absorbing of medicine.Endogenous material, refers to derive from body, with people's existence and healthy closely bound up composition.Along with molecular biology and biochemical development, increasing endogenous bioactive substance can represent its medical value, is widely used in medicine, health food and cosmetic field, and less as pharmaceutic adjuvant application of endogenous material.The endogenous material that can be used at present pharmaceutic adjuvant has lecithin, cholate, cholesterol etc.
Lecithin is the basic substance of life, is present among each cell, is more to concentrate on the vitals such as brain and nervous system, blood circulation, immune system and liver, the heart, kidney.Lecithin belongs to a kind of mixture, and its constituent comprises phosphoric acid, choline, fatty acid, glycerol, glycolipid, triglyceride and phospholipid.Lecithin is described as " the 3rd nutrient " arranged side by side with protein, vitamin, and human life be unable to do without its nourishing and protection from start to finish, has the blood glucose of adjusting, promotes the effects such as neurodevelopment and slow down aging.
Under pure lecithin (phosphatidylcholine) room temperature, be a kind of white solid of colorless and odorless, due to produce or process for purification, condition of storage different oxidized present faint yellow to brown.Lecithin content in health food, nutraceutical, also can be used as medical auxiliary materials in the major applications below 55%.60%-80% major applications, in cosmetics, pharmaceutic adjuvant, more than 90% is mainly used in pharmaceutical industry.At present, we adopt lecithin by domestic " fat milk " raw material using clinically exactly, and its essence is the product of lecithin and water emulsification, and safety is good, can be used for drug administration by injection.
Cholate is that the bile acid secreted by hepatocyte is combined with glycine or taurine and the sodium salt or the potassium salt that form.It is the main component that participates in fat digestion and absorption in bile.Cholate in bile has a lot of functions: chyle fat, promotes fat digestion; Be combined with fatty acid, promote the absorption of fatty acid; Promote the absorption of fatsoluble vitamin; Suppress the growth of intestinal bacteria; Promote the dissolving of cholesterol.
Cholesterol is extensively present in animal body, and the abundantest in You Yinaoji nervous tissue, in kidney, spleen, skin, liver and bile, content is also high.Cholesterol is the indispensable important substance of animal tissue cell, and it not only participates in forming cell membrane, and is synthetic bile acid, the raw material of vitamin D and steroid hormone.Cholesterol can be used as emulsifying agent and uses, and it act as reduction interfacial tension, forms firmly interfacial film.
Chinese medicine Herba Andrographis has antiinflammatory, analgesic activity.Current water extractions that adopt more, alcohol extracting method and alkali extraction and acid precipitation extract effective ingredient in Herba Andrographis with hyoscine, but all there is certain drawback in these methods.Water extraction generally can extract the water soluble ingredients such as flavone, polysaccharide, but because andrographolide water solublity is lower, the extraction ratio of water extraction is not high.In alcohol extract, Determination of Andrographolide is higher, and the alcohol of intermediate concentration is also suitable for extracting flavonoid glycoside, but alcohol extracting method extraction time is long, solvent load is large, troublesome poeration, and yield is lower, is unfavorable for making full use of of resource.Potass extraction method, andrographolide open-loop products is soluble in water, regulating still reducible after PH be lactone, thus to the extraction ratio of two kinds of lactones still can, but under alkaline environment, the extraction ratio of total flavones obviously declines.Visible, because the effective ingredient polarity difference of Herba Andrographis is larger, single extraction solvent is difficult to its effective ingredient more comprehensively to extract.Therefore, it is a kind of suitable to find, and the solvent of solubilising various polarity composition extracts Herba Andrographis simultaneously, is the key that guarantees to extract comprehensively, efficiently Herba Andrographis effective ingredient.
Summary of the invention
One aspect of the present invention provides a kind of biocompatibility microemulsion and preparation method thereof.
The present invention provides a kind of biocompatibility microemulsion extracting method and extract thereof of Herba Andrographis on the other hand.
Applicant has carried out research extensively and profoundly to the method for biocompatibility microemulsion and biocompatibility microemulsion extraction Herba Andrographis, thereby has completed the present invention.
The invention provides:
1. a biocompatibility microemulsion, is characterized in that, comprises lecithin, cholesterol, and cholate and water, their weight ratio is: 1:0.013:1:18.
2. according to the microemulsion of project 1, wherein also comprise isopropyl myristate,
Lecithin wherein, cholesterol, cholate, the weight ratio of isopropyl myristate and water is: 1:0.013:1:0.3:18.
3. according to the microemulsion of project 1, wherein also comprise polyoxyethylene hydrogenated Oleum Ricini,
Lecithin wherein, cholesterol, cholate, the weight ratio of polyoxyethylene hydrogenated Oleum Ricini and water is: 1:0.013:1:1:18.
4. according to the microemulsion of project 1, wherein also comprise polyoxyethylene hydrogenated Oleum Ricini and isopropyl myristate, lecithin wherein, cholesterol, cholate, polyoxyethylene hydrogenated Oleum Ricini, the weight ratio of isopropyl myristate and water is: 1:0.013:1:1:0.3:18.
5. according to the microemulsion of project 4, wherein also comprise 95% ethanol, lecithin wherein, cholesterol, cholate, polyoxyethylene hydrogenated Oleum Ricini, isopropyl myristate, 95% ethanol, the weight ratio of water is: 1:0.013:1:1:0.3:8:18.
The substituting material that can also comprise polyoxyethylene hydrogenated Oleum Ricini and/or isopropyl myristate in microemulsion of the present invention, these materials are known to those skilled in the art.
In a kind of specific embodiments of the present invention, microemulsion of the present invention is only by lecithin, cholesterol, and cholate, polyoxyethylene hydrogenated Oleum Ricini, isopropyl myristate, 95% ethanol, water forms, and their weight ratio is: 1:0.013:1:1:0.3:8:18.
Cholate/lecithin has good solubilization as mixed surfactant to slightly solubility oil phase cholesterol, and cholate/lecithin and catabolite thereof are all the compositions existing under organism physiology condition, can be absorbed completely by body, there is the safety of height, and cholate and lecithin can significantly reduce the hemolytic toxicity of lecithin catabolite (lysophosphatide) while share.So the microemulsion that cholate, lecithin, cholesterol form is good pharmaceutical carrier.
Biocompatibility micro emulsion drug carrying system of the present invention is low based on human body endogenous material (cholate, cholesterol, lecithin) and consumption; the advantage of described biocompatibility microemulsion is: can effectively solve that (content of microemulsion surfactant is generally more than 30% due to the too high untoward reaction causing of microemulsion surface-active contents 1.; common medicinal supplementary material has certain toxicity; usually can cause a series of untoward reaction, this is also the main cause of restriction microemulsion extensive use).2. formula consists of human body endogenous biocompatible material, easily by human body, is accepted, and does not produce and replys and rejection, is difficult for producing illeffects.3. dissolubility is good in vivo for biocompatible materials, easily by body, is absorbed, and contributes to improve the bioavailability of medicine.
Biocompatibility microemulsion of the present invention can be applied in health food, nutraceutical, also can be used as medical auxiliary materials.Biocompatibility microemulsion of the present invention can also, as " fat milk ", can be used for drug administration by injection.
The present invention also provides:
6. a preparation method for microemulsion described in project 1-5 any one, comprises step below:
(1) in prescription ratio, take a certain amount of lecithin and optional polyoxyethylene hydrogenated Oleum Ricini and/or 95% ethanol, dissolve;
(2) recipe quantity cholesterol and optional isopropyl myristate are mixed, dissolve;
(3) recipe quantity is added to the water cholate, dissolves;
(4) to step (1), make the solution that adds step (2) in solution, stir, then add the solution of step (3).
7. according to the preparation method of project 6, wherein step (1) takes a certain amount of lecithin and polyoxyethylene hydrogenated Oleum Ricini in prescription ratio, preferably lecithin, polyoxyethylene hydrogenated Oleum Ricini and 95% ethanol.
8. according to the preparation method of project 6 or 7, wherein step (2) is mixed recipe quantity cholesterol and isopropyl myristate.
9. according to the arbitrary preparation method of project 6-8, wherein step (1), (2) and (3) described dissolving are carried out in 37 ℃ of water-baths.
The present invention also provides:
10. a microemulsion extracting method for Herba Andrographis, is characterized in that, comprises that Herba Andrographis medical material and biocompatibility microemulsion fully contact for example 3 days a period of time, and then sucking filtration, obtains filtrate; Wherein said biocompatibility microemulsion comprises lecithin, cholesterol, and cholate and water, their weight ratio is: 1:0.013:1:18.
11. according to the microemulsion extracting method of project 10, and wherein said biocompatibility microemulsion also comprises isopropyl myristate,
Lecithin wherein, cholesterol, cholate, the weight ratio of isopropyl myristate and water is: 1:0.013:1:0.3:18.
12. according to the microemulsion extracting method of project 10, and wherein said biocompatibility microemulsion also comprises polyoxyethylene hydrogenated Oleum Ricini,
Lecithin wherein, cholesterol, cholate, the weight ratio of polyoxyethylene hydrogenated Oleum Ricini and water is: 1:0.013:1:1:18.
13. according to the microemulsion extracting method of project 10, wherein said biocompatibility microemulsion also comprises polyoxyethylene hydrogenated Oleum Ricini and isopropyl myristate, lecithin wherein, cholesterol, cholate, polyoxyethylene hydrogenated Oleum Ricini, the weight ratio of isopropyl myristate and water is: 1:0.013:1:1:0.3:18.
14. according to the microemulsion extracting method of project 13, and wherein said biocompatibility microemulsion also comprises 95% ethanol, wherein lecithin, cholesterol, cholate, polyoxyethylene hydrogenated Oleum Ricini, isopropyl myristate, 95% ethanol, the weight ratio of water is: 1:0.013:1:1:0.3:8:18.
Microemulsion extracting method described in 15. 1 kinds of project 10-14 any one, wherein said biocompatibility microemulsion adopts and comprises step method preparation below:
(1) in prescription ratio, take a certain amount of lecithin and optional polyoxyethylene hydrogenated Oleum Ricini and/or 95% ethanol, dissolve;
(2) recipe quantity cholesterol and optional isopropyl myristate are mixed, dissolve;
(3) recipe quantity cholate is added to the water, dissolves;
(4) to step (1), make the solution that adds step (2) in solution, stir, then add the solution of step (3).
16. according to the microemulsion extracting method of project 15, and wherein step (1) takes a certain amount of lecithin and polyoxyethylene hydrogenated Oleum Ricini in prescription ratio, preferably lecithin, polyoxyethylene hydrogenated Oleum Ricini and 95% ethanol; Step (1), (2) and (3) described dissolving are carried out in 37 ℃ of water-baths.
17. according to microemulsion extracting method described in project 10-14 any one, and wherein said abundant contact comprises vibration or stirs.
The microemulsion extract of 18. 1 kinds of Herba Andrographis, is characterized in that, it extracts and obtain according to project 10-17 any one microemulsion extracting method.
19. 1 kinds of medicines, health food, nutraceutical or cosmetic compositions, comprising the microemulsion extract described in project 18, preferred described compositions is oral formulations, spray or externally dressing.
The microemulsion extract of Herba Andrographis of the present invention as medicine, health food, nutraceutical can be Orally administered, as medicine can also oral cavity or nasal spray use, as cosmetics, can external application use.
Accompanying drawing explanation
Fig. 1 andrographolide standard curve
Fig. 2 blood drug level-time plot
The specific embodiment
Below by specific embodiment, describe the present invention in detail, in any case but must not be interpreted as limitation of the present invention.
Instrument and material
Instrument MD110-2 type electronic analytical balance, 81B-2 type constant temperature blender with magnetic force, nanometer particle size analyzer (Malvern), 8002 type water-baths.TGL-16G type High speed refrigerated centrifuge (Anting Scientific Instrument Factory, Shanghai), MVS-1 eddy mixer (Beijing Orient is opened thing science equipment company limited), TP-300 supersonic cleaning machine (Tian Peng New Electronic Techniques company limited), HP1100 type high performance liquid chromatograph, SPD210AV detector.
Material PC50 lecithin (the too big Pharmaceutical in Shanghai, lot number 20120301), cholesterol (U.S. SIGMA company, lot number MKBD9436V), Fel Sus domestica salt (Beijing bispin microbiological culture media products factory, lot number 20120420), isopropyl myristate (U.S. SIGMA company, lot number 20120322), polyoxyethylene hydrogenated Oleum Ricini (BASF Aktiengesellschaft, lot number 512882), 95% ethanol (analytical pure), distilled water.
Preparation example 1
(1) in prescription ratio, take 10 grams of lecithin, be placed in beaker, constant temp. heating water-bath is dissolved, after magnetic agitation is even, standby.
(2) 0.13 gram of cholesterol is dissolved in constant temp. heating water-bath, standby.
(3) 10 grams of cholate are added in 18 grams of water, constant temp. heating water-bath is dissolved, standby.
(4) to step (1), make the solution that slowly adds step (2) in solution, even by magnetic stirrer, then add the solution of step (3).Obtain microemulsion of the present invention.
Preparation example 2
(1) in the microemulsion prescription ratio of above-mentioned project 5, take 10 grams of lecithin, 10 grams of polyoxyethylene hydrogenated Oleum Ricini (RH-40), 8 gram of 95% ethanol, is placed in beaker, and 37 ℃ of water-baths are dissolved, after magnetic agitation is even, standby.
(2) by the microemulsion prescription of above-mentioned project 5,0.13 gram of cholesterol is added in 3 grams of isopropyl myristates, 37 ℃ of water-baths are dissolved, standby.
(3) by the microemulsion prescription of above-mentioned project 5,10 grams of cholate are added in 18 grams of water, 37 ℃ of water-baths are dissolved, standby.
(4) to step (1), make the solution that slowly adds step (2) in solution, even by magnetic stirrer, then the solution that adds step (3) to make.Obtain a kind of preferred microemulsion of the present invention.
Experimental example 1
The microemulsion physicochemical property of preparation example 2:
1. particle diameter and form:
Adopt Ma Erwen particle instrument to measure particle diameter and distribution of particles FACTOR P DI value, measure particle diameter 3 times and be respectively: 19.03nm, 19.23nm, 19.20nm.PDI is followed successively by: 0.165,0.147,0.147.40000 times of Electronic Speculum views demonstrations, the form of this microemulsion is spherical corpusculum, size is evenly nanoscale.Particle diameter, between 10～100nm, meets the definition of microemulsion.
2. stability:
(1) high speed centrifugation accelerates experiment: get appropriate prepared microemulsion in centrifuge tube, be placed in high speed centrifuge, under room temperature condition, with 3500rmin-1, carry out centrifugal, centrifugal twice, each 10min, centrifugal after, observe; Microemulsion still keeps clear, has no profit layering, shows that prepared microemulsion dynamic stability is good.
(2) 50 ℃ of water-baths are got appropriate prepared microemulsion in cillin bottle to the impact of microemulsion stability, and sealing bottleneck, is placed in 50 ℃ of water-baths and observes, through water-bath in 3 days, microemulsion still keeps clear, has no profit layering, shows stable in properties under prepared microemulsion high temperature.
(3) investigation of microemulsion room temperature appearance stability is placed microemulsion three months in room temperature, observe phenomena.At ambient temperature, there is not layering situation in result microemulsion, shows according to stable in properties under the microemulsion room temperature of preferred microemulsion formula preparation.(4) investigation of microemulsion cold preservation appearance stability is placed microemulsion three months in refrigerator (4 ℃), result cholesterol microemulsion is under the condition of 4 ℃, there is not layering situation, and separate out without Borneolum Syntheticum, show according to the microemulsion of preferred microemulsion formula preparation stable in properties at low temperatures.
Experimental example 2
The microemulsion biocompatibility of preparation example 2:
Sample preparation:
Culture fluid: containing the RPMI1640 culture fluid of 10% hyclone.In every liter of culture fluid, contain RPMI1640 medium powder 10.4g, hyclone 100ml, penicillin 10Wan unit, phenol red on a small quantity, sodium bicarbonate aqueous solution is adjusted to pH value 7.0-7.4, adds distilled water preparation.
Sample liquid preparation: by the microemulsion 2ml of preparation example 2 preparations, sterilizing, adds 20ml culture fluid, 37 ℃ of concussion lixiviate 24h, the aseptic filtering with microporous membrane of 0.45um, subpackage is standby.It with the RPMI1640 culture fluid that contains 10% hyclone, by lixiviating solution dilution, is 25%, 50%, 75%, 100% variable concentrations.
Negative controls: 37 ℃ of complete culture solutions of placing 24h.
Positive control solution: containing the culture fluid of 0.5% phenol.
Cell strain: l cell strain L929.
Cell culture:
Condition of culture: 37 ℃, 5%CO2, saturated humidity.
Test method: mtt assay.Get the L929 cell that is in exponential phase, with culture fluid dilution, make the L929 single cell suspension that concentration is 5 * 104/ml, be seeded in 96 holes and cultivate each hole of culture plate, every hole 0.20ml, cultivates 24h, makes L929 cell attachment.Sucking-off solution, adds each 6 holes of 0.20ml negative controls, variable concentrations microemulsion sample liquid and positive control solution successively.Cultivate 48,96, sucking-off solution after 144h, add the MTT solution of the 5mg/ml of culture fluid that 0.20ml is fresh and 20ul, shake up gently 37 ℃ of incubation 4h.Shift out solution, with 0.20ml normal saline, clean 2 times, every hole adds 0.20mlDMSO(dimethyl sulfoxide), light shaking 10min, enzyme-linked immunoassay instrument is measured absorbance OD value in 550nm place.
Result: in Table-1
Table-1
Relative cell survival rate when the biocompatibility microemulsion of MTT spectrphotometric method for measuring is cultivated L929
It is generally acknowledged, the biomaterial of cell survival rate RGR>75% does not have toxic effect to cell relatively, experimental results show that the relative cell survival rate of respectively organizing sample is all higher than 75%, proves that this microemulsion formula biocompatibility is good.
Microemulsion of the present invention can be for extracting the active component in Chinese medicinal plant.
Embodiment 1: microemulsion extracts Herba Andrographis:
(1) microemulsion dipping: take Herba Andrographis medical material 20g, measure according to the microemulsion 200ml of above-mentioned preparation example 2 preparations, the two is placed in to conical flask, vibration, fully, after contact, room temperature dipping extracts 4 days, and sucking filtration obtains filtrate.Obtain a kind of preferred extract of the present invention.
(2) microemulsion is ultrasonic: take Herba Andrographis medical material 20g, measure the microemulsion 200ml of above-mentioned preparation example 2 preparations, the two is placed in to conical flask, and vibration, after fully contacting, ultrasonic 30min, sucking filtration obtains filtrate.
Embodiment 2: microemulsion extracts Herba Andrographis:
Microemulsion dipping: take Herba Andrographis medical material 20g, measure according to the microemulsion 200ml of above-mentioned preparation example 1 preparation, the two is placed in to conical flask, stir, fully, after contact, room temperature dipping extracts 4 days, and sucking filtration obtains filtrate.Obtain extract of the present invention.
Comparative example 1: ethanol extraction Herba Andrographis:
(1) alcohol dipping: take Herba Andrographis medical material 20g, measure 60% ethanol 200ml, the two is placed in to conical flask, vibration, fully, after contact, room temperature dipping extracts 4 days, and sucking filtration obtains filtrate.
(2) ethanol is ultrasonic: take Herba Andrographis medical material 20g, measure 60% ethanol 200ml, the two is placed in to conical flask, and vibration, after fully contacting, ultrasonic 30min, sucking filtration obtains filtrate.
Experimental example 3
The comparison of different extracting solution extraction efficiency:
Precision measures above-mentioned each 2ml of Herba Andrographis extracting solution in 10ml centrifuge tube respectively, precision adds 6ml methanol, vortex shaker vibration 1min, standing, in the centrifugal 10min of 10000r, supernatant is transferred in 25ml volumetric flask to methanol constant volume, through 0.45 μ m filter membrane, filter, upper high performance liquid chromatograph is measured content.Chromatographic condition is as follows:
Chromatographic column: enlightening horse C18 chromatographic column (150mm, 5 μ m), mobile phase is acetonitrile (A)-water (B) gradient elution: 0-5min, 22%-25%A, 5-30min, 25%-30%A, 30-50min, 30%-40%A; Flow velocity: 1.0mlmin-1; Detecting wavelength employing dual wavelength is 225nm and 254nm; Sample size: 20 μ L; Column temperature: 25 ℃.
The assay result of each extracting solution is as follows:
From the above results, no matter microemulsion, as extracting solvent, is infusion process, or ultrasonic method, to the extraction efficiency of andrographolide and dehydrorographolide all higher than ethanol.Wherein the extraction of dehydrorographolide is had more to advantage function, brought into play the advantage that microemulsion of the present invention extracts for multicomponent.More outstanding advantage is, the efficiency that microemulsion extracts as solvent dipping, apparently higher than supersound extraction, illustrates that this extraction solvent is capable of reducing energy consumption, is a kind of high-quality and efficient extraction solvent.
Dehydrorographolide has antiinflammatory refrigeration function, in the time of can inflammation-inhibiting capillary permeability increase the development with inflammatory edema, and can reduce inflammatory exudation amount.Clinical have good therapeutic effect for respiratory tract and intestinal infection disease.The advantage that biocompatibility microemulsion of the present invention extracts dehydrorographolide is also the directly oral intestinal infection disease that is used for the treatment of of biocompatibility microemulsion that the present invention contains dehydrorographolide, without again by active component separation from microemulsion.
Experimental example 4
Microemulsion extracting solution and the comparison of ethanol extract oral administration biaavailability
Blood specimen collection and processing
Under rabbit waking state, auricular vein injecting narcotic urethane solution (5ml/kg), is bundled in rabbit platform after anesthesia, the total tremulous pulse of separated cervical region, pipe laying, gather respectively each about 5ml of blank blood sample, anticoagulant heparin, gathers after blank blood every group of gastric infusion embodiment 1 microemulsion dipping Herba Andrographis extracting solution and comparative example 1 each 5ml of alcohol dipping Herba Andrographis extracting solution respectively, respectively at 5min after gavage, 15min, 30min, 45min, 1h, 1.5,2h, 3h, 4h, 5h, the about 5ml of 7h blood sample collection, anticoagulant heparin.
The blood sample of above-mentioned collection is placed in to 4 ℃ of centrifugal 20min of refrigerated centrifuge 3000r/min, separated plasma, standby.Accurately draw each time point blood plasma 1ml after medicine, be placed in 10ml tool plug centrifuge tube and add 6ml chloroform, in eddy mixer, mix after 5min, the centrifugal 20min of 3000r/min, accurately draw chloroform layer 4ml, in 37 ℃ of water-baths, volatilize, 1ml methanol ultrasonic dissolution for residue, cross 0.45 μ m filter membrane, enter high performance liquid chromatogram and measure content.
The preparation of standard curve
Precision takes andrographolide reference substance 5.mg methanol in 50ml volumetric flask and is dissolved to scale, is made into storing solution standby.Get 7 blank 10ml tool plug centrifuge tubes, every pipe accurately adds respectively blank plasma 1ml, more accurately adds respectively standard substance storing solution 8,16,24,32, and 40,48,56 μ l, according to above-mentioned blood sample disposal methods, calculate standard curve equation by area normalization method.Chromatographic condition C18 post (150mm, 5 μ m, Di Ma company); Mobile phase: methanol: water (50:50); Flow velocity: 1.0ml/min, detects wavelength X=225nm, and column temperature is room temperature.
According to the compound method of standard blood sample, under the chromatographic condition of this test, the range of linearity is (0.1-2.3 μ g/ml), with sample peak area, concentration is mapped, and obtains standard curve equation: A=1.8394C-0.3068 (R2=0.9924) linear relationship better see Fig. 1 through linear regression
Result
Two kinds are extracted the andrographolide blood drug level that solution extracts
After gavage embodiment 1 microemulsion extracting solution and comparative example 1 ethanol extract, the concentration of the andrographolide in plasma in rabbit is in Table 1
Blood drug level after table 1 perfusion
The andrographolide bioavailability metrics that two kinds of solution extracts is in Table 2 and blood drug level-time plot 2.
Table 2 bioavailability metrics
The oral rear blood drug level of andrographolide that microemulsion extracts rises fast, when 30min, blood drug level reaches peak value, at 0.5h, arrive 2h time period blood drug level in a higher level, at 2h, form again an absworption peak to the 3h time period, after 3h, blood drug level starts to decline, and forms obvious biabsorption peak.After the andrographolide oral administration gavage rabbit of ethanol extraction, the blood drug level rate of climb is more slow, and when 3h, blood drug level reaches peak value, and blood drug level declines rapidly afterwards.Compare with ethanol extract, the relative bioavailability of microemulsion extracting solution can reach 142%, illustrates that the oral absorption of microemulsion extracting solution utilizes degree apparently higher than ethanol extract.
Experimental example 5
Adopt the test of dimethylbenzene induced mice ear swelling to characterize the antiinflammatory action of extracting solution; Due to employing bacteria lipopolysaccharide, rat fever test characterizes the refrigeration function of extracting solution
One, the impact of xylol induced mice ear swelling
Experiment mice is divided into model group, prednisolone acetate group, embodiment 1 microemulsion group and comparative example 1 ethanol group, 7 every group at random by body weight.Laboratory animal successive administration 4d, model group gives tap water, and prednisolone acetate group is pressed 0.2g/kg, microemulsion group and alcohol extraction group are all equivalent to crude drug by 2g/kg() and administration, fasting (can't help water) 5h before experiment.30min ether light anaesthesia after last administration, with quantitative sample injector, in mouse right ear tow sides, each is accurately coated with 0.01mL dimethylbenzene, and after 30min, de-cervical vertebra is put to death animal.With diameter 5mm card punch, at its left and right ear antimere, take off auricle, weigh, with two ear weight differences, represent swelling.Calculate respectively swelling and the suppression ratio of mice caused by dimethylbenzene xylene ear swelling.The results are shown in Table 3.
Table-3. the impact of xylol induced mice ear swelling
Note: with the comparison of blank group, * * P<0.01; With the comparison of alcohol extraction group,
△ △p<0.05
Two, the impact on rat fever due to LPS
Experiment grouping is tested with mice ear.With electronic clinical thermometer, measure rat anus temperature, every day 1 time, continuous 3 days; METHOD FOR CONTINUOUS DETERMINATION was 3 times on 1, and every minor tick 0.5h chooses qualified animal (measure mean body temperature for 3 times between 37～38 ℃, and fluctuation being in 0.3 ℃) and tests after mensuration.All animals are by 300 μ g/kg subcutaneous injection LPS normal saline solutions, and after injection, 3.5h measures body temperature, and by body temperature stratified random, is divided into 4 groups, aspirin 0.2g/kg group and model control group, microemulsion 2g/kg group, alcohol extraction 2g/kg group.During 4h, gavage gives relative medicine (model group gives ordinary water), measures anus temperature when 4.5h, 5.5h, calculates the body temperature changing value of comparing with initial body temperature.The results are shown in Table-4.
Table-4. impact on rat fever due to LPS
Note: with the comparison of blank group, * * P<0.01; With the comparison of alcohol extraction group,
△p<0.01
△ △p<0.05
To sum up, microemulsion prepared by the present invention, as extracting solvent, for extracting Herba Andrographis, has higher extraction efficiency, and the oral administration biaavailability of extracting solution is higher simultaneously.Microemulsion extracts that Herba Andrographis has that energy consumption is low, extraction efficiency is high, extract absorbs Du Gao and the outstanding advantage such as antiinflammatory ability is strong.
Above-described is only some embodiments of the present invention.For the person of ordinary skill of the art, without departing from the concept of the premise of the invention, can also make some distortion and improvement, these all belong to protection scope of the present invention.
Claims (10)
1. a microemulsion extracting method for Herba Andrographis, is characterized in that, comprises that Herba Andrographis medical material and biocompatibility microemulsion fully contact for example 3 days a period of time, and then sucking filtration, obtains filtrate; Wherein said biocompatibility microemulsion comprises lecithin, cholesterol, and cholate and water, their weight ratio is: 1:0.013:1:18.
2. according to the microemulsion extracting method of claim 1, wherein said biocompatibility microemulsion also comprises isopropyl myristate,
Lecithin wherein, cholesterol, cholate, the weight ratio of isopropyl myristate and water is: 1:0.013:1:0.3:18.
3. according to the microemulsion extracting method of claim 1, wherein said biocompatibility microemulsion also comprises polyoxyethylene hydrogenated Oleum Ricini,
Lecithin wherein, cholesterol, cholate, the weight ratio of polyoxyethylene hydrogenated Oleum Ricini and water is: 1:0.013:1:1:18.
4. according to the microemulsion extracting method of claim 1, wherein said biocompatibility microemulsion also comprises polyoxyethylene hydrogenated Oleum Ricini and isopropyl myristate, lecithin wherein, cholesterol, cholate, polyoxyethylene hydrogenated Oleum Ricini, the weight ratio of isopropyl myristate and water is: 1:0.013:1:1:0.3:18.
5. according to the microemulsion extracting method of claim 4, wherein said biocompatibility microemulsion also comprises 95% ethanol, wherein lecithin, cholesterol, cholate, polyoxyethylene hydrogenated Oleum Ricini, isopropyl myristate, 95% ethanol, the weight ratio of water is: 1:0.013:1:1:0.3:8:18.
6. a microemulsion extracting method described in claim 1-5 any one, wherein said biocompatibility microemulsion adopts and comprises step method preparation below:
(1) in prescription ratio, take a certain amount of lecithin and optional polyoxyethylene hydrogenated Oleum Ricini and/or 95% ethanol, dissolve;
(2) recipe quantity cholesterol and optional isopropyl myristate are mixed, dissolve;
(3) recipe quantity cholate is added to the water, dissolves;
(4) to step (1), make the solution that adds step (2) in solution, stir, then add the solution of step (3).
7. according to the microemulsion extracting method of claim 6, wherein step (1) takes a certain amount of lecithin and polyoxyethylene hydrogenated Oleum Ricini in prescription ratio, preferably lecithin, polyoxyethylene hydrogenated Oleum Ricini and 95% ethanol; Step (1), (2) and (3) described dissolving are carried out in 37 ℃ of water-baths.
8. according to microemulsion extracting method described in claim 1-5 any one, wherein said abundant contact comprises vibration or stirs.
9. a microemulsion extract for Herba Andrographis, is characterized in that, it extracts and obtain according to claim 1-8 any one microemulsion extracting method.
10. medicine, health food, nutraceutical or a cosmetic composition, wherein contain microemulsion extract claimed in claim 9, and preferred described compositions is oral formulations, spray or externally dressing.
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| CN107691948A (en) * | 2017-11-01 | 2018-02-16 | 陕西理工大学 | A kind of plant beverage prime cement preparation method for increasing species characteristic fragrance ingredient |
| US11224628B2 (en) | 2016-09-29 | 2022-01-18 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Method for extraction of an agent from a plant source |
| US11666618B2 (en) | 2016-09-29 | 2023-06-06 | Yissun Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Method for selective extraction of cannabinoids from a plant source |
| US11819491B2 (en) | 2016-09-29 | 2023-11-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Dilutable formulations of cannabinoids and processes for their preparation |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR101605107B1 (en) | 2014-05-02 | 2016-03-21 | 주식회사 새롬한방제약 | Method of arthritis antiinflammation product from Eucommia ulmoides and Acanthopanax sessiliflorum Seeman by applying microemusion as extraction solvent |
| US11224628B2 (en) | 2016-09-29 | 2022-01-18 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Method for extraction of an agent from a plant source |
| US11666618B2 (en) | 2016-09-29 | 2023-06-06 | Yissun Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Method for selective extraction of cannabinoids from a plant source |
| US11819491B2 (en) | 2016-09-29 | 2023-11-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Dilutable formulations of cannabinoids and processes for their preparation |
| US11819490B2 (en) | 2016-09-29 | 2023-11-21 | Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd. | Dilutable formulations of cannabinoids and processes for their preparation |
| CN107691948A (en) * | 2017-11-01 | 2018-02-16 | 陕西理工大学 | A kind of plant beverage prime cement preparation method for increasing species characteristic fragrance ingredient |
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