CN101085041B - Total alkaloid of lotus leaves extraction and its preparation method - Google Patents
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Abstract
The invention discloses total alkaloid extract extracted from lotus leaf and its preparation method. The extract mainly comprises nuciferine, N-demethylnuciferine, O-demethyl nuciferine, oxopurpureine, nuciferine, tulipiferine, N-demethyl armepavine, 2-hydroxy-1-methoxy aporphine, armepavine, N-methyl isococlaurine, N-methyl coclaurine, dehydronuciferine, dehydronuciferine and its derivant, etc. The extractcan be extracted through one or more of methods of solvent-extraction, solvent extraction, ion exchange resin process, macroporous adsorbent resins method, extraction by supercritical fluid, column chromatography, and liquid-liquid countercurrent distribution chromatography. The total amount of different kinds of alkaloids in the extract is 5-100% (w/w), wherein nuciferine content occupies 5-100% (w/w).
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of total alkaloids extract of Chinese medicine Folium Nelumbinis and preparation method, method of quality control and in the application of medicine and field of health care food.
Background technology
Folium Nelumbinis is the dried leaves of Nymphaeceae nelumbium lotus Nelumbo nucifera Gaertn..Traditional medicine thinks Folium Nelumbinis nature and flavor bitter cold to have clearing away summer-heat and eliminating dampness, and hair growth promoting is sun clearly, clears away heart-fire and reduces phlegm and internal heat, the activity of hemostasis diuretic.According to Compendium of Material Medica record " Folium Nelumbinis is obeyed it, makes us thin bad ", " hair growth promoting vigour, benefit helps taste, arresting seminal emission is turbid, the congestion that looses, the detumescence pain is sent out variola ".Modern pharmacological research show Folium Nelumbinis have lowering blood-fat and reducing weight, antioxidation, defying age, inhibition fatty liver, antibacterial, suppress effects such as HIV propagation, antiviral, antiinflammatory, antiallergic.The clinical practice of Folium Nelumbinis is more is that form with compound recipe is used for Weight-reducing and lipid-lowering, improves liver metabolism, slow down aging etc.Mainly contain compositions such as alkaloids, flavonoid, polysaccharide in the Folium Nelumbinis, pharmacological research shows that the alkaloids composition is its main active.So far, do not see the patent that relates to total alkaloid of lotus leaves extraction and its production and use.
Summary of the invention
The object of the present invention is to provide a kind of total alkaloid of lotus leaves extraction and preparation method thereof.
Another object of the present invention is to provide the salt of a kind of total alkaloid of lotus leaves extraction and some acid formation.
A further object of the present invention is to provide the salt or the complex of a kind of total alkaloid of lotus leaves extraction and the formation of some metal ion.
The total alkaloid of lotus leaves extraction that the present invention proposes is the combination that contains multiple alkaloids active component of extracting from the Chinese medicine Folium Nelumbinis, and wherein the main compound structure is as follows:
1. benzylisoquinoline class
Benzylisoquinoline constituents structure parent nucleus
2. aporphine class
Aporphine constituents structural framework
Tulipiferine
3. dehydrogenation aporphine class
Dehydrogenation aporphine constituents structural framework
Raw material Folium Nelumbinis of the present invention derives from any one plant of Nymphaeceae Nelumbo.As the raw material that extracts total alkaloid of lotus leaves, can be commercially available Folium Nelumbinis decoction pieces, also can be arbitrary position or the whole plant such as stem, leaf, spica, fruit, underground rhizome and root of these plants, wherein preferred medical material position is the dried leaves of these plants.Above-mentioned described Folium Nelumbinis comprises crude drug and the decoction pieces of handling without any process of preparing Chinese medicine, also comprises various processed products, as " stir-fry Folium Nelumbinis ", " parched with bran Folium Nelumbinis ", " burnt FOLIUM NELUMBINIS " etc.
Total alkaloid of lotus leaves extraction of the present invention, be meant to extract and obtain from any position of above-mentioned arbitrary plant, the compositions that comprises multiple lotus leaf alkaloid active component, what wherein preferably extraction prepared from Nelumbonucifera Gaertn. dried leaves, contain multiple alkaloids composition of active components.These alkaloids compositions mainly comprise nuciferine, N-demethyl nuciferine, 0-demethyl nuciferine, anonaine, nuciferine, tulipiferine, N-demethyl armepavine, 2-hydroxyl-1-methoxyl group aporphine, Ya Meipa alkali, N-methyl isococlaurine, N-methyl coclaurine, dehydronuciferine, dehydrogenation nuciferine and are other derivant of parent nucleus with it.
As total alkaloid of lotus leaves extraction, the summation of wherein various alkaloids composition percentage compositions is 5～100% (w/w), wherein 50～100% (w/w) preferably.
Total alkaloid of lotus leaves extraction of the present invention, can with organic acid such as some acid, mineral acid such as example hydrochloric acid, sulphuric acid, nitric acid, phosphoric acid or formic acid, acetic acid, citric acid, tartaric acid, succinic acid, form salt derivative.These derivants have pharmacologically active and the purposes identical or close with above-mentioned total alkaloid of lotus leaves extraction.
Total alkaloid of lotus leaves extraction of the present invention can also form slaine or complex with metal ions such as sodium, potassium, calcium, ferrum, aluminum, zinc, copper, chromium, strontiums.These slaines or complex have pharmacologically active or the purposes identical or close with above-mentioned total alkaloid of lotus leaves extraction.
Among the various alkaloids active component that total alkaloid of lotus leaves extraction of the present invention is contained, most importantly nuciferine, N-demethyl nuciferine, O-demethyl nuciferine, anonaine, nuciferine, tulipiferine, N-demethyl armepavine, 2-hydroxyl-compositions such as 1-methoxyl group aporphine.As total alkaloid of lotus leaves extraction, the content of nuciferine accounts for 5～100% (w/w) of whole total alkaloid contents.
The invention allows for the preparation technology of described total alkaloid of lotus leaves extraction, it can adopt following any one method, or the combination in any of these methods is prepared: (1) solvent extraction method; (2) solvent extraction; (3) ion-exchange-resin process; (4) macroporous adsorbent resin method; (5) supercritical fluid extraction; (6) column chromatography; (7) liquid-liquid adverse current partography.Wherein preferable methods is an ion-exchange-resin process.
When these methods of use are prepared, generally comprise following step:
(1) extract: solvent for use can be water or any one alcohols, ketone and esters solvent, or the mixed solvent formed by a certain percentage of these solvents, or the acidity or the basic solvent that are made into by these solvents and acid, alkali.Extracting method can be decoction, reflux, supersound extraction, merceration, percolation, microwave extraction, high pressure extract etc.
Preferred extraction process is: the Folium Nelumbinis medical material adds 30～90% ethanol, and reflux, extract, 2～3 times was extracted 1～2 hour at every turn, and solvent load is 15～30 times of amounts (L/kg).
(2) filter: comprise methods such as centrifugal, sucking filtration, ultrafiltration, filter pressing, use or do not use following any one clarifier or its combination: the precipitate with ethanol agent, gelatin, Kaolin, various resins, Polyethylene Glycol, poly-second triol, chitosan and natural clarifying agent finished product are as 101 fruit juice clarifiers, ZTC+1 natural clarifying agent etc.
(3) concentrate: comprise thin film evaporation, rotary evaporation and decocting and concentrating etc. under normal pressure or the reduced pressure.
(4) drying: comprise vacuum drying, spray drying, lyophilization etc.
When adopting solvent extraction to be prepared, general earlier extract mixture being suspended from the water, then with low polar esters, alkanes or ether solvent (as petroleum ether, ether, hexane, gasoline, ethyl acetate etc.) extraction weeding of grease solubility impurity, use the solvent of suitable polarity then, as chloroform, ethyl acetate, acetone, n-butyl alcohol etc., or the mixture of these solvents, extraction obtains total alkaloids composition wherein, obtains total alkaloids extract.
When using ion-exchange-resin process, used resin can be any types such as highly acid, faintly acid, macroporous type, gel-type, the code name of these resins is different because manufacturer is different, as 732 types, D001-CC, D001-SS, D061, D072 etc., strong acidic ion resin preferably wherein, as 732 types, D001-CC, D001-SS etc., used eluant is sour water and ammoniated ethanol, methanol etc., wherein 0～100% ammonia alcohol preferably.
Preferred lotus leaf alkaloid extract resin purification technology is: select 732 types for use, D001-CC, storng-acid cation exchange resins such as D001-SS are as the purification resin, the Folium Nelumbinis ethanol extraction is 0.1～1mg/mL in the sample solution concentration of total alkaloids amount, the absorption flow velocity is 3～15BV/h, the resin column blade diameter length ratio is 1: 5～1: 10, applied sample amount in the total alkaloids amount is 5～50mg/mL, 1～7 times of resin volume of 0～70% ethanol elution carries out remove impurity, the remove impurity flow velocity is 3～10BV/h, with 3～10 times of resin volumes of 40～90% ammonia ethanol (wherein ammonia concentration is 0.1%～5%) eluting, elution flow rate is 8～20BV/h.
When adopting the macroporous adsorbent resin method to be prepared, used macroporous resin can be any one types such as nonpolar, low pole, middle polarity, alkalescence or faintly acid, the code name of these resins is different because manufacturer is different, as D101, D4020, HPD400, AB-8, S-8, HZ-806 etc., the resin of low pole or middle polarity preferably wherein is as AB-8, HPD400, D101 etc.Used eluant is water and aqueous ethanol, methanol, acetone etc., wherein 0～100% ethanol preferably.
When adopting supercritical CO
2When the fluid extraction method is prepared, can directly extract, also can the product that above-mentioned arbitrary method and step obtained be extracted the Folium Nelumbinis raw material.Can use or not use following any kind solvent and solvent mixture during extraction: water, alcohols, ketone and esters solvent.
When adopting column chromatography to be prepared, the object of its processing can be the product that the said extracted step is obtained, and also can be the product behind above-mentioned solvent extraction, ion-exchange-resin process, macroporous adsorbent resin method or supercritical fluid extraction preliminary purification.Used immobile phase can be silica gel, polyamide, aluminium oxide, glucosan (Sephadex series or Sephadex LH-20 series), C-8, C-18, active carbon, cellulose etc., used eluent is different because of the difference of immobile phase, generally the mixed solvent of being made up of water, methanol, ethanol, acetone, chloroform, ethyl acetate, petroleum ether etc.
When adopting liquid-when liquid adverse current partography is prepared, the object of its processing can be the product of said extracted step, also can be the product behind above-mentioned solvent extraction, ion-exchange-resin process, macroporous adsorbent resin method or supercritical fluid extraction preliminary purification.General earlier extract mixture being suspended from the water, then with low polar esters, alkanes or ether solvent (as petroleum ether, ether, hexane, gasoline, ethyl acetate etc.) extraction weeding of grease solubility impurity, use the solvent of suitable polarity then, as chloroform, ethyl acetate, acetone, n-butyl alcohol etc., or the mixture of these solvents, extraction obtains total alkaloids composition wherein, gets total alkaloids extract.
This extract can be pressed the arbitrary proportion compatibility separately or with other any Chinese and western drugs or food, be used to prepare medicine or functional food, prepared medicine or functional food can be capsule, tablet, pill, granule, oral liquid, syrup, electuary, medicated wine, injection, unguentum, powder, beverage etc.
Method of quality control of the present invention can comprise one or both in the following content assaying method:
1. total alkaloids
Precision takes by weighing nuciferine reference substance an amount of (about 5mg), puts in the 100mL measuring bottle, adds dissolve with methanol and is diluted to scale, shakes up, in contrast product solution.Accurate nuciferine reference substance solution 0.0,0.2,0.4,0.6,0.8,1.0,1.2, the 1.5mL of drawing, evaporate to dryness, add bromocresol green buffer (pH 5.6) 4.0mL and chloroform 6.0mL, acutely earn and shake 2min, standing demix 2h in separatory funnel, divide and get chloroform layer, measure absorbance in the 413nm place.With nuciferine reference substance concentration is abscissa, and absorbance is a vertical coordinate, the drawing standard curve.
Precision takes by weighing each 3 parts in total alkaloid of lotus leaves extraction sample, and every part of 5mg puts in the 25mL measuring bottle, adds the methanol ultrasonic dissolution and is diluted to scale, shakes up.Accurate respectively above-mentioned sample solution 0.5mL, nuciferine reference substance solution 0.5mL, 1.0mL, the evaporate to dryness drawn, add bromocresol green buffer (pH 5.6) 4.0mL and chloroform 6.0mL, acutely earn and shake 2min, standing demix 2h in separatory funnel, divide and get chloroform layer, measure absorbance in the 413nm place.The external standard two-point method calculates content.
2. nuciferine
Chromatographic condition: chromatographic column: Hypersil C
18Chromatographic column (4.6mm * 150mm, 5 μ m); Mobile phase: acetonitrile-water (43: 57); Detect wavelength: 280nm; Column temperature: 30 ℃.
Standard curve is drawn: accurate respectively absorption nuciferine reference substance solution (concentration is 0.0515 μ g/ μ L) 0,2,4,6,8,10 μ L inject chromatograph of liquid, measure each chromatograph peak-to-peak area, (μ g) is abscissa with the reference substance sample size, the chromatographic peak peak area is a vertical coordinate, the drawing standard curve.
Assay: precision takes by weighing each 3 parts in 3 batches of lotus leaf alkaloid extract samples, and every part of about 5mg puts in the 25mL measuring bottle, adds the methanol ultrasonic dissolution, and is diluted to scale, shakes up, as the need testing solution of nuciferine assay.Accurate above-mentioned need testing solution 10 μ L, injection chromatograph of liquid, mensuration nuciferine peak area, the calculating content drawn.
The specific embodiment
Embodiment 1: total alkaloid of lotus leaves extraction preparation technology
Get Folium Nelumbinis decoction pieces 1kg, add 90% ethanol 16L, reflux, extract, 3 times, the each extraction 1.5 hours, decompression and solvent recovery, extract add 1% aqueous hydrochloric acid solution dispersing and dissolving, and making aqueous acid concentration is 0.165mg/mL (in the total alkaloids amount), with the centrifugal 30min of 3000 rev/mins rotating speed, get supernatant, by 300mL D001-CC macroporous type storng-acid cation exchange resin, the absorption flow velocity is 10BV/h, the resin column blade diameter length ratio is 1: 8, applied sample amount is 27.5mg/mL (in the total alkaloids amount), and 5 times of resin volumes of 50% ethanol elution carry out remove impurity, and the remove impurity flow velocity is 10BV/h, 7 times of resin volumes of 70% ammonia ethanol (ammonia concentration is 1%) eluting, elution flow rate is 10BV/h, collects 70% ammonia ethanol elution, reclaims solvent, drying under reduced pressure is total alkaloid of lotus leaves extraction.
Total alkaloid content is 56% in the mensuration total alkaloid of lotus leaves extraction, and nuciferine content is 21%.
Embodiment 2: total alkaloid of lotus leaves extraction preparation technology
Get Folium Nelumbinis decoction pieces 1kg, add 70% ethanol 20L, reflux, extract, 3 times, the each extraction 1.5 hours, decompression and solvent recovery, extract add 0.5% aqueous hydrochloric acid solution dispersing and dissolving, and making aqueous acid concentration is 0.33mg/mL (in the total alkaloids amount), with the centrifugal 30min of 3000 rev/mins rotating speed, get supernatant, by 300mL D001-CC macroporous type storng-acid cation exchange resin, the absorption flow velocity is 5BV/h, the resin column blade diameter length ratio is 1: 10, applied sample amount is 30.25mg/mL (in the total alkaloids amount), and 10 times of resin volumes of water elution carry out remove impurity, and the remove impurity flow velocity is 10BV/h, 5 times of resin volumes of 90% ammonia ethanol (ammonia concentration is 0.5%) eluting, elution flow rate is 10BV/h, collects 90% ammonia ethanol elution, reclaims solvent, drying under reduced pressure is total alkaloid of lotus leaves extraction.
Total alkaloid content is 53% in the mensuration total alkaloid of lotus leaves extraction, and nuciferine content is 20%.
Embodiment 3: total alkaloid of lotus leaves extraction preparation technology
Get Folium Nelumbinis decoction pieces 1kg, add 50% ethanol 25L, reflux, extract, 2 times, the each extraction 2 hours, decompression and solvent recovery, extract add 1.5% aqueous hydrochloric acid solution dispersing and dissolving, and making aqueous acid concentration is 0.495mg/mL (in the total alkaloids amount), with the centrifugal 30min of 3000 rev/mins rotating speed, get supernatant, by 300mL D001-CC macroporous type storng-acid cation exchange resin, the absorption flow velocity is 3BV/h, the resin column blade diameter length ratio is 1: 6, applied sample amount is 24.75mg/mL (in the total alkaloids amount), and 6 times of resin volumes of 10% ethanol elution carry out remove impurity, and the remove impurity flow velocity is 5BV/h, 7 times of resin volumes of 50% ammonia ethanol (ammonia concentration is 0.3%) eluting, elution flow rate is 10BV/h, collects 50% ethanol elution, reclaims solvent, drying under reduced pressure is total alkaloid of lotus leaves extraction.
Total alkaloid content is 55% in the mensuration total alkaloid of lotus leaves extraction, and nuciferine content is 18%.
Embodiment 4: total alkaloid of lotus leaves extraction preparation technology
Get Folium Nelumbinis decoction pieces 1kg, add 70% ethanol 18L, reflux, extract, 2 times was extracted decompression and solvent recovery 2 hours at every turn, extract adds the aqueous dispersion dissolving, making sample solution concentration is 0.165mg/mL (in the total alkaloids amount), and by 7L D101 macroporous adsorbent resin, the absorption flow velocity is 3BV/h, the resin column blade diameter length ratio is 1: 9, applied sample amount is 27.5mg/mL (in the total alkaloids amount), and 4 times of resin volumes of water elution carry out remove impurity, and the remove impurity flow velocity is 3BV/h, 4 times of resin volumes of 90% ethanol elution, elution flow rate is 6BV/h, collects 90% ethanol elution, reclaims solvent, drying under reduced pressure is total alkaloid of lotus leaves extraction.
Total alkaloid content is 58% in the mensuration total alkaloid of lotus leaves extraction, and nuciferine content is 18%.
Embodiment 5: the preparation of total alkaloid of lotus leaves sheet
Total alkaloid of lotus leaves extraction 100g
Starch 100g
The said components mix homogeneously, it is an amount of to add Pulvis Talci, is pressed into 1000.
Embodiment 6: the preparation of total alkaloid of lotus leaves compound preparation
Total alkaloid of lotus leaves extraction 50g
Fleece-flower root total stilbene extract 50g
Fructus Crataegi extract 100g
Semen Cassiae extract 300g
The said components mix homogeneously, in the hard gelatin capsule of packing into, totally 2000 capsules.
Claims (4)
1. total alkaloid of lotus leaves extraction preparation method, it is characterized in that: select 732 types for use, D001-CC, D001-SS, D061, the D072 storng-acid cation exchange resin is as the purification resin, the Folium Nelumbinis ethanol extraction is 0.1～1mg/mL in the sample solution concentration of total alkaloids amount, the absorption flow velocity is 3～15BV/h, the resin column blade diameter length ratio is 1: 5～1: 10, applied sample amount in the total alkaloids amount is 5～50mg/mL, 1～7 times of resin volume of 0～70% ethanol elution carries out remove impurity, the remove impurity flow velocity is 3～10BV/h, with 3～10 times of resin volumes of 40～90% ammonia ethanol elutions, wherein the alcoholic acid ammonia concentration of ammonia is 0.1%～5%, and elution flow rate is 8～20BV/h.
2. total alkaloid of lotus leaves extraction preparation method, it is characterized in that: get Folium Nelumbinis decoction pieces 1kg, add 90% ethanol 16L, reflux, extract, 3 times, the each extraction 1.5 hours, decompression and solvent recovery, extract adds 1% aqueous hydrochloric acid solution dispersing and dissolving, makes aqueous acid concentration count 0.165mg/mL with the total alkaloids amount, with the centrifugal 30min of 3000 rev/mins rotating speed, get supernatant, by 300mL D001-CC macroporous type storng-acid cation exchange resin, the absorption flow velocity is 10BV/h, and the resin column blade diameter length ratio is 1: 8, applied sample amount is counted 27.5mg/mL with the total alkaloids amount, 5 times of resin volumes of 50% ethanol elution carry out remove impurity, and the remove impurity flow velocity is 10BV/h, 7 times of resin volumes of 70% ammonia ethanol elution, wherein the alcoholic acid ammonia concentration of ammonia is 1%, elution flow rate is 10BV/h, collects 70% ammonia ethanol elution, reclaims solvent, drying under reduced pressure is total alkaloid of lotus leaves extraction.
3. total alkaloid of lotus leaves extraction preparation method, it is characterized in that: get Folium Nelumbinis decoction pieces 1kg, add 70% ethanol 20L, reflux, extract, 3 times, the each extraction 1.5 hours, decompression and solvent recovery, extract adds 0.5% aqueous hydrochloric acid solution dispersing and dissolving, makes aqueous acid concentration count 0.33mg/mL with the total alkaloids amount, with the centrifugal 30min of 3000 rev/mins rotating speed, get supernatant, by 300mL D001-CC macroporous type storng-acid cation exchange resin, the absorption flow velocity is 5BV/h, and the resin column blade diameter length ratio is 1: 10, applied sample amount is counted 30.25mg/mL with the total alkaloids amount, 10 times of resin volumes of water elution carry out remove impurity, and the remove impurity flow velocity is 10BV/h, 5 times of resin volumes of 90% ammonia ethanol elution, wherein the alcoholic acid ammonia concentration of ammonia is 0.5%, elution flow rate is 10BV/h, collects 90% ammonia ethanol elution, reclaims solvent, drying under reduced pressure is total alkaloid of lotus leaves extraction.
4. total alkaloid of lotus leaves extraction preparation method, it is characterized in that: get Folium Nelumbinis decoction pieces 1kg, add 50% ethanol 25L, reflux, extract, 2 times, the each extraction 2 hours, decompression and solvent recovery, extract adds 1.5% aqueous hydrochloric acid solution dispersing and dissolving, makes aqueous acid concentration count 0.495mg/mL with the total alkaloids amount, with the centrifugal 30min of 3000 rev/mins rotating speed, get supernatant, by 300mL D001-CC macroporous type storng-acid cation exchange resin, the absorption flow velocity is 3BV/h, and the resin column blade diameter length ratio is 1: 6, applied sample amount is counted 24.75mg/mL with the total alkaloids amount, 6 times of resin volumes of 10% ethanol elution carry out remove impurity, and the remove impurity flow velocity is 5BV/h, 7 times of resin volumes of 50% ammonia ethanol elution, wherein the alcoholic acid ammonia concentration of ammonia is 0.3%, elution flow rate is 10BV/h, collects 50% ammonia ethanol elution, reclaims solvent, drying under reduced pressure is total alkaloid of lotus leaves extraction.
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| 赵骏等.利用大孔吸附树脂纯化荷叶生物碱.中药材26 9.2003,26(9),669-670. * |
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