CN103566195A - Method for extracting alkaloid from dendrobium candidum - Google Patents
Method for extracting alkaloid from dendrobium candidum Download PDFInfo
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Abstract
The invention relates to a method for extracting an active component alkaloid from dendrobium candidum. The method comprises the following steps: taking fresh dendrobium candidum, degreasing, performing flash extraction and centrifuging on the fresh dendrobium candidum to obtain free crude alkaloid and synthetic crude alkaloid respectively, and separating respectively to obtain free alkaloid and synthetic alkaloid. By the method provided by the invention, the free alkaloid and the synthetic alkaloid can be effectively obtained, wherein the yield of the free alkaloid is not less than 0.35% and the extraction rate of the synthetic alkaloid is not less than 0.15%.
Description
Technical field
The present invention relates to extract the method for effective site from Herba Dendrobii, be specifically related to a kind of alkaloidal method of extracting from Herba Dendrobii.
Background technology
Herba Dendrobii is the perennial herbal herb of growing nonparasitically upon another plant of the orchid family Dendrobium, it is a kind of famous and precious Chinese herbal medicine, there is enhancing human body immunity power, antitumor, the secretion of facilitating digestion liquid, suppress the pharmacological actions such as platelet aggregation, blood fat reducing, blood sugar lowering, antioxidation, defying age and antipyretic analgesic, its chemical composition is various, mainly contains alkaloid, polysaccharide, aminoacid, luxuriant and rich with fragrance compounds etc.
Alkaloid is the nitrogenous alkaline organic compound of a class that is present in occurring in nature.Alkaloids belongs to sesquiterpene alkaloids, has sesquiterpene skeleton.In source of students, by amination or aminoacid, introduce nitrogen-atoms in skeleton.The content of Herba Dendrobii total alkaloids is low compared with other kind Dendrobium nobiles and Dendrobium Moniliforme, Herba Dendrobii etc., the non-Herba Dendrobii main component of alkaloid is described, the important function such as but alkaloid has antitumor, suppresses cardiovascular, gastroenteropathy and pain relieving are brought down a fever, the medical value of Herba Dendrobii alkaloid uniqueness, makes to study the alkaloidal separation and Extraction of Herba Dendrobii and has certain realistic meaning.In addition, for Herba Dendrobii, alkaloidal extraction research is for its free alkaloid component before, but the alkaloidal extraction of being combined with other materials does not relate to, this has just caused the raw-material waste of Herba Dendrobii, also makes alkaloidal extraction ratio undesirable simultaneously.Therefore, introduce a kind of advanced person's extractive technique, and extract free and significant for this precious raw material of Herba Dendrobii in conjunction with two kinds of alkaloids simultaneously.
Existing dendrobine or alkaloidal extracting method are as follows:
Ammonia infiltrates, ethanol ultrasonic extraction: CN201010557842.9(publication number is CN102477040A) relate to a kind of preparation method of dendrobine, it is characterized in that, by Herba Dendrobii pulverizing medicinal materials, with a small amount of ammonia, infiltrate, add again high concentration ethanol liquid supersound extraction 2-3 time, extracting solution ultrafilter membrane pressure filtration, concentrated through NF membrane again, sour water extraction 2-3 time for concentrated solution, get the alkalization of sour water layer, filter, precipitate ether dissolution, after mixing thoroughly with a small amount of silica gel, volatilize solvent, loading, employing partition chromatography is separated, thin layer monitoring, collect eluent, concentrating under reduced pressure evaporate to dryness obtains product.
Acidic ethanol extraction, cross cationic resin: CN201010300497.0(publication number is CN101735231A) relate to a kind of method of extracting purification dendrobine from Dendrobium nobile, the method is first used acidic ethanol extraction, then extract is passed through to cation exchange resin enriching and purifying, use again organic solvent extraction, finally adopt the separated preparation of HPLC.
Water dodges to be carried: CN201310026291.7(publication number is CN103113483A) a kind of method of extracting various active composition from Herba Dendrobii disclosed, specifically comprise dodge the crude extract carry, dodge after carrying centrifugal, the supernatant after centrifugal is carried out to precipitate with ethanol and three dehydrations, obtain free polysaccharide, precipitation after centrifugal is carried out extracting in conjunction with polysaccharide and alkaloid, finally from Herba Dendrobii, extract various active composition, but ungrease treatment is not carried out in this invention, larger on alkaloidal extraction ratio and purity impact.
Alcohol extraction after enzymolysis: Du's equality (Du's equality, Herba Dendrobii biology total alkali extracts purification research, Guangdong chemical industry, the 1st phase in 2013, total the 243rd phase of 40 volumes, 13-15) at Herba Dendrobii biology total alkali, extract in purification research and disclose alkaloidal extracting method, the method comprises activation, constant temperature enzymolysis, lixiviate, extraction, the assay of defat, enzyme, be specially: 1) defat: took the dry Herba Dendrobii powder 30g of 20 mesh sieves in clean flask, add about 50ml petroleum ether backflow defat 0.5h, sucking filtration; 2) activation of enzyme: take cellulase 0.15g(consumption and be Dendrobium weight 0.5%), add a small amount of 50 ℃ of warm water, be placed in 50 ℃ of calorstats and activate 2h; 3) constant temperature enzymolysis: activated cellulase solution slowly adds in the Herba Dendrobii after defat, control solid-liquid ratio is 1:10, stirring and adjusting pH value is 5.0, is warming up to immediately 90 ℃ of enzyme denaturing after constant temperature enzymolysis 1.5h, rapider cold filtration; 4) lixiviate: add 90% ethanol 50ml lixiviate 0.5h, filter, filtering residue is used 90% alcohol steep twice again, each 0.5h, filters, merging filtrate, rotary evaporation, reclaims ethanol; 5) extraction: in raffinate, add chloroform 50ml, extraction, then measure chloroform extraction twice with half, after combining extraction liquid, Distillation recovery chloroform obtains total dendrobine; 6) assay: dendrobine extractum dissolves with chloroform, is settled to 100ml, gets 10.0ml, surveys concentration by dendrobine content assaying method.
In said method, extraction efficiency is all poor, therefore, need to provide a kind of method that increases extraction ratio.
Summary of the invention
For problems of the prior art, the present invention proposes a kind of method of extracting alkaloid component of combining from Herba Dendrobii.
A kind of alkaloidal method of active component of extracting from Herba Dendrobii that the present invention proposes, specifically comprises the following steps: get fresh Herba Dendrobii defat, sudden strain of a muscle is carried, centrifugal, obtain respectively drifting alkaloids crude product and synthetic alkaloid crude product, separated respectively, obtain drifting alkaloids and synthetic alkaloid.
In said method:
Described defat: weigh-25 ℃--10 ℃ of freezing fresh Herba Dendrobii, shred, dry, pulverize, then use petroleum ether, fresh Herba Dendrobii compares 1:6-10 with the w/v g/ml of petroleum ether, reflux 4 hours, filtered, then with petroleum ether, Herba Dendrobii powder is cleaned, fresh Herba Dendrobii than for 1:5-7, obtains defat Herba Dendrobii powder with the bulking value g/ml of petroleum ether.
Herba Dendrobii dodges to carry and comprises the following steps: by defat Herba Dendrobii, adding the concentration of 40-60 times of volume of its weight is 75-90% ethanol, and after mixing, adjusting pH value is 8-10, dodges and carries twice, and the voltage that device is carried in sudden strain of a muscle is 110V-120V, and each time is 30-60 second;
Be preferably: extracting degreasing Herba Dendrobii, add the 80-85% ethanol of 45-55 times of volume of its weight, mix, adjust pH9-10, with the flash extracter that is adjusted to 110-120V, extract twice, each 30-60 second, dodge and raise alkaloid crude extract.
Described centrifugal: centrifugal speed is 5000-6000r/min, centrifugation time is 10-30min, and separation obtains supernatant A and precipitate B, and wherein drifting alkaloids is present in supernatant A, in conjunction with alkaloid, is present in precipitate B.
From supernatant A, extract the method for drifting alkaloids, comprise concentrated, adjust pH, with chloroform, dissolve ultrasonic, crystallization.
Concrete, the method for extracting drifting alkaloids from supernatant A comprises the following steps:
1) concentrated: supernatant A is evaporated to original volume 5% left and right;
2) adjust pH: to adding ammonia adjust pH in concentrated solution, be 10, evaporate to dryness;
3) extract, purification: till being then dissolved to and forming even suspension with chloroform, ultrasonic accelerate dissolution then, ultrasonic power is 300w, the ultrasonic time is 5-15min, centrifugal, and supernatant is dry, obtains solid matter;
4) crystallization: add in acetone and grind in the solid matter that step 3) is obtained, draw the supernatant, then repeatedly use acetone solution, grinding, the absorption supernatant, until no longer include precipitation, finally form suspension, use ultrasonic accelerate dissolution, then at-20 ℃ of temperature, precipitate 1 hour, after taking-up, carry out centrifugal, get precipitation, dry;
5) refining: add methanol ultrasonic dissolution, finally form suspension, suspension is centrifugal, and by the supernatant evaporate to dryness after centrifugal, remaining solid is the alkaloid of free type.
In said method:
Without particularly pointing out pH value regulator, be sodium hydroxide, hydrochloric acid
In described step 3), the w/v of solid and chloroform is 1:15-1:25g/ml;
The consumption of the acetone grinding with solid matter in described step 4), the w/v of solid matter and acetone is 1:20-1:25g/ml.
In described step 5), the w/v of solid and methanol is 1:10-1:15g/ml;
Centrifugal speed is 5000-6000r/min, and centrifugation time is 20-30min;
The method of ultrasonic accelerate dissolution is that container is put into ultrasonic cleaning instrument, utilizes ultrasonic concussion accelerate dissolution process, improves dissolved efficiency, ultrasonic time 5-15min, and ultrasonic power is 300w.
From precipitate B, extract in conjunction with alkaloidal method: water sudden strain of a muscle is carried, centrifugal, supernatant alkalization,
Specifically, from precipitate B, extract and comprise the following steps in conjunction with alkaloidal method:
1) dodge and carry: will in precipitate B, add 40-60 times of water, regulate pH to 9-10, with flash extracter, dodge and carry 1-2 time, extractor is adjusted to 110-120V, and extraction time is 30-60 second, collects extracting solution, standby;
2) centrifugal: the extracting solution that step 1) is obtained is centrifugal, get its precipitation part, add the about 40-50 of precipitation weight times water, stir evenly, then add the cellulase of its weight 0.5-0.7% and the papain of 1.3-1.7%, adjust pH5.5,50 ℃ of water-baths, enzymolysis 1.5-2h, enzymolysis post-heating to 100 ℃, boiling 3-5min, by the solution centrifugal boiling, get precipitation, standby;
3) precipitation alkalization: by step 2) obtaining adds ammonia, and adjust pH is 9-10;
4) dodge and to carry: add the dehydrated alcohol of the 40-50 times of volume of precipitation weight that step 3) obtains, adjust pH9-10, dodge and carry 1-2 time, 30-60s/ time, voltage is 110-120V, and sudden strain of a muscle is carried rear centrifugal, gets supernatant, standby;
5) evaporate to dryness: the supernatant concentration that step 4) is obtained, to 5% left and right of volume, adjust pH is 10, dry, obtains solid;
6) extract: the solid that step 5) is obtained dissolves with chloroform, and the w/v g/ml of solid and chloroform is 1:20-25, centrifugal, collects supernatant, with thermostat water bath, by its evaporate to dryness, obtains powder;
7) crystallization: the powder acetone solution that step 6) is obtained, the w/v g/ml of powder and acetone is 1:20-25, forms suspension, then uses ultrasonic accelerate dissolution, then at-20 ℃ of temperature, precipitate 1 hour, after taking-up, carry out centrifugal, get precipitation, dry, standby;
8) refining: use methanol ultrasonic dissolution, wherein precipitation is 1:10-15 with the w/v g:ml of methanol, and ultrasonic power is 300w, and time 5-15min finally forms suspension, centrifugal, collects supernatant evaporate to dryness, obtains the alkaloid of bond type.
In said method:
As nothing particularly points out, described pH adjusting agent is hydrochloric acid and sodium hydroxide;
All centrifugal conditions are all 5000-10000r/min, 15-30min.
Utilizing a kind of extraction ratio that extracts the resulting drifting alkaloids of alkaloidal method from Herba Dendrobii that the present invention proposes is 0.31-0.39%; The active component obtaining is 0.13-0.185% in conjunction with alkaloidal extraction ratio.
The advantage that the present invention has is:
1, a kind of method of extracting active component from Herba Dendrobii that the present invention proposes, by flash extracter, extract Herba Dendrobii alkaloid component, compare with traditional method, the method extraction conditions is gentle, extraction time is short, 1-3min only, and solvent load is few, extraction efficiency is high, is applicable to suitability for industrialized production.
2, a kind of method of extracting alkaloid component from Herba Dendrobii that the present invention proposes, extracts in conjunction with alkaloid in the precipitation from first centrifugal, has realized the comprehensive utilization of Herba Dendrobii, for the Chinese crude drug of this preciousness, is extremely important.
3, a kind of method of extracting alkaloid component from Herba Dendrobii that the present invention proposes, realization finishes the separation and Extraction of closing alkaloid and drifting alkaloids, in precipitation from first centrifugal, extract in conjunction with alkaloid, improve the alkaloidal effective extraction efficiency of Herba Dendrobii and comprehensive utilization ratio, avoided the waste of resource.
4, adopt flash extractive technique (be called for short to dodge and carry) in the presence of room temperature and solvent, in the several seconds, the materials such as the root of plant, stem, leaf, flower, fruit to be crushed to fine particle, make the rapid stripping of in-house effective ingredient, greatly improved extraction efficiency.
5, the present invention has used a kind of more advanced extracting method-flash extraction method, makes can reach at short notice very high extraction ratio in this way, has greatly improved work efficiency, is very feasible in commercial production; Aspect end product, the present invention has realized conjunction type and two kinds of alkaloidal extractions of form of sequestered simultaneously, method is in the past often only for sequestered alkaloid, result shows, the alkaloidal content of Herba Dendrobii conjunction type is higher, two types of alkaloidal utilization rates that greatly improved raw material of simultaneously extracting.
6, experimental result shows: the invention provides extraction separation method, can effectively obtain drifting alkaloids and in conjunction with alkaloid, wherein the yield of drifting alkaloids is not less than 0.35%, in conjunction with alkaloidal extraction ratio, be not less than 0.15%.
The specific embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.
Embodiment 1:
1, defat: weigh-20 ℃ of freezing fresh Herba Dendrobii 60g, shred, dry, pulverize, then use 360ml petroleum ether (the w/v g/ml of fresh Herba Dendrobii and petroleum ether is 1:6) to reflux 4 hours, filtered, then Herba Dendrobii powder is cleaned to (fresh Herba Dendrobii is 1:5.8 with the bulking value g/ml ratio of petroleum ether) with 350ml petroleum ether, obtain defat Herba Dendrobii powder, standby;
2, dodge and carry:
First extracting degreasing Herba Dendrobii powder 10g, adding 500ml concentration is 85% ethanol (50 times of amount volumes that are equivalent to defat Herba Dendrobii), ethanol will add gradually, limit adds ethanol limit and constantly stirs, defat Herba Dendrobii powder is evenly distributed in wherein, avoids in bulk, after mixing, adjusting pH is 10, with flash extracter, extract to dodge and to carry 2 times, the time is respectively 1min, 30 seconds, voltage 110V, collect twice and dodge extract, obtain alkaloid crude extract;
3, centrifugal:
Alkaloid crude extract is centrifugal, and it is 15min that centrifugal speed and time are respectively 5000r/min, time, and precipitation separation and supernatant, obtain supernatant A and precipitate B, and wherein drifting alkaloids is present in supernatant A, in conjunction with alkaloid, is present in precipitate B.
4, from supernatant A, extract drifting alkaloids:
4.1 is concentrated: by supernatant concentrating under reduced pressure, the volume after concentrating is 5% of original volume;
4.2 basification: add ammonia in concentrated solution, adjust pH is 10, pours evaporating dish into, then clean round-bottomed flask with dehydrated alcohol, with vacuum drying oven by its evaporate to dryness, the alkaloid of the free type that obtains slightly carrying;
4.3 extract purification: the alkaloid of the free type of slightly carrying that step 4.2 is obtained dissolves (the w/v g/ml of crude extract and chloroform is 1:20) repeatedly with 10ml chloroform, alkaloid is dissolved in chloroform, form after suspension repeatable operation, then, by ultrasonic its accelerate dissolution that makes, ultrasonic time is 6min, power is 300w, then centrifugal, centrifugal condition is that speed is 5000r/min, and the time is 15min, by supernatant thermostat water bath evaporate to dryness, obtain solid matter;
4.4 crystallizations: 4.3 solid matters that obtain are transferred among mortar, the acetone (w/v of solid matter and acetone is 1:20g/ml) that adds 10ml, constantly grind impurity is dissolved in acetone as much as possible, then with glue head straw, supernatant liquid is drawn and is transferred in the centrifugal plastic tube of acetone; Constantly repeat acetone and grind, draw the step of supernatant liquid, until no longer include precipitation, produce, finally form suspension, then to use ultrasonic accelerate dissolution, ultrasonic power and time be 300w and 10min.Then at-20 ℃ of temperature, precipitate 1 hour, carry out centrifugally after taking-up, centrifugal condition is that speed is 5000r/min, and the time is 15min, gets precipitation, dry, obtains solid, standby.
4.5 is refining: 4.4 solids that obtain are added to methanol (the w/v g/ml of solid and methanol is 1:10) ultrasonic dissolution (ultrasonic power is 300w, and the time is 10min), finally form suspension.Suspension is centrifugal, and centrifugal condition is 5000r/min, and the time is 15min, and the supernatant after centrifugal is poured in evaporating dish, with thermostat water bath, makes methanol solution evaporate to dryness, and remaining solid is the alkaloid of free type.The weight that weighing obtains drifting alkaloids is 35mg, and yield is 0.35%.
5, from precipitate B, extract in conjunction with alkaloid, be specially:
5.1 sudden strains of a muscle are carried: will in precipitate B, add the water of approximately 40 times of amounts, regulate pH value to 10, with flash extracter, extract 2 times, flash extracter is adjusted to 110V, and extraction time is respectively 60s, 30s, collects extracted twice liquid, standby;
5.2 is centrifugal: the extracting solution that 5.1 steps are obtained is centrifugal, and centrifugal condition is 5000r/min, and the time is 15min, get its precipitation part, add the approximately water of 40 times of amounts of this precipitation weight, stir evenly, then 1.5% the papain that adds 0.6% cellulase of this precipitation weight and this precipitation weight, adjusting pH is 5.5,50 ℃ of water-baths, enzymolysis 1.5h, enzymolysis post-heating to 100 ℃, boiling 5min, by the solution centrifugal boiling, centrifugal condition is 5000r/min, 15min, get precipitation, standby;
5.3 alkalization: get the precipitation of step 5.2, add ammonia, regulating pH value is 10;
5.4 sudden strains of a muscle are carried: the precipitation after the adjusted pH of step 5.3 is added to the dehydrated alcohol of 40 times of volumes of its weight, regulating pH value is 10, dodges and carries 1 time, and the time is 30s, voltage is 110V, and sudden strain of a muscle is carried rear centrifugal, and centrifugal rotational speed is 10000r/min, time is 15min, gets supernatant, standby;
5.5 evaporates to dryness: supernatant be evaporated to 5% of volume, then concentrated solution transferred in evaporating dish, and with ultra-pure water, round-bottomed flask is cleaned, transfer in evaporating dish, and to regulate pH value be 10, then with vacuum drying oven by its evaporate to dryness, obtain solid;
5.6 extract: chloroform solid for that step 5.5 is obtained (solid is 1:20 with the w/v g:ml of chloroform) dissolves that (dissolving refers to and adds identical chloroform dissolving at every turn repeatedly repeatedly, then collect supernatant, and then dissolve with chloroform, regather supernatant), alkaloid is dissolved in chloroform, ultrasonic accelerate dissolution, ultrasonic power is 300w, time is 15min, after repeatedly dissolving, obtain suspension, centrifugal, rotating speed 5000r/min, time is 15min, get supernatant, with thermostat water bath by its evaporate to dryness, obtain powder,
5.7 crystallizations: (ultrasonic power is 300w to the ultrasonic accelerate dissolution of the acetone for powder that step 5.6 is obtained (the w/v g:ml of powder and acetone is 1:20), 15min), form suspension, then be placed in-20 ℃, precipitate 1 hour, after taking-up, carry out that centrifugal (speed is 5000r/min, time is 15min), get precipitation, dry, standby.
5.8 is refining: (ultrasonic power is 300w to ultrasonic dissolution to get methanol precipitation for (the w/v g/ml of precipitation and methanol is 1:15) that step 5.7 obtains, 15min), the final suspension that forms, suspension is centrifugal, speed is 5000r/min, and the time is 15min, and the supernatant after centrifugal is poured in evaporating dish, with thermostat water bath, make methanol solution evaporate to dryness, remaining solid is the alkaloid of bond type.It is 15mg that weighing obtains in conjunction with alkaloidal weight, and yield is 0.15%.
Embodiment 2:
1, pretreatment:
Weigh certain-20 ℃ freezing fresh Herba Dendrobii 60g, shred, dry, pulverize.Then use 600ml petroleum ether (fresh Herba Dendrobii with the w/v g/ml of petroleum ether than 1:10) to reflux 4 hours, by its filtration, with 400ml petroleum ether, Herba Dendrobii powder is cleaned to (fresh Herba Dendrobii is 1:6.7 with the bulking value g/ml ratio of petroleum ether) again, obtain defat Herba Dendrobii powder, wait until use;
2, dodge and carry:
First extracting degreasing Herba Dendrobii powder 10g, adding 400ml concentration is 80% ethanol (40 times of amount volumes that are equivalent to defat Herba Dendrobii), ethanol adds gradually, constantly stirs, and defat Herba Dendrobii powder is evenly distributed in wherein, avoids in bulk.After mixing, adjusting pH is 10, with flash extracter, extracts 2 times, extracts 60 seconds at every turn, and voltage 110V, collects sudden strain of a muscle extract twice, obtains alkaloid crude extract
3, concentrated
Alkaloid crude extract is centrifugal, and centrifugal condition is 5000r/min, and the time is 20min, and precipitation separation and supernatant obtain supernatant A and precipitate B, and wherein drifting alkaloids is present in supernatant A, in conjunction with alkaloid, is present in precipitate B.
4, in supernatant A, extract drifting alkaloids:
4.1 is concentrated: by supernatant concentrating under reduced pressure, the volume after concentrating is 5% of original volume, obtains concentrated solution.
4.2 basification: add ammonia in concentrated solution, adjusting pH is 10, pours evaporating dish into, then clean round-bottomed flask with dehydrated alcohol, with vacuum drying oven by its evaporate to dryness, the alkaloid of the free type that obtains slightly carrying.
4.3 extract purification: about 10ml chloroform for the alkaloid of the free type of slightly carrying that step 4.2 is obtained (the w/v g/ml of crude extract and chloroform is 1:20) dissolves repeatedly, and alkaloid is dissolved in chloroform.After repeatable operation, ultrasonic dissolution, ultrasonic power is 300w, and ultrasonic time is 10min, and centrifugal condition is 5000r/min, and the time is 20min, by supernatant thermostat water bath evaporate to dryness, obtains solid matter;
4.4 crystallizations: 4.3 solid matters that obtain are transferred among mortar, add 10ml acetone (w/v of solid matter and acetone is 1:20g/ml), constantly grind, make impurity be dissolved in as much as possible in acetone, then with glue head straw, supernatant liquid is drawn and is transferred in the centrifugal plastic tube of acetone, constantly repeating acetone grinds, draw the step of supernatant liquid, until no longer include precipitation, the final suspension that forms, use ultrasonic accelerate dissolution, ultrasonic power and time are respectively 300w again, and the time is 15min.Then at-20 ℃ of temperature, precipitate 1 hour, carry out centrifugally after taking-up, the time is 5000r/min, and the time is 15min, gets precipitation, dry, obtains solid, standby;
4.5 is refining: the solid that step 4.4 is obtained adds methanol (the w/v g/ml of solid and methanol is 1:15) ultrasonic dissolution (ultrasonic power is 300w, time 15min), finally forms suspension.By suspension centrifugal (speed is 5000r/min, and the time is 15min), centrifugal supernatant is afterwards poured in evaporating dish, with thermostat water bath, makes methanol solution evaporate to dryness, and remaining solid is the alkaloid of free type.
5, in precipitate A, extract in conjunction with alkaloid.Specifically comprise the steps:
5.1 sudden strains of a muscle are carried: will in precipitate B, add the water of approximately 50 times of amounts, regulate pH value to 10, with flash extracter, extract 2 times, the time is 60 seconds, and flash extracter is adjusted to 110V, collects extracting solution, standby.
5.2 is centrifugal: the extracting solution that 5.1 steps are obtained is centrifugal, and speed is 10000r/min, and the time is 15min, get its precipitation part, add the approximately water of 50 times of amounts of this precipitation weight, stir evenly, then add 0.6% cellulase of this precipitation weight and the papain of this precipitation weight 1.5%, adjusting pH to 5.5,50 ℃ of water-baths, enzymolysis 2h, enzymolysis post-heating to 100 ℃, boiling 5min, by the solution centrifugal boiling, (speed is 5000r/min, time is 15min), collect precipitation, standby.
5.3 alkalization: the precipitation of getting step 5.2 adds ammonia, regulating pH value is 10;
5.4 sudden strains of a muscle are carried: the precipitation after the adjusted pH of step 5.3 is added to the dehydrated alcohol of 45 times of volumes of its weight, adjusting pH is 10, dodges and carries 2 times, and each extraction time is 60 seconds, voltage is 110V, and sudden strain of a muscle is carried rear centrifugal, and speed is 10000r/min, time is 20min, gets supernatant, standby.
5.5 evaporates to dryness: supernatant is evaporated to volume adequacy (relative density approximately 5%), then concentrated solution is transferred in evaporating dish, and with ultra-pure water, round-bottomed flask is cleaned, transfer in evaporating dish, and to regulate pH value be 10, then with vacuum drying oven by its evaporate to dryness, obtain solid;
5.6 extract: the chloroform for solid that step 5.5 is obtained (the w/v g:ml of solid and chloroform is 1:20) dissolves repeatedly, alkaloid is dissolved in chloroform, ultrasonic accelerate dissolution, ultrasonic power is 300w, time is 15min, after repeatedly dissolving, obtain suspension, centrifugal, rotating speed 5000r/min, time is 15min, get supernatant, with thermostat water bath, by its evaporate to dryness, obtain powder.
5.7 crystallizations: the acetone for powder that step 5.5 is obtained (the w/v g:ml of powder and acetone is 1:25) dissolves, form suspension, ultrasonic (ultrasonic power is 300w, 10min) accelerate dissolution.Then in-20 ℃, precipitate 1 hour, after taking-up, carry out centrifugal (5000r/min, 15min), get precipitation, dry, standby.
5.8 is refining: with methanol (precipitation is 1:15 with the w/v g/ml of methanol) ultrasonic (ultrasonic power and time are respectively 300w, 15min), dissolve, finally form suspension.By suspension centrifugal (centrifugal speed and time are respectively 5000r/min, 30min), centrifugal supernatant is afterwards poured in evaporating dish, with thermostat water bath, makes methanol solution evaporate to dryness, and remaining solid is the alkaloid of bond type.
6, end-product: the weight of drifting alkaloids is 37mg, yield 0.37% is 17.4mg in conjunction with alkaloidal weight, yield is 0.17%.
Embodiment 3:
1, pretreatment:
Weigh certain-10 ℃ freezing fresh Herba Dendrobii 90g, shred, dry, pulverize.Then use 900ml petroleum ether (fresh Herba Dendrobii with the w/v g/ml of petroleum ether than 1:10) to reflux 4 hours, by its filtration, with the petroleum ether of 450ml, Herba Dendrobii powder is cleaned to (fresh Herba Dendrobii with the w/v g/ml of petroleum ether than 1:5) again, obtain defat Herba Dendrobii powder, wait until use;
2, dodge and carry:
First extracting degreasing Herba Dendrobii powder 15g, adding 750ml concentration is 83% ethanol (45 times of amount volumes that are equivalent to defat Herba Dendrobii weight), ethanol adds gradually, constantly stir, defat Herba Dendrobii powder is evenly distributed in wherein, avoids in bulk, after mixing, adjusting pH is 10, with flash extracter, extract 2 times, each 60 seconds, voltage 110V, collect twice and dodge extract, obtain alkaloid crude extract.
3, concentrated
Alkaloid crude extract is centrifugal, and centrifugal condition is 5000r/min, and 30min makes precipitation separated with supernatant.Obtain supernatant A and precipitate B, wherein drifting alkaloids is present in supernatant A, in conjunction with alkaloid, is present in precipitate B.
4, in supernatant A, extract drifting alkaloids; Specifically comprise the steps:
4.1 is concentrated: by supernatant concentrating under reduced pressure, be concentrated into original volume 5% left and right, obtain concentrated solution.
4.2 basification: in concentrated solution, add ammonia, adjust pH10, pour in evaporating dish, then with dehydrated alcohol, round-bottomed flask is cleaned, with vacuum drying oven by its evaporate to dryness, the alkaloid of the free type that obtains slightly carrying.
4.3 extract purification: the chloroform for alkaloid of the free type of slightly carrying that step 4.2 is obtained (the w/v g/ml of crude extract and chloroform is 1:20) dissolves repeatedly, alkaloid is dissolved in chloroform, after repeatable operation, ultrasonic accelerate dissolution, ultrasonic power is 300w, ultrasonic 10min; Final object is again purification, gets rid of the material that is insoluble to chloroform.Centrifugal condition is 5000r/min, and 30min, by supernatant thermostat water bath evaporate to dryness.
4.4 crystallizations: 4.3 solid matters that obtain are transferred among mortar, add about 15ml acetone (w/v of solid matter and acetone is 1:20g/ml), constantly grind, impurity is dissolved in acetone as much as possible, then with glue head straw, supernatant liquid is drawn and is transferred in the centrifugal plastic tube of acetone; Constantly repeat above-mentioned step, until form uniform suspension, no longer include precipitation.Final formation suspension, then use ultrasonic (ultrasonic power and time are respectively 300w, 15min) accelerate dissolution.Then in-20 ℃, precipitate 1 hour, after taking-up, carry out centrifugal (centrifugal rotating speed and time are respectively 5000r/min, 15min), get precipitation, dry, obtain solid, standby.
4.5 is refining: about 10ml methanol for the solid that step 4.4 is obtained (the w/v g/ml of solid and methanol is 1:10) dissolves, and accelerates it dissolve with ultrasonic (ultrasonic power is 300w, time 15min), finally forms suspension.By suspension centrifugal (centrifugal rotational speed and time are respectively 5000r/min, 15min), the supernatant after centrifugal is poured in evaporating dish, with thermostat water bath, make methanol solution evaporate to dryness, remaining solid is the alkaloid of free type.
5, in precipitate A, extract in conjunction with alkaloid.Specifically comprise the steps:
5.1 sudden strains of a muscle are carried: will in precipitate B, add the water of approximately 45 times of amounts, regulating pH value is 9, with flash extracter, extracts 2 times, and each extraction time is 60s, and flash extracter is adjusted to 120V, collects extracted twice liquid, standby;
5.2 is centrifugal: the extracting solution that 5.1 steps are obtained is centrifugal, centrifugal rotational speed is 5000r/min, time is 30min, get its precipitation part, add the approximately water of 60 times of amounts of this precipitation weight, stir evenly, then 1.3% the papain that adds 0.5% cellulase of this precipitation weight and this precipitation weight, regulating pH is 5.5,50 ℃ of water-baths, enzymolysis 2h.Enzymolysis post-heating to 100 ℃, boiling 3min, by the solution centrifugal boiling (centrifugal rotational speed is 5000r/min, 20min), gets precipitation, standby.
5.3 alkalization: add ammonia in precipitation, regulating pH value is 9.
5.4 sudden strains of a muscle are carried: the precipitation after the adjusted pH of step 5.3 is added to the dehydrated alcohol of 50 times of volumes of its weight, regulating pH value is 9, extract each 60s/ time 2 times, voltage is 120V, and sudden strain of a muscle is carried rear centrifugal, centrifugal rotational speed 10000r/min, centrifugal 30min, gets supernatant, standby.
5.5 evaporates to dryness: supernatant is evaporated to 5% of volume, then concentrated solution is transferred in evaporating dish, and with ultra-pure water, round-bottomed flask is cleaned, transfer in evaporating dish, and to regulate pH value be 10, then with vacuum drying oven by its evaporate to dryness, obtain solid (the combination alkaloid of slightly carrying).
5.6 extract: the chloroform for solid that step 5.5 is obtained (the w/v g:ml of solid and chloroform is 1:25) dissolves repeatedly, alkaloid is dissolved in chloroform, ultrasonic accelerate dissolution, ultrasonic power is 300w, time is 15min, after repeatedly dissolving, obtain suspension, centrifugal, rotating speed 5000r/min, time is 15min, get supernatant, with thermostat water bath, by its evaporate to dryness, obtain powder.
5.7 crystallizations: the acetone for powder that step 5.6 is obtained (the solid-to-liquid ratio g/ml of powder and acetone is 1:20) dissolves, and finally forms suspension, then uses ultrasonic dissolution, and ultrasonic power is 300w, time 10min, accelerate dissolution.Then in-20 ℃, precipitate 1 hour, carry out centrifugally after taking-up, centrifugal rotational speed is 5000r/min, and 15min, gets precipitation, dry, standby.
5.8 is refining: with dissolve with methanol (precipitation is about 1:10 with the w/v g:ml of methanol), ultrasonic, ultrasonic power is 300w, and 15min accelerates it and dissolves, and finally forms suspension.Suspension is centrifugal, and centrifugal rotational speed is 5000r/min, and 15min pours the supernatant after centrifugal in evaporating dish into, with thermostat water bath, makes methanol solution evaporate to dryness, and remaining solid is the alkaloid of bond type.
6, end-product: the weight that obtains drifting alkaloids is 58.5mg, yield is 0.39%, in conjunction with alkaloidal weight, is 27.75mg, yield is 0.185%.
Embodiment 4:
1, defat: weigh-20 ℃ of freezing fresh Herba Dendrobii 60g, shred, dry, pulverize, then use 480ml petroleum ether (fresh Herba Dendrobii with the w/v g/ml of petroleum ether than 1:8) to reflux 4 hours, filtered, then Herba Dendrobii powder is cleaned to (fresh Herba Dendrobii with the w/v g/ml of petroleum ether than 1:6.7) with the petroleum ether of 400ml, obtain defat Herba Dendrobii powder, standby;
2, dodge and carry:
First extracting degreasing Herba Dendrobii powder 10g, adding 600ml concentration is 90% ethanol (60 times of amount volumes that are equivalent to defat Herba Dendrobii), ethanol will add gradually, limit adds ethanol limit and constantly stirs, defat Herba Dendrobii powder is evenly distributed in wherein, avoids in bulk, after mixing, adjusting pH is 10, with flash extracter, extract to dodge and to carry 2 times, the time is respectively 1min, 30 seconds, and voltage is adjusted to 110V, collect twice and dodge extract, obtain alkaloid crude extract;
3, centrifugal
Alkaloid crude extract is centrifugal, and centrifugal speed and time are respectively 5000r/min, 15min, make precipitation separated with supernatant, obtain supernatant A and precipitate B, and wherein drifting alkaloids is present in supernatant A, in conjunction with alkaloid, is present in precipitate B.
4, from supernatant A, extract drifting alkaloids:
4.1 is concentrated: by supernatant concentrating under reduced pressure (relative density after concentrated is 5%);
4.2 basification: add ammonia in concentrated solution, adjust pH is 10, pour in evaporating dish, then with dehydrated alcohol, round-bottomed flask is cleaned, with vacuum drying oven by its evaporate to dryness, the alkaloid of the free type that obtains slightly carrying;
4.3 extract purification: the alkaloid of the free type of slightly carrying that step 4.2 is obtained dissolves (the w/v g/ml of crude extract and chloroform is 1:25) repeatedly with about 15ml chloroform, alkaloid is dissolved in chloroform, forms suspension, by ultrasonic its accelerate dissolution that makes, ultrasonic power is 300w, ultrasonic 10min, removes the precipitation that is insoluble to chloroform, then centrifugal, centrifugal condition is 5000r/min, 15min, by supernatant thermostat water bath evaporate to dryness, obtains solid matter;
4.4 crystallizations: 4.3 solid matters that obtain are transferred among mortar, add 15ml acetone (w/v of solid matter and acetone is 1:25g/ml), constantly grind, impurity is dissolved in acetone as much as possible, then with glue head straw, supernatant liquid is drawn and is transferred in the centrifugal plastic tube of acetone; Constantly repeat above-mentioned step, until form uniform suspension, no longer include precipitation.Final form suspension, then to use ultrasonic accelerate dissolution, ultrasonic power be 300w, time 10min.Then in-20 ℃, precipitate 1 hour, carry out centrifugally after taking-up, the centrifugal speed of walking around is for 5000r/min, and 15min, gets precipitation, dry, obtains solid, standby.
4.5 is refining: 10ml methanol for the solid of step 4.4 (the w/v g/ml of solid and methanol is 1:15) is dissolved, and with ultrasonic acceleration, it dissolves, and ultrasonic power is 300w, and the time is that 15min finally forms suspension.Suspension is centrifugal, and the centrifugal speed of walking around is for 5000r/min, and 15min, pours the supernatant after centrifugal in evaporating dish into, with thermostat water bath, makes methanol solution evaporate to dryness, and remaining solid is the alkaloid of free type.The weight that weighing obtains drifting alkaloids is 36mg, and yield is 0.36%.
5, from precipitate B, extract in conjunction with alkaloid, be specially:
5.1 sudden strains of a muscle are carried: will in precipitate B, add the water of approximately 60 times of amounts, regulating pH value is 10, with flash extracter, extracts twice, and flash extracter is adjusted to 110V, and extraction time is respectively 1min, 30s, collects extracted twice liquid, standby;
5.2 is centrifugal: the extracting solution that 5.1 steps are obtained is centrifugal, and centrifugal speed is 10000r/min, and the time is 30min, get its precipitation part, add the approximately water of 40 times of amounts of this precipitation weight, stir evenly, then 1.5% the papain that adds 0.6% cellulase of this precipitation weight and precipitate B weight, regulating pH is 5.5,50 ℃ of water-baths, enzymolysis 2h, enzymolysis post-heating to 100 ℃, boiling 5min, by the solution centrifugal boiling, (centrifugal rotational speed is 5000r/min, 15min), get precipitation, standby.
5.3 alkalization: add ammonia, regulating pH value is 9;
5.4 sudden strains of a muscle are carried: the precipitation after the adjusted pH of step 5.3 is added to the dehydrated alcohol of 40 times of volumes of its weight, regulating pH value is 10, dodges and carries 1 time, and the time is 30s/ time, and voltage is 110V.Sudden strain of a muscle is carried rear centrifugal, centrifugal rotational speed 10000r/min, and 30min, gets supernatant, standby;
5.5 evaporates to dryness: it is 5% of volume that supernatant is evaporated to volume.Then concentrated solution transferred in evaporating dish, and with ultra-pure water, round-bottomed flask is cleaned, transfer in evaporating dish, and to regulate pH value be 10, then with vacuum drying oven by its evaporate to dryness, obtain solid.
5.6 extract: the chloroform for solid that step 5.5 is obtained (the w/v g:ml of solid and chloroform is 1:25) dissolves repeatedly, alkaloid is dissolved in chloroform, ultrasonic accelerate dissolution, ultrasonic power is 300w, time is 15min, after repeatedly dissolving, obtain suspension, centrifugal, rotating speed 5000r/min, time is 15min, get supernatant, with thermostat water bath, by its evaporate to dryness, obtain powder.
5.7 crystallizations: the acetone for powder that step 5.6 is obtained (the w/v g:ml of powder and acetone is 1:20) dissolves, and finally forms suspension, then to use ultrasonic accelerate dissolution, ultrasonic power be 300w, time 15min.Then in-20 ℃, precipitate 1 hour, after taking-up, carry out centrifugal (centrifugal rotational speed and time are respectively 5000r/min, 15min), get precipitation, dry, standby.
5.8 is refining: with dissolve with methanol (precipitation is 1:10 with the w/v g:ml of methanol), its dissolving of ultrasonic acceleration, ultrasonic power is 300w, and the time is 15min, finally forms suspension, suspension is centrifugal, centrifugal rotational speed is 5000r/min, and 15min pours the supernatant after centrifugal in evaporating dish into, with thermostat water bath, make methanol solution evaporate to dryness, remaining solid is the alkaloid of bond type.It is 17mg that weighing obtains in conjunction with alkaloidal weight, and yield is 0.17%.
Comparative example 1:
With reference to venturi equality (Du's equality, Herba Dendrobii biology total alkali extracts purification research, Guangdong chemical industry, the 1st phase in 2013, total the 243rd phase of 40 volumes, 13-15) at Herba Dendrobii biology total alkali, extract in purification research and disclose alkaloidal extracting method, the method comprises activation, constant temperature enzymolysis, lixiviate, extraction, the assay of defat, enzyme, be specially: 1) defat: took the dry Herba Dendrobii powder 30g of 20 mesh sieves in clean flask, add about 50ml petroleum ether backflow defat 0.5h, sucking filtration; 2) activation of enzyme: take cellulase 0.15g(consumption and be Dendrobium weight 0.5%), add a small amount of 50 ℃ of warm water, be placed in 50 ℃ of calorstats and activate 2h; 3) constant temperature enzymolysis: activated cellulase solution slowly adds in the Herba Dendrobii after defat, control solid-liquid ratio is 1:10, stirring and adjusting pH value is 5.0, is warming up to immediately 90 ℃ of enzyme denaturing after constant temperature enzymolysis 1.5h, rapider cold filtration; 4) lixiviate: add 90% ethanol 50ml lixiviate 0.5h, filter, filtering residue is used 90% alcohol steep twice again, each 0.5h, filters, merging filtrate, rotary evaporation, reclaims ethanol; 5) extraction: in raffinate, add chloroform 50ml, extraction, then measure chloroform extraction twice with half, after combining extraction liquid, Distillation recovery chloroform obtains total dendrobine; 6) assay: dendrobine extractum dissolves with chloroform, is settled to 100ml, gets 10.0ml, surveys concentration by dendrobine content assaying method.
Experimental example: alkaloidal detection
1, alkaloid embodiment being extracted is analyzed, and specific analytical method is: alkaloid detection method is acid-dye colorimetry, and selected standard substance are dendrobine standard substance
Concrete grammar: the drafting of standard curve: precision takes dendrobine standard substance 0.0200g, is mixed with the dendrobine standard solution drawing standard curve of 200ug/ml.Absorbance with spectrophotometric determination under 420nm, by absorbance X to concentration Y(ug/ml) carry out regression Calculation,, obtain standard curve equation: Y=0.6219X-0.0117, r=0.99910.
The mensuration of sample: precision takes the drifting alkaloids that this experiment makes and measures as stated above in conjunction with each 50mg of alkaloid sample.
2, result: in Table 1
Table 1: the alkaloid extraction ratio in embodiment
Table 1 result shows: this technique has not only obtained the drifting alkaloids of high level, has also isolated in conjunction with types of biological alkali simultaneously, and the extraction ratio of two kinds of compositions has all reached higher level, and effective utilization of Herba Dendrobii is had to very important directive significance.
Result shows: this experiment is aspect the extraction of Herba Dendrobii, adopted newer flash extractive technique, flash extracter, rely on high speed machine shearing force and super moving molecule diafiltration technology, under room temperature and solvent existence, medical material is crushed to fine particle, and effective ingredient is reached rapidly organize internal and external equilibrium.This experiment shows, uses flash extracter, has extraction efficiency high, the advantage that extraction time is short.Utilize this technique, realized first Herba Dendrobii drifting alkaloids, in conjunction with alkaloidal separation and Extraction, for Herba Dendrobii this precious raw-material make full use of significant.
Although, above used general explanation, the specific embodiment and test, the present invention is described in detail, on basis of the present invention, can make some modifications or improvements it, and this will be apparent to those skilled in the art.Therefore, these modifications or improvements, all belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Claims (10)
1. from Herba Dendrobii, extract the alkaloidal method of active component, it is characterized in that, comprise the following steps: get fresh Herba Dendrobii defat, sudden strain of a muscle is carried, centrifugal, obtains respectively drifting alkaloids crude product and synthetic alkaloid crude product, separated respectively, obtain drifting alkaloids and synthetic alkaloid.
2. method according to claim 1, is characterized in that, described defat: weigh-25 ℃--10 ℃ of freezing fresh Herba Dendrobii, shred, dry, pulverize, then use petroleum ether, fresh Herba Dendrobii compares 1:6-10 with the w/v g/ml of petroleum ether, reflux 4 hours, filtered, then with petroleum ether, Herba Dendrobii powder is cleaned, fresh Herba Dendrobii than for 1:5-7, obtains defat Herba Dendrobii powder with the bulking value g/ml of petroleum ether.
3. method according to claim 1, it is characterized in that, Herba Dendrobii dodges to carry and comprises the following steps: by defat Herba Dendrobii, the concentration that adds 40-60 times of volume of its weight is 75-90% ethanol, after mixing, adjusting pH value is 8-10, dodges and carries twice, the voltage that device is carried in sudden strain of a muscle is 110V-120V, and each time is 30-60 second;
Preferably, Herba Dendrobii dodges to carry and comprises the following steps: extracting degreasing Herba Dendrobii, adds the 80-85% ethanol of 45-55 times of volume of its weight, mix, adjust pH9-10, with the flash extracter that is adjusted to 110-120V, extract twice, each 30-60 second, dodge and raise alkaloid crude extract.
4. method according to claim 1, is characterized in that, described centrifugal: centrifugal speed is 5000-6000r/min, centrifugation time is 10-30min, and separation obtains supernatant A and precipitate B, wherein drifting alkaloids is present in supernatant A, in conjunction with alkaloid, is present in precipitate B.
5. according to the method described in claim 1-4 any one, it is characterized in that, from supernatant A, extract the method for drifting alkaloids, comprise concentrated, adjust pH, with chloroform, dissolve ultrasonic, crystallization.
6. method according to claim 5, is characterized in that, the method for extracting drifting alkaloids from supernatant A comprises the following steps:
1) concentrated: supernatant A is evaporated to original volume 5% left and right;
2) adjust pH: to adding ammonia adjust pH in concentrated solution, be 10, evaporate to dryness;
3) extract, purification: till being then dissolved to and forming even suspension with chloroform, ultrasonic accelerate dissolution then, ultrasonic power is 300w, the ultrasonic time is 5-15min, centrifugal, and supernatant is dry, obtains solid matter;
4) crystallization: add in acetone and grind in the solid matter that step 3) is obtained, draw the supernatant, then repeatedly use acetone solution, grinding, the absorption supernatant, until no longer include precipitation, finally form suspension, use ultrasonic accelerate dissolution, then at-20 ℃ of temperature, precipitate 1 hour, after taking-up, carry out centrifugal, get precipitation, dry;
5) refining: add methanol ultrasonic dissolution, finally form suspension, suspension is centrifugal, and by the supernatant evaporate to dryness after centrifugal, remaining solid is the alkaloid of free type.
7. method according to claim 5, is characterized in that, in described step 3), the w/v g/ml of solid and chloroform is 1:15-1:25; The consumption of the acetone grinding with solid matter in described step 4), the w/v g/ml of solid matter and acetone is 1:20-1:25; In described step 5), the w/v g/ml of solid and methanol is 1:10-1:15; Centrifugal speed is 5000-6000r/min, and centrifugation time is 20-30min.
8. according to the method described in claim 1-4 any one, it is characterized in that, from precipitate B, extract in conjunction with alkaloidal method: water dodge carry, centrifugal, alkalization, alkalization, dodge carry, evaporate to dryness, extraction, crystallization, refining.
9. method according to claim 8, is characterized in that, from precipitate B, extracts and comprises the following steps in conjunction with alkaloidal method:
1) dodge and carry: will in precipitate B, add 40-60 times of water, regulate pH to 9-10, with flash extracter, dodge and carry 1-2 time, extractor is adjusted to 110-120V, and extraction time is 30-60 second, collects extracting solution, standby;
2) centrifugal: the extracting solution that step 1) is obtained is centrifugal, get its precipitation part, add the about 40-50 of precipitation weight times water, stir evenly, then add the cellulase of its weight 0.5-0.7% and the papain of 1.3-1.7%, adjust pH5.5,50 ℃ of water-baths, enzymolysis 1.5-2h, enzymolysis post-heating to 100 ℃, boiling 3-5min, by the solution centrifugal boiling, get precipitation, standby;
3) precipitation alkalization: by step 2) obtaining adds ammonia, and adjust pH is 9-10;
4) dodge and to carry: add the dehydrated alcohol of the 40-50 times of volume of precipitation weight that step 3) obtains, adjust pH9-10, dodge and carry 1-2 time, 30-60s/ time, voltage is 110-120V, and sudden strain of a muscle is carried rear centrifugal, gets supernatant, standby;
5) evaporate to dryness: the supernatant concentration that step 4) is obtained, to 5% left and right of volume, adjust pH is 10, dry, obtains solid;
6) extract: the solid that step 5) is obtained dissolves with chloroform, and the w/v g/ml of solid and chloroform is 1:20-25, centrifugal, collects supernatant, with thermostat water bath, by its evaporate to dryness, obtains powder;
7) crystallization: the powder acetone solution that step 6) is obtained, the w/v g/ml of powder and acetone is 1:20-25, forms suspension, then uses ultrasonic accelerate dissolution, then at-20 ℃ of temperature, precipitate 1 hour, after taking-up, carry out centrifugal, get precipitation, dry, standby;
8) refining: use methanol ultrasonic dissolution, wherein precipitation is 1:10-15 with the w/v g:ml of methanol, and ultrasonic power is 300w, and time 5-15min finally forms suspension, centrifugal, collects supernatant evaporate to dryness, obtains the alkaloid of bond type.
10. method according to claim 9, is characterized in that, all centrifugal conditions are all 5000-10000r/min, 15-30min.
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| CN104232718A (en) * | 2014-09-12 | 2014-12-24 | 华南理工大学 | Preparation method and application of antitumor dendrobium officinale polypeptides |
| CN104557956A (en) * | 2014-12-24 | 2015-04-29 | 贵州师范大学 | Method for preparing dendrobine of which content is more than 95% |
| CN108645802A (en) * | 2018-05-16 | 2018-10-12 | 安徽农业大学 | A kind of detection method of dendrobium candidum total alkaloid content |
| CN111437236A (en) * | 2020-04-26 | 2020-07-24 | 华南农业大学 | Dendrobium extract, preparation method and application thereof |
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| CN104232718A (en) * | 2014-09-12 | 2014-12-24 | 华南理工大学 | Preparation method and application of antitumor dendrobium officinale polypeptides |
| CN104557956A (en) * | 2014-12-24 | 2015-04-29 | 贵州师范大学 | Method for preparing dendrobine of which content is more than 95% |
| CN108645802A (en) * | 2018-05-16 | 2018-10-12 | 安徽农业大学 | A kind of detection method of dendrobium candidum total alkaloid content |
| CN111437236A (en) * | 2020-04-26 | 2020-07-24 | 华南农业大学 | Dendrobium extract, preparation method and application thereof |
| CN111437236B (en) * | 2020-04-26 | 2021-03-26 | 华南农业大学 | Dendrobium extract, preparation method and application thereof |
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