CN110256431A - The extraction of biology total alkali and separation purifying technique in a kind of kuh-seng - Google Patents
The extraction of biology total alkali and separation purifying technique in a kind of kuh-seng Download PDFInfo
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
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- C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
- C07D471/12—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains three hetero rings
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- C07D471/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed systems contains four or more hetero rings
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
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Abstract
The present invention relates to a kind of extraction of biology total alkali in kuh-seng and separation purifying techniques, the following steps are included: pretreatment kuh-seng leaf, kuh-seng leaf powder is added in citric acid-sodium citrate buffer, and cellulase is added, it is digested, second extraction is centrifuged to obtain residue three and sour water, and sour water is incorporated into the filtrate one in step D2, after sour water and extracting solution one are merged, it is concentrated under reduced pressure, it is concentrated into the 10% of original volume, obtains total alkaloid extract, purification & isolation alkaloid.The present invention extracts kuh-seng biology total alkali from kuh-seng leaf, and flavone compound is purified simultaneously, it is disposable to extract two kinds of active principles of separation, the utilization rate of kuh-seng leaf is improved, and first uses enzymatic isolation method, softening and expansion etc. are occurred into for the cell wall in kuh-seng leaf, change, to change the permeability that must report, be conducive to various alkaloids, flavone compound is dissolved out to greatest extent, substantially increase its purification efficiency.
Description
Technical field
The present invention relates to the extractive technique fields of biology total alkali in kuh-seng, and in particular to biology total alkali mentions in a kind of kuh-seng
It takes and separation purifying technique.
Background technique
Kuh-seng is the dry root of leguminous plant kuh-seng (SoPHara flavescens Ait.), bitter in taste, cold in nature, is had
Mild toxicity.As traditional Chinese medicine, its root is clinically mainly applied, it is fixed that main component contains matrine, oxymatrine, Chinese scholartree
Alkali, sophocarpine, N-Oxysophocarpine, sophoramine.In tcm clinical practice application, kuh-seng and other Chinese herbal medicine compatibilities can be treated a variety of
Disease.Experimental study is analyzed and goed deep into comprehensively to kuh-seng pharmacological toxicity by modern medicine, makes the application range and neck of kuh-seng
Domain expands, and the dosage of kuh-seng raw material also increases year by year.The extraction of biology total alkali root mainly dry from kuh-seng in kuh-seng at present
Middle extraction has ignored the value of the total strain of kuh-seng.Each position alkaloid types are similar in kuh-seng, and content difference is obvious, stem, Ye Junhan
There is alkaloid, alkaloid is about 0.16% in cauline leaf, and the fiber content of cauline leaf is more, cell wall thickness, it is not easy to be extracted, mesh
It is preceding less for the Study on extraction of alkaloid in cauline leaf, kuh-seng total flavone, kuh-seng total flavone extract are also contained in cauline leaf
In mainly comprising flavanone kind compositions and trifolirhizin, Xanthohumol, the hardship such as Vexibinol, kurarinone, isokurarinone
Join other flavone compounds such as pyridine, be both dissolved in water or any one alcohols, ketone and esters solvent, be not readily separated,
And Banlangen is also not easily separate.
Summary of the invention
In order to solve the above technical problems, the present invention provides the extraction of biology total alkali and separation purifying technique in a kind of kuh-seng.
The technical solution adopted by the present invention to solve the technical problems is: the extraction and separation of biology total alkali in a kind of kuh-seng
Purifying process, the following steps are included:
D1 pre-processes kuh-seng leaf
By the kuh-seng leaf of picking, after magnetic separation, color sorting remove impurity, dries, is ground up, sieved, kuh-seng leaf powder particles degree
It is spare in 50-80 mesh；
D2 is tentatively extracted
Kuh-seng leaf powder is added in citric acid-sodium citrate buffer, and cellulase is added, after mixing often
Temperature is impregnated, and is impregnated three times, and each 1-4h merges residue three times and filtrate after filtering, obtains residue one, extracting solution one,
Described in the pH value of citric acid-sodium citrate buffer be 4.6-5.2,8-1.2mg cellulase is added in every 1g kuh-seng leaf powder；
Specifically, the filtering uses the methods of centrifugation, suction filtration, ultrafiltration, filters pressing, using or do not use following any one
Kind of clarifying agent or combinations thereof: alcohol precipitation agent, gelatin, kaolin, various resins, polyethylene glycol, poly- second triol, chitosan and natural
Clarifying agent finished product such as 101 fruit juice clarifiers, ZTC+1 natural clarifying agent etc..
D3 second extraction
Ethyl alcohol will be added in residue one, be centrifuged after ultrasonic dissolution, wherein concentration of alcohol is 80-95%, is obtained after centrifugation residual
Slag two and ethyl alcohol lysate, residue two discard, ethyl alcohol lysate recycling design to no alcohol taste, are added what volume was measured for 5-15 times
0.3-1%hCl ultrasonic disperse, centrifugation obtain residue three and sour water, and sour water is incorporated into the filtrate one in step D2；
Specifically, the residue three is washed to neutrality, is dried under reduced pressure, and obtains kuh-seng total flavone extract.
D4 concentrated extracting solution
After sour water and extracting solution one are merged, 15% lime alkalization is added, is layered, the water layer one after taking layering, water layer
Contain total alkaloid in one, 20% NaOH is added in water layer one, adjusts pH value to 7.5, active carbon decoloring is filtered, mentioned
Liquid two is taken, 20%NaOH is added, pH value is adjusted to 9, is concentrated under reduced pressure, is concentrated into the 10% of original volume；
D5 obtains total alkaloid extract
Concentrate is transferred in extractor, is extracted using chloroform, after separation, water layer is discarded, obtains containing total alkaloid
Chloroform layer；
D6 purification & isolation alkaloid
Ether oscillation is added in chloroform layer obtained in D5, is stood, precipitating is precipitated, precipitating one and filtrate are obtained after suction filtration
One, filtrate one is repeated into aforesaid operations in D5 and twice, is precipitated merging and obtains merging the filtrate two after precipitating, and merging,
Precipitating will be merged to be crystallized with acetone, obtain oxymatrine, by two evaporation drying of filtrate, obtain dried object, dry five kinds with stone
Oily ether refluxing extraction obtains extracting solution, and concentration and recovery petroleum ether is placed and filtered, obtains the filtrate containing N-Methylcytisine,
It is crystallized with matrine.
Specifically, dry by chloroform layer decompression, concentration in step D5, obtain biology total alkali crude extract.
Specifically, in the step D1, the fresh kuh-seng leaf of picking is put into shady and cool ground, prevents exposure dehydration and fever,
With ventilative bamboo basket or large bamboo or wicker basket, prevents from squeezing and generate mechanical damage and poor aeration and red change；Then fresh leaf is uniformly spread
On bamboo curtain splint or bamboo sieve, it is placed in air circulation, is withered at the drying of cool place, withering time 8-10h, dehydration to kuh-seng leaf contains
Water avoids kuh-seng leaf from rotting to breed bacterium, influences to purify to 60-70%.
Specifically, the condition of petroleum ether refluxing extraction is that petroleum ether is heated to 30-60 DEG C, 40 DEG C in the step D6
Water-bath refluxing extraction 4 times.
Beneficial effects of the present invention: the present invention extracts kuh-seng biology total alkali from kuh-seng leaf, and purifies flavonoids simultaneously
Object is closed, it is disposable to extract two kinds of active principles of separation, the utilization rate of kuh-seng leaf is improved, and first use enzymatic isolation method, by kuh-seng
Softening and expansion etc. occur for the cell wall in leaf, change, to change the permeability that must be reported, are conducive to various alkaloids, flavones
Class compound is dissolved out to greatest extent, substantially increases its purification efficiency, and utilize solvent extraction method, proposes total biology
Alkali, using alkaloids various in total alkaloid solubility different in petroleum ether, separating-purifying reduces the access times of chloroform
And dosage.
Detailed description of the invention
Fig. 1 is the flow diagram of one embodiment of the invention.
Specific embodiment
The present invention is described in further detail now.
Embodiment 1
The extraction of biology total alkali and separation purifying technique in a kind of kuh-seng as shown in Figure 1, the following steps are included:
D1 pre-processes kuh-seng leaf
By the kuh-seng leaf of picking, after magnetic separation, color sorting remove impurity, dries, is ground up, sieved, kuh-seng leaf powder particles degree
It is spare in 50-80 mesh；
D2 is tentatively extracted
1000g kuh-seng leaf powder is added in 5L citric acid-sodium citrate buffer, and 8g cellulase is added, is mixed
Soak at room temperature after closing uniformly impregnates three times, and each 1h merges residue three times and filtrate after filtering, obtains residue one, extracts
Liquid one, wherein the pH value of the citric acid-sodium citrate buffer is 4.6；
Specifically, in the step D2, using or without using any one following clarifying agent or combinations thereof: alcohol precipitation agent,
Gelatin, kaolin, various resins, polyethylene glycol, poly- second triol, chitosan etc..
D3 second extraction
Ethyl alcohol will be added in residue one, be centrifuged after ultrasonic dissolution, wherein concentration of alcohol is 95%, and residue two is obtained after centrifugation
With ethyl alcohol lysate, residue two is discarded, ethyl alcohol lysate recycling design to no alcohol taste, and the 0.3%hCl that volume is 5 times of amounts is added
Ultrasonic disperse, centrifugation obtain residue three and sour water, and sour water is incorporated into the filtrate one in step D2；
Specifically, the residue three is washed to neutrality, is dried under reduced pressure, and obtains kuh-seng total flavone extract.
D4 concentrated extracting solution
After sour water and extracting solution one are merged, 15% lime alkalization is added, is layered, the water layer one after taking layering, water layer
Contain total alkaloid in one, 20% NaOH is added in water layer one, adjusts pH value to 7.5, active carbon decoloring is filtered, mentioned
Liquid two is taken, 20%NaOH is added, pH value is adjusted to 9, is concentrated under reduced pressure, is concentrated into the 10% of original volume；
D5 obtains total alkaloid extract
Concentrate is transferred in extractor, is extracted using chloroform, after separation, water layer is discarded, obtains containing total alkaloid
Chloroform layer；
D6 purification & isolation alkaloid
Ether oscillation, the standing that 5 volumes are measured again is added in chloroform layer obtained in D5, precipitating is precipitated, is sunk after suction filtration
Filtrate one is repeated aforesaid operations in D5 and twice, is precipitated merging and obtained after merging precipitating, and merging by shallow lake one and filtrate one
Filtrate two, precipitating will be merged and crystallized with acetone, oxymatrine is obtained, by two evaporation drying of filtrate, obtain dried object, it is dry
Object petroleum ether refluxing extraction, obtains extracting solution, and concentration and recovery petroleum ether is placed and filtered, obtains containing N-Methylcytisine
Filtrate and matrine crystallization, wherein oxymatrine obtains 1.491g, and matrine obtains 0.098g.
Specifically, dry by chloroform layer decompression, concentration in step D5, obtain biology total alkali crude extract.
Specifically, solvent extraction is selected, concentrate is transferred in extractor, is extracted using chloroform, after separation, discards water
Layer, it is dry by chloroform layer decompression, concentration, obtain biology total alkali crude extract.
Specifically, in the step D1, the fresh kuh-seng leaf of picking is put into shady and cool ground, prevents exposure dehydration and fever,
With ventilative bamboo basket or large bamboo or wicker basket, prevents from squeezing and generate mechanical damage and poor aeration and red change；Then fresh leaf is uniformly spread
On bamboo curtain splint or bamboo sieve, it is placed in air circulation, is withered at the drying of cool place, withering time 8-10h, dehydration to kuh-seng leaf contains
Water avoids kuh-seng leaf from rotting to breed bacterium, influences to purify to 60-70%.
Specifically, the condition of petroleum ether refluxing extraction is that petroleum ether is heated to 30-60 DEG C, 40 DEG C in the step D6
Water-bath refluxing extraction 4 times.
Embodiment 2
The extraction of biology total alkali and separation purifying technique in a kind of kuh-seng as shown in Figure 1, the following steps are included:
D1 pre-processes kuh-seng leaf
By the kuh-seng leaf of picking, after magnetic separation, color sorting remove impurity, dries, is ground up, sieved, kuh-seng leaf powder particles degree
It is spare in 50-80 mesh；
D2 is tentatively extracted
1000g kuh-seng leaf powder is added in 5L citric acid-sodium citrate buffer, and 9g cellulase is added, is mixed
Soak at room temperature after closing uniformly impregnates three times, and each 2h merges residue three times and filtrate after filtering, obtains residue one, extracts
Liquid one, wherein the pH value of the citric acid-sodium citrate buffer is 5.2；
Specifically, in the step D2, using or without using any one following clarifying agent or combinations thereof: alcohol precipitation agent,
Gelatin, kaolin, various resins, polyethylene glycol, poly- second triol, chitosan and such as 101 juice clarifications of natural clarifying agent finished product
Agent, ZTC+1 natural clarifying agent etc..
D3 second extraction
Ethyl alcohol will be added in residue one, be centrifuged after ultrasonic dissolution, wherein concentration of alcohol is 90%, and residue two is obtained after centrifugation
With ethyl alcohol lysate, residue two is discarded, ethyl alcohol lysate recycling design to no alcohol taste, and the 0.5%HCl that volume is 10 times of amounts is added
Ultrasonic disperse, centrifugation obtain residue three and sour water, and sour water is incorporated into the filtrate one in step D2；
Specifically, the residue three is washed to neutrality, is dried under reduced pressure, and obtains kuh-seng total flavone extract.
D4 concentrated extracting solution
After sour water and extracting solution one are merged, 15% lime alkalization is added, alkalize 4h, static layering, after taking layering
Water layer one contains total alkaloid in water layer one, 20% NaOH is added in water layer one, adjusts pH value to 7.5, active carbon decoloring,
It filters, obtains extracting solution two, 20%NaOH is added, adjusting pH value to 9 is concentrated under reduced pressure or is concentrated in double effect evaporator, dense
It is reduced to the 10% of original volume；
D5 obtains total alkaloid extract
Concentrate is transferred in extractor, is extracted using chloroform, after separation, water layer is discarded, obtains containing total alkaloid
Chloroform layer；
D6 purification & isolation alkaloid
Ether oscillation, the standing that 7 volumes are measured again is added in chloroform layer obtained in D5, precipitating is precipitated, is sunk after suction filtration
Filtrate one is repeated aforesaid operations in D5 and twice, is precipitated merging and obtained after merging precipitating, and merging by shallow lake one and filtrate one
Filtrate two, precipitating will be merged and crystallized with acetone, oxymatrine is obtained, by two evaporation drying of filtrate, obtain dried object, it is dry
Object petroleum ether refluxing extraction, obtains extracting solution, and concentration and recovery petroleum ether is placed and filtered, obtains containing N-Methylcytisine
Filtrate and matrine crystallization, wherein oxymatrine obtains 1.322g, and matrine obtains 0.103g.
Specifically, dry by chloroform layer decompression, concentration in step D5, obtain biology total alkali crude extract.
Specifically, solvent extraction is selected, concentrate is transferred in extractor, is extracted using chloroform, after separation, discards water
Layer, it is dry by chloroform layer decompression, concentration, obtain biology total alkali crude extract.
Specifically, in the step D1, the fresh kuh-seng leaf of picking is put into shady and cool ground, prevents exposure dehydration and fever,
With ventilative bamboo basket or large bamboo or wicker basket, prevents from squeezing and generate mechanical damage and poor aeration and red change；Then fresh leaf is uniformly spread
On bamboo curtain splint or bamboo sieve, it is placed in air circulation, is withered at the drying of cool place, withering time 8-10h, dehydration to kuh-seng leaf contains
Water avoids kuh-seng leaf from rotting to breed bacterium, influences to purify to 60-70%.
Specifically, the condition of petroleum ether refluxing extraction is that petroleum ether is heated to 30-60 DEG C, 40 DEG C in the step D6
Water-bath refluxing extraction 4 times.
Embodiment 3
The extraction of biology total alkali and separation purifying technique in a kind of kuh-seng as shown in Figure 1, the following steps are included:
D1 pre-processes kuh-seng leaf
By the kuh-seng leaf of picking, after magnetic separation, color sorting remove impurity, dries, is ground up, sieved, kuh-seng leaf powder particles degree
It is spare in 50-80 mesh；
D2 is tentatively extracted
1000g kuh-seng leaf powder is added in 5L citric acid-sodium citrate buffer, and 10g cellulase is added, is mixed
Soak at room temperature after closing uniformly impregnates three times, and each 3h merges residue three times and filtrate after filtering, obtains residue one, extracts
Liquid one, wherein the pH value of the citric acid-sodium citrate buffer is 5；
Specifically, in the step D2, using or without using any one following clarifying agent or combinations thereof: alcohol precipitation agent,
Gelatin, kaolin, various resins, polyethylene glycol, poly- second triol, chitosan and such as 101 juice clarifications of natural clarifying agent finished product
Agent, ZTC+1 natural clarifying agent etc..
D3 second extraction
Ethyl alcohol will be added in residue one, be centrifuged after ultrasonic dissolution, wherein concentration of alcohol is 85%, and residue two is obtained after centrifugation
With ethyl alcohol lysate, residue two is discarded, ethyl alcohol lysate recycling design to no alcohol taste, and it is super that the 1%HCl that volume is 15 times of amounts is added
Sound dispersion, centrifugation obtain residue three and sour water, and sour water is incorporated into the filtrate one in step D2；
Specifically, the residue three is washed to neutrality, is dried under reduced pressure, and obtains kuh-seng total flavone extract.
D4 concentrated extracting solution
After sour water and extracting solution one are merged, 15% lime alkalization is added, alkalize 3h, static layering, after taking layering
Water layer one contains total alkaloid in water layer one, 20% NaOH is added in water layer one, adjusts pH value to 7.5, active carbon decoloring,
It filters, obtains extracting solution two, 20%NaOH is added, adjusting pH value to 9 is concentrated under reduced pressure or is concentrated in double effect evaporator, dense
It is reduced to the 10% of original volume；
D5 obtains total alkaloid extract
Concentrate is transferred in extractor, is extracted using chloroform, after separation, water layer is discarded, obtains containing total alkaloid
Chloroform layer；
D6 purification & isolation alkaloid
Ether oscillation, the standing that 7 volumes are measured again is added in chloroform layer obtained in D5, precipitating is precipitated, is sunk after suction filtration
Filtrate one is repeated aforesaid operations in D5 and twice, is precipitated merging and obtained after merging precipitating, and merging by shallow lake one and filtrate one
Filtrate two, precipitating will be merged and crystallized with acetone, oxymatrine is obtained, by two evaporation drying of filtrate, obtain dried object, it is dry
Object petroleum ether refluxing extraction, obtains extracting solution, and concentration and recovery petroleum ether is placed and filtered, obtains containing N-Methylcytisine
Filtrate and matrine crystallization, wherein oxymatrine obtains 1.461g, and matrine obtains 0.092g.
Specifically, dry by chloroform layer decompression, concentration in step D5, obtain biology total alkali crude extract.
Specifically, solvent extraction is selected, concentrate is transferred in extractor, is extracted using chloroform, after separation, discards water
Layer, it is dry by chloroform layer decompression, concentration, obtain biology total alkali crude extract.
Specifically, in the step D1, the fresh kuh-seng leaf of picking is put into shady and cool ground, prevents exposure dehydration and fever,
With ventilative bamboo basket or large bamboo or wicker basket, prevents from squeezing and generate mechanical damage and poor aeration and red change；Then fresh leaf is uniformly spread
On bamboo curtain splint or bamboo sieve, it is placed in air circulation, is withered at the drying of cool place, withering time 8-10h, dehydration to kuh-seng leaf contains
Water avoids kuh-seng leaf from rotting to breed bacterium, influences to purify to 60-70%.
Specifically, the condition of petroleum ether refluxing extraction is that petroleum ether is heated to 30-60 DEG C, 40 DEG C in the step D6
Water-bath refluxing extraction 4 times.
Embodiment 4
The extraction of biology total alkali and separation purifying technique in a kind of kuh-seng as shown in Figure 1, the following steps are included:
D1 pre-processes kuh-seng leaf
By the kuh-seng leaf of picking, after magnetic separation, color sorting remove impurity, dries, is ground up, sieved, kuh-seng leaf powder particles degree
It is spare in 50-80 mesh；
D2 is tentatively extracted
1000g kuh-seng leaf powder is added in 5L citric acid-sodium citrate buffer, and 12g cellulase is added, is mixed
Soak at room temperature after closing uniformly impregnates three times, and each 4h merges residue three times and filtrate after filtering, obtains residue one, extracts
Liquid one, wherein the pH value of the citric acid-sodium citrate buffer is 5；
Specifically, in the step D2, using or without using any one following clarifying agent or combinations thereof: alcohol precipitation agent,
Gelatin, kaolin, various resins, polyethylene glycol, poly- second triol, chitosan and such as 101 juice clarifications of natural clarifying agent finished product
Agent, ZTC+1 natural clarifying agent etc..
D3 second extraction
Ethyl alcohol will be added in residue one, be centrifuged after ultrasonic dissolution, wherein concentration of alcohol is 80-95%, is obtained after centrifugation residual
Slag two and ethyl alcohol lysate, residue two discard, ethyl alcohol lysate recycling design to no alcohol taste, and it is 10 times of amounts that volume, which is added,
0.8%hCl ultrasonic disperse, centrifugation obtain residue three and sour water, and sour water is incorporated into the filtrate one in step D2；
Specifically, the residue three is washed to neutrality, is dried under reduced pressure, and obtains kuh-seng total flavone extract.
D4 concentrated extracting solution
After sour water and extracting solution one are merged, 15% lime alkalization is added, alkalize 4h, static layering, after taking layering
Water layer one contains total alkaloid in water layer one, 20% NaOH is added in water layer one, adjusts pH value to 7.5, active carbon decoloring,
It filters, obtains extracting solution two, 20%NaOH is added, adjusting pH value to 9 is concentrated under reduced pressure or is concentrated in double effect evaporator, dense
It is reduced to the 10% of original volume；
D5 obtains total alkaloid extract
Concentrate is transferred in extractor, is extracted using chloroform, after separation, water layer is discarded, obtains containing total alkaloid
Chloroform layer；
D6 purification & isolation alkaloid
Ether oscillation is added in chloroform layer obtained in D5, is stood, precipitating is precipitated, precipitating one and filtrate are obtained after suction filtration
One, filtrate one is repeated into aforesaid operations in D5 and twice, is precipitated merging and obtains merging the filtrate two after precipitating, and merging,
Precipitating will be merged to be crystallized with acetone, obtain oxymatrine, by two evaporation drying of filtrate, obtain dried object, dry five kinds with stone
Oily ether refluxing extraction obtains extracting solution, and concentration and recovery petroleum ether is placed and filtered, obtains the filtrate containing N-Methylcytisine,
It is crystallized with matrine, wherein oxymatrine obtains 1.55g, and matrine obtains 0.083g.
Specifically, dry by chloroform layer decompression, concentration in step D5, obtain biology total alkali crude extract.
Specifically, in the step D1, the fresh kuh-seng leaf of picking is put into shady and cool ground, prevents exposure dehydration and fever,
With ventilative bamboo basket or large bamboo or wicker basket, prevents from squeezing and generate mechanical damage and poor aeration and red change；Then fresh leaf is uniformly spread
On bamboo curtain splint or bamboo sieve, it is placed in air circulation, is withered at the drying of cool place, withering time 8-10h, dehydration to kuh-seng leaf contains
Water avoids kuh-seng leaf from rotting to breed bacterium, influences to purify to 60-70%.
Specifically, the condition of petroleum ether refluxing extraction is that petroleum ether is heated to 30-60 DEG C, 40 DEG C in the step D6
Water-bath refluxing extraction 4 times.
The present invention is not limited to the embodiment, anyone should learn that the structure made under the inspiration of the present invention becomes
Change, the technical schemes that are same or similar to the present invention are fallen within the scope of protection of the present invention.
Technology not described in detail in the present invention, shape, construction portion are well-known technique.
Claims (6)
1. the extraction of biology total alkali and separation purifying technique in a kind of kuh-seng, it is characterised in that: the following steps are included:
D1 pre-processes kuh-seng leaf
It by the kuh-seng leaf of picking, after magnetic separation, color sorting remove impurity, dries, be ground up, sieved, kuh-seng leaf powder particles degree is in 50-
80 mesh, it is spare；
D2 is tentatively extracted
Kuh-seng leaf powder is added in citric acid-sodium citrate buffer, and cellulase is added, room temperature soaks after mixing
Bubble impregnates three times, and each 1-4h merges residue three times and filtrate after filtering, obtain residue one, extracting solution one；
D3 second extraction
Ethyl alcohol will be added in residue one, be centrifuged after ultrasonic dissolution, wherein concentration of alcohol is 80-95%, and residue two is obtained after centrifugation
With ethyl alcohol lysate, residue two is discarded, ethyl alcohol lysate recycling design to no alcohol taste, and addition volume is the 5-15 times of 0.3- measured
1%HCl ultrasonic disperse, centrifugation obtain residue three and sour water, and sour water is incorporated into the filtrate one in step D2；
D4 concentrated extracting solution
After sour water and extracting solution one are merged, 15% lime alkalization is added, is layered, the water layer one after taking layering, in water layer one
Containing total alkaloid, 20% NaOH is added in water layer one, adjusts pH value to 7.5, active carbon decoloring filters, obtains extracting solution
Two, 20%NaOH is added, adjusts pH value to 9, is concentrated under reduced pressure, is concentrated into the 10% of original volume；
D5 obtains total alkaloid extract
Concentrate is transferred in extractor, is extracted using chloroform, after separation, discards water layer, obtains the chloroform containing total alkaloid
Layer；
D6 purification & isolation alkaloid
Ether oscillation is added in chloroform layer obtained in D5, is stood, precipitating is precipitated, precipitating one and filtrate one are obtained after suction filtration, it will
Filtrate one repeats aforesaid operations in D5 and twice, is precipitated merging and obtain merging the filtrate two after precipitating, and merging, will merged
Precipitating is crystallized with acetone, obtains oxymatrine, by two evaporation drying of filtrate, obtains dried object, dry five kinds are returned with petroleum ether
Stream extracts, and obtains extracting solution, and concentration and recovery petroleum ether is placed and filtered, obtains the filtrate and kuh-seng containing N-Methylcytisine
Alkali crystallization.
2. the extraction of biology total alkali and separation purifying technique in a kind of kuh-seng according to claim 1, it is characterised in that: institute
It states in step D1, the fresh kuh-seng leaf of picking is put into shady and cool ground, then fresh leaf is uniformly spread on bamboo curtain splint or bamboo sieve, set
It withers at air circulation, cool place drying, withering time 8-10h, dehydration to kuh-seng leaf water content to 60-70%.
3. the extraction of biology total alkali and separation purifying technique in a kind of kuh-seng according to claim 1, it is characterised in that: institute
The pH value for stating citric acid-sodium citrate buffer described in step D2 is 4.6-5.2, and 8-1.2mg is added in every 1g kuh-seng leaf powder
Cellulase.
4. the extraction of biology total alkali and separation purifying technique in a kind of kuh-seng according to claim 1, it is characterised in that: institute
It states residue three obtained in step D3 and is washed to neutrality, be dried under reduced pressure, obtain kuh-seng total flavone extract.
5. the extraction of biology total alkali and separation purifying technique in a kind of kuh-seng according to claim 1, it is characterised in that: institute
It states in step D5, it is dry by chloroform layer decompression, concentration, obtain biology total alkali crude extract.
6. the extraction of biology total alkali and separation purifying technique in a kind of kuh-seng according to claim 1, it is characterised in that: institute
The condition for stating petroleum ether refluxing extraction in step D6 is that petroleum ether is heated to 30-60 DEG C, 40 DEG C of water-bath refluxing extraction 4 times.
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