CN103848920B - A kind of ultrasonic assistant extracts the method for Banlangen Polysaccharide - Google Patents
A kind of ultrasonic assistant extracts the method for Banlangen Polysaccharide Download PDFInfo
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- CN103848920B CN103848920B CN201210510656.9A CN201210510656A CN103848920B CN 103848920 B CN103848920 B CN 103848920B CN 201210510656 A CN201210510656 A CN 201210510656A CN 103848920 B CN103848920 B CN 103848920B
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Abstract
The present invention relates to a kind of method that ultrasonic assistant extracts Banlangen Polysaccharide, after chromatogram of Radix Isatidis drying, pulverizing, defat, using ultrasonic cell disruption instrument to extract, the technology such as the centrifugal supernatant obtained is concentrated, precipitate with ethanol, filtration and vacuum drying prepares Banlangen Polysaccharide.Compared with prior art, technique proposed by the invention has quick, easy, the saving energy, yield high, and is conducive to the many active substances contained in protection Banlangen Polysaccharide.The experiment proves that, use ultrasonic assistant to extract the Banlangen Polysaccharide obtained and there is good antioxidation activity in vitro.
Description
Technical field
The present invention relates to the extracting method of technical field of Chinese medicines, especially relate to a kind of method that ultrasonic assistant extracts Banlangen Polysaccharide.
Background technology
Radix Isatidis (IsatidisRadix), call indigo-blue, Isatis indigotica Fort (Indigofera tinctoria L, Baphicanthus cusia (nees) Brem. Polygonum tinctorium Ait) root, indigo root, it it is the dry root of cruciferae isatis (IsatisindigoticaFort.), there is effect of heat-clearing and toxic substances removing, removing heat from blood sore-throat relieving, be used clinically for pestilence seasonal noxious pathogen, heating pharyngalgia, maculae caused by violent heat pathogen, mumps, scarlet fever, major part pestilence, erysipelas, carbuncle etc..As the Typical Representative of China's anti virus herb, Radix Isatidis is the first Chinese medicine research project obtaining NIH's subsidy.
Polysaccharide is the biomacromolecule of the class formation complexity being formed by connecting by glycosidic bond by multiple monosaccharide molecule, is distributed widely in natural drug.Polysaccharide plays an important role in many basic life processes such as biosynthesis and the infection of intercellular identification, fertilization, embry ogenesis, neural cell development, hormone activation, cell proliferation, virus and antibacterial, Nasopharyngeal neoplasms, has therefore become the study hotspot of medical science, threpsology and field of food science.It is reported, specific immunity and nonspecific immunity are respectively provided with certain facilitation by Banlangen Polysaccharide, are a kind of more satisfactory natural immunity reinforcing agents, have DEVELOPMENT PROSPECT widely as health food and medicine.
Ultrasonic technology application development in recent years in traditional Chinese medicine extyaction relatively broad.Utilize hyperacoustic cavitation, mechanical effect and heat effect etc., accelerate the release of active substance, diffusion and dissolving in cell, can significantly shorten extraction time, improve extraction efficiency.At present, use ultrasonic cell disruption instrument to extract Banlangen Polysaccharide and there is not yet bibliographical information.
Summary of the invention
Defect that the purpose of the present invention is contemplated to overcome above-mentioned prior art to exist and provide a kind of fast and convenient, save the energy, the method extracting method utilizing ultrasonic assistant to extract Banlangen Polysaccharide that yield is high, and be conducive to the many active substances contained in protection Banlangen Polysaccharide.
The purpose of the present invention can be achieved through the following technical solutions:
A kind of ultrasonic assistant extracts the method for Banlangen Polysaccharide, comprises the following steps:
(1) Radix Isatidis dried and be ground into Isatis Root；
(2) carry out sucking filtration after adding finite concentration alcohol degreasing in Isatis Root, then filtering residue is carried out vacuum drying；
(3) Isatis Root after defat adds water, use ultrasonic cell disruption instrument to extract Banlangen Polysaccharide, cooling, centrifugation, obtain the supernatant containing Banlangen Polysaccharide；
(4) by supernatant concentrating under reduced pressure, adding equivalent or several times amount dehydrated alcohol, 4 DEG C stand overnight, and centrifugal segregation mother solution collects precipitation；
(5) successively with ethanol, washing with acetone precipitation, vacuum drying, the polysaccharide containing antioxidant activity Banlangen Polysaccharide is obtained.
Drying the temperature adopted in step (1) is 50～60 DEG C, and the granularity pulverizing the Isatis Root obtained is 60～80 orders.
Defat in step (2) adopts 5～10 times (w/v) of Isatis Root to measure 95% alcohol reflux defat twice, each 2h, and described vacuum drying temperature is 20～30 DEG C, and the time is 24～48h.
Adding water in the step (3) Isatis Root after defat, making solid-liquid ratio is 1: 10～1: 100, and the technological parameter that ultrasonic cell disruption instrument extracts is ultrasonic power 400～800W, temperature 30～60 DEG C, time 10～30min.
Step (4) adopts Rotary Evaporators, thickening temperature 55～60 DEG C when concentrating under reduced pressure, and concentration volume is the 1/5～1/10 of original volume, and dehydrated alcohol addition is 1～5 times of concentrated solution volume；Rotating speed during centrifugal treating is 3500～4500rpm, time 10～15min.
Step (5) is sequentially added into the washing with alcohol of step (4) mother solution same concentrations when washing and filters, step up concentration of alcohol, finally with dehydrated alcohol, washing with acetone, it is covered with in case moisture is brought in the evaporation of precipitate surface solvent into when sucking filtration is closely dry, the temperature that vacuum drying adopts is 40～45 DEG C, and drying time is 24～72h.
Compared with prior art; the technology such as the present invention adopts pulverizing, defat, ultrasonic cell disruption instrument to extract, centrifugal, concentration, precipitate with ethanol and vacuum drying prepare Banlangen Polysaccharide; proposed technique has quick, easy, the saving energy, yield high, and is conducive to the many active substances contained in protection Banlangen Polysaccharide.The extracting method taked is that ultrasonic cell disruption instrument extracts, compare conventional water bath reflux method, extraction time can be foreshortened to 10min by a few hours, and extraction ratio improves nearly one times, the Banlangen Polysaccharide obtained has good antioxidation activity in vitro, has good ABTS+Free radical scavenging effect, is widely used in anti-oxidation function product.
Detailed description of the invention
Below in conjunction with specific embodiment, the present invention is described in detail.
Embodiment 1
The extraction of Banlangen Polysaccharide
By Radix Isatidis in 60 DEG C of drying, pulverizing 80 mesh sieves, and added 5 times amount (w/v) 95% alcohol reflux twice, each 2h, sucking filtration removes filtrate, and filtering residue is in 25 DEG C of vacuum drying 24h.
Radix Isatidis 5g after extracting degreasing, the 200mL that adds water (solid-liquid ratio 1: 40), use ultrasonic cell disruption instrument to extract Banlangen Polysaccharide, ultrasonic power 787w, temperature 60 C, extraction time 10min.Will be cooled to the extracting solution of room temperature centrifugal (4500rpm, 15min), collect supernatant, filtering residue distilled water wash, and merge with supernatant.
Said extracted liquid Rotary Evaporators 60 DEG C is concentrated into 40mL, is added slowly with stirring 4 times amount dehydrated alcohol, and 4 DEG C stand overnight, and 4500rpm/min is centrifuged 15min, collect precipitated product；Successively by each 10mL washing precipitation of 80% ethanol, dehydrated alcohol and acetone, it is covered with when sucking filtration is closely dry；45 DEG C of vacuum drying 72h, weigh.Repeating above-mentioned experiment three times, obtain polysaccharide 1.25g, 1.24g, 1.26g respectively, calculate polysaccharide extract rate, results averaged is 25.01%.
Embodiment 2-18
Ultrasonic assistant extracts the optimization of process conditions of Banlangen Polysaccharide
Adopt the central combination design principle of Box-Benhnken, the scheme of the response surface analysis test of design Three factors-levels, with ultrasonic power, ultrasonic time, 3 technological parameters of ultrasonic temperature for independent variable, with polysaccharide extract rate for response value, the process conditions of Optimization for Ultrasonic Wave assisted extraction Banlangen Polysaccharide, EXPERIMENTAL DESIGN and result are in Table 1.
Table 1
Analyzing, by Design-Expert software (Version7.0.0), the Banlangen Polysaccharide optimum extraction condition obtained is: ultrasonic power 786.95w, ultrasonic time 10.14min, ultrasonic temperature 58.99 DEG C, the theoretical extraction of values under this condition is 25.14%.Take ultrasonic power 787w, ultrasonic time 10min, ultrasonic temperature 60 DEG C to extract Banlangen Polysaccharide, carry out three parallel laboratory tests, calculate extraction ratio respectively 24.95%, 24.88% and 25.19%, close with predictive value.
Embodiment 19
The in vitro anti-oxidation of polysaccharide measures
The Banlangen Polysaccharide obtained by embodiment 1 carries out ABTS+Free radical scavenging effect experimental.Configuration ABTS free radical working solution, is dissolved in distilled water by Banlangen Polysaccharide sample and is made into a series of concentration.Take 0.1mL sample solution and add 2.9mLABTS+Free radical working solution, mixing, measure absorbance in 734nm after placing 6min.Banlangen Polysaccharide is calculated to ABTS according to formula+The clearance rate of free radical, result is in Table 2.
Clearance rate (%)=[(A0-At)/A0] × 100%
In formulaA0 is 2.9mLABTS+Solution and the mixed absorbance of 0.1mL distilled water；AtFor 2.9mLABTS+Solution and the mixed absorbance of 0.1mL sample solution.
Table 2
Embodiment 20
A kind of ultrasonic assistant extracts the method for Banlangen Polysaccharide, comprises the following steps:
(1) Radix Isatidis dried at 50 DEG C and be ground into the Isatis Root that granularity is 60 orders；
(2) adding 5 times (w/v) in Isatis Root, concentration is the alcohol reflux defat twice of 95%, each 2h, after carry out sucking filtration, then filtering residue is carried out vacuum drying 48h at 20 DEG C；
(3) adding water in the Isatis Root after defat makes solid-liquid ratio be 1: 10, uses ultrasonic cell disruption instrument to extract Banlangen Polysaccharide, ultrasonic power 400W, temperature 30 DEG C, the response time is 30min, then cools down, centrifugation, obtains the supernatant containing Banlangen Polysaccharide；
(4) supernatant adopts Rotary Evaporators carry out concentrating under reduced pressure, and thickening temperature 55 DEG C, by the 1/5 of volume concentration to original volume, adding the dehydrated alcohol of equivalent, 4 DEG C stand overnight, centrifugal segregation mother solution, rotating speed during centrifugal treating is 3500rpm, and the process time is 15min, regathers precipitation；
(5) successively by ethanol, washing with acetone precipitation, it is sequentially added into the washing with alcohol of step (4) mother solution same concentrations during washing and filters, step up concentration of alcohol, finally with dehydrated alcohol, washing with acetone, it is covered with in case moisture is brought in the evaporation of precipitate surface solvent into when sucking filtration is closely dry, finally controlling temperature is 40 DEG C, and vacuum drying 72h obtains the polysaccharide containing antioxidant activity Banlangen Polysaccharide.
Embodiment 21
A kind of ultrasonic assistant extracts the method for Banlangen Polysaccharide, comprises the following steps:
(1) Radix Isatidis dried at 60 DEG C and be ground into the Isatis Root that granularity is 80 orders；
(2) adding 10 times (w/v) in Isatis Root, concentration is the alcohol reflux defat twice of 95%, each 2h, after carry out sucking filtration, then filtering residue is carried out vacuum drying 24h at 30 DEG C；
(3) adding water in the Isatis Root after defat makes solid-liquid ratio be 1: 100, uses ultrasonic cell disruption instrument to extract Banlangen Polysaccharide, ultrasonic power 800W, temperature 60 C, the response time is 10min, then cools down, centrifugation, obtains the supernatant containing Banlangen Polysaccharide；
(4) supernatant adopt Rotary Evaporators carry out concentrating under reduced pressure, thickening temperature 55 DEG C, by volume concentration to original volume 1/10, add the dehydrated alcohol of 5 times of volumes, 4 DEG C stand overnight, centrifugal segregation mother solution, and rotating speed during centrifugal treating is 4500rpm, the process time is 10min, regathers precipitation；
(5) successively by ethanol, washing with acetone precipitation, it is sequentially added into the washing with alcohol of step (4) mother solution same concentrations during washing and filters, step up concentration of alcohol, finally with dehydrated alcohol, washing with acetone, it is covered with in case moisture is brought in the evaporation of precipitate surface solvent into when sucking filtration is closely dry, finally controlling temperature is 45 DEG C, and vacuum drying 24h obtains the polysaccharide containing antioxidant activity Banlangen Polysaccharide.
Claims (6)
1. the method that a ultrasonic assistant extracts Banlangen Polysaccharide, it is characterised in that the method comprises the following steps:
(1) Radix Isatidis dried and be ground into Isatis Root；
(2) carry out sucking filtration after adding finite concentration alcohol degreasing in Isatis Root, then filtering residue is carried out vacuum drying；
(3) adding water in the Isatis Root after defat, use ultrasonic cell disruption instrument to extract Banlangen Polysaccharide, ultrasonic power is 400～800W, control temperature 30～60 DEG C, time 10min, then cool down, centrifugation, obtains the supernatant containing Banlangen Polysaccharide；
(4) by supernatant concentrating under reduced pressure, adding equivalent or several times amount dehydrated alcohol, 4 DEG C stand overnight, and centrifugal segregation mother solution collects precipitation；
(5) successively with ethanol, washing with acetone precipitation, vacuum drying, the polysaccharide containing antioxidant activity Banlangen Polysaccharide is obtained.
2. the method that a kind of ultrasonic assistant according to claim 1 extracts Banlangen Polysaccharide, it is characterised in that drying the temperature adopted in step (1) is 50～60 DEG C, and the granularity pulverizing the Isatis Root obtained is 60～80 orders.
3. the method that a kind of ultrasonic assistant according to claim 1 extracts Banlangen Polysaccharide, it is characterized in that, defat in step (2) adopts 5～10 times (w/v) of Isatis Root to measure 95% alcohol reflux defat twice, each 2h, described vacuum drying temperature is 20～30 DEG C, and the time is 24～48h.
4. the method that a kind of ultrasonic assistant according to claim 1 extracts Banlangen Polysaccharide, it is characterised in that adding water in the step (3) Isatis Root after defat, making solid-liquid ratio is 1:10～1:100/g:mL.
5. the method that a kind of ultrasonic assistant according to claim 1 extracts Banlangen Polysaccharide, it is characterized in that, step (4) adopts Rotary Evaporators when concentrating under reduced pressure, thickening temperature 55～60 DEG C, concentration volume is the 1/5～1/10 of original volume, and dehydrated alcohol addition is 1～5 times of concentrated solution volume；Rotating speed during centrifugal treating is 3500～4500rpm, time 10～15min.
6. the method that a kind of ultrasonic assistant according to claim 1 extracts Banlangen Polysaccharide, it is characterized in that, step (5) is sequentially added into the washing with alcohol of step (4) mother solution same concentrations when washing and filters, step up concentration of alcohol, finally with dehydrated alcohol, washing with acetone, covering when sucking filtration is closely dry in case moisture is brought in the evaporation of precipitate surface solvent into, the temperature that vacuum drying adopts is 40～45 DEG C, and drying time is 24～72h.
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| CN101367881A (en) * | 2008-09-19 | 2009-02-18 | 南京农业大学 | High-efficiency preparation method for glossy ganoderma polysaccharide |
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Patent Citations (4)
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| CN101250232A (en) * | 2008-03-27 | 2008-08-27 | 钱国英 | Extraction technique of sargassum fusiform active polysaccharides |
| CN101250234A (en) * | 2008-04-10 | 2008-08-27 | 北京联合大学生物化学工程学院 | Method for extracting ginseng polysaccharide |
| CN101367881A (en) * | 2008-09-19 | 2009-02-18 | 南京农业大学 | High-efficiency preparation method for glossy ganoderma polysaccharide |
| CN102002112A (en) * | 2010-12-02 | 2011-04-06 | 蒋熠霞 | Process for extracting ginkgo leaf polysaccharide with aid of ultrasonic |
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