Sunson Feed Enzymes Department was established in 1998. Till now, SUNSON brand has been already accepted by customers domestically and aboard by concentrating on feed enzymes production and application technology.Sunson feed enzymes are ideal solutions for: Saving feed cost; Non-starch polysaccharides (NSPs) breaking down; Endogenous enzymes supplement; Reducing the phosphorus burden on the environment.
â—SINGLE ENZYMES
Animal Feed Enzymes,Animal Liquid Feed Enzyme,Enzyme For Animal Feed,Neutral Protease Feed Additive Sunson Industry Group Co., Ltd , https://www.sunsonchinaenzymes.com
Key words: supercritical carbon dioxide; gradient crystallization; andrographolide; high performance liquid chromatography, Andrographis paniculata [4ndrographis paniculata (Burm.f) Nees] is an annual herb of the genus Rhododendron. The main active ingredients of Andrographis paniculata are diterpene lactone and flavonoids, and andrographolide compounds are clinically used for the treatment of acute gastroenteritis, tonsillitis, hepatitis and anti-cancer. Efficient separation and purification of andrographolide is an important channel to increase the added value of Andrographis paniculata.
Although supercritical fluid extraction (SCFE) technology has been in the fields of medicine and chemical industry for decades, the SCFE technology has great limitations in the extraction and separation industry. For example, SCFE technology is prone to blockage of pipes when extracting compounds of crystal structure in natural herbs. In order to improve the separation efficiency and product purity of supercritical fluid extraction, the author selected the andrographolide with crystal structure as the research object, and adopted supercritical CO2 gradient crystallization to solve the problem of high-efficiency crystallization and purification of some natural herb active ingredients. In this paper, the effects of gradient crystallization pressure, temperature and time process parameters on the separation and purification of andrographolide were studied.
1 Materials and methods
1.1 Test equipment, instruments
40MPa supercritical carbon dioxide extraction crystallization unit, 1.6L gradient crystallizer (high aspect ratio l0:1), electronic balance (sea analysis instrument factory), Waters HPLC system, including 5l5 type infusion pump, 2487 ultraviolet double beam detector, Rheodyne7725i type Manual injector and Millennium32 ChemStation.
1.2 The material of Andrographis paniculata (with a lactone content of 30%) was produced by Hefei Tuofeng Bioengineering Co., Ltd. The standard of andrographolide was purchased from China National Institute of Biological Products, carbon dioxide (99.8% food grade).
1.3 Method The test was carried out in 3 steps. * Step, raw material pretreatment, that is, first dissolve the raw material with ethanol in a warm water bath, then precipitate and filter; the second step, extract the crystalline filtrate with supercritical carbon dioxide; the third step, sample detection and analysis.
1.4 Determination of content
1.4.1 Preparation of the standard curve Accurately weigh 1.82mg of andrographolide reference substance, put it in a 5ml volumetric flask, dissolve it with methanol and dilute to the mark, and shake it to obtain the reference stock solution. Pipette 1.0 ml of the solution, transfer to a 5 ml volumetric flask, dilute to volume, and shake. The syringe draws the diluent and injects 2, 6, 10, 14 and l8u1 in sequence; high performance liquid chromatography detection conditions. Waters Symmetryshield 5μm C18 column (3.9mm × 150mm) column, methanol-water (65% -35%) as mobile phase, flow rate 1.00ml / min. The detection wavelength was 205 nm, and the area of ​​the chromatographic peak was plotted on the ordinate, and the injection amount (μg) was linearly plotted on the abscissa. Andrographolide has a linear relationship with the peak area in the range of 0.14~l.3lμg. The regression equation is: Y=3472+673X, r=0.9976.
1.4.2 Determination of andrographolide in the crystallization product Accurately weigh 2.00mg of crystalline product, place it in a 25ml volumetric flask, dissolve it in methanol and dilute to volume, shake it to obtain the sample solution, and then refer to the literature for testing. analysis.
2 Results and analysis
2.1 Effect of pressure on extraction and crystallization of andrographolide
2.1.1 Relationship between pressure and purity of different parts of crystal plate In order to study the crystal distribution of crystallized andrographolide by supercritical carbon dioxide extraction, the test material was made of 30% andrographolide extract. The temperature of the extraction crystallization kettle was controlled at 55 ° C, the extraction time was 90 min, and the flow rate of CO 2 was 15 L / min. Under the conditions, the selection pressure is l5MPa. The crystal plate is divided into five sections on the white to the top, from bottom to sampling, and the pressure-purity curve.
2.1.2 Relationship between pressure and purity of the same part of the crystal plate When the selection pressure is 8, l2, l6, 20 and 24 MPa, the crystal plate is divided into three sections from the bottom to the part, and the upper and lower parts are sampled.
2.1.3 Relationship between pressure and crystallization amount of the same part of the crystal plate When the pressure is 10, 15, 20, 25 and 30 MPa, the crystal plate is divided into 2 sections on average, and the upper part is sampled to obtain the pressure. The amount of crystallization.
The law of variation indicates that the purity of andrographolide is distributed on the crystal plate in a gradient. It may be due to the difference in the interaction of andrographolide and other impurities in supercritical CO2 due to fluid deflection, gravity, intermolecular force and surface plate adsorption force. The dominant crystallization effect forms a gradient crystal distribution; from the figure and the table, under the condition that other process parameters are unchanged, as the pressure increases, the purity of the upper part of the crystal is higher and higher, and the amount of crystallization of the upper part increases first and then decreases. Due to the increase of pressure, the solubility of SC-CO2 is enhanced, the amount of crystals deposited increases, and the purity and crystallization amount increase. When the pressure exceeds a certain value, the super-solubility of supercritical fluid will weaken the crystallization and extract into the separation kettle. The amount of andrographolide increased, so the optimum pressure value was 20 MPa, the purity of the product was over 80%, and the relative crystallization amount was 38.7%.
2.2 The effect of temperature on the extraction and crystallization of andrographolide The extraction-crystallization pressure is 20 MPa, the extraction time is 90 min, the CO2 flow rate is 15 L/min, and the raw material is 30% pure andrographolide extract. The test temperatures were 35, 45, 55 and 65 ° C, respectively. The crystal plate is divided into three parts from the bottom to the top. The upper and lower parts are sampled to obtain the temperature. Purity curve, record crystallization temperature and time data, get the temperature. Time curve. It can be seen that the proper temperature increase is beneficial to the crystallization of andrographolide, which is more beneficial to improve the aging; after the temperature exceeds 55 °C, the crystal purity of andrographolide decreases, which may be an increase in temperature and an increase in molecular activity. When the surface is more than the adsorption capacity of the crystal plate, The binding temperature of gravity, intermolecular force, etc., the extraction separation dominates, and the crystallization precipitation turns into a disadvantage. Therefore, the preferred crystallization temperature was chosen to be 55 °C.
2.3 The effect of time on the extraction and crystallization of andrographolide was 45, 60, 75, 90, 105 and 120 when the extraction crystallization pressure was 20 MPa, the extraction crystallization temperature was 55 °C, and the CO2 flow rate was 15 L/min. At min, the crystal plate is divided into two sections, and the upper section is sampled to obtain a time and purity curve.
It can be seen that the purity of lactone is positively correlated with temperature under other process parameters, especially when the time exceeds 75 min, the purity changes slowly. Considering the crystal purity and the crystallization amount, the accumulation of crystallization needs After the delay, the extraction crystallization time selected in this experiment is 90 min, and the purity is over 80.0%.
2.4 Test process and results Take 50g of raw materials, dissolve according to solid-liquid ratio 1:3 (liquid is analytical grade ethanol), stir in 50 °C water bath, let stand, layer, filter, and then extract and crystallize: set extraction The crystallization pressure was 20 MPa, the temperature was 55 ° C, the selection time was 90 min, and the CO 2 flow rate was 15 L/min. After the test, the crystallizer was taken out, and the sample was weighed and tested. It can be seen that andrographolide having a purity of up to 82.3% is obtained.
3 Conclusion Supercritical CO2 extracted synchronous and crystallization at the same time, and the crystals were distributed in the crystal plate according to the purity. With the increase of the gradient crystallization pressure, the purity of the andrographolide on the crystal plate gradually increased. The change trend is slower and slower, and the relative amount of crystallization increases first and then decreases. With the increase of gradient crystallization temperature, the purity of andrographolide first rises and then decreases; the time has a positive relationship with the purity of andrographolide. Under the experimental conditions, the optimum process parameters are extraction crystallization pressure 20 MPa, temperature 55 ° C, time 90 min, and CO 2 flow rate of 15 L/min. The results of multiple test results can reach 82.3%.
References
[1] Du QZ, Jcrz G, Winterhalter P. Separation of andrographolide and neoandrographolide from the leaves of Andrographis paniculata using high-speed counter-current chromatography. Journal of
Chromatography A, 2003, 984: l47-l5l
PHYTASE
PROTEASE
XYLANASE
AMYLASE
GLUCO OXIDASE
ALPHA GALACTOSIDASE
ACID MANNANASE
SWINE
WHEAT DIET
POULTRY
MISC.GRAIN AND MEAL
REGULAR DIET
Study on Purification Process of Andrographolide by Supercritical Carbon Dioxide Gradient Crystallization
Abstract: The effects of supercritical CO2 gradient crystallization pressure, temperature and time on the purity of andrographolide were investigated by single factor test. The purity was detected by high performance liquid chromatography . The results showed that supercritical CO2 showed simultaneous crystallization when extracting andrographolide, and showed a gradient distribution on the crystallized plate. The preferred purification process parameters were pressure 20 MPa, temperature 55 ° C, time 90 min, and CO 2 flow rate 15 L/min. At the time, the purity of andrographolide was 80% or more.
â—COMPLEX EMZYMES FOR