Include 0.6 Load Tongkat Ali tree origin rugged powder right into a 6Ton multifunctional extraction storage tank, and essence 1.5, 1.0, 0.5 h specifically with 4.8 Bunch, 4.2 Lot, and 3.6 Lot ethanol in the reflux state. The removal liquid goes to -0.08 Mpa. Focus to a certain gravity of 1.15 to acquire Tongkat Ali extract. Add 3 times the amount (w/w) of oil ether (60-90 ° C )to draw out 3 times, let it stand, divide the layers, dispose of the oil ether fluid, and retain the lower layer. Remove the degreased fluid 3 times with 5 times the quantity (w/w) of ethyl acetate, let it stand, divide the layers, discard the water liquid, and keep the ethyl acetate fluid.
Concentrate the ethyl acetate option, blend it into silica gel, completely dry it, grind it finely, include the top of the silica gel column to make sure that the surface area is 30 to 50cm far from the column mouth, very first elute with chloroform, and spot by thin layer chromatography up until the pollutants are eluted easily, and after that different Elute with chloroform: ethyl acetate = 100:10, 100:20, 100:30, detect by slim layer chromatography, collect the eluate having the flavonoid element, add 5% (w/v) turned on carbon, and reflux Decolorize by keeping cozy for thirty minutes, filter, concentrate under reduced pressure, take shape at 25 ° C. for 24 to 72 hours, filter, and acquire crude flavonoids. Include 8 times the amount (w/w) of petroleum ether (60-90 ° C )to the unrefined acanthone, gradually add ethyl acetate dropwise, and mix slowly till the pigment pollutants on the surface of the crude acanthone are washed tidy, then filter right away, get white fine Eurycomanone crystals, keep the vacuum degree at -0.08 Mpa, and completely dry at a temperature of 40 to 60 ° C for 2.5 hours to obtain great Eurycomanone crystals with a content of 98.6% (HPLC).
( 1) Enzyme soaking: crush 5kg Tongkat Ali to 10-30 mesh, take in 12.5 L water, then add 1.25 g cellulase prep work, enzymatically hydrolyze at 40 ° C for 120 mins to get enzymatic hydrolysis of Tongkat Ali;
( 2) Home heating extraction: Include 40kg of water to the enzymatically hydrolyzed Tongkat Ali obtained symphonious (1 ), and perform continuous countercurrent removal at 85 ° C for 90 mins to get 40.35 kg of Tongkat Ali extract;
( 3) Centrifugal degreasing: After cooling 40.35 kg of Tongkat Ali essence gotten symphonious (2) to 9 ° C, first perform decanter centrifugation at a rate of 3000r/min, and after that do tube centrifugation at a rate of 10000r/min. Get 39.50 kg of degreasing liquid;
( 4) Ultrafiltration and nanofiltration: Make use of an ultrafiltration membrane layer with a molecular weight cutoff of 50,000 Da to execute ultrafiltration on 39.50 kg of the degreasing fluid acquired symphonious (3) under the problems of an operating stress of 1.5 MPa and a product temperature of 30 ° C. The ultrafiltrate is then treated with a nanofiltration membrane with a molecular weight cutoff of 6000 Da. Under the conditions of an operating stress of 2.0 MPa and a product temperature level of 30 ° C, the ultrafiltrate is nanofiltrated until the conductivity of the filtrate is 300 μs/ centimeters to obtain the nanofiltration of flavonoids. Retentate 950mL;
( 5) Resin chromatography: Area 50 mL of HP20 macroporous resin on 950 mL of the broad-thin nanofiltration retentate obtained in step (4 ), and after that very first elute 2BV with an option of ethanol/pure water with a volume proportion of 10:90. After the stripping is completed, the effluent from the chromatography column is colorless and clear. The eluate is treated as waste fluid, and then 2BV is eluted with a service with an ethanol/pure water volume proportion of 80:20. All the eluates in this section are gathered, and the The area eluate was focused under vacuum at -0.08 MPa and 65 ° C to get 4.60 g of focused option;
( 6) Alcohol phase formation and drying out: Add 1.0 mL of absolute ethanol to the 4.60 g focused option acquired in step (5 ), mix thoroughly, filter snugly, then position it at 2 ° C, awesome and crystallize for 24-hour, and get 6 mL of crystals, which are then Clean two times with 2 ° C ice water, 10 mL of water each time, repeat cooling and crystallization when and clean, and completely dry under vacuum cleaner to get 1.50 g of Eurycomanone item.
The gotten eurycomanone product was in the type of white powder. After discovery by high performance fluid chromatography (the chromatogram is displayed in Figure 1), the peak elution time of eurycomanone was 9.35 minutes, the purity of eurycomanone was 98.1%, and the last yield was 94.93 %.
