Summary of the invention:
The object of the invention is to overcome the shortcomings such as the routine techniques extraction ratio is relatively low, DNA purity is low, a kind of method of utilizing Radix Astragali saponin in water vapour reflux, extract, and membrane separation technique and the macroporous adsorbent resin coupled method enriching and purifying Radix Astragali is provided.
For achieving the above object, the technical solution used in the present invention is:
A kind of method of utilizing Radix Astragali saponin in water vapour reflux, extract, and membrane separation technique and the macroporous adsorbent resin coupled method enriching and purifying Radix Astragali, its step is as follows:
(1) the dried Radix Astragali is pulverized, adding volume is the purified water of 10~20 times that the Radix Astragali is pulverized volume, carries out the water vapour reflux, extract,, extracts 2~3 hours extraction times 2~3 times;
(2) Radix Astragali extractive solution after process (1) is merged, through microfiltration, pore diameter range is at 0.1~1 micron, and pressure is controlled at 01~0.7MPa, gets filtrate;
(3) will pass through ultrafiltration through the filtrate of microfiltration, temperature is controlled at 20~40 ℃ again, and pressure is controlled at 0.1~0.6MPa, gets the Radix Astragali saponin crude extract;
(4) gained Radix Astragali saponin crude extract employment macroporous adsorbent resin is adsorbed, be adsorbed as static adsorption or dynamic adsorption;
(5) ethanol with 40%~80% variable concentrations carries out eluting to macroporous adsorbent resin, follows the tracks of with thin layer chromatography and detects, and collects each stepwise elution liquid of Radix Astragali saponin;
(6) each stepwise elution liquid of Radix Astragali saponin is concentrated, reclaim ethanol, get the Radix Astragali saponin semi-finished product, its content is more than 30%;
(7) with silica gel column chromatography on gained Radix Astragali saponin semi-finished product, and take the volume ratio of methanol and concentrated solution as 1~7: 1 ratio is carried out eluting, and thin layer chromatography is followed the tracks of and detected, and collects each stepwise elution liquid of Radix Astragali saponin;
(8) with collected Radix Astragali saponin eluent, adopt activated carbon decolorizing, recrystallization method to make with extra care to get Radix Astragali saponin, its content is more than 95%.
In described step (1), after pulverizing, the dried Radix Astragali crosses 40~80 mesh sieves.
In described step (4), macroporous adsorbent resin used is a kind of in Tianjin Nankai university X-5, the NKA-9, S-8, D3520, D4006, H103, D4020, AB-8H and the NKA-II type resin that produce.
The present invention uses water vapour reflux, extract, and membrane separation technique and macroporous adsorbent resin coupled method Seperation Purification of Astragaloside, Radix Astragali saponin is extracted fully, do not pollute, good separating effect, purity is high, the macroporous resin adsorption selectivity is good, the fast parsing of absorption is also fast, adsorption capacity is larger, separating effect is obvious, DNA purity is high, can obtain Radix Astragali saponin semi-finished product and the content Radix Astragali saponin finished product 95% or more of content more than 30%, has overcome the shortcoming that conventional extraction ratio is relatively low, DNA purity is low.
The specific embodiment:
Embodiment 1: a kind of method of utilizing Radix Astragali saponin in water vapour reflux, extract, and membrane separation technique and the macroporous adsorbent resin coupled method enriching and purifying Radix Astragali, and its step is as follows:
(1) the dried Radix Astragali of 100g is pulverized rear 60 mesh sieves of crossing, adding volume is the purified water of 10 times that the Radix Astragali is pulverized volume, carries out the water vapour reflux, extract,, extracts 2 hours extraction times 2 times;
(2) Radix Astragali extractive solution after process (1) is merged, through microfiltration, pore diameter range is at 0.1~1 micron, and pressure is controlled at 0.1MPa, gets filtrate;
(3) will pass through ultrafiltration through the filtrate of microfiltration, the ultrafiltration molecular cut off is 3000~300000 again, and temperature is controlled at 20 ℃, and pressure is controlled at 0.1MPa, gets the Radix Astragali saponin crude extract;
(4) gained Radix Astragali saponin crude extract is adsorbed with macroporous adsorbent resin, macroporous resin used is the NKA-II type resin that Tianjin Nankai university produces, and is adsorbed as static adsorption or dynamic adsorption;
(5) carry out successively gradient elution with the ethanol with 40%, 50%, 60%, 70%, 80% in macroporous adsorbent resin layer post, follow the tracks of with thin layer chromatography and detect, collect each stepwise elution liquid of Radix Astragali saponin;
(6) with the standby stepwise elution liquid concentration and recovery ethanol of Radix Astragali saponin, obtaining content is 306% Radix Astragali saponin semi-finished product;
(7) with silica gel column chromatography on gained Radix Astragali saponin semi-finished product, and be followed successively by 1: 1, carried out gradient elution in 2: 1,3: 1,4: 1,5: 1,6: 1,7: 1 with the volume ratio of methanol and concentrated solution, thin layer chromatography is followed the tracks of and is detected, and collects each stepwise elution liquid of Radix Astragali saponin:
(8) with collected Radix Astragali saponin eluent, adopt activated carbon decolorizing, refining content 95.4% Radix Astragali saponin that obtains of recrystallization method
Case study on implementation 2: a kind of method of utilizing Radix Astragali saponin in water vapour reflux, extract, and membrane separation technique and the macroporous adsorbent resin coupled method enriching and purifying Radix Astragali, its step is as follows:
(1) the dried Radix Astragali of 100g is pulverized rear 40 mesh sieves of crossing, adding volume is the purified water of 15 times that the Radix Astragali is pulverized volume, carries out the water vapour reflux, extract,, extracts 2 hours extraction times 2 times;
(2) Radix Astragali extractive solution after process (1) is merged, through microfiltration, pore diameter range is at 0.1~1 micron, and pressure is controlled at 0.2MPa, gets filtrate;
(3) will stand through super through the filtrate of microfiltration, the ultrafiltration molecular cut off is 3000~300000 again, and temperature is controlled at 30 ℃, and pressure is controlled at 0.2MPa, gets the Radix Astragali saponin crude extract;
(4) gained Radix Astragali saponin crude extract is adsorbed with macroporous adsorbent resin, macroporous resin used is the D4006 type resin that Tianjin Nankai university produces, and is adsorbed as static adsorption or dynamic adsorption;
(5) carry out successively gradient elution with 40%, 50%, 60%, 70%, 80% ethanol in macroporous adsorbent resin layer post, follow the tracks of with thin layer chromatography and detect, collect each stepwise elution liquid of Radix Astragali saponin;
(6) with each stepwise elution liquid concentration and recovery ethanol of Radix Astragali saponin, obtain content and be 31.2% Radix Astragali saponin semi-finished product;
(7) with silica gel column chromatography on gained Radix Astragali saponin semi-finished product, and be followed successively by 1: 1, carried out gradient elution in 2: 1,3: 1,4: 1,5: 1,6: 1,7: 1 with the volume ratio of methanol and concentrated solution, thin layer chromatography is followed the tracks of and is detected, and collects each stepwise elution liquid of Radix Astragali saponin;
(8) with collected Radix Astragali saponin eluent, adopt activated carbon decolorizing, the refining Radix Astragali saponin that obtains content 96.6% of recrystallization method.
Case study on implementation 3: a kind of method of utilizing Radix Astragali saponin in water vapour reflux, extract, and membrane separation technique and the macroporous adsorbent resin coupled method enriching and purifying Radix Astragali, its step is as follows:
(1) the dried Radix Astragali of 100g is pulverized rear 80 mesh sieves of crossing, adding volume is the purified water of 20 times that the Radix Astragali is pulverized volume, carries out the water vapour reflux, extract,, extracts 2 hours extraction times 3 times;
(2) Radix Astragali extractive solution after process (1) is merged, through microfiltration, pore diameter range is at 0.1~1 micron, and pressure is controlled at 0.2MPa, gets filtrate;
(3) will pass through ultrafiltration through the filtrate of microfiltration, the ultrafiltration molecular cut off is 3000~300000 again, and temperature is controlled at 40 ℃, and pressure is controlled at 0.2MPa, gets the Radix Astragali saponin crude extract;
(4) gained Radix Astragali saponin crude extract is adsorbed with macroporous adsorbent resin, macroporous resin used is the NKA-9 type resin that Tianjin Nankai university produces, and is adsorbed as static adsorption or dynamic adsorption;
(5) carry out successively gradient elution with 40%, 50%, 60%, 70%, 80% ethanol in macroporous adsorbent resin layer post, follow the tracks of with thin layer chromatography and detect, collect each stepwise elution liquid of Radix Astragali saponin;
(6) with each stepwise elution liquid concentration and recovery ethanol of Radix Astragali saponin, obtain content and be 31.7% Radix Astragali saponin semi-finished product;
(7) with silica gel column chromatography on gained Radix Astragali saponin semi-finished product, and be followed successively by 1: 1, carried out gradient elution in 2: 1,3: 1,4: 1,5: 1,6: 1,7: 1 with the volume ratio of methanol and concentrated solution, thin layer chromatography is followed the tracks of and is detected, and collects each stepwise elution liquid of Radix Astragali saponin;
(8) with collected Radix Astragali saponin eluent, adopt activated carbon decolorizing, the refining Radix Astragali saponin that obtains content 97.8% of recrystallization method.
Case study on implementation 4: a kind of method of utilizing Radix Astragali saponin in water vapour reflux, extract, and membrane separation technique and people's macroporous adsorbent resin coupled method enriching and purifying Radix Astragali, its step is as follows:
(1) the dried Radix Astragali of 100g is pulverized rear 50 mesh sieves of crossing, adding volume is the purified water of 12 times that the Radix Astragali is pulverized volume, carries out the water vapour reflux, extract,, extracts 2 hours extraction times 3 times;
(2) Radix Astragali extractive solution after process (1) is merged, through microfiltration, pore diameter range is at 0.1~1 micron, and pressure is controlled at 0.4MPa, gets filtrate;
(3) will stand through super through the filtrate of microfiltration, the ultrafiltration molecular cut off is 3000~300000 again, and temperature is controlled at 20 ℃, and pressure is controlled at 0.4MPa, gets the Radix Astragali saponin crude extract;
(4) gained Radix Astragali saponin crude extract is adsorbed with macroporous adsorbent resin, macroporous resin used is the H103 type resin that Tianjin Nankai university produces, and is adsorbed as static adsorption or dynamic adsorption;
(5) carry out successively gradient elution with 40%, 50%, 60%, 70%, 80% ethanol in macroporous adsorbent resin layer post, follow the tracks of with thin layer chromatography and detect, collect each stepwise elution liquid of Radix Astragali saponin;
(6) with each stepwise elution liquid concentration and recovery ethanol of Radix Astragali saponin, obtain content and be 30.4% Radix Astragali saponin semi-finished product;
(7) with silica gel column chromatography on gained Radix Astragali saponin semi-finished product, and be followed successively by 1: 1, carried out gradient elution in 2: 1,3: 1,4: 1,5: 1,6: 1,7: 1 with the volume ratio of methanol and concentrated solution, thin layer chromatography is followed the tracks of and is detected, and collects each stepwise elution liquid of Radix Astragali saponin:
(8) with collected Radix Astragali saponin eluent, adopt activated carbon decolorizing, the refining Radix Astragali saponin that obtains content 90.9% of recrystallization method.