CN112870271B - Preparation method of high molecular weight rehmannia root polysaccharide and method for obtaining prepared rehmannia root by high-pressure steaming of fresh rehmannia root - Google Patents
Preparation method of high molecular weight rehmannia root polysaccharide and method for obtaining prepared rehmannia root by high-pressure steaming of fresh rehmannia root Download PDFInfo
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Abstract
The invention discloses a preparation method of high molecular weight rehmannia root polysaccharide and a method for obtaining prepared rehmannia root by steaming fresh rehmannia root under high pressure. The preparation method of the prepared rehmannia root comprises the following steps: taking yellow wine as an auxiliary material, and directly steaming the fresh rehmannia under high pressure to obtain the prepared rehmannia root. The preparation method of the high molecular weight rehmannia root polysaccharide comprises the following steps: (1) extracting rehmannia root with water to obtain water extract; (2) carrying out alcohol precipitation on the water extract obtained in the step (1), and collecting precipitates; (3) dialyzing the precipitated water solution by using a dialysis bag, collecting trapped fluid, and drying to obtain the rehmannia glutinosa polysaccharide; the cut-off molecular weight of the dialysis bag is 2000-50000 Da. The method for integrally preparing the prepared rehmannia root under high pressure saves the step of airing or baking the prepared rehmannia root into the rehmannia root, has short period, high efficiency and energy conservation, and the prepared rehmannia root has the appearance quality meeting the requirement. The method can prepare the rehmannia glutinosa polysaccharide with the molecular weight of more than 8 ten thousand.
Description
Technical Field
The invention relates to a preparation method of rehmannia root polysaccharide and a preparation method of prepared rehmannia root, in particular to a preparation method of high molecular weight rehmannia root polysaccharide and a method for obtaining prepared rehmannia root by steaming fresh rehmannia root under high pressure.
Background
Rehmannia glutinosa is fresh or dried root tuber of Rehmannia glutinosa Libosch (Rehmannia glutinosa Libosch). The rehmannia root used as a medicine can be fresh rehmannia root, dried rehmannia root (radix rehmanniae) which is dried or baked to be dry, and prepared rehmannia root according to different medicine use forms.
Reviewing the traditional herbal literature of the past generation, the processing methods of radix rehmanniae preparata are different, including steaming, wine boiling, wine stewing, ginger processing, fructus amomi processing, charcoal making and the like, but the steaming is always the mainstream of the radix rehmanniae processing in each period, and the steaming includes single steaming and wine adding steaming, and the wine steaming includes steaming, pot steaming and 'nine times steaming and nine times solarization'.
As is well known, the 'nine steaming and nine sun drying' is a traditional processing method of prepared rehmannia root recorded in ancient books of a lot of Chinese herbs, and many old pharmacists advocate the 'nine steaming and nine sun drying'. Generally speaking, the prepared rehmannia root, which is steamed and dried for nine times, has the advantages of nourishing without congealing and tonifying without greasiness, and the curative effect is far better than that of the common prepared rehmannia root. However, the 'nine times steaming and sun drying' has the defects of complex process, more fuel consumption, long production period, time and labor waste and the like, and is not suitable for the requirement of large-scale industrial production.
The rehmannia polysaccharide is an important nutrient substance and a medicinal component contained in the rehmannia polysaccharide, has pharmacological actions of resisting fatigue, enhancing immunity, resisting anxiety, resisting tumors, reducing blood sugar and the like, and has small toxic and side effects and safe use. The existing preparation method of the rehmannia root polysaccharide mainly comprises water extraction and alcohol precipitation, and the obtained product is regarded as the rehmannia root polysaccharide.
Disclosure of Invention
The invention aims to provide a preparation method of high molecular weight rehmannia root polysaccharide and a method for obtaining prepared rehmannia root by steaming fresh rehmannia root under high pressure; in the method for obtaining the prepared rehmannia root by steaming the fresh rehmannia root at high pressure, the prepared rehmannia root is obtained by integrally steaming the fresh rehmannia root at high pressure, the period is short, the efficiency is high, the energy is saved, and the sensory quality of the prepared rehmannia root meets the requirement; in the preparation method of the high molecular weight rehmannia root polysaccharide, the technical bias that the rehmannia root polysaccharide can be obtained by pure water extraction and alcohol precipitation in the prior art (actually, the obtained rehmannia root polysaccharide has low purity and is doped with oligosaccharide) is overcome, and the rehmannia root polysaccharide with the molecular weight of more than 8 ten thousand can be prepared by the method.
The invention firstly provides a preparation method of prepared rehmannia root, which comprises the following steps: taking yellow wine as an auxiliary material, and directly steaming the fresh rehmannia under high pressure to obtain the prepared rehmannia root.
In the preparation method, the amount of the yellow wine can be 5-50% of the mass of the fresh rehmannia root, and specifically can be 15-50%, 15-20%, 20-50%, 20%, 15% or 50%.
In the preparation method, the high-pressure steaming time can be 2-5 h, such as 2.5-3 h, 0.5h or 3 h;
the high pressure steaming pressure can be 0.12-0.265 MPa, such as 0.12-0.19 MPa, 0.19-0.265 MPa, 0.12MPa, 0.19MPa or 0.265 MPa;
the high pressure steaming temperature can be 105-128 deg.C, such as 105-121 deg.C, 105 deg.C, 121 deg.C, 128 deg.C;
the number of times of high-pressure steaming may be 1 to 3 times, for example, 1 time.
In the preparation method, before the high-pressure steaming, the method further comprises the step of soaking the fresh rehmannia root for 2-6 hours (such as 2 hours or 6 hours) by using the yellow wine.
In the preparation method, the method further comprises the step of drying to eighty percent dry after the high-pressure steaming is finished.
The prepared rehmannia root prepared by the method for obtaining the prepared rehmannia root by steaming the fresh rehmannia root under high pressure has black and bright appearance and slightly sweet taste, and meets the standard of China pharmacopoeia of 2020.
The invention further provides a preparation method of the rehmannia polysaccharide, which comprises the following steps:
(1) extracting rehmannia root with water to obtain water extract;
(2) carrying out alcohol precipitation on the water extract obtained in the step (1), and collecting precipitates;
(3) dialyzing the precipitated water solution by using a dialysis bag, collecting trapped fluid, and drying to obtain the rehmannia glutinosa polysaccharide; the cut-off molecular weight of the dialysis bag is 2000-50000 Da.
In the above preparation method, the rehmannia root can be fresh rehmannia root, raw rehmannia root or prepared rehmannia root.
Preferably, the prepared rehmannia root is prepared by nine times of steaming and nine times of drying or the prepared rehmannia root prepared by the method of obtaining the prepared rehmannia root by steaming the fresh rehmannia root under high pressure.
The rehmannia is fresh rehmannia; the method further comprises the step of pulping the fresh rehmannia before the water extraction.
The rehmannia is rehmannia root; the method also comprises the step of powdering the dried rehmannia root before the water extraction.
The rehmannia is prepared rehmannia root; the method further comprises the step of mashing the prepared rehmannia glutinosa prior to the water extraction.
In the above preparation method, in the step (1), the method further comprises the steps of soaking the rehmannia glutinosa libosch with ethanol, filtering and removing the filtrate before the water extraction.
The ethanol may be added in the form of an aqueous ethanol solution;
the volume fraction of the ethanol water solution can be 90-98 percent, and can be 95 percent specifically;
the mass-to-volume ratio of the rehmannia glutinosa to the ethanol aqueous solution may be 1g: 20-30 mL, specifically 1g:25 mL;
the soaking time can be 5-8 h, and specifically can be 5 h.
In the above preparation method, step (1), the water extraction may be any extraction method using water as a solvent, such as hot water extraction.
The mass volume ratio of the rehmannia root to the water can be 1g: 15-35 mL, and specifically can be 1g:20 mL;
the extraction temperature can be 75-95 ℃, and specifically can be 80 ℃;
the time for each extraction can be 1.5-4 h, and specifically can be 3 h;
the number of times of extraction may be 1 to 3, specifically 2.
In the preparation method, in the step (2), the temperature of alcohol precipitation can be 0-25 ℃, and specifically can be 4 ℃; the time can be 2-12 h, specifically 4 h;
the volume fraction of ethanol in the alcohol precipitation system can be 65-85%, and specifically can be 75%. The alcohol precipitation system is a mixed system consisting of the water extract and ethanol.
The above preparation method, which may further comprise a step of subjecting the precipitated aqueous solution to a deproteinization treatment before the dialysis;
the deproteinization adopts a Sevage method.
In the water solution of the precipitate, the proportion of precipitate dry matter to water is 50-80 mg:1mL, specifically 60mg:1 mL.
In the preparation method, in the step (3), the cut-off molecular weight of 2000-50000 Da can be any cut-off molecular weight within a range of 2000-50000 Da or any cut-off molecular weight within the range, and specifically can be 2000-20000 Da, 2000-10000 Da, 3500-20000 Da, 3500-10000 Da, 3500-8000 Da, 3500-7000 Da, 3500-5000 Da, 2000Da, 3500Da, 5000Da, 7000Da, 8000-10000 Da, 8000 Da.
In the preparation method, in the step (3), the dialysis temperature is 4-25 ℃ (such as 4 ℃) for 12-24 hours (such as 24 hours) in the flowing water, and then the dialysis is performed for 12-48 hours (such as 12 hours) in the distilled water, and the water is changed every 3-6 hours (such as 3 hours).
In the above preparation method, step (3), the drying may be lyophilization.
The rehmannia polysaccharide has a weight average molecular weight of more than 8 ten thousand, such as 8-11 ten thousand, 8 ten thousand or 10 ten thousand.
The preparation method of the rehmannia root polysaccharide can prepare the rehmannia root polysaccharide with the weight average molecular weight of more than 8 ten thousand, the yield of the polysaccharide prepared by steaming and solarizing the prepared rehmannia root by an ancient method can reach 7.29 percent, and the yield of the polysaccharide prepared by the prepared rehmannia root obtained by steaming fresh rehmannia root by high pressure can reach 6.19 to 7.52 percent.
The invention also provides a method for simultaneously preparing stachyose and rehmannia root polysaccharide, which comprises the following steps:
(1) extracting rehmannia root with water to obtain water extract;
(2) carrying out alcohol precipitation on the water extract obtained in the step (1), and collecting precipitates;
(3) dialyzing the precipitated water solution by using a dialysis bag, and collecting trapped fluid and dialysate; the cut-off molecular weight of the dialysis bag is 2000-50000 Da;
drying the trapped fluid to obtain the rehmannia glutinosa polysaccharide;
and drying the dialysate to obtain the stachyose.
In the above preparation method, the rehmannia root can be fresh rehmannia root, raw rehmannia root or prepared rehmannia root.
Preferably, the prepared rehmannia root is prepared by nine times of steaming and nine times of drying or the prepared rehmannia root prepared by the method of obtaining the prepared rehmannia root by steaming the fresh rehmannia root under high pressure.
The rehmannia is fresh rehmannia; the method further comprises the step of pulping the fresh rehmannia before the water extraction.
The rehmannia is rehmannia root; the method also comprises the step of powdering the dried rehmannia root before the water extraction.
The rehmannia is prepared rehmannia root; the method further comprises the step of mashing the prepared rehmannia glutinosa prior to the water extraction.
In the above preparation method, in the step (1), the method further comprises the steps of soaking the rehmannia glutinosa libosch with ethanol, filtering and removing the filtrate before the water extraction.
The ethanol may be added in the form of an aqueous ethanol solution;
the volume fraction of the ethanol water solution can be 90-98 percent, and can be 95 percent specifically;
the mass-to-volume ratio of the rehmannia glutinosa to the ethanol aqueous solution may be 1g: 20-30 mL, specifically 1g:25 mL;
the soaking time can be 5-8 h, and specifically can be 5 h.
In the above preparation method, step (1), the water extraction may be any extraction method using water as a solvent, such as hot water extraction.
The mass volume ratio of the rehmannia root to the water can be 1g: 15-35 mL, and specifically can be 1g:20 mL;
the extraction temperature can be 75-95 ℃, and specifically can be 80 ℃;
the time for each extraction can be 1.5-4 h, and specifically can be 3 h;
the number of times of extraction may be 1 to 3, specifically 2.
In the preparation method, in the step (3), the temperature of alcohol precipitation can be 0-25 ℃, and specifically can be 4 ℃; the time can be 2-12 h, specifically 4 h;
the volume fraction of ethanol in the alcohol precipitation system can be 65-85%, and specifically can be 75%. The alcohol precipitation system is a mixed system consisting of the water extract and ethanol.
The above preparation method, which may further comprise a step of subjecting the precipitated aqueous solution to a deproteinization treatment before the dialysis;
the deproteinization adopts a Sevage method.
In the water solution of the precipitate, the proportion of precipitate dry matter to water is 50-80 mg:1mL, specifically 60mg:1 mL.
In the preparation method, in the step (3), the cut-off molecular weight of 2000-50000 Da can be any cut-off molecular weight within a range of 2000-50000 Da or any cut-off molecular weight within the range, and specifically can be 2000-20000 Da, 2000-10000 Da, 3500-20000 Da, 3500-10000 Da, 3500-8000 Da, 3500-7000 Da, 5000-5000 Da, 2000Da, 3500Da, 5000Da, 7000Da, 8000-10000 Da, 8000 Da.
In the preparation method, in the step (3), the dialysis temperature is 4-25 ℃ (such as 4 ℃) for 12-24 hours (such as 24 hours) in the flowing water, and then the dialysis is performed for 12-48 hours (such as 12 hours) in the distilled water, and the water is changed every 3-6 hours (such as 3 hours).
In the above preparation method, step (3), the drying may be lyophilization.
The rehmannia polysaccharide has a molecular weight of more than 8 ten thousand, such as 8-11 ten thousand, 8 ten thousand or 10 ten thousand.
The preparation method of the rehmannia root polysaccharide can prepare the rehmannia root polysaccharide with the molecular weight of more than 8 ten thousand, the yield of the polysaccharide prepared by steaming and solarizing the prepared rehmannia root by an ancient method for nine times can reach 7.29 percent, and the yield of the polysaccharide prepared by the prepared rehmannia root obtained by steaming the fresh rehmannia root by high pressure can reach 6.19 to 7.52 percent; the content of stachyose can reach 43.3%.
The invention has the following beneficial effects:
(1) the method of the invention directly prepares the fresh rehmannia into the mature rehmannia by using a high-pressure steaming means, omits the step of airing or baking the fresh rehmannia into the rehmannia, saves energy, ensures that the medicinal components of the prepared rehmannia do not run off, and improves the content of the medicinal components; the high-pressure steaming realizes the characteristic of high-efficiency substance transmission by improving the temperature and the pressure. As a result, the period of steaming the prepared rehmannia under high pressure is short, the efficiency is high, the energy is saved, and the like, and the appearance quality meets the requirement. The prepared rehmannia root steamed under high pressure has consistent internal and external color and luster, is soft and bright and has sweet smell. Meets the regulation of 'steaming to black moisture' regulated in Chinese pharmacopoeia (2020 edition).
(2) The present invention further provides high molecular weight rehmannia glutinosa polysaccharide and stachyose from prepared rehmannia glutinosa obtained by autoclaving fresh rehmannia glutinosa as described above, and the results show that the yield of high molecular weight rehmannia glutinosa polysaccharide in prepared rehmannia glutinosa obtained by autoclaving fresh rehmannia glutinosa is about 2 times that of prepared rehmannia glutinosa obtained by autoclaving rehmannia glutinosa as compared with prepared rehmannia glutinosa obtained by autoclaving rehmannia glutinosa; the content of stachyose in prepared rehmannia root obtained by steaming fresh rehmannia root at high pressure is about 6 times of that of prepared rehmannia root obtained by steaming the rehmannia root at high pressure, and the method for integrally steaming the fresh rehmannia root at high pressure to obtain the prepared rehmannia root can reduce the loss of high molecular weight rehmannia root polysaccharide and stachyose and improve the content of active ingredients in the prepared rehmannia root.
(3) The method overcomes the technical prejudice that the rehmannia root polysaccharide can be obtained by pure water extraction and alcohol precipitation in the prior art (actually, the obtained rehmannia root polysaccharide has low purity and is doped with oligosaccharide), and can prepare the rehmannia root polysaccharide with the molecular weight of more than 8 ten thousand.
(4) The invention provides a method for obtaining high-molecular-weight prepared rehmannia root polysaccharide for the first time. The active polysaccharide components are more defined.
Drawings
FIG. 1 is a photograph of fresh rehmannia (left) and its cut pieces (right) used in the examples.
FIG. 2 is a photograph of prepared rehmannia root obtained by high pressure integrated steaming of example 1.
FIG. 3 is a photograph of prepared rehmannia root obtained by high pressure integrated steaming of example 2.
FIG. 4 is a photograph of prepared rehmannia root obtained by high pressure integrated steaming in example 3.
FIG. 5 is a photograph of prepared rehmannia root obtained by nine times of steaming and nine times of sun-drying in comparative example 1.
FIG. 6 is a photograph (left) of raw rehmannia glutinosa used in comparative example 2 and a photograph (right) of prepared rehmannia glutinosa obtained by steaming raw rehmannia glutinosa under high pressure.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The dialysis bags with molecular weight cut-off of 3500Da in the examples below were purchased from Shanghai-derived leaf Biotechnology Ltd under the product name of general type dialysis bag (3500Da) and the product model number of MD3534-5 m.
Example 1 high pressure Integrated steaming of fresh rehmannia glutinosa Libosch to obtain prepared rehmannia glutinosa Libosch
The method for preparing the prepared rehmannia root by integrally steaming the fresh rehmannia root at high pressure comprises the following specific steps:
cutting 100g of fresh rehmanniae radix (JIUJINJIU variety JIAOZHONGWEN, Henan province) into slices; taking 50mL of yellow wine (the density of the yellow wine is about 1g/mL, the alcohol content of the yellow wine is 14.0%, and the wine age is five years); soaking fresh rehmanniae radix with yellow wine for 6 hr, and steaming at 0.19MPa for 3 hr at 121 deg.C; steaming under high pressure to black, taking out, and oven drying to obtain radix rehmanniae Preparata.
The prepared rehmannia root prepared by the embodiment has black and bright appearance and slightly sweet taste, meets the standard of Chinese pharmacopoeia in 2020, and the yield reaches more than 95%.
The photograph of fresh rehmannia glutinosa used in this example is shown in FIG. 1, and the photograph of prepared rehmannia glutinosa prepared by high pressure integrated steaming is shown in FIG. 2.
Example 2 high pressure Integrated steaming of fresh rehmannia glutinosa Libosch to obtain prepared rehmannia glutinosa Libosch
The method for preparing the prepared rehmannia root by integrally steaming the fresh rehmannia root at high pressure comprises the following specific steps:
cutting 100g of fresh rehmanniae radix (JIUJINJIU variety JIAOZHONGWEN, Henan province) into slices; taking 20mL of yellow wine (the density of the yellow wine is about 1g/mL, yellow rice wine, the alcoholic strength is 14.0%, and the wine age is five years); soaking fresh rehmanniae radix with yellow wine for 2 hr, and steaming at 0.12MPa for 2 times at 105 deg.C for 2.5 hr; steaming to black, taking out, and oven drying to obtain radix rehmanniae Preparata.
The prepared rehmannia root prepared by the embodiment has black and bright appearance and slightly sweet taste, meets the standard of Chinese pharmacopoeia in 2020, and the finished product rate reaches more than 96%.
The photograph of fresh rehmannia glutinosa used in this example is shown in FIG. 1, and the photograph of prepared rehmannia glutinosa prepared by high pressure integrated steaming is shown in FIG. 3.
Example 3 high pressure Integrated steaming of fresh rehmannia glutinosa Libosch to obtain prepared rehmannia glutinosa Libosch
The method for preparing the prepared rehmannia root by integrally steaming the fresh rehmannia root at high pressure comprises the following specific steps:
cutting 100g of fresh rehmanniae radix (JIUJINJIU variety JIAOZHONGWEN, Henan province) into slices; taking 15mL of yellow wine (the density of the yellow wine is about 1g/mL, yellow rice wine, the alcoholic strength is 14.0%, and the wine age is five years); soaking fresh rehmanniae radix with yellow wine for 2 hr, and steaming at 0.265MPa for 128 deg.C for 3 hr; steaming under high pressure to black, taking out, and oven drying to obtain radix rehmanniae Preparata.
The prepared rehmannia root prepared by the embodiment has black and bright appearance and slightly sweet taste, meets the standard of Chinese pharmacopoeia in 2020, and the yield reaches more than 97%.
The photograph of fresh rehmannia glutinosa used in the present example is shown in FIG. 1, and the photograph of prepared rehmannia glutinosa prepared by high pressure integrated steaming is shown in FIG. 4.
Comparative example 1 preparation of prepared rehmannia root by nine times of steaming and sun drying
The prepared rehmannia root is prepared by a nine-steaming and nine-sun-drying processing method, which comprises the following specific steps: steaming for 72h, and airing in sunlight until the epidermis is slightly dry, namely steaming and airing; and (3) tightly sealing the collected prepared rehmannia root juice and yellow wine (not required to be added in the steaming process) in a container, continuously steaming for 24 hours, airing, repeatedly steaming for seven times, adding fructus amomi in the ninth steaming, and steaming for nine times and drying in the sun to obtain the prepared rehmannia root.
The photograph of fresh rehmannia glutinosa used in this comparative example is shown in FIG. 1, and the photograph of prepared rehmannia glutinosa obtained by nine times of steaming and nine times of sun-drying is shown in FIG. 5.
Comparative example 2 preparation of rehmannia glutinosa Libosch by steaming under high pressure
Cleaning and draining fresh rehmannia (Jiujin Jiu variety Jiujin Xian Zhong Jiao, Henan province), placing in a drying oven, and drying at 45 ℃ until the rehmannia is eight-turn dry, which reaches the specified standard in China pharmacopoeia 2020 to obtain the rehmannia root.
Taking 100g of radix rehmanniae (Jiujin Jiu variety Jiujin Wei Zhong Jiaozhao, Henan province); taking 30mL of yellow wine (the density of the yellow wine is about 1g/mL, the alcohol content of the yellow wine is 14.0%, and the wine age is five years); soaking radix rehmanniae with yellow wine for 8h, and steaming at 0.19MPa for 121 deg.C for 3 h; steaming under high pressure to black, taking out, and oven drying to obtain radix rehmanniae Preparata.
The prepared rehmannia root prepared by the comparative example has black and bright appearance and sweetness in taste, is slightly inferior to the prepared rehmannia root obtained by steaming fresh rehmannia root under high pressure, meets the standard of Chinese pharmacopoeia of 2020, and has the yield of more than 95 percent.
The photograph of dried rehamnnia root used in this comparative example and the photograph of prepared rehamnnia root obtained by steaming dried rehamnnia root under high pressure are shown in FIG. 6.
Example 4 preparation of high molecular weight rehmannia glutinosa polysaccharides
High molecular weight polysaccharide of rehmannia glutinosa polysaccharide is prepared from fresh rehmannia glutinosa (jin Jiu variety of jiao shou Wen county, Henan province), rehmannia glutinosa (jin Jiu variety of jiao shou county, Henan province, prepared in the same manner as in comparative example 2), commercially available prepared rehmannia glutinosa (purchased from the jin Jiu county, medicinal material market), ancient method, nine times steamed and nine times steamed prepared rehmannia glutinosa (prepared in the same manner as in comparative example 1) and high-pressure steamed prepared rehmannia glutinosa, respectively, according to the following steps:
(1) fresh rehmannia root, commercially available prepared rehmannia root, ancient method nine times steaming and nine times sun drying prepared rehmannia root, prepared rehmannia root obtained by high pressure steaming in examples 1-3 are respectively selected, pulped, powdered or mashed, and the mixture is mixed with 95% (v/v) ethanol according to the weight ratio of the rehmannia root: soaking in ethanol at a ratio of 1g to 25mL for 5h, filtering, and removing the filtrate;
(2) extracting the rehmannia root in the step (1) by a hot water extraction method under the specific conditions that the ratio of material to liquid is 1g to 20mL, extracting for 3 hours at 80 ℃, extracting twice, carrying out suction filtration, and combining extracting solutions;
(3) concentrating the filtrate to 20mL, adding 3 times volume of anhydrous ethanol (the volume fraction of ethanol in the mixed solution is 75%), standing at 4 deg.C for 4h, and collecting precipitate; according to the dry matter precipitation: dissolving the precipitate in distilled water at a ratio of 60mg:1mL to obtain a crude sugar solution; deproteinizing by a Sevage reagent (Sevage reagent: chloroform: n-butanol 4: 1);
the specific operation of deproteinizing the Sevage reagent is as follows: mixing the crude sugar solution with Sevage reagent (chloroform: n-butanol-4: 1) at a volume ratio of 5: 1, magnetically stirring for 30 minutes, centrifuging at 4000rpm for 15 minutes, taking the upper layer solution, repeating the steps for 6 times until the protein at the interface of two phases is nearly absent, and finally carrying out rotary evaporation on the collected upper layer solution to remove the organic reagent.
(4) Concentrating the deproteinized crude sugar solution by using a dialysis bag with the molecular weight cutoff of 3500Da, dialyzing at 4 ℃ in flowing water for about 24 hours, putting the dialysis bag in distilled water, continuously dialyzing for 12 hours, changing the distilled water once in 3 hours, and collecting the trapped fluid (namely the solution in the bag) and the dialysate (namely the solution outside the bag) after the dialysis is finished;
concentrating the trapped solution, and lyophilizing to obtain rehmanniae radix polysaccharide.
The dialyzate was concentrated and lyophilized to obtain crude oligosaccharide.
The yield of the prepared rehmannia root polysaccharide is shown in table 1, and the molecular weight of the rehmannia root polysaccharide is more than 10 ten thousand. The method for measuring the molecular weight of the rehmannia polysaccharide comprises the following steps: size exclusion chromatography-18 degree laser light scattering (18 degree laser light scattering instrument (DAWN HELEOS II) from Huaatt corporation, USA; Shimadzu RID-20A differential detector. conditions: 0.1mol/L aqueous sodium nitrate in mobile phase, 0.5ml/min flow rate, shodex Ohpak SB-806M HQ in series with shodex Ohpak SB-804HQ, column temperature 40 ℃.
TABLE 1 yield of polysaccharide from different rehmannia glutinosa
TABLE 2 polysaccharide yields of rehmannia glutinosa Libosch and fresh rehmannia glutinosa Libosch
TABLE 3 molecular weights of rehmannia glutinosa polysaccharides prepared from different rehmannia glutinosa
The content of stachyose in the crude oligosaccharide, and the experimental results are shown in table 4. The determination method comprises the following steps: equipment: agilent 1260 high performance liquid chromatograph; a detector: RID differential refractive detector; chromatographic conditions are as follows: the mobile phase is acetonitrile: water (70:30), flow rate 1.0mL/min, column oven 40 ℃, sample volume 10 μ L, detection cell temperature 50 ℃, column model: agilent ZORBOX NH2 (4.6X 250mm,5 μm).
TABLE 4 stachyose content prepared from rehmanniae radix Preparata of different examples
Claims (2)
1. A method for simultaneously preparing stachyose and rehmannia root polysaccharide comprises the following steps:
(1) preparing prepared rehmannia root; the prepared rehmannia root is prepared by the following steps of a or b;
a. soaking fresh rehmanniae radix with yellow wine for 6 hr, and steaming at 0.19MPa for 3 hr at 121 deg.C; steaming under high pressure to black, taking out, and oven drying to obtain radix rehmanniae Preparata; the amount of the yellow wine is 50% of the mass of the fresh rehmannia;
b. soaking fresh rehmanniae radix with yellow wine for 2 hr, and steaming at 0.12MPa for 2 times at 105 deg.C for 2.5 hr; steaming to black, taking out, and drying to obtain radix rehmanniae Preparata; the amount of the yellow wine is 20 percent of the mass of the fresh rehmannia;
(2) extracting the prepared rehmannia root obtained in the step (1) with water to obtain water extract;
(3) carrying out alcohol precipitation on the water extract obtained in the step (1), and collecting precipitates;
(4) dialyzing the precipitated water solution by using a dialysis bag, and collecting trapped fluid and dialysate; the cut-off molecular weight of the dialysis bag is 3500 Da;
drying the trapped fluid to obtain the rehmannia glutinosa polysaccharide;
and drying the dialysate to obtain the stachyose.
2. The method of claim 1, wherein: in the step (2), in the step of water extraction, the mass-to-volume ratio of the prepared rehmannia root to the water is 1g: 15-35 mL, the extraction temperature is 75-95 ℃, the extraction time is 1.5-4 h each time, and the extraction times are 1-3 times; and/or the presence of a gas in the gas,
in the step (3), in the step of alcohol precipitation, the temperature of alcohol precipitation is 0-25 ℃, and the time is 2-12 hours; the volume fraction of ethanol in the ethanol precipitation system is 65-85%.
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| CN1900100A (en) * | 2006-07-20 | 2007-01-24 | 杨菁 | Rehmannia root oligose and its extracting method |
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| 崔瑛等.熟地黄粗多糖提取与初步纯化工艺研究.《时珍国医国药》.2010,第21卷(第10期),第2570页最后1段、2571页最后1段. * |
| 熟地黄粗多糖提取与初步纯化工艺研究;崔瑛等;《时珍国医国药》;20101231;第21卷(第10期);第2570页最后1段、2571页最后1段 * |
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