CN115043872B - Extraction method and application of deer blood phospholipid - Google Patents
Extraction method and application of deer blood phospholipid Download PDFInfo
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- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/10—Phosphatides, e.g. lecithin
- C07F9/103—Extraction or purification by physical or chemical treatment of natural phosphatides; Preparation of compositions containing phosphatides of unknown structure
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Abstract
A deer blood phospholipid extraction method and application thereof belong to the biotechnology field, and the specific scheme comprises the following steps: step one, preparing deer blood freeze-dried powder; step two, adding a mixed solution of dichloromethane and methanol into deer blood freeze-dried powder, carrying out ultrasonic treatment, carrying out centrifugal separation, taking supernatant, adding double distilled water, uniformly stirring, standing, collecting dichloromethane at the lower layer, and adding dichloromethane into the rest substances for secondary extraction; repeating the extraction for several times, and combining the collected dichloromethane; and thirdly, concentrating the dichloromethane collected in the second step under reduced pressure, drying the concentrated solution to obtain mixed liposome, dissolving the mixed liposome by using acetone, fully oscillating for extraction, centrifuging, and drying the precipitate to obtain deer blood phospholipid. The extraction method well preserves the biological activity of deer blood phospholipid, and makes detailed analysis on the extracted deer blood phospholipid component, further researches the antioxidant capacity of the deer blood phospholipid, and provides a material basis for discussing the pharmacological action of deer blood with anti-aging effect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an extraction method and application of deer blood phospholipid.
Background
Deer blood refers to blood of Cervus Nippon Temminck or Cervus Nippon Temminck of Cervidae, and is divided into bore blood and antler blood. As a traditional rare traditional Chinese medicine, deer blood has medical history approaching 2000, the medical value is recorded in Qianling Fang-Dingzhi in Tang Dynasutz Simiao, and the efficacy of the deer blood is described in more detail in Ben Cao gang mu in Ming Dynasty Lizhen, so that the deer blood has the effects of greatly tonifying deficiency, promoting blood circulation, removing blood stasis, removing acne toxin, treating lumbago, palpitation, insomnia and the like. Deer blood is also always taken as a good tonic product for people in folks. Along with the deep research of modern medicine, deer blood has good nourishing effects, such as antioxidation, anti-aging, blood replenishing, beautifying, kidney tonifying, essence benefiting, fatigue resisting, immunity enhancing and the like, and is rich in nutrition. The currently common phospholipid extraction methods include organic solvent extraction, supercritical extraction, complex salt precipitation, column chromatography and the like. The organic solvent extraction method is a traditional method for extracting phospholipid, the principle is to realize separation between phospholipid and other components to be separated by utilizing different solubilities of the phospholipid and other components in different organic solvents, and the key point is that a proper organic solvent extractant is difficult to find, so that the phospholipid has good solubility and selectivity. The supercritical extraction method has the defects of higher equipment cost, high-pressure operation, less sample treatment capacity and the like, so that the supercritical extraction method is not suitable for large-scale production and has larger limitation. The key point of the complex salt precipitation method is to select an effective and proper inorganic salt precipitant. Column chromatography has the limitations of long operation time, limited sample throughput, and the fact that the operation involves some toxic organic solvents.
Disclosure of Invention
The invention uses deer blood as raw material, and uses low-toxicity methylene dichloride to replace chloroform by improving the classical lipid extraction method Folch method. And an ultrasonic synergistic technology is adopted to establish an extraction method of the phospholipid in the deer blood, and the extraction process conditions in the extraction process are determined with the aim of improving the extraction efficiency of the phospholipid in the deer blood: the influence of factors such as solvent-to-liquid ratio, extraction temperature, extraction time and the like on deer blood phospholipid extraction is evaluated by adopting a standard antioxidant evaluation method, so that theoretical basis and technical support are provided for the deep development and utilization of deer blood medicinal value.
The technical scheme adopted by the invention is as follows:
a deer blood phospholipid extraction method comprises the following steps:
step one, freeze-drying deer blood to obtain deer blood freeze-dried powder;
adding a mixed solution of dichloromethane and methanol in a volume ratio of 2:1-4:1 into deer blood freeze-dried powder, uniformly mixing, performing constant-temperature ultrasonic treatment, then performing centrifugal separation, taking supernatant, adding double distilled water, uniformly stirring, standing, collecting lower dichloromethane, and adding dichloromethane into the rest substances for secondary extraction; repeating the extraction for several times, and combining the collected dichloromethane;
and thirdly, concentrating the dichloromethane collected in the second step under reduced pressure, drying the concentrated solution to obtain mixed liposome, dissolving the mixed liposome by using acetone, fully oscillating for extraction, centrifuging, and drying the precipitate to obtain deer blood phospholipid.
In the first step, the freeze drying temperature is-80 ℃ to-81 ℃ and the vacuum degree is controlled below 20 Pa.
Further, the volume mass ratio of the mixed solution of dichloromethane and methanol to the deer blood freeze-dried powder is 35-50ml:1g.
Further, in the second step, the constant temperature is 35 ℃, and the ultrasonic time is 35-45min.
And in the second step, after secondary extraction, centrifugal separation is carried out, supernatant fluid is taken, double distilled water is added, and after uniform stirring, standing is carried out, and the lower dichloromethane layer is collected.
Further, in the second step, the rotational speed of centrifugal separation is 6000-8000r/min.
Further, in the third step, the dichloromethane collected by the combination was concentrated under reduced pressure using a rotary evaporator at 25 ℃.
Further, in the third step, the drying temperature of the concentrated solution and the precipitate is 25 ℃ and the drying is carried out at constant temperature in an oven.
The application of the deer blood phospholipid prepared by the method is that in a medicament formula for removing DPPH, when the clearance rate of the deer blood phospholipid is less than or equal to 6h, the clearance rate of the deer blood phospholipid is the highest, and when the clearance rate of the deer blood phospholipid is more than 6h, the clearance rate of the deer blood phospholipid is the highest.
Compared with the prior art, the invention has the beneficial effects that:
the invention takes freeze-dried deer blood crystal as raw material to extract phospholipid component in deer blood, the extraction method well preserves the biological activity of deer blood phospholipid, the extracted deer blood phospholipid component is analyzed in detail, the oxidation resistance is further researched, and a material basis is provided for discussing the anti-aging pharmacological action of deer blood.
Drawings
FIG. 1 is a process flow diagram of deer blood phospholipid extraction;
FIG. 2 is a graph of extraction efficiency of deer blood phospholipids at various extraction temperatures;
FIG. 3 is a graph showing the extraction efficiency of deer blood phospholipids at various extraction times;
FIG. 4 is a graph showing the extraction efficiency of deer blood phospholipids by comparing different liquid materials;
FIG. 5 is a graph showing the maximum absorbance values for a specific wavelength in the UV-visible absorption spectrum of a control phosphorus standard solution;
FIG. 6 is a phosphorus standard graph;
FIG. 7 is a high performance liquid chromatogram of deer blood phospholipids;
FIG. 8 is a graph showing the results of DPPH clearance test of deer blood phospholipid extract.
Detailed Description
The invention will now be described in further detail with reference to the drawings and to specific examples. However, the present invention is not limited thereto, and modifications and equivalents may be made thereto without departing from the spirit and scope of the present invention, and the present invention shall be covered by the scope of protection.
Example 1
The extraction method of deer blood phospholipid comprises the following specific steps:
step one, pretreatment Process-Freeze drying
Opening a refrigerator switch of a freeze dryer for precooling for 30min, precooling the freeze dryer to-80 ℃ to-81 ℃, taking out frozen sika deer blood, rapidly packing the sika deer blood into a box, covering an organic glass cylinder, pressing a vacuum machine switch for vacuumizing, starting freeze drying after the vacuum degree of the freeze dryer is below 20Pa, and controlling the vacuum degree of a freeze drying box to be below 20Pa all the time in the freeze drying process. And (3) freeze-drying the deer blood for 48 hours, and taking the deer blood out of the box to obtain the freeze-dried deer blood powder which is reddish brown, loose in texture, slightly glossy and fishy.
Step two, taking a certain amount of deer blood freeze-dried powder, putting the deer blood freeze-dried powder into a beaker, adding a certain amount of dichloromethane/methanol (volume ratio is 2:1) composite extracting solution, mixing the extracting solution at present, magnetically stirring for one minute to fully and uniformly mix the extracting solution, carrying out constant-temperature ultrasonic treatment for 35-45min, centrifuging at 8000r/min after ultrasonic treatment, taking supernatant, adding a small amount of double distilled water, stirring uniformly, and standing. Collecting lower layer dichloromethane, adding 15-20mL of dichloromethane into the rest supernatant and solid matter for secondary extraction, repeating the extraction operation twice, combining the collected dichloromethane, transferring the combined dichloromethane into a round bottom flask, concentrating under reduced pressure at 25 ℃ by using a rotary evaporator, transferring the concentrated solution into a weighed centrifuge tube, washing the round bottom flask twice by using dichloromethane, and combining the washing solution with the concentrated solution to obtain a lipid mixture. Drying the lipid mixture in a vacuum oven at a constant temperature of 25 ℃, weighing again, and subtracting the mass of the empty centrifuge tube after constant weight to obtain the weight of the lipid mixture.
Deer blood phospholipid extraction and purification and quality evaluation method
After the lipid mixture is cooled, adding 30-50mL of acetone into the lipid mixture, fully oscillating and extracting the mixture, transferring the mixture into a weighed centrifuge tube, freezing and centrifuging the mixture at a high speed of 8000r/min for 3min, removing supernatant, drying the precipitate in an oven at a constant temperature of 25 ℃, weighing the precipitate again, and subtracting the empty tube mass to obtain the deer blood phospholipid weight. Storing dried deer blood phospholipid in refrigerator at 4deg.C.
Calculating deer blood phospholipid extraction rate according to a formula (1):
wherein: w (W) 1 Is the extraction rate/%; m is m 0 Is the mass/g of deer blood freeze-dried powder; m is m 2 To extract the quality/g of deer blood phospholipids.
The relative phospholipid purity was calculated according to formula (2):
wherein: w (W) 2 Relative phospholipid purity/%; m is m 2 For extracting the mass/g of deer blood phospholipid; m is m 1 To extract the quality/g of deer blood lipid mixture.
Extraction conditions of deer blood phospholipid
The liquid-material ratio, the extraction time and the extraction temperature are selected as main influencing parameters for influencing the extraction rate of the deer blood phospholipid, the extraction rate of the deer blood phospholipid and the relative phospholipid purity are used as investigation indexes, and 5 levels of each factor are selected for single-factor experiments to investigate the influence on the extraction efficiency of the deer blood phospholipid.
Optimization of extraction conditions:
temperature influence: 5 parts of deer blood freeze-dried powder are accurately weighed, 5g of each deer blood freeze-dried powder is respectively weighed, 150mL of dichloromethane-methanol (volume ratio is 2:1) mixed extractant is added, and the mixture is respectively extracted for 40min at 20 ℃, 25 ℃, 30 ℃,35 ℃ and 40 ℃, and phospholipid is easy to oxidize and deteriorate at 50 ℃, so that temperature rise investigation is not needed. And (3) washing and drying the obtained lipid mixture by using acetone, calculating the extraction rate of deer blood phospholipid and the relative phospholipid purity, and examining the influence of ultrasonic temperature on the extraction efficiency of deer blood phospholipid. The experimental results are shown in FIG. 2.
Time effects: 5 parts of deer blood freeze-dried powder are accurately weighed, 5g of each deer blood freeze-dried powder is accurately weighed, 150mL of dichloromethane-methanol (volume ratio is 2:1) mixed extractant is added, the mixture is respectively extracted for 20min, 30min, 40min, 50min and 60min at the temperature of 30 ℃, and the lipid mixture obtained after washing and drying by acetone is used for calculating the extraction rate of deer blood phospholipid and the purity of relative phospholipid, and the influence of ultrasonic time on the extraction efficiency of deer blood phospholipid is examined. The experimental results are shown in FIG. 3.
Liquid-to-material ratio effect: 5 parts of deer blood freeze-dried powder are accurately weighed, 5g of each deer blood freeze-dried powder is respectively added with 50mL (10/1), 100mL (20/1), 150mL (30/1), 175mL (35/1), 200mL (40/1) of dichloromethane-methanol (volume ratio is 2:1) mixed extractant, ultrasonic extraction is carried out at a constant temperature of 25 ℃ for 30min, the obtained lipid mixture is washed and dried by acetone, the deer blood phospholipid extraction rate and the relative phospholipid purity are calculated, the influence of the feed liquid ratio on the deer blood phospholipid extraction efficiency is examined, and the experimental result is shown in figure 4.
Therefore, under the conditions of ensuring the extraction efficiency of the deer blood phospholipid and saving the cost and the solvent, 35mL/g is selected as the optimal liquid-to-material ratio for extracting the deer blood phospholipid, the rising amplitude of the extraction rate of the deer blood phospholipid shows a trend of being obviously increased firstly and then gradually flattened along with the extension of the time, and when the ultrasonic time exceeds 40 minutes, the ultrasonic time is prolonged continuously, and no phospholipid is dissolved out; the purity of the deer blood relative phospholipid is increased and then reduced along with the extension of the ultrasonic time, when the ultrasonic time is 40min, the purity of the deer blood relative phospholipid reaches 49.75% to the maximum, and the optimal ultrasonic time for deer blood phospholipid extraction is selected for 40 min. The result of the influence of the temperature shows that the extraction rate of the phospholipid and the extraction purity of the relative phospholipid are greatly increased from 20 ℃ to 35 ℃, the extraction rate of the phospholipid is the highest at 35 ℃, the extraction rate of the phospholipid is not greatly increased when the temperature reaches 40 ℃, the purity of the phospholipid is greatly reduced, the phospholipid is not high-temperature resistant, the phospholipid can be oxidized and deteriorated to lose the physiological activity due to the excessively high temperature, and the phospholipid is oxidized and degraded to influence the purity of the phospholipid. Therefore, the temperature of 35 ℃ is selected as the optimal extraction temperature for deer blood phospholipid extraction.
Determination of phosphorus content
Wet digestion: accurately weighing 0.50g of self-made deer blood phospholipid in a laboratory, putting the self-made deer blood phospholipid into a polytetrafluoroethylene digestion tank, adding 10mL of nitric acid and 2mL of sulfuric acid into the polytetrafluoroethylene digestion tank, and putting the mixture on an electric plate at 100 ℃ for digestion reaction. If the digestive juice is brown, adding a small amount of nitric acid to continue digestion until the digestive juice is colorless and transparent or yellowish. The digestion solution was cooled at ambient temperature and 20mL double distilled water was added to remove acid. After cooling, the mixture was transferred to a 50mL volumetric flask, the digestion vessel was washed with double distilled water multiple times, the washes were combined in the volumetric flask, water was added to the scale, and mixed well. And storing the sample to be tested and simultaneously performing a reagent blank experiment.
Determination of the absorption maximum wavelength
Accurately sucking 0mL and 3.00mL of standard phosphate use solution, respectively placing into test tubes with plugs, respectively adding 2.0mL of ammonium molybdate solution, shaking, and standing. Then adding 1mL sodium sulfite solution (200 g/L), shaking 1mL10% ascorbic acid solution, standing at room temperature for 20min for color development, correcting and zeroing by taking the first part as blank control, scanning at 500-900nm wavelength by ultraviolet-visible spectrophotometry, and displaying that the sample has ultraviolet maximum absorption at 823.5nm wavelength as shown in figure 5.
Accurately sucking 0.50mL, 1.00mL, 2.00mL, 3.00mL, 4.00mL and 5.00mL of standard phosphorus using solution, which are equivalent to phosphorus content of 5.0 mug, 10.0 mug, 20.0 mug, 30.0 mug, 40.0 mug and 50.0 mug, placing the standard phosphorus using solution into a test tube with a plug, respectively adding 2.0mL of ammonium molybdate solution into the standard phosphorus using solution to shake uniformly, standing, adding 1mL of sodium sulfite solution (200 g/L) and 1mL of 10% ascorbic acid solution to shake uniformly, standing at room temperature for 25min for color development, respectively measuring absorbance values of the solutions with different concentrations at 823.5nm of molybdenum blue characteristic absorption wavelength by using an ultraviolet-visible spectrophotometer, and drawing a standard phosphorus curve.
The absorbance values corresponding to the solutions with different concentrations of phosphorus content after the reaction are shown in the following table 1.
A phosphorus standard curve was drawn according to table 1, with the phosphorus concentration as the abscissa and the absorbance value at a specific wavelength as the ordinate, and was drawn by linear fitting. As shown in FIG. 6, the standard curve obtained after measurement has good linear relationship, and the regression equation is y=0.2251x+0.0441, R 2 ＝0.9979(n＝6)。
TABLE 2 determination of total phosphorus content in sample digestate
It can be seen from Table 2 that the phosphorus content of the extract is close to that of known phospholipids, indicating that the main component of the extract is a phospholipid.
Deer blood phospholipid component analysis:
further analysis of the extracted deer blood phospholipid fraction by high performance liquid chromatography revealed that there were mainly two phospholipids, one of which had a retention time value of 34.22 and the other of which had a phospholipid fraction of 37.40, as shown in FIG. 7.
Deer blood phospholipid anti-aging activity research
DPPH clearance test: and weighing the DPPH standard substance in a 10mL volumetric flask, dissolving the DPPH standard substance in absolute ethyl alcohol, and fixing the volume to obtain a DPPH standard solution with the concentration of 0.048mg/mL, and storing the DPPH standard solution in a dark place at a low temperature for later use. Preparing a sample solution and a blank solution respectively, fully mixing 1.0mL of an LDPPH standard solution and 1.0mL of each concentration sample with a reference substance solution, shaking uniformly, and then carrying out light-proof reaction at room temperature, wherein the reactions are respectively carried out for 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 8h and 10h, absorbance values are measured at 517nm, and the parallel measurement is carried out for 3 times, so that A1 and A0 of corresponding reaction time are respectively obtained, and the clearance rate of deer blood phospholipid to DPPH and free radicals is calculated by using the following formula:
wherein:
p-clearance of deer blood phospholipids to DPPH free radical,%;
absorbance value of A0-1.0 mL of the mixed solution of the standard solution of LDPPH and 1.0mL of absolute ethyl alcohol;
a1-absorbance value of 1.0mL of mixed solution of standard solution of LDPPH and 1.0mL of sample solution;
as shown in FIG. 8, the deer blood phospholipid extract solutions with different concentrations have certain effects on DPPH free radical scavenging. In view of the short-term effect (30 min of reaction), the effect of scavenging DPPH free radicals is strongest when the concentration of deer blood phospholipid extract is 6.00mg/mL, and the DPPH free radical scavenging rate is 45.39%. And then continuously increasing the concentration of the deer blood phospholipid extract, wherein the DPPH free radical scavenging effect is reduced. In the long-term effect, along with the extension of the action time, the clearance rate of the deer blood phospholipid of 6.00mg/mL to DPPH free radicals is slowed down along with the extension of the time, and the trend is gradually gentle; the clearance effect of deer blood phosphatide with low concentration of 2.00mg/mL and 4.00mg/mL on DPPH free radical is gradually increased along with the time extension, and when the clearance rate of 4.00mg/mL on DPPH is 8h, the clearance rate of deer blood phosphatide DPPH with low concentration of 4.00mg/mL exceeds 6.00mg/mL, and the clearance rate reaches 70.69%. It was therefore concluded that 6.00mg/mL deer blood phospholipids have the best short-acting DPPH clearance effect in the experimental reaction time and concentration range; 4.00mg/mL deer blood phospholipid has the best long-acting DPPH clearance effect. It can be seen that 4mg/mL deer blood phospholipids have the highest free radical clearance.
Claims (5)
1. The extraction method of deer blood phospholipid is characterized by comprising the following steps of:
step one, freeze-drying deer blood to obtain deer blood freeze-dried powder;
adding a mixed solution of dichloromethane and methanol in a volume ratio of 2:1-4:1 into deer blood freeze-dried powder, uniformly mixing, performing constant-temperature ultrasonic treatment, then performing centrifugal separation, taking supernatant, adding double distilled water, uniformly stirring, standing, collecting lower dichloromethane, and adding dichloromethane into the rest substances for secondary extraction; repeating the extraction for several times, and combining the collected dichloromethane;
concentrating the dichloromethane collected in the second step under reduced pressure, drying the concentrated solution to obtain mixed liposome, dissolving the mixed liposome by using acetone, fully oscillating for extraction, centrifuging, and drying the precipitate to obtain deer blood phospholipid;
in the first step, the freeze drying temperature is-80 ℃ to-81 ℃ and the vacuum degree is controlled below 20 Pa;
in the second step, the volume mass ratio of the mixed solution of dichloromethane and methanol to the deer blood freeze-dried powder is 35-50ml:1g; the constant temperature is 35 ℃, and the ultrasonic time is 35-45min.
2. The extraction method of deer blood phospholipid according to claim 1, wherein the extraction method comprises the following steps: and step two, after secondary extraction, centrifugally separating, taking supernatant, adding double distilled water, uniformly stirring, standing, and collecting the lower dichloromethane layer.
3. The extraction method of deer blood phospholipid according to claim 2, wherein the method comprises the following steps: in the second step, the rotation speed of centrifugal separation is 6000-8000r/min.
4. The extraction method of deer blood phospholipid according to claim 1, wherein the extraction method comprises the following steps: in step three, the resultant methylene chloride was concentrated under reduced pressure using a rotary evaporator at 25 ℃.
5. The method for extracting deer blood phospholipid according to claim 4, wherein the steps of: in the third step, the drying temperature of the concentrated solution and the precipitate is 25 ℃ constant temperature drying.
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