Preparation method and application of sheep brain cephalin and lecithin

Abstract

The invention discloses a preparation method and application of sheep brain cephalin and lecithin. The method comprises the following steps: washing a fresh sheep brain to remove blood stains and impurities from the surface, freeze-drying the brain, crushing the freeze-dried sheep brain, sieving the brain, adding an organic solvent for extraction by stirring, combining the extracts, filtering the brain, and naturally air-drying the precipitate until the organic solvent is completely volatilized; concentrating the filtrate, adding anhydrous ethanol for reflux, filtering the filtrate, allowing the filtrate to stand overnight until stratification, filtering the filtrate, concentrating the filtrate in a vacuum or freeze-drying process to obtain lecithin; adding acetone for cholesterol removal to obtain pure lecithin; adding calcium chloride to the precipitate, stirring and extracting the precipitate, and centrifuging the precipitate to obtain a crude cephalin extract; extracting the crude extract by stirring with anhydrous ethanol, discarding the supernatant, dissolving the precipitate with ethanol, filtering the precipitate, and vacuum-drying or freeze-drying the precipitate to obtain a refined cephalin product. The method is simple, low-cost, efficient, and has a short extraction time and simple operation. The method provides raw materials and technical support for the development of drugs, preparation of health products, or application in the field of cosmetics based on active compounds such as lecithin and cephalin.

Description

Technical Field

[0001] The present invention relates to a preparation method of sheep brain cephalin and lecithin and application thereof. Background Art

[0002] Sheep brain is the brain marrow of goats or sheep, members of the bovine family. According to the Compendium of Materia Medica, sheep brain is sweet, warm, slightly toxic, and enters the heart, liver, and kidney meridians. It has the effects of tonifying the brain, moisturizing the skin, and removing wrinkles. In Traditional Chinese Medicine, it is used to treat dizziness due to physical weakness, wind-cold in the brain, persistent headaches, chapped skin, and muscle and bone fractures. Sheep brain is rich in ascorbic acid, niacin, riboflavin, lecithin, thiamin, cerebrosides, protein, and fat, as well as calcium, phosphorus, and iron. Phospholipids in sheep brain are not only important components of human biological membranes but also serve as a source of choline and essential fatty acids, and are involved in human metabolism. Therefore, developing the potential pharmaceutical value of these sheep byproducts holds profound significance.

[0003] Cephalin and lecithin are widely used in the food, pharmaceutical, and cosmetic industries. Cephalin is a natural surfactant with unique biological activities and physiological functions. It is non-toxic, non-polluting, non-irritating, and readily biodegradable, earning it high attention from both domestic and international scientific and industrial communities. Cephalin is also a novel rubber vulcanization accelerator and / or antioxidant. Demand for it is increasing annually, but my country currently relies on imports for cephalin products with a purity exceeding 90%. Therefore, developing a low-cost, high-purity cephalin preparation method suitable for my country's national conditions would be of significant economic and social value. The completion of this experiment has opened up a viable avenue for the comprehensive utilization of mammalian brain tissue.

[0004] From the perspective of resource development and utilization in Xinjiang, despite its vast territory, large-scale livestock farming, and abundant livestock resources, sheep parts such as the brain are only used for food production and processing, with some used as feed, leading to a lack of scientific and rational utilization, resulting in the loss of these abundant resources. Currently, there are limited literature reports and academic papers on sheep brain phospholipids, and research on sheep brain is in its early stages, lacking a theoretical basis for its development. Xinjiang's sheep livestock contain a vast amount of untapped raw materials, and the active ingredients extracted from these raw materials could fill the gap in the development of pharmaceuticals from livestock resources. If these untapped raw materials can be extracted and fully developed to produce active substances beneficial to human health, this could save these wasted, low-cost resources, significantly improve economic efficiency, fully utilize Xinjiang's livestock resources, provide theoretical guidance for sheep brain research, and lay the foundation for the development of new drugs using sheep brain.

[0005] If the production process is stable, it can be further promoted throughout Xinjiang. We can purchase crude products from all over the country, and then further process them into fine products for sale. We can also cooperate with pharmaceutical companies to sell them or export them to earn foreign exchange. This will not only generate good economic benefits, but also have an important impact on the rational use of animal husbandry resources and the improvement of Xinjiang's social and economic structure.

[0006] Supplementary information on domestic and foreign patent applications: Using cephalin and lecithin as keywords, a search yielded three documents on obtaining cephalin and lecithin from natural raw materials, including two on extraction methods and separation and purification methods.

[0007] Patent application number 200310111541.3 uses soybean oil as a raw material and uses high performance liquid chromatography to extract cephalin and lecithin. Patent application number 200510060501.X uses egg yolk as a raw material. This patent uses acetone as a degreasing phase to obtain crude lecithin. Alumina chromatography columns are used for separation, followed by thin layer chromatography (TLC) analysis, and silica gel column purification to obtain cephalin and lecithin. Patent application number 200410067744.1 uses egg yolk as a raw material. This patent uses acetone to degrease crude lecithin, extracts it with 95% ethanol, and purifies it with a mixed bed ion exchange resin to obtain cephalin and lecithin of relatively high purity.

[0008] Patent application No. 92107768.8 uses human fetus as raw material to separate and purify cephalin and lecithin; anhydrous ethanol precipitation method is used to obtain cephalin and lecithin by layering; then acetone precipitation is used to obtain lecithin; cephalin is refined using 0.5% calcium chloride, heated to 100°C, the precipitate is taken, concentrated, and then precipitated with three times the amount of anhydrous ethanol, at 4°C, for 4 hours, filtered, and the precipitate is again dissolved in ether, at 4°C, allowed to stand for 12 hours, filtered, and the filtrate is precipitated with an equal amount of acetone to obtain high-purity cephalin; the obtained cephalin and lecithin are used in atomized cosmetics, which are beneficial for nourishing skin cells and having anti-wrinkle and sun protection effects.

[0009] Patent application number 92107768.8 uses anhydrous ethanol precipitation method, but the repeated use of different organic solvents in the purification part of this patent will affect the activity of the product.

[0010] Patents with application numbers 200310111541.3, 200510060501.X, 200410067744.1 and 201510363668.7 use soybeans, egg yolks and fish as the main medicinal materials. The separation and purification process uses high-performance liquid chromatography, silica gel columns and ion exchange resins to obtain cephalin and lecithin. These methods have the disadvantages of high cost, long extraction time, complex operation, difficulty in expanding the preparation method, and high pollution.

[0011] Regarding the extraction and application of cephalin, the keyword cephalin was used to search for 2 documents related to obtaining cephalin from natural raw materials, including 2 articles on extraction methods and separation and purification methods, and 1 article on application;

[0012] Patent application number 201510363668.7 uses fish brain as raw material, uses papain enzymatic hydrolysis, and finally uses silica gel column chromatography to obtain cephalin. This extract has high immunomodulatory activity.

[0013] Regarding the extraction and application of lecithin, 20 documents on obtaining lecithin from natural raw materials were retrieved by searching with the keyword cephalin, including 5 articles on extraction methods and separation and purification methods; Patent application number 201611027169.1, using egg yolk as raw material, used supercritical carbon dioxide extraction method to extract lecithin with a purity of 98%; Patent application number 201410068133.2, using cottonseed oil soap stock as raw material, using petroleum ether, TBHQ, V2 and CA and other organic solvents with a certain ratio For example, lecithin was extracted by stirring, and ZnCl2 was used in the enrichment stage; Patent application number 201911392312.0, using egg yolk as raw material, lecithin was extracted by supercritical carbon dioxide fluid method; Patent application number 201810705984.1, using camellia oil cake as raw material, lecithin was extracted by silica gel column chromatography separation and purification; Patent application number 201510077403.0, using peanuts as raw material, lecithin was obtained by enzymatic hydrolysis and two supercritical extractions, with a purity of more than 96%;

[0014] The above cephalin and lecithin extraction methods are different from the raw materials and extraction methods of the present invention. The present invention is a new and simplest extraction and separation method. Summary of the Invention

[0015] The present invention aims to provide a method for preparing cephalin and lecithin from sheep brain and its application. The method comprises the following steps: washing fresh sheep brain with tap water to remove surface blood stains and impurities, freeze-drying, crushing the freeze-dried sheep brain, passing it through a 100-mesh sieve, adding an organic solvent for extraction with stirring, combining the extracts, filtering, and air-drying the precipitate until the organic solvent is completely volatilized. The filtrate is concentrated, refluxed with anhydrous ethanol, filtered, and the filtrate is allowed to stand overnight until layers are separated. The filtrate is filtered and concentrated in a vacuum or freeze-dried to obtain lecithin. Acetone is then added to remove cholesterol to obtain pure lecithin with a yield of 2.65-4.38% and a purity greater than 93.36%. Calcium chloride is added to the precipitate, extracted with stirring, and centrifuged to obtain a precipitate, i.e., a crude cephalin extract. The crude extract is then extracted with anhydrous ethanol with stirring, the supernatant is discarded, the precipitate is dissolved in 95% ethanol, filtered to obtain a precipitate, and vacuum or freeze-dried to obtain a refined cephalin product with an extraction yield of 0.57-0.77% and a purity greater than 95.28%. The present invention fully utilizes Xinjiang's unique livestock resources to produce cephalin and lecithin, achieving high-value-added utilization of livestock resources. The method is simple, low-cost, and efficient, with a short extraction time and simple operation. It provides raw materials and technical support for the development of active compounds such as lecithin and cephalin in pharmaceuticals, health products, or cosmetics.

[0016] The method for preparing sheep brain cephalin and lecithin of the present invention is carried out according to the following steps:

[0017] a. Take fresh sheep brain, wash with tap water to remove blood and impurities on the surface, freeze at -80 ℃ for 24h, then freeze-dry at a pressure of 9.5Pa and a temperature of -80 ℃ for 48h, and then lyophilize the freeze-dried sheep brain by a grinder for 30s, sieve through a 40-100 mesh sieve to obtain sheep brain powder;

[0018] b. The sheep brain powder obtained in step a was added with an organic solvent of ether, chloroform or petroleum ether in a solid-liquid ratio of 1:5-1:30, or a volume ratio of 2:1 chloroform: methanol and stirred and extracted three times, each 1-4h, and the extracts were collected and set aside;

[0019] c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, the residue was discarded, and the filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers were separated;

[0020] d. The supernatant obtained in step c was collected and the precipitate was stored for later use. The supernatant was then filtered and the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin.

[0021] e The crude lecithin obtained in step d was added 3-6 times the volume of acetone, shaken and mixed for 20-30s, placed in a temperature of 4 ° C and allowed to settle for 12h, filtered, the supernatant was discarded, and the precipitate was freeze-dried in vacuo to obtain lecithin;

[0022] f. The solid-liquid ratio was 3-8:1, and the precipitate obtained in step d was added with 0.1% -1% calcium chloride, heated to a temperature of 100 ℃, refluxed for 10min, and centrifuged at 1000-5000rpm for 10min to obtain a precipitate, ie, a crude phospholipid extract;

[0023] g. The crude phospholipid extract obtained in step f was then shaken and mixed with anhydrous ethanol for 20-30s, allowed to stand at a temperature of 4 ° C for 4h, repeated 3 times, the supernatant was discarded, the precipitate was precipitated, and then shaken and mixed with 95% ethanol for 20-30s, allowed to stand at a temperature of 4 ° C for 4h, centrifuged at 1000-5000rpm for 10min, the supernatant was discarded, and the precipitate was vacuum-dried or freeze-dried to obtain a refined phospholipid product.

[0024] The cephalin and lecithin obtained by the method are used in the preparation of antioxidant drugs.

[0025] The cephalin and lecithin obtained by the method are used in preparing food or cosmetics.

[0026] The chemical properties of the extracted cephalin and lecithin were detected by infrared characteristic functional group detection and GC-MS.

[0027] The present invention discloses a method for preparing cephalin and lecithin from sheep brain, which extracts and prepares cephalin and lecithin from sheep brain in a short period of time. Currently, there are no reports in the literature on the simultaneous extraction and separation of cephalin and lecithin from sheep brain. The present invention utilizes traditional extraction and separation methods to efficiently and thoroughly remove fat and cholesterol from the sheep brain, thereby improving the drying and extraction efficiency and purity of cephalin and lecithin from the sheep brain, shortening the extraction time, improving production efficiency, and being suitable for industrial production. The method also achieves the purpose of simultaneously separating and obtaining high-purity cephalin and lecithin with different solubility from the sheep brain. Calcium chloride is systematically utilized in the purification of cephalin, achieving the simplest and most cost-effective purification effect. Each step in the extraction process of the present invention is performed at room temperature, using conventional reagents and equipment, which can minimize the cost of the extraction process. The resulting product is free of any harmful solvent residues, and is safe, environmentally friendly, and highly efficient.

[0028] The present invention utilizes sheep brain freeze-dried powder, filling this gap in relevant research and technical fields, and the product has high purity, low cost, less solvent used, simple operation, short extraction time, high repeatability, and the preparation method has the characteristics of easy scale-up, high yield, no pollution, simple extraction conditions, low raw material cost, and rich medicinal resources. The invention provides technical support for fully utilizing sheep brain resources, avoids the waste of Xinjiang's rich sheep brain resources, provides high-quality resources and technical guarantees for fully utilizing the rich and cheap resources in Xinjiang's animal husbandry areas and improving the research and development of products with added value and technological content, and lays a foundation for developing new sheep brain drugs.

[0029] The present invention discloses a method for preparing cephalin and cerebroprotein from sheep brain and its application. The method first involves washing fresh sheep brain to remove surface impurities and blood, and then using a low-temperature vacuum freeze-drying technique to obtain crude brain dry powder with a moisture content of 77.75% (Table 1). The crude sheep brain is then defatted by stirring with an organic solvent to obtain phospholipids and defatted sheep brain powder with yields of 5.15% and 16.51%, respectively (Table 1). Three times the volume of 75-100% ethanol is added to the phospholipid component, the mixture is refluxed for 30 minutes, filtered, concentrated in a vacuum or freeze-dried state to obtain crude lecithin, and then decholesterolized with acetone to obtain a pure lecithin yield of 4.38% (Table 1).

[0030] The precipitate was added with calcium chloride, heated and extracted at 100°C, and centrifuged at 5000 rpm for 10 minutes to obtain a crude phospholipid extract. The crude extract was then extracted with anhydrous ethanol 3-4 times with stirring, filtered to obtain a precipitate, and vacuum-dried or freeze-dried to obtain a high-purity refined phospholipid product with a yield of 0.77% (Table 1).

[0031] Table 1 The proportion of each component of sheep brain

[0032]

[0033] The results of GC-MS quantitative analysis of cephalin fatty acids showed that ( Figure 1 The fatty acids of sheep brain phospholipids are mainly composed of 5.85% C16:0 (palmitic acid), 30.45% C18:0 (stearic acid), 26.05% C18:1n9c (oleic acid) (Table 3), and the contents of ARA (eicosapentaenoic acid) and DHA (eicosahexaenoic acid) are 5.01% and 4.32% respectively; the contents of EPA (docosapentaenoic acid) and DPA (docosahexaenoic acid) are 3.4% and 1.64% respectively ( Figure 2 ); The results of GC-MS quantitative analysis of lecithin fatty acids showed that ( Figure 3 ), the fatty acids of sheep brain lecithin are mainly composed of C16:0 (palmitic acid), C18:0 (stearic acid), and C18:1n9c (oleic acid), with contents of 9.73%, 18.59% and 18.47% respectively (Table 4);

[0034] Table 3 is the main fatty acid composition of sheep brain phospholipids of the present invention

[0035]

[0036] Table 4 is the main fatty acid composition of sheep brain lecithin of the present invention

[0037]

[0038] The contents of DHA (eicosahexaenoic acid), ARA (eicosapentaenoic acid), EPA (docosapentaenoic acid) and DPA (docosahexaenoic acid) were 7.65%, 5.88%, 1.96% and 1.02% respectively. Figure 4 ); the proportions of unsaturated fatty acids and saturated fatty acids in cephalin are 52.27% and 47.73% respectively ( Figure 5 ); the proportions of unsaturated fatty acids and saturated fatty acids in lecithin are 61.12% and 29.41% respectively ( Figure 5 ). Infrared spectrum analysis shows that cephalin vibrates at a frequency of 3015.21 cm -1 、2920.94cm -1 、1467.01cm -1 and 1066.45cm -1 A characteristic peak appeared at Figure 6 ); lecithin at a vibration frequency of 3010.17cm -1 、2920.60cm -1 、1232.61cm -1 、1056.13cm -1 、969.89cm -1 and 1741.23cm -1 A characteristic peak appeared at Figure 7 ).

[0039] The present invention differs from the results obtained in the master's thesis "Study on the Extraction and Activity of Phospholipids from Carp Brain" which reported that EPA and DHA accounted for 23.5% of the fatty acid composition of carp brain, while soybean lecithin and egg yolk lecithin did not contain these components (Table 2).

[0040] Table 2 shows the main fatty acid composition (%) of sheep brain phospholipids of the present invention:

[0041]

[0042] In addition to unsaturated fatty acids such as EPA and DHA, the cephalin obtained by the present invention also contains multiple unsaturated fatty acids such as ARA and DPA, accounting for 14.37% of the total fatty acids (Table 2); EPA, DHA, ARA and DPA in the lecithin obtained by the present invention account for 16.51% of the total fatty acids (Table 2), thus having richer nutritional value; carp brain fatty acids, soybean lecithin and egg yolk lecithin contain linoleic acid, while the cephalin and lecithin obtained by the present invention do not contain linoleic acid;

[0043] The document "Analysis of Phospholipid and Phospholipid Fatty Acid Composition in Sheep, Pig and Chicken Brains" reports that chicken brain has 43.35% saturated fatty acids and 56.65% unsaturated fatty acids; chicken brain has 32.22% saturated fatty acids and 67.78% unsaturated fatty acids; pig brain has 41.16% saturated fatty acids and 58.64% unsaturated fatty acids; the product cephalin obtained from sheep brain in the present invention has 47.73% saturated fatty acids and 52.27% unsaturated fatty acids; lecithin has 29.41% saturated fatty acids and 61.12% unsaturated fatty acids ( Figure 5 ). BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 The chromatogram of quantification of cephalin using GC-MS in the present invention;

[0045] Figure 2 This is a diagram showing the main fatty acid composition of sheep brain cephalin of the present invention;

[0046] Figure 3 The chromatogram of lecithin quantitatively measured using GC-MS in the present invention;

[0047] Figure 4 This is the main fatty acid composition diagram of the sheep brain lecithin of the present invention;

[0048] Figure 5 The saturation of sheep brain cephalin and lecithin of the present invention;

[0049] Figure 6 The present invention uses infrared spectroscopy to analyze the chromatogram of cephalin;

[0050] Figure 7 The chromatogram of lecithin was analyzed using infrared spectroscopy for the present invention. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below with reference to examples and drawings. However, the uses and purposes of these exemplary embodiments are only used to illustrate the present invention and do not constitute any form of limitation on the actual protection scope of the present invention, nor do they limit the protection scope of the present invention to them.

[0052] Example 1

[0053] a. Take fresh sheep brain, wash with tap water to remove blood and impurities on the surface, freeze at -80 ℃ for 24h, then freeze-dry at a pressure of 9.5Pa and a temperature of -80 ℃ for 48h, and then lyophilize the freeze-dried sheep brain by a grinder for 30s, pass through a 40-mesh sieve to obtain sheep brain powder;

[0054] b. The sheep brain powder obtained in step a was added at a solid-liquid ratio of 1:30 with an organic solvent, petroleum ether, and stirred and extracted three times, each for 4 hours, and the extracts were collected and combined for later use;

[0055] c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, and the residue was discarded. The filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers were separated;

[0056] d. The supernatant obtained in step c was collected and the precipitate was stored for later use. The supernatant was then filtered and the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin.

[0057] e. The crude lecithin obtained in step d was added 6 times the volume of acetone, shaken and mixed for 20s, allowed to settle at a temperature of 4 ° C for 12h, filtered, the supernatant was discarded, and the precipitate was freeze-dried to obtain lecithin, the extraction rate was 4.38%, and the purity was 93.36%;

[0058] f. The solid-liquid ratio was 5:1, and the precipitate obtained in step d was added with 0.5% calcium chloride, heated to a temperature of 100 ℃, refluxed for 10min, and centrifuged at 5000rpm for 10min to obtain a precipitate, ie, a crude phospholipid extract;

[0059] g. The crude phospholipid extract obtained in step f was then mixed with anhydrous ethanol for 30s, allowed to stand at 4 ° C for 4h, repeated 3 times, the supernatant was discarded, the precipitate was precipitated, and then mixed with 95% ethanol for 30s, allowed to stand at 4 ° C for 4h, centrifuged at 5000rpm for 10min, the supernatant was discarded, and the precipitate was vacuum dried to obtain a refined phospholipid product with an extraction rate of 0.77% and a purity of 95.28%;

[0060] The lecithin obtained in step d was vacuum dried and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0061] The cephalin obtained in step g was vacuum dried, and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0062] The lecithin, cholesterol and cephalin obtained in steps d and g are subjected to infrared chromatography to detect the absorbance of each phosphate group.

[0063] Example 2

[0064] a. Take fresh sheep brain, wash with tap water to remove blood and impurities on the surface, freeze at -80 ℃ for 24h, then freeze-dry at a pressure of 9.5Pa and a temperature of -80 ℃ for 48h, and then lyophilize the freeze-dried sheep brain by a grinder for 30s, pass through a 60-mesh sieve to obtain sheep brain powder;

[0065] b. The sheep brain powder obtained in step a was added with an organic solvent, ether, at a solid-liquid ratio of 1:15 and stirred and extracted three times for 2 hours each. The extracts were collected and combined for later use.

[0066] c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, and the residue was discarded. The filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers were separated;

[0067] d. The supernatant obtained in step c was collected and the precipitate was stored for later use. The supernatant was then filtered and the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin.

[0068] e. The crude lecithin obtained in step d was added 6 times the volume of acetone, shaken and mixed for 20s, allowed to settle at a temperature of 4 ° C for 12h, filtered, the supernatant was discarded, and the precipitate was freeze-dried to obtain lecithin, the extraction rate was 3.97%, and the purity was 82.37%;

[0069] f. The solid-liquid ratio was 4:1, and the precipitate obtained in step d was added with 0.4% calcium chloride, heated to a temperature of 100 ℃, refluxed for 10min, and centrifuged at 5000rpm for 10min to obtain a precipitate, ie, a crude phospholipid extract;

[0070] g. The crude phospholipid extract obtained in step f was then mixed with anhydrous ethanol for 20s, allowed to stand at 4 ° C for 4h, repeated 3 times, the supernatant was discarded, the precipitate was precipitated, and then mixed with 95% ethanol for 20s, allowed to stand at 4 ° C for 4h, centrifuged at 5000rpm for 10min, the supernatant was discarded, and the precipitate was freeze-dried to obtain a refined phospholipid product with an extraction rate of 0.64% and a purity of 86.31%.

[0071] The lecithin obtained in step d was vacuum dried and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0072] The cephalin obtained in step g was vacuum dried, and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0073] The lecithin, cholesterol and cephalin obtained in steps d and g are subjected to infrared chromatography to detect the absorbance of each phosphate group.

[0074] Example 3

[0075] a. Take fresh sheep brain, wash with tap water to remove blood and impurities on the surface, freeze at -80 ℃ for 24h, then freeze-dry at a pressure of 9.5Pa and a temperature of -80 ℃ for 48h, and then lyophilize the freeze-dried sheep brain by a grinder for 30s, pass through an 80-mesh sieve to obtain sheep brain powder;

[0076] b. The sheep brain powder obtained in step a was added with an organic solvent, ether, at a solid-liquid ratio of 1:5 and stirred and extracted three times, each for 1 h, and the extracts were combined and collected for later use;

[0077] c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, and the residue was discarded. The filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers were separated;

[0078] d. The supernatant obtained in step c was collected and the precipitate was stored for later use. The supernatant was then filtered and the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin.

[0079] e. The crude lecithin obtained in step d was added 3-6 times the volume of acetone, shaken and mixed for 30s, placed in a temperature of 4 ° C and allowed to settle for 12h, filtered, the supernatant was discarded, and the precipitate was freeze-dried in vacuo to obtain lecithin, the extraction rate was 3.69%, and the purity was 87.12%;

[0080] f. The solid-liquid ratio was 3:1, and the precipitate obtained in step d was added with 0.3% calcium chloride, heated to a temperature of 100 ℃, refluxed for 10min, and centrifuged at 4000rpm for 10min to obtain a precipitate, ie, a crude phospholipid extract;

[0081] g. The crude phospholipid extract obtained in step f was then mixed with anhydrous ethanol for 30s, allowed to stand at 4 ° C for 4h, repeated 3 times, the supernatant was discarded, the precipitate was then mixed with 95% ethanol for 30s, allowed to stand at 4 ° C for 4h, centrifuged at 5000 rpm for 10min, the supernatant was discarded, and the precipitate was vacuum-dried or freeze-dried to obtain a refined phospholipid product with an extraction rate of 0.67% and a purity of 84.72%.

[0082] The lecithin obtained in step d was vacuum dried and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0083] The cephalin obtained in step g was vacuum dried, and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0084] The lecithin, cholesterol and cephalin obtained in steps d and g are subjected to infrared chromatography to detect the absorbance of each phosphate group.

[0085] Example 4

[0086] a. Take fresh sheep brain, wash with tap water to remove surface blood and impurities, freeze at -80 ℃ for 24h, then freeze-dry at a pressure of 9.5Pa and a temperature of -80 ℃ for 48h, and then lyophilize the freeze-dried sheep brain by a grinder for 30s, pass through a 100-mesh sieve to obtain sheep brain powder;

[0087] b. The sheep brain powder obtained in step a was added with an organic solvent, ether, at a solid-liquid ratio of 1:10 and stirred and extracted three times, each for 2 hours, and the extracts were collected and set aside;

[0088] c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, and the residue was discarded. The filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers separated;

[0089] d. The supernatant obtained in step c was collected and the precipitate was stored for later use. The supernatant was then filtered and the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin.

[0090] e. The crude lecithin obtained in step d was added 3 times the volume of acetone, shaken and mixed for 25s, allowed to settle at a temperature of 4 ° C for 12h, filtered, the supernatant was discarded, and the precipitate was freeze-dried to obtain lecithin, the extraction rate was 4.03%, and the purity was 89.41%;

[0091] f. The solid-liquid ratio was 6:1, and the precipitate obtained in step d was added with 0.6% calcium chloride, heated to a temperature of 100 ℃, refluxed for 10min, and centrifuged at 5000rpm for 10min to obtain a precipitate, ie, a crude phospholipid extract;

[0092] g. The crude phospholipid extract obtained in step f was then mixed with anhydrous ethanol by shaking for 25s, allowed to stand at a temperature of 4 ° C for 4h, repeated three times, the supernatant was discarded, the precipitate was precipitated, and then mixed with 95% ethanol by shaking for 25s, allowed to stand at a temperature of 4 ° C for 4h, centrifuged at 5000rpm for 10min, the supernatant was discarded, and the precipitate was vacuum-dried or freeze-dried to obtain a refined phospholipid product with an extraction rate of 0.59% and a purity of 87.19%.

[0093] The lecithin obtained in step d was vacuum dried, and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument.

[0094] The cephalin obtained in step g was vacuum dried and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument.

[0095] The lecithin, cholesterol and cephalin obtained in steps d and g are subjected to infrared chromatography to detect the absorbance of each phosphate group.

[0096] Example 5

[0097] a. Take fresh sheep brain, wash with tap water to remove blood and impurities on the surface, freeze at -80 ℃ for 24h, then freeze-dry at a pressure of 9.5Pa and a temperature of -80 ℃ for 48h, and then lyophilize the freeze-dried sheep brain by a grinder for 30s, sieve through a 40-100 mesh sieve to obtain sheep brain powder;

[0098] b. The sheep brain powder obtained in step a was added at a solid-liquid ratio of 1:5-1:30 in a volume ratio of 2:1 chloroform: methanol and stirred and extracted three times, each for 3h, and the extracts were combined and collected for later use;

[0099] c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, the residue was discarded, and the filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers were separated;

[0100] d. The supernatant obtained in step c was collected and the precipitate was stored for later use. The supernatant was then filtered and the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin.

[0101] e. The crude lecithin obtained in step d was added 4 times the volume of acetone, shaken and mixed for 20s, allowed to settle at a temperature of 4 ° C for 12h, filtered, the supernatant was discarded, and the precipitate was freeze-dried in vacuo to obtain lecithin, an extraction rate of 3.86%, a purity of 83.21%;

[0102] f. The solid-liquid ratio was 7:1, and the precipitate obtained in step d was added with 0.7% calcium chloride, heated to a temperature of 100 ℃, refluxed for 10min, and centrifuged at 2000rpm for 10min to obtain a precipitate, ie, a crude phospholipid extract;

[0103] g. The crude phospholipid extract obtained in step f was then mixed with anhydrous ethanol by shaking for 20s, allowed to stand at a temperature of 4 ° C for 4h, repeated three times, the supernatant was discarded, the precipitate was precipitated, and then mixed with 95% ethanol by shaking for 20s, allowed to stand at a temperature of 4 ° C for 4h, centrifuged at 2000 rpm for 10min, the supernatant was discarded, and the precipitate was vacuum-dried or freeze-dried to obtain a refined phospholipid product with an extraction rate of 0.61% and a purity of 79.38%;

[0104] The lecithin obtained in step d was vacuum dried and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0105] The cephalin obtained in step g was vacuum dried, and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0106] The lecithin, cholesterol and cephalin obtained in steps d and g are subjected to infrared chromatography to detect the absorbance of each phosphate group.

[0107] Example 6

[0108] a. Take fresh sheep brain, wash with tap water to remove blood and impurities on the surface, freeze at -80 ℃ for 24h, then freeze-dry at a pressure of 9.5Pa and a temperature of -80 ℃ for 48h, and then lyophilize the freeze-dried sheep brain by a grinder for 30s, pass through a 40-mesh sieve to obtain sheep brain powder;

[0109] b. The sheep brain powder obtained in step a was added at a solid-liquid ratio of 1:20 with an organic solvent volume ratio of 2:1 of chloroform: methanol and stirred and extracted three times, each for 4 hours, and the combined extracts were collected and set aside;

[0110] c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, the residue was discarded, and the filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers were separated;

[0111] d. The supernatant obtained in step c was collected and the precipitate was stored for later use. The supernatant was then filtered and the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin.

[0112] e. The crude lecithin obtained in step d was added 5 times the volume of acetone, shaken and mixed for 30s, allowed to settle at a temperature of 4 ° C for 12h, filtered, the supernatant was discarded, and the precipitate was freeze-dried in vacuo to obtain lecithin, the extraction rate was 3.43%, and the purity was 79.64%;

[0113] f. The solid-liquid ratio was 8:1, and the precipitate obtained in step d was added with 1% calcium chloride, heated to a temperature of 100 ℃, refluxed for 10min, and centrifuged at 1000rpm for 10min to obtain a precipitate, ie, a crude phospholipid extract;

[0114] g. The crude phospholipid extract obtained in step f was then mixed with anhydrous ethanol for 30s, allowed to stand at 4 ° C for 4h, repeated 3 times, the supernatant was discarded, the precipitate was precipitated, and then mixed with 95% ethanol for 30s, allowed to stand at 4 ° C for 4h, centrifuged at 1000 rpm for 10min, the supernatant was discarded, and the precipitate was vacuum-dried or freeze-dried to obtain a refined phospholipid product with an extraction rate of 0.53% and a purity of 80.27%;

[0115] The lecithin obtained in step d was vacuum dried and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0116] The cephalin obtained in step g was vacuum dried, and different fatty acid components were detected using a gas chromatography-mass spectrometry (GC / MS) instrument;

[0117] The lecithin, cholesterol and cephalin obtained in steps d and g are subjected to infrared chromatography to detect the absorbance of each phosphate group.

[0118] Example 7

[0119] Gas chromatography-mass spectrometry (GC-MS) instrument detection:

[0120] The methylation of lecithin and cephalin is processed: take 0.1g lecithin and cephalin, be placed in a 10mL measuring flask, add a mixed solvent 2mL of petroleum ether and toluene, shake gently for 2min, dissolve the grease, then add 0.4mol / L potassium hydroxide-methanol solution 3mL, leave standstill 10-15min under room temperature, after the solution is clarified, add distil water and be settled to 10ml, rotate measuring flask, supernatant adds absolute ethanol 500ul and makes it clarify rapidly, get supernatant 600ul, 0.22um filter head filters, and GC-MS detects.

[0121] Example 8

[0122] UV and FT-IR analysis:

[0123] Cephalin and lecithin samples were dissolved in 0.1 mg / mL aqueous solution, and UV absorption spectra were collected in the wavelength range of 190 nm to 400 nm. A small amount of dry sample was taken and its infrared absorption spectrum was measured by KBr tablet method, and the measurement range was: 4000-500 cm-1.

[0124] Example 9

[0125] Phospholipid content determination:

[0126] 1. Draw a standard curve:

[0127] Take 6 colorimetric tubes and number them 0, 1, 2, 4, 6, and 8, and fill them with 0 mL, 1 mL, 2 mL, 4 mL, 6 mL, and 8 mL of standard solution in order, then add 10 mL, 9 mL, 8 mL, 6 mL, 4 mL, and 2 mL of water in order, then add 8 mL of hydrazine sulfate solution and 2 mL of sodium molybdate solution to each of the 6 colorimetric tubes, add stoppers, shake 3-4 times, remove the stoppers, place the colorimetric tubes in a boiling water bath and heat for 10 min, take them out, and cool to room temperature; dilute with water to the scale, shake well, and let stand for 10 min; pipette the solution into a dry, clean cuvette, adjust the zero point with a reagent blank on a spectrophotometer at 650 nm, and measure the absorbance respectively; draw a standard curve with absorbance as the ordinate and phosphorus content (0.01 mg, 0.02 mg, 0.04 mg, 0.06 mg, 0.08 mg) as the abscissa;

[0128] 2. Prepare test solution:

[0129] According to the phospholipid content of the sample, the prepared sample is weighed in a crucible, with 10 g of finished oil sample and 3.0 g-3.2 g (accurate to 0.001 g) of crude oil and degummed oil; 0.5 g of zinc oxide is added and slowly heated on an electric furnace until the sample becomes thick, then gradually heated until it is completely carbonized. The crucible is then placed in a muffle furnace at 550°C-600°C and burned until it is completely ash-colored (white) for about 2 hours; the crucible is removed and cooled to room temperature, the ash is dissolved with 10 mL of hydrochloric acid solution and heated to a slight boil, heating is stopped after 5 minutes, and the temperature of the solution is allowed to cool to room temperature. The solution is filtered and poured into a 100 mL volumetric flask, and the crucible and filter paper are rinsed 3-4 times with approximately 5 mL of hot water each time. After the filtrate is cooled to room temperature, it is neutralized with potassium hydroxide solution until turbidity appears, hydrochloric acid solution is slowly added dropwise to completely dissolve the zinc oxide precipitate, and then 2 drops are added. Finally, the volume is diluted with water to the mark and shaken well; a sample blank is also prepared when preparing the test solution.

[0130] 3. Colorimetry:

[0131] Use a pipette to draw 10 mL of the test solution into a 50 mL colorimetric tube. Add 8 mL of hydrazine sulfate solution and 2 mL of sodium molybdate solution. Stopper the tube and shake 3-4 times. Remove the stopper and heat the tube in a boiling water bath for 10 minutes. Remove the tube and cool it to room temperature. Dilute it to the mark with water, shake it thoroughly, and let it stand for 10 minutes. Pipette the solution into a dry, clean cuvette and measure its absorbance at 650 nm using a spectrophotometer, adjusting the zero point with a sample blank.

[0132] 4. Calculate the phospholipid content in the sample according to the formula:

[0133]

[0134] Where:

[0135] X—phospholipid content, in milligrams per gram (mg / g);

[0136] P—phosphorus content of the test solution obtained from the standard curve, in milligrams (mg);

[0137] m—sample mass, in grams (g);

[0138] V1—the volume of the sample after dilution after ashing, in milliliters (mL);

[0139] V2—the volume of the test liquid taken during colorimetry, in milliliters (mL);

[0140] 26.31—The number of milligrams of phospholipids per milligram of phosphorus.

[0141] The results of the test and potency evaluation are as follows:

[0142] The extraction rate of lecithin by stirring and extracting sheep brain powder with the organic solvent petroleum ether is 4.38%, and the purity is 93.36%; when the feed-liquid ratio is 5:1 and the calcium chloride content is 0.5%, the extraction rate of cephalin is 0.77%, and the purity is 95.28%; the content of ARA (eicosapentaenoic acid), DHA (eicosahexaenoic acid), EPA (docosapentaenoic acid) and DPA (docosahexaenoic acid) and the unsaturated fatty acid content of cephalin and lecithin are significantly higher than those of other methods.

[0143] Use of cephalin and lecithin obtained by the method in preparing food or cosmetic additives:

[0144] The present invention discloses a method for preparing sheep brain cephalin and lecithin and their use. Cephalin and lecithin extracted from sheep brain cells are additives for cosmetics. Because phospholipids are hydrophobic at one end and hydrophilic at the other, phospholipid products extracted from animal tissue are safer and more effective. Cosmetics made with these extracted lecithins as additives can directly contact the skin and nourish cells, protecting and stabilizing cell membranes. They bind to cell membranes as carriers, helping nutrients in cosmetics enter the cell membranes, promoting cell membrane permeability and metabolism, replenishing nutrients such as proteins, peptides, and amino acids within cells, extending cell lifespan, and helping to rejuvenate the skin. They also have anti-wrinkle and sun-protective properties and are emulsifiable, making them suitable as natural additives for atomized cosmetics.

Claims

1. A method for preparing sheep brain cephalin and lecithin, characterized in that Follow these steps: a. Take fresh sheep brain and wash it with tap water to remove surface blood and impurities, freeze it at -80°C for 24 h, then freeze-dry it at a pressure of 9.5 Pa and a temperature of -80°C for 48 h. Then, grind the freeze-dried sheep brain in a grinder for 30 seconds and pass it through a 40-100 mesh sieve to obtain sheep brain powder; b The sheep brain powder obtained in step a solid-liquid ratio of 1: 5-1: 30 add organic solvent ether, chloroform or petroleum ether, or a volume ratio of 2: 1 chloroform: methanol was stirred and extracted 3 times, each 1-4h, three extracts were collected and set aside; c. The extract obtained in step b was concentrated to 1 / 3 of the volume of the extract three times using a rotary evaporator, and 3 times the volume of anhydrous ethanol was added, refluxed at 70 ° C for 30 min, filtered, and the residue was discarded. The filtrate was allowed to stand at a temperature of 4 ° C overnight until the layers were separated; d The supernatant obtained in step c was collected and the precipitate was stored for later use, and then the supernatant was filtered, the filtrate was concentrated and dried in vacuo or freeze-dried to obtain crude lecithin; e. Add 3-6 volumes of acetone to the crude lecithin obtained in step d, shake and mix for 20-30 seconds, let it settle at 4°C for 12 hours, filter, discard the supernatant, and freeze-dry the precipitate under vacuum to obtain lecithin; f press the solid-liquid ratio 3-8: 1 the precipitate obtained in step d was added 0.1% -1% calcium chloride, heated to a temperature of 100 ℃, reflux extraction 10min, and centrifuged at 1000-5000rpm speed for 10min to obtain a precipitate, ie, a crude phospholipid extract; g. The crude phospholipid extract obtained in step f was then mixed with anhydrous ethanol by shaking for 20-30s, allowed to stand at a temperature of 4 ° C for 4h, repeated 3 times, the supernatant was discarded, the precipitate was precipitated, and then mixed with 95% ethanol by shaking for 20-30s, allowed to stand at a temperature of 4 ° C for 4h, centrifuged at 1000-5000rpm for 10min, the supernatant was discarded, and the precipitate was vacuum-dried or freeze-dried to obtain a refined phospholipid product.

2. Use of cephalin and lecithin obtained according to the method of claim 1 in preparing food or cosmetics.

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