CN115806860A - Preparation method and application of margaric acid-rich ruminant oil - Google Patents
Preparation method and application of margaric acid-rich ruminant oil Download PDFInfo
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- KEMQGTRYUADPNZ-UHFFFAOYSA-N heptadecanoic acid Chemical compound CCCCCCCCCCCCCCCCC(O)=O KEMQGTRYUADPNZ-UHFFFAOYSA-N 0.000 title claims abstract description 80
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/50—Reuse, recycling or recovery technologies
- Y02W30/74—Recovery of fats, fatty oils, fatty acids or other fatty substances, e.g. lanolin or waxes
Abstract
The invention discloses a preparation method and application of margaric acid-rich ruminant oil. The enrichment method comprises the step of carrying out fractionation on the sheep tail oil at a certain temperature by using an organic solvent n-hexane, wherein the content of heptadecanoic acid in the obtained solid-phase oil is increased by more than 1 time. The fatty acid mixture prepared by further combining the urea embedding method can inhibit the migration and growth of cancer cells in vitro and in vivo experiments. The heptadecanoic acid-enriched oil prepared by the method has the potential of being used for clinically and adjunctively treating cancers.
Description
Technical Field
The invention relates to the technical field of food science, in particular to a preparation method and application of ruminant grease rich in heptadecanoic acid.
Background
Cancer seriously harms human life health, and the morbidity and mortality of cancer are always high. Taking lung cancer as an example, it is clinically manifested as malignant tumor of lung bronchial mucosa or glands. The incidence and mortality of lung cancer are high in the world and in China. Lung cancer can be classified into small cell lung cancer and non-small cell lung cancer according to its pathological morphology, wherein non-small cell lung cancer accounts for about 85% of lung cancer. Clinical treatments for lung cancer currently mainly involve surgical treatment, radiotherapy and chemotherapy. Although the targeted drug therapy for lung cancer achieves certain positive therapeutic effect, drug resistance and relapse are also obstacles which are difficult to overcome by the current targeted drugs. Taking the EGFR-TKIs type of drug, which is an inhibitor targeting Epidermal Growth Factor Receptor (EGFR), clinical patients often develop acquired drug resistance, which results in the failure of the therapy. Screening and exploring more treatment means and methods, and combining with the existing drugs to be used as a treatment development direction.
Disclosure of Invention
The invention mainly aims to provide a preparation method and application of a ruminant oil rich in heptadecanoic acid, which can effectively solve the problems in the background technology.
In order to realize the purpose, the invention adopts the technical scheme that:
the invention provides a preparation method of ruminant grease rich in heptadecanoic acid, which is obtained by sequentially carrying out n-hexane fractionation and urea embedding crystallization on the ruminant grease rich in heptadecanoic acid.
The further improvement is that the ruminant oil is adeps bovis seu Bubali and adeps Caprae Seu Ovis.
The further improvement is that the ruminant oil is sheep tail oil.
The further improvement is that the n-hexane fractionation specifically comprises the following steps:
step S1, primary fractionation: melting ruminant oil, stirring, mixing with organic solvent n-hexane uniformly, standing in constant temperature water bath for a certain time to crystallize, centrifuging to separate solid-liquid phase, and removing solvent to obtain solid oil with high melting point and liquid oil with low melting point;
step S2, secondary fractionation: carrying out secondary fractionation on the solid oil according to the step S1 to obtain secondary fractionation concentrates;
wherein in the step S1, the ratio of n-hexane to ruminant grease is 0.5 to 1v/w, the fractionation temperature is-4 ℃ to 12 ℃, and the treatment time is 2 to 12 hours; in the step S2, the ratio of n-hexane to solid oil is (2) - (1-8) v/w, and the temperature is 0-24 ℃.
The further improvement is that the urea embedded crystallization specifically comprises the following steps:
mixing urea and ethanol water solution, heating in water bath and stirring until the urea is completely dissolved; adding the secondary fractionation enrichment which is subjected to saponification and decomposition, sealing, stirring, reacting, naturally cooling, embedding, centrifuging, dissolving urea, crystallizing, and adding n-hexane for extraction to obtain a fatty acid mixture.
The further improvement is that in the urea embedding crystallization treatment process, the concentration of an ethanol solvent is 50-95%, the ratio of an ethanol aqueous solution to urea is 1-12.
The application of the ruminant grease rich in heptadecanoic acid obtained by the preparation method in food for adjuvant therapy of non-small cell lung cancer is provided.
The history of food therapy has long been, and great attention is being paid to developing personalized food therapy methods for cancer patients. The fatty acids in the oil, as important nutrients in the diet, can provide energy sources, components of biological membranes, and lipid signaling molecules to the cells. Meanwhile, the fatty acid is proved to have promoting or inhibiting effect on cancer cells. Different relationships exist between fatty acids of different saturation, length and cancer. Odd-numbered fatty acid C17:0 is found in dietary oil at the early stage and shows good inhibition effect on non-small cell lung cancer cell strain PC-9 and EGFR-TKI drug-resistant strain PC-9/GR cells (Changzhi Xu, et al., oncology Reports, 2019). It was further demonstrated that margaric acid-containing sheep tail oil also significantly inhibited proliferation of non-small cell lung cancer cells in vitro and in vivo (Changzhi Xu, et al, frontiers in pharmacology, 2022). Heptadecanoic acid also has multiple biological functions, and the content level in the body is inversely related to the onset risk of cardiovascular diseases, diabetes, multiple sclerosis and cancers. The development of the functional grease for enriching the heptadecanoic acid has certain application potential. Currently, the enrichment methods for functional fatty acids in fats and oils are mainly physicochemical modification, including ester exchange method (schroe plug, etc., food Technology, 2019), fractionation method (Curtis R, et al, food Chemistry, 2019), urea embedding method (Gonzalez-fundenz MJ, et al, environmental Technology & Innovation, 2020), molecular distillation method (He J, et al, food Science & Nutrition, 2020), and the like. Wherein the fractionation method and the urea embedding method are widely used due to the advantages of low cost, convenient operation and the like. A large amount of unsaturated fatty acid obtains a functional grease product with obviously improved content by fractionation and a urea embedding method.
Although the function of heptadecanoic acid is various and important, it is contained in a low amount in natural sources. Is mainly present in ruminant oil. Through research and screening, the margaric acid is found to be higher in the sheep tail oil and accounts for about 3 percent of the total fatty acid. Therefore, the sheep tail oil is used as the starting oil, the enrichment process is optimized by utilizing a fractionation method and a urea embedding method, and the potential of the sheep tail oil for auxiliary treatment of cancer is tested.
Compared with the prior art, the invention has the following beneficial effects:
compared with the prior art, the invention has the following advantages: the invention provides a preparation method and application of margaric acid-rich ruminant grease.
Drawings
FIG. 1 is a statistical graph showing the proportion of heptadecanoic acid in the total fatty acids of various sheep tail oils;
FIG. 2 is a graph comparing the fatty acid pattern and the heptadecanoic acid content of oil and fat after organic solvent fractionation and urea embedding;
FIG. 3 is a graph of the effect of enrichment on migration of non-small cell lung cancer;
FIG. 4 is the in vivo effect of the concentrate on non-small cell lung cancer transplantable tumors.
Detailed Description
The technical scheme of the invention is clearly and completely described in the following by combining the attached drawings (tables) and the detailed description. It should be noted that the described embodiments are exemplary and not limiting. These examples are not intended to limit the scope of the present invention in any way. In addition, techniques not described or not described in detail in the following examples are conventional techniques and thus will not be described in detail.
1. Material
The experimental methods in the following examples are conventional biochemical methods unless otherwise specified; the test materials used in the following examples were all purchased from conventional biochemicals, unless otherwise specified.
(1) A549 (human lung adenocarcinoma cells), PC-9 (non-small cell lung cancer cells) and MRC-5 (human lung fibroblast cells) used for the experiment can be purchased from the market, and PC-9/GR (human lung adenocarcinoma cells gefitinib-resistant strain) is preserved in the laboratory.
(2) 24 female nude mice of 5 weeks old were purchased from Jiangsu Jiejiaokang Biotech GmbH (strain name: BALB/c-nude, strain number: D000521) and weighed 19. + -.2 g.
(3) The sheep tail oil raw material is provided by Fuyang Tianxiang food science and technology Limited, and the origin is Inmunogu Bayan Yan Kao Wuyuan county, brand: grassland leading sheep. The experimental oil is prepared by heating sheep tail fat, filtering with gauze, centrifuging, removing fine impurities, storing at 4 deg.C, and packaging. The detection proves that the animal fat food safety standard is met.
The experiment is carried out after being approved by the ethical committee of laboratory animals of the institute of fertilizer-combining and Material science of the Chinese academy of sciences (approval number: DWLL-2020-48), and meets the relevant regulations of guidance opinions on animals to be tested. Other test materials are shown in table 1. The experiments in the following examples were set up in triplicate and the results were averaged for P <0.05, P <0.01, P <0.001.
Table 1: sources of Experimental materials
2. Method of producing a composite material
2.1 screening of raw sheep tail oil and determination of melting Point
Methyl esterification of triglycerides (alkaline process): firstly, heating the grease to be measured, stirring the grease to be measured after the grease is completely melted, sucking the grease to be measured by a liquid-transferring gun from 0.04g to 15mL of a centrifuge tube, and adding 0.2mol/L KOH-CH prepared in advance 3 2mL of OH solution was added and chromatography was performedAnd 6mL of grade n-hexane is used for extracting fatty acid methyl ester, the fatty acid methyl ester is fully shaken and uniformly mixed, then the mixture is kept stand in a water bath at 40 ℃ for 40min, after the completion, the upper layer of n-hexane is absorbed by an injector, the water is removed by anhydrous sodium sulfate, the mixture is filtered by an organic filter membrane with the diameter of 0.22 mu m and then is filled in a 1.5mL sample feeding bottle, and the bottle cover is matched with a non-porous sealing gasket to wait for detection on a computer. Can be stored in refrigerator at 4 deg.C or-20 deg.C for a short period.
Gas Chromatography-Mass spectrometry (Gas Chromatography-Mass Spectrometry, GC-MS) detection conditions: SP-2560 Strong polar capillary gas chromatography column (100 m.times.0.25mm, 0.2 μm) with carrier gas: n is a radical of 2 (ii) a Vaporization chamber temperature: 280 ℃; split-flow sample injection, 20; the inlet and detector temperatures were both 250 ℃ and the column front pressure was 278.6kPa. Procedure: heating to 190 deg.C at 4 deg.C/min at 100 deg.C, heating to 220 deg.C at 2 deg.C/min, maintaining for half an hour, and maintaining for 10min at 2 deg.C/min to 250 deg.C; sample injection 1 μ L. An ionization mode: an EI source, a single quadrupole detector; electron energy: 70eV; the ion source temperature and the interface temperature were both 250 ℃.
The measurement is carried out by referring to the method of GB/T24892-2010 measurement of melting point (slip point) of animal and vegetable fats in open capillary. The sheep tail oil is always in a complete or semi-solidified state at room temperature, is heated in a water bath at 60 ℃ to be melted, and is stirred uniformly by a glass rod. The upper ends of the three capillary tubes with the same bottom are bound together by label paper, the three capillary tubes extend into the sheep tail oil to absorb grease with the height of about 1.5cm, redundant grease on the outer side tube wall is wiped off, then the three capillary tubes are quickly and horizontally placed in a refrigerator at the temperature of 20 ℃ below zero for more than 15min, and the three capillary tubes are taken out after being solidified. The capillary tube is aligned with the lower end of the thermometer, the capillary tube and the thermometer are fixed by a rubber band and then vertically placed on the bracket, the capillary tube is inserted about one inch below the liquid level of ice water in the beaker, and the magnetic stirrer is used for heating and stirring at a constant speed to ensure that the upper water temperature and the lower water temperature are consistent. Because the grease density is less than that of water, the grease floats upwards after being melted and just leaves the temperature of the capillary tube when the bottom of the capillary tube floats upwards, namely the melting point of the sheep tail oil. The experiment was set up in 3 replicates and the melting points were averaged.
The results are shown in fig. 1 as the ratio (%) of C17:0 to the total fatty acids in the various sheep tail oils. The comparison of the peak heights in the graph shows that the C17:0 content of the two tail oils of the prairie leading sheep and the Monxin Yiyi sheep is relatively high and accounts for about 3 percent. The 2 varieties are all from inner Mongolia Bayan Yankee City, and the fatty acid compositions of different sheep fats in different regions and different varieties are different through production area investigation. The melting point is measured by a capillary tube opening method, the melting point of the grassland leading sheep tail oil is (22.50 +/-0.50) DEG C, and the melting point of the Monxin Yi group STF is (24.20 +/-0.50) DEG C, which are relatively close to each other. In order to verify the stability of the sheep tail oil, through multiple tests, the content of C17:0 of different batches of prairie collar sheep varieties is basically consistent, and the stability is good, so that the method is selected for subsequent enrichment and purification experiments of C17: 0.
2.2C17 enrichment purification of
2.2.1 solvent fractionation of fats and oils
The sheep tail oil is melted and then evenly stirred, and is evenly mixed with the organic solvent according to a certain proportion, and the mixture is placed in a constant temperature water bath kettle with a set temperature for a certain time to be fully crystallized. And after complete crystallization, centrifugally separating a solid phase from a liquid phase, removing the solvent under a reduced pressure condition to obtain solid oil with a high melting point and liquid oil with a lower melting point, and detecting the fatty acid composition by GC-MS after methyl esterification treatment. The temperature, the rotating speed and the time during centrifugation need to be kept consistent, and a solid phase and a liquid phase need to be separated immediately under the low-temperature condition, so that the experimental error is reduced.
As shown in FIG. 2, under the conditions that the solvent is n-hexane, the temperature is from-4 ℃ to 12 ℃, the ratio of the solvent to the oil is 0.5. And (2) continuously carrying out secondary fractionation on the obtained solid oil, wherein the content of C17:0 in the obtained solid sheep tail oil can be maximally increased to 7.10% under the conditions that the temperature is 0-24 ℃, the proportion of normal hexane to grease is 2-8 (v: w)
1.98 percent. As can be seen from the fatty acid chromatogram, the content of C17:0 in the fractionation extract oil, the sheep tail oil and the secondary fractionation concentrate is sequentially increased, and the fractionation method has obvious enrichment effect.
2.2.2 Urea embedding method for obtaining heptadecanoic acid-enriched fatty acid mixture
Because the melting point of the heptadecanoic acid enriched grease is higher, the heptadecanoic acid enriched fatty acid mixture is obtained by decomposing the enriched grease and embedding and enriching urea. Mixing urea and an ethanol aqueous solution, adding the mixture into a triangular flask with a plug, heating the mixture in water bath at 80 ℃, and magnetically stirring the mixture until the urea is completely dissolved; weighing a certain amount of saponified and decomposed primary fractionated sheep tail oil fatty acid, adding into the system, stirring under sealed condition at 65 deg.C for reaction for 60min, and naturally cooling; embedding for a certain time at a set temperature, and then quickly centrifuging at a corresponding temperature at the rotation speed of 5000rpm for 20min to separate out a liquid part; recovering solvent from the liquid part under reduced pressure, washing with hot water to dissolve urea crystal, adding 20mL n-hexane, extracting for 3 times, removing n-hexane, and storing the obtained fatty acid at-20 deg.C. The solid phase was treated in the same manner. As shown in fig. 2, the urea embedding method is screened for the solvent type (50% -95% ethanol solution), the ratio of solvent to urea (2. The composition of various fatty acids in the starting sheep tail oil, the primary fractionation solid oil and the fatty acid mixture subjected to n-hexane fractionation and urea embedding is shown in the following table 2.
Table 2 sheep tail oil and enrichment of fatty acid component under combination conditions
2.3c17 effects of enrichment with 0 on cancer treatment
2.3.1C17
A549 cells in the logarithmic growth phase are evenly inoculated into a 6-hole plate and cultured until monolayer cells account for about 90% of the bottom of the dish, and then scratching operation can be carried out. Perpendicular to the straight line drawn in advance, two to three traces are drawn from top to bottom with a micropipette tip, and the cell is lightly washed with PBS for several times until no floating cell exists. The prepared complete culture medium containing 50 and 100 mu g/mL of sheep tail oil and the enrichment of C17:0 with the same concentration is replaced, and a blank control group and a positive control group (the high-oleic peanut oil without inhibiting effect on A549 and the concentration of 100 mu g/mL) are set at the same time. And (3) shooting the scratch area after 0h of medicine addition and 48h of culture under a fluorescence inverted microscope, obtaining a blank area by using Image J software after the acquisition is finished, and calculating the healing rate of each group according to a formula.
As shown in fig. 3A and 3B, the experimental group to which STF and C17:0 concentrates were added had a significantly smaller degree of healing of the scratched area after 2 days of culture than the blank control group and the High Oleic Peanut Oil (HOPO) group. At the same concentration, the cell-free area of the C17:0 concentrate was greater than the sheep tail oil, and the statistics of the healing rate for each group are shown in fig. 3. The healing rates of the blank control group and the high oleic acid peanut oil group have no significant difference. The 100 μ g/mL sheep tail oil and C17:0 enrichment were calculated to have a statistical difference in healing rates (p < 0.01). The experimental result shows that the sheep tail oil enriched with C17:0 can more effectively inhibit the migration of lung cancer cells.
2.3.2c17 inhibition effect of 0 enrichment on non-small cell lung cancer cell transplantable tumors
A549 cells in logarithmic growth phase were collected, washed twice with PBS to remove residual medium, resuspended with PBS, and mixed well with an equal volume of matrigel to a final volume of 2.5X 107cells/mL. Placing matrigel at 4 ℃ overnight in advance, and precooling a gun head, an Eppendorf tube and the like; the cells are placed in an ice box, and relevant experimental equipment such as an outer box, an injector and the like are carried into an animal room after being subjected to ultraviolet disinfection. And (3) reversely mixing the cell suspension, sucking 0.2mL by using a 1mL insulin syringe, performing subcutaneous injection on the right armpit of each mouse, wherein the injection cannot be performed for the second time in the process, rotating a needle after the injection is completed, pulling out the needle to prevent the liquid from overflowing, and continuously feeding the mice. To ensure cell viability, inoculation needs to be completed as soon as possible; the mice grow freely, and the tumor volume reaches 60-150mm after one week 3 The left and right are grouped and divided into 4 groups of 6; the initial administration was performed on the day of the group. A solvent control group, a sheep tail oil fatty acid group, an enriched substance group and a cis-platinum group are arranged. Solvent control group: DMSO accounts for 5% of the drug concentration; fatty acid of sheep tail oil: 200mg/kg; enrichment: 200mg/kg, all administered by intraperitoneal injection daily. Cis-platinum:5mg/kg, 1 time per 3 days, and performing intraperitoneal injection; the administration is continued for 5 weeks, the body weight is weighed daily, the tumor size is measured every 4 days with a vernier caliper, and the tumor volume is according to the formula V = (a) 2 X b)/2 (a is the short diameter, b is the long diameter). Obtaining a tumor volume curve graph and a mouse weight change graph after terminating the experiment; by using CO 2 All mice were sacrificed, tumors and liver were stripped as soon as possible and weighed, and fixed embedded after photographing; plotted and statistically analyzed. As can be seen in FIG. 4A, the control (vehicle) tumors were significantly larger than the other 3 groups. From the tumor weight statistics in fig. 4B, the difference between the solvent control group and the concentrate was most significant (P)<0.001 Superior to sheep tail oil. Fig. 4C shows that the tumor volume calculated by measurement substantially corresponds to the final tumor weight stripped, and the cancer inhibition effect of the concentrate is also proved. Also shown in figure 4D, both the sheep tail oil and the concentrate had no effect on the weight of the mice, whereas cisplatin had a greater side effect and the mice had greater weight fluctuations than the solvent control group.
The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A preparation method of ruminant grease rich in heptadecanoic acid is characterized in that: the ruminant grease rich in heptadecanoic acid is obtained by sequentially carrying out n-hexane fractionation and urea embedding crystallization.
2. The method for preparing a ruminant oil rich in heptadecanoic acid according to claim 1, wherein the method comprises the following steps: the ruminant oil is tallow oil.
3. The method for preparing the ruminant oil rich in the heptadecanoic acid according to the claim 2, is characterized in that: the ruminant oil is sheep tail oil.
4. The method for preparing a ruminant oil rich in heptadecanoic acid according to claim 1, wherein the method comprises the following steps: the n-hexane fractionation specifically comprises the following steps:
step S1, primary fractionation: melting ruminant oil, stirring, mixing with organic solvent n-hexane uniformly, standing in constant temperature water bath for a certain time to crystallize, centrifuging to separate solid-liquid phase, and removing solvent to obtain solid oil with high melting point and liquid oil with low melting point;
step S2, secondary fractionation: carrying out secondary fractionation on the solid oil according to the step S1 to obtain secondary fractionation concentrates;
wherein the proportion of the normal hexane to the ruminant oil in the step S1 is 0.5 to 1v/w, the fractionation temperature is-4 ℃ to 12 ℃, and the treatment time is 2 to 12 hours; in the step S2, the ratio of n-hexane to solid oil is 2-1 v/w, and the temperature is 0-24 ℃.
5. The method for preparing the ruminant oil rich in the heptadecanoic acid according to the claim 4, characterized in that: the urea embedded crystallization specifically comprises the following steps:
mixing urea and an ethanol water solution, heating in a water bath, and stirring until the urea is completely dissolved; adding the secondary fractionation enrichment substance subjected to saponification and decomposition, carrying out closed stirring reaction, naturally cooling, carrying out centrifugation after embedding, dissolving urea crystals, and adding n-hexane for extraction to obtain a fatty acid mixture.
6. The method for preparing a ruminant oil rich in heptadecanoic acid as claimed in claim 5, wherein the method comprises the following steps: in the urea embedding crystallization treatment process, the concentration of an ethanol solvent is 50-95%, the ratio of an ethanol aqueous solution to urea is (2).
7. Use of margaric acid-rich ruminant oil obtained by the preparation method of any one of claims 1-6 in food for adjuvant treatment of non-small cell lung cancer.
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| JP2008255022A (en) * | 2007-04-02 | 2008-10-23 | Kureha Corp | Anticancer substance |
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| JP2008255022A (en) * | 2007-04-02 | 2008-10-23 | Kureha Corp | Anticancer substance |
| CN106010798A (en) * | 2016-06-13 | 2016-10-12 | 天津科技大学 | Method for extracting palmitoleic acid from sea buckthorn fruit oil |
| CN108084020A (en) * | 2017-12-21 | 2018-05-29 | 武汉欧米嘉生物医药有限公司 | A kind of method that omega-7 aliphatic esters are prepared using grease as raw material |
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