Nervous acid is a long-chain unsaturated fatty acid, which has significant effects on the treatment of cardiovascular and human autoimmune diseases. It is also an important raw material for fragrances. The market demand is huge, but it is scarce in nature, and it is difficult to synthesize.
Malania oleifera Chun et Lee
Malania oleifera Chun et Lee, a plant belonging to the genus Garlica of the sclerophyllaceae, is a single species unique to our country. The existing population of Garlica has very few resources and is only naturally distributed in a narrow area from western Guangxi to southeastern Yunnan.
Malania oleifera Chun et Lee kernels are rich in oil, with an oil content of up to 64.5%, which is an ideal raw material for the synthesis of musk. Studies have shown that its oil contains more than 60% nervonic acid, which is currently the plant with the highest nervonic acid content found in nature. .
Therefore, it is of great significance to realize the industrialized production of nervonic acid with Malania oleifera Chun et Lee oil as the raw material.
In the past, the extraction of Malania oleifera Chun et Lee oil was mainly based on the pressing method. There is no relevant literature report on supercritical CO2 extraction of garlic oil at home and abroad.
In this study, the supercritical CO2 method was used to extract the garlic oil, the single factor method was used to explore the effect of extraction conditions on the extraction, the orthogonal test method was used to optimize the extraction process conditions, and the GC-MS method was used to analyze the fatty acid composition in the optimal process garlic oil. The saponification and crystallization method are used to separate nervonic acid.
Supercritical CO2 extraction
Supercritical CO2 extraction of Malania oleifera Chun et Lee. After crushing the Malania oleifera Chun et Lee, put it into the extraction kettle, set the temperature and pressure of the extraction kettle and the separation kettle, and then heat the extraction kettle, the separation kettle I, and the separation kettle II respectively, and cool them down. Machine refrigeration; when the temperature reaches the set value, open the CO2 cylinder to increase the pressure;
After reaching the required pressure for the experiment, close the CO2 gas cylinder, start the cyclic extraction, maintain a constant temperature and constant pressure, after reaching the selected time, discharge the material from the discharge port of the separation kettle I, and mix to obtain the golden yellow liquid, which is the Malania oleifera Chun et Lee oil, weighed Weight, calculate the yield.
Separation of nervonic acid
Take Malania oleifera Chun et Lee oil, add appropriate amount of NaOH-ethanol solution for saponification, remove part of the ethanol, heat water to make up the volume, acidify to the required pH value, take the precipitate, wash it to neutrality, and dry it to obtain crude fatty acid. Take the crude fatty acid and add an appropriate amount of acetone to remove impurities. The residue is recrystallized twice with ethanol, the crystals are evaporated to dryness, weighed, the yield is calculated, and the purity is checked.
Raw material crushing research
Shelling and crushing
Take the garlic fruit, remove the shell, crush the pulp, place it in a universal grinder to crush for 10 seconds, the crushed material will stick to the wall of the grinder. Garlic pulp contains high oil and moisture, so it is difficult to pulverize directly. Generally, it needs to be oiled with other materials that do not contain oil or have low oil content during pulverization.
Crush the garlic with its shell and crush it with a universal grinder. The result: the crushed material is granulated with a particle size of 0.5-1cm, most of which cannot pass through the No. 2 sieve. The size is uneven, no agglomeration, and dispersibility. It is better than crushing the pulp alone.
Take the garlic fruits, crush them with their shells, freeze them in a refrigerator at -20℃, and then crush them with a freezer pulverizer. The result: the crushed material is granulated, with a particle size of less than 0.3cm, which is more uniform and has better dispersibility, which is better than ordinary pulverization. .
Comparison of different crushing methods
Weigh about 200g of the raw materials obtained by the above-mentioned different crushing methods, and place them in a supercritical extraction kettle. Set the extraction pressure to 35MPa, extraction temperature 45°C, separation pressure 6MPa, separation temperature 50°C, and cycle extraction for 2 hours.
Garlic fruit with starch skewer oil has better dispersibility after crushing. When the equipment is in good condition, the fruit oil can be extracted completely.
However, garlic pulp contains a lot of oil and water, but also contains a lot of starch. The starch dissolves in the oil to form a paste, which affects the quality of the oil. Frozen crushing can also complete the extraction of garlic oil, and the quality of the oil is better.
Single factor experiment of supercritical CO2 extraction
Raw material crushing particle size
Take Malania oleifera Chun et Lee, crush them through 8, 14 and 20 mesh sieves, and take the sieved materials, put them in a supercritical extraction kettle, set extraction pressure 30MPa, extraction temperature 45℃, separation pressure 6MPa, separation temperature 50 ℃, circulate extraction for 2 hours.
The crushing particle size of 8 mesh is better, the drug residue is white, and the oil is transparent and golden; when the crushing particle size is 14 mesh, although the extraction is complete, the drug residue is also white, but there is a small amount of precipitation in the oil, which affects the quality.
Take Malania oleifera Chun et Lee and crush it into coarse powder (8 mesh). Set the extraction pressure to 25MPa, extraction temperature 45°C, separation tank I pressure 6MPa, temperature 50°C, separation tank II pressure 6MPa, temperature 45°C, and Malania oleifera Chun et Lee oil yield and free nervonic acid content as indicators, observe the change trend of extraction 1, 2 and 3 hours: under the same conditions, oil yield and free nervonic acid content both increase with the extension of supercritical CO2 cycle extraction time. Circulating extraction for 2 hours has a significant increase compared with 1 hour of cyclic extraction, while the increase of cyclic extraction for more than 2 hours is slow and not significant.
Take Malania oleifera Chun et Lee and crush it into 8 mesh coarse powder. Set the extraction time for 2 hours, extraction temperature 45℃, separation tank I pressure 6MPa, temperature 50℃, separation tank II pressure 6MPa, temperature 45℃, and Malania oleifera Chun et Lee oil yield and free nervonic acid content are indicators. Observe the change trend of extraction pressure at 15, 25, 35 and 45MPa. Under the same conditions, the supercritical cycle extraction pressure increases, and the oil yield shows an upward trend, but when the extraction pressure reaches 35MPa After that, the oil output rate increased insignificantly, and the oil output rate at an extraction pressure of 45 MPa was lower than that at 35 MPa; the extraction pressure increased, but the free nervonic acid content increased insignificantly, and the content decreased when the pressure reached 25 MPa or more.
Supercritical CO2 extraction process verification
Take about 1.5 kg of Malania oleifera Chun et Lee, crush it through an 8-mesh sieve, divide it into three portions, 500 g/portion, and perform supercritical CO2 extraction according to the optimized process parameters. The verification results of the third extraction process are similar, the oil output is about 160g, the oil recovery rate is 32%, and the free nervonic acid content is 5.45%. The extraction process is stable and feasible, with good repeatability.
The GC-MS method was used to analyze the fatty acid composition of Malania oleifera Chun et Lee oil obtained from the process verification, and the 12 fatty acids in Malania oleifera Chun et Lee oil were identified. Malania oleifera Chun et Lee oil is mainly composed of oleic acid and 22 It is composed of twelve kinds of fatty acids, such as carbosaenoic acid and docosamonoenoic acid. Among them, docosaenoic acid has a relatively high content, accounting for about 35% of the total fatty acid.
Study on the separation of nervonic acid
Weigh 68g of sodium hydroxide into a round-bottomed three-necked flask, add 912mL of ethanol, reflux at 85°C to dissolve, add 182g of Malania oleifera Chun et Lee oil, saponify at 85°C for 30 minutes, distill off part of the ethanol, and heat water to make up the volume. Add 50% sulfuric acid solution while it is hot to acidify to pH 2.5; let it stand for 48 hours, take the precipitate, wash it with water 3 times, and dry it to obtain crude fatty acid. The crude fatty acid is mixed with acetone 1 times the amount, filtered, the acetone solution is discarded, the residue is 15 times ethanol, recrystallized twice, 2 hours each time, collect the crystals, evaporate the white crystals, and separate the nerves from Malania oleifera Chun et Lee oil The purity of acid and nervonic acid can reach 96.08%, and the crystal yield is 4.6%; the separation process has good repeatability.
Malania oleifera Chun et Lee has high oil content and high water content. It is easy to stick when crushed, and needs to be crushed with other materials.
When extracting single medicinal materials, since the main components of Malania oleifera Chun et Lee oil are fatty acids and fatty acid esters, metal ion-containing excipients cannot be used, but only inert excipients can be used for pulverization.
In addition, Malania oleifera Chun et Lee also contains a lot of starch. If starch is added as an auxiliary material, the amount of starch will be too much, and the starch will dissolve in the oil to form a paste, which will affect the quality of the oil. Although the dispersibility of the materials is improved, the extraction process is prone to flushing the kettle, and the starch will block the pipeline after extraction and damage the equipment. Adopting freezing crushing method, the material is evenly dispersed and the particle size is moderate.
Crushing particle size
The crushing particle size of Malania oleifera Chun et Lee is better at 8 mesh, the extracted drug residue is white and the oil is transparent and golden; when the crushing particle size is 14 mesh, although the extraction is complete, the drug residue is also white, but there is a small amount of precipitation in the oil, which affects the quality .
Through single factor experiment and orthogonal experiment, the best extraction technology of Malania oleifera Chun et Lee supercritical CO2 extraction is: extraction pressure 35MPa, extraction temperature 45℃; separation vessel I pressure is 6MPa, temperature 55℃; separation vessel II pressure is 6MPa , The temperature is 55°C. The process is stable and feasible with good repeatability.
The fatty acids in Malania oleifera Chun et Lee oil were analyzed by GC-MS, and 12 fatty acid components were identified. The content of nervonic acid is relatively high, accounting for about 35% of the total fatty acid.
The saponification and crystallization method was used to separate nervonic acid from Malania oleifera Chun et Lee oil, and a crystal with a yield of 4.6% and a nervonic acid content of 96% was obtained. The separation process has good repeatability.