Method for extracting high-purity nervonic acid from malania oleifera oil

Abstract

The invention relates to a method for extracting high-purity nervonic acid from malania oleifera oil, which comprises the following steps: 1, saponification-metal salt precipitation: taking malania oleifera oil, adding alkali, adding a non-aqueous solvent, heating, refluxing, saponifying, cooling, precipitating, filtering, and removing a solution to obtain filter residues; step 2, acidification: adding acid water into the filter residues obtained in the step 1 for acidification, standing for layering, and discarding a water layer to obtain an oil layer; 3, washing: washing the oil layer obtained in the step 2 to obtain a washed oil layer; 4, fractional precipitation: adding a first organic solvent into the washed oil layer obtained in the step 3, carrying out first-stage precipitation, and filtering to remove filter residues; and carrying out second-stage precipitation and filtration. And 5, recrystallization: adding a second organic solvent into the filter residue obtained in the step 4, recrystallizing, filtering, and taking the filter residue to obtain nervonic acid. The nervonic acid with the content not lower than 95%, the purity not lower than 98% and the yield not lower than 70% can be obtained.

Description

Technical Field

[0001] The invention belongs to the technical field of natural product extraction, and more specifically, relates to a method for extracting high-purity nervonic acid from garlic fruit oil. Background Art

[0002] Nerve acid, also known as shark acid, was first discovered in the brain tissue of mammalian sharks. Its chemical name is cis-15-tetradecenoic acid, and its molecular formula is C 24 H 46 O2, molecular weight is 366.6, white crystalline solid or powder, easily soluble in organic solvents such as methanol and ethanol, not soluble in water. At present, the natural raw materials of neuraminic acid in my country include garlic fruit, blue vegetable and maple, among which the content of neuraminic acid in garlic fruit is relatively high.

[0003] Malania oleifera Chun et Lee is the only monospecific plant of the genus Malania in the family Olacaceae. It is also known as Malania or Yunnan Malania. It is mainly distributed in Guangnan and Funing in Wenshan Prefecture in southeastern Yunnan and parts of western Guangxi bordering Yunnan. It is a monospecific relict tree species unique to my country.

[0004] In addition to nervonic acid, the fatty acids in garlic fruit oil also include oleic acid, erucic acid, behenic acid, lignoic acid, etc. The presence of these acids affects the purity of the final product. In addition, the large amount of glycerol produced after the saponification of fatty acid glycerides and the toxic protein components naturally contained in garlic fruit oil affect the quality of the finished product and the safety of the subsequent use of the product.

[0005] At present, the method for extracting nervonic acid from garlic fruit oil mainly includes: column chromatography, molecular distillation, urea inclusion, extraction, recrystallization, and these methods basically have the disadvantages of complex processes and cannot take into account both purity and yield. For example: Chinese patent application CN105503580A discloses a method for extracting and separating nervonic acid. Although the content of nervonic acid obtained by this method can exceed 90%, the yield is extremely low, less than 30%, and the waste of resources is large; the content of the method disclosed in Chinese patent application CN107353199A can also reach 90%, but the yield is also low, and the process is complicated and the cost is high; Chinese patent CN1050117C provides two methods. Although the yield of these two methods can reach 60%, the content only exceeds 80%, and the process is complicated and the operation is difficult. Therefore, there is an urgent need for a method for extracting high-purity nervonic acid that is simple to operate, low in cost and high in yield. Summary of the invention

[0006] In order to solve the defects of the prior art, the inventors of this application have discovered, after extensive research, a method for extracting high-purity neuraminic acid with a content of not less than 95% from garlic fruit oil. This method is simple to operate, low in cost, and has industrial feasibility, and the yield is not less than 70%.

[0007] The present invention provides a method for extracting nervonic acid from garlic fruit oil, which comprises the following steps:

[0008] Step 1: saponification-metal salt precipitation: take garlic oil, add alkali, then add non-aqueous solvent, heat and reflux for saponification, cool and precipitate, filter, remove the solution, and obtain filter residue;

[0009] Step 2: Acidification: Add acid water to the filter residue obtained in step 1 for acidification, let stand for stratification, discard the water layer, and obtain the oil layer;

[0010] Step 3: Water washing: washing the oil layer obtained in step 2 with water to obtain a water-washed oil layer;

[0011] Step 4: graded precipitation: add the washed oil layer obtained in step 3 to the first organic solvent, perform the first stage precipitation, filter and discard the filter residue; perform the second stage precipitation, and filter.

[0012] Step 5: Recrystallization: Add the second organic solvent to the residue obtained in step 4, recrystallize, filter, and take the residue to obtain neuraminic acid.

[0013] Preferably, in step 1, the weight of the alkali is 0.1-0.6 times, preferably 0.2-0.4 times, the weight of the garlic fruit oil;

[0014] Preferably, in step 1, the base is selected from one or more of NaOH, KOH, NH4·H2O, Ca(OH)2, and Mg(OH)2;

[0015] Preferably, in step 1, the non-aqueous solvent is selected from one or more of methanol, acetone, ethanol, acetonitrile and tetrahydrofuran.

[0016] Preferably, in step 1, the volume (L) of the non-aqueous solvent, such as ethanol, is 8-15 times, preferably 10-15 times, the weight (kg) of the garlic fruit oil;

[0017] Preferably, in step 1, the concentration of the non-aqueous solvent, such as ethanol, is 80 v / v%-100 v / v%, preferably 85 v / v%-95 v / v%;

[0018] Preferably, in step 1, the saponification time is 30 to 90 min, preferably 45 to 75 min;

[0019] Preferably, in step 1, the temperature of the cooling precipitation is -5°C to room temperature, preferably 5°C to room temperature;

[0020] Preferably, in step 1, the precipitation time is 1 to 4 hours;

[0021] Preferably, in step 2, the acid water refers to an aqueous solution of HCl, H2SO4, or HNO3;

[0022] Preferably, in step 2, the concentration of the acid water is 0.5-2 mol / L, preferably 0.5-1.5 mol / L;

[0023] Preferably, in step 2, the volume (L) of the added acid water is 5-20 times, preferably 8-12 times, the weight (kg) of the garlic fruit oil;

[0024] Preferably, in step 2, the acidification temperature is 50 to 95°C, preferably 55 to 75°C;

[0025] Preferably, in step 2, the acidification time is 15 to 75 minutes, preferably 30 to 60 minutes;

[0026] Preferably, in step 2, the temperature of the static stratification is 0°C to room temperature, preferably 5°C to room temperature;

[0027] Preferably, in step three, the oil layer obtained in step two is washed with water 2 to 7 times, preferably 3 to 6 times, to obtain a washed oil layer;

[0028] Preferably, in step 4, the first organic solvent is selected from one or more of acetone, methanol and ethanol.

[0029] Preferably, in step 4, the concentration of the first organic solvent is not less than 80 v / v%.

[0030] Preferably, in step 4, the volume (L) of the first organic solvent added is 2-8 times, preferably 3-6 times, the weight (kg) of the garlic fruit oil.

[0031] Preferably, in the first stage precipitation of step 4, the precipitation temperature is 5°C to room temperature, preferably 10°C to room temperature.

[0032] Preferably, in the first stage precipitation of step 4, the precipitation time is 1 hour to overnight, preferably 4 hours to overnight.

[0033] Preferably, in the second stage precipitation of step 4, the precipitation temperature is -20 to 5°C, preferably -15 to 0°C.

[0034] Preferably, in the second stage precipitation of step 4, the precipitation time is 1 hour to overnight, preferably 2 to 6 hours.

[0035] Preferably, in step five, the second organic solvent is selected from one or more of methanol, ethanol and acetone.

[0036] Preferably, in step five, the volume (L) of the second organic solvent added is 2-8 times, preferably 3-6 times, the weight (kg) of the garlic fruit oil.

[0037] Preferably, in step five, the recrystallization temperature is -20°C to room temperature, preferably -15 to 10°C.

[0038] Preferably, in step five, the recrystallization temperature is 1 h to overnight, preferably 2 h to overnight.

[0039] In a specific embodiment, the method for extracting nervonic acid from garlic fruit oil of the present invention comprises the following steps, as shown in the flow chart: Figure 3 As shown:

[0040] Step 1, saponification-metal salt precipitation: take garlic fruit oil, add alkali (including NaOH, KOH, NH4·H2O, Ca(OH)2, Mg(OH)2, etc.) in an amount of 0.1-0.6 times the weight of the garlic fruit oil, then add 80-100 v / v% non-aqueous solvent, such as ethanol, whose volume (L) is 8-15 times the weight (kg) of the garlic fruit oil, heat reflux saponification for 30-90 minutes, cool and precipitate at -5°C to room temperature for 1-4 hours, and filter to remove the solution.

[0041] Step 2, acidification: add 0.5-2 mol / L acid water (including HCl, H2SO4, HNO3, etc.) to the filter residue obtained in step 1, the volume (L) of the added acid water is 5-20 times the weight (kg) of the garlic oil, acidify at 50-95° C. for 15-75 min, stand at 0° C. to room temperature for stratification, discard the water layer, and retain the oil layer.

[0042] Step 3, water washing: the oil layer obtained in step 2 is washed with water 2 to 7 times.

[0043] Step 4, graded precipitation: add 80-100 v / v% acetone, methanol, or ethanol to the oil layer obtained in step 3, the volume (L) of which is 2-8 times the weight (kg) of the garlic oil, carry out the first stage precipitation at 5°C to room temperature for 1 hour to overnight, filter and discard the filter residue, carry out the second stage precipitation at -20 to 5°C for 1 hour to overnight, and filter.

[0044] Step 5, recrystallization: Add 70-100 v / v% methanol, ethanol or acetone to the residue obtained in step 4, and its volume (L) is 2-8 times the weight (kg) of the garlic oil. Recrystallize at -20°C to room temperature for 1 hour to overnight, and filter the residue to obtain neuraminic acid.

[0045] In another specific embodiment, the method for extracting nervonic acid from garlic fruit oil of the present invention comprises the following steps:

[0046] Step 1, saponification-metal salt precipitation: take garlic fruit oil, add alkali (including NaOH, KOH, NH4·H2O, Ca(OH)2, Mg(OH)2, etc.) in an amount of 0.2-0.4 times the weight of the garlic fruit oil, then add 85-95v / v% ethanol, the volume (L) of which is 10-15 times the weight (kg) of the garlic fruit oil, heat reflux saponification for 45-75min, cool and precipitate at 5°C to room temperature for 1-4h, and filter to remove the solution.

[0047] Step 2, acidification: add 0.5-1.5 mol / L acid water (including HCl, H2SO4, HNO3, etc.) to the filter residue obtained in step 1, the volume (L) of the added acid water is 8-12 times the weight (kg) of the garlic oil, acidify at 55-75° C. for 30-60 min, stand at 5° C. to room temperature for stratification, discard the water layer, and retain the oil layer.

[0048] Step 3, water washing: the oil layer obtained in step 2 is washed with water 3 to 6 times.

[0049] Step 4, graded precipitation: After the oil layer obtained in step 3 is washed with water, 90-100 v / v% acetone, methanol, or ethanol is added to the oil layer, and its volume (L) is 3-6 times the weight (kg) of the garlic oil. The first stage precipitation is carried out at 10°C to room temperature for 4 hours to overnight, and the filter residue is discarded after filtering. The second stage precipitation is carried out at -15 to 0°C for 2 to 6 hours, and filtered.

[0050] Step 5, recrystallization: Add 80-100 v / v% methanol, ethanol or acetone to the residue obtained in step 4, and its volume (L) is 3-6 times the weight (Kg) of garlic oil. Recrystallize at -15-10°C for 2h to overnight, filter, and take the residue to obtain neuraminic acid.

[0051] Compared with the prior art, this application has at least the following beneficial technical effects:

[0052] The present invention effectively reduces the content of non-neurotropic acid salt components, especially toxic proteins and other long-chain fatty acids, by combining saponification-metal salt precipitation and graded precipitation. The quality of the neurotropic acid product finally obtained is as follows:

[0053] The content of nervonic acid is not less than 95% based on the dry product, preferably, the content of nervonic acid is not less than 97% based on the dry product.

[0054] The purity calculated by the area normalization method is not less than 98%, preferably, the purity calculated by the area normalization method is not less than 99%.

[0055] Calculated based on the content of nervonic acid in garlic fruit oil, the yield is not less than 70%. Preferably, calculated based on the content of nervonic acid in garlic fruit oil, the yield is not less than 75%.

[0056] The method for extracting neuraminic acid from garlic fruit oil provided by the present invention effectively reduces the content of non-neuraminic acid salt components, especially toxic proteins and other long-chain fatty acids through the combination of saponification and metal salt precipitation and graded precipitation, and finally obtains neuraminic acid with a content exceeding 95%, a purity exceeding 98% and a yield exceeding 70%. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The HPLC detection spectrum of nervonic acid of No. 2 in Example 11 (the preparation method refers to Chinese patent CN 107353199A);

[0058] Figure 2 The HPLC detection spectrum of nervonic acid of No. 4 in Example 11 (the preparation method refers to Chinese patent CN 1050117C);

[0059] Figure 3 This is a process flow chart for the preparation of neuraminic acid, where the multiples marked in the figure are the ratio of the volume (L) or weight (Kg) of the added solvent or reagent to the weight (Kg) of garlic fruit oil. DETAILED DESCRIPTION

[0060] Example 1: Method for extracting high-purity nervonic acid from garlic fruit oil

[0061] Step 1, saponification-metal salt precipitation: take multiple 1kg portions of garlic fruit oil from the same batch, add 0.05kg, 0.1kg, 0.2kg, 0.4kg, 0.6kg, 0.8kg, and 1.0kg of NaOH (serial numbers 1 to 7, respectively) of garlic fruit oil, add 10L of 95% ethanol, heat and reflux for saponification for 75min, cool and precipitate at 5°C for 2h, and filter to remove the solution.

[0062] Step 2, acidification: add 20L 0.5mol / L hydrochloric acid to the filter residue obtained in step 1 and acidify at 80℃ for 60min, stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0063] Step 3, water washing: The oil layer obtained in step 2 is washed with water three times.

[0064] Step 4, graded precipitation: add 6 L of 80 v / v% acetone to the oil layer obtained in step 3, carry out the first stage precipitation at 15°C for 1 hour, filter and discard the residue, carry out the second stage precipitation at -10°C for overnight, and filter.

[0065] Step 5, recrystallization: add 6L of 90v / v% acetone to the residue obtained in step 4, recrystallize at -10℃ for 1h, filter and take the residue to obtain nervonic acid.

[0066] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0067] Serial number Amount of alkali added / times Area normalized purity % Content % (based on dry product) Yield % 1 0.05 88.43 78.66 34.37 2 0.1 98.98 96.76 71.42 3 0.2 99.79 98.32 77.84 4 0.4 99.77 98.54 75.88 5 0.6 98.46 96.63 70.99 6 0.8 90.43 87.33 59.64 7 1.0 83.23 78.99 50.32

[0068] Example 2: Method for extracting high-purity nervonic acid from garlic fruit oil

[0069] Step 1, saponification-metal salt precipitation: take multiple portions of garlic oil from the same batch (1 kg), add 0.2 kg of NaOH to each portion, then add 95 v / v% methanol, 95 v / v% acetone, 95% v / v ethanol, and 10 L of water (numbered 1 to 3 respectively), heat and reflux for saponification for 75 min, cool and precipitate at 5°C for 2 h, and filter to remove the solution.

[0070] Step 2, acidification: add 15 (L) of 0.5 mol / L hydrochloric acid to the filter residue obtained in step 1 and acidify at 80°C for 60 min, stand at room temperature to separate the layers, discard the water layer, and retain the oil layer.

[0071] Step 3, water washing: The oil layer obtained in step 2 is washed with water three times.

[0072] Step 4, graded precipitation: add 6 L of 80 v / v% acetone to the oil layer obtained in step 3, carry out the first stage precipitation at 15°C for 1 hour, filter and discard the residue, carry out the second stage precipitation at -10°C for overnight, and filter.

[0073] Step 5, recrystallization: add 6 L of 90 v / v% acetone to the residue obtained in step 4, recrystallize at -10°C for 1 hour, and filter the residue to obtain nervonic acid.

[0074] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0075]

[0076] Example 3: Method for extracting high-purity nervonic acid from garlic fruit oil

[0077] Step 1, saponification-metal salt precipitation: take multiple portions of garlic oil from the same batch (1 kg), add 0.2 kg of NaOH to each portion, then add 10 L of 95 v / v% ethanol, heat and reflux for saponification for 15, 45, 75, and 105 min (serial numbers 1 to 4, respectively), cool and precipitate at 5°C for 2 h, and filter to remove the solution.

[0078] Step 2, acidification: add 8L of 0.5mol / L hydrochloric acid to the filter residue obtained in step 1 and acidify at 80℃ for 60min, stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0079] Step 3, water washing: The oil layer obtained in step 2 is washed with water three times.

[0080] Step 4, graded precipitation: add 6 L of 80 v / v% acetone to the oil layer obtained in step 3, carry out the first stage precipitation at 15°C for 1 hour, filter and discard the residue, carry out the second stage precipitation at -10°C for overnight, and filter.

[0081] Step 5, recrystallization: add 6 L of 90 v / v% acetone to the residue obtained in step 4, recrystallize at -10°C for 1 hour, and filter the residue to obtain nervonic acid.

[0082] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0083] Serial number Saponification reaction time min Area normalized purity % Content % (based on dry product) Yield % 1 15 90.33 87.22 45.67 2 45 98.67 97.65 73.37 3 75 99.79 98.32 77.84 4 105 94.32 92.33 68.23

[0084] Example 4: Method for extracting high-purity nervonic acid from garlic fruit oil

[0085] Step 1, saponification-metal salt precipitation: take multiple portions of garlic oil from the same batch (1 kg), add 0.2 kg of NaOH respectively, then add 10 L of 70, 80, 90, 100 v / v% methanol (serial numbers 1 to 4 respectively), heat reflux saponification for 75 min, cool at 5°C to precipitate for 2 h, and filter to remove the solution.

[0086] Step 2, acidification: add 9L of 0.5mol / L hydrochloric acid to the filter residue obtained in step 1 and acidify at 80℃ for 60min, stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0087] Step 3, water washing: The oil layer obtained in step 2 is washed with water three times.

[0088] Step 4, graded precipitation: add 6 L of 80 v / v% acetone to the oil layer obtained in step 3, carry out the first stage precipitation at 15°C for 1 hour, filter and discard the residue, carry out the second stage precipitation at -10°C for overnight, and filter.

[0089] Step 5, recrystallization: add 6L of 90v / v% acetone to the residue obtained in step 4, recrystallize at -10℃ for 1h, filter and take the residue to obtain nervonic acid.

[0090] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0091] Serial number Saponification reaction solvent concentration % Area normalized purity % Content % (based on dry product) Yield % 1 70 91.34 88.91 55.22 2 80 98.67 97.52 72.35 3 90 99.72 98.23 76.86 4 100 99.66 98.88 72.34

[0092] Example 5: Method for extracting high-purity nervonic acid from garlic fruit oil

[0093] Step 1, saponification-metal salt precipitation: take multiple portions of 1 kg of garlic oil from the same batch, add 0.4 kg of Mg(OH)2, KOH, and NaOH (serial numbers 1 to 3 respectively), then add 10 L of 95 v / v% ethanol, heat and reflux for saponification for 75 min, cool and precipitate at 5°C for 2 h, and filter to remove the solution.

[0094] Step 2, acidification: add 12L of 0.5mol / L hydrochloric acid to the residue obtained in step 1 and acidify at 80℃ for 60min, stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0095] Step 3, water washing: The oil layer obtained in step 2 is washed with water three times.

[0096] Step 4, graded precipitation: add 6 L of 80 v / v% acetone to the oil layer obtained in step 3, carry out the first stage precipitation at 15°C for 1 hour, filter and discard the residue, carry out the second stage precipitation at -10°C for overnight, and filter.

[0097] Step 5, recrystallization: add 6 L of 90 v / v% ethanol to the residue obtained in step 4, recrystallize at -10°C for 1 hour, filter, and take the residue to obtain neuraminic acid.

[0098] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0099] Serial number Type of alkali added Area normalized purity % Content % (based on dry product) Yield % 1 <![CDATA[Mg(OH)2]]> 99.83 98.97 76.33 2 KOH 99.79 98.32 73.84 3 NaOH 99.77 98.54 75.88

[0100] Example 6: Method for extracting high-purity nervonic acid from garlic fruit oil

[0101] Step 1, saponification-metal salt precipitation: take multiple portions of garlic oil from the same batch (1 kg), add 0.5 kg of Ca(OH)2 (serial numbers 1 to 3), then add 10 L of 95 v / v% ethanol, heat and reflux for saponification for 60 min, cool and precipitate at 5°C for 2 h, and filter to remove the solution.

[0102] Step 2, acidification: add 12L 1mol / L sulfuric acid to the filter residue obtained in step 1 and acidify at 70℃ for 60min, stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0103] Step 3, water washing: The oil layer obtained in step 2 is washed with water 5 times.

[0104] Step 4, graded precipitation: add 90v / v% acetone, 90v / v% ethanol, 90v / v% methanol, dichloromethane, and 4L of ethyl acetate (numbered 1 to 5 respectively) to the oil layer obtained in step 3, respectively, carry out the first stage precipitation at 20°C for 4 hours, filter and discard the residue, carry out the second stage precipitation at -10°C for 8 hours, and filter.

[0105] Step 5, recrystallization: add 4 L of 90 v / v% ethanol to the residue obtained in step 4, recrystallize at -10°C for 4 hours, filter, and take the residue to obtain neuraminic acid.

[0106] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0107]

[0108] Example 7: Method for extracting high-purity nervonic acid from garlic fruit oil

[0109] Step 1, saponification-metal salt precipitation: Take multiple portions of garlic oil from the same batch (1 kg), add 0.2 kg of Na(OH)2 to each portion, then add 10 L of 95 v / v% ethanol, heat and reflux for saponification for 45 min, cool and precipitate at 5°C for 2 h, and filter to remove the solution.

[0110] Step 2, acidification: add 8L of 0.8mol / L hydrochloric acid to the residue obtained in step 1 and acidify at 60℃ for 45min, stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0111] Step 3, water washing: The oil layer obtained in step 2 is washed with water 4 times.

[0112] Step 4, graded precipitation: add 4 L of methanol to the oil layer obtained in step 3, and perform the first stage precipitation (numbered 1 to 5 respectively) at 30, 20, 10, 0, and -10°C for 4 hours, filter and discard the residue, perform the second stage precipitation at -20°C for 8 hours, and filter.

[0113] Step 5, recrystallization: add 4 L of 90 v / v% ethanol to the residue obtained in step 4, recrystallize at -10°C for 4 hours, filter, and take the residue to obtain neuraminic acid.

[0114] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0115]

[0116] Example 8: Method for extracting high-purity nervonic acid from garlic fruit oil

[0117] Step 1, saponification-metal salt precipitation: Take multiple portions of garlic oil from the same batch (1 kg), add 0.4 kg of Na(OH)2 to each portion, then add 10 L of 90 v / v% ethanol, heat and reflux for saponification for 45 min, cool and precipitate at 5°C for 2 h, and filter to remove the solution.

[0118] Step 2, acidification: add 0.6L 1mol / L hydrochloric acid to the residue obtained in step 1 and acidify at 60℃ for 60min, stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0119] Step 3, water washing: The oil layer obtained in step 2 is washed with water three times.

[0120] Step 4, graded precipitation: add 4 L of methanol to the oil layer obtained in step 3, carry out the first stage precipitation at 20°C for 2, 4, 6, and 12 h (numbered 1 to 4 respectively), filter and discard the residue, carry out the second stage precipitation at -15°C for 5 h, and filter.

[0121] Step 5, recrystallization: add 5 L of 90 v / v% ethanol to the residue obtained in step 4, recrystallize at -20°C for 4 hours, filter, and take the residue to obtain neuraminic acid.

[0122] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0123]

[0124] Example 9: Method for extracting high-purity nervonic acid from garlic fruit oil

[0125] Step 1, saponification-metal salt precipitation: take 1 kg of garlic oil, add 0.2 kg of NaOH, then add 10 L of 90 v / v% ethanol, heat and reflux for saponification for 45 minutes, cool and precipitate at room temperature for 2 hours, and filter to remove the solution.

[0126] Step 2, acidification: Add 6L of 1mol / L acid water to the residue obtained in step 1 and acidify at 70℃ for 60min, let stand at room temperature to separate the layers, discard the water layer and retain the oil layer.

[0127] Step 3, water washing: The oil layer obtained in step 2 is washed with water 5 times.

[0128] Step 4, precipitation: take half of the oil layer obtained in step 3, add 95v / v% methanol, carry out the first stage precipitation at room temperature for 4 hours, filter and discard the filter residue, carry out the second stage precipitation at -10℃, overnight, filter, which is sequence number 1; take the other half of the oil layer obtained in step 3, add 95v / v% methanol at -10℃, precipitate overnight, filter, which is sequence number 2.

[0129] Step 5, recrystallization: add 90 v / v% ethanol to the residue obtained in step 4, recrystallize at -5°C for 6 hours, and filter the residue to obtain neuraminic acid.

[0130] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0131] Serial number Step 4: Precipitation method Area normalized purity % Content % (based on dry product) Yield % 1 Gradual precipitation 99.23 98.76 77.14 2 Direct precipitation 96.37 93.62 70.17

[0132] Example 10: Method for extracting high-purity nervonic acid from garlic fruit oil

[0133] Step 1, saponification-metal salt precipitation / saponification: Take 1 kg of garlic oil, add 0.2 kg of alkali, then add 10 L of 90 v / v% ethanol, heat and reflux for saponification for 45 min, cool and precipitate at room temperature for 2 h, filter and remove the solution, which is serial number 1. Take another 1 kg of garlic oil from the same batch, add 0.2 kg of alkali, then add 10 L of 90 v / v% ethanol, heat and reflux for saponification for 45 min, without cooling and filtering, which is serial number 2.

[0134] Step 2, acidification: add 6L of 1mol / L acid water to the residue / solution obtained in step 1, acidify at 70℃ for 60min, stand at room temperature to separate the layers, discard the water layer, and retain the oil layer.

[0135] Step 3, water washing: The oil layer obtained in step 2 is washed with water 5 times.

[0136] Step 4, graded precipitation: add 95 v / v% methanol to the oil layer obtained in step 3, carry out the first stage precipitation at room temperature for 4 hours, filter and discard the residue, carry out the second stage precipitation at -10°C for overnight, and filter.

[0137] Step 5, recrystallization: add 90 v / v% ethanol to the residue obtained in step 4, recrystallize at -5°C for 4 hours, filter and take the residue to obtain neuraminic acid.

[0138] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0139] Serial number Step 1: Is precipitation Area normalized purity % Content % (based on dry product) Yield % 1 Alkali metal precipitation 99.23 98.76 75.14 2 No precipitation 80.16 65.77 76.71

[0140] Example 11: Purity, content and yield of nervonic acid extracted by different methods

[0141] The preparation method of sequence number 1 is the same as sequence number 3 in Example 6.

[0142] The preparation method of No. 2 refers to the Chinese patent application with publication number CN 105503580A and invention name "A method for extracting and separating neuraminic acid", that is, 1 kg of garlic fruit oil from the same batch is added with 0.1 kg of quicklime and 10 L of 90 v / v% ethanol for extraction, the extract is concentrated to remove alcohol, 1 mol / L HCl is added to adjust the pH to 8, and 200 ml of dichloromethane is used for extraction 3 times, the dichloromethane layers are combined, the dichloromethane is concentrated to remove dichloromethane, 0.2 L of acetone is added for dissolution, C8 chromatography is used, the elution solvent is 95 v / v% ethanol, and the neuraminic acid content is tracked and monitored by a high performance liquid chromatograph, the components with higher content of neuraminic acid are mixed and concentrated, 1 L of 95 v / v% ethanol is added, and the mixture is placed at 5°C for 12 hours for recrystallization, and the neuraminic acid product is obtained by centrifugation and drying. The liquid phase detection spectrum of the obtained product is shown in the attached Figure 1 .

[0143] Preparation method No. 3 refers to the Chinese patent application with publication number CN107353199A and invention name "Method for separating and purifying neuraminic acid from garlic fruit oil", that is, take 1 kg of garlic fruit oil from the same batch and add 10 L of 10 g / v% NaOH aqueous solution, saponify at 80°C for 1 hour, let stand, extract with ether, discard the ether layer, add 10 v / v% sulfuric acid to the water layer to adjust the pH to 6, let stand and precipitate, then filter, dissolve the filter residue with 1 L of ether, discard the insoluble matter, crystallize at -10°C, filter, dissolve the filter residue with 0.8 L of acetone, discard the insoluble matter, crystallize at -10°C, filter, dissolve the filter residue with 0.6 L of petroleum ether, discard the insoluble matter, crystallize at -10°C, filter, and dry the neuraminic acid product.

[0144] The preparation method of No. 4 refers to the Chinese patent application with the authorization announcement number CN 1050117C and the invention name "Separation and Preparation Method of Cis-15-Tetradecenoic Acid", that is, take 1kg of the same batch of garlic fruit oil and add 10L of 10g / v% NaOH-ethanol solution, stir and reflux for 2h, then add 10L of 95% ethanol, place at -20℃ for 1h, filter under reduced pressure, add HCl to the filter residue to adjust the pH to 4, wash the oil layer with water to neutral after stratification, and separate the oil layer by vacuum distillation to obtain the nervonic acid product. The liquid phase detection spectrum of the obtained product is attached Figure 2 .

[0145] Preparation method No. 5 refers to the Chinese patent application with authorization announcement number CN 1050117C and invention name "Separation and Preparation Method of Cis-15-Tetracosenoic Acid", that is, take 1 kg of garlic fruit oil from the same batch and add 10 L of 10 g / v% NaOH-ethanol solution, stir and reflux for reaction for 2 hours, then add 10 L of 95% ethanol, let it stand at -20°C for 1 hour, filter under reduced pressure, add HCl to the filter residue to adjust the pH to 4, wash the oil layer with water to neutrality after stratification, discard the water layer, add 500 g of urea and 1 L of 95v / v ethanol to the oil layer to dissolve, and encapsulate at -10°C for 12 hours, filter, evaporate the filtrate to dryness, add 200 ml of water to dissolve, extract 3 times with 200 ml of dichloromethane, combine the dichloromethane layers, evaporate to dryness, and obtain the neuraminic acid product.

[0146] The purity content and yield of the obtained nervonic acid are shown in the following table:

[0147] Serial number Area normalized purity % Content % (based on dry product) Yield % 1 99.77 98.54 75.88 2 95.16 94.77 23.71 3 93.46 92.55 32.11 4 82.14 80.22 60.12 5 86.77 82.74 66.88

Claims

1. A method for extracting nervonic acid from garlic fruit oil, the method comprising the following steps: Step 1: saponification-metal salt precipitation: take garlic oil, add alkali, then add non-aqueous solvent, heat and reflux for saponification, cool and precipitate, filter, remove the solution, and obtain filter residue; Step 2: Acidification: Add acid water to the filter residue obtained in step 1 for acidification, let stand for stratification, discard the water layer, and obtain the oil layer; Step 3: Washing with water: washing the oil layer obtained in step 2 with water to obtain a water-washed oil layer; Step 4: graded precipitation: adding the washed oil layer obtained in step 3 to the first organic solvent, performing the first stage precipitation, filtering and discarding the filter residue; performing the second stage precipitation, filtering; Step 5: Recrystallization: Add the second organic solvent to the residue obtained in step 4, recrystallize, filter, and take the residue to obtain neuraminic acid.

2. The method according to claim 1, wherein: In step 1, the weight of the alkali is 0.1-0.6 times the weight of the garlic fruit oil, preferably 0.2-0.4 times; Preferably, in step 1, the base is selected from one or more of NaOH, KOH, NH4·H2O, Ca(OH)2, and Mg(OH)2; Preferably, in step 1, the non-aqueous solvent is selected from one or more of methanol, acetone, ethanol, acetonitrile and tetrahydrofuran; Preferably, in step 1, the volume (L) of the non-aqueous solvent is 8-15 times, preferably 10-15 times, the weight (kg) of the garlic fruit oil; Preferably, in step 1, the concentration of the non-aqueous solvent is 80 v / v%-100 v / v%, preferably 85 v / v%-95 v / v%.

3. The method according to claim 1 or 2, wherein: In step 1, the saponification time is 30 to 90 min, preferably 45 to 75 min; Preferably, in step 1, the temperature of the cooling precipitation is -5°C to room temperature, preferably 5°C to room temperature; Preferably, in step 1, the precipitation time is 1 to 4 hours.

4. The method according to any one of claims 1 to 3, wherein: In step 2, the acid water refers to an aqueous solution of HCl, H2SO4, or HNO3; Preferably, in step 2, the concentration of the acid water is 0.5-2 mol / L, preferably 0.5-1.5 mol / L; Preferably, in step 2, the volume (L) of the added acid water is 5-20 times, preferably 8-12 times, the weight (kg) of the garlic fruit oil.

5. The method according to any one of claims 1 to 4, wherein: In step 2, the acidification temperature is 50-95°C, preferably 55-75°C; Preferably, in step 2, the acidification time is 15 to 75 minutes, preferably 30 to 60 minutes; Preferably, in step 2, the temperature for standing and stratifying is 0°C to room temperature, preferably 5°C to room temperature.

6. The method according to any one of claims 1 to 5, wherein: In step three, the oil layer obtained in step two is washed with water for 2 to 7 times, preferably 3 to 6 times, to obtain a water-washed oil layer.

7. The method according to any one of claims 1 to 6, wherein: In step 4, the first organic solvent is selected from one or more of acetone, methanol and ethanol; Preferably, in step 4, the concentration of the first organic solvent is not less than 80 v / v%; Preferably, in step 4, the volume (L) of the first organic solvent added is 2-8 times, preferably 3-6 times, the weight (kg) of the garlic fruit oil.

8. The method according to any one of claims 1 to 7, wherein: In the first stage precipitation of step 4, the precipitation temperature is 5°C to room temperature, preferably 10°C to room temperature; Preferably, in the first stage precipitation of step 4, the precipitation time is 1 hour to overnight, preferably 4 hours to overnight; Preferably, in the second stage precipitation of step 4, the precipitation temperature is -20 to 5°C, preferably -15 to 0°C; Preferably, in the second stage precipitation of step 4, the precipitation time is 1 hour to overnight, preferably 2 to 6 hours.

9. The method according to any one of claims 1 to 8, wherein: In step 5, the second organic solvent is selected from one or more of methanol, ethanol and acetone; Preferably, in step 5, the volume (L) of the second organic solvent added is 2-8 times, preferably 3-6 times, the weight (kg) of the garlic fruit oil; Preferably, in step 5, the recrystallization temperature is -20°C to room temperature, preferably -15 to 10°C; Preferably, in step five, the recrystallization temperature is 1 h to overnight, preferably 2 h to overnight.

10. The method according to any one of claims 1 to 9, wherein: The method comprises the following steps: Step 1, saponification-metal salt precipitation: take garlic fruit oil, add alkali (including NaOH, KOH, NH4·H2O, Ca(OH)2, Mg(OH)2, etc.) in an amount of 0.1-0.6 times the weight of the garlic fruit oil, then add 80-100 v / v% non-aqueous solvent, the volume (L) of which is 8-15 times the weight (kg) of the garlic fruit oil, heat and reflux saponification for 30-90 minutes, cool and precipitate at -5°C to room temperature for 1-4 hours, and filter to remove the solution; Step 2, acidification: add 0.5-2 mol / L acid water to the filter residue obtained in step 1, the volume (L) of the added acid water is 5-20 times the weight (kg) of the garlic oil, acidify at 50-95° C. for 15-75 min, stand at 0° C. to room temperature for stratification, discard the water layer, and retain the oil layer; Step 3, water washing: the oil layer obtained in step 2 is washed with water 2 to 7 times; Step 4, graded precipitation: add 80-100 v / v% acetone, methanol, or ethanol to the oil layer obtained in step 3, the volume (L) of which is 2-8 times the weight (kg) of garlic oil, carry out the first stage precipitation at 5°C to room temperature for 1 hour to overnight, filter and discard the filter residue, carry out the second stage precipitation at -20 to 5°C for 1 hour to overnight, and filter; Step 5, recrystallization: add 70-100 v / v% methanol, ethanol or acetone to the residue obtained in step 4, the volume (L) of which is 2-8 times the weight (kg) of garlic oil, recrystallize at -20°C to room temperature for 1 hour to overnight, and filter the residue to obtain nervonic acid; Preferably, the method comprises the following steps: Step 1, saponification-metal salt precipitation: take garlic fruit oil, add alkali (including NaOH, KOH, NH4·H2O, Ca(OH)2, Mg(OH)2, etc.) in an amount of 0.2-0.4 times the weight of the garlic fruit oil, then add 85-95v / v% ethanol, the volume (L) of which is 10-15 times the weight (kg) of the garlic fruit oil, heat reflux saponification for 45-75min, cool and precipitate at 5°C to room temperature for 1-4h, and filter to remove the solution; Step 2, acidification: add 0.5-1.5 mol / L acid water (including HCl, H2SO4, HNO3, etc.) to the filter residue obtained in step 1, the volume (L) of the added acid water is 8-12 times the weight (kg) of the garlic oil, acidify at 55-75°C for 30-60min, stand at 5°C to room temperature for stratification, discard the water layer, and retain the oil layer; Step 3, water washing: the oil layer obtained in step 2 is washed with water 3 to 6 times; Step 4, graded precipitation: after washing the oil layer obtained in step 3, add 90-100 v / v% acetone, methanol, or ethanol to the oil layer, the volume (L) of which is 3-6 times the weight (kg) of garlic oil, carry out the first stage precipitation at 10°C to room temperature for 4 hours to overnight, filter and discard the filter residue, carry out the second stage precipitation at -15 to 0°C for 2 to 6 hours, and filter; Step 5, recrystallization: Add 80-100 v / v% methanol, ethanol or acetone to the residue obtained in step 4, and its volume (L) is 3-6 times the weight (kg) of garlic oil. Recrystallize at -15-10°C for 2h to overnight, filter, and take the residue to obtain neuraminic acid.

Images