Wuxi Cima Science Co., Ltd, located in Wuxi city, China, is a professional manufacturer and supplier of bulk raw ingredients for nutritional supplements. Cima Science produces the world’s first commercial AEA ingredient and owns several patents on anandamide manufacturing, purification, and applications. Below is one of Cima’s patent on the anandamide purification process.

Introduction to Anandamide

This invention is in the category of the technical field of separation and purification of amide compounds in organic chemistry and specifically relates to a method for purifying anandamide.

Anandamide is the first endocannabinoid analog discovered in humans. In these years, there have been more and more literature reports on the relationship between the change of anandamide and disease. Some researchers believe that anandamide, the primary endogenous ligand of the cannabis receptor, may play a role as a precursor of illness or a marker of disease occurrence in many conditions.

At present, most studies on the efficacy and biological metabolic pathways of anandamide are based on in vivo extraction, with low content and complicated processing. If it can be synthesized in vitro and enriched and purified to obtain higher purity arachidonoyl ethanolamide, it will not only benefit basic scientific research but will also be expected to be used in drug therapy.

According to the different synthetic pathways of the target product, the separation and purification methods are also different. The process for preparing AEA by the oil method has the characteristics of vast raw material sources, low equipment investment, and suitability for industrialization. However, in addition to the by-product glycerol and unreacted monoethanolamine, the crude product of the reaction also contains more other saturated or unsaturated fatty acid ethanol amides.

Main Steps of purifying anandamide

The invention discloses a purification method of anandamide, which includes:

  • preparing an ethanol solution of urea
  • mixing reaction crude product with an ethanol solution of urea
  • low-temperature inclusion
  • extraction of filtrate
  • extraction of the organic phase
  • water washing
  • drying
  • decompression concentration
  • anandamide product.

The separation and purification process was carried out at a relatively low temperature, avoiding the destruction of unsaturated fatty acids. The purity of the final anandamide product in AEA powder or AEA oil form was over 63%. The main components were anandamide, oleic acid monoethanolamide, linoleic acid monoethanolamide, and anandamide.

Features of this anandamide purification method:

  • Preparation of mixed fatty acid monoethanolamide
  • Urea was used to separate fatty acid monoethanolamides with different alcohol amide saturation;
  • The filtrate was obtained by separating the solid and liquid phases by vacuum filtration;
  • The filtrate is extracted and concentrated to obtain unsaturated fatty acid monoethanolamide rich in anandamide
  • The mixed fatty acid monoethanolamide is prepared, wherein the raw materials for preparing the mixed fatty acid monoethanolamide are microbial algal oil and monoethanolamides, and the content of arachidonic acid in the microbial algal oil accounts for 30% – 40%;

The main content of this anandamide patent

Because of the problems in the existing purification methods of anandamide, this paper proposes a novel method for the AEA purification.

Thus, the purpose of the present invention is to overcome the shortcomings of the prior inventions and provide a new purification method for anandamide, which provides a reference for enriching AEA from mixed fatty acid monoethanolamide.

As a preferred scheme of the purification method, the raw materials for preparing mixed fatty acid monoethanolamide are microbial algal oil and monoethanolamine, and the content of arachidonic acid in the microbial algal oil accounts for 30% – 40% by mass percentage.

As a preferred scheme of the purification method of the invention, the microbial algal oil and monoethanolamine are mixed according to the molar ratio of 1:1 ~ 1:30.

As a preferred scheme of the purification method of the invention, after the microbial algal oil is mixed with monoethanolamine, an alkaline catalyst with 0.1~ 0.5% of the total mass of the reaction substrate is added, and the alkaline catalyst is the methanol solution of sodium methoxide.

As a preferred scheme of the purification method of the invention, after adding 0.1~ 0.5% of the total mass of the reaction substrate, adding n-hexane, absolute ethanol, or a mixture of the two, stirring reaction at 25~ 60 ℃ for 0.5~ 6 H.

As a preferred scheme of the purification method of the present invention, the separation of fatty acid monoethanolamide with different saturations by urea includes: according to the volume and mass ratio, 95% ethanol: urea = 2~ 8:1, 95% ethanol and urea are respectively taken, heated and refluxed in 60 ~ 75 ℃ water bath until urea is completely dissolved, and then mixed fatty acid ethanolamide: urea = 1 according to the mass ratio. The mixture of fatty acid monoethanolamide urea ethanol was obtained by adding 2-6 crude product, heating and refluxing to clear and transparent.

As a preferred scheme of the purification method of the invention, after obtaining the mixed fatty acid monoethanolamide urea ethanol mixture, it also includes cooling the mixed fatty acid ethanolamide urea ethanol mixture to room temperature, standing at – 40 ℃ to 10 ℃ for 6 h to 24 h, and then decompressing and filtering, collecting the filtrate to obtain arachidone rich Polyunsaturated fatty acid monoethanolamide urea ethanol mixture.

As a preferred scheme of the purification method of the present invention, after obtaining the polyunsaturated fatty acid monoethanolamide urea ethanol mixed solution rich in anandamide, adding the obtained polyunsaturated fatty acid monoethanolamide urea ethanol mixed solution into the anhydrous ether and 55 ~ 75 ℃ hot water, extracting, separating and combining organic phase, drying and decompression After concentration, the liquid oil was obtained, and the solvent was removed. Anandamide product with a purity of 45% ~ 65% was obtained.

As a preferred scheme of the purification method of the invention, the drying method adopts anhydrous Na2SO4 for drying.

As a preferred scheme of the purification method of the present invention, after the extraction and before the separation and combination of organic phases, the water phase is urea solution, which is crystallized at a low temperature, and then crystallized again after the filtrate is concentrated, and the urea crystal can be recycled and dried for utilization.

The invention takes the microbial algal oil rich in arachidonic acid as the acyl of anandamide.

In addition, the crude product of mixed fatty acid monoethanolamide was purified by urea. This method has the advantages of simple operation, low cost, easy availability of reagent and urea, and mild reaction conditions. It provides a reference value for further enrichment and purification of high purity anandamide.

Figure 1 shows the analysis chart of fatty acid ethanolamide in the crude product by HPLC-ELSD, and the retention time of mixed fatty acid ethanolamide in the figure is 26.309 min.

mixed fatty acid ethanolamide retention time

Fig. 2 shows the analysis chart of fatty acid composition of mixed fatty acid monoethanolamide, in example 1 by GC; the retention time of anandamide in the figure is 23.215 min.

Anandamide retention time

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