TWI426963B - Preparation of high purity caffeic acid phenethyl ester micro - nano - powder by supercritical fluid anti - crystallization - Google Patents

Description

Method for producing high-purity caffeic acid phenethyl acrylate micro-nano powder by supercritical fluid anti-solvent crystallization

The invention relates to a method for producing phenethyl acetonate micro-nano powder, in particular to a method for producing high-purity caffeic acid phenethyl micro-nano powder by supercritical fluid anti-solvent crystallization.

Caffeic acid phenethyl ester (CAPE) is a derivative of propolis caffeic acid and is also one of the important biologically active components in propolis. According to research, phenethyl acetoacetate has antioxidant capacity, scavenges oxygen free radicals, reduces DNA damage caused by liver oxidation, anti-inflammatory, anti-viral, and a pharmaceutical composition for treating and preventing cancer in the past (Taiwan invention patent announcement) No. 561045) can inhibit the biological activity of growth of oral cancer cells, human blood cancer cells HL-60, and lymphoma cell line U937.

In the past, a method for separating natural products and a separating device thereof (Taiwan Invention Patent Publication No. I269664), the separation method uses natural propolis as a raw material, and uses a supercritical fluid anti-dissolution method to precipitate phenethyl catechol component, but The high recovery rate can be obtained by most separation tanks, and the cost of separating and purifying phenethyl acrylate from natural propolis is very high, so it is more important to synthesize phenethyl caffeate using enzymes. At present, the industry uses caffeic acid (CA) and phenethyl alcohol for enzyme synthesis, but the synthesized caffeic acid phenethyl ester is not easy to be granulated from phenylethyl alcohol. The liquid acid chromatograph is used to separate and collect caffeic acid. Phenylethyl ester requires a large amount of solvent and is time consuming, which imposes a burden on the environment and cost.

Accordingly, it is an object of the present invention to provide a method for producing high purity caffeic acid phenethyl acrylate micronized rice by using a green solvent and conveniently producing a supercritical fluid resistant to crystallization.

Thus, the method for producing high-purity caffeic acid phenethyl micro-nanofine powder by supercritical fluid anti-solvent crystallization of the present invention comprises the following steps:

(i) treating a mixture of phenethyl caffeate synthesized by an enzyme dissolved in isooctane and phenylethyl alcohol at a low temperature to obtain a mixture of first phenylethyl alcohol;

(ii) treating the first phenylethanol mixture with an ultrasonic hot water wash and centrifuging to obtain a second phenylethyl alcohol mixture;

(iii) dissolving the second phenylethyl alcohol mixture in methanol and treating it with a supercritical carbon dioxide solution crystallization to obtain a phenethyl catenate particle.

The effect of the manufacturing method of the present invention is that the supercritical carbon dioxide anti-solvent crystallization treatment can effectively reduce the use amount of the organic solvent to produce a green solvent, and can enable the caffeic acid benzene in the second phenylethyl alcohol mixture. The ethyl ester is precipitated by supersaturation to form particles, which in turn produces an effect of convenient production.

The foregoing and other objects, features, and advantages of the invention will be apparent from the

Referring to Figure 1, a preferred embodiment of the method for producing high purity caffeic acid phenethyl acrylate microalgae by supercritical fluid anti-solvent crystallization comprises the following steps:

Step 101 is to treat a mixture of phenethyl caffeate synthesized by an enzyme dissolved in isooctane (2,2,4-Trimethylpentane) and phenylethyl alcohol at a low temperature to obtain a mixture of first phenylethyl alcohol. The mixture of phenethyl caffeate and a portion of isooctane are dissolved in phenylethyl alcohol to form the first phenethyl alcohol mixture. Preferably, the temperature of the low temperature liquid separation treatment is between -10 ° C and 20 ° C, and the treatment time is between 12 hours and 24 hours.

Step 102: treating the first phenethyl alcohol mixture with an ultrasonic hot water washing process and centrifuging to obtain a second phenylethyl alcohol mixture, and removing one of the first phenethyl alcohol mixtures by the ultrasonic hot water washing treatment. Caffeic acid component. Preferably, the temperature of the ultrasonic hot water washing treatment is between 40 ° C and 60 ° C, and the processing time is between 20 minutes and 40 minutes. Further, preferably, the centrifugal processing speed is 10,000 rpm, and the processing time is 30 minutes.

Step 103 is to dissolve the second phenylethyl alcohol mixture in methanol and then treat it with a supercritical carbon dioxide solution crystallization to obtain a phenethyl catenate particle. Using supercritical anti-solvent (SAS) characteristics of carbon dioxide supercritical fluid, the ethyl phenethyl acrylate in the second phenylethyl alcohol mixture is supersaturated and precipitated in the reaction tank to form the coffee. Acid phenethyl ester particles. It is worth mentioning that the feed concentration and feed time of the second phenylethyl alcohol mixture are the main variables affecting the purity and particle size of the phenethyl catenate particles. Preferably, the second phenylethanol mixture feed rate is between 0.5 ml/min and 1.0 ml/min and the feed concentration is between 4 mg/ml and 8 mg/ml.

Hereinafter, the production method of the present invention will be further described by the following specific examples.

<Specific Example 1 - Ultrasonic washing to remove caffeic acid >

The phenethyl catecholate mixture synthesized by the enzyme was obtained from the fermentation laboratory of the ZTE University Center. The mixture of phenethyl caffeate synthesized by dissolving the enzymes of isooctane and phenylethyl alcohol was placed in a refrigerator at -10 ° C for 24 hours, and then taken out, separated by a separating funnel, and the lower first phenylolamine mixture was collected. At this time, phenethyl caffeate was all dissolved in this layer.

Under the condition of constant temperature 40 ° C, water and phenylethyl alcohol layer volume ratio of 30:1, ultrasonic washing was carried out for 20 minutes, and then centrifuged at 10,000 rpm for 30 minutes in a high-speed centrifuge to remove the lower layer for the first time. The water-washed first phenylethyl alcohol mixture was washed once with the above conditions, and then centrifuged to obtain the second layer of the second water-washed second phenylethyl alcohol mixture.

< HPLC analysis - mixture of first phenylethanol mixture and second phenylethyl alcohol >

Referring to Figure 2, a standard sample containing phenethyl caffeate and caffeic acid (99.8% CAPE + 99.9% CA) was configured and analyzed by liquid chromatography (HPLC) at a wavelength of 325 nm (Figure 2 A4), the first phenethyl alcohol mixture (a3 of Figure 2), the first water-washed second phenylethyl alcohol mixture (a2 of Figure 2), and the second water-washed second phenylethyl alcohol mixture (Figure 2 A1), and using 3,4-dimethoxybenzaldehyde (IS) as an internal standard reference, it can be observed from the HPLC analysis that the caffeic acid component cannot be measured in the second phenylethanol mixture after the second water washing, which is indicated by Ultrasonic hot water washing can effectively remove caffeic acid. Wherein, the phenylethyl alcohol in the first phenethyl alcohol mixture per ml is dissolved in 14.9 mg of phenethyl caffeate, and the total area of phenethyl caffeate is 98.3%, the total area of caffeic acid is 1.1%, and total Other unknowns with an area of 0.6%. Further, the phenylethyl alcohol in the second water-washed second phenylethanol mixture per ml was dissolved in 159.2 mg of phenethyl caffeate, and the total area of phenethyl caffeate was 99.4%.

The recovery rate of phenethyl caffeate after removal of caffeic acid by the ultrasonic hot water washing treatment was calculated to be 76%. Recovery rate = (Phenylethyl acrylate content in the second phenylethyl alcohol mixture / phenylethyl acrylate content in the first phenylethyl alcohol mixture) × 100%

<Specific Example 2 - Supercritical carbon dioxide anti-solvent recrystallization to purify phenethyl catechol in the second phenylethyl alcohol mixture >

Referring to Figure 3, 0.152 ml of this water-washed second phenylethyl alcohol mixture was metered, dissolved in 6 ml of a fixed volume of methanol solvent, and formulated into a feed solution having a concentration of 4 mg/ml, and placed in a graduated cylinder 211.

First, the carbon dioxide liquid is pressed out from the siphon tube in the cylinder 201, and after the external circulating water tank 202 is lowered to 4 ° C, the liquid carbon dioxide is sent to the preheating tank 204 through the carbon dioxide pump pressurizing module 203, and preheated into supercritical The carbon dioxide, preheating temperature is controlled by a temperature control box 205, wherein the preheating temperature is 55 °C.

By observing the pressure values of the pressure gauges 206, 207, adjusting the pressures of the back pressure valves 208, 209, the precipitation tank 210 is adjusted to a pressure of 150 bar and a temperature of 65 ° C and controlled to experimental conditions, and then the carbon dioxide supercharged mode is adjusted. Group 203 delivers the pump stroke frequency of carbon dioxide, regulates the valve opening below the settling tank 210, and records the cumulative flow rate of the wet flow meter to a carbon dioxide flow rate of 15 L/min.

After the experimental temperature, pressure, and flow rate were stabilized, the feed solution in the graduated cylinder 211 was introduced into the HPLC pump 213 through the filter tube 212 at a feed flow rate of 0.5 ml/min, and sent to the precipitation tank 210 for solvent-resistant crystallization. The bottom opening 214 of the sedimentation tank 210 is connected with a 1/4" to 1/2" tube, and a Nylon filter paper (0.45 μm) of about 1 cm outer diameter and two stainless steel mesh (37 μm) supporting filter paper are placed inside to collect the filter paper. Precipitate the particles.

After the feed solution enters, 1.5 ml of methanol solvent is pumped in and the remaining feed solution in the tube is pushed into the precipitation tank 210. After the anti-solvent time reaches (about 3 minutes to 12 minutes), the HPLC pump 210 is turned off. However, 150 L of isothermal carbon dioxide having isothermal isostatic pressure is continuously supplied to remove and dry residual solvent in the precipitation tank 210.

Finally, the back pressure valve 209 is adjusted, the pressure of the sedimentation tank 210 is released to normal pressure, and the filter is opened to take out the filter paper. The precipitate adhering to the inner wall of the tube of the precipitation tank 210 is sent to the precipitation tank 210 by HPLC pump 213, and the solvent is concentrated under reduced pressure, and combined with the precipitate on the filter paper, and weighed. To calculate the total yield.

<Specific Example 3 - Supercritical Carbon Dioxide Anti-Soluble Recrystallization Purification of Phenylethyl Caffeate in the First Phenylethanol Mixture >

The phenethyl caffeate in the first phenylethyl alcohol mixture was prepared in the procedure of Specific Example 2.

< HPLC analysis - caffeic acid phenethyl ester precipitate >

Referring to Figure 4, the phenethyl caffeate precipitate was dissolved in 1 ml of ethanol solvent and determined by high performance liquid chromatography (Specific Example 2) (a1 in Figure 4) and <Specific Example 3 In the concentration of phenethyl caffeate of the precipitate in (as in a2 of Fig. 4), it was observed that the precipitate of the specific example 2 could not measure the caffeic acid component. Here, the composition of the precipitate of the <Specific Example 2> was phenethyl caffeate having a total area of 99.4%, and the particle diameter of the precipitate was 69 nm.

The yield of caffeic acid phenethyl ester was calculated to be 73.5%. Wherein the yield = (precipitate weight / feed weight) × 100%, in addition, the feed weight = (the content of phenethyl acrylate in the second phenylethyl alcohol mixture / the phenethyl acrylate in the second phenylethyl alcohol mixture) HPLC area percentage).

In summary, the method for producing high-purity caffeic acid phenethyl micro-nanofine powder by supercritical fluid anti-solvent crystallization of the present invention can attain the following effects and advantages, thereby achieving the object of the present invention:

1. The caffeic acid component of the first phenethyl alcohol mixture is removed by the ultrasonic hot water washing treatment to produce an effect of increasing the purity of the caffeic acid phenethyl ester particles.

Second, the use of the supercritical carbon dioxide anti-solvent crystallization treatment can effectively reduce the amount of organic solvent used, and produce the effect of using a green solvent.

3. The supercritical carbon dioxide anti-solvent crystallization treatment can supersaturate the caffeic acid phenethyl ester in the second phenylethyl alcohol mixture to form particles, thereby producing a convenient production system.

The above is only the preferred embodiment of the present invention, and the scope of the invention is not limited thereto, that is, the simple equivalent changes and modifications made by the scope of the invention and the description of the invention are All remain within the scope of the invention patent.

201. . . Cylinder

202. . . Circulating sink

203. . . Pump booster module

204. . . Preheating tank

205. . . Temperature control box

206. . . Pressure gauge

207. . . Pressure gauge

208. . . Back pressure valve

209. . . Back pressure valve

210. . . Precipitation tank

211. . . Measuring cylinder

212. . . Filter tube

213. . . HPLC pump

214. . . Opening

1 is a flow chart showing a preferred embodiment of a method for producing high-purity caffeic acid phenethyl acrylate micro-nanofine powder by supercritical fluid anti-solvent crystallization according to the present invention;

Figure 2 is an HPLC analysis diagram showing the results of analysis of a standard sample, a first phenethyl alcohol mixture, a first water washed second phenylethyl alcohol mixture, and a second water washed second phenylethyl alcohol mixture;

Figure 3 is a schematic view of a device for supercritical carbon dioxide anti-solvent crystallization treatment of the preferred embodiment; and

Figure 4 is a HPLC analysis diagram showing the results of analysis of the first phenylethyl alcohol mixture and the second water-washed second phenylethyl alcohol mixture after the supercritical carbon dioxide solution crystallization treatment.

Claims (5)

The invention relates to a method for producing high-purity caffeic acid phenethyl acrylate micro-nano powder by supercritical fluid, comprising the following steps: (i) synthesizing a mixture of phenethyl caffeate synthesized by an enzyme dissolved in isooctane and phenylethyl alcohol a low temperature liquid separation treatment to obtain a first phenylethyl alcohol mixture; (ii) treating the first phenylethyl alcohol mixture with an ultrasonic hot water washing, and centrifuging to obtain a second phenylethyl alcohol mixture; and (iii) The second phenylethyl alcohol mixture is dissolved in methanol and treated with a supercritical carbon dioxide solution crystallization to obtain a phenethyl phenate particle. The method for producing high-purity caffeic acid phenethyl acrylate micro-nano powder according to the supercritical fluid anti-solvent crystal according to claim 1, wherein the temperature of the low-temperature liquid separation treatment in the step (i) is -10 ° C to Between 20 ° C, the treatment time is between 12 hours and 24 hours. The method for producing high-purity caffeic acid phenethyl acrylate micro-nano powder according to the supercritical fluid anti-solvent crystal according to claim 2, wherein the temperature of the ultrasonic hot-washing treatment in step (ii) is 40 ° C to Between 60 ° C, the treatment time is between 20 minutes and 40 minutes. A method for producing high-purity caffeic acid phenethyl micro-nanofine powder according to the supercritical fluid anti-solvent crystal according to claim 3, wherein the centrifugal treatment speed in the step (ii) is 10,000 rpm, and the treatment time is 30 minutes. The method for producing high-purity caffeic acid phenethyl acrylate micro-nano powder according to the supercritical fluid anti-solvent crystal according to claim 4, wherein the second phenylethanol mixture feed flow rate is 0.5 ml in the step (iii) The feed concentration is between 4 mg/ml and 8 mg/ml between /min and 1.0 ml/min.