KR20010105019A - Method for extracting phytosterol with high purity in a high yield - Google Patents

Abstract

The present invention relates to a high purity separation method of phytosterol, which is one of physiologically active substances. More specifically, after the dried organolpi is chopped, ethanol is added about 10 times to the weight of the organolpi sliced and extracted for about 6 hours at a temperature of 60 to 80 ℃ using an extractor equipped with a reflux cooler. 30-5 potassium hydroxide or sodium hydroxide solution was added to the extracted filtrate and heat-treated at 50-70 degreeC for about 1 hour. Subsequently, the mixture was concentrated under reduced pressure to remove alcohol, and then a mixed solvent was added so that the ratio of distilled water and hexane was about 2: 1. Then, the mixture was shaken for about 10 minutes and centrifuged to take only the supernatant. By concentrating and drying the extract under reduced pressure, a light brown phytosterol powder can be produced in high yield and high purity. The phytosterol obtained through the above process has a high calorie suppression effect as well as an anti-obesity effect, resulting in high calorie processed foods. And it can be usefully used as an additive of functional foods such as diet.

Description

Method for extracting phytosterol with high purity in a high yield}

The present invention relates to a high purity separation method of phytosterol, which is one of physiologically active substances.

Phytosterol, also known as a natural cholesterol-stopper, is known to have an excellent effect on lowering blood cholesterol levels in animals. Research reports on the separation and effect of active ingredients from plants have been widely reported, but very few cases have been directly studied on the separation and purification of phytosterols.

To date, most of the technologies applied for the separation and production of active ingredients from plants have been mainly extracted and separated from crude chemicals of low purity by using a single solvent. However, these crude extracts have a low purity and thus have insufficient effects, thus limiting their use or satisfying consumer's needs, thus failing to create new demand. That is, a method of obtaining a useful compound from existing natural products is by hot water extraction or extraction from various plants using a polar or nonpolar solvent such as methanol, ethanol, butanol, chloroform, ethyl ether and the like.

Therefore, the inventors of the present invention first extracted phytosterol, which is one of the active ingredients having a physiologically active function, and then introduced the saponification process to the extract using the property that the phytosterol was not saponified, and optimized the extract as well as using a specific solvent. Completion of the present invention by confirming that high purity phytosterols can be selectively recovered by removing impurities contained in the present invention, and that the produced phytosterols inhibit cholesterol absorption and are effective in inhibiting obesity. It was.

The present invention is to develop an efficient production method of high-purity phytosterol in view of the above situation, and studied the type and amount of each step of solvent and additives, reaction conditions, etc. for the raw material ogalpi, and the efficacy of the extracted material In this regard, the experimental animals were disclosed and their effects were investigated.

In order to achieve the above object, the present invention is primarily heat extraction with an organic solvent, an alkaline solution is added to the extract and subjected to the saponification process, characterized in that the re-extraction using a mixed solvent of distilled water and a specific organic solvent Provided is a method for extracting phytosterols from plants in high purity.

Specifically, the high purity extraction method of phytosterol of the present invention

1) heating extraction by adding an organic solvent to the dried, shredded plants;

2) adding an alkaline solution to the extract and heat treating the extract;

3) concentration under reduced pressure to remove alcohol;

4) shaking mixing by adding a mixed solvent of one or more solvents selected from hexane, methyl ether and ethyl ether with distilled water; And

5) centrifugation to obtain a supernatant.

As a plant to which the phytosterol extracting method of the present invention can be applied, Acanthopanacis Cortex Radicis is preferable, but is not limited thereto. In general, any plant known to have a high phytosterol content may be used.

In the above, the organic solvent of step 1 is preferably methanol or ethanol. In the case of using ethanol and methanol, the production yield of the extract as well as the phytosterol content of the extract is higher than in the case of using a solvent generally used for extracting the active ingredients of plants such as hexane, butanol, ethyl ether, methyl ether, chloroform, and distilled water Also, the effect is about 2 times higher.

Preferably, step 1 is to extract high purity phytosterol by adding about 10 times methanol or ethanol to the sample weight and extracting about 6 hours at a temperature of 60 ~ 80 ℃ using an extraction device equipped with a reflux condenser. Can be.

On the other hand, the alkaline solution of step 2 is preferably potassium hydroxide or sodium hydroxide, and the saponification process is preferably made by adding 3 to 5 weight of the alkaline solution and heating at 50 to 70 ° C. for about 1 hour.

More preferably, the saponification process of step 2 is advantageous in terms of production yield and purity of phytosterol, by adding about 5 weight percent of potassium hydroxide solution to the extract and heat-processing at a temperature of about 60 ° C. for about 1 hour.

The production yield tends to decrease as the amount of alkali added increases, but the phytosterol content tends to be similar or increase. Also, as the saponification temperature increases, the production yield tends to increase.

On the other hand, as the mixed solvent of step 4, the ratio of methyl ether: distilled water, ethyl ether: distilled water or hexane: distilled water is preferably 1: 1 to 1: 3, and the mixed solvent is about 5 times the sample weight. It is preferred to add and shake for about 10 minutes. Although ethyl ether is more effective than methyl ether, it has been found that the content of phytosterol is lower than that of hexane.

The high purity phytosterol extraction method of the present invention may further include a step of concentrating and drying the supernatant obtained by centrifugation under reduced pressure.

Hereinafter, the present invention will be described in detail.

The sample was used as Acanthopanacis Cortex Radicis, and the dried product was dried at a moisture of 7 or less and cut into 2 cm or less in size. As shown in Table 1, 8 kinds of solvents, such as hexane, were added 10 times the amount of each sample and 6 hours at 80 ° C. The active ingredient was first extracted using an extractor equipped with a reflux condenser. As a result, when extracted with ethanol and methanol, the highest yield was 7.2 and 7.3, respectively. The phytosterol contained in the extract was confirmed to be 38 as capillary gas chromatography (capillary GC). However, because ethanol is less toxic than methanol, methanol was selected as the primary extraction solvent. In order to remove other impurities except phytosterol from the extracted extract, an attempt was made to purify phytosterol by introducing a saponification process using properties that are not saponified since phytosterol is a kind of vegetable sterols.

As a basic process, 30 NaOH or KOH solution was added to the primary solvent extract by varying the amount of solution, and then saponified at 80 ° C. for 1 hour. After saponification, in order to separate phytosterol which is not saponified, a solvent mixed with water and ethyl ether 1: 1 is added 5 times of the sample volume, shake-mixed for 10 minutes, and centrifuged to take an ethyl ether layer. , Concentrated and dried to give the phytosterol product. The optimum conditions determined by the test of each process were adopted in the next process to determine the optimum conditions for each process.

As a method of removing phytosterol and other substances by saponification, NaOH and KOH of 30 were used as an alkali necessary for saponification for the purification test of phytosterol. As a result of measuring the production yield and phytosterol content for each addition amount was shown in Table 2. As the amount of alkali increased, the production yield tended to decrease. The content of phytosterol was the highest when added at 3 levels with 30 NaOH and the highest when added at 5 levels with 30 KOH.

As a result of measuring the yield and phytosterol content of the extract according to the saponification temperature during the separation of phytosterol by the saponification method was shown in Table 3. As the saponification temperature increased, the production yield also tended to increase, but the phytosterol content was the highest when added at the 3rd level at 60 ℃ or higher, and the highest when added at the 5th level when 30KOH was used.

Therefore, it was determined that the addition amount of 30KOH was added to the primary solvent extract 5. In addition, in order to determine the reaction temperature at the time of the saponification test in the range of 40 ℃ to 90 ℃ when the saponification for 1 hour at 60 ℃ was the highest content of phytosterol 85. However, production yield tended to increase with higher reaction temperature. Therefore, the reaction conditions during saponification were determined to be tested at 60 ° C. for 1 hour. The saponified material was shaken for 10 minutes by adding 5 times the amount of sample to a solvent mixed with water and ethyl ether, and then centrifuged and removed. The ethyl ether layer was taken, concentrated and dried to obtain a phytosterol product. . In order to more efficiently improve the separation process of phytosterol through the mixed solution of ethyl ether and water, the result of testing by adjusting the ratio of water and mixed solution by using ethyl ether, methyl ether and hexane was shown as in Table 4. Ethyl ether was more effective than methyl ether, but the phytosterol content was lower than that of hexane. Thus, the mixed solution was adjusted to a ratio of hexane and water of 1: 2 in consideration of production yield and phytosterol content. The production yield at this time was 1.8, phytosterol content was 92. The main composition of phytosterols was determined by capillary gas chromatography (Supelco's model No. SAC-5), resulting in campesterol 59, stigmasterol 23, β-sitosterol 10 and other 8 It appeared to consist.

The results of animal tests to investigate the biofunctionality of the phytosterol produced are as follows. For the experimental animals, 49 Sprague-Dawley male rats of 4 weeks old were used as general solid feed (Samyang) before the experiment, and the average weight of 325 ± 5g was used for this test for 4 weeks. The test was divided into five groups of seven animals as shown in Table 5. In other words, the control group fed the high fat diet (Table 6) and water, and the test group divided the water and the phytosterol extract into the pay group fed 0.5 mg, 1.0 mg, 3.0 mg, 5.0 mg, 7.0 mg, and 9.0 mg, respectively. The temperature of the feeding room was 22 ± 2 ℃, humidity was around 60, and the contrast was controlled every 12 hours. Water and feed were freely consumed.

Weight gain, feed intake, and feed efficiency of the rats fed the experimental diet for 4 weeks are shown in Table 7. Body weight gain decreased with increasing phytosterol supplementation, and significantly decreased when phytosterol was fed over 3mg / 100g rat / day. Feed intake was not significantly different according to phytosterol supplementation, and feed efficiency was significantly lower than control group when phytosterol was fed more than 3mg / 100g rat / day. The serum lipid content of rats fed the experimental diet for 4 weeks was shown in Table 8. Neutral lipids in serum decreased with increasing phytosterol supplementation, and the minimum concentration of phytosterols in which neutral lipids decreased was 1mg / 100g rat / day. Total cholesterol content in serum decreased with administration of phytosterol, and the minimum level of significance was 1 mg / 100 g rat / day.

The HDL-cholesterol content was higher in the diet fed phytosterol compared to the control fed high fat diet and water, and significantly improved when fed 1 mg / 100 g rat / day or more. Serum phospholipid content increased with higher levels of phytosterol and significantly increased when fed more than 1 mg / 100 g rat / day. The risk factor index (RFI), which is the difference between total cholesterol and HDL-cholesterol to total cholesterol, was significantly lower (0.09) when fed at 9 mg / 100 g rat / day compared to 0.45. Significant changes in RFI levels were identified when phytosterol levels were above 1 mg / 100 g rats / day. Therefore, when the phytosterol extract was administered to rats, the minimum concentrations of the test items showing the anti-obesity effect and the synergistic inhibitory effect of blood cholesterol level were 3mg / 100g rat / day and 1mg / 100g rat / day, respectively.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the following examples illustrate the present invention, and the scope of the present invention is not limited thereto.

Example 1 Extraction of Phytosterol from Ogalpi

1 kg of dried organolpi (water 4 or less) was cut to a size of 2 cm or less and ethanol was added 10 times to the organolpi weight, and extracted at a temperature of 80 ° C. for 6 hours using an extractor equipped with a reflux condenser. 30 potassium hydroxide solution was added to the extracted filtrate and heat-treated at 60 ° C for 1 hour. Subsequently, the resultant was concentrated under reduced pressure to remove alcohol, and then a mixed solvent was added five times to the sample weight so that the ratio of distilled water and hexane was 2: 1. The mixture was shaken for 10 minutes and centrifuged to obtain a supernatant. The extract was concentrated under reduced pressure and dried to produce 18 g of a light brown phytosterol powder.

<Example 2> Production yield of phytosterol according to the type of solvent

Cut the Acanthopanacis Cortex Radicis dried to less than 7 cm in size to 2 cm or less, and add eight solvents, such as hexane, 10 times the sample volume, as shown in Table 1, and attach the reflux cooler at 80 ° C for 6 hours. The active ingredient was first extracted using the extracted extractor. The results are shown in Table 1 below. In the following table, the production yield was calculated by dividing the total solid extracted by the weight of the sample and multiplying by 100, and the content of phytosterol was obtained from the total amount of phytosterol analyzed by capillary gas chromatography. In addition, each of the superscripts a, b, c, d, and e means that a difference (p <0.05) is recognized for an average value of different characters in the same column.

Type of solvent Production yield () Phytosterol content () Hexane 5.1 ^b 31 ^b ethanol 7.2 ^a 38 ^a Methanol 7.4 ^a 38 ^a Butanol 2.5 ^d 16 ^c Ethyl ether 2.7 ^d 17 ^c Methyl ether 3.2 ^c 17 ^c chloroform 2.3 ^d 13 ^d Distilled water 1.6 ^e 18 ^c

As a result, when extracted with ethanol and methanol, the highest yield was 7.2 and 7.3, respectively. The phytosterol contained in the extract was confirmed to be 38 as capillary gas chromatography (capillary GC). However, because ethanol is less toxic than methanol, methanol was selected as the primary extraction solvent.

<Example 3> Production yield of phytosterol according to the type and amount of alkali

For the purification test of phytosterol by the method of removing phytosterol and other substances by saponification, NaOH and KOH of 30 were used as the alkali required for saponification, and the production yield and phytosterol content were measured for each addition amount. Appeared as

Alkali type Addition amount () Production yield () Phytosterol content () 30NaOH One 3.2 ^a 52 ^b 3 2.8 ^b 57 ^a 5 2.6 ^b 53 ^b 10 1.6 ^c 54 ^b 30KOH One 4.5 ^a 57 ^c 3 4.2 ^a 61 ^b 5 3.6 ^b 67 ^a 10 2.4 ^c 58 ^c

As shown in the table, the production yield tended to decrease as the amount of alkali increased, and the content of phytosterol was the highest when added at 3 levels when 30 NaOH was used, and when added at 5 levels when 30 KOH was used. Highest.

<Example 4> Production yield of phytosterol according to the saponification temperature

In order to investigate the production yield and phytosterol content of extracts according to the saponification temperature during the separation of phytosterol by saponification method, the results of experiments varying the saponification temperature to 40, 50, 60, 70, 80 and 90 ° C. Shown in

Reaction temperature (℃) Production yield () Phytosterol content () 40 1.2 ^d 67 ^c 50 1.7 ^c 78 ^b 60 2.5 ^b 85 ^a 70 2.8 ^b 80 ^b 80 3.6 ^a 67 ^c 90 3.7 ^a 63 ^c

As a result, as the saponification temperature increased, the production yield tended to increase, but the phytosterol content was the highest when added at the 3 level at 60 ° C or higher, and the highest at the 5 level when 30 KOH was used.

Therefore, it was determined that the addition amount of 30KOH was added to the primary solvent extract 5. In addition, in order to determine the reaction temperature at the time of the saponification test in the range of 40 ℃ to 90 ℃ when the saponification for 1 hour at 60 ℃ was the highest content of phytosterol 85. However, production yield tended to increase with higher reaction temperature. Therefore, the reaction conditions during saponification were determined to be tested at 60 ° C. for 1 hour.

<Example 5> Production yield of phytosterol according to the mixing ratio of the solvent for removal of the saponification and phytosterol separation

In order to more efficiently improve the separation process of phytosterol through the mixed solution of ethyl ether and water, the test results by adjusting the ratio of water and mixed solution by using ethyl ether, methyl ether, hexane, etc. are shown in Table 4 below. .

Solvent Composition Mixing ratio of solvent Production yield () Phytosterol content () Methyl Ether: Distilled Water 1: 1 1.5 ^c 71 ^d 1: 2 1.4 ^d 72 ^d 1: 3 1.2 ^e 78 ^bc Hexane: Distilled Water 1: 1 2.3 ^a 86 ^c 1: 2 1.8 ^b 92 ^a 1: 3 1.5 ^c 93 ^a Ethyl ether: distilled water 1: 1 2.5 ^a 85 ^bc 1: 2 2.0 ^b 86 ^bc 1: 3 1.6 ^c 88 ^b

As can be seen from the above results, ethyl ether was more effective than methyl ether, but the phytosterol content was lower than hexane, so that the ratio of hexane and water was adjusted to 1: 2 in consideration of production yield and phytosterol content. The solution was determined as the final blending ratio, and the yield was 1.8 and the phytosterol content was 92. The main composition of phytosterols was determined by capillary gas chromatography (Supelco's model No. SAC-5), resulting in campesterol 59, stigmasterol 23, β-sitosterol 10 and other 8 It appeared to consist.

Experimental Example 1 Investigation of Biofunctionality of Phytosterols

For the experimental animals, 49 Sprague-Dawley male rats of 4 weeks old were used as general solid feed (Samyang) before the experiment, and the average weight of 325 ± 5g was used for this test for 4 weeks. The test was divided into five groups of seven animals as shown in Table 5. In other words, the control group fed the high fat diet (Table 6) and water, and the test group divided the water and the phytosterol extract into the pay group fed 0.5 mg, 1.0 mg, 3.0 mg, 5.0 mg, 7.0 mg, and 9.0 mg, respectively. The temperature of the feeding room was 22 ± 2 ℃, humidity was around 60, and the contrast was controlled every 12 hours. Water and feed were freely consumed. Table 5 and Table 6 show the composition of the high-fat diet for measuring the experimental dietary composition and phytosterol feeding effect, respectively.

Test treatment Experimental Dietary Composition Control High Fat Diet + Water HP 0.5 High Fat Diet + 0.5 mg / 100 g rat / day + Water HP 1.0 High Fat Diet + 1.0 mg / 100 g rat / day + Water HP 3.0 High Fat Diet + 3.0 mg / 100 g rat / day + Water HP 5.0 High Fat Diet + 5.0 mg / 100 g rat / day + Water HP 7.0 High Fat Diet + 7.0 mg / 100 g rat / day + Water HP 9.0 High Fat Diet + 9.0 mg / 100 g rat / day + Water

Ingredient content() Casein 20.0 Corn oil 10.0 Lard 10.0 Starch 49.9 cellulose 5.0 Mineral mixture 3.5 Vitamin mixture 1.0 Coline chloride 0.2 DL-methionine 0.3 cholesterol 0.1

Weight gain, feed intake and feed efficiency of the rats fed the experimental diet for 4 weeks are shown in Table 7. All values in the table below represent the mean values of 7 rats each.

Test treatment Weight gain (g / 4 weeks) Feed Intake (g / day) Feed efficiency (F.E.R) Control 101 ^a 25.1 ^a 0.14 ^a HP 0.5 98 ^a 25.3 ^a 0.14 ^a HP 1.0 96 ^a 25.2 ^a 0.14 ^a HP 3.0 78 ^b 25.7 ^a 0.11 ^b HP 5.0 75 ^b 24.8 ^a 0.11 ^b HP 7.0 70 ^c 26.1 ^a 0.10 ^c HP 9.0 69 ^c 26.0 ^a 0.10 ^c

Body weight gain decreased with increasing phytosterol supplementation, and significantly decreased when phytosterol was fed over 3mg / 100g rat / day. Feed intake was not significantly different according to phytosterol supplementation, and feed efficiency was significantly lower than control group when phytosterol was fed more than 3mg / 100g rat / day. The serum lipid content of rats fed the experimental diet for 4 weeks was shown in Table 8.

Test treatment Neutral lipids Total cholesterol HLD cholesterol Phospholipids RFI Control 124 ^a 89 ^a 49 ^c 137 ^c 0.45 ^a HP 0.5 121 ^a 83 ^a 52 ^c 136 ^c 0.37 ^a HP 1.0 113 ^b 78 ^b 60 ^b 140 ^b 0.23 ^b HP 3.0 95 ^c 79 ^b 62 ^ab 143 ^a 0.22 ^b HP 5.0 84 ^d 76 ^b 63 ^ab 144 ^a 0.17 ^bc HP 7.0 76 ^e 75 ^b 65 ^a 143 ^a 0.13 ^c HP 9.0 73 ^e 75 ^b 68 ^a 144 ^a 0.09 ^c

Unit: mg / 100ml

* Risk factor index (RFI) = (Total Cholesterol-HDL Cholesterol) / Total Cholesterol

Neutral lipids in serum decreased with increasing phytosterol supplementation, and the minimum concentration of phytosterols in which neutral lipids decreased was 1mg / 100g rat / day. Total cholesterol content in serum decreased with administration of phytosterol, and the minimum level of significance was 1 mg / 100 g rat / day.

The HDL-cholesterol content was higher in the diet fed phytosterol compared to the control fed high fat diet and water, and significantly improved when fed 1 mg / 100 g rat / day or more. Serum phospholipid content increased with higher levels of phytosterol and significantly increased when fed more than 1 mg / 100 g rat / day. The risk factor index (RFI), which is the difference between total cholesterol and HDL-cholesterol to total cholesterol, was significantly lower (0.09) when fed at 9 mg / 100 g rat / day compared to 0.45. Significant changes in RFI levels were identified when phytosterol levels were above 1 mg / 100 g rats / day. Therefore, when the phytosterol extract was administered to rats, the minimum concentrations of the test items showing the anti-obesity effect and the synergistic inhibitory effect of blood cholesterol level were 3mg / 100g rat / day and 1mg / 100g rat / day, respectively.

The production technology of phytosterols developed by the present invention is expected to provide an opportunity to mass-produce and utilize phytosterols because it is a new method that can economically produce phytosterols, which are functional materials, from plants such as organolpi in high purity.

The developed phytosterol may be used in a variety of processed foods such as seasoned bulgogi, health supplements and other beverages, alcoholic beverages. In particular, the use of phytosterol can have a cholesterol lowering effect and an obesity suppressing effect at the same time, so when it is added to high calorie processed foods and diet foods, it is expected to generate a great demand.

Claims (11)

1) heating extraction by adding an organic solvent to the dried, shredded plants; 2) adding an alkaline solution to the extract and heat treating the extract; 3) concentration under reduced pressure to remove alcohol; 4) shaking mixing by adding a mixed solvent of one or more solvents selected from hexane, methyl ether and ethyl ether with distilled water; And 5) characterized in that it comprises the step of obtaining a supernatant by centrifugation, A method of extracting high purity phytosterols from plants. The method of claim 1, wherein the plant is Acanthopanacis Cortex Radicis. The method of extracting phytosterol of high purity from a plant according to claim 1, wherein the organic solvent of step 1 is methanol or ethanol. The method according to claim 1, wherein step 1 is about 10 times of methanol or ethanol is added to the sample weight, and extraction is performed for about 6 hours at a temperature of 60 to 80 ℃ using an extraction device equipped with a reflux condenser. A method of extracting high purity phytosterols from plants. The method of extracting phytosterol of high purity from a plant according to claim 1, wherein the alkaline solution of step 2 is potassium hydroxide or sodium hydroxide. The method of extracting high purity phytosterol from the plant according to claim 1, wherein the saponification process of step 2 is performed by adding an alkaline solution 3 to 5 by weight and heating at 50 to 70 DEG C for about 1 hour. The method according to claim 1, wherein the saponification process of step 2 comprises adding about 5 weight percent of potassium hydroxide solution to the extract and heat-processing the plant for about 1 hour at a temperature of about 60 ° C. How to extract. The method of claim 1, wherein the mixed solvent of step 4 is a ratio of methyl ether: distilled water, ethyl ether: distilled water or hexane: distilled water in a ratio of 1: 1 to 1: 3, wherein the phytosterol of high purity is extracted from the plant. Way. The method of extracting high purity phytosterol from the plant according to claim 1, wherein the mixed solvent of step 4 is added about 5 times the sample weight and shaken for about 10 minutes. The method of extracting high purity phytosterol from the plant, characterized in that it further comprises the step of concentrating and drying the supernatant obtained by centrifugation under reduced pressure. Extracted by the method of claim 1, the extract of galpi containing high purity phytosterol, characterized in that it has blood cholesterol level lowering or obesity inhibitory activity.