METHOD OF SEPARATING OIL-SOLUBLE PIGMENT AND WATER-SOLUBLE PIGMENT FROM NATURAL MATERIAL USING AEROGELS AND AN APPARATUS THEREFOR

Abstract

A method of separating oil-soluble pigment and water-soluble pigment from natural material using aerogels characterized in comprising the steps of: (A) immersing a natural material in a leaching solvent to extract a pigment; (B) mixing the leaching solution from which pigment is extracted in step (A) with aerogel to adsorb oil-soluble pigment to aerogels; (C) separating oil-soluble pigment adsorbed on the surface of aerogel in step (B); (D) drying and concentrating the pigment separated in step (C), enhancing a method of separating from natural material the initial substance for pharmaceuticals, food, cosmetics, etc. facilitating a safer and more efficient production process compared to the conventional separation method.

Claims

1. A method of separating oil-soluble pigment and water-soluble pigment from natural material using aerogels characterized in comprising the steps of: (A) immersing a natural material in a leaching solvent to extract a pigment; (B) mixing the leaching solution from which pigment is extracted in step (A) with aerogel to adsorb oil-soluble pigment to aerogel; (C) separating oil-soluble pigment adsorbed on the surface of aerogels in step (B); (D) drying and concentrating the pigment separated in step (C).

2. A method of separating oil-soluble pigment and water-soluble pigment from natural material using aerogels further comprising the steps of: (E) separating water-soluble pigment which is not adsorbed on the surface of aerogel after the step (B); and (F) drying and concentrating the water-soluble pigment from step (E).

3. The method according to claim 1, wherein the leaching solvent in the step (A) is 95% ethanol, and the pigment adsorption step (B) is performed in the condition that carbon dioxide partial pressure is 36-44 mmHg; and wherein the separation of oil-soluble pigment adsorbed on the surface of aerogels in the pigment separation step (C) is practiced with hexane.

4. A method of separating oil-soluble pigment and water-soluble pigment from natural material using aerogels characterized in that the oil-soluble pigment or the water-soluble pigment separated through the method of claim 1 is any one of flavonoids, hesperidin, oil-soluble carotenoids, or hesperetin.

5. An apparatus for separating oil-soluble pigment and water-soluble pigment from natural material using aerogels characterized in comprising: a natural material tank for extracting pigment from natural material; an adsorption tank connected to the natural material tank and mixing and reacting the leaching solution leached from the natural material tank and aerogels; and a pigment separation tank provided with aerogels adsorbed with oil-soluble pigment in the adsorption tank to separate oil-soluble pigment with organic solvent, wherein a cooler is mounted for maintaining the temperature at 20 C., a regeneration tank for transferring waste aerogels from the pigment separation tank and volatilizing organic solvent is installed, and a heater is equipped in the pigment separation tank and the regeneration tank in order to remove the organic solvent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 represents the molecular structures of hesperidin (A) and hesperetin (B).

[0017] FIG. 2 discloses comparison results of (A) 50 ug hesperidin STD control, and purification efficiency of the flavonoid hesperidin purified respectively by (B) the ether extraction method of 0.0005% of citrus peel ethanol extraction liquid, and (C) the aerogel purification method of 0.0005% of citrus peel ethanol extraction liquid.

[0018] FIG. 3 shows purification efficiency of hesperidin purified respectively by the ether extraction method and the aerogel purification method.

[0019] FIG. 4 shows comparison results of (A) 50 ug Hesperetin STD control, and the cartenoid hesperetin purified respectively by (B) the ether extraction method of 5% of citrus peel ether extraction liquid and (C) the aerogel purification method of 5% of hexane-purified citrus peel adsorption liquid.

[0020] FIG. 5 shows purification efficiency of hesperetin purified by the ether extraction method and the aerogel purification method.

[0021] FIG. 6 shows a flow chart of the system for separating oil-soluble pigment and water-soluble pigment from natural material by the above experiments.

[0022] FIG. 7 is a schematic diagram showing a pigment extraction step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention.

[0023] FIG. 8 is a schematic diagram showing a pigment adsorption step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention.

[0024] FIG. 9 is a schematic diagram showing a pigment separation step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention.

[0025] FIG. 10 is a schematic diagram showing a drying and concentration step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0026] The objective of the present invention is to provide a method of separating oil-soluble substance from natural materials. In general, in order to produce a basic source for cosmetics, health food, pharmaceuticals, etc. from natural material, an organic solvent is used to purify oil-soluble and water-soluble material. The most commonly used nonpolar solvent is mostly highly inflammable and difficult to be safely dealt with, and is very harmful to the human body.

[0027] A representative organic solvent, ether, is a highly volatile and anesthetic colorless liquid which is very harmful to the human body. The vapor thereof, heavier than air, is likely to spread to the bottom of the production facility, causing a risk of fire.

[0028] In addition, the current inspection and testing standards for medicines are in line with those of the Korean Food and Drug Administration. However they only determine the content criteria, but fail to provide a standard for actual purification method. With regulations at home and abroad becoming strict, separation of the production procedure to comply with the GMP regulation leads to increase of the production cost.

[0029] The present invention provides a safer and more effective methodological standard for purifying natural materials, and also provides a comprehensive production standard for producing API such as flavonoid and carotinoid. Hereinafter, the present invention will be described in more detail with reference to specific examples.

[0030] I. Extraction of Pigment Mixture Liquid from Natural Materials

[0031] As one embodiment of the present invention, a water-soluble pigment, hesperidin (C.sub.28H.sub.34O.sub.15) and an oil-soluble pigment, hesperetin (C.sub.16H.sub.14O.sub.6) are separated and purified from Jeju citrus fruits peel. Hesperidin is water-soluble flavonoid with a molecular weight of 610 g/mol, and hesperetin is oil-soluble carotenoid with a molecular weight of 302 g/mol. FIG. 1 shows the molecular structure of hesperidin and hesperitin.

[0032] To isolate the water-soluble pigment and the oil-soluble pigment from Jeju citrus fruits, initially a pigment mixture liquid is extracted from citrus fruit peel. 1.8 kg of dried Jeju citrus fruit peel is crushed and immersed in a leaching solvent such that the peel is submerged for 30 days, wherein the leaching solution is 95% alcohol. Following the immersion step, a pigment mixture solution is obtained in which pigment of citrus fruit peel is mixed with the immersion liquid and extracted. Then, the resulting extract is filtered through a 0.45 nm membrane filter to remove impurities.

[0033] II. Comparison Experiment of Extraction Efficiency of Water-Soluble Flavonoid, Hesperidin

[0034] 1. Ether Extraction from a Pigment Mixture

[0035] 500 ml of ether was mixed with 1 L of the pigment mixture solution from which impurities are removed, followed by stirring (200 rpm). The well-stirred mixture was placed in a burrette and separated in layers to isolate the ethanol layer except ether. From the separated ethanol layer, 500 g of the ethanol layer is concentrated in a reduced pressure at 20 C. for 5 hours, and crystalized to test for water-soluble flavonoid hesperidin.

[0036] 2. Purification Using an Aerogel from a Pigment Mixture Solution

[0037] 1 L of citrus fruit peel extract from which impurities are filtered in the above procedure of extraction of pigment mixture liquid from natural materials is added with 2 g of aerogel 1-20 m (Silica aerogel, REMTECH, KOREA) and mixed to the degree that dissolved carbon dioxide (CO.sub.2) is detected as to 10% (5,000 ppm) and allowed to stand for 3 days. To perform ionization reaction of extraction solvent and physically disperse the contained material, carbon dioxide is injected into the reaction tank and airtightly sealed to react, wherein the partial pressure of carbon dioxide is 36-44 mmHg and the density is 3, 150 mg/cm.sup.3. In this process, the oil-soluble pigment is adsorbed on the porous surface of aerogels and the water-soluble pigment remains in the solution.

[0038] After 3 days, ethanol extract is filtered through hollow fiber membrane filter of 1.2 nm to remove impurities including aerogels and from the ethanol extract layer, 500 g of the ethanol extract is concentrated in a reduced pressure at 20 C. for 5 hours, and crystalized to test for water-soluble flavonoid hesperidin.

[0039] 3. Comparison of Ether Extration Method and Aerogel Purification Method

[0040] In order to compare the content of flavonoid hesperidin purified respectively with the extraction method and the aerogel purification method, the crystallization materials prepared from the previous step are diluted in methanol in 1, 5, 10, 15, and 20% of concentration, wherein the standard hesperidin for the test is 98 purchased from Sigma, SHIMADZU UFLC was used for the test, Column is C18 column (5 um), flow rate is 1 ml/min, wavelength is 280 nm, mobile phase (Methanol:water) is 3:7, and the measurement temperature is 35 C.

[0041] FIG. 2 shows comparison of flavonoid hesperidin contents (each 0.0005%) purified respectively by the ether extraction method (B) and the aerogel purification method (C) with 50 g of a control (A). As seen in FIG. 2, the peak of hesperidin was shown at an expression time of 7 minutes and 50 seconds.

[0042] FIG. 3 shows purification efficiency of hesperidin separated respectively by the ether extraction method and the aerogel purification method. The extracts separated by the aerogel purification method were diluted in concentration of 5, 10, 15, and 20%, and the extracted separated by the ether purification method were diluted in concentration of 0.005, 1, 5, 10 and 20%, and the purification efficiency of hesperidin is compared using UFLC. And the results are shown in Table 1.

TABLE-US-00001 TABLE 1 The purification efficiency of hesperidin using UFLC Data no. concentration area(Total) Data 1(Aerogel) 5% 3063098 Data 2(Aerogel) 20% 4260196 Data 3(Aerogel) 15% 3559940 Data 4(Aerogel) 10% 3502130 Data 5(Ether) 20% 3017274 Data 6(Ether) 10% 1488931 Data 7(Ether) 5% 114132 Data 8(Ether) 1% 6892 Data 9(Ether) 0.0005% Data 10(Standrad) 50 ug Hesperidin STD 226399

[0043] As shown in FIG. 3 and Table 1, the concentration of hesperidin purified by aerogel purification method was higher than that by the ether extraction method, with a difference of more than 1.5-8 times in the purification efficiency.

[0044] III. Comparison Experiments of Extraction Efficiency of Oil-Soluble Carotenoid, Hesperetin

[0045] 1. Ether Extraction from a Pigment Mixture Solution

[0046] 150 ml of oil-soluble pigment extracted in ether in the above step II.1 was concentrated under reduced pressure at 20 C. for 5 hours to be crystallized, and then the crystalized pigment was diluted in hexane respectively in 0.0005, 0.005, 0.5, 1.5, 1, 5, 10, 15, and 20% concentration for testing hesperetin.

[0047] 2. Purification Using Aerogels from Pigment Mixture Solution

[0048] In the above step II.2, aerogel immersed in the ethanol extract of citrus fruit peel are obtained and oil-soluble pigment adsorbed on the aerogel is dissolved with hexane. And the purified oil-soluble substance obtained by passing 1-20 m of aerogel layer (Silica aerogel, REMTECH, KOREA) through hexane is concentrated under reduced pressure at 20 C. for 5 hours to be crystallized. Then, these crystallized substance is diluted with hexane to have a concentration of 0.0005, 0.005, 0.5, 1.5, 1, 5, 10, 15, and 20% for testing hesperetin as in the above 111.1.

[0049] 3. Comparison of the Ether Extraction Method and the Aerogel Purification Method

[0050] In order to compare the content of carotenoid hesperetin purified respectively with the ether extraction method and the aerogel purification method, the crystallization materials prepared from the previous step are diluted in methanol in 0.0005, 0.005, 0.5, 1.5, 1, 5, 10, 15, and 20% of concentration, wherein the standard hesperetin for the test is 98, purchased from Waters.

[0051] SHIMADZU UFLC was used for the test, which is used in the same condition as in the case of the above water-soluble flavonoid as follows: column is C18 column (5 m), flow rate is 1 ml/min, wavelength is 280 nm, mobile phase (Hexane: water) is 3:7, and the measurement temperature is 35 C.

[0052] FIG. 4 shows comparison of flavonoid hesperidin contents (each 0.0005%) purified respectively by the ether extraction method (B) and the aerogel purification method (C) with a control (A). As seen in FIG. 4, the peak of hesperetin content was shown at an expression time of 15 minutes and 50 seconds.

[0053] FIG. 5 shows purification efficiency of hesperetin separated respectively by the ether extraction method and the aerogel purification method. The extracts separated by the aerogel purification method were diluted in concentration of 1, 5, 10, 15, and 20%, and the extracts separated by ether purification method were diluted in concentration of 0.005, 1, 5, 10 and 20%, and the purification efficiencies of hesperetin in both cases are compared using UFLC. And the results are shown in Table 2.

TABLE-US-00002 TABLE 2 The purification efficiency of hesperetin using UFLC Data no. concentration area(Total) Data 1(Aerogel) 5% 9050918 Data 2(Aerogel) 20% 34825184 Data 3(Aerogel) 15% 27939977 Data 4(Aerogel) 10% 23965971 Data 5(Ether) 20% 581006 Data 6(Ether) 15% 235559 Data 7(Ether) 10% 1284 Data 8(Ether) 5% 449 Data 9(Ether) 1% Data 10(Aerogel) 1% Data 11(Ether) 0.5% Data 12(Ether) 0.005% Data 13(Ether) 0.0005% Data 14(Standrad) 50 ug Hesperidin STD 54807

[0054] As shown in FIG. 5 and Table 2, the concentration of hesperetin purified by the aerogel purification method (Data 1-4, 10) was higher than that by the ether extraction method (Data 5-9). Compared with the fact that when using the ether extraction method, the separation rate of hesperetin was very low, it was confirmed that hesperetin was successfully adsorbed in higher rate when using the aerogel purification method.

[0055] IV. Separation Production System of Oil-Soluble Pigment and Water-Soluble Pigment

[0056] FIG. 6 represents a flow chart of a system for separating oil-soluble pigment and water-soluble pigment extracted from natural material through the above experiments. The process of basic separation of oil-soluble pigment and water-soluble pigment from natural material has been practiced with organic solvent extraction methods, failing to meet the GMP standard and causing various problems in safety management. However, these problems can be easily solved by separating the oil-soluble substance and the water-soluble substance from natural material using the porosity and hydrophobicity of the aerogels.

[0057] The separation method according the present invention for separating the oil-soluble substance and the water-soluble substance from natural material using aerogels comprises the steps of (A) extracting pigment, (B) adsorbing pigment, (C) separating pigment, and (D) drying and concentrating.

[0058] 1. The Step (A) of Extracting Pigment

[0059] FIG. 7 is a schematic diagram showing a pigment extraction step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention. It is desirable to use the natural material in the form of powder, which is dissolved in ethanol, and the dissolved natural material may be placed in a natural material tank for a certain period so that it has a predetermined CO2 partial pressure. To this end, the pigment extraction apparatus comprises a natural material tank (B-Tank(1)), a stirrer (M) formed inside the natural material tank, a pump (P) and an air regulator (R) connected to the natural material tank and supplying gas to maintain a proper partial pressure of CO2, and a pressure valve (pV) for regulating the same.

[0060] The natural material powder is accurately measured with a microbalance and added into the natural material tank containing ethanol, followed by completely suspending by means of a stirrer (M). After stirring, using a pump (P), an air regulator (R) and a pressure valve (pV), the natural material tank may have a stable partial pressure of CO2, and the tank pressure regulating valve becomes shut off. The above procedure can be automatically controlled.

[0061] 2. The Step (B) of Adsorbing Pigment

[0062] FIG. 8 is a schematic diagram showing a pigment adsorption step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention. When the natural material is leached for a period of time, the leaching solution is mixed with aerogels to adsorb the pigment.

[0063] First, the leaching solution is transferred to the adsorption tank (C-Tank(2)). At this time, the input valve (I) is shut off and the pressure valve (pV) and the lower drain valve (dV) are opened to put the leaching solution into the adsorption tank, wherein the leaching solution is transferred and measured coincidentally using a controlled volume pump, and the impurities therein can be removed by filtration during the transfer. When the transfer is completed, the stirring operation is performed using a stirrer (M) formed inside the adsorption tank (C-Tank(2)).

[0064] The aerogel powder previously filled in the aerogel tank (E-Tank(3)) is transferred to the adsorption tank (C-Tank(2)). In the mean time, the input valve (I) is shut off to fill the pressure, and a constant pressure is applied to transfer the aerogels to the adsorption tank (C-Tank(2)). In the adsorption tank, while the aerogels are being transferred, the natural material leaching solution is continuously stirred. And when the transfer of aerogels are completed, a stirring operation is performed during a predetermined period after shutting off the input valve(I).

[0065] 3. The Step (C) of Separating Pigment

[0066] FIG. 9 is a schematic diagram showing a pigment separation step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention. After adsorbing the hydrophobic substance with aerogels, the stirring is stopped for a predetermined period of time to allow the supernatant solution layer to separate from the aerogels. When the layer seperation is completed, the pressure valve (pV) and the drain valve (dV) are opened to transfer the supernatant solution layer to the supernant solution tank (Bulk-tank (2)) and to transfer the aerogels to the pigment separation tank (D-Tank (4)).

[0067] Hexane is added to the pigment separation tank (D-Tank(4)) containing aerogels in order to separate oil-soluble substance. At this time, a cooler (D) is mounted on the pigment separation tank to cool the reactants to remain at the temperature of 20 C., followed by stirring using a stirrer (M). After stirring at atmospheric pressure for a certain period of time, the reactants are allowed to stand at 20 C. for a predetermined time, so that the layer is separated. Then using an elevator, the tank is moved upward.

[0068] 4. The Step (D) of Concentrating Pigment

[0069] FIG. 10 is a schematic diagram showing a drying and concentration step of the system for separating oil-soluble pigment and water-soluble pigment from natural material according to the present invention. The oil-soluble substance is dissolved in a hexane layer. The waste aerogels are transferred to the regeneration tank (F-Tank(5)) cooled to 20 C. by opening a drain valve (dV) of the pigment separation tank (D-Tank(4). The upper part of the regeneration tank is equipped with a heater to heat the waste aerogels, and the stirrer (M) is operated to stir aerogels to volatilize the remaining hexane. The volatilized hexane is recovered through a heat exchanger.

[0070] The hexane layer containing the oil-soluble substance remains at 20 C. to be stored. Thereafter, the heater formed inside the pigment separation tank (D-Tank(4)) is operated to volatilize the hexane and the volatilized hexane is recovered with a heat exchanger. The tank is moved downward using an elevator and dried, and the dried substance is recovered with the opening of drain valve (dV).

[0071] 5. The Step (E) of Separating Water-Soluble Pigment

[0072] As seen in FIG. 9, non-absorbed water-soluble pigment onto the aerogels even after the pigment adsorption step (B) is moved to the bulk tank along the supernatant solution to separate the pigment. After the stirring step is completed and the layers are separated, the supernatant solution is transferred to the supernatant solution tank (bulk-tank (2)) by opening the pressure valve (pV).

[0073] 6. The Step (F) of Concentrating Water-Soluble Pigment

[0074] The water-soluble pigment from the step (E) is dried under reduced pressure, and dried and concentrated into powder.

[0075] As described above, by using the present separation method of separating oil-soluble pigment and water-soluble pigment by means of aerogels, the safety management problem possibly caused by the use of organic solvent can be solved, and at the same time, compared with the separation purification method using organic solvent, the yield is increased more than 5% in the experiment stage, and more than 800% in the production stage, confirming the economic effect.

[0076] The present method of separating oil-soluble pigment and water-soluble pigment from natural material using aerogels and the apparatus therefor enhance a method of separating the initial substance from natural material for pharmaceuticals, food, cosmetics, etc. facilitating safer and more efficient production process, compared to the conventional separation method. Therefore, the present invention is industrially useful and effective.