Method for separating collagen from liposuction effluent using supercritical process

Abstract

The present invention relates to a method for extracting collagen from a liposuction effluent, wherein collagen is extracted by treating a collagen-containing liposuction effluent in the presence of a supercritical fluid. According to the present invention, conventionally discarded collagen in a liposuction effluent can be extracted at high purity, and the extracted high purity collagen can be widely used in medical, pharmaceutical, and cosmetic products.

Claims

1. A method for extracting Type I collagen in a polymer state from a liposuction effluent using supercritical fluid extraction comprising, (a) introducing a liposuction effluent into a supercritical extraction device and supplying CO.sub.2, which is an extraction solvent, to an extraction reactor, and further adding ethanol as a co-solvent in an amount of 50 to 200% (w/w) with respect to the liposuction effluent; (b) maintaining the pressure of the CO.sub.2 supplied to the extraction reactor at 200 to 300 bar and the temperature at 30 to 35° C. to extract lipids through an outlet provided with a cooler and remove the extracted lipids; and (c) adjusting the pressure to atmospheric pressure and separating the reactor to obtain the Type I collagen in the polymer state, wherein the molecular weight of the Type I collagen in the polymer state is 140 kDa or greater and 300 kDa or less.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a process of extracting collagen from a liposuction effluent using supercritical carbon dioxide.

(2) FIG. 2 shows the results confirmed through SDS-PAGE of collagen that was separated according to the present invention.

(3) FIG. 3 shows a process of post-treating and concentrating collagen separated according to the present invention.

(4) FIG. 4 shows the results confirmed through SDS-PAGE of collagen bands after concentrating separated collagen.

(5) FIG. 5 shows the results confirmed through a Western blot analysis of a concentrated collagen sample.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

(6) Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

(7) In the present invention, in order to extract high purity collagen from high quality collagen contained in a liposuction effluent that is discarded after a conventional plastic surgical liposuction, the collagen is extracted using carbon dioxide as a supercritical fluid.

(8) Accordingly, an aspect of the present invention relates to a method for extracting collagen from a liposuction effluent characterized in that collagen is extracted by treating a liposuction effluent containing collagen in the presence of a supercritical fluid.

(9) In the present invention, ‘supercritical fluid’ refers to a fluid that is in a gaseous state under normal conditions but is a fluid at or above a critical temperature or critical pressure.

(10) The supercritical fluid suitable for use in the present invention is not particularly limited, but the supercritical fluid may be one selected from, the group consisting of a carbon dioxide gas, an ammonia gas, a nitrogen gas, a nitrogen monoxide (NO) gas, a nitrogen dioxide (NO.sub.2) gas, a nitrous oxide (N.sub.2O) gas, sulfur dioxide gas, hydrogen gas, water vapor, methane, ethylene, propane, propylene and a mixture thereof, alcohols including ethanol and methanol, and aromatic compounds including benzene and toluene. Preferably, carbon dioxide having a critical temperature of 31° C. and a critical pressure of 72.8 atm may be used.

(11) The extraction method of the present invention may include, a method for extracting collagen from a liposuction effluent including, (a) filling a liposuction effluent containing collagen into a supercritical fluid extractor; (b) introducing a supercritical fluid into the extractor to extract a collagen-containing extract through a heat exchanger; and (c) decompressing a mixture of the supercritical fluid and the collagen-containing extract in a decompression separator for separating.

(12) In the present invention, when carbon dioxide is used as a supercritical extract, it is preferable to use carbon dioxide in a condition wherein the temperature of the extraction step is preferably 10 to 50° C., and the pressure is preferably 100 to 500 bar.

(13) In an embodiment of the present invention, a step of circulating the supercritical fluid separated in step (c) in a storage tank and then decompressing and recirculating together with an externally supplied supercritical fluid, and collecting a separated extract, may be further included.

(14) In the present invention, ‘auxiliary solvent’ refers to a solvent other than the supercritical fluid that is added at the time of the supercritical fluid extraction.

(15) In the present invention, an auxiliary solvent selected from the group consisting of ethanol, methanol, acetone, hexane, ethylacetate and methylene chloride may be further added as a co-solvent.

(16) In the present invention, the supercritical fluid extraction temperature may be 10 to 50° C. and the pressure condition may be 100 to 500 bar.

(17) In the present invention, the auxiliary solvent may be used in an amount of 1 to 500% (w/w) based on a crude to be extracted. Preferably, the auxiliary solvent is used in an amount of 50 to 200% (w/w) based on the crude to be extracted.

(18) Further, the method of adding the auxiliary solvent to the extraction tank is not particularly limited. A method of a bottom-up extraction wherein the supercritical fluid and auxiliary solvent is added in a direction from the bottom of the extraction tank to the top so as to allow the extract to flow out to the top may be used, or oppositely, a method of a top-down extraction wherein the supercritical fluid and auxiliary solvent is added in a direction from the top to the bottom so as to allow the extract to flow out to the bottom of the extraction tank may be used as well.

(19) The supercritical fluid extraction condition according to the present invention preferably has an extraction pressure of 72.8 to 500 atm and an extraction temperature of 31 to 100° C. More preferably, the extraction pressure is 100 to 400 atm and the extraction temperature is 35 to 65° C.

(20) Another aspect of the present invention relates to a method for extracting collagen from a liposuction effluent using a supercritical fluid extraction including, (a) introducing a liposuction effluent into a supercritical extraction device and supplying CO.sub.2, which is an extraction solvent, to an extraction reactor; (b) maintaining the pressure of the CO.sub.2 supplied to the extraction reactor at 100 to 300 bar and the temperature at 10 to 50° C. to extract lipids through an outlet provided with a cooler; and (c) adjusting the pressure to normal pressure and separating the reactor to obtain collagen.

(21) In the present invention, a solvent selected from the group consisting of ethanol, hexane, methanol, acetone, hexane, ethylacetate and methylene chloride may be further supplied as a co-solvent (auxiliary solvent).

(22) In the present invention, the lipid is extracted in an oil phase, and the extracted lipid is lipid derived from lipid droplet of adipose tissue contained in the liposuction effluent, and includes triacylglycerol, sterol ester and the like.

(23) Looking over a collagen extracting device according to the present invention in more detail, as shown in FIG. 1, an extractor {circle around (1)} is filled with a liposuction effluent and supercritical carbon dioxide suitably heated for extraction through a heat exchanger {circle around (9)} is supplied to a lower end portion of the extractor {circle around (1)}. The supercritical carbon dioxide supplied in such a way comes into contact with the filled liposuction effluent to extract collagen and is discharged out of the extractor after rising. The extracted mixture of supercritical carbon dioxide and high purity collagen bypasses a decompression valve {circle around (2)} and is transferred to a decompressor {circle around (3)} while being decompressed. At the decompressor {circle around (3)}, the extracted collagen and carbon dioxide is separated, and the separated carbon dioxide is liquefied as it passes a heat exchanger {circle around (4)} and then is circulated to the storage tank {circle around (5)} to be reused, and the collagen separated in the decompressor {circle around (3)} is collected as a final product {circle around (10)}. In the carbon dioxide storage tank {circle around (6)} other than the carbon dioxide that is circulated and supplied, to compensate for some loss that occurs in the previous process, carbon dioxide can be supplied from the outside. The carbon dioxide stored in the storage tank {circle around (5)} is compressed through a pump {circle around (7)} and is supplied to the extractor again through the heat exchanger {circle around (9)} in a supercritical state. The auxiliary solvent is supplied though an auxiliary solvent inlet {circle around (8)} as necessary.

(24) This process can proceed continuously from the fat from liposuction until the desired collagen extraction yield is reached. Also, for continuous operation, two or more extractors {circle around (1)} are installed and they are used alternatively by controlling multiple supply valves and multiple discharge valves. In the extractor that is not being used, the crude that is done being extracted is removed and new liposuction effluent is filled to prepare for the next extraction.

(25) In this regard, a method for extracting collagen using a supercritical fluid according to the present invention will be described.

(26) A high-content, high-purity collagen extraction method using a supercritical fluid according to the present invention is configured as a process wherein, a liposuction effluent basically fills an extractor, a supercritical fluid is introduced in the extractor filled with the liposuction effluent to extract collagen, a mixture of the supercritical fluid and collagen that was extracted is separated by decompression, and the separated fluid is compressed and re-circulated by a pump.

(27) At this time, in order to improve the extraction efficiency of the collagen, it is preferable to further add a pretreatment process of breaking the liposuction effluent into fine particles. In addition, although the supercritical fluid may be of various types, it is most preferable to use carbon dioxide. The pressure is preferably 100 to 500 bar, the temperature is preferably maintained at 10 to 50° C., and more preferably, the temperature is 20 to 50° C., and the pressure is maintained at 200 to 400 bar.

(28) The collagen extracted by the present invention is advantageous in that it can be extracted in a polymer state as compared with collagen extracted by a conventional method.

(29) In another embodiment of the present invention, a collagen band of a polymer was confirmed as the result of electrophoresis of a collagen separated by supercritical extraction (FIG. 2 and FIG. 4), and it was confirmed to be a Type 1 collagen through a Western blot analysis (FIG. 5).

(30) In addition, the supercritical fluid extract prepared according to the method of the present invention can be manufactured into cosmetics such as lotion, essence and pack according to a known method. Lotions are generally based on purified water, ethanol and moisturizers. An acid, an alkali or an astringent agent is added thereto depending on the purpose. The essence refers to what contains a cosmetic ingredient having a skin moisturizing and anti-aging effects in a high concentration. The main constituents of essence are skin softener, moisturizer, solvent, emulsifier and thickener. The pack is used for the purpose of giving an appropriate tension to the skin and blocking the air from the outside to facilitate absorption of nutrients. The main components of the pack are film-forming agents, moisturizers, emulsifiers and solvents.

(31) Finally, the supercritical fluid extract prepared according to the method of the present invention can be made into cosmetics having a gel or solid form according to a known method. The gel composition can be prepared by further mixing the above-mentioned solution composition with a thickener. The composition in solid form is comprised of emollients, thickeners, emulsifiers and solvents.

(32) Hereinafter, the present invention will be described in more detail with reference to Examples. It should be apparent to those skilled in the art that these examples are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these examples.

EXAMPLE

Example 1: Separation of Collagen from a Liposuction Effluent Using a Supercritical Fluid

(33) 100 ml of a liposuction sample was introduced into an extractor of a supercritical fluid extraction device configured as shown in FIG. 1 at a normal temperature and normal pressure. As a supercritical fluid, carbon dioxide was injected using a high pressure pump at 60 bar and −40° C., and a valve of the outlet was closed to gradually increase the pressure of a stirrer. A temperature sensor was provided inside the stirrer, and the internal temperature was maintained at a supercritical condition of 31° C. When the internal pressure became 250 bar, valve of the outlet was opened and at the same time, a booster was operated to inject high pressure carbon dioxide and the internal pressure was maintained at 250 bar.

(34) In this case, the separated lipid in an oil state begins to be extracted at an outlet where the chiller is installed. If the oil spurt is low, the valve is adjusted at the outlet to raise the pressure to 300 bar and the pressure is maintained for 15 minutes. If additional oil is removed from the outlet, 30 minutes are waited for a complete removal of oil.

(35) The pressure was gradually adjusted to normal pressure and the reactor was separated to extract the remaining tissues to confirm the presence or absence of collagen.

(36) When co-solvent is used, 10 ml of 99.9% EtOH per 100 ml of fat is filled in the shear auxiliary tank of the agitator, and oil is extracted by the same process as above and collagen is separated.

Example 2: Confirmation of Supercritical Extracted Collagen by Electrophoresis

(37) As a result of electrophoresis of the collagen separated by the supercritical extraction using SDS-PAGE, a collagen band was confirmed as shown in FIG. 2.

(38) The supercritical extraction stock solution was separated by centrifugation, into a supernatant and a precipitate, and desalted and concentrated using an ultrafiltration device, and was concentrated to 20-fold (FIG. 3).

(39) The concentration of protein in the total stock solution before concentration was measured by the Bradford method. With protein quantification using the Bradford method, collagen was not measured. The protein concentration of the whole stock solution was 0.239 (mg/ml), the protein concentration of the supernatant was 0.179 mg/ml, and protein was not detected in the precipitate (Table 1).

(40) TABLE-US-00001 TABLE 1 Protein concentration (mg/ml) Positive control group 0 (positive collagen) Whole stock solution 0.239 Supernatant 0.179 Precipitate 0

(41) SDS-PAGE was performed to confirm the presence or absence of collagen in the supernatant and precipitate after concentration. As a result, collagen was confirmed in the supernatant and precipitate before and after concentration, as shown in FIG. 4.

Example 3: Confirmation of Supercritical Extracted Collagen by Western Blot Analysis

(42) Western blot analysis was performed on the collagen extract supernatant and the precipitate concentrated in Example 2 to confirm the presence or absence of Type I collagen.

(43) The supernatant and precipitate that was concentrated to 20-fold were electrophoresed on an 8% SDS-PAGE gel, and then a collagen band was moved to a nitrocellulose membrane, followed by Western blotting by treatment with a Type 1 collagen monoclonal antibody (Abcam, Cambridge, UK).

(44) As a result, as shown in FIG. 5, a distinct Type 1 collagen protein band was confirmed in the 20-fold precipitate.

INDUSTRIAL APPLICABILITY

(45) According to the present invention, collagen of a conventionally discarded liposuction effluent can be extracted in a polymer state having a high purity, and the extracted high purity collagen of the polymer can be widely used in medicine, pharmaceuticals, and cosmetics.

(46) While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments, it will be obvious that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

(47) According to the present invention, collagen of a conventionally discarded liposuction effluent can be extracted in a polymer state having a high purity, and the extracted high purity collagen of the polymer can be widely used in medicine, pharmaceuticals, and cosmetics.

DESCRIPTION OF REFERENCE NUMERALS

(48) {circle around (1)} Extractor {circle around (2)} Decompression controller {circle around (3)} CO2—collagen separator {circle around (4)} chiller {circle around (5)} Liquefied CO.sub.2 storage tank {circle around (6)} CO2 supplement supply tank {circle around (7)} CO.sub.2 circulation pump {circle around (8)} Auxiliary solvent inlet {circle around (9)} Heat exchanger {circle around (10)} High purity collagen-containing extract {circle around (11)} CO2 vent {circle around (12)} Removal of condensate