KR20040090272A - A Production method of Viscum album Coloratum extract by supercritical extraction - Google Patents

Abstract

PURPOSE: Provided is a method for extracting active ingredients from the Viscum album coloratum extract by supercritical extraction, in particular, using supercritical carbon dioxide as an extracting solvent. CONSTITUTION: The method for extracting active ingredients from Viscum album coloratum extract is characterized by mixing Viscum album coloratum with carbon dioxide and co-solvent, and followed by preheating and extraction by supercritical extraction. The extraction is performed at 30-50 deg.C under 100-300 atm, and the co-solvent is selected from ethanol, methanol. ethylacetate, normal hexane, and diethylether.

Description

A production method of Viscum album Coloratum extract by supercritical extraction

The present invention relates to a method of extracting an active ingredient from a mistletoe plant (Viscum album, Coloratum) using supercritical extraction, which is more efficient and more environmentally friendly than hot water extraction or solvent extraction. The present invention relates to a method for obtaining an active ingredient in a high yield without waste by-products after extraction by using a supercritical extraction method using a critical carbon dioxide as an extraction solvent.

Mistletoe is a half-parasitic plant that grows on many types of host trees (fir, apple, oleander, oak, pine, etc.). Currently, there are more than 1,400 species on earth, and most of them are the two families of Viscaceae and Loranthaceae. : family) (Korean Patent Publication No. 1999-0702487). It has long oval intact leaves with flowers in early spring. Fruits are spherical, usually slightly transparent yellowish green. Mucus is on the surface of the fruit, attaches to the feet of algae and germinates to other trees. It is distributed in Korea, Europe, Japan, Taiwan, China, and Africa.

In Korea, mistletoe has been commonly used as a medicinal herb called `` above life ''. It has a flat nature, tastes bitter, and has no poison.It has a strong tendon, blood vein, and skin. It is used for the treatment of low back pain, arteriosclerosis, frostbite, and miscarriage prevention.

Mistletoe's English name, mistletoe, was recommended as a tumor treatment drug by German medical doctor Rudolf Steiner in 1917, and Dr. Ita Wegmann of Switzerland accepted Steiner's claim in 1926. It began to be used for clinical trials. In the early 1960s, Lucas Hospital in Alesheim, Switzerland, was introduced into cancer treatment, resulting in breakthroughs, which led to the development of new drugs. Currently, Lucas Hospital in Switzerland, Osselbronn Hospital in Germany, Biomed Hospital and Havel Society Hospital Mistletoe therapy has been adopted for chemotherapy at Friedenweiler Hospital, Tübingen Medical University Hospital, Heidelberg Medical University Hospital, Witten-Herdecker University Hospital, Vienna Medical University Hospital in Austria and more than 400 hospitals in Central Europe.

Mistletoe therapy is widely used in affiliated hospitals, general hospitals, and cancer clinics in various universities in Korea, and more than 1,300 research papers on mistletoe therapy have been published in the German medical community. Since then, more than 700 research papers have been published, and the US FDA approval process is currently in progress.

Mistletoe X activity is currently only 596 published medically proven papers, the main efficacy is reported to be anti-cancer, immune enhancement, antihypertensive, anti-diabetic, antibacterial activity.

European mistletoe (Viscum album, Coloratum), which has been developed and used clinically as a cancer drug since 1920, has almost no side effects and shows excellent anticancer effects on all cancers. This formulation is known to have an excellent effect of improving the quality of life of cancer patients, such as reducing pain caused by cancer, reducing side effects of radiation and chemotherapy, preventing the loss and recurrence of cancer, and extending life. This agent is effective by killing cancer cells and apoptosis by apoptosis. Glycoproteins such as lectin I, II, III, etc., known as potent anticancer ingredients, are the main components of mistletoe plant extract. Component, polypeptides such as viscotoxin, polysacchride, alkaloid, flavonoids such as quercetine and the like have been reported. The most studied of mistletoe components is lectins, the main anticancer component. Mistletoe lectin is a type II ribosome-inactive protein that exhibits the activity of inhibiting protein synthesis by inactivating ribosomes in eukaryotic cells when the B-chain binds to receptors (specific carbohydrates) on the cell surface. ribosome-inactivating protein). Mistletoe lectin kills cancer cells by the same mechanism in the case of cancer cells, but the sugar of the cancer cell membrane is known to selectively kill only cancer cells because of its high affinity with lectins because the sugar structure of the normal cell membrane is different. Mistletoe lectin is also known to inhibit the growth of tumor cells by increasing the activity of macrophages and NK cells that directly and indirectly respond to tumor cells, stimulating the immune system. Cancer Invest 2002; 20 (7-8): 1069-1079, J. Ethnopharmacol 2002 Dec; 83 (3): 235-240, Immunology 2002 Oct; 107 (2): 268-274, Anticancer Res 2002 May-Jun; 22 (3) : 1399-1407}.

In addition, a peptide component such as biscotoxin is a component that causes necrosis by destroying cancer cell membranes by enhancing the cytotoxicity of T-cells and dissolving the cell membranes of cancer cells. In addition, the polysaccharide component is known to exhibit anticancer effects by participating in the proliferation of lymphocytes induced by mistletoe lectin and activating the function of NK cells. In addition, flavonosides such as alkaloids and quercetin are included, so mistletoe therapy is recognized as the most effective and safe treatment in Europe.

In Korea, the development of immunopotentiator composition using the mistletoe extract is being made (Korean Patent Publication No. 1996-0005732), and as an antioxidant cosmetic and a whitening agent (Korean Patent Publication No. 2001-0020355, 2001-0020356). In addition, the development of dietary supplements for obesity control (Korean Patent Publication No. 2000-0038820), and the development of health supplements, soft drinks, special nutrition products (Korean Patent Publication No. 1996-0007091). Korean Mistletoe native to Korea's mountains and mountains is a variety of European varieties. Unlike Europeans whose fruits are white, yellow is yellow and the shape and composition of plants differs from European ones. Therefore, the physiological activity of Korean mistletoe seems to be very different from that of existing European.

Method of extracting active ingredient from mistletoe is to wash the collected mistletoe with distilled water, freeze it at -80, then cut it and grind it again in a blender, and then extract it at 4 for 8-12 hours with 0.15M NaCl solution. After recovering the supernatant by centrifugation, the protein portion was recovered by ammonium sulfate precipitation and resuspended in a minimal volume of phosphate buffer (PBS) containing 0.15 M NaCl for 24 hours in the same solution. Purification using a column packed with Sepharose 4B after dialysis while changing the buffer solution (Korean Patent Publication No. 1999-0041288), dried mistletoe with anhydrous or hydrous alcohol having 1 to 3 carbon atoms, or Extracted with ethyl acetate for 4 to 30 days for 3 to 20 days, and then concentrated to prepare (Korean Patent Publication No. 2001-0020356), Mistletoe dried water, 1 to 3 carbon atoms Or function is known alcohol, or ethyl acetate extraction method at 50 to 100 for 3 to 24 hours and then concentrated prepared in (Republic of Korea Patent Application No. 2001-0020355 call). However, all these methods use an aqueous solution or an organic solvent, and the volume of the extraction solvent is large during the process, and a large amount of by-products are produced and the protein is denatured due to operation at high temperatures. In addition, the extraction efficiency of non-protein components is low, there are many problems in the production of mistletoe X through a large-scale process.

In the present invention, a supercritical extraction method was used to solve the problems of such extraction and purification. Supercritical extraction is a separation method based on the fact that supercritical fluids have intermediate properties of liquids or gases, and that the extraction capacity or selectivity is greatly changed by changes in pressure, temperature, or composition. Carbon dioxide is best used as a supercritical fluid and can be separated without causing denaturation at a relatively low temperature. Since there is little residue of extractant, new extraction of food and medicines such as beer hop extraction or de-caffeine processing of coffee beans It is widely used as a separation method. In addition, the pharmaceutical industry purifies and recovers enzymes and vitamins, extracts active ingredients from animals and plants (alkaloids, tocopherols, etc.), concentrates and purify pharmaceutical raw materials, isolates and purifies fat mixtures (glycerides, fatty acids, lecithins). ), Yeast, cell product extraction, animal oil extraction in the food industry (fish oil, liver oil), vegetable oil extraction (soybean oil, sunflower oil, palm oil, cocoa, coffee), defatting food (fried, potato chips, radish) Fatty starch), coffee and tea caffeine removal, spice extraction, plant pigment extraction, food decolorization, deodorization, softening of alcoholic beverages, etc. Removal of nicotine, extraction and purification of cosmetic raw materials (surfactants, fatty acid esters, monoglycerides) and hydrocarbon separation in the chemical industry (aliphatic and aromatic separation, naphthene separation, olefin separation, n-par separation of affin and iso-paraffin), purification of organic synthetic raw materials, dehydration of aqueous solutions of organic solvents, separation of azeotrope compounds and extraction of coal components in the energy industry, extraction of coal liquefied oil, removal of asphalt and heavy metals from petroleum residues, crude oil or Supercritical fluid extraction methods have been applied to various fields such as hardening of heavy oils, concentration of biomass alcohols, and dehydration {Methods in Biotechnology, Vol. 4: Natural Products Isolation, RJP Cannell}.

In order to solve the above problems, the present invention aims to extract the active ingredient from the mistletoe in high yield by using supercritical extraction in extracting the active ingredient from the mistletoe (Viscum album, coloratum) plants. .

In addition, the present invention aims to reduce the use of the extraction solvent used in the conventional extraction method to obtain a high concentration of extract and environmentally friendly production by minimizing the use of solvent.

In addition, an object of the present invention is to recover a high concentration of protein and non-protein substances with high anticancer, immune enhancing, antihypertensive, antidiabetic, antimicrobial activity contained in a high concentration of extract.

1 is a process diagram of the supercritical extraction of the present invention for the production of mistletoe extract.

2 is a standard curve of syringin, an indicator substance.

Figure 3 is an HPLC overlap chromatogram of the indicator syringin.

4 is HPLC data of mistletoe hot water extraction X.

5 is HPLC data of mistletoe 40% alcohol hot water extraction X.

In order to achieve the above object, the present invention comprises the step of putting finely chopped mistletoe in the extractor for supercritical extraction, filling the ethanol with a cosolvent for extraction and closing the reactor; Adding carbon dioxide to the supercritical fluid for supercritical extraction; Heating the temperature of the extractor to the extraction temperature; After the temperature of the extractor reaches a desired temperature, injecting carbon dioxide, which is a supercritical fluid, to pressurize until the desired pressure is reached; Extracting the active ingredient while injecting carbon dioxide which is a supercritical fluid upon reaching a desired temperature and pressure, and discharging the extract through an outline line; Provides a method for manufacturing mistletoe x using supercritical fluid extraction consisting of

The present invention is as follows.

The present invention relates to a method for preparing mistletoe X using supercritical extraction after mixing and preheating carbon dioxide and cosolvent in mistletoe.

In addition, the present invention relates to a method of manufacturing mistletoe X using the supercritical extraction method characterized in that the extraction is carried out at 30 ~ 50 ℃, 100 ~ 300 atm.

The present invention also relates to a method for preparing mistletoe extract using supercritical extraction, characterized in that at least one of ethanol, methanol ethyl acetate, normal hexane, and die ether is used as a cosolvent.

Hereinafter, the specific configuration of the present invention through the embodiment will be described in detail. However, the scope of the present invention is not limited only to the description of these examples.

Example 1 Hot Water Extraction

The dried mistletoe was finely chopped and suspended by adding 4 times the distilled water, and then the mistletoe hot water extraction X was obtained through hot water extraction for 4 hours. The obtained mistletoe extract was dried under reduced pressure to obtain a concentrate of 43brix%. To evaluate the extraction efficiency, the extraction rate was compared by analyzing the content of the indicator in the extract through PDA (photo diode array) using a syringe using a syringe as a standard component of mistletoe extract as a reference material. As a result, the content of syringin, which is an indicator substance in the mistletoe by hot water extraction, was 0.636%.

Example 2: Solvent Extraction

The dried mistletoe was finely chopped and suspended by adding 70% alcohol of 10-fold. After stirring for 4 hours at 25 ° C., mistletoe X was obtained. The extracted extract was compared with the extraction rate by the content of the syringe, a syringe in the extract content through the PDA using HPLC. As a result, the content of syringe, which is an indicator substance, by stirring extraction of 70% alcohol for 4 hours at 25 ℃ was 0.189%.

Example 3: Solvent Extraction + Ultrasonic Crushing>

70% alcohol agitation extraction was performed for 4 hours at 25 ° C. as in Example 2, and a sonication was further performed for 10 minutes with a branch sonifier 450 sonicator. The obtained mistletoe extract was compared with the extraction rate by the content of the syringe, a syringe in the extract content through the PDA using HPLC. As a result, the syringe, which is an indicator of X, was 0.212% at 70 ° C. for 4 hours.

Example 4 Hot Water Extraction + Solvent Extraction>

The dried mistletoe was finely chopped and suspended with 40% alcohol, and then the mistletoe hot water extract X was obtained through hot water extraction for 4 hours. The obtained mistletoe x was dried under reduced pressure to obtain a 15brix% concentrate. The extracted extract was compared with the extraction rate by the content of syringe (syringin) in the extract through PDA using HPLC. As a result, 0.434% of the syringe, an index substance in X, obtained by adding 40% alcohol was extracted through hot water extraction.

Example 5: Supercritical Extraction

After finely slicing the dried mistletoe, 100 ml of fine mistletoe and 30 g of ethanol with cosolvent were sealed in a 500 mL supercritical extractor, and the extractor was sealed. . After the pressurization, the temperature of the extractor was heated to 40 ° C., and then pressurized until 300 atm while injecting carbon dioxide into the extractor again. When the condition of the extractor reached 40 ℃, 300 atm while extracting a certain amount of carbon dioxide into the extractor was recovered through the outlet line (outlet line) in the upper layer (outlet line). As a result of extraction, most of the extracts (90 wt% or more) were recovered until about 1 hour, and after 2 hours, the extracts were extracted until no extracts were obtained. After extraction, the concentrated extract was recovered using HPLC using PDA. The extraction rate was compared with the content of syringin, a marker in the extract. As a result, the content of syringe, which is an indicator material, obtained by supercritical extraction using carbon dioxide as a supercritical fluid was 0.93%.

Table 1 shows the amount of syringes according to the extraction method. Mistletoe x by supercritical extraction yielded the highest yield.

Example Extraction Method Sirinazine amount (%) One Hydrothermal extraction 0.636% 2 Solvent extraction 0.189% 3 Solvent Extraction + Ultrasonic Extraction 0.212% 4 Hot Water Extraction + Solvent Extraction 0.434% 5 Supercritical Extraction 0.93%

Therefore, the present invention can obtain a high yield of mistletoe X using a supercritical extraction method.

In addition, the present invention can reduce the use of the extraction solvent to obtain a high concentration of the extract and can provide a method for producing environmentally friendly mistletoe X according to minimizing the use of solvent.

In addition, the present invention recovers high concentrations of anti-cancer, immune enhancing, antihypertensive, anti-diabetic, anti-diabetic, high-protein and non-protein substances contained in extracts of high concentration, food, beverage and health supplements with Mistletoe extract as an active ingredient Food and the like.

Claims (3)

Method of producing Mistletoe X using supercritical extraction after mixing and preheating carbon dioxide and cosolvent to mistletoe. The method of claim 1, wherein the extraction using carbon dioxide and cosolvent is performed at 30 to 50 ° C. and 100 to 300 atmospheres. The method of claim 1 or 2, wherein the cosolvent is at least one of ethanol, methanol ethyl acetate, normal hexane, and die ether.