JP2007530433A - Propolis extract and extraction method thereof - Google Patents

Abstract

Since triglycerides or fatty acids that do not contain alcohol are used as the extraction solvent, it is possible to extract a propolis extract that can be used even by persons who cannot take alcohol physically or for religious reasons. Further, by using oil as an extraction solvent, artepilin C, which is an active ingredient of propolis, can be sufficiently extracted.
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Description

  The present invention relates to a propolis extract and a method for extracting the same. More specifically, the present invention relates to a novel propolis extract using oil and an extraction method thereof.

  Propolis, which is a product of bees, not only protects bees themselves, but also is closely related to human beings and has been used for various purposes, for example, as an external medicine or an internal medicine. Nevertheless, its existence has been forgotten with the progress of modern science. However, since adverse effects such as the side effects of chemicals made in modern science were regarded as problems, propolis, a natural product, began to attract attention again.

  Phenolic acid esters such as flavonoids, phenolic acids, or caffeic acid phenethyl ester (CAPE), which are active ingredients of propolis, for example, strengthen the cell membrane, activate the immune function, prevent bleeding of the blood vessel wall It has various effects such as antibacterial action, antibacterial action, anti-inflammatory action, anticancer action, antiallergic action, antiviral action, and active oxygen scavenging action (antioxidant action). Furthermore, it is known to prevent diseases caused by LDL (low density riboprotein) and cellular oxidation. More recently, it has been attracting attention as having local anesthetic action as an analgesic, tissue regeneration action (wound healing action), plant germination inhibition action, immune function enhancement action and the like.

As described above, propolis contains a component that exhibits an excellent action and effect, and how to efficiently extract this excellent active ingredient from the propolis bulk is a problem. Therefore, as one of the methods for efficiently extracting propolis extract, for example, Patent Document 1 extracts a propolis extract by adding a mixed solution of ethanol and water with a water ratio of 10 to 35% by volume to the propolis bulk. A method is disclosed. This extraction method is improved so that the extraction efficiency is improved as compared with the propolis extract extracted with ethanol alone (extraction efficiency about 28%) and the propolis extract extracted with water alone (extraction efficiency about 7%). .
Japanese Patent Laid-Open No. 10-338641

  However, the extraction method using an extraction solvent containing alcohol has a problem that it cannot be used by those who cannot take alcohol for constitutional reasons or for religious reasons. Nevertheless, a method for extracting propolis extract with an extraction solvent not containing alcohol has not been found so far.

  Then, an object of this invention is to provide the novel propolis extract which does not contain alcohol, and its extraction method.

  As a result of intensive studies in view of the above problems, the inventors have found that a propolis extract can be extracted using oil as an extraction solvent, and have arrived at the present invention.

  The method for extracting a propolis extract according to claim 1 of the present invention is characterized in that triglyceride is added to a pulverized propolis mass and extracted.

  According to the method for extracting a propolis extract according to claim 1 of the present invention, since a triglyceride containing no alcohol is used as an extraction solvent, the propolis extract can be used even by a person who cannot take alcohol physically or for religious reasons. Can be extracted. In addition, the extraction spectrum spreads to the low polarity side compared to the conventional alcohol-containing extraction method, and a low-polar active ingredient such as artepilin C contained in propolis can be sufficiently extracted.

  The propolis extract extraction method according to claim 2 of the present invention is characterized in that the triglyceride contains oleic acid.

  According to the method for extracting a propolis extract according to claim 2 of the present invention, it is possible to extract a propolis extract having an action of further reducing blood cholesterol and various effects of propolis by reducing blood cholesterol.

  The method for extracting a propolis extract according to claim 3 of the present invention is characterized in that a fatty acid is added to a pulverized product of a propolis bulk to extract it.

  According to the method for extracting a propolis extract according to claim 3 of the present invention, since fatty acids that do not contain alcohol are used as the extraction solvent, propolis that can be used even by persons who cannot take alcohol physically or for religious reasons. Extract can be extracted. Further, compared to the conventional alcohol-containing extraction method, artepilin C, which is an active ingredient of propolis, can be sufficiently extracted.

  The method for extracting a propolis extract according to claim 4 of the present invention is characterized in that the fatty acid contains oleic acid.

  According to the method for extracting a propolis extract according to claim 4 of the present invention, it is possible to extract a propolis extract having an action of further reducing blood cholesterol and various effects of propolis by reducing blood cholesterol.

  The propolis extract according to claim 5 of the present invention is extracted by the extraction method according to claim 1.

  The propolis extract according to claim 6 of the present invention is extracted by the extraction method according to claim 2.

  The propolis extract according to claim 7 of the present invention is extracted by the extraction method according to claim 3.

  The propolis extract according to claim 8 of the present invention is extracted by the extraction method according to claim 4.

  According to the propolis extract of claims 5 to 8 of the present invention, it can be used even by a person who cannot take alcohol physically or for religious reasons. In addition, it has an active ingredient of propolis and an active ingredient of triglyceride or fatty acids, strengthening action of cell membrane, activating immune function, bleeding prevention action of blood vessel wall, antibacterial action, anti-inflammatory action, anti-cancer action, anti-allergy Various effective effects such as action, antiviral action, and active oxygen scavenging action (antioxidant action) can be exhibited, and propolis extract having these actions can be added to foods, cosmetics and pharmaceuticals. Can be used. In particular, its usefulness is demonstrated in product molding by a combination with a base or a coating material that is altered or deformed by alcohol or moisture.

  Hereinafter, the present invention will be described in detail.

  The propolis extract extraction method of the present embodiment adds a desired amount of triglycerides or fatty acids to a pulverized product obtained by pulverizing a bulk propolis bulk that is difficult to use as it is to a required particle size (within a particle size of 5 mm or less). During the temperature and time, the propolis extract is extracted by mixing and melting with stirring. Furthermore, the propolis extract obtained as described above is subjected to solid-liquid separation such as filtration or centrifugation, and is obtained as a liquid propolis extract. Furthermore, oil is added again to the residue after filtration or centrifugation, and the mixture is stirred to elute the propolis extract, and then the solution is centrifuged to collect the supernatant, which is recovered as a liquid propolis extract in the same manner as described above. May be.

  The raw material propolis is a mass, mainly resin / balsam (50-55%), beeswax (25-30%), essential oil / ether oil (10%), pollen (5%) The color is from blackish to brown, tan, yellowish green. There are odors that are slightly fragrant and those that emit a specific odor like a slightly honeyed resin. In addition, the taste is slightly bitter, somewhat astringent and paralyzes the tongue. Furthermore, as a property, it has a strong viscosity and softens when warmed by hand, but solidifies at 15 degrees and dissolves at 60 to 70 degrees. However, the composition and properties of propolis vary greatly depending on the production area.

  Propolis bulk is produced in Brazil, the United States, China, Europe, Oceania, etc., any of which can be used in the present invention. Propolis produced outside of Brazil is mainly poplar, although there are some differences in the origin, whereas Brazilian propolis has a perennial herb called “Alectrin”. There are many things, and it contains a lot of artepilin C. This artepilin C is a compound discovered and isolated at Hayashibara Biochemical Laboratories Co., Ltd., and its anticancer activity is weaker than that of chemically synthesized anticancer agents, but there are almost no side effects on animals. Artepilin C inhibits DNA synthesis, induces apoptosis, and extinguishes cancer cells. Therefore, in vitro experiments using cultured cells, human epithelial cells and leukemia cells can be added at 50-100 μg / ml. Sun treatment kills most of the various cancer cells and leukemia cells, and in animal experiments, it has not only an anticancer effect but also an immunostimulatory effect.

  Examples of active ingredients of propolis include flavonoids, artepilin C, phenolic acids, organic acids, aromatic aldehydes, aromatic acids, coumarins, etc., and their effectiveness is related to cell membrane strengthening action, immune function Appears in activating action, blood vessel wall bleeding prevention action, antibacterial action, anti-inflammatory action, anticancer action, antiallergic action, antiviral action, reactive oxygen scavenging action (antioxidant action) Accordingly, the propolis bulk mass used in the present invention contains at least an active ingredient exhibiting various useful effects described above.

  The triglycerides or fatty acids used in the present invention are preferably vegetable oils such as olive oil, rapeseed oil, corn oil, safflower oil, sunflower oil, and more preferably an oil having a high oleic acid content. Moreover, the mixed oil which mixed 2 or more types of vegetable oil may be sufficient. Further, fatty acids containing acetic acid may be used. It is known that vegetable oils contain components such as medium chain fatty acids, vegetable sterols, γ-linolenic acid, α-linolenic acid, oleic acid and linoleic acid. Oil components play a major role in maintaining health. Further, as an advantage of using vegetable oil, for example, carbohydrates and proteins are 4 kilocalories per gram, whereas vegetable oil can efficiently supply energy to the body with a small amount of about 9 kilocalories per gram. In addition, it contains a large amount of essential fatty acids that cannot be made in the human body and is essential for the normal growth and function of the body, and vegetable oil is suitable for efficient intake of these essential fatty acids. ing. In addition, vegetable oil contains a large amount of vitamin E, which can cause infertility, growth disorder and central nervous system disorder when deficient, and can increase absorption of vitamin E and other vitamins into the body. Have advantages.

  In addition, oleic acid (18 carbon atoms and 1 double bond), which is a fatty acid representative of monounsaturated fatty acids, is contained in common edible oils such as olive oil, high oleic acid type safflower oil and sunflower oil. As a typical effect of oleic acid, it is known that blood cholesterol is reduced and blood is further increased. Cholesterol in human blood exists in a form called good cholesterol (HDL) or bad cholesterol (LDL). When excessive cholesterol is present, it is oxidized, and LDL (bad) cholesterol is combined with active oxygen and becomes active. It is said that when it becomes type LDL (super bad) cholesterol and deposits in the blood vessel wall, it causes arteriosclerosis and the like. However, oleic acid reduces only this bad cholesterol without reducing good cholesterol, and is less oxidized than linoleic acid, so it is difficult to make lipid peroxides that cause carcinogenesis in the body.

  As described above, by extracting using vegetable oils having various advantages, particularly vegetable oils containing a large amount of oleic acid, not only the active ingredients of propolis but also the active ingredients of vegetable oils At the same time, a propolis extract that can be taken into the body can be extracted.

  Furthermore, since the extraction solvent is triglycerides or fatty acids, it is possible to extract a propolis extract that can be used even by those who cannot take alcohol for constitutional reasons or religious reasons, unlike conventional extraction solvents containing alcohol. it can. Moreover, compared with the conventional water-containing alcohol extraction method, an extraction spectrum spreads to the low polarity side, and artepilin C which is an active ingredient of propolis can be sufficiently extracted.

  The amount of triglycerides or fatty acids in the extraction of the propolis extract of the present invention is preferably 2 to 10 times the amount of propolis on a weight basis.

  The extraction time is preferably 1 to 24 hours, more preferably 6 to 24 hours. When extraction time is less than 1 hour, extraction efficiency falls and the target active ingredient cannot fully be extracted. On the other hand, when the extraction time exceeds 6 hours, the extraction efficiency becomes substantially constant.

  The extraction temperature is not particularly limited because there is little denaturation of the extracted component due to temperature change, but it is preferable to extract at a low temperature, specifically, about 40 to 60 ° C.

  The propolis extract of the present invention thus obtained can be used even by a person who cannot take alcohol for constitutional reasons or for religious reasons. In addition to being used alone, if necessary, other ingredients commonly used in cosmetics, foods, pharmaceuticals, etc., such as vitamins, stabilizers, colorants, fragrances, etc. may be blended. Also good.

  In addition, as a use of the propolis extract of the present invention, the propolis extract of the present invention can be used orally such as foods and drinks, for example, beverages, dairy products (specifically margarine, butter, cheese, etc.) ), Edible oils, dressings, nutritional supplements, rice cakes, confectionery, seasonings and the like.

  In addition, the propolis extract of the present invention may be used in cosmetics (for example, lotions, emulsions, creams, lip balms, sunscreens, lipsticks, massage oils), alternative medical products such as aromatherapy, and pharmaceuticals. Good.

  As a method for blending the propolis extract of the present invention into various compositions such as food compositions and cosmetics, known methods such as mixing, spraying, coating, and pouring are appropriately selected in the process until those products are completed. It is.

  EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.

  A comparative experiment of extraction by temperature change was performed when the extraction solvent was oleic acid, water, 75 volume% ethanol and 100 volume% ethanol, respectively. Details will be described below.

  First, in order to prevent variation in the components contained depending on the location of the original mass, the frozen Brazilian propolis raw mass is pulverized in small quantities (about 10 g each time) according to the capacity of the pulverizer, and the particle size is 5 mm or less. A pulverized product was obtained. Next, oleic acid (Kanto Chemical Co., Ltd., deer grade 1 oleic acid Cat. No. 31028-01), water, ethanol aqueous solution (75% by volume) and ethanol (Kanto Chemical Co., Ltd. reagent special grade ethanol Cat. No. 14033-70) 9 g of each of the four types of extraction solvents were measured and placed in tubes, and 1 g of the propolis bulk pulverized product obtained in advance was added to each tube, so that the total weight of the extraction solvent and the pulverized product was 10 g. Thereafter, each tube was placed in a water bath (Iwaki Thermoregulator, CTR-220, shaker incubator; SHK-101B) kept at 40 ° C. and extracted for 15 minutes. During extraction, the tube was always passed through a vortex mixer (Iwaki TM-152) to stir up the material accumulated at the bottom of the tube. After extraction, the propolis extract was centrifuged with a centrifuge (Hitachi, 05PR-22) at 25 ° C. and 2000 rpm for 10 minutes to obtain an extract and a residue, respectively. The same operation as above was repeated at extraction temperatures of 60 ° C., 80 ° C., and 100 ° C. to obtain respective extracts and residues.

  Next, 100 mg of the obtained extract was diluted 10-fold with 100% ethanol, applied to a vortex mixer, and centrifuged at 1000 rpm for 1 minute using a small desktop centrifuge (Millipore). The supernatant was again diluted stepwise with 100% ethanol to 300 times its volume. Absorbance was measured with a spectrophotometer (Hitachi U-1200) using 100% ethanol as a standard sample. The results are shown in Table 1.

  As shown in Table 1, the oleic acid extract at each temperature had absorption peaks in the ultraviolet region, particularly in the vicinity of wavelengths of 298 nm and 227 nm. Further, since the maximum absorption wavelength of oleic acid was around 228 nm, the peak around 227 nm of the oleic acid extract is considered to be derived from oleic acid. In addition, in water extraction, 75 volume% ethanol extraction, and 100 volume% ethanol extraction, a peak was observed in the vicinity of 298 nm, so the wavelength near 298 nm is considered to be the absorption wavelength of the component derived from propolis, and oleic acid was used as the extraction solvent. It was also found that a propolis extract containing main propolis components was produced.

  Comparison of the oleic acid extract, water extract, 100% ethanol extract, and 75% ethanol extract extracted by the method of Example 1 was evaluated by HPLC.

  First, 100 mg of the obtained extract was diluted 10-fold with 100% ethanol, applied to a vortex mixer, and centrifuged at 3000 rpm for 10 minutes with a centrifuge (Hitachi, 05PR-22). The supernatant is diluted 5 times with 75% ethanol, the diluted solution is stirred with a vortex mixer, and syringe filter (pore size 0.2um, PTFE, titanium, 42213-NP) is used with a syringe (Terumo, ss-02Sz). ) And the filtrate was placed in a sample bottle. Elution conditions of HPLC (pump: Hitachi L-7100, diode array detector: DAD, Hitachi L-7455, column oven: Hitachi l-7300, autosampler: Hitachi L-7200, column: inert sill C8 4.6x150mm, GL Sciences) Are shown in Table 2, and the results are shown in FIG.

  In the HPLC data of FIG. 1, the recovery rate of the peak with a short retention time (RT) was as low as about 20% compared to ethanol. The HPLC column inertosyl C8 used this time is a reverse phase partition column and elutes from those having high hydrophilicity, so these low peaks are considered to be highly hydrophilic components that are difficult to extract with oleic acid. However, after 12 minutes, the propolis component extracted by ethanol extraction was also recovered by oleic acid extraction. In particular, Artepilin C (retention time 24 minutes), which is an active ingredient of Brazilian propolis, can be recovered by 77% by extraction at 40 to 60 ° C., and some of the components before and after this can reach 100%.

  As shown in FIG. 1, there is little change in the composition ratio of the extracted components depending on the extraction temperature. If organism-derived material is extracted, it is preferable to perform extraction at a low temperature, and the optimum extraction temperature is preferably 40 to 60 ° C.

  The absorption spectrum of the propolis extract containing the active ingredient artepilin C was examined and the optimum extraction time was examined. Details will be described below.

  First, a crushed product of Brazilian propolis was obtained by the method of Example 2. Next, five types of samples having a weight ratio of propolis: oleic acid of 1: 9, 1: 4, 1: 3, 1: 2 and a weight ratio of propolis: 75% ethanol by volume of 1: 9 were prepared. Then, each sample was put into the water bath kept at 40 degreeC, and extracted for 0.25, 1, 6 and 24 hours. During the extraction, the tube was always subjected to a vortex mixer to stir what was collected at the bottom of the tube. After the extraction, the propolis extract was centrifuged at 25 ° C. and 3000 rpm for 10 minutes with a centrifuge (Hitachi, 05PR-22) to obtain an extract and a residue, respectively.

  Next, the obtained extract was analyzed by high performance liquid chromatography by the method of Example 1, the peak height (absorbance AU value) of Artepilin C was determined, and fluctuations due to temperature conditions during extraction were compared. The results are shown in FIG.

  Comparing oleic acid extraction (1: 9) with 75% by volume ethanol extraction (1: 9) with propolis extract extracted at the same ratio, the relative extraction amount of 75% by volume ethanol changed much over time. On the other hand, in the case of oleic acid extraction, the peak increases with time, and the relative extraction amount increases (see FIG. 2). From this result, it was found that artepilin C, which is an active ingredient of Brazilian propolis, can be extracted more efficiently by extracting oleic acid than by extracting 75% by volume of ethanol by extracting for a long time. Further, in this experiment, it was revealed that oleic acid extraction can extract an active ingredient equivalent to or more than 75% by volume ethanol extraction.

  Furthermore, since no increase in absorbance was observed after about 6 hours of extraction time, it was found that the extraction time of propolis by oleic acid extraction should be about 6 hours.

It is a chart which shows the result of HPLC in Example 2 of this invention. It shows the extraction rate of Artepilin C, which is an active ingredient of the propolis extract in Example 3 of the present invention, in terms of absorbance.

Claims (8)

  A method for extracting a propolis extract, wherein triglyceride is added to a pulverized propolis mass to extract.   The method for extracting a propolis extract according to claim 1, wherein the triglyceride contains oleic acid.   A method for extracting a propolis extract, comprising adding a fatty acid to a pulverized product of a propolis bulk and extracting it.   The method for extracting a propolis extract according to claim 3, wherein the fatty acid contains oleic acid.   A propolis extract extracted by the extraction method according to claim 1.   A propolis extract extracted by the extraction method according to claim 2.   A propolis extract extracted by the extraction method according to claim 3.   A propolis extract extracted by the extraction method according to claim 4.

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