JPH088849B2 - Method for separating squalene-containing composition - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for separating a squalene-containing composition, and more particularly to a method for separating a squalene-containing composition from the deep sea shark liver.

[0002]

2. Description of the Related Art The deep-sea shark livers such as the shark, the shark, the shark, the shark, the white shark and the like contain squalene. In addition to squalene, it also contains various components useful for humans, such as vitamins and various fatty acids, depending on the type of shark. For this reason, shark liver oils from deep-sea sharks have been conventionally used for medicinal purposes.

As a method for collecting shark liver oil from the liver of deep-sea sharks, a steam pressing method is usually applied, and an alkaline digestion method is applied when vitamin A oil is intended. Also known is a method of heating the shredded shark liver of deep-sea sharks, adding steam to this to extract oil, removing liver tissue by centrifugation, and collecting shark liver oil containing squalene. (Heilbron et.al. J. Chem. Soc. 1926 P
1630).

[0004]

However, shark liver oil containing squalene from deep-sea shark liver was prepared by a conventional method such as a steam pressing method or a method combining a steam load while heating and a centrifugal separation method. When collected, there was a problem that useful substances other than squalene were likely to undergo thermal decomposition or modification. In addition, the shark liver oil collected by the conventional method has a drawback that it has a strong odor and that it is relatively darkly colored.

Therefore, an object of the present invention is to provide a method for separating a squalene-containing composition from which both squalene and useful substances other than squalene can be stably extracted from the liver of a deep-sea shark.

[0006]

The method for separating a squalene-containing composition of the present invention which achieves the above-mentioned object, comprises the steps of slicing the liver of a deep-sea shark to allow shark liver oil to flow out from the liver, The sliver-containing shredded product and the shark liver oil are subjected to a filtration operation using at least flannel as a filter medium to separate the squalene-containing composition as a filtrate (hereinafter, this method is referred to as Method 1). .

[0007] Further, the above-mentioned object is that after slicing the liver of a deep-sea shark and letting out shark liver oil from the liver, after stirring the shredded material and the shark liver oil at a temperature of 50 ° C or lower. The method for separating a squalene-containing composition of the present invention is characterized in that the stirring mixture is subjected to a filtering operation using at least flannel as a filter medium to separate the squalene-containing composition as a filtrate (hereinafter, also achieved. Method is called method 2).

The present invention will be described in detail below. First, the method 1 of the present invention will be described. In the method 1, first, the liver of a deep-sea shark is shredded. The deep sea shark liver that is the starting material is not particularly limited as long as it is a liver of a species containing squalene, and it is possible to use a deep sea shark liver such as a shark, a shark, a shark, a shark, a white shark and the like. . When shredging the deep-sea shark liver, be careful not to hemolyze the blood in the liver. If blood hemolysate is mixed in shark liver oil, the resulting squalene-containing composition will be colored, so caution is required. The shape of the shredded material is not particularly limited, but for example, 1
Cut into small pieces of about cm square. The shark liver oil in the liver leaks with the finely chopped.

Then, the shark liver oil that has flowed out and the finely chopped pieces obtained by finely chopping are subjected to a filtering operation using at least flannel as a filter medium. As the filtering operation at this time,
For example, natural filtration or suction filtration can be applied. By using flannel as the filter medium, the tissue pieces of the liver and the foreign substances in the liver are adsorbed to the flannel, so that impurities can be removed more efficiently than when other filter mediums are used. The target squalene-containing composition can be separated from the deep-sea shark liver by obtaining the filtrate obtained by this filtration operation.

According to the method 1 for separating a squalene-containing composition as described above, the decomposition of useful substances during the separation process is suppressed, so that squalene and useful substances other than squalene (eg vitamins and various fatty acids) are separated. ) Can be taken out stably.

Next, the method 2 of the present invention will be described.
In this method 2, the liver of deep-sea shark is shredded and shark liver oil is allowed to flow out from the liver in the same manner as in method 1 described above, and then the shredded product and shark liver oil are cut at a temperature of 50 ° C or lower. Stir. For stirring at this time, use a glass rod, bamboo rod, or the like.
Alternatively, it is preferably carried out carefully with a rotary evaporator or the like. When the stirring temperature exceeds 50 ° C., the useful substance in the stirring mixture is thermally decomposed or modified, which is not preferable. Particularly preferable stirring temperature is 20 to 50 ° C.
Is. By setting the stirring temperature to 20 to 50 ° C., it is possible to particularly easily suppress the coagulation and bonding of the useful substance and the contaminants in the stirred mixture, and it becomes easy to perform filtration with flannel.

In Method 2, after the shredded product and shark liver oil are stirred in this manner, the stirring mixture is subjected to the same filtering operation as in Method 1 to separate the squalene-containing composition as a filtrate. According to this method 2, the target squalene-containing composition can be separated with higher efficiency than the above method 1.

In the method 1 and the method 2, activated carbon may be used together with flannel as the filter medium. By using the activated carbon in combination, it is possible to more efficiently remove the impurities in the shark liver oil and remove the specific odor. The type and shape of the activated carbon at this time are not particularly limited, and for example, 200 mesh coconut shell activated carbon powder can be used. When activated carbon powder is used, it is particularly preferable to use activated carbon cloth sprayed on urethane foam resin to form activated carbon cloth [for example, Kurasheet (trade name) manufactured by Kuraray Chemical Co., Ltd.].
In addition, when using activated carbon in combination, it is preferable to take measures such as holding the activated carbon or activated carbon cloth between two flannels so as not to mix the activated carbon into the filtrate.

In Method 1 and Method 2, it is preferable to carry out the step of removing the protein contained in the squalene-containing composition for the purpose of reducing the antigenicity of the obtained squalene-containing composition. This step can be performed, for example, by subjecting the obtained filtrate composed of the squalene-containing composition to centrifugation and collecting the supernatant. A cooling centrifuge is particularly preferable as the centrifugal separator used at this time. By using a cooling centrifuge, proteins can be efficiently removed while suppressing thermal decomposition and denaturation of useful substances. Centrifugation is 4,0
It is preferable to carry out at 00 to 6,000 rpm. 4,000
A rotation speed of less than 0 rpm is not preferable because it is difficult to separate the fatty acid and protein contained in the filtrate. Further, if the rotation speed exceeds 6,000 rpm, the enzyme that is a part of the protein to be removed will be decomposed, and it will be difficult to remove the decomposed product, which is not preferable. The centrifugation time is appropriately selected according to the amount of filtrate to be subjected to centrifugation.

The procedure for removing proteins by centrifugation is 1
Although it may be performed once, an incomplete layer portion remains between the supernatant and the sedimented layer in one operation, and this incomplete layer portion is mixed into the supernatant during the fractionation of the supernatant. It is preferable to perform the treatment twice or more in order to sufficiently remove the protein in the complete layer site. When the centrifugation operation is performed multiple times, the centrifugation conditions in each operation may be the same or may be appropriately changed.

In Method 1 and Method 2 of the present invention,
Even after performing the step of removing tapan quality as described above, since microorganisms derived from the starting material or microorganisms mixed in the separation process may be present in the squalene-containing composition, after the tapan quality removing step, It is preferable to perform a step of sterilizing the obtained supernatant. The sterilization treatment includes, for example, a suction filtration method using a microfilter having a pore size of about 0.45 μm as a filter material, and an ultra high temperature sterilization method (UH
T method: for example, 130 ° C. for 3 seconds), high-pressure sterilization method (for example, 120 ° C. for 20 minutes), or steam sterilization method (for example, 120)
20 ° C.) or the like. The sterilization treatment may be performed by only one method, or may be performed by combining a plurality of methods. When performing sterilization, it is preferable to select conditions so that the useful substance in the squalene-containing composition is not thermally decomposed or modified as much as possible.

The squalene-containing composition thus separated contains, in addition to squalene, other useful substances that have been stably extracted (for example, vitamins and various fatty acids), and has various immunostimulatory effects. It has a bioactive effect. The toxicity of this squalene-containing composition is extremely low, and it is substantially harmless to humans and animals. Therefore, this squalene-containing composition is useful, for example, as a raw material for pharmaceuticals or a functional food. In producing a functional food using the squalene-containing composition, for example, the obtained squalene-containing composition is directly processed into a beverage, and the obtained squalene-containing composition is enclosed in a capsule such as a gelatin capsule. The method can be applied.

[0018]

Embodiments of the present invention will be described below. Example 1 First, 100 g of liver of a white whale shark was cut into small pieces of about 1 cm square, and the shark liver oil spilled along with the small pieces and the finely cut pieces obtained by the fine cutting were carefully stirred using a rotary evaporator. did. At this time, the stirring temperature was adjusted to 50 ° C. Then, an activated carbon cloth obtained by spraying 200 mesh coconut shell activated carbon powder onto a urethane foam resin [Kuraray (trade name) manufactured by Kuraray Chemical Co., Ltd.] held with two flannel was used as a filter medium.
The stirring mixture was subjected to a natural filtration operation to separate the squalene-containing composition as a filtrate.

Next, the obtained filtrate (squalene-containing composition) was cooled and centrifuged at 5,000 rpm for 20 minutes [trade name: Universal cooling centrifuge Model 580].
0, manufactured by Kubota Manufacturing Co., Ltd.]
The supernatant was collected. Separate the collected supernatant at 5,000 rpm.
Centrifugation was performed again with a cooling centrifuge under the condition of 0 minutes to collect the supernatant. By these two centrifugation operations,
Proteins and contaminants contained in the filtrate were removed. After that, suction filtration using a microfilter having a pore size of 0.45 μm as a filter medium is performed to sterilize the supernatant obtained by the two centrifugation operations, and the squalene-containing composition subjected to the sterilization treatment. 70 g were obtained.

The squalene-containing composition thus sterilized was a slightly yellowish, almost transparent liquid having a viscosity of 32.2 cSt (measured by an Ubbelohde viscometer). The contents of squalene and total fatty acids (measured twice) were 46.7% -53.
3% (former: squalene, latter: total fatty acid), and the second time was 48.2% -51.8%. Table 1 shows the analysis results of the squalene-containing composition.

[0021]

[Table 1]

As is clear from Table 1, the squalene-containing composition finally obtained in Example 1 contained a large amount of squalene, and retinol and α-tocopherol were relatively used as useful substances other than squalene. It contains a large amount of fatty acids as well as a large amount. The amount of impurities contained in this squalene-containing composition is extremely small.

The chemical composition of the fatty acid contained in the squalene-containing composition finally obtained in Example 1 is
The analysis was carried out by gas chromatography under the following conditions.・ Condition 1 Model used: SHIMAZU GC-9A [Shimadzu Corporation] Detector: FID column: Packing agent ... 5% advance-DS / chromosolve w, 80-100 mesh glass column ... diameter 3 mm x length 2 m

Condition 2 (a condition for separating linolenic acid-eicosaenoic acid and arachidonic acid-cetrainic acid, which cannot be separated under the above-mentioned condition 1) Model used: SHIMAZU GC-9A (manufactured by Shimadzu Corporation) Detector: FID column: Packing agent ... Unisol 3,000 / Uniport C, 80-100 mesh glass column ... Diameter 3 mm × length 2 m Analysis results are shown in Table 2.

[0025]

[Table 2]

As is apparent from Table 2, the squalene-containing composition finally obtained in Example 1 contains various fatty acids, and in particular, palmitic acid, oleic acid, eicosaenoic acid, and cetoleic acid were compared. It is contained in a very high concentration.

Test for Toxicity and Utility In the same manner as in Example 1 described above, a predetermined amount of the squalene-containing composition (those subjected to sterilization treatment) was separated and
Each test was conducted.

Acute toxicity test: 50 dd mice (body weight: around 25 g) were prepared, and a squalene-containing composition was orally administered to each individual at a rate of 0.5 g / day / individual for 7 consecutive days. A toxicity test was conducted. As a result, no acute toxicity was observed in the squalene-containing composition. In addition, no side effects were observed.

Subacute toxicity test: 50 dd mice (body weight: around 25 g) were prepared, and the squalene-containing composition was prepared at a rate of 0.1 g / day / individual 60.
A sub-acute toxicity test was conducted by orally administering to each individual for consecutive days. As a result, subacute toxicity was not observed in the squalene-containing composition. In addition, no side effects were observed.

Effect on Ehrlich Ascites Cancer Two groups of dd mice (average body weight: 50 g) were prepared with 10 individuals per group, and Ehrlich ascites tumor (number of cancer cells: 1 × 10 ⁶ cells / ml) was added to the abdominal cavity of each individual. 1 ml was administered. From 3 days after the inoculation of Ehrlich's ascites cancer, each individual in one group was continuously orally administered with the squalene-containing composition at a rate of 0.1 g / day / individual, and each individual in the other group was administered with 0. 85%
The NaCl aqueous solution was continuously orally administered at a rate of 0.1 g / day / individual, and the number of surviving individuals in each group was observed. The results are shown in Fig. 1. As is clear from FIG. 1, the group to which the squalene-containing composition was administered has more immune effect days (the number of days to survive) than the group to which the 0.85% NaCl aqueous solution was administered. From this, the immunostimulatory effect of the squalene-containing composition can be confirmed.

Effect on Mouse Serum Titer One dd mouse (body weight: 50 g) was prepared, blood was collected from this individual, and serum was obtained by a conventional method. Sensitivity to infection with this serum and vesicular stomatitis virus (VSV) The interferon titer was determined by a conventional method using cells. After this,
Mice used for obtaining serum were intraperitoneally administered with 0.1 g of a squalene-containing composition, and blood was collected 24 hours and 48 hours after the administration to obtain serum by a conventional method,
The interferon titers (relative values) of these sera were similarly determined. In addition, as a control, the mouse was used as another control, except that physiological saline was intraperitoneally administered instead of the squalene-containing composition, in the same manner as above, before, 24 hours after, and 48 hours after administration of the physiological saline. The subsequent interferon titer (relative value) was determined. The results are shown in FIG. As is clear from FIG. 2, the intraperitoneal administration of the squalene-containing composition causes a relatively large increase in the interferon titer 24 hours after the administration.

Effect on Oral Administration on Immune System One dd mouse (body weight: 50 g) was prepared, and blood was collected from the bone marrow, peripheral blood vessels, and spleen of this individual.
The total number of immunocompetent cells in blood and the proportion of phagocytes and B cells in this total number were determined. In addition, the squalene-containing composition was orally administered to this individual at a rate of 0.5 g / day / individual for 7 consecutive days, and on the 1st, 3rd, 5th, and 7th days from the first administration. Blood was collected from the bone marrow, peripheral blood vessels, and spleen, and the total number of immunocompetent cells in the blood and the ratio of phagocytes and B cells to this total number were determined. Table 3 shows the results.

[0033]

[Table 3]

As is clear from Table 3, the total number of immunocompetent cells in the blood collected from the bone marrow, peripheral blood vessels, and spleen, and the proportion of phagocytes and B cells among them, are determined by the squalene-containing composition. Abnormal fluctuation did not occur before and after oral administration of the product, which was within the normal fluctuation range. From this, it is understood that the oral administration of the squalene-containing composition is harmless to the number of immunocompetent cells and the proportion of phagocytes and B cells in the cells.

[0035]

As described above, according to the present invention,
A squalene-containing composition in which both squalene and a useful substance other than squalene are stably extracted can be separated from the liver of a deep-sea shark.

[Brief description of drawings]

FIG. 1 is a graph showing the effect of a squalene-containing composition separated in the same manner as in Example 1 (those subjected to sterilization treatment) on Ehrlich ascites tumor.

FIG. 2 is a graph showing the effect of the squalene-containing composition separated in the same manner as in Example 1 (those subjected to sterilization treatment) on the interferon titer.

Claims (5)

[Claims] 1. A liver of deep-sea shark is shredded to allow shark liver oil to flow out from the liver, and then the shredded meat and the shark liver oil are filtered using at least flannel as a filter medium. A method for separating a squalene-containing composition, which comprises applying the squalene-containing composition and separating the squalene-containing composition as a filtrate. 2. The liver of deep-sea shark is shredded to let shark liver oil flow out from the liver, and the shredded meat and the shark liver oil are stirred at a temperature of 50 ° C. or lower, and then a stirred mixture is formed. A method for separating a squalene-containing composition, which comprises subjecting a squalene-containing composition to a filtrate by performing a filtering operation using at least flannel as a filter material. 3. The method for separating a squalene-containing composition according to claim 1 or 2, wherein activated carbon is used together with flannel as the filter medium. 4. The step of removing the protein contained in the filtrate by separating the squalene-containing composition as a filtrate and then subjecting the filtrate to centrifugation to separate the supernatant. A method for separating a squalene-containing composition according to claim 3. 5. The method for separating a squalene-containing composition according to claim 4, wherein the step of subjecting the supernatant to sterilization is performed.