JPS6033410B2 - Extraction and separation method of sea snake lipids - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for easily extracting and separating lipid souls, which do not have a fishy odor, from the whole body of a sea snake, especially from the lipid mass in its pelvic flukes.

The inventor of this invention first discovered that the whole sea snake or its internal organs has a medicinal effect that does not contain any phospholipids and is highly effective in the treatment of frozen shoulders, stiff lower back, aftereffects of whiplash, degenerative lumbar spondylosis, sciatica, etc. We have discovered a method for extracting and producing simple lipid substances (Hakama Seki Sho 55-1667 Matsu No. 5).

However, all the lipids obtained in this way not only have a fishy odor, but also require a long process to extract and separate.
In particular, it was necessary to use force-lamb chromatography to purify simple lipids.

The inventor of this invention subsequently conducted further research and discovered a method for extracting and separating lipids that do not have a fishy odor by extracting sea snake raw materials using an organic solvent and in a simple process. In other words, the present invention deals with the treatment of a whole sea snake, especially the entire lipid mass in its abdominal cavity, or a small piece of the sea snake, with acetone,
Extraction treatment in either a chloroform-methanol mixture or n-hexane-ethanol-water mixture,
This paper presents a method for extracting and separating sea snake lipids, which is characterized by isolating lipids from an extract. Furthermore, this invention extracts the sea snake lipids obtained by the above method by fractionation with a mixture of petroleum ether and aqueous ethanol or aqueous methanol, and extracts simple lipids from the petroleum ether fraction and lipid-like lipids from the alcohol fraction. The present invention provides a method for extracting and separating sea snake lipids, which is characterized by isolating sea snake lipids. That is,
According to this invention, lipids from sea snakes can be extracted as follows.

Captured sea serpent [Sea serpent is Eel eel wax tican ship senifasciate re plate w. and its congener, the genus Hydrophis SP.], are often extracted after transportation, temporary storage, etc. without being extracted immediately after capture. In particular, the intraperitoneal lipid mass (as it is) or cut into appropriate sizes is immersed in an extraction solvent such as acetone, chloroform-methanol mixture, or n-hexane-ethanol-water mixture, and air is removed. It is desirable to block it from

This prevents the lipids from being oxidized and deteriorated, and also allows for immersion. Then, the extraction is completed at an appropriate time. That is, for example, a sea snake soaked in this solvent is crushed into fine pieces by applying it to a homogenizer or the like. The finer the degree of pulverization, the better, but it is preferable to pulverize into a porridge shape. Acetone is preferred as the extraction solvent;
The appropriate amount of the sea snake raw material per 1 hour of the solvent is 50 to 500 hours, preferably 100 to 300 hours. When the sea snake is extracted immediately after it is captured, the process is the same as above except that the time required for soaking in the solvent after capture is shorter. The mixture of the Kaijarankyo material and the solvent obtained as described above is centrifuged or filtered to collect the transparent upper layer extract.

The following is an abbreviation of the solvent initially used in the lower sedimentation section. Add a volume of solvent and repeat the extraction process 3 to 8 times.
Combine the resulting upper transparent extract with the above extract. This extract is heated to 35qC using a rotary evaporator under reduced pressure (10 to 2 square meters of mercury).
After concentrating as described below and removing water by freeze-drying, pale yellowish brown and odorless semi-solid sea snake lipids are obtained. These lipids are stored at a temperature below -4. The above extraction may normally be carried out at room temperature, but in some cases may be carried out under humidification at a temperature below the boiling point of the solvent used.

Further, in the above extraction method, especially when intraperitoneal lipid lumps are used as a raw material, the lipids may be separated by the following method before extraction with a solvent.

That is, the lipid mass is taken out, placed in a gauze bag as soon as possible, placed in a metal container, and the metal container is placed in chick water. Once the lipid mass in the gauze bag is softened and becomes liquid, odorless crude lipids can be obtained by quickly opening the gauze bag. This lees lipid can be purified very easily by the method of the present invention using the above-mentioned solvent.
Next, the above lipids are further extracted and separated into isolated lipids and cervical lipids in the following manner.

First, petroleum ether and ethanol or methanol aqueous solution (
Mix equal amounts of 90% ethanol (preferably 90% ethanol), shake well, and leave to form two layers of solvent (top layer A, bottom layer B).
). The above-mentioned lipids are distributed between the obtained upper layer and lower layer, and simple lipids are fractionated into the upper petroleum ether layer, and similar substances are fractionated into the lower alcohol layer. Specifically, it is performed as follows. These A and B are mixed in a volume ratio of approximately 3:1, and the above-mentioned lipids are added at a ratio of 100 to 200 volumes to 1 volume of this mixed solvent, and the mixture is thoroughly shaken to dissolve. The upper layer al and lower layer bl of the obtained solvent are separated, and approximately the same amount of fresh A as the initially used A is added to the lower layer bl and shaken well. The obtained upper layer a2 and lower layer b2 are separated. On the other hand, approximately the same amount of new B as the B used initially was added to the upper layer a1, and the mixture was thoroughly mixed with the upper layer a2. and b3 is combined with b2. Next, add the same amount of new lower layer B to this upper layer a3 as that used for the first time, shake well to separate it into an upper layer a4 and a lower layer b4, and combine this lower layer b4 with the above b2 and b3. The same process as that applied to the upper layer a3 is repeated several times on the upper layer a4 obtained in this step), and the lower layer obtained each time is combined into one. The upper and lower layers thus obtained are each concentrated using a rotary evaporator or the like under reduced pressure (10 to 2 mbars of mercury) at 35° C. or lower. The water content of the lower concentrate is then removed by freeze-drying. By the method of the present invention as described above, simple lipids can be obtained from the upper layer and lipidoids can be obtained from the lower layer.

Since these methods do not require heating in water as required by conventional methods, they are excellent products with virtually no fishy odor.
Each has the following properties. Simple lipids obtained from the upper layer A pale yellow waxy substance with a softening point of 40 to 55 qo.

{o} Impervious to water, poorly soluble in ethanol, soluble in acetone, chloroform, ether, petroleum, hexane,
Easily soluble in benzene and toluene.

The liquid property of a solution prepared by dissolving this product in 100% acetone is neutral.

0 Infrared absorption spectrum: IR (HC13) 2960
・2870・173Q 1460 Skin-1 (Mura Thin Layer Chromatography (TLC) {i) For a solution of 1 part of this product dissolved in 100 parts of chloroform, attached amount: 10 pieces, carrier: Silica Gel G (Merck & Co.), Developing solvent: Petroleum ether/ether/acetic acid (85:15:1.5), Developing distance: 15 skin, Detection: 0.2%, After spraying an ethanol solution of 2-boodichlorofluorescein and drying, 36-mark skin. When subjected to TLC under the conditions of irradiation with ultraviolet rays, a large yellow spot was observed at a position with an Rf value of about 0.6 (shown at 1 in FIG. 1).

Regarding the sample solution used in 'ii''i-, attached amount: 10
K〆, carrier: silica gel G (Merck & Co.), developing solvent: n
-hexane/isopropyl ether/acetic acid (60:50
: 0.4), development distance: 15 arcs, detection: When subjected to TLC under the conditions of molybdenum reagent, a single large spot appears at an Rf value of about 0.8 (shown in 1 in Figure 2).

H The absence of any phospholipids in this substance was confirmed by the detection method modified by Dittmer-Lester.

(b) Lipid obtained from the lower layer It is a sticky oil at room temperature and is soluble in various organic solvents and vegetable oils.

{o) b The chemical composition is 37.4% phospholipid, 3. Furthermore, the fatty acid composition is C,623.9
%, C, 6:, 0.5%, C, 6, 6: 20.4%, C
,70.2%,C,84.3%,C,8:,9.4%,
C, 6: 254.3%, C, 8: 37.0%.

TLC Chromatograms 2 in FIG. 1 and 2 in FIG. 2 were obtained by TLC'i} and (ii) performed on the above-mentioned simple lipids, respectively.

Next, the method of the present invention will be explained by examples, but the present invention is not limited thereto.

Example 1 Extraction and separation of lipids from the lipid mass in the abdominal cavity of the Elabu sea snake Immediately after catching the Elabu sea snake in Okinawa, the lipid mass (approximately 2
001) The mixture was placed in a can containing acetone, then filled up to the mouth with acetone (approx. 1 volume of acetone), sealed from air, sealed tightly, stored at room temperature, and transported to Osaka.

This raw material was soaked in acetone and heated in a homogenizer at room temperature for 2 minutes to form a porridge. This was centrifuged, the upper layer of acetone solution was separated, and acetone (100%) was added to the lower layer of the sedimentation section and stirred for 2 minutes. The resulting upper acetone solution was separated and combined with the first acetone solution. This operation was repeated five times with the addition of new acetone. The acetone solution thus collected was concentrated using a rotary evaporator and a water jet pump under reduced pressure (10-2 Dyuan Mercury Co., Ltd.). Water in the concentrated extract was removed by freeze-drying to obtain a pale yellowish brown, odorless, semi-solid lipid. This was stored at -4° in the freezer. Similar products were also bound when using a chloroform-methanol mixture (volume ratio 1:2) or n-hexane-ethanol-water temperature solution (10:9:1) as a solvent instead of acetone. . Example 2 Extraction and separation of simple lipids and succulents from the lipids obtained in Example 1 Equal amounts of petroleum ether and 90% ethanol were shaken well and allowed to stand to form two upper and lower layers (upper layer A and lower layer B). The lipid obtained in Example 1 was fractionated and extracted using solvents A and B.

The lipids obtained in Example 1 (10) were combined with A (45M) and B (1
The mixture was placed in Separator Furnace 1 along with the parentheses in Step 5, and the mixture was thoroughly shaken to dissolve.

The obtained lower layer BL (approximately 15 skins) is added to a new A (45')
The upper layer a2 (approximately 45 [)] and the lower layer b2 (approximately 15
The middle and lower layers b2 of [) were taken out into flask 1. On the other hand, add new B (15 cm) to the upper layer A2 (about 45') remaining in the separation reactor 1, shake it well, and add it to the upper layer A2 (about 45') containing the upper layer A2 (about 45'). The mixture was poured into a separator funnel and thoroughly shaken to obtain an upper layer a3 (approximately 90 centimeters) and a lower layer b3 (approximately 15 centimeters), and this lower layer b3 (approximately 15 centimeters) was taken out into flask 1 and combined with lower layer b2. Next, a new B (15 units) was added to the remaining part of the upper layer A3 (approximately 90 units) and the resulting upper layer A4 (approximately 90 units) and lower layer B4 (approximately 15 units) were added and thoroughly shaken. The lower layer b4 was taken out into the flask 1 and combined with the lower layers b2 and b3. The operation of treating the remaining upper layer A4 (approximately 90') with new B (15 I) was repeated three more times. The upper layer (9
0') and the lower layer (90 [)] under reduced pressure (10 to 2 columns of mercury) using an evaporator and a water pump, respectively.
Concentrated at 30qo.

Moisture in the lower layer was removed by freeze-drying. In this way, simple lipids (90%, 90%) having the above properties are obtained from the upper layer (approximately 90%), and lipids having the above properties (0.7%) are obtained from the lower layer (approximately 90%). 7%) obtained.

[Brief explanation of the drawing]

1 and 2 in FIG. 1 and 1 and 2 in FIG. 2 are thin layer chromatograms of lipids obtained by the method of the present invention. Figure 1 Figure 2

Claims (1)

[Scope of Claims] 1. The whole sea snake, especially the lipid mass in its abdominal cavity, as it is or its fragments, in a solvent such as acetone, a chloroform-methanol mixture, or an n-hexane-ethanol-water mixture. 1. A method for extracting and separating sea snake lipids, which is characterized by performing an extraction treatment with water and isolating lipids from the extract. 2. The extraction and separation method according to claim 1, wherein the extraction solvent is a chloroform-methanol mixture (volume ratio 1:2). 3. The extraction separation method according to claim 1, wherein the extraction solvent is a mixture of n-hexane-ethanol-water (volume ratio 10:9:1). 4. The extraction and separation method according to claim 1, wherein the extraction solvent is acetone, and the lipid mass is immersed in this and then cut into pieces to complete the extraction. 5. Extract the whole sea snake, especially the lipid mass in its abdominal cavity, either as it is or in pieces, in a solvent such as acetone, chloroform-methanol mixture, or n-hexane-ethanol-water mixture. It is characterized by isolating lipids from a liquid, fractionating and eluting them with a mixture of petroleum ether and aqueous ethanol or aqueous methanol, and isolating simple lipids from the petroleum ether fraction and lipid-like substances from the alcohol fraction. Extraction and separation method of sea snake lipids. 6. The extraction and separation method according to claim 5, wherein the extraction solvent is a chloroform-methanol mixture (volume ratio 1:2). 7. The extraction and separation method according to claim 5, wherein the extraction solvent is a mixture of n-hexane-ethanol-water (volume ratio 10:9:1). 8. The extraction separation method according to claim 5, wherein the extraction solvent is acetone, and the lipid mass is immersed in this and then cut into pieces to complete the extraction. 9 The fractionation solvent is the upper layer-lower layer (volume ratio 3:
1) The extraction and separation method according to claim 5.