JPH0499776A - Extraction of chlorophyll - Google Patents

Abstract

PURPOSE:To effectively extract chlorophyll as an important organism molecule performing photosynthesis of natural substance by using carbon dioxide in a supercritical state. CONSTITUTION:A natural substance (e.g. dried powder of water cress) containing chlorophyll is brought into contact with carbon dioxide in a supercritical state in a solvent such as acetone to extract chlorophyll. Otherwise, chlorophyll is eluted from a natural substance containing chlorophyll into an organic solvent (e.g. ethanol) and resultant organic solvent containing eluted chlorophyll is brought into contact with supercritical carbon dioxide, or else, chlorophyll is eluted from a natural substance using oils and fats (e.g. shortening oil) and brought into contact with supercritical carbon dioxide to extract chlorophyll. Chlorophyll is a bluish green pigment and can be converted to a hem pigment in an animal body to be used as a tinction, a hematopoietic, a wound healing agent, bacteriostat or deodorant, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for extracting chlorophyll from natural products using supercritical carbon dioxide.

[Conventional technology]

Chlorophyll, which is an important biomolecule that performs photosynthesis, is a blue-green pigment and can be converted into heme pigment in the animal body, so it has been used as a coloring agent, as a hematopoietic agent, as a wound treatment agent, and as a bacteriostatic agent. It is used in agents, deodorizers, etc.

Among these chlorophylls, chlorophylls a and b are the most widely distributed in nature, and the conventional method for isolating them is to extract the leaves of plants such as nettle and spinach using organic solvents. . That is, first, chlorophylls a and b are extracted from these plants with an 80% acetone solution containing Na, Co3, or dilute NH. Add petroleum ether to this extract and wash out the acetone with water. Next, wash with methanol to remove rotinol. Finally, the petroleum ether is washed with water to remove acetone and methanol and precipitate the chlorophyll. It is then strained through a layer of talc and the talc is washed with petroleum ether to remove the carotene. Furthermore, chlorophyll is extracted by Niteru. The ether is dried with anhydrous Na, So, and the chlorophyll is precipitated with petroleum ether. The chlorophyll, dissolved in a small amount of pyridine and diluted with petroleum ether, is then separated by chromatography on a powdered sucrose or polyethylene column. This column is 0.5
Expand with pentane containing % inproper tools. Crystalline chlorophyll a is collected by adding water to an ether solution and removing the solvent in vacuo.

Therefore, conventional isolation of chlorophyll requires complicated and time-consuming steps. Furthermore, it has been pointed out that chlorophyll extracted using organic solvents poses a risk of cancer, which also poses a problem.

On the other hand, extraction technology using supercritical fluid has recently become known.

This extraction using supercritical fluid uses what is called a normal fluid in the supercritical region, which exceeds the critical point (critical temperature and critical pressure) unique to the substance, and this fluid is a simple fluid that cannot be called a gas or liquid without an interface. It is a single phase, has a high density (close to that of a liquid), a viscosity several times that of a gas, and a diffusion coefficient about 100 times that of a liquid. Mass transfer is fast, therefore separation is easy, and it is in a supercritical state. In this case, the isopycnal lines are densely packed, and the density can be increased by slight changes in temperature or pressure.

Therefore, the supercritical extraction method has the following excellent features.

(1) Although it is under high pressure, it can be processed at low temperature, so thermal denaturation can be avoided and high quality products can be obtained.

(2) In addition to temperature, pressure can be used as an operating factor,
Flexibility in operating conditions.

(3) Since a highly volatile fluid is used as the solvent, the amount of residual solvent in the product can be reduced to zero.

(4) Carbon dioxide can be used effectively without causing any human safety or environmental problems.

(5) Due to the excellent mobility of supercritical fluids, non-volatile substances and solid raw materials can be efficiently extracted.

Because of the above characteristics, supercritical extraction is suitable for food,
It is suitable for extracting and separating components that are easily degraded by heat from crude drugs, natural fragrances, etc., and its adoption is expected to significantly shorten the conventional complicated extraction process and reduce costs.

[Problem to be solved by the invention]

However, when the present inventor attempted to extract chlorophyll from natural products using carbon dioxide, which is commonly used in supercritical fluid extraction, it was found that chlorophyll could not be extracted at all using carbon dioxide alone, as shown in the experiments described below. found.

The present invention was made in view of the above circumstances, and an object of the present invention is to provide a method for extracting chlorophyll from natural products using a supercritical fluid extraction method.

[Means and effects for solving the problem] As a result of extensive studies to achieve the above object, the present inventor brought a natural product containing chlorophyll into contact with carbon dioxide in a supercritical state in the presence of an organic solvent. or by eluting chlorophyll from a natural product containing chlorophyll into an organic solvent and contacting the organic solvent containing the eluted chlorophyll with carbon dioxide in a supercritical state, or by eluating chlorophyll from a natural product containing chlorophyll. This method succeeded in extracting chlorophyll from natural products using the supercritical extraction method by eluting chlorophyll into fats and oils and bringing the fats and oils containing chlorophyll into contact with carbon dioxide in a supercritical state. The present inventors have discovered that chlorophyll a can be selectively extracted when chlorophyll a is present, and thus exhibits specific selectivity, leading to the present invention.

Therefore, the present invention involves contacting a natural product containing chlorophyll with carbon dioxide in a supercritical state in the presence of an organic solvent, or eluting chlorophyll from a natural product containing chlorophyll into an organic solvent, and removing the eluted chlorophyll. A method for extracting chlorophyll characterized by extracting chlorophyll by bringing an organic solvent containing it into contact with carbon dioxide in a supercritical state, and a method for extracting chlorophyll from a natural product containing chlorophyll into an oil or fat, and an oil or fat containing the eluted chlorophyll. Provided is a method for extracting chlorophyll, which is characterized in that chlorophyll is extracted by bringing the chlorophyll into contact with carbon dioxide in a supercritical state.

The present invention will be explained in more detail below.

The natural products targeted by the chlorophyll extraction method of the present invention are:
Any substance containing chlorophyll can be used, and there are no particular restrictions, but silkworm manure, comfrey, spinach, chlorella, etc. can be used, and dry watercress powder can be suitably used. .

The chlorophyll extraction method of the present invention uses a natural product that has been impregnated with an organic solvent, such as by pre-wetting the natural product with an organic solvent, or a natural product that has been mixed with an organic solvent, or extracts chlorophyll from a natural product that contains chlorophyll. It is eluted into an organic solvent, and an organic solvent containing the eluted chlorophyll is used.

Here, examples of the organic solvent include alcohol, benzene, chloroform, ether, acetone, etc.
One type of these can be used alone or two or more types can be used in combination. In addition, as a method of moistening the natural product with these organic solvents, a method of dipping the natural product in the organic solvent and pulling it up, or a method of spraying the organic solvent onto the natural product using a spray or the like can be adopted. In this case, the amount of organic solvent used is 500 to 1 part by weight per 100 parts by weight of the natural product.
It is preferable that the amount is about 0,000 parts by weight.

Further, as a method for eluting chlorophyll with an organic solvent, an extraction method using a normal organic solvent can be adopted.

According to the method using this organic solvent, chlorophyll a and b
It is possible to extract a mixture of a and b depending on the type of solvent.
The ratio of b changes, and for example, when using ethanol, the ratio of b becomes higher than when using acetone, and differences in selectivity are observed.

In the chlorophyll extraction method of the present invention, it is possible to further elute chlorophyll from a natural product containing chlorophyll into oil and fat, and use the oil and fat containing this eluted chlorophyll.

Here, as the oil or fat, one that is liquid at the extraction temperature of carbon dioxide in a supercritical state can be used. Specific examples include edible oils such as shortening oil and salad oil, and any natural product that can elute chlorophyll can be used.

In addition, as a method for eluting chlorophyll from natural products using fats and oils, ordinary extraction methods using fats and oils are adopted, such as mixing the fats and oils with natural products, stirring, and then filtering or leaving the mixture to stand to collect the supernatant liquid. can do. The content of chlorophyll in this oil is not particularly limited, but a high concentration is generally preferred from the viewpoint of efficiency.

The chlorophyll extraction method of the present invention uses carbon dioxide in a supercritical state, and the supercritical state of carbon dioxide is the shaded area in the phase diagram of carbon dioxide shown in Figure 1. Specifically, the critical concentration Tc = 304.2 or more and the critical pressure Pc = 7.38 MPa or more, preferably a temperature of 323 to 333 ° K and a pressure of 9.5 to 10.5 MPa.
It is preferred to use supercritical carbon dioxide in the range a.

The critical density Pc of carbon dioxide is 466 kg/m3, and FIG. 2 shows the relationship among temperature, pressure, and density of carbon dioxide.

Further, as a method for bringing carbon dioxide in a supercritical state into contact with a natural product, an organic solvent, or an oil or fat, a known extraction method can be adopted, and is not particularly limited. For example, as shown in FIG. After bringing the supercritical carbon dioxide into contact with the extraction target, the extraction phase (supercritical fluid and ten solutes) is depressurized and expanded at low temperatures in the separation tank 2 to separate them, and the solutes are extracted from the bottom of the separation tank 2. The fluid taken out is compressor 3
It is possible to suitably adopt a pressure change method in which the liquid is passed through and returned to the extraction tank 1 (note that 4 in FIG. 2 is an expansion valve).

) Also, as shown in Figure 4, a temperature change method is adopted, in which the temperature of the extraction phase from extraction tank 1 is raised through heat exchanger 5, the fluid and solute are separated in separation tank 2, and the solute is taken out from the bottom. Alternatively, a method may be adopted in which the fluid is cooled and compressed through the heat exchanger 6 and the compressor 3, and then returned to the extraction tank 1 for extraction. Furthermore, by adopting an adsorption method, the solute is separated from the fluid by passing the extraction phase from the extraction tank 1 through an adsorption separation tank 7 containing an adsorbent that adsorbs only the solute, as shown in FIG. Extraction can also be carried out by compressing in step 3 and then recycling to extraction tank 1.

Chlorophyll can be further isolated from the extract extracted as described above according to a conventional method.

〔Effect of the invention〕

As explained above, according to the chlorophyll extraction method of the present invention, chlorophyll can be effectively extracted from natural products using supercritical carbon dioxide.

EXAMPLES Hereinafter, examples and comparative examples will be shown to specifically illustrate the present invention, but the present invention is not limited to the following examples.

Here, the supercritical extraction apparatus used in this example is as shown in Fig. 6, and to explain this, 8 in the figure is C○, cylinder,
9 is a cool pump (CP-80 type manufactured by TAITEC), 1
0 is a liquid sending pump (MILTONROY consta
Metric III), 11 is a supercritical chromatograph (LSA-1 type supercritical chromatograph manufactured by Denki Kagaku Keiki Co., Ltd. (DKK)), and inside this chromatograph 11, a heat exchange column 12 and a cell 13 with a capacity of 2 cm3 are arranged. It is set up. Also, 14 is UV inspection instrument Waters! l
990J photodiode array detector), 15 is a computer (EPSONlij PC-286X), 16
is a fraction collector (LSA-F manufactured by DKK).

When performing extraction processing and analysis of chlorophyll in the extraction phase using this device, the carbon dioxide gas from the CO2 cylinder 8 is cooled to a liquid by the cool pump 9, and the liquid feed pump 10
and send it to the supercritical chromatograph 11. The introduced liquid carbon dioxide is heated to a predetermined temperature by the heat exchange column 12 to make it supercritical, and then sent to the cell 13, brought into contact with the object to be extracted in the cell 13, and extracted. Carbon dioxide (extraction phase) containing the extract is sent from the supercritical chromatograph 11 to the fraction collector 16, where the carbon dioxide is gasified and the extract is collected into the fraction collector. During sampling, a part of the extracted phase is sent to the UV detector 14 by the stopcock, the absorbance is measured, and this measurement value is sent to the computer 1.
5 to create a three-dimensional map and a contour map.

In this case, the cool pump 9 was set at 0°C, the flow rate of the liquid pump 10 was set at 2.00 m12/min, and the temperature of the constant temperature bath of the supercritical chromatograph 11 was set at 55°C. The carbon dioxide is passed through the heat exchange column 12, so the temperature is 55.0°C.

Note that the pressure of carbon dioxide is 10 MPa. Further, the outlet temperature of the fraction collector was set at 100.3°C. The amount of sample is determined by considering the upper concentration limit of the UV detector 14, which is determined because the cell capacity is 2 cm'. After all the analysis conditions were set as described above, the UV detector 14 was first put into the measurement state, and 20 seconds later, extraction and analysis were performed by flowing carbon dioxide into the cell.

[Comparative example 1] An attempt was made to extract chlorophyll using only carbon dioxide.

Three tablespoons of watercress powder was wrapped in filter paper, placed in a cell, and carbon dioxide was flushed through it. As a result, no peak appeared on the UV detector, and nothing was collected on the fragment collector. Furthermore, no change was observed in the watercress powder wrapped in filter paper, and from the above, it was confirmed that simply flowing carbon dioxide did not extract chlorophyll.

[Example 1] Wrap 3 pieces of watercress powder in filter paper, moisten it thoroughly with acetone, then put it in a cell and add 4 pieces of carbon dioxide.
It ran for a minute. As a result, a chlorophyll peak appeared on the UV detector. A three-dimensional diagram is shown in FIG. 7, and a contour map is shown in FIG. 8.

Additionally, the watercress powder wrapped in filter paper had lost its color from green to a grayish color.

[Example 2] In the same manner as in Example 1, watercress powder was sufficiently moistened with ethanol, and carbon dioxide was passed through it for 3.5 minutes in the same manner as in 5.

As a result, a chlorophyll peak appeared on the UV detector. A three-dimensional diagram is shown in FIG. 9, and a contour diagram is shown in FIG. 10. The shapes of these peaks are different from those when acetone is used, and it can be seen that the proportion of chlorophyll b is higher than when acetone is used.

Also, the color of watercress powder changed in the same way as acetone.

[Comparative Example 2] In the same manner as in Example 1, 3 teaspoons of watercress powder was wrapped in filter paper, thoroughly moistened with liquefied shortening oil, and then placed in a cell and carbon dioxide was flushed. As a result, no peak appeared on the UV detector, and only white shortening oil was extracted into the fraction collector.

Although the watercress powder wrapped in filter paper contained some shortening oil, no change in color was observed.

Therefore, it was confirmed that chlorophyll could not be extracted simply by moistening shortening oil in the same manner as with acetone and ethanol.

[Example 3] 1 g of watercress powder was mixed with 5 mfl of liquefied rayotoning oil, and after stirring well for 1 minute, the supernatant liquid was put into a cell and carbon dioxide was flushed. As a result, green shortening oil was squeezed into the fraction collector. The UV absorption curve diagram is shown in FIG. 11, and as shown, the peak of chlorophyll a was confirmed.

[Example 4] Similarly to Example 3, after stirring 5 mQ of salad oil and 1 g of watercress powder for 1 minute, the supernatant liquid was poured into a cell,
Carbon dioxide was flushed. As a result, green salad oil was extracted into the fraction collector. A three-dimensional diagram of the UV absorption is shown in Fig. 12, and a contour diagram is shown in Fig. 13. From this, the peak of chlorophyll a was confirmed.

[Comparative Example 3] Watercress powder was moistened with sorbitan monolaurate in the same manner as in Example 1, and carbon dioxide was passed through it. the result,
No peak recognized as chlorophyll could be confirmed.

[Brief explanation of drawings]

Figure 1 is a phase diagram of carbon dioxide, Figure 2 is a graph showing the temperature-pressure-density relationship of carbon dioxide, and Figures 3, 4, and 5 are pressure change method, temperature change method, and adsorption method, respectively. Fig. 6 is a process diagram showing the experimental equipment used in the examples, Fig. 7 is a three-dimensional diagram showing the change in absorbance with respect to extraction time when using acetone,
Figure 8 is a contour diagram of Figure 7, Figure 9 is a three-dimensional diagram showing the change in absorbance with extraction time when using ethanol, Figure 10 is a contour diagram of Figure 9, and Figure 11 is a shortening oil diagram. Absorption curve when using . FIG. 12 is a three-dimensional diagram showing changes in absorbance with respect to extraction time when salad oil is used, and FIG. 13 is a contour diagram of FIG. 12. 1... Extraction tank, 2... Separation tank, 3... Compressor, 4
... expansion valve, 5 ... heat exchanger, 6 ... heat exchanger,
7... Adsorption separation tank, 8... CO2 pump, 9...
Cool pump, 10... Liquid pump, 11... Supercritical chromatograph, 12... Heat exchange column, 13... Cell, 14...
UV detector, 15...computer 16...fraction collector

Claims (1)

[Claims] 1. A natural product containing chlorophyll is brought into contact with carbon dioxide in a supercritical state in the presence of an organic solvent, or chlorophyll is eluted from a natural product containing chlorophyll into an organic solvent. A method for extracting chlorophyll, which comprises extracting chlorophyll by bringing an organic solvent containing eluted chlorophyll into contact with carbon dioxide in a supercritical state. 2. A method for extracting chlorophyll, which comprises eluting chlorophyll from a natural product containing chlorophyll into oil and fat, and bringing the eluted chlorophyll-containing oil and fat into contact with carbon dioxide in a supercritical state to extract chlorophyll.