KR100896327B1 - A method for preparing chlorophyll a and chlorin from spirulina - Google Patents

Abstract

The present invention relates to a method for preparing chlorophyll a and chlorine from spirulina, and more particularly, to efficiently extract chlorophyll a from spirulina through a precipitation method using water and ethanol, or a precipitation method using water and acetone. Treatment of chlorophyll a with acid to remove magnesium ions from chlorophyll a yields pheophytin a, followed by treatment of base with pheophytin a to form the photosensitizer chlorine and treatment of acetonitrile and acid Acid chlorine to separate the It relates to a method of obtaining chlorine.

Spirulina, Chlorophyll a, Pheophytin, Chlorine

Description

A method for preparing chlorophyll a and chlorin from spirulina}

The present invention relates to a method for preparing chlorophyll a and chlorine from spirulina, and more particularly, to efficiently extract chlorophyll a from spirulina through a precipitation method using water and ethanol, or a precipitation method using water and acetone. Treatment of chlorophyll-a with acid to remove magnesium ions from chlorophyll-a yields pheophytin a, followed by treatment of base with pheophytin-a to form the photosensitizer chlorine and treatment of acetonitrile and acid Acid chlorine to separate the It relates to a method of obtaining chlorine.

Spirulina is a microorganism belonging to a very small algae that grows actively in the surface of tropical alkaline lakes such as Chad Lake in Africa and Texcoco Lake in Mexico. The cells contain a large amount of chlorophyll and phycocyanin, which are absorbed by the sun's rays, and are actively growing by carbon assimilation. Since these pigments have a blue-green color, they have been classified as blue-green algae since ancient times.

The name Spirulina was given because it was shaped like a spiral, and the form is primitive given the fact that DNA is a double helix.

With the development of electron microscopy, the cell structure of microorganisms has been elucidated, and the structure of green algae and brown algae has been found to be different from those of higher plants. In other words, green algae have the same eukaryote structure as those of higher plants, while cyanobacteria have a prokaryote structure similar to that of bacteria. Microbiologists have argued that cyanobacteria are closer to bacteria than algae, so they should be included in bacteria. It now accepts this claim and classifies it as blue-green bacteria. However, the industry has called the microalgae the old practice.

Chlorophyll (chlorophyll, 葉綠素, chlorophyll) is a kind of assimilation pigments of photosynthetic organisms. Four pyrroles are derivatives of forvinyl in which a cyclopentane ring is linked to a cyclic tetrapyrrole bound by a methine group —CH═, with one Mg atom coordinated in the center of the tetrapytol ring and a pyrrole or a propionyl group of the pyrrole ring IV. Farnesol is an ester bond.

Chlorophyll has been found to closely match the molecular structure of hemoglobin, also called "blue blood," and is known to perform surprisingly similar functions to the blood of animals. In particular, chlorophyll is anti-cancer, anti-inflammatory action, regenerate damaged cells, detoxification, anti-cholesterol action and a wide variety of functions.

Patents related to the conventional method for producing chlorophyll and chlorine include a method of preparing chlorophyll a comprising treating ethanol and optionally dioxane in an intact chlorella that is not crushed in Korean Patent No. 10-0841959. And a method of producing chlorine by treating acid with chlorophyll-a.

However, the method described in the registered patent is a method of extracting chlorophyll from chlorella and preparing chlorine therefrom, which has a big disadvantage in that not only chlorophyll a but also chlorophyll b is extracted. Thus, the chlorophyll a and chlorophyll b are mixed in the extract. And the reaction proceeds in the form of precipitates, the final degree of purification of the photosensitizer chlorine is a purification degree of the material derived from chlorophyll a and chlorophyll b has the disadvantage that the actual purification is low.

In addition, the use of live chlorella has the disadvantage that it is very difficult to filter and extract. Particularly, when the organic solvent (ethanol) is added to the live chlorella and filtered, the filter is hardly filtered. Therefore, the centrifugation method should be used. This takes a lot of time in the filtration process for mass production.

In addition, the Dioxane component used as a co-solvent is known to be harmful to the human body. Dioxane is a carcinogen and rarely used in the industry.

In view of the above, the present inventors have extracted chlorophyll-a from spirulina belonging to the algae in high yield, using the same, and preparing the sensitizing chlorine by using the chlorophyll-a in a manner that is suitable for mass production in a relatively safe and simple process. In order to synthesize and manufacture the photosensitizer chlorine, which has been extracted and precipitated and substituted various cations, it has been studied to complete the efficient manufacturing process.

It is therefore an object of the present invention to provide a method for efficiently extracting chlorophyll-a from spirulina.

Another object of the present invention is to provide a method for preparing various cation-substitutable photosensitive chlorine using chlorophyll a extracted from spirulina.

In one embodiment, the present invention provides a method for efficiently extracting chlorophyll a from spirulina.

The present invention is a method of efficiently extracting chlorophyll a from spirulina by (1) precipitation method using water and ethanol, (2) precipitation method using water and acetone.

In a preferred embodiment, the present invention may provide a method for extracting chlorophyll a from spirulina comprising the following steps:

Stirring and filtering spirulina with 10-60% acetone;

Extracting chlorophyll-a by adding 90-100% ethanol to the filtered spirulina; And

Precipitating chlorophyll a from spirulina by adding 1/4 to 1/2 of the total volume of water to the extract and concentrating until it is 40-50% ethanol and removing other components than chlorophyll a.

In another preferred embodiment, the present invention may provide a method for extracting chlorophyll a from spirulina comprising the following steps:

Stirring and filtering spirulina with 10-60% acetone;

Extracting chlorophyll-a by adding 90-100% ethanol to the filtered spirulina; And

Obtaining chlorophyll-a by precipitating by adding acetone and water to the extract.

Chlorophyll a-Process A

Chlorophyll a-process A was removed by stirring spirulina with 60% acetone, filtering, adding water to the chlorophyll a extract extracted with ethanol solvent, and then concentrating to remove other components except chlorophyll a and precipitating chlorophyll a. The process consists of:

Spirulina used to obtain chlorophyll a is crushed spirulina.

The main feature of the chlorophyll-a preparation was the extraction of chlorophyll-a from spirulina, which was stirred with 60% acetone and filtered, the addition of water to the chlorophyll-a extract and the concentration of ethanol to precipitate chlorophyll-a and other components other than chlorophyll-a. The removal process is to be carried out. This process allows for the precipitation of chlorophyll-a with high yield and purification.

Impurity removal is performed by treating spirulina with 10-60%, most preferably 60% acetone, and 90-100% ethanol. The reason for using acetone and ethanol is that acetone and ethanol show excellent efficiency in removing impurities. When chlorophyll-a prepared is expected to be used as food, it is recommended to use acetone and ethanol, which are organic solvents that can be used in food processing. It is because it is preferable.

When pretreatment is performed using acetone having a concentration of 60% or more, chlorophyll-a is extracted and thus cannot be performed to remove impurities. In addition, when the pretreatment is performed using acetone at 10%, 20%, 30%, 40% and 50% concentrations, the removal of impurities is less than that of 60% acetone. Thus, it can be seen that 60% acetone is the most suitable pretreatment solution for removing impurities of chlorophyll-a.

Ethanol used in the chlorophyll a extraction process has a concentration of 90-100%. If ethanol of less than 50% chlorophyll a is not extracted from spirulina, using less than 80% chlorophyll a extraction efficiency is greatly reduced. The pretreatment process using acetone is performed 1 to 3 times, most preferably 2 times, and the extraction step using ethanol 1 to 3 times, most preferably 2 times.

Ethanol was added to spirulina, stirred to extract chlorophyll-a, filtered, and filled with water 1/3 of the total volume of chlorophyll-a extract. Chlorophyll a precipitated by ethanol concentration is obtained by filtration. In the concentration process, chlorophyll-a precipitates and impurities other than chlorophyll-a are removed.

The yield for chlorophyll a in process A is 8-12%. The yield is calculated as a percentage of the weight ratio of chlorophyll a obtained to 1 g of spirulina weight used. Chlorophyll a purity of process A is 80-90% (average 80-82%).

Chlorophyll a-Process B

Chlorophyll a-process B is a method using ethanol, acetone, and water to prepare a highly purified chlorophyll a through a simple process.

The information on spirulina used in chlorophyll a-process B is the same as in process A.

The first step of chlorophyll a-process B is to wash spirulina with 60% acetone and then extract chlorophyll a from spirulina using 90-100% ethanol. In this process, if the concentration of ethanol is less than 80%, there is a problem that the extraction efficiency is greatly reduced. Extraction step using ethanol is carried out once to three times, most preferably twice.

The second step of chlorophyll-a process B is to precipitate chlorophyll-a by inducing freezing precipitation to chlorophyll-a extract using acetone and water. Through this step, chlorophyll-a can be precipitated to obtain chlorophyll-a as well as to increase the purity of chlorophyll-a. For the amount of acetone and water used, 3 L of acetone is added to 5 L of chlorophyll a extract. Acetone used here is 10-60%% acetone, most preferably 60% acetone. Acetone is added and stirred well before additional water is added. The amount of water to be used is added 1 L of water to 5 L of chlorophyll a extract and stirred sufficiently.

After the acetone and water addition steps, the reaction solution is frozen at -20 ° C or lower to allow for better precipitation of chlorophyll-a.

The yield for chlorophyll a in process B is 7-8%. The yield is calculated as a percentage of the weight ratio of chlorophyll a obtained to 1 g of spirulina weight used. The chlorophyll a purity of process A is 83-95%.

In another aspect, the present invention provides a method for preparing various cation-substitutable photosensitive chlorine using chlorophyll a extracted from spirulina.

In the present invention, the method of preparing the photosensitive chlorine is a process of synthesizing or preparing chlorine using the chlorophyll a obtained by the two chlorophyll a production methods.

In the present invention, the final material made in spirulina is the reaction proceeds only in chlorophyll a so that the degree of purification of the final material is the degree of purification of the photosensitizer chlorine, the final material derived from chlorophyll a.

In a preferred embodiment, the present invention may provide a method for preparing chlorine from spirulina comprising the following steps:

Stirring and filtering spirulina with 10-60% acetone;

Extracting chlorophyll-a by adding 90-100% ethanol to the filtered spirulina;

Precipitating chlorophyll a from spirulina by adding 1/4 to 1/2 of the total volume of water to the extract and concentrating until it is 40-50% ethanol and removing other components than chlorophyll a;

Treating chlorophyll-a obtained with an acid to remove magnesium ions from chlorophyll-a to obtain pheophytin a;

Treating base with pheophytin a to form a photosensitizer chlorine;

A solvent containing Acetonitrile: 1% TFA in a 50:50 volume ratio, a solvent in which Acetonitrile: 1% Formic acid is mixed in a 50:50 volume ratio, or a solvent in which Acetonitrile: 1% Acetic acid is mixed in a 50:50 volume ratio Isolating acid chlorine; And

^{Na + OH -, K + OH} - or _{^{NH 4 + OH - Na +,}} K + or NH ₄ ⁺ salt by treating a Obtaining chlorine.

In another preferred embodiment, the present invention may provide a method for preparing chlorine from spirulina comprising the following steps:

Stirring and filtering spirulina with 10-60% acetone;

Extracting chlorophyll-a by adding 90-100% ethanol to the filtered spirulina;

Adding acetone and water to the extract to obtain chlorophyll a by precipitating to precipitate;

Treating chlorophyll-a obtained with an acid to remove magnesium ions from chlorophyll-a to obtain pheophytin a;

Treating pheophytin a with a base to form chlorine;

A solvent containing Acetonitrile: 1% TFA in a 50:50 volume ratio, a solvent in which Acetonitrile: 1% Formic acid is mixed in a 50:50 volume ratio, or a solvent in which Acetonitrile: 1% Acetic acid is mixed in a 50:50 volume ratio Isolating acid chlorine; And

^{Na + OH -, K + OH} - or _{^{NH 4 + OH - Na +,}} K + or NH ₄ ⁺ salt by treating a Obtaining chlorine.

In the present invention, NaOH or KOH may be used as the base for treating the pheophytin a.

Photosensitizer Chlorine  Course A

Photosensitizer Chlorine-Process A is characterized in that the preparation of the photosensitizer chlorine from pheophytin a is carried out in an acetone solvent.

According to chlorine-process A, pheophytin a is obtained by treating acid with chlorophyll-a extract to remove magnesium ions from chlorophyll-a. The chemical formulas of chlorophyll a and pheophytin a are as follows:

Treatment of chlorophyll-a with acid removes magnesium ions from Formula 1 to produce pheophytin a. Treat the chlorophyll-a extract with acid to bring the pH of the chlorophyll-a extract to 1-2. In this case, when the pH exceeds 3, there is a problem that the magnesium ion removal rate is greatly reduced. Sulfuric acid and hydrochloric acid were used as the acid used in this step. Pheophytin a is usually obtained in the form of a precipitate.

In the step of forming a photosensitizer chlorine, the precipitate of pheophytin a is dissolved in acetone, and the solution of pheophytin a is treated with basic reagent to pH 13-14. As the base used in this step, various bases can be used, in which NaOH or KOH is used. Obtaining the photosensitizer chlorine is carried out in acetone solvent. Na ⁺ salt chlorine is formed when NaOH is added in acetone solvent and K ⁺ salt chlorine is formed when KOH is added.

Salt chlorine mixed with 50:50 volume of Acetonitrile: 1% TFA, 50:50 volume of Acetonitrile: 1% Formic acid, or 50:50 volume of Acetonitrile: 1% Acetic acid After treating the solvent to stir to form the acid chlorine and put it in -20 ℃ freezer to separate the water layer and the organic solvent layer to form the acid chlorine. Na ⁺ OH by filtration and the organic solvent (acetonitrile) layer ^{-, K + OH -, NH} 4 + OH - treating the _{^{Na +, K +, NH 4}} + salt Obtain chlorine.

The purity of the Na ⁺ salt chlorine or K ⁺ salt chlorine in the photosensitizer chlorine-process A was 90-95%, a solvent containing Acetonitrile: 1% TFA in a 50:50 volume ratio, or Acetonitrile: 1% Formic acid. a mixed solvent of 50 volume ratio, or Acetonitrile: 1% Acetic acid to the processes the solvent is mixed in a volume ratio of 50: 50 separating the acid chlorin ^{Na + OH -, K + OH} -, NH 4 + OH - treatment and the Na ⁺ , K ⁺ , NH ₄ ⁺ salt Salt obtained with chlorine The degree of purification for chlorine is 99.9%.

The yield for chlorine is 3-7% with spirulina as starting material. The yield is calculated as a percentage of the weight ratio of chlorine obtained to 1 g dry weight of spirulina used.

The chemical formula of chlorine is as shown in the following formula (3).

Extraction method of chlorophyll a using spirulina of the present invention has the advantage of a simple and fast filtration process. As a result, the cost and yield are much better. In addition, spirulina contains only chlorophyll-a, so the purity of pure chlorophyll-a is very high.

In addition, the pheupinin synthesized in spirulina consists only of pheupintine a and the final substance is also a substance formed in pheupinin a. The degree of purification of various cation-substituted chlorine is 95% to 99%, which indicates the degree of purification that can be used as a medicine in a very easy way.

That is, in spirulina, only chlorophyll-a is extracted and precipitated, and the synthesized pheophytin is pure photosensitive chlorine, which is composed only of pheophytin a and the final substance is also formed in pheophytin a. A solvent containing Acetonitrile: 1% TFA in a 50:50 volume ratio, a solvent in which Acetonitrile: 1% Formic acid is mixed in a 50:50 volume ratio, or a solvent in which Acetonitrile: 1% Acetic acid is mixed in a 50:50 volume ratio the acid then left in a freezer at -20 ℃ for chlorine formed was separated to an aqueous layer and an organic solvent layer to thereby form acid chlorine organic solvent (acetonitrile) layer was filtrated to ^{Na + OH -, K + OH} -, NH 4 + Treatment of OH ^- with Na ⁺ , K ⁺ , NH ₄ ⁺ salts Purity degree of various cation-substituted chlorine by the method of obtaining chlorine is 95% ~ 99% shows the degree of purification that can be used as a medicine in a very easy way. Synthesis and preparation of the photosensitizer chlorine using spirulina is more suitable for mass production process with more streamlined process, higher yield and higher purification rate than the existing method.

The present invention efficiently extracts chlorophyll-a from spirulina using a precipitation method using water and ethanol, or a precipitation method using water and acetone, and removes magnesium ions from chlorophyll-a by treating acid with chlorophyll-a. Pheophytin a is obtained, followed by treatment of base with peophytin a to form a photosensitive chlorine, and treatment with acetonitrile and acid to separate acid chlorine, followed by base treatment with salt By obtaining chlorine, it has a very good effect that only chlorophyll a can be extracted purely and pure sensitizer chlorine which is formed only from pheophytin a can be produced.

In addition, the present invention has the advantage of synthesizing and preparing a photosensitive chlorine substituted with chlorophyll a extraction and precipitation and various cations in a manner suitable for mass production in a relatively simple process using spirulina.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to examples, but these examples are merely illustrative of the present invention, and the scope of the present invention is not limited only to these examples.

Example  1: suitable as starting material Spirulina  Selection

An ultrasonically ground spirulina sample was prepared as a starting material for obtaining chlorophyll a and a photosensitive chlorine. It was then checked how much chlorophyll a and other ingredients were contained in the ultrasonic milled spirulina.

20 g of ultrasonically ground spirulina was taken, 100 ml of ethanol was added, stirred for 30 minutes, 1 hour 30 minutes, 3 hours, 6 hours, and 12 hours, followed by filtration to obtain an extract solution. The extraction solution was analyzed by HPLC (high pressure liquid chromatography) and mass spectrometry, and performed using an agilent 1200, agilent 6320 ion trap LC / MS system. The sample injection volume was 10 µl and the solvent flow rate was 1 ml / min. As a developing solution, a mixture of methanol and acetonitrile in a 1: 1 ratio was used. As a reference, chlorophyll a from Sigma-aldrich company was used. In Figure 1, the peak corresponding to the retention time of about 20.883 minutes corresponds to chlorophyll a.

As can be seen in Figure 1, when using spirulina was not included chlorophyll b purely contained only chlorophyll a was confirmed that it can increase the yield for chlorophyll a. As a result, it was found that the process of removing chlorophyll b was not necessary at all.

Example  2: From spirulina  Obtaining Chlorophyll a

Course A

A-1: Precipitation and Impurity Removal of Chlorophyll a from Spirulina

Shandong Binzhou Tianjian Biotechnology Co., Ltd. In order to remove components other than chlorophyll a from spirulina purchased from Ltd, spirulina was stirred with 60% acetone and filtered, ethanol was added to spirulina and stirred to extract chlorophyll a, and water was added to the total volume of chlorophyll a extract. 1/3 filled. Chlorophyll a precipitated by ethanol concentration was obtained by filtration. Extraction precipitation process with ethanol and water was performed once.

After taking a small amount of the precipitate, high pressure liquid chromatography (HPLC) confirmed how much chlorophyll a and other ingredients were contained. HPLC was performed using an agilent 1200 system with a sample injection volume of 10 μl and solvent flow rate of 1 ml / min. As a developing solution, a mixture of methanol and acetonitrile in a 1: 1 ratio was used.

As a result, as shown in FIG. 2, a peak corresponding to chlorophyll a was observed at a retention time of about 19.076 min, and it was found that a considerable amount of impurities were removed from the retention time of 00-13 min. In addition, it was confirmed that the purification degree of chlorophyll-a was 82.19% in the HPLC chromatogram. The yield for chlorophyll a in Process A was 9.32%. As a reference, chlorophyll a from Sigma-aldrich company was used.

Course B

B-1: Precipitation and Impurity Removal of Chlorophyll a from Spirulina

Shandong Binzhou Tianjian Biotechnology Co., Ltd. In order to remove components other than chlorophyll a from spirulina purchased from Ltd, spirulina was washed with 60% acetone, filtered, ethanol was added to spirulina and stirred to extract chlorophyll a. Extraction with ethanol was carried out twice. Subsequently, after adding 1/2 acetone and 1/5 distilled water of the total volume of the chlorophyll a extract, the mixture was sufficiently stirred and stored in a freezer for at least 24 hours to induce precipitation. The precipitate was then filtered to give chlorophyll a.

After taking a small amount of the chlorophyll a precipitate, high pressure liquid chromatography (HPLC) confirmed how much chlorophyll a and other components were contained.

As a result, as shown in FIG. 3, a peak corresponding to chlorophyll a was observed at a retention time of about 19.206 minutes. In addition, it was confirmed that the purification degree of chlorophyll-a was 84.617% in the HPLC chromatogram. The yield for process B chlorophyll a was 7.52%. As a result, it can be seen that when chlorophyll-a precipitate is formed using acetone and water, chlorophyll-a can be obtained with higher purification.

Example  3: from chlorophyll a Photosensitizer Chlorine  Produce

3-1: Production of pheophytin a

Acetone was added to chlorophyll-a obtained in Process A of Example 2, and the mixture was sufficiently stirred. Thereafter, the chlorophyll-a extract was treated with 5N HCl or 5N H ₂ SO _4, and the pH of the extract was added to 1-2, and the reaction of removing magnesium ions from chlorophyll-a was stirred for 3 hours. After stirring, the pH was adjusted to 6 ~ 7 (neutralized) with NaOH, and the black phephytin a solution was stored at -20 ° C. in the freezer for 6 hours to form a precipitate. A precipitate was obtained.

3-2: Production of the photosensitizer chlorine

Course A

After dissolving the 3-1 pheophytin a precipitate with acetone, 5N NaOH or 5N KOH was added thereto to adjust the pH of the pheophytin a solution to 13-14, and stirred for 6 hours to remove light from the pheophytin a. A sensitized chlorine precipitate was formed. After filtration, acetone was completely removed.

After taking a small amount of the chlorine precipitate obtained above, high pressure liquid chromatography (HPLC) confirmed how much chlorine and other ingredients were contained.

As a result, as shown in FIG. 4, a peak corresponding to chlorine was observed at the retention time of about 6.729 minutes, and the purity of chlorine was 98.822%.

Na ⁺ salt chlorine or K ⁺ salt chlorine is prepared and then a mixture of Acetonitrile: 1% TFA in a 50:50 volume ratio, or a solvent in which Acetonitrile: 1% Formic acid is mixed in a 50:50 volume ratio, or Acetonitrile: 1% and processes the solvent mixture of Acetic acid 50: 50 by volume as the acid separation chlorin ^{Na + OH -, K + OH} -, NH 4 + OH - , and process the _{^{Na +, K +, NH 4}} + salt Chlorine was obtained. Chlorine was obtained. The yield of chlorine in salt form yielded 5.8 g with spirulina 100 g treatment and yield 5.8%. In addition, high pressure liquid chromatography (HPLC) was performed on chlorine in salt form. As a result, as can be seen in Figure 5, the degree of purification of the salt form chlorine was 99.99%.

Example  4: according to acetone concentration Spirulina  Pretreatment Efficiency Survey

To investigate the efficiency of the pretreatment process for removing impurities depending on the acetone concentration, HPLC was performed on the extract obtained after stirring and filtering spirulina with acetone at 10%, 20%, 30%, 40%, 50% and 60% concentrations. (high pressure liquid chromatography) was performed. As a result, as can be seen in Figures 6a to 6f it was confirmed that as the concentration of acetone increases, the components extracted and removed from spirulina are more varied and more.

Through the above results, it was confirmed that 60% acetone is the most suitable pretreatment solution for removing impurities of chlorophyll-a.

1 is a result of HPLC analysis of spirulina before extraction used in the present invention.

FIG. 2 is an HPLC chromatogram showing the result of extracting chlorophyll-a through the precipitation method using water and ethanol using the spirulina in the present invention, that is, process A. The arrow is the peak corresponding to chlorophyll a.

3 is an HPLC chromatogram showing the result of extracting chlorophyll-a through the precipitation method using water and acetone using the spirulina in the present invention, that is, process B. The arrow is the peak corresponding to chlorophyll a.

Figure 4 is an HPLC chromatogram for chlorine prepared through chlorophyll a process A or B using spirulina in the present invention. The arrow is the peak corresponding to chlorine.

FIG. 5 is an HPLC chromatogram for final synthesized chlorine prepared via Process A or B using spirulina in the present invention. The arrow is the peak corresponding to chlorine.

Figure 6a to 6f is a pretreatment process using water and acetone using spirulina in the present invention 10% (Fig. 6a), 20% (Fig. 6b), 30% (Fig. 6c), 40% (Fig. 6d), HPLC chromatogram of the extract obtained after extraction with 50% (FIG. 6E) and 60% (FIG. 6F) acetone to remove components other than chlorophyll.

Claims (6)

Washing spirulina by stirring with 60% acetone and filtering; Extracting chlorophyll-a by adding 90-100% ethanol to the washed spirulina; And Precipitating chlorophyll a from spirulina by adding 1/4 to 1/2 of the total volume of water to the extract and concentrating until it is 40-50% ethanol and removing components other than chlorophyll a. A method of extracting chlorophyll a from spirulina. Washing spirulina by stirring with 60% acetone and filtering; Extracting chlorophyll-a by adding 90-100% ethanol to the washed spirulina; And A method of extracting chlorophyll a from spirulina comprising the step of obtaining precipitated chlorophyll a by adding 60% acetone and water to the extract liquid by freezing precipitation. Treating the chlorophyll-a obtained in claim 1 or 2 with an acid to remove magnesium ions from the chlorophyll-a to obtain pheophytin a; Treating base with pheophytin a to form a photosensitizer chlorine; A solvent containing Acetonitrile: 1% TFA in a 50:50 volume ratio, a solvent in which Acetonitrile: 1% Formic acid is mixed in a 50:50 volume ratio, or a solvent in which Acetonitrile: 1% Acetic acid is mixed in a 50:50 volume ratio Isolating acid chlorine; And ^{Na + OH -, K + OH} - or _{^{NH 4 + OH - Na +,}} K + or NH ₄ ⁺ salt by treating a A method of making chlorine from spirulina comprising the step of obtaining chlorine. delete The method for extracting chlorophyll a from spirulina according to claim 1, wherein the pretreatment using acetone is performed 1 to 3 times, and the extraction using ethanol is performed 1 to 3 times. The method for extracting chlorophyll a from spirulina according to claim 2, wherein the extraction with ethanol is carried out once to three times.

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