KR20120021798A - Methods for preparing high purity chlorophyll and chlorin e6 from chlorophyll extracts - Google Patents

Abstract

PURPOSE: A method for preparing chlorophyll and chlorine e6 from chlorophyll extract is provided to improve the purity of chlorine e6. CONSTITUTION: A method for preparing chlorohpyll of high purity comprises: a step of extracting green plants or green algae containing chlorophyll using methanol, ethanol, acetone or solution thereof; a step of adding hexane and water to the chlorophyll extract; and a step of placinhg the extract at -20°C to 0°C and purifying the chlorophyll of high purity. A method for preparing chlororine e6 comprises: a step of extracting the green plants or microalgae with methanol, ethanol, acetone or solution thereof; a step of mixing the chlorophyll extract with hexane and water; a step of placing the solution at -20°C to 0°C to purify chlorophyll of high purity; a step of dissolving the purified chlorophyll in solvent and adding acid; a step of adding water and obtaining pheophytin precipitate; and a step of adding base and reacting at 0°C-120°C.

Description

Methods for Preparing high purity chlorophyll and Chlorin e6 from Chlorophyll extracts}

The present invention relates to a method for efficiently preparing high purity chlorophyll and chlorine e6 from chlorophyll extract.

Chlorophyll is a compound found in leaves and microalgae of green plants. These chlorophylls are green pigments in chloroplasts, which are elliptical structures, and play an important role in carbon assimilation. Chlorophyll is named by chlorophyll a, b, c, d, e and bacterio chlorophyll a, b due to its chemical structural difference. These chlorophylls are commonly used forbin derivatives containing one magnesium (Mg) atom in one molecule and having a cyclopentane ring linked to cyclic tetrapyrrole in which four pyrroles are bonded by a methane group. Since the molecular structure of chlorophyll is almost similar to that of hemoglobin, it is called 'blue blood' and is known to perform a function similar to that of animal blood.

On the other hand, chlorine e6 is well known as a photosensitizer that is used in the photodynamic therapy (PDT) of malignancies. Photosensitizers require a characteristic that selectively accumulates only in tumor tissues, that is, selectivity in tumor tissues and normal tissues. Selective photosensitizers can increase the efficacy of PDT, shorten the treatment time, and reduce the side effects of drugs injected into the body. When irradiated and activated by light of specific wavelength, the photosensitive agent generates single oxygen and radical species, which is a kind of active oxygen, which directly kills tumor cells and induces immune inflammatory reactions. Or damage the tumor microvascular system. Conventional photosensitizers have been known to accumulate selectively to some extent in tumors, but some also accumulate in normal tissues including skin. However, chlorine e6 is known to have high selectivity for malignant cells and no toxicity to normal cells as compared to other photosensitizers used for tumor therapy.

Many patents and papers reported to date disclose a method of making chlorine e6 from chlorophyll.

Korean Patent No. 628548 discloses chlorine e6 through complex processes such as chlorophyll extraction, acid treatment, neutralization, hydrolysis, extraction of pheophovida, dissolution in acetone, addition of strong base, neutralization, and reprecipitation in spirulina biomass. A method of preparing is disclosed. The method has limitations for commercial use due to a long reaction time and a complicated reaction process, and is not suitable for treatment methods and the like in the pharmaceutical or food industry.

Republic of Korea Patent No. 841959 discloses a process for the preparation of chlorophyll-a comprising treating crushed intact chlorella with ethanol and optionally dioxane. Also disclosed is a process of treating chlorophyll-a with an acid to obtain a pheophitin a precipitate, which is dissolved in an ethanol solvent or acetone solvent and then treated with a base to produce chlorine e6. In the above method, 1,2-dioxane is used for the precipitation of chlorophyll-a, and 1,2-dioxane is a carcinogen, which limits its use in food and pharmaceutical manufacturing. In addition, the purification process for chlorophyll a and pheophytin a obtained separately in the process of preparing chlorine e6 from chlorophyll a is not separately performed. Therefore, since chlorophyll-a and pheophytin-a are not fully purified, the reaction proceeds in the form of extract and extract. Thus, the final substance, chlorine e6, is a mixture of impurities derived from chlorophyll and pheophytin. It resulted.

Republic of Korea Patent No. 994441 is to extract spirulina using ethanol or ethanol aqueous solution, to freeze precipitate the extract to obtain chlorophyll a, and to dissolve the chlorophyll a in hexane and then purified by glucamine A method for preparing chlorophyll, comprising the step of obtaining chlorophyll-a, is disclosed. The method takes a long reaction time to obtain a high purity chlorophyll, there is a disadvantage that consists of a complex process such as performing an impurity removal process by glucamine treatment. In addition, in the case of cryoprecipitation, as the capacity increases, it is difficult to realize reproducibility for removing impurities.

Republic of Korea Patent No. 896327 discloses a step of extracting spirulina using ethanol or an ethanol aqueous solution, a method of obtaining a chlorophyll precipitate by adding water alone or acetone aqueous solution to the ethanol extract. In addition, chlorophyll a acid treatment and base treatment to form chlorine; Separating the chlorine by treating the chlorine with a solvent in which acetonitrile and 1% trifluoroacetic acid, 1% formic acid or 1% acetic acid are mixed in a 50:50 volume ratio; Disclosed is a method for preparing the chlorine salt, which comprises treating the acid chlorine with a base to obtain a chlorine salt. The method applies the precipitation method using water and ethanol and the precipitation method using water and acetone for extraction of chlorophyll a. The crystallization of chlorophyll using water may be highly reproducible in the laboratory, but crystal formation in a high-volume process This is difficult and has low reproducibility. The patent describes that the yield of chlorophyll crystals relative to the amount of spirulina used is about 4.5% or more. However, given that the amount of chlorophyll contained in 1 g of spirulina is about 10-20 mg (corresponding to a maximum yield of about 1-2%), the chlorophyll crystal obtained by the above patent method contains indirect amounts of impurities. It seems to be suggested.

As described above, the method for producing chlorine e6 from chlorophyll so far known uses a limited amount of hazardous reactants in the food or pharmaceutical field, has a long reaction time and complexity, and is not reproducible. There is a limit to this.

It is an object of the present invention to provide a method for preparing high purity chlorophyll comprising the step of selectively separating and purifying chlorophyll from chlorophyll extract.

It is an object of the present invention to provide a process for the preparation of chlorine e6 which comprises a simplified purification process with short reaction time and high reproducibility.

It is another object of the invention to provide a process for the preparation of chlorine e6 purified to a high degree of purity suitable for use as a food and pharmaceutical ingredient.

In order to achieve the above object, the present invention is characterized by a method of preparing high-purity chlorophyll comprising the following process:

Iii) extracting the chlorophyll-containing green plant or microalgae with an extraction solvent selected from methanol, ethanol, acetone or their respective aqueous solutions;

Ii) adding hexane and water to the extract of chlorophyll and stirring;

V) stabilizing the stirred solution at a temperature of -20 ° C to 0 ° C, and then separating the hexane layer to obtain high-purity chlorophyll;

In addition, the present invention is characterized by a method for preparing chlorine e6 comprising the following steps:

Iii) extracting the chlorophyll-containing green plant or microalgae with an extraction solvent selected from methanol, ethanol, acetone or their respective aqueous solutions;

Ii) adding hexane and water to the extract of chlorophyll and stirring;

V) stabilizing the stirred solution at a temperature of -20 ° C to 0 ° C, and then separating the hexane layer to obtain high-purity chlorophyll;

Iii) dissolving the purified chlorophyll in a solvent selected from methanol, ethanol, acetone and a mixed solvent thereof, and then acidifying the mixture to an pH of 1-3 by adding an acid;

Iii) adding water to the acidified solution, and then stagnating at -20 ° C to 0 ° C to obtain a produced pheophytin precipitate; And

V) dissolving the pheophytin precipitate in acetone, adding a base, and reacting at a temperature of 60 ° C. to 120 ° C. to produce chlorine e6.

In the present invention, in performing a series of processes for preparing chlorine e6 from chlorophyll extract, the purification process of chlorophyll and pheophytin is included as an essential process, thereby obtaining an effect of greatly improving the purity of chlorine e6 finally produced.

The present invention uses the difference in the solubility of hexane and water in chlorophyll to obtain the effect of obtaining high purity chlorophyll as a precipitate from chlorophyll extract.

The present invention is to neutralize the reaction solution by adding water directly to the solution converted to pheopine by acid treatment of chlorophyll, as well as to improve the precipitation rate and precipitation rate of pheopine to obtain a high-purity pheopite Get at the same time.

The present invention performs the process of converting pheophytin to chlorine e6 under the presence of a base and heating conditions, thereby obtaining the effect of shortening the reaction time and improving the production yield.

Therefore, the manufacturing method of this invention acquires the effect which can mass-produce the chlorine e6 which is a photosensitizer.

1 is a result of HPLC analysis of chlorophyll extract.
2 is a result of HPLC analysis of high purity chlorophyll chlorophyll extract separated and purified by hexane and water solubility difference and cryoprecipitation method.
FIG. 3 shows the results of nuclear magnetic resonance (NMR) analysis of high-purity chlorophyll, in which chlorophyll extract is separated and purified by hexane and water solubility and freeze precipitation.
Figure 4 is an HPLC analysis of pheophytin obtained by acid treatment of high-purity chlorophyll and then precipitated after storage by adding water.
FIG. 5 shows the results of nuclear magnetic resonance (NMR) analysis of pheophytin obtained by acid treatment of high purity chlorophyll and adding water to precipitate after freezing storage.
FIG. 6 shows the results of HPLC analysis of chlorine e6 obtained by freeze storage of pheophytin after heating in the presence of a base.
7 is a result of nuclear magnetic resonance (NMR) analysis of chlorine e6 obtained by freeze storage of pheophitin in the presence of a base.

The present invention relates to a method for preparing high purity chlorophyll and chlorine e6 from chlorophyll extract.

The method for preparing chlorine e6 according to the present invention will be described in more detail by dividing each process as follows.

Extraction and Purification of Chlorophyll

This manufacturing process is a process of obtaining chlorophyll by extracting green plants or microalgae containing chlorophyll.

The present invention places no particular limitation on the selection of green plants or microalgae used to obtain chlorophyll, and can be applied to all plants or microalgae containing chlorophyll. Specific examples of green plants or microalgae containing chlorophyll may include mulberry leaves, pine needles, jujube, sesame root, kelp, sesame leaf, tofu peel, spinach, kale, chlorella, spirulina and the like.

In the present invention, the extraction solvent used to extract chlorophyll from green plants or microalgae and the purification solvent used to obtain high-purity chlorophyll from extracts of chlorophyll are selected from one another. The extraction solvent and the purification solvent are selected from the group of solvents commonly used in the food and pharmaceutical fields, and are selected in consideration of the polarity of the solvent and the difference in solubility in chlorophyll.

Thus, in the present invention, the extraction solvent may be selected from methanol, ethanol, acetone or their respective aqueous solutions, and the purification solvent may be selected from hexane and water. That is, in the present invention, a green plant or microalgae is extracted with a solvent having a relatively high polarity such as water, methanol, ethanol, acetone and an aqueous solution thereof, and concentrated to obtain a chlorophyll extract. Then, hexane with high solubility in chlorophyll and relatively low polarity is added to the chlorophyll extract and stirred to move the chlorophyll to the hexane layer as much as possible. High purity chlorophyll is obtained from the hexane layer through a purification process that transfers impurities to the water layer.

Looking at the dielectric constant of the solvent selected and used in the extraction and purification of chlorophyll in the present invention, water 78, methanol 32.7, ethanol 24.5, acetone 21, hexane 1.9 degree. That is, water has the strongest polarity, and the polarity decreases in the order of water> methanol> ethanol> acetone> hexane. Hexane has the same nonpolar characteristics as chlorophyll, so that it has high solubility in chlorophyll, and hexane has the property of being separated without mixing with methanol, ethanol, acetone or their respective aqueous solutions. Therefore, by purifying the chlorophyll extract using hexane and water, chlorophyll is dissolved and distributed in the hexane layer, and impurities other than chlorophyll are separated and distributed in layers of water, methanol, ethanol or acetone, thereby obtaining high-purity chlorophyll. This is possible.

When methanol, ethanol, acetone or their respective aqueous solutions are used as the extraction solvent for chlorophyll, it is more advantageous to control the water content in the extraction solvent to less than 15% by volume. This is because the presence of excess water in the extraction solvent may cause decomposition and denaturation of chlorophyll. In addition, the chlorophyll extract is concentrated in the range of 1 to 10% by volume to obtain a chlorophyll extract. At this time, if the concentration of the chlorophyll extract is less than the above range, the purification efficiency in the purification process using hexane and water is lowered, as well as a large volume of the extraction container is required, so the economic scale may be reduced by increasing the process scale.

Hexane used as a purification solvent is used in the range of 3 to 10 times, preferably 4 to 6 times the volume of the concentrated chlorophyll extract. If the amount of hexane used is less than the above range, the chlorophyll may not be easily transferred to the hexane layer, which may eventually cause the yield of chlorophyll to be lowered. Moving to the bed can eventually cause the chlorophyll to lose its purity. In the extraction process for hexane, the stirring time is 1 to 5 hours, preferably stirred for 1 to 3 hours. If the stirring time is too short, the transfer of chlorophyll to the hexane layer may not be completed. If the stirring is too long, impurities other than chlorophyll may move to the hexane layer, which is not preferable.

In addition, in the present invention, after extraction with hexane for the purification of chlorophyll, the extraction effect of the addition of water can be further maximized the purification effect of chlorophyll. That is, hexane may be added to the chlorophyll extract and stirred, and then water may be further added to transfer impurities to the aqueous layer to obtain high-purity chlorophyll from the hexane layer. In this case, the water to be added is preferably used in the range of 10 to 30% by volume based on the amount of hexane used. In addition, when the water is added and stirred for an excessively long time, it may be a problem that the separation of the layer is not easy due to the formation of an emulsion, it is preferable to stir for a short time within 5 to 30 minutes.

In addition, the present invention is maintained in a state of stagnation for about 1 to 5 hours for selective movement of chlorophyll and crystallization of impurities. The stagnation may induce selective movement of chlorophyll and crystallization of impurities even at room temperature, but it is preferable to maintain a low temperature condition of -20 ° C to 0 ° C for faster movement and precipitation induction. However, if the storage temperature is kept lower than -20 ° C migration and precipitates can be produced rapidly to reduce the removal efficiency of impurities.

In addition, the present invention does not place a special limitation on the number of solvent extraction in performing the extraction and purification process of the chlorophyll, two or more repeated extraction is uneconomical because the extraction efficiency is not significant over time, and also the modification of chlorophyll It is not preferable because it may cause.

High purity chlorophyll obtained by performing the above extraction and purification process is obtained in the form of a high concentrate or dry powder, it is preferable to obtain and use in the form of a dry powder containing no maximum moisture. Chlorophyll extracted and purified by the method of the present invention has a purity of 85% or more, preferably 90% or more, and can be extracted in a yield of 90% or more relative to the amount of chlorophyll contained in green plants or microalgae. That is, since the content of chlorophyll contained in 1 g of spirulina is about 20 mg, when the extraction method proposed by the present invention is applied, about 16 mg of chlorophyll can be obtained.

Pheophytin Synthesis

The manufacturing process is a process of obtaining pheopine represented by the following formula (2) to remove the magnesium by treating the chlorophyll represented by the formula (1) with an acid.

More specifically, after dissolving chlorophyll in a solvent, an acid is added and stirred to desorb the magnesium atom contained in the chlorophyll molecule, thereby converting it into phefitin.

The solvent for dissolving the chlorophyll is selected from methanol, ethanol, acetone and a mixed solvent thereof, preferably using acetone. Acetone has an advantage in that it is easy to dissolve chlorophyll even when a small amount is used because of its excellent solubility in chlorophyll. The acid added for the desorption of magnesium is used at least 3 equivalents, preferably 3 to 5 equivalents, per molecule of chlorophyll, to adjust the pH of the reaction solution in the range of 1 to 3. At this time, if the amount of the acid used is excessively excessive, since the side reaction may form the pheophoride by-products, care should be taken in adjusting the amount of acid added. There is no restriction | limiting in particular about the kind of acid added for magnesium desorption, It can select from normal inorganic acids, such as hydrochloric acid, a sulfuric acid, phosphoric acid, or normal organic acids, such as acetic acid. Preferably, inorganic acids such as hydrochloric acid or sulfuric acid are used.

In addition, the present invention is characterized by increasing the purity of the final product by separating and purifying the pheophytin produced by the addition of an acid used in the next chlorine e6 manufacturing process. Therefore, in the present invention, water is added to the reaction solution for separation and purification of the pheophytin, and the suspension is maintained while maintaining a low temperature of -20 ° C to 0 ° C to form a pheophytin precipitate. Although water has low solubility in nonpolar pheophytin, it promotes precipitation of pheophytin, but another effect of removing polar by-products and excess acid contained in the reaction solution is expected. The amount of water added is in the range of 1 to 3 times the volume of the reaction solution. When water is added and stirred for about 10 to 30 minutes, the pheophytin precipitate is formed at a high speed.

The pheophytin precipitate obtained by the above acid treatment method has a purity of 90% or more and is obtained in a yield of 95% or more based on the weight of chlorophyll used.

If necessary, the pheophytin precipitate obtained by the above method can be crystallized using methanol and hexane to obtain a pheophytin crystal. In other words, after dissolving the pheophytin precipitate in methanol, hexane was added and stirred to completely dissolve, and then stored for 5 to 30 minutes while maintaining a low temperature condition of -20 ℃ to 0 ℃ to produce a pheophytin crystals do. As the solvent used for the crystallization, it is more preferable for methanol and hexane to maintain a volume ratio of 1: 3 to 6 in order to improve crystallization efficiency and purity of pheophytin.

Chlorine e6 synthesis

This preparation process is a process of obtaining a chlorine e6 represented by the following formula 3 by heating the base in the pheopytin.

Synthesis of chlorine e6 of the present invention is carried out by 'bimolecular nucleophilic substitution reaction (SN2)'. The bimolecular nucleophilic substitution reaction is carried out under the condition that 2-3 equivalents of base per molecule of pheophytin are added to the solution in which the pheophytin is dissolved. At this time, the base is commonly used in the art may be used an inorganic base such as an alkali metal or alkaline earth metal hydroxide, or an organic base such as ammonium salt, there is no particular limitation in the selection of the base of the present invention, but preferably Sodium hydroxide is used. Methanol, ethanol, acetone, etc. can be used as a reaction solvent. The present invention is characterized by maintaining the conditions for heating the above-mentioned two-molecule nucleophilic substitution reaction (SN2) to 60 ℃ to 120 ℃ bar, by maintaining the heating conditions to obtain the effect of promoting the reaction rate and yield.

Upon completion of the SN2 reaction, the resulting chlorine e6 crystals are obtained by filtration. When the obtained chlorine e6 crystal is added to ethanol and stirred, impurities other than chlorine e6 are removed to obtain chlorine e6 as a high-purity product, and the effect of neutralizing the base remaining in the crystal can be obtained.

The chlorine e6 obtained by the above method has a purity of 95% or more, preferably 98% or more, and is obtained in a yield of 90% or more based on the weight of pheofin used.

If necessary, chlorine e6 obtained by performing the above method may be performed by adding a conventional purification process such as HPLC to increase the purity. In particular, most of the chlorophyll used as a raw material is chlorophyll a and b coexist in a 3: 1-5: 1 molar ratio, these compounds are known to be difficult to completely separate except the high separation method using a column. The high purity chlorophyll obtained through the preparation method of the present invention may be chlorophyll a or b, or a mixture thereof, and if it is desired to separate by chlorophyll a or b, it may be separated by a conventional purification method such as HPLC. .

As described above, the present invention is a simple and efficient method of extracting chlorophyll from the starting material containing chlorophyll without restriction of a particular raw material, and using a solvent solubility difference and polarity to remove substances other than chlorophyll at low temperature Chlorophyll of high purity can be obtained. Through this, in the synthesis of pheophytin and chlorine e6, a very effective method for shortening the reaction time and improving the purity was developed. In addition, the present invention has a process of synthesizing and manufacturing in a manner suitable for mass production by improving the reaction rate and the degree of purification, and the yield in a relatively simple process than the conventional method.

EXAMPLE

Example 1. Obtaining Chlorophyll Extract

2 g of acetone was added to 20 g of chlorella as an extraction solvent, stirred for 4 hours, and filtered to obtain a chlorophyll extract. The chlorophyll extract obtained above was analyzed by HPLC (High Pressure Liquid Chromatography) under the following conditions.

[HPLC Analysis Conditions]

Analyzer: agilent 1200 system

Sample injection volume: 10 μl

Developing solvent: methanol / acetonitrile / dichloromethane / water = 67.5 / 9.5 / 22.5 / 0.5 by volume

Solvent flow rate: 1 mL / min

Analysis wavelength: 450 nm

HPLC analysis of the chlorophyll extract obtained above is shown in FIG. According to the HPLC analysis results shown in FIG. 1, a peak corresponding to chlorophyll was observed at a retention time of 3 to 4 minutes, and it was confirmed that a considerable amount of impurities existed between 0 to 3 minutes. At this time, chlorophyll from Sigma-aldrich company was used as a reference.

Example 2. Obtaining High Purity Chlorophyll

The volume of the chlorophyll extract obtained in Example 1 was concentrated to about 5%, 500 mL of hexane was added to 100 mL of the concentrated chlorophyll extract and stirred for 1 hour. Additional 200 mL of water was added and stirring continued for 10 minutes. After standing for 3 hours at -10 ℃ condition, the reaction solution was separated by layer to obtain an organic layer. The organic layer was distilled under reduced pressure to obtain high purity chlorophyll (HPLC purity 85%, yield 90%).

The high purity chlorophyll obtained above was analyzed by HPLC and NMR under the conditions of Example 1, and the results are shown in FIGS. 2 and 3, respectively.

According to the HPLC analysis results shown in FIG. 2, the corresponding peak of chlorophyll was confirmed at a retention time of 3 to 4 minutes, and it was confirmed that impurities between 0 and 3 minutes were almost removed. In addition, it was confirmed that the purification degree of chlorophyll is about 85%. Its yield was extracted at least 90% relative to the amount of chlorophyll contained in the starting material containing chlorophyll. Qualitative analysis was performed through the NMR analysis shown in FIG. 3.

Example 3. Obtaining High Purity Pheophytin from Chlorophyll

After dissolving 1 g of the high-purity chlorophyll obtained in Example 2 in 50 mL of acetone, 3.365 mL of 1M hydrochloric acid solution was added (the pH of the solution became 2) so that the amount of hydrochloric acid added was about 3 equivalents, and the mixture was 2 hours. Was stirred. After the reaction was completed, 100 mL of water was added to the solution, stirred for 10 minutes, and then, precipitated for 3 hours at -10 ° C. The precipitate was filtered off to give phefitin (HPLC purity 90%, yield 95%).

The obtained pheophytin was analyzed by HPLC and NMR under the conditions of Example 1, and the results are shown in FIGS. 4 and 5, respectively.

According to the HPLC analysis results shown in FIG. 4, the corresponding peak of pheophytin was confirmed at a retention time of 7.5 to 8.5 minutes, and impurities were hardly identified at other time points. The purity of pheophytin confirmed by HPLC analysis was about 90%. The yield of pheophytin was at least 95% relative to the amount of high purity chlorophyll. Qualitative analysis was performed through the NMR analysis shown in FIG. 5.

1 g of the above was dissolved in 50 mL of methanol, then 250 mL of hexane was added and stirred for 10 minutes. After stirring for 3 hours at -10 ° C, 0.9 g (HPLC purity 92.5%) of partially crystallized pheophytin were obtained.

Example 4. Synthesis of Chlorin e6 from Pheophytin

After dissolving 1 g of pheophytin obtained in Example 3 in 50 mL of acetone, after adding 2.297 mL of about 2 equivalents of a 1M aqueous sodium hydroxide solution, the pH of the solution is about 12. Then, the mixture was stirred for 1 hour under the condition of heating to 80 ° C. Upon completion of the reaction, filtration gave 0.653 g (90% HPLC purity, 90% yield) of chlorine e6 crystals.

Further, in order to purify the chlorine e6 crystals obtained above with high purity, it was added to 100 mL of ethanol with respect to 1 g of chlorine e6, stirred for 2 hours, and filtered to obtain 0.95 g of high purity chlorine e6 crystals (HPLC purity 95%, yield 95%). Or more).

The obtained chlorine e6 crystals were analyzed by HPLC and NMR under the above conditions, and the results are shown in FIGS. 6 and 7, respectively.

[HPLC Analysis Conditions]

Analyzer: agilent 1200 system

Sample injection volume: 10 μl

Developing solvent: acetonitrile / 0.1% trifluoroacetic acid = 50/50 by volume

Solvent flow rate: 1 mL / min

Analysis wavelength: 407 nm

According to the HPLC analysis results shown in FIG. 6, a peak corresponding to chlorophyll was observed at a retention time of 3 to 4 minutes, and impurities were hardly identified at other time points. In addition, the purity of chlorine e6 was confirmed to be about 95% or more. Its yield was about 90% higher than the theoretical yield after reacting 1 g of pheophytin. The obtained chlorine e6 was completed qualitative analysis through NMR as shown in FIG.

As described above, chlorine e6 obtained by the manufacturing method according to the present invention is high in purity and can be used directly as food and medicine.

Therefore, the present invention is useful as a mass production method of chlorine e6 used in food and medicine.

Claims (11)

Iii) extracting the chlorophyll-containing green plant or microalgae with an extraction solvent selected from methanol, ethanol, acetone or their respective aqueous solutions;
Ii) adding hexane and water to the extract of chlorophyll and stirring; And
V) stabilizing the stirred solution at a temperature of -20 ° C to 0 ° C, and then separating the hexane layer to obtain high-purity chlorophyll;
Method for producing high purity chlorophyll comprising a.
Iii) extracting the chlorophyll-containing green plant or microalgae with an extraction solvent selected from methanol, ethanol, acetone or their respective aqueous solutions;
Ii) adding hexane and water to the extract of chlorophyll and stirring;
V) stabilizing the stirred solution at a temperature of -20 ° C to 0 ° C, and then separating the hexane layer to obtain high-purity chlorophyll;
Iii) dissolving the purified chlorophyll in a solvent selected from methanol, ethanol, acetone and a mixed solvent thereof, and then acidifying the mixture to an pH of 1-3 by adding an acid;
Iii) adding water to the acidified solution, and then stagnating at -20 ° C to 0 ° C to obtain a produced pheophytin precipitate; And
Iii) dissolving the pheophytin precipitate in acetone, adding a base, and reacting at a temperature of 60 ° C. to 120 ° C. to produce chlorine e6;
Method for producing chlorine e6 comprising a.
The method according to claim 1 or 2,
The extraction solvent used in the step iii) is ethanol, acetone or their respective aqueous solution.
The method according to claim 1 or 2,
Chlorophyll extract used in the ii) process is characterized in that the concentration of 1 to 10% by volume concentration.
The method according to claim 1 or 2,
Ii) the process comprises the step of adding hexane to the extract of the chlorophyll and stirring, further adding water and stirring.
The method according to claim 1 or 2,
High purity chlorophyll in the process iii) is obtained as a dry powder or a concentrate.
The method according to claim 2,
And adding water to the pheophytin precipitate obtained in step iv) and stagnating at −20 ° C. to 0 ° C. to obtain the produced pheophytin crystals.
The method according to claim 7,
The process of dissolving the pheophytin precipitate obtained in step iii) in methanol, followed by addition of hexane, stirring and dissolving, followed by stagnation at a temperature of −20 ° C. to 0 ° C. to obtain the resulting pheophytin crystals Manufacturing method characterized in that it is included.
The method according to claim 8,
Methanol and hexane is a manufacturing method, characterized in that used in 1: 3 to 6 volume ratio range.
The method according to claim 2,
Iii) a process used in which the base used in the process is sodium hydroxide.
The method according to claim 2,
Chlorine e6 obtained in the step iii) is washed with ethanol and further comprising the step of purifying.

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