KR20190053490A - Process for preparing high purity pheophytin, Chlorin e6, and Chlorin e6 and PVP complex from natural products containing chlorophyll - Google Patents

Abstract

The present invention relates to a manufacturing method of high purity pheophytin from a natural product containing chlorophyll, and a manufacturing method of chlorin e6 using pheophytin and composite of chlorin e6 and polyvinylpyrrolidone (PVP). According to one embodiment of the present invention, disclosed is a manufacturing method of pheophytin comprising: a step of dissolving green plants or microalgae containing chlorophyll in an extraction solvent comprising any one of methanol, ethanol, acetone or each aqueous solution of the same, adding acids, stirring the same and extracting a pheophytin extract; a step filtering a mixture of mixing nucleic acids and water with the pheophytin extract; a storage step of adding carbon black to the filtered solution, stirring the same and storing the same for a predetermined time; and a step of separating a nucleic acid layer from a resulting product of the storage step, to obtain pheophytin.

Description

TECHNICAL FIELD [0001] The present invention relates to a process for preparing high purity pheophytin from natural products containing chlorophyll, and a process for preparing a complex of chlorin e6 and chlorphene e6 with polyvinylpyrrolidone (PVP) using pheophytin, and a process for preparing high purity pheophytin, Chlorin e6, natural products containing chlorophyll}

The present invention relates to a process for producing high purity pheophytin from chlorophyll-containing natural products and a process for producing a complex of chlorin e6, chlorin e6 and polyvinylpyrrolidone (PVP) using pheophytin.

Chlorine e6 is well known as a photosensitizer used in the photodynamic therapy (PDT) of malignant tumors. In the tumor tissue and normal tissue, the photosensitizer selectively accumulates only in the tumor tissue, that is, the selectivity is required. The selectivity of the photosensitizer substance which is commercialized so far and the PDT effect is increased, And the side effects of drugs injected into the body can be reduced.

When chlorin e6 accumulated in the tumor is activated by irradiation with light of a specific wavelength of 660 nm, it reacts with a molecule having oxygen in the tumor to generate singlet oxygen and radical species, which are active oxygen species . This directly kills tumor cells, causes an immune inflammatory response, or damages the tumor's microvessel system to remove the tumor.

It has been previously known that most of the photosensitizers accumulate selectively in tumors, but some accumulate in normal tissues including skin. However, chlorin e6 has a high selectivity for malignant cells compared to other photosensitizers used in tumor therapy But not toxic to normal cells.

A method for producing chlorin e6 described in the following conventional patents discloses a method for producing chlorophyll e6 by extracting chlorophyll from Spirulina or Chlorella, obtaining porphytin through acid treatment, and then treating with base under acetone have.

Korean Patent No. 10-0628548 discloses a method for producing chlorin e6. This patent discloses a method for producing chlorin e6, which comprises the steps of extracting chlorophyll, performing acid treatment, neutralization, hydrolysis, extraction of perofovid a, dissolving in acetone, , Neutralization, and re-precipitation, to obtain chlorin e6.

The patent has problems with long reaction times and complicated reaction processes, and is unsuitable for mass production of chlorin e6.

Korean Patent No. 10-0841959 discloses a method for producing chlorophyll-a, comprising the step of extracting ethanol with chlorotrimethylsilane, which is not pulverized, and selectively treating dioxane, and a process for producing chlorophyll-a by treating an acid with chlorophyll, Is disclosed.

The patent discloses a complicated manufacturing process for making chlorophyll e6 after producing chlorophyll extraction / purification to obtain porphytin to obtain chlorin e6. For the purification of chlorophyll, chlorophyll was obtained using 1,2-Dioxane, which is known to be harmful to human body.

In addition, there is a disadvantage in that the raw material storage, filtration and extraction are very difficult when using the raw chlorella, which can be a limitation of mass production.

Korean Patent Nos. 10-0896327 and 10-0950441 disclose a method for producing chlorophyll-a and chlorin or photodiazine from spirulina by a method of obtaining chlorophyll by precipitation using water and ethanol or acetone, The removal of impurities by precipitation and the selective treatment of chlorophyll through glucamine treatment were suggested. The chlorophyll was obtained by acid treatment, base treatment and recrystallization.

Chlorophyll crystallization using water has a high reproducibility when the volume is small, but has low reproducibility, in which crystals are not formed well in extracts of about 25 L or more. Also, in the case of freeze precipitation, it is difficult to realize the reproducibility of the impurity removal as the amount increases.

It is also confirmed by the yield that the degree of refining of chlorophyll is low. The amount of chlorophyll contained in the weight of 1 g of spirulina has about 10 to 20 mg, which can be confirmed in various documents having a content of about 1 to 2%. In this patent, having a yield of about 4.5% or more means that the spirulina has an excess amount of chlorophyll, which means that chlorophyll and other substances are coexisting and extracted or mass analyzed.

In addition, when the yields of pheophytin and chlorine e6 are observed, the yields are smaller than the theoretical yields in comparison with the obtained chlorophyll, which means that the impurities are removed or the reaction is lost when the crystallization occurs.

In the extraction of high purity chlorophyll and the synthesis of chlorin e6, the reproducibility is deteriorated as the reaction time and the external environment (humidity, temperature, etc.) are changed, and there are disadvantages such as low temperature storage and glucamine treatment.

That is, the method of the conventional patents must undergo a complicated process of obtaining salt chlorin e6 through various processes such as chlorophyll extraction, acid treatment, base treatment, hydrolysis, neutralization, reprecipitation, separation and purification, As a single compound, it is unsuitable for use in cosmetics, food or medicine.

According to one embodiment of the present invention, there is provided a process for producing high purity pheophytin comprising the step of obtaining pheophytin from chlorophyll-containing plants or green algae without chlorophyll extraction.

According to one embodiment of the present invention, a method for efficiently producing chlorin e6, which is a photosensitizer, is provided utilizing high purity pheophytin.

According to one embodiment of the present invention, a method for producing chlorin e6 having a short production time, high reproducibility and purification degree is provided.

According to one embodiment of the present invention, there is provided a method for producing a complex of chlorin e6 and PVP (polyvinylpyrrolidone), which is purified to a high purity and suitable for use as a raw material for cosmetics, foods and pharmaceuticals.

According to one embodiment of the present invention, there is provided a method for producing pheophytin comprising dissolving green plants or microalgae containing chlorophyll in an extraction solvent composed of methanol, ethanol, acetone or an aqueous solution of each of them Adding an acid, and stirring to extract a pheophytin extract; Filtering the mixture obtained by adding hexane and water to the pheophytin extract; A storage step of adding carbon black to the filtered solution, stirring the solution, and storing the solution for a predetermined time; Separating the hexane layer from the result of the storing step to obtain pheophytin; Wherein the method comprises the steps of: (a) preparing a solution of pheophytin;

According to one embodiment of the present invention, there is provided a method for producing chlorin e6, wherein chlorophyll-containing green plants or microalgae are dissolved in an extraction solvent composed of methanol, ethanol, acetone or an aqueous solution of each of them, And stirring to extract pheophytin extract; Filtering the mixture obtained by adding hexane and water to the pheophytin extract; A storage step of adding carbon black to the filtered solution, stirring the solution, and storing the solution for a predetermined time; Separating the hexane layer from the result of the storing step to obtain pheophytin; Dissolving pheophytin, which is the result of the step of obtaining, in acetone, and then basifying the solution by adding a base; And stirring the resultant of the basicization step while applying heat thereto; And neutralizing the resulting product of the stirring step with a pH of 7 to 9 to produce a salt chlorine e6 of formula (3).

(Formula 3)

According to an embodiment of the present invention, there is provided a method for producing a complex of acid chlorin e6 with polyvinylpyrrolidone (PVP), comprising the steps of: mixing acid chlorin e6 having formula 4 with polyvinylpyrrolidone (PVP);

(Formula 4)

Adjusting the pH by adding a base to the result of performing the mixing step; Filtering the result of the step of adjusting the pH using a membrane filter; And lyophilizing the result of the filtration to form a powder; A method for producing a complex of acid chlorin e6 with polyvinylpyrrolidone (PVP) can be provided.

According to an embodiment of the present invention, chlorophyll-containing green plants or microalgae containing chlorophyll may be subjected to chlorophyll extraction without any additional steps, without addition of hydrochloric acid, appropriate ratio of solvent and addition of carbon black And a method for preparing high purity pheophytin having a high purity and yield than the conventional method through purification separation process for extracting high purity pheophytin through low temperature storage treatment and removing substances other than pheophytin do.

According to one embodiment of the present invention, a method for efficiently producing chlorin e6, which is a photosensitizer, is provided utilizing high purity pheophytin.

According to one embodiment of the present invention, by extracting pheophytin directly without extracting chlorophyll from a natural product containing chlorophyll, a very simplified process with a shorter production time and a high reproducibility is presented and mass production And a process for producing pheophytin and chlorin e6 having high purification degree.

According to one embodiment of the present invention, there is provided a method for producing a complex of chlorin e6 and PVP (polyvinylpyrrolidone), which is purified to a high purity and suitable for use as a raw material for cosmetics, foods and pharmaceuticals.

FIG. 1 shows HPLC analysis results of pheophytin prepared according to one embodiment of the present invention.
2 shows the nuclear magnetic resonance (NMR) analysis results of pheophytin prepared according to one embodiment of the present invention.
Figure 3 shows the HPLC analysis of chlorin e6 prepared according to one embodiment of the present invention.
Figure 4 shows mass spectrometry results of chlorin e6 prepared according to one embodiment of the present invention.
FIG. 5 shows the results of comparative analysis of absorbance using UV-vis in chlorin e6 according to an embodiment of the present invention.
6 shows the nuclear magnetic resonance (NMR) analysis results of chlorin e6 prepared according to one embodiment of the present invention.
7 shows the results of comparative analysis of absorbance using UV-vis in chlorin e6 and polyvinylpyrrolidone (PVP) complexes according to an embodiment of the present invention.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

The terminology used herein is for the purpose of illustrating embodiments and is not intended to be limiting of the present invention. In the present specification, the singular form includes plural forms unless otherwise specified in the specification. The terms "comprises" and / or "comprising" used in the specification do not exclude the presence or addition of one or more other elements.

As used herein, the terms " cancer " and " tumor " refer to or describe physiological pathologies in mammals that are typically characterized by uncontrolled cell growth. Benign and malignant cancers, and dormant tumors or micro metastasis are included in this definition. Examples of cancer include, but are not limited to, carcinoma, lymphoma, bloomer, sarcoma, and leukemia. Other examples of such cancers include skin cancer, oral cancer, melanoma (skin metastasis), breast cancer (skin metastasis), skin basal cell carcinoma, deformed skin cancer, squamous cell squamous cell carcinoma, and squamous cell carcinoma of the lower lips. . Examples of other cancers include breast cancer, squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous cell carcinoma of the lung), total neuroblastoma, neuroblastoma, typical glioblastoma and mesenoproblastoma, Cancer of the peritoneum, gastrointestinal cancer, gastric cancer (including gastrointestinal cancer), pancreatic cancer, ovarian cancer, cervical cancer, liver cancer, bladder cancer, colorectal cancer, colorectal cancer, endometrial cancer, uterine carcinoma, salivary cancer, kidney cancer, prostate cancer, , Thyroid cancer, various types of head and neck cancer, B-cell lymphoma (non-Hodgkin's lymphoma (NHL), small lymphocytic (SL) NHL, middle / follicular NHL, Diffuse NHL, high grade immune subtype NHL, high grade lymphocyte NHL, high grade small undifferentiated cell NHL, bulky disease NHL, mantle cell lymphoma, AIDS related lymphoma, and valdendrogmaglobulinemia), chronic lymphocytic leukemia (CLL), chronic lymphocytic leukemia (CML), acute lymphoblastic leukemia (ALL), hair cell leukemia, chronic myelogenous leukemia, post-transplant lymphoproliferative disorder (PTLD), abnormal vascular proliferation associated with maternal hyperplasia, edema Such as those associated with brain tumors, and Meigs' syndrome.

In addition, " cancer " and " tumor ", as used herein, refers to all neoplastic cell growth and proliferation (whether malignant or benign) and all pre-cancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are mutually exclusive as referred to herein.

As used herein, the term " microalgae " is a concept that includes plants that are photosynthetic to chlorophyll, that is, algae, among the lower plants in which roots, stems and leaves are not systemically differentiated, and preferably algae ), Which can not be seen with the naked eye and can be seen only through a microscope. It means a single-celled algae with a size of 50 ㎛ or less, living as a creature floating freely in water.

As used herein, the term 'chlorin e6' is meant to include both 'salt chlorin e6' and 'acid chlorin e6', and distinguish between salt chlorin e6 and acid chlorin e6 only when there is a distinct benefit. .

As used herein, the term 'complex' refers to a complex of 'acid chlorin e6' and polyvinylpyrrolidone (hereinafter "PVP"), and the complex is a pharmacological composition for photodynamic therapy .

Hereinafter, a method for preparing 'pheophytin' and 'chlorin e6' using the same according to an embodiment of the present invention will be described. The chlorin e6 prepared according to this method can be used to prepare a complex of 'chlorin e6 and polyvinylpyrrolidone (PVP)' to be described later.

First Embodiment

According to the first embodiment, a method for producing chlorin e6 comprises dissolving chlorophyll-containing green plants or microalgae in an extraction solvent composed of methanol, ethanol, acetone or an aqueous solution of them, adding an acid, Stirring the mixture to extract pheophytin extract (S10); Filtering the mixed mixture of hexane and water into the pheophytin extract (S20); A storage step (S30) of adding carbon black to the filtered solution, stirring the solution, and storing the solution for a predetermined time; Separating the hexane layer from the result of the storing step (S40) to obtain pheophytin; Dissolving pheophytin as a result of the obtaining step in acetone, and then basifying (P50) the base by adding a base; And a step (S60) of stirring the result of performing the basicization step while applying heat thereto; A step (S70) of neutralizing the result of the stirring step to adjust pH to 7 to 9 to produce salt chlorine e6 of formula (3); . Namely, according to the first embodiment, the salt chlorin e6 is produced.

(Formula 3)

According to the first embodiment, there is no particular limitation on the selection of green plants or microalgae used as a starting material for extracting pheophytin in step S10, and any plant or microalgae containing chlorophyll can be applied . All plants or microalgae containing chlorophyll may include, for example, mulberry leaves, pine needles, jujubes, mung bean sprouts, kelp, sesame leaves, pea bark, spinach, kale, chlorella, spirulina and the like.

According to the first embodiment, the extraction solvent used in the step (S10) of extracting the pheophytin extract may be any one of methanol, ethanol, acetone, or an aqueous solution thereof, Is 85% to 100%.

According to the first embodiment, the acid used in step S10 of extracting the pheophytin extract may be hydrochloric acid (HCl). The addition of the acid in step S10 is for removing the magnesium atom contained in the chlorophyll molecule. There is no particular limitation on the kind of the acid to be added, and the addition of an inorganic acid such as hydrochloric acid, sulfuric acid, phosphoric acid, Organic acids may be selected and used.

In step S20 according to the first embodiment, for example, the amount of hexane may be 1/10 to 2/10 times the volume of the extraction solvent, and the amount of water may be 1/3 to 1/2 times the volume of the extraction solvent .

According to the first embodiment, even when pheophytin is directly extracted from the starting material (raw material) containing chlorophyll without performing a separate step of extracting and purifying chlorophyll, purification of pheophytin by carbon black Can be improved.

That is, carbon black serves as a seed for forming crystallization through agglomeration of impurities, thereby enhancing the removal efficiency of materials other than pheophytin, thereby enhancing the degree of purification.

In the storage step S30, for example, 1/20 to 1/10 volume of carbon black of green plants or microalgae is added, and the mixture is stirred for 1 hour to 2 hours and then stored for a predetermined time Lt; / RTI > In the present embodiment, the predetermined time means a time for allowing the impurities to be agglomerated by the carbon black.

Alternatively, the storage step S30 may be a step of storing at least 4 hours or more at a temperature of -20 DEG C to -10 DEG C. That is, it is possible to improve the flocculation efficiency by carbon black by carrying out the storage step (S30) at a low temperature of -20 ° C to -10 ° C.

According to the first embodiment, the step (S40) of acquiring pheophytin includes a step of washing the result of the storing step (S30) with ethanol, and then removing hexane from the purified solution do. For example, ethanol is added to the result of the storage step (S30) and stirred. Then, the mixture is allowed to stand until the layer separation occurs, impurities remaining in the hexane layer are removed, and the hexane layer is concentrated by rotary vacuum decompression to remove the solvent .

According to the first embodiment, for example, potassium hydroxide, calcium hydroxide or sodium hydroxide can be used as the base to be added in the basicization step (S50), and sodium hydroxide is preferably used.

In the step S60 according to the first embodiment, for example, the operation of stirring for about 4 hours to 8 hours while applying heat of 60 DEG C or more can be performed. Here, by applying heat at 60 DEG C or more, an effect of promoting the reaction rate and increasing the yield can be obtained. In this embodiment, a cooling device may be separately provided so that the solvent does not flow.

According to the first embodiment, the neutralization treatment in step S70 is to adjust the pH to 7 to 9, preferably to 8 to 9, using a weak base to obtain salt chlorine e6.

According to the first embodiment, the step S70 may further include, after the neutralization treatment, washing the result of performing the neutralization treatment with acetone and washing the result of performing the washing with acetone with ethanol. Thereby, the impurities other than the salt chlorin e6 can be removed, and the salt chlorine e6 of high purity can be obtained.

Second Embodiment

According to the second embodiment, the step of dissolving the salt chlorin e6 in a solvent of any one of methanol, ethanol, acetone, and a mixed solvent thereof after the step S10 to S70 of the first embodiment, (S80); (S90) of concentrating the result of the acidification step under reduced pressure to remove the solvent; Removing the solvent, dissolving the resultant in ethyl acetate, and adding water to produce acid chlorine e6 (S100); . That is, according to the second embodiment, acid chlorin e6 is produced.

(Formula 4)

According to the second embodiment, it is possible to use any of trifluoroacetic acid and acetic acid, which is easy to remove after the reaction, in the step of adding acid (S80) and acidification, preferably trifluoroacetic acid do.

According to the second embodiment, the step (S100) of producing acid chlorin e6 comprises the steps of adding water and then removing the ethyl acetate by concentration under reduced pressure; And vacuum drying the result of performing the step of concentrating and removing under reduced pressure; As shown in FIG.

Hereinafter, a method for producing a complex of 'acid chlorin e6 and polyvinylpyrrolidone (PVP)' according to an embodiment of the present invention will be described. A " complex " according to the present method can be prepared by the above-mentioned acid chlorin e6.

A method for preparing a composite according to an embodiment of the present invention comprises mixing (S200) polyvinylpyrrolidone (PVP) with acid chlorine e6 having the formula (4);

(Formula 4)

Adjusting the pH by adding a base to the result of performing the mixing step (S210); Filtering the result of the step of adjusting the pH using a membrane filter (S220); And a step (S230) of lyophilizing the result of the filtration step to form a powder; .

According to this embodiment, step S210 may be a step of adjusting the pH to 8-9, using basic NaOH.

According to this embodiment, in step S200, acid chlorin e6 and polyvinylpyrrolidone (PVP) may be mixed in a weight ratio of 1: 1.

Hereinafter, a method for producing pheophytin, a method for producing chlorin e6, and a method for producing a complex of chlorin e6 and polyvinylpyrrolidone (PVP) according to an embodiment of the present invention will be described in detail do.

Pheophytin  Extraction and purification

The method is a method for producing " pheophytin " according to an embodiment of the present invention. The pheophytin obtained according to the extraction and purification method of pheophytin of the present method can be used to prepare the above-mentioned 'chlorin e6' and 'complex'.

According to the present method, there is no particular limitation on the selection of green plants or microalgae used as a starting material for extracting pheophytin, and any plant or microalgae containing chlorophyll can be applied. All plants or microalgae containing chlorophyll may include, for example, mulberry leaves, pine needles, jujubes, mung bean sprouts, kelp, sesame leaves, pea bark, spinach, kale, chlorella, spirulina and the like.

According to this method, when a green plant or microalgae containing chlorophyll is dissolved in an extraction solvent, and an acid is added and stirred, the magnesium atom contained in the chlorophyll molecule is desorbed by the added acid and is converted into pheophytin.

That is, the present method can extract pheophytin of high purity represented by the following formula (2) without going through a separate procedure for extracting chlorophyll represented by the following formula (1) from green plants or microalgae.

≪ Formula 1 >

(2)

According to the present method, the extraction solvent may be any one of organic solvents such as methanol, ethanol, and acetone, or an aqueous solution thereof, preferably ethanol. It is preferable that the purity of the extraction solvent has a concentration of about 85% or more. When the concentration of the extraction solvent is less than 85%, the proportion of water as the polar solvent increases in the extraction solvent and the content of the impurities increases. Lt; RTI ID = 0.0 > pheophytin < / RTI >

According to this method, there is no particular limitation on the type of acid to be added for the magnesium desorption, and any one of ordinary inorganic acids such as hydrochloric acid, sulfuric acid, and phosphoric acid, or common organic acids such as acetic acid can be selected and used. Preferably, hydrochloric acid is used.

According to this method, by using hydrochloric acid as a kind of acid to be added for magnesium desorption, pectin constituting the cell wall is melted to make the cell wall free, and the effect of increasing the extraction efficiency of pheophytin can be obtained.

Conventionally, it took 8 hours to 12 hours or more to extract pheophytin by a separate process of extracting chlorophyll from green plants or microalgae. However, it has been known that the extraction of chlorophyll by addition of hydrochloric acid It is possible to shorten the extraction time because it takes about 4 hours for pheophytin to be extracted at a yield of 90% or more.

Then, hexane in a ratio of 1/10 to 2/10 volume of the extraction solvent and 1/3 to 1/2 volume of water of the extraction solvent are added to the pheophytin extract, stirred, and then filtered. According to this method, the pheophytin migrates to the hexane layer to the greatest extent through the separation of the layer by the solubility and the specific gravity of the solvent, and the impurities other than the pheophytin which can be contained in the hexane layer migrate to the water layer.

Subsequently, 1/20 to 1/10 volume of carbon black of a green plant or microalgae is added to the resultant to which the filtering process is applied, and the mixture is stirred for about 1 hour to 2 hours, and then cooled at a low temperature For 4 hours or more. Thereafter, the mixture is allowed to stand at room temperature and left to stand until the temperature of the solution to which the carbon black is added is room temperature. Then, the layer separation takes place with an aqueous solution of hexane-extraction solvent, and only the hexane layer can be separated.

That is, in the storage step, carbon black serves as a seed for forming crystallization through aggregation of impurities, and in particular, by storing at low temperatures of -20 ° C to -10 ° C, impurities other than pheophytin (For example, lipid, beta-carotene, etc.) can be improved and the purity of high purity can be obtained.

 As described above, according to the present method, even when pheophytin is extracted directly from the starting material (raw material) containing chlorophyll without extracting and purifying chlorophyll, it is possible to obtain a pheophytin- There is a characteristic that refining degree can be improved.

According to the present method, the temperature of the storage step is preferably -20 DEG C to -10 DEG C, and the storage time is preferably 4 hours or more, and more preferably 3 hours or more. When the temperature of the storage step is higher than -10 ° C, the cooling rate of the solvent is slow, so that the efficiency of separating the impurities contained in the extraction solvent and the water layer and the pheophytin contained in the hexane layer is lowered and included in the extraction solvent and water layer The resulting impurities are difficult to crystallize, which may hinder the obtaining of pheophytin. If the temperature of the storage step is lower than -20 占 폚, the separation rate between the impurity layer and the hexane layer may be lowered and the impurity removal efficiency may be lowered.

The pheophytin extracted and purified by the method of the present invention described above has a purity of 85% or more, and can be extracted at a yield of 90% or more in comparison with the amount of chlorophyll contained in green plants or microalgae.

salt Chlorine  Conversion of e6

The method is a method for producing 'salt chlorine e6' according to an embodiment of the present invention.

According to the present method, a base is added to pheophytin dissolved in acetone, and a reaction is carried out by heating to obtain a salt chlorine e6 represented by the following general formula (3).

(3)

The process for preparing the salt chlorin e6 of the present invention is carried out by a 'bis nucleophilic substitution reaction (SN2)'. The bi nucleophilic substitution reaction is carried out under the condition that 2 to 3 equivalents of base per molecule of pheophytin is added to a solution of pheophytin dissolved in acetone.

As the base according to the present method, for example, potassium hydroxide, calcium hydroxide or sodium hydroxide can be used, and sodium hydroxide is preferably used.

According to the present method, pheophytin is dissolved in acetone. Sodium hydroxide is added to the dissolved acetone solution, and the mixture is stirred for about 4 to 8 hours while heating at 60 DEG C or more. The effect of promoting the reaction rate and increasing the yield can be obtained by applying heat of 60 DEG C or more.

After completion of the reaction, the pH is adjusted to 7 to 9 with a weak base to obtain salt chlorine e6, and the pH is preferably adjusted to 8 to 9. When the pH is less than 8, the carboxyl group of chlorine e6 has less than 2 Na substitution. When the pH is 9 or more, the chlorine e6 is substituted with Na at the center of the porphyrin.

When the neutralization treatment is finished, the resulting salt chlorine e6 crystal is obtained. The obtained salt chlorine e6 is washed with acetone and ethanol in this order to remove impurities other than the salt chlorine e6, thereby obtaining salt chlorine e6 of high purity.

The salt chlorin e6 thus obtained has a purity of 90% or more and is obtained in a yield of 90% or more as compared with the theoretical yield.

If necessary, the salt chlorin e6 obtained by the above method can be further purified by an ordinary purification process such as HPLC to increase its purity. In particular, the chlorophyll contained in the raw material coexists with a molar ratio of chlorophyll a and b of 3: 1 to 5: 1. These compounds are known to be difficult to separate completely except for the high separation method using a column. Therefore, since the chlorophyll contained in the raw material reacts with the chlorin reaction to coexist, a column should be added to improve the purification degree of chlorin.

mountain Chlorine  Conversion of e6

The method is a method for producing 'acid chlorin e6' according to an embodiment of the present invention.

According to the present method, acid chloride is added to the salt chlorine e6 dissolved in the reaction solvent, and then ethyl chloride and water are added to obtain acid chlorine e6 represented by the following general formula (4).

≪ Formula 4 >

For example, the reaction solvent may be any one of methanol, ethanol, acetone and a mixed solvent thereof, preferably acetone.

For example, the acid may be any one of trifluoroacetic acid and acetic acid, preferably trifluoroacetic acid. This is because they are easily removed by vacuum depressurization and extraction after completion of the reaction. If hydrochloric acid or sulfuric acid is used, a salt can be formed in the NH of the porphyrin molecule of chlorin e6 to reduce the purification degree.

According to the present method, after the salt chlorine e6 is dissolved in acetone, trifluoroacetic acid is added to the dissolved acetone solution to obtain acid chlorine e6. Thereafter, ethyl chloride and water are added to the acid chloride e6 to remove impurities, whereby high purity acid chloride e6 can be obtained.

The acid chlorine e6 obtained by the above method has a purity of 95% or more and is obtained in a yield of 90% or more as compared with the theoretical yield.

mountain Chlorine  e6 and PVP  Composite manufacturing

The method is a method for producing 'acid chlorin e6 and PVP complex' according to an embodiment of the present invention. The production of the acid chlorin e6 and the PVP complex of the present invention means inducing molecular-molecular coordination of the acid chlorin e6 and PVP. Acid chlorin e6 has a problem of lowering photoactivities due to aggregation in an aqueous solution. For this purpose, it is possible to prevent the aggregation formation by using PVP and to increase the stability through coordination with acid chlorin e6 . Here, although PVP is used as a polymer used for inhibiting aggregation formation, it is not limited thereto, and it is possible to use a polymer having water-solubility and a substance having a coordination bond such as cyclodextrin.

According to the present method, the sterilized water-bacterial cells were killed with an electric vapor incineration drug or the like, and the acid chlorin e6 and PVP were weighed in a weight ratio of 1: 1, and 1N NaOH was adjusted to pH 8-9. do. The complex is sterile filtered through a 0.22 μm membrane filter. The sterile filtered complex is then lyophilized to a powder.

For example, the freeze-drying method may be a method of cooling while maintaining a low temperature of -20 캜 to -40 캜 and a low pressure of 20 mba, and removing water using a cold trap (-70 캜 to -80 캜). At the above-mentioned low temperature and low pressure, the vacuum state becomes such that water can be sublimated into a vapor state and removed. The surface of the composite is not cured or shrunk because it is sublimed and dried in the frozen state. Keep the final amount of moisture to dry between 1.5% and 2%, as it can be damaged by excessive drying.

In the present invention, the complex formation rate depends on the degree of purification of the acid chlorin e6, and the degree of purification of the acid chlorin e6 should be 95% or more so that the formation of the complex reaches 100%.

As described above, according to the embodiments of the present invention, it is possible to extract pheophytin directly from the starting material containing chlorophyll without purification of chlorophyll, and thus it is suitable for mass production. That is, the present invention has a process of synthesizing and preparing in a manner suitable for mass production by improving the reaction rate and purification degree by a simpler process than the conventional process, and improving the yield, and as the purification and yield of pheophytin is higher, and the yield and purification degree of e6 are also increased.

In addition, according to the embodiments of the present invention, pheophytin is extracted from a starting material containing chlorophyll without any restriction of a specific raw material by using an extraction solvent and hydrochloric acid. Thereafter, the addition of carbon black, the difference in solubility and polarity of the solvent , It is possible to obtain high purity pheophytin by a simple and efficient method of removing pheophytin and its substance through a low temperature storage treatment. Thus, in the synthesis of salt chlorin e6, the reaction time is shortened and the purification degree is improved.

Therefore, by using the pheophytin produced by the present invention, it is possible to improve the yield and purification of chlorin e6, and thus it is possible to produce a high-purity complex.

Hereinafter, experimental examples will be described according to embodiments of the present invention.

Experiment 1. Pheophytin  Extraction and purification

10 L of ethanol and 1 L of a hydrochloric acid solution are added to 1 kg of a natural substance containing chlorophyll (spirulina, chlorella, and mulberry leaves), and the mixture is stirred for 4 hours. After stirring, 2 L of hexane is added, 2 L of water is added, and the mixture is stirred for about 1 hour and then filtered to remove spirulina residue. To this solution, about 100 g of carbon black is added, and the storage step of stirring for about 1 hour and storing at a low temperature of -20 캜 for about 4 hours is carried out. After the storage step is carried out, the solution is allowed to stand at room temperature. The layer separation takes place with an aqueous hexane-ethanol solution, and only the hexane layer is separated.

1 L of 70% ethanol was added to the separated hexane solution, and the mixture was stirred for about 30 minutes until the layer separation occurred. After removing impurities remaining in the hexane layer, the hexane layer was removed by rotary vacuum decompression to remove the solvent.

In the case of spirulina, about 40 g of pheophytin obtained by the above process can be obtained, about 35 g in case of chlorella, and 20 g in case of mulberry leaf. It was confirmed that the yield of this extract was about 90% or more as compared with the amount of chlorophyll contained in the corresponding starting material containing chlorophyll.

After a small amount of pheophytin was obtained, it was confirmed by HPLC (High Pressure Liquid Chromatography) how much pheophytin and other components were contained under the following conditions.

[HPLC analysis conditions]

- Analytical instrument: agilent 1200 system

- Sample injection amount: 10 μl

Eluent: methanol: acetonitrile: dichloromethane: water = 67.5: 9.5: 22.5: 0.5 (volume ratio)

- Solvent flow rate: 1 ml / min

- Analysis wavelength: 450 nm

The results of HPLC analysis of pheophytin obtained under the above conditions are shown in Fig. As shown in FIG. 1, pectin was confirmed at a retention time of about 15 to 25 minutes, and it was confirmed that impurities of 0 to 15 minutes were almost completely removed. It was also confirmed that the purity of pheophytin was about 85% or more.

As shown in Fig. 2, the obtained pheophytin was subjected to qualitative analysis through NMR. Unlike the conventional method, purification of pheophytin was performed by a simpler process without chlorophyll extraction, Pheophytin was obtained.

Experiment 2. Salt Chlorine  Conversion of e6

10 g of the pheophytin obtained in Experiment 1 was dissolved in 500 ml of acetone and treated with a 1 M sodium hydroxide solution as a base reagent so that the pH of the solution became about 12 and the solution was stirred for about 4 hours while heating at about 60 ° C . In this case, the added base reagent is a mixture of 3 to 5 equivalents of base solution, which is sufficient for the amount of pheophytin, so that the base reagent can react with one molecule of pheophytin, and the base reagent contains 35 ml to 55 ml of 1N NaOH Lt; / RTI >

After the reaction is complete, adjust the pH to 8 ~ 9 and observe no pH change for about 1 hour.

The crystals were washed with ethanol and acetone, and the resulting crystals were stirred in an ethanol solution for about 2 hours to remove impurities other than chlorine. The remaining sodium hydroxide was removed to remove the pH Can be lowered.

After taking a small amount of salt chlorine e6 obtained above, the amount of salt chlorin e6 and other components was determined by HPLC. The results are shown in FIG.

[HPLC analysis conditions]

- Analytical instrument: agilent 1200 system

- Sample injection amount: 10 μl

- Developing liquid: methanol: acetonitrile: 0.1% trifluoroacetic acid = 50: 50 (volume ratio)

- Solvent flow rate: 1 ml / min

- Analysis wavelength: 407 nm

According to the results of HPLC analysis of the salt chlorin e6 obtained under the above conditions, as shown in FIG. 3, the corresponding peak of chlorine was confirmed at a retention time of about 4 minutes, and almost no impurities were observed at other times .

It was also confirmed that the degree of purification of the salt chlorin e6 was about 90% or more, and 10 g of pheophytin was reacted to obtain about 7.54 g of chlorin e6. This corresponds to a yield of about 90% or more as compared to the theoretical yield.

The qualitative analysis of the high-purity salt chlorin e6 obtained above from the mass spectrum is shown in FIG.

Experiment 3. Mountain Chlorine  Conversion of e6

Add 250 ml of acetone and 25 ml of trifluoroacetic acid to 5 g of salt chlorine e6 obtained in Experiment 2 and stir for about 4 hours or longer. After stirring, the solvent and the acid are removed by rotary vacuum concentration under reduced pressure. It is dissolved in about 500 ml of ethyl acetate, and 500 ml of water is added to wash it. After stirring for about 1 hour, the mixture is allowed to stand until layer separation takes place. The water layer is discarded and the ethyl acetate layer is collected to remove the solvent.

A UV-vis spectrometer was used to confirm the purification of the obtained acid chlorine e6, and the results are shown in FIG.

 The reference material utilized> 95% of chlorine e6 sold to Frontier. Purification was confirmed by absolute comparison of the 10 ppm solution of acid chlorin e6 prepared from the reference material with calibration curve at concentrations of 8, 10 and 12 ppm.

As a result, the purification degree of the acid chlorin e6 was 95% or more, and about 4.15 g of the acid chlorin e6 was obtained. This corresponds to 90% of the theoretical yield.

NMR was analyzed for its qualitative analysis, and the results are shown in FIG.

Experiment 4. Mountain Chlorine  e6 and PVP  Composite manufacturing

5 g of the obtained acid chlorine e6 and 5 g of PVP are added to 50 ml of water, and 1N NaOH is added thereto while stirring to adjust the pH to 8 to 9. Stir for about 2 hours and filter through a 0.22um membrane filter. The filtered solution is freeze-dried to remove water.

A UV-vis spectrometer was used for the quantitative analysis of the compound forming the complex (complex), and the result is shown in FIG. The reference material utilized> 95% of the triosodium chloride e6 sold by Fronter. The calibration curve of the reference material was confirmed at 6, 8, and 10 ppm, and the reference compound and the complex-forming compound prepared were each prepared to have chlorine e6 of 8 ppm.

As a result, the degree of purification of the composite was 95% or more, and about 9.5 g of the composite was obtained. This corresponds to about 95% of the theoretical yield.

While the present invention has been described with reference to the particular embodiments and drawings, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims. This is possible. Therefore, the present invention should not be construed as limited to the embodiments described, but should be determined by the scope of the appended claims as well as the appended claims.

Claims (16)

In the method for producing pheophytin,
Dissolving a green plant or microalgae containing chlorophyll in an extraction solvent composed of methanol, ethanol, acetone or an aqueous solution of each of them, adding an acid, and stirring to extract a pheophytin extract;
Filtering the mixture obtained by adding hexane and water to the pheophytin extract;
A storage step of adding carbon black to the filtered solution, stirring the solution, and storing the solution for a predetermined time;
Separating the hexane layer from the result of the storing step to obtain pheophytin; ≪ RTI ID = 0.0 > pheophytin. ≪ / RTI > The method according to claim 1,
The step of obtaining the pheophytin comprises:
And washing the hexane layer separated from the result of performing the storing step with ethanol to remove hexane from the purified solution. 3. The method according to claim 1 or 2,
In the storing step,
Wherein the storage temperature is from -20 占 폚 to -10 占 폚 and the storage time is at least 4 hours or more. The method of claim 3,
The extraction solvent used is one having a concentration of methanol, ethanol or acetone of 85% to 100%
Wherein the acid is hydrochloric acid (HCl). The method of claim 3,
The hexane is used in an amount of 1/10 to 2/10 by volume of the extraction solvent,
Wherein the water is used at 1/3 to 1/2 volume times the volume of the extraction solvent. The method of claim 3,
Wherein the carbon black uses 1/20 to 1/10 times the volume of the green plant or the microalgae. In the method for producing chlorin e6,
Dissolving a green plant or microalgae containing chlorophyll in an extraction solvent composed of methanol, ethanol, acetone or an aqueous solution of each of them, adding an acid, and stirring to extract a pheophytin extract;
Filtering the mixture obtained by adding hexane and water to the pheophytin extract;
A storage step of adding carbon black to the filtered solution, stirring the solution, and storing the solution for a predetermined time;
Separating the hexane layer from the result of the storing step to obtain pheophytin;
Dissolving pheophytin, which is the result of the step of obtaining, in acetone, and then basifying the solution by adding a base; And
Stirring the resultant of the basicization step while applying heat thereto;
Neutralizing the result of the stirring step by adjusting the pH to 7 to 9 to produce salt chlorine e6 of formula (3); Lt; RTI ID = 0.0 > e6. ≪ / RTI >
(Formula 3)

8. The method of claim 7,
The step of producing the salt chlorin e6 comprises:
Washing the resultant of the neutralization treatment with acetone after the neutralization treatment; And
Washing the resultant of washing with acetone with ethanol; ≪ RTI ID = 0.0 > e6. ≪ / RTI > 9. The method according to claim 7 or 8,
After the step of producing the salt chlorin e6,
Dissolving the salt chlorine e6 in a solvent selected from the group consisting of methanol, ethanol, acetone and a mixed solvent thereof, and acidifying the mixture by adding an acid;
Concentrating the result of the acidification step to remove the solvent;
Dissolving the resultant of the step of removing the solvent with ethyl acetate, and then adding water to produce acid chlorine e6 of formula (4); ≪ RTI ID = 0.0 > e6. ≪ / RTI >
(Formula 4)

10. The method of claim 9,
In the storing step,
Wherein the storage temperature is from -20 占 폚 to -10 占 폚, and the storage time is at least 4 hours or longer. 11. The method of claim 10,
The step of producing the acid chlorin e6 comprises:
Adding the water and concentrating the ethyl acetate under reduced pressure to remove the ethyl acetate; And
Vacuum drying the result of performing the step of concentrating and removing under reduced pressure; ≪ RTI ID = 0.0 > e6. ≪ / RTI > 12. The method of claim 11,
The base is any one of potassium hydroxide, calcium hydroxide, and sodium hydroxide,
Wherein the acid is trifluoroacetic acid. 12. The method of claim 11,
The base is any one of potassium hydroxide, calcium hydroxide, and sodium hydroxide,
Wherein the acid is trifluoroacetic acid. In a method for producing a complex of acid chlorin e6 with polyvinylpyrrolidone (PVP)
Mixing polychlorinated e6 with polyvinylpyrrolidone (PVP) having formula (4);
(Formula 4)

Adjusting the pH by adding a base to the result of performing the mixing step;
Filtering the result of the step of adjusting the pH using a membrane filter; And
Lyophilizing the result of the filtration to produce a powder; (Polyvinylpyrrolidone). ≪ / RTI > 15. The method of claim 14,
The base is NaOH,
Wherein the step of adjusting the pH controls the pH to 8-9. 16. The method according to claim 14 or 15,
Wherein the mixing comprises:
Wherein said acid chlorin e6 and polyvinylpyrrolidone (PVP) are mixed at a weight ratio of 1: 1.

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