WO2018110777A1 - Method for producing composite of high purity trisodium chlorin e6 and polyvinylpyrrolidone, and method for producing chlorin e6 - Google Patents

Abstract

According to an embodiment, a method for producing a composite of trisodium chlorin e6 and polyvinylpyrrolidone (PVP) is disclosed, the method comprising: a step for mixing chlorin e6 represented by the molecular formula C34H36N4O6, polyvinylpyrrolidone (PVP), NaOH, and HCl; and a step for freeze-drying a material obtained as a result of performing the previous step for mixing, thereby making said material into a powder, wherein the step for mixing is a step for generating a composite of trisodium chlorin e6 and polyvinylpyrrolidone (PVP).

Description

Method for producing a complex of high purity trisodium chlorine eixix and polyvinylpyrrolidone and method for preparing chlorine eix

The present invention relates to a method for producing a complex of high purity trisodium chlorin e6 (trisodium chlorin e6) and polyvinylpyrrolidone (PVP) and a method for producing chlorin e6.

Conventional patents for producing chlorine e6 include complex chlorophyll extraction, acid treatment, neutralization, hydrolysis, extraction of pheophovida, dissolution with acetone, strong base addition, neutralization, and reprecipitation in spirulina or chlorella. A method for preparing chlorine six (hereinafter referred to as 'chlorine e6') through a long time extraction, separation or purification process is disclosed. In this method, chlorophyll a and pheophytin a are not completely purified, and then the reaction proceeds in the form of extracts and extracts, so the purity of the final material chlorine e6 is lowered.

In addition, chlorine e6 prepared by conventional methods is not suitable for use in foods or pharmaceuticals, and also has a limitation in commercial use due to long reaction time and complicated reaction process. In particular, conventionally known methods for producing chlorine e6 from spirulina use harmful organic solvents, which are prohibited from use in the food or pharmaceutical fields, have a long reaction time and complexity, and do not have sterilization processes, and thus are not suitable for application to food or pharmaceutical production processes. There is a limit.

According to one embodiment of the present invention, a method of preparing chlorine e6 is provided.

According to an embodiment of the present invention, there is provided a method for preparing a composite of high purity trisodium chlorin e6 (PVSO) and polyvinylpyrrolidone (PVP) having a short production time and high reproducibility.

According to one embodiment of the present invention, a method for preparing a complex of high purity trisodium chlorin e6 (polysodium chlorin e6) and polyvinylpyrrolidone (PVP) suitable for use as a food and pharmaceutical raw material by being sanitized and sanitized is provided.

According to an embodiment of the present invention the following structure

Mixing chlorine e6 (Chlorin e6), PVP (polyvinylpyrrolidone), NaOH, and HCl represented by a molecular formula C ₃₄ H ₃₆ N ₄ O ₆ ; And

And freeze-drying the resultant of the mixing step into powder.

The mixing step is the following structure

Trisodium Chlorin e6 (trisodium Chlorin e6) represented by the molecular formula C ₃₄ Н ₃₃ О ₆ N ₄ Na ₃ and the step of generating a complex of the polyvinylpyrrolidone (PVP), trisodium Chlorin e6 (trisodium Chlorin e6) And a method for preparing a composite of polyvinylpyrrolidone (PVP) may be provided.

According to another embodiment of the present invention, in the method for preparing chlorine e6 (Chlorin e6),

Extracting a chlorophyll extract by adding a chlorophyll-containing green plant or microalgae to methanol, ethanol, acetone or their respective aqueous solutions;

Adding hexane and water to the chlorophyll extract and stirring;

Purifying chlorophyll by separating the hexane layer from the stirred solution;

Dissolving the purified chlorophyll in a solvent of any one of methanol, ethanol, acetone, and a mixed solvent thereof, and then acidifying by adding an acid;

Adding water to the resultant of the acidifying step to obtain a pheophytin precipitate; And

Dissolving the pheophytin precipitate in acetone and adding a base to produce chlorine e6; which may include providing a method for producing chlorine e6 (Chlorin e6).

According to one or more embodiments of the present invention, in the process of preparing a complex of trisodium chlorin e6 and PVP from spirulina, purification of chlorophyll and pheophytin is included as an essential process. I) purity and refining process period of i) high purity trisodium chlorin e6 or ii) complex of high purity trisodium chlorin e6 and PVP Get the effect.

In addition, according to one or more embodiments of the present invention, the effect of obtaining a high purity chlorophyll from the chlorophyll extract by using the difference in the solubility of hexane and water in the chlorophyll, dynamic compound wavelength ultrasonic process, porous alumina ceramic filter.

In addition, according to one or more embodiments of the present invention, in the preparation of high-purity pheophytin, water is added directly to the solution converted to pheophytin by adding acid to chlorophyll, neutralizing the reaction solution, and water By applying an ultrasonic process to one reaction solution, the effect of improving the precipitation rate and precipitation rate of pheophytin and the effect of obtaining high purity pheophytin are simultaneously obtained.

In addition, according to one or more embodiments of the present invention, the process for preparing peotitin from chlorine e6 is carried out in the presence of a base, heating conditions, and sterile filtration process, thereby shortening the reaction time, improving the hygienic process and production yield. Get the effect.

In addition, according to one or more embodiments of the present invention, the process of converting the high purity chlorine e6 and PVP into a complex of water-soluble trisodium chlorine e6 and PVP by carrying out at a specific temperature, stirring, acid, and base presence conditions Get the effect of improving

Further, according to one or more embodiments of the present invention, a trisodium chlorine e6 solution converted to water-soluble is subjected to a process of converting to a complex of trisodium chlorine e6 and PVP in the presence of an acid, a base, and at specific pH conditions and performing specific lyophilization Conditions are used to achieve the effect of drying into complexes usable for food and medicine.

Further, according to one or more embodiments of the present invention, the converted trisodium chlorine e6 is sterilized and filtered and then lyophilized to be converted into a powder form of a complex of high purity trisodium chlorin e6 and PVP to food and pharmaceuticals. The effect of improving the production yield is obtained by producing a composite usable in

Accordingly, according to one or more embodiments of the present invention, mass production of a complex of high purity trisodium chlorin e6 and PVP is possible.

Figure 1 shows the HPLC analysis of the chlorophyll extract prepared according to an embodiment of the present invention.

Figure 2 shows the HPLC analysis of the high purity chlorophyll extract prepared according to an embodiment of the present invention.

Figure 3 shows the results of HPLC analysis for pheophytin prepared according to an embodiment of the present invention.

Figure 4 shows the results of nuclear magnetic resonance (NMR) analysis of high purity Chlorin e6 (Chlorin e6) prepared according to an embodiment of the present invention.

Figure 5 shows the results of HPLC analysis of Chlorin e6 (Chlorin e6) prepared according to an embodiment of the present invention.

FIG. 6 shows the results of Liquid Chromatography Mass Spectrometry LC / MS analysis of Chlorin e6 prepared according to one embodiment of the present invention.

FIG. 7 shows the results of HPLC comparison analysis of a high purity trisodium chlorin e6 (sample) prepared in accordance with an embodiment of the present invention and a commercial product (standard-Frontiers).

Figure 8 shows the measurement results of the ultraviolet visible absorbance measurement method of trisodium chlorin e6 (trisodium Chlorin e6) and the molecular weight of 10,000 or less PVP composite prepared according to an embodiment of the present invention.

Figure 9 shows the formula for calculating the content of trisodium Chlorin e6 (trisodium Chlorin e6) according to an embodiment of the present invention.

FIG. 10 shows the results of comparative absorbance analysis using UV / VIS Spectrophotometry in trisodium chlorin e6 and PVP composite according to an embodiment of the present invention.

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.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In this specification, the singular also includes the plural unless specifically stated otherwise in the phrase. As used herein, the words 'comprises' and / or 'comprising' do not exclude the presence or addition of one or more other components.

As used herein, the terms “cancer” and “tumor” generally mean or describe physiological conditions in a mammal characterized by uncontrolled cell growth. Benign and malignant cancers, and dormant tumors or micrometastases are included in this definition. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. Other examples of such cancers include skin cancer, cleft lip cancer, melanoma (skin metastasis), breast cancer (skin metastasis), skin basal cell cancer, deformed skin cancer, skin squamous cell carcinoma, and squamous cell carcinoma of the lower lip. It is not limited to. Examples of other cancers include breast cancer, squamous cell cancer, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous carcinoma of the lung), systemic glioblastoma, glioblastoma, typical glioblastoma and mesenchymal glioblastoma, Cancer of the peritoneum, gastric cancer, gastric cancer (including gastrointestinal cancer), pancreatic cancer, ovarian cancer, cervical cancer, liver cancer, bladder cancer, colon cancer, colorectal cancer, endometrial cancer, uterine carcinoma, salivary gland carcinoma, kidney cancer, prostate cancer, vulvar cancer , Thyroid cancer, various types of head and neck cancer, B cell lymphoma (non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; medium / follicular NHL; medium grade) Diffuse NHL; High Grade Immunoblastic NHL; High Grade Lymphoblastic NHL; High Grade Small Non-dividing Cell NHL; Bulky Disease NHL; Mantle Cell Lymphoma; AIDS-Related Lymphoma; and Valdenstrom Giant Globulinemia), Chronic Lymphocytic Leukemia (chronic lymphocytic leukemia: CLL), Acute lymphoblastic leukemia (ALL), hair cell leukemia, chronic myeloblastic leukemia, post-transplant lymphoproliferative disorder (PTLD), abnormal blood vessel proliferation associated with nevus, edema (Eg, those associated with brain tumors), and Meigs' syndrome.

Also, as used herein, "cancer" and "tumor" refer to all neoplastic growth and proliferation (either malignant or benign), and all precancerous and cancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are referred to herein and are not mutually exclusive.

In the present specification, the term 'chlorine e six' may be represented by 'Chlorin e6' or 'chlorine e6'.

In the present specification, the term 'composite' is used to mean a complex of trisodium Chlorin e6 and polyvinylpyrrolidone (hereinafter referred to as "PVP") (polyvinylpyrrolidone) having a molecular weight of 15,000 or less.

The complex of trisodium Chlorin e6 and trivinylpyrrolidone (PVP) prepared according to an embodiment of the present invention may be used as a pharmaceutical composition for treating cancer photodynamically.

According to one embodiment of the present invention, trisodium Chlorin e6 and PVP (polyvinylpyrrolidone) composite production method, Chlorin e6, polyvinylpyrrolidone (PVP), NaOH, HCl and the result of the step of mixing the frozen result Drying to powder.

In this embodiment, Chlorin e6 has the structure

It is represented by the molecular formula C ₃₄ H ₃₆ N ₄ O ₆ .

In this embodiment, the mixing step has the following structure

It is a step of generating a complex of trisodium Chlorin e6 and the polyvinylpyrrolidone (PVP) having a molecular formula C ₃₄ Н ₃₃ О ₆ N ₄ Na ₃ .

First embodiment

According to the first embodiment, the method for producing a complex of trisodium Chlorin e6 and polyvinylpyrrolidone (PVP), sterilizing and filtering Chlorin e6 (S50), mixing Chlorin e6, polyvinylpyrrolidone (PVP), NaOH, and HCl (S100), and lyophilizing the resultant of the performing of the step of mixing to make a powder (S110).

That is, according to the first embodiment, before performing the mixing step (S100), the step of sterilizing and filtering Chlorin e6 (S50) is performed.

According to a first embodiment, the step of sterilization and filtration (S50), the first pH adjustment step of dissolving Chlorin e6 in water, adjusting the pH to less than 7 pH of the lysate; a second pH adjusting step of adjusting the pH of the lysate having a pH less than 7 to 7 or more; And sterilizing and filtering the lysate having a pH of 7 or more using a micro filter. Here, the micro filter may be, for example, having a diameter of 0.22 μm, but this value is exemplary.

According to the first embodiment, for example, the first pH adjusting step is adjusting the pH of the lysate to 2, and the second pH adjusting step is adjusting the pH of the lysate to 7.1.

According to the first embodiment, the mixing step (S100) includes adjusting the pH of the mixture as a result of performing the mixing step (S100) to 7 to 9.

According to the first embodiment, the mixing step (S100), the step of adjusting the pH of the mixture resulting from the mixing step (S100) to 7 to 9 and sterilizing and filtering the mixture using a micro filter It can be implemented to include.

According to the first embodiment, the mixing step S100 may be performed such that the weight percentage of NaCl in the mixture is 5% or less.

Second embodiment

According to a second embodiment, the step of mixing Chlorin e6, polyvinylpyrrolidone (PVP), NaOH, and HCl (S100), and lyophilizing the resultant of the mixing step to make a powder (S110), Here, the mixing step S110 may include adjusting the pH of the mixture resulting from the mixing step S110 to 7 to 9 and sterilizing and filtering the mixture using a micro filter. The micro filter may be, for example, having a diameter of 0.22 μm but this value is exemplary.

According to the second embodiment, the mixing step S100 may be performed such that the weight percentage of NaCl in the mixture is 5% or less.

As used herein, the term 'composite' refers to a complex of trisodium Chlorin e6 and polyvinylpyrrolidone (PVP).

In the composite according to an embodiment of the present invention, trisodium chlorin e6 and polyvinylpyrrolidone (PVP) may be composed of 1: 9, 5: 5, 4: 6, or 6: 4, 9: 1. These figures are exemplary, and the scope of the present invention is not limited only to those figures.

 According to embodiments of the present invention, the mixing step S100 is performed such that the pH of the mixture, which is a result of performing the mixing step, becomes 7-9.

On the other hand, the mixing step (S100)

or

It can be expressed as

According to one embodiment of the invention, the mixing step (S100),

Chlorin e6 and a molecular weight of 15,000 or less polyvinylpyrrolidone (PVP) may be mixed first, and then a mixture of chlorin e6 and polyvinylpyrrolidone (PVP) may be implemented by mixing NaOH and HCl. Here, the number of moles of NaOH and HCl is set so that the pH of the result of the mixing step is 7 to 9.

 According to another embodiment of the present invention, the mixing step (S100) is

Chlorin e6 and NaOH and HCl may be mixed first, and then a mixture of Chlorin e6 and NaOH and HCl and polyvinylpyrrolidone (PVP) may be implemented. Here, the number of moles of NaOH and HCl is set so that the pH of the result of the mixing step is 7 to 9.

According to another embodiment of the present invention, the mixing step (S100)

Chlorin e6 and NaOH and HCl and PVP (polyvinylpyrrolidone) may be mixed at the same time, the pH of the mixture can be implemented by a method of 7 to 9.

Hereinafter, a method of preparing chlorin e6 according to an embodiment of the present invention will be described. Chlorin e6 prepared according to the present method can be used to prepare the complexes described above.

Method for preparing chlorin e6 according to an embodiment of the present invention, the green plant or microalgae containing chlorophyll in a methanol, ethanol, acetone or each of these aqueous solution to extract the chlorophyll extract, chlorophyll extract hexane and Adding water and stirring, separating the hexane layer from the stirred solution to purify the chlorophyll, dissolving the purified chlorophyll in a solvent of any one of methanol, ethanol, acetone and a mixed solvent thereof, and then adding an acid. Acidifying, adding water to the result of the acidifying step to obtain a pheophytin precipitate, and dissolving the pheophytin precipitate in acetone and adding a base to produce chlorine e6. .

According to the present embodiment, the extracting may include adding green plants or microalgae to methanol, ethanol, acetone or their respective aqueous solutions, and applying ultrasonic waves to the aqueous solutions. Here, the ultrasound may have a frequency of 10 to 50 kHz.

The present embodiment may further include a step of separating and concentrating the result of performing the extracting step using a filter before performing the stirring step.

According to this embodiment, the stirring may include adding hexane and water to the chlorophyll extract to generate a first mixture, and stirring the first mixture while applying ultrasonic waves to the mixture. Here, the ultrasound may have a frequency of 10 to 50 kHz.

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

Chlorophyll Extraction and Purification Methods

Chlorophyll a and b extracted and purified according to the method for extracting and purifying chlorophyll a and b according to an embodiment of the present invention may be used to prepare the above-mentioned complex.

According to this method, a green plant or microalgae containing spirulina is obtained by extracting chlorophyll using a dynamic compound wavelength ultrasonic extraction technique.

According to the method, no particular limitation is placed on the selection of the green plant or microalgae used to obtain chlorophyll, and any plant or microalgae containing chlorophyll may be applied.

According to the present method, green plants or microalgae are stirred with a solvent having a relatively high polarity, such as water, methanol, ethanol, acetone, and an aqueous solution thereof, and an ultrasonic extraction method is applied to the stirred solution. In addition, a porous alumina ceramic filter (5-20 μm) is applied to the resultant obtained by applying the ultrasonic extraction method, and separated and concentrated to obtain a chlorophyll extract. Then, hexane with high solubility in chlorophyll and relatively low polarity is added to the chlorophyll extract. The chlorophyll extract to which hexane is added is subjected to ultrasonic extraction and stirring to move the chlorophyll to the hexane layer as much as possible. Thereafter, when water is added to the resultant to which the ultrasonic extraction and stirring process is applied and stirred, impurities other than chlorophyll, which may be included in the hexane layer, are transferred to the water layer, thereby obtaining high purity chlorophyll from the hexane layer.

 According to the method, hexane has a nonpolar characteristic such as chlorophyll, so that it has high solubility in chlorophyll, and hexane has a property of being separated without mixing with methanol, ethanol, acetone or their respective aqueous solutions. Therefore, by purifying chlorophyll extract using ultrasonic wave, hexane and water, chlorophyll is more easily dissolved and distributed in the hexane layer, and impurities other than chlorophyll are easily separated and distributed into water, methanol, ethanol or acetone layers. It is possible to obtain high purity chlorophyll.

According to the method, the ultrasonic wave used to extract chlorophyll in spirulina can be adjusted to 10-50 kHz, 100-500 W, and the time taken to reach 100% extraction rate is up to 2 hours, which is more effective than the existing 6 to 12 hours. It is economical.

According to this method, when methanol, ethanol, acetone or their respective aqueous solutions are used as the chlorophyll extractant, the water content in the extractant can be adjusted to less than 30% by volume. The reason is that the use of ultrasonic extraction equipment enables the extraction of chlorophyll in a short time, thereby preventing the decomposition and denaturation of chlorophyll.

According to the method, the purified solvent hexane used in conjunction with the ultrasonic wave can be reduced to within 3 times the volume of the concentrated chlorophyll extract. The use of ultrasound allows the chlorophyll to move easily into the hexane layer with a relatively small amount of hexane, thereby economically increasing the yield of chlorophyll. In the extraction process with hexane accompanied by ultrasonic waves, the stirring time is less than 1 hour, and when ultrasonic waves are not used, the stirring time is shorter than 3 hours.

In addition, according to the method, after the addition of hexane to the chlorophyll extract, if the chlorophyll is further extracted by using ultrasonic waves and water can be further maximized the purification effect of chlorophyll. That is, it is possible to easily transfer impurities to the aqueous layer by sonication and water to obtain high purity chlorophyll from the hexane layer. In this case, a filtration process using a porous alumina ceramic filter (5-20 μm) may be performed. The frequency of the ultrasonic wave is 10 to 50 kHz, the power is adjusted to 100 to 500 W, and a short time treatment such as within 5 to 10 minutes is preferable. The use of ultrasonic waves in this process is economical since emulsion formation can be prevented.

In the method for extracting and purifying chlorophyll according to the present invention described above, the conventional three-day process period can be shortened within 12 hours, and the purified chlorophyll a and b have a purity of 90% or more.

Pheophytin Synthesis Method

This method is a method for obtaining pheophytin according to an embodiment of the present invention.

According to the present method, chlorophyll-a represented by the following formula (1) is treated with an acid to obtain a phephytin represented by the following formula (2) from which magnesium is removed.

<Formula 1>

<Formula 2>

According to the method, after the chlorophyll is dissolved in the solvent, the acid is added and sonicated (10-50 kHz, adjustable to 100-500 W) and stirred at room temperature. As a result, the magnesium atom contained in the chlorophyll molecule is released and converted into phefitin within 30 minutes to 1 hour.

 According to this method, the acid added for magnesium desorption is within 3 equivalents per molecule of chlorophyll, and the pH of the reaction solution is adjusted in the range of 1 to 3. At this time, the amount of acid used is economical because it is used in a small amount together with the sonication, and there is a characteristic that can prevent the formation of the pheopovid by-products due to overreaction. There is no particular restriction on the kind of acid added for the desorption of magnesium, and it may be selected from conventional inorganic acids such as hydrochloric acid, sulfuric acid, or phosphoric acid, or organic acids such as acetic acid and the like.

According to this method, the purity of the intermediate chlorine e6 can be increased by economically separating and purifying the pheophytin produced by adding acid in addition to sonication economically.

According to the method, the pheophytin produced by sonication and acid treatment as described above is filtered by a porous alumina ceramic filter (5-20 μm). The filtered pheophytin precipitate is then cooled while maintaining at a low temperature of -20 ° C to -40 ° C and a low pressure of 20mbar and water is removed using a cold trap (-70 ° C to -80 ° C). At low temperature and low pressure in the above-described range, a vacuum state is formed in which moisture can be sublimed and removed. As such, sublimation drying in the frozen state does not cure or shrink the surface of the pheophytin particles. Many new drugs can be damaged by overdrying, so the final amount of water is maintained at the best level when the final moisture is dried between 1.5% and 2.0%.

According to the method for the purification of pheophytin, the ultrasonic wave (adjustable to 10 to 50 kHz, 100 to 500 W) is applied to the chlorophyll-dissolved solution and washing with ethanol and water is performed, The purification effect of tin can be further maximized. That is, by the sonication and the addition of ethanol and water, impurities can be easily transferred to the aqueous solution layer to obtain a high purity pheophytin from the hexane layer. In this case, the ultrasonic waves to be treated can be adjusted to 10 to 50 kHz, 100 to 500 W, and a short time is processed within 5 to 10 minutes.

The pheophytin precipitate obtained through the above acid treatment method and obtained through the porous alumina ceramic filter has a purity of 95% or more and is obtained in a yield of 95% or more based on the weight of chlorophyll used.

Chlorine e6 manufacturers

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

According to the method, chlorine e6 represented by the following Chemical Formula 3 is obtained by adding a base to pheophytin and heating the reaction.

<Formula 3>

According to this method, the concentrated phephytin dry matter is dissolved in acetone. The remaining moisture of the dissolved acetone solution is removed using Na ₂ SO ₄ , and added to the reflux reactor together with ethanol. The pH is adjusted to 13 or higher using sodium hydroxide at 60 ° C. to 70 ° C. reflux. In this case, the base is commonly used, and may be an inorganic base such as a hydroxide of an alkali metal or an alkaline earth metal, or an organic base such as an ammonium salt. pH titration can be rapidly titrated in real time using a potentiometric automatic titrator.

After the reaction is completed, chlorine e6 is obtained from the reactants using a porous alumina ceramic filter (5-20 μm), and the obtained chlorine e6 is dried by vacuum and drying process. Chlorin e6 obtained by the above method is at least 90% and is obtained in a yield of at least 90% based on the weight of the pheophytin used.

Chlorine e6 and trisodium chlorine e6 tablets

This method is a chlorine e6 purification method according to an embodiment of the present invention.

According to this method, it is a process of obtaining the high purity chlorine e6 by performing acid treatment and alkali treatment on the dried chlorine e6.

According to the method, after dissolving the dry chlorine e6 in water, the pH is adjusted to 7-9 using a potentiometric autotitrator, and microparticles which are not dissolved in water are removed by sterile filtration using a 0.22 μm microfilter. And, while acid-treating the sterile filtered lysate, the pH of the lysate was adjusted to 2 using a potentiometric autotitrator and chlorine e6 was obtained by sterile filtration using a 0.22 μm microfilter. To purify chlorine e6, it is added to 1% by weight in ethanol and stirred for at least 1 hour, followed by vacuum drying to obtain high purity chlorine e6. Chlorine e6 obtained by the above method is 95% or more.

The dried high purity chlorine e6 is then dissolved in acetone or water, the pH is slowly adjusted to 8.5, sterilized using a 0.22 μm microfilter and dried by lyophilization process. By the above method, high purity trisodium chlorine e6 is obtained.

Complex preparation of trisodium chlorine e6 with PVP

The method is a method for producing trisodium chlorine e6 and PVP complex according to an embodiment of the present invention. According to one embodiment of the method, high purity chlorine e6 or high purity trisodium chlorine e6 is stirred and mixed with PVP in an aqueous solution at room temperature in one-to-one or one-to-one or teenage days. At this time, the pH of the mixture is adjusted to 7 to 9 using a potentiometric automatic titrator to maintain a minimum amount of NaCl to prepare trisodium chlorine e6 and PVP complex. In this process, stirring is carried out within 1 hour. The complex is sterile filtered with a 0.22 μm microfilter. The sterile filtered complex is then cooled for freeze-drying while maintaining a low temperature of -20 ° C to -40 ° C and a low pressure of 20 mbar and water is removed using a cold trap (-70 ° C to -80 ° C). At the low temperature and low pressure described above, the water becomes a vacuum state in which water can be sublimated and removed. Sublimation drying in the frozen state does not cure or shrink the surface of the composite.

The final amount of moisture is kept dry between 1.5% and 2% as damage may be caused by excessive drying.

As described above, according to the embodiments of the present invention, trisodium chlorine e6 is separated, extracted, and purified from a chlorophyll-containing starting material economically using a short-term process without any limitation of specific raw materials.

In addition, according to embodiments of the present invention, it is possible to obtain a high-purity chlorophyll by a simple and efficient method of removing a substance other than chlorophyll using a solubility difference and polarity of the solvent from the raw material, and an ultrasonic process. In addition, through this, in the synthesis of pheophytin and chlorine e6, an economical process for shortening the reaction time and the amount of the organic solvent was used, and separation and purification were improved. In addition, according to the embodiments of the present invention by using a sterile filtration and automatic titrator superior to the conventional method, highly purified trisodium chlorine e6 and PVP complexes were prepared. In particular, the new hygienic process through sterile filtration greatly improves the physiological activity, and by using ultrasonic waves to significantly improve the yield for a short time has the characteristics of synthesizing trisodium chlorine e6 complex in a process suitable for mass drug production.

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

Experiment 1. Obtaining Chlorophyll Extract

Extraction solvent (750 liters of ethanol) was added to 75 kg of spirulina, sonicated (30 kHz, 300 W), stirred for 1 hour, and filtered using a porous alumina ceramic filter (5-20 μm) to obtain a chlorophyll extract. The chlorophyll extract obtained above was analyzed by HPLC (High Pressure Liquid Chromatography) under the following conditions. The solvent used was methanol, acetonitrile, dichloromethane, water and the like, and HPLC analysis was performed at 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 5 to 8 minutes, and it was confirmed that a considerable amount of impurities existed between 0 and 3 minutes. At this time, chlorophyll from Sigma-aldrich company was used as a reference.

Experiment 2. Obtaining High Purity Chlorophyll

The chlorophyll extract obtained in Experiment 2 was concentrated to a maximum volume of 10%, and the same volume of hexane was added to the concentrated chlorophyll extract, followed by stirring with sonication (20 kHz, 300 W) for 30 minutes. An additional volume of water equal to hexane was added and stirring continued for 10 minutes with sonication. After stirring the reaction solution at -10 ℃ for 5 hours, the reaction solution was separated by layers to obtain an organic layer. Thereafter, the organic layer was distilled under reduced pressure to obtain high purity chlorophyll. HPLC analysis of the obtained chlorophyll extract is shown in FIG. According to the HPLC analysis results shown in FIG . 2 , 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 90%. The yield of this means that 90% or more of the extract was extracted relative to the amount of chlorophyll contained in the starting material.

Experiment 3. Obtaining High Purity Pheophytin from Chlorophyll

Hydrochloric acid or sulfuric acid was administered to the chlorophyll hexane solution obtained in Experiment 2 so that the pH of the solution was 2, and stirred for 1 hour while sonicating (20 kHz, 300 W). After the reaction was completed, 70% ethanol in the same volume as the chlorophyll hexane solution was added to the solution, stirred for 10 minutes, and then, precipitated for 3 hours at -10 ° C. The precipitate was lyophilized to give pheoptine (HPLC purity 90%, yield 95%) (alternatively, drying by filtration with a porous alumina ceramic filter (5-20 μm) is possible).

According to the HPLC analysis results shown in FIG. 3, the corresponding peak of pheophytin was confirmed, and impurities were hardly identified at other time points. The pheophytin precipitate obtained by the above acid treatment method has a purity of 95% or more and is obtained in a yield of 95% or more based on the weight of chlorophyll used.

Experiment 4. Synthesis of Chlorin e6 from Pheophytin

After dissolving 1 g of pheophytin obtained in Experiment 3 in 50 mL of acetone, when 2.297 mL of 1 M aqueous sodium hydroxide solution was added, the pH of the solution was about 12. This solution was stirred for 1 h while heating to 60 ° C. Upon completion of the reaction, sterilization and filtration using a 0.22 μm microfilter yielded 0.653 g of chlorine e6 (90% HPLC purity, 90% yield).

In addition, in order to purify the chlorine e6 obtained in high purity, 1 g of chlorine e6 was added to 100 mL of ethanol, stirred for 1 hour, sterilized and filtered to obtain 0.95 g of high purity chlorine e6 (HPLC purity 95%, yield 95% or more). Obtained. Nuclear magnetic resonance (NMR) analysis of the purified high-purity chlorine e6 was confirmed (Fig. 4).

In addition, the obtained chlorine e6 was analyzed by HPLC at 407 nm using acetonitrile and 0.1% trifluoroacetic acid solvent (Fig. 5). As a result of HPLC analysis, peaks corresponding to chlorine e6 were observed at 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. In addition, the results of liquid chromatography mass spectrometry (Liquid Chromatography Mass Spectrometry LC / MS) confirmed that the corresponding peak of chlorine e6, it was confirmed that the impurities are almost removed (Fig. 6).

Experiment 5. Preparation and Purification of Trisodium Chlorine e6

After dissolving 1 g of chlorine e6 obtained in Experiment 4, the pH was adjusted to 7.1 using a potentiometric autotitrator, and microparticles not dissolved in water were removed by sterilization and filtration using a 0.22 μm microfilter. Acid treatment was again performed on the sterilized and filtered solution to adjust the pH up to 2 using a potentiometer.

From the chlorine e6 solution, either high purity chlorine e6 is obtained or high purity trisodium chlorine e6 is obtained.

First, the process of obtaining high purity chlorine e6 is demonstrated.

The chlorine e6 solution is sterilized and filtered using a 0.22 μm microfilter to obtain chlorine e6. The obtained chlorine e6 was dissolved in 95% or more of acetone, then the pH was adjusted to 7.1, sterilized, filtered and vacuum dried to obtain high purity chlorine e6.

Now, a process for obtaining high purity trisodium chlorine e6 will be described.

The chlorine e6 solution is adjusted to pH 7-9 to form trisodium chlorine e6, then sterilized, filtered and lyophilized using a 0.22 μm micro filter to obtain high purity trisodium chlorine e6. HPLC analysis of the obtained high-purity trisodium chlorine e6 confirmed the corresponding peak of chlorine e6 at retention time of 4 minutes, and has a purity of at least 95% compared to commercially available chlorine e6 reagent (Frontiers). It was confirmed that the impurities were almost removed (Fig. 7).

Experiment 6. Preparation of Trisodium Chlorine e6 and PVP Complex

After dissolving 1 g of trisodium chlorine e6 obtained in Experiment 5 in water, 1 g of molecular weight of 10,000 or less PVP is added and the pH is adjusted to 7 to 9 using a potentiometric autotitrator while stirring for 1 hour. After the complex is formed, the solution is sterilized and filtered using a 0.22 μm micro filter. The sterile and filtered solution is lyophilized in aseptic state to obtain a complex. Sterilization and filtration prevent the growth of microorganisms and exclude the potential for increased entotoxin.

The complex is sterile filtered with a 0.22 μm microfilter. The sterile filtered complex is then cooled for freeze-drying while maintaining a low temperature of -20 ° C to -40 ° C and a low pressure of 20 mbar and water is removed using a cold trap (-70 ° C to -80 ° C). At the low temperature and low pressure described above, the water becomes a vacuum state in which water can be sublimated and removed. Sublimation drying in the frozen state does not cure or shrink the surface of the composite. On the other hand, since it may be damaged by excessive drying, the amount of final moisture is maintained to be dried between 1.5%-2%.

Experiment 7. Trisodium Chlorine e6 and PVP Complex Identification Test

The composite according to an embodiment of the present invention is well dissolved in water, and the solution prepared for the content test is black green. 200 mg of the complex according to one embodiment of the present invention is diluted with 25 mL of water and left for 10 minutes. Take 0.2 mL of this solution into a 100 mL volumetric flask and add a diluent (95% ethanol) to dissolve completely. Absorption is shown at wavelengths of 402.4, 503.2, 531.7, 608.0, and 662.7 when the absorption spectrum is measured at 400-700 nm according to the ultraviolet visible absorbance measurement method (see FIG. 8).

Experiment 8. Trisodium Chlorine e6 and PVP Complex Content Test

A solution of 200 mg of the complex according to one embodiment of the present invention diluted with 25 mL of water is left for 10 minutes. After leaving for 10 minutes, take 0.2 mL of the solution, place it in a 100 mL flask, add diluent (95% ethanol) to the mark, and dissolve to prepare a sample solution (standard solution: 95% ethanol). The sample solution and the standard solution were put into a 1 cm spectrophotometer cell and calculated by measuring the transmittance at 662 nm according to the UV-visible absorbance measurement method (refer to the formula of FIG. 9). Preparation of all test substances is carried out under shading. Accuracy is calculated as recovery at each concentration and average recovery should be within 100% of each concentration. Precision should be calculated as a coefficient of variation for the recovery of each concentration and should be less than 5%.

The formula for calculating the recovery rate is as follows.

Recovery (or content) = inverted concentration / theoretical concentration × 100

Experiment 8. Quantitative NaCl Test in Trisodium Chlorine e6 and PVP Complex

After measuring about 30 mg of the composite sample according to an embodiment of the present invention, it is transferred to a beaker, 50 ml of water is dissolved. After adding indicator 10% potassium chromate to 50 ml of water, reddish brown appears in the solution with 0.1 N silver nitrate. Through the above method, NaCl content of the complex can be calculated based on the quantitative titration data of NaCl, and it can be confirmed that NaCl is contained in the complex.

Experiment 9.Chlorin e6 assay in trisodium chlorine e6 and PVP complex

The chlorine e6 in physiological saline was analyzed using UV / VIS Spectrophotometry. Through this analysis, the accuracy (complex 1), precision (complex 2) and stability (composite 3) were investigated.

To prepare an injection preparation of 100 mg / mL (as Chlorin e6), add excipients to the complex and dissolve by ultrasonic shaking for about 30 minutes. Low concentration preparations are prepared by dilution with excipients from high concentration preparations (100 mg / mL) to the desired concentration of 1 mg / mL. Preparation of all test substances is carried out under shading. Chlorin e6 at 2.5, 5 and 10 mg / mL in excipients are assessed by intra-day and inter-day analysis. In-day analysis is performed at three concentrations by preparing three samples per concentration. The daily analysis is evaluated with a result of at least 3 days in the same manner as the daily analysis, and one sample is performed by another experimenter to confirm the reproducibility between the experimenters. Accuracy is calculated as recovery at each concentration and average recovery should be within 100% of each concentration. Precision is calculated as the coefficient of variation for the recovery of each concentration and should be less than 5%. The stability of the complex is taken three times in the middle layer after storage of 2.5, 10 mg / mL of the complex sample in excipients at each storage condition. Each sample is shaded and stored for about 7 days in refrigeration. The conformance criteria should be within 100% of the theoretical concentration. Referring to FIG. 10, reference may be made to an analysis result of chlorine e6 for accuracy, precision, and stability.

As described above, the present invention has been described by way of limited embodiments and drawings, but the present invention is not limited to the above embodiments, and those skilled in the art to which the present invention pertains various modifications and variations from such descriptions. This is possible.

Therefore, the present invention should not be limited to the described embodiments, but should be defined not only by the claims below but also by equivalents thereof.

Claims (16)

Structure

Mixing chlorine e6 (Chlorin e6), molecular weight 15,000 or less PVP (polyvinylpyrrolidone) represented by molecular formula C ₃₄ H ₃₆ N ₄ O ₆ , NaOH, and HCl; And And freeze-drying the resultant of the mixing step into powder. The mixing step is the following structure

Trisodium chlorin e6 represented by the molecular formula C ₃₄ Н ₃₃ О ₆ N ₄ Na ₃ and the molecular weight of 15,000 or less polyvinylpyrrolidone (PVP), which is a step of producing a trisodium chlorine e6 (trisodium chlorin e6) and PVP. Method for preparing a complex of (polyvinylpyrrolidone). The method of claim 1, Before performing the step of mixing, sterilizing and filtering the Chlorin e6; further comprising, trisodium Chlorin e6 (trisodium Chlorin e6) and PVP (polyvinylpyrrolidone) complex manufacturing method. The method of claim 2, The sterilization and filtration step, A first pH adjusting step of dissolving the Chlorin e6 in water and adjusting the pH of the lysate to less than 7 before performing the mixing step; a second pH adjusting step of adjusting the pH of the lysate having a pH less than 7 to 7 or more; And Sterilizing and filtering the lysate having a pH of 7 or more using a micro filter; comprising, trisodium Chlorin e6 (trisodium Chlorin e6) and PVP (polyvinylpyrrolidone) complex manufacturing method. The method of claim 3, The first pH adjusting step is to adjust the pH of the lysate to 2, the second pH adjusting step is to adjust the pH of the lysate to 7.1, trisodium Chlorin e6 ) And PVP (polyvinylpyrrolidone). The method of claim 4, wherein The mixing step Adjusting the pH of the mixture as a result of performing the mixing to 7 to 9; And Sterilizing and filtering the mixture using a micro filter; comprising, trisodium chlorin e6 (trisodium Chlorin e6) and PVP (polyvinylpyrrolidone) complex manufacturing method. The method of claim 1, The mixing step Adjusting the pH of the mixture as a result of performing the mixing to 7-9; And Sterilizing and filtering the mixture using a micro filter; comprising, trisodium chlorin e6 (trisodium Chlorin e6) and PVP (polyvinylpyrrolidone) complex manufacturing method. The method of claim 1, The mixing step Method for producing a complex of trisodium chlorin e6 (trisodium Chlorin e6) and polyvinylpyrrolidone (PVP) in the mixture of the result of the mixing step that the weight percent of NaCl is 5% or less. The method of claim 1, The mixing step, Mixing the chlorin e6 and polyvinylpyrrolidone (PVP); A method of manufacturing a complex of trisodium chlorin e6 and polyvinylpyrrolidone (PVP), which is a step of sequentially performing a step of mixing the mixture of chlorin e6 and polyvinylpyrrolidone (PVP) and NaOH and HCl. The method of claim 1, The mixing step, Mixing the chlorin e6 with NaOH and HCl; The method of sequentially performing the step of mixing the mixture of Chlorin e6 and NaOH and HCl and PVP (polyvinylpyrrolidone), trisodium chlorine e6 (trisodium Chlorin e6) and PVP (polyvinylpyrrolidone) composite manufacturing method. The method of claim 1, The mixing step The chlorin e6 and NaOH and HCl and PVP (polyvinylpyrrolidone) is a step of mixing at the same time, trisodium chlorine e6 (trisodium Chlorin e6) and PVP (polyvinylpyrrolidone) composite manufacturing method. In the method for producing chlorine e6 (Chlorin e6), Extracting a chlorophyll extract by adding a chlorophyll-containing green plant or microalgae to methanol, ethanol, acetone or their respective aqueous solutions; Adding hexane and water to the chlorophyll extract and stirring; Purifying chlorophyll by separating the hexane layer from the stirred solution; Dissolving the purified chlorophyll in a solvent of any one of methanol, ethanol, acetone, and a mixed solvent thereof, and then acidifying by adding an acid; Adding water to the resultant of the acidifying step to obtain a pheophytin precipitate; And Dissolving the pheophytin precipitate in acetone and adding a base to produce chlorine e6 (Chlorin e6). The method of claim 11, The extracting step, Adding green plants or microalgae to methanol, ethanol, acetone or their respective aqueous solutions; And Applying ultrasonic waves to the aqueous solution; comprising, chlorine e6 (Chlorin e6) method for producing a. The method of claim 12, The ultrasound is a frequency of 10 to 50 kHz, how to produce Chlorin e6 (Chlorin e6). The method of claim 11, Before performing the stirring step, further comprising the step of separating and concentrating the result of performing the extraction step using a filter, Chlorin e6 (Chlorin e6) method for producing a. The method of claim 11, The stirring step Adding hexane and water to the chlorophyll extract to produce a first mixture; And Stirring the first mixture while applying ultrasonic waves to the first mixture; That includes, a method for producing chlorine e6 (Chlorin e6). The method of claim 15, The ultrasound is a frequency of 10 to 50 kHz, how to produce Chlorin e6 (Chlorin e6).

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