High-purity hematoporphyrin and preparation method thereof

Abstract

The invention relates to a high-purity hematoporphyrin preparation and a preparation method thereof.The preparation method comprises the steps that S1, hemin and a saturated hydrobromic acid glacial acetic acid solution are mixed and stirred for 15-24 h, and a reaction solution is obtained; s2, adding a sodium hydroxide solution into the reaction solution, and filtering and collecting a first precipitate; s3, dissolving the first precipitate in a hydrochloric acid methanol solution, adding an ammonium carbonate solution after water bath reflux, and filtering and collecting a second precipitate; s4, dissolving the second precipitate in chloroform, enabling the second precipitate to pass through a chromatographic column filled with neutral aluminum oxide, and performing elution by adopting a mixed solution of chloroform and petroleum ether to obtain an eluent; s5, crystallizing; s6, adding the crystals into a sodium hydroxide solution, heating in a water bath, and filtering and collecting a third precipitate; s7, dissolving the third precipitate in water, adding a polylactic acid-glycolic acid copolymer, and filtering and collecting a fourth precipitate; and S8, putting the fourth precipitate and water-soluble nano titanium dioxide into water, stirring, standing, and filtering to obtain filtrate. The impurity content in the hematoporphyrin preparation can be reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of hematoporphyrin production, in particular to high-purity hematoporphyrin and a preparation method thereof. Background Art

[0002] Hematoporphyrin is a common photosensitizer used for photodynamic therapy of tumor diseases. The existing hematoporphyrin and its preparation method can refer to the invention patent CN200510134599.9 - a hematoporphyrin derivative with stable composition and its preparation method and injection, the preparation process of which is as follows: (1) using hemoglobin as the initial raw material, stirring in a closed environment in hydrogen bromide saturated glacial acetic acid for 18-24 hours; (2) adding water and adjusting the pH with NaOH solution, filtering and collecting the precipitate, and washing with water; (3) dissolving the precipitate obtained in step (2) in a dilute hydrochloric acid solution, standing, precipitating the precipitate, filtering and collecting the precipitate; (4) dissolving the precipitate obtained in step (3) in water, filtering, adjusting the pH of the filtrate with sodium acetate solution to precipitate the precipitate, filtering and collecting the precipitate and washing with water until neutral, and drying; (5) dissolving the dried precipitate in a mixture of concentrated sulfuric acid and glacial acetic acid, stirring, filtering, adding the filtrate to a sodium acetate solution, adjusting the pH with dilute hydrochloric acid to precipitate the precipitate, filtering and collecting the precipitate and washing with water until neutral, and drying. The hematoporphyrin prepared by this patented technology contains a large number of impurities, including: protoporphyrin (3-(1-hydroxyethyl)-8-vinylprophyrin and 3-vinyl-8-(1-hydroxyethyl)prophyrin), hematoporphyrin dimers (dimer-1 and dimer-2), protoporphyrin and other impurities. Once these impurities enter the human body, they are difficult to decompose quickly and the side effects last for a long time. Summary of the invention

[0003] The technical problem to be solved by the present invention is to provide a high-purity hematoporphyrin and a preparation method thereof, so as to reduce the impurity content and reduce the negative effects on the human body.

[0004] To solve the above problems, the technical solution adopted by the present invention is: a method for preparing high-purity hematoporphyrin, comprising the following steps:

[0005] S1, mixing hemin with saturated hydrobromic acid and glacial acetic acid solution, stirring for 15-24h in an inert atmosphere to obtain a reaction solution;

[0006] S2, adding sodium hydroxide solution to the reaction solution, adjusting the pH to 4-6, precipitating a first precipitate, and filtering the first precipitate;

[0007] S3, dissolving the first precipitate in a hydrochloric acid methanol solution, reflux in a water bath for 12-18 minutes, adding an ammonium carbonate solution after cooling, precipitating a second precipitate, and collecting the second precipitate by filtration;

[0008] S4. Dissolve the second precipitate in chloroform and pass it through a chromatography column filled with neutral alumina, and elute it with a mixed solution of chloroform and petroleum ether to obtain an eluate;

[0009] S5. Evaporate the solvent of the eluate to obtain a third precipitate;

[0010] S6. Dissolve the third precipitate in water, add poly (lactic - glycolic acid) copolymer powder to the water, and slowly stir in an inert atmosphere for 0.5 - 1 h, and then filter to collect a fourth precipitate;

[0011] S7. Put the fourth precipitate and water - soluble nano - titanium dioxide into water, stir ultrasonically for 0.5 - 1 h and then stand for 0.5 - 1 h, and the filtrate obtained by filtration is high - purity hematoporphyrin.

[0012] Further, in step S1, the ratio of the weight of hemin to the volume of saturated hydrobromic acid glacial acetic acid solution is 1:(1.5 - 2.5);

[0013] In step S2, the mass fraction of the sodium hydroxide solution is 30% - 50%;

[0014] In step S3, mix a 1% hydrochloric acid solution and a 30% - 40% methanol solution in a volume ratio of 1:1 to obtain a hydrochloric acid - methanol solution; the mass fraction of the ammonium carbonate solution is 1%;

[0015] In step S4, the chloroform - petroleum ether mixed solution is obtained by mixing chloroform and petroleum ether in a volume ratio of 5:1;

[0016] In step S6, the volume ratio of the added fourth precipitate to water - soluble nano - titanium dioxide is 1:(1 - 2).

[0017] Further, in step S1, the ratio of the weight of hemin to the volume of saturated hydrobromic acid glacial acetic acid solution is 1:2;

[0018] In step S2, the mass fraction of the sodium hydroxide solution is 40%;

[0019] In step S3, mix a 1% hydrochloric acid solution and a 30% methanol solution in a volume ratio of 1:1 to obtain a hydrochloric acid - methanol solution; the mass fraction of the ammonium carbonate solution is 1%;

[0020] In step S4, the chloroform - petroleum ether mixed solution is obtained by mixing chloroform and petroleum ether in a volume ratio of 5:1;

[0021] In step S6, the volume ratio of the added fourth precipitate to water - soluble nano - titanium dioxide is 1:2.

[0022] Further, in step S5, it is carried out in a vacuum crystallization tank.

[0023] Further, in step S7, the water-soluble nano-titanium dioxide is a powder with a particle size of 30-60 nm.

[0024] Further, in step S4, the chromatography column includes a tube body, a liquid outlet is provided at the lower end of the tube body, a detachable packing assembly is arranged inside the tube body, and the neutral alumina is arranged in the packing assembly; a detachable top cover is provided at the top of the tube body, and a liquid inlet valve is arranged on the top cover.

[0025] Further, the packing assembly includes a cylindrical support mesh cylinder, a central cylinder coaxial with the support mesh cylinder is arranged on the bottom plate of the support mesh cylinder, a positioning plate is arranged at the upper end of the support mesh cylinder, a plurality of filter holes are arranged on the positioning plate, the positioning plate is sleeved on the outer wall of the central cylinder, and the positioning plate is fixedly connected with a rotating sleeve, and the inner wall of the rotating sleeve is in threaded fit with the outer wall of the central cylinder;

[0026] During loading, the neutral alumina is loaded into the support mesh cylinder, the rotating sleeve and the positioning plate are installed at the upper port of the support mesh cylinder, the whole packing assembly is placed in the oscillation box and the support mesh cylinder is fixed, the top of the rotating sleeve is connected to a rotating drive mechanism with a fixed output torque, then liquid is added to the oscillation box until the liquid submerges the rotating sleeve, the liquid is stirred by a stirring device, the liquid impacts the neutral alumina, and at the same time the rotating drive mechanism applies torque to the rotating sleeve, so that the positioning plate applies a constant pressure to the neutral alumina to eliminate the cavities inside the neutral alumina;

[0027] After the cavities are eliminated, the packing assembly is loaded into the tube body.

[0028] Further, a filter plate is fixedly arranged at the bottom of the inner cavity of the tube body, a vertical connecting column is arranged on the upper surface of the filter plate, the connecting column penetrates through the central cylinder and is connected to the top plate of the rotating sleeve by screws; a guide cover with a central upward protrusion is arranged inside the top cover, and uniformly distributed liquid distribution holes are arranged on the guide cover.

[0029] Further, after step S7, the obtained filtrate is sterilized.

[0030] High-purity hematoporphyrin is prepared by the above method.

[0031] The beneficial effects of the present invention are as follows: Based on the existing preparation and purification processes of hematoporphyrin, the present invention also uses poly(lactic-co-glycolic acid) to adsorb and purify hematoporphyrin. Then, the hematoporphyrin adsorbed by poly(lactic-co-glycolic acid) is transferred to water-soluble nano-titanium dioxide. Since the adsorption effect of water-soluble nano-titanium dioxide is better, it can effectively capture the hematoporphyrin that detaches from poly(lactic-co-glycolic acid). By adding a purification process, the impurity content in hematoporphyrin is reduced, and the negative effects on the human body are minimized. In addition, water-soluble nano-titanium dioxide has a wrapping effect, which can wrap hematoporphyrin molecules, improve the stability of hematoporphyrin, and avoid the denaturation of hematoporphyrin caused by weak light that may exist during packaging, transportation, and storage. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 is a schematic diagram of the preparation process of the present invention;

[0033] Figure 2 is a schematic diagram of the packing component of the chromatography column;

[0034] Figure 3 is a schematic diagram of eliminating cavities in the packing component;

[0035] Figure 4 is a schematic diagram of the chromatography column;

[0036] Reference numerals: 1—tube body; 2—liquid outlet; 3—top cover; 4—inlet valve; 5—supporting mesh cylinder; 6—central cylinder; 7—positioning plate; 8—rotating sleeve; 9—oscillation box; 10—rotating drive mechanism; 11—filter plate; 12—connecting column; 13—screw; 14—flow guiding cover. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The present invention will be further described below with reference to the drawings and embodiments.

[0038] The high-purity hematoporphyrin of the present invention, as Figure 1 shown, is prepared by the following method:

[0039] S1. Mix hemin with a saturated hydrobromic acid glacial acetic acid solution, and stir in an inert atmosphere for 15 - 24 h to obtain a reaction solution;

[0040] S2. Add a sodium hydroxide solution to the reaction solution, adjust the pH to 4 - 6, precipitate a first precipitate, and filter and collect the first precipitate;

[0041] S3. Dissolve the first precipitate in a hydrochloric acid methanol solution, reflux in a water bath for 12 - 18 min, cool, add an ammonium carbonate solution, precipitate a second precipitate, and filter and collect the second precipitate.

[0042] The above steps S1 to S3 can prepare a crude product of hematoporphyrin.

[0043] S4. Dissolve the second precipitate in chloroform and pass it through a chromatography column filled with neutral alumina. Use a mixed solution of chloroform and petroleum ether for elution to obtain an eluate. Through the chromatography purification process, the preliminary purification of hematoporphyrin is achieved.

[0044] S5. Evaporate the solvent of the eluate to obtain crystals, which are the third precipitate. The solvent is chloroform and petroleum ether. After evaporation, the solvent can be separated from hematoporphyrin.

[0045] S6. Dissolve the third precipitate in water, add poly (lactic - glycolic acid) copolymer powder to the water, and slowly stir in an inert atmosphere for 0.5 - 1 h, then filter to collect the fourth precipitate. Poly (lactic - glycolic acid) copolymer is insoluble in water and can adsorb hematoporphyrin, so hematoporphyrin can be separated from the solution. In this step, components insoluble in water can be removed, and at the same time, components not adsorbed by poly (lactic - glycolic acid) copolymer can be removed.

[0046] S7. Put the fourth precipitate and water - soluble nano - titanium dioxide into water, stir ultrasonically for 0.5 - 1 h and then stand for 0.5 - 1 h. The filtrate obtained by filtration is high - purity hematoporphyrin. Water - soluble nano - titanium dioxide can also adsorb hematoporphyrin, and its adsorption capacity is stronger than that of poly (lactic - glycolic acid) copolymer. Therefore, during the stirring process, the hematoporphyrin adsorbed by poly (lactic - glycolic acid) copolymer gradually transfers to the water and is adsorbed by water - soluble nano - titanium dioxide, realizing the transfer of hematoporphyrin.

[0047] It can be seen that based on the existing preparation and purification processes of hematoporphyrin, the present invention also uses poly (lactic - glycolic acid) copolymer to adsorb and purify hematoporphyrin, and then transfers the hematoporphyrin adsorbed by poly (lactic - glycolic acid) copolymer to water - soluble nano - titanium dioxide. Since the adsorption effect of water - soluble nano - titanium dioxide is better, it can effectively capture the hematoporphyrin that has detached from poly (lactic - glycolic acid) copolymer. By adding purification processes, the impurity content in hematoporphyrin is reduced, and the negative effects on the human body are reduced. In addition, water - soluble nano - titanium dioxide has a wrapping effect, which can wrap hematoporphyrin molecules, improve the stability of hematoporphyrin, and avoid the denaturation of hematoporphyrin caused by weak light that may exist during packaging, transportation, and storage.

[0048] As a preferred embodiment, in step S1, the ratio of the weight (g) of hemin to the volume (ml) of saturated hydrobromic acid glacial acetic acid solution is 1:(1.5 - 2.5);

[0049] In step S2, the mass fraction of the sodium hydroxide solution is 30% - 50%;

[0050] In step S3, mix a 1% hydrochloric acid solution and a 30% - 40% methanol solution in a volume ratio of 1:1 to obtain a hydrochloric acid methanol solution; the mass fraction of the ammonium carbonate solution is 1%.

[0051] In step S4, the chloroform and petroleum ether mixture is obtained by mixing chloroform and petroleum ether at a volume ratio of 5:1;

[0052] In step S6, the volume ratio of the added fourth precipitate to the water-soluble nano-titanium dioxide is 1:(1 - 2).

[0053] As a further preferred embodiment, in step S1, the weight ratio of hemin to saturated hydrobromic acid glacial acetic acid solution is 1:2;

[0054] In step S2, the mass fraction of the sodium hydroxide solution is 40%;

[0055] In step S3, a hydrochloric acid methanol solution is obtained by mixing a hydrochloric acid solution with a mass fraction of 1% and a methanol solution with a mass fraction of 30% at a volume ratio of 1:1; the mass fraction of the ammonium carbonate solution is 1%;

[0056] In step S4, the chloroform and petroleum ether mixture is obtained by mixing chloroform and petroleum ether at a volume ratio of 5:1;

[0057] In step S6, the volume ratio of the added fourth precipitate to the water-soluble nano-titanium dioxide is 1:2.

[0058] The above step S5 is carried out in a vacuum crystallization tank, and chloroform can be recovered.

[0059] In step S7, the water-soluble nano-titanium dioxide is a powder with a particle size of 30 - 60 nm.

[0060] Chromatography columns are commonly used separation and purification equipment, but existing chromatography columns have problems of inconvenient column packing and unloading. In step S4 of the present invention, the chromatography column used is as Figure 2 shown, including a tube body 1, the tube body 1 can be an organic glass tube, a liquid outlet 2 is arranged at the lower end of the tube body 1, a detachable packing assembly is arranged inside the tube body 1, and neutral alumina is arranged in the packing assembly; a detachable top cover 3 is arranged at the top of the tube body 1, and a liquid inlet valve 4 is arranged on the top cover 3.

[0061] Since the packing assembly is detachable, during packing, the neutral alumina particles can be first loaded into the packing assembly, and then the packing assembly can be loaded into the tube body 1; during unloading, the packing assembly can be taken out as a whole and the packing can be replaced, and the operation is relatively convenient.

[0062] The neutral alumina uses granular materials. During the packing process, there are gaps between the particles, and it is difficult to keep the gap size uniform, resulting in cavities inside the packing, and the purification effect cannot be guaranteed. Currently, it is usually by oscillating the packed chromatography column to make the gaps between the packings uniform, which requires a special oscillating device, the operation is troublesome, and due to the poor fluidity of the solid particles, the oscillation efficiency is low.

[0063] In the present invention, in order to quickly eliminate the voids in the packing, the packing assembly includes a cylindrical support mesh cylinder 5. The support mesh cylinder 5 includes a cylindrical barrel body and a circular bottom plate. Filter holes are provided on both the barrel body and the bottom plate to allow liquid to enter and exit. A central cylinder 6 coaxial with the support mesh cylinder 5 is provided on the bottom plate of the support mesh cylinder 5, and no filter holes are provided on the central cylinder 6. A positioning plate 7 is provided at the upper end of the support mesh cylinder 5. A plurality of filter holes are provided on the positioning plate 7. The positioning plate 7 is used to compress the packing inside the support mesh cylinder 5 to prevent the packing from becoming loose after being impacted during the chromatography process. The positioning plate 7 is sleeved on the outer wall of the central cylinder 6, and the positioning plate 7 is fixedly connected with a rotating sleeve 8. The inner wall of the rotating sleeve 8 is in threaded cooperation with the outer wall of the central cylinder 6. Rotating the rotating sleeve 8 can drive the positioning plate 7 to move axially, and at the same time facilitate the disassembly and assembly of the rotating sleeve 8 and the positioning plate 7.

[0064] During loading, neutral alumina is loaded into the support mesh cylinder 5, and the rotating sleeve 8 and the positioning plate 7 are installed at the upper port of the support mesh cylinder 5. The positioning plate 7 seals the support mesh cylinder 5. The whole packing assembly is placed in an oscillation box 9 and the support mesh cylinder 5 is fixed. A clamping mechanism can be arranged in the oscillation box 9 to clamp the lower end of the support mesh cylinder 5 by using the clamping mechanism, and the clamping mechanism can adopt the existing technology. The top of the rotating sleeve 8 is connected to a rotation driving mechanism 10 with a fixed output torque. The rotation driving mechanism 10 can adopt a motor. Then, liquid is added to the oscillation box 9. The liquid can be water until the liquid submerges the rotating sleeve 8. The liquid is stirred by a stirring device. When the stirring device stirs the liquid, the liquid flows. The flowing liquid impacts the neutral alumina to promote the flow of neutral alumina particles, so as to fill the internal voids and discharge the gas in the voids. At the same time, the rotation driving mechanism 10 applies torque to the rotating sleeve 8, so that the positioning plate 7 applies a constant pressure to the neutral alumina to eliminate the voids inside the neutral alumina. By applying a constant pressure to the neutral alumina, the neutral alumina particles are in close contact with each other, and will not become loose after being scoured by the liquid during chromatography. The stirring device can specifically adopt a pump, and the outlet of the pump faces the support mesh cylinder 5, and the liquid can be sprayed onto the support mesh cylinder 5 to make the liquid impact the neutral alumina particles inside the support mesh cylinder 5.

[0065] After the voids are eliminated, the packing assembly is loaded into the tube body 1.

[0066] In the present invention, due to the strong fluidity of the liquid and its ability to fill the gaps between neutral alumina particles, by stirring the liquid to make it move, the liquid scours the neutral alumina particles, which can drive the neutral alumina particles to flow and promote the neutral alumina particles to fill the voids, so that the voids can be eliminated faster and the effect of chromatographic purification can be guaranteed.

[0067] At the bottom of the inner cavity of the pipe body 1, a filter plate 11 is fixedly arranged. The filter plate 11 is provided with filter holes. On the upper surface of the filter plate 11, vertical connecting columns 12 are arranged. When loading the packing assembly into the pipe body 1, the connecting columns 12 are inserted into the central cylinder 6. The lower end of the packing assembly is supported by the filter plate 11. The upper end of the connecting column 12 penetrates through the central cylinder 6 and is connected to the top plate of the rotating sleeve 8 by screws 13, so that the whole packing assembly is kept fixed. In order to ensure that the liquid uniformly enters the neutral alumina at each part, a flow guide cover 14 with a central upward protrusion is arranged inside the top cover 3. The flow guide cover 14 is provided with uniformly distributed liquid distribution holes. The liquid distribution holes can be arranged in the area directly above the positioning plate 7, and no liquid distribution holes are arranged in the area above the central cylinder 6.

[0068] After step S7, the obtained filtrate is sterilized, and specifically, the method of microfiltration membrane filtration can be used to remove bacteria.

[0069] Example 1

[0070] Mix 10 g of hemin with 15 ml of saturated hydrobromic acid glacial acetic acid solution, and stir in an inert atmosphere for 15 h to obtain a reaction solution;

[0071] Add 600 ml of sodium hydroxide solution with a mass fraction of 30% to the reaction solution to precipitate a first precipitate, and filter and collect the first precipitate;

[0072] Mix a hydrochloric acid solution with a mass fraction of 1% and a methanol solution with a mass fraction of 30% according to a volume ratio of 1:1 to obtain a hydrochloric acid methanol solution; dissolve the first precipitate in 200 ml of the hydrochloric acid methanol solution, reflux in a water bath for 15 min, cool, and then add 200 ml of ammonium carbonate solution with a mass fraction of 1% to precipitate a second precipitate, and filter and collect the second precipitate;

[0073] Dissolve the second precipitate in 100 ml of chloroform, and pass it through a chromatography column filled with neutral alumina, and use a mixed solution of chloroform and petroleum ether for elution to obtain an eluate;

[0074] Evaporate the solvent of the eluate to obtain a third precipitate;

[0075] Dissolve the third precipitate in water, add 20 g of poly(lactic-co-glycolic acid) copolymer powder to the water, and slowly stir in an inert atmosphere for 40 min, and then filter and collect a fourth precipitate;

[0076] Put the fourth precipitate and water-soluble nano-titanium dioxide into water. The volume ratio of the fourth precipitate to the water-soluble nano-titanium dioxide is 1:1. After ultrasonic stirring for 40 min and standing for 30 min, the filtered filtrate is used as Preparation 1.

[0077] Example 2

[0078] Mix 10 g of hemin with 20 ml of saturated hydrobromic acid glacial acetic acid solution, and stir for 20 h in an inert atmosphere to obtain a reaction solution;

[0079] Add 600 ml of 35% sodium hydroxide solution by mass to the reaction solution to precipitate a first precipitate, and filter and collect the first precipitate;

[0080] Mix a 1% hydrochloric acid solution by mass with a 30% methanol solution by volume at a ratio of 1:1 to obtain a hydrochloric acid methanol solution; dissolve the first precipitate in 200 ml of the hydrochloric acid methanol solution, reflux in a water bath for 15 min, cool, and then add 200 ml of 1% ammonium carbonate solution by mass to precipitate a second precipitate, and filter and collect the second precipitate;

[0081] Dissolve the second precipitate in 100 ml of chloroform, and pass it through a chromatography column filled with neutral alumina, and perform elution with a mixed solution of chloroform and petroleum ether to obtain an eluate;

[0082] Evaporate the solvent of the eluate to obtain a third precipitate;

[0083] Dissolve the third precipitate in water, add 25 g of poly (lactic-co-glycolic acid) copolymer powder to the water, and slowly stir for 60 min in an inert atmosphere, and then filter and collect a fourth precipitate;

[0084] Put the fourth precipitate and water-soluble nano-titanium dioxide into water, and the volume ratio of the fourth precipitate to water-soluble nano-titanium dioxide is 1:1.5. After ultrasonic stirring for 50 min and standing for 30 min, the filtered filtrate is used as Formulation II.

[0085] Example III

[0086] Mix 10 g of hemin with 25 ml of saturated hydrobromic acid glacial acetic acid solution, and stir for 24 h in an inert atmosphere to obtain a reaction solution;

[0087] Add 450 ml of 50% sodium hydroxide solution by mass to the reaction solution to precipitate a first precipitate, and filter and collect the first precipitate;

[0088] Mix a 1% hydrochloric acid solution by mass with a 30% methanol solution by volume at a ratio of 1:1 to obtain a hydrochloric acid methanol solution; dissolve the first precipitate in 200 ml of the hydrochloric acid methanol solution, reflux in a water bath for 20 min, cool, and then add 200 ml of 1% ammonium carbonate solution by mass to precipitate a second precipitate, and filter and collect the second precipitate;

[0089] Dissolve the second precipitate in 100 ml of chloroform, and pass it through a chromatography column filled with neutral alumina, and perform elution with a mixed solution of chloroform and petroleum ether to obtain an eluate;

[0090] Evaporate the solvent of the eluate to obtain a third precipitate;

[0091] Dissolve the third precipitate in water, add 25 g of poly(lactic-co-glycolic acid) copolymer powder to the water, and slowly stir for 60 min in an inert atmosphere, and then filter and collect a fourth precipitate;

[0092] Put the fourth precipitate and water-soluble nano-titanium dioxide into water, and the volume ratio of the fourth precipitate to water-soluble nano-titanium dioxide is 1:2. After ultrasonic stirring for 60 min and standing for 60 min, the filtered filtrate is used as Preparation III.

[0093] Impurity content detection

[0094] Detection instrument: Agilent 1200 high performance liquid chromatography system, including the following parts:

[0095] G1322A Degasser

[0096] G1311A QuatPump

[0097] G1367B Hip-ALS

[0098] G1316A TCC

[0099] G1315D DAD

[0100] ChemStation for LC 3D Systems Rev.B.04.02

[96]

[0101] Pipette (Eppendorf, 100 μL, 1000 μL, 5000 μL)

[0102] Milli-Q ultrapure water purification system (Millipore Corporation)

[0103] pH meter (Mettler Corporation)

[0104] YB-Z type clarity detector (TianDa TianFa Technology Co., Ltd.)

[0105] DZKW-S-8 electrothermal constant temperature water bath (Beijing Guangming Medical Instrument Factory)

[0106] CP225D electronic balance (Sartorius, d = 0.00001 g)

[0107] Reagents:

[0108]

[0109] Accurately weigh 0.00985 g of hematoporphyrin, place it in a 10 mL volumetric flask, dissolve it with mobile phase (tetrahydrofuran) and dilute to the mark, shake well to obtain a solution with a concentration of 0.9653 mg / mL. Accurately measure 0.5 mL of the above solution and place it in a 10 mL volumetric flask, dissolve it with mobile phase and dilute to the mark, shake well to obtain a solution with a concentration of 48.27 μg / mL as the first control solution.

[0110] Take 2 mL of Preparation 1, Preparation 2 and Preparation 3 respectively, place them in 10 mL volumetric flasks, dissolve them with mobile phase and dilute to the mark, shake well to obtain Test Sample 1, Test Sample 2 and Test Sample 3.

[0111] Take 2 mL of the existing hematoporphyrin injection, place it in a 10 mL volumetric flask, dissolve it with mobile phase and dilute to the mark, shake well as the second control solution.

[0112] Chromatographic conditions:

[0113] Chromatographic column: Agela Venusil XBP C18 150×4.6 mm, 5 μm

[0114] Mobile phase: A: 5 mM ammonium acetate buffer solution (adjust the pH value to 4.0 with glacial acetic acid)

[0115] B: Tetrahydrofuran - water (9:1, v:v)

[0116] C: Tetrahydrofuran

[0117] Gradient:

[0118]

[0119] Flow rate: 1.0 mL / min

[0120] Injection volume: 10 μL

[0121] Column temperature: 25 °C

[0122] Detection wavelength: 400 nm.

[0123] Detection: Respectively take 10 μL of the first control solution, the second control solution, Test Sample 1, Test Sample 2 and Test Sample 3 and inject them into a high performance liquid chromatograph to obtain the chromatograms of the first control solution, the second control solution, Test Sample 1, Test Sample 2 and Test Sample 3 respectively.

[0124] Each reagent is detected 6 times respectively, and the chromatograms are recorded respectively.

[0125] Calculate the multiples (%) of the peak areas of each impurity in the second control solution, Test Sample 1, Test Sample 2, and Test Sample 3 relative to the peak area of the main peak in the first control solution. Among them, the multiples (%) of the peak areas of each impurity in the second control solution relative to the peak area of the main peak in the first control solution are shown in the following table:

[0126]

[0127] The multiples (%) of the peak areas of each impurity in Test Sample 1 relative to the peak area of the main peak in the first control solution are shown in the following table:

[0128]

[0129] The multiples (%) of the peak areas of each impurity in Test Sample 2 relative to the peak area of the main peak in the first control solution are shown in the following table:

[0130]

[0131] The multiples (%) of the peak areas of each impurity in Test Sample 3 relative to the peak area of the main peak in the first control solution are shown in the following table:

[0132]

[0133]

[0134] It can be seen that compared with the second control solution, the impurity contents in Test Sample 1, Test Sample 2, and Test Sample 3 are reduced, and after entering the human body, the side effects can be reduced.

[0135] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for preparing high-purity hematoporphyrin, characterized in that, It includes the following steps: S1. Mix hemin with a saturated hydrobromic acid glacial acetic acid solution, and stir in an inert atmosphere for 15 - 24 h to obtain a reaction solution; S2. Add a sodium hydroxide solution to the reaction solution, adjust the pH to 4 - 6, precipitate a first precipitate, and filter and collect the first precipitate; S3. Dissolve the first precipitate in a hydrochloric acid methanol solution, reflux in a water bath for 12 - 18 min, add an ammonium carbonate solution after cooling, precipitate a second precipitate, and filter and collect the second precipitate; S4. Dissolve the second precipitate in chloroform, and pass it through a chromatography column filled with neutral alumina, and perform elution with a mixed solution of chloroform and petroleum ether to obtain an eluate; S5. Evaporate the solvent of the eluate to obtain a third precipitate; S6. Dissolve the third precipitate in water, add poly(lactic - glycolic acid) copolymer powder to the water, and slowly stir in an inert atmosphere for 0.5 - 1 h, and then filter and collect a fourth precipitate; S7. Put the fourth precipitate and water - soluble nano - titanium dioxide into water, ultrasonically stir for 0.5 - 1 h and then stand for 0.5 - 1 h, and the filtrate obtained by filtration is a high - purity hematoporphyrin preparation.

2. The preparation method of high - purity hematoporphyrin according to claim 1, wherein in step S1, the ratio of the weight of hemin to the volume of the saturated hydrobromic acid glacial acetic acid solution is 1:(1.5 - 2.5); in step S2, the mass fraction of the sodium hydroxide solution is 30% - 50%; in step S3, mix a hydrochloric acid solution with a mass fraction of 1% and a methanol solution with a mass fraction of 30% - 40% according to a volume ratio of 1:1 to obtain a hydrochloric acid methanol solution; the mass fraction of the ammonium carbonate solution is 1%; in step S4, the mixed solution of chloroform and petroleum ether is obtained by mixing chloroform and petroleum ether according to a volume ratio of 5:1; in step S6, the volume ratio of the added fourth precipitate to water - soluble nano - titanium dioxide is 1:(1 - 2).

3. The preparation method of high - purity hematoporphyrin according to claim 2, wherein in step S1, the ratio of the weight of hemin to the volume of the saturated hydrobromic acid glacial acetic acid solution is 1:2; in step S2, the mass fraction of the sodium hydroxide solution is 40%; in step S3, mix a hydrochloric acid solution with a mass fraction of 1% and a methanol solution with a mass fraction of 30% according to a volume ratio of 1:1 to obtain a hydrochloric acid methanol solution; the mass fraction of the ammonium carbonate solution is 1%; in step S4, the mixed solution of chloroform and petroleum ether is obtained by mixing chloroform and petroleum ether according to a volume ratio of 5:1; in step S6, the volume ratio of the added fourth precipitate to water - soluble nano - titanium dioxide is 1:

2.

4. The preparation method of high-purity hematoporphyrin according to claim 1, characterized in that, In step S5, it is carried out in a vacuum crystallization tank.

5. The preparation method of high-purity hematoporphyrin according to claim 1, characterized in that, In step S6, the water - soluble nano - titanium dioxide is a powder with a particle size of 30 - 60 nm.

6. The preparation method of high-purity hematoporphyrin according to claim 1, characterized in that, In step S4, the chromatography column includes a tube body (1), a liquid outlet (2) is arranged at the lower end of the tube body (1), a detachable packing assembly is arranged in the tube body (1), and the neutral alumina is arranged in the packing assembly; a detachable top cover (3) is arranged at the top of the tube body (1), and a liquid inlet valve (4) is arranged on the top cover (3).

7. The preparation method of high-purity hematoporphyrin according to claim 6, characterized in that, The packing assembly includes a cylindrical support mesh cylinder (5). A central cylinder (6) coaxial with the support mesh cylinder (5) is arranged on the bottom plate of the support mesh cylinder (5). A positioning plate (7) is arranged at the upper end of the support mesh cylinder (5). A plurality of filter holes are arranged on the positioning plate (7). The positioning plate (7) is sleeved on the outer wall of the central cylinder (6), and the positioning plate (7) is fixedly connected with a rotating sleeve (8). The inner wall of the rotating sleeve (8) is in threaded cooperation with the outer wall of the central cylinder (6). During filling, neutral alumina is loaded into the support mesh cylinder (5). The rotating sleeve (8) and the positioning plate (7) are installed at the upper port of the support mesh cylinder (5). The whole packing assembly is placed into an oscillation box (9) and the support mesh cylinder (5) is fixed. The top of the rotating sleeve (8) is connected to a rotation driving mechanism (10) with a fixed output torque. Then, liquid is added into the oscillation box (9) until the liquid submerges the rotating sleeve (8). The liquid is stirred by a stirring device. The liquid impacts the neutral alumina. At the same time, the rotation driving mechanism (10) applies torque to the rotating sleeve (8), so that the positioning plate (7) applies a constant pressure to the neutral alumina to eliminate the cavities inside the neutral alumina. After the cavities are eliminated, the packing assembly is loaded into the tube body (1).

8. The preparation method of high-purity hematoporphyrin according to claim 7, characterized in that, A filter plate (11) is fixedly arranged at the bottom of the inner cavity of the tube body (1). Vertical connecting columns (12) are arranged on the upper surface of the filter plate (11). The connecting columns (12) penetrate through the central cylinder (6) and are connected to the top plate of the rotating sleeve (8) by screws (13). A flow guiding cover (14) with a central upward protrusion is arranged inside the top cover (3). Uniformly distributed liquid distribution holes are arranged on the flow guiding cover (14).

9. The preparation method of high-purity hematoporphyrin according to claim 1, characterized in that, After step S7, the obtained filtrate is sterilized.

10. High-purity hematoporphyrin, characterized in that, Prepared by the method according to any one of claims 1 to 9.

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