A kind of use of ethyl pheophorbide

Abstract

The present invention belongs to the field of medical technology, and specifically relates to a use of ethyl pheophorbide. Experiments have confirmed that ethyl pheophorbide a and ethyl pheophorbide b of the present invention can significantly inhibit the proliferation and differentiation of T and B lymphocytes induced by mitogens in vitro, showing a significant immunosuppressive effect. The compounds of the present invention are further expected to be developed into immunosuppressive drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of medicines, and in particular relates to a use of ethyl pheophorbide. Background Art

[0002] Autoimmune diseases are diseases in which the body's immune system malfunctions, causing the body to attack its own tissues. It is characterized by the immune system producing a large number of autoantibodies and immune complexes against self-antigens, which ultimately cause damage to the functions of multiple tissues and organs. Symptoms vary depending on the disease and the affected organs. Autoimmune diseases are controlled by host genes and the environment, both of which can increase susceptibility to autoimmunity by affecting the overall reactivity and quality of immune system cells. Cytokines and chemokines produced by innate immune cells including macrophages, natural killer cells, dendritic cells, mast cells and different granulocyte subsets, as well as the complement system, provide an inflammatory microenvironment, affect the differentiation of immune cells, lead to the activation of T and B cells, and plasma cells release autoantibodies, which in turn stimulate dendritic cells to produce type I interferon, thereby creating a closed loop of positive feedback between innate immunity and adaptive immunity.

[0003] Autoimmune diseases are treated with drugs that suppress the activity of the immune system. Currently, there are immunosuppressants, hormones, etc. However, a considerable number of patients in clinical treatment have low or no response to current treatment methods, so finding new treatment methods is still an urgent demand for the treatment of autoimmune diseases.

[0004] T and B lymphocytes have receptors that recognize antigens and mitogen receptors on their surfaces, which can cause the corresponding lymphocyte clones to proliferate under specific antigen stimulation. Concanavalin A (ConA) and bacterial lipopolysaccharide (LPS) are mitogens that can stimulate T and B lymphocytes to proliferate and differentiate, respectively. This process is similar to the activation process of lymphocytes in the body. Therefore, mitogen-induced lymphocyte proliferation is often used as an indicator to evaluate lymphocyte function.

[0005] Chlorophyll is the main component of photosynthesis in the stems and leaves of green plants. During the digestion of vegetables, humans convert chlorophyll into fucophytin, pyrofucophytin or pheophorbide. Chlorophyll and its derivatives are well known for their health benefits. They not only act as colorants, but also have antioxidant and potential therapeutic properties. Due to their special photosensitivity, pheophorbide and its derivatives have been used as pioneer molecules in photodynamic therapy in recent years. They can selectively kill tumor cells through phototoxicity. Some studies have pointed out that after the C-17 position of pheophorbide is replaced by ethyl ester, the phototoxicity is reduced (CHEMISTRY&BIODIVERSITY, 2005, 2, 1648). In addition, pheophorbide and its derivatives also have a variety of biological activities, including antiviral, anti-inflammatory, antioxidant and other wide-ranging biological activities. So far, studies have not mentioned the immunosuppressive effect of such ingredients. The present invention first discovered that pheophorbide a ethyl ester and pheophorbide b ethyl ester can significantly inhibit the proliferation and differentiation of T and B lymphocytes induced by mitogens in vitro, showing an immunosuppressive effect. Summary of the invention

[0006] The purpose of the present invention is to provide a use of ethyl pheophorbide and to report for the first time its immunosuppressive function regulation in vitro.

[0007] The specific technical solutions of the present invention are as follows:

[0008] A use of ethyl pheophorbide alpha or a pharmaceutically acceptable salt thereof in the preparation of an immunosuppressant, wherein the structural formula of ethyl pheophorbide alpha is as shown in Formula I:

[0009]

[0010] A use of ethyl pheophorbide b or a pharmaceutically acceptable salt thereof in the preparation of an immunosuppressant, wherein the structural formula of ethyl pheophorbide b is as shown in Formula II:

[0011]

[0012] By adopting the above technical solution: the present invention is through [ 3 H]-thymidine ( 3 The results showed that ethyl pheophorbide a (Formula I) and ethyl pheophorbide b (Formula II) could significantly inhibit the proliferation and differentiation of T and B lymphocytes induced by mitogens, showing a significant immunosuppressive effect.

[0013] Another object of the present invention is to provide a compound of formula I and its pharmaceutically acceptable salts (including but not limited to magnesium salts, sodium copper salts, etc.) for use in the preparation of autoimmune inhibitors.

[0014] Another object of the present invention is to provide a compound of formula II and its pharmaceutically acceptable salts (including but not limited to magnesium salts, sodium copper salts, etc.) for use in the preparation of autoimmune inhibitors.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] The present invention is achieved by 3 H]-thymidine ( 3 The results showed that ethyl pheophorbide a (Formula I) and ethyl pheophorbide b (Formula II) could significantly inhibit the proliferation and differentiation of T and B lymphocytes induced by mitogens, showing a significant immunosuppressive effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The hydrogen nuclear magnetic resonance spectrum of the compound pheophorbide a ethyl ester (Formula I) of the present invention;

[0018] Figure 2 The carbon-NMR spectrum of the compound pheophorbide alpha ethyl ester (Formula I) of the present invention;

[0019] Figure 3 The hydrogen nuclear magnetic resonance spectrum of the compound pheophorbide b ethyl ester (Formula II) of the present invention;

[0020] Figure 4 The carbon-NMR spectrum of the compound pheophorbide b ethyl ester (Formula II) of the present invention. DETAILED DESCRIPTION

[0021] In order to facilitate the understanding of the technical solution of the present invention, a detailed description is given below in conjunction with the accompanying drawings and specific embodiments.

[0022] The terms used in the present invention generally have the meanings commonly understood by those of ordinary skill in the art unless otherwise specified.

[0023] The present invention is further described in detail below in conjunction with specific examples and with reference to data. It should be understood that these examples are only intended to illustrate the present invention, and are not intended to limit the scope of the present invention in any way.

[0024] In the following examples, various processes and methods not described in detail are conventional methods well known in the art.

[0025] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0026] As described in the following examples, one skilled in the art can prepare the compounds of the present invention.

[0027] Example 1

[0028] Preparation of the compound pheophorbide a ethyl ester (Formula I) of the present invention:

[0029] 10 kg of dried Euonymus fortunei (Turcz.) Hand.-Mazz. stems and leaves were crushed and extracted three times with 95% ethanol by percolation, each time for 2 to 3 days, the extracts were combined and concentrated under reduced pressure to obtain an alcohol extract of Euonymus fortunei stems and leaves, the extracts were suspended in water, extracted with ethyl acetate, and the ethyl acetate extract was obtained. The sample was mixed with 100 mesh silica gel, separated by silica gel column chromatography (200 to 300 mesh), and gradient eluted with petroleum ether / ethyl acetate (10:1→5:1→2:1→0:1) to obtain components Fr.1-Fr.8. Fr.5 was further purified repeatedly by MCI column chromatography and preparative HPLC (98% acetonitrile) to obtain pure compound pheophorbide a ethyl ester.

[0030] The physical and chemical data of the compound pheophorbide a ethyl ester are as follows: dark green amorphous powder, easily soluble in organic solvents such as methanol, ethanol, acetone, chloroform, etc., with a molecular formula of C 37 H 40 N4O5, ESIMS m / z 621([M+H] + The spectral data of the compound are shown in Table 1 and the NMR spectrum is shown in Appendix Figure 1 and attached Figure 2 .

[0031] Table 1 H-spectrum (400 MHz) and C-spectrum (100 MHz) NMR signals of compound pheophorbide a ethyl ester (Formula I) (measured in deuterated chloroform)

[0032]

[0033] Example 2

[0034] Preparation of the compound pheophorbide b ethyl ester (Formula II) of the present invention:

[0035] 10 kg of dried Euonymus fortunei (Turcz.) Hand.-Mazz. stems and leaves were crushed and extracted three times with 95% ethanol by percolation, each time for 2 to 3 days, the extracts were combined and concentrated under reduced pressure to obtain an alcohol extract of Euonymus fortunei stems and leaves, the extracts were suspended in water, extracted with ethyl acetate, and the ethyl acetate extract was obtained. The sample was mixed with 100 mesh silica gel, separated by silica gel column chromatography (200 to 300 mesh), and gradient eluted with petroleum ether / ethyl acetate (10:1→5:1→2:1→0:1) to obtain components Fr.1-Fr.8. Fr.7 was further purified repeatedly by MCI column chromatography and preparative HPLC (98% acetonitrile) to obtain pure compound pheophorbide b ethyl ester.

[0036] The physical and chemical data of the compound pheophorbide a ethyl ester are as follows: dark green amorphous powder, easily soluble in organic solvents such as methanol, ethanol, acetone, chloroform, etc., with a molecular formula of C 37 H 38 N4O6, ESIMS m / z 635 ([M+H] + The spectral data of the compound are shown in Table 2, and the NMR spectrum is shown in Appendix Figure 3 and attached Figure 4 .

[0037] Table 2 H-spectrum (400 MHz) and C-spectrum (100 MHz) NMR signals of compound pheophorbide b ethyl ester (Formula II) (measured in deuterated chloroform)

[0038]

[0039] Example 3

[0040] Experiment on the non-specific toxicity and proliferation reaction of pheophorbide a ethyl ester (Formula I) of the present invention on mouse lymphocytes:

[0041] Animal source: BALB / c pure strain mice, female, 18-20 g, purchased from the animal room of our unit at 4-5 weeks of age, and raised for at least one week before use. Temperature 22±1℃, humidity 55±5%, 12-hour light-dark cycle. Feed and water were sterilized and freely available to the animals. All experiments were conducted in strict accordance with the relevant regulations on experimental animals.

[0042] Preparation of mouse spleen lymphocytes: Mice were sacrificed by spinal cord dislocation, and the spleen was aseptically removed to prepare a single cell suspension. Red blood cells were removed with red blood cell lysis buffer and the cell concentration was adjusted.

[0043] CCK-8 method was used to detect the effect of compounds on the activity of mouse spleen lymphocytes: mouse spleen lymphocyte suspension 1×10 6 / well was inoculated in a 96-well plate, and different concentrations of ethyl pheophorbide a (Compound I) were added at the same time, and corresponding solvent controls and culture medium background controls were set up, with a total volume of 200 μL. Cultured in a 37°C, 5% CO2 incubator for 48 hours. CCK-8 solution was added 8-10 hours before the end of the culture. At the end of the culture, the OD value was measured at 450nm (reference 650nm) on an ELISA reader.

[0044] [ 3 H]-thymidine ( 3 The effect of ethyl pheophorbide a (compound I) on the proliferation of splenic T and B lymphocytes induced by bacterial lipopolysaccharide (LPS) and concanavalin A (ConA) was evaluated by H-TdR incorporation method: 5×10 mouse spleen lymphocyte suspension was used for 5 / well was inoculated in a 96-well plate, and ConA (final concentration 5 μg / mL) or LPS (final concentration 10 μg / mL) and different concentrations of pheophorbide a ethyl ester (Compound I) were added, and corresponding control wells without ConA, LPS and drug were set up. Cultured in a 37°C, 5% CO2 incubator for 48 hours. 8 hours before the end of the culture, 25 μL of 3 H-thymidine nucleotide (10 μCi / mL). Continue to culture until the end of the experiment. Collect the cells onto a glass fiber membrane using a cell harvester, add scintillation fluid, and read the number of cells incorporated into the cell DNA using a Beta counter (MicroBeta Trilux, PerkinElmer). 3 The H-TdR amount is expressed as cpm, which represents the cell proliferation. The proliferation response of lymphocytes is calculated by subtracting the cpm value of the positive control sample from the cpm value of the sample being tested, and then dividing the cpm value of the positive control sample, and marked as % enhancement / inhibition.

[0045] T and B lymphocytes have receptors that recognize antigens and mitogen receptors on their surfaces, which can cause the corresponding lymphocyte clones to proliferate under specific antigen stimulation. Concanavalin A (ConA) and bacterial lipopolysaccharide (LPS) are mitogens that can stimulate T and B lymphocytes to proliferate and differentiate, respectively. This process is similar to the activation process of lymphocytes in the body. Therefore, mitogen-induced lymphocyte proliferation is often used as an indicator to evaluate lymphocyte function.

[0046] The results are shown in Table 1. The IC values ​​of ethyl pheophorbide a (Compound I) on immunosuppression of LPS-induced B lymphocytes are 50 The IC value of the immunosuppression of ConA-induced T lymphocytes was 0.538 μM and SI was 9.18. 50 The concentration of 1.426 μM and SI of 3.46 indicated that it had good immunosuppressive activity.

[0047] Example 4

[0048] Experiment on the non-specific toxicity and proliferation reaction of pheophorbide b ethyl ester (Formula II) on mouse lymphocytes:

[0049] Animal source: BALB / c pure strain mice, female, 18-20 g, purchased from the animal room of our unit at 4-5 weeks of age, and raised for at least one week before use. Temperature 22±1℃, humidity 55±5%, 12-hour light-dark cycle. Feed and water were sterilized and freely available to the animals. All experiments were conducted in strict accordance with the relevant regulations on experimental animals.

[0050] Preparation of mouse spleen lymphocytes: Mice were sacrificed by spinal cord dislocation, and the spleen was aseptically removed to prepare a single cell suspension. Red blood cells were removed with red blood cell lysis buffer and the cell concentration was adjusted.

[0051] CCK-8 method was used to detect the effect of compounds on the activity of mouse spleen lymphocytes: mouse spleen lymphocyte suspension 1×10 6 / well was inoculated in a 96-well plate, and different concentrations of ethyl pheophorbide a (Compound I) were added at the same time, and corresponding solvent controls and culture medium background controls were set up, with a total volume of 200 μL. Cultured in a 37°C, 5% CO2 incubator for 48 hours. CCK-8 solution was added 8-10 hours before the end of the culture. At the end of the culture, the OD value was measured at 450nm (reference 650nm) on an ELISA reader.

[0052] [ 3 H]-thymidine ( 3 The effect of ethyl pheophorbide a (compound I) on the proliferation of splenic T and B lymphocytes induced by bacterial lipopolysaccharide (LPS) and concanavalin A (ConA) was evaluated by H-TdR incorporation method: 5×10 mouse spleen lymphocyte suspension was used for 5 / well was inoculated in a 96-well plate, and ConA (final concentration 5 μg / mL) or LPS (final concentration 10 μg / mL) and different concentrations of pheophorbide a ethyl ester (Compound I) were added, and corresponding control wells without ConA, LPS and drug were set up. Cultured in a 37°C, 5% CO2 incubator for 48 hours. 8 hours before the end of the culture, 25 μL of 3 H-thymidine nucleotide (10 μCi / mL). Continue to culture until the end of the experiment. Collect the cells onto a glass fiber membrane using a cell harvester, add scintillation fluid, and read the number of cells incorporated into the cell DNA using a Beta counter (MicroBeta Trilux, PerkinElmer). 3 The H-TdR amount is expressed as cpm, which represents the cell proliferation. The proliferation response of lymphocytes is calculated by subtracting the cpm value of the positive control sample from the cpm value of the sample being tested, and then dividing the cpm value of the positive control sample, and marked as % enhancement / inhibition.

[0053] T and B lymphocytes have receptors that recognize antigens and mitogen receptors on their surfaces, which can cause the corresponding lymphocyte clones to proliferate under specific antigen stimulation. Concanavalin A (ConA) and bacterial lipopolysaccharide (LPS) are mitogens that can stimulate T and B lymphocytes to proliferate and differentiate, respectively. This process is similar to the activation process of lymphocytes in the body. Therefore, mitogen-induced lymphocyte proliferation is often used as an indicator to evaluate lymphocyte function.

[0054] The results are shown in Table 3. The IC values ​​of ethyl pheophorbide (Formula II) on the immunosuppression of LPS-induced B lymphocytes are 50The IC value of the immunosuppression of ConA-induced T lymphocytes was 1.579 μM and the SI value was 9.97. 50 It was 4.411 μM and SI was 3.57, indicating that it had good immunosuppressive activity.

[0055] Table 3 Ethyl pheophorbide a (Compound I) and ethyl pheophorbide b (Compound II) inhibit mitogen-induced proliferation of whole spleen lymphocytes in mice

[0056]

[0057] Note: CC 50 , the concentration of compound at which the cell viability is 50%; IC 50 , the concentration of compound at which cell proliferation is reduced by 50%; SI = CC 50 / IC 50

[0058] In summary, the present invention is 3 H]-thymidine ( 3 The results showed that ethyl pheophorbide a (Formula I) and ethyl pheophorbide b (Formula II) could significantly inhibit the proliferation and differentiation of T and B lymphocytes induced by mitogens, showing a significant immunosuppressive effect.

[0059] The above are only preferred embodiments of the present invention. The protection scope of the present invention shall be based on the scope defined by the claims. Several improvements and modifications made by those skilled in the art without departing from the spirit and scope of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A use of ethyl pheophorbide a or a pharmaceutically acceptable salt thereof in the preparation of an immunosuppressant, characterized in that: The structural formula of the ethyl pheophorbide a is shown in Formula I: The preparation method of the ethyl pheophorbide a is as follows: 10 kg of dried Euphorbia fortunei stems and leaves were crushed and extracted three times with 95% ethanol by percolation, each time for 2 to 3 days. The extracts were combined and concentrated under reduced pressure to obtain an alcohol extract of Euphorbia fortunei stems and leaves. The extract was suspended in water and extracted with ethyl acetate to obtain an ethyl acetate extract. The sample was mixed with 100 mesh silica gel, separated by silica gel column chromatography with 200 to 300 mesh, and eluted with petroleum ether / ethyl acetate gradient to obtain components Fr.1-Fr.8; Fr.5 was repeatedly purified by MCI column chromatography and preparative HPLC to obtain the pure compound ethyl pheophorbide a.

2. A use of ethyl pheophorbide b or a pharmaceutically acceptable salt thereof in the preparation of an immunosuppressant, characterized in that: The structural formula of the ethyl pheophorbide b is shown in Formula II: The preparation method of the pheophorbide b ethyl ester is as follows: 10 kg of dried stems and leaves of Euonymus fortunei were crushed and extracted three times with 95% ethanol by percolation, each time for 2 to 3 days. The extracts were combined and concentrated under reduced pressure to obtain an alcohol extract of stems and leaves of Euonymus fortunei. The extract was suspended in water and extracted with ethyl acetate to obtain an ethyl acetate extract. The sample was mixed with 100 mesh silica gel, separated by silica gel column chromatography with 200 to 300 mesh, and eluted with petroleum ether / ethyl acetate gradient to obtain components Fr.1-Fr.8; Fr.7 was repeatedly purified by MCI column chromatography and preparative HPLC to obtain the pure compound ethyl pheophorbide b.

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