Medical application of meroterpenoids

Abstract

The invention belongs to the technical field of biological medicine, and relates to medical application of a meroterpenoid compound, in particular to application of the meroterpenoid compound in preparation of immunomodulatory and antioxidant drugs or food. The meroterpenoid compound is a compound shown in the following structure or a pharmaceutically acceptable salt thereof, and R1, R2 and R3 are described in the claims and the specification. The meroterpenoid compound or the composition containing the meroterpenoid compound provided by the invention has an anti-oxidation or immunoregulation effect, and can be used for preparing anti-oxidation and immunoregulation medicines or foods. # imgabs0 #

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the medical use of a terpene compound, and specifically relates to the use of a terpene compound in the preparation of immunomodulatory and antioxidant drugs or foods. Background Art

[0002] The function of the immune system is closely related to human health, such as inflammation, infection, tumors, aging, reproduction, allergic diseases, autoimmune diseases, etc. The pathogenesis of most human diseases involves immune function. With the accelerated pace of modern life, irregular work and rest, unbalanced diet, lack of exercise, and air pollution, the incidence of human immune system dysfunction is on the rise, and the ability to resist pathogens is also declining. Therefore, improving immunity is of great significance for preventing diseases, coping with viral infections, and promoting overall health.

[0003] Macrophages are important innate immune cells in the immune system. They are specialized cells of the natural immune system that have a long survival time and phagocytic function. They are the first responders to pathogens. Macrophages mediate a bridge between innate and adaptive immune responses. Macrophages participate in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vivo. Macrophages participate in the recognition, phagocytosis and degradation of cell fragments and pathogens. Macrophages also play a role in presenting antigens to T cells and inducing other antigen-presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses. In addition, in the early stages of pathogen invasion, they play an important role by releasing cytokines and chemokines, which in turn recruit other immune cells to the site of inflammation. In addition to initiating immune and inflammatory responses to pathogens, macrophages also play a role in maintaining tissue homeostasis and repairing and remodeling tissues. Unfortunately, this function is associated with many diseases, including metabolic and autoimmune diseases, cancer, infection, obesity and fibrosis. Therefore, macrophages have become therapeutic targets for a variety of diseases, and the development of drugs that regulate macrophage function has important clinical value.

[0004] Reactive oxygen species (ROS) such as hydrogen peroxide, singlet oxygen, hydroxyl radicals and superoxide anions are natural byproducts of normal oxygen metabolism in the body. ROS can act as signaling molecules to regulate various physiological functions of cells and play an important role in cell signal transduction and homeostasis. ROS can directly affect the activity of pathogens, cancer cells and various immune cells.

[0005] The generation and clearance of ROS are strictly regulated to maintain homeostasis. The appropriate level of ROS in the body is a key signaling molecule that regulates various physiological functions (including inflammatory responses), and can also activate the immune system to produce immune protection against pathogens or tumor cells. However, excessive and uncontrolled ROS may trigger harmful oxidative stress responses in the body, including lipid peroxidation, oxidative damage to DNA, protein oxidation, and monosaccharide oxidation, and destroy cell structures such as mitochondrial structures, leading to various diseases. Reactive oxygen species have been widely found to play an important role in a variety of diseases that threaten human health, including cancer, cardiovascular disease, diabetes, neurodegeneration (such as Parkinson's disease and Alzheimer's disease), autoimmune diseases, stroke, aging, inflammatory diseases, etc. Therefore, finding ways to remove intracellular reactive oxygen species has become an important solution to restore ROS homeostasis, limit inflammatory responses, immune imbalance, and treat diseases.

[0006] ROS plays an important role in regulating macrophage function. However, excessive ROS can cause mitochondrial structural damage, lipid peroxidation, promote macrophage immunosuppression or inflammatory response, and lead to the occurrence and development of diseases such as tumors, aging, and inflammation. Therefore, targeted regulation of ROS levels in cells is an important strategy for the treatment of various diseases. However, currently, there are limited reagents and drugs that regulate the production or clearance of intracellular ROS.

[0007] Certain small molecule compounds play an important regulatory role in disease treatment. They have the advantage of entering cells to regulate ROS levels and can better regulate intracellular ROS production or clearance. They can be used to prepare immunomodulatory and antioxidant drugs or foods. Summary of the invention

[0008] In order to overcome the defects in the prior art, the present invention provides an application of a heteroterpenoid compound in the preparation of immunomodulatory and antioxidant drugs or foods.

[0009] The present invention is achieved through the following technical solutions:

[0010] Application of terpenoid compounds in the preparation of immunomodulatory drugs or foods.

[0011] Application of terpenoid compounds in the preparation of antioxidant drugs or foods.

[0012] Furthermore, the food is a health food.

[0013] The structure of the heteroterpenoid compound is as follows:

[0014]

[0015] in,

[0016] R 1 , R2 , R 3 is H, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, halogen, amino, cyano;

[0017] Further,

[0018] R 1 , R 2 , R 3 is H, C1-C4 alkyl, C1-C4 alkoxy;

[0019] Furthermore,

[0020] The structure of the heteroterpenoid compound is:

[0021]

[0022] The heteroterpenoid compounds achieve anti-oxidation and enhance immune response by reducing the generation of reactive oxygen species (ROS) in cells.

[0023] The heteroterpenoid compounds achieve the effects of anti-oxidation and enhancing immunity by regulating the function of macrophages.

[0024] The heteroterpenoid compounds can regulate the functions of macrophages, including regulating the redox homeostasis of macrophages, regulating the ROS level of macrophages, reducing the ROS level in macrophage mitochondria, and regulating the production of macrophage cytokines.

[0025] The heteroterpenoid compounds of the present invention can be prepared into compositions with acceptable carriers or excipients.

[0026] The terpenoid compound or the composition containing the terpenoid compound provided by the present invention has an antioxidant or immunomodulatory effect and can be used for preparing an antioxidant or immunomodulatory drug or food. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The enhancement effect of the terpenoid compound CND on cellular immune response;

[0028] A is a terpenoid compound CND that enhances CD8 + Schematic diagram of the T cell-mediated immune response assay;

[0029] B shows the expression of CD8 in macrophages after treatment with terpenoid compound CND. + Representative peak graph of T cell proliferation;

[0030] C shows the macrophages and CD8 + T cells co-cultured, CD8 + Statistical analysis of the proportion of T cells;

[0031] D is the macrophages and CD8 + After co-culture of T cells, CD8 + Statistical analysis of changes in T cell numbers.

[0032] Figure 2 The effect of CND on CD8 + The level of interferon IFN expressed by T cells.

[0033] Figure 3 This is a statistical analysis of the effects of CND treatment on the expression levels of different inflammatory factors and chemokines in mouse macrophages;

[0034] A: Inflammatory factors B: Chemokines.

[0035] Figure 4 This is a graph showing the regulation and statistical analysis of the terpene compound CND on ROS levels in macrophages. DETAILED DESCRIPTION

[0036] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0037] Intracellular reactive oxygen species have an important regulatory effect on cell function and the development of diseases. Macrophages are an important subset of immune cells that are involved in a variety of inflammatory-related diseases. The maintenance of redox homeostasis in macrophages is the key to the normal physiological function of macrophages. Under stress conditions, macrophages become dysfunctional, leading to the development of diseases. Intracellular reactive oxygen species can regulate the polarization of macrophages, and polarized macrophages have different abilities to produce reactive oxygen species. The present invention is in H 2 O 2 The antioxidant and immunomodulatory activities of terpenoids were evaluated after treatment of macrophages.

[0038] Tumor-associated macrophages (TAMs) in the tumor microenvironment inhibit anti-tumor immune responses under the regulation of reactive oxygen species. The present invention treats macrophages with tumor cell culture supernatant (TCM) to induce the production of reactive oxygen species in macrophages, and further evaluates the inhibitory activity of the compound on the level of reactive oxygen species, its ability to regulate the redox homeostasis of macrophages, and the activity of heteroterpenoid compounds in protecting macrophages from reactive oxygen damage, resulting in the destruction of cytoplasmic membrane structure and mitochondrial structure.

[0039] Macrophages regulate the type and intensity of immune responses and affect the progression of diseases by expressing various cytokines. Furthermore, the changes in the expression of different cytokines in macrophages were evaluated after treating macrophages with terpenoids, and the ability of terpenoids to regulate inflammatory responses was evaluated.

[0040] The preparation method of the ameroterpenoid compound CND involved in each embodiment of the present invention can be referred to: Construction of ameroterpenoid-like compound collection by precursor-assisted biosynthesis [J]. Organic & Biomolecular Chemistry, 2020, 18. DOI: 10.1039 / D0OB01235A.

[0041] Example 1

[0042] Enhancement of terpenoid compound CND on cellular immune response

[0043] 1.1 Take C57BL / 6J wild-type adult mice, kill them and soak them in 75% alcohol for 2-3 minutes for disinfection. Make an incision in the abdomen, separate the muscles on the bones with scissors, take the mouse tibia and femur and then soak them in phosphate buffered saline (PBS). Subsequent steps are completed in a sterile operating table.

[0044] 1.2 After removing the tibia and femur with sterile tweezers, cut off small pieces on both sides with scissors, and use a 20mL syringe filled with PBS to repeatedly flush the bone marrow up and down alternately. Collect the flushed bone marrow cells into a 50mL sterile centrifuge tube through a 70μm cell sieve and centrifuge at 2000rpm for 4min at 4℃.

[0045] 1.3 Add 1 mL of 1× red blood cell lysis buffer to the cell pellet to suspend the cells. After standing for 2 minutes, add 10 mL of PBS for washing and centrifuge at 4°C, 2000 rpm for 4 minutes.

[0046] 1.4 The cells were resuspended in RPMI 1640 medium (containing 5% mouse fibroblast L929 conditioned medium) and the cell density of bone marrow-derived macrophages (BMDM) was adjusted to 2×10 6 / well, plated in untreated 6-well cell culture plates and incubated at 37°C, 5% CO 2 The cells were cultured under the conditions of 4 days, and the bone marrow-differentiated macrophages were observed under a microscope.

[0047] 1.5 BMDM cells were cultured at 1×10 4 / wells were plated in 96-well plates and divided into three groups for the following treatments:

[0048] A: Fresh culture medium (FCM) was used for 12 hours, and then DMSO (0.1%) was added for 24 hours.

[0049] B: Tumor cell culture supernatant (TCM) was used for 12 h, and then DMSO (0.1%) was added for 24 h.

[0050] C: Tumor cell culture supernatant (TCM) was treated for 12 h, and then 1 μM compound CND was added for 24 h.

[0051] The TCM preparation method is as follows: 3×10 6 After mixing 100 Hepa1-6LM cells in 10 ml of culture medium, they were plated in a 10 cm culture dish and cultured for 36 h before collecting the cell culture supernatant.

[0052] Compound CND was dissolved in DMSO (dimethyl sulfoxide).

[0053] 1.6 Discard the culture medium in the above step, add 100 ng / ml ovalbumin (OVA) to stimulate BMDM cells for 2 h, and wash twice with PBS.

[0054] 1.7 Labeling of OT1 mouse derived CD8 + T cells and BMDM were co-cultured at a ratio of 1:10 for 2-3 days, and flow cytometry was used to detect the expression of CTV in CD8 + Expression intensity in T cells, evaluating CD8 + T cell proliferation effect.

[0055] 1.8 CD8 T cells isolated from OT1 mice by flow cytometry + T cells were labeled with CTV, and then CD8 + T cells and BMDM were co-cultured at a ratio of 1:10 for 2-3 days.

[0056] 1.9 Flow cytometry detection of CTV in CD8 + Expression intensity in T cells and calculation of CD8 + The number of T cells was used to evaluate CD8 + T cell proliferation.

[0057] 1.10 Collect the supernatant of the co-cultured cells and detect the IFN level using ELISA.

[0058] Results Figure 1-2 .

[0059] like Figure 1As shown in B, treatment of macrophages with tumor supernatant inhibited the expression of macrophages to CD8 + The activation ability of T cells (simulating poor immune status of the body), the addition of compound CND-treated macrophages significantly promoted OT1 CD8 + T cell proliferation (ability to divide).

[0060] Figure 1 C shows the statistical analysis of CD8 proliferation more than 4 times + The proportion of T cells;

[0061] Figure 1 D in shows CD8 + Total number of T cells;

[0062] Figure 2 Shows that tumor cell culture supernatant can inactivate CD8 + The ability of T cells to produce IFNγ, and CND treatment reversed this effect;

[0063] The above results show that the terpenoid compound CND of the present invention can significantly promote the proliferation of CD8 + The proliferation capacity of T cells is manifested by a significant increase in the number of divisions and the total number. It can be seen that CND can relieve the macrophages in the tumor microenvironment from CD8 + T cell suppression, thereby promoting CD8 + T cells produce effector molecules that boost the immune response to cells.

[0064] Example 2

[0065] Terpenoid compound CND regulates the expression of different cytokines in macrophages

[0066] The specific steps are as follows:

[0067] 2.1 The method for preparing macrophages is the same as steps 1.1-1.4 of Example 1.

[0068] 2.2 Place BMDM cells in a 6-well plate and perform the following treatments:

[0069] A: control (0.1% DMSO)

[0070] B: Lipopolysaccharide LPS solution (final concentration 500ng / ml) + 0.1% DMSO

[0071] C: Lipopolysaccharide LPS solution (500ng / ml) + CND (1μM)

[0072] 2.3 After 6 hours of cell treatment, the culture medium was discarded, the cells were washed with PBS, Trizol was added, the cells were collected by pipetting, and mRNA was extracted.

[0073] 2.3.1 Discard the cell culture supernatant, add PBS buffer to the cell culture dish, wash the cells three times, add 1 mL of Trizol solution, blow the cells until they are completely removed, and transfer the mixture into a 1.5 mL nuclease-free centrifuge tube.

[0074] 2.3.2 Incubate at room temperature for 5 min, add 200 μL of chloroform, vortex for 15 s, and incubate on ice for 3 min.

[0075] 2.3.3 Centrifuge at 12,000 rpm for 15 min at 4°C in a high-speed centrifuge.

[0076] 2.3.4 Pipette the supernatant into a new 1.5 mL nuclease-free centrifuge tube, add isopropanol in the same volume as the supernatant, mix by inverting 8-10 times, and place on ice for 30 min.

[0077] 2.3.5 Centrifuge at 4°C, 12,000 rpm for 10 min.

[0078] 2.3.6 Discard the supernatant, add 1 mL of 75% alcohol, gently blow up the cell pellet, and centrifuge at 12,000 rpm at 4°C for 3 min.

[0079] 2.3.7 Discard the supernatant, place on ice for 10 min, add 30 μL DEPC water to dissolve and mix.

[0080] 2.3.8 Use a spectrophotometer to detect RNA concentration and OD value and store at -80℃.

[0081] 2.4 Convert mRNA to cDNA.

[0082] 2.4.1 Using reverse transcription reagents, mix the reactants in a 0.2 mL nuclease-free centrifuge tube according to the system in Table 1:

[0083] Table 1

[0084]

[0085] 2.4.2 Place the 0.2 mL centrifuge tube in a 42°C water bath, react for 2 min, add 8 μL 5×Hiscript IIqRT Supermix II, and perform reverse transcription according to the procedure in Table 2:

[0086] Table 2

[0087]

[0088] 2.4.3 After the reaction is completed, store the cDNA sample at -20℃.

[0089] 2.5 Use Roche LightCycler480II real-time fluorescence quantitative PCR instrument to detect the expression of different factors.

[0090] 2.5.1 Use of high pressure ddH 2 O, dilute the cDNA template 5 times, mix well and set aside.

[0091] 2.5.2 Using the qPCR plate, mix the reaction components according to the system in Table 3:

[0092] Table 3

[0093]

[0094] 2.5.3 Centrifuge at 4°C, 1500 rpm for 3 min.

[0095] 2.5.4 Place the qPCR plate in the instrument and amplify according to the reaction program in Table 4:

[0096] Table 4

[0097]

[0098]

[0099] qPCR result calculation method: Actb gene was used as the internal reference, the Ct value of the target gene of each sample was corrected, the ΔCt value was calculated, and the relative expression level of the gene was calculated using 2-ΔΔCt value.

[0100] The qPCR primer sequences used are shown in Table 5:

[0101] Table 5

[0102]

[0103] Results Figure 3 .like Figure 3 As shown in A and 3B, CND significantly increased the expression levels of different inflammatory-related factors (IL6, IL12b, NOS2, COX2) and chemokines (CCL2, CXCL9) in macrophages, indicating that CND has the ability to enhance the inflammatory response of macrophages to pathogens and recruit other immune cells to the lesion site to control pathogens. CND is a potential immunomodulatory active molecule.

[0104] Table 6

[0105]

[0106] Example 3 Antioxidant activity of heteroterpenoid compound CND

[0107] The experimental steps are as follows:

[0108] 1.1 Preparation and treatment of mouse macrophages

[0109] 1.1.1 Take C57BL / 6J wild-type adult mice, kill them and soak them in 75% alcohol for 2-3 minutes for disinfection. Make an incision in the abdomen, separate the muscles on the bones with scissors, take the tibia and femur of the mouse and then soak them in PBS buffer. The subsequent steps are completed in a sterile operating table.

[0110] 1.1.2 After removing the tibia and femur with sterile forceps, cut off small pieces on both sides with scissors, and repeatedly flush the bone marrow up and down with a 20mL syringe filled with PBS. Collect the flushed bone marrow cells through a 70μm cell sieve into a 50mL sterile centrifuge tube and centrifuge at 2000rpm for 4min at 4℃.

[0111] 1.1.3 Add 1 mL of 1× red blood cell lysis buffer to the cell pellet to suspend the cells. After standing for 2 min, add 10 mL of PBS for washing and centrifuge at 2000 rpm at 4°C for 4 min.

[0112] 1.1.4 The cells were resuspended in RPMI 1640 culture medium (containing 5% L929 supernatant) and the cell density was adjusted to 2×106 / well. The cells were plated into untreated 6-well cell culture plates and cultured at 37°C and 5% CO2. On the fourth day of cell culture, bone marrow-differentiated macrophages (BMDM) were observed under a microscope.

[0113] 1.1.5 Place BMDM cells in a 6-well plate and perform the following treatments:

[0114] Control (0.1% DMSO) and terpenoid compound CND (1 μM) were treated for 12 h.

[0115] 1.1.6 Each group joins H 2 O 2 (0.25 μM), treated for 1 h.

[0116] 1.1.7. Discard the supernatant, add PBS to wash once, resuspend in 200ul 2% FACS Buffer per well in a 96-well U-bottom plate, and centrifuge at 2000rpm for 4min.

[0117] 1.1.8. Measure ROS: discard the supernatant, add PBS to wash once, add ROS probe (1000x) and treat at 37 degrees for 30 minutes (1640 serum-free and double antibody-free)

[0118] 1.1.9. Wash once with PBS, add 150ul of 7AAD (400x) to each well of 2% FACS Buffer, mix well, filter into flow tube, detect on Canto II flow cytometer, and analyze with Flowjo software.

[0119] The results are as follows Figure 4 As shown in Table 7, the terpenoid compound CND can effectively reduce H 2 O 2 The level of reactive oxygen species (ROS) in macrophages after treatment indicated that CND had good antioxidant activity and was an effective antioxidant molecule.

[0120] Table 7

[0121]

[0122]

[0123] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. Use of the following heteroterpenoid compounds or pharmaceutically acceptable salts thereof in the preparation of antioxidant drugs or foods: in, R1, R2, and R3 are H, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, halogen, amino, and cyano.

2. Use of the following heteroterpenoid compounds or pharmaceutically acceptable salts thereof in the preparation of immune-regulating drugs or foods: in, R1, R2, and R3 are H, C1-C6 alkyl, C1-C6 alkoxy, hydroxy, halogen, amino, and cyano.

3. The use according to claim 1 or 2, characterized in that: R1, R2, and R3 are H, C1-C4 alkyl, and C1-C4 alkoxy.

4. The use according to claim 1 or 2, characterized in that: The structure of the heteroterpenoid compound or a pharmaceutically acceptable salt thereof is as follows:

5. The use according to claim 1 or 2, characterized in that: The heteroterpenoid compound or a pharmaceutically acceptable salt thereof realizes an antioxidant or immune-regulating effect by regulating the function of macrophages.

6. The use according to claim 1 or 2, characterized in that: The heteroterpenoid compound or a pharmaceutically acceptable salt thereof achieves an antioxidant or immune-regulating effect by reducing the generation of active oxygen in cells.

7. The use according to claim 1 or 2, characterized in that: The heteroterpenoid compound or a pharmaceutically acceptable salt thereof enhances the immune response mediated by T cells by enhancing the immune activation ability of macrophages.

8. The use according to claim 1 or 2, characterized in that: The heteroterpenoid compound or a pharmaceutically acceptable salt thereof achieves an anti-oxidative or immune-regulating effect by increasing the cytokines and chemokines produced by macrophages.

9. The use according to claim 1 or 2, characterized in that: The heteroterpenoid compound or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or excipient are prepared into a composition.

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