Novel immunosuppressant and application thereof

Abstract

The invention belongs to the technical field of medicines, and particularly discloses a novel immunosuppressant and application thereof, and the novel immunosuppressant is pinomoic acid separated and identified from glossy privet fruits. The invention relates to pomolic acid (PA) separated and identified from glossy privet fruit, which has an immunosuppressive effect, plays an immunosuppressive role by inhibiting the expression of proinflammatory factors and promoting the expression of anti-inflammatory factors, and also has application in preparation medicines for treating inflammatory disease models (such as acute lung injury). The PA can obviously inhibit the expression of proinflammatory factors (IL-1beta, IL-6, TNF-alpha and iNOS) and promote the expression of anti-inflammatory factors (IL-10), and can obviously improve the survival rate of acute lung injury model mice, reduce lung tissue pathological injury and relieve inflammatory response, which indicates that the PA has a very good immunosuppression effect. The invention provides a theoretical basis for developing novel immunosuppressants, and has potential application value.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to a novel immunosuppressant and its application. Background Technology

[0002] Immunosuppressants play a vital role in the treatment of inflammatory diseases, autoimmune diseases, and as adjuvant therapy for tumors. Currently, clinically used immunosuppressants mainly fall into three categories: first, glucocorticoids, such as dexamethasone; second, chemically synthesized drugs, such as methotrexate, calcineurin, and cyclosporine A; and third, biological agents, such as anti-IL-6 receptor monoclonal antibodies and TNF-α antagonists. While these immunosuppressants offer some therapeutic benefits, long-term use can easily lead to problems such as osteoporosis and metabolic disorders. Therefore, the development of highly effective and safe immunosuppressants has become a hot topic in the field.

[0003] Clinically used immunosuppressants mainly exert their effects through the following mechanisms:

[0004] Glucocorticoids reduce the release of pro-inflammatory factors (such as IL-1β and TNF-α) by inhibiting the NF-κB and MAPK pathways, but long-term use can lead to adrenal suppression and abnormal glucose metabolism.

[0005] Chemically synthesized drugs, such as methotrexate, inhibit the proliferation of immune cells by interfering with folic acid metabolism, but have side effects such as hepatotoxicity and bone marrow suppression.

[0006] Biologics: such as anti-IL-6 receptor monoclonal antibodies (tocilizumab) can block inflammatory signals, but they are expensive and may increase the risk of pathogen infection.

[0007] These immunosuppressants generally suffer from significant side effects or high costs, highlighting the urgent need to discover safer new immunosuppressants from natural products.

[0008] Ligustrum lucidum Ait., the fruit of the Oleaceae plant Ligustrum lucidum, is also known as "Wintergreen Fruit." The *Shennong Bencao Jing* (Divine Farmer's Materia Medica) records that "Ligustrum lucidum fruit primarily nourishes the middle jiao (spleen and stomach), calms the five internal organs, nourishes the spirit, and eliminates a hundred diseases." Modern research shows that its active ingredients include polysaccharides, flavonoids, and triterpenoids. Ligustrum lucidum contains various active ingredients, such as pomolic acid, oleanolic acid, privetin, rhodioloside, and verbascoside, which have immunomodulatory, anticancer, and antitumor effects. After extraction with an organic solvent, the privet fruit was filtered through a microporous membrane and then analyzed by UPLC-MS / MS. This invention investigated the immunomodulatory function of its component, pomolic acid (PA, CAS: 13849-91-7). Summary of the Invention

[0009] The purpose of this invention is to provide a novel immunosuppressant and its application. Experiments have shown that pomolic acid (PA, CAS: 13849-91-7) can significantly inhibit the expression of pro-inflammatory factors in macrophages while promoting the expression of anti-inflammatory factors. In vivo, PA can significantly alleviate lung tissue pathological damage in mice with acute lung injury, reduce inflammatory response, and improve survival rate.

[0010] The first aspect of this invention involves determining the components of *Ligustrum lucidum* using ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS / MS). Based on this, a database of traditional Chinese medicine systems pharmacology and an integrated platform for traditional Chinese medicine were used to search for compounds (with CAS numbers) in *Ligustrum lucidum* using oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 as screening criteria. Known compounds including PA, quercetin, and neolignoside were identified from *Ligustrum lucidum*.

[0011] The second aspect of this invention is the in vitro analysis of the immunosuppressive effect of PA. PA, at concentrations ranging from 6.25 µM to 25 µM, can inhibit the expression of iNOS and the release of NO from macrophages, as well as inhibit the expression of IL-1β, IL-6, and TNF-α, and promote the expression of IL-10, significantly reducing the production of inflammatory mediators, indicating that PA has potential immunosuppressive effects.

[0012] The third aspect of this invention is to provide the application of PA in the treatment of inflammatory diseases (such as acute lung injury). First, an acute lung injury mouse model was established by intraperitoneal injection of LPS. By observing changes in mouse body weight and survival rate throughout the drug intervention period, it was found that the survival rate of mice without PA intervention was significantly reduced to 33.3%, while the survival rate of the PA intervention group was significantly increased to 83.3%. Compared with the control group, the decrease in body weight of mice in the PA intervention group was significantly alleviated, suggesting that PA has a certain improving effect on the survival rate and body weight of mice with acute lung injury. Furthermore, the lung tissue of mice in the control group showed extensive infiltration of inflammatory cells, alveolar collapse, alveolar congestion and edema accompanied by hemorrhage, while the lung tissue lesions of mice in the PA intervention group were reduced, and lung tissue damage was decreased. Compared with the LPS group, the NO content in the serum and bronchoalveolar lavage fluid of mice in the PA group was significantly reduced, and the expression levels of IL-1β, IL-6, TNFα, and iNOS in the lung tissue of mice were significantly downregulated, while the expression level of IL-10 was significantly upregulated. The above results further demonstrate that PA has a good anti-inflammatory effect, which may exert its immunosuppressive effect by inhibiting the expression of pro-inflammatory factors and promoting the expression of anti-inflammatory factors.

[0013] To achieve the above objectives, the specific technical solution of the present invention is as follows:

[0014] A novel immunosuppressant, wherein the chemical composition of the immunosuppressant is pomodoric acid, and the molecular formula is... .

[0015] Preferably, the immunosuppressant pomolytic acid exerts its immunosuppressive effect by inhibiting the expression of pro-inflammatory factors and promoting the expression of anti-inflammatory factors.

[0016] Preferably, the pro-inflammatory factors include IL-1β, IL-6, TNF-α, and iNOS, and the anti-inflammatory factors include IL-10.

[0017] Preferably, the immunosuppressant polimoic acid is used in medicaments for treating inflammatory diseases.

[0018] Preferably, the inflammatory disease includes LPS-induced acute lung injury.

[0019] Pomolic acid, abbreviated as PA, has the CAS number 13849-91-7 and its molecular formula is... Its structure is as follows:

[0020] .

[0021] This invention relates to a polysaccharide (PA) isolated and identified from privet fruit, which exhibits immunosuppressive effects by inhibiting the expression of pro-inflammatory factors and promoting the expression of anti-inflammatory factors. It also has potential application value in the formulation of drugs for treating inflammatory disease models (such as acute lung injury). PA significantly inhibits the expression of pro-inflammatory factors (IL-1β, IL-6, TNF-α, iNOS) and promotes the expression of anti-inflammatory factor (IL-10), and significantly alleviates lung tissue pathological damage in mice with acute lung injury, reduces inflammatory responses, and improves survival rates, indicating that PA has a good immunosuppressive effect. This invention provides a theoretical basis for the development of novel immunosuppressants and has potential application value. Attached Figure Description

[0022] Figure 1 The effect of PA on NO release from RAW 264.7 cells in Example 2;

[0023] Figure 2 The effect of PA on cytokine synthesis in Example 2;

[0024] Figure 3 This refers to the effect of PA on the survival rate of mice with acute lung injury in Example 2.

[0025] Figure 4 The effect of PA on pathological changes in mice with acute lung injury in Example 3 (where A is the gross pathological examination of lung tissue in the acute lung injury model; B is the pathological observation of lung tissue (HE)).

[0026] Figure 5 The NO levels in mouse serum and lung lavage fluid in Example 3 are (where A is the NO level in lung lavage fluid; B is the NO level in mouse serum).

[0027] Figure 6 The effects of PA on the transcriptional levels of cytokines and the protein levels of IL-1β in mouse lung tissue in Example 3 are shown in Figure 3 (where A represents the effect of PA on the transcriptional levels of cytokines in mouse lung tissue; and B represents the effect of PA on the protein levels of IL-1β in lung tissue). Detailed Implementation

[0028] The following embodiments are further illustrations of the present invention, but not the only limitations thereof.

[0029] Example 1: Identification of components in privet fruit

[0030] Experimental techniques: The privet fruit purchased from a Chinese medicine store was ground into powder, extracted with alcohol or alcohol solution, filtered through a microporous membrane, and then analyzed by UPLC-MS / MS.

[0031] Mass spectrometry analysis employed electrospray ionization (ESI) as the ionization method, with the operating temperature controlled at 500℃. In positive ion detection mode, the ion spray voltage was set to 5500V, while in negative ion mode, a voltage of -4500V was used. To obtain optimal ionization efficiency, the gas path system was configured as follows: ion source gas I (GSI) pressure 50 psi, ion source gas II (GSII) pressure 60 psi, and curtain gas (CUR) pressure 25 psi, with the collision-induced ionization intensity adjusted to a high-sensitivity state. During detection, triple quadrupole mass spectrometry was used in multiple reaction monitoring (MRM) mode, with nitrogen at medium pressure purged into the collision chamber as the collision gas. The declustering voltage and collision energy parameters were optimized for different metabolites to ensure optimal ionization efficiency for each target compound. Based on the differences in retention time of metabolites on the chromatographic column, the entire analysis process was divided into multiple time periods. Each time period selectively monitored its corresponding characteristic ion pairs, thereby achieving simultaneous and accurate quantitative analysis of multiple components in complex samples.

[0032] Using the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP) and the Integrative Pharmacology-based Research Platform of Traditional Chinese Medicine (ITCM), compounds (with CAS numbers) in Ligustrum lucidum were searched based on oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18.

[0033] Experimental results: Pomocarboxylic acid (CAS: 13849-91-7), quercetin (CAS: 117-39-5), and neolignan (CAS: 96382-91-1) were identified from privet fruit.

[0034] Example 2: In vitro immunosuppressive effect of PA

[0035] Experimental Methods: RAW 264.7 cells were cultured in complete medium containing 10% FBS and 1% penicillin-drug antibiotics. Cells in good growth condition were collected, centrifuged, and the supernatant was discarded. The cells were resuspended in an appropriate amount of medium and counted. The cell density was adjusted to the desired concentration, and equal volumes of cell suspension were evenly seeded into 6-well or 96-well plates, ensuring a consistent cell count per well. After seeding, the cells were incubated at 37°C with 5% CO2 for 24 hours. The culture medium was then discarded, and complete medium containing the appropriate concentration of the drug was added to each well, mixed well, and the cells were incubated again.

[0036] NO was detected using the Griess method. RAW 264.7 cells in the logarithmic growth phase were passaged, centrifuged, and the supernatant was removed. The cells were then resuspended in fresh culture medium and thoroughly mixed. The cell density was adjusted to 2 × 10⁶ cells / year using a cell counting method. 5Cells were inoculated at a rate of 1 cell / mL, and then the cell suspension was transferred to 6-well plates at a seeding rate of 2 mL per well. The plates were incubated at 37°C and 5% CO2 for 24 hours, after which the culture medium was discarded. The following treatment groups were set up: groups with different concentrations of PA (2.5 μM, 5 μM, 10 μM); a 100 ng / mL lipopolysaccharide (LPS) positive control group; and a PBS blank control group. Each group had three replicates, with each well containing 2 mL. The treated cells were cultured at 37°C and 5% CO2 for another 24 hours. After collecting the cell culture supernatant, 150 μL of the supernatant was transferred to a 96-well plate (three technical replicates per sample), and 130 μL of deionized water was added to each well. Finally, 20 μL of Griess reagent was added to each well, and the reaction was carried out at room temperature in the dark for 30 minutes. The absorbance of each well was measured at 548 nm using a microplate reader, and the nitric oxide (NO) content in the sample was calculated based on the standard curve.

[0037] Real-time quantitative PCR analysis. Specific primers were designed for the target genes (IL-1β, IL-6, TNF-α, and iNOS). The PCR reaction system (20 μL) contained: 10 μL 2×SYBR qPCR Master Mix, 1 μL each of forward and reverse primers (Table 1), 2 μL cDNA template, and 6 μL RNase-free water. The amplification program was set as follows: 95℃ pre-denaturation for 2 minutes; followed by 41 cycles of amplification (95℃ for 5 seconds, 60℃ for 30 seconds). Using GAPDH as an internal control, the relative expression level of each target gene was calculated using the 2[-ΔΔCt] method.

[0038] Experimental results: Compared with the blank control group (NC), the LPS group showed a significant increase in NO content (P < 0.05); compared with the LPS group, the NO content in all PA groups with different concentrations was significantly decreased (P < 0.05). Figure 1 Compared with the control, PA (6.25 μM, 12.5 μM, 25 μM) significantly downregulated the gene expression levels of IL-1β, IL-6, TNF-α, and iNOS (P < 0.05); simultaneously, PA (6.25 μM, 12.5 μM) significantly upregulated the expression levels of IL-10 and TGF-β, but at a concentration of 25 μM, PA significantly inhibited TGF-β expression (P < 0.05), while there was no significant difference in IL-10 expression level (P > 0.05). Figure 2 ).

[0039] Example 3: Efficacy analysis of PA in mice with acute lung injury

[0040] Experimental Methods: BALB / c mice were acclimatized for one week and then randomly divided into a blank control group, a control group (LPS 5 mg / kg), and a PA group (0.4 mg / kg), with six mice in each group. An acute lung injury mouse model was established. Except for the blank control group, all other groups received an intraperitoneal injection (ip) of LPS (5 mg / kg). One hour later, the PA group received an intraperitoneal injection of the corresponding dose of PA. Initial mouse weight was recorded, and mouse weight was recorded every 24 hours, and mouse survival was observed.

[0041] Lung tissues from mice in each group were fixed in 4% paraformaldehyde, dehydrated, cleared, and embedded in paraffin to prepare tissue sections with a thickness of 3-5 μm. After hematoxylin-eosin staining and mounting, the pathological changes of the lung tissues were observed under a microscope.

[0042] The Griess method was used to detect NO levels in mouse serum and mouse bronchoalveolar lavage fluid. Before the experiment, mice were fasted for 6 hours and anesthetized with ether. A capillary tube was gently inserted into the inner orbit of the mouse, allowing blood to flow out naturally. 500-1000 μL of whole blood was collected into an EP tube and allowed to stand at room temperature for 2 hours. The tube was then centrifuged at 4°C and 4000 r / min for 10 minutes. The supernatant was collected and placed on ice for later use. Bronchoalveolar lavage fluid (BALF) was collected: Mice were fixed in a supine position on a sterile operating table. After confirming the depth of anesthesia by the pinch reflex, a longitudinal incision (approximately 1 cm) was made in the midline of the neck. The muscle tissue was bluntly dissected to expose the trachea. A T-shaped incision was made 2-3 mm below the thyroid cartilage, and a 22G indwelling needle (approximately 5 mm deep) was inserted and secured with sutures. Irrigation and recovery: 0.5 mL of pre-cooled PBS was slowly injected initially, followed by gentle chest compressions three times. The lavage fluid was then aspirated. Inject 0.3 mL PBS again, repeating the process three times. Combine the recovered solutions and place them on ice. Each irrigation volume should not exceed 0.5 mL to avoid mechanical damage to lung tissue; maintain a stable negative pressure during aspiration. Sample processing: Centrifuge at 4°C for 10 min, aliquot the supernatant into EP tubes and place on ice. Measure NO levels in serum and BALF strictly according to the instructions of the Thermo Fisher Griess kit (G7921).

[0043] The expression of IL-1β protein in mouse lung tissue was detected by immunoblotting.

[0044] Experimental results: The survival rate of mice in the LPS group was 33.33% on day 1 and 16.67% on day 2, with no change in survival rate in the following days, while the survival rate of mice in the PA group was 100% throughout the day. Figure 3After LPS injection, the body weight of mice decreased significantly, but the weight loss in the PA group was significantly less severe compared to the LPS group (P < 0.05). Furthermore, the LPS group mice exhibited lung congestion, severe inflammation, and lung tissue necrosis, while the lung damage in the PA group mice was significantly alleviated. Figure 4 A). HE staining results also showed that the lung tissue of mice in the LPS group had extensive inflammatory cell infiltration, alveolar collapse, alveolar congestion and edema accompanied by hemorrhage. In contrast, the lung lesions of mice in the PA group were significantly reduced, and lung tissue damage was decreased. Figure 4 B). The results also showed that, compared with the NC group, the NO content in the serum and lung lavage fluid of mice in the LPS group was significantly increased (P < 0.01), while compared with the LPS group, the NO content in the PA group was significantly decreased (P < 0.01). Figure 5 Compared with the control group (NC), the expression levels of IL-1β, IL-6, TNFα, and iNOS in the PA group were significantly downregulated (P < 0.01), while the expression level of IL-10 was significantly upregulated (P < 0.05). Figure 5 ).

[0045] The above examples demonstrate that PA has a good immunosuppressive effect and has potential application value.

[0046]

Claims

1. A novel immunosuppressant, characterized in that, The immunosuppressant is composed of pomodoroic acid, and its molecular formula is... .

2. The novel immunosuppressant according to claim 1, characterized in that, The immunosuppressant poliomyelic acid exerts its immunosuppressive effect by inhibiting the expression of pro-inflammatory factors and promoting the expression of anti-inflammatory factors.

3. A novel immunosuppressant according to claim 2, characterized in that, The pro-inflammatory factors include IL-1β, IL-6, TNF-α, and iNOS, and the anti-inflammatory factors include IL-10.

4. The application of a novel immunosuppressant according to any one of claims 1-3, characterized in that, The application of the immunosuppressant poliomyelic acid in the formulation of drugs for treating inflammatory diseases.

5. The application of the novel immunosuppressant according to any claim 4, characterized in that, The inflammatory diseases include LPS-induced acute lung injury.

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