CN114209812B - Application of alpha-Momordica charantia extract in preparation of anti-inflammatory drugs - Google Patents

Abstract

The invention provides an application of alpha-Momordica charantia extract in preparation of anti-inflammatory drugs, and belongs to the technical field of medicines. The amino acid sequence of the alpha-Momordica charantia essence is shown in SEQ ID NO. 1. The research of the invention discovers that the alpha-balsam pear essence has excellent anti-inflammatory activity, can be used for preparing anti-inflammatory drugs, such as drugs for treating pneumonia, and has excellent application prospect.

Description

Application of alpha-Momordica charantia extract in preparation of anti-inflammatory drugs

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of alpha-Momordica charantia in preparation of anti-inflammatory medicines.

Background

Cytokine storm is a pathological immune phenomenon which has been attracting attention in recent years, and refers to a pathological process in which body immune cells, particularly macrophages, are over-expressed with proinflammatory cytokines and cause serious inflammatory reactions after being infected and activated by bacteria or viruses. When the anti-inflammatory immune response of the body cannot weaken the inflammation trend, serious pathological damage of organs at an infection part is caused, and multi-organ failure is induced to cause death of patients. Such as coronavirus pneumonia (covd-19), the rapidly increasing cytokines trigger inflammatory storms, which are important causes of Acute Respiratory Distress Syndrome (ARDS) and respiratory failure in infected individuals. In addition to acute infections, cytokine storms are also common endpoints for autoimmune diseases such as rheumatoid and systemic lupus erythematosus, and for diseases such as tumors and aids. Thus, therapeutic measures to inhibit "cytokine storm" are key to reducing the excessive inflammatory response of the body. The anti-inflammatory drug can inhibit inflammatory reaction and relieve corresponding symptoms of patients.

Alpha-Momordica grosvenori (Alpha-Momordica grosvenori) is a type I ribosome inactivating protein isolated from Momordica charantia seeds. alpha-Momordica charantia extract is not known at present as an anti-inflammatory drug.

Disclosure of Invention

The invention aims to provide an application of alpha-Momordica charantia in preparing anti-inflammatory drugs.

The invention provides an application of alpha-Momordica charantia extract in preparing anti-inflammatory drugs.

Further, the amino acid sequence of the alpha-Momordica charantia essence is shown in SEQ ID NO. 1.

Further, the drug is a drug that inhibits the proliferation activity of THP-1 monocytes and/or macrophages.

Further, the medicine is a medicine for treating pneumonia.

Further, the medicine is a preparation prepared by taking alpha-balsam pear essence as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Further, the preparation is an external preparation, an oral preparation or an injection preparation.

Further, the formulation is an injectable formulation.

The invention also provides a medicine which is a preparation prepared by taking alpha-balsam pear element as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

Further, the preparation is an external preparation, an oral preparation or an injection preparation.

Further, the formulation is an injectable formulation.

The research of the invention discovers that the alpha-balsam pear essence has excellent anti-inflammatory activity, can be used for preparing anti-inflammatory drugs, such as drugs for treating pneumonia, and has excellent application prospect.

It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.

Drawings

FIG. 1 is a three-level structure of an alpha-MMC and a quality test result of an alpha-MMC protein sample; wherein A is a three-level structure schematic diagram of alpha-MMC; b is an HPLC chromatogram of an alpha-MMC protein sample, and the purity is higher than 97% through analysis and verification; c is the uniformity of alpha-MMC by natural PAGE; d is SDS-PAGE analysis of alpha-MMC purity and relative size, lane1 is protein molecular weight Marker; lanes 2, 3, 4 and 5 are four consecutive batches of a-MMC samples having a molecular weight of about 28kDa.

FIG. 2 shows the results of determining LRP1 receptor density by Western blotting and CCK-8 assay; wherein A is the Western blot analysis result of LRP1 receptor protein density in normal WIL2-S B cells, normal H9T cells, THP-1 monocytes and M0 macrophages; b is CCK-8 assay survival of four cell lines after treatment with a dose of α -MMC of 0 μg/ml,0.5 μg/ml,1 μg/ml,2 μg/ml,4 μg/ml,8 μg/ml,16 μg/ml,32 μg/ml and 64 μg/ml; the data shown are mean ± single values of SEM, n=5; * P <0.05 was significantly different from the control group; significant differences were found between the group with <0.05 > and 0 μg/ml α -MMC; no significant difference from the control; one-way analysis of variance, multiple comparison test of Tukey.

FIG. 3 is a histopathological evaluation (H & E staining) of LPS-induced Acute Lung Injury (ALI) in mice; wherein, A is a negative control group (sham group); b is LPS induction model group; c is LPS and dexamethasone treatment group; d is LPS and alpha-MMC treated group (0.1 mg/kg); e is LPS and alpha-MMC treated group (0.3 mg/kg); f is lung injury score; the data shown are mean ± single values of SEM, n=8; * P <0.05 was significantly different from the sham surgery group; significant differences were observed between #p <0.05 and LPS-induced model groups; no significant difference from the control; one-way analysis of variance, multiple comparison test of Tukey; scale bar, 200 μm for 5-fold zoom level image and 10 μm for 10-fold zoom level image.

Detailed Description

Unless otherwise indicated, the materials and equipment used in the embodiments of the present invention are all known products and are obtained by purchasing commercially available products.

EXAMPLE 1 preparation of alpha-Momordica Charantia extract

The preparation method of the alpha-Momordica charantia essence (alpha-MMC) comprises the following steps: the peeled, dried mature balsam pear seeds were ground in cold 50mM HAC-NaAC buffer (pH 5.0) and homogenized. The resulting slurry was stirred at 48℃for 12 hours and then centrifuged at 15000g at 48℃for 30 minutes. After filtration through cheesecloth, the supernatant was separated by precipitation with an ammonium sulfate solution (30-60% strength). The precipitate was dissolved in water and dialyzed against 50mM sodium phosphate solution (pH 7.0) at 48℃for 12 hours. The dialyzed sample was passed through an SP-Sepharose FF column equilibrated with dialysis buffer. The retained protein was eluted with a 50mM sodium phosphate solution of 0.1M NaCl, pH7.0, flow rate 1ml/min. Fractions were monitored by UV detector at 280nm and analyzed by 12% SDS-PAGE. Fractions containing significant 30kDa were collected and concentrated appropriately. The concentrated sample was further applied to a Superdex75 column and eluted with 50mM sodium phosphate (pH 7.0) containing 0.15M NaCl at a flow rate of 1.5 ml/min. Fractions containing ribosome inactivating protein analyzed by 12% SDS-PAGE were pooled, concentrated and then dialyzed against 50mM sodium phosphate solution, pH 7.0. The concentrated ribosome inactivating protein passes through a 0.22mm filter membrane to obtain alpha-balsam pear essence, and the alpha-balsam pear essence is preserved at 4-8 ℃ for standby.

The amino acid sequence of the alpha-balsam pear is shown as SEQ ID NO. 1:

MSRFSVLSFLILAIFLGGSIVKGDVSFRLSGADPRSYGMFIKDLRNALPFREKVYNIPLLLPSVSGAGRYLLMHLFNYDGKTITVAVDVTNVYIMGYLADTTSYFFNEPAAELASQYVFRDARRKITLPYSGNYERLQIAAGKPREKIPIGLPALDSAISTLLHYDSTAAAGALLVLIQTTAEAARFKYIEQQIQERAYRDEVPSLATISLENSWSGLSKQIQLAQGNNGIFRTPIVLVDNKGNRVQITNVTSKVVTSNIQLLLNTRNIAEGDNGDVSTTHGFSSY(SEQ ID NO.1)

EXAMPLE 2 anti-inflammatory Activity study of alpha-Momordica charantia

1. Experimental method

Cell assay

1. Cell culture

The human monocyte THP-1 cell line, the human normal B lymphocyte line WIL2-S and the human normal T lymphocyte line H9 were purchased from the China academy of sciences typical culture protection Committee cell bank. They were grown in complete RPMI-1640 medium containing 10% heat-inactivated fetal bovine serum, 100U/ml penicillin and 100. Mu.g/ml streptomycin (Thermo USA). Maintaining the cells at 37deg.C with 5% CO ₂ Is a wet air. Suspension culture, liquid exchange and passage in conventional method. PMA-induced macrophages (M0 type): will be 1X 10 ⁶ And (3) inoculating the THP-1 cells into a 6-hole plate, adding PMA (30 ng/ml) for culturing for 72 hours, completely washing floating cells by changing liquid, and adding fresh culture medium (without PMA) for culturing for 24 hours to obtain the compound feed.

2. CCK8 analysis

The conventional in vitro cultured logarithmic growth phase B cell strain WIL2-S, T cell strain H9 cells, monocyte strain THP-1 and M0 type macrophages were inoculated into 96-well plates in an amount of 12000 cells/well, and alpha-MMC was added to each well cell at a dose of 0. Mu.g/ml, 0.5. Mu.g/ml, 1. Mu.g/ml, 2. Mu.g/ml, 4. Mu.g/ml, 8. Mu.g/ml, 16. Mu.g/ml, 32. Mu.g/ml and 64. Mu.g/ml, respectively, and 3 parallel multiplex wells were made per dose group for each cell strain. After incubation for 24h 20. Mu.l of CCK8 solution was added to each well, incubated for 4h at 37℃and 20. Mu.l of stop solution was added to each well, and the OD450 of each well was measured by an ELISA reader.

3. Western blot analysis (Western blotting analysis) LRP1 receptor Density

WIL2-S, H9, THP-1 and M0 type macrophages in the logarithmic growth phase are inoculated into a six-hole cell culture plate, 5 parallel compound holes are arranged, and the cells are harvested when the cells grow until the fusion degree reaches 75%. The cell samples from each well were added to freshly prepared RIPA lysate, lysed on ice for 30min, sonicated in an ice bath for 3min, and centrifuged at 12000rpm for 10min at 4℃and the supernatant transferred to a fresh EP tube. The BCA method measures the protein concentration by adding 5 x SDS loading bufer according to the volume of the lysate and boiling at 100 ℃ for 5min. Protein samples (20. Mu.g) were separated on 10% SDS polyacrylamide gel and 5% SDS concentrate gel. Samples were transferred electrically to PVDF and blocked with 5% mill/TBST. After washing, the primary anti-cotranslative PVDF diluted with 1% BSA/PBST was placed in a hybridization bag, refrigerator overnight at 4 ℃. The primary antibody is rabbitAnti-human LRP1 antibodies (Abcam, ab92544, US) and mouse anti-human beta-action antibodies (Proteintech, 60008-1-Ig) were diluted in 1:1000 and 1:5000, respectively. The PVDF membranes were then placed in goat anti-rabbit secondary antibodies (Wohan three eagles, 1:5000) and goat anti-mouse secondary antibodies (Wohan three eagles, 1:5000) respectively, each labeled with horseradish peroxidase diluted with 5% mill/PBST, and incubated for 1h at room temperature in a shaker. The chromogenic substrates Enhanced Luminol Reagent and Oxidizing Reagent (ECL Millipore, USA) were used. Using a chemiluminescent gel imager (ChemiDoc) ^TM XRS+, bio-Rad, USA) and rabbit anti-human β -actin antibodies (Novus, USA) were selected as reference antibodies. Image J software was used to scan each band of gray values and calculate the ratio to beta-actin.

(II) animal experiments

1. Experimental animal

40 SPF-grade Balb/c mice (Male, 6-8 w) were supplied by the institute of laboratory animals, academy of sciences of Sichuan medicine. All animal studies were conducted according to the institutional animal care guidelines and according to protocols approved by the institutional ethics committee of the institutional. Animals were housed in SPF animal rooms maintained at 22-25℃for 12 hours of light and dark cycles and 50-70% relative humidity. Fresh water and standardized foods were provided and acclimatized for at least 7 days prior to starting the study.

2. Injury induction of Acute Lung Injury (ALI) in mice

Animals were randomly divided into 5 groups (n=8 per group): (1) Normal mouse control group (Normal sample, NS), (2) LPS model group (8.0 mg/kg), (3) LPS (8.0 mg/kg) +dexamethasone treatment group (3.0 mg/kg), (4) LPS (8.0 mg/kg) +α -MMC treatment group (0.3 mg/kg), (5) LPS (8.0 mg/kg) +α -MMC treatment group (0.1 mg/kg). The mice were weighed and anesthetized with 1% sodium pentobarbital (50 mg/kg), 50 μl of 3.2mg/ml LPS (normal control group mice normal saline) was taken with a microsyringe, and the mice were lifted up and shaken left and right after intratracheal injection to homogenize the pulmonary drug and returned to the cage after the mice breathed normally, enhancing care. The mice in the treatment group are respectively injected with dexamethasone or alpha-MMC medicine in the abdominal cavity 1h after LPS injection, and the mice in the model group and the normal control group are injected with the physiological saline in the same volume in the abdominal cavity. After 24h of the 1 st moulding, the 2 nd LPS moulding and the administration of each group of mice were performed in exactly the same way as the 1 st. Animals were sacrificed 24h after the second molding and administration, and the left lung was fixed with paraformaldehyde for paraffin embedding.

3. Histopathological evaluation of ALI

The left lung was used for microscopic analysis of pathological changes. It was fixed in 4% buffered formalin for 24 hours, dehydrated, paraffin embedded, sectioned and stained with hematoxylin and eosin for analysis by Olympus BX53 microscope (Olympus, tokyo, japan). From each slide, five fields containing 400 alveoli (×200 times) were selected, and the extent of lung injury was assessed by a researcher unaware of the treatment method. The severity of lung inflammation was semi-quantitatively assessed. Briefly, the grade of lung inflammation was scored on a zero to five scale. The lung injury grading criteria were as follows: level 0, normal organization; grade 1, slight inflammatory changes; grade 2, mild to moderate inflammatory changes (no apparent damage to lung structure); grade 3, moderate inflammatory injury (thickening of alveolar spaces); grade 4, moderate to severe inflammatory lesions (forming nodular or pneumonia areas that distort normal structures); grade 5, severe inflammatory injury, complete disappearance of visual field. The images were collected and processed using caseviewer2.4 software.

(III) data and statistical analysis

Data analysis was performed in a blind manner. Data are expressed as mean ± SD unless otherwise indicated. The animal experiment sample size is n=8 for each group, and the cell CCK8 experiment is repeated 6 times, and at least 18 measures are included. Unidirectional analysis of variance was performed using GraphPad Prism 5.0 (GraphPad Prism, RRID: scr_ 002798), with a value of P <0.05 considered significant at a 95% confidence level and considered statistically significant. The data and statistical analysis are in accordance with the recommendations of the journal of pharmacology in the uk for pharmacological experimental design and analysis.

2. Experimental results

1. alpha-Momordica charantia essence sample identification

The three-level structure of the alpha-MMC is schematically shown in FIG. 1A, the HPLC purity of the alpha-MMC is >97% (FIG. 1B), the homogeneity of the alpha-MMC is analyzed by natural PAGE, the purity and the relative size are analyzed by SDS, and the SDS-PAGE is schematically shown in FIG. 1C and FIG. 1D, and the molecular weight of the alpha-MMC is about 28kDa.

2. Selective cytotoxicity of alpha-MMC on mononuclear-macrophages

Western blot analysis of LRP1 receptor protein density in normal WIL2-S B cells, normal H9T cells, THP-1 monocytes and M0 macrophages is shown in FIG. 2A. WIL2-S and H9 cells are in light and fuzzy bands, while THP-1 cells and M0 macrophages are in deep and dense bands; the relative densities of LRP 1/beta-actin values (average of 5 tests) for THP-1 cells and M0 macrophages were significantly different from those of WIL2-S and H9 cells.

The results of the viability of α -MMC for these four cells at doses of 0 μg/ml,0.5 μg/ml,1 μg/ml,2 μg/ml,4 μg/ml,8 μg/ml,16 μg/ml,32 μg/ml and 64 μg/ml are shown in FIG. 2B. The results show that the cytotoxicity of alpha-MMC on the cells of the normal B cell line WIL2-S and the T cell line H9 is significantly lower than that of the monocyte line THP-1 and M0 type macrophages. At doses below 8 μg/ml, no significant cytotoxicity could be found; above 16 μg/ml, α -MMC showed cytotoxicity to mononuclear-macrophages; at doses of 32. Mu.g/ml and 64. Mu.g/ml, the α -MMC cytotoxicity to THP-1 and M0 cells was more pronounced (cell viability < 90%) and the differences were significant (P < 0.05) compared to WIL2-S cells and H9 cells. The cytotoxic effect of alpha-MMC on THP-1 and M0 macrophages was much higher than for WIL2-S and H9 cells, and this difference in cytotoxicity was consistent with the difference in distribution density of the LRP1 receptor proteins in the four cells.

3. Anti-inflammatory effect of alpha-MMC on LPS-induced mouse pneumonia model

The results of the lung histopathological analysis (H & E staining) of each group of mice are shown in fig. 3. In fig. 3A, the lung tissue of the negative control (sham) mice showed intact alveolar structure, and the cavities were separated by a monolayer of alveolar epithelial cells, transparent and clear. Whereas in fig. 3B, severe pneumonia appears in the lung tissue of mice in the LPS-induced model group: alveolar space is reduced, alveolar space is obviously thickened, and inflammatory cells such as neutrophils, macrophages and the like which infiltrate in a large quantity and diffuse erythrocytes are present in a large quantity. Some exudates were also observed in the alveolar spaces. The microscopic damage severity index was 3.44±0.56, which was significantly different from the sham surgery group (0.13±0.23) (fig. 3F). In the LPS and dexamethasone treated group, only mild inflammation, such as alveolar wall thickening and inflammatory cell infiltration (fig. 3C), was observed, with a microscopic injury index of 1.64±0.85. In the LPS and α -MMC treated groups, various degrees of alveolar contractions and deformations, mild thickening of the alveolar compartments, bleeding and inflammatory cell infiltration were observed (fig. 3D and 3E). However, compared with the LPS model group, the two groups have obviously reduced inflammatory lesions and no effusion in the alveolar space. The microscopic damage index was 2.06.+ -. 0.76 (0.1 mg/kg) and 1.71.+ -. 0.81 (0.3 mg/kg), as shown in FIG. 3F. Experimental results show that the alpha-Momordica charantia extract has anti-inflammatory effect and can be used for treating pneumonia.

In conclusion, the research of the invention discovers that the alpha-balsam pear essence has excellent anti-inflammatory activity, can be used for preparing anti-inflammatory drugs, such as drugs for treating pneumonia, and has excellent application prospect.

SEQUENCE LISTING

<110> Chengdu medical college

Application of <120> alpha-Momordica charantia in preparation of anti-inflammatory drugs

<130> GY044-2021P0114346CCR4

<160> 1

<170> PatentIn version 3.5

<210> 1

<211> 286

<212> PRT

<213> artificial sequence

<400> 1

Met Ser Arg Phe Ser Val Leu Ser Phe Leu Ile Leu Ala Ile Phe Leu

1 5 10 15

Gly Gly Ser Ile Val Lys Gly Asp Val Ser Phe Arg Leu Ser Gly Ala

20 25 30

Asp Pro Arg Ser Tyr Gly Met Phe Ile Lys Asp Leu Arg Asn Ala Leu

35 40 45

Pro Phe Arg Glu Lys Val Tyr Asn Ile Pro Leu Leu Leu Pro Ser Val

50 55 60

Ser Gly Ala Gly Arg Tyr Leu Leu Met His Leu Phe Asn Tyr Asp Gly

65 70 75 80

Lys Thr Ile Thr Val Ala Val Asp Val Thr Asn Val Tyr Ile Met Gly

85 90 95

Tyr Leu Ala Asp Thr Thr Ser Tyr Phe Phe Asn Glu Pro Ala Ala Glu

100 105 110

Leu Ala Ser Gln Tyr Val Phe Arg Asp Ala Arg Arg Lys Ile Thr Leu

115 120 125

Pro Tyr Ser Gly Asn Tyr Glu Arg Leu Gln Ile Ala Ala Gly Lys Pro

130 135 140

Arg Glu Lys Ile Pro Ile Gly Leu Pro Ala Leu Asp Ser Ala Ile Ser

145 150 155 160

Thr Leu Leu His Tyr Asp Ser Thr Ala Ala Ala Gly Ala Leu Leu Val

165 170 175

Leu Ile Gln Thr Thr Ala Glu Ala Ala Arg Phe Lys Tyr Ile Glu Gln

180 185 190

Gln Ile Gln Glu Arg Ala Tyr Arg Asp Glu Val Pro Ser Leu Ala Thr

195 200 205

Ile Ser Leu Glu Asn Ser Trp Ser Gly Leu Ser Lys Gln Ile Gln Leu

210 215 220

Ala Gln Gly Asn Asn Gly Ile Phe Arg Thr Pro Ile Val Leu Val Asp

225 230 235 240

Asn Lys Gly Asn Arg Val Gln Ile Thr Asn Val Thr Ser Lys Val Val

245 250 255

Thr Ser Asn Ile Gln Leu Leu Leu Asn Thr Arg Asn Ile Ala Glu Gly

260 265 270

Asp Asn Gly Asp Val Ser Thr Thr His Gly Phe Ser Ser Tyr

275 280 285

Claims (4)

1. The application of alpha-Momordica charantia in preparing anti-inflammatory drugs;

the amino acid sequence of the alpha-balsam pear element is shown as SEQ ID NO. 1;

the anti-inflammatory drug is a drug for treating pneumonia.

2. Use according to claim 1, characterized in that: the medicine is a preparation prepared by taking alpha-balsam pear essence as an active ingredient and adding pharmaceutically acceptable auxiliary materials or auxiliary ingredients.

3. Use according to claim 2, characterized in that: the preparation is an oral preparation and an injection preparation.

4. Use according to claim 3, characterized in that: the preparation is injection preparation.