Application of Impatiens balsamina extract in the preparation of antibacterial and anti-inflammatory drugs

Abstract

The present invention provides an application of an Impatiens balsamina extract in the preparation of an antibacterial and anti-inflammatory drug, and relates to the field of biomedicine technology. The present invention provides an application of an Impatiens balsamina extract in the preparation of an antibacterial and anti-inflammatory drug, wherein the Impatiens balsamina extract 2-methoxy-1,4-naphthoquinone (MNQ) is an active substance in the plant, which avoids the disadvantages that traditional anti-inflammatory drugs can cause immune disorders in the body accompanied by adverse reactions and drug resistance of microorganisms to traditional drugs. It has been confirmed by experiments that the Impatiens balsamina extract MNQ has a good inhibitory effect on Staphylococcus aureus, and at the same time has a good protective effect on LPS-induced inflammatory response of bovine ovarian follicle granulosa cells (GCs) cultured in vitro, thereby causing their functional disorders, providing a theoretical basis for effectively treating bovine reproductive tract inflammation and improving the reproductive health of cattle.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to application of an Impatiens balsamina extract in preparing an antibacterial and anti-inflammatory drug. Background Art

[0002] Inflammation of the reproductive tract of dairy cows is one of the major challenges facing contemporary animal husbandry production. Gram-negative bacteria are the main pathogens causing inflammation of the reproductive tract. Bacterial infection can cause diseases such as oophoritis and metritis. In particular, the postpartum uterus is more susceptible to bacterial infection and inflammation. This leads to decreased milk production, delayed ovulation, decreased fertility and even infertility in dairy cows, causing huge economic losses to the animal husbandry industry. Granulosa cells (GCs) in the ovaries are the main functional cells in the formation and development of follicles. They can provide the necessary nutrients for the growth and maturation of oocytes, and they also proliferate and differentiate themselves, while synthesizing and secreting steroid hormones such as estradiol (E2) and progesterone (P4).

[0003] LPS is a lipid component with a special structure in the cell wall of Gram-negative bacteria, which can cause the synthesis and release of a variety of cytokines and inflammatory mediators, thereby triggering a series of inflammatory injuries. Studies have shown that lipopolysaccharide (LPS) from Gram-negative bacteria accumulates in the follicular fluid of animals with genital tract inflammation, leading to ovarian dysfunction. When bovine GCs are cultured in a medium containing LPS, the secretion of estradiol (E2) will decrease, and animals with oophoritis will change the gene expression of follicular granulosa cells and reduce the amount of E2 they produce. Genital tract inflammation can cause functional disorders in GCs. At the same time, there are a large number of methicillin-resistant Staphylococcus aureus (MRSA) in cattle with genital tract inflammation, as well as in humans and other animals. Its pathogenicity is extremely strong, which has brought great pressure to the global health system. Many strains of this species have evolved antibiotic resistance genes, posing a huge challenge to public health. Therefore, it is crucial to eliminate the inflammation of GCs caused by LPS while inhibiting bacteria to ensure public health. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an application of an Impatiens balsamina extract in the preparation of antibacterial and anti-inflammatory drugs, wherein the Impatiens balsamina extract has a good inhibitory effect on Staphylococcus aureus and has a certain degree of protective effect on the LPS-induced GCs inflammatory response.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] The invention provides an application of an Impatiens balsamina extract in inhibiting bacteria. The Impatiens balsamina extract is 2-methoxy-1,4-naphthoquinone.

[0007] Preferably, the bacteria include Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus anthracis, and Salmonella enteritidis.

[0008] The present invention also provides an application of an Impatiens balsamina extract in preparing a medicine for eliminating inflammation of follicular granulosa cells, wherein the Impatiens balsamina extract is 2-methoxy-1,4-naphthoquinone.

[0009] Preferably, the granulosa cell inflammation is an inflammatory response of the granulosa cells induced by LPS.

[0010] Preferably, the concentration of the Impatiens balsamina extract is 1-3 μM.

[0011] The present invention also provides a drug for inhibiting bacteria and reducing inflammation, and the drug comprises 2-methoxy-1,4-naphthoquinone extracted from Impatiens balsamina.

[0012] Preferably, the bacteria include Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus anthracis, and Salmonella enteritidis.

[0013] Preferably, the inflammation is an in vitro inflammatory response of granulosa cells induced by LPS.

[0014] The present invention provides an application of an Impatiens balsamina extract in the preparation of an antibacterial and anti-inflammatory drug. The Impatiens balsamina extract MNQ is an active substance in the plant, which avoids the disadvantages of traditional anti-inflammatory drugs that can cause immune disorders in the body, accompanied by adverse reactions, and drug resistance of microorganisms to traditional drugs. It has been confirmed by experiments that the Impatiens balsamina extract MNQ has a good inhibitory effect on Staphylococcus aureus, and has a good protective effect on LPS-induced inflammatory reactions in bovine ovarian granulosa cells (GCs) cultured in vitro, thereby causing functional disorders, providing a theoretical basis for effectively treating bovine reproductive tract inflammation and improving the reproductive health of cattle. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 These are the results of isolation and culture of follicular granulosa cells.

[0016] Figure 2 In the figure, A shows the effect of different concentrations of MNQ treatment for different time periods (12, 24, 48 h) on the survival rate of GCs; B shows the effect of different concentrations of LPS treatment for different time periods (12, 24, 48 h) on the survival rate of GCs.

[0017] Figure 3 The relative expression results of different genes after GCs were treated with different concentrations of LPS for 12 h; A represents the relative expression of IL-6, B represents the relative expression of IL-1β, and C represents the relative expression of TNF-α.

[0018] Figure 4 The morphological photographs of GCs in the LPS group and the MNQ group after 24 hours of treatment observed under an optical microscope, where Scale bar = 100 μm.

[0019] Figure 5 The relative expression levels of IL-6, IL-1β and TNF-α in GCs treated with LPS group and MNQ group for 24 h, where A is the relative expression level of IL-6, B is the relative expression level of IL-1β, and C is the relative expression level of TNF-α.

[0020] Figure 6 A is the content of E2 in the supernatant of the culture medium of different treatment groups, B is the content of P4 in the supernatant of the culture medium of different treatment groups, C is the relative expression level of steroid synthesis related gene CYP19A1 in different treatment groups, D is the relative expression level of CYP11A1 gene in different treatment groups, E is the relative expression level of 3β-HSD in different treatment groups, and F is the relative expression level of STAR in different treatment groups. DETAILED DESCRIPTION

[0021] The invention provides an application of an Impatiens balsamina extract in inhibiting bacteria. The Impatiens balsamina extract is 2-methoxy-1,4-naphthoquinone.

[0022] The present invention also provides an application of an Impatiens balsamina extract in preparing a medicine for eliminating inflammation of follicular granulosa cells, wherein the Impatiens balsamina extract is 2-methoxy-1,4-naphthoquinone.

[0023] In the present invention, the bacteria are preferably Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus anthracis, and Salmonella enteritidis. In the present invention, the inflammation of follicular granulosa cells is preferably an inflammatory response of follicular granulosa cells induced by LPS; the concentration of LPS is preferably 10 μg / mL. In the present invention, the Impatiens balsamina extract is preferably an extract of Impatiens balsamina stems, and the extract of Impatiens balsamina stems is preferably 2-methoxy-1,4-naphthoquinone (MNQ); the present invention does not specifically limit the source of the 2-methoxy-1,4-naphthoquinone (MNQ), and the extraction can be carried out by the commonly used preparation method in the prior art. The molecular formula of the 2-methoxy-1,4-naphthoquinone is as follows:

[0024]

[0025] In the present invention, the concentration of the Impatiens balsamina extract is preferably 1-3 μM; when the concentration of the Impatiens balsamina extract MNQ is 3 μM, it can effectively eliminate the inflammatory response of GCs induced by LPS in vitro and restore the functional disorder of GCs.

[0026] The present invention also provides a drug for inhibiting bacteria and reducing inflammation, and the drug comprises 2-methoxy-1,4-naphthoquinone extracted from Impatiens balsamina.

[0027] In the present invention, the bacteria are preferably Staphylococcus aureus, Bacillus subtilis, Escherichia coli, Bacillus anthracis, and Salmonella enteritidis; and the inflammation is preferably an in vitro inflammatory response of follicular granulosa cells induced by LPS.

[0028] In the present invention, the superscript "*" indicates that the difference between the LPS group and the CK group is significant (P<0.05), and "**" indicates that the difference between the LPS group and the CK group is extremely significant (P<0.01); "#" indicates that the difference between the MNQ group and the LPS group is significant (P<0.05), and "##" indicates that the difference between the MNQ group and the LPS group is extremely significant (P<0.01).

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0030] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0032] Example 1

[0033] Isolation and culture of granulosa cells

[0034] The ovaries of sexually mature and healthy Holstein cows were collected from the slaughterhouse, and the follicles were cut off with sterile surgical instruments. Follicles of appropriate size were selected, cut open, and the follicle granulosa cells were scraped and collected into complete culture medium. The follicle granulosa cells were cultured in vitro at 37°C and 5% CO2 until the follicle granulosa cells covered 50-60% of the culture dish. During the culture period, fresh complete culture medium was replaced every 24 hours. The final follicle granulosa cells were placed under a microscope and observed. Figure 1 .

[0035] Example 2

[0036] MTT assay

[0037] The logarithmic phase GCs cells were collected, their concentration was adjusted, and they were seeded into 96-well plates, with 5000 cells per well, 100 μL per well, and cultured in a 37° C., 5% CO 2 incubator for 24 h.

[0038] After the cells were completely attached, the supernatant was removed, and 100 μL of complete culture medium containing MNQ at final concentrations of 1 μM, 1.5 μM, 3 μM, 6 μM, 12 μM, 25 μM, and 50 μM was added (6 replicates were set for each concentration), and a control group (no drug was added) was set up. The culture was continued for 12 h, 24 h, and 48 h, respectively.

[0039] Take another 96-well plate, add 100 μL of complete culture medium containing LPS at final concentrations of 0.01 μg / mL, 0.1 μg / mL, 1 μg / mL, 10 μg / mL, and 100 μg / mL (set 6 replicates for each concentration), and set up a control group (no drug added). Continue culturing for 12 h, 24 h, and 48 h, respectively.

[0040] The culture medium was discarded, and 110 μL of Formazan dissolving solution (i.e., dimethyl sulfoxide) was added to each well, and the cells were shaken at a low speed for 10 to 15 minutes to fully dissolve the crystals. The cells were shaken for 15 seconds on an enzyme-linked immunosorbent assay (Thermo Fisher FC), and the OD value was measured at 490 nm to calculate the cell survival rate.

[0041] Cell survival rate = (OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0042] It can be seen that the survival rate of GCs is related to the concentration of MNQ and the treatment time. As the concentration of MNQ increases, the survival rate of GCs gradually decreases. Within the experimental time range, as the time increases, the survival rate of GCs decreases to a certain extent. However, when the concentration of MNQ is less than 3μM and the treatment time is 12h, the survival rate of GCs is above 90%, which has almost no effect on the activity of GCs ( Figure 2 A). Therefore, the MNQ concentration in the subsequent group tests was 3 μM and the GCs treatment time was 12 h.

[0043] When the concentration of LPS was less than 10 μg / mL, it had little effect on the survival rate of GCs. However, when the concentration of LPS reached 100 μg / mL, the survival rate of GCs decreased significantly. Different treatment times had little effect on the survival rate of GCs ( Figure 2 B). Therefore, the subsequent LPS concentration was selected to be less than 10 μg / mL and the treatment time was 12 h.

[0044] Example 3

[0045] Real-time qRT-PCR

[0046] The follicle granulosa cells treated as described in Example 2 were taken, and total RNA was extracted using the Trizol method, and then reverse transcription was performed according to the instructions of the PrimeScriptTMRT reagent Kit with gDNAEraser kit. The reaction conditions were 37°C, 15min; 85°C, 5s; and then placed on ice. After the reaction was completed, the product was placed in a -20°C refrigerator for standby use.

[0047] Based on the predicted sequence of bovine on NCBI, primers of target genes were designed using Primer 5.0 and Oligo 6 software (Table 1) and synthesized by Shanghai Biotechnology Co., Ltd. Bovine β-actin was used as the internal reference gene.

[0048] Table 1 Target gene primers

[0049]

[0050] According to the instructions of TB GreenTM Premix Ex TaqTM II kit, cDNA was used as template to construct a 10μL qRT-PCR reaction system: 0.4μL (10μmol / L) of PCR upstream and downstream primers, 5μL of TB Green Premix Ex Taq II, 2μL of cDNA template, and 2.2μL of RNase Free dH2O. The reaction conditions were 95℃, 30s; 95℃, 5s; 60℃, 30s, for a total of 45 cycles. After amplification, amplification curves and melting curves were generated, and the results were analyzed using CT values.

[0051] After GCs were treated with different concentrations of LPS (0.01, 0.1, 1, 10 μg / mL) for 12 h, the relative expression levels of IL-6, IL-1β, and TNF-α gradually increased with the increase of concentration ( Figure 3 ).

[0052] Using a microscope to observe the cell morphology, we can see that there are obvious changes in the cell morphology, such as blurred cell membrane boundaries, severe cell differentiation, and pseudopodia. Figure 4 A and B). The LPS concentration of 10 μg / mL was selected for subsequent group experiments.

[0053] Example 4

[0054] Trial Groups

[0055] According to the test results obtained in Examples 1-3, the test was divided into three groups: CK group: control group; LPS group: the cells were treated the same as the CK group for the first 12 hours, and the cells were treated with LPS at a concentration of 10 μg / mL for the next 12 hours; MNQ group: the cells were treated with MNQ at a concentration of 3 μM for the first 12 hours, and the cells were treated with LPS at a concentration of 10 μg / mL for the next 12 hours. Three replicates (1, 2, 3) were set for each group, and the total action time was 24 hours.

[0056] Using a microscope to observe the cell morphology, we can see that compared with the CK group, the morphology of GCs in the LPS group changed significantly, but the cell morphology in the MNQ group basically returned to its original state ( Figure 4 B and C).

[0057] The expression levels of IL-6, IL-1β and TNF-α were determined by the Real-time qRT-PCR method described in Example 3. Figure 5 . It can be seen that the expression levels of inflammatory factors IL-6, IL-1β and TNF-α in the LPS group were significantly increased compared with the CK group (P<0.01), but the expression levels of inflammatory factors IL-6, IL-1β and TNF-α in the MNQ group were significantly decreased compared with the LPS group (P<0.01). Figure 5 ) experiments showed that MNQ at a concentration of 3 μM could effectively inhibit the inflammatory response induced by LPS.

[0058] Example 5

[0059] ELISA experiment

[0060] The concentration of E2 and P4 in the supernatant of each culture solution was detected using an ELISA kit (purchased from Wuhan Boster Bioengineering Co., Ltd.). The cell culture medium was collected and the supernatant was taken after centrifugation. The samples to be tested and the standard were added to the 96-well ELISA plate, reacted at 37°C for 30 minutes, and then the enzyme label solution was added to the test wells and standard wells after washing, reacted at 37°C for 30 minutes, and washed again. After mixing equal volumes of color developer A and color developer B, 100 μL was added to each well, and the color was developed at 37°C in the dark for 10 minutes. Stop solution was added to the color developer, and the OD value of each well at a wavelength of 450 nm was detected using an ELISA reader.

[0061] LPS inhibited the secretion of E2 and P4 by GCs, especially the secretion of P4 (P<0.01). MNQ had a certain degree of protective effect on the inhibition of GCs secretion of steroids by LPS ( Figure 6 A, B).

[0062] Corresponding to the above results, the expression levels of the E2 synthesis-related gene CYP19A1 and the P4 synthesis-related gene CYP11A1 showed a trend of significant decrease in the LPS group and significant increase in the MNQ group ( Figure 6 C, D). Steroid synthesis-related genes 3β-HSD and STAR also showed the same trend of change ( Figure 6 E, F). These results indicate that MNQ has a certain degree of protective effect on the disorder of steroid secretion in GCs induced by LPS.

[0063] Example 6

[0064] Antibacterial effect of MNQ

[0065] The minimum inhibitory concentration (MIC) of MNQ against bacteria was determined by agar dilution method. The specific steps are as follows:

[0066] Agar liquid with a final concentration of MNQ of 1, 2, 4, 8, 16, 32, 64, and 128 mg / L was prepared to prepare plates containing different concentration gradients of antimicrobial drugs, inoculated with bacteria, and incubated at 35°C for 16-20 hours to observe bacterial growth. Compared with the control group, the lowest drug concentration that inhibited more than 80% of bacterial growth was taken as the endpoint concentration MIC.

[0067] The test results show that the MIC of MNQ against Staphylococcus aureus is 4-8mg / L, the MIC against Bacillus subtilis is 2-4mg / L, and the MIC against Escherichia coli, Bacillus anthracis, and Salmonella enteritidis is 64-128mg / L. The results show that MNQ has a good antibacterial effect on different bacteria, especially for the inhibition of Staphylococcus aureus, which is beneficial to avoid various inflammations caused by Staphylococcus aureus.

[0068] The above descriptions are merely embodiments of the present invention and are not intended to limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made using the contents of the present invention specification, or directly or indirectly applied in other related technical fields, are also included in the patent protection scope of the present invention.

Claims

1. Use of an Impatiens balsamina extract in the preparation of a drug for eliminating inflammation of follicular granulosa cells, characterized in that: The Impatiens balsamina extract is 2-methoxy-1,4-naphthoquinone; the follicular granulosa cell inflammation is an inflammatory response of follicular granulosa cells induced by LPS; and the concentration of the Impatiens balsamina extract is 3 μM.

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