Derivative of impatiens balsamina plant extract 2-methoxy-1, 4-naphthoquinone and application of derivative in preparation of anti-inflammatory drugs

Abstract

The invention provides a derivative of an impatiens balsamina plant extract 2-methoxy-1, 4-naphthoquinone and an application of the derivative in preparation of an anti-inflammatory drug. Belongs to the technical field of chemical pharmacy. The derivative of 2-methoxy-1, 4-naphthoquinone provided by the invention has a good treatment effect on inflammation, and due to the fact that the derivative of 2-methoxy-1, 4-naphthoquinone is a plant active component, immune disorder, adverse reaction and drug resistance caused by traditional drugs can be avoided. And compared with 2-methoxy-1, 4 naphthoquinone, the 2-methoxy-1, 4 naphthoquinone has the advantages of lower cytotoxicity and higher safety.

Description

Technical Field

[0001] The invention relates to the technical field of chemical pharmacy, and more specifically to a derivative of 2-methoxy-1,4-naphthoquinone of an Impatiens balsamina plant extract and application thereof in the preparation of anti-inflammatory drugs. Background Art

[0002] Inflammation is a complex biological response of vascular tissue to harmful stimuli, such as pathogens, damaged cells, or irritants (chemical), or autoimmunity. Classic manifestations of acute inflammation include pain, heat, redness, swelling, and limited function. Inflammation is a protective response of the organism and also a patterned response, so it is considered to be an innate (intrinsic) immune mechanism, as opposed to adaptive immunity, which varies depending on the specific pathogen. Inflammation can not only help the collective resist harmful stimuli, but also play a role in preventing infection. However, if the inflammation lasts too long, that is, chronic inflammation, it will become the basis for the further development of many diseases, such as trauma, tumors, gastroenteritis, pneumonia, encephalitis, reproductive system inflammation, etc. Therefore, effectively controlling the duration of inflammation and appropriately intervening in the development of inflammation have positive significance for preventing and treating the occurrence of various inflammatory diseases in humans and animals.

[0003] Currently commonly used anti-inflammatory drugs include penicillins, cephalosporins, macrolides, aminoglycosides, tetracyclines, quinolones and sulfonamides, but the above traditional anti-inflammatory drugs may cause immune disorders in the body, accompanied by a series of adverse reactions and microbial resistance to drugs.

[0004] Therefore, how to provide a plant-derived active anti-inflammatory drug with higher safety is a technical problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention provides a derivative of 2-methoxy-1,4-naphthoquinone of Impatiens balsamina plant extract and its use in the preparation of anti-inflammatory drugs, which can effectively regulate the expression of inflammatory factors and have a good therapeutic effect on inflammatory reactions. Since it is a plant active ingredient, it can avoid immune disorders, adverse reactions and drug resistance caused by traditional drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A derivative of 2-methoxy-1,4-naphthoquinone from an Impatiens balsamina plant extract, wherein the general structural formula of the derivative is as shown in formula (I):

[0008]

[0009] Preferably, R in the formula (I) can be selected from the following structures:

[0010]

[0011] Another object of the present invention is to provide the use of the above-mentioned Impatiens balsamina plant extract 2-methoxy-1,4-naphthoquinone derivatives in the preparation of anti-inflammatory drugs.

[0012] Preferably, the anti-inflammatory drug includes a human anti-inflammatory drug or an animal anti-inflammatory drug.

[0013] Another object of the present invention is to provide the use of the above-mentioned Impatiens balsamina plant extract 2-methoxy-1,4-naphthoquinone derivatives in the preparation of drugs for treating inflammation of the animal reproductive system.

[0014] It can be seen from the above technical solution that compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention provides a derivative of 2-methoxy-1,4-naphthoquinone (MNQ) of Impatiens balsamina plant extract, the structure of which is shown in formula (I), which has a good intervention and therapeutic effect on various inflammatory reactions, can effectively regulate the expression levels of various inflammatory factors, is safer than MNQ, and because it is a plant-derived active ingredient, it can avoid immune disorders, adverse reactions and drug resistance caused by traditional drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments or the prior art description are briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on the provided drawings without creative work.

[0017] Figure 1 Figure 1: Analysis of the effects of MNQ and its derivatives on the survival rate of GCs at different concentrations and different action times; AH represent MNQ and its seven derivatives respectively;

[0018] Figure 2 Figure 3: Effects of MNQ and its derivatives on the relative expression of inflammatory factors in GCs. AC represents three inflammatory factors, respectively. DETAILED DESCRIPTION

[0019] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0020] The screening test was conducted with seven synthetic MNQ derivatives, the structural formulas of which are shown in Table 1:

[0021] Table 1 Molecular structures of MNQ and its derivatives

[0022]

[0023]

[0024] In the following examples, we selected cattle as the research subjects and conducted relevant research on the anti-inflammatory effects of MNQ and its derivatives.

[0025] Example 1

[0026] 1. Safety study of MNQ and its derivatives

[0027] (1) Isolation and culture of granulosa cells (GCs):

[0028] Follicles were separated from normal bovine ovarian tissue using pre-sterilized scissors, follicles with a diameter of 4 to 8 mm were selected, and follicle GCs were gently scraped from their inner walls; GCs were collected and washed; and cultured in a medium containing 10% fetal bovine serum at 37°C, saturated humidity, and 5% CO2;

[0029] (2) MTT test

[0030] ① Collect logarithmic phase cells, adjust the concentration of cell suspension, inoculate into 96-well plates, make 5000 cells per well, 100 μL per well, and culture in a 37°C, saturated humidity, 5% CO2 incubator for 24 h;

[0031] ② After the cells are completely attached, the supernatant is aspirated, and 100 μL of complete culture medium containing MNQ and MNQ derivatives with final concentrations of 1, 2, 4, 8, 16, 32, 64, and 128 μM are added (6 replicates are set for each concentration), and a control group (no drug is added) is set up; the culture is continued for 12 h, 24 h, and 48 h, respectively.

[0032] ③ Remove the supernatant, add 90 μL of culture medium to each well, add 10 μL of MTT solution, and continue to culture for 4 h; discard the culture medium, add 110 μL of DMSO to each well to dissolve the crystals; measure the OD value at 490 nm and calculate the cell survival rate ( Figure 1 ).

[0033] Cell survival rate = (OD value of drug group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%

[0034] Results: MNQ and MNQ derivatives (1, 2, 4, 8, 16, 32, 64, 128 μM) were added to GCs for different time periods (12 h, 24 h, 48 h). The survival rate of GCs gradually decreased with the increase of the added concentration; the survival rate of GCs also decreased with the extension of the culture time. Among them, MNQ, A1 and A2 were more toxic to GCs. When the concentration of A1 was above 16 μM, the survival rate of GCs was less than 50%; when the concentration of A2 was above 24 μM, the survival rate of GCs was less than 50%; derivatives D18, D19 and D21 all showed less toxicity to GCs, and even promoted the proliferation of GCs within a certain concentration range (1-8 μM); derivatives C17 and D5 were at an intermediate level of toxicity to GCs.

[0035] 2. Explore the maximum safe concentration of MNQ and its derivatives for GCs at different treatment times

[0036] according to Figure 1 The analysis of the survival rate of CGs at different concentrations and different action times can determine the MNTC (maximum safe concentration, the concentration that can make more than 90% of GCs survive) of MNQ and its derivatives on GCs at different treatment times. The experimental results are shown in Table 2:

[0037] Table 2 MNTC (μM) of MNQ and its derivatives on GCs at different treatment times

[0038]

[0039]

[0040] According to the maximum safe concentration MNTC of MNQ and its derivatives on GCs at different times (12h, 24h, 48h), subsequent experiments were carried out within the MNTC range of MNQ and its derivatives.

[0041] Example 2

[0042] Verification of the anti-inflammatory effects of MNQ and its derivatives

[0043] According to the maximum safe concentration at different times determined in Example 1, we selected 12 hours as the test time, and used the maximum safe concentration of MNQ and its derivatives corresponding to 12 hours as the drug concentration. Cattle infected with reproductive system inflammation were used as research subjects, and MNQ and its different derivatives were respectively administered to conduct anti-inflammatory effect verification tests of MNQ and its derivatives:

[0044] (1) The main pathogenic component of bovine reproductive system inflammation is lipopolysaccharide (LPS), an endotoxin in the cell wall of Gram-negative bacteria. It can destroy the function of the uterus and ovaries, leading to functional disorders of the follicular granulosa cells (GCs) in the ovaries, thereby reducing the fertility of cattle or even causing infertility. LPS can induce inflammatory responses in follicular GCs in vitro. We divided the experiment into ten groups, namely the control group (CK group), LPS treatment group (LPS group), MNQ combined with LPS treatment group (M+L group) and MNQ derivative combined with LPS treatment group (A1~D21+L group). Each group was set up with three replicates (n=3), and the total exposure time of each group was 24 hours. The grouping, treatment method, treatment time and treatment concentration are shown in Table 3:

[0045] Table 3 Grouping of the anti-inflammatory effect verification of MNQ and its derivatives

[0046]

[0047] (2) Real-time qRT-PCR: After the above treatment, total RNA was extracted using the Trizol method and reverse transcribed according to the instructions of the PrimeScriptTMRT reagent Kit with gDNA Eraser kit. According to the predicted sequence of bovine on NCBI, the target gene primers were designed using Primer 5.0 and synthesized by Shanghai Biotechnology Co., Ltd. Bovine β-actin was used as the internal reference gene. The primer sequences and product lengths are shown in Table 4. According to the instructions of the TB GreenTM Premix Ex TaqTMⅡ kit, qRT-PCR reactions were performed using cDNA as a template. 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 CT value analysis results were calculated. MNQ and its derivatives reduced the relative expression of inflammatory factors in GCs ( Figure 2 ).

[0048] Table 4 Amplified sequences of related inflammatory factors and internal reference genes

[0049]

[0050]

[0051] Results: LPS treatment of follicular GCs with a concentration of 10 μg / mL for 12 hours produced a strong inflammatory response, which was manifested by a significant increase in the relative expression of inflammatory cytokines (IL-6, IL-1β and TNF-α) (P<0.01). Follicular GCs were first exposed to 4 μM MNQ for 12 hours, and then exposed to 10 μg / mL LPS for 12 hours (M+L group). Compared with exposure to LPS alone, the relative expression of inflammatory factors was significantly reduced (P<0.05), indicating that MNQ does have an anti-inflammatory effect.

[0052] The seven derivatives of MNQ have very different protective effects on the inflammatory response induced by LPS. As can be seen from the figure, the anti-inflammatory effects of class A derivatives (A1, A2) and class C derivatives (C17) are average, or even non-existent; the anti-inflammatory effects of class D derivatives are better, especially D5, D18, D19 and D21.

[0053] The various embodiments in the specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0054] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A derivative of 2-methoxy-1,4-naphthoquinone of Impatiens balsamina plant extract, characterized in that: The general structural formula of the derivative is shown in formula (I):

2. The derivative of 2-methoxy-1,4-naphthoquinone of Impatiens balsamina plant extract according to claim 1, characterized in that: In the formula (I), R can be selected from the following structures:

3. Use of the derivative of 2-methoxy-1,4-naphthoquinone of the Impatiens balsamina plant extract according to any one of claims 1 or 2 in the preparation of anti-inflammatory drugs.

4. The use according to claim 3, characterized in that: The anti-inflammatory drugs include human anti-inflammatory drugs or animal anti-inflammatory drugs.

5. Use of the derivative of 2-methoxy-1,4-naphthoquinone of the Impatiens balsamina plant extract according to any one of claims 1 or 2 in the preparation of a drug for treating inflammation of the animal reproductive system.

Images