LU504956B1

LU504956B1 - Radix lithospermi naphthoquinone derivative and use of radix lithospermi naphthoquinone derivative, combined with antibiotic, in preparation of drug for treating bacterial infectious disease

Info

Publication number: LU504956B1
Application number: LU504956A
Authority: LU
Inventors: Xia Xiao
Original assignee: Univ Yangzhou
Priority date: 2023-03-02
Filing date: 2023-08-18
Publication date: 2024-02-20
Also published as: CN115969824A; CN115969824B

Abstract

The present invention discloses a Radix lithospermi naphthoquinone derivative and a use of the Radix lithospermi naphthoquinone derivative, combined with an antibiotic, in the preparation of a drug for treating a bacterial infectious disease. The Radix lithospermi naphthoquinone derivative has a good antibacterial effect on Gram-positive bacteria in vitro and in vivo, may restore the sensitivity of polymyxin or meropenem resistant bacteria, and is one of strategies to achieve antibiotic substitution and solve the problem of bacterial resistance.

Description

RADIX LITHOSPERMI NAPHTHOQUINONE DERIVATIVE AND USE OF RADH#504956

LITHOSPERMI NAPHTHOQUINONE DERIVATIVE, COMBINED WITH ANTIBIOTIC,

IN PREPARATION OF DRUG FOR TREATING BACTERIAL INFECTIOUS DISEASE

TECHNICAL FIELD

The present invention relates to a novel pharmaceutical use of a Radix lithospermi naphthoquinone derivative, and in particular to a Radix lithospermi naphthoquinone derivative and a use of the Radix lithospermi naphthoquinone derivative, combined with an antibiotic, in the preparation of a drug for treating a bacterial infectious disease.

BACKGROUND

In recent years, with the wide application of antibacterial drugs, the problem of drug resistance has become increasingly prominent. The rapid emergence and rapid spread of multidrug resistant bacteria have made the global public health system face a major threat. More seriously, due to a long development time, a high cost, a low return on investment and the like of a novel antibacterial drug, a development pipeline for the novel antibacterial drug has been depleted since the late 1990s. A development speed of the novel antibacterial drug has lagged far behind that of the drug resistance, making human beings enter the post-antibiotic era. Based on the concept of “one health”, a series of action plans for “reducing resistance and limiting resistance” have been implemented all over the world. In the context of the rapid development of the drug resistance, the “post-antibiotic era” with a development shortage of a novel antibiotic and “reducing resistance and limiting resistance”, there is an urgent need for a novel antibacterial strategy to cope with the growing antibiotic crisis.

Plants occupy the largest biomass on the earth, and have evolved many secondary metabolites similar to drug functions to respond to infection. It has been reported that from 1981 to 2010, about 65% of approved drugs were either natural compounds or their semi-synthetic derivatives.

By 2018, FDA has received more than 800 research or pre-conference applications for botanical drugs, and approved two novel botanical drug applications (tea polyphenol and Fulyzaq). 4504956 indicates that a botanical small molecule is a promising source of an antibacterial lead compound.

Moreover, definite effects, diversified structures, rich sources and safe effects of the natural compounds suggest that finding small molecular natural compounds with the “antibacterial” or “synergistic” activity from plants, and combining them with existing important antibacterial drugs to improve their antibacterial effects and restore their sensitivity to multidrug resistant pathogens are important strategies to solve the problem of bacterial resistance at present.

The main components of Radix lithospermi may be classified into two categories: one is fatty acids; and the other is naphthoquinone and its derivatives such as shikonin, acetylshikonin (ASK), deoxyshikonin and isobutyrylshikonin. The antibacterial activity of the Radix lithospermi naphthoquinone derivative is still unknown. Whether there is a synergistic effect between the

Radix lithospermi naphthoquinone derivative and other antibiotics remains to be further explained.

SUMMARY

Invention objective: an objective of the present invention is to provide a use of a Radix lithospermi naphthoquinone derivative in the preparation of a drug for treating a bacterial infectious disease.

An objective of the present invention is to provide a Radix lithospermi naphthoquinone derivative and a use of the Radix lithospermi naphthoquinone derivative, combined with an antibiotic, in the preparation of a drug for treating a bacterial infectious disease.

Technical solution: the present invention provides the use of the Radix lithospermi naphthoquinone derivative in the preparation of the drug for treating the bacterial infectious disease.

Further, the Radix lithospermi naphthoquinone derivative includes deoxyshikonin, shikonin, acetylshikonin, B, B-dimethylacrylshikonin and B-hydroxyisovalerylshikonin. Its structure is as follows:

En ve a i 2e = Da His a = ; > i € 5 15 De 3 fl a | SA $ à 8 3 it i tt 1 Pa 9 BE OH) Coa 8 Ey ENE Nee

I ah CREA RS TY Pa a yy

T1 ET PET ss yo *; ye + 3 1 i i 8 £ 8 ER > : DR Oo Sine a

Shikonin Acetyl afhannin Besxy<hihouit

Lx HR + ass 2 oo ENE

TOR Tw “ip froma ous 38 ti PT a ET

BW, SET pr

Fad ie à | Bye um É han Aa &

Sani ee TT © rs NT >

Brin-Hydrosyhovalerylshikonin Reta beta-Dimethylacrylshibonin

Further, bacteria are Gram-positive bacteria, which include Staphylococcus aureus,

Enterococcus faecalis, E. faecium, Staphylococcus epidermidis and the like.

A use of the Radix lithospermi naphthoquinone derivative, combined with an antibiotic, in the preparation of a drug for treating a disease caused by infection with multidrug resistant

Gram-negative bacteria.

Further, the Radix lithospermi naphthoquinone derivative includes the deoxyshikonin, the shikonin, the acetylshikonin, the B, B-dimethylacrylshikonin and the

P-hydroxyisovalerylshikonin.

Further, the multidrug resistant Gram-negative bacteria resist polymyxin (COL) or meropenem (COL).

Beneficial effect: compared with the prior art, the present invention has the following advantages: the present invention provides the use of the Radix lithospermi naphthoquinone derivative as the antibacterial drug. The Radix lithospermi naphthoquinone derivative has a good antibacterial effect on the Gram-positive bacteria in vitro and in vivo, may restore the sensitivity of polymyxin or meropenem resistant bacteria, and is one of strategies to achieve antibiotic substitution and solve the problem of the bacterial resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to describe the technical solutions in the embodiments of the present invention or the prior art more clearly, the following briefly introduces the accompanying drawings required for describing the embodiments or the prior art. Apparently, those of ordinary skill in the art may still derive other drawings from these drawings without any creative efforts.

FIG. 1 1s a diagram showing a result of acetylshikonin rapidly killing Staphylococcus aureus,

FIG. 2 is a diagram showing results of acetylshikonin being capable of enhancing killing activities of polymyxin and meropenem against drug resistant Escherichia coli;

FIG. 3 is a diagram showing results of acetylshikonin having or not having weak hemolytic activity to red blood cells;

FIG. 4 is a diagram showing a result of acetylshikonin significantly increasing a survival rate of larvae of Galleria mellonella;

FIG. 5 is a diagram showing results of acetylshikonin accelerating healing of a wound infection model and significantly reducing a bacterial load;

FIG. 6 is a diagram showing results of acetylshikonin destroying a morphology of

Staphylococcus aureus; and

FIG. 7 is a diagram showing results of acetylshikonin destroying a cell membrane of

Staphylococcus aureus, dissipating a membrane potential and lowering an intracellular ATP level.

DETAILED DESCRIPTION

1. Antimicrobial spectrum of Radix lithospermi naphthoquinone derivative and determination on minimal inhibitory concentration

The antibacterial activity of a Radix lithospermi naphthoquinone derivative was determined by a broth microdilution method. The Radix lithospermi naphthoquinone derivative was purchased from Chengdu Biopurify Phytochemicals Ltd., including shikonin (with an Article Number being

517-89-5), deoxyshikonin (with an Article Number being 43043-74-9), acetylshikonin (with 4504956

Article Number being 43043-74-9), B-hydroxyisovalerylshikonin (with an Article Number being 7415-78-3), and PB, B-dimethylacrylshikonin (with an Article Number being 24502-79-2). Test strains are shown in Table 1, including sensitive bacteria and multidrug resistant bacteria 5 carrying different drug resistance genes, especially the most clinically severe methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus (VRE), and linezolid-resistant Enterococcus carrying optrA or poxtA, which all have good antibacterial activity.

Table 1 Information About Strain pes

The broth microdilution method specifically includes the following steps: (1) A to-be-tested bacterial solution was diluted with a CAMHB broth medium (Haibo

Biotechnology Co., Ltd., HB6231-1) to make a concentration of a bacterial suspension be 1x106

CFU/mL. LU504956 (2) The Radix lithospermi naphthoquinone derivative was dissolved with dimethyl sulfoxide (DMSO) and diluted with the CAMHB broth medium to obtain an antibacterial drug solution (with a concentration of 512 pg/mL) of shikonin, deoxyshikonin, acetylshikonin,

P-hydroxyisovalerylshikonin or PB, B-dimethylacrylshikonin. Linezolid and vancomycin were dissolved into ultrapure water to prepare a stock solution with a concentration of 2560 pg/mL, which was diluted with CAMHB to a concentration required for the test when in use. (3) A 96-well plate was taken; 100 uL of a CAMHB broth medium was added to each well; 100 uL of the antibacterial drug solution prepared in step (2) was added to each well in the first column, and subjected to doubling dilution from the first column to the tenth column; and then 100 pL of a bacterial suspension prepared in step (1) was added to each well and left for standing for culture at 37°C for 18 h. Positive control wells and negative control wells were set; 100 uL of the bacterial suspension prepared in step (1) was added to each positive control well; and each negative control well only contained the CAMHB broth medium.

Test results are shown in Table 2. Five Radix lithospermi naphthoquinone derivatives all show certain antibacterial activity against the Gram-positive sensitive bacteria and the drug-resistant bacteria, and a minimal inhibitory concentration is between 0.5 pg/mL and 64 pg/mL. The antibacterial activity of the acetylshikonin is the strongest, and its minimal inhibitory concentration is between 0.5 ug/mL and 8 ug/mL. By comparing the structures of several compounds, it is found that hydroxyl acetylation on carbon in the 11-position is of great significance for improving the antibacterial activity of the shikonin.

Table 2 Antibacterial Effects of Five Naphthoquinone Compounds from Radix lithospermi on G+ ~ Minimal inhibitory concentration (ng/mL) anh Linezoli fen Shikonin shikon P-dimethylac valerylshikon Vancomycin d

Staphylococcus aureus S5iiiiiiiii LU504956 ns os 8 3264 16 1 2

ATCC 29213 a os 516 05 0 . « 328 8 781 1816 3264 162 1632 2 16 18-243 oo i em 32 16 16 2 8 >256 >256 >256 7256 >256 — ——- 25922

Embodiment 2 Synergistic antibacterial activity of Radix lithospermi naphthoquinone derivative and antibiotic

The synergistic antibacterial activity of the Radix lithospermi naphthoquinone derivative, combined with the antibiotic, against multidrug resistant Escherichia coli B2 (blanpy-s + mer-1),

Salmonella 15E343 (mer-3), K. Pneumoniae 19-2-1 (mcr-8) and Escherichia coli ATCC 25922 was determined using a chessboard dilution method.

The chessboard dilution method specifically includes the following steps: (1) A to-be-tested bacterial solution was diluted with a CAMHB broth medium to make a concentration of a bacterial suspension be 1 x 106 CFU/mL. (2) The antibiotic was dissolved with a solvent recommended by the CLSI criterion, and diluted with the CAMHB broth medium to obtain an antibiotic solution with a concentration of 256 ug/mL. Specifically, colistin, meropenem, tetracycline, tigecycline, kanamycin, and ciprofloxacin were dissolved into ultrapure water to prepare stock solutions with a concentration of 2560 pg/mL respectively; ampicillin was dissolved into 0.1 M PBS (pH = 8), and then dilutéd/504956 with the ultrapure water to obtain a stock solution with a concentration of 2560 ug/mL; and rifampicin was dissolved with methanol, and then diluted with the ultrapure water to 2560 ug/mL. When in use, the stock solutions were diluted with the CAMHB to a concentration required for the test. (3) Preparation of a Radix lithospermi naphthoquinone derivative solution: the Radix lithospermi naphthoquinone derivative was dissolved with dimethyl sulfoxide (DMSO) and diluted with the

CAMHB broth medium to obtain an antibacterial drug solution (with a concentration of 256 ug/mL) of shikonin, deoxyshikonin, acetylshikonin, P-hydroxyisovalerylshikonin or J,

P-dimethylacrylshikonin. (4) A 96-well flat plate was taken, and 100 uL of a CAMHB broth medium was added to each well, and 100 pL of antibiotic solution prepared in step (2) was added to each well in the last row, and subjected to doubling dilution from the eighth column to the second column; the Radix lithospermi naphthoquinone derivative solution prepared in step (3) was added to each well in the first column (with 100 uL per well), and subjected to doubling dilution to the seventh column; and then 100 uL of the bacterial suspension prepared in step (1) was added to each well, and left for standing for culture at 37°C for 18 h. A lowest concentration combination of the Radix lithospermi naphthoquinone derivative and polymyxin for inhibiting bacterial growth was observed.

A method of calculating a fractional inhibitory concentration (FICI) is as follows:

FIC= MIC(combined use of A drug)/MIC(single use of A drug) + MIC(combined use of B drug)/MIC(single use of B drug)

Experimental results are shown in Table 3. The acetylshikonin has no synergistic effect on the ampicillin, the tetracycline, the tigecycline, the rifampicin, the ciprofloxacin and the kanamycin; and its fractional inhibitory concentration index (FICI) is 2. The FICIs of the acetylshikonin with the polymyxin and the meropenem are 0.04 and 0.09 respectively, which may make the MICs of the polymyxin and the meropenem reduced by 32 times; and the synergistic effects of th&504956 acetylshikonin with the polymyxin and the meropenem are obvious.

Table 3 Synergistic Antibacterial Effects of Acetylshikonin and Different Types of Antibiotics on

Escherichia Coli B2

BE Symergism

Antibiotic MIC? (ug/mL) FIC index MIC® (ug/mL) multiple“

Ampicillin >128 2 >128 1

Tetracycline > 128 2 > 128 1

Tigecycline 0.5 2 0.5 1

Rifampicin >128 2 >128 1

Ciprofloxacin 32 2 32 1

Kanamycin >128 2 >128 1 a represents a minimal inhibitory concentration of the antibiotic against the drug-resistant bacteria when used alone; b represents minimal inhibitory concentrations of different types of antibiotics against the drug-resistant bacteria after addtion of the acetylshikonin; c represents a multiple of the antibacterial activity increased of each antibiotic.

Embodiment 3 Time sterilization curve of acetylshikonin

Staphylococcus aureus ATCC 29213 was cultured in a CAMHB broth medium at 37°C to an exponential phase, and a bacterial solution was diluted with a CAMHB broth to an ideal concentration of 106 CFU/mL. 4 ng/mL acetylshikonin (4 x MIC) was added for culture at 37°C; 50 uL of bacterial solutions were taken at 0 min, 2 min, 5 min, 10 min, and 15 min, and diluted in a 10-fold order; resultants were dropped on an LB agar (Haibo Biotechnology Co., Ltd,

HBO0129) plate for culture at 37°C for 24 h; and then a colony forming unit (CFU/mL) was calculated. LU504956

Escherichia coli B2 was cultured in a CAMHB broth medium at 37°C to an exponential phase, and a bacterial solution was diluted with a CAMHB broth to an ideal concentration of 106

CFU/mL. Then the bacteria were treated with the acetylshikonin (32 pg/mL) and the polymyxin (2 pg/mL) or the meropenem (8 pg/mL) alone or in combination respectively. 50 pL of bacterial solutions were taken at Oh, 4 h, 8 h, 12 h, and 24 h, respectively, and diluted in a 10-fold order; resultants were dropped on an LB agar plate for culture at 37°C for 24 h; and then a colony forming unit (CFU/mL) was calculated. All the experiments were biologically repeated at least 3 times.

Results show that the acetylshikonin has a rapid bactericidal effect on the Staphylococcus aureus, killing 99% of the bacteria at 15 min (FIG. 1). Also, the acetylshikonin (ASK) may enhance the bactericidal effect of the polymyxin (COL) and the meropenem (MER) on the drug-resistant bacteria, and all the bacteria may be killed within 4 h under their combined effect (FIG. 2).

Embodiment 4 Analysis on haemolytic activity of acetylshikonin

In order to evaluate the safety of the acetylshikonin, the hemolytic activity of the acetylshikonin is evaluated. The acetylshikonin was subjected to doubling dilution with a phosphate buffer (PBS) (10 mM, pH = 7.4) from the 1st well to the 10th well in a 96-well plate; and sterile PBS served as a negative control in the 11th well, and double distilled water (ddH2O) served as a positive control in the 12th well. Fresh sheep red blood cells (RBC) (Beijing Solarbio Science &

Technology Co., Ltd., TX0030) were washed with the PBS (10 mM, pH = 7.4) twice, and then resuspended with the PBS (10 mM, pH = 7.4) to obtain a 8% red blood cell suspension; and the red blood cell suspension was mixed with the acetylshikonin with concentrations of 0.5 ng/mL, 1 ng/mL, 2 pg/mL, 4 pg/mL, 8 pg/mL, 16 pg/mL, 32 pg/mL, 64 pg/mL and 128 pg/mL for incubation at 37°C for 1 h. Subsequently, 120 pL of a supernatant was sucked, and centrifuged at 3000 rmp/min for 10 min; and 100 pL of released hemoglobin was sucked to measure its absorbance value at 576 nm (OD576); and a corresponding hemolysis rate was calculated. The hemolysis rate is calculated as follows: hemolysis rate (%) = [(OD576 sample - OD576 negatit&/504956 control )/(OD576 positive control - OD576 negative control)] x 100%. Experimental results are shown in FIG. 3. Even at a high concentration of 128 pg/mL, the acetylshikonin shows a low hemolysis rate (5.5%).

Embodiment 5 Treatment on infection of methicillin-resistant Staphylococcus aureus in larvae of

Galleria mellonella with acetylshikonin

The acetylshikonin was dissolved with DMSO to prepare 2560 ng/mL stock solution; and the stock solution was diluted with the PBS (10 mM, pH = 7.4) to prepare 50 pg/mL working solution and 150 pg/mL working solution.

Staphylococcus aureus MRSA T144 was inoculated to an LB broth medium for culture at 37°C to a logarithmic growth phase. MRSA T144 was resuspended with the PBS (10 mM, pH = 7.4) to make a concentration of a bacterial solution be 105 CFU/mL. 50 larvae of Galleria mellonella weighed about 300 mg were randomly divided into 4 groups, and 10 pL of the bacterial solution in step (2) was injected into each last lower left abdominal foot; after 1 h of infection, 10 pL of acetylshikonin with concentrations of 10 pg/mL, 50 pg/mL and 150 pg/mL was injected into the last lower right abdominal feet respectively; and the control group was given 10 uL of PBS (10 mM, pH = 7.4).

Survival rates of the larvae of the Galleria mellonella were counted at 12 h, 24 h, 36 h and 48 h.

Experimental results are shown in FIG. 4. Treatment with 50 pg/mL acetylshikonin may increase the survival rate of the MRSA-infected Galleria mellonella to 70%; and after treatment with 150 ng/mL acetylshikonin, 90% of the MRSA-infected Galleria mellonella may survive.

Embodiment 6 Treatment on rat skin wound infection with acetylshikonin

A rat skin wound infection model was constructed with Staphylococcus aureus MRSA T144 as model bacteria, to evaluate the therapeutic effect of the acetylshikonin on Gram-positive bacterial infection. The acetylshikonin was dissolved with DMSO to prepare 2560 pg/mL stock solution; and the stock solution was diluted with the PBS (10 mM, pH = 7.4) to prepare 10 ng/mL working solution, 20 pg/mL working solution and 50 pg/mL working solution/504956

Staphylococcus aureus MRSA T144 was inoculated to an LB broth medium (Haibo

Biotechnology Co., Ltd., HB6231-1) for culture at 37°C to a logarithmic growth phase. MRSA

T144 was resuspended with the PBS (10 mM, pH = 7.4) to make a concentration of a bacterial solution be 108 CFU/mL. Wistar rats (Comparative Medical Center of Yangzhou University) were divided into 4 groups, i.e, a 10 ug/mL acetylshikonin treatment group, a 20 ug/mL acetylshikonin treatment group, a 50 pg/mL acetylshikonin treatment group and a control group.

A wound of about 1 cm2 was cut on the back of each rat with a sterile surgical scissor.

Subsequently, each wound was infected with 100 uL (108 CFUs/mL) of MRSA T144 bacterial suspension; after 1 h of infection, each of 100 pL of acetylshikonin with different concentrations (10 pg/ml, 20 pg/ml, and 50 pg/ml respectively) was given to each rat; and the control group was given the PBS (10 mM, pH = 7.4). Healing of the wound of the back of each rat was observed for 12 consecutive days, and a change on a wound size was measured. On the last day, each rat was euthanized, and right wound skin (1 cm2) of each rat was homogenized, diluted and dropped into a plate for colony counting. Experimental results are shown in FIG. 5. The acetylshikonin may significantly promote wound healing, and reduce a bacterial load of each skin wound by about 100 times (from 107 CFUs/mL to 105 CFUs/mL).

Embodiment 7 Observation on morphology of Staphylococcus aureus

Staphylococcus aureus MRSA T144 was cultured at 37°C in 25 mL of an MHB medium (Haibo

Biotechnology Co., Ltd., HB6231-1) overnight, then centrifuged at 5000 rpm and 4°C for 5 min, and washed with PBS (10 mM, pH = 7.4). Then, a resultant was co-incubated with 5 pg/ml (10 *

MIC) or 10 pg/ml (20 * MIC) acetylshikonin (ASK). Subsequently, the bacteria were washed with the PBS (10 mM, pH = 7.4) for three times, fixed with 2.5% glutaraldehyde, and left at 4°C overnight. The next day, the bacteria were dehydrated with ethanol with different concentrations (30%, 50%, 70%, 90% and 100%). Finally, the bacteria were dried, plated and adhered, and samples were observed by a GeminiSEM 300 electron microscope. Experimental results are shown in FIG. 6. The morphological structure of the Staphylococcus aureus MRSA T144 wh$/504956 destroyed after treatment with the acetylshikonin.

Embodiment 8 Research on bactericidal mechanism of acetylshikonin

Staphylococcus aureus MRSA T144 was cultured in an LB medium at 37°C overnight, diluted by 1: 100 in 25 ml of an LB medium, and subjected to spreading cultivation to a logarithmic growth phase. The bacteria were washed with a PBS (10 mM, pH = 7.4) for 3 times, and a

OD600 value of a bacterial suspension was adjusted to about 0.5. Incubation was performed using a fluorescent probe at 37°C in dark for 30 min to obtain probe-labeled bacteria. 190 uL of probe-labeled bacteria were added to a 96-well plate, and incubated with 10 uL of ASK with different concentrations (0.5 pg/ml, 1 ug/ml, 2 ug/ml and 4 ug/ml) for 1 h. Fluorescence values were measured using an Infinite M200 microplate reader (Tecan). Propidium iodide (PI) (Shanghai Beyotime Biotechnology Co., Ltd., ST512) was used to measure the permeability of bacterial cell membranes. An excitation wavelength was 535 nm, and an emission wavelength was 615 nm. 3,3-sodium dipropyl thiocyanoiodide (disc3 (5)) (Shanghai Aladdin Biochemical

Technology Co., Ltd., 53213-94-8) was used to measure a membrane potential of the bacteria.

An excitation wavelength was 622 nm, and an emission wavelength was 670 nm. A control was the PBS. A pH-sensitive fluorescent probe BCECF-AM (Shanghai Beyotime Biotechnology Co.,

Ltd, S1006) was used to evaluate ApH of the bacteria. An excitation wavelength was 488 nm, and an emission wavelength was 535 nm.

Determination on intracellular ATP in bacteria: an intracellular ATP level of Staphylococcus aureus MRSA T144 after the effect of the ASK was determined using an enhanced ATP detection kit (Shanghai Beyotime Biotechnology Co., Ltd., S0027). Staphylococcus aureus MRSA T144 was shaken for culture at 37°C overnight, then washed with the PBS (10 mM, pH = 7.4), and resuspended to make a OD600 nm be around 0.5. Then, a bacterial suspension was treated with

ASK with different concentrations (0.5 pg/ml, 1 ug/ml, 2 pg/ml and 4 pg/ml) for 10 min, and then centrifuged at 10000 rpm and 4°C for 5 min; and bacterial precipitates lysed and centrifuged by lysozyme were used to determine the intracellular ATP level. A detection solution was addéd/504956 to a 96-well plate, and incubated at a room temperature for 5 min. A supernatant was added for rapid mixing, and luminescence was measured using an Infinite M200 microplate reader (Tecan).

Results are shown in FIG. 7. The acetylshikonin improves the permeability of the bacterial cell membranes, dissipates proton driving force, and reduces the intracellular ATP level. It is suggested that the acetylshikonin mediates the killing of the bacteria through membrane destroying.

Those of ordinary skill in the art should understand: the discussion of any of the above embodiments is exemplary only and is not intended to imply that the scope of the present invention (including the claims) is limited to these examples; and in the idea of the present invention, technical features in the above embodiments or different embodiments may also be combined, steps may be implemented in any order, and many other variations of different aspects of the present invention as described above, which are not provided in detail for the sake of brevity, are available.

The present invention is intended to cover all such substitutions, modifications and variations that fall within the broad scope of the claims. Therefore, any omission, modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

CLAIMS LU504956

1. A use of a Radix lithospermi naphthoquinone derivative in the preparation of a drug for treating a bacterial infectious disease.

2. The use according to claim 1, characterized in that the Radix lithospermi naphthoquinone derivative comprises one or more of deoxyshikonin, shikonin, acetylshikonin, P, B-dimethylacrylshikonin or B-hydroxyisovalerylshikonin.

3. The use according to claim 1, characterized in that bacteria are Gram-positive bacteria.

4. A use of the Radix lithospermi naphthoquinone derivative, combined with an antibiotic, in the preparation of a drug for treating a disease caused by infection with multidrug resistant Gram-negative bacteria.

5. The use according to claim 4, characterized in that the Radix lithospermi naphthoquinone derivative comprises one or more of the deoxyshikonin, the shikonin, the acetylshikonin, the P, B-dimethylacrylshikonin and the B-hydroxyisovalerylshikonin.

6. The use according to claim 4, characterized in that the multidrug resistant Gram-negative bacteria comprise polymyxin or meropenem resistant bacteria.

7. The use according to claim 6, characterized in that the multidrug resistant Gram-negative bacteria comprise Staphylococcus aureus.