WO2018107614A1

WO2018107614A1 - Use of luteolin in preparation of medicament for preventing and treating dengue virus infection

Info

Publication number: WO2018107614A1
Application number: PCT/CN2017/078685
Authority: WO
Inventors: 李耿; 彭敏桦; 吴建国; 萨博哈什•瓦苏德凡; 渡边哲; 赖小平
Original assignee: 东莞广州中医药大学中医药数理工程研究院
Priority date: 2016-12-12
Filing date: 2017-03-30
Publication date: 2018-06-21
Also published as: CN106692127B; CN106692127A

Abstract

The present invention relating to a use of luteolin in the preparation of a medicament for preventing and treating dengue virus infection. The medicament in the use consists of luteolin and medically acceptable adjuvants, the mass percentage of the luteolin used in the medicament being 0.1-90%. The medicament in the present invention has an effect of resisting dengue virus and can be used for preventing and treating dengue virus infection.

Description

Application of luteolin in the preparation of drugs for preventing and treating dengue virus infection

Technical field

The present invention relates to a pharmaceutical preparation containing an organic active ingredient, and in particular to a medicament containing a flavonoid compound.

Background technique

The dengue virus is classified into the Flaviviridae and the Flavivirus genus. It includes 4 different serotypes, namely type I, II, III and IV dengue viruses. Depending on the severity of the disease, clinically it can be divided into dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue fever spreads rapidly and the incidence rate is high. The dengue hemorrhagic fever and dengue shock syndrome are fierce and the mortality rate is high. With the acceleration of global economic integration and the continued warming of the climate, coupled with the accelerated spread of mosquito vectors and widening of distribution, the disease has spread rapidly around the world. Dengue fever has become a worldwide economic burden, with approximately 500,000 critically ill dengue patients requiring hospitalization each year, with a mortality rate of approximately 2.5%.

Although the world's first dengue vaccine was approved in three countries, the vaccine is not particularly effective, reducing the risk of developing dengue fever to only 60%, and children under 9 are not eligible. Within the scope. Therefore, prevention and treatment of dengue virus infection and transmission has become a top priority in the medical profession. Since the pathogenic mechanism of dengue virus has not been elucidated, there is no vaccine for human body and effective therapeutic drugs. It is extremely urgent to find a drug effective for treating dengue fever.

Luteolin, a flavonoid, wood yellow needle crystal, melting point 328-330 ° C. Slightly soluble in water, weakly acidic, soluble in alkaline solution, stable under normal conditions. Density: 1.654 g/cm3, melting point: 330 ° C, boiling point: 616.1 ° Cat 760 mmHg, flash point: 239.5 ° C. Steam pressure: 9.03E-16mmHg at 25 °C.

The chemical structure of luteolin is as shown in the following formula (I).

Luteolin, originally named from the leaves, stems and branches of the genus Rhododendron, Resedaodorata L., can be isolated from a variety of natural medicinal materials and vegetable fruits. It is found mainly in natural medicinal materials such as honeysuckle, chrysanthemum, schizonepeta, Prunella vulgaris, Artichoke, Perilla, Astragalus, and Naked Violet, as well as Brussels sprouts, cabbage, cauliflower, beet, broccoli and carrot. , celery, sweet pepper, pepper, groundnut and other vegetable fruits. It has anti-tumor, anti-inflammatory, anti-allergic, anti-fibrosis, anti-fertility and immune regulation. However, there is no report in the literature that luteolin has anti-dengue virus activity.

Summary of the invention

The technical problem to be solved by the present invention is to provide a new use of luteolin, a new application in pharmaceuticals.

The above new application in pharmaceuticals is the use of luteolin in the preparation of a medicament for the prevention and treatment of dengue virus infection.

In the above application, the dengue virus is a dengue type I, II, III or IV virus.

In the above application, the luteolin can be extracted from natural plants by a conventional method, or can be obtained by synthesis or other methods.

In the above application, the drug is composed of luteolin and a medically acceptable adjuvant, wherein the luteolin is 0.1% to 90% by mass in the drug.

In the above application, the drug may be a clinically acceptable injection, capsule or tablet.

The injections, capsules and tablets of the present invention have an anti-dengue virus action and can be used for controlling dengue infection.

The use of luteolin against dengue virus in the present invention is to determine the inhibitory effect of luteolin on dengue fever type I, II, III and IV viruses by virus plaque titer method, and to observe the toxic effect of luteolin on Huh-7 cells and Inhibition of dengue fever type I, II, III, and IV viruses

The detection reagents measured half of the toxic concentration (CC50) of the test drug was 45.89, and the maximum effect concentration (EC50) of the half-type I, II, III, and IV dengue viruses were 7.2, 5.19, 5.40, and 8.38, respectively. They are 6.38, 8.84, 8.07, and 5.48, respectively, which are low-toxic and efficient.

The invention uses the luteolin against dengue virus and also adopts AG129 mouse animal experiment to determine the inhibitory effect of luteolin on dengue type II virus, and observes the protective effect of luteolin on the death of AG129 mice infected with dengue type II virus. The death protection rate was 33.33%.

Experimental material

1.1 cell line

Human liver cancer cells (Huh-7) were purchased from the American Model Culture Collection (ATCC), T5 generation. Stored by Duke-NUS Medical School.

Milk hamster kidney cells (BHK-21) were purchased from the American Model Culture Collection (ATCC), T6 generation. Stored by Duke-NUS Medical School.

1.2 experimental group and its corresponding test drugs

Test sample: The luteolin purchased from Baoji Chenguang Biotechnology Co., Ltd. (Lot: 0151024) was added to DMSO to prepare a stock solution having a concentration of 20 mM. NITD008 was used as a positive control and was donated by the Novartis Institute for Tropical Disease (Singapore) to make a stock solution with a concentration of 20 mM.

1.3 virus strain

DENV-1 (EDEN-1, GenBank accession EU081230)

DENV-2 (EDEN-2, GenBank accession EU081177)

DENV-3 (EDEN-3, GenBank accession EU081190)

DENV-4 (EDEN-4, GenBank accession GQ398256)

Presented by Professor Luo Dahai from Nanyang Technological University, Singapore, the DENV strain was expanded in C6/36 cells, and the supernatant was filtered through a 0.45 μm microporous membrane and stored at -80 °C. Viral titers were determined by plaque assay in BHK-21 cells.

1.4 animals

Sv/129 mice lack the type I and type II interferon receptor (AG129) and are purchased from B&K Universal (UK). All animal experiments were in compliance with the Singapore National guidelines (protocol 2012/SHS/713), approved by the Institutional Animal Care and Use Committee at Singapore Health Services. All experiments were performed in a Duke-NUS BSL-2 laboratory.

1.5 Reagents and instruments Newborn calf serum and DMEM medium (Gibco); DMSO (Sigma); trypsin (DIFCO, USA). Olympus PM-6 inverted microscope (Japan OLYMPUS company).

2. Method

2.1 Determination of the toxicity concentration of luteolin on Huh-7 cells

Huh-7 grown in pellets in cell vials was counted by EDTA-pancreatin digestion, cells were seeded at 2 x 10 ⁴ /100 μl/well in 96-well plates (white flat bottom), and incubated overnight at 37 ° C 5% CO ₂ . The drug was diluted with cell growth medium (containing 10% serum) at a final concentration of 50, 25, 12.5, 6.25, 3.125, 1, 0.1, 0.01 μM. The medium was aspirated, 100 μl of the drug solution was added to each well, and a normal cell control was set, and 100 μl of the cell growth medium was added to each well. Incubate for 48 h at 37 ° C in 5% CO ₂ . CellTiter-GloBuffer was added to the CellTiter-Glo Substrate to form a mixture of enzyme and substrate, CellTiter-Glo reagent. An equal volume (100 μl) of CellTiter-Glo reagent was added to each well, incubated for 10 minutes at room temperature, and Luminescence values were read under a microplate reader (Tecan Infinite 200PRO).

Cell viability = drug group Luminescence / normal group Luminescence × 100%

2.2 Determination of antiviral effect in vitro

Huh-7 grown in pellets in cell vials was counted by EDTA-pancreatin digestion, and cells were seeded at 1 x 10 ⁵ /500 μl/well in 24-well plates. Incubate overnight at 37 ° C 5% CO ₂ . The drug was diluted with cell growth medium (containing 10% serum) at a final concentration of 50, 25, 12.5, 6.25, 3.125, 1, 0.1, 0.01 μM. EDEN-1, EDEN-2, EDEN-3 and EDEN-4 virus solutions were diluted with serum-free medium at a multiplicity of infection MOI=0.3. The medium was aspirated, and 100 μl of each of the drug solution and the virus solution was added to each well, and cultured at 37 ° C for 5% CO 2 for 1 h. The virus-drug mixture was aspirated, 400 μl of the drug-containing medium was added to each well, and cultured at 37 ° C for 5% CO 2 for 48 hours. The supernatant was collected and centrifuged at 9000 rpm for 3 minutes to remove cell debris. Store at -80 ° C for later use. Virus titers were determined by plaque assay.

2.3 Determination of virus titer

BHK-21 grown in pellets in cell vials was counted by EDTA-pancreatin digestion, and cells were seeded at 2 x 10 ⁵ /500 μl/well in 24-well plates. Incubate overnight at 37 ° C 5% CO ₂ . The virus sample was diluted with serum-free medium and two dilution factors were set. Two replicate wells were set for each dilution factor, and the dilution factor was 10 ^-2 -10 ^-7 . The medium was aspirated, 200 μl of virus solution was added to each well, and cultured at 37 ° C for 5% CO 2 for 1 h. The inoculum was removed, 500 μl of 0.8% CMC containing 2% serum was added to each well, and cultured for 4-5 days at 37 ° C 5% CO ₂ (EDEN-2, EDEN-3, EDEN-4 culture for 5 days, EDEN-1 culture for 4 days). ). Fix with 3.7% paraformaldehyde, stain with 1% crystal violet, and count plaques. Pfu/ml = number of plaques × 1000/200 × reciprocal of the dilution factor.

2.4 Determination of antiviral effects in vivo

Mice from 7 to 11 weeks were injected intraperitoneally with 50 μg of 4G2 antibody one day prior to infection. Infection was carried out by intravenous injection of 1×10 ⁸ pfu EDEN-1, EDEN-2, EDEN-3, EDEN-4 virus, and the mice were intragastrically administered with 100 mg/kg luteolin 4 times a day for 4 times. /4/4/12h (9AM, 1PM, 5PM and 9PM) cycle administration. The tenth day of mouse survival was observed. Six mice were placed in each group.

3. Results

3.1 Drug toxicity results are shown in Table 1.

Table 1 Evaluation of the toxicity of luteolin on Huh-7 cells

Drawing with the software GraphPad Prism 5.0, the results are shown in Figure 1.

3.2 The results of the antiviral effect of the drug are shown in Table 2 below.

Table 2 Determination of virus titer

Drawing with the software GraphPad Prism 5.0, the results are shown in Figure 2.

3.3 The protective effect of luteolin on death of AG129 mice is shown in Table 3-6.

Table 3 Survival time of luteolin against EDEN-1 virus-infected AG129 mice

Table 4 Survival time of luteolin against EDEN-2 virus-infected AG129 mice

Table 5 Survival time of luteolin against EDEN-3 virus-infected AG129 mice

Table 6 Survival time of luteolin against EDEN-4 virus-infected AG129 mice

Drawing with the software GraphPad Prism 5.0, the results are shown in Figures 3-6.

DRAWINGS

Figure 1 is a graph showing the toxic effects of luteolin on Huh-7 cells.

Figure 2 is a graph showing the inhibitory effect of luteolin on dengue virus.

Figure 3 is a graph showing the survival curve of luteolin against dengue virus I-infected mice.

Figure 4 is a graph showing the survival curve of luteolin against dengue virus II-infected mice.

Figure 5 is a graph showing the survival curve of luteolin against dengue virus III-infected mice.

Figure 6 is a graph showing the survival curve of luteolin against dengue virus IV-infected mice.

detailed description

Example 1: (injection)

Take luteolin 1000mg, add citric acid 1000mg, sodium citrate 500mg, sodium chloride 1800mg, add 1000ml of water for injection, stir to dissolve, sterilize filtered, potted, steam sterilized at 100 ° C for 15 minutes , each 2mg / 2ml injection is made for injection.

Example 2: (capsule)

4500mg of luteolin is mixed with 200mg microcrystalline cellulose, 200mg sodium carboxymethyl starch, 100mg sodium lauryl sulfate and other auxiliary materials, dry granulation by roll pressing, and then mixed with appropriate amount of magnesium stearate. The capsule was filled into 3# hollow capsules and made into a capsule of 100 mg/granule for oral use.

Example 3: (tablet)

Take luteolin 5000mg and 4000mg starch, 200mg cross-linked PVP, 300mg carboxymethyl starch sodium, mix well, use 5% PVP 75% ethanol solution as binder, make soft material, granulate with 18 mesh sieve, 60 °C 1 h after drying, after 20 mesh granules, add appropriate amount of talc powder, mix and compress, and prepare tablets of 100 mg/tablet for oral use.

Claims

The use of luteolin in the preparation of a medicament for controlling dengue virus infection.
The use according to Claim 1, characterized in that the medicament consists of luteolin and a medically acceptable adjuvant, wherein the luteolin is used in the medicament in an amount of from 0.1% to 90% by mass.
The use according to claim 2, wherein the drug is an injection, a capsule or a tablet.