CN102266389A

CN102266389A - Application of cortex meliae in preparation of drug for inhibiting influenza A H1N1 virus

Info

Publication number: CN102266389A
Application number: CN2011102531069A
Authority: CN
Inventors: 徐经纬; 杨艳琴; 杨卫; 房学迅; 田莉; 肖延蒙; 王晨旭
Original assignee: Changchun Institute of Applied Chemistry of CAS
Current assignee: Changzhou Institute of Energy Storage Materials & Devices
Priority date: 2011-08-30
Filing date: 2011-08-30
Publication date: 2011-12-07
Anticipated expiration: 2031-08-30
Also published as: CN102266389B

Abstract

The invention provides application of cortex meliae in preparation of a drug for inhibiting influenza A H1N1 virus, belonging to the field of traditional Chinese medicine application. The invention is implemented according to the following technical schemes: firstly preparing a cortex meliae solution, incubating a neuraminidase solution and the cortex meliae solution in a 37 DEG C incubator for 30 minutes, then adding an MUNANA (2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid) buffer solution, and detecting with a Bio-Red FLx-800 fluorescence microplate reader. In the invention, new medical application of cortex meliae is discovered and a new application field is developed; and the extract of cortex meliae crude drug or cortex meliae has significant effect on inhibiting influenza A H1N1 virus, thereby laying a foundation for future development of a new drug for inhibiting influenza A H1N1 virus.

Description

The application of Cortex Meliae in preparation inhibition influenza A H1N1 influenza virus medicine

Technical field

The invention belongs to the Chinese medicine application, be specifically related to the application of Cortex Meliae in preparation inhibition influenza A H1N1 influenza virus medicine.

Background technology

Mexico found the first influenza A H1N1 case since on March 18th, 2009, the virus gene sequence that the U.S. obtained on one's body and separated to obtain a California patient April 27 in 2009.Studies show that: this viral genome is mixed by bird flu, swine flue and human influenza virus's gene, is a kind of novel influenza A H1N1 influenza virus, and its caused influenza has hyperinfection, propagates rapid, easy popular characteristics

Influenza A virus infectiousness height, propagate rapidly, easily take place popular, speed of mutation is fast, and vaccine research and speed of production are slower, make chemicals and influenza virus vaccine be difficult to bring into play its optimal treatment and preventive effect.Chinese herbal medicine has the influenza virus of inhibition to be duplicated, stops pathological changes caused by virus, regulate comprehensive effect such as immunity, antalgic and inflammation relieving, has special advantages aspect the control influenza virus.

Cortex Meliae derives from the dry bark and the root bark of Meliaceae plant Fructus Toosendan, has following effect:

1, anthelmintic action: contained Melia azedarach L. have and drives the ascarid work in root bark of Fructus Toosendan, Melia azedarach L. or the dried bark.Its alcoholic extract to the pig ascarid, has the paralysis effect to its head external especially.25%～50% Cortex Meliae medicinal liquid also has the paralysis effect external to the mice pinworm.Cortex Meliae extractive liquid still has certain anti-schistosome function.

2, antifungic action: Fructus Toosendan ethanol immersion (1: 4) in vitro to common pathogenic funguses such as yellow trichophyton, Trichophyton concentricunm, trichophyton, Du's big belly microspore tinea bacterium difficult to understand, rust microspore's tinea bacterium, ulotrichy microspore's tinea bacterium, red dermatophytosis, star-shaped nocardias, has than the obvious suppression effect.

3, to the effect of muscle contraction: experiment shows that Toosendanin has potentiation to skeletal muscle by the contractile response that direct stimulation causes.

Studies show that 4, Fructus Toosendan have anti-botulinal effect. Toosendanin energy significant prolongation botulism specimen points out it can resist the effect of checking of meat poison at the neuromuscular junction place to the paralysis time of indirect stimulation contractile response.

Up to now, do not preparing the report that suppresses the application in the influenza A H1N1 influenza virus medicine as yet relevant for Cortex Meliae.

Summary of the invention

The object of the present invention is to provide the application of Cortex Meliae in preparation inhibition influenza A H1N1 influenza virus medicine.The present invention implements by the following technical programs: the application of Cortex Meliae in preparation inhibition influenza A H1N1 influenza virus medicine.Described Cortex Meliae is a kind of in raw medicinal herbs or the Cortex Meliae extract.The preparation method of described Cortex Meliae extract is: after Cortex Meliae is spent the night with distilled water immersion, filter, Cortex Meliae after time immersion of winning is filtered back gained liquid and filtered, Cortex Meliae after filtering is added after distilled water immersion spends the night again, boiled Chinese medicine 3-5 hour with the slow fire heating, filter, get the liquid after infusion is filtered, back gained liquid is filtered in immersion for the first time and back gained liquid mixing is filtered in infusion, obtain mixed solution, obtain Szechwan Chinaberry Bark behind the lyophilizing mixed solution, the solution of described immersion Cortex Meliae is a kind of in methanol or the ethanol.

Is 10mg/mL with the dissolving of the Szechwan Chinaberry Bark after the described lyophilizing with dissolved in distilled water to concentration, gets Cortex Meliae solution.Neuraminidase is dissolved in MES buffer (PH 6.5) and CaCl ₂In, get neural propylhomoserin enzymatic solution, getting neuraminic acid enzymatic solution and Cortex Meliae solution hatched 30 minutes in 37 ℃ of incubators, the buffer solution that adds MUNANA then, detect with Bio-Red FLx-800 fluorescence microplate reader immediately, excitation wavelength 360nm, emission wavelength 450nm, minute are 8min.

To the neuraminidase of 0.001-0.1U, be 31.25-250ug/mL to its inhibited Cortex Meliae solution concentration.

Principle of the present invention: effect of the present invention mainly is by (neuraminidase, inhibition ability NA) is verified to neuraminidase.NA is one of most important albumen of influenza A H1N1 influenza virus, and in the process of the release of virus from infection cell, particularly the later stage in virus replication cycle, NA plays a major role.Because it has the enzyme function, the sialic acid of various glycoprotein receptor ends on the energy hydrolysis cell membrane, therefore, NA can remove the sialic acid of virion surface glycoprotein end, prevents that the oneself of virus from building up, and impels progeny virus to discharge.In addition, NA is an important antigen, and it is necessary to be that virus penetrates the airway epithelial mucus.In a single day NA undergos mutation, and can cause influenza A H1N1 influenza virus that neuraminidase inhibitor is produced the resistance effect.

Beneficial effect of the present invention:

1, the present invention has excavated new medical application for Cortex Meliae, has opened up a new application.

2, the present invention suppresses A (H 1 N 1) virus with Cortex Meliae raw medicinal herbs, Cortex Meliae extract, and effect is remarkable, and Cortex Meliae is to the half-inhibition concentration (IC of neuraminidase ₅₀) be IC ₅₀(μ g/mL)=184.3 ± 13.1 and IC ₅₀(μ g/mL)=0.76 ± 0.27 the invention provides the application of Cortex Meliae active component in as the medicine that suppresses influenza A H1N1 influenza virus, for the medicine of developing a kind of new inhibition influenza A H1N1 influenza virus from now on lays the foundation.

Description of drawings

Fig. 1 is the relation of relative vigor of cell and Cortex Meliae solution concentration;

Fig. 2 is that the MTT of Cortex Meliae solution concentration and viral yield detects figure.

The specific embodiment

Below the present invention is described in further detail for the specific embodiment by example forms.

Embodiment 1

Take by weighing the Cortex Meliae that 10g pulverizes, the 100ml distilled water immersion spends the night, and filters, and the Cortex Meliae after back gained liquid and the filtration is filtered in the inferior immersion of winning, add the 100ml distilled water in the Cortex Meliae after filtration again, after the soaked overnight, boiled Chinese medicine 4 hours, filter with the slow fire heating, get the liquid after infusion is filtered, back gained liquid is filtered in immersion for the first time and back gained liquid mixing is filtered in infusion, obtains mixed solution, obtains Szechwan Chinaberry Bark behind the lyophilizing mixed solution.Is 10mg/mL with Szechwan Chinaberry Bark with dissolved in distilled water to concentration, gets Cortex Meliae solution.

Embodiment 2

Neuraminidase (A/California/7/2009 (H1N1) NYMC X-179A c strains of influenza viruses goes down to posterity through Embryo Gallus domesticus, receives allantoic fluid, and-70 ℃ of preservations are standby) is dissolved in MES buffer (PH 6.5) and the 4mmol/L CaCl of 32.5mmol/L ₂In, get neural propylhomoserin enzymatic solution, get 49 μ L neuraminic acid enzymatic solution and 1 μ l Cortex Meliae solution was hatched 30 minutes in 37 ℃ of incubators, adding final concentration then is the buffer solution 50 μ l of the MUNANA of 20 μ mol/L, detect with Bio-Red FLx-800 fluorescence microplate reader immediately, excitation wavelength 360nm, emission wavelength 450nm, minute are 8min.Testing result is that Cortex Meliae is to the half-inhibition concentration (IC of neuraminidase ₅₀) be IC ₅₀(μ g/mL)=184.3 ± 13.1.

Embodiment 3

Neuraminidase (Clostridium perfringens (C.welchii), N2876 purchases in Sigma) is dissolved in MES buffer (PH 6.5) and the 4mmol/L CaCl of 32.5mmol/L ₂In, get neural propylhomoserin enzymatic solution, get 49 μ l neuraminic acid enzymatic solution and 1 μ l Cortex Meliae solution was hatched 30 minutes in 37 ℃ of incubators, adding final concentration then is the buffer solution 50 μ l of the MUNANA of 20 μ mol/L, detect with Bio-Red FLx-800 fluorescence microplate reader immediately, excitation wavelength 360nm, emission wavelength 450nm, minute are 8min.The power of inhibition degree can decide testing result to be from the initial reaction rate that adds or do not add Cortex Meliae, Cortex Meliae is to the half-inhibition concentration (IC of neuraminidase ₅₀) be IC ₅₀(μ g/mL)=0.76 ± 0.27.

Embodiment 4

In conjunction with Fig. 1 Fig. 2 present embodiment is described.

In order to detect the influenza effect of Cortex Meliae at cellular level, we will become different concentration by gradient dilution by the Cortex Meliae solution that embodiment 1 obtains, be respectively 31.25ug/ML, 62.5ug/ML, 125ug/ML and 250ug/ML, above-mentioned Cortex Meliae solution was hatched 2 hours with the viral equal-volume of 100TCID50 respectively, cumulative volume is 200 μ L, and diluent is all used and kept liquid (is serum-free, the DMEM that contains the 10ug/ml pancreatin), mixture with the virus of above-mentioned a series of Cortex Meliae solution and 100TCID50 is inoculated on the mdck cell culture plate that grows up to monolayer then, after 2 hours, change the liquid of keeping that does not contain FBS, change the liquid of keeping that contains 2%FBS after 24 hours, observed 2-3 days continuously, detect the cytopathy effect with mtt assay.Establish simultaneously only to add and keep liquid, only add or only add variable concentrations drug dilution liquid and organize in contrast, in 37 ℃, 5%CO with dilution viral liquid ₂Cultivate, the Chinese medicine Cortex Meliae is carried out antiviral activity research.

The result as shown in Figure 1, when not adding H1N1 virus, cell viability illustrates that along with the increase of Cortex Meliae concentration presents reduction trend Cortex Meliae extract has cytotoxicity; When adding Cortex Meliae extract and H1N1 virus at the same time, along with the increase of Cortex Meliae extract concentration, cell viability is also along with increase.Illustrate that Cortex Meliae can suppress influenza virus, the protection mdck cell is avoided virus damage, and protective effect increases along with the increase of Cortex Meliae extract concentration and promptly presents concentration dependent.

Fig. 2 is that the MTT of Cortex Meliae solution concentration and viral yield detects figure, and as seen from the figure, with the raising of drug level, viral yield descends gradually, and this is auxilliary has simultaneously demonstrate,proved Cortex Meliae better resisiting influenza virus effect.

Embodiment 5

In order to further specify the activity that Cortex Meliae has resisiting influenza virus, we have done zoopery.The female BALB/c mouse (being provided by Changchun Biological Products Institute) that six weeks are big is anaesthetized with 50 μ l, 0.2% pentobarbital sodium, and 50 μ l A/FM/1/47 (H1N1) influenza virus (being provided by Norman Bethune Medical University's molecular biosciences laboratory) are provided collunarium.Mice is divided into five groups, ten every group, one, two, three group of per 15 days Cortex Meliae extract of feed 40,80 and 160mg/kg respectively, feed is mice a few days ago implementing by influenza infection for the first time.Observed mortality of mice in per 15 days.The result is as shown in table 3, and therefrom the book number of elements of the death of three of the feed Cortex Meliae extract groups of mices is significantly less than the virus group as can be seen, and the existence natural law also obviously is longer than the virus group, illustrates that Cortex Meliae can increase by the survival rate of the mice of influenza infection.

Table 3

Claims

1. the application of Cortex Meliae in preparation inhibition influenza A H1N1 influenza virus medicine.

2. Cortex Meliae according to claim 1 suppresses application in the medicine of influenza A H1N1 influenza virus in preparation, it is characterized in that described Cortex Meliae is a kind of in raw medicinal herbs or the Cortex Meliae extract.

3. the application of Cortex Meliae according to claim 2 in the medicine of preparation inhibition influenza A H1N1 influenza virus, it is characterized in that, the preparation method of described Cortex Meliae extract is for after spending the night Cortex Meliae with distilled water immersion, filter, Cortex Meliae after time immersion of winning is filtered back gained liquid and filtered, Cortex Meliae after filtering is added after distilled water immersion spends the night again, boiled Chinese medicine 3-5 hour with the slow fire heating, filter, get the liquid after infusion is filtered, back gained liquid is filtered in immersion for the first time and back gained liquid mixing is filtered in infusion, obtains mixed solution, obtains Szechwan Chinaberry Bark behind the lyophilizing mixed solution.

4. the application of Cortex Meliae according to claim 3 in the medicine of preparation inhibition influenza A H1N1 influenza virus, it is characterized in that, in the preparation method of described Cortex Meliae extract, described with Cortex Meliae with distilled water immersion spend the night the back and will filter after Cortex Meliae add distilled water in the step after distilled water immersion spends the night again and replace with a kind of in methanol or the ethanol.