CN102920686A - New application of patchoulic alcohol - Google Patents

Abstract

The invention discloses an application of patchoulic alcohol in the preparation of drugs against coxsackie virus. The patchoulic alcohol can effectively restrain synthesis of coxsackie virus B type 3, has a certain function against the coxsackie virus, and can be used in the treatment of coxsackie virus infectious diseases, especially coxsackie virus B type 3 infectious diseases; and simultaneously, the patchoulic alcohol is far less poisonous to host cells compared with positive drug ribavirin, and lower in toxic and side effects, thus providing a safe and effective new choice for clinical medication.

Description

The new purposes of patchouli alcohol

Technical field

The present invention relates to the new purposes of patchouli alcohol.

Background technology

Coxsackie virus (Coxsaekieviurs) belongs to Picornaviridae (Picomaviridea), and enterovirus genus (Enterroviurs) can be divided into A group and B group according to its difference to the neonatal rat pathogenecity.This viroid does not have mantle, underlying stock RNA, and the virus of positive 20 body protein housings, it is the prime that is used as rna replicon that its genosome has an albumen at 5 ' end.Wherein, Coxsackie B virus (Coxsackievirusgroup B, CVB) can cause epidemic pleurodynia, aseptic encephalitis, meningoencephalitis and pericarditis, especially CVB3 is the main pathogens of viral myocarditis, can cause that focal necrosis occurs in cardiac muscular tissue, and with inflammatory cell infiltration, the pathological changes such as myocardial cell cracking, persistent viral infection can cause DCM (dilated cardiomyopathy), has brought great threat to the mankind.

Since the nineties in 20th century, research worker is found many new antiviral drugs, and the antiviral drugs of using has clinically at present surpassed more than 20 plants, but its definite curative effect still has very large dispute.Because virus is to colonize in the host cell, guarantee that antiviral drug has high selectivity, should enter cell, the optionally effectively reproduction process of viral interference, avoid again injuring host cell, this is that antiviral especially exists selectivity height and the high main cause of toxic and side effects in the Respirovirus Western medicine more.Such as ribavirin, can damage erythrocytic cell membrane, cause hemolytic anemia.Therefore, the research of antiviral drugs has important practical significance, and the research of antiviral drugs of seeking a kind of efficient, safety, few side effects is imperative.

Patchouli alcohol is the main component of Herba Pogostemonis Volatile oil, is to go through version " the evaluation Herba Pogostemonis medical material of Chinese pharmacopoeia regulation and the index composition of patchouli oil quality standard.Patchouli alcohol claims again patchouli alcohol, is a kind of tricyclic sesquiterpene chemical compound that exists in the natural plants, and its molecular formula is C ₁₅H ₂₆O, molecular weight is 222.37, structure is as follows:

The patchouli alcohol monomer is clear crystal, and lighter patchouli aroma is arranged, and its fusing point is 55～56 ℃, 280 ℃ of boiling points (under the normal pressure), and relative density is 1.0284, optical rotation-97.4 ° (c=24, chloroform); Water insoluble, be dissolved in alcohol, ether and organic solvent commonly used.Modern pharmacology is tested and is shown, patchouli alcohol has the pharmacologically actives such as antiinflammatory, antibacterial, anti-young worm, antioxidation, the unusual rising of antagonism calcium ion, has certain new drug development to be worth.

At present, yet there are no the relevant report of using the anti-Coxsackie virus of patchouli alcohol and treatment Coxsackie virus infection disease.

Summary of the invention

The object of the present invention is to provide the new medical use of patchouli alcohol.

The invention provides the purposes of patchouli alcohol in the medicine of the anti-enterovirus genus virus of preparation.

Wherein, described enterovirus genus virus is Coxsackie virus.

Further, described Coxsackie virus is the Coxsackie B virus group.

Further preferably, described Coxsackie virus is Coxsackie B virus group 3 type CoxsackievirusB3.

Wherein, described medicine is the medicine for the treatment of Coxsackie virus infection disease.

Further, described Coxsackie virus infection disease is epidemic pleurodynia, aseptic encephalitis, meningoencephalitis, pericarditis or viral myocarditis.

Wherein, described medicine is to be active component by patchouli alcohol, adds the preparation that adjuvant pharmaceutically commonly used is prepared from.

Further, described preparation is oral formulations, ejection preparation or external preparation.

Patchouli alcohol can synthesize by establishment Coxsackie B virus group 3 types, has certain anti-Coxsackie virus effect, can be used for treating the Coxsackie virus infection disease, especially Coxsackie B virus group 3 type infectious disease; Simultaneously, far below the positive drug ribavirin, toxic and side effects is lower to host cell toxicity for patchouli alcohol, newly selects safely and effectively for clinical application provides a kind of.

The specific embodiment

Patchouli alcohol can obtain by buying the commercial goods, also can prepare by existing isolation and purification method.Used patchouli alcohol in the specific embodiment of the invention, after identifying, purity is greater than 95%.

The effect of the anti-Coxsackie virus of embodiment 1 patchouli alcohol

1, material and instrument

1.1 cell strain and Strain

1.1.1 cell strain:

Hela cell (cervical cancer cell) is available from Chinese medicine and biological products assay institute.

1.1.2 Strain:

Coxsackie B virus group 3 types (the following CVB-3 that is called for short) are derived from the clinical separation strain that visiting center, Sichuan Province provides.

1.2 medicine and control drug

Trial drug: patchouli alcohol.

Positive drug: ribavirin injection: specification is 100mg/ml, and Tianjin Pharmaceutical Jiaozuo Co., Ltd. produces.

Authentication code: the accurate word H19992467 of traditional Chinese medicines, product batch number: 10110521;

1.3 culture medium, reagent and consumptive material

RPMI-1640 culture medium (Gibco ^TMCompany, lot number 1313945); Hyclone (HyClone biochemistry goods (Beijing) company limited, lot number NVM0347); Trypsin Amresco company); Penicillin G sodium, the aseptic Tissue Culture Flask of streptomycin and 96 porocyte culture plates (U.S. Corning company).1,5, the 10ml Dispette, the disposable rifle head of 200 μ l, 1ml (the biological consumptive material in Haimen, Jiangsu company)

1.4 key instrument

Water isolation type constant temperature CO ₂Incubator (model MCO-15AC, SANYO GS company);

Microplate reader (model Varioskan Flash, U.S. Thermo Scientific company);

OLYMPUS inverted microscope (model C KX41, Japanese Olympus company);

Micropipettor (French GILSON company produce);

Water isolation type electro-heating standing-temperature cultivator (model GSV-DA-1, Huangshi, Hubei Province medical apparatus and instruments factory);

Horizontal centrifuge (model LXJ-II, Shanghai medical analytical instrument factory);

Electronic balance (model JA-2603, Shanghai balance equipment factory)

2, test method

2.1 the pre-treatment of medicine and positive control

Patchouli alcohol is mixed with the volume ratio of 2:1 with soybean oil first, and then adding 0.5% tween 80 hydrotropy is the white liquid preparation, and its initial concentration is 100mg/ml, and is for subsequent use after the filtration sterilization.

To do 1/10 dilution (being 10mg/ml) with for subsequent use with sterilized water for the positive control drug 'Libaweilin ' injection of Coxsackie virus.

The mensuration of 2.250% histiocyte infective dose

Coxsackie B virus 3 usefulness 3% hyclone RPMI-1640 is diluted to the virus liquid of variable concentrations.

Coxsackie B virus 3 strains inoculation Hela cell, in 37 ℃, 5%CO ₂Observe cytopathy (cytopathiceffect, the CPE) situations such as the infection cell circle contracts, comes off after cultivating 7d in the incubator, cause infective dose (50%tissue culture infective dose, the TCID of 50% pathogenic histiocyte to measure each virus ₅₀).

2.3 the cytotoxic assay of patchouli alcohol

In advance 100 times of dilutions of patchouli alcohol for subsequent use will be prepared, join in the Hela cell monolayer with 7 concentration of the continuous two-fold dilution of RPMI-1640 cell culture fluid configuring patchouli alcohol for subsequent use and positive control drug ribavirin, each medicine of different dilution factors all repeats 3 holes.37 ℃, 5%CO ₂Cultivated observation of cell pathological changes situation every day (CPE) 5 days.Be judged to be half toxic concentration (TC with the drug level that causes half cell generation pathological changes ₅₀), be judged to be maximal non-toxic concentration (TC with the drug level that does not cause cell generation pathological changes fully ₀).Continuous two-fold dilution's 0.5% tween 80 and normal cell contrast established in experiment.

2.4 antiviral experiment

According to the replicative cycle of virus, three kinds of approach of design patchouli alcohol antivirus action, every kind of virus is carried out three groups of route of administration experiments, and each group is all established normal cell matched group, virus control group and positive drug matched group.Concrete operations are as follows:

2.4.1 Antiviral Effect Biotechnology Compose Experiment (going up first poison)

The corresponding virus liquid of 100 μ l * TCID50 is added in the intact corresponding monolayer cell culture plate of growth, 37 ℃, 5%CO ₂After hatching 2h, discard virus liquid, the patchouli alcohol liquid 100 μ l and the cell maintenance medium that add variable concentrations continue to cultivate again.

2.4.2 Antiviral Effect adsorption experiment (first medicine-feeding)

The patchouli alcohol culture fluid 100 μ l of variable concentrations are added in the intact monolayer cell culture plate of growth, 37 ℃, 5%CO ₂After hatching 2h, discard the pastille culture fluid, add again the corresponding virus liquid of 100 μ l * TCID50,37 ℃, 5%CO ₂After hatching 2h, discard virus liquid, add again cell maintenance medium and continue to cultivate.

2.4.3 medicine direct effect experiment (neutralization)

100 μ l patchouli alcohol culture fluid of variable concentrations and the corresponding virus liquid of 100 μ l * TCID50 are mixed, 37 ℃, 5%CO ₂After hatching 2h, join again in the intact corresponding monolayer cell culture plate of growth, 37 ℃, 5%CO ₂After hatching 2h, discard pastille and virus-culturing fluid, add again cell maintenance medium and continue to cultivate.

Each culture plate is in 37 ℃, 5%CO2 continuation cultivation 5d, and every day, microscopically was observed CPE.Use the Reed-Muench method and calculate the 50% cytopathic effect inhibition concentration (50%inhibiting concentration, IC50) of each medicine, the IC50 value that occurs with the high dilution of medicine is as the terminal point of antiviral effect judgement.Each concentration of above patchouli alcohol is done 3 multiple holes, and every group of experiment repeats 3 times,

3. experimental result and pharmacodynamic index calculate

3.1 the TCID of virus ₅₀Value

CPE approximately appearred in 5 days behind the Coxsackie B virus 3 inoculation Hela cells.The TCID of Coxsackie B virus 3 ₅₀Tire and be 10 ^-12

3.2 the cytotoxicity of patchouli alcohol

Within the tested time, the cellular morphology in normal control hole is normal.Patchouli alcohol the results are shown in Table 1 to the toxicity test of three kinds of cells.

The cytotoxic assay result of table 1 patchouli alcohol and ribavirin

Shown by the cytotoxicity experiment result, patchouli alcohol is consistent to the toxicity result of three kinds of cells.Wherein: patchouli alcohol is to the TC of cell ₅₀Concentration is 1/800(0.125mg/ml), TC ₀Concentration is 1/3200(0.031mg/ml); Positive control drug 'Libaweilin ' injection is to the TC of cell ₅₀Concentration is 1/20(5.00mg/ml), TC ₀Concentration is 1/80(1.25mg/ml); 0.5% tween after doing 1/4 dilution (namely 0.125%) namely to three kinds of equal avirulences of cell.

Experiment shows, patchouli alcohol is extremely low to cytotoxicity, much smaller than the cytotoxic effect of positive drug ribavirin.

3.3 patchouli alcohol antiviral experiment effect and pharmacodynamic index calculate

3.3.1 anti-Coxsackie virus 3 type experimental result and the pharmacodynamic indexs of patchouli alcohol:

Normal control porocyte form is all normal within the under test time, CVB ₃Infect the control wells cell and obvious CPE within the time of estimating, occurs.Three kinds of route of administration experimental results of patchouli alcohol see Table 2.

The anti-CVB of table 2 patchouli alcohol ₃Experimental result (n=3)

Shown by upper table, ribavirin injection has anti-CVB ₃Effect, IC ₅₀Concentration is 1/1280(0.078mg/ml); Patchouli alcohol is to CVB ₃Certain inhibitory action is arranged, and the effect of its Chinese medicine direct effect (neutralization) experiment is relatively best, IC ₅₀Concentration is 1/3200(31.25mg/ml); The effect of Antiviral Effect biosynthesis (going up first poison) and Antiviral Effect absorption (first medicine-feeding) experiment is suitable, IC ₅₀Concentration is 1/1600(62.50mg/ml).

In sum, patchouli alcohol can synthesize by establishment Coxsackie B virus group 3 types, has certain anti-Coxsackie virus effect, can be used for treating the Coxsackie virus infection disease, especially Coxsackie B virus group 3 type infectious disease; Simultaneously, far below the positive drug ribavirin, toxic and side effects is lower to host cell toxicity for patchouli alcohol, newly selects safely and effectively for clinical application provides a kind of.

Claims (8)

1. Patchouli alcohol the preparation anti-enterovirus genus virus medicine in purposes.
2. 2. purposes according to claim 1, it is characterized in that: described enterovirus genus virus is Coxsackie virus.
3. 3. purposes according to claim 2, it is characterized in that: described Coxsackie virus is the Coxsackie B virus group.
4. 4. purposes according to claim 3, it is characterized in that: described Coxsackie virus is Coxsackie B virus group 3 type Coxsackievirus B3.
5. 5. purposes according to claim 2 is characterized in that: described medicine is the medicine for the treatment of Coxsackie virus infection disease.
6. 6. purposes according to claim 5, it is characterized in that: described Coxsackie virus infection disease is epidemic pleurodynia, aseptic encephalitis, meningoencephalitis, pericarditis or viral myocarditis.
7. 7. purposes according to claim 1 is characterized in that: described medicine is to be active component by patchouli alcohol, adds the preparation that adjuvant pharmaceutically commonly used is prepared from.
8. 8. purposes according to claim 7, it is characterized in that: described preparation is oral formulations, ejection preparation or external preparation.