JP2007254319A - Influenza-preventing/treating agent - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an influenza-preventing/treating agent which contains a compound isolated from plant ingredients, has high influenza virus-killing and wounding activities, and little produces side effects. <P>SOLUTION: This influenza-preventing/treating agent is characterized by containing a Meliaceae plant or its extract. The present invention is based on a knowledge found in a process of researching the activities of plant ingredients, that the Meliaceae plant or its extract contains an ingredient for strongly killing and wounding human influenza viruses, avian influenza viruses, and type B influenza virus, and can be used to solve the problems. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to an influenza preventive / therapeutic agent, and more particularly, to an influenza preventive / therapeutic agent containing a plant-derived component and having an excellent effect on human influenza.

Influenza viruses are widely distributed in animals such as humans, horses, pigs, and birds and cause great damage. In particular, the distribution area of type A virus is deeply rooted in the human territory with roots as a root, and it is prevalent worldwide every year, especially in the winter, and its damage is immeasurable. Harm is no exception. For example, today's H5 bird flu epidemic in the territory of chickens has caused tremendous economic losses, and the appearance of this virus in the human world is a great fear. Furthermore, not only type A viruses but also type B viruses are prevalent every year in the human domain.
For virus infections, prevention by vaccine is the basic strategy rather than treatment, but no exception is given, and the basic influenza strategy is centered on prevention by vaccine. In addition to this method, a therapeutic drug represented by Tamiflu is now available, but this is a therapeutic drug and cannot be used as a preventive drug. In addition to Tamiflu, amandadine and rimandazine are also approved as therapeutic drugs, but the speed of the emergence of resistant viruses against this drug is high, and it is difficult to say that it is practically useful. Under such circumstances, effective influenza preventive drugs have not yet been developed in today's world. In that sense, the development of preventive anti-influenza substances effective against human and avian influenza as well as influenza B is urgently needed.
On the other hand, the present inventor has found that a plant belonging to the family Sendanidae or an extract thereof has an antitumor activity (Patent Document 1), but it is not yet known that these have an influenza virus killing activity.

JP 2004-256426 A

  The object of the present invention is to provide an influenza preventive / therapeutic agent having a high influenza virus killing activity and few side effects, comprising a compound isolated from plant components as an active ingredient.

In the process of studying the activity in plant components, the present invention includes a component of the family Sendanidae or an extract thereof that strongly kills human and avian influenza viruses and influenza B viruses. This is based on the knowledge that the above problems can be solved.
That is, the present invention provides an influenza preventive / therapeutic agent characterized by containing a ginseng plant or an extract thereof.

  According to the present invention, an influenza preventive / therapeutic agent having an excellent influenza virus killing effect and few side effects can be provided.

  The Sendai family used in the present invention is a plant belonging to the family Medeliaceae (Meliaceae), which is mainly an evergreen or deciduous high tree having a feathery compound leaf on a thick branch tip and having a conical inflorescence, but is not shrub or herbaceous. There are also. Of these, it is particularly preferable to use Sendan (Melia Azedarach L. or Melia Azedarach var. Subtripinnata). In the present invention, leaves, stems, branches, bark, and fruits of the family Sendanidae can be used. A root bark can also be used. In the case of using the ginseng plant itself as an active ingredient of the influenza preventive / therapeutic agent, it is preferable to dry these and then finely pulverize them or extract them directly.

In the present invention, the leaves, stems, branches, bark and berries of the gypsidaceae plant, for example, after being dried and pulverized, or in an undried raw state, with water, such as distilled water or ion-exchanged water, or The liquid itself extracted with a hydrophilic or hydrophobic organic solvent or a dried product thereof can be used. Examples of the organic solvent used here include ethyl acetate, carbon tetrachloride, chloroform, dichloromethane, methanol, ethanol, (iso) propyl alcohol, butanol, acetone, or DMSO. Here, the hydrophilic solvent can also be used in a water-containing form. The amount of water or solvent to be used can be set arbitrarily, but it is preferably used in an amount of 1/5 to 10 times, particularly preferably in an equivalent amount. The extraction is preferably performed at a temperature of 60 ° C. or lower, more preferably at room temperature, and particularly with stirring by a mixer or the like.
It is preferable that the active ingredient in the extract of the gypsidaceae plant has a molecular weight of 10,000 or less, more preferably a molecular weight of 4000 to 10,000 or 3000, and most preferably about 5,000.
The water or solvent extract can be used in the liquid state as it is, but it can also be dried and used in a solid form such as powder or granule.
In addition, when it is used as an influenza preventive / therapeutic agent containing a herbaceous plant or an extract thereof, in addition to these, various pharmaceutically acceptable substances for formulation, such as excipients, diluents, disintegrants, binding agents Agents, coating agents, lubricants, lubricants, lubricants, flavors, sweeteners, solubilizers and the like can be included as auxiliary agents. Specific examples include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, cellulose and derivatives thereof, animal and vegetable oils, polyethylene glycol, glycerol and the like.

The influenza preventive / therapeutic agent of the present invention is preferably by spray administration, but is not limited thereto. The influenza preventive / therapeutic agent of the present invention is preferably used in an amount of about 0.01 to 2 g per 1 kg body weight.
EXAMPLES Next, an Example demonstrates this invention in detail.

Example 1
Sendan liquid extraction method Sendan leaves were dried at 60 ° C, placed in a fabric bag, extracted with hot water at 60 ° C, and cold water extracted. This sendan extract was further partially purified stepwise using filter membranes of 1 million or more, 300,000, 100,000, 50,000, and 10,000 molecules after removing leaf contaminants with a centrifuge (6500 rpm, 30 minutes). The extract thus obtained (hereinafter referred to as “extracted extract”) was sterilized with 200 nm and 450 nm minopore filters and subjected to the following tests.
Virus inactivation test For virus inactivation, MD / CK / 8/34 (H1N1), A / Moscow / 1/00 (H3N2), B / Yamagata / 16/88 and avian influenza in canine kidney cells It was carried out by infecting the virus strain A / duck / Singapore-Q / F119-3 / 97 (H5N3). The infection method is to wash MDCK cells grown on the whole surface of a petri dish with a diameter of 60 mm once with minus PBS (phosphate buffered saline without divalent ions), inoculate with a virus with an infectivity of about 300 PFU, 30 minutes later After the adsorption time, the MEM medium for multi-layer containing agar was added, and the number of plaques that appeared after 3 days was counted. At the time of inoculation with the virus, a sendan partial purified component (extract extract) that had been diluted 2-fold in advance was mixed with the virus in an equal amount. The reaction time after mixing was set at 30 minutes. The virus infection titer was determined by dividing the average number of plaques that appeared in two dishes at each sample dilution point by the control virus, and calculating the plaque inhibition rate.
Specifically, two petri dishes were set at one dilution point, the number of plaques that appeared on the two sheets was calculated, and the plaque formation inhibition rate was examined based on the average value. The results are shown in Table 1.

表中、＃は、プラーク感染価は１希釈点で２枚のシャーレに形成された平均プラーク数で
ある。−＊は試験せずを意味する。 Table 1 shows the results when Sendend extract was diluted to various magnifications.

In the table, # is the average number of plaques formed in two petri dishes at 1 dilution point. -* Means not tested.

表中、*ウイルスの増殖はＨＡ活性で調べた。被検体１には抽出エキスの16倍希釈品、被検体２には抽出エキスの32倍希釈品を与えて検査に供した。増殖実験にはA/PR/8/34ウイルスを使用した。 Table 2. Effect of extract on influenza virus growth

In the table, * virus growth was examined by HA activity. The specimen 1 was given a 16-fold diluted product of the extract, and the specimen 2 was given a 32-fold diluted product of the extract. A / PR / 8/34 virus was used for propagation experiments.

From the results shown in Table 1, the sendan component is 100% -inhibited by A / PR / 8/34 virus plaques even when diluted to 4-fold, 8-fold, 16-fold, 32-fold, or 64-fold, and 128-fold. The virus inactivation effect was as high as 90% even at a dilution of 1. Similarly, A / Moscow / 1/00 of A / Hong Kong virus, and B virus also showed a plaque formation inhibition rate of 98% or more at a dilution point of 64 times, and was diluted 256 times. However, it was revealed that the virus blocking rate was over 80%. In addition to these viruses, H5 avian influenza virus was also used in the experiment, but it was found that even if the sample was diluted 128 times, it still showed high virus inactivation effect (91.1%).
From the above, it was shown that the components of Sendan can kill all influenza A and B viruses distributed in human and animal areas, and the possibility of use as preventive and disinfectant for influenza virus infection was shown. become. In addition to this plaque formation inhibition test, a sendan component was also added to the place where the virus was growing, and the influence on the growth curve progressed with time was also examined. The results are shown in Table 2, and it is clear that the growth of the virus control without added sendan component has already increased the HA activity by a factor of 8 on the second day after infection and reached the peak of 1024 on the second day. Became. In contrast, when the Sendan solution was diluted 16-fold and 32-fold, the amount of virus was not detected at all even after 4 days. Judging from this growth curve, Sendan extract has the ability to effectively kill viruses, so it is likely that it can be used as an anti-influenza drug in a wide range of product development.

Example 2: Influenza virus infection prevention test
Four-week female ddY mice were anesthetized with barbital and 20-40 microliters of test samples were inoculated intranasally and infected. Each sample contains only about 1000 PFU (plaque infectivity) virus solution, a mixture of virus solution and Sendan extract (extracted extract obtained in Example 1: undiluted product), and Sendan solution to the nose of mice before infection. Virus was inoculated into the nasal cavity of the sprayed mice. Each group of test mice was composed of 5 to 7 mice, and the evaluation was finally conducted by examining the body weight and survival rate of the mice. In addition, the incidence of pneumonia was also examined during the study period.
Table 3 shows the results of the survival rate of the mice after administration of the extract (ML1) after inoculation with influenza virus (A / PR / 8/34).

表中の数字１、２、３、−−−は、インフルエンザ感染後の日数を示す。
表３に示されている結果から、ウイルスだけを接種したマウス群は、接種後3日目から体重が急激に減少し肺炎が強く進行していることが示唆された。その後も体重は減少し続け、7日目におけるマウスの生存率は60％、さらに9日目には40％の生存率となっていた。体重の減少から判断して、その後も生存率は小さくなっていくものと考えられる。しかしながら、感染前にセンダン成分を鼻腔内に吹きつけたマウス群の体重は、全実験期間中にわたって体重の増加は順調に推移し、肺炎の発生は未然に食い止められていることが明らかになった。また、ウイルスとセンダン成分を混合して感染させたマウス群には体重の減少は認められず、センダンの成分によるウイルスの不活化がマウスへの感染が成立させなかったことが明らかになった。このことを反映して、結局、マウスの鼻腔内へのセンダン成分の吹きつけ、さらに、感染前のウイルスへの同成分を混合されたマウスの生存率は100％となった。このようなことから、センダン成分に著しいインフルエンザウイルス感染予防効果のあることが確認された。また、感染病理学的側面から調べてみても、ウイルスとセンダン成分を混合した場合、あるいは感染前にセンダン成分を鼻腔内に吹きつけてからウイルスを感染させたマウスには肺炎所見は見られなかった。このときのウイルスだけを鼻腔に接種したマウスの肺炎所見は100％の肺面積に広がり、センダン成分で処置したマウス群の肺炎発生が全くなかったこととは対象的な違いを見せた。

Numbers 1, 2, 3, and-in the table indicate the number of days after influenza infection.
From the results shown in Table 3, it was suggested that in the group of mice inoculated with the virus alone, the body weight rapidly decreased from the third day after the inoculation, and pneumonia progressed strongly. After that, the body weight continued to decrease, and the survival rate of mice on the 7th day was 60%, and on the 9th day, the survival rate was 40%. Judging from weight loss, the survival rate is expected to decrease thereafter. However, the body weight of the group of mice sprayed with the sendan component into the nasal cavity before infection showed that the increase in body weight was steady over the whole experiment period, and the occurrence of pneumonia was stopped. . In addition, the group of mice infected with a mixture of virus and sendan component did not lose weight, and it became clear that inactivation of the virus by the sendan component did not establish infection in mice. Reflecting this, in the end, the survival rate of mice that were sprayed with the sendan component into the nasal cavity of the mouse and further mixed with the same component to the virus before infection was 100%. From these facts, it was confirmed that the sendan component has a remarkable effect of preventing influenza virus infection. In addition, even when examined from the pathological aspect of infection, there was no pneumonia observed in mice infected with the virus after mixing the virus and the sendan component or by spraying the sendan component into the nasal cavity before infection. It was. At this time, the pneumonia findings of the mice inoculated with only the virus in the nasal cavity spread to 100% of the lung area, showing that there was no occurrence of pneumonia in the mice treated with the sendan component.

  From the above results, it was evaluated that the influenza virus preventive effect of the sendan component is extremely significant. Based on this finding, the advantage of the availability of influenza drugs such as vaccines and Tamiflu to date is certainly a major advance. However, it is still more important to stop infection in advance. In this sense, it is strongly suggested that the anti-influenza virus agent containing the sendan component of the present invention has the ability to kill all influenza viruses and directly inactivates viruses that enter from the nasal cavity and mouth. It can be used for the development of various forms of influenza prevention drugs. For example, it can be used for sprays for preventing influenza and gargles for preventing influenza that enter from the nasal cavity. In addition, there is a possibility that it can be used as a spray for home influenza prevention, as well as a disinfectant in a large-scale facility such as a company or hospital, or a station or poultry farm. Is extremely expensive.

Claims (3)

  An influenza preventive / therapeutic agent characterized by containing a plant of the family Sendanidae or an extract thereof.   The influenza preventive / therapeutic agent according to claim 1, wherein the extract of the family Sendanaceae is an aqueous extract of leaves, berries, branches or barks of the family Sendanaceae.   The influenza preventive / therapeutic agent according to claim 1, wherein the extract of the plant family is an organic solvent extract of leaves, fruits, branches or bark of the plant family.