JP2001010967A - Antichlamidial agent - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an antichlamidial agent which shows antimicrobial effect, e.g. detergency, disinfection or the like, on chlamidia, exhibits increased antimicrobial effect on chlamidia particularly before or just after infection and exerts excellent infection prophylactic effect or detergent effect. SOLUTION: This antichlamidial agent is obtained by including an extract obtained by using a bamboo, e.g. Phyllostachys pubescens or the like, and an alcohol or ether as an extraction raw material and an extracting solvent, respectively, as an active ingredient. This antichlamidial agent contains benzoquinone in the extract, increases the infection preventive effect of somatic cells on infectious elementary bodies(EB) in the life cycle of chlamidia and exhibits a certain antimicrobial effect due to annihilating intracellular reticular bodies(RB). Further, the more certain antimicrobial effect is exhibited when the extract contains liquid benzoquinone in a formation amount of 0.2-5 vol.%.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anti-Chlamydia agent which suppresses the infection of humans with Chlamydia, which is a pathogenic microorganism, and the growth after infection.

[0002]

2. Description of the Related Art Chlamydia, a pathogenic microorganism
The genus dia) has been classified as an independent genus, neither a bacterium nor a virus, since 1974, including Chlamydia trachomatis (Chlamydia pneumoniae)
(C. pneumoniae), 3 of Chlamydia psittaci
Seeds are known. Of these, Chlamydia Shitashi is
It is a common zoonotic pathogen, a pathogen of parrot disease that infects birds and infects humans and causes pneumonia, and Chlamydia pneumoniae is known as a pathogen of respiratory infections that transmit humans from human to human.

[0003] Chlamydia trachomatis is a causative agent of eye infection and urinary tract infection, and causes birth canal infection in the eyes and respiratory tract of newborn babies. In Japan, sexually transmitted diseases: STD)
Is the main etiological agent.

The species of the genus Chlamydia, which is such a pathogenic microorganism, has a life cycle that proliferates in living cells, unlike bacteria, and is a virus in that it has both DNA and RNA nucleic acids. Is a different pathogen.

[0005] Incidentally, the life cycle of Chlamydia (48 to 72 hours per cycle) is schematically shown in FIG. Chlamydia includes elementary bodies (EBs) that propagate from solid to solid or from host cell to cell, and reticulate bodies (RBs), which are non-infectious and mitotically proliferative. There is a time. When an EB (a sphere having a diameter of about 0.3 μm) is invaded into a host cell and infected, the EB is converted into an RB (a sphere having a diameter of about 0.5 μm) inside the cell, and the EB is further converted into an RB (a sphere having a diameter of about 0.5 μm).
After mitosis, they mature through intermediates, are released again as EBs, and infect other hosts or transfer to other cells of the same host.

[0006] As a conventional anti-chlamydia agent which suppresses the infection of humans with the pathogenic microorganisms of the genus Chlamydia and the proliferation after infection, tetracycline antibiotics such as tetracycline, doxycycline and minocycline are most recommended. Then, macrolide antibiotics such as erythromycin and rifampicin are excellent in antibacterial activity. In addition, clindamycin, penicillin-G, ampicillin and the like are known to exhibit some anti-chlamydia action.

[0007] The mechanism of action of antibiotics such as tetracyclines on chlamydia utilizes the specificity of tetracycline for ribosomes in host cells and ribosomes in chlamydia cells. That is, the ribosome of the host animal has a protein precipitation constant of 8
0S, which is composed of 40S and 60S subunits, whereas the intracellular ribosome of Chlamydia has a protein precipitation constant of 70S,
It is composed of 0S and 50S subunits.

[0008] In response to these differences in ribosome structure, tetracycline antibiotics act on smaller subunits of ribosomes in Chlamydia cells,
It is said to inhibit its protein synthesis.

As an extract of a general-purpose natural material, which has antibacterial properties against bacteria, yeasts and molds, and which can be used in foods and cosmetics, it can be used as a molybdenum (Phyllo) extract.
stachys heterocycla var pubescens), a water extract, an ether extract, and an organic solvent extract are disclosed in
-290825.

[0010]

However, from the above publications, there is no example of the use of Moso bamboo-derived antibacterial extracts other than foods and cosmetics, and in particular, antibacterial properties against Chlamydia, a pathogenic microorganism different from bacteria and viruses. Cannot be found, and there is no confirmation that bamboo-derived antibacterial extracts can be actually used for use as anti-chlamydia agents.

Further, conventional tetracycline antibiotics are remedy after Chlamydia infection, but they are not anti-Chlamydia agents which have positively improved EB infection prevention in the Chlamydia life cycle, but have an effect of preventing infection. For example, there is no cleaning agent that can be applied as a genital cleaning agent or a medical device cleaning agent for preventing STD infection by enhancing the cleaning effect immediately after infection.

[0012] Therefore, an object of the present invention is to solve the above-mentioned problems of the prior art and to make sure the antibacterial action such as washing and disinfection against Chlamydia, and particularly to the antibacterial activity before or immediately after infection with Chlamydia. And to provide an anti-Chlamydia agent excellent in infection prevention effect and cleaning effect.

[0013] It is another object of the present invention to develop a new use of an antibacterial extract derived from bamboo and to provide an anti-chlamydia agent which is safe and surely has a preventive effect against infection, in response to the frequent demands of consumers who prefer natural products.

[0014]

In order to solve the above-mentioned problems, the present invention provides an anti-chlamydia agent containing bamboo as an extraction raw material and an extract obtained using alcohol or ether as a solvent as an active ingredient. It is. This anti-chlamydia agent contains an antibacterial substance such as benzoquinone as an extract, and has an enhanced antibacterial effect on somatic cells against EB, and has a reliable antibacterial effect by killing intracellular RB. Moreover, when the compounding amount of the extract is a compounding amount containing 0.2 to 5% by volume of liquid benzoquinone, more reliable antibacterial action is obtained.

That is, the anti-Chlamydia agent of the present invention comprises:
Alcohol or ether extract is the active ingredient. Such extracts contain benzoquinone as one of the active ingredients, and such plant-derived benzoquinone, as approved in Japan as a food additive, can be in contact with the human body or ingested. It is a substance that has been confirmed to be safe when used, and can be used safely as an ingredient for preventing and treating Chlamydia infection.

Accordingly, the anti-chlamydia agent of the present invention may be used, for example, as a genitalia-washing disinfectant such as an eye washing solution, a vaginal suppository or an intravaginal washing solution, an antibacterial agent to be added to a laundry detergent during washing, There are antibacterial agents and the like used while being held by antibacterial fiber products.

Bamboo-derived benzoquinone obtained by using ethanol or ether as an extraction solvent includes p-benzoquinone, which is a yellow needle-like crystal having a melting point of 116 ° C. and has an odor similar to chlorine, It is a physical property that a compound is easily added to oxygen and thus has an electron supply function. Incidentally, o-benzoquinone decomposes in one day when left to stand, and rapidly decomposes in an aqueous solution, so that the amount contained in the bamboo-derived ethanol or alcohol extract decreases with time after production.

The antibacterial action mechanism of such an extract containing benzoquinone against Chlamydia is considered as follows.

Chlamydia requires ATP (adenosine triphosphate) as energy when synthesizing proteins with intracellular ribosomes.
Is thought to be obtained from parasitic cells. Since bamboo extract containing benzoquinone has a reducing effect to prevent electron transfer in energy metabolism using ATP, ATP, which is a protein synthesis energy, is cut off, and chlamydia becomes elementary bodies (EB) and net-like. It is thought to die at any stage of the body (RB).

In addition, for the Chlamydia basic body (EB), the cell membrane potential of the infected cells is negatively charged by the benzoquinone-containing bamboo extract.
Is considered to be unable to attach and enter the infected cells. By the way, there is a knowledge that if the membrane potential of the cell membrane is negatively charged by a polysaccharide such as heparin, Chlamydia EBs cannot adhere to and enter the infected cells (HeLa cells) (Cell N
o.69, p861-869,1992).

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION Bamboo, an extraction raw material of the present invention, is a plant belonging to the subfamily Bamboo, which has antibacterial properties, and includes stems (aboveground parts), rhizomes,
Sites such as roots, leaves, and branches can be appropriately selected or mixed for use.

Examples of suitable types of bamboo include bamboo, mushroom, bee, black croak, yam, and medaka. Among them, Moso bamboo (Phyllostachys heterocycla var pubescens) is recognized as an extract having good antibacterial activity.

Using such bamboo as an extraction raw material, extraction is performed with alcohol or ether under normal pressure or reduced pressure. Alcohol and ether used in the extraction operation include
It is easy to handle liquid at normal temperature and pressure. A well-known extraction solvent (ethyl alcohol, methyl alcohol, ethyl ether, isopropyl ether, etc.) may be used without particular limitation, and thereafter, the alcohol or ether may be distilled off. Ethyl alcohol may be used as a solvent at the stage of the production of anti-chlamydia drugs, but in that case, it is necessary to take into account the irritation and safety of contact with the human skin, etc. Is formulated so as to satisfy general safety standards.

In particular, when Moso bamboo is used as an extraction raw material, it is preferable to employ an ether extraction operation disclosed in JP-A-63-290825. In this method, the stem of the Moso bamboo or the trunk or the epidermis of the trunk is directly or crushed and extracted with ether, and then the ether is distilled off. More specifically, Moso bamboo treated at 160 ° C. and 5 kg / cm ² for 40 minutes is powdered, and the powder is extracted with ether such as ethyl ether, and then the ether is distilled off. Can be used.

When the concentration is adjusted at the stage of the final product, about 5 to 30% of a solvent having cell permeability, such as ethanol, methanol or physiological saline, may be added.

In order to stabilize the anti-Chlamydia effect of the extract, 1000 μl
2 μl of the extract (the solvent was distilled off)
Concentration, that is, 0.2% by volume or more (for example,
It is preferable to contain quinones (benzoquinone) in an amount of 0.2 to 5% by volume or 0.2 to 1.6% by volume, and the concentration is adjusted according to the form of the preparation or the purpose. For example, when used as a vaginal suppository, the effective concentration of quinones added to gelatin is preferably adjusted to 0.5 to 10% by volume. Further, when another antibiotic such as minomycin is used in combination, the amount of the other antibiotic can be reduced by adding the extract.

Other uses and uses of the anti-chlamydial agent containing the obtained extract as an active ingredient include cleaning agents for gynecological, urological and ophthalmic examination instruments, particularly medical examinations such as endoscopes. Cleaning and disinfecting agents. When used as a medical cleaning liquid such as an esophageal cleaning liquid or a bidet cleaning liquid, about 0.2 to 1.6% by volume of isotonic or hypotonic physiological saline is added to a living body. .

Further, the obtained extract can be impregnated into a fiber product such as a knitted fabric or a nonwoven fabric, and used in combination with a well-known anti-inflammatory agent, if necessary, and applied to the affected area of inflammation. In that case, the preferred effective concentration of benzoquinone or a derivative thereof is 400-800 μl / ml. When the extract is applied to the fibers, the fibers are mixed with a physiologically active peptide or a functional protein such as collagen or lactoferrin, and the physiologically active peptide is cross-linked as necessary, and the active ingredient is fixed to the fiber product. You can also put it.

Further, when washing clothes which may be infected with Chlamydia, the anti-Chlamydia agent of the present invention may be added together with a detergent, that is, sterilization and removal of Chlamydia adhering to clothes such as underwear. it can. When the target of sterilization is Chlamydia trachomatis, the concentration of benzoquinone is 0.5 to 2% by volume in eye drops for treating conjunctivitis.
The extract may be blended so that

[0030]

[Examples 1 to 4, Comparative Examples 1 to 4] Ethyl ether was obtained from an extract obtained by pulverizing raw moso bamboo (Phyllostachys heterocycla var pubescens) as an extraction raw material and using ethyl ether as a solvent. The extract obtained by distillation was used as an active ingredient, and the extract was added to physiological saline at a ratio (volume%) shown in Table 1 to obtain an anti-chlamydia agent (cleaning agent) of Examples 1 to 4.

The determination of the anti-Chlamydia effect (MLC measurement) is carried out by the standard method of the Japan Society for Chemotherapy or a modified method based thereon, as described below.
The evaluations were (1) MLC method (whether or not the reticular body was broken), (2) modified method (whether or not the basic body was broken), and (3) MLC method (whether or not the basic body and reticular body were broken) ), And these results are also shown in Table 1.

(1) <Chlamydia MLC measurement method (Standard method of the Japan Society for Chemotherapy)> A 24-well cell culture plastic plate was used, and a culture solution containing 1500-2000 / ml Hela cells was used. Was. The composition of the culture solution was prepared by using EagleMEM and heat-inactivated fetal bovine serum in a volume of 1 ml per well (hole) at 37 ° C and 5% carbon dioxide (CO2).
_The cells were cultured in the incubator in ₂ ) for 24 hours. After confirming the formation of the confluent monomer in the cells, the culture solution was removed by suction.

In this well 4000 / ml chlamydia
0.25 ml of the trachomatis strain (D / UW-3 / CX)
After inoculation, centrifugal adsorption was performed for 1 hour at room temperature by applying a gravitational acceleration of 500 to 900 G.

The dilution of the antimicrobial solution, performed in the master da illusion, diluent using Eagle and heat-inactivated fetal bovine serum, which was added 1ml of wells inoculated with Chlamydia trachomatis, 37 ° C., in 5% CO ₂ 72 After incubation for a period of time, the drug in the wells is removed by suction, and serum-free Eagle ME
After washing three times with M, 1 ml of heat-inactivated fetal bovine serum supplemented with Eagle MEM was added thereto, followed by culturing at 37 ° C and 5% CO ₂ for 24 hours.

The determination of the antibacterial effect was carried out by arbitrarily measuring 200 reticulated bodies (R
In B), the number of destroyed RBs was counted and the percentage (%) was used.

(2) Chlamydia MLC of the above (1)
In the measurement method (standard method of the Japan Society for Chemotherapy), antibacterial judgment was performed based on whether or not the basic body was destroyed.

The antibacterial effect was determined by counting the number of destroyed EBs in any 200 elementary bodies (EBs) observed in one visual field with a fluorescence microscope at a magnification of 200. Performed by percentage (%).

(3) <Variant of Chlamydia MLC> A 24-well plastic plate for cell culture was used.
A culture solution containing ２０００2000 / ml of Hela cells was used.
The composition of the culture solution was prepared by using EagleMEM and heat-inactivated fetal bovine serum in a volume of 1 ml in each well (hole) of the plate, and culturing for 24 hours at 37 ° C. in an incubator of 5% carbon dioxide (CO ₂ ). Then, after confirming the formation of the confluent monomer in the cells, the culture solution was removed by suction.

In this well 4000 / ml chlamydia
0.25 ml of the trachomatis strain (D / UW-3 / CX)
And the anti-Chlamydia agent to be tested (Examples 1-4,
Eagle MEM and heat-inactivated fetal calf serum were used. ) Was inoculated into a well in which 1 ml of an antimicrobial-contacted chlamydial culture was formed in a Hela cell for 1 hour at 37 ° C and 5% CO ₂ in a confluent monomer-formed Hela cell, and this was inoculated at room temperature for 500 to 9 hours.
Centrifugal adsorption was performed for 1 hour while applying a gravitational acceleration of 00G.

After culturing at 37 ° C. and 5% CO ₂ for 72 hours, the drug in the wells was removed by suction, and serum-free Eagle ME was added.
After washing three times with M, 1 ml of heat-inactivated fetal bovine serum supplemented with Eagle MEM was added thereto, followed by culturing at 37 ° C and 5% CO ₂ for 24 hours.

The determination of the antibacterial effect was determined by examining the number of destroyed EBs and EBs in any 200 elementary bodies (EB) or reticulate bodies (RB) observed in one visual field with a fluorescence microscope at 200 ×. The number of RBs was counted, and the measurement was performed based on the ratio (%).

[0042]

[Table 1]

As is clear from the results in Table 1, the anti-Chlamydia agents of Examples 1 to 4 were used at a concentration of 0.2 to 1.6% by volume, corresponding to the EB and Chlamydia corresponding to the time of growth or infection. Destruction of RB, showing obvious antibacterial properties.

If the concentration of the extract of Moso bamboo is too high, the cell membrane is destroyed, which hinders the judgment of the drug effect. Therefore, the experiment was carried out at a concentration that does not cause cell disruption. That is, although the benzoquinone concentration in the example was set to 1.6% by volume or less, it was clear that an antibacterial effect was obtained even at a higher concentration.

[0045]

As described above, the present invention provides an anti-chlamydia agent containing bamboo as an extraction raw material and an extract obtained using alcohol or ether as a solvent as an active ingredient. Body (EB),
They die at any stage of the reticulum (RB), and the basic body of Chlamydia (EB) is unable to attach and enter the infected cells, and has a better antibacterial effect on Chlamydia such as washing and disinfection. It is a reliable anti-Chlamydia agent.

Therefore, the anti-Chlamydia agent of the present invention has a high antibacterial property before or immediately after infection with Chlamydia, and has the advantage of being an anti-Chlamydia agent having excellent infection-preventing and cleaning effects.

Further, the anti-Chlamydia agent of the present invention provides a new use of an antibacterial extract derived from bamboo, and according to the diversified demands of consumers who prefer natural products, an anti-Chlamydia agent which is safe and has a reliable infection-preventing effect. There is also the advantage of providing an agent.

The advantage of the anti-Chlamydia agent of the present invention with respect to the above-mentioned antibacterial property is that when the extract contains benzoquinone, especially when the compounding amount of the extract is 0.2 to 5 volumes of liquid benzoquinone. %, It can be obtained more reliably.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram schematically showing the life cycle of Chlamydia

Continued on front page F-term (reference) 4C088 AB76 AC02 AC05 AC06 AC11 AC12 BA08 BA10 BA11 MA01 NA14 ZA33 ZA81 ZA82 ZB32 4C206 AA01 AA02 CB28 KA18 MA01 MA04 NA14 ZA33 ZA81 ZA82 ZB22

Claims (3)

[Claims] 1. An anti-chlamydia agent comprising, as an active ingredient, an extract obtained using bamboo as an extraction raw material and alcohol or ether as a solvent. 2. The anti-Chlamydia agent according to claim 1, wherein the extract is an extract containing benzoquinone. 3. The anti-chlamydia agent according to claim 2, wherein the compounding amount of the extract is a compounding amount containing 0.2 to 5% by volume of liquid benzoquinone.