JP3746705B2 - Methicillin-resistant Staphylococcus aureus antibacterial agent - Google Patents

Description

【００２６】
【表２】

【００２７】
これらの結果より、表に示す多種類の台湾産茶葉及び中国産茶葉のうち、２．５％の極く低濃度濃度であっても、ＭＲＳＡに対して優れた抗菌作用を発揮したのは、白豪烏龍の茶葉及び杉林渓高山茶の茶葉のみであることが分かる。
【００２８】
これに対し、他の茶葉については抗菌作用は殆ど認められず、１２．５〜１５．０％と、極めて高濃度に調整した場合にのみ、僅かに抗菌作用を示したに過ぎなかった。
【００２９】
尚、表には示していないが、ＭＲＳＡ以外の他のブドウ球菌についても、同様の試験結果が得られたことを実験により確認している。
【００３０】
実施例２：各種菌に対する杉林渓高山茶の感受性試験
（１）供試菌
供試菌として、メチシリン耐性黄色ブドウ球菌（ＭＲＳＡ），黄色ブドウ球菌（Staphylococcus aureus野生株、およびStaphylococcus aureus 209P），表皮ブドウ球菌（Staphylococcus epidermidis、およびStaphylococcus haemolyticus），腐生ブドウ球菌（Staphylococcus saprophyticus）のブドウ球菌；その他に、Streptococcus pneumoniae，Streptococcus agalactiaeの連鎖球菌；Enterococcus faealisの連鎖腸球菌；更に、Escehrichia coli大腸菌，enterohemohemorrhagic E. coli（O157）；Salmonella enteritidis，Salmonella thyphimuriumなどのサルモネラ菌等合計１３菌種を使用した。
【００３１】
（２）試験方法
上記供試菌を予め、各菌種毎に共通の最適栄養培地内で増殖培養したこと以外は、前述した実施例１と同様にして感受性試験に供した。その結果を表３に示す。
【００３２】
【表３】

【００３３】
表３より、杉林渓高山茶の茶葉を用いれば、ブドウ球菌、なかでもＭＲＳＡに対して極めて優れた抗菌作用を示すことが分かった。同一の茶葉抽出物における抗菌作用を比較すると一目瞭然であるが、ブドウ球菌の標準株２０９Ｐや野生株に対する阻止円幅よりも、ＭＲＳＡに対する阻止円幅は極めて大きかった。
【００３４】
これに対し、ブドウ球菌以外の他の菌種については抗菌作用は殆ど認められず、高濃度に高めた場合にのみ、僅かに抗菌作用を示したに過ぎなかった。
【００３５】
尚、参考までに、ブドウ球菌のうちＭＲＳＡ、黄色ブドウ球菌（Staphylococcus aureus野生株、およびStaphylococcus aureus 209P）、並びにO157に対する阻止円の結果を示す写真を図１に示す。
【００３６】
実施例３：白豪烏龍の各菌種に対する感受性試験
実施例２において、杉林渓高山茶の代わりに白豪烏龍を用いたこと以外は実施例２と同様の感受性試験を行った。得られた結果を表４に示す。
【００３７】
【表４】

【００３８】
表４より、白豪烏龍の茶葉を用いた場合にも、ブドウ球菌に対し、優れた抗菌作用を発揮した。杉林渓高山茶の場合と同様、ＭＲＳＡに対する抗菌作用が極めて強く観察された。
【００３９】
参考までに、ブドウ球菌のうちＭＲＳＡ、黄色ブドウ球菌（Staphylococcus aureus野生株、およびStaphylococcus aureus 209P）、並びにO157に対する阻止円の結果を示す写真を図２に示す。
【００４０】
実施例４：殺菌効果確認試験（in vitro試験）
本発明に用いられる杉林渓高山茶及び白豪烏龍が、ブドウ球菌に対する殺菌作用を有するか否かを最終的に確認する目的で、生菌浮遊液に夫々の台湾産茶葉液を加え、一定時間後に菌が死滅するかどうか調べた。
【００４１】
詳細には、表３に示す三種の由来株ＭＲＳＡ浮遊液５ｃｃを容れた各試験管に、杉林渓高山茶葉／白豪烏龍茶葉の５％溶液を５ｃｃ加えた後、３７℃のふ卵器内に２４時間放置した後、遠心分離し、沈渣を採取してスタヒロコッカスNo.１１０培地に塗抹した。更に、上記ふ卵器で２４時間培養し、コロニーが発生しなかったときには「−」と判定した。
【００４２】
得られた結果を表５に併記する。
【００４３】
【表５】

【００４４】
表５より、台湾産の杉林渓高山茶・白豪烏龍の茶葉抽出物を添加した場合はいずれも、コロニーの発生が全く観察されなかったことから、各ＭＲＳＡ菌株の死滅が確認された。
【００４５】
実施例５：殺菌効果確認試験（in vivo試験）
本発明抗菌剤による殺菌作用を更に実証すべく、上記のin vitro試験に加え、ラットを用いたin vivo試験を行った。
【００４６】
詳細には、ＳＰＦ由来のＳＤ系成熟健康雄性ラット合計２４匹を４匹／群に分け、表６に示す三種のＭＲＳＡ菌浮遊液を５ｃｃ／匹ずつラット用ゾンデ針で直接胃内投与した。投与後３０分経過してから、各台湾産の５．０％茶葉抽出物を５ｃｃ／匹ずつ同様に胃内投与した。２４〜４８時間経過後、採取した糞便から釣菌し、スタヒロコッカスNo.１１０培地に塗抹して３７℃のふ卵器に２４〜４８時間培養した。発生したコロニーについてはグラム染色を施し、鏡検してブドウ球菌であるかどうかを観察した。その結果を表６に併記する。尚、本実験を行う前に、実験に供したラットの糞便中にはブドウ球菌は存在していないことを確認している。
【００４７】
【表６】

【００４８】
表６より、いずれの台湾産の茶葉抽出物においても、コロニーの発生が全く観察されなかったことから、各ＭＲＳＡ菌株の死滅が確認された。
【００４９】
【発明の効果】
本発明は上記の様に構成されており、ブドウ球菌、特にＭＲＳＡに対して、極めて顕著な抗菌作用を示す抗菌剤を提供することができた。本発明の抗菌剤は、上述した２種の茶葉／茎から抽出される熱水抽出液を使用しているので、副作用もなく、安全且つ手軽に服用できる抗菌剤として極めて有用である。
【図面の簡単な説明】
【図１】実施例２において、ＭＲＳＡ、黄色ブドウ球菌（Staphylococcus aureus野生株、およびStaphylococcus aureus 209P）、及びO157に対する阻止円の結果を示す写真である。
【図２】実施例３において、ＭＲＳＡ、黄色ブドウ球菌（Staphylococcus aureus野生株、およびStaphylococcus aureus 209P）、及びO157に対する阻止円の結果を示す写真である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to methicillin resistant Staphylococcus aureus (hereinafter referred to as MRSA), which is a problem as a pathogen of nosocomial infection among staphylococci (genus Staphylococcus) having pathogenicity to humans, livestock, pets and the like. It relates to an antibacterial agent that exhibits antibacterial action.
[0002]
[Prior art]
Staphylococci are known as pathogenic bacteria for various inflammations in humans and animals, such as cutaneous suppuration, conjunctivitis, sinusitis, otitis media, cystitis, pneumonia, osteomyelitis, arthritis, visceral abscess. Among them, Staphylococcus aureus is an important causative bacterium of human and animal suppuration, and enterotoxin enterotoxin produced thereby also causes food poisoning. In recent years, in particular, nosocomial infections caused by MRSA, which is resistant to many types of antibiotics, or severe enteritis due to fungal complications have become a problem.
[0003]
MRSA, as the name suggests, is a Staphylococcus aureus that is resistant to the antibiotic methicillin. That is, MRSA has acquired resistance against methicillin skeleton drugs (for example, penicillin and cephalosporin drugs) having a strong antibacterial action against Staphylococcus aureus.
[0004]
MRSA is broad for all commercially available β-lactam drugs, including all penicillin drugs; all cephem drugs called first, second, and third generation; monobactam drugs; carbapenem drugs, etc. Demonstrate drug resistance. Furthermore, it is known to show resistance to new quinolone drugs. Recently, it has been eagerly desired to provide an antibacterial agent effective for MRSA, for example, it has been reported that vancomycin, which has been used for the purpose of treating infections caused by MRSA, has also emerged.
[0005]
Furthermore, MRSA not only exhibits multidrug resistance, but may be very strongly expressed in pathogenicity. In particular, MRSA infection in easily infected patients who have become less resistant to infection is a serious problem. For example, when a patient who is immunocompromised due to leukemia or cancer, a severely burned patient, a bedridden elderly person, a premature baby, or other serious patient is infected with MRSA, it is actually difficult to cure.
[0006]
[Problems to be solved by the invention]
The present invention has been made paying attention to the above circumstances, and an object thereof is to provide an antibacterial agent that exhibits an effective antibacterial action against MRSA.
[0007]
[Means for Solving the Problems]
The methicillin-resistant Staphylococcus aureus antibacterial agent of the present invention that has achieved the above-mentioned object is hot water extraction extracted from the tea leaves and / or stems of cedar forest Takayama tea native to Taiwan and / or white gourd dragon as an active ingredient It has a gist where it contains the product.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
In order to provide an antibacterial agent capable of exhibiting an effective antibacterial action against staphylococci, particularly MRSA, the present inventors have intensively studied with a particular focus on tea. Specifically, 35 kinds of tea leaves originating in Japan, 15 kinds of tea leaves originating in Taiwan, and 5 kinds of tea leaves originating in China were examined in detail for antibacterial activity against various staphylococci including MRSA. As a result, it has been found that two types of tea leaves and / or stems of Sugininkei Takayama tea and Hakuo Oolong native to Taiwan can extremely effectively inhibit the growth of staphylococci, especially MRSA, and completed the present invention. .
[0009]
In general, tea is prepared by collecting young shoots (leaves) of tea plants belonging to the camellia family, and is widely used for non-fermented tea (green tea), semi-fermented tea (such as oolong tea), and fermented tea (black tea) by the production method. Separated. Furthermore, for example, semi-fermented tea is broadly classified into oolong tea and baked tea depending on the manufacturing method, the type and shape of the raw materials.
[0010]
It is known that tea is not only preferred as a favorite beverage due to its elegant fragrance and taste, but also has various physiological functions. For example, it is known that catechin (tannin) contained in a large amount in tea leaves has an anticancer effect, an anti-aging effect, a lowering effect of blood cholesterol level, and the like.
[0011]
According to recent research, the usefulness of catechins contained in tea has been actively promoted. For example, a Japanese tea exudate has the effect of sterilizing the virulent pathogenic E. coli O157 and detoxifying verotoxin. Some reports have been made. The present inventors also focused on catechins and examined whether they have antibacterial activity against staphylococci, particularly MRSA, but it was found that the desired antibacterial effect could not be obtained with a simple concentration of catechin. .
[0012]
Furthermore, various teas were examined while paying attention to the content of catechin, but no significant correlation was found between catechin and staphylococcal antibacterial activity. For example, Japanese green tea is drunk by pouring hot water at a relatively low temperature, so catechins, which are bitter components, dissolve in a relatively large amount in the tea. In addition, roasted tea is roasted sencha, but catechins are reduced by roasting. Furthermore, oolong tea and semi-fermented tea of baked tea are poured after pouring boiling water and steamed for 3-5 minutes, so catechins are reduced to less than half of green tea. As a result of examining the antibacterial action against MRSA for various teas with different catechins content as described above, even oolong tea, which is considered to have a low catechin content as described above, is a tea leaf of Taiwanese white gourd dragon. It was found that excellent antibacterial action can be obtained by drinking. In contrast, Taiwan's frozen crested dragon has no antibacterial action against MRSA. On the other hand, Suginbayashi Takayama tea from Taiwan is used as oolong tea. Therefore, it can be seen that the antibacterial action according to the present invention is not necessarily due to the action of catechin alone.
[0013]
Incidentally, when the amount of catechin was examined for the two types of tea leaves used in the present invention, it was found to be very small or hardly detectable. For example, in the case of white gourd, 15 g of this tea leaf and / or stem was immersed in 650 mL of water at 90 ° C., left at room temperature for 30 seconds, and the amount of catechin in the filtrate was measured by the FOLIN-DENIS method. No catechin (detected as tannic acid) was detected (detection limit 0.01 g / 100 g). On the other hand, Sugibayashi Takayama tea was also measured by the same method, and only 0.03 g / 100 g of tannic acid was detected.
[0014]
Although it is unclear in detail why the above-mentioned Taiwan tea leaves used in the present invention can exert an extremely excellent antibacterial action against MRSA, at least from the above basic experiment, medicinal ingredients in tea leaves have been conventionally used. In view of the fact that catechins alone, which were considered to be antagonism, have no antibacterial activity against MRSA and other staphylococci, Taiwan's soil, climate, cultivation conditions, and active ingredients in tea leaves are intricately intertwined. It is thought that it showed antibacterial action.
[0015]
Here, the tea used in the present invention will be described.
[0016]
First, Hakuo Oolong is produced only in Taiwan [Manufacturer: Xinhua Tea Co., Ltd.] is a green tea (Oolong tea) with a fermentation degree of about 60-70%, and is known as an Oolong tea type that is close to black tea It has been. Of the semi-fermented teas, the only one produced from tea buds is Hakuo Oolong, which is characterized by a higher degree of fermentation than ordinary semi-fermented teas. The tea buds are preferably those with enlarged heart buds, and mainly tea trees such as Seishin Daiyu, Taicha No. 17 and Shirage-an are used. The main production areas are Taiwan's Hsinchu North Miaoli, Miaoli, and head portion. In preparing the white gourd dragon, the tea leaves and / or stalks are first exposed to weak sunlight for a long time. Stir until fragrant. Next, the obtained tea buds are fried at a low temperature, covered with a wet cloth, and then kneaded to such an extent that they do not become shattered, and then dried by hot air at about 90 ° C.
[0017]
Sugibayashi Takayama tea is a green tea (Oolong tea) with a degree of fermentation of about 15-30%, which is cultivated in Sugininkei in the Central Mountains of Taiwan (main production areas are Chiayi and Nanfang). A hemispherical dragon (for example, a blue heart dragon) is used. Due to the effects of alpine cultivation, the tea leaves are bluish green in color, have little bitterness and astringency, and are known as high-quality incense teas that have a long-lasting scent. The production method is the same as that of ordinary oolong tea, and after being exposed to weak sunlight, it is transferred to the room, stirred, fermented, and then obtained through the steps of fried tea, heating, twisting and drying. Specifically, what is manufactured by Xinhua Tea Co., Ltd. may be used.
[0018]
The antibacterial agent of the present invention contains the above-mentioned tea leaf and / or stem hot water extract as an active ingredient. In order to obtain a desired antibacterial effect, it is recommended to adjust the concentration of the hot water extract to about 2 to 15%, preferably 2 to 10%. As will be apparent from the examples to be described later, the higher the concentration of the hot water extract, the more antibacterial action tends to increase, but usually considering the balance with ease of taking, etc. It is recommended that about 100 cc of hot water be poured into about 2 to 10 g of tea leaves for adjustment. For example, when used as a tea pack, it is preferable to pack 2.5 g or 5.0 g per pack and pour about 100 cc of hot water of 65 to 95 ° C. into the pack. It is recommended to take about 1.2 to 2.0 cc / kg of hot water extract adjusted to such a concentration 3 to 4 times a day.
[0019]
In the present invention, the hot water extract of the above-mentioned Taiwan-grown Sugibayashi Takayama tea and white gourd tea leaves and / or stems may be used alone or in combination of two kinds. Absent. When both are used in combination, it is preferable to use approximately equal amounts so that the total content falls within the above range. Of course, the content is not intended to be limited to this amount, and the content can be appropriately adjusted according to the condition, sex, weight, etc. of the subject within a range in which a desired antibacterial action can be obtained.
[0020]
In addition, the antibacterial agent may appropriately contain vitamin C, vitamin E, vitamin K, folic acid, sweetener, excipient, stabilizer, and the like as long as the effects of the present invention are not impaired.
[0021]
The antibacterial agent of the present invention is widely applied to staphylococci, that is, bacteria belonging to the genus Staphylococcus, for example, S. aureus containing MRSA, S. epidermidis, S. saprophyticus, etc. In particular, it exhibits an extremely strong antibacterial action against MRSA.
[0022]
Hereinafter, the present invention will be described in detail based on examples. However, the following embodiments are not intended to limit the present invention, and all modifications made without departing from the spirit of the preceding and following descriptions are encompassed within the technical scope of the present invention.
[0023]
【Example】
Example 1: Sensitivity test for MRSA using various Taiwan teas and Chinese oolong teas Various Taiwan teas shown in Table 1 and various Chinese oolong teas shown in Table 2 were used for the following tests.
[0024]
First, methicillin-resistant Staphylococcus aureus (MRSA) was grown and cultured in a nutrient medium and then subjected to a halo test (paper disk sensitivity test). Specifically, tea of various concentrations shown in Tables 1 and 2 was soaked in a disc of 8 mm diameter made by Showa Disc. What was picked up from each fungal colony was smeared on a flat plate medium, and the above-mentioned three discs were set on it. After culturing in a 37 ° C. incubator for 24 to 48 hours, the width of the growth inhibition circle was measured. The results are shown in Table 1.
[0025]
[Table 1]

[0026]
[Table 2]

[0027]
From these results, among the various types of Taiwan tea leaves and Chinese tea leaves shown in the table, the antibacterial action excellent against MRSA was demonstrated even at a very low concentration of 2.5%. It can be seen that there are only tea leaves of Hakuo Oolong and Sugibayashi Takayama tea.
[0028]
On the other hand, almost no antibacterial action was observed for the other tea leaves, and only a slight antibacterial action was shown when adjusted to a very high concentration of 12.5 to 15.0%.
[0029]
Although not shown in the table, it was confirmed by experiments that the same test results were obtained for staphylococci other than MRSA.
[0030]
Example 2: Susceptibility test of Sugibayashi Takayama tea to various bacteria (1) As test bacteria, methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus wild strain, and Staphylococcus aureus 209P, epidermis Staphylococcus epidermidis and Staphylococcus haemolyticus, Staphylococcus saprophyticus staphylococci; in addition, Streptococcus pneumoniae, Streptococcus agalactiae streptococci; Enterococcus faealis streptococcus eh E. coli (O157): Salmonella enteritidis, Salmonella thyphimurium, and other Salmonella bacteria were used in total.
[0031]
(2) Test method The test bacteria were subjected to a sensitivity test in the same manner as in Example 1 except that the test bacteria were previously grown and cultured in an optimal nutrient medium common to each bacterial species. The results are shown in Table 3.
[0032]
[Table 3]

[0033]
From Table 3, it was found that if the tea leaves of Sugibayashi Takayama tea were used, they exhibited an excellent antibacterial action against staphylococci, especially MRSA. When the antibacterial action of the same tea leaf extract was compared, it was obvious that the inhibition circle width for MRSA was much larger than the inhibition circle width for staphylococcal standard strain 209P and wild strain.
[0034]
On the other hand, the antibacterial activity was hardly observed for other bacterial species other than staphylococci, and only a slight antibacterial activity was exhibited only when the concentration was increased to a high concentration.
[0035]
For reference, FIG. 1 shows a photograph showing the results of inhibition circles for MRSA, Staphylococcus aureus wild strain, and Staphylococcus aureus 209P among staphylococci, and O157.
[0036]
Example 3: Sensitivity test for each species of white gourd dragon In Example 2, the same susceptibility test as in Example 2 was performed except that white gourd dragon was used instead of Sugibayashi Takayama tea. Table 4 shows the obtained results.
[0037]
[Table 4]

[0038]
From Table 4, even when using the tea leaves of white gourd dragon, it exhibited an excellent antibacterial action against staphylococci. As with Sugibayashi Takayama tea, the antibacterial action against MRSA was observed very strongly.
[0039]
For reference, FIG. 2 shows photographs showing the results of inhibition circles for MRSA, Staphylococcus aureus wild strains and Staphylococcus aureus 209P among staphylococci, and O157.
[0040]
Example 4: Bactericidal effect confirmation test (in vitro test)
For the purpose of finally confirming whether Sugibayashi Takayama tea and white gourd dragon used in the present invention have a bactericidal action against staphylococci, add each Taiwanese tea leaf liquid to the viable bacterial suspension, constant It was examined whether the bacteria were killed after a time.
[0041]
Specifically, 5 cc of a 5% solution of Sugibayashi Takayama tea leaves / Shirago oolong tea leaves was added to each test tube containing 5 cc of the three types of strain MRSA suspension shown in Table 3, and then placed in an incubator at 37 ° C. After standing for 24 hours, the mixture was centrifuged, and the sediment was collected and smeared on Staphylococcus No. 110 medium. Furthermore, it culture | cultivated with the said incubator for 24 hours, and when the colony did not generate | occur | produced, it determined with "-".
[0042]
The obtained results are also shown in Table 5.
[0043]
[Table 5]

[0044]
From Table 5, the occurrence of colonies was not observed at all in the case where the tea leaves extract of Taiwan-grown Sugibayashi Takayama tea and white gourd tea was added, and thus the death of each MRSA strain was confirmed.
[0045]
Example 5: Bactericidal effect confirmation test (in vivo test)
In order to further verify the bactericidal action of the antibacterial agent of the present invention, in vivo tests using rats were conducted in addition to the in vitro tests described above.
[0046]
Specifically, a total of 24 SPF-derived SD-type mature healthy male rats were divided into 4 / group, and 3 kinds of MRSA bacteria suspensions shown in Table 6 were directly administered intragastrically at 5 cc / mouse using a rat needle for rats. 30 minutes after the administration, 5.0 cc tea leaf extract from Taiwan was administered intragastrically in the same manner at 5 cc / animal. After 24-48 hours, fish were collected from the collected feces, smeared on Staphylococcus No. 110 medium, and cultured in an incubator at 37 ° C. for 24-48 hours. The generated colonies were subjected to Gram staining and microscopically observed for staphylococci. The results are also shown in Table 6. Before conducting this experiment, it was confirmed that staphylococci were not present in the feces of rats subjected to the experiment.
[0047]
[Table 6]

[0048]
From Table 6, since no colony was observed in any Taiwan tea leaf extract, the death of each MRSA strain was confirmed.
[0049]
【The invention's effect】
The present invention is configured as described above, and can provide an antibacterial agent exhibiting a very remarkable antibacterial action against staphylococci, particularly MRSA. Since the antibacterial agent of the present invention uses the hot water extract extracted from the two types of tea leaves / stems described above, it is extremely useful as an antibacterial agent that can be safely and easily taken without side effects.
[Brief description of the drawings]
FIG. 1 is a photograph showing the results of inhibition circles for MRSA, Staphylococcus aureus wild strains and Staphylococcus aureus 209P, and O157 in Example 2.
FIG. 2 is a photograph showing the results of inhibition circles for MRSA, Staphylococcus aureus (Staphylococcus aureus wild strain, and Staphylococcus aureus 209P), and O157 in Example 3.

Claims (1)

A methicillin-resistant Staphylococcus aureus antibacterial agent comprising, as an active ingredient, a hot water extract of cedar forest Takayama tea and / or white gourd tea leaves and / or stems produced in Taiwan.

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