TW201914440A - Composition for order decapoda, including 5-aminolevulinic acid - Google Patents
Composition for order decapoda, including 5-aminolevulinic acid Download PDFInfo
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本發明涉及含有選自5-氨基乙醯丙酸(5-ALA)或者其酯、或它們的鹽中的至少一種的十足目用口服給藥組合物、飼料和飼料用添加劑,更詳細而言,涉及含有選自5-ALA或者其酯、或它們的鹽中的至少一種的十足目早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)的預防.治療用口服給藥組合物。另外,本發明涉及包括使十足目的生物攝取選自5-ALA或者其酯、或它們的鹽中的至少一種的方法,更詳細而言,涉及包括使十足目的生物攝取選自5-ALA或者其酯、或它們的鹽中的至少一種的預防.治療十足目早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)的方法。 The present invention relates to an oral administration composition, a feed and a feed additive containing at least one selected from the group consisting of 5-aminoacetic acid (5-ALA) or an ester thereof, or a salt thereof, and more particularly And relates to the prevention of decapod early death syndrome/acute hepatopancreatic necrosis (EMS/AHPND) containing at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof. Oral administration compositions for therapeutic use. Further, the present invention relates to a method comprising the step of ingesting at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof, and more particularly, to include ingesting a target organism for selection from 5-ALA or Prevention of at least one of esters, or salts thereof. A method for treating decapod early death syndrome/acute hepatic pancreatic necrosis (EMS/AHPND).
世界的蝦的生產量從平成4(1992)年的301萬噸激增到平成24(2012)年的768萬噸。若觀察其中基於養殖的生產量的比例,則從平成4(1992)年的30%到平成24(2012)年超過生產量的過半數而達到56%等,近年來的蝦的養殖的發展引人注目(非專利文獻1)。蝦的養殖與天然的環境不同,一般而言,由於以高密度進行飼養、施加過大的壓力等理由,在蝦的養殖場中確認到各種疾病的發生。養殖水產動植物的存活率對養殖經營有很大影響,因此在養殖業中要求對疾病的適當的措施。 The world's shrimp production has increased from 3.01 million tons in Heisei 4 (1992) to 7.68 million tons in Heisei 24 (2012). If we observe the proportion of production based on aquaculture, it will reach 56% from 30% of the year of Heisei 4 (1992) to more than half of the production volume of Heisei 24 (2012). Attention is paid to people (Non-Patent Document 1). Shrimp culture is different from the natural environment. In general, various diseases are confirmed in shrimp farms due to high density feeding and excessive pressure. The survival rate of cultured aquatic animals and plants has a great impact on aquaculture operations, so appropriate measures for diseases are required in the aquaculture industry.
近年來,由於在幼蝦中發生,死亡率大致為100%的被稱為EMS(Early Mortality Syndrome:早期死亡綜合症)的蝦的疾病,在一部分國家,蝦養殖業直面危機的狀況。該疾病2009年在中國首先被報告,接下來還擴散到越南、泰國、馬來西亞等東南亞,在2013年報告了在墨西哥發生。對於EMS,蝦的肝胰臟出現變色等症狀,產生壞死,因此該早期死亡綜合症也被稱為EMS/AHPND(Acute Hepatopancreatic Necrosis Disease:急性肝胰臟壞死病)。而 且,還已知該EMS/AHPND是由特殊類型的副溶血弧菌(Vibrio parahaemolyticus)的感染引起的(非專利文獻2)。 In recent years, shrimp disease has been facing the crisis in some countries due to the disease of shrimp, which is called EMS (Early Mortality Syndrome), which occurs in juvenile shrimp. The disease was first reported in China in 2009, and then spread to Southeast Asia such as Vietnam, Thailand, and Malaysia, and reported in Mexico in 2013. For EMS, the liver and pancreas of shrimp have symptoms such as discoloration and necrosis, so the early death syndrome is also called EMS/AHPND (Acute Hepatopancreatic Necrosis Disease). Further, it is also known that the EMS/AHPND is caused by infection of a special type of Vibrio parahaemolyticus (Non-Patent Document 2).
開發出了對於蝦的弧菌感染使用疫苗的方法(專利文獻1)。但是,該專利文獻1中,作為物件菌,啟示了副溶血弧菌(Vibrio parahaemolyticus),但是,具體而言,並沒有公開針對副溶血弧菌(Vibrio parahaemolyticus)的疫苗的製造,況且完全不清楚該疫苗療法對預防.治療EMS/AHPND是有效的。進而,明確若是利用比特殊的疫苗廉價且能夠簡單地獲得的物質預防.治療EMS/AHPND的方法,則優選該方法。另外,若有除EMS/AHPND的預防.治療以外還帶來在蝦的養殖中有利的效果的物質,則進一步優選。 A method of using a vaccine for Vibrio infection of shrimp has been developed (Patent Document 1). However, in Patent Document 1, Vibrio parahaemolyticus is suggested as an object bacteria, but specifically, the production of a vaccine against Vibrio parahaemolyticus is not disclosed, and it is completely unclear. The vaccine therapy is for prevention. It is effective to treat EMS/AHPND. Furthermore, it is clear that if it is cheaper than a special vaccine and can be easily obtained, it can be prevented. This method is preferred for the treatment of EMS/AHPND. In addition, if there is prevention of EMS / AHPND. A substance which has an advantageous effect in the culture of shrimp in addition to the treatment is more preferable.
已知5-ALA存在於細胞的線粒體,對動物而言,是在線粒體進行生物合成,與鐵分結合成為血紅素、細胞色素的原料等代謝需要的成分,對植物而言,是在葉綠體進行生物合成,與鎂結合成為葉綠素,對光合成需要的成分。而且,專利文獻2中公開了5-ALA磷酸鹽的製造方法,進而,還記載了已知5-ALA鹽酸鹽的合成方法。另外,還已知利用微生物的5-ALA的製造方法(專利文獻3)。 It is known that 5-ALA is present in the mitochondria of cells, and in animals, it is a mitochondria for biosynthesis, and it is combined with iron to become a component required for metabolism of heme and cytochrome. For plants, it is carried out in chloroplasts. Biosynthesis, combined with magnesium to become chlorophyll, a component required for photosynthetic synthesis. Further, Patent Document 2 discloses a method for producing 5-ALA phosphate, and further discloses a method for synthesizing 5-ALA hydrochloride. Further, a method for producing 5-ALA using a microorganism is also known (Patent Document 3).
專利文獻4中記載了含有5-ALA作為有效成分的魚類病原性微生物的感染預防和治療用組合物,進而,作為上述魚類病原性微生物,記載了遲緩愛德華氏菌(Edwardsiella tarda)、鏈球菌屬細菌(Streptococcus sp.)、葡萄球菌屬細菌(Staphylococcus sp.)、表皮葡萄球菌細菌(Staphilococcus epidermidis)、假單胞菌屬細菌(Pseudomonas sp.)或鰻弧菌細菌(Vibrio anguillarum)。但是,專利文獻4中,對於5-ALA對十足目的生物的的影響,沒有任何研究。進而,專利文獻4所記載的鰻弧菌(Vibrio anguillarum)與十足目的EMS/AHPND的致病菌即副溶血弧菌(Vibrio parahaemolyticus)不同,因此在十足目的生物中不會發生EMS/AHPND。因此,根據專利文獻4的記載,完全無法預期十足目的生物中的基於5-ALA的EMS/AHPND的預防.治療效果。另外,尚不知道5-ALA促進十足目的生物的生長。 Patent Document 4 describes a composition for preventing and treating infections of fish pathogenic microorganisms containing 5-ALA as an active ingredient, and further, as the fish pathogenic microorganism, Edwards ellada and Streptococcus are described. Streptococcus sp., Staphylococcus sp., Staphilococcus epidermidis, Pseudomonas sp. or Vibrio anguillarum. However, in Patent Document 4, there is no research on the influence of 5-ALA on a full-scale organism. Further, Vibrio anguillarum described in Patent Document 4 is different from Vibrio parahaemolyticus, which is a pathogen of EMS/AHPND, so that EMS/AHPND does not occur in a full-scale organism. Therefore, according to the description of Patent Document 4, the prevention of 5-ALA-based EMS/AHPND in a full-scale organism is completely unpredictable. treatment effect. In addition, it is not known that 5-ALA promotes the growth of a full-scale organism.
現有技術文獻 Prior art literature
專利文獻 Patent literature
專利文獻1:日本特開2015-137254號公報 Patent Document 1: Japanese Laid-Open Patent Publication No. 2015-137254
專利文獻2:日本特開2006-182753號公報 Patent Document 2: Japanese Laid-Open Patent Publication No. 2006-182753
專利文獻3:日本特開2005-333907號公報 Patent Document 3: Japanese Laid-Open Patent Publication No. 2005-333907
專利文獻4:日本特開2001-316255號公報 Patent Document 4: Japanese Laid-Open Patent Publication No. 2001-316255
非專利文獻 Non-patent literature
非專利文獻1:平成25年度水產白皮書,(6)世界的養殖業的生產狀況 Non-Patent Document 1: Heisei 25 Aquatic White Paper, (6) Production Status of the World's Aquaculture Industry
非專利文獻2:Mohammad Jalil Zorriehzahra, Reza Banaederakhshan; Early Mortality Syndrome (EMS) as new Emerging Threat in Shrimp Industry; Advances in Animal and Veterinary Sciences, MarcH2015, Volume 3, Special issue 2, Pages 64-72 Non-Patent Document 2: Mohammad Jalil Zorriehzahra, Reza Banaederakhshan; Early Mortality Syndrome (EMS) as new Emerging Threat in Shrimp Industry; Advances in Animal and Veterinary Sciences, MarcH2015, Volume 3, Special issue 2, Pages 64-72
因此,多年來一直強烈要求在以對蝦科為代表的十足目的生物的飼養、養殖中有用的、特別是能夠預防.治療EMS/AHPND的十足目用口服給藥組合物的開發。另外,還要求不僅能夠預防.治療十足目的生物的EMS/AHPND,而且還能夠促進其生長的十足目用口服給藥組合物的開發。但是,這樣的十足目用口服給藥組合物尚未實現。 Therefore, for many years, it has been strongly requested to be useful in the breeding and breeding of the full-scale organisms represented by the shrimp family, especially for prevention. Development of a decapitated oral administration composition for the treatment of EMS/AHPND. In addition, it is also required to be able to prevent not only. EMS/AHPND for the treatment of a full-length organism, and the development of a full-course oral administration composition capable of promoting its growth. However, such a full-course oral administration composition has not been achieved.
本發明的發明人等對能夠解決上述問題這樣的十足目用口服給藥組合物進行了深入研究,結果發現,含有選自5-ALA或者其酯、或它們的鹽中的至少一種的組合物是極其有用的,基於該發現完成了本發明。 The inventors of the present invention have conducted intensive studies on a thorough oral administration composition capable of solving the above problems, and as a result, have found that a composition containing at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof It is extremely useful to complete the present invention based on this finding.
即,本發明如下所述。 That is, the present invention is as follows.
〔1〕一種十足目用口服給藥組合物,含有選自5-氨基乙醯丙酸(5-ALA)或者其酯、或它們的鹽中的至少一種。 [1] A therapeutic composition for oral administration comprising at least one selected from the group consisting of 5-aminoacetic acid (5-ALA) or an ester thereof or a salt thereof.
〔2〕一種十足目早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)的預防.治療用口服給藥組合物,含有選自5-氨基乙醯丙酸(5-ALA)或者其酯、或它們的鹽中的至少一種。 [2] Prevention of a decapod early death syndrome / acute hepatic pancreatic necrosis (EMS / AHPND). The therapeutic oral administration composition contains at least one selected from the group consisting of 5-aminoacetic acid (5-ALA) or an ester thereof or a salt thereof.
〔3〕根據上述〔1〕或〔2〕所述的組合物,其中,十足目為對蝦科。 [3] The composition according to the above [1] or [2] wherein the decapod is a prawn family.
〔4〕根據上述〔1〕~〔3〕中任一項所述的組合物,其中,組合物為飼料或飼料用添加劑。 [4] The composition according to any one of the above [1] to [3] wherein the composition is an additive for feed or feed.
〔5〕一種方法,包括使十足目的生物攝取選自5-氨基乙醯丙酸(5-ALA)或者其酯、或它們的鹽中的至少一種。 [5] A method comprising ingesting at least one selected from the group consisting of 5-aminoacetic acid (5-ALA) or an ester thereof, or a salt thereof.
〔6〕一種預防.治療十足目早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)的方法,包括使十足目的生物攝取選自5-氨基乙醯丙酸(5-ALA)或者其酯、或它們的鹽中的至少一種。 [6] A prevention. A method for treating decapod early death syndrome/acute hepatic pancreatic necrosis (EMS/AHPND), comprising inducing a full-scale organism to be selected from the group consisting of 5-aminoacetic acid (5-ALA) or an ester thereof, or a salt thereof At least one of them.
〔7〕根據上述〔5〕或〔6〕所述的方法,其中,十足目為對蝦科。 [7] The method according to the above [5] or [6] wherein the decapod is a prawn family.
〔8〕一種促進十足目的生物的生長的方法,包括使十足目的生物以按照5-ALA磷酸鹽換算計,十足目的生物的體重每1g且每1天為0.25μg/g.天~2.5μg/g.天的量攝取選自5-氨基乙醯丙酸(5-ALA)或者其酯、或它們的鹽中的至少一種。 [8] A method for promoting the growth of a full-length organism, comprising: counting the target organism in a weight ratio of 5-ALA phosphate, the weight of the organism of interest is 1 g per day and 0.25 μg/g per day. Day ~2.5μg/g. The amount of the day is selected from at least one selected from the group consisting of 5-aminoacetic acid (5-ALA) or an ester thereof, or a salt thereof.
本發明的含有選自5-ALA或者其酯、或它們的鹽中的至少一種的十足目用口服給藥組合物可帶來在十足目的生物的飼養、養殖中能夠預防.治療十足目EMS/AHPND這樣的效果。其結果,能夠預防.治療若在以往則死亡率大致為100%的EMS/AHPND,能夠對十足目的生物的飼養、養殖帶來經濟貢獻。另外,本發明的十足目用口服給藥組合物以規定的給藥量對十足目的生物給藥時,帶來促進生長這樣的有利的效果。該有利的效果是以往未知的效果,且是十足目的生物的飼養、養殖的技術領域的本領域技術人員無法預期的效果。 The above-mentioned oral administration composition containing at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof can be prevented in the breeding and breeding of a full-scale organism. Treats the effects of the decapod EMS/AHPND. The result can be prevented. EMS/AHPND, which has a mortality rate of approximately 100% in the past, can contribute economically to the breeding and breeding of the target organisms. Further, the above-mentioned oral administration composition of the present invention has an advantageous effect of promoting growth when administered to a specific target organism in a predetermined administration amount. This advantageous effect is an effect which has not been known in the past, and is an effect which cannot be expected by those skilled in the art in the field of breeding and breeding of a full-scale organism.
第1圖是將對給藥了5-ALA磷酸鹽的凡納濱對蝦進行利用EMS/AHPND的致病菌(Vibrio parahaemolyticus)的攻擊試驗的結果與未給藥5-ALA磷酸鹽的對照組相比並隨時間表示的圖。 Figure 1 is a comparison of the results of an challenge test using Vibrio parahaemolyticus against EMS/AHPND of Penaeus vannamei to which 5-ALA phosphate was administered, and a control group not administered with 5-ALA phosphate. A graph that is more than and expressed over time.
第2圖是將3個月的飼養期間中的給藥了5-ALA的凡納濱對蝦的脫皮的累積頻率與未給藥5-ALA的對照組相比而示出的圖。 Fig. 2 is a graph showing the cumulative frequency of peeling of L. vannamei to which 5-ALA was administered during the three-month feeding period as compared with the control group not administered with 5-ALA.
第3圖是將給藥2周5-ALA的凡納濱對蝦的肝胰臟的ATP水準與未給藥5-ALA的對照組相比而表示的圖。 Fig. 3 is a graph showing the ATP level of the hepatopancreas of Penaeus vannamei administered with 5-ALA for 2 weeks compared with the control group not administered with 5-ALA.
第4圖是將對給藥3個月5-ALA的凡納濱對蝦進行利用高用量的EMS/AHPND的致病菌(Vibrio parahaemolyticus)的攻擊試驗的結果與未給藥5-ALA的對照組相比並隨時間表示的圖。 Figure 4 is a comparison of the results of an challenge test using Vibrio parahaemolyticus with high doses of EMS/AHPND for 5-month 5-ALA-administered P. vannamei and a control group not administered with 5-ALA. A graph compared to and expressed over time.
第5圖是將對給藥3個月5-ALA的凡納濱對蝦進行利用低用量的EMS/AHPND的致病菌(Vibrio parahaemolyticus)的攻擊試驗的結果與未給藥5-ALA的對照組相比並隨時間表示的圖。 Figure 5 is a comparison of the results of an challenge test using Vibrio parahaemolyticus with low doses of EMS/AHPND for 5-month-old 5-ALA-administered shrimps and a 5-ALA-free control group. A graph compared to and expressed over time.
第6圖是將對給藥3個月5-ALA的凡納濱對蝦進行利用EMS/AHPND的致病菌(Vibrio parahaemolyticus)的感染處理時的血淋巴中的總血球數與未給藥5-ALA的對照組相比而表示的圖。 Figure 6 is a diagram showing the total number of hematocrit in the hemolymph when the infection of the pathogen (Vibrio parahaemolyticus) using EMS/AHPND was administered to the 5-ALA 5-ALA-administered 5-ALA. A graph represented by the ALA control group.
第7圖是將對給藥3個月5-ALA的凡納濱對蝦進行利用EMS/AHPND的致病菌(Vibrio parahaemolyticus)的感染處理時的肝胰臟中的血紅素加氧酶-1(HO-1)的基因表達與未給藥5-ALA的對照組相比而表示的圖。 Figure 7 is a diagram showing the heme oxygenase-1 in the hepatopancreas when the infection of the pathogen (Vibrio parahaemolyticus) using EMS/AHPND is administered to the 5-ALA-treated 5-ALA. The gene expression of HO-1) is shown in comparison with the control group in which 5-ALA was not administered.
第8圖是將對給藥3個月5-ALA的凡納濱對蝦進行利用EMS/AHPND的致病菌(Vibrio parahaemolyticus)的感染處理時的肝胰臟中的酚氧化酶前體(proPO)的基因表達與未給藥5-ALA的對照組相比而表示的圖。 Figure 8 is a diagram showing the phenol oxidase precursor (proPO) in the hepatopancreas when the infection of the pathogen (Vibrio parahaemolyticus) using EMS/AHPND was carried out on 5-ALA-administered 5-ALA. The gene expression was compared to the control group in which 5-ALA was not administered.
第9圖是將給藥3個月5-ALA的凡納濱對蝦的肝胰臟中的核受體E75的基因表達與未給藥5-ALA的對照組相比而表示的圖。 Fig. 9 is a graph showing the gene expression of nuclear receptor E75 in the hepatopancreas of the L. vannamei administered with 5-ALA for 3 months as compared with the control group not administered with 5-ALA.
第10圖是將給藥3個月5-ALA的凡納濱對蝦的肝胰臟中的一氧化氮合成酶的基因表達與未給藥5-ALA的對照組相比而表示的圖。 Fig. 10 is a graph showing the expression of nitric oxide synthase gene in the hepatopancreas of the L. vannamei administered with 5-ALA for 3 months as compared with the control group not administered with 5-ALA.
第11圖是將給藥2周5-ALA的凡納濱對蝦的肝胰臟中的一氧化氮合成酶的基因表達與未給藥5-ALA的對照組相比而表示的圖。 Fig. 11 is a graph showing the expression of nitric oxide synthase gene in the hepatopancreas of the L. vannamei administered with 5-ALA for 2 weeks as compared with the control group not administered with 5-ALA.
第12圖是將給藥2周5-ALA的凡納濱對蝦的肝胰臟中的C型凝集素的基因表達與未給藥5-ALA的對照組相比而表示的圖。 Fig. 12 is a graph showing the gene expression of C-type lectin in the hepatopancreas of the L. vannamei administered with 5-ALA for 2 weeks as compared with the control group not administered with 5-ALA.
本發明的一個實施方式是含有選自5-ALA或者其酯、或它們的鹽中的至少一種的十足目用口服給藥組合物。 One embodiment of the present invention is an orthotopic composition for oral administration containing at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof.
本發明中,5-氨基乙醯丙酸(5-ALA)是也被稱為δ-氨基乙醯丙酸的化合物。本發明中,“5-ALA或者其酯”是“5-ALA或者5-ALA酯”,可由下述式(I)表示。本發明中,“5-ALA或者其酯、或它們的鹽”的記載中的“它們的鹽”是指5-ALA的鹽、或者5-ALA酯的鹽。作為該鹽,例如可舉出鹽酸鹽、氫溴酸鹽、氫碘酸鹽、磷酸鹽、甲基磷酸、乙基磷酸、亞磷酸鹽、次磷酸鹽、硝酸鹽、硫酸鹽、乙酸鹽、丙酸鹽、甲苯磺酸鹽、琥珀酸鹽、草酸鹽、乳酸鹽、酒石酸鹽、乙醇酸鹽、甲磺酸鹽、丁酸鹽、戊酸鹽、檸檬酸鹽、富馬酸鹽、馬來酸鹽、蘋果酸鹽等酸加成鹽以及鈉鹽、鉀鹽、鈣鹽等金屬鹽、銨鹽、烷基銨鹽等,但並不限定於這些。 In the present invention, 5-aminoacetic acid (5-ALA) is a compound also called δ-aminoacetic acid. In the present invention, "5-ALA or an ester thereof" is "5-ALA or 5-ALA ester" and can be represented by the following formula (I). In the present invention, "the salt thereof" in the description of "5-ALA or an ester thereof or a salt thereof" means a salt of 5-ALA or a salt of a 5-ALA ester. Examples of the salt include a hydrochloride, a hydrobromide, a hydroiodide, a phosphate, a methylphosphoric acid, an ethylphosphoric acid, a phosphite, a hypophosphite, a nitrate, a sulfate, and an acetate. Propionate, tosylate, succinate, oxalate, lactate, tartrate, glycolate, methanesulfonate, butyrate, valerate, citrate, fumarate, horse An acid addition salt such as a salt or a malate or a metal salt such as a sodium salt, a potassium salt or a calcium salt, an ammonium salt or an alkyl ammonium salt is not limited thereto.
上述式(I)中,R1為氫原子、直鏈或支鏈烷基、環烷基、芳基或芳烷基。R1為氫時,上述式(I)表示5-ALA。R1為直鏈或支鏈烷基、環烷基、芳基或芳烷基時,上述式(I)表示5-ALA酯。 In the above formula (I), R 1 is a hydrogen atom, a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group. When R 1 is hydrogen, the above formula (I) represents 5-ALA. When R 1 is a linear or branched alkyl group, a cycloalkyl group, an aryl group or an aralkyl group, the above formula (I) represents a 5-ALA ester.
R1中示出的直鏈或支鏈的烷基優選為碳原子數為1~18的烷基,例如可舉出甲基、乙基、正丙基、異丙基、正丁基、異丁基、叔丁基、正戊基、異戊基、新戊基、叔戊基、2-甲基丁基、正己基、異己基、3-甲基戊基、乙 基丁基、正庚基、2-甲基己基、正辛基、異辛基、叔辛基、2-乙基己基、3-甲基庚基、正壬基、異壬基、1-甲基辛基、乙基庚基、正癸基、1-甲基壬基、正十一烷基、1,1-二甲基壬基、正十二烷基、正十三烷基、正十四烷基、正十五烷基、正十六烷基、正十七烷基、正十八烷基等。作為環烷基,不僅可舉出例如環丙基、環丁基、環戊基、環己基、環庚基、環辛基等,還可舉出具有烷基取代基的環烷基、例如具有碳原子數1~6的烷基取代基的環烷基、例如3-甲基環己基、4-甲基環己基、4-乙基環己基、2-甲基環辛基等。作為直鏈或支鏈的烷基,更優選碳原子數1~16的烷基,特別優選甲基、乙基、正丁基、正十六烷基或2-乙基己基。 The linear or branched alkyl group represented by R 1 is preferably an alkyl group having 1 to 18 carbon atoms, and examples thereof include a methyl group, an ethyl group, a n-propyl group, an isopropyl group, an n-butyl group, and a different alkyl group. Butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl, 2-methylbutyl, n-hexyl, isohexyl, 3-methylpentyl, ethylbutyl, n-glycol Base, 2-methylhexyl, n-octyl, isooctyl, tert-octyl, 2-ethylhexyl, 3-methylheptyl, n-decyl, isodecyl, 1-methyloctyl, ethyl Heptyl, n-decyl, 1-methylindenyl, n-undecyl, 1,1-dimethylindenyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-ten Pentaalkyl, n-hexadecyl, n-heptadecyl, n-octadecyl and the like. The cycloalkyl group includes, for example, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a cyclohexyl group, a cycloheptyl group, a cyclooctyl group, and the like, and a cycloalkyl group having an alkyl substituent, for example, A cycloalkyl group having an alkyl group having 1 to 6 carbon atoms, for example, a 3-methylcyclohexyl group, a 4-methylcyclohexyl group, a 4-ethylcyclohexyl group, a 2-methylcyclooctyl group or the like. As the linear or branched alkyl group, an alkyl group having 1 to 16 carbon atoms is more preferable, and a methyl group, an ethyl group, a n-butyl group, a n-hexadecyl group or a 2-ethylhexyl group is particularly preferable.
作為R1中示出的芳基,可舉出苯基、萘基等。該芳基例如也可以被1~3個甲基、乙基、正丙基、異丙基、正丁基、異丁基、叔丁基、正戊基、正己基、環丙基、環丁基、環己基等碳原子數1~6的烷基、甲氧基、乙氧基、正丙氧基、正丁氧基、異丁氧基、叔丁氧基等碳原子數1~6的烷氧基、羥基、氨基、硝基、氰基、氟、氯、溴、碘等鹵素原子、羧基等取代基取代。 The aryl group shown by R 1 may, for example, be a phenyl group or a naphthyl group. The aryl group may, for example, also be 1 to 3 methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, n-hexyl, cyclopropyl, cyclobutyl a group having 1 to 6 carbon atoms such as an alkyl group, a methoxy group, an ethoxy group, a n-butoxy group, a n-butoxy group, an isobutoxy group or a t-butoxy group having 1 to 6 carbon atoms; A substituent such as a halogen atom such as an alkoxy group, a hydroxyl group, an amino group, a nitro group, a cyano group, a fluorine group, a chlorine group, a bromine group or a iodine group, or a carboxyl group.
作為R1中示出的芳烷基,優選由碳原子數1~6的烷基和碳原子數6~20的芳基構成。作為碳原子數1~6的烷基,例如可舉出甲基、乙基、正丙基、異丙基、正丁基、異丁基、叔丁基、正戊基、正己基、環丙基、環丁基、環己基等,作為碳原子數6~20的芳基,可舉出苯基、萘基等。芳烷基中,優選苄基或苯乙基,特別優選苄基。該芳烷基的芳基也可以被1~3個上述記載的碳原子數1~6的烷基、甲氧基、乙氧基、正丙氧基、正丁氧基、異丁氧基、叔丁氧基等碳原子數1~6的烷氧基、羥基、氨基、硝基、氰基、氟、氯、溴、碘等鹵素原子、羧基等取代基取代。 The aralkyl group shown by R 1 is preferably composed of an alkyl group having 1 to 6 carbon atoms and an aryl group having 6 to 20 carbon atoms. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, a n-propyl group, an isopropyl group, a n-butyl group, an isobutyl group, a t-butyl group, a n-pentyl group, a n-hexyl group, and a cyclopropyl group. Examples of the aryl group having 6 to 20 carbon atoms, such as a phenyl group and a cyclohexyl group, include a phenyl group and a naphthyl group. Among the aralkyl groups, a benzyl group or a phenethyl group is preferred, and a benzyl group is particularly preferred. The aryl group of the aralkyl group may be one to three alkyl groups having 1 to 6 carbon atoms as described above, a methoxy group, an ethoxy group, a n-propoxy group, a n-butoxy group, an isobutoxy group, or the like. A substituent such as a halogen atom having 1 to 6 carbon atoms such as a tert-butoxy group, a hydroxyl group, an amino group, a nitro group, a cyano group, a fluorine atom, a chlorine atom, a bromine group or a carbonyl group, or a carboxyl group.
本發明中,只要使用選自5-ALA或者其酯、或它們的鹽中的至少一種作為有效成分即可,該有效成分可以僅使用一種,或者該有效成分可以是多種的組合的形態。例如,本發明中使用的有效成分可以是5-ALA、5-ALA酯、5-ALA鹽、或5-ALA酯的鹽中的任一者。另外,例如,也可以是5-ALA與5-ALA酯的鹽的組合等。本發明中使用的選自5-ALA或者酯、或它們的鹽中的至少一種可以是純化後的狀態,也可以是粗純化的狀態或合成 而得到的混合物的狀態。本發明中優選使用5-ALA鹽作為有效成分,更優選使用5-ALA鹽酸鹽和/或5-ALA磷酸鹽作為有效成分。 In the present invention, at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof may be used as the active ingredient, and only one of the active ingredients may be used, or the active ingredient may be in a combination of a plurality of types. For example, the active ingredient used in the present invention may be any of 5-ALA, 5-ALA ester, 5-ALA salt, or 5-ALA ester salt. Further, for example, a combination of a salt of 5-ALA and a 5-ALA ester or the like may be used. At least one selected from the group consisting of 5-ALA or an ester or a salt thereof used in the present invention may be in a purified state, or may be in a crude purified state or a state of a mixture obtained by synthesis. In the present invention, a 5-ALA salt is preferably used as an active ingredient, and it is more preferred to use 5-ALA hydrochloride and/or 5-ALA phosphate as an active ingredient.
本發明中“十足目”是也被稱為十腳目(Decapoda)的甲殼類的分類群之一,是包含蝦.蟹.寄居蟹的生物群。作為本發明的物件,十足目的生物優選為蝦,更優選為也被稱為枝鰓亞目(Dendrobranchiata)的對蝦亞目的生物。更優選作為本發明的物件的十足目的生物是屬於對蝦亞目的對蝦科(Penaeidae)的生物。進一步更優選作為本發明的物件的十足目的生物可舉出對蝦科的凡納濱對蝦(Litopenaeus vannamei)、日本對蝦(Marsupenaeus japonicus)、草蝦(黑虎)(Penaeus monodon)、中國對蝦(明蝦)(Fenneropenaeus chinensis)、寬溝對蝦(Melicertus latisulcatus)、刀額新對蝦(Metapenaeus ensis)、須赤蝦(Metapenaeopsis barbata)、短溝對蝦(Penaeus semisulcatus)等,但不限定於這些。另外,進一步更優選作為本發明的物件的十足目的生物為凡納濱對蝦。另外,從十足目EMS/AHPND在幼蝦發生的觀點出發,優選作為本發明的物件的十足目的生物為幼蝦,進一步更優選作為本發明的物件的十足目的生物為對蝦科的幼蝦。 In the present invention, "Decapod" is one of the taxa of the crustaceans also known as Decapoda , which contains shrimp. crab. Herd of hermit crabs. As the article of the present invention, the organism of interest is preferably a shrimp, and more preferably an organism of the shrimp subfamily also known as Dendrobranchiata . More preferably, the organism of interest as an object of the present invention is an organism belonging to the family Penaeidae of the prawn subspecies. Further preferably, as a target organism of the present invention, Litopenaeus vannamei , Marsupenaeus japonicus , Penaeus monodon , and Chinese shrimp (prawn) ( Fenneropenaeus chinensis ), Penicillium latisulcatus , Metapenaeus ensis , Metapenaeopsis barbata , Penaeus semisulcatus , etc., but are not limited thereto. Further, it is still more preferable that the organism of interest as the article of the present invention is P. vannamei. Further, from the viewpoint of the occurrence of juvenile shrimp in the case of the decapod EMS/AHPND, it is preferable that the organism which is the object of the present invention is a juvenile, and it is more preferable that the organism which is a target of the present invention is a juvenile shrimp of the prawn family.
本發明中,十足目用口服給藥組合物只要是對十足目的生物口服給藥的組合物就沒有特別限定。例如,可以是將作為有效成分的選自5-ALA或者其酯、或它們的鹽中的至少一種溶解於水等介質,給藥到飼養十足目的生物的環境中這樣的形態的組合物。此時,通過所給藥的環境中的有效成分被十足目的生物口服攝取,可帶來其效果。 In the present invention, the oral administration composition for decapod is not particularly limited as long as it is a composition for oral administration to a full-sized organism. For example, a composition selected from the group consisting of 5-ALA or an ester thereof or a salt thereof, which is an active ingredient, is dissolved in a medium such as water, and is administered to an environment in which a living organism is raised. At this time, the active ingredient in the administered environment can be orally ingested by a target organism, and the effect can be brought about.
從使十足目的生物更有效地攝取選自5-ALA或者其酯、或它們的鹽中的至少一種的觀點出發,本發明的口服給藥組合物優選為十足目用飼料或十足目用飼料添加劑。十足目用飼料只要是通常在十足目的生物的飼養、養殖中使用的成分就可以含有任意的成分,也可以是在任意的製造方法中製造的飼料。本發明的飼料中,可使用與以往的蝦用飼料的原材料大致相同的原材料,例如,可以含有一般的蝦用飼料中使用的魷魚粉、磷蝦粉、白魚粉、豆油渣、玉米蛋白粉等蛋白質源、穀蛋白、澱粉等粘合劑類、其它維生素混合物、礦物混合物、微量金屬,但不限定於這些。另外,本發明的十足目用飼料可以根據 所飼養的十足目的生物的種類和大小等為任意的形狀、大小。另外,本發明的十足目用飼料可以製造成各種形態。例如,本發明的十足目用飼料可以是將乾燥原料混合、粉碎而成的粉末狀飼料、將粉體固化而成的固化飼料,例如幹顆粒或含有水分的固化飼料,例如糊狀的飼料、或者濕顆粒等。例如,將一般的粉末的蝦養殖用飼料和選自5-ALA或者其酯、或它們的鹽中的至少一種的有效成分任意地與混合用的介質、例如水等一起混合,將該混合物成型,例如,將該混合物從50mL的注射器擠出成型,使成型物乾燥,例如,在60~65℃乾燥2小時左右,從而能夠形成本發明的含有選自5-ALA或者其酯、或它們的鹽中的至少一種的十足目用飼料。飼料的形式、乾燥的程度等只要對給藥沒有不便就沒有特別限定。從在十足目的生物內充分發揮該有效成分的效果這樣的觀點出發,飼料中所含的有效成分(即,飼料中可含有的5-ALA、5-ALA酯、5-ALA的鹽和5-ALA酯的鹽)的合計量以5-ALA磷酸鹽換算計,優選為1~100ppm,更優選為2~50ppm,進一步優選為3~20ppm。特別是從促進十足目的生體的生長的觀點出發,飼料中所含的有效成分的合計量以5-ALA磷酸鹽換算計,優選為5~50ppm,更優選為10~40ppm,進一步更優選為15~30ppm。 The oral administration composition of the present invention is preferably a feed for a full-eyed feed or a feed additive for a full-eyed purpose from the viewpoint of more efficiently ingesting at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof. . The feed for the decapod may be any component as long as it is a component which is usually used for raising and breeding a full-scale organism, and may be a feed produced by any production method. In the feed of the present invention, substantially the same raw materials as those of the conventional shrimp feed can be used, and for example, salmon powder, krill powder, white fish meal, soybean oil residue, corn protein powder, etc., which are used in general shrimp feed, can be used. Binders such as protein sources, gluten, and starch, other vitamin mixtures, mineral mixtures, and trace metals are not limited thereto. Further, the tequila feed of the present invention can be of any shape and size depending on the type and size of the target organism to be raised. Further, the decapod feed of the present invention can be produced in various forms. For example, the feed for the purpose of the present invention may be a powdery feed obtained by mixing and pulverizing dry raw materials, or a solidified feed obtained by solidifying the powder, such as dry granules or a solidified feed containing moisture, such as a paste-like feed. Or wet granules, etc. For example, a feed for shrimp culture of a general powder and an active ingredient of at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof are optionally mixed with a medium for mixing, for example, water or the like, and the mixture is molded. For example, the mixture is extruded from a 50 mL syringe, and the molded product is dried, for example, at 60 to 65 ° C for about 2 hours, thereby being able to form the present invention containing a selected from 5-ALA or an ester thereof, or At least one of the salts of the decapod feed. The form of the feed, the degree of drying, and the like are not particularly limited as long as there is no inconvenience in administration. The active ingredient contained in the feed (that is, the 5-ALA, 5-ALA ester, 5-ALA salt, and 5-- contained in the feed) from the viewpoint of sufficiently exerting the effect of the active ingredient in the full-scale organism. The total amount of the salt of the ALA ester is preferably from 1 to 100 ppm, more preferably from 2 to 50 ppm, even more preferably from 3 to 20 ppm, in terms of 5-ALA phosphate. In particular, the total amount of the active ingredients contained in the feed is preferably 5 to 50 ppm, more preferably 10 to 40 ppm, more preferably 10 to 40 ppm, more preferably from the viewpoint of promoting the growth of the living body. 15~30ppm.
另外,十足目用口服給藥組合物可以是十足目用飼料添加劑。這裡的飼料添加劑只要是能夠添加於通常的十足目用的飼料的添加劑即可,沒有特別限定。例如,本發明中的飼料添加劑可以含有選自5-ALA或者其酯、或它們的鹽中的至少一種以及能夠使該有效成分附著於十足目用飼料的擴展劑等,也可以含有使該有效成分吸收到十足目用飼料這樣的介質,或者也可以含有使該有效成分容易混合於十足目用飼料的原料這樣的介質。本發明的飼料添加劑優選以飼料中所含的有效成分的合計量為上述範圍的方式添加於飼料。 Alternatively, the versatile oral administration composition may be a feed additive for the full purpose. The feed additive herein is not particularly limited as long as it is an additive that can be added to a normal fortune feed. For example, the feed additive of the present invention may contain at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof, and an extender capable of attaching the active ingredient to a feed for decapod, or the like, or may be effective. The component is absorbed into a medium such as a feed for the full-eyed feed, or may be a medium containing a raw material which allows the active ingredient to be easily mixed with the feed for the full-eyed feed. The feed additive of the present invention is preferably added to the feed so that the total amount of the active ingredients contained in the feed is in the above range.
本發明的其它實施方式是包括使十足目的生物攝取選自5-ALA或者其酯、或它們的鹽中的至少一種的方法。這裡的攝取是口服攝取。只要能夠使十足目的生物攝取作為有效成分的選自5-ALA或者其酯、或它們的鹽中的至少一種,則其方法沒有特別限定。例如,有如下方法:在飼養十足目的生物的環境下,添加選自5-ALA或者其酯、或它們的鹽中的至少一種,使十足 目攝取該有效成分。但是,從使十足目更有效地攝取作為有效成分的選自5-ALA或者其酯、或它們的鹽中的至少一種這樣的觀點出發,優選使十足目的生物攝取含有選自5-ALA或者其酯、或它們的鹽中的至少一種的飼料。 Another embodiment of the present invention is a method comprising ingesting at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof. The intake here is oral intake. The method is not particularly limited as long as it is capable of ingesting at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof as an active ingredient. For example, there is a method of adding at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof in an environment in which a full-bodied organism is raised, so that the active ingredient is ingested. However, from the viewpoint of allowing the decapod to more efficiently ingest at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof as an active ingredient, it is preferred that the biological ingestion of the purpose is selected to be selected from 5-ALA or A feed of at least one of an ester, or a salt thereof.
本發明的其它實施方式是包括使十足目的生物攝取規定的量的選自5-ALA或者其酯、或它們的鹽中的至少一種的、促進十足目的生物的生長的方法。該實施方式中,使十足目的生物攝取的作為有效成分的選自5-ALA或者其酯、或它們的鹽中的至少一種的合計量優選以5-ALA磷酸鹽換算計,十足目的生物的體重每1g且每1天計為0.25μg/g.天~2.5μg/g.天,更優選為0.5μg/g.天~2.0μg/g.天,進一步更優選為0.75~1.5μg/g.天。雖然不受理論的束縛,但作為本發明的該實施方式的十足目的生物的生長促進的機理之一,認為在十足目的生物的生體內,5-ALA會提高從攝取的餌提取能量的效率。 Other embodiments of the present invention include a method of promoting the growth of a target organism by selecting a predetermined amount of a selected organism selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof. In this embodiment, the total amount of at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof, which is an active ingredient ingested by the target organism, is preferably in the form of 5-ALA phosphate, and the body weight of the target organism It is 0.25 μg/g per 1 g and every 1 day. Day ~2.5μg/g. Day, more preferably 0.5 μg / g. Day ~2.0μg/g. Further, it is more preferably 0.75 to 1.5 μg/g. day. Although not bound by theory, as one of the mechanisms for promoting growth of a full-scale organism of this embodiment of the present invention, it is considered that 5-ALA improves the efficiency of extracting energy from the ingested bait in the living organism of the full-scale organism.
本發明的其它實施方式是含有選自5-ALA或者其酯、或它們的鹽中的至少一種的十足目早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)的預防.治療用口服給藥組合物。另外,本發明的另一其它實施方式是包括使十足目的生物攝取選自5-ALA或者其酯、或它們的鹽中的至少一種的、預防.治療十足目早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)的方法。本發明中的“十足目早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)”的“預防.治療”是指在十足目的生物的養殖中成為嚴重問題的以副溶血弧菌(Vibrio parahaemolyticus)為致病菌的被稱為早期死亡綜合症/急性肝胰臟壞死病(EMS/AHPND)的蝦的疾病的預防和/或治療。這裡,預防是指在選自5-ALA或者其酯、或它們的鹽中的至少一種的給藥下抑制EMS/AHPND的發病,即完全地抑制發病或降低發病率。另外,治療是指使感染了副溶血弧菌(Vibrio parahaemolyticus)或發病了EMS/AHPND的十足目的生物的該感染和EMS/AHPND治癒。本發明的該實施方式中,作為有效成分的選自5-ALA或者其酯、或它們的鹽中的至少一種發揮能夠預防和/或治療該十足目的EMS/AHPND這樣的有利的效果。該效果在實施例中以致病菌攻擊後的死亡率降低這樣的形式明確地示出。通過作為有效成分的選自5-ALA或者其酯、或它們的鹽中的至少一種帶來的該有利的效果是以往未知的效果,是 十足目的飼養、養殖的技術領域的本領域技術人員無法預期的效果。十足目EMS/AHPND的預防.治療中,使十足目的生物攝取的作為有效成分的選自5-ALA或者其酯、或它們的鹽中的至少一種的合計量以5-ALA磷酸鹽換算計,十足目的生物的體重每1g且每1天計優選為0.05μg/g.天~5μg/g.天,更優選為0.1μg/g.天~2.5μg/g.天,進一步更優選為0.15~1μg/g.天。 Another embodiment of the present invention is the prevention of Decapod Early Death Syndrome/Acute Hepatopancreatic Necrosis (EMS/AHPND) comprising at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof. Oral administration compositions for therapeutic use. In addition, another embodiment of the present invention includes the prevention of at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof. A method for treating decapod early death syndrome/acute hepatic pancreatic necrosis (EMS/AHPND). "Prevention, treatment" of "Decaptor Early Death Syndrome/Acute Hepatopancreatic Necrosis (EMS/AHPND)" in the present invention refers to Vibrio parahaemolyticus which is a serious problem in the cultivation of a full-scale organism ( Vibrio) parahaemolyticus) for the pathogen is known as prevention of early death syndrome / acute liver necrosis pancreas disease (EMS / AHPND) of shrimp disease and / or therapy. Here, prevention means inhibiting the onset of EMS/AHPND under administration of at least one selected from the group consisting of 5-ALA or an ester thereof, or a salt thereof, that is, completely inhibiting the onset or reducing the incidence. In addition, treatment refers to the infection and EMS/AHPND healing of a full-scale organism infected with Vibrio parahaemolyticus or EMS/AHPND. In the embodiment of the present invention, at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof as an active ingredient exerts an advantageous effect of preventing and/or treating the EMS/AHPND of the full purpose. This effect is clearly shown in the form of a decrease in mortality after attack by pathogenic bacteria in the examples. The advantageous effect by at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof as an active ingredient is a previously unknown effect, and those skilled in the art who are skilled in breeding and breeding can not Expected effect. Prevention of Decapod EMS/AHPND. In the treatment, the total amount of at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof, which is an active ingredient ingested by a full-scale organism, is in a weight ratio of 5-ALA phosphate, and the weight of the objective organism is 1 g per 1 g. It is preferably 0.05 μg/g per day. Day ~5μg/g. Day, more preferably 0.1 μg / g. Day ~2.5μg/g. Further, it is more preferably 0.15 to 1 μg/g. day.
應用有本發明的十足目的生物對飼養、養殖的環境沒有特別限定,本發明也能夠應用於在東南亞等大規模進行的池中的養殖,另外,本發明也能夠應用於水槽等的小規模的飼養中。以下, 通過實施例詳述本發明,但本發明不限定於實施例的範圍。 The environment in which the organism of the present invention is used for feeding and breeding is not particularly limited, and the present invention can also be applied to cultivation in a pond which is carried out on a large scale such as Southeast Asia, and the present invention can also be applied to a small scale such as a water tank. Feeding. Hereinafter, the present invention will be described in detail by way of examples, but the invention is not limited to the scope of the examples.
實施例 Example
實施例1:含有5-ALA的飼料的製成 Example 1: Preparation of a feed containing 5-ALA
以5-ALA磷酸鹽(C5H9NO3.H3PO4)為濃度15ppm的方式與粉末飼料(將在泰國為了養殖凡納濱對蝦而使用的一般的市售餌進行粉末化而使用)充分混合,添加與粉末飼料等量的蒸餾水並充分混合。接下來,將所得的混合物裝入50mL的注射器,進行擠出,從而製成義大利麵條狀的成型飼料。將其在60~65℃乾燥2小時左右。乾燥後,將義大利麵條狀的成型飼料較小地粉碎製成顆粒狀以便容易給藥。該顆粒保存在冰箱中直至使用。 Using 5-ALA phosphate (C 5 H 9 NO 3 .H 3 PO 4 ) at a concentration of 15 ppm and powdered feed (used in order to pulverize a general commercial bait used in Thailand for the cultivation of P. vannamei) ) Mix well, add equal amount of distilled water to the powder feed and mix well. Next, the obtained mixture was placed in a 50 mL syringe and extruded to prepare a shaped feed of Italian pasta. It is dried at 60 to 65 ° C for about 2 hours. After drying, the pasta-shaped shaped feed is pulverized into small particles to facilitate administration. The granules are kept in the refrigerator until use.
實施例2:5-ALA對受到利用副溶血弧菌(Vibrio parahaemolyticus)的攻擊的凡納濱對蝦的存活率產生的影響 Example 2: Effect of 5-ALA on the survival rate of Penaeus vannamei challenged by Vibrio parahaemolyticus
使用體重約2g的凡納濱對蝦。將每組20只的凡納濱對蝦放入100L的水槽中,進行28天飼養。在5-ALA投藥組中,給藥實施例1中製成的飼料,在對照組中,除了不含5-ALA以外,給藥相同的飼料。投餌量設為蝦的體重的5%,投餌為1天4次,使用自動投餌器進行。 Use a prawn with a body weight of about 2g. Twenty per cent of each group of P. vannamei was placed in a 100 L water tank for 28 days. In the 5-ALA administration group, the feed prepared in Example 1 was administered, and in the control group, the same feed was administered except that 5-ALA was not contained. The amount of feeding was set to 5% of the weight of the shrimp, and the feeding was performed 4 times a day, using an automatic feeder.
從试验开始第2周,將凡納濱對蝦轉移到放入了以3×105cfu/ml的量含有副溶血弧菌(Vibrio parahaemolyticus)的海水的15L水槽,進行副溶血弧菌(Vibrio parahaemolyticus)對凡納濱對蝦的感染處理。從感染處理起經過2周確認凡納濱對蝦的存活率。將結果示於第1圖。第1圖中,“Control”表 示對照組,“ALA”表示5-ALA投藥組。 From the second week of the test, the P. vannamei was transferred to a 15 L water tank containing seawater containing Vibrio parahaemolyticus in an amount of 3 × 10 5 cfu/ml for Vibrio parahaemolyticus. ) Infection treatment of P. vannamei. The survival rate of P. vannamei was confirmed 2 weeks after the infection treatment. The results are shown in Fig. 1. In Fig. 1, "Control" indicates a control group, and "ALA" indicates a 5-ALA administration group.
如第1圖所示,在未給藥5-ALA的對照組中,從感染後第7天,顯示存活率的顯著降低,在感染後第13天,全部凡納濱對蝦死亡。另一方面,在預先給藥5-ALA的投藥組中,在觀察期間中,僅死亡1只,相對於對照組,顯示顯著的存活率(p值<0.0001)。因此,明確了5-ALA是在十足目的生物中對以副溶血弧菌(Vibrio parahaemolyticus)為致病菌的EMS/AHPND有效的預防.治療藥。應予說明,認為若在實際的蝦養殖場發生EMS/AHPND,則大致100%的蝦會死亡,因此,實施例2的實驗條件模擬了在實際的蝦養殖場的EMS/AHPND的發生。因此,推測5-ALA對在實際的蝦養殖場的EMS/AHPND的預防.治療是有效的。 As shown in Fig. 1, in the control group to which 5-ALA was not administered, a significant decrease in the survival rate was observed from the 7th day after the infection, and on the 13th day after the infection, all the P. vannamei died. On the other hand, in the administration group to which 5-ALA was administered in advance, only one patient died during the observation period, and a significant survival rate (p value <0.0001) was shown with respect to the control group. Therefore, it is clear that 5-ALA is an effective preventive against EMS/AHPND which is a pathogenic bacterium of Vibrio parahaemolyticus in a full-scale organism. Therapeutic medicine. It should be noted that if EMS/AHPND occurs in an actual shrimp farm, approximately 100% of the shrimp will die. Therefore, the experimental conditions of Example 2 simulate the occurrence of EMS/AHPND in the actual shrimp farm. Therefore, it is speculated that 5-ALA is against the prevention of EMS/AHPND in actual shrimp farms. Treatment is effective.
實施例3:以下的試驗中使用的凡納濱對蝦的飼養條件 Example 3: Feeding conditions of Penaeus vannamei used in the following tests
將平均體重0.84±0.33克的凡納濱對蝦400只每100只分成以下的4組,各組在不同的水槽中飼養。在開始飼養時、從飼養開始起第2周和第3個月,測試以下所示的各種項目。 400 of the average body weight of 0.84 ± 0.33 g of 400 vanilla lobster were divided into the following 4 groups, and each group was kept in a different tank. The various items shown below were tested at the beginning of the breeding, from the start of the second week and the third month from the start of the breeding.
(a)給藥含有15ppm的5-ALA的飼料的15ppm的5-ALA給藥組。 (a) A 15 ppm 5-ALA administration group containing 15 ppm of 5-ALA feed was administered.
(b)給藥含有30ppm的5-ALA的飼料的30ppm的5-ALA給藥組。 (b) A 30 ppm 5-ALA administration group containing 30 ppm of 5-ALA feed was administered.
(c)給藥含有60ppm的5-ALA的飼料的60ppm的5-ALA給藥組。 (c) A 60 ppm 5-ALA administration group containing 60 ppm of 5-ALA feed was administered.
(d)給藥不含5-ALA的飼料的對照組。 (d) A control group in which a feed containing no 5-ALA was administered.
對凡納濱對蝦的每一天的投餌量設為凡納濱對蝦的平均體重的5%。將每一天的投餌量分成4次(8:00、13:00、18:00和23:00),對凡納濱對蝦給予飼料。通過每週測定各組的凡納濱對蝦整體的重量來監測凡納濱對蝦的體重,基於所測定的體重調節每一天的投餌量。吃剩的飼料和***物每天去除一次。飼養中也監測水質參數。 The amount of feed per day for the prawn was set at 5% of the average body weight of the prawn. The amount of feed per day was divided into 4 times (8:00, 13:00, 18:00, and 23:00), and the feed was given to the shrimp. The body weight of L. vannamei was monitored by measuring the overall weight of each group of L. vannamei per week, and the amount of feed per day was adjusted based on the measured body weight. The leftover feed and excreta are removed once a day. Water quality parameters are also monitored during rearing.
含有15ppm的5-ALA的飼料以如下方式製成。將150mg的1%5-ALA粉體(含有5-ALA磷酸鹽(C5H9NO3.H3PO4))溶解於100ml的水中,將所得的溶液與100克的蝦用粉末飼料(將在泰國為了養殖凡納濱對蝦而使用的一般的市售餌粉末化而使用)充分混合,得到飼料混合物。接下來,使用磨碎機將飼料混合物製成顆粒。將該顆粒在恒溫箱內在60~65℃乾燥2~3小時左右,得到含有15ppm的5-ALA的飼料。該飼料在冰箱內在4℃保存直至使用。含有30ppm的5-ALA的飼料和含有60ppm的5-ALA的飼料除了所添加的1%5-ALA粉體的量分別為300mg和600mg以外,通過與含有15ppm的5-ALA的飼料的製造方法相同的方法製造。對照組中使用的不含5-ALA的飼料除了不添加1%5-ALA粉體以外,通過與含有15ppm的5-ALA的飼料的製造方法相同的方法製造。 A feed containing 15 ppm of 5-ALA was prepared in the following manner. 150 mg of 1% 5-ALA powder (containing 5-ALA phosphate (C 5 H 9 NO 3 .H 3 PO 4 )) was dissolved in 100 ml of water, and the resulting solution was mixed with 100 g of shrimp powder feed ( The mixture was thoroughly mixed to pulverize a general commercial bait used in Thailand for the cultivation of P. vannamei to obtain a feed mixture. Next, the feed mixture is granulated using an attritor. The pellet was dried in an incubator at 60 to 65 ° C for about 2 to 3 hours to obtain a feed containing 15 ppm of 5-ALA. The feed was stored in the refrigerator at 4 ° C until use. A feed containing 30 ppm of 5-ALA and a feed containing 60 ppm of 5-ALA, except for the amount of 1% 5-ALA powder added, respectively, 300 mg and 600 mg, and a method of producing a feed containing 15 ppm of 5-ALA Manufactured in the same way. The 5-ALA-free feed used in the control group was produced by the same method as the method of producing a feed containing 15 ppm of 5-ALA except that 1% of the 5-ALA powder was not added.
實施例4:5-ALA對凡納濱對蝦的生長的影響 Example 4: Effect of 5-ALA on the growth of Penaeus vannamei
在實施例3所記載的條件下的凡納濱對蝦的飼養中,通過測定3個月的飼養期間的體重增加,研究通過5-ALA對凡納濱對蝦的生長產生的影響。飼養開始時和從飼養開始起第3個月,測定各組的凡納濱對蝦的各個體重。結果示於以下的表1。表中,初始體重是飼養開始時(第0天)的體重,最終體重是第3個月的體重,體重增加為最終體重-初始體重,SGR為瞬間生長率(Specific Growth Rate)(%),根據SGR=〔(ln最終重量-ln初始重量)/投餌天數〕×100的式子算出,FCR為飼料要求率(Feed Conversion Rate),FCR=飼料消耗/體重增加。表中的初始體重、最終體重、體重增加和SGR的數值為平均±標準差(mean±SD)。表中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。 In the feeding of L. vannamei under the conditions described in Example 3, the effect of 5-ALA on the growth of P. vannamei was examined by measuring the body weight gain during the three-month feeding period. The respective body weights of each group of P. vannamei were measured at the start of feeding and at the third month from the start of feeding. The results are shown in Table 1 below. In the table, the initial body weight is the body weight at the start of feeding (day 0), the final body weight is the weight of the third month, the weight gain is the final body weight - the initial body weight, and the SGR is the Specific Growth Rate (%). Calculated according to the formula of SGR = [(infinal weight - ln initial weight) / feeding days] × 100, FCR is the feed conversion rate (Feed Conversion Rate), FCR = feed consumption / weight gain. The values of initial body weight, final body weight, weight gain, and SGR in the table are mean ± standard deviation (mean ± SD). The records of 15 ppm, 30 ppm, and 60 ppm in the table indicate 15 ppm of the 5-ALA administration group, 30 ppm of the 5-ALA administration group, and 60 ppm of the 5-ALA administration group, respectively.
如表1所示,15ppm的5-ALA給藥組和30ppm的5-ALA給藥組與對照組相比,體重增加和瞬間生長率(SGR)大。由此明確了對凡納濱對蝦的15ppm的5-ALA給藥和30ppm的5-ALA給藥促進凡納濱對蝦的生長。另外,15ppm的5-ALA給藥組和30ppm的5-ALA給藥組與對照組相比,飼料要求率(FCR)稍微降低。由此,雖然不受理論的束縛,但推測15ppm的5-ALA給藥組和30ppm的5-ALA給藥組的通過5-ALA的凡納濱對蝦的生長促進的原因之一有可能是在凡納濱對蝦生體內通過5-ALA而提高從飼料的能量提取效率。 As shown in Table 1, the 15 ppm 5-ALA administration group and the 30 ppm 5-ALA administration group had a larger body weight gain and instantaneous growth rate (SGR) than the control group. It was thus confirmed that 15 ppm of 5-ALA administration and 30 ppm of 5-ALA administration to P. vannamei promoted the growth of P. vannamei. In addition, the feed requirement rate (FCR) of the 15 ppm 5-ALA administration group and the 30 ppm 5-ALA administration group was slightly lower than that of the control group. Thus, although not bound by theory, it is presumed that one of the reasons for the growth promotion of the 5-ALA-administered penaeus vannamei in the 15-ppm 5-ALA-administered group and the 30-ppm 5-ALA-administered group may be The efficiency of energy extraction from feed is improved by 5-ALA in the body of the shrimp.
另外,測定3個月的飼養期間中的凡納濱對蝦的脫皮的頻率,將其脫皮的累積頻率示於第2圖。第2圖中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。第2圖中,將脫皮的累積頻率最高的30ppm的5-ALA給藥組的第12周的脫皮的累積頻率設為100%來表示各組的脫皮的累積頻率。如第2圖所示,脫皮的累積頻率在30ppm的5-ALA給藥組中最高,接下來為15ppm的5-ALA給藥組,脫皮的頻率最低的是對照組。蝦隨著生長而脫皮,因此推測通過30ppm和15ppm的5-ALA給藥帶來的脫皮的累積頻率的提高也表示通過5-ALA給藥促進了凡納濱對蝦的生長。 Further, the frequency of peeling of P. vannamei during the feeding period of 3 months was measured, and the cumulative frequency of peeling was shown in Fig. 2 . The descriptions of 15 ppm, 30 ppm, and 60 ppm in Fig. 2 indicate a 15 ppm 5-ALA administration group, a 30 ppm 5-ALA administration group, and a 60 ppm 5-ALA administration group, respectively. In Fig. 2, the cumulative frequency of peeling at the 12th week of the 5-ALA administration group having the highest cumulative frequency of peeling was set to 100%, and the cumulative frequency of peeling of each group was shown. As shown in Fig. 2, the cumulative frequency of peeling was highest in the 5-ALA administration group of 30 ppm, followed by the 15-ALA administration group of 15 ppm, and the lowest frequency of peeling was the control group. Shrimp peeled off as it grew, so it is speculated that an increase in the cumulative frequency of peeling by administration of 30- and 15 ppm of 5-ALA also indicates that the growth of Penaeus vannamei is promoted by 5-ALA administration.
實施例5:5-ALA對肝胰臟的ATP水準的影響 Example 5: Effect of 5-ALA on ATP level of hepatopancreas
從飼養開始起第2周,測定凡納濱對蝦的肝胰臟中的ATP水準。該ATP水準的測定是對每組3只的凡納濱對蝦進行的。ATP濃度測定用樣品以 如下方式製備。從各個凡納濱對蝦採取約10mg的肝胰臟。所採取的肝胰臟用磷酸緩衝生理鹽水(1×PBS)清洗。所清洗的肝胰臟在冰冷的2N的高氯酸(PCA)100μl中進行勻漿化,均漿在冰上保持30分鐘,接下來以13000×g、4℃離心分離2分鐘,得到上清液。將該上清液用ATP反應緩衝液稀釋成500μl。接下來,在稀釋的上清液中添加50~100μl的冰冷KOH(2M),使過量的PCA沉澱。根據需要在該上清液中添加0.1M的KOH或PCA,調節pH。所得的樣品接下來以13000×g離心分離15分鐘,回收上清液,用作ATP濃度測定用樣品。ATP濃度測定使用ATP比色分析試劑盒(目錄編號:ab83355;(Abcam)(註冊商標),劍橋,馬薩諸塞州,美國)。ATP濃度的測定根據該試劑盒的製造者的指示進行。結果示於第3圖。第3圖中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。 The ATP level in the hepatopancreas of L. vannamei was measured from the second week from the start of feeding. The ATP level was determined for each group of 3 P. vannamei. A sample for ATP concentration measurement was prepared in the following manner. About 10 mg of hepatopancreas was taken from each shrimp. The hepatopancreas taken was washed with phosphate buffered saline (1 x PBS). The washed hepatopancreas was homogenized in 100 μl of ice-cold 2N perchloric acid (PCA), and the homogenate was kept on ice for 30 minutes, followed by centrifugation at 13,000 × g for 2 minutes at 4 ° C to obtain a supernatant. liquid. The supernatant was diluted to 500 μl with ATP reaction buffer. Next, 50 to 100 μl of ice-cold KOH (2 M) was added to the diluted supernatant to precipitate an excess of PCA. 0.1 M KOH or PCA was added to the supernatant as needed to adjust the pH. The obtained sample was centrifuged at 13,000 × g for 15 minutes, and the supernatant was collected and used as a sample for ATP concentration measurement. ATP concentration assay using ATP colorimetric assay kit (Catalog No.: ab83355; (Abcam) (registered trademark), Cambridge, Massachusetts, USA). The ATP concentration was measured according to the instructions of the manufacturer of the kit. The results are shown in Figure 3. The descriptions of 15 ppm, 30 ppm, and 60 ppm in Fig. 3 indicate a 15 ppm 5-ALA administration group, a 30 ppm 5-ALA administration group, and a 60 ppm 5-ALA administration group, respectively.
如第3圖所示,凡納濱對蝦的肝胰臟中的ATP水準與對照組相比,在全部5-ALA給藥組中高。凡納濱對蝦的肝胰臟中的ATP水準依賴於5-ALA的用量地增大。30ppm和60ppm的5-ALA給藥組中,對於相對於對照組的ATP水準的上升,在統計學上確認到顯著性差異。顯著性差異檢驗使用t檢驗法,第3圖中的星號表示p<0.05。60ppm的5-ALA給藥組中,ATP水準的上升與體重增加不相關。雖然不受理論的束縛,但認為這是因為由於過量的5-ALA,電子傳遞系統被過量地活化,通過對電子傳遞系統供給還原力的TCA回路消耗了大量的脂質、碳水化合物。但是,15ppm和30ppm的5-ALA給藥組中的ATP水準的上升支持了推測為通過5-ALA的凡納濱對蝦的生長促進的原因之一的在凡納濱對蝦生體內通過5-ALA提高了從飼料的能量提取效率。 As shown in Fig. 3, the ATP level in the hepatopancreas of L. vannamei was higher in all 5-ALA-administered groups than in the control group. The ATP level in the hepatopancreas of L. vannamei depends on the amount of 5-ALA. In the 5-ALA administration group of 30 ppm and 60 ppm, a statistically significant difference was statistically confirmed for the rise in the ATP level with respect to the control group. The significance test was performed using the t test, and the asterisk in Fig. 3 indicates p < 0.05. In the 60-ppm 5-ALA-administered group, the increase in ATP level was not associated with weight gain. While not being bound by theory, it is believed that this is because the electron transfer system is excessively activated due to excess 5-ALA, and a large amount of lipids, carbohydrates are consumed by the TCA loop that supplies reducing power to the electron transport system. However, the increase in ATP level in the 5-ALA-administered group of 15 ppm and 30 ppm supported the passage of 5-ALA in the body of P. vannamei which was presumed to be one of the causes of growth promotion of P. vannamei by 5-ALA. Improved energy extraction efficiency from feed.
實施例6:利用副溶血弧菌(Vibrio parahaemolyticus)的EMS/AHPND感染試驗 Example 6: EMS/AHPND infection test using Vibrio parahaemolyticus
對5-ALA對受到利用副溶血弧菌(Vibrio parahaemolyticus)的攻擊的凡納濱對蝦的存活率產生的影響進行研究。 The effect of 5-ALA on the survival rate of P. vannamei challenged by Vibrio parahaemolyticus was investigated.
對各個組準備放入了以3×106cfu/ml的量(高用量)含有副溶血 弧菌(Vibrio parahaemolyticus)的海水的10L水槽和放入了以3×105cfu/ml的量(低用量)含有副溶血弧菌(Vibrio parahaemolyticus)的海水的10L水槽。將從飼養開始起第3個月的凡納濱對蝦各組各10只轉移到這些水槽中,進行副溶血弧菌(Vibrio parahaemolyticus)對凡納濱對蝦的感染處理。從感染處理起經過2周對各組的凡納濱對蝦投餌與在感染處理前給藥的相同的飼料(含有15ppm、30ppm或60ppm的5-ALA的飼料,或不含5-ALA的飼料)。從感染處理起經過2周每天確認各組和各副溶血弧菌(Vibrio parahaemolyticus)用量下的凡納濱對蝦的存活率。將用以3×106cfu/ml的量(高用量)含有副溶血弧菌(Vibrio parahaemolyticus)的海水處理凡納濱對蝦的結果示於第4圖,將用以3×105cfu/ml的量(低用量)含有副溶血弧菌(Vibrio parahaemolyticus)的海水處理凡納濱對蝦的結果示於第5圖。第4圖和第5圖中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。 For each group, a 10 L water tank containing seawater containing Vibrio parahaemolyticus in an amount of 3 × 10 6 cfu/ml (highly used) was placed and contained in an amount of 3 × 105 cfu/ml (low dosage). A 10L water tank of seawater of Vibrio parahaemolyticus. Ten groups of each of the groups of the five-month-old prawn were transferred to the water tanks for Vibrio parahaemolyticus infection treatment of the prawn. The same feed (containing 15 ppm, 30 ppm or 60 ppm of 5-ALA feed, or 5-ALA-free feed) was administered to each group of prawn peninsula for 2 weeks from the infection treatment. ). The survival rate of P. vannamei at each dose of Vibrio parahaemolyticus was confirmed daily for 2 weeks from the infection treatment. The results of seawater treatment of P. vannamei with an amount of 3 x 106 cfu/ml (high dose) containing Vibrio parahaemolyticus are shown in Fig. 4 and will be used in an amount of 3 x 105 cfu/ml (low The amount of the seawater-treated P. vannamei containing Vibrio parahaemolyticus is shown in Fig. 5. The records of 15 ppm, 30 ppm, and 60 ppm in Figs. 4 and 5 represent a 15 ppm 5-ALA administration group, a 30 ppm 5-ALA administration group, and a 60 ppm 5-ALA administration group, respectively.
如第4圖所示,用以3×106cfu/ml的量(高用量)含有副溶血弧菌(Vibrio parahaemolyticus)的海水處理凡納濱對蝦時,60ppm的5-ALA給藥組與對照組相比,顯示非常高的存活率。15ppm和30ppm的5-ALA給藥組與60ppm的5-ALA給藥組相比,存活率低,但與對照組相比,顯示高的存活率。用以3×105cfu/ml的量(低用量)含有副溶血弧菌(Vibrio parahaemolyticus)的海水處理凡納濱對蝦時,如5圖所示,15ppm、30ppm和60ppm的5-ALA給藥組與對照組相比,均顯示非常高的存活率。因此,明確了5-ALA是在十足目的生物中對以副溶血弧菌(Vibrio parahaemolyticus)為致病菌的EMS/AHPND有效的預防.治療藥。 As shown in Fig. 4, 60 ppm of 5-ALA-administered group and control were used to treat L. vannamei with seawater containing Vibrio parahaemolyticus in an amount of 3 × 10 6 cfu/ml (high dose). Compared to the group, it shows a very high survival rate. The 5-ALA administration group of 15 ppm and 30 ppm showed a lower survival rate than the 60 ppm 5-ALA administration group, but showed a high survival rate as compared with the control group. When the L. vannamei was treated with seawater containing Vibrio parahaemolyticus in an amount of 3 × 10 5 cfu/ml (as shown in Figure 5), 15 ppm, 30 ppm and 60 ppm of 5-ALA were administered. The groups showed very high survival rates compared to the control group. Therefore, it is clear that 5-ALA is an effective preventive against EMS/AHPND which is a pathogenic bacterium of Vibrio parahaemolyticus in a full-scale organism. Therapeutic medicine.
實施例7:利用副溶血弧菌(Vibrio parahaemolyticus)的感染處理中的5-ALA對總血球數的影響 Example 7: Effect of 5-ALA on the total number of blood cells in an infection treatment using Vibrio parahaemolyticus
通過與實施例6中的方法相同的方法利用副溶血弧菌(Vibrio parahaemolyticus)(高用量)對從飼養開始起第3個月的凡納濱對蝦進行感染處理。在感染處理前(0小時)、從感染處理起6小時後以及從感染處理起12小時後,從各組各3只凡納濱對蝦的腹竇(ventral sinus)採取200μl的血淋巴 (hemolymph),用800μl的抗凝劑稀釋。使用C-晶片(C-chip)血球計(NanoEntek,德國)測定血淋巴中的總血球數。第6圖中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。如第6圖所示,在感染處理前和感染處理後的全部時刻,在5-ALA給藥組中,與對照組相比,總血球數增加。從感染處理起6小時後的60ppm的5-ALA給藥組中,與其它組相比,顯示統計學上優勢高的總血球數。顯著性差異檢驗使用t檢驗法。雖然不受理論的束縛,但認為在蝦中粒細胞等血球作為免疫擔當細胞承擔重要的作用,因此5-ALA給藥對EMS/AHPND的預防.治療效果的原因之一有可能是通過基於5-ALA的總血球數的增大而引起自然免疫系統的活化。 In the same manner as in the method of Example 6, Vibrio parahaemolyticus (high amount) was used to infect the P. vannamei in the third month from the start of the feeding. 200 μl of hemolymph was taken from the ventral sinus of each of the three groups of each of the three groups of ventral sinus before the infection treatment (0 hours), 6 hours after the infection treatment, and 12 hours after the infection treatment. , diluted with 800 μl of anticoagulant. The total number of hematocrit in the hemolymph was measured using a C-chip hemacytometer (NanoEntek, Germany). The records of 15 ppm, 30 ppm, and 60 ppm in Fig. 6 indicate a 15 ppm 5-ALA administration group, a 30 ppm 5-ALA administration group, and a 60 ppm 5-ALA administration group, respectively. As shown in Fig. 6, the total number of blood cells was increased in the 5-ALA-administered group compared with the control group at all times before the infection treatment and after the infection treatment. In the 60 ppm 5-ALA administration group 6 hours after the infection treatment, the statistically superior total number of blood cells was shown as compared with the other groups. The significance test was performed using the t test. Although not bound by theory, it is believed that blood cells such as granulocytes in shrimp play an important role as immune-bearing cells, so 5-ALA administration is the prevention of EMS/AHPND. One of the reasons for the therapeutic effect may be the activation of the natural immune system by an increase in the total number of blood cells based on 5-ALA.
實施例8:利用副溶血弧菌(Vibrio parahaemolyticus)的感染處理中的5-ALA對血紅素加氧酶-1和酚氧化酶前體的基因表達的影響 Example 8: Effect of 5-ALA on infection of heme oxygenase-1 and phenoloxidase precursors in infection treatment with Vibrio parahaemolyticus
通過與實施例6中的方法相同的方法利用副溶血弧菌(Vibrio parahaemolyticus)(高用量)對從飼養開始起第3個月的凡納濱對蝦進行感染處理。在感染處理前(0小時)、從感染處理起6小時後以及從感染處理起12小時後,從各組各5只凡納濱對蝦採取肝胰臟,使用RNAiso Plus試劑(Takara Bio,日本)根據該試劑的製造者的指示從各個凡納濱對蝦的肝胰臟提取總RNA。使用高容量逆轉錄試劑盒(High-Capacity Reverse Transcription kit)(應用生物系統(Applied Biosystems),美國)根據該試劑盒的製造者的指示由1μg的總RNA提取物合成cDNA。所合成的cDNA稀釋為5倍,作為用於qPCR的範本使用。通過對該cDNA施加使用SYBR綠色螢光色素的即時聚合酶鏈反應(PCR),測定血紅素加氧酶-1(HO-1)和酚氧化酶前體(proPO)的基因表達。該測定使用Thunderbird(註冊商標)SYBR qPCR Mix(東洋紡,日本)進行。擴增反應使用MicroAmp Optical 96-孔反應板(應用生物系統,美國)進行。各孔收容10μl的qPCRMix、0.6μl的各引物、0.4μl的ROX參照色素和2μl的cDNA範本。迴圈條件如下所述。將在95℃1分鐘,接下來,在90℃15秒和在60℃60秒進行40次迴圈。各qPCR反應結束時,進行解離分析,確認為僅1種生成物的檢測。使用2-ΔΔCt法(Livak和Schmittgen,2001)確定基 於qPCR的基因表達資料的相對變化。為了計算Ct值(迴圈閾值:Threshold Cycle),測定EF1α的基因表達作為內部標準。計算使用對照組的相對表達進行標準化的比率。資料在分析前進行對數(底=2)轉換。為了測定血紅素加氧酶-1的表達,使用具有序列1(5’-CTGAGGAGCTCGATGAGGAG-3’)的正向引物和具有序列2(5’-CATGGCCACAACACTACCAG-3’)的反向引物。為了測定酚氧化酶前體的表達,使用具有序列3(5’-GGAATTGTTTTACTACATGCATCAGC-3’)的正向引物和具有序列4(5’-GGAACAAGTCATCCACGAGCTT-3’)的反向引物。為了測定EF1α的表達,使用具有序列5(5’-ATTGCCACACCGCTCACA-3’)的正向引物和具有序列6(5’-TCGATCTTGGTCAGCAGTTCA-3’)的反向引物。結果示於第7~8圖。各時刻的對照組的基因表達的平均值設為0,各組的基因表達是相對於該平均值的相對值。第7~8圖中的誤差棒為標準差。第7~8圖中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。 In the same manner as in the method of Example 6, Vibrio parahaemolyticus (high amount) was used to infect the P. vannamei in the third month from the start of the feeding. Before the infection treatment (0 hours), 6 hours after the infection treatment, and 12 hours after the infection treatment, hepatic pancreas was taken from each of the 5 groups of each of the five species of vannamei, using RNAiso Plus reagent (Takara Bio, Japan) Total RNA was extracted from the hepatopancreas of each P. vannamei according to the instructions of the manufacturer of the reagent. cDNA was synthesized from 1 μg of total RNA extract using a High-Capacity Reverse Transcription kit (Applied Biosystems, USA) according to the manufacturer's instructions. The synthesized cDNA was diluted 5 times and used as a template for qPCR. Gene expression of heme oxygenase-1 (HO-1) and phenol oxidase precursor (proPO) was measured by applying real-time polymerase chain reaction (PCR) using SYBR green fluorescent pigment to the cDNA. This measurement was carried out using Thunderbird (registered trademark) SYBR qPCR Mix (Toyobo, Japan). The amplification reaction was carried out using a MicroAmp Optical 96-well reaction plate (Applied Biosystems, USA). Each well contained 10 μl of qPCRMix, 0.6 μl of each primer, 0.4 μl of ROX reference dye, and 2 μl of cDNA template. The loop conditions are as follows. 40 cycles were performed at 95 ° C for 1 minute, followed by 90 ° C for 15 seconds and 60 ° C for 60 seconds. At the end of each qPCR reaction, dissociation analysis was performed, and it was confirmed that only one product was detected. The relative change in qPCR-based gene expression data was determined using the 2- ΔΔCt method (Livak and Schmittgen, 2001). To calculate the Ct value (Threshold Cycle), the gene expression of EF1α was determined as an internal standard. The ratios normalized using the relative expression of the control group were calculated. The data was converted to logarithmic (bottom = 2) before analysis. To determine the expression of heme oxygenase-1, a forward primer with sequence 1 (5'-CTGAGGAGCTCGATGAGGAG-3') and a reverse primer with sequence 2 (5'-CATGGCCACAACACTACCAG-3') were used. To determine the expression of the phenol oxidase precursor, a forward primer with sequence 3 (5'-GGAATTGTTTTACTACATGCATCAGC-3') and a reverse primer with sequence 4 (5'-GGAACAAGTCATCCACGAGCTT-3') were used. To determine the expression of EF1α, a forward primer with sequence 5 (5'-ATTGCCACACCGCTCACA-3') and a reverse primer with sequence 6 (5'-TCGATCTTGGTCAGCAGTTCA-3') were used. The results are shown in Figures 7-8. The average value of the gene expression of the control group at each time was set to 0, and the gene expression of each group was a relative value with respect to the average value. The error bars in Figures 7-8 are standard deviations. The records of 15 ppm, 30 ppm, and 60 ppm in Figs. 7 to 8 represent a 15 ppm 5-ALA administration group, a 30 ppm 5-ALA administration group, and a 60 ppm 5-ALA administration group, respectively.
如第7圖所示,從感染處理起6小時後,在5-ALA給藥組中,與對照組相比,血紅素加氧酶-1的基因表達依賴於用量地增加。如第8圖所示,從感染處理起6小時後,在5-ALA給藥組中,與對照組相比,酚氧化酶前體的基因表達增加。已知作為血紅素蛋白的1種的血紅素加氧酶-1是與血紅素代謝相關的酶,並且是使細胞免受因氧化應激所致的損害的細胞保護蛋白質。啟示了酚氧化酶前體有可能參與識別真菌、細菌的細胞壁成分並將其識別結果與toll受容體的配體生成聯繫起來的機制。因此,雖然不受理論的束縛,但認為通過5-ALA給藥的對EMS/AHPND的預防.治療效果的原因之一有可能是通過5-ALA帶來的血紅素加氧酶-1和酚氧化酶前體的基因表達的增加。 As shown in Fig. 7, after 6 hours from the infection treatment, the gene expression of heme oxygenase-1 was increased in a dose-dependent manner in the 5-ALA-administered group as compared with the control group. As shown in Fig. 8, after 6 hours from the infection treatment, the gene expression of the phenol oxidase precursor was increased in the 5-ALA-administered group as compared with the control group. Heme oxygenase-1, which is one type of heme protein, is known to be an enzyme related to heme metabolism, and is a cell protection protein that protects cells from damage caused by oxidative stress. It is suggested that the phenol oxidase precursor may be involved in the recognition of the cell wall components of fungi and bacteria and the association between their recognition results and the ligand production of the toll receptor. Therefore, although not bound by theory, it is considered that the prevention of EMS/AHPND by 5-ALA administration. One of the reasons for the therapeutic effect may be an increase in gene expression of heme oxygenase-1 and phenoloxidase precursor by 5-ALA.
實施例9:5-ALA對核受體E75和一氧化氮合成酶的基因表達的影響 Example 9: Effect of 5-ALA on gene expression of nuclear receptor E75 and nitric oxide synthase
從飼養開始起第3個月,從各組3~4只的凡納濱對蝦採取肝胰臟,從各個凡納濱對蝦的肝胰臟提取總RNA,由總RNA合成cDNA。通過對該cDNA施加使用SYBR綠色螢光色素的即時聚合酶鏈反應(PCR),測定核 受體基因E75和一氧化氮合成酶的基因表達。為了計算Ct值,測定EF1α的基因表達作為內部標準。為了測定核受體基因E75的表達,使用具有序列7(5’-GCCTACAACAAGCCCCATAA-3’)的正向引物和具有序列8(5’-GCCAGAGAGGAAGTCTGGTG-3’)的反向引物。為了測定一氧化氮合成酶的表達,使用具有序列9(5’-GGAAGACCCACGTCTGGAAG-3’)的正向引物和具有序列10(5’-TCGAGCGATCTCCTTGAAGC-3’)的反向引物。測定中使用的裝置、條件等與實施例8中的相同。結果示於第9~10圖。對照組的基因表達的平均值設為0,各組的基因表達是相對於該平均值的相對值。第9~10圖中的誤差棒為標準差。第9和10圖中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。 In the third month from the start of breeding, hepatic pancreas was taken from 3 to 4 pairs of vannamei in each group, and total RNA was extracted from the hepatopancreas of each of the shrimps, and cDNA was synthesized from total RNA. Gene expression of the nuclear receptor gene E75 and nitric oxide synthase was determined by applying real-time polymerase chain reaction (PCR) using SYBR green fluorescent pigment to the cDNA. To calculate the Ct value, the gene expression of EF1α was determined as an internal standard. For the determination of the expression of the nuclear receptor gene E75, a forward primer having the sequence 7 (5'-GCCTACAACAAGCCCCATAA-3') and a reverse primer having the sequence 8 (5'-GCCAGAGAGGAAGTCTGGTG-3') were used. To determine the expression of nitric oxide synthase, a forward primer having the sequence 9 (5'-GGAAGACCCACGTCTGGAAG-3') and a reverse primer having the sequence 10 (5'-TCGAGCGATCTCCTTGAAGC-3') were used. The apparatus, conditions, and the like used in the measurement were the same as those in Example 8. The results are shown in Figures 9-10. The average value of gene expression in the control group was set to 0, and the gene expression of each group was a relative value with respect to the average value. The error bars in Figures 9-10 are standard deviations. The 15 ppm, 30 ppm, and 60 ppm records in Figures 9 and 10 represent a 15 ppm 5-ALA administration group, a 30 ppm 5-ALA administration group, and a 60 ppm 5-ALA administration group, respectively.
如第9圖所示,在全部5-ALA給藥組中,與對照組相比,核受體E75的基因表達在統計學上顯著地增加。顯著性差異檢驗使用t檢驗法。如第10圖所示,在5-ALA給藥組中,與對照組相比,一氧化氮合成酶的基因表達增加。已知核受體E75是蛻皮激素合成所需的蛋白質,含有血紅素作為輔基,作為細胞內的血紅素濃度的感測器發揮功能,除此以外,還有可能感知一氧化氮作為細胞內信號傳遞分子。已知一氧化氮合成酶是參與在細菌感染等方面具有強力的抗菌活性的過氧亞硝酸鹽的產生所需的一氧化氮的合成的酶,是血紅素蛋白。E75為了穩定其結構而需要血紅素,進而成為血紅素濃度的感測器,一氧化氮合成酶其自身是血紅素蛋白,因此,通過研究它們的表達,有可能成為用於確認5-ALA的給藥與蝦的體內的血紅素合成相關的指標。 As shown in Fig. 9, gene expression of nuclear receptor E75 was statistically significantly increased in all 5-ALA-administered groups as compared with the control group. The significance test was performed using the t test. As shown in Fig. 10, in the 5-ALA-administered group, the gene expression of nitric oxide synthase was increased as compared with the control group. It is known that the nuclear receptor E75 is a protein required for ecdysone synthesis, contains heme as a prosthetic group, and functions as a sensor for intracellular heme concentration. In addition, it is possible to perceive nitric oxide as intracellular. Signal transfer molecule. Nitric oxide synthase is known as an enzyme which synthesizes nitric oxide which is required for the production of peroxynitrite which has strong antibacterial activity in bacterial infection and the like, and is a heme protein. E75 requires heme in order to stabilize its structure, and thus becomes a sensor of hemoglobin concentration. Nitric oxide synthase itself is a heme protein, and therefore, by studying their expression, it is possible to confirm 5-ALA. Administration is related to indicators of heme synthesis in the body of shrimp.
實施例10:5-ALA對一氧化氮合成酶和C型凝集素的基因表達的影響 Example 10: Effect of 5-ALA on gene expression of nitric oxide synthase and C-type lectin
從飼養開始起第2周,從各組3~4只的凡納濱對蝦採取肝胰臟,從各個凡納濱對蝦的肝胰臟提取總RNA,由總RNA合成cDNA。通過對該cDNA施加使用SYBR綠色螢光色素的即時聚合酶鏈反應(PCR),測定核受體基因E75和一氧化氮合成酶的基因表達。為了計算Ct值,測定EF1α的基因表達作為內部標準。為了測定一氧化氮合成酶的表達,使用具有序列9(5’- GGAAGACCCACGTCTGGAAG-3’)的正向引物和具有序列10(5’-TCGAGCGATCTCCTTGAAGC-3’)的反向引物。為了測定C型凝集素的表達,使用具有序列11(5’-CAAGATGGCTCCCACCAACA-3’)的正向引物和具有序列12(5’-GTCGAACTCGGCGTTATCGG-3’)的反向引物。測定中使用的裝置、條件等與實施例8中的相同。結果示於第11~12圖。對照組的基因表達的平均值設為0,各組的基因表達是相對於該平均值的相對值。第11~12圖中的誤差棒為標準差。第11~12圖中的15ppm、30ppm和60ppm的記載分別表示15ppm的5-ALA給藥組、30ppm的5-ALA給藥組和60ppm的5-ALA給藥組。 From the beginning of the second week of the breeding, hepatic pancreas was taken from 3 to 4 of each group of prawn, and total RNA was extracted from the hepatopancreas of each prawn, and cDNA was synthesized from total RNA. Gene expression of the nuclear receptor gene E75 and nitric oxide synthase was determined by applying real-time polymerase chain reaction (PCR) using SYBR green fluorescent pigment to the cDNA. To calculate the Ct value, the gene expression of EF1α was determined as an internal standard. To determine the expression of nitric oxide synthase, a forward primer having the sequence 9 (5'-GGAAGACCCACGTCTGGAAG-3') and a reverse primer having the sequence 10 (5'-TCGAGCGATCTCCTTGAAGC-3') were used. To determine the expression of C-type lectin, a forward primer having the sequence 11 (5'-CAAGATGGCTCCCACCAACA-3') and a reverse primer having the sequence 12 (5'-GTCGAACTCGGCGTTATCGG-3') were used. The apparatus, conditions, and the like used in the measurement were the same as those in Example 8. The results are shown in Figures 11-12. The average value of gene expression in the control group was set to 0, and the gene expression of each group was a relative value with respect to the average value. The error bars in Figures 11 to 12 are standard deviations. The records of 15 ppm, 30 ppm, and 60 ppm in Figs. 11 to 12 represent a 15 ppm 5-ALA administration group, a 30 ppm 5-ALA administration group, and a 60 ppm 5-ALA administration group, respectively.
如第11圖所示,在5-ALA給藥組中,與對照組相比,一氧化氮合成酶的基因表達依賴於用量地增加。如第12圖所示,在15ppm的5-ALA給藥組中,與對照組相比C型凝集素的基因表達增加。啟示了C型凝集素有可能誘發在初期的免疫應答中最重要的結節狀反應。雖然不受理論的束縛,但認為啟示了C型凝集素由於顆粒細胞捕獲細菌,並且在阻止細菌的擴散的反應即結節形成反應中承擔重要的作用,因此,啟示了對EMS的有效性的一個原因有可能是通過基於5-ALA給藥的C型凝集素的表達增加而帶來的結節反應促進作用。一氧化氮合成酶是參與一氧化氮的合成的酶,是血紅素蛋白,通過5-ALA的給藥促進其合成。 As shown in Fig. 11, in the 5-ALA-administered group, the gene expression of nitric oxide synthase was increased depending on the amount of administration as compared with the control group. As shown in Fig. 12, in the 15-ppm 5-ALA-administered group, gene expression of C-type lectin was increased as compared with the control group. It is suggested that C-type lectin may induce the most important nodular response in the initial immune response. Although not bound by theory, it is believed that C-type lectin plays an important role in the reaction of preventing bacterial proliferation, that is, in the nodule formation reaction, because of the granulocyte-captured bacteria, and therefore, a revelation of the effectiveness of EMS is revealed. The reason may be a nodule response promoting effect by an increase in the expression of a C-type lectin based on 5-ALA administration. Nitric oxide synthase is an enzyme involved in the synthesis of nitric oxide and is a heme protein that promotes its synthesis by administration of 5-ALA.
產業上的可利用性 Industrial availability
本申請發明的含有選自5-ALA或者其酯、或它們的鹽中的至少一種的十足目用口服給藥組合物能夠用於十足目的生物的飼養、養殖,能夠有效地預防和治療以副溶血弧菌(Vibrio parahaemolyticus)為致病菌的EMS/AHPND。另外,本申請發明的含有選自5-ALA或者其酯、或它們的鹽中的至少一種的十足目用口服給藥組合物能夠以規定的給藥量促進十足目的生物的生長。 The above-mentioned oral administration composition containing at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof can be used for the breeding and breeding of a full-scale organism, and can be effectively prevented and treated. Vibrio parahaemolyticus is the EMS/AHPND of pathogenic bacteria. Further, the oral administration composition of the present invention containing at least one selected from the group consisting of 5-ALA or an ester thereof or a salt thereof can promote the growth of a target organism in a predetermined dose.
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- 2018-09-10 BR BR112020005255-8A patent/BR112020005255A2/en not_active IP Right Cessation
- 2018-09-10 JP JP2018168668A patent/JP2019055943A/en active Pending
- 2018-09-19 TW TW107133024A patent/TWI791045B/en active
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CN111132676A (en) | 2020-05-08 |
JP2019055943A (en) | 2019-04-11 |
BR112020005255A2 (en) | 2020-09-24 |
TWI791045B (en) | 2023-02-01 |
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