TWI580355B - Compounds α The use of celene for the preparation of drugs to increase the survival rate of shrimp - Google Patents

Compounds α The use of celene for the preparation of drugs to increase the survival rate of shrimp Download PDF

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TWI580355B
TWI580355B TW104142379A TW104142379A TWI580355B TW I580355 B TWI580355 B TW I580355B TW 104142379 A TW104142379 A TW 104142379A TW 104142379 A TW104142379 A TW 104142379A TW I580355 B TWI580355 B TW I580355B
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shrimp
phellandrene
increasing
compound
medicament
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TW201722277A (en
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Ya-Ting Chen
shu-ling Xie
zhi-zhong Wu
Pei-Xuan Luo
Shu Chen Hsieh
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化合物 α -水芹烯用於製備增加蝦類存活率之藥物的用途Use of the compound α-phellandrene for the preparation of a medicament for increasing the survival rate of shrimps

本發明係有關於一種化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,尤指涉及一種α-水芹烯對蝦類感染細菌性疾病及病毒性疾病進行免疫反應之調控,特別係指以α-水芹烯提升免疫能力達到抗病毒效力者。 The present invention relates to the use of a compound α-phellandrene for the preparation of a medicament for increasing the survival rate of shrimps, and more particularly to the regulation of an immune response of a kind of α-phellandene to shrimp-infected bacterial diseases and viral diseases. In particular, it refers to those who use α-phellandrene to enhance immunity and achieve antiviral efficacy.

在熱帶地區,特別是亞洲次大陸,蝦類之水產養殖,由於其具出口市場及高收益性,已成長為主要的商業項目之一。自1970年開始蝦子之產量急遽增加,而目前全球蝦類養殖中,以價值數十億美元之南美白蝦養殖為主要產業,具有高達74%之產量。近年來,台灣蝦類養殖亦蓬勃發展,在2010年時,南美白蝦在台灣水產養殖產量已達到7,978公噸(mt),而至2014年,南美白蝦在台灣水產養殖產量已高達12,643公噸。由於蝦類養殖快速發展,而礙於土地價格高,養殖業者須採用高密度養殖,以降低成本。 In the tropics, especially in the Asian subcontinent, shrimp aquaculture has grown into one of the major commercial projects due to its export market and high profitability. Since the beginning of 1970, the production of shrimp has increased rapidly. At present, in the world of shrimp farming, the multi-billion dollar South American white shrimp culture is the main industry, with a yield of up to 74%. In recent years, shrimp farming in Taiwan has also flourished. In 2010, the production of South American white shrimp in Taiwan has reached 7,978 metric tons (mt), and by 2014, the production of South American white shrimp in Taiwan has reached 12,643 metric tons. Due to the rapid development of shrimp farming and the high price of land, aquaculture operators must adopt high-density farming to reduce costs.

然而,因養殖密度(Aquaculture density)之增加,加上水土資源使用管理不當,造成養殖環境及沿岸水資源嚴重被破壞及惡化,進而引起病原蔓延,使得近年來蝦類養殖產業發展在疾病侵害下跌受重創,且蝦疾病問題不僅出現在台灣,世界各主要蝦類養殖國家亦相繼發生大規模感染,造成蝦類大量死亡,對養殖產業損失嚴重;估計每年之損失近10億美元。因此解決蝦病問題已屬世界性之共同問題,為了克服養殖蝦類遭受病原侵襲,除了要維持良好之養殖環境與抑制養殖池中 病原滋生外,了解蝦類免疫抗病作用機轉,並強化蝦體本身對於病原之抵抗力,藉以提高存活率,係最為重要之解決策略,藉以提升繁殖養殖技術,增進養殖效益。 However, due to the increase in aquaculture density and the improper management of water and soil resources, the culture environment and coastal water resources have been seriously damaged and deteriorated, which has caused the spread of pathogens. It has been hit hard, and shrimp disease problems have not only appeared in Taiwan, but also major infections in major shrimp farming countries in the world, resulting in a large number of shrimp deaths and serious losses to the aquaculture industry; the estimated annual loss is nearly 1 billion US dollars. Therefore, solving the problem of shrimp disease has become a common problem in the world. In order to overcome the pathogen invasion of cultured shrimps, in addition to maintaining a good breeding environment and inhibiting the breeding ponds In addition to the pathogen breeding, understand the role of shrimp immune and disease resistance, and strengthen the resistance of the shrimp body itself to the pathogen, in order to improve the survival rate, is the most important solution strategy, in order to enhance breeding and breeding technology and enhance the breeding efficiency.

感染蝦子之病原菌主要包括有霍亂弧菌(Vibrio cholerae)、哈威弧菌(Vibrio harveyi)、熱帶魚弧菌(Vibrio damsela)及溶藻弧菌(Vibrio alginolyticus)之弧菌病、桿狀病毒中腸腺壞死(Baculoviral midgut gland necrosis,BMN)、草蝦桿狀病毒(Penaeus monodon baculovirus,MBV)、傳染性上皮組織及造血組織壞死病毒(Infectious hypodermal and haematopoietic necrosis virus,IHHNV)、黃頭病(Yellow head disease caused by Yellowhead virus,YHV)、桃拉綜合症病毒(Taura syndrome virus,TSV)、及白斑綜合症病毒(White spot syndrome virus,WSSV)等。其中弧菌病屬細菌型,可能會造成各種嚴重綜合症,死亡率高;而WSSV係白斑綜合症桿狀病毒(White spot syndrome baculovirus,WSBV)複雜之一部分,屬病毒型,在3~10天,病死率高達100%。 The pathogens infected with shrimp mainly include Vibrio cholerae, Vibrio harveyi, Vibrio damsela and Vibrio alginolyticus, and baculovirus midgut. Baculoviral midgut gland necrosis (BMN), Penaeus monodon baculovirus (MBV), infectious epithelial tissue and hematopoietic necrosis virus (IHHNV), yellow head disease (Yellow head) Disease caused by Yellowhead virus (YHV), Taura syndrome virus (TSV), and White spot syndrome virus (WSSV). Among them, Vibrio is a bacterial type, which may cause various serious syndromes with high mortality. WSSV is a part of the complex of white spot syndrome baculovirus (WSBV), which is a viral type, in 3 to 10 days. The case fatality rate is as high as 100%.

針對上述蝦疾病問題,有業者使用抗生素及其他治療藥劑作為感染後實施之普遍治療方法,但因為這些治療性化學物質之應用已造成抗生素抗藥性之發展與蔓延,不僅降低抗生素治療之效果,更造成在組織中殘留物之累積與潛在對環境之危害。 In view of the above-mentioned shrimp disease problems, some people use antibiotics and other therapeutic agents as a general treatment method after infection, but because the application of these therapeutic chemicals has caused the development and spread of antibiotic resistance, it not only reduces the effect of antibiotic treatment, but also Causes the accumulation of residues in the tissue and potential environmental hazards.

日常生活中,許多食用性蔬菜、香辛料及草本植物(herb)之精油中皆含有天然抗氧化劑,可有助於免疫力之提升。其中特別係α-水芹烯(α-phellandrene,α-PA),其屬於一種環狀之單萜類(cyclic monoterpenes)化合物,為青蔥(green onion)、薑(ginger)、蒜(garlic)、肉桂(cinnamon)及時蘿(dill)等蔬菜、香辛料與藥草(herb)之精油(essential oil)中重要的活性成分之組成。 In daily life, many edible vegetables, spices and herbal essential oils contain natural antioxidants, which can help improve immunity. Specifically, it is α-phellandrene (α-PA), which belongs to a cyclic monoterpenes compound, which is green onion, ginger, garlic, Cinnamon is a composition of important active ingredients in vegetables, spices, and essential oils of the herb.

過去有許多學者利用含有α-PA之植物、香辛料與藥草或是以α-PA純品進行不同實驗模式之研究來探討α-PA之作用機制。其中發現α-PA具有多種功效,包含降血糖(Hypoglycemic)、抗菌(Antibacterial)、抗癌(Anticancer;如本申請人曾以α-PA純品進行細胞試驗,結果發現α-PA具有抑制人類肝腫瘤細胞生長之功效)、降血脂(Hypolipidemic)、抗氧化(Antioxidant)、及提高免疫力(Improve immune)。而在2013年有學者利用α-PA純品研究能促進正常小鼠之免疫反應,增強巨噬細胞之吞噬能力與自然殺手細胞之活性。然而,這些α-PA之植物、香辛料與藥草或是α-PA純品並未曾針對感染蝦類之病原菌(尤指溶藻弧菌與WSSV)試驗過。 In the past, many scholars have used the experiments of α-PA containing plants, spices and herbs or different experimental models of α-PA to explore the mechanism of action of α-PA. Among them, α-PA has been found to have various functions, including Hypoglycemic, Antibacterial, and Anticancer; as the applicant has performed cell tests on α-PA pure product, it was found that α-PA inhibits human liver. The effect of tumor cell growth), hypolipidemic, antioxidant, and immune immunity. In 2013, some scholars used α-PA pure product research to promote the immune response of normal mice, enhance the phagocytic capacity of macrophages and the activity of natural killer cells. However, these α-PA plants, spices and herbs, or α-PA pure products have not been tested against pathogens that infect shrimp (especially Vibrio alginolyticus and WSSV).

有鑑於此,基於「蝦體內抗生素危害人體健康與環境安全」及「α-PA是否可以提升蝦類之免疫力」等問題,因此極須提供一種利用適合之天然物質(即α-PA)來取代抗生素及化學物質之使用,並改善上述受病原感染造成蝦類大量死亡情形之問題。 In view of this, based on the question of "antibiotics in shrimps endanger human health and environmental safety" and "whether alpha-PA can enhance the immunity of shrimps", it is extremely necessary to provide a suitable natural substance (ie α-PA). It replaces the use of antibiotics and chemicals and improves the above-mentioned problems caused by a large number of shrimp deaths caused by pathogenic infections.

本發明之主要目的係在於,克服習知技藝所遭遇之上述問題並提供一種α-水芹烯能在南美白蝦之先天免疫中發揮重要作用之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途。 The main object of the present invention is to overcome the above problems encountered in the prior art and to provide a compound α-phellandene which can play an important role in the innate immunity of P. vannamei for the preparation of increased shrimps. The use of a drug for survival.

本發明之次要目的係在於,提供一種以南美白蝦接受濃度大於4μg/g之α-水芹烯即可有效增加免疫能力與抵抗溶藻弧菌感染之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途。 A secondary object of the present invention is to provide a compound α-phellandene which can effectively increase immunity and resist infection by Vibrio alginolyticus with a concentration of more than 4 μg/g of α-phellandene. The use of drugs that increase the survival rate of shrimps.

本發明之另一目的係在於,提供一種當α-水芹烯濃度提升至10μg/g時,即可有效調節白斑綜合症病毒引起之細胞凋亡作用機制之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途。 Another object of the present invention is to provide a compound α-phellandrene which can effectively regulate the mechanism of apoptosis caused by white spot syndrome virus when the concentration of α-phellandene is increased to 10 μg/g. The use of drugs that increase the survival rate of shrimps.

為達以上之目的,本發明係一種化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,係將含有4~12μg/g有效量之α-水芹烯(α-phellandrene,α-PA)投予一蝦體,以有效地誘導增強在該蝦體中對抗該細菌性疾病及該病毒性疾病之免疫反應。 For the purpose of the above, the present invention is a use of a compound α-phellandrene for the preparation of a medicament for increasing the survival rate of shrimps, which comprises an effective amount of α-phellandrene (α-phellandrene, 4 to 12 μg/g, α-PA) is administered to a shrimp body to effectively induce an immune response against the bacterial disease and the viral disease in the shrimp body.

於本發明上述實施例中,該蝦體係為南美白蝦(Litopenaeus vannamei)。 In the above embodiment of the invention, the shrimp system is Litopenaeus vannamei.

於本發明上述實施例中,該α-水芹烯係以注射方式投予該蝦體。 In the above embodiment of the invention, the α-phellandene is administered to the shrimp body by injection.

於本發明上述實施例中,該細菌性疾病係為溶藻弧菌(Vibrio alginolyticus)。 In the above embodiment of the invention, the bacterial disease is Vibrio alginolyticus.

於本發明上述實施例中,該病毒性疾病係為白斑綜合症病毒(White spot syndrome virus,WSSV)。 In the above embodiment of the present invention, the viral disease is White spot syndrome virus (WSSV).

於本發明上述實施例中,該濃度4~12μg/g之α-水芹烯係可增加受溶藻弧菌感染蝦體之存活率,並使非先天特異性免疫反應之數值回復至受損前之正常值。 In the above embodiment of the present invention, the α-phellandene having a concentration of 4 to 12 μg/g can increase the survival rate of the shrimp infected by Vibrio alginolyticus and restore the value of the non-innate specific immune response to the damaged The normal value before.

於本發明上述實施例中,該非先天特異性免疫反應包括總血球數(total haemocyte counts,THC)、酚氧化酶活性(phenoloxidase,PO)、呼吸爆活性(respiratory burst,RB)、及超氧化物歧化酶活性(superoxide dismutase,SOD)。 In the above embodiments of the present invention, the non-innate specific immune response includes total haemocyte counts (THC), phenoloxidase (PO), respiratory burst (RB), and superoxide. Dismutase activity (superoxide dismutase, SOD).

於本發明上述實施例中,該濃度10μg/g之α-水芹烯係可調節受白斑綜合症病毒感染蝦體之細胞凋亡相關基因及細胞凋亡相關因數。 In the above embodiment of the present invention, the α-phellandene having a concentration of 10 μg/g can regulate apoptosis-related genes and apoptosis-related factors of the white spot syndrome virus-infected shrimp.

於本發明上述實施例中,該細胞凋亡相關基因包括電壓依賴性陰離子通道(voltage-dependent anion channel,VDAC)、胱天蛋白酶(caspase)、及腫瘤抑制類蛋白(tumor suppressor-like protein,TSL)。 於本發明上述實施例中,該細胞凋亡相關因數包括活性氧(reactive oxygen species,ROS)、一氧化氮(nitric oxide,NO)、非特異性酯酶 活性(non-specific esterase activity)、及細胞凋亡比例(Apoptosis cell ratio)。 In the above embodiments of the present invention, the apoptosis-related genes include a voltage-dependent anion channel (VDAC), a caspase, and a tumor suppressor-like protein (TSL). ). In the above embodiments of the present invention, the apoptosis related factors include reactive oxygen species (ROS), nitric oxide (NO), and non-specific esterase. Non-specific esterase activity and Apoptosis cell ratio.

第1圖,係本發明α-水芹烯對蝦類感染細菌性疾病之生理及免疫反應調控流程示意圖。 Fig. 1 is a schematic diagram showing the physiological and immune response regulation process of the α -phellandene-infected bacterial disease of the shrimp of the present invention.

第2圖,係本發明α-水芹烯對感染溶藻弧菌之南美白蝦之總血球數之影響示意圖。 Fig. 2 is a schematic view showing the effect of α -phellandene of the present invention on the total number of blood cells of the white shrimp infected with Vibrio alginolyticus.

第3圖,係本發明α-水芹烯對感染溶藻弧菌之南美白蝦之酚氧化酶之影響示意圖。 Fig. 3 is a schematic view showing the effect of α -phellandene of the present invention on phenoloxidase of P. sinensis infected with Vibrio alginolyticus.

第4圖,係本發明α-水芹烯對感染溶藻弧菌之南美白蝦之呼吸爆活性之影響示意圖。 Fig. 4 is a view showing the effect of α -phellandene of the present invention on the respiratory burst activity of the white shrimp infected with Vibrio alginolyticus.

第5圖,係本發明α-水芹烯對感染溶藻弧菌之南美白蝦之超氧化物歧化酶之影響示意圖。 Fig. 5 is a schematic view showing the effect of α -phellandrene of the present invention on superoxide dismutase of S. cerevisiae infected with Vibrio alginolyticus.

第6圖,係本發明α-水芹烯對蝦類感染病毒性疾病引起細胞凋亡調控流程示意圖。 Fig. 6 is a schematic diagram showing the regulation process of apoptosis induced by the viral infection of α -phellandene in the present invention.

第7圖,係本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦血球之活性氧含量示意圖。 Fig. 7 is a schematic view showing the active oxygen content of the α -phellandene of the present invention against the white blood shrimp of the white spot syndrome virus infected with the white spot syndrome virus.

第8圖,係本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦血球之一氧化氮含量示意圖。 FIG. 8, the present invention is based α - phellandrene nitrogen oxide content of the blood cell infected with one White shrimp white spot syndrome virus of Fig.

第9圖,係本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦之電壓依賴性陰離子通道之mRNA相對表現示意圖。 Fig. 9 is a graph showing the relative expression of mRNA of the voltage-dependent anion channel of the white spotted shrimp of the white spot syndrome virus of the present invention by α -phellandrene.

第10圖,係本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦之胱天蛋白酶之mRNA相對表現示意圖。 Fig. 10 is a graph showing the relative expression of α -phellandrene of the present invention against caspase mRNA of P. vannamei infected with white spot syndrome virus.

第11圖,係本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦之腫瘤抑制類蛋白之mRNA相對表現示意圖。 Fig. 11 is a graph showing the relative expression of α -phellandrene of the present invention against mRNA of tumor suppressor protein of P. vannamei infected with white spot syndrome virus.

第12圖,係本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦血球之非特異性酯酶活性示意圖。 Fig. 12 is a view showing the non-specific esterase activity of α -phellandene of the present invention against the white blood shrimp of the white spot syndrome virus infected with the white spot syndrome virus.

第13圖,係本發明於南美白蝦體內注射10μg/g之α-水芹烯在12小時及24小時後之細胞凋亡率示意圖。 FIG. 13, the present invention is based on the in vivo injection vannamei 10 μ g / g of α - schematic view of apoptotic cells after 12 hours and 24 hours phellandrene.

請參閱『第1圖~第5圖』所示,係分別為本發明α-水芹烯對蝦類感染細菌性疾病之生理及免疫反應調控流程示意圖、本發明α-水芹烯對感染溶藻弧菌之南美白蝦之總血球數之影響示意圖、本發明α-水芹烯對感染溶藻弧菌之南美白蝦之酚氧化酶之影響示意圖、本發明α-水芹烯對感染溶藻弧菌之南美白蝦之呼吸爆活性之影響示意圖、及本發明α-水芹烯對感染溶藻弧菌之南美白蝦之超氧化物歧化酶之影響示意圖。如圖所示:本發明係一種化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,係將含有效量之α-水芹烯(α-phellandrene,α-PA)投予一蝦體,以有效地誘導增強在該蝦體中對抗細菌性疾病及病毒性疾病之免疫反應。其中該α-水芹烯(α-phellandrene,α-PA)又稱5-異丙基-2-甲基-1,3-環己二烯,為一種環狀之單萜類化合物,係日常生活中使用之食用性蔬菜、香辛料及草本植物(herb)之精油中常見之組成分。 Please refer to the "Fig. 1 to Figure 5", which are schematic diagrams showing the physiological and immune response regulation processes of the α-phellandene-infected bacterial diseases of the present invention, and the α-phellandene of the present invention is used for the infection of algae Schematic diagram of the influence of the total number of blood cells of Vibrio sinensis on the total number of blood cells of the white shrimp, the effect of the α-phellandene of the present invention on the phenoloxidase of the white shrimp infected with Vibrio alginolyticus, and the α-phellandene of the present invention Schematic diagram of the effect of the respiratory blast activity of Vibrio sinensis on white shrimp, and the effect of α-phellandene of the present invention on superoxide dismutase of S. cerevisiae infected with Vibrio alginolyticus. As shown in the figure: The present invention is a use of a compound α-phellandrene for the preparation of a medicament for increasing the survival rate of shrimps, which comprises administering an effective amount of α-phellandrene (α-PA). A shrimp body to effectively induce an immune response against bacterial diseases and viral diseases in the shrimp body. Wherein the α-phellandrene (α-PA), also known as 5-isopropyl-2-methyl-1,3-cyclohexadiene, is a cyclic monoterpenoid compound, which is a daily A common ingredient in edible vegetables, spices, and herbal essential oils used in life.

上述蝦體係選用目前全球養殖高達74%之南美白蝦(Litopenaeus vannamei)。 The above shrimp system uses Litopenaeus vannamei, which is currently cultured in the world by up to 74%.

於一實施例中,為瞭解蝦類是否可藉由α-水芹烯提升免疫能力達到抗病毒效力,因此以α-水芹烯為主要材料進行一系列α-水芹烯對南美白蝦生理與免疫反應之影響之實驗。 In one embodiment, in order to understand whether the shrimp can enhance the immunity by α-phellandrene, the α-phellandene is used as a main material to carry out a series of α-phellandene physiology against the white shrimp of South America. Experiment with the effects of immune response.

當操作時,其流程如第1圖所示,本發明係給予南美白蝦注射4、8、及12μg/g等不同濃度之α-水芹烯,於24小時後以溶藻弧菌(Vibrio alginolyticus)進行感染,分別在0、12、24、36、48、60及72小時等不同時間進行生理反應測定,包含檢測蝦類存活率及其非先天特異性免疫反應,該非先天特異性免疫反應包含總血球數(total haemocyte counts,THC)、酚氧化酶活性(phenoloxidase,PO)、呼吸爆活性(respiratory burst,RB)、及超氧化物歧化酶活性(superoxide dismutase,SOD)之測定。 When operating, the flow is as shown in Fig. 1. The present invention is applied to different concentrations of α-phellandene, such as 4, 8, and 12 μg/g, to Vibrio alginolyticus after 24 hours. Alginolyticus) is used for physiological responses at 0, 12, 24, 36, 48, 60, and 72 hours, including the detection of shrimp survival rate and its non-innate specific immune response. The determination includes total haemocyte counts (THC), phenoloxidase (PO), respiratory burst (RB), and superoxide dismutase (SOD).

在總血球數之計數方法中,本發明係抽取血淋巴50μl與950μl之抗凝血劑混合後,取100μl混合稀釋液,置於血球計數器中,以相位差顯微鏡計算總血球數。如第2圖所示,為對照組蝦隻與接受生理食鹽水、接受α-水芹烯4μg/g、8μg/g、及12μg/g濃度之蝦隻在0、12、24、36、48、60及72小時後之總血球數。其中每直條線代表來自5隻蝦之平均值與標準誤差(mean±SE)。 In the method for counting the total number of blood cells, the present invention extracts 50 μl of hemolymph and 950 μl of anticoagulant, and then takes 100 μl of the mixed diluent, placed in a blood cell counter, and counts the total number of blood cells by a phase contrast microscope. As shown in Fig. 2, the shrimps in the control group were only at 0, 12, 24, 36, 48 with shrimps receiving physiological saline and receiving α-phellandene at 4 μg/g, 8 μg/g, and 12 μg/g. The total number of blood cells after 60 and 72 hours. Each of the straight lines represents the mean and standard error (mean ± SE) from 5 shrimps.

在酚氧化酶活性之測定方法中,本發明係使用酵素免疫分析儀(ELISA Reader)紀錄來自左旋多巴(L-DOPA)還原成多巴色素(Dopachrome)含量,以測量酚氧化酶活性。簡言之,將1000μl之稀釋血淋巴樣品在4℃下以300xg離心30分鐘,去除上液後,將沉澱物重新懸浮於100μl甲次砷酸鹽-檸檬酸緩衝液(cacodylate citrate buffer)中。再次離心20分鐘後,去除上液後,將沉澱物重新懸浮100μl二甲次砷酸鹽緩衝液(cacodylate buffer)中,取懸浮液加入100μl濃度0.1%之胰蛋白(Trypsin)溶液作為誘發組,控制組以100μl之二 甲次砷酸鹽緩衝液代替誘發劑Trypsin溶液,於37℃下作用10分鐘,再加入50μl之L-DOPA溶液作為受質,在37℃下作用5分鐘,以酵素免疫分析儀在波長490nm下測量南美白蝦之酚氧化酶活性,其係以每50μl血淋巴之多巴色素之形成表示之。如第3圖所示,為對照組蝦隻與接受生理食鹽水、接受α-水芹烯4μg/g、8μg/g、及12μg/g濃度之蝦隻在0、12、24、36、48、60及72小時後之酚氧化酶活性。其中每直條線代表來自5隻蝦之平均值與標準誤差。帶有不同字母(a、b、c)之相同暴露時間之數據表示各處理之間有顯著差異(P<0.05)。圖中結果顯示,不同濃度之α-水芹烯注射南美白蝦12小時後,皆會顯著增加酚氧化酶活性;而且,以8μg/g與12μg/g之α-水芹烯注射24~60小時後,其酚氧化酶活性顯著高於其他組別。 In the method for measuring phenol oxidase activity, the present invention uses an enzyme immunoassay analyzer (ELISA Reader) to record the reduction of levodopa (L-DOPA) into a Dopachrome content to measure phenol oxidase activity. Briefly, 1000 μl of the diluted hemolymph sample was centrifuged at 300 x g for 30 minutes at 4 ° C. After removing the supernatant, the pellet was resuspended in 100 μl of cacodylate citrate buffer. After centrifugation for another 20 minutes, after removing the supernatant, the pellet was resuspended in 100 μl of cacadylate buffer, and 100 μl of 0.1% trypsin solution was added as the induction group. Control group with 100μl A arsenate buffer was used instead of the inducer Trypsin solution for 10 minutes at 37 ° C, then 50 μl of L-DOPA solution was added as a substrate, and it was applied at 37 ° C for 5 minutes to obtain an enzyme immunoassay at a wavelength of 490 nm. The phenoloxidase activity of P. vannamei was measured and expressed as the formation of dopachrome per 50 μl of hemolymph. As shown in Fig. 3, shrimps in the control group were treated with physiological saline, 4 μg/g, 8 μg/g, and 12 μg/g shrimp at 0, 12, 24, 36, 48. Phenol oxidase activity after 60 and 72 hours. Each of the straight lines represents the mean and standard error from 5 shrimps. Data with the same exposure time with different letters (a, b, c) indicated a significant difference (P < 0.05) between treatments. The results showed that different concentrations of α-phellandene injected with P. vannamei significantly increased phenol oxidase activity after 12 hours; moreover, injection of 24-μg/g and 12 μg/g α-phellandene 24~60 After an hour, its phenol oxidase activity was significantly higher than in other groups.

在呼吸爆活性之測定方法中,本發明係以硝基藍四氮唑(NBT)還原成甲瓚(Formazan),測量產生之超氧化陰離子,以定量血球之呼吸爆發。簡言之,將100μl之稀釋血淋巴液,置於預先塗覆0.2%之多聚賴氨酸(poly-L-lysine)之96孔盤中,室溫下作用30分鐘後取出,去除聚賴氨酸後,將沉澱物重新懸浮100μl甲次砷酸鹽-檸檬酸緩衝液中,在4℃下以300xg離心20分鐘,去除上液後,取懸浮液加入100μl濃度0.1%之酵母聚糖(zymosam)溶液作為誘發組,控制組以100μl之MCHBSS代替誘發劑zymosam溶液,於37℃下作用30分鐘後,加入100μl濃度0.3%之NBT於37℃中作用30分鐘,再加入100μl濃度100%甲醇終止反應,並以100μl濃度70%甲醇沖洗三次,風乾後加入120μl濃度2M氫氧化鉀(KOH)及140μl二甲基亞碸(dimethylsulfoxide,DMSO)混合液以溶解細胞質內之不溶性Formazan(還原態NBT),然後,以ELISA分析儀在630nm波長測量南美白蝦之呼吸爆活性,其係以每10μl血淋巴之NBT還原表示之。如第4圖所示,為對照組蝦隻 與接受生理食鹽水、接受α-水芹烯4μg/g、8μg/g、及12μg/g濃度之蝦隻在0、12、24、36、48、60及72小時後之呼吸爆活性。其中每直條線代表來自5隻蝦之平均值與標準誤差。帶有不同字母(a、b、c)之相同暴露時間之數據表示各處理之間有顯著差異(P<0.05)。圖中結果顯示,不同濃度之α-水芹烯注射南美白蝦12小時後,皆會顯著增加呼吸爆活性;而且,以8μg/g與12μg/g之α-水芹烯在注射36~60小時後,呼吸爆活性也顯著高於其他組別。 In the method for measuring respiratory burst activity, the present invention reduces nitroblue tetrazolium (NBT) to formazan and measures the superoxide anion produced to quantify the respiratory burst of the blood cell. Briefly, 100 μl of diluted hemolymph was placed in a 96-well plate pre-coated with 0.2% poly-L-lysine. After 30 minutes at room temperature, the solution was removed and removed. After the acid, the precipitate was resuspended in 100 μl of arsenate-citrate buffer and centrifuged at 300 x g for 20 minutes at 4 ° C. After removing the supernatant, the suspension was added to 100 μl of 0.1% zymosan ( The zymosam) solution was used as the induction group. The control group was replaced with 100 μl of MCHBSS in place of the inducer zymosam solution. After 30 minutes at 37 ° C, 100 μl of 0.3% NBT was added for 30 minutes at 37 ° C, and then 100 μl of 100% methanol was added. The reaction was terminated and washed three times with 100 μl of 70% methanol. After air drying, 120 μl of 2M potassium hydroxide (KOH) and 140 μl of dimethylsulfoxide (DMSO) were added to dissolve the insoluble solvatin in the cytoplasm (reduced NBT). Then, the respiratory burst activity of P. vannamei was measured by an ELISA analyzer at a wavelength of 630 nm, which was expressed as NBT reduction per 10 μl of hemolymph. As shown in Figure 4, the control group is only shrimp The respiratory burst activity was only after 0, 12, 24, 36, 48, 60 and 72 hours of shrimp receiving physiological saline solution, 4 μg/g of α-phellandene, 8 μg/g, and 12 μg/g. Each of the straight lines represents the mean and standard error from 5 shrimps. Data with the same exposure time with different letters (a, b, c) indicated a significant difference (P < 0.05) between treatments. The results show that the different concentrations of α-phellandene injection of South American white shrimp for 12 hours, will significantly increase the respiratory burst activity; and, 8μg / g and 12μg / g of α-phellandene in the injection of 36 ~ 60 After a few hours, respiratory burst activity was also significantly higher than in other groups.

在超氧化物歧化酶活性之測定方法中,本發明係使用市售套組,透過抑制超氧自由基依賴反應,以檢測超氧化物歧化酶活性。簡言之,抽取血淋巴50μl加入950μl之抗凝血劑混合,在4℃下以860xg離心30分鐘,去除上液後,以1ml濃度0.85%生理食鹽水清洗後,再次離心30分鐘,去除上液後,加入100μl預冷雙蒸水,在4℃下靜置15分鐘,以脹破血球,再加入96孔盤中。取已稀釋完全之標準品10μl加入96孔盤中,並立即以ELISA分析儀分析,在37℃下測量波長505nm之光學密度,並從添加黃嘌呤氧化酶後0.5至3.5分鐘之吸光值,測量動力學反應。市售套組提供超氧化物歧化酶活性參考標準物。每單位超氧化物歧化酶活性係定義為抑制黃嘌呤還原率50%所需量。超氧化物歧化酶活性係以每毫升超氧化物歧化酶活性單位表示之。如第5圖所示,為對照組蝦隻與接受生理食鹽水、接受α-水芹烯4μg/g、8μg/g、及12μg/g濃度之蝦隻在0、12、24、36、48、60及72小時後之超氧化物歧化酶活性。其中每直條線代表來自5隻蝦之平均值與標準誤差。帶有不同字母(a、b、c)之相同暴露時間之數據表示各處理之間有顯著差異(P<0.05)。圖中結果顯示,以8μg/g與12μg/g之α-水芹烯注射12小時後,超氧化物歧化酶活性則顯著高於生理食鹽水組。 In the method for measuring superoxide dismutase activity, the present invention uses a commercially available kit to detect superoxide dismutase activity by inhibiting superoxide radical dependent reaction. Briefly, 50 μl of hemolymph was added and 950 μl of anticoagulant was added and mixed at 860×g for 30 minutes at 4° C. After removing the supernatant, it was washed with 1 ml of 0.85% physiological saline, centrifuged again for 30 minutes, and removed. After the solution, 100 μl of pre-cooled double distilled water was added, and allowed to stand at 4 ° C for 15 minutes to rupture the blood cells, and then added to a 96-well plate. 10 μl of the diluted standard was added to a 96-well plate and immediately analyzed by an ELISA analyzer. The optical density at a wavelength of 505 nm was measured at 37 ° C, and the absorbance was measured from 0.5 to 3.5 minutes after the addition of xanthine oxidase. Kinetic reaction. Commercially available kits provide superoxide dismutase activity reference standards. Each unit of superoxide dismutase activity is defined as the amount required to inhibit the reduction of xanthine by 50%. Superoxide dismutase activity is expressed in units of superoxide dismutase activity per ml. As shown in Fig. 5, the shrimps in the control group were only at 0, 12, 24, 36, 48 with shrimps receiving physiological saline and receiving α-phellandene at 4 μg/g, 8 μg/g, and 12 μg/g. Superoxide dismutase activity after 60 and 72 hours. Each of the straight lines represents the mean and standard error from 5 shrimps. Data with the same exposure time with different letters (a, b, c) indicated a significant difference (P < 0.05) between treatments. The results showed that superoxide dismutase activity was significantly higher than that of the physiological saline group after 12 hours of injection of 8 μg/g and 12 μg/g of α-phellandene.

而在蝦類存活率方面,不同濃度α-水芹烯注射南美白蝦後,再感染溶藻弧菌,觀察南美白蝦72小時之存活率,如表一所示,為對照組蝦隻與接受生理食鹽水、接受α-水芹烯4μg/g、8μg/g、及12μg/g濃度之蝦隻在12、24、36、48、60及72小時後之存活率。表中數據為平均值±標準誤差(mean±SE),且每種處理中之蝦隻樣本數目為30(n=30)。帶有不同字母(a、b、c)且在相同時間週期之數據表示各處理之間有顯著差異(P<0.05)。表一結果顯示,於α-水芹烯各組之存活率皆顯著高於生理食鹽水與對照組。 In terms of shrimp survival rate, different concentrations of α-phellandene were injected into South American white shrimp, and then infected with Vibrio alginolyticus to observe the 72-hour survival rate of South American white shrimp. As shown in Table 1, the control group was only Survival rates of shrimps receiving physiological saline and receiving α-phellandrene at 4 μg/g, 8 μg/g, and 12 μg/g concentrations after 12, 24, 36, 48, 60, and 72 hours. The data in the table are mean ± standard error (mean ± SE), and the number of shrimp samples per treatment was 30 (n = 30). Data with different letters (a, b, c) and at the same time period indicated a significant difference (P < 0.05) between treatments. The results in Table 1 show that the survival rates of the α-phellandene groups were significantly higher than those of the physiological saline and the control group.

由以上結果顯示,給予南美白蝦大於4μg/g之α-水芹烯濃度,可增加南美白蝦免疫能力與抗溶藻弧菌感染之能力;由此可知,α-水芹烯在南美白蝦之先天免疫能力中扮演重要角色。 From the above results, the concentration of α-phellandene of the white shrimp of greater than 4 μg/g can increase the immunity of the white shrimp and the ability to resist infection by Vibrio alginolyticus; thus, it can be seen that α-phellandrene is in South America white. Shrimp plays an important role in the innate immunity.

請參閱『第6圖~第13圖』所示,係分別為本發明α-水芹烯對蝦類感染病毒性疾病引起細胞凋亡調控流程示意圖、本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦血球之活性氧含量示意圖、本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦血球之一氧化氮含量示意圖、本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦之電壓依賴性陰離子通道之mRNA相對表現示意圖、本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦之胱天蛋白酶之mRNA相對表現示意圖、本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦之腫瘤抑制類蛋白之mRNA相對表現示意圖、本發明α-水芹烯對感染白斑綜合症病毒之南美白蝦血球之非特異性酯酶活性示意圖、及本發明於南美白蝦體內注 射10μg/g之α-水芹烯在12小時及24小時後之細胞凋亡率示意圖。如圖所示:本發明於另一實施例中,為瞭解α-水芹烯在甲殼類動物之功能及其調節作用,因此利用α-水芹烯為主要材料進行一系列α-水芹烯對南美白蝦細胞凋亡之機制之實驗。 Please refer to the "Fig. 6 to Fig. 13", which are schematic diagrams showing the regulation process of apoptosis induced by viral diseases of the α-phellandene shrimp in the present invention, and the α-phellandene of the present invention against white spot syndrome. Schematic diagram of the active oxygen content of the white shrimp blood cell of the virus, the α-phellandene of the present invention, the nitrogen oxide content of one of the white blood shrimp blood cells of the white spot syndrome virus, and the α-phellandene of the present invention against the white spot syndrome virus Schematic diagram of relative expression of mRNA of voltage-dependent anion channel of P. vannamei, relative expression of α-phellandrene of caspase of P. vannamei infected with white spot syndrome virus, α-phelene of the present invention Schematic diagram of the relative expression of mRNA of tumor suppressor protein of P. vannamei infected with white spot syndrome virus, and non-specific esterase activity of α-phellandene of the present invention against white spotted bloodsuckle virus of white spot syndrome virus, and Invented in the South American White Shrimp A schematic diagram of the apoptotic rate of 10 μg/g of α-phellandene after 12 hours and 24 hours. As shown in the figure: in another embodiment, in order to understand the function and regulation of α-phellandrene in crustaceans, a series of α-phellandene is carried out by using α-phellandene as a main material. Experiment on the mechanism of apoptosis of white shrimp in South America.

當操作時,其流程如第6圖所示,本發明係給予南美白蝦注射濃度10μg/g之α-水芹烯,於24小時後以白斑綜合症病毒(White spot syndrome virus,WSSV)進行感染,再抽取血淋巴液,進行血球分離,分別在12及24小時針對促進凋亡蛋白質與抗凋亡蛋白質進行分析測定,包含檢測蝦類細胞凋亡相關基因及細胞凋亡相關因數,該細胞凋亡相關基因包括以反轉錄酶-聚合酶連鎖反應(RT-PCR)檢測之電壓依賴性陰離子通道(voltage-dependent anion channel,VDAC)、胱天蛋白酶(caspase)、及腫瘤抑制類蛋白(tumor suppressor-like protein,TSL);而該細胞凋亡相關因數包括以流動式細胞測量術(Flow Cytometry)檢測之活性氧(reactive oxygen species,ROS)、一氧化氮(nitric oxide,NO)、非特異性酯酶活性(non-specific esterase activity)、細胞凋亡比例(Apoptosis cell ratio)之分析。 When operating, the flow is as shown in Fig. 6. The present invention is administered with a concentration of 10 μg/g of α-phellandene in the white shrimp of South America, and after 24 hours with White spot syndrome virus (WSSV). Infection, blood lymphatic fluid was taken, blood cell separation was performed, and apoptosis-promoting proteins and anti-apoptotic proteins were analyzed at 12 and 24 hours, respectively, including detection of apoptosis-related genes and apoptosis-related factors of shrimp cells. Apoptosis-related genes include voltage-dependent anion channels (VDACs), caspases, and tumor suppressor proteins (tumor) detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Suppressor-like protein (TSL); and the apoptosis-related factors include reactive oxygen species (ROS), nitric oxide (NO), and non-specificity measured by Flow Cytometry. Analysis of non-specific esterase activity and Apoptosis cell ratio.

以下本發明將藉由第7圖~第13圖依序說明檢測活性氧、一氧化氮、電壓依賴性陰離子通道、胱天蛋白酶、腫瘤抑制類蛋白、非特異性酯酶活性、及細胞凋亡比例之分析結果。其中各實驗所得資料為通過T-檢驗(T-test)統計分析同一實驗重複三次之平均值±標準差(mean±SD);帶有不同字母(a、b)之相同暴露時間之數據表示各處理之間有顯著差異(P<0.05)。 Hereinafter, the present invention will sequentially describe the detection of active oxygen, nitric oxide, voltage-dependent anion channels, caspase, tumor suppressor protein, non-specific esterase activity, and apoptosis by means of Figures 7 to 13 The analysis results of the ratio. The data obtained from each experiment is the mean ± standard deviation (mean ± SD) of the same experiment repeated three times by T-test (T-test); the data of the same exposure time with different letters (a, b) indicates each There was a significant difference between treatments (P < 0.05).

如第7圖所示,為對照組蝦隻與接受α-水芹烯10μg/g濃度之蝦隻在12小時及24小時後之活性氧含量。結果顯示,活性氧在12小時及24小時與對照組相比係有增加之情形。 As shown in Fig. 7, the active oxygen content of the shrimp in the control group and the shrimp receiving the concentration of α-phellandene at 10 μg/g was only 12 hours and 24 hours later. The results showed that the active oxygen was increased in comparison with the control group at 12 hours and 24 hours.

如第8圖所示,為對照組蝦隻與接受α-水芹烯10μg/g濃度之蝦隻在12小時及24小時後之一氧化氮含量。結果顯示,一氧化氮在12小時及24小時與對照組相比係有增加之情形。 As shown in Fig. 8, one of the control shrimps had a nitrogen oxide content of only 12 hours and 24 hours after receiving the shrimp at a concentration of 10 μg/g of α-phellandene. The results showed that nitric oxide was increased in comparison with the control group at 12 hours and 24 hours.

如第9圖所示,為接受生理食鹽水之對照組蝦隻與接受α-水芹烯10μg/g濃度之蝦隻在12小時及24小時後之電壓依賴性陰離子通道之mRNA相對表現。結果顯示,給予南美白蝦注射10μg/g之α-水芹烯在12小時及24小時與對照組相比並沒有顯著差異。 As shown in Fig. 9, the control shrimp which received the physiological saline showed a relative expression of the mRNA of the voltage-dependent anion channel only after 12 hours and 24 hours of the shrimp receiving the α-phellandene concentration of 10 μg/g. The results showed that there was no significant difference in the administration of 10 μg/g of α-phellandrene to the white shrimp of South America at 12 hours and 24 hours.

如第10圖所示,為接受生理食鹽水之對照組蝦隻與接受α-水芹烯10μg/g濃度之蝦隻在12小時及24小時後之胱天蛋白酶之mRNA相對表現。結果顯示,給予南美白蝦注射10μg/g之α-水芹烯在12小時及24小時表現之mRNA相對表現與對照組相比皆有增加之情形。 As shown in Fig. 10, the shrimps in the control group receiving physiological saline showed a relative expression of caspase mRNA only at 12 hours and 24 hours after the shrimps receiving the α-phellandene concentration of 10 μg/g. The results showed that the relative mRNA expression of 10 μg/g of α-phellandene administered to South American white shrimp at 12 hours and 24 hours was increased compared with the control group.

如第11圖所示,為接受生理食鹽水之對照組蝦隻與接受α-水芹烯10μg/g濃度之蝦隻在12小時及24小時後之腫瘤抑制類蛋白之mRNA相對表現。結果顯示,給予南美白蝦注射10μg/g之α-水芹烯在12小時及24小時表現之mRNA相對表現與對照組相比皆有增加之情形,但只有在12小時有顯著差異。 As shown in Fig. 11, the control group shrimp receiving physiological saline showed a relative expression of tumor suppressor-like protein mRNA only at 12 hours and 24 hours after the shrimp receiving the α-phellandene concentration of 10 μg/g. The results showed that the relative performance of mRNA for the injection of 10 μg/g of α-phellandene at 12 hours and 24 hours was significantly increased compared with the control group, but only significantly at 12 hours.

如第12圖所示,為對照組蝦隻與接受α-水芹烯10μg/g濃度之蝦隻在12小時及24小時後之非特異性酯酶活性。結果顯示,非特異性酯酶活性與對照組相比,在12小時係有增加之情形,在24小時反而下降;鑑於酯酶活性與細胞生存力相關,故非特異性酯酶活性到比較後面會下降,其原因可能係因為發生細胞凋亡之細胞增加。 As shown in Fig. 12, the control group had non-specific esterase activity only after 12 hours and 24 hours with shrimps receiving α-phellandene at a concentration of 10 μg/g. The results showed that the non-specific esterase activity increased compared with the control group at 12 hours, but decreased at 24 hours; since the esterase activity was related to cell viability, the non-specific esterase activity was compared to the latter. It will decline, probably because of the increase in cells that have undergone apoptosis.

如第13圖所示,為接受α-水芹烯10μg/g濃度之蝦隻在12小時及24小時後之細胞凋亡率。 As shown in Fig. 13, the apoptosis rate of shrimps at a concentration of 10 μg/g of α-phellandene was observed only after 12 hours and 24 hours.

由以上數據顯示,腫瘤抑制類蛋白、活性氧、一氧化氮、胱天蛋白酶、及非特異性酯酶活性之上升,有助於細胞凋亡產生,故推測α-水芹烯係具有調節南美白蝦細胞凋亡之作用。 From the above data, the increase in tumor suppressor protein, reactive oxygen species, nitric oxide, caspase, and non-specific esterase activity contributes to apoptosis, so it is speculated that α-phellandene has regulation of South America. The role of white shrimp cell apoptosis.

綜上所述,本發明係一種化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,可有效改善習用之種種缺點,以南美白蝦接受濃度大於4μg/g之α-水芹烯即可有效增加免疫能力與抵抗溶藻弧菌之感染,且當α-水芹烯濃度提升至10μg/g時,亦可有效調節白斑綜合症病毒引起之細胞凋亡作用機制之功效,顯見α-水芹烯能在南美白蝦之先天免疫中發揮重要作用,進而使本發明之產生能更進步、更實用、更符合使用者之所須,確已符合發明專利申請之要件,爰依法提出專利申請。 In summary, the present invention is a use of a compound α-phellandrene for the preparation of a medicament for increasing the survival rate of shrimps, and can effectively improve various disadvantages of the conventional use, and the white shrimp of the South American white shrimp receives an α-water having a concentration of more than 4 μg/g. Celery can effectively increase immunity and resist the infection of Vibrio alginolyticus, and when the concentration of α-phellandene is increased to 10 μg/g, it can also effectively regulate the effect of the mechanism of apoptosis caused by white spot syndrome virus. It is obvious that α-phellandene can play an important role in the innate immunity of South American white shrimp, which makes the invention more progressive, more practical and more suitable for users. It has indeed met the requirements of the invention patent application. File a patent application according to law.

惟以上所述者,僅為本發明之較佳實施例而已,當不能以此限定本發明實施之範圍;故,凡依本發明申請專利範圍及發明說明書內容所作之簡單的等效變化與修飾,皆應仍屬本發明專利涵蓋之範圍內。 However, the above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto; therefore, the simple equivalent changes and modifications made in accordance with the scope of the present invention and the contents of the invention are modified. All should remain within the scope of the invention patent.

Claims (10)

一種化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,係將含有4~12μg/g有效量之α-水芹烯(α-phellandrene,α-PA)投予一蝦體,以有效地誘導增強在該蝦體中對抗細菌性疾病及病毒性疾病之免疫反應。 Administration phellandrene -phellandrene, α -PA) a - one compound [alpha] - phellandrene for the preparation of a medicament for increasing the survival of shrimp, the system comprising 4 ~ 12 μ g / g effective amount of [alpha] Shrimp body to effectively induce an immune response against bacterial diseases and viral diseases in the shrimp body. 依申請專利範圍第1項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該蝦體係為南美白蝦(Litopenaeus vannamei)。 The use of the compound α -phellandene according to claim 1 of the patent application for the preparation of a medicament for increasing the survival rate of shrimp, wherein the shrimp system is Litopenaeus vannamei. 依申請專利範圍第1項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該α-水芹烯係以注射方式投予該蝦體。 The use of the compound α -phellandrene according to the first aspect of the patent application for the preparation of a medicament for increasing the survival rate of shrimp, wherein the α -phellandene is administered to the shrimp by injection. 依申請專利範圍第1項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該細菌性疾病係為溶藻弧菌(Vibrio alginolyticus)。 The use of the compound α -phellandrene according to the first aspect of the patent application for the preparation of a medicament for increasing the survival rate of shrimp, wherein the bacterial disease is Vibrio alginolyticus. 依申請專利範圍第1項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該病毒性疾病係為白斑綜合症病毒(White spot syndrome virus,WSSV)。 The use of the compound α -phellandrene according to the first aspect of the patent application for the preparation of a medicament for increasing the survival rate of shrimp, wherein the viral disease is White spot syndrome virus (WSSV). 依申請專利範圍第1或4項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該濃度4~12μg/g之α-水芹烯係可增加受溶藻弧菌感染蝦體之存活率,並使非先天特異性免疫反應之數值回復至受損前之正常值。 Or the compound according to [alpha] 4 of the first range patent - phellandrene for the preparation of a medicament for increasing the survival of shrimp, wherein the concentration of 4 ~ 12 μ g / g of [alpha] - phellandrene line may Increasing the survival rate of the shrimp infected with Vibrio alginolyticus and returning the value of the non-innate specific immune response to the normal value before the damage. 依申請專利範圍第6項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該非先天特異性免疫反應包括總血球數(total haemocyte counts,THC)、酚氧化酶活性(phenoloxidase, PO)、呼吸爆活性(respiratory burst,RB)、及超氧化物歧化酶活性(superoxide dismutase,SOD)。 The use of the compound α -phellandrene according to claim 6 of the patent application for the preparation of a medicament for increasing the survival rate of shrimp, wherein the non-innate specific immune reaction comprises total haemocyte counts (THC), phenol Oxidase activity (PO), respiratory burst (RB), and superoxide dismutase (SOD). 依申請專利範圍第1或5項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該濃度10μg/g之α-水芹烯係可調節受白斑綜合症病毒感染蝦體之細胞凋亡相關基因及細胞凋亡相關因數。 Or the compound according to the [alpha] 1 5 range patent - phellandrene for the preparation of a medicament for increasing the survival of shrimp, wherein the concentration of 10 μ g / g of [alpha] - phellandrene can be regulated by the Department of Apoptosis-related genes and apoptosis-related factors of white spot syndrome virus-infected shrimp. 依申請專利範圍第8項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該細胞凋亡相關基因包括電壓依賴性陰離子通道(voltage-dependent anion channel,VDAC)、胱天蛋白酶(caspase)、及腫瘤抑制類蛋白(tumor suppressor-like protein,TSL)。 The use of the compound α -phellandrene according to claim 8 of the patent application for the preparation of a medicament for increasing the survival rate of shrimp, wherein the apoptosis-related gene comprises a voltage-dependent anion channel (voltage-dependent anion channel, VDAC), caspase, and tumor suppressor-like protein (TSL). 依申請專利範圍第8項所述之化合物α-水芹烯用於製備增加蝦類存活率之藥物的用途,其中,該細胞凋亡相關因數包括活性氧(reactive oxygen species,ROS)、一氧化氮(nitric oxide,NO)、非特異性酯酶活性(non-specific esterase activity)、及細胞凋亡比例(Apoptosis cell ratio)。 The use of the compound α -phellandrene according to claim 8 of the patent application for the preparation of a medicament for increasing the survival rate of shrimps, wherein the apoptosis-related factors include reactive oxygen species (ROS), monooxygenation Nitric oxide (NO), non-specific esterase activity, and Apoptosis cell ratio.
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