Study on Effect of Essential Oils from Houttuynia cordata Thunb Supplemented Diets on Growth Performance and Immune Response of Hybrid Red Tilapia (Oreochromis mossambicus Linn. × Oreochromis niloticus Linn.) 著者 Supranee Wigraiboon year 2016 その他のタイトル Houttuynia cordata Thunb（ドクダミ）から抽出さ れたエッセンシャルオイルを添加した 飼料の Hybrid Red Tilapia(Oreochromis mossambicus Linn. × Oreochromis niloticus Linn.) に対する 増殖および免疫賦活作用に関する基礎的研究 学位授与大学 筑波大学 (University of Tsukuba) 学位授与年度 2016 報告番号 12102甲第7943号 URL http://hdl.handle.net/2241/00145394 Study on Effect of Essential Oils from Houttuynia cordata Thunb Supplemented Diets on Growth Performance and Immune Response of Hybrid Red Tilapia (Oreochromis mossambicus Linn. × Oreochromis niloticus Linn.) July 2016 Supranee Wigraiboon Study on Effect of Essential Oils from Houttuynia cordata Thunb Supplemented Diets on Growth Performance and Immune Response of Hybrid Red Tilapia (Oreochromis mossambicus Linn. × Oreochromis niloticus Linn.) A Dissertation Submitted to the Graduate School of Life and Environmental Sciences, the University of Tsukuba in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Agricultural Science (Doctoral Program in Bioindustrial Sciences) Supranee Wigraiboon CONTENTS Page CONTENTS ……………………………………………………………………………..……… i LIST OF TABLES ………………………………………………………………………..…… iv LIST OF FIGURES ……………………………………………………………………...…….. v ABSTRACT ………………………………………………………………………...……….. viii CHAPTER 1 General Introduction ……………………………………………...…………… 1 1.1 Concepts ……………………………...……………………………………………… 1 1.2 Objective and outline of the thesis …………………………………………...……… 2 1.3 Overview of Hybrid red tilapia …………………………………………….…...…… 4 1.4 Herb in aquaculture ………………………………………….…………………..… 16 1.5 Houttuynia cordata Thunb (dokudami) ………….…………………..…………… 24 1.6 Activity of essential oils (EOs) against bacteria …….…………….....…………… 27 CHAPTER 2 Gas Chromatography-Mass Spectrometry (GC-MS) for Analysis of Chemical Composition of Essential Oils (EOs) from Houttuynia cordata Thunb (dokudami) ……...……....……………………… 34 2.1 Introduction ………………………………………………………………………… 34 2.2 Methods …………………………………………………………………………….. 35 2.2.1 Plant material and EOs extraction ………………......……………...…… 35 2.2.2 GC-MS analysis ……………………………………………………….. 35 2.3 Results …………………………………………………………………………….... 38 2.4 Discussion ……………………………………………………………………...…… 38 i 2.5 Conclusion ……………………………………………………………..…………… 39 CHAPTER 3 Antibacterial Activities of Essential Oils (EOs) from Houttuynia cordata Thunb (dokudami) Against Fish Pathogens ……..…… 44 3.1 Introduction ……………………………………………………………………….... 44 3.2 Methods ……………………………………………………………………..……… 45 3.2.1 EOs extraction ……...……………...…………………………….….… 45 3.2.2 Determination of the antibacterial activity (in vitro study) ………….… 46 3.2.3 Statistical analysis ……………………………………………...…...….. 48 3.3 Results ……………………………………………………………………..……….. 49 3.4 Discussion ………………………………………………………..……………….… 53 3.5 Conclusion …………………………………………………..…………………….... 54 CHAPTER 4 Effect of Essential Oils (EOs) from Houttuynia cordata Thunb (dokudami) Supplemented Diets on Growth Performance and Immune Response in Hybrid Red Tilapia (Oreochromis mossambicus Linn. × Oreochromis niloticus Linn.) ..................................................................................................................... 55 4.1 Introduction ……..……………………..…………………………………………… 55 4.2 Methods ……..………..………………..…………………………………………… 56 4.2.1 Essential oils (EOs) extraction ……….……...…..……………………… 56 4.2.2 Fish study (In vivo study) ……….……………………………...……… 57 4.2.3 Statistical analysis ……….……………...……………………………… 68 4.3 Results ……..………..…………..……..…………………………………………… 69 4.4 Discussion ……..………..……………..…………………………………………… 77 4.5 Conclusion ……..……..………………..…………………………………………… 79 ii CHAPTER 5 Summary …………………………………………………………………….. 80 CHAPTER 6 Conclusion and Future prospects …………….……………………………… 82 REFERENCES ………..……………………………………….…………………………….. 83 ADDENDUM …..………...….………………………………..…………………………….... 102 ACKNOWLEDGEMENTS ….…………….………………….…………………………….. 103 iii LIST OF TABLES Page Table 1 Herbs or herbal extracts that improve aquatic animal performance …………………. 21 Table 2 Herbs or herbal extracts that improve aquatic animal immunostimulants ……………. 22 Table 3 Herbs or herbal extracts that improve antimicrobial (fish and shrimp pathogen) …….. 23 Table 4 Pharmacology of Houttuynia cordata Thunb applied to animals …………………….. 26 Table 5 Chemical composition of EOs from Houttuynia cordata Thunb extract ………….….. 43 Table 6 Antibacterial activity against fish pathogens …………………………………….…… 50 Table 7 Composition of soybean oils and HEO on diets ……………………………………. 58 Table 8 Effect of fish diets on growth performance of Hybrid red tilapia ………………….…. 70 Table 9 Proximate composition of fillet of Hybrid red tilapia (%; on fresh weight basis) ………73 Table 10 Immunological of Hybrid red tilapia after 60 days feeding period ………………….. 74 Table 11 Differential morphology counting of white blood cells (%) …………………………. 75 iv LIST OF FIGURES Page Figure 1 Total production of tilapia in Thailand (1950-2014) ……………………………….…. 5 Figure 2 Map of Thailand showing the major sites of tilapia hatcheries, pond and cages contacted/visited for the collection of information …………………………………… 7 Figure 3 Major diseases affecting tilapia ……………………………………………………… 9 Figure 4 In Oreochromis niloticus fish bilateral showing exophthalmia (A), ulcer on skin and erosion on fins and tail (B), severe abdominal ascites (C) and congestion and hemorrhages in all internal organs (D) ………………………………………………. 13 Figure 5 Bilateral exophthalmia in infected Streptococcus sp. tilapia ………………………… 14 Figure 6 Naturally diseased Nile tilapia exhibited discoloration and hemorrhage on body surface (A), necrotic gills, swollen gall bladder, hemorrhagic liver (B), and enteritis (C). Red tilapia challenging to F. columnare NK-Fc01 exhibited typical skin lesion (D) and severe epidermis ulcer (E). Red tilapia exposed to A. veronii NK01 or NK06 showing hemorrhage at basal fins (F) and liver (G) …………………………………………… 15 Figure 7 Number of published papers about the use of plant or natural products in aquaculture ………………………………………………………………………… 17 Figure 8 Houttuynia cordata Thunb …………………………………………………………. 25 Figure 9 The decanoyl acetaldehyde (houttuynin) …………………………………………….. 28 Figure 10 Mechanism of action and target sites of EOs on microbial cells ……..……………. 31 v Figure 11 The difference between cell membranes of Gram-positive (on the right) and Gram- negative bacteria (on the left) ……………………………………………………. 32 Figure 12 Identified bacterial cell structures and cellular processes disrupted by the action of EOs or their components. EOs treated cells are more permeable to protons, experience an ATP imbalance and induced synthesis of chaperones. OMP (Outer membrane protein); QS (quorum sensing). Metabolic pathways can be disrupted ……………………… 33 Figure 13 Map of study area in Pasao district of Lamphun province, Thailand. Arrow indicates Houttuynia cordata Thunb culture locations ……………………………………… 36 Figure 14 Preparation of sample for hydrodistillation ………………………………………… 37 Figure 15 EOs of Houttuynia cordata Thunb extract ………………………………………… 40 Figure 16 Chromatogram of EOs components of Houttuynia cordata Thunb ………………… 41 Figure 17 Structure of beta-myrcene (A) and 2-undecanone (B) ……………………………… 42 Figure 18 The minimum inhibitory concentration and minimum bactericidal concentration testing. In this example, MIC of the Houttuynia cordata Thunb essential oils (HEO) is 250 ppm (tube A) and MBC is 1,000 ppm (tube B) …………………………………….…….. 47 Figure 19 Minimum inhibitory concentration (MIC) of HEO compared with ampicillin sodium ……………………………………………………………. 51 Figure 20 Complete absence of Aeromonas hydrophila (A) Streptococcus sp. (B) and Flavobacterium columnare (C) were compared with control ……………………… 52 Figure 21 Preparation of fish diets …………………………………………………………… 59 Figure 22 Floating cages in Mae Taeng reservoir, Thailand …………………………………. 61 vi Figure 23 Hybrid red tilapia were randomly chosen into cages ……………………………… 62 Figure 24 The body weight was monitored every 15 days …………………………………… 63 Figure 25 The blood samples were collected from the caudal vein of the fish (A) and microplate reader (Metertech M965+) (B) ……………………………………………………… 66 Figure 26 The Biofuge haemo and hematocrit dish for read hematocrit values ……………… 67 Figure 27 Total weight (g/cage) of Hybrid red tilapia monitored every 15 days ……………. 71 Figure 28 Hybrid red tilapia after 60 days experimental; Control (A), HEO 1.5 g/kg (B) and HEO 3.0 g/kg (C) ………………………………………………………….……………… 72 Figure 29 The hematological profile of Hybrid red tilapia during 60 days experimental period; monocyte and eosinophil (A), small lymphocyte (B), monocyte and lymphocyte (C), neutrophil (D) and thrombocyte (E) ………………………………………………. 76 vii ABSTRACT Houttuynia cordata Thunb (dokudami) is a flowering plant native to Japan, Southern China and Southeast Asia. It contains volatile oil, fatty acids, sterols, flavonoid and alkaloids. Moreover, it possesses a variety of pharmacological functions. This study investigated the effects of essential oils (EOs) from H. cordata Thunb as diet supplements on growth performance and immune responses of Hybrid red tilapia (Oreochromis mossambicus Linn. × Oreochromis niloticus Linn.). H. cordata Thunb were extracted using hydrodistillation produced a clear colorless to pale yellow oil with a yield of 0.057%v/w and analyzed by gas chromatograph-mass spectrometer (GC-MS). Beta-myrcene (44.220%) and 2-undecadone (21.985%) were the major components