Antioxidation activity and total phenolic contents of various Toona sinensis extracts

Products Processing Division, LRI, COA, Tainan, Taiwan. Accepted 1 August, 2012 The purpose of this study was to analyze the antioxidation activities and total phenolic contents of Toona sinensis. An extractive method of the antioxidant activities of local T. sinensis leaf extracts and various antioxidation models were analyzed. The effects of various solvent concentrations to extract the T. sinensis leaf on the antioxidation activity were compared. The results showed that the T. sinensis leaf extracts of various solvent concentrations had good antioxidant activities. The chelating abilities of ferrous ion in the T. sinensis leaf extracts obtained from various solvent concentrations were above 80%. It is concluded that T. sinensis leaf extracts could be used as biopreservatives of food products. The total antioxidant activities, chelating abilities of ferrous ions and reducing capacities of the T. sinensis leaf extracts might provide a substitute for a natural antioxidant.Keywords: Antioxidative activity, bio-preservatives, extracts, Toona sinensi

Effect of Free-range Rearing on Meat Composition, Physical Properties and Sensory Evaluation in Taiwan Game Hens

Experiments were conducted to evaluate the effect of an outdoor-grazed raising model on meat composition, physical properties and sensory attributes of Taiwan game hens. Six hundred 1-d old female chicks were raised on a floor for 8 weeks. On day 57, 600 healthy birds, with similar body weight, were selected and randomly assigned to three treatment groups (cage, floor-pen and free-range). The results showed that different feeding models had no effect on drip loss, cooking loss, moisture, crude protein, crude fat, crude ash, zinc and calorie contents in breast meat and moisture content in thigh meat. The free-range group had the lowest fat content in both breast and thigh meat, and the lowest calorie content in thigh meat. The firmness and toughness in both thigh and breast of the free-range group were the highest values (p<0.05). The crude protein, total collagen, zinc and iron contents in thigh meat and total collagen content in breast meat of the free-range group were significantly higher than those of the cage-feeding group (p<0.05). The meat sensory scores of flavor, chewiness and overall acceptability of both thigh and breast meat of the free-range group were significantly (p<0.05) better than those of the other two groups. Moreover, the current findings also indicate that the Taiwan game hens of the free-range feeding model displayed well-received carcass traits and meat quality, with higher scores for flavor, chewiness and overall acceptability for greater sensory satisfaction in both breast and thigh meat. In addition, the thigh meat contained high protein and total collage but low fat, offering a healthier diet choice

Effects of Angelica dahurica

The main objective of wound treatments is to restore the functional skin properties and prevent infection. Traditional Chinese medicine provides alternative anti-inflammatory, antimicrobial, and wound healing therapies. Both Angelica dahurica extract (AE) and Rheum officinale extract (RE) possess antimicrobial activity. In this study, AE and RE were applied in wound treatment to investigate their healing effects. Thirty Sprague-Dawley rats with dorsal full-thickness skin excision were divided into normal saline (NS), AE, RE, AE plus RE (ARE), and Biomycin (BM) groups. The treatment and area measurement of wounds were applied daily for 21 days. Wound biopsies and blood samples were obtained for histology examinations and cytokine analysis. Results showed that wound contraction in ARE group was significantly higher than that in NS and BM groups (P 0.05), and plasma TGF-β1 levels were significantly lower than those in the NS group on days 3-4 (P < 0.05). In conclusion, ARE accelerates wound healing during inflammation and proliferation phases

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Correction to: Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

The original version of this article unfortunately contained a mistake

Prevalence, associated factors and outcomes of pressure injuries in adult intensive care unit patients: the DecubICUs study

Funder: European Society of Intensive Care Medicine; doi: http://dx.doi.org/10.13039/501100013347Funder: Flemish Society for Critical Care NursesAbstract: Purpose: Intensive care unit (ICU) patients are particularly susceptible to developing pressure injuries. Epidemiologic data is however unavailable. We aimed to provide an international picture of the extent of pressure injuries and factors associated with ICU-acquired pressure injuries in adult ICU patients. Methods: International 1-day point-prevalence study; follow-up for outcome assessment until hospital discharge (maximum 12 weeks). Factors associated with ICU-acquired pressure injury and hospital mortality were assessed by generalised linear mixed-effects regression analysis. Results: Data from 13,254 patients in 1117 ICUs (90 countries) revealed 6747 pressure injuries; 3997 (59.2%) were ICU-acquired. Overall prevalence was 26.6% (95% confidence interval [CI] 25.9–27.3). ICU-acquired prevalence was 16.2% (95% CI 15.6–16.8). Sacrum (37%) and heels (19.5%) were most affected. Factors independently associated with ICU-acquired pressure injuries were older age, male sex, being underweight, emergency surgery, higher Simplified Acute Physiology Score II, Braden score 3 days, comorbidities (chronic obstructive pulmonary disease, immunodeficiency), organ support (renal replacement, mechanical ventilation on ICU admission), and being in a low or lower-middle income-economy. Gradually increasing associations with mortality were identified for increasing severity of pressure injury: stage I (odds ratio [OR] 1.5; 95% CI 1.2–1.8), stage II (OR 1.6; 95% CI 1.4–1.9), and stage III or worse (OR 2.8; 95% CI 2.3–3.3). Conclusion: Pressure injuries are common in adult ICU patients. ICU-acquired pressure injuries are associated with mainly intrinsic factors and mortality. Optimal care standards, increased awareness, appropriate resource allocation, and further research into optimal prevention are pivotal to tackle this important patient safety threat

Bacterial isolation and identification of natural Calculus bovis and study of in vitro cultured Calculus bovis and natural Calculus bovis on biochemical characteristics

本研究目的為篩選與鑑定天然牛黃的菌株、分析體外培養牛黃與天然牛黃之主要成分及其抗氧化能力。並進一步以二乙基亞硝胺誘發大鼠的肝臟損傷，探討上述兩種牛黃對肝臟的保護效果。 運用不同的方法鑑定分離得到的菌株，例如微生物的型態特性、鑑定細菌脂肪酸使用微生物鑑定系統（Microbial Identification System）、判斷微生物碳源利用生化特性（Biolog Identification System）與16S rDNA 的基因序列分析。本試驗分離得到一株短桿狀格蘭氏陰性菌。使用微生物鑑定系統鑑定細菌脂肪酸與判斷微 生物碳源利用生化特性的鑑定方法得到的結果，在資料庫中已知的微生物，並無此相符合的微生物資料，因此有可能為一株新型微生物，所以更進一步進行基因序列分析。從16S rDNA 基因序列分析得到的結果，在基因庫中亦無法找到相同基因序列的微生物，因此可確認為一株新型的桿菌。 天然牛黃中含有高量膽紅素、膽綠素與必需胺基酸，而且必需胺基酸占全部胺基酸的比值亦較高。體外培養牛黃與天然牛黃的脂肪酸組成皆以飽和脂肪酸為主。天然牛黃中微量元素鋅、鐵與錳的含量亦顯著高於體外培養牛黃。此二種牛黃的主要膽汁成分為膽酸、去氫膽汁酸與鵝去氧膽汁酸。將分離得到的微生物添加入豬膽汁中培養，培養期間樣品pH 值有稍微下降的趨勢。培養一日後微生物迅速增值。研究顯示，體外培養牛黃與天然牛黃皆具有良好的抗氧化能力。天然牛黃的還原力顯著高於體外培養牛黃。兩種牛黃皆具有高的DPPH 自由基清除與螯合亞鐵離子能力。兩種的牛黃皆有強抗氧化能力的抗 氧化劑。因為天然牛黃長期供不應求且價格昂貴，體外培養牛黃具有作為天然牛黃替代品的潛力。 在動物試驗中，大鼠經二乙基亞硝胺處理以誘發肝臟損傷，分析其血清中天門冬胺酸鹽轉胺酶、胺基丙酸轉胺酶、伽瑪麩胺基酸轉胺酶、鹼性磷酶與乳酸去氫酶。大鼠經二乙基亞硝胺處理後，血清中的天門冬胺酸鹽轉胺酶、胺基丙酸轉胺酶、伽瑪麩胺基酸轉胺酶、鹼性磷酶、與乳酸去氫酶皆顯著提高。體外培養牛黃與天然牛黃處理皆具有預防二乙基亞硝胺誘發肝損傷與氧化壓力之作用；顯示試驗動物經服用體外培養牛黃與天然牛黃均具有保護肝臟抵抗二乙基亞硝胺造成 的肝臟損傷的作用。The objectives of the study were to isolate and identify of bacteria of natural Calculus bovis, to analyze the major components of in vitro cultured Calculus bovis and natural Calculus bovis, the antioxidative capacities of both kinds of Calculus bovis and effects of Calculus bovis on diethylnirosamine induced hepatic injury in rats. Morphological features, bacterial fatty acid analysis of bacterium using Microbial Identification System, carbon source utilization profiles using Biolog system and 16S rDNA sequencing were employed to identify isolated bacterium. This isolated bacterium was observed to be Gram-negative, rod-shaped and short chain. The results of bacterial fatty acid analysis and physiological characteristics were not matched to the database. Based on 16S rDNA sequence analysis, this bacterium was classified as a novel Bacillus species. The product of natural Calculus bovis was rich in bilirubin and biliverdin. The natural Calculus bovis had higher content of essential amino acids and a higher ratio of essential amino acids to total amino acids. The dominant fatty acid composition of in vitro cultured Calculus bovis and natural Calculus bovis was saturated fatty acids. The mineral contents of zinc, iron and manganese of natural Calculus bovis were significantly higher than those of in vitro cultured Calculus bovis. The major bile acids of both kinds of Calculus bovis were cholic acid, dehydrocholic acid and chenodeoxycholic acid, respectively. The results showed that the changes of pH values were slightly decreased throughout inoculation with starter. The initial microbial counts increased rapidly after inoculation at d 1. The in vitro cultured Calculus bovis groups and the natural Calculus bovis group had good antioxidant activities. The reducing ability of natural Calculus bovis was significantly higher than that of in vitro cultured Calculus bovis. The in vitro cultured Calculus bovis and natural Calculus bovis showed high activities of the scavenging activities of DPPH radical and chelating abilities of ferrous ion. The in vitro cultured Calculus bovis and natural Calculus bovis were natural antioxidants with potent antioxidative activities. The in vitro cultured Calculus bovis might therefore be an alternative of natural Calculus bovis because of expensive and deficient natural Calculus bovis. Aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase were determined in serum of rats. The previous enzymes were estimated as markers of hepatoprotective studies. All the experimental animals administered with diethylnitrosamine were obviously by elevated serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase. The treatment of the in vitro cultured Calculus bovis and natural Calculus bovis prevented the diethylnitrosamine induced hepatic damage and oxidative stress. In conclusion, the results were shown that both of the experimental rats administered with the in vitro cultured Calculus bovis and natural Calculus bovis protected the liver against diethylnitrosamine induced hepatic injury.1 Chapter One -Abstract 1 1.1 Chinese abstract 1 1.2 English abstract 3 2. Chapter Two -Introduction 5 2.1 Introduction 7 2.2 Identification method of bacteria 8 2.2.1 Morphological properties 8 2.2.2 Bacterial fatty acid analysis 8 2.2.3 Determination of physiological characteristics 10 2.2.4 Isolation of 16S rDNA and sequencing 11 2.3 Role of major components in in vitro cultured Calculus bovis and natural Calculus bovis 14 2.3.1 Importance of Calculus bovis as Chinese medical material 14 2.3.2 Utilization of byproduct 15 2.3.3 Formation of natural Calculus bovis 16 2.3.4 Major components 17 2.3.4.1 Bile pigment 17 2.3.4.2 Zinc as an antioxidant 18 2.4 Antioxidative capacity of in vitro cultured Calculus bovis and natural Calculus bovis 21 2.4.1 Damage of reactive oxygen species 21 2.4.2 Antioxidant capacity 22 2.4.3 Natural antioxidant substances 23 2.4.4 Functional foods 24 2.5 Perspectives of chemoprevention of hepatoprotective properties 26 2.5.1 Chemoprevention 26 2.5.2 Hepatoinjury 30 2.5.3 Liver injury induced by diethylnitrosamine 31 2.5.4 Hepatoprotection 31 2.6 Objectives of this thesis research 34 2.7 References 35 Chapter Three -Identification of a novel Bacillus species isolated from Calculus bovis 42 3.1 Abstract 43 3.2 Introduction 44 3.3 Materials and methods 46 3.4 Results and discussion 52 3.4.1 Morphological properties 52 3.4.2 Bacterial fatty acid analysis 55 3.4.3 Determination of physiological characteristics 57 3.4.4 Isolation of 16S rDNA and sequencing 60 3.5 Conclusion 64 3.6 References 65 Chapter Four -Study of in vitro cultured Calculus bovis and natural Calculus bovis on major components 68 4.1 Abstract 69 4.2 Introduction 70 4.3 Materials and methods 72 4.4 Results and discussion 79 4.4.1 Yield percentage of in vitro cultured Calculus bovis 79 4.4.2 Bilirubin and biliverdin determination 82 4.4.3 Amino acid determination of Calculus bovis 84 4.4.4 Ratio of essential amino acids to total amino acids of Calculus bovis 87 4.4.5 Fatty acid analysis of Calculus bovis 89 4.4.6 Mineral content of Calculus bovis 92 4.4.7 Bile acid content of Calculus bovis 94 4.5 Conclusion 96 4.6 References 97 Chapter Five -Antioxidative capacity of in vitro cultured Calculus bovis and natural Calculus bovis 101 5.1 Abstract 101 5.2 Introduction 103 5.3 Materials and methods 105 5.4 Results and discussion 110 5.4.1 Determination of pH values 110 5.4.2 Microbiological determination 110 5.4.3 Measurement of antioxidant activity 114 5.4.4 Determination of reducing capacity 117 5.4.5 Analysis of DPPH radical scavenging activity 118 5.4.6 Chelating ability of ferrous ion 122 5.5 Conclusion 125 5.6 References 126 Chapter Six -Study of in vitro cultured Calculus bovis and natural Calculus bovis on diethylnitrosamine induced hepatic injury in rats 130 6.1 Abstract 131 6.2 Introduction 132 6.3 Materials and methods 134 6.4 Results and discussion 138 6.4.1 Aspartate aminotransferase 138 6.4.2 Alanine aminotransferase 142 6.4.3 Gamma glutamyl transferase 146 6.4.4 Alkaline phosphatase 149 6.4.5 Lactate dehydrogenase 152 6.5 Conclusion 155 6.6 References 156 Chapter Seven -Conclusion 15

Study of treatment with organic acids on microorganisms growth and biogenic amines formation in raw meat and Chinese-style sausage

不同有機酸處理對原料肉及中式香腸內微生物生長及生物胺形成之研究， 主要探討微生物生長與生物胺形成。原料肉處理組之總生菌數、乳酸菌數 、葡萄球菌數和細球菌皆顯著低於對照組(P<0.05)；處理組之腸內桿菌數 和李斯特菌數低於對照組；明度以對照組較低(P<0.05)。中式香腸冷藏儲 存30日，對照組之總生菌數、乳酸菌、葡萄球菌和細球菌皆顯著高於三個 處理組(P<0.05)；對照組之李斯特菌儲存至30日高於處理組；對照組之腸 內桿菌除儲存20日外，皆高於處理組；然而， TBA值隨儲存時間增加有上 升之趨勢；儲存期間風味品評在可接受範圍。中式香腸冷藏儲存0-20日對 照組之pH值較高(P<0.05)，儲存30-40日則無顯著差異；儲存0日起對照組 之VBN值高於處理組(P<0.05)，儲存20日起對照組即高於處理組(P<0.05) ；儲存期40日之色胺和胺基乙苯均未檢測出；儲存30日之屍胺對照組高於 處理組(P<0.05)；組織胺在儲存期 5日以檸檬酸和乳酸處理組較對照組為 高(P<0.05)；酪胺在儲存30日對照組較高(P<0.05)。Study of treatment with organic acids on the microbial counts and the biogenic amines formation of raw meat and Chi- nese- style sausage contained two parts, which were the micro- bial growth and biogenic amines formation. The total plate counts, lactic acid bacteria, Staphylococ- cus and Micrococcus of the treated groups on raw meat were si- gnificantly smaller than those of the controls (P<0.05). Ente- robacteriaceae and Listeria spp. of the treated groups were smaller than those of the controls. The L value of the controls were significantly (P<0.05) smaller than those of the other treated groups. The total plate counts, lactic acid bacteria and Staphylo- coccus and Micrococcus of the treated groups were significantly (P<0.05) greater than those of the treated groups in the Chi- nese-style sausage after storage for 30 days at 4℃. The Lis- teria spp. counts of the controls were greater than those of the treated groups after storage for 30 days. The Enterobacte- riaceae counts of the controls were greater than those of the treated groups on days 0, 5, 10, 30 and 40. Although the TBA value increased after storage, the level was still acceptable. The pH of the controls in Chinese-style sausage had a sig- nificant difference after storage from day 0 to 20 at 4℃ (P< 0.05), but it hadn''t a significant difference after storage from days 30 to 40. VBN value of the controls were greater than that of the treated groups after storage except day 10. Trypta- mine, phenylethylamine and spermidine were not detected after storage for 40 days. Cadaverine of the controls were greater than that of the treated groups after storage on day 30. Hista- mine of the treated groups with lactic and citric acid were greater than that of the other treatments after storage on day 5 (P<0.05). Tyramine of the controls were significantly greater than that of treated groups after storage on day 30

Bacterial isolation and identification of natural Calculus bovis and study of in vitro cultured Calculus bovis and natural Calculus bovis on biochemical characteristics

本研究目的為篩選與鑑定天然牛黃的菌株、分析體外培養牛黃與天然牛黃之主要成分及其抗氧化能力。並進一步以二乙基亞硝胺誘發大鼠的肝臟損傷，探討上述兩種牛黃對肝臟的保護效果。 運用不同的方法鑑定分離得到的菌株，例如微生物的型態特性、鑑定細菌脂肪酸使用微生物鑑定系統（Microbial Identification System）、判斷微生物碳源利用生化特性（Biolog Identification System）與16S rDNA 的基因序列分析。本試驗分離得到一株短桿狀格蘭氏陰性菌。使用微生物鑑定系統鑑定細菌脂肪酸與判斷微 生物碳源利用生化特性的鑑定方法得到的結果，在資料庫中已知的微生物，並無此相符合的微生物資料，因此有可能為一株新型微生物，所以更進一步進行基因序列分析。從16S rDNA 基因序列分析得到的結果，在基因庫中亦無法找到相同基因序列的微生物，因此可確認為一株新型的桿菌。 天然牛黃中含有高量膽紅素、膽綠素與必需胺基酸，而且必需胺基酸占全部胺基酸的比值亦較高。體外培養牛黃與天然牛黃的脂肪酸組成皆以飽和脂肪酸為主。天然牛黃中微量元素鋅、鐵與錳的含量亦顯著高於體外培養牛黃。此二種牛黃的主要膽汁成分為膽酸、去氫膽汁酸與鵝去氧膽汁酸。將分離得到的微生物添加入豬膽汁中培養，培養期間樣品pH 值有稍微下降的趨勢。培養一日後微生物迅速增值。研究顯示，體外培養牛黃與天然牛黃皆具有良好的抗氧化能力。天然牛黃的還原力顯著高於體外培養牛黃。兩種牛黃皆具有高的DPPH 自由基清除與螯合亞鐵離子能力。兩種的牛黃皆有強抗氧化能力的抗 氧化劑。因為天然牛黃長期供不應求且價格昂貴，體外培養牛黃具有作為天然牛黃替代品的潛力。 在動物試驗中，大鼠經二乙基亞硝胺處理以誘發肝臟損傷，分析其血清中天門冬胺酸鹽轉胺酶、胺基丙酸轉胺酶、伽瑪麩胺基酸轉胺酶、鹼性磷酶與乳酸去氫酶。大鼠經二乙基亞硝胺處理後，血清中的天門冬胺酸鹽轉胺酶、胺基丙酸轉胺酶、伽瑪麩胺基酸轉胺酶、鹼性磷酶、與乳酸去氫酶皆顯著提高。體外培養牛黃與天然牛黃處理皆具有預防二乙基亞硝胺誘發肝損傷與氧化壓力之作用；顯示試驗動物經服用體外培養牛黃與天然牛黃均具有保護肝臟抵抗二乙基亞硝胺造成 的肝臟損傷的作用。The objectives of the study were to isolate and identify of bacteria of natural Calculus bovis, to analyze the major components of in vitro cultured Calculus bovis and natural Calculus bovis, the antioxidative capacities of both kinds of Calculus bovis and effects of Calculus bovis on diethylnirosamine induced hepatic injury in rats. Morphological features, bacterial fatty acid analysis of bacterium using Microbial Identification System, carbon source utilization profiles using Biolog system and 16S rDNA sequencing were employed to identify isolated bacterium. This isolated bacterium was observed to be Gram-negative, rod-shaped and short chain. The results of bacterial fatty acid analysis and physiological characteristics were not matched to the database. Based on 16S rDNA sequence analysis, this bacterium was classified as a novel Bacillus species. The product of natural Calculus bovis was rich in bilirubin and biliverdin. The natural Calculus bovis had higher content of essential amino acids and a higher ratio of essential amino acids to total amino acids. The dominant fatty acid composition of in vitro cultured Calculus bovis and natural Calculus bovis was saturated fatty acids. The mineral contents of zinc, iron and manganese of natural Calculus bovis were significantly higher than those of in vitro cultured Calculus bovis. The major bile acids of both kinds of Calculus bovis were cholic acid, dehydrocholic acid and chenodeoxycholic acid, respectively. The results showed that the changes of pH values were slightly decreased throughout inoculation with starter. The initial microbial counts increased rapidly after inoculation at d 1. The in vitro cultured Calculus bovis groups and the natural Calculus bovis group had good antioxidant activities. The reducing ability of natural Calculus bovis was significantly higher than that of in vitro cultured Calculus bovis. The in vitro cultured Calculus bovis and natural Calculus bovis showed high activities of the scavenging activities of DPPH radical and chelating abilities of ferrous ion. The in vitro cultured Calculus bovis and natural Calculus bovis were natural antioxidants with potent antioxidative activities. The in vitro cultured Calculus bovis might therefore be an alternative of natural Calculus bovis because of expensive and deficient natural Calculus bovis. Aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase were determined in serum of rats. The previous enzymes were estimated as markers of hepatoprotective studies. All the experimental animals administered with diethylnitrosamine were obviously by elevated serum aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase, alkaline phosphatase and lactate dehydrogenase. The treatment of the in vitro cultured Calculus bovis and natural Calculus bovis prevented the diethylnitrosamine induced hepatic damage and oxidative stress. In conclusion, the results were shown that both of the experimental rats administered with the in vitro cultured Calculus bovis and natural Calculus bovis protected the liver against diethylnitrosamine induced hepatic injury.1 Chapter One -Abstract 1 1.1 Chinese abstract 1 1.2 English abstract 3 2. Chapter Two -Introduction 5 2.1 Introduction 7 2.2 Identification method of bacteria 8 2.2.1 Morphological properties 8 2.2.2 Bacterial fatty acid analysis 8 2.2.3 Determination of physiological characteristics 10 2.2.4 Isolation of 16S rDNA and sequencing 11 2.3 Role of major components in in vitro cultured Calculus bovis and natural Calculus bovis 14 2.3.1 Importance of Calculus bovis as Chinese medical material 14 2.3.2 Utilization of byproduct 15 2.3.3 Formation of natural Calculus bovis 16 2.3.4 Major components 17 2.3.4.1 Bile pigment 17 2.3.4.2 Zinc as an antioxidant 18 2.4 Antioxidative capacity of in vitro cultured Calculus bovis and natural Calculus bovis 21 2.4.1 Damage of reactive oxygen species 21 2.4.2 Antioxidant capacity 22 2.4.3 Natural antioxidant substances 23 2.4.4 Functional foods 24 2.5 Perspectives of chemoprevention of hepatoprotective properties 26 2.5.1 Chemoprevention 26 2.5.2 Hepatoinjury 30 2.5.3 Liver injury induced by diethylnitrosamine 31 2.5.4 Hepatoprotection 31 2.6 Objectives of this thesis research 34 2.7 References 35 Chapter Three -Identification of a novel Bacillus species isolated from Calculus bovis 42 3.1 Abstract 43 3.2 Introduction 44 3.3 Materials and methods 46 3.4 Results and discussion 52 3.4.1 Morphological properties 52 3.4.2 Bacterial fatty acid analysis 55 3.4.3 Determination of physiological characteristics 57 3.4.4 Isolation of 16S rDNA and sequencing 60 3.5 Conclusion 64 3.6 References 65 Chapter Four -Study of in vitro cultured Calculus bovis and natural Calculus bovis on major components 68 4.1 Abstract 69 4.2 Introduction 70 4.3 Materials and methods 72 4.4 Results and discussion 79 4.4.1 Yield percentage of in vitro cultured Calculus bovis 79 4.4.2 Bilirubin and biliverdin determination 82 4.4.3 Amino acid determination of Calculus bovis 84 4.4.4 Ratio of essential amino acids to total amino acids of Calculus bovis 87 4.4.5 Fatty acid analysis of Calculus bovis 89 4.4.6 Mineral content of Calculus bovis 92 4.4.7 Bile acid content of Calculus bovis 94 4.5 Conclusion 96 4.6 References 97 Chapter Five -Antioxidative capacity of in vitro cultured Calculus bovis and natural Calculus bovis 101 5.1 Abstract 101 5.2 Introduction 103 5.3 Materials and methods 105 5.4 Results and discussion 110 5.4.1 Determination of pH values 110 5.4.2 Microbiological determination 110 5.4.3 Measurement of antioxidant activity 114 5.4.4 Determination of reducing capacity 117 5.4.5 Analysis of DPPH radical scavenging activity 118 5.4.6 Chelating ability of ferrous ion 122 5.5 Conclusion 125 5.6 References 126 Chapter Six -Study of in vitro cultured Calculus bovis and natural Calculus bovis on diethylnitrosamine induced hepatic injury in rats 130 6.1 Abstract 131 6.2 Introduction 132 6.3 Materials and methods 134 6.4 Results and discussion 138 6.4.1 Aspartate aminotransferase 138 6.4.2 Alanine aminotransferase 142 6.4.3 Gamma glutamyl transferase 146 6.4.4 Alkaline phosphatase 149 6.4.5 Lactate dehydrogenase 152 6.5 Conclusion 155 6.6 References 156 Chapter Seven -Conclusion 15