以同位素比值質譜儀測定補充chrysin對人體尿液中睪固酮及δ13C‰之影響

行政院國家科學委員會補助專題研究計畫

▉ 成 果 報 告

□期中進度報告

以同位素比值質譜儀測定補充chrysin對人體尿液中睪固酮及δ

C

之影響

The effects of chrysin supplementation on uric testosterone and δ

C

by

isotope ratio mass spectrometry

一、中文摘要 由於在離體實驗 (in vivo) 中發現 chrysin (5,7-dihydroxyflavone) 具有與芳 香族酶抑制劑相似的作用，因此研究學者 認為chrysin 可能具有提高睪固酮濃度的 功效。然而，過去關於補充chrysin 的研究 相當少數，且皆是以補充天然複方為主， 並沒有補充單方chrysin 的研究，因此本研 究目的在探討補充市售單方chrysin 是否 影響睪固酮的合成，進而改變身體組成、 肌力與運動表現，並藉由尿液檢測了解是 否有違反運動禁藥規範之疑慮。受試對象 為20 位國立體育大學投擲隊隊員（男性 14 位；女性 6 位），實驗設計採配對設計 (matched pairs design)，依照競賽項目將受 試者配對分組為chrysin 組（C 組；20.9 ± 1.3 歲）及 placebo 組（P 組；20.7 ± 1.5 歲）。 受試者每餐飯後服用2 顆 chrysin 膠囊或等 量之安慰劑膠囊（3000mg/天），持續 21 天。於第1 日、第 8 日、第 22 日早上進行 身體組成、肌力、運動表現檢測與血液、 尿液樣本之採集。身體組成測量：體重、 體脂率、皮脂厚度、體圍。肌力及運動表 現檢測項目：股四頭肌最大等長肌力、肱 二頭肌最大等長肌力、垂直跳、立定跳遠、 藥球胸前傳球。血液樣本檢測：睪固酮、 游離睪固酮、可體松、睪固酮/可體松比值 (T/C 比值)、雌二醇。尿液樣本檢測：睪 固酮/表睪固酮比值（T/E 比值）與睪固酮 類緣物δ13C‰數值。統計方式以混合式二

因子變異數分析 (two-way mixed design ANOVA) 檢驗 C 組及 P 組之各項數值是

否具有組間或組內之差異性，並以LSD 法

進行事後比較各項數值以平均數 ± 標準 差表示，顯著水準訂為 (p＜.05)。T/E 比

值與δ13C‰數值則以世界反運動禁藥機

構 (World Anti-Doping Agency) 所訂定之 檢測標準判斷是否違反運動禁藥規範。研 究結果顯示各項指標並無發現組間之顯著 差異 (p＞.05)。尿液檢測方面，T/E 比值 皆低於4，而 δ13C‰數值分佈於正常範圍 （大於-28‰）。本研究結果顯示，連續服 用21 天 chrysin 並不會提升體內荷爾蒙的 濃度，因此對於身體組成、肌力與運動表 現並無正面幫助，且無違反運動禁藥規範 之疑慮。 關鍵字：chrysin、睪固酮、T/E 比值、同 位素比值質譜儀、δ13C

Previous studies had suggested that chrysin (5,7-dihydroxyflavone) in vitro to be an aromatase inhibitor. However, there was no study to investigate effects of the single constituent chrysin supplementation in human. The purpose of this study was to investigate whether single constituent chrysin supplementation would increase serum testosterone, change body

二、計畫緣由 included body weight, body fat percentage,

skinfold thickness, and body circumference. Muscle strength and exercise performance tests contained maximal isometric force of quadriceps and biceps, vertical jump, standing broad jump, and medicine ball chest pass. Blood samples were measured for serum testosterone, free testosterone, estradiol, cortisol, and testosterone/cortisol ratio (T/C ratio). Urine samples were analysed for testosterone/epitestosterone ratio (T/E ratio) and δ13C‰ for

differentiating the exogenous and endogenous steroids. A two-way mixed designed analysis of variance (ANOVA) was used to determine changes over times and between groups. Statistic significance was set at P < 0.05. Data were presented as the mean ± SD.Determining whether or not an anti-doping violation occurred on urine analysis according to the anti-doping rules of World Anti-Doping Agency (WADA). The present data showed no significant difference on body composition, muscle strength, exercise performances, and blood biomarkers between group C and P (P >.05). All data of T/E ratio and δ13C‰ conformed to the anti-doping rules of WADA (T/E ratio ＜4; δ13C‰＞-28‰). The results of this study suggested that single constituent chrysin supplementation for 3 weeks would not affect body composition, muscle

strength and exercise performance; and further, chrysin supplementation would also not cause an anti-doping violation.

運動員濫用同化性類固醇

(androgenic-anabolic steroids; AAS)情形甚

為嚴重，1984 年洛杉磯奧林匹克運動會藥 檢中，檢驗出AAS 佔全部陽性反應總數的 91.6%（許美智、詹貴惠，2005）。除了直 接服用睪固酮等 AAS 外，亦有些運動 員服用抑制芳香化作用的物質，如 chrysin，以維持睪固酮在體內的高濃 度。chrysin 是從一種叫做 Passiflora coerulea 植物萃取出來的，也可以在蜂 巢中找到，此外，中藥黃芩也含有 chrysin (劉雄、高建德，2007)。chrysin 為 5,7 二羥基黃酮 (5,7 -dihydroxyflavone)，因此被認為具有降 低芳香族酶活性 (aromatase inhibition) 的作用 (Ibrahim & Abul-Hajj, 1990)。 過去關於補充 chrysin 的研究相當少 數，皆是以補充複方為主 (Brown, et al., 2001a; Brown, et al., 2001b; Gambelunghe, et al., 2003)，並沒有補充單方 chrysin 的 研究，因此使用市售單方 chrysin 增補 劑是否影響睪固酮的生成，進而改變身 體組成、肌力與運動表現，以及是否有 違反運動禁藥規範之疑慮，為本研究要 釐清的問題。 世界反運動禁藥組織 (World

Anti-Doping Agency; WADA) 於2009年所 頒布的細則中提到，若T/E值超過4，則可 以利用同位素比值質譜儀 (Isotope Ratio Mass Spectrometry; IRMS) 進行外源性或 內生性的確認。因此，本研究將探討補充 chrysin對運動禁藥δ13C‰之影響。 三、研究目的 本研究目的為探討補充市售單方 chrysin是否影響睪固酮的合成，進而改 變身體組成、肌力與運動表現，並藉由

液，再加入10 mL乙醚萃取5 min，收集有 機層，於40℃下以氮氣吹乾，加入100 μL 衍生試劑MSTFA，加熱60℃，30分鐘，冷 卻至室溫後，將萃取物取1 μL注入GC-MS 上分析。以GC-MS為分析儀器工具，建立 一精密且準確之分析方法，以定量雞睪丸 中睪固酮之濃度。經過分析方法的尋找及 確效之後，其分析條件如下：使用儀器為 Agilent technologies HP7890A gas

chromatograph結合5975C mass selective detector。使用管柱為DB-5MS capillary column (0.25 mm×15 m, 0.25 um)，升溫條 件為初始溫度120℃停留時間2分鐘，以 15℃/min升溫至230℃，再以5℃/min升溫 至250℃，停留時間2分鐘。注射口溫度為 250℃，氣體流速1 ml/min，偵測模式為 selection ion monitor (SIM)，睪固酮與表睪 固酮之特徵離子同為432及433。確效的內 容包括再現性測試、精密度及準確度之測 定。 尿液中δ13C‰數值分析：檢體前處理 方式為取10 mL 尿液檢體，使用固相萃取 管Bakerbond octadecyl C18在自動萃取機 上(HAMILTON ML4000)執行固相萃取。 萃取步驟如下：在管柱中加入1 mL 的甲 醇，等待2 分鐘，吹氣，在管柱中加入檢 體。吹氣，加入8 mL 的去離子水清洗液， 吹氣， 加入 8 mL 甲醇沖提溶液，吹氣。 將沖提液以氮氣吹乾後加入50 μL β-glucuronidase 溶液和 1mL 0.2 M phosphate buffer(pH=7.0)，於 50℃下水解 60 分鐘，再次使用固相萃取，萃取步驟如 下：在管柱中加入1 mL 的甲醇，等待 2 分鐘，吹氣，在管柱中加入檢體。吹氣， 加入6 mL 的去離子水/乙腈(80:20)與去離 子水/乙腈(65:35)清洗液，吹氣，加入 12 mL 乙腈沖提溶液，吹氣。將沖提液用氮氣吹 乾，各加入50 μL 醋酸酐與 pyridine 於 60℃ 下乙醯化1 小時，冷卻至室溫後用氮氣吹 乾，加入3 mL 去離子水/乙腈(50:50)回 溶，再次使用固相萃取，萃取步驟如下： 在管柱中加入1 mL 的甲醇，等待 2 分鐘， 吹氣，在管柱中加入檢體，吹氣，加入12 mL 的去離子水/乙腈(50:50)清洗液，吹氣， 加入第一段(F1)沖提液 15 mL 去離子水/乙 腈(25:75)與第二段(F2)沖提液 15 mL 乙 腈，吹氣。在沖提液中分別加入80 uL(F1) 與10 uL(F2)內標(5α-androstan-3β-ol)用氮 氣吹乾將萃取物，分別加入500 uL(F1)與 40 uL(F2) cyclohexane 回溶，取 2μL 注入 GC/IRMS 進行分析。以氣相層析同位素比 值質譜儀為分析儀器工具，以測定δ13C‰ 之數值，其分析條件如下：使用儀器為 Agilent technologies HP6890A gas

伍、結果 身體組成各項數據如表ㄧ所示，此部 分僅C 組的第 22 天腰圍較第 1 天腰圍顯 著下降 (p＜.05)，其他數據於組間或組內 比較皆無顯著差異。肌力與運動表現方面 數據於組間或組內皆無顯著變化（表二）。 血液生化值部分可發現肌酸酐於C 組及 P 組的第8、22 天皆顯著高於其第 1 天之數 值 (p＜.05)，而其他數據則均無組間或組 內的顯著差異（表三）。補充chrysin 並未 對尿液分析中之各項數據造成陽性反應 （表四、表五）。 六、討論 本次研究結果顯示連續補充 3 週市售 單方chrysin 膠囊並未影響血液中睪固酮 之生成，同時亦無降低血液中雌二醇的濃 度。在尿液檢測方面，T/E 比值與 δ13C‰ 數值皆無違反運動禁藥檢測之標準。本次 研究是第一個以單方chrysin 作為增補物 質來研究其芳香族酶抑制作用的人體試 驗，過去相關的人體試驗皆為複方的補充 方式 (Brown, et al., 2001a; Brown, et al., 2001b)，並且過去的研究設計中 chrysin 的 劑量遠低於本次研究所使用的劑量。過去 的相關研究中發現chrysin 具有降低芳香 族酶活性的實驗主要為離體細胞實驗 (in vitro) (Ibrahim & Abul-Hajj, 1990; Gambelunghe, et al, 2003 ; Ta, & Thomas, 2007)，是否在非離體實驗 (in vivo) 的模 式下同樣具有降低芳香族酶活性，影響睪 固酮的生成，尚未獲得證實。本次研究與 過去研究相較下，雖以較高的劑量來進行 實驗，不過在人體試驗的模式中並無法證 實chrysin 具有降低芳香族酶活性之作 用，進而降低雌二醇的濃度及相對地提升 血液中睪固酮濃度。因此，對於身體組成、 肌力、運動表現並無顯著影響。同時也未 影響尿液中T/E 比值及睪固酮類緣物 δ13C‰之數值，因此並無違反運動禁藥規 範之疑慮。本次研究中在肝腎功能的檢測 結果證實，連續攝取21 天單方 chrysin 膠 囊 (3000mg/天) 並不會對肝腎功能造成 顯著的副作用。 七、結論與建議 本研究結果顯示連續 3 週補充市售單 方chrysin 並不會使身體組成、最大肌力、 運動表現、血液荷爾蒙及肝腎功能產生顯 著變化，且並無違反運動禁藥之疑慮。 八、參考文獻 （一）中文部分 1. 許美智、詹貴惠 (2005)：運動藥物 學。高雄：春橋田股份有限公司。 2. 劉雄、高建德 (2007)。黃岑研究進 展。甘肅中醫學院學報，24(2)，46-51 頁。 （二）英文部分

1. Brown, G. A., Vukovich, M. D., Martini, E. R., Kohut, M. L., Franke W.D., Jackson, D.A. et al. (2001a). Effects of androstenedione-herbal supplementation on serum sex hormone concentrations in 30- to 59-year-old men. International Journal for Vitamin and Nutrition Research, 71(5), 293-301. 2. Brown, G. A., Vukovich, M. D.,

Martini, E. R., Kohut, M. L., Franke, W. D., Jackson, D. A. et al. (2001b). Endocrine and lipid responses to chronic androstenediol herbal supplementation in 30 to 58 year old men.Journal of the American

4. Gambelunghe, C., Rossi, R.,

Sommavilla, M., Ferranti, C., Rossi, R., Ciculi, C. et al., (2003). Effects of chrysin on urinary testosterone levels in human males. Journal of Medicinal Food, 6(4), 387-390

5. Ta, N., & Thomas, W. (2007).

表四 補充 chrysin 對 T/E 比值之影響 編號 第1 天 第8 天 第22 天 1 N.D. 0.39 N.D. 2 N.D. N.D. N.D. 3 2.75 2.58 2.18 4 N.D. 0.12 N.D. 5 N.D. N.D. N.D. 6 N.D. 0.18 N.D. 7 N.D. 0.49 N.D. 8 N.D. N.D. N.D. 9 N.D. N.D. N.D. 10 0.36 0.45 N.D. N.D.：檢測濃度小於最低偵測極限 (2ng/mL) 濃度單位：ng/mL 表五 補充 21 天後睪固酮類緣物 δ13C‰之數值

編號 etiocholanolone androsterone 5βA 5αA 5βP

參加2010年第57屆

美國大學運動醫學會年會報告

一、 會議時間：2010 年 6 月 1 日至 6 月 5 日

二、 會議地點：Baltimore, Maryland, USA (美國)

三、 出席人員：許美智教授

四、 主辦單位：美國大學運動醫學會（ACSM）

五、 會議內容

ACSM是世界上最大的運動醫學及訓練科學組織機構，會員

近2萬名，推展及整合運動醫學及運動科學的研究，大會除邀

請專家學者做專題演講外，口頭發表、海報發表同ㄧ時段之內

探討的議題很多，座談會也有許多有趣的主題。其目的在提升

並整合運動醫學與運動科學之研究、教育與實際應用，以維持

與增進身體活動表現、適能、健康與生活品質。

第57屆年會之研究議題有基礎與應用科學、教育、醫學、

公共衛生、健康適能與運動表現。每天之主題細分為運動員照

護與臨床醫學；生物力學與移動之神經控制；心血管、腎臟、

呼吸生理；臨床運動生理；環境與職業生理；流行病、生統與

健康促進；運動醫學；適能評估與訓練；免疫、基因、内分泌；

代謝與營養；心理行為與神經學；骨骼肌、骨頭與結締組織；

專業發展、組織資訊。

1.大會會長邀請演講

大會會長邀請在運動醫學及運動科學領域具貢獻並具研究

領導地位的學者 Dr. Joseph B. Wolffe 給予 75 分鐘的演

reform and lifestyle reform revisited.

2.口頭發表

3.臨床案例報告

4.海報發表

5.座談會

6.廠商展覽: 運動器材、醫療器材、營養增補劑、書籍等。

六、個人海報發表：共 5 篇

1. Mei-Chich Hsu, Shu-Li Chen, Cheng-Chen Hsu, Borcherng Su

(2010) Effects of American Ginseng Supplementation on

Cytokines and Oxidative Stress Following an Acute

Downhill Running. Paper presented at the 57th Annual

Meeting of the American College of Sports Medicine, June

1-3, Baltimore, Maryland, USA. Medicine & Science in

Sports & Exercise, 42 (5), S556.

(國科會補助出國)

2. Sharon Hsu, Mei-Chich Hsu. Health Promotion Program on

Reduction of Cardiovascular Disease Risk Factors for Adults

with Intellectual Disabilities. 57th Annual Meeting of the

American College of Sports Medicine. June 1-3, Baltimore,

Maryland, USA. Medicine & Science in Sports & Exercise,

42 (5), S462. NSC

96-2413-H-179-009

3. Chung-Chen Huang, Sheng-An Lo, Mei-Chich Hsu, Li-Lan Fu,

Kuei-Hui Chan, Feng-Yi Chang, Kuei-Yu Chien. Effects of

modified Tai Chi training program on hematological

4. Kuei-Yu Chien, Ku-Fu Hsu, Chia-Hua Kuo, Mei-Chich Hsu.

Swimming training reduced metformin concentration after

single dosage of administration in insulin resistance rats.

57th Annual Meeting of the American College of Sports

Medicine. June 1-3, Baltimore, Maryland, USA. Medicine &

Science in Sports & Exercise, 42 (5), S567.

5. Chia-Jung Chung, Yi-An Lin, Mei-Chich Hsu (2010) Effects

of Branch-Chain Amino Acids and Arginine Drink on

Post-Exercise Metabolic Response Paper presented at the

57th Annual Meeting of the American College of Sports

Medicine, June 1-3,Baltimore, Maryland, USA. Medicine &

Science in Sports & Exercise, 42 (5), S545.

七、心得

Effects of American Ginseng Supplementation on Cytokines and

Oxidative Stress Following an Acute Downhill Running

Mei-Chich Hsu, Shu-Li Chen, Cheng-Chen Hsu, Borcherng Su National Taiwan Sport University, Taiwan

Taipei Medical University, Taiwan Buddhist Tzu Chi General Hospital, Taiwan

American ginseng (AG) supplementation has been shown to reduce the leakage of creatine kinase from skeletal sarcoplasm into blood stream during an exhaustive treadmill run. There is relatively little research that showed the effects of AG on cytokines and oxidative stress in human beings.PURPOSE: This study aimed to investigate the effects of 4 wk of AG supplementation on exercise-induced changes in immune function and oxidative stress. METHODS: Fourteen physically active male college students (mean +/- SD for age, 22.1 +/- 1.8 yr; height, 174.2 +/- 5.7 cm; weight, 70.0 +/- 7.1 kg; V‧O2max, 53.1 +/- 4.0 ml / kg/ min) supplemented either 4 AG

capsule (AG, 400 mg per capsule) or placebo (PL) capsules daily for 4 wk in this randomized, placebo-controlled, double-blinded, cross-over study. After 4 wk of supplementation, subjects performed a 10% grade downhill 60-min treadmill run at 60% V‧O2max. Venous blood samples were collected at pre-supplement, pre-exercise,

immediately after exercise, and 1h, 2h, and 24 h after exercise. RESULTS: No group differences were observed in plasma TNF-α, IL-1β, and IL-10. However, 4-wk AG supplementation resulted in a significant increase in plasma IL-4. Furthermore, serum lipid peroxidative biomarker, 8-iso-PGF2α concentration of the AG group was

significantly lower than that of the PL group at immediately, and 1, 2, and 72 hr after exercise. AG group showed a less muscle soreness than that of PL group at 72 hr after exercise. CONCLUSION: Four-week supplementation with AG may attenuate lipid peroxidation caused by downhill running in male young adults.

Effects of a 12-Week Health Promotion Program on Reduction of

Cardiovascular Disease Risk Factors for Older Adults with

Intellectual Disabilities (less than 15)

Sharon Hsu, Konstantinos Vrongistinos, Yu Tseng, Mei-Chich Hsu California State University, Northridge, CA

National Taiwan Sport University, Taiwan

Disease prevention for persons with intellectual disabilities (ID) has been recognized as an under researched area. Health promotion programs integrating structured and multiple approaches to reduce cardiovascular disease (CVD) risk factors for persons with ID are imperative and greatly needed. Little is known in the application of theoretical-based health promotion program on reduction of CVD risk factors for the population with ID. PURPOSE: The purpose of this study was to examine the effect of a 12-Week theoretical-based health promotion program in reduction of CVD risk factors for older adults with ID. METHOD: A total of 33 older adults with ID (Age in yr.: M = 39.18, SD = 9.49, Range = 30 to 70; Gender, Males = 33.3%, Females = 66.7%) participated in a 12-week health promotion program (3 day per week and 90 minutes per day) at a local educational and vocational center in Taiwan. The 12-week health promotion program consisted health education and exercise components using Transtheoretical Model (TTM) as a framework. The direct caregivers of participants with ID were closely involved throughout the

intervention program. Pre- and post-outcome measures include weight, height, waist circumference, resting blood pressure, skinfolds, as well as fasting plasma

triglycerides, total cholesterol, and glucose (sampled after a 12-hour overnight fast). A multivariate analysis of variance (MANOVA) for repeated measures was used to examine statistically significant differences between pre and post outcome measures.

RESULTS: The findings of repeated MANOVA revealed statistically significant

differences (Pillai's Trace = .681, F(5,23) =7.844, p=.000) as results of the 12-week

intervention program. The univariate tests suggested that weight, waist

Effects of Modified Tai Chi Training Program on Hematological

Parameters in the Elderly

Chung-Chen Huang, Sheng-An Lo, Mei-Chich Hsu, Li-Lan Fu, Kuei-Hui Chan, Feng-Yi Chang, Kuei-Yu Chien

National Taiwan Sport University, Taiwan, Kainan University, Taiwan

National Penghu University, Taiwan

There are several evidences to support health benefits of Tai Chi exercise. However, Tai Chi gait has an increased shear force and frontal plane torque at lower extremity joints than normal gait. That is not suitable for elderly with degenerative joint dysfunction. Therefore, we develop the modified tai chi training program (MTC), which is low impact training program. PURPOSE: This study was to investigate effects of hematological parameters by MTC training program for elderly people.

METHODS: Before the intervention, disease history, exercise habit and medicine use

Swimming Training Reduced Metformin Concentration after Single

Dosage of Administration in Fructose-induced Insulin Resistance

Rats

Kuei-Yu Chien1, Ku-Fu Hsu, Chia-Hua Kuo, Mei-Chich Hsu

Kainan University, Taiwan.

National Taiwan Sport University, Taiwan. Taipei Physical Education College, Taiwan.

Previous study has shown that performing a single bout of swimming before administration of metformin can improve insulin sensitivity and the rate of metformin absorption. We here extended knowledge to examine the effects of swimming training on the absorption, distribution, metabolism, and excretion for the use of metformin.

PURPOSE: To investigate the effect of 28 days swimming training (5days/week,

45min/day) on the pharmacokinetics for the use of metformin in fructose-induced insulin resistance rats. METHODS: Twelve fructose-induced rats were assigned to two groups: administration single dose of metformin without swimming training (M, n=6) and administration single dose of metformin with swimming training (MS, n=6). After 12 hrs fasting, blood samples (0.5 mL) were collected before administration of metformin and at 15, 45, 60, 120, 180, 240, 360, 480, 600, 720-min after dosing (450mg/kg). Plasma metformin concentrations were determined by high performance liquid chromatography (HPLC, Hypersil Cps 3-0679 column (250mm×4.6mm I.D.). The flow rate kept constant at 1mL/min, and the temperature maintained at 40℃. A mixture of acetonitrile and 0.01M potassium dihydrogen phosphate (40:60) (pH 5.5) was used as the mobile phase with UV detection at 234 nm. RESULTS: The

maximum concentration (Cmax) and area under the serum concentration-time curve

(AUC) for the MS group were significantly lower than M group. Apparent volume of the distribution (Vd) and the time-averaged total body clearance (CL) for the MS group were significantly faster than M group. There were no significant differences in the time to maximum concentration (Tmax) and the time to half-life concentration (t1/2)

Effects of Branch-Chain Amino Acids and Arginine Drink on

Post-Exercise Metabolic Response

Chia-Jung Chung,Yi-An Lin, Mei-Chich Hsu National Taiwan Sport University, Taiwan

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