QSAR分析

第五章實驗結果與討論

5.6 QSAR分析

本實驗將 16 種一級炔丙基醇的EC₅₀值與不同參數來進行相關性之探討，過去的文獻資料對於此類化合物的研究較少，也增加本篇研究的重要性。

首先以單純的Log p對於一級炔丙基醇進行QSAR模式的建立,首先先將其分成兩大部分(Primary and homo) ，並以三個實驗終點分別討論，針對模式之適合度，在統計回歸分析中，模式通常採用回歸係數之平方( r²，代表模式所解釋之方差)或自由度校正的r²_adj，標準偏差 (Standard error)及檢驗值F等來評判。

Primary propargylic alcohols 的 QSAR 回歸方程式:

log(1/EC50) = 0.49 Log p-0.27 ; n=9 ;

r² = 0.73; Q²= 0.69 ; S=0.47 ; F =19 (DO)

log(1/EC50) = 0.47 Log p-0.093 ; n=9 ;

r² = 0.81; Q²= 0.78 ; S=0.36 ; F =29.04 (FY)

log(1/EC50) = 0.41 Log p-0.46 ; n=9 ;

r² = 0.63; Q²= 0.5 ; S=0.36 ; F =12.14 (GR)

Primary homopropargylic alcohols 的 QSAR 回歸方程式:

log(1/EC50) = 0.58 Log p-0.61 ; n=7 ;

r² = 0.88 ; Q²= 0.85 ; S=0.28 ; F =34.95 (DO)

log(1/EC50) = 0.62 Log p-0.46 ; n=7 ;

r² = 0.92 ; Q²= 0.9 ; S=0.23 ; F =55.26 (FY)

log(1/EC50) = 0.77 Log p-1.2; n=9 ;

r² = 0.84; Q²= 0.81 ; S=0.41 ; F =27.07 (GR)

All primary propargylic alcohols 的 QSAR 回歸方程式: homopropargylic alcohols與LogP有不錯的相關性(r²=0.84~0.92) ，此結果與( T. Wayne Schultz, 2004)結果相似，Primary propargylic alcohols的部份(r²=0.63~0.81)由以上結果也可發現其作用機制和一般傳統的非極性

Log p

Fig 5.10 一級炔丙基醇 Log p 與 Log(1/EC50)之關係圖(based on Final Yield)

Fig 5.11 一級炔丙基醇 Log p 與 Log(1/EC50)之關係圖(based on Growth Rate)

由圖可發現，三個反應終點呈現的趨勢大致相同，其中 2-propyn-1-ol 這項化合物在圖上明顯偏離許多; 由結果看來和文獻中 (Veith et al., 1989)比較中發現推斷在代謝活性的過程中，2-propyn-1-ol

產生的未飽和之醛類化合物，而此化合物為 Michael-addition reaction

而所有回歸的點再去除掉2-propyn-1-ol 這個 outlier 之後，其回歸的圖形也都落在 95%信賴區間附近(Fig 5.11)，說明了此一級炔丙基醇類

Fig 5.12 一級炔丙基醇去除掉 2-propyn-1-ol Log p 與 Log(1/EC50)之關

係圖(based on Final Yield)

模式回歸分析法亦為判定準確性之一種方法，即將模式之預測值與實驗值進行回歸分析，當回歸公式之斜率愈接近於 1 且截距愈接近於 0 時，其模式之準確性也相對愈高。由Fig 5.12 可知在本研究中，以三種參數回歸之模式其斜率皆接近於 1，且截矩接近 0，由此可知以此反應

y = 1.0044x + 0.0063

Fig 5.13 log(1/EC50) calc. V.S. log (1/EC50)exp. (based on Final Yield)

在其他QSAR的參數選擇方面，雖然可以參考的數據有限，我們利

第六章結論與建議 6.1 結論

本研究為針對 16 種炔丙基醇進行密閉式之藻類毒性試驗，其所得之結論如下:

1. 三個終點參數的敏感度比較， Final yield，依序為∆DO 與 Growth Rate。

2. 炔基接在 2 的位置(Primary propargylic alcohols)毒性比炔基接在 3 的位置(Primary homopropargylic alcohols)毒性要高，大約相差 1.1~7.4 倍左右。

3. 毒性最高的化合物為 2-decyn-1-ol，由結果可顯示隨著碳數越高，取代基分子量越大，毒性會增強。

4. 2-propyn-1-ol 雖然在測試物種中碳數最低( 3 個碳) ，分子量最小 (56.06) ，但也具有相對高的毒性，原因可能和其代謝活性過程的機制有關

。

5. 利用ANOVA(analysis of variance)檢定三項重要參數：Sw、F ration以及中斷值(cut-off value)，發現對於本研究的毒性物質而言，NOEC值比EC10能提供更佳的保護標準。

6. 在 cut of value 的比較上，發現∆DO 的變異性在三個參數來說是最高的，而Final Yield 和 Growth rate 敏感度及變異性，都很低，具有很高的再現性，由於每個參數都有其優點所在，故在本實驗中所選用的反應終點都是很好的參考標準。

7. 以物種比較方面看來，敏感度以魚類較佳，其次為月芽藻，較差的為纖毛蟲，而造成的原因可能是此一級炔丙基醇類對於魚類的選擇性較高，其在體內代謝活性酵素與之反應較為激烈所造成的結果。

8. QSAR之研究方面，利用簡單的Log P對 16 個一級炔丙基醇做回歸，

以結果看來，以Final Yield的回歸結果最好(r² = 0.83)，其次為∆DO (r² = 0.76)和Growth Rate (r² = 0.67)，將殘值較高的 2-propyn-1-ol當作 outlier去除之後，大幅提升回歸的效果r² value最高可達 0.9。

9. 過去針對此類化合物機制的研究，將一級炔丙基醇類分類於具前親電性機制(proelectrophile mechanism)的反應性物質，從 QSAR 的分析及與藻類基線毒性的比較結果看來，低分子量的Primary propargylic alcohols 與 Log p 相關性較不佳，可能的原因和其 Michael-addition 反應的效應較為明顯有關，而其餘的一級炔丙基醇則是與Log p 有不錯的相關性。

10. 針對此一級炔丙基醇類所建立的QSAR模式，r² value達 0.9 以上、斜率接近1 及截距趨近 0，說明此模式有不錯的預測性。

11. 在急慢毒性數據的研究方面，建議 ACR ratio 為 20 做為保護水體的指標。

12. 針對一級炔丙基醇類，將本研究結果納入藻類毒性資料庫中，以供此類化合物對於水體生物毒性分析的參考依據。

6.2 建議

1. 由於對於此類一級炔丙基醇的水體生物的毒性數據很稀少，在未來的研究方面應從其他物種進行試驗，以增加對於替代物種的研究。

2. 增加更多的物化參數及電子性參數進一步提高 QSAR 模式建立的準確性。

3. 針對此類化合物對於藻類特殊的作用機制更加深入的探討。

4. 加強對於一級炔丙基醇類慢毒性的影響之研究。

5. 進一步討論對於此類一級炔丙基醇在作用途徑中產生的中間產物及其造成的毒性影響。

6. 增加對於此類化合物生物降解性的研究。

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附錄一:原始數據

Primary propargylic alcohols

實驗毒物 : 2-propyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.22 8.94 328600 7.72 1.54340 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 2-butyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.7 7.93 274800 6.23 1.45400 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 2-butyn-1,4-diol 初始細胞密度

(cells/mL) : 15000

MCV (μm3) : 39.2 D (μm) : Initial pH : 7.46 EDTA(%) : 0 T(℃) : 24.1 Test duration : 48-h

Conc Initial DO Final DO Final cells Delta DO IR IR IR

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative

( growth rate) (Biomass) (DO)

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative

( growth rate) (Biomass) (DO)

Control 0.8 8.42 318300 7.62 1.52747 1 0 0 0

2.41 0.98 8.21 316700 7.23 1.52495 0.99835 0.00165 0.00528 0.05118 4.83 0.87 7.55 305300 6.68 1.50662 0.98635 0.01365 0.04286 0.12336 9.66 1.11 8.72 278300 7.61 1.46032 0.95604 0.04396 0.13188 0.00131 19.32 1.02 7.74 242200 6.72 1.39086 0.91056 0.08944 0.25091 0.11811 38.63 1.28 6.64 184700 5.36 1.25534 0.82184 0.17816 0.44049 0.29659 77.27 1.43 5.49 109400 4.06 0.99348 0.65041 0.34959 0.68876 0.46719 154.53 1.94 3.02 43900 1.08 0.53693 0.35152 0.64848 0.90471 0.85827

Control 1.04 8.44 318500 7.4 1.52779 1 0 0 0

2.41 1.13 8.19 316900 7.06 1.52527 0.99835 0.00165 0.00527 0.04595 4.83 1.03 7.86 304200 6.83 1.50482 0.98497 0.01503 0.04712 0.07703 9.66 1.26 8.2 278900 6.94 1.46140 0.95655 0.04345 0.13048 0.06216 19.32 1.12 7.77 241300 6.65 1.38900 0.90916 0.09084 0.25437 0.10135 38.63 1.44 6.79 183900 5.35 1.25317 0.82025 0.17975 0.44349 0.27703 77.27 1.41 5.52 109900 4.11 0.99576 0.65177 0.34823 0.68731 0.44459 154.53 1.95 3.16 45400 1.21 0.55373 0.36244 0.63756 0.89984 0.83649

Control 1.02 8.39 317900 7.37 1.52684 1 0 0 0

2.41 1.11 8.16 316400 7.05 1.52448 0.99845 0.00155 0.00495 0.04342 4.83 1.05 7.79 304800 6.74 1.50580 0.98622 0.01378 0.04325 0.08548 9.66 1.24 8.14 280300 6.9 1.46391 0.95878 0.04122 0.12413 0.06377 19.32 1.18 7.68 242600 6.5 1.39168 0.91148 0.08852 0.24860 0.11805 38.63 1.39 6.76 183200 5.37 1.25126 0.81951 0.18049 0.44470 0.27137 77.27 1.42 5.48 108700 4.06 0.99027 0.64857 0.35143 0.69066 0.44912 154.53 1.92 3.09 44600 1.17 0.54484 0.35684 0.64316 0.90228 0.84125

Control 0.95333 8.41667 318233.33 7.46333 1.52737 1 0 0 0

2.41 1.07333 8.18667 316666.67 7.11333 1.52490 0.99838 0.00162 0.00517 0.04690 4.83 0.98333 7.73333 304766.67 6.75000 1.50575 0.98585 0.01415 0.04441 0.09558 9.66 1.20333 8.35333 279166.67 7.15000 1.46188 0.95712 0.04288 0.12883 0.04198 19.32 1.10667 7.73000 242033.33 6.62333 1.39051 0.91040 0.08960 0.25129 0.11255 38.63 1.37000 6.73000 183933.33 5.36000 1.25326 0.82054 0.17946 0.44289 0.28182 77.27 1.42000 5.49667 109333.33 4.07667 0.99317 0.65025 0.34975 0.68891 0.45377 154.53 1.93667 3.09000 44633.33 1.15333 0.54517 0.35693 0.64304 0.90228 0.84547

IR : Inhibition rate

實驗毒物 : 2-hexyn-1-ol 初始細胞密度

(cells/mL) : 15000

MCV (μm3) : 40.7 D (μm) : Initial pH : 7.54 EDTA(%) : 0

T(℃) : 23.7 Test duration : 48-h

Conc Initial DO Final DO Final cells Delta DO IR IR IR

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative

( growth rate) (Biomass) (DO)

Control 1.27 6.54 271300 5.27 1.44759 1 0 0 0

2.43 1.23 6.43 270500 5.2 1.44611 0.99898 0.00102 0.00312 0.01328 4.86 1.24 6.34 249000 5.1 1.40470 0.97037 0.02963 0.08701 0.03226 9.72 1.31 6.28 215600 4.97 1.33269 0.92063 0.07937 0.21732 0.05693 19.43 1.33 5.75 186600 4.42 1.26046 0.87073 0.12927 0.33047 0.16129 38.87 1.34 5.15 129500 3.81 1.07782 0.74456 0.25544 0.55326 0.27704 77.73 1.56 4.06 86000 2.5 0.87315 0.60317 0.39683 0.72298 0.52562 155.47 2.41 3.23 31100 0.82 0.36458 0.25185 0.74815 0.93718 0.84440

Control 1.22 6.52 271600 5.3 1.44814 1 0 0 0

2.43 1.24 6.39 270300 5.15 1.44574 0.99834 0.00166 0.00507 0.02830 4.86 1.25 6.32 248600 5.07 1.40390 0.96945 0.03055 0.08963 0.04340 9.72 1.27 6.25 214700 4.98 1.33060 0.91883 0.08117 0.22175 0.06038 19.43 1.32 5.66 187200 4.34 1.26206 0.87151 0.12849 0.32892 0.18113 38.87 1.34 5.22 129800 3.88 1.07897 0.74507 0.25493 0.55261 0.26792 77.73 1.48 4.12 85400 2.64 0.86965 0.60053 0.39947 0.72564 0.50189 155.47 2.39 3.18 31600 0.79 0.37255 0.25726 0.74274 0.93531 0.85094

Control 1.2 6.53 271400 5.33 1.44777 1 0 0 0

2.43 1.24 6.4 270400 5.16 1.44593 0.99873 0.00127 0.00390 0.03189 4.86 1.31 6.34 248100 5.03 1.40289 0.96900 0.03100 0.09087 0.05629 9.72 1.3 6.23 215900 4.93 1.33338 0.92099 0.07901 0.21646 0.07505 19.43 1.27 5.78 185300 4.51 1.25696 0.86821 0.13179 0.33580 0.15385 38.87 1.35 5.19 128900 3.84 1.07549 0.74286 0.25714 0.55577 0.27955 77.73 1.49 4.17 85800 2.68 0.87198 0.60229 0.39771 0.72387 0.49719 155.47 2.38 3.21 30900 0.83 0.36135 0.24959 0.75041 0.93799 0.84428

Control 1.23000 6.53000 271433.33 5.30000 1.44783 1 0 0 0

2.43 1.23667 6.40667 270400.00 5.17000 1.44593 0.99868 0.00132 0.00403 0.02453 4.86 1.26667 6.33333 248566.67 5.06667 1.40383 0.96961 0.03039 0.08917 0.04403 9.72 1.29333 6.25333 215400.00 4.96000 1.33222 0.92015 0.07985 0.21851 0.06415 19.43 1.30667 5.73000 186366.67 4.42333 1.25983 0.87015 0.12985 0.33173 0.16541 38.87 1.34333 5.18667 129400.00 3.84333 1.07743 0.74417 0.25583 0.55388 0.27484 77.73 1.51000 4.11667 85733.33 2.60667 0.87159 0.60200 0.39800 0.72416 0.50818 155.47 2.39333 3.20667 31200.00 0.81333 0.36616 0.25290 0.74708 0.93683 0.84654

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 2-heptyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.15 5.23 253400 4.08 1.41346 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 2-octyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 2.21 7.01 232900 4.8 1.37128 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

初始細胞密度

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.09 8.31 290600 7.22 1.48195 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 2-decyn-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.26 8.86 263600 7.6 1.43319 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

Primary homopropargylic alcohols

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.37 8.72 302600 7.35 1.50218 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 3-pentyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.65 6.09 257800 4.44 1.42207 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 3-hexyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.43 5.59 235400 4.16 1.37662 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 3-heptyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.58 9.43 281200 7.85 1.46551 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 3-octyn-1-ol 初始細胞密度

(cells/mL) : 15000

MCV (μm3) : 43.3 D (μm) : Initial pH : 7.55 EDTA(%) : 0 T(℃) : 23.2 Test duration : 48-h

Conc Initial DO Final DO Final cells Delta DO IR IR IR

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative

( growth rate) (Biomass) (DO)

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 3-nonyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.09 9.81 403100 8.72 1.64557 1 0 0 0

IR : Inhibition rate

Biomass : Yield f (Final yield based on cell density)

實驗毒物 : 3-decyn-1-ol

mg/L mg/L mg/L cells/ml mg/L μspecific μrelative ( growth rate) (Biomass) (DO)

Control 1.22 9.29 345200 8.07 1.56804 1 0 0 0

IR : Inhibition rate

附錄二:劑量反應曲線圖

Primary propargylic alcohols

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.00 2-propyn-1-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 1 The Dose-response Curve of 2-propyn-1-ol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.00 2-butyn-1-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 2 The Dose-response Curve of 2-butyn-1-ol

0.00 0.00 0.10 10.00 1000.00 100000.00

2-butyn-1,4-diol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 3 The Dose-response Curve of 2-butyn-1,4-diol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.0 0

Final Yield. data Growth Rate. data

Fig 4 The Dose-response Curve of 2-pentyn-1-ol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.0 0

Final Yield. data Growth Rate. data

Fig 5 The Dose-response Curve of 2-hexyn-1-ol

0.00 0.00 0.10 10.00 1000.00 100000.00

2-heptyn-1-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 6 The Dose-response Curve of 2-heptyn-1-ol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.0 0

Final Yield. data Growth Rate. data

Fig 7 The Dose-response Curve of 2-octyn-1-ol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.0 0

Final Yield. data Growth Rate. data

Fig 8 The Dose-response Curve of 2-nonyn-1-ol

0.00 0.20 0.40 0.60 0.80 1.00 1.20

0.00 0.01 0.10 1.00 10.00 100.00 1000.00 2-decyn-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 9 The Dose-response Curve of 2-decyn-1-ol

Primary homopropargylic alcohols

0.00 0.10 10.00 1000.00 100000.00 10000000.

00 3-butyn-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 10 The Dose-response Curve of 3-butyn-1-ol

0.00 0.00 0.10 10.00 1000.00 100000.00

3-pentyn-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 11 The Dose-response Curve of 3-pentyn-1-ol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.0 0

Final Yield. data Growth Rate. data

Fig 12 The Dose-response Curve of 3-hexyn-1-ol

0.00 0.00 0.10 10.00 1000.00 100000.00

3-heptyn-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data Growth Rate. data

Fig 13 The Dose-response Curve of 3-heptyn-1-ol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.0 0

Final Yield. data Growth Rate. data

Fig 14 The Dose-response Curve of 3-octyn-1-ol

0.00 0.00 0.01 0.10 1.00 10.00 100.00 1000.00 3-nonyn-1-ol conc. (mg/L)

Inhibition Rate

DO. data

Final Yield. data

Growth Rate. data

0.00 0.20 0.40 0.60 0.80 1.00 1.20

0.00 0.01 0.10 1.00 10.00 100.00 3-decyn-1-ol conc. (mg/L)

Inhi bi tion R at e

DO. data

Final Yield. data Growth Rate. data

Fig 16 The Dose-response Curve of 3-decyn-1-ol

在文檔中以密閉式藻類毒性試驗方法評估一級炔丙基醇類之毒性 (頁 120-0)

第五章 實驗結果與討論

5.6 QSAR分析

Fig 5.10 一級炔丙基醇 Log p 與 Log(1/EC50)之關係圖(based on Final Yield)

Fig 5.11 一級炔丙基醇 Log p 與 Log(1/EC50)之關係圖(based on Growth Rate)

Fig 5.12 一級炔丙基醇去除掉 2-propyn-1-ol Log p 與 Log(1/EC50)之關

Fig 5.13 log(1/EC50) calc. V.S. log (1/EC50)exp. (based on Final Yield)

第六章 結論與建議 6.1 結論

。

6.2 建議

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第五章實驗結果與討論

第六章結論與建議 6.1 結論