臺灣博碩士論文加值系統

第一部份：
累積之研究顯示(+)-pinitol具有抗黴菌、抗昆蟲攝食、抑制昆蟲生長及對白化症的老鼠具有降血糖及抗糖尿病之活性。最近的研究發現，pinitol可與錳螯合形成一個pH 2.0具有胰島素作用之中間活性物，這些活性指出(+)-pinitol具有發展潛力，由於pinitol廣泛存在於各類植物中，特別以豆科最常見，本論文的第一部份擬自大豆葉建立一套簡易分離(+)-pinitol之方法，以期大量分離作為生物活性之研究。
乾燥大豆葉之甲醇抽提物，劃分為正丁醇和水可溶兩部分，水可溶部分經由活性碳管柱分離，可得到含大量pinitol的粗萃取物，此部份經由乙醯化後，再藉由快速矽膠管柱層析及一般層析分離得到pentaacetylpinitol及2,3-diacetyl-2-methylbutyrolactone。Pentaacetyl- pinitol在鹼性下水解即得到(+)-pinitol (2)。因此本研究之分離方法在人力需求與相關的運用，皆有更佳的經濟效益並容易執行。這些化合物的結構鑑定可由光譜解析。
第二部份：
本實驗是針對植物中所含pinitol進行定性與定量研究，所使用之分析方法為對碳水化合物具高效率及高靈敏度(達picomole)之脈衝式安培偵測陰離子交換法(High Performance anion exchange and pulsed amperometric detector)。
將待測植物以熱水抽提，劃分為正丁醇與水可溶兩部分，水可溶部分通過活性碳管柱，得到含pinitol的粗萃取物，以醣類分析儀做pinitol檢量線後，再測定這些植物中pinitol的含量，以pinitol做外標準品，使用的分析條件為：陰離子交換管柱－CarboPac MA1 4 × 250mm；偵測器－積分和脈衝式安培偵測器；移動相－0.45N氫氧化鈉；滯留時間－8.47分鐘。研究結果顯示大豆葉、二葉松、華山松、五葉松pinitol含量分別為1.72﹪w/w(乾重)、1.12﹪、0.77﹪、0.50 w/w (新鮮葉部重)。

Part Ⅰ：
Cumulative studies indicate that (+)-pinitol possesses anti-fungal, insect anti-feeding and anti-growth, and significant hypoglycemic and antidiabetic in diabetic albino mice. Its activity could be potentiated by manganese ion (Mn2+) through a chelation mechanism. These activities reveal the potential of (+)-pinitol. Pinitol widely distributed in various plants, especially common in Leguminosae. In this study, we tried to develop a simple separation method for (+)-pinitol from soybean leaves to provide large quantity for further biological study and chemical modification.
The methanolic extract of the dry soybean leaves was divided into n-butanol- and water - soluble fractions by liquid-liquid partitioning process. The water layer was passed through charcoal columns eluted with water. The pinitol major fraction after peracetylation was chromatographed on a flash column to give pentaacetylpinitol and 2,3-diacetyl-2-methylbutyrolactone. Hydrolysis of pentaacetylpinitol under basic conditions yielded (+)-pinitol (2). The developed method is facile since it’s more economic both in manpower and regards used.
Part Ⅱ：
This part of study was aimed to analyze the content of pinitol in plants both qualitatively and quantitatively via an improved carbohydrate chromatographic technique known as High Performance Anion Exchange (HPAE) and Pulsed Amperometric Detection (PAD), which possesses high separation efficiency and high sensitivity (picomole level) toward carbohydrates.
The hot water extract of plant materials was divided into n-butanol- and water-soluble fractions. The water-soluble fraction was fractionated through a charcoal column to give a pinitol containing fraction. The pinitol content of these plants was determined by calibration curve which was established by using sugar chromatography (anion-exchange column: CarboPac MA1, 4×250 mm, detection: Integrated and pulsed amperometric detector, mobile phase: 0.45 N NaOH, flow rate: 0.4 ml/min, retention time: 8.47 min) with is (+)-pinitol external standard. The experimental results indicated that the pinitol contents of the leaves of soybean, Pinus taiwanensis, Pinus armandii and Pinus morrisonicola is 1.72% w/w (dry weight), 1.12%, 0.77% and 0.50% w/w (weight of fresh leaves), respectively.

總目錄
中文摘要i
英文摘要iii
總目錄v
表目錄(List of Tables)viii
圖目錄(List of Figures)ix
流程圖目錄(List of Schemes)xi
第一部分：自大豆葉分離(+)-pinitol之方法研究1
壹、植物簡介與研究目的1
1.1 植物簡介1
1.2 研究目的3
1.3 Pinitol 之相關研究3
貳、實驗結果與討論9
2.1 結 果9
2.1.1Pentaacetylpinitol (1)10
2.1.2 (+)-Pinitol (2)12
2.1.3 2,3-Diacetyl-2-methylbutyrolactone (3)14
2.3討論18
參、實驗部分20
3.1 儀器與器材20
3.1.1 理化性質測定儀器20
3.1.2 試劑、材料及溶劑20
3.1.3 薄層層析展開溶媒系統21
3.2 抽取與分離21
3.2.1 (+)-Pinitol之分離與製備21
3.2.2 2,3-Diacetyl-2-methylbutyrolactone之分離與製備22
第二部分：大豆葉及松科屬植物所含(+)-pinitol成分之定量研究27
壹、緒論與研究目的27
1.1 松屬植物簡介27
1.2 緒論31
1.3 研究目的33
1.4 原理33
貳、實驗部分36
2.1 儀器與器材36
2.1.1 理化性質測定儀器36
2.1.2 試劑、材料及溶劑36
2.1.3 薄層層析分析系統36
2.1.4 醣類分析儀37
2.2 (+)-Pinitol原料溶液(stock solution)與各濃度標準溶液之製備38
2.2.1 (+)-Pinitol原料溶液之製備38
2.2.2 各濃度 (+)-pinitol標準溶液的製備38
2.3 (+)-Pinitol之醣類分析儀定性分析38
2.4 檢量線之製作38
2.5 大豆葉(+)-pinitol成份之定量39
2.5.1 大豆葉抽提物含(+)-pinitol檢品之製備39
2.5.2 大豆葉樣品之配製39
2.6 五葉松(+)-pinitol成份之定量39
2.6.1 五葉松抽提物含(+)-pinitol檢品之製備39
2.6.2 五葉松樣品之配製40
2.7 二葉松(+)-pinitol成份之定量40
2.7.1 二葉松抽提物含(+)-pinitol檢品之製備40
2.7.2 二葉松樣品之配製40
2.8 華山松(+)-pinitol成份之定量40
2.8.1 華山松抽提物含(+)-pinitol檢品之製備40
2.8.2 華山松樣品之配製41
2.9 準確度測試41
2.10 大豆葉渣(+)-pinitol成分之定性分析41
2.10.1 大豆葉渣(+)-pinitol成分之製備41
2.10.2 大豆葉渣抽提物樣品之配置42
2.11 大豆葉中其他成分之定性42
2.11.1 各種醣類標準溶液之配製42
參、實驗結果與討論44
3.1 結 果44
3.2 討 論46
3.2.1 大豆葉中存在之蔗糖46
3.2.2 氫氧化鈉濃度與pinitol及其它醣(醇)層析滯留時間之關係46
3.2.3 氫氧化鈉濃度與pinitol及其它醣(醇)層析解析度之關係46
3.3 結論47
參考文獻 (References)48
附圖 (Spectra Appendies)52
表目錄 (List of Tables)
Table 1. Antigrowth activity of soybean extracts and
constituents7
Table 2. 1H and 13C NMR data of Pentaacetylpinitol (1)(CDCl3,
1H-NMR 400 MHz; CDCl3, 13C-NMR 50MHz)11
Table 3.1H and 13C NMR data of (+)-Pinitol (2) (D2O, 1H-NMR
400 MHz; D2O+CD3OD, 13C-NMR 50MHz)13
Table 4.1H and 13C NMR data of 2,3-Diacetyl-2-
methylbutyrolactone
(3) (CDCl3, 1H-NMR 200MHz；CDCl3, 13C-NMR 50MHz)15
Table 5.Comparison of physical properties of compound 3 and
(-)-2-C-methyl-D-erythrono-1,4-lactone16
Table 6.pka value of some common carbohydrates (in water at
25℃)34
Table 7. The calibration curve of pinitol in five different
weight67
Table 8. The pinitol content in the leaves of soybean, Pinus
taiwanensis, Pinus armandii and Pinus morrisonicola.67
Table 9.The pinitol content of crude sample plants (w/w)67
Table 10. Accuracy tests for (+)-pinitol assay in the Pinus
taiwanensis68
Table 11.Accuracy tests for (+)-pinitol assay in the Pinus
morrisonicola68
Table 12. Accuracy tests for (+)-pinitol assay in the Pinus
armandii 68
Table 13. Accuracy tests for the pinitol assay in of soybean
leaves68
Table 14.Carbohydrate contents in the leaves of soybean and
various Pinus species.69
圖目錄 (List of Figures)
Fig. 1.Glycine max Merrill2
Fig. 2.Biogenesis of the optically active inositols and some
inositolmethyl ethers4
Fig. 3.Effects of plasma glucose of NaCl, 3-O-methyl-D-chiro-
inositol associated with manganese6
Fig. 4.Structure of compound (1)-- 10
Fig. 5.Structure of compound (2)12
Fig. 6.Structure of compound (3)14
Fig. 7.(-)-2-C-methyl-D-erythrono-1,4-lactone16
Fig. 8.Mass spectral fragmentation of 2,3 -diacetyl-2-methyl- butyrolactone (3)17
Fig. 9.1H-NMR spectrum of pentaacetylpinitol (1)
(CDCl3, 400 MHz)52
Fig.10.13C-NMR and DEPT-135 spectra of pentaacetylpinitol
(1)(CDCl3, 50 MHz)53
Fig. 11.1H-NMR spectrum of pinitol (2) (D2O, 400 MHz)54
Fig. 12.13C-NMR and DEPT-135 spectra of pinitol (2)
(D2O+CD3OD, 50 MHz)55
Fig. 13.1H-NMR spectrum of 2,3-Diacetyl-2-methylbutyrolactone
(3)(CDCl3, 200 MHZ)56
Fig. 14.13C-NMR and DEPT-135 spectra of 2,3 - Diacetyl -2-
methylbutyrolactone (3) (CDCl3, 200 MHz)57
Fig. 15.EIMS spectrum of 2,3-Diacetyl-2-methylbutyrolactone
(3)(20e V)58
Fig. 16.Pinus morrisonicola Hayata (五葉松)28
Fig. 17.Pinus taiwanensis Hayata (二葉松)29
Fig. 18.Pinus armandii Franchet var. masteriana Hayata (華山
松)30
Fig. 19.Pellicular anion exchange resin bead34
Fig. 20.Chromatogram of sugar analysis by HPAE-PAD method34
Fig. 21.Diagram of the triple-pulse potential waveform for
carbohydrate detection35
Fig. 22.HPAEC-PAD chromatogram of the (+)-pinitol59
Fig. 23.HPAEC-PAD chromatogram of the soybean leaves60
Fig. 24.HPAEC-PAD chromatogram of the Pinus armandii61
Fig. 25.HPAEC-PAD chromatogram of the Pinus morrisonicola62
Fig. 26.HPAEC-PAD chromatogram of the Pinus tawanensisi63
Fig. 27.Calibration curve for five different concentration of
pinitol standard solution64
Fig. 28.HPAEC-PAD chromatogram of the 7 standard compounds
peak65
Fig. 29.HPAEC-PAD chromatogram of the water soluble fraction
of the residue of soybean leaves after MeOH extraction66
流程圖目錄 (List of Schemes)
Scheme 1.Fractionation procedure of the MeOH-extract of
soybean leaves21
Scheme 2.Separation of compound (1), (2) and (3) from the
water-soluble fraction22
Scheme 3.Isolation and preparation of pinitol from Pinus
Morrisonicola40

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