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Isolation and Characterization Procedure for Indole Alkaloids from the Marquesan Plant Rauvolfia Nukuhivensis
马克萨斯植物萝芙木中吲哚生物碱的分离和特性描述步骤   

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Abstract

A plethora of natural products, mostly secondary metabolites, are isolated and purified from many different organisms, like plants, fungi, algae, marine invertebrates, etc. The extraction procedure is specific to each organism, but some guidelines are usually followed for any purification procedure regarding targeted metabolites, such as alkaloids. Alkaloids are secondary metabolites that contain basic nitrogen in their structures and they are often associated with interesting biological properties especially in pharmacology field. This protocol describes the isolation procedure of indole alkaloids from Rauvolfia nukuhivensis directly from the ethanol extract of the plant material yielding different skeleton-type compounds including non-basic derivatives (ajmaline, sarpagine, macroline and β-carboline). The procedure details the guidelines and the steps to characterize new or known isolated compounds, beginning from the plant collection to the molecule level with the use of spectroscopic techniques (NMR, MS, UV). We detailed the extraction and fractionation procedures followed by the purification of compounds, as well as their physico-chemical characterizations. The procedure is illustrated by the example of the purification of a large array of indole alkaloids from the bark of Rauvolfia nukuhivensis.

Materials and Reagents

  1. Rauvolfia nukuhivensis (Voucher JFB 2808): In our example, the bark of Rauvolfia nukuhivensis was collected at Maauu in “Terre Déserte” area on Nuku Hiva Island, Marquesas archipelago, French Polynesia, at an altitude of 477 m and identified by Dr Jean-François Butaud. A voucher specimen (JFB 2808) has been deposited at the Herbarium of French Polynesia. The sample was dried after collection at 30 °C using an air dryer device and stored in the room until grinding and extraction.
  2. Ethanol (95%) (Thermo Fisher Scientific, Fisher chemical)
  3. Methanol (HPLC grade from Fisher chemical)
  4. Dichloromethane (HPLC grade from Fisher chemical)
  5. Cyclohexane (HPLC grade from Fisher chemical)
  6. Ethyl acetate (HPLC grade from Fisher chemical)

Equipment

  1. SEB Prep'line grinder (SEB)
  2. Semi-preparative HPLC column Luna 5 μm C18 (50 mm x 10 mm) (Phenomenex, model: RP-C18 )
  3. Vacuum liquid chromatography (Buchner filter from Buchi, chromatography performed with vacuum pump) (BÜCHI Labortechnik AG)
  4. High Pressure Liquid Chromatography (HPLC) Waters 600 system equipped with a Waters 717 Plus autosampler, a Waters 998 photodiode array detector (WATERS, model: Waters 998 photodiode array detector ), and a Sedex 75 evaporative light-scattering detector (SEDERE, model: Sedex 75)
  5. NMR spectrometer (Bruker Corporation, model: Bruker Avance 500 MHz )
  6. Polarimeter (jascoinc, model: Jasco P-2000 )
  7. Spectropolarimeter (jascoinc, model: Jasco J-810 )
  8. Mass spectrometer (Bruker Corporation, model: Bruker ESQUIRE 3000 plus )
  9. Thermo Finnigan LTQ Orbitrap mass spectrometer (Thermo Fisher Scientific)

Procedure

  1. Sample collection
    The isolation procedure starts with the collection of the sample which is identified by a botanist and certified with the record of a voucher specimen. In our example, the bark of Rauvolfia nukuhivensis was collected at Maauu in “Terre Déserte” area on Nuku Hiva Island, Marquesas archipelago, French Polynesia, at an altitude of 477 m and identified by Dr Jean-François Butaud. A voucher specimen (JFB 2808) has been deposited at the Herbarium of French Polynesia. The sample was dried after collection at 30 °C using an air dryer device and stored in the room until grinding and extraction.

  2. Extraction and isolation
    Prior to extraction, batches of 50 g of bark pieces were ground into powder during at least 5 periods of 1 min repeated grinding with the rotor setting at 10,000 rpm. In order to avoid overheating of the grinder, the system was covered with plastic and cooled in ice between each grinding process.
    The procedure can be divided into three steps, the extraction process, one or more fractionation steps, according to the chemical complexity of the crude extract, and finally the isolation process. For our samples, two successive fractionations were performed, with a range of solvents described in Tables 1 and 2.


    Figure 1. Bark of Rauvolfia nukuhivensis after and before the grinding process

    Table 1. Solvent composition of the first fractionation
    Fraction
    Solvent
    Proportion
    FA
    Water
    -
    FB
    Water/Methanol
    1:1
    FC
    Methanol
    -
    FD
    Methanol/Dichloromethane
    1:1
    FE
    Dichloromethane
    -

    1. For our example of the plant Rauvolfia nukuhivensis, its dried bark (250 g) was ground by industrial equipment.
    2. The dried ground bark was macerated in 750 ml of ethanol during 16 h at room temperature and then the solvent was filtered (0.45 µm) and concentrated by evaporation to yield a crude oil.
    3. The resulting extract (21 g) was dissolved by sonication for 10 min in a mixture of 200 ml methanol/dichloromethane (1:1) and then filtered.
    4. The fractionation was carried out by vacuum liquid chromatography on RP-C18 column and eluted with solvents of decreasing polarity as shown in Table 1.
    5. Fraction FC provided 8.43 g of an oily residue and showed the presence of indole alkaloids by HPLC-DAD.
    6. Consequently, a portion of fraction FC (600 mg) was further fractionated by normal phase (diol) flash column chromatography using solvents of increasing polarity as shown in Table 2.

      Table 2. Solvent composition of the second fractionation
      Sub-fraction
      Solvent
      Proportion
      f1
      Cyclohexane
      -
      f2
      Cyclohexane/Ethyl acetate
      3:1
      f3
      Cyclohexane/Ethyl acetate
      1:1
      f4
      Cyclohexane/Ethyl acetate
      1:3
      f5
      Ethyl acetate
      -
      f6
      Ethyl acetate/Methanol
      3:1
      f7
      Ethyl acetate/Methanol
      1:1
      f8
      Ethyl acetate/Methanol
      1:3
      f9
      Methanol
      -
      f10
      Methanol/Dichloromethane
      1:1
    7. Each obtained fraction contains a lower compound diversity than the extract and thus facilitates the isolation and purification procedure.
    8. Fraction FCf7 was selected for final purification on HPLC mainly because of its weight but also the high chemical diversity.
    9. Purification of FCf7 (50.2 mg) major compounds was performed by RP-C18 semi-preparative HPLC (Phenomenex, Luna 5 μm C18, 250 mm x 10 mm) with a gradient of water/methanol/trifluoro acetic acid (from 70:30:0.1 to 30:70:0.1, flow 3 ml/min) to afford, after a drying step under nitrogen gas flow, pure compound 1 (4.7 mg), 2 (2.8 mg) 3 (1.83 mg), 4 (2.17 mg), 5 (3.25 mg), 6 (3.24 mg), 7 (1.23 mg), 8 (6.43 mg), 9 (1.82 mg), 10 (1.71 mg), 11 (1.39 mg), 12 (2.00 mg) and 13 (2.20 mg).

  3. Characterization
    The last step of the procedure involves different spectral and chromatographic techniques for a full description of the purified compound. To date, Nuclear Magnetic Resonance (NMR) spectroscopy is the most common technique to elucidate the structure of a compound. However, other techniques are needed to along with NMR for a better characterization, including high-resolution mass spectroscopy, UV spectra, optical rotation, infrared spectra, and circular dichroism if needed. NMR spectroscopy is frequently used in two major ways, 1D and 2D. For a better understanding of NMR spectroscopy see for example Keeler (2011); Lewitt (2001) and Fan (1996).
    In our case, we chose to study and describe several physicochemical characteristics of each compound, listed here: UV-Vis spectra were recorded by HPLC-DAD. NMR spectra were measured on a Bruker Avance 500 MHz spectrometer with pulsed field gradient and signals were referenced to the residual solvent signals (CD3OD, at δH 3.31 and δC 49.0 ppm). For this step, each was diluted in 700 µl of CD3OD. Electrospray Ionization Mass spectrometry (ESIMS) and High Resolution ESIMS (HRESIMS) data were measured with a Bruker ESQUIRE 3000 plus mass spectrometer and a Thermo Finnigan LTQ Orbitrap mass spectrometer, respectively. HPLC purification was carried out on a Waters 600 system equipped with a Waters 717 Plus autosampler, a Waters 998 photodiode array detector, and a Sedex 75 evaporative light-scattering detector (Sedere, France). Optical rotation and circular dichroism were measured on a Jasco P-2000 polarimeter and a Jasco J-810 spectropolarimeter, respectively.
    The structures for some compounds have been reported in Martin et al. (2012) and Martin et al. (2015). Some examples are provided here (Figure 2). We have also provided NMR spectroscopic data summarized in Tables 3 and 4.


    Figure 2. Structure of compounds 1-13 isolated from the bark extract of Rauvolfia nukuhivensis. Nukuhivensium (1). Yellow-coloured amorphous powder; UV measured by HPLC/DAD (MeOH/H2O/TFA) λmax 223,6 ; 241,3 ; 289,9 ; 340,0 ; 381,7 nm; 1H and 13C NMR see Table 3; HRESIMS m/z 261.13953 [M]+ (calcd for C18H17N2+, 261.13917). N12-methylnukuhivensium (2). Yellow-coloured amorphous powder; UV measured by HPLC/DAD (MeOH/H2O/TFA) λmax 224,8 ; 236,6 ; 248,4 ; 286,7 ; 335,2 ; 392,6 nm; 1H and 13C NMR see Table 3; HRESIMS m/z 275.15417 [M]+ (calcd for C19H19N2+, 275.15482). Nortueiaoine (11): Light yellow amorphous powder; [α]20D - 1.1 (MeOH); 1H NMR and 13C NMR spectroscopic data, see Table 4; ESIMS m/z 343.1 [M+H]+ ; HRESIMS m/z 343.16611 [M+H]+ (calcd for C19H23N2O4+, 343.16578). Tueiaoine (12) Light yellow amorphous powder ; [α]20D + 1.2 (MeOH); 1H NMR and 13C NMR spectroscopic data, see Table 4; ESIMS m/z 357.2 [M+H]+ ; HRESIMS m/z 357.18289 [M+H]+ (calcd for C20H25N2O4+, 357.18143).

    Table 3. 1H (500 MHz) and 13C (125 MHz) NMR data for indoloquinolizinium derivatives 1 and 2 in CD3OD

    Nukuhivensium (1)
    N12-methylnukuhivensium (2)
    Position
    δC in ppm
    δH in ppm, mult. (J in Hz)
    δC in ppm
    δH in ppm, mult. (J in Hz)
    1
    120.5   
    8.65, s
    121.5
    8.96, s
    2
    154.4
    -
    154.1
    -
    3
    124.4
    7.83, d (7.0)
    123.9
    7.86, d (7.0)
    4
    137.6
    9.19, d (7.0)
    138.4
    9.24, d (7.0)
    6
    128.1
    8.84, d (7.0)
    128.1
    8.90, d (7.0)
    7
    117.2
    8.61, d (7.0)
    116.8
    8.66, d (7.0)
    7a
    125.0
    -
    125.6
    -
    7b
    122.5
     
    128.9

    8
    123.3
    8.36, d (8.0)
    122.8
    8.41, d (8.0)
    9
    122.9
    7.50, dd (8.0; 7.0)
    123.5
    7.54, dd (8.0, 7.0)
    10
    131.0
    7.74, dd (8.0; 7.0)
    131.2
    7.83, dd (8.0,7.0)   
    11
    113.6
    7.84, d (8.0)
    112.1
    7.95, d (8.0)
    11a
    143.3

    144.9

    12
     
     
    34.6
    4.58, s
    12a
    131.7, C
     
    131.4   

    12b
    134.3, C

    135.2   

    13
    38.6, CH2
    3.06, t (7.7)
    38.7
    3.09, t (7.7)   
    14
    23.9, CH2
    1.94, tq (7.7; 7.5)
    24.4
    1.93, tq (7.7, 7.5)
    15
    14.0, CH3
    1.12, t (7.5)
    14.0
    1.12, t (7.5)

    Table 4. 1H (500 MHz) and 13C (125 MHz) NMR data for macroline derivatives 11 and 12 in CD3OD

    Nortueiaoine (11)
    Tueiaoine (12)
    Position
    δC in ppm
    δH in ppm, mult. (J in Hz)
    δC in ppm
    δH in ppm, mult. (J in Hz)

    128.9
    -
    n.d.
    -
    3
    49.1
    4.89, br s
    47.8
    5.08, br s
    5
    50.9
    4.31, d (8.0)
    51.2
    4.26, d (7.5)

    25.5
    3.52, dd (17.5, 7.5)
    25.0
    3.50, dd (17.5, 7.5)

    2.99, d (17.5)

    7
    107.7
    -
    108.2
    -
    8
    127.0
    -
    127.2
    -
    9
    119.2
    7.51, d (8.0)
    119.8
    7.51, d (8.0)
    10
    120.8
    7.09, ddd (8.0, 7.0, 1.0)
    121.3
    7.09, br dd (8.0, 7.0)
    11
    123.7
    7.18, ddd (8.0, 7.0, 1.0)
    124.2
    7.25, br dd (8.0, 7.0)
    12
    112.5
    7.38, br d (8.0)
    110.9
    7.43, d (8.0)
    13
    138.1
    -
    139.8
    -
    14α
    28.4
    2.16, td (12.5, 3.0)
    28.3
    2.20, td (12.5, 3.0)
    14β
    2.02, br d (12.5)

    15
    30.2
    2.17, m
    30.6
    2.11, br t (11.5 Hz)
    16
    48.0
    3.08, br s
    49.3
    2.93, m
    17
    177.1
    -
    n.d.
    -
    18
    10.3
    0.66, t (7.5)
    10.7
    0.71, t (7.5)
    19
    23.1
    1.64, m
    22.9
    1.58, m
    1.38, m
    1.31, m
    20
    49.0
    2.42, br t (7.5)
    50.2
    2.41, m
    21
    177.9
    -
    n.d.
    -
    CH3-N1


    29.3
    3.73, s

Conclusion

Our protocol shows a significant improvement for the isolation of indole alkaloids. Indeed, with the classical extraction, the non-basic compounds would not have been extracted such as ionic compounds like nukuhivensiums. In a continuous work for improvements of structural, physical and chemical characteristics, additional NMR spectroscopy can be performed, in order to describe the relative configuration of some compounds, like NOESY experiments.
This protocol allowed the characterization of different structure of indole alkaloids different skeletons as well as stereoisomers (normal and iso compounds such as sandwicine and isosandwicine) and analogous series (normal and Nor- compounds such as sandwicine and Norsandwicine). Therefore, the method described here is more efficient and requires less time solvent consuming.

Acknowledgments

We would like to thank Fanglian He for inviting us to write this manuscript. The authors are grateful to Diren (Direction de l’Environnement de la Polynésie française) department for financial aid. This protocol have been written with the great help of Pr. Olivier P. Thomas, and the authors thank him for his work and advice.

References

  1. Fan, T. (1996). Metabolite profiling by one-and two-dimensional NMR analysis of complex mixtures. Prog Nucl Mag Res Sp 28(2): 161-219.
  2. Keeler, J. (2011). Understanding NMR spectroscopy. John Wiley & Sons.
  3. Lewitt, M. H. (2001). Spin dynamics: basics of nuclear magnetic resonance. John Wiley & Sons.
  4. Martin, N. J., Ferreiro, S. F., Barbault, F., Nicolas, M., Lecellier, G., Paetz, C., Gaysinski, M., Alonso, E., Thomas, O. P., Botana, L. M. and Raharivelomanana, P. (2015). Indole alkaloids from the Marquesan plant Rauvolfia nukuhivensis and their effects on ion channels. Phytochemistry 109: 84-95.
  5. Martin, N. J., Prado, S., Lecellier, G., Thomas, O. P. and Raharivelomanana, P. (2012). Nukuhivensiums, indolo[2,3-a]quinoliziniums from the Marquesan plant Rauvolfia nukuhivensis. Molecules 17(10): 12015-12022.

简介

从许多不同的生物体如植物,真菌,藻类,海洋无脊椎动物等分离和纯化了大量天然产物,主要是次级代谢物。提取程序对每个生物体是特异性的,但是关于靶向代谢物(例如生物碱)的任何纯化程序通常遵循一些指导。生物碱是在其结构中含有碱性氮的次级代谢物,并且它们通常与有趣的生物学性质相关,特别是在药理学领域。该方案描述了直接从植物材料的乙醇提取物中的来自真鞭毛虫的吲哚生物碱的分离过程,产生不同的骨架类化合物,包括非碱性衍生物(水杨酸,胭脂碱,大线和β-咔啉)。该程序详述了使用光谱技术(NMR,MS,UV)从植物收集到分子水平表征新的或已知的分离的化合物的指南和步骤。我们详细描述了化合物纯化之后的萃取和分馏方法,以及它们的物理化学表征。该程序通过从大白菜Rauvolfia nukuhivensis的树皮中纯化大量吲哚生物碱的实例来说明。

材料和试剂

  1. Rauvolfia nukuhivensis (Voucher JFB 2808):在我们的示例中, R auvolfia nukuhivensis的树皮是在Maauu"TerreDéserte" Nuku Hiva Island,Marquesas群岛,法属波利尼西亚,海拔477米,由Jean-Fran?oisJean Butaud博士确认。凭证样本(JFB 2808)已存放在法属波利尼西亚的植物标本馆。使用空气干燥装置在30℃下收集样品后干燥,并储存在室内直到研磨和提取
  2. 乙醇(95%)(Thermo Fisher Scientific,Fisher chemical)
  3. 甲醇(来自Fisher chemical的HPLC级)
  4. 二氯甲烷(HPLC化学品的HPLC级)
  5. 环己烷(来自Fisher chemical的HPLC级)
  6. 乙酸乙酯(来自Fisher chemical的HPLC级)

设备

  1. SEB预研磨机(SEB)
  2. 半制备型HPLC柱Luna 5μmC18(50mm x 10mm)(Phenomenex,型号:RP-C18)
  3. 真空液相色谱(Buchner过滤器,Buchi,用真空泵进行色谱)(BüCHILabortechnik AG)
  4. 高压液相色谱(HPLC)Waters 600系统,其配备有Waters 717 Plus自动进样器,Waters 998光电二极管阵列检测器(WATERS,型号:Waters 998光电二极管阵列检测器)和Sedex 75蒸发光散射检测器(SEDERE,型号: Sedex 75)
  5. NMR光谱仪(Bruker公司,型号:Bruker Avance 500MHz)
  6. 偏光计(jascoinc,型号:Jasco P-2000)
  7. 分光偏光计(jascoinc,型号:Jasco J-810)
  8. 质谱仪(Bruker Corporation,型号:Bruker ESQUIRE 3000 plus)
  9. Thermo Finnigan LTQ Orbitrap质谱仪(Thermo Fisher Scientific)

程序

  1. 样品收集
    分离程序开始于收集由植物学家识别并且用凭证样本的记录证明的样品。在我们的实施例中,在Maauu,在Nuku Hiva岛,Marquesas群岛,法属波利尼西亚的"TerreDéserte"地区,在477m的高度收集了毛霉菌的树皮,并由Jean-Fran?ois博士鉴定布托。凭证样本(JFB 2808)已存放在法属波利尼西亚的植物标本馆。使用空气干燥装置在30℃下收集样品后干燥,并储存在室内直到研磨和提取
  2. 提取和隔离
    在萃取之前,将批次的50g树皮片在至少5个1分钟的重复研磨期间以转子设定在10,000rpm下研磨成粉末。为了避免研磨机过热,系统用塑料覆盖,并在每个研磨过程之间在冰中冷却。
    根据粗提取物的化学复杂性,该过程可以分为三个步骤,即提取过程,一个或多个分馏步骤,以及最后的分离过程。对于我们的样品,进行两次连续分馏,使用表1和2中所述的一系列溶剂

    图1.研磨过程之后和之前的Rauvolfia nukuhivensis的树皮

    表1.第一次分馏的溶剂组成
    分数
    溶剂
    比例
    FA

    -
    FB
    水/甲醇
    1:1
    FC
    甲醇
    -
    FD
    甲醇/二氯甲烷
    1:1
    FE
    二氯甲烷
    -

    1. 对于我们的植物 的例子,其干燥的树皮(250g)设备
    2. 将干燥的树皮在750ml乙醇中浸渍16小时 ?,然后过滤(0.45μm)溶剂 通过蒸发浓缩,得到粗油。
    3. 所结果的 提取物(21g)通过在混合物中超声处理10分钟而溶解 200ml甲醇/二氯甲烷(1:1),然后过滤。
    4. 的 在RP-C18 C18柱上通过真空液相色谱进行分离,并用极性递减的溶剂洗脱,如表1所示 ?1.
    5. 部分FC提供8.43g油状残留物,并通过HPLC-DAD显示吲哚生物碱的存在。
    6. 因此,进一步加入一部分级分FC(600mg) 通过正相(二醇)快速柱色谱法分离 增加极性的溶剂如表2所示。

      表2.第二次分馏的溶剂组成
      小部分
      溶剂
      Pro 部分
      f1
      环己烷
      -
      f2
      环己烷/乙酸乙酯
      3:1
      f3
      环己烷/乙酸乙酯
      1:1
      f4
      环己烷/乙酸乙酯
      1:3
      f 5
      乙酸乙酯
      -
      f6
      乙酸乙酯/甲醇
      3:1
      f7
      乙酸乙酯/甲醇
      1:1
      f8
      乙酸乙酯/甲醇
      1:3
      f9
      甲醇
      -
      f10
      甲醇/二氯甲烷
      1:1
    7. 每个获得的级分含有比提取物更低的化合物多样性,因此促进分离和纯化过程
    8. 选择级分FCf7在HPLC上进行最终纯化,主要是因为其重量,还具有高的化学多样性
    9. FCf7(50.2mg)主要化合物的纯化通过RP-C18半制备型HPLC(Phenomenex,Luna 5μmC18,250mm×10mm)进行,用水/甲醇/三氟乙酸的梯度(从70:30: 0.1至30:70:0.1,流速3ml/min),在氮气流下干燥步骤后,得到纯的化合物1(4.7mg),2(2.8mg)3(1.83mg),4(2.17mg) ,5(3.25mg),6(3.24mg),7(1.23mg),8(6.43mg),9(1.82mg),10(1.71mg),11(1.39mg),12 (2.20mg)
  3. 表征
    该方法的最后一步涉及不同的光谱和色谱技术,用于纯化化合物的完整描述。到目前为止,核磁共振(NMR)光谱是阐明化合物结构的最常用的技术。然而,需要其它技术与NMR一起以获得更好的表征,包括高分辨率质谱,UV光谱,旋光度,红外光谱和圆二色性(如果需要)。 NMR光谱通常以两种主要方式使用,1D和2D。为了更好地理解NMR光谱,参见例如Keeler(2011); Lewitt(2001)和Fan(1996)。
    在我们的情况下,我们选择研究并描述每种化合物的几种物理化学特性,如下所列:通过HPLC-DAD记录UV-Vis光谱。 NMR光谱在具有脉冲场梯度的Bruker Avance 500MHz光谱仪上测量,并且信号参照残余溶剂信号(CD 3 OD,at H < /s);3.31和δem} 49.0ppm)。对于该步骤,将每个稀释在700μlCD 3 OD中。分别使用Bruker ESQUIRE 3000 plus质谱仪和Thermo Finnigan LTQ Orbitrap质谱仪测量电喷雾离子化质谱(ESIMS)和高分辨率ESIMS(HRESIMS)数据。在配备有Waters 717 Plus自动进样器,Waters 998光电二极管阵列检测器和Sedex 75蒸发光散射检测器(Sedere,France)的Waters 600系统上进行HPLC纯化。在Jasco P-2000偏振计和Jasco J-810分光偏振计上分别测量旋光度和圆形显微镜。
    Martin等人(2012)和Martin等人(2015)已??经报道了一些化合物的结构。这里提供了一些示例(图2)。我们还提供了在表3和4中总结的NMR光谱数据

    图2.从伊红蠕虫的树皮提取物中分离的化合物1-13的结构。 Nukuhivensium(1)。黄色无定形粉末;通过HPLC/DAD(MeOH/H 2 O/TFA)测量的UVλmax 223,6; 241,3; 289,9; 340,0; 381.7nm; 1 H和13 C NMR,参见表3; HRESIMS m/z 261.13953 [M] + sup /(C 18 H 17 17 N 2的计算值)/sub> + ,261.13917)。 N 12 -methylnukuhivensium(2)。黄色无定形粉末;通过HPLC/DAD(MeOH/H 2 O/TFA)测量的UVλmax 224.8; 236,6; 248,4; 286,7; 335,2; 392.6nm; 1 H和13 C NMR,参见表3; HRESIMS m/z 275.15417 [M] + sup /(C 19 H 19 N 2 N 2的计算值)/sub> + ,275.15482)。诺维胺(11):淡黄色无定形粉末; [α] 20 D-1.1(MeOH); 1 H NMR和13 CNMR光谱数据,参见表4; ESIMS m/z 343.1 [M + H] +; HRESIMS m/z 343.16611 [M + H] + sup /(C 19 H 23 N 23 N的计算值) 2 + ,343.16578)。淡黄色(12)浅黄色无定形粉末; [α] 20 D + 1.2(MeOH); 1 H NMR和13 CNMR光谱数据,参见表4; ESIMS m/z 357.2 [M + H] +。 HRESIMS m/z 357.18289 [M + H] +的计算值(C 20 H 25 N 2 O 3) 2 4 + ,357.18143)。

    表3. 1 H(500 MHz)和 13 3 OD
    中的吲哚喹啉衍生物1和2的C(125MHz) />

    Nukuhivensium(1)
    N12 -methylnukuhivensium(2)
    位置
    δ 以ppm为单位
    以ppm计,ppm。 ( J 以Hz为单位)
    δ 以ppm为单位
    以ppm计,ppm。 ( J 以Hz为单位)
    1
    120.5   
    8.65,s
    121.5
    8.96,s
    2
    154.4
    -
    154.1
    -
    3
    124.4
    7.83,d(7.0)
    123.9
    7.86,d(7.0)
    4
    137.6
    9.19,d(7.0)
    138.4
    9.24,d(7.0)
    6
    128.1
    8.84,d(7.0)
    128.1
    8.90,d(7.0)
    7
    117.2
    8.61,d(7.0)
    116.8
    8.66,d(7.0)
    7a
    125.0
    -
    125.6
    -
    7b
    122.5
     
    128.9

    8
    123.3
    8.36,d(8.0)
    122.8
    8.41,d(8.0)
    9
    122.9
    7.50,dd(8.0; 7.0)
    123.5
    7.54,dd(8.0,7.0)
    10
    131.0
    7.74,dd(8.0; 7.0)
    131.2
    7.83,dd(8.0,7.0)   
    11
    113.6
    7.84,d(8.0)
    112.1
    7.95,d(8.0)
    11a
    143.3

    144.9

    12
     
     
    34.6
    4.58,s
    12a
    131.7,C
     
    131.4   

    12b
    134.3,C

    135.2   

    13
    38.6,CH 2
    3.06,t(7.7)
    38.7
    3.09,t(7.7)   
    14
    23.9,CH 2
    1.94,tq(7.7; 7.5)
    24.4
    1.93,tq(7.7,7.5)
    15
    14.0,CH 3
    1.12,t(7.5)
    14.0
    1.12,t(7.5)

    表4. 1 H(500 MHz)和 /strong> 3 OD
    中的大线衍生物11和12的C(125MHz) />

    北京市(11)
    。(12)
    位置
    δ 以ppm为单位
    以ppm计,ppm。 ( J 以Hz为单位)
    δ 以ppm为单位
    δ 以ppm计,mult。 ( J 以Hz为单位)

    126.9
    -
    n.d.
    -
    3
    49.1
    4.89,br s
    47.8
    5.08,br s
    5
    50.9
    4.31,d(8.0)
    51.2
    4.26,d(7.5)

    25.5
    3.52,dd(17.5,7.5)
    25.0
    3.50,dd(17.5,7.5)

    2.99,d(17.5)

    7
    107.7 / -
    108.2
    -
    8
    127.0
    -
    127.2
    -
    9
    119.2
    7.51,d(8.0)
    119.8
    7.51,d(8.0)
    10
    120.8
    7.09,ddd(8.0,7.0,1.0)
    121.3
    7.09,brdd(8.0,7.0)
    11
    123.7
    7.18,ddd(8.0,7.0,1.0)
    124.2
    7.25,br dd(8.0,7.0)
    12
    112.5
    7.38,br d(8.0)
    110.9
    7.43,d(8.0)
    13
    138.1
    -
    139.8
    -
    14α
    28.4
    2.16,td(12.5,3.0)
    28.3
    2.20,td(12.5,3.0)
    14β
    2.02,br d(12.5)

    15
    30.2
    2.17,m
    30.6
    2.11,br t(11.5Hz)
    16
    48.0
    3.08,br s
    49.3
    2.93,m
    17

    -
    n.d.
    -
    18
    10.3
    0.66,t(7.5)
    10.7
    0.71,t(7.5)
    19
    23.1
    1.64,m
    22.9
    1.58,m
    1.38,m
    1.31,m
    20
    49.0
    2.42,br t(7.5)
    50.2
    2.41,m
    21
    177.9
    -
    n.d.
    -
    CH 3 - N 1


    29.3
    3.73,s

结论

我们的协议表明吲哚生物碱的隔离的重大改进。事实上,使用经典提取,非碱性化合物不会被提取,例如离子化合物如nukuhivensium。在改进结构,物理和化学特性的连续工作中,可以进行另外的NMR光谱,以描述一些化合物的相对构型,如NOESY实验。
该方案允许吲哚生物碱不同骨架以及立体异构体(正常和异构化合物,例如三明治和异维生素)和类似系列(正常和正化合物,如三明治和Norsandwicine)的不同结构的表征。因此,本文所述的方法更有效,并且需要更少的溶剂消耗时间。

致谢

我们要感谢方长安邀请我们写这篇手稿。作者感谢Diren(法国方面环境部)财政援助部门。这个协议已经写了Pr的伟大帮助。 Olivier P. Thomas,作者感谢他的工作和建议。

参考文献

  1. Fan,T。(1996)。 一维和二维的代谢分析复杂混合物的NMR分析。 161-219。
  2. Keeler,J。(2011)。了解NMR光谱。 John Wiley&儿子。
  3. Lewitt,M.H。(2001)。自旋动力学:核磁共振的基础。 儿子。
  4. Martin,N.J.,Ferreiro,S.F.,Barbault,F.,Nicolas,M.,Lecellier, G.,Paetz,C.,Gaysinski,M.,Alonso,E.,Thomas,O.P.,Botana, 和Raharivelomanana,P.(2015)。 来自Marquesan植物的Rauvolfia nukuhivensis的吲哚生物碱及其对离子通道的影响。 109:84-95。
  5. Martin,N.J.,Prado,S.,Lecellier,G.,Thomas,O.P.and Raharivelomanana,P.(2012)。 来自Marquesan植物的Rhovolfia nukuhivensis的Nukuhivensiums,indolo [2,3-a] quinoliziniums/em> 分子 17(10):12015-12022。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
引用:Martin, N. J., Nicolas, M., Lecellier, G. and Raharivelomanana, P. (2015). Isolation and Characterization Procedure for Indole Alkaloids from the Marquesan Plant Rauvolfia Nukuhivensis. Bio-protocol 5(20): e1625. DOI: 10.21769/BioProtoc.1625.
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