Chromatographic Separation of the Codonocarpine Type Alkaloids from the Root Bark of Capparis decidua

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Aug 2016



Various parts of the caper tree Capparis decidua have found application in traditional medicine. The isolation and structural elucidation of the codonocarpine type alkaloids contained in the root bark, however, is not trivial and has probably led to misinterpretation in the past. This protocol describes the extraction and chromatographic separation of the four major alkaloids of the root bark of Capparis decidua. The delivered samples of cadabicine, codonocarpine, isocodonocarpine and capparidisinine were suitable for their unambiguous structural elucidation by NMR, MS and MS/MS.

Keywords: Capparis decidua (凯尔树), Capparaceae (山柑科), Spermidine alkaloid (亚精胺生物碱), Cadabicine (Cadabicine), Codonocarpine (Codonocarpine), Isocodonocarpine (Isocodonocarpine), Capparidisinine (Capparidisinine), Chromatographic separation (色谱法分离)


The tree Capparis decidua is widely distributed in the arid regions of Africa, the Middle East and Southern Asia, where various parts of the plant are commonly used in local folk medicines for the treatment of various disorders. The root bark, for example, is applied as anthelmintic and purgative, and it has been shown that its alcoholic extract possesses significant antibacterial and antifungal activities (Singh et al., 2011; Singh and Singh, 2011; Tlili et al., 2011; Mohammed et al., 2015).

Ahmad et al. (1985; 1986; 1987; 1989; Arif, 1986) intensively studied the root bark extract of Capparis decidua and published several structures of codonocarpine type alkaloids. Some of these structures did not fit with our understanding of the biosynthesis of such alkaloids. Further, we were not convinced that the given analytic data and its interpretation gives unambiguous proof for the structural claims (Bienz et al., 2002). Therefore, we initiated our own investigation. We developed a protocol for the extraction and chromatographic separation of codonocarpine type alkaloids and could isolate three alkaloid fractions that allowed the identification and structural elucidation of the four major spermidine alkaloids of the root bark of Capparis decidua: cadabicine (1), codonocarpine (2), isocodonocarpine (3), and capparidisinine (4) (Figure 1) (Forster et al., 2016).

Figure 1. The four major codonocarpine type alkaloids found in the root bark of Capparis decidua. (1) Cadabicine; (2) Codonocarpine; (3) Isocodonocarpine; and (4) Capparidisinine.

The isolation and structure elucidation of alkaloids is not trivial. The class of alkaloids, defined as nitrogen containing organic compounds from natural sources with a most basic character, comprises more than 10,000 compounds. As the properties and structures of alkaloids differ strongly from compound to compound, there is no general way to isolate them. Nevertheless, most alkaloids exist naturally in their protonated form. Hence, the methanolic extraction of dried and crushed plant material is often applied. As alkaloids usually possess amino and other functional groups that could react with solvents and additives, as well as with CO2 or oxygen from air, the formation of artefacts is prevented as far as possible by the choice of appropriate conditions. In general, artefacts can be recognized by comparing the analytical data of the isolated fractions and the original sample. Therefore, some of the original sample should always be kept as reference material (Hesse, 2000).

Materials and Reagents

  1. Cotton wool (e.g., Thomas Scientific, catalog number: 2904W25 )
  2. Sea sand (cristobalite, Brenntag Schweizerhall AG) (e.g., Grogg Chemie, catalog number: G890 )
  3. Round bottom test tubes, ca. 100 x 16 mm (Assistant, catalog number: 42775045 )
  4. TLC plates (TLC Silica gel 60 F254 on aluminum 20 x 20 cm) (EMD Millipore, catalog number: 100390 )
  5. Glass capillaries (MARCHEREY-NAGEL, catalog number: 814022 )
  6. 2 ml 9 mm ScreVial, clear glass 12 x 32 mm (Infochroma, catalog number: G004-HP-H )
  7. 9 mm screw cap with 1 mm pigment-free ms-pure PTFE/Silicone/PTFE-Septum (Infochroma, catalog number: G004-HP-CB-FKSKFK10 )
  8. 1.5 ml Eppendorf tubes (Eppendorf, catalog number: 0030120086 )
  9. Plant material: Capparis decidua root bark (collected in Sahiwal, Pakistan and identified by M. Waris, Department of Cholistan Institute of Desert Studies, The Islamia University of Bahawalpur, Pakistan)
  10. MeOH (methanol, technical grade, distilled prior to use) (Thommen Furler, catalog number: 203-VL54TE )
  11. SiO2 (Merck silica gel 60 [40-63 μm]) (EMD Millipore, catalog number: 109385 )
  12. Ammonium hydroxide (NH3, aqueous ammonia solution 25%, puriss p.a.) (Honeywell, catalog number: 30501 )
  13. Chloroform (CHCl3, stabilized with EtOH) (Scharlab, catalog number: CL02181000 )
  14. Dichloromethane (CH2Cl2, technical grade, distilled prior to use) (Thommen-Furler, catalog number: 739-VL54TE )
  15. Diethylether (Et2O, puriss. p.a., stabilized with BTH, distilled from NaOH prior to use) (Honeywell, catalog number: 32203 )
    1. Pure ether is sensitive towards oxidation and can form explosive peroxides upon prolonged exposure to air and light. It therefore is usually stabilized with the antioxidant butylhydroxytoluene (BHT), which is removed upon distillation. Thus, distilled ether is again prone to form epoxides, and it is necessary to test your distilled ether for peroxides prior to its use if the solvent was exposed to air and light for several days.
    2. To test for peroxides use: Quantofix® peroxides test sticks (Sigma-Aldrich, catalog number: Z101680 ).
  16. Methylamine solution (MeNH2, 40 wt. %) (Sigma-Aldrich, catalog number: 426466 )
  17. Hydrochloric acid (HCl, 37%) (for analysis, Merck) (EMD Millipore, catalog number: 100317 )
  18. Acetonitrile (MeCN, LC-MS Ultra CHROMASOLV®) (Honeywell, catalog number: 14261 )
  19. Formic acid (HCO2H, 99%, ULC/MS) (Biosolve, catalog number: 069141 )
  20. 2-propanol (LC-MS Ultra CHROMASOLV®) (Honeywell, catalog number: 650447 )
  21. Sodium hydroxide solution (NaOH for HPCE, 0.1 N in H2O) (e.g., MSP KOFEL, catalog number: 5062-8575 )
  22. H2PtCl6
  23. KI
  24. Ce(SO4)2
  25. H2SO4
  26. Schlittler reagent (see Recipes)
  27. Ce(SO4)2 solution (see Recipes)


  1. Sharp axe (e.g., TRANSA, catalog number: 040929-001001 )
  2. Capped fermentation tank (50 L, e.g., Braupartner, catalog number: 141-0 )
  3. Filter strainer cloth (75 x 75 cm, e.g., Erwin Müller, catalog number: i10014278 )
  4. Stainless steel spatula (e.g., Thomas Scientific, catalog number: 1232X12 )
  5. Rotary evaporator (Büchi Rotavapor R-134 with Büchi Waterbath B-481) (Büchi, Flawil, Switzerland)
  6. Glass funnels (e.g., Duran, catalog number: 21 351 23 )
  7. Round bottom flasks of several sizes (5 ml, 10 ml, 25 ml, 50 ml, 250 ml, 1 L) (e.g., Schott, Germany)
  8. Steel spring clips (e.g., Thomas Scientific)
    NS 14.5 (e.g., Thomas Scientific, catalog number: 1178Z55 )
    NS 29 (e.g., Thomas Scientific, catalog number: 1178Z57 )
  9. Magnetic stir bars (e.g., Thomas Scientific, catalog number: 8608S78 )
  10. Magnetic stirrer (Heidolph Instrument, catalog number: 505-30080-00 )
  11. Glass bottle 1 L (e.g., Thomas Scientific, catalog number: 1228R95 )
  12. Glass chromatography columns of different length and diameters with stopcock at the bottom and a ground socket joint at the top to fit a solvent reservoir (e.g., Neubert Glas, catalog number: 1196-29-20400 )
  13. Glass beaker of several sizes (e.g., Duran, Germany)
  14. A piece of vacuum hose (e.g., Thomas Scientific, catalog number: 9544T15 )
  15. Solvent reservoirs: glass round bottom flasks of several sizes with oppositely attached ground cone and ground socket joints (e.g., Thomas Scientific, catalog number: 1197C03 )
  16. Adaptors:
    Ground cone joint NS 14.5 with hose barbe to tubing, bent (e.g., Thomas Scientific, catalog number: 1195C09 )
    Ground cone joint NS 29 with hose barbe to tubing, bent (e.g., LabMarket, catalog number: 1681029 )
  17. Hand pump (VWR, catalog number: 612-9952 )
  18. 125 ml Erlenmeyer flasks (e.g., Thomas Scientific, catalog number: 4907F23 )
  19. Test tube racks (e.g., Thomas Scientific)
  20. TLC chamber (screw cap glass jar, 250 ml)
  21. UV lamp (254 nm), e.g., UV lamp 4, dual (Camag, model: 022.9160 )
  22. High vacuum pump (Alcatel Pascal 2015 SD) (e.g., Ideal Vaccum Products, catalog number: P102310 )
  23. CortecsTM UPLC® C18+, 1.6 μm, 2.1 x 150 mm (Waters, catalog number: 186007117 ), equipped with a CortecsTM UPLC® C18+ 1.6 μm, 2.1 x 5 mm VanGuardTM precolumn (Waters, catalog number: 186008713 )
  24. AquityTM Ultra Performance LC (Waters, Milford MA, USA)
  25. Bruker maXis Q-Tof HR-MS (Bruker Daltonics, Bremen, Germany)
  26. Eppendorf refrigerated microcentrifuge (Eppendorf, model: 5417R )
  27. MilliQ gradient apparatus (deionized water, for HPLC) (EMD Millipore, catalog number: Z00Q0V0WW )


  1. Extraction of the root bark
    1. Dry the root bark in the shade for one week at 30-35 °C.
      Note: If this is not your regular ambient temperature you may use a drying oven.
    2. Shred the bark finely with a sharp axe and extract it with MeOH (1:1, w/v) in a capped fermentation tank for one week. Stir the mixture several times a day. If possible: stir your extraction mixture permanently with a mechanical stirrer.
      Note: In our case, the extraction was performed under regular laboratory condition at ambient temperature (30-35 °C in Pakistan).
    3. Filter off the solid through a tightly woven cloth and evaporate the MeOH on the rotary evaporator at 30 °C and 70 mbar for several hours, upon which the extract becomes highly viscous. Since it is not possible to completely remove the MeOH, stop with the evaporation of the solvent when no longer the formation of bubbles in the extract can be observed.
    4. Store the almost black, viscous extract at 4 °C until it is used.
    Note: In our experiment, we extracted 45 kg of root bark with 45 L of MeOH and obtained 215 g of the black, viscous extract.

  2. Chromatographic separation of the alkaloids: general procedures
    The chromatographic separation of the codonocarpine type alkaloids on silica gel (SiO2) involves at least four chromatographic separations that follow all the main principles described below:
    1. Adsorption of the sample on SiO2
      The crude extract and the alkaloid fractions are poorly soluble in small amounts of MeOH and even less soluble in the eluent that is used for the chromatography. It is, however, very important to apply the extract onto the chromatography column within the smallest possible volume. Therefore, the mixtures were adsorbed on SiO2 before they were brought onto the column.
      1. Transfer the sample into a round bottom flask and add MeOH (1:4, w/v). Take a large round bottom flask (we used a 250 ml flask for 10.8 g of extract) to ensure that the glass surface is large enough to secure later a rapid and efficient evaporation of the solvent. Add a magnetic stir bar and stir vigorously until you obtain a uniform (still heterogeneous) mixture.
      2. While still stirring, add SiO2 (sample/SiO2 1:2, w/w), and continue with stirring for another 5 min.
      3. Remove the stir bar and then remove the MeOH on the rotary evaporator (30 °C, 160 mbar) until you obtain a bright brown powder.
    2. Preparation of the eluent mixture and the column
      1. Mix the eluent components in the given ratio (Tables 1-5) in a clean glass bottle. It is crucial to distil the solvents prior to use.
      2. Prepare a column as shown in Figure 2.

        Figure 2. Packing of a chromatographic column. The columns were stacked layers of cotton wool (A), sea sand (B), SiO2 (C), additional sea sand (D) and the adsorbed sample on SiO2 (E).

        1. Fill the column with approximately 5 cm of the eluent and seal it loosely and free of air at the bottom with some cotton wool (Figure 2A) to prevent the SiO2 from flowing out.
        2. Add 1 cm of sea sand (Figure 2B) so that you obtain a plane surface.
        3. In a beaker, prepare a slightly viscous, bubble-free suspension of SiO2 in the eluent. Add eluent until the SiO2 is well covered and stir with a spatula until all air has escaped.
        4. Fill the SiO2 suspension through a glass funnel carefully, not disturbing the surface of the sea sand, into your glass column to obtain a loose column of SiO2 (Figure 2C).
        5. Open the stopcock and let the eluent escape until the solvent surface exactly reaches the surface of the SiO2. Close the stopcock.
        6. Compact the SiO2 column by tapping against the glass column with a piece of vacuum hose. Upon tapping you can observe that the SiO2 level drops. Open the stopcock from time to time to drain the solvent again to the SiO2 surface. Continue compacting until the SiO2 surface no longer sinks.
        7. Add approximately 5 cm of solvent and trickle approximately 2 cm of sea sand (Figure 2D) through the solvent onto the surface of your column to protect the column against mechanical perturbations. Drain the solvent until its surface reaches the surface of the sea sand.
        8. Apply the brown powder of your adsorbed substance onto the column (Figure 2E), add carefully 2 cm of eluent, then drain the eluent carefully until it reaches the surface of your SiO2 with the adsorbed extract. Repeat the addition and draining of solvent three times.
        9. Carefully fill up the column with the eluent, connect the solvent reservoir, fix it with a clamp, and fill the reservoir with eluent.
    3. Chromatographic separation
      1. To start your separation, open the stopcock and adjust the flow of the eluent to an appropriate rate. Depending on the size of the column, this shall be approx. 12 ml min-1 (coarse fractionation) or approx. 4 ml min-1 (fine fractionation). If the flow rate is too low with a completely opened stopcock, apply slight but constant pressure with a manual pump.
      2. Collect fractions of reasonable volumes. The volumes to choose depend on the size of the column and the intended purpose of the chromatography. For a preliminary, rough separation of the original crude extract on a large column, 100 ml fractions, collected in Erlenmeyer flasks, are appropriate. For fine separations on smaller columns, preferential fractions of approx. 10 ml are collected in round bottom test tubes. Since the alkaloids of interest are readily recognizable as yellow bands on the column, the clearly colourless pre-fractions are usually collected in Erlenmeyer flasks.
    4. Fraction control by TLC
      1. Transfer a small amount of your fraction with a glass capillary on a TLC plate and let the spot dry. You can re-use the same capillary several times when you clean it in-between with some MeOH.
      2. Place the TLC plate in a TLC chamber (Figure 3) with 3-5 mm of one of the following eluents:
        1. CH2Cl2/MeOH/NH3 40:13:1: to test if your fraction contains any spermidine alkaloids at all. The typical Rf value for the spermidine alkaloids is shown in Figure 3.
        2. MeOH/H2O/NH3 15:2:1: to test which spermidine alkaloid is in your fraction. This eluent was developed by Kyburz (1985) and was the only one that was found to distinguish between the alkaloids of the different masses. The typical Rf values for the spermidine alkaloids are given in Figure 3.
      3. Let the TLC develop till the solvent front reaches almost the top of the plate, remove the TLC plate from the chamber, and let it dry until the plate no longer smells of NH3.
      4. Check for UV-active spots under a UV lamp (254 nm) and mark them with a pencil.
      5. Stain your TLC plate with Schlittler reagent and dry it. The spermidine alkaloids give red/violet spots. When the dry Schlittler-active spots are stained subsequently with Ce(SO4)2 solution, the colour of the spots deepens. According to our experience, the detection by UV is the most sensitive method. Combined with the staining with Schlittler reagent, the spots for the alkaloids of interest are very specifically and clearly recognized. The treatment with Ce(SO4)2 is helpful but not necessary.

        Figure 3. TLC chamber and the schematic TLCs with the two most useful eluents for the detection of spermidine alkaloids. For the detection of the alkaloid fractions in the preliminary rough chromatography use the eluent system CH2Cl2/MeOH/NH3 40:13:1 (gives no separation of the individual alkaloids, left), and for fine separation of the alkaloids use the eluent system MeOH/H2O/NH3 15:2:1 (separation of the individual alkaloids, right). However, codonocarpine (2) and isocodonocarpine (3) remain indistinguishable by TLC.

    5. Combination of the fractions and evaporation of the solvent
      1. Combine the fractions according to their TLCs. Pay attention not to pool pure alkaloid fractions with fractions that contain alkaloid mixtures. Mind that the alkaloids are chromatographing quite closely, and a lowly abundant alkaloid next to a major component may not be easily recognized by TLC! Very small amounts of an alkaloid are only detected by UV light but not with the Schlittler staining reagent.
      2. Evaporate the solvent on the rotary evaporator at 30 °C (pressure depending on your eluent), and remove residual solvent at high vacuum (23 °C, 4 x 10-4 mbar).
        Note: To determine the net mass of your sample subtract the tare mass (empty flask) from the gross mass (sample + flask).
      3. Control your dried samples by HPLC-MS (see Data analysis). Check their final alkaloid composition with the extracted ion chromatograms (EIC) m/z 436.2231 (1), m/z 466.2237 (2/3) and m/z 496.2442 (4).

  3. Chromatographic separation of the four major alkaloids in detail
    Notes: This section contains a detailed description of what was done in our laboratory. For an improved separation, we recommend the following steps:
    1. Start with a rough alkaloid isolation as done in column B (Table 3).
    2. Separate the alkaloids 1 and 2/3 from alkaloid 4 as described for column B1 (Table 4).
    3. Combine all fractions containing 1 and 2/3 to one sample and chromatograph this as described in column A1.2 to obtain pure 1 and 2/3 (Table 2).
    4. Wash the fraction containing 4 with MeOH as described for B1.4.1.
    The four alkaloids cadabicine (1, 435 Da), codonocarpine (2, 465 Da), isocodonocarpine (3, 465 Da) and capparidisinine (4, 495 Da) have very similar chromatographic behaviours. For this reason, we obtained at the beginning of our project many mixed fractions upon preliminary chromatographies of the crude original extract as well as of partially separated fractions. For the isolation of 1 and 2/3 we started with an alkaloid mixture (A1, 576 mg) that was obtained by the combination of several of these fractions. A pre-purified alkaloid fraction that could be used likewise is fraction B1, employed below for the isolation of capparidisine (4).
    1. Path A: Isolation of cadabicine (1) and codonocarpine (2)/isocodonocarpine (3)
      The separation path A is schematically shown in Figure 4. The alkaloid-containing fractions of various preliminary chromatographies (A) were combined in A1. The chromatographic separation of A1 led to four major fractions. Three of them contained mixtures of 1 and 2/3 in diverse ratios (A1.1 mainly 1, A1.2 comparable amounts of 1 and 2/3, and A1.3 mainly 2/3). The fourth fraction (A1.4) contained mainly alkaloid 4 but also some 2/3. As none of the obtained fractions contained a pure alkaloid, the samples A1.1 and A1.3 were kept as reserve material and the largest fraction (A1.2) was further separated on SiO2 to obtain the alkaloids 1 (A1.2.1) and 2/3 (A1.2.3) in high purity (> 98%).
      Unfortunately, fraction A1.4 contained not enough material for the final isolation of pure 4.
      The chromatic details and results are summarized in the Tables 1 and 2. In fractions of alkaloid mixtures, the major alkaloid is underlined.

      Figure 4. Separation path A. Overview over the chromatographic steps from an alkaloid mixture (A1) to the pure sample of cadabicine (1, A1.2.1) and the mixed sample of codonocarpine and isocodonocarpine (2/3, A1.2.3) that were used to elucidate their structures. The major alkaloid in a mixed fraction is underlined.

      Table 1. Chromatographic separation of A1

      Table 2. Chromatographic separation of A1.2

    2. Path B: Isolation of capparidisinine (4)
      The separation path B is schematically shown in Figure 5. The crude methanolic extract (B) was roughly fractionated on SiO2 to separate the alkaloid-containing fractions (B1) from the further extracted material. The alkaloid mixture B1 was chromatographed to obtain three fractions containing different ratios of 1 and 2/3 (B1.1-B1.3) and one fraction containing only one alkaloid (4). Alkaloid 4 in fraction B1.4 however, was contaminated with some unidentified Schlittler-inactive compounds (detected by 1H-NMR). An additional chromatographic step (B1.4.1) could not separate the alkaloid 4 from its contaminates. Capparidisinine (4, B1.4.1.1) was finally gained by washing sample B1.4.1 with small amounts of MeOH.
      The chromatic details and results are summarized in the Tables 3-5. In fractions of alkaloid mixtures, the major alkaloid is underlined.

      Figure 5. Separation path B. Overview over the chromatographic steps from the crude extract (B) to alkaloid 4 (B1.4.1.) inclusive the washing step to obtain the pure form of capparidisinine (4, B1.4.1.1) from which its structure was elucidated. The major alkaloid in a mixed fraction is underlined.

      Table 3. Chromatographic separation of B

      Table 4. Chromatographic separation of B1

      Table 5. Chromatographic separation of B1.4

    3. Further purification of B1.4.1
      The yellow solid of B1.4.1 (77 mg) was placed in a 1.5 ml Eppendorf tube, and MeOH (0.3 ml) was added. The cloudy mixture was shaken carefully and then centrifuged (1 min, 20,817 x g). The yellow solution was carefully separated from the remaining solid with a pipette. The solid was washed three more times with MeOH (0.2 ml each time). The remaining solid was transferred to a round bottom flask, and dried in vacuo (30 °C, 160 mbar then 23 °C, 0.2 mbar) to deliver a bright yellow solid B1.4.1.1 (4, 15 mg).

  4. Conversion to the hydrochloride salts
    1. Suspend the alkaloid in MeOH.
    2. Add dropwise 1.5 eq of aq. HCl (0.1 or 1.0 N) to the cloudy solution. The solution becomes clear.
    3. Evaporate the solvent in vacuo (30 °C, 160 mbar then 23 °C, 4 x 10-4 mbar).

Data analysis

    1. The alkaloid fractions were analysed by HPLC-ESI-MS. The samples were dissolved in MeOH + 0.1% HCO2H at 0.5 mg ml-1 for the crude extract and 0.1 mg ml-1 for the purified alkaloids. The samples were injected at a volume of 1 μl and chromatographed at a flow rate of 0.3 ml min-1 on a RP C18+ column with the solvents A (H2O + 0.1% HCO2H) and B (MeCN + 0.1% HCO2H) at 25 °C. The gradient started isocratic at 10% B for 5 min and raised then to 25% B within 4 min. The column was washed with 100% B for 2 min and then equilibrated at 5% B for 6 min.
    2. The connected Q-Tof MS was equipped with an ESI source. The MS was recorded in positive ionisation mode from 50-800 m/z under the following conditions: HV end plate offset 500 V, HV capillary 3,000 V, nebulizer gas (N2) 2.0 bar, dry gas (N2) 10.0 bar, and dry temperature 200 °C. The mass spectrometer was calibrated for mass accuracy with 2 mM sodium formate (980 μl H2O/2-propanol 1:1, 20 μl 0.1 N NaOH and 1 μl HCO2H), the relative mass error being typically lower than 2 ppm (externally).
    3. The MS/MS spectra were recorded with 35.00 eV collision energy and 4.00 isolation width.
    4. The chromatograms and spectra were evaluated with DataAnalysis. The extracted ion chromatograms (EIC) were educed based on the exact mass ± 0.02 m/z.
    As an example, the base peak chromatogram (BPC) of the crude extract B is shown in Figure 6. The alkaloid-related EIC’s are displayed below. The extract contains three forms of codonocarpine type alkaloids: (1) the glycosidic derivatives (Rt 2.3-2.7 min), (2) the (E/Z)-derivatives (Rt 3.4-3.7 min), which may be artefacts, and (3) the major (E/E)-alkaloids (Rt 4.2-4.4 min) that were isolated with our protocol.
    While cadabicine (1) and capparidisinine (4) can be distinguished with HR-MS, this is not the case for codonocarpine (2) and isocodonocarpine (3). We found that codonocarpine (2) has a slightly smaller retention time (4.2 min) than isocodonocarpine (3, 4.3 min). The MS/MS spectra for (2) and (3) are virtually the same. They differ only by one low-intense signal at m/z 218.08058 that is unique for codonocarpine (2). However, be very careful; both compounds show a signal at m/z 218.11713.
    Note: For a detailed discussion of the MS/MS spectra and the interpretation of the NMR spectra check ‘A new sight on the Codonocarpine Type Alkaloids of Capparis decidua’ (Forster et al., 2016).

    Figure 6. BPC of the crude extract and the EICs of the codoncarpine type alkaloid related masses. The crude extract contains three different forms of alkaloid derivatives. The four major alkaloids cadabicine (1), codonocarpine (2), isocodonocarpine (3) and capparidisinine (4), all (E/E) derivatives, can be identified by their exact mass and retention time.


We found that the most stable form to store the codonocarpine type alkaloids is in their solid forms. The fractions were therefore always dried on the rotary evaporator immediately after chromatography.


  1. Schlittler reagent (Schlittler and Hohl, 1952)
    The mixture of H2PtCl6 (1.0 g in H2O [6 ml]), KI (22.5 g in H2O [225 ml]), aq. HCl solution (1 N, 20 ml) was diluted with deionized H2O to a total volume of 1,000 ml
    Amines: brown in diverse shades (yellow-red-blue-grey)
    Amides: bright yellow, followed by Ce(SO4)2: brown
  2. Ce(SO4)2 solution
    A solution of Ce(SO4)2 (10.0 g) in conc. H2SO4 (55 ml) was diluted with deionized H2O to at total volume of 1,000 ml
    Amines: (together with Schlittler reagent) brown
    Amides: (together with Schlittler reagent) intensifies the staining colour of the Schlittler reagent


We are grateful to PD Dr. L. Bigler for his great support in the acquisition of the MS and MS/MS data and we thank the Higher Education Commission of Pakistan (HEC) (1-8/HEC/HRD/2013/2582, PIN:IRSIP 23 Ps 48) for funding a six-month research visit of A. Ghaffar to the University of Zurich.


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  12. Singh, D. and Singh, R. K. (2011). Kair (Capparis decidua): A potential ethnobotanical weather predictor and livelihood security shrub of the arid zone of Rajasthan and Gujrat. Indian J Tradit Knowl 10: 146-155.
  13. Singh, P., Mishra, G., Srivastava, S., Jha, K. K. and Khosa, R. L. (2011). Traditional uses, phytochemistry and pharmacological properties of Capparis decidua: An overview. Der Pharm Lett 3: 71-82.
  14. Tlili, N., Elfalleh, W., Saadaoui, E., Khaldi, A., Triki, S. and Nasri, N. (2011). The caper (Capparis L.): Ethnopharmacology, phytochemical and pharmacological properties. Fitoterapia 82(2): 93-101.


刺槐树Capparis蜕膜的各个部分已经在传统医学中得到应用。然而,根皮中所含的密码子类生物碱的分离和结构阐释并不是微不足道的,并且可能导致过去的误解。该方案描述了Capparis decidua的根皮的四种主要生物碱的提取和色谱分离。递送的cadabicine,codonocarpine,异香豆香碱和capparidisinine的样品适合通过NMR,MS和MS / MS明确的结构阐明。

背景 Capparis id藜树分布在非洲,中东和南亚的干旱地区,其中植物的各个部分通常用于当地民间药物治疗各种疾病。例如,根皮被应用为驱肠和泻药,并且已经显示其酒精提取物具有显着的抗菌和抗真菌活性(Singh等人,2011; Singh和Singh,2011; Tlili等人,2011; Mohammed等人,2015)。
    Ahmad 等人。 (1985; 1986; 1987; 1989; Arif,1986)深入研究了Capparis decidua的根皮提取物,并发表了几种结构的密码子生物碱。这些结构中的一些不符合我们对这种生物碱的生物合成的理解。此外,我们不相信给定的分析数据及其解释给出了结构性声明的明确证据(Bienz et al。,2002)。所以我们开始了自己的调查。我们制定了密码子生物碱提取和色谱分离方案,并且可以分离三种生物碱级分,这些生物碱级分允许鉴定和结构阐明卡拉底菌葛根根皮的四种主要亚精胺生物碱:cadabicine( 1),密酮皂草苷(2),异香豆素(3)和capparidisinine(4)(图1)(Forster等人,2016)。

图1.卡帕斯藜藜根皮中发现的四种主要的密码子草酮生物碱。(1)加巴二菌素; (2)Codonocarpine; (3)异臭果香;和(4)卡帕西星。

 生物碱的分离和结构解释不是微不足道的。生物碱类,定义为含有大多数基本特征的天然来源的含氮有机化合物,包含超过10,000种化合物。由于生物碱的性质和结构与化合物的差异很大,所以没有一般​​的方法来分离它们。然而,大多数生物碱天然存在于其质子化形式中。因此,经常应用干燥和粉碎的植物材料的甲醇萃取。由于生物碱通常具有可与溶剂和添加剂以及CO 2或空气中的氧气反应的氨基和其它官能团,所以通过选择合适的方法可以尽可能地防止人造物的形成条件。通常,通过比较分离级分和原始样品的分析数据可以识别人为的物质。因此,一些原始样本应始终作为参考资料(Hesse,2000)。

关键字:凯尔树, 山柑科, 亚精胺生物碱, Cadabicine, Codonocarpine, Isocodonocarpine, Capparidisinine, 色谱法分离


  1. 棉绒(例如,Thomas Scientific,目录号:2904W25)
  2. 海砂(方英石,Brenntag Schweizerhall AG)(例如,格罗格化学公司,目录号:G890)
  3. 圆底试管,约100 x 16毫米(助手,目录号:42775045)
  4. TLC板(在20×20cm铝上的TLC硅胶60°F 254°)(EMD Millipore,目录号:100390)
  5. 玻璃毛细管(MARCHEREY-NAGEL,目录号:814022)
  6. 2 ml 9 mm ScreVial,透明玻璃12 x 32 mm(Infochroma,目录号:G004-HP-H)
  7. 9毫米毫米无色无氟PTFE /硅胶/PTFE隔膜的螺帽(Infochroma,目录号:G004-HP-CB-FKSKFK10)
  8. 1.5ml Eppendorf管(Eppendorf,目录号:0030120086)
  9. 植物材料:Capparis蜕膜根瘤皮(巴基斯坦Sahiwal收集),巴基斯坦巴哈瓦尔布尔伊斯兰教大学Cholistan沙漠研究所M。Waris先生鉴定;
  10. MeOH(甲醇,技术级,使用前蒸馏)(Thommen Furler,目录号:203-VL54TE)
  11. SiO 2(Merck硅胶60 [40-63μm])(EMD Millipore,目录号:109385)
  12. 氢氧化铵(NH 3,氨水溶液25%,puriss p.a.)(Honeywell,目录号:30501)
  13. 氯仿(CHCl 3,用EtOH稳定)(Scharlab,目录号:CL02181000)
  14. 二氯甲烷(CH 2/2 Cl 2),技术级,使用前蒸馏)(Thommen-Furler,目录号:739-VL54TE)
  15. (Honeywell,目录号:32203)
    的Diethylether(Et 2 O,puris p.a.,用BTH稳定,用NaOH蒸馏) 注意:
    1. 纯乙醚对氧化敏感,长时间暴露于空气和光线时可形成爆炸性过氧化物。因此,通常用抗氧化剂丁基羟基甲苯(BHT)稳定,蒸馏除去。因此,蒸馏醚再次易于形成环氧化物,如果溶剂暴露于空气和光照几天,则需要在使用前对蒸馏醚进行过氧化物测试。
    2. 为了测试过氧化物,请使用:Quantofix ®过氧化物试棒(Sigma-Aldrich,目录号:Z101680)。
  16. 甲胺溶液(MeNH 2 O,40wt。%)(Sigma-Aldrich,目录号:426466)
  17. 盐酸(HCl,37%)(用于分析,默克)(EMD Millipore,目录号:100317)
  18. 乙腈(MeCN,LC-MS Ultra CHROMASOLV )(Honeywell,目录号:14261)
  19. 甲酸(HCO 2 H,99%,ULC/MS)(Biosolve,目录号:069141)
  20. 2-丙醇(LC-MS Ultra CHROMASOLV ®)(Honeywell,目录号:650447)
  21. 氢氧化钠溶液(HPCE的NaOH,H 2 O的0.1N)(例如,MSP KOFEL,目录号:5062-8575)
  22. H 2 PtCl 6
  23. KI
  24. Ce(SO 4)2
  25. H 2 2< 4>< 4>
  26. Schlittler试剂(参见食谱)
  27. Ce(SO 4)2< 2>溶液(参见食谱)


  1. 锋利的斧头(例如,,TRANSA,目录号:040929-001001)
  2. 包覆的发酵罐(50L,例如Braupartner,目录号:141-0)
  3. 过滤器滤布(75×75cm,例如,ErwinMüller,目录号:i10014278)
  4. 不锈钢刮刀(例如,Thomas Scientific,目录号:1232X12)
  5. 旋转蒸发器(BüchiRotavapor R-134,BüchiWaterbath B-481)(Büchi,Flawil,瑞士)
  6. 玻璃漏斗(例如,,Duran,目录号:21 351 23)
  7. 多种尺寸的圆底烧瓶(5ml,10ml,25ml,50ml,250ml,1L)(例如Schott,德国)
  8. 钢弹簧夹(例如,,托马斯科学)
    NS 14.5(例如,Thomas Scientific,目录号:1178Z55)
    NS 29(例如,Thomas Scientific,目录号:1178Z57)
  9. 磁力搅拌棒(例如,Thomas Scientific,目录号:8608S78)
  10. 磁力搅拌器(Heidolph Instrument,目录号:505-30080-00)
  11. 玻璃瓶1升(例如,Thomas Scientific,目录号:1228R95)
  12. 具有不同长度和直径的玻璃色谱柱,底部带有旋塞,顶部具有接地插座接头以安装溶剂容器(例如,Neubert Glas,目录号:1196-29-20400)< br />
  13. 几种尺寸的玻璃烧杯(例如德国杜兰德国)
  14. 一件真空软管(例如,Thomas Scientific,目录号:9544T15)
  15. 溶剂储存器:具有相反连接的地锥和接地插座接头的几种尺寸的玻璃圆底烧瓶(例如,Thomas Scientific,目录号:1197C03)
  16. 适配器:
    地面锥形接头NS 14.5,带有管道的管道,弯曲(例如,Thomas Scientific,目录号:1195C09)
    接地锥形接头NS 29,带有管道的软管,弯曲(例如,LabMarket,目录号:1681029)
  17. 手动泵(VWR,目录号:612-9952)
  18. 125ml锥形瓶(例如,Thomas Scientific,目录号:4907F23)
  19. 试管架(例如,Thomas Scientific)
  20. TLC室(螺帽玻璃罐,250毫升)
  21. UV灯(254nm),例如UV灯4,双(Camag,型号:022.9160)
  22. 高真空泵(Alcatel Pascal 2015 SD)(例如,,Ideal Vaccum Products,目录号:P102310)
  23. Cortecs TM 具有Cortecs 的Cortecs 18mm,1.6μm,2.1×150mm(Waters,目录号:186007117) sup> TM UPLC > C 18>1.6μm,2.1×5mm VanGuard TM预柱(Waters,目录号:186008713 )
  24. Aquity TM Ultra Performance LC(Waters,Milford MA,USA)
  25. Bruker maXis Q-Tof HR-MS(Bruker Daltonics,Bremen,Germany)
  26. Eppendorf反渗透微量离心机(Eppendorf,型号:5417R)
  27. MilliQ梯度仪(用于HPLC的去离子水)(EMD Millipore,目录号:Z00Q0V0WW)


  1. 根皮提取
    1. 在树荫下将根皮干燥30-35°C一周。
    2. 用锋利的斧头细碎树皮,并用封盖的发酵罐中的MeOH(1:1,w/v)提取1周。每天搅拌混合几次。如果可能,请用机械搅拌器永久搅拌您的萃取混合物 注意:在我们的例子中,提取是在环境温度(巴基斯坦30-35°C)的常规实验室条件下进行的。
    3. 通过紧密编织的布过滤掉固体,并将旋转蒸发器上的MeOH在30℃和70毫巴下蒸发数小时,其中提取物变得高粘度。由于不可能完全除去MeOH,因此当不再可以观察到提取液中形成气泡时,停止溶剂的蒸发。
    4. 将几乎黑色粘稠的提取物储存在4°C直至使用。

  2. 生物碱的色谱分离:一般程序
    二氧化硅(SiO 2)上密码子香草型生物碱的色谱分离涉及至少四个色谱分离,遵循以下所有主要原理:
    1. 样品在SiO 2上的吸附
      粗提取物和生物碱馏分在少量的MeOH中难溶,甚至在用于色谱的洗脱液中溶解性较差。然而,将提取物以尽可能小的体积应用到色谱柱上是非常重要的。因此,混合物在被带到柱上之前被吸附在SiO 2上。
      1. 将样品转移到圆底烧瓶中并加入MeOH(1:4,w/v)。取大圆底烧瓶(我们为10.8克提取物使用250毫升烧瓶),以确保玻璃表面足够大以确保溶剂快速有效的蒸发。加入磁力搅拌棒并剧烈搅拌,直到获得均匀(仍然是非均质的)混合物。
      2. 在搅拌的同时,加入SiO 2(样品/SiO 2 <1:2,w/w),继续搅拌5分钟。
      3. 取出搅拌棒,然后取出旋转蒸发器上的MeOH(30℃,160毫巴),直到得到明亮的棕色粉末。
    2. 制备洗脱剂混合物和色谱柱
      1. 以干净的玻璃瓶将洗脱组分以给定的比例混合(表1-5)。在使用前蒸馏溶剂至关重要。
      2. 准备一列,如图2所示。

      图2.色谱柱的包装柱子是棉绒(A),海砂(B),SiO 2(C),附加海砂(D)和SiO 2(E)上的吸附样品。

      1. 用大约5厘米的洗脱液填充柱子,并用一些棉绒松散地密封底部的空气(图2A),以防止SiO 2流出。
      2. 添加1厘米的海沙(图2B),以便您获得一个平面。
      3. 在烧杯中,在洗脱液中制备轻微粘稠,无气泡的SiO 2悬浮液。加入洗脱剂,直到SiO 2覆盖良好,并用刮刀搅拌,直到所有空气逸出。
      4. 将SiO 2悬浮液通过玻璃漏斗仔细地填充到玻璃柱中,不会干扰海砂的表面,以获得松散的SiO 2柱(图2C) 。
      5. 打开旋塞阀,让洗脱液逸出,直到溶剂表面正好到达SiO 2的表面。关闭旋塞。
      6. 通过用一块真空软管攻丝玻璃柱,压制SiO 2柱。敲击时可以观察到SiO 2水平下降。不时打开旋塞阀将溶剂再次排出至SiO 2表面。继续压实,直到SiO 2表面不再沉没。
      7. 加入约5厘米的溶剂,并将大约2厘米的海砂(图2D)通过溶剂滴入到柱的表面上,以保护色谱柱免受机械扰动。排出溶剂,直到其表面到达海沙表面。
      8. 将吸附物质的棕色粉末涂抹在色谱柱上(图2E),仔细加入2厘米的洗脱液,然后小心地排出洗脱液,直至吸附的萃取物达到SiO 2的表面。重复添加和排出溶剂三次。
      9. 小心地用洗脱液填满色谱柱,连接溶剂储存器,用夹子固定,并用洗脱液填充储存器。
    3. 色谱分离
      1. 要开始分离,打开旋塞并将洗脱液的流量调整到适当的速率。根据列的大小,这应该是大约。 12ml min -1(粗分馏)或约4ml min -1(精细分馏)。如果使用完全打开的旋塞阀,流量太低,用手动泵施加轻微但恒定的压力。
      2. 收集合理体积的分数。要选择的体积取决于色谱柱的大小和色谱的预期目的。对于粗大的原始提取物在大柱上进行初步粗略分离,将100ml级分收集在锥形瓶中是合适的。对于较小的色谱柱进行精细分离,将10毫升收集在圆底试管中。由于感兴趣的生物碱可以容易地识别为柱上的黄色条带,所以通常在锥形瓶中收集明确无色的前馏分。
    4. TLC分级控制
      1. 将一小部分用玻璃毛细管转移到TLC板上,让斑点干燥。您可以在几次使用相同的毛细管之间重复使用相同的毛细管。
      2. 将TLC板置于TLC室(图3)中,3-5毫米以下洗脱液之一:
        1. CH 2 2 Cl 2/MeOH/NH 3 40:13:1,以测试您的级分是否含有任何亚精胺生物碱。亚精胺生物碱的典型的 R 值如图3所示。
        2. MeOH/H 2 O/NH 3 15:2:1,以测试您的馏分中的哪些亚精胺生物碱。这种洗脱液由Kyburz(1985)开发,是唯一被发现区分不同质量的生物碱的洗脱液。图3给出了亚精胺生物碱的典型值 f 值。
      3. 使TLC发展直到溶剂前端几乎达到板的顶部,从室中取出TLC板,使其干燥直到板不再具有NH 3的气味。
      4. 在紫外线灯(254 nm)下检查UV活性点,并用铅笔标记。
      5. 用Schlittler试剂染色您的TLC板并干燥。亚精胺生物碱会产生红色/紫色斑点。当干燥的Schlittler活性斑点随后用Ce(SO 4)2< 2>溶液染色时,斑点的颜色变深。根据我们的经验,紫外线检测是最敏感的方法。结合Schlittler试剂的染色,感兴趣的生物碱的斑点非常明确和清楚。 Ce(SO 4)2 的处理是有帮助的,但不是必需的。

        图3. TLC室和具有两种最有用的洗脱液的示意图TLCs用于检测亚精胺生物碱。为了在初步粗量色谱中检测生物碱级分,使用洗脱液系统CH 2 (不产生单独的生物碱的分离),并且为了细分离生物碱,使用洗脱液系统MeOH/H 2 O/NH 3 15:2:1(分离各种生物碱,右)。然而,密码子(2)和异香瓜素(3)通过TLC仍然无法区分。

    5. 馏分的组合和溶剂的蒸发
      1. 根据它们的TLC组合级分。注意不要将含有生物碱混合物的馏分与纯生物碱馏分进行混合。注意生物碱的色谱非常接近,并且主要成分旁边的低丰度生物碱可能不容易被TLC识别!非常少量的生物碱只能通过紫外光检测,但不能与Schlittler染色试剂一起检测
      2. 在旋转蒸发器上在30°C下蒸发溶剂(取决于洗脱液的压力),并在高真空(23°C,4×10 -4)上清除残留溶剂。
      3. 通过HPLC-MS控制您的干燥样品(参见数据分析)。用提取的离子色谱(EIC)m/z 436.2231(1),m/z 466.2237(2/3)和m/z 496.2442(4)检查其最终生物碱组合物。

  3. 四种主要生物碱的色谱分离细节
    1. 路径A:分离cadabicine(1)和codonocarpine(2)/异香草香(3)
      分离路径A在图4中示意性地示出。将各种初步色谱(A)的含生物碱的级分在A1中合并。 A1的色谱分离导致四个主要部分。其中三个含有不同比例的1和2/3的混合物(A1.1主要是1,A1.2可比量为1和2/3,A1.3主要为2/3)。第四部分(A1.4)主要含有生物碱4,还有2/3。由于所得到的级分均不含有纯生物碱,所以将样品A1.1和A1.3作为储备材料保存,并且在SiO 2上进一步分离最大级分(A1.2),得到生物碱1(A1.2.1)和2/3(A1.2.3),纯度高(> 98%)。


      表1. A1的色谱分离

      表2. A1.2的色谱分离

    2. 路径B:Capparidisinine(4)的分离
      分离路径B在图5中示意性地示出。粗的甲醇提取物(B)在SiO 2上大致分馏,以将含生物碱的级分(B1)与进一步提取的物质分离。将生物碱混合物B1进行色谱分离,得到含有不同比例的1和2/3(B1.1-B1.3)和1份仅含有一种生物碱(4)的三种级分。部分B1.4中的生物碱4被一些未鉴定的Schlittler无活性化合物(通过1 H-NMR检测)污染。额外的色谱步骤(B1.4.1)不能将生物碱4与其污染物分离。最终通过用少量的MeOH洗涤样品B1.4.1来获得卡派里沙星(4,B1.4.1.1)。

      图5.分离途径B。概述从粗提物(B)到生物碱4(B1.4.1。)的色谱步骤,包括洗涤步骤,以获得纯形式的capparidisinine(4,B1 .4.1.1),其结构被阐明。混合部分中的主要生物碱被加下划线。

      表3. B的色谱分离

      表4. B1的色谱分离

      表5. B1.4的色谱分离

    3. 进一步净化B1.4.1
      将B1.4.1(77mg)的黄色固体置于1.5ml Eppendorf管中,加入MeOH(0.3ml)。将混浊混合物小心摇动,然后离心(1分钟,20,817×g)。用吸管将黄色溶液与剩余的固体小心分离。将固体用MeOH洗涤三次(每次0.2ml)。将剩余的固体转移到圆底烧瓶中,真空干燥(30℃,160mbar,然后23℃,0.2mbar),得到明亮的黄色固体B1.4.1.1(4 ,15mg)。

  4. 转化为盐酸盐
    1. 将生物碱悬浮于MeOH中。
    2. 滴加1.5当量的水溶液HCl(0.1或1.0N)至浑浊溶液。解决方案变得清晰。
    3. 真空蒸发溶剂(30℃,160毫巴,然后23℃,4×10 -4平方毫米)。


    1. 通过HPLC-ESI-MS分析生物碱级分。将样品溶于MeOH + 0.1%HCO 2 H,0.5ml/min,粗提取物和0.1mg ml -1重量份,用于纯化的生物碱。以1μl的体积注射样品,并在RP C 18+柱上以0.3ml/min的流速进行色谱分离,用溶剂A(H <亚O 2 + 0.1%HCO 2 H)和B(MeCN + 0.1%HCO 2 H)在25℃下反应。梯度开始于10%B等静5分钟,然后在4分钟内升至25%B。柱用100%B洗涤2分钟,然后在5%B下平衡6分钟
    2. 连接的Q-Tof MS装有ESI源。在50-800m/z的正电离模式下,在以下条件下记录MS:HV端板偏移500V,HV毛细管3,000V,喷雾气体(N 2)2.0巴,干燥气体(N 2)10.0巴,干燥温度200℃。用2mM甲酸钠(980μlH 2 O/2-丙醇1:1,20μl0.1N NaOH和1μlHCO 2 H),相对质量误差通常低于2ppm(外部)
    3. 用35.00eV的碰撞能量和4.00的隔离宽度记录MS/MS谱
    4. 使用DataAnalysis评估色谱图和光谱。基于精确质量±0.02 m/z,提取萃取离子色谱图(EIC)。
    例如,粗提物B的基峰色谱图(BPC)如图6所示。生物碱相关EIC如下所示。提取物含有三种形式的密码子型生物碱:(1)糖苷衍生物(2.3-2.7分钟),(2)(E/Z) - 衍生物(3/3),其可以是人工制品,和(3)主要(E/E) - 生物碱(R t) 4.2-4.4 min),用我们的方案分离。
    虽然cadabicine(1)和capparidisinine(4)可以与HR-MS区别,但是对于密码子(2)和异香豆素(3)而言并不是这样。我们发现密码子(2)的保留时间(4.2分钟)比异香豆素(3,4.3分钟)略小。 (2)和(3)的MS/MS光谱几乎相同。它们仅在m/z 218.08058处仅有一个低密度信号,这对于密码子(2)是唯一的。但要小心;两种化合物都显示在m/z 218.11713处的信号。
    注意:有关MS/MS光谱和NMR光谱解释的详细讨论,请检查"卡帕斯藜甙的Codonocarpine类型生物碱的新视野"(Forster 等> ,2016)。





  1. Schlittler试剂(Schlittler和Hohl,1952)
    将H 2 PtCl 6 N(1.0g,在H 2 O [6ml])中的混合物,KI(22.5g, 2 O [225ml]),水溶液HCl溶液(1N,20ml)用去离子H 2 O 2稀释至总体积为1000ml
    胺类:棕色,多种色调(黄 - 红 - 蓝灰色)
    酰胺:亮黄色,其次是Ce(SO 4)2 :棕色
  2. Ce(SO 4)2< 2><><<> 将Ce(SO 4)2(10.0g)的浓度溶液将H 2 SO 4(55ml)用去离子H 2 O 2稀释至总体积为1000ml


我们非常感谢李博士博士在收购MS和MS/MS数据方面的大力支持,我们感谢巴基斯坦高等教育委员会(HEC)(1-8/HEC/HRD/2013/2582, PIN:IRSIP 23 Ps 48),为A. Ghaffar对苏黎世大学进行为期6个月的研究考察。


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  2. Ahmad,VU,Arif,S.,Amber,AR和Nasir,MA(1986)。来自Capparis decidua的根皮的新生物碱 ZeitschriftfürNaturforschung B 41(8):1033-1035。
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引用:Forster, Y., Ghaffar, A. and Bienz, S. (2017). Chromatographic Separation of the Codonocarpine Type Alkaloids from the Root Bark of Capparis decidua. Bio-protocol 7(4): e2144. DOI: 10.21769/BioProtoc.2144.