Materials and Reagents

Cotton wool (e.g., Thomas Scientific, catalog number: 2904W25 )
Sea sand (cristobalite, Brenntag Schweizerhall AG) (e.g., Grogg Chemie, catalog number: G890 )
Round bottom test tubes, ca. 100 x 16 mm (Assistant, catalog number: 42775045 )
TLC plates (TLC Silica gel 60 F₂₅₄ on aluminum 20 x 20 cm) (EMD Millipore, catalog number: 100390 )
Glass capillaries (MARCHEREY-NAGEL, catalog number: 814022 )
2 ml 9 mm ScreVial, clear glass 12 x 32 mm (Infochroma, catalog number: G004-HP-H )
9 mm screw cap with 1 mm pigment-free ms-pure PTFE/Silicone/PTFE-Septum (Infochroma, catalog number: G004-HP-CB-FKSKFK10 )
1.5 ml Eppendorf tubes (Eppendorf, catalog number: 0030120086 )
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)
MeOH (methanol, technical grade, distilled prior to use) (Thommen Furler, catalog number: 203-VL54TE )
SiO₂ (Merck silica gel 60 [40-63 μm]) (EMD Millipore, catalog number: 109385 )
Ammonium hydroxide (NH₃, aqueous ammonia solution 25%, puriss p.a.) (Honeywell, catalog number: 30501 )
Chloroform (CHCl₃, stabilized with EtOH) (Scharlab, catalog number: CL02181000 )
Dichloromethane (CH₂Cl₂, technical grade, distilled prior to use) (Thommen-Furler, catalog number: 739-VL54TE )
Diethylether (Et₂O, puriss. p.a., stabilized with BTH, distilled from NaOH prior to use) (Honeywell, catalog number: 32203 )
Notes:

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.
To test for peroxides use: Quantofix^® peroxides test sticks (Sigma-Aldrich, catalog number: Z101680 ).

Methylamine solution (MeNH₂, 40 wt. %) (Sigma-Aldrich, catalog number: 426466 )
Hydrochloric acid (HCl, 37%) (for analysis, Merck) (EMD Millipore, catalog number: 100317 )
Acetonitrile (MeCN, LC-MS Ultra CHROMASOLV^®) (Honeywell, catalog number: 14261 )
Formic acid (HCO₂H, 99%, ULC/MS) (Biosolve, catalog number: 069141 )
2-propanol (LC-MS Ultra CHROMASOLV^®) (Honeywell, catalog number: 650447 )
Sodium hydroxide solution (NaOH for HPCE, 0.1 N in H₂O) (e.g., MSP KOFEL, catalog number: 5062-8575 )
H₂PtCl₆
KI
Ce(SO₄)₂
H₂SO₄
Schlittler reagent (see Recipes)
Ce(SO₄)₂ solution (see Recipes)

Equipment

Sharp axe (e.g., TRANSA, catalog number: 040929-001001 )
Capped fermentation tank (50 L, e.g., Braupartner, catalog number: 141-0 )
Filter strainer cloth (75 x 75 cm, e.g., Erwin Müller, catalog number: i10014278 )
Stainless steel spatula (e.g., Thomas Scientific, catalog number: 1232X12 )
Rotary evaporator (Büchi Rotavapor R-134 with Büchi Waterbath B-481) (Büchi, Flawil, Switzerland)
Glass funnels (e.g., Duran, catalog number: 21 351 23 )
Round bottom flasks of several sizes (5 ml, 10 ml, 25 ml, 50 ml, 250 ml, 1 L) (e.g., Schott, Germany)
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 )
Magnetic stir bars (e.g., Thomas Scientific, catalog number: 8608S78 )
Magnetic stirrer (Heidolph Instrument, catalog number: 505-30080-00 )
Glass bottle 1 L (e.g., Thomas Scientific, catalog number: 1228R95 )
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 )
Glass beaker of several sizes (e.g., Duran, Germany)
A piece of vacuum hose (e.g., Thomas Scientific, catalog number: 9544T15 )
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 )
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 )
Hand pump (VWR, catalog number: 612-9952 )
125 ml Erlenmeyer flasks (e.g., Thomas Scientific, catalog number: 4907F23 )
Test tube racks (e.g., Thomas Scientific)
TLC chamber (screw cap glass jar, 250 ml)
UV lamp (254 nm), e.g., UV lamp 4, dual (Camag, model: 022.9160 )
High vacuum pump (Alcatel Pascal 2015 SD) (e.g., Ideal Vaccum Products, catalog number: P102310 )
Cortecs^TM UPLC^® C₁₈₊, 1.6 μm, 2.1 x 150 mm (Waters, catalog number: 186007117 ), equipped with a Cortecs^TM UPLC^® C₁₈₊ 1.6 μm, 2.1 x 5 mm VanGuard^TM precolumn (Waters, catalog number: 186008713 )
Aquity^TM Ultra Performance LC (Waters, Milford MA, USA)
Bruker maXis Q-Tof HR-MS (Bruker Daltonics, Bremen, Germany)
Eppendorf refrigerated microcentrifuge (Eppendorf, model: 5417R )
MilliQ gradient apparatus (deionized water, for HPLC) (EMD Millipore, catalog number: Z00Q0V0WW )

Procedure

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.
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.
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
4. Fraction control by TLC
5. Combination of the fractions and evaporation of the solvent
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).
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

HPLC-ESI-MS
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 (R_t 2.3-2.7 min), (2) the (E/Z)-derivatives (R_t 3.4-3.7 min), which may be artefacts, and (3) the major (E/E)-alkaloids (R_t 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.

Notes

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.

Recipes

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
Colouring,
Amines: brown in diverse shades (yellow-red-blue-grey)
Amides: bright yellow, followed by Ce(SO₄)₂: brown
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
Colouring,
Amines: (together with Schlittler reagent) brown
Amides: (together with Schlittler reagent) intensifies the staining colour of the Schlittler reagent

Acknowledgments

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.

References

Ahmad, V. U., Arif, S., Amber, A. R. and Fizza, K. (1987). Capparisinine, a new alkaloid from Capparis decidua. Liebigs Ann der Chemie 2: 161-162.
Ahmad, V. U., Arif, S., Amber, A. R. and Nasir, M. A. (1986). A new alkaloid from root bark of Capparis decidua. Zeitschrift für Naturforschung B 41(8): 1033-1035.
Ahmad, V. U., Arif, S. Amber, A. R., Usmanghani, K. and Miana, C. A. (1985). A new spermidine alkaloid from Capparis decidua. Heterocycles 23: 3015-3020.
Ahmad, V. U., Ismail, N. and Amber, A. R. (1989). Isocodonocarpine from Capparis decidua. Phytochemistry 28: 2493-2495.
Arif, S. (1986). Studies on alkaloids of Capparis decidua. Dissertation.
Bienz, S., Detterbeck, R., Ensch, C., Guggisberg, A., Häusermann, U., Meisterhans, C., Wendt, B., Werner, C. and Hesse, M. (2002). The Alkaloids, Vol 58. Academic Press pp: 83-338.
Forster, Y., Ghaffar, A. and Bienz, S. (2016). A new view on the codonocarpine type alkaloids of Capparis decidua. Phytochemistry 128: 50-59.
Hesse, M. (2000). Alkaloide - Fluch oder Segen der Natur? Verlag Helvetica Chimica Acta.
Kyburz, R. (1985). Alkaloide aus Aristotelia peduncularis (Labill.) Hook. F. und aus Capparis decidua (Forsk.) Edgew. Dissertation.
Mohammed, M. S., Khalid, H. S., Muddathir, A. E., K. Tahir, El, Khan, A. A., Algadir, H. A., Ahmed Osman, J. W. and Siddiqui, N. A. (2015). Effect of some plants’ extracts used in Sudanese folkloric medicines on carrageenan-induced inflammation. Pak J Pharm Sci 28(1): 159-165.
Schlittler, E. and Hohl, J. (1952). Über die Alkaloide aus Strychnos melinoniana Baillon. Helv Cimica Acta 35: 29-45.
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.
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.
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.