**4.4. High, Medium and Low Performance Liquid Chromatography (HPLC, MPLC and LPLC)**

Studies on HPLC with iridoids of Apocynaceae focus mainly on the separation of components from extracts or fraction. The chromatography profile, the identification and quantification of these terpenes in the extracts are described. Table 4 shows the principal references on iridoids isolated from Apocynaceae by HPLC, MPLC and LPLC.



form and butanol. Plumericin (420 mg) was directly obtained from the benzene fraction (0.02% yield). The butanol fraction was subjected to sequential chromatographic steps, using XAD-2 column and gradient of water/methanol (mobile phase), silica gel column and solvent system containing chloroform/methanol/water or chloroform/methanol, followed by droplet current chromatography with chloroform/methanol/ water. Plumieride (1.3 g) and protoplumericin (13.2 g) were obtained with yields of 0.08% and 0.83%, respectively.

A General Description of Apocynaceae Iridoids Chromatography

http://dx.doi.org/10.5772/55784

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The iridoids allamcidin B *β*-D-glucoside, plumiepoxide and protoplumericin B were isolated from *Allamanda neriifolia* extract obtained by percolation with methanol, also by droplet counter-current chromatography [39]. The crude methanol extracts from 2.6 kg of stem and 6.7 kg of leaves were sequentially fractionated with benzene, chloroform and butanol. Previous chromatographic treatment with MCI gel (elution with water/methanol), silica gel column (mobile phases: chloroform/methanol/water; benzene/acetone; ethyl acetate/methanol/ water and ethyl acetate/hexane) and Sephadex LH20 column (mobile phase: chloroform/methanol) led to the isolation of isoallamandicin (10 mg from the stem), allamcin (230 mg from the leaves), 3-*O*-methylallamcin (30 mg from the leaves), allamancin (102 mg from the stem), 3-*O*methylallamancin (41 mg from the leaves), allamcidin (125 mg from the leaves), plumieride 13-*O*-acetate (760 mg from the stem and *ca.* 2 g from the leaves). Fractions of butanol extracts from the stem and leaves were subjected to droplet counter-current chromatography using chloroform/methanol/water (5:6:4, ascending mode) to obtain allamcidin B *β*-D-glucoside (17 mg from the stem), plumiepoxide (7 mg from the stem and 374 mg from the leaves) and

Iridoids can be isolated from *Plumeria acutifolia* roots following a similar methodology [66]. Successive liquid-liquid partitions of the crude methanol extract (obtained from 6 kg of plant material) with benzene, chloroform and butanol, followed by several chromatographic steps for fractionation of the butanol fraction (using polystyrene, silica gel and octadecyl silica columns) led to the isolation of 13-*O-*coumaroylplumieride (43 g), plumieride (7.5 g), 13-Ocaffeoylplumieride (60 mg), 1*α*-plumieride (20 mg) and protoplumericin A (9 g). A further purification step involving droplet counter-current chromatography and a mixture of chloro‐ form/methanol/water (4:6:5, ascending mode) led to the isolation of 13-deoxyplumieride

For analytical purposes, iridoids can be analyzed by capillary electrophoresis. A method to separate nine iridoids described in [131] uses a Hewlett-Packard (HP3D CE) capillary electro‐ phoresis system coupled to a photodiode array detector (210 nm and 230 nm) and equipped with a fused-silica capillary tube (90 cm × 75µm I.D.). The distance to the detector was 81.5 cm. Other conditions: sample injection at 50 mbar for 3 s and further deionized water injection at 50 mbar for 3 s; constant voltage, 16 kV (positive to negative); cartridge temperature, 30 °C; electrolyte (buffer), 50 mM sodium borate and 30 mg/mL 2,6-di-*O*-methyl-*β*-cyclodextrin (DMβ-CD); run time, 32 min. Before the analyses, the capillary column was sequentially purged with 0.5 M NaOH, 0.1 M NaOH, deionized water and buffer solution. The iridoids studied eluted in the following order: geniposide, loganin, shanzhiside, aucubin, catalpol, harpago‐

protoplumericin B (70 mg from the leaves).

**4.6. Capillary electrophoresis**

(200mg), plumenoside (50 mg) and 8-isoplumieride (700 mg).

**Table 4.** Separation by Pressure Liquid Chromatography

### **4.5. Counterflow**

Protoplumericin and plumieride can be extracted from the methanol extract of *Allamanda neriifolina* stems [117] by droplet counter-current chromatography. Crude extract, ob‐ tained from 1.6 kg of plant material, was successively partitioned with benzene, chloro‐ form and butanol. Plumericin (420 mg) was directly obtained from the benzene fraction (0.02% yield). The butanol fraction was subjected to sequential chromatographic steps, using XAD-2 column and gradient of water/methanol (mobile phase), silica gel column and solvent system containing chloroform/methanol/water or chloroform/methanol, followed by droplet current chromatography with chloroform/methanol/ water. Plumieride (1.3 g) and protoplumericin (13.2 g) were obtained with yields of 0.08% and 0.83%, respectively.

The iridoids allamcidin B *β*-D-glucoside, plumiepoxide and protoplumericin B were isolated from *Allamanda neriifolia* extract obtained by percolation with methanol, also by droplet counter-current chromatography [39]. The crude methanol extracts from 2.6 kg of stem and 6.7 kg of leaves were sequentially fractionated with benzene, chloroform and butanol. Previous chromatographic treatment with MCI gel (elution with water/methanol), silica gel column (mobile phases: chloroform/methanol/water; benzene/acetone; ethyl acetate/methanol/ water and ethyl acetate/hexane) and Sephadex LH20 column (mobile phase: chloroform/methanol) led to the isolation of isoallamandicin (10 mg from the stem), allamcin (230 mg from the leaves), 3-*O*-methylallamcin (30 mg from the leaves), allamancin (102 mg from the stem), 3-*O*methylallamancin (41 mg from the leaves), allamcidin (125 mg from the leaves), plumieride 13-*O*-acetate (760 mg from the stem and *ca.* 2 g from the leaves). Fractions of butanol extracts from the stem and leaves were subjected to droplet counter-current chromatography using chloroform/methanol/water (5:6:4, ascending mode) to obtain allamcidin B *β*-D-glucoside (17 mg from the stem), plumiepoxide (7 mg from the stem and 374 mg from the leaves) and protoplumericin B (70 mg from the leaves).

Iridoids can be isolated from *Plumeria acutifolia* roots following a similar methodology [66]. Successive liquid-liquid partitions of the crude methanol extract (obtained from 6 kg of plant material) with benzene, chloroform and butanol, followed by several chromatographic steps for fractionation of the butanol fraction (using polystyrene, silica gel and octadecyl silica columns) led to the isolation of 13-*O-*coumaroylplumieride (43 g), plumieride (7.5 g), 13-Ocaffeoylplumieride (60 mg), 1*α*-plumieride (20 mg) and protoplumericin A (9 g). A further purification step involving droplet counter-current chromatography and a mixture of chloro‐ form/methanol/water (4:6:5, ascending mode) led to the isolation of 13-deoxyplumieride (200mg), plumenoside (50 mg) and 8-isoplumieride (700 mg).
