**4.1. Thin layer chromatography**

Preparative chromatography was performed with thin layer chromatography (TLC) alumi‐ num sheets and 8:2 chloroform/ methanol as mobile phase of a fraction from the 95% EtOH extract of stems of *Alstonia macrophylla*. This procedure led to the isolation of the compounds sweroside (2 mg) and naresuanoside (3 mg) [128]

**SPECIES PLANT MATERIAL IRIDOIDS STATIONARY**

Isoplumericin

Isoplumericin

Roots Plumericin

cyclocerberidol-3-*O*-*β*-D-allopyranoside (14 mg).

**Table 3.** TLC analyses of Apocynaceae iridoids.

**4.2. Open column chromatography**

4-*epi*-alyxialactone

*Himatanthus sucuuba*

*Alyxia reinwardti*

*Allamanda cathartica*

**PHASE**

Latex Plumericin Si gel GF254 Hexane-EtOAc (6:4) [92]

Leaves Alyxialactone Si gel GF254 CHCl3-MeOH (95:5) [59]

Allamandicin 2- Si gel 2- CHCl3-MeOH (95:5) Allamdin 3- Si gel 3- ether-hexane (1:1)

The methanol extract of the leaves of *Cerbera manghas* and its fruit contain the iridoids theve‐ sideandtheviridoside,asdescribedin[124].Themethanolextractoftheleaves,aftertheaddition ofwater,was sequentiallypartitionedwithchloroform, acetic acidandbutanol.This extract and the aqueous phase were submitted to column chromatography with charcoal and water/ methanol as eluent. Theveside was isolated from the aqueous phase. Fractions of the butanol extract, which turn blue after heating withmineral acid, were chromatographedover a silica gel column with a gradient of increasing polarity of chloroform/methanol to afford theviridoside.

Cerberidol, epoxycerberidol, cyclocerberidol, cerberidol-3-*O*-*β*-D-allopyranoside, cerberi‐ dol-3,10-bis-*O*-*β*-D-allopyranoside, epoxycerberidol-3-*O*-*β*-D-allopyranoside and cyclocerber‐ idol-3-*O*-*β*-D-allopyranoside are present in the leaves of *C. manghas* [55]. The methanol extract from 1.45 kg of dried leaves was concentrated, re-suspended in water, and sequentially partitioned with benzene and chloroform. The aqueous phase was chromatographed using MCI-gel column (elution with gradients of water/methanol) and the fraction eluted with 20% methanol was chromatographed in two steps: using RQ-1 (Fuji-gel phase) column (elution with water/acetonitrile) and using a silica gel column (elution with gradient of chloroform/ methanol/water 7:3:1 to 7:3:1.2). Fractionation afforded cerberidol (75 mg), epoxycerberidol (10 mg), cyclocerberidol (160 mg), cerberidol-3-*O*-*β*-D-allopyranoside (135 mg), cerberi‐ dol-3,10-bis-*O*-*β*-D-allopyranoside (20 mg), epoxycerberidol-3-*O*-*β*-D-allopyranoside (27 mg) and cyclocerberidol-3-*O*-*β*-D-allopyranoside (250 mg). Some of these iridoids are also found in dried leaves of *Cerbera odollam* Gaertn [55]. The same methodology was used to isolate cerberidol (15 mg), cyclocerberidol (48 mg), cerberidol-3-*O*-*β*-D-allopyranoside (28 mg) and

The iridoids cerbinal, cerberic acid and cerberinic acid are found in the methanol extract of the bark of *C. manghas* [67]. The crude extract obtained by percolation from 4 kg of stem bark and 1.9 kg of root bark were diluted with water to 50% water:methanol. The mixture was washed with hexane and partitioned with benzene. Benzene fractions were re-suspended in methanol and cerbinal (120 mg from stem bark extract and 300 mg from root bark extract) precipitated.

**MOBILE PHASE REF.**

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163

1- Si gel 1- CHCl3 [41]

A General Description of Apocynaceae Iridoids Chromatography

Another interesting application of preparative chromatography is described in [47] with ethanol extract of ground stems of *A. schottii*. This extract was fractionated by bioassay monitoring and after successive liquid-liquid partition and flash column chromatography; the authors obtained the iridoids allamandin; allamcin and a mixture of plumericin and isoplu‐ mericin. This separation was performed with a Chromatotron rotor (silica gel (Si gel), 2-mm) with 1 % methanol in chloroform as solvent system.

The bioassay-guided fractionation of the extracts of *Plumeria rubra* barks also proved to be a successful strategy, leading to the isolation of eleven substances, of which eight were iridoids. In this context, the aqueous extract of *Plumeria rubra* bark has iridoids such as the epimers, *α*allamcidin and *β*-allamcidin, which were resolved by preparative TLC on Si-gel G plates (20 × 20 cm, 250µm,Merck®),usingchloroform/ ethyl acetate/methanol(3:3:1) as solvent system[44].

Ferreira and coworkers [95] describe the HPTLC analysis of the bark, latex and leaf extracts and substances of *Himatanthus sucuuba*. The solutions of the extracts in methanol (10 mg mL -1) and the isolated iridoids (3 mg mL-1 - plumieride; 1 mg mL -1 - isoplumieride) were applied (5 µL) using Linomat IV, a Camag semi-automatic spotter. The analysis was carried out on a precoated silica gel 60 F254 (Merck) HPTLC plate (0.2 mm of layer thickness and 10 × 10 cm size) using chloroform/ methanol (8:2) as developing system. The resulting chromatogram was dried and the spots were visualized by spraying with vanillin – sulfuric acid solution, followed by heating at 100 °C. Table 3 shows other studies using TLC analysis of Apocynaceae iridoids.



**Table 3.** TLC analyses of Apocynaceae iridoids.

**4. Chromatographic separation**

sweroside (2 mg) and naresuanoside (3 mg) [128]

with 1 % methanol in chloroform as solvent system.

**SPECIES PLANT MATERIAL IRIDOIDS STATIONARY**

6"-*O*-acetylplumieride *p*-Z-

Leaves 6"-*O*-acetylplumieride *p*-Ecoumarate

Stems Plumericin

coumarate

Isoplumericin

*Plumeria obtusa*

*Plumeria obtusa*

*Himatanthus fallax*

Preparative chromatography was performed with thin layer chromatography (TLC) alumi‐ num sheets and 8:2 chloroform/ methanol as mobile phase of a fraction from the 95% EtOH extract of stems of *Alstonia macrophylla*. This procedure led to the isolation of the compounds

Another interesting application of preparative chromatography is described in [47] with ethanol extract of ground stems of *A. schottii*. This extract was fractionated by bioassay monitoring and after successive liquid-liquid partition and flash column chromatography; the authors obtained the iridoids allamandin; allamcin and a mixture of plumericin and isoplu‐ mericin. This separation was performed with a Chromatotron rotor (silica gel (Si gel), 2-mm)

The bioassay-guided fractionation of the extracts of *Plumeria rubra* barks also proved to be a successful strategy, leading to the isolation of eleven substances, of which eight were iridoids. In this context, the aqueous extract of *Plumeria rubra* bark has iridoids such as the epimers, *α*allamcidin and *β*-allamcidin, which were resolved by preparative TLC on Si-gel G plates (20 × 20 cm, 250µm,Merck®),usingchloroform/ ethyl acetate/methanol(3:3:1) as solvent system[44].

Ferreira and coworkers [95] describe the HPTLC analysis of the bark, latex and leaf extracts and substances of *Himatanthus sucuuba*. The solutions of the extracts in methanol (10 mg mL -1) and the isolated iridoids (3 mg mL-1 - plumieride; 1 mg mL -1 - isoplumieride) were applied (5 µL) using Linomat IV, a Camag semi-automatic spotter. The analysis was carried out on a precoated silica gel 60 F254 (Merck) HPTLC plate (0.2 mm of layer thickness and 10 × 10 cm size) using chloroform/ methanol (8:2) as developing system. The resulting chromatogram was dried and the spots were visualized by spraying with vanillin – sulfuric acid solution, followed by heating at 100 °C. Table 3 shows other studies using TLC analysis of Apocynaceae iridoids.

**PHASE**

Flowers Plumieride coumarate glucoside Si gel 60G F254 CHCl3-MeOH (3:1) [118]

Plumeridoid B 2- Si gel GF254 2- Hexane-EtOAc (4%)

Plumieride CHCl3-MeOH (85:15)

*Plumeria rubra* Stem bark Plumeridoid A 1- Si gel GF254 1- Hexane-EtOAc (20%) [48]

**MOBILE PHASE REF.**

1- Si gel GF254 1- CHCl3-MeOH (8.5:1.5) [37]

Si gel 10% Me2CO in CHCl3 [89]

2- Si gel 2- Gradients of Petrol-EtOAc

**4.1. Thin layer chromatography**

162 Column Chromatography

### **4.2. Open column chromatography**

The methanol extract of the leaves of *Cerbera manghas* and its fruit contain the iridoids theve‐ sideandtheviridoside,asdescribedin[124].Themethanolextractoftheleaves,aftertheaddition ofwater,was sequentiallypartitionedwithchloroform, acetic acidandbutanol.This extract and the aqueous phase were submitted to column chromatography with charcoal and water/ methanol as eluent. Theveside was isolated from the aqueous phase. Fractions of the butanol extract, which turn blue after heating withmineral acid, were chromatographedover a silica gel column with a gradient of increasing polarity of chloroform/methanol to afford theviridoside.

Cerberidol, epoxycerberidol, cyclocerberidol, cerberidol-3-*O*-*β*-D-allopyranoside, cerberi‐ dol-3,10-bis-*O*-*β*-D-allopyranoside, epoxycerberidol-3-*O*-*β*-D-allopyranoside and cyclocerber‐ idol-3-*O*-*β*-D-allopyranoside are present in the leaves of *C. manghas* [55]. The methanol extract from 1.45 kg of dried leaves was concentrated, re-suspended in water, and sequentially partitioned with benzene and chloroform. The aqueous phase was chromatographed using MCI-gel column (elution with gradients of water/methanol) and the fraction eluted with 20% methanol was chromatographed in two steps: using RQ-1 (Fuji-gel phase) column (elution with water/acetonitrile) and using a silica gel column (elution with gradient of chloroform/ methanol/water 7:3:1 to 7:3:1.2). Fractionation afforded cerberidol (75 mg), epoxycerberidol (10 mg), cyclocerberidol (160 mg), cerberidol-3-*O*-*β*-D-allopyranoside (135 mg), cerberi‐ dol-3,10-bis-*O*-*β*-D-allopyranoside (20 mg), epoxycerberidol-3-*O*-*β*-D-allopyranoside (27 mg) and cyclocerberidol-3-*O*-*β*-D-allopyranoside (250 mg). Some of these iridoids are also found in dried leaves of *Cerbera odollam* Gaertn [55]. The same methodology was used to isolate cerberidol (15 mg), cyclocerberidol (48 mg), cerberidol-3-*O*-*β*-D-allopyranoside (28 mg) and cyclocerberidol-3-*O*-*β*-D-allopyranoside (14 mg).

The iridoids cerbinal, cerberic acid and cerberinic acid are found in the methanol extract of the bark of *C. manghas* [67]. The crude extract obtained by percolation from 4 kg of stem bark and 1.9 kg of root bark were diluted with water to 50% water:methanol. The mixture was washed with hexane and partitioned with benzene. Benzene fractions were re-suspended in methanol and cerbinal (120 mg from stem bark extract and 300 mg from root bark extract) precipitated. The supernatant was chromatographed on a silica gel column and cerbinal, cerberic acid and cerberinic acid were eluted with benzene/acetone.

gate the cytotoxic activity against various cancer cells, particularly murine lymphocytic leukemia cells (P-388). Petroleum ether extract (30 g) was submitted to flash column chroma‐ tography over silica gel (750 g, 230-400 mesh) with chloroform/petroleum ether (1:1) as eluent. Fraction 002 (200 mg) was rechromatographed using silica gel (120 g) and chloroform/ methanol (99:1) to isolate fulvoplumierin (25 mg after recrystallization from petroleum ether/ chloroform 1:1). The methanol extract (295 g) was partitioned between chloroform and water. The chloroform extract (60 g) was successively chromatographed in a silica gel column (1.5 kg) with gradients of increasing polarity, with chloroform and methanol, and then in a silica gel (600 g) with petroleum ether/chloroform/ethyl acetate (1:3:1) as eluent to furnish allamandin (12 mg after recrystallization from chloroform). Another fraction from the first column chromatography of the methanol extract (1.2 g) was purified in a silica gel column (400 g) and ethyl acetate/ methanol (97:3) and recrystallyzed from chloroform to furnish 9 mg of allamcin. The aqueous extract (200 g) was also subjected to column chromatography using silica gel column (2 kg) and gradient of chloroform and methanol. Plumieride (55 g) was obtained directly from fraction F 022 after recrystallization from methanol. Fraction F022 (29 g) also furnished 15-demethylplumieride and 13-*O*-*trans*-*p*-coumaroylplumieride (3 g) by column chromatography over silica gel and gradient of chloroform and methanol. Fraction F018 (800 mg) was purified over silica gel (400 g) with gradient of ethyl acetate and methanol to afford plumericin (18 mg after recrystallization with ethyl acetate). Another fraction (F019) was purified over silica gel (250 g) with ethyl acetate/chloroform/methanol (6:6:1) solvent system to furnish an unstable iridoid aldehyde and a mixture of *α*- and *β*-allamcidin (12 mg and 16 mg, respectively) further isolated by TLC on silica gel plates (20 cm x 20 cm, 250 µm) with ethyl

A General Description of Apocynaceae Iridoids Chromatography

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165

Plumericin, isoplumericin, plumieride, 13-*O*-coumaroylplumieride and protoplumericine A were isolated from the ethyl acetate extract of *P. rubra* heartwood [85]. The extract (44 g) obtained by percolation was submitted to column chromatography on silica gel with light petroleum ether/ethyl acetate (1:1) and methanol as eluent to furnish four fractions (A-D). Fraction C directly afforded plumericin (1.22 g) after recrystallization from toluene/ethyl acetate. Fraction B was rechromatographed on a silica gel with toluene/ethyl acetate (9:1), and isoplumericin (140 mg) was isolated from fraction 2. Fraction D was also rechromatographed on a silica gel column, but with chloroform/methanol/water (90:10:0.5 to 70:30:10) as solvent system. Fraction 3' was 13-*O*-coumaroylplumieride (2.0 g) and fraction 7' was protoplumeri‐ cine (1.6 g). Fraction 5' was subjected to Sephadex LH20 column eluted with methanol, followed by silica gel column eluted with chloroform/ methanol/water (85:15:0.7) to furnish

The iridoids, plumericin, isoplumericin, plumieride and fulvoplumierin, were present in the extracts of *Plumeria rubra* bark. After maceration of the powdered bark (3.5 kg) with dichloro‐ methane/methanol (1:1) and pure methanol, the combined extracts were partitioned between water and ethyl acetate. To isolate the four iridoids, the organic layer was chromatographed twice in a column using silica gel and gradient of increasing polarity with hexane and ethyl acetate, ethyl acetate and methanol, and then pure methanol. The amounts of the compounds

acetate/chloroform/methanol (3:3:1).

plumieride (120 mg).

isolated were not reported [75].

The iridoids isoplumericin, plumericin, plumieride, plumieride coumarate and plumieride coumarate glucoside can be detected and quantified in several species of *Plumeria* and *Allamanda* by TLC using silica gel 60 and the following mobile phases: benzene/ethyl acetate 4:1, chloroform/methanol 4:1, chloroform/methanol 7:3, propanol/ethyl acetate/water 7:2:1 [84]. Visualization of the chromatograms is achieved by spraying with 50% sulfuric acid/ ethanol solution and heating. For the analyses, it is necessary to use iridoids as standards that can be isolated from the roots of *Allamanda cathartica*. For the isolation of iridoids during the development of the method, chloroform and methanol extracts, sequentially obtained in a Soxhlet apparatus from the root bark and inner roots of *Allamanda cathartica* (15.0 g for each extract) were successively fractionated on column chromatography. In the first chromatogra‐ phy step, the chloroform extract from the bark (1.7 g) was applied to a silica gel column (80 g) and eluted with gradient of petrol, ethyl ether, chloroform and methanol. This procedure yielded a mixture containing isoplumericin and plumericin (160 mg). Fractions eluted with chloroform/ methanol (3:2) were mixed with the methanol extract of the bark (total mass: 2.8 g) and chromatographed on a silica gel column (150 g deactivated with water) with gradient of chloroform and methanol as mobile phase to furnish plumieride coumarate (150 mg), plumieride coumarate glucoside (480 mg) and two mixtures: one containing plumieride and plumieride coumarate, and the other containing plumieride and plumieride coumarate glucoside. The first mixture was further resolved on partition between water and ethyl acetate and afforded 180 mg of plumieride and 150 mg of plumieride coumarate. The other mixture was rechromatographed to afford plumieride (300 mg) and plumieride coumarate glucoside (410 mg). Neither the chloroform (810 mg) nor the methanol (920 mg) extracts from the inner part of the roots contained isoplumericin and plumericin. These extracts were purified on silica gel column (75 g) deactivated with water and eluted with chloroform and methanol gradient, to give plumieride (70 mg), plumieride coumarate (80 mg) and plumieride coumarate gluco‐ side (200 mg).

The fresh leaves of *Cerbera manghas* contain the iridoids 10-*O*-benzoyltheveside, 10-dehydro‐ geniposide, loganin, theviridoside and theveside [124]. For the isolation, the methanol extract obtained by percolation using 2.6 kg of fresh leaves was extracted with butanol, and this extract was partitioned with benzene. After partition, the remaining butanol fraction (22.8 g) was subjected to column chromatography using MCI-gel (CHP-20) as stationary phase and a gradient of methanol/water as eluent. The fraction eluted with 20% methanol was subjected to C-18 column (elution with acetonitrile/water) to afford 20 mg of 10-dehidrogeniposide and 20 mg of loganin. The fraction eluted with pure water (2.6g) in the first chromatographic step was also subjected to C-18 column (elution with acetonitrile/ water) to furnish 23 mg of 10-*O*benzoyltheveside and 270 mg of theveside.

According to [44] the iridoids fulvoplumierin, allamandin, *α*- and *β*-allamcidin, plumieride, 15-demethylplumieride, 13-*O*-*trans*-*p*-coumaroylplumieride and plumericin are present in extracts of *Plumeria rubra*. For isolation of these iridoids, the stem bark (2.5 kg) was successively extracted with petroleum ether and methanol followed by bioguided fractionation to investi‐ gate the cytotoxic activity against various cancer cells, particularly murine lymphocytic leukemia cells (P-388). Petroleum ether extract (30 g) was submitted to flash column chroma‐ tography over silica gel (750 g, 230-400 mesh) with chloroform/petroleum ether (1:1) as eluent. Fraction 002 (200 mg) was rechromatographed using silica gel (120 g) and chloroform/ methanol (99:1) to isolate fulvoplumierin (25 mg after recrystallization from petroleum ether/ chloroform 1:1). The methanol extract (295 g) was partitioned between chloroform and water. The chloroform extract (60 g) was successively chromatographed in a silica gel column (1.5 kg) with gradients of increasing polarity, with chloroform and methanol, and then in a silica gel (600 g) with petroleum ether/chloroform/ethyl acetate (1:3:1) as eluent to furnish allamandin (12 mg after recrystallization from chloroform). Another fraction from the first column chromatography of the methanol extract (1.2 g) was purified in a silica gel column (400 g) and ethyl acetate/ methanol (97:3) and recrystallyzed from chloroform to furnish 9 mg of allamcin. The aqueous extract (200 g) was also subjected to column chromatography using silica gel column (2 kg) and gradient of chloroform and methanol. Plumieride (55 g) was obtained directly from fraction F 022 after recrystallization from methanol. Fraction F022 (29 g) also furnished 15-demethylplumieride and 13-*O*-*trans*-*p*-coumaroylplumieride (3 g) by column chromatography over silica gel and gradient of chloroform and methanol. Fraction F018 (800 mg) was purified over silica gel (400 g) with gradient of ethyl acetate and methanol to afford plumericin (18 mg after recrystallization with ethyl acetate). Another fraction (F019) was purified over silica gel (250 g) with ethyl acetate/chloroform/methanol (6:6:1) solvent system to furnish an unstable iridoid aldehyde and a mixture of *α*- and *β*-allamcidin (12 mg and 16 mg, respectively) further isolated by TLC on silica gel plates (20 cm x 20 cm, 250 µm) with ethyl acetate/chloroform/methanol (3:3:1).

The supernatant was chromatographed on a silica gel column and cerbinal, cerberic acid and

The iridoids isoplumericin, plumericin, plumieride, plumieride coumarate and plumieride coumarate glucoside can be detected and quantified in several species of *Plumeria* and *Allamanda* by TLC using silica gel 60 and the following mobile phases: benzene/ethyl acetate 4:1, chloroform/methanol 4:1, chloroform/methanol 7:3, propanol/ethyl acetate/water 7:2:1 [84]. Visualization of the chromatograms is achieved by spraying with 50% sulfuric acid/ ethanol solution and heating. For the analyses, it is necessary to use iridoids as standards that can be isolated from the roots of *Allamanda cathartica*. For the isolation of iridoids during the development of the method, chloroform and methanol extracts, sequentially obtained in a Soxhlet apparatus from the root bark and inner roots of *Allamanda cathartica* (15.0 g for each extract) were successively fractionated on column chromatography. In the first chromatogra‐ phy step, the chloroform extract from the bark (1.7 g) was applied to a silica gel column (80 g) and eluted with gradient of petrol, ethyl ether, chloroform and methanol. This procedure yielded a mixture containing isoplumericin and plumericin (160 mg). Fractions eluted with chloroform/ methanol (3:2) were mixed with the methanol extract of the bark (total mass: 2.8 g) and chromatographed on a silica gel column (150 g deactivated with water) with gradient of chloroform and methanol as mobile phase to furnish plumieride coumarate (150 mg), plumieride coumarate glucoside (480 mg) and two mixtures: one containing plumieride and plumieride coumarate, and the other containing plumieride and plumieride coumarate glucoside. The first mixture was further resolved on partition between water and ethyl acetate and afforded 180 mg of plumieride and 150 mg of plumieride coumarate. The other mixture was rechromatographed to afford plumieride (300 mg) and plumieride coumarate glucoside (410 mg). Neither the chloroform (810 mg) nor the methanol (920 mg) extracts from the inner part of the roots contained isoplumericin and plumericin. These extracts were purified on silica gel column (75 g) deactivated with water and eluted with chloroform and methanol gradient, to give plumieride (70 mg), plumieride coumarate (80 mg) and plumieride coumarate gluco‐

The fresh leaves of *Cerbera manghas* contain the iridoids 10-*O*-benzoyltheveside, 10-dehydro‐ geniposide, loganin, theviridoside and theveside [124]. For the isolation, the methanol extract obtained by percolation using 2.6 kg of fresh leaves was extracted with butanol, and this extract was partitioned with benzene. After partition, the remaining butanol fraction (22.8 g) was subjected to column chromatography using MCI-gel (CHP-20) as stationary phase and a gradient of methanol/water as eluent. The fraction eluted with 20% methanol was subjected to C-18 column (elution with acetonitrile/water) to afford 20 mg of 10-dehidrogeniposide and 20 mg of loganin. The fraction eluted with pure water (2.6g) in the first chromatographic step was also subjected to C-18 column (elution with acetonitrile/ water) to furnish 23 mg of 10-*O*-

According to [44] the iridoids fulvoplumierin, allamandin, *α*- and *β*-allamcidin, plumieride, 15-demethylplumieride, 13-*O*-*trans*-*p*-coumaroylplumieride and plumericin are present in extracts of *Plumeria rubra*. For isolation of these iridoids, the stem bark (2.5 kg) was successively extracted with petroleum ether and methanol followed by bioguided fractionation to investi‐

cerberinic acid were eluted with benzene/acetone.

side (200 mg).

164 Column Chromatography

benzoyltheveside and 270 mg of theveside.

Plumericin, isoplumericin, plumieride, 13-*O*-coumaroylplumieride and protoplumericine A were isolated from the ethyl acetate extract of *P. rubra* heartwood [85]. The extract (44 g) obtained by percolation was submitted to column chromatography on silica gel with light petroleum ether/ethyl acetate (1:1) and methanol as eluent to furnish four fractions (A-D). Fraction C directly afforded plumericin (1.22 g) after recrystallization from toluene/ethyl acetate. Fraction B was rechromatographed on a silica gel with toluene/ethyl acetate (9:1), and isoplumericin (140 mg) was isolated from fraction 2. Fraction D was also rechromatographed on a silica gel column, but with chloroform/methanol/water (90:10:0.5 to 70:30:10) as solvent system. Fraction 3' was 13-*O*-coumaroylplumieride (2.0 g) and fraction 7' was protoplumeri‐ cine (1.6 g). Fraction 5' was subjected to Sephadex LH20 column eluted with methanol, followed by silica gel column eluted with chloroform/ methanol/water (85:15:0.7) to furnish plumieride (120 mg).

The iridoids, plumericin, isoplumericin, plumieride and fulvoplumierin, were present in the extracts of *Plumeria rubra* bark. After maceration of the powdered bark (3.5 kg) with dichloro‐ methane/methanol (1:1) and pure methanol, the combined extracts were partitioned between water and ethyl acetate. To isolate the four iridoids, the organic layer was chromatographed twice in a column using silica gel and gradient of increasing polarity with hexane and ethyl acetate, ethyl acetate and methanol, and then pure methanol. The amounts of the compounds isolated were not reported [75].

The flowers of *Plumeria rubra* L. cv. acutifolia can provide plumericidine, as described by [97]. The ethanol (95%) extract, obtained from 2.9 kg of flowers, was successively partitioned with petroleum ether, ethyl acetate and butanol. The ethyl acetate fraction was sequentially submitted twice to column chromatography using silica gel and gradient of chloroform/ methanol as mobile phase. Chromatography on a Sephadex LH20 column yielded 20 mg of plumericidine.

sequentially chromatographed on a column. The first chromatography, over silica gel (2.1 kg) and using gradient of chloroform and ethyl ether, furnished five fractions (1-5). From fraction 2, 8 mg of scholarein B and 7 mg of scholarein D were isolated after silica gel chromatography and elution with petroleum ether/ethyl ether (3:1). From fraction 3, 25 mg of scholarein A and 60 mg of scholarein C were obtained after successive columns using silica gel and chloroform/

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In the study on iridoids, the technique of gas chromatography is generally used for analytical purposes. Gas chromatography represents an advantage over thin layer chromatography, particularly for detecting substances in small amounts, and mass spectrometry can be used to

Methods have been developed by [112] for the detection of 33 iridoids and secoiridoid glucosides in mixtures and plant extracts using gas chromatography sometimes coupled to mass spectrometry. For the gas chromatography analyses, a Shimadzu Model GC-1C gas chromatograph with hydrogen FID-1B flame ionization detector was used. Columns were packed with 1.5% OV-1, 1.5% OV-17, 2% OV-210 and 2% OV-225 on 80-100-mesh Shimalite W AW/DMCS. In all, 33 iridoids and secoiridoids glucosides are analyzed as TMS-derivatives. Using the 1.5% OV-17 column with 1.8 m in length and 4 mm in I.D. at 270 ºC, the elution order was: aucubin, 7-deoxyloganic acid and catalpol (retention time = 1.37 min), 7-deoxyloganin, monotropein, gardenoside, secologanin, loganin, scandoside, theviridoside, geniposide, scandoside methyl ester, 7-dehydrologanin, morroniside, hastatoside and forsythide (reten‐ tion time = 2.55 min), forsythide 10-methyl ester, verbenalin, sweroside, gentiopicroside and swertiamarin (retention time = 3.08 min), bankakosin, kingiside, amaroswerin, amarogentin and asperuloside. The 1.5% OV-17 column, 0.5 m in length and 3 mm in I.D., at 230ºC, furnished the same elution order as above, but forsythide 10-methyl ester, verbenalin and sweroside eluted together (retention time = 3.20 min), while the separation of hastatoside and forsythide, gentiopicroside and swertiamarin were better. When the non-polar 1.5% OV-1 column with 1.8 m in length and 4 mm in I.D. was used at 270 ºC, better separation between loganin and secologanin occurred. Better results were achieved for the 7-deoxyloganic, 7-deoxyloganic acid and catalpol. However, verbenalin and sweroside eluted together, and amaroswerin and amarogentin were not detected. The OV-17 column was slightly polar and, in general, it influenced the larger range of the retention times. The more polar columns with 2% OV-210 at 215 ºC, and 2% OV-225 at 230 ºC, both with 0.5 m in length, showed important differences from keto compounds, such as 7-dehydrologanin and verbenalin, and lactonic compounds, such as sweroside, gentiopicroside and gentiopicroside, reflected in their longer retention times. Amaroswerin and amarogentin were not detected. Sweroside and gentiopicroside were well-separated on OV-210 column, which was not observed using other columns. Paederoside, ligustroside, catalposide, oleuropein, 10-acetoxyligustroside and 10-acetoxyoleuropein were only detected and well-separated in OV-17 and OV-1 columns with 0.5m in length at 270 °C.

For the gas chromatography-mass spectrometry studies, a Hitachi K-53 gas chromatograph and a Hitachi RMU-6 E mass spectrometer were used. The glass columns, 0.5 m x 3 mm in I.D.,

distinguish most iridoid and secoiridoid glucosides by fragmentation patterns [112].

ethyl ether as stationary and mobile phases, respectively.

**4.3. Gas Chromatography (GC)**

According to [72], several iridoids can be isolated from the aerial parts of *Plumeria obtusa*: obtusadoid A, obtusadoid B, plumieridin A, 1α-plumieride, 15-demethylplumieride and plumieridine. The methanol extract (400 g) was obtained from 10 kg of the plant material and sequentially partitioned with hexane and ethyl acetate. The ethyl acetate extract was chroma‐ tographed using a silica gel column and gradients of hexane, ethyl acetate and methanol. The less polar fractions were rechromatographed in the same stationary phase, and eluted with hexane/dichloromethane (1:1) to afford obtusadoid A (6 mg), obtusadoid B (11.5 mg), plu‐ mieridin A (8 mg) and plumieridine (12 mg). The more polar fraction obtained in the first chromatography was filtered on a Sephadex LH20 column using methanol, and further submitted to RP-8 flash column chromatography. Elution with 50% methanol afforded 1αplumieride (22 mg) and 15-demethylplumieride (13 mg).

Plumieride also can be isolated from the bark of *Plumeria bicolor* [114]. Powdered bark (4 kg) was extracted in methanol, and the crude extract was washed with acetonitrile. The material was re-extracted with chloroform, and this extract was fractionated in column chromatogra‐ phy using silica gel (900 g) and different solvents of increasing polarity. Plumieride was eluted with chloroform/ethyl acetate (1:1) and recrystallized from methanol.

The bark of *Plumeria bicolor* also contains plumericin and isoplumericin, as described in [86]. The methanol extract (100 g), after washing with acetonitrile, was extracted with chloroform and chromatographed on a column containing 800 g of silica gel G (60-120 mesh). Elution was carried out using gradients of increasing polarities with benzene, chloroform and methanol. Plumericin and isoplumericin was recrystallized from methanol.

Isoplumericin and plumericin are present in the bark of *Himatanthus sucuuba* [88]. For the isolation of these iridoids, 95% ethanol extract (2 g), obtained from 50 g of plant material was submitted to column chromatography using silica gel and gradients of increasing polarities with hexane, ethyl acetate and methanol. After recrystallization, isoplumericin (18 mg) was obtained from ethyl acetate and plumericin (70 mg) from methanol.

The stem bark of *Winchia calophylla* contains loganin (1.25 g) [129]. The 95% ethanol extract (600 g) from the dried stem bark (10.5 kg) was partitioned between petroleum ether and water. The petroleum ether extract was submitted to acid-base extraction and after adjustment to pH 9-10 with ammonium hydroxide; the aqueous layer was extracted with petroleum ether, chloro‐ form and butanol. The chromatography of the butanol fraction using silica gel H column led to the isolation of loganin.

The iridoids, scholarein A, B, C and D, can be obtained by the fractionation of the ethanol extract from bark of *Alstonia scholaris* [60]. The crude extract, obtained from 15 kg of the plant material, was partitioned between ethyl acetate and water. The organic layer (190 g) was sequentially chromatographed on a column. The first chromatography, over silica gel (2.1 kg) and using gradient of chloroform and ethyl ether, furnished five fractions (1-5). From fraction 2, 8 mg of scholarein B and 7 mg of scholarein D were isolated after silica gel chromatography and elution with petroleum ether/ethyl ether (3:1). From fraction 3, 25 mg of scholarein A and 60 mg of scholarein C were obtained after successive columns using silica gel and chloroform/ ethyl ether as stationary and mobile phases, respectively.
