**4.3. Gas Chromatography (GC)**

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

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 1α-

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

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.

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

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

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

plumieride (22 mg) and 15-demethylplumieride (13 mg).

with chloroform/ethyl acetate (1:1) and recrystallized from methanol.

Plumericin and isoplumericin was recrystallized from methanol.

obtained from ethyl acetate and plumericin (70 mg) from methanol.

to the isolation of loganin.

plumericidine.

166 Column Chromatography

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

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., were packed with 1.5% OV-17 on 80-100-mesh Shimalite W AW/DMCS and 1.5 % OV-1 on 80-100-mesh Shimalite W AW/DMCS, and were used to the oleuropein-type glucosides detection. The authors considered the GC-MS identification of some iridoids was not satisfac‐ tory: any important peak different from the sugar moiety was detected in asperuloside and paederoside TMS-derivatives of amaroswerin, amarogaentin asperuloside and paederoside exhibited different retention times, but the same fragmentation pattern; separation using this technique was unsuccessful [112].

mieride coumarate glucoside, respectively. Furthermore, plumieride, plumieride coumarate and plumieride glucoside were hydrolyzed under heating and acid conditions (sulfuric acid, 1 N for 2-3 h), extracted with ethyl acetate and both organic phase and aqueous phase (after neutralization with Amberlite) were analyzed by TLC and GC. Analyses were performed on a 1.5% OV-17 glass column with 0.4 m length and 4 mm I.D. Other analytical conditions were: nitrogen as carrier gas at 50 mL/min; detector temperature, 320 ºC; column temperatures: 190 ºC for isoplumericin and plumericin, 240 ºC for TMS-plumieride, 300 ºC TMS-plumieride coumarate. Glucose was detected in aqueous phases from acid hydrolyses of plumieride, plumieride coumarate and plumieride coumarate glucoside, while *p*-coumaric acid was detected in the organic phases of plumieride coumarate and plumieride coumarate glucoside. GC analyses also showed that plumieride coumarate was isolated as an isomer mixture (20%

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

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

> **SPECIES/ PLANT MATERIAL**

> *Rauwolfia grandiflora*/ bark

*Thevetia peruviana/* root

*Thevetia peruviana/* leaves

*Rauwolfia* / barks

ethanol extract

methanol extract

methanol extract

> ethanol extract

**SAMPLE IRIDOID REF**

A General Description of Apocynaceae Iridoids Chromatography

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

169

theviridoside, 10-*O*-β-Dfructofuranosyltheviridoside, 6'- *O*-β-Dglucopyranosyltheviridoside, 10- *O*-β-Dglucopyranosyltheviridoside and 3'-*O*-β-Dglucopyranosyltheviridoside

10-*O*-β-Dfructofuranosyltheviridoside and 6'-*O*-β-Dglucopyranosyltheviridoside

> loganin, loganic acid and gardenoside

boonein and isoboonein [1]

[125]

[127]

[131]

cis and 80% trans isomers) [116].

isolated from Apocynaceae by HPLC, MPLC and LPLC.

μBondapack C-18

Octadecylsilane (ODS)

Octadecylsilane (ODS)

Cosmosil 5 C18- AR (5 μm, 25 cm × 4.6 mm I.D.)

**TECHNIQUE MOBILE PHASE COLUMN**

50% methanol in water

HPLC 10% acetonitrile

HPLC 10% acetonitrile

20 mM KH2PO4 and acetonitrile Flow: 0.8 mL/min

**LPLC)**

HPLC

HPLC

Aqueous extracts of different plant species with known presence of iridoid and secoridoid glucosides were analyzed by GC-FID and GC-MS [112]. One of these species was *Allamanda cathartica* var. schottii (Pohl) Rafill (Apocynaceae) cultivated in a greenhouse. Aqueous extracts obtained from 3-5 g of fresh plant material and hot water were treated in a column of charcoal (active carbon for column chromatography) for removal of sugars by elution with water. The sample was eluted with methanol and concentrated under reduced pressure. TMS-derivatives were prepared. For the analyses, a 1.5% OV-17 column with 1.8 m in length at 280 ºC was used. GC-FID and GC-MS (70 eV) showed the presence of one iridoid glycoside. The fragmentation pattern indicated that the original glucoside was plumieride, and that relative retention time was the same as that of asperuloside.

Isoplumericin, plumericin, plumieride, plumieride coumarate and plumieride coumarate glucoside can be detected by GC-FID [116]. For the development of the method, it was necessary to isolate these iridoids for use as standard. The methanol extract of *Allamanda cathartica* L. roots, obtained with 500 g of the dried plant material and boiling methanol, was submitted to silica gel 60 column (2.5 kg) with the eluents: petrol, petrol/ethyl ether, ethyl ether, ethyl ether/chloroform, chloroform/ methanol and pure methanol [116]. Fractionation was monitored by TLC [84]. The fractions were eluted with petrol/ethyl ether until chloroform/ methanol contained isoplumericin and plumericin (4.2 g). This mixture (1.0 g) was suspended in ethyl ether and rechromatographed on silica gel (40 g). Elution with petrol and a gradient of increasing polarity with petrol/ethyl ether furnished isoplumericin (250 mg), plumericin (140 mg) and a mixture of both (290 mg). A second fraction eluted with chloroform/methanol in the first chromatographic step (8.0 g) was resubmitted to column chromatography on silica gel (300 g). Elution with a gradient of chloroform and methanol led to the isolation of plu‐ mieride coumarate (5.1 g). The third fraction of the first chromatograph step (10.0 g), eluted with chloroform/methanol, was partitioned between water and ethyl acetate to give plumier‐ ide coumarate in the organic phase (3.8 g) and plumieride in the aqueous phase (1.8 g). Finally, 30.0 g of the forth fraction of the first chromatograph step, eluted with chloroform/methanol to methanol, was submitted to column chromatography on silica gel (1.0 kg) deactivated with water. The elution was carried out with a gradient of chloroform and methanol and furnished plumieride coumarate glucoside (6.2 g). GC-FID analyses were used to evaluate the pure grade of fractions and isolated substances. For these analyses, trimethylsilylation of iridoids using HMDS-TMCS and pyridine, and acetylations with acetic anidride and pyridine, were neces‐ sary. Plumieride coumarate (isomer mixture) and plumieride coumarate glucoside were acetylated and their products were purified on chromatography with silica gel column and ethyl ether as eluents, to afford pure penta-acetylplumieride coumarate and octa-acetylplu‐ mieride coumarate glucoside, respectively. Furthermore, plumieride, plumieride coumarate and plumieride glucoside were hydrolyzed under heating and acid conditions (sulfuric acid, 1 N for 2-3 h), extracted with ethyl acetate and both organic phase and aqueous phase (after neutralization with Amberlite) were analyzed by TLC and GC. Analyses were performed on a 1.5% OV-17 glass column with 0.4 m length and 4 mm I.D. Other analytical conditions were: nitrogen as carrier gas at 50 mL/min; detector temperature, 320 ºC; column temperatures: 190 ºC for isoplumericin and plumericin, 240 ºC for TMS-plumieride, 300 ºC TMS-plumieride coumarate. Glucose was detected in aqueous phases from acid hydrolyses of plumieride, plumieride coumarate and plumieride coumarate glucoside, while *p*-coumaric acid was detected in the organic phases of plumieride coumarate and plumieride coumarate glucoside. GC analyses also showed that plumieride coumarate was isolated as an isomer mixture (20% cis and 80% trans isomers) [116].

were packed with 1.5% OV-17 on 80-100-mesh Shimalite W AW/DMCS and 1.5 % OV-1 on 80-100-mesh Shimalite W AW/DMCS, and were used to the oleuropein-type glucosides detection. The authors considered the GC-MS identification of some iridoids was not satisfac‐ tory: any important peak different from the sugar moiety was detected in asperuloside and paederoside TMS-derivatives of amaroswerin, amarogaentin asperuloside and paederoside exhibited different retention times, but the same fragmentation pattern; separation using this

Aqueous extracts of different plant species with known presence of iridoid and secoridoid glucosides were analyzed by GC-FID and GC-MS [112]. One of these species was *Allamanda cathartica* var. schottii (Pohl) Rafill (Apocynaceae) cultivated in a greenhouse. Aqueous extracts obtained from 3-5 g of fresh plant material and hot water were treated in a column of charcoal (active carbon for column chromatography) for removal of sugars by elution with water. The sample was eluted with methanol and concentrated under reduced pressure. TMS-derivatives were prepared. For the analyses, a 1.5% OV-17 column with 1.8 m in length at 280 ºC was used. GC-FID and GC-MS (70 eV) showed the presence of one iridoid glycoside. The fragmentation pattern indicated that the original glucoside was plumieride, and that relative retention time

Isoplumericin, plumericin, plumieride, plumieride coumarate and plumieride coumarate glucoside can be detected by GC-FID [116]. For the development of the method, it was necessary to isolate these iridoids for use as standard. The methanol extract of *Allamanda cathartica* L. roots, obtained with 500 g of the dried plant material and boiling methanol, was submitted to silica gel 60 column (2.5 kg) with the eluents: petrol, petrol/ethyl ether, ethyl ether, ethyl ether/chloroform, chloroform/ methanol and pure methanol [116]. Fractionation was monitored by TLC [84]. The fractions were eluted with petrol/ethyl ether until chloroform/ methanol contained isoplumericin and plumericin (4.2 g). This mixture (1.0 g) was suspended in ethyl ether and rechromatographed on silica gel (40 g). Elution with petrol and a gradient of increasing polarity with petrol/ethyl ether furnished isoplumericin (250 mg), plumericin (140 mg) and a mixture of both (290 mg). A second fraction eluted with chloroform/methanol in the first chromatographic step (8.0 g) was resubmitted to column chromatography on silica gel (300 g). Elution with a gradient of chloroform and methanol led to the isolation of plu‐ mieride coumarate (5.1 g). The third fraction of the first chromatograph step (10.0 g), eluted with chloroform/methanol, was partitioned between water and ethyl acetate to give plumier‐ ide coumarate in the organic phase (3.8 g) and plumieride in the aqueous phase (1.8 g). Finally, 30.0 g of the forth fraction of the first chromatograph step, eluted with chloroform/methanol to methanol, was submitted to column chromatography on silica gel (1.0 kg) deactivated with water. The elution was carried out with a gradient of chloroform and methanol and furnished plumieride coumarate glucoside (6.2 g). GC-FID analyses were used to evaluate the pure grade of fractions and isolated substances. For these analyses, trimethylsilylation of iridoids using HMDS-TMCS and pyridine, and acetylations with acetic anidride and pyridine, were neces‐ sary. Plumieride coumarate (isomer mixture) and plumieride coumarate glucoside were acetylated and their products were purified on chromatography with silica gel column and ethyl ether as eluents, to afford pure penta-acetylplumieride coumarate and octa-acetylplu‐

technique was unsuccessful [112].

168 Column Chromatography

was the same as that of asperuloside.
